Aspectos sobresalientes en la celebración del contrato

L m e ta l (Liuet a l, 1997) had carried out mutational analysis of Leu 1 in 180 of their B-cell CLL patients with heterozygous loss of 13ql4 and found no abnormalities. It was, however, relevant to analyse a small cohort of our patients with heterozygous deletion of 13ql4.3 as the sequence of our clone had some minor differences to Leu 1.

According to Knudson’s 'two hit' theory of tumour suppressor genes (Knudson, 1971), both copies of the gene must be inactivated within a cell for malignant potential to occur. Six patients known to have heterozygous deletion of 13ql4.3 were chosen for analysis. The patients chosen were numbers 4, 14, 48, 54,62 and patient K. The data showing the full genomic status of these patients are described in chapter 7. Subsequent to this work, patients 4 and 62 were shown not to have heterozygous deletion of 13ql4.3. Patients 14,48 and 54 were shown to have heterozygous deletion of all markers tested at 13ql4.3. Patient K had heterozygous deletion of D13S25 and D13S319. The data for patient K are not shown as they were from work done by Dr Jabbar from our group.

cDNA was made from the total RNA isolated from malignant, purified B lymphocytes in the selected patients. The PCR reaction described in section 5.9. to amplify across the open reading frame of cDNA clone 2:2 was repeated using patient cDNA. Both splice versions of the clone were seen in all patients when the RT PCR products were analysed by electrophoresis. This was an indication that the 3' splice site of exon 1 and the 5' splice site of exon 2 were intact. The existence of the two splice versions also indicated that both splice sites of the extra 53bp exon were present within the RNA of these patients. The entire PCR product for each patient was purified, digested and cloned into a Bluescript plasmid (KS+) vector. Despite all samples having been monocyte and T cell depleted (see chapter 7), at least 3 clones were sequenced for each patient to minimise the chance of sequencing cDNA amplified from normal contaminating cells, and to account for error that may arise from PCR amplification. When all the

clones were analysed for each patient using DNAStar software, no base changes were consistently seen for any patient. The sequence of cDNA clone 2:2 seen in all the patients was the same as our original cDNA clone and differed from the published sequence of Leu 1 at base pair 443 of cDNA clone 2:2 as already described. It was concluded that the postulated open reading frame of cDNA clone 2:2 was not mutated in any of these patients. It should be noted that we had not analysed the full non coding 5' and 3' regions in these patients. This data was in keeping with the data from Liu et al (Liu et a i, 1997) and, more recently. Rondeau et al (Rondeau et ai, 1999).

5.11. Conclusion

This chapter has shown how a novel gene was isolated by cDNA library screening with a probe initially designed from a longer, more complex cDNA. Both cDNAs had different 3' regions with poly adénylation sites and poly A tails. Only the first 5' exon was shared between the 2 transcripts. The subsequent 53 base pairs of cDNA clone 1 (exon la) also existed in the cDNA clone 2:2 as an alternative splice version. This sequence was not contained within the clone isolated from cDNA library screening, but was only seen when normal lymphocyte cDNA was amplified by RT PGR using primers designed to amplify across the two exons. The EST homologous to clone 2:2 identified through the BLAST N database also contained this additional sequence.

cDNA clone 2:2 was 960bp in length and the TOObp probe containing exon 1 of this clone identified a message on Northern blot analysis of 1.1 kb (corresponding with the Northern blot data published by Liu et a/.(Liu et al.,

1997)). The original clone isolated by cDNA library screening consisted of 2 exons which mapped to 13ql4.3, centromeric of D13S319. Both of these exons were shown to lie within the deleted area in our patients with homozygous deletion of the markers D13S319 andD13S25 as described. The cDNA existed as two splice forms. The smaller version had a postulated open reading frame encoding for a 78 amino acid protein. The larger version would give rise to a very short open reading frame of 44 amino acids. No mutations could be found within the open reading frame of this gene on the retained allele in any of the four patients with heterozygous deletion of 13ql4.3 that were tested. Although no mutations were detected, this gene remains a candidate tumour suppressor gene from I3ql4.3 because expression of the retained allele could be reduced or absent due to

mutations in other sections of the gene such as the promoter region, which has yet to be identified (discussed further in chapter 8), or the 3' and 5' untranslated regions.

In view of the data available at this stage of the study, we also analysed clone 14:2 and 19:3 (chapter 4) which were found to be 100% homologous to cDNA clone 2:2. Clones 3,4 and 5, isolated from the peripheral leucocyte library (Gibco) discussed above, were contained within cDNA clone 2:2, starting at position 40.

The relationship between cDNA clone 1 and cDNA clone 2:2 remained to be clarified. It was possible that cDNA clone 1 was a hybrid clone, formed during the construction of the cDNA library by abnormal fusion of part of cDNA clone 2:2 with a further transcript from 13ql4.3, comprising the 3' part of the sequence. However, the RT-PCR reaction using primers F2 and B7 from cDNA clone 1 successfully amplified the expected product, linking these two sequences (chapter 4).

Another explanation for the relationship between cDNA clones 1 and 2 was that the first 5’ exon of cDNA clone 2:2 was part of a second transcript, due to alternative splicing. It had already been demonstrated that this exon could be alternatively spliced either to exon 2 of cDNA 2:2 or to the third 53bp exon, exon la, which was part of the sequence of cDNA clone 1. Alternative splicing o f RNA transcripts is a common, normal phenomenon. There are 3 main forms of alternative splice versions seen in RNA processing (Lewin, 1994);

1. The first form of alternative splicing occurs when the 5' and 3' exons remain the same but the number of exons within the message may change; exon 'skipping' (figure 5.11.A). All variants may be functional and translated into a protein, or only some spliced forms may be translated. In some genes, the exon that is 'spliced in' contains a stop codon causing premature termination of the protein e.g. P elements of D. melanogaster show a tissue specific splicing pattern causing a longer protein to be translated in germline cells but not in somatic cells due to the splicing out of a termination codon. In the case of cDNA clone 2:2, the 2 alternative splice versions that have been demonstrated to occur would fit into this category. The shorter splice variant has a postulated open reading frame which is disrupted when the third exon is spliced in.

2. The second form of alternative splicing occurs when the 5' part of the gene remains constant, but the 3' part is completely different (figure 5.11.B). An example of this type of alternative splicing is seen in sex specific exons being

Figure 5.11; The different forms of altermative splicing which may generate a variety of protein products from an individual gene.

Figure A: Type 1 The 5' and 3’ exons remain the same but the exons in the middle can vary. 5 ' exon

□

The 5 ’ region remains constant but the 3’ part of the gene is completely different.

3 'e x o n

5 ' exon Figure C: Type 3

The 5’ region of the gene varies but the 3’ region remains constant.

/ \

3 ' exon

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spliced to the 3' region of the dsx mRNA in D. melanogaster. If cDNA clones 1 and 2:2 do share the same first two 5' exons, then their existence could be explained by this form of alternative splicing.

3. The third form of alternative splicing is similar to that in example 2, except that the 3' part of the gene remains constant and the 5' part changes. The

large T/t antigens of SV40 are generated by connecting a varying 5' site to a constant 3' site (figure 5.ll.C).

Despite the attractiveness of this theory, some of our expression data remained to be clarified. The message identified by TA 6.35 was 3.5kb. cDNA clone 1 was 1.7kb in length. If the 5' exon of clone 1 was shared with clone 2:2 then the model of splicing adopted would suggest that there were no further exons upstream. That would leave 1.8kb of message unaccounted for.

cDNA clone 2:2 appeared to be a genuine gene fi'om the area of deletion. We had identified it from more than one library and it had been published by others. cDNA clone 1 had only been isolated from one cDNA library even though 3 other libraries had been extensively screened with probes containing the TA 6.35 sequence, including a second peripheral leucocyte library. Therefore, it was decided to go back to the original putative exon, TA 6.35 to try and identify the transcript that it identified on Northern blot by using RACE PCR and further cDNA library screening. This work will be discussed in chapter 6.

Chapter 6. Identification of a novel coding