The structure of nuclear pre-mRNA. VII. The hybridization and renaturation properties of double-stranded sequences of nuclear pre-mRNA]

Some properties of the double-stranded regions of pre-mRNA are discribed. 1. The double-stranded regions contain approximately 80 base pairs. 2. The material contains the heterogeneous populations of sequences and some homogenous material which renatures with a COT1/2 value of (1.5-3) X 10(-4). 3. Identical sequences of fast-renaturing "hairpins" may be found in various tissues. 4. Double-stranded RNA and mRNA have some sequences complementary to each other. These results consistent with the view that the hairpin sequences may act as specific recognition sites for ribonucleases involved in processing of pre-mRNA.     

Phylogenetic relationships in Pleurothallidinae (Orchidaceae): combined evidence from nuclear and plastid DNA sequences

To evaluate the monophyly of subtribe Pleurothallidinae (Epidendreae: Orchidaceae) and the component genera and to reveal evolutionary relationships and trends, we sequenced the nuclear ribosomal DNA internal transcribed spacers (ITS1 and ITS2) and 5.8S gene for 185 taxa. In addition, to improve the overall assessments along the spine of the topology, we added plastid sequences from matK, the trnL intron, and the trnL-F intergenic spacer for a representative subset of those taxa in the ITS study. All results were highly congruent, and so we then combined the sequence data from all three data sets in a separate analysis of 58 representative taxa. There is strong support in most analyses for the monophyly of Pleurothallidinae and in some for inclusion of Dilomilis and Neocognauxia of Laeliinae. Although most genera in the nine clades identified in the analyses are monophyletic, all data sets are highly congruent in revealing the polyphyly of Pleurothallis and its constitutent subgenera as presently understood. The high degree of homoplasy in morphological characters, especially floral characters, limits their usefulness in phylogenetic reconstruction of the subtribe.     

Endogenous oncornaviral DNA sequences: evidence for two classes of viral DNA sequences in guinea pig cells.

The nature of the endogenous viral DNA sequences in guinea pig cells was studied by hybridization. A segment of the viral RNA (r-VRNA) hybridizing to abundant (or reiterated) DNA sequences (R-VDNA) was isolated by recycling to a Cot of 300. The hybridization of the recycled VRNA, as well as the total VRNA, was followed by determining their kinetics and by Wetmur-Davidson analysis. The kinetics of hybridization of total VRNA were complex, did not follow a second-order kinetics, and revealed two slopes by Wetmur-Davidson analysis. The recycled RNA, on the other hand, had a second-order reaction rate expected of the hybridization between a single species of RNA and DNA sequences and yielded a single straight line in a Wetmur-Davidson plot. The Cot1/2 and slope of the recycled r-VRNA was almost identical to that of the abundant VDNA sequences obtained from the hybridization data of the total VRNA. Guinea pig 28S rRNA with or without recycling was used in monitoring hybridization rate. The kinetics of hybridization of 28S RNA followed a second-order reaction and produced a single straight line by Wetmur-Davidson plot, with a second-order reassociation rate constant of 9.6 x 10(-3) liters/mol-s, a Cot1/2 of 104 mol-s/liter, and reiteration frequency of 146. There was no difference in the kinetics of hybridization of 28S RNA before and after recycling. These experiments showed that guinea pig cells contain two classes of VDNA sequences. (i) R-VDNA sequences with a second-order reassociation rate constant of 8.2 x 10(-4) liters/mol-s, a Cot1/2 of 1,219 mol-s/liter, and a reiteration frequency of 12 represent 37.5% of the viral genome. (ii) Unique VDNA sequences with a second-order reassociation rate constant of 1.2 x 10(-4) liters/mol-s, a Cot1/2 of 7,692 mol-s/liter, and a reiteration frequency of 2 represent 62.5% of the viral genome.     

Structure of nuclear pre-mRNA. VI. "Reversed repeats" in animal DNA and their hybridization with double-stranded regions of pre-mRNA.

About 2% of mouse DNA reassociates at extremely low Cot values (10-7-10-6 mole-liter-1-sec). This DNA fraction was isolated with the aid of nuclease S1 and purified by chromatography on hydroxyapatite. The study of this fraction suggests that it has a structure of hairpin type. The DNA of the hairpins can hybridize with sequences forming the double-stranded regions in pre-mRNA. Probably at least some of the DNA of the hairpins, described as "reversed repeating sequences" of DNA, is transcribed with the formation of structures of hairpin type in pre-mRNA.     

Globin mRNA contains a sequence complementary to double-stranded region of nuclear pre-mRNA.

Melted ds RNA isolated from rabbit bone marrow pre-mRNA was hybridized with excess of globin mRNA which was prepared from rabbit reticulocytes. 7-9% of ds sequences became RNAase-stable and about 30% of the sequences could be bound to poly(U)-Sepharose through poly (A) of mRNA. The size of RNAase-stable hybrid is about 30 nucleotides, that is one fourth of the length of one strand of the ds RNA.     

Radiation of the Australian Salicornioideae (Chenopodiaceae)--based on evidence from nuclear and chloroplast DNA sequences

In phylogenetic analyses of nuclear ITS and chloroplast trnL DNA sequences, the mostly endemic Australian genera; Halosarcia, Pachycornia, Sclerostegia, Tecticornia, and Tegicornia of the subfamily Salicornioideae (Chenopodiaceae) together form a monophyletic group, congruent with the hypothesis that they evolved from a common ancestor. However, limited genetic differentiation evident in both nrDNA and cpDNA sequences among these taxa suggests a possible rapid radiation. Based on fossil pollen records and climatic models of other authors, it is hypothesized that the expansion of the Australian endemic Salicornioideae most likely occurred during the Late Miocene to Pliocene, when increasing aridity caused the formation of extensive salt lakes along endorheic paleodrainage channels. Moreover, Australian Sarcocornia representatives were supported as monophyletic, nested within a paraphyletic Sarcocornia clade that also comprised European Salicornia in the ITS analysis. This suggests that Sarcocornia arrived in Australia subsequent to the ancestor of the Australian endemic genera most likely via long-distance dispersal.     

Phylogeny of psychodid subfamilies (Diptera: Psychodidae) inferred from nuclear DNA sequences with a review of morphological evidence for relationships

Psychodidae is a diverse family of flies with approximately 3000 described species in six subfamilies, including Phlebotominae vectors of human disease. Psychodidae has been the subject of few phylogenetic investigations and development of a stable classification has been hampered by poor understanding of the morphology of larvae, pupae and adults. Specimens were collected, and we analysed DNA sequence data from two nuclear genes for one or more representatives of all subfamilies. The subfamilies with multiple representatives included were resolved as monophyletic with good support. Placement of Horaiellinae, Sycoracinae and Trichomyiinae remains unclear, whereas Bruchomyiinae is hypothesized as the sister group to (Phlebotominae + Psychodinae). Representatives of some psychodine tribes were resolved in agreement with previous hypotheses. Relationships among and within subfamilies are discussed, and morphological characters supporting these relationships are reviewed. One compelling synapomorphy of the male genitalia supporting a relationship between Phlebotominae and Psychodinae is the presence of articulated surstyli with apical retinacula. Only cerci are present and sometimes developed into clasping structures in males of other subfamilies.     

基于ITS序列探讨山茶属金花茶组的系统发育关系

测定了分布于我国的 2 2个山茶属金花茶组的种或变种的nrDNAITS区序列 ,它们的序列长度在 476～ 496之间。GC含量都超过了 70 % ,应用Kimura2 模型计算了序列间的分化程度 ,构建了最大简约树、邻接树和最大似然树 ,分析结果表明 :( 1 )淡黄金花茶、毛籽金花茶、陇瑞金花茶、弄岗金花茶、大样金花茶和凹脉金花茶的关系较近 ;( 2 )小瓣金花茶、小花金花茶、薄叶金花茶、多瓣金花茶、夏石金花茶和龙州金花茶的关系较近。ITS区序列分析结果与AFLP分析结果相近     

Phylogenetics of Cranichideae with emphasis on Spiranthinae (Orchidaceae, Orchidoideae): evidence from plastid and nuclear DNA sequences

DNA sequences from plastid rbcL and matK genes and the trnL-F region, as well as the nuclear ribosomal ITS region, were used to evaluate monophyly and subtribal delimitation of Cranichideae and generic relationships in Spiranthinae. Cranichideae are moderately supported as monophyletic, with Chloraeinae and Pterostylis-Megastylis indicated as their collective sisters. Within Cranichideae, Pachyplectroninae and Goodyerinae form a well-supported monophyletic group sister to a "core spiranthid" clade that includes, according to their branching order, Galeottiellinae, Manniellinae, and a Prescottiinae-Cranichidinae-Spiranthinae subclade. Inclusion of Galeottiella in Spiranthinae, as in previous classifications, renders the latter paraphyletic to all other spiranthid subtribes. Cranichidinae and Spiranthinae (minus Galeottiella) are monophyletic and strongly supported, but Prescottiinae form a grade that includes a strongly supported prescottioid Andean clade and a weakly supported Prescottia-Cranichidinae clade sister to Spiranthinae. Well-supported major clades in Spiranthinae identified in this study do not correspond to previous alliances or the narrowly defined subtribes in which they have been divided recently. Morphological characters, especially those that have been used for taxonomic delimitation in Cranichideae, are discussed against the framework of the molecular trees, emphasizing putative synapomorphies and problems derived from lack of information or inadequate interpretation of the characters.     

a molecular phylogeny of apiaceae subfamily Apioideae: evidence from nuclear ribosomal DNA internal transcribed spacer sequences

Phylogenetic relationships among 40 New World and Old World members of Apiaceae subfamily Apioideae, representing seven of the eight tribes and eight of the ten subtribes commonly recognized in the subfamily, were inferred from nucleotide sequence variation in the internal transcribed spacer (ITS) regions of 18-26S nuclear ribosomal DNA. Although the sequences are alignable, with only 11% of sites excluded from the analyses because of alignment ambiguity, divergence values in pairwise comparisons of unambiguous positions among all taxa were high and ranged from 0.5 to 33.2% of nucleotides in ITS 1 and from 0 to 33.2% of nucleotides in ITS 2. Average sequence divergence across both spacer regions was 18.4% of nucleotides. Phylogenies derived from ITS sequences estimated using neighbor-joining analysis of substitution rates, and maximum likelihood and parsimony methods give trees of essentially similar topology and indicate that: (1) there is little support for any existing system of classification of the subfamily that is based largely on morphological and anatomical features of the mericarp; (2) there is a major phylogenetic division within the subfamily, with one clade comprising the genus Smyrnium and those taxa belonging to Drude's tribes Dauceae, Scandiceae, and Laserpitieae and the other clade comprising all other examined taxa; and (3) the genera Arracacia, Coaxana, Coulterophytum, Enantiophylla, Myrrhidendron, Prionosciadium, and Rhodosciadium, all endemic to Mexico and Central America, comprise a clade but their relationships to other New World taxa are equivocal. A phylogeny derived from parsimony analysis of chloroplast DNA rpoC1 intron sequences is consistent with, but considerably less resolved than, relationships derived from these ITS regions. This study affirms that ITS sequences are useful for phylogenetic inference among closely related members of Apioideae but, owing to high rates of nucleotide substitution, are less useful in resolving relationships among the more ancestral nodes of the phylogeny.     

The infrageneric taxonomy of Chaerophyllum (Apiaceae) revisited: new evidence from nuclear ribosomal DNA ITS sequences and fruit anatomy

Evolutionary relationships among 65 Chaerophyllum spp. were inferred from nuclear ribosomal DNA internal transcribed spacer (ITS) sequence variation. Thirty‐one species, represented by 158 mericarp samples, were analysed for fruit anatomical character variation, employing phylogenetic and phenetic methods to explore their congruence with infrageneric divisions based on molecular data. Phylogenetic trees inferred from molecular data using maximum likelihood (ML) and Bayesian inference (BI) methods corroborated the division of the genus into four sections: Chaerophyllum, Dasypetalon, Physocaulis and Chrysocarpum. From among the newly sequenced species, the Greek endemic C. heldreichii was grouped with section Chaerophyllum, whereas the highly variable Asian C. reflexum–C. villosum complex formed an early‐branching paraphyletic assemblage in section Chrysocarpum. The recently described C. karsianum has an identical ITS sequence to C. bulbosum, whereas C. aksekiense was clearly separated from the morphologically similar C. macrospermum. Our study confirmed the postulated synonymy of several species on the basis of morphology, but also demonstrated distant relationships between some morphologically similar species. With the exception of the monotypic section Physocaulis, we were unable to find carpological traits matching sectional divisions. We hypothesize that fruit characters evolved rapidly as a result of diversification of members of the genus in different habitats. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 178 , 298–313.     

Phylogeny of swallows (Aves: Hirundinidae) estimated from nuclear and mitochondrial DNA sequences

The phylogeny of swallows was reconstructed by comparing segments of three genes, nuclear beta-fibrinogen intron 7 (betafib7), mitochondrial cytochrome b (cytb), and mitochondrial ND2, in a variety of combinations using maximum likelihood and Bayesian methods. betafib7 was sequenced for 47 species, cytb for 74 species, and ND2 for 61 species to yield comparisons among 75 of the 84 currently recognized swallow species. The family Hirundinidae was confirmed to consist of two clades, Pseudochelidoninae (river martins) and Hirundininae (typical swallows). The Hirundininae is further divided into mud nesters (Hirundo sensu lato), core martins (Phedina, Riparia, and New World endemic genera), and basal relicts (Psalidoprocne, Cheramoeca, and Pseudhirundo). We did not resolve the hierarchy among these three hirundinine groups, but discovered many relationships within them. Mud-nesting genera have the following relationships: (Hirundo sensu stricto, Ptyonoprogne), (Delichon, (Petrochelidon, Cecropis)). Core martins have the following topology: (Phedina, Riparia cincta), (Riparia sensu stricto, Tachycineta, ((Stelgidopteryx, Progne), (Neotropical endemic genera))). Interspecific relationships among the Neotropical endemics were resolved completely; Atticora and Notiochelidon are paraphyletic, and all Neotropical endemics probably should be lumped into one or two genera. The final group of hirundinines, the basal relicts, consists of a sister pair, the Australian Cheramoeca and African Pseudhirundo. The African saw-wings (Psalidoprocne) are their likely sister group.     

Population history of Manihot esculenta (Euphorbiaceae) inferred from nuclear DNA sequences

The nature of gene flow in plants -- including the propensity for interspecific introgression -- makes them interesting candidates for phylogeographical analysis. Plant phylogeography studies have been limited, however, by the availability of suitable intraspecific variation. In this study, DNA sequence variation from a nuclear gene [Glyceraldehyde 3-phosphate dehydrogenase; (G3pdh)] was used to examine the population history of Manihot esculenta ssp. flabellifolia and a potentially hybridizing species, M. pruinosa. These species occur in the rainforest-savanna ecotone adjoining the Amazon basin, a region believed to have undergone major habitat shifts since the Pleistocene. Geographical distributions of the G3pdh haplotypes indicate genetic isolation-by-distance across the range of M. esculenta ssp. flabellifolia. However, there is greater genetic similarity between northeastern and western populations than would be expected given the present species distribution. A nested clade analysis suggests that northeastern and western populations were connected by gene flow until relatively recently, when they became fragmented. This inferred fragmentation event is consistent with post-Pleistocene habitat shifts proposed for the Amazon basin. At the interspecific level, haplotype sharing with M. pruinosa may reflect either recent interspecific introgression or incomplete lineage sorting between these closely related species.     

The influenza virus haemagglutinin gene: cloning and characterisation of a double-stranded DNA copy.

A protocol has been developed for the synthesis of a double-stranded DNA (dsDNA) copy of the influenza virus RNA genome segment which codes for the major surface antigen, haemagglutinin (HA). This dsDNA copy was inserted, after digestion with S1 nuclease and poly (dC) tailing with terminal transferase, into poly(dG)-tailed, PstI-cut, pBR322 DNA, and used to transform E. coli RR1. Tetracycline-resistant bacterial colonies were screened for the presence of plasmid containing the copied HA gene by testing their ability to hybridise to a specific, 32P-labelled, single-stranded DNA probe. Four cloned hybrid plasmids, containing DNA complementary to the HA gene of the influenza strain 29C (a laboratory derivative of influenza A/NT/60/68 (1)) were analysed by restriction enzyme mapping. Each contained a dsDNA insert equivalent to a full length copy of the HA gene. The nucleotide sequence of a selected restriction fragment from the DNA inserted in one of these cloned plasmids (C89) was determined. The amino acid sequence deduced from these data agreed with the amino acid sequence determined for the corresponding region of HA from the influenza strain A/Mem/102/72, another member of the Hong Kong subtype, identifying the inserted dsDNA of C89 as an authentic copy of the influenza HA gene.     

The complex history of the genus Ilex L. (Aquifoliaceae): evidence from the comparison of plastid and nuclear DNA sequences and from fossil data

 A plastid phylogeny of the genus Ilex based on three different loci (the atpB-rbcL spacer, trnL-trnF and rbcL) is compared with its nuclear phylogeny based on two different loci (the ribosomal ITS and the 5S RNA spacer). These two sets of molecular data are then compared to geographical and temporal data from the fossil record. The plastid phylogeny is strongly correlated with the geographic distribution of extant species. However, the nuclear phylogeny is strongly incongruent with the plastid phylogeny, suggesting frequent interlineage hybridizations. Moreover, the comparison of the ribosomal ITS tree and the 5S RNA spacer tree indicates also possible lineage sorting. Particularly interesting is the finding of two different Ilex lineages in the plastid American clade showing different biogeographic patterns in South America. One of them has a simple North American/South American biogeographical relationship. The other has complex biogeographical relationships, some species showing direct Asian/South American biogeographical relationships. During its history, the genus Ilex probably experienced frequent lineage sorting and interlineage hybridization with subsequent nuclear or cytoplasmic introgression, making the study of its history very complex. Received September 24, 2001; accepted August 19, 2002 Published online: November 28, 2002 Addresses of the authors: Jean-Fran?ois Manen (e-mail: manen@cjb.ville-ge.ch), Yamama Naciri-Graven, Conservatoire et Jardin Botaniques, Impératrice 1, CH-1292 Chambésy/Genève, Switzerland. Michael C. Boulter, Palaeobiology Research Unit, University of East London, Romford Road, London E15 4LZ, UK.     

The phylogenetic position of Anomochilidae (Reptilia: Serpentes): first evidence from DNA sequences

Previously, anilioids (Aniliidae, Anomochilidae, Cylindrophiidae and Uropeltidae) were considered the only extant, non‐macrostomatan alethinophidian snakes. Although their monophyly and intrarelationships remained poorly established, their fossoriality, small gape, and inferred phylogenetic position have been important evidence in orthodox scenarios about early snake evolution. Recent molecular studies including aniliids, cylindrophiids and uropeltids indicate anilioid polyphyly, with the latter two families comprising a clade nested within Macrostomata. We carried out the first molecular phylogenetic analysis to include the very poorly known and seemingly rare Anomochilidae. Only partial sequences of 12S and 16S rRNA mitochondrial genes could be amplified from tissue collected from a single dead specimen of Anomochilus leonardi. Amplification failed for nuclear and other mitochondrial genes, and for all the investigated genes for the holotype and paratype of A. leonardi. Analyses recovered a para‐ or polyphyletic Anilioidea. Anomochilus is recovered as most closely related to Cylindrophis maculatus (rendering Cylindrophiidae possibly paraphyletic). The relatively small amount of available data produces only moderate levels of support, but the stability of taxa and agreement across different analytical methods and with larger analyses of snake phylogeny support the abandonment of Anilioidea as a natural taxon, and the recognition of a higher category for a clade comprising Asian anilioids (Anomochilidae, Cylindrophiidae and Uropeltidae).     

Evolution of modern humans: evidence from nuclear DNA polymorphisms.

Previously we have described studies of the evolution of modern humans based upon data for classical genetic markers and for nuclear DNA polymorphisms. Such polymorphisms provide a different point of view regarding human evolution than do mitochondrial DNA sequences. Here we compare revised dates for major migrations of anatomically modern humans, estimated from archaeological data, with separations suggested by a genetic tree constructed from classical marker allele frequencies. Analyses of DNA polymorphisms have now been extended and compared with those of classical markers; genetic trees continue to support the hypothesis of an initial African and non-African divergence for modern humans. We have also begun testing non-human primates for a set of human DNA polymorphisms. For most polymorphisms tested so far, humans share a single allele with other primates; such shared alleles are likely to be ancestral. Populations living in humid tropical environments have significantly higher frequencies of ancestral alleles than do other populations, supporting the hypothesis that natural selection acts to maintain high frequencies of particular alleles in some environments.     

A phylogeny of Apiaceae tribe Scandiceae: evidence from nuclear ribosomal DNA internal transcribed spacer sequences

The evolutionary relationships among members of Apiaceae (Umbelliferae) tribe Scandiceae and representatives of all major lineages of Apioideae (including putatively allied Caucalideae) identified in earlier molecular studies were inferred from nucleotide sequence variation in the internal transcribed spacer regions (ITS1 and ITS2) of nuclear ribosomal DNA. In all, 134 accessions representing 18 genera commonly treated in Scandiceae were analyzed. Phylogenies estimated using maximum parsimony and distance methods were generally similar and suggest that: (1) Scandiceae form a well-supported clade, consisting of the genera Anthriscus, Athamanta (in part), Balansaea, Chaerophyllum, Conopodium, Geocaryum, Kozlovia, Krasnovia, Myrrhis, Myrrhoides, Neoconopodium, Osmorhiza, Scandix, Sphallerocarpus, and Tinguarra; (2) Athamanta is polyphyletic, with A. della-cellae allied with Daucus and A. macedonica placed close to Pimpinella; and (3) Rhabdosciadium and Grammosciadium find affinity with the Aegopodium group of umbellifers, whereas the placement of the monotypic Molopospermum cannot be inferred because of its high sequence divergence. The genus Bubon has been restored with two new combinations, B. macedonicum subsp. albanicum and B. macedonicum subsp. arachnoideum. Scandiceae arise within paraphyletic Caucalideae, the latter comprising two major lineages whose relationships to Scandiceae are not clear. Therefore, a broad treatment of Scandiceae is proposed, with subtribes Scandicinae, Daucinae, and Torilidinae (the latter two representing the Daucus and Torilis subgroups, respectively, of recent molecular systematic investigations).     

Incongruence in Veroniceae (Plantaginaceae): evidence from two plastid and a nuclear ribosomal DNA region

Nucleotide data from nuclear ribosomal and plastid DNA did not agree on all relationships within Veroniceae (Plantaginaceae) in previous analyses. We present here additional data from a second plastid DNA marker, the rps16 intron, and evaluate incongruence between the ribosomal and plastid DNA matrices. Based on these evaluations and analysis of secondary structure in ITS, we conclude that there is a biological cause for incongruence among the genera of Veroniceae. The sequence data imply a history of hybridization and polyploidization, in which ancestors of Wulfeniopsis and Veronica+Paederota gave rise to Picrorhiza and ancestors of Wulfeniopsis and Wulfenia gave rise to Veronicastrum.     

Ancient allopolyploid speciation in Geinae (Rosaceae): evidence from nuclear granule-bound starch synthase (GBSSI) gene sequences

A nuclear low-copy gene phylogeny provides strong evidence for the hybrid origin of seven polyploid species in Geinae (Rosaceae). In a gene tree, alleles at homologous loci in an allopolyploid species are expected to be sisters to orthologues in the ancestral taxa rather than to each other. Alleles at a duplicated locus in an autopolyploid, however, are expected to be more closely related to each other than they are to any orthologous copies in closely related species. We cloned and sequenced about 1.9 kilobases from the 5' end of the GBSSI-1 gene from two diploid, one tetraploid, and six hexaploid species. Each of the three loci in the hexaploid species forms a separate group, two of which are more closely related to copies in other species than they are to each other. This finding indicates that the hexaploid lineage evolved through two consecutive allopolyploidization events. Based on the GBSSI-1 gene tree, we hypothesized that there was an initial hybridization between a diploid species from the ancestral lineage of Coluria and Waldsteinia and an unknown diploid species to form the tetraploid Geum heterocarpum lineage. Backcrossing of G. heterocarpum with a representative of the unknown diploid lineage then resulted in a hexaploid lineage that has radiated considerably since its origin, comprising at least 40 extant species with various morphologies. A penalized likelihood analysis indicated that Geinae may be about 17 million years old, implying that the hypothesized allopolyploid speciation events are relatively ancient. Six of the 22 cloned Geinae GBSSI-1 copies in this study, which all are duplicate copies in polyploid taxa, may have become pseudogenes. We compared the GBSSI-1 phylogeny with one from chloroplast data and explored implications for the evolution of some fruit characters.