Progress 10/01/02 to 09/30/05
Outputs
Transposons have long been viewed as molecular parasites, mobile genetic elements that reside in host genomes but play no functional role in these genomes. That view must be reassessed with the recent discovery in eukaryotes of noncoding or microRNAs that regulate gene expression on a genome-wide level. This regulation is effected by the RNAi machinery and acts at least in part at the level of chromatin. Transposons have been shown to be a natural target of RNAi. Together, these features suggest a possible functional role for transposons in regulation of the host genome, acting as the cis elements that mediate chromatin level control of gene expression induced by noncoding RNAs. We tested this idea in two ways. First, we used the complete genome sequence of the related nematodes C. elegans and C. briggsae to identify transposon resident sites conserved between the two genomes. We previously identified all sites for each of eight transposon families in C. elegans. In
this study we identified homologous transposon sequences in the C. briggsae genome. Conserved transposon resident sites might reflect functional roles such as that described above. Second, we attemped to compile all available expression data from C. elegans microarrays to identify clusters of genes that are up- or down-regulated in concert in response to developmental or other ques. These two avenues of investigation have led us to our understanding of the role of resident transposon sequences in host genome regulatory events.
Impacts
These studies provided insight into the role of transposons in regulation of host functions in eukaryotic genomes, and the mechanism by which RNAi regulates gene expression and chromatin density. Our bioinformatic analysis of trasposon distribution provides a detailed view of the genome architecture of this important model organism.
Publications
- No publications reported this period
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Progress 10/01/03 to 09/29/04
Outputs
Transposons have long been viewed as molecular parasites, mobile genetic elements that reside in host genomes but play no functional role in these genomes. That view must be reassessed with the recent discovery in eukaryotes of noncoding or microRNAs that regulate gene expression on a genome-wide level. This regulation is effected by the RNAi machinery and acts at least in part at the level of chromatin. Transposons have been shown to be a natural target of RNAi. Together, these features suggest a possible functional role for transposons in regulation of the host genome, acting as the cis elements that mediate chromatin level control of gene expression induced by noncoding RNAs. We are testing this idea in two ways. First, we are using the complete genome sequence of the related nematodes C. elegans and C. briggsae to identify transposon resident sites conserved between the two genomes. We previously identified all sites for each of eight transposon families in C.
elegans. In the current reporting period we have identified homologous transposon sequences in the C. briggsae genome and we are determining syntenic regions in briggsae and elegans. Conserved transposon resident sites might reflect functional roles such as that described above. Second, we are compiling all available expression data from C. elegans microarrays to identify clusters of genes that are up- or down-regulated in concert in response to developmental or other ques. These two avenues of investigation represent a start point for the design of experiments to test directly the role of resident transposon sequences in host genome regulatory events.
Impacts
These studies will provide insight into the role of transposons in regulation of host gene functions in eukaryotic genomes, and the mechanism by which RNAi regulates gene expression and chromatin density. Our bioinformatic analysis of transposon distribution provides a detailed view of the genome architecture of this important model organism.
Publications
- No publications reported this period
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Progress 10/01/02 to 09/29/03
Outputs
We mapped the mut-2 mutator to a small interval near the middle of LG I in the C. elegans genome. Attempts to identify the mut-2 gene by standard cosmid rescue approaches have not succeeded, to date. Current efforts are focused on sequencing candidate genes to identify the lesion corresponding to mut-2(r459). We have exploited the complete genome sequence to determine the precise location of all copies of each transposon family in the C. elegans genome. We have also identified and determined the position of hundreds of related sequences, representing subfamilies of these elements.
Impacts
These studies bring us closer to cloning mut-2, which will provide insight into the control of transposons in eukaryotic genomes, and the mechanism by which RNAi regulates gene expression and chromatin density. Our bioinformatic analysis of transposon distribution provides a detailed view of the genome architecture of this important model organism.
Publications
- Bei, X.,* Hogan, J.* , Berkowitz, Soto, M., Rocheleau, C., Pang, K.M., Collins, J. and Mello, C. Phosphotyrosine signaling acts in parallel with Wnt signaling to specify endoderm and to control cleavage orientation in early C. elegans embryos. Developmental Cell 3:1-20, (2002). (* signifies both authors contributed equally to this work)
- Pandey, A., Peri, S., Thacker, C., Pugh, C. A., Collins, J. J. and Mann, M. Computational and experimental analysis reveals a novel Src family kinase in the C. elegans genome. Bioinformatics 19(2):169-172 (2003).
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Progress 10/01/01 to 09/30/02
Outputs
We confirmed preliminary evidence indicating that mut-2(r459) confers an RNAi-resistant phenotype, using an unc-22 dsRNA. We are currently trying to exploit this feature to clone mut-2, using an RNAi assay to rescue r459. In addition, we have mapped another mutator allele, r461, to LGX. This mutator allele does not disrupt RNAi for; mutants are sensitive to unc-22 dsRNA. We are using pos-1 dsRNA to determine if this allele affects RNAi for germline-expressed genes (unc-22 is expressed in muscle). We will use RNAi and ts-sterile phenotypes to clone these genes by rescue and we will determine if r459 and r461 represent new alleles of known mut or rde genes.
Impacts
This work will enhance our understanding of the genetic mechanisms that ensure genome stability in metazoans. It may serve to identify new components of the eukaryotic RNAi machinery, and may reveal links between RNAi and other aspects of genome stability such as transposon control.
Publications
- No publications reported this period
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Progress 10/01/00 to 09/30/01
Outputs
Work during the project period focused on characterization of several mutator genes implicated in transposon control in the nematode Caenorhabditis elegans. We showed that a mutant allele (r459) of one mutator gene, mut-2, confers a recessive, temperature-sensitive sterile phenotype. Exploiting this phenotype, mapped and rescued r459, setting the stage for cloning the gene. We (and others) also showed that mut-2 plays a role in gene silencing in response to double-stranded RNA (RNAi) in this organism. We also began characterization of seven other mutator mutants. We determined that five exhibit a ts-sterile phenotype like mut-2(r459). We are using this phenotype to perform complementation tests and to map the genes identified by these mutants. We also showed that none of these seven mutants are affected in the ability to respond to interfering ds-RNA, suggesting that these mutants may define genes that play a role specifically in transposon control, unlike mut-2.
Impacts
Cloning and further characterization of mut genes will elucidate the function of each gene and the role of each in transposon control, RNAi and other processes in the C. elegans germ line. This work also has direct implications for agriculturally important nematodes. Unlike C. elegans, these organisms are not amenable to genetic analysis. Characterization of the mut genes will enhance our understanding of transposons and their control in one nematode. This knowledge should help in efforts to use transposon-mediated approaches to do genetics in other nematodes.
Publications
- No publications reported this period
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Progress 01/01/00 to 12/31/00
Outputs
We have established that mut-2 is involved in the process of RNA interference (RNAi); our mut-2 mutant stain is resistant to dsRNA-mediated gene disruption. We tested this by feeding mut-2 mutants a bacterial strain expressing both strands of a segment of the unc-22 gene. These results add to our understanding of the roles of mut-2 and provide another suitable phenotype for mapping and cloning efforts. Complementation analysis indicates that mut-2 and rde-3 are likely the same gene. rde-3 was identified by virtue of its RNAi-resistant phenotype, and it maps to the same genetic interval as mut-2. This further supports the idea that mut-2 plays an important role in dsRNA interference. Experiments are ongoing to clone mut-2/rde-3, exploiting both the RNAi and the ts-sterile phenotypes of these mutants.
Impacts
Characterization of mut-2 should provide insight into the mechanism and role of RNAi, and its relation to transposon control, in C. elegans. We want to know the role of this gene and the function of the encoded product in RNAi, transposon control and other aspects of genomic integrity in this organism.
Publications
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Progress 01/01/99 to 12/31/99
Outputs
Using the temperature-sensitive (ts) sterile phenotype conferred by mut-2(r459), we have localized mut-2 on the C. elegans physical map to the region covered by the cosmid H06O01. This position is established by rescue of ts sterility with the free duplications sDp2 and hDp65, but not by hDp62, and by rescue with the cosmid H06O01. This cosmid contains four predicted ORFs. Current efforts are focused on identifying a single rescuing ORF and identifying the lesion associated with r459.
Impacts
Identification of the mut-2 gene will provide immediate information on the predicted function of the encoded protein. This in turn will shed light on the mechanisms that maintain genome integrity in eukaryotes.
Publications
- No publications reported this period
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Progress 01/01/98 to 12/31/98
Outputs
We identified one new polymorphism in the dpy-14 sem-4 region, a Tc1 element present in Bergerac and absent in Bristol. Scoring our recombinants for this polymorphism established that mut-2 maps to the right of it. This further narrows the mut-2 containing interval to a region covered by three cosmids, containing 19 ORFs. Current efforts are concentrated on determining which ORF encodes mut-2. To aid in this effort we have characterized an additional phenotype conferred by mut-2(r459); temperature-sensitive sterility. This r459 is recessive for this phenotype, making it potentially very useful in our efforts to clone mut-2 by rescue approaches.
Impacts
(N/A)
Publications
- No publications reported this period
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Progress 01/01/97 to 12/31/97
Outputs
The goal of this project is to identify factors involved in regulating transposon activity in C. elegans. Work over the past year has focused on mapping and cloning mut-2, a gene implicated in control of all known transposons in this organism. Our previous work positioned this gene in the dpy-5 region of LG I. To map mut-2 more precisely, we crossed TW404 (dpy-5(e61); mut-2(r459)) by a sem-4 unc-13 mutant and picked Dpy Sem Unc and Dpy Unc recombinants. Each was screened for the presence of the mut-2 mutator, using the Himphenotype (high incidence of males) and Tc5 activation as indicators. The results place mut-2 to the left of sem-4 and closer to sem-4 than to dpy-5 To obtain even higher resolution, we crossed a mut-2 sem-4 unc-13 strain by a dpy-14 mutant, picked Dpy Sem Unc recombinants and screened each for the mut-2 mutator. The results place mut-2 between dpy-14 and sem-4. To position mut-2 with high precision within this interval, we will isolate a large
number of independent Dpy Sem Unc recombinants and map mut-2 with respect to physical markers in this region.
Impacts
(N/A)
Publications
- No publications reported this period
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Progress 01/01/96 to 12/30/96
Outputs
Work over the past year has focused on two areas. First, we have demonstrated that the ORF contained with the C. elegans transposon Tc5 encodes a factor involved in transposition of Tc5. Forced expression of this ORF from a heat-shock promoter fusion in transgenic strains activates transposition of Tc5, monitored by a PCR assay of new insertions of this element into a target gene. This information opens the door to analysis of the interaction of the Tc5-encoded transposase with regions of the terminal inverted repeats (TIR) of Tc5 (Aim 1). Second, we have identified several clones in a one-hybrid assay that encode factors that interact with the Tc5 TIR. These clones represent candidates for genes encoding factors involved in transposition of Tc5. Experiments are underway to characterize each clone to determine for each if the encoded protein interacts with the Tc5 TIR in vivo, and to investigate the role of each in Tc5 transposition.
Impacts
(N/A)
Publications
- NO PUBLICATIONS REPORTED THIS PERIOD.
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Progress 01/01/95 to 12/30/95
Outputs
Work over the past year has focused on two questions about the C. elegans transposon Tc5. To test for function of the putative Tc5-encoded protein, we monitored Tc5 activity in response to forced expression of the Tc5 ORF fused to a heat shock promoter (hsp-Tc5ORF). A PCR-based molecular assay was used to detect new insertions of Tc5 into the src-1 gene. We detect a significant increase in Tc5 transposition into src-1 upon heat shock-induction of hsp-Tc5ORF transgenic animals. Experiments are in progress to confirm that the PCR products detected represent bona fide Tc5 insertions in src-1 and to test for their dependence on Tc5ORF expression. To identify factors that bind to the terminal inverted repeats (TIR) of Tc5, we have amplified and cloned the entire 492-bp Tc5 TIR into a yeast vector for use in a "one-hybrid" screen. We are now using this modification of the two-hybrid system to screen for C. elegans proteins that recognize and bind to the Tc5 TIR. At the same
time, we are amplifying and cloning smaller segments of this DNA region into the same vector. These fragments will be used to define the binding site within the Tc5 TIR for each factor identified.
Impacts
(N/A)
Publications
- GLASNER, J.D., KOCHER, T.D. AND COLLINS, J.J. 1995. Caenorhabditis elegans contains genes encoding two new members of the Zn-containing alcohol dehydrogenase family. J. Mol. Evol. 41:46-53.
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Progress 01/01/94 to 12/30/94
Outputs
The focus of our studies over the past year has been the in vivo function of thepredicted protein encoded by Tc5; we are testing the idea that this protein represents the Tc5 transposase. To do this we are monitoring Tc5 movement in response to forced expression of the Tc5-encoded factor. The Tc5 ORF was fused to the promoter of a C. elegans heat shock gene, hsp16-2. This construct (hsp-Tc5ORF) was introduced into the C. elegans germ line and stable transgenic lines were established. RT-PCR and Northern analyses demonstrate that Tc5 transcripts are induced to high levels by heat shock in these strains. We used a genetic approach to determine if Tc5 is activated in these conditions. We crossed the mutation unc-22(r741:Tc5) into a hsp-Tc5ORF transgenic line and screened for reversion of this Tc5-induced mutant following heat shock. No reversion was observed. We are currently investigating two possible explanations for these initial negative results. First, we are
repeating these experiments with strains containing unc-22(r741:Tc5) and hsp-Tc5ORF in the mut-2 mutant background. To date, Tc5 activity has been detected only in this mutator background. Second, we are using a PCR-based molecular approach to monitor Tc5 transposition. This assay will enable us to detect both somatic and germ-line events. Lack of reversion in the experiments described above may reflect lack of expression of our hsp-Tc5ORF fusion in the germ line.
Impacts
(N/A)
Publications
- COLLINS, J.J., AND ANDERSON, P. 1994. The Tc5 family of transposable elements in Caenorhabditis elegans. Genetics 137:771-781.
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Progress 01/01/93 to 12/30/93
Outputs
My current Hatch Project has two objectives: 1) to test for expression of ORFs contained within C. elegans transposons; 2) to test element-encoded ORFs in vivo for function. Focusing on the transposon Tc5, we have completed the first objective and made good progress on the second one. Using reverse transcription and the PCR, we identified and characterized a Tc5 cDNA. The sequence of this cDNa predicts a 532 amino acid protein. Intriguingly, the predicted Tc5-encoded protein is very similar (33% amino acid identity) to the predicted protein encoded by another C. elegans transposon, Tc4v. another interesting feature of the Tc5 transcript is that we detect it only in mut-2 mutant strains, not in wild-type strains. This correlates with Tc5 activity: transposition and excision of Tc5 are detected only in the mut-2 mutant background. To address the question of functional significance, our approach has been to express the Tc5-encoded factor from a heterologous promoter --
forcing expression of this gene at high levels. To date, such approaches in C. elegans have been stymied in cases where germ-line expression is required. Such is the case in our experiments to overcome this problem we obtained a cloning/transformation vector containing a promoter that will drive expression of a transgene in the germ line, the C. elegans heat shock promoter hsp16-2. We fused the Tc5-encoded gene to this promoter and generated transgenic C. elegans lines containing this fusion construct integrated at random sites in the C. elegans genome.
Impacts
(N/A)
Publications
- No publications reported this period.
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Progress 01/01/92 to 12/30/92
Outputs
Specific objectives of this Hatch Project (H344) are to determine if ORFs encoded by C. elegans transposons are expressed, and if this expression has functional significance for transposon activity. Work has focused on Tc5. Our previous DNA sequence analysis revealed a long ORF composed of five exons, encoding a predicted protein of 495 amino acids. To determine if this ORF is expressed and processed as predicted, we screened total RNA from wild-type worms and transgenic animals overexpressing the Tc5 ORF from a heterologous promoter, using reverse transcription combined with PCR. We also screened a cDNA library via PCR for Tc5 clones. Each approach detected Tc5-specific cDNAs, and in each case DNA sequence analysis revealed that the Tc5 transcript contains six exons, the five we originally predicted plus a small exon between the second and third. This additional exon has important implications for the hypothetical Tc5-encoded gene product; it alters the reading frame,
resulting in a dramatically truncated protein. We are currently investigating whether alternative splicing, translational frame-shifting, or heterogeneity in different genomic copies of Tc5 might allow for expression of the full-length Tc5 protein. To address the question of functional significance, we are determining if forced expression of Tc5 ORF stimulates element activity. Starting with a mutant strain containing a Tc5 insertion in the unc-22 gene, we have generated transgenic lines in which Tc5 ORF is expressed from the C.
Impacts
(N/A)
Publications
- No publications reported this period.
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Progress 01/01/91 to 12/30/91
Outputs
Expression of element-encoded ORFs: We have completed nucleotide sequence determination for the transposon Tc5. The sequence reveals an ORF composed of four or five exons. Appropriate oligonucleotide primers have been synthesized and experiments are in progress using reverse transcriptase and PCR to identify the predicted Tc5 transcript. Similar experiments are underway aimed at identifying the predicted Tc1 and Tc3 transcripts. Once identified, full-length cDNAs will be obtained to determine the exact structure of the element-encoded proteins. This information will enable us to design and synthesize peptides for antibody production and detection of the protein products in C. elegans extracts. Transpose activity in vivo: Once available, full-length element-encoded cDNAs will be cloned into available c. elegans expression vectors to test in vivo for their ability to supply transposase activity.
Impacts
(N/A)
Publications
- No publications reported this period.
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Progress 01/01/90 to 12/30/90
Outputs
Identification of additional C. elegans transposons: Two novel insertions in the unc-22 gene are under investigation. As previously reported, the resulting mutations are unstable, reverting to wild type at high frequency. Using total genomic Southern blots, we tested whether reversion resulted from excision of the inserted DNA, a feature expected of transposon-induced mutations. Our results indicate that one of the novel insertions has excised in the corresponding revertant. We are now cloning this insertion to determine its structure and genome organization. DNA sequence analysis of Tc3 and Tc5: This objective is nearly completed. We are analyzing two C. elegans transposons that we recently discovered, designated Tc3 and Tc5. The sequence of Tc3 has been completed, as reported last year. The sequence of Tc5 is nearly finished: we have determined over 2900 base pairs (bp) of approx. 3200 total. Noteworthy features are: a) the termini of Tc5 are inverted repeats
(IR) of 488 bp. The most terminal ten nucleotides are identical to the most terminal bases of the C. elegans transposon Tc4, suggesting that common factor(s) might regulate the activity of these two element families. b) the Tc5 sequence contains a long open reading frame (ORF) with the capacity to encode a protein of 256 amino acids. Collectively, the results described in this and the previous progress report reveal that both Tc3 and Tc5 have the capacity to encode proteins that regulate their activity.
Impacts
(N/A)
Publications
- NO PUBLICATIONS REPORTED THIS PERIOD.
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Progress 01/01/89 to 12/30/89
Outputs
Identification of Additional C. elegans Transposons: We are analyzing two novel insertions in the unc-22 gene to determine if they represent previously uncharacterized C. elegans transposons. To test this hypothesis, we have asked if the mutations caused by these insertions are unstable. Both revert to wild type at high frequency (greater than 10). Experiments are in progress to determine if reversion is caused by loss of the insertion from unc-22 (excision of the putative transposon). DNA Sequence Analysis: The complete nucleotide sequence of the newly discovered transposable element Tc3 has been determined. Two features of this sequence are noteworthy. First, Tc3 has 450 base pair inverted repeat termini (IR). As described previously (Collins et al. 1989, Genetics 121:47-55), the most terminal nine nucleotides are nearly identical to the terminal nine nucleotides of the C. elegans transposon Tc1. However, the overall length of the Tc3 IR is considerably longer than
the 54 base pair IR of Tc1, and rather long for members of the class of eukaryotic transposons that have short inverted repeats at their ends. Second, the internal sequence of Tc3 contains a single long open reading frame (ORF) capable of encoding a protein of 319 amino acids. This predicted protein contains regions of significant amino acid identity with the protein predicted by an ORF of approximately the same size contained in Tc1. The functional significance of these structural features is under investigation.
Impacts
(N/A)
Publications
- NO PUBLICATIONS REPORTED THIS PERIOD.
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