Search

When bacteriophage lambdapga18 DNA is transcribed in a purified in vitro system by E. coli RNA polymerase (nucleoside triphosphate: RNA nucleotidyl-transferase, EC 2.7.7.6), several major transcripts are synthesized. We have investigated transcriptional termination of one of these transcripts, the 4S, or "oop" RNA. Analysis by two-dimensional "fingerprinting" of T1 oligonucleotides reveals that transcription of the 4S RNA terminates at a specific site on the lambdapga18 DNA template, t-L with an efficiency of approximately 80%, i.e. 20% of transcripts are extended into larger RNAs. Addition of the E. coli protein rho to our transcription reactions has two effects: a) the efficiency of termination at the t-L site is increased to 100%; b) the number of 4S transcripts synthesized is increased by greater than 5-fold. Rho appears to stimulate 4S RNA synthesis by facilitating more rapid release of RNA polymerase from the t-L' termination site.

The efficiency of transfection and subsequent expression of recombinant DNA plasmids in monolayers of CV-1 monkey kidney cells was analyzed by immunoperoxidase and in situ hybridization with biotin-nucleotide-labeled DNA molecular probes. Two recombinant plasmids were used for transfection. Both contained the 3' long terminal repeat (LTR) of Rous sarcoma virus (RSV) as the transcriptional promoter, but two different coding sequences were employed [bacterial chloramphenicol acetyltransferase (pRSVcat) and mouse casein alpha (pRSVcsn alpha)]. In our experiments up to 25% of the transfected cells were positive for pRSVcat expression by indirect immunoperoxidase assay with affinity-purified, biotinylated anti-goat gamma-globulin after exposure to goat anti-chloramphenicol acetyltransferase antibody. In duplicate cultures, where pRSVcat expression was monitored by in situ hybridization signal that was restricted to the cytoplasm in positive cells was identified as pRSVcat RNA by its sensitivity to alkali. Although transfection of CV-1 cells with pRSVcsn-alpha did not result in immunologically detectable alpha casein, greater than 14% of the cells possessed cytoplasmic RNA concentrations detectable by in situ hybridization. These observations provide comparative information on in situ hybridization and immunoperoxidase techniques. They further indicate that in situ hybridization can be used to evaluate the effectiveness of transfection with recombinant expression vectors.

Normal resting T cells were stimulated through the alternate CD2 pathway. A CD3 mAb VIT3 completely blocked their proliferative response. The time interval for 50% inhibition lasted for 24 h after the onset of CD2 stimulation. Mitogen-activated cloned long term cultured T cells could also be stimulated via CD2. This proliferative response was again inhibitable by VIT3, indicating that CD3 regulates the CD2 pathway not only in resting cells, but also in lymphocytes actively involved in an Ir. T cells were further loaded with Quin2 and their free cytoplasmic Ca2+ levels were monitored in response to CD3 and CD2 stimulation. Antibodies directed against both surface R triggered a rapid elevation of Ca2+ levels. Both responses were abrogated when the cells had been treated overnight with VIT3. The free cytoplasmic Ca2+ levels of VIT3-pretreated cells, however, were not higher than those of control cells. These results point to a functional interaction between CD3 and CD2 possibly at the level of signal transducing proteins. Finally, cholera toxin was found to inhibit the Ca2+ response in Jurkat T cells. Both the CD3 and CD2 stimulation were sensitive to cholera toxin, indicating that a GTP-binding protein may be involved in signal transduction for both surface structures.

A sulfate-reducing bacterium has been isolated in pure culture from sheep rumen contents. Its properties agree in all respects tested with those ascribed to Desulfovibrio desulfuricans. The populations observed (about 10(8)/ml) are sufficient to account for published rates of ruminal sulfide production.

The Escherichia coli galactokinase gene (galK) was inserted into a modified early region transcription unit of simian virus 40 (SV40) contained on a bacterial plasmid. Introduction of this pSVK vector into monkey, mouse, and hamster cell lines by transfection resulted in efficient expression of the bacterial galK gene. This expression was shown to be dependent upon fusion of the galK gene to the early promoter of SV40 and did not appear to require SV40 splice signals. Moreover, expression in these cells could be obtained either transiently, 24--72 hr after transfection, or continuously, after stable transformation. In particular, pSVK-dependent galK expression was obtained in a hamster cell line genetically deficient in galactokinase activity. Expression of the bacterial enzyme was shown to complement the galactosemic defect of these cells, thereby allowing their selective survival and growth on galactose as the only carbon source. The ability to readily assay, select for, and potentially select against galK expression from pSVK and its derivatives should prove extremely useful in studying eukaryotic gene regulatory signals.

A method was developed to purify transiently transfected HeLa cells or African green monkey kidney CV-1 cells by magnetic affinity cell sorting. Monolayer cultures were transfected with mammalian expression vectors coding for either of two novel cell surface antigens, the Tac subunit of the human IL-2 receptor or vesicular stomatitis virus G protein. During the transient expression phase, cell populations were placed in suspension and mixed with monoclonal-antibody-coated magnetic particles in the presence of a sorting solution designed to minimize nonspecific cell/cell and cell/particle interactions. Transfected cells expressing the vector-encoded cell surface antigen were then isolated by application of a magnetic field. Reconstruction experiments indicated that IL-2 receptor-positive cells were bound about 100-fold more efficiently than receptor-negative cells. In transient transfection experiments, populations of greater than 90% antigen-positive cells were reproducibly obtained.

A variation of the classical DEAE-dextran method of gene transfer was developed for efficient transfection of HeLa cells with plasmid DNA. A brief exposure of the cells to medium containing DEAE-dextran was found to be sufficient for subsequent uptake of pRSVcat and to be superior to cocultivation of the cells with DEAE-dextran plus DNA. This sequential method of gene transfer is nontoxic and yielded up to 60% of HeLa cells positive for a surface protein encoded by the transfected sequence. The implications of this sequential transfection technique regarding the mechanisms of gene transfer are discussed.

We have found that double-stranded cDNA synthesized in extended reactions by avian myeloblastosis virus reverse transcriptase is suitable substrate for a variety of restriction endonucleases. Experiments in which rabbit reticulocyte mRNA was reverse-transcribed and restricted to generate beta-globin-specifihe nucleotide sequence of beta-globin mRNA. This method has been applied to study collagen mRNA synthesis in normal and Rous sarcoma virus (RSV)-transformed chick embryo fibroblasts. Characteristic sets of collagen cDNA restriction fragments were produced from RNA fractions rich in collagen message activity. These sets of cDNA fragments, generated by the restriction endonucleases Hae III and Hap II, provided a convenient marker for the presence of collagen mRNA sequences. Equal quantities of high molecular weight mRNA from chick embryo fibroblasts (CEF) and RSV-CEF were reverse-transcribed and the resulting cDNA was restricted. The relative yields of collagen cDNA fragments from such reactions strongly suggest that the decrease in functional collagen RNA following RSV-induced transformation of CEF represents a decrease in the copy number of collagen messenger sequences. The potential of this approach for the study of regulation in other systems is discussed.

Extinction of rat growth hormone (rGH) gene expression occurs in somatic cell hybrids of GH3 cells (rat pituitary adenoma) and L cells (mouse transformed fibroblast). We have suggested that this is due to transacting factors contributed directly or indirectly by the L cells. To study this in more detail, the cloned rGH gene was introduced into clones of these hybrid cells by microinjection or calcium phosphate transfection. GH peptides were detected 24 hr later in the microinjected cells by immunofluorescence. Production of rGH gene transcripts in the stably transformed hybrid cells was also detected by Northern hybridization. Although the overall patterns of transcripts differed from those present in GH3 cells, normal-sized rGH mRNA species were detected, whereas these had not been observed in previous studies in which the GH gene was transfected into L cells and other mouse fibroblasts. No more than one or two extra GH gene copies per cell were required to restore GH gene expression in the hybrids. Whereas GH gene expression in the hybrid cells transfected with the GH gene might reflect activation of the endogenous GH gene, this was not the case in at least one transfected hybrid cell line which had lost the endogenous gene. These results suggest: (i) the putative transactive repressors of GH gene expression in the hybrid cells do not recognize the transfected genes; and (ii) elements in the hybrid cells, unlike the case with L cells, allow for the production of normal-sized GH mRNA that can also produce GH peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

The sequence poly(dT-dG).poly(dC-dA) (TG-element) is a ubiquitous component of eucaryotic genomes and has the potential to adopt a left-handed DNA conformation (Z-DNA). In this report, we have tested the hypothesis that the TG-element can modulate gene expression. Human genomic DNA fragments (1 to 1.5 kilobases) containing a (dT-dG)n.(dC-dA)n tract (30, 40, or 50 base pairs) or chemically synthesized (dT-dG)n.(dC-dA)n fragments (50 to 130 base pairs) were inserted in the pSV2-cat (simian virus 40 enhancer plus) or pA10-cat (enhancer minus) expression vector plasmid. These constructs were transfected into CV-1 cells or HeLa cells, and their transcription was monitored by assaying chloramphenicol acetyltransferase activity. The results showed that pSV2-cat with the TG-element and pA10-cat with the TG-element synthesized more chloramphenicol acetyltransferase activity (2 to 10 times, depending on the location of the TG-element) than did parental pSV2-cat and pA10-cat DNAs, respectively. Furthermore, the TG-element appeared to have characteristics similar to those of viral enhancers: (i) the TG-element enhanced transcription from a distance, (ii) its closer location to the promoter was more effective, and (iii) its orientation was not crucial. However, its enhancer-like activity was much weaker than that of the simian virus 40 enhancer, and, unlike many viral enhancers, it was equally active in monkey and in human cells. These results suggest that the TG-element may influence the expression of cellular genes.

Tissue culture cells from several mammalian species, including three primate lines, were transfected with recombinant vectors carrying Escherichia coli xanthine-guanine phosphoribosyltransferase or Tn5 aminoglycoside phosphotransferase dominant selectable markers. Human HeLa and SV40-transformed xeroderma pigmentosum cells exhibited stable transformation frequencies of at least 10(-3) (0.1 percent). CV-1, an African green monkey kidney cell line, could be stably transformed with the exceptionally high frequency of 6 X 10(-2) (6 percent).

Transformation of chick embryo fibroblasts by Rous sarcoma virus results in decreased amounts of a major cell surface protein and of collagen. To determine the mechanism accounting for the decreased production of these proteins, we have measured the relative amounts of functional mRNAs for these and other transformation-sensitive proteins. Total cellular RNAs extracted from normal cells and from cells transformed by the Schmidt-Ruppin strain of Rous sarcoma virus were translated in a cell-free system derived from wheat germ. Analysis of the in vitro translation products of RNAs from normal and transformed chick embryo fibroblasts shows a 5-fold reduction in the translatable mRNA for cell surface protein and a 10-fold reduction in translatable mRNA for two collagen precursors. In addition, increases in functional mRNA are observed for myosin and for two membrane polypeptides with molecular weights of 95,000 and 78,000; the latter two proteins increase on transformation, but the increases are in large part secondary to the depletion of glucose from the medium of transformed cells. Our data suggest that some of the major cellular changes induced by oncogenic viruses are due to changes in the activity of specific cellular genes.

We have used the lambda SV2 system [Howard and Gottesman. In Gluzman (Ed.), Eukaryotic Viral Vectors. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982, pp. 211-216; in Inouye, M. (Ed.) Experimental Manipulations of Gene Expression. Academic Press, New York, 1983, pp. 137-153] to reconstitute the Salmonella typhimurium his operon from overlapping fragments. lambda SV2 can be propagated as an autonomously replicating plasmid or as a prophage integrated in the Escherichia coli chromosome at the lambda attachment site; our reconstitution was accomplished in the integrated state. We first inserted a portion of the his operon into lambda SV2 and integrated the resulting plasmid by site-specific recombination into the E. coli chromosome. This was achieved by brief induction of a resident prophage. The lysogen was then transformed with DNA from a lambda SV2 clone carrying the remainder of the his operon on an overlapping DNA fragment. The second plasmid was forced to integrate into the first by homologous recombination. When this recombination occurs at the his overlap, a lysogen carrying two lambda SV2 prophages is produced. One prophage carries the entire his operon and the other carries the his overlap region. The latter is removed by site-specific recombination, permitting further contiguous sequences to be sequentially added to the remaining prophage. This method should be applicable for the reconstitution and maintenance of large genes or gene clusters in the E. coli genome.