Worm Breeder's Gazette 6(1): 23

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Attempts to Map Actin

D. Hirsh, S. Carr, M. Krause

We reported earlier that we had cloned four DNA fragments that 
hybridize strongly to Dictyostelium actin cDNA (WBG, Vol.  5, No.  1). 
We refer to these four fragments as C.  elegans actin genes although 
we do not know that each is expressed as actin.  Three of these genes 
are in a cluster.  When the DNA's from Bristol and Bergerac strains 
are cut with several different restriction endonucleases and the DNA's 
hybridized on a Southern filter to 32P-labeled actin probe, distinctly 
different actin patterns can be observed between the two strains.
We have used these interstrain differences in restriction 
endonuclease cleavage patterns as a phenotype for mapping the actin 
genes.  We crossed Bergerac hemaphrodites with heterozygous dumpy 
males with dumpy markers representing the five different autosomal 
linkage groups.  From each cross, about 70 to 100 F2 dumpy animals 
were picked, and grown up.  Each dumpy stock therefore is assumed to 
be homozygous for the Bristol chromosome represented by the particular 
dumpy but randomly heterozygous for the other four unselected 
autosomes.  F2 non-dumpy animals that did not segregate dumpy progeny 
were also collected from each cross and they are assumed to represent 
the recovery of the corresponding homozygous Bergerac autosomes.  
Because linkage group III is so big, two different dumpy markers were 
individually used.  DNA was extracted from each of the twelve stocks, 
cut with HindIII, transferred to a nitrocellulose filter by the 
Southern technique, and hybridized to a 32P nick-translated C.  
elegans actin probe.  DNA from strains in which chromosome V was 
retrieved by recovery of the dumpy marker showed only the Bristol 
pattern and the corresponding non-dumpy segregants displayed only the 
Bergerac pattern.  DNA from all other ten strains displayed a hybrid 
pattern indicating random assortment of chromosome V.  The X-linkage 
is being done with lon-1 but the experiment is not yet finished.  We 
conclude at this time that the cluster of three C.  elegans actin 
genes are on linkage group V.
Three factor crosses have been carried out by crossing dpy-11 
gerac, recovering the recombinant phenotypes, 
growing them to be sure each one represents a single recombination 
event, i.e.  they initially segregate parental phenotypes, then 
picking the homozygous recombinant phenotype segregants and growing 
them up for DNA preparations.  After restriction endonuclease cleavage,
DNA was analyzed for the Bristol versus Bergerac pattern by 
hybridization on a Southern blot.  Seven of eight dpy-11 recombinants 
showed the Bristol pattern and one showed the Bergerac pattern.  One 
unc-76 recombinant showed the Bristol pattern and one showed the 
Bergerac pattern.  We tentatively conclude at this point that the 
three linked actin genes are between dpy-11 and unc-76, near dpy-11.  
More three factor crosses are in progress.
Bob Waterston has alerted us to unc-92 and we are going to try to 
zero it via Df-1.  We still have not located the fourth unliked actin 
gene.  We believe that this interstrain cross method will be generally 
applicable to mapping any gene for which a cloned probe exists and we 
are trying others.