Human hypervariable minisatellite probes detect DNA polymorphisms in the fungus Colletotrichum gloeosporioides

Summary The human minisatellite probes for hypervariable regions within the genome are able to detect restriction fragment length polymorphisms between subgroups of the filamentous fungus Colletotrichum gloeosporioides. The two types of this pathogen that cause anthracnose on Stylosanthes spp. in Australia were distinguishable using the polycore probes 33.6 and 33.15. This is the first report of the use of these versatile probes for detecting polymorphic DNA regions in fungi.     

Inheritance of chromosome length polymorphisms in Saccharomyces cerevisiae

Summary Although Saccharomyces cerevisiae strains generally have similar chromosomal band patterns as revealed by pulsed field gel electrophoresis, individual bands often move slightly differently from one strain to the other. Surveying strains from our stock collection, we found that nearly all the bands of a certain pair of strains differed in their mobility. Some of these chromosome length polymorphisms segregated in a 2:2 ratio, indicating that they resulted from single structural alterations (i.e. additions or deletions). One of these was mapped on the right arm of chromosome 1. Others did not segrate in a simple 2:2 ratio. That is, there were progenies which had bands not present in either parent. We suggest that these new bands are the products of recombination between homologous chromosomes having two or more structural alterations.     

Genetic polymorphism of Leishmania species using kinetoplast DNA restriction fragment length polymorphism and cDNA probe of Leishmania donovani

Leishmaniasis represents a group of diseases that range from simple cutaneous lesions through metastasizing diffused cutaneous to severe systemic infection depending upon the taxon to which the causative parasite belongs. Therefore, it is important to identify the infecting Leishmania. Methods presently being used, including immunology, biochemistry and molecular biology have one or the other limitations, leaving scope for the search for newer probes. This study reports the characterization of leishmania isolates both by restriction fragment length polymorphism of kinetoplast DNA (kDNA) and genomic DNA. The genomic DNA was probed with a cDNA probe B₂a₁. Using a kDNA restriction pattern technique, different isolates of Leishmania donovani could be differentiated from the UR6 strain of L. tropica, but it was not possible to differentiate between newer local isolates of L. donovani with most of the restriction enzymes except AluI. However, the B₂a₁ cDNA probe was able to differentiate these isolates effectively. Both of these techniques could differentiate newer local isolates of L. donovani from the older isolates of L. donovani from India, i.e., DD8, RMRI and SS. The Indian isolates of L. donovani could also be differentiated from isolates of L. donovani from Jeddah and Germany using both techniques. The present study indicates that the cDNA probe B₂a₁ can be used as an important adjunct to kDNA restriction analysis for the characterization of Leishmania species. Received: 15 October 1996     

Identification of mycobacteria to the species level by automated restriction enzyme fragment length polymorphism analysis

An automated method for the restriction fragment length polymorphism (RFLP) analysis for the differentiation of mycobacteria to the species level is described. After polymerase chain reaction (PCR) amplification of a sequence of the gene encoding the 65-kDa surface antigen common to all mycobacteria the product was investigated by RFLP analysis. For accurate determination of fragment sizes the asymmetrically fluorescein-labelled PCR product was partially digested with restriction site enzymes BstEII and HaeIII. The fragments obtained were analysed electrophoretically using an automated laser fluorescence DNA sequencer. Determination of fragment sizes revealed a deviation of ±1 base pair (bp; 0.6%) when compared to expected sizes. The validity of this approach was confirmed by analysing mycobacterial DNA obtained from pure cultures of Mycobacterium (M.) tuberculosis and alcohol-fixed smears as well as paraffin-embedded sputa of patients with culture-proven tuberculosis. Additionally a diagnostic algorithm was established by investigation of cultured M. bovis, M. bovis bacille Calmette-Guérin, M. avium, M. intracellulare and M. fortuitum. The method allows the identification of restriction enzyme sites which are only 40 bp apart. Partial restriction enzyme digestion of asymmetrically fluorescence-labelled PCR products will presumably lead to the discovery of new restriction enzyme sites.     

An<Emphasis Type="Italic"> ospA</Emphasis>-polymerase chain reaction/restriction fragment length polymorphism-based method for sensitive detection and reliable differentiation of all European<Emphasis Type="Italic"> Borrelia burgdorferi</Emphasis> sensu lato species and OspA types

We describe a sensitive and reliable method for detection and differentiation of the five relevant European Borrelia burgdorferi sensu lato species (B. burgdorferi sensu stricto, B. afzelii, B. garinii, B. valaisiana, and B. lusitaniae), based on a heminested ospA-PCR followed by restriction enzyme analysis. Sensitivity was one borrelia per PCR except for B. afzelii, where it was five per PCR. None of seven relapsing fever borreliae, eight Leptospira serovars or two Treponema species were amplified. Except B. garinii, each of the five B. burgdorferi s.l. species is represented by one or two characteristic restriction fragment length polymorphism (RFLP) patterns. Analysis of the heterogeneous group of B. garinii resulted in five different RFLP patterns, corresponding to the OspA types 3–7 associated with this species. In a pilot study on 529 Ixodes ricinus ticks from three different regions in Southern Germany, all species and OspA types were found except B. lusitaniae and B. garinii OspA type 7, arguing for a broad distribution of almost all OspA types. A further notable finding was the focal prevalence of OspA type 4, which has rarely been detected in ticks previously. Thus, the developed method provides a fast and simple tool for epidemiological studies on the heterogeneity of species and OspA types in Europe which has important implications for the development of vaccines and (microbiological) test systems for Europe.     

Major chromosomal length polymorphisms are evident after meiosis in the phytopathogenic fungus Leptosphaeria maculans

Chromosomal DNA of Australian field-isolates of the phytopathogenic ascomycete Leptosphaeria maculans was resolved by pulsed-field gel electrophoresis. All isolates examined had highly variable karyotypes. Ascospores (sexual spores) derived from single pseudothecia (sexual fruiting bodies) isolated from Brassica napus (oilseed rape) stubble were analyzed. In two tetrads four distinct karyotypes were observed, with only one chromosomal DNA band in common to all the members of each tetrad. Although isolates had highly variable karyotypes, two overall patterns were present. In one pattern there were at least 12 chromosomal DNA bands, the largest being greater than 2.2 Mb in size; in the other there were more than 15 chromosomal DNA bands, the largest being about 2.0 Mb. The chromosomal DNA preparations included mitochondrial DNA which migrated as a diffuse band between 0.10 and 0.15 Mb in size, and DNA molecules of 8 and 9 kb in size.     

Chloroplast gene inheritance studied by somatic fusion in Chlamydomonas reinhardtii

Summary Somatic fusion between strains of Chlamydomonas containing complementing cell-wall and auxotrophic mutations, having the same mating-type (mt) and bearing chloroplast markers, have been performed to study the mode of chloroplast gene inheritance in the fusion products. About one third of the fusion products (mt ⁺/mt ⁺ or mt ^–/mt ^–) transmitted chloroplast markers from both parents (= biparental fusion products). The rest of the population was equally distributed between fusion products transmitting the chloroplast marker of one parent or the other (uniparental fusion products) exclusively. Incubation of the fusion products in the dark for 48 hours, immediately after the fusion, decreases the frequency of biparental fusion products. The results indicate that the general process of elimination of chloroplast alleles is independent of the presence of both mt ⁺ and mt ^– alleles in the cell. In contrast, directional elimination (i.e. preferential elimination of paternal chloroplast alleles) does appear to depend upon heterozygosity at the mt locus. These results are discussed in relation to the models which have been proposed to explain the maternal inheritance of chloroplast genes in Chlamydomonas.     

A uniparental mutant of Chlamydomonas reinhardtii resistant to chloramphenicol

Summary A uniparental mutant of Chlamydomonas resistant to chloramphenicol was selected following treatment of wild-type cells with 5-fluorodeoxyuridine. Under heterotrophic conditions, growth and chloroplast protein synthesis of this mutant (CAP1) are resistant to chloramphenicol. Under phototrophic conditions, CAP1 is sensitive to chloramphenicol. In addition CAP1 displays thermosensitivity when grown phototrophically in the absence of antibiotics: at the restrictive temperature, a specific reduction of those thylakoid membrane polypeptides which are synthesized inside the chloroplast is observed. Alternative explanations for the pleiotropic phenotype of CAP1 are discussed.     

乌鲁木齐地区人巨细胞病毒临床分离株PCR-RFLP分析研究

目的 通过对人巨细胞病毒临床分离株基因结构的分析，了解乌鲁木齐地区人巨细胞病毒(HCMV)的分子流行病学情况。方法 分别扩增28株HCMV临床分离株的3个相对保守基因：DNA聚合酶基因(pol)，糖蛋白H基因(gH)和主要即刻早期蛋白基因(MIE)片段并进行限制性片段长度多态性(PCR-RFLP)分析。结果 非流行病学相关毒株限制性图谱差异较大，流行病学相关毒株如母婴配对株限制性图谱有很大的相似性。8对母婴配对HCMV毒株，4对限制性图谱一致，4对存在差异。结论 本地HCMV分离株基因变异广泛存在。PCR-RFLP分析可揭示HCMV感染的传播方式，可用于病毒的分子流行病学研究。     

Cytological demonstration of chloroplast DNA behavior during gametogenesis and zygote formation in Chlamydomonas reinhardtii

Summary We used the flourescent dye DAPI to visualize nucleoids of chloroplast DNA and follow their behavior through sexual reproduction by counting nucleoids in fixed cells at various stages. Nucleoid number varied greatly among cells at each stage. The mean number of nucleoids per cell was similar in mt ⁺ and mt ^– vegetative cells, and declined similarly during gametogenesis. Longer periods of nitrogen starvation reduced the mean nucleoid number further. Mean nucleoid number declined again in mating pairs, and continued to drop in zygotes up to the latest stage that can be examined (24-h zygotes). The oldest zygotes had means of about 2 to 3 nucleoids in different experiments, significantly fewer than in the mt ⁺ gametes (usually 4 to 5). The quantitative data on nucleoid number, mating efficiency, and germination efficiency allowed us to show that the decrease in nucleoid number is not limited to gametes that do not mate, or to zygotes that do not germinate. These data are consistant with earlier biochemical studies showing loss of chloroplast DNA during gametogenesis in both mating types, and with the degradation of paternal chloroplast DNA detected biochemically and (in non-quantitative studies) by DAPI staining. There may also be some fusion of nucleoids, although if it occurs it is not complete by 24 h of zygote maturation.     

Deletions, duplications and novel restriction fragment length polymorphism in Duchenne and Becker muscular dystrophies

To determine the mutations of Southern Chinese with Duchenne and Becker muscular dystrophies (DMD, BMD), we analysed 28 DMD and BMD patients in 24 unrelated families for intragenic deletions and duplications by using cDNA probes covering the entire 14 kb of the dystrophin gene. Deletions were detected in nine unrelated patients (seven patients by probe 8 and two by probe 2b-3). Gene duplications were detected by probe 1-2a in two patients with the duplication bands confirmed in both Hind III and Bgl II digests and by densitometry. A third patient was found to have a junction fragment with Bgl II and a duplication band with Hind III by probe 5b-7. Therefore 50% of the 24 unrelated families were found to have either deletions or duplications. A previously undescribed restriction fragment length polymorphism (RFLP) was found in one family with probe 5b-7 in Bgl II digests which was found to segregate with the disease phenotype. This new RFLP was not detected in over 70 unrelated X chromosomes we have examined so far, and appeared to be "private" for this family. The presence of this new restriction site may or may not be the mutation responsible for the disease phenotype.     

Changes in chloroplast genome composition and recombination during the maturation of zygospores of Chlamydomonas reinhardtii

Summary In crosses of the unicellular green alga Chlamydomonas reinhardtii, the chloroplast genes are normally transmitted exclusively by the maternal parent to zygospore progeny. However, transmission of the paternal chloroplast alleles can be increased markedly by certain pretreatments of the maternal parent prior to mating. As zygospores age prior to induction of meiosis, they display decreased biparental transmission of chloroplast alleles and increased transmission of chloroplast alleles from only the maternal or paternal parent. In this report, chloroplast genome composition of biparental zygospores is shown to change in several ways during zygospore maturation. Allelic ratios of chloroplast genes within biparental zygospore clones become maternally or paternally skewed as the zygospores age, cotransmission of chloroplast alleles is reduced, and recombination increases, resulting in an expansion of genetic map distances between chloroplast markers used in this cross. The recovery of unequal frequencies of zygospore progeny expressing reciprocal recombinant genotypes confirms and extends other reports of the predominance of nonreciprocal recombination in organelle genetic systems.     

Mitochondrial DNAs of the fungus Armillaria ostoyae: restriction map and length variation

A restriction-enzyme-site map is presented for the 147-kb mtDNA of North American Armillaria ostoyae. The locations of five structural genes, atp6, atp8, coxI, coxIII, and cob, along with the location and orientation of the large and small ribosomal RNA genes, were determined through Southern hybridizations with cloned genes from other fungal mtDNAs. Based on this map, the variation in mtDNA suggested geographic structure at two different levels. On a large geographic scale, 17 mtDNA types from North America were distinct, with respect to both size and restriction maps, from three mtDNA types from Europe. At the local scale, identical mtDNA types were evident among several different genetic individuals located no more than 1 km apart at a site in Michigan. No mtDNA type occurred more than once among genetic individuals from different regions of North America, although the occurrence of similar mtDNAs in isolates from distant regions suggested that this may occur at a low frequency with large sample sizes. Among the North American mtDNA types, analysis of discrete length variants was inconsistent with the hypothesis that the mtDNA of A. ostoyae evolves as a clonal lineage in which each length mutation represents a unique event. The two remaining hypotheses, that similar mutational events have occurred independently and that genetic exchange and recombination occurs among mtDNAs in natural populations of this species, remain to be tested.     

阿尔茨海默病患者环氧化酶-2基因启动子区多态性分析

目的检测环氧化酶-2(COX-2)基因的1195和765两个多态位点,探讨其与阿尔茨海默病(AD)的可能相关性。方法采用Qiagen DNA提取试剂盒提取外周血白细胞DNA,根据位点基因序列设计引物,采用PCR-限制性片段长度多态分析(PCR-RFLP)分别对90例AD患者(AD组)和110例正常人(对照组)进行基因分型,PCR扩增出各样本的目的片断,对扩增产物进行酶切,酶切产物经2%琼脂糖凝胶电泳,EB染色,凝胶成像系统判定结果。结果AD组共检出3种COX-2-1195基因型,GG基因型频率为21.0%,GA基因型频率为50.0%,AA基因型频率为29.0%,G、A等位基因频率分别为46.1%和53.9%,与对照组比较,差异均无统计学意义;GG基因型频率为77.8%,GC基因型频率为20.0%,CC基因型频率为2.2%,G、C等位基因频率分别为87.8%和12.2%,与对照组比较,AD组中CC基因型频率、C等位基因频率差异均有统计学意义。COX-2蛋白水平在AD组和对照组分别为(124.95±8.73)IU/L、(51.81±7.33)IU/L,差异有统计学意义(P<0.05)。结论①COX-2基因启动子区-1195G/A多态性可能与中国南方人群AD的发生无关;②COX-2基因～765G/C与AD的发生有关,-765G/C多态性可能是AD发生的一风险因素;③AD患者血浆中COX-2蛋白水平升高,可能是由于COX-2基因-765G/C多态性引起。     

Direct recombinogenic effect of dimethylnitrosamine (DMN) on zygotes of Chlamydomonas reinhardtii

Summary We developed a test system with mutant strains of Chlamydomonas reinhardtii requiring arginine. Here, the meiotic recombination between two arginine loci served as an indicator of genotoxicity. After crossing, zygotes were treated with the alkylating agent dimethylnitrosamine (DMN) for 30 min. A dose dependent increase of wild type recombinants was found for DMN concentrations ranging from 68 M to 680 M. Like in cells of some higher plants, the agent was active in Chlamydomonas zygotes without any exogenous metabolic activation.     

A new assay system to classify non-mating mutants and to distinguish between vegetative cell and gamete in Chlamydomonas reinhardtii

Summary A new system is described for the classification of various types of non-mating mutants of Chlamydomonas reinhardtii. This system makes use of two simple assays: one for cell body-agglutinin (Saito et al. 1985), and another for the cell wall lytic enzyme (Matsuda et al. 1987). Both assays use the soluble fraction of homogenates from nitrogen-starved cells. Many non-mating mutants previously isolated were analysed and divided into four main phenotypic classes. This assay method is also a valuable tool for distinguishing between vegetative cells and gametes. Without this assay, verifying gametogenesis is especially difficult in agglutinin-and flagella-deficient cells. We found that wild-type cells, which had been grown for 7 days on TAP plates and lacked flagella, contain neither cell body-agglutinin activity nor lytic enzyme activity in the soluble fraction of their cell homogenates, indicating that these cells can be classified as vegetative. When suspended in nitrogen-free liquid medium, the two activities developed rapidly in parallel to the acquisition of mating ability: a maximum level was reached within two hours.     

Isolation and characterization of dominant,pleiotropic drug-resistance mutants in Chlamydomonas reinhardtii

Summary Three independent pleiotropic drug-resistance (pdr) mutants were isolated by selecting for resistance to the anti-microtubule herbicides amiprophos-methyl (APM) and oryzalin (ORY). These three mutants and a previously isolated mutant, ani1 (anisomycin resistance), were semi-dominant in heterozygous diploids, and they displayed varying degrees of resistance to structurally and functionally unrelated inhibitors such as cycloheximide, cryptopleurine, emetine, atrazine, and nonidet P-40. Linkage analysis and genetic mapping suggested that three of the four mutants, including ani1, define a single locus, here named pdr1. The fourth mutant defined a new locus, pdr2, which is located on the left arm of linkage group VI. One pdr1 mutant exhibited unusual genetic interactions, including enhanced ts-lethality and synergistic increases in drug resistance, when combined with pdr2-1 and with herbicide-resistant alleles of three other genes.     

Studies on homologous recombination in the green alga Chlamydomonas reinhardtii

The introduction of exogenous DNA into the nuclear genome of Chlamydomonas reinhardtii occurs predominantly via non-homologous (illegitimate) recombination and results in integration at apparently-random loci. Using truncated and modified versions of the C. reinhardtii ARG7 gene in a series of transformation experiments, we demonstrate that homologous recombination between introduced DNA molecules occurs readily in C. reinhardtii, requires a region of homology of no more than 230 bp, and gives rise to intact copies of ARG7 in the nuclear genome. Evidence is presented for homologous recombination between introduced ARG7 DNA and the resident copy of the gene, and for the de-novo synthesis of the ARG7 sequence during transformation.     

Outcrossing in the homothallic oomycete,Pythium ultimum,detected with molecular markers

The oomycete Pythium ultimum is homothallic, thus a single isolate completes the sexual stage in pure culture. It has been generally assumed that homothallic oomycetes are predominantly inbreeding. In P. ultimum, antheridia occasionally develop from hyphae not directly connected to the oogonium and appear to participate in fertilization, suggesting a possible mechanism for outcrossing. We have used molecular markers to confirm that outcrossing can occur between isolates of P. ultimum. Genetic markers based on randomly amplified polymorphic DNA (RAPD) and restriction fragment length polymorphisms (RFLP) were used to distinguish isolates in a collection of P. ultimum. Two isolates displaying a high level of polymorphism were mixed and placed on media which allows the development of the sexual stage. RAPD markers were used to screen single oospore progeny to identify potential hybrids between the two parental isolates. Subsequent self-fertilization of one putative F₁ yielded a F₂ population which demonstrated segregation and independent assortment of RAPD and RFLP markers. A similar strategy was used to show that an isolate which is incapable of producing oospores in pure culture can outcross when mixed with a homothallic isolate. These results suggest that other homothallic oomycetes may be capable of outcrossing, and sexual reproduction may, therefore, play an important role in the generation of variation in homothallic oomycetes.     

Evidence for persistence of chloroplast markers in the heteroplasmic state in Chlamydomonas reinhardtii

Summary In the green alga Chlamydomonas reinhardtii, reciprocal crosses between strains carrying non-allelic chloroplast mutations to streptomycin dependence (sd-u) produce streptomycin sensitive (sd-u ⁺) recombinant progeny. Transfer of these sd-u ⁺progeny to streptomycin-containing medium results in a much higher frequency of recovery of streptomycin dependent isolates than expected by mutation. Failure to recover the more commonly encountered class of streptomycin resistant mutants also suggests that mutation is not responsible for appearance of the new dependent isolates. Backcrosses of these new sd-u isolates to strains carrying the original sd-u mutations demonstrate their allelism with the sd-u mutation contributed by the mt ⁺parent. Earlier work by Schimmer and Arnold (1969, 1970a-d) indicated that newly isolated sensitive revertants of the streptomycin dependent mutant sd-u-3-18 also yielded high frequencies of sd-u cells but these were never analyzed genetically. We have now obtained new sd-u. isolates from streptomycin sensitive revertants of sd-u-318 and shown them all to be allelic with the original sd-u3-18 mutation. Thus hidden sd-u alleles can coexist with sd-u ⁺alleles in heteroplasmic cells. These heteroplasmic cells are streptomycin sensitive in phenotype and may arise in crosses or from new mutation.