To analyse the 3’ end of the BKB] receptor cDNA, 3’RACE was performed. RACE cDNA was synthesised from control and IE-ip-treated (4 hour) JTC-19 cells. Products were analysed by Southern blotting and hybridisation with a primer located within the PCR products. PCR products were cloned into pGEM-T and DNA was prepared from six recombinants and sequenced. Two of the clones appeared to be PCR artifacts. Three of the clones showed the same 3’ end as identified previously (Figure 3.8). These corresponded to a coding exon of 1244 bp and contain the poly(A) addition site (CATAAA). The remaining clone contained an additional 372 bp of sequence which was contiguous with the genomic sequence. Sequence analysis revealed the presence of a poly(A) addition site (ACTAAA) within this region located 374 bp downstream of the first identified poly(A) site. Assuming the poly(A) tails are of equivalent length, utilisation of the downstream poly(A) addition site would generate mRNA transcripts 372 bp longer than those generated using the upstream first identified poly(A) addition
site. However, alternative mRNA transcripts exhibiting this size difference were not detected by Northern blot analysis. The functional significance of these alternative 3’ UTRs thus remains to be determined.
The 1.6 kb transcript corresponds to the predicted BKBi receptor mRNA size, however, the origin of the 4 and 8 kb transcripts could not be explained. RACE studies and primer extension analysis showed no evidence for the large transcripts. These large, hybridising bands were present after washing the blots to high stringency (data not shown) suggesting that they are due to transcription of the BKB] receptor gene. Therefore, transcription must be occurring either further upstream, due to alternative TISs, or proceeding further downstream, due to read-through at the 3’ end of the gene. To elucidate the nature of the 4 and 8 kb transcripts, probes were synthesised spanning the 5’ and 3’ flanking regions of the BKB] receptor gene and used in Northern blot studies. BKBi receptor mRNA levels were analysed from JTC-19 cells treated with IL- IP for 2 hours. Using a 594 bp probe located -1941 to -2535 bp, hybridisation products were not detected (data not shown). Figure 3.9B shows the Northern blot analysis using a 450 bp probe spanning the genomic DNA starting at the sequence located 6 bp downstream of the most 3’ poly(A) addition site. Two mRNA transcripts of 4 and 8 kb were detected using this probe. To confirm the existence of all three transcripts the same blot was stripped and reprobed with probe A, the canonical rat BKBi receptor probe (Figure 3.9A). Thus, these findings suggest that the large transcripts are not generated by differential 5’ processing but are produced by 3’ read- through and inefficient termination of the transcript.
3.2.6 Comparison of the rat and human BKBi receptor gene structure
Whilst this work was carried out, the human BKBi receptor gene structure was reported (Bachvarov et al, 1996, Yang and Polgar., 1996). In comparison with the rat, the human BKBi receptor gene contains 3 exons of 92, 119 and 1086 bp separated by introns of approximately 7000 bp and 900 bp (Figure 3.11 A). Consequently, comparative Southern blot analysis of restriction digests from rat genomic DNA and cosmid RB1 DNA were carried out to eliminate the possibility of a deletion within the cosmid clone. Restriction digests were separated by electrophoresis on a 1% agarose gel and transferred to nylon membranes by Southern blotting. Hybridisation with probe A showed the presence of equivalent cosmid and genomic Bam HI and Eco RI fragments of 2.2 kb and 5.2 kb, respectively (Figure 3.10). A map corresponding to the rat BKBi receptor genomic structure is illustrated in Figure 3.II A.
To elucidate the nature of the differences in gene structure between the rat and human BKBi receptor genes, the non-coding exon sequences were aligned (Figure 3.1 IB). Alignment of the rat and human BKBi receptor non-coding exon sequences revealed 64.7% identity between exon 1 of the rat with exon 1 of the human. In contrast, alignment of exon I of the rat with exon 2 of the human shows 35.2% identity. These data suggests that exon 1 of the rat BKB] receptor gene is equivalent to exon I of the human BKBi receptor gene and one can further speculate that the human exon 2 arose by duplication and divergent evolution of the primordial precursor of exon I.
Figure 3.1 BKBi receptor amino acid alignment.
Predicted ORFs from the rat (this study, Jones et al, 1999; Ni et ai, 1998b), mouse (Pesquero et al, 1996; Hess et al, 1996), rabbit (MacNeil et al, 1995) and human (Menke et al, 1994; Jones et al, 1999) were aligned using the GCG PILEUP program. Predicted transmembrane domains are underlined. Potential sites of N-linked glycosylation are shown by an asterisk. The conserved cysteine residues of the second and third extracellular loops ,which may form a disulphide bond, are joined by the dotted line.
M u r B l R a b B l H um B l R a t B l M u r B l R a b B l H um B l M A S.Q A SLK L QPSNQSQQAP P N IT S C E G A P EAWDLLCRVL M A S.Q G PL E L QPSNQSQLAP PNATSCSGAP DAWDLLHRLL MASSWPPLEL Q SSN Q SQ LFP QNATACDNAP EAWDLLHRVL
P G F V IT V C F F GLLGNLLVLS FFLLPWRRWW . . . QQRRQRL P T F I I A I F T L GLLGNSFVLS V F L L A R R ... RL P T F I I S I C F F GLLGNLFVLL V F L L P R R ... QL 1 00 T IA E IY L A N L AASDLVFVLG LPFW AENIGN RFNWPFGTDL T IA E IY L A N L AASDLVFVLG LPFWAENVGN RFNWPFGSDL SV AEIYLA NL AASDLVFVLG LPFWAENVRN QFDWPFGAAL NVAEIYLANL AASDLVFVLG LPFWAENIWN QFNWPFGALL_
TM2
TM l
C R W S G V IK A N L F V S I F L W A ISQ D R Y R LL VYPMTSWGYR C R W S G V IK A N L F I S I F L W A ISQ D R Y R LL VYPMTSWGNR CRIV N G V IK A N L F I S I F L W A ISQ D R Y SV L VHPMASRRGR CR V IN G V IK A N L F I S I F L W A IS Q DRYRVL VHPMASRRQQ
TM3
2 0 0 * I
R a t B l RRRQAQATCL LIWVAGGLLS IP T F L L R S V K W PD L N V SA C ILLFPHEAW H FARMVELNVL G F L L P V T A II F F N Y H IL A S L M u r B l RRRQAQVTCL LIWVAGGLLS TPT FLLR SV K W P D L N IS A C ILLFPHEAW H FVRMVELNVL G F L L P L A A IL Y F N F H IL A S L R a b B l RRRQAQATCA LIW LAGGLLS TPTFV LR SV R AVPELNVSAC ILLLPHEAW H WLRMVELNLL G F L L P L A A IL F F N C H IL A S L H um B l RRRQARVTCV L IW W G G L L S I P T F L L R S I Q AVPDLNITAC ILLLPHEAW H F A R IV E L N IL G F L L P L A A IV FFN Y H IL A S L
oo TM4 TM5
3 0 0
R a t B l RGQKEASRTR CGGPKGSKTT G L IL T L V A S F LVCWCPYHFF AFLDFLVQVR V IQD CSW K EI TDLGLQLANF FA FVN SCLNP M u r B l RGQKEASRTR CGGPKDSKTM G L IL T L V A S F LVCWAPYHFF AFLDFLVQVR VIQDCFWKEL TDLGLQLANF FA FVN SCLNP R a b B l RRRGERVPSR CGGPRDSKST A L IL T L V A S F LVCWAPYHFF AFLECLWQVH AIGGCFW EEF TDLGLQLSNF SA FVN SCLNP H um B l RTREEV SRTR CGGRKDSKTT A L IL T L W A F LVCWAPYHFF A FLEFLFQVQ AVRGCFWEDF ID LG LQLA NF FA FT N S SL N P
TM6 TM7
R a t B l LIY V FA G RLL K T R V L G TL... M u r B l L IY V FA G R LF K T R V L G TL... R a b B l V IY V FV G RLF RTKVWELCQQ C SPR SL A PV S SSRRKEMLWG FWRN. H um B l V IY V FV G RLF RTKVWELYKQ C T P K S L A P IS S SH R K E IF Q L FWRN.