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PubMed1994

Structure and inducible regulation of the human MET promoter.

The MET oncogene, encoding the tyrosine kinase receptor for the hepatocyte growth factor/scatter factor, is expressed in epithelial cells and overexpressed in a significant proportion of human epithelial cancers, suggesting the occurrence of transcriptional alteration(s). To identify the MET promoter, we isolated recombinant cDNA clones encompassing the entire 5'-noncoding sequence of MET messenger RNAs. Using probes derived from this region, we cloned the entire genomic region spanning the first MET exon and the flanking regulatory sequences. The first exon, containing the entire untranslated sequence, is present in the MET mRNAs of 7.1, 5.9, and 4.6 kilobases, showing that the expression of the multiple transcripts is regulated by a single promoter. The start site of transcription was determined by primer extension and by rapid amplification of cDNA ends. We show that a 300-base pair fragment, containing sequences upstream from the start site, efficiently drives the expression of a reporter gene in transfected epithelial cells. This promoter fragment also contains the cis-acting elements responsible for phorbol-ester induction.

Base Sequence↗
PubMed2001

Sequence comparison and secondary structure analysis of the 3' noncoding region of flavivirus genomes reveals multiple pseudoknots.

Sequences of 191 flavivirus RNAs belonging to four sero-groups were used to predict the secondary structure of the 3' noncoding region (3' NCR) directly upstream of the conserved terminal hairpin. In mosquito-borne flavivirus RNAs (n = 164) a characteristic structure element was identified that includes a phylogenetically well-supported pseudoknot. This element is repeated in the dengue and Japanese encephalitis RNAs and centers around the conserved sequences CS2 and RCS2. In yellow fever virus RNAs that contain one CS2 motif, only one copy of this pseudoknotted structure was found. The conserved pseudoknotted element is absent from the 3' NCR of tick-borne virus RNAs, which altogether adopt a secondary structure that is very different from that of mosquito-borne virus RNAs. The strong conservation of the pseudoknot in mosquito-borne flavivirus RNAs implies a stronger relationship between these viruses than concluded from previous secondary structure analyses. The role of the (tandem) pseudoknots in flavivirus replication is discussed.

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↗
PubMed1992

Second-site suppressor mutations assist in studying the function of the 3' noncoding region of turnip yellow mosaic virus RNA.

The 3' noncoding region of turnip yellow mosaic virus RNA includes an 82-nucleotide-long tRNA-like structure domain and a short upstream region that includes a potential pseudoknot overlapping the coat protein termination codon. Genomic RNAs with point mutations in the 3' noncoding region that result in poor replication in protoplasts and no systemic symptoms in planta were inoculated onto Chinese cabbage plants in an effort to obtain second-site suppressor mutations. Putative second-site suppressor mutations were identified by RNase protection and sequencing and were then introduced into genomic cDNA clones to permit their characterization. A C-57----U mutation in the tRNA-like structure was a strong suppressor of the C-55----A mutation which prevented both systemic infection and in vitro valylation of the viral RNA. Both of these phenotypes were rescued in the double mutant. An A-107----C mutation was a strong second-site suppressor of the U-96----G mutation, permitting the double mutant to establish systemic infection. The C-107 and G-96 mutations are located on opposite strands of one helix of a potential pseudoknot, and the results support a functional role for the pseudoknot structure. A mutation near the 5' end of the genome (G + 92----A), at position -3 relative to the initiation codon of the essential open reading frame 206, was found to be a general potentiator of viral replication, probably as a result of enhanced expression of open reading frame 206. The A + 92 mutation enhanced the replication of mutant TYMC-G96 in protoplasts but was not a sufficiently potent suppressor to permit systemic spread of the A + 92/G-96 double mutant in plants.

Anticodon↗
PubMed1995

Interactions between the IBV nucleocapsid protein and RNA sequences specific for the 3' end of the genome.

The infectious bronchitis virus (IBV) nucleocapsid protein was expressed as a fusion protein in bacteria. The coding sequence differed from the native protein only in the addition of six histidine residues at the amino terminus which were used for enrichment with a nickel affinity column. In gel shift assays, the mobility of labelled G RNA was decreased with increasing concentrations of the fusion protein. Competitive gel shift assays with labelled G RNA indicated that the protein interacted with relatively high avidities to several unlabelled RNAs representing sequences at the 3' noncoding end of the IBV genome. Cache Valley virus (a bunyavirus) mRNA transcribed from the smaller segment cDNA also inhibited the interaction with IBV G RNA to the same extent as homologous unlabelled G RNA. In contrast, interactions of the fusion proteins with a region from 99 to 249 bases from the 3' terminus of the IBV genome and bovine liver RNA were relatively weak. The binding of IBV nucleocapsid protein with RNA probably requires specific sequences and/or structures that are present at a number of sites on the genome, and may represent a common mechanism used by similar viral proteins whose functions depend on binding to RNA.

Animals↗
PubMed2003

The redusome hypothesis of aging and the control of biological time during individual development.

The redusome hypothesis of aging and the control of biological time in individual development is proposed. Redusomes are hypothetical perichromosomal particles arising in differentiation events during morphogenesis of an organism. The linear molecule of DNA covered with proteins in the redusome is assumed to be a copy of a segment of chromosomal DNA. Redusomes are located mainly in subtelomeric regions of chromosomes. The redusome does not leave the body of a chromosome even in the course of cellular divisions, being kept in its chromosomal nest. Like telomeric DNA, redusome linear DNA is shortened step by step. Thus, tiny redusomes progressively decrease in size; it is from here their name originates. Together with loss of the length of DNA in a redusome, the number of different genes contained in it also decreases. Shortening of the redusomal DNA molecules (and, coupled to it, changes of the sets of genes in redusomes) is responsible for age-dependent shifts in the level of expression of different chromosomal genes. Owing to this, redusome DNA shortening serves as a key means of measuring biological time in individual development. The main part of DNA of most redusomes is postulated to be occupied by noncoding genes. Low-molecular-weight RNAs (micro RNAs and fountain RNAs, or fRNAs) are assumed to be transcribed from them. These RNAs are involved in regulation of various chromatin repackings that are specific to certain differentiations, while others modulate the levels of expression of chromosomal genes. Hypothetical fountain RNAs can quantitatively regulate the expression levels of chromosomal genes, forming specific complexes with fions. Fions are suggested to be specific sites of a chromosomal DNA which are complementary to different fRNAs. Fions reside in the vicinity of usual chromosomal genes. A complex of the fRNA-fion, specifically interacting with a closed gate of the corresponding ion channel of the internal nuclear membrane, initiates the opening of the gate for a very short time, thus organizing activity of an ion fountain which appears to be automatically aimed at the chromosomal gene nearest to the fion involved. The ion fountain creates, depending on specificity of matching fRNA, fion, and ion channel, a distinctive ionic environment near certain structural genes. Ion fountains exert their action on the configuration of corresponding segments of chromatin and on the transcriptional efficiency of chromosomal genes in a topographically specific manner. Hence, the fountain system of the nucleus is able to regulate the quantitative traits both of cells and organism; it can control dominance of alleles and plays a role in individual development. Significant and escalating truncation of the redusome DNA causes cell aging due to an arising and increasing deficit of fRNAs and, for this reason, the lack of required ions near certain structural genes. Progressive shortening of DNA of redusomes is proposed to result in cellular aging because of a constantly growing shortage of low-molecular-weight RNAs transcribed from redusomal genes. Two types of redusomes are postulated: chronosomes and printosomes. Linear molecules of DNA in these two types of redusomes are called chronomeres and printomeres, respectively. Chronosomes are responsible for measurement of biological time in nondividing cells of the CNS. Printosomes remember positions of cells in the course of interpretation of the positional information in morphogenesis. In accordance with the position of a cell in a morphogenetic field, printomeres do change cellular properties and remember the change made (this is a so-called printomere mechanism of interpretation of positional information). Besides, printomeres participate in maintaining the achieved state of cellular differentiation. Normally, the chronomere is shortened only on the maximum of infradian hormonal rhythm (T-rhythm) which initiates the act of a superhigh velocity of its transcription that is finished with truncation of the end of a chronomere (an effect called scrupting). Theprintomere can be shortened due to the effect of DNA end underreplication and owing to scrupting. The effect of the end underreplication of DNA in doubling cells occurs simultaneously both in printomeres and telomeres. Shortening of telomeres is just a bystander process of aging of cells, whereas the true cause of biological aging is only the shortening of redusome DNA. Processing of certain redusomes in terminally differentiating cells is a cause of a proliferation arrest. Linkage of genes in a eukaryotic chromosome is determined by the distances between genes and redusomes.

Aging↗
PubMed1998

Interaction of poly(rC) binding protein 2 with the 5' noncoding region of hepatitis A virus RNA and its effects on translation.

Utilization of internal ribosome entry segment (IRES) structures in the 5' noncoding region (5'NCR) of picornavirus RNAs for initiation of translation requires a number of host cell factors whose distribution may vary in different cells and whose requirement may vary for different picornaviruses. We have examined the requirement of the cellular protein poly(rC) binding protein 2 (PCBP2) for hepatitis A virus (HAV) RNA translation. PCBP2 has recently been identified as a factor required for translation and replication of poliovirus (PV) RNA. PCBP2 was shown to be present in FRhK-4 cells, which are permissive for growth of HAV, as it is in HeLa cells, which support translation of HAV RNA but which have not been reported to host replication of the virus. Competition RNA mobility shift assays showed that the 5'NCR of HAV RNA competed for binding of PCBP2 with a probe representing stem-loop IV of the PV 5'NCR. The binding site on HAV RNA was mapped to nucleotides 1 to 157, which includes a pyrimidine-rich sequence. HeLa cell extracts that had been depleted of PCBP2 by passage over a PV stem-loop IV RNA affinity column supported only low levels of HAV RNA translation. Translation activity was restored upon addition of recombinant PCBP2 to the depleted extract. Removal of the 5'-terminal 138 nucleotides of the HAV RNA, or removal of the entire IRES, eliminated the dependence of HAV RNA translation on PCBP2.

Binding Sites↗
PubMed2006

Host species-dependent population structure of a pollen-borne plant virus, Cherry leaf roll virus.

Cherry leaf roll virus (CLRV) belongs to the Nepovirus genus within the family Comoviridae. It has a host range which includes a number of wild tree and shrub species. The serological and molecular diversity of CLRV was assessed using a collection of isolates and samples recovered from woody and herbaceous host plants from different geographical origins. Molecular diversity was assessed by sequencing a short (375-bp) region of the 3' noncoding region (NCR) of the genomic RNAs while serological diversity was assessed using a panel of seven monoclonal antibodies raised initially against a walnut isolate of CLRV. The genomic region analyzed was shown to exhibit a significant degree of molecular variability with an average pairwise divergence of 8.5% (nucleotide identity). Similarly, serological variability proved to be high, with no single monoclonal antibody being able to recognize all isolates analyzed. Serological and molecular phylogenetic reconstructions showed a strong correlation. Remarkably, the diversity of CLRV populations is to a large extent defined by the host plant from which the viral samples are originally obtained. There are relatively few reports of plant viruses for which the genetic diversity is structured by the host plant. In the case of CLRV, we hypothesize that this situation may reflect the exclusive mode of transmission in natural plant populations by pollen and by seeds. These modes of transmission are likely to impose barriers to host change by the virus, leading to rapid biological and genetic separation of CLRV variants coevolving with different plant host species.

3' Untranslated Regions↗
PubMed1996

Nonconserved nucleotides at the 3' and 5' ends of an influenza A virus RNA play an important role in viral RNA replication.

The genome of influenza A viruses is composed of eight negative-strand RNA segments which contain short noncoding regions at their 3' and 5' ends. The signals required for replication, transcription, and packaging of the viral RNAs are thought to be located in these regions. The highly conserved noncoding nucleotides, which form "panhandle" or "fork" structures by partial complementarity, are important for the transcriptional activity of the viral RNA polymerase. In contrast, the nonconserved noncoding nucleotides located close to the open reading frame of the viral RNAs had not been implicated in RNA transcription. Using a reverse-genetics system, we have now rescued influenza A/WSN/33 viruses whose NA-specific RNA segments have deletions in these nonconserved noncoding regions. Deletion either of the nucleotide residues between the poly(U) stretch and the stop codon at the 5' end or of the nucleotides between position 15 and the start codon at the 3' end did not affect the amount of NA-RNA species found in virions or infected cells. However, a combination of deletions at both the 3' and the 5' ends decreased by 60 times the levels of NA-specific viral RNA found in infected cells at late periods of infection and in virions. This double deletion was also responsible for a fourfold reduction of the steady-state levels of the NA-specific mRNA in infected cells. Viruses whose NA-specific open reading frames were flanked by the noncoding regions of the PB1- or the NS-RNA segments of infuenza A/WSN/33 virus also showed a reduction in the NA-specific viral RNA in virions and in infected cells. The present results demonstrate that the nonconserved nucleotides at the 3' and 5' ends of the NA-RNA segment of influenza A virus play an important role in the replication of this segment.

Animals↗
PubMed1995

Long, nearly identical untranslated sequences at the 3' terminal regions of the genomic RNAs of cherry leafroll virus (walnut strain).

Hybridization analyses of cDNA clones derived from the two genomic RNAs, RNA1 and RNA2, of the walnut strain of the nepovirus cherry leafroll nepovirus (wCLRV) demonstrated a long region of high homology between the two viral RNAs. Subsequent mapping and nucleotide sequencing revealed a long, noncoding, presumably untranslated, region (3' UTR) immediately 5' of the terminal polyadenylate, a region that is almost identical in the two RNAs. This 3' UTR is 1567 nucleotide residues long in RNA1. Homologies of about 80% were found with corresponding regions of genomic RNAs from other strains of CLRV, but not with the corresponding regions of other nepovirus genomic RNAs.

Base Sequence↗
PubMed2002

Noncoding RNA genes identified in AT-rich hyperthermophiles.

Noncoding RNA (ncRNA) genes that produce functional RNAs instead of encoding proteins seem to be somewhat more prevalent than previously thought. However, estimating their number and importance is difficult because systematic identification of ncRNA genes remains challenging. Here, we exploit a strong, surprising DNA composition bias in genomes of some hyperthermophilic organisms: simply screening for GC-rich regions in the AT-rich Methanococcus jannaschii and Pyrococcus furiosus genomes efficiently detects both known and new RNA genes with a high degree of secondary structure. A separate screen based on comparative analysis also successfully identifies noncoding RNA genes in P. furiosus. Nine of the 30 new candidate genes predicted by these screens have been verified to produce discrete, apparently noncoding transcripts with sizes ranging from 97 to 277 nucleotides.

Adenine↗
PubMed2001

A novel brain-specific box C/D small nucleolar RNA processed from tandemly repeated introns of a noncoding RNA gene in rats.

Antisense box C/D small nucleolar RNAs (snoRNAs) guide the 2'-O-ribose methylations of eukaryotic rRNAs and small nuclear RNAs (snRNAs) through formation of a specific base pairing at each RNA methylation site. By analysis of a box C/D snoRNA cDNA library constructed from rat brain RNAs, we have identified a novel box C/D snoRNA, RBII-36, which is devoid of complementarity to rRNA or an snRNA and exhibits a brain-specific expression pattern. It is uniformly expressed in all major areas of adult rat brain (except for choroid plexus) and throughout rat brain ontogeny but exclusively detected in neurons in which it exhibits a nucleolar localization. In vertebrates, known methylation guide snoRNAs are intron-encoded and processed from transcripts of housekeeping genes. In contrast, RBII-36 snoRNA is intron-encoded in a gene preferentially expressed in the rat central nervous system and not in proliferating cells. Remarkably, this host gene, which encodes a previously reported noncoding RNA, Bsr, spans tandemly repeated 0.9-kilobase units including the snoRNA-containing intron. The novel brain-specific snoRNA appears to result not only from processing of the debranched lariat but also from endonucleolytic cleavages of unspliced Bsr RNA (i.e. an alternative splicing-independent pathway unreported so far for mammalian intronic snoRNAs). Sequences homologous to RBII-36 snoRNA were exclusively detected in the Rattus genus of rodents, suggesting a very recent origin of this brain-specific snoRNA.

Animals↗
PubMed1997

Mutational analysis of the 5' noncoding region of human immunodeficiency virus type 1 genome.

Retrovirus particles are released by budding from the membranes of infected cells. In the course of virus production, particularly during the late stage, viral genomic RNA is incorporated specifically into virion particles. This specific incorporation of the genomic RNA requires a packaging signal sequence. A region that functions as the packaging signal was mapped to a location upstream of the gag open reading frame on the HIV-1 viral genome. In addition of this packaging signal, other cis-acting elements that are scattered throughout the genome are also required for efficient packaging. The region upstream of the splice donor site is probably important for dimer formation. Therefore, we focused on one region located between the 3' end of the primer binding site and the 5' splice donor site of HIV-1. Experiments were conducted to investigate how deletions or point mutations in this region affect both dimerization in vitro and the production of infectious virus particles. A series of RNAs of varying lengths containing the 5' noncoding region were generated, and genomic dimerization of the altered viral RNA was analyzed in vitro. One RNA construct which consisted of 112 nucleotides (nt) from nt 639 to nt 750 formed a heterodimeric complex with the RNA which consisted of 200 nucleotides from nt 551 to nt 750. We then constructed proviruses with mutations in the 639 to 750 nt region and assayed for virus production. Several mutants that lacked the complementarity necessary to form a possible stem-loop structure in this region showed decreased production of infectious virus particles. Moreover, both deletion of this region and randomization of its nucleotide sequence completely impaired infectious virus production. Thus, the way that this region affects infectious virus production may be through its RNA secondary structure.

Animals↗
PubMed1997

Replication-competent picornaviruses with complete genomic RNA 3' noncoding region deletions.

The genomic RNA 3' noncoding region is believed to be a major cis-acting molecular genetic determinant for regulating picornavirus negative-strand RNA synthesis by promoting replication complex recognition. We report the replication of two picornavirus RNAs harboring complete deletions of the genomic RNA 3' noncoding regions. Our results suggest that while specific 3'-terminal RNA sequences and/or secondary structures may have evolved to promote or regulate negative-strand RNA synthesis, the basic mechanism of replication initiation is not strictly template specific and may rely primarily upon the proximity of newly translated viral replication proteins to the 3' terminus of template RNAs within tight membranous replication complexes.

Gene Expression Regulation, Viral↗
PubMed1995

Foreign complementary sequences facilitate genetic RNA recombination in brome mosaic virus.

We have demonstrated that local antisense sequences can mediate genetic recombination within the 3' noncoding region among brome mosaic virus (BMV) RNAs (P. Nagy and J. J. Bujarski, 1993, Proc. Natl. Acad. Sci. USA 90, 6390-6394). Here we show that foreign complementary inserts can direct crossovers between BMV RNA3 components within an internal region. A 170-nt polynucleotide derived from the cowpea chlorotic mottle virus (CCMV) RNA3 was inserted just upstream of the initiation codon of the BMV coat protein open reading frame in either sense or antisense orientations. The resulting respective mutants, BCC+ and BCC-, maintained unchanged CCMV inserts when inoculated separately on leaves of a local lesion host for BMV. In contrast, when a mixture containing both mutated RNAs3 was inoculated, a significant fraction of lesions accumulated the BMV RNA3 lacking the CCMV insert. The presence of a 3' marker mutation confirmed that the BMV RNA3 progeny arose due to crossovers between BCC+ and BCC- within the complementary sequences. The highest frequency of recombinant appearance was observed when the RNA mixtures were annealed prior to inoculation on the host plants. Our results confirm a concept predicting the general nature of the heteroduplex-mediated recombination functioning in RNA viruses. Examples of possible applications of this approach in recombinant RNA technology are discussed.

Antisense Elements (Genetics)↗
PubMed2003

Posttranscriptional gene silencing is not compromised in the Arabidopsis CARPEL FACTORY (DICER-LIKE1) mutant, a homolog of Dicer-1 from Drosophila.

Posttranscriptional silencing (PTGS) in plants, nematodes, Drosophila, and perhaps all eukaryotes operates by sequence-specific degradation or translational inhibition of the target mRNA. These processes are mediated by duplexed RNA. In Drosophila and nematodes, double-stranded (ds)RNA or self-complementary RNA is processed into fragments of approximately 21 nt by Dicer-1. These small interfering RNAs (siRNAs) serve as guides to target degradation of homologous single-stranded (ss)RNA. In some cases, the approximately 21 nt guide fragments derived from endogenous, imperfectly self-complementary RNAs cause translational inhibition of their target mRNAs, with which they have substantial, but not perfect sequence complementarity. These small temporal RNAs (stRNAs) belong to a class of noncoding microRNAs (miRNAs), 20-24 nt in length, that are found in flies, plants, nematodes, and mammals. In nematodes, the Dicer-1 enzyme catalyzes the production of both siRNA and stRNA. Mutation of the Arabidopsis Dicer-1 homolog, CARPEL FACTORY (CAF), blocks miRNA production. Here, we report that the same caf mutant does not block either PTGS or siRNA production induced by self-complementary hairpin RNA. This suggests either that this mutation only impairs miRNA formation or, more interestingly, that plants have two distinct dicer-like enzymes, one for miRNA and another for siRNAi production.

Animals↗
PubMed2004

Primary acute lymphoblastic leukemia cells use a novel promoter and 5'noncoding exon for the human reduced folate carrier that encodes a modified carrier translated from an upstream translational start.

The human reduced folate carrier (hRFC) is reported to be regulated by up to seven alternatively spliced noncoding exons (A1, A2, A, B, C, D, and E). Noncoding exon and promoter usage was analyzed in RNAs from 27 childhood acute lymphoblastic leukemia (ALL) specimens by real-time PCR and/or 5' rapid amplification of cDNA ends (5' RACE) assay. By real-time PCR, total hRFC transcripts in ALL spanned a 289-fold range. Over 90% of hRFC transcripts were transcribed with A1, A2, and B 5' untranslated regions (UTRs). Analysis of 5' RACE clones showed that the A1 + A2 5'UTRs contained A1 sequence alone or a fusion of A1 and A2, implying the existence of a single, alternatively spliced 1021-bp A1/A2 noncoding region. High frequency sequence polymorphisms (AGG deletion, C/T transition) identified in the A1/A2 region by 5'RACE were confirmed in normal DNAs. By reporter assays in HepG2 hepatoma and Jurkat leukemia cells, A1/A2 promoter activity was localized to a 134-bp minimal region. Translation from an upstream AUG in the A1/A2 noncoding region in-frame with the normal translation start resulted in synthesis of a larger ( approximately 7 kDa) hRFC protein with transport properties altered from those for wild-type hRFC. Although there was no effect on transcript or protein stabilities, in vitro translation from A1/A2 transcripts was decreased compared with those with the B 5'UTR. Our results document the importance of the hRFC A1/A2 upstream region in childhood ALL and an intricate transcriptional and posttranscriptional regulation of hRFC-A1/A2 mRNAs. Furthermore, they suggest that use of the A1/A2 5'UTR may confer a transport phenotype distinct from the other 5'UTRs due to altered translation efficiency and transport properties.

5' Untranslated Regions↗
PubMed1989

Cloning and synthesis of infectious cardiovirus RNAs containing short, discrete poly(C) tracts.

Mengovirus RNA transcripts with 5' noncoding poly(C) tracts of C8, C12, and C13UC10 have been synthesized in vitro from cDNA clones and shown to be infectious to HeLa cells. A chimeric clone has also been constructed which links the 5' end from one mengovirus clone (299 nucleotides, containing C13UC10) to a 7,424-base fragment derived from the 3' end of encephalomyocarditis (EMC) virus. Progeny virus isolated after transfection with the clone-derived RNAs had the same poly(C) tracts, mengovirus-specific sequences, or EMC virus-specific sequences as the transcript from which it was derived. Although the cloned poly(C) tracts were considerably shorter than those found in viral RNA from mengovirus (C50UC10) or EMC virus (C115UCUC3UC10), the growth characteristics of the progeny viruses in HeLa cells were indistinguishable from those of the parental viruses, indicating the length of this tract does not play a significant restrictive role for cardiovirus infectivity in tissue culture.

Cloning, Molecular↗