Transfer of the Pea Symbiotic Plasmid pJB5JI in Nonsterile Soil

Transfer of the pea (Pisum sativum L.) symbiotic plasmid pJB5JI between strains of rhizobia was examined in sterile and nonsterile silt loam soil. Sinorhizobium fredii USDA 201 and HH003 were used as plasmid donors, and symbiotic plasmid-cured Rhizobium leguminosarum 6015 was used as the recipient. The plasmid was carried but not expressed in S. fredii strains, whereas transfer of the plasmid to R. leguminosarum 6015 rendered the recipient capable of nodulating pea plants. Confirmation of plasmid transfer was obtained by acquisition of plasmid-encoded antibiotic resistance genes, nodulation of pea plants, and plasmid profiles. Plasmid transfer in nonsterile soil occurred at frequencies of up to 10⁻⁴ per recipient and appeared to be highest at soil temperatures and soil moisture levels optimal for rhizobial growth. Conjugation frequencies were usually higher in sterile soil than in nonsterile soil. In nonsterile soil, transconjugants were recovered only with strain USDA 201 as the plasmid donor. Increasing the inoculum levels of donor and recipient strains up to 10⁹ cells g of soil⁻¹ increased the number of transconjugants; peak plasmid transfer frequencies, however, were found at the lower inoculum level of 10⁷ cells g of soil⁻¹. Plasmid transfer frequencies were raised in the presence of the pea rhizosphere or by additions of plant material. Transconjugants formed by the USDA 201(pJB5JI) × 6015 mating in soil formed effective nodules on peas.     

Genetic analysis of the gentamicin resistance region of pPH1JI and incorporation into a wide host range cloning vehicle

A region of the IncP plasmid pPH1JI encoding resistance to gentamicin, spectinomycin, and streptomycin was characterized by subcloning, deletion, and insertion mutagenesis. Approximate locations of these resistance determinants were established. A 1.6-kb HindIII-SphI segment of this region expresses gentamicin resistance (Gmr) in Escherichia coli when inserted into various plasmid vectors; this DNA segment encodes a polypeptide of 17.5 kDa. Incorporation of this fragment into an IncP cloning vehicle produced a Gmr wide host range vector, pRAR209, which confers levels of Gmr comparable to those expressed by pPH1JI in E. coli, Agrobacterium tumefaciens, and Rhizobium meliloti.     

Behavior of plasmid pJB5JI in Rhizobium huakuii under free-living and symbiotic conditions

The Rhizobium leguminosarum biovar viceae host-range plasmid pJB5JI was transferred into Rhizobium huakuii strains, both wild-type 7653R and its sym plasmid-cured mutant 7653R-1. Transconjugant 7653R-1 (pJB5JI) acquired the ability to form ineffective nodules on pea plants, whereas transconjugant 7653R (pJB5JI) could not do so, indicating that the indigenous symbiotic plasmid could restrict the functional expression of pJB5JI. On the other hand, transconjugant 7653R (pJB5JI) showed higher nitrogenase activity on A. sinicus and higher shoot dry weight than the recipient strain 7653R. The alien plasmid pJB5JI in both kinds of transconjugants remained stable during frequent transfer on culture media, but in part of the isolates from nodules formed by them the pJB5JI was not visualized on gel by the Eckhardt procedure. Southern hybridization with Tn5 and nod gene probes showed that these isolates still reserved, at least in part, DNA of pJB5JI, which was probably intergrated onto the chromosome of cells.     

A physical map of the permuted genome of bacteriophage T1

Summary A restriction map has been constructed for the DNA of coliphage T1 which locates the cleavage sites of the restriction endonucleases, BglI (6 cuts), BglII (16 cuts), EcoRI (2 cuts), HindIII (2 cuts) and PstI (2 cuts). Digestions with BglI and BglII reveal fragments which are present in sub-molar quantities. Two methods, one using the selective removal of molecular ends with exonuclease III and the other involving the comparison of digestion patterns of concatemeric and virion DNA, have shown that the submolar fragments are at or close to the ends of the molecules. Digestions with BglI show that one terminal fragment has a very precise molecular weight whereas all the others are of heterogenous molecular weight. These results are consistent with the model for DNA packaging in which maturation is initiated at a precise site on a concatemeric precursor and proceeds by the encapsidation of up to four successive headfuls of 1.065 genome equivalents (MacHattie and Gill 1977).     

Genetic and physical map of a P1 miniplasmid

The prophage form of bacteriophage P1 is a unit-copy plasmid which is maintained with great fidelity in its Escherichia coli host. The plasmid maintenance functions of P1 are clustered in one region of the genome. An 11.5-kilobase fragment from this region has been cloned into a lambda delta att vector and promotes stable unit-copy plasmid maintenance. The properties of the lambda vector facilitated the isolation of deletion mutants affecting the P1 DNA. Twenty-eight deletion mutants were isolated, and their lesions were mapped by physical techniques. The genetic properties of the mutants with respect to plasmid replication, stability of plasmid maintenance, and ability to exert incompatibility effects against P1 and P7 plasmids were determined. These properties, along with those of several subfragments of the P1 insert cloned into high-copy-number plasmid vectors, allow the construction of an unambiguous genetic and physical map of the maintenance functions. A region of less than 3 kilobases, the rep region, is essential for plasmid replication and contains the incA incompatibility determinant within an 800-base-pair segment. Immediately adjacent to rep is a second region of approximately 3 kilobases which is required for stable plasmid maintenance, but not replication. This region, par, contains a second incompatibility element incB which is approximately 1 kilobase in size. The par region appears to specify equipartition of plasmid copies to daughter cells during cell division.     

A physical and genetic map of the Stigmatella aurantiaca DW4/3.1 chromosome

A physical map of the myxobacterium Stigmatella aurantiaca DW4/3.1 chromosome was constructed by pulsed-field gel (PFG) long-range mapping. One-and two-dimensional pulsed-field gel analyses were used together with reciprocal double-restriction, cross-hybridization and hybridization fingerprint analysis. These PFG results were confirmed by Smith-Birnstiel analysis, by Southern hybridization using linking clones and clones of a λ genomic library for the determination of adjacent restriction fragments and by transposon insertion mapping using defined genomic sequences for hybridization. It was thus possible to construct a circular restriction map of the single 9.35 Mbp chromosome of S. aurantiaca based on the endonucleases Asel and Spel. Genetic loci as well as the replication origin were located on the physical map by Southern hybridization using heterologous (derived from Myxococcus xanthus, Escherichia coli and Streptomyces lividans) and homologous probes that are mainly involved in development and ceil motility.     

A physical map of the bovine genome

Background

Cattle are important agriculturally and relevant as a model organism. Previously described genetic and radiation hybrid (RH) maps of the bovine genome have been used to identify genomic regions and genes affecting specific traits. Application of these maps to identify influential genetic polymorphisms will be enhanced by integration with each other and with bacterial artificial chromosome (BAC) libraries. The BAC libraries and clone maps are essential for the hybrid clone-by-clone/whole-genome shotgun sequencing approach taken by the bovine genome sequencing project.

Results

A bovine BAC map was constructed with HindIII restriction digest fragments of 290,797 BAC clones from animals of three different breeds. Comparative mapping of 422,522 BAC end sequences assisted with BAC map ordering and assembly. Genotypes and pedigree from two genetic maps and marker scores from three whole-genome RH panels were consolidated on a 17,254-marker composite map. Sequence similarity allowed integrating the BAC and composite maps with the bovine draft assembly (Btau3.1), establishing a comprehensive resource describing the bovine genome. Agreement between the marker and BAC maps and the draft assembly is high, although discrepancies exist. The composite and BAC maps are more similar than either is to the draft assembly.

Conclusion

Further refinement of the maps and greater integration into the genome assembly process may contribute to a high quality assembly. The maps provide resources to associate phenotypic variation with underlying genomic variation, and are crucial resources for understanding the biology underpinning this important ruminant species so closely associated with humans.     

Genetic and physical map of the pLAFR1 vector.

This paper presents the complete sequencing and annotation of the pLAFR1 vector. pLAFR is a tetracycline-resistant "cosmid" cloning vector, which is derived from the 20 kb plasmid pRK290, a RK2-derivative. Due to its broad host range, the pLAFR1 vector has been widely used in the genetic analysis of a broad number of gram-negative bacterial species. The availability of the complete pLAFR1 sequence will most definitely help in the construction and analysis of clone librares based on pRK290 or pLAFR vectors.     

A physical map of the genome of ethanol fermentative bacterium Zymomonas mobilis ZM4 and localization of genes on the map

A physical map of the Zymomonas mobilis ZM4 genome has been constructed from the results of reciprocal Southern hybridization with PmeI, PacI, and NotI-digested genomic DNA fragments and linking cosmid clones. Restriction enzyme-digested Z. mobilis ZM4 genome was electrophoresed with phage lambda DNA concatemers as a size standard in a Bio-Rad CHEF-DRII pulsed-field gel electrophoresis (PFGE) system. The restriction enzyme PmeI generated 15 fragments (3–625 kb), and PacI produced 19 fragments (7–525 kb). Each size of restriction fragment was calculated by comparison to the size of phage lambda DNA concatemers, and the genome size of Z. mobilis ZM4 was estimated to be 2085.5 kb. The 19 known genes and three rrn operons were localized on the map.     

A genetic and physical map of bovine chromosome 3

This paper reports a map of nine polymorphic microsatellite markers previously assigned to bovine chromosome 3 (BTA3) by somatic cell genetics. The linkage group covers 101 cM on the chromosome with an average intermarker distance of 13-9 cM. One marker (INRA200) was isolated from a peak of flow sorted chromosomes 2 and 3. Another marker (INRA197) was derived from a cosmid. The localization of the cosmid by in situ hybridization enabled the orientation of the linkage group on BTA3. Markers were relatively evenly spaced and consequently can be used to complement other mapping data about this chromosome. This establishes a framework of polymorphic markers that can be used to search for quantitative trait loci (QTL).     

A genetic and physical map of bovine Chromosome 11

A genetic map of bovine Chromosome (Chr) 11 (BTA11, synteny group U16) has been constructed from 330 animals belonging to 21 families, which constitute the international bovine reference panel (IBRP). This map is based on 13 polymorphic microsatellite markers, two of which were chosen in previously published maps. Three markers have been isolated from cosmids. Two of the three cosmids have been physically localized by fluorescence in situ hybridization (FISH), to anchor the genetic map on the chromosome. In addition, a biallelic polymorphism in the -lactoglobulin gene (LGB) has been genetically positioned relative to the microsatellite markers. The most probable order of the markers is: cen-INRA044-BM716-INRA177-(TGLA 327, INRA198, INRA131)-INRA111-INRABERN169-(INRA115, INRA032)-INRA108-INRABERN162-INRA195-LGB. T The total linkage group spans 126 cM, which probably corresponds to most of the chromosome length. The average intermarker distance is about 10.5 cM, allowing the potential detection of a genetic linkage with any Economic Trait Loci (ETL) of this chromosome.     

A cytogenetically based physical map of chromosome 1B in common wheat.

We have constructed a cytogenetically based physical map of chromosome 1B in common wheat by utilizing a total of 18 homozygous deletion stocks. It was possible to divide chromosome 1B into 17 subregions. Nineteen genetic markers are physically mapped to nine subregions of chromosome 1B. Comparison of the cytological map of chromosome 1B with an RFLP-based genetic linkage map of Triticum tauschii revealed that the linear order of the genetic markers was maintained between chromosome 1B of hexaploid wheat and 1D of T. tauschii. Striking differences were observed between the physical and genetic maps in relation to the relative distances between the genetic markers. The genetic markers clustered in the middle of the genetic map were physically located in the distal regions of both arms of chromosome 1B. It is unclear whether the increased recombination in the distal regions of chromosome 1B is due to specific regions of increased recombination or a more broadly distributed increase in recombination in the distal regions of Triticeae chromosomes.     

A physical map of plasmid pDU1 from the cyanobacterium Nostoc PCC 7524

Nostoc 7524 contains three different plasmids of molecular weight, 4, 8, and 28 Mdal. The smallest plasmid, designated pDU1, because of its size and ease of isolation, may prove to be useful as a cloning vector. Plasmid pDU1 was incubated separately with 26 different restriction enzymes and only 8 of the enzymes tested cut pDU1. A composite restriction enzyme map consisting of a total of 17 restriction sites was constructed for BglI, HindIII, HpaI, and XbaI. The sites of restriction enzyme cleavage were determined by single, double, and partial digests of plasmid DNA or redigestion of isolated restriction fragments. All the restriction sites were aligned relative to the single BglI site. This is the first restriction enzyme map of a plasmid from a filamentous cyanobacterium.     

A first generation bovine BAC-based physical map

A first generation clone-based physical map for the bovine genome was constructed combining, fluorescent double digestion fingerprinting and sequence tagged site (STS) marker screening. The BAC clones were selected from an Inra BAC library (105 984 clones) and a part of the CHORI-240 BAC library (26 500 clones). The contigs were anchored using the screening information for a total of 1303 markers (451 microsatellites, 471 genes, 127 EST, and 254 BAC ends). The final map, which consists of 6615 contigs assembled from 100 923 clones, will be a valuable tool for genomic research in ruminants, including targeted marker production, positional cloning or targeted sequencing of regions of specific interest.     

The function of three indigenous plasmids in Mtesorhizobium huakuii 2020 and its symbiotic interaction with Sym pJB5JI of Rhizobium leguminosarum

A Mesorhizobium huakuii strain 2020, isolated from a rice-growing field in southern China, contains three indigenous plasmids named p2020a, p2020b and p2020c, respectively. The plasmids were deleted via Tn5-sacB insertion, and two cured derivatives were obtained. Interestingly, the mutant 2020D29 curing of p2020c could significantly enhance the capacity of symbiotic nitrogen fixation. But the mutant 2020D8 curing of p2020b lost the ability to nodulate Astragalus sinicus. Furthermore, the third plasmid p2020a could be hardly eliminated, suggesting that some house-keeping genes necessary for strain growth located on this plasmid. Then the Sym plasmid pJB5JI of R. leguminosarum bv. viciae was transferred into 2020 and its cured derivatives. The pot plant test showed that the ability of competition and symbiotic nitrogen fixation of transconjugant 2020-137 (pJB5JI) was increased evidently in con-trast to 2020. pJB5JI could not restore the ability of 2020D8 to nodulate Astragalus sinicus. 2020D8-8 (pJB5JI) could form ineffective nodules on peas, which implied that the symbiotic plasmid pJB5JI could express its function at the chromosomal background of Mesorhizobium huakuii 2020. The plas-mid stability was checked in transconjugants under free-living and during symbiosis. The results indi-cated that pJB5JI failed to be detected in some nodule isolates. That Km resistance gene could be am-plified from all transconjugants and nodule isolates suggested that pJB5JI was fully or partially inte-grated into the chromosome of recipients.     

A physical map of the Mycoplasma genitalium genome

We report the construction of a physical map of the genome of the human pathogen Mycoplasma genitalium through the use of pulse-field gel electrophoresis. The small size and relative simplicity of this genome permit the arrangement of restriction fragments without having to construct linking clones. The size of the genome has been calculated to be approximately 600 kb and several important genetic determinants have been assigned specific loci on the map.     

A physical map of the sorghum chloroplast genome

Summary The chloroplast genome of the IS1112C cytoplasm of sorghum was mapped by the construction of a Bam-HI library in pUC8, and hybridization with BamHI, SalI, and PstI digests of chloroplast DNA (ctDNA) of sorghum and maize. The molecules are extensively colinear, with only one of 13 SalI fragments differing slightly from maize. Seven of 70 restriction sites differed in the two species. A total molecular size of ca. 138 kb was estimated for sorghum. The inverted repeat was not conserved between sorghum and maize, as revealed by a slightly larger BamHI 16S rDNA fragment in sorghum. Homology of a sequence adjacent to the bcl gene and one end of the inverted repeat was detected. These homologies were also observed in maize, and suggest that the ctDNA genomes of sorghum and maize share small reiterations of sequences of the inverted repeat.USDA-ARS     

A BAC-based physical map of the apple genome

Genome-wide physical mapping is an essential step toward investigating the genetic basis of complex traits as well as pursuing genomics research of virtually all plant and animal species. We have constructed a physical map of the apple genome from a total of 74,281 BAC clones representing approximately 10.5x haploid genome equivalents. The physical map consists of 2702 contigs, and it is estimated to span approximately 927 Mb in physical length. The reliability of contig assembly was evaluated by several methods, including assembling contigs using variable stringencies, assembling contigs using fingerprints from individual libraries, checking consensus maps of contigs, and using DNA markers. Altogether, the results demonstrated that the contigs were properly assembled. The apple genome-wide BAC-based physical map represents the first draft genome sequence not only for any member of the large Rosaceae family, but also for all tree species. This map will play a critical role in advanced genomics research for apple and other tree species, including marker development in targeted chromosome regions, fine-mapping and isolation of genes/QTL, conducting comparative genomics analyses of plant chromosomes, and large-scale genomics sequencing.     

Establishment of a physical and genetic map for bacteriophage PRD1

DNA was isolated from the lipid-containing bacteriophage PRD1 and subjected to restriction endonuclease analysis. The total genome size is 14.7 kb. PRD1 DNA was resistant to cutting by fifteen restriction endonucleases with six base specificity. HaeII made thirty-seven cuts in the DNA, MboI made one cut, and MnlI made six cuts. DNA that was not treated with protease yielded two fewer fragments when treated with HaeII. Evidence is presented to indicate that the PRD1 DNA has protein at the ends of the DNA. The thirty-eight HaeII fragments were ordered using the ladder technique of Smith and Birnstiel (1976) on MboI and MnlI fragments of the genome. Clones of HaeII partial digests of PRD1 DNA in pBR322 were analyzed by HaeII digestion and were then assigned to specific regions of the genome by their HaeII fragment composition. A comparison of the marker rescue characteristics of the cloned DNA with the overall restriction fragment map generated a physical map of the genome. Some genes that have not been mapped because of a lack of mutants or leakiness at restrictive conditions were mapped by studying the in vitro protein synthesis of restriction endonuclease fragments.