Non-coding End Research Articles

Design of primers and optimization of PCR conditions for the detection of alternatively spliced isoforms of mouse ChAT mRNA

Acetylcholine plays an important role as a neurotransmitter in the central and peripheral nervous systems. Its biosynthesis is catalyzed by choline acetyltransferase (ChAT). In mice, the ChAT gene has three 5′-noncoding (R, N, and M) and 14 coding exons, from which seven mRNA isoforms (M, N1, N2, R1, R2, R3, and R4) are transcribed. They differ in the 5′-noncoding ends and encode the same protein (common ChAT). An additional isoform that lacks coding exons from 5 to 8 encodes a smaller protein (peripheral ChAT). This research aimed to design and check the specificity of primers targeting R3, N1, N2, M, peripheral ChAT, and common ChAT isoforms. The optimal PCR conditions and specificity of the primers were tested in a series of PCR reactions. Specially designed DNA fragments or plasmids containing isoform-specific nucleotide sequences were used as templates. The appropriate annealing temperature, which yields sufficient specificity for each tested primer pair, was as follows: R3 – 60 °C; N1 – 63 °C; N2 – 65 °C; M – 65 °C; peripheral ChAT – 65 °C and common ChAT – 63 °C.

An 82 bp tandem repeat family typical of 3' non-coding end of Gypsy/TAT LTR retrotransposons is conserved in Coffea spp. pericentromeres.

Coffea spp. chromosomes are very small and accumulate a variety of repetitive DNA families around the centromeres. However, the proximal regions of Coffea chromosomes remain poorly understood, especially regarding the nature and organisation of the sequences. Taking advantage of the genome sequences of C. arabica (2n = 44), C. canephora, and C. eugenioides (C. arabica progenitors with 2n = 22) and good coverage genome sequencing of dozens of other wild Coffea spp., repetitive DNA sequences were identified, and the genomes were compared to decipher particularities of pericentromeric structures. The searches revealed a short tandem repeat (82 bp length) typical of Gypsy/TAT LTR retrotransposons, named Coffea_sat11. This repeat organises clusters with fragments of other transposable elements, comprising regions of non-coding RNA production. Cytogenomic analyses showed that Coffea_sat11 extends from the pericentromeres towards the middle of the chromosomal arms. This arrangement was observed in the allotetraploid C. arabica chromosomes, as well as in its progenitors. This study improves our understanding of the role of the Gypsy/TAT LTR retrotransposon lineage in the organisation of Coffea pericentromeres, as well as the conservation of Coffea_sat11 within the genus. The relationships between fragments of other transposable elements and the functional aspects of these sequences on the pericentromere chromatin were also evaluated. Highlights: A scattered short tandem repeat, typical of Gypsy/TAT LTR retrotransposons, associated with several fragments of other transposable elements, accumulates in the pericentromeres of Coffea chromosomes. This arrangement is preserved in all clades of the genus and appears to have a strong regulatory role in the organisation of chromatin around centromeres.

Telomeres and Age-Related Diseases.

Telomeres are at the non-coding ends of linear chromosomes. Through a complex 3-dimensional structure, they protect the coding DNA and ensure appropriate separation of chromosomes. Aging is characterized by a progressive shortening of telomeres, which compromises their structure and function. Because of their protective function for genomic DNA, telomeres appear to play an important role in the development and progression of many age-related diseases, such as cardiovascular disease (CVD), malignancies, dementia, and osteoporosis. Despite substantial evidence that links telomere length with these conditions, the nature of these observations remains insufficiently understood. Therefore, future studies should address the question of causality. Furthermore, analytical methods should be further improved with the aim to provide informative and comparable results. This review summarize the actual knowledge of telomere biology and the possible implications of telomere dysfunction for the development and progression of age-related diseases. Furthermore, we provide an overview of analytical techniques for the measurement of telomere length and telomerase activity.

Experimental manipulation of telomere length: does it reveal a corner-stone role for telomerase in the natural variability of individual fitness?

Telomeres, the non-coding ends of linear chromosomes, are thought to be an important mechanism of individual variability in performance. Research suggests that longer telomeres are indicative of better health and increased fitness; however, many of these data are correlational and whether these effects are causal are poorly understood. Experimental tests are emerging in medical and laboratory-based studies, but these types of experiments are rare in natural populations, which precludes conclusions at an evolutionary level. At the crossroads between telomere length and fitness is telomerase, an enzyme that can lengthen telomeres. Experimental modulation of telomerase activity is a powerful tool to manipulate telomere length, and to look at the covariation of telomerase, telomeres and individual life-history traits. Here, we review studies that manipulate telomerase activity in laboratory conditions and emphasize the associated physiological and fitness consequences. We then discuss how telomerase's impact on ageing may go beyond telomere maintenance. Based on this overview, we then propose several research avenues for future studies to explore how individual variability in health, reproduction and survival may have coevolved with different patterns of telomerase activity and expression. Such knowledge is of prime importance to fully understand the role that telomere dynamics play in the evolution of animal ageing.This article is part of the theme issue 'Understanding diversity in telomere dynamics'.

Isolation and genomic characterization of Culex theileri flaviviruses in field-collected mosquitoes from Turkey

Vector surveillance for the arthropod-borne infections has resulted in the isolation of a growing number of novel viruses, including several flavivirus strains that exclusively replicate in insects. This report describes the isolation and genomic characterization of four insect-specific flaviviruses from mosquitoes, previously collected from various locations in Turkey. C6/36 Aedes albopictus and Vero cell lines were inoculated with mosquito pools. On C6/36 cells, mild cytopathic effects, characterized as rounding and detachment, were observed in four pools that comprised female Culex theileri mosquitoes. Complete (3 isolates, 10,697 nucleotides) or near-complete (1 isolate, 10,452 nucleotides) genomic characterization was performed in these culture supernatants via next generation sequencing. All strains demonstrated high genetic similarities, with over 99% identity match on nucleotide and amino acid alignments, revealing them to be different isolates of the same virus. Sequence comparisons identified the closest relative to be the Culex theileri flavivirus (CTFV) strains, originally characterized in Portugal. Phylogenetic analyses demonstrated that the isolates remained distinct as a cluster but formed a monophyletic group with CTFV strains, and shared a common ancestor with Quang Binh or related Culex flaviviruses. The organization of the viral genome was consistent with the universal flavivirus structure and stem-loops; conserved motifs and imperfect tandem repeats were identified in the non-coding ends of the viral genomes. A potential ribosomal shifting site, resulting in the translation of an additional reading frame, was detected. The deduced viral polyprotein comprised 3357 amino acids and was highly-conserved. Amino acid variations, presumably associated with adaptive environmental pressures, were identified. These isolates comprise the first fully characterized insect-specific flaviviruses in Turkey. Their impact on West Nile virus circulation, which is also endemic in the study region, remains to be explored.

Grapevine leafroll associated virus 4 strain 9: complete genome and quantitative analysis of virus-derived small interfering RNA populations.

The complete genome sequence of one isolate of Grapevine leafroll-associated virus 4 strain 9 (GLRaV-4 strain 9) was determined using a library constructed from the sequencing of the small RNA population. The RNA genome consists of 13,858 nucleotides and contains seven open reading frames. A 5’ non coding end (218 nt) is followed by ORF1 encoding the 261.5 kDa methyltransferase/helicase domains then, through a +1 ribosomal frameshift, by ORF1b encoding a 58.28 kDa product corresponding to the replicase RdRp. The ORFs that follow encode the 5.32 kDa P5 protein, the 58.12 kDa HSP70h protein, the 60.18 kDa protein, the 29.9 kDa coat protein and finally the 23.2 kDa p23 protein followed by a 127 nt non coding 3’ end. The analysis of virus-derived siRNA populations aligning to the GLRaV-4 strain 9 genome showed a major representation of 21-nt reads, suggesting the involvement of DCL4 in virus silencing. The profile of the vsiRNAs density along the viral genome differed clearly from that of GLRaV-3, the representative member of subgroup I of the genus Ampelovirus, that was present in the same plant.

Sequencing artifacts in the type A influenza databases and attempts to correct them

BackgroundThere are over 276 000 influenza gene sequences in public databases, with the quality of the sequences determined by the contributor.ObjectiveAs part of a high school class project, influenza sequences with possible errors were identified in the public databases based on the size of the gene being longer than expected, with the hypothesis that these sequences would have an error. Students contacted sequence submitters alerting them of the possible sequence issue(s) and requested they the suspect sequence(s) be correct as appropriate.MethodsType A influenza viruses were screened, and gene segments longer than the accepted size were identified for further analysis. Attention was placed on sequences with additional nucleotides upstream or downstream of the highly conserved non-coding ends of the viral segments.Results and ConclusionsA total of 1081 sequences were identified that met this criterion. Three types of errors were commonly observed: non-influenza primer sequence wasn't removed from the sequence; PCR product was cloned and plasmid sequence was included in the sequence; and Taq polymerase added an adenine at the end of the PCR product. Internal insertions of nucleotide sequence were also commonly observed, but in many cases it was unclear if the sequence was correct or actually contained an error. A total of 215 sequences, or 22.8% of the suspect sequences, were corrected in the public databases in the first year of the student project. Unfortunately 138 additional sequences with possible errors were added to the databases in the second year. Additional awareness of the need for data integrity of sequences submitted to public databases is needed to fully reap the benefits of these large data sets.

A seven plasmid-based system for the rescue of influenza C virus

We report the establishment of a reverse-genetics system for the rescue of recombinant influenza C/JJ/50 virus from seven plasmids. The nucleotide sequence of the whole C/JJ/50 genome was determined and full-length cDNAs were cloned into an RNA pol I/pol II-based bidirectional vector. Transfection of Vero cells and subsequent amplification on MDCK cells yielded viral HA titres of 128. The utility of this bidirectional approach was proved by generating a reassortant virus encoding the NS segment from strain C/JHB/1/66 and a virus with mutations in the noncoding ends of PB1. The latter virus, which has a base-pair mutation within the proposed double-stranded region of the PB1 termini, exhibited impaired replication. In conclusion, our efficient seven-plasmid system for the rescue of recombinant influenza C virus may be used to study the influenza C virus life cycle in more detail and for generation of influenza C virus-based vectors.

Establishment of multiple RT-PCR diagnostic techniques forAvian influenza virus(AIV),Newcastle disease virus(NDV),Classical swine fever virus(CSFV) andFoot-and-mouth disease virus(FMDV)

AbstractThe homology of the sequences, reported and registered in GenBank, of different strains ofAvian influenza virus(AIV),Newcastle disease virus(NDV),Classical swine fever virus(CSFV) andFoot-and-mouth disease virus(FMDV), was analysed and compared with each other. According to the properties of these viruses, the conservative domain of theMgene for AIV, theFgene for NDV, the 5' non-coding domain end for CSFV and the2Bgene for FMDV were selected for polymerase chain reaction (PCR) amplification. In order to prevent the formation of conformational dimers between different primers, four pairs of primers designed with the DNAsis system under the condition of G+C (50–60%),18–25 bp in length andTm(72–85), were analysed using the VNTI5.5 system. The specific fragments amplified were as follows: 141 bp for FMDV, 200 bp for CSFV, 319 bp for NDV and 471 bp for AIV. The optimal conditions of PCR for each virus mentioned above were determined by orthogonal assay, and two or four of the four pairs of primers were then combined and used for amplification trials. The results showed that four specific fragments of different lengths would be successfully amplified in one tube at the same time. The products of PCR were tested to be specific by sequencing. Out of 46 pathological samples detected with the multiple PCR, there were 5 AIVs, 7 NDVs, 15 CSFVs and 6 FMDVs. The amplification above was identified with a single PCR. On the other hand, the results corresponded to those of electronic microscopy, haemagglutination (HA) and enzyme-linked immunosorbent assay (ELISA). The method described here is practicable, sensitive, specific, simple and cheap. It could be used for diagnosing AIV, NDV, CSFV and FMDV in different animals.

Differential mRNA fingerprinting by preferential amplification of coding sequences

The need for rapid identification of differentially expressed genes will persist even after the complete human genomic sequence becomes available. The most popular method for identifying differentially expressed genes acquires expressed sequence tags (ESTs) from the extreme 3′ non-coding end of mRNAs. Such ESTs have limitations for downstream applications. We have developed a method, termed preferential amplification of coding sequences (PACS), that was applied to identify differentially expressed coding sequence tags (dCSTs) between osteoblasts and osteosarcoma cells. PACS was achieved by PCR with a set of primers to anchor at sequences complementary to AUG sequences in mRNAs and another set of primers to anchor at a PCR-amplifiable distance from AUG sequences. An initial screen identified 103 candidate dCSTs after screening ∼15% of the expressed genes between the two cell types. Of these sequences, 27 represent CSTs of known genes and two are from 3′-ESTs of known mRNAs. Thus, PACS identified CSTs ∼13.5 times more often than it identified 3′ ESTs, attesting to the objective of the method. Since many of the dCSTs represent known genes, their identity and potential relevance to osteosarcoma could be immediately hypothesized. Differential expression of many of the dCSTs was further demonstrated by northern blotting or RT-PCR. Since PACS is not dependent on the existence of a poly A tail on an mRNA, it should have application to identify dCSTs for both prokaryotic and eukaryotic organisms. Additionally, PACS should aid in the identification of cell-specific or tissue-specific genes and bidirectional acquisition of cDNA sequence enabling rapid retrieval of full-length cDNA sequence of novel genes.

Detailed characterization of the posttranscriptional gene-silencing-related small RNA in a GUS gene-silenced tobacco

Posttranscriptional gene-silencing phenomena such as cosuppression and RNA interference are associated with the occurrence of small, about 21-23 nt short RNA species homologous to the silenced gene. We here show that the small RNA present in silenced transgenic plants can easily be detected in total RNA isolated according to standard procedures. This will allow for the development of routine and early screenings for the presence of small RNA species and, therefore, gene silencing in transgenic plants. We further demonstrate that the small RNA fraction can be visualized with the SYBR Green II RNA stain, isolated from a gel, labeled and used as a specific probe. Using these approaches, we have fine-mapped the sequences of the GUS gene that are represented in the small RNA fraction of a GUS-silenced tobacco line containing an inverted repeat of the GUS gene. In this tobacco line, the silencing-associated small RNA is a mixture of fragments that cover the 3' two-thirds of the GUS coding region. The 5' coding and the 3' noncoding ends of the GUS mRNA are not represented in the small RNA fraction. The RNA fragments are not likely to be a primary synthesis product of an RNA-dependent RNA polymerase, but rather degradation products from nuclease activity. Surprisingly, RNA isolated from wild-type, untransformed plants showed the presence of a similar-sized small RNA pool. This might indicate the existence of small RNA species from putative endogenous genes that are down regulated by a similar posttranscriptional gene-silencing mechanism. The possibility of isolating and labeling the small RNA pool from wild-type plants will provide a way to identify and study such putative genes.

Complete sequence of two tick-borne flaviviruses isolated from Siberia and the UK: analysis and significance of the 5′ and 3′-UTRs

The complete nucleotide sequence of two tick-transmitted flaviviruses, Vasilchenko (Vs) from Siberia and louping ill (LI) from the UK, have been determined. The genomes were respectively, 10928 and 10871 nucleotides (nt) in length. The coding strategy and functional protein sequence motifs of tick-borne flaviviruses are presented in both Vs and LI viruses. The phylogenies based on maximum likelihood, maximum parsimony and distance analysis of the polyproteins, identified Vs virus as a member of the tick-borne encephalitis virus subgroup within the tick-borne serocomplex, genus Flavivirus, family Flaviviridae. Comparative alignment of the 3′-untranslated regions revealed deletions of different lengths essentially at the same position downstream of the stop codon for all tick-borne viruses. Two direct 27 nucleotide repeats at the 3′-end were found only for Vs and LI virus. Immediately following the deletions a region of 332–334 nt with relatively conserved primary structure (67–94% identity) was observed at the 3′-non-coding end of the virus genome. Pairwise comparisons of the nucleotide sequence data revealed similar levels of variation between the coding region, and the 5′ and 3′-termini of the genome, implying an equivalent strong selective control for translated and untranslated regions. Indeed the predicted folding of the 5′ and 3′-untranslated regions revealed patterns of stem and loop structures conserved for all tick-borne flaviviruses suggesting a purifying selection for preservation of essential RNA secondary structures which could be involved in translational control and replication. The possible implications of these findings are discussed.

The Infectious Bronchitis Virus Nucleocapsid Protein Binds RNA Sequences in the 3′ Terminus of the Genome

The infectious bronchitis virus (IBV) nucleocapsid protein was expressed as a bacterial fusion protein which differed from the native protein only in the addition of six amino terminus histidine residues. Using RNA overlay protein blot assays, the recombinant protein was shown to bind to RNA fragments specific for the positive sense 3′ noncoding end of the IBV genome. At greater concentrations of sodium chloride, the native and fusion nucleocapsid proteins similarly bound to G RNA, representing the terminal 1805 3′ nt of the genome, whereas bovine serum albumin and allantoic fluid protein did not bind to labeled G RNA. Competitive gel shift assays with labeled G RNA indicated that the protein interacted with several unlabeled RNA representing sequences at the 3′ noncoding end of the IBV genome. Cache Valley virus (a bunyavirus) mRNA transcribed from the small segment cDNA also inhibited the interaction with IBV G RNA to approximately the same extent as homologous unlabeled G RNA, whereas reactions with bovine liver RNA and yeast tRNA were considerably weaker. Whereas yeast tRNA did not inhibit the interaction with the labeled large G RNA, interactions of the fusion protein with EF, a region from 78 to 217 nt from the 3′ terminus of the IBV genome, were also apparently weaker than interactions with fragment CD which consisted of the 3′ terminal 155 nt. On a molar basis, the latter interacted in an identical nature to a RNA consisting of CD and an additional 1053 nt of plasmid sequences. Compared to bovine liver RNA, unlabeled G specifically inhibited binding to the two smaller labeled IBV fragments in gel shift assays. The binding of IBV nucleocapsid protein with RNA probably requires specific sequences and/or structures that are present on the genome, and may represent a common mechanism used by similar viral nucleoproteins whose functions depend on binding to RNA.

Serological and molecular biology screening techniques for HVC infection.

This is a review of the main biological tests that can be requested by nephrologists to diagnose hepatitis C virus infection, together with a tentative specification of their indications and interpretation methods. This diagnosis is mainly based on serological tests (ELISA). The second and third generation tests contain, in addition to the NS4, proteins of the core and NS3 region. The latter two proteins provided great specificity and are antigenically well preserved in most virus types. They constitute the backbone of serological screening systems. In parallel with ELISA, analytical tests have been developed. In the form of immunoblots, they use proteins similar to those of ELISA and permit separate reading of antibody response to each protein. To achieve serological results, biologists must use at least two different techniques. Several strategies for using the various tests are thus available. However, serological diagnosis has its limitations: acute hepatitis most often precedes seroconversion; the results do not date the disease nor do they specify infectivity, evolution or recovery; seronegativity does not exclude a positive diagnosis with any certainty, especially in such populations as haemodialysis patients. To make up for the shortcomings of serological tests, direct viral detection can be achieved by genic amplification, most often by polymerase chain reaction in the best preserved part of the genome (5' non-coding end). PCR is currently becoming the real reference test for hepatitis C diagnosis. It is also possible to quantify viraemia and to determine the viral genotype. The relevancy of these two factors to therapy is controversial. Immunohistochemical study of liver biopsy specimens is still little used.

Sequence of the Genome of Lactate Dehydrogenase-Elevating Virus: Heterogenicity between Strains P and C

The complete nucleotide sequence of genomic RNA (14104 nt) of one strain of lactate dehydrogenase-elevating virus (LDV), LDV-P, is reported. It exhibits only about 80% nucleotide identity with the sequence reported for another LDV strain, LDV-C (Godeny et al., Virology 194, 585-596 (1993), and is 68 nucleotides shorter than the reported LDV-C sequence. The difference in length is largely due to the lack of a 59-nucleotide-long direct repeat in ORF 1a of the reported LDV-C sequence. Sequence analysis of a total of 1.4 kb of ORF 1a of LDV-C via reverse transcription/polymerase chain reaction (RT/PCR) technology failed to confirm the presence of this repeat in the LDV-C genome as well as of 24 deletions/insertions of single nucleotides that give rise to apparent transient reading frame differences between the LDV-P and LDV-C genomes and might have represented frameshift mutations. An additional 35 nucleotides in ORF 1a of the RT/PCR LDV-C products were the same as in the LDV-P rather than the reported LDV-C genome. The nucleotide sequences of the 5′ leader and the 3′ noncoding ends of the two genomes and the heptanucleotides involved in joining the 5′ leader to the bodies of the subgenomic mRNAs were highly conserved or identical. The predicted LDV-P proteins, however, differed from those predicted for the LDV-C proteins between 25% for the ORF 2 protein and 1% for the ORF 7 nuoleocapsid protein. All functional motifs of the ORF 1a and ORF 1b proteins were conserved. The ORF 1a protein possesses 11 potential transmembrane segments that flank the serine protease domain.

Comparison of cDNA probe hybridizations and RT-PCR detection methods for the identification and differentiation of enteroviruses isolated from sea water samples

Fifteen enteroviruses (EVs) previously isolated from Tyrrhenian sea water samples were used. They were first identified by traditional dot-blot and Northern-blot hybridizations with a group of cDNA probes from cloned Poliovirus 1 and Coxsackievirus B4 and oligodeoxynucleotides complementary to echovirus 6 and 9 sequences. Using both wild viruses and known enteroviruses a reverse-PCR protocol was then set up followed by cDNA sequencing of the fragments generated. The sequences of primers were selected from a consensus of several 5′ non-coding ends of enterovirus genomes, representing highly conserved regions. The downstream (region 577–603) and the upstream (region 436–465) oligonucleotide primers carried an extra sequence in order to generate BamHI and a HindIII restriction sites at the 5′ and 3′ end respectively of the amplified cDNA fragments for directional cloning in a plasmid. The downstream 5′NC primer was 5′-biotinylated in order to allow direct sequencing of the amplicon, when possible, after strand separations on streptavidin coated magnetic beads. The PCR of reverse transcribed viral RNAs resulted in a 167–170 b.p. cDNA product on ethidium bromide-stained 2% agarose gels in all the samples and reference viruses. The test is negative on reoviruses, hepatitis A and uninfected BGM cells and detects < 1 TCID 50 viral particles. Sequences of cloned fragments were compared with sequences of cloned enteroviruses stored in commercial data banks. The 5′NC region of a reference echovirus 5 was also cloned and sequenced to improve the comparison. On the basis of deduced genetic distances, three poliovirus 1, eight coxsakievirus B5, four coxsakievirus B1 were diagnosed. One poliovirus Sabin 2 was isolated together with a coxsackievirus-related strain in the same lysate sample. The reliability and sensitivity of this RT-PCR method makes it an attractive approach to virus detection in environmental samples.

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.

Fine mapping of a replication origin of human DNA.

A highly sensitive procedure was developed for the identification of the origin of bidirectional DNA synthesis in single-copy replicons of mammalian cells. The method, which does not require cell synchronization or permeabilization, entails the absolute quantification, by a competitive PCR procedure in newly synthesized DNA samples, of the abundance of neighboring DNA fragments distributed along a given genomic region. This procedure was utilized for mapping the start site of DNA replication in a 13.7-kb region of human chromosome 19 coding for lamin B2, which is replicated immediately after the onset of S phase in HL-60 cells. Within this region, DNA replication initiates in a 474-bp area corresponding to the 3' noncoding end of the lamin B2 gene and the nontranscribed spacer between this gene and the 5' end of another highly transcribed one. This localization was obtained both in aphidicolin-synchronized and in exponentially growing HL-60 cells.

Molecular techniques for the identification of enteric viruses in marine waters

The RNAs extracted from 28 high spin pellets of clarified lysates from BGM cells, previously infected by concentrated Adriatic Sea water samples, were analyzed by a dot-blot test. We used a 32P-labeled cDNA probe ( BamHI 220–670) from the 5′ non-coding end of the cloned poliovirus I (Mahoney) genome. The probe was selected for its broad range of genetic specificities. Based on the density of the hybridization signals on the autoradiograph, our dot-blot results showed a high degree of reactivity of the probe to homologous (poliovirus 1) and heterologous (echovirus 6 and coxsackievirus B3) reference RNA, as predicted by a computer analysis of their sequences, and a low reactivity to that of the samples leading us to exclude the presence of enteroviruses like the reference strains. In order to understand if there were other enteroviral serotypes or not, dot-blot tests were supplemented with Northern-blot hybridization assays. Results from the Northern-blot showed a series of fragments ranging from 4.3 to 1.2 kb but no signal corresponding to 7.5 kb (as did the positive control RNAs). These features suggested that the tested RNAs might derive from viruses of the Reoviridae family, which includes members with segmented genome. Traditional PAGE analysis showed clearly the 10-fragments pattern characteristic of reoviruses. Twenty-three out of the twenty-eight samples tested showed the presence of viruses, and this confirms the previously noted cytopathic effect (CPE) of the samples on the BGM cells.

Cis- and Trans-acting Elements in Cowpea Mosaic Virus RNA Replication

Cowpea mosaic virus (CPMV) B-RNA encodes the viral proteins required for viral RNA replication while M-RNA does so for the capsid proteins and functions required in cell-to-cell movement of the virus. Accordingly, B-RNA can replicate by itself, whereas M-RNA can only replicate in the presence of B-RNA. We have made heterologous sequence insertions at different positions in the open reading frame of B-RNA, leaving the 5′ and 3′ non-coding ends intact. None of these mutant B-RNAs were able to replicate. Furthermore, it was not possible to support replication of these mutant B-RNAs by co-inoculating wild-type B-RNA as a helper, indicating that B-RNA can not be replicated in trans. In contrast, replication of M-RNA must occur in trans, as the viral replicative proteins are encoded by B-RNA. Mutant M-RNA transcripts containing 5′ and 3′ non-coding regions of B-RNA are still efficiently replicated in protoplasts if co-inoculated with B-RNA, indicating that in cis or in trans replication of the CPMV RNAs is not primarily determined by the non-coding regions. Remarkably, for replication of M-RNA, the N-terminal domain of the 58K protein encoded by M-RNA was found to be required.