Molecular Biology and Biochemistry, Department of

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High-Throughput In Vivo Analysis of Gene Expression in Caenorhabditis elegans

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2007-09
Abstract: 

Using DNA sequences 5′ to open reading frames, we have constructed green fluorescent protein (GFP) fusions and generated spatial and temporal tissue expression profiles for 1,886 specific genes in the nematode Caenorhabditis elegans. This effort encompasses about 10% of all genes identified in this organism. GFP-expressing wild-type animals were analyzed at each stage of development from embryo to adult. We have identified 5′ DNA regions regulating expression at all developmental stages and in 38 different cell and tissue types in this organism. Among the regulatory regions identified are sequences that regulate expression in all cells, in specific tissues, in combinations of tissues, and in single cells. Most of the genes we have examined in C. elegans have human orthologs. All the images and expression pattern data generated by this project are available at WormAtlas (http://gfpweb.aecom.yu.edu/index) and through WormBase (http://www.wormbase.org).

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Article
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Evidence of a Large Novel Gene Pool Associated with Prokaryotic Genomic Islands

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2005
Abstract: 

Microbial genes that are “novel” (no detectable homologs in other species) have become of increasing interest as environmental sampling suggests that there are many more such novel genes in yet-to-be-cultured microorganisms. By analyzing known microbial genomic islands and prophages, we developed criteria for systematic identification of putative genomic islands (clusters of genes of probable horizontal origin in a prokaryotic genome) in 63 prokaryotic genomes, and then characterized the distribution of novel genes and other features. All but a few of the genomes examined contained significantly higher proportions of novel genes in their predicted genomic islands compared with the rest of their genome (Paired t test = 4.43E-14 to 1.27E-18, depending on method). Moreover, the reverse observation (i.e., higher proportions of novel genes outside of islands) never reached statistical significance in any organism examined. We show that this higher proportion of novel genes in predicted genomic islands is not due to less accurate gene prediction in genomic island regions, but likely reflects a genuine increase in novel genes in these regions for both bacteria and archaea. This represents the first comprehensive analysis of novel genes in prokaryotic genomic islands and provides clues regarding the origin of novel genes. Our collective results imply that there are different gene pools associated with recently horizontally transmitted genomic regions versus regions that are primarily vertically inherited. Moreover, there are more novel genes within the gene pool associated with genomic islands. Since genomic islands are frequently associated with a particular microbial adaptation, such as antibiotic resistance, pathogen virulence, or metal resistance, this suggests that microbes may have access to a larger “arsenal” of novel genes for adaptation than previously thought.

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Article
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Evolution of Duplicated IgH Loci in Atlantic salmon, Salmo salar

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2010
Abstract: 

Background: The Atlantic salmon (Salmo salar) immunoglobulin heavy chain (IgH) locus possesses two parallel IgHisoloci (IGH-A and IGH-B), that are related to the genomic duplication event in the family Salmonidae. Theseduplicated IgH loci in Atlantic salmon provide a unique opportunity to examine the mechanisms of genomediversity and genome evolution of the IgH loci in vertebrates. In this study, we defined the structure of these lociin Atlantic salmon, and sequenced 24 bacterial artificial chromosome (BAC) clones that were assembled into theIGH-A (1.1 Mb) and IGH-B (0.9 Mb) loci. In addition, over 7,000 cDNA clones from the IgH variable (VH) region havebeen sequenced and analyzed.Results: The present study shows that the genomic organization of the duplicated IgH loci in Atlantic salmondiffers from that in other teleosts and other vertebrates. The loci possess multiple Cτ genes upstream of the Cμregion, with three of the Cτ genes being functional. Moreover, the duplicated loci possess over 300 VH segmentswhich could be classified into 18 families. This is the largest number of VH families currently defined in anyvertebrate. There were significant structural differences between the two loci, indicating that both IGH-A and -B locihave evolved independently in the short time after the recent genome duplication approximately 60 mya.Conclusions: Our results indicate that the duplication of the IgH loci in Atlantic salmon significantly contributes tothe increased diversity of the antibody repertoire, as compared with the single IgH locus in other vertebrates.

Document type: 
Article

The NIMA-family Kinase, Nek1 Affects the Stability of Centrosomes and Ciliogenesis

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2008
Abstract: 

Background: Mutations in Nek1 (NIMA-Related Kinase 1) are causal in the murine models ofpolycystic kidney disease kat and kat2J. The Neks are known as cell cycle kinases, but recent workin protists has revealed that in addition to roles in the regulation of cell cycle progression, someNeks also regulate cilia. In most cells, cilia are disassembled prior to mitosis and are regeneratedafter cytokinesis. We propose that Neks participate in the coordination of ciliogenesis with cellcycle progression. Mammalian Nek1 is a candidate for this activity because renal cysts form inresponse to dysfunctional ciliary signalling.Results: Here we report that over-expression of full-length mNek1 inhibited ciliogenesis withoutdisrupting centrosomes in the murine renal epithelial cell line IMCD3. In contrast, over-expressionof the kinase domain with its associated basic region, but without the acidic domain, caused loss ofcentrosomes. As expected, these cells also failed to grow cilia. Both defective ciliogenesis inresponse to too much mNek1 and disassembly of centrosomes in response to expression of thekinase lacking the presumptive regulatory domain was abrogated by kinase-inactivating mutationsor by removal of the coiled-coil domain. We observed that kinase-inactive, C-terminal truncationsof mNek1 retaining the coiled-coil domain localized to the cilium, and we define a ciliary targetingregion within the coiled-coil domain.Conclusion: Based on our data, we propose that Nek1 plays a role in centrosome integrity,affecting both ciliogenesis and centrosome stability.

Document type: 
Article

Genomic Organization of Duplicated Short Wave-Sensitive and Long Wave-Sensitive Opsin Genes in the Green Swordtail, Xiphophorus helleri

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2010
Abstract: 

Background: Long wave-sensitive (LWS) opsin genes have undergone multiple lineage-specific duplication eventsthroughout the evolution of teleost fishes. LWS repertoire expansions in live-bearing fishes (family Poeciliidae) haveequipped multiple species in this family with up to four LWS genes. Given that color vision, especially attraction toorange male coloration, is important to mate choice within poeciliids, LWS opsins have been proposed ascandidate genes driving sexual selection in this family. To date the genomic organization of these genes has notbeen described in the family Poeciliidae, and little is known about the mechanisms regulating the expression ofLWS opsins in any teleost.Results: Two BAC clones containing the complete genomic repertoire of LWS opsin genes in the green swordtailfish, Xiphophorus helleri, were identified and sequenced. Three of the four LWS loci identified here were linked in atandem array downstream of two tightly linked short wave-sensitive 2 (SWS2) opsin genes. The fourth LWS opsingene, containing only a single intron, was not linked to the other three and is the product of a retrotranspositionevent. Genomic and phylogenetic results demonstrate that the LWS genes described here share a commonevolutionary origin with those previously characterized in other poeciliids. Using qualitative RT-PCR and MSP weshowed that each of the LWS and SWS2 opsins, as well as three other cone opsin genes and a single rod opsingene, were expressed in the eyes of adult female and male X. helleri, contributing to six separate classes of adultretinal cone and rod cells with average lmax values of 365 nm, 405 nm, 459 nm, 499 nm, 534 nm and 568 nm.Comparative genomic analysis identified two candidate teleost opsin regulatory regions containing putative CRXbinding sites and hormone response elements in upstream sequences of LWS gene regions of seven teleostspecies, including X. helleri.Conclusions: We report the first complete genomic description of LWS and SWS2 genes in poeciliids. These datawill serve as a reference for future work seeking to understand the relationship between LWS opsin genomicorganization, gene expression, gene family evolution, sexual selection and speciation in this fish family.

Document type: 
Article

Isolation, Characterization and Comparison of Atlantic and Chinook salmon Growth Hormone 1 and 2

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2008
Abstract: 

Background: Growth hormone (GH) is an important regulator of skeletal growth, as well as other adapted processesin salmonids. The GH gene (gh) in salmonids is represented by duplicated, non-allelic isoforms designated as gh1 and gh2.We have isolated and characterized gh-containing bacterial artificial chromosomes (BACs) of both Atlantic and Chinooksalmon (Salmo salar and Oncorhynchus tshawytscha) in order to further elucidate our understanding of the conservationand regulation of these loci.Results: BACs containing gh1 and gh2 from both Atlantic and Chinook salmon were assembled, annotated, andcompared to each other in their coding, intronic, regulatory, and flanking regions. These BACs also contain the genesfor skeletal muscle sodium channel oriented in the same direction. The sequences of the genes for interferon alpha-1,myosin alkali light chain and microtubule associated protein Tau were also identified, and found in opposite orientationsrelative to gh1 and gh2. Viability of each of these genes was examined by PCR. We show that transposon insertions haveoccurred differently in the promoters of gh, within and between each species. Other differences within the promotersand intronic and 3'-flanking regions of the four gh genes provide evidence that they have distinct regulatory modes andpossibly act to function differently and/or during different times of salmonid development.Conclusion: A core proximal promoter for transcription of both gh1 and gh2 is conserved between the two species ofsalmon. Nevertheless, transposon integration and regulatory element differences do exist between the promoters of gh1and gh2. Additionally, organization of transposon families into the BACs containing gh1 and for the BACs containing gh2,are very similar within orthologous regions, but much less clear conservation is apparent in comparisons between thegh1- and gh2-containing paralogous BACs for the two fish species. This is consistent with the hypothesis that a burst oftransposition activity occurred during the speciation events which led to Atlantic and Pacific salmon. The Chinook andother Oncorhynchus GH1s are strikingly different in comparison to the other GHs and this change is not apparent in thesurrounding non-coding sequences.

Document type: 
Article

Regulation and Expression of Sexual Differentiation Factors in Embryonic and Extragonadal Tissues of Atlantic salmon

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2011
Abstract: 

Background: The products of cyp19, dax, foxl2, mis, sf1 and sox9 have each been associated with sex-determiningprocesses among vertebrates. We provide evidence for expression of these regulators very early in salmoniddevelopment and in tissues outside of the hypothalamic-pituitary-adrenal/gonadal (HPAG) axis. Although thefunction of these factors in sexual differentiation have been defined, their roles in early development before sexualfate decisions and in tissues beyond the brain or gonad are essentially unknown.Results: Bacterial artificial chromosomes containing salmon dax1 and dax2, foxl2b and mis were isolated and theregulatory regions that control their expression were characterized. Transposon integrations are implicated in theshaping of the dax and foxl2 loci. Splice variants for cyp19b1 and mis in both embryonic and adult tissues weredetected and characterized. We found that cyp19b1 transcripts are generated that contain 5’-untranslated regionsof different lengths due to cryptic splicing of the 3’-end of intron 1. We also demonstrate that salmon mistranscripts can encode prodomain products that present different C-termini and terminate before translation of theMIS hormone. Regulatory differences in the expression of two distinct aromatases cyp19a and cyp19b1 are exerted,despite transcription of their transactivators (ie; dax1, foxl2, sf1) occurring much earlier during embryonicdevelopment.Conclusions: We report the embryonic and extragonadal expression of dax, foxl2, mis and other differentiationfactors that indicate that they have functions that are more general and not restricted to steroidogenesis andgonadogenesis. Spliced cyp19b1 and mis transcripts are generated that may provide regulatory controls for tissueordevelopment-specific activities. Selection of cyp19b1 transcripts may be regulated by DAX-1, FOXL2 and SF-1complexes that bind motifs in intron 1, or by signals within exon 2 that recruit splicing factors, or both. Thepotential translation of proteins bearing only the N-terminal MIS prodomain may modulate the functions of otherTGF b family members in different tissues. The expression patterns of dax1 early in salmon embryogenesisimplicate its role as a lineage determination factor. Other roles for these factors during embryogenesis and outsidethe HPAG axis are discussed.

Document type: 
Article

Fish and Chips: Various Methodologies Demonstrate Utility of a 16,006-Gene Salmonid Microarray

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2005
Abstract: 

Background: We have developed and fabricated a salmonid microarray containing cDNAsrepresenting 16,006 genes. The genes spotted on the array have been stringently selected fromAtlantic salmon and rainbow trout expressed sequence tag (EST) databases. The EST databasespresently contain over 300,000 sequences from over 175 salmonid cDNA libraries derived from awide variety of tissues and different developmental stages. In order to evaluate the utility of themicroarray, a number of hybridization techniques and screening methods have been developed andtested.Results: We have analyzed and evaluated the utility of a microarray containing 16,006 (16K)salmonid cDNAs in a variety of potential experimental settings. We quantified the amount oftranscriptome binding that occurred in cross-species, organ complexity and intraspecific variationhybridization studies. We also developed a methodology to rapidly identify and confirm thecontents of a bacterial artificial chromosome (BAC) library containing Atlantic salmon genomicDNA.Conclusion: We validate and demonstrate the usefulness of the 16K microarray over a widerange of teleosts, even for transcriptome targets from species distantly related to salmonids. Weshow the potential of the use of the microarray in a variety of experimental settings throughhybridization studies that examine the binding of targets derived from different organs and tissues.Intraspecific variation in transcriptome expression is evaluated and discussed. Finally, BAChybridizations are demonstrated as a rapid and accurate means to identify gene content.

Document type: 
Article

Polymorphic Segmental Duplication In The Nematode Caenorhabditis elegans

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2009
Abstract: 

Background: The nematode Caenorhabditis elegans was the first multicellular organism to have itsgenome fully sequenced. Over the last 10 years since the original publication in 1998, the C. elegansgenome has been scrutinized and the last gaps were filled in November 2002, which present aunique opportunity for examining genome-wide segmental duplications.Results: Here, we performed analysis of the C. elegans genome in search for segmental duplicationsusing a new tool–OrthoCluster–we have recently developed. We detected 3,484 duplicatedsegments–duplicons–ranging in size from 234 bp to 108 Kb. The largest pair of duplicons, 108 kbin length located on the left arm of Chromosome V, was further characterized. They are nearlyidentical at the DNA level (99.7% identity) and each duplicon contains 26 putative protein codinggenes. Genotyping of 76 wild-type strains obtained from different labs in the C. elegans communityrevealed that not all strains contain this duplication. In fact, only 29 strains carry this large segmentalduplication, suggesting a very recent duplication event in the C. elegans genome.Conclusion: This report represents the first demonstration that the C. elegans laboratory wildtypeN2 strains has acquired large-scale differences.

Document type: 
Article

Large Synteny Blocks Revealed Between Caenorhabditis elegans And Caenorhabditis briggsae Genomes Using OrthoCluster

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2010
Abstract: 

Background: Accurate identification of synteny blocks is an important step in comparative genomics towards theunderstanding of genome architecture and expression. Most computer programs developed in the last decade foridentifying synteny blocks have limitations. To address these limitations, we recently developed a robust programcalled OrthoCluster, and an online database OrthoClusterDB. In this work, we have demonstrated the application ofOrthoCluster in identifying synteny blocks between the genomes of Caenorhabditis elegans and Caenorhabditisbriggsae, two closely related hermaphrodite nematodes.Results: Initial identification and analysis of synteny blocks using OrthoCluster enabled us to systematically improvethe genome annotation of C. elegans and C. briggsae, identifying 52 potential novel genes in C. elegans, 582 inC. briggsae, and 949 novel orthologous relationships between these two species. Using the improved annotation,we have detected 3,058 perfect synteny blocks that contain no mismatches between C. elegans and C. briggsae.Among these synteny blocks, the majority are mapped to homologous chromosomes, as previously reported. Thelargest perfect synteny block contains 42 genes, which spans 201.2 kb in Chromosome V of C. elegans. On average,perfect synteny blocks span 18.8 kb in length. When some mismatches (interruptions) are allowed, synteny blocks(“imperfect synteny blocks”) that are much larger in size are identified. We have shown that the majority (80%) ofthe C. elegans and C. briggsae genomes are covered by imperfect synteny blocks. The largest imperfect syntenyblock spans 6.14 Mb in Chromosome X of C. elegans and there are 11 synteny blocks that are larger than 1 Mb insize. On average, imperfect synteny blocks span 63.6 kb in length, larger than previously reported.Conclusions: We have demonstrated that OrthoCluster can be used to accurately identify synteny blocks and havefound that synteny blocks between C. elegans and C. briggsae are almost three-folds larger than previouslyidentified.

Document type: 
Article