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At least 595 records · Page 33Linked to original sources
PubMed1997

Expression patterns and promoter activity of the cold-regulated gene ci21A of potato.

Storage of potato (Solanum tuberosum) tubers at 4 degrees C is associated with the accumulation of several transcripts. DNA sequence analysis of cDNA clone CI21, which corresponds to one of the cold-induced transcripts, revealed high homology to transcripts of tomato (Lycopersicon esculentum) and wild potato (Solanum chacoense) induced by ripening and water stress. Two homologous, nonallelic genes, ci21A and ci21B, were isolated and sequenced. Northern blot analysis showed that CI21 transcripts were present at the highest levels in cold-stored tubers, at lower levels in stems and roots, and at the lowest levels in leaves and tubers stored at room temperature. Treatment with abscisic acid, heat, and a high concentration of salt had no marked effect on CI21 transcript levels in tubers and leaves. Drought was the only stress treatment that induced CI21 transcripts in leaves, but it did not do so in tubers. Western blot analysis detected CI21 protein only in tubers. Chimeric gene constructs between the putative ci21A promoter region and the uidA reporter gene were tested in transgenic potato plants for induction of beta-glucuronidase activity by low temperature. A 2-fold increase of beta-glucuronidase activity in response to tuber storage at 4 degrees C was observed for fragments between 380 and 2000 bp of the ci21A promoter region.

Amino Acid Sequence↗
PubMed1993

The minCD locus of Bacillus subtilis lacks the minE determinant that provides topological specificity to cell division.

A key event of the sporulation process in Bacillus subtilis is the asymmetric cell division that divides the developing cell into two unequal compartments. To examine the function of vegetative cell division genes in this developmental division, we isolated and characterized the B. subtilis counterpart to the Escherichia coli minicell operon minB, which governs correct placement of the division septum. Starting from the closely linked spoIVF locus, we used walking methods to isolate the region of the B. subtilis chromosome proximate to the divIVB minicell locus. DNA sequence analysis found two open reading frames whose predicted products had significant identity to the E. coli MinC cell division inhibitor and the MinD ATPase activator of MinC, and disruption of minCD function generated a minicell phenotype in B. subtilis. Notably, no homologue to the E. coli MinE topological specificity element was found in the B. subtilis minCD region. The B. subtilis min genes were part of an operon transcribed from a major promoter more than 2.5 kb upstream from minC. An internal promoter immediately upstream from minC was dependent on RNA polymerase containing sigma-H and was active at the onset of sporulation. However, neither minC nor minD function was absolutely required for sporulation and, by implication, for asymmetric septum formation.

Adenosine Triphosphatases↗
PubMed1993

Characterization of anf genes specific for the alternative nitrogenase and identification of nif genes required for both nitrogenases in Rhodobacter capsulatus.

To identify Rhodobacter capsulatus nif genes necessary for the alternative nitrogenase, strains carrying defined mutations in 32 genes and open reading frames of nif region A, B or C were constructed. The ability of these mutants to grow on nitrogen-free medium with molybdenum (Nif phenotype) or in a nifHDK deletion background on medium without molybdenum (Anf phenotype) was tested. Nine nif genes and nif-associated coding regions are absolutely essential for the alternative nitrogenase. These genes comprise nifV and nifB, the nif-specific ntr system (nifR1, R2, R4) and four open reading frames, which exhibit no homology to known genes. In addition, a significantly reduced activity of both the alternative nitrogenase and the molybdenum-dependent nitrogenase was found for fdxN mutants. By random Tn5 mutagenesis of a nifHDK deletion strain 42 Anf- mutants were isolated. Southern hybridization experiments demonstrated that 17 of these Tn5 mutants were localized in at least 13 different restriction fragments outside of known nif regions. Ten different Anf- Tn5 mutations are clustered on a 6 kb DNA fragment of the chromosome designated anf region A. DNA sequence analysis revealed that this region contained the structural genes of the alternative nitrogenase (anfHDGK). The identification of several Tn5 insertions mapping outside of anf region A indicated that at least 10 genes specific for the alternative nitrogenase are present in R. capsulatus.

Amino Acid Sequence↗
PubMed1995

glmS of Thermus thermophilus HB8: an essential gene for cell-wall synthesis identified immediately upstream of the S-layer gene.

A 30 kbp chromosomal region containing the S-layer gene (slpA) from Thermus thermophilus HB8 was cloned from a lambda phage gene library. DNA sequence analysis of the region upstream to the slpA gene revealed the presence of an open reading frame (ORF) which coded for a 604-amino-acid protein highly homologous to the glucosamine-6-P synthases (EC 2.6.1.16) of both prokaryotic and eukaryotic origin. The identification of this ORF as the glucosamine-6-P synthase gene from T. thermophilus (glmSth) has been carried out using three different strategies: (i) complementation of an Escherichia coli glmS mutant; (ii) in vivo insertional inactivation of the gene; and (iii) in vitro synthesis of glucosamine-6-P at 60 degrees C by a cytoplasmic extract of an overproducing E. coli strain. The glmSth gene is transcribed divergently from slpA in a 2.0 kb mRNA which probably also includes a tryptophan tRNA gene (trpTth) identified at its 3' extreme. As the products of both the glmSth and the slpA genes are main components of the cell envelope of T. thermophilus, their unusual clustering in the chromosome could be related to the existence of specific mechanisms for their coordinate expression.

Amino Acid Sequence↗
PubMed1995

Characterization of the superoxide dismutase gene and its upstream region from Methanobacterium thermoautotrophicum Marburg.

A gene (sod) encoding superoxide dismutase (SOD) was isolated from the strictly anaerobic archaeon Methanobacterium thermoautotrophicum Marburg. Its identify was confirmed by functional complementation of an Escherichia coli mutant strain lacking SOD activity and by DNA sequence analysis of a cloned fragment. Upstream of sod, separated by a 5-bp intergenic region, lies the open reading frame orfk which potentially codes for a protein of 209 amino acid residues. The amino acid sequence for this presumptive product had a similarity coefficient of 55.5% to a subunit of the alkyl hydroperoxide reductase (encoded by the ahpC gene) from Salmonella typhimurium.

Amino Acid Sequence↗
PubMed2000

Characterization of adherence of nontypeable Haemophilus influenzae to human epithelial cells.

The adherence of 58 nontypeable Haemophilus influenzae isolates obtained from patients with otitis media or chronic obstructive pulmonary disease (COPD) and obtained from the throats of healthy individuals to Chang and NCI-H292 epithelial cells was compared. Otitis media isolates, but not COPD isolates, adhered significantly more to both cell lines than did throat isolates. Since high-molecular-weight (HMW) proteins are major adhesins of nontypeable H. influenzae, the isolates were screened for HMW protein expression by Western blotting with two polyclonal sera and PCR with hmw-specific primers. Twenty-three of the 32 adhering isolates (72%) and only 1 of the 26 nonadherent strains were HMW protein or hmw gene positive. Among the 32 isolates adhering to either cell line, 5 different adherence patterns were distinguished based on the inhibiting effect of dextran sulfate. Using H. influenzae strain 12 expressing two well-defined HMW proteins (HMW1 and HMW2) and its isogenic mutants as a reference, we observed HMW1-like adherence to both cell lines for 16 of the 32 adherent isolates. Four others showed HMW2-like adherence to NCI-H292. Of the three other patterns of adherence, one probably also involved HMW protein. Screening of the isolates with six HMW-specific monoclonal antibodies in a whole-cell enzyme-linked immunosorbent assay showed that the HMW proteins of COPD isolates and carrier isolates were more distinct from the HMW proteins from H. influenzae strain 12 than those from otitis media isolates. Characterization of the HMW protein of a COPD isolate by adherence and DNA sequence analysis showed that despite large sequence diversity in the hmwA gene, probably resulting in the antigenic differences, the HMW protein mediated the HMW2-like adherence of this strain.

Adhesins, Bacterial↗
PubMed1994

Role of phosphoglucomutase in lipooligosaccharide biosynthesis in Neisseria gonorrhoeae.

A region of pSG30 that complements the pyocin-derived gonococcal lipooligosaccharide (LOS) mutants 1291d and 1291e was characterized by DNA sequence analysis and an open reading frame of 1,380 bases was identified that is 89% similar and 56% identical over 452 amino acids to the algC gene product from Pseudomonas aeruginosa that encodes phosphomannomutase. Enzymatic analysis of gonococcal crude protein extracts demonstrated that pSG30 encodes phosphoglucomutase (PGM) and phosphomannomutase activity. This activity is absent in 1291d and 1291e but is restored upon introduction of pSG30. PGM encoded by pSG34, a subclone of pSG30, was able to complement Escherichia coli PGM1, a strain deficient in PGM, as determined by bacteriophage C21 plaque formation. A revertant of 1291d that binds monoclonal antibody 2-1-L8 (specific for a 3.6-kDa LOS component) was isolated. The construction of a site-specific deletion of this region in the chromosome of 1291 confirms the role of this open reading frame in LOS biosynthesis.

Amino Acid Sequence↗
PubMed1994

Molecular characterization of a conserved 20-kilodalton membrane-associated lipoprotein antigen of Helicobacter pylori.

Antisera raised in rabbits to whole cells of Helicobacter pylori recognized as a major antigen a protein with an apparent molecular weight of 20,000. The antigen was purified by differential solubilization with N-octyl-beta-D-glucopyranoside, urea, and sodium dodecyl sulfate followed by molecular sieving. The mass of the protein, Lpp20, was 18,283 Da as determined by mass spectrometry. The lpp20 gene encoding this protein was cloned in Escherichia coli by using the vector lambda EMBL3, and plasmid subclones expressed the full-length protein from the native H. pylori promoter. lpp20 was mapped to the same 358-kb NruI fragment as flaB. DNA sequence analysis showed that the gene was 525 bp long and encoded a 175-amino-acid protein with a molecular weight of 19,094 containing a 21-residue typical lipoprotein signal peptide and consensus prolipoprotein processing site. The mass of the deduced 154-residue mature protein was 16,865 Da. Growth of E. coli cells expressing the cloned H. pylori lpp20 gene in the presence of [3H]palmitic acid resulted in radiolabelled Lpp20 while treatment of the E. coli cells with globomycin caused accumulation of unprocessed Lpp20, consistent with Lpp20 being a lipoprotein. Lpp20 cofractionated with the cytoplasmic membrane fraction, although a proportion of the protein was also found in the outer membrane. A mutant generated by mutant-allele exchange displayed normal viability, showing that Lpp20 belonged to the nonessential class of lipoproteins.

Amino Acid Sequence↗
PubMed1995

Characterization of a 2,3-dihydroxybiphenyl dioxygenase from the naphthalenesulfonate-degrading bacterium strain BN6.

An extradiol dioxygenase was cloned from the naphthalenesulfonate-degrading bacterial strain BN6 by screening a gene bank for colonies with 2,3-dihydroxybiphenyl dioxygenase activity. DNA sequence analysis of a 1,358-bp fragment revealed an open reading frame of only 486 bp. This is the smallest gene encoding an extradiol dioxygenase found until now. Expression of the gene in a T7 expression vector enabled purification of the enzyme. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the protein was a dimer with a subunit size of 21.7 kDa. The enzyme oxidized 2,3-dihydroxybiphenyl, 3-isopropylcatechol, 3- and 4-chlorocatechol, and 3- and 4-methylcatechol. Since the ability to convert 3-chlorocatechol is an unusual characteristic for an extradiol-cleaving dioxygenase, this reaction was analyzed in more detail. The deduced amino-terminal amino acid sequence differed from the corresponding sequence of the 1,2-dihydroxynaphthalene dioxygenase, which had been determined earlier from the enzyme purified from this strain. This indicates that strain BN6 carries at least two different extradiol dioxygenases.

Amino Acid Sequence↗
PubMed1995

A mutation in the Neisseria gonorrhoeae rfaD homolog results in altered lipooligosaccharide expression.

The gonococcal lsi-6 locus was cloned and shown by DNA sequence analysis to have homology with the E. coli rfaD gene, which encodes ADP-L-glycero-D-mannoheptose epimerase. This enzyme is involved in the biosynthesis of the lipopolysaccharide precursor ADP-L-glycero-D-mannoheptose. A site-directed frameshift mutation in lsi-6 was constructed by PCR amplification and introduced into the chromosome of Neisseria gonorrhoeae MS11 P+ by transformation. The lipooligosaccharides (LOS) of mutant and parental strains were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The lsi-6 mutant produced LOS components with apparent molecular masses of 2.6 and 3.6 kDa as compared with a 3.6-kDa band of the MS11 P+ strain. The parental LOS phenotype was expressed when a revertant was constructed by transformation of the cloned wild-type gene into the lsi-6 mutant. The immunoreactivity of LOS from parental and constructed strains was examined by SDS-PAGE and Western blotting. Only the parental and reconstructed wild-type strains produced a 3.6-kDa LOS component that reacted with monoclonal antibody 2-1-L8. These results suggest that the lsi-6 locus is involved in gonococcal LOS biosynthesis and that the nonreactive mutant 3.6-kDa LOS component contains a conformational change or altered saccharide composition that interferes with immunoreactivity.

Amino Acid Sequence↗
PubMed1996

Molecular and evolutionary analyses of a variable series of genes in Borrelia burgdorferi that are related to ospE and ospF, constitute a gene family, and share a common upstream homology box.

In this study we report on the molecular characterization of a series of genes that constitute a gene family related to ospE and ospF. Some members of this family appear to represent recombined or variant forms of ospE and ospF. Variant ospE and ospF genes were found in several Borrelia burgdorferi isolates, demonstrating that their occurrence is not a phenomenon relevant to only a single isolate. Hybridization analyses revealed that the upstream sequence originally identified 5' of the full-length ospEF operon exists in multiple copies ranging in number from two to six depending on the isolate. This repeated sequence, which we refer to as the upstream homology box (UHB), carries a putative promoter element. In some isolates, UHB elements were found to flank copies of ospE and ospF that exist independently of each other. We refer to this group of UHB-flanked genes collectively as the UHB gene family. The evolutionary relationships among UHB gene family members were assessed through DNA sequence analysis and gene tree construction. These analyses suggest that some UHB-flanked genes might actually represent divergent forms of other previously described genes. Analysis of the restriction fragment length polymorphism patterns of the UHB-flanked genes among B. burgdorferi isolates demonstrated that these patterns are highly variable among isolates, suggesting that these genes are not phylogenetically conserved. The variable restriction fragment length polymorphism patterns could indicate recombinational activity in these sequences. The presence of numerous copies of the UHB elements and the high degree of homology among UHB-flanked genes could provide the necessary elements to allow for homologous recombination, leading to the generation of recombination variants of UHB gene family members.

Amino Acid Sequence↗
PubMed1997

An endoglucanase from the anaerobic fungus Orpinomyces joyonii: characterization of the gene and its product.

An endoglucanase gene (celA) was isolated from a genomic library of the ruminal fungus Orpinomyces joyonii. DNA sequence analysis of celA revealed an intronless gene encoding a typical signal sequence, an N-terminal catalytic domain, two repeated regions linked by a short Ser/Thr-rich linker and a domain of unknown function. The deduced amino acid sequence of the catalytic domain showed homology with the family 5 cellulases. While the catalytic domain of CelA was not homologous to the catalytic domain of the endoglucanase gene (EG3) from the ruminal bacterium Fibrobacter succinogenes, the repeated regions of CelA were very similar to the noncatalytic domain of EG3. This suggests that evolutionary shuffling of endoglucanase domains might occur among bacteria and fungi within the anaerobic ecosystem of the rumen. The celA gene was expressed in Escherichia coli, and the periplasmic endoglucanase was used for the characterization studies of the enzyme. CelA exhibited both endoglucanase and xylanase activities. Its pH optimum was 4 and the temperature optimum was 40 degrees C. Deletion analysis showed that the repeated sequences and C-terminal domain of CelA were not required for enzyme activity.

Amino Acid Sequence↗
PubMed1988

Murine A type retroviruses promote high levels of gene expression in embryonal carcinoma cells.

The expression of Intracisternal A Particle (IAP) genes in the mouse embryonal carcinoma cell line PCC3 was investigated by cDNA cloning and transient gene expression assays. A group of 26 IAP cDNA clones, products of transcriptionally active IAP proviruses, were selected from a cDNA library made from undifferentiated PCC3 cell RNA. Several of these clones were characterized by restriction enzyme mapping and DNA sequence analysis. The DNA sequence in both the promoter and structural regions of two cDNAs closely resembles those of IAP genomic clones. Three new sequence elements were identified within the U3 region, an Sp1 transcription-factor-binding site, an adenovirus E1a enhancer sequence and a region of homology to a promoter element of adenovirus E4 gene. Hybrid constructs were made that place the U3/R region of the IAP cDNAs immediately 5' to the chloramphenicol acetyl transferase (CAT) gene. IAP-CAT constructs were transfected into PCC3 cells, and cell extracts prepared and analysed for CAT enzyme activity and CAT RNA levels. IAP-CAT transfected cells were shown to contain substantial levels of CAT enzyme activity and to accumulate much greater levels of CAT RNA than two standard promoters, pRSVcat and pSV2cat. The ability of these A type retroviral promoters to function in PCC3 cells is in direct contrast to the near total restriction of normal C type retroviral expression in EC cells.

Animals↗
PubMed1998

MEN1 gene mutations in 12 MEN1 families and their associated tumors.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant inherited tumor syndrome characterized by the development of multiple endocrine tumors. The gene responsible for the disease, termed MEN1 gene. has recently been isolated and germline mutations have been described in affected MEN1 individuals. Twelve unrelated (German MEN1 families and their associated tumors (5 parathyroid tumors, 1 vipoma, 1 gastrinoma, 1 insulinoma) were characterized for MEN1 gene mutations by single-strand conformational variant (SSCV) analysis and DNA sequence analysis as well as for loss of heterozygosity on chromosome 11q13. We identified nine different heterozygous germline mutations (6 frameshift, 2 missense, 1 nonsense), eight of them were novel. Four of five informative MEN1-associated tumors revealed deletion of the second MEN1 allele, supporting the concept of a tumor suppressor gene. Furthermore. SSCV analysis proved an effective and sensitive method for the detection of menin mutations providing a reliable genetic screening approach supporting genetic counseling and clinical management of MEN1 family members.

Adenoma↗
PubMedDate not supplied

Isolation and characterization of a 6/85-like Mycoplasma gallisepticum from commercial laying hens.

Eighty-three-week-old table egg layers with swollen sinuses were presented with a history of increased mortality. Serology revealed positive titers to Mycoplasma gallisepticum (MG). The birds were part of a flock in which some birds had been vaccinated with 6/85 live MG vaccine at 18 wk of age. Tracheal cultures were obtained from both vaccinated and unvaccinated birds within the flock. The cultures were indistinguishable from 6/85 vaccine by both random amplified polymorphic DNA analysis and DNA sequence analysis. Challenge studies were performed to compare the field isolates with 6/85 vaccine and the R strain of MG. The field isolates produced a greater antibody response by serum plate agglutination than did the 6/85 vaccine. The isolates effectively colonized the trachea without increasing the tracheal mucosal thickness; however, they did not extensively colonize the air sacs or cause airsacculitis in the experimental birds.

Air Sacs↗
PubMed2004

[Identification of a novel mutation of the SEDL gene in X-linked spondyloepiphyseal dysplasia tarda].

OBJECTIVE: To investigate further the genetic basis of hereditary X-linked spondyloepiphyseal dysplasia tarda (SEDL). METHODS: Single strand conformation polymorphism (SSCP) combined with polymerase chain reaction and denaturing polyacrylamide gel electrophoresis were used to detect the mutation for the coding exons of SEDL gene as well as their exon/intron boundaries in 5 unrelated Chinese boys clinically diagnosed as having SEDL. DNA sequencing analysis was further used to identify the mutation. RESULTS: The 13 bp deletion mutation consisting of IVS5-2-1delAG and 322-332del TTTTCAATGAA was identified in one of SEDL patients, but not detected in 30 chromosomes from 30 unrelated normal male individuals. CONCLUSION: This is a novel mutation cosegregated with the patient's skeletal disease. The intragenic deletion occurred in the acceptor splice site of the 3' region of intron 5 and the 5' coding region of exon 6, which may result in one or a combination of splicing defects. The results of this study expand the spectrum of SEDL mutations associated with SEDL, and this will help to elucidate further the role of this novel protein in the etiology of this form of osteochondrodysplasia.

Adolescent↗
PubMed1996

Polymorphisms of the human alpha 2A-adrenergic receptor gene in a Catalan population: description of a new polymorphism in the promoter region.

Alpha 2A-adrenergic receptor polymorphisms were investigated in 55 unrelated Caucasian Catalan individuals. Phenotypes were expressed by alpha 2-adrenoreceptor density and affinity determined by radioligand binding. In addition to the two previously described biallelic restriction fragment length polymorphisms (RFLP) (DraI and Bsu36I) [Hoehe et al 1988; Sun et al 1992], a transition was located in the promoter region of the human alpha 2A-adrenergic receptor using a non-radioisotopic single-strand conformational polymorphism (SSCP) analysis. DNA sequence analysis of this region showed that this transition produces the loss of a HhaI site. In our sample, the estimates of the frequency of this allele was 11% and those of DraI alleles were similar to those reported for other Caucasian populations but different from those observed in Afro-Americans. In addition, frequencies of the Bsu36I alleles in the Catalan population differed from those of Japanese and American White populations. A slight linkage disequilibrium between Bsu36I-DraI was found in our population. No association between phenotypes and RFLP genotypes was observed.

Alleles↗
PubMed1996

Wavelet analysis of DNA sequences.

Wavelet decomposition is applied to the analysis of the nucleotide sequence of the rhodopsin gene of Chinese Hamster cells. The Lipschitz-Hölder exponents for the probability measurements of adenine, guanine, thymine and cytosine distributions are obtained. The local scaling found by means of wavelet analysis is argued to be an indication of long-range correlations.

Animals↗