Identification of a novel aviadenovirus, designated pigeon adenovirus 2 in domestic pigeons (Columba livia).

The young pigeon disease syndrome (YPDS) affects mainly young pigeons of less than one year of age and leads to crop stasis, vomitus, diarrhea, anorexia and occasionally death. This disease is internationally a major health problem because of its seasonal appearance during competitions such as homing pigeon races or exhibitions of ornamental birds. While the etiology of YPDS is still unclear, adenoviruses are frequently discussed as potential causative agents. Electron microscopy of feces from a YPDS outbreak revealed massive shedding of adenovirus-like particles. Whole genome sequencing of this sample identified a novel adenovirus tentatively named pigeon adenovirus 2 (PiAdV-2). Phylogenetic and comparative genome analysis suggest PiAdV-2 to belong to a new species within the genus Aviadenovirus, for which we propose the name Pigeon aviadenovirus B. The PiAdV-2 genome shares 54.9% nucleotide sequence identity with pigeon adenovirus 1 (PiAdV-1). In a screening of further YPDS-affected flocks two variants of PiAdV-2 (variant A and B) were detected which shared 97.6% nucleotide identity of partial polymerase sequences, but only 79.7% nucleotide identity of partial hexon sequences. The distribution of both PiAdV-2 variants was further investigated in fecal samples collected between 2008 and 2015 from healthy or YPDS-affected racing pigeons of different lofts. Independent of their health status, approximately 20% of young and 13% of adult pigeon flocks harbored PiAdV-2 variants. Birds were free of PiAdV-1 or other aviadenoviruses as determined by PCRs targeting the aviadenovirus polymerase or the PiAdV-1 fiber gene, respectively. In conclusion, there is no indication of a correlation between YPDS outbreaks and the presence of PiAdV-2 or other aviadenoviruses, arguing against an causative role in this disease complex.

In vitro characterization of the tobacco rpoB promoter reveals a core sequence motif conserved between phage-type plastid and plant mitochondrial promoters.

We report here the in vitro characterization of PrpoB-345, the tobacco rpoB promoter recognized by NEP, the phage-type plastid RNA polymerase. Transcription extracts were prepared from mutant tobacco plants lacking PEP, the Escherichia coli-like plastid-encoded RNA polymerase. Systematic dissection of a approximately 1 kb fragment determined that the rpoB promoter is contained in a 15-nucleotide segment (-14 to +1) upstream of the transcription initiation site (+1). Point mutations at every nucleotide reduced transcription, except at the -5 position which was neutral. Critical for rpoB promoter function was a CRT-motif (CAT or CGT) at -8 to -6 (transcription <30%), defining it as the promoter core. The core CAT sequence is also present in the maize rpoB promoter, which is faithfully recognized by tobacco extracts. Alignment of NEP promoters identified a CATA or TATA (=YATA) sequence at the rpoB core position, also present in plant mitochondrial promoters. Furthermore, NEP and the phage T7 RNA polymerase exhibit similar sensitivity to inhibitors of transcription. These data indicate that the nuclear RpoZ gene, identified by sequence conservation with mitochondrial RNA polymerases, encodes the NEP catalytic subunit.

Chloroplast endoribonuclease p54 involved in RNA 3'-end processing is regulated by phosphorylation and redox state.

Chloroplast RNA-binding protein p54 is an endoribonuclease required for 3'end-processing of plastid precursor transcripts. We find that purified p54 can serve as a phosphate acceptor for protein kinases in vitro. Both the processing and RNA-binding activities of p54 are enhanced by phosphorylation and decreased by dephosphorylation. In addition, the enzyme is activated by the oxidized form of glutathione and inhibited by the reduced form, whereas other redox reagents that were tested showed no effect. Kinase treatment of p54 prior to oxidation by glutathione resulted in highest levels of activation, suggesting that phosphorylation and redox state act together to control p54 activity in vitro and possibly also in vivo.

Identification and characterization of the Arabidopsis thaliana chloroplast DNA region containing the genes psbA, trnH and rps19'.

A 1887-nucleotide chloroplast-DNA region from Arabidopsis thaliana was analyzed. It contains the conserved genes psbA for the precursor of the D1 reaction-centre protein of photosystem II, trnH for tRNAHis, and rps19' for the 6.8-kDa protein of the small ribosomal subunit. Northern hybridization and RNase protection experiments suggest co-transcription of a minor RNA fraction over the full lengths of psbA and the preceding trnK-UUU gene, but not including downstream trnH sequences. In front of the mapped 5' end of the major 1.2-kb psbA transcript is a DNA region that shows the typical architecture of a psbA promoter, consisting of the prokaryotic-type '-35' and '-10' elements as well as the eukaryotic-type 'TATA' motif. The common 3' end of psbA transcripts seems to be located immediately after a stem-loop structure downstream from the coding region.

RNA-binding activity of the matK protein encoded by the chloroplast trnK intron from mustard (Sinapis alba L.).

The chloroplast trnK gene for tRNALys(UUU) from mustard contains a 2574 bp group II intron with a long open reading frame for 524 amino acids. The encoded polypeptide appears to be structurally related to mitochondrial maturases which are involved in splicing. To study the properties of the intron encoded protein, we overexpressed the trnK ORF as a beta-galactosidase fusion protein in E. coli and carried out RNA-protein binding experiments with crude bacterial extracts and the purified fusion protein. Both gel-shift and UV-crosslinking experiments revealed preferential binding to the trnK precursor transcript. Of two other RNA probes containing chloroplast group II introns, the trnG precursor was recognized by the trnK ORF protein, but the rps16 precursor was not. Competition binding experiments indicate that G-residues seem to play a role in RNA-protein interaction. RNA-binding activity of the trnK intron encoded polypeptide is consistent with its suggested function as a plastid maturase, hence justifying the assignment matK for this gene.

Structure and expression characteristics of the chloroplast DNA region containing the split gene for tRNA(Gly) (UCC) from mustard (Sinapis alba L.).

The mustard chloroplast gene trnG-UCC is split by a 717-bp group-II intron. Northern hybridization and RNase protection experiments suggest cotranscription with the upstream psbK-psbI operon, but not with the downstream trnR-UCU gene. The ends of most RNase-protected fragments between psbI and trnG correlate with the position of two potential stem-loop structures in this region, which could act as RNA processing elements. However, one RNA 5' end, approximately 75 bp upstream of the trnG 5' exon, does not so correlate and is preceded by prokaryotic-type '-10' and '-35' sequence elements. This suggests the possibility that a fraction of the trnG transcripts is initiated here. All precursor transcripts spanning the trnG region seem to have a common 3' end, which was located 117 bp downstream from the 3' exon, immediately after a stem-loop region. During seedling development, the major 0.8-0.9-kb trnG precursor transcripts show a transient maximum level at around 48 h after sowing, at a time when the mature tRNA begins to accumulate to constant levels. No significant differences in transcript patterns were observed either in the light or in darkness.