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Biomedical subjects

D W Stacey

Publications and source records attributed to D W Stacey.

68 records · Page 4Linked to original sources
PubMed1982

Analysis by microinjection of the biological effects of site-directed mutagenesis in cloned avian leukosis viral DNAs.

Cloned avian leukosis viral DNAs were mutagenized in the long terminal repeat, in the leader sequence for env mRNA, and at the poly-env junction. The effect of these mutations in the viral DNA upon its ability to direct virus production or env mRNA synthesis was analyzed by microinjecting the mutant DNAs into chicken embryo fibroblasts and into chicken cells transformed by the env-deficient Bryan strain of Rous sarcoma virus, respectively. The results indicated that: (i) addition of up to 8 base pairs 19 nucleotides upstream of the Hogness box did not block transcription; (ii) deletion of 26 base pairs, including the tRNA primer binding site, allowed synthesis of all viral products and participation in recombination, but replication was blocked; (iii) deletion of fewer than 50 base pairs 250 bases downstream of the long terminal repeat depressed expression of all viral genes; and (iv) deletion of most of the gag and pol genes did not inhibit env mRNA synthesis, but virion packaging of the unspliced transcript was inefficient.

Animals↗
PubMed1981

Biological activity of cloned retroviral DNA in microinjected cells.

Avian retroviral DNA molecules that had been cloned from infected cells by using recombinant DNA techniques were microinjected into either uninfected chicken embryo fibroblasts (CEF) or CEF transformed by the envelope glycoprotein-deficient Bryan strain of Rous sarcoma virus [RSV(--)cells]. Retroviral DNA injected into RSV(--) cells directed transcription of envelope mRNA, which was then able to complement the RSV(--) env deficiency and promote the production of infectious transforming virus. The retroviral DNA also directed the production of fully infectious virus after injection into uninfected cells or RSV(--) cells. Virus production began within 3--4 hr after microinjections. When 100 DNA molecules per cell were injected, almost all injected cells produced infectious virus. As the number of injected molecules per cell was decreased, a corresponding decrease was observed in the number of cells that produced infectious virus. DNA injected into the cytoplasm was 1/50th to 1/10th as effective in virus production as DNA molecules injected into the nucleus. DNA molecules containing one or two tandem copies of the viral long terminal repeat were equally effective in virus production.

Animals↗
PubMed1980

Expression of a subgenomic retroviral messenger RNA.

When mRNA for avian retroviral envolope glycoprotein (env) was injected into cells transformed by env-deficient Bryan Rous sarcoma virus, the env deficiency of the injected cells was complemented to allow the release of transforming virus for up to 40 hr. When virus spread within the injected culture was allowed to occur, a second phase of transforming virus production by the injected culture began approximately 2 days following injection, continued for many days and often increased to titers well above those seen soon after injection. The requirement for virus spread, along with the genetic properties of virus released long after injection, supported the hypothesis that the second phase of virus production resulted when injected env mRNA was packaged into virus released by injected cells. When this virus infected other cells within the culture the env mRNA was reverse-transcribed to form a subgenomic, proviral-like molecule able to direct the synthesis of env mRNA. Accordingly, it was shown tht neither DNA nor full genomic viral RNA contaminating injected mRNA preparations could account for the results. Evidence that an mRNA can be reverse-transcribed into an active, proviral-like molecule may be of importance in the relationship between retroviruses and their hosts.

Animals↗
PubMed1979

Messenger activity of virion RNA for avian leukosis viral envelope glycoprotein.

An intracellular assay for viral envelope glycoprotein (env) messenger was employed to analyze the RNA from virus particles of Rous-associated virus type 2. For this assay RNA was microinjected into cells infected by the env-deficient Bryan strain of Rous sarcoma virus [RSV(-) cells]. Only when the injected RNA could be translated by the recipient cells to produce viral envelope glycoprotein was the env deficiency of the RSV(-) cells complemented, enabling them to release focus-forming virus. RNA in a 21S size fraction from the Rous-associated virus particle promoted the release of numerous focus-forming virus from RSV(-) cells, whereas the major 35S virion RNA species was inactive. The env messenger activity sedimented as a sharp peak with high specific activity. RNase T1-generated fragments of virion 35S RNA were unable to promote the release of infectious virus from RSV(-) cells. Consequently, the active molecule was most likely to be env messenger which had been encapsulated by the virus particle from the cytoplasm of infected cells. Approximately 95% of the env messenger within the virion was associated with the virion high-molecular-weight RNA complex. The temperature required to dissociate env messenger from the high-molecular-weight complex was indistinguishable from the temperature required to disrupt the complex itself. Virion high-molecular-weight RNA that was associated with env messenger sedimented slightly more rapidly than the bulk virion RNA; this was the strongest evidence that the 21S messenger had been encapsulated directly from the infected cells. These data are considered along with a related observation [concerning the prolonged expression of env messenger after injection into RSV(-) cells] to raise the possibility that virus-encapsulated env messenger can become expressed within subsequently infected cells.

Avian Leukosis Virus↗
PubMed1977

Microinjection analysis of envelope-glycoprotein messenger activities of avian leukosis viral RNAs.

Virion RNA from the avian leukosis virus Rous-associated virus 2 (RAV-2) and poly(A)-containing RNAs from RAV-2-infected chick embryo fibroblasts were microinjected into fibroblasts transformed by the Bryan high-titer strain of Rous sarcoma virus (RSV), which is deficient in viral envelope glycoprotein. Production of infectious RSV following these injections depended upon the viral envelope-messenger activity of the injected RNA. This system constituted a sensitive and rigorous assay system for viral envelope-messenger RNA. It was found that 21S mRNA from RAV-2-infected cells expressed the highest activity, while 35S mRNA expressed comparatively little. In addition, RAV-2-virion RNA expressed little messenger activity. The rate of formation of infectious RSV following 21S mRNA injections reached a peak near 9 hr, which was followed by a rapid decline. Evidence has been obtained that a small fraction of both 35S virion RNA and 35S mRNA from virus-infected cells was encapsulated into virus particles following their injection into virus-producing cells.

Animals↗
PubMed1977

Evidence for the autophagy of microinjected proteins in HeLA cells.

Rhodamine-conjugated proteins were microinjected into living HeLa cells. Fluorescence microscopy was then employed to study their segregation from the cytoplasm into lysosomes. Results obtained in this way were verified when the corresponding unconjugated proteins were localized by autoradiographic, histological, and antibody-staining methods after their microinjection. Most injected proteins were segregated into cytoplasmic granular structures during their removal from cells. As evidence that these were autophagic vacuoles, they were found to contain no detectable acid phosphatase activity upon formation, after which they moved to the juxtanuclear position of lysosomes and appeared to fuse with them. The segregation of microinjected proteins exhibited a high degree of selectivity. The half-times of placement of individual exogenous proteins into cytoplasmic granules varied from 3 h to nearly 3 days, and one protein, hemoglobin, was never observed to enter them. Furthermore, endogenous HeLa proteins in a size fraction near 200,000 daltons were segregated much more rapidly than those in a fraction near 40,000 daltons. In these studies, rapid protein segregation appeared to take place by a mechanism of exclusion of the injected protein from numerous cytoplasmic domains.

Acid Phosphatase↗
PubMed1976

Microinjection studies of duck globin messenger RNA translation in human and avian cells.

Microinjection techniques were used for the study of the translation of heterospecific messenger RNAs in living cells. Duck globin messenger RNAs, in the form of polysomes, messenger ribonucleoprotein particles, or free mRNAs, were injected into cells of human origin (HeLa), and the subsequent appearance and accumulation of avian globin were measured by specific immunofluorescence techniques. Globin accumulated within a high proportion of HeLa cells for at least 25 hr following injection, indicating the intracellular stability of the heterospecific globin messenger RNA and the fact that mRNA initially associated with more complex structures could be translated in the HeLa cytoplasm. In similar experiments, duck reticulocyte polysomes were shown to be translated in duck embryo cells but not in embryonic chick liver cells. The latter, however, could be shown to withstand the microinjection procedure as judged by their capacity to respond normally to inducers and inhibitors of the synthesis of alpha-aminolevulinic acid synthetase. HeLa cells injected with duck hemoglobin divide at normal or nearly normal rates following the injection, but their globin content diminishes, in contrast with the increasing globin content of cells receiving duck reticulocyte polysomes, mRNP particles, or free mRNA.

5-Aminolevulinate Synthetase↗
PubMedDate not supplied

Transformation of NIH 3T3 cells by microinjection of Ha-ras p21 protein.

Alteration in gene structure has been shown to occur in some human tumours. These altered genes, termed oncogenes, were originally identified by their ability to induce foci of transformed cells on transfected mouse 3T3 cultures. The oncogene identified in the EJ/T24 human bladder carcinoma is similar to the transforming gene of BALB and Harvey murine sarcoma virus (MSV) and differs from its counterpart in normal cells by a single amino acid. All three of these Ha-ras genes direct the production of similar proteins (p21). While the ras gene appears to be involved in tumour formation in some situations, its role is unclear. The ras protein product (p21) binds guanine nucleotides and has a unique autophosphorylating activity, but no other enzymatic activity has been found. We report here the injection of purified Ha-ras p21 protein, made in Escherichia coli from the gene of BALB-MSV, into NIH 3T3 cells and show that the purified protein itself is sufficient to induce a transformed morphology. In addition, the injected protein stimulates quiescent cells to enter the S-phase of the cell cycle. This result clearly demonstrates that the ras gene functions directly through the protein product. It also establishes an assay for the protein which depends on its activity within a living cell. The transforming activity of a p21 ras protein equivalent to the product of the normal cellular ras gene, is also demonstrated.

Animals↗
PubMedDate not supplied

Requirement for ras proto-oncogene function during serum-stimulated growth of NIH 3T3 cells.

Human tumours often contain DNA sequences not found in normal tissues which are able to transform cultured NIH 3T3 cells. In some tumours the gene responsible for this transformation belongs to the cellular ras gene family. A specific type of mutation is responsible for converting the cellular proto-oncogene into a ras oncogene capable of inducing transformation. In a study of the function of a cellular ras gene, its protein product (produced in a bacterial cell) was microinjected into NIH 3T3 cells; the recipient cells became morphologically transformed and were induced to initiate DNA synthesis in the absence of added serum, but only when cellular ras protein was injected at much higher concentrations than required with protein of the transforming ras gene. To further analyse the function of the cellular ras gene, we have now injected monoclonal antibodies against ras proteins into NIH 3T3 cells. We report here that NIH 3T3 cells induced to divide by adding serum to the culture medium are unable to enter the S phase of the cell cycle after microinjection of anti-ras antibody, showing that the protein product of the ras proto-oncogene is required for initiation of the S-phase in NIH 3T3 cells.

Animals↗
PubMedDate not supplied

Requirement for c-ras proteins during viral oncogene transformation.

Many retroviral oncogenes have been classified into one of several categories based on structure, enzymology and cellular localization. These genes originated from host cells and are probably derived from genes normally involved in the control of cell proliferation. The cellular counterparts of three oncogenes have been identified as a growth factor or growth factor receptor; related oncogenes include receptor-like membrane proteins which often express tyrosine kinase activity. These growth factor-related oncogenes are structurally and biochemically distinct from the membrane-associated ras gene family, which bind and hydrolyse GTP. Oncogenes localized primarily in the cytoplasm which probably have serine kinase activity, have also been identified. Although the structure and biochemistry of many oncogenes have been extensively studied, relatively little is known about the functional relationships of oncogene proteins within the cell. An opportunity to study such interaction is provided by the identification of a monoclonal antibody that neutralizes cellular ras proteins when microinjected into cells. It has been shown previously that the injected antibody inhibits the initiation of S-phase in NIH 3T3 cells. In the present study we injected this monoclonal antibody into NIH 3T3 cells transformed by a variety of oncogenes. The results show that transformation by three growth factor receptor-like oncogenes depends on c-ras proteins, while transformation by two cytoplasmic oncogenes appears to be independent of c-ras protein.

Animals↗
PubMedDate not supplied

Transient blockage of proliferative signalling: a novel strategy for protective chemotherapy.

An intact proliferative signalling pathway is essential to the growth of all normal cells, but is often not required by tumor cells. This fact was used to devise a protective chemotherapeutic protocol potentially applicable to all tissues. Four treatments were chosen to temporarily disrupt proliferative signalling. They acted either upstream, at, or downstream of cellular ras activity. As expected, the cell cycle progression of normal cells was temporarily interrupted, while those cells transformed by tumor genes, or tumor cells themselves often were not affected. During these cell cycle blocking treatments the cells were exposed to the topoisomerase inhibitor m-AMSA. This anti-cancer drug is selectively toxic to cycling cells. In each case the tumor cells were selectively killed as judged either by their ability to incorporate labeled thymidine, replate, or grow. These studies suggest new ways to utilize current drugs or search for new ones.

3T3 Cells↗