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Biomedical subjects

C F Garon

Publications and source records attributed to C F Garon.

At least 73 records · Page 4Linked to original sources
PubMed1981

The transforming sequences of avian myelocytomatosis virus (MC29).

Avian myelocytomatosis virus (MC29), a defective acute leukemia virus, has a broad oncogenic spectrum in vivo, and transforms fibroblasts and hematopoietic target cells in vitro, We have used recombinant DNA technology to isolate and characterize the sequences that are essential in the transformation process. Integrated MC29 proviral DNA was isolated from a library of recombinant phage containing DNA from the MC29-transformed nonproducer quail cell line Q5. The cloned DNA was analyzed by Southern blotting of restriction endonuclease digests and by electron microscopic visualization of R-loops formed between the cloned DNA and MC29 or helper virus RNA. It was found that the 9.2 kb cloned DNA insert contains approximately 4 kb of viral sequences and 5.2 kb of quail cellular sequences. The viral sequences contain all of the MC29 specific sequences and 5' helper related sequences as well as part of the envelope region. The size of the cloned EcoRI fragment is the same as that of the major band in EcoRI-cleaved Q5 DNA that hybridizes to viral sequences. Transfection of the cloned DNA into NIH 3T3 cells revealed that the MC29-specific sequences are functional in that they induce foci of transformed cells with high efficiency.

Avian Leukosis Virus↗
PubMed1981

Molecular cloning of avian myelocytomatosis virus (MC29) transforming sequences.

Avian myelocytomatosis virus (MC29), a defective acute leukemia virus, has a broad oncogenic spectrum in vivo and transforms fibroblasts and hematopoietic target cells in vitro. We have used recombinant DNA technology to isolate and to characterize the sequences that are essential in the transformation process. Integrated MC29 proviral DNA was isolated from a library of recombinant phage containing DNA from the MC29-transformed nonproducer quail cell line Q5. The cloned DNA was analyzed by Southern blotting of restriction endonuclease digests and by electron microscopic visualization of R loops formed between the cloned DNA and MC29 or helper virus RNA. It was found that the 9.2-kilobase cloned DNA insert contains approximately 4 kilobases of viral sequences and 5.2 kilobases of quail cellular sequences. The viral sequences contain all of the MC29-specific sequences and 5' helper-related sequences as well as part of the envelope region. The size of the cloned EcoRI fragment is the same as that of the major band in EcoRI-cleaved Q5 DNA that hybridizes to viral sequences. Transfection of the cloned DNA into NIH 3T3 cells revealed that the MC29-specific sequences are functional in that they induce foci of transformed cells with high efficiency.

Animals↗
PubMed1981

Recombinant bacteriophages containing the integrated transforming provirus of Gardner--Arnstein feline sarcoma virus.

The integrated DNA provirus of the Gardner-Arnstein (GA) strain of feline sarcoma virus (FeSV) was molecularly cloned in a bacteriophage lambda vector. The cloned DNA fragment is 14.4 kilobase pairs long and contains a 6.7-kilobase provirus flanked by cellular sequences derived from nonproductively transformed mink cells. Transfection of mouse NIH/3T3 cells with the cloned DNA fragment induced foci of transformation at efficiencies of 10(4) focus-forming units/pmol of sarcoma virus DNA. Restriction endonuclease mapping and heteroduplex analyses were used to compare the GA-FeSV provirus with that of Snyder-Theilen (ST)-FeSV, a second strain that contains homologous transformation-specific sequences (v-fes). Both viruses have the general structure 5'-gag-fes-env-c region-3', each having retained portions of the feline leukemia virus (FeLV) gag and env genes. In addition to segments shared by the two sarcoma viruses, GA-FeSV contains 1.7 kilobases of extra sequences not found in ST-FeSV. Of these, at least 400-500 base pairs located near the 5' end of v-fes encode a portion of the GA-FeSV polyprotein; the remaining 1.2 kilobases are derived from the FeLV env gene but do not appear to encode any detectable product related to the FeLV envelope glycoprotein. The close homology of the v-fes sequences shows that GA- and ST-FeSV were formed by recombination of FeLV with similar portions of a cat cellular gene (c-fes).

Bacteriophage lambda↗
PubMed1980

Inverted terminal repetition in vaccinia virus DNA encodes early mRNAs.

Vaccinia virus DNA contains a long inverted terminal repetition of MW approximately 6.8 x 10(6). A fragment of MW 6.3 X 10(6) from this repetition has been cloned in coliphage lambda and used to isolate RNA from virus-infected cells. Electron microscopy indicates that early RNAs are transcribed from the repeated sequence and cell-free translation shows that the RNAs code for polypeptides.

Bacteriophage lambda↗
PubMed1980

Molecular cloning of infectious integrated murine leukemia virus DNA from infected mouse cells.

The lack of an endonuclease EcoRI site in the AKR murine leukemia virus (MuLV) DNA genome was utilized to molecularly clone, in Charon 4A lambda DNA, integrated infectious AKR MuLV DNA isolated from productively infected mouse cells. Three lambda-mouse recombinants (clones 614, 621, and 623) were selected by virtue of their reactivity with AKR MuLV [32P]cDNA. Clones 614 and 623 contained the complete AKR MuLV DNA flanked by nonviral cell sequences of which no more than 100 base pairs beyond the viral DNA appear to be shared. DNAs from both clones 614 and 623 were highly infectious for mouse cells and yielded N-tropic ecotropic MuLV; the specific infectivity of the DNA and the titer of the derived virus was more than 10-fold higher with 623. Clone 621 contained only some viral DNA and was not infectious under similar conditions.

AKR murine leukemia virus↗
PubMed1980

Native ribonucleoprotein is an efficient transcriptional complex of avian myeloblastosis virus.

A native ribonucleoprotein (RNP) complex of avian myeloblastosis virus was prepared under conditions that gave optimal cDNA synthesis. The complex was an autonomous transcriptional unit capable of synthesizing DNA complementary to the RNA virus genome in the absence of exogenous reverse transcriptase (RNA-dependent DNA nucleotidyltransferase), genomic RNA, and primer. The RNA of the RNP complex cannot be translated in an in vitro cell-free translational system. The RNP contains intact viral RNA, the two subunits of the reverse transcriptase (beta and alpha), the p32 polypeptide resulting from the cleavage of the beta subunit into the alpha subunit, and p12. The principal polypeptide constituent of the RNP complex is the highly basic protein p12, which occurs at a molar ratio of 40:1 in relation to the beta subunit of the polymerase. When examined by the electron microscope, the RNP complex appears similar to the beaded structure of chromatin fiber. A significant portion of these molecules are circular, with headlike structures attached. The circular nature of the proviral DNA and the ability of the RNP complex to generate large intact cDNA copies from the natural primer end suggest that the 5' and 3' ends of the viral RNA are in proximity when in the RNP complex.

Avian Leukosis Virus↗
PubMed1980

Molecular cloning of the Harvey sarcoma virus circular DNA intermediates. II. Further structural analyses.

Three species of unintegrated supercoiled Harvey sarcoma virus DNA (6.6, 6.0, and 5.4 kilobase pairs) have been molecularly cloned from Harvey sarcoma virus-infected cells. On the basis of restriction enzyme analyses, the 6.6- and 6.0-kilobase pair viral DNAs contain two and one copies, respectively, of a 650-base pair DNA segment which contains sequences present at the 3' and 5' termini of the viral genome. R-loop structures formed between Moloney leukemia virus RNA and the cloned Harvey sarcoma virus DNA indicated that about 500 base pairs of the 650-base pair repeating segment was complementary to the 3' end of the viral RNA. During amplification in the Escherichia coli host, some recombinants containing the 6.6- or the 6.0-kilobase pair Harvey sarcoma virus DNA insert acquired or lost the complete 650-base pair DNA segment. These changes occurred in both recA+ and recA- E. coli.

Animals↗
PubMed1980

A cloned polyoma DNA fragment representing the 5' half of the early gene region is oncogenic.

The two polyoma DNA fragments generated by cleavage with BamHI and EcoRI were cloned in pBR322, and their oncogenic potential was tested in vivo and in vitro. Only recombinant plasmid DNA containing a polyoma DNA fragment which extends clockwise from 58 to 0 map units and include approximately the 5'-proximal half of the early gene region produced tumors in newborn hamsters and transformed rat embryo cells in tissue culture. Southern blotting analysis indicated that the entire 2.2-kilobase polyoma BamHI-EcoRI fragment was intact in both a tumor cell line and a cell line transformed in culture which we examined. The presence of polyoma middle and small T antigen in these lines was demonstrated by immunoprecipitation and tryptic peptide mapping. DNA from a recombinant plasmid containing a polyoma genome deleted between 90 and 4 map units failed to induce tumors or transform cells.

Animals↗
PubMed1979

Molecular cloning of polyoma virus DNA in Escherichia coli: lambda phage vector system.

The biological activity of recombinant phage and recombinant phage DNA containing monomeric or dimeric polyoma DNA inserts was examined in mice and cultured mouse cells. Recombinant preparations containing a single copy of viral DNA were invariably noninfectious; molecules containing a dimeric polyoma DNA insert were at least seven orders of magnitude less infectious than polyoma virions after parenteral inoculation. No infection was detected with any recombinant preparation after oral administration.

Animals↗
PubMed1979

Influence of the lactose plasmid on the metabolism of galactose by Streptococcus lactis.

Streptococcus lactis strain DR1251 was capable of growth on lactose and galactose with generation times, at 30 degrees C, of 42 and 52 min, respectively. Phosphoenolpyruvate-dependent phosphotransferase activity for lactose and galactose was induced during growth on either substrate. This activity had an apparent K(m) of 5 x 10(-5) M for lactose and 2 x 10(-2) M for galactose. beta-d-Phosphogalactoside galactohydrolase activity was synthesized constitutively by these cells. Strain DR1251 lost the ability to grow on lactose at a high frequency when incubated at 37 degrees C with glucose as the growth substrate. Loss of ability to metabolize lactose was accompanied by the loss of a 32-megadalton plasmid, pDR(1), and Lac(-) isolates did not revert to a Lac(+) phenotype. Lac(-) strains were able to grow on galactose but with a longer generation time. Galactose-grown Lac(-) strains were deficient in beta-d-phosphogalactoside galactohydrolase activity and phosphoenolpyruvate phosphotransferase activity for both lactose and galactose. There was also a shift from a predominantly homolactic to a heterolactic fermentation and a fivefold increase in galactokinase activity, relative to the Lac(+) parent strain grown on galactose. These results suggest that S. lactis strain DR1251 metabolizes galactose primarily via the tagatose-6-phosphate pathway, using a lactose phosphoenolpyruvate phosphotransferase activity to transport this substrate into the cell. Lac(-) derivatives of strain DR1251, deficient in the lactose phosphoenolpyruvate phosphotransferase activity, appeared to utilize galactose via the Leloir pathway.

Fermentation↗
PubMed1979

Molecular cloning of the Harvey sarcoma virus closed circular DNA intermediates: initial structural and biological characterization.

Supercoiled Harvey sarcoma virus (Ha-SV) DNA was extracted from newly infected cells by the Hirt procedure, enriched by preparative agarose gel electrophoresis, and digested with EcoRI, which cleaved the viral DNA at a unique site. The linearized Ha-SV DNA was then inserted into lambda gtWESlambda B at the EcoRI site and cloned in an approved EK2 host. Ha-SV DNA inserts from six independently derived recombinant clones have been analyzed by restriction endonuclease digestion, molecular hybridization, electron microscopy, and infectivity. Four of the Ha-SV DNA inserts were identical, contained about 6.0 kilobase pairs (kbp), and comigrated in agarose gels with the infectious, unintegrated, linear Ha-SV DNA. One insert was approximately 0.65 kbp smaller (5.35 kbp) and one was approximately 0.65 kpb larger (6.65 kpb) than the 6.0 kpb inserts. R-looping with Ha-SV RNA revealed that the small (5.35 kbp) insert contained one copy of the Ha-SV RNA. Preliminary restriction endonuclease digestion of the recombinant DNAs suggested that the middle-size inserts contained a 0.65-kbp tandem duplication of sequences present only one in the small-size insert; this duplication corresponded to the 0.65-kpb terminal duplication of the unintegrated linear Ha-SV DNA. The large-size insert apparently contained a tandem triplication of these terminally located sequences. DNA of all three sized inserts induced foci in NIH 3T3 cells, and focus-forming activity could be rescued from the transformed cells by superinfection with helper virus. Infectivity followed single-hit kinetics, suggesting that the foci were induced by a single molecule.

Animals↗
PubMed1978

Visualization of an inverted terminal repetition in vaccinia virus DNA.

An inverted terminal repetition was observed in DNA molecules extracted from vaccinia virus. The repeated sequence was visualized by (i) nicking the hairpin loops present of the ends of vaccinia virus DNA, (ii) separating the strands of DNA by alkali denaturation, (iii) allowing the single strands to self-anneal, and (iv) examining the DNA with an electron microscope. Single-stranded circular molecules, each of which contained a duplex projection (3.54 +/- 0.12 micron) representing the terminal repetition, readily formed. Similar size projections were also seen in heteroduplex structures formed by crosshybridization of the separated strands of the two terminal HindIII restriction fragments. Based on contour length measurements and the electrophoretic mobility of the isolated inverted terminal repetition, a molecular weight of approximately 6.9 X 10(6), equivalent to about 10,500 nucleotide base pairs, was estimated. Evidence was obtained from DNA-RNA hybridization studies that the terminal repetition is transcribed.

Base Sequence↗
PubMed1978

The relationship of SV40 replicating chromosomes to two forms of the non-replicating SV40.

SV40 replicating chromosomes were extracted from infected cells using a detergent free extraction method. This procedure also extracts 2 forms of the non-replicating chromosome, one of which corresponds to the well characterized 50-55S SV40 minichromosome. The other is a more compact structure which has a sedimentation coefficient of 80-85S. The replicating chromosomes sediment between the 2 conformations of the mature chromosome. Electron microscopy of the replicating chromosomes suggests an overall conformation that resembles the 50-55S form of the mature chromosome rather than that of the 80-85S structure. Nucleosomes are present on both sides of the replication forks. When the replicating chromosomes were incubated in an in vitro DNA synthesis assay all regions of the SV40 genome were synthesized and a significant fraction of the replicating chromosomes completed replication. The progeny chromosomes co-sedimented with the 50-55S chromosomes which were present prior to the incubation. The sedimentation coefficients and relative amounts of the two forms of the mature chromosome were unaffected by the incubation.

Centrifugation, Density Gradient↗
PubMed1977

Spiroplasmavirus citri 3: propagation, purification, proteins, and nucleic acid.

SVC3 is a short-tailed polyhedral virus particle morphologically detectable in many spiroplasmas. It was isolated from two different spiroplasmas (Spiroplasma citri and the suckling mouse cataract agent) by infecting lawns and broth culture of another strain of Spiroplasmavirus citri. Virions from either donor strain had a buoyant density of 1.26 grams per cubic centimeter (metrizamide) or 1.45 grams per cubic centimeter (cesium chloride), and contained five proteins and linear double-stranded DNA with a molecular weight of 14 X 10(6). Other spiroplasmaviruses have not been propagated, and the molecular weights of double-stranded DNA from other mycoplasma (Acholeplasma) viruses are unknown.

Bacteriophages↗