SP6 RNA Polymerase Research Articles

Infectivity studies on different potato spindle tuber viroid (PSTV) RNAs synthesized in vitro with the SP6 transcription system

We have constructed two sets of clones in which one to six head-to-tail connected DNA copies of the potato spindle tuber viroid (PSTV) RNA genome were inserted into the plasmid pSP62- Pl downstream of the promoter for SP6 RNA polymerase. In vitro transcription of these constructs with the promoter-specific SP6 RNA polymerase yielded the corresponding oligomeric single-stranded linear PSTV RNA molecules of (+) and (-) polarity. Except for short vector-derived terminal sequences these in vitro synthesized PSTV RNA forms are equivalent to the RNA intermediates of the PSTV replication cycle which are present in vivo only in extremely low concentrations. From each DNA template molecule up to 600 RNA copies could be transcribed in vitro and yields > 100 microg were obtained. When mechanically inoculated to tomato seedlings the PSTV (+) RNA oligomers were as infectious as the natural PSTV (+) RNA monomers. Surprisingly, the corresponding oligomeric PSTV (-) RNAs wer approximately 10(4)-fold less infectious. However, when these (-) RNAs were partially protected prior to inoculation by mixing or hybridizing them with non-infectious (+) RNA fragments or by 'capping' their 5' terminus, an increase in the number of infections was observed. The in vitro synthesis of infectious RNA from cloned cDNA means that, in principle, it should be possible to develop vector systems from pathogens with RNA genomes.

Translation of mRNA injected into Xenopus oocytes is specifically inhibited by antisense RNA.

The bacteriophage SP6 promoter and RNA polymerase were used to synthesize sense and antisense RNAs coding for the enzymes thymidine kinase (TK) and chloramphenicol acetyl transferase (CAT). Injection of antisense CAT RNA into frog oocytes inhibited expression of sense CAT mRNA. Similarly, antisense TK RNA inhibited expression of sense TK mRNA. Antisense RNAs were stable in oocytes and had no detectable effect on either the expression of endogenous proteins or on the expression of nonhomologous RNA transcripts. CAT activity expressed from a plasmid transcribed in the oocyte nucleus was also inhibited by antisense RNA injected into the oocyte cytoplasm. The data suggest that antisense RNA will be useful in identifying the function of specific mRNA sequences during early development of the frog.

Mouse-Torpedo hybrid acetylcholine receptors: functional homology does not equal sequence homology.

The nicotinic acetylcholine (AcCho) receptor (AcChoR) is a multisubunit protein complex of stoichiometry alpha 2 beta gamma delta. The several subunits show homology with each other within a given species; in addition, homology is found between analogous subunits between species. We have used the phage SP6 RNA polymerase transcription system to produce single-species RNA in vitro for various AcChoR subunits from cDNAs. Injection of an equimolar mixture of RNA for the alpha, beta, gamma, and delta subunits of Torpedo californica AcChoR into Xenopus oocytes results in the appearance of functional receptors in the oocyte membrane. No response to AcCho is detected when the beta or gamma subunit RNA is omitted, and a small response is seen when the delta subunit RNA is omitted. Replacement of Torpedo delta subunit RNA by the mouse BC3H-1 cell line AcChoR delta subunit RNA leads to the formation of functional receptors that show a 3-4-fold greater response to AcCho than does the full Torpedo complex. No response is seen when the mouse delta RNA replaces Torpedo gamma RNA. By amino acid homology profile comparisons, the mouse delta subunit appears to be moderately but not highly similar to the Torpedo delta subunit; the apparent similarity to the Torpedo gamma subunit is only slightly less. Therefore, the features of the primary sequence that determine the functional delta character of the mouse polypeptide are not revealed by simple homology comparisons.

Molecular cloning of cDNA for human von willebrand factor: Authentication by a new method

We have identified a 2.4 kb partial cDNA clone (pDL34) for human von Willebrand factor (vWf) mRNA. pDL34 was selected by screening an endothelial cell cDNA library with a radiolabeled reverse transcript of mRNA obtained by specific immunoisolation of vWf polysomes from endothelial cells. pDL34 selectively hybridized to an endothelial cell-associated 9.5 kb mRNA. To confirm its identity, SP6 RNA polymerase was used to generate in vitro transcripts of the cDNA. This synthetic RNA, truncated at its 5′ end, directed the synthesis of several unique polypeptides in rabbit reticulocyte lysates. These polypeptides were immunoprecipitated by polyclonal and monoclonal anti-vWf antibodies. These results indicate that pDL34 contains an authentic partial copy of vWf mRNA. In vitro transcription of partial cDNA clones and translation of the resulting RNAs may be a useful general method of verifying the identity of various cDNA clones in circumstances where antibodies are available and protein sequence is not.

A transcription termination signal immediately precedes the coding sequence for the chloramphenicol-inducible plasmid gene cat-86.

The plasmid gene cat-86 specifies chloramphenicol-inducible, chloramphenicol acetyltransferase in Bacillus subtilis. The inducible regulation is independent of the promoter that is used to activate cat-86 and is independent of the cat-86 coding sequence. We have proposed that the regulation of cat-86 results from the transcription of a pair of inverted-repeat sequences that immediately precede the coding sequence. These transcripts are predicted to sequester the cat-86 ribosome binding site in a stable RNA stem-loop which, in theory, should block the ribosome binding site from pairing with 16S rRNA. Inducible expression of cat-86 may therefore result in part from regulation of the translation of cat-86 mRNA. However, chloramphenicol-induction correlates with increased levels of cat-86 mRNA and the RNA stem-loop preceding the cat-86 coding sequence structurally resembles a rho-independent transcription terminator. We have therefore tested the inverted-repeats as a potential site of transcription termination. Transcription studies performed in vitro using SP6 RNA polymerase and in vivo by S1 mapping demonstrate that a substantial fraction of the potential cat-86 transcripts terminate at a site immediately 3' to the inverted-repeats. The results of the in vivo experiments suggest that the termination signal may be partially relieved by growth of cells in chloramphenicol.

Cell-free synthesis and processing of an infectious dimeric transcript of potato spindle tuber viroid RNA

A full-length cloned cDNA insert containing the sequence of potato spindle tuber viroid (PSTV) was dimerized and placed in the plasmid vector pSP65 adjacent to a promoter for bacteriophage SP6 RNA polymerase. In vitro transcription of this region yielded a single linear RNA chain 760 bases in length containing two copies of PSTV RNA with about 20 bases of vector sequence at each end. Bioassay on tomato plants revealed that this transcript has infectivity comparable to that of PSTV itself, yielding circular progeny RNAs indistinguishable from PSTV grown in vivo Comparative RNA fingerprinting analysis revealed that the viroid sequence in the dimeric transcript breeds true (compared to control viroid strains of different sequence grown in parallel) but loses the vector-specific sequences during growth in plants. Incubation of 32P-labeled dimeric PSTV RNA at neutral pH and 39° gave a 1–5% yield of three RNA segments, one comigrating with unit-length linear PSTV and two smaller fragments. Reaction of the unit-length cleavage product with wheat germ RNA ligase gave a high yield of circular molecules indistinguishable from PSTV circles arising in vivo, suggesting that the cleavage reaction yields 2′,3′ cyclic phosphate termini and could represent a part of the in vivo PSTV replication cycle.

A novel transcription property of SP6 and T7 RNA polymerases: dependence on template structure.

The in vitro synthesis of extraneous RNA sequences by SP6 and T7 RNA polymerases from specific DNA templates is described. Transcription of templates prepared by digestion with restriction enzymes that leave 3' protruding ends resulted in the production of significant amounts of long, template-sized RNA transcripts which hybridized to vector DNA. Sequences copied from the noncoding template strand were among the extraneous transcripts. The presence of these sequences in probe preparations were detected in Southern and RNase protection hybridization assays. In contrast, transcription of DNA templates with blunt or 5' protruding ends yielded few RNA products as extraneous sequences.

The effect of capping and polyadenylation on the stability, movement and translation of synthetic messenger RNAs in Xenopus oocytes.

Synthetic RNAs coding for chicken lysozyme, calf preprochymosin and Xenopus globin were transcribed in vitro using Sp6 RNA polymerase. The effects of capping and adding a poly(A) tail on the stability, movement and translation of these RNAs in Xenopus oocytes was examined. Capping and polyadenylation increased stability of the transcripts, with at least 40% remaining intact 48 h after injection into oocytes. Capped poly(A)- transcripts moved more rapidly in oocytes than either capped poly(A)+ transcripts or naturally occurring mRNAs. The translational efficiency of most of the synthetic RNAs in oocytes increased with both capping and polyadenylation. The exception was one Xenopus globin transcript which had an unusual 3' end of 20As and 30Cs, where further polyadenylation decreased translational efficiency. Polyadenylation was essential for detectable expression of the synthetic RNAs in cultured cells, but decreased translation of the synthetic RNAs in vitro.

Secondary structure of splice sites in adenovirus mRNA precursors.

In order to investigate the possible role of RNA secondary structure in determining the efficiency and specificity of mRNA splicing, the structures of sequences at three acceptor splice sites in adenovirus were studied. Transcripts spanning intron-exon junctions were synthesized using SP6 RNA polymerase and analyzed using single and double-strand specific nucleases. Distinctive patterns of nuclease cleavage were observed for each of the 3 sites examined. At both sites in the E2a region sequences adjacent to the splice sites were particularly susceptible to digestion with T1 and S1 nucleases. In contrast, a splice site for hexon mRNA was largely resistant to these nucleases. The results obtained suggest that the conformation of the RNA at some, but not all, acceptor sites may enhance the accessibility of these sites to factors involved in splicing nuclear RNA and confirm the presence of a large, previously predicted hairpin structure centered on the acceptor site at 67 map units.

Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter.

A simple and efficient method for synthesizing pure single stranded RNAs of virtually any structure is described. This in vitro transcription system is based on the unusually specific RNA synthesis by bacteriophage SP6 RNA polymerase which initiates transcription exclusively at an SP6 promoter. We have constructed convenient cloning vectors that contain an SP6 promoter immediately upstream from a polylinker sequence. Using these SP6 vectors, optimal conditions have been established for in vitro RNA synthesis. The advantages and uses of SP6 derived RNAs as probes for nucleic acid blot and solution hybridizations are demonstrated. We show that single stranded RNA probes of a high specific activity are easy to prepare and can significantly increase the sensitivity of nucleic acid hybridization methods. Furthermore, the SP6 transcription system can be used to prepare RNA substrates for studies on RNA processing (1,5,9) and translation (see accompanying paper).

Bacteriophage SP6-specific RNA polymerase. I. Isolation and characterization of the enzyme.

SP6 is a small, virulent bacteriophage which grows on Salmonella typhimurium LT2. It is morphologically similar to Escherichia coli bacteriophage T7 and its relatives, but appears to be genetically distinct. After infection a bacteriophage-specific RNA polymerase is induced in infected cells. SP6 RNA polymerase is a stable enzyme and is easily purified to homogeneity in good overall yield. The activity resides in a single polypeptide chain of Mr = 96,000. Synthesis of RNA by SP6 RNA polymerase requires a DNA template and Mg2+ ion and is strongly stimulated by either bovine serum albumin of spermidine. Thiol-reactive reagents inhibit the enzyme, suggesting the presence of essential sulfhydryl residues. RNA synthesis requires native SP6 RNA as template; DNAs from other bacteriophages including T3 and T7 are inert; hence, SP6 RNA polymerase possesses a stringent promoter specificity similar to, but distinct from that of the other phage RNA polymerases. The SP6 RNA polymerase is also highly active in synthesis of poly(rG) with poly(dI) . (dC) as template. This reaction is unlikely to involve promoter-like sites, but it appears to reflect a general catalytic capacity of the polymerase, since cleavage of the SP6 RNA polymerase with trypsin, which completely eliminates SP6-transcribing activity, has little effect on poly(rG) synthesis. Hence, it appears that the catalytic portion of the polymerase can be separated from the RNA polymerase holoenzyme.