R-protein Genes Research Articles

Ribosomal Protein Cluster Organization in Asgard Archaea.

It has been proposed that the superphylum of Asgard Archaea may represent a historical link between the Archaea and Eukarya. Following the discovery of the Archaea, it was soon appreciated that archaeal ribosomes were more similar to those of Eukarya rather than Bacteria. Coupled with other eukaryotic-like features, it has been suggested that the Asgard Archaea may be directly linked to eukaryotes. However, the genomes of Bacteria and non-Asgard Archaea generally organize ribosome-related genes into clusters that likely function as operons. In contrast, eukaryotes typically do not employ an operon strategy. To gain further insight into conservation of the r-protein genes, the genome order of conserved ribosomal protein (r-protein) coding genes was identified in 17 Asgard genomes (thirteen complete genomes and four genomes with less than 20 contigs) and compared with those found previously in non-Asgard archaeal and bacterial genomes. A universal core of two clusters of 14 and 4 cooccurring r-proteins, respectively, was identified in both the Asgard and non-Asgard Archaea. The equivalent genes in the E. coli version of the cluster are found in the S10 and spc operons. The large cluster of 14 r-protein genes (uS19-uL22-uS3-uL29-uS17 from the S10 operon and uL14-uL24-uL5-uS14-uS8-uL6-uL18-uS5-uL30-uL15 from the spc operon) occurs as a complete set in the genomes of thirteen Asgard genomes (five Lokiarchaeotes, three Heimdallarchaeotes, one Odinarchaeote, and four Thorarchaeotes). Four less conserved clusters with partial bacterial equivalents were found in the Asgard. These were the L30e (str operon in Bacteria) cluster, the L18e (alpha operon in Bacteria) cluster, the S24e-S27ae-rpoE1 cluster, and the L31e, L12..L1 cluster. Finally, a new cluster referred to as L7ae was identified. In many cases, r-protein gene clusters/operons are less conserved in their organization in the Asgard group than in other Archaea. If this is generally true for nonribosomal gene clusters, the results may have implications for the history of genome organization. In particular, there may have been an early transition to or from the operon approach to genome organization. Other nonribosomal cellular features may support different relationships. For this reason, it may be important to consider ribosome features separately.

Discovery of 20 novel ribosomal leader candidates in bacteria and archaea

BackgroundRNAs perform many functions in addition to supplying coding templates, such as binding proteins. RNA-protein interactions are important in multiple processes in all domains of life, and the discovery of additional protein-binding RNAs expands the scope for studying such interactions. To find such RNAs, we exploited a form of ribosomal regulation. Ribosome biosynthesis must be tightly regulated to ensure that concentrations of rRNAs and ribosomal proteins (r-proteins) match. One regulatory mechanism is a ribosomal leader (r-leader), which is a domain in the 5′ UTR of an mRNA whose genes encode r-proteins. When the concentration of one of these r-proteins is high, the protein binds the r-leader in its own mRNA, reducing gene expression and thus protein concentrations. To date, 35 types of r-leaders have been validated or predicted.ResultsBy analyzing additional conserved RNA structures on a multi-genome scale, we identified 20 novel r-leader structures. Surprisingly, these included new r-leaders in the highly studied organisms Escherichia coli and Bacillus subtilis. Our results reveal several cases where multiple unrelated RNA structures likely bind the same r-protein ligand, and uncover previously unknown r-protein ligands. Each r-leader consistently occurs upstream of r-protein genes, suggesting a regulatory function. That the predicted r-leaders function as RNAs is supported by evolutionary correlations in the nucleotide sequences that are characteristic of a conserved RNA secondary structure. The r-leader predictions are also consistent with the locations of experimentally determined transcription start sites.ConclusionsThis work increases the number of known or predicted r-leader structures by more than 50%, providing additional opportunities to study structural and evolutionary aspects of RNA-protein interactions. These results provide a starting point for detailed experimental studies.

Interaction between the assembly of the ribosomal subunits: Disruption of 40S ribosomal assembly causes accumulation of extra-ribosomal 60S ribosomal protein uL18/L5.

Inhibition of the synthesis of an essential ribosomal protein (r-protein) abrogates the assembly of its cognate subunit, while assembly of the other subunit continues. Ribosomal components that are not stably incorporated into ribosomal particles due to the disrupted assembly are rapidly degraded. The 60S protein uL18/L5 is an exception and this protein accumulates extra-ribosomally during inhibition of 60S assembly. Since the r-proteins in each ribosomal subunit are essential only for the formation of their cognate subunit, it would be predicted that accumulation of extra-ribosomal uL18/L5 is specific to restriction of 60S assembly and does not occur abolition of 40S assembly. Contrary to this prediction, we report here that repression of 40S r-protein genes does lead to accumulation of uL18/L5 outside of the ribosome. Furthermore, the effect varies depending on which 40S ribosomal protein is repressed. Our results also show extra-ribosomal uL18/L5 is formed during 60S assembly, not during degradation of mature cytoplasmic 60S subunits. Finally, we propose a model for the accumulation of extra-ribosomal uL18 in response to the abolition of 40S r-proteins.

Proteomic LC–MS analysis of Arabidopsis cytosolic ribosomes: Identification of ribosomal protein paralogs and re-annotation of the ribosomal protein genes

Arabidopsis thaliana cytosolic ribosomes are large complexes containing eighty-one distinct ribosomal proteins (r-proteins), four ribosomal RNAs (rRNA) and a plethora of associated (non-ribosomal) proteins. In plants, r-proteins of cytosolic ribosomes are each encoded by two to seven different expressed and similar genes, forming an r-protein family.Distinctions in the r-protein coding sequences of gene family members are a source of variation between ribosomes. We performed proteomic investigation of actively translating cytosolic ribosomes purified using both immunopurification and a classic sucrose cushion centrifugation-based protocol from plants of different developmental stages. Both 1D and 2D LC–MSE with data-independent acquisition as well as conventional data-dependent MS/MS procedures were applied. This approach provided detailed identification of 165 r-protein paralogs with high coverage based on proteotypic peptides. The detected r-proteins were the products of the majority (68%) of the 242 cytosolic r-protein genes encoded by the genome. A total of 70 distinct r-proteins were identified. Based on these results and information from DNA microarray and ribosome footprint profiling studies a re-annotation of Arabidopsis r-proteins and genes is proposed. This compendium of the cytosolic r-protein proteome will serve as a template for future investigations on the dynamic structure and function of plant ribosomes. Biological significanceTranslation is one of the most energy demanding processes in a living cell and is therefore carefully regulated. Translational activity is tightly linked to growth control and growth regulating mechanism. Recently established translational profiling technologies, including the profiling of mRNAs associated with polysomes and the mapping of ribosome footprints on mRNAs, have revealed that the expression of gene expression is often fine-tuned by differential translation of gene transcripts. The eukaryotic ribosome, the hub of these important processes, consists of close to eighty different proteins (depending on species) and four large RNAs assembled into two highly conserved subunits.In plants and to lesser extent in yeast, the r-proteins are encoded by more than one actively transcribed gene. As r-protein gene paralogs frequently do not encode identical proteins and are regulated by growth conditions and development, in vivo ribosomes are heterogeneous in their protein content. The regulatory and physiological importance of this heterogeneity is unknown. Here, an improved annotation of the more than two hundred r-protein genes of Arabidopsis is presented that combines proteomic and advanced mRNA expression data. This proteomic investigation and re-annotation of Arabidopsis ribosomes establish a base for future investigations of translational control in plants.

Repressed synthesis of ribosomal proteins generates protein-specific cell cycle and morphological phenotypes

The biogenesis of ribosomes is coordinated with cell growth and proliferation. Distortion of the coordinated synthesis of ribosomal components affects not only ribosome formation, but also cell fate. However, the connection between ribosome biogenesis and cell fate is not well understood. To establish a model system for inquiries into these processes, we systematically analyzed cell cycle progression, cell morphology, and bud site selection after repression of 54 individual ribosomal protein (r-protein) genes in Saccharomyces cerevisiae. We found that repression of nine 60S r-protein genes results in arrest in the G2/M phase, whereas repression of nine other 60S and 22 40S r-protein genes causes arrest in the G1 phase. Furthermore, bud morphology changes after repression of some r-protein genes. For example, very elongated buds form after repression of seven 60S r-protein genes. These genes overlap with, but are not identical to, those causing the G2/M cell cycle phenotype. Finally, repression of most r-protein genes results in changed sites of bud formation. Strikingly, the r-proteins whose repression generates similar effects on cell cycle progression cluster in the ribosome physical structure, suggesting that different topological areas of the precursor and/or mature ribosome are mechanistically connected to separate aspects of the cell cycle.

Phylogenomics of Prokaryotic Ribosomal Proteins

Archaeal and bacterial ribosomes contain more than 50 proteins, including 34 that are universally conserved in the three domains of cellular life (bacteria, archaea, and eukaryotes). Despite the high sequence conservation, annotation of ribosomal (r-) protein genes is often difficult because of their short lengths and biased sequence composition. We developed an automated computational pipeline for identification of r-protein genes and applied it to 995 completely sequenced bacterial and 87 archaeal genomes available in the RefSeq database. The pipeline employs curated seed alignments of r-proteins to run position-specific scoring matrix (PSSM)-based BLAST searches against six-frame genome translations, mitigating possible gene annotation errors. As a result of this analysis, we performed a census of prokaryotic r-protein complements, enumerated missing and paralogous r-proteins, and analyzed the distributions of ribosomal protein genes among chromosomal partitions. Phyletic patterns of bacterial and archaeal r-protein genes were mapped to phylogenetic trees reconstructed from concatenated alignments of r-proteins to reveal the history of likely multiple independent gains and losses. These alignments, available for download, can be used as search profiles to improve genome annotation of r-proteins and for further comparative genomics studies.

Differential contributions of ribosomal protein genes to Arabidopsis thaliana leaf development

In Arabidopsis thaliana, mutations in genes encoding ribosomal proteins (r-proteins) perturb various developmental processes. Whether these perturbations are caused by overall ribosome insufficiency or partial dysfunction of the ribosome caused by deficiency of a particular ribosomal protein is not known. To distinguish these possibilities, a comparative study using several r-protein mutants was required. Here, we identified mutations in 11 r-protein genes from previously isolated denticulata and pointed-leaves mutants. Most of these mutations were associated with pointed leaves, with reduced growth due to a decrease in the number or size of palisade mesophyll and pavement cells. In addition, leaf abaxialization was usually observed when these r-protein mutations were combined with asymmetric leaves1 (as1) and as2 mutations. These results suggest that the establishment of leaf polarity is highly sensitive to ribosome functionality in general. However, several r-protein mutants showed a preference towards a specific developmental defect. For example, rpl4d mutations did not affect cell proliferation but caused strong abaxialization of leaves in the as1 and as2 backgrounds. On the other hand, rps28b enhanced leaf abaxialization of as2 to a weaker extent than expected on the basis of its negative effect on cell proliferation. In addition, hypomorphic rps6a alleles had the strongest effects on most of the phenotypes examined. These findings suggest that deficiencies in these three r-protein genes lead to production of dysfunctional ribosomes. Depending on their structural abnormalities, dysfunctional ribosomes may affect translation of specific transcripts involved in the regulation of some leaf developmental processes.

Transcript Profiling Provides Evidence of Functional Divergence and Expression Networks among Ribosomal Protein Gene Paralogs inBrassica napus

The plant ribosome is composed of 80 distinct ribosomal (r)-proteins. In Arabidopsis thaliana, each r-protein is encoded by two or more highly similar paralogous genes, although only one copy of each r-protein is incorporated into the ribosome. Brassica napus is especially suited to the comparative study of r-protein gene paralogs due to its documented history of genome duplication as well as the recent availability of large EST data sets. We have identified 996 putative r-protein genes spanning 79 distinct r-proteins in B. napus using EST data from 16 tissue collections. A total of 23,408 tissue-specific r-protein ESTs are associated with this gene set. Comparative analysis of the transcript levels for these unigenes reveals that a large fraction of r-protein genes are differentially expressed and that the number of paralogs expressed for each r-protein varies extensively with tissue type in B. napus. In addition, in many cases the paralogous genes for a specific r-protein are not transcribed in concert and have highly contrasting expression patterns among tissues. Thus, each tissue examined has a novel r-protein transcript population. Furthermore, hierarchical clustering reveals that particular paralogs for nonhomologous r-protein genes cluster together, suggesting that r-protein paralog combinations are associated with specific tissues in B. napus and, thus, may contribute to tissue differentiation and/or specialization. Altogether, the data suggest that duplicated r-protein genes undergo functional divergence into highly specialized paralogs and coexpression networks and that, similar to recent reports for yeast, these are likely actively involved in differentiation, development, and/or tissue-specific processes.

Differential requirement of ribosomal protein S6 by plant RNA viruses with different translation initiation strategies

Potyvirus infection has been reported to cause an increase in the mRNA transcripts of many plant ribosomal proteins (r-proteins). In this study, increased expression of r-protein mRNA transcripts was determined to occur in Nicotiana benthamiana during infection by potyviruses as well as a tobamovirus demonstrating that this response is not unique to potyviruses. Five r-protein genes, RPS6, RPL19, RPL13, RPL7, and RPS2, were silenced in N. benthamiana to test their roles in viral infection. The accumulation of both Turnip mosaic virus (TuMV), a potyvirus, and Tobacco mosaic virus (TMV), a tobamovirus, was dependent on RPL19, RPL13, RPL7, and RPS2. However, TMV was able to accumulate in RPS6-silenced plants while accumulation of TuMV and Tomato bushy stunt virus (TBSV) was abolished. These results demonstrate that cap-independent TuMV and TBSV require RPS6 for their accumulation, whereas accumulation of TMV is independent of RPS6.

Many nonuniversal archaeal ribosomal proteins are found in conserved gene clusters.

The genomic associations of the archaeal ribosomal proteins, (r-proteins), were examined in detail. The archaeal versions of the universal r-protein genes are typically in clusters similar or identical and to those found in bacteria. Of the 35 nonuniversal archaeal r-protein genes examined, the gene encoding L18e was found to be associated with the conserved L13 cluster, whereas the genes for S4e, L32e and L19e were found in the archaeal version of the spc operon. Eleven nonuniversal protein genes were not associated with any common genomic context. Of the remaining 19 protein genes, 17 were convincingly assigned to one of 10 previously unrecognized gene clusters. Examination of the gene content of these clusters revealed multiple associations with genes involved in the initiation of protein synthesis, transcription or other cellular processes. The lack of such associations in the universal clusters suggests that initially the ribosome evolved largely independently of other processes. More recently it likely has evolved in concert with other cellular systems. It was also verified that a second copy of the gene encoding L7ae found in some bacteria is actually a homolog of the gene encoding L30e and should be annotated as such.

Arabidopsis Ribosomal Proteins RPL23aA and RPL23aB Are Differentially Targeted to the Nucleolus and Are Disparately Required for Normal Development

Protein synthesis is catalyzed by the ribosome, a two-subunit enzyme comprised of four ribosomal RNAs and, in Arabidopsis (Arabidopsis thaliana), 81 ribosomal proteins (r-proteins). Plant r-protein genes exist as families of multiple expressed members, yet only one r-protein from each family is incorporated into any given ribosome, suggesting that many r-protein genes may be functionally redundant or development/tissue/stress specific. Here, we characterized the localization and gene-silencing phenotypes of a large subunit r-protein family, RPL23a, containing two expressed genes (RPL23aA and RPL23aB). Live cell imaging of RPL23aA and RPL23aB in tobacco with a C-terminal fluorescent-protein tag demonstrated that both isoforms accumulated in the nucleolus; however, only RPL23aA was targeted to the nucleolus with an N-terminal fluorescent protein tag, suggesting divergence in targeting efficiency of localization signals. Independent knockdowns of endogenous RPL23aA and RPL23aB transcript levels using RNA interference determined that an RPL23aB knockdown did not alter plant growth or development. Conversely, a knockdown of RPL23aA produced a pleiotropic phenotype characterized by growth retardation, irregular leaf and root morphology, abnormal phyllotaxy and vasculature, and loss of apical dominance. Comparison to other mutants suggests that the phenotype results from reduced ribosome biogenesis, and we postulate a link between biogenesis, microRNA-target degradation, and maintenance of auxin homeostasis. An additional RNA interference construct that coordinately silenced both RPL23aA and RPL23aB demonstrated that this family is essential for viability.

Analysis of the Arabidopsis Cytosolic Ribosome Proteome Provides Detailed Insights into Its Components and Their Post-translational Modification

Finding gene-specific peptides by mass spectrometry analysis to pinpoint gene loci responsible for particular protein products is a major challenge in proteomics especially in highly conserved gene families in higher eukaryotes. We used a combination of in silico approaches coupled to mass spectrometry analysis to advance the proteomics insight into Arabidopsis cytosolic ribosomal composition and its post-translational modifications. In silico digestion of all 409 ribosomal protein sequences in Arabidopsis defined the proportion of theoretical gene-specific peptides for each gene family and highlighted the need for low m/z cutoffs of MS ion selection for MS/MS to characterize low molecular weight, highly basic ribosomal proteins. We undertook an extensive MS/MS survey of the cytosolic ribosome using trypsin and, when required, chymotrypsin and pepsin. We then used custom software to extract and filter peptide match information from Mascot result files and implement high confidence criteria for calling gene-specific identifications based on the highest quality unambiguous spectra matching exclusively to certain in silico predicted gene- or gene family-specific peptides. This provided an in-depth analysis of the protein composition based on 1446 high quality MS/MS spectra matching to 795 peptide sequences from ribosomal proteins. These identified peptides from five gene families of ribosomal proteins not identified previously, providing experimental data on 79 of the 80 different types of ribosomal subunits. We provide strong evidence for gene-specific identification of 87 different ribosomal proteins from these 79 families. We also provide new information on 30 specific sites of co- and post-translational modification of ribosomal proteins in Arabidopsis by initiator methionine removal, N-terminal acetylation, N-terminal methylation, lysine N-methylation, and phosphorylation. These site-specific modification data provide a wealth of resources for further assessment of the role of ribosome modification in influencing translation in Arabidopsis.

Molecular evolutionary analyses of the Arabidopsis L7 ribosomal protein gene family

Cytoplasmic ribosomal protein (r-protein) genes in Arabidopsis thaliana are encoded by 80 multigene families that contain between two and seven members. Gene family members are typically similar at the protein sequence level, with the most divergent members of any gene family retaining 94% identity, on average. However, three Arabidopsis r-protein families – S15a, L7 and P2 – contain highly divergent family members. Here, we investigated the organization, structure, expression and molecular evolution of the L7 r-protein family. Phylogenetic analyses showed that L7 r-protein gene family members constitute two distinct phylogenetic groups. The first group including RPL7B, RPL7C and RPL7D has homologs in plants, animals and fungi. The second group represented by RPL7A is found in plants but has no orthologs from other fully-sequenced eukaryotic genomes. These two groups may have derived from a duplication event prior to the divergence of animals and plants. All four L7 r-protein genes are expressed and all exhibit a differential expression in inflorescence and flowers. RPL7A and RPL7B are less expressed than the other genes in all tissues analyzed. Molecular characterization of nucleic and protein sequences of L7 r-protein genes and analysis of their codon usage did not indicate any functional divergence. The probable evolution of an extra-ribosomal function of group 2 genes is discussed.

Ribosomal protein gene regulation: what about plants?

The ribosome is an intricate ribonucleoprotein complex with a multitude of protein constituents present in equimolar amounts. Coordination of the synthesis of these ribosomal proteins (r-proteins) presents a major challenge to the cell. Although most r-proteins are highly conserved, the mechanisms by which r-protein gene expression is regulated often differ widely among species. While the primary regulatory mechanisms coordinating r-protein synthesis in bacteria, yeast, and animals have been identified, the mechanisms governing the coordination of plant r-protein expression remain largely unexplored. In addition, plants are unique among eukaryotes in carrying multiple (often more than two) functional genes encoding each r-protein, which substantially complicates coordinate expression. A survey of the current knowledge regarding coordinated systems of r-protein gene expression in different model organisms suggests that vertebrate r-protein gene regulation provides a valuable comparison for plants.

Variation in transcript abundance among the four members of the Arabidopsis thaliana RIBOSOMAL PROTEIN S15a gene family

As a ribonucleoprotein complex, the plant ribosome consists of four ribosomal RNAs (rRNAs) and 75–92 ribosomal proteins (r-proteins), depending on the species. Arabidopsis thaliana r-protein genes exist in multi-gene families ranging in size from two to seven transcriptionally active members. The cytosolic RPS15a gene family consists of four members that, at the amino acid level, share 87–100% identity. The 5′ regulatory regions of the four genes contain many of the same putative regulatory elements but share only 46–49% nucleotide sequence identity. Semi-quantitative RT-PCR (sqRT-PCR) was used to determine RPS15a transcript abundance patterns and identify possible regulatory differences among the four genes. RPS15aC expression was not detected in any tissue under any treatment while transcript abundance for RPS15aA, - D and - F was highest in mitotically active tissues e.g. bud and flower. Seedlings showed increased transcript abundance following treatment with the cytokinin 6-benzylaminopurine (BAP) while the auxin indole acetic acid (IAA) induced an increase in RPS15aF abundance only. Abscisic acid (ABA) treatment resulted in decreased transcript abundance while gibberellic acid (GA 3) had little effect on all four genes. Similar trends were established for RPS15aA, -D and - F transcript abundance as a result of temperature mechanical stress. This study reports the differences in transcript abundance of the four cytosolic RPS15a genes in Arabidopsis and we suggest that RPS15a genes should no longer be considered ‘housekeeping’ genes.

Proteomic Characterization of Evolutionarily Conserved and Variable Proteins of Arabidopsis Cytosolic Ribosomes

Analysis of 80S ribosomes of Arabidopsis (Arabidopsis thaliana) by use of high-speed centrifugation, sucrose gradient fractionation, one- and two-dimensional gel electrophoresis, liquid chromatography purification, and mass spectrometry (matrix-assisted laser desorption/ionization time-of-flight and electrospray ionization) identified 74 ribosomal proteins (r-proteins), of which 73 are orthologs of rat r-proteins and one is the plant-specific r-protein P3. Thirty small (40S) subunit and 44 large (60S) subunit r-proteins were confirmed. In addition, an ortholog of the mammalian receptor for activated protein kinase C, a tryptophan-aspartic acid-domain repeat protein, was found to be associated with the 40S subunit and polysomes. Based on the prediction that each r-protein is present in a single copy, the mass of the Arabidopsis 80S ribosome was estimated as 3.2 MD (1,159 kD 40S; 2,010 kD 60S), with the 4 single-copy rRNAs (18S, 26S, 5.8S, and 5S) contributing 53% of the mass. Despite strong evolutionary conservation in r-protein composition among eukaryotes, Arabidopsis 80S ribosomes are variable in composition due to distinctions in mass or charge of approximately 25% of the r-proteins. This is a consequence of amino acid sequence divergence within r-protein gene families and posttranslational modification of individual r-proteins (e.g. amino-terminal acetylation, phosphorylation). For example, distinct types of r-proteins S15a and P2 accumulate in ribosomes due to evolutionarily divergence of r-protein genes. Ribosome variation is also due to amino acid sequence divergence and differential phosphorylation of the carboxy terminus of r-protein S6. The role of ribosome heterogeneity in differential mRNA translation is discussed.

Sequence variations of theS7 ribosomal protein gene in primitive cyprinid fishes: Implication on phylogenetic analysis

Cyprinidae is the largest fish family in the world and contains about 210 genera and 2010 species. Appropriate DNA markers must be selected for the phylogenetic analyses of Cyprinidae. In present study, the 1st intron of the S7 ribosomal protein (r-protein) gene is first used to examine the relationships among cyprinid fishes. The length of the 1st intron obtained by PCR amplification ranges from 655 to 859 bp in the 16 cyprinid species investigated, and is 602 bp inMyxocyprinus asiaticus. Out of the alignment of 925 nucleotide sites obtained, the parsimony informative sites are 499 and occupy 54% of the total sites. The results indicate that the 1st intron sequences of the S7 r-protein gene in cyprinids are rich in informative sites and vary remarkably in sequence divergence from 2.3% between close species to 66.6% between distant species. The bootstrap values of the interior nodes in the NJ (neighbor-joining) and MP (mostparsimony) trees based on the present S7 r-protein gene data are higher than those based on cytochrome b and the d-loop region respectively. Therefore, the 1st intron sequences of the S7 r-protein gene in cyprinids are sensitive enough for phylogenetic analyses, and the 1st intron is an appropriate genetic marker for the phylogenetic reconstruction of the taxa in different cyprinid subfamilies. However, attempts to discuss whether the present S7 r-protein gene data can be applied to the phylogeny of the taxa at the level of the family or the higher categories in Cypriniformes need further studies.

The Organization of Cytoplasmic Ribosomal Protein Genes in the Arabidopsis Genome

Eukaryotic ribosomes are made of two components, four ribosomal RNAs, and approximately 80 ribosomal proteins (r-proteins). The exact number of r-proteins and r-protein genes in higher plants is not known. The strong conservation in eukaryotic r-protein primary sequence allowed us to use the well-characterized rat (Rattus norvegicus) r-protein set to identify orthologues on the five haploid chromosomes of Arabidopsis. By use of the numerous expressed sequence tag (EST) accessions and the complete genomic sequence of this species, we identified 249 genes (including some pseudogenes) corresponding to 80 (32 small subunit and 48 large subunit) cytoplasmic r-protein types. None of the r-protein genes are single copy and most are encoded by three or four expressed genes, indicative of the internal duplication of the Arabidopsis genome. The r-proteins are distributed throughout the genome. Inspection of genes in the vicinity of r-protein gene family members confirms extensive duplications of large chromosome fragments and sheds light on the evolutionary history of the Arabidopsis genome. Examination of large duplicated regions indicated that a significant fraction of the r-protein genes have been either lost from one of the duplicated fragments or inserted after the initial duplication event. Only 52 r-protein genes lack a matching EST accession, and 19 of these contain incomplete open reading frames, confirming that most genes are expressed. Assessment of cognate EST numbers suggests that r-protein gene family members are differentially expressed.

Cross-genomic analysis of the translational systems of various organisms.

We have characterized the genes encoding ribosomal proteins (r-proteins) as well as other translation-related factors of 15 eubacteria and four archaebacteria, and the genes for the mitochondrial r-proteins of Saccharomyces cerevisiae by using the complete genomic nucleotide sequence data of these organisms. In eubacteria, including two species of Mycoplasma, the operon structure of the r-protein genes is well conserved, while their relative orientation and chromosomal location are quite divergent. The operon structure of the r-protein genes in archaebacteria, on the other hand, is quite different from eubacteria and also among themselves. In addition, many archaebacterial r-proteins show similarity to rat cytoplasmic r-proteins. Nonetheless, characteristic features of several genes encoding proteins of functional importance are well conserved throughout the bacterial species including archaebacteria, as well as in S. cerevisiae. We searched for the genes encoding mitochondrial r-proteins in yeast by combining informatics and genetic experiments. Furthermore, we characterized some of the r-proteins genes by exchanging portions between Escherichia coli and S. cerevisiae and performed functional analysis of some of the genes from different evolutionary points of view. Our work may be extended towards phylogenetic analysis of organisms producing secondary metabolites of various sorts.

Characterization and expression of a Neurospora crassa ribosomal protein gene, crp-7.

We have isolated and characterized a Neurospora crassa cytoplasmic ribosomal protein gene, named crp-7, which is found upstream of the photolyase gene. The deduced amino-acid sequence of this gene is highly homologous to the YS25 ribosomal protein of Saccharomyces cerevisiae. The crp-7 ORF consists of two exons which are separated by a short intron. The deduced polypeptide contains 87 amino acid residues and has a calculated molecular weight of 9.7 kDa. RFLP mapping showed that the crp-7 gene is located on the right arm of linkage group I. Southern blot hybridization analyses indicated that there is only one copy of the crp-7 gene in the N. crassa genome. Transcriptional elements, the Dde box, the Taq box and the CG element, that have been identified in other N. crassa ribosomal protein genes are observed in the promoter region of the crp-7 gene. The crp-7 mRNA levels were low in conidia and highest in young mycelia during vegetative growth. The mRNA levels of four r-protein genes, including the crp-7 gene, as well as the tef-1 gene encoding translational elongation factor 1 alpha, were raised following the treatment of mycelia with a low concentration of cycloheximide. This indicates that the expression of r-protein genes is under the control of so-called super-induction.