A. 84% similarity in a 117 nucleotide overlap with chicken L7a (accession no. D14522)
SURF Bam B 120 CTTGGCGGTCTTGATGATCTCGGTGAAGTTGTCCATGA Chicken L7a 468 CTTAGCGGTCTTAATGATCTCGGTAAAGTTGTCCATAA SURF Bam B 82 TGGACTTAATGTCGTCCTTCAGCCGCTTGTTGTAGGAC Chicken L7a 506 TGGATTTGACGTCATCCTTAAGGCGCTTGTTGTAGGAC SURF Bam B 43 TGCAGCAGCGTCTCCTTGCTCTGGGGCAGTGCTCTCTG Chicken L7a 545 TGCAGCAGGGTCTCTTTACTCTGCGGCAGAACCCGCTG SURF Bam B 2 CTG 4
Chicken L7a 582 CTG 584
B. 100% identity in a 81 nucleotide overlap with human L7a (accession no. X52138)
SURF Bam B 120 CTTGGCGGTCTTGATGATCTCGGTGAAGTTGTCCATGA Human L7a 398 CTTGGCGGTCTTGATGATCTCGGTGAAGTTGTCCATGa' SURF Bam B 82 TGGACTTAATGTCGTCCTTCAGCCGCTTGTTGTAGGAC Human L7a 436 TGGACTTAATGTCGTCCTTCAGCCGCTTGTTGTAGGAC
TGCAG 39 TGCAG 479 SURF Bam B 120 Human L7a 398 SURF Bam B 82 Human L7a 436 SURF Bam B 43 Human L7a 475
Figure 3.41
O rgan isation o f the hum an and m ouse Surfeit gene clu ster
A. H um an Surfeit locus
CpG 8kb CpG
SURF-5 SURF-3 SURF-1 SURF-2 SURF-4
B. Mouse Surfeit locus
CpG 8kbCpG 6kb CpG 18kb CpG
u L J U l_ l
70 70 15-73 133
bp bp bp bp
Surf-6 Surf-5 Surf-3 Surf-1 Surf-2 Surf-4
Taken from Yon et al. (1993). Arrow heads show the direction of transcription, dotted lines indicate the uncertainty of extent and position of the SURF-1, SURF-2 and SURF- 4 genes. Putative CpG islands are shown as black boxes with the distances between in kilobase pairs.
3' end. As SURF Bam B was the product which was positioned the most 3' on SURF-3, it would be the most 5' on SURF-5. SURF Bam A and SURF Pst D begin at approximately the same position but the former is 189bp larger in size. Therefore it is suggested that the next complete exon amplified is SURF Pst D. Between the end of SURF Pst B and the start of SURF Pst D within SURF Bam A, there is 66bp sequence which may represent the third exon. The region from the beginning of SURF Pst B to the end of SURF Bam A could represent the fourth exon amplified. These results suggest the minimum number of exons within SURF-5 is six.
Figure 3.42
Possible organ isation o f SU R F Bam A, SU RF Bam B, SU R F Pst B and SU R F Pst D w ithin the hum an Surfeit gene cluster
SURF Pst B SURF Pst D
3' -5' 3' — 5'
SURF Bam A SURF Bam B
4 3 2 1
3' 5' 3'— 5'
5' flanking sequence coding region SURF-3
SURF-5
The direction of transcription for SURF-3 is shown by an arrow, the presumed direction for SURF-5 is shown by a dotted arrow. The boxes represent products cloned and the numbers inside indicate the different possible exons which have been amplified. The shaded box represents HIV-1 tat intron sequence.
Although SURF Bam C was derived from the same cosmid (255 A6) as the products with strong similarity to ribosomal protein L7a, it showed strong similarity to human L21 (96% on lOlbp, p value e-30). This is also a ribosomal gene but it has not been localised to the Surfeit gene cluster. SURF Bam C also had strong similarity to clone P94_70 (p value e-36) which was an amplified exon isolated by Church et al. (1994) and was most probably identical. In addition, there was strong similarity to a cDNA clone (p value e-33) from the Human Genome Mapping Project Resource Centre (HGMP-RC) (M-02-H01, human foetal organ mixture tissue 3' end). These results suggest that it could be a true exon. However it could also be a processed pseudogene which is localised between SURF-5 and SURF-6 as the distance between these genes has not been published. Other possibilities include it being derived from SURF-6 as no sequence information is available for this gene. Alternatively it could be derived from the untranslated leader sequence of SURF-5 or SURF-6 or from an aberrant splicing event within the intronic sequences.
The remaining two products from the SURF contig were deduced to be artefacts as they contained either entirely HIV-1 sequence (SURF Pst C) or pSPL3 vector sequence (SURF Pst A).
3.3,2.1.b. Contig D9S10
Six different sized products were cloned from contig D9S10. Two products (D9S10 Bam C and D9S10 Bam D) showed strong similarity to human VAV mRNA (accession number X I6316) and provided further confirmation of a VAV-like gene (VAV2) on human chromosome 9q34. D9S10 Bam C had identical sequence to exon e which was
amplified from cosmid 17BE using vector pSPLl. Figure 3.22 showed the similarity between exon e and the mouse and human VAV sequence. D9S10 Bam D was found to overlap the human VAV mRNA immediately upstream of this sequence. Therefore it was assumed that the intronic sequence between these two exons contained a BamYH
restriction enzyme site which resulted in them being cloned into pSPL3 on separate genomic fragments. Sequence data for a VAV2 cDNA was provided by Dr. D. Kwiatkowski prior to publication (Henske et ah, 1995) and D9S10 Bam C was localised to base pairs 455-557, the same position as exon e. D9S10 Bam C was localised immediately upstream at basepairs 386-454
D9S10 Pst A and D9S10 Pst B showed similarity to human sequences (see table 3.9) and D9S10 Bam A showed similarity to the mouse otxl homeobox gene. D9S10 Bam B was deduced to be an artefact as it was composed of entirely HIV-1 sequence. The 116bp product was produced by activation of vector cryptic splice donor and acceptor sites. Figure 3.43 shows this sequence with the flanking vector sequence which acts as splice junctions.