The c-terminal DNA-binding domain ofChironomus BR gene products shows preferential affinity for (dA,dT)-rich sequences

Balbiani ring genes (BRs), the most active loci in the polytene chromosomes of the salivary gland of the midgeChironomus (Diptera), code for secretory giant peptides (the sp-I family). Evidence previously reported indicated that the conserved C-terminal region of proteins of the sp-I family had DNA-binding properties (assayed with sp-Ia), and one such region, derived fromBR2.2, which codes for the product sp-Ib, might occur as a stable independent peptide, being transferred to the nucleus where it is detectable in the largeBRs (BR1 andBR2), among other structures, by immunostaining. Here, we show that the C-terminal portion of one of theBR gene products, expressed as a glutathione-S-transferase fusion protein shows preferential affinity for A.T-rich sequences and binds with varying affinity to restriction fragments of the A.T-rich BR1 promoter. The binding was inhibited by distamycin, suggesting that the interaction involves the minor groove of the DNA. Analysis of the promoter fragments by gel electrophoresis indicated that most appeared to present a conspicuous bend, as deduced from their anomalous electrophoretic mobilities. Furthermore, the affinity of the C-terminal domain for the different promoter fragments appeared to correlate with the degree of bending. Thus, the C-terminal domain might play a role in controlling gene expression by binding to A.T-rich sequences, including those of theBR genes.     

Molecular structure and flanking nucleotide sequences of the natural chicken ovomucoid gene

Five independent clones containing the natural chicken ovomucoid gene have been isolated from a chicken gene library. One of these clones, CL21, contains the complete ovomucoid gene and includes more than 3 kb of DNA sequences flanking both termini of the gene. Restriction endonuclease mapping, electron microscopy and direct DNA sequencing analyses of this clone have revealed that the ovomucoid gene is 5.6 kb long and codes for a messenger RNA of 821 nucleotides. The structural gene sequence coding Ifor the mature messenger RNA is split into at least eight segments by a minimum of seven intervening sequences of various sizes. The shortest structural gene segment is only 20 nucleotides long. All seven intervening sequences are located within the peptide coding region of the gene, and the sequences at the 5' and 3' untranslated regions of the mRNA are not interrupted by intervening sequences. The DNA sequences of the regions flanking the 5' and 3' termini of the gene have been determined. Thirty nucleotides before the start of the messenger RNA coding sequence is the heptanucleotide TATATAT, which is also present in a similar location relative to the chicken ovalbumin gene and other unique sequence eucaryotic genes. This sequence resembles that of the Pribnow box in procaryotic genes where a promoter function has been implicated. Seven nucleotides past the 3' end of the gene is the tetranucleotide TTGT, a sequence found to be present at identical locations as either TTTT or TTGT in other eucaryotic genes that have been sequenced. These conserved DNA sequences flanking eucaryotic genes may serve some regulator function in the expression of these genes.     

The c-terminal DNA-binding domain ofChironomus BR gene products shows preferential affinity for (dA,dT)-rich sequences

Balbiani ring genes (BRs), the most active loci in the polytene chromosomes of the salivary gland of the midgeChironomus (Diptera), code for secretory giant peptides (the sp-I family). Evidence previously reported indicated that the conserved C-terminal region of proteins of the sp-I family had DNA-binding properties (assayed with sp-Ia), and one such region, derived fromBR2.2, which codes for the product sp-Ib, might occur as a stable independent peptide, being transferred to the nucleus where it is detectable in the largeBRs (BR1 andBR2), among other structures, by immunostaining. Here, we show that the C-terminal portion of one of theBR gene products, expressed as a glutathione-S-transferase fusion protein shows preferential affinity for A.T-rich sequences and binds with varying affinity to restriction fragments of the A.T-rich BR1 promoter. The binding was inhibited by distamycin, suggesting that the interaction involves the minor groove of the DNA. Analysis of the promoter fragments by gel electrophoresis indicated that most appeared to present a conspicuous bend, as deduced from their anomalous electrophoretic mobilities. Furthermore, the affinity of the C-terminal domain for the different promoter fragments appeared to correlate with the degree of bending. Thus, the C-terminal domain might play a role in controlling gene expression by binding to A.T-rich sequences, including those of theBR genes.     

Comparison of the solution and crystal conformations of (G + C)-rich fragments of DNA.

DNA fragments crystallize in an unpredictable manner, and relationships between their crystal and solution conformations still are not known. We have studied, using circular dichroism spectroscopy, solution conformations of (G + C)-rich DNA fragments, the crystal structures of which were solved in the laboratory of one of the present authors. In aqueous trifluorethanol (TFE) solutions, all of the examined oligonucleotides adopted the same type of double helix as in the crystal. Specifically, the dodecamer d(CCCCCGCGGGGG) crystalized as A-DNA and isomerized into A-DNA at high TFE concentrations. On the other hand, the hexamer d(CCGCGG) crystallized in Z-form containing tilted base pairs, and high TFE concentrations cooperatively transformed it into the same Z-form as adopted by the RNA hexamer r(CGCGCG), although d(CCGCGG) could isomerize into Z-DNA in the NaCl + NiCl2) aqueous solution. The fragments crystallizing as B-DNA remained B-DNA, regardless of the solution conditions, unless they denatured or aggregated. Effects on the oligonucleotide conformation of 2-methyl-2,4-pentanediol and other crystallization agents were also studied. 2-Methyl-2,4-pentanediol induced the same conformational transitions as TFE but, in addition, caused an oligonucleotide condensation that was also promoted by the other crystallization agents. The present results indicate that the crystal double helices of DNA are stable in aqueous TFE rather than aqueous solution.     

Sequences in human cytomegalovirus which hybridize with the avian retrovirus oncogene v-myc are G + C rich and do not hybridize with the human c-myc gene.

The degree of relatedness between previously identified cross-hybridizing regions within human cytomegalovirus strain AD169 and the avian retrovirus oncogene v-myc were investigated by nucleotide sequence comparison. We found that the homologous regions between the human cytomegalovirus genome and v-myc are limited to short G + C-rich regions in each genome and that the human cytomegalovirus genome shares little or no homology with the human c-myc gene.     

Identification of three sequence-specific DNA-binding proteins which interact with the Rous sarcoma virus enhancer and upstream promoter elements.

Three avian nuclear proteins which bind to the Rous sarcoma virus long terminal repeat have been detected. Two of the proteins bind to sequences within the enhancer, and the third protein binds to a sequence spanning the enhancer and an upstream promoter region.     

Identification of Archaea-specific chemotaxis proteins which interact with the flagellar apparatus

Background

Prevotella intermedia (P. intermedia), a gram-negative, black-pigmented anaerobic rod, has been implicated in the development of chronic oral infection. P. intermedia strain 17 was isolated from a chronic periodontitis lesion in our laboratory and described as a viscous material producing strain. The stock cultures of this strain still maintain the ability to produce large amounts of viscous materials in the spent culture media and form biofilm-like structures. Chemical analyses of this viscous material showed that they were mainly composed of neutral sugars with mannose constituting 83% of the polysaccharides. To examine the biological effect of the extracellular viscous materials, we identified and obtained a naturally-occurring variant strain that lacked the ability to produce viscous materials in vitro from our stock culture collections of strain 17, designated as 17-2. We compared these two strains (strains 17 versus 17-2) in terms of their capacities to form biofilms and to induce abscess formation in mice as an indication of their pathogeniCity. Further, gene expression profiles between these two strains in planktonic condition and gene expression patterns of strain 17 in solid and liquid cultures were also compared using microarray assays.

Results

Strain 17 induced greater abscess formation in mice as compared to that of the variant. Strain 17, but not 17-2 showed an ability to interfere with the phagocytic activity of human neutrophils. Expression of several genes which including those for heat shock proteins (DnaJ, DnaK, ClpB, GroEL and GroES) were up-regulated two to four-fold with statistical significance in biofilm-forming strain 17 as compared to the variant strain 17-2. Strain 17 in solid culture condition exhibited more than eight-fold up-regulated expression levels of several genes which including those for levanase, extracytoplasmic function-subfamily sigma factor (σ^E; putative) and polysialic acid transport protein (KpsD), as compared to those of strain 17 in liquid culture media.

Conclusion

These results demonstrate that the capaCity to form biofilm in P. intermedia contribute to their resistance against host innate defence responses.     

Identification of Archaea-specific chemotaxis proteins which interact with the flagellar apparatus

Background  

Archaea share with bacteria the ability to bias their movement towards more favorable locations, a process known as taxis. Two molecular systems drive this process: the motility apparatus and the chemotaxis signal transduction system. The first consists of the flagellum, the flagellar motor, and its switch, which allows cells to reverse the rotation of flagella. The second targets the flagellar motor switch in order to modulate the switching frequency in response to external stimuli. While the signal transduction system is conserved throughout archaea and bacteria, the archaeal flagellar apparatus is different from the bacterial one. The proteins constituting the flagellar motor and its switch in archaea have not yet been identified, and the connection between the bacterial-like chemotaxis signal transduction system and the archaeal motility apparatus is unknown.     

Identification of nuclear factors which interact with the 5' flanking region of the EF-1 alpha O gene in Xenopus laevis.

The EF-1 alpha O gene of Xenopus laevis is a stage-specific gene, being transcribed in oogonia and oocytes, but not in postmeiotic germ cells and terminally differentiated cells. We found that two trans-acting factors from oocyte nuclear extract are able to interact with a DNA sequence in the 5'-upstream region of the EF-1 alpha O gene. Methylation interference experiments suggested that the two factors recognised the same DNA element. Gel retardation assays indicated that part of the protein binding site could be confined to a 21 bp sequence, located between -51 and -72, relative to the cap site. Interestingly, this region shares great homology to a negative regulatory segment in the promoter of the TFIIIA gene, another developmentally regulated gene.     

cDNA clones encoding leucine-zipper proteins which interact with G-CSF gene promoter element 1-binding protein.

The gene expression of granulocyte colony-stimulating factor (G-CSF) is induced by lipopolysaccharide (LPS). GPE1, a cis-controlling element of the G-CSF gene, functions as an LPS-responsive element. GPE1-binding protein (GPE1-BP), a leucine-zipper protein, did not independently activate G-CSF gene expression. Protein blot analysis with biotinylated GPE1-BP revealed that there were nuclear proteins that interact specifically with GPE1-BP. Three leucine-zipper proteins were isolated from mouse cDNA expression libraries by this method: NF-IL6, ATF4, and a novel ATF4-related ATFx. The interactions of these proteins with GPE1-BP may play key roles in G-CSF gene expression.     

Novel G proteins, Rag C and Rag D, interact with GTP-binding proteins, Rag A and Rag B

Rag A/Gtr1p are G proteins and are known to be involved in the RCC1-Ran pathway. We employed the two-hybrid method using Rag A as the bait to identify proteins binding to Rag A, and we isolated two novel human G proteins, Rag C and Rag D. Rag C demonstrates homology with Rag D (81.1% identity) and with Gtr2p of Saccharomyces cerevisiae (46.1% identity), and it belongs to the Rag A subfamily of the Ras family. Rag C and Rag D contain conserved GTP-binding motifs (PM-1, -2, and -3) in their N-terminal regions. Recombinant glutathione S-transferase fusion protein of Rag C efficiently bound to both [(3)H]GTP and [(3)H]GDP. Rag A was associated with both Rag C and Rag D in their C-terminal regions where a potential leucine zipper motif and a coiled-coil structure were found. Rag C and D were associated with both the GDP and GTP forms of Rag A. Both Rag C and Rag D changed their subcellular localization, depending on the nucleotide-bound state of Rag A. In a similar way, the disruption of S. cerevisiae GTR1 resulted in a change in the localization of Gtr2p.