CA1340733C - Regulatory region for heterologous gene expression in yeast - Google Patents

Abstract

Novel DNA sequences which are responsive to the presence of methanol, catabolite non-repressing carbon sources and carbon source starvation are provided. In addition, novel constructs including these DNA sequences, as well as transformed organisms therewith are provided. Processes for producing the DNA sequences and constructs of the invention are detailed. The production of polypeptide product under the control of the regulatory regions of the invention is demonstrated.

Description

~~~v73~

REGULATORY REGION FOR
HETEROLOGOUS GENE EXPRESSION IN YEAST
' Background This invention relates to the field of recombinant DNA biotechnology. In one of its aspects, the invention relates to DNA fragments which regulate the transcription of DNA into messenger RNA, and the initiation and termination of the translation of messenger RNA into protein. In another aspect, the invention relates to expression vectors which incorporate the above-described DNA fragments. In yet another aspect, the invention relates to novel microorganisms transformed with the above-described expression vectors. In a further aspect, the invention relates to the production of polypeptides.
As :recombinant DNA technology has developed in recent years, the controlled production by microorganisms of an enormous variety of useful polypeptides has become possible. Many eukaryotic polypeptides, such as for example human growth :hormone, leukocyte interferons, human insulin and human proinsulin have already been produced by various microorganisms. The continued application of techniques already in hand is expected in the future to permit production by microorganisms of a variety of other useful polypeptide products.

.---2 1~~0'~33 The basic techniques employed in the field of recombinant DNA technology are known by those of skill in the art. The elements desirably present in order for a host microorganism to be useful for the practice of recombinant DNA technology include, but are not limited to:
(1) a gene encoding one or more desired polypeptide(s) and provided with adequate control sequences required for expression in the host microorganism, (2) a vector, usually a plasmid, into which the gene can be inserted. The vector serves to guarantee transfer of the gene into the cell and maintenance of DNA
sequences in i~he cell as well as a high level of expression of the above-mentioned gene, and (3) a suitable host microorganism into which the vector carrying the desired gene can be transformed, where the host microorganism also has the cellular apparatus to allow expression of the information coded for by the inserted gene.
A basic element employed in recombinant DNA
technology is. the plasmid, which is extrachromosomal, double-stranded DNA found in some microorganisms. Where plasmids have been found to naturally occur in microorganisms, they are often found to occur in multiple copies per cell. In addition to naturally occurring plasmids, a variety of man-made plasmids, or hybrid vectors, have been prepared. Included in the information encoded in plasmid DNA i.s that required to reproduce the plasmid in daughter cells, i.e., an autonomously replicating sequence or an origin of replication. One or more phenotypic selection characteristics must also be included in the information encoded in t:he plasmid DNA. The phenotypic selection characteristics permit clones of the host cell containing the plasmid of interest to be recognized and selected by preferential growth of the cells in selective media.

3 ~.~~~'~133 The utility of plasmids lies in the fact that they can be specifically cleaved by one or another restriction endonuclease or restriction enzyme, each of which recognizes a specific, unique site on the plasmid DNA. Thereafter, homologous genes, heterologous genes, i.e., genes derived from organisms. other than the host, or gene fragments may be inserted into the plasmid by endwise joining of the cleaved plasmid and desired genetic material at the cleavage site or at reconstruci~ed ends adjacent to the cleavage site. The resulting recombined DNA material can be referred to as a hybrid vector.
DNA recoiribination is performed outside the host microorganism. The resulting hybrid vector can be introduced into the host microorganism by a process known as transformation. By growing the transformed microorganism, large quantities of the hybrid vector can be obtained. When the gene is properly inserted with reference to the portions of the plasmid which govern transcription and translation of the encoded DNA message, the resulting hybrid vector can be used to direct the production of the polypeptide sequence for which the inserted gene codes. The production of polypeptide in this fashion is referred to as gene expression.
Gene expression is initiated in a DNA region known as the promoter region. In the transcription phase of expression, the DNA. unwinds exposing it as a template for synthesis of messenger RNA. RNA polymerase binds to the promoter region and travels along the unwound DNA from its 3~
end to its 5~ end, transcribing the information contained in the coding strand into messenger RNA (mRNA) from the S~ end to the 3~ end of the mRNA. The messenger RNA is, in turn, bound by ribosomes, where the mRNA is translated into the polypeptide chain. Each amino acid is encoded by a nucleotide triplet or codon within what may be referred to as the structural gene, i.e., that part of the gene which encodes the amino acid sequence of the expressed product.

~~ 4 ___ Since three nucleotides code for the production of each amino acid, it is possible for a nucleotide sequence to be "read"
in three different ways. The specific reading frame which encodes the desired polypeptide product is referred to as the proper reading frame.
After binding to the promoter, RNA polymerase first transcribes a 5~ leader region of mRNA, then a translation initiation or start codon, followed by the nucleotide codons within the structural gene itself. In order to obtain the desired gene product, it is necessary for the initiation or start codon to correctly initiate the translation of messenger RNA by the ribosome in the proper reading frame.
Finally, stop codons are transcribed at the end of the structural gene which cause any additional sequences of mRNA
to remain untranslated into peptide by the ribosomes, even though additional sequences of mRNA had been formed by the interaction of RNA polymerase with the DNA template. Thus, stop codons determine the end of translation and therefore the end of further incorporation of amino acids into the polypeptide product. The polypeptide product can be obtained by lysing the host cell and recovering the product by appropriate purification from other microbial protein, or, in certain circumstances, by purification of the fermentation medium in which the host cells have been grown and into which the polypeptide product has been secreted.
In practice, the use of recombinant DNA technology can create m:icroorganisms capable of expressing entirely heterologous polypeptides, i.e., polypeptides not ordinarily found in, or produced by, a given microorganism --- so called direct expression. Alternatively, there may be expressed a fusion protein, i.e., a heterologous polypeptide fused to a portion of the amino acid sequence of a homologous polypeptide, i.e., polypeptides found in, or produced by, the wild-type (non.-transformed) host microorganism --- so called indirect expression. With indirect expression, the initially ~.~~(1'~33 obtained fusion protein product is sometimes rendered inactive foz: its intended use until the fused homologous/het.erologous polypeptide is cleaved in an extracellular environment. Thus, for example, cyanogen 5 bromide cleavage of methionine residues has yielded somatostatin, thymosin alpha 1 and the component A and B
chains of human insulin from fused homologous/heterologous polypeptides, while enzymatic cleavage of defined residues has yielded beta endorphin.
Up to now, commercial efforts employing recombinant DNA technology for producing various polypeptides have centered on Escherichia coli as a host organism. However, in some situations E. coli may prove to be unsuitable as a host.
For example, E. coli contains a number of toxic pyrogenic factors that must be eliminated from any polypeptide useful as a pharmaceutical product. The efficiency with which this purification c:an be achieved will, of course, vary with the particular polypeptide. In addition, the proteolytic activities of E. coli can seriously limit yields of some useful products. These and other considerations have led to increased interest in alternative hosts, in particular, the use of eukaryotic organisms for the production of polypeptide products is appealing.
The availability of means for the production of polypeptide products in eukaryotic systems, e.g., yeast, could provide significant advantages relative to the use of prokaryotic systems such as E. coli for the production of polypeptides encoded by recombinant DNA. Yeast has been employed in :Large scale fermentations for centuries, as compared to the relatively recent advent of large scale E.
coli fermentat:ions. Yeast can generally be grown to higher cell densities than bacteria and are readily adaptable to continuous fermentation processing. In fact, growth of yeast such as Pichia pastoris to ultra-high cell densities, i.e., cell densities in excess of 100 g/L, is disclosed by Wegner in U.S. 4,414,329 (assigned to Phillips Petroleum Co.).
Additional advantages of yeast hosts include the fact that many critical functions of the organism, e.g., oxidative phosphorylatio~n, are located within organelles, and hence not exposed to the possible deleterious effects of the organism's production of polypeptides foreign to the wild-type host cells. As a eukaryotic organism, yeast may prove capable of glycosylating expressed polypeptide products where such glycosylation is important to the bioactivity of the polypeptide product. It is also possible that as a eukaryotic organism, yeast will exhibit the same codon preferences as higher organisms, thus tending toward more efficient pro<iuction of expression products from mammalian genes or from complementary DNA (cDNA) obtained by reverse transcription from, for example, mammalian mRNA.
The development of poorly characterized yeast species as host/vector systems is severely hampered by the lack of knowledge about transformation conditions and suitable vectors. In addition, auxotrophic mutations are often not available, precluding a direct selection for transformants by auxotrophic complementation. If recombinant DNA technology is to fully sustain its promise, new host/vector systems must be devised which facilitate the manipulation of DNA as well as optimize expression of inserted DNA sequences so that the desired polypeptide products can be prepared under controlled conditions and in high yield.
Obyjects of the Invention An c>bject of our invention is therefore a novel regulatory region responsive to the presence of methanol.
A further object of the invention is a novel catabolite sensitive regulatory region which is responsive to the presence of some carbon sources but which is not responsive to 'the presence of other carbon sources.

Another object of the invention is a novel regulatory region responsive to carbon source starvation.
Yet another object of our invention is novel vectors capable of expressing an inserted polypeptide coding sequence.
Still another object of our invention is novel yeast strains of the genus Pichia and Saccharomyces.
A further object of our invention is a process for producing polypeptides employing novel yeast strains as described hereinabove.
These and other objects of our invention will become apparent from the disclosure and claims herein provided.
Statement of the Invention In accordance with the present invention, we have discovered, isolated and characterized DNA sequences which control the ltranscription of DNA into messenger RNA and translation of the messenger RNA to give a polypeptide product. The novel DNA sequences of this invention are useful for the production of polypeptide products by (a) yeast strains which are capable of growth on methanol as a carbon and energy source, (b) yeast strains which are capable of growth on glucose, ethanol, fructose and the like; and (c) yeast strains which are capable of growth on glycerol, galactose, acetate and the like.
Brief Description of the Figures Figure 1 is a correlation of the relationship between the ge:nomic clone (pPG 6.0) and cDNA clone (pPC 15.0) for protein p76.
Figure 2 is a correlation of the relationship between the ge:nomic clone (pPG 4.0) and cDNA clones (pPC 8.3 and pPC 8.0) for protein p72 (alcohol oxidase).

~3~0~33 Figure 3 is a correlation of the relationship be-tween the genomic clone (pPG 4.8) and cDNA clone (pPC 6.7) for protein p40.

Figure 4 provi des restriction maps of regulatory regions of the invention. from clone pPG 6Ø

Figure 5 is a restriction map of the regulatory re-gion of the invention fr om clone pPG 4Ø

Figure 6 is a restriction map of the regulatory re-gion of the invention fr om clone pPG 4.8.

Figure 7 is a restriction map of a sequence of DNA

obtained from the 3~ end of the p76 structural gene.

Figure 8 is a restriction map of a sequence of DNA

obtained from the 3~ end. of the p72 (alcohol oxidase) struc-tural gene.

Figure 9 is a restriction map Qf a sequence of DNA

~

obtained from the 3 of the p40 structural gene.
end Figure 10 is a restriction map of the protein p76 ~

structural gene and the 5 regulatory region therefor.

Figure 11 is a restriction map of the protein p40 ~

structural gene and the 5 regulatory region therefor.

Figure 12 is a restriction map of the protein p76 cDNA.
Figure 13 is a restriction map of the protein p72 (alcohol oxidase) ~~DNA.
Figure 14 is a restriction map of the protein p40 cDNA.
Figure 15 provides restriction maps of two novel p76 regulatory region-ZacZ DNA constructs of the invention.
Figure 16 is a restriction map of a novel p72 (al-cohol oxidase) regulatory region~ZacZ DNA construct of the invention.
Figure 17 is a restriction map of plasmid pSAOHl.
Figure 1~B is a restriction map of plasmid pSAOH5.
Figure 19 is a restriction map of plasmid pSAOHIO.
!1B

r~ 9 134~'~33 Figure 20 is a restriction map of plasmid pTAFH.85.
Figure 21 is a restriction map of plasmid pT76H1.
Figure 22 is a. restriction map of plasmid pT76H2.
Figure 22a is a.restriction map of plasmid pT76H3.
Figure 22b is a restriction map of plasmid pT76H4.
Figure 23 is a restriction map of plasmid pYA2.
Figure 24 is a restriction map of plasmid pYA4.
Figure 25 is a restriction map of plasmid pYJ8.
Figure 26 is a restriction map of plasmid pYJ8~CZa.
Figure 27 is a restriction map of plasmid pYJ30.
Figure 28 provides a restriction map of plasmid pTAFHl and shows how the plasmid was derived.
Figure 29 provides a restriction map of plasmid pTA012 and shows how the plasmid was derived.
Figure 30 is a restriction map of plasmid pTA013.
Figure 30a is a restriction map of plasmid pT76U1.
Figure 31 provides a restriction map of plasmid pTA01 and shows how the plasmid was derived:
Figure 32 provides a restriction map of plasmid pTAF.85 and shows :how the plasmid was derived.
Figure 33 provides a restriction map of plasmid YEpl3.
Figure 3~4 is a restriction map of pBPfl.
The following abbreviations are used throughout this application to represent the restriction enzymes em ployed:
H3 ~- HindIII
Rl :- EeoRI

H2 :- HincLI

Xh :- XhoT

Ps :- Ps~I

Pvl:= PvuI

Pv2:= PvuII

B ~- BamHI

K ~- KpnI

St :- StuI

Ndl:= Nde I

B2 :_ BgZII

>.a .

w l~~v'~3 Sc = SacI
S - SaZI
R5 = EcoRV
Xb = Xbal 5 Rs = RsaI:
C - CZaI
Xm = Xmal Ss = SstI
Bc = BeZI
10 A - AsuII
Nr = NruI
Sm = SmaI
Th = Thai S3 = Sau3AI
Sp = SphI
T - TaqI
In the attached figures, restriction sites employed for mani pulation of DNA fragments, but which are destroyed upon liga tion are indicated by enclosing the abbreviation for the des troyed site in parentheses.
Detailed Description of the Invention In accordance with the present invention, there is provided a novel DNA fragment comprising a regulatory region responsive to at least one of the following conditions: the presence of methanol, carbon source starvation when cells are grown on some substrates other than methanol, and the presence of non-catabolite :repressing carbon sources other than metha-nol. The regulatory region of the DNA fragment of this inven-tion is capable of controlling the transcription of messenger RNA when positioned at the 5~ end o~ the DNA which codes for the production of messenger RNA. Also included within the scope of our invention axe mutants o~ the above~described DNA
fragment.
Further in accordance with the present invention, there is provided a DNA fragment which comprises a regulatory ".
~' ~4x4'~~~r'.:

region which :is capable of controlling the polyadenylation, termination oi: transcription and termination of translation of messenger RNA when positioned at the 3~ end of the polypeptide coding region which codes for the production of messenger RNA, wherein the transcription and translation of the messenger RNA is controlled by a regulatory region which is responsive to at least one of the following conditions:
the presence of methanol, carbon source starvation when cells are grown on some substrates other than methanol and the presence of non-catabolite repressing carbon sources other than methanol. Also included within the scope of our invention are mutants of the above-described DNA fragment.
Still further in accordance with a specific embodiment of the invention, there are provided DNA fragments which direct the incorporation of encoded polypeptide into peroxisomes. Peroxisomes are intracellular bodies present in large amount: in methanol grown yeast cells. These intracellular bodies serve to isolate the incorporated polypeptide product from intracellular fluids and enzymes such as proteases.
In accordance with another embodiment of the invention, genes coding for the production of alcohol oxidase, a protein of about 40 kilodaltons and a protein of about 76 kilodaltons are provided.
In accordance with yet another embodiment of the present invention, plasmids and transformed organisms containing the: above-described DNA fragments are provided.
In accordance with still another embodiment of the invention, methods are provided for producing the plasmids and DNA fragments of the invention, as well as heterologous polypeptides, i.e., polypeptides not native to the host organisms.

Isolation of Regulatable Genes from Pichia pastoris An .approximately 20,000 member cDNA library was prepared in E, coli: with poly A+ RNA isolated from Pichia pas~oris cell:a grown on methanol as the sole carbon source (See Example III). The library was screened by hybridization using kinased poly A+ RNA isolated from Pichia pastoris grown either in the presence of methanol or ethanol. After several rounds of this plus-minus screening, three distinct, non-homologous cDNA clones were identified as being copies of methanol specific messenger RNA's. These clones were designated as pPC 6.4, pPC 8.0, and pPC 15.0 and were determined to contain inserts of 470, 750 and 1100 nucleotides in length, respectively.
In an attempt to obtain cDNA clones of longer length, a second cDNA library was prepared using milder S1 nuclease dige~~tion conditions than used for the preparation of the first cDNA library and the members of this new library screened individually with 32P-labeled cDNA clones pPC 6.4, pPC 8.0, and pPC 15Ø As a result, larger cDNA clones were isolated corresponding to cDNA clones pPC 6.4 and pPC 8Ø
The larger clones, pPC 6.7 and pPC 8.3, were found to contain inserts measuring 1200 and 2100 nucleotides, respectively (See Figures 2 and 3). A cDNA clone possessing an insert larger than the 1100 nucleotides for pPC 15.0 has not been observed after screening more than 40,000 cDNA clones.
The isolation of the genomic DNA fragments corresponding to each of these cDNA clones was accomplished by first cutting out and electroeluting from agarose gels Pichia pastor_is DNA fragments of restriction endonuclease treated chromosomal DNA that hybridized with 32P-labeled pPC
15.0, pPC 8.0, or pPC 6.4. Then the eluted genomic DNA
fragments wei:e cloned into Escherichia coli and the appropriate genomic clones identified by screening several times with each of the above cDNA probes.

13 ~3~~~~3 The relationship of each cDNA clone to its corresponding genomic clone is illustrated in Figures 1, 2, and 3. pPC 15.0 is encoded within a 6000 nucleotide HindIII
genomic fragment present in clone pPG 6.0 (Figure 1). The 5~
end of the gene encoded by pPC 15.0 is oriented toward the 1300 by HindIII-EcaRI fragment contained in pPG 6.0, while the 3~ end of the gene is proximal to the PstI sites in pPG
6Ø
The cDNA clone pPC 8.3 is included within the genomic clone pPG 4.0 (Figure 2). pPG 4.0 contains an EcoRI-PvuII insert of 4000 nucleotides of contiguous genomic DNA. The orientation of pPC 8.3 within pFG 4.0 places the 5~
end of the gene for this cDNA clone close to the BamHI sites while the 3~ end of this gene is located near the PvuII site.
The orientation of pPC 8.0 (a related cDNA clone) within pPG

4.0 places the 5~ end of this cDNA clone close to the RpnI
site at the 3~~ end of gPG 4.0 and the 3~ end of the cDNA
clone is located near the PvuII site.
The cDNA clone pPC 6.7 is located entirely within a 4800 nucleotide EcaRI-BamFiI genomic fragment (Figure 3).
Clone pPC 6.4 is in turn located completely within cDNA clone pPC 6.7. Since pPC 6.7 was a more complete copy than pPC
6.4, the latter was not investigated further. The 5~ end of the gene is positioned closer to the BamHI end than to the EcoRI end of t:he genomic clone pPG 4.8 (Figure 3).
In all of these above-described genomic clones, there are at least 1.2 kilobase pairs of flanking genomic DNA
sequence which are 5~ to the structural genes copied in each of the cDNA clones.
Each of the genomic and cDNA clones described above have been deposited with the Northern Regional Research Center of the United States of America, Peoria, Illinois, to insure access to the public upon issuance of this application as a patent. All clones have been deposited in E. coli hosts:

Plasmid Host Accession No.

pPG 6.0 E. coli LE392-pPG 6.0 NRRL B-15867 pPG 4.0 E. coli LE392-pPG 4.0 NRRL B-15868 pPG 4.8 E. coli LE392-pPG 4.8 NRRL B-15869 pPC 15.0 E. coli LE392-pPC 15.0NRRL B-15870 pPC 8.3 E. coli LE392-pPC 8.3 NRRL B-15871 pPC 6.7 E. coli LE392-pPC 6.7 NR.RL B-15872 pPC 8.0 E. coli Nll~'I294-pPC8.0 NR.RL B-15873 All of the above organisms have been evocably irr deposited and made available to the August 1984.
public 31, as of Uniqueness of pPG 6.0, pPG 4.0 and pPG 4.8 to Methanol Assimilating Yeasts Each of the cDNA clones described above have been labeled and employed as probes of chromosomal DNA sequences from a number of methanol assimilating yeasts and a methanol non-assimilating yeast. Homologous genes for all three of the cDNAs were found to exist in essentially all methanol assimilating yeasts, but were clearly not present in methanol non-assimilating yeast (S. cerevisiae). It is thus believed that these genes are unique to methanol assimilating yeast.
In addition, t:he Southern hybridization experiments detailed in Example XV:fI demonstrate that a high degree of homology exists between these unique methanol responsive genes from various methanol assimilating yeasts.
Characterization of the RNA Transcripts of the pPG 6.0, pPG 4.0 and pP~G 4.8 Genes The influence of methanol on the expression of each of these cloned genes can be observed by studying the effects on transcription of these genes. Isolated poly A+ RNA from Pichia pastoris cells grown with ethanol or methanol as sole carbon source was used to prepare Northern hybridization filters (See Example IV). Three identical pairs of filters from methanol and ethanol grown cells (See Example I) were probed separately with 32P-labeled pPC 15.0, pPC 8.0 and pPC

15 1~~~'~33 6.4. The clones pPC 15.0, pPC 8.0, and pPC 6.4 hybridized to RNA molecules (of approximately 2400, 2300, and 1300 nucleotides, :respectively) from methanol grown cells. No hybridization of clones pPC 15.0 and pPC 8.0 with the hybridization probes was observed with RNA obtained from cells grown in the presence of ethanol. However, when RNA
isolated from cells grown on ethanol was probed with pPC 6.4, the clone hybridized to a 1300-nucleotide RNA molecule identical to 'that seen with methanol-grown cells but at an estimated (qualitatively) 5-fold lower level.
Size Determination of Protein Products Encoded by pPG 6.0, pPG 4.0 and pPG 4.8 To determine what protein products were encoded by each of the above-identified cDNA clones, poly A+ RNA from Pichia pastoris cells grown on methanol was selectively hybridized to each of the cDNA clones. The hybrid-selected mRNA, i.e., mRNA which hybridized to each of the cDNA clones, was then translated in vitro and each of the protein products resolved by electrophoresis using SDS-denaturing conditions (See Example V). The results of these in vitro positive hybridization-translation experiments indicated that clones pPC 15.0, pPC 8.3, and pPC 6.7 select mRNAs which encode information for polypeptides of 76,000 (p76), 72,000 (p72) and 40,000 (p4~0) daltons, respectively. These same proteins are observed when total poly A+ RNA (i.e., not hybrid-selected) from methanol grown Pichia pastoris cells is translated in the same in vitro system.
Identification of p72 as Alcohol Oxidase A. Molecular 'We_ iqht Comparison A sample highly enriched for alcohol oxidase protein was prepared by dialysis of cleared cell lysates against HZO (;>ee Example VII). The crystalline precipitate resulting from this dialysis was shown by SDS electrophoresis to contain predominantly two polypeptides of 76,000 and 72,000 daltons;, respectively. The precipitate was subjected to additional purification by chromotography through Sephacryl 200 (See Example VII), which demonstrated that alcohol oxida~~e activity corresponded to the activity of the purified 72,000 dalton polypeptide. The size of this polypeptide was identical to that of the protein product selected by cDNA clane pPC 8.3 (See Example X).
B. Immunoprecipitation Additional. support that clones pPC 8.3 and pPG 4.0 encode the alcohol oxidase structural gene was obtained by means of an immunological approach (Example XI). The protein preparation isolated from Pichia pastoris containing both the 76,000 and 72,000 dalton polypeptides was used to raise specific antisera for these polypeptides in rabbits. When the hybrid-selected poly A+ RNA from clone pPC 8.3 was translated in vitro, only the 72,000 dalton translation product was precipitated by the antisera made against the protein preparation from Pichia pastoris cells.
C. Predicted/Actual Amino Acid Sequence Comparison To further verify that clone pPC 8.3 is in fact the cDNA clone encoding Pichia pastoris alcohol oxidase, the amino acid sequence for the amino terminal end of the protein was compared with the predicted amino acid sequence encoded by pPC 8.3. Thus,, the NH2-terminal amino acid sequence (Sequence A) of the isolated 72,000 dalton protein was determined (Example VIII) to be:
Ala-Ile-Pro-Glu-Glu-Phe-Asp-Ile-Leu-Val-Leu-Gly-Gly-Gly-Ser-Ser-Gly-Ser.
Sequence A
In parallel, the nucleotide sequence of the 5~ end of the gene encoded in pPC 8.3 and pPG 4.0 was determined.

The predicted amino acid sequence for amino acids 2-19 (See Sequence B) derived from the DNA sequences of both the genomic and cIDNA clones agreed perfectly with the first 18 amino acids o f the above determined amino acid sequence (Sequence A) for isolated Pichia pastoris alcohol oxidase:
Predicted amino acid sequence: Met a1a ile pro gZv glu phe Nucleotide sequence 5~-ATG GCT ATC CCC GAA GAG TTT
(pPC 8.3 and pPG 4.0):3-TAC CGA TAG GGG CTT CTC AAA
asp i1e Ieu vat Zeu g1y g1y glr~ ser ser gIg ser GAT ATC CTA GTT CTA GGT GGT GGA TCC AGT GGA TCC-3~
CTA TAG GAT CAA GAT CCA CCA CCT AGG TCA CCT AGG-5~
Sequence B
DNA Fragments Containing Regulatable Promoters from Pichia pastoris The 5~ regulatory regions of the invention are detailed in restriction maps presented in Figures 4, 5 and 6.
The 5~ regulatory region which controls the expression of polypeptide pT6 is located within the DNA fragment depicted in Figure 4.a. The approximately 2.9 kilobase pair HindIII-XhoI fragment has been clearly demonstrated to contain the regulatory function as detailed more fully below.
Since cDNA clone pPC 15.0 is not a full copy cDNA, it is most likely that air least a portion of the DNA fragment depicted in Figure 4a. includes structural coding sequences for polypeptide p76. Thus, the regulatory function is believed to reside in the approximately 1300 base pair HindIII-EcoRI

..-, 1$ ~3~Q'~33 fragment shown in Figure 4b. Novel ~-galactosidase gene containing constructs, to be discussed in greater detail below, support. this suggestion.
The 5' regulatory region which controls the expression of polypeptide p72 (alcohol oxidase) is located within the approximately 2000 base pair EcoRI-BamHI DNA
fragment illustrated in Figure 5. Novel ~-galactosidase gene containing constructs discussed below demonstrate the regulatable nature of this DNA fragment.
Figure 6 provides a restriction map for the approximately 3 kilobase pair BamHI-SalI DNA fragment which includes the 5~ regulatory region which controls the production of polypeptide p40. This fragment is clearly distinguishable from the 5~ regulatory regions detailed in Figures 4 and 5 based, inter alia, on the different restriction sites located within the DNA fragment.
Figures 10, 2a and 11 provide restriction enzyme data for the regulatory regions plus structural genes for polypeptides p76, p72(alcohol oxidase) and p40, respectively.
Hence, Figure 10 provides detail for the 3.8 kilobase pair HindIII-PstI i:ragment of Pichia pastoris genomic DNA which controls and codes for the production of polypeptide p76.
Figure 2a deals with the 4.0 kilobase pair EcoRI-PvuII
fragment of Pichia pastoris genomic DNA which controls and codes for the ;production of polypeptide p72(alcohol oxidase).
Figure 11 presents the 3.7 kilobase pair BamHI-EcoRV fragment of Pichia pastoris genomic DNA which controls and codes for the production of polypeptide p40.
The genomic clones, pPG 6.0, pPG 4.0 and pPG 4.8 have also been characterized by restriction mapping. Thus, clone pPG 6.0 is detailed in Figure la. As a point of reference the 5~ end of the DNA fragment is deemed the origin. Clone pPG 6.0 is a HindIII fragment of Pichia pastoris chromosomal DNA which is about 6 kilobase pairs in 19 1~~~~~3 length, and is cleaved as follows by various restriction enzymes:
Cleavage Distance Restriction Enzyme Sites From Origin (bp) HancII 5 1070, 1740, 1890, 3320, 5520 EcoRI 2 1300, 3450 XhoI 1 2860, PstI 2 3820, 4200 PvuII 1 4120 PvuI 1 4950 Clone pPG 4.0 is illustrated in detail in Figure 2a. The clone is an EcoRI-HindIII fragment of chromosomal DNA which is about 4 kilobase pairs in length. Referring to the 5~ end of the clone as the origin, the following restriction data was obtained for pPG 4.0:
Cleavage Distance Restriction Enzyme Sites From Origin (bp) HindIII 3 400, 600, 1840 PstI 1 850 BamHI 2 1960, 1970 SalI 1 2620 BglII 2 1040, 2700 KpnI 2 500, 2730 XbaI 1 3330 StuI 1 3880 NdeI 1 420 HincII 2 870; 2430 SstI 1 1200 20 ~~~07~
BcII 2 1710, 4080 AsuII 2 1900, 2300 EcoRV 1 1930 PvtrI I 1 4120 Clone pPG 4.8 is illustrated in detail in Figure 3a. The clone is a 4.8 kilobase pair BamHI-EcoRI fragment of Pichia pastoris chromosomal DNA with the following additional restriction sites:
Cleavage Distance Restriction Enzyme Sites From Origin bp) CIaI 1 410 KpnI 3 500, 3890, 4280 Pvul 1 1120 SalI 1 2900 PvuII 1 4135 EcoRV 2 3690, 3890 BglII 1 4500 XmaI 1 4800 The genomic clones pPG 6.0, pPG 4.0 and pPG 4.8 were manipulated by insertion into unique restriction sites on the E. co~'i plasmid pBR322. Clone pPG 6.0, which is a HindIII fragment, was conveniently cloned into the HindLII
site of pBR:322. Clone pPG 4.0 was cloned into the EcoRI-PvuII sites of pBR322 and clone pPG 4.8 was cloned into the EcoRI-BamH;I sites of pBR322. (See Example VI ) . E. coli strains transformed with these plasmids have been deposited with the Northern Regional Research Center, Peoria, Illinois, to insure free access to the public upon issuance of a patent on this application. The deposited strains have been given the following accession numbers:

Laboratory Genomic Class Designation Accession No.
pPG 6.0 LE392-pPG 6.0 NRRL B-15867 pPG 4.0 LE392-pPG 4.0 NRRL B-15868 pPG 4.8 LE392-pPG 4.8 NRRL B-15869 Figures 7, 8 and 9 provide restriction map data for the 3~ regulatory regions of the polypeptides p76, p72 (alco-hol oxidase) and p40, respectively. The 3~ regulatory regions are useful in controlling the polyadenylation, termination of transcription and termination of translation of messenger RNA
which is coded for by preceding nucelotide sequences. Thus, the 3~ regulatory region. from the polypeptide p76 gene, a 2.7 kilobase pair EcoRI-HindIII fragment illustrated in Figure 7, is useful in controlling the polyadenylation as well as termi-nation of transcription and termination of translation of the mRNA which codes for polypeptide p76, or any other mRNA derived from a gene inserted upstream of the 3~ regulatory region. The 0.2 kilobase pair StuI-PvuII fragment from the p72 gene detailed in Figure 8a, the,0.3 kilobase pair StuI-HindIII fragment from the p72 gene detailed in Figure 8b, the 3:2 kilobase pair SaZI-EcoRI fragment from the p72 gene detailed in Figure 8c, and the 1.9 kilobase pair PvuII-EcoRI fragment from the p40 gene de-tailed in Figure 9 have similar utility, both with respect to the structural genes with which they are associated in the wild type Pichia pastoris and any foreign (i.e. heterologous) genes which may be inserted upstream of these 3~ regulatory regions.
Characterization of cDNA Clones The cDNA clones for the regulatable genes from Pichia pastoris have also been characterized by restriction mapping.
In Figure 12, the p76 cDNA, a 1.1 kilobase pair fragment is detailed. Referring to the 5~ end of the DNA sequence as the origin, restriction enzyme XhoI cleaves p76 cDNA about 500 base pairs from the origin, FIincII cleaves about 950 base pairs from the origin and EeaRI cleaves p76 k. S,~ ';
~4.i :r p. s cDNA about 1050-1100 base pairs from the origin. The cDNA
clone shown in. Figure 12, as well as the cDNA clones shown in Figures 13 and. 14 are all shown with PstI termini. These are artificially created restriction sites produced by G-C
tailing of the initially obtained complementary DNA to facilitate cloning of the DNA fragments into pBR322. Based on Northern hybridization studies and the size of the polypeptide product, it is estimated that the cDNA clone pPC
15.0 is an incomplete copy of p76 mRNA, representing only about half of the total messenger RNA sequence.
In Figure 13, a composite restriction map for p72 (alcohol oxidase) cDNA, constructed by overlap of clones pPC
8.3 and pPC 8..0, is presented. As above, the 5~ end of the DNA sequence :is referred to as the origin. Thus, treating alcohol oxidase cDNA with a variety of restriction enzymes gives the following size fragments:
Cleavage Distance Restriction Enzyme Sites From Oriq_in (bp) AsuII 2 20, 420 EcoRV 1 50 BamHI 2 80, 90 HincII 1 550 SalI 1 820 BglII 1 820 KpnI 1 850 XbaI 1 1450 RsaI 1 1760 StuI 1 2000 Restriction enzyme mapping of the 3~ end of the alcohol oxida.~e gene in clones pPC 8.0 and pPC 8.3 revealed that cDNA clone pPC 8.3 is missing approximately 250 nucleotides of the alcohol oxidase mRNA sequence (Figure 2).

~3~~ fi~~

The sequences present at the 3~ end of the alcohol oxidase mRNA are present in cDNA clone pPC 8.0 which overlaps pPC 8.3 by approximately 500 nucleotides.
Figure 14 presents a restriction map for the cDNA
of polypeptide: p40, a 1.2 kilobase pair fragment. Referring to the 5~ end of the cDNA clone as the origin, clone pPC 6.7 is cleaved by SalI (and HincII) about 1000 bases from the origin.
Each of the cDNA fragments have been cloned into pBR322, which. is then transformed into E. coli. The transformed strains have been deposited with the Northern Regional Research Center in Peoria, Illinois to insure free access to the public upon issuance of this application as a patent. The deposited strains have been assigned the following accession numbers:
Laboratory cDNA clone Description Accession No.
pPC 15.0 LE392-pPC 15.0 NRRL B-15870 pPC 8.3 LE392-pPC 8.3 NRRL B-15871 pPC 8.0 MM294-pPC 8.0 NRRL B-15873 pPC 6.7 LE392-pPC 6.7 NRRL B-15872 Each of the above-described cDNA clones are useful as probes for the identification and isolation of chromosomal DNA encoding the production of polypeptides unique to the growth of yeast on methanol as a carbon and energy source.
Hence as already described, these clones were used to identify P. pa.storis chromosomal DNA fragments containing the regulatory regions and structural coding information for the unique polype~>tides which are observed when P. pastoris is grown on methanol. In a similar fashion, these cDNA clones have utility as probes for the identification and isolation of analogous genes from other methanol assimilating yeasts such as, for example, Torulopsis molischiana, Hansenula capsvlatUm, H. nonfermantens and the like (See Example XVII).

r_ .

Detailed Analysis of the Alcohol Oxidase Gene The 5~ regulatory region of clone pPG 4.0 was further characterized by determining the nucleotide sequence of the clone upstream (5~) of the point where the structural information for p72 (alcohol oxidase) is encoded. The first 250 nucleotides prior to the mRNA translation start site (ATG
codon) are believed to be:

5~-ATGCTTCCAA GATTCTGGTG GGAATACTGC TGATAGCCTA

ACGTTCATGA TCAAAATTTA ACTGTTCTAA CCCCTACTTG

GACAGGCAATA TATAAACAGA AGGAAGCTGC CCTGTCTTAA

ACCTTTTTTT TTATCATCAT TATTAGCTTA CTTTCATAAT

TGCGACTGGT TCCAATTGAC AAGCTTTTGA TTTTAACGAC

TTTTAACGAC AACTTGAGAA GATCAAAAAA CAACTAATTA

TTCGAAACG-3~.

Sequence C
The promoter function of clone pPG 4.0 is believed to be contained within this sequence of nucleotide bases.
In order to more fully describe this novel DNA
fragment, an additional 301 nucleotides further upstream of the sequence detailed in Sequence C above have been determined. Thus, the first 551 nucleotides prior to the mRNA translation start site are believed to be:

1~4Q"~3~

5~-AATGGCCCAAA ACTGACAGTTT AAACGCTGTC TTGGAACCTA

ATATGACAAi~ AGCGTGATCT CATCCAAGAT GAACTAAGTT

TGGTTCGTT(= AAATGCTAAC GGCCAGTTGG TCAAAAAGAA

ACTTCCAAAA GTCGCCATAC CGTTTGTCTT GTTTGGTATT

GATTGACGAA TGCTCAAAAA TAATCTCATT AATGCTTAGC

GCAGTCTCT(: TATCGCTTCT GAACCCGGTG GCACCTGTGC

CGAAACGCAA ATGGGGAAAC AACCCGCTTT TTGGATGATT

ATGCATTGT(: CTCCACATTGT ATGCTTCCAA GATTCTGGTG

GGAATACTGC: TGATAGCCTA ACGTTCATGA TCAAAATTTA

ACTGTTCTAA CCCCTACTTG GACAGGCAATA TATAAACAGA

AGGAAGCTGC: CCTGTCTTAA ACCTTTTTTT TTATCATCAT

TATTAGCTTA CTTTCATAAT TGCGACTGGT TCCAATTGAC

AAGCTTTTGA TTTTAACGAC TTTTAACGAC AACTTGAGAA

GATCAAAAAA CAACTAATTA TTCGAAACG-3~ .

Sequence D
The additional nucleotides contained in Sequence D (compared to Sequence f) are believed to impart, by an unknown mechanism, additional regulatory functions to the promoter region contained within Sequence C. It should be recognized that Sequence D represents only partial DNA sequencing for the 1.1 kbp DNA fragment shown in Examples XIV and XV to be capable of controlling gene expression in yeast. It may be that additional, control functions are encoded in the portion of the 1.1 kbp DNA fragment not detailed in Sequence D.
To determine where RNA transcription for the alcohol oxidase gene is initiated, the DNA sequences around the S~ end of this gene from the genomic clone pPG 4.0 and the cDNA clone pPC 8.3 were compared. cDNA clone pPC 8.3 contains about 100 nucleotides~of an untranslated region 5~
to the alcohol oxidase gene. Based upon this sequence, an --w 26 1340?'33 oligonucleotic~.e of 15 bases (5~-CTTCTCAAGTTGTCG-3~);
complementary with respect to nucleotides -29 to -43, where the A of the translation start site (ATG codon) is designated as +1 and the G in the 5~ direction is designated as -1, was synthesized ( :3ee Example IX ) and used as a primer to extend along the alcohol oxidase mRNA to reach the 5~ end. The sequence of cDNA obtained from this primer-extension experiment revealed three different transcriptional initiation points for Pichia pastoris alcohol oxidase mRNA.
The major transcript begins 114 nucleotides from the translational initiation codon. Two minor alternative transcripts begin 1:L7 and 111 nucleotides upstream (5~) from the alcohol oxidase AUG codon.
The 55 nucleotides preceding the start of alcohol oxidase mRNA contain a putative Goldberg-Hogness box (TATAA
box). The sequence TATAAA occurs at position -40 from the 5~
end of the predominant transcript for alcohol oxidase mRNA
and therefore 165 nucleotides upstream from the initiation codon for this protein.
Expression in Transformed Yeast The above-described plasmids of the present invention have utility in yeast strains which can be transformed. Regulation of gene expression in yeast by the novel DNA fragments of the present invention can be accomplished by subjecting the transformed organisms to carbon source starvation. Carbon source starvation after growth on a variety of both catabolite repressing and non-catabolite repressing carbon sources induces expression of the gene ;product maintained under the control of the regulatory regions of the invention. Another means to achieve expression of the desired gene product in appropriate species of transformed yeast is to grow transformed yeasts on ..-..

methanol. Yet another means to induce expression of the desired gene product is to grow transformed yeast on media containing non-catabolite repressing carbon sources.
The regulatory regions of this invention are useful for expression in all yeast strains, since the regulatory regions have been shown to be induced under a variety of conditions. Thus, yeasts capable of growth on methanol or on non-catabolite repressing carbon sources can be caused to produce foreign, i.e., heterologous, polypeptides directly;
while yeasts capable of growth on catabolite repressing carbon sources can be caused to produce foreign polypeptides by subjecting yeast cells so grown to conditions of carbon source starvation.
Transformed yeast strains which are preferred in the process o:E the present invention include members of the genera:
Candida, Kloeckera, Saccharomyces, Schi zosaccharomryces, Rhodotorula, Hansenula, Torulopsis, Pichia, and 2 5 KI urweromyces .
Yeasts from these genera are preferred because their safety of handling, growth conditions and the like have been established and are well known to those of skill in the art.
Especially preferred yeast strains for use in one embodiment of the process of the present invention are those yeast strains which are capable of growth on methanol as carbon and energy source. Yeasts known to be capable of growth on methanol include members of the genera:

28 13~4?'33 Candida, Kloeckera, saccharom~ces, Rhodotorula, Hansenula, Torulopsis, and Pichia.
Since the regulatory regions of the present invention are also induced by growth on non-catabolite repressing carbon sources as well as conditions of carbon source starvation, yeast strains which are capable of growth on such non-methanolic substrates as:
glucose, acetate, glycerol, ethanol, lactose, galactose, fructose, sucrose, and the like and mixtures of any two or more thereof are also useful in the practice of the invention. By growing the host organism on a suitable non-catabolite repressable non-met:hanolic carbon source such as, for example, glycerol and galactose, or by growing the host organism on a suitable catabolite repressable carbon source such as, for example, ethanol, glucose and fructose, then subjecting the host organism to c<~rbon source starvation conditions, expression of a gene product under the control of the regulatory regions of the invention can be achieved.
An especially preferred host yeast strain is the mutant Pichia pastoris GS115, which is a mutant defective in the ability to produce histidine, and has thus been designated as having the mutant genotype his4. GS115 is derived from Pichia pastoris NRRL Y-11430 and has been deposited with the Northern Regional Research Center of the United States Department of Agriculture in Peoria, Illinois, in order to ensure free access of the host to the public upon issuance of this application as a patent. Pichia pastoris GS115 has been assigned the accession number NRRL Y-15851, as of August 31, 1984. This particular host is useful because it is an au:xotrophic mutant deficient in the histidine pathway. Transformation of this host with a vector containing, ~unong other DNA sequences, the HIS4 gene function, allows ready selection for transformed host.
Escherichia coli is also a suitable host for the plasmids of the invention. Those of skill in the art recognize that: many strains of E. coli are suitable hosts.
Several strains employed in the present work are summarized below:
Strain designation Accession Number MC1061 None known LE392 ATCC #33572 MM294 ATCC #33625 Pichia pastoris Transformation Procedure The transformation of Pichia pastoris has not been previously described. The experimental procedures for transformation of Pichia pastoris are presented in greater detail below (Example XII). In order to develop a transformation system for P. pastoris, the auxotrophic mutant GS115 (NRRL 'Y-15851) was isolated and determined to be defective in t:he histidine pathway in that the strain has no detectable histidinol dehydrogenase activity.
GS115 (NRRL Y-15851) can be transformed by enzymatic digestion of the cell walls to give spheroplasts;
the spheroplasts are then mixed with the transforming DNA and incubated in the presence of calcium ions and polyethylene glycol, then regenerated in selective growth medium deficient in histidine. The transforming DNA includes the HIS4 gene in which the host strain is deficient, thus only transformed cells survive on the selective growth medium employed.
Isolation of P~ichia pastoris HIS4 Gene The HIS4 gene was isolated from the strain P.
pastoris NRRL Y-11430 by partial digestion of total chromosomal DNA with Sau3A followed by centrifugation through sucrose gradients. (See Example XIII). Fragments of 5 to 20 kbp were cloned into the BamHI cleavage site of the S.
cerevis.iae-E. colt shuttle vector YEpl3 (ATCC 37115; Figure 33) and transformed into E. coli. Approximately 50,000 colonies were combined and total plasmid DNA extracted.
Spheroplasts o~f S. cerevisiae strain 5799-4D (NRRL Y-15859), a his4ABC mutant, were mixed with about 1 ~g of the YEpl3 Pichia DNA library by the procedure of Hinnen et a1 (1978) and allowed to regenerate in a medium deficient in histidine.
The transformation resulted in about 1x103 prototrophic yeast colonies from. a population of 5x10 total regenerable spheroplasts. A parallel control sample incubated without DNA produced no colonies. Total yeast DNA was extracted from 20 of the His+ colonies and transformed back into E. coli.
Seventeen of the yeast DNA preparations produced ampicillin resistant colonies. These cloned fragments were further characterized by restriction enzyme sizing and mapping as well as by their ability to cross hybridize with a labelled S. cerevisiaE~ HIS4 fragment at low stringency (post hybridization 'washes in 2xSSC at 55°) by the method described in Example X:III, ~G. The HIS4-containing plasmids each contained one or more fragments which hybridized to the S.
cerevisiae HIS4 gene. One such HIS4-containing plasmid was recloned to give a HIS4-containing plasmid designated pYJ8 and is shown in Figure 25. Plasmid pYJ8 contains pBR325 sequences, including chloramphenicol and ampicillin resistance genes, as well as the Pichia HIS4 gene.

Isolation of Pichia pastoris Autonomous Re lication Sequences Another useful component of the vectors of the present inveni:,ion are Pichia-derived autonomous replication sequences (PARS), which enhance both the transformation frequency of GS115 (NRRL Y-15851) and the maintenance of plasmid as a stable extrachromosomal element.
To search for Pichia ARSs, DNA from Pichia pastoris GS115 (NRRL Y~-15851) was partially digested with TaqI and 5 to 10 kbp fragments were isolated and cloned into the unique CIaI site of pYJ8aCla. (See Figure 2b). Plasmid DNA was recovered from, about 10,000 His+ Pichia colonies and used to transform E. coli. Plasmids from about 10,000 ampicillin resistant colonies were isolated and then transformed back into GS115. Forty of the His+ yeast colonies from this sublibrary transformation were separately streaked onto selective medium and grown in separate cultures in selective medium. Total yeast DNA was extracted from each of these 40 cultures and i~ransformed into E. coli. Two plasmids, pYA63 (PARS1) and ;pYA90 (PARS2) whose yeast DNA preparations produced the most ampicillin resistant E. coli colonies, were selected for further analysis. Both of these plasmids transformed Pi:chia pastoris 65115 (NRRL Y-15851) at a very high frequency and each contained an insert of foreign DNA.
As a. measure the ability of the ARSs to maintain plasmids as autonomous elements in Pichia, cultures of yeast cells which had been transformed with each plasmid were grown in selective medium and periodically sampled. The state of the plasmid sequences in the cells was determined by Southern hybridization of unrestricted yeast DNAs to radioactively labeled pBR325. Plasmids pYA63 and pYA90 were maintained in Pichia for at, least 10 generations in the selective medium (but had integrated by 50 generations).

1344?~3 Novel S-Galactosidase Gene Containing Constructs In order to demonstrate the ability of the regula-tory regions of the present invention to control the produc-tion of protein products, novel DNA constructs were prepared.
Thus the E. coZi ZacZ gene was placed in several plasmids un-der the control of the regulatory regions of the genes enco-ding polypeptide p72 (alcohol oxidase) or p76. The prepara-tion of plasmids pSAOHl, pSAOH5, pSAOHIO, pTAFH.85, pT76Hl, pT76H2, pT76H3 and pT76H4 is described in Example XIV.
Although the introduction of the regulatory region -S-galactosidase gene fusions of the invention into host yeast cells is described herein employing plasmids as the vehicle for introduction, those of skill in the art recognize that it is not necessary for the regulatory region-structural gene construct to be introduced into the cell via a plasmid.
Hence, any molecule capable of being maintained in yeast can be employed. Therefore, the regulatory region-structural gene constructs of the invention can be manipulated via vec-tors other than plasmids. Alternatively, the regulatory region-structural gene construct can be integrated into the chromosome of the :host yeast cell.
Those of skill in the art also recognize that the scope of the present invention is not limited to the produc-tion of ~-galactosidase under the regulation of the regula-tory regions disclosed herein. The variety of polypeptides which can be produced under the regulation of the regulatory regions of the invention is limited only by the imagination of the reader. Many procedures exist for the preparation of DNA sequences which code for desired polypeptides. For ex-ample, oligonucleotides of various lengths can be synthesized by known procedures. Several such oligonucleotides can be assembled, in consequence of the specific base pairing pro-perties thereof, into longer, double-stranded molecules. The component oligonuc:Leotides of this double-stranded molecule can be joined (ligated) by the enzyme DNA ligase. Alterna-tively, DNA molecu:Les having the desired coding sequence can be synthesized by ruse of the enzyme reverse transcriptase, '~ 33 using messenger RICA related to the desired polypeptide as a template for the action of reverse transcriptase. Yet an-other possibility is the cloning of genomic DNA fragments and observing whether direct expression of the desired product occurs.
The DNA sequence which codes for the desired poly-peptide can be modified for preparation of the regulatory region-structural gene construct by a variety of procedures.
For example, the ends of the DNA prepared as described above can be ligated with the enzyme DNA ligase to short double-stranded DNA molecules which contain the nucleotide sequence recognized by specific restriction endonucleases, so called linker molecules. Digestion of these molecules with a spe-cific restriction ~endonuclease following the ligation will generate termini corresponding to the specified restriction endonuclease recognition site at the ends of the prepared DNA sequence.
Three specific regulatory region-S-galactosidase gene constructs prepared in the course of this work are des-cribed in terms of restriction mapping data presented in Figures 15 and 16. The restriction map presented in Figure 15 describes a construct comprising a 0.85 kilobase pair HindIII-BamHI portion derived from the 5~ regulatory region of pPG 6.0 and the ZaeZ gene fxom E'. eoZi (the 3.6 kilobase pair BamHI-NruI fragment shown), This same construct is present in each of the plasmids pTAFH.85, pT76H1 and pT76H2, to be described in greater detail below. (See Example XIV).
The restriction ma:p presented in Figure 15b describes a con-struct comprising .a 1.3 kilobase pair HindIII-EcoRI portion derived from the 5~ regulatory region of pPG 6,0 and the ZaeZ gene from E. eoZi. This same construct is present in each of the plasmids pT76U1, pT76H3 and pT76H4, to be des-cribed in greater detail below. (See Example XIV).
Figure 16 is a restriction map of a construct com-prising a 1.1 kilo:base pair EeoRI-BamHI fragment derived from a portion of the 5~ regulatoxy region of pPG 4.0 and the ZaeZ
gene from E. coZi. This construct is present in each of the 134a'~~~
..

plasmids pSAOHl, pSAOH5 and pSAOHIO, to be described in greater detail below. (See Example XIV).
Plamid pSAOHl is illustrated schematically in Fig-ure 17. In addition to containing the regulatory region-S-galactosidase gene fusion detailed in Figure 16, the plasmid is shown to contain:
(a) pBR322 sequences, including the ampR gene;
(b) Pic.izia pastoris HIS4 gene;
(c) S. cerevisiae 2u circle DNA; and (d) the interrupted URA3 gene from S, cerevisiae.
The plasmid therefore has the capability to transform and replicate in E. coZi hosts and yeast hosts. Selectable mark-ers are present fo:r manipulation of the DNA in either E. coZi or yeast hosts.
Plasmid :pSAOH5 is illustrated schematically in Fig-ure 18. The plasmid is similar to pSAOHI described above, except the S. cerewisiae 2u circle DNA and some of the Pichia pastoris HIS4 gene flanking DNA has been deleted while a Pi-chia pastoris autonomously replicating sequence (PARS1 from pYA63) has been added.
Plasmid :pSAOHIO is illustrated schematically in Figure 19. The pl,asmid contains:
(a) regulatory region-S-galactosidase gene fusion;
(b) pBR325 sequences, including genes conferring tetracycline resistance, chloramphenicol resistance and amp-icillin resistance (tetR, camR and ampR, respectively); and (c) S. cerevisiae HIS4 gene (obtained from plas mid pYA2 as described below).
Plasmids pTAFH.85, pT76H1 and pT76H2 are analogous to the above three described plasmids, except the regulatory region-S-galactosidase gene fusion employed was that described in Figure 15a (instead of the fusion described in Figure 16).
Plasmids pT76H3 and pT76H4 are analogous to pSAOHl and pSAOH5, respectively, except the regulatory region-S-galactosidase gene fusion employed was that described in Fig ure 15b (instead of the fusion described in Figure 16).
;,' 1340?33 '~' 3 5 Plasmid pTAFH.85 is illustrated schematically in Figure 20 and comprises:
(a) the: regulatory region-S-galactosidase gene fu-sion shown in Figure 15;
(b) pBF;322 sequences, including the ampR gene;
(c) Pic~hia pastoris HIS4 gene;
(d) S. cerevisiae 2u circle DNA; and (e) the interrupted URA3 gene from S. cerevisiae.
Plasmid pT76H1 is illustrated schematically in Fig-ure 21 and comprises:
(a) the: regulatory region-S-galactosidase gene fu-sion shown in Figure 15;
(b) pBF:322 sequences, including the ampR gene; and (c) Pichia pastoris HIS4 gene and autonomously rep-licating sequence (PARS1).
Plasmid pT76H2 is illustrated schematically in Fig-ure 22 and comprises:
(a) the: regulatory region-B~galactosidase gene fu-sion shown in Figure 15;
(b) pBR:325 sequences, including genes conferring tetracycline resistance, chlora;nphenicol resistance and amp-icillin resistance:; and (c) S. eerevisiae HIS4 gene.
Plasmid pT76H3 is illustrated sck~ematically in Fig-ure 22a and comprises:
(a) the regulatory region-~-galactosidase gene fu-sion shown in Figure 15b;
(b) pB1~.322 sequences, including the ampR gene;
(c) Pichia pastoris HIS4 gene;
(d) S. eerevisiae 2u circle DNA; and (e) the: interrupted UF;A3 gene from S. cex~eviszae.
Plasmid pT76H4 is illustrated schematically in Fig-ure 22b and comprises:
(a) the. regulatory region-S-galactosidase gene fu-sion in Figure 15b~;
(b) pBR:322 sequences, including the ampR gene;
(c) Piehia pas~oxis HIS4 gene; and ''~' 3 5 a (d) Pichia pastoris autonomous replication sequence ( PARS 1 ) .
Expression of S-Galactosidase in Yeast Pichia pastoris GS115 (NRRL Y-15851) was transformed with the novel ~-g~alactosidase gene-containing constructs des-cribed above. Several of the resulting transformed yeast strains have been deposited with the Northern Regional Research Center of the United States Department of Agriculture and as-signed deposit accession numbers as follows:

Accession Number of Host Plasmid Transformed Strain GS115 pSAOHl NRRL Y-15852 GS115 pSAOHS NRRL Y-15853 GS115 pSAOHIO NRRL Y-15854 GS115 pTAFH.85 NRRL Y-15855 GS115 pT76H1 NRRL Y-15856 GS115 pT76H2 NRRL Y-15857 The novel S-galactosidase gene-containing constructs were also used to transform E. coZi. Transformed bacterial strains ha ve also been depo sited with the Northern Regional Research Center Illinois to insure availability in Peoria, to the public upon issuance of this application as a patent. The transforme d strains have been assigned the following accession numbers:

Accession Number of Host Plasmid Transformed Strain MC1061 pSAOHl NRRL B-15861 MC1061 pSAOH5 NRRL B-15862 MC1061 pSAOHIO NRRL B~15863 MC1061 pTAFH.85 NRRL B-15864 MC1061 pT76H1 ~1RRL B~15865 MC1061 pT76H2 NRRL B-15866 MC1061 pTA013 NRRL B-15875 MC1061 pT76H3 NRRL B~18000 MC1061 pT76H4 NRRL B-18001 MC1061 pT76Ul NRRL B~18002 Pichia pastoris GS115 (~T~tRL Y~15851) transformed with each of the first eight pl asmids described above which contain the alcohol p76 regulatory region-ZacZ
oxidase and gene fusions of the invention were grown to stationary phase on minimal medium with biotin plus glucose supplemented as carbon source. Once cells reached stationary phase, they were shifted to minimal medium supplemented with biotin plus methanol as carb on source. Aft er cells had grown for about 3-5 generations at 30C, they were shifted to fresh minimal ~< ~A'i:...
'y %., medium supplemented with biotin and grown on glucose or meth-anol as carbon source. At distinct time points, culture sam-ples were withdrawn and analyzed for the presence of ~-galac-tosidase and alcohol oxidase by methods detailed in Examples VII and XV.
It was found that cells grown on glucose as carbon source produced no detectable levels of ~-galactosidase or alcohol oxidase, while cells grown on methanol as sole carbon source expressed significant levels of both alcohol oxidase and S-galactosidase. It. was also found that the glucose grown cells, when subjected to conditions of carbon source starvation, also expressed measurable quantities of alcohol oxidase as well as S-galactosidase. Thus, it is clear that the regulatory regions of the invention are responsive to both the presence of methanol as well as conditions of carbon source starvation.
As verification that the regulatory regions of the invention are responsive to growth on non-catabolite repres-sing carbon sources as well as conditions of carbon source starvation, a plasmid containing the alcohol oxidase regula-tory region, PTA013; and a plasmid containing the p76 regula-tory region, pT76U1, was used to transform a non-methanol utilizing strain of yeast, Saecharomyees cerevisiae. The transformed strains employed, having the laboratory designa-tion of SEY2102-pTA013, has been deposited with the Northern Regional Research Center in Peoria, Illinois to insure access to the public upon granting of a patent on this application.
The transformed strain has been assigned accession number NRRL Y-15858. Saccharom~ces ee~e~isiae ~TRRL Y-,15858 and SEY2102-pT76U1 were grown up on glucose, fructose, ethanol, glycerol and galactose for about five generations then sub-jected to conditions of carbon source starvation. The usual assay for S-galactosidase (See Example XV) a~tex five gener-ations indicated that glycerol and galactose grown cells pro-duced large amounts of ~-gal.actosidase while glucose and fructose grown cells produced essentially no 38 ~34~~'33 S-galactosidase. When ~-galactosidase was measured after 6 hours under ~:arbon source starvation, the production of moderate quantities of ~-galactosidase by the transformed organisms grown on glucose and fructose as well as substantial quantities of ~-galactosidase produced by glycerol and galactose grown cells was observed. Thus, the regulatory regions of the invention are capable of controlling the production of protein products in genetically very diverse yeast hosts and are not limited to utilization in methanol utilizing strains.
~srnrrtvr_~c The buffers and solutions employed in the following examples have the compositions given below:
1M Tris buffer 121.1 g Tris base in 800 mL of H20;
adjust pH to the desired value by adding concentrated (35%) aqueous HC1;
allow solution to cool to room temperature before final pH adjustment, dilute to a final volume of 1L.
S-buffer 1.5 M sorbitol in 0.04 M sodium phosphate buffer at pH 6.6.
PK buffer 0.14 M NaCl 1% Sodium dodecylsulfate (SDS) 0.01 M EDTA
in 0.05 M (pH 8.4) Tris buffer ETS buffer lOmM EDTA
0.2 % SDS
in 0.01 M (pH 7.4) Tris buffer '~ 3 9 TE buffer Z.0 mM EDTA
in 0.01 M (pH 7.4) Tris buffer SSC 0.15 M NaCl 15 mM sodium citrate adjusted to pH 7.0 with NaOH
TAE 40 mM acetic acid 5 mM EDTA
in 0.02 M (pH 8.3) Tris buffer PBS (Phosphate 10 mM sodium phosphate (pH 7.0) buffered saline) 0.15 M NaCl Laemmli Loading 62.5 mM Tris-HCl (pH 6.8) Buffer 2% SDS
10% glycerol 5% 2-mercaptoethanol 0.01% bromphenol blue RIPA Buffer 1% NP40 (Sigma) 1% sodium deoxycholate 0.1% sDs in PBS
20xSSPE 20 mM EDTA
0.16 M NaOH
0.2 M NaH2P04~H20 3.6 M NaCl adjusted pH to 7.0 with NaOH

~~~0733 Denhardts'Solution (50x) 5 g Ficoll (trademark) g pol.yvinylpyrrolidone 5 g Bovine serum albumin (BSA; Pentax 5 Fractian V) brought: to a total volume of 500 mL
with water Prehydridization buffer 5x SSPE
5x Denhardt's solution 50% dei.onized formamide 0.2Y SDS
200 pg/mL sheared and denatured herring sperm DNA
LB (Luria-Bertani) 5 g Bac.to-tryptone Medium 5 g Bacto-yeast extract 2.5 g NaCl in 1 L of water, adjusted to pH 7.5 with NaOH
YPD Medium ly Bacto-yeast extract 29~ Bacto-peptone 29~ Dextrose SD Medium 6.75 g yeast nitrogen base without amino acids (DIFCO) 29~ Dextrose in 1 L of water SED 1 M Sorbitol 25 mM EDTA
50 mM DTT

'~ 41 SCE Buffer 9 .1 g Sorbitol 1 ~ 4 4'~ ~ 3 1.47 g Sodium citrate 0.168 g EDTA
50 mL HZO
--pH to 5.8 with HC1 CaS 1 M Sorbitol mM CaCl2 --filter sterilize PEG Solution 20% polyethylene glycol-3350 10 lOmM CaCl2 IOmM Tris-HC1 (pH ?.4) --filter sterilize SOS 1 M Sorbitol 0.3x YPD medium 10 mM CaCl2 Formamide dye :mix 0.1% xylene cylenol FF
0.2% bromphenol blue 10 mM EDTA
95% deionized formamide Top gel 76.8 gm urea 24 mL acrylamide stock 8 mL lOx TBE
bring to final volume of 160 mL
Acrylamide stock 38 gm acrylamide 2 gm bis(N,N-methylenebisacrylamide) add water to total volume of 100 mL

''" 42 Bottom gel 14.4 gm urea 3.0 gm sucrose 7.5 mL lOx TBE
4.5 mL acrylamide stock 0.3 mL bromphenol blue solution (0.01 g/mL) add water to give total volume of 30 mL
Prehybridization Buffer for hybridization selection 50% formamide 0.75% M NaCI
0.1 M TRIS, pH 7.4 0.008 M EDTA
0.5% SDS
200 Ng/mL rabbit liver tRNAs (Sigma) 0.5 M NETS
Buffer 0.5 M NaCl 10 mM EDTA
10 mM TRIS, pH 7.4 0.2% SDS
lOX RT Buffer 500 mM NaCl 340 mM TRIS, pH 8.3 60 mM MgCl2 50 mM DTT (dithiothreitol) dil RT 4 NL H20 1 ~L lOX RT Buffer 5 ~L reverse transcriptase, 15 U/~L
(Life Sciences, Inc.) t dideoxy:
dd ATP 0.49 mM
dd CTP 0.1165 mM
dd GTP 0.369 mM
dd TTP 0.82 mM
dNTP mix 0.625 mM dGTP
0.625 mM dATP
0.625 mM TTP
Chase 1.125 mM dATP

1.125 mM dCTP

1.125 mM dGTP

1.125 mM TTP

in 1X RT buffer Unless otherwise specified, the above solutions represent the basic (lx) concentration employed. Throughout the examples, where the different concentration levels are employed, that fact is indicated by referring to the solution as a multiple of the basic (1x) concentration.
The following abbreviations are used throughout the examples, with the following meaning:
EDTA ethy~enediamine tetraacetic acid TEMED N,N,N~,N~-tetramethylenediamine DTT dithiothreitol BSA bovine serum albumin EtBr ethidium bromide Ci Curie dATP deoxyadenosine triphosphate dGTP deoxyguanosine triphosphate TTP thymidine triphosphate dCTP deoxycytidine triphosphate dXTP "generic" deoxy triphosphate nucleotide oligo(dT)12-18 Source: Collaborative Research, Inc.
Zymolyase 60,00 (Trademark) Source: Miles Laboratories Several procedures carried out on a routine basis follow a standard protocol which will be detailed here.
Centrifugation is carried out for a period of time and at a spin rate sufficient to provide a clear supernatant.
Generally, centrifugation of yeast cells is carried out at at least 1500 g for at least 5 minutes.
Nucleic acid extractions with phenol/chloroform/
isoamyl alcohol involve contacting the nucleic acid containing solution with an equal volume of a 50:48:2 ratio by volume mixture of phenol., chloroform and isoamyl alcohol, respectively. Extractions with chloroform/isoamyl alcohol involve contacting the solution to be treated with an equal volume of 48:2 ratio by volume mixture of chloroform and isoamyl alcohol.
When gels, filters, etc. are described as being washed or soaked in a specified solution, the entire gel, filter, or the like was immersed in an appropriate vessel (pan, dish, vial, etc.) in order to contact the entire surface of the gel, filter, or the like with the solution of interest.
Ethanol precipitation of nucleic acids involves first adjusting the salt content of the nucleic acid-containing solution, then contacting the solution with two volumes of cold ethanol.
c . "'- 45 ~340~~~
EXAMPLE I
Growth and Preparation of Yeast Cells Pich.ia pastoris NRRL Y-11430 was grown under carbon limited conditions in continuous culture at 30°C with either methanol or ethanol as sole carbon source in IM1 salts minimal medium as described by Wegner in U.S. 4,414,329. IM1 minimal media contains, per liter of media, 36 mM KHZP04, 23mM (NH4 ) 2 S04 , 2mM MgS04 , 6 . 7 mM KC1, 0 . 7 mM CaCl2 , 0 . 2 ~M
CUS04~5 H20, 1.25 NM KI, 4.5 NM MnS04, 2 ~M Na2Mo04, 0.75 ~M
H3B03, 17.5 NN1 ZnSO,~, 44.5 ~M FeCl2 and 1.6 NM biotin. The cells grown on methanol were grown up to a cell density of 140 g/L (dry weight) with a retention time of about 12 hours.
The cells grown on ethanol were grown up to a cell density of 90 g/L with a retention time of about 11.5 hours. When methanol or ethanol were fed into the fermenter, feed stocks containing concentrations of 20% and 45% alcohol, respectively, 'were used.
Ten grams of fermenter grown Pichia pastoris cells were collectE:d by centrifugation and resuspended at approximately 108 cells/mL in 0.1 M Tris (pH 8.0) containing 1% 2-mercaptoethanol. These cells were incubated for 5 to 10 minutes at 37°C and collected by centrifugation. The pellet was washed once with 30 mL of S-buffer and resuspended in 5 mL of S-buffer per gram of cells. Zymolyase (Miles Biochemicals) was added to the cell suspension to give a final concentration of 500 ~g/mL. The cells were incubated at 37°C for 20 minutes and then centrifuged; supernatant discarded and the cell pellet collected. This pellet was frozen in liquid nitrogen and stored at -70°C for later use.
EXAMPLE II
Isolation of Yeast RNA
Total cell RNA was prepared by pulverizing the frozen pellet prepared as described in Example I with a w ~.3~fl?3~
mortar and pe:>tle and further disrupting the frozen pellet for about 2-5 minutes in a blaring (trademark of Dynamics Corporation of America) blender in the presence of liquid nitrogen. 7.'he pulverized pellet was added to PK
buffer at a .concentration of 7.5 mL per gram of cells.
Proteinase K (Boehringer Mannheim) was added to the resuspended pellet to give a final concentration of 400 ~g/mL, and the suspension was incubated at room temperature for 10 minutes. This mixture was extracted with phenol/chloroform/isoamyl alcohol followed by a chloroform/iso~imyl alcohol extraction. Nucleic acids were precipitated by adjusting the solution to be 0.25 M NaCl and adding ethanol. The pellet was resuspended in a minimum volume of ETS buffer, i.e. that volume of buffer sufficient to dissolve the nucleic acids; generally, about 100 ~g up to about 1 mg of DNA per mL of solution. This solution was re-extracted with phenol/chloroform/isoamyl alcohol, then chloroform/isoamyl alcohol and finally precipitated with ethanol.
The nucleic acids were redissolved in a minimum volume of TE buffer. The RNA present in this solution was enriched either by centrifugation through a 4 mL CsCl cushion (1 g CsCl/mL, 7L mM EDTA, in 10 mM Tris (pH 7.4) buffer, or by precipitation by making the solution 2 M LiCl, maintaining at 4-8°C overnight. and collected by centrifugation. The poly A+
RNA was selected from the solution by affinity chromatography on oligo(dT)cellulose columns. Generally, 0.25 gm of oligo (dT) cellulose, type 3 (Collaborative Research) was prepared for chromatography per 5 to 10 mg of total RNA. 0.25 g of oligo (dT) cellulose was slurried in 2 mL of ETS buffer and poured into a small, siliconized glass column. This oligo (dT) cellulose column was washed by layering 10 mL of 0.1 M
NaOH over the oligo (dT) cellulose and allowing the wash solution to f7Low through the oligo (dT) cellulose matrix.
The oligo (dT) cellulose was then washed in the same manner 4~ 1~~~'~33 with 10 mL of ETS buffer and washed a final time with 10 mL
of 0.5 M NETS buffer.
Total RNA (5 to 10 mg) was resuspended in ETS
buffer at a concentration not greater than about 10 mg/mL, placed in a E~5°C water bath for 2 minutes and then placed immediately on ice. The RNA solution was then allowed to warm to room temperature and a stock solution of 5 M NaCl was added to give a final salt concentration in the RNA solution of 0.5 M NaCl. The resulting RNA solution was layered onto the prepared oligo (dT) cellulose column and allowed to slowly flow through the column at a rate of about 1 drop/5 seconds. The material flowing out the column bottom was collected in a tube and relayered onto the top of the column.
The material collected from the column bottom was relayered on top a second time, resulting in the RNA solution being passed through the oligo (dT) cellulose column a total of three times. After' the last pass through the column, the material was collected and labelled as the poly A-, i.e., non-poly A RNA. The column was then washed with 30 mL of 0.5 M NETS and finally the poly A+ RNA was eluted from the column by loading 5 :mL of ETS buffer onto the column and allowing this buffer to flow through slowly, collecting the poly A+
RNA fraction i.n the 5 mL fraction flowing from the bottom of the column. Assuming that there was no NaCl in the poly A+
RNA fraction, the NaCI concentration of this fraction was adjusted to 0..25 M NaCl and RNA precipitated with ethanol.
EXAMPLE III
Construction of cDNA Library Complementary DNA (cDNA) clones were synthesized as follows. Ten ~g of poly A+ RNA prepared as described in Example II was resuspended in 7 ~L H20 and brought to a final concentration of 2.7 mM CH3HgOH, then incubated at room temperature for 5 minutes. The first strand of cDNA was synthesized at: 42°C fox 15 minutes in 50 ~L of a solution 134~7~~
containing 50 mM Tri.s, (pH 8.3) at 42°C, 10 mM MgCl2, 30mM
2-mercaptoethanol, i'OmM KCI, 500 pM each of dATP, dGTP, and TTP, 200 pM dCTP, 2f~ pg/mL oligo(dT), 60pg/mL actinomycin D, 25 units RNasin (Trademark Biotec, Inc.), 25 pCi a-32P dCTP (32.5) pmoles), and 120 units of reverse transcriptase (Life Sciences Inc.). This reaction mix was incubated at 37°C for an additional 15 minutes. The reaction was terminated by the addition of 2 pL of 0.5 M EDTA and 0.5 pL 20% SDS. The reaction was adjusted to 0.3 M NaOH and incubated at 65°C for 30 minutes. The reaction mix was then neutralized by the addition of 10 pL of 1 M Tris, (pH 7.4) and adjusting the reaction mix to 0.21. M HC1. The reaction mix was phenol/chloroform/isoamyl alcohol extracted, then chlorofrom/isoamyl alcohol extracted and finally chromatographed over a Sephadex (Trademark) G50 column in TE buffer.
The radioactive single-stranded cDNA was pooled into one fraction and concentrated to 100 pL either by butanol extraction or evaporation by centrifugation under vacuum. The single stranded cDNA was ethanol precipitated from the concentrated solution, cDNA collected by centrifugation and resuspended in 100 pL of water.
The aqueous single-stranded cDNA solution was adjusted to 2.5 M ammonium acetate, ethanol precipitated, collected by centrifugation. and resuspended in 20 pL of water. This single stranded DNA solution was brought to a final volume of 50 pL with 50 mM potassium phosphate buffer (pH 7.4) containing 5 mM MgCl2, 1 mM 2-mercaptoethanol, 250 pM each of dATP, dGTP, and TTP, 125 pM dCTP, 25 pCi-a-32P-dCTP
(32.5 pmoles), and 8 units of Klenow fragment DNA Poll (New England Biolabs). The resulting reaction mixture was incubated at 37° for one hour in order to synthesize the complementary second DNA strand to the single stranded cDNA.
The reaction was terminated by the addition of 2pL of 0.5 M
EDTA. The double stranded cDNA was phenol/chlorofrom/isoamyl alcohol extracted, chloroform/isoamyl alcohol extracted and CJ

49 134~~~3 chromatographed over a Sephadex G50 column in TE buffer. The double stranded cDNA fractions were pooled and the pool was concentrated and precipitated as described for the single-stranded cDNA.
After the final ethanol precipitation and the collection of the double stranded cDNA by centrifugation, the pellet was re~:uspended in 20.25 ~L of water, then brought to a final volume of 50 ~L with 50 mM Tris, (pH 8.3 at 42°C), containing 10 mM MgCl2, 30 mM 2-mercaptoethanol, 70 mM KC1, 500 ~M of dXTP, and 150 units of reverse transcriptase. The resulting solution was incubated at 42°C for 15 minutes in order to insure completion of the synthesis of the second strand of cDNP~. The reaction was terminated by the addition of 2 ~L of 0.5 M EDTA and concentrated and precipitated as described for the single stranded cDNA reaction.
The double stranded cDNA pellet was resuspended in 42 ~L of H20 and the solution brought to a final volume of 47 ~L by the addition of 5 ~L of a stock solution containing 2.8 M NaCl, 200 mM NaOAc and 45 mM ZnSO~, then adjusted to a pH
of 4.5 at 22° with HC1. In order to digest the hairpin loop, three separate reactions were done with three different concentrations of S1 nuclease (Sigma). One unit, 10 units or 100 units of S1 nuclease were added to bring the reaction volume to 50 ~L, and the reaction incubated at 22°C for 30 minutes. The reaction was terminated by the addition of 2 ~L
of 0.5 M EDTA and 2.67 ~L of 2 M Tris base. Six Ng of rabbit liver tRNA were added as a carrier, and the reaction mixture was concentrated and precipitated as described above except the DNA pellets were resuspended in TE buffer rather than water. After the final precipitation, the pellet was resuspended in 20 ~L of TE buffer and brought to a final volume of 50~ ~L in terminal transferase buffer (BRL) containing 10 ;pmoles of a-32P-dCTP, 2 NM dCTP and 21 units of terminal transferase (Ratliff Biochem). The resulting solution was incubated at 3?°C for 30 minutes in order to add -~ 50 1~4a7 poly d(C) tail. to the 3~-OH end of the double-stranded cDNA.
The reaction was terminated by the addition of 5 NL of 0.5 M
EDTA, extracted, chromatographed, and stored as an ethanol precipitate.
The double stranded, d(C) tailed cDNA was either reannealed directly to poly d(G) tailed pBR322 opened at the PstI site or i=first size fractionated on a Sepharose CL4B-200 column (25 ~L fractions). For the unfractionated library, 150 ng of double-stranded poly d(C) tailed cDNA were annealed in 180 ~L of :LO mM Tris, (pH 7.4) which is 0.1 M in NaCl and 1 mM in EDTA 1to 900 ng of d(G) tailed pBR322 opened at the PstI site. Each 25 ~L fraction of the fractionated library was annealed t:o 125 ng of poly d(G) tailed pBR322 in a 50 ~L
final volume of the same annealing mixture described above.
The annealing reactions were incubated at 65°C for 3 minutes, then 42°C for 2 hours and allowed to cool slowly to room temperature.
The annealed cDNA library was transformed into competent E. coli LE392 (ATCC 33572) prepared as follows: An inoculum of LE392 was grown overnight at 37°C in 2x LB media.
Five mL of this overnight culture was inoculated into 200 mL
of fresh 2x LB media and grown to an ODsoo of 0.2-0.3 at 37°C. This culture was placed on ice for 10 minutes and the cells were then collected by centrifugation at 4°C. The cell pellet was resuspended in 80 mL of ice cold 0.1 M CaCl2 and incubated for 25 minutes at 4°C. The cells were collected by centrifugation at 4°C, the cell pellet resuspended in 2mL of ice cold 0.1 ;M CaCl2 and incubated for at least 2 hours at 4°C prior to use. Then 200 ~L of competent cells per 50 ~L
of annealing mix were used for the transformation. The competent cells and the DNA were combined and incubated at about 4°C for ten minutes, followed by an incubation at 37°C
for 5 minutes and finally placed on ice for 10 minutes. An equal volume of 2X LB media was added to the transformation mix and incub<~ted at 37°C for 45 minutes. The transformed ''~ 51 1340733 cells were plated at 250 NL/plate on 150 mm 2x LB plates containing 15 Ng/mL of tetracycline. The plates were incubated at 37°C for 24 hours and stored at 4°C.
Replica filters were prepared by stamping nitrocellulose filters onto an original filter used to lift the colonies off of the plate. These replica filters were incubated on 2x LB-Tet (15 ~g/mL of tetracycline) plates.
The colonies on the filters were prepared for probing by transferring t:he filters to 2x LB-Tet plates containing 200 ~g/mL of chloramphenicol, incubating the filters at 37°C for at least 12 hours, then lysing the colonies by floating the filters on an aqueous pool which is 1.5 M NaCl and 0.5 M_ NaOH
for 10 minutes. The filters were then neutralized by floating them on an aqueous pool which is 1.5 M NaCL and 0.5 M Tris, (pH 7.4) for 15 minutes and repeating this neutralization again. The filters were then air dried and finally dried under vacuum for 2 hours at 70°C.
EXAMPLE IV
Colony Hybridization The vacuum dried nitrocellulose filters containing the cDNA library {prepared as described in the previous example) were' prehyridized at 42°C. for 5 hours in prehybridization buffer. The filters were removed from the prehybridization buffer and lightly rubbed with a gloved hand in 5x SSPE in order to remove cell debris. The filters were placed in hydridization buffer (same as prehybridization buffer except lx Denhardt's). Either 32P-labelled single-strand cDNA (106 cpm/mL) or end-labeled poly A+ RNA
was hybridized to the filters for 17 hours at 42°C. After hybridization, the filters were washed briefly in 2x SSPE at 22°C, followed by two washes at 65°C in O.lx SSPE, 10 minutes each.
End-labeling of poly A+ mRNA was performed by adding 2 ~g of: poly A+ mRNA to a volume of 50 NL containing 52 ~.3~~~33 50 M Tris, (pH 9.5) heating to 100°C for three minutes, and rapidly chilling on ice. This RNA solution was diluted to a final volume of 200 NL and adjusted to SO mM Tris, (pH 9. 5 ) mM MgCl2, 5mM DDT and 50 pmoles of 32P-a-ATP. Ten units 5 of T4 polynuc:Leotide kinase (Boehringer Mannheim) was added and the mixture incubated at 37°C for one hour. The kinasing reaction was terminated by the addition of 10 NL of 0.5 M
EDTA, extractE~d with phenol/chloroform/isaomyl alcohol and chromatographed through Sephadex G50 to remove the 10 unincorporated radioactive label.
~vn~rvr_~ v Northern Hybridizations Two to five ~g of poly A+ mRNA were heated at 65°C
for 5 minute.. in 10 mM sodium phosphate buffer (pH 7.4) containing 50;o formamide, 2.2 M formaldehyde, and 0.5 mM
EDTA. The re~;ulting solution was cooled to room temperature and an appropriate amount (generally about 0.2 volumes based on the volume of sample treated) of 5x sample buffer (0.5%
SDS, 0.025% bromophenol blue, 25% glycerol, 25 mM EDTA) was added. The samples were loaded on a 1.5% agarose gel prepared in 10 mM sodium phosphate buffer (pH 7.4), containing 1.:L M formaldehyde, and electrophoresed in the same buffer. The gel was stained with acridine orange (33 Ng/mL) in 10 mM sodium phosphate buffer (pH 7.4), destained by soaking they gel in the same buffer for 10 minutes, soaked in 10x SSPE far at least 10 minutes, and the RNA transferred to nitrocellulose as described in Example VI.
EXAMPLE VI
Isolation Of Genomic DNA And Clones Pichia genomic DNA was isolated using the method described in Example II for Pichia RNA isolation. The nucleic acid pellet was resuspended in a minimum volume TE
buffer, and incubated with 20 ~g/mL RNase A for 30 minutes at 53 1340?3~
37°C. The solution was brought to 0.14 M ~aC~ and'treated with proteinase K at 200 ~g/mL for 15 minutes at 22°C. The resulting solution was first extracted with phenol/chlorof~orm/isoamyl alcohol and then with chloroform/isoamyl alcohol and finally ethanol precipitated.
The precipitated DNA was resuspended in a minimum volume of TE buffer, a:nd centrifuged in order to clear the DNA
solution.
Ten ~g of P:ichia genomic DNA prepared as described in the previous paragraph was digested with various restriction enzymes (BRL) and electrophoresed on a 1% agarose gel containing TAE. The DNA fragments in the gel were denatured by soaking the gel in 1.5 M NaCl, 0.5 M NaOH for 20 minutes. The gel was neutralized by soaking in 1.5 M NaCl, 0.5 M Tris, (~>H 7.4) for 20 minutes. Prior to transfer, the gel was soakedl in 10x SSPE for at least 5 minutes . A sheet of nitrocellulose was cut to the size of the gel, wetted in water and soal~,ed briefly in lOx SSPE. This filter was laid on top of the gel which in turn had been placed on a piece of parafilm. A sheet of Whatman filter paper and a stack of paper towels were placed on top of the nitrocellulose in order to draw the DNA out of the gel and transfer it to the nitrocellulose. A weight was placed on the stack to facilitate transfer. The DNA was allowed to transfer in this manner for at least 3 hours. After the transfer, the filter was soaked in 5x SSPE briefly, air dried, and dried under vacuum at 70°C'. for 2 hours. Complementary genomic fragments were identified by hybridization to nick-translated cDNA
clones pPC 8.0, pPC 6.4 and pPC 15.0 using the same prehybridization, hybridization, and washing buffers described in Example IV.
200 ng of the cDNA clones were nick-translated for 90 minutes at 14°C in 30 ~L of a solution containing 50 mM
Tris-HCl (pH i'.4), 10 mM MgS04, 100 NM DTT, 50 Ng/mL BSA, 20 ~M each of dG9"P, TTP and dATP, 31.25 pmoles 32P-a-dCTP (3200 Ci/mmol, NEN), 2 units E. cola DNA Poll (BRL), and 0.02 ng DNaseI. The reaction was germinated by the addition of 1pL
of 0.5 M EDTA and 1 pL of 20% SDS. The labelled DNA solution was brought to a finial concentration of 0.3 M NaOH and placed in boiling water for 3 minutes. This mixture was chromatographed on a~ Sephades G50 column. The labelled DNA
fractions were pooled, the specific activity determined and the probe used in hydridization experiments.
Genomic fragments which hydridized to the cDNA
probes were isolatef. by digesting 200 pg of Pichia genomic DNA with various restriction enzymes (BRL) and electrophoresing the: digest: on a 1% agarose gel in TAE
buffer. The appropriate sized band was sliced from the agarose gel, the DNA, electroeluted, passed through an Elutip.
column (Trademark Sc.hleicher and Schuell) and ethanol precipitated.
The electroeluted fragments were resuspended in water and 200 ng fra.gments were ligated to 500 ng of pBR322 which was cleaved at. the appropriate restriction site and dephosphorylated when necessary. The ligation reaction was carried out in 300 p~L of 66 mM Tris, (pH 7.4) containing 6.6 mM MgCl2, lOmM DTT, 0.4 mM ATP, 25 pg/mL BSA, and 40-80 units of T4 DNA ligase, then incubated at 4°C for 24 hours.
The ligation mix was transformed directly into competent LE 392 E. coli cells. The cells were made competent and the transformation done as described in Example III. A series of three transformations were done with 10, 40, and 100 ng of pBR322 (plus insert), each transformation in 100 pL of competent cells. The cells were plated as described in Example III except the antibiotic selection was 50 pg/mL of ampicillin. The clones were transferred to nitrocellulose, replicated and prepared for hybridization as described in Example III. The filters were probed with the appropriate nick-translated cDNA fragment. Streak-purified colonies which were positive in the hydridization were used to prepare additional plasmid, as follows.
c, The plasmi.d bearing LE392 E. coli was grown to a ~ 3 4 0 7 3 OD600 °f 1.0 in lx I,B media containing 50 pg/mL of ampicillin and amplified overnight by the addition of chloramphenicol to a final concentration of 200 pg/mL. The cells were washed in 0.8% NaCl, 20 mM Tri.s, (pH 8.0) 20 mM EDTA, then lysozome treated in 259 sucrose, 50 mM Tris, (pH 7.4) and 20 mM EDTA
with 450 pg/mL lysoz;ome. hysis was achieved by adding 5 M
NaCl to a final conc.entrati.on of 2.0 M followed by the *
addition of an equal. volume of 0.2% Triton X-100 and 40mM
EDTA. The preparation was cleared by spinning at 20,000 RPM
for 45 minutes. The: supernatant was then phenol/chloroform/isoamyl alcohol extracted, chloroform/isoamyl alcohol extracted and EtOH precipitated.
The pellet was resuspended in TE buffer, RNase A treated, phenol/chloroform/isoamyl alcohol extracted and chloroform/isoamyl alcohol extracted. Solid CsCl was added to give a final concentration of 800 pg/mL plus EtBr was added to give a final concentration of 1 mg/mL. The resulting solution was spun in a Vti 50 rotor at 49,000 RPM
for 18-20 hours at 20°C.
The plasmi.d band was visualized by UV fluorescence and drawn from the tube using a needle and syringe. The plasmid solution was n-buta.nol extracted four times and ethanol precipitated at -20°C as an ethanol precipitate.
EXAMPLE VII
Purification Of Alcohol Oxidase Protein samples from Pichia pastoris cells grown on methanol as described in Example I were prepared by lysis of yeast cells, followed by a clearing spin to remove cell debris, as follows: A portion of the fermenter effluent was removed and adjusted to pH 7.5 with ammonion hydroxide, and was homogenized on a Dyno-Mill Model KDL using a 0.6 liter *Trademark r vessel in a continuous operation at 30°C. using belt combination ~~3 and a. flow of 20-30 mL/hr. The beads in the mill were lead free glass beads with a diameter of 0.3-0.5 mm. The resulting homogenate was centrifuged at 5°C. and 20,OOOXg for 30 minutes to yield a cell-free supernatant.
Six 130 mL~ portions of the cell-free supernatant were placed in cellulose acetate dialysis bags and dialyzed at 5°C. against about 8 liters of distilled water. After 4 days, the aqueous phase of each bag was decanted. The solids remaining in the bags consisted of two types of solid. The thin upper white layer was carefully removed and discarded.
The bottom solid was brown-yellow and was crystalline alcohol oxidase. A portion of the crystalline alcohol oxidase was dissolved in distilled water (about 10 times the volume of the solid) and an assay by the dye-peroxidase method showed an activity of 94 EU/mL. The specific activity of the alcohol oxidase was 10.4 EU/mg of protein.
The crystalline precipitate resulting from the above-described dialysis was dialyzed against 0.05 M
potassium phosphate buffer (pH 7.5), and applied to a 2x 200 cm Sepachryl 200 (trademark Pharmacia) column equilibrated with the same buffer. Fractions of 3.5 mL were collected at a flow rate of 10 mL/hr and assayed for alcohol oxidase activity.
The alcohol oxidase activity for reaction with methanol was determined by the following assay procedure (dye-peroxidase method). A dye-buffer mixture was prepared by mixing 0.1 mL of an o-dianisidine solution (1 weight 9 o-dianisidine in water) with 12 mL of aerated 0.1 M sodium phosphate buffer (pH 7.5). The assay mixture was prepared with 2.5 mL of the dye-buffer mixture, 50 pL of methanol, 10 pL of a peroxidase solution (1 mg of horse-radish peroxidase-Sigma, Type II), and 25 pL of the alcohol oxidase solution. The assay mixture was maintained at 25°C in a r ,...., 57 ~~~~733 4x1x1 cm cuvette and the increase in absorbance by the dye at 460 nm was recorded for 2 to 4 minutes. The enzyme activity was calculated by DA
Activity (N mole/min/mL or Enzyme Units/mL) = min x 11.5 wherein 11.5 :is a factor based on a standard curve prepared with known aliquots of H202 and DA is the change in absorbance during the experimental interval.
A total of 0.1 Ng of total protein from each fraction was also assayed for alcohol oxidase content by gel electrophoresis with SDS-polyacrylamide (12%).
EXAMPLE VIII
DNA And Protein Sequencing Determination of DNA sequences was performed by the dideoxy chain. elongation method using bacteriophage M13 (Sanger et al, 1980) or by the chemical modification method (Maxam and Gilbert, 1980). The DNA fragments corresponding to the 5~ end of the alcohol oxidase gene were inserted into the M13mp8 a.nd M13mp9 vectors or end-labelled for the chemical modiiEication method using restriction enzyme sites available in this region.
The 710bp HindIII/SaII fragment from pPG 4.0 was end-labelled f:or Maxam-Gilbert sequencing by first digesting 33 Ng of the ;plasmid with HindIII. The reaction mixture was phenol/chloroform/isoamyl alcohol extracted, chloroform/isoamyl alcohol extracted and ethanol precipitated. The DNA was collected by centrifugation and resuspended in 31 ~L of water. 100 NCi of 32P-a-dCTP (3200 Ci/mmol) and 2. units of Klenow fragment DNA Poll was added to the reaction mixture to give a final volume of 50 ~L
containing 400 NM dATP, 400 ~M dGTP, 50 mM Tris, (pH 7.4), 59 13~0'~33 (b) above. (d) the C (cytosine) reaction was incubated for minutes at. 22°C and contained 1 NL (50,000 cpm) of the labelled DNA i:ragment, 4 NL of water, 15 NL of 5 M NaCl, and 30 NL of hydrazine. The reaction was terminated as described 5 in (b) above.
The DNA pellets were collected by centrifugation, resuspended in 250 NL of 0.3 M sodium acetate, pH 5.5 and ethanol precipitated with 750 ~L of 95% ethanol. The pellets were collected by centrifugation, dried under vacuum for 5 10 minutes, and 'the DNA cleaved by resuspending the pellets in 100 ~L of a 1 to 10 (v/v) dilution of piperidine. The cleavage reaci:ion was incubated at 90°C for 30 minutes and terminated by the addition of 500 ~L of 98% ethanol, 60 mM
sodium acetate (pH 5.5) and 10 ~g/mL tRNA. The reaction mixture was placed in a dry-ice/ethanol bath (about -70°C) for about 5 minutes and the DNA fragments were collected by centrifugation.. The fragments were resuspended in 50 ~L of 0.3 M sodium acetate (pH 5.5) and then ethanol precipitated with 100 NL o~f 95% ethanol. This ethanol precipitation was repeated, the: pellets were washed with 95% ethanol and evaporated under vacuum during centrifugation. The pellet was resuspende:d in 10 ~L of 80% formamide, 10 mM NaOH, 1 mM
EDTA, 0.1% xylene c;yanol and 0.1% bromphenol blue. Two to three ~L were electrophoresed on a 10% 0.4 mm thick polyacrylamide: gel in TBE buffer.
The amino acid sequence of alcohol oxidase was determined by Sequemat, Inc. (Watertown, Mass.) using 2 mg of purified alcoriol oxidase from Pichia pastoris. The first 18 amino acids of the mature protein were determined to be:
Ala-Ile-Pro-Gl.u-Glu-Phe-Asp-Ile-Leu-Val-Leu-Gly-Gly-Gly-Ser-Gly-Ser.

''~ 5$ 134Q 733 mM MgS04, and 1 mM DTT. The reaction mixture was incubated at 37°C for 1 hour and stopped by the addition of 2 NL of 0.:5 M EDTA. The mixture was then phenol/chloroform/isoamyl alcohol extracted, 5 chloroform/isoamyl alcohol extracted, chromatographed on a Sephadex G-50 column, and the labelled nucleic acid fractions pooled and ethanol precipitated. After centrifugation, the DNA pellet waa resuspended in water and digested with SalI.
The digest wa,s electrophoresed on a 1% agarose gel in TAE
10 buffer, and the 710 by band was cut from the gel, the DNA
electroeluted, butanol extracted, and ethanol precipitated.
The fragment was resuspended in 100 ~L of TE buffer, adjusted to 2.5 M ammonium acetate and then ethanol precipitated. The resulting DNA fragment was resuspended in TE buffer at a concentration of 50,000 cpm/NL.
The four base modification reactions were performed as follows: I;a) the G (guanine) reaction was incubated for 8 minutes at 22°C and contained 1 ~L (50,000 CPM) of the labelled DNA i:ragment, 4 ~L of water, 200 ~L of 50 mM sodium cacodylate, pH 8.0, 1 mM EDTA (DMS buffer) and 1 ~L dimethyl sulfate. The reaction was terminated by the addition of 50 NL of DMS stop buffer containing 1.5 M sodium acetate, (pH
7 . 0 ) , 1 M 2-mercaptoethanol and 100 ~ g/mL tRNA, then ethanol (750 NL) was added and the reaction mixture was held at -70°C
for at least 15 minutes. (b) the G/A (guanine/adenine) reaction was incubated for 10 minutes at 22°C and contained 2 ~L (100,000 cpm) of the labelled DNA fragment, 8 NL of water and 30 NL of formic acid. The reaction was terminated by the addition of 200 ~L of Hz stop buffer (0.3 M sodium acetate, pH 5.5, 0.1 M EDTA and 25 ~g/mL tRNA), then ethanol (750 NL) was added and the reaction mixture held at -70°C for at least 15 minutes. (c) t:he T/C (thymine/cystosine) reaction was incubated for 10 minutes at 22°C and contained 2 ~L (100,000 cpm) of the labelled DNA fragment, 18 ~L of water and 30 ~L
of hydrazine. The reaction was terminated as described in 1340?33 EXAMPLE IX
Determination Of Transcriptional Initiation Site To determf.ne where the start of the mRNA for alcohol oxidase was located, a primer extension experiment was performed using a synthetic oligonucleotide copied from the DNA sequences of the 5' end of the alcohol oxidase gene as primer and 10 pg of poly A+ Pichia pastoris mRNA as template. Ten pg of Pichia pastoris poly A+ mRNA was combined with 3 ng of primer (5' -CTT CTC AAG TTG TCG-3') in a final volume of 9.3 pL which was 43 mM NaCl, 29.2 mM Tris (pH
8.3), 5.2 mM MgCl2 a.nd 4.3 mM DTT. The nucleic acids were denatured at 70°C for 5 minutes and reannealled by allowing to slowly cool to 22.°C. The reannealling mix was added to a tube containing 4 pL~ of dNTP mix 0.8 pL RT buffer, and 1 pL
32P-a-dCTP (800 Ci/mmol). Three pL of this mixture was added to 1 pL of each respective ddNTP. The final 3 pL in the mixture was added to 1 pL of water. The reactions were started by the addition of 1 pL of dil RT and incubated at 42°C for 15 minutes. The reactions were chased with 3 pL of Chase RT at 42°C for 15 minutes. The reactions were stopped by the addition of 7.5 pL formamide dye mix and 4-5 pL were electrophoresed on a. 0.4 mm thick gradient geI in lx TBE.
After electrophoresis the gel was fixed in 109 acetic acid with l0y methanol for 20 minutes. The gel was dried under vacuum and used to expose a.n XAR (trademark) x-ray film.
The gradient gel was prepared as follows: 300 pL
of l0y ammonium persulphate and 14 pL of TEMED were added to mL of top gel; 75 pL of 109 ammonium persulfate and 3.5 pL
TEMED were added to 7 mL of bottom gel, 6 mL of top gel were 30 drawn up into a pipet and then 6 mL of bottom gel were drawn into the same pipet. The gel was poured between the gel plates followed by 22 mL of top gel.
i ;

..-. 61 134~'~33 EXAMPLE X
mRNA Hybridization-Selection And In Vitro Translations Positive hybridization-translation experiments were performed by :Linearizing twenty ~g of cloned Pichia genomic DNA (prepared as described in Example VI) by digestion with various restriction endonucleases. This DNA was denatured by making the so7.ution 0.3 M NaOH and incubating at 65°C for 10 minutes. The denatured DNA-containing solution was quickly chilled on ice and neutralized by adjusting to 0.5 M Tris~HC1 (pH 7.4). An equal volume of 20x SSPE was added to the denatured DNA immediately prior to binding the DNA to the nitrocellulose filters. Prior to applying the DNA to the nitrocellulose filters (Schleicher and Schuell BA83, 9 mm dia.), the filters were prepared by wetting with HZO, boiling for 10 minutes and rinsing three times in lOx SSPE. Ten Ng of DNA was then bound to each filter by applying the DNA to the filter, a.ir drying and finally drying the filters under vacuum at 70°C for 2 hours.
Prior to prehybridization, the filters with the bound DNA were placed in 1 mL of sterile water and heated for one minute at 100°C, cooled on ice, rinsed by vortexing in 1 mL of sterile water and rinsed with 1 mL of prehybridization buffer. The filters were prehybridized in 1 mL of prehybridization buffer, then 40 ~g (2~g/mL ET5) of poly A+
mRNA was added directly to the prehybridization buffer. The hybridization mixture was heated at 65°C for 10 minutes and then incubated. at 42°C for 24 hours.
Following the hydridization, filters were washed briefly 2 time's in lx SSPE which contained 0.5% SDS at 22°C, 7 times in lx: SSPE which contained 0.5% SDS at 50°C for 5 minutes each, 3 times in O.lx SSPE at 50°C for 5 minutes each, and once in O.lx SSPE at 65°C for 10 minutes. The RNA
was eluted from the filters by boiling for 1 minute in 300 ~L
of H20 containing 15 ~g of rabbit liver tRNA. The eluted RNA
was quickly frozen. in a dry-ice ethanol bath. The RNA

mixture was allowed to warm to room temperature and the ~ 3 4 0 7 3 3 filters removed. Th.e RNA mixture was then precipitated by adjusting the medium to 2.5 M ammonium acetate and precipitating with eahanol 2 times, and finally resuspended in 100 pL of H20 before being lyophilized.
Translations were performed according to standard techniques known by those of skill in the art, such as for example, instructions provided by New England Nuclear in vitro rabbit reticulocyte lysate translation kits. Protein products were electrophoresed on 8% polyacrylamide gels containing a 4.5% stacking gel.
T~YAMDT.~ YT
Antisera Preparations And Immunoprecipitations Antisera raised in rabbits against an extract from Pichia pastoris cells containing both p72 (alcohol oxidase) and p76 polypeptides were prepared using standard protocols.
Extracts were dialyzed against PBS before injections. Over a course of 8 weeks, each rabbit received 3 injections each of which consisted of 1 mg of total protein in a volume of 0.1 mL with 0.2 mL of Freunds complete adjuvant. Injections were intradermally delivered to 6-10 sites in the rabbit. At the end of eight weeks, the rabbits were bled, and their sera tested against purified Pichia pastoris alcohol oxidase by the Ouchterlony double diffusion procedure.
Purified rabbit anti-p72 (alcohol oxidase) and anti-p76 protein antibodies were prepared by affinity chromatography of whole antisera through a CNBr coupled p72 (alcohol oxidase)-p76 Sepharose 4B column (trademark Pharmacia). One gram of CNBr activated gel was prepared by hydrating the gel for 15 minutes in 200 mL of 1 mM HC1 followed by 3x 50 mL
washes in coupling buffer (0.1 M sodium carbonate (pH 8) and 0.5 M NaCl). Five mL of a 6 mg/mL solution of p72-p76 in coupling buffer was added to the gel and gently agitated overnight at 4°C. Unbound protein was removed by washing 3x G

50 mL with coupling buffer. The remaining active groups were eliminated by a 2-hour incubation in 1 M ethanolamine (pH 8).
Non-covalently bound material was removed from the gel by a 50 mL wash with 2 M sodium thiocyanate in PBS. Prior to chromatography of the antisera, the gel was finally washed with 3x 50 mL of PBS. Five mL of clarified anti p72-p76 antisera were mixed with the gel and incubated with gentle agitation for 2 hours at 4°C. The antisera-gel mixture was then pipetted into a lx 8 cm column and washed with 150 mL of PBS. Purified antibody was eluted from the column with 6 mL
of 2 M sodium thiocynate in PBS. After elution from the gel, the purified antibody was dialyzed extensively against PBS
which contained 0.02y sodium azide.
The affinity-purified antisera was added to an in vitro translation mix in PBS, ly NP40 and incubated overnight at 4°C. The antibody-antigen complex was precipitated with Pansorbin (trademark Calbiochem) on ice for 2.5 hours. Pansorbin was prepared by wahsing in RIPA buffer. Pansorbin precipitates were washed 4 times in RIPA buffer and dissolved in Laemmli loading buffer before electrophoresis.
RYAMDT.F YTT
Pichia nastoris Transformation Procedure A. Cell Growth 1. Inoculate a colony of Pichia pastoris GS115 (NRRL
Y-15851) into about 10 mL of YPD medium and shake culture at 30°C for 12-20 hrs.
2. After about 12-20 hrs., dilute cells to an OD600 of about 0.01-0.1 and maintain cells in log growth phase in YPD
medium at 30°C for about 6-8 hrs.
3. After about 6-8 hrs, inoculate 100 mL of YPD medium with 0.5 mL of the seed culture at an OD600 of about 0.1 (or equivalent amount). Shake at 30°C for about 12-20 hrs.

64 ~344~33 4 . Harve;st culture when ODs o o is about 0 . 2-0 . 3 ( after approximately 16-20 hrs) by centrifugation at 1500 g for 5 minutes.
B. Preparation of Spheroplasts 1. Wash cells once in 10 mL of sterile water. (All centrifugation.s for steps 1-5 are at 1500 g for 5 minutes.) 2. Wash cells once in 10 mL of freshly prepared SED.
3. Wash cells twice in 10 mL of sterile 1 M Sorbitol.
4. Resuspend cells in 10 mL SCE buffer.
5. Add 5-10 ~L of 4 mg/mL Zymolyase 60,000 (Miles Laboratories). Incubate cells at 30°C for about 30-60 minutes.
Since the preparation of spheroplasts is a critical step in the transformation procedure, one should monitor spheroplast formation as follows: add 100 ~L aliquots of cells to 900 ~L of 5% SDS and 900 ~L of 1 M Sorbitol before or just after the addition of zymolyase and at various times during the incubation period. Stop the incubation at the point where cells l.yse in SDS but not in sorbitol (usually between 30 and. 60 minutes of incubation).

6. Wash spheroplasts twice in 10 mL of sterile 1 M
Sorbitol by cE:ntrifugation at 1000 g for 5-10 minutes. (The time and speed. for centrifugation may vary; centrifuge enough to pellet sphe:roplasts but not so much that they rupture from the force.) 7. Wash cells once in 10 mL of sterile CaS.

8. Resuspend cells in total of 0.6 mL of CaS.
C . Transforrr~ation 1. Add I)NA samples (up to 20 ~L volume) to 12 X 75 mm sterile polypropylene tubes. (DNA should be in water or TE
buffer; for maximum transformation frequencies with small amounts of DNA, it is advisable to add about 1 ~L of 5 mg/mL
sonicated E. c~oli DNA to each sample.) 65 X340 ~3~
2. Add :L00 ~L of spheroplasts to each DNA sample and incubate at room temperature for about 20 minutes.
3. Add 1 mL of PEG solution to each sample and incubate at room temperature for about 15 minutes.
4. Centrifuge samples at 1000 g for 5-10 minutes and decant PEG solution.
5. Resuspend samples in 150 NL of SOS and incubate for 30 minutes at room temperature.
6. Add 850 ~L of sterile 1 M Sorbitol and plate aliquots of samples as described below.
D. Regeneration of:~heroplasts 1. Recipe for Regeneration Agar Medium:
a. Agar-Sorbitol- 9 g Bacto-agar, 54.6 g Sorbitol, 240 mL H20, autoclave.
b. lOX Glucose- 20 g Dextrose, 100 mL H20, autoclave.
c. lOX SC- 6.75 g Yeast Nitrogen Base without amino acids, 100 mL H20, autoclave. (Add any desired amino acid or nucleic acid up to a concentration of 200 Ng/mL before or after autoclaving.) d. Add 30 mL of lOX Glucose and 30 mL of lOX SC to 240 mL of the melted Agar-Sorbitol solution. Add 0.6 mL of 0.2 mg/mL biotin and any other desired amino acid or nucleic acid to a concentration of 20 ~g/mL. Hold melted Regeneration Agar at: 55-60°C.
2. Plating of Transformation Samples:
Pour bottom agar layer of 10 mL Regeneration Agar per plate at least 30 minutes before transformation samples are ready. Distribute 10 mL aliquots of Regeneration Agar to tubes in a 45-~50°C bath during the period that transformation samples are in SOS.. Add aliquots of transformation samples described above to tubes with Regeneration Agar and pour onto bottom agar layer of plates. Add a quantity of each sample to 10 mL al.iquots of melted Regeneration Agar held at 45-50°C and pour each onto plates containing a solid 10 mL
bottom agar layer of Regenation Agar.

66 ~34p733 3. Determination of Quality of Spheroplast Preparation:
Remove 10 NL of one sample and dilute 100 times by addition to 990 ~L of 1 M Sorbitol. Remove 10 NL of the 100 fold dilution and dilute an additional 100 times by addition to a second 990 ~L aliquot of 1 M Sorbitol. Spread plate 100 NL of both dilutions on YPD agar medium to determine the concentration of unspheroplasted whole cells remaining in the preparation. Add 100 ~L of each dilution to 10 mL of Regeneration Agar supplemented with 40 ~g/mL histidine to determine total regeneratable spheroplasts. Good values for a transformation experiment are 1-3 X 10~ total regeneratable spheroplasts/mL and about 1 X 103 whole cells/mL.
4. Incubate plates at 30°C for 3-5 days.
Example XIII
Isolation Of P~ichia Pastoris HIS4 Gene And Autonomous Replication Sequences A. Strains The strains employed include:
(a) Pich.ia pastoris strain NRRL Y-11430;
(b) Pich.ia pastoris GS115 (his4; NRRL Y-15851);
(c) S. ~~erevisiae strain 5799-4D (a his4-260 his4-39;
NRRL Y-15859); and (d) E. coli strain 848 (F met thi gal T1R X805 hsdR
hsdM+).
B. Plasmids pYA2 (see Figure 23; consists of the S. cerevisiae HIS4 gene on a 9.3 kbp Pstl fragment inserted at the PstI
site of pBR325) was the source of the S. cerevisiae HIS4 gene fragments and has been deposited in an E. coli host and is available to t:he public as NRRL B-15874.

.~. 134~'~3~
6?
YEpl3 is available from the American Type Culture Collection and has been assigned accession number ATCC 37115.
C. Media Pichia pastoris was grown in YPD (rich) or IMG
(minimal) media. IMG, a minimal medium, consists of the following:
1. IM1 Salts at a final concentration of 36.7 mM
KH2P04, 22.? mM (NH4)2S04, 2.0 mM MgS04~7H20, 6.7 mM KC1, 0.7 mM CaCl2~2H20, prepared as a lOx stock solution and autoclaved;
2. Trace. Salts at a final concentration of 0.2 ~M
CuS04~5H20, 1.25 ~M KI, 4.5 ~M MnS04~H20, 2.0 NM NaMo04~2H20, 0.75 NM H3B03, 17.5 NM ZnS04~7H20, 44.5 ~M FeCl3~6H20, prepared as a 400x stock solution and filter sterilized;
3. 0.4 ~g/mL biotin; and 4. 2% dextrose.
E. coli was cultured in either LB medium or 2B
medium (0.2% DB4PO4,, 1.2% Na2HPO4, 0.013% MgS04~7H20, 0.074%
CaCl2~2H20, 1 ~g/mL thiamine and 0.4% dextrose) supplemented with 100 ~g/mL trypt:ophan, and 0.2% Casamino acids.
D. DNA Isolation 1. Lar a Scale Preparations of Yeast DNA.
Both Pichia pastoris and S. cerevisiae DNA
preparations were carried out by growing yeast cells in 100 mL of minimal medium until Asoo equals 1-2 and then harvesting the cells by centrifugation at 2,000 g for 5 minutes. The cells were washed once in H20, once in SED, once in 1 M sorbitol and then suspended in 5 mL of 0.1 M
Tris-HC1 (pH ',~ . 0 ) which is 1 M in sorbitol . The cells were mixed with 50-100 ~L of a 4 mg/mL solution of Zymolase 60,000 (Miles Laboratories) and incubated at 30°C for 1 hour to digest the cell walls. The spheroplast preparation was then centrifuged at 1000 g for 5-10 minutes and suspended in Lysis buffer (0.1% SDS, 10 mM Tris-HC1, (pH 7.4), 5 mM EDTA and 50 mM NaCl). Proteinase K (Boeringer Mannheim) and RNase A
(Sigma) were each added to 100 Ng/mL and the mixtures incubated at :37°C for 30 minutes. DNA was deproteinized by gently mixing the preparation with an equal volume of chloroform containing isoamyl alcohol (24:1, v/v) and the phases were separated by centrifugation at 12,000 g for 20 minutes. The upper (aqueous) phase was drawn off into a fresh tube and extracted with an equal volume of phenol/
chloroform/isoamyl alcohol. The phases were separated as before and th.e top phase placed in a tube containing 2-3 volumes of cold 100% ethanol. The sample was gently mixed and DNA was collected by spooling onto a plastic rod. The DNA was immediately dissolved in 1 mL of TE buffer and dialyzed overnight at 4°C against 100 volumes TE buffer.
2. Small Scale Yeast DNA Preparations.
Five mL of yeast cultures in minimal medium were grown until A~oo equals 1-5 and harvested by centrifugation at 2,000 g for 5 minutes. Cells were suspended in 1 mL of SED and transferred to a 1.5 mL microfuge tube, washed once in 1 M sorbitol and resuspended in 0.5 mL of 0.1 M Tris-HC1 (pH 7.4) which. is 1 M sorbitol Zymolyase 60,000 (10 ~L of a 4 mg/mL solution) was added and the cells were incubated for 30-60 minutes at 30°C. Cells were then centrifuged for 1 minute, suspended in the Lysis buffer and incubated at 65-70°C. After 15 minutes the samples were mixed with 100 ~L
of 5 M potassium acetate, held in an ice bath for 15 minutes and centrifuged for 5 minutes. The supernatants were decanted into a fresh microfuge tube containing 1 mL of 100%
ethanol, mixed and centrifuged for 10 seconds. Finally, the DNA pellets were aii: dried for 10-15 minutes and dissolved in 50 NL of TE buffer.
3. Large: Scale E. coli DNA Isolations.
E. coli cultures for large scale (0.5-1 L) plasmid preparations were grown at 37°C with shaking in 2B medium supplemented as described above and with the appropriate 1 3 4 0 7 3 3 antibiotic. For cells which contained pBR322 derived plasmids, cultures were grown to an A500 of about 0.7 at which time sufficient chloramphenicol was added to give a concentration of 100 pg/mL and cells harvested approximately hours later. Strains which contained pBR325 derived plasmids were inoculated into the supplemented 2B medium at a starting A550 of about 0.01-0.05 and incubated with shaking at 37°C for 20-24 hours before harvesting.
10 4. Small Scale E. cola DNA Preparations.
For small scale rapid plasmid isolations, 2 mL
cultures in the supplemented 2B medium with antibiotic were grown overnight at 37°C with shaking and harvested by centrifugation in 1.5 mL microfuge tubes. Plasmids from all 15 preparations were isolated by the alkaline lysis method described by Birnboim and Doly (1979).
E. Restriction DNA and Fragment Isolation.
Restriction enzymes were obtained from New England Biolabs and Bethesda Research Laboratories and digestions were performed by routine techniques. Restriction mappings were carried out by comparing parallel digestions of plasmids with and without insert DNA. Restriction fragments were purified by electroelution from agarose gels into Whatman (trademark) 3 MM paper strips backed by dialysis tubing. The fragments were recovered from the paper and tubing by 3-4 washings with 0.1-0.2 mL volumes of a solution which contained 0.1 M NaCl, 50 mM Tris-HC1 (pH 8.0) and 1 mM EDTA. Finally, the fragments were extracted with phenol/chloroform/isoamyl alcohol, precipitated with ethanol and redissolved in a small volume of TE buffer.
F. P. pastoris Library Construction in E. Coli.
For the Pichia pastoris DNA-YEpl3 library construction, 100 pg of YEpl3 was digested to completion with ,.., BamHI and treated with calf intestinal alkaline phosphatase to remove the terminal 5~ phosphate from the DNA. A 100 ~g aliquot of wild type Pichia pastoris DNA from Pichia pastoris NRRL Y-11430) was partially digested with 10 units of Sau3A I
5 by incubation for 5 minutes at 37°C in a total volume of 1 mL. Fragmenta of 5 to 10 kb were size selected by centrifugation through 5-20% sucrose gradients. One ~g of the vector and 2 ~g of the Pichia Sau3A I fragments were mixed with 20 units o.f T4 DNA ligase (Bethesda Research 10 Laboratories) in a total volume of 200 ~L and incubated overnight at 4°C. The ligated DNAs were transformed into E.
coli by adding the entire ligation reaction mix to 2 mL of competent E. coli 848 cells and incubating for 15 minutes at 0°C. The mixture was warmed to 37°C for 5 minutes after 15 which time 4~~ mL of LB medium was added and the 37°C
incubation continued for another 1 hour. Ampicillin was then added to give a total concentration of 100 ~g/mL and the incubation continued for a second hour. Finally, the cells were centrifuged for 10 minutes at 3,000 g, resuspended in 1 20 mL of fresh LB medium and spread in equal aliquots on 10 LB
agar plates containing 100 ~g/mL of ampicillin. The approximately 50,000 colonies which resulted were scraped from the plates and a portion of the cells was inoculated into 500 mL o:E the supplemented 2B medium at a starting A5so 25 of about 0.1. The culture was grown and plasmid was extracted as described above. Of the colonies which were pooled for 'the library, 96 out of 100 tested were tetracycline sensitive and 7 out of 10 examined contained plasmids with insert DNA.
30 For the Pichia pastoris DNA-pYJ8~Cla library construction, 50 Ng of pYJ8~Cla was digested to completion with ClaI a.nd treated with calf intestinal alkaline phosphatase to~ remove the terminal 5~ phosphate from the DNA.
A 100 Ng aliqu.ot of DNA from Pichia pastoris NRRL Y-15851 was 35 partially digeated with 20 units of TaqI by incubation for 5 minutes at 65°C in a total volume of 1 mL. Fragments of 5 to kbp were size selected by electroelution from a 0.5%
agarose gel (See Example II, Section E). One ~g of the vector and 2 Eag of the Pichia TaqI fragments were mixed with 5 20 units of T4 DNA ligase (Bethesda Research Laboratories) in a total volume of 200 ~L and incubated overnight at 4°C. The ligated DNAs were transformed into E. coli by adding the entire ligation reaction mix to 2 mL of competent E. coli 848 cells and incubating for 15 minutes at 0°C. The mixture was 10 warmed to 37°C for 5 minutes after which time 40 mL of LB
medium was added and the 37°C incubation continued for another 1 hour. Ampicillin was then added to give a total concentration of 100 ~g/mL and the incubation continued for a second hour. Fina:Lly, the cells were centrifuged for 10 minutes at 3,000 g, resuspended in 1 mL of fresh LB medium and spread in equal aliquots on 10 LB agar plates containing 100 ~g/mL of ampicillin. The approximately 10,000 colonies which resulted were scraped from the plates and a portion of the cells was inoculated into 500 mL of the supplemented 2B
medium at a starting A5so of about 0.1. The culture was grown and plasmid was extracted as described above.
G. Southern Hybridizations.
For transfer of large or supercoiled DNA molecules to nitrocellulose, DNA was first partially hydrolyzed by soaking agarose gels in 0.25 M HC1 for 10 minutes prior to alkali denaturation.. The hybridization of labelled fragments from the S. cerevisiae HIS4 gene to Pichia pastoris DNA was performed in the presence of 50% formamide, 6x SSC, 5x Denhardt's, 0.1% SDS, 1 mM EDTA, and 100 ~g/mL denatured herring sperm DNA at 42°C. Post-hybridization washes were in lx SSC, 1 mM :EDTA, 0.1% SDS and 1.0% sodium pyrophosphate at 65°C. DNA was. s2p-labelled as described in Example IV.

H. DNA Sequencing DNA sequencing was by the dideoxynucleotide chain termination method of Sanger et a1 (1980).
I. Yeast Transformations S. cerevisiae transformations were carried out by the spheroplast generation method of Hinnen et a1 (1978).
Pichia pastoris transformations were performed following the procedure described above.
J. Analysis of Pichia pastoris Transformants The ability of each plasmid to be maintained as an autonomous element in Pichia pastoris cells was determined as follows: A transformant colony was picked from the regeneration agar plate and streaked onto an SD medium agar plate and inoculated into liquid IMG medium. The SD plate was incubated at 30°C for 3 days after which time a single colony was picked from this plate, streaked onto a second SD
plate and inoculated into a second flask of IMG medium. This process was repeated a third time. The 3 IMG cultures were grown at 30 ° C with shaking to an As o 0 of about 1-2 and then harvested by centrifugation. DNA from the yeast cultures was extracted as described above, electrophoresed at 30 Volts and mAmps for :l0-15 hours into 0.8% agarose gels, transferred to nitrocellulose and hybridized to 32P-labelled pBR322 or pBR325 as described above. As controls, a sample containing 25 10 ng of plasmid isolated from E. coli and a sample containing 1-2. ~g of untransformed Pichia pastoris GS115 DNA
were electrophoresed in parallel with the experimental samples.
K. Isolation of Pichia DNA Sequences.
30 DNA fragments which contained the Pichia HIS4 gene were isolated from a Pichia DNA library by their ability to complement S. cerevisiae his4 strains. The library was 13~fl'~33 composed of 5-20 kb Sau3AI partial digestion fragments of wild type Pichia DNA inserted into the BamHI site of the S.
cerevisiae-E. coli shuttle vector YEpl3. Spheroplasts of S.
cerevisiae NRRL Y-15859 (5799-4D; a his4ABC~ strain) were generated by the technique of Hinnen et a1 (1978), mixed with the Pich.ia DNA library and allowed to regenerate in a medium deficient in histidine. The transformation resulted in about 1x10~~ prototrophic yeast colonies from a population of 5x10 total regeneratable spheroplasts. Total yeast DNA
was extracted from 20 of the His+ colonies and transformed into E. coli. Seventeen of the yeast DNA preparations produced ampi~cillin resistant colonies and each contained plasmid comprised o:f YEpl3 plus insert DNA. To confirm that the His+ transforming plasmids contained the Pichia HIS4 gene and not. a I>NA fragment with suppressor activity, restriction digests of the plasmids were hybridized to a labelled DNA fragment containing a large portion of the S.
cerevisiae HIS4 gene and washed at low stringency. Each of the plasmids which complemented the his4 S. cerevisiae strains contained sequences which hybridized to the S.
cerevisiae HIS4 gene.
To ;search for DNA fragments which contain Pichia ARS activity, DNA from Pichia pastoris GS115 (NRRL Y-15851) was partially digested with TaqI and 5 to 10 kbp fragments were isolated and cloned into the unique CZaI site of pYJ8~Cla. (See Figure 26). Plasmid DNA was recovered from about 10,000 His+ Pichia colonies and used to transform E. coli. Plasmids from about 10,000 ampicillin resistant colonies were isolated and then transformed back into GS115. Forty of the His+ yeast colonies from this sublibrary transformation were separately streaked onto selective med~_um and grown in separate cultures in selective medium. TotaT~ yeast DNA was extracted from each of these 40 cultures and transformed into E. coli. Two plasmids, pYA63 (PARS1) and pYA90 (PARS2) whose yeast DNA preparations produced the most ampicillin resistant E. coli colonies, were -w~ ~4 1340733 selected for further analysis. Both of these plasmids transformed P~~chia pastoris GS115 (NRRL Y-15851) at a very high frequency and each contained an insert of foreign DNA.
EXAMPLE XIV
Construction Of Regulatory Region-ZacZ Gene Fusions A. p72 (Alcohol Oxidase) Regulatory Region Constructs Plasmid pPG 4.0, a pBR322 vector which contains the 4 kilobase pair EcoRI-PvuII genomic DNA fragment from Pichia pastoris was cut with PstI, treated with S1 nuclease to produce blunt ends where cleavage occurred, then cut with BamHI to give a 1.12 kbp DNA fragment which contains the alcohol oxidas;e regulatory region and the coding information for the first 15 amino acids of alcohol oxidase. This 1.12 kbp DNA fragment has the following nucleotide sequence:
1.12 kbp "S1 nuclease".................CTA GGT GGT G
treated end ..................GAT CCA CCA CCT AG
B
alcohol oxidase leuil glyi2 glYi3 This 1.12 kbp~ was ligated into the EcoRI/SmaI/BamHI linker (cleaved with BamHI and SmaI) of the E. coli-S. cerevisiae shuttle vector pSEY:101, Douglas et a1 (1984). Vector pSEY101 contains the E. coli lacZ gene and a polylinker with the following nucleotide sequence:
R1 Sm B
y ....GAATTCCCGGGGATCCC GTC GTT....
CTTAAGGGCC'CCTAGGG CAG CAA....
T T T
R1 Sm B Val9 Vallo..~-galactosidase to give hybrid plasmid pTA011 (See Figure 29).
Since the regulatory region-lacZ gene fusion of pTA011 is out of phase with respect to production of ~-galactosidas;e as shown in Sequence E:

,...
75 _ H
V
t~ t~ b H ~ b~
C~ G
G C~ rtt V c~7 ~ ~
H~
W
~ H ~, U ~ Cq ~ C7 U b~
H~
U b~
N
~i H ~ ~r U b~
H~

. .
a~
N
t~ C9 H ~ E-L~
H
C~~~ V

'' 76 ~34~1'~33 Vector pTA011 is cleaved at the unique BamHI site and the following SmaI linker inserted:
Sm G A T C A C C CJrGG G T

T G G G C C A C T A
C G

~
-I' m S

thus producing hybrid vector pTA012, which has the following nucleotide sequence with respect to the regulatory region-ZacZ fusion:

c~ c~
G t9 V C9 b G b~
rt H
~U
~E-~

O

w U

N

N~

V
w m H W

~

U

cm H

U
b'~

~i H
~C

U
b'~

U

p,.

N

.
.

.
.
.
.

V

H

H
-~' ~

V
tY

1340'33 and thus, the: regulatory region-ZacZ fusion of pTA012 is still out of phase with respect to the LacZ reading frame. In order to bring the N-terminal coding information for the alcohol oxidase structural gene into an open reading frame with the structural ZacZ gene, pTA012 was treated with EcoRI-SmaI anal the resulting DNA fragment ligated into pSEY101 which had similarly been treated with EcoRI and SmaI
thus producing hybrid vector pTA013 (See Figure 30 and the nucleotide seguence below:

1~40'~33 m C9 C) m t~ C9 C9 ~ 5 V C~'E' ~ p, b C9 C~

C9 C) C~ CJ
tr C~ ~

C9 t, f3~

tv m ~r ~

C~ tr M

'Jv C~ b~

N

H Ca Dr C~ CT

H

U i9 r-i p,.

N

C~ C9 Ey ~

E~

i3~~733 The vector pTA013 was then used to transform S. cerevisiae SEY2102 for further studies described below in Example XV.
Vector pTA013 was then cleaved with PstI or PstI
NruI and the regulatory region-ZacZ fusion contained in the 5 cleaved fragment ligated with the HIS4 gene-containing frag ment of shuttle vectors pTAFHl (See Figure 28), pYJ30 (See Figure 27) or pYA2 (See Figure 23) to give, respectively, plasmids pSAOHl, pSAOH5 and pSAOHIO.
pTA013 plus Resulting Plasmids 10 pTAFHl pSAOHl pYJ30 pSAOH5 pYA2 pSAOHIO
B. X76 Regulatory Region CoriStructs Regulatory region-ZacZ gene fusions were prepared 15 as follows with the p76 regulatory region.
1. Using the Entire 5~ Portion of pPG 6.0 The 1.35 kb pair EcoRI fragment of pPG 6.0 was cloned into the unique EeoRI site of pSEY101, an E. coZi-S.
cerevisiae shuttle vector, giving plasmid pT76U1 (See Figure 20 30a). Vector pT76U1 was then cleaved with PstI-NruI, and the larger DNA fragment ligated with the HIS4 gene-containing frag-ment of shuttle vector pTAFHl (Figure 28) as described above to give pT76H3; or with the EcoRI-end filled in PstI-EeoRI
fragment of shuttle vector pBPfl (See Figure 34) to give pT76H4.
25 2. Using a BaZ31 Digest of 5~-pPG 6.0 A 1.35 kb pair EeoRI fragment of pPG 6.0 was cloned into the unique EcoRI site of pSEY8, an E, coZi-S. cerevisiae shuttle vector, which also has a unique SaZI site adjacent to the EeoRI site into which the Piehia DNA was inserted, thus 30 giving plasmid pTA01 (See Figure 31). The plasmid pTA01 was cleaved with SaZI, treated with BaZ31 exonuclease to produce blunt-ended fragments of polypeptide p76 regulatory region of various lengths. The blunt-ended fragments were freed from the remainder of plasmid pSEY8 by cleavage with EcoRI. The ,, ;;
-; r, .

81 ~3~0733 resulting DNA fragments were cloned into the EcoRI-SmaI linker of pSEY101 to give, among others, plasmid pTAF.85 (See Figure 32). Plasmid pTAF.85 is analogous to pTA011 shown in Figure 29, with the p76 regulatory region instead of the p72 (alcohol oxidase) regulatory region.
Vector pTAF.85 was then treated in an analogous fa-shion as vector pTA013 to give plasmids pTAFH.85, pT76H1 and pT76H2. Thus, the following vectors were cleaved and ligated:
pTAF.85 plus Resulting Plasmid pTAFHl pTAFH.85 pT76H7.
pYJ30 pYA2 pT76H2 EXAMPLE XV
Regulation Of ~-Galactosidase Production In Piehia pastorzs The production of S-galactosidase by several Pichia pastoris GS115 (NRRL Y-15851) transformants grown on different carbon sources and under different conditions was investigated.
The transformed strains were grown in minimal medium contain-ing 0.5 ug/mL of biotin and. 0.1~ glucose at 30°C until they reached stationary phase. The cells were then collected by centrifugation and transferred to minimal medium containing 0.5 ug/mL of biotin and 0.5~ methanol and grown for about 3-5 generations at 30°C. After this initial growth on methanol, cells were collected by centrifugation and transferred to fresh minimal medium containing 0.5 ug/mL of biotin and either 0.1~ glucose or 0.3$ methanol as carbon source. The cells were then incubated at 30°C for about 80 hours, with samples being periodically withdrawn to determine the alcohol oxidase and ~-galactosidase levels. After about 20-50 houxs, the car-bon source was depleted and thereafter the cells were main-tained under these carbon source starvation conditions. The results are summarized in Table I.
r: Y

M c~ O O t~ ~ '~ 'Tj rl ''~ b :~ C; G'..
f"'., f.', ~ lf1 Wit' r-~

O

- - - - - - - O

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10 ., >~ s~
>-r~

~ z rn ~ ui ,~

o N
b .

Nw rti Fa ~ -_ --- ---- U1 >C

v O ~t ~ rd N

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O

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Cf r tr '~'' -I .

chi ~ m. rl b~ e~

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-rd ,~

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df7 v v v ~' N M t~$ ~

_ W U7 -IJ ~, ri 00 O O ~; .~
a ~ ~ d1 O ~' o-N ~mn o00 d o M ~ M N U1 ~

r -~ ~
-I r-I

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b s~ >'a O ~~ ..

_ N N N ~ ~ O 5-I

''' 'b "~ ~ N v (($ rl U .'~ O l~ lfl ~ O 4-I
f, C~
a r' ~' rl tT ~ to ~ oo 'd' b N
I

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N

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' 1 ~ 3 - - - - i - - -N

U7 \ U N ~

ca cd rl N N .

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ri .. r-I

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- - -- ----k b N +~

cd W n ~

ri tll rl N M ~ U1 .-~

00o w~~~~

v~

V~ V~ En Ei E-~ U zi Q' cn Ei H

f.~ s~ f3~ i~ C~
fl.. t.~ s~

cd ~r1 o m w.

Alcohol oxidase was de~erm ~ ~d ~ - by the dye-peroxidase method described above (See Example VII) and ~-galactosidase was determined as follows:
~-Galactosidase Assay A. Solutions required:
Z-buffer: Final concentration Na2HP04 ~ 7H20~ 16.1 g 0.06 M
NaH2P04 5.5 g 0.04 M
KC1 0.75 g 0.01 M
MgS04 ~ 7H20 0.246 g 0.001 M
2-mercaptoethanol 2.7 mL 0.05 M
fill up to 1L; pH should be 7 0-Nitrophenyl-S-D-galactoside (ONPG):
Dissolve 400 mg ONPG (Sigma N-1127) in 100 mL of distilled water to make a 4 mg/mL ONPG solution.
B. Assay Procedure:
1. Withdraw an aliquot from the culture medium (0.1-0.5 ODsoo of yeast cells), centrifuge and wash cell pellet with water.
2. Add 1 mL of Z buffer to the cell pellet, 30 NL of CHC13 and 30 NL of 0.1% SDS, vortex, incubate 5 minutes at 30°C.
3. Start reaction by adding 0.2 mL of ONPG (4 mg/mL), vortex.

4. Stop reaction by adding 0.5 mL of a 1 M Na2C03 solution at appropriate time points (A4ao<1).
5. Read absorbance of supernatant at 420 nm.
C. Calculation of ~-Galactosidase Activity Units:
1 U = 1 nmole of orthonitrophenol (ONP) formed per min-ute at 30°C and a pH 7.
1 nmole of ONP has an absorbance at 420 nm (A42o1 of 0.0045 with a 1 cm pathlength; thus, an absorbance of 1 at 420 nm represents 222 nmole ONP/mL, or 378 nmole/1.7 mL since the total volume of supernatant being analyzed is 1.7 mL. Hence, Units expressed in the Tables are calculated:
U = A420 X 378 t(min) The results presented in Table I indicate that a protein foreign to yeast, i.e., ~-galactosidase, can be pro-duced in Piehia pastoris regulated either by the presence of methanol in the nutrient. medium or by carbon source starva-tion after growth on a catabolite repressing carbon source.
EXAMPLE XVI
Regulation Of ~-Galactosidase Production In S. cerevisiae Saecharomyees eerevisiae SEY2102, a strain requir-ing uracil, leucine and histidine supplementation for survi-val, was transformed with plasmids pTA013 and pT76U1. Trans-formed organisms were readily isolated by selecting for colo-nies which did not require uracil supplementation for growth.
The isolated transformants, have been given the laboratory designation SEY2102-pTA013, and SEY2102-pT76U1, respectively.
SEY2102-pTA013 has been deposited with the Northern Regional Research Center in Peoria, Illinois to insure access to the public as of the deposit. date of August 31, 1984. This strain has been assigned accession number NRRL Y-15858.

r'"' 85 ~.34Q~3~ _ Cells of NRRL Y-15858 and SEY2102-pT76U1 were incu-bated at 30°C for about 3-4 generations in minimal medium con-taining 20 Wg/mL of histidine and leucine and 5$ glucose.
Cells were then shifted, i.e., collected by centrifugation and transferred into YP medium with 3~ of the carbon source indi-cated in Table II and grown for about 5 generations at 30°C.
Cells were then incubated for an additional 50 hours under carbon source starvation conditions, and periodically sampled for S-galactosidase. The results are summarized in Table II.
TABLE II
Production of S-Galactosidase by S. eereyisiae ~-gal:actosidase, Units/ODsoo A. Alcohol oxidase regulatory pTA013) region ( Carbon .After 5 Sta rvation Conditions Source (3$) Generations 6 hrs. 20 hrs. 50 hrs.

glucose 0.2 9 11 11 fructose 0.3 30 31 28 ethanol 23 137 115 77 glycerol 640 806 656 788 galactose 982 960 651 766 B. p76 Reg ulatory region T76U1) (p Carbon .After 5 Starvation tions Condi Source (5~) Generations 12 hrs. 25 hrs. 30 hrs.

glucose 2.3 254 815 803 glycerol 470 nd nd nd These results indicate that a protein foreign to yeast, i.e., S-galactosidase, can be produced by Saeeharom~ees cerevisiae regulated by the p72 (alcohol oxidase) and p76 reg-ulatory regions under conditions of carbon source starvation when a catabolite repressing carbon source is employed for ~.3~0733 85a growth, or by growth of transformed S. eerevisiae cells on a relatively non-catabolite repressing carbon source such as glycerol or galactose.
The expression levels for s-galactosidase in S.
cerevisiae under the control of the regulatory regions of this invention can be compared to the expression levels pos-sible with other S. eerevisiae regulated promoters.
~-galactosidase, Promoter Carbon Source Units/OD6oo cytochrome C-ZacZ Raffinose (3$) 460 fusion (CYC1) galactose permease- Galactose (2~) 450 ZaeZ fusion (GAL2) Invertase-ZacZ Glucose (0.1$) 160 fusion (SUC2) It is seen that the regulatory region of the invention sur-prisingly are at least as effective as, or more effective as S. cerevisiae promoters than promoters native to S. cerevi-siae.
x ~3~0'~33 EXANIP LE XV I I
Southern Hybridizations With Yeast Genomic DNA
Nine different methanol assimilating yeasts and one methanol non-assimilating yeast were grown on minimal media (IM1, See Example 1) plus 0.75% methanol or 1.0% glucose, respectively. Total chromosomal DNA was isolated as described in Example VI, i.e., total nucleic acids were isolated, treated with RNase A, extracted first with phenol/chloroform/isoamyl alcohol, then with chloroform/isoamyl alcohol and finally ethanol precipitated.
The precipitated DNA was resuspended in a minimum volume of TE buffer and centrifuged to clean the DNA solution.
Southern hybridization filters were prepared as described in Example VI, i.e., 10 Ng of total DNA from various yeast species was digested with excess HindIII, electrophoresed, DNA denatured, the gel neutralized and DNA
transferred t:o nitrocellulose filters. Prehybridization conditions for these filters included treatment with 50%
deionized formamide, 6x SSC, 5x Denhardt's, 1 mM EDTA, 0.1%
SDS and 100 ~rg/mI~ denatured salmon sperm DNA, at 42°C
overnight. Th.e same conditions were used as a hybridization medium using 32P-nick-translated probes at a final concentration of 106 cpm/mL. The probes included the cDNA
inserts (PstI fragments) from clones pPC 8.3, pPC 15.0 and pPC 6.7, as well as a 2.7 kbp BglII DNA fragment of the P.
pastoris HIS4 gene. Each of these probes were separately used on identical filters for hybridization lasting 24 hours at 42°C. After hybridization the filters were washed twice for 15 minutes at room temperature, and three times at 65°C
for 15 minutes in a solution containing 2x SSC, 1 mM EDTA, 0.1% SDS and 0.1% sodium pyrophosphate. Other washes were tried at lower stringency, i.e., at 55°C and 42°C, to confirm the hybridization results. The filters were then autoradiographed far 72 hours. The results of these hybridizations are summarized in Table III.

87 I344?'33 TABLE III

Hybridization of P. pastoris Genes to Various Yeast Chromosomal DNA *

P. pastoris HIS4 pPC 8.3 pPC 15.0 pPC 6.7 1 ) P. pastoris + + + +

2 ) P. pastoris + + + +

NRRL Y-1b03 3) Hansenula + + + +

capsulatum 4) H. henricii + + (+) +

5) X. nonfermentans + + + +

6) H. polr~morpha (+) + (+) +

7) H. wickerhamii + + + +

8) Torulopsis + + + +

molischiana 9) Saccharomyces (+) - - -cerevisiae 10 ) P. pastoris + + + +

*Legend: + hybridi zation (+) weak hybridization n.o hybridization observed under the conditions employed The results presented in Table III indicate that genes for polypeptides analogous to p76, p72 and p40 are pre-sent in virtually all methanol-assimilating yeasts. It is notable that none of these three genes vaere observed by hy-bridization of DNA from a methanol non-assimilating yeast, S. cerevisiae, while homology between the Pichia pastoris HIS4 gene and the HIS4 gene from S. cerevisiae was readily observed.
The examples have been provided merely to illus-trate the practice of our invention and should not be read so as to limit the scope of our invention or the appended claims in any way. Reasonable variation and modification, not departing from the essence and spirit of our invention, are contemplated to be within the scope of patent protection desired and sought.
SUPPLEMENTARY DISCLOSURE
Further investigation of the invention as herein-before described has now been carried out and the following matter sets forth additional description and exemplification of the invention.
With respect to the nucleotide sequence of the 5~
end of the gene en~~oded in pPC 8.3 and pPG 4.0 as described in Sequences A and B on foregoing pages l6 to 24, the fol-lowing information and results are provided.
Sequence B
In addition, the entire nucleotide sequence for the coding region of t:he alcohol oxidase gene was determined. The nucleotide sequence determined and the predicted amino acid sequence are set forth in Sequence B~ and are believed to be:
Predicted amino acid sequence: Met aZa iZe pro gZu gZu phe ,~..,' ~~"
~~k Nucleotide 5~ -ATG GCT ATC CCC GAA GAG TTT
sequence (pPC 8.3 and 0):3 -TAC CGA TAG GGG CTT CTC AAA
pPG
4.

asp iZe Zeu vaZ Zeu gZy gZy gZy ser ser gZy ser GAT ATC CTA GTT CTA GGT GGT GGA TTC AGT GGA TCC

cys iZe ser gZy arg Zeu aZa asn Zeu asp his ser TGT ATT TCC GGA AGA TTG GCA AAC TTG GAC CAC TCC

ACA TAA AGG CCT TCT AAC CGT TTG AAC CTG GTG AGG

Zeu Zys vaZ gZy Zeu iZe gZu aZa gZy gZu asn gZn AAC TTT CAA CCA GAA TAG CTT CGT CCA CTC TTG GTT

pro gZn g2n pro met gZy Zeu pro ser arg tyr Zeu CCT CAA CAA CCC ATG GGT CTA CCT TCC AGG TAT TTA

GGA GTT GTT GGG TAC CCA GAT GGA AGG TCC ATA AAT

15 pro Zys Zys gZn Zys Zeu asp ser Zys thr aZa ser CCC AAG AAA CAG AAG TTG GAC TCC AAG ACT GCT TCC

GGG TTC TTT GTC TTC AAC CTG AGG TTC TGA CGA AGG

phe tyr thr ser asn pro ser pro his Zeu asn gZy TTC TAC ACT TCT AAC CCA TCT CCT CAC TTG AAT GGT

arg arg aZa iZe vaZ pro cys aZa asn vaZ Zeu gZy AGA AGA GCC ATC GTT CCA TGT GCT AAC GTC TTG GGT

TCT TCT CGG TAG CAA GGT ACA CGA TTG CAG AAC CCA

gZy gZy ser ser iZe asn phe met met tyr thr arg CCA CCA AGA AGA TAG TTG AAG TAC TAC ATG TGG TCT

gZy ser aZa ser asp ser asp asp ? gZn aZa gZu GGT TCT GCT TGT GAT TCT GAT GAC TTN CAA GCC GAG

CCA AGA CGA AGA CTA AGA CTA CTG AAN GTT CGG CTC

30 gZy ser Zys thr gZu asp Zeu Zeu pro Zeu met Zys GGC TCG AAA ACA GAG GAC TTG CTT CCA TTG ATG AAA

CCG AGC TTT TGT CTC CTG AAC GAA GGT AAC TAC TTT

Zys thr gZu thr tyr gZn arg aZa ? gZn ? tyr AAG ACT GAG ACC TAC CAA AGA GCT TGN CAA CNA TAC

35 TTC. TGA CTC TGG ATG GTT TCT CGA ACN GTT GNT ATG

pro asp iZe his gZy phe gZu gZy pxo iZe Zys vaZ

CCT GAC ATT CAC GGT TTC GAA GGT CCA ATC AAG GTT

GGA CTG TAA GTG CCA AAG CTT CCA GGT TAG TTC CAA

ser phe gZy asn tyr thr tyr pro vaZ cys gZn asp AGA AAG CCA TTG ATG TGG ATG GGT CAA ACG GTC CTG

.1,..

phe Zeu arg aZa ser gZu ser gZn gZy iZe pro tyr TTC TTG AGG GCT TCT GAG TCC CAA GGT ATT CCA TAC

AAG AAC TCC CGA AGA CTC AGG GTT CCA TAA CGT ATG

vaZ asp asp Zeu gZu asp Zeu vaZ Zeu thr his gZy GTT GAC GAT CTG GAA GAC TTG GTA CTG ACT CAC GGT

CAA CTG CTA GAC CTT CTG AAC CAT GAC TGA GTG CCA

ata gZu his trp Zeu Zys trp iZe asn arg asp thr GCT GAA CAC TGG TTG AAG TGG ATC AAC AGA GAC ACT

CGA CTT GTG ACC AAC TTC ACC TAG TTG TCT CTG TGA

gZy arg arg ser asp ser aZa his aZa phe vaZ his CGT CGT TCC GAC TCT GCT CAT GCA TTT GTC CAC TCT

GCA GCA AGG CTG AGA CGA GTA CGT AAA CAG GTG AGA

ser thr met arg asn his asp asn Zeu tyr Zeu iZe TCT ACT ATG AGA AAC CAC GAC AAC TTG TAC TTG ATC

AGA TGA TAC TCT TTG GTG CTG TTG AAC ATG AAC TAG

cys asn thr Zys vaZ asp Zys iZe iZe vaZ gZu asp TGT AAC ACG AAG GTC GAC AAA ATT ATT GTC GAA GAC

ACA TTG TGC TTC CAG CTG TTT TAA TAA CAG CTT CTG

gZy arg aZa aZa aZa vaZ arg thr vaZ pro ser Zys GGA AGA GCT GCT GCT GTT AGA ACC GTT CCA AGC AAG

CCT TCT CGA CGA CGA CAA TCT TGG CAA GGT TCG TTC

pro Zeu asn pro Zz~s Zys pro ser his Zys iZe tyr CCT TTG AAC CCA AAG AAG CCA AGT CAC AAG ATC TAC

GCA AAC TTG GGT TTC TTG GGT TCA GTG TTC TAG ATG

arg aZa arg Zys gZn iZe phe Zeu ser cys gZy thr CGT GCT AGA AAG CAA ATC TTT TTG TCT TGT GGT ACC

GCA CGA TCT TTC GTT TAG AAA AAC AGA ACA CCA TGG

iZe ser ser pro Zeu vaZ Zeu gZn arg ser gZy phe ATC TCC TCT CCA TTG GTT TTG CAA AGA TCC GGT TTT

TAG AGG AGA GGT AAC CAA AAC GTT TCT AGG CCA AAA

gZy asp pro iZe Zys Zeu arg aZa aZa gZy vaZ Zys GGT GAC CCA ATC AAG TTG AGA GCC GCT GGT GTT AAG

CCA CTG GGT TAG TTC AAC TCT CGG CGA CCA CAA TTC

pro Zeu vaZ asn Zeu pro gZy vaZ gZy arg asn phe CCT TTG GTC AAC TTG CCA GGT GTC GGA AGA AAC TTC

GGA AAC CAG TTG AAC GGT CCA CAG CCT TCT TTG AAG

gZn asp his tyr eys phe phe ser pro tyr arg iZe CAA GAC CAT TAT TGT TTC TTC AGT CCT TAC AGA ATC

GTT CTG GTA ATA ACA AAG AAG TCA GGA ATG TCT TAG

Zys pro gZn tyr gZu ser phe asp asp phe vaZ arg AAG CCT CAG TAC GAG TCT TTC GAT GAC TTC GTC CGT

TTC GCA GTC ATG CTC AGA AAG CTA CTG AAG CAG GCA
j .r 134a~33 .,.,~..

gZy asp aZa gtu iZe gZn Zys arg vat vaZ asp gZn GGT GAT GCT GAG ATT CAA AAG AGA GTC GTT GAC CAA

CCA CTA CGA CTC TAA GTT TTC TCT CAG CAA CTG GTT

trp tyr aZa asn gZy thr gZy pro Zeu aZa thr asn TGG TAC GCC AAT GGT ACT GGT CCT CTT GCC ACT AAC

ACC ATG CGG TTA CCA TGA CCA GGA GAA CGG TGA TTG

gZy iZe gZu aZa gZy vaZ Zys iZe arg pro thr pro GGT ATC GAA GCT GGT GTC AAG ATC AGA CCA ACA CCA

CCA TAG CTT CGA CCA CAG TTC TAG TCT GGT TGT GGT

gZu gZu Zeu ser gZn met asp gZu ser phe gZn gZu GAA GAA CTC TCT CAA ATG GAC GAA TCC TTC CAG GAG

CTT CTT GAG AGA GTT TAC CTG CTT AGG AAG GTC CTC

gZy tyr arg gZu tyr phe gZu asp Zys pro asp Zys GGT TAC AGA GAA TAC TTC GAA GAC AAG CCA GAC AAG

CCA ATG TCT CTT ATG AAG CTT CTG TTC GGT CTG TTC

pro vaZ met his tyr ser iZe iZe aZa gZy phe phe CCA GTT ATG CAC TAC TCC ATC ATT GCT GGT TTC TTC

GGT CAA TAC GTG ATG AGG TAG TAA CGA CCA AAG AAG

gZy asp his thr Zys iZe pro pro gZy Zy-s tyr met GGT GAC CAC ACC AAG ATT CCT CCT GGA AAG TAC ATG

CCA CTG GTG TGG TTG TAA GGA GGA CCT TTC ATG TAC

thr met phe his phe Zeu gZu tyr pro phe ser arg ACT ATG TTC CAC TTC TTG GAA TAC CCA TTC TCC AGA

TGA TAC AAG GTG AAG AAC CTT ATG GGT AAG AGG TCT

gZy ser iZe his iZe thr sex pro asp pro tyr aZa GGT TCC ATT CAC ATT ACC TCC CCA GAC CCA TAC GCA

CCA AGG TAA GTG TAA TGG AGG GGT CTG GGT ATG CGT

aZa pro asp phe asp arg gZy phe met asn asp gZu GCT CCA GAC TTC GAC CGA GGT TTC ATG AAC GAT GAA

CGA GGT CTG AAG CTG GCT CCA AAG TAC TTG CTA CTT

arg asp met aZa pro met vaZ trp aZa tyr Zys ser AGA GAC ATG GCT CCT ATG GTT TGG GCT TAC AAG TCT

TCT CTG TAC CGA GGA TAC CAA ACC CGA ATG TTC TTC

ser arg gZu thr aZa arg arg ser asp his phe aZa TCT AGA GAA ACC GCT AGA AGA AGT GAC CAC TTT GCC

AGA TCT CTT TGG CGA TCT TCT TCA CTG GTG AAA CGG

gZy gZu vaZ, thr ser his his pro Zeu phe pro tyr GGT GAG GTC ACT TCT CAC CAC CCT CTG TTC CCA TAC

CCA CTC CAG TGA AGA GTG GTG GGA GAC AAG GGT ATG

ser ser gZu aZa arg aZa Zeu gZu met asp Zeu gZu TCA TCC GAG GCC AGA GCC TTG GAA ATG GAT TTG GAG

AGT AGG CTC CGG TCT CGG AAC CTT TAC CTA AAC CTC

~34U'~3~
~''.' 9 3 thr ser asn aZa tyr gZy gZy pro Zeu asn Zeu ser ACC TCT AAT GCC TAC GGT GGA CCT TTG AAC TTG TCT

TGG AGA TTA CGG ATG CCA CCT GGA AAC TTG AAC AGA

aZa gZy Zeu aZa his gZy ser trp thr gZn pro Zeu GCT GGT CTT GCT CAC GGT TCT TGG ACT CAA CCT TTG

CGA CCA GAA CGA GTG CCA AGA ACC TGA GTT GGA AAC

Zys Zys pro thr aZa Zys asn gZu gZy his vaZ thr AAG AAG CCA ACT GCA AAG AAC GAA GGC CAC GIT ACT

TTC TTC GGT TGA CGT TTC TTG CTT CCG GTG CAA TGA

ser asn gZn vaZ gZu Zeu his pro asp iZe gZu tyr TCG AAC CAG GTC GAG CTT CAT CCA GAC ATC GAG TAC

AGC TTG GTC CAG CTC GAA GTA GGT CTG TAG CTC ATG

asp gZu gZu asp asp Zys aZa iZe gZu asn tyr iZe GAT GAG GAG GAT GAC AAG GCC ATT GAG ACC TAC ATT

CTA CTC CTC CTA CTG TTC CGG TAA CTC TTG ATG TAA

arg gZu his thr gZu thr thr trp his cys Zeu gZy CGT GAG CAC ACT GAG ACC ACA TGG CAC TGT CTG GGA

GCA CTC GTG TGA CTC TGG TGT ACC GTG ACA CCA GGT

thr eys ser iZe gZy pro arg gZu gZy ser Zys iZe ACC TGT TCC ATC GGT CCA AGA GAA GGT TCC AAG ATC

TGG ACA AGG TAG CCA GGT TCT CTT CCA AGG TTC TAG

vaZ Zys trp gZy gZy vaZ Zeu asp his arg ser asn GTC AAA TGG GGT GGT GTT TIG GAC GAC AGA TCC AAC

CAG TTT ACC CCA CCA CAA AAC CTG GTG TCT AGG TTG

vaZ tyr gZy vaZ Zys gZy Zeu Zys vaZ gZy asp Zeu GTT TAC GGA GTC AAG GGC TIG AAG GTT GGT GAC TTG

CAA ATG CCT CAG TTC CCG AAC TTC CAA CCA CTG AAC

ser vaZ. eys pro asp asn vaZ gZy cys asn thr tyr TCC GTG TGC CCA GAC AAT GTT GGT TGT A~1C ACC TAC

AGG CAC ACG GGT CTG TTA CAA CCA ACA TTG TGG ATG

thr thr aZa Zeu Zeu iZe gZy gZu Zys thr aZa thr ACC ACC GCT CTT TTG ATC GGT GAA AAG ACT GCC ACT

TGG TGG CGA GAA AAC TAG CCA GTT TTC TGA CGG TGA

Zeu vaZ, gZy gZu asp Zeu gZy tyr ser gZy gZu aZa TTG GTT GGA GAA CAT TTA GGA TAC TCT GGT GAG GCC

AAC CAA. CCT CTT CTA AAT CCT ATG AGA CCA CTC CGG

Zeu asp met thr vaZ pro gZn phe Zys Zeu gZy thr TTA GAC ATG ACT GTT CCT CAG TTC AAG TTG GGC ACT

AAT CTG TAC TGA CAA GGA GTC AAG TTC AAC CCG TGA

tyr gZu Zys thr gZy Zeu aZa arg phe stop TAC GAG AAG ACC GGT CTT GCT AGA TTC TAA- 3~

r""' 9 4 Sequence B~
A comparison of the above nucleotide sequence with the published (Ledeboer et al.) nucleotide sequence for the previously described alcohol oxidase from HansenuZa poZ~morpha reveals numerous significant differences, including the pre-dicted amino acid sequence, the actual size of the gene (and the resulting protein), codon usage bias, and the like.
Identification of p76 as Dihydroxyacetone Synthase The nucleotide sequence for the first 51 nucleo-tides of the p76 gene was determined by standard techniques.
From this sequence, the amino acid sequence for the amino terminal end of the p76 protein can be predicted:
Amino acid sequence: met aZa arg iZe pro Zys Nucleotide sequence: 5~-ATG GCT AGA ATT CCA AAA
3~-TAC CGA TCT TAA GGT TTT
pro vaZ ser thr gZn asp asp iZe his gZr~ Zeu CCA GTA TCG ACA CAA GAT GAC ATT CAT GAA TTG-3~

This predicted amino acid sequence for p76 can be compared with the published amino acid sequence for the di-hydroxyacetone synthase (DHAS) protein from HansenuZa poZz~mor-pha (Manowicz et al..). Although several differences in the sequences are apparent, there are similarities between the two proteins which can be discerned:
Pichia DHAS: met-aZa- arg-iZe-pro-Z~s-pro-HansenuZa DHAS: met-ser-met-arg-iZe-pro-Z~s-aZa-vaZ-ser-thr-gZn-asp-asp-iZe-his-g2u- -Zeu-aZa-ser-vaZ-asn-asp-gZu-gZn-his-gZn-arg-iZe-Based on. the significant degree of homology and the similar protein size (about 76,000 daltons) of Piehia p76 and HansenuZa DHAS, p76 has been tentatively identified as DHAS
from Piehia.
As above with the alcohol oxidase gene, a compari-son of the nucleotide sequence for the first 51 nucleotides of e, ~~4~~'33 -'"' 9 5 the Pichia DHAS protein with the previously published (Jano-wicz et al.) nucleotide sequence of HansenuZa DHAS suggests numerous differences in codon usage bias, the predicted amino acid sequence, the total size of the gene, etc.
Figure 8c shows a restriction endonuclease cleavage site map of the Piehia DNA fragment from pPG 3.2.
Since the alcohol oxidase gene in pPG 4.0 terminates within a few hundred base pairs of the AO gene transcription termination site, t:he additional 3~ sequence detailed in Figure 8c was obtained as follows. The first step was to digest Pichia chromosomal DNA with EcoRI and SaZI and hybridize the digested DNA with a 2.0 kbp 32P-labelled BamHI-HindIII fragment from the AO gene by the Southern blot method. Among the Pichia EcoRI-SaZI
digestion fragments which hybridized with the AO gene probe was a 3.2 kbp fragment 'which encodes the 3~ portion of the AO gene and sequences flanking the 3~ terminus of the gene.
The 3~ AO gene fragment was then cloned by recovering EcoRI-SaZI-cut Pichia DNA fragments of about 3.2 kbp by gel elu-tion and inserting the fragments into EcoRI and SaZI-digested pBR322. Finally, a recombinant plasmid, pPG 3.2, which contains the 3~ AO gene fragment was identified by colony hybridization using the labelled .AO gene fragment as probe. An E. coZi strain transformed with plasmid pPG 3.2 has been deposited with the Northern Regional Research Center, Peoria, Illinois, to insure free access to the 'public upon issuance of a patent on this ap-plication. Th.e deposited strain has been assigned accession number ~1RRL B~15999. Figure 8c shows a restriction endonuclease cleavage site map of the Pichia DNA fragment from pPG 3.2. The fragment contains about 1.5 kbp encoding the 3~ portion of the AO gene (fxom SaZI to HindIII) and about 1.7 kbp of sequence 3~
of the AO gene.
In referring to the detailed analysis of the alcohol oxidase gene and the Sequence C and Sequence D described at pages 24 to 26 hereinbefore, further information was obtained with respect to the novel 1.1 kbp DNA fragment and the 3~ regu-latory region of the alcohol oxidase gene was further charac-terized.

In order to further describe this novel 1.1 kbp DNA
fragment, additional was carried out to nucleotide sequencing fully deline ate the nucleotide sequence the entire 1.1 kbp of DNA fragment shown in Exampl es XIV and to be capable of XV

controlling gene expression in yeast. The nucleotide sequence is set forth as Sequence D~:

5~- C
TCT
G
A

A ATCCAAAGA
A
A

CGAAAGGTTG AATG.AAACCT TTTTGCCATC CGACATCCAC

AGGTCCATTC TCAC.ACATAA GTGCCAAACG CAACAGGAGG

GGATACACTA GCAGCAGACG TTGCAAACGC AGGACTCATC

CTCTTCTCTA ACACCATTTT GCATGAAAAC AGCCAGTTAT

GGGCTTGATG GAGCTCGCTC ATTCCAATTC CTTCTATTAG

GCTACTAACA CCATGACTTT ATTAGCCTGT CTATCCTGGC

CCCCCTGGCG AGGTCATGTT TGTTTATTTC CGAATGCAAC

AAGCTCCGCA TTAC.ACCCGA ACATCACTCC AGATGAGGGC

TTTCTGAGTG TGGGGTCAAA. TAGTTTCATG TTCCCAAATG

GCCCAAAACT GACAGTTTAA ACGCTGTCTT GGAACCTAAT

ATGACAAAAG CGTG.ATCTCA TCCAAGATGA ACTAAGTTTG

GTTCGTTGAA ATCCTAACGG CCAGTTGGTC AAAAAGAAAC

TTCCAAAAGT CGCC.ATACCG TTTGTCTTGT TTGGTATTGA

TTGACGAATG CTCAAAAATA ATCTCATTAA TGCTTAGCGC

AGTCTCTCTA TCGCTTCTGA ACCCGGTGGC ACCTGTGCCG

AAACGCAAAT GGGG.AAACAA CCCGCTTTTT GGATGATTAT

GCATTGTCCT CCAC.ATTGTA TGCTTCCAAG ATTCTGGTGG

GAATACTGCT GATAGCCTAA CGTTCATGAT CAAAATTTAA

CTGTTCTAAC CCCT.ACTTGG ACAGGCAATA TATAAACAGA

AGGAAGCTGC CCTGTCTTAA ACCTTTTTTT TTATCATCAT

TATTAGCTTA CTTTCATAAT TGGGACTGGT TCCAATTGAC

AAGCTTTTGA TTTT.AACGAC TTTTAACGAC AACTTGAGAA

~
-GATCAAAAAA CAACTAATTA 3 .
TTCGAAACG

Sequence D~
It is recognized by those of skill in the art that additional control functions, relative to Sequences C and D, may be encoded in that portion of Sequence D~ which is further "' 9 7 upstream (i.e., in the 5~ direction) of the nucleotide sequence detailed in Sequences C and D.
The 3~ regulatory region of the alcohol oxidase gene was further characterized by determining the nucleotide sequence for about 120 nucleotides downstream of the point where the structural information for p72 (alcohol oxidase) is encoded.
The sequence is set forth below as Sequence D~~:
5~-TCAAGAGGAT GTCAGAATGC CATTTGCCTG AGAGATGCAG
GCTTCATTTT TGATACTTTT TTATTTGTAA CCTATATAGT
ATAGGATTTT TTTTGTCAAA AAAAAAA.AAA AAAAAAAAAA-3~
Sequence D~~
Detailed Analysis of the p76 Gene The regulatory region of the clone pPG 6.0 was also further characterized by determining the nucleotide se-quence of the clone upstream (5~) of the point where the struc-tural information for p76 is encoded. The first 622 nucleo-tides prior to the mRNA translation start site (ATG codon) are believed to be:
5~-TT
CACCCATACA ACTATAAACC TTAGCAATTG AAATAACCCC

AATTCATTGT TCCGAGTTTA ATATACTTGC CCCTATAAGA

AACCAAGGGA TTTCAGCTTC CTTACCCCAT GAACAGAATC

TTCCATTTAC CCCCCACTGG AGAGATCCGC CCAAACGAAC

AGATAATAGA AAAAAACAAT TCGGACAAAT AGAACACTTT

CTCAGCCAAT TAAAGTCATT CCATGCACTC CCTTTAGCTG

CCGTTCCATC CCTTTGTTGA GCAACACCAT CGTTAGCCAG

TACGAAAGAG GAAACTTAAC CGATACCTTG GAGAAATCTA

AGGCGCGAAT GAGTTTAGCC TAGATATCCT TAGTGAAGGG

TGTCCGATAC TTCTCCACAT TCAGTCATAG ATGGGCAGCT

TGTATCATGA AGAGACGGAA ACGGGCATAA GGGTAACCGC

CAAATTATAT AAAGACAACA TGCCCCAGTT TAAAGTTTTT

CTTTCCTATT CTTGTATCCT GAGTGACCGT TGTGTTTAAT

ATAAAAAGTT CGTTTTAACT TAAGACCAAA ACCAGTTACA

98 1340'33 ACAAATTATA ACCCCTCTAA ACACTAAAGT TCACTCTTAT
CAAACTATCA AACATCAAAA-3~
Sequence D~~~
The promoter function of clone pPG 6.0 is believed to be contained within this sequence of nucleotide bases, al-though those of skill in the art recognize that additional regulatory properties may be imparted by sequences further up-stream than the sequences presented as Sequence D~
The 3~ regulatory region of clone pPG 6.0 was fur-ther characterized :by determining the nucleotide sequence for about 180 nucleotides downstream (3~) of the point where the p76 structural information is encoded. The sequence is set forth below as Sequence D~~~~:
5~-GTCAGCAGTG TTTCCTGCCA AAGCGATCAA GAGGACGTAC
ATGCTCTCAT TTTTTGGTTT TCTATGTCCG ACGGGGTTCG
TAAACTGGCT TCCTCCTTTT CCTTTCCTGT TGCATTTTAT
TTGGTCAAAC AAAACTAGGG TCTTTTCCTA AAACCTTATG
TCAATGGACC T.ACCACATAG-3~
Sequence D~~~~
We have earlier described the regulatory regions of this expression and particularly transformed yeast strains which are preferred in the process of the present invention be they yeasts known to be capable of growth on methanol or on non-methanolic substrates as given in pages 27 and 28 hexein-before.
In addition, since the regulatory regions of the invention are responsive to a variety of growth conditions, both in terms of induction and repression of expression, the regulated expression of a gene product under the control of the regulatory regions of the invention can be achieved. Thus, for example, cells can be grown on a carbon source which in-duces only low levels of foreign gene expression, then switched to methanol which will strongly induce gene expression. Al-ternatively, regulated gene expression can be achieved by em-ploying mixtures of inducing/repressing feeds such as, for ex-ample, methanol-glu~~ose mixtures. As yet another alternative, high expression levels produced by growth on methanol can be reduced as desired :by addition to the growth media of a re-pressing carbon source such as glucose or ethanol. Of course, those of skill in t:he art recognize that other variations of feed mixtures and order of feed introduction are possible, and afford a great deal of control over the level of gene expres-sion obtained from the invention regulatory regions.
Since the regulatory regions of the present inven-tion have also been demonstrated to be useful for the regulated expression of heter~ologous gene products in yeast strains of the genus Saceharom;yces, for which a large number of auxotro-phic mutants are known. Additional preferred host yeast strains include ATCC 24683 (a trpl, adel, his2, Zeul, gall, ural mutant), ATCC 24684 (a trpl, adel, his7, gall, ural mutant), ATCC 32810 (a trp5, arg4, hiss, Zysl, ade2, gaZ2 mutant), ATCC 34182 (an ade3, his, Zys, ura mutant), ATCC 34352 (an ura2 mutant), ATCC
34353 (an ura2 muta:nt), ATCC 38523 (an argl, thrl mutant), ATCC
38626 (a Zeu2, his4 mutant), ATCC 38660 (a his4, Zeu2, thx4 mutant), ATCC 42243 (an ura3 mutant), ATCC 42336 (an adel, his4, thr4 mutant), ATCC 42403 (an a~g4, Zys7 mutant), ATCC
42404 (an adel, his4, Zeu2 mutant), ATCC 42564 (an ural, his6 mutant), ATCC 42596 (a his4, Zeu2, Zysl mutant), ATCC 42957 (a his4, Zeu2, thr4, t.rp5 mutant), ATCC 42950 (an ade mutant), ATCC 42951 (an ade, Zeu mutant), ATCC 44069 (an ural mutant), ATCC 44070 (a Zeu2, his4 mutant), ATCC 44222 (a his4 mutant), ATCC 44376 (a his4, ade2 mutant), ATCC 44377 (a his4, Zeul mu-tant), and the like axe readily accessible to those of skill in the art.
It is recognized by those of skill in the art that useful host strains are not limited to auxotrophic mutants.
Thus, transformation of prototrophic strains with positive sel-ection markers, such as, for example, antibiotic resistance genes, also provides a useful means for the detection and iso-1340~~~
'"' 10 0 lation of transformed strains.
Following the isolation of Pichia pastoris HIS4 gene, as described on page 42 hereinbefore, it can now be disclosed that the ARG4 gene 'was isolated from Pichia pastoris NRRL Y-11430 employing an analogous protocol and the Arg S. cerevisiae strain S2072A (an a:rg4 Zeu2 trpl gaZ2; obtained from the Yeast Genetic Stock Center, Berkeley, CA).
Those of skill in the art recognize that other marker genes from Pichia c,an similarly be isolated employing appropri-ately deficient S. ~~erevisiae strains.
In the isolation of Piehia pastoris autonomous rep-lication sequences, as described on pages 43 and 44 hereinbefore, one of the putative Pichia autonomous replication sequences (PARSl) was cloned into several other Piehia vectors to examine its ability to maintain the transforming DNA as an autonomous element. Plasmids :pYJ30 (Figure 27) and pBPfl (Figure 34) were still present as autonomous elements after 20 generations of growth on selective media (His-) and were present in multi-copies per cell. Southern blot analysis of cells transformed with pYJ30 indicate about 10 copies per cell.
To determine if plasmids pSA0H5 (See Figure 18) axed pT76H4 (See Figure 22b), which contain PARS1 contributed by pYJ30 and pBPfl, respectively, display similar stability to the plasmids from which they were derived. Cells containing these vectors were grown 'under selective conditions for about 50 gen-erations under selective conditions (His-) in the presence of glucose. The cells were then shifted to non-selective condi-tions (His+) and the loss of prototrophy was monitored. The stability of these :plasmids was comparable to the stability of pYJ30, including the rapid loss of His prototrophy upon shift to non-selective media. Thus, it is believed that ex-periments carried out with plasmids containing the autonomous replication sequence, PARSI, provide results of gene expression from autonomous plasmid DNA.

~~40~~~

Bibliography Birnboim and Doly (1979) Nucl. Acids Res. 7, 1513-1523.
M. G. Douglas et aZ (1984) Proc. Nat. Acad. Sci. U.S. 81, 3983-3987.
Hinnen et aZ (1978) Proc. Nat. Acad. Sci., USA 75, 1929-1933.
Z. A. Janowicz et aZ (1985) Nucl. Acids Res. 13, 3043-3062.
A. M. Ledeboer et aZ (1985) Nucl. Acids Res. 13, 3063-3082.
Maxam and Gilbert (1980) in Methods in Enzymology 65, 499-560.
Southern (1975) J. Mol. Biol. 98, 503-517.
Sanger et aZ (1980) J. Mol. Biol. 143, 161-178.
:a . m:.

Claims (93)

1. An isolated DNA fragment derived from a yeast of the species Pichia pastoris comprising a regulatory region of the alcohol oxidase gene wherein said regulatory region is responsive to the presence of methanol in the culture medium with which a host microorganism is in contact, wherein said regulatory region is capable of controlling the transcription of messenger RNA when positioned at the 5' end of the DNA which codes for the production of said mRNA and wherein said fragment is characterized by the restriction map in Fig. 5 of the drawings or a functional equivalent thereof having one or more bases of a mutation such as an insertion, deletion, or substitution while maintaining substantially the same regulatory activity as said DNA fragment.

2. A DNA fragment in accordance with claim 1 wherein said yeast is Pichia pastoris~NRRL Y-11430.

3. An isolated DNA fragment derived from a yeast of the species Pichia pastoris comprising a regulatory region of the gene coding for the production of polypeptide p76 wherein said regulatory region is responsive to the presence of methanol in the culture medium with which a host microorganism is in contact, wherein said regulatory region is capable of controlling the transcription of messenger RNA when positioned at the 5' end of the DNA which codes for the production of said mRNA and wherein said regulatory region is characterized by the restriction map in Fig. 4 of the drawings or functional equivalent thereof having one or more bases of a mutation such as an insertion, deletion, or substitution while maintaining substantially the same regulatory activity as said DNA
fragment.

4. An isolated DNA fragment derived from a yeast of the species Pichia pastoris which comprises a regulatory region derived from the gene coding for the production of polypeptide p40 wherein said regulatory region is responsive to the presence of methanol in the culture medium with which a host microorganism is in contact, wherein said regulatory region is capable of controlling the transcription of messenger RNA when positioned at the 5' end of the DNA which codes for the production of said mRNA and wherein said regulatory region is characterized by the restriction map in Fig. 6 of the drawings or a functional equivalent thereof having one or more bases of a mutation such as an insertion, deletion, or substitution while maintaining substantially the same regulatory activity as said DNA fragment.

5. An isolated gene from a yeast of the species Pichia pastoris coding for the production of alcohol oxidase having the following amino acid sequence:
Met ala ile pro glu glu phe asp ile leu val leu gly gly gly ser ser gly ser cys ile ser gly arg leu ala asn leu asp his ser leu lys val gly leu ile glu ala gly glu asn gln pro gln gln pro met gly leu pro ser arg tyr leu pro lys lys gln lys leu asp ser lys thr ala ser phe tyr thr ser asn pro ser pro his leu asn gly art arg ala ile val pro cys ala asn val leu gly gly gly ser ser ile asn phe met met tyr thr arg gly ser ala ser asp ser asp asp ? gln ala glu gly ser lys thr glu asp leu leu pro leu met lys lys thr glu thr tyr gln arg ala ? gln ? tyr pro asp ile his gly phe glu gly pro ile lys val ser phe gly asn tyr thr tyr pro val cys gln asp phe leu arg ala ser glu ser gln gly ile pro tyr val asp asp leu glu asp leu val leu thr his gly ala glu his trp leu lys trp ile asn arg asp thr gly arg arg ser asp ser ala his ala phe val his ser thr met arg asn his asp asn leu tyr leu ile cys asn thr lys val asp lys ile ile val glu asp gly arg ala ala ala val arg thr val pro ser lys pro leu asn pro lys lys pro ser his lys ile tyr arg ala arg lys gln ile phe leu ser cys gly thr ile ser ser pro leu val leu gln arg ser gly phe gly asp pro ile lys leu arg ala ala gly val lys pro leu val asn leu pro gly val gly arg asn phe gln asp his tyr cys phe phe ser pro tyr arg ile lys pro gln tyr glu ser phe asp asp phe val arg gly asp ala glu ile gln lys arg val val asp gln trp tyr ala asn gly thr gly pro leu ala thr asn gly ile glu ala gly val lys ile arg pro thr pro glu glu leu ser gln met asp glu ser phe gln glu gly tyr arg glu tyr phe glu asp lys pro asp lys pro val met his tyr ser ile ile ala gly phe phe gly asp his thr lys ile pro pro gly lys tyr met thr met phe lys phe leu glu tyr pro phe ser arg gly ser ile his ile thr ser pro asp pro tyr ala ala pro asp phe asp arg gly phe met asn asp glu arg asp met ala pro met val trp ala tyr lys ser ser arg glu thr ala arg arg ser asp his phe ala gly glu val thr ser his his pro leu phe pro tyr ser ser glu ala arg ala leu glu met asp leu glu thr ser asn ala tyr gly gly pro leu asn leu ser ala gly leu ala his gly ser trp thr gln pro leu lys lys pro thr ala lys asn glu gly his val thr ser asn gln val glu leu his pro asp ile glu tyr asp glu glu asp asp lys ala ile glu asn tyr ile arg glu his thr glu thr thr trp his cys leu gly thr cys ser ile gly pro arg glu gly ser lys ile val lys trp gly gly val leu asp his arg ser asn val tyr gly val lys gly leu lys val gly asp leu ser val cys pro asp asn val gly cys asn thr tyr thr thr ala leu leu ile gly glu lys thr ala thr leu val gly glu asp leu gly tyr ser gly glu ala leu asp met thr val pro gln phe lys leu gly thr tyr glu lys thr gly leu ala arg phe stop

6. An isolated DNA fragment from Pichia pastoris coding for the production of polypeptide p76 and characterized by the restriction map in Fig. 12 of the drawings.

7. An isolated DNA fragment coding for the production of polypeptide p40 in Pichia pastoris wherein said DNA is characterized by the restriction map in Fig. 14 of the drawings.

8. An isolated DNA fragment in accordance with any of claims 1, 3 or 4 wherein said messenger RNA codes for the production of a heterologous polypeptide.

9. An isolated DNA fragment in accordance with claim 1 having the nucleotide sequence:
5'-ATGCTTCCAA GATTCTGGTG GGAATACTGC TGATAGCCTA
ACGTTCATGA TCAAAATTTA ACTGTTCTAA CCCCTACTTG
GACAGGCAAT ATATAAACAG AAGGAAGCTG CCCTGTCTTA
AACCTTTTTT TTTATCATCA TTATTAGCTT ACTTTCATAA
TTGCGACTGG TTCCAATTGA CAAGCTTTTG ATTTTAACGA
CTTTTAACGA CAACTTGAGA AGATCAAAAA ACAACTAATT
ATTCGAAACG-3'.

10. A DNA fragment in accordance with claim 1 further comprising the additional fragments characterized by the restriction map in Fig. 8 of the drawings.

11. A DNA fragment in accordance with claim 3 further comprising the additional fragment characterized by the restriction map in Fig. 7 of the drawings.

12. A DNA fragment in accordance with claim 4 further comprising the additional fragment as characterized by the restriction map in Fig. 9 of the drawings.

13. A DNA fragment in accordance with claim 8 further comprising an additional fragment positioned at the 3' end of the DNA which codes for the production of messenger RNA, wherein said additional fragments is at least one fragment characterized by the restriction map in Fig. 8 of the drawings.

14. A DNA fragment in accordance with claim 13 wherein said additional fragment has the nucleotide sequence:
5'-AATGGCCCAA ACTGACAGTT AAACGCTGTC TTGGAACCTA
ATATGACAAA AGCGTGATCT CATCCAAGAT GAACTAAGTT
TGGTTCGTTG AAATGCTAAC GGCCAGTTGG TCAAAAAGAA
ACTTCCAAAA GTCGGCATAC CGTTTGTCTT GTTTGGTATT
GATTGACGAA TGCTCAAAAA TAATCTCATT AATGCTTAGC
GCAGTCTCTC TATCGCTTCT GAACCCGGTG GCACCTGTGC
CGAAACGCAA ATGGGGAAAC AACCCGCTTT TTGGATGATT
ATGCATTGTC TCCACATTGT ATGCTTCCAA TATTCTGGTG
GGAATACTGC TGATAGCCTA ACGTTCATGA TCAAAATTTA
ACTGTTCTAA CCCCTACTTG ACAGGCAATA TATAAACAGA
AGGAAGCTGC CCTGTCTTAA ACCTTTTTTT TTATCATCAT
TATTAGCTTA CTTTCATAAT TGCGACTGGT TCCAATTGAC
AAGCTTTTGA TTTTAACGAC AACTTGAGAA
GATCAAAAAA CAACTAATTA TTCGAAACG-3'.

15. A DNA fragment in accordance with claim 8 further comprising a polypeptide coding region wherein said regulatory region is positioned at the 5' end of said polypeptide coding region.

16. A DNA fragment in accordance with claim 15 wherein said polypeptide coding region codes for the production of alcohol oxidase.

17. A DNA fragment in accordance with claim 15 wherein said polypeptide coding region codes for the production of polvpeptide p76.

18. A DNA fragment in accordance with claim 15 wherein said polypeptide coding region codes for the production of polypeptide p40.

19. A DNA fragment in accordance with claim 15 wherein said polypeptide coding region codes for the production of a heterologous polypeptide.

20. A DNA fragment in accordance with claim 19 wherein said heterologous polypeptide is beta-galactosidase.

21. A DNA fragment in accordance with claim 20 wherein said DNA fragment is characterized by the restriction map in Fig. 15 of the drawings.

22. A DNA fragment in accordance with claim 20 wherein said DNA fragment is characterized by the restriction map in Fig. 16 of the drawings.

23. A DNA fragment in accordance with claim 16 wherein said DNA fragment is characterized by the restriction map in Fig. 2 of the drawings.

24. A DNA fragment in accordance with claim 17 wherein said DNA fragment is characterized by the restriction map in Fig. 10 of the drawings.

25. A DNA fragment in accordance with claim 18 wherein said DNA fragment is characterized by the restriction map in Fig. 11 of the drawings.

26. A DNA fragment in accordance with claim 15 further comprising a sequence of DNA downstream of the polypeptide coding region, wherein said sequence of DNA is capable of controlling the polyadenylation, termination of transcription and termination of translation of messenger RNA coded for by said polypeptide coding region.

27. A DNA fragment in accordance with claim 15 wherein said DNA fragment further comprises one or more additional DNA sequence derived from the group consisting of bacterial plasmid DNA, and isolated yeast chromosomal DNA.

28. A DNA fragment in accordance with claim 27 wherein said yeast chromosomal DNA comprises an autonomously replicating DNA sequence and a marker gene.

29. A DNA fragment in accordance with claim 26 wherein said DNA fragment further comprises one or more additional DNA sequences derived from the group consisting of bacterial plasmid DNA, and isolated yeast chromosomal DNA.

30. A DNA fragment in accordance with claim 26 wherein said yeast chromosomal DNA comprises an autonomously replicating DNA sequence and a marker gene.

31. A DNA fragment comprising a first regulatory region wherein said first regulatory region is capable of controlling the polyadenylation, termination of transcription and germination of translation of messenger RNA
when positioned at the 3' end of the polypeptide coding region which codes for the production of said messenger RNA, wherein the transcription and translation of said messenger RNA is controlled by a second regulatory region of any one of claims 1, 3 or 4 and which is responsive to the presence of methanol in the culture medium with which a host microorganism for said DNA fragment is in contact, wherein said second regulatory region is capable of controlling the transcription of messenger RNA when positioned at the 5' end of the DNA which codes for the production of said messenger RNA.

32. A DNA fragment in accordance with claim 31 wherein said first DNA fragment is characterized by the restriction map in Fig. 7 of the drawings.

33. A DNA fragment in accordance with claim 31 wherein said first DNA fragment is one of the fragments characterized by the restriction map in Fig. 8 of the drawings.

34. A DNA fragment in accordance with claim 31 wherein said first DNA fragment is characterized by the restriction map in Fig. 9 of the drawings.

35. An isolated gene from a yeast of the species Pichia pastoris coding for the production of alcohol oxidase wherein said gene is characterized by the restriction map in Fig. 13 of the drawings.

36. A gene in accordance with claim 35 further comprising flanking regions of chromosomal DNA as characterized by the restriction map in Fig. 2a of the drawings.

37. A gene in accordance with claim 6 further comprising flanking regions of chromosomal DNA as characterized by the restriction map in Fig. 1a of the drawings.

38. A gene in accordance with claim 8 further comprising flanking regions of chromosomal DNA as characterized by the restriction map in Fig. 3a of the drawings.

39. Hybrid plasmid pPG 6Ø

40. Hybrid plasmid pPG 4Ø

41. Hybrid plasmid pPG 4.8.

42. Hybrid plasmid pPC 15Ø

43. Hybrid plasmid pPC 8.3.

44. Hybrid plasmid pPC 8Ø

45. Hybrid plasmid pPC 6.7.

46. Hybrid plasmid pSAOH 1.

47. Hybrid plasmid pSAOH 5.

48. Hybrid plasmid pSAOH 10.

49. Hybrid plasmid pTAFH 85.

50. Hybrid plasmid pT76H 1.

51. Hybrid plasmid pT76H2.

52. Hybrid plasmid pTA013.

53. A transformed yeast strain wherein said transformed yeast strain is a host for recombinant DNA
material and wherein said recombinant DNA material comprises:
1) a DNA fragment comprising a regulatory region according to any one of claims 1, 3 or 4; and 2) a polypeptide coding region;
wherein said DNA fragment is responsive to the presence of methanol in the culture medium with which a host microorganism for said DNA fragment is in contact wherein said regulatory region is positioned at the 5' end of said polypeptide coding region; and wherein said transformed yeast is capable of expressing the polypeptide coded for by said polypeptide coding region.

54. A transformed yeast strain in accordance with claim 53 wherein said transformed yeast strain is capable of growth on methanol as carbon and energy source.

55. A transformed yeast strain in accordance with claim 54 wherein said transformed yeast strain is selected from the group consisting of the genera:
Candida, Kloeckera, Saccharomyces, Rhodotorula, Hansenula, Torulopsis, Pichia, Schizosaccharomyces, and Kluyveromyces.

56. A transformed yeast strain in accordance with claim 55 wherein said transformed yeast strain is selected from the genus Pichia.

57. A transformed yeast strain in accordance with claim 53 wherein said recombinant DNA material further comprises a second DNA fragment, wherein said second DNA
fragment is capable of controlling the polyadenylation, termination of transcription and termination of translation of messenger RNA when positioned at the 3' end of the polypeptide coding region which codes for the production of said messenger RNA.

58. A transformed yeast strain in accordance with claim 53 wherein said transformed yeast is capable of growth on at least one carbon and energy source selected from the group consisting of:
methanol, glucose, acetate, ethanol, glycerol, sucrose, lactose, fructose, and galactose.

59. A transformed yeast strain in accordance with claim 58 wherein said transformed yeast strain is selected from group consisting of:
Candida, Kloeckera, Saccharomyces, Rhodotorula, Hansenula, Torulopsis, Pichia, Schizosaccharomyces, and Kluyveromyces.

60. A transformed yeast strain in accordance with claim 59 wherein said yeast strain is selected from the genus Saccharomyces.

61. Pichia pastoris NRRL Y-15852 (GS115-pSAOH) 1.

62. Pichia pastoris NRRL Y-15853 (GS115-pSAOH) 5.

63. Pichia pastoris NRRL Y-15854 (GS115-pSAOH) 10.

64. Pichia pastoris NRRL Y-15855 (GS115-pTAFH.85).

65. Pichia pastoris NRRL Y-15856 (GS115-pT76H1).

66. Pichia pastoris NRRL Y-15857 (GS115-pT76H2).

67. Saccharomyces cerevisiae NRRL Y-15858 (SEY
2102-pTA013).

68. Escherichia coli NRRL B-15861 (MC1061-pSAOH

1).

69. Escherichia coli NRRL B-15862 (MC1061-pSAOH
5).

70. Escherichia coli NRRL B-15863 (MC1061-pSAOH
10).

71. Escherichia coli NRRL B-15864 (MC1061-pTAFH
85).

72. Escherichia coli NRRL B-15865 (MC1061-pT76H
1).

73. Escherichia coli NRRL B-15866 (MC1060-pT76H

2).

74. Escherichi a coli NRRL B-15875 (MC1061-pTA013).

75. Escherichia coli NRRL B-15867 (LE392-pPG
6.0).

76. Escherichia coli NRRL B-15868 (LE392-pPG
4.0).

77. Escherichia coli NRRL B-15869 (LE392-pPG
4.8).

78. Escherichia coli NRRL B-15870 (LE392-pPC
15.0).

79. Escherichia coli NRRL B-15871 (LE392-pPC
8.3).

80. Escherichia coli NRRL B-15873 (MM294-pPC
8.0).

81. Escherichia coli NRRL B-15872 (LE392-pPC
6.7).

82. A process for preparing polypeptides comprising cultivating a transformed yeast strain in a nutrient medium containing methanol wherein said transformed yeast strain is capable of expressing an inserted polypeptide coding sequence derived from recombinant DNA
material, wherein said recombinant DNA material comprises:
1) a methanol responsive DNA fragment comprising a regulatory region of any one of claims 1, 3 or 4; and 2) a polypeptide coding region;
wherein said methanol responsive DNA fragment is positioned at the 5' end of said polypeptide coding region.

83. A process in accordance with claim 82 further comprising isolating and purifying said polypeptide.

84. A process in accordance with claim 82 wherein said transformed yeast strain is selected from the group consisting of members of the genera:
Candida, Kloeckera, Saccharomyces, Rhodotorula, Hansenula, Torulopsis, Pichia, Schizosaccharomyces, and Kluyveromyces.

85. A process for preparing polypeptides comprising:
a) cultivating a transformed yeast strain in a nutrient medium wherein said nutrient medium comprises at least one catabolite non-repressing carbon source, wherein said transformed yeast strain is capable of expressing an inserted polypeptide coding sequence derived from recombinant DNA material, wherein said recombinant DNA material comprises:
1) a DNA fragment comprising a regulatory region of any one of claims 1, 4 or 5 wherein said fragment is responsive to the presence of a catabolite non-repressing carbon source in the culture medium with which the transformed yeast strain is in contact; and 2) a polypeptide coding region wherein said DNA fragment is positioned at the 5' end of said polypeptide coding region.

86. A process in accordance with claim 85 wherein said catabolite non-repressing carbon source is selected from the group consisting of glycerol and galactose.

87. A process in accordance with claim 85 wherein said transformed yeast strain is selected from the group consisting of:
Candida, Kloeckera, Saccharomyces, Rhodatorula, Hansenula, Torulopsis, Pichia, Schizosaccharomyces, and Kluyveromyces.

88. A process for preparing polypeptides comprising:
a) cultivating a transformed yeast strain in a nutrient medium wherein said nutrient medium comprises at least one carbon and energy source wherein said transformed yeast strain is capable of expressing an inserted polypeptide coding sequence derived from recombinant DNA material wherein said recombinant DNA material comprises:
1) the regulatory region of any one of claims 1, 4 or 5; and 2) a polypeptide coding region wherein said regulatory region is responsive to carbon source starvation in the culture medium with which the transformed yeast strain is in contact after growth of the transformed yeast strain on at least one catabolite repressing carbon and energy source and wherein said regulatory region is positioned at the 5' end of said polypeptide coding region; and b) subjecting the product of step a) to conditions of carbon source starvation.

89. A process in accordance with claim 88 wherein said at least one catabolite repressing carbon and energy source is selected from the group consisting of glucose, ethanol and fructose.

90. A process in accordance with claim 88 wherein said catabolite repressing carbon source is glucose.

91. A process in accordance with claim 88 wherein said transformed yeast strain is selected from the group consisting of the genera:
Candida, Kloeckera, Saccharomyces, Rhodotorula, Hansenula, Torulopsis, Pichia, Schizosaccharomyces, and Kluyveromyces.

92. An isolated DNA fragment in accordance with claim 6 wherein p76 has the following 17 amino acids at the N-terminal:
met ala arg ile pro lys pro val ser thr gln asp asp ile his gly leu.

93. A DNA fragment in accordance with claim 8 wherein said messenger RNA codes for the production of a heterologous polypeptide.