Full-length cDNA Synthesis Research Articles

Synthesis of full-length potyvirus cDNA copies suitable for the analysis of genome polymorphism

New methods facilitating the synthesis and amplification of full-length cDNA copies of single-stranded viral RNA genomes have been developed. A method is described for the efficient purification of potyviral RNA and total RNA from infected plants and it is shown that they can serve as templates for the efficient synthesis of a full-length, 10 kb long, genomic cDNA. Two different reverse transcriptases were used (AMV-RT and MMLV-RT); only the first reverse transcriptase produced a good quality, full-length cDNA using viral RNA as a template. Surprisingly, MMLV-RT allowed for the full-length cDNA synthesis on virions rather than viral RNA. The PVY cDNA, synthesized using either RNA or virions, can be amplified successfully by PCR with high yields of full-length products. Such products are good substrates for the study by RFLP of the total genome polymorphism of virus isolates.

The multimerization state of retroviral RNA is modulated by ammonium ions and affects HIV-1 full-length cDNA synthesis in vitro.

Genomic human immunodeficiency virus type 1 (HIV-1) RNA fragments containing the dimer linkage structure (DLS) can be dimerized and multimerized in the presence of NH4+ and in the absence of any other cation and any viral or cellular protein. This effect strongly supports the notion that dimerization and multimerization of genomic RNA occurs via purine-quartet formation in quadruple helical RNA structures. The efficiency of RNA dimerization and multimerization in the presence of ammonium ions is about 400 fold increased as compared to alkali metal ions such as potassium. Dimerized retroviral RNA representing a pseudodiploid genome could account for genetic recombination within the virion and during reverse transcription. Application of a novel South-Northern-Blotting procedure with biotinylated RNA and digoxigenin-labelled cDNA in vitro reveals that efficient human- and bovine tRNA(Lys3) primed full-length cDNA-synthesis only takes place with a predominantly monomerized RNA template. Dimerization and multimerization of the RNA significantly reduces full-length cDNA-synthesis. This suggests that monomerization of the dimerized RNA, effected by deionization in vitro, is essential for efficient retroviral reverse transcription in vivo.

Immunoreactivity and protective effects in mice of a recombinant dengue 2 Tonga virus NS1 protein produced in a baculovirus expression system

The double-stranded replicative form of dengue 2 virus (DEN-2) RNA (Tonga strain) was used as a substrate to produce DNA clones of the NS1-NS2A genes via reverse transcriptase synthesis of full length cDNA followed by polymerase chain reaction amplification of the NS1-NS2A region. Products were cloned into pTZ18R for sequencing and into baculovirus for expression studies. The deduced amino acid sequence of the NS1-NS2A was almost identical to that of the S1 attenuated strain of DEN-2 Puerto Rico 159, differing in only four amino acids. The NS1 protein expressed in insect cells [Spodoptera frugiperda (Sf-9)] from the baculovirus recombinant was indistinguishable from authentic NS1 of DEN-infected Aedes albopictus cells in glycosylation, dimerization, cellular presentation and antigenicity. Mice injected with the expressed protein developed NS1-specific complement-fixing antibodies and were partially protected against neurological residua after intracranial challenge. The protective effect was mouse strain- and gender-specific.

An assay for full-length cDNA synthesis from a heterogeneous population of mRNA, exemplified by opsin and the retina

An assay for full-length cDNA synthesis from a heterogeneous population of mRNA, exemplified by opsin and the retina

Reverse transcriptase and its associated ribonuclease H: interplay of two enzyme activities controls the yield of single-stranded complementary deoxyribonucleic acid.

The synthesis of single-stranded globin cDNA by the RNA-directed DNA polymerase activity of reverse transcriptase in the presence of oligothymidylate primers was investigated in order to determine the limitations to higher yields. The results indicated that the associated ribonuclease H activity, an integral part of reverse transcriptase, plays a large role in the synthesis of the first strand of cDNA and that the interplay of the two enzyme activities for any specific set of conditions determines the yield of single-stranded products. In both the presence and the absence of polymerization, the associated ribonuclease H catalyzed the deadenylation of mRNA, producing molecules that were somewhat shorter, highly homogeneous in size, and fully translatable into globin protein. They were also entirely lacking in the ability to serve as templates for cDNA synthesis. The reaction was completely dependent on oligothymidylate and completely independent of deoxyribonucleoside triphosphates. The initial rate of deadenylation was one-fourth the initial rate of initiation of polymerization when saturating levels of deoxyribonucleoside triphosphates were used in the polymerase reaction. In the presence of ribonuclease H activity, the DNA polymerase catalyzed the synthesis of an array of cDNAs including some that were full length. The initiation of polymerization was rate limiting: once synthesis had begun, it required 1-1.5 min to transcribe globin mRNA. However, most primers that were elongated were aborted prematurely. Maximum synthesis of full-length cDNA required stoichiometric levels of enzyme and high triphosphate levels, but regardless of conditions, the sum of completed cDNA and deadenylated mRNA accounted for only 50% of the input mRNA. The data fit a model in which synthesis of full-length cDNA molecules depends on the arrangement of primers and transcription initiation complexes on the poly(A) "tail" of mRNA.

Methylmercury hydroxide enhancement of translation and transcription of ovalbumin and conalbumin mRNA's.

Translation of total mRNA in heterologous protein-synthesizing systems is often employed as an indirect means of assessing relative mRNA concentrations. However, it is well known that the efficiency of translation of specific mRNAs differs. One such example is the poor translational efficiency of conalbumin mRNA relative to ovalbumin mRNA. In this report we have studied the translation of conalbumin and ovalbumin mRNAs in crude mRNA preparations and with highly purified mRNA preparations. We find that treatment of RNA with methylmercury hydroxide prior to translation improves the translational efficiency of both mRNAs and preferentially improves translational efficiency of conalbumin mRNA to the point where it more correctly reflects the relative concentration of these two mRNAs in crude mRNA preparations. Conalbumin mRNA is also a poor template for the synthesis of full length cDNA synthesis by avian myeloblastosis virus reverse transcriptase, and treatment of this mRNA with methylmercury hydroxide increases the size of DNA sequences synthesized. We conclude that treatment with methylmercury hydroxide produces a partial denaturation of mRNA complexed with either itself or with other RNA molecules and results in more efficient utilization in both translational assays and DNA polymerization reactions.