Worm Breeder's Gazette 12(3): 56 (June 15, 1992)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Gamma tubulin is a recent addition to the tubulin family that has sequence similarities to both alpha and beta tubulins, and is highly conserved from yeast to humans (1-3). In C. elegans, we have been looking at the molecular and cellular specificity of gamma tubulin. A polyclonal anti-gamma tubulin, antiserum JH 46, which was raised against a 17 amino-acid oligopeptide corresponding to a conserved region of gamma tubulin in all known species (4), was used in immuno-blots of crude homogenates of mixed population of C. elegans. We identified a single band of a 50 K protein, that corresponds to the size of gamma tubulin from other species (M. Shakir). Immunocytochemical staining of wholemount worm preparations with JH 46 antibody shows bright staining of developing embryos. There is a little background staining of non-divding cells in larval and adult animals. These results are consistent with the role of gamma tubulin in dividing cells for microtubule nucleation (4).
To identify gamma tubulin gene in C. elegans. we screened a nematode lambda gt11 cDNA library with a mouse gamma tubulin cDNA probe, that provided a few positive clones. However. during this work we came to know about the clone # cm13 g11 .from the Chris Martin's sorted cDNA library, that encoded gamma tubulin, reported by C. elegans Sequencing Consortium, WBG: 12,(2). p28 ,1992. We have obtained the cDNA clone cm13 g11 from St. Louis group, and used it to identify two overlapping YACs on polytene filter, namely Y41D1 ant Y1G11 .This places gamma tubulin gene in the vicinity of III chromosome gene cluster. We received confirmation of this result from A. Coulson, as they had also placed cm13 g11 in the identical region. This is a gene rich region, including emb-9 , unc-32 , unc-50 , unc-69 ,and ced-9 .We are looking at the RFLP of various embryonic arrest mutants in this region, to identify mutations affecting gamma tubulin in C. elegans. Genomic Southern blot analysis of N2 DNA, gives a single band with BamHl and two bands with EcoRI digests. Northern blot analysis of C. elegans poly A RNA from different developmental stages, using the gamma tubulin cDNA probe, reveals a single 1.5 Kb band. The sequencing of the cDNA and genomic clones of gamma tubulin is in progress. We also intend to see the expression of gamma tubulin gene during C. elegans development using gene fusion with the marker lac-z gene.
(2) Stearns, T. et al., Cell 65, 825-836, 1991
(3) Horio, T. et al., Cell Sci. 99, 693-700. 1991
(4) Joshi, H. C. et al., Nature 356, 80-83. 1992
With generous help from the C. elegans Sequencing Consortium