Differential expression of individual suppressor tRNA(Trp) gene gene family members in vitro and in vivo in the nematode Caenorhabditis elegans

Abstract

Eight different amber suppressor tRNA (suptRNA) mutations in the nematode Caenorhabditis elegans have been isolated; all are derived from members of the tRNA(Trp) gene family (K. Kondo, B. Makovec, R. H. Waterston, and J. Hodgkin, J. Mol. Biol. 215:7-19, 1990). Genetic assays of suppressor activity suggested that individual tRNA genes were differentially expressed, probably in a tissue- or developmental stage-specific manner. We have now examined the expression of representative members of this gene family both in vitro, using transcription in embryonic cell extracts, and in vivo, by assaying suppression of an amber-mutated lacZ reporter gene in animals carrying different suptRNA mutations. Individual wild-type tRNA(Trp) genes and their amber-suppressing counterparts appear to be transcribed and processed identically in vitro, suggesting that the behavior of suptRNAs should reflect wild-type tRNA expression. The levels of transcription of different suptRNA genes closely parallel the extent of genetic suppression in vivo. The results suggest that differential expression of tRNA genes is most likely at the transcriptional rather than the posttranscriptional level and that 5' flanking sequences play a role in vitro, and probably in vivo as well. Using suppression of a lacZ(Am) reporter gene as a more direct assay of suptRNA activity in individual cell types, we have again observed differential expression which correlates with genetic and in vitro transcription results. This provides a model system to more extensively study the basis for differential expression of this tRNA gene family.

FIG. 1

Transcription of different suptRNA^Trp genes in vitro. (A) This autoradiograph presents a typical experiment, with samples electrophoresed until the bromophenol blue marker was only about halfway down the gel to reduce sample spreading for convenience. (B) Quantitation of transcript labelling for different suptRNA genes. The amount of incorporation into bands excised from gels such as those shown in panel A was quantitated by Cerenkov counting, as indicated in Materials and Methods.

FIG. 2

Transcription of sup-7 deletion clones in vitro. Representative autoradioagraph of transcripts from the indicated sup-7 deletion derivatives are shown. The top row shows results for the 5′ deletion constructs; the bottom row shows those for the 3′ constructs. Numbers above the lanes identify the endpoints of the deletion constructs used.

FIG. 3

Graphical representation of transcription of sup-7 deletion clones in vitro. Transcript labelling was quantitated as for Fig. 2. The line below the graph shows the tRNA, with 5′ and 3′ boundaries, A and B box internal promoters, and the TTTT termination sequence. The extent of DNA remaining after deletions is shown under the tRNA map.

FIG. 4

Graphical representation of transcription of sup-24 (A) and sup-29 (B) deletion clones in vitro. See Fig. 3 and Materials and Methods.

FIG. 5

Staining of beta-galactosidase activity from lacZ(Am) genes in suppressor-containing strains. suptRNA genes were introduced into lacZ(Am)-containing animals as described. Animals were heat shocked, stained for beta-galactosidase activity, and photographed. Representatives from those animals with the maximum numbers of cells stained are shown; because each cross generates both males and females, in some cases the animals shown are males (e.g., 24/+, upper animal; 29/+, both animals).

FIG. 6

Staining of beta-galactosidase activity from lacZ(Am) genes in the head regions of three sup-24/+ animals. See the legend to Fig. 5 and Materials and Methods for details.