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Science 222:740-748, 1983). Chromatography of the programmed extract over a biotin-celiulose affinity resin resulted in the selective, and virtually quantitative, retention of one of two stable preinitiation complexes, either VA-IIIC or VA-IIIC-IIIB, depending on the length of template incubation in the S100 extract. After washing the resin with 0.10 M and 0.25 M KCI to remove RNA polymerase HI and nonspecifically bound proteins, respectively, TFIIIC was eluted from the VA-IIIC complex by the addition of 1.5 M KCI. The VA-IIIC-IIIB complex exhibited a higher salt stability. Most of TFIIIB and some TFIIIC were released by the addition of 1.5 M KCI; however, the majority of TFIIIC activity was recovered only after a subsequent 3.0 M KCI elution. The specific activity of the TFIIIC in the 3.0 M KCI fraction was 770-fold higher than that in the S100 extract, while the protein content of the 1.5 and 3.0 M KCI fractions was reduced 7,500-and 100,000-fold, respectively.The elucidation of the molecular mechanisms underlying eucaryotic gene expression has been facilitated by the development of soluble cell extracts which direct the accurate transcription of purified DNA templates. Studies analyzing the transcription of class I, II, and III genes by RNA polymerases I, II, and III (pol III), respectively, have identified both specific DNA sequences and cellular factors as key elements of these processes (16). Our present study is focused on the development of rapid procedures for the isolation of those cellular factors involved in the transcription of class III genes, which include the tRNA, 5S RNA, and adenovirus VA RNA genes.Functional dissections of class III genes have identified intragenic sequences as essential elements for the initiation of transcription. The promoter of tRNA genes is split into two regions of about 10 nucleotides each (termed the A and B blocks), which are separated by 30 to 40 base pairs and are highly conserved in all tRNA genes (12, 17). The VA RNA gene promoter appears to share the same structure (11). The 5S RNA genes contain a 34-nucleotide internal promoter consisting of two separable but contiguous elements (4, 26); the first 11 nucleotides are homologous to the A block of tRNA genes, and the remainder-are specific to 5S genes (7).Chromatographic fractionation of crude cellular extracts has shown that in addition to pol III, tRNA and VA RNA gene transcription requires at least two separate components (IIIB and IIIC), whereas 5S RNA gene transcription requires these two together with another gene-specific factor (IIIA) (27, 28). Although factor IIIA, a 38,000-molecular-weight protein, has been purified to homogeneity from Xenopus oocytes (9), the purification and characterization of factors IIIB and IIIC has proven to be much more difficult, in large * Corresponding author. t Present address: Department of Microbiology, University of Missouri Medical School, Columbia, MO 65201. part owing to their relatively low abundance and instability during purification.Detailed analyses of gene-fact...

We analyzed the long control regions (LCRs) of seven human papillomavirus type 6b (HPV-6b) clones, which contained prototype HPV-6b sequences recloned into various plasmid vectors and propagated in different strains of Escherichia coli. Southern blot analysis and DNA sequencing demonstrated three different sequences, each distinct from the published prototype HPV-6b sequence. Two of the plasmids contained insertions of 24 and 94 base pairs (bp) and a 1-bp deletion. Four plasmids contained insertions of 24 and 58 bp and a deletion of 49 bp. One plasmid contained a single insertion of 77 bp. The 94-, 77-, and 58-bp insertions occurred at the same site and had 100% positional identity across their shared lengths. All changes were located in the purine-thymidine-rich region of the LCR (nucleotides 7292 to 7400). Two additional LCR sequences were detected by restriction analysis of two other HPV-6b clones. We conclude that the purine-thymidine-rich region of the LCR is a hot spot for recombination in E. coli and that the alterations are the result of recA-independent events. These results emphasize the need to rigorously prove that a cloned isolate is an authentic copy of the genomic DNA present in the original lesion. In addition, the data indicate that the HPV-6b LCR sequences employed in different laboratories may be different, even if their parental DNAs were identical. Finally, we discuss the need for caution in assigning biological significance to alterations in this region, in view of the limited data available on the true identity of the HPV-6b LCR.

Plasmid DNA containing the adenovirus type 2 genes for VA RNA was linearized at a site distal to the gene, end labeled with a biotin-nucleotide analog of TTP, and incubated with avidin to form an avidin-biotinylated DNA complex. HeLa cell S100 extracts containing crude RNA polymerase III and transcription factors (TFs) IIIB and IIIC were programmed with the avidin-biotin-VA DNA to allow stable complex formation (A.B. Lassar, P.L. Martin, and R.G. Roeder, Science 222:740-748, 1983). Chromatography of the programmed extract over a biotin-cellulose affinity resin resulted in the selective, and virtually quantitative, retention of one of two stable preinitiation complexes, either VA-IIIC or VA-IIIC-IIIB, depending on the length of template incubation in the S100 extract. After washing the resin with 0.10 M and 0.25 M KCl to remove RNA polymerase III and nonspecifically bound proteins, respectively, TFIIIC was eluted from the VA-IIIC complex by the addition of 1.5 M KCl. The VA-IIIC-IIIB complex exhibited a higher salt stability. Most of TFIIIB and some TFIIIC were released by the addition of 1.5 M KCl; however, the majority of TFIIIC activity was recovered only after a subsequent 3.0 M KCl elution. The specific activity of the TFIIIC in the 3.0 M KCl fraction was 770-fold higher than that in the S100 extract, while the protein content of the 1.5 and 3.0 M KCl fractions was reduced 7,500- and 100,000-fold, respectively.