16S rRNA及rpoC1基因用于螺旋藻、节旋藻系统发育的比较北大核心CSCD

【目的】利用16S rRNA和rpoC1基因分子标记研究螺旋藻、节旋藻的系统发育关系,并对其区分能力进行比较。【方法】以84株螺旋藻、节旋藻为研究对象,对其进行16S rRNA、rpoC1基因序列的扩增、测序及分析,并对构建的系统发育树进行对比。【结果】rpoC1基因序列保守位点所占比例49.7%、平均G+C百分含量47.7%和序列相似度76%–100%明显低于16S rRNA基因序列的79.4%、55.6%和91%–100%,其变异程度高于16S rRNA基因;基于16S rRNA、rpoC1基因构建的系统发育NJ树拓扑结构基本一致,84株实验藻株分为2个属3个类群,其中仅F-351、F-904-2、F-1070和TJBC14-1藻株为螺旋藻,其余均为节旋藻;虽然2个基因都不能区分形态种和地理种,但rpoC1基因NJ树的置信度(100%)高于16S rRNA基因(99%),属内分群效果也明显优于16S rRNA基因。【结论】支持了螺旋藻、节旋藻为两个不同属的结论,且在属内分类时rpoC1基因比16S rRNA基因具有更高的区分度。     

Examination of the Intron in the meiosis-specific recombination gene REC114 in Saccharomyces

REC114 is one of 10 genes known to be required for the initiation of meiotic recombination in Saccharomyces cerevisiae. It is transcribed only in meiosis, and our previous sequence analysis suggested the presence of an intron in the 3′ end of the gene. Hypotheses in the literature have suggested, because of its unusual location, either that the putative intron in REC114 is likely to be necessary for expression, or that there may actually be no intron present. This work demonstrates that REC114 does have an intron and is one of only three genes in yeast with introns located in the 3′ end. Furthermore, the 3′ splice site utilized in REC114 is a very rare AAG sequence; only three other genes in yeast use this nonconsensus sequence. The splicing of REC114 does not require MER1, a gene known to be involved in meiosis-specific RNA processing. In fact, an intronless copy of REC114 can complement a null rec114 mutation. Thus, it does not appear that the intron is essential for expression of REC114. Although the intron is not absolutely required for meiotic function, it is conserved in evolution; two other species of yeast contain an intron at the same location in their REC114 genes. Received: 16 October 1996 / Accepted: 10 February 1997     

Oenothera mitochondrial orf454, a gene involved in cytochrome c biogenesis corresponds to orf169 and orf322 of Marchantia

We have characterized a mitochondrial gene in Oenothera, designated orf454, capable of encoding a component of the cytochrome c biogenesis system. This open reading frame is interrupted by an intron of 941 nucleotides showing high similarity to a group II intron residing in the rpl2 gene. RNA editing, which is observed at 18 cytidine positions within the orf454 reading frame, improves the similarity to protein-coding sequences in bacteria and higher plants and removes the last 16 amino acids. orf454 also shows high sequence similarity to two overlapping reading frames (orf169 and orf322) of Marchantia mitochondria. These ORFs belong to an operon-like cluster of genes in the liverwort that is not conserved in Oenothera mitochondria. However, in bacteria these reading frames are organized like the Marchantia gene cluster. It has been shown by genetical analysis in Rhodobacter capsulatus that these genes are essential for cytochrome c biogenesis. Genes of bacterial operons — ccl1 in Rhodobacter and yejR and nrfE in Escherichia coli — show high sequence similarity to the mitochondrial reading frames orf577 and orf454 of Oenothera. orf454, which we describe here, is homologous to the C-terminal region of these bacterial genes, while the previously described orf577 is homologous to the N-terminal region.     

Characterization of the pyruvate kinase-encoding gene (pki1) of Trichoderma reesei

The pyruvate kinase-encoding gene (pki1) from Trichoderma reesei was isolated by hybridization to the corresponding Aspergillus nidulans pkiA gene. The 1614-bp nucleotide (nt) sequence of the cloned gene codes for a 538-amino-acid protein. The coding sequence contains a single intron of 246 nt at a position identical to that of intron E in the A. nidulans gene. The PKI protein shows extensive homology to the PKIs of A. nidulans and A. niger (67%) and Saccharomyces cerevisiae (59%). The 5' non-coding sequence contains a number of motifs typical for yeast glycolytic genes, but so far only rarely found in filamentous fungi.     

An evolutionarily conserved nested gene pair — Mab21 and Lrba/Nbea in metazoan

The embedding of one gene in another as a nested gene pair is a unique phenomenon of gene clustering in the metazoan genome. A gene-centric paralogous genomic sequence comparison strategy was used in this study to align these paralogous nested pairs, Mab21l2-Lrba and Mab21l1-Nbea, to identify the associated paralogous non-coding elements (pNEs) they shared. A majority of these pNEs in the Mab21l2-Lrba locus display tissue-specific enhancer activities recapitulating the expression profiles of Mab21l2 and Mab21l1. Since these enhancers are spread into the introns of Lrba, dissociation of the two genes will likely disrupt the function of at least one of them. Phylogenetic analysis of this complex locus in different species suggests that Mab21 was probably locked in the Lrba/Nbea intron in the ancestral metazoan species, in which the cis-elements uncovered in this study may act as a selective force to prevent the dissociation of this gene pair in vertebrates.     

Isolation and analysis of functional homologues of the secretion-related SAR1 gene of Saccharomyces cerevisiae from Aspergillus niger and Trichoderma reesei

The Aspergillusniger and Trichodermareesei genes encoding the functional homologues of the small GTP-binding protein SAR1p, which is involved in the secretion pathway in Saccharomyces cerevisiae, have been cloned and characterised. The A. niger gene (sarA) contains five introns, whereas the T. reesei gene (sar1) has only four. In both cases the first intron is at the same position as the single S. cerevisiae SAR1 intron. The encoded proteins show 70–80% identity to the SAR1 protein. Complementation of S. cerevisiaesar1 and sec12 mutants by expression vectors carrying the A. nigersarA and T. reesei sar1 cDNA clones confirmed that the cloned genes are functional homologues of the S. cerevisiae SAR1 gene. Three mutant alleles of the A. nigersarA gene (D29G, E109K, D29G/E109K), generated by site-directed mutagenesis, revealed a thermosensitive dominant-negative phenotype in the presence of the wild-type sarA allele. This result contrasts with the situation in S. cerevisiae, where similar mutations have a thermosensitive phenotype. Taken together, our results indicate that the sarA gene is involved in an essential function in A. niger. Received: 21 January 1997 / Accepted: 21 June 1997     

Cloning and characterization of chloroplast ribosomal protein-encoding genes, rpl16 and rps3, of the marine macro-algae, Gracilaria tenuistipitata

In Gracilaria tenuistipitata, a highly differentiated multicellular member of the marine red algae, Rhodophyta, chloroplast (cp) DNA can be separated as a satellite band from the nuclear DNA in a CsCl gradient. Using a heterologous probe from Chlamydomonas, the ribosomal protein-encoding gene, rpl16, was located on a 4.5-kb EcoRI fragment of cp DNA. The fragment was cloned and a 1365-bp region around rpl16 was sequenced. The gene order around rpl16, 5′ rpl22-rps3-rpl16, is identical to that detected in the chloroplast DNA of liverwort, tobacco and maize. Both the nucleotide sequence and the amino-acid sequence of rpl16 are more conserved than that of rps3. The rpl16 gene contains no intron, a feature which shows more similarity to the unicellular green algae, Chlamydomonas, than the other land plants. Sequences that may form a stable stem-loop structure were detected within the coding sequence of rpl16.     

鹅掌楸LcUGE基因的克隆和组织表达分析

为了解鹅掌楸(Liriodendron chinense)的UGE基因功能,采用RACE和EPIC-PCR技术克隆到2个UGE基因,命名为LcUGE1和LcUGE2。结果表明,LcUGE1基因的c DNA全长为1 531 bp,包含1 050 bp的开放阅读框,编码349个氨基酸, gDNA长度为11 920 bp;LcUGE2基因的c DNA长度为1 378 bp,包含1 056 bp的开放阅读框,编码351个氨基酸,g DNA长度为6544 bp。LcUGE1和LcUGE2基因均含有9个外显子和8个内含子,且外显子长度和内含子剪切位点序列几乎一致,但内含子片段长度存在显著差异。编码的LcUGE1和LcUGE2蛋白高度保守,保守性达到82%。LcUGE1基因在雄蕊中表达量最高,而LcUGE2基因则在花萼中表达量最高。这表明LcUGEs基因可能参与鹅掌楸的生殖发育过程。     

Molecular characterization of the rbcS multi-gene family of Petunia (Mitchell)

Summary The small subunit (RbcS) of ribulose bisphosphate carboxylase (RuBPCase) is encoded by eight genes in Petunia (Mitchell). These genes can be divided into three subfamilies (51, 117 and 71) based upon hybridization to three petunia rbcS cDNA clones. The nucleotide sequence of six of the eight petunia rbcS genes is presented here and the structure of the genes is discussed with respect to their genomic linkage and their expression levels in petunia leaf tissue. The rbcS genes belonging to the same subfamily encode an identical mature RbcS polypeptide, however the different subfamilies encode distinguishable polypeptides. All the genes, except one, contian two introns within the mature subunit coding region; one gene contains one extra intron within the coding region. There are large regions of nucleotide sequence homology within the introns of genes within a subfamily, but significantly less homology between the introns of genes of different subfamilies. A complex pattern of homology within the multiple genes of the 51 subfamily is observed. There are regions within these genes which share high levels of sequence homology; this homology does not extend throughout the whole gene and the regions of homology do not always occur in adjacent genes. Two 3 rbcS gene fragments which we isolated from the petunia genome show high levels of homology to two of the intact rbcS genes.     

The chorion genes of the medfly. II. DNA sequence evolution of the autosomal chorion genes s18, s15, s19 and s16 in Diptera

We present a total of approximately 15 kb of DNA sequences, encompassing four chorion genes Ccs18, Ccs15, Ccs19, Cc16 and their flanking DNA in the medfly C. capitata. Comparison of coding regions, introns and intergenic sequences in five Dipteran species, D. melanogaster, D. subobscura, D. virilis, D. grimshawi and C. capitata documented an extensive divergence in introns and coding regions, but few well conserved elements in the proximal 5′ flanking regions in all species. These elements are related to conserved regulatory features of three of the genes, including tissue- and temporal regulation. In the fourth, gene s15, significant alterations in the 5′ flanking region may be responsible for its changed temporal regulation in C. capitata. One long intergenic sequence, located in the distal 5′ flanking region of gene s18, is homologous to ACE3, a major amplification control element and contains an 80-bp A/T-rich sequence, known to stimulate strong binding of the origin recognition complex (ORC) in D. melanogaster. Analysis of the nucleotide composition of all chorion genes in C. capitata and D. melanogaster showed that C. capitata exhibit less biased representation of synonymous codons than does D. melanogaster.     

Characterization and expression in Streptomyces lividans of cefD and cefE genes from Nocardia lactamdurans: the organization of the cephamycin gene cluster differs from that in Streptomyces clavuligerus

Summary The cefD and cefE genes of Nocardia lactamdurans, which encode isopenicillin N epimerase and deacetoxycephalosporin C synthase respectively, have been located 0.63 kb upstream from the lysine-6-amino-transferase (lat) gene. cefD contains an open reading frame (ORF) of 1197 nucleotides (nt) encoding a protein of 398 amino acids with a M_r of 43 622. The deduced amino acid sequence exhibits 62.2% identity to the cefD gene product of Streptomyces clavuligerus. The sequence SXHKXL in isopenicillin N epimerase resembles the consensus sequence for pyridoxal phosphate binding found in several amino acid decarboxylases from Enterobacteria. cefE contains an ORF of 945 nt encoding a protein of 314 amino acids with a M_r of 34532, which is similar to the deacetoxycephalosporin C synthase of S. clavuligerus. Expression of both genes, cefD and cefE, in S. lividans transformants, results in deacetoxycephalosporin C synthase and isopenicillin N epimerase activities that are 10–12 times higher than those in N. lactamdurans. The cefD and cefE genes of N. lactamdurans are closely linked but the overall organization of the cephamycin gene cluster differs in N. lactamdurans and S. clavuligerus.     

Slipped-strand mispairing in a plastid gene: rpoC2 in grasses (Poaceae)

An exception to the generally conservative nature of plastid gene evolution is the gene coding for the beta" subunit of RNA polymerase, rpoC2. Previous work by others has shown that maize and rice have an insertion in the coding region of rpoC2, relative to spinach and tobacco. To assess the distribution of this extra coding sequence, we surveyed a broad phylogenetic sample comprising 55 species from 17 angiosperm families by using Southern hybridization. The extra coding sequence is restricted to the grasses (Poaceae). DNA sequence analysis of 11 species from all five subfamilies within the grass family demonstrates that the extra sequence in the coding region of rpoC2 is a repetitive array that exhibits more than a twofold increase in nucleotide substitution, as well as a large number of insertion/deletion events, relative to the adjacent flanking sequences. The structure of the array suggests that slipped-strand mispairing causes the repeated motifs and adds to the mechanisms through which the coding sequence of plastid genes are known to evolve. Phylogenetic analyses based on the sequence data from grass species support several relationships previously suggested by morphological work, but they are ambiguous about broad relationships within the family.      

Molecular analysis of two genes between let-653 and let-56 in the unc 22(IV) region of Caenorhabditis elegans

Summary A previous study of genomic organization described the identification of nine potential coding regions in 150 kb of genomic DNA from the unc-22(IV) region of Caenorhabditis elegans. In this study, we focus on the genomic organization of a small interval of 0.1 map unit bordered on the right by unc-22 and on the left by the left-hand breakpoints of the deficiencies sDf9, sDf19 and sDf65. This small interval at present contains a single mutagenically defined locus, the essential gene let-56. The cosmid C11F2 has previously been used to rescue let-56. Therefore, at least some of C11F2 must reside in the interval. In this paper, we report the characterization of two coding elements that reside on C11F2. Analysis of nucleotide sequence data obtained from cDNAs and cosmid subclones revealed that one of the coding elements closely resembles aromatic amino acid decarboxylases from several species. The other of these coding elements was found to closely resemble a human growth factor activatable Na⁺/H⁺ antiporter. Pairs of oligonucleotide primers, predicted from both coding elements, have been used in PCR experiments to position these coding elements between the left breakpoint of sDf19 and the left breakpoint of sDf65, between the essential genes let-653 and let-56.     

Orthologues of a plant-specific At-4/1 gene in the genus Nicotiana and the structural properties of bacterially expressed 4/1 protein

Arabidopsis thaliana At-4/1 is the protein of unknown function capable of polar localization in plant cells and intercellular trafficking. In this work, we cloned cDNAs and chromosomal genes of At-4/1 orthologues from several Nicotiana species. Similarly to the 4/1 genes of A. thaliana and Oryza sativa, Nicotiana 4/1 genes have eight exons and seven introns but are considerably longer due to their larger introns. The allotetraploid genome of Nicotiana tabacum, which is known to consist of the ‘S genome’ originated from Nicotiana sylvestris and the ‘T genome’ derived from Nicotiana tomentosiformis, encodes two 4/1 genes. The T genome-encoded 4/1 gene, but not that of the S genome, contains a SINE-like transposable element in its intron 2. The 4/1 genes of Nicotiana hesperis and Nicotiana benthamiana lack such an element in the intron 2, but possess a related SINE-like sequence in their intron 4. Collectively, the sequence analysis data provide an insight into the organization of 4/1 genes in flowering plants and the patterns of evolution in the genus Nicotiana. The Nicotiana 4/1 proteins and those of other flowering plants show a significant level of sequence similarity. Computer-assisted analysis was further used to compare their predicted secondary structures. Several algorithms confidently predicted the presence of several coiled-coil domains occupying similar positions in different 4/1 proteins. Analysis of circular dichroism spectra carried out for bacterially expressed N. tabacum 4/1 protein (Nt-4/1) and its N- and C-terminally truncated mutants confirmed that the secondary structure of Nt-4/1 is generally alpha-helical. The C-terminal region of Nt-4/1 was found to undergo a partial proteolysis in Escherichia coli cells. Differential scanning calorimetry of Nt-4/1 protein and its mutants revealed three calorimetric domains most probably corresponding to the N-terminal, central, and C-terminal structural domains of the protein.     

A complete sequence of the rice sucrose synthase-1 (RSs1) gene

Using a fragment of the maize sucrose synthase gene Sh-1 as probe, the rice genome was shown to contain at least three genes encoding sucrose synthase. One of these genes was isolated from a genomic library, and its full sequence, including 1.7 kb of 5 flanking sequence and 0.9 kb of 3 flanking sequence, is reported. The new rice gene, designated RSs1, is highly homologous to maize Sh-1 (approx. 94% identity in derived amino acid sequence), and contains an identical intron-exon structure (16 exons and 15 introns). Both RSs1 and maize Sh-1 show similar sequence homologies to a second rice sucrose synthase gene described recently (designated RSs2, Yu et al. (1992) Plant Mol Biol 18: 139–142), although both the rice genes predict an extra 6 amino acids at the C-terminus of the protein when compared to the maize gene. The RSs1 5 flanking sequence contains a number of promoter-like sequences, including putative protein-binding regions similar to maize zein genes.     

Nuclear genes encoding chloroplast hemoglobins in the unicellular green alga Chlamydomonas eugametos

When the green unicellular alga Chlamydomonas eugametos is grown under light/dark regimes, nuclear genes are periodically activated in response to the changes in light conditions. These genetic responses are dependent upon the activation of genes associated with photosynthesis (LI616 and LI637), nonphotosynthetic photoreceptors (LI410 and LI818) and the biological clock (LI818). We report here that the LI410 and LI637 genes are part of a small gene family encoding hemoglobins (Hbs) related to those from two unicellular eukaryotes, the ciliated protozoa Paramecium caudatum and Tetrahymena pyriformis, and from the cyanobacterium Nostoc commune. Investigations of the intracellular localization of C. eugametos Hbs by means of immunogold electron microscopy indicate that these proteins are predominantly located in the chloroplast, particularly in the pyrenoid and the thylakoid region. To our knowledge, this constitutes the first evidence for the presence of Hbs in chloroplasts. Alignment of the LI637 cDNA nucleotide sequence with its corresponding genomic sequence indicates that the L1637 gene contains three introns, the positions of which are compared with those in the Hb genes of plants, animals and the ciliate P. caudatum. Although the LI637 gene possesses a three-intron/four-exon pattern similar to that of plant leghemoglobin genes, introns are inserted at different positions. Similarly the position of the single intron in the P. caudatum gene differs from the intron sites in the LI637 gene. The latter observations argue against the current view that all eukaryotic Hbs have evolved from a common ancestor having a gene structure identical to that of plant or animal Hbs.     

Multiple copies of functional, Tet(M)-encoding Tn916-like elements in a clinical Enterococcus faecium isolate

Tn916 and similar elements are very common in clinical enterococcal isolates, and are responsible for transmission of a variety of resistance determinants. It is commonly assumed that clinical strains carrying Tn916 have a single copy, although the actual number of copies in clinical isolates has never been systematically studied. We report a clinical isolate of Enterococcus faecium in which three distinct and excision-proficient copies of Tn916-like elements are present in the genome. All of the elements contain tet(M) genes, at least one of which confers resistance to tetracycline and minocycline. Two elements (Tn6085a, Tn6085b) are indistinguishable, containing an inserted 2758 bp Group II intron at the start of open reading frame Tn916ORF_06. The third (Tn6084) also contains the intron, but also has an ISEfa11 integrated upstream of tet(M). All three copies are able to excise from plasmid vectors when cloned in E. coli, and at least two of the elements can transfer to an E. faecium recipient strain. These data indicate that nearly identical Tn916-like elements encoding Tet(M)-mediated tetracycline/minocycline resistance can coexist in clinical E. faecium isolates.