A primer vector system that allows temperature dependent gene amplification and expression in mammalian cells: regulation of the influenza virus NS1 gene expression.

Influenza virus RNA segment 8 has been cloned into primer-vector pSLts1. This vector was designed to replicate in simian cells in a temperature dependent fashion by use of the SV40 tsA209 T-antigen gene. The oriented synthesis of cDNA on dT-tailed pSLts1 was performed on in vitro synthesized mRNA, and the second DNA strand was primed with an influenza-specific terminal oligodeoxynucleotide. Recombinant pSLVa232 contained the RNA segment 8 sequence directly fused to the SV40 late promoter contained in pSLts1, and followed by the SV40 polyadenylation signal. Expression of NS1 gene in transfected COS cells took place at a level comparable to that found in infected cells. When VERO cell cultures were transfected with recombinant pSLVa232, expression of the NS1 gene was temperature dependent. Close to one hundred fold increase in the amplification and expression of the cloned gene was observed after shift down of the transfected cells to permissive temperature. Vector pSLts1 and the cloning strategy described may be useful for the specific cloning and regulated expression of mRNAs of known 5'-terminal sequence.     

Expression of adenovirus type 12 E1A gene in monkey cells, using a simian virus 40 vector.

Simian virus 40 (SV40) recombinants carrying the adenovirus type 12 E1A gene were constructed. The SV40 expression vector was constructed by removing most of the VP1 gene and an internal part of the intervening sequence for late 16S RNA and by joining the 5' and 3' splice sites into a small segment. The adenovirus type 12 E1A gene with or without its own promoter was inserted downstream from the SV40 late promoter and the splicing junctions. The recombinant DNA was propagated and packaged in monkey cells by cotransfection with an early temperature-sensitive mutant (tsA58) DNA as helper. Immunofluorescent staining of the monkey cells infected with the resulting virus stocks showed that up to 20% of the cells overproduced the E1A gene products in the nuclei. Two-dimensional gel electrophoresis of the products indicated that the products were very similar or identical to the authentic polypeptides synthesized in adenovirus type 12-infected human embryo kidney cells. The E1A mRNA was initiated at the SV40 late promoter irrespective of the presence of the E1A promoter and terminated at either the E1A or the SV40 polyadenylation signal. These hybrid mRNAs were correctly spliced in the E1A coding region.     

Clone banks of cDNA from the parasite Schistosoma mansoni: isolation of clones containing a potentially immunodiagnostic antigen gene

We have constructed a small vector specifically for blunt-end cloning of fragments of DNA. Both the PvuII site and the EcoRI site allow the detection of recombinants using a simple and inexpensive colour screen. We have used this vector to construct cDNA clone banks from polyadenylated messenger RNA [poly(A)+mRNA] from several life cycle stages of the human parasite Schistosoma mansoni and have identified clones encoding an immunodiagnostic antigen gene by a combination of Southern blotting and mRNA hybrid-selection and in vitro translation. Antibodies against this antigen are only present in patients infected with S. mansoni.     

SV40 recombinants carrying a functional RNA splice junction and polyadenylation site from the chromosomal mouse beta maj globin gene.

We have introduced a fragment of the chromosomal mouse beta major globin gene into SV40 and used the resultant hybrid virus to infect cultured monkey kidney cells. The mouse DNA fragment, which contains an intervening sequence and a poly(A) addition site, has been inserted in both possible orientations relative to the SV40 late region promoter. While the fragment is transcribed regardless of orientation, the RNA splice signal and poly(A) addition site are utilized only when the fragment is inserted in the "sense" orientation. Thus genomic mouse signals for both splicing and polyadenylation are recognized across species boundaries. Furthermore, since only an 18 bp segment was included on the 5' border of the intervening sequence, we can define a maximum length for this splice signal.     

A procedure for selective full length cDNA cloning of specific RNA species.

A method allowing routine establishment of full length and functionally competent cDNA clones of particular mRNAs from small preparations of polyadenylated RNA is described. Pairs of synthetic primers are used for first and second strand synthesis. They include sequences complementary to the 3' terminal regions of the mRNAs and of the full length first cDNA strands, respectively and bear a few additional nucleotides at their 5' ends. After synthesis of both cDNA strands in one tube, they are precisely trimmed back with T4 DNA polymerase in presence of only two nucleoside triphosphates, to yield sticky ends fitting into a vector plasmid cleaved with two restriction endonucleases. The procedure was first applied to the simultaneous cloning of all five major measles virus (MV) mRNA species from a persistently infected cell line. Two thirds of all clones contained full length MV-specific cDNAs. Screening of less than 200 clones was sufficient to obtain several independent clones corresponding to each mRNA, except for gene F which was represented only once.