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PubMedDate not supplied

Repetitive sequences with homology to Bradyrhizobium japonicum DNA and the T-DNA of Agrobacterium rhizogenes are closely linked to nodABC of Rhizobium fredii USDA257.

We have detected strong homology between a 9.2-kb EcoRI restriction fragment from Rhizobium fredii USDA257 that contains nodABC and eight additional EcoRI fragments in DNA digests from this organism. A series of repetitive sequences responsible for this hybridization lies within a 0.95-kb HindIII/SalI subfragment about 1-kb upstream of nodA. This subfragment also hybridizes to multiple restriction fragments from nine other strains of R. fredii, but only one is common to all strains. The 0.95-kb subfragment does not hybridize to genomic DNA from 17 other strains of fast-growing rhizobia, but there is weak homology to two fragments from Rhizobium sp. strain NGR234. We sequenced 2,432 base pairs (bp) of the region encompassing the repetitive sequences. It contains 65 separate 8- to 11-bp inverted and direct repeats, as well as two large open reading frames (ORFs) that overlap on opposite strands. ORF1 reads in the same direction as nodABC, contains 1,071 bp, and encodes a 40.6-kD protein. It has 74% sequence homology to an ORF within the T-DNA of Agrobacterium rhizogenes and similar homology to a series of repetitive sequences from Bradyrhizobium japonicum. ORF2 (981 bp) reads in the opposite direction, encodes a 34.7-kD protein, and has partial identity with a second ORF from A. rhizogenes. We could detect no poly(A)+ nodule transcripts with homology to ORF1 and ORF2. The eight sets of repetitive sequences found in other EcoRI fragments of the genome were cloned from USDA257 on separate cosmids. Some of these cosmids appear to overlap, and two have fragments with homology to nifKDH.

Amino Acid Sequence↗

PubMedDate not supplied

Genetic analysis of the aggA locus involved in agglutination and adherence of Pseudomonas putida, a beneficial fluorescent pseudomonad.

An isolate of Pseudomonas putida, which rapidly adheres to plant roots, is agglutinated by a glycoprotein from root surfaces. Agglutination is prevented and adherence to the root surface is diminished by Tn5 insertion in mutant 5123. Two cosmid clones from wild type P. putida and a 2.7-kbp EcoRI-HindIII subclone present in both cosmid clones restored agglutinable to wild type levels in transconjugants of the nonagglutinable (Agg-) Tn5 mutant 5123. These three clones increased agglutinability in transconjugants of the parental Agg+ isolate. The 2.7-kbp EcoRI-HindIII subclone restored adherence to bean root surfaces of 5123 to wild type levels in a short-term binding assay. Deletion analysis of the 2.7-kbp fragment indicated only 1.45 kbp was necessary for complementation of agglutinability in 5123. This sequence, termed the aggA locus, contains an open reading frame of 1,356 nucleotides encoding a predicted 50,509-Da protein. The distribution of the aggA locus in plant-associated bacteria, as detected through Southern hybridization, is limited to bacteria that express the agglutination phenotype.

Agglutination Tests↗

PubMedDate not supplied

Cloning and heterologous expression of the phenazine biosynthetic locus from Pseudomonas aureofaciens 30-84.

Pseudomonas aureofaciens strain 30-84 suppresses take-all disease of wheat caused by Gaeumannomyces graminis var. tritici. Three antibiotics, phenazine-1-carboxylic acid, 2-hydroxyphenazine-1-carboxylic acid, and 2-hydroxyphenazine, were responsible for disease suppression. Tn5-induced mutants deficient in production of one or more of the antibiotics (Phz-) were significantly less suppressive than the parental strain. Cosmids pLSP259 and pLSP282 from a genomic library of strain 30-84 restored phenazine production and fungal inhibition to 10 different Phz- mutants. Sequences required for production of the phenazines were localized to a segment of approximately 2.8 kilobases that was present in both cosmids. Expression of this locus in Escherichia coli required the introduction of a functional promoter, was orientation-specific, and resulted in the production of all three phenazine antibiotics. These results strongly suggest that the cloned sequences encode a major portion of the phenazine biosynthetic pathway.

Antifungal Agents↗

PubMedDate not supplied

Identification and complementation of a mutation to constitutive filamentous growth in Ustilago maydis.

Pathogenicity of the corn smut fungus Ustilago maydis involves the formation of a filamentous, infectious dikaryon by fusion of compatible, yeastlike haploid cells. The mating-type loci, a and b, regulate cell fusion and establishment of the dikaryotic cell type, respectively. On solid medium, compatibility at the mating-type loci, in particular heterozygosity at the b locus, is manifested by the formation of aerial hyphae on colonies formed by mating cells. We have employed this "fuzzy" phenotype to identify haploid mutants that constitutively form hyphal filaments and forego cell division by budding. A total of 125 such mutants have been isolated; characterization of one mutant (termed rem1-1) revealed that it can participate in infection of the host plant, although it must be paired with a compatible, wild-type mating partner. That is, mutation to the mycelial phenotype is not sufficient to allow a haploid strain to be pathogenic by itself. A cosmid has been isolated that restores the ability of an rem1-1 mutant to grow with a budding phenotype. Localization of the complementing region on cosmid DNA allowed the construction of an additional mutation by gene disruption. Coinoculation of plants with two compatible strains, each carrying the disruption mutation, gave greatly reduced disease symptoms. The analysis of the rem1 gene should contribute to an understanding of dimorphic growth and pathogenesis in U. maydis.

Base Sequence↗

PubMed1996

Isolation and characterization of a pSym locus of Rhizobium sp. BR816 that extends nodulation ability of narrow host range Phaseolus vulgaris symbionts to Leucaena leucocephala.

Introduction of a cosmid library of megaplasmid DNA of Rhizobium sp. BR816, a broad host range Rhizobium strain, into R. etli CE3, a narrow host range bean symbiont, resulted in the isolation of a transconjugant that could effectively nodulate Leucaena leucocephala. Analysis of the corresponding cosmid, pBRF2, revealed the presence of genes required for elicting nitrogen-fixing nodules on L. leucoephala. Subcloning and Tn5 tagging identified a locus responsible for the host range extension. Sequence analysis of this locus revealed an ORF that shows significant identity with NodO of R. leguminosarum bv. viciae.

Amino Acid Sequence↗

PubMed1996

Cloning and characterization of a melanin biosynthetic THR1 reductase gene essential for appressorial penetration of Colletotrichum lagenarium.

Melanin biosynthesis of Colletotrichum lagenarium is essential for appressorial penetration of the host plant. A melanin deficient mutant 9141 (Thr-) has a defect in the conversion of 1,3,8-trihydroxynaphthalene to vermelone in the melanin biosynthetic pathway. The mutant formed nonmelanized appressoria and had little infectivity on cucumber leaves. A cosmid clone pCR1 was selected from a cosmid library of wild-type C. lagenarium by means of a heterologous probe BRM2, one of the clustered genes involved in melanin biosynthesis of Alternaria alternata. pCR1 transformed the Thr- mutant 9141 to wild-type phenotype. A DNA fragment (THR1) homologous to BRM2 was subcloned from pCR1 and the nucleotide sequence determined. THR1 contains one open reading frame that encodes a protein of 282 amino acids. A transformant resulting from gene disruption showed a light brown phenotype different from the dark brown phenotype of the wild-type 104-T. The transformant formed nonmelanized appressoria and had little infectivity. The THR1 amino acid sequence contains a region highly similar to the Ver1 gene involved in the conversion of versicolorin A to sterigmatocystin in aflatoxin biosynthesis by Aspergillus parasiticus and to the T4HN reductase gene involved in the conversion of 1,3,6,8-tetrahydroxynaphthalene to scytalone and 1,3,8-trihydroxynaphthalene to vermelone in melanin biosynthesis by Magnaporthe grisea. Expression of the THR1 gene during spore germination of C. lagenarium was detected by RNA blotting. We propose that the C. lagenarium THR1 gene encodes a reductase involved in conversion of 1,3,8-trihydroxynaphthalene to vermelone.

Amino Acid Sequence↗

PubMed2000

A second T-region of the soybean-supervirulent chrysopine-type Ti plasmid pTiChry5, and construction of a fully disarmed vir helper plasmid.

Agrobacterium tumefaciens Chry5, which is particularly virulent on soybeans, induces tumors that produce a family of Amadori-type opines that includes deoxyfructosyl glutamine (Dfg) and its lactone, chrysopine (Chy). Cosmid clones mapping to the right of the known oncogenic T-region of pTiChry5 conferred Amadori opine production on tumors induced by the nopaline strain C58. Sequence analysis of DNA held in common among these cosmids identified two 25-bp, direct repeats flanking an 8.5-kb segment of pTiChry5. These probable border sequences are closely related to those of other known T-regions and define a second T-region of pTiChry5, called T-right (TR), that confers production of the Amadoriopines. The oncogenic T-left region (TL) was located precisely by identifying and sequencing the likely border repeats defining this segment. The two T-regions are separated by approximately 15 kb of plasmid DNA. Based on these results, we predicted that pKYRT1, a vir helper plasmid derived from pTiChry5, still contains all of TR and the leftmost 9 kb of TL. Consistent with this hypothesis, transgenic Arabidopsis thaliana plants selected for with a marker encoded by a binary plasmid following transformation with KYRT1 co-inherited production of the Amadori opines at high frequency. All opine-positive transgenic plants also contained TR-DNA, while those plants that lacked TR-DNA failed to produce the opines. Moreover, A. thaliana infected with KYRT1 in which an nptII gene driven by the 35S promoter of Cauliflower mosaic virus was inserted directly into the vir helper plasmid yielded kanamycin-resistant transformants at a low but detectable frequency. These results demonstrate that pKYRT1 is not disarmed, and can transfer Ti plasmid DNA to plants. A new vir helper plasmid was constructed from pTiChry5 by two rounds of sacB-mediated selection for deletion events. This plasmid, called pKPSF2, lacks both of the known T-regions and their borders. pKPSF2 failed to transfer Ti plasmid DNA to plants, but mobilized the T-region of a binary plasmid at an efficiency indistinguishable from those of pKYRT1 and the nopaline-type vir helper plasmid pMP90.

Agrobacterium tumefaciens↗

PubMed2000

Restriction enzyme-mediated integration used to produce pathogenicity mutants of Colletotrichum graminicola.

We have developed a restriction enzyme-mediated insertional mutagenesis (REMI) system for the maize pathogen Colletotrichum graminicola. In this report, we demonstrate the utility of a REMI-based mutagenesis approach to identify novel pathogenicity genes. Use of REMI increased transformation efficiency by as much as 27-fold over transformations with linearized plasmid alone. Ninety-nine transformants were examined by Southern analysis, and 51% contained simple integrations consisting of one copy of the vector integrated at a single site in the genome. All appeared to have a plasmid integration at a unique site. Sequencing across the integration sites of six transformants demonstrated that in all cases the plasmid integration occurred at the corresponding restriction enzyme-recognition site. We used an in vitro bioassay to identify two pathogenicity mutants among 660 transformants. Genomic DNA flanking the plasmid integration sites was used to identify corresponding cosmids in a wild-type genomic library. The pathogenicity of one of the mutants was restored when it was transformed with the cosmids.

Blotting, Southern↗

PubMed2000

Mapping of avirulence genes in the rice blast fungus, Magnaporthe grisea, with RFLP and RAPD markers.

Three genetically independent avirulence genes, AVR1-Irat7, AVRI-MedNoi; and AVR1-Ku86, were identified in a cross involving isolates Guy11 and 2/0/3 of the rice blast fungus, Magnaporthe grisea. Using 76 random progeny, we constructed a partial genetic map with restriction fragment length polymorphism (RFLP) markers revealed by probes such as the repeated sequences MGL/MGR583 and Pot3/MGR586, cosmids from the M. grisea genetic map, and a telomere sequence oligonucleotide. Avirulence genes AVR1-MedNoi and AVR1-Ku86 were closely linked to telomere RFLPs such as marker TelG (6 cM from AVR1-MedNoi) and TelF (4.5 cM from AVR1-Ku86). Avirulence gene AVR1-Irat7 was linked to a cosmid RFLP located on chromosome 1 and mapped at 20 cM from the avirulence gene AVR1-CO39. Using bulked segregant analysis, we identified 11 random amplified polymorphic DNA (RAPD) markers closely linked (0 to 10 cM) to the avirulence genes segregating in this cross. Most of these RAPD markers corresponded to junction fragments between known or new transposons and a single-copy sequence. Such junctions or the whole sequences of single-copy RAPD markers were frequently absent in one parental isolate. Single-copy sequences from RAPD markers tightly linked to avirulence genes will be used for positional cloning.

Amino Acid Sequence↗

PubMed2000

Structural and functional complexity of the genomic region controlling AK-toxin biosynthesis and pathogenicity in the Japanese pear pathotype of Alternaria alternata.

The Japanese pear pathotype of Alternaria alternata produces host-specific AK-toxin and causes black spot of Japanese pear. Previously, a cosmid clone, pcAKT-1, was isolated that contains two genes, AKT1 and AKT2, within a 5.0-kb region required for AK-toxin biosynthesis. The wild-type strain has multiple, nonfunctional copies of these genes. In the present study, two additional genes, AKTR-1 and AKT3-1, downstream of AKT2 were identified. Transformation of the wild type with AKTR-1- and AKT3-1-targeting vectors produced toxin-deficient (Tox-), nonpathogenic mutants. DNA gel blot analysis, however, demonstrated that the fragments targeted in Tox- mutants were different from those containing AKTR-1 and AKT3-1 on the transforming vectors. A cosmid clone, pcAKT-2, containing the targeted DNA was isolated and shown to carry two genes, AKTR-2 and AKT3-2, with high similarity to AKTR-1 and AKT3-1, respectively. Transcripts from not only AKTR-2 and AKT3-2 but also AKTR-1 and AKT3-1 were found in the wild type. DNA gel blot analysis with pulsed-field gel electrophoresis showed that AKT1, AKT2, AKT3, and AKTR and their homologues are on a single chromosome. These results indicate the structural and functional complexity of the genomic region controlling AK-toxin biosynthesis.

Alternaria↗

PubMed2002

Transformation and transposon mutagenesis of Leifsonia xyli subsp. xyli, causal organism of ratoon stunting disease of sugarcane.

Conditions have been developed for genetic transformation and insertional mutagenesis in Leifsonia xyli subsp. xyli (Lxx), the causal organism of ratoon stunting disease (RSD), one of the most damaging and intractable diseases of sugarcane internationally. Transformation frequencies ranged from 1 to 10 colony forming units (CFU)/microg of plasmid DNA using Clavibacter/Escherichia coli shuttle vectors pCG188, pDM302, and pDM306 and ranged from 50 to 500 CFU/microg using cosmid cloning vectors pLAFR3 and pLAFR5-km. The transformation/transposition frequency was 0 to 70 CFU/microg of DNA, using suicide vectors pUCD623 and pSUP2021 containing transposable elements Tn4431 and Tn5, respectively. It was necessary to grow Lxx in media containing 0.1% glycine for electroporation and to amplify large plasmids in a dam-/dcm- E. coli strain and purify the DNA by anion exchange. To keep selection pressure at an optimum, the transformants were grown on nitrocellulose filters (0.2-microm pore size) on media containing the appropriate antibiotics. Transposon Tn4431 containing a promoterless lux operon from Vibrio fischeri and a tetracycline-resistance gene was introduced on the suicide vector pUCD623. All but 1% of the putative transposon mutants produce light, indicating transposition into functional Lxx genes. Southern blot analysis of these transformants indicates predominantly single transposon insertions at unique sites. The cosmid cloning vector pLAFR5-km was stably maintained in Lxx. The development of a transformation and transposon mutagenesis system opens the way for molecular analysis of pathogenicity determinants in Lxx.

Base Sequence↗

PubMed1995

Detection of a human DNA sequence correlated with melanocyte-like differentiation and tumour suppression after transfection into murine melanoma cells.

Since there is much indirect evidence for dominant suppressor genes for melanoma, we sought to isolate such a gene. Metastatic B16-F10 murine melanoma cells were lipofected with a normal human genomic library in a cosmid vector that also confers resistance to the drug G418. A few of the G418-resistant colonies acquired combinations of properties resembling those of normal melanocytes, including differentiated features (pigmentation, dendricity), slower growth, flat shape, monolayered colony form, stimulation of proliferation by a phorbol ester, and anchorage dependence. Four out of eight also showed reduced tumorigenicity in mice. Southern blotting indicated the presence of numerous cosmids in the melanocyte-like transfectants. DNA from one such line was used for secondary transfection. One secondary G418-resistant line showed pronounced melanocytic properties and marked tumour suppression in syngeneic and nude mice. A human repetitive sequence detected in this line was used in the polymerase chain reaction (PCR) to isolate intervening unique DNA sequences. One unique human sequence was attenuated in all tumors arising from both primary and secondary transfectants, suggesting close linkage with the sequence responsible for tumour suppression.

Animals↗

PubMed1984

The factor B and C2 genes.

Factor B and C2, components of the complement system, are novel types of serine proteinase that are encoded by genes in the major histocompatibility complex. cDNA probes specific for these two proteins have been used to isolate cosmid clones of genomic DNA which contain the Factor B and C2 genes. Southern blot analysis of the cosmid clones and of uncloned genomic DNA has shown that there are single Factor B and C2 loci that are less than 1 kilobase apart. The Factor B gene has been further characterized by DNA sequence analysis. It is approximately 6 kilobases in length, and is split into 18 exons. The amino acid sequence of the Ba fragment contains three homologous regions which are encoded by separate exons, suggesting that they arose by DNA duplication events. In the serine proteinase domain each of the functionally important parts of the active site are encoded on separate exons. Comparison of this region of the gene with the exon organization of other serine proteinases shows a close correlation between them, but also reveals the presence of an exon in Factor B with no homologous counterpart in the other serine proteinases. The possible functional significance of the peptide encoded by this exon and the evolution of this novel serine proteinase are discussed.

Amino Acid Sequence↗

PubMed1985

Demonstration of the colinearity of human cytomegalovirus genomes and construction of restriction maps of unknown isolates using cloned subgenomic fragments.

In this study, we have established the colinearity of human cytomegalovirus (HCMV) genomes using stringent conditions of DNA-DNA filter hybridization of HCMV HindIII fragments and cosmid-cloned AD169 strain HCMV DNA fragments. Large cosmid-cloned fragments of AD169 DNA were used for the preparation of radioactive probes by nick translation. These probes were hybridized to HindIII digests of DNA from three fresh isolates of HCMV and to that of the Davis strain. Using published HindIII restriction maps for the AD169 strain as a reference, the results obtained by hybridization allowed us to construct HindIII restriction maps for the genomes of the three fresh isolates. Confirmation of our methodology was found in the correspondence between the HindIII map we constructed for the Davis strain and that published previously. Furthermore, it is shown that variation in the restriction profiles of the unique regions of the genome are due to the absence or gain of restriction sites, and not to a rearrangement of fragments. This technique allows rapid construction of physical maps of the DNA of any fresh isolate for a given restriction enzyme provided the corresponding restriction map of a strain to be used as reference is available.

Base Sequence↗

PubMed1988

Restriction mapping of Lymantria dispar nuclear polyhedrosis virus DNA: localization of the polyhedrin gene and identification of four homologous regions.

The genome of the multiple-embedded nuclear polyhedrosis virus (MNPV) of Lymantria dispar (LdMNPV) was partially characterized by restriction endonuclease analysis and a physical map was constructed using cosmid cloning and Southern cross blot hybridization. Using BamHI, BglII, EcoRI and HindIII, the size of the genome was estimated to be 88.5 x 10(6) Mr or 134.04 kbp. LdMNPV DNA was also analysed using methylation-sensitive restriction enzymes. The resulting restriction profiles suggested that extensive methylation did not occur at the nucleotide sequence recognized by HpaII and MspI. A BamHI restriction map was constructed by comparing overlapping BamHI fragments between cosmid clones containing partial digests of viral DNA. The positions of the BglII, EcoRI and HindIII sites were determined by Southern cross blot hybridizations and aligned to the BamHI restriction map. At least four homologous regions were identified by cross blot hybridizations of BglII-digested LdMNPV DNA and such regions were found to be interspersed along the genome in a fashion similar to that reported for other baculoviruses. Using recombinant plasmids containing the HindIII-V fragment of Autographa californica MNPV to probe Southern blots of LdMNPV DNA, the restriction fragment(s) that contain the polyhedrin gene were identified. Based on these findings the map was oriented with the polyhedrin gene of LdMNPV as the zero point.

Chromosome Mapping↗

PubMed1986

Molecular cloning of a gene coding for a Vibrio cholerae haemagglutinin.

Recombinant plasmids encoding a Vibrio cholerae haemagglutinin were isolated from the highly virulent V. cholerae strain C5 by cosmid cloning. Both Escherichia coli HB101 containing the recombinant plasmids and V. cholerae C5 were able to agglutinate a variety of erythrocytes from human and animal origin; this haemagglutination was not inhibited by D-mannose or L-fucose. Subcloning of the recombinant cosmid DNA revealed that a 1.3 kb DNA fragment was sufficient for haemagglutinin production in E. coli HB101. Under direction of this 1.3 kb Vibrio DNA fragment, two proteins were made in E. coli minicells, of 27 and 10 kDa. Haemagglutinin-encoding sequences were not detected in every V. cholerae strain.

Agglutination↗

PubMed1986

Overlapping deletion in two spontaneous phase variants of Coxiella burnetii.

Chromosomal DNA from the Nine Mile phase I strain of Coxiella burnetii (CB9MIC7) was cloned into the cosmid vector pHC79. The resulting gene library was probed with a radiolabelled HaeIII fragment present in the parental strain but absent from a spontaneously derived Nine Mile phase II strain (CB9MIIC4). The insert, which includes the missing HaeIII fragment, was 38.5 kb in length. When DNA from this cosmid clone was hybridized to genomic DNA of the parental CB9MIC7 and its derivative CB9MIIC4, a number of fragments were missing or altered in the latter strain. Restriction mapping localized the fragments to a contiguous portion of the chromosomal DNA fragment. The data were consistent with an 18 kb deletion in the chromosome of CB9MIIC4. Another intrastrain spontaneous derivative, CB9MI514, also lacked the sentinel HaeIII fragment and carried a deletion of approximately 29 kb within the same cloned insert. Both deletions appeared to share a common terminus, within the limits of resolution. In all other strains investigated, both phase I and phase II, the DNA represented by the insert seemed intact. The strains examined were representative of various stages of phase variation. The relationship between the observed deletions and the mechanism of phase transition in Nine Mile strains is discussed.

Chromosome Deletion↗

PubMed1989

Transposon mutagenesis and complementation of the fructokinase gene in Rhizobium leguminosarum biovar trifolii.

Transposon Tn5 was used to generate a fructokinase mutation in Rhizobium leguminosarum biovar trifolii BAL. The section of the genome containing Tn5 was cloned into the EcoRI site of the vector pHC79 and isolated by direct selection on medium containing kanamycin and tetracycline. Total EcoRI digestion was used to obtain a single fragment containing Tn5 and flanking DNA sequences. The flanking DNA was used as a probe to isolate an intact fructokinase gene from a pLAFR1 cosmid clone bank of the parental strain. A cosmid showing homology to the probe was tri-parentally conjugated into the fructokinase-negative strain, complementing the mutation. The complemented mutant exhibited the wild-type phenotype, with an increase in fructokinase production presumably due to multiple copies of the gene.

Cloning, Molecular↗