Elimination of the Sarcoma Genome from Murine Sarcoma Virus Transformed Cat Cells

Our previous studies have shown that, while a persistent but potentially reversible suppression of the tumorous state appears to be a characteristic feature of the vegetative phase of teratoma shoot growth in the crown gafl disease of plants, a recovery from that state occurs during the reproductive phase. An analysis has now been made of the reproductive process in an attempt to define the precise stage at which recovery occurs. The results of this analysis have shown that diploid somatic cells of teratoma-derived flower parts such as those found in petals and filaments are inherently neoplastic. On the other hand, haploid cells of plants grown from anthers of the same flowers and diploid cells of F1 generation plants grown from teratoma-derived seed have, by generally accepted criteria, recovered from the tumorous state. These findings have been interpreted to mean that the loss of neoplastic properties occurs in crown gall teratoma cells during meiosis rather than during fertilization or later stages of the reproductive process.An analysis of more than 2000 teratoma-derived tumor shoots has shown, moreover, that a recovery from the tumorous state may also occur, although apparently as a very rare event, during the vegetative phase of teratoma shoot growth.Cloned lines of crown gall teratoma tissues of tobacco (Nicotiana tabacum L.) are characterized by a~capacity to organize tumor shoots when grown on a basic culture medium. When such teratoma-derived shoots are isolated and grafted at cambial level into appropriate morphologically distinct stock plants a broad spectrum of responses may be obtained (1). These responses range at the one extreme to a breakdown of the implanted shoot with a resulting development of typical disorganized teratomatous growths, while at the other extreme in this spectrum of examples are found shoots that develop quite normally, some of which ultimately flower and set viable seed. The leaves that develop from such teratoma shoots are composed of all of the specialized cell types found in normal leaves and they appear by all generally accepted criteria to be normal. It has been found, however, that when tissues are isolated from the teratoma-derived leaves and planted on a basic culture medium they again assume their neoplastic properties. formed by the T37 strain of the crown gall bacterium Agrobacterium tumefaciens. Procedures used in these studies for grafting teratoma shoots and for culturing cells have previously been described elsewhere (1). Briefly, shoots produced by teratoma tissue grown in liquid culture were grafted at cambial level to detopped stems of healthy tobacco plants of the morphologically distinguishable Turkish cultivar. Pieces of leaf, flower, and pith tissue derived from grafted teratoma and normal shoots were cultured on the agar-containing basic medium of Linsmaier and Skoog (2). The growth hormones naphthalene acetic acid (NAA) (0.5 mg/liter) and kinetin (6-furfurylaminopurine) (0.5 mg/liter) were added to the medium in certain cases as described in ...

Section: Discussionmentioning

confidence: 99%

Studies on the recovery of crown gall tumor cells

Turgeon¹,

Wood²,

Braun³

1976

“…The molecular biology of the transforming viruses, such as Rous sarcoma virus, Moloney murine sarcoma virus (Mo-MuSV), Abelson murine leukemia virus, and Harvey murine sarcoma virus (Ha-MuSV), has been of special interest because viruses ofthis class enable cellular transformation related to a defined genetic sequence and its gene product(s) to be studied in great detail and under a high degree ofexperimental control. Accordingly, the genetic origin and transforming proteins of these viruses have come under close scrutiny (2)(3)(4)(5)(6)(7)(8)(9)(10).…”

mentioning

confidence: 99%

Analysis of two divergent rat genomic clones homologous to the transforming gene of Harvey murine sarcoma virus.

DeFeo¹,

Gonda²,

Young³

et al. 1981

270

Harvey murine sarcoma virus (Ha-MuSV) is a mouse-rat recombinant retrovirus that encodes a protein designated p21, required for virally induced transformation. Using a radiolabeled DNA fragment from the p21 coding region, we have detected homologous DNA sequences in the normal DNA of rats and of several other vertebrate species. Moreover, many tested cells from these species contain low levels of a p21 protein that is highly related to viral 21. Now we report two independent fragments from normal rat DNA containing sequences (sarc) homologous to the Ha-MuSV transforming region that were cloned in the bacteriophage vector Charon 4A. Sarc sequences in the one fragment are completely colinear with the viral sequences and share apparently all restriction endonuclease sites. Sarc sequences in the second fragment have several sets of intervening sequences and lack some restriction endonuclease sites found in the viral transforming region. Despite the presence of these intervening sequences in the second sarc fragment, we have been able to ligate this sarc fragment to the long terminal repeat sequence of HaMuSV and to induce cellular transformation and high levels of p21 expression upon transfection of this DNA to NIH 3T3 mouse cells. These results suggest that elevated levels of p21, normally expressed at low levels in a variety of cells, can induce cellular transformation.Retroviruses can be grouped into two broad classes based upon their biological activities: the nontransforming leukemia or leukosis viruses and the transforming sarcoma or acute leukemia viruses (1). The molecular biology of the transforming viruses, such as Rous sarcoma virus, Moloney murine sarcoma virus (Mo-MuSV), Abelson murine leukemia virus, and Harvey murine sarcoma virus (Ha-MuSV), has been of special interest because viruses ofthis class enable cellular transformation related to a defined genetic sequence and its gene product(s) to be studied in great detail and under a high degree ofexperimental control. Accordingly, the genetic origin and transforming proteins of these viruses have come under close scrutiny (2-10).Our laboratory has been investigating the molecular biology of Ha-MuSV, a replication-defective retrovirus derived after injection of a mouse leukemia virus into a rat (11). Similar to sequences in the above-mentioned viruses, the Ha-MuSV src gene sequences are derived from (rat) cellular unique sequences (10). These viral src sequences encode the transformation-specific p21 protein (12, 13) and the p21 biochemical activities, which specifically use guanine nucleotides (14,15). Consistent with the conserved nature of these sequences is the expression ofa related, but distinguishable p21 sarc protein in normal cells from a wide variety of species (16).In order to understand better any differences in the structure, biology, and regulation of expression between the viral p21 src and the related cellular p21 sarc protein, we have initiated studies into the structure and function of rat sarc genes. In this report, we describ...

“…It has now been well established that MSV arose by recombination between Moloney murine leukemia virus and a sequence derived from mouse cells (1)(2)(3). This mouse cell-derived segment of MSV, termed v-mos, is required for the induction and maintenance of viral transformation (3)(4)(5).…”

mentioning

confidence: 99%

Molecular cloning and chromosomal mapping of a human locus related to the transforming gene of Moloney murine sarcoma virus.

Prakash¹,

McBride²,

Swan³

et al. 1982

Human DNA was analyzed for the presence of sequences homologous to the transforming gene (v-mos) of Moloney murine sarcoma virus. A single 2.5-kilobase pair (kbp) EcoRIgenerated fragment of human DNA was identified by using cloned v-mo8 as probe. This DNA was molecularly cloned in a bacteriophage vector. By heteroduplex and restriction enzyme analyses, this human DNA fragment, designated c-mos (human), contained a 0.65-kbp region ofcontinuous homology with v-mos and was present as a single copy in human DNA. By testing for the presence of c-mos (human) in somatic cell hybrids possessing various numbers of human chromosomes, as well as in subelones of such hybrids, it was possible to assign c-mos (human) Cit/10% dextran sulfate (10). Filters were washed at 450C in 0.1 X NaCi/Cit.Enrichment and Molecular Cloning ofc-mos (Human). High molecular weight DNA was extracted from human placenta tissue as reported (11). EcoRI digested DNA was subjected to RPC-5 column chromatography (12) and agarose gel electrophoresis followed by blotting analysis (13). Hybridization was performed under relaxed conditions. A nick-translated, molecularly cloned v-mos fragment (14) composed of sequences extending from the Bgl I site to the HindIII site was used to probe the fractions. This probe contained 80% of the 1. 15-kbp v-mos gene. The appropriate RPC-5 fractions were pooled and subjected to further fractionation in a 1.0% preparative agarose gel. Those fractions hybridizing to v-mos were ligated to EcoRIcut A Charon 16A DNA, packaged in vitro (15), and plated onto Escherichia coli LE 392. Plaques containing recombinant phage were detected (16) and purified.Restriction Enzyme Mapping. Restriction endonuclease mapping was carried out by single and double digestion analysis and the partial digestion method of Smith and Birnstiel (17).Electron Microscopy. Heteroduplex analysis was performed according to the method of Davis et al. (18). Equal amounts of the appropriate DNAs (1-2 pRg) were mixed, denatured with 0.1 M NaOH/0.02 M EDTA (pH 8.5), neutralized with 0.2 M Tris HCI (pH 7.2), and hybridized in 50% formamide. After dialysis against 10 mM Tris.HCI/1 mM EDTA (pH 8.5) at 40C overnight, a portion was mixed with 100 mM Tris-HCl (pH 7.8)! 10 mM EDTA and cytochrome c at 50 Ag/ml and spread onto a water hypophase. The heteroduplexes were transferred to parlodion-coated grids, stained with 0.1 mM uranyl acetate, and shadowed with platinum/palladium. By using a Tektronix graphics system, DNA contour lengths were measured, with single-and double-stranded 4X174 DNA serving as internal length standards.Cell Hybridization. Human and rodent cells were cocultivated (1: 1 mixture) for 24 hr before induction ofcell fusion with 52.5% (vol/vol) polyethylene glycol 1000 (19)