Invariant Sites Research Articles

Appropriate Likelihood Ratio Tests and Marginal Distributions for Evolutionary Tree Models with Constraints on Parameters

We show how to make appropriate likelihood ratio tests for evolutionary tree models when parameters such as edge (internodes or branches) lengths have nonnegativity constraints. In such cases, under the null model of an edge length being zero, the marginal distribution of this parameter is proven to be a "half-normal", that is, 50% zero values and 50% the positive half of a normal distribution. Other constrained parameters, such as the proportion of invariant sites, give similar results. To make likelihood ratio tests between nested models, e.g., H(0): homogeneous site rates, and H(1): site rates follow a gamma distribution with variance 1/k, then asymptotically as sequence length increases, the distribution under H(0) becomes a mixture of chi distributions, in this case 50% chi(0), and 50% chi(1) (where the subscript denotes degrees of freedom, i.e. , not the usually assumed 100% chi(1); which leads to a conservative test). Such mixtures are sometimes called distributions. Simulations show that even with sequences as short as 125 sites, some parameters, including the proportion of invariant sites, fit asymptotic distributions closely.

A phylogeographical analysis of the bemisia tabaci species complex based on mitochondrial DNA markers

Mitochondrial 16S ( approximately 550 bp) and cytochrome oxidase I (COI) ( approximately 700 bp) sequences were utilized as markers to reconstruct a phylogeography for representative populations or biotypes of Bemisia tabaci. 16S sequences exhibited less divergence than COI sequences. Of the 429 characters examined for COI sequences, 185 sites were invariant, 244 were variable and 108 were informative. COI sequence identities yielded distances ranging from less than 1% to greater than 17%. Whitefly 16S sequences of 456 characters were analysed which consisted of 298 invariant sites, 158 variable sites and 53 informative sites. Phylogenetic analyses conducted by maximum parsimony, maximum-likelihood and neighbour-joining methods yielded almost identical phylogenetic reconstructions of trees that separated whiteflies based on geographical origin. The 16S and COI sequence data indicate that the B-biotype originated in the Old World (Europe, Asia and Africa) and is most closely related to B-like variants from Israel and Yemen, with the next closest relative being a biotype from Sudan. These data confirm the biochemical, genetic and behavioural polymorphisms described previously for B. tabaci. The consideration of all global variants of B. tabaci as a highly cryptic group of sibling species is argued.

Structural constraints on RNA virus evolution.

The recently discovered hepatitis G virus (HGV) or GB virus C (GBV-C) is widely distributed in human populations, and homologues such as HGV/GBV-CCPZ and GBV-A are found in a variety of different primate species. Both epidemiological and phylogenetic analyses support the hypothesis that GB viruses coevolved with their primate hosts, although their degree of sequence similarity appears incompatible with the high rate of sequence change of HGV/GBV-C over short observation periods. Comparison of complete coding sequences (8,500 bases) of different genotypes of HGV/GBV-C showed an excess of invariant synonymous sites (at 23% of all codons) compared with the frequency expected by chance (10%). To investigate the hypothesis that RNA secondary-structure formation through internal base pairing limited sequence variability at these sites, an algorithm was developed to detect covariant sites among HGV/GBV-C sequences of different genotypes. At least 35 covariant sites that were spatially associated with potential stem-loop structures were detected, whose positions correlated with positions in the genome that showed reductions in synonymous variability. Although the functional roles of the predicted secondary structures remain unclear, the restriction of sequence change imposed by secondary-structure formation provides a mechanism for differences in net rate of accumulation of nucleotide substitutions at different sites. However, the resulting disparity between short- and long-term rates of sequence change of HGV/GBV-C violates the assumptions of the "molecular clock." This places a major restriction on the use of nucleotide or amino acid sequence comparisons to calculate times of divergence of other viruses evolving under the same structural constraints as GB viruses.

Using novel phylogenetic methods to evaluate mammalian mtDNA, including amino acid-invariant sites-LogDet plus site stripping, to detect internal conflicts in the data, with special reference to the positions of hedgehog, armadillo, and elephant.

We look at the higher-order phylogeny of mammals, analyzing in detail the complete mtDNA sequences of more than 40 species. We test the support for several proposed superordinal relationships. To this end, we apply a number of recently programmed methods and approaches, plus better-established methods. New pairwise tests show highly significant evidence that amino acid frequencies are changing among nearly all the genomes studied when unvaried sites are ignored. LogDet amino acid distances, with modifications to take into account invariant sites, are combined with bootstrapping and the Neighbor Joining algorithm to account for these violations of standard models. To weight the more slowly evolving sites, we exclude the more rapidly evolving sites from the data by using "site stripping". This leads to changing optimal trees with nearly all methods. The bootstrap support for many hypotheses varies widely between methods, and few hypotheses can claim unanimous support from these data. Rather, we uncover good evidence that many of the earlier branching patterns in the placental subtree could be incorrect, including the placement of the root. The tRNA genes, for example, favor a split between the group hedgehog, rodents, and primates versus all other sequenced placentals. Such a grouping is not ruled out by the amino acid sequence data. A grouping of all rodents plus rabbit, the old Glires hypothesis, is also feasible with stripped amino acid data, and rodent monophyly is also common. The elephant sequence allows confident rejection of the older taxon Ferungulata (Simpson, 1945). In its place, the new taxa Scrotifera and Fereuungulata are defined. A new likelihood ratio test is used to detect differences between the optimal tree for tRNA versus that for amino acids. While not clearly significant as made, some results indicate the test is tending towards significance with more general models of evolution. Individual placement tests suggest alternative positions for hedgehog and elephant. Congruence arguments to support elephant and armadillo together are striking, suggesting a superordinal group composed of Xenarthra and African endemic mammals, which in turn may be near the root of the placental subtree. Thus, while casting doubt on some recent conclusions, the analyses are also unveiling some interesting new possibilities.

Maximum-Likelihood Analysis of the Tethythere Hypothesis Based on a Multigene Data Set and a Comparison of Different Models of Sequence Evolution

Hyracoids have been allied with either perissodactyls or tethytheres (i.e., Proboscidea + Sirenia) based on morphological data. The latter hypothesis, termed Paenungulata, is corroborated by numerous molecular studies. However, molecular studies have failed to support Tethytheria, a group that is supported by morphological data. We examined relationships among living paenungulate orders using a multigene data set that included sequences from four mitochondrial genes (12S rRNA, tRNA valine, 16S rRNA, cytochrome b) and four nuclear genes (aquaporin, A2AB, IRBP, vWF). Nineteen maximum-likelihood models were employed, including models with process partitions for base composition and substitution parameterizations. With the inclusion of partitions with a heterogeneous base composition, 18 of 19 models favored Hyracoidea + Sirenia. All 19 models favored Hyracoidea + Sirenia after excluding heterogeneous base composition partitions. Most of the support for Hyracoidea + Sirenia derived from the mitochondrial genes (bootstrap support ranged from 51 to 99%); Tethytheria, in turn, received 0 to 19% support in different analyses. Bootstrap support deriving from the nuclear genes was more evenly split among the competing hypotheses (3 to 45% for Tethytheria; 17.5 to 62% for Hyracoidea + Sirenia). Lineage-specific rate variation among both mitochondrial and nuclear genes may contribute to the different results that were obtained with mitochondrial versus nuclear data. Whether Tethytheria or a competing hypothesis is correct, short internodes on the molecular phylogenies suggest that paenungulate orders diverged from each other over a 5- to 8-million-year time window extending from the late Paleocene into the early Eocene. We also used likelihood-ratio tests to compare different models of sequence evolution. A gamma distribution of rates results in a greater improvement in likelihood scores than does an allowance for invariant sites. Twenty-one rate partitions corresponding to stems, loops, and codon positions of different genes result in higher likelihood scores than a gamma distribution of rates and/or an allowance for invariant sites. Process partitions of the data that incorporate base composition and substitution parameterizations result in significant improvements in likelihood scores in comparison to models that allow only for relative rate differences among partitions.

Hierarchical analysis of variation in the mitochondrial 16S rRNA gene among Hymenoptera.

Nucleotide sequences from a 434-bp region of the 16S rRNA gene were analyzed for 65 taxa of Hymenoptera (ants, bees, wasps, parasitoid wasps, sawflies) to examine the patterns of variation within the gene fragment and the taxonomic levels for which it shows maximum utility in phylogeny estimation. A hierarchical approach was adopted in the study through comparison of levels of sequence variation among taxa at different taxonomic levels. As previously reported for many holometabolous insects, the 16S data reported here for Hymenoptera are highly AT-rich and exhibit strong site-to-site variation in substitution rate. More precise estimates of the shape parameter (alpha) of the gamma distribution and the proportion of invariant sites were obtained in this study by employing a reference phylogeny and utilizing maximum-likelihood estimation. The effectiveness of this approach to recovering expected phylogenies of selected hymenopteran taxa has been tested against the use of maximum parsimony. This study finds that the 16S gene is most informative for phylogenetic analysis at two different levels: among closely related species or populations, and among tribes, subfamilies, and families. Maximization of the phylogenetic signal extracted from the 16S gene at higher taxonomic levels may require consideration of the base composition bias and the site-to-site rate variation in a maximum-likelihood framework.

Conserved in VivoPhosphorylation of Calnexin at Casein Kinase II Sites as Well as a Protein Kinase C/Proline-directed Kinase Site

Calnexin is a lectin-like chaperone of the endoplasmic reticulum (ER) that couples temporally and spatially N-linked oligosaccharide modifications with the productive folding of newly synthesized glycoproteins. Calnexin was originally identified as a major type I integral membrane protein substrate of kinase(s) associated with the ER. Casein kinase II (CK2) was subsequently identified as an ER-associated kinase responsible for the in vitro phosphorylation of calnexin in microsomes (Ou, W-J., Thomas, D. Y., Bell, A. W., and Bergeron, J. J. M. (1992) J. Biol. Chem. 267, 23789-23796). We now report on the in vivo sites of calnexin phosphorylation. After 32PO4 labeling of HepG2 and Madin-Darby canine kidney cells, immunoprecipitated calnexin was phosphorylated exclusively on serine residues. Using nonradiolabeled cells, we subjected calnexin immunoprecipitates to in gel tryptic digestion followed by nanoelectrospray mass spectrometry employing selective scans specific for detection of phosphorylated fragments. Mass analyses identified three phosphorylated sites in calnexin from either HepG2 or Madin-Darby canine kidney cells. The three sites were localized to the more carboxyl-terminal half of the cytosolic domain: S534DAE (CK2 motif), S544QEE (CK2 motif), and S563PR. We conclude that CK2 is a kinase that phosphorylates calnexin in vivo as well as in microsomes in vitro. Another yet to be identified kinase (protein kinase C and/or proline-directed kinase) is directed toward the most COOH-terminal serine residue. Elucidation of the signaling cascade responsible for calnexin phosphorylation at these sites in vivo may define a novel regulatory function for calnexin in cargo folding and transport to the ER exit sites.

Structural determinants in the sequences of immunoglobulin variable domain

To determine the general relation between the sequence and structure of variable domains of immunoglobulins we have carried out an analysis of their atomic structures, some 5300 different expressed sequences and the human germline gene segments. Variable domains are formed by two β-sheets, packed face to face, and the inter-strand turns. Comparison of the different known structures shows that they have a core of 76 residues which has the same main-chain conformation in all structures. This common core contains almost all of the β-sheet structure and three inter-strand turns. The regions that differ in conformation are the three hypervariable regions, three other inter-strand turns and a few adjacent residues.The 5300 expressed sequences currently known for variable domains were examined to determine the residues that occur at the 76 sites. Ignoring site conservations that occur for functional reasons, there are eight sites that have the same residue in almost all sequences; 12 that have one of a small group of very similar residues, and 52 where the chemical character of the residues is strongly conserved but not their volume.The role of residues at each site in the core was determined from the examination of their accessible surface areas, contacts, packing and buried side-chain hydrogen bonds. The most strongly conserved sites form the “deep” structure of the domain at the centre of the interface between the β-sheets. It includes eight invariant sites and 11 sites that have one of a set of very similar residues. Around the deep structure there are buried hydrophobic residues that, in different variable domains, can differ greatly in volume. These differences in volume are accommodated by conformational changes in turn regions that are outside the common core. On the surface nearly all residues not involved in function or turn conformations strongly conserve hydrophilic or neutral residues.The implications of these results for the general relations between the sequence and structure of proteins are discussed.

General Time-Reversible Distances with Unequal Rates across Sites: Mixing Γ and Inverse Gaussian Distributions with Invariant Sites

A series of new results useful to the study of DNA sequences using Markov models of substitution are presented with proofs. General time-reversible distances can be extended to accommodate any fixed distribution of rates across sites by replacing the logarithmic function of a matrix with the inverse of a moment generating function. Estimators are presented assuming a gamma distribution, the inverse Gaussian distribution, or a mixture of either of these with invariant sites. Also considered are the different ways invariant sites may be removed and how these differences may affect estimated distances. Through collaboration, we implemented these distances into PAUP* in 1994. The variance of these new distances is approximated via the delta method. It is also shown how to predict the divergence expected for a pair of sequences given a rate matrix and a distribution of rates across sites, allowing iterated ML estimates of distances under any reversible model. A simple test of whether a rate matrix is time reversible is also presented. These new methods are used to estimate the divergence time of humans and chimps from mtDNA sequence data. These analyses support suggestions that the human lineage has an enhanced transition rate relative to other hominoids. These studies also show that transversion distances differ substantially from the overall distances which are dominated by transitions. Transversions alone apparently suggest a very recent divergence time for humans versus chimps and/or a very old (>16 myr) divergence time for humans versus organgutans. This work illustrates graphically ways to interpret the reliability of distance-based transformations, using the corrected transition to transversion ratio returned for pairs of sequences which are successively more diverged.

Hadamard Conjugations and Modeling Sequence Evolution with Unequal Rates across Sites

This paper considers the many different distributions that may approximate the distribution of site rates in DNA sequences and shows how the Hadamard conjugation may be modified to take these into account. This is done for both 2-state and 4-state data. Distributions which give simple closed forms include the gamma (Γ) distribution, the inverse Gaussian distribution (which is similar to the lognormal), and a mixture of either of these with a proportion of sites which cannot change (invariant sites). It is seen that the tail of a distribution can have major effects upon the coefficient of variation of site rates. Because the Hadamard conjugation can be used to either correct data or predict the data given the model (i.e., the likelihood of site patterns), light is shed on properties of maximum likelihood tree selection with unequal site rates. Analysis of rRNA shows how unequal rates across sites can change the optimal tree. Maximum likelihood analysis also shows that distinct distributions fit each data set, with the Γ often not being the best. Analyzing both these data and a long stretch of primate mtDNA reveals evidence of many “hidden” multiple substitutions, while signals not corresponding to the preferred biological tree generally decrease as unequal rates are allowed for. Last, we discuss the expected behavior of sequences evolving by models where stabilizing selection alone explains unequal site rates. Such models do not explain “synapomorphies” or informative changes in ancient molecules, because while stabilizing selection can vastly decrease change at a site, it will also vastly accelerate back-substitution (leaving only a covarion model to explain old synapomorphies). When and why models allowing a continuous distribution of site rates (e.g., Γ) will approximate covarion evolution requires further study.

Molecular evolution of ependymin and the phylogenetic resolution of early divergences among euteleost fishes.

The rate and pattern of DNA evolution of ependymin, a single-copy gene coding for a highly expressed glycoprotein in the brain matrix of teleost fishes, is characterized and its phylogenetic utility for fish systematics is assessed. DNA sequences were determined from catfish, electric fish, and characiforms and compared with published ependymin sequences from cyprinids, salmon, pike, and herring. Among these groups, ependymin amino acid sequences were highly divergent (up to 60% sequence difference), but had surprisingly similar hydropathy profiles and invariant glycosylation sites, suggesting that functional properties of the proteins are conserved. Comparison of base composition at third codon positions and introns revealed AT-rich introns and GC-rich third codon positions, suggesting that the biased codon usage observed might not be due to mutational bias. Phylogenetic information content of third codon positions was surprisingly high and sufficient to recover the most basal nodes of the tree, in spite of the observation that pairwise distances (at third codon positions) were well above the presumed saturation level. This finding can be explained by the high proportion of phylogenetically informative nonsynonymous changes at third codon positions among these highly divergent proteins. Ependymin DNA sequences have established the first molecular evidence for the monophyly of a group containing salmonids and esociforms. In addition, ependymin suggests a sister group relationship of electric fish (Gymnotiformes) and Characiformes, constituting a significant departure from currently accepted classifications. However, relationships among characiform lineages were not completely resolved by ependymin sequences in spite of seemingly appropriate levels of variation among taxa and considerably low levels of homoplasy in the data (consistency index = 0.7). If the diversification of Characiformes took place in an "explosive" manner, over a relatively short period of time this pattern should also be observed using other phylogenetic markers. Poor conservation of ependymin's primary structure hinders the design of efficient primers for PCR that could be used in wide-ranging fish systematic studies. However, alternative methods like PCR amplification from cDNA used here should provide promising comparative sequence data for the resolution of phylogenetic relationships among other basal lineages of teleost fishes.

Evolution of chlorophyll and bacteriochlorophyll: the problem of invariant sites in sequence analysis.

Competing hypotheses seek to explain the evolution of oxygenic and anoxygenic processes of photosynthesis. Since chlorophyll is less reduced and precedes bacteriochlorophyll on the modern biosynthetic pathway, it has been proposed that chlorophyll preceded bacteriochlorophyll in its evolution. However, recent analyses of nucleotide sequences that encode chlorophyll and bacteriochlorophyll biosynthetic enzymes appear to provide support for an alternative hypothesis. This is that the evolution of bacteriochlorophyll occurred earlier than the evolution of chlorophyll. Here we demonstrate that the presence of invariant sites in sequence datasets leads to inconsistency in tree building (including maximum-likelihood methods). Homologous sequences with different biological functions often share invariant sites at the same nucleotide positions. However, different constraints can also result in additional invariant sites unique to the genes, which have specific and different biological functions. Consequently, the distribution of these sites can be uneven between the different types of homologous genes. The presence of invariant sites, shared by related biosynthetic genes as well as those unique to only some of these genes, has misled the recent evolutionary analysis of oxygenic and anoxygenic photosynthetic pigments. We evaluate an alternative scheme for the evolution of chlorophyll and bacteriochlorophyll.

Mutation accumulation in transfer RNAs: molecular evidence for Muller's ratchet in mitochondrial genomes.

The accumulation of deleterious mutations is thought to be a major factor preventing the long-term persistence of obligately asexual lineages relative to their sexual ancestors. This phenomenon is also of potential relevance to sexual species that harbor asexually propagating organelle genomes. A comparative study of the transfer RNA genes in animal mitochondrial and nuclear genomes demonstrates that the former accumulate nucleotide substitutions much more rapidly than do the latter, and several lines of evidence are consistent with the idea that the excess substitutions are mildly deleterious. First, the average binding stability between complementary strands in the stems of mitochondrial tRNAs is less than half that in nuclear tRNAs. Second, most loop sizes in the mitochondrial tRNAs have experienced a net reduction in size over evolutionary time, and they are nearly 50 times more variable in the mitochondrial than in the nuclear genome. Third, although nearly 20% of the nucleotides in nuclear tRNA genes (particularly those involved in tertiary interactions) are invariant across all animal taxa and all tRNA species, there are no invariant sites in the mitochondrial tRNAs. These observations, as well as results from recent laboratory experiments, are consistent with the hypothesis that nonrecombining organelle genomes are subject to gradual loss of fitness due to the cumulative chance fixation of mildly deleterious mutations.

22] Selection of aminoacylated tRNAs from RNA libraries having randomized acceptor stem sequences: Using old dogs to perform new tricks

Publisher Summary Most systematic evolution of ligands by exponential enrichment (SELEX) schemes, involving nucleic acid ligands, require some type of postselection amplification of the nucleic acid sequence. This is usually accomplished by the polymerase chain reaction (PCR) that requires the use of short DNA oligonucleotide primers. When RNA ligands are the subject of study, PCR is also used to incorporate an RNA polymerase promoter into the DNA template for the subsequent transcription of more RNA ligand. Because the 5' and 3' termini of the polynucleotide serve as primer-binding sites, their sequence must be defined and invariant. Therefore, only internal stretches of the polynucleotide sequence can be randomized and studied, using typical SELEX schemes. Because this chapter is interested in using SELEX to explore whether unique combinations of acceptor stem nucleotides that can functionally mimic the conserved sequence of wild type Escherichi coli serine tRNAs exist, it is critical that the nucleotides at both termini be randomized. It, therefore, developed an approach that circumvents restrictions on the randomization of the transfer RNA (tRNA) acceptor stem nucleotides. This entailed extending the tRNA 5' and 3' termini to create defined and invariant primer-binding sites and, thus, allowing it to randomize the acceptor stem nucleotides so that their recognition by E. coli seryl-tRNA synthetase (SRS) could be studied, using a SELEX approach. The steps of the selection scheme outlined in this chapter, in total or in combination, are generally applicable to other problems that also involve selection for RNA ligands that are active for reversable chemical reactions and to situations, in which it would be advantageous to randomize the 3' terminus of a RNA.

Subcellular calcium oscillators and calcium influx support agonist-induced calcium waves in cultured astrocytes.

We have analysed Ca2+ waves induced by norepinephrine in rat cortical astrocytes in primary culture using fluorescent indicators fura-2 or fluo-3. The temporal pattern of the average [Ca2+]i responses were heterogeneous from cell to cell and most cells showed an oscillatory response at concentrations of agonist around EC50 (200 nM). Upon receptor activation, [Ca2+]i signals originated from a single cellular locus and propagated throughout the cell as a wave. Wave propagation was supported by specialized regenerative calcium release loci along the length of the cell. The periods of oscillations, amplitudes, and the rates of [Ca2+]i rise of these subcellular oscillators differ from each other. These intrinsic kinetic properties of the regenerative loci support local waves when stimulation is continued over long periods of time. The presence of local waves at specific, invariant cellular sites and their inherent kinetic properties provide for the unique and reproducible pattern of response seen in a given cell. We hypothesize that these loci are local specializations in the endoplasmic reticulum where the magnitude of the regenerative Ca2+ release is higher than other regions of the cell. Removal of extracellular Ca2+ or blockade of Ca2+ channels by inorganic cations (Cd2+ and Ni2+) during stimulation of adrenergic receptors alter the sustained plateau component of the [Ca2+]i response. In the absence of Ca2+ release, due to store depletion with thapsigargin, agonist occupation alone does not induce Ca2+ influx in astrocytes. This finding suggests that, under these conditions, receptor-operated Ca2+ entry is not operative. Furthermore, our experiments provide evidence for local Ca2+ oscillations in cells which can support both wave propagation as well as spatially discrete Ca2+ signalling.

Relationships among native and introduced North American species of Hordeum, based on chloroplast DNA restriction-site variation

Restriction-site analysis of chloroplast DNA was carried out on a total of 13 North American species of Hordeum, 7 native and 6 introduced. Sites associated with polymorphic banding patterns were omitted so that only invariant sites within species were used in the analyses. A number of taxa whose species status is in dispute can indeed be supported at species rank, i.e., H. brachyantherum, H. californicum, H. pusillum, H. intercedens, H. marinum, and H. geniculatum. The maternal progenitor of H. arizonicum is H. pusillum, and this is further discussed in light of other possibilities. Both phenetic and cladistic analyses indicate a tripartite relationship whereby H. vulgare is distant from the group of H. leporinum, H. glaucum, and H. murinum and from a second group comprising the native species H. brachyantherum, H. californicum, H. arizonicum, H. jubatum, H. pusillum, H. intercedens, and H. depressum. Hordeum marinum and H. geniculatum occupy a central position in this relationship. Speculation that the putative ancient form of Hordeum resembled a H. murinum – H. geniculatum-like species is discussed. Key words: chloroplast DNA, Hordeum, phylogenetic relationships.

Polymerase chain reaction (PCR) mutagenesis enabling rapid non‐radioactive detection of common β‐thalassaemia mutations in Mediterraneans

The IVS-1-110 (G----A) and IVS-1-1 (G----A) mutations occur in approximately 33% and 9% respectively of beta-thalassaemia alleles in Mediterraneans (Kazazian & Boehm, 1988). They are generally detected in polymerase chain reaction (PCR)-amplified material by allele-specific oligonucleotide (ASO) hybridization patterns. In this study, artificial base substitutions in amplified material have been created to distinguish normal from mutant alleles on the basis of restriction enzyme digestion patterns. Invariant target sites provide an internal control for restriction enzyme activity. Mutagenesis was achieved by 3' base mismatches in primers selected to anneal immediately adjacent to target sites. Digestion of PCR products from normal and thalassaemic alleles with the restriction enzymes MboI (IVS-1-110) and HinfI (IVS-1-1) produced different fragments on electrophoresis. The above strategy was validated by allele-specific oligonucleotide probing. Identification of the three commonest mutations in this population (IVS-1-110, codon 39 and IVS-1-1), which account for approximately 69% of thalassaemic alleles (Kazazian & Boehm, 1988), was subsequently undertaken in seven chorion villus biopsies.

Peculiar sequence organization of kinetoplast DNA minicircles from Trypanosoma cruzi

The sequences of two minicircles from the kinetoplast DNA of the CL strain and one of the Y strain of Trypanosoma cruzi are reported. These 1.4 kb molecules have a peculiar sequence organization, the most distinctive feature being the occurrence of a 120 bp sequence repeated four times, located at 0, 90, 180 and 270 degrees along each circle. We have termed these conserved regions in this species ‘minirepeats’. Minirepeats have a 3-fold higher concentration of cytosine residues in comparison with the variable regions and contain the universal 12-mer motif GGGGTTGGTGTA present in all sequenced minicircles and which was shown to be involved in DNA replication. A consensus sequence of T. cruzi minirepeats was determined using the 20 minirepeats present in five known T. cruzi minicircle sequences. This consensus sequence contains regions which have been remarkably well preserved in strains which show great biological diversity. In addition a low level of intraminicircle sequence similarity was also observed within the variable region, but this similarity did not extend between strains. The abundance of conserved minirepeat sequences containing invariant restriction sites in T. cruzi cells may prove valuable for the development of new direct diagnostic methods for Chagas' disease based on DNA probe technology.

Cloning and sequence analysis of the mouse genomic locus encoding the largest subunit of RNA polymerase II.

The genomic locus (RPII215) encoding the largest subunit of mouse RNA polymerase II has been cloned by low stringency hybridization to a Drosophila RPII215 probe. The mouse gene consists of 28 exons which span 30 kilobases. Analysis of the nucleotide and predicted protein sequences indicates that the protein is comprised of two domains. There is a 1500 residue amino-terminal domain which contains seven regions strikingly similar to those in the beta' subunit of Escherichia coli RNA polymerase, and a carboxyl-terminal domain comprised of 52 repeats of a 7-amino-acid consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. Among the seven highly conserved regions are a strongly basic domain consistent with a DNA-binding site and a consensus sequence characteristic of a potential zinc-binding domain. The 5' upstream region contains three tandem sequences similar to binding sites for the transcription factor SP1. Two of the introns in this gene splice at donor GC dinucleotides as opposed to previously described invariant GT sites. The identification of regions which are highly conserved as compared with bacterial and yeast RNA polymerase and other regions which are unique to the mouse protein suggests which domains of RNA polymerase large subunits are involved in aspects of transcription common to both procaryotes and eucaryotes and which are characteristic of transcription in higher organisms.

EVOLUTIONARY INFERENCES FROM RESTRICTION MAPS OF MITOCHONDRIAL DNA FROM NINE TAXA OF XENOPUS FROGS

Restriction endonuclease cleavage maps were prepared by the double digestion method for mitochondrial DNAs (mtDNAs) purified from Xenopus borealis, X. clivii, X. fraseri, X. muelleri, X. ruwenzoriensis, X. vestitus, X. laevis victorianus, X. l. laevis, and a variant of X. laevis designated X. laevis "davis." An average of 21 cleavage sites per genome were mapped with 11 restriction endonucleases. Among the four invariant sites found are three conserved not only among the Xenopus mtDNAs tested but also among nearly all vertebrate mtDNAs examined to date. Two of these are Sac II sites in the 12S and 16S ribosomal RNA genes, and one is a Hpa I site in the gene for asparagine transfer RNA. These three sites permit the alignment and comparison of mtDNAs from different vertebrate classes. Although most of the differences observed among the Xenopus maps are attributable to point mutations causing gain or loss of restriction sites, the maps also differ by three large length mutations in or near the displacement loop. Phylogenetic analysis of 30 informative sites suggests that those members of the laevis species-group that have 36 chromosomes per somatic cell can be divided into three subgroups: 1) X. borealis, X. clivii, and perhaps X. fraseri (the "borealis" subgroup), 2) X. muelleri, and 3) the subspecies of X, laevis. The mtDNA of the hexaploid (2n = 108) species, X. ruwenzoriensis, is most similar to that of taxa in the latter two subgroups, which contrasts with the morphological similarity of this species to X. fraseri. X. ruwenzoriensis may be an allopolyploid with a mother (the contributor of the cytoplasmic mtDNA genome) on the X. laevis or X. muelleri lineage and a father on the X. fraseri lineage. We present a model showing how mtDNA and nuclear genomes can yield contrasting phytogenies for species-groups that have undergone several rounds of interspecific hybridization. Comparison of mitochondrial and nuclear sequence divergences suggests that Xenopus mtDNA, like that of mammals and birds, evolves faster than nuclear DNA. Genetic distances among mtDNAs of Xenopus species are very large, generally approaching or exceeding one substitution per nucleotide.