Nonessential plastid-encoded ribosomal proteins in tobacco: a developmental role for plastid translation and implications for reductive genome evolution

Plastid genomes of higher plants contain a conserved set of ribosomal protein genes. Although plastid translational activity is essential for cell survival in tobacco (Nicotiana tabacum), individual plastid ribosomal proteins can be nonessential. Candidates for nonessential plastid ribosomal proteins are ribosomal proteins identified as nonessential in bacteria and those whose genes were lost from the highly reduced plastid genomes of nonphotosynthetic plastid-bearing lineages (parasitic plants, apicomplexan protozoa). Here we report the reverse genetic analysis of seven plastid-encoded ribosomal proteins that meet these criteria. We have introduced knockout alleles for the corresponding genes into the tobacco plastid genome. Five of the targeted genes (ribosomal protein of the large subunit22 [rpl22], rpl23, rpl32, ribosomal protein of the small subunit3 [rps3], and rps16) were shown to be essential even under heterotrophic conditions, despite their loss in at least some parasitic plastid-bearing lineages. This suggests that nonphotosynthetic plastids show elevated rates of gene transfer to the nuclear genome. Knockout of two ribosomal protein genes, rps15 and rpl36, yielded homoplasmic transplastomic mutants, thus indicating nonessentiality. Whereas Δrps15 plants showed only a mild phenotype, Δrpl36 plants were severely impaired in photosynthesis and growth and, moreover, displayed greatly altered leaf morphology. This finding provides strong genetic evidence that chloroplast translational activity influences leaf development, presumably via a retrograde signaling pathway.     

Differential expression of house-keeping genes of Aspergillus nidulans during sexual development

The rpl3 gene and the rpl37 gene for Aspergillus nidulans ribosomal protein L3 (RPL3) and RPL37, which were identified as located on chromosome I and chromosome III, respectively, were isolated from chromosome-specific cosmid libraries. The nucleotide sequences of both of the rpl3 gene and the rpl37 gene identified the ORFs of 392 amino acids and 92 amino acids, respectively. Both of the two genes were present in a single copy. The expression of both genes together with two other house-keeping genes, the rps16 gene for RPS16 and the gene for gamma-actin, was analyzed during sexual development. All four genes showed nearly identical expression patterns in that each gene expression reached its maximum after 2 h, decreased thereafter, and increased again after 30-40 h of induction of sexual development.     

Complete nucleotide sequence of the S10-spc operon of phytoplasma: gene organization and genetic code resemble those of Bacillus subtilis

An 11.4-kbp region of genomic DNA containing the complete S10-spc operon was constructed by an integrative mapping technique with eight plasmid vectors carrying ribosomal protein sequences from onion yellows phytoplasma. Southern hybridization analysis indicated that phytoplasmal S10-spc is a single-copy operon. This is the first complete S10-spc operon of a phytoplasma to be reported, although only a part of six serial genes of the S10 operon is reported previously. The operon has a context of 5'-rps10, rpl3, rpl4, rpl23, rpl2, rps19, rpl22, rps3, rpl16, rpl29, rps17, rpl14, rpl24, rpl5, rps14, rps8, rpl6, rpl18, rps5, rpl30, rpl15, SecY-3', and is composed of 21 ribosomal protein subunit genes and a SecY protein translocase subunit gene. Resembling Bacillus, this operon contains an rpl30 gene that other mollicutes (Mycoplasma genitalium, M. pneumoniae, and M. pulmonis) lack. A phylogenetic tree based on the rps3 sequence showed that phytoplasmas are phylogenetically closer to acholeplasmas and bacillus than to mycoplasmas. In the S10-spc operon, translation may start from either a GTG codon or an ATG codon, and stop at a TGA codon, as has been reported for acholeplasmas and bacillus. However, in mycoplasmas, GTG was found as a start codon, and TGA was found not as a stop codon, but instead as a tryptophan codon. These data derived from the gene organization, and the genetic code deviation support the hypothesis that phytoplasmal genes resemble those of acholeplasmas and Bacillus more than those of other mollicutes.     

Cotranscription of the S10- and spc-like operons in spinach chloroplasts and identification of three of their gene products

The organisation and expression of the rpl22, rps3, rpl16 and rpl14 genes, which belong to the S10- and spc-like operons of spinach chloroplasts, have been studied. Northern experiments and nuclease S1 mapping show that the two operon-like groups of genes are cotranscribed. It is demonstrated that the intron-containing rpl16 gene is spliced in vivo. Based on amino acid composition and protein sequence data, the products of the rpl22, rpl16 and rpl14 genes are identified respectively as the spinach chloroplast ribosomal proteins CS-L13, CS-L24 and CS-L29. The rpl22 gene product is a 5S rRNA binding protein and therefore is distinguishable from the homologous Escherichia coli L22 ribosomal protein.     

The mapping of seven intron-containing ribosomal protein genes shows they are unlinked in the human genome.

Mammalian ribosomal protein (rp) genes are members of multigene families which are composed predominantly of multiple processed pseudogenes and one functional intron-containing gene. The presence of multiple pseudogenes has hampered the isolation and study of the functional rp genes. We have recently developed a polymerase chain reaction (PCR)-based strategy for the detection of intron-containing genes in the presence of multiple pseudogenes (B. Davies, S. Feo, E. Heard, and M. Fried, 1989, Proc. Natl. Acad. Sci. USA 86: 6691-6695). We have used this technique to identify the intron-containing PCR products of seven human rp genes (rpL19, rpL30, rpL35a, rpL36a, rpS6, rpS11, rpS17) and to map their chromosomal locations. No linkage was found between any of these seven rp genes nor was linkage found to the three other rp genes previously mapped. The wide distribution of the rp genes throughout the human genome strongly suggests that the coordinate regulation of the expression of mammalian ribosomal proteins in response to the cell's varying requirements for protein synthesis is not a result of cis activation of chromosomal regions but is mediated by trans-acting factors.     

Ras-related genes in Dictyostelium discoideum

Dictyostelium discoideum, like other eukaryotes, has been shown to express several ras-related genes. Two gene products, Ddras and DdrasG, are highly conserved relative to the human ras proteins. Ddras is expressed at the pseudoplasmodial stage of development, whereas DdrasG is expressed in vegetative cells and during early development. In addition, Dictyostelium possesses three ras-related genes, SAS1, SAS2 and Ddrap1, whose gene products are only partially conserved relative to those of the ras genes. The expression of these three genes is also developmentally regulated.     

Cytoplasmic ribosomal protein genes of the fission yeast Schizosaccharomyces pombe display a unique promoter type: a suggestion for nomenclature of cytoplasmic ribosomal proteins in databases.

We identified 34 new ribosomal protein genes in the Schizosaccharomyces pombe database at the Sanger Centre coding for 30 different ribosomal proteins. All contain the Homol D-box in their promoter. We have shown that Homol D is, in this promoter type, the TATA-analogue. Many promoters contain the Homol E-box, which serves as a proximal activation sequence. Furthermore, comparative sequence analysis revealed a ribosomal protein gene encoding a protein which is the equivalent of the mammalian ribosomal protein L28. The budding yeast Saccharomyces cerevisiae has no L28 equivalent. Over the past 10 years we have isolated and characterized nine ribosomal protein (rp) genes from the fission yeast S.pombe . This endeavor yielded promoters which we have used to investigate the regulation of rp genes. Since eukaryotic ribosomal proteins are remarkably conserved and several rp genes of the budding yeast S.cerevisiae were sequenced in 1985, we probed DNA fragments encoding S.cerevisiae ribosomal proteins with genomic libraries of S.pombe . The deduced amino acid sequence of the different isolated rp genes of fission yeast share between 65 and 85% identical amino acids with their counterparts of budding yeast.     

Transfer of rpl22 to the nucleus greatly preceded its loss from the chloroplast and involved the gain of an intron.

Most chloroplast and mitochondrial proteins are encoded by nuclear genes that once resided in the organellar genomes. Transfer of most of these genes appears to have occurred soon after the endosymbiotic origin of organelles, and so little is known about the process. Our efforts to understand how chloroplast genes are functionally transferred to the nuclear genome have led us to discover the most recent evolutionary gene transfer yet described. The gene rpl22, encoding chloroplast ribosomal protein CL22, is present in the chloroplast genome of all plants examined except legumes, while a functional copy of rpl22 is located in the nucleus of the legume pea. The nuclear rpl22 gene has acquired two additional domains relative to its chloroplast ancestor: an exon encoding a putative N-terminal transit peptide, followed by an intron which separates this first exon from the evolutionarily conserved, chloroplast-derived portion of the gene. This gene structure suggests that the transferred region may have acquired its transit peptide by a form of exon shuffling. Surprisingly, phylogenetic analysis shows that rpl22 was transferred to the nucleus in a common ancestor of all flowering plants, at least 100 million years preceding its loss from the legume chloroplast lineage.     

Effect of RP51 gene dosage alterations on ribosome synthesis in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae ribosomal protein rp51 is encoded by two interchangeable genes, RP51A and RP51B. We altered the RP51 gene dose by creating deletions of the RP51A or RP51B genes or both. Deletions of both genes led to spore inviability, indicating that rp51 is an essential ribosomal protein. From single deletion studies in haploid cells, we concluded that there was no intergenic dosage compensation at the level of mRNA abundance or mRNA utilization (translational efficiency), although phenotypic analysis had previously indicated a small compensation effect on growth rate. Similarly, deletions in diploid strains indicated that no strong mechanisms exist for intragenic dosage compensation; in all cases, a decreased dose of RP51 genes was characterized by a slow growth phenotype. A decreased dose of RP51 genes also led to insufficient amounts of 40S ribosomal subunits, as evidenced by a dramatic accumulation of excess 60S ribosomal subunits. We conclude that inhibition of 40S synthesis had little or no effect on the synthesis of the 60S subunit components. Addition of extra copies of rp51 genes led to extra rp51 protein synthesis. The additional rp51 protein was rapidly degraded. We propose that rp51 and perhaps many ribosomal proteins are normally oversynthesized, but the unassembled excess is degraded, and that the apparent compensation seen in haploids, i.e., the fact that the growth rate of mutant strains is less depressed than the actual reduction in mRNA, is a consequence of this excess which is spared from proteolysis under this circumstance.     

Discovery of the rpl10 Gene in Diverse Plant Mitochondrial Genomes and Its Probable Replacement by the Nuclear Gene for Chloroplast RPL10 in Two Lineages of Angiosperms

Mitochondrial genomes of plants are much larger than those of mammals and often contain conserved open reading frames (ORFs) of unknown function. Here, we show that one of these conserved ORFs is actually the gene for ribosomal protein L10 (rpl10) in plant. No rpl10 gene has heretofore been reported in any mitochondrial genome other than the exceptionally gene-rich genome of the protist Reclinomonas americana. Conserved ORFs corresponding to rpl10 are present in a wide diversity of land plant and green algal mitochondrial genomes. The mitochondrial rpl10 genes are transcribed in all nine land plants examined, with five seed plant genes subject to RNA editing. In addition, mitochondrial-rpl10-like cDNAs were identified in EST libraries from numerous land plants. In three lineages of angiosperms, rpl10 is either lost from the mitochondrial genome or a pseudogene. In two of them (Brassicaceae and monocots), no nuclear copy of mitochondrial rpl10 is identifiably present, and instead a second copy of nuclear-encoded chloroplast rpl10 is present. Transient assays using green fluorescent protein indicate that this duplicate gene is dual targeted to mitochondria and chloroplasts. We infer that mitochondrial rpl10 has been functionally replaced by duplicated chloroplast counterparts in Brassicaceae and monocots.     

Euglena gracilis chloroplast ribosomal protein operon: a new chloroplast gene for ribosomal protein L5 and description of a novel organelle intron category designated group III

We describe the structure (3840 bp) of a novel Euglena gracilis chloroplast ribosomal protein operon that encodes the five genes rpl16-rpl14-rpl5-rps8-rpl36. The gene organization resembles the spc and the 3'-end of the S10 ribosomal protein operons of E. coli. The rpl5 is a new chloroplast gene not previously reported for any chloroplast genome to date and also not described as a nuclear-encoded, chloroplast protein gene. The operon contains at least 7 introns. We present evidence from primer extension analysis of chloroplast RNA for the correct in vivo splicing of five of the introns. Two of the introns within the rps8 gene flank an 8 bp exon, the smallest exon yet characterized in a chloroplast gene. Three introns resemble the classical group II introns of organelle genomes. The remaining 4 introns appear to be unique to the Euglena chloroplast DNA. They are uniform in size (95-109 nt), share common features with each other and are distinct from both group I and group II introns. We designate this new intron category as 'group III'.