Purification and partial characterization of a novel baculovirus RNA polymerase complex /
An in vitro transcription system was developed for the
| Main Author: | |
|---|---|
| Format: | Thesis Book |
| Language: | English |
| Published: |
[Place of publication not identified] :
[publisher not identified] ;
1995.
|
| Subjects: | |
| Online Access: | Link to OAKTrust copy http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=742536501&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | An in vitro transcription system was developed for the Autographa califomica nuclear polyhedrosis virus (ACNPV) late 39k gene and the very late polyhedrin gene in order to identify and purify trans-acting factors involved in ACNPV late and very late gene transcription. This system was shown to reproduce in vivo control mechanisms for baculovirus late and very late transcription. Optimization of transcription conditions was achieved empirically, and it was shown that a preincubation step was not required for in vitro transcription of the 39k and polyhedrin promoters. The nuclear extract prepared from virus-infected insect cells at 36 h postinfection was fractionated by phosphocellulose chromatography. Both the polyhedrin and 39k constructs were transcribed by two of the five fractions. However, the 0.3 M KCI fraction preferred the 39k late promoter, while the 0.5 M KCI fraction preferred the very late polyhedrin promoter. Further fractionation of both phosphocellulose fractions on four additional chromatography steps revealed that transcription activity eluted as a single peak. This strongly suggested that there are two distinct stable baculovirus RNA polymerase complexes in the 36-h nuclear extract. Gel filtration of these two complexes indicated that their molecular weights were approximately 750,000 for the very late complex, and 640,000 for the late complex. The ACNPV very late complex was purified to near homogeneity by conventional chromatography. The complex consists of at least five different subunits, with apparent molecular weights of 138,000, 102,000, 59,000, 58,000, and 47,000. lmmunochemical analysis indicated that the 102,000 subunit was encoded by the ACNPV lef-8 gene. The purified complex alone specifically transcribed both the 39k late and the very late polyhedrin promoters, although it preferred the polyhedrin constructs. The optimal Mg2+ concentration of in vitro transcription with this RNA polymerase complex is 2 mM, lower than those of most eukaryotic RNA polymerases (6 mM). The sensitive in vitro transcription assay established here, and the identification and purification of the two stable RNA polymerase complexes, will help to understand the mechanisms for differential transcription of baculovirus late and very late genes. |
|---|---|
| Item Description: | Vita. "Major Subject: Biochemistry". |
| Physical Description: | xii, 135 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilms Inc. |
| Bibliography: | Includes bibliographical references. |