Function of Oct-1 protein in basal transcription from the mouse mammary tumor virus promoter /
The steroid hormone-inducible promoter of mouse mammary tumor virus (MMTV) contains three overlapping sequences related to the consensus octamer motif (ATGCAAAT). Basal promoter activity in the absence of hormone induction from a template in which all three octamer elements were mutated was decreas...
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
1995.
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| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=742161591&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | The steroid hormone-inducible promoter of mouse mammary tumor virus (MMTV) contains three overlapping sequences related to the consensus octamer motif (ATGCAAAT). Basal promoter activity in the absence of hormone induction from a template in which all three octamer elements were mutated was decreased by 2-3 fold in vitro transcription assays. Oct- I protein purified from HeLa cell nuclear extracts, as well as recombinant Oct-1 expressed in bacteria, recognized MMTV octamer-related sequences as shown by DNase I footprinting. Furthermore, rabbit polyclonal antiserum directed against recombinant Oct-1 completely inhibited the formation of specific complexes between MMTV octamer-related sequences and proteins present in nuclear extracts of HeLa cells, indicating that Oct-I is the major protein in HeLa nuclear extracts that recognizes octamer-related sequences in the MMTV promoter. In addition, depletion of Oct-I from the nuclear extract using Oct-l-specific antiserum or a sequence-specific DNA affinity resin decreased in vitro transcription from the wild-type MMTV promoter to a level identical to that obtained from a promoter in which all three octamer-related sequences were mutated. Addition of purified HeLa Oct-1 to the depleted extract increased transcription from the wild-type but not the mutated promoter, demonstrating that Oct-I transcription factor stimulates basal transcription from the MMTV promoter. Addition of an oligonucleotide containing an Oct-I binding site inhibited MMTV transcription in crude nuclear extract. However, following preincubation of the MMTV promoter in the nuclear extract, transcription was refractory to inhibition by the octamer oligonucleotide. Furthermore, addition of Oct-I to the Oct-depleted extract after preincubation of the MMTV promoter had no stimulatory effect on the transcription. Kinetic experiments showed that Oct-1 increased the extent of the assembly of functional preinitiation complexes and not the rate of assembly. Finally, Sarkosyl-inhibition experiments clearly demonstrated that Oct-I affected transcription assembly at the template-commitment step during preinitiation complex assembly. Overall, these results indicate that Oct-I acts at the early stage of preinitiation complex assembly to increase basal transcription activity from the MMTV promoter. |
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| Item Description: | Vita. "Major Subject: Biochemistry". |
| Physical Description: | xiii, 146 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilms Inc. |
| Bibliography: | Includes bibliographical references. |