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At least 757 records · Page 42Linked to original sources
PubMed2004

Selection of unusual actinomycetal primary sigma70 factors by plant-colonizing Frankia strains.

Functional adaptations of sigma70 transcriptional factors led to the emergence of several paralogous lineages, each one being specialized for gene transcription under particular growth conditions. Screening of a Frankia strain EaI-12 gene library by sigma70 DNA probing allowed the detection and characterization of a novel actinomycetal primary (housekeeping) sigma70 factor. Phylogenetic analysis positioned this factor in the RpoD cluster of proteobacterial and low-G+C-content gram-positive factors, a cluster previously free of any actinobacterial sequences. sigma70 DNA probing of Frankia total DNA blots and PCR screening detected one or two rpoD-like DNA regions per species. rpoD matched the conserved region in all of the species tested. The other region was found to contain sigA, an alternative primary factor. sigA appeared to be strictly distributed among Frankia species infecting plants by the root hair infection process. Both genes were transcribed by Frankia strain ACN14a grown in liquid cultures. The molecular phylogeny of the sigma70 family determined with Frankia sequences showed that the alternative actinomycetal factors and the essential ones belonged to the same radiation. At least seven distinct paralogous lineages were observed among this radiation, and gene transfers were detected in the HrdB actinomycetal lineage.

Base Sequence↗
PubMed2005

Development of zinc finger domains for recognition of the 5'-CNN-3' family DNA sequences and their use in the construction of artificial transcription factors.

Considerable progress has been made in recent years in the design of transcription factors for the directed regulation of endogenous genes. Although many strategies involve selection methods that must be applied for each new target sequence, we have developed an approach based on linkage of predefined zinc finger domains that each recognize a three-base pair DNA sequence to construct artificial transcription factors that bind to a desired sequence. These domains can be assembled to recognize unique 18-base pair DNA sequences with high specificity. Here we report the development and characterization of zinc finger domains that bind to 15 of the 16 5'-CNN-3' subsites. These domains were created through a combination of phage display selection, site-directed mutagenesis, and de novo design. Furthermore, these domains were used to generate a highly specific six-finger protein targeting the ERBB-2 promoter. When fused to regulatory domains, this protein was capable of up- and down-regulating the expression of the endogenous ERBB-2 gene. With the addition of this collection of predefined zinc finger domains, most 5'-CNN-3'-, 5'-GNN-3'-, and 5'-ANN-3'-containing sequences can now be rapidly targeted for directed gene regulation and nuclease cleavage.

Amino Acid Sequence↗
PubMed1982

Cloning of the complete human cytomegalovirus genome in cosmids.

Purified virion DNA (155 X 10(6) Mr) of human cytomegalovirus (CMV) strain Ad169 was partially cleaved with restriction endonucleases HindIII and EcoRI and cloned in the respective cleavage sites of cosmid pHC79. A complete gene library was established in a set of clones containing the viral DNA in long overlapping segments. Restriction maps for HindIII (29 fragments) and EcoRI (36 fragments) were constructed from the linkage of cosmid-cloned fragments, from double digestions of cloned DNA, and from blot hybridization of labeled cloned viral DNA with restriction fragments of virion DNA and singly or doubly cleaved cosmid clones.

Cloning, Molecular↗
PubMed1992

Isolation and characterization of an ndvB locus from Rhizobium fredii.

A gene (ndvB) in Rhizobium meliloti that is essential for nodule development in Medicago sativa (alfalfa), specifies synthesis of a large membrane protein. This protein appears to be an intermediate in beta-1,2-glucan synthesis by the microsymbiont. Southern hybridization analysis showed strong homology between an ndvB (chvB) probe and genomic DNA of R. fredii but not from Bradyrhizobium japonicum. A cosmid clone containing the putative ndvB locus was isolated from a Rhizobium fredii gene library. The cosmid clone which complemented R. meliloti ndvB mutants for synthesis of beta-1,2-glucans and effective nodulation of alfalfa was mapped and subcloned. Fragment-specific Tn5 mutagenesis followed by homologous recombination into the R. fredii genome indicated that the region was essential for beta-1,2-glucan synthesis and for formation of an effective symbiosis with Glycine max (soybean).

DNA, Bacterial↗
PubMed1985

Evolution of the single copy alpha A-crystallin gene: differently sized mRNAs of mammals and birds show homology in their 3' non-coding regions.

alpha A-Crystallin, a major structural polypeptide of the vertebrate eye lens, is evolutionarily highly conserved. We have analyzed the corresponding nucleic acid sequences in both genomic DNA digests as well as in lens cytoplasmic RNA preparations from a wide variety of vertebrates by blot hybridization with cloned rat alpha A2-crystallin cDNA probes. The probes are not able to hybridize under any conditions to RNA and DNA derived from fishes and amphibia, but do show substantial homology with the sequences of mammals, birds and reptiles. The alpha A-crystallin gene, which has been isolated from a hamster gene library occurs only once in the haploid genome. Coding and 3'-untranslated regions of alpha A2-crystallin mRNA are conserved among all mammals and birds examined. However, the regions comprising the conserved sequences are differently represented in the ultimate mRNA. The alpha A2-mRNA 3'-non-coding regions of reptiles and birds are 300-550 bases longer than those of mammals. Some rodents produce next to the alpha A2-mRNA another messenger that encodes the alpha AIns-polypeptide possessing an insertion of 22/23 amino acid residues between positions 63 and 64 of the alpha A2-polypeptide chain. alpha A2 and alpha AIns-mRNA are generated from a single gene as major and minor species, respectively, in a proportion which is similar to the ratio of the polypeptides found in vivo and in vitro. The size heterogeneity of the alpha A2-mRNA from most mammals examined is due to the variable size of the poly(A) tail.

Animals↗
PubMed1989

Cloning and expression of a cohemolysin, the CAMP factor of Actinobacillus pleuropneumoniae.

The genetic determinant of the cohemolysin which is responsible for the CAMP phenomenon, a cohemolysis, of Actinobacillus pleuropneumoniae was cloned in Escherichia coli. Total DNA from the A. pleuropneumoniae serotype 1 type strain 4074 was used to construct a gene library in plasmid pUC18 in E. coli JM83. A total of 10,500 clones containing recombinant plasmids have been screened for hemolysis on blood plates. Fifty-five clones which showed a weak hemolytic response after 24 to 48 h of incubation were screened for the CAMP reaction with Staphylococcus aureus. This led to the identification of one clone which showed a positive CAMP reaction. Immunoblot analysis revealed that the recombinant strain expressed a protein with a molecular mass of 27,000 daltons, similar in size to the CAMP protein of the group B streptococci. Rabbit antibodies against the CAMP+ clone neutralized the CAMP reaction mediated by the E. coli strain containing the cloned CAMP gene as well as that of A. pleuropneumoniae. Antibodies raised against the cloned CAMP cohemolysin cross-reacted with Streptococcus agalactiae protein B. We designate the 27,000-dalton molecule CAMP factor protein and name its corresponding gene cfp.

Actinobacillus↗
PubMedDate not supplied

[Regions of human genome containing analogs of oncogenes and retrovirus genes. I. A family of c-mos genes and unusual structure of ORA-gp5 locus].

The structural organization of a number of recombinant phages previously selected from the human gene library has been studied. On the basis of comparison of physical maps and hybridization to cloned probes it was deduced that different human loci with the homology to v-mos are represented in lambda recombinants. The physical map of the cloned region of the human genome designated as ORA-gp5 was constructed. The sequences of three different genetical elements v-mos-related oncogene, mammalian type C retrovirus and Alu type repeat are interspersed in this structure. The hypothesis concerning the probable origin of this locus has been proposed. The mosaical structure of ORA-gp5 could be the result of the integration of mammalian retrovirus in the vicinity to c-mos gene with subsequent recombination and transposition. The resulting potentially oncogenic structure was later inactivated by the integration of Alu-type repeats.

Animals↗
PubMed1990

The structural analysis of the mitochondrial SSUrRNA implies a close phylogenetic relationship between mitochondria from plants and from the heterotrophic alga Prototheca wickerhamii.

The gene for the mitochondrial small subunit rRNA (SSUrRNA) from the heterotrophic alga Prototheca wickerhamii has been isolated from a gene library of extranuclear DNA. Sequence and structural analyses allow the determination of a secondary structure model for this rRNA. In addition, several sequence motifs are present which are typically found in SSUrRNAs of various mitochondrial origins. Unexpectedly, the Prototheca RNA sequence has more features in common with mitochondrial SSUrRNAs from plants than with that from the green alga Chlamydomonas reinhardtii. The phylogenetic relationship between mitochondria from plants and algae is discussed.

Base Sequence↗
PubMed2001

Analysis of the pmsCEAB gene cluster involved in biosynthesis of salicylic acid and the siderophore pseudomonine in the biocontrol strain Pseudomonas fluorescens WCS374.

Mutants of Pseudomonas fluorescens WCS374 defective in biosynthesis of the fluorescent siderophore pseudobactin still display siderophore activity, indicating the production of a second siderophore. A recombinant cosmid clone (pMB374-07) of a WCS374 gene library harboring loci necessary for the biosynthesis of salicylic acid (SA) and this second siderophore pseudomonine was isolated. The salicylate biosynthesis region of WCS374 was localized in a 5-kb EcoRI fragment of pMB374-07. The SA and pseudomonine biosynthesis region was identified by transfer of cosmid pMB374-07 to a pseudobactin-deficient strain of P. putida. Sequence analysis of the 5-kb subclone revealed the presence of four open reading frames (ORFs). Products of two ORFs (pmsC and pmsB) showed homologies with chorismate-utilizing enzymes; a third ORF (pmsE) encoded a protein with strong similarity with enzymes involved in the biosynthesis of siderophores in other bacterial species. The region also contained a putative histidine decarboxylase gene (pmsA). A putative promoter region and two predicted iron boxes were localized upstream of pmsC. We determined by reverse transcriptase-mediated PCR that the pmsCEAB genes are cotranscribed and that expression is iron regulated. In vivo expression of SA genes was achieved in P. putida and Escherichia coli cells. In E. coli, deletions affecting the first ORF (pmsC) diminished SA production, whereas deletion of pmsB abolished it completely. The pmsB gene induced low levels of SA production in E. coli when expressed under control of the lacZ promoter. Several lines of evidence indicate that SA and pseudomonine biosynthesis are related. Moreover, we isolated a Tn5 mutant (374-05) that is simultaneously impaired in SA and pseudomonine production.

Bacterial Proteins↗
PubMed1987

Molecular cloning and nucleotide sequence of the 3-isopropylmalate dehydrogenase gene of Candida utilis.

A 3-isopropylmalate dehydrogenase (3-IMDH, EC 1.1.1.85) gene was cloned from a gene library of Candida utilis. One of the plasmids, pYKL30, could complement Escherichia coli leuB and Saccharomyces cerevisiae leu2 auxotrophs; a 2.2 kb HindIII fragment subcloned in pBR322 could still complement the leuB mutation. Southern hybridization confirmed that this fragment was derived from C. utilis. An open reading frame of 1089 bp that corresponded to a polypeptide of 363 amino acids, one residue shorter than the 3-IMDH of S. cerevisiae, was found in the cloned fragment. The homology between the 3-IMDHs of C. utilis and S. cerevisiae was 76.2% in nucleotides and 85.4% in amino acids. In contrast, the homology between the 3-IMDHs of C. utilis and Thermus thermophilus was much smaller and was restricted to some regions of the gene.

3-Isopropylmalate Dehydrogenase↗
PubMed2004

[Genetic diversity of microsatellite loci in captive Amur tigers].

The tiger is one of the most threatened wildlife species since the abundance and distribution of tiger have decreased dramatically in the last century. The wild Amur tiger (Panthera tigris altaica) only distributed in northeast China, the far east area of Russia and the north Korea and its size of wild population is about 450 in the world and 20 in China. Several hundred captive populations of Amur tigers are the main source to protect gene library of tiger and the source of recovering the wild populations. The Breeding Center for Felidae at Hengdaohezi and Haoerbin Tiger Park in Heilongjiang Province is the biggest captive breeding base in China. How to make clear the genetic pedigree and establish reasonable breeding system is the urgent issues. So we use the microsatellite DNA markers and non-invasive technology to research on the genetic diversity of captive Amur tiger in this study. Ten microsatellite loci (Fca005, Fca075, Fca094, Fca152, Fca161, Fca294, Pti002, Pti003, Pti007 and Pti010), highly variable nuclear markers, were studied their genetic diversity in 113 captive Amur tigers. The PCR amplified products of microsatellite loci were detected by non-denatured polyacrylamide gel electrophoresis. Allele numbers, allelic frequency, gene heterozygosity(H(e)), polymorphism information content(PIC) and effective number of allele(N(e)) were calculated. 41 alleles were found and their size were ranged from 110bp to 250bp in ten microsatellite loci, Fca152 had 6 alleles, Fca075, Fca094 and Fca294 had 5 alleles, Fca005 and Pti002 had 4 alleles and the others had 3 alleles in all tiger samples, respectively. The allelic frequencies were from 0.009 to 0.767; The He ranged from 0.385 to 0.707, and Fca294 and Pti010 locus had the highest and lowest value; the PIC were from 0.353 to 0.658, Fca294 and Pti010 locus had the highest and lowest value; and N(e) were from 1.626 to 3.409, Fca294 and Pti010 locus had the highest and lowest value, which showed the ten microsatellie loci had high or medium polymorphism in these Amur tigers and had high genetic diversity. At the same time, we only found even bases variability which showed the even bases repeat sequence (CA/GT) maybe the basic unit for length variability of microsatellite in all loci. In this study, the samples were made up of 75 hair specimens, 23 blood specimens and 15 tissue specimens, we obtained the genome DNA from hairs using the non-invasive DNA technology and demonstrated that DNA derived from hair samples is as good as that obtained from blood samples for the analysis of microsatellite polymorphism. These results imply that microsatellite DNA markers and non-invasive DNA technology can help study the genetic diversity of Amur tiger. This method could be used in the captive management of other endangered species.

Alleles↗
PubMed1984

Cloning of genomic DNA for human atrial natriuretic factor.

A human genomic DNA clone for atrial natriuretic factor (ANF) gene was isolated from a human gene library using the previously cloned cDNA for rat ANF as a probe. Partial nucleotide sequencing of the cloned DNA revealed the location of a 120-bp long intron between Lys-41 and Asn-42 of the ANF precursor. Restriction mapping also suggested the existence of at least one other intron in or proximal to the 3' -untranslated region.

Amino Acid Sequence↗
PubMed1985

Molecular cloning and a stable amplification of the DNA molecules heavily methylated at CpG sequences using a new E. coli cell system (GC3).

Fish lymphocystis disease virus (FLDV) DNA is heavily methylated in CpG sequences and therefore the amplification of recombinant DNA of FLDV inserted into a bacterial plasmid vector and/or a bacteriophage system poses severe problems. The problem was solved using a newly constructed and developed bacterial system (E. coli GC-3 system), which allowed the amplification of foreign DNA material heavily methylated at CpG sequences. E. coli GC-3 (Gc-1, rifd) is derived from E. coli GC-1 (K 12, hsd-, MDU) after transformation with phage lambda drifd 18 Hind III-B-fragment. A defined and complete gene library of the FLDV DNA sequences was established by insertion of FLDV DNA fragments (Eco RI, Eco RI/Bam HI, Eco RI/Hind III) into the corresponding restriction sites of bacterial plasmid vector pAT 153. Bacterial colonies harbouring recombinant plasmids were individually identified by digestion of the recombinant plasmid DNA with different restriction enzymes and screened by hybridizing plasmid DNA to viral DNA. The analyses revealed that sequences representative for the complete viral genome were cloned.

Animals↗
PubMed1984

Characterization of Rhizobium japonicum hydrogen uptake genes.

Recombinant cosmids from a gene library of the DNA from Hup+ Rhizobium japonicum 122DES previously have been shown to restore hydrogenase activity when transferred by conjugation into certain Hup- mutants of R. japonicum. We generated a restriction map covering 32.2 kilobases of this cosmid DNA. At least 25.3 kilobases of the cosmid pHU1 were shown to have the same arrangement as those in the genome of strain 122DES. Analysis of Tn5 insertions into the 122DES genome indicates that hup-specific sequences occur in a region spanning about 15 kilobases of insert DNA within pHU1. Introduction of pHU1 into five out of six R. japonicum Hup- mutants resulted in a Hup+ phenotype in some transconjugants. Three of the mutations appear to be in transcriptional units completely contained within pHU1, whereas the other two must be in genes that are at least partially contained within pHU1. pBR235 derivatives containing fragments of hup DNA can be transferred into the R. japonicum Hup- mutant PJ18nal if the derivatives contain a region of homology with the R. japonicum genome. The hup mutation in strain PJ18nal appears to be dominant. The hup genes in R. japonicum strain 122DES appear to be organized in at least two, and probably three, transcriptional units.

Base Sequence↗
PubMed1989

Expression of Bradyrhizobium japonicum nodulation gene in Rhizobium fredii nod mutants.

The B. japonicum USDA 110 pLAFR1 gene library was transferred to Tn5-induced Rhizobium fredii USDA 191-4 Nod- mutants with helper plasmid pRK2013. SmR TcR transconjugants occurred with a frequency of 5 x 10(-4). The transconjugants were purified and used to inoculate germinated soybean seeds. Seven nodules were obtained in the nodulation experiment. The fast-growing Nod+ SmR TcR Rhizobium fredii strain was isolated from all nodules. Each isolate had acquired a new plasmid with a molecular mass of approximately 51 kb. This recombinant plasmid was transferred to E. coli HB101 by helper pRK2013 at a frequency of 1 x 10(-4). In addition to the vector fragment, all the recombinant plasmids gave 23 kb and 6.5 kb fragments on EcoRI digestion. A DNA-DNA hybridization test with a 32P-labelled nod gene probe (prepared from pRmSL26) confirmed that the 20 kb EcoRI-BamHI DNA fragment of the plasmids exhibited sequence homology with an R. meliloti nod gene probe.

Blotting, Southern↗
PubMed1985

Cloning of the newt Pleurodeles waltlii chromosomal DNA.

Pleurodeles waltlii genomic DNA has been cloned using several phage lambda vectors. We have isolated approx. 600 000 clones, which correspond to about 20% of the total DNA sequences of this organism. This constitutes the first large gene library of a Urodele. The low yield of cloning was attributable to the abundance of highly repetitive sequences, since recombinations in the bacterial host could lead to the loss of clones. Indeed, the existence of highly repetitive sequences was directly demonstrated by hybridization between recombinants and the total genome, and some of the cloned DNA was found to be unstable. We suggest new methods for cloning the highly repetitive sequences.

Animals↗
PubMed1988

Molecular cloning of the penicillin G acylase gene from Arthrobacter viscosus.

Penicillin G acylase was purified from the cultured filtrate of Arthrobacter viscosus 8895GU and was found to consist of two distinct subunits with apparent molecular weights of 24,000 (alpha) and 60,000 (beta). The partial N-terminal amino acid sequences of the alpha and beta subunits were determined with a protein gas phase sequencer, and a 29-base oligonucleotide corresponding to the partial amino acid sequence of the alpha subunit was synthesized. An Escherichia coli transformant having the penicillin G acylase gene was isolated from an A. viscosus gene library by hybridization with the 29-base probe. The resulting positive clone was further screened by the Serratia marcescens overlay technique. E. coli carrying a plasmid designated pHYM-1 was found to produce penicillin G acylase in the cells. This plasmid had an 8.0-kilobase pair DNA fragment inserted in the EcoRI site of pACYC184.

Amidohydrolases↗
PubMed2000

[Subcloning and sequencing of DNA fragment related to salt tolerance in Sinorhizobium fredii RT19].

A 23 kb DNA fragment related to salt tolerance was obtained from the gene library of S. fredii strain RT19. In this study, BamH I was selected to digest 23 kb DNA fragment into different length of DNA fragments. The resulting fragments were ligated with plasmid pML122, then the recombinant plasmids were transformed to competent cells of E. coli S17-1 on selective medium and three transformants TR were obtained. Two-parental mating experiments were carried out with these transformants as donor and salt sensitive S. fredii strain RC3-3 as recipient, and the transconjugant BR2 was selected on FY plates containing gentamycin and 0.4 mol/L NaCl. Thus, a 4.4 kb DNA fragment related to salt tolerance was obtained. Based on its physical map, six restriction fragments were subcloned into plasmid pUC18 for DNA sequencing. Subsequently, sequencing and analysis of 4.4 kb DNA fragment showed that fixO, fixN genes and three ORFs were obtained.

Amino Acid Sequence↗