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PubMed2006

Biological function of the pld gene product that degrades epsilon-poly-L-lysine in Streptomyces albulus.

Epsilon-poly-L-lysine (epsilon-PL) is one of the few naturally occurring biopolymers and is characterized by a peptide bond between the alpha-carboxyl and epsilon-amino groups. Previously, we purified and characterized the epsilon-PL-degrading enzyme (Pld) from Streptomyces albulus, which is an epsilon-PL producer, and this enzyme was expected to confer self-resistance to the epsilon-PL produced by the organism itself. The gene encoding Pld was cloned based on the N-terminal amino acid sequence determined in this study, and a sequencing analysis revealed eight open reading frames (ORFs), i.e., ORF1 to ORF8 in the flanking region surrounding the pld gene (present in ORF5). To investigate the biological function of Pld, we constructed a knockout mutant in which the pld gene is inactivated. Studies on epsilon-PL susceptibility, epsilon-PL-degrading activity, and epsilon-PL productivity demonstrated that the pld gene does play a partial role in self-resistance and that S. albulus was found to produce other epsilon-PL-degrading enzyme(s) in addition to Pld. To the best of our knowledge, this is the first report on a self-resistance gene for a biopolymer possessing antibacterial activity.

Amino Acid Sequence↗
PubMed1998

Evolutionarily conserved promoter region containing CArG*-like elements is crucial for smooth muscle myosin heavy chain gene expression.

In recent years, significant progress has been made toward understanding skeletal muscle development. However, the mechanisms that regulate smooth muscle development and differentiation are presently unknown. To better understand smooth muscle-specific gene expression, we have focused our studies on the smooth muscle myosin heavy chain (SMHC) gene, a highly specific marker of differentiated smooth muscle cells. The goal of the present study was to isolate and characterize the mouse SMHC gene promoter, since the mouse promoter would be particularly suited for in vivo promoter analyses in transgenic mice and would serve as a tool for targeting genes of interest into smooth muscle cells. We report here the isolation and characterization of the mouse SMHC promoter and its 5' flanking region. DNA sequence analysis of a 2.6-kb portion of the promoter identified several potential binding sites for known transcription factors. Transient transfection analysis of promoter deletion constructs in primary cultures of smooth muscle cells showed that the region between -1208 and -1050 bp is critical for maximal SMHC promoter activity. A comparison of SMHC promoter sequences from mouse, rat, and rabbit revealed the presence of a highly conserved region located between -967 and -1208 bp. This region includes three CArG/CArG*-like elements, two SP-1 binding sites, a NF-1-like element, an Nkx2-5 binding site, and an Elk-1 binding site. Gel mobility shift assay and DNase I footprinting analyses show that all three CArG/CArG*-like elements can form DNA-protein complexes with nuclear extract from vascular smooth muscle cells. Protein binding to the CArG* elements can be competed out by either serum response element or by an authentic CArG element from the cardiac alpha-actin gene. Using a serum response factor (SRF) antibody, we demonstrate that SRF is part of the protein complex. In addition, we show that cotransfection with the SRF dominant-negative mutant expression vector abolishes SMHC promoter activity, suggesting that SRF protein plays a critical role in SMHC gene regulation.

Animals↗
PubMed1997

Genomic organization and promoter structure of the human EXT1 gene.

Hereditary predisposition to multiple exostoses is a genetically heterogeneous disease. Recently, we have reported the identification of the EXT1 gene on human chromosome 8. We have now isolated a cDNA clone from a human adult lung cDNA library and have determined the genomic organization and promoter structure of the EXT1 gene. The gene is composed of 11 exons, ranging from 90 to 1735 bp, and spans approximately 350 kb of genomic DNA. Sequence analysis of the promoter region revealed the presence of a CpG island containing GC and CAAT boxes, but no TATA box. Such a promoter is characteristic for housekeeping genes. This finding is in good agreement with the ubiquitous expression of the EXT1 gene.

Adult↗
PubMed2000

The dual function in virulence and host range restriction of a gene isolated from the pPATH (Ehg) plasmid of Erwinia herbicola pv. gypsophilae.

The host range of the gall-forming bacterium Erwinia herbicola pv. gypsophilae (Ehg) is restricted to gypsophila whereas Erwinia herbicola pv. betae (Ehb) attacks beet as well as gypsophila. Both pathovars contain an indigenous plasmid (pPATH(Ehg or pPATH(Ehb)) that harbors pathogenicity genes, including the hrp gene cluster. A cosmid library of Ehg824-1 plasmid DNA was mobilized into Ehb4188 and the transconjugants were screened for pathogenicity on beet. One Ehb transconjugant harboring the cosmid pLA173 of pPATHEb induced a hypersensitive-like response and abolished pathogenicity on beet. Transposon mutagenesis of an open reading frame (ORF) located on this cosmid eliminated its affect on pathogenicity. Marker exchange of this mutation into Ehg824-1 caused a substantial reduction in gall size on gypsophila and caused Ehg824-1 to extend its host range and incite galls on beet. The ORF (1.5 kb) was designated as pthG (pathogenicity gene on gypsophila). DNA sequence analysis of pthG revealed no significant homology to known genes in the data bank. Only remnants of the pthG sequences were identified on the pPATH of Ehb4188. The deduced protein lacked an N-terminal signal peptide but contained a short trans-membrane helix in its C terminus. The gene product, as determined by expression in Escherichia coli and Western blots (immunoblots), was a 56-kDa protein.

Amino Acid Sequence↗
PubMed2001

Genetic and biochemical characterization of glutamyl endopeptidase of Staphylococcus warneri M.

A Staphylococcus warneri strain M, newly isolated from processed seafood (smoked Watasenia scintillans), produced an extracellular protease. The protease, designated to as m-PROM (the mature form of PROM), selectively cleaved the carbonyl side of glutamic acid residues in beta-casein. Sequence of N-terminal 27 amino acids of m-PROM, RANVILPNNDRHQINDTTLGHYAPVTF, was found to be similar to those of other glutamyl endopeptidases, V8 protease (Staphylococcus aureus strain V8) and SPase (S. aureus ATCC 12600). To determine the complete primary structure and precursor of PROM, its gene (proM) was cloned and sequenced. The gene proM was found to encode for a protein of 316 amino acids. The amino acid residues from 64 to 90 completely coincided with the N-terminal 27 amino acids of the m-PROM, suggesting that the N-terminal 63 amino acids region of p-PROM (the precursor form of PROM) might be processed posttranslationally. Moreover, the whole amino acid sequence deduced from the primary structure of proM shows significant similarity to those of other glutamyl endopeptidases, V8 protease and SPase. These results suggested that PROM belongs to the glutamyl endopeptidase class. PROM, however, differs from V8 and SPase proteases in the processing site and the C-terminal region.

Amino Acid Sequence↗
PubMed1997

Identification of a novel Escherichia coli gene whose expression is dependent on the flagellum-specific sigma factor, FliA, but dispensable for motility development.

FliA is an alternative sigma factor specific for class 3 flagellar operons. Using a promoter-probe vector, we randomly cloned Escherichia coli DNA fragments, which showed FliA-dependent promoter activities. Among the DNA fragments cloned, one was found to be derived from a non-flagellar region. Hybridization analysis with the Kohara E. coli library indicated that this DNA fragment is located at around 35.4 min on the E. coli chromosome where no flagellar gene has been reported yet. DNA sequence analysis revealed that it contains an FliA-dependent promoter-like sequence followed by an open reading frame (ORF) that can encode a 110-amino-acid protein. A rho-independent terminator-like sequence follows this ORF. This putative gene was named flxA. A gene disruptant was constructed by inserting the kan gene cassette into the flxA gene on the chromosome. This mutant was found to be actively motile, suggesting that this gene is unlikely to be involved in the motility phenotype of E. coli.

Amino Acid Sequence↗
PubMed2001

Photoaffinity labelling with the cytokinin agonist azido-CPPU of a 34 kDa peptide of the intracellular pathogenesis-related protein family in the moss Physcomitrella patens.

As in higher plants, the development of the moss Physcomitrella patens is regulated by environmental signals and phytohormones. At the protonema level transition from chloronema to caulonema cells is under auxin control. The formation on second sub-apical caulonema cells of buds that will give rise to the leafy gametophore requires cytokinins. Using [3H]azidoCPPU (1-(2-azido-6-chloropyrid-4-yl)-3-(4-[3H])phenylurea), a photoactivatable cytokinin agonist, we have specifically photolabelled a soluble 34 kDa protein of P. patens. Urea derivatives were very efficient competitors of photolabelling while purine-type cytokinins were poor competitors. The protein UBP34 was purified by affinity chromatography and the sequences of six internal peptides obtained. A cDNA encoding UBP34 was cloned by screening a P. patens protonema cDNA library with a probe amplified by PCR using degenerate primers designed from the peptide sequences. The UBP34 amino acid sequence shows an average sequence identity of 42% with both intracellular PR proteins and the BetV1-related family of plant allergens. Recombinant UBP34 expressed in Escherichia coli was confirmed to bind azidoCPPU.

Amino Acid Sequence↗
PubMed2005

Statistics of protein library construction.

UNLABELLED: We have investigated the statistics associated with constructing and sampling large protein-encoding libraries. Using fairly simple statistics we have written algorithms for estimating the diversity in libraries generated by the most commonly used protocols, including error-prone PCR, DNA shuffling, StEP PCR, oligonucleotide-directed randomization, MAX randomization, synthetic shuffling, DHR, ADO and SISDC. AVAILABILITY: Web interface and C++ source code available at http://guinevere.otago.ac.nz/stats.html. SUPPLEMENTARY INFORMATION: Complete mathematical notes, model assumptions and justification, users' guide and worked examples at above website.

Algorithms↗
PubMed1994

The DNA polymerase genes of several HMU-bacteriophages have similar group I introns with highly divergent open reading frames.

A previous report described the discovery of a group I, self-splicing intron in the DNA polymerase gene of the Bacillus subtilis bacteriophage SPO1 (1). In this study, the DNA polymerase genes of three close relatives of SPO1: SP82, 2C and phi e, were also found to be interrupted by an intron. All of these introns have group I secondary structures that are extremely similar to one another in primary sequence. Each is interrupted by an open reading frame (ORF) that, unlike the intron core or exon sequences, are highly diverged. Unlike the relatives of Escherichia coli bacteriophage T4, most of which do not have introns (2), this intron seems to be common among the relatives of SPO1.

Amino Acid Sequence↗
PubMed1994

Nucleotide sequence, genomic organization and synthesis of infectious transcripts from a full-length clone of artichoke mottle crinkle virus.

The complete nucleotide sequence of the genome of artichoke mottle crinkle virus (AMCV), a member of the tombusvirus group, has been determined. The genome is 4790 nucleotides (nt) in length. A full-length cDNA of the AMCV genome has been cloned in pUC9 downstream of the T7 RNA polymerase promoter. Transcripts were infective when inoculated onto Nicotiana clevelandii and N. benthamiana plants. The AMCV genome contains five open reading frames (ORFs). The first ORF from the 5' terminus (ORF1) encodes a protein with a predicted M(r) of 33K. ORF2 extends through the amber termination codon of ORF1 to yield a polypeptide of predicted M(r) 92K and which is the putative RNA-dependent RNA polymerase. ORF3 codes for the coat protein (41K). Two nested ORFs in different reading frames (ORFs 4 and 5) code for a 22K and a 19K polypeptide respectively. Sequence homologies suggest that the 22K protein could be involved in cell-to-cell movement of virus. ORFs 3, 4 and 5 are translated from two 3' coterminal subgenomic (sg) RNAs, the 5' termini of which have been mapped. The two sg RNAs are 2155 (sg1) and 934 (sg2) nt in length. ORF3 is expressed from sg1 RNA whereas ORFs 4 and 5 are potentially expressed from sg2 RNA. Time course experiments with Cynara scolymus protoplasts indicate that during AMCV infection both positive and negative strands of genomic and sg RNAs are produced and that sg2 RNA is produced before and at a higher level than sg1 RNA.

Amino Acid Sequence↗
PubMed2006

Exogean: a framework for annotating protein-coding genes in eukaryotic genomic DNA.

BACKGROUND: Accurate and automatic gene identification in eukaryotic genomic DNA is more than ever of crucial importance to efficiently exploit the large volume of assembled genome sequences available to the community. Automatic methods have always been considered less reliable than human expertise. This is illustrated in the EGASP project, where reference annotations against which all automatic methods are measured are generated by human annotators and experimentally verified. We hypothesized that replicating the accuracy of human annotators in an automatic method could be achieved by formalizing the rules and decisions that they use, in a mathematical formalism. RESULTS: We have developed Exogean, a flexible framework based on directed acyclic colored multigraphs (DACMs) that can represent biological objects (for example, mRNA, ESTs, protein alignments, exons) and relationships between them. Graphs are analyzed to process the information according to rules that replicate those used by human annotators. Simple individual starting objects given as input to Exogean are thus combined and synthesized into complex objects such as protein coding transcripts. CONCLUSION: We show here, in the context of the EGASP project, that Exogean is currently the method that best reproduces protein coding gene annotations from human experts, in terms of identifying at least one exact coding sequence per gene. We discuss current limitations of the method and several avenues for improvement.

Amino Acid Sequence↗
PubMed2003

Cloning and characterization of the 82 kDa tyrosine-rich sexual stage glycoprotein, GAM82, and its role in oocyst wall formation in the apicomplexan parasite, Eimeria maxima.

The sexual (macrogamete/macrogametocyte) stage antigen, GAM82, in the apicomplexan parasite Eimeria maxima, has an apparent molecular mass of 82 kDa, and has been implicated in protective immunity against coccidiosis in poultry. The gene encoding this protein, gam82, was cloned and sequenced. It is a single-copy, intronless gene, which localizes to a 2145 bp transcript, and is first detected at 130 h post-infection. The gene predicts two distinct domains rich in the residues tyrosine and serine, amino acids that have been implicated in oocyst wall formation in other Eimeria spp., and in the extraorganismic sclerotization of structural proteins throughout the animal kingdom. A high number of small amino acids, predominantly alanine and proline, were detected in the intervening sequence between these two domains. The inference that GAM82 is involved in oocyst wall formation in Eimeria was confirmed when it was shown that a specific antibody to a recombinant version of GAM82 recognized the wall forming bodies in macrogametes, and the walls of oocysts in E. maxima. A closer biochemical analysis of the role of GAM82 in oocyst wall formation by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting showed that the antibodies to the recombinant version of GAM82 recognized an 82 kDa protein in macrogametocyte extracts, and a 30 kDa protein in unsporulated and sporulated oocyst extracts, as well as in purified oocyst wall fragments. Together, these findings indicate that the 82 kDa macrogametocyte antigen, GAM82, is a tyrosine and serine rich precursor protein that is proteolytically processed during development to give rise to a 30 kDa protein, that is incorporated into the oocyst wall. In addition, these findings provide evidence that the oocyst wall of Eimeria species is composed of a family of tyrosine rich proteins, that arise from precursor proteins found in the wall forming bodies of macrogametes.

Amino Acid Sequence↗
PubMed2002

DNA polymerase beta mRNA determination by relative quantitative RT-PCR from Leishmania infantum intracellular amastigotes.

Gene expression level is extremely difficult to assess in the intracellular form of Leishmania infantum, the amastigote, due to host mRNA contamination, low supply of parasites and stress degradation problems. We obtained and analyzed L. infantum DNA polymerase beta (Li Pol beta), a suitable enzyme for differential expression studies. The amount of Li Pol beta mRNA was determined in different forms of the parasite (extracellular promastigote, intracellular amastigote) by Northern blot and by relative quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Amastigote transcript levels were determined from both gradient-purified parasites and directly from a population of infected macrophages. The mRNA, undetectable when obtained from amastigotes by the classic gradient centrifugation method and subsequent Northern analysis, was clearly and specifically detectable by this quantitative RT-PCR method from the mixed macrophage/parasite cell population. Li Pol beta displays a different pattern of expression in the distinct forms of the parasite cycle that correlate with the infectivity of the protozoon. Thus, Li Pol beta mRNA expression is developmentally regulated, being clearly higher in the more infective forms of the parasite.

Animals↗
PubMed2000

Gall mite molecular phylogeny and its relationship to the evolution of plant host specificity.

The phylogenetic relationships of all seven known species of Cecidophyopsis mites (Acari: Eriophyidae) with Ribes hosts have been inferred from ribosomal DNA sequences. This analysis found groups of closely related mites. The five gall-forming species, four of which are monophagous and one which has two hosts, were found in two groups. Another group consisted of the two non gall-forming species, one of which has two hosts, while the other is monophagous. The molecular phylogeny of their known Ribes host plants was calculated using the equivalent ribosomal regions as the mites. The structure of the two trees (mites vs hosts) was clearly different, implying that mite speciation did not closely follow speciation events in the plant hosts. Instead, the three groups of Ribes-infesting Cecidophyopsis mites have derived from a common galling ancestor millions of years ago. Each mite group has recently diversified onto different primary hosts. One group of mites has also lost the galling habit. The results have implications for host range changes and the durability of mite-resistance genes in cultivated Ribes.

Animals↗
PubMed2005

Mitochondrial voltage-dependent anion channel gene family in Drosophila melanogaster: complex patterns of evolution, genomic organization, and developmental expression.

Voltage-dependent anion channels (VDACs), also known as mitochondrial porins, are a family of small pore-forming proteins of the mitochondrial outer membrane found in all eukaryotes. VDACs play important roles in the regulated flux of metabolites between the cytosolic and mitochondrial compartments, energy metabolism, and apoptosis. Annotation of the genome sequence of Drosophila melanogaster revealed three genes (CG17137, CG31722-A, and CG31722-B) with homology to porin, the previously described Drosophila VDAC. Molecular analysis reveals a complex pattern of organization and expression. The genomic organization of these four genes and sequence comparisons with other insect VDAC homologs indicate that this gene family evolved through a mechanism of duplication and divergence from an ancestral VDAC gene during the radiation of the genus Drosophila. CG17137, CG31722-A, and CG31722-B are expressed in a male-specific pattern on both transcriptional and translational levels, while porin is equally expressed in both male and female flies. Additionally, CG31722-A and CG31722-B are expressed as a dicistronic transcript. Western blot analysis and immunofluorescence microscopy confirm that these proteins localize to the mitochondrion. Further expression analysis showed that CG17137 and CG31722-B are abundant in testes, while porin is ubiquitously expressed. While porin, CG17137, and CG31722-B are expressed to different degrees during embryogenesis, all of these proteins are dramatically reduced relative to cytochrome c content during larvogenesis. These studies illustrate a complex genomic organization and spatiotemporal pattern of expression for Drosophila VDACs as well as an evolutionary history consistent with either a partitioning of VDAC functions or an acquisition of novel functions among isoforms.

Amino Acid Sequence↗
PubMed1998

Isolation of carbon- and nitrogen-deprivation-induced loci of Sinorhizobium meliloti 1021 by Tn5-luxAB mutagenesis.

Soil bacteria, such as Sinorhizobium meliloti, are subject to variation in environmental conditions, including carbon- and nitrogen-deprivation. The ability of bacteria to sense changes in their environment and respond accordingly is of vital importance to their survival and persistence in the soil and rhizosphere. A derivative of Tn5 which creates transcriptional fusions to the promoterless luxAB genes was used to mutagenize S. meliloti 1021 and 5000 insertion mutants were subsequently screened for gene fusions induced by selected environmental stresses. The isolation of 21 gene fusions induced by nitrogen-deprivation and 12 induced by carbon-deprivation is described. Cloning and partial DNA sequence analysis of the transposon-tagged loci revealed a variety of novel genes, as well as S. meliloti genes with significant similarity to known bacterial loci. In addition, nodule occupancy studies were carried out with selected Tn5-luxAB insertion mutants to examine the role of the tagged genes in competition.

Amino Acid Sequence↗
PubMed2004

Crassostrea gigas ferritin: cDNA sequence analysis for two heavy chain type subunits and protein purification.

Ferritin has been shown as being the principal iron storage in the majority of living organisms. In marine species, ferritin is also involved in high-level accumulation of (210)Po. As part of our work on the investigation of these radionuclides' concentration in natural environment, ferritin was searched at the gene and protein level. Ferritin was purified from the visceral mass of the oyster Crassostrea gigas by ion-exchange chromatography and HPLC. SDS-PAGE revealed one band of 20 kDa. An Expressed Sequence Tag (EST) library was screened and led to the identification of two complementary DNA (cDNA) involved in ferritin subunit expression. The complete coding sequences and the untranslated regions (UTRs) of the two genes were obtained and a 5' Rapid Amplification of cDNA Ends (RACE) was used to obtain the two iron-responsive elements (IREs) with the predicted stem-loop structures usually present in the 5'-UTR of ferritin mRNA. Sequence alignment in amino acid of the two new cDNA showed an identity with Pinctada fucata (85.4-88.3%), Lymnaea stagnalis (79.3-82.2%) and Helix pomatia (79.1-79.1%). The residues responsible for the ferroxidase center, conserved in all vertebrate H-ferritins, are present in the two oyster ferritin subunits. Oyster ferritins do not present the special characteristics of other invertebrate ferritins like insect ferritins but have some functional similarities with the vertebrate H chains ferritin.

Amino Acid Sequence↗
PubMed1999

Identification and characterization of three cDNAs that encode putative novel hyaluronan-binding proteins, including an endothelial cell-specific hyaluronan receptor.

The glycosaminoglycan hyaluronan (HA) and HA-binding proteins (HABPs) serve important structural and regulatory functions during development and in maintaining adult tissue homeostasis. Here we have identified and partially characterized the sequence and expression pattern of three putative novel HABPs. DNA sequence analysis revealed that two of the novel HABPs, WF-HABP and BM-HABP, form a unique HA-binding subfamily, whereas the third protein, OE-HABP, is more closely related to the LINK subfamily of HABPs. Northern blotting experiments revealed that the expression of BM-HABP was highly restricted, with substantial expression detected only in human fetal liver. In contrast, WF-HABP and OE-HABP mRNAs were detected in a number of tissues, with particularly prominent expression in highly vascularized tissues such as the heart, placenta, and lung. Additional studies showed that OE-HABP was expressed by cultured human endothelial cells, smooth muscle cells, and differentiated monocytes. However, only endothelial cells expressed WF-HABP mRNA, and its expression was regulated by growth state, being most prominent in quiescent endothelial cells. We further characterized the expression of WF-HABP in vivo and found that its expression colocalized with CD31-positive cells and was prominently expressed in microvessels in the human aorta and in atherectomy samples. Our data suggest that WF-HABP is an endothelial cell-specific HA receptor and that it may serve a unique function in these cells. The WF-HABP gene was localized to chromosome 3p21.31 and the OE-HABP gene to 15q25.2-25.3.

Adult↗