Restriction Fragment Length Polymorphisms in the Mushrooms Agaricus brunnescens and Agaricus bitorquis

[This corrects the article on p. 817 in vol. 53.].     

Restriction fragment length polymorphisms in the mushrooms Agaricus brunnescens and Agaricus bitorquis

Two Agaricus species, A. brunnescens (a commercial mushroom) and A. bitorquis (a wild, edible species), were examined for restriction fragment length polymorphisms. EcoRI-digested nuclear DNA from isolates of both species were cloned in plasmid vector pUC18. Ten random recombinant clones were used in Southern DNA-DNA hybridizations to probe EcoRI-digested DNA from 11 A. brunnescens isolates (7 commercial, 2 wild type, and 2 homokaryotic) and 7 A. bitorquis isolates. Most cloned fragments were polymorphic in both species. There were fewer different genotypes than expected, however, in the sample of commercial A. brunnescens strains. DNA from homokaryotic strains showed fewer bands in most hybridizations than DNA from heterokaryotic strains. All A. bitorquis isolates could be distinguished from each other as well as from every A. brunnescens strain. Putative homokaryons were detected by the loss of polymorphic bands among protoplast regenerates from one commercial strain and two strains collected in the wild.     

Restriction Fragment Length Polymorphisms in the Mushrooms Agaricus brunnescens and Agaricus bitorquis

Two Agaricus species, A. brunnescens (a commercial mushroom) and A. bitorquis (a wild, edible species), were examined for restriction fragment length polymorphisms. EcoRI-digested nuclear DNA from isolates of both species were cloned in plasmid vector pUC18. Ten random recombinant clones were used in Southern DNA-DNA hybridizations to probe EcoRI-digested DNA from 11 A. brunnescens isolates (7 commercial, 2 wild type, and 2 homokaryotic) and 7 A. bitorquis isolates. Most cloned fragments were polymorphic in both species. There were fewer different genotypes than expected, however, in the sample of commercial A. brunnescens strains. DNA from homokaryotic strains showed fewer bands in most hybridizations than DNA from heterokaryotic strains. All A. bitorquis isolates could be distinguished from each other as well as from every A. brunnescens strain. Putative homokaryons were detected by the loss of polymorphic bands among protoplast regenerates from one commercial strain and two strains collected in the wild.     

Ti Plasmid Homologous Sequences Present in Tissues from Agrobacterium Plasmid-transformed Petunia Protoplasts

Suspension cell protoplasts of albino Petunia hybrida have beentransformed by isolated Agrobacterium tumefaciens Ti plasmid.Uptake of octopine Ti plasmid (pTiACH5) into protoplasts wasstimulated by poly-L-ornithine and polyethylene glycol (PEG).The frequency and efficiency of transformation of protoplaststo phytohormone autotrophy was compared using the two uptakeagents with various concentrations of plasmid. Transformationwas most efficient with PEG-mediated uptake, 5 µg of Tiplasmid per 10⁶ protoplasts giving a frequency of 6?10^–5.Octopine was not synthesised in any of the transformants afterthe second subculture on hormone-free medium. DNA-DNA hybridisationshowed the presence of DNA homologous to the T-DNA region ofpTiACH5 in all clones analysed. (Received November 9, 1981; Accepted January 29, 1982)     

Maize Mitochondrial Plasmid S-1 Sequences Share Homology with Chloroplast Gene psbA

The linear, 6397-base pair (bp), mitochondrial S-1 DNA molecule from maize contains a 420-bp segment that is homologous with the chloroplast gene (psbA) that codes for the quinone binding protein of photosystem II. This is the first report of a chloroplast sequence in a naturally occurring viral-like or plasmid DNA. The complete sequence of the S-1 chloroplast segment has been compared with homologous regions of six different chloroplast genes. The S-1 segment has diverged from the other genes both by length mutation and base substitution. Several of the length mutations are exact adjacent tandem duplications of 4 and 5 bp similar to "footprints" left after excision of transposable elements in maize nuclear DNA.     

Occurrence of Verticillium fungicola var. fungicola on Agaricus bitorquis Mushroom Crops in Spain

Diseased fruit bodies of Agaricus bitorquis, with similar symptoms to those caused by dry bubble on Agaricus bisporus, were observed in some Spanish crops during summer 1999. Isolates of Verticillium fungicola from A. bitorquis and A. bisporus were submitted to different temperatures and to prochloraz–Mn sensitivity tests. All the isolates collected from A. bitorquis and A. bisporus were identified as V. fungicola var. fungicola. Artificial infections of A. bisporus and A. bitorquis with V. fungicola var. fungicola are also described in the present study. The appearance of natural infections of V. fungicola var. fungicola in A. bitorquis crops could well be due to the growing temperatures used in Spain, which are considerably below those used in other countries.     

植物线粒体DNA质粒研究进展

本文列出了已发现的高等植物中的线粒体DNA质粒,按分子形状分为线粒体环状DNA质粒和线粒体线状DNA质粒,环状线粒体DNA质粒的特征是分子较小, 序列中有正向/反向重复序列,ORF一般较小。线状线粒体DNA质粒的特征是分子较大,末端有重复序列,5'端与蛋白质共价结合,有较长的ORF。还分别介绍了它们的复制机制、转录和起源。质粒间及质粒与核基因组、线粒体基因组、叶绿体基因组的同源性也作了介绍。最后,综述了植物线粒体DNA质粒与植物的细胞质雄性不育（CMS）之间的关系。     

植物线粒体DNA质料研究进展

本文列出了已发现的高等植物中的线粒体ＤＮＡ质粒,按分子形状分为线粒体环状ＤＮＡ质粒和线粒体线状ＤＮＡ质粒,环状线粒体ＤＮＡ质粒的特征是分子较小,序列中有正向／反向重复序列,ＯＲＦ一般较小。线状线粒体ＤＮＡ质粒的特征是分子较大,末端有重复序列,５’端与蛋白质共价结合,有较长的ＯＲＦ。还分别介绍了它们的复制机制、转录和起源。质粒间及质粒及核基因组、线粒体基因组、叶绿体基因组的同源性也作了介绍。最后,综     

Evaluation of the Extent of Homologous Chloroplast DNA Sequences in the Mitochondrial Genome of Cowpea (Vigna unguiculata L.)

Southern blot hybridization techniques were used to estimate the extent of chloroplast DNA sequences present in the mitochondrial genome of cowpea (Vigna unguiculata L.) The entire mitochondrial chromosome was homogeneously labeled and used to probe blotted DNA fragments obtained by extensive restriction of the tobacco chloroplast genome. The strongest cross-homologies were obtained with fragments derived from the inverted repeat and the atpBE cluster regions, although most of the clones tested (spanning 85% of the tobacco plastid genome) hybridized to mitochondrial DNA. Homologous chloroplast DNA restriction fragments represent a total of 30 to 68 kilobase pairs, depending upon the presence or absence of tRNA-encoding fragments. Plastid genes showing homology with mitochondrial DNA include those encoding ribosomal proteins, RNA polymerase, subunits of photosynthetic complexes, and the two major rRNAs.     

Loss of UCP2 Attenuates Mitochondrial Dysfunction without Altering ROS Production and Uncoupling Activity

Although mitochondrial dysfunction is often accompanied by excessive reactive oxygen species (ROS) production, we previously showed that an increase in random somatic mtDNA mutations does not result in increased oxidative stress. Normal levels of ROS and oxidative stress could also be a result of an active compensatory mechanism such as a mild increase in proton leak. Uncoupling protein 2 (UCP2) was proposed to play such a role in many physiological situations. However, we show that upregulation of UCP2 in mtDNA mutator mice is not associated with altered proton leak kinetics or ROS production, challenging the current view on the role of UCP2 in energy metabolism. Instead, our results argue that high UCP2 levels allow better utilization of fatty acid oxidation resulting in a beneficial effect on mitochondrial function in heart, postponing systemic lactic acidosis and resulting in longer lifespan in these mice. This study proposes a novel mechanism for an adaptive response to mitochondrial cardiomyopathy that links changes in metabolism to amelioration of respiratory chain deficiency and longer lifespan.     

Mitochondrial DNA variation in natural populations of the mushroom Agaricus bisporus

We investigated the patterns of mitochondrial DNA variation in the global population of the commercial mushroom Agaricus bisporus . Through the analysis of RFLP's among 441 isolates from nine countries in North America and Eurasia, we found a total of 140 mtDNA haplotypes. Based on population genetic analysis, there are four genetically distinct natural populations in this species, found in coastal California, desert California, France and Alberta (Canada). While 134 of the 140 mtDNA haplotypes were unique to single geographical regions, two mtDNA haplotypes, mt001 and mt002, were found in almost every population surveyed. These two mtDNA haplotypes also predominate among cultivars used throughout the world for at least the last two decades. These two mtDNA haplotypes are more similar to the cosmopolitan groups of mtDNA haplotypes than to the indigeneous clusters of mtDNA haplotypes from the two Californian regions.     

Uniparental Mitochondrial Transmission in the Cultivated Button Mushroom, Agaricus bisporus

A uniparental mitochondrial (mt) transmission pattern has been previously observed in laboratory matings of the cultivated mushroom Agaricus bisporus on petri dishes. In this study, four sets of specific matings were further examined by taking mycelial plugs from the confluent zone of mated homokaryons and inoculating these plugs into rye grain for laboratory fruiting and for fruiting under industrial conditions. Examination of the mt genotype of each individual fruit body for mt-specific restriction fragment length polymorphisms further confirmed that the mt genome was inherited uniparentally. The vegetative radial growth and the fruiting activity of two pairs of intraspecific heterokaryons, each pair carrying the same combination of nuclear genomes but different mt genotypes, were compared. Our results suggested that the mt genotype did not appreciably affect radial growth or fruiting activity. The failure to recover both heterokaryons, each carrying either parental mt genotype in any given cross, therefore clearly indicated that in matings of A. bisporus, the mt genome from one of the parental homokaryons is either selectively excluded in the newly formed heterokaryon or selectively eliminated in the immediate heterokaryotic mitotic progeny of the newly formed heterokaryon.     

Conservation of Plasmid DNA Sequences in Coronatine-Producing Pathovars of Pseudomonas syringae

In Pseudomonas syringae pv. tomato PT23.2, plasmid pPT23A (101 kb) is involved in synthesis of the phytotoxin coronatine (C. L. Bender, D. K. Malvick, and R. E. Mitchell, J. Bacteriol. 171:807-812, 1989). The physical characterization of mutations that abolished coronatine production indicated that at least 30 kb of pPT23A DNA are required for toxin synthesis. In the present study, ³²P-labeled DNA fragments from the 30-kb region of pPT23A hybridized to plasmid DNAs from several coronatine-producing pathovars of P. syringae under conditions of high stringency. These experiments indicated that this region of pPT23A was strongly conserved in large plasmids (90 to 105 kb) that reside in P. syringae pv. atropurpurea, glycinea, and morsprunorum. The functional significance of the observed homology was demonstrated in marker-exchange experiments in which Tn5-inactivated sequences from the 30-kb region of pPT23A were used to mutate coronatine synthesis genes in the three heterologous pathovars. Physical characterization of the Tn5 insertions generated by marker exchange indicated that genes controlling coronatine synthesis in P. syringae pv. atropurpurea 1304, glycinea 4180, and morsprunorum 567 and 3714 were located on the large indigenous plasmids where homology was originally detected. Therefore, coronatine biosynthesis genes are strongly conserved in the plasmid DNAs of four producing pathovars, despite their disparate origins (California, Japan, New Zealand, Great Britain, and Italy).     

Silencing Homologous RNA Recombination Hot Spots with GC-Rich Sequences in Brome Mosaic Virus

It has been observed that AU-rich sequences form homologous recombination hot spots in brome mosaic virus (BMV), a tripartite positive-stranded RNA virus of plants (P. D. Nagy and J. J. Bujarski, J. Virol. 71:3799–3810, 1997). To study the effect of GC-rich sequences on the recombination hot spots, we inserted 30-nucleotide-long GC-rich sequences downstream of AU-rich homologous recombination hot spot regions in parental BMV RNAs (RNA2 and RNA3). Although these insertions doubled the length of sequence identity in RNA2 and RNA3, the incidence of homologous RNA2 and RNA3 recombination was reduced markedly. Four different, both highly structured and nonstructured downstream GC-rich sequences had a similar “homologous recombination silencing” effect on the nearby hot spots. The GC-rich sequence-mediated recombination silencing mapped to RNA2, as it was observed when the GC-rich sequence was inserted at downstream locations in both RNA2 and RNA3 or only in the RNA2 component. On the contrary, when the downstream GC-rich sequence was present only in the RNA3 component, it increased the incidence of homologous recombination. In addition, upstream insertions of similar GC-rich sequences increased the incidence of homologous recombination within downstream hot spot regions. Overall, this study reveals the complex nature of homologous recombination in BMV, where sequences flanking the common hot spot regions affect recombination frequency. A replicase-driven template-switching model is presented to explain recombination silencing by GC-rich sequences.     

西藏小型猪的线粒体DNA控制区分析

目的研究西藏小型猪的遗传标记以及与其他国内地方猪的亲缘关系。方法扩增102头西藏小型猪以及16头巴马小型猪、17头贵州香猪的线粒体DNA控制区,测序并与国内其他猪进行比较。结果西藏小型猪线粒体DNAD-loop区分三个区域。串联重复序列区处于中间位置,包含有15～29个10 bp的重复片段,分为A、B两种类型。D-loop 3′端340 bp,与国内其他猪的序列相同比较保守;5′端704 bp,共有22个变异位点。由22个变异位点中归纳出25个单倍型,其中有两种主要的单倍型,分别占34.4%和36.6%。根据三个转换位点：305、500、691,将西藏小型猪分成了两组,几乎与串联重复序列所分的A、B两组类型相对应。与西藏小型猪相比,巴马小型猪和贵州香猪D-loop 5′端变异位点较少,分别只有4种和2种单倍型,串联重复区也只有一个类型。结论西藏小型猪可能有两个母系祖先并且与我国西南地区的品种猪有较近的亲缘关系;不同的串联重复片段类型和5′端的变异位点可以联合组建西藏小型猪的遗传标记。     

F-Like Plasmid Sequences in Enteric Bacteria of Diverse Origin, with Implication of Horizontal Transfer and Plasmid Host Range

Seventy-eight bacterial isolates from human, animal, and plant hosts, representing eight species of the family Enterobacteriaceae, were screened for F-like plasmid sequences. Of the examined human Escherichia coli strains, 28% harbored one or two of the three F-like, RepFI replication regions, while 35% of the examined animal and all phytopathogenic strains harbored RepFIA-specific sequences. Comparative analysis of Salmonella, Shigella, Erwinia, and E. coli plasmid RepFI sequences showed 100% or very high homology, indicating frequent and recent interspecies gene transfer. The high incidence of RepFIA sequences in enteric bacterial species, including Klebsiella and Erwinia, showed that F-like plasmids are successful in avoiding natural barriers to establishment of horizontally transferred DNA and that in the natural environment conjugal transfer is efficient in diverse ecological niches. Received: 26 March 2001 / Accepted 12 July 2001     

Mitochondrial Sequences as Indicators of Genetic Classification in Bush Babies

Systematic relationships among the African bush babies are not well understood. Various generic designations are currently in use. Some authors refer all species to a single genus (Galago), while others recognize 4 genera. Phylogenetic reconstructions based on morphology, karyology, allozymes and vocal repertoires have generated inconsistent hypotheses of relationship. We analyzed partial sequences of three mitochondrial genes (270 bp from cytochrome b, 387 bp from 12S rRNA, and 241 bp from 16S rRNA, total 898 bp) to resolve some uncertainties. We sampled taxa from each of three genera: Galagoides alleni, G. demidoff and G. zanzibaricus; Galago senegalensis, G. gallarum and G. moholi; and Otolemur crassicaudatus and O. garnettii. Outgroup taxa were Asian lorises: Nycticebus coucang and Loris tardigradus. We analyzed sequences separately and in combination, and modeled phylogenies using maximum parsimony, weighted parsimony, neighbor-joining and maximum-likelihood. We obtained some variation in phylogenetic inference depending on sequence and analytical method, but the results also gave strong phylogenetic signals. The lesser bush babies invariably formed a clade, showing evidence of very recent radiation. The greater bush babies also formed a clade, marked by somewhat greater interspecific genetic distances, which was allied with Galagoides alleni in most instances. Galagoides demidoff and G. zanzibaricus are not closely related, though both diverged early in the history of the group. A genus comprising Galagoides alleni, G. demidoff and G. zanzibaricus is not supported by our data. The most likely alliance for Galagoides alleni is within the genus Otolemur. Of the three partial sequences employed in the study, 16S rRNA gave the most consistent results, while cytochrome b was least informative.     

Sequences Essential for Replication of Plasmid pIJ101 in Streptomyces lividans

A 2.1-kb SacII DNA fragment of the high-copy-number Streptomyces lividans plasmid pIJ101 previously has been shown to include the functions required for maintenance of pIJ101 as an extrachromosomal replicon. This fragment contains the open translational reading frames rep and orf 56 plus an intervening segment believed to be noncoding. Using deletion mutations, we show that the pIJ101 replication origin and other cis -acting sites necessary and sufficient for replication map to a DNA segment that extends 515 bp 5′ to the translational start of Rep and lacks orf56. Plasmids that include this segment are maintained in S. lividans as extrachromosomal replicons when complemented in trans by the rep gene product.     

Conserved Symbiotic Plasmid DNA Sequences in the Multireplicon Pangenomic Structure of Rhizobium etli

Strains of the same bacterial species often show considerable genomic variation. To examine the extent of such variation in Rhizobium etli, the complete genome sequence of R. etli CIAT652 and the partial genomic sequences of six additional R. etli strains having different geographical origins were determined. The sequences were compared with each other and with the previously reported genome sequence of R. etli CFN42. DNA sequences common to all strains constituted the greater part of these genomes and were localized in both the chromosome and large plasmids. About 700 to 1,000 kb of DNA that did not match sequences of the complete genomes of strains CIAT652 and CFN42 was unique to each R. etli strain. These sequences were distributed throughout the chromosome as individual genes or chromosomal islands and in plasmids, and they encoded accessory functions, such as transport of sugars and amino acids, or secondary metabolism; they also included mobile elements and hypothetical genes. Sequences corresponding to symbiotic plasmids showed high levels of nucleotide identity (about 98 to 99%), whereas chromosomal sequences and the sequences with matches to other plasmids showed lower levels of identity (on average, about 90 to 95%). We concluded that R. etli has a pangenomic structure with a core genome composed of both chromosomal and plasmid sequences, including a highly conserved symbiotic plasmid, despite the overall genomic divergence.It is becoming clear that bacterial genomes of strains of the same species vary widely both in size and in gene composition (39). An unexpected degree of genomic diversity has been found by comparing whole genomes (39). For instance, in Escherichia coli strains, differences of up to 1,400 kb account for some strain-specific pathogenic traits (5, 56). The extent of intraspecies genome diversity varies in different bacterial lineages. Some species have a wide range of variation; these species include E. coli (42), Streptococcus agalactiae (53), and Haloquadratum walsbyi (34). Other bacteria display only limited gene content diversity; an example is Ureaplasma urealyticum (1, 54). Tettelin and colleagues have suggested that bacterial species can be characterized by the presence of a pangenome consisting of a core genome containing genes present in all strains and a dispensable genome consisting of partially shared and strain-specific genes (53, 54). This concept is rooted in the earlier ideas of Reanney (43) and Campbell (7) concerning the structure of bacterial populations, and it indicates both that there is a pool of accessory genetic information in bacterial species and that strains of the same or even different species can obtain this information by horizontal transfer mechanisms (7, 43).Genome size and diversity are related to bacterial lifestyle. Small genomes are typical of strict pathogens such as Rickettsia prowazekii (2) and endosymbionts such as Buchnera aphidicola (44a). In contrast, free-living bacteria, such as Pseudomonas syringae and Streptomyces coelicolor, have large genomes (4, 6). The bacteria with the largest genomes are common inhabitants of heterogeneous environments, such as soil, where energy sources are limited but diverse (32). An increase in genome size is attributable mainly to expansion of functions such as secondary metabolism, transport of metabolites, and gene regulation. All these features are common to the nitrogen-fixing symbiotic bacteria of legumes, which are collectively known as rhizobia, and their close relative the plant pathogen Agrobacterium. The genomes of such bacterial species have diverse architectures with circular chromosomes that are different sizes or linear chromosomes, like that in Agrobacterium species, and the organisms contain variable numbers of large plasmids (31, 49). Comparative genomic studies have highlighted the conservation of gene content and order among the chromosomes of some species of rhizobia (22, 23, 25, 40). Furthermore, Guerrero and colleagues (25) observed that most essential genes occur in syntenic arrangements and display a higher level of sequence identity than nonsyntenic genes. In contrast, plasmids, including symbiotic plasmids and symbiotic chromosomal islands (like those in Mesorhizobium loti and Bradyrhizobium japonicum) are poorly conserved in terms of both gene content and gene order (21). It is not clear what evolutionary advantage, if any, is provided by multipartite genomes, but some authors have speculated that such genomes may allow further accumulation of genes independent of the chromosome. Recently, Slater and coworkers (46) proposed a model for the origin of secondary chromosomes. Their idea is based on the notion of intragenomic gene transfers that might occur from primary chromosomes to ancestral plasmids of the repABC type. Observations of conservation of clusters of genes in secondary chromosomes or in large plasmids that retain synteny with respect to the main chromosome support this hypothesis (46).We have been studying Rhizobium etli as a multipartite genome model species (23). This organism is a free-living soil bacterium that is able to form nodules and fix nitrogen in the roots of bean plants. The genome of R. etli is partitioned into several replicons, a circular chromosome, and several large plasmids. In the reference strain R. etli CFN42, the genome is composed of a circular chromosome consisting of about 4,381 kb and 6 large plasmids whose total size is 2,148 kb (23). A 371-kb plasmid, termed pSym or the symbiotic plasmid, contains most of the genes required for symbiosis (21). Previous studies have described the high level of genetic diversity among geographically different R. etli isolates (41). The strains are also variable with respect to the number and size of plasmids. Nevertheless, there has been no direct measurement of diversity at the genomic level, nor have comparative studies of shared and particular genomic features of R. etli strains been reported. Therefore, to assess the degrees of genomic difference and genomic similarity in R. etli, we obtained the complete genomic sequence of an additional R. etli strain and partial genomic sequences of six other R. etli strains isolated worldwide. Our results support the concept of a pangenomic structure at the multireplicon level and show that a highly conserved symbiotic plasmid is present in divergent R. etli isolates.