Worm Breeder's Gazette 12(4): 42 (October 1, 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.

Progress in the Characterization of the msp Gene Family in Caenorhabditis vulgariensis

Robert A. Setterquist, G. Kenneth Smith, Ramya Natarajan, George E. Fox

Department of Biochemical Sciences, University of Houston, Houston, Texas 77204-5934

We are utilizing the major sperm protein (MSP) gene family found in nematodes as a model system to gain insight to genomic evolution. Gene families provide a unique window on evolution because the loss of functional genes is not necessarily deleterious. Therefore gene families can and do accumulate "molecular fossils" of events such as insertions/deletions, duplications, recombinations, etc. The msp family is especially interesting in that its intermediate size, approximately 50 copies in C. elegans, is large enough that a number of such events can accumulate and are small enough that they can be characterized in detail in a number of nematode strains and species. We are currently conducting such a comparative study of the msp family. We have begun characterization of a male/female strain WS9 -6(C. vulgariensis). This work has been facilitated by a lambda library kindly provided to us by Chris Link.

A combination of msp specific PCR amplification and lambda library screening for msp genes was used to obtain C. vulgariensis msp sequences. Msp specific PCR products were used as both probes to find msp lambda clones from the library and to quickly obtain a sample of msp genes which could be cloned and sequenced. PCR products were cloned into TA vector (Invitrogen Inc.) and then sequenced. Four unique clones were sequenced using this PCR strategy. Six lambda clones hybridized to the msp probe, five of these were unique (based on restriction digests). Msp coding fragments were further subcloned into Bluescript (Stratagene Inc.) and these were used for double-stranded sequencing.

All sequenced msp genes from C. vulgariensis encode proteins 96-99% similar to the C. elegans MSP proteins. Southern blot analysis indicates that the msp copy number is probably less than the 50 seen in C. elegans. The 5' upstream region (150 bases) immediately flanking the ATG start codon has some recognizably similar sequence elements suggesting similar MSP regulation. One obvious difference between the C. vulgariensis and C. elegans msp genes are that all of the msp genes in C. vulgariensis contain a conserved intron. A PCR based experiment designed to screen chromosomal DNA for "intronless" genes shows that all amplified msp genes contain introns after resolution on a polyacrylamide gel. Although most of the introns are highly divergent, two of the PCR sequenced genes contain introns which can be easily aligned with only a few nucleotide changes. It will be interesting to see if these genes are located near each other, perhaps even on the same lambda clone. The intron sequence from these clones is being used as a probe to answer this question. The orientation of msp genes which are less than 10Kb apart can be determined by PCR analysis using a combination of specifically oriented msp primers. This technique is currently being tested with C. elegans and C. briggsae clones. It is important to note that none of the sequenced C. vulgariensis msp genes can be considered analogous to any of the C. elegans msp genes. Other nematodes are currently being studied in order to better describe the expansion and evolution of this family.