A survey of vectors for regulating expression of cloned DNA in E. coli.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Biomedical subjects
D T Denhardt
Publications and source records attributed to D T Denhardt.
Specialized plasmid vectors for cloning cDNA.
Explore the source record for details and available documents.
Cloning, Molecular↗
Mechanism of replication of bacteriophage phi X174. XXII. Site-specific mutagenesis of the A* gene reveals that A* protein is not essential for phi X174 DNA replication.
The A and A* proteins of phage phi X174 are encoded in the same reading frame in the viral genome; the smaller A protein is the result of a translational start signal with the A gene. To differentiate their respective functions, oligonucleotide-directed site-specific mutagenesis was used to change the ATG start codon of the phi X 174 A* gene, previously cloned into pCQV2 under lambda repressor control, into a TAG stop codon. The altered A gene was then inserted back into phi X replicative form DNA to produce an amber mutant, phi XamA*. Two different Escherichia coli amber suppressor strains infected with this mutant produced viable progeny phage with only a slight reduction in yield. In Su+ cells infected with phi XamA*, phi X gene A protein, altered at one amino acid, was synthesized at normal levels; A* protein was not detectable. These observations indicate that the A* protein increases the replicative efficiency of the phage, perhaps by shutting down host DNA replication, but is not required for replication of phi X174 DNA or the packaging of the viral strand under the conditions tested.
Bacteriophage phi X 174↗
Evidence that post-transcriptional changes in the expression of mitogen regulated protein accompany immortalization of mouse cells.
Primary mouse embryo fibroblasts passed in culture go through a period of declining growth rate and then after a period of minimal proliferation begin to grow again, generating, under appropriate conditions, an immortal 3T3 line. The 3T3 cells, but not the primary mouse embryo fibroblasts, are able to synthesize and to secrete mitogen regulated protein (MRP, also known as proliferin). We report here that the level at which the change in gene expression occurs during this spontaneous immortalization process is post-transcriptional, reflecting a change in either RNA processing or transcript stability. To our knowledge, this is the first report of an alteration at the post-transcriptional level of gene expression that accompanies immortalization.
Animals↗
Escherichia coli rep gene: sequence of the gene, the encoded helicase, and its homology with uvrD.
The sequence of a 2.67-kilobase section of the Escherichia coli chromosome that contains the rep gene has been determined. This gene codes for a protein of predicted Mr 72,800, a DNA helicase, which is also a single-stranded DNA-dependent ATPase. The sequenced region contains an open reading frame of the correct length and orientation to encode the Rep protein. A secondary structure for the protein can be formulated from the amino acid sequence. We have compared both the primary and the secondary structures of Rep with other proteins and find the greatest homology between Rep and E. coli helicase II, the product of the uvrD gene.
Adenosine Triphosphatases↗
Molecular cloning of a tumor promoter-inducible mRNA found in JB6 mouse epidermal cells: induction is stable at high, but not at low, cell densities.
From the mouse JB6 epidermal cell line C122 we have isolated a cDNA clone representing a 1.6-kilobase mRNA, called 2ar, that exhibits a biphasic induction in response to 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The first phase of induction in subconfluent cells is transient, peaking at 6 h after the addition of TPA and returning to noninduced levels by 24 h. When the cells reach plateau density, in the continued presence of TPA, this mRNA is reinduced and remains so upon continued exposure to the tumor promoter. Serum and certain growth factors also induce 2ar mRNA in serum-deprived quiescent fibroblasts. In vitro nuclear "run-on" transcription experiments indicate that the induction of 2ar mRNA is controlled at the transcriptional level.
Animals↗
The Escherichia coli rep mutation. X. Consequences of increased and decreased Rep protein levels.
Recombinant DNA techniques were used to study various aspects of rep gene function in Escherichia coli. In order to enhance expression of the Rep protein, the rep gene was cloned into the vector pKC30 under the control of the lambda pL promoter. By trimming away a portion of the DNA sequence immediately upstream of the translational start site of rep, we were able to obtain very high levels of Rep protein upon induction. Cells carrying such plasmids showed no ill effects from the high concentration of the protein. To ascertain the consequence of the absence of Rep protein on the cell, the chromosomal copy of the gene was deleted using a homologous recombination technique. The viability of E. coli strains completely lacking the rep gene proves that the Rep function is not essential, at least in wild-type cells under laboratory conditions. We confirmed that in the absence of Rep function there is an increase in the average number of growing forks in exponentially growing cells; augmentation of Rep protein levels above normal, however, did not detectably decrease the number of growing forks.
Adenosine Triphosphatases↗
Cysteine proteinase cathepsin L expression correlates closely with the metastatic potential of H-ras-transformed murine fibroblasts.
A set of H-ras-transfected mouse C3H 10T1/2 cell lines that vary in their experimental and spontaneous metastatic potential has been analysed for the level of expression of the cysteine proteinase cathepsin L, also known as the major excreted protein (MEP). We found a good positive correlation between the extent of ras expression, the metastatic potential, and the amount of the secreted protease in nine independently isolated lines. There was an increased abundance of the MEP mRNA in cells with increased ras expression, suggesting that this gene is under ras control. Expression of glyceraldehyde phosphate dehydrogenase mRNA was also elevated.
Animals↗
A growth-responsive gene (16C8) in normal mouse fibroblasts homologous to a human collagenase inhibitor with erythroid-potentiating activity: evidence for inducible and constitutive transcripts.
We present the DNA sequence of an essentially full-length cDNA clone of 16C8, a growth factor-inducible gene isolated from a mouse embryo fibroblast cDNA library. The 0.9-kb mRNA encodes an Mr 22,500 protein that has substantial homology to a human protein with the reported abilities to potentiate erythroid differentiation and to inhibit collagenases and other tissue metalloproteinases. The N-terminus of the predicted protein has a hydrophobic nature characteristic of secreted proteins, and two potential sites for N-linked glycosylation are present. The cytoplasmic concentration of 16C8 mRNA is maximal in mid G1 at about 6 h after serum stimulation of quiescent fibroblasts. Northern blot analysis showed a progressive reduction in the size of the induced 16C8 transcripts with increasing time after serum stimulation. This was shown to be due to the reduction in length of the poly(A) tails. S1 analysis of the 5' portion of the mRNA revealed the presence of three different species of transcript, only one of which was inducible.
Amino Acid Sequence↗
Gene expression during the mammalian cell cycle.
Explore the source record for details and available documents.
Animals↗
Close relationship of the major excreted protein of transformed murine fibroblasts to thiol-dependent cathepsins.
Complementary DNA clones corresponding to 638 nucleotides of the messenger RNA encoding the major portion of murine major excreted protein have been isolated and sequenced. The amino acid sequence of a part of the murine major excreted protein deduced from the DNA sequence reveals substantial and significant homology with the cysteine proteases actinidin, rat cathepsin H, and papain. Since the amount of murine major excreted protein secreted by cultured cells is often enhanced by transformation, it is implicated in oncogenic phenomena and may play a role in the metastatic process by virtue of its proteolytic activity.
Amino Acid Sequence↗
A study of mitochondrial and nuclear transcription with cloned cDNA probes. Changes in the relative abundance of mitochondrial transcripts after stimulation of quiescent mouse fibroblasts.
From a cDNA library constructed in pBR322 we have isolated and studied a set of clones corresponding to mRNAs whose abundance changes when serum-deprived murine fibroblasts are stimulated to enter the cell cycle. A subset of these clones was derived from mRNA species whose abundance decreased during the G1 period following serum stimulation; all but one of these clones turned out to be clones of mitochondrial poly(A)mRNAs. There was no detectable change in the rate of transcription of the mitochondrial genome compared with the nuclear genome, and the lengths of the poly(A) segments on both mRNA species did not change significantly after serum stimulation. We conclude that the apparent decline in the relative abundance of the mitochondrial mRNAs is the result of a relative increase in the processing and/or transport of nuclear mRNA.
Animals↗
Vectors for the direct selection of cDNA clones corresponding to mammalian cell mRNA of low abundance.
We have constructed two cDNA cloning vectors which (1) carry the intergenic region of phage f1 and (2) permit efficient cloning (by the Okayama-Berg procedure) of full-length copies of mammalian mRNA in either orientation. Infection of cells harboring these vectors with f1 phage results in the encapsidation of single-stranded (ss) plasmid DNA carrying either the sense or the anti-sense sequence of the cDNA inserts. The complementary nature of the cDNA inserts in two such cDNA libraries facilitates preparative hybridization procedures. These vectors have general applicability to any eukaryotic system where changes in the abundance of mRNA transcripts are to be measured and the corresponding cDNA clones isolated.
Cloning, Molecular↗
A pBR322-derived vector for cloning blunt-ended cDNA: its use to detect molecular clones of low-abundance mRNAs.
In place of the unique Pst I site in pBR322, we have engineered by GC tailing a unique Sma I site bracketed by Pst I sites. The resulting vector, pDE61, and an improved derivative with greater symmetry around the Sma I site, pDE613, have been used to clone blunt-ended duplex cDNA molecules in Escherichia coli in an efficient manner (5 X 10(5) clones from 1 microgram of double-stranded cDNA). When DNA is cloned into the Sma I site, the ability of both vectors to confer ampicillin resistance is lost. Evidence suggests that functional beta-lactamase is made only after the GC-rich sequence containing the Sma I site is deleted: an insert in the Sma I site prevents this. Libraries in either vector, with single or multiple inserts, can be used to generate amplified amounts of cloned heterogeneous cDNA for screening other "target" libraries in a non-homologous vector (e.g., a Bacillus subtilis vector) for cDNA clones of low-abundance mRNAs. Species as infrequent as 0.003% can be readily detected by colony hybridization.
Cloning, Molecular↗
Escherichia coli rep gene: identification of the promoter and N terminus of the rep protein.
The functional Escherichia coli rep gene, which encodes the Mr 67,000 Rep helicase, has been localized within a 2.55-kilobase sequence. Its regulatory region has been characterized by the use of rep-lacZ fusions. The direction of transcription of the rep gene is clockwise on the E. coli chromosome, as are the nearby ilvC and rho genes. The sequence of the rep control region was determined, and putative regulatory sequences were identified; no evidence for autoregulation of expression was obtained. Transcription of the gene was not enhanced during the SOS response. The location of the promoter and the beginning of the protein were confirmed by S1 nuclease mapping of the 5' end of rep mRNA and determination of the NH2-terminal sequence of the rep protein.
Amino Acid Sequence↗
Expression of the cloned bacteriophage phi X174 A* gene in Escherichia coli inhibits DNA replication and cell division.
The A* gene of bacteriophage phi X174 has been cloned into the inducible expression vector pCQV2 under conditions allowing its lethal action to be controlled by the lambda cI857 repressor. Upon induction of expression, DNA synthesis in Escherichia coli carrying the recombinant plasmid is severely inhibited; however, these same cells permit beta-galactosidase induction at a rate similar to that observed in control cells at the inducing (for A*) temperature. Cells in which A* is expressed form filaments and produce more RecA protein, indicating at least a partial induction of the SOS response; however, there is no evidence of damage to the bacterial chromosome. It appears that the A* protein has as one function the inhibition of cell division and DNA replication but not transcription or protein synthesis during phage infection.
Bacteriophage phi X 174↗
Transcriptional regulation of two serum-induced RNAs in mouse fibroblasts: equivalence of one species to B2 repetitive elements.
We obtained eight cDNA clones that define five genes whose expression (appearance of transcripts in the cytoplasm) is enhanced when quiescent mouse fibroblasts are stimulated with serum to divide. Two of these clones (designated 49C8 and 16C8) correspond to RNA species that are present in the cytoplasm of quiescent cells at very low levels. After serum stimulation, the level of 16C8 mRNA rose more rapidly than that of 49C8 RNA, reaching a maximum around 6 to 12 h. The data suggest that 49C8 and 16C8 RNAs are induced as a result of independent stimuli. Either fibroblast growth factor or 12-tetradecanoylphorbol-13-acetate alone could induce 16C8 expression almost as effectively as serum; in contrast, 49C8 was not efficiently induced by epidermal growth factor, fibroblast growth factor, insulin, or 12-tetradecanoylphorbol-13-acetate. Inhibitors of transcription and translation diminished the induction of 16C8, while 49C8 expression was sensitive to actinomycin D but not cycloheximide or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole. In vitro transcription experiments with isolated nuclei revealed a peak in transcriptional activity of the 16C8 gene at around 3 h after serum stimulation. Sequence analysis of the 49C8 cDNA clone showed greater than 90% homology of a large portion to a consensus rodent B2 repetitive element.
Animals↗
Characterization of a cDNA clone encoding murine mitogen-regulated protein: regulation of mRNA levels in mortal and immortal cell lines.
Mitogen-regulated protein (MRP) is secreted by certain immortal murine cell lines (Swiss 3T3, BNL) stimulated with serum or particular growth factors. We have identified a cDNA clone that encodes part of the protein and have confirmed that MRP is closely related to, if not identical to, the prolactin-related protein designated proliferin. MRP is not produced by primary mouse embryo fibroblasts to nearly the same extent as it is produced by many immortal or transformed lines. Control of expression of this protein by growth factors is achieved both by regulating the extent of transcription and by regulating the processing of the protein.
Animals↗