Characteristics and utility of nuclear-encoded large-subunit ribosomal gene sequences in phylogenetic studies of red algae

Primer sequences are described for amplifying and sequencing a large fragment (approximately 2500 b.p.) of the nuclear-encoded large-subunit ribosomal RNA gene (LSU) from red algae. In comparison to RuBisCo large-subunit gene (rbcL) and nuclear-encoded small-subunit ribosomal RNA gene (SSU) sequence data, LSU sequence data was intermediate in the number of phylogenetically informative positions and sequence divergence. Parsimony analysis of LSU sequences for 16 Gelidiales species resolved some nodes unresolved in rbcL and SSU parsimony trees. An analysis of LSU sequences from 13 species of red algae classified in 11 orders suggests that this gene may be useful in studies of higher-level relationships of red algae.     

Comparison of the nucleotide sequence of soybean 18S rRNA with the sequences of other small-subunit rRNAs

Summary We present the sequence of the nuclearencoded ribosomal small-subunit RNA from soybean. The soybean 18S rRNA sequence of 1807 nucleotides (nt) is contained in a gene family of approximately 800 closely related members per haploid genome. This sequence is compared with the ribosomal small-subunit RNAs of maize (1805 nt), yeast (1789 nt),Xenopus (1825 nt), rat (1869 nt), andEscherichia coli (1541 nt). Significant sequence homology is observed among the eukaryotic small-subunit rRNAs examined, and some sequence homology is observed between eukaryotic and prokaryotic small-subunit rRNAs. Conserved regions are found to be interspersed among highly diverged sequences. The significance of these comparisons is evaluated using computer simulation of a random sequence model. A tentative model of the secondary structure of soybean 18S rRNA is presented and discussed in the context of the functions of the various conserved regions within the sequence. On the basis of this model, the short basepaired sequences defining the four structural and functional domains of all 18S rRNAs are seen to be well conserved. The potential roles of other conserved soybean 18S rRNA sequences in protein synthesis are discussed.     

Nucleotide sequence of the gene coding for non-proteolyticClostridium botulinum type B neurotoxin: Comparison with other clostridial neurotoxins

The neurotoxin gene of non-proteolyticClostridium botulinum type B (strain Eklund 17B) was cloned as a series of overlapping polymerase chain reaction (PCR) fragments generated with primers designed to conserved regions of published botulinal toxin (BoNT) sequences. The 3 end of the gene was obtained by using primers designed to the determined sequence of non-proteolytic BoNT/B and a published downstream region of BoNT/B gene from a proteolytic strain. Translation of the nucleotide sequence derived from cloned PCR fragments demonstrated the toxin gene encodes a protein of 1291 amino acid residues. Comparative alignment of the derived BoNT/B sequence with those of other published botulinal neurotoxins revealed highest sequence relatedness with BoNT/B of proteolyticC. botulinum. The sequence identity between non-proteolytic and proteolytic BoNT/B was 97.7% for the light chain (corresponding to 10 amino acid changes) and 90.2% for the heavy chain (corresponding to 81 amino acid changes), with most differences occurring at the C-terminal end. A genealogical tree constructed from all known botulinal neurotoxin sequences revealed marked topological differences with a phylogenetic tree ofC. botulinum types based upon small-subunit (16S) ribosomal RNA sequences.     

Molecular and cytological characterization of repetitive DNA sequences in Brassica

Summary We isolated three different repetitive DNA sequences from B. campestris and determined their nucleotide sequences. In order to analyze organization of these repetitive sequences in Brassica, Southern blot hybridization and in situ hybridization with metaphase chromosomes were performed. The sequence cloned in the plasmid pCS1 represented a middle repetitive sequence present only in B. campestris and not detected in closely related B. Oleracea. This sequence was localized at centromeric regions of six specific chromosomes of B. campestris. The second plasmid, pBT4, contained a part of the 25S ribosomal RNA gene, and its copy number was estimated to be 1,590 and 1,300 per haploid genome for B. campestris and B. oleracea, respectively. In situ hybridization with this sequence showed a clear signal at the NOR region found in the second largest chromosome of B. Campestris. The third plasmid, pBT11, contained a 175-bp insert that belongs to a major family of tandem repeats found in all the Brassica species. This sequence was detected at centromeric regions of all the B. campestris chromosomes. Our study indicates that in situ hybridization with various types of repetitive sequences should give important information on the evolution of repetitive DNA in Brassica species.     

IDENTIFICATION OF GROUP- AND STRAIN-SPECIFIC GENETIC MARKERS FOR GLOBALLY DISTRIBUTED ALEXANDRIUM (DINOPHYCEAE). I. RFLP ANALYSIS OF SSU rRNA GENES1

Two distinct small-subunit ribosomal RNA genes (SSU rDNAs), termed the “A gene” and “B gene,” were recently found in the toxic dinoflagellate Alexandrium fundyense Balech. A restriction fragment length polymorphism (RFLP) assay was developed to rapidly detect the A and B genetic markers. SSU rDNA from 58 cultures with species designations of A. tamarense (Lebour) Balech, A. catenella (Whedon et Kofoid) Balech, A. fundyense, A. affine (Fukuyo et Inoue)Balech, A. minutum Halim, A. lusitanicum Balech, and A. andersoni Balech were screened. These cultures represent toxic and non-toxic isolates from North America, western Europe, Thailand, Japan, Australia, and the ballast water of several cargo ships. The RFLP assay revealed five distinct groups. Three subdivided the A. tamarense/catenella/fundyense“species complex” into clusters defined by geographic origin, not by morphospecies designations. The fourth group consisted of A. affine, whereas the fifth group was represented by A. minutum, A. lusitanicum, and A. andersoni. The B gene was only found in A. tamarense, A. catenella, and A. fundyense, but not in all isolates. However, all North American isolates of this closely related group harbored this gene, and it also was found in some A. tamarense from scattered locations in Japan and in the ballast water of one ship that operated exclusively between Japan and Australia. Isolates without the B gene appeared to have only a single class of SSU rDNA. The B sequence was not essential for toxin production, but thus far those organisms harboring it were toxic. The A. tamarense/catenella/fundyense complex is composed of genetically distinct populations, within which may exist two or all three of the mophotypically defined species. The B gene is a promising taxonomic and biogeographic marker and may be useful for tracking the regional and/or global dispersal of particular populations.     

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.     

Ribosomal DNA and ITS-2 sequence comparisons as a tool for predicting genetic relatedness

The determination of the secondary structure of the internal transcribed spacer (ITS) regions separating nuclear ribosomal RNA genes of Chlorophytes has improved the fidelity of alignment of nuclear ribosomal ITS sequences from related organisms. Application of this information to sequences from green algae and plants suggested that a subset of the ITS-2 positions is relatively conserved. Organisms that can mate are identical at all of these 116 positions, or differ by at most, one nucleotide change. Here we sequenced and compared the ITS-1 and ITS-2 of 40 green flagellates in search of the nearest relative to Chlamydomonas reinhardtii. The analysis clearly revealed one unique candidate, C. incerta. Several ancillary benefits of the analysis included the identification of mislabelled cultures, the resolution of confusion concerning C. smithii, the discovery of misidentified sequences in GenBank derived from a green algal contaminant, and an overview of evolutionary relationships among the Volvocales, which is congruent with that derived from rDNA gene sequence comparisons but improves upon its resolution. The study further delineates the taxonomic level at which ITS sequences, in comparison to ribosomal gene sequences, are most useful in systematic and other studies. Received: 14 February 1997 / Accepted: 28 March 1997     

Homology between the ribosomal DNA of Escherichia coli and mitochondrial DNA preparations of maize is principally to sequences other than mitochondrial rRNA genes

E. coli ribosomal DNA has been used to probe maize mitochondrial DNA. It hybridizes primarily with chloroplast ribosomal DNA sequences and with fungal and bacterial sequences which may contaminate the mtDNA preparations. It also hybridizes to the chloroplast 16S ribosomal RNA gene sequence present in the mitochondrial genome (1) as well as to the mitochondrial 18S ribosomal RNA gene sequence. Weak sequence homology was detected between E. coli rDNA and the mitochondrial 26S ribosomal RNA gene.     

Evidence for a relationship between the Bombyx mori middle repetitive Bm1 sequence family and U1 snRNA

Several genomic library equivalents of Bombyx mori were constructed in the EMBL-4 lambda derivative. The genomic bank was screened with purified Bombyx mori U1 RNA and twenty positive clones for the U1 gene were isolated. Three U1-related sequences were subcloned and sequenced. Two of the sequences are U1 pseudogenes while a third sequence represents a member of the Bm1 family of repetitive elements of B.mori with significant sequence similarity to U1 small nuclear RNA. The U1-related Bm1 element exhibits 82% sequence similarity with the Bm1 consensus sequence and, under less stringent computer comparison parameters, 60% similarity with a composite B.mori/Drosophila melanogaster U1 gene. The Bm1 family consensus sequence exhibits 53% sequence similarity with the composite U1 gene. The two pseudogenes possess highly conserved sequences with the B.mori U1 gene only for the first 101 nucleotides. These findings are indicative of at least two different categories of U1-related sequences in B.mori, one with a possible evolutionary relationship to the Bm1 family of repetitive elements and the other representing characteristic processed pseudogenes with retroposon mode of dispersion and target selection for the TTTA hotspot. In addition, the U1-related Bm1 element may demonstrate for the first time that a family of retroposons is ultimately derived from a U snRNA.This article is dedicated in memory of Ms. Deborah Lampert who helped so much in the preparation of this paper.     

The contribution of DNA slippage to eukaryotic nuclear 18S rRNA evolution

Six of 204 eukaryotic nuclear small-subunit ribosomal RNA sequences analyzed show a highly significant degree of clustering of short sequence motifs that indicates the fixation of products of replication slippage within them in their recent evolutionary history. A further 72 sequences show weaker indications of sequence repetition. Repetitive sequences in SSU rRNAs are preferentially located in variable regions and in particular in V4 and V7. The conserved region immediately 5 to V7 (C7) is also consistently repetitive. Whereas variable regions vary in length and appear to have evolved by the fixation of slippage products, C7 shows no indication of length variation. Repetition within C7 is therefore either not a consequence of slippage or reflects very ancient slippage events. The phylogenetic distribution of sequence simplicity in small-subunit rRNAs is patchy, being largely confined to the Mammalia, Apicomplexa, Tetrahymenidae, and Trypanosomatidae. The regions of the molecule associated with sequence simplicity vary with taxonomic grouping as do the sequence motifs undergoing slippage. Comparison of rates of insertion and substitution in a lineage within the genus Plasmodium confirms that both rates are higher in variable regions than in conserved regions. The insertion rate in variable regions is substantially lower than the substitution rate, suggesting that selection acts more strongly on slippage products than on point mutations in these regions. Patterns of coevolution between variable regions may reflect the consequences of selection acting on the incorporation of slippage-derived sequences across the gene.     

A preliminary investigation of the order Bangiales (Bangiophycidae, Rhodophyta) based on sequences of nuclear small-subunit ribosomal RNA genes

We investigated phylogenetic relationships among red algae of the order Bangiales by analysis of sequences of the nuclear gene encoding cytosolic small-subunit ribosomal RNA in Bangia atropurpurea (Roth) C. Ag. and eight samples representing seven species of Porphyra. The ssu-rDNA range from 1818 to 1845 nucleotides in length, with guanosine plus cytosine ratios between 47.0% and 48.6%. A group IC1 intron occurs in the B atropurpurea ssu-rDNAs at the same position as in P. spiralis var. amplifolia Oliveira Filho et Coll and several other eukaryote ssu-rDNAs. The nine sequences form a stable monophyletic group upon phylogenetic analysis. The ssu-rDNA from B. atropurpurea nests stably within the Porphyra group and is closely related to P. amplissima (Kjellm.) Setchell et Hus in Hus, making the genus Porphyra paraphyletic. No correlation is seen between phylogenetic position and number of cell layers in the Porphyra thallus. We discuss possible taxonomic and evolutionary implications of these observations.     

Small-subunit ribosomal RNA sequence from Naegleria gruberi supports the polyphyletic origin of amoebas

We have sequenced the small-subunit ribosomal RNA gene of the amoebo- flagellate protozoan Naegleria gruberi. Comparison of this sequence with the rRNA sequences of other eukaryotes resulted in a phylogenetic tree that supports the suggested polyphyletic origin of amoebas and suggests a flagellate ancestry for Naegleria.      

Characterization of a novel small RNA encoded by Dictyostelium discoideum mitochondrial DNA

In this study, we analyzed a mitochondrial small (ms) RNA in Dictyostelium discoideum, which is 129 nucleotides long and has a GC content of only 22.5%. In the mitochondrial DNA, a single-copy gene (msr) for the ms RNA was located downstream of the gene for large-subunit rRNA. The location of msr was similar to that of the 5S rRNA gene in prokaryotes and chloroplasts, but clearly different from that in mitochondria of plants, liverwort and the chlorophycean alga Prototheca wikerhamii, in which small-subunit rRNA and 5S rRNA genes are closely linked. The primary sequence of ms RNA showed low homology with mitochondrial 5S rRNA from plants, liverwort and the chlorophycean alga, but the proposed secondary structure of ms RNA was similar to that of cytoplasmic 5S rRNA. In addition, ms RNA showed a highly conserved GAAC sequence in the same loop as in common 5S rRNA. However, ms RNA was detected mainly in the mitochondrial 25 000 × g supernatant fraction which was devoid of ribosomes. It is possible that ms RNA is an evolutionary derivative of mitochondrial 5S rRNA. Received: 17 May 1997 / Accepted: 26 August 1997     

SEQUENCES OF THE RRN18 GENES OF CHLAMYDOMONAS HUMICOLA AND C. DYSOSMOS ARE IDENTICAL,IN AGREEMENT WITH THEIR COMBINATION IN THE SPECIES C. APPLANATA (CHLOROPHYTA)1

The nuclear Rrn18 gene coding for small-subunit ribosomal RNA was amplified from Chlamydomonas humicola and C. dysosmos. The sequences were identical, in agreement with the combination of these two species under the name C. applanata on morphological and physiological grounds by Ettl and Schlösser (1992).     

Sequence evolution in and around the mitochondrial control region in birds

By cloning and sequencing 3.4 kilobases of snow goose mtDNA we found that the ND5 gene is followed by the genes for cytochrome b, tRNA^Thr, tRNA^Pro, ND6, tRNA^Glu, the control region, tRNA^Phe, and srRNA. This order is identical to that of chicken, quail, and duck mtDNA but differs from that of mammals and a frog (Xenopus). The mean extent of difference due to base substitution between goose and chicken is generally closer to the same comparison between rat and mouse but less than that between human and cow. For one of the nine regions compared (tRNA^Glu), the bird differences appear to be anomalous, possibly implicating altered functional constraints. Within the control region, several short sequences common to mammals are also conserved in the birds. Comparison of the goose control region with that of quail and chicken suggests that a sequence element with similarity to CSB-1 duplicated once prior to the divergence of goose and chicken and again on the lineage leading to chicken. Between goose (or duck) and chicken there are four times more transversions at the third positions of fourfold-degenerate codons in mitochondrial than in nuclear genes.Abbreviations CSB conserved sequence block - cytb cytochrome b - ND NADH dehydrogenase - srRNA small-subunit ribosomal RNA Deceased July 21, 1991 Correspondence to: T.W. Quinn at the University of Denver     

Antigenic structure of the influenza B virus hemagglutinin: nucleotide sequence analysis of antigenic variants selected with monoclonal antibodies.

We report here the complete nucleotide sequence of the hemagglutinin (HA) gene of influenza B virus B/Oregon/5/80 and, through comparative sequence analysis, identify amino acid substitutions in the HA1 polypeptide responsible for the antigenic alterations in laboratory-selected antigenic variants of this virus. The complete nucleotide sequence of the B/Oregon/5/80 HA gene was established by a combination of chemical sequencing of a full-length cDNA clone and dideoxy sequencing of the virion RNA. The nucleotide sequence is very similar to previously reported influenza B virus HA gene sequences and differs at only nine nucleotide positions from the B/Singapore/222/79 HA gene (Verhoeyen et al., Nucleic Acids Res. 11:4703-4712, 1983). The nucleotide sequences of the HA1 portions of the HA genes of 18 laboratory-selected antigenic variants were determined by the dideoxy method. Comparison of the deduced amino acid sequences of the parental and variant HA1 polypeptides revealed 16 different amino acid substitutions at nine positions. All amino acid substitutions resulted from single-point mutations, and no double mutants were detected, demonstrating that as in the influenza A viruses, single amino acid substitutions are sufficient to alter the antigenicity of the HA molecule. Many of the amino acid substitutions in the variants occurred at positions also observed to change in natural drift strains. The substitutions appear to identify at least two immunodominant regions which correspond to proposed antigenic sites A and B on the influenza A virus H3 HA.     

Heterogeneity of the genes coding for 5 S RNA in three related strains of the genus Bacillus.

Summary Three related strains of the genus Bacillus, viz. B. licheniformis, B. subtilis and Bacillus Q were all found to contain a minor species of 5 S RNA in an amount indicating that it is transcribed from only one of the multiple 5 S RNA cistrons known to be present in these strains. The major and minor types of 5 S RNA from each of the three strains could be separated from each other by polyacrylamide gel electrophoresis in the presence of urea. The complete nucleotide sequences of the minor B. subtilis and Bacillus Q 5 S RNAs have been determined. Comparison of these sequences to the previously determined sequence of the minor 5 S RNA from B. licheniformis (Raué et al., 1976) showed the three minor types of 5 S RNA to be identical except for the residues at positions 79, 85 and 95. Moreover, seven out of the eight sequence differences between the major and the minor 5 S RNA are the same in all three strains. Thus, the single cistron coding for minor 5 S RNA has been strongly conserved in all three strains, which may indicate a biological significance for the minor 5 S RNA species.     

Comparison of the nucleotide sequence of soybean 18S rRNA with the sequences of other small-subunit rRNAs

We present the sequence of the nuclear-encoded ribosomal small-subunit RNA from soybean. The soybean 18S rRNA sequence of 1807 nucleotides (nt) is contained in a gene family of approximately 800 closely related members per haploid genome. This sequence is compared with the ribosomal small-subunit RNAs of maize (1805 nt), yeast (1789 nt), Xenopus (1825 nt), rat (1869 nt), and Escherichia coli (1541 nt). Significant sequence homology is observed among the eukaryotic small-subunit rRNAs examined, and some sequence homology is observed between eukaryotic and prokaryotic small-subunit rRNAs. Conserved regions are found to be interspersed among highly diverged sequences. The significance of these comparisons is evaluated using computer simulation of a random sequence model. A tentative model of the secondary structure of soybean 18S rRNA is presented and discussed in the context of the functions of the various conserved regions within the sequence. On the basis of this model, the short base-paired sequences defining the four structural and functional domains of all 18S rRNAs are seen to be well conserved. The potential roles of other conserved soybean 18S rRNA sequences in protein synthesis are discussed.     

The Giardia lamblia actin gene and the phylogeny of eukaryotes

The single-copy actin gene of Giardia lamblia lacks introns; it has an average of 58% amino acid identity with the actin of other species; and 49 of its amino acids can be aligned with the amino acids of a consensus sequence of heat shock protein 70. Analysis of the potential RNA secondary structure in the transcribed region of the G. lamblia actin gene and of the single-copy actin gene of nine other species did not reveal any conserved structures. The G. lamblia actin sequence was used to root the phylogenetic trees based on 65 actin protein sequences from 43 species. This tree is congruent with small-subunit rRNA trees in that it shows that oomycetes are not related to higher fungi; that kinetoplatid protozoans, green plants, fungi and animals are monophyletic groups; and that the animal and fungal lineages share a more recent common ancestor than either does with the plant lineage. In contrast to smalls-ubunit rRNA trees, this tree shows that slime molds diverged after the plant lineage. The slower rate of evolution of actin genes of slime molds relative to those of plants, fungi, and animals species might be responsible for this incongruent branching. Correspondence to: G. Drouin