Vectors for expression and secretion of FLAG epitope-tagged proteins in mammalian cells.

The FLAG peptide, AspTyrLysAspAspAspAspLys, has been used as an epitope tag in a variety of cell types. The modification of the cytomegalovirus (CMV) promoter containing vector, pCMV5, to create two transient expression vectors designed for secretion and intracellular expression of FLAG-fusion proteins in mammalian cells is described. As a functional test, the bacterial alkaline phosphatase gene was cloned into both vectors, and anti-FLAG monoclonal antibodies were used for detection of FLAG epitope-tagged bacterial alkaline phosphatase in mammalian cells. In addition, secreted bacterial alkaline phosphatase was purified from the extracellular medium by anti-FLAG affinity chromatography.

Immunoaffinity purification of FLAG epitope-tagged bacterial alkaline phosphatase using a novel monoclonal antibody and peptide elution.

The FLAG epitope is an eight amino acid peptide (AspTyrLysAspAspAspAspLys) that is useful for immunoaffinity purification of fusion proteins. A monoclonal antibody (anti-FLAG M1) that binds the FLAG epitope in a calcium-dependent manner and requires an N-terminal FLAG sequence has been described previously. We describe the use of a second anti-FLAG monoclonal antibody (anti-FLAG M2) in immunoaffinity purification of N-terminal Met-FLAG and C-terminal FLAG fusion to bacterial alkaline phosphatase. Although binding of an anti-FLAG M2 monoclonal antibody to the FLAG epitope is not calcium-dependent, bound fusion proteins can be eluted by competition with FLAG peptide.

Expression of the cryIB crystal protein gene of Bacillus thuringiensis.

The cryIB gene of Bacillus thuringiensis subsp. thuringiensis HD-2 codes for a Mr 139492 protein that is lethal to certain lepidopteran larvae. We used primer extension to map transcriptional initiation sites and found that cryIB was transcribed from two sites that are activated at different times during sporulation. The presumed promoter regions for the two start sites are very similar to the two promoters preceding the cryIA (a) gene, and the in vivo transcriptional start sites were found to be identical. Variable amounts of the full-length cryIB protein were detected by immunoblotting of extracts of recombinant cells of Escherichia coli; larger amounts were found when the TTG translational start codon was changed to ATG and when an htpR- strain of E. coli was used as the recipient for transformation. When expressed in E. coli, the cryIB protein was found to be toxic to the larvae of Artogeia rapae (LC50 of 58 ng/cm2) and exhibited little toxicity to the larvae of Manduca sexta (LC50 greater than 5000 ng/cm2).

Temperature-sensitive viral RNA expression in Moloney murine sarcoma virus ts110-infected cells.

We examined the mos-specific intracellular RNA species in 6m2 cells, an NRK cell line nonproductively infected with the ts110 mutant of Moloney murine sarcoma virus. These cells present a normal phenotype at 39 degrees C and a transformed phenotype at 28 or 33 degrees C, expressing two viral proteins, termed P85gag-mos and P58gag, at 28 to 33 degrees C, whereas only P58gag is expressed at 39 degrees C. It has been previously shown that 6m2 cells contain two virus-specific RNA species, a 4.0-kilobase (kb) RNA coding for P58gag and a 3.5-kb RNA coding for P85gag-mos. Using both Northern blot and S1 nuclease analyses, we show here that the 3.5-kb RNA is the predominant viral RNA species in 6m2 cells grown at 28 degrees C, whereas only the 4.0-kb RNA is detected at 39 degrees C. During temperature shift experiments, the 3.5-kb RNA species disappears after a shift from 28 to 39 degrees C and is detected again after a shift back from 39 to 28 degrees C. By Southern blot analysis, we have detected only one ts110 proviral DNA in the 6m2 genome. This observation, as well as previously published heteroduplex and S1 nuclease analyses which showed that the 3.5-kb RNA species lacks about 430 bases found at the gag gene-mos gene junction in the 4.0-kb RNA, suggests that the 3.5-kb RNA is a splicing product of the 4.0-kb RNA. The absence of the 3.5-kb RNA when 6m2 cells are grown at 39 degrees C indicates that the splicing reaction is thermosensitive. The splicing defect of the ts110 Moloney murine sarcoma virus viral RNA in 6m2 cells cannot be complemented by acute Moloney murine leukemia virus superinfection, since no 3.5-kb ts110 RNA was detected in acutely superinfected 6m2 cells maintained at 39 degrees C. The spliced Moloney murine leukemia virus env mRNA, however, is found in acutely infected cells maintained at 39 degrees C, suggesting that the lack of ts110 viral RNA splicing at 39 degrees C is not due to an obvious host defect. In sharp contrast, however, 6m2 cells chronically superinfected with Moloney murine leukemia virus produce a 3.5-kb RNA species at 39 degrees C as well as at 28 degrees C and contain proviral DNAs corresponding to the two viral RNA species.(ABSTRACT TRUNCATED AT 400 WORDS)

Murine sarcoma virus ts110 RNA transcripts: origin from a single proviral DNA and sequence of the gag-mos junctions in both the precursor and spliced viral RNAs.

Our previous studies have argued persuasively that in murine sarcoma virus ts110 (MuSVts110) the gag and mos genes are fused out of frame due to a approximately 1.5-kilobase (kb) deletion of wild-type murine sarcoma virus 349 (MuSV-349) viral information. As a consequence of this deletion, infected cells grown at 39 degrees C appear morphologically normal, producing a 4-kb viral RNA and a truncated gag gene product, P58gag. At 33 degrees C, however, MuSVts110-infected cells appear transformed, producing two viral RNAs, about 4 and 3.5 kb in length, and two viral proteins, P58gag and P85gag-mos. Recent S1 nuclease analyses (Nash et al., J. Virol. 50:478-488, 1984) suggested strongly that at 33 degrees C about 430 bases surrounding the out-of-frame gag-mos junction and bounded by consensus splice donor and acceptor sites are excised from the 4-kb RNA to form the 3.5-kb RNA. As a result of this apparent splicing event, the gag and mos genes seemed to be fused in frame and allowed the translation of P85gag-mos. In the present study, DNA primers hybridizing to the MuSVts110 4- and 3.5-kb RNAs just downstream of the gag-mos junction points were used to sequence these junctions by the primer extension method. We observed that, relative to wild-type MuSV-349 5.2-kb RNA, the MuSVts110 4-kb RNA had suffered a 1,488-base deletion as a result of the fusion of wild-type gag gene nucleotide 2404 to wild-type mos gene nucleotide 3892. This gag-mos junction is out of frame, containing both TAG and TGA termination codons in the reading frame 42 and 50 bases downstream of the gag-mos junction, respectively. Thus, the MuSVts110 4-kb RNA can only be translated into a truncated gag precursor containing an additional C-terminal 14 amino acid residues derived from an alternate mos gene reading frame. Similar analyses of the MuSVts110 3.5-kb RNA showed a further loss of both gag and mos sequences over those deleted in the original 1,488-base deletion. In the MuSVts110 3.5-kb RNA, we found that gag nucleotide 2017 was fused to mos nucleotide 3936 (nucleotide 2449 in the MuSVts110 4-kb genome). This 431-base excised fragment is bounded exactly by in-frame consensus splice donor and acceptor sequences. As a consequence of this splice event, the TAG codon is excised and the restoration of the original mos gene reading frame allows the TGA codon to be bypassed.(ABSTRACT TRUNCATED AT 400 WORDS)

Reverse transcription of yeast double-stranded RNA and ribosomal RNA using synthetic oligonucleotide primers.

The ability of the four oligodeoxyribonucleotide primers oligo(dT)12-18, oligo(dA)12-18, oligo(dG)12-18 and oligo(dC)12-18 to act as primers for avian myeloblastosis virus reverse transcriptase on denatured yeast double-stranded (ds) RNA templates was investigated. Oligo(dT) and oligo(dA) were found to prime the synthesis of 1.1 and 1.0 kb reverse transcripts, respectively, using denatured M dsRNA as a template. The oligo(dT)- and oligo(dA)-primed cDNAs of M dsRNA hybridized to the region of the M dsRNA that encoded the killer toxin and to each other. Addition of oligo(dT) to reverse transcription reactions of denatured L dsRNA produced a 4.3 kb cDNA. During the course of this investigation oligo(dC) was observed to be a highly efficient primer for reverse transcription of yeast 18 S ribosomal RNA. Oligo(dC) primed the synthesis of a 1.0 kb transcript of 18 S rRNA which hybridized to the large Eco RI fragment of the 18 S rRNA gene. Reverse transcription of double-stranded RNA and 25 S ribosomal RNA was found to occur to some extent in the absence of added oligonucleotide primer.