Characterization and in vitro expression of the cytochromeb-559 genes of barley I. Localization and sequence of the genes

Abstract

The psbE and psbF genes encoding the 9.4 and 4.4 kD apoproteins of cytochrome b-559 have been located in the chloroplast genome of barley. As in other plant species they are found adjacent to each other in the large single copy region of the chloroplast DNA. Both the nucleotide sequence and the deduced amino acid sequence for the two polypeptides are identical to that of wheat and more than 95% similar to those of spinach, tobacco and Oenothera. The region between the two genes spans 10 nucleotides (excluding the stopcodon) and contains a typical procaryotic ribosomal binding site. A dicistronic transcript is identified, but the presence of a ribosomal binding site between the two genes may allow independent translation.

Characterization and in vitro expression of the cytochromeb-559 genes of barley. II. In vitro transcription and translation

“…Whereas immunoprecipitation of translation products obtained in the $30 extract of strain Y1090 included also several proteins due to endogenous protein synthesis when the insert was in the correct orientation, there is no comparable background with the insert in reverse orientation or if translates obtained in the E. coli PR7 lysate were immunoprecipitated ( Figure 2). The fact that the 9 kD immunoprecipitable cytochrome b-559 translate is obtained by inverse orientation of the insert indicates that transcription is from the chloroplast DNA promoter, included in the 1150 bp AccI-BamHI fragment (11). Transcripts derived from pGem3 containing the 1150 bp fragment in an orientation appropriate for the SP6 polymerase gave with this enzyme rise to the same three polypeptide bands as obtained with pDS6 ( Figure 3).…”

“…coli HB 101 was the recipient strain for the expression vectors pDS6, pDS6-RBSII and pGem3. The bacteria were prepared for transformation as described in part I (11). Minimal medium (12) supplied with 0.4% casamino acids and ampicillin ( 100 ~tgxml ~) was used to propagate bacteria with recombinant plasmids.…”

“…plasmids DNA was isolated and purified as described in (11). The orientation of fragments inserted into pDS6, pDS6-RBSII and pGem3 was checked by digesting the recombinant plasmid with appropriate restriction enzymes, or by sequencing with the dideoxy chain termination method of SANGER et al (16) using synthetic 15-mer oligonucleotide primers complementary to the binding sites of the E. coli, SP6 and T7 RNApolymerases, respectively.…”

“…A possible variation in the stoichiometry of the two proteins among plant species of different tissues or developmental stages is, however, not excluded. The genes coding for the two apoproteins, psbE and psbF, are bicistronically transcribed into a single mRNA species (9,11). At present it is not known, if a coordinate synthesis of the corresponding proteins takes place or if a precursor polypeptide is made which subsequently is cleaved into the two polypeptides.…”

In vitro transcription and translation of the psbD gene encoding the D-2 protein of photosystem II in barley

“…mRNA in a cell-free E. coli $30 and wheat germ extracts Since the protein expression machinery in chloroplasts resembles the bacterial one more than the eukaryotic one it was assumed that translation of psbD-derived mRNA should be more efficient in a cell-free E. coli lysate than in an eukaryoti'c translation system, mRNA, obtained by transcription of pGem 101-7 with SP6 RNA polymerase, was added to an $30 E. coli lysate (12,25) in the presence of 35S-methionine, but no D-2-related polypeptide could be detected (not shown). This negative result is in contrast to the successful translation of transcripts from psbE and psbF genes encoding the cytochrome b-559 polypeptides in the same $30 E. coli lysate (l 2) as used in the present work.…”