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We identified in the nuclear extract of tobacco seedlings a 43-kDa DNA-binding protein RCS2 that interacted with the 5′-upstream region of the rolC gene of the Agrobacterium rhizogenes Ri plasmid . DNA-protein gel shift and competition assays demonstrated that RCS2 bound to single-stranded DNA in a sequence-specific manner. A five-base direct repeat is important for the DNA binding of RCS2. South-Western blot analysis was employed to determine the size of RCS2 which appears to be approx. 43 kDa.

A CArG box motif [CC(A+T-rich)6GG] is one of the DNA elements required for muscle-specific gene transcription. Nuclear factors in mouse C2 myogenic cells strongly bind to the CArG box in the first intron of the gene (Sm alpha-A) encoding human smooth muscle alpha-actin. To clone cDNAs of the CArG box-binding factor (CBF), lambda gt11 cDNA expression libraries from C2 cells were screened for in situ DNA binding specific for this CArG box sequence. The 1.6-kb cDNA (CBF-A) encoding 285 amino acids (aa) was obtained, and a beta-galactosidase fusion protein, bacterially produced from the cDNA, bound to DNA fragments containing several CArG boxes. When the expression level of CBF-A in C2 cells increased by transfection of CBF-A expression plasmids, Sm alpha-A transcription was repressed. The deduced aa sequence of CBF-A is similar to some single-stranded (ss) nucleic acid-binding proteins. The fusion protein could bind to ssDNA, whereas CBF in C2 cell nuclear extracts could not. From these results, CBF-A is a novel CArG box-, ssDNA- and RNA-binding protein, as well as a repressive transcriptional factor.

The gene for the rat mitochondrial single-stranded DNA-binding protein (mtSSB) was amplified by PCR and isolated as several overlapping genomic clones. The clones encompassed the 5′ untranslated sequence and all intron/exon junctions. The gene contained seven exons and six introns. The first exon contained only 5′ untranslated sequence. The 16-amino acid mitochondrial targeting presequence, encoded by the second and third exons, was precisely bisected by intron 2. All intron donor and acceptor sites were consistent with the GT/AG consensus. The transcription start site was determined by primer-extension analysis to be 69 bp upstream of the translation start codon. The upstream sequence lacked TATA and CCAAT boxes at the expected locations, but did contain several other potential regulatory elements including a GC box (Sp1-binding site) and three NRF-2 sites, one of which was located precisely beside the transcription start site. A 10 out of 12 imperfect NRF-1 site was located within the first exon. The 5′ flanking sequence (−546 to +30) was shown to have strong promoter activity in transient transfection assays in primary rat hepatocytes and HepG2 cells. In addition, evidence for the existence of several mtSSB processed pseudogenes was obtained. These pseudogenes lacked introns and contained substitution and deletion mutations compared to the cDNA sequence. The 5′ upstream region of one of the pseudogenes was analyzed and found to contain negligible promoter activity.

The ssb gene of Haemophilus influenzae was cloned and sequenced. The deduced protein possessed 61 and 60% identity with the Serratia marcescens and Escherichia coli SSB proteins, respectively. H. influenzae ssb was also shown to complement an E. coli ssb-1 mutation.

The recombinant plasmid pJA01 contains, besides the uvrA gene, the genes lexA, ubiA and ssb. This plasmid does not fully complement a uvrA mutation in a Rec+ background. Plasmids which contain the uvrA and ssb genes, but not the lexA gene, show a higher but still only partial complementation. Full complementation is achieved when the ssb gene is inactivated by insertion of Tn5. Furthermore, it appears that the presence of the ssb gene on a multicopy plasmid sensitizes wild-type cells to UV light. The effect of Ssb (single-strand DNA binding protein) overproduction on UV survival is discussed.

Single-strand DNA binding protein (SSB) from Escherichia coli abolishes transfection of E.coli by viral M13mp2 DNA at levels that inhibit transfection by M13mp2 replicative form (RF) DNA by approx. 25%. Synthesis of M13mp2 RF DNA (SS → DS) has been carried out using DNA polymerase I (Klenow fragment) and a unique 15-nucleotide primer. A time course for in vitro synthesis showed that the increase in transfection in the presence of SSB paralleled DNA synthesis after an initial lag period for transfection. Digestion of replication products with restriction endonucleases and Sl endonuclease indicates that only those molecules that are fully or almost fully duplex transfect competent cells in the presence of SSB.