Worm Breeder's Gazette 11(4): 13

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.

Isolation of cDNAs Differentially Expressed During the Adult Life Span

Tom Fabian and Tom Johnson

We are exploring age-dependent changes in gene expression during the 
adult life span.  To identify any abundantly-expressed genes whose 
expression increases or decreases with age, we differentially screened 
a cDNA library with age-specific cDNA probes.
To generate the library, RNA was isolated from a synchronous culture 
of strain BA671, which carries a temperature-sensitive allele of the 
gene spe-9.  At 25.5 C, a synchronous population of BA671 adults 
remains largely free from progeny contamination.  The mean life span 
of about 16 days for BA671 under our mass culturing conditions is 
similar to that for N2, indicating that processes limiting life span 
are not altered in BA671.
Worms were harvested for RNA isolation 14 days from hatching, i.e., 
near the end of the life span.  cDNA was synthesized from poly(A)+ RNA 
according to standard methods and ligated to the vector LambdaGEM-2.  
The resulting 'old worm' cDNA library contained 6.3x10+E6 recombinants,
0.1% of which hybridized to an act-4-specific probe.  The library was 
screened in duplicate with cDNA probes synthesized from 'young' or 
'old' poly(A)+ RNA isolated from synchronous adults 4 and 14 days, 
respectively, from the time of hatching.  Of 2x10+E4 plaques screened, 
about 3000 (15%) hybridized detectably with one or both probes.  
Approximately 2.5-3 % of these gave a differential signal.  Replicate 
filter lifts were probed with act-3- or act-4-specific probes to 
determine if these abundantly-expressed genes were represented, as 
expected, among the plaques yielding detectable signals in the 
differential screen.  Plaques hybridizing to the act-3 or act-4 probes 
were identified among those detected with the age-specific cDNA probes.
Thus, the differential screen was capable of detecting sequences 
corresponding to transcripts present in the poly(A)+ RNA fraction at 
frequencies of at least those of act-3 and act-4 (about 0.1%).  
Neither the act-3- nor the act-4-hybridizing plaques, however, 
exhibited differential signals with the age-specific cDNA probes.  
Four clones (two hybridizing more strongly to the 'young' cDNA probe 
and two hybridizing more strongly to the 'old' cDNA probe) were chosen 
for further analysis.  The inserts from these four clones were 
isolated and used to probe Northern blots of age-specific total RNAs 
isolated from several synchronous cultures.  Two of the clones (y1 and 
y16) detected a transcript of 2.4 kb which decreased with increasing 
age in RNA from three different cultures.  The extent of the decrease 
varied somewhat among these cultures, with the greatest drop observed 
to be about tenfold.  The other two clones, s3 and s20, hybridized to 
transcripts of 1.2 and 1.6 kb, respectively.  Both of these 
transcripts exhibited increases of a few-fold in total RNA from old vs.
young cultures.