Analysis of the 2,4-dichlorophenoxyacetic acid-degradative plasmid pEST4011 of Achromobacter xylosoxidans subsp. denitrificans strain EST4002

The 2,4-dichlorophenoxyacetic acid (2,4-D)-degradative bacterium Achromobacter xylosoxidans subsp. denitrificans strain EST4002, isolated in Estonia more than 10years ago, was found to contain the 70kb plasmid pEST4011 that is responsible for the bacterium having had obtained a stable 2,4-D(+) phenotype. The tfd-like genes for 2, 4-D degradation of the strain EST4002 were located on a 10.5kb region of pEST4011, but without functional genes coding for chloromuconate cycloisomerase and chlorodienelactone hydrolase. The latter two genes are probably encoded by homologous, tcb-like genes, located elsewhere on pEST4011. We also present evidence of two copies of insertion element IS1071-like sequences on pEST4011. IS1071 is a class II (Tn3 family) insertion element, associated with different catabolic genes and operons and globally distributed in the recent past. We speculate that this insertion element might have had a role in the formation of plasmid pEST4011. The 28kb plasmid pEST4012 is generated by deletion from pEST4011 when cells of A. xylosoxidans EST4002 are grown in the absence of 2,4-D in growth medium. We propose that this is the result of homologous recombination between the two putative copies of IS1071-like sequences on pEST4011.     

The Mitochondrial Genome of Soybean Reveals Complex Genome Structures and Gene Evolution at Intercellular and Phylogenetic Levels

Determining mitochondrial genomes is important for elucidating vital activities of seed plants. Mitochondrial genomes are specific to each plant species because of their variable size, complex structures and patterns of gene losses and gains during evolution. This complexity has made research on the soybean mitochondrial genome difficult compared with its nuclear and chloroplast genomes. The present study helps to solve a 30-year mystery regarding the most complex mitochondrial genome structure, showing that pairwise rearrangements among the many large repeats may produce an enriched molecular pool of 760 circles in seed plants. The soybean mitochondrial genome harbors 58 genes of known function in addition to 52 predicted open reading frames of unknown function. The genome contains sequences of multiple identifiable origins, including 6.8 kb and 7.1 kb DNA fragments that have been transferred from the nuclear and chloroplast genomes, respectively, and some horizontal DNA transfers. The soybean mitochondrial genome has lost 16 genes, including nine protein-coding genes and seven tRNA genes; however, it has acquired five chloroplast-derived genes during evolution. Four tRNA genes, common among the three genomes, are derived from the chloroplast. Sizeable DNA transfers to the nucleus, with pericentromeric regions as hotspots, are observed, including DNA transfers of 125.0 kb and 151.6 kb identified unambiguously from the soybean mitochondrial and chloroplast genomes, respectively. The soybean nuclear genome has acquired five genes from its mitochondrial genome. These results provide biological insights into the mitochondrial genome of seed plants, and are especially helpful for deciphering vital activities in soybean.     

新疆军垦型细毛羊基因组BAC文库的构建

选用新疆军垦型细毛羊为材料,.构建了含有190 464个克隆的BAC文库,文库平均插入片段大小为133 kb,同时文库92.5%的克隆插入片段大于100 kb,而且有部分克隆甚至大于300 kb,这将满足大多数基因筛选的要求.假定绵羊的基因组含有3x106 kb,根据文库的平均插入片段大小,计算的文库基因组覆盖率为8倍.因此,从文库筛选到目的片段的概率为98.2%.由于该文库中插入的外源片段来自新疆军垦型细毛羊的基因组,这对于研究新疆军垦型细毛羊的特殊性状的基因与其他绵羊品种和物种之间的差异,及构建其全基因组物理图谱和基因图谱地完善是非常有利的.     

Plastid translation and transcription genes in a non-photosynthetic plant: intact, missing and pseudo genes

The non-photosynthetic, parasitic flowering plant Epifagus virginiana has recently been shown to contain a grossly reduced plastid genome that has lost many photosynthetic and chloro-respiratory genes. We have cloned and sequenced a 3.9 kb domain of plastid DNA from Epifagus to investigate the patterns of evolutionary change in such a reduced genome and to determine which genes are still present and likely to be functional. This 3.9 kb domain is colinear with a 35.4 kb region of tobacco chloroplast DNA, differing from it by a minimum of 11 large deletions varying in length from 354 bp to 11.5 kb, as well as by a number of small deletions and insertions. The nine genes retained in Epifagus encode seven tRNAs and two ribosomal proteins and are coextensive and highly conserved in sequence with homologs in photosynthetic plants. This suggests that these genes are functional in Epifagus and, together with evidence that the Epifagus plastid genome is transcribed, implies that plastid gene products play a role in processes other than photosynthesis and gene expression. Genes that are completely absent include not only photosynthetic genes, but surprisingly, genes encoding three subunits of RNA polymerase, four tRNAs and one ribosomal protein. In addition, only pseudogenes are found for two other tRNAs. Despite these defunct tRNA genes, codon and amino acid usage in Epifagus protein genes is normal. We therefore hypothesize that the expression of plastid genes in Epifagus relies on the import of nuclear encoded tRNAs and RNA polymerase from the cytoplasm.     

Tripartite mitochondrial genome of spinach: physical structure, mitochondrial gene mapping, and locations of transposed chloroplast DNA sequences.

A complete physical map of the spinach mitochondrial genome has been established. The entire sequence content of 327 kilobase pairs (kb) is postulated to occur as a single circular molecule. Two directly repeated elements of approximately 6 kb, located on this "master chromosome", are proposed to participate in an intragenomic recombination event that reversibly generates two "subgenomic" circles of 93 kb and 234 kb. The positions of protein and ribosomal RNA-encoding genes, determined by heterologous filter hybridizations, are scattered throughout the genome, with duplicate 26S rRNA genes located partially or entirely within the 6 kb repeat elements. Filter hybridizations between spinach mitochondrial DNA and cloned segments of spinach chloroplast DNA reveal at least twelve dispersed regions of inter-organellar sequence homology.     

Physical and gene mapping of chloroplast DNA from Atriplex triangularis and Cucumis sativa.

A rapid and simple method for constructing restriction maps of large DNAs (100-200 kb) is presented. The utility of this method is illustrated by mapping the Sal I, Sac I, and Hpa I sites of the 152 kb Atriplex triangularis chloroplast genome, and the Sal I and Pvu II sites of the 155 kb Cucumis sativa chloroplast genome. These two chloroplast DNAs are very similar in organization; both feature the near-universal chloroplast DNA inverted repeat sequence of 22-25 kb. The positions of four different genes have been localized on these chloroplast DNAs. In both genomes the 16S and 23S ribosomal RNAs are encoded by duplicate genes situated at one end of the inverted repeat, while genes for the large subunit of ribulose-1,5-bisphosphate carboxylase and a 32 kilodalton photosystem II polypeptide are separated by 55 kb of DNA within the large single copy region. The physical and genetic organization of these DNAs is compared to that of spinach chloroplast DNA.     

Presence of 16S rRNA genes in multiple replicons in Azospirillum brasilense

Rhizosphere and endophytic Azospirillum brasilense isolates recovered from sugarcane plants and the reference strains Sp7 and Cd were analyzed for plasmid occurrence. All of the 26 A. brasilense isolates analyzed harbored from five to eight replicons. Several strains contained small plasmids from 45 to 70 kb, but all of the isolates harbored other plasmids ranging from 100 to 290 kb and two megareplicons of approximately 1700 and over 1800 kb. Most of the strains contained a replicon with a size of either 570 or 630 kb, and another large 910- or 980-kb replicon. The 1700-kb megareplicon and some others around 600 kb strongly hybridized to 16S rDNA genes, while the 910- or 980-kb replicons hybridized only slightly. This suggests that the A. brasilense genome is composed of multiple minichromosomes instead of a single circular chromosome. The apparent genome complexity of A. brasilense deserves to eventually be resolved by complete genome sequencing.     

The linear 20 kb mitochondrial genome of Pandorina morum (Volvocaceae,Chlorophyta)

A physical restriction map of the mitochondrial genome from one clone (TCC 854) of the sexually isolated populations (syngens) of the morphologically uniform species Pandorina morum Bory has been constructed using restriction endonucleases Ava I, Bam HI, Bgl II, Eco RI, Kpn I, and Pst I. The 20 kb linear genome can easily be separated from plastid DNA, nuclear satellite rDNA, and main band (nuclear) DNA on a Hoechst/CsCl buoyant density gradient. The Pandorina mitochondrial DNA shows sufficient similarity to the 16 kb mitochondrial genome of Chlamydomonas reinhardtii to cross-hybridize, and also hybridizes with a probe containing maize mitochondrial 18S rRNA genes. Double digests, self-probing, and Bal31 exonuclease experiments suggest that 1.8 to 3.3 kb of sequence is repeated at each end of the genome as an inverted repeat. Mitochondrial genome sizes of other P. morum syngens were found to range from ca. 20 to ca. 38 kb. The mitochondrial genome should be valuable for taxonomic studies; it can be used for comparative organellar studies; and it should be of interest to compare with that of other plant and animal mitochondrial genomes.     

Complete genomic sequence of nitrogen-fixing symbiotic bacterium Bradyrhizobium japonicum USDA110.

The complete nucleotide sequence of the genome of a symbiotic bacterium Bradyrhizobium japonicum USDA110 was determined. The genome of B. japonicum was a single circular chromosome 9,105,828 bp in length with an average GC content of 64.1%. No plasmid was detected. The chromosome comprises 8317 potential protein-coding genes, one set of rRNA genes and 50 tRNA genes. Fifty-two percent of the potential protein genes showed sequence similarity to genes of known function and 30% to hypothetical genes. The remaining 18% had no apparent similarity to reported genes. Thirty-four percent of the B. japonicum genes showed significant sequence similarity to those of both Mesorhizobium loti and Sinorhizobium meliloti, while 23% were unique to this species. A presumptive symbiosis island 681 kb in length, which includes a 410-kb symbiotic region previously reported by G?ttfert et al., was identified. Six hundred fifty-five putative protein-coding genes were assigned in this region, and the functions of 301 genes, including those related to symbiotic nitrogen fixation and DNA transmission, were deduced. A total of 167 genes for transposases/104 copies of insertion sequences were identified in the genome. It was remarkable that 100 out of 167 transposase genes are located in the presumptive symbiotic island. DNA segments of 4 to 97 kb inserted into tRNA genes were found at 14 locations in the genome, which generates partial duplication of the target tRNA genes. These observations suggest plasticity of the B. japonicum genome, which is probably due to complex genome rearrangements such as horizontal transfer and insertion of various DNA elements, and to homologous recombination.     

Unusual characteristics of Codium fragile chloroplast DNA revealed by physical and gene mapping

Summary A complete physical map of the Codium fragile chloroplast genome was constructed and the locations of a number of chloroplast genes were determined. Several features of this circular genome are unusual. At 89 kb in size, it is the smallest chloroplast genome known. Unlike most chloroplast genomes it lacks any large repeat elements. The 8 kb spacer region between the 16 S and 23 S rRNA genes is the largest such spacer characterized to date in chloroplast DNA. This spacer region is also unusual in that it contains the rps12 gene or at least a portion thereof. Three regions polymorphic for size are present in the Codium chloroplast genome. The psbA and psbC genes map closely to one of these regions, another region is in the spacer between the 16 S and 23 S rRNA genes and the third is very close to or possibly within the 16 S rRNA gene. The gene order in the Codium genome bears no marked resemblance to either the consensus vascular plant order or to that of any green algal or bryophyte genome. Present address: Department of Biology, Texas A&M University, College Station, TX 77843; USA     

Construction of the temperature-sensitive vectors pLUCH80 and pLUCH88 for delivery of Tn917::NotI/SmaI and use of these vectors to derive a circular map of Listeria monocytogenes Scott A, a serotype 4b isolate.

A physical map of Listeria monocytogenes Scott A was generated by the pulsed-field technique of contour-clamped-homogeneous-electric-field (CHEF) electrophoresis. The circular genome of this serotype 4b strain contains 12 AscI fragments (38 to 790 kb), 5 NotI fragments (55 to 1,400 kb), 3 SrfI fragments (110, 1,110, and 2,000 kb), and 2 SfiI fragments (1,320 and 1,920 kb). Summation of individually sized fragments derived by digestion of Scott A genomic DNA with each of these four enzymes provided an average estimated genome length of 3,210 +/- 60 kb. Efforts to assemble the macrorestriction map benefited greatly from the construction and use of pLUCH80 and pLUCH88, temperature-sensitive vectors for delivering transposon Tn917::NotI/SmaI to the chromosome of Scott A. As another component of this study, the positions of four known virulence genes (inlA, mpl, hly, and prf) and three L. monocytogenes-specific sequences (lisM44, lisM51, and lisM52) were localized on the physical map of Scott A by hybridization. Probes prepared from lisM44, lisM51, and the four virulence genes hybridized within a cluster on a 150-kb fragment of the Scott A genome that overlaps part of the NotI-B and AscI-D fragments. The lisM52 probe hybridized with the AscI-F2 (120-kb) fragment of Scott A, which is separated from the NotI-B-AscI-D region by about 300 kb. These results established the first physical and genetic map of a serotype 4b strain of L. monocytogenes and provided further insight on this important food-borne pathogen at the genome level.     

Abundance of plastid DNA insertions in nuclear genomes of rice and Arabidopsis

Pairwise comparison of whole plastid and draft nuclear genomic sequences of Arabidopsis thaliana and Oryza sativa L. ssp. indica shows that rice nuclear genomic sequences contain homologs of plastid DNA covering about 94 kb (83%) of plastid genome and including one or more full-length intact (without mutations resulting in premature stop codons) homologues of 26 known protein-coding (KPC) plastid genes. By contrast, only about 20 kb (16%) of chloroplast DNA, including a single intact plastid-derived KPC gene, is presented in the nucleus of A. thaliana. Sixteen rice plastid genes have at least one nuclear copy without any mutation or with only synonymous substitutions. Nuclear copies for other ten plastid genes contain both synonymous and non-synonymous substitutions. Multiple ESTs for 25 out of 26 KPC genes were also found, as well as putative promoters for some of them. The study of substitutions pattern shows that some of nuclear homologues of plastid genes may be functional and/or are under the pressure of the positive natural selection. The similar comparative analysis performed on rice chromosome 1 revealed 27 contigs containing plastid-derived sequences, totalling about 84 kb and covering two thirds of chloroplast DNA, with the intact nuclear copies of 26 different KPC genes. One of these contigs, AP003280, includes almost 57 kb (45%) of chloroplast genome with the intact copies of 22 KPC genes. At the same time, we observed that relative locations of homologues in plastid DNA and the nuclear genome are significantly different.     

Molecular cloning of retrovirus-like genes present in multiple copies in the Syrian hamster genome.

Endogenous retrovirus-like sequences homologous to intracisternal type-A particle (IAP) genes, which are present in the inbred mouse (Mus musculus) genome, were cloned from a Syrian hamster gene library. A typical hamster IAP gene was 7 kb long and segments homologous to long terminal repeat (IAP) sequences present in Mus musculus IAP genes were located at both ends of the gene. Contrary to the pattern found in the Mus musculus IAP genes, the organization of the cloned hamster IAP genes was not markedly polymorphic and deletion was not observed among these cloned genes. A sequence about 0.8 kb long and located close to the 3' end of the hamster IAP gene was well conserved in both IAP gene families, although they showed less overall homology with one another. The reiteration frequency of the hamster IAP genes was calculated to be 950 copies per haploid genome. Since such IAP genes with the above properties were not found in the genome of the Chinese hamster, whose progenitors diverged from those of the Syrian hamster about 7.5 Myr ago, the integration of a huge number of Syrian hamster IAP genes must have occurred subsequent to such divergence.     

A combined molecular and cytogenetic approach to genome evolution in Drosophila using large-fragment DNA cloning

Methods of genome analysis, including the cloning and manipulation of large fragments of DNA, have opened new strategies for uniting molecular evolutionary genetics with chromosome evolution. We have begun the development of a physical map of the genome of Drosophila virilis based on large DNA fragments cloned in bacteriophage P1. A library of 10,080 P1 clones with average insert sizes of 65.8 kb, containing approximately 3.7 copies of the haploid genome of D. virilis, has been constructed and characterized. Approximately 75% of the clones have inserts exceeding 50 kb, and approximately 25% have inserts exceeding 80 kb. A sample of 186 randomly selected clones was mapped by in situ hybridization with the salivary gland chromosomes. A method for identifying D. virilis clones containing homologs of D. melanogaster genes has also been developed using hybridization with specific probes obtained from D. melanogaster by means of the polymerase chain reaction. This method proved successful for nine of ten genes and resulted in the recovery of 14 clones. The hybridization patterns of a sample of P1 clones containing repetitive DNA were also determined. A significant fraction of these clones hybridizes to multiple euchromatic sites but not to the chromocenter, which is a pattern of hybridization that is very rare among clones derived from D. melanogaster. The materials and methods described will make it possible to carry out a direct study of molecular evolution at the level of chromosome structure and organization as well as at the level of individual genes.