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Results for “Nuclease Protection Assays”

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PubMed1992

Developmental expression of the S35-S45/SGP-2/TRPM-2 gene in rat testis and epididymis.

Testosterone-repressed prostate message-2 (TRPM-2) was originally isolated and cloned from the regressing ventral prostate of the rat. In this tissue, and in other hormone-dependent tissues such as the mammary gland, this gene is induced in the absence of the appropriate trophic hormone. Sequence analysis of the cDNA and genomic clones of TRPM-2 have demonstrated that the coding sequence of this gene is identical to S35-S45 (also known as SGP-2 and clusterin), which is constitutively expressed by the Sertoli cells of the adult testis. Using Northern, slot blot, S1-nuclease analysis, and in situ hybridization, we have investigated the regulation of TRPM-2 expression in the testis and epididymis during development. Slot blot analysis of RNA extracted from the testis and epididymis of 7-, 14-, 28-, 35-, and 91-day-old rats demonstrates that the gene is induced to detectable levels between days 7 and 14 and that the relative level of expression does not change significantly after day 14. In situ hybridization using frozen sections of testis from day 2-, 7-, 14-, 28-, 35-, and 91-day-old rats confirms that there is little expression of TPRM-2 in the seminiferous epithelium of 7-day-old rats, but this increases considerably after 14 days, primarily in Sertoli cells but also in association with meiotic developing spermatogenic cells. However, TRPM-2 mRNA is expressed in the rete testis at 2 days of age, reaches a peak at 35 days of age, and continues to be expressed in the adult. Slot blot analysis demonstrates that TRPM-2 is also induced in the epididymis between 7 and 14 days of age, although, as has been demonstrated by in situ hybridization, TRPM-2 mRNA is detectable in the epithelial cells in the head of the epididymis but is barely detectable in the midportion or tail regions. Northern analysis suggests that the size of the TRPM-2 transcript in the testis also changes during development. In the early stages of testicular development, the TRPM-2 transcript appears to be a broad band of approximately 1.5 kb, while the transcript in the adult appears to be approximately 1.8 kb in length. S1-nuclease protection assays suggest that this increase in size is not due to differential splicing of the first exon of TRPM-2/SGP-2 and most probably reflects a difference in the polyadenylation of the mRNA in the testis at different times during development.

Animals↗

PubMed1994

Transcription of circular and noncircular forms of Sry in mouse testes.

Although its expression in adult testis was immediately apparent, the role for Sry (sex determining region, Y) in testicular function remains elusive. We have performed transcriptional studies in an effort to elucidate potential roles of Sry by studying the time and location of its transcription in mouse testes. Northern analyses and more sensitive nuclease protection assays detected transcripts in 28-day-old testes and beyond. The highly sensitive technique of reverse transcription polymerase chain reaction (RTPCR) could not detect Sry expression in 14-day testes when primers for the most conserved portion of the gene, the high mobility group (HMG) box, were used, but primers for the circular form detected Sry transcription at all postnatal stages studied. The same HMG box primers were able to detect expression of Sry in XX, Sxra or Sxrb testes. This suggested that Sry is expressed in cells other than germ cells, which was confirmed with studies on fractionated cells--RTPCR detected transcription of Sry in the highly pure interstitial cell fraction. However, Leydig cells and a Leydig cell tumor were negative for Sry expression. We performed in situ studies in an attempt to localize the expression of Sry in the testes. Abundant expression of an Sry cross-hybridizing transcript was found in spermatogonia, in early spermatocytes, and in some interstitial cells with antisense probes to the HMG box or a more specific, 3' region, whereas the sense probe gave little or no hybridization. It is probable that the circular transcripts, which are seen in reverse transcriptase positive (RT+) and RT- reactions by PCR because of the RT activity of Taq polymerase, are responsible for the hybridization seen in spermatogonia and spermatocytes, whereas linear and circular forms are detected later. Thus Sry is expressed in pre- and postmeiotic germ cells and in somatic cells of the testes.

Animals↗

PubMed1999

Complex transcriptional initiation pattern of the thymidylate synthase promoter in mouse tissues.

Thymidylate synthase (TS) is an essential enzyme that must be expressed in all proliferating cells. The mouse TS promoter lacks a TATA box and an initiator element and initiates transcription over a broad region in cultured fibroblasts. The goal of this study was to determine if expression of the TS gene in a variety of cells and tissues involves the use of alternative promoters or different patterns of transcriptional initiation sites. The amount of TS mRNA and the pattern of initiation sites were determined using S1 nuclease protection assays. We found that even though the amount of TS mRNA varied over a wide range, reflecting differences in cell proliferation rates, the pattern of initiation sites was nearly identical in all of the cell lines and tissues that were examined. Therefore transcription of the TS gene is directed by a single promoter that is capable of being expressed in a wide variety of cellular environments.

Aging↗

PubMed1993

Expression of ionotropic glutamate receptor genes by P19 embryonal carcinoma cells.

P19 embryonal carcinoma cells can be induced to differentiate into neuron-like cells by retinoic acid. P19 neurons were recently shown to express both NMDA and non-NMDA type glutamate receptor-mediated currents and be susceptible to glutamate excitotoxicity. In this study, we used RT-PCR to survey differentiated P19 cultures for glutamate receptor transcript expression. The following transcripts were detected: at least one member of the GluR1-4 family, GluR5, GluR6, GluR7, KA1, KA2, NMDAR1, and NMDAR2B. Nuclease protection assays revealed a large quantitative induction of GluR6 transcripts following retinoic acid treatment. Inotropic glutamate receptors are a fundamental and major feature of CNS neurons which are not expressed by the cell lines commonly used as experimental models for mammalian neurons. The present results show that P19 cells express multiple genes involved in glutamate receptor biology. Since the stem cells can be manipulated genetically, the system has the basic requirements for analyzing mechanisms involved in glutamate receptor gene expression.

Animals↗

PubMed1997

Differentiation-dependent expression of a human carboxylesterase in monocytic cells and transcription factor binding to the promoter.

Carboxylesterases play an important role in defense and clearance mechanisms of the monocyte/macrophage system. During the differentiation process of cells from the monocytic cell line THP-1 we observed a transient transcriptional upregulation of a human carboxylesterase analyzed by means of Northern blots. In PMA-treated THP-1 cells we could detect three major transcription initiation sites as revealed by Nuclease Protection Assay carried out with two overlapping antisense RNA probes. We have recently cloned the carboxylesterase upstream sequence and showed its basal promoter activity in CHO cells. Using electrophoretic mobility shift analysis we demonstrated that the promoter region spanning base pairs -1 to -275, which contains several putative binding sites for transcription factors, is bound by nuclear factors Sp1 and IRBP but not by C/EBPs. Taken together these data indicate that carboxylesterase gene transcription in THP-1 cells starts at multiple initiation sites and that Sp1 and IRBP may be critical factors for modulating the differentiation-dependent transcription of this human carboxylesterase gene.

Animals↗

PubMed1998

Silencing of CYP1A1 expression in rabbits by DNA methylation.

Unlike most experimental animals, treatment of adult rabbits with 3-methylcholanthrene (MC) does not induce the expression of the CYP1A1 gene. In this study, we show that DNA methylation plays one of the key roles in the suppression of CYP1A1 gene expression. S1 nuclease protection assay showed that the induction of CYP1A1 mRNA by MC occurred in rabbit kidney RK13 cells but not in rabbit lung R9ab cells, while aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (Arnt) mRNAs were expressed in both cells at similar levels. Interestingly, the treatment of R9ab cells with a DNA demethylating agent, 5-aza-2'-deoxycitidine, resulted in the induction of the expression of the CYP1A1 gene by MC. The results indicate that DNA methylation is one of the factors involved in the loss of the MC-induced expression of the CYP1A1 gene. Thus, it seemed that the binding of the AhR/Arnt complex to the xenobiotic-responsive element (XRE) was inhibited by the hypermethylation of CpG dinucleotides within an XRE core sequence (5'-CGTG-3'). To explore this possibility, we compared the methylation status of XRE in R9ab cells with that in RK13 cells. A bisulfite sequence analysis using genomic DNAs from R9ab cells showed that the CpG site within XRE was highly methylated on both coding and non-coding strands. In contrast to this result, the hypomethylation of XRE was seen in RK13 cells. To examine whether or not the binding of the AhR/Arnt heterodimer to XRE is affected by the methylation status of XRE, a gel shift assay using a methylated XRE as a probe was carried out. As expected, the AhR/Arnt complex could not bind to the methylated XRE. From these results, we conclude that the cell type-specific transcription of the rabbit CYP1A1 gene is caused by DNA methylation.

Animals↗

PubMed2000

Differential expression of sense and antisense transcripts of the mitochondrial DNA region coding for ATPase 6 in fetal and adult porcine brain: identification of novel unusually assembled mitochondrial RNAs.

The mammalian mitochondrial genome is a double-stranded circular DNA molecule, which is transcribed from both strands as polycistronic RNAs, which are further processed to yield the mature polyadenylated mRNAs, rRNAs and tRNAs. We compared the gene expression patterns of foetal and adult porcine brains and identified a sequence tag from the ATPase 6 region of the mitochondrial genome which, in adult brain, was more abundant in the sense (H-strand) form, but, in foetal brain, more abundant in the antisense form (L-strand). By means of solution hybridisation/S1 nuclease protection assay, Northern blotting, and PCR based techniques, we demonstrated that the ATPase 6 region of the porcine mitochondrial genome is transcribed as co-existing, stable sense and antisense RNAs. Furthermore, we identified sense and antisense transcripts from this region consisting of inversely assembled fragments joined together at a direct repeat of 7 nucleotides. Our results suggest that transcription and post-transcriptional processing of mitochondrial RNAs are much more complex than presently thought.

Adenosine Triphosphatases↗

PubMed1995

Alternative splicing of HLA class I transcripts induced by IFN-gamma and TNF in fibroblasts: release of soluble HLA class I heavy chain and an associate protein.

FS-4 fibroblasts were found to produce 37-kDa HLA class I heavy chain in response to IFN-gamma or TNF in a time- and dose-dependent fashion, and a synergism between IFN-gamma and TNF was observed. Immunoprecipitation of IFN-gamma- or TNF-induced FS-4 cell culture supernatants by mAb A1.4 revealed an additional 33-kDa protein in association with the 37-kDa heavy chain. The 33-kDa protein appeared to be expressed in a 38-kDa form on the membrane of FS-4 cells induced by IFN-gamma or TNF, as A1.4 immunoprecipitated the 38-kDa band in association with the 44-kDa transmembrane HLA class I heavy chain. Release of the 37-kDa heavy chain could well be due to an alternative RNA splicing with the deletion of exon 5 encoding the hydrophobic transmembrane region of membrane-anchored HLA class I heavy chain. Northern blot analysis and S1 nuclease protection assay suggested the existence of HLA class I heavy chain mRNA lacking exon 5 in IFN-gamma- or TNF-induced FS-4 cells. Southern blot analysis on the products of reverse transcription-polymerase chain reaction amplification from cytoplasmic RNA confirmed induction of alternative splicing by these cytokines. Our results suggest that cytokine-induced production of soluble HLA class I molecules may play important roles in the regulation of T cell interaction with antigen-presenting cells.

Alternative Splicing↗

PubMed1993

Beta-1,4-galactosyltransferase expression during spermatogenesis: stage-specific regulation by t alleles and uniform distribution in + -spermatids and t-spermatids.

In the mouse, gamete recognition is mediated in part by the binding of sperm surface beta-1,4-galactosyltransferase (GalTase) to specific oligosaccharide residues on the zona pellucida glycoprotein ZP3 (D. J. Miller, M. B. Macek, and B. D. Shur. Nature 357, 589-593, 1992). The expression of GalTase on the sperm surface is regulated by alleles within the distal segment of the T/t complex and results in a haploid-specific increase in GalTase expression on spermatids and sperm from t-bearing males, suggesting that differences in sperm GalTase activity may contribute to t-sperm transmission ratio distortion (B.D. Shur and N. F. Scully. Genet. Res. Camb. 55, 177-181, 1990). In this study, we characterized the expression of GalTase RNA during wild-type and T/t-mutant spermatogenesis and analyzed the potential role of GalTase in transmission ratio distortion. Using northern blot analysis, S1 nuclease protection assays, and in situ hybridization, it was found that spermatogenic cells predominantly express the long form of the GalTase RNA, which encodes the GalTase protein that is preferentially targeted to the cell surface in somatic cells. In wild-type testes, GalTase RNA accumulates during the maturation of primary spermatocytes, reaches peak levels prior to meiosis, and decreases at meiosis. GalTase RNA accumulates to similar levels during the maturation of +/t and t/t primary spermatocytes, but unlike wild-type, the level of GalTase RNA in t-bearing spermatocytes remains elevated during meiotic division. Consequently, spermatids in t-mutant testes inherit higher levels of GalTase RNA than do wild-type spermatids, which likely accounts for the haploid-specific increase in surface GalTase activity characteristic of spermatids from t-bearing mice. The functional significance of the increased GalTase activity during t-sperm transmission ratio distortion was determined by examining the distribution of GalTase RNA and surface GalTase protein in haploid spermatids from heterozygous +/t males. Result show that+and t spermatids have similar levels of GalTase RNA assayed by quantitative in situ hybridization and similar levels of surface GalTase protein assayed by PCR genotyping of spermatids separated by fluorescence-activated cell sorting. These results indicate that although the expression of GalTase is regulated by alleles within the distal segment of the T/t complex, transmission ratio distortion in +/t mice is not likely due to haploid-specific differences in sperm surface GalTase activity.

Alleles↗

PubMed1998

Analysis of transcripts derived from sequences upstream of the bidirectional mouse thymidylate synthase promoter.

The promoter of the mouse thymidylate synthase (TS) gene lacks a TATAA box and an initiator element and is capable of directing transcriptional initiation with approximately equal strength and over broad initiation windows in both directions. The goal of the present study was to determine if the TS promoter directs the transcription of a second gene that is upstream of the TS gene by characterizing the transcripts that correspond to the upstream sequences. RNA blot analyses revealed the presence of 1.4 and 5 kb cytoplasmic, polyadenylated transcripts that include sequences upstream of the TS promoter. The transcripts were much more abundant in a cell line in which the TS gene is amplified. S1 nuclease protection assays showed that the transcripts have multiple 5' termini. An exon trap approach identified a potential splice donor site that might correspond to the 3' end of the first exon of the upstream gene. A cDNA library was probed with a sequence from the putative first exon, and six different cDNA clones were isolated. However, analysis of the sequences of the cDNAs revealed that the upstream transcripts were not spliced at the potential 3' donor site but instead extended into a repetitive LINE (long interspersed nuclear element) sequence that begins 0.3 kb upstream of the TS promoter. RNase protection assays confirmed that the in vivo transcripts extend into the LINE element. Therefore it appears that the upstream transcripts are unlikely to correspond to a functional mRNA molecule.

Amino Acid Sequence↗

PubMed1995

Schistosoma mansoni: cloning and characterization of a gene encoding cytosolic Cu/Zn superoxide dismutase.

Two forms of Cu/Zn superoxide dismutase (SOD) have been identified in Schistosoma mansoni, signal peptide-containing SOD (SP-SOD, Simurda et al., 1988, Exp. Parasitol. 67, 73-84) and cytosolic SOD (CT-SOD, Hong et al., 1992, Exp. Parasitol. 75, 308-322). The SP-SOD gene has been previously characterized. We report the isolation and characterization of the gene encoding CT-SOD. A cDNA clone of CT-SOD was radiolabeled and used to screen an EMBL3 genomic library. Of 100,000 plaques, 1 hybridizing phage contained an insert of 14.6 kb. The CT-SOD gene spans 8.5 kb of chromosomal DNA and possesses three exons (150, 285, and 234 bp, respectively) and two introns (4.9 and 2.7 kb, respectively) which interrupt the coding region. Separate copies of the gene as determined by restriction fragment patterns on Southern blots are found on two YAC clones. Using YAC clones that contain the CT-SOD gene, fluorescence in situ hybridization on chromosomes identified a copy of the gene located on the middle region of chromosome 1 and another copy localized to the middle region of the long arm of chromosome 3. Primer extension and nuclease protection assay performed on total RNA define the transcription initiation site and allow the identification of potential cis-acting sequences, such as a CAAT box and a hexanucleotide GCCCGG. Comparison of this gene with Cu/Zn SOD genes from Drosophila, humans, and mice identifies conserved exons that represent functional domains in the SOD protein.

Amino Acid Sequence↗

PubMed1993

Characterization of the gene encoding human cone transducin alpha-subunit (GNAT2).

The human cone transducin alpha-subunit (GNAT2) gene has been completely characterized. The human GNAT2 transcription unit is 9967-bp in length and consists of eight exons with seven introns. The eight exons are identical to the reported cDNA sequence (Lerea et al., 1989). Northern blot analysis of RNA from human retinas and a retinoblastoma cell line, WERI-RB1, reveals a 1.7-kb transcript for GNAT2. Multiple transcription initiation sites were mapped for human retina and WERI-RB1 RNA by primer extension and S1 nuclease protection assays. This gene has seven initiation sites spanning 31 bp. The sequence upstream of the GNAT2 gene shows a TATA box consensus sequence at -29, a CCAAT box consensus sequence at -58 (reverse orientation), and a sequence (CCATAT) similar to the CCAAT box consensus at -76. The GNAT2 upstream sequence shows no significant identity with the upstream region of the human rod transducin alpha-subunit gene (GNAT1) or with the upstream regions of the color visual pigment genes, indicating that the expression of GNAT2 may be regulated differently than these other rod- and cone-specific proteins.

Amino Acid Sequence↗

PubMed1997

Human fatty aldehyde dehydrogenase gene (ALDH10): organization and tissue-dependent expression.

Mutations in the fatty aldehyde dehydrogenase gene (ALDH10) are responsible for Sjögren-Larsson syndrome (De Laurenzi et al., 1996). In this study, the expression and the genomic organization of the ALDH10 gene are reported. The gene spans approximately 31 kb and consists of 10 exons and 9 introns. All exon-intron junction sequences match the classical GT/AG rule. Both S1 nuclease protection assay and primer extension study suggest that the transcription initiation site is located 195 nucleotides upstream from the ATG codon. No canonical TATA box can be found in the 5'-flanking sequence of the gene, but a CCAAT-like box was found 58 bp upstream of the putative transcription start site. Sequence analysis of the 5'-flanking region revealed numerous potential binding sites for transcription factors Sp1 and AP-2 and one putative HIP-1 binding site. Northern blot analysis of poly(A)+ RNA from various tissues revealed two mRNA species, with sizes around 4.0 and 2.0 kb, that are derived from the differential use of two polyadenylation sites. Although this gene is expressed in a variety of human tissues, the expression level of ALDH10 in the liver and skeletal muscle appears to be higher than that in other tissues examined.

Aldehyde Oxidoreductases↗

PubMed1997

Molecular characterization of the gene for human cartilage gp-39 (CHI3L1), a member of the chitinase protein family and marker for late stages of macrophage differentiation.

We have previously reported that the expression of HC gp-39, a 39-kDa secretory glycoprotein and member of the chitinase protein family, is associated with late stages of monocyte to macrophage maturation. To allow further investigations of its unique expression pattern and to facilitate studies on the regulation of this "marker gene," we characterized the genomic organization of the HC gp-39 gene (HGMW-approved symbol CHI3L1) and cloned its 5'-proximal promotor region. The gene is composed of 10 exons distributed over an 8-kb region. Two transcriptional initiation sites located 82 and 126 nucleotides upstream of the ATG start codon were identified using primer extension and S1 nuclease protection assays. We sequenced 1.3 kb of the 5'-flanking region and identified a variety of putative regulatory elements.

Adipokines↗

PubMed1996

Prognostic significance of c-erB-2 mRNA in ovarian carcinoma.

The oncogene specific mRNA of c-erbB-2 was detected by the S1 nuclease protection assay in 95 ovarian cancer specimens. In 79 primary carcinomas, we found 16 (20%) with strong expression, 13 (17%) with weak expression, 4 (5%) with very weak expression, and 46 (58%) with no expression. In 3 of 16 recurrencies (19%) a strong expression of c-erbB-2 mRNA was detected, in 2 (12%) weak expression was detected, and in 11 (69%) no expression of c-erbB-2 mRNA was detected. Kaplan-Meier analysis revealed no significant association between strong expression of c-erbB-2 mRNA and survival of the 79 patients with primary cancer. However, in the subgroup of patients with FIGO (International Federation of Gynecology and Obstetrics) stages III and IV (n = 60) a shorter median survival time (12 months) was obtained for patients with strong c-erbB-2 mRNA expression compared to patients with no, very weak, and weak c-erbB-2 mRNA expression (25 months, P = 0.04). In addition, strong expression of c-erbB-2 mRNA did not depend on histologic grade, histologic type, and FIGO stage. Adverse prognostic factors include histologic type (serous carcinoma), high grade, high stage (FIGO stages III and IV), and residual of tumor after surgery. From our results we conclude that for all patients (FIGO stages I-IV) strong expression of c-erbB-2 mRNA is not a prognostic parameter, but in the subpopulation of patients with FIGO stage III and IV only, an association between strong c-erbB-2 mRNA expression and shorter median survival time was found, although statistical significance was weak (P = 0.04).

Female↗

PubMed1995

Rapid conversion from beta-MHC to alpha-MHC mRNA expression in embryonic rat ventricle cultured in oculo is not dependent on thyroid hormone or testosterone.

We investigated the expression of myosin heavy chain (MHC) isoenzymes in embryonic rat ventricles cultured in the anterior eye chamber of an adult rat. In oculo, these grafts beat and mature in an environment where the hormonal milieu can be manipulated. S1 nuclease protection assays were performed on pooled samples of ventricle grafts and compared to normally growing ventricles. At the time of grafting (embryonic day 12, E-12), 23 +/- 4% of the MHC mRNA was of the alpha isoform. While the proportion of ventricular alpha-MHC mRNA did not increase in utero, embryonic ventricles cultured in oculo showed a rapid increase in the relative amount of alpha-MHC mRNA expression (to 84 +/- 10% by 3 days and 86 +/- 5% by 8 days in oculo). alpha-MHC mRNA expression predominated through 8 weeks of culture in oculo, being 76% at 8 weeks in oculo. Additional experiments were performed to determine whether the rapid conversion to alpha-MHC expression resulted from exposure to adult levels of testosterone or thyroid hormone. Reduction of testosterone exposure to nondetectable levels by host orchiectomy did not affect the rapid conversion to alpha-MHC mRNA expression. Exposure to a hypothyroid milieu (i.e., PTU-treated hosts) decreased but did not prevent the conversion from beta- to alpha-MHC mRNA expression at 8 days in oculo; with 83% of the MHC mRNA being of the alpha isoform in hypothyroid hosts compared to 95% in euthyroid hosts. After 8 weeks of culture in hypothyroid hosts, however, alpha-MHC mRNA expression was undetectable in grafted ventricles. These data suggest that E-12 myocardial grafts respond to the hormonal milieu of an adult rat with rapid conversion from beta- to alpha-MHC mRNA expression and that alpha-MHC expression in early developing heart may show reduced sensitivity to downward modulation by a hypothyroid hormonal milieu.

Animals↗

PubMed1994

Regional distinction in age-related changes of preoptic regulatory factor-1 and preoptic regulatory factor-2 mRNA expression in the rat brain.

Preoptic regulatory factor-1 (porf-1) and preoptic regulatory factor-2 (porf-2) are two novel neuropeptide genes expressed in the central nervous system and peripheral tissues. Other studies have shown that these genes may play a role in steroid-dependent brain development and functions. In this study, nuclease protection assays were employed to investigate Porf-1 and Porf-2 mRNA expression in male rat brains of different ages. The preoptic area (POA), cerebral cortex (CC), and hippocampus (HIPP) expressed both Porf-1 and Porf-2 mRNA, while only Porf-2 mRNA was detectable in the medial basal hypothalamus (MBH). Porf-1 mRNA in the POA was highest at the age of 2 months (young adult), decreased at the age of 6 months (mature adult), and remained low at the ages of 12 (middle aged) and 24 months (aged). Porf-1 mRNA in the CC was also the highest at the age of 2 months and decreased with age. However, there were no age-related changes for Porf-1 mRNA in the HIPP. Porf-2 mRNA in the HIPP was found to be low at the age of 2 months, increased at the ages of 6 and 12 months, and decreased at the age of 24 months. The effect of age on Porf-2 mRNA in the POA was similar to that seen for Porf-1, with the highest expression observed in the 2-month-old rats. There were no age-related Porf-2 mRNA changes in the MBH and CC. These results indicate differential regulation of expression of the porf-1 and porf-2 genes in the MBH, POA, HIPP, and CC. The possible roles of these two genes in maturation and aging of male rats are discussed.

Aging↗

PubMed1993

Nucleotide sequence and genomic organization of rice tungro spherical virus.

Rice tungro disease is caused by a combination of two viruses: rice tungro spherical virus (RTSV) and rice tungro bacilliform virus (Jones et al. (1991) J. Gen. Virol. 72, 757-761.). The genome of RTSV is a single-stranded polyadenylated RNA. We present here the 12,433-nucleotide complete sequence of RTSV genomic RNA and its deduced coding regions. This sequence contains a large open reading frame (ORF) which initiates following a 514-nucleotide 5' leader sequence and is capable of encoding a viral polyprotein of 390.3 kDa. Two viral subgenomic RNAs of ca. 1.2 and 1.4 kb, respectively, were detected in RTSV-infected leaf tissues and mapped by S1 nuclease protection assay. These RNAs were determined to be congruent with the genomic RNA sequence proximal to the 3' terminus and could contain up to two small ORFs in their 5' to 3' orientation. There are at least three capsid protein subunit cistrons near the N-terminus of the large ORF. A computer-aided search of the C-terminal half of the large ORF revealed conserved protein sequence motifs for a viral RNA polymerase, proteinase, and a putative NTP-binding protein. These sequence motifs are arranged in a manner that resembles those of picorna-like viruses. Taken together, these data indicate that RTSV is a distinct type of positive-strand RNA virus. The evolutionary relationships between RTSV and other picorna-like plant viruses are discussed.

Amino Acid Sequence↗