Identifying novel genes in <Emphasis Type="Italic">C. elegans</Emphasis> using SAGE tags

Background  

Despite extensive efforts devoted to predicting protein-coding genes in genome sequences, many bona fide genes have not been found and many existing gene models are not accurate in all sequenced eukaryote genomes. This situation is partly explained by the fact that gene prediction programs have been developed based on our incomplete understanding of gene feature information such as splicing and promoter characteristics. Additionally, full-length cDNAs of many genes and their isoforms are hard to obtain due to their low level or rare expression. In order to obtain full-length sequences of all protein-coding genes, alternative approaches are required.     

Complexity of rice Hsp100 gene family: lessons from rice genome sequence data

Elucidation of genome sequence provides an excellent platform to understand detailed complexity of the various gene families. Hsp100 is an important family of chaperones in diverse living systems. There are eight putative gene loci encoding for Hsp100 proteins in Arabidopsis genome. In rice, two full-length Hsp100 cDNAs have been isolated and sequenced so far. Analysis of rice genomic sequence by in silico approach showed that two isolated rice Hsp100 cDNAs correspond to Os05g44340 and Os02g32520 genes in the rice genome database. There appears to be three additional proteins (encoded by Os03g31300, Os04g32560 and Os04g33210 gene loci) that are variably homologous to Os05g44340 and Os02g32520 throughout the entire amino acid sequence. The above five rice Hsp100 genes show significant similarities in the signature sequences known to be conserved among Hsp100 proteins. While Os05g44340 encodes cytoplasmic Hsp100 protein, those encoded by the other four genes are predicted to have chloroplast transit peptides.     

Mating,male Ixodes scapularis express several genes including those with sequence similarity to immunoglobulin-binding proteins and metalloproteases

We created a cDNA library from mating, male Ixodes scapularis ticks and screened the library with a subtracted probe to eliminate genes common to feeding female and mating male I. scapularis ticks. A total of seven unique cDNAs were identified in this screen. One cDNA had sequence similarity to the IGBP-MC gene from Rhipicephalus appendiculatus, and another cDNA potentially encodes a protein with similarity to metalloproteases. RT-PCR, using RNA isolated from male and female I. scapularis ticks, confirmed that these genes are expressed in male, but not female ticks. The remaining five cDNAs did not match any sequences in the GenBank database. This revised version was published online in July 2006 with corrections to the Cover Date.     

Multiple genes are transcribed in Hordeum vulgare and Zea mays that carry the DNA binding domain of the myb oncoproteins

Summary cDNA clones were isolated from tissue specific cDNA libraries of barley and maize using as a probe the cDNA of the maize gene C1, a regulator of anthocyanin gene expression. C1-related homology for all of the four cDNAs characterized by sequence analysis is restricted to the N-terminal 120 amino acids of the putative proteins. This region shows striking homology to the N-proximal domain of the myb oncoproteins from vertebrates and invertebrates. Within the myb proto-oncogene family this part of the respective gene products functions as a DNA binding domain. Acidic domains are present in the C-proximal protein segments. Conservation of these sequences, together with the genetically defined regulator function of the C1 gene product, suggest that myb-related plant genes code for trans-acting factors which regulate gene expression in a given biosynthetic pathway.     

<Emphasis Type="Italic">Drosophila</Emphasis> retinophilin contains MORN repeats and is conserved in humans

The function of conserved novel human genes can be efficiently addressed in genetic model organisms. From a collection of genes expressed in the Drosophila visual system, cDNAs expressed in vertebrates were identified and one similar to a novel human gene was chosen for further investigation. The results reported here characterize the Drosophila retinophilin gene and demonstrate that a similar gene is expressed in the human retina. The Drosophila and human retinophilin sequences are 50% identical, and they share an additional 16% conserved substitutions. Examination of the cDNA and genomic sequence indicates that it corresponds to the gene CG10233 of the annotated genome and predicts a 22.7 kDa protein. Polyclonal antibodies generated to a predicted retinophilin peptide recognize an antigen in Drosophila photoreceptor cells. The retinophilins encode 4 copies of a repeat associated with a Membrane Occupation and Recognition Nexus (MORN) function first discovered in junctophilins, which may interact with the plasma membrane. These results therefore show that Drosophila retinophilin is expressed in fly photoreceptor cells, demonstrate that a conserved human gene is expressed in human retina, and suggest that a mutational analysis of the Drosophila gene would be valuable.     

Identification and expression analysis of cold-regulated genes from the cold-hardy Citrus relative Poncirus trifoliata (L.) Raf.

Citrus is a cold-sensitive genus and most commercially important varieties of citrus are susceptible to freezes. On the other hand, Poncirus trifoliata (L.) Raf. is an interfertile Citrus relative that can tolerate temperatures as low as −26°C when fully cold acclimated. Therefore, it has been used for improving cold tolerance in cold-sensitive commercial citrus rootstock varieties and in attempts to improve scion varieties. In this study, cDNA libraries were constructed from both 2-day cold-acclimated and from non-acclimated Poncirus seedlings using a subtractive hybridization method with the objective of identifying cold-regulated genes. A total of 192 randomly picked clones, 136 from the cold-induced library and 56 from the cold-repressed library, were sequenced. The majority of these clones showed sequence homology to previously identified cold-induced and/or environmental stress-regulated genes in Arabidopsis. In addition, some of them shared homology with cold and/or environmental stress-induced genes previously identified in other herbaceous and woody perennial plants and some showed no homology with sequences in GenBank. When these 192 cDNAs were analyzed by reverse northern blot with cold-acclimated and non-acclimated probes, 92 of the cDNAs displayed significantly increased expression, ranging from 2 to 49-fold, during cold acclimation; all 92 were from the cold-induced library. Surprisingly no clones displayed significantly repressed expression in response to cold. Analysis of a number of selected genes individually in northern blots of mRNA from cold-acclimated and non-acclimated plants largely confirmed the reverse northern analysis, verifying induction of expression of selected cDNAs in response to cold. The results showed that subtractive hybridization is an efficient method for identification of cold-induced genes in plants with limited sequence information available. This study also revealed that genes induced during cold acclimation of the cold-hardy citrus relative P. trifoliata are similar to those in Arabidopsis, indicating that similar pathways may be present and activated during cold acclimation in woody perennial plants.     

分离自印度洋深海沉积物的部分链霉菌分离株多位点序列分析

【目的】采用多位点序列分析方法,研究印度洋3 000 m以下深海沉积物中分离得到的16S rRNA基因比对高度相似的链霉菌菌株的种间系统发育关系,同时探讨各管家基因及多基因聚类分析后的种间区分能力。【方法】以分离自印度洋深海沉积物的7株Streptomyces albidoflavus,11株Streptomyces cavourensis,16株Streptomyces pratensis为研究对象,以16S rRNA、atpD、recA和rpoB基因片段为标记,通过PCR扩增、测序,获得序列。同时从NCBI上下载5株S.pratensis上述4个基因的序列,将所有序列在MLST网站进行比对,并构建系统进化树进行比较。【结果】S.pratensis各菌株种内比较发现,16S rRNA基因构建的系统进化树中相同基因型的菌株没有聚在一起,系统进化树不稳定,区分度不高。其余3个构建的系统进化树稳定,菌株的聚类关系与MLST数据库得到的基因型一致。同时,多基因聚类分析后将菌株分为6个类群。在3个种的种间多位点序列比较中,除区分度明显增加、进化树更加稳定以外,还发现rec A基因进化上比较特殊的菌株。【结论】多位点序列分析将实验菌株分为很多不同的类型,成功地将所分离的链霉菌进行了更细的分类,同时也找到部分菌株在个别基因上差异较大。此方法可以用于相近种的快速鉴定。     

Identification of MHC class I sequences in Chinese-origin rhesus macaques

The rhesus macaque (Macaca mulatta) is an excellent model for human disease and vaccine research. Two populations exhibiting distinctive morphological and physiological characteristics, Indian- and Chinese-origin rhesus macaques, are commonly used in research. Genetic analysis has focused on the Indian macaque population, but the accessibility of these animals for research is limited. Due to their greater availability, Chinese rhesus macaques are now being used more frequently, particularly in vaccine and biodefense studies, although relatively little is known about their immunogenetics. In this study, we discovered major histocompatibility complex (MHC) class I cDNAs in 12 Chinese rhesus macaques and detected 41 distinct Mamu-A and Mamu-B sequences. Twenty-seven of these class I cDNAs were novel, while six and eight of these sequences were previously reported in Chinese and Indian rhesus macaques, respectively. We then performed microsatellite analysis on DNA from these 12 animals, as well as an additional 18 animals, and developed sequence specific primer PCR (PCR-SSP) assays for eight cDNAs found in multiple animals. We also examined our cohort for potential admixture of Chinese and Indian origin animals using a recently developed panel of single nucleotide polymorphisms (SNPs). The discovery of 27 novel MHC class I sequences in this analysis underscores the genetic diversity of Chinese rhesus macaques and contributes reagents that will be valuable for studying cellular immunology in this population.     

Nitric oxide synthase sequences in the marine fish Stenotomus chrysops and the sea urchin Arbacia punctulata, and phylogenetic analysis of nitric oxide synthase calmodulin-binding domains

The phylogenetic distribution and structural diversity of the nitric oxide synthases (NOS) remain important and issues that are little understood. We present sequence information, as well as phylogenetic analysis, for three NOS cDNAs identified in two non-mammalian species: the vertebrate marine teleost fish Stenotomus chrysops (scup) and the invertebrate echinoderm Arbacia punctulata (sea urchin). Partial gene sequences containing the well-conserved calmodulin (CaM)-binding domain were amplified by RT-PCR. Identical 375-bp cDNAs were amplified from scup brain, heart, liver and spleen; this sequence shares 82% nucleic acid and 91% predicted amino acid identity with the corresponding region of human neuronal NOS. A 387-bp cDNA was amplified from sea urchin ovary and testes; this sequence shares 72% nucleic acid identity and 65% deduced amino acid identity with human neuronal NOS. A second cDNA of 381 bp was amplified from sea urchin ovary and it shares 66% nucleic acid and 57% deduced amino acid identity with the first sea urchin sequence. Together with earlier reports of neuronal and inducible NOS sequences in fish, these data indicate that multiple NOS isoforms exist in non-mammalian species. Phylogenetic analysis of these sequences confirms the conserved nature of NOS, particularly of the calmodulin-binding domains.     

Functional analysis of the <Emphasis Type="Italic">BIN2</Emphasis> genes of cotton

Brassinosteroids (BR) promote the elongation of cotton fibers and may be a factor in determining their final length. To begin to understand the role of BR-mediated responses in the development of cotton fibers we have characterized the BIN2 genes of cotton. BIN2 is a member of the shaggy-like protein kinase family that has been identified as a negative regulator of BR signaling in Arabidopsis. Sequence analyses indicate that the tetraploid cotton genome includes four genes with strong sequence similarity to BIN2. These genes fall into two distinct subclasses based on sequence and expression patterns. Sequence comparisons with corresponding genes from cotton species that have the diploid A and D genomes, respectively, shows that each pair of genes comprises homeologs derived from the A and D sub-genomes. Transgenic Arabidopsis plants that express these cotton BIN2 cDNAs show reduced growth and similar phenotypes to the semi-dominant bin2 mutant plants. These results indicate that the cotton BIN2 genes encode functional BIN2 isoforms that can inhibit BR signaling. Further analyses of the function of BIN2 genes and their possible roles in determining fiber yield and quality are underway.     

Characterization of long cDNA clones from human adult spleen. II. The complete sequences of 81 cDNA clones.

To accumulate information on the coding sequences (CDSs) of unidentified genes, we have conducted a sequencing project of human long cDNA clones. Both the end sequences of approximately 10,000 cDNA clones from two size-fractionated human spleen cDNA libraries (average sizes of 4.5 kb and 5.6 kb) were determined by single-pass sequencing to select cDNAs with unidentified sequences. We herein present the entire sequences of 81 cDNA clones, most of which were selected by two approaches based on their protein-coding potentialities in silico: Fifty-eight cDNA clones were selected as those having protein-coding potentialities at the 5'-end of single-pass sequences by applying the GeneMark analysis; and 20 cDNA clones were selected as those expected to encode proteins larger than 100 amino acid residues by analysis of the human genome sequences flanked by both the end sequences of cDNAs using the GENSCAN gene prediction program. In addition to these newly identified cDNAs, three cDNA clones were isolated by colony hybridization experiments using probes corresponding to known gene sequences since these cDNAs are likely to contain considerable amounts of new information regarding the genes already annotated. The sequence data indicated that the average sizes of the inserts and corresponding CDSs of cDNA clones analyzed here were 5.0 kb and 2.0 kb (670 amino acid residues), respectively. From the results of homology and motif searches against the public databases, functional categories of the 29 predicted gene products could be assigned; 86% of these predicted gene products (25 gene products) were classified into proteins relating to cell signaling/communication, nucleic acid management, and cell structure/motility.     

Concurrent evolution of nitrogenase genes and 16S rRNA in Rhizobium species and other nitrogen fixing bacteria

It was known that nitrogenase genes and proteins are well conserved even though they are present in a large variety of phylogenetically diverse nitrogen fixing bacteria. This has lead to the speculation, among others, that nitrogen fixation (nif) genes were spread by lateral gene transfer relatively late in evolution. Here we report an attempt to test this hypothesis.We had previously established the complete nucleotide sequences of the three nitrogenase genes from Bradyrhizobium japonicum, and have now analyzed their homologies (or the amino acid sequence homologies of their gene products) with corresponding genes (and proteins) from other nitrogen fixing bacteria. There was a considerable sequence conservation which certainly reflects the strict structural requirements of the nitrogenase iron-sulfur proteins for catalytic functioning. Despite this, the sequences were divergent enough to classify them into an evolutionary scheme that was conceptually not different from the phylogenetic positions, based on 16S rRNA homology, of the species or genera harboring these genes. Only the relation of nif genes of slow-growing rhizobia (to which B. japonicum belongs) and fast-growing rhizobia was unexpectedly distant. We have, therefore, performed oligonucleotide cataloguing of their 16S rRNA, and found that there was indeed only a similarity of S _AB=0.53 between fast- and slowgrowing rhizobia.In conclusion, the results suggest that nif genes may have evolved to a large degree in a similar fashion as the bacteria which carry them. This interpretation would speak against the idea of a recent lateral distribution of nif genes among microorganisms.     

The dcr gene family of Desulfovibrio: Implications from the sequence of dcrH and phylogenetic comparison with other mcp genes

Desulfovibrio vulgaris Hildenborough contains a family of genes for methyl-accepting chemotaxis proteins (MCPs). Here we report the complete sequence of the gene for Desulfovibrio chemoreceptor H (dcrH). The deduced amino acid sequence of DcrH protein, which has an enlarged N-terminal, ligand binding domain, indicates a structure similar to that of other MCPs. Comparison of the sequences for DcrA, determined earlier, and DcrH indicated that similarity is essentially limited to the C-terminal excitation region. The dcr gene family differs, in this respect, from mcp gene families in other eubacteria (e.g. Escherichia coli and Bacillus subtilis), where MCPs share significant homology throughout their C-terminal signal transduction domains. This may point to an ancient evolutionary origin of the dcr gene family, which is widely distributed throughout the genus Desulfovibrio. The evolutionary origin of mcp genes was traced by comparing nucleotide sequences for the excitation region that is common to all MCPs. Phylogenetic analysis of sequences for thirty mcp genes from nine eubacterial and one archaebacterial species suggested that multiplication of mcp genes has occurred at least twice since the eubacteria diverged from the archaebacteria.Nucleotide accession number: The nucleotide sequence reported in this paper has been entered into GenBank under accession number U30319. Phone: 403-220-6388. Fax: 403-289-9311. Electronic mail address: voordouw@acs.ucalgary.ca.     

Moss (Physcomitrella patens) Expressed Sequence Tags Include Several Sequences which are Novel for Plants*

The moss Physcomitrella patens (Hedw.) B.S.G. is the first land plant in which gene disruption by homologous recombination Is directly accessible. In order to obtain cloned sequences which may be used in such an approach, complementary DNAs (cDNAs) have been isolated by subtractlve hybridisation of representative cDNA libraries from cytoklnin-treated tissue. Sequencing of these clones from both ends yielded over 35 kb of non-redundant sequence Information, of which 20 kb results from clones which appear to be novel to plants. Database comparisons have revealed that 39 of the expressed sequence tags (ESTs) generated show significant homology to identified sequences. Analysis of these ESTs shows a high degree of conservation between Physcomitrella and seed plant sequences, and codon usage is found to be very similar to that In dicotyledonous species. Furthermore, 43 sequences showing no significant homology to sequences in the databases represent previously unidentified expressed genes.     

Anther-specific,developmentally regulated expression of genes encoding a new class of proline-rich proteins in sunflower

We have used RNA gel blot analysis to demonstrate the anther-specific expression of three genes in sunflower. Expression of these genes was first detected shortly before flower opening, which occurs sequentially on the sunflower inflorescence, and continues during pollination. In contrast, these genes are not expressed (or only weakly expressed) in a male-sterile line in which anther development aborts. In situ hybridization experiments showed that these genes are only expressed in the single cell layer of the sunflower anther epidermis. In the case of one of these genes, which codes for an abundant mRNA, we report the peptide sequences deduced from the sequence of two similar but non identical cDNAs. These proteins contain a potential signal peptide and are characterized by the presence of a proline-rich region which reads KPSTPAPPPPPP(PP)K. Our results also suggest that several proline-rich proteins of unknown functions are specifically synthesized during the maturation of anthers in sunflower.     

Identification of two differentially regulated isoforms of protochlorophyllide oxidoreductase (POR) from tobacco revealed a wide variety of light- and development-dependent regulations of POR gene expression among angiosperms

NADPH-protochlorophyllide oxidoreductase (POR) catalyzes the light-dependent reduction of protochlorophyllide a in the chlorophyll biosynthetic pathway. Here, we identified two distinct POR cDNAs from tobacco. Both POR isoforms are encoded by a respective single copy gene in tobacco genome. The overall deduced amino acid sequences of two tobacco cDNAs, designated here POR1 and POR2, displayed significant identities (∼75%), but showed different patterns of light and developmental regulation. In contrast to the previously isolated POR isoforms of Arabidopsis thaliana and barley, the expression of both tobacco POR isoforms were not negatively regulated by light and persisted in matured green tissues. Furthermore, the expression of both genes appeared to be regulated by a diurnal regulation. These results show a wide variety of light- and development-dependent regulations of POR gene expression among angiosperms. Furthermore, phylogenetic analysis including tobacco revealed that POR gene family is differentially represented by angiosperms, most of which is probably caused by independent gene duplication in individual plant. Present results imply a modification of the previous concept that chlorophyll biosynthesis and chloroplast differentiation in angiosperms are ubiquitously controlled by unique functions of two POR isoforms. This revised version was published online in June 2006 with corrections to the Cover Date.