Evidence for a Conjugation-Like Mechanism of DNA Transfer in Helicobacter pylori

Many strains of Helicobacter pylori are naturally competent for transformation in vitro. Since there is a high degree of genetic variation among H. pylori strains, we sought to determine whether mechanisms of DNA exchange other than transformation exist in these organisms. Studies were done with H. pylori cells that each were resistant to two different antibiotics; the procedure used involved mating of cells on plates or in broth, in the absence or presence of DNase. In each experiment, such matings produced progeny with the markers of both parents. Examination of the full resistance profile and random arbitrarily primed DNA PCR (RAPD-PCR) profiles of the progeny indicated that DNA transfer was bidirectional. DNase treatment reduced but did not eliminate transfer; only the presence of both DNase and a membrane separating the cells did so. For progeny derived from matings in the presence of DNase, antibiotic resistance and RAPD profiles indicated that transfer was unidirectional. DNase-treated cell-free supernatants also did not transform, ruling out transduction. These experiments indicate that both a DNase-sensitive mechanism (transformation) and a DNase-resistant conjugation-like mechanism involving cell-to-cell contact may contribute to DNA transfer between H. pylori cells.     

Transferable lincosamide-macrolide resistance in Bacteroides.

Inter- and intraspecies transfer of resistance to clindamycin, lincomycin, and erythromycin in the strict anaerobe, Bacteroides, is described. This lincosamide-macrolide resistance was found to be specified by a 27 × 10⁶-dalton plasmid, designated pBF4, originally identified in a clinical Bacteroides fragilis isolate. Transfer of this plasmid to a strain of Bacteroides uniformis was demonstrated to occur by a deoxyribonuclease insensitive process which required cell-to-cell contact. Chloroform sterilized donor cell supernatants or filtrates of donor cells did not mediate resistance transfer. Transfer of the antibiotic resistance and pBF4 plasmid deoxyribonucleic acid (DNA) were always coincident. Drug resistant progeny recovered from such matings were able to transfer the pBF4 plasmid and its associated resistance markers to a suitable B.fragilis recipient strain. Compared to interspecies matings, resistance transfer was 100- to 1000-fold greater between isogenic donor and recipient strains, suggesting the possibility of a host controlled restriction-modification system.     

Genetic exchanges in the macrocysts of Dictyostelium discoideum.

Crosses were made between strains of Dictyostelium discoideum involving two drug resistance markers and the mating-type locus. Over 6000 progeny from 263 individual germinated macrocysts from four single-factor crosses, five two-factor crosses and one three-factor cross were characterized. In most cases the progeny from a single macrocyst were of one genotype, although in the population of macrocysts from any two-factor cross all possible parental and recombinant genotypes were recovered. There was no evidence of linkage between any of the markers examined. No selection against progeny carrying the methanol or the cycloheximide resistance markers was found in two-factor crosses, but selection against progeny carrying both resistance markers was found in the three-factor cross. Germination of macrocysts in all crosses was poor, only once exceeding 2.5% of the total macrocyst population. A variety of crosses and back-crosses with different parental strains indicated that germination might be influenced by both extrinsic (environmental) and multiple genetic factors. About 10% of the macrocysts yielded progeny spores that were ambivalent in their mating reactions. After extensive recloning these populations could be resolved to the normal matA (formerly A1) and mata (formerly A2) mating-types and might therefore have represented aneuploids. The results obtained with D. discoideum macrocysts differ from those obtained with other cellular slime moulds--Dictyostelium mucoroides, Dictyostelium giganteum and Polysphondylium pallidum--and are reminiscent of the results reported for germinated zygospores of Phycomyces blakesleeanus.     

Polyresistant Enterobacteriaceae strains and their plasmids in a hospital. Medical and theoretical aspects

The properties and origin of multiple resistant strains of Enterobacteriaceae found in the intestine and nasopharynx of infants admitted to the hospital for premature infants were studied. The strains of E. coli of different serovars isolated at various periods contained similar conjugative R plasmids with a molecular weight of 80 Md belonging to the O incompatibility group controlling resistance to kanamycin and physically independent small plasmids controlling resistance to ampicillin (7 Md) and streptomycin-sulfanilamides (4 Md). Multiple drug resistance in the strains of K. pneumoniae was controlled by single large (100-120 Md) plasmid cointegrates with 6-8 resistance markers. Such cointegrates consisted of several potentially independent plasmids, sometimes dividing on transformation of plasmid DNA of the recipient strains of E. coli K12. The small plasmids controlling resistance to ampicillin and streptomycin-sulfanilamides similar to the respective plasmids of E. coli were the constant components of the plasmids cointegrates. The multiple drug resistance in the above strains was combined with high capacity for colonization in premature infants. The medical staff and mothers were the sources of bacterial strains with single plasmids controlling definite types of resistance. It is suggested that the multiple resistant strains of Enterobacteriaceae are formed in hospital as a result of accumulation of the plasmids or plasmid markers and selection. One of the conditions for successive acquisition of new plasmid markers by definite bacterial strains was their high capacity for colonization in patients, which provided constant contacts and genetic exchange of such strains with a wide range of immigrant strains during colonization in the newly admitted patients.     

Discrimination of species and strains of basidiomycete genusCoprinus by random amplified polymorphic DNA (RAPD) analysis

All five examined strains ofCoprinus cinereus could be clearly discriminated from the strains of five otherCoprinus species by RAPD patterns with 12 of 13 primers. Also one specimen of unknownCoprinus strain was identified to beC. cinereus by this method. The RAPD patterns were similar among the strains in the same species; many common DNA fragments were recognized as well as some strain-specific DNA fragments. Thus all seven strains ofC. cinereus and all four strains ofC. angulatus examined could be distinguished individually. Diakryotic strains showed the combined RAPD patterns of the two monokaryotic strains constituting the dikaryon. The combined RAPD markers observed in the dikaryons were segregated in their basidiospore progeny. All 18 randomly picked progeny showed different combinations of RAPD markers from the parental strains.     

Fine-scale genetic mapping of two Pierce’s disease resistance loci and a major segregation distortion region on chromosome 14 of grape

A refined genetic map of chromosome 14, which contains the Pierce's disease (PD) resistance locus, was created from three grape mapping populations. The source of PD resistance in these populations was b43-17, a male form of Vitis arizonica Engelm. that is homozygous resistant. The resistance locus segregated as a single dominant gene and mapped as PdR1a in the F1 selection F8909-17 (9621 population) and as PdR1b in a sibling F1 selection F8909-08 (04190 population). These two full sibs inherited either allele of the Pierce's disease resistance locus from the b43-17 parent, which is homozygous at that locus. The 9621 population consisted of 425 progeny and PdR1a mapped between markers VvCh14-56/VvCh14-02 and UDV095 within a 0.6 cM genetic distance. The 04190 population consisted of 361 progeny and PdR1b mapped between markers VvCh14-02 and UDV095/VvCh14-10 within a 0.4 cM distance. Many of the markers present on chromosome 14 were distorted with an excess of female alleles in the 04190 and 04373 population (developed from a cross of V. vinifera L. F2-35 x b43-17) indicating that potential gametophytic factors are present in this region. Common markers from this region within the 9621 population were not distorted except Scu15. When these markers were compared to V. vinifera-based maps of chromosome 14 they were also distorted suggesting the involvement of gametophytic factors, and prompting the identification of this region as Vitis-segregation distortion region 1 (V-SDR1). The refined genetic maps developed from this study can be used to identify and clone genes that confer resistance to Pierce's disease.     

Chromosomal Integration of F′ Factors in Recombination-Deficient Hfr Strains of Escherichia coli

In matings between F′ donors and recombination-deficient Hfr recipients, we isolated progeny which transferred both episomal markers and Hfr markers early and with high frequency. A number of these progeny had two integrated sex factors. Investigation of these double Hfr strains showed that the F′ nearly always integrated in a homologous region of the chromosome. In any particular mating system integration was specific as to location and direction of chromosome transfer.     

Evidence of Tandem Duplication of Genes in a Merodiploid Region of Pneumococcal Mutants Resistant to Sulfonamide

A Pneumococcal mutant, sulr-c, resistant to sulfonamides, and three transformants bearing associated d or d+ resistance markers have earlier been reported to be unstable and show distinct patterns and frequencies of segregating stable progeny lacking the c marker. Each of the four strains showed a characteristic dosage of the genes involved in the merodiploidy. Complementary strands of DNA's from these stable and unstable strains were resolved and homoduplex and heteroduplex hybrids made from the separated DNA strands were used as donors in genetic transformations. Activities of a normal marker (streptomycin resistance) and those involved in the heterozygosity (c, d and d+) were quantitatively measured. From those heteroduplexes made up of opposite strands derived from a heterozygote and a stable strain, the normal marker is transferred efficiently, but the heterozygous markers are not. On the other hand, if both strands of a heteroduplex are derived from different heterozygotic strains, all markers can be transferred with usual efficiency to a stable recipient strain. The lowered efficiency in the former type of heteroduplex is attributed to an inhomology resulting from a tandem duplication in the merodiploid strains, and a postulated DNA repair process stimulated by it while in the form of the donor duplex. The inhomology probably includes (a) a microheterogeneity between the c site and the wild type locus, and (b) a more extensive incompatibility attributable to an extra segment of genome in a tandem duplication covering the c and d sites. The first of these inhomologies produces a lowered efficiency of transfer from all configurations of the particular d allele associated with the mutant c marker, and therefore accounts for the characteristic transfer patterns even from the native merodiploid DNA's.     

A test for genetic exchange in mixed infections of Leishmania major in the sand fly Phlebotomus papatasi

We tested if genetic exchange was observable between two strains of Leishmania major (Trypanosomatidae) during mixed infection of the sand fly Phlebotomus papatasi. Previous studies suggested that genetic exchange may occur in natural populations of Leishmania at a low frequency, but experimental crosses examining small numbers of progeny (less than 60) did not reveal hybrid parasites. Accordingly, a strategy was devised to increase the number of progeny that could be screened by 100-fold. Clonal derivatives from two strains that were infective to flies and contained numerous restriction fragment length polymorphisms were characterized and selected for resistance to methotrexate or tunicamycin by gene amplification. A successfully mixed infection of P. papatasi was obtained, and a method was developed for directly plating promastigotes from the gut contents of infected flies onto selective media. Twenty-five hundred independent progeny were scored for the presence of both drug resistance markers. No hybrid parasites were observed, indicating that the frequency of genetic exchange in this cross must be less than 4 x 10(-4). The lines and methods established in this work may prove useful in future studies of the mechanism and frequency of gene exchange in Leishmania.     

Genetic mapping in Streptomyces clavuligerus by protoplast fusion

The development of a protoplast manipulation protocol for the industrially important bacterium Streptomyces clavuligerus, which produces the beta-lactamase inhibitor clavulanic acid, made possible a preliminary genetic mapping study based on protoplast fusion crosses. A preliminary position for 11 markers on the S. clavuligerus genetic map is proposed. Fusion progeny were characterized by random spore analysis because the markers present in the strains were not amenable to the conventional four-on-four selection procedure. Whilst the resulting map is similar to that derived by conjugation for S. clavuligerus and S. coelicolor, further analysis of the markers is required to confirm these observations.     

Mobilization of Haemophilus influenzae chromosomal markers by an Escherichia coli F'' factor.

Filter matings between E. coli K-12 strains carrying an F'::Tn5,Tn9 factor with H. influenzae Rd strains gave rise to kanamycin-chloramphenicol-resistant H. influenzae strains at a frequency of approximately 10(-6). Transfer of the F' factor to H. influenzae was verified by expression of unselected markers in H. influenzae (lac+ or cotransfer of the nonselected antibiotic resistance), physical presence of a high-molecular-weight plasmid in recipient H. influenzae cells, and detection by Southern hybridization analysis of DNA sequences specific for the F' factor replication and partition functions in recipient H. influenzae cells. H. influenzae (F' Tn5,Tn9) strains were capable of transferring kanamycin and chloramphenicol resistances to other H. influenzae strains and were capable of mobilizing H. influenzae chromosomal markers at a low frequency. Insertion of a Tn5 element in the H. influenzae genome near the novobiocin resistance gene increased the frequency of transfer of novobiocin resistance about 30-fold. Transfer of other chromosomal markers also increased, although to a lesser extent, and ordered transfer of chromosomal markers could be demonstrated. Gene transfer was insensitive to DNase I, and transfer of chromosomal (but not F' factor) markers was dependent on the H. influenzae rec-1 and rec-2 gene functions.     

Investigation of the het genes that control heterokaryon incompatibility between members of heterokaryon-compatibility (h-c) groups A and G1 of Aspergillus nidulans

A chromosome assay method was used to determine the heterokaryon compatibility relationships between strains belonging to heterokaryon-compatibility (h-c) groups A and G1 of Aspergillus nidulans. A hybrid strain (RD15) was isolated following protoplast fusion of strains 65-5 (h-cA) and 7-141 (h-cG1). The morphology of RD15 was severely abnormal compared to diploid strains of A. nidulans produced from heterokaryon-compatible haploid parents. Inocula of RD15 were induced to haploidize on medium containing Benlate and a parasexual progeny sample of 291 haploid segregants was obtained. The progeny strains were genotyped for standard markers. Allelic ratios and pairwise marker segregations were determined. Pairs of progeny strains that carried different alleles for the standard markers on each linkage group in turn were tested for compatibility. Strain pairs that possessed different alleles for the markers on linkage groups II, III, V, VI and VII were incompatible indicating the presence of heterokaryon-incompatible (het) genes on these linkage groups. Backcrosses to an h-cGl strain showed that two het genes were located on linkage group III and confirmed a total of six het gene differences between the h-cA and h-cGl strains.     

A genetic map of mouse chromosome 1 near the Lsh-Ity-Bcg disease resistance locus

Isozyme and restriction fragment length polymorphism (RFLP) analyses of backcross progeny, recombinant inbred strains, and congenic strains of mice positioned eight genetic markers with respect to the Lsh-Ity-Bcg disease resistance locus. Allelic isoforms of Idh-1 and Pep-3 and RFLPs detected by Southern hybridization for Myl-1, Cryg, Vil, Achrg, bcl-2, and Ren-1,2, between BALB/cAnPt and DBA/2NPt mice, were utilized to examine the cosegregation of these markers with the Lsh-Ity-Bcg resistance phenotype in 103 backcross progeny. An additional 47 backcross progeny from a cross between C57BL/10ScSn and B10.L-Lshr/s mice were examined for the cosegregation of Myl-1 and Vil RFLPs with Lsh phenotypic differences. Similarly, BXD recombinant inbred strains were typed for RFLPs upon hybridization with Vil and Achrg. Recombination frequencies generated in the different test systems were statistically similar, and villin (Vil) was identified as the molecular marker closest (1.7 +/- 0.8 cM) to the Lsh-Ity-Bcg locus. Two other DNA sequences, nebulin (Neb) and an anonymous DNA fragment (D2S3), which map to a region of human chromosome 2q that is homologous to proximal mouse chromosome 1, were not closely linked to the Lsh-Ity-Bcg locus. This multipoint linkage analysis of chromosome 1 surrounding the Lsh-Ity-Bcg locus provides a basis for the eventual isolation of the disease gene.     

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.     

Conjugation transfer of plasmid resistance to gentamycin and other antibiotics in clinical strains of Ps. aeruginosa]

A possibility of conjugation transfer of the markers of the plasmid resistance to gentamicin and other antibiotics from 10 clinical strains of Ps. aeruginosa, isolated from burn patients to the recipient strain of Ps. aeruginosa PTO 629 Rfr was shown. The marker of gentamicin resistance was transferred to 100 out of 110 of the exconjugants, i.e. 86.2 per cent. The rate of the conjugation transfer in the crosses between the clincal strains of Ps. aeruginosa and the recipient strain PTO 629 Rfr with respect to the gentamicin marker was about 10--7. The plasmid resistance markers in the clincal strains Ps. aeruginosa were transferred in various combinations. Transfer of the markers of resistance to streptomycin, carbenicillin, neomycin and combinations Sm, Nm and Sm, Nm, Cm was not achieved.     

A Test for Genetic Exchange in Mixed Infections of Leishmania major in the Sand Fly Phlebotomus papatasi

We tested if genetic exchange was observable between two strains of Leishmania major (Trypanosomatidae) during mixed infection of the sand fly Phlebotomus papatasi. Previous studies suggested that genetic exchange may occur in natural populations of Leishmania at a low frequency, but experimental crosses examining small numbers of progeny (<60) did not reveal hybrid parasites. Accordingly, a strategy was devised to increase the number of progeny that could be screened by 100-fold. Clonal derivatives from two strains that were infective to flies and contained numerous restriction fragment length polymorphisms were characterized and selected for resistance to methotrexate or tunicamycin by gene amplification. A successfully mixed infection of P. papatasi was obtained, and a method was developed for directly plating promastigotes from the gut contents of infected flies onto selective media. Twenty-five hundred independent progeny were scored for the presence of both drug resistance markers. No hybrid parasites were observed, indicating that the frequency of genetic exchange in this cross must be less than 4 times 10^-4. The lines and methods established in this work may prove useful in future studies of the mechanism and frequency of gene exchange in Leishmania.     

Fine mapping of quantitative trait loci for mastitis resistance on bovine chromosome 11

Quantitative trait loci (QTL) affecting clinical mastitis (CM) and somatic cell score (SCS) were mapped on bovine chromosome 11. The mapping population consisted of 14 grandsire families belonging to three Nordic red cattle breeds: Finnish Ayrshire (FA), Swedish Red and White (SRB) and Danish Red. The families had previously been shown to segregate for udder health QTL. A total of 524 progeny tested bulls were included in the analysis. A linkage map including 33 microsatellite and five SNP markers was constructed. We performed combined linkage disequilibrium and linkage analysis (LDLA) using the whole data set. Further analyses were performed for FA and SRB separately to study the origin of the identified QTL/haplotype and to examine if it was common in both populations. Finally, different two-trait models were fitted. These postulated either a pleiotropic QTL affecting both traits; two linked QTL, each affecting one trait; or one QTL affecting a single trait. A QTL affecting CM was fine-mapped. In FA, a haplotype having a strong association with a high negative effect on mastitis resistance was identified. The mapping precision of an earlier detected SCS-QTL was not improved by the LDLA analysis because of lack of linkage disequilibrium between the markers used and the QTL in the region.     

美洲黑杨抗黑斑病基因的RAPD标记筛选和连锁分析

以美洲黑杨（Populus deltoides Bartr.cv.“Lux”(I-69/55)）为母本,欧美杨（P.euramericana cv.I-45）为父本得到的F1为材料,其中I-69对黑斑病表现为高度抗病,I-45为高度感病。利用分离群体混合分析（bulked segregrants analysis,BSA）技术建立两个DNA池（感病池和抗病池）,筛选出了与抗黑斑病性状基因相连锁的RAPD标记:OPAI17―1550、OPAI13―900。利用选择性基因型分析法进行标记―抗黑斑病基因连锁分析,两标记与抗黑斑病基因遗传距离分别为29.9cM和37.4cM。 Identification of Markers Linked to Resistance Locus of Marssonina Leaf Spot in Poplars by Bulked Segregant Analysis(BSA) ZHANG Bo1,HUANG Min-ren1,ZHUGE Qiang1,HAN Zheng-min1,YIN Tong-ming1,PAN Hui-xin1,ZHU Li-huang2,WU Rong-ling3,WANG ming-xiu１ 1.The Key Laboratory of Forest Tree Genetic Engineering,Nanjing Forestry University,Nanjing,210037,China; 2.The Institute of Genetics and Developmental Biology,Chinese Academy of Science,Beijing 100101,China; 3.Department of Statistics,University of Florida,Gainesville,Florida 32611,USA Abstract:DNA markers linked to resistance locus of Marssonina leaf spot in poplars were found by bulked segregant analysis(BSA).The bulks consisted of individual with a extreme phenotype taken from a population of 91 F1 clones,which is a progeny of Populus deltoides Bartr.cv.“Lux”(I-69/55)(Resistance) and P.euramericana cv.I-45(Susceptible).Out of 114 RAPD primers,four markers showed polymorphisms between the resistance-bulk and the susceptible-bulk.By using selective genotype linkage analysis,OPAI17-1550 and OPAI13-900 were found linked to the resistance locus.The genetic distances between the two markers and the resistance locus were 29.9cM and 37.4cM,respectively. Key words：BSA; RAPD; selective genotype analysis; Marssonina leaf spot     

Effect of ouabain resistance on human diploid fibroblasts carrying other genetic variants.

Human skin fibroblasts from a Lesch-Nyhan male with the G6PD A variant and those from a female with the cri-du-chat deletion have been treated with ethyl methane sulfonate and subjected to ouabain selection. Sixteen ouabain-resistant clones were obtained from 1.2 × l0⁸ cells plated in 10^?6 M ouabain. Some variation in degree of ouabain resistance was noted among subclones. The ouabain-resistant phenotype was stable and was associated with G6PD and HGPRT phenotypes and karyotypes similar to the parent strains except that tetraploidy characterized 2 of 5 of the clones karyotyped. The ouabain-resistant cells were used to study the influence of the mutation leading to ouabain resistance on the transfer of nucleotides from cell to cell. Results indicate that contact-feeding of nucleotides is not dependent upon the ouabain-sensitive Na⁺K⁺ transport system. It is suggested that ouabain resistance may provide a simple assay to detect agents mutagenic for human cells, and that cells carrying both HGPRT as well as ouabain-resistant markers are ideal for cell hybridization with strains having no selectable markers.