Recombinant Plasmid Vector Research Articles

Growth and plasmid stability of recombinantE. coli cells producing hepatitis B surface antigen

The self-made recombinant plasmid vector, pKSB292, harboring the amino terminal-deleted surface antigen gene of hepatitis B virus (HBsAg) under the control ofIpp + lac double promoters with the signal peptide region of the lipoprotein gene was tested for its effect on host cell growth and for the stability behaviour under various environmental conditions. The selection pressure imposed by the antibiotic ampicillin resulted in the more hindered cell growth as compared with the metabolic load caused by the recombinant plasmid in the host cell induced by the IPTG in the antibiotic-free medium. However the simultaneous application of both the antibiotic selection pressure and the metabolic load from the 1PTG induction exhibited the lowest cell growth rate. The host cells harboring the recombinant plasmid, pKSB292, accounted for the majority of cell population under antibiotic selection pressure showing the apparently higher stability. The better growth rate of the recombinant cells and the higher HBsAg production rate could be achieved in the nonselective complex medium.

Interferon-stimulated transcription: isolation of an inducible gene and identification of its regulatory region.

A human gene (termed ISG-54K) that is induced from near undetectable levels to high transcriptional activity by alpha- and beta-interferons has been cloned. The genomic structure and nucleotide sequence of the coding region were determined and the RNA initiation site was identified. The 5' portion of the gene was fused with a heterologous gene lacking an active promoter in recombinant plasmid and adenoviral vectors. These fusion genes were used to assess the activity of the ISG-54K promoter in response to interferon. RNA was formed in HeLa cells from recombinant plasmids only in response to interferon. Furthermore, in human diploid fibroblasts, infection with the recombinant adenovirus vector resulted in a 50-fold increase in specific RNA in response to interferon, followed by a subsequent decrease, imitating the natural regulated transcriptional cycle of the endogenous gene.

Vectors for expression and amplification of cDNA in mammalian cells: expression of rat phenylalanine hydroxylase.

We have constructed two recombinant plasmid vectors for direct expression and amplification of cDNA in mammalian cells. Each vector carries two dominant selectable markers (the bacterial neo gene and the mouse DHFR gene), a promoter sequence (viral LTR in pAV009/A+, and sheep metallothionein promoter in pMT010/A+), a polyadenylation signal sequence, and a Bam HI site to allow insertion of cDNA. We have used these vectors to prepare recombinant clones for the expression of rat phenylalanine hydroxylase (PH) in LTK- cells. Selection of transformants with neomycin followed by selection of the transformants in methotrexate led to a 30- to 60-fold amplification of the DHFR marker and co-amplification of the PH cDNA, with a corresponding increase in the level of PH mRNA and enzyme polypeptide. The expressed enzyme has a subunit molecular weight of 50,000 which corresponds to the W- allele of rat liver PH. PH activity was detected in the transfected cells by enzymatic measurement of the conversion of [14C]phenylalanine to [14C]tyrosine, and by growth of these cells in a tyrosine-free culture medium. Expression of rat PH in cell culture should facilitate the analysis of the biochemical properties of this enzyme.

Expression of Hepatitis B Viral Core Region in Mammalian Cells

We studied the expression of the core region of the hepatitis B virus genome in mammalian cells with recombinant plasmid vectors. Stably transformed rat fibroblast cell lines were established by transfection with vectors containing subgenomic and genome-length hepatitis B virus DNA, followed by G418 selection. The RNA transcripts directed by the core region were characterized by Northern blot hybridization and S1 nuclease mapping. Using the chloramphenicol acetyltransferase gene expression system, the promoter activity located upstream of the core open reading frame was confirmed. The synthesis of core and e polypeptides was studied with a commercial radioimmunoassay. These studies show that partial deletion of the precore sequences abolished secretion of the e antigen, but there was pronounced synthesis of the core antigen in transfected cells.

Expression of hepatitis B viral core region in mammalian cells.

We studied the expression of the core region of the hepatitis B virus genome in mammalian cells with recombinant plasmid vectors. Stably transformed rat fibroblast cell lines were established by transfection with vectors containing subgenomic and genome-length hepatitis B virus DNA, followed by G418 selection. The RNA transcripts directed by the core region were characterized by Northern blot hybridization and S1 nuclease mapping. Using the chloramphenicol acetyltransferase gene expression system, the promoter activity located upstream of the core open reading frame was confirmed. The synthesis of core and e polypeptides was studied with a commercial radioimmunoassay. These studies show that partial deletion of the precore sequences abolished secretion of the e antigen, but there was pronounced synthesis of the core antigen in transfected cells.

Transcriptional control elements of hepatitis B surface antigen gene.

A series of recombinant plasmid vectors containing hepatitis B virus (HBV) DNA sequences was constructed to study the biosynthesis of the hepatitis B virus surface antigen (HBsAg) RNA and to locate transcriptional control elements involved in the regulation of the S and pre-S DNA sequences. We examined the transcription of the HBsAg gene in permanent cell lines that were developed by transfecting with recombinant vectors containing HBV sequences and the neomycin gene followed by G418 selection. We further defined the promoter activities upstream of and within the pre-S sequences using the assayable chloramphenicol acetyltransferase gene. Results obtained from S1 nuclease digestion and primer extension suggest that HBsAg transcripts are initiated at multiple sites in the pre-S region and from a site upstream of the pre-S region. Chloramphenicol acetyltransferase assays indicate that DNA sequences within and upstream of the pre-S region contain promoter activities and that the "TATA" sequence-containing promoter and the internal promoter show similar levels of activities in CV-1 cells and several other cell lines tested.

Transformation of Streptomyces granaticolor with natural and recombinant plasmid vectors.

Protoplasts of Streptomyces granaticolor were found to be transformable by the broad-host-range plasmid pIJ350 but no transformants were detected when the narrow-host-range plasmid pIJ2 or the shuttle vector pPM66 (pIJ350--pBR322) isolated from E. coli cells were used. The onset of blue colour granaticin production by S. granaticolor cells was used as a marker to prepare protoplasts with a high transformation capacity. The presence of a restriction system is discussed.

Somatic cell hybrid selection with a transfectable dominant marker.

A recombinant plasmid vector, pSV2-neo, coding for resistance to neomycin and the related antibiotic G-418, was transfected into the mouse myeloma line X63-Ag8.653 by a modification of the protoplast fusion technique. The time interval required to obtain 10(6) G-418 resistant cells was 20 days and the efficiency was 10(-4)-10(-5), which represents a significant advantage over classical methods of selecting mutant cells bearing a dominant selection marker. To investigate the efficiency of this marker in somatic cell hybrid selection, these cells were fused to the human myeloma line U-266 and the hybrids were selected either in HAT + G-418 or HAT + ouabain. The pSV2-neo vector was as efficient as ouabain as a dominant marker with respect to the number of viable hybrid colonies selected and their levels of immunoglobulin secretion. The reciprocal experiment was also performed: HAT-sensitive, mutant U-266 cells were transfected with pSV2-neo, clones selected in G-418 and fused with X63-Ag8.653 cells, and hybrids selected in ouabain plus G-418, yielding HAT-sensitive hybrid "heteromyelomas" that were effective fusion partners with human B lymphocytes.

Construction and testing of mouse--human heteromyelomas for human monoclonal antibody production.

FU-266, a mutant human myeloma cell line sensitive to hypoxanthine/aminopterin/thymidine (HAT), was transfected by protoplast fusion with DNA of the recombinant plasmid vector pSV2-neoR, thus acquiring a dominant marker conferring resistance to the antibiotic G-418. One of the resultant neoR clones, E-1, was fused to irradiated (500 rads) or unirradiated cells of the HAT-sensitive, G-418-sensitive, nonproducer mouse myeloma line X63-Ag8.653. Hybrid clones were selected in G-418 plus ouabain, thus preserving their HAT sensitivity. Small numbers of human chromosomes were retained in all such hybrids, but most of them ceased secreting human myeloma (IgE(lambda). Selected hybrid clones were then tested as malignant fusion partners in a series of fusions with polyclonally activated human B lymphocytes and with antigen-primed human B lymphocytes, in some instances after transformation of the latter with Epstein-Barr virus. The yield of viable chimeric hybridomas has been consistently high, as has the proportion of hybridomas secreting human immunoglobulin molecules unpermuted with mouse or human myeloma heavy or light chains. Secretion by many subcloned hybridomas has been stable for over 6 months, and several antigen-specific human monoclonal antibodies have been generated. Thus these heteromyeloma cell lines appear to have significant advantages for human monoclonal antibody production.

Expression of hepatitis B virus surface antigen gene in cultured cells by using recombinant plasmid vectors.

By using a new host-vector system, expression of the gene coding for hepatitis B surface antigen has been studied. A subgenomic fragment of cloned hepatitis B viral DNA was inserted into the plasmid vector pSV010. Transfection of COS cells with the recombinant plasmid vector containing hepatitis sequences leads to the synthesis of hepatitis B surface antigen, which is released in the culture medium in the form of 22-nm particles similar to those found in the sera of hepatitis carriers.

Expression of Hepatitis B Virus Surface Antigen Gene in Cultured Cells by Using Recombinant Plasmid Vectors

By using a new host-vector system, expression of the gene coding for hepatitis B surface antigen has been studied. A subgenomic fragment of cloned hepatitis B viral DNA was inserted into the plasmid vector pSV010. Transfection of COS cells with the recombinant plasmid vector containing hepatitis sequences leads to the synthesis of hepatitis B surface antigen, which is released in the culture medium in the form of 22-nm particles similar to those found in the sera of hepatitis carriers.

Extrachromosomal genetics of Cephalosporium acremonium

In order to facilitate strain improvement by concerted breeding in the cephalosporin producing imperfect filamentous fungus Cephalo sporium acremonium, it is attempted to develop a eukaryotic vector for molecular cloning based on mitochondrial (mt) DNA.Fragments of mtDNA from C. acremonium were inserted into a yeast/bacterial hybrid plasmid (pDAM1) lacking a eukaryotic replicon. Six hybrid plasmids were obtained each containing a different mt fragment which together comprise about 60% of the total mtDNA. One of these recombinant plasmid vectors (pCP2) showed a high replication efficiency which is comparable to that of vectors containing yeast 2 μm DNA. This plasmid therefore fulfills the requirements for practical application.

Base insertion and deletion mutations induced in an Escherichia coli plasmid by benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide.

Mutations induced by (+/-)trans-benzo-[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BaP-diol epoxide-1) were selected by using a recombinant plasmid vector system designed for the study of transcription termination signals. The plasmid contains a transcription terminator positioned between a promotor signal and the Escherichia coli galactokinase structural gene (galK). By selections for the expression of galK (i.e., galK- to galK+), mutations are obtained in the terminator region that allow transcription from the promotor to read the galK gene. These mutations were characterized by direct DNA sequence of the terminator region. The DNA sequence changes caused by BaP-diol epoxide-1 were demonstrated for three different mutants. Two were found to be single-base-pair insertions of T.A into a cluster of consecutive T.A base pairs and the other change was a single-base-pair deletion of G.C from a cluster of consecutive G.C base pairs.