Transcript Secondary Structure Research Articles

The RNA secondary structure of androgen receptor-FL and V7 transcripts reveals novel regulatory regions.

The androgen receptor (AR) is a ligand-dependent nuclear transcription factor belonging to the steroid hormone nuclear receptor family. Due to its roles in regulating cell proliferation and differentiation, AR is tightly regulated to maintain proper levels of itself and the many genes it controls. AR dysregulation is a driver of many human diseases including prostate cancer. Though this dysregulation often occurs at the RNA level, there are many unknowns surrounding post-transcriptional regulation of AR mRNA, particularly the role that RNA secondary structure plays. Thus, a comprehensive analysis of AR transcript secondary structure is needed. We address this through the computational and experimental analyses of two key isoforms, full length (AR-FL) and truncated (AR-V7). Here, a combination of in-cell RNA secondary structure probing experiments (targeted DMS-MaPseq) and computational predictions were used to characterize the static structural landscape and conformational dynamics of both isoforms. Additionally, in-cell assays were used to identify functionally relevant structures in the 5' and 3' UTRs of AR-FL. A notable example is a conserved stem loop structure in the 5'UTR of AR-FL that can bind to Poly(RC) Binding Protein 2 (PCBP2). Taken together, our results reveal novel features that regulate AR expression.

RNA structure profiling at single-cell resolution reveals new determinants of cell identity.

RNA structure is critical for multiple steps in gene regulation. However, how the structures of transcripts differ both within and between individual cells is unknown. Here we develop a SHAPE-inspired method called single-cell structure probing of RNA transcripts that enables simultaneous determination of transcript secondary structure and abundance at single-cell resolution. We apply single-cell structure probing of RNA transcripts to human embryonic stem cells and differentiating neurons. Remarkably, RNA structure is more homogeneous in human embryonic stem cells compared with neurons, with the greatest homogeneity found in coding regions. More extensive heterogeneity is found within 3' untranslated regions and is determined by specific RNA-binding proteins. Overall RNA structure profiles better discriminate cell type identity and differentiation stage than gene expression profiles alone. We further discover a cell-type variable region of 18S ribosomal RNA that is associated with cell cycle and translation control. Our method opens the door to the systematic characterization of RNA structure-function relationships at single-cell resolution.

Molecular bases for strong phenotypic effects of single synonymous codon substitutions in the E. coli ccdB toxin gene

BackgroundSingle synonymous codon mutations typically have only minor or no effects on gene function. Here, we estimate the effects on cell growth of ~ 200 single synonymous codon mutations in an operonic context by mutating almost all positions of ccdB, the 101-residue long cytotoxin of the ccdAB Toxin-Antitoxin (TA) operon to most degenerate codons. Phenotypes were assayed by transforming the mutant library into CcdB sensitive and resistant E. coli strains, isolating plasmid pools, and subjecting them to deep sequencing. Since autoregulation is a hallmark of TA operons, phenotypes obtained for ccdB synonymous mutants after transformation in a RelE toxin reporter strain followed by deep sequencing provided information on the amount of CcdAB complex formed.ResultsSynonymous mutations in the N-terminal region involved in translation initiation showed the strongest non-neutral phenotypic effects. We observe an interplay of numerous factors, namely, location of the codon, codon usage, t-RNA abundance, formation of anti-Shine Dalgarno sequences, predicted transcript secondary structure, and evolutionary conservation in determining phenotypic effects of ccdB synonymous mutations. Incorporation of an N-terminal, hyperactive synonymous mutation, in the background of the single synonymous codon mutant library sufficiently increased translation initiation, such that mutational effects on either folding or termination of translation became more apparent. Introduction of putative pause sites not only affects the translational rate, but might also alter the folding kinetics of the protein in vivo.ConclusionIn summary, the study provides novel insights into diverse mechanisms by which synonymous mutations modulate gene function. This information is useful in optimizing heterologous gene expression in E. coli and understanding the molecular bases for alteration in gene expression that arise due to synonymous mutations.

Mutant Allele of CD44 (rs8193C>T) and Pum2 Regulatory Element as A Prognosis Factor of Prostate Neoplasms: A Case-Control and In Silico Studies.

Expression of CD44 variant 6 (CD44v6) as a homing-associated cell adhesion molecule (HCAM), has proved to change most cancer cells. Aim of the study is the effect of mutant allele of CD44 (rs8193C>T) and Pum2 regulatory element as a prognosis factor of prostate neoplasms: a case-control and in silico studies in the Mazandaran province-Iran. In a case-control study, CD44-rs8193C>T genotyping of the 420 prostate neoplasms (210 benign prostatic hyperplasia (BPH) patients and 210 prostate cancer patients) and 150 healthy samples are performed by the touchdown polymerase chain reaction with confronting two-pair primers (PCR-CTPP) method. The T mutant allele effects on the mRNA structure and cell pathways were also investigated in silico methods. Our results showed that the increase of T mutant allele frequency was significantly associated with BPH compared with prostate cancer. Furthermore, results showed TT genotype was significantly associated with BPH [odds ratio (OR)=0.572 and P=0.015], and also influenced the CD44v6 transcript secondary structure, miRNA binding, and regulatory element-binding site for Pum2 protein. Attachment of Pum2 to standard CD44 transcript may lead to transcript isoform-switching and shift-expression to a variety of CD44 isoforms, which can trigger some of the cell signaling pathways, such as Nanog-Stat, PKC-Nanog, and PKC-Twist. Based on this, the presence of the T mutant allele of CD44 (rs8193C>T) in the populations may create a regulatory element-binding site for Pum2. So, it could be known as a prognosis factor and prediction of prostate neoplasms. However, more comprehensive studies in different populations (with various ethnicities and large population sizes), and also CD44v6 gene expression studies in protein and transcript levels are required to confirm our data.

Genome-wide analysis of lncRNA stability in human.

Transcript stability is associated with many biological processes, and the factors affecting mRNA stability have been extensively studied. However, little is known about the features related to human long noncoding RNA (lncRNA) stability. By inhibiting transcription and collecting samples in 10 time points, genome-wide RNA-seq studies was performed in human lung adenocarcinoma cells (A549) and RNA half-life datasets were constructed. The following observations were obtained. First, the half-life distributions of both lncRNAs and messanger RNAs (mRNAs) with one exon (lnc-human1 and m-human1) were significantly different from those of both lncRNAs and mRNAs with more than one exon (lnc-human2 and m-human2). Furthermore, some factors such as full-length transcript secondary structures played a contrary role in lnc-human1 and m-human2. Second, through the half-life comparisons of nucleus- and cytoplasm-specific and common lncRNAs and mRNAs, lncRNAs (mRNAs) in the nucleus were found to be less stable than those in the cytoplasm, which was derived from transcripts themselves rather than cellular location. Third, kmers-based protein-RNA or RNA-RNA interactions promoted lncRNA stability from lnc-human1 and decreased mRNA stability from m-human2 with high probability. Finally, through applying deep learning-based regression, a non-linear relationship was found to exist between the half-lives of lncRNAs (mRNAs) and related factors. The present study established lncRNA and mRNA half-life regulation networks in the A549 cell line and shed new light on the degradation behaviors of both lncRNAs and mRNAs.

Posttranscriptional Control of Gene Expression and Role of Small RNAs in <i>Streptococcus mutans</i>

Streptococcus mutansis a key contributor to the formation of biofilms associated with dental caries disease. Living in the oral environment and developing of disease require tight gene regulation. In bacteria, gene expression is most commonly regulated at the level of transcription initiation. This control involved interactions of specific DNA sequences with regulatory proteins. A second mechanism of control of gene expression is mediated at the RNA level by several mechanisms and is generally called posttranscriptional regulation. These mechanisms include cis- and trans-acting small, non-coding RNAs, RNA-binding proteins, riboswitches, thermosensors, RNases, and Type I toxin-antitoxin systems, and may result in changes in RNA stability, efficiency of ribosome binding, translation initiation, and transcript secondary structures. Here I review the posttranscriptional regulation in S. mutans.

Isolation and sequence analysis of pCS36-4CPA, a small plasmid from Citrobacter sp. 36-4CPA.

A small plasmid designated pCS36-4CPA with a size of 5217 base pairs and G-C content of 50.74% was isolated from Citrobacter sp. 36-4CPA. The origin of replication (ori) of the plasmid was identified as a region of about 800 bp in length with an identity of 67.1% to the ColE1 plasmid at the nucleotide level. The replication region contained typical elements of ColE1-like plasmids: RNA I and RNA II with their corresponding −10 and −35 boxes, a single-strand initiation site (ssi), and a lagging-strand termination site (terH). As seen in other ColE1-like plasmids, pCS36-4CPA carried mobilisation machinery that include mobABCD genes but it did not possess the rom gene. Analysis of the multimer resolution site (mrs) was performed and XerC and XerD binding sites were identified. Also, the 70-nt transcript Rcd of pCS36-4CPA was predicted and similarity of the transcript’s secondary structure with those of the ColE1-family was shown. The cargo module of pCS36-4CPA contained three open reading frames (ORFs). Two of them (ORF5 and ORF6) showed no significant homology to any known gene sequences but contained putative THAP DNA-binding (DBD) and type II restriction endonuclease EcoO109I domains. The seventh open reading frame (ORF7) encodes YhdJ-like DNA modification methylase. The region highly homologous to pCS36-4CPA was found in the Salmonella phage SE2 genome.

Cryptic diversity in a cosmopolitan diatom known as Asterionellopsis glacialis (Fragilariaceae): Implications for ecology, biogeography, and taxonomy

Diatoms have long been known as the most species-rich of algal groups, with a wide range of estimates for species number (20-200 k) due to differing species concepts. The fine valve structure in Asterionellopsis glacialis, a diatom believed cosmopolitan and eurytopic, has never been systematically examined using modern microscopy and is an excellent candidate to genetically test morphology-based conspecificity among its geographically distant culture isolates. Isolates from the Atlantic and Pacific Oceans that were morphologically delineated as A. glacialis were genetically characterized using three nuclear DNA regions (two 18S rDNA fragments and ITS region) and one plastidal (rbcL) and one mitochondrial gene (cox1) and related to SEM-based morphometrics. Five genetically distinct groupings were found, four of which are new to science. ITS2 RNA transcript secondary structure was species specific as were plastidal and mitochondrial genes, while the 18S gene fragments did not diverge sufficiently to segregate new species efficiently. We genetically circumscribed the A. glacialis epitype. The morphological diversification of the species examined in this study lags behind their genetic divergence. The currently accepted 2% cutoff level of operational taxonomic unit (OTU) clustering in 18S rDNA environmental sequencing is too high to recognize genetic diversity in Asterionellopsis and very likely in many other species. Our results support the notion that a considerable number of species and diversity remain to be discovered among diatoms and that species number may be more in line with higher estimates. Molecular signatures of the species discovered here will aid in their globally consistent identification and ultimate understanding of their ecology.

Staufen1 senses overall transcript secondary structure to regulate translation.

Human Staufen1 (Stau1) is a double-stranded RNA (dsRNA)-binding protein implicated in multiple post-transcriptional gene-regulatory processes. Here we combined RNA immunoprecipitation in tandem (RIPiT) with RNase footprinting, formaldehyde cross-linking, sonication-mediated RNA fragmentation and deep sequencing to map Staufen1-binding sites transcriptome wide. We find that Stau1 binds complex secondary structures containing multiple short helices, many of which are formed by inverted Alu elements in annotated 3' untranslated regions (UTRs) or in 'strongly distal' 3' UTRs. Stau1 also interacts with actively translating ribosomes and with mRNA coding sequences (CDSs) and 3' UTRs in proportion to their GC content and propensity to form internal secondary structure. On mRNAs with high CDS GC content, higher Stau1 levels lead to greater ribosome densities, thus suggesting a general role for Stau1 in modulating translation elongation through structured CDS regions. Our results also indicate that Stau1 regulates translation of transcription-regulatory proteins.

Analysis of Mammalian rDNA Internal Transcribed Spacers

Nuclear rDNA Internal Transcribed Spacers, ITS1 and ITS2, are widely used for eukaryote phylogenetic studies from the ordinal level to the species level, and there is even a database for ITS2 sequences. However, ITS regions have been ignored in mammalian phylogenetic studies, and only a few rodent and ape sequences are represented in GenBank. The reasons for this dearth, and the remedies, are described here. We have recovered these sequences, mostly >1 kb in length, for 36 mammalian species. Sequence alignment and transcript folding comparisons reveal the rRNA transcript secondary structure. Mammalian ITS regions, though quite long, still fold into the recognizable secondary structure of other eukaryotes. The ITS2 in particular bears the four standard helix loops, and loops II and III have the hallmark characters universal to eukaryotes. Both sequence and insertions/deletions of transcript secondary structure helices observed here support the four superorder taxonomy of Placentalia. On the family level, major unique indels, neatly excising entire helices, will be useful when additional species are represented, resulting in significant further understanding of the details of mammalian evolutionary history. Furthermore, the identification of a highly conserved element of ITS1 common to warm-blooded vertebrates may aid in deciphering the complex mechanism of RNA transcript processing. This is the last major group of terrestrial vertebrates for which rRNA ITS secondary structure has been resolved.

Catalyzing plant science research with RNA-seq.

Next generation DNA sequencing technologies are driving increasingly rapid, affordable and high resolution analyses of plant transcriptomes through sequencing of their associated cDNA (complementary DNA) populations; an analytical platform commonly referred to as RNA-sequencing (RNA-seq). Since entering the arena of whole genome profiling technologies only a few years ago, RNA-seq has proven itself to be a powerful tool with a remarkably diverse range of applications, from detailed studies of biological processes at the cell type-specific level, to providing insights into fundamental questions in plant biology on an evolutionary time scale. Applications include generating genomic data for heretofore unsequenced species, thus expanding the boundaries of what had been considered “model organisms,” elucidating structural and regulatory gene networks, revealing how plants respond to developmental cues and their environment, allowing a better understanding of the relationships between genes and their products, and uniting the “omics” fields of transcriptomics, proteomics, and metabolomics into a now common systems biology paradigm. We provide an overview of the breadth of such studies and summarize the range of RNA-seq protocols that have been developed to address questions spanning cell type-specific-based transcriptomics, transcript secondary structure and gene mapping.

Secondary Structure of the rRNA ITS2 Region Reveals Key Evolutionary Patterns in Acroporid Corals

This study investigates the ribosomal RNA transcript secondary structure in corals as confirmed by compensatory base changes in Isopora/Acropora species. These species are unique versus all other corals in the absence of a eukaryote-wide conserved structural component, the helix III in internal transcriber spacer (ITS) 2, and their variability in the 5.8S-LSU helix basal to ITS2, a helix with pairings identical among all other scleractinian corals. Furthermore, Isopora/Acropora individuals display at least two, and as many as three, ITS sequence isotypes in their genome which appear to be capable of function. From consideration of the conserved elements in ITS2 and flanking regions, it appears that there are three major groups within the IsoporaAcropora lineage: the Isopora + Acropora "longi" group, the large group including Caribbean Acropora + the Acropora "carib" types plus the bulk of the Indo-Pacific Acropora species, and the remaining enigmatic "pseudo" group found in the Pacific. Interbreeding is possible among Caribbean A. palmata and A. cervicornis and among some species of Indo-Pacific Acropora. Recombinant ITS sequences are obvious among these latter, such that morphology (as represented by species name) does not correlate with common ITS sequence. The combination of characters revealed by RNA secondary structure analyses suggests a recent past/current history of interbreeding among the Indo-Pacific Acropora species and a shared ancestry of some of these with the Caribbean Acropora. The unusual absence of helix III of ITS2 of Isopora/Acropora species may have some causative role in the equally unusual instability in the 5.8S-LSU helix basal to ITS2 of this species complex.

Posttranscriptional regulation ofcspEinEscherichia coli: involvement of the short 5′-untranslated region

Escherichia coli K-12 contains nine paralogs of CspA, namely CspA-CspI. In spite of considerable sequence similarity among these genes, the individual members of this family show significant differences in their expression regulation. Among these nine members, cspA, B, G and I have been reported to be cold-induced. The unusually long 5'-untranslated region (5'-UTR) of these four and other cold-induced genes has often been associated with their inducibility. Sequence analysis of the cspE upstream region revealed two promoter-like motifs having high scores. We identified the promoter site and established that cspE has a much shorter 5'-UTR compared to other cold-induced genes. Our results showed that cspE is induced to about threefold at both the transcript and the protein level in response to cold-shock. Its transcript half-life increases significantly upon cold-shock. Furthermore, we demonstrated that RNase E, a key endonuclease responsible for mRNA degradation in E. coli, regulates cspE transcript stability, possibly through the assembly of a degradosome. In silico analysis of the cspE 5'-UTR revealed alternative secondary structures at 37 and 15 degrees C. A point mutation that was predicted to relax the secondary structure of the 5'-UTR at 15 degrees C showed considerable reduction in transcript stability, indicating that alternative transcript secondary structures might be the cause of the differential stability.

Pan-eukaryote ITS2 homologies revealed by RNA secondary structure

For evolutionary comparisons, phylogenetics and evaluation of potential interbreeding taxa of a species, various loci have served for animals and plants and protistans. One [second internal transcribed spacer (ITS2) of the nuclear ribosomal DNA] is highly suitable for all. Its sequence is species specific. It has already been used extensively and very successfully for plants and some protistans, and a few animals (where historically, the mitochondrial genes have dominated species studies). Despite initial impressions that ITS2 is too variable, it has proven to provide useful biological information at higher taxonomic levels, even across all eukaryotes, thanks to the conserved aspects of its transcript secondary structure. The review of all eukaryote groups reveals that ITS2 is expandable, but always retains in its RNA transcript a common core structure of two helices with hallmark characteristics important for ribosomal RNA processing. This aspect of its RNA transcript secondary structure can rescue difficult alignment problems, making the ITS2 a more powerful tool for phylogenetics. Equally important, the recognition of eukaryote-wide homology regions provides extensive and detailed information to test experimental studies of ribosomal rRNA processing.

Paramecium aureliaRevisited

The species Paramecium aurelia sensu latu, containing 15 sexually isolated subspecies (syngens), is the classic example of a sibling species complex in the ciliates. Using DNA sequence comparison, it is now possible to see whether this example parallels other studied sibling species complexes. We sequenced the internal transcribed spacer (ITS) region of the nuclear ribosomal cistron for 13 of the syngens plus two other Paramecium species and several Tetrahymena spp. Using available spirotrich sequences of the internal transcribed spacer 2 (ITS2), we established the RNA transcript folding pattern for ciliates. Ciliates exhibit the two highly conserved helices in their RNA transcript folding pattern in common with other eukaryotes, despite their unusual nuclear behavior and their presumed low copy number of micronuclear ribosomal repeats. Consequently, the set of 111-116 ITS2 nucleotide positions that are relatively conserved in evolution can be derived and used for comparative analysis. Mating behavior (i.e. gamete agglutination and fusion) is the character showing greatest correlation with the degree of ITS2 evolution in the P. aurelia complex, as also found in other eukaryotes. The degree of change in the ITS2 relatively conserved sequences found among the sibling species of P. aurelia is the same degree as found among the sibling species of the Drosophila melanogaster-mauritania-sechellia-simulans-yakuba species complex. The relatively conserved subregion of ITS2, determined from transcript secondary structure, is a tool for identifying the level of the biological species in the absence of knowledge of sexual compatibility in both micro- and macro-eukaryote species complexes.

ITS2 is a double-edged tool for eukaryote evolutionary comparisons

The internal transcribed spacer (ITS) region of the nuclear rDNA cistrons is one of the more frequently utilized regions for phylogenetic analyses at the genus and species levels. It has proven valuable for phylogenetic reconstruction of species and genus relationships, using comparisons of primary sequence. Although potential transcript secondary structure homology is often utilized to aid alignment in comparisons of ribosomal gene sequences, such consideration has rarely been applied to ITS primarily because secondary structures for its transcript were not available. Here, we describe the value of applying ITS2 RNA transcript secondary structure information to improve alignments, which then allows comparisons at even deeper taxonomic levels. The evolutionarily conserved subportions of ITS2, apparently necessary for positioning of the multimolecular transcript processing machinery, also provide material for distinguishing evolutionarily rare events, ‘Compensatory Base Changes’ in the relatively conserved regions, that might be useful in recognizing how arbitrary are the assignments of classical taxonomic ranks in disparate groups of eukaryotes. This relatively short and easily sequenced region of DNA – ITS2 – has yet to be fully exploited in phylogenetics.

Comparison of Eudorina/Pleodorina ITS sequences of isolates from nature with those from experimental hybrids

Internal transcribed spacer (ITS) regions of nuclear ribosomal repeats were compared among 50 Eudorina and Pleodorina isolates and two Volvox species known to clade with Eudorina species. Of the six major subclades found, four containing Eudorina and Pleodorina illinoisensis isolates, one containing Eudorina and Pleodorina indica, and one containing Volvox gigas and V. powersii, the basal branching order remains uncertain, but the positioning of isolates known to mate was always as nearest neighbors on the terminal branches of the tree. Four hybrid clones from a cross of E. elegans with P. illinoisensis, known from chromosome counts to be products of the failure of meiosis at zygote germination, contain both parental ITS repeat regions, as expected. However, they have in addition both crossover and other variant ITS cistrons among their many repeats of ITS. Such variation is limited to terminal regions of helices, as recognized from knowledge of RNA transcript secondary structure. Proper alignment then utilizes all of the nucleotide positions; the hybrid variants appear in positions intermediate between their parents in the tree. In fact, such variants seem to be hallmarks of recent hybridization events, since they were not found in any of the other 50 isolates.

Exploring the phylogenetic utility of ITS sequences for animals: a test case for abalone (Haliotis).

To evaluate the general utility of sequences of the nuclear rDNA internal transcribed spacer (ITS) regions for phylogenetic analyses of animal species groups and their broader relationships, sequences were obtained for 19 species of the genus Haliotis plus a keyhole limpet and a more distantly related gastropod, the Chilean abalone. Three subclades of Haliotis species appear consistently, each encompassing little variation. They are (A) the North Pacific species, (B) the European species, and (C) the Australia species. The one Caribbean species examined clearly groups with the North Pacific clade, not the European clade. H. midae (South Africa) and H. diversicolor supertexta (Taiwan) both diverge basal to the European and Australian species groups in the phylogenetic trees. Sequence comparisons showed that one species of Haliotis, H. iris from New Zealand, is quite distant from the remaining Haliotis species, almost as much as the more obvious outgroup, the keyhole limpet, an observation common to other DNA sequence analyses of these taxa. Using the rate of nucleotide change calculated from the sister Caribbean-Pacific pair, the length of the H. iris long branch is compatible with the suggestion that its ancestry became isolated on New Zealand at Gondwandan breakup. Use of ITS permits a totally independent estimate of the phylogenetic relationships, yet branching order was very similar to that established using other DNA regions studied previously, including those under strong positive selection. Knowledge of the RNA transcript secondary structure is particularly useful in the optimal alignment of more distantly related taxa. The RNA transcript secondary structure of Haliotis ITS2 shows conservation of features found also in ITS2 of angiosperms and algal taxa. Since ITS, particularly ITS2, is not saturated with nucleotide changes even at the family level, it should be useful for phylogenetic reconstruction of animal groups, not just at the species and genus levels but perhaps also for families and above.

A REASSESSMENT OF PHYLOGENETIC SIGNAL IN DNA SEQUENCES FROM THE ITS/5.8S REGION OF THE SYNUROPHYCEAE

Wee1, J. L. , Fasone1,2, L. D., Sattler1,3, A., Starks1,4, W. W. & Hurley5, D. L. 1Department of Biological Sciences, Loyola University, New Orleans, LA 70118‐6195, USA; 2Current address, Department of Biological Sciences, University of New Orleans, New Orleans, LA 70148, USA; 3Current address, Department of Zoology, Duke University, Durham, NC, 27708‐0325, USA; 4Current address, University of Tennessee‐Memphis, 255 South Dunlap, Box 108, Memphis, TN, 38126, USA; 5Department of Cell and Molecular Biology, Tulane University, New Orleans, LA 70118, USAThe objective of this study was to enhance the phylogenetic signal contained in the nuclear‐encoded ITS/5.8S region in the Synurophyceae. DNA sequences were determined for internal transcribed spacers 1 and 2, the 5.8S coding region as well as the 18S and 26S flanking regions annealing to PCR primers. DNA sequences were obtained from 15 Synura petersenii strains, 5 additional Synura species, 3 Mallomonas species, Tessellaria volvocina and Chrysodidymus synuroideus. Sequences from the S. petersenii strains and S. uvella aligned unambiguously. A parsimony analysis using S. uvella as the designated outgroup sorted the S. petersenii strains into two well‐supported clades or ITS variants. Sequence variation was low, and sequence divergence values ranged from 0‐1.4% within the ITS variants, to 3%‐4% between the two S. petersenii ITS variants and from 8.5%‐9% between S. uvella and the fifteen S. petersenii isolates. Extensive length variation occurred among the remaining synurophycean sequences (e.g. 495 bp in S. sphagnicola ‐ 598 bp in Tessellaria) and precluded reliable alignments. In the 5.8S coding region, changes at eight positions differed between S. uvella and all of the S. petersenii strains, suggesting that 5.8S sequences should contribute characters with low homoplasy for resolving interspecific relationships in the Synurophyceae. Other workers have used derived RNA transcript secondary structure to facilitate alignments and increase phylogenetic signal in ITS 1 and 2. This presentation summarizes the previous study at the intraspecific level and investigates the interspecific phylogenetic signal in the 5.8S region and in ITS 2 alignments ascertained from secondary structure.

A REASSESSMENT OF ITS/5.8S DNA SEQUENCE VARIATION AND PHYLOGENETIC SIGNAL IN THE SYNUROPHYCEAE

Previously, DNA sequences were determined for the entire ITS/5.8S region by direct sequencing of PCR amplification products. 28 sequences were obtained from 12 species of Synurophyceae, including Tessellaria volvocina, Chrysodidymus synuroideus, six species of Synura and four species of Mallomonas. Sequences also were determined from multiple strains of some species, including 15 strains of Synura petersenii. The length of these sequences varied from 495–598 bp. The large amount of ITS/5.8S length variation made initial attempts in determining reliable sequence alignment difficult and precluded their use in phylogenetic analyses of the class. Subsequently, the approach of Bogler & Simpson (1996) was investigated where reiterative, computer assisted alignments were generated and the quality of the alignment was evaluated by homoplasy index and consistency index values. However, these alignments also were equivocal. ITS/5.8S sequences sorted the 15 S. petersenii strains into two well supported ITS lineages. One ITS type included ten isolates from North America while isolates from the second ITS type included one each from Germany and Australia and three from North America. Results are presented here where the sequences from the 15 S. petersenii isolates were used to characterize ITS‐1 and ITS‐2 RNA transcript secondary structure. Next, this information was expanded to include ITS/5.8S sequences from the other taxa in the class. Finally, the characteristics of ITS 1 and ITS 2 RNA transcript secondary structure were used as a guide to align all of the ITS/5.8S sequences from the Synurophyceae in a final phylogenetic analysis.