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Background: DIALIGN-T is a reimplementation of the multiple-alignment program DIALIGN. Due to several algorithmic improvements, it produces significantly better alignments on locally and globally related sequence sets than previous versions of DIALIGN. However, like the original implementation of the program, DIALIGN-T uses a a straight-forward greedy approach to assemble multiple alignments from local pairwise sequence similarities. Such greedy approaches may be vulnerable to spurious random similarities and can therefore lead to suboptimal results. In this paper, we present DIALIGN-TX, a substantial improvement of DIALIGN-T that combines our previous greedy algorithm with a progressive alignment approach. Results:Our new heuristic produces significantly better alignments, especially on globally related sequences, without increasing the CPU time and memory consumption exceedingly. The new method is based on a guide tree; to detect possible spurious sequence similarities, it employs a vertex-cover approximation on a conflict graph. We performed benchmarking tests on a large set of nucleic acid and protein sequences For protein benchmarks we used the benchmark database BALIBASE 3 and an updated release of the database IRMBASE 2 for assessing the quality on globally and locally related sequences, respectively. For alignment of nucleic acid sequences, we used BRAliBase II for global alignment and a newly developed database of locally related sequences called DIRM-BASE 1. IRMBASE 2 and DIRMBASE 1 are constructed by implanting highly conserved motives at random positions in long unalignable sequences. Conclusion:On BALIBASE3, our new program performs significantly better than the previous program DIALIGN-T and outperforms the popular global aligner CLUSTAL W, though it is still outperformed by programs that focus on global alignment like MAFFT, MUSCLE and T-COFFEE. On the locally related test sets in IRMBASE 2 and DIRM-BASE 1, our method outperforms all other programs while MAFFT E-INSi is the only method that comes close to the performance of

Transcription of plastid genes and transcript accumulation were investigated in white leaves of the albostrians mutant of barley (Hordeum vulgare) and in heat‐bleached leaves of rye (Secale cereale) as well as in normal green leaves of both species. Cells of white leaves of the mutant and cells of heat‐bleached leaves bear undifferentiated plastids lacking ribosomes and, consequently, plastid translation products, among them the subunits of a putative chloroplast RNA polymerase encoded by the plastid genes rpoA, B, C1 and C2. The following results were obtained. (i) Plastid genes are transcribed despite the lack of chloroplast gene‐encoded RNA polymerase subunits. The plastid origin of these transcripts was proven. This finding provides evidence for the existence of a plastid RNA polymerase encoded entirely by nuclear genes. (ii) Transcripts of the rpo genes and of rps15, but not of genes involved in photosynthesis and related processes (psbA, rbcL, atpI‐H), were abundantly accumulated in ribosome‐deficient plastids. In contrast, chloroplasts accumulated transcripts of photosynthetic, but not of the rpo genes. (iii) Differences in transcript accumulation between chloroplasts and ribosome‐deficient plastids are due to different relative transcription rates and different transcript stability. (iv) The observed differences in transcription are not caused by an altered pattern of methylation of plastid DNA. Thus, the prokaryotic plastid genome of higher plants is transcribed by two RNA polymerases. The observed differences in transcription between chloroplasts and undifferentiated plastids might reflect different functions of the two enzymes.

We have completed identification of all the ribosomal proteins (RPs) in spinach plastid (chloroplast) ribosomal 50 S subunit via a proteomic approach using twodimensional electrophoresis, electroblotting/protein sequencing, high performance liquid chromatography purification, polymerase chain reaction-based screening of cDNA library/nucleotide sequencing, and mass spectrometry (reversed-phase HPLC coupled to electrospray ionization mass spectrometry and electrospray ionization mass spectrometry). Spinach plastid 50 S subunit comprises 33 proteins, of which 31 are orthologues of Escherichia coli RPs and two are plastid-specific RPs The plastid (chloroplast) ribosome is a plant-specific, organelle ribosome that produces proteins encoded by the plastid genome. Plastid ribosomes are responsible for the synthesis of huge amounts of biomass, since the large subunit of ribulose 1,5-bisphophate carboxylase/oxygenase (a most abundant protein in the biosphere) is synthesized in plastids. Plastid ribosomes are very similar to the eubacterial 70 S-type ribosome, in composition and general mode of function (1-4). The rRNAs and most of the characterized ribosomal proteins (RPs) 1 in plastid ribosomes also bear close resemblance to the corresponding components so far identified in cyanobacteria, a correlation supporting the importance of endosymbiotic theory in plastid evolution (5).The Escherichia coli ribosome, the most well studied of the eubacterial ribosomes (6), is composed of 21 RPs in the 30 S subunit and 33 RPs in the 50 S subunit. Two more possible E. coli RPs have been suggested: protein Y, the product of E. coli yfia gene (7), bearing a distant sequence homology to a chloroplast-specific RP (PSRP-1), and a protein designated S22 (8). Post-translational modifications are found in many E. coli RPs, although a modification in L16 (Arg 81 ) remains yet to be characterized (see "Results" for plastid L16). We have recently identified all the RPs in spinach plastid 30 S ribosomal subunit, including all its PSRPs and many post-translational modifications (83). The number of RPs in plastid 50 S subunits has so far only been estimated (ϳ35-39; reviewed in Ref.2) and has not been determined.Although the constituents of plastid translational machinery in general are similar to those of E. coli, the genes are distributed in two genome compartments: the plastid and the nucleus. The rRNA and tRNA genes are located in the plastid genome, whereas the genes for processing/modification enzymes, aminoacyl tRNA synthetases, and 60% of the RPs are located in the nuclear genome (1-4). The plastid translation system also differs from the eubacterial system in other significant ways, e.g. chloroplast mRNA is often edited (9); about 60% of chloroplast mRNAs lack canonical ribosome-binding sites found in E. coli mRNAs (10); mRNA levels in chloroplasts remain relatively unchanged through dark/light transitions, whereas protein

Six ribosomal proteins are specific to higher plant chloroplast ribosomes [Subramanian, A.R. (1993) Trends Biochem. Sci.18, 177–180]. Three of them have been fully characterized [Yamaguchi, K., von Knoblauch, K. & Subramanian, A. R. (2000) J. Biol. Chem. 275, 28455–28465; Yamaguchi, K. & Subramanian, A. R. (2000) J. Biol. Chem. 275, 28466–28482]. The remaining three plastid‐specific ribosomal proteins (PSRPs), all on the small subunit, have now been characterized (2D PAGE, HPLC, N‐terminal/internal peptide sequencing, electrospray ionization MS, cloning/ sequencing of precursor cDNAs). PSRP‐3 exists in two forms (α/β, N‐terminus free and blocked by post‐translational modification), whereas PSRP‐2 and PSRP‐4 appear, from MS data, to be unmodified. PSRP‐2 contains two RNA‐binding domains which occur in mRNA processing/stabilizing proteins (e.g. U1A snRNP, poly(A)‐binding proteins), suggesting a possible role for it in the recruiting of stored chloroplast mRNAs for active protein synthesis. PSRP‐3 is the higher plant orthologue of a hypothetical protein (ycf65 gene product), first reported in the chloroplast genome of a red alga. The ycf65 gene is absent from the chloroplast genomes of higher plants. Therefore, we suggest that Psrp‐3/ycf65, encoding an evolutionarily conserved chloroplast ribosomal protein, represents an example of organelle‐to‐nucleus gene transfer in chloroplast evolution. PSRP‐4 shows strong homology with Thx, a small basic ribosomal protein of Thermus thermophilus 30S subunit (with a specific structural role in the subunit crystallographic structure), but its orthologues are absent from Escherichia coli and the photosynthetic bacterium Synechocystis. We would therefore suggest that PSRP‐4 is an example of gene capture (via horizontal gene transfer) during chloro‐ribosome emergence. Orthologues of all six PSRPs are identifiable in the complete genome sequence of Arabidopsis thaliana and in the higher plant expressed sequence tag database. All six PSRPs are nucleus‐encoded. The cytosolic precursors of PSRP‐2, PSRP‐3, and PSRP‐4 have average targeting peptides (62, 58, and 54 residues long), and the mature proteins are of 196, 121, and 47 residues length (molar masses, 21.7, 13.8 and 5.2 kDa), respectively. Functions of the PSRPs as active participants in translational regulation, the key feature of chloroplast protein synthesis, are discussed and a model is proposed.

Identification of all the protein components of a plastid (chloroplast) ribosomal 30 S subunit has been achieved, using two-dimensional gel electropholesis, high performance liquid chromatography purification, N-terminal sequencing, polymerase chain reactionbased screening of cDNA library, nucleotide sequencing, and mass spectrometry (electrospray ionization, matrixassisted laser desorption/ionization time-of-flight, and reversed-phase HPLC coupled with electrospray ionization mass spectrometry). 25 proteins were identified, of which 21 are orthologues of all Escherichia coli 30 S ribosomal proteins (S1-S21), and 4 are plastid-specific ribosomal proteins (PSRPs) that have no homologues in the mitochondrial, archaebacterial, or cytosolic ribosomal protein sequences in data bases. 12 of the 25 plastid 30 S ribosomal proteins (PRPs) are encoded in the plastid genome, whereas the remaining 13 are encoded by the nuclear genome. Post-translational transit peptide cleavage sites for the maturation of the 13 cytosolically synthesized PRPs, and post-translational N-terminal processing in the maturation of the 12 plastid synthesized PRPs are described. Post-translational modifications in several PRPs were observed: ␣-N-acetylation of S9, N-terminal processings leading to five mature forms of S6 and two mature forms of S10, C-terminal and/or internal modifications in S1, S14, S18, and S19, leading to two distinct forms differing in mass and/or charge (the corresponding modifications are not observed in E. coli). The four PSRPs in spinach plastid 30 S ribosomal subunit (PSRP-1, 26.8 kDa, pI 6.2; PSRP-2, 21.7 kDa, pI 5.0; PSRP-3, 13.8 kDa, pI 4.9; PSRP-4, 5.2 kDa, pI 11.8) comprise 16% (67.6 kDa) of the total protein mass of the 30 S subunit (429.3 kDa). PSRP-1 and PSRP-3 show sequence similarities with hypothetical photosynthetic bacterial proteins, indicating their possible origins in photosynthetic bacteria. We propose the hypothesis that PSRPs form a "plastid translational regulatory module" on the 30 S ribosomal subunit structure for the possible mediation of nuclear factors on plastid translation.

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Background: We present a complete re-implementation of the segment-based approach to multiple protein alignment that contains a number of improvements compared to the previous version 2.2 of DIALIGN. This previous version is superior to Needleman-Wunsch-based multialignment programs on locally related sequence sets. However, it is often outperformed by these methods on data sets with global but weak similarity at the primary-sequence level.