Regulators responsible for the pervasive, nonsex-specific alternative pre-mRNA splicing characteristic of metazoans are almost entirely unknown or uncertain. We describe here a novel family of splicing regulators present throughout metazoans. Specifically, we analyze two nematode (Caenorhabditis elegans) genes. One, CeSWAP, is a cognate of the suppressor-of-white-apricot (DmSWAP) splicing regulator from the arthropod Drosophila. Our results define the ancient, conserved SWAP protein family whose members share a colinearly arrayed series of novel sequence motifs. Further, we describe evidence that the CeSWAP protein autoregulates its levels by feedback control of splicing of its own pre-mRNA analogously to the DmSWAP protein and as expected of a splicing regulator. The second nematode gene, Ceprp21, encodes an abundant nuclear cognate of the constitutive yeast splicing protein, prp21, on the basis of several lines of evidence. Our analysis defines prp21 as a second novel, ancient protein family. One of the motifs conserved in prp21 proteins--designated surp--is shared with SWAP proteins. Several lines of evidence indicate that both new families of surp-containing proteins act at the same (or very similar) step in early prespliceosome assembly. We discuss implications of our results for regulated metazoan pre-mRNA splicing.

Plasmids pNovl and pNovls, coding for resistance and sensitivity to novobiocin, respectively, were readily lost from wild-type Haemophilus influenzae but retained in a strain lacking an inducible defective prophage. The plasmid loss could be partly or wholly eliminated by a low-copy-number mutation in the plasmid or by the presence of certain antibiotic resistance markers in the host chromosome. Release of both phage HP1cl, measured by plaque assay, and defective phage, measured by electron microscopy, was increased when the plasmids were present. The frequency of recombination between pNovl and the chromosome, causing the plasmid to be converted to pNovls, could under some circumstances be decreased from the normal 60 to 70% to below 10% by the presence of a kanamycin resistance marker in the chromosome. This suggested that a gene product coded for by the plasmid, the expression of which was affected by the kanamycin resistance marker, was responsible for the high recombination frequency. Evidence was obtained from in vitro experiments that the gene product was a gyrase.

The rec-l gene of Haemophilus influenzae was cloned into a shuttle vector that replicates in Escherichia coli as well as in H. influenzae. The plasmid, called pRecl, complemented the defects of a rec-l mutant in repair of UV damage, transformation, and ability of prophage to be induced by UV radiation. Although UV resistance and recombination were caused by pRecl in E. coli recA mutants, UV induction of lambda and UV mutagenesis were not. We suggest that the ability of the H. influenzae Rec-1 protein to cause cleavage of repressors but not the recombinase function differs from that of the E. coli RecA protein.Mutants carrying mutations in the rec-J gene of Haemophilus influenzae have the following properties: (i) transformation frequencies are -106 those of the wild type (20, 36), (ii) phage recombination is not measurable in the mutants (6,8,37), (iii) there is no UV induction of prophage (8), (iv) the cells are sensitive to UV radiation (20, 38) because of their inability to carry out postreplication repair (22, 41), (v) UV-induced degradation of DNA is somewhat greater than in the wild type, and there is some degradation without UV (20-22), (vi) the cells are sensitive to X rays and mitomycin C (38, 42), (vii) following transformation but not conjugal transfer, the establishment of plasmids containing a cloned segment of H. influenzae DNA is profoundly depressed (3, 39), although without the insert the plasmid is established as well as in the wild type (30), and (viii) plasmid-to-chromosome gene transfer, chromosome-to-plasmid gene transfer, and plasmid-to-plasmid recombination are greatly depressed (1, 4, 39).Evidence was previously presented that the recombination defect and the defect in repair of UV damage result from a single mutation (20,37). Subsequent to the isolation of the first strain containing a rec-l mutation, called DB117 (38), two other such mutations were obtained that conferred the same phenotypic properties and were mapped to similar but slightly different loci on the chromosome (20, 21).In some respects the H. influenzae rec-J mutants resemble some Escherichia coli recA strains (9). In particular, they are both sensitive to UV and mitomycin C, lack prophage inducibility by UV, exhibit enhanced degradation of DNA induced by UV, and are defective in recombination. However, there are quantitative differences. The UV sensitivity of at least some recA strains is much greater relative to the wild-type E. coli (15) than is the rec-J mutant relative to its isogenic strain. The difference between recA mutants and wild type in UV-induced DNA degradation is considerably greater than in rec-J mutants (16,20,48 In order to investigate further these differences between the rec-J and recA mutations and wild-type alleles, we needed to determine how the H. influenzae and E. coli genes interact. Previous work (34) suggested that the wild-type rec-J gene could function on a piece of transforming DNA without being integrated into the chromosome. Thus, we concluded that this gene could also readily function on...

We characterize spliceosome assembly in Drosophila embryonic nuclear extracts. Further, we show that these extracts contain high levels of a 5' to 3' exoribonuclease activity allowing rapid, convenient protection mapping of 5' splice site and branchpoint sequences. We use this assay to show, for the first time, that a regulated arthropod intron uses a remote branchpoint strikingly similar in structure to those observed previously in regulated vertebrate introns. These results provide new evidence that both regulated and constitutive splicing are similar in detail in vertebrates and arthropods indicating that the powerful genetic systems for analysis of splicing regulation in Drosophila are likely to be directly informative for regulated splicing throughout metazoa. In addition, we report formation of a novel class of intron-dependent complexes. Behavior of these complexes indicates that they represent a mutually exclusive, kinetically competing pathway with spliceosome assembly. We propose that this competition represents the basis for a kinetic proofreading mechanism enhancing fidelity of intron recognition. We also discuss possible implications of this model for regulated splicing.