Molecular analysis of chromosome-mediated gene transfer.

Scangos, George; Huttner, Kenneth; Silverstein, Saul; Ruddle, Frank H.

doi:10.1073/pnas.76.8.3987

Cited by 32 publications

(23 citation statements)

References 23 publications

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“…Hanahan et al (7) recently reported the transformation of a plasmid containing the whole early region of SV40 (pOT) into L cells and described a heterogeneity in expression similar to that observed by us for the expression of plasmid p102 containing only a fragment of the early SV40 gene region. Heterogeneity offoreign gene expression in mammalian cells can arise for various reasons, as can be deduced from experimental results (7,18,19,(30)(31)(32)(33). As shown by our results, lack of a signal sequence such as a polyadenylylation site seems to contribute to heterogeneous and significantly lower expression of a foreign gene introduced into a mammalian cell.…”

Section: Fig 2 Expression Of Sv40 T-antigen-related Proteins In Celsupporting

confidence: 49%

Integration and expression of a truncated simian virus 40 early gene fragment in mammalian cells.

Horst¹,

Weckler²,

Jacob³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

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The recombinant plasmid p102 based on pBR322 carrying -50% of the replicator proximal early region of simian virus 40 (SV40) DNA, including the viral origin of replication, has been constructed. It lacks a major part of the large tumor (T) antigen 3'-coding region, the T-antigen termination codon, and the polyadenylylation site. The plasmid was transferred together with the herpes simplex virus thymidine kinase (TK) gene as a selectable marker to mouse LTK-cells. TKV cell clones were isolated and their high molecular weight DNAs were shown by DNA blotting and hybridization experiments to contain the SV40 DNA fragment from the recombinant. In some of these clones, heterogeneous expression of the SV40 DNA fragment could be detected by immunofluorescence while, in control experiments in which a plasmid containing the complete SV40 early DNA region was used, this extensive heterogeneity of T-antigen expression was not observed. RNA-DNA hybridization experiments showed that the SV40-specific RNA of those clones is polyadenylylated. The molecular weight of the T-antigen-related protein coded by p102 corresponded well to the expected coding capacity of the SV40 DNA fragment. Small tumor antigen was not expressed.The small DNA tumor virus simian virus 40 (SV40) codes for two early proteins: large tumor (T) antigen (Mr, =96,000) and small tumor (t) antigen (Mr~z20,000). Possible functions of t antigen in viral replication and transformation have not yet been elucidated, but multiple and diverse functions in both events have been inferred for SV40 T antigen (1). SV40 T antigen, therefore, seems to be a pleiotropic molecule (2). Studies with adenovirus serotype 2-SV40 hybrids have shown that certain functions can be performed by COOH-terminal fragments of T antigen, indicating that T antigen contains a variety of functional domains (3). Whereas analysis of adenovirus 2-SV40 hybrids has allowed the characterization of some biological properties ofthe COOH-terminal domains of SV40 T antigen, so far little is known about its NH2-terminal domains. Recent data, however, suggested that the 5'-coding region of T antigen may be involved in its transformation functions but that its lytic functions require in addition the 3'-coding region (4, 5). Therefore, it seems possible that cellular transformation by SV40 might be achieved, at least in part, by NH2-terminal fragments of this molecule. However, all SV40-transformed cells analyzed so far seem to contain all sequences of the SV40 early genome region (6) and express T antigen or related molecules containing duplications of certain T-antigen sequences (super T antigen). In addition, some SV40-transformed cells express truncated T-antigen molecules.A prerequisite for studying potential functions of NH2-terminal T-antigen fragments is the expression of such fragments in the absence of functional T antigen. This might be achieved by constructing a recombinant plasmid containing NH2-terminal coding sequences of T antigen and analyzing their expression after transfection and st...

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Section: Fig 2 Expression Of Sv40 T-antigen-related Proteins In Celsupporting

confidence: 49%

Integration and expression of a truncated simian virus 40 early gene fragment in mammalian cells.

Horst¹,

Weckler²,

Jacob³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

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“…Le présent rapport ne saurait prétendre considérer la totalité des données relatives à ces questions dont la masse est écrasante. Ce sont préférentiellement certains aspects ayant reçu un intense développement ces dernières années que nous envisagerons en nous limitant volontairement aux étapes précédant l'assemblage des acides aminés en polypeptides qui est traité conjointement dans un autre rapport (Pétrissant, 1981 (Breathnach et al, 1977 ;Jeffryes et Flavell, 1977 ;Gilbert, 1978 ;Crick, 1979 ;Kolata, 1980 (Wigler et al,1977(Wigler et al, , 1978(Wigler et al, , 1979Pellicer et al, 1978 ;Wold et al, 1979 ;Scangos et al, 1979 ;Mantei et al, 1979 ;Hamer et al, 1979 ;Mulligan et al, 1979 ; Leder, 19796 ;Lai et al, 1980 ;Breathnach et al, 1980 (Knapp et al, 1978 ;Melton et al, 1980). Le fait que des introns n'aient jamais pu être mis en évidence dans les gènes des procaryotes a conduit à postuler que les introns (Kolata, 1980 ;Gilbert, 1978) étaient apparus au cours de l'évolution, ou qu'au contraire deux voies relativement indépen-dantes auraient été suivies par les procaryotes et les eucaryotes (Darnell, 1978 ;Doolittle, 1978 Ross, 1976 ;Kinninburgh et al, 1978 ;Horowitz et al, 1978 ;Roop et al, 1978Roop et al, , 1980Harpold et al, 1979 ;Scherrer et al, 1979 ;Ryffel et al, 1980).…”

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Le contrôle de la synthèse protéique dans les cellules des eucaryotes

Houdebiné¹

1981

Reprod. Nutr. Dévelop.

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“…In this paper, the initial drug resistance was obtained by DNA-mediated gene transfer. Observations of initial instability of such resistance (33,49,61), suggestions of amplification of the transferred genes during this period (18,50), probable extrachromosomal maintenance of the transferred DNA during the unstable phase (33,44), and evidence for stabilization of the resistance by chromosomal integration of the transferred DNA (33,47,49) mirror the results obtained when cell mutants are selected in vitro for increasing drug resistance (1, 8, 14, 30, 41, 51, 58, 60). By applying an initial mild selection pressure after exposure to DNA, followed quickly by stepwise increases in the selection stringency ( (42).…”

Section: Discussionmentioning

confidence: 50%

Co-amplification of double minute chromosomes, multiple drug resistance, and cell surface P-glycoprotein in DNA-mediated transformants of mouse cells.

Robertson¹,

Ling²,

Stanners³

1984

Mol. Cell. Biol.

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A genetic system comprised of mammalian cell mutants which demonstrate concomitant resistance to a number of unrelated drugs has been described previously. The resistance is due to reduced cell membrane permeability and is correlated with the presence of large amounts of a plasma membrane glycoprotein termed P-glycoprotein. This system could represent a model for multiple drug resistance which develops in cancer patients treated with chemotherapeutic drugs. We demonstrate here that the multiple drug resistance phenotype can be transferred to mouse cells with DNA from a drug-resistant mutant and then amplified quantitatively by culture in media containing increasing concentrations of drug. The amount of Pglycoprotein was correlated directly with the degree of drug resistance in the transformants and amplified transformants. In addition, the drug resistance and expression of P-glycoprotein of the transformants were unstable and associated quantitatively with the number of double minute chromosomes. We suggest that the gene for multiple drug resistance and P-glycoprotein is contained in these extrachromosomal particles and is amplified by increases in double minute chromosome number. The potential use of this system for manipulation of mammalian genes in general is discussed.A genetic system of clinical relevance involving mammalian cell mutants resistant to a wide variety of cytocidal drugs has recently emerged (36). This system began with the isolation of a number of CHO cell mutants which were resistant to colchicine (38). These mutants were shown to be resistant to the drug as a result of a membrane permeability barrier, which reduced the cellular uptake of colchicine (38). They were also found to be cross-resistant to a number of structurally diverse and functionally unrelated drugs, some of which are used in chemotherapy of cancer, again, by virtue of reduced uptake of these compounds (10). Further analysis of the membranes of these cells, in conjunction with hybrid cell studies (37) and selection of revertants (35), clearly demonstrated the association of the multiple drug resistance phenotype and the reduced membrane permeability with the presence in the cell membrane of a glycoprotein of 160,000 to 180,000 daltons termed P-glycoprotein (27, 28). P-glycoprotein was undetectable in the membrane of the drug-sensitive parental CHO cells but present in drugresistant mutants in amounts directly correlated with the level of resistance. The overexpression of a 160,000-to 180,000-dalton protein has also been observed in several drug resistance systems where resistance has been selected for drugs other than colchicine (13,22,45), including a human leukemic cell line selected for vinblastine resistance (11). As it appears likely that the development of resistance to chemotherapy of human tumors after repeated treatment with these drugs involves, at least in part, the emergence of such mutants (10, 36, 52), it seems important to carry their genetic analysis further. As a first step in this direction, we * Correspondin...

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Molecular analysis of chromosome-mediated gene transfer.

Abstract: Metaphase chromosomes isolated from a cell line carrying the thymidine kinase (TI) gene of herpes simplex virus type 1 were used to transform the TK-deficient cell line LMTK-to the TK+ phenotype. Four independent transformants were isolated, all of which expressed virus-specific TI.

Cited by 32 publications

References 23 publications

Integration and expression of a truncated simian virus 40 early gene fragment in mammalian cells.

Integration and expression of a truncated simian virus 40 early gene fragment in mammalian cells.

Le contrôle de la synthèse protéique dans les cellules des eucaryotes

Co-amplification of double minute chromosomes, multiple drug resistance, and cell surface P-glycoprotein in DNA-mediated transformants of mouse cells.

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