Worm Breeder's Gazette 15(3): 11 (June 1, 1998)
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.
|1||Dept. of Molecular & Cellular Biology, University of Arizona, Tucson, AZ 85721.|
|2||Dept. of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305.|
In the Ward lab, we are interested in understanding the genes that control the development and motility of C. elegans spermatozoa. Over the years we and others have used genetic and molecular methods to identify genes that are necessary for spermatogenesis. Recently we collaborated with Stuart Kim to screen a microarray of about 1200 random ESTs to identify additional genes that are expressed preferentially in sperm and to assess how reliably the microarray can detect differential germ-line gene expression.
To screen the array, we prepared RNA from young adult fem-3(q23ts) (gain-of-function) hermaphrodites, which produce only sperm, and RNA from fem-1(hc17ts) hermaphrodites, which produce only oocytes. Both mutants have hermaphrodite soma, so any major difference in mRNA expression is likely due to either sperm- or oocyte- specific gene regulation. Synchronized worms of each genotype were grown in parallel on rich plates at 25C and monitored to ensure they were not starved. RNA was prepared by homogenizing worms with glass beads and by collecting poly(A)+ RNA using oligo(dT) cellulose and spin columns.
The Kim lab prepared cDNA probes labeled with different fluorochromes from the fem-1 and fem-3 RNAs, and then hybridized these simultaneously to the microarray of ESTs. The microarrays were then scanned with a confocal scanner at two wavelengths. The results were analyzed by determining the ratio between the scans after subtracting background. To ensure reproducibility, duplicate microarrays were hybridized with independently-prepared probes from the same RNA samples; three independently-grown fem-1 and fem-3 worm populations were tested in all. Differentially-regulated genes were identified by criteria requiring reproducibility between microarray scans and at least a two-fold difference in expression ratio between the two probes.
Of the nearly 1200 ESTs analyzed, a subset was expressed preferentially in fem-3 (spermatogenesis-enriched) and a larger subset was expressed preferentially in fem-1 (oogenesis-enriched). ESTs with strong expression ratios in one microarray scan were nearly always highly regulated in all scans. ESTs with weak expression ratios tended to show more variability. In general, where there were ESTs from the same gene on the microarray, they showed similar regulation.
Each of the eight major sperm protein (msp) ESTs present on the microarray was among the top 20 spermatogenesis-enriched genes. This family produces the most abundant protein in the sperm and has been shown previously to be sperm-specific. No other previously-identified sperm-specific genes are on the array. The remaining newly-identified spermatogenesis-enriched genes are uncharacterized, although some have homologues, including two protein-tyrosine phosphatases. The oocyte-enriched genes include mei-1, a C-type lectin, several DNA replication enzymes, possible transcription factors, and a likely G-protein. A complete list of the regulated clones, ranked by reproducibility, can be obtained at http://www.acaciabio.com/bin/wormxcmp4?ex1=38&ex2=100&ex3=102&min=0.75 . Expression ratios are listed as ln(fem-1 probe/fem-3 probe).
These results demonstrate that microarray technology can yield valuable information about C. elegans gene regulation. If similar arrays are probed with RNA from other stages of worm development and from other mutants it will be possible to learn more about the expression of these genes outside of the germ line. The development of microarrays containing every C. elegans gene would enable labs to comprehensively survey gene expression patterns in the worm.