A fragment of the yeast DNA repair protein Rad4 confers toxicity to E. coli and is required for its interaction with Rad7 protein

The RAD4 gene of Saccharomyces cerevisiae is required for the incision of damaged DNA during nucleotide excision repair. Plasmids carrying the wild-type RAD4 gene cannot be propagated in Escherichia coli. In this study, a rad4 mutant that can be grown in E. coli was isolated. This rad4 allele is deleted of a large positively charged segment of the RAD4 coding region which is toxic to E. coli when expressed alone. The deletion mutant retains its ability to interact with Rad23 protein but not with Rad7 protein and is defective in nucleotide excision repair. The smallest Rad4 fragment that is toxic to E. coli consists of 336 amino acids with a calculated pI = 9.99.     

Cloning, expression and characterization of thymidylate synthase from Crypto coccus neoformans

The thymidylate synthase (TS)-encoding gene from Cryptococcus neoformans (Cn) has been isolated from cDNA and genomic libraries. The 1127-bp gene contains three introns and a 951-bp open reading frame encoding a 35844-Da protein. The cDNA clones lack 324 bp of the 5' coding region of the gene. The complete coding sequence was assembled as an expression cassette in pUC19 using parts of the coding sequence from the cDNA and genomic DNA and completing the sequence using synthetic DNA. Production of active TS from Cn (CnTS) was first demonstrated by complementation of a thymine(Thy)-requiring Escherichia coli strain. The expression cassette was subsequently subcloned into the T7 polymerase vector pET15-b. In this construct, CnTS is produced as approximately 10% of the total soluble protein in E. coli. Homogeneous enzyme was obtained at a 36% yield after consecutive chromatography on DEAE-cellulose, Q-Sepharose, phenyl-Sepharose and Affi-Gel Blue. Steady-state kinetic analysis showed that the K_m values for dUMP and CH₂H₄-folate were 2.7 ± 0.5 μM and 38.2 ± 2.5 μM, respectively, and the K_cat was 5.1 s⁻¹. The enzyme was stable upon storage at −80°C in Tris-HCl pH 7.4 and thiol.     

Genomic organization of mouse Dishevelled genes

We report the isolation and characterization of two new genomic loci corresponding to the mouse Dishevelled (Dvl) genes Dvl2 and Dvl3. The Dvl genes are homologs of the Drosophila dsh segment polarity gene, and are involved in the Wnt/wingless signal transduction pathway. Dvl2 and Dvl3 genomic clones were isolated from a mouse 129 strain λFIXII genomic library and have identical exon/intron organization to Dvll. All three Dishevelled genes span 15 exons and 14 introns and have a number of conserved splice junction sites.     

Cloning and characterisation of the Schizosaccharomyces pombe rad8 gene, a member of the SNF2 helicase family.

The Schizosaccharomyces pombe rad8 mutant is sensitive to both UV and gamma irradiation. We have cloned the rad8 gene by complementation of the UV sensitivity of a rad8.190 mutant strain. The gene comprises an open reading frame of 3.4 kb which does not contain any introns and is capable of encoding a 1133 amino acid protein of 129 kDa. Deletion of the gene indicates that it is not essential for cell viability. Recognisable motifs are present for a nuclear localisation signal, a RING finger and helicase domains. The predicted protein is a member of the SNF2 subfamily of proteins and shows particular homology to the Saccharomyces cerevisiae RAD5 protein. Double mutant analysis demonstrated that the rad8 mutant is not epistatic to mutants in the excision repair pathway (rad13) or checkpoint pathway (rad9). Analysis of radiation sensitivity though the cell cycle indicates that, unlike most other rad mutants, rad8 is most sensitive to irradiation during the G1/S period.     

RAD51 homologues in Xenopus laevis: two distinct genes are highly expressed in ovary and testis

The RAD51 gene is a eukaryotic counterpart of the Escherichia coli recA gene which is involved in genetic recombination. Two distinct Xenopus laevis RAD51 cDNA clones (XRAD51.1 and XRAD51.2) were isolated from an oocyte cDNA library using the human RAD51 cDNA (HsRAD51) as a probe. Sequence analysis revealed that 98.2% of the amino-acid residues were identical between XRAD51.1 and XRAD51.2, and that both were 95% identical to HsRAD51. Both of the XRAD51 genes were expressed at a higher level in ovary and testis than in other somatic tissues, suggesting their involvement in meiotic recombination. The expression of XRAD51.1 was about eightfold in excess of that of XRAD51.2 in all of the tissues examined. Analysis of the rates of synonymous substitution in the coding sequences of the two XRAD51 suggests that these two genes diverged about 50 million years ago. The structural similarities of the XRAD51 proteins to RecA in E. coli and Rad51 in yeasts or vertebrates are discussed.     

The Aspergillus niger carbon catabolite repressor encoding gene, creA

In order to undertake a comparative analysis of carbon catabolite repression in two Aspergillus species, the creA gene has been isolated from A. niger by cross hybridization, using the cloned A. nidulans gene. The A. niger gene has been shown to be functional in A. nidulans by heterologous complementation of the creA204 mutation of A. nidulans. Overall, the genes show 90% sequence similarity (82% identity) at the amino acid (aa) level. There were some striking similarities between the aa sequences encoded by the two fungal creA genes and two genes involved in carbon catabolite repression in Saccharomyces cerevisiae. The zinc-finger regions showed 96% similarity (84% identity) with the zinc-finger region of the MIG1 gene of S. cerevisiae. The CREA protein contains a stretch of 42 aa that is identical in A. niger and A. nidulans, and these show 81% similarity (33% identity) with a region of the S. cerevisiae RGR1 gene.     

Chromosomal localization, gene structure and transcription pattern of the ORFX gene, a homologue of the MHC-linked RING3 gene

We have mapped the human ORFX gene to chromosome 9q34 and determined its complete gene structure. Comparison with RING3, the human MHC-linked homologue on 6p21.3, shows the two gene structures to be highly conserved but with an approximate threefold expansion in the ORFX introns. RING3 and ORFX are found to be ubiquitously expressed in human adult and foetal tissues. Evidence suggests that the two genes may have arisen from an ancient duplication in a common ancestral chromosome.     

Structural organisation of the rat genes encoding liver- and heart-type of cytochrome c oxidase subunit VIa and a pseudogene related to the COXVIa-L cDNA

To study the tissue-specific expression of the heart(H)- and liver(L)-type of rat cytochrome-c oxidase subunit VIa (rCOXVIa), we have screened and sequenced the genes for the two isoforms. Both genes contain three exons and two introns, spanning 880 bp (rCOXVIa-H) and 3089 bp (rCOXVIa-L), respectively. In both genes, exon I codes for the whole leader sequence comprising 12 (rCOXVIa-H) or 26 (rCOXVIa-L) amino acids and for 12 (rCOXVIa-H) or 10 (rCOXVIa-L) amino acids of the corresponding mature protein, while the remaining amino acids for the mature proteins are encoded by exons II and III. The 5′ region of the genes lack both TATA and CAAT boxes, but show a high G+C content in the early 5′-upstream region. We have identified in upstream regions and in the introns of both genes several putative binding sites associated with respiratory function, muscle gene activation and housekeeping function. In rCOXVIa-H, we identified a CCAC/Myo-D motif, known to be required for muscle-specific expression of the human myoglobin-encoding gene, which is not present in rCOXVIa-L. In addition, we have analyzed a pseudogene, showing 84% homology to the COXVIa-L cDNA sequence.     

Opioid-binding cell adhesion molecule (OBCAM)-related clones from a rat brain cDNA library.

The AKin10 gene from Arabidopsis thaliana encoding a putative Ser/Thr protein kinase (PK) has been isolated and characterized. The AKin10-encoding gene is located on a genomic 5.4-kb BamHI fragment and contains ten introns, one being located in the 5' untranslated region. The deduced amino acid sequence of AKin10 is 65% identical over the catalytic domain to the yeast PK (SNF1). SNF1 is essential for the derepression of many glucose-repressible genes, including Suc2 which encodes invertase. Southern blot hybridization experiments suggested the presence of one copy of the gene per haploid genome of A. thaliana. Northern hybridization experiments indicated that this gene is expressed in roots, shoots and leaves. AKin10 may play an important role in a signal transduction cascade regulating gene expression and carbohydrate metabolism in higher plants.     

The rad16 gene of Schizosaccharomyces pombe: a homolog of the RAD1 gene of Saccharomyces cerevisiae.

The rad10, rad16, rad20, and swi9 mutants of the fission yeast Schizosaccharomyces pombe, isolated by their radiation sensitivity or abnormal mating-type switching, have been shown previously to be allelic. We have cloned DNA correcting the UV sensitivity or mating-type switching phenotype of these mutants and shown that the correcting DNA is encompassed in a single open reading frame. The gene, which we will refer to as rad16, is approximately 3 kb in length, contains seven introns, and encodes a protein of 892 amino acids. It is not essential for viability of S. pombe. The predicted protein is the homolog of the Saccharomyces cerevisiae RAD1 protein, which is involved in an early step in excision-repair of UV damage from DNA. The approximately 30% sequence identity between the predicted proteins from the two yeasts is distributed throughout the protein. Two-hybrid experiments indicate a strong protein-protein interaction between the products of the rad16 and swi10 genes of S. pombe, which mirrors that reported for RAD1 and RAD10 in S. cerevisiae. We have identified the mutations in the four alleles of rad16. They mapped to the N-terminal (rad10), central (rad20), and C-terminal (rad16 and swi9) regions. The rad10 and rad20 mutations are in the splice donor sequences of introns 2 and 4, respectively. The plasmid correcting the UV sensitivity of the rad20 mutation was missing the sequence corresponding to the 335 N-terminal amino acids of the predicted protein. Neither smaller nor larger truncations were, however, able to correct its UV sensitivity.     

Ester production in E. coli and C. acetobutylicum

Genetic engineering has improved the product yield of a variety of compounds by overexpressing, inactivating, or introducing new genes in microbial systems. The production of flavor-enhancing ester compounds is an emerging area of heterologous gene expression for desired product yield in Escherichia coli. Isoamyl acetate, butyl acetate, ethyl acetate, and butyl butyrate are reported here to be produced by expressing Saccharomyces cerevisiae genes ATF1 or ATF2 and the strawberry gene SAAT in E. coli when the appropriate substrates are provided. Increasing the concentration of alcohol added to the reaction generally resulted in increased ester production. ATF1 expression was found to produce more isoamyl acetate and butyl acetate than ATF2 expression or SAAT expression in the strains and culture conditions examined. Additionally, SAAT expression resulted in greater isoamyl acetate and butyl acetate production than ATF2 expression. Butyl butyrate is produced by cell-free extracts of E. coli harboring SAAT but not ATF1 or ATF2.     

Cloning and characterization of femA and femB from Staphylococcus epidermidis

A DNA fragment was identified and cloned from Staphylococcus epidermidis (Se) using femA from S. aureus (Sa) as a heterologous hybridization probe. DNA sequence analysis of a portion of this clone revealed two complete ORFs highly related to femA and femB of Sa. The genomic arrangement of the Se femA/B complex was nearly identical to that observed in Sa. Intra- and interspecies relatedness of these genes and conservation of genomic organization were consistent with gene duplication of one of these genes in an ancestral organism. Recombinant FEMA, produced in Escherichia coli (Ec), was purified to near homogeneity. Identity of the purified protein was verified by N-terminal amino acid (aa) sequence analysis.     

The rad2 mutation affects the molecular nature of UV and acridine-mustard-induced mutations in the ADE2 gene of Saccharomyces cerevisiae

We have studied the molecular nature of ade2 mutations induced by UV light and bifunctional acridine-mustard (BAM) in wild-type (RAD) and in excision-deficient (rad2) strains of the yeast, Saccharomyces cerevisiae. In the RAD strain, UV causes 45% GC → AT transitions among all mutations; in the rad2 strain this value is 77%. BAM was shown to be highly specific for frameshift mutagenesis: 60% frameshifts in the RAD strain, and as many as 84% frameshifts in the rad2 strain were induced. Therefore, the rad2 mutation affects the specificity of UV- and BAM-induced mutagenesis in yeast. Experimental data agree with the view that the majority of mutations in the RAD strain are induced by a prereplicative mechanism, whereas mutations in the RAD strain are induced by a prereplicative mechanism, whereas mutations in the rad2 strain are predominantly postreplicative events. Our results also suggest that: (1) cytosine-containing photoproducts are the substances responsible for major premutational damage to DNA; (2) a fraction of the mutations may arise in the course of excision repair of UV photoproducts.