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At least 1,027 records · Page 57Linked to original sources
PubMed1998

Identification of differentially expressed genes in human hepatoblastoma cell line (HepG2) and HBV-X transfected hepatoblastoma cell line (HepG2-4x).

Hepatitis B virus-X protein (HBV-X) is known to be an important factor in the formation of hepatocellular carcinoma by acting as a transcriptional activator on viral or cellular genes. To identify differentially expressed genes between the human hepatoblastoma cell line HepG2 and HBV-X gene transfected hepatoblastoma cell line HepG2-4X, we used a differential display polymerase chain reaction technique. The technique produced numerous up-regulated and down-regulated bands, each representing a partial cDNA fragment. We isolated 23 different kinds of cDNA fragments that showed marked differences in two cell lines. The fragments were used as templates for DNA sequencing analysis and as probes for Northern blot analysis. This analysis revealed that eight cDNA clones were differentially expressed in each cell line but fifteen cDNA clones were not. Among the 8 clones, 3 clones showed sequence similarities with human mitochondrial ATPase subunit 6 (mtATPase 6) and the human amidophosphoribosyl transferase (ATase) precursor, whereas 5 other clones were human novel protein encoding genes. Two genes having similarity with known genes were repressed by HBV-X. These results reflect that complex alterations of the expression of enzymes concerning the energy-generating system in mitochondria and metabolite synthesis are closely associated with the HBV-X function during the formation of hepatocellular carcinoma. These newly obtained genes will be useful for analyzing HBV-X functions. We are in the process of further characterizing these genes.

Blotting, Northern↗
PubMed1997

Double-strand breaks may initiate the inversion mutation causing the Hunter syndrome.

We have previously shown that patients with the Hunter syndrome frequently have suffered from a recombination event between the IDS gene and its putative pseudogene, IDS-2, resulting in an inversion of the intervening DNA. The inversion, which might be the consequence of an intrachromosomal mispairing, is caused by homologous recombination between sequences located in intron 7 of the IDS gene and sequences located distal of exon 3 in IDS-2. In order to gain insight into the mechanisms causing the inversion, we have isolated both inversion junctions in six unrelated patients. DNA sequence analysis of the junctions showed that all recombinations have taken place within a 1 kb region where the sequence identity is >98%. An interesting finding was the identification of regions with alternating IDS gene and IDS-2 sequences present at one inversion junction, suggesting that the recombination event has been initiated by a double-strand break in intron 7 of the IDS gene. The results from this study suggest that homologous recombination in man could be explained by mechanisms similar to those described for Saccharomyces cerevisiae. The results also have practical implications for diagnosis of patients with the Hunter syndrome.

Chromosome Inversion↗
PubMed2001

Mapping of resistance to vegetable polyphenols among Aedes taxa (Diptera, Culicidae) on a molecular phylogeny.

To recover some evolutionary aspects of the interaction between culicine larvae and dietary polyphenols of the vegetation surrounding mosquito breeding sites, we constructed a phylogeny of the most common French Aedes species, chosen as reference species. We also evaluated the differential resistance of these larval taxa to the polyphenols of leaf litter from the riparian vegetation used as a food source. Mitochondrial DNA sequence analysis was performed among 14 different taxa and ecotypes (Aedes aegypti, Ae. albopictus, Ae. cantans, Ae. caspius, Ae. cataphylla, Ae. cinereus, Ae. detritus, Ae. geniculatus, Ae. mariae, Ae. pullatus, Ae. punctor, Ae. rusticus, Ae. sticticus, and Ae. vexans) through direct sequencing of a 763-base segment of the cytochrome oxidase subunit I gene. Phylogenetic analysis, based on nucleotide and amino acid sequences, was conducted by means of parsimony and distance methods. The differential tolerance of larvae to vegetable leaf litter was comparatively tested by use of 10-month-old alder leaf litter as an experimental standard. The absence of correlation between resistance to polyphenols and molecular phylogeny suggests that larval adaptation to polyphenol-rich vegetable breeding sites is a labile character. The acquisition of such resistance appears not to be ancestrally inherited, but rather to be a dynamic adaptation to the environment. Molecular data also support the classical morphological classification within the Aedes genus.

Aedes↗
PubMed2000

Genetic analysis of a bacterial genetic exchange element: the gene transfer agent of Rhodobacter capsulatus.

An unusual system of genetic exchange exists in the purple nonsulfur bacterium Rhodobacter capsulatus. DNA transmission is mediated by a small bacteriophage-like particle called the gene transfer agent (GTA) that transfers random 4.5-kb segments of the producing cell's genome to recipient cells, where allelic replacement occurs. This paper presents the results of gene cloning, analysis, and mutagenesis experiments that show that GTA resembles a defective prophage related to bacteriophages from diverse genera of bacteria, which has been adopted by R. capsulatus for genetic exchange. A pair of cellular proteins, CckA and CtrA, appear to constitute part of a sensor kinase/response regulator signaling pathway that is required for expression of GTA structural genes. This signaling pathway controls growth-phase-dependent regulation of GTA gene messages, yielding maximal gene expression in the stationary phase. We suggest that GTA is an ancient prophage remnant that has evolved in concert with the bacterial genome, resulting in a genetic exchange process controlled by the bacterial cell.

Amino Acid Sequence↗
PubMed1991

Apolipoprotein E-4Philadelphia (Glu13----Lys,Arg145----Cys). Homozygosity for two rare point mutations in the apolipoprotein E gene combined with severe type III hyperlipoproteinemia.

The molecular defect in a 24-year-old white female with severe type III hyperlipoproteinemia has been elucidated. The patient's apolipoprotein (apo) E migrated in the apoE-4 position on isoelectric focusing gels. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the apoE-4 variant had a smaller apparent molecular weight than apoE-4(Cys112----Arg). Sequence analysis of DNA amplified with the polymerase chain reaction revealed two nucleotide substitutions in the proband's apoE gene. A C to T mutation converted arginine (CGT) at position 145 of the mature protein to cysteine (TGT) thus creating the apoE-2 variant. A second G to A substitution at amino acid 13 led to the exchange of lysine (AAG) for glutamic acid (GAG), thereby adding 2 positive charge units to the protein and producing the apoE-5 variant. Computer analysis of the apoE-4Philadelphia gene revealed that the G to A mutation in exon 3 resulted in the loss of an AvaI restriction enzyme site. The second mutation, a C to T substitution in the fourth exon of the apoE gene, eliminated a cleavage site for the enzyme BbvI. Using these restriction fragment length polymorphisms as well as DNA sequence analysis we have demonstrated that the patient is homozygous for both point mutations in the apoE gene.

Adult↗
PubMed2004

Isolation and characterization of the Rhodococcus opacus thiostrepton-inducible genes tipAL and tipAS: application for recombinant protein expression in Rhodococcus.

We cloned the Rhodococcus opacus (strain DSM 44193) tipA gene, which encodes two translation products, TipAL and TipAS. The gene products are homologous to the Streptomyces spp. TipAL and TipAS proteins, respectively. The tipA promoter is highly active and TipAS protein is predominantly accumulated in R. opacus cells when the inducer of transcription, thiostrepton, was presented in culture medium. We found that thiostrepton is also induced the expression of an endogenous TipA-family protein in Rhodococcus erythropolis (strain JCM3201). The minimal tipA promoter region was defined (57 bp) and the conserved nucleotide sequence of the putative TipAL protein binding site (TipA-box) was identified in that region. The tipA gene is presumed to be transcribed into a leaderless mRNA. We applied the tipA promoter successfully for recombinant protein expression in R. erythropolis cells.

Artificial Gene Fusion↗
PubMed2004

LDDist: a Perl module for calculating LogDet pair-wise distances for protein and nucleotide sequences.

LDDist is a Perl module implemented in C++ that allows the user to calculate LogDet pair-wise genetic distances for amino acid as well as nucleotide sequence data. It can handle site-to-site rate variation by treating a proportion of the sites as invariant and/or by assigning sites to different, presumably homogenous, rate categories. The rate-class assignments and invariant proportion can be set explicitly, or estimated by the program; the latter using either of two different capture-recapture methods. The assignment to rate categories in lieu of a phylogeny can be done using Shannon-Wiener index as a crude token for relative rate.

Algorithms↗
PubMed2000

Molecular cloning and overexpression of the gene encoding an NADPH-dependent carbonyl reductase from Candida magnoliae, involved in stereoselective reduction of ethyl 4-chloro-3-oxobutanoate.

An NADPH-dependent carbonyl reductase (S1) isolated from Candida magnoliae catalyzed the reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate (CHBE), with a 100% enantiomeric excess, which is a useful chiral building block for the synthesis of pharmaceuticals. The gene encoding the enzyme was cloned and sequenced. The S1 gene comprises 849 bp and encodes a polypeptide of 30,420 Da. The deduced amino acid sequence showed a high degree of similarity to those of the other members of the short-chain alcohol dehydrogenase superfamily. The S1 gene was overexpressed in Escherichia coli under the control of the lac promoter. The enzyme expressed in E. coli was purified to homogeneity and had the same catalytic properties as the enzyme from C. magnoliae did. An E. coli transformant reduced COBE to 125 g/l of (S)-CHBE, with an optical purity of 100% enantiomeric excess, in an organic solvent two-phase system.

Acetoacetates↗
PubMed1995

Unusual sequences of group 3 LEA mRNA inducible by maturation or drying in soybean seeds.

Two cDNA clones, pGmPM8 and pGmPM10, which correspond to two mRNA species in mature or dry soybean seeds, were characterized. The deduced proteins, based on DNA sequence analysis, have a molecular mass of 49 and 51 kDa for pGmPM8 and pGmPM10, respectively. These two cDNA clones share a high homology with an amino acid identity of about 90% between the two deduced proteins. Both proteins appear to be extremely hydrophilic except at their N-termini that contain a 29 amino acid hydrophobic region at the N-terminus and the sizes of proteins decrease after co-incubating with ER membranes. These two proteins contain more than 30 similar, contiguous repeats of 11 amino acids, which is characteristic of group 3 LEA proteins. The mRNAs corresponding to pGmPM8 and pGmPM10 were expressed at high levels in dried or mature soybean seeds, but not in fresh immature seeds. The RNAs were also present in abscisic acid (ABA) treated leaves or cultured cells, and in tissues subjected to water stress or low temperatures.

Amino Acid Sequence↗
PubMed2000

Characterization and cDNA cloning of two glycine- and histidine-rich antimicrobial peptides from the roots of shepherd's purse, Capsella bursa-pastoris.

Two novel antimicrobial peptides were isolated and characterized from the roots of shepherd's purse, Capsella bursa-pastoris. These antimicrobial peptides, named shepherin I and shepherin II, consist of 28 and 38 amino acids, respectively, and are glycine- and histidine-rich peptides. Shepherin I and shepherin II have 67.9% and 65.8% (mol/mol) glycine, respectively, and 28.6% and 21.1% (mol/mol) histidine, respectively. Both shepherins have a Gly-Gly-His motif. These antimicrobial peptides exhibit antimicrobial activity against Gram-negative bacteria and fungi. Circular dichroism spectra of shepherin I and shepherin II showed that shepherin I and shepherin II in 50% trifluoroethanol have 66.7% and 75% random coils, respectively, without any alpha-helices. cDNA sequence analysis revealed that shepherin I and shepherin II are produced from a single polypeptide, designated shep-GRP, consisting of 120 amino acids; shep-GRP has five distinct domains, an amino-terminal putative signal peptide, a shepherin I, a linker dipeptide, a shepherin II and a carboxy-terminal peptide. Southern blot analysis indicates that the gene encoding shepherins belongs to a low-complexity gene family. Northern blot analysis revealed that transcripts of shep-GRP are present in roots but not in leaves and stems.

Amino Acid Sequence↗
PubMed2002

Substrate specificity and sequence analysis define a polyphyletic origin of betanidin 5- and 6-O-glucosyltransferase from Dorotheanthus bellidiformis.

Betanidin 6-O-glucosyltransferase (6-GT) is involved in the glycosylation of betacyanins, which replace the chromogenic anthocyanins as flower colorants in the Caryophyllales. The 6-GT cDNA was cloned from a cDNA library of Dorotheanthus bellidiformis (Burm. f.) N.E. Br., and the amino acid and nucleotide sequences were shown to be distinctly different from the corresponding betanidin 5-O-glucosyltransferase (5-GT) from the same plant species. Although both enzymes share very similar substrates, the proteins show only 19% amino acid sequence identity. In contrast, the protein sequence of the 6-GT showed significant identity to GTs from other species and may identify a new cluster of putative anthocyanidin GTs. Therefore, 6-GT and 5-GT apparently have evolved independently from ancestral glucosyltransferases involved in flavonoid biosynthesis.

Anthocyanins↗
PubMed1999

Methylmalonyl-CoA mutase encoding gene of Sinorhizobium meliloti.

A cluster of genes on megaplasmid pRmeSU47b, bhbA-D, is required for growth on the polyhydroxyalkanoate degradation pathway intermediates 3-hydroxybutyrate and acetoacetate as sole carbon source. DNA sequence analysis of the bhbA gene indicated that it encoded a protein of 712 amino acids (aa) (78kDa) which appeared to be a homodimeric methylmalonyl-CoA mutase enzyme (EC 5.4.99.2). Cell-free extract of a bhbA::Tn5 mutant was devoid of methylmalonyl-CoA mutase activity, thus confirming the identity of the bhbA-encoded enzyme. The reason for the requirement of methylmalonyl-CoA mutase activity for operation of the polyhydroxyalkanoate degradation pathway is not immediately apparent. Situated immediately upstream of bhbA, in the same orientation, is a gene which is predicted to encode a protein that exhibits remarkable sequence similarity to the alpha subunit of propionyl-CoA carboxylase (EC 6.4.1.3). A mutation in this gene did not affect ability to grow on 3-hydroxybutyrate as sole carbon source. Downstream of, and oriented towards bhbA, was identified a member of the GNTR class of transcriptional regulator-encoding genes. It is not yet known whether this regulatory protein is directly involved in modulation of bhbA expression.

3-Hydroxybutyric Acid↗
PubMed1996

Use of messenger RNA differential display to identify interleukin-11-responsive genes in human umbilical cord blood mononuclear cells: IL-11 upregulates the expression of the hMAL gene.

Human umbilical cord blood (HUCB) mononuclear cells represent a source of hematopoietic stem and progenitor cells, including cells responsive to interleukin-11 (IL-11). To investigate the molecular mechanisms associated with IL-11 action, we have used HUCB mononuclear cells as a model system to identify genes that are transcriptional targets of IL-11. Using the technique of messenger RNA differential display, we have identified 17 candidate cDNA differentially expressed in mononuclear cells incubated without and with IL-11. Fifteen of these cDNA were recovered, and 11 were sequenced. DNA sequence analysis has identified one of these cDNA as being the human MAL gene, originally identified as a marker for intermediate stages of T cell differentiation. Northern analysis using a MAL-specific probe confirms the upregulation of MAL by IL-11 in HUCB cells.

Cells, Cultured↗
PubMed1998

Chlorella virus PBCV-1 encodes functional glutamine: fructose-6-phosphate amidotransferase and UDP-glucose dehydrogenase enzymes.

DNA sequence analysis of the 330-kb Chlorella virus PBCV-1 genome unexpectedly revealed several open reading frames which encode proteins that are homologous to sugar-manipulating enzymes including glutamine:fructose-6-phosphate amidotransferase (GFAT), UDP-glucose dehydrogenase (UDP-GlcDH), and hyaluronan synthase (HAS). PBCV-1 genes encoding the putative GFAT and UDP-GlcDH enzymes were expressed in Escherichia coli, and both recombinant proteins have the predicted enzyme activity in cell free extracts. These same two genes are transcribed early in PBCV-1 infection, and both genes are widespread among the Chlorella viruses. The products of the reactions catalyzed by these two enzymes are precursors in the biosynthesis of hyaluronan polysaccharide. Previous experiments established that, like the GFAT and UDP-GlcDH genes, the HAS gene is transcribed early and encodes a functional enzyme (DeAngelis, P. L., Jing. W., Graves, M. V., Burbank, D. E., and Van Etten, J. L. (1997) Science 278, 1800-1803). Interestingly, the predicted amino-acid sequences of the PBCV-1 GFAT and UDP-GlcDH enzymes are more similar to bacterial GFAT and UDP-GlcDH enzymes than to their eukaryotic counterparts. In contrast, the amino-acid sequence of the PBCV-1 HAS enzyme more closely resembles eukaryotic enzymes.

Animals↗
PubMed2006

Analysis of a xyloglucan endotransglycosylase/hydrolase (XTH) from the lycopodiophyte Selaginella kraussiana suggests that XTH sequence characteristics and function are highly conserved during the evolution of vascular plants.

A tissue print followed by a xyloglucan endotransglycosylase assay revealed that XET activity is present at sites of cell elongation in both roots and shoots of the lycopodiophyte Selaginella kraussiana. This paper provides the first report and analysis of a xyloglucan endotransglycosylase/hydrolase (XTH) cDNA sequence, isolated from a club moss. In silico analysis of the deduced amino acid sequence revealed a strong conservation of the XET-domain described in higher plants. The catalytic site (DEIDLEFLG) varies in only one amino acid compared with the consensus sequence and was shown to be functional after recombinant expression of Sk-XTH1 in Pichia pastoris. Sk-XTH1 displays xyloglucan endotransglycosylase activity over a broad pH (4.5-7.5) and temperature range (4-30 degrees C), but it shows no hydrolase activity. The catalytic site is followed by a consensus sequence for N-linked glycosylation. Four terminal cysteines were shown to stabilize a putative XET-C terminal extension region, which includes conserved amino acids, involved in the recognition and binding of the substrates. The N-linked sugar interactions as well as the disulphide bridges were shown to be necessary to perform XET activity. The presence of a highly conserved XTH sequence and function in a microphyllophyte suggests that XTHs were present before the divergence of lycopodiophytes and euphyllophytes. It also points to a possible key role for XTHs in the production of a cell wall that allowed the further evolution of land plants.

Amino Acid Sequence↗
PubMed2004

The 5'-upstream region of human programmed cell death 5 gene contains a highly active TATA-less promoter that is up-regulated by etoposide.

The PDCD5 (programmed cell death 5), a novel apoptosis related gene, is functionally associated with cell apoptosis, exhibits a ubiquitous expression pattern and is up-regulated in some types of tumor cells undergoing apoptosis. To study the transcriptional regulation of the PDCD5 gene, we have cloned 1.1 kb of its 5'-upstream region. The DNA sequencing analysis revealed a major transcriptional start site at 72 base pairs in front of the ATG translational start codon. The upstream of the transcriptional start site lacks a canonical TATA box and CAAT box. Transient transfection and luciferase assay demonstrate that this region presents extremely strong promoter activity. The 5'-deleted sequences fused to a luciferase reporter gene demonstrated that the -555/-383 region from the transcription start site is crucial for transcriptional regulation, and the luciferase reporter gene's expression significantly increased in the early stage of cell apoptosis induced by etoposide. These results imply that the PDCD5 gene may be a target gene under the control of some important apoptosis-related transcriptional factors during the cell apoptosis.

5' Flanking Region↗
PubMed1999

Identification of a novel nutrient-deprivation-induced Sinorhizobium meliloti gene (hmgA) involved in the degradation of tyrosine.

Sinorhizobium meliloti strain N4 carries a Tn5luxAB insertion in a gene which is induced by nitrogen and carbon deprivation as well as in the presence of tyrosine. The Tn5luxAB-tagged locus was found to share significant similarity with the human hmgA gene and the corresponding Aspergillus nidulans gene, encoding the enzyme homogentisate dioxygenase, which is involved in the degradation of tyrosine. Extended DNA sequence analysis of the tagged locus revealed the presence of several ORFs, including one encoding a polypeptide sharing a high degree of similarity with human and fungal maleylacetoacetate isomerases. Strain N4 was found to be unable to use tyrosine as carbon source, to lack homogentisate dioxygenase activity, to produce a melanin-like pigment and to be affected in stationary-phase survival. This is believed to be the first report of a hmgA-homologous gene in bacteria.

Amino Acid Sequence↗
PubMed2001

A gene family expressing a host-protective antigen of Echinococcus granulosus.

Echinococcus granulosus causes cystic hydatidosis in humans. A recombinant antigen vaccine has been developed, for use in the parasite's natural animal intermediate hosts, that may provide a new tool for control of hydatid disease transmission. The antigen, designated EG95, is encoded by a cDNA the features of which indicate it to be an incomplete copy of the associated mRNA. Characterisation of the gene(s) encoding the antigen was undertaken in order to enable subsequent study of genetic variability in the gene and associated protein in different parasite isolates. Southern hybridisation studies of E. granulosus genomic DNA probed with the eg95 cDNA revealed that the gene belonged to a gene family. DNA sequence analysis of cloned genomic fragments indicated that the gene family consists of at least seven members, one of which is a pseudogene. The gene having identity with the eg95 cDNA was cloned and sequenced, and the full length mRNA characterised. Genomic sequence and structure of the eg95 gene family members are highly conserved with respect to the gene encoding EG95. Four eg95-related genes are predicted to express an identical EG95 protein and all four were shown to be expressed in the oncosphere life-cycle stage. The full length EG95 protein has a predicted molecular mass of 16.9 kDa, secretory signal sequence, carboxy-terminal glycosylphosphatidylinositol hydrophobic anchor motif and a fibronectin type III domain. PCR amplification conditions were established which allow gene-specific characterisation of the eg95 gene in E. granulosus isolates from different host species and geographical locations.

5' Untranslated Regions↗