Transcriptional regulation of baculovirus late and very late genes /
Baculovirus late genes are transcribed by a viral-encoded RNA polymerase (RNAP). In addition to RNAP, sixteen proteins, called late expression factors or LEFs, have been identified that support transcription from late and very late promoters. Three of these proteins, PP31, LEF-12 and VLF-1, are th...
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
2002.
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| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=765105921&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | Baculovirus late genes are transcribed by a viral-encoded RNA polymerase (RNAP). In addition to RNAP, sixteen proteins, called late expression factors or LEFs, have been identified that support transcription from late and very late promoters. Three of these proteins, PP31, LEF-12 and VLF-1, are the topic of this dissertation. Native PP31 was purified from infected insect cells using a combination of column chromotagraphies. PP31 bound to single-stranded and double-stranded DNA with equal affinities. Addition of PP31 to in vitro transcription assays with purified baculovirus RNAP resulted in a strong inhibition of transcription indicating that the viral RNAP was not able to displace PP31. The baculovirus lef-12 gene is required for transient expression of baculovirus late genes. The role of LEF-12 in viral infection was analyzed by constructing two mutant viruses. The mutants were viable, although yields were somewhat reduced in the absence of LEF-12. Pulse-labeling of infected cells revealed that LEF-12 mutant viruses entered the late phase and synthesized late proteins at levels equivalent to, or two-fold lower than, wildtype virus-infected cells. In wild type virus-infected cells, LEF-12 was not detected until 18 hours post infection, and accumulation of LEF-12 peaked at 24-36 hours post infection. Primer extension mapping revealed that lef-12 mRNA was synthesized by 12 hours post infection, and peaked between 18 and 24 hours post infection. Furthermore synthesis of lef-12 message was inhibited by the addition of aphidicolin, confirming that lef-12 is expressed after DNA replication and should be classified as a late gene. The very late factor-1 gene (vlf-1) is required for baculovirus very late gene expression but its mechanism of action is unknown. Native and histidine tagged VLF-1 were purified using baculovirus and bacterial systems. In vitro transcription assays with purified components showed that VLF-1 acted directly at the level of transcription. The burst sequence of the very late polyhedrin gene promoters was required for this activity. EMSA results indicated that VLF-1 bound equally well to DNA fragments containing the polh promoter, the 39k promoter or non-specific DNA fragments. We also presented evidence of an antitermination activity of VLF-1. |
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| Item Description: | Vita. "Major Subject: Biochemistry". |
| Physical Description: | xiii, 130 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilm Inc. |
| Bibliography: | Includes bibliographical references (leaves 115-129). |