P element transposition in Drosophila melanogaster is limited to the germ line because the third intron (the ORF2-ORF3 intron) of the P element transcript is spliced only in germ line cells. We describe a systematic search for P element sequences that are required to regulate the splicing of the ORF2-ORF3 intron. We have identified three adjacent mutations that abolish the germ line specificity and allow splicing of this intron in all tissues. These mutations define a 20-base regulatory region located in the exon, 12 to 31 bases from the 5' splice site. Our data show that this cis-acting regulatory sequence is required to inhibit the splicing of the ORF2-ORF3 intron in somatic cells.The splicing of pre-mRNA is one of the key regulatory events of gene expression in eukaryotic cells. Although much progress has been made toward understanding the biochemistry of splicing, little is known about its regulation. To further our understanding of this process, we are studying the germ line-specific splicing of the P element.P elements are transposable elements found in Drosophila melanogaster. They are the causal agents of P-M hybrid dysgenesis, a syndrome whose traits include high rates of sterility, mutation, and chromosomal rearrangement (12; for reviews see references 1, 5, and 7). P element transposition is tissue specific; transposition occurs at high rates in the germ line but is not detected in somatic tissue. Previously we have shown that the germ line specificity of P element transposition is due to germ line-specific splicing that joins open reading frame 2 (ORF2) to ORF3 (14). The first two introns of the P element are spliced in all tissues, but the ORF2-ORF3 intron is spliced only in the germ line, not in the soma (Fig. 1). cis-acting sequences must be present in the pre-mRNA which instruct the splicing machinery to splice the ORF2-ORF3 intron only in the germ line. The identification of these regulatory sequences may suggest a mechanism by which the germ line-specific splicing is regulated.Previously we have shown that a 240-base fragment that includes the third intron of the P element contains sufficient information to confer germ line specificity on the splice (15). For those experiments the ORF2-ORF3 intron of the P element was positioned between the Drosophila hsp70 (heat shock) promoter (20), plus about 1 kb of hsp70 proteincoding sequence, and the Escherichia coli lacZ gene, which encodes P-galactosidase (P-gal) (Fig. 2A). The hsp70 coding sequence was joined in frame to ORF2, and the lacZ gene was joined in frame following ORF3. For p-gal to be expressed from this construct, the hsp70 promoter must be activated and the ORF2-ORF3 intron must be spliced. The presence of P-gal activity in a heat-shocked fly containing this construct identifies tissues in which the ORF2-ORF3 intron is spliced. Because Transformants were also generated with the control construct hsp70-P(1911-[A2-3]-2183)1acZ, which differs from hsp70-P(1911-2183)-lacZ in that it contains the A2-3 deletion (a precise deletion of the ORF2...
P element transposition in Drosophila melanogaster is limited to the germ line because the third intron (the ORF2-ORF3 intron) of the P element transcript is spliced only in germ line cells. We describe a systematic search for P element sequences that are required to regulate the splicing of the ORF2-ORF3 intron. We have identified three adjacent mutations that abolish the germ line specificity and allow splicing of this intron in all tissues. These mutations define a 20-base regulatory region located in the exon, 12 to 31 bases from the 5' splice site. Our data show that this cis-acting regulatory sequence is required to inhibit the splicing of the ORF2-ORF3 intron in somatic cells.
Upon woundig of sweet potato (Ipomea batatas, Lam. var. Puerto Rico) RNase activity increases rapidly following a 4-hour lag, peaks in 24 hours, and then declines. Cyclobexhide inhibits Induction indcating that increased activity is probably due to de nowv synthesis. The half-time (t.,) (19, 20), storage tissues RNase activity peaks in 6 to 8 h and then declines. The RNase in these tissues has a long-lived mRNA that is made during the initial 30-to 45-min period after cutting (18,21, 24 Matsushita and Uritani (13) have shown that wounding ofsweet potato results in an increase in both the respiration rate, and the mitochondrial fraction, as well as de novo synthesis of peroxidase isozymes, phenylalanine ammonia lyase, the dehydrogenase activities of the pentase phosphate pathway, starch breakdown, and the level of RNA. In this paper, we report that cutting of sections of sweet potato causes a large increase in RNase activity over a period of 24 h.The results indicate that RNase mRNA in sweet potato is shortlived and that both the induction and subsequent decline in RNase activity are regulated at the transcription of RNase mRNA. MATERIALS AND METHODSPreparation of Tissue. Sweet potatoes (Ipomea batatas, Lam. var. Puerto Rico) were obtained from local markets and stored at 15°C until used. Tissue discs (1.5 x 5 mm) were cut, randomized, washed briefly in running distilled H20, and then blotted gently. Duplicate samples of20 discs (0.644 g fresh weight) were incubated in the dark at 220C in 5-cm-diameter Petri dishes on Whatman No. 1 filter paper wetted with 0.7 ml of a solution of0.5 mm CaCl2 and 34 AM streptomycin sulfate. In experiments in which inhibitors were added, the volume of the incubation medium was 2.0 ml.Enzyme Extraction. The samples of tissue were homogenized in 10 ml of cold 0.02 M K-phosphate (pH 7) for 0.5 min at 25,000 rpm in a VirTis model 23 homogenizer. The homogenate was centrifuged at 25,000g for 15 min, and the supernatant fraction was used as the source of enzyme. All steps were conducted at 1 to 40C.Enzyme Assay. The standard reaction mixture (2 ml volume) contained enzyme (32 mg fresh weight equivalent containing approximately 0.2 mg of protein) and 2 mg/ml Torula yeast RNA (Calbiochem) buffered to pH 5.4 with 0.1 M acetate buffer. Incubation was at 370C. Aliquots of 0.5 ml were removed at various times and mixed with 5 volumes (v/v) of 95% ethanol and stored overnight at 50C. After centrifugation at 12,000g for 20 min, the supernatant fractions were assayed for absorption at 260 nm against a zero time blank using a double-beam spectrophotometer.A series of reaction mixtures was prepared in which enzyme from both freshly cut and 24-h-aged tissues was diluted up to 16-fold with buffer with all other factors constant. Total RNase activity did not change significantly with dilution indicating that the enzyme solutions were free of any significant amount of either inhibitors or enhancers of enzyme activity. In the standard reaction mixture, the reaction rate was linear until ab...
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