HIV-1 Evolution and Disease Progression

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The published 5' splice site sequence of intron 2 of human CACNL1 Al, the fibroblast voltage-gated Ltype calcium channel, precisely matches the AT-AC consensus, whereas the reported sequences of two other calcium channels, CACNL1 A2 and CACNL1 A3, have GTATCC (rather than ATATCC) at the corresponding 5' splice site; however, all three calcium channel introns reportedly end with the conventional AG 3' splice site [N. M. Soldatov, Genomics 22, 77 (1994); Y. Yamada et al., ibid. 27, 312 (1995); K. Hogan, R. G. Gregg, P. A. Powers, ibid. 31, 392 (1996)]. The sodium and calcium channels are thought to derive from a common ancestral gene: they have considerable nucleotide and amino acid sequence homology, and the unusual intron interrupts a homologous position of the coding sequence in all five genes. Unless there are errors in some of the reported sequences, it will be interesting to determine whether the calcium channel transcripts are processed via the major pathway, the AT-AC pathway, or a hybrid pathway. 8. A portion of the human SCN4A gene was amplified by PCR from human total genomic DNA (Promega) with primers containing restriction sites and matching exons 2 and 3, to generate a fragment comprising nt 886 to 1229 (numbering according to GenBank accession number L04216). This fragment was digested with Hind IlIl and Xba and subcloned into the corresponding sites of pSP64 (Promega) to generate the pSP64-SCN4A plasmid. For construction of pSP64-SCN4AM, a different downstream PCR primer containing the mutations and an Eco RI site was used to amplify a mutant fragment from the cloned wild-type template, which was then subcloned as a Hind III-Eco RI fragment in pSP64. All constructs were confirmed by sequence analysis. pSP64-SCN4A and pSP64-SCN4AM were linearized with Xba or Eco RI, respectively, for use as templates for in vitro transcription with SP6 RNA polymerase. The transcripts contain short extensions at both ends, derived from the vector. 9. Nuclear extract preparation and conditions for in vitro transcription and for 3-globin splicing were as described [A. Mayeda and A. R. Krainer, Cell 68, 365 (1992)]. The SCN4A in vitro splicing reaction was optimized by varying individual parameters. The optimum condition for SCN4A splicing was 60% (v/v) nuclear extract [giving final concentrations of 12% (v/v) glycerol, 12 mM Hepes-K+ (pH 8.0), 60 mM KCI, 0.6 mM dithiothreitol, and 0.3 mM EDTA[ plus an additional 32 mM Hepes-K+ (pH 7.3), 3.5 mM MgCI2, 0.5 mM ATP, 20 mM creatine phosphate, 2.6% (w/v) polyvinyl alcohol, and 1.6 nM SCN4A pre-mRNA, incubated at 300C for 6 hours. No splicing was detectable when ATP or MgCI2 was omitted. RNA was recovered and analyzed on 4.5% denaturing polyacrylamide gels, followed by autoradiography. Splicing efficiency, defined as the molar ratio mRNA/(pre-mRNA + mRNA), was estimated by phosphor image analysis (Fujix, BAS2000). 10. Q. Wu and A. R. Krainer, data not shown. 11. To sequence the authentic and cryptic spliced mRNAs across the spliced junctions, we amplified each gel-purified RNA by RT-PCR with exon 2 and exon 3 primers (TCATCGTACTCAACAAGG and TACTCCACATTCTTGGAC). The amplified fragment, subcloned into PCR2.1 (Invitrogen), was sequenced with T7 Sequenase 2.0 (USB). 12. P. J. Grabowski, R. A. Padgett, P. A. Sharp, Cell 37, 415 (1984); B. Ruskin, A. R. Krainer, T. Maniatis, M. R. Green, ibid. 38, 317 (1984). 13. W. -Y. Tarn and J. A. Steitz, ibid. 84, 801 (1996). 14. A. Kramer, W. Keller, B. Appel, R. Luhrmann, ibid. 38, 299 (1984); A. R. Krainer and T. Maniatis, ibid. 42, 725 (1985); D. L. Black, B. Chabot, J. A. Steitz, ibid., p. 737; S. M. Berget and B. L. 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