Comparative genomics and proteomics of Drosophila,Brenner's nematode,and Arabidopsis: identification of functionally similar genes and proteins of meiotic chromosome synapsis

The published principles of computer analysis of genomes and protein sets in taxonomically distant eukaryotes are expounded. The authors developed a search strategy to identify in genomes of such organisms genes and proteins nonhomologous in primary structure but having similar functions in cells dividing by meiosis. This strategy based on the combined principles of genomics, proteomics, and morphometric analysis of subcellular structures was applied to a computer search for genes encoding the proteins of synaptonemal complexes in genomes of Drosophila melanogaster, the nematode Caenorhabditis elegans, and the plant Arabidopsis thaliana. These proteins proved to be functionally similar to their counterparts in yeast Saccharomyces cerevisiae (protein Zip1p) and mammals (protein SCP1).     

Putative DNA-(amino)methyltransferases in eucaryotes

By computer analysis of the known data bases, we have established that the open reading frames (ORF) coding for proteins that possess high degree of homology with procaryotic DNA-(amino)methyltransferases are present in the genomes of Leishmania major, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Arabidopsis thaliana, Drosophila melanogaster, Caenorhabditis elegans, and Homo sapiens. Conservative motifs typical for bacterial DNA-(amino)methyltransferases are detected in the amino acid sequences of these putative proteins. The ORF of all putative eucaryotic DNA-(amino)methyl-transferases found are encoded in nuclear DNA. In mitochondrial genomes including a few fully sequenced higher plant mtDNA, nucleotide sequences significantly homologous to genes of procaryotic DNA-(amino)methyltransferases are not found. Thus, ORF homologous to bacterial adenine DNA-methyltransferases are present in nuclei of protozoa, yeasts, insects, nematodes, vertebrates, higher plants, and other eucaryotes. A special search for corresponding proteins and, in particular, adenine DNA-methyltransferases in these organisms and a study of their functions are quite promising.     

Distribution and Expression of Elicitin‐like Protein Genes of the Biocontrol Agent Pythium oligandrum

Pythium oligandrum has the ability to induce plant defence reactions, and four elicitin‐like proteins (POD‐1, POD‐2, POS‐1 and oligandrin) that are produced by this oomycete have been identified as elicitor proteins. The first three are cell wall protein elicitors (CWPs), and the latter is an extracellular protein. Pythium oligandrum isolates have been previously divided into two groups based on the CWPs: the D‐type isolate containing POD‐1 and POD‐2, and the S‐type isolate containing POS‐1. We identified the genes encoding these elicitin‐like proteins and analyzed the distribution of these genes among 10 P. oligandrum isolates. A genomic fosmid library of the D‐type isolate MMR2 was constructed and genomic regions containing the elicitin‐like protein genes were identified. Southern blot analyses with probes derived from pod‐1 and an oligandrin gene indicated that the 10 P. oligandrum isolates could be divided into the same groups as those based on the CWPs. The D‐type isolates carried pod‐1, pod‐2 and two oligandrin genes, termed oli‐d1 and oli‐d2, while the S‐type isolates carried pos‐1 and one oligandrin gene termed oli‐s1. Phylogenetic analysis of POD‐1, POD‐2, POS‐1, Oli‐D1, Oli‐D2 and Oli‐S1 with the previously defined elicitins and elicitin‐like proteins of Phytophthora and Pythium species showed the specific clade. These genes occurred as single copies and were present in the P. oligandrum genomes but not in the other nine Pythium species (Pythium iwayamai, Pythium volutum, Pythium vanterpoolii, Pythium spinosum, Pythium torulosum, Pythium irregulare, Pythium ultimum, Pythium aphanidermutum and Pythium butleri). Furthermore, RT‐PCR analysis demonstrated that all of these genes were expressed during the colonization of tomato roots by P. oligandrum, supporting the idea that they encode potential elicitor proteins. To investigate the genetic relationships between the D‐type and the S‐type isolates, physical maps of the flanking regions around pod‐1, pod‐2, pos‐1 and the oligandrin genes were constructed. The maps suggest that the D‐type isolates may be derived from the S‐type isolates due to gene duplication and deletion events.     

Constant relative rate of protein evolution and detection of functional diversification among bacterial, archaeal and eukaryotic proteins

Background  

Detection of changes in a protein's evolutionary rate may reveal cases of change in that protein's function. We developed and implemented a simple relative rates test in an attempt to assess the rate constancy of protein evolution and to detect cases of functional diversification between orthologous proteins. The test was performed on clusters of orthologous protein sequences from complete bacterial genomes (Chlamydia trachomatis, C. muridarum and Chlamydophila pneumoniae), complete archaeal genomes (Pyrococcus horikoshii, P. abyssi and P. furiosus) and partially sequenced mammalian genomes (human, mouse and rat).     

Detection and Characterization of Fungal-Specific Proteins in Saccharomyces cerevisiae

In this study, I searched for fungal-specific proteins in the genome of the budding yeast Saccharomyces cerevisiae, inferred from a comparison of amino acid sequences. I used the GTOP (Genomes to Protein structures and functions) database of the DDBJ (DNA Data Bank of Japan), which consists of 21 genomes from Archaea, 203 genomes from Bacteria, and 50 genomes from Eucarya (including 18 fungal genomes). Among 5,874 proteins of S. cerevisiae, 1,551 have homologs only in Eucarya, and 504 of the 1,551 have homologs only in fungi. To find fungal-specific proteins, homologs of the homologs have been searched repeatedly. As a result, 132 of the 504 are characterized as fungal-specific proteins. The genes encoding the 132 fungal-specific proteins are not included in the list of essential genes for viability in the S. cerevisiae genome deletion project. Among the 132 proteins, 99 are S. cerevisiae-specific, and no protein that is distributed among 10 or more of the 18 fungal species exists. In addition, most of the fungal-specific proteins are very small and functionally unknown. My results show that the fungal-specific proteins have short evolutionary histories, suggesting that S. cerevisiae produces novel proteins and that ancestral fungi also produced small proteins most of which have disappeared or have been combined with other proteins during fungal evolution.     

ATG Genes Involved in Non-Selective Autophagy are Conserved from Yeast to Man,but the Selective Cvt and Pexophagy Pathways also Require Organism-Specific Genes

ATG genes encode proteins that are required for macroautophagy, the Cvt pathway and/or pexophagy. Using the published Atg protein sequences, we have screened protein and DNA databases to identify putative functional homologs (orthologs) in 21 fungal species (yeast and filamentous fungi) of which the genome sequences were available. For comparison with Atg proteins in higher eukaryotes, also the genomes of Arabidopsis thaliana and Homo sapiens were included. This analysis demonstrated that Atg proteins required for non-selective macroautophagy are conserved from yeast to man, stressing the importance of this process in cell survival and viability. Remarkably, the A. thaliana and human genomes encode multiple proteins highly similar to specific Atg proteins (paralogs), the function of which is unknown. The Atg proteins specifically involved in the Cvt pathway and/or pexophagy showed poor conservation, and were generally not present in A. thaliana and man. Furthermore, the receptor of Cvt cargo, Atg19, was only detected in S. cerevisiae. Nevertheless, Atg11, a protein that links receptor-bound cargo (peroxisomes, Cvt bodies) to the autophagic machinery was identified in all yeast species and filamentous fungi under study. This suggests that in fungi an organism-specific form of selective autophagy may occur, for which specialized Atg proteins have evolved.     

Patterns of protein-fold usage in eight microbial genomes: A comprehensive structural census

Eight microbial genomes are compared in terms of protein structure. Specifically, yeast, H. influenzae, M. genitalium, M. jannaschii, Synechocystis, M. pneumoniae, H. pylori, and E. coli are compared in terms of patterns of fold usage—whether a given fold occurs in a particular organism. Of the ∼340 soluble protein folds currently in the structure databank (PDB), 240 occur in at least one of the eight genomes, and 30 are shared amongst all eight. The shared folds are depleted in all-helical structure and enriched in mixed helix-sheet structure compared to the folds in the PDB. The top-10 most common of the shared 30 are enriched in superfolds, uniting many non-homologous sequence families, and are especially similar in overall architecture—eight having helices packed onto a central sheet. They are also very different from the common folds in the PBD, highlighting databank biases. Folds can be ranked in terms of expression as well as genome duplication. In yeast the top-10 most highly expressed folds are considerably different from the most highly duplicated folds. A tree can be constructed grouping genomes in terms of their shared folds. This has a remarkably similar topology to more conventional classifications, based on very different measures of relatedness. Finally, folds of membrane proteins can be analyzed through transmembrane-helix (TM) prediction. All the genomes appear to have similar usage patterns for these folds, with the occurrence of a particular fold falling off rapidly with increasing numbers of TM-elements, according to a “Zipf-like” law. This implies there are no marked preferences for proteins with particular numbers of TM-helices (e.g. 7-TM) in microbial genomes. Further information pertinent to this analysis is available at http://bioinfo.mbb.yale.edu/genome. Proteins 33:518–534, 1998. © 1998 Wiley-Liss, Inc.     

The complete chloroplast genome of Gracilariopsis lemaneiformis (Rhodophyta) gives new insight into the evolution of family Gracilariaceae

The complete chloroplast genome of Gracilariopsis lemaneiformis was recovered from a Next Generation Sequencing data set. Without quadripartite structure, this chloroplast genome (183,013 bp, 27.40% GC content) contains 202 protein‐coding genes, 34 tRNA genes, 3 rRNA genes, and 1 tmRNA gene. Synteny analysis showed plasmid incorporation regions in chloroplast genomes of three species of family Gracilariaceae and in Grateloupia taiwanensis of family Halymeniaceae. Combined with reported red algal plasmid sequences in nuclear and mitochondrial genomes, we postulated that red algal plasmids may have played an important role in ancient horizontal gene transfer among nuclear, chloroplast, and mitochondrial genomes. Substitution rate analysis showed that purifying selective forces maintaining stability of protein‐coding genes of nine red algal chloroplast genomes over long periods must be strong and that the forces acting on gene groups and single genes of nine red algal chloroplast genomes were similar and consistent. The divergence of Gp. lemaneiformis occurred ~447.98 million years ago (Mya), close to the divergence time of genus Pyropia and Porphyra (443.62 Mya).     

The Mdm2 and p53 genes are conserved in the Arachnids

The p53 protein and its negative regulator the ubiquitin E3 ligase Mdm2 have been shown to be conserved from the Placazoan to man. In common with D.melanogaster and C.elegans, there is a single copy of the p53 gene in T.adhaerens, while in the vertebrates three p53-like genes can be found: p53 , p63 and p73. The Mdm2 gene is not present within the fully sequenced and highly annotated genomes of C.elegans and D.melanogaster. However, it is present in the Placazoan and the presence of multiple distinct p53 genes in the Sea anemone N.vectensis led us to examine the genomes of other phyla for p53 and Mdm2-like genes. We report here the discovery of an Mdm2-like gene and two distinct p53 like genes in the Arachnid Ioxodes scapularis (Northern Deer Tick). The two predicted Deer Tick p53 proteins are much more highly related to the human p53 protein in sequence than are the fruit fly and nematode proteins. One of the Deer tick genes encodes a p53 protein that is initiated within the DNA binding domain of p53 and shows remarkable homology to the newly described N-terminally truncated delta isoforms of human and zebrafish p53.     

GUSP1 and GUSP2, Two Drought-Responsive Genes in Gossypium arboreum Have Homology to Universal Stress Proteins

Two closely related genes GUSP1 and GUSP2, within the universal stress protein (USP) family, were identified and cloned from water-stressed leaves of Gossypium arboreum. GUSP1 and GUSP2 genes code for proteins with predicted molecular weights of 18.2 and 19.1 kDa, respectively. Sequence analysis showed that GUSP1 and GUSP2 are highly similar to the bacterial MJ0577-type of adenosine-triphosphate-binding Usp proteins, which have been proposed to function as a molecular switch. Nucleotide sequences of these two genes showed 81% sequence similarity while their encoded proteins share 75% amino acid homology. Both proteins have high percentages of similarity (17% to 61%) to the USPs from a variety of bacteria and plants. Real-time polymerase chain reaction expression analysis revealed a high level of GUSP gene expression in leaves, roots, and stems exclusively in plants following water stress. The highest levels of drought-inducible expression were found in the leaves. A progressive decrease in expression was observed in the stem and roots compared to very low expression in control tissues.     

Structural characterization of CA1462, the <Emphasis Type="Italic">Candida albicans</Emphasis> thiamine pyrophosphokinase

Background  

In search of new antifungal targets of potential interest for pharmaceutical companies, we initiated a comparative genomics study to identify the most promising protein-coding genes in fungal genomes. One criterion was the protein sequence conservation between reference pathogenic genomes. A second criterion was that the corresponding gene in Saccharomyces cerevisiae should be essential. Since thiamine pyrophosphate is an essential product involved in a variety of metabolic pathways, proteins responsible for its production satisfied these two criteria.     

The complete mitochondrial genome of the entomopathogenic fungus Metarhizium anisopliae var. anisopliae: gene order and trn gene clusters reveal a common evolutionary course for all Sordariomycetes, while intergenic regions show variation

The mitochondrial genome (mtDNA) of the entomopathogenic fungus Metarhizium anisopliae var. anisopliae, with a total size of 24,673 bp, was one of the smallest known mtDNAs of Pezizomycotina. It contained the 14 typical genes coding for proteins related to oxidative phosphorylation, the two rRNA genes, a single intron that harbored an intronic ORF coding for a putative ribosomal protein (rps) within the large rRNA gene (rnl), and a set of 24 tRNA genes which recognized codons for all amino acids, except proline and valine. Gene order comparison with all known mtDNAs of Sordariomycetes illustrated a highly conserved genome organization for all the protein- and rRNA-coding genes, as well as three clusters of tRNA genes. By considering all mitochondrial essential protein-coding genes as one unit a phylogenetic study of these small genomes strongly supported the common evolutionary course of Sordariomycetes (100% bootstrap support) and highlighted the advantages of analyzing small genomes (mtDNA) over single genes. In addition, comparative analysis of three intergenic regions demonstrated sequence variability that can be exploited for intra- and inter-specific identification of Metarhizium. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Purification and Characterization of NfrA1, a Bacillus subtilis Nitro/flavin Reductase Capable of Interacting with the Bacterial Luciferase

ipa-43d is a hypothetical gene identified by the Bacillus subtilis genome project (Mol. Microbiol. 10, 371-384 1993; Nature 390, 249-256 1997). The ipa-43d protein overexpressed in E. coli was purified to homogeneity and its properties were analyzed biochemically. The ipa-43d protein was found to be tightly associated with FMN and to be capable of reducing both nitrofurazone and FMN effectively. Although the ipa-43d protein catalysis obeys the ping-pong Bi-Bi mechanism, catalysis mode was changed to the sequential mechanism upon coupling with the bioluminescent reaction. Database search showed that B. subtilis possessed four genes (ipa-44d, ytmO, yddN, and yvbT), encoding proteins similar in amino acid sequence to LuxA and LuxB of Photobacterium fischeri, and, in particular, ipa-44d is immediately adjacent to the ipa-43d gene on the chromosome.     

Identification,chromosomal mapping and conserved synteny of porcine Argonaute family of genes

The Argonaute proteins are recently identified and evolutionarily conserved family with two subfamilies Ago and Piwi, which play important roles in small RNA pathways. Most species have eight Argonaute members in their genomes, ranging from 1 to 27. Here we report identification of six Argonaute genes in pig, four members of the Ago subfamily (Ago1, Ago2, Ago3 and Ago4) and two members of the Piwi subfamily (Piwil1 and Piwil2), which were predicted to encode proteins of 857, 860, 860, 861, 861 and 985 amino acids, respectively. Phylogenetic analysis showed that the porcine Ago and Piwi genes were clustered into relevant branch of mammalian Argonaute members. The porcine Ago4- Ago1-Ago3 genes are linked together at the p12 of the chromosome 6, while Ago2 is located at the p15 of the chromosome 4. The porcine Piwil1 and Piwil2 are mapped together onto the chromosome 14, at the q14 and q11 respectively. Comparatively mapping of the Argonaute members on chromosomes showed that linkage group of the Ago4-Ago1-Ago3 and several neighborhood genes is evolutionarily conserved from chicken to mammals. The genes Piwil1 and Piwil2 are separated onto different chromosomes from fish to mammals, with exception to this tendency in both pig and stickleback, indicating an opposite tendency of recombination together or non-disjunction of these two genes during speciation. Further expression analysis showed an ubiquitous expression pattern of Ago members, oppositely a restricted expression pattern in gonads of the Piwi members, suggesting distinct potential roles of the porcine Argonaute genes.     

The mitochondrial DNA of Dictyostelium discoideum: complete sequence, gene content and genome organization

We present an overview of the gene content and organization of the mitochondrial genome of Dictyostelium discoideum. The mitochondria genome consists of 55,564 bp with an A + T content of 72.6%. The identified genes include those for two ribosomal RNAs (rnl and rns), 18 tRNAs, ten subunits of the NADH dehydrogenase complex (nad1, 2, 3, 4, 4L, 5, 6, 7, 9 and 11), apocytochrome b (cytb), three subunits of the cytochrome oxidase (cox1/2 and 3), four subunits of the ATP synthase complex (atp1, 6, 8 and 9), 15 ribosomal proteins, and five other ORFs, excluding intronic ORFs. Notable features of D. discoideum mtDNA include the following. (1) All genes are encoded on the same strand of the DNA and a universal genetic code is used. (2) The cox1 gene has no termination codon and is fused to the downstream cox2 gene. The 13 genes for ribosomal proteins and four ORF genes form a cluster 15.4 kb long with several gene overlaps. (3) The number of tRNAs encoded in the genome is not sufficient to support the synthesis of mitochondrial protein. (4) In total, five group I introns reside in rnl and cox1/2, and three of those in cox1/2 contain four free-standing ORFs. We compare the genome to other sequenced mitochondrial genomes, particularly that of Acanthamoeba castellanii. Received: 5 July 1999 / Accepted: 17 January 2000     

鹅掌楸DHHC型锌指蛋白家族基因的克隆及表达分析

棕榈酰化修饰是一种最普遍且唯一可逆的翻译后脂质修饰方式,赋予蛋白质多样化的生理功能。DHHC( Asp-His-His-Cys)蛋白家族是一类与棕榈酰化修饰相关的蛋白,多数DHHC蛋白家族成员具有蛋白质酰基转移酶( protein S-acyltransferase,PAT)活性。该研究以鹅掌楸叶芽为材料,采用RT-PCR和RACE技术,克隆获得了3个鹅掌楸DHHC蛋白家族基因cDNA全长,命名为LcPAT7、LcPAT22、LcPAT23。序列分析结果表明：LcPAT7、LcPAT22、LcPAT23基因全长分别为1933、2592、2217 bp,各包含1332、1839、1662 bp的开放阅读框( Open Reading Frame,ORF),编码433、612、533个氨基酸,预测蛋白分子量分别为40.04、67.3、60.57 kDa,理论等电点为9.15、9.03、7.29。3个基因编码的蛋白均有4个跨膜区,并且都在跨膜域( transmembrane domain, TM) TM2和 TM3之间存在一个 DHHC 蛋白家族典型的 DHHC-CRD 结构域。同源性分析表明：鹅掌楸LcPAT7、LcPAT22、LcPAT23编码的氨基酸序列与其他植物中预测的PAT具有较高的相似性。利用荧光定量PCR技术检测3个基因在鹅掌楸不同组织中的表达特性,发现3个基因在不同组织中均有表达,但表达量具有明显区别。同一家族基因表达模式的变化表明其功能非冗余。该研究结果将为鹅掌楸生长发育与形态建成,以及逆境响应信号传导等相关基因的调控研究提供了参考。