Shaham S et al. (1992) Worm Breeder's Gazette "Three Classes of Repeats in the C. elegans Genome: Clues About IntronEvolution?"

Horvitz HR, Shaham S, Hengartner MO

[Worm Breeder's Gazette, 1992]

While determining the sequence of the genomic regions of ced-3 ,ced-9 and unc-50 ,several classes of inverted repeats were discovered in non-coding regions both upstream of the ced-3 start site and within some of the introns (see article this issue by Shaham and Horvitz). We searched the GenBank database for similar sequences using the "Blast" program and identified three sequences similar to other regions of the C. elegans genome. The sequence comparisons of these various repeats are shown below. Repeats a and c [See Figres 1 & 2] are found as inverted repeats, thus, we have compared each arm (forward) to its partner (reverse) and to the other sequences. Repeat d [See Figure 3] was found as a single (as opposed to inverted) repeat in both ced-3 and lin-12 .The four largest introns of ced-3 contain repetitive elements, suggesting that large introns (>200 bp) in C. elegans might be a consequence of the presence of defective transposons, which might not affect the function of the gene. We are interested in obtaining more information concerning these or other repeats in other regions of the genome.

Rezsohazy R et al. (1996) Worm Breeder's Gazette "Tc7, a new family of transposable elements in C. elegans."

Plasterk RHA, Durbin RM, Rezsohazy R

[Worm Breeder's Gazette, 1996]

Six families of transposable elements (Tc1-Tc6) have been described until now in C. elegans . They are class II transposons which presumably transpose via a DNA intermediate, and are delimited by inverted repeat sequences. Using the terminal 24 bp of the Tc1 inverted repeats as a probe to screen the whole genomic dataset, we found 8 hits correeponding to a new family of elements, which we call Tc7. They are 921- to 923-bp elements made up of two 327-bp inverted repeats and a conserved middle section which contains no large open reading frame. The outer 38 bp of their inverted repeats show 36 matches with the last 38 bp of Tc1. Furthermore, like Tc1, they are flanked by TA dinucleotides which correspond presumably to the target duplication generated upon transposition. The elements show less than 1% sequence polymorphism, but three copies of Tc7 show one small deletion (6- to 45-bp long) at different locations. Extrapolating the number of these sequences to the entire genome, we expect approximately 30 copies of Tc7, which is similar to the copy number of Tc1 in Bristol N2. The binding site of the Tc1 transposase (Tc1A) within Tc1 inverted repeats (Vos et al. , Genes Dev. <b> 7 , 1244-1253,1993) is fully contained within the 38-bp sequence shared with Tc7, suggesting that Tc7 could be a parasitic family of elements using Tc1A for their transposition. To test the ability of Tc7 to jump upon Tc1A expression, we used an integrated line harbouring the Tc1A gene under the control of an heat-shock promoter (NL818, Vos et al. , submitted for publication). Using Tc7 primers, we visualized by PCR Tc7 insertions in a defined genomic target ( gpa-2 ). After heat-shock, several insertions were found in NL818, whereas no jumps were detected in Bristol N2. Comparison with control experiments using Tc1 specific primers suggest that Tc7 transposes at a slightly lower frequency than Tc1 when Tc1A is provided in trans in somatic cells. Altogether these data support the idea that Tc7 is a hitch-hiking transposon whose mobility depends on Tc1A expression. However, whether or not Tc7, like Tc1, is able to transpose in the germ line of mutator strains remains to be investigated.

Vos JC et al. (1993) Worm Breeder's Gazette "Tc1 Transposase has Separate Domains for Site-Specific and General DNA Binding"

Vos JC, Plasterk RHA, van Luenen HGAM

[Worm Breeder's Gazette, 1993]

We have previously reported the intron-exon structure of the putative transposase gene Tc1A )of the transposable element Tc1 and the binding of Tc1A to the inverted repeat of Tc1 (WBG 12 (3), pg. 87). In a manner similar to Tc3 (see WBG 12(4), pg. 15), we analyzed somatic transposition of endogenous Tc1 elements in transgenic N2 animals in which we induced Tc1 A expression. In non- transgenic, or in non-induced transgenic N2 ,we found approximately 3 Tc1 insertions in the gpa-2 gene per 1 ug of worm DNA (equivalent to approximately 10.000 genomes). A 15 fold increase in the number of Tc1 insertions is seen after Tc1 A expression in the transgenic strain. Therefore, we conclude that the Tc1A gene product is a transposase and that it is a limiting factor for transposition in N2 .Tc1 A induction does not result in Tc3 transposition, which requires expression of its own Tc3 transposase. We expressed Tc1A in E. coli and could show the appearance of a polypeptide which binds specifically to the inverted repeat of Tc1 .We have characterized the DNA binding further by DNase I footprinting and observed protection between basepairs 3 and 30 relative to the end of the transposon. This suggested that the terminal 6 nucleotides shared between most members of the Tc1 family (TACAGT) are not involved in sequence specific binding of the transposase to the ends of the element. Mutation analysis of the inverted repeat showed that the TAGATC sequence is indeed not important for bindinq of Tc1A to the inverted repeat. Perhaps they play a role in a later step in the transposition pathway. Purification of the polypeptide used for these assays revealed that a (probably proteolytic) derivative of Tc1A was responsible for the high- affinity DNA binding activity. In contrast, the full-length Tc1A protein has a very low affinity for the inverted repeat, which suggests the presence of a domain in Tc1 A which regulates the DNA binding potential. A similar result was obtained when we analyzed Tc1 A DNA binding activity in nuclear extracts prepared from induced transgenic animals. In a gel retardation assay we detected a complex which migrates at a position expected for a smaller version of Tc1A .We do not know what the significance of this observation is, but one may speculate about the occurrence of a smaller version of Tc1A which acts as a repressor in vivo. Analysis of carboxy- terminal deletion mutants of Tc1A showed that the domain required for site-specific binding is contained within the first 63 amino acids. Thus, the coding information for inverted repeat binding is mostly contained within the first exon of Tc1A .Furthermore, there is a second DNA binding domain, because a construct in which the first 70 amino acids of Tc1 A are absent, was previously shown to contain a strong general DNA binding domain (Schukkink and Plasterk, NAR 1990). In summary, we conclude that Tc1A is a transposase and has two independent DNA binding domains, one N- terminal for the binding to the inverted repeat of Tc1 and a second non-specific DNA binding domain which possibly interacts with target DNA in the transposition reaction.

Felsenstein KM et al. (1982) Worm Breeder's Gazette "the analysis of repetitive DNA sequences and their relationship to gene expression."

Emmons SW, Felsenstein KM

[Worm Breeder's Gazette, 1982]

The presence of interspersed repetitive DNA sequences has been shown to be universal in all eukaryotic genomes that have been examined. In Caenorhabditis een shown that the genome is interspersed with short repeated sequences of an average length of 300 bp (Emmons et al., 1980). The function of these sequences, at present, is unknown. In addition, via hybridization of random genomic fragments back to the genome, it was shown that no single major family of repeated sequences exists, such as, for example, the 300,000 member 'Alu' family of primates (Emmons et al., 1979). The C. elegans genome seems to contain many small families with on the average 10 members or less, which are more easily studied. It is therefore possible to isolate and analyze all the members of a single repetitive family. By looking at the structure, organization, and possible expression of these sequences, it may be possible to ascertain their role in gene expression. In our studies a 450bp inverted repeat was isolated from a randomly cloned 10 kb BamHI restriction endonuclease fragment from the Bristol genome. via S1 nuclease digestion. This sequence was shown to hybridize to 14 other BamHI fragments in the genome. The fragments ranged in size from less than 0.7 kb to greater than 15 kb. All the fragments were shown to be conserved throughout the developmental stages as well as in the germ line. The inverted repeat isolated above hybridizes to Bergerac genomic DNA with the same repetition frequency and to the same size fragments as in Bristol. In addition, hybridization was shown to C. briggsae genomic DNA at three locations. It is noted, however, that the original 10 kb fragment carrying the inverted repeat is not detected in C. briggsae. The inverted repeat sequence was used to screen a Bristol genomic clone bank in the phage vector lambda1059 for the other hybridizing sequences, which constitute a family of repetitive sequences. Preliminary EM results indicate that very few of the hybrid phages isolated contain any secondary structure. This observation indicates that most of the family members exist as isolated repeats. In a case where secondary structure can be demonstrated, the size and structure of the inverted repeat is quite distinct from the 450bp inverted repeat used to define the family. Present experiments are concentrating on the genomic location of the repetitive sequences of the isolated family, that is, are they clustered or widely dispersed in the genome? Sequence analysis will soon be undertaken, as well as an examination of the possibility that members of this repetitive family may hybridize to members of other repetitive families. In addition, an attempt will be made to determine whether these sequences are transcribed.

Robertson B et al. (2000) Worm Breeder's Gazette "e917 is an inversion that should balance the center of LGV"

Van Auken K, Wood WB, Robertson B

[Worm Breeder's Gazette, 2000]

In attempts to find additional alleles of unc-62 (pka ceh-25), we tested e917 on a suggestion from Andrew Chisholm, who had found linkage to LGVL. We confirmed his observations that e917 is a male-rescuable, maternal-effect, partially penetrant, early larval lethal and showed that it failed to complement the maternal-effect lethal allele unc-62(ct344). In experiments to define the e917 molecular lesion, we identified it as a probable inversion by inverse PCR sequencing of religated Sau3AI fragments of e917 genomic DNA. Using further direct PCR sequencing we have confirmed the inversion by sequencing across the break points and further defining its structure. One break point is in the unc-62 region of LGVL, included in cosmid T08H10. The other is on the right arm of V, in a region included in the YAC Y04H4A. The inverted segment near the left break point includes a duplication of 94bp from a region about 700bp to the left of the break point. Therefore the e917 rearrangement, which was originally isolated following 32P-decay mutagenesis, should be designated C (for complex) rather than In. It is possible that there are additional complex features in the inverted region of LGV, and although the strain we have worked with has been backcrossed 3 times, it could possibly still carry lesions on other chromosomes.

Felsenstein KM et al. (1984) Worm Breeder's Gazette "the genome of Caenorhabditis elegans: organizational analysis of interspersed repetitive DNA sequences (progress report)."

Emmons SW, Felsenstein KM

[Worm Breeder's Gazette, 1984]

We have previously reported the isolation of three distinct, non- homologous repetitive DNA families termed CeRep1, CeRep2, and CeRep3, respectively. The structure, organization and interrelationships of individual family members are being analyzed by DNA sequencing, electron microscopy, and cross-hybridization experiments. We have found that each family has a distinct and conserved structure with similarities to repetitive families that have been described for other organisms. CeRep1, present in 8-10 copies per genome, is organized with 450 bp terminal inverted repeats with variably-sized separating loop regions (500 bp to greater than 2.5 kb). The inverted repeats are 75-100% conserved and a 500 bp region in the loop is 80-90% conserved. This organizational pattern is reminiscent to Drosophila foldback elements, which are known to be transposable sequences. CeRep2 and CeRep3 are organized as 400-500 bp units that are present in about 15 and 100 copies in the genome, respectively. The elements are highly dispersed though extensive clusters have been detected. The CeRep3 element is extremely A-T-rich and has been found to share homology with known centromeric regions in yeast and other eukaryotes. To test for possible centromeric function, CeRep3 elements have been placed into yeast via an E. coli-yeast shuttle vector, and in addition the CeRep3 element has been microinjected into C. elegans. These experiments are in progress. With respect to transcriptional activity of these sequences, no RNA has been detected that is homologous to these families on Northern hybridizations.

Fodor A et al. (1981) Worm Breeder's Gazette "a technique for C elegans mass cultures."

Fodor A, Deak P, Csepely C

[Worm Breeder's Gazette, 1981]

A 4 liter glass 'fermenter' has been used instead of Roux bottles in our lab for C. elegans mass liquid cultures. It is made of glass plates stuck together with silicone rubber. It is autoclavable at 120 C, and provided with an aluminium cover containing two noles, into which tubes, are sealed by silicone rubber. One tube (the shorter one) includes a 'weighted' valve, supplied with a movable but hole- covering glass ball. The other an inverted T, the horizontal arms of which are perforated with holes, the prefiltered condensed air entering via the vertical section. This apparatus is practical for both bacteria and C. elegans. New cultures are started by transferring the cover to a new, sterilized culture from an old one. [See Figure 1]

Li WQ et al. (1992) Worm Breeder's Gazette "A Variant Tc4 Element"

Shaw JE, Li WQ

[Worm Breeder's Gazette, 1992]

Two TR679 -derivedunc-33 mutations, mn260 and rh1 O30(the latter given to us by Ed Hedgecock), are insertions of Tc4 elements at identical sites within the unc-33 coding sequence. The mn260-Tc4 element is 1.6 kb in size and appears to be very similar to the Tc4 elements in ced-4 and unc-86 described by Yuan et al. (1991, Proc. Natl. Acad. Sci. USA 88: 3334-3338), who showed that the 1.6-kb elements contain almost perfect inverted terminal repeats of 774 base pairs. Because the rh1O30-Tc4 element was 3.5 kb in size, we cloned and sequenced it. We found a 2342-bp sequence in rh1O30-Tc4 that was not present in the previously-studied Tc4 elements; it replaced a 477-bp segment in the middle of one of the inverted repeats of the l.6kb Tc4 element [See Figure 1]. When a 2-kb fragment from within the novel 2342-bp sequence was used as a probe on a Southern blot with N2 and TR679 genomic DNA digested with enzymes that do not cut any known Tc4 elements, 4-5 bands in each strain were identified. N2 and TR679 have about 25 and 30 total Tc4 -hybridizingbands, respectively; the 4-5 bands recognized by the 2-kb probe were a subset of these Tc4 -hybridizingbands. On northern blots with poly(A)-enriched RNAs prepared from N2 and TR679 ,the 2-kb probe did not detect any message, even though the 1.6-kb Tc4 element hybridized to a number of bands on northern blots with N2 and TR679 RNA. We also used the 2-kb probe to screen two different N2 cDNA libraries (provided by J. Ahringer and J. Kimble and by R. Barstead and R. Waterston). Three cDNA clones were isolated and together they represent 2.1 kb of a message, including a poly(A) tail. The 5' end of the cDNA sequence lies within the Tc4 inverted repeat and extends through most of the novel 2342-bp sequence (see figure). We have not mapped the 5' end(s) of the transcript(s) that the cDNA clones represent. The poly(A) tail begins 11-12 nucleotides downstream from a potential poly(A) signal AATAAA. The 2342-bp cDNA sequence contains five exons (all four introns are very short) within the 3.5-kb Tc4 element; an open reading frame with a coding capacity for 564 amino acids is entirely within the novel 2342-bp sequence. The putative Tc4 protein is very hydrophilic and positively charged with a pI of 8.83. It does not have high similarity to any other polypeptide in the databanks.

van Luenen HGAM et al. (1997) Worm Breeder's Gazette "Tc1 as a tool for transgenesis of vertebrates"

Raz E, van Luenen HGAM, Plasterk RHA, Driever W, Shaerringer B

[Worm Breeder's Gazette, 1997]

Members of the Tc1/mariner transposon family are present in many species from different phyla. Interestingly, distantly related species have been shown to carry closely related Tc1- like sequences, while different Tc1-like elements can be found in the same species. These findings strongly suggest horizontal transmission of Tc1-like elements during evolution. We have sown previously that the requirements for Tc1 transposition are limited to the transposase protein and the terminal part of the inverted repeats of the transposon (Vos et. al. (1996) Genes & Development 10:755-761). In vitro transposition is observed when purified transposase is incubated with an artificial transposon that carries only the last 26 basepairs of the inverted repeats. These limited requirements might explain why the family of Tc1/mariner elements has so successfully spread through the animal kingdom and might make it possible to use Tc1 for the transgenesis of different species. We coinjected plasmids containing a modified Tc3 transposon together with Tc3 transposase mRNA into one cell stage zebrafish embryos. The progeny of the injected fish were screened for the presence of the transposon and absence of the plasmid that carried the transposon. Three founder fish were isolated which transmitted the transposon sequence through their germ line. These fish also express the GFP gene, which was cloned into the Tc3 element. The insertion site of one of the Tc3 elements was sequenced. The element had correctly integrated into a TA sequences. Upon injection of Tc3 mRNA into progeny embryos of this founder fish the integrated element was correctly excised, resulting in a characteristic footprint at the site of excision. These results show that Tc3, and most likely all Tc1-like elements, can transpose in a wide range of hosts as long as the minimal requirements for transposition are met. Preliminary results show that Tc1 can also transpose in human cell lines. We will continue to develop this new tool for transgenesis.

Ruvolo VR et al. (1990) Worm Breeder's Gazette "SEQUENCE ANALYSIS OF THE TRANSPOSABLE ELEMENT Tc2"

Levitt A, Ruvolo VR

[Worm Breeder's Gazette, 1990]

A transposed copy of the mobile element Tc2 has been cloned and sequenced from the Bergerac/Bristol recombinant strain RW7406. The element is 2074 base pairs in length and contains both inverted and direct repeats at each end. The termini of the element consist of a 24 base pair perfect inverted repeat, and there is a 60-80 base pair AT-rich sequence approximately 150 base pairs in from each end. Between the ends of the element and the AT-rich region is a degenerate direct repeat that consists of overlapping tandem repeats based on a 10 base pair motif. In addition, our analysis suggests that Tc2 may have the capacity to code for a transposase protein and/or for regulatory functions. The element contains 9 potential open reading frames of 75 codons or longer, six on one strand and three on the other. We have defined the ends of the Tc2 element by examining the sequence of the corresponding 'empty site' fragment from the Bergerac strain. Our analysis indicates that this copy of the element has transposed into a highly repetitive chromosomal region. The transposon's insertion site lies at the beginning of a 120 base pair triple tandem repeat which is followed by two shorter multiple repeats (of fourteen and six copies respectively), which resemble the satellite sequences of other eukaryotes. Like Tc1, the Tc2 element is flanked by a two-base pair (AT) target site duplication. Satellite DNA has not previously been reported in C. elegans. In other eukaryotes, satellite sequences are typically found near centromeres. The chromosomes of C. elegans, however, are holocentric, and the distribution of these sequences in the genome if they have a structural function may be novel. Surprisingly, an open reading frame analysis of the repeat region suggests that it may contain one or more transcription units. We are currently analyzing polymorphic copies of Tc2; studying Tc2 transcripts; and determining the number and distribution of satellite- like sequences in the worm genome.