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.

Dosage Compensation Operates at the Level of Transcription in C.elegans

B. Meyer, W. Champness

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.