At the beginning of this study, it was intended that the region proximal to the transcription start site would be screened first, as it contains a number of elements essential for stromelysin-lgene transcription, including the TATA box and the AP-1 site (see Figure 3.2). Thus, sequence changes in this region might be expected to have a significant effect on gene expression. Primers 3 and 4 (Figure 3.2) were designed to analyse this region, but PCRs persistently failed. Consequently, primers 9 and 11 were synthesized, and PCRs with this pair of primers were successful, generating a 501 bp fragment. The PCR products were then digested with Ava I and subjected to SSCP analysis as described earlier (section 3.2). No sequence variation was detected in this region of the gene in the 55 individuals (40 patients with coronary artery diseaes and 15 healthy individuals) tested.
PCRs using primers 7 and 10, or any combinations of primers seeking to amplify the entire 5’-flanking region (see Figure 3.2), also persistently failed, although amplification of the upstream sequence (-1259 to -826, using primers 2 and 5) or the proximal region (-237 to +264, using primers 9 and 11) alone were successful. The reason for the persistent failure to amplify this region remained unsolved until early 1995 when we were informed by Dr Makku Kurkinen that there were cloning errors in the Quinones et al (1989) stromelysin-1 5'-flanking sequence.
Because of the cloning artifact, it was uncertain, to begin with, whether the sequence was a part of the stromelysin-1 gene or whether it was an unrelated sequence. As described previously (see Introduction, section 1.4.3c), stromelysin-1 had been localized to chromosome 11. If this anonymous sequence was located at the stromelysin-1 gene locus, it would also be on this chromosome. Therefore, PCR analyses of a human-rodent somatic cell hybrid panel were carried out to determine whether this was the case. PCR primers 2 and 5 (see Figure 3.2) were used in this analyses, as they had worked successfully on human genomic DNA (see above). As shown in Figure 3.7a, a clear PCR product was seen using template DNA from a hybrid cell line containing human chromosome 11 (lane 5), and its size equated with
that of PCR product from human genomic DNA (lanes 1, 2, and 3). This PCR product was not detected using template DNAs from hybrid cell lines that did not contain human chromosome 11 (lanes 6, 7, 9 and 10). Similar experiments were carried out using another pair of primers, i.e. primers 9 and 11 (see Figure 3.2), which had also been found to amplify human genomic DNA, and the results were consistent with those described above (Figure 3.7.b). These data indicated that the Quinones' sequence was located on chromosome 11, as is the stromelysin-1 gene.
(A) (B)
1 2 3 4 5 6 7 8 9 10 ^ 1 2 3 4 5 6 7 8 9 10 Z
Figure 3.7 PCR analysis of a human-rodent somatic cell hybrid panel - Results indicating the Quinones' sequence is located on human chromosome 11
Lanes 1, 2, and 3: human gen om ic D N A ; lane 4: hamster D N A (a23); lane 5: hum an-ham ster hybrid D N A (J1C L 4) containing human ch rom osom es
11 ; lanes 6 and 7: hum an-hamster hybrid D N A (TW IN 19C5 and T W IN 19D 12, resp ectively), not containing hum an chrom osom e 11 (see Table 3.1 );
lane 8: rat D N A (RAG); lanes 9 and 10: human-rat hybrid D N A (M O G 2E5 and E D A G 3 R , resp ectively), not containing human chrom osom e 11 (see
Table 3 .1 ).
Panel (A): PC R s using primers 2 and 5 w hich generate a 43 3 bp fragment. PCR products detected only w hen u sin g human gen om ic D N A (lanes 1, 2, and 3) or hum an-hamster hybrid D N A containing human chrom osom e 11 (lane 5).
Panel (B): PC R s using primers 9 and 11 w hich generate a 501bp fragment. PC R products detected w hen using hum an gen om ic D N A (lanes 1, 2 , and 3) or hum an-ham ster hybrid D N A containing human ch rom osom e 11 (lane 5). Sm aller PC R bands seen in lanes 8 and 9 m ay result from sequence h om ology b etw een human and rat.
Table 3.1 Human chromosomal content in 5 human-rodent somatic cell hybrids
Adapted from Dolphin et al (1991)
Hybrid PCR product chromosome number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X J1CL4 + - - 4- - - TWIN19C5 - - - + + - - - + - - - + O + - - 4- 4- - + 4- 4- - TWIN19D12 - + - + + - + - -t- - - ■ + - + - + 4- 4- - 4- 4- 4- - MOG2E5 - + - + + + + + + + + - + + + + 4- + 4- - O 4- 4- 4- EDAG3R - + - + - + 0 0 0 + - + + + 4- 4- 4- 4- 4- - 4- O -
A 5 '-flanking fragment from -257 to +1 in the stromelysin-1 gene had been cloned previously by Sirum and Brinckerhoff (1989) and this sequence shared complete homology with that of the corresponding region in the Quinones et al (1989) clone. The consistency of results from two independent groups suggested that this fragment (-257 and 4-1) was likely to be in the correct location, i.e. immediately upstream from the transcriptional start site of the stromelysin-1 gene. The nature of the remainder (from -1304 to -258) of the fragment remained obscure.
In the original paper, the cloning strategy adopted by Quinones et al (1989) was as follows (Figure 3.8): The 1.3kbp stromelysin-1 promoter sequence was isolated by Sst I and partial Xba I digestion from a X genomic clone, and subcloned into a Bluescript plasmid before DNA sequence was determined. This 1.3kbp sequence contained an Xba I restriction site at position -480, raising the possibility that errors could have occurred during the subcloning procedure: The -1304 -480 fragment might not be adjacent to the -479 4-1 sequence in the original clone, but the two fragments could have become ligated after partial digestion with Xba I, creating a rearrangement. If this proved to be the case, the persistent failure in PCR amplification of fragment from upstream to downstream of the Xba site (-480) could be due to one of the following three possibilities: 1) in the human genome, the sequence numbered as -1304 to -480 by Quinones et al (1989) was distant from the stromelysin-1 gene and thus beyond the range of PCR; 2) in the human genome, this fragment was located 3' rather than 5' of the stromelysin-1 gene; or 3) in the human genome, this fragment was located in the 5'-flanking region of the stromelysin-1 gene but was in the reverse orientation. If the third possibility was correct, PCR using two primers both in the reverse direction, but one within the -479 to 4-1 region and the other within the -1304 to -480 sequence, should be able to amplify a fragment containing these two regions.
g e n o m ic c lo n e HAF 10
S s t ! S s t !
Xba I X ba I
Xba I Xba I
S s t I d ig e s tio n Partial Xba I
d ig e s tio n
S s t I ( -1304) Xba I (-480) +1 Xba I
I.S k b p fr a g m e n t in