WO2000070945A2 - Fatty acid elongation genes and uses thereof - Google Patents

Abstract

Human and mouse genes involved in fatty acid elongation and related to CIG30. Nucleic acids, polypeptides, oligonucleotide probes and primers, methods of diagnosis or prognosis, and other methods relating to and based on the genes, and generation of animal models for deficiencies in one or more of these genes, useful particularly in study of various disorders, especially skin or eye disorders, and in assay methods for obtaining agents of therapeutic potential in such disorders.

Description

FATTY ACID ELONGATION GENES AND USES THEREOF

The present invention relates to nucleic acids, polypeptides, oligonucleotide probes and primers, methods of diagnosis or

prognosis, and other methods relating to and based on the

identification of several genes involved in fatty acid

elongation, and generation of animal models for deficiencies

in one or more of these genes, useful particularly in study of

various disorders, especially skin or eye disorders, and in assay methods for obtaining agents of therapeutic potential in

such disorders.

More particularly, the present invention is based on cloning

and characterisation of genes which the present inventors have

termed Sscl and Ssc2. Mouse and human homologues are

provided. Further work providing basis for aspects of the

present invention involved characterisation and use of these

genes and the previously cloned related gene Cig30.

A number of human disorders are known to be related to

abnormal levels of very long chain fatty acids (VLCFA) such as peroxisomal disorders (nine out of fifteen) , myelin

deficiency. There are also several pathological cases, such as e.g. tumor invasion, which are related to a disturbed metabolism of sphingolipid and ceramides, in which VLCFA are

part of. A rational analysis of VLCFA and sphingolipid

metabolism has not been possible since the condensing enzymes which are responsible for the elongation process have not been

isolated.

Fatty acyl chains account for more than half of the mass of

most major phospholipids and are primarily responsible for the apolar nature of the membrane bilayer. Depending on their

chain length and degree of unsaturation, they contribute to

fluidity and other physical and chemical properties of the

membrane. De novo synthesis of fatty acids by the soluble, cytosolic enzymes of the acetyl-CoA carboxylase and fatty acid

synthase complexes produces mainly palmitate (16-carbons) ,

with minor amounts of stearate (18-carbons) . Quantitatively,

these chain lengths are major components of many membrane

lipids and qualitatively appear to be related to the optimum

width of the membrane lipid bilayer. On the other hand, in membranes many major acyl chains are longer than 16-carbons.

For example, in the myelin surrounding axonal processes of

neuronal cells, fatty acyl chains of 18-carbons or greater

make up more than 60% of the total, and in sphingolipids in

particular, acyl chains of 24-carbons are prominent. In liver, brain and other tissues there are two primary systems for elongation, one in the endoplasmic reticulum, and the other in mitochondria.

Microsomal chain elongation appears to be the major source of

acyl chains greater than 16-carbons during growth and maturation, when required long chain acids may not be supplied

in the diet. For example, 18- to 24-carbons are required for

normal brain myelination, regardless of dietary fluctuations

during development .

Furthermore, as major membrane components, acyl chains influence a variety of membrane functions, such as ion

channels and transport, endocytosis and exocytosis, and the

activities of membrane-associated enzymes. These processes

are proposed to occur through dynamic, so called, "rafts"

which are made up specific sphingolipids and cholesterol

within the plasma membrane. Rafts, together with the

structural proteins caveolins, are the major structure of

caveolae which are vesicular imaginations of the plasma

membrane. In these caveolae many classes of signalling molecules are assembled to generate functional signal transducers, including e.g. G-proteins, Ras- and Src family

tyrosine kinases, Protein Kinase C isoforms, EGF receptors, Nitric Oxide Synthase, etc, within the plasma membrane. The regulation of the eukaryotic stress response involves the

operation of multiple and poorly defined regulatory and signal

transduction pathways that sense the degree and type of stress and reprogram it to a cell response. Multiple lines of

evidence implicate sphingolipids, ceramides and sphingosines as key regulators of the eukaryotic stress response, e.g.

ceramides formed after activation of sphingomyelinase (by TNFa and IL-1) decrease cell division and induce apoptosis which on

the other hand can be counteracted by sphingosine-phosphate which stimulate cell growth and inhibit apoptosis.

The inventors isolated a full-length cDNA of a previously uncharacterised gene now termed Cig30 (Tvrdik et al . , J. Biol.

Chem. (1997) 272: 31738-31746).

In the public databases, there are no mammalian proteins

resembling Cig30 , but three yeast proteins (J0343, FEN1 and

SUR4 ) are significantly homologous to Cig30 . Interestingly,

the yeast genes have recently been suggested to function as membrane-bound fatty acid elongases and designated

ELOKJ0343), EL02(FEN1) and EL03(SUR4) where ELOl elongates

fatty acids specifically up to 16-carbons and EL02 and EL03 specifically elongate up to very long chain fatty acids, 24- carbons and 26-carbons respectively (C. Oh, D.A. Toke, S. Mandala and C.E. Martin, J. Biol. Chem. 272; 17376-84, 1997)

mutations in the corresponding genes give rise to disorders in several metabolic pathways as well as disorganised

cytoskeleton, impaired regulation of cellgrowth and budding deficiency (L. Desfarge et al . , Yeast 9, 267-77, 1993; E.

Revardel et al . , Biochem. Biophys . Acta 1263, 261-265, 1995;

S. Silve et al . , Mol. Cell Bio., 16, 2719-27, 1996).

Disruption of either EL02 or EL03 is associated with lower

levels of sphingolipids as a result of reduced ceramide

synthesis (Oh et al . , (1997) J. Biol . Chem . 272, 17376-17384).

This can provide a rational explanation to all the pleiotropic phenotypes seen in SUR4 and FEN1 mutations. The three yeast

members have been characterised by several independent groups and therefor been given different names. SUR4 was originally

selected as a suppressor of the phenotypes of the rvs-strains

(reduced viability upon starvation) . Mutants for RVS161 and

RSV167 are identified by heterologous cell morphology upon starvation followed by deficient bud localisation and cell

lysis. Further, the SUR4 mutant cells show abnormal

morphologies in stationary phase as well as a decrease in phospholipids (Desfarges et al.(1993) Yeast 9, 267-277). It

was also observed that the same mutant confers resistance when exposed to the immunosuppressor SR31747 in yeast cells which inhibits sterol isomeraεe in the synthesis of ergosterol

(Silve et al . , (1996) Mol . Cell . Biol . 16. 2719-2727). The

SUR4 mutation also show a decreased level of plasma membrane

H+-ATPaεe and several genes related to glucose metabolism (Garcia-Arranz et al . , (1994) J. Biol . Chem . 269, 18076-

18082) .

The FENl mutation was initially selected due to its ability to confer resistance to inhibition of sterol synthesis,

particular by fenpropimorph (Ladeveze et al . , (1993) Lipids 28, 907-912). The same resistance towards fenpropimorph is

also seen by SUR4 . Mutation of FENl exhibit similar

phenotypes like the SUR4 mutations, i.e. the ability to

suppress RVS161 and RSV167 , increased generation time, bud

localisation defect and resistance to SR31747. Also, yeast

cells deficient in FENl show a decrease in 1,3-β-glucan

synthesis which catalyses the polymerisation of glucose

necessary for the cell wall (El-Sherbeini and Clemas . , (1995) J. Bacteriol . Il l , 3227-3234). Simultaneous disruption of

SUR4 and FENl produces a lethal phenotype, which indicates

that their encoded proteins have related functions (Silve et al., (1996) Mol . Cell . Biol . 16, 2719-2727). The third

mutation, in the gene called J0343, shows no different phenotype from wild-type cells and had recently no detectable function (Revardel et al , (1995) Biochim . Biophys . Acta 1263,

261-265)

The inventors have now cloned two genes, Sscl and Ssc2 (human

and mouse) Sscl mRNA seems to be ubiquitous present m all

tissues, while Ssc2 mRNA has only been detected m testiε and

liver

The inventors subcloned Cιg30 , as well as Sscl and Ssc2 , into a yeast expression vector, and transformed the genes into sur4

and fenl yeast mutants The transformants were tested for

wildtype phenotype The data show that complementation

clearly occurs and indicates that Cιg30 and Ssc2 are

functional equivalents to EL02 , whereas Sscl is equivalent to

EL03

This provides indication that Cιg30 , Sscl and Sεc2 are

involved m the formation of very long fatty acids (VLCFA

As VLCFA are mainly precursors for ceramide and sphingolipid

synthesis, the inventors measured the levels of specific ceramides and sphingolipids m the transformants by thm- layer- chromatography after incubation with radioactive seπne as precursor. The data confirm that Cιg30 and Sscl can

restore the levels of specific ceramides and sphingolipids m the corresponding yeast mutants.

Further work described below has established deficiency m

Sscl gene expression in myelm deficient mice. Furthermore,

Cιg30 knockout mice have surprisingly been found to provide

phenotypes representing a good model for skin disease, the

gene apparently being involved m skin development. Such mice

and equivalent Sscl and Ssc2 knockout mice provide for assays for identifying and obtaining agents which may be used for

treatment of skin disorders, disorders of hair growth,

fertility (especially Ssc2) , multiple sclerosis (MS -

especially Sscl ) , obesity, cachexia, and thermoregulation

(especially Cιg30) . Additionally, the m ce show eye problems

within the first 3-5 weeks after birth. This provides for

testing of substances for treatment of eye disorders or other

problems (e.g. when contact lenses are used) .

Further assays are provided by the invention, employing the

Sscl and Ssc2 polynucleotides and encoded polypeptides in identifying and obtaining agents of therapeutic potential in peroxisomal disorders, cancers, and other disorders as

discussed herein and will be apparent to those skilled m the art in view of the present disclosure.

The peroxisomal disorders display frequently the phenomena of

several genetic defects/mechanisms, leading to multiple

phenotypic effects such as multiple congenital anomalies and severe neurological deficits . All these are associated with

defects in peroxisomal beta oxidation.

Pathologically high levels of very long chain fatty acids are

often found in plasma of patients with peroxisomal disorders,

such as Zellweger syndrome, neonatal adrenoleukodystrophy

(NALD) , infantile Resu disease (IRD) and the rhizomelic

chondrodysplasia puncata (RCDP) because of the impaired

oxidation of very long chain fatty acids by peroxisomes.

Saturated VLCFA accumulate in ALD, appear to disrupt membrane

structure, and may play a role in the pathogenesis of bain inflammatory response. The glyceryl trierucate oil, Lorenzo's

oil, combined with a reduced dietary fat intake, normalises

levels of VLCFA in plasma within 4 wk . The oil is thought to

act by reducing endogenous synthesis of saturated VLCFA by microsomal elongation system (see review H. Moser and A.

Moser, 1996, Lipids 31, S141-144. Furthermore, in the adrenoleukodystrophy case, dietary

restriction of very long chain fatty acids has been tried as

therapy but with very little improvement of clinical symptoms. In addition to the aberrant degradation activity of VLCFA in

this disease, there has been reported a significant increased synthesis of VLCFA in ALD fibroblasts which suggests that this

is of importance for the accumulation of the VLCFA in this

disease as well (Tsuji et al . , J. Biochem. 96, 1241-1247,

1984) .

Gangliosides and sphingolipids modulate transmembrane

signalling essential for tumour cell growth, invasion and metastasis. The transducer molecules susceptible to

gangliosides and sphingolipids include integrin receptors,

tyrosine kinase-linked growth factor receptors, protein kinase

C, and G-protein-linked receptor affecting protein kinase A. Some glycosphingolipids, ceramides and sphingosine induce

tumour cell differentiation and subsequently apoptosis.

Ceramide accumulation has been reported in Rous sarcoma

transformed fibroblasts. In addition, natural killer T (NKT)

lymphocytes express an invariant T cell receptor which uses

glycosylceramides with very long chain fatty acid (C26) as ligand which exist in restricted mammalian tissues or expressed on cells after activation or during malignancy (T. Kawano et al . , 1997, Science 278, 1626-1629).

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows mouse Sscl cDNA sequence, including coding sequence .

Figure 2 shows mouse SSCl amino acid sequence.

Figure 3 shows human Sscl cDNA sequence, including coding

sequence .

Figure 4 shows human SSCl amino acid sequence.

Figure 5 shows mouse Ssc2 cDNA sequence, including coding

sequence .

Figure 6 shows mouse SSC2 amino acid sequence .

Figure 7 shows an alignment of SSCl, SSC2 and CIG30 protein

and yeast protein sequences .

Figure 8 shows a schematic overview of genomic Cig30 , identified as Plaque A in plaque hybridisation as described below. The insert of approximately 14kb is in the Lambda

FIXII vector. The boxes indicate exon 1-4 of Cig30 . Below

are the two plasmids shown which correspond to the two arms in

the gene targeting vector. The dotted lines show to which

part in Plaque A the plasmid corresponds and which restriction

enzymes were used to digest Plaque A. The lower plasmid,

large right arm 5.30 kb, was initially used to create the gene

targeting vector. The inserts are cloned into the Bluescript

plasmid, pBS .

Figure 9 illustrates sequence replacement of Cig30 by the

corresponding gen targeting construct .

Figure 9A shows the knock-out vector for Cig30 (top)

replaces the transcription start and exon 1 and 2 of the

genomic DNA (below) upon homologous recombination with the neo

gene (crosshatched box) . The neo¹ replaces exon 1 and 2 (bend

part) in the endogenous sequence. The black box indicates the

thymidine kinase ( tk) gene with its corresponding PYF

enhancer. The light grey areas indicate the left (LA) and right (RA) arm which pair with a chromosomal copy of Cig30

(dotted lines) . Medium grey shows the 5 ' UTR and the 3 ' UTR regions and dark grey exon 1 to exon 4. Above the gene

targeting construct are the positions of restriction enzymes. WO 00/70945 PCTtEPOO/04371

13

Restriction enzymes within brackets were deleted during the cloning procedure .

Figure 9B shows a schematic model of the final

incorporation of the gene targeting constructs into the

genome .

Figure 10 illustrates a flow scheme with the pathway used in

making knock-out mice.

The Figure 1A of Tvrdik et al . , J. Biol. Chem. (1997) 272:

31738-31746 is also specifically incorporated herein by

reference as if it appears in full. It shows the nucleotide sequence of mouse Cig30 cDNA, including coding region, and

predicted amino acid sequence of CIG30 protein. Putative membrane-spanning segments are underlined with continuous

lines and numbered with Roman numerals . An N-glycosylation

consensus site (amino acids 6-9) is underlined with a dashed

line. An arbitrary stretch of amino acids in the central part of the polypeptide (amino acids 116-150), which displays the

highest degree of homology throughout the family, is marked by a dotted line. The four leucines defining the leucine zipper

within transmembrane domain IV are italicized, and the poly (A)

signal is marked by a grey box. Sequence data deposited with the GenBank Data Library under Accession No. AF054504 are also incorporated herein by

reference .

According to one aspect of the present invention there is

provided a nucleic acid molecule or polynucleotide which has a

nucleotide sequence encoding a polypeptide which includes an

ammo acid sequence selected from the group consisting of ^• (1) the mouse Sscl ammo acid sequence shown herein,-

(2) the mouse Ssc2 ammo acid sequence shown herein;

(3) the human Sscl ammo acid sequence shown herein;

(4) a fragment or active portion of any of (1) , (2) or (3) as

explained further below;

(5) a variant or derivative of any of (1) , (2) or (3) as

explained further below.

The coding sequence may be the relevant one shown herein, or

it may be a mutant, variant, derivative or allele of the

sequence shown. The sequence may differ from that shown by a

change which is one or more of addition, insertion, deletion

and substitution of one or more nucleotides of the sequence

shown Changes to a nucleotide sequence may result m an

amino acid change at the protein level, or not, as determined

by the genetic code. Thus, nucleic acid according to the present invention may

include a sequence different from a sequence specifically

shown herein, yet encode a polypeptide with the same amino

acid sequence.

On the other hand the encoded polypeptide may comprise an

amino acid sequence which differs by one or more amino acid

residues from the amino acid sequence shown herein. Nucleic

acid encoding a polypeptide which is an amino acid sequence

mutant, variant or derivative of the sequence shown is further provided by the present invention. Such polypeptides are

discussed below. Nucleic acid encoding such a polypeptide may

show at the nucleotide sequence and/or encoded amino acid

level greater than about 60% homology with the relevant coding

or encoded sequence shown herein, greater than about 70%

homology, greater than about 80% homology, greater than about

90% homology or greater than about 95% homology. For amino

acid "homology" , this may be understood to be similarity (according to the established principles of amino acid similarity, e.g. as determined using the algorithm GAP

(Genetics Computer Group, Madison, WI) or identity. GAP uses the Needleman and unsch algorithm to align two complete

sequences that maximizes the number of matches and minimizes

the number of gaps. Generally, the default parameters are used, with a gap creation penalty = 12 and gap extension

penalty = 4. Use of GAP may be preferred but other algorithms

may be used, e.g. BLAST (which uses the method of Altschul et al . (1990) J. Mol . Biol . 215: 405-410), FASTA (which uses the

method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448),

or the Smith-Waterman algorithm (Smith and Waterman (1981) J.

Mol Biol . 147 : 195-197), generally employing default

parameters. Use of either of the terms "homology" and

"homologous" herein does not imply any necessary evolutionary

relationship between compared sequences, in keeping for

example with standard use of terms such as "homologous

recombination" which merely requires that two nucleotide sequences are sufficiently similar to recombine under the

appropriate conditions. Further discussion of polypeptides

according to the present invention, which may be encoded by

nucleic acid according to the present invention, is found

below .

The present invention extends to nucleic acid that hybridizes with any one or more of the specific sequences disclosed

herein under stringent conditions . Suitable conditions

include, e.g. for detection of sequences that are about 80-90% identical suitable conditions include hybridization overnight at 42°C in 0.25M NaHPO-,, pH 7.2, 6.5% SDS , 10% dextran sulfate and a final wash at 55°C in 0. IX SSC, 0.1% SDS . For detection of sequences that are greater than about 90% identical,

suitable conditions include hybridization overnight at 65 °C in

0.25M Na_..HP0 _l pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 60°C in 0. IX SSC, 0.1% SDS.

Thus, for example the present invention further extends to a

polynucleotide which hybridizes under stringent conditions

with a Cig30 , Sscl and/or Ssc2 sequence as disclosed herein.

An example of an embodiment of a polynucleotide encoding a

Sscl human homologue of a mouse sequence provided herein is

shown herein.

The human and mouse SSCl have 92.3% similarity as calculated using BLAST with standard algorithm parameters W, T and X and

the BLOSUM62 matrix (Altschul, et al . , supra) , 92.5% identity using BESTFIT (see below) over the full length 279 amino

acids. Suitably stringent conditions for selective

hybridisation between the mouse and human homologues may be provided in accordance with the explanation and formula as

follows .

Preliminary experiments may be performed by hybridising under low stringency conditions. For probing, preferred conditions are those which are stringent enough for there to be a simple

pattern with a small number of hybridisations identified as positive which can be investigated further.

For example, hybridizations may be performed, according to the method of Sambrook et al . (below) using a hybridization

solution comprising: 5X SSC (wherein "SSC = 0.15 M sodium

chloride; 0.15 M sodium citrate; pH 7 ) , 5X Denhardt ' s reagent,

0.5-1.0% SDS, 100 μg/ml denatured, fragmented salmon sperm

DNA, 0.05% sodium pyrophosphate and up to 50% formamide. Hybridization is carried out at 37-42°C for at least six

hours. Following hybridization, filters are washed as

follows: (1) 5 minutes at room temperature in 2X SSC and 1%

SDS; (2) 15 minutes at room temperature in 2X SSC and 0.1%

SDS; (3) 30 minutes - 1 hour at 37°C in IX SSC and 1% SDS; (4)

2 hours at 42-65°C in IX SSC and 1% SDS, changing the solution

every 30 minutes.

One common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid

molecules of a specified sequence homology is (Sambrook et al., 1989): T, = 81.5°C + 16.6Log [Na+] + 0.41 (% G+C) - 0.63

(% formamide) - 600/#bp in duplex. As an illustration of the above formula, using [Na+] = [0.368^ and 50-% formamide, with GC content of 42% and an average probe size of 200 bases, the T_r is 57°C. The T, of a DNA

duplex decreases by 1 - 1.5°C with every 1% decrease in

homology. Thus, targets with greater than about 75% sequence

identity would be observed using a hybridization temperature

of 42°C. Such a sequence would be considered substantially homologous to the nucleic acid sequence of the present invention .

It is well known in the art to increase stringency of

hybridisation gradually until only a few positive clones remain. Other suitable conditions include, e.g. for detection

of sequences that are about 80-90% identical, hybridization

overnight at 42°C in 0.25M Na₂HP0₄ , pH 7.2 , 6.5% SDS, 10%

dextran sulfate and a final wash at 55°C in 0. IX SSC, 0.1%

SDS. For detection of sequences that are greater than about 90% identical, suitable conditions include hybridization

overnight at 65 °C in 0.25M Na₂HP0, , pH 7.2 , 6.5% SDS, 10% dextran sulfate and a final wash at 60°C in 0. IX SSC, 0.1%

SDS.

Generally, nucleic acid according to the present invention is

provided as an isolate, in isolated and/or purified form, or free or substantially free of material with which it is

naturally associated, such as free or substantially free of

nucleic acid flanking the gene in the human genome, except

possibly one or more regulatory sequence (s) for expression. Nucleic acid may be wholly or partially synthetic and may

include genomic DNA, cDNA or RNA. The coding sequence shown

herein is a DNA sequence. Where nucleic acid according to the

invention includes RNA, reference to the sequence shown should

be construed as encompassing reference to the RNA equivalent,

with U substituted for T.

Nucleic acid may be provided as part of a replicable vector, and also provided by the present invention are a vector

including nucleic acid as set out above, particularly any

expression vector from which the encoded polypeptide can be expressed under appropriate conditions, and a host cell

containing any such vector or nucleic acid. An expression

vector in this context is a nucleic acid molecule including

nucleic acid encoding a polypeptide of interest and appropriate regulatory sequences for expression of the

polypeptide, in an in vi tro expression system, e.g.

reticulocyte lysate, or in vivo , e.g. in eukaryotic cells such

as COS or CHO cells or in prokaryotic cells such as E. coli . This is discussed further below. The nucleic acid sequence provided in accordance with the present invention is useful for identifying nucleic acid of

interest (and which may be according to the present invention)

in a test sample. The present invention provides a method of

5 obtaining nucleic acid of interest, the method including

hybridisation of a probe having the an Sscl or Ssc2 sequence

shown herein, or a complementary sequence, to target nucleic acid. Hybridisation is generally followed by identification

of successful hybridisation and isolation of nucleic acid

10 which has hybridised to the probe, which may involve one or

more steps of PCR. It will not usually be necessary to use a

probe with the complete sequence shown in any of these

figures. Shorter fragments may be used, e.g. fragments of

about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,

15 150, 160, 170, 180, 190 or 200 nucleotides.

Nucleic acid according to the present invention is obtainable using one or more oligonucleotide probes or primers designed

to hybridise with one or more fragments of the nucleic acid

20 sequence shown in any of the figures, particularly fragments

of relatively rare sequence, based on codon usage or statistical analysis. A primer designed to hybridise with a

fragment of the nucleic acid sequence shown in any of the figures may be used in conjunction with one or more WO 00/70945 PCTtEPOO/04371

22 oligonucleotides designed to hybridise to a sequence in a cloning vector within which target nucleic acid has been

cloned, or in so-called "RACE" (rapid amplification of cDNA ends) in which cDNA's in a library are ligated to an

oligonucleotide linker and PCR is performed using a primer

which hybridises with a sequence shown and a primer which

hybridises to the oligonucleotide linker.

Such oligonucleotide probes or primers, as well as the full-

length sequence (and mutants, alleles, variants and

derivatives) are also useful in screening a test sample

containing nucleic acid for the presence of alleles, mutants

and variants, with diagnostic and/or prognostic implications as discussed in more detail below.

Nucleic acid isolated and/or purified from one or more cells

(e.g. human) or a nucleic acid library derived from nucleic

acid isolated and/or purified from cells (e.g. a cDNA library

derived from mRNA isolated from the cells) , may be probed under conditions for selective hybridisation and/or subjected

to a specific nucleic acid amplification reaction such as the polymerase chain reaction (PCR) (reviewed for instance in "PCR

protocols; A Guide to Methods and Applications", Eds. Innis et al, 1990, Academic Press, New York, Mullis et al, Cold Spring Harbor Symp . Quant. Biol., 51:263, (1987), Ehrlich

(ed) , PCR technology, Stockton Press, NY, 1989, and Ehrlich et

al, Science, 252:1643-1650, (1991)) . PCR comprises steps of

denaturation of template nucleic acid (if double- stranded) , annealing of primer to target, and polymerisation. The nucleic acid probed or used as template in the amplification

reaction may be genomic DNA, cDNA or RNA. Other specific

nucleic acid amplification techniques include strand

displacement activation, the QB replicase system, the repair

chain reaction, the ligase chain reaction and ligation

activated transcription. For convenience, and because it is

generally preferred, the term PCR is used herein in contexts

where other nucleic acid amplification techniques may be

applied by those skilled in the art . Unless the context requires otherwise, reference to PCR should be taken to cover

use of any suitable nucleic amplification reaction available

in the art .

In the context of cloning, it may be necessary for one or more

gene fragments to be ligated to generate a full-length coding sequence. Also, where a full-length encoding nucleic acid

molecule has not been obtained, a smaller molecule representing part of the full molecule, may be used to obtain

full-length clones. Inserts may be prepared from partial cDNA clones and used to screen cDNA libraries. The full-length clones isolated may be subcloned into expression vectors and activity assayed by transfection into suitable host cells,

e.g. with a reporter plasmid.

A method may include hybridisation of one or more (e.g. two)

probes or primers to target nucleic acid. Where the nucleic acid is double-stranded DNA, hybridisation will generally be

preceded by denaturation to produce single- stranded DNA. The

hybridisation may be as part of a PCR procedure, or as part of

a probing procedure not involving PCR. An example procedure

would be a combination of PCR and low stringency hybridisation. A screening procedure, chosen from the many

available to those skilled in the art, is used to identify

successful hybridisation events and isolated hybridised

nucleic acid.

Binding of a probe to target nucleic acid (e.g. DNA) may be

measured using any of a variety of techniques at the disposal

of those skilled in the art. For instance, probes may be

radioactively , fluorescently or enzymatically labelled. Other

methods not employing labelling of probe include examination of restriction fragment length polymorphisms, amplification using PCR, RN'ase cleavage and allele specific oligonucleotide probing. Probing may employ the standard Southern blotting

technique. For instance DNA may be extracted from cells and digested with different restriction enzymes. Restriction

fragments may then be separated by electrophoresis on an agarose gel, before denaturation and transfer to a

nitrocellulose filter. Labelled probe may be hybridised to

the DNA fragments on the filter and binding determined. DNA

for probing may be prepared from RNA preparations from cells.

Preliminary experiments may be performed by hybridising under

low stringency conditions various probes to Southern blots of

DNA digested with restriction enzymes . Suitable conditions

would be achieved when a large number of hybridising fragments

were obtained while the background hybridisation was low. Using these conditions nucleic acid libraries, e.g. cDNA

libraries representative of expressed sequences, may be

searched. Those skilled in the art are well able to employ

suitable conditions of the desired stringency for selective hybridisation, taking into account factors such as

oligonucleotide length and base composition, temperature and

so on. On the basis of amino acid sequence information, oligonucleotide probes or primers may be designed, taking into

account the degeneracy of the genetic code, and, where

appropriate, codon usage of the organism from the candidate nucleic acid is derived. An oligonucleotide for use in nucleic acid amplification may have about 10 or fewer codons

(e.g. 6, 7 or 8) , i.e. be about 30 or fewer nucleotides in

length (e.g. 18, 21 or 24, 10-15, 10-20, 15-20 or 20-30) .

Generally specific primers are upwards of 14 nucleotides in length, but need not be than 18-20. Those skilled in the art

are well versed in the design of primers for use processes

such as PCR. Various techniques for synthesizing

oligonucleotide primers are well known in the art, including

phosphotriester and phosphodiester synthesis methods.

A further aspect of the present invention provides an

oligonucleotide or polynucleotide fragment of the nucleotide

sequence shown in any of the figures herein providing nucleic

acid according to the present invention, or a complementary

sequence, in particular for use in a method of obtaining

and/or screening nucleic acid. Some preferred

oligonucleotides have a sequence shown herein, or a sequence

which differs from any of the sequences shown by addition, substitution, insertion or deletion of one or more

nucleotides, but preferably without abolition of ability to hybridise selectively with nucleic acid in accordance with the present invention, that is wherein the degree of similarity of the oligonucleotide or polynucleotide with one of the sequences given is sufficiently high.

In some preferred embodiments, oligonucleotides according to the present invention that are fragments of any of the

sequences shown, or any allele associated with a disorder or other disease susceptibility, may be or consist of at least

about 10 nucleotides in length, more preferably at least about 15 nucleotides in length, more preferably at least about 20

nucleotides in length, at least about 30 nucleotides in

length, more preferably at least about 40 nucleotides in

length, more preferably at least about 50 nucleotides in

length. Fragments may be 10-20 nucleotides, 10-30, 20-30, 30-

40, 30-50, 40-50, 50-60, 50-100, 50-150 or 100-150 nucleotides in length. Such fragments themselves individually represent

aspects of the present invention. Fragments and other

oligonucleotides may be used as primers or probes as discussed

but may also be generated (e.g. by PCR) in methods concerned with determining the presence in a test sample of a sequence

indicative of a disorder or disease susceptibility.

Methods may involve use of nucleic acid in diagnostic and/or

prognostic contexts, for instance in determining susceptibility to a disease, and other methods are concerned with determining the presence of sequences indicative of a defect in VLCFA biosynthesis or other disease susceptibility.

Further embodiments of oligonucleotides according to the

present invention are anti-sense oligonucleotide sequences

based on the nucleic acid sequences described herein. Anti- sense oligonucleotides may be designed to hybridise to the

complementary sequence of nucleic acid, pre-mRNA or mature

mRNA, interfering with the production of polypeptide encoded

by a given DNA sequence (e.g. either native polypeptide or a mutant form thereof) , so that its expression is reduce or

prevented altogether. Anti-sense techniques may be used to

target a coding sequence, a control sequence of a gene, e.g.

in the 5' flanking sequence, whereby the antisense

oligonucleotides can interfere with control sequences. Anti- sense oligonucleotides may be DNA or RNA and may be of around

14-23 nucleotides, particularly around 15-18 nucleotides, in

length. Fragments of lengths identified above may also be

employed (in the antisense orientation.) The construction of

antisense sequences and their use is described in Peyman and Ulman, Chemical Reviews, 90:543-584, (1990), and Crooke, Ann.

Rev. Pharmacol. Toxicol . , 32:329-376, (1992).

Nucleic acid according to the present invention may be used in

methods of gene therapy, for instance in treatment of individuals with the aim of preventing or curing (wholly or partially) a disorder or defect in VLCFA biosynthesis or other disease. This may ease one or more symptoms of the disease.

This is discussed below.

Nucleic acid according to the present invention, such as a

full-length coding sequence or oligonucleotide probe or primer, may be provided as part of a kit, e.g. in a suitable

container such as a vial in which the contents are protected

from the external environment. The kit may include

instructions for use of the nucleic acid, e.g. in PCR and/or a method for determining the presence of nucleic acid of

interest in a test sample. A kit wherein the nucleic acid is intended for use in PCR may include one or more other reagents

required for the reaction, such as polymerase, nucleosides,

buffer solution etc. The nucleic acid may be labelled. A kit

for use in determining the presence or absence of nucleic acid

of interest may include one or more articles and/or reagents

for performance of the method, such as means for providing the

test sample itself, e.g. a swab for removing cells from the

buccal cavity or a syringe for removing a blood sample (such components generally being sterile) .

A further aspect of the present invention provides a polypeptide which has the amino acid sequence of a Sscl or

Ssc2 polypeptide shown herein, which may be in isolated and/or

purified form, free or substantially free of material with which it is naturally associated, such as other polypeptides

or such as human or mouse polypeptides other than that for

which the relevant amino acid sequence is shown herein, or

(for example if produced by expression in a prokaryotic cell) lacking in native glycosylation, e.g. unglycosylated .

Polypeptides which are amino acid sequence variants, alleles,

derivatives or mutants are also provided by the present

invention. A polypeptide which is a variant, allele,

derivative or mutant may have an amino acid sequence which

differs from that given in a figure herein by one or more of addition, substitution, deletion and insertion of one or more

amino acids. Preferred such polypeptides have whichever is

the relevant function out of CIG30, SSCl and SSC2 function,

that is to say have one or more of the following properties:

immunological cross-reactivity with an antibody reactive with the relevant polypeptide for which the sequence is given herein; sharing an epitope with the relevant polypeptide for

which the amino acid sequence is shown herein (as determined

for example by immunological cross-reactivity between the two polypeptides) ; a biological activity which is inhibited by an antibody raised against the polypeptide whose sequence is shown in a figure herein; ability to complement ELOl, EL02

and/or EL03 mutations in yeast; enzymatic activity in common

with CIG 30, SSCl and/or SSC2. Alteration of sequence may

change the nature and/or level of activity and/or stability of the relevant protein.

A polypeptide which is an amino acid sequence variant, allele,

derivative or mutant of the amino acid sequence shown in a figure herein may comprise an amino acid sequence which shares

greater than about 35% sequence identity with the sequence

shown, greater than about 40%, greater than about 50%,

greater than about 60%, greater than about 70%, greater than

about 80%, greater than about 90% or greater than about 95%.

The sequence may share greater than about 60% similarity,

greater than about 70% similarity, greater than about 80%

similarity or greater than about 90% similarity with the amino

acid sequence shown in the relevant figure. Amino acid

similarity is generally defined with reference to the algorithm GAP (Genetics Computer Group, Madison, WI) as noted

above, or the TBLASTN program, of Altschul et al . (1990) J.

Mol. Biol. 215: 403-10. Similarity allows for "conservative variation", i.e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as

arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine . Particular amino acid sequence variants may differ from that shown in a figure herein by insertion,

addition, substitution or deletion of 1 amino acid, 2, 3, 4,

5-10, 10-20 20-30, 30-50, 50-100, 100-150, or more than 150

amino acids .

Sequence comparison may be made over the full -length of the

relevant sequence shown herein, or may more preferably be over

a contiguous sequence of about or greater than about 20, 25, 30, 33, 40, 50, 67, 133, 167, 200, 233, 267 or more amino acids or nucleotide triplets, compared with the relevant amino

acid sequence or nucleotide sequence as the case may be.

The present invention also includes peptides which include or

consist of fragments of a polypeptide of the invention.

The skilled person can use the techniques described herein and

others well known in the art to produce large amounts of peptides, for instance by expression from encoding nucleic

acid.

Peptides can also be generated wholly or partly by chemical synthesis . The compounds of the present invention can be

readily prepared according to well-established, standard

liquid or, preferably, solid-phase peptide synthesis methods, general descriptions of which are broadly available (see, for

example, in J.M. Stewart and J.D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford,

Illinois (1984) , in M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York

(1984) ; and Applied Biosystems 430A Users Manual, ABI Inc., Foster City, California) , or they may be prepared in solution,

by the liquid phase method or by any combination of solid-

phase, liquid phase and solution chemistry, e.g. by first

completing the respective peptide portion and then, if desired

and appropriate, after removal of any protecting groups being present, by introduction of the residue X by reaction of the

respective carbonic or sulfonic acid or a reactive derivative

thereof .

The present invention also includes active portions, fragments, derivatives and functional mimetics of the

polypeptides of the invention. An "active portion" of a polypeptide means a peptide which is less than said full

length polypeptide, but which retains a biological activity, such as ability to complement ELOl, EL02 and/or EL03 mutations in S . cerevisiae . Such an active fragment may be included as part of a fusion protein

A "fragment" of a polypeptide generally means a stretch of

amino acid residues of at least about five contiguous amino acids, often at least about seven contiguous amino acids,

typically at least about nine contiguous amino acids, more preferably at least about 13 contiguous amino acids, and, more

preferably, at least about 20 to 30, at least about 30-40, at

least about 40-50, at least bout 5-60, at least about 60-70,

at least about 70-80, at least about 80-90, at least about 90- 100, or more contiguous amino acids. Fragments of the

relevant polypeptide sequence may include antigenic

determinants or epitopes useful for raising antibodies to a

portion of the amino acid sequence. Alanine scans are

commonly used to find and refine peptide motifs within

polypeptides, this involving the systematic replacement of

each residue in turn with the amino acid alanine, followed by an assessment of biological activity.

Preferred fragments of polypeptides according to the present invention include those with sequences which may be used for instance in raising or isolating antibodies, for instance amino acids 116-150 of any of CIG30, SSCl and SSC2 (mouse or human) . Variant and derivative peptides, peptides which have an amino acid sequence which differs from one of these sequences by way of addition, insertion, deletion or

substitution of one or more amino acids are also provided by

the present invention, generally with the proviso that the variant or derivative peptide is bound by an antibody or other

specific binding member which binds one of the peptides whose sequence is shown. A peptide which is a variant or

derivative of one of the shown peptides may compete with the

shown peptide for binding to a specific binding member, such

as an antibody or antigen-binding fragment thereof .

Where additional amino acids are included in a peptide, these may be heterologous or foreign to the polypeptide of the

invention, and the peptide may be about 20, 25, 30 or 35 amino

acids in length. A peptide according to this aspect may be

included within a larger fusion protein, particularly where

the peptide is fused to a heterologous or foreign sequence, such as a polypeptide or protein domain.

A "derivative" of a polypeptide or a fragment thereof may include a polypeptide modified by varying the amino acid

sequence of the protein, e.g. by manipulation of the nucleic acid encoding the protein or by altering the protein itself . Such derivatives of the natural amino acid sequence may

involve one or more of insertion, addition, deletion or

substitution of one or more amino acids, which may be without fundamentally altering the qualitative nature of biological

activity of the wild type polypeptide. Also encompassed

within the scope of the present invention are functional

mimetics of active fragments of the polypeptides provided

(including alleles, mutants, derivatives and variants) . The term "functional mimetic" means a substance which may not

contain an active portion of the relevant amino acid sequence,

and probably is not a peptide at all, but which retains in

qualitative terms biological activity of the natural

polypeptide. The design and screening of candidate mimetics

is described in detail below.

Other fragments of the polypeptides for which sequence

information is provided herein are provided as aspects of the

present invention, for instance corresponding to functional

domains. One such functional domain may be amino acids 126-150 or amino acids 175-196 of the relevant CIG30, SSCl or SSC2

protein .

A polypeptide according to the present invention may be isolated and/or purified (e.g. using an antibody) for instance after production by expression from encoding nucleic acid (for which see below) Thus, a polypeptide may be provided free or

substantially free from contaminants with which it is

naturally associated (if it is a naturally-occurring

polypeptide) A polypeptide may be provided free or

substantially free of other polypeptides Polypeptides according to the present invention may be generated wholly or

partly by chemical synthesis The isolated and/or purified

polypeptide may be used m formulation of a composition, which

may include at least one additional component, for example a

pharmaceutical composition including a pharmaceutically

acceptable excipient, vehicle or carrier A composition including a polypeptide according to the invention may be used

m prophylactic and/or therapeutic treatment as discussed

below

A polypeptide, peptide, allele, mutant, derivative or variant according to the present invention may be used as an immunogen

or otherwise in obtaining specific antioodies Antibodies are

useful purification and other manipulation of polypeptides

and peptides, diagnostic screening and therapeutic contexts This is discussed further below

A polypeptide accordmg to the present mvention may be used m screening for molecules which affect or modulate its activity or function Such molecules may interact with a

portion of the polypeptide, and may be useful m a therapeutic (possibly including prophylactic) context

It is well known that pharmaceutical research leading to the

identification of a new drug may involve the screening of very large numbers of candidate substances, both before and even after a lead compound has been found This is one factor

which makes pharmaceutical research very expensive and time-

consuming Means for assisting m the screening process can

have considerable commercial importance and utility Such means for screening for substances potentially useful m

treating or preventing a disorder or disease is provided by

polypeptides according to the present invention Substances

identified as modulators of the polypeptide represent an

advance the fight agamst disease smce they provide basis for design and investigation of therapeutics for in vivo use

Furthermore, they may be useful m any of a number of conditions, including diseases and disorders involving sk

development, hair growth, body fat mass (subcutaneous),

fertility, thermoregulation, and/or the eye, given the

functional indications discussed elsewhere herein As noted elsewhere, polypeptides, fragments thereof, and nucleic acid according to the invention may also be useful m combatting

any of these diseases and disorders

In various further aspects the present invention relates to

screening and assay methods and means, and substances

identified thereby

Thus, further aspects of the present invention provide the use of a polypeptide or peptide (particularly a fragment of a

polypeptide of the invention as disclosed, and/or encoding

nucleic acid therefor, m screening or searching for and/or obtaining/identifying a substance, e g peptide or chemical

compound, which interacts and/or binds with the polypeptide or peptide and/or interferes with its function or activity or

that of another substance, e g polypeptide or peptide, which

interacts and/or binds with the polypeptide or peptide of the

invention For instance, a method according to one aspect of

the invention includes providing a polypeptide or peptide of

the invention and bringing it mto contact with a substance, which contact may result m binding between the polypeptide or

peptide and the substance Binding may be determined by any of a number of techniques available m the art, both qualitative and quantitative In various aspects the present invention is concerned with provision of assays for substances which inhibit interaction

between a polypeptide of the invention and another polypeptide

or molecule able to interact with it .

Further assays are for substances which interact with or bind a polypeptide of the invention and/or modulate one or more of

its activities.

One aspect of the present invention provides an assay which

includes :

(a) bringing into contact a polypeptide or peptide according to the invention and a putative binding molecule or other test

substance; and

(b) determining interaction or binding between the

polypeptide or peptide and the test substance.

A substance which interacts with the polypeptide or peptide of the invention may be isolated and/or purified, manufactured

and/or used to modulate its activity as discussed. A test

substance found to be able to bind is a candidate modulator of activity, either inhibitor or promoter of activity, and can be tested for ability to modulate the activity. A further aspect of the present invention provides an assay

method which includes :

(a) bringing into contact a substance including a polypeptide

or fragment, mutant, variant or derivative thereof, in

accordance with the present invention, a substance including a

fragment of a second polypeptide or a fragment, mutant,

variant or derivative of a second molecule, which is able to

bind the relevant polypeptide of the invention,- and a test

compound, under conditions in which in the absence of the test

compound being an inhibitor, the two said substances interact;

(b) determining interaction between said substance.

It is not necessary to use the entire proteins for assays of

the invention which test for binding between two molecules.

Fragments may be generated and used in any suitable way known

to those of skill in the art . Suitable ways of generating

fragments include, but are not limited to, recombinant

expression of a fragment from encoding DNA. Such fragments

may be generated by taking encoding DNA, identifying suitable

restriction enzyme recognition sites either side of the

portion to be expressed, and cutting out said portion from the

DNA. The portion may then be operably linked to a suitable

promoter in a standard commercially available expression

system. Another recombinant approach is to amplify the relevant portion of the DNA with suitable PCR primers. Small

fragments (e.g. up to about 20 or 30 amino acids) may also be

generated using peptide synthesis methods which are well known

in the art .

The precise format of the assay of the invention may be varied by those of skill in the art using routine skill and

knowledge. For example, the interaction between the

polypeptides may be studied in vi tro by labelling one with a

detectable label and bringing it into contact with the other

which has been immobilised on a solid support. Suitable

detectable labels include ^' S-methionine which may be

incorporated into recombinantly produced peptides and

polypeptides. Recombinantly produced peptides and

polypeptides may also be expressed as a fusion protein

containing an epitope which can be labelled with an antibody.

Fusion proteins may be generated that incorporate six

histidine residues at either the N-terminus or C-terminus of the recombinant protein. Such a histidine tag may be used for

purification of the protein by using commercially available columns which contain a metal ion, either nickel or cobalt

(Clontech, Palo Alto, CA, USA) . These tags also serve for

detecting the protein using commercially available monoclonal antibodies directed against the six histidine residues (Clontech, Palo Alto, CA, USA) .

The protein which is immobilized on a solid support may be

immobilized using an antibody against that protein bound to a solid support or via other technologies which are known per

se . A preferred in vi tro interaction may utilise a fusion

protein including glutathione-S- transferase (GST) . This may

be immobilized on glutathione agarose beads. In an in vi tro

assay format of the type described above a test compound can

be assayed by determining its ability to diminish the amount

of labelled peptide or polypeptide which binds to the immobilized GST-fusion polypeptide. This may be determined by

fractionating the glutathione-agarose beads by SDS-

polyacrylamide gel electrophoresis. Alternatively, the beads

may be rinsed to remove unbound protein and the amount of protein which has bound can be determined by counting the

amount of label present in, for example, a suitable

scintillation counter.

An assay according to the present invention may also take the form of an in vivo assay. The in vivo assay may be performed

in a cell line such as a yeast strain in which the relevant polypeptides or peptides are expressed from one or more vectors introduced into the cell .

A method of screening for a substance which modulates activity of a polypeptide may include contacting one or more test substances with the polypeptide in a suitable reaction medium,

testing the activity of the treated polypeptide and comparing

that activity with the activity of the polypeptide in comparable reaction medium untreated with the test substance or substances. A difference in activity between the treated

and untreated polypeptides is indicative of a modulating

effect of the relevant test substance or substances .

Combinatorial library technology (Schultz, JS (1996)

Biotechnol. Prog. 12:729-743) provides an efficient way of

testing a potentially vast number of different substances for

ability to modulate activity of a polypeptide. Prior to or as

well as being screened for modulation of activity, test

substances may be screened for ability to interact with the

polypeptide, e.g. in a yeast two-hybrid system (which requires that both the polypeptide and the test substance can be expressed in yeast from encoding nucleic acid) . This may be

used as a coarse screen prior to testing a substance for actual ability to modulate activity of the polypeptide. The amount of test substance or compound which may be added to an assay of the invention will normally be determined by trial

and error depending upon the type of compound used.

Typically, from about 0.01 to 100 nM concentrations of

putative inhibitor compound may be used, for example from 0.1 to 10 nM . Greater concentrations may be used when a peptide

is the test substance .

Compounds which may be used may be natural or synthetic

chemical compounds used in drug screening programmes.

Extracts of plants which contain several characterised or

uncharacterised components may also be used. A further class

of putative inhibitor compounds can be derived from the

relevant polypeptide of the invention. Peptide fragments of

from 5 to 40 amino acids, for example from 6 to 10 amino acids from the region of the relevant polypeptide responsible for

interaction with another molecule, may be tested for their

ability to disrupt such interaction.

Other candidate inhibitor compounds may be based on modelling

the 3 -dimensional structure of a polypeptide or peptide fragment and using rational drug design to provide potential inhibitor compounds with particular molecular shape, size and

charge characteristics . Important assay methods of the invention employ an animal model, such as a Cιg30, Sscl or Sεc2 transgenic or knockout

mouse or other rodent Such animal models and their generation are discussed m detail below.

A further aspect of the present invention therefore provides

an assay method which comprises :

(a) treating an animal model as disclosed herein with a test

substance ;

(b) determining the presence or absence of an effect on the

animal as a result of the treatment with the test substance.

The nature of an effect when detected may be investigated.

Potential end-points for detection include visual effects, effects determined immunologically or biochemically, and

effects determined by means of determination of gene

expression, for instance by means of Southern or Northern

blotting of nucleic acid extracts or derivatives from

appropriate cells . The skilled person is well aware of the need for control experiments and well able to design

appropriate controls, both positive and negative An end- point indicative of a positive result may be chosen view of the therapeutic application m mmd . In certain embodiments of this aspect of the invention, the

animal model is a knockout for one or more of Cιg30 , Sscl and

Ssc2, the animal generally being a rodent, preferably mouse

The purpose of an assay employing an animal model m

accordance with the invention may be for identifying 01

obtaining an agent with therapeutic potential in treatrent of a disorder resulting from a defect m C g30, Sscl and/cr Ssc2

function Such disorders may include any mentioned herein

Of particular interest are agents useful m treatment cf a

skm disorder (such as liquid pharmaceuticals or others

suitable for topical application), or eye problems, for which a Cιg30 knockout mouse is well suited

Further embodiments employ animal models m which heterologous

Cιg30 , Sscl and/or Ssc2 sequences are expressed m the animal, for instance particular mutant sequences or human sequences

(e.g a mouse m which endogenous Cιg30 , Sscl and/or Ssc2 has been knocked out) This allows for screening for

inhibitors and other modulators of the relevant function

These may be useful m treatment of e g a disease or αisorder mentioned herein

An animal may be treated with a test substance at an appropriate dosage, depending on the site of administration (e.g. topically or to the eye) , any known potency of the

substance, solubility and other factors routinely taken into

account by those skilled in the art.

Similar assay methods may employ host cells transformed with

nucleic acid of the invention and expressing a polypeptide of

the invention, or may employ cells or cell lines derived from

transgenic animals (including knock-outs) generated as described further elsewhere herein.

Following identification of a substance which modulates or

affects polypeptide activity, the substance may be

investigated further. Furthermore, it may be manufactured and/or used in preparation, i.e. manufacture or formulation,

of a composition such as a medicament, pharmaceutical composition or drug. These may be administered to

individuals .

Thus, the present invention extends in various aspects not

only to a substance identified as a modulator of polypeptide

activity, in accordance with what is disclosed herein, but also a pharmaceutical composition, medicament, drug or other

composition comprising such a substance, a method comprising administration of such a composition to a patient, e.g. for

treatment (which may include preventative treatment) of a

disorder disease, use of such a substance in manufacture of a composition for administration, e.g. for treatment of a

disorder or disease, and a method of making a pharmaceutical

composition comprising admixing such a substance with a

pharmaceutically acceptable excipient, vehicle or carrier, and optionally other ingredients.

Disorders and diseases which may be treated in accordance with

aspects of the present invention have been discussed and

mentioned already herein.

A substance identified using as a modulator of polypeptide or promoter function may be peptide or non-peptide in nature.

Non-peptide "small molecules" are often preferred for many in

vivo pharmaceutical uses. Accordingly, a mimetic or mimick of

the substance (particularly if a peptide) may be designed for pharmaceutical use. The designing of mimetics to a known

pharmaceutically active compound is a known approach to the

development of pharmaceuticals based on a "lead" compound.

This might be desirable where the active compound is difficult or expensive to synthesise or where it is unsuitable for a particular method of administration, e.g. peptides are not well suited as active agents for oral compositions as they

tend to be quickly degraded by proteases the alimentary canal Mimetic design, synthesis and testing may be used to avoid randomly screening large number of molecules for a

target property

There are several steps commonly taken m the design of a

mimetic from a compound having a given target property

Firstly, the particular parts of the compound that are

critical and/or important m determining the target property

are determined In the case of a peptide, this can be done by

systematically varying the ammo acid residues m the peptide, e.g by substituting each residue m turn These parts or

residues constituting the active region of the compound are known as its "pharmacophore"

Once the pharmacophore has been found, its structure is

modelled to according its physical properties, e g

stereochemistry, bonding, size and/or charge, using data from

a range of sources, e g spectroscopic techniques, X-ray

diffraction data and NMR Computational analysis, similarity mapping (which models the charge and/or volume of a

pharmacophore, rather than the bonding between atoms) and other techniques can be used m this modelling process WO 00/70945 PCTtEPOO/04371

51

In a variant of this approach, the three-dimensional structure of the ligand and its binding partner are modelled. This can

be especially useful where the ligand and/or binding partner

change conformation on binding, allowing the model to take account of this the design of the mimetic.

A template molecule is then selected onto which chemical

groups which mimic the pharmacophore can be grafted. The

template molecule and the chemical groups grafted on to it can

conveniently be selected so that the mimetic is easy to

synthesise, is likely to be pharmacologically acceptable, and

does not degrade in vivo, while retaining the biological

activity of the lead compound. The mimetic or mimetics found by this approach can then be screened to see whether they have

the target property, or to what extent they exhibit it.

Further optimisation or modification can then be carried out

to arrive at one or more final mimetics for in vivo or clinical testing.

Mimetics of substances identified as having ability to

modulate polypeptide or activity using a screening method as

disclosed herein are included within the scope of the present invention. A polypeptide, peptide or substance able to

modulate activity of a polypeptide according to the present WO 00/70945 PCTtEPOO/04371

52 invention may be provided in a kit, e.g. sealed in a suitable container which protects its contents from the external

environment. Such a kit may include instructions for use.

A convenient way of producing a polypeptide according to the

present invention is to express nucleic acid encoding it, by

use of the nucleic acid in an expression system. Accordingly, the present invention also encompasses a method of making a

polypeptide (as disclosed), the method including expression

from nucleic acid encoding the polypeptide (generally nucleic

acid according to the invention) . This may conveniently be

achieved by growing a host cell in culture, containing such a vector, under appropriate conditions which cause or allow

expression of the polypeptide. Polypeptides may also be

expressed in in vi tro systems, such as reticulocyte lysate.

Systems for cloning and expression of a polypeptide in a

variety of different host cells are well known. Suitable host

cells include bacteria, eukaryotic cells such as mammalian and yeast, and baculovirus systems. Mammalian cell lines

available in the art for expression of a heterologous

polypeptide include Chinese hamster ovary cells, HeLa cells,

baby hamster kidney cells, COS cells and many others. A common, preferred bacterial host is E. coli . Suitable vectors can be chosen or constructed, containing appropriate

regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences,

marker genes and other sequences as appropriate. Vectors may

be plasmids, viral e.g. ' hage , or phagemid, as appropriate. For further details see, for example, Molecular Cloning: a

Laboratory Manual: 2nd edition, Sambrook et al . , 1989, Cold

Spring Harbor Laboratory Press . Many known techniques and

protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis,

sequencing, introduction of DNA into cells and gene

expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Ausubel et al .

eds., John Wiley & Sons, 1992.

Thus, a further aspect of the present invention provides a

host cell containing nucleic acid as disclosed herein. The

nucleic acid of the invention may be integrated into the

genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences which promote recombination

with the genome, in accordance with standard techniques. The nucleic acid may be on an extra- chromosomal vector within the

cell. A still further aspect provides a method which includes introducing the nucleic acid into a host cell. The

introduction, which may (particularly for in vitro

introduction) be generally referred to without limitation as

"transformation", may employ any available technique. For

eukaryotic cells, suitable techniques may include calcium

phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using

retrovirus or other virus, e.g. vaccinia or, for insect cells,

baculoviruε . For bacterial cells, suitable techniques may

include calcium chloride transformation, electroporation and

transfection using bacteriophage .

Marker genes such as antibiotic resiεtance or εenεitivity

geneε may be uεed in identifying clones containing nucleic

acid of interest, aε iε well known in the art.

The introduction may be followed by causing or allowing

expression from the nucleic acid, e.g. by culturing host cells

(which may include cells actually transformed although more

likely the cells will be deεcendantε of the transformed

cells) under conditions for expression of the gene, so that the encoded polypeptide is produced. If the polypeptide iε expressed coupled to an appropriate signal leader peptide it WO 00/70945 PCTtEPOO/04371

55 may be secreted from the cell into the culture medium.

Following production by expresεion, a polypeptide may be

isolated and/or purified from the host cell and/or culture

medium, aε the caεe may be, and subsequently used as desired, e.g. in the formulation of a composition which may include one

or more additional components, such as a pharmaceutical

composition which includes one or more pharmaceutically

acceptable excipients, vehicles or carriers (e.g. see below) .

Introduction of nucleic acid may take place in vivo by way of

gene therapy, as discuεεed below. A host cell containing

nucleic acid according to the present invention, e.g. aε a

result of introduction of the nucleic acid into the cell or

into an ancestor of the cell and/or genetic alteration of the

sequence endogenous to the cell or ancestor (which introduction or alteration may take place in vivo or ex vivo) ,

may be comprised (e.g. in the soma) within an organism which

is an animal, particularly a mammal, which may be human or non-human, such as rabbit, guinea pig, rat, mouse or other

rodent, cat, dog, pig, sheep, goat, cattle or horse, or which

iε a bird, such aε a chicken. Genetically modified or transgenic animals or birds comprising such a cell are also

provided as further aspects of the present invention. Thus, in various further aspects, the present invention

provides a non-human animal with a Cig30 , Sscl or Ssc2

transgene within its genome. The transgene may have the

sequence of any of the isoformε identified herein or a mutant, derivative, allele or variant thereof aε diεcloεed. In one

preferred embodiment, a heterologouε human sequence replaceε

the endogenouε animal εequence . In other preferred

embodimentε, one or more copieε of the human εequence are

added to the animal genome .

Preferably the animal iε a rodent, and most preferably mouse

or rat .

This may have a therapeutic aim. (Gene therapy is diεcuεsed

below.) The presence of a mutant, allele or variant εequence

within cells of an organism, particularly when in place of a

homologous endogenous sequence, may allow the organism to be used as a model in testing and/or studying the role of the

gene or substanceε which modulate activity of the encoded

polypeptide and/or promoter in vi tro or are otherwiεe

indicated to be of therapeutic potential .

Further important aεpects of the present invention are based

on the inventors' construction of animal modelε for diεeaεe. Animal modelε for the relevant gene deficiency may be conεtructed using standard techniques for introducing

mutations into an animal germ-line. In one example of this

approach, using a mouse, a vector carrying an insertional

mutation within the gene may be transfected into embryonic

stem cells. A selectable marker, for example an antibiotic

resistance gene such as neoR, may be included to facilitate

selection of clones in which the mutant gene has replaced the

endogenouε wild type homologue . Such cloneε may be alεo be

identified or further inveεtigated by Southern blot

hybridisation. The cloneε may then be expanded and cells

injected into mouse blastocyst stage embryos. Mice in which

the injected cells have contributed to the development of the mouse may be identified by Southern blotting. These chimeric

mice may then be bred to produce mice which carry one copy of the mutation in the germ line. These heterozygous mutant

animalε may then be bred to produce mice carrying mutationε in

the gene homozygously . The mice having a heterozygous mutation in the gene may be a suitable model for human

individuals having one copy of the gene mutated in the germ

line who are at risk of developing a disorder or disease.

The invention therefore further provides a non-human transgenic animal which harbours at least one copy of a tranεgene either homologously or nonhomologously integrated into a chromosomal location and encoding a heterologouε polypeptide of the invention, e.g. human εequence, or a

mutant, variant, derivative, or fragment, of a human or mouεe εequence .

In a further aεpect the invention provides a non-human

transgenic animal which harbours one or more integrated constructs or targeted mutations that disrupt the function of

endogenous Cig30 , Sscl and/or Ssc2 genes. Such animals are

referred to as "knock-outs", although it is not required by

this aspect of the invention that function be totally ablated

(this may be preferred) . The invention provides a non-human animal with at least one inactivated endogenous Cig30 , Sscl or Ssc2 allele, and which is preferably homozygouε for inactivated Cig30 , Sscl and/or Ssc2 alleles.

Various approaches for targeting conεtructε or mutationε

(generally deletionε) to chromosomal locationε are available in the art, and generally make uεe of homologous recombination between a target sequence in the chromosome and a region in a

vector which iε εubεtantially complementary to the target

sequence. Sequences flanking a target gene or a portion thereof may be employed, allowing for deletion of the target gene or the relevant portion.

Tranεgenic mutationε, including deletionε, in a gene locuε in

accordance with the present invention may be detected using

conventional techniqueε, such as fluoreεcent in situ hybridisation (FISH) with appropriate probeε appropriately

labelled, PCR analyεis etc.

A tranεgenic animal according to the preεent invention is

generally selected from mammals such as rabbit, guinea pig,

rat, mouse or other rodent, cat, dog, pig, sheep, goat, cattle or horse, and preferably rodent, most preferably mouεe.

Animal models may alεo be useful for any of the various

diseases discussed elsewhere herein.

However, a non-obvious application based on the inventors'

work is use in identifying and obtaining agents for treatment

of eye problems, and skin disorders, which may be selected

from disorders such as atopic dermatits, hair loss, psoriasiε, cachexia and others disclosed herein and apparent to the skilled person based on the present disclosure. Assay methods employing animals in accordance with the present invention

have been described already above. Further aεpectε of the preεent invention provide cells of

transgenic animals aε diεcloεed, whether iεolated cells or cell lines derived from the animals and optionally

immortalised using standard techniques.

Host cells transformed with a polynucleotide of the invention

provide further aspects of the invention, and are useful for example in production of an encoded polypeptide. Instead of

or as well as being used for the production of a polypeptide

encoded by a tranεgene, host cells may be used aε a nucleic

acid factory to replicate the nucleic acid of intereεt in

order to generate large amounts of it . Multiple copies of

nucleic acid of intereεt may be made within a cell when coupled to an amplifiable gene such as dihyrofolate reductase

(DHFR) , as is well known. Host cells transformed with nucleic

acid of interest, or which are descended from host cells into

which nucleic acid was introduced, may be cultured under

suitable conditions, e.g. in a fermentor, taken from the culture and subjected to proceεεing to purifiy the nucleic

acid. Following purification, the nucleic acid or one or more fragmentε thereof may be used as desired, for inεtance in a

diagnostic or prognostic assay as discussed elsewhere herein.

The provision of the novel polypeptides enables for the first time the production of isolated antibodies able to bind these molecules specifically.

Accordingly, a further aspect of the present invention

provides an antibody able to bind specifically to the

polypeptide whose sequence is given in a figure herein. Such an antibody may be specific in the sense of being able to

distinguiεh between the polypeptide it is able to bind and other human polypeptides for which it haε no or substantially

no binding affinity (e.g. a binding affinity of about lOOOx

less) . Specific antibodies bind an epitope on the molecule

which is either not present or is not accessible on other

molecules . Antibodies according to the preεent invention may

be specific for the wild-type polypeptide. Antibodies according to the invention may be specific for a particular

mutant, variant, allele or derivative polypeptide as between

that molecule and the wild-type polypeptide, so as to be

useful in diagnostic and prognostic methods aε diεcuεsed below. Antibodieε are also uεeful in purifying the

polypeptide or polypeptides to which they bind, e.g. following production by recombinant expression from encoding nucleic

acid.

Preferred antibodies according to the invention are iεolated, WO 00/70945 PCTtEPOO/04371

62 in the sense of being free from contaminants such aε antibodieε able to bind other polypeptides and/or free of

serum components. Monoclonal antibodies are preferred for

some purposeε, though polyclonal antibodieε are within the

εcope of the present invention.

Antibodies may be obtained using techniques which are standard

in the art . Methods of producing antibodieε include

immuniεing a mammal (e.g. mouεe, rat, rabbit, horse, goat,

sheep or monkey) with the protein or a fragment thereof .

Antibodieε may be obtained from immunised animals using any of

a variety of techniques known in the art, and screened,

preferably using binding of antibody to antigen of interest.

For instance, Western blotting techniques or

immunoprecipitation may be used (Armitage et al . , 1992,

Nature 357: 80-82). Isolation of antibodies and/or antibody- producing cells from an animal may be accompanied by a step of

sacrificing the animal.

As an alternative or supplement to immunising a mammal with a peptide, an antibody specific for a protein may be obtained

from a recombinantly produced library of expreεεed immunoglobulin variable domains, e.g. using lambda

bacteriophage or filamentous bacteriophage which display WO 00/70945 PCTtEPOO/04371

63 functional immunoglobulin binding domains on their surfaces,-

for instance see WO92/01047. The library may be naive, that

iε conεtructed from sequences obtained from an organism which has not been immunised with any of the proteins (or

fragmentε) , or may be one conεtructed uεing εequenceε obtained from an organiεm which haε been expoεed to the antigen of

intereεt .

Suitable peptideε for uεe in immunising an animal and/or

isolating anti-Sscl or anti-Ssc2 antibody especially include

C-terminal fragments.

Antibodies according to the present invention may be modified in a number of ways. Indeed the term "antibody" should be

construed as covering any binding substance having a binding

domain with the required specificity. Thus the invention

covers antibody fragmentε, derivativeε, functional equivalentε

and homologues of antibodies, including synthetic molecules and molecules whose shape mimickε that of an antibody enabling it to bind an antigen or epitope.

Example antibody fragmentε, capable of binding an antigen or other binding partner are the Fab fragment consisting of the

VL, VH, Cl and CHI domains,- the Fd fragment consisting of the WO 00/70945 PCTtEPOO/04371

64

VH and CHI domains; the Fv fragment consisting of the VL and

VH domains of a single arm of an antibody; the dAb fragment which consists of a VH domain; isolated CDR regions and

F(ab')2 fragments, a bivalent fragment including two Fab

fragments linked by a disulphide bridge at the hinge region. Single chain Fv fragmentε are alεo included.

A hybridoma producing a monoclonal antibody according to the

preεent invention may be εubject to genetic mutation or other

changes. It will further be understood by those skilled in

the art that a monoclonal antibody can be subjected to the techniques of recombinant DNA technology to produce other

antibodies or chimeric molecules which retain the specificity of the original antibody. Such techniqueε may involve introducing DNA encoding the immunoglobulin variable region,

or the complementarity determining regions (CDRε) , of an

antibody to the conεtant regions, or constant regions plus

framework regions, of a different immunoglobulin. See, for instance, EP184187A, GB 2188638A or EP-A-0239400. Cloning and

expression of chimeric antibodies are described in EP-A-

0120694 and EP-A-0125023.

Hybridomas capable of producing antibody with desired binding characteristics are within the scope of the present invention, as are hoεt cells, eukaryotic or prokaryotic, containing nucleic acid encoding antibodies (including

antibody fragments) and capable of their expression. The

invention also provides methods of production of the antibodieε including growing a cell capable of producing the

antibody under conditionε in which the antibody is produced,

and preferably secreted.

The reactivities of antibodies on a sample may be determined by any appropriate means . Tagging with individual reporter

molecules is one possibility. The reporter molecules may

directly or indirectly generate detectable, and preferably measurable, signals. The linkage of reporter molecules may be

directly or indirectly, covalently, e.g. via a peptide bond or

non-covalently . Linkage via a peptide bond may be as a result of recombinant expression of a gene fusion encoding antibody

and reporter molecule.

One favoured mode is by covalent linkage of each antibody with an individual fluorochrome, phosphor or laser dye with

spectrally isolated absorption or emission characteristics. Suitable fluorochromeε include fluorescein, rhodamine, phycoerythrin and Texas Red. Suitable chromogenic dyes

include diaminobenzidine . 66

Other reporters include macromolecular colloidal particles or particulate material such aε latex beadε that are coloured,

magnetic or paramagnetic, and biologically or chemically

active agentε that can directly or indirectly cauεe detectable εignalε to be visually observed, electronically detected or

otherwiεe recorded. Theεe moleculeε may be enzymes which

catalyse reactionε that develop or change colourε or cauεe changeε in electrical propertieε, for example. They may be molecularly excitable, εuch that electronic tranεitions

between energy states result in characteristic εpectral

absorptions or emissions. They may include chemical entities used in conjunction with biosenεors . Biotin/avidin or biotin/streptavidin and alkaline phosphatase detection systems

may be employed.

The mode of determining binding is not a feature of the

present invention and thoεe skilled in the art are able to

choose a suitable mode according to their preference and

general knowledge. Particular embodiments of antibodies according to the preεent invention include antibodieε able to

bind and/or which bind εpecifically, e.g. with an affinity of at least 10^~7 M , to a peptides fragment of a polypeptide as diεclosed herein, especially a C-terminal fragment. Antibodies according to the present invention may be used in screening for the presence of a polypeptide, for example in a

test sample containing cells or cell lysate aε discusεed, and

may be uεed in purifying and/or isolating a polypeptide according to the preεent invention, for inεtance following production of the polypeptide by expression from encoding

nucleic acid therefor. Antibodieε may modulate the activity

of the polypeptide to which they bind and εo, if that

polypeptide haε a deleteriouε effect in an individual, may be

useful in a therapeutic context (which may include

prophylaxis) .

An antibody may be provided in a kit, which may include

instructions for use of the antibody, e.g. in determining the presence of a particular substance in a test sample. One or

more other reagents may be included, εuch aε labelling

molecules, buffer solutions, elutants and so on. Reagents may be provided within containerε which protect them from the

external environment, such as a sealed vial.

Whether it iε a polypeptide, antibody, peptide, nucleic acid

molecule, small molecule or other pharmaceutically useful compound according to the present invention that is to be given to an individual, administration is preferably in a

"prophylactically effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis

may be considered therapy) , this being sufficient to εhow

benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the

nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical

doctors .

A composition may be administered alone or in combination with other treatments, either εimultaneouεly or sequentially

dependent upon the condition to be treated.

Pharmaceutical compositions according to the present

invention, and for use in accordance with the present invention, may include, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer,

stabiliser or other materials well known to those skilled in

the art. Such materials should be non-toxic and should not

interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneouε or intravenouε .

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier εuch aε gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such aε water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline εolution, dextroεe or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included .

For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicleε such as Sodium Chloride Injection, Ringer's Injection, or Lactated Ringer's Injection.

Preservatives, stabilisers, bufferε, antioxidants and/or other additives may be included, as required.

Targeting therapies may be used to deliver the active agent more specifically to certain types of cell, by the use of targeting systems such aε antibody or cell εpecific ligandε . Targeting may be desirable for a variety of reasons ; for

example if the agent is unacceptably toxic, or if it would otherwise require too high a doεage, or if it would not otherwiεe be able to enter the target cells.

Instead of administering an agent directly, it may be be

produced in target cells by expression from an encoding gene

introduced into the cells, e.g. in a viral vector (see below) . The vector may be targeted to the specific cells to be

treated, or it may contain regulatory elements which are

switched on more or lesε selectively by the target cells. Viral vectors may be targeted using specific binding

molecules, such aε a εugar, glycolipid or protein such as an antibody or binding fragment thereo . Nucleic acid may be

targeted by means of linkage to a protein ligand (such as an

antibody or binding fragment thereof) via polylysine, with the

ligand being specific for a receptor present on the surface of

the target cells.

An agent may be administered in a precursor form, for conversion to an active form by an activating agent produced

in, or targeted to, the cells to be treated. This type of approach iε sometimes known aε ADEPT or VDEPT; the former involving targeting the activating agent to the cells by

conjugation to a cell-specific antibody, while the latter involves producing the activating agent, e.g. an enzyme, in a

vector by expression from encoding DNA in a viral vector (see

for example, EP-A-415731 and WO 90/07936) .

Nucleic acid according to the present invention, e.g. encoding

the authentic biologically active Sscl or Ssc2 polypeptide or

a functional fragment thereof, may be used in a method of gene

therapy, to treat a patient who is unable to syntheεize the active polypeptide or unable to synthesize it at the normal

level, thereby providing the effect provided by the wild- type with the aim of treating and/or preventing one or more

symptoms of a disorder or disease.

Vectors such as viral vectors have been used to introduce

genes into a wide variety of different target cells. Typically the vectors are exposed to the target cells so that transfection can take place in a sufficient proportion of the

cells to provide a useful therapeutic or prophylactic effect from the expresεion of the desired polypeptide. The

transfected nucleic acid may be permanently incorporated into the genome of each of the targeted cells, providing long WO 00/70945 PCTtEPOO/04371

72 laεting effect, or alternatively the treatment may have to be repeated periodically.

A variety of vectors, both viral vectors and plasmid vectors, are known in the art, see e.g. US Patent No. 5,252,479 and WO

93/07282. In particular, a number of viruseε have been uεed

aε gene tranεfer vectors, including adenovirus, papovaviruseε , such as SV40, vaccinia virus, herpeεviruseε, including HSV and EBV, and retroviruseε , including gibbon ape leukaemia viruε,

Rous Sarcoma Virus, Venezualian equine enchephalitis virus,

Moloney murine leukaemia viruε and murine mammary tumourvirus .

Many gene therapy protocols in the prior art have used

disabled murine retroviruses .

Disabled virus vectors are produced in helper cell lines in which genes required for production of infectious viral

particles are expressed. Helper cell lines are generally

missing a εequence which is recognised by the mechanism which

packageε the viral genome and produce virionε which contain no nucleic acid. A viral vector which containε an intact packaging εignal along with the gene or other εequence to be

delivered (e.g. encoding the polypeptide or a fragment thereof) is packaged in the helper cells into infectious virion particles, which may then be used for the gene delivery .

Other known methods of introducing nucleic acid into cells

include electroporation, calcium phosphate co-precipitation,

mechanical techniques such as microinjection, transfer

mediated by lipoεomes and direct DNA uptake and receptor- mediated DNA transfer. Liposomes can encapεulate RNA, DNA and

virionε for delivery to cells . Depending on factors such as

pH, ionic strength and divalent cations being present, the

compoεition of lipoεomeε may be tailored for targeting of

particular cells or tisεue . Liposomes include phospholipids

and may include lipids and steroids and the compoεition of each such component may be altered. Targeting of liposomes may also be achieved using a specific binding pair member such

as an antibody or binding fragment thereof, a sugar or a

glycolipid.

The aim of gene therapy using nucleic acid encoding the

polypeptide, or an active portion thereof, is to increase the amount of the expression product of the nucleic acid in cells

in which the level of the wild-type polypeptide is absent or present only at reduced levels. Such treatment may be therapeutic or prophylactic, particularly in the treatment of individuals known through screening or testing to have a WO 00/70945 PCTtEPOO/04371

74 disease suεceptibility allele and hence a prediεposition to the diεeaεe.

Similar techiqueε may be uεed for anti-sense regulation of gene expression, e.g. targeting an antiεenεe nucleic acid

molecule to cells in which a mutant form of the gene iε

expreεεed, the aim being to reduce production of the mutant gene product. Other approaches to specific down-regulation of

genes are well known, including the uεe of ribozymeε deεigned to cleave specific nucleic acid sequences. Ribozymeε are

nuceic acid molecules, actually RNA, which specifically cleave

single- stranded RNA, such as mRNA, at defined sequences, and

their specificity can be engineered. Hammerhead ribozymes may be preferred because they recognise base sequences of about

11-18 bases in length, and εo have greater specificity than ribozymes of the Tetrahymena type which recognise εequenceε of

about 4 baεes in length, though the latter type of ribozymes are useful in certain circumstances . References on the use of

ribozymes include Marschall, et al . Cellular and Molecular Neurobiology, 1994. 14(5): 523; Hasεelhoff, Nature 334: 585

(1988) and Cech, J. Amer. Med. Assn., 260: 3030 (1988).

Aspects of the present invention will now be illustrated with reference to the accompanying figures described already above and experimental exemplification, by way of example and not

limitation. Further aspects and embodiments will be apparent to those of ordinary skill in the art. All documents

mentioned in this εpecification are hereby incorporated herein

by reference.

EXAMPLE 1 - Cloning and characterisation of Sscl and Ssc2

Mouεe and human cDNA εequences were cloned aε described below

in Materials and Methods.

The deduced SSCl and SSC2 polypeptides consiεt of 279 and 292 amino acidε respectively. Relevant similarities and

identitieε are aε follows, calculated using BLAST with

standard algorithm parameters W, T and X and the BLOSUM62 matrix (Altschul, et al . , supra) : CIG30-SSC1 44% εimilarity,

34% identity; CIG30-SSC2 43% εimilarity and 31% identity;

SSC1-SSC2 50% εimilarity and 37% identity. Human SSCl is 95% similar to and 92% identical with mouεe SSCl.

At the nucleic acid level, using the program BESTFIT with gap creation penalty of 50, gap extension penalty of 3 the

following results were obtained: mouse Sscl and human Sscl - 89% identity with quality 6397, quality ratio 7.947, length 805, no gaps ;

mouse Sscl and Ssc2 - 59% identity with quality 586, ratio

2.071, length 286, one gap,-

mouse Sεc2 and human Sscl - 61% identity with quality 633, quality ratio 2.035, length 320, 3 gaps;

mouse Sscl and Cig30 - 63% identity with quality 229, quality

ratio 2.290, length 106, one gap;

mouse Ssc2 and Cig30 - 67% identity with quality 154, quality

ratio 3.667, length 42, no gaps ;

human Sscl and mouse Cig30 - 64% identity with quality 214,

quality ratio 3.194, length 67, no gap .

BESTFIT uεeε the local homology algorithm of Smith and

Waterman (Advanceε in Applied Mathematicε (1981) 2: 482-489)

to find the beεt εegment of similarity between two εequenceε . The quality score for the best alignment to any point is equal

to the εum of the scoring matrix values of the matches in that alignment, less the gap creation penalty times the number of gaps in that alignment, leεε the gap extenεion penalty timeε the total length of all gapε in that alignment.

Northern blotting waε used to show the preεence of Ssc 1 in

liver, brown adipose tissue (BAT), heart, kidney, skin, spleen testiε, brain, stomach, lung, and skeletal muscle, with

higheεt levelε in stomach, lung, kidney and skin, and Ssc2

mRNA in only testiε and liver.

Computer analyεiε of the Cig30 cDNA εequence εuggests it to be

a transmembrane glycoprotein with a size of about 30 kD and

with five putative transmembrane domains.

Twelve tissues were analysed for expression of Cig30 mRNA, but detectable levels were only found in liver, brown adipose

tissue and skin, where the expreεεion iε affected during

tissue development. In hepatectomized mice suppression in

Cig30 expreεsion was found in the liver whereaε in skin an increased expression during fur development was detected. In

brown adipose tissue the Cig30 expresεion waε εelectively elevated about 200 timeε when mice were exposed to a three day

cold stress. A similar increase, although to a lesεer extent, was brought about in two other conditions of brown fat activation, namely during perinatal development and after WO 00/70945 PCTtEPOO/04371

78 cafeteria diet. The magnitude of Cig30 mRNA induction in the

cold could be mimicked almoεt fully by chronic norepinephrine treatment indicating that the gene expreεεion iε mainly

modulated by catecholamineε in vivo . However, in primary 5 cultureε of brown adipocyteε, a εynergiεtic action of both

norepinephrine and glucocorticoidε waε required for full

expreεεion of the gene. See Tvrdik et al . , J. Biol. Chem. (1997) 272: 31738-31746.

10 EXAMPLE 2 - Cig30 , Sscl and Ssc2 function

Three yeaεt proteinε (J0343, FENl and SUR4 ) are εignificantly

homologouε to each other (Revardel et al, Biochimica et

Biophyεica Acta (1995) 1263: 261-265) and Cig30: SUR4-FEN1

15 50.3%, SUR4-J0343 47.2%, FEN1-J0343 66.8%. The yeaεt genes

are indicated to function as membrane-bound fatty acid elongaεeε and designated ELOl (J0343), EL02 (FENl) and EL03

(SUR4) , where ELOl elongates fatty acids specifically up to 16 carbons, and EL02 and EL03 specifically elongate up to very-

20 long-chain fatty acids, 24-carbons and 26-carbonε respectively. Mutations in the corresponding genes give rise

to disorders in several metabolic pathwayε, aε well aε disorganised cytoskeleton, impaired regulation of cell growth and budding deficiency. Cig30, Sscl and Ssc2 were individually expressed in ELOl, EL02 and EL03 yeast cells. Complementation analysiε revealed that Cig30 and Ssc2 are functional equivalents to EL02 , while Sscl

is equivalent to EL03 : the correεponding geneε were able to

reεcue phenotypic diεorderε in the reεpective mutantε, namely their incapacity to grow on lactate and inεenεitivity to inhibitorε of εterol synthesis.

The inventors investigated whether the mouse geneε would revert SR31747 reεiεtance of the Δsur4 and Δfenl yeaεt

mutants. Aε demonεtrated earlier, yeast vegetative growth waε inhibited by the εterol εyntheεiε inhibitor SR31747 in a dose-

dependent manner (Silve et al . , 1996, Mol . Cell Biol . 16, 2719-2727) . Growth of the parental yeast strain EMA6 waε

severely inhibited by 2 μM SR31747. Following disruption of

either SUR4 or FENl , however, SR31747 tolerance was greatly

enhanced and the resulting mutants were able to grow in the

presence of >35 μM SR31747.

In a first series of experiments, the Δsur4 mutant waε transformed with yeast expression plasmids containing the

genes of intereεt, and εelected colonieε were εcored for their ability to grow in increasing concentrations of SR31747. Control transformation SUR4 restored SR31747 senεitivity to the wild type levels, while FENl overexpression had no effect. In this expreεεion εyεtem, Sscl clearly conferred SR31747

sensitivity, even though less well than SUR4 , and inhibited growth of the Δsur4 mutant at SR31747 concentrationε of 20-25 5 μM . Tranεformation with Cig30 did not affect SR31747

tolerance in the mutant .

A εecond series of experiments performed in the Δfenl mutant

revealed that FENl restored wild type SR31747 sensitivity, but

10 also that SUR4 overexpression conferred nearly aε low level of

SR31747 εenεitivity aε FENl . Of the homologouε mouse genes,

Cig30 consiεtently complemented best the Δfenl mutant, conferring growth arrest at 15-20 μM SR31747, but Sscl could also mediate SR31747 sensitivity at higher concentrations (25-

15 30 μM) . In addition to being resiεtant to SR31747, the L\sur4

mutant waε alεo found to be unable to grow on glycerol/ethanol

(Silve et al . , 1996, Mol . Cell Biol . 16, 2719-2727). In good

agreement with the above results, SUR4 or Sscl rescued the

Δsur4 mutant's capacity to grow on glycerol/ethanol, while the

20 other genes did not .

Sscl rescues lethali ty of the Δsur4Δfenl double mutant

EMY22, a sur4 mutant, waε transformed to Ura+ with the plasmid WO 00/70945 PCTtEPOO/04371

81 pSSCl. Transformant were crossed with EMA41, a fenl

disruptant . Diploid was sporulated and spores analysed for

uracile prototrophy, SR31747 resiεtance, fenl deletion -

replacement with LEU2 , and for sur4 mutation by PCR followed

by εequence analysis. Among 67 sporeε analyεed, only 3 were

diεrupted for FENl . Among the three, one waε an aneuploid,

and one contained a wild type allele of the SUR4 gene. The laεt εpore was resistant to 25 μM SR31747. PCR analyεiε

confirmed that the fenl gene waε diεrupted, and sequence

analysiε revealed that sur4 was mutated at the ATG starting

codon, aε for the original mutant.

To further test this double mutant strain, it was verified

that the presence of the plasmid was required for growth.

When the medium is supplemented with uracile, URA3 cells are

sensitive to the analog 5 fluoroorotic acid (5FOA) , whereas

ura3 mutant are able to grow in the presence of this

inhibitor. A Ura' spore with a wild type allele of FENl and

the double mutant sur4 fenl, Ura^* and therefore expreεεing SSCl, were incubated in the presence of 5FOA (lmg/ml) and

uracil for 48 hours. Only the wild-type cells were able to

proliferate in these conditions, whereas the double mutant

neve grew. Therefore, SSCl is required for the double mutant growth. Even in the presence of fatty acids C20, C22 and C24, WO 00/70945 PCTtEPOO/04371

82 or ceramides, the double mutant was not able to grow in the presence of the analog.

Sscl is involved in biosynthesis of C26

Recently, strong evidence has been provided that ELOl ,

FEN1/EL02 and SUR4/EL03 are involved in fatty acid chain

elongation. In particular, FEN1/EL02 and SUR4/EL03 were

reported to be neceεεary for the synthesis of fatty acids of

up to 24 and 26 carbons, respectively. As a result, Asur4 and

-.fenl mutants had modified εphingolipid composition.

In view of this, the same range of yeast tranεformantε uεed in

the previouε experimentε were metabolically labelled with [³H]εerine, [^JH] sphinganine or [³H] inoεitol , and sphingolipids

were isolated and separated by thin- layer chromatography. No

significant difference was seen in εphingolipid synthesis between wild- type and the A fenl strain. The Asur4 mutant

showed, however, a modified sphingolipid pattern as compared to the parental wild type strain. Most notably, the band

corresponding to M(IP)_C was absent in the mutant. This band

could not be restored by overexpression of the FENl gene, but complementation with the wild type SUR4 gene restored the normal εphingolipid composition. Transformation with Sscl alεo ameliorated sphingolipid synthesiε in the Δsur4Δfenl

double mutant. On the other hand, Ssc2 or Cig30 had no

effect. As M(IP) C contains almost exclusively cerotoyl

(26:0), this experiment shows that Sscl is similar to SUR4 in

5 its capacity to catalyse the synthesis of cerotic acid.

EXAMPLE 3 - Sscl expression in myel in -defi cient mutant mi ce

Fatty acid elongation activity in mammalian tisεueε has been

10 mainly studied in brain and in liver. The microεomal

elongation εyεtem, especially in the brain, has been

extensively studied because the VLCFA are essential

constituents of brain sphingolipids such as cerebrosides and

sulfatides (J.M. Bourre et al . , 1978, Adv. Exp . Med. Biol.

15 101, 17-26) . However, the relative physiological significance of the elongation system is not fully elucidated.

In early studies, Sidman et al . (Sidman et al . , 1964, Science

144, 309-311) described two separate mutant mice that were

20 deficient in myelin throughout the CNS . One was described as an autosomal, recesεive mutation and termed quaking (qk)

whereaε the other more εevere myelin deficient mouεe waε an X- chromosome linked recessive mutation named j impy (jp) . In the

j impy mutation the primary genetic leεion resides in a defect WO 00/70945 PCTtEPOO/04371

84 in the myelin proteolipid protein (PLP) gene. The primary molecular defect in quaking remainε to be established.

Analysis of the lipid composition of brain in the quaking

mouse showed a marked decrease in the VLCFA (Baumann et al . ,

5 1968, Eur. J. Biochem. 4, 340-344) and it was later

eεtabliεhed that the brain microεomal fatty acid elongation εyεtem waε deficient in providing the fatty acids necesεary

for myelin formation (Goldberg et al . , 1973, Science 182, 497-

499) . A decreaεe in overall fatty acid chain elongation 0 activity was observed in both the quaking and j impy mouεe

brain microεomeε relative to controlε (Bourre et al . , 1975,

Biochem. Biophyε . Res. Commun. 63, 1027-1034).

Sscl is marked downregulated in the brains of myelin -defi cient

5 mouse mutants Quaking and Jimpy

Sscl mRNA levels in the brains of 19 -day-old quaking mutantε were reduced by 40% aε compared to their normal littermateε,

while the actin mRNA levelε in the εame mutants were only 0 insignificantly reduced by 10%. In the j impy mutant, cerebral

Sscl mRNA levelε were downregulated even more dramatically,

being reduced by 70%. Thuε, the reduced fatty acid chain elongation activity in the brains of quaking and j impy mutants appears to correlate with expression of the Sscl gene. EXAMPLE 4 - Transgeni c overexpression of Cig30

Cig30 overexpression in testis does not infl uence the mRNA

level of Ssc2

The inventorε generated a tranεgenic line of mice which

expresses Cig30 cDNA under CMV (Cytomegalovirus) promoter.

Curiouεly, deεpite the broad εpecificity of CMV expreεεion,

the transgene was only expresεed only in the teεtiε and (more

weakly) in ovarie . Mo morphological deviationε were found on

the electron microεcopy level, and both the transgenic males

and females were fertile. To address the posεibility whether there was a feed-back inhibition of Ssc2 transcription by

ectopic expression of Cig30, Northern blot analysis of Ssc2

mRNA levels waε performed in the CMV- Cig30 tranεgenic mice and

in their non-tranεgenic littermates . Transgenic expresεion of

Cig30 did not exert any change in endogenouε Ssc2 mRNa levels,

suggeεting that there iε no feed-back interaction between the

two paralogouε geneε .

Cig30 mRNA induction coincides wi th enhanced fatty acid chain

elongation activi ty during brown fat recrui tment

Among the members of the novel mouεe gene family deεcribed here, the most dramatic regulatory change so far observed waε

that of Cig30 gene expression during brown fat recruitment.

The inventors isolated microsomal membranes from brown fat of thermoneutral and cold εtimulated mice, and determined fatty

acid chain elongation capacity in theεe samples. The results

show that elongation activity waε indeed significantly

increaεed in brown fat of mice εtimulated by low ambient

temperature (4°C) for 3 dayε, as compared to thermoneutral

controls. The highest increase (4.7x) waε observed when

palmitoyl-CoA (16:0) was used as substrate. Lower values were

obtained with arachidoyl-CoA (20:0) used as substrate (4.3x), whereas elongation activity on lignoceroyl-CoA (24:0) was not

statistically different.

Hence, the data demonstrate that elongation of two acyl-CoA

substrates is strongly enhanced in the recruiting brown fat, paralleling the induction of Cig30 expression.

MATERIAL AND METHODS

Animals and treatments

For comparison of Sscl, Ssc2 and Cig30 expression profiles and for fatty acid chain elongation εtudies, NMRI male mice (6-8 weeks old) were obtained from the local supplier (Eklunds) and kept at thermoneutral temperature (28°C) for 1 week. After

this period, some animals were exposed to 4°C when indicated.

The mice were εacrificed by cervical diεlocation and the

tiεεueε were diεεected and directly subjected to RNA extraction or homogenisation and differential centrifugation .

To analyse cerebral Sscl mRNA levelε dyεmyelinating mutantε,

heterozygous breeder pairs of Quaking (B6C3Fe-a/a-g.J) and

Jimpy (B6CBACa-A^w~ /A- Ta jp) were obtained from the Jackson

Laboratory (Bar Harbor, ME, USA) and bred in an animal facility. The mutant pups and their healthy littermate

controlε were εacrificed at the age of 18 days and their

brains were quickly dissected and frozen in liquid nitrogen

before RNA extraction.

Transgenic mouse techniques

The CMV- Cig30 transgenic line of mice waε created by

pronuclear microinjection according to the εtandard protocolε

(Hogan et al . , (1986) Cold Spring Harbor Laboratory Preεε, Cold Spring Harbor, NY) . The DNA conεtruct waε prepared by subcloning of a cDNA fragment corresponding to nt 162 to 1056 WO 00/70945 PCTtEPOO/04371

88 in the Cig30 mRNA into Xhol and Xbal sites of the pCI-neo vector (Promega) . The construct was digested with Bgll l and

NgoMI , and the 2.5-kb fragment containing the CMV promoter,

chimeric intron, Cig30 open reading frame and the synthetic

polyadenylation signal was gel-purified and injected (0.2

ng/μl) into pronuclei of fertilised mouse eggs collected from

4-week-old C57BL/6J x CBA/J females (Bommice) . Microinj ected

eggε were implanted at two- cell εtage into oviductε of

pseudopregnant foster mothers of the FVB/Ν strain. One

founder was obtained, which was bred to establish a mouse line

homozygous for the transgenic locus. The line expressed the

transgene only in testis and, to a lesεer degree, in ovaries.

Mouse cDNA cloning and sequencing

Primers were designed to PCR amplify the protein coding

regions of Sscl , Ssc2 and Cig30 from mouse liver Marathon-

Ready cDΝA library (Clontech) . The primers used were:

Primer 1

5 ' -GGACGTCGACTGAGTCCTTAGCCAGGATGGAGGCTGTTGT-3 ' , and primer 2

5 ' -GAGCAGATCTGTCCTGAGGCACTTAGGTGGGCAATGTCTA-3 ' , for Sscl ORF amplification; primer 3

5 ' -GGACGTCGACCGCGGCCGCGCGGCCATGGAGCAGCTGAA-3 ' , and

primer 4

5 ' -GAGCAGATCTCCACCTCAGTTTGTGTTCCCCGGCACTTCA-3 ' ,

for Ssc2 ORF amplification;

primer 5

5 ' -GGACGTCGACCGTCTGCAAAATCGAAATGGACACATCCAT-3 ' , and

primer 6 5 ' -GAGCAGATCTACGGAGGAACGGCTGAGGCTCCATCTTTCT-3 ' .

for Cig30 ORF amplification.

These may be useful for amplifying and cloning the human

homologueε .

Specific primers useful for cloning human SSCl are shown below.

To facilitate cloning, all forward primers contain exogenous Sail siteε and all reverεe primerε contain foreign Bglll εiteε

(both εhown in bold face) . The touch-down PCR reactionε were performed with the Pfu polymerase (Stratagene) for 32 cycles totally (after denaturation at 94°C for 1 min, 5 cycles 94°C for 30 sec, 72°C for 3 min, 5 cycles 94°C for 30 sec, 70°C for 3 min, and 22 cycles 94°C for 30 sec, 68°C for 3 min) . For

each gene, at least two independent PCR productε were

εequenced to check that no amplification errors occurred.

In order to obtain the full-length mRNA sequenceε of Sscl and

Ssc2 , RACE experimentε were performed using:

primer 7

5' -GGCTATTGGAAAAGTCTATGGGGTCACA-3 ' for Sscl 5' RACE,

primer 8

5' -GGCACCATCTTCTTCATACTGTTCTCCA-3' for Sscl 3 ' RACE ,

primer 9

5' -CCAGCATATACGCAGAAAGAAGTGTG-3 ' for Ssc2 5' RACE, and

primer 10

5' -GACATACCGGAAAAAGCCAGTGAAGAAA-3' for Ssc2 3' RACE.

The template and PCR conditions were same as above .

The PCR products were subcloned into pCR-XL-TOPO vector

(Invitrogen) and sequenced using ABI Prismε Dye Terminator

Cycle Sequencing Ready Reaction kit (Perkin-Elmer) with an

ABI373A automatic DNA εequencer (Applied Biosystems) .

Human Sscl waε cloned in an analogous fashion from a human

liver Marthon-Ready cDNA library (Clontech) . The following primers were employed: WO 00/70945 PCTtEPOO/04371

91

Human- sscl-9Op

5 ' -GCCCCTACGGAGTCCTTAGCCA-3 '

Human- sscl-1007p

5 ' -GGTGCAGTCCTGAGGCACTTAGG-3 '

RNA isolation and Northern blotting

Total RNA was iεolated from freεh or frozen εpecimens of mouse

tissues (ca 50mg) using the Ultraspec RNA isolation protocol

(Biotech Laboratories) . RNA electrophoresiε , blotting and

hybridisation was performed as previously described (Tvrdik et

al., (1997) J. Biol . Chem . 272, 31738-31746). The cDNA probes

for Sscl , Ssc2 and Cig30 were Sall-Bgrlll fragments isolated

from their corresponding pEMR yeast expression vectors . The

actin probe waε as in (Tvrdik et al . , (1997) J. Biol . Chem .

272, 31738-31746). The probes were labelled using random-

primed DNA labelling kit (Boehringer Mannheim) with [³¹P]dCTP.

Yeast strains and cul ture condi tions

Yeast strainε uεed in this study and their genotypes are presented in Table 1. They are all FL100 and FL200 derivatives. For metabolic labelling and growth studies, all

strains were grown on YNBG medium (Difco) , supplemented with tryptophan (20 mg/1) (for EMA41 transformantε) , tryptophane

and leucine (for EMY58 transformants) and tryptophan and

uracil (for EMY3 ) .

Yeast plasmids and DNA manipulation

pEMR1023, a multicopy plasmid containing the selection marker

URA3 , was described previously (Silve et al . , (1996) Mol .

Cell . Biol . 16, 2719-2727). The coding sequence of Sscl , Ssc2 and Cig30 were obtained by PCR amplification as described

above, digested with Sail and Bg--.II, and ligated into Sail and

Bglll sites of the pEMR1023 vector. Yeast transformation waε carried out aε deεcribed (Gietz et al . , (1992) Nucleic Acids

Res 20, 1425) . The SUR4 gene disruption was performed by

replacing the 1. lkb fragment between the PvuII and Hpal εiteε

of SUR4 with a 1.4kb Dral -MIuI fragment encompassing the gene

conferring geneticin resistance. The resulting 3-kb BamHI cassette was used to replace the SULR4 gene in EMY30 cells. Disruption was checked by PCR analysiε . Diεruptant cells were resistant to 200 μg/ml geneticin and 25 μM SR31747. Sphingolipid analysis

Overnight cultures of yeast transformants were diluted to approx. 2 x 10^e cells/ml (OD₆₀₀=0.066) and grown in 2 ml of

appropriately supplemented YNBG at 30°C in the presence of the

radioactive precursor. [³H] serine (20 μCi/ml) waε added

immediately after dilution and the cells were labelled for 6 h. [H] εphinganine and [³H] inoεitol (each at 1 μCi/ml in the

total chemical concentration of 10 μM) were added 4.5 h after

dilution and the cells were labelled for 1.5 h. Incubations

were terminated by chilling on ice and 0.5 ml of unlabeled stationary phase cells was added. The cultures were

sedimented at 2800 x g for 10 min at 4°C, treated with 5% TCA

at 4°C for 20 min and washed once with 5 ml of ice-cold H₂0.

Lipids were prepared as described (Hanson and Lester., (1980)

J Lipid Res 21, 309-15) . Briefly, each pellet was extracted

twice with 1 ml of ethanol/water/diethyl ether/pyridine/4.2 N

NH₄0H (15:15:5:1:0.018) at 60°C for 15 min. In order to

destroy glycerophoεpholipids , the pooled extracts were

optionally treated with 1 ml of monomethylamine reagent (33%

monomethylamine in ethanol, diluted by 30%(v/v) with water) at 52°C for 30 min (Clarke and Dawson. , (1981) Biochem J 195,

301-6) . The extracts were then dried in SpeedVac and dissolved in 120 μl of chloroform/methanol/water (16:16:5) Thirty μl of each sample were applied to Whatman LK5D silica gel TLC plates and resolved in

chloroform/methanol/4.2NH₄OH (9 : 7 : 2) . When the runε were

completed, the plateε were dried at 100°C for 5 min and

sprayed several times with EN³HANCE (NEN Life Science

Products) . The signal was viεualised by exposing the TLC

plates to DuPont Cronex X-ray films at -80°C for several

weeks .

Preparation of microsomes and fatty acid elongation assay

Interεcapular brown fat was dissected and homogenised in 4 ml of ice-cold 0.25 M sucrose. Following a 30-min stepwise

centrifugation (10 min at each 700 g, 8000g and 17000 g) at 4 to 10°C, the supernatant was carefully transferred to fresh

tubes and microsomes were sedimented at 105000 g for 45 min.

The pellet was resuεpended in 20 mM Tris-HCl, pH7.4 ,

containing 0.4 M KCL, and centrifuged at 105000 g 45 min. The

final microsomal pellet was resuspended in 200 μl of 0.1 M Tris-HCl, pH 7.4, and the protein was measured with the BCA

protein assay (Pierce) .

Total fatty acid elongation activity was measured essentially

according to Suneja et al . , (1991) J. Neurochem . 57, 140-146. The assay mixtures (1 ml total, including protein addition)

contained 0.1 M Tris-HCl, pH 7.4; either 50 μM palmitoyl-CoA,

15 μM arachidoyl-CoA or 15 μM lignoceroyl -CoA; substrate/BSA

ratio 2:1; 1 mM NADPH; and 50 μM malonyl-CoA (containing 0.20

μCi of [2-¹⁴C]malonyl-CoA) . After 1 min of preincubation at 37°C, the reaction was initiated by the addition of 1 mg of

microsomal protein and carried out for 20 min at 37°C. The

incubation was terminated by addition of 1 ml of 15% KOH in

methanol and saponified at 65°C for 45 min. Then the sampleε were cooled and acidified with 1 ml of cold 5 M HC1. Free

fatty acid were extracted from the mixture three timeε with 3

ml of n-hexane and dried under vacuum. The extract was dissolved in 1 ml of chloroform and measured after addition of

10 ml of scintillation mixture in a Beckman liquid

scintillation system 3801.

EXAMPLE 5 - Generation of Cig30 knockout mice

MATERIAL AND METHODS

Gene targeting vector

A genomic liver DNA library from mouse strain 129/Sv cloned

into the Lambda FIXII vector waε used to isolate a DNA fragment containing the entire Cig30 gene.

Mouse CJ 30 genomic cloning

Genomic clones of Cig30 were isolated by plaque hybridisation of a commercial mouse 129 strain liver genomic DNA library in

the Lambda FIX II vector (Stratagene Catalogue # 946308) with a ³"P- labelled probe corresponding to 1.2 kb from the 5' end of

the Cig30 cDNA (GenBank U97107, Tvrdik et al . Figure la,

supra) . Hybridisation waε carried out overnight at 45 °C in

50% formamide, 5 x SSC, 5 x Denhardt ' ε solution, 50 mM sodium

phosphate, pH 6.5 , 0.5% SDS and 100 μg/ml degraded herring sperm DNA. The membranes were first washed twice for 15 min

at room temperature in 2 x SSC, 0.1% SDS, and a high

stringency was then performed at 55 °C in 0.1 x SSC, 0.1% SDS

for 15-30 min. Phage DNA was prepared on a large scale by the

PEG precipitation method. Cig30 gene was checked by

hybridisation with a 3' UTR probe (0.8 kb from the 3' end of

the Cig30 cDNA) .

DNA sequencing and sequence analysis

Using recombinant phage DNA as a template, the genomic insert

waε sequenced by the primer walking strategy. Sequencing waε performed with an ABI373A automatic DNA sequencer (Applied

Biosystemε) on reactionε prepared by the dye- termination

method, using the ABI Prisms Dye Terminator Cycle Sequencing

Ready Reaction kit (Perkin-Elmer) . The sequence information

was compiled and analysed with the use of the University of Wiεconsin Genetics Computer Group software. The complete

nucleotide sequence of the 13,869-bp genomic fragment has been

deposited in GenBank (Accesεion No. AF054504) .

The DNA probeε used were as follows :

(i) the Cig30 ORF probe - a 871-bp fragment corresponding to

nt 162-1056 in the Cig30 cDNA;

(ii) the Cig30 3' probe - a 294-bp Pstl-Stul fragment corresponding to nt 10646-10939 in the genomic clone; and

(iii) the Pitx3 probe - a 897-bp Pstl-Pstl fragment

corresponding to nt 11443-12340 in the genomic clone.

The probeε were labelled with [ -³'P]dCTP using a random primed DNA labelling kit (Boehringer Mannheim) .

A genomic liver DNA library from mouse strain 129/Sv cloned into the Lambda FIXII vector waε used to isolate a DNA

fragment containing the entire Cig30 gene. A 2.75 kb fragment between Sad and Sail upstream of the first exon was subcloned into the polylinker of the pBluescript SK(+/-) plasmid in order to become the left arm in the gene targeting vector. The Sad site was blunt-ended to T4 DNA polymerase (New England Biolab) and turned into a Notl site by ligation of Notl primers (Bio Source International) . A 1.2 kb fragment containing a neomycin' (neo¹ ) gene was cut out with Xhol from the plasmid KT1 LoxA (Thomas and Capecchi, (1987) Cell 51, 503-512) and inserted into the compatible Sail εite of the plasmid containing the 2.75 kb left arm fragment. The ligation between Sail and Xhol ends consequently destroyed the recognition site for respective digestion enzyme. A 5.30 kb Sail/Sail fragment downstream of the second exon of Cig30 was ligated into the pBluescript SK(+/-) plasmid and further

digested with Sail and Xhol to receive a 2.77 kb fragment corresponding to the right arm in the gene targeting vector. This fragment was subcloned into the Xhol site of the vector containing the 2.75 kb left arm fragment and the neo^r gene. The Notl/Xhol fragment from the resulting plasmid was ligated into the corresponding sites in the TK1-TK2 vector (Barrow, J.R. and Capecchi, M.R. (1996) Development 122, 3817-3828) .

Gene - targeting in ES cell cul tures The targeting vector waε linearized with Sail and electroporated into the Rl embryonic stem (ES) cells (Nagy, A. et al (1993) Proc . Natl . Acad, Sci . USA 90, 8424-8428) derived from male 129/Sv agouti mice. ES cells were cultured aε earlier described (Thomas, K.R. and Capecchi, M.R. (1987) Cell 51, 503-512) and expoεed to 250 μg/ml G418 and 2 μM GANC (Gangcyclovir) aε poεitive and negative εelection respectively for three weeks (Manεour, S.L et al (1988) Nature 336, 348- 352) . G418/GANC resistant ES cells were screened for targeting events by Southern blot analysiε (εee below) .

Generation and screening of Cig30 knock-out mice

Cig30 targeted ES cells were injected into C57BL/6J blastocysts and implanted into white foster mothers (Fl,

CBAxC57BL6) according to standard procedures (Gossler, A. et al , (1986) Proc . Natl . Acad . Sci . USA 83, 9065-9069) . The male offspring being the most chimeric, approximately 80% agouti and 20% black in the coat colour, were bred with C57B1/6J female in order to generate offspring heterozygous for the mutation. Genotypic analysis was performed by Southern blotting with Seal and probing with a 897 bp Pstl genomic right arm fragment (see under DNA analysis) . RNA analysis

Total RNA was isolated using Ultraεpec (Biotech lab.) from 50-

100 mg (w/w) of each tissue. For Northern blot analysis, 20

μg of total RNA was separated on a 1.2% (w/v) formaldehyde agarose gel and blotted onto Hybond-N membrane (Amersham) in

20 x SSC. The Hybond-N membrane was prehybridiεed with a

solution containing 5 x SSC, 5 x Denhardt's, 0.5%, SDS, 50 mM

εodium phoεphate, 50% formamide and 100 μg/ml of degraded DNA

from herring sperm (Sigma) at 45°C. After prehybridisation,

the Hybond-N membrane was transferred to a similar solution

containing the denatured probe. The hybridisation waε carried

out over night at 45°C. The Hybond-N membrane waε then washed

twice in 2 x SSC, 0.2% SDS at 30°C for 20-30 min each and then

twice in 0.1 x SSC, 0.2% SDS at 50°C for 45 min. The filters

were analysed on a Molecular Dynamics Phoεphorlmager with the

ImageQuant program. When the same membrane was analysed for several mRNA species, the previous probe was removed by

boiling the membrane twice for 20 min in 0.1% SDS solution.

DNA analysis

Genomic DNA waε prepared from mouεe tailε by the simplified

mammalian DNA isolation procedure published by Laird et al . (Laird et al . , (1991) Nucleic Acids Res . 19, 4293-4293) . Tail biopsies were collected from 3-week-old mice and used directly for DNA isolation. ES cell DNA was prepared according to

standard procedureε (Nagy, A. et al (1993) Proc . Natl , Acad .

Sci USA 90, 8424-8428) , DNA from ES cells were digested with Xbal and Xbal/Sall respectively and analysed with a 752 bp

Ahdl/Sall probe from the 2.75 kb left arm fragment. Two further probeε, the 1.2 kb neo¹ (Xhol/Xhol) iεolated from KT1

LoxA plasmid and the Cig30 probe which binds to Cig30

downstream of exon 4, were used to confirm or exclude ES cell

clones from the first screening. The DNA for the probeε were purified according to the Jetsorb Gel extraction kit (Genomed

Inc.) and were labelled with [α-³²P]dCTP using a random primed labelling kit (Boehringer Mannheim) . Ten μg of digested DNA

was separated on 0.8% agarose gel and transferred to Hybond-N

membrane (Amersham) in 20 x SSC. The hybridisation procedure

were identical to those for Northern blot analysis, except that hybridiεation was carried out over night at 55°C. Hybond-

N membrane waε then washed twice in 2 x SSC, 0.2% SDS at 30°C for 20 min each and then once in 0.1 x SSC, 0.2% SDS at 55"C

for 30 min. The filters were analysed on a Molecular Dynamics

Phosphorlmager with the ImageQuant program.

RESULTS WO 00/70945 PCTtEPOO/04371

102

Targeting in ES cells

A Cig30 genomic DNA fragment waε initially isolated from a

liver genomic lambda library (Figure 8) . A 2.75 kb fragment downstream of the transcription start site waε εubcloned into

a Bluescript vector as described in Materials and Methods.

The following subcloning of the neo' gene and a 2.77 kb

fragment aε the right arm fulfilled the criteria for a gene

targeting vector againεt Cig30 . By incorporating a negative

εelection marker, the thymidine kinase ( tk) gene, it became

possible to enrich for homologous recombination events (Figure 9) . The gene targeting vector was linearized with Sail and

electroporated into Rl ES cells (Nagy, A. et al . (1993), Proc . Natl . Acad . Sci . USA 90, 8424-8428). Upon selection on G418

and GAΝC resistance 144 ES cell clones were chosen for

Southern blot analysis.

DΝA from the ES cells were digested with Xbal and hybridised with a probe which binds upstream of the transcription start

site but still resides in the left arm of the targeting vector. In case of a homologous recombination event, the

probe should hybridise with two fragments, the wild-type

fragment of 5.836 bp and the targeted fragment of 4.703 bp in size which is due to the fact that the neo^: gene will

εpecifically replace a 2.1 kb endogenous fragment including the transcription start site and the first two exons upon a

correct targeting event (Figure 9B) . Seventeen putative

targeted clones were investigated further by extended Southern

blot analysi .

Thiε time, DNA from ES cell cloneε were digeεted with Seal and

Clal respectively and hybridised with one probe εpecifically

towardε the neo^: gene and a 897bp genomic probe binding

downεtream of the right arm fragment (εee Materials and

Methods) . Two of the seventeen ES clones were positive for the targeting event. Digestion with Seal of one of these

clones, lhl, resulted in a distinct single fragment of 11.644

bp when hybridiεing with the neo^r probe. Fragments of other

sizes were due to random integration of the gene targeting vector as in clone 2e5. Digestion with Clal showε one

fragment of 5.639 upon homologouε recombination. The fragment

εize in the randomly integrated ES cells iε unpredictable,

since there is only one Clal site within the Cig30 gene.

When the same blot was probed with the Cig30 gendmic fragment downstream of the right arm it gave rise to two fragments of

11.644 bp (Seal) and 5.639 bp (Clal) respectively, i.e. the WO 00/70945 PCTtEPOO/04371

104 same two fragmentε εeen with the neo^' gene probe. The wild- type allele showed a distinct band of 5.603 bp which was not

εeen in the previouε hybridisation with the neo¹ probe. In

summary, two targeted Cig30 ES cloneε from the initial 144

cloneε were identified.

Cig30 - defi cien t mi ce

A εchematic picture of how the Cig30-deficient mice were obtained is illustrated in Figure 10. ES cell clones were

injected into blastocystε from C57B1/6 and implanted into

white foεter mothers. The resulting male chimeraε which were

approximately 80% agouti and 20% black in the coat colour were

bred with C57BL/6J femaleε . The resulting heterozygote

offspring were bred to homozygosity and DNA was isolated and

genotypically analysed by Southern blot analysis. The

homozygote diεrupted Cig30 mice were eaεily diεtinguiεhed from wild-type and heterozygouε mice by εhowing a εingle 11.644 bp

fragment .

Phenotype of Cig30 - defi cient mice

In order to confirm the Cig30 knock-out expression in mouse,

Northern blot analysis were performed with total RNA iεolated from brown adipose tissue, skin and liver, i.e. organε which

expresε Cig30, from both knock-out and wild-type mice kept at

30°C and 4°C for 6 days. Northern blot analysis showed there

iε no detectable Cig30 mRNA in theεe organs in the Cig30

knock-out mice at any temperature compared with wild-type mice where there is detectable amounts in all three tissueε

especially in cold induced brown adipose tissue. Mice

heterozygous for Cig30 show an expresεion pattern εimilar to wild-type litter mateε indicating that the gene iε receεεive.

Cig30 knock-out mice show a retarded growth during the first

four-five weeks of age which in disεection studies can be seen

as less muscle maεε and leεs subcutaneouε white fat mass.

After approximately two weeks of age there iε a more obvious

phenotypic distinction between knock-out and wild-type mice.

The fur is tousled and there is a marked decrease in hair over

the whole body. These observations are confirmed by histology

studies of the skin. The hairs, hair follicles and associated

sebaceous glands are leεε numerouε and show a rudimentary and inactive appearance in the knock-out mice compared to control

mice. Further, the skin of Cig30-deficient mice waε several times thinner than in the control mice which is due a

underdevelopment of dermiε and all dermal εtructureε (adnexa) . Another distinct phenotype iε the cloεed the eye lidε, i.e. the eye lidε open at a later stage during growth and can even in some mice be closed after weaning.

However, aε indicated, Cig30-deficient mice tolerate cold exposure as well as the control mice which implies that the

function of the brown fact in theεe animalε iε intact . The UCP1 mRNA levels were found not to change upon cold

stimulation in Cig30 knock-out mice compared with wild-type

mice. In addition, histological studieε revealed no

difference between brown fat tiεεue from knock-out and control

animals .

Since Cig30 is expressed in liver as well, total liver from

Cig30-deficient mice was analysed hiεtologically with no

indicativeε of a different phenotype than from control mice.

DJSCCJSSJON

Cig30 , Sscl and Ssc2 belong to a novel mammalian gene family

which haε been shown to be implicated in the elongation of VLCFA aε well as in the formation of specific sphingolipids.

Since Cig30 and the other mammalian family members shows a tissue specific expression it can be assumed that there is a special need for VLCFA of a specific length in different cells at a specific time. This is verified by the experiments

described above in which the inventors conεtructed a gene

targeting vector for Cig30 to diεrupt the gene in mice.

The method employed for disrupting Cig30 was baεed on the

Positive-Negative-Selection (PNS) method originally described

by Capecchi and co-workers (Mansour, S.L. et al (1988) Nature 336, 348-352) . The method can target and select for homologous recombination of any endogenous gene.

The gene targeting vector which haε two fragments of 2.75 kb and 2.77 kb, the left and right arm respectively, flanking the

neo^r gene, facilitateε replacement of exon 1 and 2 with the

neo¹ gene (Figure 9) . Two out of 144 ES cell lineε had

undergone homologouε recombination, which waε confirmed by

Southern blot analysis. The most important factor determining

targeting frequency iε the length of the target homology. In

a study by Hasty et al . there was a 200-fold increase in gene- targeting efficiency associated with an increase of homology

from 1.3 to 6.8 kb for replacement vectors (Hasty, P. et al

(1991) Mol . Cell . Biol . 11, 5586-5591).

In the work described here, the inventors were unable to ascribe the significance of the tk selection. However, the

uεe of the tk εelection aε deεcribed by Mansour et al . showed

a 200-fold enrichment of ES cells that contained homologous vε . non-homologous integration of the target vector (Mansour,

S.L. et al (1988) Nature 336, 348-352) . Most groups however, have obεerved only a 5-20 fold enrichment with thiε method

(Roller, B.H. et al (1992) Annu . Rev. Immunol . 10, 705-730).

Analyεis of the targeted clones was by Southern blot analysis

since the inventors were unable to successfully amplify PCR fragments corresponding to homologous recombination of DNA

from targeted ES cells. Initial difficulties were also

encountered with probes binding unεpecifically to the genome

and restriction enzymes, i.e. Sail and Clal, being unable to

digest in an eukaryotic environment . The left arm-probe of

752 bp waε chosen in order to perform the first screening of

the 144 cloneε resulting in 17 putative correctly targeted ES

cell cloneε. A final screening of these 17 clones was achieved by hybridising the same membranes with the neo¹ probe

and a probe that binds outside and upstream of exon 4 in the targeting vector ( Cig30 probe) . DNA from the remaining 17

clones was digested with Seal and Clal . Although Clal iε methylated in an eucaryotic environment aε in the ES cells

(Nelson, M. (1991) Nuclei c Acids Res . 19 Suppl . , 2045-2071), still 10% of the DNA as seen on Southern blots were digested.

By these means two ES cell clones with a disrupted Cig30 gene were finally detected. Both hybridisationε gave riεe to the

expected fragmentε, aε well aε to the extra fragment of 5.603

bp with the latter probe.

When mice heterozygote for the targeting event were bred, a Mendelian 1:2:1 distribution was observed. This implies that

Cig30 expression is not essential for prenatal development.

The relevance of VLCFA in general iε indisputable. Depending

on their chain length and degree of unsaturation, they

regulate the fluidity and other physical and chemical properties of the membrane such as cell-cell contact, transport over the membrane, activities of membrane-asεociated

enzymes and key regulators of the eukaryotic stress response

(signal transduction molecules) . The streεε response iε

particular interesting in view of the strong and εelective

induction of Cig30 in recruiting brown adipoεe tiεsue, suggesting that syntheεiε of a specific VLCFA iε required for

tissue recruitment and/or cell proliferation. Multiple lines

of evidence are beginning to implicate sphingolipidε ,

ceramides and sphingosines as key regulators of the eucaryotic stress response, e.g. ceramides formed after activation of sphingomyelinase (by TNF- and IL-1) decrease cell division and induce apoptosis (Kim, M.Y. et al (1991) J. Biol .

Chem . 266, 484-489; Obeid, L.M. et al (1993) Science 259,

1769-1771) which on the other hand can be counteracted by sphingosine-phosphate which εtimulate cell growth and inhibit

apoptosis (Zhang, H. et al (1991) J. Cell Biol . 114, 155-167)

A marked increase can be seen in the microsomal elongation

activity of long chain and very long chain fatty acids from cold εtimulated brown adipoεe tiεsue compared with warm

controls .

The work clearly demonεtrateε involvement of VLCFA in εkin

development. Skin lipidε are an important determinant for both water- retention function and permeability-barrier

function. Sphingolipidε (ceramides) have been reported to

comprise up to 50% of the total amount lipids in epidermis

which is the highest amount present in any mammalian tissue

(Forslind, B. (1997) J. Dermatol . Sci . 14, 115-125). Another

intriguing fact iε that more than one-third of human εtratum

corneum lipidε have chain lengthε longer than 22-carbonε, i.e VLCFA . Disorders such as atopic dermatitis correspond to

impaired barrier function due to reduced ceramide content

which leads to dry and irritated εkin (Di Nardo, A. et al

(1998) Acta Derm . Venereol . 78, 27-30) . The alterations seen in the Cig30 knock-out mice may be regarded as a dysplastic skin decease, characterised by

abnormal growth and development of the dermiε and adnexa .

Alternatively, the changes observed could be considered aε an atrophic diεease of the skin characterised by hair growth

arrest. The skin changes observed in Cig30-deficient mice are

moεt dramatic within the dermiε layer, although it is not

clear yet whether the skin barrier in itself is defect.

Further analysis of the skin will reveal which cells are

normally expreεεing Cig30 and the εpectrum of VLCFA and

sphingolipidε in theεe mice.

The mice show impaired hair growth, dry skin and much less

subcutaneous fat than normal mice, and this appearε to be due

to a general developmental problem of the εkin layerε .

However, despite the high Cig30 expression in brown fat in

wild-type mice during cold exposure, the Cig30 knockout mice

are fully capable of maintaining their thermoregulatory

function including high UCP1 expression upon cold exposure.

The mice also show eye problems within the first 3-5 weeks

after birth. This iε thought to be due to undeveloped membonious glands in their eye lids which normally "oil" the

eye so the lid is smoothly mobile. This provideε for teεting of substances for treatment of eye disorders or other problems (e.g. when contact lenses are used), for instance ceramideε with specific fatty acids.

CONCLUDING DISCUSSION

The inventors have characterized three paralogouε geneε in

mice and homologueε in humans, which geneε are indicated to be the firεt mammalian genes directly involved in fatty acid

elongation.

Cig30 was previously isolated in the laboratory as a gene

specifically expressed during brown fat recruitment, and in liver and skin. The inventors have now identified and cloned

two new full-length cDNA'ε, Sscl and Ssc2 . Analysiε of

expression in mice revealed that Sscl mRNA is ubiquitously

expressed in all tissues tested, with higheεt levelε in

stomach, lung, kidney, and skin. In contrast, Ssc2 mRNA was

found to be rather tissue specific, being predominantly expressed in teεtis and liver. The deduced SSCl and SSC2

polypeptides are approximately 30% identical with CIG30.

Rescue experiments of phenotypes of yeaεt null mutantε demonεtrate involvement in biosynthesiε of very long chain

fatty acidε and consequently the formation of sphingolipids. Cig30 deficient mice have been made and these have markedly diminished subcutaneous fat and abnormal hair growth. This is correlated with suppressed development of all epidermal and dermal structures .

The present invention allows for screening of agents for identification of candidate pharmaceuticals and other active agents, which may for instance be used in treatment of atopic dermatitis and other skin disorderε, or in coεmetic applicationε, for inεtance in improving hair growth, or in treating eye problems .

Furthermore, Sscl mRNA levels were found to be markedly reduced in brains of myelin-deficient mice which have decreased cerebral fatty acid chain elongation activity. This provides for screening for agents which may be used in

treatment of MS .

Ssc2 was found to be highly expressed in testis, providing indication of a role in fertility given that ceramides (sphingolipids) are essential for function of sperm. TABLE 1.

Yeast strains used in this study

EMY58 Matα ura3 tipl Ieu2 sur4::GenR (for SR31747 resistance)

EMA41 Mata ura3 trpl Ieα2 fenl::LEU2 (for SR31747 resistance and crosses

EMA3 Mata ura3 trpl (for SR31747 resistance)

EMY30 Matα ura3 trpl leu2 (for SUR4 disruption)

EMY22 Ivxaiα ura3 sur4-232 (for crosses)

AU these strains are FL100 or FL200 derivatives.

Claims

CLAIMS :

1. A non-human transgenic animal which is a knock-out for nucleic acid encoding a polypeptide which has at least 80%

amino acid sequence identity with a polypeptide εelected from

i CIG30, of which the amino acid sequence is εhown

in Tvrdik et al . , J. Biol. Chem. (1997) 272: 31738-31746;

ii human SSCl, of which the amino acid εequence is

shown in Figure 4 ;

iii mouεe SSCl, of which the amino acid sequence iε

εhown in Figure 2 ; iv mouse SSC2, of which the amino acid sequence iε

shown Figure 6.

2. A non-human transgenic animal which has at least one

copy of a polynucleotide according to any one of claims 28 to

30 below which is heterologous to said animal integrated into

a chromosomal location.

3. An animal according to claim 1 or claim 2 that is a

mouεe or other rodent .

4. An assay method for obtaining an agent able to modulate fatty acid elongation, the method comprising contacting an animal according to any one of claims 1 to 3 with a test

compound, and determining ability of the test compound to

affect a tissue of the animal.

5. An assay method for obtaining an agent that is a

candidate pharmaceutical for use in therapeutic treatment of a

εkin or hair diεorder or eye problem, the method compriεing

contacting an animal according to any one of claims 1 to 3

with a test compound and determining ability of the test

compound to affect the skin, hair or eyes of the animal.

6. A method comprising:

(a) performing an assay method according to claim 4 or

claim 5 to obtain a said agent; (b) formulating said agent into a composition which

comprises one or more additional components .

7. An agent obtained by a method according to claim 4 or

claim 5.

8. Use of an animal according to any one of claims 1 to 3 for identifying a substance which modulates fatty acid elongation.

9. Use of an animal according to any one of claims 1 to 3 for identifying a substance which has a therapeutic effect on a disorder of the skin, hair or eyes.

5 10. An isolated polypeptide compriεing an amino acid

εequence selected from the group consisting of:

(1) the human Sscl amino acid sequence shown in Figure 4;

(2) the mouse Sscl amino acid sequence shown in Figure 2;

(3) the mouse Sεc2 amino acid sequence shown Figure 6.

10

11. An isolated polypeptide according to claim 10 conεisting

of the amino acid sequence shown in Figure 4, Figure 2 or

Figure 6.

15 12. An isolated polypeptide which has an amino acid sequence

which shares at least 80% identity with a polypeptide

according to claim 11.

13. An isolated polypeptide which has an amino acid εequence

20 which shares at least 90% identity with a polypeptide

according to claim 11.

14. An isolated fragment, active portion, variant or

derivative of a polypeptide according to claim 11, which has WO 00/70945 PCTtEPOO/04371

118 one or more of the following properties:

immunological crosε-reactivity with an antibody reactive with εaid polypeptide;

sharing an epitope with said polypeptide;

5 a biological activity which is inhibited by an antibody

raiεed against said polypeptide;

ability to complement ELOl, EL02 and/or EL03 mutationε

in yeast;

enzymatic activity in common with said human Sεcl, mouse 0 Sscl and/or mouse Ssc2.

15. An iεolated fragment of a polypeptide according to claim 11, which fragment iε at least 5 amino acids in length.

15 16. An isolated fragment according to claim 15 which is less

than about 40 amino acids in length.

17. An assay method for obtaining an agent able to interact with a polypeptide, fragment, active portion, variant or 20 derivative according to any one of claims 10 to 16, the method comprising:

(a) bringing into contact a substance which comprises a said polypeptide, fragment, active portion, variant or

derivative , and a test compound; and (b) determining interaction between said substance and the test compound.

18. An assay method for obtaining an agent with ability to

5 modulate interaction between a polypeptide, fragment, active

portion, variant or derivative according to any one of claims

10 to 16, and a εecond molecule, the method comprising:

(a) bringing into contact a subεtance which comprises

said polypeptide, fragment, active portion, variant or

10 derivative, a substance comprising a molecule which interacts

with said polypeptide, fragment, active portion, variant or

derivative, and a test compound; and

(b) determining interaction between said substances.

15 19. An assay method according to claim 17 or claim 18, the

method further comprising determining ability of a test

compound or an agent obtained in the assay to affect fatty

acid elongation.

20 20. An assay method for obtaining an agent able to affect

fatty acid elongation, the method comprising:

(a) bringing into contact a substance which comprises

a polypeptide, fragment, active portion, variant or derivative

according to any one of claims 10 to 16, and a test compound; and

(b) determining fatty acid elongation activity.

21. An assay method according to any one of claimε 17 to 20

5 further compriεing determining ability of a test compound or

agent obtained in the assay to affect mammalian skin, hair or

eyes .

22. A method comprising:

10 (a) performing an assay method according to any one of

claims 17 to 21 to obtain a said agent;

(b) formulating said agent into a composition which

comprises one or more additional components.

15 23. An agent obtained by a method according to any one of

claims 17 to 21.

24. Use of a polypeptide, fragment, active portion, variant

or derivative according to any one of claims 10 to 16, for

20 identifying a substance which interacts with said a

polypeptide, fragment, active portion, variant or derivative.

25. Use of a polypeptide, fragment, active portion, variant

or derivative according to any one of claims 10 to 16, for identifying a substance which modulates fatty acid elongation.

26. Uεe of a polypeptide, fragment, active portion, variant or derivative according to any one of claimε 10 to 16, for

5 identifying a substance which has a therapeutic effect on a

disorder of mammalian skin, hair or eyeε .

27. An iεolated εpecific binding member comprising an

antigen-binding domain of an antibody specific for a 0 polypeptide according to claim 10.

28. An isolated polynucleotide encoding a polypeptide

according to any one of claims 10 to 13.

15 29. An isolated polynucleotide according to claim 28 of

which the sequence encoding said polypeptide is shown in

Figure 3 , Figure 1 or Figure 5.

30. An isolated polynucleotide encoding a fragment, active 20 portion, variant or derivative according to any one of claims

14 to 16.

31. An expression vector comprising a polynucleotide according to claim 29 or claim 30 operably linked to regulatory sequences for expression of said polypeptide, fragment, active portion, variant or derivative.

32. A host cell transformed with an expression vector according to claim 31.

33. A method of making a product, the method comprising

culturing a host cell according to claim 32 under conditionε

for expression of said polypeptide, fragment, active portion,

variant or derivative to provide said product, and isolating or purifying εaid product.

34. A method according to claim 33, further comprising

testing the product for ability to modulate fatty acid elongation.

35. A method according to claim 33 or claim 34, further

comprising testing the product for therapeutic effect on a

disorder of mammalian skin, hair or eyes.

36. A method according to any one of claims 33 to 35 wherein

the isolated or purified product is formulated into a composition comprising one or more additional components.

37. A method which comprises determining in a sample the

presence or absence of a polypeptide, fragment, active

portion, variant or derivative according to any one of claims

10 to 16.

38. A method which comprises determining in a sample the

presence or absence of nucleic acid encoding a polypeptide,

fragment, active portion, variant or derivative according to

any one of claimε 10 to 16.