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PubMed2000

The tryptophan synthase-encoding trpB gene of Aspergillus nidulans is regulated by the cross-pathway control system.

The tryptophan synthase-encoding gene, trpB, of Aspergillus nidulans was cloned and characterized. It was mapped to chromosome I, between the gene medA, which is required for sexual and asexual development, and an ORF encoding a protein with significant similarity to subunit B of vacuolar ATP synthases. The 5' untranslated region was found to be at least 142 nucleotides (nt) long, the poly(A) addition site was localized at position + 216 relative to the stop codon by sequencing of several independent cDNA clones. The trpB gene contains two exons separated by an intron of 105 nt, which is located close to the 5' end of the ORF. Directly upstream of the transcriptional start site, one well conserved potential binding site for the cross-pathway control transcriptional activator CPCA was found. The level of trpB transcript was shown to be regulated by cross-pathway control. A knockout mutant for trpB displays tryptophan auxotrophy, no trpB transcript is detectable, and development is perturbed to an extent that is dependent on the amount of tryptophan added to the medium. The trpB gene encodes a protein of 723 amino acids, with a calculated molecular weight of 77.6 kDa. The deduced amino acid sequence shows 72.6% similarity to the tryptophan synthase of Neurospora crassa. Most amino acid residues essential for catalytic activity in the tryptophan synthase of Salmonella typhimurium are conserved. The linker region joining the two domains of the enzyme is 13 residues longer than the longest connector found so far in tryptophan synthases from fungi.

Amino Acid Sequence↗
PubMed1990

The E7 protein of human papillomavirus 8 is a nonphosphorylated protein of 17 kDa and can be generated by two different mechanisms.

The E7 protein of human papillomavirus (HPV) 8 shows no in vitro transforming activity, in contrast to E7 of the genital HPVs, but seems to be involved in the control of viral DNA replication. To determine whether functional differences between the E7 proteins of HPV16 and HPV8 were reflected in differences in their biochemical properties, we characterized the E7 protein of HPV8 expressed from the late simian virus 40 promoter in COS 7 cells. An E7-specific antiserum was obtained by immunizing rabbits with a beta-gal-E7 fusion protein containing all of the E7 polypeptide. This antiserum specifically precipitated from [35S]cysteine but not from 32PO4-labeled transiently transfected COS 7 cells a protein with a low mobility of 17 kDa in SDS-PAGE. The high sedimentation rate in nondenaturing glycerol gradients pointed to an interaction with other proteins or to the existence of E7 oligomers. An association with the retinoblastoma protein could, however, be excluded. The 5' ends of HPV8 transcripts derived from the E6-E7 region in G418 selected rodent cells, which were mapped by nuclease S1 digestion, are located upstream of ORFs E6 and E7, respectively. No evidence for an E6*I-like mRNA was found. In COS 7 cells transfected with a plasmid expressing the E6-E7 region of HPV8 under the control of the late SV40 promoter, however, the E7 protein was translated from a polycistronic mRNA potentially encoding E6 and E7. These data indicate that the E7 protein of HPV8 may be expressed in two ways: (i) through translation of an E7-specific mRNA, as with HPV6 and HPV11, or (ii) through internal initiation of translation at the ATG of E7.

Animals↗
PubMed1991

The oac gene encoding a lipopolysaccharide O-antigen acetylase maps adjacent to the integrase-encoding gene on the genome of Shigella flexneri bacteriophage Sf6.

Lysogens of Shigella flexneri harbouring the temperate bacteriophage, Sf6, have been previously shown to undergo a serotype conversion due to O-acetylation of the O-antigen of the lipopolysaccharide. A partial physical map of the phage genome has been constructed. Analysis of the phage DNA suggests that the phage packages by a headful mechanism and that the mature DNA molecules are terminally redundant. Cloning of the PstI fragments of Sf6 enabled the region encoding the serotype conversion to be localized, showing that this was clearly phage-encoded. The gene was further localized by mutagenesis with Tn5 and the nucleotide sequence of the entire 2693-bp PstI fragment was determined. Two major open reading frames (ORFs) were found capable of encoding proteins of 44.1 and 37.2 kDa. The latter corresponds to the O-antigen acetylase and its gene has been designated oac. The oac gene is capable of converting Sh. flexneri serotypes X, Y, 1a and 4a to 3a, 3b, 1b and 4b, respectively. The Oac protein bears a high degree of homology to the NodX protein of Rhizobium leguminosarum suggesting that it, too, may be a sugar acetylase. The second ORF immediately upstream from oac corresponds to the bacteriophage Sf6 integrase responsible for chromosomal integration and is highly homologous to the integrases of Escherichia coli bacteriophages P4 and phi 80, but less closely related to those of P1, P2, P22, 186 and lambda.

Acetylesterase↗
PubMed1993

The pab gene of Streptomyces griseus, encoding p-aminobenzoic acid synthase, is located between genes possibly involved in candicidin biosynthesis.

The nucleotide (nt) sequence of the gene (pab) encoding p-aminobenzoic acid (PABA) synthase, a key enzyme in the biosynthesis of candicidin by Streptomyces griseus IMRU3570, was determined and an open reading frame (ORF) of 2171 nt was found. The predicted amino acid sequence demonstrated extensive sequence identity with PABA synthases (Pab) from Gram-negative Enterobacteria. The protein encoded by ORF pab shows a clear relationship at the N terminus with PabA and at the C terminus with PabB from Escherichia coli, Serratia and Klebsiella. We also determined the extent of a spontaneous deletion that removed the ORF located upstream from pab near the 5' end of the cloned fragment. The deletion occurred when the gene was cloned in the BamHI site of pBR322 and allowed pab expression in E. coli.

Amino Acid Sequence↗
PubMed1994

Biosynthesis of butirosin in Bacillus circulans NRRL B3312: identification by sequence analysis and insertional mutagenesis of the butB gene involved in antibiotic production.

As an approach to an analysis of the biosynthesis of the aminoglycoside antibiotic butirosin (But), we investigated the chromosomal regions flanking the ButR gene (aphA4/butA) of Bacillus circulans NRRL-B3312, and have identified, by nucleotide sequence analysis, a large open reading frame (ORF; ButB) upstream from the ButR gene. Hybridization was detected between butB and chromosomal DNA from other Bacillaceae that produce But-like compounds (but not from non-producers). Interruption of this sequence by insertion of an erythromycin-resistance-encoding gene (erm) at either of two distinct sites eliminated the production (biosynthesis or export) of But, thus indicating a role for butB in antibiotic production. Gene butB is transcribed in the same direction as butA and encodes a protein of 1616 amino acid (aa) residues with a 30-aa N-terminal signal peptide. Comparison of the sequence for the translation product (ButB) with the aa compositions and sequences of known bacterial surface proteins, such as S-layer proteins, suggests that this protein is cell-wall associated. It is proposed that ButB plays a role in the export of But from the producing organism.

Amino Acid Sequence↗
PubMed1994

Conservation of the 168 divIB gene in Bacillus subtilis W23 and B. licheniformis, and evidence for homology to ftsQ of Escherichia coli.

The chromosomal regions of Bacillus subtilis (Bs) W23 and Bacillus licheniformis (Bl), which span the sequence encoding the homolog of the division initiation gene, divIB, of Bs168 were cloned and sequenced. The high level of conservation of the amino acid (aa) sequence of the DivIB protein (99 and 68% identity for BsW23 and Bl, respectively) was consistent with a significant role for this protein in the cell cycle of the two species. The hydropathy profile for DivIB of Bl was almost identical to that of Bs168 and consistent with a membrane location, as previously established for the latter. The higher than average level of identity (87%) of the 31-aa N-terminal cytoplasmic domain of DivIB between Bs168 and Bl raised the possibility of a special role for this domain. Database analyses using the Bl DivIB sequence and similarity analyses also strongly suggested that DivIB, of Bl and Bs, is a homolog of FtsQ of Escherichia coli. The flanking sequences extending into the unidentified orfs both upstream and downstream from divIB were highly conserved between Bs168 and Bl at both the nucleotide and aa levels. It was confirmed that orf4 of Bs168 is dispensable.

Amino Acid Sequence↗
PubMed1997

Polypurine/polypyrimidine sequences as cis-acting transcriptional regulators.

Genome sequence information has generated increasing evidence for the claim that repetitive DNA sequences present within and around genes could play a important role in the regulation of gene expression. Polypurine/polypyrimidine sequences [poly(Pu/Py)] have been observed in the vicinity of promoters and within the transcribed regions of many genes. To understand whether such sequences influence the level of gene expression, we constructed several prokaryotic and eukaryotic expression vectors incorporating poly(Pu/Py) repeats both within and upstream of a reporter gene, lacZ (encoding beta-galactosidase), and studied its expression in vivo. We find that, in contrast to the situation in Escherichia coli, the presence of poly(Pu/Py) sequences within the gene does not significantly inhibit gene expression in mammalian cells. On the other hand, the presence of such sequences upstream of lacZ leads to a several-fold reduction of gene expression in mammalian cells. Similar down-regulation was observed when a structural cassette containing poly(Pu/Py) sequences upstream of lacZ was integrated into yeast chromosome V. Sequence analysis of the nine totally sequenced yeast chromosomes shows that a large number of such sequences occur upstream of ORFs. On the basis of our experimental results and DNA sequence analysis, we propose that these sequences can function as cis-acting transcriptional regulators.

Animals↗
PubMed2003

Identity and conservation of mating type genes in geographically diverse isolates of Phaeosphaeria nodorum.

Mating type idiomorphs (MAT1-1 and MAT1-2) were identified from the heterothallic loculoascomycete Phaeosphaeria nodorum (wheat biotype) using DNA from a pair of isolates from Poland and Georgia, USA that are known to mate. MAT predicted proteins of P. nodorum are similar in sequence and in phylogenetic relationship to those described for other loculoascomycetes such as Cochliobolus spp., Alternaria alternata, and Didymella zeae-maydis. The organization of the MAT locus of the P. nodorum differs from these species in that its idiomorph begins within an adjacent upstream conserved ORF of unknown function. MAT-specific primers were used to identify isolates of both mating types in field populations, demonstrating that an absence of either mating type is not the reason that the teleomorph has not been found in New York. Portions of MAT1-1 and MAT1-2 were sequenced from geographically diverse isolates, including those from regions where the teleomorph has been reported. MAT was highly conserved and no significant differences in sequence were found.

Amino Acid Sequence↗
PubMed2003

Highly abundant pea LTR retrotransposon Ogre is constitutively transcribed and partially spliced.

We have isolated and characterized a novel giant retroelement, named Ogre, which is over 22 kb long and makes up at least 5% of the pea (Pisum sativum L.) genome. This element can be classified as a Ty3/gypsy-like LTR retrotransposon based on the presence of long terminal repeats (LTRs) and the order of the domains coding for typical retrotransposon proteins. In addition to its extreme length, it has several features which make it unique among the retroelements described so far: (1) the sequences coding for gag and prot proteins are separated from the rt/rh-int domains by several stop codons; (2) the region containing these stop codons is removed from the element transcripts by splicing which results in reconstitution of the complete gag-pol coding sequence; (3) only a part of the transcripts is spliced which probably determines the ratio of translated proteins; (4) the element contains an extra ORF located upstream the gag-pol coding sequences, potentially coding for a protein of 546-562 amino acids with unknown function. The transcriptional activity of the Ogre elements has been detected in all organs tested (leaves, roots, flowers) as well as in wounded leaves and protoplasts. Considering this retroelement's constitutive expression and observed high mutual similarity of the element genomic sequences, it is possible to speculate about its recent amplification in the genomes of pea and other legume plants.

Alternative Splicing↗
PubMed1999

A protein residing at the subunit interface of the bacterial ribosome.

Surface labeling of Escherichia coli ribosomes with the use of the tritium bombardment technique has revealed a minor unidentified ribosome-bound protein (spot Y) that is hidden in the 70S ribosome and becomes highly labeled on dissociation of the 70S ribosome into subunits. In the present work, the N-terminal sequence of the protein Y was determined and its gene was identified as yfia, an ORF located upstream the phe operon of E. coli. This 12.7-kDa protein was isolated and characterized. An affinity of the purified protein Y for the 30S subunit, but not for the 50S ribosomal subunit, was shown. The protein proved to be exposed on the surface of the 30S subunit. The attachment of the 50S subunit resulted in hiding the protein Y, thus suggesting the protein location at the subunit interface in the 70S ribosome. The protein was shown to stabilize ribosomes against dissociation. The possible role of the protein Y as ribosome association factor in translation is discussed.

Amino Acid Sequence↗
PubMed2000

Complex promoter and coding region beta 2-adrenergic receptor haplotypes alter receptor expression and predict in vivo responsiveness.

The human beta(2)-adrenergic receptor gene has multiple single-nucleotide polymorphisms (SNPs), but the relevance of chromosomally phased SNPs (haplotypes) is not known. The phylogeny and the in vitro and in vivo consequences of variations in the 5' upstream and ORF were delineated in a multiethnic reference population and an asthmatic cohort. Thirteen SNPs were found organized into 12 haplotypes out of the theoretically possible 8,192 combinations. Deep divergence in the distribution of some haplotypes was noted in Caucasian, African-American, Asian, and Hispanic-Latino ethnic groups with >20-fold differences among the frequencies of the four major haplotypes. The relevance of the five most common beta(2)-adrenergic receptor haplotype pairs was determined in vivo by assessing the bronchodilator response to beta agonist in asthmatics. Mean responses by haplotype pair varied by >2-fold, and response was significantly related to the haplotype pair (P = 0.007) but not to individual SNPs. Expression vectors representing two of the haplotypes differing at eight of the SNP loci and associated with divergent in vivo responsiveness to agonist were used to transfect HEK293 cells. beta(2)-adrenergic receptor mRNA levels and receptor density in cells transfected with the haplotype associated with the greater physiologic response were approximately 50% greater than those transfected with the lower response haplotype. The results indicate that the unique interactions of multiple SNPs within a haplotype ultimately can affect biologic and therapeutic phenotype and that individual SNPs may have poor predictive power as pharmacogenetic loci.

Base Sequence↗
PubMedDate not supplied

Azorhizobium caulinodans nitrogen fixation (nif/fix) gene regulation: mutagenesis of the nifA -24/-12 promoter element, characterization of a ntrA(rpoN) gene, and derivation of a model.

Using site-directed mutagenesis, mutations were introduced in the -24/-12 promoter element of the Azorhizobium caulinodans nifA gene, and chimeric nifA-lacZ reporter gene fusions were constructed. Single base-pair mutations in the conserved -25 or -13 G residues were found to reduce or abolish nifA promoter activity, respectively, demonstrating that the -24/-12 promoter element is important for nifA gene expression and suggesting the involvement of a sigma 54 (NtrA/RpoN)-type transcription factor in nifA gene regulation. A 2-bp mutation at positions -25 and -16 was found to create a relatively nitrogen-control-independent, highly expressed nifA promoter. Using a heterologous ntrA(rpoN) gene probe, an A. caulinodans ntrA(rpoN)-like gene was cloned and the DNA sequence of this gene and flanking regions was determined. The presence of three open reading frames (ORF1-3) was demonstrated. ORF2 was found to contain regions sharing a high degree of homology with all characterized bacterial ntrA(rpoN) genes. ORF1 was found to share homology with ORFs found upstream of other bacterial ntrA(rpoN) genes, which have been postulated to encode members of a superfamily of ATP-binding proteins. Transposon Tn5 insertion mutations were introduced into the cloned ntrA(rpoN) gene, and chromosomal ntrA(rpoN)::Tn5 A. caulinodans mutants were created. The resulting mutants were found to be unable to fix nitrogen in the free-living state (Nif- in culture) or in stem or root nodules induced on Sesbania rostrata (Fix- in planta), and to be unable to grow aerobically in the presence of nitrate as sole nitrogen source (Ntr-). A nifH-lacZ gene fusion was found to be silent in ntrA(rpoN)::Tn5 mutant strains, but a nifA-lacZ gene fusion was found to be expressed at a wild-type level, suggesting that the ntrA(rpoN) gene identified here controls the expression of some of the A. caulinodans nif genes, but not the central nif regulatory gene nifA. Based on these results, a new model for the regulation of nif/fix gene expression in A. caulinodans is proposed.

Amino Acid Sequence↗
PubMed1988

Sequence analysis of an 11.2 kilobase, near-terminal, BamHI fragment of fowlpox virus.

The nucleotide sequence of an 11.2 kilobase fragment of the fowlpox virus genome is presented. The fragment comes from near one end of the genome and contains part of the terminal inverted repeat. Twenty open reading frames (ORFs) are predicted from the sequence and are classified into 13 major and seven minor ORFs. The 100 base pairs immediately upstream of each ORF are up to 83% AT-rich, with some motifs similar to those seen in vaccinia virus early gene promoters. The TTTTTNT element which has been identified as a termination signal for vaccinia virus early genes is also found downstream of several ORFs. Three ORFs are predicted to specify polypeptides with significant homology to proteins coded by genes near termini of orthopoxvirus genomes: the vaccinia virus 42K early gene and 32.5K host range gene, and the cowpox virus 38K red pock gene. In addition, there are two families of ORFs within the fragment which potentially encode related polypeptides. One of these, family B, contains three ORFs which are related to those of chicken and rat hepatic lectins.

Amino Acid Sequence↗
PubMed2000

Identification of new loci involved in adhesion of Listeria monocytogenes to eukaryotic cells. European Listeria Genome Consortium.

Insertional mutagenesis was performed with Tn1545 in the genetic background of an inIAB deletion mutant to identify new adhesion determinants in Listeria monocytogenes. Four insertion mutants defective in adhesion to eukaryotic cells were identified. Insertion sites were cloned by inverse-PCR and sequenced. The genetic organization of insertion regions was further analysed by screening and sequencing DNA fragments from a HindIII library and by searching databases. Three adhesion-defective mutants each had one copy of Tn1545 inserted into their chromosome. The insertion sites were different in the three mutants: (i) upstream from two ORFs in tandem, similar to dfp and priA of Bacillus subtilis, respectively; (ii) within an ORF encoding a putative 126 amino-acid-polypeptide with no significant similarity to any known protein; (iii) within an ORF similar to a B. subtilis ORF with no known function, just upstream from an operon similar to an ABC (ATP-binding cassette) transporter operon from B. subtilis. The excisants obtained from these mutants using the excision reporter plasmid pTCR9 recovered full adhesion capacity. A fourth mutant was the most severely defective in adhesion. It had five Tn1545 insertions, one of which was upstream from dfp and priA, and another of which was upstream from ami, a gene encoding a surface-exposed autolysin with a C terminus similar to that of InIB. Ami was clearly involved because an ami null mutant constructed in an EGDdeltainIA-F background was adhesion-defective. Thus new regions involved in the adhesion of L. monocytogenes to eukaryotic cells were identified. Further study is required to define more accurately the roles of these regions in the adhesion process itself.

Bacterial Adhesion↗
PubMed1996

Complete cDNA sequence of the preproform of human pregnancy-associated plasma protein-A. Evidence for expression in the brain and induction by cAMP.

A cDNA that encodes the prepropeptide of pregnancy-associated plasma protein-A (preproPAPP-A), a putative metalloproteinase, has been cloned and sequenced. PAPP-A is synthesized in the placenta as a 1627-residue precursor preproprotein with a putative 22-residue signal peptide and a highly basic propeptide of 58 residues. The prepro-PAPP-A-encoding transcript contains a region with an extremely high G+C content and has an unusually long 5' untranslated region with several upstream short ORF. No alternatively spliced products could be identified by means of Northern blotting experiments or with rapid amplification of 5' cDNA ends experiments. A stretch within the 5' untranslated region shows sequence identities to a partial cDNA isolated from brain and a to cAMP-inducible sequence from a choriocarcinoma cell line.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1992

Molecular analyses of the lactococcin A gene cluster from Lactococcus lactis subsp. lactis biovar diacetylactis WM4.

The genes responsible for bacteriocin production and immunity in Lactococcus lactis subsp. lactis biovar diacetylactis WM4 were localized and characterized by DNA restriction fragment deletion, subcloning, and nucleotide sequence analysis. The nucleotide sequence of a 5.6-kb AvaII restriction fragment revealed a cluster with five complete open reading frames (ORFs) in the same orientation. DNA and protein homology analyses, combined with deletion and Tn5 insertion mutagenesis, implicated four of the ORFs in the production of and immunity to lactococcin A. The last two ORFs in the cluster were the lactococcin A structural and immunity genes, lcnA and lciA. The two ORFs immediately upstream of lcnA and lciA were designated lcnC and lcnD, and the proteins that they encoded showed similarities to proteins of signal sequence-independent secretion systems. lcnC encodes a protein of 716 amino acids that could belong to the HlyB family of ATP-dependent membrane translocators. LcnC contains an ATP binding domain in a conserved C-terminal stretch of approximately 200 amino acids and three putative hydrophobic segments in the N terminus. The lcnD product, LcnD, of 474 amino acids, is essential for lactococcin A expression and shows structural similarities to HlyD and its homologs. On the basis of these results, a secretion apparatus that is essential for the full expression of active lactococcin A is postulated.

Adenosine Triphosphate↗
PubMed1996

Circular and linear plasmids of Lyme disease spirochetes have extensive homology: characterization of a repeated DNA element.

We have cloned three copies of a repeated DNA segment from Borrelia burgdorferi sensu stricto strain B31, present on both circular and linear plasmids of this and other B. burgdorferi sensu lato strains. The DNA sequences are characterized by a highly homologous segment containing two open reading frames (ORFs), ORF-A and ORF-B. Five additional ORFs can be found on the slightly less homologous flanking sequences: ORF-G on the opposite strand upstream of ORF-A, and ORF-C, ORF-D, ORF-E, and ORF-F downstream of ORF-B. The 4.6-kb-long element containing ORF-A through ORF-E is flanked by approximately 180-bp-long imperfect inverted repeats (IRs). The putative gene product of ORF-C displays homology to proteins involved in plasmid maintenance in a number of gram-positive and gram-negative bacteria. ORF-E features several short, highly homologous direct repeats. ORF-A, ORF-B, and ORF-D are homologous to three ORFs on a recently described 8.3-kb circular plasmid of Borrelia afzelii Ip21 that are flanked by similar IRs (J. J. Dunn, S. R. Buchstein, L.-L. Butler, S. Fisenne, D. S. Polin, B. N. Lade, and B. J. Luft, J. Bacteriol. 176:2706-2717,1994). ORF-C and ORF-E, however, are missing from this region on the Ip21 plasmid. Furthermore, the repeated DNA element as defined by the IRs is present in opposite orientations relative to the flanking sequences on the B31 and Ip21 plasmids.

Base Sequence↗