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At least 559 records · Page 31Linked to original sources
PubMed1989

Cloning of chromosomal genes of Lactococcus by heterologous complementation: partial characterisation of a putative lactose transport gene.

A cosmid gene library of the genome of Lactococcus lactis subsp. lactis 712 has been constructed in the broad host range plasmid pLAFR1 in Escherichia coli LE392. Three lactococcal genes from the bank were identified by heterologous complementation of specific mutations in strains of E. coli. A cosmid clone encoding a putative lactose transport gene was identified by complementing an E. coli lacY mutant. The complemented clone supported the uptake of 14C lactose in transport assays. The DNA fragment responsible was subcloned and localised to a 1.28 kb fragment of the lactococcal chromosome.

Biological Transport↗
PubMed1985

Molecular cloning of WHI2, a gene involved in the regulation of cell proliferation in Saccharomyces cerevisiae.

The WHI2 gene of Saccharomyces cerevisiae plays a key role in coordinating cell proliferation and nutrient availability. A 2.6 kb yeast DNA sequence has been cloned which fully suppresses the whi2 mutation. Integration of this sequence to demonstrate that the structural gene itself had been cloned proved difficult. Integration occurred only rarely and only at the LEU2 locus which was also present on the integrating plasmid. To circumvent these difficulties an adjacent sequence, present on the original isolate from the gene library, was subcloned onto the integrating vector YIp5, after which directed integration proved straightforward. The integrated sequence was closely linked to WHI2, confirming that the structural gene had been cloned. A chromosomal restriction map of the WHI2 region is presented; no gross changes were observed in the region as cells entered stationary phase.

Autoradiography↗
PubMed2005

Diversity of green-like and red-like ribulose-1,5-bisphosphate carboxylase/oxygenase large-subunit genes (cbbL) in differently managed agricultural soils.

A PCR-based approach was developed to detect ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) form I large-subunit genes (cbbL) as a functional marker of autotrophic bacteria that fix carbon dioxide via the Calvin-Benson-Bassham cycle. We constructed two different primer sets, targeting the green-like and red-like phylogenetic groups of cbbL genes. The diversity of these cbbL genes was analyzed by the use of three differently managed agricultural soils from a long-term field experiment. cbbL gene fragments were amplified from extracted soil DNAs, and PCR products were cloned and screened by restriction fragment length polymorphism analysis. Selected unique cbbL clones were sequenced and analyzed phylogenetically. The green-like cbbL sequences revealed a very low level of diversity, being closely related to the cbbL genes of Nitrobacter winogradskyi and Nitrobacter vulgaris. In contrast, the red-like cbbL gene libraries revealed a high level of diversity in the two fertilized soils and less diversity in unfertilized soil. The majority of environmental red-like cbbL genes were only distantly related to already known cbbL sequences and even formed separate clusters. In order to extend the database of available red-like cbbL sequences, we amplified cbbL sequences from bacterial type culture strains and from bacterial isolates obtained from the investigated soils. Bacterial isolates harboring the cbbL gene were analyzed phylogenetically on the basis of their 16S rRNA gene sequences. These analyses revealed that bacterial genera such as Bacillus, Streptomyces, and Arthrobacter harbor red-like cbbL genes which fall into the cbbL gene clusters retrieved from the investigated soils.

Agriculture↗
PubMed1986

Hemolysin production and cloning of two hemolysin determinants from classical Vibrio cholerae.

The hemolytic activity of 20 classical and 3 El Tor strains of V. cholerae O1 was examined phenotypically and genetically. The El Tor strains lysed bovine, chicken, human, rabbit, and sheep erythrocytes (RBCs), while the classical strains lysed only chicken and rabbit RBCs. The assay was done with RBCs in Tris-NaCl buffer, since phosphate-buffered saline was found to inhibit hemolytic activity. Hemolytic activity in culture supernatants from El Tor strains was more sensitive to heat inactivation than that in supernatants from the classical strain 395. A gene library of strain 395 was examined for hemolytic activity, and two distinct hemolytic clones were identified. One clone appeared identical to the previously cloned hemolysin structural gene from El Tor V. cholerae, while the other did not hybridize to the El Tor hemolysin probe, had a unique restriction enzyme digestion pattern, and encoded a hemolysin whose activity differed from that of the El Tor hemolysin clones. We suggest that the hemolysin specified by the determinant originally cloned from an El Tor vibrio be designated hemolysin I and the second hemolysin, cloned from the classical vibrio, be designated hemolysin II.

Animals↗
PubMed1989

Isolation, characterization, and nucleotide sequence of appY, a regulatory gene for growth-phase-dependent gene expression in Escherichia coli.

A plasmid carrying a regulator gene, designated appY, was found in the screening of an Escherichia coli gene library for clones overproducing AppA, an acid phosphatase which is induced as a culture approaches the stationary phase. In cells containing multicopy plasmids carrying the appY gene, the expression of the chromosomal appY gene was stimulated 10- to 40-fold in the stationary phase and more than 100-fold during exponential growth. The appA plasmid also changed the rate of synthesis of more than 30 other proteins in a growth-phase-dependent way. The appY gene was mapped to 13 min on the E. coli genetic map. The position of the appY gene on the 4.9-kilobase HindIII fragment of the original clone was located by Tn5 mutagenesis and deletion analysis, and the nucleotide sequence of a 1.9-kilobase region containing the gene was determined. The appY gene product was identified as a weakly expressed 243-amino-acid polypeptide which contains a stretch of 20 amino acids showing very good similarity to the conserved DNA-binding domain of repressors and transcriptional activators.

Amino Acid Sequence↗
PubMed1994

Isolation, characterization, and expression in Escherichia coli of the Pseudomonas aeruginosa rhlAB genes encoding a rhamnosyltransferase involved in rhamnolipid biosurfactant synthesis.

Transposon Tn5-GM-induced mutant strains of Pseudomonas aeruginosa which are unable to produce rhamnolipid biosurfactants and lack rhamnosyltransferase activity have been isolated. The DNA regions flanking the transposon were cloned and used as specific probes for the isolation of the corresponding wild-type genes from a P. aeruginosa wild-type cosmid gene library. Single cosmid clones capable of restoring rhamnolipid synthesis in the mutant strains were isolated and further subcloned and sequenced, resulting in the identification of two genes (rhlAB) which are organized as an operon upstream of the previously identified rhlR regulatory gene. The RhlA protein (32.5 kDa) harbors a putative signal sequence, suggesting that this protein is located in the periplasm, while the RhlB protein (47 kDa) contains at least two putative membrane-spanning domains. The expression of the rhlAB genes was found to be enhanced 20-fold during the stationary phase of growth under conditions of nitrogen limitation, as measured by using rhlA::lacZ fusions. Moreover, the transcriptional activation of the rhlAB genes appears to depend on a functional RhlR regulatory protein. The sequence upstream of the rhlA promoter contains two inverted repeats which define putative binding sites for the RhlR regulator. The controlled expression of the rhlAB genes in Escherichia coli led to the formation of active rhamnosyltransferase. This provides direct evidence for the fact that the rhamnosyltransferase encoding genes have been identified.

Amino Acid Sequence↗
PubMed1988

A yeast gene required for the G1-to-S transition encodes a protein containing an A-kinase target site and GTPase domain.

A new temperature-sensitive mutant of Saccharomyces cerevisiae, gst1 (G1-to-S transition) was isolated. At nonpermissive temperature the mutant cells with large buds accumulated and DNA synthesis was substantially arrested. From the reciprocal experiment of temperature-shift and mating-factor treatment, it was shown that the execution point was post 'START'. This suggested that the mutation affected the G1-to-S phase transition in the cell cycle. A DNA clone complementing the gst1-1 mutation was isolated from a yeast gene library, and gst1 was mapped in chr4R, by Southern blotting of cloned sequence to the individual yeast chromosome DNA by OFAGE system and by genetic analysis. The gene product was tentatively assigned from DNA sequencing analysis, as a protein of mol. wt 76,565 which contained consensus sequences for a target site of cAMP-dependent protein kinase(s) and for GTPase with extensive homology to polypeptide chain elongation factor EF1 alpha.

Amino Acid Sequence↗
PubMed1983

Genes for elongation factor EF-1 alpha in the brine shrimp Artemia.

A plasmid carrying a cDNA sequence coding for elongation factor EF-1 alpha from Artemia was used to probe blots of mRNA and chromosomal DNA from Artemia. A messenger length for EF-1 alpha corresponding to 1850 nucleotides was found. Southern blots pointed to a limited number (1-4) of genes, coding for EF-1 alpha. From an Artemia gene library a recombinant phage was isolated, which contains genomic sequences of EF-1 alpha. S1-nuclease mapping indicated the presence of intervening sequences within this cloned gene.

Animals↗
PubMed1993

A gene encoding a protein related to eukaryotic protein kinases from the filamentous heterocystous cyanobacterium Anabaena PCC 7120.

Protein kinases play essential roles in the development of eukaryotic cells. These enzymes display various degrees of sequence similarity in their catalytic domains. This conservation has allowed the identification of protein kinases in a variety of organisms, including the Gram-negative bacterium Myxococcus xanthus. In this study, sequences related to those encoding eukaryotic protein kinases were amplified by PCR from DNA of Anabaena PCC 7120, a filamentous cyanobacterium that differentiates cells specifically for nitrogen fixation, called heterocysts, under conditions of combined nitrogen limitation. Results from Southern hybridization and sequencing of PCR products suggest the presence of a family of similar protein kinases in this strain. One of the corresponding genes (pknA) was isolated from a gene library. The N-terminal region of its amino acid sequence shows significant similarity to the catalytic domains of eukaryotic-type protein kinases. Expression of this gene was found to be developmentally regulated. Inactivation of pknA led to colonies that appeared light green and rough in the absence of combined nitrogen. Mutant filaments produce fewer heterocysts than wild-type ones. These results suggest that pknA is required for both normal cellular growth and differentiation of Anabaena PCC 7120.

Amino Acid Sequence↗
PubMed1979

Isolation of the lysozyme gene of chicken.

The lysozyme gene has been purified by molecular cloning from two chicken gene libraries. Several recombinant phages harbouring sequences homologous to a plasmid carrying a double stranded lysozyme cDNA have been isolated. One recombinant appears to carry an entire lysozyme gene. Electron microscopic studies show that the latter is split by at least three introns. The length of the gene is about 3.9 kb, 6 times longer than lysozyme mRNA.

Animals↗
PubMed2003

Molecular evidence for novel planctomycete diversity in a municipal wastewater treatment plant.

We examined anoxic and aerobic basins and an anaerobic digestor of a municipal wastewater treatment plant for the presence of novel planctomycete-like diversity. Three 16S rRNA gene libraries were constructed by using a 16S rRNA-targeted universal reverse primer and a forward PCR primer specific for Planctomyces: Phylogenetic analysis of 234 16S rRNA gene sequences defined 110 operational taxonomic units. The majority of these sequences clustered with the four known genera, Pirellula (32%), Planctomyces (18.4%), Gemmata (3.8%), and Isosphaera (0.4%). More interestingly, 42.3% of the sequences appeared to define two distantly separated monophyletic groups. The first group, represented by 35.5% of the sequences, was related to the Planctomyces group and branched as a monophyletic cluster. It exhibited between 11.9 and 20.3% 16S rRNA gene sequence dissimilarity in comparisons with cultivated planctomycetes. The second group, represented by 6.8% of the sequences, was deeply rooted within the Planctomycetales tree. It was distantly related to the anammox sequences (level of dissimilarity, 20.3 to 24.4%) and was a monophyletic cluster. The retrieved sequences extended the intralineage phylogenetic depth of the Plantomycetales from 23 to 30.6%. The lineages described here may have a broad diversity of undiscovered biochemical and metabolic novelty. We developed a new 16S rRNA-targeted oligonucleotide probe and localized members of one of the phylogenetic groups using the fluorescent in situ hybridization technique. Our results indicate that activated sludge contains very diverse representatives of this group, which grow under aerobic and anoxic conditions and even under anaerobic conditions. The majority of species in this group remain poorly characterized.

Bacteria↗
PubMed2001

The coxBAC operon encodes a cytochrome c oxidase required for heterotrophic growth in the cyanobacterium Anabaena variabilis strain ATCC 29413.

Three genes, coxB, coxA, and coxC, found in a clone from a gene library of the cyanobacterium Anabaena variabilis strain ATCC 29413, were identified by hybridization with an oligonucleotide specific for aa(3)-type cytochrome c oxidases. Deletion of these genes from the genome of A. variabilis strain ATCC 29413 FD yielded strain CSW1, which displayed no chemoheterotrophic growth and an impaired cytochrome c oxidase activity. Photoautotrophic growth of CSW1, however, was unchanged, even with dinitrogen as the nitrogen source. A higher cytochrome c oxidase activity was detected in membrane preparations from dinitrogen-grown CSW1 than from nitrate-grown CSW1, but comparable activities of respiratory oxygen uptake were found in the wild type and in CSW1. Our data indicate that the identified cox gene cluster is essential for fructose-dependent growth in the dark, but not for growth on dinitrogen, and that other terminal respiratory oxidases are expressed in this cyanobacterium. Transcription analysis showed that coxBAC constitutes an operon which is expressed from two transcriptional start points. The use of one of them was stimulated by fructose.

Anabaena↗
PubMed1988

[Gene amplification and rearrangement of murine ribosomal RNA in virus-induced Rauscher leukemia].

Using as a hybridization probe cDNA 35s RNA from the Rauscher leukemia cells, a part of rRNA gene cluster from the gene library of mice C-1 erythroleukemia cells has been cloned. Fragment 6.7 kb recloned into pUC19 rRNA was used as a probe to analyse organization of rRNA genes of mice with RL. The observed amplification and rearrangement in genome DNA of spleen cells are determined by a new type of their rRNA genes rearrangement in the nontranscribed as well as in the transcribed part of rRNA.

Animals↗
PubMed1992

Elongation factor 3 (EF-3) from Candida albicans shows both structural and functional similarity to EF-3 from Saccharomyces cerevisiae.

As with many other fungi, including the budding yeast Saccharomyces cerevisiae, the dimorphic fungus Candida albicans encodes the novel translation factor, elongation factor 3 (EF-3). Using a rapid affinity chromatography protocol, EF-3 was purified to homogeneity from C. albicans and shown to have an apparent molecular mass of 128 kDa. A polyclonal antibody raised against C. albicans EF-3 also showed cross-reactivity with EF-3 from S. cerevisiae. Similarly, the S. cerevisiae TEF3 gene (encoding EF-3) showed cross-hybridization with genomic DNA from C. albicans in Southern hybridization analysis, demonstrating the existence of a single gene closely related to TEF3 in the C. albicans genome. This gene was cloned by using a 0.7 kb polymerase chain reaction-amplified DNA fragment to screen to C. albicans gene library. DNA sequence analysis of 200 bp of the cloned fragment demonstrated an open reading frame showing 51% predicted amino acid identity between the putative C. albicans EF-3 gene and its S. cerevisiae counterpart over the encoded 65-amino-acid stretch. That the cloned C. albicans sequence did indeed encode EF-3 was confirmed by demonstrating its ability to rescue an otherwise non-viable S. cerevisiae tef3:HIS3 null mutant. Thus EF-3 from C. albicans shows both structural and functional similarity to EF-3 from S. cerevisiae.

Amino Acid Sequence↗
PubMed1983

Isolation and sequence analysis of the human corticotropin-releasing factor precursor gene.

A human genomic DNA segment containing the gene for the corticotropin-releasing factor precursor has been isolated by screening a gene library with an ovine cDNA probe. The cloned DNA segment has been subjected to restriction endonuclease mapping and nucleotide sequence analysis. Comparison of the nucleotide sequence of the gene with that of the ovine cDNA indicates that an intron of 800 bp is inserted in the segment encoding the 5'-untranslated region of the mRNA. The segment corresponding to the protein-coding and the 3'-untranslated region of the mRNA is uninterrupted. The mRNA and amino acid sequences of the human corticotropin-releasing factor precursor have been deduced from the corresponding gene sequence. The deduced amino acid sequence of human corticotropin-releasing factor exhibits seven amino acid substitutions in comparison with the ovine counterpart.

Amino Acid Sequence↗
PubMed1999

A beta-1,4-endoglucanase-encoding gene from Cellulomonas pachnodae.

A gene library of Cellulomonas pachnodae was constructed in Escherichia coli and was screened for endoglucanase activity. Five endoglucanase-positive clones were isolated that carried identical DNA fragments. The gene, designated cel6A, encoding an endoglucanase enzyme, belongs to the glycosyl hydrolase family 6 (cellulase family B). The recombinant Cel6A had a molecular mass of 53 kDa, a pH optimum of 5.5, and a temperature optimum of 50-55 degrees C. The recombinant endoglucanase Cel6A bound to crystalline cellulose and beech litter. Based on amino acid sequence similarity, a clear cellulose-binding domain was not distinguished. However, the regions in the Cel6A amino acid sequence at the positions 262-319 and 448-473, which did not show similarity to any of the known family-6 glycosyl hydrolases, may be involved in substrate binding.

Amino Acid Sequence↗
PubMed2004

Optimal construction of non-immune scFv phage display libraries from mouse bone marrow and spleen established to select specific scFvs efficiently binding to antigen.

Monoclonal antibodies (MAbs) are widely applied in basic research, medicine, and the pharmaceutical industry. Recently, applications and generations of MAbs have been increasingly attracting attention in many research areas since MAbs could be produced in large quantities with the development of genetic technology and antibody engineering. On the other hand, in recent years, phage display system has been developed for high-throughput isolation and generation of novel MAbs that have high affinity with various antigens. This technology is capable of constructing "Library" containing billions of phage repertoires displaying various antibody fragments, and rapid selection of a specific MAb from this phage library. Additionally, this technology has a great advantage that MAbs can be generated without immunization to animals. However, there are still relatively few reports confirming that useful MAbs can be derived from non-immune antibody libraries. The latter, as undertaken by current methods, seem unable to achieve the high quality required to produce useful MAbs for any desired antigen because cloning of antibody gene from non-immune donors is inefficient. This problem is caused by the fact that their RT-PCR primer sets, PCR conditions, and efficiency of subcloning through construction of antibody gene library cannot encompass all the antibody diversity. In an attempt to overcome some of these earlier problems, here we describe an optimized method to establish a high quality, non-immune library from mouse bone-marrow and spleen, and assess its diversity in terms of content of multiple antibodies for a wide antigenic repertoire. As an example of the application of the methodology, we describe the selection of specific MAbs binding to Luciferase and identify at least 18 different clones. Using this non-immune mouse antibody library, we also obtained MAbs for VEGF, VEGF receptor 2, TNF-alpha, and Pseudomonas Exotoxin, confirming the high quality of the library and its suitability for this application.

Animals↗
PubMed2005

Seasonal variation in gene expression for loblolly pines (Pinus taeda) from different geographical regions.

In developing xylem, gene expression levels vary in different genotypes, at different stages of development, throughout a growing season, and in response to stresses. Commercially important characteristics such as wood-specific gravity are known to differ with seed source. For example, when grown on a common site, the specific gravity of Arkansas loblolly pine (Pinus taeda L.) trees is greater than that of Louisiana loblolly pine, and Texas loblolly pines have a greater specific gravity than loblolly pines from the Atlantic coast. A microarray analysis was performed to examine variation in gene expression among trees from different geographical sources when grown on a common site, and seasonal variation in gene expression in each seed source. We used microarrays containing 2171 expressed sequence tags (ESTs) with putative functions of interest, selected from several loblolly pine xylem partial cDNA libraries and a shoot tip library. Genes with significant variation in expression for each factor were identified. Many genes preferentially expressed in latewood compared with earlywood were for proteins involved in cell wall biosynthesis. Variation in gene expression among trees from the two seed sources in each growing season suggests that there may be more differences between South Arkansas trees and South Louisiana trees in latewood than in earlywood. Variation in gene expression among trees from different regions may reflect adaptation to different environments.

Gene Expression Profiling↗