Worm Breeder's Gazette 13(2): 55 (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.

Identification and characterization of act-5 (III), a new conventional actin gene in C. elegans.

Lawrence A. Schriefer, James A. Waddle, Robert H. Waterston

Dept. of Genetics, Washington Univ. Sch. of Med., St. Louis, MO 63110

C. elegans contains four known actin genes, act-1 ,2, 3, and 4 (1) and three divergent actin-like genes, arp-1 , arp-2 ,and arp-3 (for actin related protein).(2) act-1 ,2, and 3 are located in a 12 kbp stretch of DNA on chromosome V whereas act-4 resides on the X chromosome V. Confirmation of the physical map positions of the three actin-like genes is underway (See abstract by Shrimankar et al.). Existing data suggests that the act-1 ,2, 3, and 4 genes function only in myofilament containing tissues but not in non-muscle tissues. Phenotypic analysis of 8 dominant mutations and various deletions affecting the act-1 ,2,3cluster suggest that these genes are used only in the pharynx, body wall muscles and somatic gonad.(3) While mutations in act-4 have yet to be identified, the deduced amino acid sequence of act-4 is 98% identical to act-1 ,2 and 3 and the extreme N-termini of act-1 ,2, 3 and act-4 are nearly identical to one another and resemble those found in muscle actins in vertebrates.(4) Moreover, act-4 is highly expressed in body wall muscle tissues suggesting that act-4 function may also be restricted to myofilament containing tissues.

In an effort to find potential cytoplasmic C. elegans actin genes, we searched C. elegans expressed-sequence tags (ESTs) for sequence similarity to the four known C. elegans actin genes. Five ESTs contain sequence distinct from act-1 ,2, 3, and 4; of these, three ESTs show sequence similarity with divergent actin-related proteins from other species (See abstract by Shrimankar et al.) whereas two (wEST02108(6) and cml9a 6(7))are more closely related to the conventional actin gene family. To determine whether wEST02108 and cml9a 6were made from the same or different genes, we subcloned and sequenced eml9a 6,the larger of the two cDNAs. A comparison of cml9a 6and wEST02108 revealed these two cDNAs are derived from the same gene; cml9a 6is apparently full length and wEST02108 is a partial clone. Because the DNA sequence of cml9a 6is related but not identical to act-1 ,2, 3 and 4, we named this new gene act-5 .A comparison of the deduced amino acid sequence of act-5 to existing actin sequences reveals that act-5 is more closely related to vertebrate cytoplasmic actins (89% identical) than to act-1 ,2, 3, and 4 (87% identical). Moreover, the N-termini of known muscle actins including act-1 ,2, 3, 4 contain a cysteine at amino acid position 2, whereas act-5 and most nonmuscle actins lack this residue.(4) An act-5 specific probe to the polytene YAC grids hybridized to YACs Y30E6 and Y76A2 on chromosome III. The wEST02108 -specificPCR product amplified from yeast strains carrying Y30E6 , Y76A2 ,or a larger overlapping clone, Y107A12 ,but not adjacent YAC clones, confirming the assignment. To obtain a full length act-5 genomic clone we are subcloning and sequencing YAC restriction fragments that hybridize to the cml9a 6clone.

To test the possibility that act-5 encodes a non-muscle or cytoplasmic actin, we plan to compare the expression patterns of act-1 ,2, 3, 4 and 5 by in situ hybridization of embryos with specific nucleic acid probes. A cytoplasmic actin gene could be a strong, ubiquitous C. elegans promoter that is useful for molecular genetic experiments. In addition, drug studies implicate a role for actin filaments in key developmental events in early embryogen esis8 .Temperature sensitive alleles of the act-5 gene might be used to test hypotheses about actin function in early development.

References: (l) Krause, M. et al 1989, J Mol Biol 208:381 (2) J. Lees-Miller and D. Helfman, CSHL, pers comm

(3) Waterson et al, 1984, J Mol Biol 180:473; Schriefer and Waterston, 1993 Worm Mtg, # 398

(4) Herman, I.A. 1993, Curr Opin in Cell Biol 5:48

(5) Stone and Shaw, WBG 10(3):27(1988)

(6) McCombie et al, 1992, Nature Genet 1:124

(7)Waterston et al 1992 Nature Genetics 1:114.

(8)Hill and Strome,1988,Dev Biol 125:75;Hyman and White, 1987, J Cell Biol 105: 2123; Priess and Hirsh, 1986, Dev Biol 117: 156.