PUNO Y PLATERÍA EN EL CONTEXTO POLÍTICO, SOCIAL Y

Having established the genetic maps with respect to wst and Ra, it became apparent that D2Mit266 and Acra4 were the closest genetic markers to wst and were the only molecular markers available on the extreme end of distal MMU2. To obtain a distal flanking marker for both wst and Ra and provide a resource for obtaining expressed sequences, I initiated a ‘chromosome walking' strategy towards the telomere. Both these markers were used as probes in an attempt to isolate Yeast Artificial Chromosomes (YACs). It was anticipated that the three recombination events observed between wst, one of which was believed to be a true recombinant, would be instrumental in defining the direction of the walk. For clarity, the identification numbers of these three animals are w1, w3 and w84, the former two being the ‘dubious’ wst/wst homozygotes.

2.3.1 The isolation of genomic clones from the Acra4 gene region

The first objective was to obtain a specific set of hybridisation probes and sequence tagged sites (STSs), which define the Acra4 gene. The only hybridisation probe available for Acra4 was the rat cDNA which shares up to 57% overall sequence homology with other members of the nicotinic acetylcholine receptor subunit genes (Goldman et al., 1987). From a partial screening (about 150,000 clones) of a bacteriophage X mouse genomic library, eighteen clones (CDA1-18) were isolated. To distinguish which clones were specific to Acra4, PCR oligonucleotide primers were designed (by Dr Cathy Abbott) to correspond to the 5’ coding end of the rat cDNA. The expected PCR product size was 139 bp. The PCR product sizes of the C57BL/6J and Mus spretus parents were c. 218 and 214 bp respectively. I have followed the segregation of this locus in the BSB backcross and assigned it to distal MMU2 coincident with D2M/Y74,^r . 7"' Four bacteriophage X clones contained this locus, as shown by PCR analysis (data not shown). One of them, CDA4, was further subcloned in pBluescript and several clones partially sequenced. All the sequences obtained were checked for homology with other sequences in the EMBL database but none shared significant homology to any cloned sequences. PCR primers were designed for one of these STSs, G85/86, to amplify a region of 206 bp (fig.3.11; table 3.6). The alleles that segregate in the wasted stock and Mas spretus, at G85/86, showed length variation (fig 3.12). I tested the phenotypes of w1, w3 and w84 at this locus and found that w84 was homozygous, suggesting that a recombination event took place between the segregating RFLV, previously used to score the phenotype of the wasted animals for Acra4, and the locus

defined by G85/86. Animal w84 is heterozygous at D2Ucl29 as well so this recombination event took place between D2Ucl29 and G85/86, both loci being on the same clone. Therefore, additional STSs from clone CDA4 were analysed to confirm this scoring. The loci G85/86 and D2Ucl29 define a 9 kb region which is presented in figure 3.11. The phenotypes of animals w1, w3 and w84 across this region are shown in figure 3.12. Partial DNA sequencing and restriction site analysis revealed that this region contains the recognition sites for five restriction enzymes with (G+C)-rich recognition sites, suggesting the presence of a possible ‘CpG island'. Probes flanking these sites were obtained by PCR and used for a long range physical map (see later).

Figure 3.11 Partial physical map of a 9 kilobase region derived from clone CDA4. The oligonucleotide primers are denoted by the letter G. The orientation of this clone with respect to the centromere is based on the data presented in figure 3.12 and it is depicted in the 5’ to 3’ orientation. The two sequence tagged sites A1/2 are defined by the oligonucleotide pairs G19/20 and G85/86. The primers Gs/a/s define D2Ucl29. The sequence of the oligonucleotide Ga/s corresponds to the antisense strand of the rat Acra4 gene (transcript 4-1 Goldman ef a/., 1987). The physical distances were determined through the analysis of PCR products obtained from pair-wise primer combinations, on G.8%-1.5% agarose. The size of the ‘CpG island' was estimated to be approximately 500 bp. The recognition sites for the restriction enzymes Sst n, BssH n and Nar I occur twice in this region.

C8i G96 G66 TEL p = p r G«S CEN G#/i C8< G9S G20 G23 0.7 kb 3.2 kb 0.8 kb 4.3 kb X -X h o l, S - S s tl,S t - S s tI,E - E a g I,N - Not I, B - B u H II,N a - Nar I

Table 3.6 PCR primer sequences and PCR product sizes in clone CD A4

P rim e r Name Prim er sequence PCR p ro d u c t size (bp)

G66 5’-TGTGGGAGTCTGGCATACCA-3‘ 210® G67 5’-GTGACCTAGCTCTCTTGCCT-3‘ G85 5-CTCGAGCACCTTCTCCTTAG-3' 206 G86 5-AGCTAGGACCAAGGTCCATC-3' G95 5-AAGTTTCCTTGTCGTCCTGAGG-3' _450* G96 5’-TGACAGTTTACCGAGAAGGCAG-3’ G19 5’-TCAGTACCACCCAGGGTTCT-3’ 262 G20 5-TGAGCCAACACTTCCCCTTG-3' G23 5-ACTGGATGCAGACCTCAGAG-3' 200 G24 5-TGCTGGTTCTAGGTTGGCAA-3' Gs 5-GACGAGAAGAACCAGATGAT-3' 215® Ga/s 5’-ATGTCAGGCCTCCAGATGAG-3‘

The PCR conditions for the Gs-Ga/s, which define D2Ucl29, are given in table 2.1 (page 64). These conditions apply to all possible pair-wise combinations using 58°C annealing temperature. ‘e‘- estimated PCR product size.

F ig u re 3.12 Phenotypes of critical recombinant mice across clone CDA4. a. Silver-stained polyacrylamide gels used to determine the phenotypes of the three recombinant animals (w1, w3 and w84) in the wasted backcross at sequence tagged sites A1 (G85/86) and D2Ucl29 (fig 3.11). The bands indicated by arrows represent the segregating homoduplexes. Note that animal w84 is heterozygous (i.e. carries a M.spretus allele) at D2Ucl29, but homozygous (i.e. does not carry a M.spretus allele) at STS Ai (this animal is also homozygous at STS A2: data not shown). Animals w1 and w3 are included as controls for the Mus spretus allele. Abbreviations- ‘kb’- DNA molecular weight markers; ‘C3H’- C3H/HeH; 'Spr' - Mus spretus] ‘HRS’ - HRS/J; ‘10T - 101/H {wst arose on HRS/J and is maintained on 3H1 background; ‘HM‘ - homozygous, b. The chromosomal haplotype of the distal part of MMU2 of animal w84, given these data. The markers shown are those presented in section 3.1.2. The boxes are labeled w and s to indicate the alleles from the wasted stock and the Mus spretus parent.

(a)