Mobilization of the Proteus mirabilis chromosome by R plasmid R772.

The P-1 incompatibility group plasmid R772 can mobilize the chromosome of Proteus mirabilis strain PM5006. The decreasing gradient of recombinant recovery frequencies found for markers which were increasingly distal to 0 min with plasmid D donors was not found with R772. Instead, it produced recombinants for all markers at frequencies of about 5 X 10(-5) per donor. This is about 10-fold lower than the plasmid transfer frequency. Recombinants were stable and recombination was only detected over short segments of the chromosome which corresponded to about 10 min on the D plasmid map of the chromosome. All recombinants had inherited R772 and expressed all properties of the plasmid. Attempts to isolate variant plasmids with increased frequencies of recombinant formation were unsuccessful.     

Patterns of mobilization of the Proteus mirabilis chromosome by R plasmids.

R plasmids R40a, Rip69, R447b, R769 belonging to incompatibility groups A-C, M, N, V, respectively, were investigated for chromosomal mobilizing ability in Proteus mirabilis. Plasmids R40a, Rip69 and R447b mediated polarized transfer of markers in a clockwise direction from origins near tyr-1, metF and ser-2, respectively, on the linkage map. The recovery frequency per donor cell of proximal markers approached 1 x 10(-4) for these three plasmids and the efficiency of chromosomal transfer was higher than that of the previously studied plasmid D. The plasmid-guided chromosomal trajectories overlap and it was possible to complement results obtained with plasmid D to assemble a time-of-entry chromosomal map and directly establish the circularity of the linkage group. The map comprises a length of 93 min in terms of transfer time. Plasmid R769 had a different pattern of chromosome transfer. This plasmid produced recombinants for all markers at frequencies of about 4 x 10(-6) per donor. It effected multiple and more or less simultaneous entry of markers and produced recombination over lengths of chromosome rarely corresponding to more than 10 min on the linkage map.     

Extension of a chromosome linkage group of Proteus mirabilis.

Mating procedures for detection of mobilization of the Proteus mirabilis chromosome were re-investigated. The chromosome was mobilized by plasmid D, the previously used hybrid between plasmids P-lac and R1drd19. About a 40-fold increase in recombinant recovery correlated with the absence of swarming during mating and a lower temperature of incubation. The modification introduced was that conjugation was allowed to proceed on a non-selective supplemented minimal medium at 30 degrees C before washing and plating on selective media. Final incubation was also at 30 degrees C. This technique enabled eight additional chromosomal markers to be mapped. Polarized transfer of the chromosome was shown by gradient of transmission experiments using a previously described marker as reference, by linkage analysis with reference to proximal and distal markers and (less successfully) by interrupted mating on solid medium. Markers of plasmid D transferred at high frequency to all recombinants. The plasmid was stable in recombinants and could transfer itself and chromosomal markers of the new hosts in further matings. Resulting recombination of markers occurred at usual frequencies. The marker order, his-1, ser-2, ura-2, pyrB1, trp-3, cysA1, ade-2, ilv-2, cysG1, gly-1, cysC1, argA2, metF2, nalA1, thr-1, leuB2, did not resemble the order of these markers in Escherichia coli.     

Phage t: a group T plasmid-dependent bacteriophage

Phage t was isolated from sewage from Pretoria. It formed plaques only on Escherichia coli and Salmonella typhimurium strains that carried plasmids belonging to incompatibility group T. Five of six group T plasmids permitted visible lysis of R+ host strains. There was no visible lysis of E. coli J53-2 or S. typhimurium LT2trpA8 carrying the T plasmid Rts1 although the strains supported phage growth as indicated by at least a 10-fold increase in phage titre. The latter strains transferred the plasmid at high frequency to E. coli strain CSH2 and the resulting transconjugants plated the phage. Proteus mirabilis strain PM5006(R402) failed to support phage growth although it transferred the plasmid and concomitant phage sensitivity to E. coli J53-2. The phage was hexagonal in outline, RNA-containing, resistant to chloroform and adsorbed to the shafts of pili determined by T plasmids.     

Properties of R plasmid R772 and the corresponding pilus-specific phage PR772.

R plasmid R772 was isolated from a strain of Proteus mirabilis and is a self-transmissible P-1 incompatibility group plasmid having a molecular weight of about 27 x 10(6). It renders bacterial hosts resistant to kanamycin. Phage PR772 was isolated as a phage dependent on the presence of R772 in bacterial hosts. It is hexagonal-shaped with a diameter of 53 nm, has a thick inner membrane and no tail. Vaguely defined appendages are sometimes apparent at some vertices and the phage possesses double-stranded DNA. The DNA has a guanine plus cytosine molar content of 48%. The phage is sensitive to chloroform and has a buoyant density of 1.26 g cm(-3). These observations suggested that the inner membrane of the phage could contain lipid. Phage PR772 differs in morphology from the double-stranded DNA plasmid-specific phages PR4 and PRR1 which adsorb to tips and sides, respectively, of sex pili coded for by P-1 incompatibility group plasmids. Phage PR772 formed clear plaques which varied in diameter. Serologically, phages PR772 and PR4 are possibly related though very distantly, but the two phages have identical host ranges. Phage PR772 adsorbed by one of its apices to tips of sex pili coded for by plasmid R772 in Escherichia coli. It also formed plaques on Salmonella typhimurium Proteus morganii and Providence strains harbouring this plasmid as well as strains of E. coli carrying plasmids of incompatibility groups N or W. The phage produced areas of partial clearing on lawns of P. mirabilis PM5006 harbouring plasmid R772, the P-1 incompatibility group plasmid RP4, the W group plasmid RSa or the N group plasmid N3, and on lawns of Providence strain P29 carrying plasmid RP4.     

Identification and mapping of the replication genes of an R factor, R100-1, integrated into the chromosome of Escherichia coli K-12

A stable Hfr strain of Escherichia coli K-12 was obtained by integrative suppression by an R factor, R100-1. The R factor was integrated into the right of 81 min, and chromosome transfer occurred counterclockwise. Mating experiments revealed two linkage groups of genes on the R factor. Drug-resistant transductants of a dnaA-ts recipient from an R-factor Hfr and from an R(+) strain differ in their drug resistance patterns, temperature sensitivity, and transferability of drug resistance as well as chromosome markers. Transductants that transferred chromosome markers were further classified as to the origin and direction of chromosome transfer. For temperature-sensitive transductants, the reversion frequency to temperature resistance was determined, and these revertants were scored for transfer of drug resistance as well as chromosome markers. Two genes responsible for integrative suppression (designated as repA) and the other for autonomous replication (designated as repB) were identified and mapped. The arrangement of genes on the R factor is... (sul, str, cml)... repA... tra... (tet, repB).... The map of the autonomously replicating R factor is probably a circle connecting both sides of this linear map. Thus, a method has been established to map a plasmid that could not finely be analyzed under autonomous state by transduction. It also permits genetic analysis of genes responsible for replication of the plasmid without making use of a conditional mutant of itself but with that of the host, dnaA.     

R factor variants with enhanced sex factor activity in Pseudomonas aeruginosa

Summary The R factor R68 readily promotes chromosome transfer in Pseudomonas aeruginosa strain PAT, but shows little such sex factor activity in strain PAO. A variant of this plasmid, R68.45, has been isolated which produces recombinants in PAO plate matings at frequencies of 10^-3–10^-5 per donor cell for markers in the 0–60 min region of the chromosome. Little or no chromosome transfer was shown in liquid media. The kinetics of chromosome transfer were studied by interrupting matings on solid media with nalidixic acid. Five chromosomal markers, mapping in widely spaced regions of the chromosome all entered 3–5 min after initiation of mating. These results, combined with linkage studies, indicate that R68.45, unlike the Pseudomonas sex factors FP2 and FP39, promotes chromosome transfer from a range of origin sites and can thus be used for mapping the region of the P. aeruginosa chromosome later than 40 min.R68.45 and other similar variants were isolated from rare chromosomal recombinants appearing in crosses between PAO(R68) donors and PAO recipients in which selection for argB ⁺ was made. Selection for other chromosomal markers did not result in such variants suggesting that plasmids of the R68.45 type arise by recombination of genetic material between the R68 plasmid and certain regions of the bacterial chromosome.     

Isolation of transposon Tn551 insertions near chromosomal markers of interest in Staphylococcus aureus.

A procedure was developed to isolate insertions of transposon Tn551 near other markers of interest on the chromosome of Staphylococcus aureus NCTC 8325. When an inoculum of strain 8325-4 carrying a thermosensitive mutant of plasmid pI258 (on which Tn551 resides) was inoculated into brain heart infusion agar plus erythromycin and grown to saturation at 43 degrees C, the transforming DNA extracted from this population of cells contained a random collection of different chromosomal insertions of Tn551; this DNA is referred to as pooled Tn551 DNA. When erythromycin-sensitive recipient strains containing chromosomal markers of interest were transformed with pooled Tn551 DNA, and the resulting Emr transformants were screened for coinheritance of the donor allele of the marker of interest, insertions of Tn551 were isolated near several markers, including fus-149, tet-3490, mec-4916, pig-131, ilv-129, pur-140, and uraA141. Many of the insertions were within the linkage groups that contained these markers, and several insertions occupied different positions between the linkage groups in heretofore undefined regions of the circular chromosomal map of S. aureus. These insertions of transposon Tn551 extend the known limits of the existing linkage groups, provide linkage data and map locations for markers not previously mapped, and provide a means to map markers which cannot be directly selected.     

An integrated genetic and physical map for the malaria vector Anopheles funestus

We have constructed a genetic map of the major African malaria vector, Anopheles funestus, using genetic markers segregating in F(2) progeny from crosses between two strains colonized from different field sites. Genotyping was performed on 174 progeny from three families using 33 microsatellite markers, a single RFLP, and 15 single nucleotide polymorphism (SNP) loci. Four linkage groups were resolved and these were anchored to chromosomes X and 2 and chromosomal arms 3R and 3L by comparison with a physical map of this species. Five markers were linked to the X chromosome, 16 markers to chromosome 2, and 10 and 11 markers to chromosomal arms 3R and 3L, respectively. This significantly increases the number of chromosomally defined genetic markers for this species and will facilitate the identification of genes controlling epidemiologically important traits such as resistance to insecticides or vector competence.     

R68.45 mediated chromosomal gene transfer in Agrobacterium tumefaciens

A large plasmid enables its host Agrobacterium tumefaciens to cause tumorous condition in a wide variety of dicotyledonous plants [see Ooms et al. Gene 14:33–50 (1981)]. The location and role of chromosomal genes in this phenomenon are not known. As the first stage in studying this aspect, a project was initiated to investigate the chromosomal genetics of the bacterium. R68.45, a P group plasmid, was chosen as a transmission agent. After a preliminary assessment it was decided to use C58 as a standard strain to carry out the mapping. The plasmid itself, as judged by the presence of antibiotic markers, appears to be stable in a. tumefaciens; its ability to promote chromosomal mobilisation, however, remains only in 60–80% transconjugants. Good Agrobacterium donors are capable of transferring chromosomal genes at a frequency varying between 10^-5 to 10^-6 per recipient. The recombinants are stable even under non-selective conditions. A linear linkage map consisting of 16 markers was built using coinheritance frequencies obtained from 21 four-point crosses.     

Suppression of an Escherichia coli dnaA mutation by the integrated R factor R.100.1: Change of chromosome replication origin in synchronized cultures.

We have followed, by deoxyribonucleic acid-deoxyribonucleic acid hybridization, the order of replication of three chromosomal markers during a synchronous round of replication in three strains of Escherichia coli carrying a dnaAts mutation: one strain in which the F-like R factor R.100.1 was established as a plasmid and two strains in which the dnaA mutation was suppressed by the integration of R.100.1 into the chromosome. In the R+ strain at 30C, replication of the plasmid took place simultaneously with the initiation of chromosome replication at the normal origin. In the integratively suppressed Hfr strains, at 42.5 C, chromosome replication was initiated preferentially from the integrated plasmid; little or no initiation occurred at the normal origin. Similar results were obtained for the one strain tested at 30 C. For both Hfr strains at 42.5 C, the data suggest that at least part of the population replicated bidirectionally. This conclusion had been confirmed using an autoradiographic procedure. Both types of experiment indicate a wide variation in the rate of travel of individual replication forks within the population.     

Genome analysis of Agrobacterium tumefaciens: linkage map and genetic features of the left region of the linear chromosome

In addition to a unique tumor-inducing (Ti) plasmid, the plant pathogenic bacterium Agrobacterium tumefaciens has an unconventional chromosomal organization. Our previous studies on A. tumefaciens MAFF301001 revealed that it possesses a 2 Mb linear and a 2.8 Mb circular chromosome plus a 206.479 kbp Ti plasmid (pTi-SAKURA). In this study, a linkage map for the left half of its linear chromosome covering a 900 kbp region was constructed and the number of potential genes existing in the region was estimated. The linkage map consists of 31 BAC and 8 lambda phage recombinants without any gaps. It confirmed the size and all the structural landmarks indicated in the corresponding region of our previously constructed physical map for the linear chromosome. Sequencing analysis of the end-regions of each linking clone led to the identification of 6 genes and another 27 potential genes or ORFs, including genes and/or gene clusters responsible for homologous recombination (ruvB), trehalose/maltose sugar transport (thuR, thuG) and alanine catabolism (dadR). Two virulence-related gene homologues (attK and celB), previously reported in the circular chromosome of a different strain of A. tumefaciens were found in this region. These findings will provide a ready-to-use linkage map for further functional analysis of the linear chromosome.     

Prime plasmids derived from the IncP-10 plasmid R91-5 in Pseudomonas putida

Abstract Plasmid primes carrying various fragments of Pseudomonas putida chromosome have been derived from pMO22, a derivative of R91-5 loaded with Tn 501 . These prime plasmids transfer efficiently to P. aeruginosa where they effectively complement various auxotrophic markers. Proof of prime plasmid formation has been provided by the high-frequency transfer of plasmid and chromosomal markers, the unselected cotransfer of either plasmid or chromosomal markers into P. aeruginosa and by transformation of both plasmid and chromosomal markers using prime plasmid DNA. Such prime plasmids have been used to map the location of new markers on the P. putida chromosome.     

Use of plasmid R68.45 for constructing a circular linkage map of the Rhizobium trifolii chromosome.

Plasmid R68.45 was used to promote conjugal transfer of chromosomal markers in Rhizobium trifolii RS55. Analysis of two-factor and three-factor crosses among R. trifolii strains enabled construction of a circular linkage map of the R. trifolii chromosome, containing 17 nutritional and resistance markers.     

High-density sex-specific linkage maps of a European tree frog (Hyla arborea) identify the sex chromosome without information on offspring sex

Identifying homology between sex chromosomes of different species is essential to understanding the evolution of sex determination. Here, we show that the identity of a homomorphic sex chromosome pair can be established using a linkage map, without information on offspring sex. By comparing sex-specific maps of the European tree frog Hyla arborea, we find that the sex chromosome (linkage group 1) shows a threefold difference in marker number between the male and female maps. In contrast, the number of markers on each autosome is similar between the two maps. We also find strongly conserved synteny between H. arborea and Xenopus tropicalis across 200 million years of evolution, suggesting that the rate of chromosomal rearrangement in anurans is low. Finally, we show that recombination in males is greatly reduced at the centers of large chromosomes, consistent with previous cytogenetic findings. Our research shows the importance of high-density linkage maps for studies of recombination, chromosomal rearrangement and the genetic architecture of ecologically or economically important traits.     

Establishment of gene transfer systems for and construction of the genetic map of a marine Vibrio strain.

Two gene transfer systems were established for a marine bacterium, Vibrio sp. strain 60. One was generalized transduction with a newly isolated bacteriophage, As3, and the other was conjugal gene transfer by the use of newly constructed transposon-facilitated recombination (Tfr) donors. As3 transduced various chromosomal markers at frequencies of 10(-4) to 10(-6). Tfr donors, which were constructed by introducing transposon Tn10 into both plasmid RP4 and the chromosome, mediated the polarized transfer of chromosomal genes from the sites of Tn10 insertion on the chromosome. By means of these gene transfer systems, a genetic map of the vibrio chromosome was constructed.     

对家蚕第18连锁群隐性基因elp、ch-2和mln测交系的分子定位分析

家蚕长形卵(elp)、第二隐性赤蚁(ch-2)、暗化型(mln)均为第18染色体上的隐性突变, 在经典连锁图谱上的顺序和遗传距离已经排定。文章采用正常卵、正常黑蚁及正常白蛾品种P50与包含此3个隐性突变的三隐性测交系W18组配正反交群体, F1回交W18后获得回交群体(P50×W18)♀×W18♂ 和W18♀×(P50×W18)♂, 分别记作BC1F和BC1M, 利用已构建的家蚕SSR分子连锁图谱和根据家蚕基因组精细图设计的STS标记, 对这3个突变基因elp、ch-2、mln进行了分子定位研究, 并根据家蚕基因组精细图, 将第18连锁群的经典遗传图、分子连锁图和基因组物理图进行了对应。整合后的图谱遗传距离为94.2 cM, 突变基因和分子标记的排列顺序分别与形态标记连锁图和基因组精细图相一致, 研究结果对家蚕第18 染色体上其他突变的定位与克隆有重要的借鉴作用。     

Genetic linkage mapping of multiple epiphyseal dysplasia to the pericentromeric region of chromosome 19.

Multiple epiphyseal dysplasia (MED) is an inherited chondrodystrophy that results in deformity of articular surfaces and in subsequent degenerative joint disease. The disease is inherited as an autosomal dominant trait with high penetrance. An MED mutation has been mapped by genetic linkage analysis of DNA polymorphisms in a single large pedigree. Close linkage of MED to 130 tested chromosomal markers was ruled out by discordant inheritance patterns. However, strong evidence for linkage of MED to markers in the pericentromeric region of chromosome 19 was obtained. The most closely linked marker was D19S215, with a maximum LOD score of 6.37 at theta = .05. Multipoint linkage analysis indicated that MED is located between D19S212 and D19S215, a map interval of 1.7 cM. Discovery of the map location of MED in this family will facilitate identification of the mutant gene. The closely linked DNA polymorphisms will also provide the means to determine whether other inherited chondrodystrophies have underlying defects in the same gene.     

Transposon-facilitated chromosome mobilization in Agrobacterium tumefaciens.

We improved chromosomal gene transfer in Agrobacterium tumefaciens strain 15955 by constructing donors containing homologous transposons on both the sex factor plasmid and chromosome. First, we constructed plasmid pDP35, a kanamycin-sensitive derivative of R68.45. We then constructed derivatives of pDP35 that contained insertions of the kanamycin resistance transposon Tn5. By restriction endonuclease analysis, we identified two plasmids, pDP37 and pDP38, in which Tn5 was inserted in the same region of the plasmid but in opposite orientations. We also constructed isolates of A. tumefaciens containing an insertion of Tn5 in the chromosome. We transferred pDP37 or pDP38 into these chromosomal Tn5 strains and tested their ability to mobilize chromosomal markers to a series of auxotrophic recipients. Mobilization was observed at frequencies ranging from 10(-4) to 10(-7) recombinants per input donor for most markers tested. Both the plasmid and the chromosomal Tn5 elements were found to be required for mobilization at these higher frequencies. Donors were shown to transfer chromosomal markers in a polarized fashion. Recombinants coinherited unselected markers at frequencies of from 100 to 0.3 percent. The improved transfer frequencies and the observed polarity in chromosome transfer suggest that with this method we can genetically characterize A. tumefaciens chromosomal functions.