1992 Fiscal Year Final Research Report Summary
Analysis of Marek's disease virus (MDV) genes responsible for oncogenes is by MDV and protection of chickens from Marek's disease.
| Project/Area Number |
03454187
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Virology
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| Research Institution | TOKYO MEDICAL AND DENTAL UNIVERSITY |
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Principal Investigator |
HIRAI Kanji TOKYO MEDICAL AND DENTAL UNIVERSITY, MEDICAL RESEARCH INSTITUTE, PROFESSOR, 難治疾患研究所, 教授 (00100991)
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| Project Period (FY) |
1991 – 1992
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| Keywords | Marek's disease virus(MDV) / Tumor / Sequence determination / PCR / Recombinant MDV / Restriction endonuclease map / Protein kinase / Vaccine |
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| Research Abstract |
(1).Sequence determination of the tumor-associated region of the Marek's disease virus type 1 (MDV1) genome and isolation of cDNA clones of transcripts from the region : (a)Twelve clones carrying the 132bp tandem direct repeat, tumor associated region, within the long inverted repeat region of MDV1 genome were isolated from a cDNA library constructed from chick embryo fibro-blasts infected with MDV1. Two open reading frames (ORF) were found in a cDNA clone encoding the mRNA from the 132bp repeat by sequence analysis. (b)Nucleotide sequence analysis of 9.3kb DNA of MDV1, including the tumor-associated 132bp repeat revealed the presence of 14 potential ORFs. Identification of the proteins encoded from these ORFs is under investigation.
(2).Analysis of MDV1 genes related to vaccinal immunity: (a)The DNA sequence (8.9kb) covering about 70% of the short unique region (Us) and part of the short inverted repeat of MDV1-GA strain was determined.Out of nine potential ORFs within the 8.9kb sequen
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ce, four were found to be homologous to US10, US3, US2, and US6 (gD) of herpes simplex virus type 1. (b)The US3 was shown to encode MDV1 specific protein kinase by using a baculovirus vector
(3).Construction of recombinant MDV1 expressing the lacZ gene of Escherichia coli: We attempted to insert the Escherichia coli lacZ gene at 22 sites into the MDV1 genome by homologous recombination. Among them, expression of the lacZ gene at four sites were found to be stable during serial passages in culture. Especially, the US10 and US3 genes were nonessential for induction of protective immunity as well as for in vitro viral growth.
(4).Construction of the restriction endonuclease map of MDV2: A BamHI, EcoRI, and XhoI restriction endonuclease map of MDV2 DNA was constructed. We showed the presence of the collinear relationship among viral genomes of three serotypes.
(5).Differentiation of oncogenic and nononcogenic strains of MDV1 by using PCR: Differentiation of oncogenic and nononcogenic strains of MDV1 was possible by PCR using the primers chosen from the sequence including the 132bp repeat. Less
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