S1 Nuclease Protection Analysis Research Articles

Differential expression of the multiple forms of rat prekininogen mRNAs after acute inflammation.

Responses of the rat liver prekininogen mRNAs after induction of acute inflammation were examined by blot-hybridization and S1 nuclease protection analyses with the aid of cDNA probes specific for rat kininogens. Marked changes in the relative levels of the low molecular weight (LMW) prekininogen mRNAs were observed after administration of Escherichia coli lipopolysaccharide, and the mRNA levels increased with a half-maximal dose of approximately 100 ng of lipopolysaccharide/100 g body weight. At maximum level of induction, the LMW prekininogen mRNAs comprised about 1% of total liver mRNA, thus representing a major component of the liver mRNA in the acutely inflamed rat. Differences in the inflammatory responses of various forms of the prekininogen mRNAs were then investigated by S1 nuclease protection analysis with the use of three different cDNA probes, each specific for either K-prekininogen or two types of T-prekininogens. Both of the T-prekininogen mRNAs increased progressively during the first 24 h after induction of inflammation, and at maximum level of induction, these two mRNAs increased about 10- and 13-fold over their normal level. In contrast, neither of the high molecular weight and LMW K-prekininogen mRNAs exhibited such an increase after induction of inflammation. Thus, the expressions of the rat T- and K-prekininogen mRNAs are differentially regulated in response to the induction of acute inflammation.

Determination of a splice acceptor site of pX gene in HTLV-I infected cells

The splice acceptor site of pX gene of HTLV-I has been determined to be at base position 7301 using S1 nuclease protection analysis. This splice acceptor site is used in all HTLV-I immortalized T-cell clones studied clones despite variation in the abundance levels of pX mRNA. Our results confirmed the proposal by Haseltine et al. (W. A. Haseltine, J. Sodroski, R. Patarca, D. Briggs, D. Perkins, and F. Wong-Staal, Science (Washington, D. C. 225, 421–424 (1984); K. Shimotono, W. Wachsman, Y. Takahashi, D. W. Golde, M. Miwa, T. Sigimura, and I. S. Y. Chen, Proc. Natl. Acad. Sci. USA 81, 6657–6661 (1984)) that a pX protein with a molecular weight of at least 38,000 could be synthesized. Generation of a 2.0-kb pX mRNA may involve a double-splicing event.

Analysis of the major transcripts encoded by the long repeat of human cytomegalovirus strain AD169.

In this report, we describe the size and kinetics of appearance of RNAs from the long repeat of human cytomegalovirus. The most abundant RNA from this region was a 2.7-kilobase (kb) species that was detected throughout the infection and was most abundant at 27 and 72 h after infection. The 2.7-kb RNA was the only major species detected with a probe that included the terminus of the long repeat and the heterogeneous L-S junction region. Other transcripts were detected with probes from the internal portion of the long repeat, including an immediate-early RNA of 1.3 kb, early and late RNAs of 1.2 kb, and minor late transcripts of 4.4, 3.6, 3.3, and 1.8 kb. S1 nuclease and exonuclease VII protection analyses of RNA from immediate-early, early, midpoint, and late times in the infection indicated that the major 2.7-kb RNA was not spliced and that the RNA mapped within the long repeat, 1.6 kb from the heterogeneous region. No evidence for temporally regulated changes in transcription initiation, splicing, or choice of 3' end of this RNA was observed. Nuclease protection analysis also demonstrated that the second most abundant late RNA from this region, the 1.2-kb species, was not spliced and had the same polarity as the 2.7-kb RNA. The 1.2-kb also mapped entirely within the long repeat, with its 3' terminus 1.7 kb upstream from the 5' terminus of the 2.7-kb RNA.

Multiple polyadenylation sites in a Drosophila tropomyosin gene are used to generate functional mRNAs.

The gene encoding muscle tropomyosin I in Drosophila is alternatively spliced in embryonic and thoracic muscle to generate two sizes classes of RNAs. By Northern blot analysis, the embryonic RNA class shows a broad RNA band of hybridization of 1.3 kb and a more sharply defined, less abundant RNA band at 1.6 kb. The thoracic class of RNAs, on the other hand, consists of a broad hybridization band at 1.7 kb and a more sharply defined band at 1.9 kb. Each size class of RNA encodes a different tropomyosin isoform. The two classes of alternatively spliced RNAs utilize the same 3' terminal exon of the gene. The DNA sequence of this exon reveals a cluster of several polyadenylation signals (AAUAAA) or polyadenylation-like signals. We show here by S1 nuclease protection analysis that at least five and possibly seven of these polyadenylation or polyadenylation-like sequences are associated with in vivo embryonic and thoracic mRNA cleavage processing sites. Six of these S1 sites are clustered within 119 bp and a seventh is located 255 bp downstream. At least one of the polyadenylation-like signal sequences appears to be an unusual AACAAA sequence. In addition we also show that these mRNAs function in vitro to synthesize muscle tropomyosins.

Multiple transcripts for higher plantrbcL andatpB genes and localization of the transcription initiation site of therbcL gene

We have compared therbcL andatpB transcription units from spinach, maize, and pea. In most cases multiple transcripts were found for a given chloroplast gene. The 5' termini of these transcripts were determined by S1 nuclease protection and primer extension analyses. TherbcL transcripts have 5' termini 178-179 and 64 nucleotides (spinach), 300 and 59-63 nucleotides (maize), and 178 and 65 nucleotides (pea) upstream from their respective protein coding regions. TheatpB transcripts have 5' termini (453-454, 272-273, 179, and 99 nucleotides (spinach), 298-302 nucleotides (maize), and 351-355 nucleotides (pea) upstream from their respective protein coding regions. The intergenic distance between therbcL andatpB genes is relatively constant (152 to 157 base pairs) among the three chloroplast genomes. In spinach, maize, and pea, the 80 base pairs surrounding the 5' end of therbcL gene (±40 base pairs) have 85% sequence homology. Similarly, the 60 base pairs preceding theatpB gene have 48% sequence homology. Both genes have '-10' and '-35' regions that resemble the prokaryotic consensus promoter sequence. The larger, but not smaller,rbcL transcripts from spinach and pea can be labeled with alpha-(32)P-GTP by guanylyltransferase. These data suggest that DNA sequences 178-179 (spinach), 300 (maize), and 178 (pea) base pairs before therbcL protein coding regions represent sites of transcription initiation. The sequences 59-65 base pairs before therbcL protein coding regions may correspond to sites of RNA cleavage.

Ordered processing of Escherichia coli 23S rRNA in vitro.

In an RNase III-deficient strain of E. coli 23S pre-rRNA accumulates unprocessed in 50S ribosomes and in polysomes. These ribosomes provide a substrate for the analysis of rRNA maturation in vitro. S1 nuclease protection analysis of the products obtained in in vitro processing reactions demonstrates that 23S rRNA processing is ordered. The double stranded stem of 23S rRNA is cleaved by RNase III in vitro to two intermediate RNAs at the 5' end and one at the 3' end. Mature termini are then produced by other enzyme(s) in a soluble protein fraction from wild-type cells. The nature of the reaction at the 5' end is not clear, but the reaction at the 3' end is exonucleolytic, producing three heterogeneous mature termini. The two reactions are coordinated; 3' end maturation progresses concurrently with cleavages at the 5' end. Two results suggest a possible link between final maturation and translation: in vitro, mature termini are formed efficiently in the presence of additives required for protein synthesis; and all the processing intermediates detected from in vitro reactions are also found in polysomes from wild-type cells.

In Vitro and in Vivo transcription initiation sites on the TK-encoding BamHI Q fragment of HSV-1 DNA

The sites of in vitro transcription initiation on the BamHI Q fragment of herpes simplex virus DNA have been compared with the sites of 5′ ends of RNAs made in vivo after virus infection. S1-nuclease protection analysis of these RNAs shows that there are in vivo counterparts for each of the five previously identified in vitro transcripts. The whole-cell-extract RNA polymerase II transcription system faithfully initiates RNAs predominantly at bona fide in vivo start sites and gives few, if any, false positive start sites. Further, antiparallel, self-complementary transcripts from the BamHI Q fragment were observed in the coding region of the HSV thymidine kinase gene.

DNA sequence and transcription of a DNA minicircle isolated from male-fertile sugar beet mitochondria.

A minicircle consisting of 1440 bp was isolated from male fertile sugar beet mitochondria. The entire nucleotide sequence of the minicircle was determined. There are several stop codons in all three reading frames and it is therefore unlikely that the minicircle codes for a polypeptide product. However, the minicircle is transcribed yielding a major RNA product consisting of 850 bases in addition to two very minor RNA products of 1100 and 950 bases, respectively. All three RNA products are transcribed from the same strand. The starting point for transcription of the major RNA product was determined by S1 nuclease protection analysis.