A comparative map of the zebrafish genome

Zebrafish mutations define the functions of hundreds of essential genes in the vertebrate genome. To accelerate the molecular analysis of zebrafish mutations and to facilitate comparisons among the genomes of zebrafish and other vertebrates, we used a homozygous diploid meiotic mapping panel to localize polymorphisms in 691 previously unmapped genes and expressed sequence tags (ESTs). Together with earlier efforts, this work raises the total number of markers scored in the mapping panel to 2119, including 1503 genes and ESTs and 616 previously characterized simple-sequence length polymorphisms. Sequence analysis of zebrafish genes mapped in this study and in prior work identified putative human orthologs for 804 zebrafish genes and ESTs. Map comparisons revealed 139 new conserved syntenies, in which two or more genes are on the same chromosome in zebrafish and human. Although some conserved syntenies are quite large, there were changes in gene order within conserved groups, apparently reflecting the relatively frequent occurrence of inversions and other intrachromosomal rearrangements since the divergence of teleost and tetrapod ancestors. Comparative mapping also shows that there is not a one-to-one correspondence between zebrafish and human chromosomes. Mapping of duplicate gene pairs identified segments of 20 linkage groups that may have arisen during a genome duplication that occurred early in the evolution of teleosts after the divergence of teleost and mammalian ancestors. This comparative map will accelerate the molecular analysis of zebrafish mutations and enhance the understanding of the evolution of the vertebrate genome.     

Genetic linkage mapping of zebrafish genes and ESTs

Genetic screens in zebrafish (Danio rerio) have isolated mutations in hundreds of genes essential for vertebrate development, physiology, and behavior. We have constructed a genetic linkage map that will facilitate the identification of candidate genes for these mutations and allow comparisons among the genomes of zebrafish and other vertebrates. On this map, we have localized 771 zebrafish genes and expressed sequence tags (ESTs) by scoring single-stranded conformational polymorphisms (SSCPs) in a meiotic mapping panel. Of these sequences, 642 represent previously unmapped genes and ESTs. The mapping panel was comprised of 42 homozygous diploid individuals produced by heat shock treatment of haploid embryos at the one-cell stage (HS diploids). This "doubled haploid" strategy combines the advantages of mapping in haploid and standard diploid systems, because heat shock diploid individuals have only one allele at each locus and can survive to adulthood, enabling a relatively large quantity of genomic DNA to be prepared from each individual in the mapping panel. To integrate this map with others, we also scored 593 previously mapped simple-sequence length polymorphisms (SSLPs) in the mapping panel. This map will accelerate the molecular analysis of zebrafish mutations and facilitate comparative analysis of vertebrate genomes.     

A genetic linkage map of the chromosome 4 short arm

We have generated an 18-interval contiguous genetic linkage map of human chromosome 4 spanning the entire short arm and proximal long arm. Fifty-seven polymorphisms, representing 42 loci, were analyzed in the Venezuelan reference pedigree. The markers included seven genes (ADRA2C, ALB, GABRB1, GC, HOX7, IDUA, QDPR), one pseudogene (RAF1P1), and 34 anonymous DNA loci. Four loci were represented by microsatellite polymorphisms and one (GC) was expressed as a protein polymorphism. The remainder were genotyped based on restriction fragment length polymorphism. The sex-averaged map covered 123 cM. Significant differences in sex-specific rates of recombination were observed only in the pericentromeric and proximal long arm regions, but these contributed to different overall map lengths of 115 cM in males and 138 cM in females. This map provides 19 reference points along chromosome 4 that will be particularly useful in anchoring and seeding physical mapping studies and in aiding in disease studies.     

A novel PCR-based technique using expressed sequence tags and gene homology for murine genetic mapping: localization of the complement genes

The complement system is a cascade of serum proteins and receptors which forms a vital arm of innate immunity and enhances the adaptive immune response. This work establishes the chromosomal localization of four key genes of the murine complement system. Mapping was performed using a novel and rapid PCR restriction length polymorphism method which was developed to exploit the murine expressed sequence tag (EST) database. This technique circumvents the laborious cDNA or genomic cloning steps of other mapping methods by relying on EST data and the prediction of exon-intron boundaries. This method can be easily applied to the genes of other systems, ranging from the interests of the individual researcher to large-scale gene localization projects. Here the complement system, probably one of the most well-characterized areas of immunology, was used as a model system. It was shown that the C3a receptor C1r and C1s genes form an unexpected complement gene cluster towards the telomeric end of chromosome 6. The second mannose binding lectin-associated serine protease gene was mapped to the telomeric end of chromosome 4, which is distinct from other complement-activating serine proteases. These results provide new insights into the evolution of this group of proteins.     

A genetic map of chromosome 19 based on family linkage data

A large linkage group has been assigned to chromosome 19. The loci have been ordered using a two-point mapping analysis based on all available family linkage data summarized in the form of lod score tables.     

A genetic linkage map of the apicomplexan protozoan parasite Eimeria tenella

Apicomplexan protozoan parasites have complex life cycles that involve phases of asexual and sexual reproduction. Some genera have intermediate insect hosts, for example, Plasmodium spp. (the cause of malaria), but related genera such as Eimeria spp. (causative agents of coccidiosis in poultry) have a direct life cycle occurring in only a single host. Mechanisms that regulate the life cycles of apicomplexan parasites are unknown, but the intracellular growth of avian Eimeria spp. is easily shortened by serial selection for the first parasites to complete the transition from asexual to sexual reproduction (to yield so-called precocious lines). To investigate the genetic basis of such an abbreviated life cycle, we have used the species E. tenella and analyzed the inheritance of 443 polymorphic DNA markers in 22 recombinant cloned progeny derived from a cross between parents that had selectable phenotypes of precocious development or resistance to an anticoccidial drug. The markers were placed in 16 linkage groups (which defined 12 chromosomes) and a further 57 unlinked groups. Two linkage groups showed an association (P =.0105) with the traits of precocious development or drug-resistance and were mapped to chromosome 2 (ca 1.2 Mbp) and chromosome 1 (ca 1.0 Mbp), respectively. The map provides a framework for further studies on the identification of genetic loci implicated in the regulation of the life cycle of an important protozoan parasite and a representative of a major taxonomic group. [A table with the segregation data is available as an online supplement at http://www.genome.org.]     

Development of a molecular genetic linkage map for <Emphasis Type="Italic">Colletotrichum lindemuthianum</Emphasis> and segregation analysis of two avirulence genes

A framework genetic map was developed for the fungal pathogen Colletotrichum lindemuthianum, the causal agent of anthracnose of common bean (Phaseolus vulgaris L.). This is the first genetic map for any species within the family Melanconiaceae and the genus Colletotrichum and provides the first estimate of genome length for C. lindemuthianum. The map was generated using 106 haploid F1 progeny derived from crossing two Mexican C. lindemuthianum isolates differing in two avirulence genes (AvrclMex and AvrclTO). The map comprises 165 AFLP markers covering 1,897 cM with an average spacing of 11.49 cM. The markers are distributed over 19 major linkage groups containing between 5 and 25 markers each and the genome length was estimated to be approximately 3,241 cM. The avirulence genes AvrclMex and AvrclTO segregate in a 1:1 ratio supporting the gene for gene hypothesis for the incompatible reaction between C. lindemuthianum and P. vulgaris, but could not be incorporated into the genetic map. This initial outline map forms the basis for the development of a more detailed C. lindemuthianum linkage map, which would include other types of molecular markers and allow the location of genes previously isolated and characterized in this species.     

The inheritance of immunoglobulin E: genetic linkage analysis

Linkage analyses between 21 genetic markers including HLA-A, B, and the postulated locus for determining total serum IgE levels were done to try to clarify the inheritance of total IgE levels and to map the locus. A total of 316 individuals from five Mormon kindreds were studied, and data from an additional 204 Amish individuals from 11 families were analyzed for possible HLA linkage. Segregation analyses of both data sets did not give clear definition of the mode of inheritance of total IgE levels, but purely environmental models were rejected. Linkage analyses gave significant evidence against HLA linkage with the codominant, recessive, or dominant model of inheritance for total IgE levels. No significant evidence for linkage with any of the genetic markers was obtained. Since total serum IgE levels are correlated with allergies, understanding the genetics of total IgE levels is important to understanding the genetics of allergic disease in man.     

Emilin genes are duplicated and dynamically expressed during zebrafish embryonic development.

Emilins are a family of extracellular matrix proteins with common structural organization and containing a characteristic N-terminal cysteine-rich domain. The prototype of this family, Emilin-1, is found in human and murine organs in association with elastic fibers, and other emilins were recently isolated in mammals. To gain insight into these proteins in lower vertebrates, we investigated the expression of emilins in the fish Danio rerio. Using sequence similarity tools, we identified eight members of this family in zebrafish. Each emilin gene has two paralogs in zebrafish, showing conserved structure with the human ortholog. In situ hybridization revealed that expression of zebrafish emilin genes is regulated in a spatiotemporal manner during embryonic development, with overlapping and site-specific patterns mostly including mesenchymal structures. Expression of certain emilin genes in peculiar areas, such as the central nervous system or the posterior notochord, suggests that they may play a role in key morphogenetic processes.     

Marfan syndrome: exclusion of genetic linkage to three major collagen genes

The Marfan syndrome is an autosomal dominant connective tissue disorder with pleiotropic manifestations affecting skeletal, ocular and cardiovascular systems. Because the fibrillar collagens are major structural components of connective tissue, the hypothesis has long been set forth that the Marfan syndrome is a disorder of fibrillar collagen. We have investigated this hypothesis by performing linkage studies in 12 multiplex families with the Marfan syndrome, using restriction fragment length polymorphisms (RFLP's) associated with 3 genes encoding chains of fibrillar collagens. The data exclude linkage to all 3 candidate genes in 2 families and at least 1 of the candidates is excluded in 6 additional families. Each candidate was excluded in at least 3 families. In no case was strong evidence in favor of linkage of the Marfan syndrome to any of the 3 genes observed. These data speak against the hypothesis that mutations in one or more of these 3 fibrillar collagens cause the classic Marfan syndrome.     

Identification and linkage of the proteasome activator complex PA28 subunit genes in zebrafish

PA28 is an activator of the latent 20S proteasome, a large multisubunit complex involved in intracellular proteolysis. Two forms of hexameric PA28 have been identified, PA28-(alphabeta)3 and PA28-(gamma)6, of which the former is of immunological importance. Both the PA28-alpha and PA28-beta subunits are inducible by interferon-gamma (IFN-gamma) and the PA28-(alphabeta)3 complex enhances the ability of the 20S proteasome to produce peptides suited for binding to major histocompatibility complex (Mhc) class I molecules. To identify the homologues of the PA28 subunits in zebrafish we screened a cDNA library and obtained full-length cDNA sequences of the genes PSME1, PSME2 and PSME3 coding for the PA28-alpha, PA28-beta and PA28-gamma subunits, respectively. Phylogenetic analysis indicates the existence of the ancestors of all three genes prior to the divergence of tetrapods and bony fishes. The IFN-gamma-inducible subunits, PA28-alpha and PA28-beta, evolve faster than the presumably older PA28-gamma subunit. Using zebrafish radiation hybrid panels, the genes PSME2 and PSME3 were mapped to linkage group 12 and shown to be separated by a distance of less than 2.4 cM. This observation suggests that an intrachromosomal duplication event created the precursor of the IFN-gamma-inducible genes from a PA28-gamma-like ancestor prior to their recruitment into the Mhc class I peptide presentation pathway.     

Characterization and comparative expression of zebrafish calpain system genes during early development

The classic calpain system has been implicated in regulating a variety of cellular processes including cell adhesion, migration, and intracellular signaling; however, little is known regarding the function of this system in vivo. Two heterodimeric Ca(2+)-dependent cysteine proteases, mu-calpain (CAPN1) and m-calpain (CAPN2), and the endogenous inhibitor calpastatin (CAST) comprise the classic/ubiquitous calpain system in mammals. Recently, knockout of two murine classic calpain genes, Capn2 and Capn4/Capns1, revealed that components of the classic system are indispensable for preimplantation development. We identified four classic calpain catalytic subunit genes (capn1a, 1b, 2a, 2b), two regulatory subunit genes (capns1a, 1b), and calpastatin (cast) from the zebrafish. Our data suggest that the components of the classic mammalian system are both conserved and expanded in the teleost lineage. In contrast to the classic/ubiquitous mammalian system, zebrafish calpain system genes acquire unique, tissue-specific patterns of expression within the first 2 days of development.     

Identification of genes expressed by zebrafish oligodendrocytes using a differential microarray screen

Myelination of central nervous system axons requires that oligodendrocytes extend multiple membrane processes that specifically recognize and wrap axons, which is followed by expression of proteins necessary for formation of myelin sheaths. To identify new genes that might be important for myelination, we used microarrays to analyze the expression profiles of cells sorted from transgenic zebrafish embryos and larvae under conditions that permitted or blocked oligodendrocyte development. Here, we describe eight genes that have not been previously implicated in oligodendrocyte development. Among the predicted functions of proteins encoded by these genes are lipid sensing, cell–cell junction formation, cytoskeleton regulation, and intracellular signaling. The predicted functions raise the possibility that these genes are involved in multiple cellular events during oligodendrocyte differentiation and myelin formation. Developmental Dynamics 239:2041–2047, 2010. © 2010 Wiley‐Liss, Inc.     

Physical and genetic mapping of Candida albicans: several genes previously assigned to chromosome 1 map to chromosome R, the rDNA-containing linkage group.

Analysis of the karyotypes of multiple Candida albicans isolates by pulsed-field electrophoresis confirms the observation by Lasker et al. of eight chromosomes. The genes previously assigned to chromosome 1 in fact fall into two groups, one (including ADE1, SOR9, and CDC10) is linked to the ribosomal DNA genes on a chromosome called R, whereas the others are found on chromosome 1. Chromosome R varies in electrophoretic mobility among strains, usually running equal to or faster than chromosome 1 but in rare cases running slower than chromosome 1. In strain 1012A, the decreased mobility of one homolog is associated with the very large majority of the rDNA genes being on that homolog; the second homolog, with only a few copies, migrates with chromosome 2. Linkage analysis by using spheroplast fusion confirms the gene assignments made by hybridization to blots of the electrophoretic karyotype. A newly cloned gene, LYS2, hybridizes to chromosome 1.     

A consensus linkage map of the chicken genome

A consensus linkage map has been developed in the chicken that combines all of the genotyping data from the three available chicken mapping populations. Genotyping data were contributed by the laboratories that have been using the East Lansing and Compton reference populations and from the Animal Breeding and Genetics Group of the Wageningen University using the Wageningen/Euribrid population. The resulting linkage map of the chicken genome contains 1889 loci. A framework map is presented that contains 480 loci ordered on 50 linkage groups. Framework loci are defined as loci whose order relative to one another is supported by odds greater then 3. The possible positions of the remaining 1409 loci are indicated relative to these framework loci. The total map spans 3800 cM, which is considerably larger than previous estimates for the chicken genome. Furthermore, although the physical size of the chicken genome is threefold smaller then that of mammals, its genetic map is comparable in size to that of most mammals. The map contains 350 markers within expressed sequences, 235 of which represent identified genes or sequences that have significant sequence identity to known genes. This improves the contribution of the chicken linkage map to comparative gene mapping considerably and clearly shows the conservation of large syntenic regions between the human and chicken genomes. The compact physical size of the chicken genome, combined with the large size of its genetic map and the observed degree of conserved synteny, makes the chicken a valuable model organism in the genomics as well as the postgenomics era. The linkage maps, the two-point lod scores, and additional information about the loci are available at web sites in Wageningen (http://www.zod.wau.nl/vf/ research/chicken/frame_chicken.html) and East Lansing (http://poultry.mph.msu.edu/).     

A set of genes selectively expressed in murine dendritic cells: utility of related cis-acting sequences for lentiviral gene transfer

Professional antigen presenting cells such as dendritic cells (DC) and macrophages (Mphi) share similar characteristics; however, they differ in their ability to initiate an immune response. DCs are much more potent in priming and stimulating nai;ve T-cells. Thus, DCs are good targets for the expression of foreign genes to elicit and specifically modify immune responses. To identify DC markers cDNA subtraction was performed using murine MHC class II(high), B7(high) bone marrow derived DCs as tester and interferon-gamma/E. coli lipopolysaccaride (LPS) treated bone marrow derived macrophages as driver. Analysis of 114 resulting clones revealed a diverse pattern of DC selective (DC(DeltaMphi)) gene expression including known genes whose expression in DCs had not been previously demonstrated as well as multiple novel genes. For several identified DC(DeltaMphi) genes, proximal promoter elements were isolated and incorporated into self-inactivating lentiviral GFP reporter vectors. Promoter activity was measured in bone marrow derived macrophages or dendritic cells. Of the promoters analyzed those for B7-DC and CCL17 drove strong GFP expression in DCs but not in resting or activated macrophages. The CCL17 promoter offered the highest level of expression in DCs and was further activated by culture with LPS or interleukin-4 (IL-4). In contrast, the B7-DC promoter was induced by IL-4 but not by LPS. Endogenous CCL17 and B7-DC mRNAs were increased similarly in IL-4 cultured DCs but only CCL17 was induced by LPS. Additionally, IL-4 increased cell surface expression of B7-DC in both immature and mature DCs.     

连锁分析在血友病甲基因诊断中的应用

目的综述及讨论近几年以来连锁分析在血友病甲基因诊断中的应用的研究进展,为血友病甲连锁分析位点的选择提供有利的参考。方法以网络数据库资源为主,查询ScienceDirect,pubmed等数据库关于血友病甲连锁分析研究进展等方面的资料。结果共收集到多篇文献,选择其中16篇进行归纳总结与讨论。结论连锁分析现在在世界范围内都应用广泛,对血友病甲携带者的检出与产前诊断不失为一种简便、快捷实用的方法。但是依然有其局限性,比如,有些多态性标记并不能提供诊断信息,对于一些散发病例,连锁分析则不一定有效。对于基因外的多态性标记,细胞减数分裂期间基因重组会导致连锁分析错误。因此,有待发现更多有效的遗传多态性标记用于血友病甲的连锁分析。