γ-Glutamylcysteine Production by Escherichia coli B Cells Dosed with the γ-Glutamylcysteine Synthetase Gene

Molecular studies on the evolution and systematics of fungi have been established primarily based on the neutral theory by analyzing neutral mutations in some defined segments of housekeeping genes as genetic markers. Such an approach is, however, hardly applicable for analyzing ancient evolutionary radiations. In the present study, we looked for DNA sequences characterizing higher taxa, and discovered a unique macroevolutionary genomic marker, megB1, that specifies the phylum Basidiomycota. megB1 is an approximately 500-bp DNA element, which is defined by terminal sequences and five internal segments conserved throughout the phylum. megB1 resides on the rDNA intergenic spacer 1 (IGS1) from 27 species of 10 Basidiomycota genera examined. While megB1 was not found in IGS1 from the other 92 species of the 27 Basidiomycota genera, several genera representing them carry megB1 in some other genomic regions. No known taxonomic criteria fit into the classification on the basis of whether megB1 resides on rDNA. Neighbor-joining analysis of the megB1 sequence, however, properly assigned species to their respective genera. Thus far, megB1 has not been found in any genomic or genetic databases currently available for other phyla. These results suggest that megB1 may have emerged upon the occurrence of Basidiomycota, and that this phylum evolved thereafter leaving this element conserved throughout their further differentiation. megB1 may be a novel genomic marker useful in the analysis of ancient through the latest evolutionary radiation in Basidiomycota.     

The structure of a single unit of ribosomal RNA gene (rDNA) including intergenic subrepeats in the Australian bulldog ant Myrmecia croslandi (Hymenoptera: Formicidae)

A complete single unit of a ribosomal RNA gene (rDNA) of M. croslandi was sequenced. The ends of the 18S, 5.8S and 28S rRNA genes were determined by using the sequences of D. melanogaster rDNAs as references. Each of the tandemly repeated rDNA units consists of coding and non-coding regions whose arrangement is the same as that of D. melanogaster rDNA. The intergenic spacer (IGS) contains, as in other species, a region with subrepeats, of which the sequences are different from those previously reported in other insect species. The length of IGSs was estimated to be 7-12 kb by genomic Southern hybridization, showing that an rDNA repeating unit of M. croslandi is 14-19 kb-long. The sequences of the coding regions are highly conserved, whereas IGS and ITS (internal transcribed spacer) sequences are not. We obtained clones with insertions of various sizes of R2 elements, the target sequence of which was found in the 28S rRNA coding region. A short segment in the IGS that follows the 3' end of the 28S rRNA gene was predicted to form a secondary structure with long stems.     

Unraveling the sequence dynamics of the formation of genus-specific satellite DNAs in the family solanaceae

Tandemly repeated DNAs, referred to as satellite DNAs, often occur in a genome in a genus-specific manner. However, the mechanisms for generation and evolution for these sequences are largely unknown because of the uncertain origins of the satellite DNAs. We found highly divergent genus-specific satellite DNAs that showed sequence similarity with genus-specific intergenic spacers (IGSs) in the family Solanaceae, which includes the genera Nicotiana, Solanum and Capsicum. The conserved position of the IGS between 25S and 18S rDNA facilitates comparison of IGS sequences across genera, even in the presence of very low sequence similarity. Sequence comparison of IGS may elucidate the procedure of the genesis of complex monomer units of the satellite DNAs. Within the IGS of Capsicum species, base substitutions and copy number variation of subrepeat monomers were causes of monomer divergence in IGS sequences. At the level of inter-generic IGS sequences of the family Solanaceae, however, genus-specific motif selection, motif shuffling between subrepeats and differential amplification among motifs were involved in formation of genus-specific IGS. Therefore, the genus-specific satellite DNAs in Solanaceae plants can be generated from differentially organized repeat monomers of the IGS rather than by accumulation of mutations from pre-existent satellite DNAs.     

Phylogeny of the glomeromycota (arbuscular mycorrhizal fungi): recent developments and new gene markers

The fungal symbionts of arbuscular mycorrhiza form a monophyletic group in the true Fungi, the phylum Glomeromycota. Fewer than 200 described species currently are included in this group. The only member of this clade known to form a different type of symbiosis is Geosiphon pyriformis, which associates with cyanobacteria. Because none of these fungi has been cultivated without their plant hosts or cyanobacterial partners, progress in obtaining multigene phylogenies has been slow and the nuclear-encoded ribosomal RNA genes have remained the only widely accessible molecular markers. rDNA phylogenies have revealed considerable polyphyly of some glomeromycotan genera that has been used to reassess taxonomic concepts. Environmental studies using phylogenetic methods for molecular identification have recovered an amazing diversity of unknown phylotypes, suggesting considerable cryptic species diversity. Protein gene sequences that have become available recently have challenged the rDNA-supported sister group relationship of the Glomeromycota with Asco/Basidiomycota. However the number of taxa analyzed with these new markers is still too small to provide a comprehensive picture of intraphylum relationships. We use nuclear-encoded rDNA and rpb1 protein gene sequences to reassess the phylogeny of the Glomeromycota and discuss possible implications.     

Diversity and specific composition of microscopic fungi on synthetic polymeric materials

We summarized experimental data on species diversity of fungi decomposing synthetic polymeric materials. Most of the fungi were anamorphs of the phylum Ascomycota, class Ascomycetes (231 species and 85 genera). Teleomorphs of ascomycetes were represented by 18 species and 7 genera. We revealed a smaller number of fungi belonging to the phylum Zygomycota, class Zygomycetes (31 species and 15 genera), or the phylum Basidiomycota, class Basidiomycetes (5 species and 5 genera). The specific composition of fungi was assessed on polymeric materials of various classes.     

毛木耳rDNA序列结构及IGS鉴别菌种初探

rDNA序列中的ITS作为DNA barcode广泛应用于真菌的系统发育与物种辅助鉴定,IGS被认为可以用于种内水平不同菌株的鉴别。有关食用菌rDNA序列的报道较少。本研究对毛木耳Auricularia cornea单核菌株B02进行三代测序与组装,然后用二代测序数据进行校正,得到一个组装效果较好的基因组序列。比对Fibroporia vaillantiir的rDNA序列获得毛木耳rDNA重复单元的完整序列,每个重复单元包含ETS、18S rDNA、ITS1、5.8S rDNA、ITS2、28S rDNA、IGS1、5S rDNA和IGS2,长度分别为398bp、1 790bp、156bp、156bp、206bp、3 432bp、2 247bp、121bp和2 135bp,总长度10 641bp,毛木耳rDNA有310个串联重复单元,转录组和系统发育分析均支持这一结果。与其他已报道的食用菌不同,毛木耳的IGS1、IGS2序列高度保守,其中IGS1的1 400-2 200bp区域在各拷贝之间没有多态性、而在品种之间有较高频率的SNP,这一片段序列有望用于品种鉴别研究。     

Molecular evolution of mitochondrial ribosomal DNA in the fungal genus Tricholoma: barcoding implications

The molecular evolution of the V6 and V9 domains of the mitochondrial SSU-rDNA was investigated to evaluate the use of these sequences for DNA barcodes in the Basidiomycota division. The PCR products from 27 isolates belonging to 11 Tricholoma species were sequenced. Both domains in the isolates belonging to the same species had identical sequences. All the species possess distinctive V9 sequences due to point mutations and insertion/deletion events. Secondary structures revealed that the insertion-deletion events occurred in regions not directly involved in the maintenance of the standard SSU-rRNA structure. The inserted sequences possess conserved motifs that enable their alignment among phylogenetically distant species. Hence, the V9 domain by displaying identical sequences within species, an adequate divergence level, easy amplification, and alignment represents an alternative molecular marker for the Basidiomycota division and opens the way for this sequence to be used as specific molecular markers of the fungal kingdom.     

A two-locus DNA sequence database for typing plant and human pathogens within the Fusarium oxysporum species complex

We constructed a two-locus database, comprising partial translation elongation factor (EF-1α) gene sequences and nearly full-length sequences of the nuclear ribosomal intergenic spacer region (IGS rDNA) for 850 isolates spanning the phylogenetic breadth of the Fusarium oxysporum species complex (FOSC). Of the 850 isolates typed, 101 EF-1α, 203 IGS rDNA, and 256 two-locus sequence types (STs) were differentiated. Analysis of the combined dataset suggests that two-thirds of the STs might be associated with a single host plant. This analysis also revealed that the 26 STs associated with human mycoses were genetically diverse, including several which appear to be nosocomial in origin. A congruence analysis, comparing partial EF-1α and IGS rDNA bootstrap consensus, identified a significant number of conflicting relationships dispersed throughout the bipartitions, suggesting that some of the IGS rDNA sequences may be non-orthologous. We also evaluated enniatin, fumonisin and moniliformin mycotoxin production in vitro within a phylogenetic framework.     

Polymorphism at the ribosomal DNA spacers and its relation to breeding structure of the widespread mushroom Schizophyllum commune

The common split-gilled mushroom Schizophyllum commune is found throughout the world on woody substrates. This study addresses the dispersal and population structure of this fungal species by studying the phylogeny and evolutionary dynamics of ribosomal DNA (rDNA) spacer regions. Extensive sampling (n = 195) of sequences of the intergenic spacer region (IGS1) revealed a large number of unique haplotypes (n = 143). The phylogeny of these IGS1 sequences revealed strong geographic patterns and supported three evolutionarily distinct lineages within the global population. The same three geographic lineages were found in phylogenetic analysis of both other rDNA spacer regions (IGS2 and ITS). However, nested clade analysis of the IGS1 phylogeny suggested the population structure of S. commune has undergone recent changes, such as a long distance colonization of western North America from Europe as well as a recent range expansion in the Caribbean. Among all spacer regions, variation in length and nucleotide sequence was observed between but not within the tandem rDNA repeats (arrays). This pattern is consistent with strong within-array and weak among-array homogenizing forces. We present evidence for the suppression of recombination between rDNA arrays on homologous chromosomes that may account for this pattern of concerted evolution.     

The evolution of eukaryotic ribosomal DNA

Mutations occur randomly throughout the ribosomal DNA (rDNA) sequence. Molecular drive (unequal crossing-over, gene conversion, and transposition) spreads these variations through the multiple copies of rDNA. Forces of selection act upon the variants to favor and fix them or disfavor and eliminate them. Selection has not permitted changes in regions within rRNA vital for its function; these sequences are evolutionarily conserved between diverse species. Possible functions for some of these conserved sequences are discussed. The secondary structure of rRNA is also highly conserved during evolution. However, eukaryotic rRNA is larger than prokaryotic rRNA due to blocks of "expansion segments". Arguments are put forward that expansion segments might not play any functional role. Other examples are reviewed of rDNA sequence insertion or deletion, including introns and the internal transcribed spacer 2.     

贵州赤水桫椤自然保护区桫椤内生真菌多样性研究

桫椤Alsophila spinulosa是白垩纪时期遗留的古老孑遗蕨类植物,具有较高的药用价值和观赏价值。本研究采用形态特征和ITS序列分析方法,从250个组织块中共分离获得84个菌株,隶属于2门3纲10目18科25属。子囊菌门Ascomycota为优势菌门,其相对丰度高达92.86%,而担子菌门Basidiomycota仅为2.38%;粪壳菌纲Sordariomycetes和座囊菌纲Dothideomycetes为优势纲,其相对丰度分别为78.57%和13.10%;小丛壳目Glomerellales和炭角菌目Xylariales为优势目,其相对丰度分别为33.33%和25.00%;小丛壳科Glomerellaceae和炭角菌科Xylariaceae为优势科,其相对丰度分别为32.14%和19.05%;刺盘孢属 Colletotrichum和炭角菌属Xylaria为优势属,其相对丰度分别为32.14%和14.29%。这些菌株存在一定的组织差异,属水平上,叶轴中共获得15属,特有属9属;羽状复叶中共获得16属,特有属10属,两组织间共有属为6属,其中刺盘孢属Colletotrichum和炭角菌属Xylaria均为两组织的优势属,但其相对丰度存在一定差异。从不同组织多样性来看,桫椤叶轴的Shannon、Simpson多样性指数和Pielou均匀度均高于桫椤叶。     

Phylogenetic analysis of enteric species of the family Enterobacteriaceae using the oriC-locus

Phylogenetic analysis of 38 enteric species belonging to the Enterobacteraceae family was carried out using the non-coding locus oriC, the chromosomal replication origin. The oriC loci were amplified with conserved oligonucleotides and the PCR fragments were sequenced directly. The results establish a phylogenetic tree for the classification of different species of the genera Escherichia, Shigella, Salmonella, Enterobacter, Citrobacter, Klebsiella, Raoultella, Kluyvera, Cedecea and Buttiauxella. Functional important protein-binding sites located in oriC are well conserved throughout the enteric group. More over, due to a high overall divergence value phylogenetic trees were robust and well supported by bootstrap analysis. In comparison with 16S rDNA analysis, the oriC sequences indicated a greater evolutionary divergence for bacteria. We propose that the oriC locus might be a suitable phylogenetic marker for the identification and classification of bacteria, in particular for closely related species.     

Completion of the sequence of the nuclear ribosomal DNA subunit of Simulium sanctipauli,with descriptions of the 18S, 28S genes and the IGS

We describe the IGS-ETS, 18S and 28S ribosomal gene sequences of Simulium sanctipauli Vajime & Dunbar, a member of the S. damnosum Theobald (Diptera: Simuliidae) complex of blackflies (Diptera: Simuliidae). These regions, together with the ITS-1, ITS-2 and 5.8S rDNA presented elsewhere (accession number U36206), constitute the composite sequence of the entire rDNA unit, making S. sanctipauli the second dipteran species of medical importance for which the entire rDNA has been sequenced. Despite the lack of sequence identity, the IGS of S. sanctipauli showed some structural similarities to other Diptera, i.e. the mosquito Aedes albopictus Skuse (Culicidae), the fruitfly Drosophila melanogaster Meigen (Drosophilidae) and the tsetse Glossina (Glossinidae). Two blocks of tandemly repeated subunits were present in the IGS of S. sanctipauli and, unlike other species of Diptera, they contained no duplications of promoter-like sequences. However, two promoter-like sequences were identified in the unique DNA stretches of the IGS by their sequence similarity to the promoter of Aedes aegypti L. (Diptera: Culicidae). The observed sequence variation can be explained, as in the case of Drosophila spp., by the occurrence of slippage-like and point mutation processes, with unequal crossing-over homogenizing (to a certain extent) the region throughout the gene family and blackfly population. The 18S and 28S rDNA genes show more intraspecific variability within the expansion segments than in the core regions. This is also the case in the interspecific comparison of these genes from S. sanctipauli with those of Simulium vittatum, Ae. albopictus and D. melanogaster. This pattern is typical of many eukaryotes and likely to be the result of a more relaxed functional selection in the expansion segments than on the core regions. The A + T content of the S. sanctipauli genes is high and similar to those of other Diptera. This could be the result of a change in the mutation pressure towards AT in the Diptera lineage.     

Phylogeographic congruence between mtDNA and rDNA ITS markers in brown trout

Variation in the internal transcribed spacer (ITS) of rDNA was examined throughout the range of the brown trout (Salmo trutta) to analyze the usefulness of this molecular marker for phylogeographic analysis. The results were compared with those previously obtained with mtDNA, a region exhaustively analyzed along the brown trout distribution. ITS2 was essentially conserved at all populations sampled, no informative characters being detected across the main lineages described in this species. Conversely, ITS1 showed a greater homogenization than other genetic markers at a microgeographic scale, with variation partitioning into several major phylogenetic groups. Phylogeographic patterns were partially congruent between both ITS1 and mtDNA. The main discrepancies were the detection of intra-individual variation and putative recombinant ITS1 sequences in hybridization areas between genetically different, yet historically overlapping, assemblages. Also, the existence of an ancient ITS1 sequence in the Mediterranean-southeastern area (rMEDA), not revealed by mtDNA analysis, was evidenced after rDNA ITS1 analysis.     

金针菇rDNA序列结构特征分析

rDNA序列中的ITS作为DNA barcoding广泛应用于真菌的系统发育与物种辅助鉴定,IGS被认为可以用于种内水平不同菌株的鉴别。食用菌中还没有完整的rDNA序列的报道。本研究采用二代和三代测序技术分别对金针菇单核菌株“6-3”进行测序,用二代测序的数据对三代测序组装得到的基因组序列进行修正,得到一个在基因完整性、连续性和准确性均较好的基因组序列,对比Fibroporia vaillantii rDNA序列,获得金针菇完整的rDNA序列。金针菇rDNA序列结构分析表明,它有8个rDNA转录单元,长度均为5 903bp,有9个基因间隔区,其长度有较大差异,3 909-4 566bp。rDNA转录单元中,各元件的序列长度分别为：18S rDNA 1 796bp、ITS1 234bp、5.8S rDNA 173bp、ITS2 291bp、28S rDNA 3 410bp。基因间间隔区中,IGS1 1 351-1 399bp、5S rDNA 124bp、IGS2 2 435-3 092bp。金针菇的5S、5.8S、18S、28S rDNA序列准确性得到转录组数据的验证,也得到系统发育分析结果的支持。多序列比对发现,不同拷贝的基因间间隔区序列（IGS1和IGS2）存在丰富的多态性,多态性来源于SNP、InDel和TRS（串联重复序列）,而TRS来源于重复单元的类型和数量。9个基因间间隔区之间,IGS1只有少量的SNP和InDel,IGS2不仅有SNP和InDel,还有TRS。本研究结果提示,在应用IGS进行种内水平不同菌株之间的鉴别时,需要选取不同拷贝之间的保守IGS序列。     

Sequencing and analysis of the internal transcribed spacers (ITSs) and coding regions in the EcoR I fragment of the ribosomal DNA of the Japanese pond frog Rana nigromaculata

The rDNA of eukaryotic organisms is transcribed as the 40S-45S rRNA precursor, and this precursor contains the following segments: 5' - ETS - 18S rRNA - ITS 1 - 5.8S rRNA - ITS 2 - 28S rRNA - 3'. In amphibians, the nucleotide sequences of the rRNA precursor have been completely determined in only two species of Xenopus. In the other amphibian species investigated so far, only the short nucleotide sequences of some rDNA fragments have been reported. We obtained a genomic clone containing the rDNA precursor from the Japanese pond frog Rana nigromaculata and analyzed its nucleotide sequence. The cloned genomic fragment was 4,806 bp long and included the 3'-terminus of 18S rRNA, ITS 1, 5.8S rRNA, ITS 2, and a long portion of 28S rRNA. A comparison of nucleotide sequences among Rana, the two species of Xenopus, and human revealed the following: (1) The 3'-terminus of 18S rRNA and the complete 5.8S rRNA were highly conserved among these four taxa. (2) The regions corresponding to the stem and loop of the secondary structure in 28S rRNA were conserved between Xenopus and Rana, but the rate of substitutions in the loop was higher than that in the stem. Many of the human loop regions had large insertions not seen in amphibians. (3) Two ITS regions had highly diverged sequences that made it difficult to compare the sequences not only between human and frogs, but also between Xenopus and Rana. (4) The short tracts in the ITS regions were strictly conserved between the two Xenopus species, and there was a corresponding sequence for Rana. Our data on the nucleotide sequence of the rRNA precursor from the Japanese pond frog Rana nigromaculata were used to examine the potential usefulness of the rRNA genes and ITS regions for evolutionary studies on frogs, because the rRNA precursor contains both highly conserved regions and rapidly evolving regions.