A comparative study of the immunity region of lambdoid phages including Shiga-toxin-converting phages: molecular basis for cross immunity.

Comparison of eight lambdoid phages, including three Shiga-toxin converting phages, has been carried out with respect to the immunity region, especially the recognition helices of their repressor and CRO proteins on the one hand, and operator sequences on the other. Some as yet unassigned components of the regulatory circuits have been inferred by computer search. The cross immunity phenomenon shown by phages VT2-Sa and lambda is explained on the basis of similarity in their sequences. In addition, the similarity of 933W and HK022 in the sequences of their recognition helices of repressor and CRO, on the one hand, and operators, on the other, has led us to predict that they will have identical or similar immunity specificity. This homology has enabled us also to locate the OL (and consequently PL) of phage 933W that has been thought to be non-existent.

Complementary DNA Cloning and Characterization of Pearlin, a New Class of Matrix Protein in the Nacreous Layer of Oyster Pearls.

Calcified shell layer is composed of two polymorphs of CaCO(3), aragonite or calcite, and an organic matrix. The organic matrix consists of EDTA-soluble and insoluble fractions. These fractions are thought to regulate the formation of the elaborate shell structure. After decalcification of powdered pearl with 0.3 M EDTA, an EDTA-insoluble fraction was extracted with 0.3 M EDTA/8 M urea. This extraction step enabled us to purify a new class of EDTA-insoluble protein, Pearlin, almost homogeneously. Pearlin has a molecular weight of about 15 kDa and contains a sulfated mucopolysaccharide. We cloned the complementary DNA coding for Pearlin and deduced its complete amino acid sequence. Sequence analysis reveals that Pearlin is composed of 129 amino acids with a high proportion of Gly (10.8%), Tyr (10.0%), Cys (8.5%), Asn (7.7%), Asp (7.7%), and Arg (7.7%). Northern blot analysis showed that Pearlin messenger RNA was expressed specifically in mantle epithelium. From the sequencing data, Pearlin was shown to be quite different from the fibrous protein rich in Ala and Gly. The function of this protein in biomineralization is discussed.

Sequence analysis of Stx2-converting phage VT2-Sa shows a great divergence in early regulation and replication regions.

In enterohemorrhagic Escherichia coli, Shiga toxin is produced by lysogenic prophages. We have isolated the prophage VT2-Sa that is responsible for production of Shiga toxin type 2 protein, and determined the complete nucleotide sequence of this phage DNA. The entire DNA sequence consisted of 60,942 bp, exhibiting marked similarity to the 933W phage genome. However, several differences were observed in the immunity and replication regions, where cI, cII, cIII, N, cro, O, and P genes were present: Predicted amino acid sequences of N, cI, cro, O and P in the VT2-Sa genome did not show significant similarity to the counterparts of the 933W genome; however its cI showed higher similarity to lambda. Furthermore, O and P closely resembled those of phage HK022. These observations suggest that the various degrees of homology observed in the immunity and replication regions of VT2-Sa could have resulted from frequent recombination events among the lambdoid phages, and that these regions play a key role as a functional unit for phage propagation in competition with other lambdoid phages.

Induction of prophages of enterohemorrhagic Escherichia coli O157:H7 with norfloxacin.

Norfloxacin (NFLX) caused induction of prophages VT1 and VT2 of enterohemorrhagic Escherichia coli O157 at subinhibitory concentrations. In time course experiments, we observed the following sequential events: upon induction, the phage genomes underwent multiplication; the amount of stx genes increased; and subsequently, large quantities of toxins VT1 and VT2 were produced. Further studies showed that the molecular mechanism of prophage induction is closely related to the RecA system since the prophage VT2 was not induced with NFLX in a recA mutant strain.

A carbonic anhydrase from the nacreous layer in oyster pearls.

It is believed that the polymorphism observed in calcium carbonate crystals, such as aragonite and calcite in mollusk shells, is controlled by organic matrix proteins secreted from the mantle epithelia. However, the fine structures of these proteins are still unknown, and to understand the molecular mechanisms of mineralization process, detailed structural analyses of the organic matrix proteins are essential. For this, we have carried out purification, characterization, and cDNA cloning of nacrein, which is a soluble organic matrix protein in the nacreous layer of oyster pearls. Northern blot analysis showed that the nacrein transcript was specifically expressed in mantle pallial. Analysis of the deduced amino acid sequence revealed that the protein contained two functional domains: one was a carbonic anhydrase and another was a Gly-Xaa-Asn (Xaa = Asp, Asn, or Glu) repeat domain; however, the carbonic anhydrase domain was split into two subdomains with insertion of the Gly-Xaa-Asn repeat domain between them. Our findings suggest that nacrein actually functions as a matrix protein whose repeated Gly-Xaa-Asn domain possibly binds calcium and as a carbonic anhydrase that catalyzes the HCO3- formation, thus participating in calcium carbonate crystal formation of the nacreous layer.

Targeted disruption of the Rad51 gene leads to lethality in embryonic mice.

The mouse Rad51 gene is a mammalian homologue of the Escherichia coli recA and yeast RAD51 genes, both of which are involved in homologous recombination and DNA repair. To elucidate the physiological role of RAD51 protein, the gene was targeted in embryonic stem (ES) cells. Mice heterozygous for the Rad51 null mutation were intercrossed and their offspring were genotyped. There were no homozygous (Rad51-/-) pups among 148 neonates examined but a few Rad51-/- embryos were identified when examined during the early stages of embryonic development. Doubly knocked-out ES cells were not detected under conditions of selective growth. These results are interpreted to mean that RAD51 protein plays an essential role in the proliferation of cell. The homozygous Rad51 null mutation can be categorized in cell-autonomous defects. Pre-implantational lethal mutations that disrupt basic molecular functions will thus interfere with cell viability.

Cell cycle-dependent expression of the mouse Rad51 gene in proliferating cells.

The mouse Rad51 gene is a mammalian homologue of the Escherichia coli recA and yeast RAD51 genes, both of which are involved in homologous recombination and DNA repair in mitosis and meiosis. The expression of mouse Rad51 mRNA was examined in synchronized mouse m5S cells. The Rad51 transcript was observed from late G1 phase through to M phase. During the period of late G1-S-G2, the RAD51 proteins were observed exclusively in nuclei. Activation by mitogens of T cell and B cell proliferation in spleen induced the expression of Rad51 mRNA. By immunohistochemical analyses, in mouse RAD51 protein was detected in proliferating cells: spermatogonia in testis, immature T cells in thymus, germinal center cells of the secondary lymphatic nodules of spleen and intestine, follicle cells in ovary and epithelial cells in uterus and intestine. It was also expressed in spermatocytes during early and mid-prophase of meiosis and in resting oocytes before maturation. Thus, mouse Rad51 expression is closely related to the state of cell proliferation and is presumably involved in DNA repair coupled with DNA replication, as well as in meiotic DNA recombination in spermatocytes.

Regulation of the mouse histone H2A.X gene promoter by the transcription factor E2F and CCAAT binding protein.

We have molecularly cloned the genomic gene encoding the mouse histone variant H2A.X and characterized the promoter. The promoter region of the H2A.X gene was characterized by chloramphenicol acetyltransferase analysis using Balb/c 3T3 cells. Maximal promoter activity was found in the construct containing up to -282 base pairs H2A.X upstream region. Within this region, we found two sequences regulating the promoter activation; one was an E2F site and another was a CCAAT box. These sequences were also required for the DNA/protein binding activities. Thus, these activities corresponded to the promoter activities, implying that the promoter activity H2A.X gene was controlled by both the transcription factor E2F and H1TF2 through the E2F and CCAAT element. The CCAAT box binding activity was constitutive when cell cycle was progressed by release from G1 arrest, but transiently transfected chloramphenicol acetyltransferase activity slightly increased when cells entered S phase. Similarly, the level of the smallest form of E2F (free E2F) became higher when cells reentered the cell cycle, indicating that the free E2F was one capable of inducing the promoter activation. Thus, the free E2F and CCAAT DNA binding activity correlated with regulation of the promoter activity.

Cloning and sequencing of the gene coding for dextranase from Streptococcus salivarius.

We cloned and sequenced the dextranase (Dex) (1,6-alpha-glucanhydrolase; EC 3.2.1.11)-encoding gene from Streptococcus salivarius (Ss) strain M-33. Recombinant clones from an Ss genomic library specifying Dex activity were identified as colonies surrounded by transparent halos on blue dextran plates. One of the clones had a 4.3-kb KpnI fragment containing the gene coding for an 826-amino-acid polypeptide with a molecular mass of 87.9 kDa, which corresponds well to that of native Dex from the Ss culture supernatant. There was no sequence homology between the gene encoding Ss Dex and the gene encoding dextran glucosidase of S. mutans, or between their protein products.

Activation of the mouse cytokeratin A (endo A) gene in teratocarcinoma F9 cells by the histone deacetylase inhibitor Trichostatin A.

Treatment of cultured cells with sodium butyrate, that is the histone deacetylase inhibitor, induces the histone hyperacetylation and the expressions of various mammalian genes without affecting the level of protein synthesis. However, butyrate is a non-specific inhibitor of deacetylase because of its effects on various other enzymes and nuclear proteins other than histones. On the other hand, Trichostatin A (TSA) was recently found to be a potent and specific inhibitor of histone deacetylase. We examined the effect of TSA on the expression of mouse cytokeratin A (endo A). TSA increased endoA expression in F9 cells, and was effective at a much lower concentration than sodium butyrate. We also examined the changes of chromatin structure induced by the two drugs by a DNase I-hypersensitivity assay. Both drugs induced the formation of a DNase I-hypersensitive site (DH site) in only the promoter region. The precise mechanism(s) by which the two drugs increase endoA gene expression is unknown, but these results suggest that endoA expression is induced by inhibition of histone deacetylase and that the effect is at the transcriptional level.

Dynamic properties of nucleic acids in biosupramolecular systems, as studied by 31P NMR.

The dynamic properties of nucleic acids in five different types of intact supramolecular systems, namely, chicken erythrocyte chromatin, the wild type and a deletion mutant of the lambda phage, lipid-containing phage PM2, and Alteromonas espejiana ribosomes, were investigated by means of 31P solid-state NMR. The nucleic acids in the different supramolecular systems showed unique dynamic properties, which are closely connected with their functions. The total anisotropy of the phosphorus chemical shift (delta sigma = sigma 33-sigma 11) of the ribosomes was 210 ppm at 5 degrees C. This anisotropy was much larger than those of any DNA complexes, suggesting the highly rigid structure of ribosomal RNA. In contrast, 160 ppm was the largest chemical shift anisotropy at 5 degrees C for B-form DNA in the supramolecular systems. This flexibility would be essential for DNAs to exert their functions. The involvement of a condensation protein in the PM2 phage was supported by the chemical shift anisotropy. The spin-lattice relaxation time in the proton rotating frame [T1 rho(H)] of the nucleic acids became shorter with the increase in the effective field in the rotating frame for all systems examined, showing that the motions of the nucleic acids effective for the relaxation are in the slow motional regime or in the range of omega 1 tau c = 1 at 5 degrees C. The motional state of DNA of the lambda phage was found to change at about 20 degrees C on the basis of the temperature dependence of the spin-lattice relaxation time of phosphorus (T1).(ABSTRACT TRUNCATED AT 250 WORDS)

The complete sequence of the gene encoding mouse cytokeratin 15.

To characterize the type-I keratin-encoding gene family around the mouse keratin 19-encoding gene (K19, EndoC), which encodes simple epithelial-type cytokeratin (CK), we screened a mouse genomic library by hybridization to a K19 cDNA probe. One clone of 16 kb contained the second to the sixth exons of K19 and the other keratin-encoding gene was located about 4 kb downstream from K19. Sequencing, Northern hybridization and genomic Southern blotting revealed that the downstream gene encodes the mouse K15 gene. This gene consists of eight exons and the positions of the introns coincide with those of other type-I keratin-encoding genes. The 5' upstream regions of the mouse and human K15 genes contain homologous sequences around the respective TATA boxes, suggesting that the same factors are involved in the regulation of their transcription.

Chromosome mapping of the human (RECA) and mouse (Reca) homologs of the yeast RAD51 and Escherichia coli recA genes to human (15q15.1) and mouse (2F1) chromosomes by direct R-banding fluorescence in situ hybridization.

We mapped the human (RECA) and mouse (Reca) homologs of the yeast RAD51 and Escherichia coli recA genes to human and mouse chromosomes by direct R-banding fluorescence in situ hybridization. This gene was assigned to human chromosome 15q15.1 and to mouse chromosome 2F1, respectively. This is the first report on the precise localization of this gene to human and mouse chromosomes. This gene was mapped to a region on human chromosome 15q15.1 and mouse 2F1 that is believed to be a conserved syntenic group.

Expression and secretion of an Arthrobacter dextranase in the oral bacterium Streptococcus gordonii.

We have constructed a plasmid to express and secrete dextranase in the oral bacterium Streptococcus gordonii. The dextranase gene from Arthrobacter sp. strain CB-8 was linked to a promoter and a DNA sequence encoding the signal peptide of Streptococcus downei glucosyltransferase I (gtfI) followed by the Escherichia coli rrnBt1t2 terminator and inserted in the shuttle vector pVA838. S. gordonii transformed with this plasmid (pMNK-4) expressed and secreted mature Arthrobacter dextranase. The transformant was found to repress the firm adherence of water-insoluble glucan in a coculture experiment with cariogenic bacteria, Streptococcus sobrinus, in the presence of sucrose. Such genetically engineered oral bacteria could provide a therapy to prevent dental caries.

A mouse homolog of the Escherichia coli recA and Saccharomyces cerevisiae RAD51 genes.

Analysis of mitotic and meiotic recombination in mammalian cells has been hampered by the complexity of the reactions involved as well as lack of mutants. Furthermore, none of the genes involved in the process has yet been identified. In budding yeast, Saccharomyces cerevisiae, the RAD51 gene is essential along with other genes of the RAD52 epistasis group for mitotic and meiotic recombination and DNA repair. The Rad51 protein is structurally similar to Escherichia coli RecA protein, which is required in homologous recombination and SOS responses in bacteria. Here we report the isolation of a mouse homolog of the yeast RAD51 gene. The amino acid sequence predicted from the gene shows 83% and 55% homology with those of the yeast RAD51 and the E. coli recA product, respectively. The mouse gene complemented a rad51 mutation of S. cerevisiae with sensitivity to methyl-methanesulfonate, which produces double-strand breaks of DNA. This gene is expressed in the thymus, testis, ovary, spleen, and intestine, suggesting that its product is involved in mitotic and meiotic recombination in addition to DNA repair.

Sequence specific binding of Ets-1 to the mouse cytokeratin EndoA gene enhancer.

Expression of the mouse cytokeratin EndoA gene is regulated by an enhancer that is located 1 kilobases (kb) 3' downstream from the gene. The EndoA enhancer consists of six direct repeats with homology to the PEA3 motif in the polyoma virus enhancer core. The PEA3 motif binds to the proto-oncogene Ets product. In this report we show that GST-c-Ets-1 fusion protein binds specifically to the EndoA enhancer unit sequence by gel shift analysis. Footprinting showed that the Ets-1 binding site consisted of 20 bases in each repeat. Ets-1 also moderately activated the EndoA enhancer. These results demonstrate that Ets-1 binds in a sequence specific fashion to the mouse EndoA enhancer and suggest that c-Ets-1 or other related Ets family genes function in regulating EndoA gene expression.

Identification of differentiation-dependent DNase I-hypersensitive sites in the mouse EndoA gene.

DNase I hypersensitive (DH) sites in a 12-kb genomic fragment carrying the mouse EndoA gene were examined to obtain information on the changes in chromatin structure associated with activation of this gene encoding extra-endodermal cytoskeletal protein A (EndoA) during early mouse embryogenesis. Seven DH sites were found in this locus in parietal yolk-sac-like cells, PYS-2, which produce EndoA constitutively. In differentiated mouse teratocarcinoma F9 cells that produce EndoA inductively, this locus has three DH sites. In both cell lines, these sites were mapped to the upstream region of the promoter, the promoter and the 3' enhancer region. The DNA of PYS-2 cells has one more DH site within the first exon and three additional DH sites within the first intron. These DH sites are not present in DNA from BALB/c 3T3 cells and undifferentiated F9 cells that do not produce EndoA. Thus, the formation of these differentiation-dependent DH sites is required for the differentiation-specific expression of the mouse EndoA. In addition, another strong DH site, which may be associated with the B2 element expression, was detected in the third intron of the gene in undifferentiated F9 cells.

Molecular cloning of a novel mRNA sequence expressed in cleavage stage mouse embryos.

In an approach to study genes transcribed during early mouse development, a cDNA library was constructed from poly(A) RNA isolated from the 8-cell morula. The cDNA library was differentially screened with labelled cDNA probes synthesized on poly(A) RNA isolated from the 8-cell morula or unfertilized eggs. Six clones which increased in abundance in the 8-cell morula were selected and further analyzed. Sequencing analyses showed that some of these clones corresponded to RNA transcripts from B1 and B2 repetitive sequences, as well as mRNA for cytochrome C oxidase I and NADH dehydrogenase III derived from the mitochondrial genome. One clone was not identical to any known sequences. The unidentified sequence (MO25) was found at low levels in the unfertilized egg, but increased at the 2-cell stage. The predicted amino acid sequence revealed that the MO25 gene may encode a Ca2+ binding protein.