Ederle S et al. (1999) Worm Breeder's Gazette "Searching the genome for DNA sites recognized by binding proteins."

Pulitzer JF, La Volpe A, Ederle S

[Worm Breeder's Gazette, 1999]

In the past two decades much effort in biology has gone into cloning and sequencing of genes. The accomplishment of genome sequencing projects, such as that of Caenorhabditis elegans, is leading to a new scenario, where all genomic information is available, and the remaining challenge is to interpret it in biological terms. It is becoming essential to develop strategies to link non coding sequence to function. We have set up a one hybrid system kit, using C. elegans as model, to search the DNA for sites of binding of structural and regulatory proteins. We have constructed yeast strains in which C. elegans non-coding DNA sequences (baits) are inserted, in place of the UAS, upstream the two test genes HIS-3 and LacZ. The recipient strain is a diploid ura- yeast strain carrying a deletion of the HIS-3 gene on one chromosome and a wild type copy on the other. We have chosen to use diploid strains because the most frequent cause of HIS spontaneous reversion is the transposition of a Ty element upstream the HIS coding region; in diploid strains the transposable element Ty is quiescent. Our bait sequences are cloned upstream the HIS-3 gene in an integrative plasmid and transformed in yeast after restriction digestion. The integrative plasmid had been constructed by inserting a polylinker into the partially deleted HIS-3 promoter for ease of manipulation and it contains homology cassettes appropriate for gene replacement and to facilitate the screening of the integrants. It contains, in fact, HIS-3 flanking regions for proper integration of the plasmid, and the selectable marker URA3 in such a position to be deleted, after integration, via homologous recombination together with the wild type copy of HIS-3. Such event can be selected by plating the recombinant strains on FOA (5-fluor-orotic acid), only the ura- cells will growth in such conditions. Most pop-outs occur by recombination in the extensive tracts of homology between the recombinant HIS-3 sequence and the wild type. The obtained yeast strains are auxotrophic for histidin in the presence of only 5mM aminotriazol unless a protein binds to the bait sequence and activates transcription by mean of an activation domain such as that of GAL-4. This is the selection tool for the screening of our cDNA library (described in Ederle et al. European C. elegans Meeting 1996). In all cases the second test gene (LacZ) is carried as an episome by the cell. We have tested our tools by cloning the GATA-box as DNA bait and successfully screening our library. We are now proceeding in using this kit for the analysis of gene promoter regions and other DNA sequences potentially target of binding proteins. 1) Ederle S, De Felice B, Pulitzer JF, La Volpe A (1996) A C. elegans cDNA library for one hybrid/two hybrid system screening in yeast. 2nd European C. elegans Meeting Mont Saint-Odile, France. p.23.

Sookhareea S et al. (1997) Worm Breeder's Gazette "Desperate about getting the Tc1 to jump out from your favourite gene? Try first to mutagenize your Tc1 insertion strain with trimethylpsoralen/UV"

Dufourcq P, Sookhareea S, Labouesse M

[Worm Breeder's Gazette, 1997]

We want here to share our experience with Tc1 mutagenesis. We described at the last Worm Meeting the existence of a gene related to lin-26 immediately upstream of lin-26, which we called lir-1 for lin-26 related gene. To determine the function of lir-1, we set out to isolate a mutation using the Tc1 insertion strategy designed by Ronald Plasterk and his collaborators (1). The Plasterk lab was able to identify a Tc1 within the first lir-1 intron, lir-1(pk96), which was inserted approximately 1.7 kb from the first lir-1 exon. Not surprisingly, we found that lir-1(pk96) animals are wild-type. Starting from lir-1(pk96), we decided to try and get a jump-out event that would generate a concomitant excision of lir-1 sequences. Using a 5' oligonucleotide located approximately 1 kb upstream of the first lir-1 exon and a 3' oligonucleotide located 0.4 kb downstream of the Tc1 (about 3.3 kb downstream of the 5' oligo in a wild-type strain), we screened pools of animals to find deletion events. We first screened the progeny of 10,000 F0 lir-1(pk96) animals in pools of three to ten animals per plate and did not obtain any deletion. While we were conducting that screen, Piali Sengupta and her colleagues published a paper in which they mentioned that to get odr-10(ky255) they treated animals with UV prior to screening (2). Apparently, they had done so because in initial screens they were unsuccessful using a standard protocol (Cori Bargmann, personal communication). We decided to use a somewhat similar strategy, except that we subjected animals to mutagenesis with trimethylpsoralen and UV (3). We screened 2500 pools of F0 lir-1(pk96) animals that had first been mutagenized with TMP/UV and obtained a 779 nucleotide deletion that takes out only intronic sequences, lir-1(pk96 mc32) (the phenotype induced by mc32 is still under investigation). The deletion does not break at the Tc1 insertion point but rather about 380 pb from the original insertion point of pk96; it starts or finishes with an AT dinucleotide. We do not know what was the actual effect of psoralen mutagenesis: it could have increased the frequency of Tc1 mobilization and excision, or it could have induced the deletion by itself. Still, mutagenizing with TMP/UV and Tc1 can be an alternative strategy in certain difficult cases. Our thanks to Ronald Plasterk for isolating lir-1(pk96). (1) Zwaal R. R, Broeks A., van Meurs J., Groenen J. T. M. and Plasterk R. H. A. (1993). Proc. Natl. Acad. Sci. USA 90: 7431-7435. (2) Sengupta P., Chou J. H. and Bargmann C. I. (1996). Cell 84: 899-909 1996. (3) Yandell M. D., Edgar L. G. and Wood W. B. (1994). Proc. Natl. Acad. Sci. USA 91: 1381-1385.

Bird DM et al. (1991) Worm Breeder's Gazette "A Model for the Structure of the dpy-13 Collagen"

Bird DM, Chen TY

[Worm Breeder's Gazette, 1991]

Using a combination of computer and hand alignments, we have generated a model for the dpy-13 product in which the peptide folds back on itself to form a triple helical structure with non-helical termini (telopeptides) and a region of potential interaction with other collagens. This model is consistent with a proposal based on a classical, physical biochemical analysis of Ascaris collagen (1) ( McBride and Harrington, Biochem., 6: 1484, 1967). Key features of our model are: 1) The four stretches of sequence able to form triple-helix define three domains (3, 5 and 7 in Fig. 1). Domains 5 and 7 are available to participate in helix formation along most of their length, but domain 3 is sufficient to interact with only the amino-half of 5 and the carboxy half of 7. Perhaps the hypervariable domain (2) interacts with the remainder of domains 5 and 7. Alternatively, another peptide (8) with a gly-X-Y motif might contribute to triple-helix. The discontinuity in 5 possibly is required for such interactions. This folding arrangement places the second gly-X-Y region (5) in anti-parallel to the other two (3 and 7), effectively reversing X and Y order. This reversal enhances the preferential distribution of the bulky residues in the X position (Traub, Israel J. Chem., 12: 435, 1974) and places six of the seven proline residues in suitable locations for X/Y pairing (Jones and Miller, JMB, 218: 209, 1991). 2) Rather than being formed from the termini of peptides, the telopeptides are generated by turns in the non-gly-X-Y regions (4, 6 in Fig. l) and stabilized by disulfide linkages; the location of cysteines is consistent with formation of disulfide bridges between the ends of 1 and 5, and start of 5 and the end of 7. Chou/Fasman algorithms predict alpha-helix at both turns. The distribution of lysine residues in these turns is characteristic of other telopeptides. . 3) Sequences towards the amino terminus are not shown as being involved in folding as they presumably include a signal sequence and pro-collagen domain that are absent in the mature protein. It is not inconceivable that the putative pro-collagen peptidase target is the 'Box A consensus' (Levy, et al., WBG 11(5): 36) in the center of the otherwise hypervariable region 2. 'Box A' also contains a putative glycosylation site. It has been suggested that the semi-dominant alleles of dpy-13 (e.g. e184) might have products with aberrant collagen (triple helix?) formation but normal sites of interaction with other cuticle components, whereas the products of recessive alleles (e.g. e225) might have normal collagen helix but an altered ability to interact with other components (von Mende and Riddle,WBG 11(4): 50). The molecular location of these defects is consistent with our folding model. 1. The size of collagen monomers calculated by these workers is not in accord with that predicted from C. elegans gene sequence data. We feel that the conditions used for their sedimentation studies (low concentrations of reducing agents) were conducive to cross-linking of monomers. [See Figure 1]

Mori I et al. (1985) Worm Breeder's Gazette "observations on the biology on Tc1."

Waterston RH, Moerman DG, Mori I, Benian GM

[Worm Breeder's Gazette, 1985]

A consensus sequence for Tc1 insertions: Using an oligonucleotide complementary to part of the Tc1 inverted repeat as a primer, we have sequenced the insertion sites of unc-22(st136::Tc1), 137::Tc1), and stP1: stP1 is a Tc1 dimorphism located about 0.05 mu left of unc-22 and is due to a recent transposition event. A comparison of these three sites and two sites described by Hirsh's laboratory (NAR 11:4201 and 7137) and a repeatedly targeted site in unc-54 described by Eide and Anderson ( PNAS 82:1756; pers. comm.) reveals the following common sequence motif: T(5/6) N N N N A(6/6) T(5/6) N T(6/6) [Tc1] A(6/6) N N T(5/6) The frequency of tagging a gene of interest probably reflects both the number and the location of this motif within a gene. For example, unc-22 is a much 'hotter' target than unc-54 for Tc1 insertion, but the difference in gene size, about three-fold, cannot really account for the difference in insertion frequency. A search for this motif in unc-54 reveals that it occurs ten times but only one of these is in an exon and this is the insertional 'hot spot' recovered by Eide and Anderson. For st136 and st137 of unc-22, 12 of 15 nucleotides around the insertion sites are identical. These two sites are out of reading frame, so this homology does not merely reflect a constraint imposed at the protein level. This observation suggests that the six bases identified above are perhaps only part of a preferred target site. We are sequencing more Tc1-induced unc-22 mutants to further examine this idea of sequence specificity. Heterogeneity in the Tc1 family: The Tc1 element at stP1 lacks the 3' Tha [site present in the Tc1 sequenced by Rosenzweig, et. al., (NAR 11:4201)]. We surveyed Tc1 elements isolated from a lambda 1059-BO library and found that about 10% of the Tc1 elements lacked this site. The restriction site heterogeneity reported by others (Harris, et. al. , WBG 8, 1 : 48; Eide and Anderson, pers. comm.) for HindIII and EcoRI is not correlated with this ThaI difference, which suggests there may be a great deal of microheterogeneity within the Tc1 family in the BO strain. Mapping a mutator: We have continued to genetically localize a factor which is necessary for germ-line excision and transposition of Tc1. From three-factor crosses we have positioned the factor to a region right of dpy-5, left of unc-29 and possibly near unc-37 on chromosome I. At present, we do not know whether we are mapping a single unit or a cluster of active factors but we have evidence the factor on chromosome I can itself transpose. RW7037, which has unc-22( st136::Tc1) as a marker and has been used for the mutator localization on chromosome I, was further outcrossed to N2 and RW7080 was isolated after recovering st136 animals in the F2. This strain has fewer copies of Tc1 (probably about 70), but greater activity for unc-22 reversion. Mapping experiments show that RW7080 has the activity on LGI as well as on at least one other chromosome. Using Ac/Ds and P factor/elements as our paradigm, we are searching through the LGI recombinants for a Tc1 that may be acting as an activator or a factor. A region of Tc1 homologous to the SV40 enhancer core: Recent results in yeast demonstrate that at least part of the transcriptional control exerted by Ty elements on nearby genes is due to an enhancer sequence (PNAS 82:5423 and 5428). For a lark, we examined Tc1 and found that there is a sequence that differs by 1 bp from the SV40 enhancer 'core' at position-107 relative to the putative Met start site at bp 523. The sequence in Tc1 is TTTGGAAAGT, and in SV40 is TGTGGAAAGT. Does it mean anything? Obviously, we do not know, but the fact that Tc1 can be incorporated into the mRNA of unc-22, leads one to wonder.