scholarly journals Threonine synthesis from homoserine as a selectable marker in mammalian cells

1994 ◽  
Vol 299 (3) ◽  
pp. 637-644 ◽  
Author(s):  
W D Rees ◽  
S D Grant ◽  
S M Hay ◽  
K M Saqib
Keyword(s):  
Escherichia Coli ◽  
Active Region ◽  
Cell Lines ◽  
Hela Cells ◽  
Mammalian Cells ◽  
Selectable Marker ◽  
Threonine Synthase ◽  
Homoserine Kinase ◽  
Mouse Cells ◽  
High Concentrations

The plasmid pSVthrBC expresses the Escherichia coli thrB (homoserine kinase) and thrC (threonine synthase) genes in mouse cells and enables them to synthesize threonine from homoserine. After transfection with pSVthrBC and culture in medium containing homoserine, only cells that have incorporated pSVthrBC survive. Homoserine at concentrations greater than 1 mM is toxic to mammalian cells. Mouse cells selected from medium containing 5 mM homoserine had incorporated 20-100 copies of the plasmid per cell and had homoserine kinase activities of 0.001-0.012 nmol/min per mg of protein per copy. Cells selected from medium containing 10 mM homoserine had incorporated one or two copies of the plasmid per cell and had homoserine kinase activities of 0.06-0.39 nmol/min per mg of protein per copy. By using high concentrations of homoserine, it is possible to use pSVthrBC to select and isolate cell lines that have one or two copies of the plasmid incorporated into an active region of chromatin. CHO and HeLa cells have also been successfully transfected with pSVthrBC. COS-7 cells are naturally resistant to homoserine as they are able to metabolize homoserine.

Attachment of Salmonella to mammalian cells in vitro

1983 ◽  
Vol 29 (12) ◽  
pp. 1731-1735 ◽  
Author(s):  
Clifford S. Mintz ◽  
Dean O. Cliver ◽  
R. H. Deibel
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
Culture Cell ◽  
Intestinal Cell ◽  
Tissue Culture Cell ◽  
Bacterial Cells ◽  
Intestinal Cell Line ◽  
Hela Cell Lines ◽  
High Concentrations

The attachment of Salmonella typhimurium strain PHL67342 to several mammalian tissue culture cell lines was investigated. Strain PHL67342 failed to attach in significant numbers to the Buffalo green monkey (BGM), swine testicular (ST), and HeLa cell lines. Significant attachment was observed with the Henle intestinal cell line. Log-phase cells of strain PHL67342 attached in greatest numbers to the Henle cells after 45 min of incubation at 37 °C. Attachment to the Henle cells was not affected by D-mannose or D-galactose, but was markedly inhibited by high concentrations of alpha-methyl-D-mannoside. Also, Salmonella lipopolysaccharide had no effect on the attachment of strain PHL67342 to the Henle cells. Fimbriae were not detected on the bacterial cells used in the adherence experiments. These results suggest that some bacterial factor(s) other than fimbriae and lipopolysaccharide mediate the attachment of strain PHL67342 to the Henle cells.

scholarly journals Plasmids containing mouse rDNA do not recombine with cellular ribosomal genes when introduced into cultured mouse cells.

1984 ◽  
Vol 4 (4) ◽  
pp. 576-582 ◽  
Author(s):  
R E Steele ◽  
A H Bakken ◽  
R H Reeder
Keyword(s):  
Homologous Recombination ◽  
Host Cell ◽  
Thymidine Kinase ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Gene Clusters ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Kinase Gene ◽  
Mouse Cells

We have examined the fate of plasmids containing a segment of a mouse rDNA repeat after they were introduced by transfection into cultured mouse cells. In addition to the rDNA segment, the plasmids contained the thymidine kinase gene from herpes simplex virus 1 to allow for selection of the plasmid after transfection into thymidine kinase-deficient mouse cells. Thus far, no cases of homologous recombination between transfected plasmid DNAs and host cell sequences have been documented. We reasoned that the high repetition frequency of the rRNA genes in the mouse genome (200 copies per diploid cell) might create a favorable situation for obtaining homologous recombination events between the plasmids containing rDNA and host cell rDNA sequences. The plasmids were introduced into cells in both the presence and the absence of carrier DNA and both as covalently closed circles and linear molecules. The sites of plasmid integration in the genomes of various cell lines were examined by DNA restriction digests and hybridization, molecular cloning, and nuclear fractionation. In the seven cell lines examined, there was no evidence that the plasmids had integrated into the rRNA gene clusters of the cell. Thus, the apparent absence of site-specific integration of cloned DNAs introduced into mammalian cells does not appear to be due simply to the small target presented by most host cell sequences.

scholarly journals The expression of Escherichia coli threonine synthase and the production of threonine from homoserine in mouse 3T3 cells

1993 ◽  
Vol 291 (1) ◽  
pp. 315-322 ◽  
Author(s):  
W D Rees ◽  
S M Hay
Keyword(s):  
Escherichia Coli ◽  
Protein A ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Eukaryotic Expression ◽  
Cell Protein ◽  
3T3 Cells ◽  
Threonine Synthase ◽  
Homoserine Kinase ◽  
A Cell

We have subcloned the coding sequence for the Escherichia coli threonine synthase gene into a eukaryotic expression vector based on the simian-virus-40 early promoter. When mouse 3T3 cells which already expressed homoserine kinase were transfected with the new plasmid, the cells were able to incorporate radioactivity from [14C]homoserine into their cell proteins. Stable cell lines were established by co-transfecting 3T3 cells with the plasmid coding for threonine synthase and another coding for homoserine kinase and G-418 (Geneticin) resistance. Cells were selected for G-418 resistance and then screened for an ability to synthesize threonine from homoserine and incorporate it into the cell protein. A cell line which expressed both the homoserine kinase and threonine synthase genes was capable of growth in a threonine-deficient medium containing homoserine.

scholarly journals Selection against expression of the Escherichia coli gene gpt in hprt+ mouse teratocarcinoma and hybrid cells.

1987 ◽  
Vol 7 (11) ◽  
pp. 4139-4141 ◽  
Author(s):  
C Besnard ◽  
E Monthioux ◽  
J Jami
Keyword(s):  
Escherichia Coli ◽  
Positive Selection ◽  
Mammalian Cells ◽  
Selectable Marker ◽  
Hybrid Cells ◽  
Genetic Studies ◽  
Dominant Selectable Marker ◽  
Hypoxanthine Guanine Phosphoribosyltransferase ◽  
Mouse Teratocarcinoma

Thioxanthine is toxic for mammalian cells transformed by the dominant selectable marker gpt. It allowed us to select, in the presence of the endogenous hypoxanthine-guanine phosphoribosyltransferase gene, mutants that did not express gpt any more and also hybrid cells that had lost the chromosome carrying it. The gpt marker is thus dominant in negative as well as in positive selection, which makes it potentially very useful for genetic studies of mammalian cells.

Selection against expression of the Escherichia coli gene gpt in hprt+ mouse teratocarcinoma and hybrid cells

1987 ◽  
Vol 7 (11) ◽  
pp. 4139-4141
Author(s):  
C Besnard ◽  
E Monthioux ◽  
J Jami
Keyword(s):  
Escherichia Coli ◽  
Positive Selection ◽  
Mammalian Cells ◽  
Selectable Marker ◽  
Hybrid Cells ◽  
Genetic Studies ◽  
Dominant Selectable Marker ◽  
Hypoxanthine Guanine Phosphoribosyltransferase ◽  
Mouse Teratocarcinoma

Thioxanthine is toxic for mammalian cells transformed by the dominant selectable marker gpt. It allowed us to select, in the presence of the endogenous hypoxanthine-guanine phosphoribosyltransferase gene, mutants that did not express gpt any more and also hybrid cells that had lost the chromosome carrying it. The gpt marker is thus dominant in negative as well as in positive selection, which makes it potentially very useful for genetic studies of mammalian cells.

scholarly journals Mechanical Fractionation Reveals Structural Requirements for Enteropathogenic Escherichia coli Tir Insertion into Host Membranes

2000 ◽  
Vol 68 (7) ◽  
pp. 4344-4348 ◽  
Author(s):  
Annick Gauthier ◽  
Myriam de Grado ◽  
B. Brett Finlay
Keyword(s):  
Escherichia Coli ◽  
Host Cell ◽  
Hela Cells ◽  
Protein Delivery ◽  
Mammalian Cells ◽  
Host Cells ◽  
Host Cell Membrane ◽  
Bacterial Proteins ◽  
Host Cell Membranes ◽  
Triton X

ABSTRACT Enteropathogenic Escherichia coli (EPEC) inserts its receptor for intimate adherence (Tir) into host cell membranes by using a type III secretion system. Detergents are frequently used to fractionate infected host cells to investigate bacterial protein delivery into mammalian cells. In this study, we found that the Triton X-100-soluble membrane fraction from EPEC-infected HeLa cells was contaminated with bacterial proteins. We therefore applied a mechanical method of cell lysis and ultracentrifugation to fractionate infected HeLa cells to investigate the biology and biochemistry of Tir delivery and translocation. This method demonstrates that the translocation of Tir into the host cell membrane requires its transmembrane domains, but not tyrosine phosphorylation or binding to Tir's ligand, intimin.

scholarly journals Escherichia coli aspartate transcarbamylase: a novel marker for studies of gene amplification and expression in mammalian cells.

1986 ◽  
Vol 6 (9) ◽  
pp. 3050-3058 ◽  
Author(s):  
J C Ruiz ◽  
G M Wahl
Keyword(s):  
Escherichia Coli ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Expression Vectors ◽  
Aspartate Transcarbamylase ◽  
Multifunctional Protein ◽  
High Concentrations ◽  
Atcase Activity ◽  
Ovary Cell Line

Eucaryotic expression vectors containing the Escherichia coli pyrB gene (pyrB encodes the catalytic subunit of aspartate transcarbamylase [ATCase]) and the Tn5 phosphotransferase gene (G418 resistance module) were transfected into a mutant Chinese hamster ovary cell line possessing a CAD multifunctional protein lacking ATCase activity. G418-resistant transformants were isolated and analyzed for ATCase activity, the ability to complement the CAD ATCase defect, and the ability to resist high concentrations of the ATCase inhibitor N-(phosphonacetyl)-L-aspartate (PALA) by amplifying the donated pyrB gene sequences. We report that bacterial ATCase is expressed in these lines, that it complements the CAD ATCase defect in trans, and that its amplification engenders PALA resistance. In addition, we derived rapid and sensitive assay conditions which enable the determination of bacterial ATCase enzyme activity in the presence of mammalian ATCase.

Plasmids containing mouse rDNA do not recombine with cellular ribosomal genes when introduced into cultured mouse cells

1984 ◽  
Vol 4 (4) ◽  
pp. 576-582
Author(s):  
R E Steele ◽  
A H Bakken ◽  
R H Reeder
Keyword(s):  
Homologous Recombination ◽  
Host Cell ◽  
Thymidine Kinase ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Gene Clusters ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Kinase Gene ◽  
Mouse Cells

We have examined the fate of plasmids containing a segment of a mouse rDNA repeat after they were introduced by transfection into cultured mouse cells. In addition to the rDNA segment, the plasmids contained the thymidine kinase gene from herpes simplex virus 1 to allow for selection of the plasmid after transfection into thymidine kinase-deficient mouse cells. Thus far, no cases of homologous recombination between transfected plasmid DNAs and host cell sequences have been documented. We reasoned that the high repetition frequency of the rRNA genes in the mouse genome (200 copies per diploid cell) might create a favorable situation for obtaining homologous recombination events between the plasmids containing rDNA and host cell rDNA sequences. The plasmids were introduced into cells in both the presence and the absence of carrier DNA and both as covalently closed circles and linear molecules. The sites of plasmid integration in the genomes of various cell lines were examined by DNA restriction digests and hybridization, molecular cloning, and nuclear fractionation. In the seven cell lines examined, there was no evidence that the plasmids had integrated into the rRNA gene clusters of the cell. Thus, the apparent absence of site-specific integration of cloned DNAs introduced into mammalian cells does not appear to be due simply to the small target presented by most host cell sequences.

scholarly journals Adhesion of Escherichia coli to HeLa Cells Mediated byTrypanosoma cruzi Surface Glycoprotein-Derived Peptides Inserted in the Outer Membrane Protein LamB

1999 ◽  
Vol 67 (9) ◽  
pp. 4908-4911 ◽  
Author(s):  
Cátia M. Pereira ◽  
Sílvio Favoreto ◽  
José Franco da Silveira ◽  
Nobuko Yoshida ◽  
Beatriz A. Castilho
Keyword(s):  
Escherichia Coli ◽  
Membrane Protein ◽  
Cell Surface ◽  
Hela Cells ◽  
Outer Membrane Protein ◽  
Fusion Proteins ◽  
Mammalian Cells ◽  
Host Cells ◽  
Surface Glycoprotein ◽  
Parasite Invasion

ABSTRACT Peptides derived from the surface glycoprotein gp82 ofTrypanosoma cruzi, previously implicated in the parasite’s invasion of host cells, were expressed as fusions to the protein LamB of Escherichia coli in a region known to be exposed on the cell surface. Bacteria expressing these proteins adhered to HeLa cells in a manner that mimics the pattern of parasite invasion of mammalian cells. Purified LamB fusion proteins were shown to bind to HeLa cells and to inhibit infection by T. cruzi, supporting the notion that these gp82-derived peptides can mediate interaction of the parasite with its host.

NATURAL CYTOTOXIC ANTIBODIES IN HUMAN BLOOD SERA WHICH REACT WITH MAMMALIAN CELLS AND BACTERIA: II. EFFECT OF HEATED HUMAN SERUM ON MICROORGANISMS

1963 ◽  
Vol 9 (2) ◽  
pp. 155-162 ◽  
Author(s):  
S. J. Webb ◽  
S. Fedoroff
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Human Serum ◽  
Human Blood ◽  
Mammalian Cells ◽  
Antibacterial Effect ◽  
Mycobacterium Phlei ◽  
Receptor Sites ◽  
Human Sera ◽  
Mouse Cells

It was found that certain bacterial and mammalian cells have similar receptor sites which react with natural antibodies present in human blood sera. Heated human sera which contain these antibodies are bacteriostatic to Escherichia coli and Mycobacterium phlei and bactericidal to Bacillus subtilis. This antibacterial effect is eliminated from heated human serum when the serum is treated with mouse cells.

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