果蝇细胞凋亡核心机制的基因组比较

基因组比较研究是从基因组序列推测调控网络的主要途径。细胞凋亡信号网络是调控网络的一个典型代表。ＥＧＬ１、ＣＥＤ３、ＣＥＤ４和ＣＥＤ９及其同源蛋白质的线虫和哺乳动物构成保守的凋亡核心机制。目前果蝇细胞凋亡核心机制尚不完整,还未找到ＥＧＬ１和ＣＥＤ９类似蛋白质。通过一系列基于生物信息学的基因组比较分析,在果蝇的基因组数据库中发现了两个ＢＣＬ２／ＣＥＤ９和一个ＥＧＬ１的同源蛋白质的编码基因,并重构了果蝇     

More genes in vertebrates?

With the acquisition of complete genome sequences from several animals, there is renewed interest in the pattern of genome evolution on our own lineage. One key question is whether gene number increased during chordate or vertebrate evolution. It is argued here that comparing the total number of genes between a fly, a nematode and human is not appropriate to address this question. Extensive gene loss after duplication is one complication; another is the problem of comparing taxa that are phylogenetically very distant. Amphioxus and tunicates are more appropriate animals for comparison to vertebrates. Comparisons of clustered homeobox genes, where gene loss can be identified, reveals a one to four mode of evolution for Hox and ParaHox genes. Analyses of other gene families in amphioxus and vertebrates confirm that gene duplication was very widespread on the vertebrate lineage. These data confirm that vertebrates have more genes than their closest invertebrate relatives, acquired through gene duplication. abbreviations IHGSC, International Human Genome Sequencing Consortium; TCESC, The C. elegans Sequencing Consortium.     

Ribosomal proteins L34 and S29 of amphioxus Branchiostoma belcheri tsingtauense: cDNAs cloning and gene copy number

The complete cDNA and deduced amino-acid sequences of ribosomal proteins L34 (AmphiL34) and S29 (AmphiS29) from the amphioxus Branchiostoma belcheri tsingtauense were identified in this study. The AmphiL34 cDNA is 435 nucleotides in length and encodes a 118 amino-acid protein with calculated molecular mass of 13.6 kDa. It shares 53.6-67.5% amino-acid sequence identity with its eukaryotic counterparts including human, mouse, rat, pig, frog, catfish, fruit fly, mosquito, armyworm, nematode and yeast. The AmphiS29 cDNA comprises 453 nucleotides and codes for a 56 amino-acid protein with a calculated molecular mass of 6.6 kDa. It shows 66.1-78.6% amino-acid sequence identity to eukaryotic S29 proteins from human, mouse, rat, pig, zebrafish, seahorse, fruit fly, nematode, sea hare and yeast. AmphiL34 contains a putative nucleolar localization signal, while AmphiS29 has a zinc finger-like domain. A phylogenetic tree deduced from the conserved sequences of AmphiL34 and AmphiS29 and other known counterparts indicates that the positions of AmphiL34/AmphiS29 are intermediate between the vertebrate and invertebrate L34/S29. Southern blot analysis demonstrates the presence of one copy of the L34 gene and 2-3 copies of the S29 gene in the genome of the amphioxus B. belcheri tsingtauense. This is in sharp contrast to the existence of 7-9 copies of the L34 gene and 14-17 copies of the S29 gene in the rat genome. These date suggest that housekeeping genes like AmphiL34 and AmphiS29 have undergone large-scale duplication in the chordate lineage.     

Human and nematode orthologs--lessons from the analysis of 1800 human genes and the proteome of Caenorhabditis elegans

Recently, we have defined and analyzed over 1800 orthologous human and rodent genes. Here we extend this work to compare human and Caenorhabditis elegans coding sequences. 1880 human proteins were compared with about 20000 predicted nematode proteins presumably comprising nearly the complete proteome of C. elegans. We found that 44% of human/rodent orthologs have convincing nematode counterparts. On average, the amino acid similarity and identity between aligned human and C. elegans orthologous gene products are 69.3% and 49.1% respectively, and the nucleotide identity is 49.8%. Detailed investigation of our results suggests that some nematode gene predictions are incorrect, leading to erroneous pairing with human genes (e.g. calcineurin and polymerase II elongation factor III). Furthermore, other proteins (i.e. homologs of human ribosomal proteins S20 and L41, thymosin) are missing entirely from the nematode proteome, suggesting that it may not be complete. These results underscore the fact that metazoan gene prediction is a very challenging task and that most computer-predicted nematode genes require supporting evidence of their existence from comparative genomics and/or laboratory investigation.     

Comparisons of the complete sequences of two collagen genes from Caenorhabditis elegans

Several collagen genes have been isolated from the nematode Caenorhabditis elegans. The complete nucleotide sequences of two of these genes, col-1 and col-2, have been determined. These collagen genes differ from vertebrate collagen genes in that they contain only one or two introns, their triple-helical regions are interrupted by nonhelical amino acid sequences and they are smaller. A high degree of nucleotide and amino acid homology exists between col-1 and col-2. In particular, the regions around cysteines and lysines are most highly conserved. The C. elegans genome contains 50 or more collagen genes, the majority of which probably encode cuticle collagens; col-1 and col-2 apparently are members of this large family of cuticle collagen genes.     

Genes coding for 5S ribosomal RNA of the nematode Caenorhabditis elegans

We have identified a 1-kb genomic sequence that represents the major class of 5S rRNA genes in the nematode Caenorhabditis elegans. This 1-kb sequence is tandemly repeated 110 times in the haploid genome forming a single homogeneous gene family. Other nematode genomic sequences, distinct from the major 1-kb repeat class but homologous to it, may represent dispersed 5S rRNA genes or the ends of a gene cluster. One such fragment shows a restriction fragment length difference between two C. elegans strains. This should allow the genetic analysis of 5S rRNA-coding DNA (5S X rDNA) and its flanking regions in C. elegans.     

The genome of Oscheius tipulae: determination of size, complexity, and structure by DNA reassociation using fluorescent dye.

This work describes the physicochemical characterization of the genome and telomere structure from the nematode Oscheius tipulae CEW1. Oscheius tipulae is a free-living nematode belonging to the family Rhabditidae and has been used as a model system for comparative genetic studies. A new protocol that combines fluorescent detection of double-stranded DNA and S1 nuclease was used to determine the genome size of O. tipulae as 100.8 Mb (approximately 0.1 pg DNA/haploid nucleus). The genome of this nematode is made up of 83.4% unique copy sequences, 9.4% intermediate repetitive sequences, and 7.2% highly repetitive sequences, suggesting that its structure is similar to those of other nematodes of the genus Caenorhabditis. We also showed that O. tipulae has the same telomere repeats already found in Caenorhabditis elegans at the ends and in internal regions of the chromosomes. Using a cassette-ligation-mediated PCR protocol we were able to obtain 5 different putative subtelomeric sequences of O. tipulae, which show no similarity to C. elegans or C. briggsae subtelomeric regions. DAPI staining of hermaphrodite gonad cells show that, as detected in C. elegans and other rhabditids, O. tipulae have a haploid complement of 6 chromosomes.     

Changes in Nuclear Receptor and Vitellogenin Gene Expression in Response to Steroids and Heavy Metal in Caenorhabditis elegans

To gain basic understanding of the reproductive and developmental effects of endocrine disrupting chemicals in invertebrates, we have used C. elegans as an animal model. The completion of the C. elegans genome sequence brings to bear microarray analysis as a tool for these studies. We previously showed that the C. elegans genome was responsive to vertebrate steroid hormones, and changes in gene expression of traditional biomarkers used in environmental studies were detected; i.e., vitellogenin (vtg), cytochrome P450 (cyp450), glutathione-S-transferase (gst) and heat shock proteins (hsp). The data were interpreted to suggest that exogenous lipophilic compounds can be metabolized via cytochrome P450 proteins, and that the resulting metabolites can bind to members of the Nuclear Receptor (NR) class of proteins and regulate gene expression. In the present study, using DNA microarrays, we examined the pattern of gene expression after progesterone (10(-5), 10(-7) M), estradiol (10(-5) M), cholesterol (10(-9) M) and cadmium (0.1, 1 and 10 μM) exposure, with special attention to the members of NRs. Of approximately 284 NRs in C. elegans, expression of 25 NR genes (representing 9% of the total NRs in C. elegans) was altered after exposure to steroids. Of note, each steroid activated or inhibited different subsets of NR genes, and only estradiol regulated NR genes implicated in neurogenesis. These results suggest that NRs respond to a variety of exogenous steroids, which regulate important metabolic and developmental pathways. The response of the C. elegans genome to cholesterol and cadmium was analyzed in more detail. Cholesterol is a probable precursor to signaling molecules that may interact with NRs and we focused on expression of genes related to lipid metabolism (cyp450), transport and storage (i.e., vitellogenin). Worms exposed to cadmium respond principally by activating the expression of genes encoding stress-responsive proteins, such as mtl-2 and cdr-1, and no significant changes in expression of NRs or vtg genes were observed. The possible implications of these results with regard to the evolution of steroid receptors, endocrine disruption and the role of vitellogenin as a lipid transporter are discussed.     

High-throughput RNAi in Caenorhabditis elegans: genome-wide screens and functional genomics

The phenomenon of RNA-mediated interference (RNAi) was first discovered in the nematode Caenorhabditis elegans, in which introduction of double-stranded RNA causes specific inactivation of genes with corresponding sequences. Technical advances in RNAi methodology and the availability of the complete genome sequence have enabled the high-throughput, genome-wide RNAi analysis of this organism. Several groups have used large-scale RNAi to systematically examine every C. elegans gene for knock-down phenotypes, providing basal information to be mined in more detailed studies. Now, in addition to functional genomic RNAi analyses, high-throughput RNAi is also routinely used for rapid, genome-wide screens for genes involved in specific biological processes. The integration of high-throughput RNAi experiments with other large-scale data, such as DNA microarrays and protein-protein interaction maps, enhances the speed and reliability of such screens. The accumulation of RNAi phenotype data dramatically accelerates our understanding of this organism at the genetic level.     

Reverse genetics of Caenorhabditis elegans.

It is somewhat ironic that animals that are the prime choice for detailed genetic analysis, such as the fruit fly and the nematode, have thus far been largely refractory to reverse genetic analysis. Their detailed genetic map, and small genome size have made them subjects of ambitious genome analysis projects, but there is still no strategy to introduce desired changes into their genomes by homologous recombination. Some alternative approaches have recently become available; this review describes possibilities and unsolved problems for reverse genetics in the nematode Caenorhabditis elegans. The transposon Tc1 could prove to be very useful for the isolation of knock out mutants, and possibly also for introduction of more subtle alterations.     

Functional genomic approaches using the nematode Caenorhabditis elegans as a model system

Since the completion of the genome project of the nematode C. elegans in 1998, functional genomic approaches have been applied to elucidate the gene and protein networks in this model organism. The recent completion of the whole genome of C. briggsae, a close sister species of C. elegans, now makes it possible to employ the comparative genomic approaches for identifying regulatory mechanisms that are conserved in these species and to make more precise annotation of the predicted genes. RNA interference (RNAi) screenings in C. elegans have been performed to screen the whole genome for the genes whose mutations give rise to specific phenotypes of interest. RNAi screens can also be used to identify genes that act genetically together with a gene of interest. Microarray experiments have been very useful in identifying genes that exhibit co-regulated expression profiles in given genetic or environmental conditions. Proteomic approaches also can be applied to the nematode, just as in other species whose genomes are known. With all these functional genomic tools, genetics will still remain an important tool for gene function studies in the post genome era. New breakthroughs in C. elegans biology, such as establishing a feasible gene knockout method, immortalized cell lines, or identifying viruses that can be used as vectors for introducing exogenous gene constructs into the worms, will augment the usage of this small organism for genome-wide biology.     

Consistency of genome-based methods in measuring Metazoan evolution

Seven distinct genome-wide divergence measures were applied pairwise to the nine sequenced animal genomes of human, mouse, rat, chicken, pufferfish, fruit fly, mosquito, and two nematode worms (Caenorhabditis briggsae and Caenorhabditis elegans). Qualitatively, all of these divergence measures are found to correlate with the estimated time since speciation; however, marked deviations are observed in a few lineages. The distinct genome divergence measures also correlate well among themselves, indicating that most of the processes shaping genomes are dominated by neutral events. The deviations from the clock-like scenario in some lineages are observed consistently by several measures, implicitly confirming their reliability.     

Extent of gene duplication in the genomes of Drosophila, nematode, and yeast

We conducted a detailed analysis of duplicate genes in three complete genomes: yeast, Drosophila, and Caenorhabditis elegans. For two proteins belonging to the same family we used the criteria: (1) their similarity is > or =I (I = 30% if L > or = 150 a.a. and I = 0.01n + 4.8L(-0.32(1 + exp(-L/1000))) if L < 150 a.a., where n = 6 and L is the length of the alignable region), and (2) the length of the alignable region between the two sequences is > or = 80% of the longer protein. We found it very important to delete isoforms (caused by alternative splicing), same genes with different names, and proteins derived from repetitive elements. We estimated that there were 530, 674, and 1,219 protein families in yeast, Drosophila, and C. elegans, respectively, so, as expected, yeast has the smallest number of duplicate genes. However, for the duplicate pairs with the number of substitutions per synonymous site (K(S)) < 0.01, Drosophila has only seven pairs, whereas yeast has 58 pairs and nematode has 153 pairs. After considering the possible effects of codon usage bias and gene conversion, these numbers became 6, 55, and 147, respectively. Thus, Drosophila appears to have much fewer young duplicate genes than do yeast and nematode. The larger numbers of duplicate pairs with K(S) < 0.01 in yeast and C. elegans were probably largely caused by block duplications. At any rate, it is clear that the genome of Drosophila melanogaster has undergone few gene duplications in the recent past and has much fewer gene families than C. elegans.     

Cuticle collagen genes. Expression in Caenorhabditis elegans

Collagen is a structural protein used in the generation of a wide variety of animal extracellular matrices. The exoskeleton of the free-living nematode, Caenorhabditis elegans, is a complex collagen matrix that is tractable to genetic research. Mutations in individual cuticle collagen genes can cause exoskeletal defects that alter the shape of the animal. The complete sequence of the C. elegans genome indicates upwards of 150 distinct collagen genes that probably contribute to this structure. During the synthesis of this matrix, individual collagen genes are expressed in distinct temporal periods, which might facilitate the formation of specific interactions between distinct collagens.     

Nematode repetitive DNA with ARS and segregation function in Saccharomyces cerevisiae.

Several members of a repetitive DNA family in the nematode Caenorhabditis elegans have been shown to express ARS and centromeric function in Saccharomyces cerevisiae. The repetitive family, denoted CeRep3, consists of dispersed repeated elements about 1 kilobase in length, present 50 to 100 times in the nematode genome. Three elements were sequenced and found to contain DNA sequences homologous to yeast ARS and CEN consensus sequences. Nematode DNA segments containing these repeats were tested for ARS and CEN (or SEG) function after ligation to shuttle vectors and introduction into yeast cells. Such nematode segments conferred ARS function to the plasmid, as judged by an increased frequency of transformation compared with control plasmids without ARS function. Some, but not all, also conferred to the plasmid increased mitotic stability, increased frequency of 2+:2- segregation in meiosis, and decreased plasmid copy number. These effects are similar to those of yeast centromeric DNA. In view of these results, we suggest that the CeRep3 repetitive family may have replication and centromeric functions in C. elegans.