Construction of recombination-deficient strains of Streptococcus gordonii by disruption of the recA gene

Degenerate oligonucleotide primers were used in a polymerase chain reaction (PCR) to amplify a region of the recA sequence of Streptococcus gordonii Challis. The resulting PCR fragment was cloned into the suicide vector pAM6199 and introduced into strain Challis, giving rise to recombination-deficient strains in which the recA gene was specifically inactivated.     

Activin from secondary follicles causes small preantral follicles to remain dormant at the resting stage

The sequences controlling the expression of the Rhodobacter capsulatus recA and uvrA genes belonging to the SOS DNA repair system have been identified by PCR mutagenesis. Data obtained demonstrated that the GTTCN7GTAC and GAACN7GAAC motifs present upstream of the recA gene and the GTTCN7GTTC motif found upstream of the uvrA gene are required for their respective DNA damage-mediated induction. Alignment of recA promoters of R. capsulatus, Rhodobacter sphaeroides and Rhodopseudomonas viridis with the uvrA promoters of R. capsulatus and R. sphaeroides has identified the consensus sequence GTTCVYVYTWTGTTC as the SOS operator site of the Rhodospirillaceae family.     

Characterization of the recA gene regions of Spiroplasma citri and Spiroplasma melliferum

In previous studies (A. Marais, J. M. Bove, and J. Renaudin, J. Bacteriol. 178:862-870, 1996), we have shown that the recA gene of Spiroplasma citri R8A2 was restricted to the first 390 nucleotides of the N-terminal part. PCR amplification and sequencing studies of five additional strains of S. citri have revealed that these strains had the same organization at the recA region as the R8A2 strain. In contrast to S. citri, Spiroplasma melliferum was found to contain a full-length recA gene. However, in all five S. melliferum strains tested, a TAA stop codon was found within the N-terminal region of the recA reading frame. Our results suggest that S. melliferum, as well as S. citri, is RecA deficient. In agreement with the recA mutant genotype of S. citri and S. melliferum, we have shown that these organisms are highly sensitive to UV irradiation.     

Isolation and characterization of the recA gene of Xanthomonas campestris pv. campestris

A 1.8-kb NsiI-StuI fragment containing the recA gene of Xanthomonas campestris pv. campestris was cloned by a PCR-based approach and complementation of Escherichia coli HB 101. Sequence analysis of this fragment revealed an ORF (orf343) of 1,032 bp able to encode a protein of 343 amino acids with a calculated MW of 37,021 Da, a size similar to the values detected by in vitro system and Western blotting. It showed 69.6% identity to the E. coli RecA in amino acid sequence. Amino acid residues of the E coli RecA associated with functional activities are conserved in this Xc17 RecA. The recA mutant, L1, constructed by gene replacement, was sensitive to ultraviolet irradiation and methyl methanesulfonate, and deficient in homologous recombination.     

Chromosomal changes and correspondingly altered proto-oncogene expression in human gliomas. Value of combined cytogenetic and molecular genetic analysis

The recA gene from the bacterium Xanthomonas oryzae pv. oryzae (Xoo), a rice pathogen, was cloned based on its ability to complement DNA repair defects of Escherichia coli recA- mutants. The Xoo recA was localized to a 1.3-kb Sau3AI-XhoI fragment and, when cloned into pBR322, specifies increased methylmethanesulfonate and mitomycin C resistance to E. coli recA mutants and allows lambda red- gam- to plaque on an E. coli recA- host. An E. coli recA- strain harboring a plasmid containing the Xoo recA-like gene was shown to produce a 40-kDa protein which cross-reacted with an anti-E. coli RecA antibody. A similar molecular mass protein to RecA has been detected in several Xanthomonas pathovars using an anti-E. coli RecA antibody. Furthermore, the cloned Xoo recA was shown to hybridize to genomic DNA from various Xanthomonas pathovars, but not to genomic DNA from other bacteria species under high-stringency hybridization conditions. These results indicate the isolation of the Xoo recA gene.     

Cloning, characterization and construction of htrA and htrA-like mutants of Brucella abortus and their survival in BALB/c mice

A genomic library of Brucella abortus S2308 was screened for expression of recombinant proteins recognized by sera from mice and from cattle infected with B. abortus. A positive clone, BA1, expressing a 50 kDa peptide was recognized by both sera. Plasmid pBA1, isolated from BA1, was shown by restriction enzyme digestion to possess a 1.9 kb insert. The nucleotide sequence of the pBA1 insert revealed an open reading frame with of 1539 bases with a coding capacity of 513 amino acids and a predicted molecular weight of 50,992. The predicted amino acid sequence showed 37% identity to E. coli HtrA, a temperature inducible serine protease. A second B. abortus htrA gene, designated htrA-like, was identified on a different cloned fragment that also encoded B. abortus recA. The nucleotide sequence of the htrA-like gene revealed an open reading frame of 1422 nucleotides with a coding capacity of 474 amino acids and a predicted molecular weight of 50,155. The deduced amino acid sequence of the htrA-like gene showed 42% and 36% identity with B. abortus and E. coli HtrAs respectively. Western blotting of E. coli lysate containing the htrA-like gene was not recognized by sera from B. abortus-infected cattle or mice. B. abortus htrA but not htrA-like relieved the temperature sensitive phenotype and permitted growth of an E. coli htrA mutant at 42 degrees C. B. abortus htrA and htrA-like mutants were constructed and their survival and growth in BALB/c mice was compared to the parental strain S2308.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inversin, a novel gene in the vertebrate left-right axis pathway, is partially deleted in the inv mouse

A DNA fragment containing the recA gene of Gluconobacter oxydans was isolated and further characterized for its nucleotide sequence and ability to functionally complement various recA mutations. When expressed in an Escherichia coli recA host, the G. oxydans recA protein could efficiently function in homologous recombination and DNA damage repair. The recA gene's nucleotide sequence analysis revealed a protein of 344 amino acids with a molecular mass of 38 kDa. We observed an E. coli-like LexA repressor-binding site in the G. oxydans recA gene promoter region, suggesting that a LexA-like mediated response system may exist in G. oxydans. The expression of G. oxydans recA in E. coli RR1, a recA+ strain, surprisingly caused a remarkable reduction of the host wild-type recA gene function, whereas the expression of both Serratia marcescens recA and Pseudomonas aeruginosa recA gene caused only a slight inhibitory effect on function of the host wild-type recA gene product. Compared with the E. coli RecA protein, the identity of the amino acid sequence of G. oxydans RecA protein is much lower than those RecA proteins of both S. marcescens and Pseudomonas aeruginosa. This result suggests that the expression of another wild-type RecA could interfere with host wild-type recA gene's function, and the extent of such an interference is possibly correlated to the identity of the amino acid sequence between the two classes of RecA protein.     

RecA protein from an extremely thermophilic bacterium, Thermus thermophilus HB8

The recA gene of a thermophilic eubacterial strain, Thermus thermophilus (T.th.) HB8, was cloned from a genomic DNA library by Southern hybridization using a gene-internal fragment amplified by the polymerase chain reaction (PCR) method as the probe. The gene encoded a 36 kDa polypeptide whose amino acid sequence showed 61% identity with that of the Escherichia coli RecA protein. Characteristic amino acid changes between the two RecA proteins were found. In the amino acid composition of the T.th. RecA protein, the number of Pro residues was increased, the number of Cys residues was decreased, and Lys residues were replaced by Arg, Asp by Glu, Thr by Val, and Ile by Val or Leu. These changes are supposed to stabilize the native protein conformation against heat denaturation. The amino acid residues in the nucleotide binding site of the protein and in the protein-protein interaction site responsible for the oligomer formation were well conserved. The T.th. recA gene has the ability to complement the ultraviolet light (UV) sensitivity of a E. coli recA deletion mutant. Thus, the thermophilic bacterium has a RecA protein whose function will be common to the E. coli RecA protein.     

Repressor titration: a novel system for selection and stable maintenance of recombinant plasmids

The propagation of recombinant plasmids in bacterial hosts, particularly in Escherichia coli, is essential for the amplification and manipulation of cloned DNA and the production of recombinant proteins. The isolation of bacterial transformants and subsequent stable plasmid maintenance have traditionally been accomplished using plasmid-borne selectable marker genes. Here we describe a novel system that employs plasmid-mediated repressor titration to activate a chromosomal selectable marker, removing the requirement for a plasmid-borne marker gene. A modified E.coli host strain containing a conditionally essential chromosomal gene (kan) under the control of the lac operator/promoter, lac O/P, has been constructed. In the absence of an inducer (allolactose or IPTG) this strain, DH1 lackan , cannot grow on kanamycin-containing media due to the repression of kan expression by LacI protein binding to lac O/P. Transformation with a high copy-number plasmid containing the lac operator, lac O, effectively induces kan expression by titrating LacI from the operator. This strain thus allows the selection of plasmids without antibiotic resistance genes (they need only contain lac O and an origin of replication) which have clear advantages for use as gene therapy vectors. Regulation in the same way of an essential, endogenous bacterial gene will allow the production of recombinant therapeutics devoid of residual antibiotic contamination.     

Cloning and expression of a plasmid pBS195 gene, determining oxygenase activity, in Escherichia coli cells

Plasmid pBS195, detected in a strain of Lactobacillus sp. isolated from long-living persons, has a broad host range, including Gram-positive and Gram-negative microorganisms [1]. Plasmid-harboring colonies of the strain Escherichia coli HB101 give a color reaction with catechol. This indicates that genes mediating the activity of oxygenase are present in this plasmid. The high activity level of this enzyme, mediated by pBS195, and substrate specificity, which has not bee detected in any known metapyrocatechases, were found in cells of E. coli. Hybridization with a 32P-labeled fragment containing the NahC gene revealed a region of homology with a 1.6-kb EcoR I- BamH I fragment of plasmid pBS195. Deletion variants of this plasmid that lost oxygenase activity confirmed the location of the oxygenase gene in this region. The gene responsible for oxygenase activity in the plasmid was cloned on the pUC19 vector in E. coli cells. The expression of the cloned gene is controlled by the lac promoter of this vector. Physical, hybridization, and deletion analyses as well as analysis of polypeptides, which are synthesized in E. coli mini-cells, showed that this activity requires the participation of a polypeptide with molecular mass of 34 kDa.     

Mechanism of thermotolerance induction by split-dose hyperthermia in Deinococcus radiodurans DNA repair deficient mutants

We examined the phenomenon of thermotolerance induction in the radioresistant prokaryote, Deinococcus radiodurans, which was initially exposed to 30 min at 52 degrees C followed by various intervals up to 6 h at 30 degrees C in TGY medium and then re-exposed to 52 degrees C for various periods, i.e., split-dose hyperthermia. This thermotolerance induction was analyzed in DNA repair deficient mutants (strain 302, 251, UVS25, rec30 and KH840) and the wild-type strain MR1. The strain UVS25 is a double mutant for the mtcA and uvsD genes, and strain rec30 is a mutant for the deinococcal recA gene. The induction was suppressed to 1/10 and 1/25 in strains UVS25 and rec30 respectively, as compared with the maximum level in the wild-type strain MR1. However, the induction in strain 302 (mutant for the uvrA gene) was not suppressed. Therefore, we conclude that proteins synthesized during the interexposure interval, i.e., the products of the uvsD (UV endonuclease beta) and recA (RecA protein) genes contribute to the induction of thermotolerance in D. radiodurans.     

Vectors for the expression of PCR-amplified immunoglobulin variable domains with human constant regions

Cassette vectors have been constructed for mammalian expression of complete immunoglobulin heavy and light chain genes whose variable regions are produced by the polymerase chain reaction (PCR). The light and heavy chain vectors have promoter, leader, partial intron, enhancer and constant region segments within modified pSV2-gpt and pSV2-neo plasmids, respectively. Variable (V) regions are obtained by PCR using a two step process: 1) the V gene is amplified from genomic or cDNA, cloned into an intermediate vector and sequenced; 2) the first PCR product serves as the template for a second amplification in which restriction enzyme recognition sites and limited flanking intron sequence are added. The second PCR product is inserted into the expression vector, which is then transfected into mouse myeloma cells. These vectors contain human constant regions and may be used to express chimeric, humanized or human Ig genes. This report describes the design of these vectors and their application for the expression of chimeric 60.3, an anti-CD18 antibody.     

The isfA mutation inhibits mutator activity and processing of UmuD protein in Escherichia coli recA730 strains

Further studies on the isfA mutation responsible for anti-SOS and antimutagenic activities in Escherichia coli are described. We have previously shown that the isfA mutation inhibits mutagenesis and other SOS-dependent phenomena, possibly by interfering with RecA coprotease activity. The isfA mutation has now been demonstrated also to suppress mutator activity in E. coli recA730 and recA730 lexA51(Def) strains that constitutively express RecA coprotease activity. We further show that the antimutator activity of the isfA mutation is related to inhibition of RecA coprotease-dependent processing of UmuD. Expression of UmuD' from plasmid pGW2122 efficiently restores UV-induced mutagenesis in the recA730 isfA strain and partially restores its mutator activity. On the other hand, overproduction of UmuD'C proteins from pGW2123 plasmid markedly enhances UV sensitivity with no restoration of mutability.