Biomedical subjects
Publications and source records attributed to P B Heifetz.
The genome of the plastid has generated much interest as a target for plant transformation. The characteristics of plastid transgenes both reflect the prokaryotic origin of plastid organelles and provide a unique set of features that are currently lacking in genes introduced into the plant nucleus. Recent progress has been made in understanding plastid expression of recombinant proteins.
Controlled down-regulation of endogenous plant gene expression is a useful tool, but antisense and sense silencing lack predictability. Recent studies show that expression of both antisense and sense RNA together is an effective means of inactivating reporter and viral genes in plants. We created transgenic plants expressing antisense and sense RNA together in a single 'double-stranded RNA' (dsRNA) transcript. This approach shows great promise as a highly effective means for reducing gene function. With this approach, we demonstrated that the Arabidopsis cystathionine beta-lyase gene, which encodes a methionine biosynthetic enzyme, is essential for viability. Inactivation of this gene was rescued by the addition of methionine to the growth medium. Compared to antisense and sense constructs, the dsRNA construct showed a much more consistent and complete suppression of gene activity. Additionally, expression of a transcript with a spacer sequence containing an unrelated gene between antisense and sense luciferase gene fragments led to stronger inactivation of a second luciferase transgene than did constructs with a minimal spacing between sense and antisense fragments. However, the gene in the spacer region was neither functionally expressed nor functional in silencing a second, unlinked homologous transgene.
The green alga Chlamydomonas reinhardtii can grow photoautotrophically utilizing CO(2), heterotrophically utilizing acetate, and mixotrophically utilizing both carbon sources. Growth of cells in increasing concentrations of acetate plus 5% CO(2) in liquid culture progressively reduced photosynthetic CO(2) fixation and net O(2) evolution without effects on respiration, photosystem II efficiency (as measured by chlorophyll fluorescence), or growth. Using the technique of on-line oxygen isotope ratio mass spectrometry, we found that mixotrophic growth in acetate is not associated with activation of the cyanide-insensitive alternative oxidase pathway. The fraction of carbon biomass resulting from photosynthesis, determined by stable carbon isotope ratio mass spectrometry, declined dramatically (about 50%) in cells grown in acetate with saturating light and CO(2). Under these conditions, photosynthetic CO(2) fixation and O(2) evolution were also reduced by about 50%. Some growth conditions (e.g. limiting light, high acetate, solid medium in air) virtually abolished photosynthetic carbon gain. These effects of acetate were exacerbated in mutants with slowed electron transfer through the D1 reaction center protein of photosystem II or impaired chloroplast protein synthesis. Therefore, in mixotrophically grown cells of C. reinhardtii, interpretations of the effects of environmental or genetic manipulations of photosynthesis are likely to be confounded by acetate in the medium.
Transfer of Chlamydomonas reinhardtii cells grown photoautotrophically in low light to higher light intensities has a dramatic transient effect on the differential expression of the two major chloroplast encoded photosynthetic proteins. Synthesis of the D1 protein of Photosystem II increases more than 10-fold during the first six hours in high light (HL), whereas synthesis of the large subunit (LSU) of Rubisco drops dramatically within 15 min and only gradually resumes at about 6 h. Synthesis of the chloroplast-encoded ATP synthase beta subunit, the nuclear-encoded Rubisco small subunit and the nuclear-encoded beta-tubulin is not noticeably affected. Up regulation of psbA mRNA translation accounts for a substantial fraction of the increased D1 synthesis, since accumulation of psbA mRNA increases 4.2- and 6.3-fold less than D1 synthesis at 6 and 18 h in HL. Down-regulation of LSU synthesis is not correlated with a reduction in the steady-state level of the rbcL transcript. Primer extension mapping of the 5' ends of the rbcL mRNAs reveals transcripts with start points located at -93 and -168 relative to the first translated ATG. Transfer of low light (LL)-grown cells to HL temporarily decreases the ratio of the -93 to -168 transcripts, but this ratio normalizes after 6 h in HL, coincident with the recovery in the synthesis of LSU. These several distinct effects of temporary light stress were correlated with a rapid, sustained increase in the reduction state of QA, a transient decline in photosynthetic efficiency, a less rapid drop in total chlorophyll content and a delay in cell division.
Synthesis of the photosystem II D1 protein as a precursor with a carboxyl-terminal extension occurs in almost all eukaryotic photosynthetic organisms examined so far, as well as in cyanobacteria. Processing of the D1 precursor has been recently postulated to play a regulatory role in the light-dependent migration of photosystem II units from the unstacked to the stacked thylakoids (Bowyer, J. M., Packer, J. C. L., McCormack, B. A., Whitelegge, J. P., Robinson, C., and Taylor, M. A. (1992) J. Biol. Chem. 267, 5424-5433). To test this hypothesis, site-directed mutagenesis and chloroplast transformation have been used to create a "preprocessed" mutant Chlamydomonas strain which synthesizes mature D1 protein directly. We have found that this strain is indistinguishable from wild type in terms of photosynthetic performance and cell doubling time under CO2- and light-saturated photoautotrophic growth conditions.
Transformation of the plastid genome has a number of inherent advantages for the engineering of gene expression in plants. These advantages include: 10-50 times higher transgene expression levels; the absence of gene silencing and position effect variation; the ability to express polycistronic messages from a single promoter; uniparental plastid gene inheritance in most crop plants that prevents pollen transmission of foreign DNA; integration via a homologous recombination process that facilitates targeted gene replacement and precise transgene control; and sequestration of foreign proteins in the organelle which prevents adverse interactions with the cytoplasmic environment. It is now 12 years since the first conclusive demonstration of stable introduction of cloned DNA into the Chlamydomonas chloroplast by the Boynton and Gillham laboratory, and 10 years since the laboratory of Pal Maliga successfully extended these approaches to tobacco. Since then, technical developments in plastid transformation and advances in our understanding of the rules of plastid gene expression have facilitated tremendous progress towards the goal of establishing the chloroplast as a feasible platform for genetic modification of plants.