Annette W. Coleman

The Internal Transcribed Spacer 2 Exhibits a Common Secondary Structure in Green Algae and Flowering Plants

Abstract. Sequences of the Internal Transcribed Spacer 2 (ITS-2) regions of the nuclear rDNA repeats from 111 organisms of the family Volvocaceae (Chlorophyta) and unicellular organisms of the Volvocales, including Chlamydomonas reinhardtii, were determined. The use of thermodynamic energy optimization to generate secondary structures and phylogenetic comparative analysis of the spacer regions revealed a common secondary structure that is conserved despite wide intra- and interfamilial primary sequence divergence. The existence of this conserved higher-order structure is supported by the presence of numerous compensating basepair changes as well as by an evolutionary history of insertions and deletions that nevertheless maintains major aspects of the overall structure. Furthermore, this general structure is preserved across broad phylogenetic lines, as it is observed in the ITS-2s of other chlorophytes, including flowering plants; previous reports of common ITS-2 secondary structures in other eukaryotes were restricted to the order level. The reported ITS-2 structure possesses important conserved structural motifs which may help to mediate cleavages in the ITS-2 that occur during rRNA transcript processing. Their recognition can guide further studies of eukaryotic rRNA processing, and their application to sequence alignments may contribute significantly to the value of ITS-2 sequences in phylogenetic analyses at several taxonomic levels, but particularly in characterizing populations and species.

Applications of Fluorochromes to Pollen Biology. I. Mithramycin and 4′,6-Diamidino-2-Phenylindole (Dapi) as Vital Stains and for Quantitation of Nuclear Dna

The two DNA-specific fluorochromes DAPI and mithramycin have been found to be extremely useful dyes in studies of pollen development and growth. Both fluorochromes stain nuclei brilliantly either in fixed or in living tricellular and bicellular angiosperm pollen, thereby permitting rapid scanning for pollen abnormalities and easy observation of nuclear details. These water soluble dyes can be incorporated into the germination medium for studies of pollen germination in vitro, facilitating observation of the movement of generative, sperm and tube nuclei during pollen growth. In fixed pollen, the fluorochromes bind quantitatively with DNA and thus may be used to quantitate ploidy changes and to study cell cycles during pollen development, germination and fertilization.

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.

MOLECULAR DELINEATION OF SPECIES AND SPECIES RELATIONSHIPS IN THE RED ALGAL AGAROPHYTES GRACILARIOPSIS AND GRACILARIA (GRACILARIALES)1

Delineation of species in the economically important agarophyte genera Gracilaria and Gracilariopsis has proven extremely difficult using available morphological characteristics. In this study, we examine the usefulness of two transcribed spacers for molecular systematic studies of these genera. The polymerase chain reaction was used to amplify the internal transcribed spacers (ITSs) and the intervening 5.8S ribosomal DNA of the nuclear ribosomal repeat region. In addition, a plastid spacer region and flanking regions of coding genes were amplified from the RUBISCO operon. Both regions were sequenced for individuals and populations of Gracilariopsis lemaneiformis (Bory) Dawson, Acleto, et Foldvik to determine the usefulness of these spacers in delimiting populations. These studies reveal that there is as much variation among individuals of a population as there is between individuals of geographically separate populations. In addition, the ITS spacer regions were compared between different species of Gracilariopsis and Gracilaria. The nuclear ITS spacer region is conserved at a species level in both genera and provides phylogenetically informative characters that can be used to examine species interrelationships among relatively closely related taxa. However, because of the difficulties of aligning this entire region among species from the two genera, the ITS region is not useful for examining intergenera relationships. ITS interspecies sequence comparisons indicate that Gracilariopsis lemaneiformis from California is significantly different from G. lemaneiformis from China and that a species of Gracilariopsis from Peru is more closely related to G. lemaneiformis from North Carolina than it is to the other Gracilariopsis species examined. In addition, these studies indicate that Gracilaria chilensis Bird, McLachlan, et Oliveira from New Zealand and Gracilaria tenuistipitata Chang et Xia from southeast Asia are as closely related as are Gracilaria verrucosa (Hudson) Papenfuss, G. pacifica Abbott, and Gracilaria robusta Kylin. Phylogenetic analysis of aligned plastid spacer sequences from Gracilaria and Gracilariopsis taxa provide similar conclusions about species relationships.

MOLECULAR DELINEATION OF SPECIES AND SYNGENS IN VOLVOCACEAN GREEN ALGAE (CHLOROPHYTA)1

Two species of the colonial green flagellate family Volvocaceae are worldwide in distribution yet exhibit contrasting species structure. Geographically disparate isolates of Gonium pectorale Mueller can interbreed while isolates of Pandorina morum Bory behave quite differently. More than 20 sexually isolated subpopulations occur within this species; these have been termed “syngens” (sensu Sonneborn). Because prezygotic barriers to mating cause intersyngen pairings to fail, breeding analyses cannot be used to estimate genetic relatedness among the syngens of P. morum. DNA comparisons provide an alternative method of assessing genetic relatedness. We compared the nucleotide sequence of the internal transcribed spacer (ITS) region of the nuclear ribosomal repeat among clones of P. morum and of G. pectorale. Members of syngens of P. morum with distribution restricted to one small geographical area show great similarity. Likewise, members of any syngen of worldwide distribution show near uniformity, even those from different continents. However, the ITS sequence of each syngen differs from that of other syngens. In contrast, G. pectorale, which has an ITS region that is remarkably uniform throughout the world, appears to consist of a single syngen within North America and Europe by mating tests. The molecular data are in complete conformity with previous syngen assignment. Because the latter is based on mating affinity, with two complementary mating types per syngen, the evolution of new mating type pairs appears to be the basis of microevolution in these algae. We infer that either P. morum is a more ancient species than G. pectorale or that P. morum has a less stable genome. In either case, the biogeographic distribution of certain syngens may reflect climatological changes of the past.

RIBOSOMAL DNA ITS-1 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 mis-identified 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.

The reversible control of animal cell differentiation by the thymidine analog, 5-bromodeoxyuridine☆

Abstract In addition to suppressing reversibly the differentiation of chicken embryo skeletal muscle cells in culture, BUdR also reversibly suppresses the resumption of specialized synthesis by sternal cartilage and pigmented retina cells in culture. This effect is demonstrated best at concentrations of the drug which do not interfere with cell proliferation. Only mitotically active cells are sensitive, and treatment for less than one generation time is sufficient, at least for chondrocytes. Inhibition of DNA synthesis during exposure to BUdR prevents the suppression, as does competition by equimolar concentrations of TdR. Suppressed cells exhibit an indistinctive morphology, produce little or no myosin, acid muco-polysaccharide or melanin, but retain their commitment to differentiate into the specific cell type for which they were previously determined. The findings are discussed with respect to their implications for the mechanisms controlling cellular differentiation.

Paramecium aurelia revisited

Abstract. 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.

A clonal study of the reversible inhibition of muscle differentiation by the halogenated thymidine analog 5-bromodeoxyuridine☆

Abstract The thymidine analog 5-bromodeoxyuridine (BUdR) is well known for its mutagenic effects when incorporated in DNA. Similarly, BUdR has been reported by several workers to inhibit cytodifferentiation when applied during the cell proliferative phase. We have observed that relatively low levels (ca 10−6M) of BUdR, IUdR, ClUdR, and BCdR all cause a gradual change in cell morphology, and inhibit the fusion of cells to form syncytia in muscle clones, but do not interfere with proliferation of mononuclear cells. The inhibition of differentiation can be reversed by further cell division in the absence of the analog. After several days, this results in clonal cultures which are indistinguishable from untreated controls both in terms of total number of clones and of the proportion of clones which are myogenic; thus the drugs are not acting either by cell selection or mutagenesis. BUdR is incorporated into DNA in place of thymine, its effect on differentiation can be competitively prevented by simultaneous inclusion in the medium of TdR or compounds readily convertible to TdR, and it has no detectable effect on postmitotic cells (muscle fibers). Although this suggests that BUdR exerts its effect on differentiation from a site in the DNA, an effect on cell metabolism at some other level cannot be ruled out.

The advantages of the ITS2 region of the nuclear rDNA cistron for analysis of phylogenetic relationships of insects: a Drosophila example.

We examined the utility for phylogenetic reconstruction of the second internal transcribed spacer (ITS2), lying between the nuclear 5.8S gene and the gene for large subunit ribosomal RNA, using sequences of Ceratitis, Bactrocera, Musca, and Drosophila. We aligned and analyzed 13 sequences from GenBank and 11 new sequences from diverse species of Drosophila. Derivation of the secondary structure of the ITS2, the RNA transcript folding pattern required for transcript processing into functional RNA units, revealed the facets of sequence conservation common to all the sequences, that then allowed alignment of all the genera. The resultant tree, though including only a sparse representation of the enormous Drosophila diversity, conforms generally with the consensus of all prior phylogenetic reconstructions, using eight other nuclear and mitochondrial gene regions; where species representation is greater, as in the melanogaster subgroup of the Sophophora subgenus representatives, it conforms exactly. The paradigm ITS2 secondary structure presented can now be used to assess the genus more thoroughly, since its base pairing pattern makes alignment of sequences obvious. In addition, it shows that these insects share the ITS2 secondary structure characteristics of the other major animal groups as well as the green line of eukaryote evolution. The relatively short (<400 bp) ITS2 region seems ideal for reconstructing evolutionary relationships at the levels of species, genera, and perhaps even higher.