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PubMed1997

Cloning and characterization of the extreme 5'-terminal sequences of the RNA genomes of GB virus C/hepatitis G virus.

The extreme 5'-terminal sequences of the GB virus C/hepatitis G virus (GBV-C/HGV), containing elements essential for regulation of viral gene expression and replication, have not been determined. By using a RNA-ligase-mediated RACE (rapid amplification of the cDNA ends) procedure, we have cloned the extreme 5'-terminal sequences of the viral genome from the serum of three Taiwanese patients. Sequence analysis of the 5' noncoding region in alignment with one West African and two American isolates showed that (i) a consensus 5'-end sequence was cloned; (ii) about 97% of sequences were homologous among the three Taiwan isolates and also between the two American isolates, whereas about 90% of sequences were homologous among the isolates from the three different geographic areas; (iii) the sequence heterogeneity related to geographic separation is confined mainly to three domains; and (iv) a potential hairpin structure, resembling the hairpin structure found in the 5' end of hepatitis C virus genome, was detected in the 5' end of the noncoding region. Our data support the hypotheses that (i) the extreme 5' end of the hepatitis GBV-C/HGV viral genome has been cloned, (ii) there are different genotypes correlated with geographic separation, and (iii) the viral translation and replication mechanisms may be similar to that of hepatitis C virus and pestiviruses. Our data have not only shed light on the viral replication mechanism but also offer information for selection of optimal primer sequences for the detection and genotyping of the hepatitis GBV-C/HGV virus by PCR assays.

Base Sequence↗
PubMed1995

Early transcription of the ie-1 transregulator gene of Autographa californica nuclear polyhedrosis virus is regulated by DNA sequences within its 5' noncoding leader region.

The ie-1 gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) encodes a transregulatory protein (IE1) which accelerates the expression of early and late virus genes. Transcription of ie-1 occurs immediately upon infection from a conserved CAGT motif and continues into the late phases. To examine the mechanisms by which ie-1 expression is regulated, cis-acting control elements within the ie-1 promoter were identified by constructing hybrid early promoters and by using site-directed mutagenesis. The ie-1 upstream activating region, extending from nucleotide -546 to the TATA element at -34, stimulated ie-1 basal promoter activity more than 1,000-fold when transfected into uninfected Spodoptera frugiperda SF21 cells. However, when introduced into the genome of AcMNPV recombinants, the ie-1 upstream activating region had only a minimal twofold effect early in infection. Instead, maximum steady-state levels of early ie-1 RNAs required sequences within the 5' noncoding leader region extending from +11 to +24 relative to the RNA start site (+1). The +11 to +24 noncoding region did not influence the stability of ie-1 transcripts. When assayed by in vitro transcription, deletion of the +11 to +24 region reduced the levels of ie-1 runoff RNAs. Thus, this downstream activating sequence controlled the rate of early ie-1 transcription. A larger overlapping region from +11 to +36 affected steady-state levels of ie-1 RNAs late (24 h) in infection. Deletion of sequences that included the conserved CAGT start site abolished early ie-1 transcription. Thus, ie-1 is the first example of an early baculovirus gene in which essential cis-acting regulatory elements reside within the 5' noncoding region and include sequences comprising the RNA start site.

Animals↗
PubMed1986

Molecular cloning and sequence analysis of the human parainfluenza 3 virus RNA encoding the nucleocapsid protein.

The sequence of 1690 nucleotides from the 5' end of the viral complementary RNA for the human parainfluenza 3 virus was determined by molecular cloning. One large open reading frame consisting of 1548 nucleotides was demonstrated. The encoded protein, the nucleocapsid protein (NP), consists of 515 amino acids, and has a predicted molecular weight of 57,819. A noncoding 5' sequence of 51 nucleotides is present at the end of the NP-mRNA. Two consensus sequences were identified which are homologous with sequences found in Sendai virus. One of these sequences, AGGATTAAAG, was located at the 5' end of the nucleocapsid mRNA and may function in transcription initiation. The other consensus sequence, GTAAGGGAA, was found in the viral genomic leader sequence. The nucleocapsid protein amino acid sequence was compared to other members of the Paramyxoviridae family. The parainfluenza 3 virus protein nucleocapsid amino acid sequence demonstrated a high degree of homology with the Sendai virus nucleocapsid protein. Seventy percent of the first 387 amino acids from the amino termini were identical. Little homology was observed in the distal carboxy termini.

Amino Acid Sequence↗
PubMed1992

Detection of hepatitis C virus RNA sequences by polymerase chain reaction in fixed liver tissue.

Currently, the most reliable method for the diagnosis of hepatitis C virus (HCV) infection is the detection of viral sequences by the reverse transcription double polymerase chain reaction (RT/PCR) in serum or liver samples. We demonstrate here that noncoding region sequences (NT) of HCV were amplifiable by RT/PCR in guanidinium extracts of formalin-fixed (for 6 to 48 h), paraffin-embedded liver sections of patients with chronic hepatitis C. In contrast, core and nonstructural region sequences of HCV were not detectable in fixed tissues by PCR amplification. Boiling of routinely processed tissue sections in water containing Chelex-100, a method for extraction of amplifiable hepatitis B virus DNA, was not successful. The amount of nucleic acid extracts from fixed liver sections needed for amplification of NT region sequences was over 1000 times larger than that of extracts from frozen tissue. This method will be useful for diagnostic and investigative studies of HCV infection.

Aged↗
PubMed1997

Five new or recently discovered (GBV-A) virus species are indigenous to New World monkeys and may constitute a separate genus of the Flaviviridae.

In previous studies, human hepatitis viruses have been experimentally transmitted to New World monkeys of the genus Saguinus (tamarins). Recently, two Flaviviridae-like agents (GBV-A and GBV-B) were identified in tamarins that developed hepatitis following inoculation with serum of the 11th tamarin passage of a potentially new human hepatitis agent. However, it was not shown that these viruses originated from the initial inoculum. We here report the discovery of indigenous species-specific viruses related to GBV-A in several species of New World monkeys and suggest that GBV-A virus was fortuitously acquired during passage in tamarins. Sera or plasma from 98 wild-caught New World monkeys representing 10 different species was tested by RT-PCR with conserved degenerate primers to the 5' noncoding region of the genome. Viral sequences were identified in 33 animals and sequence analysis was performed on the amplicons. In addition, the genomic region corresponding to the putative NS3 RNA helicase of GBV-A was amplified from most positive animals and sequenced. We detected GBV-A-like viruses in 13 (35%) of 37 S. mystax, 7 (78%) of 9 S. nigricollis, 3 (25%) of 12 S. labiatus, 2 (50%) of 4 S. oedipus, 2 (100%) of 2 Callithrix jacchus, and 6 (50%) of 12 Aotus trivirgatus monkeys. Each positive animal was infected with a unique strain of the GBV-A-like viruses. Analysis of the 5' NC and NS3 helicase sequences revealed that these viruses could be classified into 5 major genetic groups with genetic distances equivalent to or greater than those found among major genetic groups of hepatitis C virus. Species-specific GBV-A-like viruses were found in S. mystax, S. nigricollis, S. oedipus, C. jacchus, and A. trivirgatus species. The viruses specific for S. nigricollis were closely related to GBV-A, suggesting that GBV-A was acquired by passage through this species during the initial transmission studies. The natural history of the GBV-A-like viruses was studied in serial serum samples from 9 S. mystax and 2 A. trivirgatus monkeys. Each animal was chronically infected and the viral strain did not vary during 9-27 months of follow-up. Finally, we demonstrated that four S. mystax were positive upon arrival to the United States from the country of origin. No apparent disease was associated with chronic infection of the GBV-A-like viruses. In conclusion, many New World monkeys are persistently infected with indigenous species-specific viruses that may represent a new genus within the virus family Flaviviridae.

Animals↗
PubMed1998

A cluster of latently expressed genes in Kaposi's sarcoma-associated herpesvirus.

Infection with Kaposi's sarcoma-associated herpesvirus (KSHV) is closely associated with Kaposi's sarcoma (KS) and primary effusion lymphoma, with viral genomes present in a latent state in the majority of tumor cells. Here we describe a cluster of latently expressed viral genes whose mRNAs are generated from a common promoter. Two mRNAs in this region encode the latency-associated nuclear antigen, the product of open reading frame 73 (ORF73). The larger RNA, of 5.8 kb, is an unspliced transcript that includes ORF72 and -71 at its 3' end; it initiates at nucleotides (nt) 127880 to 127886 from a promoter lacking recognizable TATA elements. A less abundant mRNA, of 5.4 kb, is a variant of this transcript, in which 336 nt of 5' noncoding information has been removed by RNA splicing. A third, more abundant RNA is generated from the same promoter region via splicing from the common splice donor at nt 127813 to an acceptor 5' to ORF72; this transcript is the presumed mRNA for ORF72, which encodes the viral cyclin D homolog. All three RNAs are 3' coterminal. In situ hybridization analysis with probes that can detect all three transcripts shows that the RNAs are detectable in a large fraction of BCBL-1 cells prior to lytic induction and in >70% of KS spindle cells in primary KS tumors. This confirms that these transcripts are indeed latent RNAs and suggests a role for their products in viral persistence and/or KSHV-associated proliferation.

Alternative Splicing↗
PubMed1987

Photosensitization of SV 40 DNA mediated by promazine derivatives and 4'-hydroxymethyl-4,5',8-trimethylpsoralen. Inhibition of the in vitro transcription.

In vitro transcription by E. coli RNA polymerase was carried out on SV40 DNA photoreacted with various promazine derivatives. Inhibition of the template activity was recorded with increasing irradiation times in the presence of promazine derivatives. Promazine covalent adducts on guanine did not terminate RNA synthesis and seemed to be bypassed by the enzyme. HMT (4'-hydroxymethyl-4,5',8-trimethylpsoralen) photoreaction with DNA was carried out under two conditions: irradiation with lambda greater than 395 nm favouring monoadduction on pyrimidine residues and irradiation at 360 nm inducing a maximum of interstrand diadducts. Both adducts were able to terminate RNA synthesis on the phototreated SV40 DNA and using the O-methyl-nucleotide sequencing procedure, the termination sites were precisely mapped. Monoadducts on the coding strand and cross-links induced termination two bases away from the covalent adduct, but monoadducts on the noncoding strand did not half RNA polymerase.

Base Sequence↗
PubMed1991

Presence and distribution of human papillomavirus sense and antisense RNA transcripts in genital cancers.

RNA transcription in eight human papillomavirus (HPV) type 16-positive genital carcinomas and in two cervical squamous cell carcinoma (SCC)-derived cell lines was analysed by in situ hybridization using 125I-labelled subgenomic riboprobes. Transcripts corresponding to the E6 and E7 open reading frames were always present, except within the keratinizing layers of differentiated SCCs. Intranuclear E1 gene transcripts were detectable in both cell lines and some tumours whereas E2/E4 transcripts were absent from five of seven assessable tumours, suggesting transcription from integrated viral DNA. When mRNA sense riboprobes were used as controls, no signal was seen in the two cell lines; however, three tumours contained a focal, intense nuclear RNase-sensitive signal using mRNA sense riboprobes. This antisense RNA signal mapped across the whole genome including the noncoding region. In one tumour, the presence of antisense RNA was independently confirmed by using E7 and E2/E4 RNA probes of both orientations in RNase protection assays. Transcription of antisense RNA may be a natural feature of some HPV-positive genital tumours and its possible role in modulating cell behaviour in vivo requires further investigation.

Carcinoma, Squamous Cell↗
PubMed1986

Structure and transforming function of transduced mutant alleles of the chicken c-myc gene.

A small retroviral vector carrying an oncogenic myc allele was isolated as a spontaneous variant (MH2E21) of avian oncovirus MH2. The MH2E21 genome, measuring only 2.3 kilobases, can be replicated like larger retroviral genomes and hence contains all cis-acting sequence elements essential for encapsidation and reverse transcription of retroviral RNA or for integration and transcription of proviral DNA. The MH2E21 genome contains 5' and 3' noncoding retroviral vector elements and a coding region comprising the first six codons of the viral gag gene and 417 v-myc codons. The gag-myc junction corresponds precisely to the presumed splice junction on subgenomic MH2 v-myc mRNA, the possible origin of MH2E21. Among the v-myc codons, the first 5 are derived from the noncoding 5' terminus of the second c-myc exon, and 412 codons correspond to the c-myc coding region. The predicted sequence of the MH2E21 protein product differs from that of the chicken c-myc protein by 11 additional amino-terminal residues and by 25 amino acid substitutions and a deletion of 4 residues within the shared domains. To investigate the functional significance of these structural changes, the MH2E21 genome was modified in vitro. The gag translational initiation codon was inactivated by oligonucleotide-directed mutagenesis. Furthermore, all but two of the missense mutations were reverted, and the deleted sequences were restored by replacing most of the MH2E21 v-myc allele by the corresponding segment of the CMII v-myc allele which is isogenic to c-myc in that region. The remaining two mutations have not been found in the v-myc alleles of avian oncoviruses MC29, CMII, and OK10. Like MH2 and MH2E21, modified MH2E21 (MH2E21m1c1) transforms avian embryo cells. Like c-myc, it encodes a 416-amino-acid protein initiated at the myc translational initiation codon. We conclude that neither major structural changes, such as in-frame fusion with virion genes or internal deletions, nor specific, if any, missense mutations of the c-myc coding region are necessary for activation of the basic oncogenic function of transduced myc alleles.

Alleles↗
PubMed1982

Complete nucleotide sequences of the coat protein messenger RNAs of brome mosaic virus and cowpea chlorotic mottle virus.

The nucleotide sequences of the subgenomic coat protein messengers (RNA4's) of two related bromoviruses, brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV), have been determined by direct RNA and CDNA sequencing without cloning. BMV RNA4 is 876 b long including a 5' noncoding region of nine nucleotides and a 3' noncoding region of 300 nucleotides. CCMV RNA 4 is 824 b long, including a 5' noncoding region of 10 nucleotides and a 3' noncoding region of 244 nucleotides. The encoded coat proteins are similar in length (188 amino acids for BMV and 189 amino acids for CCMV) and display about 70% homology in their amino acid sequences. Length difference between the two RNAs is due mostly to a single deletion, in CCMV with respect to BMV, of about 57 b immediately following the coding region. Allowing for this deletion the RNAs are indicate that mutations leading to divergence were constrained in the coding region primarily by the requirement of maintaining a favorable coat protein structure and in the 3' noncoding region primarily by the requirement of maintaining a favorable RNA spatial configuration.

Amino Acid Sequence↗
PubMed1995

Tripartite genome organization of a natural type 2 vaccine/nonvaccine recombinant poliovirus.

Intertypic vaccine/vaccine recombinant polioviruses are frequently isolated from vaccine-associated paralytic poliomyelitis cases (VAPP). We identified a vaccine/nonvaccine poliovirus recombinant as the causative agent of a lethal VAPP. Partial RNA sequencing revealed a tripartite recombinant structure of the viral genome. This consisted of a central capsid core of vaccine origin flanked by two units of nonvaccine origin. The first nonvaccine genomic unit spanned the whole 5' noncoding region, and the second one almost the entire nonstructural protein-coding region and the 3' noncoding region. Amino acid and nucleotide sequence similarities in the 3' and 5' unidentified regions indicated that the viral donor(s) were poliovirus species, suggesting recombination between a vaccine-derived and a wild poliovirus. The nonvaccine donor(s) could not be identified among the investigated wild polioviruses cocirculating in the same geographical area. This is the first report of a natural recombination event occurring in the 5' genomic extremity of poliovirus. The neurovirulence for transgenic mice and the pathogenicity for humans of the recombinant suggested that the modular genomic organization of this virus might have conferred a selective advantage over its vaccine parent.

Amino Acid Sequence↗
PubMed1982

A topological model for transcription based on unwinding angle analysis of E. coli RNA polymerase binary, initiation and ternary complexes.

DNA unwinding induced by Escherichia coli RNA polymerase is measured for binary, initiation and ternary complexes formed from a unique promoter sequence on simian virus 40 DNA. At 37 degrees C the complexes all have an unwinding angle of 17 +/- 1 base pairs (580 degrees +/- 30 degrees). This unwinding is attributed to an enzyme-stabilized separation of the double helix at the promoter site, which is maintained throughout initiation and elongation. There is no heterogeneity in the unwinding angle of the ternary complex as it progresses down the helical template. The constant DNA unwinding during all phases of transcription leads us to propose the existence of unwindase and rewindase activities on the enzyme that allow it to travel down the helix like a nut on a DNA bolt. During elongation, the unwindase unwinds the DNA helix while the rewindase, lagging by 17 base pairs, displaces the RNA transcript and reseals the helix. Both activities induce a rotation in the DNA double helix relative to the polymerase. The RNA-DNA hybrid also rotates, maintaining both ends of that helix fixed relative to the catalytic and windase sites. Formation of an RNA-DNA hybrid which spans the distal end of the DNA unwound region is proposed as a possible mechanism for polymerase pausing and termination. This model requires that the polymerase direct the transcript past the noncoding DNA strand. Pausing occurs 16-20 nucleotides downstream from the centers of appropriately sized dyad symmetry elements.

Base Sequence↗
PubMed1993

Identification of a trans-acting activity from liver that stimulates hepatitis A virus translation in vitro.

Hepatitis A virus (HAV), a picornavirus, is the causative agent of infectious hepatitis, generally a self-limiting disease of the liver. Recently, sequences within the 5' noncoding region that affect the translation of the viral genome have been identified using in vitro systems. In this report we demonstrate that extracts prepared from mouse liver cytoplasm specifically stimulate HAV RNA translation in a rabbit reticulocyte lysate in vitro. This activity appears to act specifically on HAV sequences and is not found in other mouse tissue and several cell lines of tissue culture origin.

Animals↗
PubMed1998

Cloning, expression and sequence analysis of the classical swine fever virus nucleocapsid protein.

The DNA complementary to the 5'-terminal 1929 nucleotides of classical swine fever virus (CSFV; alias hog cholera virus, HCV) LPC vaccine strain RNA was cloned and sequenced. The sequence encompasses a 5'-noncoding region (NCR) of 264 nucleotides and an open reading frame (ORF) of 1665 nucleotides. The cloned sequence contains genes of four viral proteins, P23, nucleocapsid (core) protein, E0 and part of E1 proteins. Alignment of the 5'-terminal 1929 nucleotides of LPC strain with other strains of CSFV showed well conservation and a homology as high as 84-95% was found between these strains. The cDNA of CSFV-LPC core was cloned into an expression vector, and a fusion protein of 38.5 kDa was obtained which reacted strongly to CSFV antiserum. Purification of the core fusion protein was achieved by a single-step affinity chromatography and the purified product could be recognized by the sera of CSFV-infected swine in ELISA assay. Phylogenetic analysis of the 5'-terminal 1929 nucleotides between pestiviruses revealed that the 5'-end region seems to be suitable for differentiation of different strains of CSFV.

Amino Acid Sequence↗
PubMed2007

A structured viroid RNA serves as a substrate for dicer-like cleavage to produce biologically active small RNAs but is resistant to RNA-induced silencing complex-mediated degradation.

RNA silencing is a potent means of antiviral defense in plants and animals. A hallmark of this defense response is the production of 21- to 24-nucleotide viral small RNAs via mechanisms that remain to be fully understood. Many viruses encode suppressors of RNA silencing, and some viral RNAs function directly as silencing suppressors as counterdefense. The occurrence of viroid-specific small RNAs in infected plants suggests that viroids can trigger RNA silencing in a host, raising the question of how these noncoding and unencapsidated RNAs survive cellular RNA-silencing systems. We address this question by characterizing the production of small RNAs of Potato spindle tuber viroid (srPSTVds) and investigating how PSTVd responds to RNA silencing. Our molecular and biochemical studies provide evidence that srPSTVds were derived mostly from the secondary structure of viroid RNAs. Replication of PSTVd was resistant to RNA silencing, although the srPSTVds were biologically active in guiding RNA-induced silencing complex (RISC)-mediated cleavage, as shown with a sensor system. Further analyses showed that without possessing or triggering silencing suppressor activities, the PSTVd secondary structure played a critical role in resistance to RISC-mediated cleavage. These findings support the hypothesis that some infectious RNAs may have evolved specific secondary structures as an effective means to evade RNA silencing in addition to encoding silencing suppressor activities. Our results should have important implications in further studies on RNA-based mechanisms of host-pathogen interactions and the biological constraints that shape the evolution of infectious RNA structures.

Arabidopsis↗
PubMed1994

Mutational analysis of the tobacco vein mottling virus genome.

We have used a cDNA clone of the potyvirus, tobacco vein mottling virus, to construct 19 mutants bearing 12-nt insertions in the viral genome. These mutants display a variety of phenotypes in inoculated tobacco plants or protoplasts. All mutants with insertions in P3, CI, 6K, NIa, or NIb failed to produce detectable amounts of progeny viral RNA in protoplasts or plants which suggests that they all may be directly involved in replication. Mutants (one in P1 and one in HCpro) presumably affected in polyprotein processing also did not replicate in plants or protoplasts. Seven mutants, with insertions in the 5' noncoding region, P1, HCpro, or CP regions of the genome, were able to infect protoplasts. Three of the 7 mutants (1 in the 5' noncoding region and 2 in HCpro) were able to infect protoplasts but not plants. The remaining 4 mutants replicated in protoplasts and were able to cause systemic infection in plants. The mutation in the CP had no effect on virus accumulation or symptom development in inoculated plants, whereas the other 3 (1 in P1 and 2 in HCpro) induced cyclical patterns of symptom expression. These symptoms ranged from very mild to wild-type-like as new leaves emerged and, as the plants continued to grow, this pattern was repeated. These results support the assignment of roles in replication to five coding regions in the genome and demonstrate that sequence alterations in many parts of other regions of the viral genome may have pronounced effects on replication and the expression of disease symptoms.

Amino Acid Sequence↗
PubMed1995

The small cysteine-rich protein P14 of beet necrotic yellow vein virus regulates accumulation of RNA 2 in cis and coat protein in trans.

The effect of null mutations of the small cysteine-rich protein P14 encoded by RNA 2 of beet necrotic yellow vein virus has been investigated using in vitro transcripts of viral RNA to infect Chenopodium quinoa protoplasts. The P14 mutations down-regulated RNA 2 accumulation by approximately 10- to 50-fold. Accumulation of minus-strand RNA 2 was also diminished but RNA 1 accumulation was much less affected. The inhibition of RNA 2 accumulation could not be complemented in trans by providing P14 from another source (either a second molecule of RNA 2 or an RNA 3-based replicon) containing and expressing the P14 gene. The P14 null mutations dramatically inhibited accumulation of viral coat protein, which is encoded by the 5'-proximal gene on RNA 2, but this effect could be complemented in trans, indicating that it occurs by a mechanism distinct from that affecting RNA 2 accumulation. Transient expression experiments were also carried out in which a plasmid expressing P14 and plasmids expressing a reporter gene placed downstream of potential translational control sequences (the 5'-noncoding sequences of RNAs 2, 3, or 4) were introduced into C. quinoa or Nicotiana tabacum leaves by microprojectile bombardment. Coexpression of P14 produced a 3- to 4-fold stimulation of reporter gene expression levels for all the constructs. The lack of sequence specificity suggests that this phenomenon is not directly related to the RNA 2-specific stimulation of coat protein accumulation observed in a viral infection.

Base Sequence↗
PubMed1982

Hybrid selection of small RNAs by using simian virus 40 DNA: evidence that the simian virus 40-associated small RNA is synthesized by specific cleavage from large viral transcripts.

The simian virus 40 (SV40)-associated small RNA (SAS-RNA), approximately 64 nucleotides, is virally encoded within a region of the viral late (+) DNA strand which encodes no known protein. The SAS-RNA arises in abundance late in SV40 lytic infection. Previous data indicate that the synthesis of the SAS-RNA may be under the control of the normal late viral promoter; i.e., inhibition of transcription from the late promoter results in cessation of SAS-RNA synthesis. The synthesis of SAS-RNA was examined to determine whether the SAS-RNA is the product of cleavage from noncoding regions of nuclear late transcripts or an independent transcription product like 5S RNA, or the adenovirus VA-RNAs. The data described below suggest that SAS-RNA is cleaved from large late transcripts. In vitro transcription of DNA fragments containing the SAS-RNA coding region yielded no SAS-RNA synthesis; this result was supported by DNA sequence analysis, which indicated no promoter-like regions either within or flanking the SAS-RNA coding region. In support of a cleavage mechanism, the SAS-RNA has a 3'-phosphate end, an occurrence which is indicative of nuclease cleavage. In addition, 5'-end labeling of the SAS-RNA was possible only after calf alkaline phosphatase treatment; this indicates that the SAS-RNA is not capped. Hybrid selection analysis was used to demonstrate that separation of the SAS-RNA coding region from the normal late promoter resulted in elimination of SAS-RNA synthesis. This was demonstrated in SV40-transformed cells in which integration of a single copy of SV40 breaks the continuity of the late coding region, so that the SAS-RNA coding region is physically separated from the normal late promoter. The lack of SAS-RNA synthesis indicates that the SAS-RNA coding region cannot function as a primary transcription unit. The same result and conclusion were obtained by using a permissive cell line transformed by SV40 (COS-1 cells); here it was found that the integrated SAS-RNA coding region was not expressed even during a viable lytic infection in which the SAS-RNA could be expressed from the infecting viral genomes. The simplest conclusion drawn from the data is that the SAS-RNA is cleaved from larger late transcripts which initiate at the normal late promoter. This conclusion suggests that many of the small RNAs found in normal eucaryotic cells may be synthesized by specific cleavage rather than by primary transcription. In the course of these studies several small cellular RNAs were detected, due to their specific hybrid selection, by using SV40 DNA. Primary mapping and characterization data of these RNAs are also presented.

Animals↗