Ali Khan L et al. (1996) Worm Breeder's Gazette "emb-8 mislocalizes Glp-1 and P-granules"

Siddiqui SS, Ali Khan L

[Worm Breeder's Gazette, 1996]

We have previously reported the transformation rescue of the emb-8(hc69) mutant with a cosmid containing the gene cej-1(C. elegans Meeting 1995, abstract 310). emb-8 is an embryonic lethal mutant at nonpermissive temperature (25oC). Cell division arrest between one cell to 50 cells stage. The egg shell is osmotically sensitive. Recently we have come to know from Sarah Crittenden( Judith Kimble Lab.) that emb-8 mislocalizes Glp-1 & P-granules specific antigens. We have earlier observed symmetric distribution of P-granules in a fraction of embryos using a monoclonal antibody specific to germ cells. These observation suggest a role of emb-8 in early cell divisions of the embryos.

Ali Khan L et al. (1997) International C. elegans Meeting "THE KINESIN SUPERFAMILY OF C. ELEGANS"

Gogonea CB, Siddiqui ZK, Shakir MA, Hori F, Hori Y, Ohnishi H, Ali Khan L, Siddiqui SS, Ali MY

[International C. elegans Meeting, 1997]

Kinesins are cytoplasmic microtubule based ATPase, that mediate intracellular transport of a variety of cargo, such as vesicles, chromosomes and morphogens in eukaryotic cells. Previously in C. elegans four genetic loci have been identified that encode members of the kinesin family, osm-3, unc-104, unc-116, and vab-8. To identify the entire kinesin gene family, we have used degenerate primers to the conserved ATP and microtubule binding sites in the motor domain of kinesins to screens C. elegans genomic and cDNA libraries. A total of 12 new kinesin genes have been identified klp-3 to klp-14, which have been mapped on different chromosomes corresponding to the cosmid clones that harbor these genes. Based on their primary sequence homology, secondary structure predictions, and the location of the motor domain within the kinesin molecule, these novel kinesins can be divided into seven distinct classes, and an outgroup. Data will be presented that may help to deduce the cellular functions of the novel kinesins during development. We thank J. Miwa, Y. Kohara, K. Nishikawa, A. Otsuka and R. Kikuno, and the nematode genome and cDNA sequencing groups for their generous support of this project.

Ali Khan L et al. (1994) Worm Breeder's Gazette "cej-1 Encodes a Novel Protein With Poly-Threonine Motif"

Siddiqui SS, Fukushige T, Ali Khan L, Tsukita Sh, Tabish M, Itoh M

[Worm Breeder's Gazette, 1994]

cej-1 Encodes a Novel Protein with Poly-Threonine Motif M. L. A. Khanl, M. Tabish, T. Fukushigel1 S. Tsukita2, M. Itoh , Sh. Tsukita , and S. S. Siddiqui. (1): Lab. of Molecular Biology, Dept of Ecological Engg. Toyohashi Univ. Technology, Toyohashi 441, and (2). National Institute for Physiological Sciences, Okazaki 444, Japan.

Ali Khan L et al. (1995) International C. elegans Meeting "PARENTAL EFFECT MUTANT emb-8 RESCUED, IS IT REALLY THE SAME AS cej-1 ?"

Siddiqui SS, Ali Khan L

[International C. elegans Meeting, 1995]

Earlier, we have identified a gene cej-1 (cell junction-1) on LGIII by screening a C. elegans cDNA expression library with a McAb T4192 raised against the mouse tight junction 220 KDa protein (Itoh et al., 1993). The nematode cDNA encodes a novel protein, with no homology to known proteins, but has conserved residues in SH3 and GK domains, and it is rich in threonine re peats. The cDNA probe identifies a single YAC clone on polyte- nes Y50C3. Subsequently we have narrowed cej-1's location to the cosmid ZC235, between the pat-3 and mab-21 loci. To identi- fy mutants in the cej-1 gene, we have been able to rescue the temp. sensitive emb-8(hc69)III mutant by germline transformat- ion using DNA from the cosmid ZC235. Mutants in emb-8 strict paternal effect, i.e. maternal gene function is both necessary and sufficient. Mutant embryos have defects in cell division time, osmotic sensitivity, cytoplasmic streaming. We are trying germline rescue of emb-8 with different regions of ZC235 to de- termine whether the cej-1 and emb-8 encode the same gene, and characterize them further.

Ali Khan L et al. (1996) Worm Breeder's Gazette "Expression of Klp-3, a Kinesin with a C-terminal motor, is in the pharynx and sphincter cells."

Siddiqui SS, Nishikawa K, Siddiqui ZK, Ali Khan L

[Worm Breeder's Gazette, 1996]

We have characterized more than a dozen members of kinesin superfamily of the motor proteins, based on the data and clones obtained from sequencing project (Y. Kohara and Genomic consortium). One of these, klp-3 has an unusual of the motor domain (which is typically found in the N-terminus region), located in the C-terminal region. Primary structure analysis shows that its length is 598 aa, it contains consensus ATP and MT binding sites at the C-terminal region. Secondary structure analysis indicates that this protein contains three distinct regions, a C-terminal globular motor domain, a coiled-coil rod and the tail which is not globular like typical kinesins. In the C. elegans so far there is only one C-terminal motor containing kinesin. To see the expression, a klp-3::lacZ construct was made. HindIII/BglII 3.5 kb fragment, containing about 1kb of the klp-3 promotor region, was put into the HindIII/BamHI site of the vector pPD16.51. The klp-3::lacZ construct was coinjected with the rol-6 marker DNA into the wild-type animals, the germ line transformants were histochemically stained for the beta-galactosidase activity. The staining is seen in pharynx, intestinal muscle and in the rectal sphincter. In the pharynx it apparently expresses in the mc1 and mc2 marginal cells (Leon Avery kindly helped us in cell identification). Previously we have reported in the west coast meeting abstract that the klp-3 (located on cosmid T09A5) can rescue the him-14 mutant phenotype partially, i. e., the number of males in the transformed progeny are reduced significantly. Also, if the klp-3 gene is injected into the wild-type animals, resulting transformants show sick worms, and about 5-10% males in the progeny. Our two factor cross data places him-14 very close to another kinesin gene unc-104, and thus it can not be in the same region as T09A5. Thus, although Klp-3 is not encoded by the him-14 gene, our results suggest that klp-3 and him-14 genes have some strong genetic interactions. Ken Kemphues and Ann Villeneuve have cloned the him-14 gene by rescueing with the cosmid ZK1127. We are interested to unravel the molecular and genetic basis of these interactions. We would like to thank the C. elegans genome sequencing consortium, Leon Avery, Y. Kohara, Ken Kemphues, Ann Villeneuve, A. Fire, M. Y. Ali, and the members of siddiqui Lab for their help.

Ali Khan L et al. (1997) International C. elegans Meeting "MATERNAL GENE EMB-8 IS NECESSARY FOR ASYMMETRIC DISTRIBUTION OF DEVELOPMENTAL POTENTIALS IN EARLY C. ELEGANS EMBRYOS"

Siddiqui SS, Ali Khan L, Ali MY, Miwa J, Kikuno R, Kohara Y, Siddiqui ZK

[International C. elegans Meeting, 1997]

A number of observations suggest that emb-8 gene plays critical roles in C. elegans development. At non-permissive temperature, emb-8 embryos divides symmetrically; in 50% the first division produces two equal sized cells, and in the second cleavage both of these cells divide symmetrically again to produce four equal sized cells. In some embryos, these four cells fuse togather and produce two asymmetric cells. emb-8 variably mislocalizes P-granules, they are uniformly distributed in 1-cell embryos, are localized on both anterior AB, and posterior P1 cells of the 2-cell embryos, and are found in all four blastomeres of the 4-cell embryos. Crittenden et al., (1996) also observed mislocalization of the P-granules and Glp-1 in emb-8 embryos. emb-8 also appears to control the orientation of early mitotic spindles, similar to the par-3 muatnts (K. Kemphues and others). However complementation tests show that emb-8 and par-3 are different genes. We thank Sarah Crittenden, Judith Kimble and Ken Kemphues for sharing unpublished results.

Ali Khan L et al. (1995) International C. elegans Meeting "A NEW MEMBER OF C. ELEGANS MECHANOCHEMICAL ENZYMES FAMILY ENCODES A KINESIN LIKE PROTEIN WITH C-TERMINAS MOTOR."

Siddiqui SS, Nishikawa K, Ali Khan L

[International C. elegans Meeting, 1995]

Kinesin is a microtubule based mechanochemical enzyme (ATP ase) that mediates transport of cellular cargo, and exists in a wide range of cell types and organisms. In C. elegans at least three members of kinesin superfamily have been characterized, osm-3, unc-104, and unc-116 ( e.g. see the abstract by M. Tabish et al., in this meeting, & JMB 247 377-389, 1995). We have been studying other members of the kinesin family, some of whichhave been identified by the cDNA or genome sequencing projects (Kohara et al., and the sequencing consortium). Among these are cDNA clones which encode KLPs similar to mouse KIF2 (LGIII), KIF4 (LGIV), human mitotic KLP-1 (LGV), and genomic sequences correspon ding to encoded homologs such as the KAR3, and XKLP2 kine- sins. In particular we are studying a KLP-lkt (LGII, not the unc-104), that has a globular motor domain at the car- boxyl terminus, simalar to that of other C-terminus KLPs such as the kar3,pkl1,klp2,katA, family. Since most of these mediate chromosomal dynamics, we speculate a similar role for the KLP-lkt.

Ali Khan L et al. (1997) International C. elegans Meeting "C-DNA CLONING, SENSE AND ANTISENSE EXPRESSION OF THE C. ELEGANS KLP-3, AN ORTHOLOG OF THE NCD AND KAR3 KINESINS"

Kikuno R, Ali MY, Nishikawa K, Ali Khan L, Siddiqui ZK, Siddiqui SS, Gogonea CB

[International C. elegans Meeting, 1997]

We have undertaken a project to molecularly clone and study cellular focus of the kinesin gene family of C. elegans. Among these we have isolated a novel cDNA (klp-3) that maps on LGII. Northern blot analysis reveals a 1.9 kb band which is expressed at a low level during development. Temporal and spatial expression of the reporter gene klp-3::lacZ is limited to the marginal cells in the pharynx and a group of muscle cells in the posterior gut region. The nucleotide sequence of the cDNA and genomic clone, reveals a kinesin like protein that shares extensive homology in the motor domain with the yeast KAR3 and Drosophila NCD kinesins, that are retrograde intracellular transport motors, mediating chromosome movement. An antisense klp-3 construct under the control of heat shock vector appears to arrest embryonic cell division and chromosome segregation, resulting in dead eggs and abnormal segregation of chromosomes. These results suggest that KLP-3 acts both during embryonic and post embryonic development and affects among others chromosomal movement in C. elegans. We thank A. Fire for the expression vectors, L. Avery for advice and J. Miwa for his support.

Ali Khan L et al. (1997) J Mol Biol "Molecular cloning and expression of the Caenorhabditis elegans klp-3, an ortholog of C terminus motor kinesins Kar3 and ncd."

Ali MY, Siddiqui SS, Nishikawa K, Ali Khan L, Siddiqui ZK, Gogonea CB, Kikuno R

[J Mol Biol, 1997]

Common to all eukaryotes, kinesins are cytoskeletal motor proteins that mediate intracellular transport on microtubule tracks, using ATP hydrolysis. A Caenorhabditis elegans cDNA clone corresponding to the klp-3 gene, encoding a novel kinesin, was isolated, and mapped on LGII. Northern blot analysis using the klp-3 cDNA probe reveals a 1.9 kb mRNA that is transcribed at a low level during development. Temporal and spatial expression of the klp-3::lacZ fusion gene is limited to the marginal cells in the pharynx, and a group of muscle cells in the posterior gut region. The nucleotide sequence of klp-3 has been deduced from the cDNA and nematode genome sequencing consortium data. Conceptual translation of the klp-3 gene reveals a kinesin-like protein with its conserved motor domain containing the ATP binding and microtubule binding sites located in the C terminus. KLP-3 shares extensive homology with the yeast Kar3 and Drosophila ncd kinesins, which have previously been shown to mediate chromosomal movement and segregation during meiosis and mitosis. Overexpression of the klp-3 gene partially rescues the lethal phenotype of the maternal lethal him-14 ts(it44) mutants at non-permissive temperatures, and reduces the incidence of males caused by non-disjunction of the X-chromosome. Similarly, expression of a klp-3 antisense RNA, under the control of a heat shock promoter, causes embryonic arrest, dead eggs and polyploid cells in transgenic lines, suggesting a critical role for the klp-3 function in chromosome segregation. Further analysis of the klp-3 gene in C. elegans may elucidate diverse functions of the C terminus mitotic motor proteins during development.

Tabish M et al. (1994) Worm Breeder's Gazette "On Rescuing the Maternal Effect emb-30 Mutant."

Siddiqui SS, Tabish M, Ali Khan L

[Worm Breeder's Gazette, 1994]

On Rescuing the Maternal Effect emb-30 Mutant. M. Tabish, M. L. A. Khan, and S. S. Siddiqui Laboratory of Molecular Biology, Dept of Ecological Engineering, Toyohashi University of Technology, Toyohashi 441, JAPAN A large set of genes required for embryogenesis has been characterized among temperature sensitive lethal mutants following EMS mutagenesis, using 25C as non- permissive and 16C as permissive tempertaures (Reviewed by Wood, 1988). To understand molecular basis of such developmental defects, we have used germline transformation technique to rescue the emb- 30(g53)III, a strict parental effect mutant for which maternal gene function is necessary and sufficient (Cassada et al., 1981; Isnenghi et al., 1983; Denich et al. 1984). We used cosmid clones located in the emb-30 region of chromosome III together with the rol-6(su1006) marker genomic DNA, and screened for emb-30(g53) roller transformants. From a selection of several cosmid clones in that region only two overlapping cosmids W07H4 and F58A4 produced roller transformants that were viable at the non permissive temperature of 25C. Since both cosmid clones contain the gene encoding a novel member of the gamma tubulin gene, we investigated if an antisense construct from such a gamma tubulin cDNA may produce a phenotype resembling the emb-30 phene. A gamma tubulin cDNA clone (from Chris Martin's library) cml3gll which produced excision plasmid pRATII containing a 1.5 kb insert was used to obtain a 0.5 kb fragment of SacI-ScaI fragment of gamma tubulin cDNA, that was ligated in opposite orientation in the hspl6-2 heat shock expression vector pPD49.78 (A. Fire). The resulting construct called pAGO.5 contains the ligated fragment, downstream of the hspl6-2 in the opposite orientation and can produce antisense transcript for the 5' end of the gamma tubulin. Marker DNA from dPY-20 ( D. Baillie and D. Suleman) together with the antisense pAG 0.5 plasmid DNA was microinjected into dPy- 20(e2017) animals, and non-dpy germline transgenic animals were tested after given a 30C heat shock treatment for eight hours. There was great reduction in the brood size only for the non-DPY animals compared to the control set in which no heat shock was administered, when screened at the the permissive and non permissive temperatures of 16 and 25C, respectively. Reduction in brood size of the heat shock treated transgenic non-dpy animals clearly suggested that antisense construct disrupted the gamma tubulin function resulting in the embryonic arrest phenotype similar to the emb-30 mutants. Finally using gamma tubulin specific flanking primers, PCR amplification, subcloning in pUC118 plasmid vector, we are in the process of sequencing emb-30(g53) mutant DNA to identify the mutation site of this allele. We thank A. Coulson, J. Sulston, R. Cassada, T. Fukushige, A. Fire, J. Kramer, D. Baillie, D. Suleman, J. Miwa, Y. Kohara, R. Durbin, Y. Hotta , R. Holmgren and the Caenorhabditis Genetics Center, for their help in this project.