Database Analysis Using The Isolated Junction Fragments

A database search using the FASTA program (Pearson and Lipman, 1988), via the HGMP resource centre, with TgVlO, Tg300 and wt300 sequences was perfonned. The search results revealed that the reverse complement of TgVlO has 59.5%

identity with the bovine beta-casein gene in a 269 nucleotide overlap and Tg300 and wt300 have 64.2% identity with the H. sapiens casK mRNA for kappa-casein in a

134 nucleotide overlap. 5.9 Conclusions

The database search with TgTlO, Tg300 and wt300 revealed a degree of homology with the bovine beta-casein gene and the human casK mRNA for kappa-casein, respectively. In 4.2.4, TgVlO {DSUcll) was shown to be located on mouse Chr 5,

1.9 ± 0.5cM distal to DSNdsl and 6 . 8 ± 1.0cM proximal to DSNdsô {Gus). The Chr 5 committee concensus map shows that the murine alpha- and beta-casein genes {Csna and Csnh) lie in this region. Csna and Csnb are placed 3cM and 4cM distal to DSNds2, respectively (Kozak et a l, 1996). The caseins are the major mammalian milk proteins encoded by a small gene family, which in cows and sheep consists of four members, p, %% and K, and in mice and rats five members, a, p, y, e and k. The spatial arrangement of the genes is largely conserved between cows and mice, bovine %!, p, and k caseins are homologous to murine a, P, £, and k caseins

respectively; cattle have no known y casein (Tomlinson et a l, 1996). Since TgTlO shows a degree of homology with bovine beta-casein and the concensus map places it in close proximity to murine beta-casein, the TgTlO sequence was aligned to the

Figure 21. Southern Analysis Of Fi Hybrid Progeny Carrying The Deletion And A Partial Map Of Mouse Chr 5 Showing The Known Extent Of The

Deletion, (a) Southern analysis of interspecific Fi hybrids between the laboratory- derived mutant stock and the wild-derived M. spretus stock. DNA samples were digested with Taql and sequentially hybridised with the TgVlO and MR310 probes. Left: When hybridised with TgVlO, the M. spretus allele (+^) is defined by a fragment size of 2.5kb, whereas the laboratory strain (C57BL/6) allele (4-^) is identified by a fragment of 6.2kb. Right: When hybridised with MR310, the M. spretus allele (+^) is defined by a fragment of 2.7kb, and the laboratory strain (C57BL/6) allele (+^) is defined by a fragment of 4.5kb. Control M. spretus DNA shows an additional RFLV compared with the M. spretus DNA used to generate the Fj hybrids (fragment sizes 2.9kb, 2.7kb and 2.1 kb). This can be attributed to

different sources of M. spretus animals, (b) Partial maps of mouse Chr 5 showing the known extent of the deletion. Upper: Map positions (cM) of loci on mouse Chr 5 taken from the Chr 5 committee concensus map (Kozak et a i, 1996). Lower: Map positions of loci on mouse Chr 5 presented in Figure 20. The solid line indicates the known extent of the deletion and includes the Ph and W loci but not Rw (Lyon et a i, 1984). The broken lines indicate the region of the proximal and distal breakpoints of the deletion. The proximal breakpoint has been defined as lying between Tec and D5M nl25 (Nagle et al., 1995). The position of the distal

breakpoint is relatively uncertain but is thought to be close to the alpha-casein (Csna) gene which is known to be outside the deletion (Geissler et al., 1988b).

Taql Taql

4 i

I

4 ^ + 1

I I 4 I-

1 1 " : ^

DNA sequence for the murine beta-casein gene and revealed 52.6% identity in a 344 nucleotide overlap.

The data in 5.6 showed that D5Ucll and Elsl map to a region of mouse Chr 5 close to the PhlRwlW loci. The Rw backcross analysis refined the position of these two markers distal to the PhlRwlW loci but proximal to Csnb (Bentley et a l, 1996). The current concensus genetic map from the mouse Chr 5 committee (Kozak et al., 1996) places Elsl in the same position as Csnb so this new mapping data defines the position of Elsl with respect to Csnb.

The data in 5.7 revealed that both D5Ucll and Elsl lie outside the W^^^ deletion. The proximal breakpoint of the W^^^ deletion has recently been defined as lying between Tec and D5M nl25 (Nagle et a l, 1995), the position of the distal breakpoint is still relatively uncertain but is thought to be close to the alpha-casein {Csna) gene which is known to be outside the W^^^ deletion (Figure 21b) (Geissler et a l, 1988b). DSUcll and Elsl have not been mapped with respect to Csna, but since like Csna they both lie proximal to Csnb and outside the W^^^ deletion (Geissler et a l, 1988b), the three loci must be closely linked. The mapping of DSUcll and Elsl to this region may help to define more precisely the distal breakpoint of the deletion.

Whilst these studies were in progress a number of blebbed {bl) animals were obtained and used to perform a test of allelism with the my^'^^lmy^’^^ animals. The

animals are the ‘ ‘insertional’ ’ mutant animals described in chapter 3 which were originally thought to have arisen as a result of an insertional mutation at or near the myelencephalic blebs (my) locus on mouse Chr 3. Subsequent analysis revealed that the ‘ ‘insertional’ ’ mutant phenotype was not associated with the (3- actin lacZ transgene insertion but the test of allelism with my and subsequent genetic mapping studies (described in 6.2) confirmed that a new allele of my had arisen, my^^\ Homozygous and heterozygous bl mice were mated with homozygous and heterozygous my^""^ mice; wild type progeny were seen in all litters and litter size was normal. The complementation of these two mutations indicates that bl and my^^^ are not allelic and provides strong evidence against the theory of non-allelic non­

complementation.

The positions of DSUcll and Elsl with respect to Csna, Csnb and other loci in this region will be further refined by the analysis of a backcross segregating the blebbed

(bl) mutation which lies on mouse Chr 5. A backcross segregating the blebbed {bl) mutation has been set up. Homozygous bUbl animals were crossed with Mus musculus castaneus mice to produce animals. These F| animals are now being backcrossed to bUbl animals to produce N2 backcross progeny. A positional cloning strategy, as utilised for the x castaneus backcross (described in 6.4), will be used to clone the bl mutation. The loci, DSUcll and E lsl, which were

positioned on mouse Chr 5 using backcrosses segregating and Rw mutations will be mapped using the bl backcross. This will be particularly interesting with regard to E lsl, as this has been proposed as a possible candidate gene for bl (Inglis and Lee,

CHAPTER SIX

GENETIC MAPPING OF mjy™

6.1 Introduction

As we were not able to clone the my locus using the insertional mutant, a different strategy was required. As described in 1.4.2, the identification of mutations purely by map or c h ro m o ^ e location is termed positional cloning. This is achieved by

constructing a fine genetic map of the region spanning the mutation and then using this to construct a physical map of the region. Further analysis of the physical map with SSLPs and RFLVs enables loci to be identified which produce no

recombination events between them and the mutant locus, thereby defining a ‘ ‘non­ recombinant region” ; a number of methods can be employed to select putative genes from this region and these will be discussed in 6.9.3. The genetic map can also be used to evaluate putative candidate genes.

As stated above, the first step in positional cloning is to construct a genetic map and one method of doing this is by analysing the progeny of an intersubspecific backcross segregating the mutation using SSLPs and RFLVs. The data generated in this

analysis can then be used to produce a genetic map by utilising the Map Manager programme.

6.1.1 Map Manager

Map Manager is a programme which assists in the analysis of data generated in animal genetic mapping experiments using intercrosses, backcrosses and recombinant inbred (RI) strains (Manly, 1993). Datais entered into the programme for all the progeny in a particular cross for each locus analysed, including the mutant locus of interest. This data indicates whether a particular progeny has inherited a paternal or maternal allele for a particular locus thus producing a “ strain distribution pattern” which in our case represents the pattern of genotypes in the progeny of a backcross. Manly (1993) describes the analysis functions of Map Manager as addressing “ the three basic problems of genetic analysis: the establishment of linkage, the ordering of groups of linked loci and the estimation of distance between linked loci” . A

summary of how these problems are addressed by Map Manager is described below and details of the statistical analyses are described in Appendix III-A.