Worm Breeder's Gazette 13(2): 90 (February 1, 1994)
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.
The view that protein folding in the cell is a spontaneous process guided entirely by the primary amino acid sequence has been revised by new findings which implicate molecular chaperones in the maturation of polypeptides into their final tertiary structures. Interestingly, most of the heat shock proteins (HSPs) are believed to have potential roles in protein folding. Recently, a gene encoding murine T-complex polypeptide 1 ( TCP-1 )was shown to have weak amino acid sequence similarity to H sp60 ,which suggested that the protein might function as a cytoplasmic molecular chaperones. Structural and functional studies of TCP-1 seem to support this hypoth esis2 .
We are currently characterizing the gene which corresponds to the C. elegans orthologue to mouse TCP-1 .The complete cDNA, clone cmO8 g1 Oobtained from Bob Waterston's lab, consists of a 1894-nt sequence whose ORF encodes a protein of 549 amino acids (59.5-kDa) with 66% a.a. identity to murine TCP-1 .Northern analysis using the cDNA as a probe reveals a single transcript of about 1.9-kb. The level of this message is invariant after heat shock, in contrast to the heat-inducible hsp genes. Southern analysis indicates that TCP-1 ais a single copy gene contained within a single 3.7-kb PstI restriction fragment. In order to clone TCP-1 agenomic DNA, the gene was first mapped to two overlapping YACs on chromosome II, then to a single cosmid, T05C12 .The PstI genomic fragment was isolated from the cosmid and sequenced. The structure of the tcp-1 agene is depicted in figure 1. The tcp-1 atranscript undergoes both cis- and trans-splicing to yield the mature 1.9-kb m RNA. Trans-splicing to SL1 was confirmed by performing PCR on a cDNA library using a SL1 oligonucleotide primer and a primer within the coding region; the amplication product indeed corresponds to tcp-1 a.
Previous studies have shown that the vertebrate TCP-1 chaperone is a hetero-oligomeric double-ring complex comprised of 8 different polypeptides, including several distinct proteins related to TCP-1 (3).Hence, we were not surprised to uncover at least four different, closely related TCP-1 genes in the C. elegans sequence database. To study both the structural and functional properties of the TCP-1 chaperone in the nematode, we are attempting to purify the native protein complex.
This work is supported the Medical research Council of Canada.
1. Gupta, R. S. Biochem. lnt. 4, 833-839 (1990).
2. Gao, Y., et. al. Cell, 69,1043-1050 (1992).
3. V. A. Lewis et. al. Nature, 358, 249-252 (1992).