Plasterk RHA (1988) Worm Breeder's Gazette "A Tc1 homologue in Drosophila."

Plasterk RHA

[Worm Breeder's Gazette, 1988]

Recently a homology was found between the TcA gene in Tc1 and an ORF in the Drosophila low repetitive sequence HB (Dr. S. Henikoff pers. comm. and manuscript in prep.). If one assumes that the Tc1 TcA gene encodes the transposase of the element, then it would follow that the target sequence of this enzyme (the ends of Tc1) could be homologous to those of the HB sequences as well. Therefore I compared the ends of the two elements. Since HB has not been shown to jump, the exact ends of the element are not defined. I looked at 8 ends of 4 elements (1), and took the point up to where they are identical or 7 out of 8 identical to be the assumed end of HB. In the figure the assumed end of HB and the end of Tc I (2) are compared. [See Figure 1] As can be seen they have 16 out of 24 basepairs in common. This is sufficient to conclude that Tc1 and HB elements are related transposable elements (or in the case of HB perhaps relics of transposable elements). To my knowledge this is the first case of similarity between DNA transposons of not directly related species. I'd like to thank Dr. S. Henikoff for communicating his observation prior to publication.

McCoubrey WR et al. (1988) Worm Breeder's Gazette "thirty something."

Nordstrom KD, Meneely PM, McCoubrey WR

[Worm Breeder's Gazette, 1988]

In previous Gazettes (WBG 10(1):62-63; 10(2):25-26) we have described a series of experiments in which we have used microinjection to assay cloned regions of the X-chromosome for feminizing elements. Injection of three different regions of the X into tetraploid hermaphrodites led to feminization of their chromosomal male progeny when those progeny also carried the X-chromosome duplication mnDp8. Such animals frequently developed as mosaic intersexes, possessing both male and hermaphrodite characteristics. We are further defining the feminizing element(s) using two approaches: reducing the size of the injected region and comparing feminizing clones. A 130 bp sequence consisting of the 3' end of the first intron and the first nucleotide of the second exon of act-4 had been shown to containing a feminizing element. Exonuclease III deletions indicated that the 5' half of this sequence was not required for feminization. An oligonucleotide was synthesized which matched 36 nucleotides at the 3' end of the intron but did not contain the nucleotide from the exon and therefore lacked the splice acceptor site. When this oligo was cloned and tested in our assay, 5 of 7 injected hermaphrodites produced a total of 15 intersexual progeny among a total of 82 3A; 2X+Dp animals. Thus, for this region, the effect has been confined to a region of less than 40 bp. Pairwise comparisons of the three X-chromosomal regions which produce intersexes in our assay, act-4, a single 8 nucleotide sequence common to all positive clones. This sequence, TATTGAAA, is present within noncoding regions in all cases. We are testing an oligonucleotide containing only this octamer. Interestingly, the identical 8 base sequence is found in the regulatory region of X-linked genes in Drosophila melanogaster (E. Jaffe, personal communication). A clone containing this element from the white locus of Drosophila (kindly provided by R. Levis) has been tested by the microinjection procedure. Although only 20 3A;2X+Dp progeny arose from nine injected animals, three hermaphrodites produced a total of five intersexual progeny. A clone of the related Drosophila autosomal gene, brown (kindly provided by S. Henikoff), did not produce any intersexual animals among 106 duplication-bearing males from 18 injected hermaphrodites. The apparent relationship between X-chromosomal sequences from C. elegans and D. melanogaster is particularly striking because of their great evolutionary separation. Whether there is any functional significance to this sequence similarity remains to be explored.

Heschl MFP et al. (1985) Worm Breeder's Gazette "sequencing the heat-inducible hsp70 gene."

Baillie DL, Heschl MFP

[Worm Breeder's Gazette, 1985]

Three genes showing homology to a Drosophila melanogaster hsp70 gene have been previously isolated [Snutch and Baillie, MGG 195, 329 (1984) ]. One of these three genes, the class A gene,is heat inducible and has been suggested to be the major heat inducible hsp70 gene in C. elegans. We have started to sequence the 5' flanking regions and the 5' coding region of the class A gene which is carried in the plasmid pUC19 (Nevins, pers. comm.). We have generated several exonuclease III deletions [Henikoff, Gene 28, 351 (1984)] to facilitate the sequencing. Several interesting observations have been made. 1) There are four Pelham consensus sequences (a 14 nucleotide sequence with dyad symmetry important for heat shock inducibility [Pelham, Cell 30, 517 (1982)]). One at approximately -280 with respect to the presumptive start of transcription (about 30 bp downstream of the TATA box) and one at approximately +4 show 70% homology. Sequences at -65 and one within the coding region show 90% homology with the consensus sequence. 2) The amino acid sequence, as deduced from the nucleotide sequence, shares a 75% homology with both D. melanogaster and human hsp70 amino acid sequences [Ignolia et al., Cell 21, 669 (1980); Voellmy et al., PNAS 82, 4948 (1985)]. 3) The presence of an intron of 46 bp has been detected at least 92 amino acids into the coding region (the start of translation has not been sequenced yet). The intron was detected by a sharp drop in amino acid homology with the human sequence only to have the homology continue further on down the sequence in a different reading frame. It was also evident when a homology matrix was done against the human hsp70 nucleotide sequence. 4) The intron shares several features with the introns from the hsp16 genes [Russnack and Candido, MCB 5, 1268 (1985)]. The sequence TTTTCAA is found 15 bp into the intron of the hsp70 gene and 17 bp into the introns of the 16-1 and 16-48 hsp genes. This sequence shares homology with the lariat consensus sequence for removal of introns [Bangeford et al., Cell 36, 645 (1984)]. At the 3' intron boundary, the sequence TTTCAG (as reported by Blumenthal et al., C. elegans newsletter 8,1) is conserved in all three introns. At the 5' boundary, the hsp70 and 16 genes share a 60% homology with the 5' consensus sequence described by Blumenthal et al. (GTRAGTTTTT; 70: GTGctTaaTT; 16-1: GTAAGTaaca; 16-48: GTAAGaaaaT). The presence of this intron is interesting in that this is the first heat inducible hsp70 gene to be described which contains an intron This suggests that the class A gene may not be the major heat inducible hsp70 gene in C. elegans or, alternatively, the intron may be used for some form of post-transcriptional control.

van Luenen HGAM et al. (1994) Worm Breeder's Gazette "Site Directed Mutagenesis of the Tc3 Transposase Gene Suggests Functional Conservation Between Retro-Elements, IS Elements and the Tc1-Like Family"

van Luenen HGAM, Plasterk RHA

[Worm Breeder's Gazette, 1994]

Tc1 and Tc3 are members of a family of transposable elements found in many different species (for example: Henikoff (1992), New Biologist 4:382-388). The Tc1 -likefamily shares no homology with other groups of transposable elements, except for a motif within the coding region of the transposase gene which it has in common with integrases of retroviruses and transposases of bacterial IS elements (Doak et al. (1994), Proc. natl. Acad. Sci. USA in press). This motif includes 2 conserved aspartic acid residues and a glutamic acid residue: the latter two are separated by 35 amino acids. This motif was first identified in a comparison of retroviral/retrotransposon integrase proteins and IS3 transposases (Fayet et al. (1990), Mol. Microbiol. 4:1771-1777; Khan et al. ( 1990), Nucl. Acids Res. 19:851-860). A mutation of each of these amino acids abolishes the catalytic functions of these proteins as shown in vitro (Drelich et al. (1992), Virology 188:459-468; Engelman and Craigie (1992), J. Virol. 66:6361-6369; Van Gent et al. (1992), Proc. Natl. Acad. Sci. USA 89:9598-9602; Kulkosky et al. (1992), Mol. Cell. Biol. 12:2331-2338). We have assayed the biological relevance of the D-D35E motif for Tc3 transposition in vivo by mutagenising each of the three conserved amino acids. The aspartic acid residues at position 144 and 231 and the glutamic acid residue at position 266 were changed to valine residues. As controls we made two additional mutations outside the D-D35E motif: a glutamic acid residue at position 213 and an aspartic acid residue at position 254 were both changed to a valine residue. The first residue (E213) is not conserved between Tc1 and Tc3 or any other member of the Tc1 -likefamily, the second residue (D254) is conserved in several transposable elements (Tc1 ,C. briggsae Tc2 and Minos). The mutant genes and the wild type Tc3A gene were fused to the hsp-16 promoter and assayed in transgenic nematodes as described before (Van Luenen et al., (1993), EMBO J. 12:2513-2520). The activity of the transposase protein was determined in two different assays. Transposition of endogenous Tc3 elements was detected using PCR, and excision of Tc3 elements was detected by Southern blot analysis (Van Luenen et al. (1993), EMB0 J. 12:2513-2520). The three mutants within the D-D35E motif were no longer capable of transposition or excision. The non-conserved mutant (E213V) was normally active. The conserved, non-D-D35E mutant (D254V) was also active, but somewhat reduced in activity compared to the wild type transposase. The presence of a motif that is similar between bacterial IS transposases, retroviral integrases and transposases of the Tc1 -likefamily suggests a possible common ancestry. Our mutagenesis data show that these residues are as important for Tc1 jumping as they are for the other transposases and integrases. This may suggest not only a conservation in sequence but also a conservation in function.

Zhen M et al. (1997) Worm Breeder's Gazette "A genetic screen for mutations affecting D neuron synapse formation and synaptic plasticity"

Jin YS, Zhen M

[Worm Breeder's Gazette, 1997]

Long-term synaptic plasticity is widely observed in diverse organisms from insects to mammals. It often involves two aspects of concurrent changes in the neurons: the structural change and the change in synaptic strength. In C. elegans, one example of such neuronal plasticity is the synaptic rewiring of the DD motor neurons. DD motor neurons are GABAergic inhibitory neurons born during embryogenesis. In L1 larval stage, DDs receive synaptic inputs from DA and DB motor neurons on the dorsal side and innervate ventral body wall muscles. However in older larvae and adults, DD neurons receive inputs from VA and VB neurons on the ventral side and form NMJs on the dorsal side. The original DD neuron connectivity is replaced by another class of GABAergic neurons, VDs, which are generated by postembryonic cell division (l). Little is known about either the detailed process of the synaptic rewiring of DD neurons or the molecular mechanism underlying such a synaptic polarity switch. We are interested in elucidating the molecular mechanisms regulating the remodeling of DD synapses. We are using a synapse specific GFP marker to visualize the synaptic rewiring (2), and to perform a genetic screen for genes regulating the structural and synaptic changes of DD neurons during the rewiring. The GFP marker, Punc-25-VAMP-GFP, is specifically expressed in the pre-synaptic zones of DD and VD neurons (together called type D neurons). VAMP-GFP chimeric protein is localized to the presynaptic zone of neurons (3). The promoter of the unc-25 gene, which encodes GABA biosynthetic enzyme glutamic acid decarboxylase, is used to specifically activate the expression of VAMP-GFP in DD neurons in L1 stage and DD&VD neurons in L2 to adult stages. In this screen we are recovering mutant animals showing any abnormal GFP expression patterns in the D neurons so that a wide spectrum of genes involved in the synapse formation of the D neurons can be isolated. But the further characterization will be focused on those disrupting the DD neuron synaptic rewiring (most likely to be the ones with little or abnormal GFP expression in the dorsal cord from L2 stage onward). So far we have screened 8,000 haploid genomes using EMS as the mutagen. New alleles of known genes required for axonal growth and synaptic formation, such as unc-5, unc-6, unc-11, unc-30, unc-33, unc-51, unc-76, and unc-104 have been isolated. The new allele (ul9) of unc-104 gene, which encodes the kinesin heavy chain and is essential for vesicle transport, is particularly interesting since the mutant animals show very little abnormality in movement. The GFP expression pattern in ju19 indicates that this mutant is able to make nearly normal synapses in the ventral cord, but fail to make synapses in the dorsal code. This suggests that ju19 may be defective in long-distance vesicle transport. More than 100 lethal/sterile mutants, most of which are defective in postembryonic cell division (thus fail to generate VD neurons) were also isolated from the screen. Intriguingly we have isolated a number of viable mutants with severely disrupted GFP expression pattern in either the DD or VD neurons (or in both), yet only subtle abnormality in locomotion. Because of the subtle visible phenotype of these mutants, they might represent new genes which are essential for the normal D neuron synaptic formation, but fail to be uncovered by previous unc screens. We are in the process of characterizing and mapping these mutations. Ref: 1) White JG, Albertson DG and Anness M. Nature 271: 764-766, 1978 2) Jin YS and Horvitz HR. 1995 Worm Meeting Abstract: 291. 3) Nonet ML WBG 13(5): 40, 1995