Analysis of expressed sequence tags from two starvation, time-of-day-specific libraries of Neurospora crassa reveals novel clock-controlled genes

In an effort to determine genes that are expressed in mycelial cultures of Neurospora crassa over the course of the circadian day, we have sequenced 1 3,000 cDNA clones from two time-of-day-specific libraries (morning and even ing library) generating similar to 20,000 sequences. Contig analysis allowe d the identification of 445 unique expressed sequence tags (ESTs) and 986 E STs present in multiple cDNA clones. For similar to 50% of the sequences (7 10 of 1431), significant matches to sequences in the National Center for Bi otechnology Information database (of known or unknown function) were detect ed. About 50% of the ESTs (721 of 1431) showed no similarity to previously identified genes. We hybridized Northern blots with probes derived from 26 clones chosen from contigs identified by multiple cDNA clones and EST seque nces. Using these sequences, the representation of genes among the morning and evening sequences, respectively, in most cases does not reflect their e xpression patterns over the course of the day. Nevertheless, we were able t o identify four new clock-controlled genes. On the basis of these data we p redict that a significant proportion of the expressed Neurospora genes may be regulated by the circadian clock. The mRNA levels of all four genes peak in the subjective morning as is the case with previously identified ccgs.