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Biomedical subjects

E Gilboa

Publications and source records attributed to E Gilboa.

104 records · Page 6Linked to original sources
PubMed1979

A detailed model of reverse transcription and tests of crucial aspects.

A model of reverse transcription has been devised by which the detailed architecture of ten molecular structures is predicted. The model includes a number of novel features for which experimental evidence is presented. First, growing minus DNA strand is copied from the viral RNA only up to a position about 150 nucleotides from the 5' end of the RNA. Second, plus-strand DNA, after being copied from approximately 600 nucleotides at the 5' end of the minus-strand DNA, then transcribes the first approximately 20 nucleotides of the tRNApro primer (which is covalently attaced to the 5' end of the minus DNA strand). The 3' ends of the minus and plus DNA probably form a hybrid through the homology conferred by the primer binding site sequences. Third, the minus and plus DNA strands are elongated in a continuous fashion resulting in a linear double-stranded DNA molecule containing a 600 nucleotide direct repeat at both ends. The most of the features of the model have experimental support, and it appears to provide a credible description of reverse transcription.

DNA, Viral↗
PubMed1979

Synthesis of a 600-nucleotide-long plus-strand DNA by virions of Moloney murine leukemia virus.

A discrete, 600-nucleotide-long plus-strand DNA has been identified among the products of reverse transcription by virions of Moloney murine leukemia virus. Its polarity was shown by hybridization to minus-strand DNA. It appears to be copied from the right end of minus-strand DNA because (i) its restriction endonuclease cleavage pattern corresponds to the redundant 600-base segment found at either end of the ultimate double-stranded reverse transcription products, (ii) its synthesis is actinomycin D sensitive, and (iii) its synthesis begins during the first hour of a reverse transcription reaction when only the right-hand end of minus-strand DNA is available as template. We therefore call this DNA plus-strong-stop DNA by analogy with the minus-strong-stop DNA copied from the left end of the viral RNA. Both strong-stop DNAs are made early during in vitro reactions and decline in concentration later, consistent with postulated roles as initiators of long minus- and plus-strand DNA. Unlike minus-strong-stop DNA, plus-strong-stop DNA remains as a double-stranded nucleic acid after its synthesis, as shown by S1 nuclease resistance. A primer to initiate plus-strong-stop DNA synthesis has not been identified; the product found thus far has no detectable RNA attached to it.

Chromosome Mapping↗
PubMed1978

Methylation and capping of RNA polymerase II primary transcripts by HeLa nuclear homogenates.

HeLa nuclear homogenates incubated in vitro incorporate [beta-32P]ATP and S-[methyl-3H]-adenosylmeth-ionine ([3H]SAM) into blocked methylated 5' termini of newly synthesized RNA. Approximately 10% of the RNA chains initiated in vitro with [beta-32P]ATP are subsequently blocked by condensation of GMP to di- or triphosphate terminated RNA. The blocked termini can then be methylated by transfer of methyl groups from [3H]SAM to the 7 position of the guanosine and 2'-O position of the adenosine to form m7Gpp*pAm- capped terminus. In addition to conventional triphosphate caps, HeLa nuclear homogenates produce capping structures containing two phosphate residues in the pyrophosphate bridge. The two distinct cap forms were separated by DEAE-cellulose chromatography and analyzed. In contrast to triphosphate caps (m7GpppXm) in which X can be any one of the four nucleosides (G, A, C, or U), in diphosphate caps (m7GppXm), more than 95% of the penultimate nucleoside Xm is G. Incorporation of both [beta-32P]ATP and [3H]SAM into caps was markedly reduced by low concentrations of alpha-amanitin. However, an ammonium sulfate fraction of the nuclear homogenate can cap beta-32P-labeled RNA (pp*pA-RNA) to form m7Gpp*pA-RNA, in the presence of 0.5 microgram/mL of alpha-amanitin. Therefore, the nuclear capping enzyme is resistant to this drug. Our results indicate that RNA polymerase II primary transcripts are the substrate for the cellular capping enzyme and that the beta phosphate in the pyrophosphate bridge (m7GgammapbetapalphapXm) is derived from the 5' ends of the RNA chains.

Amanitins↗
PubMed1977

Initiation of RNA synthesis in isolated nuclei.

Ribonucleotide triphosphates, labeled at the beta position, were synthesized and used directly to quantify RNA chain initiation in nuclei isolated from Friend cells grown in tissue culture. At the optimal salt concentration, low-molecular-weight RNAs (4-5 S) synthesized by RNA polymerase III were the predominant species initiated. Less than 5% of the molecules were initiated by polymerase II. We calculate that 50-80% of the small RNA molecules synthesized in vitro were also initiated in vitro. Assuming that a substantial fraction of the nuclei were active in vitro, the number of 4-5 S RNA molecules initiated per nucleus was about 100 molecules/min.

Acetates↗
PubMed1977

Cell-free translation of simian virus 40 early messenger RNA coding for viral T-antigen.

Simian virus 40 (SV40) mRNA was isolated by hybridization of cytoplasmic RNA, from SV40-infected BS-C-1 monkey cells early in lytic infection, to SV40 DNA immobilized on Sepharose. The early viral mRNA, when added to a wheat-germ translation system, directed the synthesis of a unique class of products including a 90,000 molecular weight (Mr) polypeptide. It was found that this 90,000 Mr product as well as a prominent 17,000 Mr polypeptide could be specifically immunoprecipitated with hamster antiserum to SV40 T-antigen, but not with hamster control serum. Similar immunoprecipitation of extracts of SV40-infected cells with hamster anti-T serum yielded 90,000 Mr and 17,000 Mr polypeptides; these polypeptides were not found in immunoprecipitates of uninfected cell extracts. SV40 cRNA, prepared by asymmetric transcription of plaque-purified SV40 DNA, directed the cell-free synthesis of several products, including a 70,000 Mr polypeptide that could be specifically immunoprecipitated with anti-T serum. However, no T-antigen-related polypeptide was found in infected cells that corresponded in size to the major immunoprecipitated cRNA product.

Animals↗
PubMed1976

Preferential synthesis of viral late RNA by nuclei isolated from SV40 lytically infected cells.

Nuclei from SV40-infected monkey cells were isolated late in lytic infection and their cell-free transcriptional activity was characterized. 3H-RNA synthesized in vitro was hybridized to excess quantities of separated SV40 DNA strands which were each covalently bound to Sepharose. It was found that 3-5% of the newly synthesized RNA is virus-specific and that the plus-strand DNA, coding for late RNA sequences, is transcribed at a rate about 15 times higher than that of the minus-strand DNA, which codes for early RNA sequences. This indicates that transcriptional control has a major role in determining the relative abundancy of early and late RNA classes in lytically infected cells.

Amanitins↗
PubMed1975

Purification of SV-40 messenger RNA by hybridization to SV-40 DNA covalently bound to Sepharose.

SV-40 DNA sheared form was coupled in a stable covalent bond to cyanogen bromide activated Sepharose. Under the conditions used at least 80% of the DNA was bound to Sepharose. The T 1/2 of hybridization of 0.5 mug/ml of SV-40 cRNA to SV-40 DNA-Sepharose was 1 hr. This rate of hybridization is sufficiently rapid to purify SV-40 sequences from solutions containing as little as 0.05-0.1 mug/ml. Nonspecific hybridization of RNA is in the range of 0.1-0.2% of the total input RNA. The DNA-Sepharose is fairly stable and can be reused several times to purify RNA. The SV-40 DNA-Sepharose was used to select large quantities of virus specific RNA from SV-40 infected BS-C-1 cells. The virus specific RNA when added to cell-free extracts from wheat germ was shown to direct the synthesis of the major viral structural protein VP-1.

Cells, Cultured↗
PubMedDate not supplied

Expression of a foreign gene in myeloid and lymphoid cells derived from multipotent haematopoietic precursors.

Bone marrow cells infected with retroviral vectors carrying the bacterial neomycin resistance (neo) gene as a marker were used for long-term reconstitution of the haematopoietic system of irradiated mice. The neo gene is expressed in the myeloid and lymphoid lineages of these animals and an analysis of the sites of viral integration indicates that these lineages are derived from the same primitive multipotent cells.

Animals↗
PubMedDate not supplied

Induction of cytotoxic T lymphocytes with dendritic cells transfected with human papillomavirus E6 and E7 RNA: implications for cervical cancer immunotherapy.

Human papillomavirus (HPV) infection is associated with cervical cancer. The high-risk HPV E6 and E7 oncoproteins are constitutively expressed in most cervical carcinoma cells, and are, therefore, attractive antigens for cytotoxic T-lymphocyte (CTL)-mediated immunotherapy. The objective of this study was to evaluate the use of dendritic cells (DCs) transfected with RNA encoding the E6 and E7 protein for cervical cancer immunotherapy. The authors have shown that DCs transfected with RNA-encoding antigen stimulate potent antigen-specific CTL responses in vitro and in vivo. In this study, they tried to determine whether DCs transfected with E6 and E7 RNA stimulate primary, antigen-specific CTL responses in vitro. The results show that DCs pulsed with E6 or E7 RNA stimulate antigen-specific CTL responses that recognize and lyse DCs transfected with E6 and E7 RNA and human cervical carcinoma cells expressing the E6 and E7 products, and the lysis was comparable to that achieved with E6 and E7 peptide-pulsed DCs. Dendritic cells cotransfected with both E6 and E7 RNA stimulate CTLs that are more effective at lysing human cervical cancer cells. This study provides a rationale for the development of cervical carcinoma immunotherapy using DCs transfected with HPV E6 and E7 RNA.

Cytotoxicity Tests, Immunologic↗
PubMedDate not supplied

Inhibition of established pancreatic cancers following specific active immunotherapy with interleukin-2 gene-transduced tumor cells.

Pancreatic cancer has a poor prognosis even when complete resection can be accomplished. Recent studies have demonstrated that the immune system is capable of mounting effective tumor-specific immune responses even against "nonimmunogenic" tumors. The studies reported herein were conducted to determine if induction of tumor-specific immune responses of inhibiting in vivo pancreatic tumor growth could be achieved through active immunization with pancreatic tumor cells genetically engineered to secrete interleukin-2 (IL-2). A relevant poorly immunogenic subcutaneous model of murine ductal pancreatic cancer was first developed using an implantable tumor cell line Panc02 in C57BL/6 mice. Panc02 cells were then genetically engineered to secrete human IL-2 (Panc02/IL2). The ability of irradiated Panc02/IL2 cells to stimulate an immune response capable of rejecting a subsequent tumor challenge was first demonstrated. Ninety percent of animals vaccinated with irradiated parental Panc02 and subsequently challenged with parental Panc02 cells developed tumors by 48 days (mean tumor volume of 234 mm3) compared to only 40% (P < .05, chi square) of animals vaccinated with irradiated Panc02/IL2 and challenged with parental Panc02 (14 mm3, P < .004, tau test). The therapeutic benefit of active immunization in tumor-bearing animals was then examined. Mice with 3-day-old established subcutaneous tumors were administered a series of 4 weekly vaccinations with irradiated Panc02 or Panc02/IL2 cells. A significant reduction in tumor growth was present in those animals vaccinated with Panc02/IL2 (P < .005, tau test) versus Panc02 or saline. Animals whose established tumors regressed following vaccinations with IL-2-secreting Panc02 cells were found to have long-lasting immunity as demonstrated by rejection of a tumor challenge presented over 140 days following inoculation of the primary tumor. We conclude that an immune response capable of inhibiting established pancreatic tumors can be generated by vaccination with IL-2-secreting tumor cells. Furthermore, long-term immunological memory was established in mice that rejected the original established tumor. These studies provide preclinical evidence to support the use of cytokine gene-transduced tumor cell vaccinations in patients with pancreatic cancer.

Adenocarcinoma↗