Jeffrey C. Rapp

Plastid Genes Encoding the Transcription/Translation Apparatus Are Differentially Transcribed Early in Barley (Hordeum vulgare) Chloroplast Development (Evidence for Selective Stabilization of psbA mRNA)

Chloroplast genomes encode rRNAs, tRNAs, and proteins involved in transcription, translation, and photosynthesis. The expression of 15 plastid genes representing each of these functions was quantitated during chloroplast development in barley (Hordeum vulgare). The transcription of all plastid genes increased during the initial phase of chloroplast development and then declined during chloroplast maturation. RNAs corresponding to rpoB- rpoC1-rpoC2, which encode subunits of a plastid RNA polymerase, and rps16, which encodes a ribosomal protein, reached maximal abundance early in chloroplast development prior to genes encoding subunits of the photosynthetic apparatus (rbcL, atpB, psaA, petB). Transcription of rpoB as well as 16S rRNA, trnfM-trnG, and trnK was high early in chloroplast development and declined 10-fold relative to rbcL transcription during chloroplast maturation. RNA hybridizing to psbA and psbD, genes encoding reaction center proteins of photosystem II, was differentially maintained in mature chloroplasts of illuminated barley. Differential accumulation of psbD mRNA relative to rbcL mRNA was due to light-stimulated transcription of psbD. In contrast, enhanced levels of psbA mRNA in mature chloroplasts were due primarily to selective stabilization of the psbA mRNA. These data document dynamic modulation of plastid gene transcription and mRNA stability during barley chloroplast development.

Chloroplast transcription is required to express the nuclear genes rbcS and cab. Plastid DNA copy number is regulated independently

RbcL and rbcS mRNA levels and plastid transcription activity are low in the basal meristematic region of barley primary leaves and increase coordinately during leaf cell development with a similar time course in dark-grown or illuminated seedlings. The capacity of light to cause cab mRNA accumulation shows a similar dependence on leaf cell development. These results indicate that the initial activation of chloroplast gene expression and the expression of some nuclear genes encoding plastid proteins are coupled to leaf cell development. RbcL and rbcS mRNA levels and plastid transcription activity decline in older leaf sections of dark-grown or illuminated barley. The decreases in plastid transcription and rbcS and rbcL mRNA levels in older dark-grown seedlings could be reversed by plant illumination. Therefore, while the initial activation of plastid transcription and accumulation of rbcS mRNA are largely light-independent, these events become light-dependent in older leaves of dark-grown barley. If the initial increase in plastid transcription which occurs early in leaf cell development is prevented by tagetitoxin, a specific inhibitor of the plastid RNA polymerase, rbcS mRNA does not accumulate and cab mRNA accumulation cannot be induced by light. The effect of tagetitoxin is selective because this compound does not inhibit barley leaf growth, or the normal accumulation of nuclear-encoded actin and BN3 transcripts and plastid DNA which occurs during chloroplast development. Furthermore, a barley pigment-deficient mutant, alb-f17, and plants containing photo-oxidized plastids show parallel reductions in plastid transcription activity and levels of rbcS and cab mRNA. This suggests that the activation of plastid transcription during the early stages of chloroplast biogenesis is necessary for the expression of rbcS and cab.

Biologically Active Human Interferon α-2b Produced in the Egg White of Transgenic Hens

We have previously described the expression of a bacterial protein in the egg white of transgenic chickens using a replication-deficient retroviral vector. Here we report the expression of a glycosylated human protein, interferon α-2b (hIFN), in the egg white of transgenic hens. The hIFN secreted into the egg white was biologically active as determined by a viral inhibition assay. Purification and carbohydrate analysis of the hIFN expressed in egg white revealed that two of the six major glycosylated hIFN species match the naturally occurring human hIFN glycovariants. These results support the potential of the hen as a bioreactor for the production of commercially valuable, biologically active, and glycosylated proteins in egg white.

Regulation of Chloroplast Biogenesis in Barley

Higher plant plastids contain multiple copies of a circular genome which encodes between 120 to 140 genes1,2,3. Many of these genes encode proteins or RNAs involved in transcription or translation. Among this group of genes are rpoA, rpoB, rpoC1, and rpoC2 which encode RNA polymerase subunits, 18 genes encoding ribosomal proteins, 4 rRNA-encoding genes and 30 tRNA genes. Another large group of plastid genes encode proteins involved in photosynthesis. These include rbcL, which encodes the large subunit of Rubisco, psaA, psaB and psaC which encode subunits of Photosystem I (PSI), psbA through psbF and psbH through psbN, which encode subunits of Photosystem II (PSII), and other genes encoding proteins of the chloroplast ATP synthase and cytochrome b6/f complexes. While the plastid genome contributes subunits to Rubisco and the electron transport units, these complexes also contain proteins encoded by nuclear genes. As a consequence, biosynthesis of these protein complexes requires coordination of plastid and nuclear gene expression.