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[
Worm Breeder's Gazette,
1991]
After the introduction of YAC libraries into the C. elegans mapping project, we pursued reciprocal hybridization of YACs and cosmids to link together the cosmid contigs (see Coulson et al., Nature 335, 184- 186). The YAC libraries have been increased to over 9000 clones, with over 3000 used in hybridization. A steady reduction in contig number resulted so that by the beginning of this year the number of contigs had fallen to 170 (from a starting number of 700) and the average size had risen from 125kb to more than 550kb. This greater continuity, combined with the ever increasing number of genetic loci placed on the physical map, increased the amount of DNA positioned on the genome map from less than 20 Mb to more than 75 Mb. The YAC coverage of the contigs was almost complete, and less than 1000 were required to give redundant (2 fold) coverage of the physical map. A grid of these clones (genomically ordered at the time of selection) has been prepared on a postcard sized piece of nylon (958 clones in all) and replicas made for distribution. Using the key provided and the electronic map (see WBG#10;3) it is now a straightforward procedure for a lab to locate any given DNA sequence on the genome map. They have been used successfully in a number of labs. These 'polytene' filters may be obtained from either St. Louis or Cambridge by request. By the beginning of the year, it had become clear that the reciprocal hybridization strategy was decreasingly useful and another approach was needed. The remaining contigs were largely of two classes: (1) contigs with YACs as the likely endmost clones; and (2) small contigs lacking YACs. The contigs with YAC ends were blocked from growth because the reciprocal hybridization strategy provides no way to recognize YAC-YAC overlaps. To obtain an end probe from the YACs, sequence was obtained via an adaptation of Engelke's genomic sequencing method (Methods in Enzymology). Primers were made and the PCR product used to probe to the YAC grids. Hybridizing YACs were tested with the same primer pairs by PCR to confirm the overlap. A total of 34 joins and rearrangements have resulted from the analysis of 186 ends. Because these joins involve just the ends of YACs they are particularly vulnerable to artifact; please inquire if you need to know the status of particular areas. The small YACless contigs were of a variety of types. Some contained only cosmids made in the pJB8 vector that had been left out of the above grids because sequences share with the YAC vector. Some had repeated sequences that led to ambiguous positioning. Others simply had failed to hybridize with YACs tested. To assign these cosmids to YACs, end sequence was obtained by direct sequencing of cosmid DNA, PCR was used to generate small probes and positive YACs were tested by PCR to confirm assignment. Of 50 cosmids from which we have useful data, 38 have been assigned to YACs. Currently, for 31 cases these YACs either had previously been placed on the physical map or have been subsequently linked to larger contigs, thereby locating the small contigs. Many of the remaining clones failed to give a PCR- product from nematode DNA and have been set aside. Only 2 clones were found which gave a PCR product from worm DNA and failed to identify any overlapping YAC. Together these efforts have reduced the contig number to 102. With the joins obtained and the continuing efforts of the community to clone and assign genetically mapped fragments on the physical map, 85 Mb of DNA is now aligned with the genetic map. [See Figures 1-4]
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[
International C. elegans Meeting,
1991]
The C. elegans physical map has been assembled with a combination of cosmid and yeast artificial chromosome (YAC) clones. In the first phase, cosmid clone overlaps were detected using a high resolution fingerprinting technique. This produced a map with more than 700 contigs. Next, YACs were used to link together the multiple cosmid contigs via hybridization of the YACs to colony grids of representative cosmids. By the time this approach reached its practical limits, the map had been reduced to about 170 contigs. The current phase has involved detecting overlaps between protruding YAC clones at the ends of existing contigs, and also between small cosmid contigs and YACs. End sequence from the clones was obtained by using flanking vector primers either on total yeast genomic DNA for YACs or miniprep DNA for cosmids. PCR was then used to get unique hybridization probes. Hybridizing YACs were checked by PCR to confirm overlaps. From these efforts, the map now contains less than 90 contigs. Through the supporting evidence of genetic and physical information from other sources, more than 85 Mb of the assembled DNA has been assigned to chromosomes in 35 large contigs. Since C. elegans genes are preferentially located in the centers of chromosomes and the continuity of the map is greatest in these locations, all but a few coding sequences are now covered by contigs. The results of the physical map effort are available to the community in three forms: The database can be accessed either via network or modem from several locations throughout the world; a colony grid of 958 YACs, genomically ordered at the time of selection, has been prepared in a postcard sized array and replicas have been distributed to interested labs; YACs and cosmid clones covering any region are available upon request.
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[
Mol Biol Evol,
2007]
The Y genes encode small non-coding RNAs whose functions remain elusive, whose numbers vary between species, and whose major property is to be bound by the Ro60 protein (or its ortholog in other species). To better understand the evolution of the Y gene family, we performed a homology search in 27 different genomes along with a structural search using Y RNA specific motifs. These searches confirmed that Y RNAs are well conserved in the animal kingdom and resulted in the detection of several new Y RNA genes, including the first Y RNAs in insects and a second Y RNA detected in Caenorhabditis elegans. Unexpectedly, Y5 genes were retrieved almost as frequently as Y1 and Y3 genes, and, consequently are not the result of a relatively recent apparition as is generally believed. Investigation of the organization of the Y genes demonstrated that the synteny was conserved among species. Interestingly, it revealed the presence of six putative "fossil" Y genes, all of which were Y4 and Y5 related. Sequence analysis led to inference of the ancestral sequences for all Y RNAs. In addition, the evolution of existing Y RNAs was deduced for many families, orders and classes. Moreover, a consensus sequence and secondary structure for each Y species was determined. Further evolutionary insight was obtained from the analysis of several thousand Y retropseudogenes among various species. Taken together, these results confirm the rich and diversified evolution history of Y RNAs.
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[
Biochim Biophys Acta,
2016]
BothDrosophila melanogaster and Caenorhabditis elegans (C. elegans) are useful model organisms to study in vivo roles of NF-Y during development. Drosophila NF-Y (dNF-Y) consists of three subunits dNF-YA, dNF-YB and dNF-YC. In some tissues, dNF-YC-related protein Mes4 may replace dNF-YC in dNF-Y complex. Studies with eye imaginal disc-specific dNF-Y-knockdown flies revealed that dNF-Y positively regulates the sevenless gene encoding a receptor tyrosine kinase, a component of the ERK pathway and negatively regulates the Sensless gene encoding a transcription factor to ensure proper development of R7 photoreceptor cells together with proper R7 axon targeting. dNF-Y also controls the Drosophila Bcl-2 (debcl) to regulate apoptosis. In thorax development, dNF-Y is necessary for both proper Drosophila JNK (basket) expression and JNK signaling activity that is responsible for thorax development. Drosophila
p53 gene was also identified as one of the dNF-Y target genes in this system. C. elegans contains two forms of NF-YA subunit, CeNF-YA1 and CeNF-YA2. C. elegans NF-Y (CeNF-Y) therefore consists of CeNF-YB, CeNF-YC and either CeNF-YA1 or CeNF-YA2. CeNF-Y negatively regulates expression of the Hox gene
egl-5 (ortholog of Drosophila Abdominal-B) that is involved in tail patterning. CeNF-Y also negatively regulates expression of the
tbx-2 gene that is essential for development of the pharyngeal muscles, specification of neural cell fate and adaptation in olfactory neurons. Negative regulation of the expression of
egl-5 and
tbx-2 by CeNF-Y provides new insight into the physiological meaning of negative regulation of gene expression by NF-Y during development. In addition, studies on NF-Y in platyhelminths are also summarized.
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[
Trends in Cell Biology,
1996]
Keeling and Logsdon propose that the y-like sequences from Caenorhabditis elegans and Saccharomyces cerevisiae are bona fide y-tubulins that have undergone rapid evolutionary divergence. Indeed, genetic and localization studies with the yeast epsilon-tubulin (encoded by the TUB4 gene) reveal striking similarities to the bona fide y-tubulins, whereas there is no apparent human analogue to the C. elegans delta-tubulin among the 60 available human y-tubulin expressed-sequence tags. (ESTs).
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[
RNA,
2009]
Noncoding Y RNAs are required for the reconstitution of chromosomal DNA replication in late G1 phase template nuclei in a human cell-free system. Y RNA genes are present in all vertebrates and in some isolated nonvertebrates, but the conservation of Y RNA function and key determinants for its function are unknown. Here, we identify a determinant of Y RNA function in DNA replication, which is conserved throughout vertebrate evolution. Vertebrate Y RNAs are able to reconstitute chromosomal DNA replication in the human cell-free DNA replication system, but nonvertebrate Y RNAs are not. A conserved nucleotide sequence motif in the double-stranded stem of vertebrate Y RNAs correlates with Y RNA function. A functional screen of human Y1 RNA mutants identified this conserved motif as an essential determinant for reconstituting DNA replication in vitro. Double-stranded RNA oligonucleotides comprising this RNA motif are sufficient to reconstitute DNA replication, but corresponding DNA or random sequence RNA oligonucleotides are not. In intact cells, wild-type hY1 or the conserved RNA duplex can rescue an inhibition of DNA replication after RNA interference against hY3 RNA. Therefore, we have identified a new RNA motif that is conserved in vertebrate Y RNA evolution, and essential and sufficient for Y RNA function in human chromosomal DNA replication.
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[
J Bacteriol,
2006]
Yersinia pestis, the agent of plague, is usually transmitted by fleas. To produce a transmissible infection, Y. pestis colonizes the flea midgut and forms a biofilm in the proventricular valve, which blocks normal blood feeding. The enteropathogen Yersinia pseudotuberculosis, from which Y. pestis recently evolved, is not transmitted by fleas. However, both Y. pestis and Y. pseudotuberculosis form biofilms that adhere to the external mouthparts and block feeding of Caenorhabditis elegans nematodes, which has been proposed as a model of Y. pestis-flea interactions. We compared the ability of Y. pestis and Y. pseudotuberculosis to infect the rat flea Xenopsylla cheopis and to produce biofilms in the flea and in vitro. Five of 18 Y. pseudotuberculosis strains, encompassing seven serotypes, including all three serotype O3 strains tested, were unable to stably colonize the flea midgut. The other strains persisted in the flea midgut for 4 weeks but did not increase in numbers, and none of the 18 strains colonized the proventriculus or produced a biofilm in the flea. Y. pseudotuberculosis strains also varied greatly in their ability to produce biofilms in vitro, but there was no correlation between biofilm phenotype in vitro or on the surface of C. elegans and the ability to colonize or block fleas. Our results support a model in which a genetic change in the Y. pseudotuberculosis progenitor of Y. pestis extended its pre-existing ex vivo biofilm-forming ability to the flea gut environment, thus enabling proventricular blockage and efficient flea-borne transmission.
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[
International Worm Meeting,
2015]
Y RNA is a small structured ncRNA of about 100 nt in length. This RNA binds to Ro60 protein, which is a target of autoimmune disease antibody in patients with systemic lupus erythematosus and Sjogren's syndrome. Several lines of evidence suggest that the role of Y RNA and Ro60 function in the quality control of structured ncRNAs in cells under stress conditions. It is also indicated that vertebrate Y RNAs function in the initiation of DNA replication without Ro60. However, the molecular mechanisms of these functions and the contribution of Ro60/Y RNP to the autoimmune disease are still unclear. C. elegans genome encodes one Ro60 homolog (ROP-1) and 19 Y RNA homologs (1 CeY RNA and 18 sbRNAs). Other animals also have several Y RNA homologs, but C. elegans is the first example which has more than 5 Y RNA homologs encoded in the genome. Here we show the expression pattern and the cellular localization of these Y RNA homologs in C. elegans examined by the RNA fluorescent in situ hybridization (RNA-FISH). The signals of 14 homologs were detected in the intestinal cytoplasm. The signals of two other homologs were detected in the germ cytoplasm. The remaining three could not be detected, probably because they present in too low abundance to be detected by RNA-FISH. All 19 Y RNA homologs have the structural elements required for the binding of ROP-1. In other organisms, Ro60 binding stabilizes Y RNAs in cells. To know whether C. elegans Y RNA homologs also stabilized by the presence of ROP-1, we examined RNA-FISH of the Y RNA homologs against a mutant strain MQ470, which has a transposon insertion in the middle of the ROP-1 gene and lacks ROP-1 proteins in the cell. As expected, all Y RNAs examined so far decreased extensively. These were confirmed by northern hybridization. The results suggest that several C. elegans Y RNA homologs are expressed in a tissue-specific manner and most Y RNA homologs are stabilized by ROP-1 binding as well as those in other organisms.
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[
Protein Cell,
2011]
Flea-borne transmission is a recent evolutionary adaptation that distinguishes the deadly Yersinia pestis from its progenitor Y. Pseudotuberculosis, a mild pathogen transmitted via the food-borne route. Y. Pestis synthesizes biofilms in the flea gut, which is important for fleaborne transmission. Yersinia biofilms are bacterial colonies surrounded by extracellular matrix primarily containing a homopolymer of N-acetyl-D-glucosamine that are synthesized by a set of specific enzymes. Yersinia biofilm production is tightly regulated at both transcriptional and post-transcriptional levels. All the known structural genes responsible for biofilm production are harbored in both Y. Pseudotuberculosis and Y. Pestis, but Y. Pestis has evolved changes in the regulation of biofilm development, thereby acquiring efficient arthropod-borne transmission.
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[
Mol Cell Biol,
2001]
Weak hypomorph mutations in the enhancer of yellow genes, e(y)1 and e(y)2, of Drosophila melanogaster were discovered during the search for genes involved in the organization of interaction between enhancers and promoters. Previously, the e(y)1 gene was cloned and found to encode TAF(II)40 protein. Here we cloned the e(y)2 gene and demonstrated that it encoded a new ubiquitous evolutionarily conserved transcription factor. The e(y)2 gene is located at 10C3 (36.67) region and is expressed at all stages of Drosophila development. It encodes a 101-amino-acid protein, e(y)2. Vertebrates, insects, protozoa, and plants have proteins which demonstrate a high degree of homology to e(y)2. The e(y)2 protein is localized exclusively to the nuclei and is associated with numerous sites along the entire length of the salivary gland polytene chromosomes. Both genetic and biochemical experiments demonstrate an interaction between e(y)2 and TAF(II)40, while immunoprecipitation studies demonstrate that the major complex, including both proteins, appears to be distinct from TFIID. Furthermore, we provide genetic evidence suggesting that the carboxy terminus of dTAF(II)40 is important for mediating this interaction. Finally, using an in vitro transcription system, we demonstrate that recombinant e(y)2 is able to enhance transactivation by GAL4-VP16 on chromatin but not on naked DNA templates, suggesting that this novel protein is involved in the regulation of transcription.