[74] Microinjection of early SV40 DNA fragments and T antigen

Graessmann, A.; Graessmann, M.; Mueller, Christian

doi:10.1016/s0076-6879(80)65076-9

Cited by 170 publications

(62 citation statements)

References 9 publications

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“…GST protein alone was generated as above to serve as a control. Cells plated on coverslips were microinjected by using the method described by Graessmann et al (1980). Control injections were performed by using GST alone or bovine serum albumin (BSA) in the same microinjection buffer.…”

Section: Recombinantmentioning

confidence: 99%

Rho-stimulated Contractility Contributes to the Fibroblastic Phenotype of Ras-transformed Epithelial Cells

Zhong

Kinch

Burridge

1997

MBoC

148

131

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Oncogenic transformation of cells alters their morphology, cytoskeletal organization, and adhesive interactions. When the mammary epithelial cell line MCF10A is transformed by activated H-Ras, the cells display a mesenchymal/fibroblastic morphology with decreased cell–cell junctions but increased focal adhesions and stress fibers. We have investigated whether the transformed phenotype is due to Rho activation. The Ras-transformed MCF10A cells have elevated levels of myosin light chain phosphorylation and are more contractile than their normal counterparts, consistent with the activation of Rho. Furthermore, inhibitors of contractility restore a more normal epithelial phenotype to the Ras-transformed MCF10A cells. However, inhibiting Rho by microinjection of C3 exotransferase or dominant negative RhoA only partially restores the normal phenotype, in that it fails to restore normal junctional organization. This result prompted us to examine the effect that inhibiting Rho would have on the junctions of normal MCF10A cells. We have found that inhibiting Rho by C3 microinjection leads to a disruption of E-cadherin cytoskeletal links in adherens junctions and blocks the assembly of new adherens junctions. The introduction of constitutively active Rho into normal MCF10A cells did not mimic the Ras-transformed phenotype. Thus, these results lead us to conclude that some, but not all, characteristics of Ras-transformed epithelial cells are due to activated Rho. Whereas Rho is needed for the assembly of adherens junctions, high levels of activated Rho in Ras-transformed cells contribute to their altered cytoskeletal organization. However, additional events triggered by Ras must also be required for the disruption of adherens junctions and the full development of the transformed epithelial phenotype.

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Section: Recombinantmentioning

confidence: 99%

Rho-stimulated Contractility Contributes to the Fibroblastic Phenotype of Ras-transformed Epithelial Cells

Zhong

Kinch

Burridge

1997

MBoC

148

131

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“…Cells were released from confluence by trypsin digestion and seeded onto gridded coverslips 2 days before microinjection. The microinjection system has been described previously (3), and it was implemented by using procedures originally described by Grässmann et al (15). For each pool of cDNA transcripts, approximately 200 cells were injected, and 30 to 40 of these were in S phase at the time of the assay for DNA synthesis rates.…”

Section: Methodsmentioning

confidence: 99%

Irreversible Repression of DNA Synthesis in Fanconi Anemia Cells Is Alleviated by the Product of a Novel Cyclin-Related Gene

Digweed

Günthert

Schneider

et al. 1995

Molecular and Cellular Biology

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Primary fibroblasts from patients with the genetic disease Fanconi anemia, which are hypersensitive to cross-linking agents, were used to screen a cDNA library for sequences involved in their abnormal cellular response to a cross-linking challenge. By using library partition and microinjection of in vitro-transcribed RNA, a cDNA clone, pSPHAR (S-phase response), which is able to correct the permanent repression of semiconservative DNA synthesis rates characteristic of these cells, was isolated. Wild-type SPHAR mRNA is expressed in all fibroblasts so far analyzed, including those of Fanconi anemia patients. Correction of the abnormal response in these cells appears therefore to be due to overexpression after cDNA transfer rather than to genetic complementation. The cDNA contains an open reading frame coding for a polypeptide of 7.5 kDa. Rabbit antiserum directed against a SPHAR peptide detects a protein of 7.9 kDa in Western blots (immunoblots) of whole-cell extracts from proliferating, but not resting, fibroblasts. The deduced amino acid sequence of SPHAR contains a motif found in the cyclins, and it is proposed that SPHAR acts within the injected cell by interfering with the cyclin-controlled maintenance of S phase. In agreement with this proposal, normal cells transfected with an antisense SPHAR expression vector have a significantly reduced rate of DNA synthesis during S phase and a prolonged G 2 phase, reflecting the need for postreplicative DNA processing before entry into mitosis.

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“…A/NT/60/68 at a m.o.i, of 30, or microinjected with pTKNP DNA at 1 mg/ml in distilled water (Graessmann et al, 1980). Monolayers were cultured at 37 °C for the required time before fixing in paraformaldehyde and staining by indirectimmunofluorescence as described by Ash et al (1977).…”

Section: Immunofluorescent Staining Oj Injected and Microinjected Culmentioning

confidence: 99%

“…They also have the ability to correctly modify, process and segregate many foreign proteins introduced as the protein itself or encoded in RNA or DNA (Asselbergs, 1979;Gurdon & Melton, 1981;Wickens & Laskey, 1981;Lane, 1983;Colman, 1984a;Soreq, 1985). However, mammalian cells in culture are more similar to those of the natural host and, although much smaller than amphibian oocytes, can also be injected with foreign macromolecules (Graessmann et a~., 1980) whose behaviour can then be compared with the same molecules produced in parallel batches of cells infected with virus. Thus, there are advantages in complementary studies employing both oocytes and mammalian cells.…”

Section: Introductionmentioning

confidence: 99%

Location of Influenza Virus M, NP and NS1 Proteins in Microinjected Cells

Davey

Colman

Dimmock

1985

Journal of General Virology

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SUMMARYWhen microinjected as cloned DNA, the nucleoprotein (NP) of influenza virus A/NT/60/68 (H3N2) accumulated in the nuclei of Xenopus laevis oocytes, and cultured cells of rodent and primate origin. This accumulation appeared to be specific and a property of the NP itself (or conceivably NP in association with unknown cellular constituents) since no other influenza virus components were present in DNA-injected cells. In the oocyte nucleus, clonally derived NP achieved an eightfold concentration over that in the cytoplasm. Such NP was full-length as judged by its mobility during PAGE and had the native conformation of H3N2 virus NP according to its reaction with a panel of monoclonal antibodies. NP appeared to be in the soluble fraction of the nucleus as it did not sediment under conditions which removed particulate matter from nuclear extracts. Microinjection of extracts of chick embryo fibroblast cells infected with A/FPV/Rostock/34 (H7N1) showed that exogenous NP had an affinity for the nucleus similar to that synthesized intracellularly from cloned NP DNA. This conclusion was supported by an experiment in which cloned NP from the oocyte nucleus re-entered the nucleus after injection into the cytoplasm of fresh oocytes. Injection of mRNA, extracted from chick embryo fibroblast cells infected with A/FPV/Rostock/34, into oocytes directed the synthesis of the viral proteins M (Mr 28000), and NS1 (MT 27000) as well as NP (Mr 56000). While NP from this source concentrated in the nucleus as before, M merely associated with the nucleus without exceeding the cytoplasmic level. Even more remarkable was NS1 ; although in injected cells this protein is concentrated in nucleoli, in microinjected oocytes its nuclear concentration was threefold less than that in the cytoplasm, despite the very large number (> 1500) of nucleoli present in Xenopus oocytes. It seems likely that the karyophilic nature of M and NS1, unlike that of NP, is a property not of the proteins themselves, but of a complex which they form with some other product of the infected cell. These findings were repeated when extracts from infected chick embryo cells containing NP, M and NS1 proteins radiolabelled in vivo, were injected into the cytoplasm of oocytes.

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[74] Microinjection of early SV40 DNA fragments and T antigen

Cited by 170 publications

References 9 publications

Rho-stimulated Contractility Contributes to the Fibroblastic Phenotype of Ras-transformed Epithelial Cells

Rho-stimulated Contractility Contributes to the Fibroblastic Phenotype of Ras-transformed Epithelial Cells

Irreversible Repression of DNA Synthesis in Fanconi Anemia Cells Is Alleviated by the Product of a Novel Cyclin-Related Gene

Location of Influenza Virus M, NP and NS1 Proteins in Microinjected Cells

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