Genome Wide Survey of the Entire C: elegans

alpha-Tubulin Gene Family: cDNA Cloning,Expression, and Structure-Function Analysis
Camelia B. Gogonea

1M. Y. Ali

1Tetsunari Fukushige

camelia@eco1.eco.tut.ac.jp

yusuf@eco1.eco.tut.ac.jp

tfukushi@ucalgary.ca

M. Afaq Shakir

Takashi Matuso

Shahid S. Siddiqui3

mafg@warren.med.harvard.edu

1matsuo@eco.tut.ac.jp

2

1

sdq@tut.ac.jp

1 Laboratory of Molecular Biology, Dept. of Ecological Engineering
Toyohashi University of Technology, Toyohashi 441, Japan

2 Department of Medical Biochemistry, University of Calgary, Medical School
Calgary, Alberta T2N 4N1, Canada

3 Department of Pathology, Harvard Medical School, Mass. General Hospital, Boston,MA 02115, USA

1 Introduction
With the aim of molecularly and functionally characterizing each and every member of the alphatubulin gene family in a metazoan, previously we have cloned and characterized four alpha-tubulin
genes in Caenorhabditis elegans. In a genome wide search, we have now discovered four novel alphatubulin genes. Using a size selected C. elegans cDNA library, we have identi ed cDNA clones corresponding to three new alpha-tubulin isotypes. The new alpha-tubulin genes, tba-5, tba-6, tba-7 andtba-8 are transcribed at a low level. The expression pattern of the C. elegans alpha-tubulin genes
are quite complex. To study tubulins at a high molecular resolution, a comparative study between
the crystal structure of the pig brain alpha-tubulin [1] was done, using computational mutagenesis tomutate the pig alpha-tubulin to C. elegans isotypes and we analysed the changes in the 3D structuresusing MIDAS graphic package. We use CLUSTALW program to determine the homology between C.
elegans and other alpha-tubulin from bacteria, fungi, insects, and mammalians. We also constructeda phylogeny tree for the alpha-tubulin families. Our study of the promoter region of the tubulin genes
show divergence in the frequency and distribution of regulatory sequences. These data provide strong
evidence that although the alpha-tubulin appear to have very similar protein structure, nevertheless,their expression is highly regulated during animal development. Based on these data we propose, thatthe genomic architecture of tubulins and the regulation of its expression could directly contribute to
the speci city of tubulin function during animal development.

2 Results and discussion
To isolate the novel alpha-tubulin cDNA clones, a C. elegans cDNA library in the lambda ZAP vector(Y. Kohara) was screened at a low stringency using the tba-1 cDNA probe [2], 27 positive clones wereisolated, majority of which encoded the tba-1 gene. However, nucleotide sequence of the 5' upstream
region of the three positive clones revealed three new alpha-tubulin cDNAs, which were mapped on
C. elegans chromosomes, in silico, i.e. by computer based homology search, and were assigned tothree di erent cosmid clones. The sequence of the tubulin cDNA clones was also compared with theset of EST tagged clones in the C. elegans cDNA library (Y. Kohara). The genomic sequence of
the alpha-tubulin cDNA clones was obtained from the nematode sequencing consortium [3]. No fulllength cDNA clone corresponding to the tba-5 (encoded by the cosmid F16D3.1), tba-7 (encoded by386

the cosmid T28D6.2), and tba-8 (encoded by the cosmid ZK899.4) has been obtained, but two clones
for the tba-6 (encoded by the cosmid F32H2.9) isotype, namely yk450g5, and SQ#CBG32 have been
isolated, and partially sequenced. The tba-5 and tba-6 genes are located on the chromosome I, tba-7 islocated on chromosome III, and tba-8 on chromosome X. How much homology exist among the new C.
elegans alpha-tubulin and the homologous ones from other species? The CLUSTALW program showsthat the homology ranges between 63the nematode), and 82the nematode). In our search for motifs,
we nd that key features of the alpha-tubulin such as the GTP nucleotide binding site GGGTGSG, ishighly conserved in all C. elegans isotypes. Similarly, the cell attachment RDG and the amidation site,
YGKK, which are speci c only to the alpha, but not beta-tubulin, are present. Using computationalmutagenesis we mutate the TBA PIG to the new C. elegans alpha-tubulin proteins and analysed thechanges in the 3D structures, in the active site, and the morphological changes in the secondarystructures, and correlated these changes in the structure and functionality of the TBA PIG with thechange in the parent gene. The computational mutagenesis was done in the following way: we used
MIDAS graphic package [4] to replace the aminoacids from the TBA PIG with those of our isotypes,
then we added the hydrogen atoms and performed a minimisation of the energy with the molecularmechanics force eld of AMBER [5].

Acknowledgements
We thank A. Coulson, R. Durbin, S. Jones and J. E. Sulston of the Sanger Centre, Nematode Consortium, Y. Kohara, J. Spieth, for their kind assistance. We are particularly grateful to E. Nogales(which provided us with the pdb structure of the pig brain, before its releasein the PDB databank)and V. Gogonea for the help in generating the structures. Funds were generously provided by theMinistry of Education, Science, Sports & Culture, Japan, and the NEC Corp. Japan, to SSS.

References[1] Nogales, E., Wolf, S.G. and Downing, K., Structure of the alpha-beta tubulin dimer by electron
crystalography, Nature, 391:199{203, 1998.
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[3] Wilson, R., 2.2 Mb of contiguous nucleotide sequence from chromosome III of C. elegans, Nature,
368:32{38, 1994.
[4] Perlman, D.A., Case, D.A., Caldwell, J.C., Seibel, G.L., Sigh, U.C., Weiner, P. and Kollman, P.A.,
AMBER 4.0, University of California, San Francisco, 1990.
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