Characterization of Mitochondrial Small-Subunit Ribosomal RNAs from Holoparasitic Plants

Mitochondrial small-subunit (19S) rDNA sequences were obtained from 10 angiosperms to further characterize sequence divergence levels and structural variation in this molecule. These sequences were derived from seven holoparasitic (nonphotosynthetic) angiosperms as well as three photosynthetic plants. 19S rRNA is composed of a conservative core region (ca. 1450 nucleotides) as well as two variable regions (V1 and V7). In pairwise comparisons of photosynthetic angiosperms to Glycine, the core 19S rDNA sequences differed by less than 1.4%, thus supporting the observation that variation in mitochondrial rDNA is 3–4 times lower than seen in protein coding and rDNA genes of other subcellular organelles. Sequences representing four distinct lineages of nonasterid holoparasites showed significantly increased numbers of substitutions in their core 19S rDNA sequences (2.3–7.6%), thus paralleling previous findings that showed accelerated rates in nuclear (18S) and plastid (16S) rDNA from the same plants. Relative rate tests confirmed the accelerated nucleotide substitution rates in the holoparasites whereas rates in nonparasitic plants were not significantly increased. Among comparisons of both parasitic and nonparasitic plants, transversions outnumbered transitions, in many cases more than two to one. The core 19S rRNA is conserved in sequence and structure among all nonparasitic angiosperms whereas 19S rRNA from members of holoparasitic Balanophoraceae have unique extensions to the V5 and V6 variable domains. Substitution and insertion/deletion mutations characterized the V1 and V7 regions of the nonasterid holoparasites. The V7 sequence of one holoparasite (Scybalium) contained repeat motifs. The cause of substitution rate increases in the holoparasites does not appear to be a result of RNA editing, hence the underlying molecular mechanism remains to be fully documented. Received: 18 May 1997 / Accepted: 11 July 1997     

The rates of evolution in some ribosomal components

Summary The rate of nucleotide substitution (k(nuc)) of 5s RNA was estimated to be (1.8 ± 0.5) × 10^–10 per site per year by comparing the nucleotide sequences of human andXenopus 5s RNA and using the geological time elapsed since the separation of mammals and amphibians. Similarly, k(nuc) of 5.8s rRNA was calculated to be 0.93 – 1.4 × 10^–10 per site per year from the sequences of rat hepatoma cells andSaccharomyces cerevisiae. For the comparison of these data with the amino acid substitution rate of known proteins, the k(nuc) values of 5s rRNA and 5.8s rRNA were converted to the rate of amino acid substitution (k(aa)). The k(aa) values in pauling units were 0.4 and 0.2 – 0.3, respectively.The average k(aa) of ribosomal proteins was also estimated to be 0.2 – 0.3 pauling from the N-terminal amino acid sequences of seventeen 30s ribosomal proteins ofBacillus stearothermophilus andEscherichia coli. Thus, the evolutionary rates of these ribosomal components studied here are similar to each other; they are considerably slower than that of the known cellular proteins. Most, if not all, of the replacements in ribosomal proteins occurred between amino acids of a chemically similar nature.     

Structural analyses of plastid-derived 16S rRNAs in holoparasitic angiosperms

Higher-order structures have been constructed for plastid-encoded small-subunit (SSU, 16S), rRNAs from representatives of seven nonphotosynthetic holoparasitic angiosperm families: Apodanthaceae, Cynomoriaceae, Cytinaceae, Balanophoraceae, Hydnoraceae, Mitrastemonaceae, and Rafflesiaceae. Whereas most pairwise comparisons among angiosperms differ by 2–3% in substitutions, the 16S rRNAs of the holoparasites show an increasingly greater number of mutations: Cynomorium (7.3%), Cytinus (8.0%), Bdallophyton (12.7%), Mitrastema (14.9%), Hydnora (19.4%), Pilostyles (30.4%) and Corynaea (35.9%). Despite this high level of sequence variation, SSU structures constructed for all species except Pilostyles possess the typical complement of 50 helices (that contain numerous compensatory mutations) thereby providing indirect evidence supporting their functionality. Pilostyles, likely with the most unusual plastid 16S rRNA yet documented, lacks four major helices and contains lengthy insertions for four others. Sequences of products generated via RT-PCR show that these structural modifications are present on a mature (transcribed) rRNA. The trend toward increasing numbers of base substitutions in the holoparasites is accompanied by a marked increase in AA+U content of the rRNA. This A/T drift phenomenon of rDNA is especially apparent in Corynaea whose SSU rDNA sequence is 72% A+T. A comparison of Cytinus to tobacco showed that substitution rates appear to be dependent upon the composition of neighboring bases. Transversions represented 26% of the mutations when flanking bases were G or C whereas transversions increased to 36% when the flanking bases were A to T. The underlying molecular mechanism associated with these high substitution rates is presently unknown, however, relaxation of selection pressure on ribosome function resulting in altered DNA replication and/or repair systems may be involved.     

The complete nucleotide sequence of the small-subunit ribosomal RNA coding region for the cycadZamia pumila: Phylogenetic implications

Summary The DNA sequence of the small-subunit ribosomal RNA coding region for the cycadZamia pumila L. was determined. TheZamia smallsubunit rRNA was found to be 1813 nucleotides in length and approximately 92% identical to published angiosperm small-subunit rRNA sequences. Conserved regions interspersed with variable regions are observed corresponding to those found in other eukaryotic small-subunit sequences. Using representatives from protist, fungal, plant, and animal groups, a distance matrix was constructed of average nucleotide substitution rates for pairs of organisms. Phylogenetic trees were inferred from similarities between sequences. The sequence ofZamia represents the earliest divergence from the higher plant lineage reported to date for small-subunit rRNA data. Inferred phylogenies also support a monophyletic origin for the angiosperms consistent with studies citing phenotypic characters.     

Evolution in bacteria: Evidence for a universal substitution rate in cellular genomes

Summary This paper constructs a temporal scale for bacterial evolution by tying ecological events that took place at known times in the geological past to specific branch points in the genealogical tree relating the 16S ribosomal RNAs of eubacteria, mitochondria, and chloroplasts. One thus obtains a relationship between time and bacterial RNA divergence which can be used to estimate times of divergence between other branches in the bacterial tree. According to this approach,Salmonella typhimurium andEscherichia coli diverged between 120 and 160 million years (Myr) ago, a date which fits with evidence that the chief habitats occupied now by these two enteric species became available that long ago.The median extent of divergence betweenS. typhimurium andE. coli at synonymous sites for 21 kilobases of protein-coding DNA is 100%. This implies a silent substitution rate of 0.7–0.8%/Myr—a rate remarkably similar to that observed in the nuclear genes of mammals, invertebrates, and flowering plants. Similarities in the substitution rates of eucaryotes and procaryotes are not limited to silent substitutions in protein-coding regions. The average substitution rate for 16S rRNA in eubacteria is about 1%/50 Myr, similar to the average rate for 18S rRNA in vertebrates and flowering plants. Likewise, we estimate a mean rate of roughly 1%/25 Myr for 5S rRNA in both eubacteria and eucaryotes.For a few protein-coding genes of these enteric bacteria, the extent of silent substitution since the divergence ofS. typhimurium andE. coli is much lower than 100%, owing to extreme bias in the usage of synonymous codons. Furthermore, in these bacteria, rates of amino acid replacement were about 20 times lower, on average, than the silent rate. By contranst, for the mammalian genes studied to date, the average replacement rate is only four to five times lower than the rate of silent substitution.     

Reassessment of the bioenergetic yield of Arthrospira platensis using continuous culture

Reassessement of bioenergetic growth yield of Arthrospira platensis was performed by using continuous culture under both autotrophic and mixotrophic conditions. Continuous culture was carried out at dilution rates of 0.017, 0.023 and 0.030 h^–1. Under these dilution rates bioenergetic yields ranged between 4.45–6.03 × 10^–3 g biomass kJ^–1 and between 5.42–7.46 × 10^–3 g biomass kJ^–1, under autotrophic and mixotrophic conditions respectively. A maximum bioenergetic yield of 8.1 × 10^–3 g biomass kJ^–1 using an autotrophic culture can be calculated. Pigment accumulation (chlorophyll a and carotenoids) may be related to light irradiance, reaching a maximum pigment concentration under light saturation irradiance. Phycocyanin concentration increased during light limitation.     

Using frequency of dividing cells in estimating autotrophic picoplankton growth and productivity in the Chesapeake Bay

In situ incubations of natural autotrophic picoplankton populations during a 15 month study were used to test the frequency of dividing cells proceduresin estimating phototrophic picoplankton growth rates. These rates were estimated using dilution experiments and compared to the average frequency of dividing cells over the same time interval. The regression equation of µ = 2.85 × 10^–3 (FDC) + 0.022 was calculated to relate autotrophic picoplankton growth rate and the frequency of dividing cells in this study. The resulting relationship was compared to ¹⁴C-bicarbonate derived growth rates. Productivity estimates using frequency of dividing cells correlated closely to sodium ¹⁴C-bicarbonate results and indicated a range of productivity by autotrophic picoplankton of 55.6% the total phytoplankton primary productivity in July to a January rate of 2.3%. Annual autotrophic picoplankton abundance varied seasonally in the lower Chesapeake Bay ranging from 7.26 × 10⁶ cells 1^–1 in winter to 9.28 × 10⁸ cells 1^–1 during late summer.     

Evolution of the nad3-rps12 Gene Cluster in Angiosperm Mitochondria: Comparison of Edited and Unedited Sequences

We have analyzed the nad3-rps12 locus for eight angiosperms in order to compare the utility of mitochondrial DNA and edited mRNA sequences in phylogenetic reconstruction. The two coding regions, containing from 25 to 35 editing sites in the various plants, have been concatenated in order to increase the significance of the analysis. Differing from the corresponding chloroplast sequences, unedited mitochondrial DNA sequences seem to evolve under a quasi-neutral substitution process which undifferentiates the nucleotide substitution rates for the three codon positions. By using complete gene sequences (all codon positions) we found that genomic sequences provide a classical angiosperm phylogenetic tree with a clear-cut grouping of monocotyledons and dicotyledons with Magnoliidae at the basal branch of the tree. Conversely, owing to their low nucleotide substitution rates, edited mRNA sequences were found not to be suitable for studying phylogenetic relationships among angiosperms. Received: 24 January 1996 / Accepted: 5 June 1996     

Phylogenetic position of an obligately chemoautotrophic, marine hydrogen-oxidizing bacterium, Hydrogenovibrio marinus, on the basis of 16S rRNA gene sequences and two form I RuBisCO gene sequences

Two form ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) genes from the obligately autotrophic, marine hydrogen oxidizer Hydrogenovibrio marinus were sequenced. The deduced amino acid sequences of both RuBisCOs revealed that they are similar to those of sulfur oxidizers (Thiobacillus) and a purple sulfur bacterium (Chromatium vinosum). According to the 16S rRNA gene sequences, H. marinus is also affiliated with these microorganisms, members of Thiomicrospira being the closest relatives. Sequence similarities of the 16S rRNA genes and of the RuBisCO genes among these γ-Proteobacteria suggest a common autotrophic ancestry. An ancestor of purple sulfur bacteria might be a common root of H. marinus and related sulfur oxidizers. Received: 17 June 1997 / Accepted: 14 November 1997     

Ribosomal DNA repeat unit polymorphism in 25 Hordeum species

Summary Tandemly repeated DNA sequences containing structural genes encoding ribosomal RNA (rDNA) were investigated in 25 species of Hordeum using the wheat rDNA probe pTA71. The rDNA repeat unit lengths were shown to vary between 8.5 and 10.7 kb. The number of length classes (1–3) per accession generally corresponded to the number of nucleolar organizing regions (NORs). Intraspecific variation was found in H. parodii, H. spontaneum and H. leporinum, but not in H. bulbosum. Restriction analysis showed that the positions of EcoRI, SacI and certain BamHI cleavage sites in the rRNA structural genes were highly conserved, and that repeat unit length variation was generally attributable to the intergenic spacer region. Five rDNA BamHI restriction site maps corresponded to the following groups of species: Map A — H. murinum, H. glaucum, H. leporinum, H. bulbosum, H. marinum, H. geniculatum; Map B — H. leporinum; Map C — H. vulgare, H. spontaneum, H. agriocrithon; Map D — H. chilense, H. bogdanii; and Map E — remaining 14 Hordeum species. The repeat unit of H. bulbosum differed from all other species by the presence of a HindIII site. The closer relationship of H. bulbosum to H. leporinum, H. murinum and H. glaucum than to H. vulgare was indicated by their BamHI restriction maps.Contribution No. 1169, Plant Research Centre     

Estimation of the number of amino acid substitutions per site when the substitution rate varies among sites

A general model for estimating the number of amino acid substitutions per site (d) from the fraction of identical residues between two sequences (q) is proposed. The well-known Poisson-correction formula q = e ^–d corresponds to a site-independent and amino-acid-independent substitution rate. Equation q = (1 – e ^–2d )/2d, derived for the case of substitution rates that are site-independent, but vary among amino acids, approximates closely the empirical method, suggested by Dayhoff et al. (1978). Equation q = 1/(1 + d) describes the case of substitution rates that are amino acid-independent but vary among sites. Lastly, equation q = [ln(1 + 2d)]/2d accounts for the general case where substitution rates can differ for both amino acids and sites.     

Induced mutations and barley improvement

Summary Seven barley varieties, originating from three X-ray induced mutations, have been officially approved in Sweden since 1958. Some have gained a wide area of cultivation. The list is as follows: Pallas, isolated 1947, approved 1958, mutant ert-k ³² of Bonus barley. — Mari, isolated 1950, approved 1960, mutant mat-a ⁸ of Bonus. — Hellas, approved 1967, mutant cross of Pallas × Herta. — Kristina, approved 1969, mutant cross of Domen × Mari. — Visir, approved 1970, Pallas × Long Glumes back-crossed to Pallas. — Mona, approved 1970, mutant cross of Mari × Monte Cristo back-crossed to Mari. — Gunilla, approved 1970, hybrid cross of the mutant 44/3 arisen from Gull barley in 1939; evolved in a series of steps, using one six-row and four two-row varieties, with mutant characters prevailing and Gull genes reiterated. — After the first approval of Pallas in 1958, 12 more years have led to the approval of a second mutant case and five mutant crosses. In addition, chromosome translocations, induced by irradiation in Bonus, have been instrumental in the production of hybrid barley in USA and are used in the barley improvement program of Sweden, as well as for theoretical analysis in numerous countries.     

Use of microbial peptide inhibitors for characterization of the substrate specificity of thermitase,a thermostable serine protease fromThermoactinomyces vulgaris

Seven microbial peptide inhibitors—chymostatin, antipain, elastatinal, leupeptin, pepstatin, bestatin, and phosphoramidon—were tested for their efficiency to inhibit thermitase, a thermostable serine protease fromThermoactinomyces vulgaris. Chymostatin and antipain were the most effective inhibitors, with K_i values of 7×10^–8 M and 2×10^–7 M, respectively. Except for leupeptin, all inhibitors resist hydrolysis by thermitase. Leupeptin, however, is cleaved by thermitase between the two leucylresidues. Further, a close relationship in specificity between thermitase and subtilisin BPN and their distinct discrimination from elastase specificity was demonstrated by using these inhibitors.     

Microbial diversity in acid mine drainage of Xiang Mountain sulfide mine, Anhui Province, China

To understand the composition and structure of microbial communities in acid (pH 3.0) mine drainage (AMD) associated with pyrite mine tailings in Anhui Province, China, molecular diversities of 16S rRNA and 18S rRNA genes were examined using a PCR-based cloning approach. Bacterial, archaeal and microeukaryotic clone libraries were constructed. In contrast to typical dominance of autotrophic acidophiles, genus Acidiphilium, which consists of mixotrophic acidophiles capable of chemoorganotrophic and photosynthetic metabolisms, was the largest group in the bacterial clone library. These mixotrophic organisms may be advantageous in the oligotrophic AMD environment of the study site (certain amounts of dissolved organic carbon and light) by switching between two modes of metabolisms. Unexpectedly, a large fraction of bacterial clones (12.7%) were related to the neutrophilic genus Legionella, which can cause Legionnaires’ disease, a potentially lethal pneumonia. The eukaryotic 18S rRNA gene sequences were mostly related to Oxytricha, Nuclearia, and Penicillium. In the archaeal clone library, all the sequences were affiliated to the phylum Crenarchaeota, while the Euryarchaeota was not present.     

Cloning and sequence analysis of the 18 S ribosomal RNA gene of tomato and a secondary structure model for the 18 S rRNA of angiosperms

Summary The gene of a cytoplasmic 18 S ribosomal RNA (18 S rDNA) of the dicotyledonous plant tomato (ycopersicon esculentum) cv. Rentita has been cloned, and its complete primary structure has been determined. The tomato 18 S rDNA is 1805 by long with a G+C content of 49.6%. Its sequence exhibits 94%–96% positional identity when it is colinearly aligned with the previously reported sequences of the 17–18 S rDNAs of the dicot soybean and the monocots maize and rice. A model of the secondary structure of the 18 S rRNA of angiosperms is presented and its genera-specific structural features are compared with a current eukaryotic 18 S rRNA consensus model.     

Chromosome mapping in barley by means of telotrisomic analysis

Summary A total of 37 genetic markers located in chromosomes 2, 3, 4 and 5 were associated with specific arms by means of telotrisomic analysis in five telotrisomics (Triplo 2 L, 2 S, 3 S, 4 S, 5 L) of barley (Hordeum vulgare L.). The genes v, gp (= gp 2), li, gs 5, tr and msg2 showed a trisomic ratio with Triplo 2 L indicating that these genes were on the long arm of chromosome 2. A disomic ratio was obtained for genes wst 4, gs 5, and v with Triplo 2 S, confirming that these genes were on the long arm of chromosome 2(2 L). A disomic ratio was observed for genes e, f(= lg), sk, and gs6 with Triplo 2 L. Two genes, f(= lg) and gs6 showed a trisomic ratio with Triplo 2S. These results indicated that genes e, f(= lg), sk, and gs 6 are on the short arm of chromosome 2 (2S). Since only one telocentric chromosome was available for chromosome 3, 4 and 5, most of the well-mapped marker genes were tested with those telocentric chromosomes. The genes cu 2, uz, wst, als, gs 2, zb,f2, and cer-zn ³⁴⁸ showed trisomic ratio with the telocentric for chromosome 3. These genes were located on the short arm of chromosome 3 (Robertson 1971). This indicated that the telocentric chromosome is for the short arm of chromosome 3(3 S). A disomic ratio was obtained for genes yst, x _c, al, yst2, a _n, ari-a ⁶ and x _s, indicating that these genes are on the long arm of chromosome 3. Two genes, f9 and K, showed trisomic ratio with the telocentric chromosome for 4, while genes gl(= gl2), br2, yh, lg 3, lg 4 and lk 5 showed disomic ratios. This indicated that the telocentric chromosome is for the short arm of chromosome 4. Two genes, fs 2 and g, were studied with Triplo 5 L. Both showed trisomic ratio, indicating that fs 2 and g are located on Triplo 5 L. The centromere position (C) on chromosome 2, 3 and 4 was thus located as (the left side of C is the short arm and the right is the long arm): chromosome 2: f — sk — gs6 — e — C — gs5 — msg2 — wst4 — v — gp — li — tr; chromosome 3: f2 — cer-zn ³⁴⁸ — uz — gs2 — als — cu2 — wst — zb — C — yst — x _c — al — yst2 — a _n — ari-a ⁶ — x _s; chromosome 4: f9 — K — C — lg4 — lg ³ — gl2 — br2 — lk5 — yh. The centromere position on chromosome 5 was not precisely located.Contribution from the Department of Agronomy, Published with the approval of the director of the Colorado State University Experiment Station as Scientific Series Paper No. 2606. This research was supported in part by by NSF Grant GB 4482X and GB 30 493 to T. Tsuchiya and Colorado State University Experiment Station Hatch Project     

Oxygen tolerance of strictly aerobic hydrogen-oxidizing bacteria

Growth of various bacteria, especially aerobic hydrogen-oxidizing bacteria, in the presence of 2 to 100% (v/v) oxygen in the gas atmosphere was evaluated. The bacterial strains included Alcaligenes eutrophus, A. paradoxus, Aquaspirillum autotrophicum, Arthrobacter spec. strain 11X, Escherichia coli, Arthrobacter globiformis, Nocardia opaca, N. autotrophica, Paracoccus denitrificans, Pseudomonas facilis, P. putida, and Xanthobacter autotrophicus. Under heterotrophic conditions with fructose or gluconate as substrates neither colony formation on solid medium nor the growth rates in liquid media were drastically impaired by up to 100% oxygen. In contrast, autotrophic growth — with hydrogen, carbon dioxide and up to 80% oxygen in the gas atmosphere — was strongly depressed by high oxygen concentrations. However, only the growth rate, not the viability of the cells, was decreased. Growth retardation was accompanied by a decrease of hydrogenase activity.The work was supported by the Deutsche Forschungsgemeinschaft.     

Ancestral Population Sizes and Species Divergence Times in the Primate Lineage on the Basis of Intron and BAC End Sequences

The effective sizes of ancestral populations and species divergence times of six primate species (humans, chimpanzees, gorillas, orangutans, and representatives of Old World monkeys and New World monkeys) are estimated by applying the two-species maximum likelihood (ML) method to intron sequences of 20 different loci. Examination of rate heterogeneity of nucleotide substitutions and intragenic recombination identifies five outrageous loci (ODC1, GHR, HBE, INS, and HBG). The estimated ancestral polymorphism ranges from 0.21 to 0.96% at major divergences in primate evolution. One exceptionally low polymorphism occurs when African and Asian apes diverged. However, taking into consideration the possible short generation times in primate ancestors, it is concluded that the ancestral population size in the primate lineage was no smaller than that of extant humans. Furthermore, under the assumption of 6 million years (myr) divergence between humans and chimpanzees, the divergence time of humans from gorillas, orangutans, Old World monkeys, and New World monkeys is estimated as 7.2, 18, 34, and 65 myr ago, respectively, which are generally older than traditional estimates. Beside the intron sequences, three other data sets of orthologous sequences are used between the human and the chimpanzee comparison. The ML application to these data sets including 58,156 random BAC end sequences (BES) shows that the nucleotide substitution rate is as low as 0.6–0.8 × 10^–9 per site per year and the extent of ancestral polymorphism is 0.33–0.51%. With such a low substitution rate and short generation time, the relatively high extent of polymorphism suggests a fairly large effective population size in the ancestral lineage common to humans and chimpanzees.[Reviewing Editor: Dr. Magnus Nordborg]     

Complex Patterns of Plastid 16S rRNA Gene Evolution in Nonphotosynthetic Green Algae

This study provides a phylogenetic/comparative approach to deciphering the processes underlying the evolution of plastid rRNA genes in genomes under relaxed functional constraints. Nonphotosynthetic green algal taxa that belong to two distinct classes, Chlorophyceae (Polytoma) and Trebouxiophyceae (Prototheca), were investigated. Similar to the situation described previously for plastid 16S rRNA genes in nonphotosynthetic land plants, nucleotide substitution levels, extent of structural variations, and percentage AT values are increased in nonphotosynthetic green algae compared to their closest photosynthetic relatives. However, the mutational processes appear to be different in many respects. First, with the increase in AT content, more transversions are noted in Polytoma and holoparasite angiosperms, while more transitions characterize the evolution of the 16S rDNA sequences in Prototheca. Second, although structural variations do accumulate in both Polytoma and Prototheca (as well as holoparasitic plastid 16S rRNAs), insertions as large as 1.6 kb characterize the plastid 16S rRNA genes in the former, whereas significantly smaller indels (not exceeding 24 bp) seem to be more prevalent in the latter group. The differences in evolutionary rates and patterns within and between lineages might be due to mutations in replication/repair-related genes; slipped-strand mispairing is likely the mechanism responsible for the expansion of insertions in Polytoma plastid 16S rRNA genes. Received: 29 December 2000 / Accepted: 18 May 2001