Nucleotide sequences ofCyanophora paradoxa cellular and cyanelle-associated 5S ribosomal RNAs: The cyanelle as a potential intermediate in plastid evolution

Summary The 5S ribosomal RNAs from the cell cytoplasm and cyanelle (photosynthetic organelle) ofCyanophora paradoxa have been isolated and sequenced. The cellular and cyanelle 5S rRNAs were 119 and 118 nucleotides in length, respectively. Both RNAs exhibited typical 5S secondary structure, but the primary sequence of the cellular species was clearly eukaryotic in nature, while that of the organellar species was prokaryotelike. The primary sequence of the cyanellar 5S rRNA was most homologous to cyanobacterial 5S sequences, yet possessed secondary-structural features characteristic of higher-plant chloroplast 5S rRNAs. Both sequence comparison and structural analysis indicated an evolutionary position for cyanelle 5S rRNA intermediate between blue-green alga and chloroplast 5S rRNAs.Contribution from the Department of Biochemistry, School of Agriculture and Life Sciences and School of Physical and Mathematical Sciences, North Carolina State University, Raleigh, North Carolina. This is paper no. 10259 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina 27695-7601, USA     

Cyanelle DNA from Cyanophora paradoxa exists in two forms due to intramolecular recombination

The DNA of cyanelles which are described as endosymbiotic cyanobacteria from Cyanophora paradoxa (strain LB555UTEX) is equivalent to 127 kilobasepairs. It is characterized by two inverted repeat segments, 10 kilobasepairs in size, which are separated from each other by long single-copy DNA segments of unequal size. This morphology of the chromosome is also found in the chloroplast DNA of most higher plants and some green algae. The cyanelle DNA exists in two forms of circular molecules which differ only in the orientation of the two single-copy DNA segments relative to each other. This is likely due to intramolecular recombination within the inverted repeat segments.     

Substitutional bias confounds inference of cyanelle origins from sequence data

Summary Available molecular and biochemical data offer conflicting evidence for the origin of the cyanelle of Cyanophora paradoxa. We show that the similarity of cyanelle and green chloroplast sequences is probably a result of these two lineages independently developing the same pattern of directional nucleotide change (substitutional bias). This finding suggests caution should be exercised in the interpretation of nucleotide sequence analyses that appear to favor the view of a common endosymbiont for the cyanelle and chlorophyll-b-containing chloroplasts. The data and approaches needed to resolve the issue of cyanelle origins are discussed. Our findings also have general implications for phylogenetic inference under conditions where the base compositions (compositional bias) of the sequences analyzed differ. Offprint requests to: C.J. Howe     

Organisation of the S10, spc and alpha ribosomal protein gene clusters in prokaryotic genomes

Although it is well known that there is no long range colinearity in gene order in bacterial genomes, it is thought that there are several regions that are under strong structural constraints during evolution, in which gene order is extremely conserved. One such region is the str locus, containing the S10-spc-alpha operons. These operons contain genes coding for ribosomal proteins and for a number of housekeeping genes. We compared the organisation of these gene clusters in 111 sequenced prokaryotic genomes (99 bacterial and 12 archaeal genomes). We also compared the organisation to the phylogeny based on 16S ribosomal RNA gene sequences and the sequences of the ribosomal proteins L22, L16 and S14. Our data indicate that there is much variation in gene order and content in these gene clusters, both in bacterial as well as in archaeal genomes. Our data indicate that differential gene loss has occurred on multiple occasions during evolution. We also noted several discrepancies between phylogenetic trees based on 16S rRNA gene sequences and sequences of ribosomal proteins L16, L22 and S14, suggesting that horizontal gene transfer did play a significant role in the evolution of the S10-spc-alpha gene clusters.     

RNase P of the Cyanophora paradoxa cyanelle: a plastid ribozyme

Ribonuclease P (RNase P) is a ribonucleoprotein enzyme that generates the mature 5' ends of tRNAs. Ubiquitous across all three kingdoms of life, the composition and functional contributions of the RNA and protein components of RNase P differ between the kingdoms. RNA-alone catalytic activity has been reported throughout bacteria, but only for some archaea, and only as trace activity for eukarya. Available information for RNase P from photosynthetic organelles points to large differences to bacterial as well as to eukaryotic RNase P: for spinach chloroplasts, protein-alone activity has been discussed; for RNase P from the cyanelle of the glaucophyte Cyanophora paradoxa, a type of organelle sharing properties of both cyanobacteria and chloroplasts, the proportion of protein was found to be around 80% rather than the usual 10% in bacteria. Furthermore, the latter RNase P was previously found catalytically inactive in the absence of protein under a variety of conditions; however, the RNA could be activated by a cyanobacterial protein, but not by the bacterial RNase P protein from Escherichia coli. Here we demonstrate that, under very high enzyme concentrations, the RNase P RNA from the cyanelle of C. paradoxa displays RNA-alone activity well above the detection level. Moreover, the RNA can be complemented to a functional holoenzyme by the E. coli RNase P protein, further supporting its overall bacterial-like architecture. Mutational analysis and domain swaps revealed that this A,U-rich cyanelle RNase P RNA is globally optimized but conformationally unstable, since changes as little as a single point mutation or a base pair identity switch at positions that are not part of the universally conserved catalytic core led to a complete loss of RNA-alone activity. Likely related to this low robustness, extensive structural changes towards an E. coli-type P5-7/P15-17 subdomain as a canonical interaction site for tRNA 3'-CCA termini could not be coaxed into increased ribozyme activity.     

A regulatory element within a gene of a ribosomal protein operon of Escherichia coli negatively controls expression by decreasing the translational efficiency

Summary The trmD operon of Escherichia coli consists of the genes for the ribosomal protein (r-protein) S16, a 21 kDa protein (21K) of unknown function, the tRNA(m¹G37)methyltransferase (TrmD), and r-protein L19, in this order. Previously we have shown that the steady-state amount of the two r-proteins exceeds that of the 21K and TrmD proteins 12- and 40-fold, respectively, and that this differential expression is solely explained by translational regulation. Here we have constructed translational gene fusions of the trmD operon and lacZ. The expression of a lacZ fusion containing the first 18 codons of the 21K protein gene is 15-fold higher than the expression of fusions containing 49 or 72 codons of the gene. This suggests that sequences between the 18th and the 49th codon may act as a negative element controlling the expression of the 21K protein gene. Evidence is presented which demonstrates that this regulation is achieved by reducing the efficiency of translation.     

Isolation of the ribosomal protein gene S6 from Schizosaccharomyces pombe

Summary We screened a Schizosaccharomyces pombe genomic library using the ribosomal protein gene SI0 from Saccharomyces cerevisiae as a probe. Hybrid-selected translation of the positive clones revealed a ribosomal protein of S. pombe which is probably equivalent to the ribosomal protein SI0 from S. cerevisiae.     

Sequence and characterisation of a ribosomal RNA operon fromAgrobacterium vitis

One of the four ribosomal RNA operons (rrnA) from theAgrobacterium vitis vitopine strain S4 was sequenced.rrnA is most closely related to therrn operons ofBradyrhizobium japonicum andRhodobacter sphaeroides and carries an fMet-tRNA gene downstream of its 5S gene, as in the case ofR. sphaeroides. The 16S rRNA sequence of S4 differs from theA. vitis K309 type strain sequence by only one nucleotide, in spite of the fact that S4 and K309 have very different Ti plasmids. The predicted secondary structure of the S4 23S rRNA shows several features that are specific for the alpha proteobacteria, and an unusual branched structure in the universal B8 stem. The 3 ends of the three otherrrn copies of S4 were also cloned and sequenced. Sequence comparison delimits the 3 ends of the four repeats and defines two groups:rrnA/rrnB andrrnC/rrnD.     

Amino acid sequences of ribosomal proteins S11 from Bacillus stearothermophilus and S19 from Halobacterium marismortui Comparison of the ribosomal protein S11 family

The complete amino acid sequences of ribosomal proteins S11 from the Gram-positive eubacterium Bacillus stearothermophilus and of S19 from the archaebacterium Halobacterium marismortui have been determined. A search for homologous sequences of these proteins revealed that they belong to the ribosomal protein S11 family. Homologous proteins have previously been sequenced from Escherichia coli as well as from chloroplast, yeast and mammalian ribosomes. A pairwise comparison of the amino acid sequences showed that Bacillus protein S11 shares 68% identical residues with S11 from Escherichia coli and a slightly lower homology (52%) with the homologous chloroplast protein. The halophilic protein S19 is more related to the eukaryotic (45–49%) than to the eubacterial counterparts (35%)     

Hydroxylation of steroids by Fusarium oxysporum, Exophiala jeanselmei and Ceratocystis paradoxa

The potential of Fusarium oxysporum var. cubense UAMH 9013 to perform steroid biotransformations was reinvestigated using single phase and pulse feed conditions. The following natural steroids served as substrates: dehydroepiandrosterone (1), pregnenolone (2), testosterone (3), progesterone (4), cortisone (5), prednisone (6), estrone (7) and sarsasapogenin (8). The results showed the possible presence of C-7 and C-15 hydroxylase enzymes. This hypothesis was explored using three synthetic androstanes: androstane-3,17-dione (9), androsta-4,6-diene-3,17-dione (10) and 3α,5α-cycloandrost-6-en-17-one (11). These fermentations of non-natural steroids showed that C-7 hydroxylation was as a result of that position being allylic. The evidence also pointed towards the presence of a C-15 hydroxylase enzyme.The eleven steroids were also fed to Exophialajeanselmei var. lecanii-corni UAMH 8783. The results showed that the fungus appears to have very active 5α and 14α-hydroxylase enzymes, and is also capable of carrying out allylic oxidations.Ceratocystis paradoxa UAMH 8784 was grown in the presence of the above-mentioned steroids. The results showed that monooxygenases which effect allylic hydroxylation and Baeyer–Villiger rearrangement were active. However, redox reactions predominated.     

The chloroplast gene encoding ribosomal protein S4 in Chlamydomonas reinhardtii spans an inverted repeat — unique sequence junction and can be mutated to suppress a streptomycin dependence mutation in ribosomal protein S12

The ribosomal protein gene rps4 was cloned and sequenced from the chloroplast genome of Chlamydomonas reinhardtii. The N-terminal 213 amino acid residues of the S4 protein are encoded in the single-copy region (SCR) of the genome, while the C-terminal 44 amino acid residues are encoded in the inverted repeat (IR). The deduced 257 amino acid sequence of C. reinhardtii S4 is considerably longer (by 51–59 residues) than S4 proteins of other photosynthetic species and Escherichia coli, due to the presence of two internal insertions and a C-terminal extension. A short conserved C-terminal motif found in all other S4 proteins examined is missing from the C. reinhardtii protein. In E. coli, mutations in the S4 protein suppress the streptomycin-dependent (sd) phenotype of mutations in the S12 protein. Because we have been unable to identify similar S4 mutations among suppressors of an sd mutation in C. reinhardtii S12 obtained using UV mutagenesis, we made site-directed mutations [Arg₆₈ (CGT) to Len (CTG and CTT)] in the wild-type rps4 gene equivalent to an E. coli Gln₅₃ to Len ribosomal ambiguity mutation (ram), which suppresses the sd phenotype and decreases translational accuracy. These mutants were tested for their ability to transform the sd S 12 mutation of C. reinhardtii to streptomycin independence. The streptomycin-independent isolates obtained by biolistic transformation all possessed the original sd mutation in rps12, but none had the expected donor Leu₆₈ mutations in rps4. Instead, six of 15 contained a Gln₇₃ (CAA) to Pro (CCA) mutation five amino acids downstream from the predicted mutant codon, irrespective of rps4 donor DNA. Two others contained six- and ten-amino acid, in-frame insertions at S4 positions 90 and 92 that appear to have been induced by the biolistic process itself. Eight streptomycin-independent isolates analyzed had wild-type rps4 genes and may possess mutations identical to previously isolated suppressors of sd that define at least two additional chloroplast loci. Cloned rps4 genes from streptomycin-independent isolates containing the Gln₇₃ to Pro mutation and the 6-amino acid insertion in r-protein S4 transform the sd strain to streptomycin independence.     

Characterization of the str operon genes from Spirulina platensis and their evolutionary relationship to those of other prokaryotes

Summary A 5.3 kb DNA segment containing the str operon (ca. 4.5 kb) of the cyanobacterium Spirulina platensis has been sequenced. The str operon includes the structural genes rpsL (ribosomal protein S12), rpsG (ribosomal protein S7), fus (translation elngation factor EF-G) and tuf (translation elongation factor EF-Tu). From the nucleotide sequence of this operon, the primary structures of the four gene products have been derived and compared with the available corresponding structures from eubacteria, archaebacteria and chloroplasts. Extensive homologies were found in almost all cases and in the order S12>EF-Tu>EF-G>S7; the largest homologies were generally found between the cyanobacterial proteins and the corresponding chloroplast gene products. Overall codon usage in S. platensis was found to be rather unbiased.     

Fragmentation heterogeneity of 23S ribosomal RNA in Haemophilus species

The fragmentation of 23S rRNA of 22 Haemophilus influenzae strains and eight strains belonging to other Haemophilus species was investigated. Instead of intact molecules, the 23S rRNA molecules were found to be cleaved into two to five smaller conserved fragments in most strains examined, especially in H. influenzae type b (5/6) and nontypeable strains (5/5). One or two conserved potential cleavage sites were identified by PCR analysis of the strains showing a fragmented 23S rRNA pattern. The relevant nucleotide sequences were determined and compared to H. influenzae Rd, which contains intact 23S rRNA molecules. An identical 112 bp long intervening sequence (IVS) at position 542 and a conserved 121–123 bp IVS sequence at position 1171 were found in two H. influenzae type b strains and one nontypeable strain. Among the strains with fragmented 23S rRNA, nearly half showed a heterogeneous cleavage pattern due to the dispersion of IVSs among different 23S rRNA operons. The localization of the conserved H. influenzae IVSs coincided well with the extensively studied IVSs among other bacteria, but differed in nucleotide sequence from any other reported IVSs. Therefore, the IVSs of Haemophilus 23S rRNA may originate from a common source that is independent of other bacteria.     

Nucleotide sequence of the cyanelle genome fromCyanophora paradoxa

The complete nucleotide sequence of the cyanelle genome ofCyanophora paradoxa Pringsheim strain LB 555 was determined (accession number U30821). The circular molecule is 135,599 base pairs in length. The physical map of this DNA molecule is shown along with identified genes and open reading frames.     

The gene for mitochondrial ribosomal protein S14 has been transferred to the nucleus in Arabidopsis thaliana

The transfer of genetic information from the mitochondrion to the nucleus is thought to be still underway in higher plants. The mitochondrial genome of Arabidopsis thaliana contains only one rps14 pseudogene. In this paper we show that the functional gene encoding mitochondrial ribosomal protein S14 has been translocated to the nucleus. This gene transfer is a recent evolutionary event, which occurred within Cruciferae, probably after the divergence of Arabidopsis and Brassica napus. A 5′ extension of the rps14 reading frame encodes a presequence which, in vitro, targets the polypeptide to isolated mitochondria and is cleaved off during or after import. No intron was found at the junction of the targeting presequence with the mitochondrially derived sequence, which are directly connected. By contrast, a 90-bp intron, which is removed by splicing to give a mature poly(A)⁺mRNA of 0.9 kb, is located in the 3′ non-coding region. To our knowledge, this is the first report of an intron in such a position in a functional transferred gene in higher plants, and suggests that exon shuffling may have been involved in the acquisition of elements necessary for expression in the nucleus. Putative roles of this intron in polyadenylation and enhancement of gene expression are discussed. Received: 11 January 1999 / Accepted: 27 April 1999     

The nucleotide sequence of five ribosomal protein genes from the cyanelles ofCyanophora paradoxa: Implications concerning the phylogenetic relationship between cyanelles and chloroplasts

Summary The nucleotide sequences of the ribosomal protein genesrps18, rps19, rpl2, rpl33, and partial sequence ofrpl22 from cyanelles, the photosynthetic organelles of the protistCyanophora paradoxa, have been determined. These genes form two clusters oriented in opposite and divergent directions. One cluster contains therpl33 andrps18 genes; the other contains therpl2, rps19, andrpl22 genes, in that order. Phylogenetic trees were constructed from both the DNA sequences and the deduced protein sequences of cyanelles,Euglena gracilis and land plant chloroplasts, andEscherichia coli, using parsimony or maximum likelihood methods. In addition, a phylogenetic tree was built from a distance matrix comparing the number of nucleotide substitutions per site. The phylogeny inferred from all these methods suggests that cyanelles fall within the chloroplast line of evolution and that the evolutionary distances between cyanelles and land plant chloroplasts are shorter than betweenE. gracilis chloroplasts and land plant chloroplasts.