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At least 775 records · Page 43Linked to original sources
PubMed1999

Molecular cloning and biological activity of a novel Ha-Ras suppressor gene predominantly expressed in skeletal muscle, heart, brain, and bone marrow by differential display using clonal mouse EC cells, ATDC5.

We cloned a cDNA encoding a novel mouse protein, named A-C1, by differential display between two mouse cell lines: embryonic fibroblast C3H10T1/2 and chondrogenic ATDC5. The deduced amino acid sequence of A-C1 consists of 167 amino acids and shows 46% identity with that of a ras-responsive gene, rat Ha-rev107. Northern blot analysis showed a distinct hybridization band of 3.2 kilobases. Expression of A-C1 mRNA was detected in undifferentiated ATDC5 cells and myoblastic C2C12 cells, while none of C3H10T1/2 cells, NIH3T3 fibroblasts, Balb/c 3T3 fibroblasts, osteoblastic MC3T3-E1 cells, and ST2 bone marrow stromal cells expressed A-C1 mRNA in vitro. Moreover, A-C1 mRNA was expressed in skeletal muscle, heart, brain, and bone marrow in adult mice. By in situ hybridization, A-C1 gene expression was localized in hippocampus as well as bone marrow cells. By immunocytochemistry, A-C1 protein was detected in the cytoplasm as well as perinuclear region of the cells. Transfection of A-C1 cDNA into Ha-ras-transformed NIH3T3 cell line caused increase in the number of flat colonies and inhibition of cell growth. Our data indicate that A-C1 is expressed in some specific tissues in vivo and modulates Ha-ras-mediated signaling pathway.

3T3 Cells↗
PubMed1994

Analysis of the sequences within and flanking the cyanoglobin-encoding gene, glbN, of the cyanobacterium Nostoc commune UTEX 584.

A 3.5-kb segment of DNA containing nifU glbN nifH nifD was cloned from a gene library of Nostoc 584 and sequenced. The nifU-glbN intergenic region contains short tandemly repeated repetitive sequences (5'-AATTACG). A sequence corresponding to a NifA-like upstream activator sequence (with the consensus recognition sequence for BifA in Anabaena 7120), elements of a nifH promoter and a sequence that may function as a transcription terminator, were identified downstream from glbN. GlbN, unique to certain Nostoc spp., is more homologous to protozoan myoglobins than to any other prokaryotic, vertebrate or plant globins.

Amino Acid Sequence↗
PubMed1990

Mapping and molecular cloning of the phn (psiD) locus for phosphonate utilization in Escherichia coli.

The Escherichia coli phn (psiD) locus encodes genes for phosphonate (Pn) utilization, for phn (psiD) mutations abolish the ability to use as a sole P source a Pn with a substituted C-2 or unsubstituted hydrocarbon group such as 2-aminoethylphosphonate (AEPn) or methylphosphonate (MPn), respectively. Even though the E. coli K-12 phosphate starvation-inducible (psi) phn (psiD) gene(s) shows normal phosphate (Pi) control, Pn utilization is cryptic in E. coli K-12, as well as in several members of the E. coli reference (ECOR) collection which are closely related to K-12. For these bacteria, an activating mutation near the phn (psiD) gene is necessary for growth on a Pn as the sole P source. Most E. coli strains, including E. coli B, are naturally Phn+; a few E. coli strains are Phn- and are deleted for phn DNA sequences. The Phn+ phn(EcoB) DNA was molecularly cloned by using the mini-Mu in vivo cloning procedure and complementation of an E. coli K-12 delta phn mutant. The phn(EcoB) DNA hybridized to overlapping lambda clones in the E. coli K-12 gene library (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987) which contain the 93-min region, thus showing that the phn (psiD) locus was itself cloned and verifying our genetic data on its map location. The cryptic phn(EcoK) DNA has an additional 100 base pairs that is absent in the naturally Phn+ phn(EcoB) sequence. However, no gross structural change was detected in independent Phn+ phn(EcoK) mutants that have activating mutations near the phn locus.

Cloning, Molecular↗
PubMed2000

Characterization of a bordetella pertussis diaminopimelate (DAP) biosynthesis locus identifies dapC, a novel gene coding for an N-succinyl-L,L-DAP aminotransferase.

The functional complementation of two Escherichia coli strains defective in the succinylase pathway of meso-diaminopimelate (meso-DAP) biosynthesis with a Bordetella pertussis gene library resulted in the isolation of a putative dap operon containing three open reading frames (ORFs). In line with the successful complementation of the E. coli dapD and dapE mutants, the deduced amino acid sequences of two ORFs revealed significant sequence similarities with the DapD and DapE proteins of E. coli and many other bacteria which exhibit tetrahydrodipicolinate succinylase and N-succinyl-L,L-DAP desuccinylase activity, respectively. The first ORF within the operon showed significant sequence similarities with transaminases and contains the characteristic pyridoxal-5'-phosphate binding motif. Enzymatic studies revealed that this ORF encodes a protein with N-succinyl-L,L-DAP aminotransferase activity converting N-succinyl-2-amino-6-ketopimelate, the product of the succinylase DapD, to N-succinyl-L,L-DAP, the substrate of the desuccinylase DapE. Therefore, this gene appears to encode the DapC protein of B. pertussis. Apart from the pyridoxal-5'-phosphate binding motif, the DapC protein does not show further amino acid sequence similarities with the only other known enzyme with N-succinyl-L,L-DAP aminotransferase activity, ArgD of E. coli.

Acyltransferases↗
PubMed1984

Structure and expression in Escherichia coli of a cloned rat interferon-alpha gene.

DNA synthesized by in vitro transcription on rat interferon (IFN) mRNA has been cloned and amplified as recombinant DNA. The nucleotide sequence of these rat IFN cDNA clones revealed i. the partial presence of the coding region of the gene and ii all cDNA clones were derived from the same subtype of rat IFN-alpha mRNA. Purified inserted fragments were used as a hybridisation probe against chromosomal "Southern blots" to show that at least twelve rat IFN-alpha-related sequences are present in the genome. A lambda-linked rat gene library was screened with the cDNA probes, resulting in an equivalent number of rat IFN-alpha-related hybrid phages. By use of a 3'-noncoding region as a probe, the chromosomal counterpart of the cDNA clones could be detected and the nucleotide sequence of its coding region has been determined. Expression of the coding region in E. coli yielded biologically active IFN, when tested for in vitro or in vivo antiviral activity.

Animals↗
PubMed1989

Molecular cloning and expression in Escherichia coli of a cellodextrinase gene from Bacteroides succinogenes S85.

A DNA fragment coding for a cellodextrinase of Bacteroides succinogenes S85 was isolated by screening of a pBR322 gene library in Escherichia coli HB101. Of 100,000 colonies screened on a complex medium with methylumbelliferyl-beta-D-cellobioside as the indicator substrate, two cellodextrinase-positive clones (CB1 and CB2) were isolated. The DNA inserts from the two recombinant plasmids were 7.7 kilobase pairs in size and had similar restriction maps. After subcloning from pCB2, a 2.5-kilobase-pair insert which coded for cellodextrinase activity was isolated. The enzyme was located in the cytoplasm of the E. coli host. It exhibited no activity on carboxymethyl cellulose, Avicel microcrystalline cellulose, acid-swollen cellulose, or cellobiose but hydrolyzed p-nitrophenyl-beta-D-cellobioside and p-nitrophenyl-beta-D-lactoside. The Km (0.1 mM) for the hydrolysis of p-nitrophenyl-cellobioside by the enzyme expressed in E. coli was similar to that reported for the purified enzyme from B. succinogenes. Expression of the cellodextrinase gene was subjected to catabolite repression by glucose and was not induced by cellobiose. The origin of the DNA insert from B. succinogenes was confirmed by Southern blot analysis. Western blotting (immunoblotting) using antibodies raised against the purified B. succinogenes cellodextrinase revealed a protein with a molecular weight of approximately 50,000 in E. coli clones which comigrated with the native enzyme isolated from B. succinogenes. These data indicate that the cellodextrinase gene expressed in E. coli is fully functional and codes for an enzyme with properties similar to those of the native enzyme.

Bacteroides↗
PubMed1994

Isolation and characterization of a regulatory gene affecting rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa.

A mutant strain (65E12) of Pseudomonas aeruginosa that is unable to produce rhamnolipid biosurfactants and lacks rhamnosyltransferase activity was genetically complemented by using a P. aeruginosa PG201 wild-type gene library. A single complementing cosmid was isolated on the basis of surface tension measurements of subcultures of the transconjugants by using a sib selection strategy. The subcloning of the complementing cosmid clone yielded a 2-kb fragment capable of restoring rhamnolipid biosynthesis, rhamnosyltransferase activity, and utilization of hexadecane as a C source in mutant 65E12. The nucleotide sequence of the complementing 2-kb fragment was determined, and a single open reading frame (rhlR) of 723 bp specifying a putative 28-kDa protein (RhlR) was identified. Sequence homologies between the RhlR protein and some regulatory proteins such as LasR of P. aeruginosa, LuxR of Vibrio fischeri, RhiR of Rhizobium leguminosarum, and the putative activator 28-kDa UvrC of Escherichia coli suggest that the RhlR protein is a transcriptional activator. A putative target promoter which is regulated by the RhlR protein has been identified 2.5 kb upstream of the rhlR gene. Multiple plasmid-based rhlR gene copies had a stimulating effect on the growth of the P. aeruginosa wild-type strain in hexadecane-containing minimal medium, on rhamnolipid production, and on the production of pyocyanin chromophores. Disruption of the P. aeruginosa wild-type rhlR locus led to rhamnolipid-deficient mutant strains, thus confirming directly that this gene is necessary for rhamnolipid biosynthesis. Additionally, such PG201::'rhlR' mutant strains lacked elastase activity, indicating that the RhlR protein is a pleiotropic regulator.

Alkanes↗
PubMed1992

Identification of the cellulose-binding domain of the cellulosome subunit S1 from Clostridium thermocellum YS.

The 3' region of a gene designated cipB, which shows strong homology with cipA that encodes the cellulosome SL subunit of Clostridium thermocellum ATCC 27405, was isolated from a gene library of C. thermocellum strain YS. The truncated S1 protein encoded by the cipB derivative bound tightly to cellulose. The cellulose-binding domain in this polypeptide consisted of a C-terminal proximal 167 residue sequence which showed complete identity with residues 337-503 of mature SL from C. thermocellum strain ATCC 27405. The cellulose-binding domain interacted with both crystalline and amorphous cellulose, but not with xylan.

Amino Acid Sequence↗
PubMed1996

Desferrioxamine-dependent iron transport in Erwinia amylovora CFBP1430: cloning of the gene encoding the ferrioxamine receptor FoxR.

Iron deprivation of Erwinia amylovora CFBP1430, a species causing fire blight on Pomoïdeae, was shown to induce the production of siderophores of the desferrioxamine (dfo) family and two outer membrane polypeptides with apparent molecular weight of about 70 and 80 kDa, respectively. Cyclic dfo E was characterized as the major metabolite. Phage MudIIpR13 insertional mutagenesis and screening on CAS-agar medium yielded three dfo non-producing and one overproducing clones. These clones failed to grow in the presence of the Fe(III) chelator EDDHA and were determined further as dfo and ferrioxamine transport negative mutants, respectively. The transport mutant which appeared to lack the 70 kDa polypeptide in the outer membrane allowed the purification of dfo E. Growth under iron limitation of dfo negative mutants was stimulated with ferrioxamine E and B but not with other ferrisiderophores tested. The host DNA sequence flanking the left terminal part of the MudIIpR13 prophage responsible for the transport mutation was cloned and used to probe a parental gene library by DNA-DNA hybridization. Two recombinant cosmids restoring the transport mutation to normal were identified. Both cosmids also conferred the ability to utilize ferrioxamine B and E as iron sources on a FhuE- mutant of Escherichia coli. This correlated with the production of an additional polypeptide of 70 kDa in the outer membrane of E. coli transconjugants, thus confirming that this protein serves the ferrioxamine receptor function (FoxR) in E. amylovora.

Bacterial Outer Membrane Proteins↗
PubMed1988

New aerobactin-mediated iron uptake system in a septicemia-causing strain of Enterobacter cloacae.

Unlike the great majority of the aerobactin-producing enteric bacteria documented in the literature, Enterobacter cloacae EK33, isolated from a case of human neonatal meningitis, did not show any homology at the DNA level with the prototype aerobactin system encoded by the ColV-K30 plasmid. However, both the nuclear magnetic resonance spectrum and fast-atom bombardment mass spectrometry of the siderophore purified from EK33 confirmed its identity with aerobactin. Bioassay screening of a gene library of total DNA of EK33 led to the isolation of several aerobactin-positive clones. Under conditions of iron limitation, these clones expressed in Escherichia coli a protein of 72 kilodaltons that reacted with antiserum raised against the pColV-K30 74-kilodalton aerobactin receptor, while the original E. cloacae strain synthesized an 85-kilodalton protein which also cross-reacted with the antiserum. Restriction endonuclease analysis of the cloned DNA confirmed the structural differences between the two aerobactin genetic systems.

Conjugation, Genetic↗
PubMed1986

Analysis of the genome of molluscum contagiosum virus by restriction endonuclease analysis and molecular cloning.

Virions of molluscum contagiosum virus (MCV), a member of the poxviridae, were isolated directly from lesions of individual patients and characterized by restriction enzyme analysis. The comparative analysis of the cleavage patterns and Southern blot hybridization of 14 independently isolated virus samples revealed that MCV isolates can be classified into two different types. The majority of MCV isolated from clinically typical skin lesions (13 of 14) showed similar DNA cleavage patterns and were termed MCV type 1, whereas one isolate derived from a vaginal lesion showed a completely different DNA cleavage pattern and therefore was termed MCV type 2. For detailed investigation of the viral genome, a defined gene library of MCV DNA sequences was established. The Bam HI DNA fragments of the viral genome of MCV type 1 prototype isolate 1/80 was inserted into the bacterial plasmid vector pAT153. With the exception of terminal fragments (fragments A and B) of the viral genome, all other DNA fragments were cloned. All cloned Bam HI DNA fragments were individually identified by digestion of the recombinant plasmid DNA with different restriction enzymes and screened by hybridization of plasmid DNA to viral DNA.

Adult↗
PubMed2006

Supplementation of the diet with high-viscosity beta-glucan results in enrichment for lactobacilli in the rat cecum.

BBn (BioBreeding) rats were fed casein-based diets supplemented with barley flour, oatmeal flour, cellulose, or barley beta-glucans of high [HV] or low viscosity [LV] in order to measure the prebiotic effects of these different sources of dietary fiber. The dietary impact on the composition of the cecal microbiota was determined by the generation of denaturing gradient gel electrophoresis (DGGE) profiles of PCR-amplified 16S rRNA gene sequences. The DGGE profiles produced from the cecal microbiota of rats within each dietary group were similar, but consensus profiles generated from pooled bacterial DNAs showed differences between rat groups. Animals fed HV glucans (HV-fed rats) had DGGE consensus profiles that were 30% dissimilar from those of the other rat groups. A 16S rRNA gene fragment that was more conspicuous in the profiles of HV-fed animals than in those of cellulose-fed rats had sequence identity with Lactobacillus acidophilus. Measurements of L. acidophilus rRNA abundance (DNA-RNA hybridization), the preparation of cloned 16S rRNA gene libraries, and the enumeration of Lactobacillus cells (fluorescent in situ hybridization) showed that lactobacilli formed a greater proportion of the cecal microbiota in HV-fed rats. In vitro experiments confirmed that some lactobacilli utilize oligosaccharides (degree of polymerization, 3 or 4) present in beta-glucan hydrolysates. The results of this study have relevance to the use of purified beta-glucan products as dietary supplements for human consumption.

Animals↗
PubMed1986

Selection of bacterial pac sites recognized by Salmonella phage P22.

A gene library of chromosomal PstI fragments from Salmonella typhimurium strain DB5575 has been established. By means of phage P22 mediated transduction, ten different clones which contained inserts that promoted plasmid transduction were selected out of a total of about 7,000 clones. Seven of these clones carried inserts that stimulated transduction independently of general and int-promoted recombination and were interpreted as carrying pac analogous signals. The remaining three clones carried inserts that promoted transduction under recombination proficient conditions, whereas transduction occurred at reduced rate in the absence of recombination. These were believed to have short regions of homology with P22 DNA.

Cloning, Molecular↗
PubMed1988

A heat shock operon in Coxiella burnetti produces a major antigen homologous to a protein in both mycobacteria and Escherichia coli.

A gene library from the DNA of Coxiella burnetii has been constructed in the cosmid vector pHC79. A particular clone, pJB196, reacted strongly with Coxiella-specific antibodies elicited in a number of different species of animals. This clone produced two abundant C. burnetii-specific polypeptides, a 14-kilodalton nonimmunoreactive protein and a 62-kilodalton immunoreactive protein. Sequencing identified two open reading frames, encoding polypeptides of 10.5 and 58.3 kilodaltons. The only transcriptional control element observed on the 5' side of the initiation codon resembled a heat shock promoter. This heat shock promoter was functionally regulated in Escherichia coli, since both proteins were produced by growth conditions at 37 degrees C and neither protein was detected at 23 degrees C. There were four sequences from the literature that were highly homologous (greater than 50%) to the 62-kilodalton protein from C. burnetii. Three were from Mycobacterium species and represent the immunodominant antigen of this genus. The other was from E. coli, detected as a gene that complements or suppresses a temperature-sensitive RNase activity. Since the recombinant protein was immunogenic, it may serve as an efficacious vaccine against C. burnetii and other pathogenic microorganisms that express the conserved antigen.

Amino Acid Sequence↗
PubMed1994

Cloning and sequencing of a Rhodothermus marinus gene, bglA, coding for a thermostable beta-glucanase and its expression in Escherichia coli.

A gene library of the thermophilic eubacterium, Rhodothermus marinus, strain 21, was prepared in pUC18 and used to transform Escherichia coli. Of 5400 transformants, two produced halos on lichenan plates after Congo-red staining. Restriction mapping showed that the two clones shared an overlapping 1200-bp DNA fragment, which was used for DNA sequencing. Five potential methionine (Met) translational-initiation codons were identified. A putative signal peptide of 30 amino acids was identified with a hydrophobic core of nine hydrophobic amino acids. The molecular mass of the mature enzyme was estimated to be 29.7 kDa. A comparison of the primary protein sequence of beta-glucanase of Rhodothermus marinus with other glycosyl hydrolases showed 38.5% identity to the C-terminal part of the beta-1,3-glucanase of Bacillus circulans and limited identity to bacterial endo-beta-1,3-1,4-glucanases. The amino acid sequence showed high similarity to regions surrounding the catalytic Glu residue of bacterial beta-glucanases. A gene fragment of 889 bp containing the catalytic domain was overexpressed in E. coli using the pET23, T7-phage RNA polymerase system. The enzyme showed activity on lichenan, beta-glucan and laminarin but not on CMC cellulose or xylan. The expressed enzyme was purified by heat treatment of the host. The enzyme had a temperature and pH optima of 85 degrees C and pH 7.0, respectively, and was shown to retain full activity after incubation for 16 h at 80 degrees C and have a half life of 3 h at 85 degrees C.

Amino Acid Sequence↗
PubMed1994

Characterization and expression of the Lactobacillus helveticus pepC gene encoding a general aminopeptidase.

An aminopeptidase C gene (pepC) was detected by nucleic acid hybridization from an industrially important Lactobacillus helveticus strain. Three hybridization positive clones were isolated from a gene library of this L. helveticus strain, and one of them was characterized in more detail. Deletion mapping localized the hybridization positivity into a 2.8-kb fragment, which also encoded aminopeptidase activity. This fragment was sequenced and two open reading frames (ORF1 and 2) of 1347 and 840 base pairs were identified. The ORF1 was preceded by a typical prokaryotic promoter region, and an inverted repeat structure with delta G of -49.0 kJ mol-1 was found downstream of the coding region. The deduced amino acid sequence of ORF1, with an encoding capacity for a 51.4-kDa protein, was shown to share 48.3% and 98.0% identities with the PepC proteins from Lactococcus lactis and L. helveticus CNRZ32, respectively, thus confirming that ORF1 codes for an aminopeptidase C. mRNA size analyses revealed 1.7-kb and 2.7-kb transcripts in Northern blot with the pepC-specific probe. A further analysis with the pepC- and ORF2-specific probes showed that downstream ORF2 is co-transcribed with the pepC gene at the exponential phase of growth whereas, at the stationary growth phase, transcripts derived from the pepC promoter were below the detection limit, and the ORF2 was expressed by its own promoter. The 5' end mapping of the pepC transcripts with primer extension revealed one transcription start site suggesting a new position for the pepC promoter region when compared to that predicted for the L. helveticus CNRZ32 pepC gene. Expression of pepC was also studied in L. helveticus as the function of growth in a bioreactor study. Transcription of pepC was typical to exponential growth phase expression. The level of total thiol-aminopeptidase activity, however, remained nearly constant throughout the stationary growth phase.

Amino Acid Sequence↗
PubMed1991

Isolation and developmentally regulated expression of an Aspergillus nidulans phenol oxidase-encoding gene, ivoB.

Ivory (ivo) mutants of Aspergillus nidulans lack conidiophore pigmentation. We have cloned ivoB which codes for a conidiophore-specific phenol oxidase (AHTase) via the adjacent selectable ureD gene. Gene-library transformants of a ureD4 strain proved defective for the vector, but we recovered both ureD and ivoB from a lambda library of transformant DNA. The ivoB transcription unit was localized to a SalI-XbaI 3-kb fragment and its 5' end was located by hybridization with an oligodeoxyribonucleotide corresponding to the N-terminal polypeptide sequence of AHTase. Expression of the ivoB 1.4-kb mRNA corresponded temporally with AHTase in conidiating cultures, and the levels of both mRNA and AHTase in leaky brlA mutants implied transcriptional control by brlA. A second developmentally regulated locus of unknown function adjacent to ivoB was also transcriptionally dependent on brlA, but was expressed 4 h later.

Aspergillus nidulans↗
PubMed1989

Characterization of native and recombinant 75-kilodalton immunogens from Chlamydia trachomatis serovar L2.

A 75-kilodalton (kDa) immunogen from Chlamydia trachomatis serovar L2 was characterized. The 75-kDa protein was localized in the cytoplasm of chlamydiae and was shown to be a protein synthesized early in the developmental cycle of chlamydiae. A gene library was made by the recombinant DNA technique, using the expression vectors pEX1, pEX2, and pEX3. From this library one clone was found which reacted with a monoclonal antibody against the 75-kDa immunogen of C. trachomatis. The 75-kDa protein produced by the recombinant Escherichia coli was expressed independently of the promoter for the hybrid protein cro-betagalactosidase. Thus it is not produced as a fusion protein. Epitope mapping of the 75-kDa protein from C. trachomatis L2 and from the recombinant E. coli performed by Staphylococcus aureus V8 protease digestion showed that the two proteins are identical. Furthermore, patient sera reacted with both proteins.

Animals↗