Programs were developed under the UNIX operating system and are available by anonymous ftp from ftp://beagle.colorado.edu/pub/consensus.

A database of 209 Drosophila introns was extracted from Genbank (release number 64.0) and examined by a number of methods in order to characterize features that might serve as signals for messenger RNA splicing. A tight distribution of sizes was observed: while the smallest introns in the database are 51 nucleotides, more than half are less than 80 nucleotides in length, and most of these have lengths in the range of 59 -67 nucleotides. Drosophila splice sites found in large and small introns differ in only minor ways from each other and from those found in vertebrate introns. However, larger introns have greater pyrimidine-richness in the region between 11 and 21 nucleotides upstream of 3' splice sites. The Drosophila branchpoint consensus matrix resembles C T A A T (in which branch formation occurs at the underlined A), and differs from the corresponding mammalian signal in the absence of G at the position immediately preceding the branchpoint. The distribution of occurrences of this sequence suggests a minimum distance between 5' splice sites and branchpoints of about 38 nucleotides, and a minimum distance between 3' splice sites and branchpoints of 15 nucleotides. The methods we have used detect no information in exon sequences other than in the few nucleotides immediately adjacent to the splice sites. However, Drosophila resembles many other species in that there is a discontinuity in A+ T content between exons and introns, which are A + T rich.

The immunoglobulin heavy chain switch from synthesis of IgM to IgG, IgA or IgE is mediated by a DNA recombination event. Recombination occurs within switch regions, 2-10 kb segments of DNA that lie upstream of heavy chain constant region genes. A compilation of DNA sequences at more than 150 recombination sites within heavy chain switch regions is presented. Switch recombination does not appear to occur by homologous recombination. An extensive search for a recognition motif failed to find such a sequence, implying that switch recombination is not a site-specific event. A model for switch recombination that involves illegitimate priming of one switch region on another, followed by error-prone DNA synthesis, is proposed.

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Comparative sequence analysis addresses the problem of RNA folding and RNA structural diversity, and is responsible for determining the folding of many RNA molecules, including 5S, 16S, and 23S rRNAs, tRNA, RNAse P RNA, and Group I and II introns. Initially this method was utilized to fold these sequences into their secondary structures. More recently, this method has revealed numerous tertiary correlations, elucidating novel RNA structural motifs, several of which have been experimentally tested and verified, substantiating the general application of this approach. As successful as the comparative methods have been in elucidating higher-order structure, it is clear that additional structure constraints remain to be found. Deciphering such constraints requires more sensitive and rigorous protocols, in addition to RNA sequence datasets that contain additional phylogenetic diversity and an overall increase in the number of sequences. Various RNA databases, including the tRNA and rRNA sequence datasets, continue to grow in number as well as diversity. Described herein is the development of more rigorous comparative analysis protocols. Our initial development and applications on different RNA datasets have been very encouraging. Such analyses on tRNA, 16S and 23S rRNA are substantiating previously proposed associations and are now beginning to reveal additional constraints on these molecules. A subset of these involve several positions that correlate simultaneously with one another, implying units larger than a basepair can be under a phylogenetic constraint.

Mutants of simian virus 40 (SV40) lacking parts of the 72- and 21-base-pair repeat regions were made deficient in large T antigen by recombination with dlA 4000, a mutant containing a frameshift deletion near the amino terminus of the T antigen genes. These double mutants were transfected into COS cells, and the amounts of replicated viral DNA were measured at various times thereafter. It was found that deletion of either the 72- or 21-base-pair repeat region did not significantly reduce the accumulation of viral DNA. However, cells transfected with mutants lacking both of these promoter elements accumulated 100-fold less viral DNA than cells transfected with wild-type SV40. This indicates that the 72- and 21-base-pair repeat regions are each sufficient for supplying a function required for efficient replication of SV40 DNA. In addition, the ability of either of these regions to support efficient replication was gradually reduced as the number of promoter elements within each was decreased. Since the 72- and 21-base-pair repeat regions bidirectionally induce transcription, our results indicate that bidirectional promoter elements play a role in the replication of viral DNA. However, fewer of these elements are required for efficient replication than for efficient transcription.

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