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.     

Binding of the recA protein of Escherichia coli to single- and double-stranded DNA

The recA protein of Escherichia coli binds both single- (SS) and double-stranded (DS) DNA; however, the optimal conditions differ for interaction with these DNA substrates. Binding of DS DNA by recA protein is pH dependent (optimum near pH 6.2) and requires a nucleoside triphosphate (ATP) and divalent cation. Substitution of the 5'-O-3'-thiotriphosphate (ATP(gamma S)) for ATP leads to formation of stable complexes of recA protein and DNA that dissociate very slowly. Formation of these complexes is extremely sensitive to ionic strength and pH. However, once formed, the complexes resist changes in pH and high salt concentrations. SS DNA binds to recA protein in the absence of a nucleoside triphosphate, but recA protein-SS DNA complexes are stabilized by ATP(gamma S). At high recA protein/DNA ratios (1 recA protein monomer/30 nucleotides), these complexes sediment in sucrose gradients as large protein-DNA aggregates. Although ATP(gamma S) blocks dissociation of recA protein from DNA, ATP stimulates the release of recA protein from SS DNA. Hydrolysis of the ATP is not required for dissociation since it is also enhanced by ADP and certain nucleoside triphosphates that are not hydrolyzed by recA protein. recA protein binds with different affinities to ribohomopolymers and deoxyhomopolymers. It preferentially binds polydeoxythymidylate and polydeoxycytidylate but does not bind short oligonucleotides, indicating that there is a minimum size requirement for the binding step. The recA protein exists as a heterogeneous aggregate at pH 7.5 and at low ionic strength. At pH 6.2 in the presence of Mg2+, the protein sediments homogeneously as a dimer. At pH 6.2, ATP or ATP(gamma S) promotes an oligomerization of the recA protein which can be observed as filamentous structures by electron microscopy. Oligomerization is not induced by UTP, a nucleoside triphosphate that is efficiently hydrolyzed by the recA protein, but fails to stimulate efficiently recA protein-promoted annealing and assimilation of single-stranded DNA.     

Cloning, sequencing, and characterization of the recA gene from Rhodopseudomonas viridis and construction of a recA strain

A recombination-deficient strain of the phototrophic bacterium Rhodopseudomonas viridis was constructed for the homologous expression of modified photosynthetic reaction center genes. The R. viridis recA gene was cloned and subsequently deleted from the R. viridis genome. The cloned R. viridis recA gene shows high identity to known recA genes and was able to complement the Rec- phenotype of a Rhizobium meliloti recA strain. The constructed R. viridis recA strain showed the general Rec- phenotype, i.e., increased sensitivity to DNA damage and severely impaired recombination ability. The latter property of this strain will be of advantage in particular for expression of modified, nonfunctional photosynthetic reaction centers which are not as yet available.     

Cloning and characterization of the rpoH gene of Rhodobacter capsulatus

By using an oligonucleotide mixture corresponding to a region highly conserved among alternative sigma factors we identified a new sigma factor gene (rpoH) from Rhodobacter capsulatus. This gene encodes a protein of 34 kDa with strong similarity to the RpoH (sigma32) factors from other bacterial species. It was not possible to inactivate the R. capsulatus rpoH gene by introducing a resistance cassette, implying that it is essential for growth. The 5' ends of the mRNAs were mapped to two sequences with similarity to an rpoH- and an rpoD-dependent promoter, respectively. The amounts of both these mRNAs increased after heat shock, but were unaffected by a decrease in oxygen tension. Western analysis using a sigma factor-specific antibody revealed the accumulation of a protein of about 34 kDa after heat shock, and an increase in the amounts of a protein with the same size after reduction of oxygen tension in R. capsulatus cultures.     

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.     

Aerobic repression of the Rhodobacter capsulatus bchC promoter involves cooperative interactions between CrtJ bound to neighboring palindromes

Previous studies demonstrated that bacteriochlorophyll, carotenoid, and light harvesting gene expression in Rhodobacter capsulatus is repressed under aerobic growth conditions by the repressor CrtJ. Isolated CrtJ is known to bind to the palindrome TGTN12ACA, which is present in two copies in the bchC promoter, one of which spans the -35 and the other the -10 sigma-70 recognition sequences. In this study, we demonstrate that CrtJ binds to the two palindromic sites in the bchC promoter in a cooperative manner. The level of cooperativity of CrtJ binding to the -35 palindrome was shown to be 26-fold. A distance of 8 base pairs between the two palindromic sites was shown to be critical for cooperative binding, as evidenced by the disruption of binding that resulted when +6 and +11 base pairs were inserted between the palindromes.