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PubMed1988

Small gene family encoding an eggshell (chorion) protein of the human parasite Schistosoma mansoni.

We have isolated six independent genomic clones encoding schistosome chorion or eggshell proteins from a Schistosoma mansoni genomic library. A linkage may of five of the clones spanning 35 kilobase pair (kbp) of the S. mansoni genome was constructed. The region contained two eggshell protein genes closely linked, separated by 7.5 kbp of intergenic DNA. The two genes of the cluster were arranged in the same orientation, that is, they were transcribed from the same strand. The sixth clone probably represents a third copy of the eggshell gene that is not contained within the 35-kbp region. The 5' end of the mRNA transcribed from these genes was defined by primer extension directly off the RNA. The ATCAT cap site sequence was homologous to a silkmoth chorion PuTCATT cap site sequence, where Pu indicates any purine. DNA sequence analysis showed that there were no introns in these genes. The DNA sequences of the three genes were very homologous to each other and to a cDNA clone, pSMf61-46, differing only in three or four nucleotides. A multiple TATA box was located at positions -23 to -31, and a CAAAT sequence was located at -52 upstream of the eggshell transcription unit. Comparison of sequences in regions further upstream with silkmoth and Drosophila sequences revealed several very short elements that were shared. One such element, TCACGT, recently shown to be an essential cis-regulatory element for silkmoth chorion gene promoter function, was found at a similar position in all three organisms.

Amino Acid Sequence↗
PubMed1989

Stable maintenance of a 35-base-pair yeast mitochondrial genome.

Small deletion variants ([rho-] mutants) derived from the wild-type ([ rho+]) Saccharomyces cerevisiae mitochondrial genome were isolated and characterized. The mutant mitochondrial DNAs (mtDNAs) examined retained as little as 35 base pairs of one section of intergenic DNA, were composed entirely of A.T base pairs, and were stably maintained. These simple mtDNAs existed in tandemly repeated arrays at an amplified level that made up approximately 15% of the total cellular DNA and, as judged by fluorescence microscopy, had a nearly normal mitochondrial arrangement throughout the cell cytoplasm. The simple nature of these [rho-] genomes indicates that the sequences required to maintain mtDNA must be extremely simple.

Base Sequence↗
PubMed2001

Organization of 5S rDNA in species of the fish Leporinus: two different genomic locations are characterized by distinct nontranscribed spacers.

To address understanding the organization of the 5S rRNA multigene family in the fish genome, the nucleotide sequence and organization array of 5S rDNA were investigated in the genus Leporinus, a representative freshwater fish group of South American fauna. PCR, subgenomic library screening, genomic blotting, fluorescence in situ hybridization, and DNA sequencing were employed in this study. Two arrays of 5S rDNA were identified for all species investigated, one consisting of monomeric repeat units of around 200 bp and another one with monomers of 900 bp. These 5S rDNA arrays were characterized by distinct NTS sequences (designated NTS-I and NTS-II for the 200- and 900-bp monomers, respectively); however, their coding sequences were nearly identical. The 5S rRNA genes were clustered in two chromosome loci, a major one corresponding to the NTS-I sites and a minor one corresponding to the NTS-II sites. The NTS-I sequence was variable among Leporinus spp., whereas the NTS-II was conserved among them and even in the related genus Schizodon. The distinct 5S rDNA arrays might characterize two 5S rRNA gene subfamilies that have been evolving independently in the genome.

Animals↗
PubMed2005

Evolution of genome size across some cultivated Allium species.

Allium L. (Alliaceae), a genus of major economic importance, exhibits a great diversity in various morphological characters and particularly in life form, with bulbs and rhizomes. Allium species show variation in several cytogenetic characters such as basic chromosome number, ploidy level, and genome size. The purpose of the present investigation was to study the evolution of nuclear DNA amount, GC content, and life form. A phylogenetic approach was used on a sample of 30 Allium species, including major vegetable crops and their wild allies, belonging to the 3 major subgenera Allium, Amerallium, and Rhizirideum and 14 sections. A phylogeny was constructed using internal transcribed spacer (ITS) sequences of 43 accessions representing 30 species, and the nuclear DNA amount and the GC content of 24 Allium species were investigated by flow cytometry. For the first time, the nuclear DNA content of Allium cyaneum and Allium vavilovii was measured, and the GC content of 16 species was measured. We addressed the following questions: (i) Is the variation in nuclear DNA amount and GC content linked to the evolutionary history of these edible Allium species and their wild relatives? (ii) How did life form (rhizome or bulb) evolve in edible Allium? Our results revealed significant interspecific variation in the nuclear DNA amount as well as in the GC content. No correlation was found between the GC content and the nuclear DNA amount. The reconstruction of nuclear DNA amount on the phylogeny showed a tendency towards a decrease in genome size within the genus. The reconstruction of life form history showed that rhizomes evolved in the subgenus Rhizirideum from an ancestral bulbous life form and were subsequently lost at least twice independently in this subgenus.

Allium↗
PubMed2000

Molecular evolution and phylogeny of the atpB-rbcL spacer of chloroplast DNA in the true mosses.

The nucleotide variation of a noncoding region between the atpB and rbcL genes of the chloroplast genome was used to estimate the phylogeny of 11 species of true mosses (subclass Bryidae). The A+T rich (82.6%) spacer sequence is conserved with 48% of bases showing no variation between the ingroup and outgroup. Rooted at liverworts, Marchantia and Bazzania, the monophyly of true mosses was supported cladistically and statistically. A nonparametric Wilcoxon Signed-Ranks test Ts statistic for testing the taxonomic congruence showed no significant differences between gene trees and organism trees as well as between parsimony trees and neighbor-joining trees. The reconstructed phylogeny based on the atpB-rbcL spacer sequences indicated the validity of the division of acrocarpous and pleurocarpous mosses. The size of the chloroplast spacer in mosses fits into an evolutionary trend of increasing spacer length from liverworts through ferns to seed plants. According to the relative rate tests, the hypothesis of a molecular clock was supported in all species except for Thuidium, which evolved relatively fast. The evolutionary rate of the chloroplast DNA spacer in mosses was estimated to be (1.12 +/- 0.019) x 10(-10) nucleotides per site per year, which is close to the nonsynonymous substitution rates of the rbcL gene in the vascular plants. The constrained molecular evolution (total nucleotide substitutions, K approximately 0.0248) of the chloroplast DNA spacer is consistent with the slow evolution in morphological traits of mosses. Based on the calibrated evolutionary rate, the time of the divergence of true mosses was estimated to have been as early as 220 million years ago.

AT Rich Sequence↗
PubMed2006

Genetic differentiation in the soil-feeding termite Cubitermes sp. affinis subarquatus: occurrence of cryptic species revealed by nuclear and mitochondrial markers.

BACKGROUND: Soil-feeding termites are particularly interesting models for studying the effects of fragmentation, a natural or anthropic phenomenon described as promoting genetic differentiation. However, studying the link between fragmentation and genetics requires a method for identifying species unambiguously, especially when morphological diagnostic characters are lacking. In humivorous termites, which contribute to the fertility of tropical soils, molecular taxonomy and phylogenetic relationships are rarely studied, though mitochondrial and nuclear molecular markers are widely used in studies of pest termites. Here, we attempt to clarify the taxonomy of soil-feeding colonies collected throughout the naturally fragmented Lopé Reserve area (Gabon) and morphologically affiliated to Cubitermes sp. affinis subarquatus. The mitochondrial gene of cytochrome oxidase II (COII), the second nuclear rDNA internal transcribed spacer (ITS2) and five microsatellites were analyzed in 19 colonies. RESULTS: Bayesian Inference, Maximum Likelihood and Maximum Parsimony phylogenetic analyses, which were applied to the COII and ITS2 sequences, and Neighbor-Joining reconstructions, applied to the microsatellite data, reveal four major lineages in the Cubitermes sp. affinis subarquatus colonies. The concordant genealogical pattern of these unlinked markers strongly supports the existence of four cryptic species. Three are sympatric in the Reserve and are probably able to disperse within a mosaic of forests of variable ages and savannahs. One is limited to a very restricted gallery forest patch located in the North, outside the Reserve. CONCLUSION: Our survey highlights the value of combined mitochondrial and nuclear markers for exploring unknown groups such as soil-feeding termites, and their relevance for resolving the taxonomy of organisms with ambiguous morphological diagnostic characters.

Animals↗
PubMed2004

Complete mitochondrial genome sequence of Urechis caupo, a representative of the phylum Echiura.

BACKGROUND: Mitochondria contain small genomes that are physically separate from those of nuclei. Their comparison serves as a model system for understanding the processes of genome evolution. Although hundreds of these genome sequences have been reported, the taxonomic sampling is highly biased toward vertebrates and arthropods, with many whole phyla remaining unstudied. This is the first description of a complete mitochondrial genome sequence of a representative of the phylum Echiura, that of the fat innkeeper worm, Urechis caupo. RESULTS: This mtDNA is 15,113 nts in length and 62% A+T. It contains the 37 genes that are typical for animal mtDNAs in an arrangement somewhat similar to that of annelid worms. All genes are encoded by the same DNA strand which is rich in A and C relative to the opposite strand. Codons ending with the dinucleotide GG are more frequent than would be expected from apparent mutational biases. The largest non-coding region is only 282 nts long, is 71% A+T, and has potential for secondary structures. CONCLUSIONS: Urechis caupo mtDNA shares many features with those of the few studied annelids, including the common usage of ATG start codons, unusual among animal mtDNAs, as well as gene arrangements, tRNA structures, and codon usage biases.

Amino Acid Sequence↗
PubMed2006

Complete mitochondrial genome sequences for Crown-of-thorns starfish Acanthaster planci and Acanthaster brevispinus.

BACKGROUND: The crown-of-thorns starfish, Acanthaster planci (L.), has been blamed for coral mortality in a large number of coral reef systems situated in the Indo-Pacific region. Because of its high fecundity and the long duration of the pelagic larval stage, the mechanism of outbreaks may be related to its meta-population dynamics, which should be examined by larval sampling and population genetic analysis. However, A. planci larvae have undistinguished morphological features compared with other asteroid larvae, hence it has been difficult to discriminate A. planci larvae in plankton samples without species-specific markers. Also, no tools are available to reveal the dispersal pathway of A. planci larvae. Therefore the development of highly polymorphic genetic markers has the potential to overcome these difficulties. To obtain genomic information for these purposes, the complete nucleotide sequences of the mitochondrial genome of A. planci and its putative sibling species, A. brevispinus were determined and their characteristics discussed. RESULTS: The complete mtDNA of A. planci and A. brevispinus are 16,234 bp and 16,254 bp in size, respectively. These values fall within the length variation range reported for other metazoan mitochondrial genomes. They contain 13 proteins, 2 rRNA, and 22 tRNA genes and the putative control region in the same order as the asteroid, Asterina pectinifera. The A + T contents of A. planci and A. brevispinus on their L strands that encode the majority of protein-coding genes are 56.3% and 56.4% respectively and are lower than that of A. pectinifera (61.2%). The percent similarity of nucleotide sequences between A. planci and A. brevispinus is found to be highest in the CO2 and CO3 regions (both 90.6%) and lowest in ND2 gene (84.2%) among the 13 protein-coding genes. In the deduced putative amino acid sequences, CO1 is highly conserved (99.2%), and ATP8 apparently evolves faster any of the other protein-coding gene (85.2%). CONCLUSION: The gene arrangement, base composition, codon usage and tRNA structure of A. planci are similar to those of A. brevispinus. However, there are significant variations between A. planci and A. brevispinus. Complete mtDNA sequences are useful for the study of phylogeny, larval detection and population genetics.

Animals↗
PubMed2004

Sequencing and analysis of the internal transcribed spacers (ITSs) and coding regions in the EcoR I fragment of the ribosomal DNA of the Japanese pond frog Rana nigromaculata.

The rDNA of eukaryotic organisms is transcribed as the 40S-45S rRNA precursor, and this precursor contains the following segments: 5' - ETS - 18S rRNA - ITS 1 - 5.8S rRNA - ITS 2 - 28S rRNA - 3'. In amphibians, the nucleotide sequences of the rRNA precursor have been completely determined in only two species of Xenopus. In the other amphibian species investigated so far, only the short nucleotide sequences of some rDNA fragments have been reported. We obtained a genomic clone containing the rDNA precursor from the Japanese pond frog Rana nigromaculata and analyzed its nucleotide sequence. The cloned genomic fragment was 4,806 bp long and included the 3'-terminus of 18S rRNA, ITS 1, 5.8S rRNA, ITS 2, and a long portion of 28S rRNA. A comparison of nucleotide sequences among Rana, the two species of Xenopus, and human revealed the following: (1) The 3'-terminus of 18S rRNA and the complete 5.8S rRNA were highly conserved among these four taxa. (2) The regions corresponding to the stem and loop of the secondary structure in 28S rRNA were conserved between Xenopus and Rana, but the rate of substitutions in the loop was higher than that in the stem. Many of the human loop regions had large insertions not seen in amphibians. (3) Two ITS regions had highly diverged sequences that made it difficult to compare the sequences not only between human and frogs, but also between Xenopus and Rana. (4) The short tracts in the ITS regions were strictly conserved between the two Xenopus species, and there was a corresponding sequence for Rana. Our data on the nucleotide sequence of the rRNA precursor from the Japanese pond frog Rana nigromaculata were used to examine the potential usefulness of the rRNA genes and ITS regions for evolutionary studies on frogs, because the rRNA precursor contains both highly conserved regions and rapidly evolving regions.

Animals↗
PubMed2004

Complete nucleotide sequence and gene rearrangement of the mitochondrial genome of the bell-ring frog, Buergeria buergeri (family Rhacophoridae).

In this study we determined the complete nucleotide sequence (19,959 bp) of the mitochondrial DNA of the rhacophorid frog Buergeria buergeri. The gene content, nucleotide composition, and codon usage of B. buergeri conformed to those of typical vertebrate patterns. However, due to an accumulation of lengthy repetitive sequences in the D-loop region, this species possesses the largest mitochondrial genome among all the vertebrates examined so far. Comparison of the gene organizations among amphibian species (Rana, Xenopus, salamanders and caecilians) revealed that the positioning of four tRNA genes and the ND5 gene in the mtDNA of B. buergeri diverged from the common vertebrate gene arrangement shared by Xenopus, salamanders and caecilians. The unique positions of the tRNA genes in B. buergeri are shared by ranid frogs, indicating that the rearrangements of the tRNA genes occurred in a common ancestral lineage of ranids and rhacophorids. On the other hand, the novel position of the ND5 gene seems to have arisen in a lineage leading to rhacophorids (and other closely related taxa) after ranid divergence. Phylogenetic analysis based on nucleotide sequence data of all mitochondrial genes also supported the gene rearrangement pathway.

Animals↗
PubMed2006

PCR-based species-specific amplification of ITS of Mecistocirrus digitatus and its application in identification of GI nematode eggs in bovine faeces.

Differential diagnosis of Mecistocirrus digitatus infection relies on morphological examination of either eggs in faecal samples or L3 larvae developed in vitro. Technical limitations hinder the practicability of these approaches. Hence, in order to develop a specific diagnostic measure for M. digitatus infection, we determined the sequence of the internal transcribed spacer (ITS) of its ribosomal DNA (rDNA) and designed primers for PCR-based species-specific amplification of the ITS to differentiate between M. digitatus and other common gastrointestinal (GI) nematode species. The newly designed primers amplified a single specific 520 base pair (bp) fragment from the M. digitatus ITS, and its detection limit was as low as 0.001 ng. Further, this sensitivity suggested that the specific fragment could be amplified even from a unicellular egg that collected directly from uteri of an adult M. digitatus female. In fact, we designed a method that employs a small piece of a cover slip and a filter paper by which we could differentially amplify a PCR fragment from a unicellular egg. The reliability of the specific PCR assay was also demonstrated with 10 oval samples that collected from bovine faeces by using sugar flotation method. These data suggested that the specific PCR assay of the ITS region of M. digitatus rDNA could be useful for the identification of GI nematodes.

Animals↗
PubMed2005

Cis-effects on meiotic recombination across distinct a1-sh2 intervals in a common Zea genetic background.

Genetic distances across the a1-sh2 interval varied threefold in three near-isogenic stocks that carry structurally distinct teosinte A1 Sh2 haplotypes (from Z. mays spp. mexicana Chalco, Z. mays spp. parviglumis, and Z. luxurians) and a common maize a1::rdt sh2 haplotype. In each haplotype >85% of recombination events resolved in the proximal 10% of the approximately 130-kb a1-sh2 interval. Even so, significant differences in the distributions of recombination breakpoints were observed across subintervals among haplotypes. Each of the three previously detected recombination hot spots was detected in at least one of the three teosinte haplotypes and two of these hot spots were not detected in at least one teosinte haplotype. Moreover, novel hot spots were detected in two teosinte haplotypes. Due to the near-isogenic nature of the three stocks, the observed variation in the distribution of recombination events is the consequence of cis-modifications. Although generally negatively correlated with rates of recombination per megabase, levels of sequence polymorphisms do not fully account for the nonrandom distribution of recombination breakpoints. This study also suggests that estimates of linkage disequilibrium must be interpreted with caution when considering whether a gene has been under selection.

Chi-Square Distribution↗
PubMed2005

Minos as a genetic and genomic tool in Drosophila melanogaster.

Much of the information about the function of D. melanogaster genes has come from P-element mutagenesis. The major drawback of the P element, however, is its strong bias for insertion into some genes (hotspots) and against insertion into others (coldspots). Within genes, 5'-UTRs are preferential targets. For the successful completion of the Drosophila Genome Disruption Project, the use of transposon vectors other than P will be necessary. We examined here the suitability of the Minos element from Drosophila hydei as a tool for Drosophila genomics. Previous work has shown that Minos, a member of the Tc1/mariner family of transposable elements, is active in diverse organisms and cultured cells; it produces stable integrants in the germ line of several insect species, in the mouse, and in human cells. We generated and analyzed 96 Minos integrations into the Drosophila genome and devised an efficient "jump-starting" scheme for production of single insertions. The ratio of insertions into genes vs. intergenic DNA is consistent with a random distribution. Within genes, there is a statistically significant preference for insertion into introns rather than into exons. About 30% of all insertions were in introns and approximately 55% of insertions were into or next to genes that have so far not been hit by the P element. The insertion sites exhibit, in contrast to other transposons, little sequence requirement beyond the TA dinucleotide insertion target. We further demonstrate that induced remobilization of Minos insertions can delete nearby sequences. Our results suggest that Minos is a useful tool complementing the P element for insertional mutagenesis and genomic analysis in Drosophila.

Animals↗
PubMed2006

Low variability of internal transcribed spacer rDNA and trnL (UAA) intron sequences of several taxa in the Festuca ovina aggregate (POACEAE).

Identification and classification of numerous Festuca species is still a difficult problem due to the close morphological resemblance. The most difficult fine fescues to identify belong to the Festuca ovina aggregate, which is the largest group in the genus Festuca. Many taxons are considered to be separate species based on quantitative taxonomic characters, differences in ploidy level or the structure of sclerenchyma cells. In order to evaluate the taxonomic value of DNA-based markers, sequence analysis of the internal transcribed spacer (ITS1-5.8S-ITS2) region and the chloroplast trnL (UAA) intron was performed in the ten most problematic fine fescues belonging to the Festuca ovina aggregate. Intraspecific ITS variants were found in a single case while in other cases only intragenomic ITS polymorphisms were detected with 1-2 ambiguous positions. Among the sequences of the trnL (UAA) intron even intragenomic polymorphisms were not detected in any of the Festuca species studied. Thus, the results do not support the species status of these ten taxa.

Base Sequence↗
PubMed2004

Ribosomal DNA ITS2 sequences differentiate six species in the Anopheles crucians complex (Diptera: Culicidae).

Anopheles crucians Wiedemann (sensu lato) was investigated for the presence of cryptic species using rDNA ITS2 sequences. This complex of species presently contains the named species An. crucians, An. bradleyi King, and An. georgianus King. Adult female mosquitoes were collected at 28 sites in Alabama, Florida, Georgia, North Carolina, Mississippi, and Louisiana, resulting in 245 progeny broods. Species were identified using preliminary morphological characters, and the internal transcribed spacer two (ITS2) was amplified from all broods. The result was five distinct sizes of amplification product, and based on morphological characters, one of the size classes was suspected to consist of two species. All six putative species were then sequenced: five directly, and the sixth, because of extreme intragenomic (each individual with many variants) size variability, cloned. The ITS2 sequences were markedly distinct for all six species. Species designations and ITS2 sequence lengths (base pairs in parentheses) were A (461), B (1,000+), C (204), D (293), E (195), and An. bradleyi (208). Species B showed both large intraspecific and intragenomic sequence variability and is distinguished by having the longest ITS2 found so far in an Anopheles. Based on these data, we found that all species could be identified with polymerase chain reaction (PCR) using a mixture of four primers in a single reaction. Members of this complex were often found in sympatry, with the adults of five species collected at a single site in central Florida.

Animals↗
PubMed2004

Differential propagation of the metazoan parasite Myxobolus cerebralis by Limnodrilus hoffmeisteri, Ilyodrilus templetoni, and genetically distinct strains of Tubifex tubifex.

Whirling disease, caused by the parasite Myxobolus cerebralis, has infected rainbow trout (Oncorhynchus mykiss) and other salmonid fish in the western United States, often with devastating results to native populations but without a discernible spatial pattern. The parasite develops in a complex 2-host system in which the aquatic oligochaete Tubifex tubifex is an obligate host. Because substantial differences in whirling disease severity in different areas of North America did not seem explainable by environmental factors or features of the parasite or its fish host, we sought to determine whether ecological or genetic variation within oligochaete host populations may be responsible. We found large differences in compatibility between the parasite and various laboratory strains of T. tubifex that were established from geographic regions with different whirling disease histories. Moreover, 2 closely related species of tubificids, Limnodrilus hoffmeisteri and Ilyodrilus templetoni, which occur naturally in mixed species assemblages with T. tubifex, were incompatible with M. cerebralis. Virulence of the parasite was directly correlated with the numbers of triactinomyxon spores that developed within each strain of T. tubifex. Thus, the level of virulence was directly related to the compatibility between the host strain and the parasite. Genetic analyses revealed relationships that were in agreement with the level of parasite production. Differences in compatibilities between oligochaetes and M. cerebralis may contribute to the spatial variance in the severity of the disease among salmonid populations.

Analysis of Variance↗
PubMed2005

Systematics of Mesocestoides (Cestoda: Mesocestoididae): evaluation of molecular and morphological variation among isolates.

A hypothesis-based framework was used to test if 3 genetic strains of Mesocestoides (clades A, B, and C) are distinct evolutionary lineages, thereby supporting their delimitation as species. For comparative purposes, 3 established cestode species, Taenia pisiformis, Taenia serialis, and Taenia crassiceps were assessed using the same methods. Sequence data from mitochondrial rDNA (12S) and the second internal transcribed spacer of nuclear rDNA (ITS-2) revealed derived (autapomorphic) characters for lineages representing clade A (n = 6 autapomorphies), clade B (n = 4), and clade C (n = 9) as well as T. pisiformis (n = 15) and T. serialis (n = 12). Furthermore, multivariate analysis of morphological data revealed significant differences among the 3 genetic strains of Mesocestoides and between T. pisiformis and T. serialis. The level of phenotypic variation within evolutionary lineages of Mesocestoides and Taenia spp. tapeworms was similar. Results from this study support recognizing Mesocestoides clades A, B, and C as separate species, and provide evidence that clade B and Mesocestoides vogae are conspecific.

Animals↗