Worm Breeder's Gazette 8(3): 38
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.
One of our approaches to studying dosage compensation in the nematode has been to establish a biochemical assay for the expression of X-linked genes. This has involved isolating genes from the X chromosome that are transcriptionally active and using the DNA as probes to measure mRNA levels in XO males and XX hermaphrodites. The expectation was that the level of mRNA from X-linked genes would be equal in the two sexes (when normalized to mRNA from autosomal genes) if a dosage compensation mechanism were operational. In general, such an assay is a powerful tool to demonstrate the involvement of various genes in either dosage compensation or the mechanism of assessing the ratio of X chromosomes to autosomes, the presumed basis for setting dosage compensation. In particular, this assay has the potential to determine directly whether mutations in the genes, dpy-21, dpy-23, dpy-27, sult in phenotypes dependent on the X dosage, affect the level of X-linked gene expression. Cloning of Transcriptionally Active X-Specific Genes. At the time this work was begun no methods were available for cloning nematode genes identified solely by genetic criteria. Thus we devised such a strategy in order to clone genes on X. Our approach involved differentially probing a nematode cDNA library with total genomic DNA from males (haploid for X) and hermaphrodites homozygous for a duplication of the right end of the X chromosome (mnDp27) (tetraploid for this region). cDNA clones exhibiting a four-fold difference in intensity between the hybridizations were isolated and analyzed more completely to prove their linkage to the X chromosome. Two cDNA libraries, each containing five million independent cDNA clones, were constructed using the vectors lambda gt10 and lambda gt11. We chose to construct cDNA, rather than genomic, libraries to avoid the problem that in probing a genomic library, probe sequences containing repetitive but highly dispersed DNA as well as unique DNA would preferentially hybridize to clones containing copies of the repeated sequences. This would interfere with the sensitive quantitation required by our method. cDNA libraries contain much less repetitive DNA, and using them also guarantees that selected clones represent transcriptionally active genes. These libraries were screened as indicated above and the clones with the desired properties were isolated. These clones were proven to represent cDNA from the region of the genetic duplication of X as follows: The clones were used to probe a Southern blot of genomic DNA from males (1X), hermaphrodites (2X), hermaphrodites heterozygous for a deficiency of the region, mnDf42, ( 1X), and hermaphrodites homozygous for the duplication of the region, mnDp27, (4X). A single band of restricted DNA was observed in each lane, with intensities in the expected ratio 1:2:1:4. (As a control to ensure that equal amounts of the different DNAs were in the lanes, the filters were probed simultaneously with a cDNA clone of actin gene) . Among the clones that passed this test, one (which was homologous to five separate isolates) was selected and shown genetically to map to the X chromosome. First, a clone-specific restriction fragment length polymorphism was identified between Bristol and Bergerac. Then Bristol-Bergerac hybrids homozygous for one or the other X chromosome were constructed. In each set of hybrids probed, only one of the two restriction fragment patterns was observed and the pattern corresponded completely with the strain from which the X chromosome was derived. (With this X-specific clone in hand, it was most efficient to obtain further clones by 'walking' to adjacent regions.) In addition, we felt it was important to confirm any conclusions reached about dosage compensation by using clones from different regions of the X chromosome. Thus, when X-specific clones were isolated by other laboratories, we incorporated their use into our assay. In particular, we have used transcriptionally active DNA from genomic 'walks' in the region surrounding the amber suppressor tRNA gene, sup-7, (R. Waterston) and from the region around a locus on the left end of X encoding yolk proteins (T. Blumenthal). As autosomal controls, we have used a gene-specific probe for actin- 1 on chromosome V( Kraus and Hirsh) and a transcriptionally active sequence found in a 'walk' in the region of the amber suppressor tRNA gene, sup-5 on III (R. Waterston). Results of Biochemical Analysis. The various X-specific and autosomal DNA sequences were used to probe Northern blots of RNA from adult wild-type hermaphrodites (XX animals), adult wild-type males (XO animals) and adult dpy-21 hermaphrodites (X animals). Mutations in dpy-21 appear to produce wild-type XO animals but morphologically dumpy XX animals. By genetic criteria XX dpy-21 animals behave as if they express their X chromosomes to the level expected of animals with three X chromosomes. Specifically, in a dpy-21 background triplo-X animals are dead. (J. Hodgkin [1983] Molec. Gen. Genet. 192 452- 458)}. Our results were: 1. The phenomenon of dosage compensation exists at the level of transcription in the nematode. Specifically, mRNA levels transcribed from X-specific sequences were found to be identical in XX and XO animals (normalized to mRNA levels associated with autosomal genes). 2. dpy-21 disrupts proper dosage compensation. Specifically, the level of X-transcription in dpy-21 hermaphrodites was found to be three-fold higher than in wild-type hermaphrodites, again controlling for autosomal mRNA levels. These studies are now being extended by the inclusion of more X- specific and autosomal probes and are being used to investigate mRNA levels in males and in hermaphrodites containing mutations in dpy-21, dpy-23, dpy-27, als containing double and triple combinations of these mutations. With this information we hope to sort out the patterns of interactions among these genes which are excellent candidates for being involved in dosage compensation.