WO1999053090A2

WO1999053090A2 - Genotypic screening

Info

Publication number: WO1999053090A2
Application number: PCT/NZ1999/000044
Authority: WO
Inventors: Garth James Smith Cooper
Original assignee: Auckland Uniservices Limited
Priority date: 1998-04-08
Filing date: 1999-04-08
Publication date: 1999-10-21
Also published as: WO1999053090A3; AU3541399A

Abstract

The invention provides a screening method by which bovines can be selected for as having desirable genotypes, or selected against as having undesirable genotypes. Selection is with reference to allelic variations in exon 1 of the MLC-1/3f gene.

Description

GENOTΎPIC SCREENING

The present invention relates to methods of selecting for a desirable genotype, particularly by screening nucleic acid, and to kits for use in carrying out such methods

INTRODUCTION

In the farming industry where animals are farmed for their flesh and milk production, there are a number of genotypes which it is desirable for the farmed animal to possess One example is a predisposition towards lean muscle mass, lower fat content and/ or higher feed efficiency in animals such as cattle where the muscle is ultimately intended for human consumption

It would therefore be useful to be able to select for desirable genotypic characteristics It is generally towards this goal that the present invention is directed

The protein myosin light chain- 1 -fast (MLC- lf), also known as alkali light chain- 1 (alkali-LC I), is present in striated muscle containing white (glycolytic/ anaerobic) fast twitch muscle fibres It is one of two products derived from a single gene in LC- l/3f {MLC-1/ ' 3j) which by differential gene splicing gives πse to both MLC- lf (approximately 21 kDa) and MLC-3f (approximately 17 kDa) Exon 1 of the gene is expressed only in MLC- lf The physiological role of MLC- lf is to modulate the interaction between actin and the myosin head duπng muscle contraction (Rushbrook et al (1988). Stepkowki ( 1995))

Polymorphic variations have been reported for alleles of MLC-1/ 3f in the tropical cichlid (a fish) (Crockford et al (1995)) and in chickens (Rushbrook et al ( 1988)) However, no association has been drawn between these observed polymorphisms and anv desirable characteristic

What the applicants have now determined is that a polymorphism also exists in MLC-1/ 3f of bovines Further, the apphcants have determined an association between one of the allelic variations of MLC-1/ 3f and a number of desirable genotypes, including muscle mass It is this association which forms the basis of the invention

SUMMARY OF THE INVENTION

Accordingly, in one aspect, the invention provides a method of selecting for a bovine having a desirable genotype, which method includes the step of determining the presence or absence of additional codon(s) in exon 1 in at least one allele of MLC- 1/3/ for that bovine

As used herein, the words "additional codon(s)" mean in excess of 123 nucleotides in exon 1 of MLC-1/3J, in a group or groups of 3 Such group(s) need not necessarily be included in frame

In a further aspect the invention provides a method of selecting for a bovine having a desirable genotype comprising the steps of

(a) determining the presence or absence of additional codon(s) in exon 1 m at least one allele of MLC-1/ 3f for that bovine, and

(b) selecting for said bovine where no additional codon(s) are present in exon 1 in at least one allele of MLC-1/ 3f

Most preferabh the method involves selecting for said bovine where no additional codon(s) are present in exon 1 in both alleles of MLC-1/ 3f

In yet a further aspect, the invention provides a method of selecting for a bovine having a desirable genotype, which method includes the step of determining the presence or absence, in at least one allele of MLC-1/ 3f, of the following nucleotide sequence for exon 1

CGA CCA. AAG AAA GAT GTG AAG AAA CCT GCT GCT GCC GCT GCC CCC GCC CCG GCC CCG GCC CCT GCC CCA GCA CCT GCA CCT GCC CCA CCC AAA GAA GAA AAG ATT GAC CTC TCT GCC ATC AAG

Preferably, the method selects for bovines in which both alleles of MLC-1/ 3f have the above sequence in exon 1 Most conveniently, the presence or absence of additional codon(s) is determined by analysis of nucleic acid (DNA or RNA) obtained from said bovine

Alternatively, the presence or absence of additional codon(s) is determined by analysis of the amino acid sequence of MLC- If protein obtained from said bovine

Preferably, the desirable genotype selected for is a genetic predisposition to larger musculature, reduced fat, faster growth rate and/ or improved feed efficiency

Where the bovine is farmed to produce milk for human consumption, the desirable genotype is a predisposition to increased milk production

In a further aspect, the invention provides a method of selecting against a bovine having an undesirable genotype, said method including the step of determining the presence or absence of additional codon(s) in exon 1 in at least one allele of MLC- l/3fioτ that bovine

In a further aspect, the invention provides a method of selecting against a bovine having an undesirable genotype which comprises the steps of

(a) determining the presence or absence of additional codon(s) in exon

1 m at least one allele of MLC 1/3/ for that bovine, and

(b) selecting against said bovine where additional codon(s) are present in exon 1 in both alleles of MLC-1/ 3f

Conveniently, said additional codon(s), upon expression, result in one or more additional amino acids being present in the amino acid sequence of MLC- If protein

More conveniently, the additional codon(s). upon expression, result in the inclusion of two additional ammo acids in the amino acid sequence of MLC- If protein

Most conveniently said two additional amino acids are alanine (A) and proline (P) Most conveniently, the additional codon(s) have the following nucleotide sequence:

GG C CCC

Preferably, said two additional amino acids are located in the amino acid sequence of MLC-lf protein such that the sequence resulting from expression of exon 1 is, or includes, the following:

APKKDVKKPAAAAAPAPAPAPAPAPAPAPAPPKEEKID LSAIK

(additional amino acids in bold)

Preferably, said additional nucleotides are located between nucleotides 62 and 63 of the nucleic acid sequence of exon 1 of MLC-1/ 3f such that the sequence is as follows:

GCA CCA AAG AAA GAC GTG AAG AAA CCT GCT GCT GCC GCT GCC CCC GCC CCG GCC CCG GCC CCG GCC CCT GCC CCA GCA CCT GCA CCT GCC CCA CCC AAA GAA GAA AAG ATT GAC CTC TCT GCC ATC AAG

In a further embodiment, the invention provides a nucleic acid probe which hybridizes, under high stringency conditions, to the nucleotide sequence:

but not to the nucleotide sequence:

CGA CCA AAG AAA GAT GTG AAG AAA CCT GCT GCT GCC GCT GCC CCC GCC CCG GCC CCG GCC CCT GCC CCA GCA CCT GCA CCT GCC CCA CCC AAA GAA GAA AAG ATT GAC CTC TCT GCC ATC AAG.

In yet a further embodiment, the invention provides a nucleic acid probe which hybridizes, under high stringency conditions, to the nucleotide sequence: CGA CCA AAG AAA GAT GTG AAG AAA CCT GCT GCT GCC GCT GCC CCC GCC CCG GCC CCG GCC CCT GCC CCA GCA CCT GCA CCT GCC CCA CCC AAA GAA GAA AAG ATT GAC CTC TCT GCC ATC AAG

but not to the nucleotide sequence

In still a further embodiment, the invention provides a paired set of PCR amplification primers, one of said primers hybridizing, at its 3' or 5' end, to nucleotides 63-68 of the following sequence

In a final embodiment, the invention provides a kit suitable for use in a method as defined above which comprises a nucleic acid probe, or PCR primers, as defined above

Most convementl} said kit includes nucleic acid probes specific to both the long allele and short allele

BRIEF DESCRIPTION OF THE DRAWINGS

Those persons skilled in the art will appreciate that while the invention is broadly as outlined above, it also includes embodiments of which the following descπption provides examples In particular, a better understanding of the invention will be gained by reference to the accompanying drawings in which

Figure 1 is a portion of a gel showing the presence of both variants of MLC- If in a normal muscled (NM) bovine

Figure 2 is a portion of a gel showing the presence of only a single lower molecular weight isoform of MLC- If in a double-muscled (DM) animal Figure 3 shows portions of partially focused gels of four NM animals and an under- muscled animal (NM*) showing in the case of the NM animals expression of both MLC- lf isoforms and, in the case of the NM* animal, expression of only the higher molecular weight MLC- If isoform

Figure 4 shows the three isoforms on one gel lane 1 - molecular weight marker, lane 2 is the lower molecular weight isoform (homozygous short), lane 3 is the higher molecular weight isoform (homozygous long), and lane 4 is heterozygous long and short Note the band above the heterozygous doublet band is a heteroduplex of the two products

DESCRIPTION OF THE INVENTION

As described above, the primary focus of the invention is on selection for desirable genotypes in bovines The invention has particular application to bovines which are farmed for their flesh or for their milk

The desirable genotypes which are the particular focus of the invention include a genetic predisposition to increased muscle mass, to increased milk production (females) to increased ratios of protein to fat deposition, to increased growth rates and to increased feed efficiency However, it will be appreciated that these are but representative, and should not be regarded az a limitation on the invention

The invention is oased upon the apphcants finding that such desirable genotypes are associated with MLC-1/ 3f in bovines The apphcants have found that there are two allelic variations in exon 1 of MLC-1/ 3f, one being associated with increased muscle mass and the other associated with decreased muscle mass In turn, the allelic variation associated with increased muscle mass has been associated with a predisposition to increased feed efficiency and with greater growth rates and milk production

The allelic variations are found in the length of the nucleotide sequence of exon 1 of the gene In terms of muscle mass, the allele for decreased muscle mass contains 129 nucleic acids (herein the "long" allele) whereas the allele for increased muscle mass contains 123 nucleic acids (herein the "short" allele) The long allele therefore includes two additional codons when compared to the short allele This leads to the following association

homozygous long - under-muscled or a "runt" heterozygous (both long and short) - with normal musculature homozygous short - muscle mass above normal

The specific long allele identified by the apphcants includes the following additional nucleotides

GG C C C C

These are nucleotides 63 to 68 in the follov.ing sequence which is of exon 1 in the long allele

It will be noted that these nucleotides are inserted at a point where the insertion disrupts two codons in frame, but without affecting the amino acids encoded at the point of insertion due to the degeneracy of the genetic code

In comparison, the sequence of exon 1 in the short allele is as follows

CGA CCA AAG AAA GAT GTG AAG AAA CCT GCT GCT GCC GCT GCC CCC GCC CCG GCC CCG GCC CCT GCC CCA GCA CCT GCA CCT GCC CCA CCC AAA GAA GAA AAG ATT GAC CTC TCT GCC ATC AAG

The net result of expression of the additional nucleotides is that the MLC- If protein contains two additional amino acids - alanine (A) and proline (P) The resultant amino acid sequence of exon 1 as expressed is as follows

PKKDVKKPAAAAAPAPAPAPAPAPAPAPAPPKEEKID LSAIK

(Long) A P K K D V K K P A A A A A P A P A P A P A P A P A P A P P K E E K I D L S A I K

(Short)

The presence or absence of the additional nucleotides in exon 1 can be detected using any conventional approach, including those described in Sambrook et al (1989) For example, detection can be through reference to indirect indicators such as mRNA or analysis of the amino acid sequence of the expressed protein (eg by the visualisation of expressed MLC- lf protein extracted from tissue by 2D gel electrophoresis or by immunological techniques using monoclonal antibodies (Harlow & Lane (1988))

If detection at an mRNA level is the selected approach, it is preferred that RNase protection assays be employed (Finkelstein et al (1990), Kinsler et al (1991)) This method involves the use of a labelled riboprobe which is complementary to the long allele The riboprobe and mRNA isolated from the test tissue are annealed and digested with the enzyme RNase A which detects mismatches in a duplex RNA structure, and cleaves at the site of the mismatch Following separation of the annealed duplex into single strands, this in turn results in a smaller RNA product, which can be detected by gel electrophoresis

It is however preferred that the presence or absence of additional nucleotides be detected directly in relation to the DNA itself Direct analysis can be through a number of conventional techniques and can use DNA extracted from the bovine at any stage of development (foetal or postnatal) For example, the entire sequence of exon 1 of MLC-1/ 3f can be obtained from a post-natal bovine and the length determined by direct sequencing or electrophoresis on a high resolution agarose or polyacrylamide gel

Another approach is restriction fragment length polymorphism (RFLP) This involves digestion of the DNA using restπction-endonucleases and analysis of the length of the resulting fragments, including by techniques such as pulsed field gel electrophoresis (PFGE) Yet another approach is to employ allele-specific ohgonucleotides (Conner et al (1983) or nucleic acid probes under high stringency Such probes will have a sequence which will hybridise only to the target sequence (the region containing the additional nucleotides Probes can be used in relation to DNA preparations transferred to suitable matrices for hybridisation such as nylon membranes (eg Hybond N Amersha International) by Southern, Northern or dot blotting The amount of bound probe can then be quantified through labelling the probes, for example with radioactive isotopes, or fluorescent dyes

The reverse of this procedure involving hybridisation of nucleic acid to the matrix is also possible In this fashion, the nucleic acid probe is bound to the matrix and used to capture, through a hybridisation protocol, genomic DNA containing the target sequence

Still another approach, which is presently most preferred, is to amplify exon 1 from genomic DNA Any standard amplification technique (such as the ligase chain reaction or methods based upon the use of Q-beta rephcase) can be employed, although the more widely available technique of polymerase chain reaction is preferred These methods are well known and widely practised (see, for example, Innis et al (1990) and Wu et al (1989))

The polymerase chain reaction or PCR is a widely used procedure in which a defined region of a DNA molecule can be amplified in vitro using a thermostable version of the enzyme DNA polymerase Two known sequences that define the regions to be amplified are selected and priming ohgonucleotides synthesised to correspond to these regions If the primers are located sufficiently close together in the same piece of DNA, the region between them will be amplified PCR consists of a number of cycles of amplification Each cycle begins with a denaturation step, typically at 94°C, in which the two stands of the template DNA molecule are separated The temperature is then dropped to a temperature at which the synthetic oligonucleotide primers can anneal to the template (typically 50-60°C) Through the high concentration of primers relative to template, the primers anneal to the template before template-template hybrids form The annealing temperature is chosen such that annealing occurs to the complementary regions of DNA within the template, and not to other regions of imperfect complementarity The temperature is then raised to 72°C at which the thermostable DNA polymerase can extend the bound primer, thus producing a strand of DNA complementary to the template In the earlier stages of the reaction, each cycle results in two-fold amplification of the template As each of these newly synthesised strands can function as a template, the increase in molecules corresponding to the defined region is exponential

For use in amplification in accordance with the present invention, PCR primers can be selected such that only one allele is amplified (ie by selecting one primer to bind via its 3' end to the nucleotides unique to the long allele - the technique of allele- specific PCR (Rano & Kidd ( 1989)) Alternatively, the primers can be selected so that both alleles are amplified, if present The amplified regions can then be sorted, eg by reference to the size of the amplified sequence using electrophoresis or bv direct sequencing if desired

Yet another method of quantifying the products of a differential PCR in which two PCR products are produced is the Taqman system (Perkin Elmer Cetus) In this system in addition to the two ohgonucleotide primers flanking the region to be amplified a third ohgonucleotide probe is used that binds to the amplified region The flanking primers are unlabelled while the probe carries two fluorescent labels On the 3' end of the probe is a reporter dye, the fluorescence of which is quenched by a separate fiuorophore attached to the 5' end of the probe During PCR this probe bmds to the product of DNA molecules As PCR proceeds, these products are used as templates duπng which the Taq DNA polymerase cleaves off the 5' quenching dye of the probe as it displaces it This removal of the quenching agent allows fluorescence from the reporter dye to be detected The degree of fluorescence is proportional to and therefore a measure of, the amount of PCR product produced In this way, the relative amounts of the separate PCR products produced can be measured

Still a further approach (although not preferred) is direct sequencing of the PCR products Such sequencing will determine which allele is present (ie the short allele, the long allele or both)

In summary, any approach to detecting the presence of additional nucleotides m exon 1 of the MLC- 1/ 3f gene can be employed, whether analysis be of the DNA itself. mRNA transcnbed from the DNA or the protein which is the ultimate expression product of the DNA The invention will now be illustrated with reference to the following experimental section, which is to be regarded as exemplary only rather than limiting.

EXPERIMENTAL

Section 1

The focus of the following experimental section is on a study performed in relation to bovines Three phenotype Bos taurus were used in the stud}' Normal muscled (NM), double muscled (DM) (generated from the Belgium Blue breed that displays "double muscling") and under muscled (NM*)

Physical data was first collected from bovine foetuses which were sacrificed at 260 days of foetal development, two weeks before the expected date of partuntion The data is summansed below for individual animals.

Animal Sex mass (gj CR length VH (I) V (L) Weight VM% weight VL% weight

Ho. (cm) Weight (£) (£)

NM33 M 29900 93.2 58.43 113.91 0.0019542 0.00381

NM*34 M 23100 87.2 54.78 83.23 0.0023714 0.003603

NM35 F 28400 91.6 62.84 104.29 0.0022 127 0.003672

NM36 M 33800 88.3 73.25 134.97 0.0021672 0.003993

NM37 F 29800 88.4 63.63 107. 15 0.0021352 0.003596 mean 30666 7 89 4 66 573 115 47 0 002172 0 003754 sem 17₂7 1 1 134 3 107 8 330 0 000067 0 000075

DM29 M 35700 93.8 60.36 189.77 0.0016908 0.005316

DM30 F 41 100 96.5 81.96 236.95 0.0019942 0.005765

DM31 F 37600 96.2 66.82 196.89 0.0017771 0.005236 mean 38133 3 95 5 69 713 207 87 0 001821 0 005439 sem 1581 5 0 854 6 401 14 685 0 000090 0 000165

VM = vastus mediahs VL = vastus laterahs Protein was then extracted from the vastus laterahs of individual animals and separated on the basis of charge by isoelectnc focusing (IEF) followed by separation on the basis of size using SDS-PAGE The protocol employed was as follows

Tissue samples were homogenised in lysis buffer (8 9M Urea 130mM DTT, 8mM PMSF, 2% Tnton X- 100, 2% ampholyte 3- 10) at a ratio of lmg tissue 3μL lysis buffer

0 5μL (analytical gel) or 20μL (sample preparation gel) was made up to 60μL with sample solution (8 9M Urea, 65 mM DTT, 2% Pharmalyte 3- 10, 0 5% Tnton X- 100) and loaded onto a [Pharmacia Biotech Immobihne Drystnp pH 3- 10 NL 180mm] that had been rehvdrated in rehvdration solution (8M Urea, 0 5% Tnton X- 100, 13mM DTT, 0 5% Pharmalyte 3- 10) for a first dimension separation bv isoelectnc focusing (IEF) The IEF was performed on Pharmacia Biotech Multiphor II electrophoresis unit at 20°C for (500Vx 5 Olh + 3500Vxl4 5h)

After the IEF step the gel stnp was immersed in equilibration solution 1 (0 1M HCL pH6 8, 6M Urea, 30% glycerol, 2% SDS, 50mM DTT) for 10 minutes at room temperatyre (RT) followed by immersion in equilibration solution 2 (0 1M HCL pH6 8, 6M Urea, 30% glycerol, 2% SDS, 250mM lodoacetamide) for 10 mmutes at RT

Equilibrated IEF gel stnps were then laid face down on a [Pharmacia Biotech ExecelGel XL SDS 12- 14%] or [Pharmacia Biotech ExcelGel SDS 8- 18%] and run on a Pharmacia Multiphor II electrophoresis unit for (20mA x 0 7h + 40mA x 2 75h) or 20mA x 0 5h + 50mA x 1 25h) respectively

Analytical gels were silver stained to visualise proteins

Step Solution Time

Fixation 10% glacial acetic acid 30 mm

40% methanol Sensitizing 30% methanol, 6 8% w/v sodium acetate,

5% glutarylaldehyde (25% w/v), 2% w/v 30 mm sodium thiosulphate

Washing distilled water 3x5 mm Silver Reaction 0 25% w/v silver nitrate, 0 2% formaldehyde 20 mm

(37% w/v) Washmg distilled water 2x1 mm

Developing 25% w/v sodium carbonate, 0 1% formaldehyde (37% w/v) Stoppmg 15% w/v EDTA-Na₂ 2H₂0 10 min

Washing distilled water 3x5 mm

Preserving 10% glycerol 20 mm

Sample preparation gels were stained using coomassie blue to visuahse protems of interest

Step Solution Time

Stain 4% w/v coomassie brilliant blue R250 5% 2 hours glacial acetic acid, 30% methanol Destain 20% methanol 3x45 min

The results are shown in Figures 1, 2 and 3 Figure 1 shows a portion of an gel showing two protein vanants obtained from an NM animal The vanants differ in terms of molecular weight

Figure 2 shows a portion of a gel showing one protein only obtained from a DM animal This was found to be the lower molecular weight protein of Figure 1

Figure 3 shows portions of partially focused gels of four NM animals (Figures 3A, 3B, 3C and 3D) displaying the expression of both protein isoforms, and one NM* animal (Figure 3E) showing one protein vaπant only (the higher molecular weight protein of Figure 1)

The protems of interest from the gels were identified as MLC- lf isoforms by sequencing digested fragments obtained by m-gel tryptic digest and purified by reverse phase HPLC The digest and purification protocols were as follows

In gel digestion Proteins of mterest were excised from sample preparation gels us g a sterile scapel For tryptic digest and HPLC peptide separation four protem "spots" were used Gel pieces containing protein of interest were washed twice in lOOμl of wash solution (50% acetonitπle, lOOmM tns-HCL pH 8 0) for 20 mmutes The wash solution was then discarded and the gel pieces aspirated completely The gel pieces were diced into 1 mm² sizes and then incubated in 30μl digest solution (100 mM tns-HCL pH 80, 10% acetonitπle, 02% reduce tnton, 4μg modified Trypsin) overnight at 37°C Peptide fragments were extracted twice by adding 100 μl extraction buffer (60% acetonitπle, 01% tπfluoroacetic acid, 002% reduced tnton) and mcubated, shaking, at 37°C for 20 mmutes

Digested peptide fragments were separated by reverse phase HPLC on a 5-65% solution B (80% acetonitnle) gradient usmg a C18 column Purified peptide fragments were N-terminal sequenced using an Apphed Biosystems Procise Protem Sequencer Sequence data was compared to Swiss Pit databases to identify proteins

The complete protein sequence of the two bovme MLC- If isoforms is as follows

APKKDVKKPAAAAAPAPAPAPAPAPAPAPAPPKEEKID LSAIKIEFSKQQ (Q/R) DEFKEAFLLFDRTGECKIYEDFV EGLRVEDKEGNGTCMGAELRHVLATLGEKMKEEEVE ALMAGQEDSNGCINYEAFVKHIMSN

Bold denotes inserted amino acids in long bovine MLC-lf

Bracketed amino acids are vaπations between Belgian Blue and Fnesian breeds

The high molecular weight MLC-lf isoform includes two additional amino acids, proline and alanine as shown in bold

To obtain the nucleotide sequence encoding the isoforms, cDNA of MLC-1/ 3f was obtained from animals N35, N34 and B30 by RT-PCR of extracted total RNA RNA was extracted usmg Qiagen RNeasy RNA extraction kit The RT-PCR protocol was as follows

RT 05ml reaction tube containing [4μl first strand buffer, 2ul lOmM dNTPs, 2μl 100 mM DTT, 4μl IU/μl DNasel, 0 lμl 40U/μl Rnase inhibitor, 59μl total RNA] was incubated at 37°C for 30 minutes, 80°C for 5 minutes and then placed on ice for 5 mmutes lμl 200U_/ ul MLC-RT and lμl lOOμM T18 primer was added to mixture and the tube incubated at 37°C for 1 hour The reaction mix was inactivated by being heated to 94°C for 3 mmutes

PCR 2μl of reaction mix or extracted genomic DNA was added to a 500 μl reaction tube contammg lOμl 10 [7 5mM Mg²⁺, 300mM Tns-S0₄ (pH 9 1 at RT), 90mM (NH4)Sθ4], 0 5μl lOμM forward pn er (5'-atggcaccaaagaaaga(t/c)gtgaag-3'j, 0 5μl lOμM reverse pnmer (S'-ttagacatgatgtgcttgacaaaag-S^*) for cDNA. amplification or (5 - cttgatggcagagagaggtcaaatctt) for exon 1 amplification), lul dNTPs, lμl Taq polymerase enzyme, 37μl H2O], 40μl paraffin overlay and subjected to the following thermocvcle lx [94°C 2 mmute], 35x [55°C 30s, 68°C 30s, 94°C 30s] lx [55°C 1 mm, 68°C 3 mm]

The resulting cDNA was cloned into pGEM plasmid vectors for sequencing (Promega)

Sequencing was performed using the fluorescence-activated chain terminating method and provided the following results

CGA CCA AAG AAA GAT GTG AAG AAA CCT GCT GCT GCC GCT GCC CCC GCC CCG GCC CCG GCC CCT GCC CCA GCA CCT GCA CCT GCC CCA

CCC AAA GAA GAA AAG ATT GAC CTC TCT GCC ATC AAG

Low MW MLC- 1/ 3f exon 1 cDNA

High MW MLC-1/ 3f exon 1 cDNA

Companson of the sequences revealed that the two additional amino acids result from the insertion of nucleic acids G G C C C C in exon 1 While the nucleic acids are not inserted in frame (they disrupt two codons at the point of insertion), tne amino acids at the point of insertion remain unchanged due to the degeneracy of the genetic code Conclusion

Collectively, the above results clearly show that DM animals only express lower molecular weight MLC- lf vanants, NM animals express both variants and NM* animals express only the higher molecular weight MLC- lf vanant.

Section 2

Semen samples from twelve bulls of high genetic ment were obtained from Livestock Improvement Corporation, Newstead, Hamilton, New Zealand Six samples were from sires (five Holstem-Fresian and one Jersey) of high genetic meπt with regard to milk production (of their female progeny) , whereas the other six samples were from sires of breeds maintained for beef production (Belgian Blue. Simmental, Angus, Hereford, Limosin and Piedmontese).

DNA extracted from the samples was amplified by PCR usmg the protocol and primers of Section 1 , with results as follows

Sires (Mlk Production) Homozygous short 6 Heterozygous

Homozygous long

Sires (Beef production) Homozygous short 5 Heterozygous 1 (Hereford)

Homozygous long

Conclusion

These results establish a clear linkage between the short allele and cattle possessing desirable traits The linkage between the short allele and milk production is particularly pronounced; the linkage with beef production only marginally less so

Section 3

Tissue samples from 95 beef cattle (of Hereford, Angus and Black and White breeds) processed at an abattoir were obtained, and the DNA extracted PCR amplification was performed on these samples using the protocol of Secuon 1 but with the following reverse pnmer

5' - cttttcttctttgggtggggc-3'

The results were as follows

Homozygous short 57

Heterozygous 37 Homozygous long 1

The allehc frequencies are 'Short' - 0 795 'Long' - 0 205

Conclusion These animals, while not of high genetic ment, show evidence of selection towards the short allele and desirable traits for beef cattle However, the long allele remains present in a significant number of animals

INDUSTRIAL APPLICATION

Thus, m accordance with the present invention, there is provided a method by which protein and/ or nucleic acid obtained from bovines can be screened for genotype The screening can either be in favour of a desirable genotype or to exclude an unfavourable genotype

In a specific application, the methods can be employed to screen nucleic acid obtained from bovines to select for a predisposition to increased muscle mass, an improved protein to fat deposition ratio, improved feed efficiency, improved growth rate and increased milk production

Kits for use in practising the preferred methods of the invention can also be provided Such kits will include nucleic acid probes or PCR primers, depending upon the detection method to be employed The probes/pnmers will be capable of distinguishing between the long and short alleles so that the genotype of the bovine from which the sample is extracted can be determined (ie homozygous short, heterozygous, or homozygous long) The nucleic acid probes will conveniently be ohgonucleotides of approximately 20-30 nucleotides in length They will also generally carry a revealing label which may be fluorescent, luminescent or radioactive Such labels are conventional, and are a matter of routine choice for the skilled artisan

The PCR primers will be a paired set, with one pnmer hybndising at its 3' end to the unique nucleotide sequence present in the long allele (G G C C C C) Again, the selection and synthesis of such primers will be routine to the skilled artisan once the target sequence is known

It will be appreciated by those persons skilled in the art that while the mvention is descnbed above with reference to specific embodiments, it is not limited thereto and that it also includes all apparent vanations, modifications and alternatives tnereof with the scope being limited only bv the appended claims

REFERENCES

Rushbrook, J I Wadewitz, A G , Ehzinga, M , Yao, T and Somes, R G (1988) Vanabmty in the Amino Terminus of Myosm Light Chain 1 Biochemistry 27 8953- 8958

Crockford, T , Johnston, I A and McAndrew, B J (1995) Functionally Significant Allehc Vanation in Myosin Light Chain Composition in a Tropical Cichlid The Journal of Experimental Biology 198 2501-2508

Sambrook, J et al , ( 1989) Molecular cloning A Laboratory Manual, 2^nd Ed (Cold Spring Harbour Laboratory, Cold Spring Harbour, New York)

Stepkowski, D The Role of the Skeletal Muscle Myosm Light Chain N-terminal Fragments (1995) FEBS Letters 374 6- 1 1

Conner, B J et al (1983) Proc Natl Acad Sci USA, 80 278-282

Fmkelstem, J et al , ( 1990) Genomis 7 167- 172

Kinsler K W et al ( 1991) Science 251 1366- 1370 Innis et al, ( 1990) PCR Protocols: A Guide to Methods and Apphcations (Academic Press, San Diego).

Harlow & Lane (1988). Antibodies: A Laboratory Manual (Cold Spring Harbour Laboratory, Cold Spring Harbour, New York).

Rano & Kidd (1989) Nucl. Acid. Res. 17: 8392

Wu et al, ( 1989) Genomics 4: 560-569.

Claims

1 method of selecting for a bovine having a desirable genotype, which method mcludes the step of determining the presence or absence of additional codon(s) in exon 1 in at least one allele of MLC-1/ 3/ for that bovine

2 A method of selecting for a bovine having a desirable genotype compnsing the steps of

(a) determining the presence or absence of additional codon(s) m exon

1 in at least one allele of MLC- 1/3/ for that bovine, and

3 method as claimed in claim 2 which involves selecting for said bovine where no additional codon(s) are present in exon 1 in both alleles of MLC-1/ 3/

4 A method of selecting for a bovine having a desirable genotype, which method includes the step of determining the presence or absence, in at least one allele of MLC-1/ 3f, of the following nucleotide sequence for exon 1

5 \ method as claimed in claim 4 wherein the presence of said nucleotide sequence in both alleles of MLC-1/ 3f is selected for

6 method as claimed in any one of claims 1 to 5 wherein the presence or absence of additional codon(s) is determined by analysis of nucleic acid obtained from said bovine

A method as claimed in claim 6 wherein the nucleic acid is DNA

\ method as claimed in claim 6 wherein the nucleic acid is mRNA 9 A method as claimed in any one of claims 1 to 5 wherein the presence or absence of additional codon(s) is determined by analysis of the amino acid sequence of MLC- If protein obtained from said bovine

10 A method as claimed in any preceding claim wherein the desirable genotype selected for is a genetic predisposition to large musculature

1 1 A method as claimed in any one of claims 1 to 9 wherein the desirable genotype selected for is a genetic predisposition towards low fat

12 A method as claimed in any one of claims 1 to 9 wherein the desirable genotype selected for is a genetic predisposition towards high feed efficiency

13 A method as claimed in any one of claims 1 to 9 wherein the desirable genotype selected for is a genetic predisposition towards a high growth rate

14 A method as claimed in any one of claims 1 to 9 wherein the desirable genotype is a genetic predisposition for high milk production

15 A method of selecting against a bovine having an undesirable genotype, said method including the step of determining the presence or absence of additional codon(s) in exon 1 m at least one allele of MLC-1/ 3fϊoτ that bovine

16 A method for selecting against a bovine having an undesirable genotype which compnses the steps of

(a) determining the presence or absence of additional codon(s) in exon

1 m at least one allele of MLC- 1/3/ for that bovme, and

17 A method as claimed in claim 15 or claim 16 wherein the presence or absence of additional codon(s) is determined by analysis of nucleic acid obtained from said bovine

18 A method as claimed in claim 17 wherein the nucleic acid is DNA 19 A method as claimed in claim 17 wherein the nucleic acid is mRNA

20 A method as claimed in claim 15 or claim 16 wherein the presence or absence of additional codon(s) is determined by analysis of the amino acid sequence of MLC- If protem obtained from said bovine

21 A method as claimed m any one of claims 1 to 3, 15 and 16 wherein said additional codon(s), upon expression, result in one or more additional amino acids being present in the amino acid sequence of MLC- If protein

22 A method as claimed in claim 21 wherein the additional codon(s), upon expression, result in the inclusion of two additional amino acids in the amino acid sequence of MLC- If protein

23 A method as claimed m claim 22 wherein said two additional amino acids are alanine (A) and proline (P)

24 A method as claimed in claim 23 wherein said two additional amino acids are located in the amino acid sequence of MLC-lf protein such that the sequence resulting from expression of exon 1 is as follows

APKKDVKKPAAAAAPAPAPAPAPAPAPAPAPPKEEKID LS AIK

25 A method as claimed in any one of claims 1 to 20 wherein the additional codon(s) have the following nucleotide sequence

GG C C C C

26 A method as claimed m claim 25 wherein said additional nucleotides are located between nucleotides 62 and 63 of the nucleic acid sequence of exon 1 of MLC- .2/3/ such that the sequence is as follows

GCA CCA AAG AAA GAC GTG AAG AAA CCT GCT GCT GCC GCT GCC CCC GCC CCG GCC CCG GCC CCG GCC CCT GCC CCA GCA CCT GCA CCT GCC CCΛ CCC AAA GAA GAA AAG ATT GAC CTC TCT GCC ATC AAG 27 A nucleic acid probe which hybndizes, under high stringency conditions, to the nucleotide sequence

but not to the nucleotide sequence

CGA CCA AAG AAA GAT GTG AAG AAA CCT GCT GCT GCC GCT GCC CCC GCC CCG GCC CCG GCC CCT GCC CCA GCA CCT GCA CCT GCC CCA CCC AAA GAA GAΛ AAG ATT GAC CTC TCT GCC ATC AAG

28 A nucleic acid probe which hybndizes under high stringency conditions to the nucleotide sequence

but not to the nucleotide sequence

29 A paired set of PCR amplification primers, one of said primers hybndizing, at its 3 or 5' end, to nucleotides 63-68 of the following sequence

GCA CCA AAG AAA GAC GTG AAG AAA CCT GCT GCT GCC GCT GCC CCC GCC CCG GCC CCG GCC CCG GCC CCT GCC CCA GCA CCT GCA CCT GCC CCA CCC AAA GAA GAAAAG ATT GAC CTC TCT GCC ATC AAG

30 A kit suitable for use in a method as claimed in any one of claims 1 to 26 which mcludes a nucleic acid probe as claimed in claim 27 or 28, or a set of PCR primers as claimed in claim 29

31. A kit as claimed in claim 30 which includes nucleic acid probes as claimed in both claims 27 and 28.