Sequence and transcriptional analysis of the barley ctDNA region upstream of psbD-psbC encoding trnK(UUU), rps16, trnQ(UUG), psbK, psbI, and trnS(GCU)

Abstract

A 6.25 kbp barley plastid DNA region located between psbA and psbD-psbC were sequenced and RNAs produced from this DNA were analyzed. TrnK(UUU), rps16 and trnQ(UUG) were located upstream of psbA. These genes were transcribed from the same DNA strand as psbA and multiple RNAs hybridized to them. TrnK and rsp16 contained introns; a 504 amino acid open reading frame (ORF504) was located within the trnK intron. Between trnQ and psbD-psbC was a 2.24 kbp region encoding psbK, psbI and trnS(GCU). PsbK and psbI are encoded on the same DNA strand as psbD-psbC whereas trnS(GCU) is transcribed from the opposite strand. Two large RNAs accumulate in barley etioplasts which contain psbK, psbI, anti-sense trnS(GCU) and psbD-psbC sequences. Other RNAs encode psbK and psbI only, or psbK only. The divergent trnS(GCU) located upstream of psbD-psbC and a second divergent trnS(UGA) located downstream of psbD-psbC were both expressed. Furthermore, RNA complementary to psbK and psbI mRNA was detected, suggesting that transcription from divergent overlapping transcription units may modulate expression from this DNA region.

Inefficient rpl2 splicing in barley mutants with ribosome-deficient plastids.

“…This ORF is located in the intron of the tRNA gene for lysine (tmK; cf. Figure 1) and is well conserved in mustard (Neuhaus and Link, 1987), rice (Hiratsuka et al, 1989), tobacco (Shinozaki et al, 1986), barley (Sexton et al, 1990), potato (Jardin et al, 1994), liverwort (Ohyamaet al, 1986), and maize (R. Maier and H. Kossel, unpublished data). Its presence in the reduced plastid genome of the parasitic, nonphotosynthetic plant Epifagus viginiana suggests an important function of the encoded peptide (Wolfe et al, 1992).…”

Involvement of Protein Kinase and Extraplastidic Serine/Threonine Protein Phosphatases in Signaling Pathways Regulating Plastid Transcription and the psbD Blue Light- Responsive Promoter in Barley

“…The deoxyribonucleotide 5'-GAATTCAGCATTATTCC-AAG spans nucleotides 6226 to 6245 of the psbD mRNA leader (Sexton et al, 1990b). It was labeled at the 5' terminus using T4 polynucleotide kinase and [Y-~'P]ATP (ICN).…”

“…The radiolabeled primer was used in primer extension analysis experiments, as described previously (Christopher et al, 1992). The deoxyoligonucleotide 5'-GCGTGGTC-TAAGTCTAAGG, which spans nucleotides 5971 to 5989 of the psbD mRNA leader (Sexton et al, 1990b), was used for mapping psbD-psbC mRNA 5' ends by primer extension analysis. Sequencing reactions were conducted on the plasmid pBE613 (Sexton et al, 1990a) in parallel with primer extension assays.…”

Identification of a sequence-specific DNA binding factor required for transcription of the barley chloroplast blue light-responsive psbD-psbC promoter.

“…For the construction of pLRP185/MCS @ultkloning _site), the previously cloned pBE3-5, which contains 837-bp EcoRI-EcoRI DNA fragment spanning the LRP185 region (5372 to 6244; Sexton et al, 1990a) in pTZl8 vector (Pharmacia), was first linearízed by cutting a BstXl site, which is located 65 to 76 bp upstream of the transcription initiation site of the psbD LRP in vitro. Then, the BstXl3'overhangs were removed by S1 nuclease digestion and ligated with 175-bp BssHII-BssHII MCS DNA fragment, which was released from pBluescript KS+, gel purified, and 3'filled in by the Klenow DNA polymerase I.…”

“…Because D2 is degraded when plants are exposed to high light, it has been proposed that blue light activation of psbD transcription helps to maintain D2 levels under these conditions (Christopher and Mullet, 1994). The mechanism that activates psbD transcription was difficult initially to delineate because this gene is located in a complex operon that also encodes psbC, psbK, psbl, orf62, and trnG (Sexton et al, 1990a). Two frnS genes are encoded within this DNA region on the opposite strand.…”