The main objectives of the proposed research are (1) to determine the relationship between the rpoA, rpoB, rpoC DNA-dependent RNA polymerase subunit genes encoded in chloroplast DNA of Euglena gracilis and higher plants and the RNA polymerase activities of chloroplasts, (2) to define how the complex rpoB- rpoC transcription units are transcribed, and the resulting pre- mRNAs are processed and translated to yield polypeptide subunits, (3) to identify, isolate and characterize any additional chloroplast or nuclear localized genes for chloroplast RNA polymerases, and (4) to determine how the expression of the chloroplast RNA polymerase subunit genes is regulated during light induced organelle development. Emphasis will be placed on the importance of transcriptional vs post-transcriptional events in regulating the transcriptional vs post-transcriptional events in regulating the transcriptional status of the developig plastid. Chloroplast contain two types of transcriptional activity. In order to determine if the rpoA, B, and C genes encode subunits of either or both polymerase activities, polypeptide coding domains of the chloroplast rpoA, B, and C genes will being expressed in E. coli to yield fusion polypeptides for use in generating monospecific antibodies directed against the putatative RNA polymerase subunits. Antibodies will be used to immunopurify the enzymes, to identify subunits by Western blotting, and to specifically inhibit the different transcriptional reactions. Homogenous preparations of these enzymes should be obtained via immunoaffinity chromatography and FPLC, in addition to currently available purification procedures. Genes for RNA polymerase subunits that are not encoded by the rpoA, B, and C loci will also be studied. In order to determine how rpoC transcripts are processed, and if there are unusual features to this process such as alternat splice sites, cDNA clones of rpoC mRNA will be prepared and sequenced. Through the types of experiments described above and studies on the regulation of expression of the RNA polymerase subunit genes, and of the resulting enzyme activities, it will be possible to define many important fundamental aspects of RNA synthesis in cell organelles.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM035665-06
Application #
3288664
Study Section
Molecular Biology Study Section (MBY)
Project Start
1985-02-01
Project End
1993-03-31
Budget Start
1990-04-01
Budget End
1991-03-31
Support Year
6
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Arizona
Department
Type
Schools of Arts and Sciences
DUNS #
City
Tucson
State
AZ
Country
United States
Zip Code
85721
Sheveleva, Elena V; Hallick, Richard B (2004) Recent horizontal intron transfer to a chloroplast genome. Nucleic Acids Res 32:803-10
Sheveleva, Elena V; Giordani, Nicole V; Hallick, Richard B (2002) Identification and comparative analysis of the chloroplast alpha-subunit gene of DNA-dependent RNA polymerase from seven Euglena species. Nucleic Acids Res 30:1247-54
Doetsch, N A; Favreau, M R; Kuscuoglu, N et al. (2001) Chloroplast transformation in Euglena gracilis: splicing of a group III twintron transcribed from a transgenic psbK operon. Curr Genet 39:49-60
Doetsch, N A; Thompson, M D; Favreau, M R et al. (2001) Comparison of psbK operon organization and group III intron content in chloroplast genomes of 12 Euglenoid species. Mol Gen Genet 264:682-90
Doetsch, N A; Thompson, M D; Hallick, R B (1998) A maturase-encoding group III twintron is conserved in deeply rooted euglenoid species: are group III introns the chicken or the egg? Mol Biol Evol 15:76-86
Stevenson, J K; Hallick, R B (1994) The psaA operon pre-mRNA of the Euglena gracilis chloroplast is processed into photosystem I and II mRNAs that accumulate differentially depending on the conditions of cell growth. Plant J 5:247-60
Hong, L; Hallick, R B (1994) Gene structure and expression of a novel Euglena gracilis chloroplast operon encoding cytochrome b6 and the beta and epsilon subunits of the H(+)-ATP synthase complex. Curr Genet 25:270-81
Drager, R G; Hallick, R B (1993) A novel Euglena gracilis chloroplast operon encoding four ATP synthase subunits and two ribosomal proteins contains 17 introns. Curr Genet 23:271-80
Drager, R G; Hallick, R B (1993) A complex twintron is excised as four individual introns. Nucleic Acids Res 21:2389-94
Copertino, D W; Shigeoka, S; Hallick, R B (1992) Chloroplast group III twintron excision utilizing multiple 5'- and 3'-splice sites. EMBO J 11:5041-50

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