Dictyostelium discoideum cobB mutants show reduced heavy metal accumulation associated with gene amplification

 Wild-type Dictyostelium discoideum cells grow- ing on non-toxic levels of nickel chloride or cobaltous chloride accumulate 2–3.5 times as much nickel and at least 1.5 times as much cobalt as cobB mutants. The cobB trait is dominant, confers unstable cobalt and nickel resistance and is correlated with the presence of up to 50 copies of a linear extrachromosomal DNA, approximately 100 kb in length, derived from linkage group III. Independent cobB mutants can be obtained by selection on medium containing either cobalt or nickel. The amplified DNA can be transferred to wild-type strains by electroporation. Strains with mutations at a second cobalt resistance locus, cobA, accumulate the same amount of cobalt, but more nickel than wild-type strains. Our results are consistent with the cobA mutant phenotype being due to internal sequestration of cobalt, and the cobB mutant phenotype being due to reduced net uptake of cobalt and nickel. Energy-dependent nickel export was detectable in wild-type and cobB mutant strains but its role in heavy metal resistance has not yet been proved. Received: 21 December 1995/Accepted: 10 June 1996     

Dictyostelium discoideum cobB mutants show reduced heavy metal accumulation associated with gene amplification

Wild-type Dictyostelium discoideum cells grow- ing on non-toxic levels of nickel chloride or cobaltous chloride accumulate 2–3.5 times as much nickel and at least 1.5 times as much cobalt as cobB mutants. The cobB trait is dominant, confers unstable cobalt and nickel resistance and is correlated with the presence of up to 50 copies of a linear extrachromosomal DNA, approximately 100?kb in length, derived from linkage group III. Independent cobB mutants can be obtained by selection on medium containing either cobalt or nickel. The amplified DNA can be transferred to wild-type strains by electroporation. Strains with mutations at a second cobalt resistance locus, cobA, accumulate the same amount of cobalt, but more nickel than wild-type strains. Our results are consistent with the cobA mutant phenotype being due to internal sequestration of cobalt, and the cobB mutant phenotype being due to reduced net uptake of cobalt and nickel. Energy-dependent nickel export was detectable in wild-type and cobB mutant strains but its role in heavy metal resistance has not yet been proved.     

Genome of Azotobacter vinelandii: counting of chromosomes by utilizing copy number of a selectable genetic marker

Studies utilizing several physical, biochemical and spectroscopic methods have suggested that Azotobacter vinelandii contains multiple copies (40–80) of its chromosome per cell, whereas genetic analysis indicated that these cells function like haploid cells. To further verify if A. vinelandii indeed contains 40–80 copies of its chromosome per cell, we have developed an in vivo chromosome counting technique. The basic principle of this technique is to introduce the same genetic marker on the chromosome and on an extrachromosomal element of known copy number into the bacterium. The copy number of the chromosome can be determined by comparing the intensity of the hybridization signal generated by the DNA fragment carrying the chromosomal marker with that of the extrachromosomal marker when the total DNA isolated from this strain is hybridized with a probe made of the same genetic marker DNA. To do this we used an A. vinelandii BG102 strain which carries a kanamycin resistance marker gene integrated into the nifY locus on its chromosome(s). The plasmids pRK293 and pKT230, which can replicate in A. vinelandii and carry the kanamycin resistance gene (similar to the one present on the chromosome of A. vinelandii BG102), served as the extrachromosomal elements with known copy number. Southern blotting and hybridization analysis of the total DNA, isolated from A. vinelandii BG102 containing these plasmids, with a probe made of the kanamycin resistance gene clearly indicated that the copy number of A. vinelandii chromosome is slightly lower than the copy number of the low-copy plasmid pRK293 and about 21-fold lower than the copy number of the high copy plasmid pKT230. We believe that this In vivo chromosome counting technique can be used for determination of the copy number of the chromosome in other cells with appropriate modifications in the nature of the extrachromosomal element and the genetic marker.     

Genetic mapping reveals a single major QTL for bacterial wilt resistance in Italian ryegrass (Lolium multiflorum Lam.)

Bacterial wilt caused by Xanthomonas translucens pv. graminis (Xtg) is a major disease of economically important forage crops such as ryegrasses and fescues. Targeted breeding based on seedling inoculation has resulted in cultivars with considerable levels of resistance. However, the mechanisms of inheritance of resistance are poorly understood and further breeding progress is difficult to obtain. This study aimed to assess the relevance of the seedling screening in the glasshouse for adult plant resistance in the field and to investigate genetic control of resistance to bacterial wilt in Italian ryegrass (Lolium multiflorum Lam.). A mapping population consisting of 306 F₁ individuals was established and resistance to bacterial wilt was assessed in glasshouse and field experiments. Highly correlated data (r = 0.67–0.77, P < 0.01) between trial locations demonstrated the suitability of glasshouse screens for phenotypic selection. Analysis of quantitative trait loci (QTL) based on a high density genetic linkage map consisting of 368 amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers revealed a single major QTL on linkage group (LG) 4 explaining 67% of the total phenotypic variance (Vp). In addition, a minor QTL was observed on LG 5. Field experiments confirmed the major QTL on LG 4 to explain 43% (in 2004) to 84% (in 2005) of Vp and also revealed additional minor QTLs on LG 1, LG 4 and LG 6. The identified QTLs and the closely linked markers represent important targets for marker-assisted selection of Italian ryegrass.     

A PCR-based marker tightly linked to the nematode resistance gene,Mi, in tomato

A PCR-based codominant marker has been developed which is tightly linked to Mi, a dominant genetic locus in tomato that confers resistance to several species of root-knot nematode. DNA from tomato lines differing in nematode resistance was screened for random amplified polymorphic DNA markers linked to Mi using decamer primers. Several markers were identified. One amplified product, REX-1, obtained using a pair of decamer primers, was present as a dominant marker in all nematode-resistant tomato lines tested. REX-1 was cloned and the DNA sequences of its ends were determined and used to develop 20-mer primers. PCR amplification with the 20-mer primers produced a single amplified band in both susceptible and resistant tomato lines. The amplified bands from susceptible and resistant lines were distinguishable after cleavage with the restriction enzyme Taq I. The linkage of REX-1 to Mi was verified in an F₂ population. This marker is more tightly linked to Mi than is Aps-1, the currently-used isozyme marker, and allows screening of germplasm where the linkage between Mi and Aps-1 has been lost. Homozygous and heterozygous individuals can be distinguished and the procedure can be used for rapid, routine screening. The strategy used to obtain REX-1 is applicable to obtaining tightly-linked markers to other genetic loci. Such markers would allow rapid, concurrent screening for the segregation of several loci of interest.     

The degradation profile of extrachromosomal circular DNA during cisplatin-induced apoptosis is consistent with preferential cleavage at matrix attachment regions

Extrachromosomal circular DNA molecules are prevalent in cancer cells and harbor amplified genes, such as oncogenes and drug resistance genes, that can provide a selective growth advantage to cancer cells. These circular DNA structures include double minute chromosomes (dmin), which can be detected with light microscopy following Giemsa staining, and submicroscopic circular DNA structures referred to as episomes. In this study, we investigated the fate of dmin and episomes in multidrug-resistant human epidermoid KB-V1 cells undergoing cisplatin-induced apoptosis – a mode of cell death initially characterized by the fragmentation of chromosomal DNA, while the nuclear membrane remains intact. The circular DNA structures carry amplified copies of the multidrug resistance gene (MDR1). During cisplatin-induced apoptotic cell death, episomes and dmin, as well as native chromosomes, were degraded into high molecular weight DNA fragments of approximately 50 kb in length. DNA fragments in this size range appear to result from the preferential cleavage of matrix-associated regions in chromatin with the subsequent release of 20–30 nm loop domains of chromatin from the nuclear scaffold. Scanning electron microscopy studies were performed and confirmed the presence of 30 nm filaments in a higher-order DNA packing of MDR1-containing dmin and episomes. These combined data provide strong evidence that the higher-order DNA packing of episomes, as well as dmin, is similar to that of native chromosomes and underscore the potential for extrachromosomal DNA amplicons to study the structural and functional organization of chromatin. We discuss the implications of extrachromosomal DNA matrix associated regions competing with native chromosomal DNA for binding to the nuclear matrix in tumor cells. Received: 18 August 1998; in revised form: 6 December 1998 / Accepted: 21 January 1999     

Elimination of extrachromosomal c-myc genes by hydroxyurea induces apoptosis

Hydroxyurea (HU) increases extrachromosomal DNA elimination in tumor cell lines. The c-myc oncogene is one of the many relevant amplified genes contained within the extrachromosomal DNA compartment. Spontaneous loss of amplified copies of c-myc induces terminal differentiation and apoptosis in the human HL-60 leukemia cell lines. In the present study, we evaluate HU's ability to induce apoptosis by eliminating extrachromosomally located c-myc oncogene in human tumor cell lines. The consequences of eliminating extrachromosomal DNA by HU were explored in two different cell lines using the TdT assay and acridine orange/ethidium bromide labeling. COLO 320 clone 3 and COLO 320 clone 21 cell lines contain the same number of amplified copies of c-myc oncogene, but located respectively on extrachromosomal DNA, and intrachromosomally in homogeneously staining regions. HU induced apoptosis in the COLO 320 clone 3 cell line by a time and concentration dependent mechanism but could not induce apoptosis in the COLO 320 clone 21 cell line. These results suggested that HU-induced apoptosis in COLO 320 cell lines depends on elimination of extrachromosomal amplified copies of the c-myc oncogene. The ability of HU to eliminate extrachromosomally amplified copies of the c-myc oncogene and to induce apoptosis should be considered when targeting malignancies with amplification of the c-myc oncogene in an extrachromosomal site.     

Extrachromosomal elements in the lower eukaryote Leishmania

Extrachromosomal DNA elements have been identified in wild-type populations of the parasitic protozoan Leishmania. Elements from L. major and L. tropica were detected using orthogonal-field-alternation-gel electrophoresis. They are nonhomologous, supercoiled circular DNA molecules derived from different chromosomes in the Leishmania genome. Electron microscopy revealed that the elements have very similar physical properties; both are 80-kilobase supercoiled DNA molecules that contain large inverted repeat structures. The extrachromosomal DNAs are amplified in the Leishmania populations and show a fluctuation in copy number, from undetectable to around 20 copies per cell. After exposure of the L. tropica population to the drug methotrexate (MTX), a second amplified DNA was observed that is homologous to the extrachromosomal DNA found in L. major. Furthermore, wild-type Leishmania populations containing extrachromosomal DNA adapt more readily to MTX selection than populations with no amplified DNA. From these observations, there appears to be a relationship between the presence of extrachromosomal elements in wild-type Leishmania and the genesis and maintenance of MTX resistance in these organisms.     

Mapping quantitative trait loci (QTLs) for resistance to Gibberella zeae infection in maize

The basic prerequisite for an efficient breeding program to improve levels of resistance to pathogens in plants is the identification of genes controlling the resistance character. If the response to pathogens is under the control of a multilocus system, the utilization of molecular markers becomes essential. Stalk and ear rot caused by Gibberella zeae is a widespread disease of corn: resistance to G. zeae is quantitatively inherited. Our experimental approach to understanding the genetic basis of resistance to Gibberella is to estimate the genetic linkage between available molecular markers and the character, measured as the amount of diseased tissue 40 days after inoculation of a suspension of Fusarium graminearum, the conidial form of G. zeae, into the first stalk internode. Sensitive and resistant parental inbreds were crossed to obtain F₁ and F₂ populations: the analysis of the segregation of 95 RFLP (restriction fragment length polymorphism) clones and 10 RAPD (random amplified polymorphic DNA) markers was performed on a population of 150 F₂ individuals. Analysis of resistance was performed on the F₃ families obtained by selfing the F₂ plants. Quantitative trait loci (QTL) detection was based either on analysis of regression coefficients between family mean value and allele values in the F₂ population, or by means of interval mapping, using MAPMAKER-QTL. A linkage map of maize was obtained, in which four to five genomic regions are shown to carry factors involved in the resistance to G. zeae.     

Single-stranded DNA transforms plant protoplasts

Summary The transfer of the Agrobacterium T-DNA to plant cells involves the induction of the Ti plasmid virulence genes. This induction results in the generation of linear single-stranded (ss) copies of the T-DNA inside Agrobacterium and such molecules might be directly transferred to the plant cell. A central requirement of this ss transfer model is that the plant cell must generate a second strand and integrate the resulting double-stranded (ds) molecule into its genome. Here we report that incubating plant protoplasts with ss or ds DNA under conditions favouring DNA uptake results in transformation. The frequencies of transformation are similar and analysis of ss transformants suggests that the introduced DNA becomes double stranded and integrated. Analysis of transient expression from introduced ss DNA suggests that generation of the second strand is rapid and extrachromosomal.     

A detailed linkage map around an apple scab resistance gene demonstrates that two disease resistance classes both carry the V f gene

A detailed genetic map has been constructed in apple (Malus x domestica Borkh.) in the region of the v _f gene. This gene confers resistance to the apple scab fungus Venturia inaequalis (Cooke) Wint. Linkage data on four RAPD (random amplified polymorphic DNA) markers and the isoenzyme marker PGM-1, previously reported to be linked to the v _f gene, are integrated using two populations segregating for resistance to apple scab. Two new RAPD markers linked to v _f (identified by bulked segregant analysis) and a third marker previously reported as being present in several cultivars containing v _f are also placed on the map. The map around v _f now contains eight genetic markers spread over approximately 28 cM, with markers on both sides of the resistance gene. The study indicates that RAPD markers in the region of crab apple DNA introgressed with resistance are often transportable between apple clones carrying resistance from the same source. Analysis of co-segregation of the resistance classes 3A (weakly resistant) and 3B (weakly susceptible) with the linked set of genetic markers demonstrates that progeny of both classes carry the resistance gene.This work was supported in part by grants from the New Zealand Foundation for Research Science and Technology (FoRST) Programme 94-HRT-07-366 and ENZA New Zealand (International)     

Allele-specific CAPS markers based on point mutations in resistance alleles at the pvr1 locus encoding eIF4E in Capsicum

Marker-assisted selection has been widely implemented in crop breeding and can be especially useful in cases where the traits of interest show recessive or polygenic inheritance and/or are difficult or impossible to select directly. Most indirect selection is based on DNA polymorphism linked to the target trait, resulting in error when the polymorphism recombines away from the mutation responsible for the trait and/or when the linkage between the mutation and the polymorphism is not conserved in all relevant genetic backgrounds. In this paper, we report the generation and use of molecular markers that define loci for selection using cleaved amplified polymorphic sequences (CAPS). These CAPS markers are based on nucleotide polymorphisms in the resistance gene that are perfectly correlated with disease resistance, the trait of interest. As a consequence, the possibility that the marker will not be linked to the trait in all backgrounds or that the marker will recombine away from the trait is eliminated. We have generated CAPS markers for three recessive viral resistance alleles used widely in pepper breeding, pvr1, pvr1 ¹, and pvr1 ². These markers are based on single nucleotide polymorphisms (SNPs) within the coding region of the pvr1 locus encoding an eIF4E homolog on chromosome 3. These three markers define a system of indirect selection for potyvirus resistance in Capsicum based on genomic sequence. We demonstrate the utility of this marker system using commercially significant germplasm representing two Capsicum species. Application of these markers to Capsicum improvement is discussed.     

In VivoAddition of Telomeric Repeats to Foreign DNA Generates Extrachromosomal DNAs in the Taxol-Producing FungusPestalotiopsis microspora

Transformation of the taxol-producing filamentous fungusPestalotiopsis microsporawith a plasmid containing the bacterial hygromycin resistance gene fused toAspergillusregulatory sequences resulted in thein vivoformation of extrachromosomal DNAs with telomeric repeats in the majority of transformants. Repeats of the telomeric sequence 5′-TTAGGG-3′ were appended to nontelomeric transforming DNA termini. No fungal sequences other than telomeric repeats were detected in extrachromosomal DNAs. Transformants contained three to six different sizes or conformational forms of extrachromosomal DNAs. The DNAs showed no change in size or internal structure during 6 months of growth with selection, but were lost after 20 days of growth without selection. Transformation of wild-typeP. microsporawith a PCR-amplified extrachromosomal DNA having terminal telomeric repeats produced up to 50-fold more transformants than the original transformation vector. The addition of telomeric repeats to foreign DNA is unusual among fungi and may have important adaptive or developmental implications.     

Synthesis of ribosomal DNA in conjugating Tetrahymena.

In the ciliate protozoan Tetrahymena thermophila, a single integrated gene coding for ribosomal RNA in the micronucleus is amplified during the sexual cycle to yield many copies of extrachromosomal palindromic rDNA in the macronucleus. Hybridization of newly synthesized DNA with rRNA has shown that extensive rDNA synthesis takes place early in the sexual cycle of Tetrahymena. The number of genes synthesized during this period is sufficient to account for gene amplification. A later period of rDNA synthesis occurs when new macronuclear anlagen are beginning to develop. This synthesis may represent preferential polyploidization of already amplified rDNA.     

29个香蕉基因型遗传多样性的SRAP分析

采用SRAP分子标记技术对29个香蕉品种(系)的多样性进行研究,结果显示,64对SRAP引物中筛选出25个多态性较高的引物组合,共扩增出324条条带;UPGAM聚类图显示所有供试的29个香蕉品种(系)可分为2个类群且与基因型相一致;实验结果与形态、农艺性状标记分类基本一致。研究表明,SRAP技术可有效运用于香蕉基因型的遗传和育种研究。     

Isolation and characterization of RAPD-based markers linked to the beet cyst nematode resistance locus (Hs1 pat-1) on chromosome 1 of B. patellaris

A beet cyst nematode (BCN)-resistant telosomic addition of B. patellaris chromosome 1 in B. vulgaris was used to isolate 6 RAPD markers linked to the BCN resistance locus Hs1 ^pat-1. Southern analysis showed that the analyzed RAPD products contain either low-, middle or high-repetitive DNA. The relative positions of the random amplified polymorphic DNA (RAPD) markers and of the restriction fragment length polymorphism (RFLP) loci corresponding to the low-repetitive RAPD products were determined by deletion mapping using a panel of seven nematode-resistant B. patellaris chromosome-1 fragment additions. One RAPD marker, OPB11₈₀₀, was found to be present in two copies on the long arm telosome of B. patellaris chromosome 1. These copies are closely linked to the BCN resistance gene and flank the gene on both sides. On the basis of the nucleotide sequence of OPB11₈₀₀, sequence-tagged site (STS) primers were developed that amplify specific fragments derived from the two OPB11₈₀₀ loci. These STS markers can be used in the map-based cloning of the BCN gene, as they define start and finishing points of a chromosomal walk towards the Hs1 ^pat-1 locus. Two copies of the middle-repetitive OPX2₁₁₀₀ marker were mapped in the same interval of the deletion mapping panel as the resistance gene locus and thereby belong to the nearest markers as yet found for the BCN gene in B. patellaris.     

Use of asymmetric somatic hybridization for transfer of the bacterial blight resistance trait from Oryza meyeriana L. to O. sativa L. ssp. japonica

Bacterial blight is one of the major diseases affecting rice productivity. To improve the resistance of cultivated rice to bacterial blight, we introduced a bacterial blight resistance trait from Oryza meyeriana, a wild rice species, into an elite japonica rice cultivar (Dalixiang) using asymmetric somatic hybridization. One hundred and thirty-two independent lines were regenerated. The hybrid plants possessed several morphological features of the donor species, O. meyeriana. Random amplified polymorphic DNA analysis revealed that hybrid plants exhibited banding patterns derived from their parental genotypes. For the majority of the hybrids, resistance to bacterial blight pathogens was intermediate to that observed for O. meyeriana and O. sativa (cv. Dalixiang). Four of the hybrid lines exhibited a high bacterial blight resistance, but it was less than that observed for O. meyeriana. These results demonstrate that O. meyeriana can be used as a good genetic source for improving bacterial blight resistance in commercial rice cultivars through asymmetric somatic hybridization.Abbreviations 2,4-D: 2,4-Dichlorophenoxyacetic acid - IOA: Iodoacetamide - NAA: -Naphthaleneacetic acid - PEG: Polyethylene glycol Communicated by P. Lakshmanan     

Genetic variability of resistance to Eimeria acervulina and E. tenella in chickens

Summary The genetic variability of 18 sire families of the Athens-Canadian randombred population infected with coccidiosis was assessed by examining the response variables of weight gain, packed red blood cell volume, mortality and coccidial lesions. A significant gain and PCV depression and high lesion scores for Eimeria tenella and E. acervulina were produced in the infected group compared to the noninfected group. Significant variation among the sire families was observed for all of the response variables except E. acervulina lesions and a significant sex x sire interaction was observed for weight gain. The heritability (h²) estimates for the response variables revealed that resistance to coccidiosis in chickens is moderately heritable. The h² estimates for gain and PCV increased with the coccidial infections indicating that maximum progress in selecting for resistance should be made when the population was exposed to coccidial infection. Gain was positively correlated to the other measures of resistance and thus selecting for coccidial resistance should not reduce growth rates. PCV was similarly correlated but had higher positive correlation with E. tenella lesion. Percent mortality which is the selection parameter in most coccidial selection programs was correlated with resistance to coccidiosis. The phenotypic and genotypic correlations demonstrated that chickens susceptible to E. tenella were also susceptible to E. acervulina. Total lesion scores were moderately to highly correlated with the other variables and would be a suitable variable to use in coccidiosis experimentation including a genetic selection program for resistance. This study shows that progress could be made in selecting for resistance to coccidiosis in chickens using one or a combination of these response variables.     

QTL analysis for aphid resistance and growth traits in apple

The rosy apple aphid (Dysaphis plantaginea), the leaf-curling aphid (Dysaphis cf. devecta) and the green apple aphid (Aphis pomi) are widespread pest insects that reduce growth of leaves, fruits and shoots in apple (Malus × domestica). Aphid control in apple orchards is generally achieved by insecticides, but alternative management options like growing resistant cultivars are needed for a more sustainable integrated pest management (IPM). A linkage map available for a segregating F₁-cross of the apple cultivars ‘Fiesta’ and ‘Discovery’ was used to investigate the genetic basis of resistance to aphids. Aphid infestation and plant growth characteristics were repeatedly assessed for the same 160 apple genotypes in three different environments and 2 consecutive years. We identified amplified fragment length polymorphism (AFLP) markers linked to quantitative trait loci (QTLs) for resistance to D. plantaginea (‘Fiesta’ linkage group 17, locus 57.7, marker E33M35–0269; heritability: 28.3%), and to D. cf. devecta (‘Fiesta’ linkage group 7, locus 4.5, marker E32M39–0195; heritability: 50.2%). Interactions between aphid species, differences in climatic conditions and the spatial distribution of aphid infestation were identified as possible factors impeding the detection of QTLs. A pedigree analysis of simple sequence repeat (SSR) marker alleles closely associated with the QTL markers revealed the presence of the alleles in other apple cultivars with reported aphid resistance (‘Wagener’, ‘Cox’s Orange Pippin’), highlighting the genetic basis and also the potential for gene pyramiding of aphid resistance in apple. Finally, significant QTLs for shoot length and stem diameter were identified, while there was no relationship between aphid resistance and plant trait QTLs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Co-amplification of double minute chromosomes, multiple drug resistance, and cell surface P-glycoprotein in DNA-mediated transformants of mouse cells.

A genetic system comprised of mammalian cell mutants which demonstrate concomitant resistance to a number of unrelated drugs has been described previously. The resistance is due to reduced cell membrane permeability and is correlated with the presence of large amounts of a plasma membrane glycoprotein termed P-glycoprotein. This system could represent a model for multiple drug resistance which develops in cancer patients treated with chemotherapeutic drugs. We demonstrate here that the multiple drug resistance phenotype can be transferred to mouse cells with DNA from a drug-resistant mutant and then amplified quantitatively by culture in media containing increasing concentrations of drug. The amount of P-glycoprotein was correlated directly with the degree of drug resistance in the transformants and amplified transformants. In addition, the drug resistance and expression of P-glycoprotein of the transformants were unstable and associated quantitatively with the number of double minute chromosomes. We suggest that the gene for multiple drug resistance and P-glycoprotein is contained in these extrachromosomal particles and is amplified by increases in double minute chromosome number. The potential use of this system for manipulation of mammalian genes in general is discussed.