Structural Analysis of a recA-like Gene in the Genome of Arabidopsis thaliana

A recA-like gene was identified in the genome of Arabidopsisthaliana by means of PCR using primers designed on the basisof previously reported amino acid sequences of eukaryotic RecA-likeproteins. The structure of the gene, termed ArLIM15, was investigatedby comparing the primary structure of the genomic DNA with thatof the corresponding cDNA. The open reading frame, which wassplit into 15 exons, was established to have the capacity forencoding a 37.3-kDa polypeptide. The amino acid sequence ofthe putative product of ArLIM15 showed a high degree of similaritytothat of LIM15 in the monocotyledonous plant Lilium, includinga 93% identity, and to those of other recA-like genes in yeastsand vertebrates with identities of 69–71%. Phylogeneticanalysis indicated ArLIM15 to be much closer to meiosis-specificLIM15 and DMC1 in Saccharomyces cerevisiae than to RAD51 inS. cerevisiae and its homologues on an evolutionary scale.     

Cloning, sequencing and expression of a recA gene from Amycolatopsis mediterranei

The 3.5 kb nucleotide fragment, including the recA gene and its downstream recX-like gene, has been isolated from a genomic library by dot-blot hybridization with the Mycobacterium smegmatis recA gene. The recA gene, consisting of 1047 base pairs (bp), encodes a polypeptide of 348 amino acids while the recX-like gene, consisting of 450 bp, encodes a shorter polypeptide of 149 amino acids. Both the deduced amino acid sequences of recA and recX resemble those of the recA and recX genes from other bacteria. The cloned Amycolatopsis mediterranei U32 recA gene conferred partial resistance to ethyl methane sulfonate when expressed in E. coli with the lacZ promoter.     

Isolation of a LIM15/DMC1 homolog from the basidiomycete Coprinus cinereus and its expression in relation to meiotic chromosome pairing

The Escherichia coli gene recA is essential for homologous recombination and DNA repair, and homologs have been identified in eukaryotes. A basidiomycete, Coprinus cinereus, which has many advantages for the study of meiosis, was recently reported to have a homolog of one of these, RAD51. In the yeast Saccharomyces, mutations in the RAD5I gene cause defects in both somatic and meiotic cells. Based on this finding, we screened for a meiosis-specific homolog of recA, equivalent to Lilium LIM15 or Saccharomyces DMC1, in C. cinereus, and isolated a clone containing a 1.2-kb DNA fragment from a cDNA library constructed with Coprinus poly(A)⁺ RNA isolated from cells undergoing meiosis. The predicted amino acid sequence was 52% identical to the putative gene product of the lily cDNA clone LIM15 and 61% identical to Saccharomyces DMC1, and showed limited sequence similarity to the products of RAD52, 55, and 57. The synchrony of meiosis in Coprinus provides an ideal system for the investigation of differential gene expression in relation to meiosis and fruiting body development. Northern analysis indicated that Coprinus LIM15/DMC1 was expressed at meiotic prophase within 8 h after the onset of karyogamy, suggesting that the gene functions mostly at the stage at which the homologous chromosomes pair, but may not be essential at the point at which they recombine. The gene is not expressed in somatic cells. Received: 8 October 1998 / Accepted: 22 July 1999     

Cloning and Characterization of HumanSep1 (hSEP1) Gene and Cytoplasmic Localization of its Product

We isolatedand sequenced a human cDNA (designated as hSEP1)encoding both a homologue of mouse Dhm2 and budding yeast SEP1.The gene was shown to be locatedon the long arm of chromosome3 (3q25-26.1). The putative hSEP1 product (hSEP1p) consistedof 1694 amino acid residues with a molecular mass of about 190kDa. Northern blot analysis showeda major 10-kb mRNA expressedubiquitously in variousorgans as well as a minor 5.5-kb mRNAexpressed relatively highly in the testis and placenta. hSEP1pis localizedin the cytoplasm as examinedb y cytochemical andWestern blot analyses of fractionated cellular extracts. Thebiological function of hSEP1p was discussed in correlation withits cytoplasmic localization.     

Cloning and Bacterial Expression of Sesquiterpene Cyclase, a Key Branch Point Enzyme for the Synthesis of Sesquiterpenoid Phytoalexin Capsidiol in UV-Challenged Leaves of Capsicum annuum

Sesquiterpene cyclase, a branch point enzyme in the generalisoprenoid pathway for the synthesis of phytoalexin capsidiol,was induced in detached leaves of Capsicum annuum (pepper) byUV treatment. The inducibility of cyclase enzyme activitiesparalleled the absolute amount of cyclase protein(s) of pepperimmunodetected by monoclonal antibodies raised against tobaccosesquiterpene cyclase. A cDNA library was constructed with poly(A)⁺RNA isolated from 24 h UV-challenged leaves of pepper. A cDNAclone for sesquiterpene cyclase in pepper was isolated by usinga tobacco 5-epi aristolochene synthase gene as a hetero-logousprobe. The predicted protein encoded by this cDNA was comprisedof 559 amino acids and had a relative molecular mass of 65,095.The primary structural information from the cDNA clone revealedthat it shared 77%, 72% and 49% identity with 5-epi aristolochene,vetispiradiene, and cadinene synthase, respectively. The enzymaticproduct catalyzed by the cDNA clone in bacteria was identifiedas 5-epi aristolochene, as judged by argentation TLC. RNA blothybridization demonstrated the induction of an mRNA consistentwith the induction of cyclase enzyme activity in UV-treatedpepper. (Received March 2, 1998; Accepted June 15, 1998)     

Construction of a fused operon consisting of the recA and kan (Kanamycin resistance) genes and regulation of its expression by the lexA gene

Summary The kanamycin resistance gene (kan) of transposon Tn5 was cloned into a derivative of plasmid pBR322. A DNA fragment containing the promoter-operator region of the recA gene was inserted into the promoter region of the cloned kan gene to produce a fused operon, recA-kan. Plasmid pMCR685 carrying recA-kan expressed a low level of activity of the kan gene product (kanamycin phosphotransferase; KPT) in the wildtype cells of Escherichia coli, while the plasmid showed an increased level of the activity in the Spr^- mutant cells which produce the inactive lexA protein. The KPT activity in the wildtype cells harboring the plasmid increased 6-to 11-fold upon treatment of the cells with mitomycin C or nalidixic acid, both of which are known to induce synthesis of recA protein.Expression of the recA-kan operon fusion was remakably repressed by the lexA gene cloned into a plasmid carrying the operon fusion. Higher concentrations of mitomycin C were required for maximal induction of KPT activity in the cells harboring the resulting plasmid pMCR687. These results strongly suggest that the lexA gene product can by itself repress the recA gene, and that pMCR687 is a useful vector to clone genes whose expression is harmful to the host cell growth.     

Transoceanic Spreading of Pathogenic Strains of Vibrio parahaemolyticus with Distinctive Genetic Signatures in the recA Gene

Vibrio parahaemolyticus is an important human pathogen whose transmission is associated with the consumption of contaminated seafood. Consistent multilocus sequence typing for V. parahaemolyticus has shown difficulties in the amplification of the recA gene by PCR associated with a lack of amplification or a larger PCR product than expected. In one strain (090–96, Peru, 1996), the produced PCR product was determined to be composed of two recA fragments derived from different Vibrio species. To better understand this phenomenon, we sequenced the whole genome of this strain. The hybrid recA gene was found to be the result of a fragmentation of the original lineage-specific recA gene resulting from a DNA insertion of approximately 30 kb in length. This insert had a G+C content of 38.8%, lower than that of the average G+C content of V. parahaemolyticus (45.2%), and contained 19 ORFs, including a complete recA gene. This new acquired recA gene deviated 24% in sequence from the original recA and was distantly related to recA genes from bacteria of the Vibrionaceae family. The reconstruction of the original recA gene (recA3) identified the precursor as belonging to ST189, a sequence type reported previously only in Asian countries. The identification of this singular genetic feature in strains from Asia reveals new evidence for genetic connectivity between V. parahaemolyticus populations at both sides of the Pacific Ocean that, in addition to the previously described pandemic clone, supports the existence of a recurrent transoceanic spreading of pathogenic V. parahaemolyticus with the corresponding potential risk of pandemic expansion.     

Identification and Characterization of a Novel Human Phosphatidylinositol 4-kinase

The extensive sequence homology that exists among the catalyticdomains of phosphatidylinositol 3- and 4-kinases allowed usto clone a novel human gene encoding a putative phosphatidylinositolkinase, NPIK. Among other known phosphatidylinositol 3- and4-kinases, NPIK was most closely related to yeast PIK1 phosphatidylinositol4-kinase. Several forms of NPIK cDNAs were isolated, and expressionof NPIK message was detected in a wide variety of tissues. Fluorescencein situ hybridization and radiation hybrid analyses assignedthe NPIK gene to human chromosome 1. Recombinant NPIK proteincatalyzed a conversion from phosphatidylinositol to phosphatidylinositol4-phosphate. The catalytic activity of NPIK was augmented byTriton X-100, and was reduced in the presence of adenosine.Using green .uorescent protein system we determined that NPIKis localized in the cytoplasm. Taken together, the data suggestthat NPIK may play a pivotal role in regulating the synthesisof phosphatidylinositol 4-phosphate at the site(s) accessiblefrom cytoplasm.     

Molecular cloning, sequence and regulation of expression of the recA gene of the phototrophic bacterium Rhodobacter sphaeroides

The recA gene of Rhodobacter sphaeroides 2.4.1 has been isolated by complementation of a UV-sensitive RecA^– mutant of Pseudomonas aeruginosa. Its complete nucleotide sequence consists of 1032 bp, encoding a polypeptide of 343 amino acids. The deduced amino acid sequence displayed highest identity to the RecA proteins from Rhizobium mehloti, Rhizobium phaseoli, and Agrobacterium tumefaciens. An Escherichia coli-like SOS consensus region, which functions as a binding site for the LexA repressor molecule was not present in the 215 by upstream region of the R. sphaeroides recA gene. Nevertheless, by using a recA-lacZ fusion, we have shown that expression of the recA gene of R. sphaeroides is inducible by DNA damage. A recA-defective strain of R. sphaeroides was obtained by replacement of the active recA gene by a gene copy inactived in vitro. The resulting recA mutant exhibited increased sensitivity to UV irradiation, and was impaired in its ability to perform homologous recombination as well as to trigger DNA damage-mediated expression. This is the first recA gene from a Gram-negative bacterium that lacks an E. coli-like SOS box but whose expression has been shown to be DNA damage-inducible and auto-regulated.     

Rhipicephalus appendiculatus: characterization of a testis-associated protein

A novel gene coding for Rhipicephalus appendiculatus Male-specific Protein (RAMP) was identified in a cDNA library constructed from the testis/vas deferens of R. appendiculatus ticks. This gene encodes a secreted protein exclusively expressed in the testis/vas deferens. The putative RAMP amino acid sequence contains a signal peptide and has 29% amino acid identity with male-specific Is5 gene of Ixodes scapularis. Gene expression studies revealed that RAMP mRNA was up-regulated in male ticks during blood feeding. RAMP was detected not only in the testis/vas deferens of males but also in postcoitum female ticks based on Western blotting, indicating that this protein is transferred to the female tick during copulation. Virgin female ticks, microinjected with recombinant RAMP, had significantly prolonged attachment duration during feeding, but there was no effect on fed weight. These results suggest that RAMP is a male-specific molecule in the spermatophore, and is related to female attachment behavior in R. appendiculatus ticks.     

Molecular cloning,sequence and regulation of expression of the recA gene of the phototrophic bacterium Rhodobacter sphaeroides

The recA gene of Rhodobacter sphaeroides 2.4.1 has been isolated by complementation of a UV-sensitive RecA^? mutant of Pseudomonas aeruginosa. Its complete nucleotide sequence consists of 1032 bp, encoding a polypeptide of 343 amino acids. The deduced amino acid sequence displayed highest identity to the RecA proteins from Rhizobium mehloti, Rhizobium phaseoli, and Agrobacterium tumefaciens. An Escherichia coli-like SOS consensus region, which functions as a binding site for the LexA repressor molecule was not present in the 215 by upstream region of the R. sphaeroides recA gene. Nevertheless, by using a recA-lacZ fusion, we have shown that expression of the recA gene of R. sphaeroides is inducible by DNA damage. A recA-defective strain of R. sphaeroides was obtained by replacement of the active recA gene by a gene copy inactived in vitro. The resulting recA mutant exhibited increased sensitivity to UV irradiation, and was impaired in its ability to perform homologous recombination as well as to trigger DNA damage-mediated expression. This is the first recA gene from a Gram-negative bacterium that lacks an E. coli-like SOS box but whose expression has been shown to be DNA damage-inducible and auto-regulated.     

Molecular Cloning and Chromosomal Localization of the MouseGpr37Gene Encoding an Orphan G-Protein-Coupled Peptide Receptor Expressed in Brain and Testis

We report the cloning of the mouse ortholog of the humanGPR37gene, which encodes an orphan G-protein-coupled receptor highly expressed in brain tissues and homologous to neuropeptide-specific receptors ([20],Genomics 45:68–77;[45],Biochem. Biophys. Res. Commun. 233:559–567). The genomic organization of theGPR37gene is conserved in both mouse and human species with a single intron interrupting the receptor-coding sequence within the presumed third transmembrane domain. Comparative genetic mapping of theGPR37gene showed that it maps to a conserved chromosomal segment on proximal mouse chromosome 6 and human chromosome 7q31. The mouseGpr37gene contains an open reading frame coding for a 600-amino-acid protein 83% identical to the humanGPR37gene product. The predicted mouse GPR37 protein contains seven putative hydrophobic transmembrane domains, as well as a long (249 amino acid residues), arginine- and proline-rich amino-terminal extracellular domain, which is also a distinctive feature of the human GPR37 receptor. Northern blot analysis of mouse tissues withGpr37-specific probes revealed a main 3.8-kb mRNA and a much less abundant 8-kb mRNA, both expressed in the brain. A 3-kb mRNA is also expressed in the testis. Both the mouse and the humanGPR37genes may belong to a class of highly conserved mammalian genes encoding a novel type of G-protein-coupled receptor predominantly expressed in the brain.     

Cloning of a recA-like gene of Proteus mirabilis

A gene of Proteus mirabilis that can substitute for functions of the recA gene of Escherichia coli has been cloned into the plasmid pBR322, using shotgun experiments. The recA-like gene (recA_P.m.) has been localized by restriction mapping within a 1.5-Md PstI fragment that is a part of two cloned HindIII fragments of the chromosome of P. mirabilis.The restriction map of the recA_P.m. gene differs from that of the recA gene of E. coli. Functionally, the recombinant plasmids containing the recA_P.m. gene restore a nearly wild-type level of UV-resistance to several point and deletion mutants in the recA gene of E. coli.     

Investigation of a phage resistantSerratia entomophila strain (BC4B), establishment of generalised transduction and construction ofS. entomophila RecA mutants

ArecA clone was isolated from a cosmid library ofSerratia entomophila constructed in theEscherichia coli strain HB101. Subcloning and transposon mutagenesis were used to identify a 1.36 kb fragment containing therecA gene. A clonedrecA mutation, generated by transposon mutagenesis and the replacement of a portion of therecA gene with an antibiotic resistance cassette, was introduced into the chromosome via a marker exchange technique. TherecA strains created were deficient in DNA repair, homologous recombination and both the spontaneous and UV induction of prophages.S. entomophila recA strains showed continued pathogenicity towards the New Zealand grass grub,Costelytra zealandica. Simple procedures for further construction ofS. entomophila recA strains have been demonstrated.