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PubMed2005

Residues in the conserved His domain of fruit fly tRNase Z that function in catalysis are not involved in substrate recognition or binding.

Transfer RNAs are transcribed as precursors with extensions at both the 5' and 3' ends. RNase P removes endonucleolytically the 5' end leader. tRNase Z can remove endonucleolytically the 3' end trailer as a necessary step in tRNA maturation. CCA is not transcriptionally encoded in the tRNAs of eukaryotes, archaebacteria and some bacteria and must be added by a CCA-adding enzyme after removal of the 3' end trailer. tRNase Z is a member of the beta-lactamase family of metal-dependent hydrolases, the signature sequence of which, the conserved histidine cluster (HxHxDH), is essential for activity. Starting with baculovirus-expressed fruit fly tRNase Z, we completed an 18 residue Ala scan of the His cluster to analyze the functional landscape of this critical region. Residues in and around the His cluster fall into three categories based on effects of the substitutions on processing efficiency: substitutions in eight residues have little effect, five substitutions reduce efficiency moderately (approximately 5-50-fold), while substitutions in five conserved residues, one serine, three histidine and one aspartate, severely reduce efficiency (approximately 500-5000-fold). Wild-type and mutant dissociation constants (Kd values), determined using gel shifts, displayed no substantial differences, and were of the same order as kM (2-20 nM). Lower processing efficiencies arising from substitutions in the His domain are almost entirely due to reduced kcat values; conserved, functionally important residues within the His cluster of tRNase Z are thus involved in catalysis, and substrate recognition and binding functions must reside elsewhere in the protein.

Amino Acid Sequence↗

PubMed1995

Conversion of mammalian tRNA 3' processing endoribonuclease to four-base-recognizing RNA cutters.

The spermidine-dependent, sequence-specific endoribonuclease (RNase 65) activities in mammalian cell extracts require both protein and 3' truncated tRNA, species of which direct their substrate sequence specificity. Computer analysis for searching possible base pairing between substrate RNAs and their corresponding 3' truncated tRNA, suggested a unified model for substrate recognition mechanism, in which a four-nucleotide (nt) sequence in the target tRNAs 1 nt upstream of their cleavage site, base pairs with the 5' terminal 4 nt sequence of their corresponding 3' truncated tRNA. This model was supported by experiments with several RNA substrates containing a substituted nucleotide in the target 4 nt sequence. In this model, the tRNA substrates and their corresponding 3' truncated tRNA form a complex resembling a 5' processed tRNA precursor containing a 3' trailer, suggesting that the protein component of RNase 65 is identical to tRNA 3' processing endoribonuclease (3' tRNase). Actually, 3' tRNase purified from pig liver cleaved the target RNAs at the expected sites only in the presence of their corresponding 3' truncated tRNA. These results show that the 3' tRNase can be converted to 4 nt specific RNA cutters using the 3' truncated tRNAs.

Animals↗

PubMed1996

The genome sequence of the virulent Kabete 'O' strain of rinderpest virus: comparison with the derived vaccine.

We have compared the complete genome sequences of the vaccine strain of rinderpest virus and the virulent strain from which it was derived. Only 87 bases differed between the two genomes (0.55%). Possibly significant differences in amino acid sequence were found in the N, P, F, H and L proteins. A number of differences were also found in the leader region (3' end of the genome), whilst the trailer region appears to be more conserved. In addition, the length of the genome was found in both cases to be 15882, an exact multiple of six, fulfilling predictions made earlier based on work with Sendai and measles viruses.

Base Sequence↗

PubMed1982

Ribonuclease E is involved in the processing of 5-S rRNA from a number of rRNA transcription units.

Strains of Escherichia coli having a thermosensitive RNase E produce a number of 5-S ribosomal RNA precursors at a non-permissive temperature. One of these precursors, 9-S RNA, was reported earlier [Ghora, B.K. and Apirion, D. (1978) Cell, 15, 1055-1066]. Here we show the existence of additional precursors to 5-S rRNA, originating from a number of rRNA genes. All the precursors are very similar in the first 200 nucleotides and the last part of this sequence contains the mature 5-S rRNA. Precursors that contain only these nucleotides (8-S RNA) accumulate. They probably originate from the rrn genes C, D and F which contain trailer tRNAs. The 9-S RNA precursors contain in addition a termination stem and loop structure and are derived from genes which do not contain trailer tRNA (Singh and Apirion, unpublished results). In addition, a 10-S precursor was identified. It contains distal to the 5-S rRNA a trailer tRNA, tRNAAsp, and a transcription termination signal. It is derived from the rrnF gene. The accumulation of an RNA precursor containing an RNase P site in an rne mutant suggests that the efficiency of one RNA processing enzyme depends on the activity of other RNA processing enzymes.

Base Composition↗

PubMed1996

Effects of mutations in the gene-start and gene-end sequence motifs on transcription of monocistronic and dicistronic minigenomes of respiratory syncytial virus.

Preceding and following each gene of respiratory syncytial virus (RSV) are two conserved sequences, the gene-start (GS) and gene-end (GE) motifs, respectively, which are thought to be transcription signals. The functions and boundaries of these signals and the process of sequential transcription were analyzed with cDNA-encoded RNA analogs (minigenomes) of nonsegmented negative-sense RSV genomic RNA. Two minigenomes were used. The monocistronic RSV-CAT minigenome consists of the chloramphenicol acetyltransferase (CAT) translational open reading frame (ORF) bordered by the GS and GE motifs and flanked by the 3' leader and 5' trailer extragenic regions of genomic RNA. The dicistronic RSV-CAT-LUC minigenome is a derivative of RSV-CAT into which the ORF for luciferase (LUC), bordered by GS and GE motifs, was inserted downstream of the CAT gene with an intergenic region positioned between the two genes. Each minigenome was synthesized in vitro and transfected into RSV-infected cells, where it was replicated and transcribed to yield the predicted polyadenylated subgenomic mRNA(s). The only RSV sequences required for efficient transcription and RNA replication were the 44-nucleotide 3' leader region, the last 40 nucleotides of the 5' trailer region, and the 9- to 10-nucleotide GS and 12- to 13-nucleotide GE motifs. The GS and GE motifs functioned as self-contained, transportable transcription signals which could be attached to foreign sequences to direct their transcription into subgenomic mRNAs. Removal of the GS motif greatly reduced transcription of its gene, and the requirement for this element was particularly strict for the gene in the downstream position. Ablation of the promoter-proximal GS signal was not associated with increased antigenome synthesis. Consistent with its proposed role in termination and polyadenylation, removal of the CAT GE signal in RSV-CAT resulted in the synthesis of a nonpolyadenylated CAT mRNA, and in RSV-CAT-LUC the same mutation resulted in readthrough transcription to yield a dicistronic CAT-LUC mRNA. The latter result showed that a downstream GS signal is not recognized for reinitiation by the polymerase if it is already engaged in mRNA synthesis; instead, it is recognized only if the polymerase first terminates transcription at an upstream termination signal. This result also showed that ongoing transcription did not open the downstream LUC gene for internal polymerase entry. Removal of both the GS and GE signals of the upstream CAT gene in RSV-CAT-LUC silenced expression of both genes, confirming that independent polymerase entry at an internal gene is insignificant. Remarkably, whereas both genes were silent when the CAT GS and GE signals were both absent, restoration of the CAT GE signal alone restored a significant level (approximately 10 to 12% of the wild-type level) of synthesis of both subgenomic mRNAs. This analysis identified a component of sequential transcription that was independent of the promoter-proximal GS signal and appeared to involve readthrough from the leader region.

Animals↗

PubMed2004

Substrate tRNA recognition mechanism of tRNA (m7G46) methyltransferase from Aquifex aeolicus.

Transfer RNA (m7G46) methyltransferase catalyzes the methyl transfer from S-adenosylmethionine to N7 atom of the guanine 46 residue in tRNA. Analysis of the Aquifex aeolicus genome revealed one candidate open reading frame, aq065, encoding this gene. The aq065 protein was expressed in Escherichia coli and purified to homogeneity on 15% SDS-polyacrylamide gel electrophoresis. Although the overall amino acid sequence of the aq065 protein differs considerably from that of E. coli YggH, the purified aq065 protein possessed a tRNA (m7G46) methyltransferase activity. The modified nucleoside and its location were determined by liquid chromatography-mass spectroscopy. To clarify the RNA recognition mechanism of the enzyme, we investigated the methyl transfer activity to 28 variants of yeast tRNAPhe and E. coli tRNAThr. It was confirmed that 5'-leader and 3'-trailer RNAs of tRNA precursor are not required for the methyl transfer. We found that the enzyme specificity was critically dependent on the size of the variable loop. Experiments using truncated variants showed that the variable loop sequence inserted between two stems is recognized as a substrate, and the most important recognition site is contained within the T stem. These results indicate that the L-shaped tRNA structure is not required for methyl acceptance activity. It was also found that nucleotide substitutions around G46 in three-dimensional core decrease the activity.

Amino Acid Sequence↗

PubMed2005

Plant-derived transfer DNAs.

The transfer of DNA from Agrobacterium to plant cell nuclei is initiated by a cleavage reaction within the 25-bp right border of Ti plasmids. In an effort to develop all-native DNA transformation vectors, 50 putative right border alternatives were identified in both plant expressed sequence tags and genomic DNA. Efficacy tests in a tobacco (Nicotiana tabacum) model system demonstrated that 14 of these elements displayed at least 50% of the activity of conventional Agrobacterium transfer DNA borders. Four of the most effective plant-derived right border alternatives were found to be associated with intron-exon junctions. Additional elements were embedded within introns, exons, untranslated trailers, and intergenic DNA. Based on the identification of a single right border alternative in Arabidopsis and three in rice (Oryza sativa), the occurrence of this motif was estimated at a frequency of at least 0.8x10(-8). Modification of plasmid DNA sequences flanking the alternative borders demonstrated that both upstream and downstream sequences play an important role in initiating DNA transfer. Optimal DNA transfer required the elements to be preceded by pyrimidine residues interspaced by AC-rich trinucleotides. Alteration of this organization lowered transformation frequencies by 46% to 93%. Despite their weaker resemblance with left borders, right border alternatives also functioned effectively in terminating DNA transfer, if both associated with an upstream A[C/T]T[C/G]A[A/T]T[G/T][C/T][G/T][C/G]A[C/T][C/T][A/T] domain and tightly linked cytosine clusters at their junctions with downstream DNA. New insights in border region requirements were used to construct an all-native alfalfa (Medicago sativa) transfer DNA vector that can be used for the production of intragenic plants.

Amino Acid Sequence↗

PubMed1999

The 5' terminal trailer region of vesicular stomatitis virus contains a position-dependent cis-acting signal for assembly of RNA into infectious particles.

The cis-acting genomic RNA requirements for the assembly of vesicular stomatitis virus (VSV) ribonucleocapsids into infectious particles were investigated. Using a biological assay based on particle infectivity, we demonstrated that subgenomic replicons that contained all four possible combinations of the natural genomic termini, the 3' leader (Le) and 5' trailer (Tr) regions, were replication competent; however, a 3' copyback replicon (3'CB), containing the natural 3' terminus but having the 5' Tr replaced by a sequence complementary to the 3' Le for 46 nucleotides, was unable to assemble infectious particles, despite efficient replication. When a copy of Tr was inserted 51 nucleotides from the 5' end of 3'CB, infectious particles were produced. However, analysis of the replication products of these particles showed that the 51 nucleotides which corresponded to the Le complement sequences at the 5' terminus were removed during RNA replication, thus restoring the wild-type 5' Tr to the exact 5' terminus. These data showed that a cis-acting signal was necessary for assembly of VSV RNAs into infectious particles and that this signal was supplied by Tr when located at the 5' end. The regions within Tr required for assembly were analyzed by a series of deletions and exchanges for Le complement sequences, which demonstrated that the 5' terminal 29 nucleotides of Tr allowed assembly of infectious particles but that the 5' terminal 22 nucleotides functioned poorly. Deletions in Tr also altered the balance between negative- and positive-strand genomic RNA and affected levels of replication. RNAs that retained fewer than 45 but at least 22 nucleotides of the 5' terminus could replicate but were impaired in RNA replication, and RNAs that retained only 14 nucleotides of the 5' terminus were severely reduced in ability to replicate. These data define the VSV Tr as a position-dependent, cis-acting element for the assembly of RNAs into infectious particles, and they delineate RNA sequences that are essential for negative-strand RNA synthesis. These observations are consistent with, and offer an explanation for, the absence of 3' copyback defective interfering particles in nature.

5' Untranslated Regions↗

PubMed2005

Optimized SYBR green real-time PCR assay to quantify the absolute copy number of measles virus RNAs using gene specific primers.

A sensitive and specific RT-QPCR based on real-time analysis of PCR products stained with SYBR green, was designed and carefully optimised to quantify individual measles virus RNA species. Pairs of specific primers were designed to detect N, P, M, F, H, or L sequences. To detect the genome and/or antigenome, two primers were chosen so as to amplify a 221 nt fragment (L-Tr) encompassing L gene end and trailer. Every gene-specific PCR assay was able to detect = 10 copies/sample, with a dynamic range of 4-5 log10 copies. No significant fluorescent signal was detected from non-infected cell cDNA template. When measles virus microccocal nuclease resistant genomic RNA was reverse transcribed, a 1:1 ratio was observed between single gene amplicons except for L-Tr which displayed a 2.6-fold excess over the other genes. This likely reflects the presence of some shorter abortive genome since the use of a plasmid encoding the entire virus genome resulted in 1:1 ratio for L-Tr segment when compared to others amplicons. Thus, this RT-QPCR assay appears suitable for follow-up studies of viral RNA populations during infection and may also be useful for reliable detection of measles virus in clinical samples.

Animals↗

PubMed1987

Cloning and nucleotide sequence of rat ornithine decarboxylase cDNA.

The enzyme ornithine decarboxylase (ODC; EC 4.1.1.17) catalyses the first and rate-limiting step in polyamine biosynthesis. Its activity is markedly increased in rapidly growing or regenerating tissue and is subject to regulation by a variety of trophic and mitogenic stimuli. ODC is therefore believed to play an essential role in the onset of cellular proliferation. In a molecular-biological approach to investigate ODC regulation upon induction by tumor promoters in rat liver we isolated an almost full-length rat ODC cDNA clone of 2.4 kb (designated pODC.E10) from a cDNA library of testosterone-induced rat kidney poly(A)+ RNA. Characterization by restriction-endonuclease mapping and sequence analysis showed strong homology to mouse ODC cDNA sequences previously published [Gupta and Coffino, J. Biol. Chem. 260 (1985) 2941-2944; Kahana and Nathans, Proc. Natl. Acad. Sci. USA 82 (1985) 1673-1677; Hickok et al., Proc. Natl. Acad. Sci. USA 83 (1986) 594-598]. This homology is most pronounced in the 461-aa-spanning coding region, amounting to 94% and 97% at the DNA and protein levels, respectively. In the 423-nt 5' leader the rat-mouse homology (approx. 75%) is most pronounced in a region of about 175 nt directly upstream from the translational start site. The leader sequence also contains a perfect inverted repeat of 54 nt and ten additional upstream ATG triplets, which are all followed by nonsense codons before the initiating ATG. In the 633-nt 3' trailer region of pODC.E10 an additional polyadenylation signal is observed more than 300 nt upstream from the 3' end. Rat-mouse homology is about 80% up to this first polyadenylation signal and is considerably less thereafter. The presence of two alternate polyadenylation sites most likely accounts for the 3' size heterogeneity observed in the two ODC mRNAs of 2.1 and 2.6 kb, respectively. In rat liver both mRNAs are coordinately induced by different tumor promoters. Finally, Southern blot analysis of normal rat liver and rat hepatoma DNA revealed that rat ODC, as in other rodents, belongs to a multigene family.

Amino Acid Sequence↗

PubMed1997

Rescue of synthetic minireplicons establishes the absence of the NS1 and NS2 genes from avian pneumovirus.

We have determined the nucleotide sequences of the regions 3' and 5' proximal to the avian pneumovirus (APV) N and L genes, respectively. These sequences were used in the construction of a synthetic minireplicon construct in which the chloramphenicol acetyltransferase (CAT) reporter gene was flanked at its 3' end with the APV leader together with the APV N gene start signal and at its 5' end with the APV L gene end signal and the genome trailer region. The ability of T7 RNA polymerase runoff transcripts to direct the replication and expression of the CAT reporter gene in APV-infected cells demonstrated the ability of the putative leader and trailer regions to direct genome replication and gene expression. Furthermore, this confirms the absence of the NS1 and NS2 gene analogs within the APV genome. We were able to detect the expression of CAT protein from cells that had been infected with supernatants from the initially infected and transfected cells. These results have identified the cis-acting sequences of APV responsible for viral replication, gene expression, and packaging into virus-like particles.

Animals↗

PubMed1981

Yeast tRNA3Leu gene transcribed and spliced in a HeLa cell extract.

A cloned yeast tRNA3Leu gene containing a 33-base intervening sequence (IVS) is selectively transcribed by a soluble extract from HeLa cells. The 130-nucleotide tRNA3Leu precursor RNA formed is colinear with the gene and contains approximately 4 leader nucleotides and up to 9 trailer nucleotides. The IVS is accurately and efficiently removed by an endogenous HeLa excision-ligase activity to yield the spliced tRNA, the free IVS, and the half-tRNA intermediates. The splicing reaction occurs without prior 5' and 3' maturation of the precursor but, with this exception, this pattern of synthesis and subsequent maturation of the tRNA3Leu precursor conforms to the scheme for tRNA biosynthesis deduced for the xenopus system. Indeed, the two systems utilize similar or identical tRNA3Leu precursors. Our results stress the extraordinary conservation of tRNA biosynthesis in eukaryotes and demonstrate that a HeLa extract provides a useful system for investigating this process.

Amino Acyl-tRNA Synthetases↗

PubMed2005

The Ebola virus genomic replication promoter is bipartite and follows the rule of six.

In this work we investigated the cis-acting signals involved in replication of Ebola virus (EBOV) genomic RNA. A set of mingenomes with mutant 3' ends were generated and used in a reconstituted replication and transcription system. Our results suggest that the EBOV genomic replication promoter is bipartite, consisting of a first element located within the leader region of the genome and a second, downstream element separated by a spacer region. While proper spacing of the two promoter elements is a prerequisite for replication, the nucleotide sequence of the spacer is not important. Replication activity was only observed when six or a multiple of six nucleotides were deleted or inserted, while all other changes in length abolished replication completely. These data indicate that the EBOV replication promoter obeys the rule of six, although the genome length is not divisible by six. The second promoter element is located in the 3' nontranslated region of the first gene and consists of eight UN5 hexamer repeats, where N is any nucleotide. However, three consecutive hexamers, which could be located anywhere within the promoter element, were sufficient to support replication as long as the hexameric phase was preserved. By using chemical modification assays, we could demonstrate that nucleotides 5 to 44 of the EBOV leader are involved in the formation of a stable secondary structure. Formation of the RNA stem-loop occurred independently of the presence of the trailer, indicating that a panhandle structure is not formed between the 3' and 5' ends.

Base Sequence↗

PubMed1997

Nucleotide sequence of nuclear tRNA(Gly) genes and tRNA(Gly) pseudogenes from yellow lupin (Lupinus luteus): expression of the tRNA(Gly) genes in vitro and in vivo.

A nuclear DNA fragment (7.8 kb) from yellow lupin (L. luteus) was sequenced and shown to contain tRNA(Gly) (GGC) genes and tRNAGly (GGC) pseudogenes organized in three tandemly repeated units: of 2565 bp and 2564 bp, and one, truncated from its 3' end, of 1212 bp. Each unit contains an identical pair of a tRNA(Gly) gene and a pseudogene, both having the same polarity. The nucleotide sequence of the gene appears colinear to L. luteus cytoplasmic tRNA(Gly) (GGC) primary structure. All three genes are efficiently transcribed in HeLa-cell nuclear extract giving two primary transcripts. The main, longer primary transcripts have each an extremely long 3' trailer of about 100 nucleotides, the structure of which is specific only for tRNAGly genes and pseudogenes (80% homology) of the studied tandem (but not for other tRNA(Gly) genes of the yellow lupin genome) as it has been shown by Southern hybridization. This distinctive feature allowed to isolate putative tRNAGly precursor(s) encoded by at least one of the three tRNA(Gly) (GGC) genes from L. luteus seedlings.

Base Sequence↗

PubMed1994

Synthesis of circular RNA in bacteria and yeast using RNA cyclase ribozymes derived from a group I intron of phage T4.

Studies on the function of circular RNA and RNA topology in vivo have been limited by the difficulty in expressing circular RNA of desired sequence. To overcome this, the group I intron from the phage T4 td gene was split in a peripheral loop (L6a) and rearranged so that the 3' half intron and 3' splice site are upstream and a 5' splice site and 5' half intron are downstream of a single exon. The group I splicing reactions excise the internal exon RNA as a circle (RNA cyclase ribozyme activity). We show that foreign sequences can be placed in the exon and made circular in vitro. Expression of such constructs (RNA cyclase ribozymes) in Escherichia coli and yeast results in the accumulation of circular RNA in these organisms. In yeast, RNA cyclase ribozymes can be expressed from a regulated promoter like an mRNA, containing 5' leader and 3' trailer regions, and a nuclear pre-mRNA intron. RNA cyclase ribozymes have broad application to questions of RNA structure and function including end requirements for RNA transport or function, RNA topology, efficacy of antisense or ribozyme gene control elements, and the biosynthesis of extremely long polypeptides.

Bacteriophage T4↗

PubMed1987

Unusual sequence conservation in the 5' and 3' untranslated regions of the sea urchin spec mRNAs.

The Spec1 and Spec2 mRNAs (Strongylocentrotus purpuratus ectoderm mRNAs) represent a small gene family that encodes 10-12 members of the troponin C superfamily of calcium-binding proteins. These mRNAs and proteins accumulate in the aboral (dorsal) ectoderm of sea urchin embryos and larvae. Using genomic and cDNA clones, we have compared the sequences of four Spec mRNAs: Spec1, Spec2a, Spec2c, and Spec2d. The mRNAs all have at least 120 bases of 5' untranslated leader, approximately 450 bases of open reading frame, and 900 bases (Spec1) or 1250 bases (Spec2a, 2c, 2d) of 3' untranslated trailer. Unexpectedly, when long stretches of 5' untranslated regions or 3' untranslated regions are compared to one another, they are found to be less divergent than the protein-coding regions. Comparing Spec2d, the most divergent member of the family, with the other Spec mRNAs shows that while the protein-coding regions are 60-62% matched, the untranslated regions are greater than 80% matched. Comparisons among Spec1, Spec2a, and Spec2c demonstrate similar but less dramatic conservation of untranslated regions. Our data imply that the Spec gene family has evolved differently from most gene families, with mutations accumulating most rapidly in intron regions, less rapidly in protein-coding regions, and least rapidly in 5' and 3' untranslated regions.

Animals↗

PubMed2002

Molecular characterization of an isolate from the 1989/90 epizootic of Ebola virus Reston among macaques imported into the United States.

We have determined the entire genomic sequence of the Pennsylvania strain, which was isolated along with the Virginia strain during the emergence of Ebola virus Reston in 1989/90 in the United States. Thus, either the Pennsylvania or Virginia strain, neither of which had been previously molecularly characterized, can be considered as the prototype for Ebola virus Reston. Comparative analysis showed a high degree of homology to the concomitantly analyzed and recently published Philippine strain of EBOV Reston from 1996 (Ikegami et al., Arch. Virol., 146 (2001) 2021). In comparison to EBOV Zaire, strain Mayinga, conservation could be found within the open reading frames, the 3' leader and 5' trailer region and the transcriptional signals, whereas the non-coding and intergenic regions did not show any homology. This clearly supports that EBOV Reston is a distinct species within the genus Ebola-like virus but which seems to be similar to other members with respect to transcription and replication strategies. The sequence determination provides the basis for the development of a reverse genetics system for Ebola virus Reston, which is needed to study differences in pathogenicity among filoviruses.

Amino Acid Sequence↗

PubMed1993

Rescue of synthetic analogs of genome RNA of human parainfluenza virus type 3.

A simple system that allows expression and packaging of a foreign gene by human parainfluenza virus type 3 (HPIV-3) has been described. First, a cDNA was constructed to encode an internally deleted version of HPIV-3 genome RNA. The viral genes were replaced with a negative sense copy of the bacterial chloramphenicol acetyl transferase (CAT) reporter gene. In vitro run-off transcription with T7 RNA polymerase synthesized an 870 nucleotide RNA that contained the antisense coding region of the CAT gene flanked by the transcription regulatory sequences and the 3' and 5' end extracistronic sequences of the HPIV-3 genome. When introduced into cells that are infected with HPIV-3, this RNA was amplified and the reporter gene was expressed, as measured by the CAT activity in the cell extract. Furthermore, the synthetic RNA was packaged into infectious virions. The addition of two extra nucleotides at the 5' end of the parental trailer region decreased the CAT activity by more than 90%, suggesting a requirement for the intact 5'-regulatory domain in the viral replicative cycle. Interestingly, the addition of one extra nucleotide to the 3' end totally abolished the CAT activity indicating that an exact 3' terminus is critical in this process.

Animals↗