A steroid/thyroid hormone receptor superfamily member in Drosophila melanogaster that shares extensive sequence similarity with a mammalian homologue

A gene in Drosophila melanogaster that maps cytologically to 2C1-3 on the distal portion of the X-chromosome encodes a member of the steroid/thyroid hormone receptor superfamily. The gene was isolated from an embryonic cDNA library using an oligonucleotide probe that specifies the consensus amino acid sequence in the DNA-binding domain of several human receptors. The conceptual amino acid sequence of 2C reveals at least four regions of homology that are shared with all identified vertebrate receptors. Region I includes the two cysteine-cysteine zinc fingers that comprise a DNA-binding domain which typifies all members of the superfamily. In addition, three regions (Regions II-IV) in the carboxy-terminal portion of the protein that encode the putative hormone-binding domain of the 2C gene product resemble similar sequences in vertebrate steroid/thyroid hormone receptors. The similarity suggests that this Drosophila receptor possesses many of the regulatory functions attributed to these regions in vertebrate counterparts. A portion of Region II also resembles part of the human c-jun oncoprotein's leucine zipper, which in turn, has been demonstrated to be the heterodimerization site between the jun and fos oncoproteins. The 2C receptor-like protein most resembles the mouse H2RII binding protein, a member of the superfamily which has been implicated in the regulation of major histocompatibility complex (MHC) class I gene expression. These two gene products are 83% identical in the DNA-binding domain and 50% identical in the putative hormone-binding domain, although no ligand has been identified for either protein. The high degree of similarity in the hormone-binding domain between the 2C protein and the H2RII binding protein outside regions II-IV suggests specific functional roles which are not shared by other members of the superfamily.     

The fidelity of DNA synthesis by yeast DNA polymerase zeta alone and with accessory proteins

DNA polymerase zeta (pol ζ) participates in several DNA transactions in eukaryotic cells that increase spontaneous and damage-induced mutagenesis. To better understand this central role in mutagenesis in vivo, here we report the fidelity of DNA synthesis in vitro by yeast pol ζ alone and with RFC, PCNA and RPA. Overall, the accessory proteins have little effect on the fidelity of pol ζ. Pol ζ is relatively accurate for single base insertion/deletion errors. However, the average base substitution fidelity of pol ζ is substantially lower than that of homologous B family pols α, δ and . Pol ζ is particularly error prone for substitutions in specific sequence contexts and generates multiple single base errors clustered in short patches at a rate that is unprecedented in comparison with other polymerases. The unique error specificity of pol ζ in vitro is consistent with Pol ζ-dependent mutagenic specificity reported in vivo. This fact, combined with the high rate of single base substitution errors and complex mutations observed here, indicates that pol ζ contributes to mutagenesis in vivo not only by extending mismatches made by other polymerases, but also by directly generating its own mismatches and then extending them.     

Fission yeast with DNA polymerase delta temperature-sensitive alleles exhibits cell division cycle phenotype.

DNA polymerases alpha and delta are essential enzymes believed to play critical roles in initiation and replication of chromosome DNA. In this study, we show that the genes for Schizosaccharomyces pombe (S.pombe) DNA polymerase alpha and delta (pol alpha+ and pol delta+) are essential for cell viability. Disruption of either the pol alpha+ or pol delta+ gene results in distinct terminal phenotypes. The S.pombe pol delta+ gene is able to complement the thermosensitive cdc2-2 allele of Saccharomyces cerevisiae (S.cerevisiae) at the restrictive temperature. By random mutagenesis in vitro, we generated three pol delta conditional lethal alleles. We replaced the wild type chromosomal copy of pol delta+ gene with the mutagenized sequence and characterized the thermosensitive alleles in vivo. All three thermosensitive mutants exhibit a typical cell division cycle (cdc) terminal phenotype similar to that of the disrupted pol delta+ gene. Flow cytometric analysis showed that at the nonpermissive temperature all three mutants were arrested in S phase of the cell cycle. The three S.pombe conditional pol delta alleles were recovered and sequenced. The mutations causing the thermosensitive phenotype are missense mutations. The altered amino acid residues are uniquely conserved among the known polymerase delta sequences.     

The fidelity of DNA synthesis by the catalytic subunit of yeast DNA polymerase alpha alone and with accessory proteins

The fidelity of DNA synthesis catalyzed by the 180-kDa catalytic subunit (p180) of DNA polymerase alpha from Saccharomyces cerevisiae has been determined. Despite the presence of a 3'----5' exonuclease activity (Brooke et al., 1991, J. Biol. Chem., 266, 3005-3015), its accuracy is similar to several exonuclease-deficient DNA polymerases and much lower than other DNA polymerases that have associated exonucleolytic proofreading activity. Average error rates are 1/9900 and 1/12,000, respectively, for single base-substitution and minus-one nucleotide frameshift errors; the polymerase generates deletions as well. Similar error rates are observed with reactions containing the 180-kDa subunit plus an 86-kDa subunit (p86), or with these two polypeptides plus two additional subunits (p58 and p49) comprising the DNA primase activity required for DNA replication. Finally, addition of yeast replication factor-A (RF-A), a protein preparation that stimulates DNA synthesis and has single-stranded DNA-binding activity, yields a polymerization reaction with 7 polypeptides required for replication, yet fidelity remains low relative to error rates for semiconservative replication. The data suggest that neither exonucleolytic proofreading activity, the beta subunit, the DNA primase subunits nor RF-A contributes substantially to base substitution or frameshift error discrimination by the DNA polymerase alpha catalytic subunit.     

RNase X2, a pistil-specific ribonuclease from Petunia inflata,shares sequence similarity with solanaceous S proteins

Petunia inflata, a species with gametophytic self-incompatibility, has previously been found to contain a large number of ribonucleases in the pistil. The best characterized of the pistil ribonucleases are the products of the S alleles, the S proteins, which are thought to be involved in self-incompatibility interactions. Here we report the characterization of a gene encoding another pistil ribonuclease of P. inflata, RNase X2. Degenerate oligonucleotides, synthesized based on the amino-terminal sequence of RNase X2, were used as probes to isolate cDNA clones, one of which was in turn used as a probe to isolate genomic clones containing the gene for RNase X2, rnx2. The deduced amino acid sequence of RNase X2 shows 42% to 71% identity to the 20 solanaceous S proteins reported so far, with the highest degree of similarity being to S₃ and S₆ proteins of Nicotiana alata. The cDNA sequence predicts a leader peptide of 22 amino acids, suggesting that RNase X2, like S proteins, is an extracellular ribonuclease. Also, similar to the S gene, rnx2 is expressed only in the pistil, and contains a single intron comparable in size and identical in location to that of the S gene. However, rnx2 is not linked to the S locus, and, in contrast to the highly polymorphic S gene, it is monomorphic. The possible biological function of RNase X2 is discussed.     

Drosophila fibronectin: a protein that shares properties similar to those of its mammalian homologue.

This is the first report on the existence in Drosophila of a protein with properties similar to those of vertebrate fibronectin that we shall refer to as Drosophila fibronectin. Rabbit antibodies against human plasma fibronectin have allowed the detection of this molecule in Drosophila haemolymph; common epitopes are shared by the two proteins. Drosophila fibronectin with a subunit mol. wt of approximately 230 kd is a glycoprotein which binds to denatured mammalian collagen. It is present throughout development and is as abundant in embryos as in larvae and adult flies. Drosophila fibronectin is differentially expressed during embryogenesis, a small amount being present before the blastoderm stage. Its concentration increases at gastrulation and reaches a steady-state value at the end of organogenesis. Drosophila fibronectin is predominantly detected by immunofluorescence on frozen sections of 16 h embryos in the extracellular spaces lying between the different tissues and organs. In mature third instar larvae, most of the staining is concentrated in fat body and imaginal discs, and the pattern strongly supports an extracellular localization of the protein. In addition, it is shown that Drosophila embryonic cells can functionally utilize vertebrate fibronectin for their spreading and differentiation. Finally, injection of antihuman plasma fibronectin antibodies in early embryos leads to the same phenotype as injection of Arg-Gly-Asp-containing peptides. This result suggests that one of the Arg-Gly-Asp-bearing protein(s) involved in gastrulation might be fibronectin.     

Fission yeast and a plant have functional homologues of the Sar1 and Sec12 proteins involved in ER to Golgi traffic in budding yeast.

Sec12p and Sar1p are required for the formation of transport vesicles generated from the endoplasmic reticulum (ER) in the yeast Saccharomyces cerevisiae. Sec12p is an ER type II membrane protein that mediates the membrane attachment of the GTP-binding Sar1 protein. The SAR1 gene is a multi-copy suppressor of a thermosensitive sec12 mutation. In an attempt to identify functional homologues of Sec12p and Sar1p from other eukaryotic organisms, we screened cDNA expression libraries derived from the fission yeast Schizosaccharomyces pombe and from the plant Arabidopsis thaliana for complementation of the sec12ts mutation. Four individual cDNAs were isolated, two of which encode the S. pombe and A. thaliana homologues of Sar1p. The three Sar1 proteins are 67% identical on average. The two other cDNAs encode type II membrane proteins which were designated Stl1p for the S. pombe protein and Stl2p for the A. thaliana protein (Stl stands for Sec12p-like). Both proteins have NH2-terminal cytoplasmic domains which resemble that of Sec12p: they are similar in size and present a significant degree of amino acid identity with the cytoplasmic domain of Sec12p. In contrast, the lumenal domains of Sec12p, Stl1p and Stl2p are very different in size and do not show any appreciable homology. That Stl1p and Stl2p are functional homologues of Sec12p was confirmed by showing that expression of either cloned gene complements a sec12 null mutation. Our results indicate that some of the mechanisms regulating vesicle formation at the ER are conserved not only in yeasts, but also in plants.     

Isolation of a yeast single-strand deoxyribonucleic acid binding protein that specifically stimulates yeast DNA polymerase I

We sought a protein from yeast that would bind more strongly to single-stranded DNA than to duplex DNA and would stimulate the activity of the major yeast DNA polymerase, but not polymerases from other organisms. We isolated a protein that binds about 200 times more strongly to single-stranded DNA than duplex DNA and stimulates yeast DNA polymerase I activity 4-5-fold. It inhibits synthesis catalyzed by calf thymus DNA polymerase alpha and has little effect on T4 DNA polymerase. This yeast protein, SSB-1, has a molecular weight of approximately 40 000. At apparent saturation there is one protein molecule bound per 40 nucleotides. Protein binding causes the single-stranded DNA molecule to assume a relatively extended conformation. It binds to single-stranded RNA as strongly as to DNA. SSB-1 increases the initial rate of polymerization catalyzed by yeast DNA polymerase I apparently by increasing the processivity of the enzyme. We estimate there are 7500-30 000 molecules of SSB-1 per yeast cell, enough to bind at least 400-1600 nucleotides per replication fork. Thus it is present in sufficient abundance to participate in DNA replication in vivo in the manner suggested by these in vitro experiments.     

Repair of plasmid and genomic DNA in a rad7 delta mutant of yeast.

Repair of UV-induced cyclobutane pyrimidine dimers (CPDs) was examined in a yeast plasmid of known chromatin structure and in genomic DNA in a radiation-sensitive deletion mutant of yeast, rad7 delta, and its isogenic wild-type strain. A whole plasmid repair assay revealed that only approximately 50% of the CPDs in plasmid DNA are repaired after 6 h in this mutant, compared with almost 90% repaired in wild-type. Using a site-specific repair assay on 44 individual CPD sites within the plasmid we found that repair in the rad7 delta mutant occurred primarily in the transcribed regions of each strand of the plasmid, however, the rate of repair at nearly all sites measured was less than in the wild-type. There was no apparent correlation between repair rate and nucleosome position. In addition, approximately 55% of the CPDs in genomic DNA of the mutant are repaired during the 6 h period, compared with > 80% in the wild-type.     

Cloning and characterization of the rad4 gene of Schizosaccharomyces pombe; a gene showing short regions of sequence similarity to the human XRCC1 gene.

The rad4.116 mutant of the fission yeast Schizosaccharomyces pombe is temperature-sensitive for growth, as well as being sensitive to the killing actions of both ultraviolet light and ionizing radiation. We have cloned the rad4 gene by complementation of the temperature sensitive phenotype of the rad4.116 mutant with a S. pombe gene bank. The rad4 gene fully complemented the UV sensitivity of the rad4.116 mutant. The gene is predicted to encode a protein of 579 amino acids with a basic tail, a possible zinc finger and a nuclear location signal. The amino terminal part of the predicted rad4 ORF contains two short regions of similarity to the C-terminal part of the human XRCC1 gene. Codon usage suggests that the gene is very poorly expressed, and this was confirmed by RNA studies. Gene disruption showed that the rad4 gene was essential for the mitotic growth of S. pombe.     

The sen1(+) gene of Schizosaccharomyces pombe, a homologue of budding yeast SEN1, encodes an RNA and DNA helicase.

Two polynucleotide-dependent ATPases, 95 and 181 kDa in size, have been purified to near homogeneity from cell-free extracts of Schizosaccharomyces pombe. Despite their size differences, their biochemical properties were strikingly similar. Both enzymes were capable of unwinding RNA and DNA duplexes in keeping with their ability to hydrolyze ATP in the presence of either ribo- or deoxyribopolynucleotide. In addition, they were capable of unwinding DNA/RNA or RNA/DNA hybrid duplexes and translocated in the 5' to 3' direction. These results strongly indicate that they are closely related to each other. Determination of the partial amino acid sequence of the 95-kDa enzyme revealed that it is encoded by the sen1(+)() gene, an S. pombe homologue of yeast SEN1, a protein essential for the processing of small nucleolar RNA, transfer RNA, and ribosomal RNA. The molecular weight of the S. pombe Sen1 protein (SpSen1p) predicted from the sen1(+)() open reading frame was 192.5 kDa, suggesting that the 181-kDa enzyme is likely to be a full-length protein, whereas the 95-kDa polypeptide has arisen by proteolysis. In accord with this possibility, polyclonal antibodies specific to the C-terminal region of sen1(+)() cross-reacted with both 95- and 181-kDa polypeptides. We discuss the biochemical activities associated with SpSen1p and their relevance to the apparently divergent functions ascribed to the yeast Sen1 protein in RNA metabolism.