Molecular phylogeny and karyotype differentiation in <Emphasis Type="Italic">Paratelmatobius</Emphasis> and <Emphasis Type="Italic">Scythrophrys</Emphasis> (Anura,Leptodactylidae)

Paratelmatobius and Scythrophrys are leptodactylid frogs endemic to the Brazilian Atlantic forest and their close phylogenetic relationship was recently inferred in an analysis that included Paratelmatobius sp. and S. sawayae. To investigate the interspecific relationships among Paratelmatobius and Scythrophrys species, we analyzed a mitochondrial region (approximately 2.4 kb) that included the ribosomal genes 12S and 16S and the tRNAval in representatives of all known localities of these genera and in 54 other species. Maximum parsimony inferences were done using PAUP* and support for the clades was evaluated by bootstrapping. A cytogenetic analysis using Giemsa staining, C-banding and silver staining was also done for those populations of Paratelmatobius not included in previous cytogenetic studies of this genus in order to assess their karyotype differentiation. Our results suggested Paratelmatobius and Scythrophrys formed a clade strongly supported by bootstrapping, which corroborated their very close phylogenetic relationship. Among the Paratelmatobius species, two clades were identified and corroborated the groups P. mantiqueira and P. cardosoi previously proposed based on morphological characters. The karyotypes of Paratelmatobius sp. 2 and Paratelmatobius sp. 3 described here had diploid chromosome number 2n = 24 and showed many similarities with karyotypes of other Paratelmatobius representatives. The cytogenetic data and the phylogenetic analysis allowed the proposal/corroboration of several hypotheses for the karyotype differentiation within Paratelmatobius and Scythrophrys. Namely the telocentric pair No. 4 represented a synapomorphy of P. cardosoi and Paratelmatobius sp. 2, while chromosome pair No. 5 with interstitial C-bands could be interpreted as a synapomorphy of the P. cardosoi group. The NOR-bearing chromosome No. 10 in the karyotype of P. poecilogaster was considered homeologous to chromosome No. 10 in the karyotype of Scythrophrys sp., chromosome No. 9 in the karyotype of Paratelmatobius sp. 1, chromosome No. 8 in the karyotypes of Paratelmatobius sp. 2 and of Paratelmatobius sp. 3, and chromosome No. 7 in the karyotype of P. cardosoi. A hypothesis for the evolutionary divergence of these NOR-bearing chromosomes, which probably involved events like gain in heteochromatin, was proposed.     

Genomic localization of endogenous mobile CACTA family transposons in natural variants of<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

The differentiation between gene-rich and transposon-rich (gene-poor) regions is a common feature of plant genomes. This may be due to preferential integration of transposons into gene-poor regions or may be due to purifying selection against transposon insertion into gene-rich regions. We examined the distribution of a low-copy-number mobile subfamily of Arabidopsis CACTA transposons in the genomes of 19 natural variants (ecotypes) of A. thaliana, and compared that to the pattern of integrations induced in the laboratory by mutation of the DDM1 ( Decrease in DNA Methylation) gene. Sequences similar to mobile CACTA1 copies were distributed among the ecotypes and showed high degrees of polymorphism in genomic localization. Despite the high level of polymorphism, the copy number was low in all the ecotypes examined, and the elements were localized preferentially in pericentromeric and transposon-rich regions. This contrasts with the pattern of transposition induced by the ddm1 mutation, in which the range of integration sites is less biased and the copy number frequently increases. Based on these observations, we discuss the possible contribution of natural selection and chromatin structure to the distribution of transposons.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by M.-A. Grandbastien     

Polymorphism of canonical and noncanonical <Emphasis Type="Italic">gypsy</Emphasis> sequences in different species of <Emphasis Type="Italic">Drosophila </Emphasis><Emphasis Type="Italic">melanogaster</Emphasis> subgroup: possible evolutionary relations

Mobile genetic elements constitute a substantial part of eukaryotic genome and play an important role in its organization and functioning. Co-evolution of retrotransposons and their hosts resulted in the establishment of control systems employing mechanisms of RNA interference that seem to be impossible to evade. However, “active” copies of endogenous retrovirus gypsy escape cellular control in some cases, while its evolutionary elder “inactive” variants do not. To clarify the evolutionary relationship between “active” and “inactive” gypsy we combined two approaches: the analysis of gypsy sequences, isolated from G32 Drosophila melanogaster strain and from different Drosophila species of the melanogaster subgroup, as well as the study of databases, available on the Internet. No signs of “intermediate” (between “active” and “inactive”) gypsy form were found in GenBank, and four full-size G32 gypsy copies demonstrated a convergence that presumably involves gene conversion. No “active” gypsy were revealed among PCR generated gypsy ORF3 sequences from the various Drosophila species indicating that “active” gypsy appeared in some population of D. melanogaster and then started to spread out. Analysis of sequences flanking gypsy variants in G32 revealed their predominantly heterochromatic location. Discrepancy between the structure of actual gypsy sites in G32 and corresponding sequences in database might indicate significant inter-strain heterochromatin diversity. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Accumulation of Y-specific satellite DNAs during the evolution of <Emphasis Type="Italic">Rumex acetosa</Emphasis> sex chromosomes

The study of the molecular structure of young heteromorphic sex chromosomes of plants has shed light on the evolutionary forces that control the differentiation of the X and Y during the earlier stages of their evolution. We have used the model plant Rumex acetosa, a dioecious species with multiple sex chromosomes, 2n = 12 + XX female and 2n = 12 + XY₁Y₂ male, to analyse the significance of repetitive DNA accumulation during the differentiation of the Y. A bulk segregant analysis (BSA) approach allowed us to identify and isolate random amplified polymorphic DNA (RAPD) markers linked to the sex chromosomes. From a total of 86 RAPD markers in the parents, 6 markers were found to be linked to the Ys and 1 to the X. Two of the Y-linked markers represent two AT-rich satellite DNAs (satDNAs), named RAYSII and RAYSIII, that share about 80% homology, as well as with RAYSI, another satDNA of R. acetosa. Fluorescent in situ hybridisation demonstrated that RAYSII is specific for Y₁, whilst RAYSIII is located in different clusters along Y₁ and Y₂. The two satDNAs were only detected in the genome of the dioecious species with XX/XY₁Y₂ multiple sex chromosome systems in the subgenus Acetosa, but were absent from other dioecious species with an XX/XY system of the subgenera Acetosa or Acetosella, as well as in gynodioecious or hermaphrodite species of the subgenera Acetosa, Rumex and Platypodium. Phylogenetic analysis with different cloned monomers of RAYSII and RAYSIII from both R. acetosa and R. papillaris indicate that these two satDNAs are completely separated from each other, and from RAYSI, in both species. The three Y-specific satDNAs, however, evolved from an ancestral satDNA with repeating units of 120 bp, through intermediate satDNAs of 360 bp. The data therefore support the idea that Y-chromosome differentiation and heterochromatinisation in the Rumex species having a multiple sex chromosome system have occurred by different amplification events from a common ancestral satDNA. Since dioecious species with multiple XX/XY₁Y₂ sex chromosome systems of the section Acetosa appear to have evolved from dioecious species with an XX/XY system, the amplification of tandemly repetitive elements in the Ys of the section Acetosa is a recent evolutionary process that has contributed to an increase in the size and differentiation of the already non-recombining Y chromosomes.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Metabolic and ionoregulatory responses of the Amazonian cichlid, <Emphasis Type="Italic">Astronotus ocellatus</Emphasis>, to severe hypoxia

We examined the metabolic and ionoregulatory responses of the Amazonian cichlid, Astronotus ocellatus, to 20 h exposure to severe hypoxia (0.37 ± 0.19 mg O₂/l; 4.6% air saturation) or 8 h severe hypoxia followed by 12 h recovery in normoxic water. During 20 h exposure to hypoxia, white muscle [ATP] was maintained at normoxic levels primarily through a 20% decrease in [creatine phosphate] (CrP) and an activation of glycolysis yielding lactate accumulation. Muscle lactate accumulation maintained cytoplasmic redox state ([NAD⁺]/[NADH]) and was associated with an inactivation of the mitochondrial enzyme pyruvate dehydrogenase (PDH). The inactivation of PDH was not associated with significant changes in cytoplasmic allosteric modulators ([ADP_free], redox state, or [pyruvate]). Hypoxia exposure caused a ∼65% decrease in gill Na⁺/K⁺ ATPase activity, which was not matched by changes in Na⁺/K⁺ ATPase α-subunit protein abundance indicating post-translational modification of Na⁺/K⁺ ATPase was responsible for the decrease in activity. Despite decreases in gill Na⁺/K⁺ ATPase activity, plasma [Na⁺] increased, but this increase was possibly due to a significant hemoconcentration and fluid shift out of the extracellular space. Hypoxia caused an increase in Na⁺/K⁺ ATPase α-subunit mRNA abundance pointing to either reduced mRNA degradation during exposure to hypoxia or enhanced expression of Na⁺/K⁺ ATPase α-subunit relative to other genes.     

Localization of replication forks in wild-type and <Emphasis Type="Italic">mukB</Emphasis> mutant cells of <Emphasis Type="Italic">Escherichia coli</Emphasis>

To examine the subcellular localization of the replication machinery in Escherichia coli, we have developed an immunofluorescence method that allows us to determine the subcellular location of newly synthesized DNA pulse-labeled with 5-bromo-2′-deoxyuridine (BrdU). Using this technique, we have analyzed growing cells. In wild-type cells that showed a single BrdU fluorescence signal, the focus was located in the middle of the cell; in cells with two signals, the foci were localized at positions equivalent to 1/4 and 3/4 of the cell length. The formation of BrdU foci was dependent upon ongoing chromosomal replication. A mutant lacking MukB, which is required for proper partitioning of sister chromosomes, failed to maintain the ordered localization of BrdU foci: (1) a single BrdU focus tended to be localized at a pole-proximal region of the nucleoid, and (2) a focus was often found to consist of two replicating chromosomes. Thus, the positioning of replication forks is affected by the disruption of the mukB gene.     

The karyology of the cyprinid genera <Emphasis Type="Italic">Scardinius</Emphasis> and <Emphasis Type="Italic">Rutilus</Emphasis> in southern Europe

The chromosomes of eight species of Rutilus and Scardinius, mostly endemic to the Italian and the Balkan peninsula, were analyzed by conventional and other banding techniques. Parallel analyses were conducted also on two leuciscine species, Alburnus albidus, for which we provide the first karyological analysis, and Leuciscus cephalus. All species examined displayed the same karyotype (2n=50 chromosomes, 8 metacentric+13 submetacentric+4 subtelo/acrocentric pairs) with nucleolus organizer regions (NORs) on the ends of the shorter arms of a medium-sized submetacentric pair. In contrast, interspecific variation was observed in the distribution of constitutive heterochromatin. The variation observed in this genomic material proved to be systematically and phylogenetically informative. Indeed, a peritelomeric C-band on the first telocentric pair characterizes species of Rutilus and Scardinius. In both genera heterochromatin differentiation appears to be directed to a centromere–telomere direction, particularly evident along the metacentric elements of their karyotypes.An erratum to this article can be found at     

Structure Analysis of Two <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> and <Emphasis Type="BoldItalic">Neospora caninum</Emphasis> Satellite DNA Families and Evolution of Their Common Monomeric Sequence

A family of repetitive DNA elements of approximately 350 bp—Sat350—that are members of Toxoplasma gondii satellite DNA was further analyzed. Sequence analysis identified at least three distinct repeat types within this family, called types A, B, and C. B repeats were divided into the subtypes B1 and B2. A search for internal repetitions within this family permitted the identification of conserved regions and the design of PCR primers that amplify almost all these repetitive elements. These primers amplified the expected 350-bp repeats and a novel 680-bp repetitive element (Sat680) related to this family. Two additional tandemly repeated high-order structures corresponding to this satellite DNA family were found by searching the Toxoplasma genome database with these sequences. These studies were confirmed by sequence analysis and identified: (1) an arrangement of AB1CB2 350-bp repeats and (2) an arrangement of two 350-bp-like repeats, resulting in a 680-bp monomer. Sequence comparison and phylogenetic analysis indicated that both high-order structures may have originated from the same ancestral 350-bp repeat. PCR amplification, sequence analysis and Southern blot showed that similar high-order structures were also found in the Toxoplasma-sister taxon Neospora caninum. The Toxoplasma genome database ( ) permitted the assembly of a contig harboring Sat350 elements at one end and a long nonrepetitive DNA sequence flanking this satellite DNA. The region bordering the Sat350 repeats contained two differentially expressed sequence-related regions and interstitial telomeric sequences.     

The bacterial Tn<Emphasis Type="Italic">9</Emphasis> chloramphenicol resistance gene: an attractive DNA segment for <Emphasis Type="Italic">Mos1 mariner</Emphasis> insertions

The eukaryotic mariner transposons are currently thought to have no sequence specificity for integration other than to insert within a TA contained in a degenerated [TA]_1–4 tract, either in vitro or in vivo. We have investigated the properties of a suspected hotspot for the integration of the mariner Mos1 element, namely the Tn9 cat gene that encodes a chloramphenicol acetyl transferase. Using in vitro and bacterial transposition assays, we confirmed that the cat gene is a preferential target for MOS1 integration, whatever its sequence environment, copy number or chromosomal locus. We also observed that its presence increases transposition rates both in vitro and in bacterial assays. The structural and sequence features that constitute the attractiveness of cat were also investigated. We first demonstrated that supercoiling is essential for the cat gene to be a hot spot. In contrast to the situation for Tc1-like elements, DNA curvature and bendability were not found to affect integration target preferences. We found that Mos1 integrations do not occur randomly along the cat gene. All TA dinucleotides that are preferred for integration were found within either TATA or TA×TA motifs. However, these motifs are not sufficient to constitute an attractive dinucleotide, since four TATA and TA×TA sites are cold spots. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Comparative genetics at the gene and chromosome levels between rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>) and wildrice (<Emphasis Type="Italic">Zizania palustris</Emphasis>)

Using comparative genetics, genes, repetitive DNA sequences and chromosomes were studied in the Oryzeae in order to more fully exploit the rice genome sequence data. Of particular focus was Zizania palustris L., n = 15, commonly known as American wildrice. Previous work has shown that rice chromosomes 1, 4 and 9 are duplicated in wildrice. The Adh1 and Adh2 genes were sequenced and, based on phylogenetic analyses, found to be duplicated in wildrice. The majority of the sequence diversity in the Adh sequences was in intron 3, in which were found several MITE insertions. Cytological and molecular approaches were used to analyze the evolution of rDNA and centromeric repetitive sequences in the Oryzeae. In wildrice, copies of the 5S rDNA monomer were found at two loci on two different chromosomes near the centromeres, as in rice. One nucleolar organizer region (NOR) locus was found adjacent to the telomere, as in rice. RCS1, a middle repetitive sequence in rice, was present in all of the centromeres of wildrice. RCS2/CentO, the highly repetitive component of Oryza sativa L. centromeres, was conserved in eight of the Oryza species examined, but was not found in wildrice. Three other middle repetitive centromeric sequences (RCH1, RCH2/CentO and RCH3) were also examined and found to have variable evolutionary patterns between species of Oryza and Zizania.Communicated by B. Friebe     

Mining <Emphasis Type="Italic">Xanthomonas</Emphasis> and <Emphasis Type="Italic">Streptomyces</Emphasis> genomes for new pectinase-encoding sequences and their heterologous expression in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Microbial genome sequencing has left a legacy of annotated yet uncharacterized genes or open reading frames, activities that may have useful applications in health and/or the environment. We are interested in the discovery and characterization of potentially new pectinolytic activities for the enzymatic retting of natural bast fibers such as hemp and flax. A highlight in this study is the discovery of a cold-active pectate lyase among five pectate-lyase-encoding sequences and two polygalacturonase-encoding sequences that we have cloned from the genomes of Xanthomonas campestris pv. campestris and Streptomyces coelicolor A3(2). Heterologous expression of these sequences as active pectate lyases and polygalacturonases required their subcloning in Escherichia coli Rosetta™ cells. The most active recombinant pectate lyase (XcPL NP_638163), a cold-active pectate lyase (XcPL NP_636037), and a polygalacturonase (XcPG NP_638805) were purified to near homogeneity and their kinetic parameters were determined. A significant amount of pectin degradation products was shown to be released by the two pectate lyases but not the polygalacturonase when hemp fiber pectin was used as substrate. Results of this study showed that genome data mining, besides an economical approach to new gene acquisition, may uncover new findings such as the discovery of a cold-active pectate-lyase-encoding sequence from X. campestris, a mesophilic microorganism.     

Plasmids captured in <Emphasis Type="Italic">C</Emphasis>. <Emphasis Type="Italic">metallidurans</Emphasis> CH34: defining the PromA family of broad-host-range plasmids

The self-transmissible, broad-host-range (BHR) plasmid pMOL98 was previously isolated from polluted soil using a triparental plasmid capture approach and shown to possess a replicon similar to that of the BHR plasmids pSB102 and pIPO2. Here, complete sequence analysis and comparative genomics reveal that the 55.5 kb nucleotide sequence of pMOL98 shows extensive sequence similarity and synteny with the BHR plasmid family that now includes pIPO2, pSB102, pTER331, and pMRAD02. They share a plasmid backbone comprising replication, partitioning and conjugative transfer functions. Comparison of the variable accessory regions of these plasmids shows that the majority of natural transposons, as well as the mini-transposon used to mark the plasmids, are inserted in the parA locus. The transposon unique to pMOL98 appears to have inserted from the chromosome of the recipient strain used in the plasmid capture procedure. This demonstrates the necessity for careful screening of plasmids and host chromosomes to avoid mis-interpretation of plasmid genome content. The presence of very similar BHR plasmids with different accessory genes in geographically distinct locations suggests an important role in horizontal gene exchange and bacterial adaptation for this recently defined plasmid group, which we propose to name “PromA”. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Organization of the<Emphasis Type="Italic"> Kpn</Emphasis>I family of chromosomal distal-end satellite DNAs in<Emphasis Type="Italic"> Silene latifolia</Emphasis>

The major satellite DNAs of the dioecious plant Silene latifolia are represented by the repetitive sequences X43.1, RMY1 and members of the SacI family, which are located at the distal ends of chromosomes. To characterize the satellite DNAs at the distal ends of the chromosomes in S. latifolia (Sl-distal-satDNA), we isolated a bacterial artificial chromosome clone (number 15B12) that contained multiple repeat sequences with KpnI restriction sites, and subcloned a portion of this sequence into a plasmid vector. Sequencing analysis confirmed that recognition or degenerate sites for KpnI were repeated 26 times at intervals of 310–324 bp in the inserted DNA. The phylogenetic tree that was constructed with the 26 KpnI repeat units contained clustered branches that were independent of the SacI family. It is clear that the KpnI repeat belongs to an Sl-distal-satDNA family that is distinct from the SacI family. We designated this family as "KpnI" after the restriction enzyme that does not have a site in the SacI family. Multi-colored fluorescent in situ hybridization was performed with the KpnI family and RMY1 probes under high stringency conditions. The results suggest that chromosome 7 is unique and that it carries the KpnI family at only one end.     

Simple sequence repeat analyses of interspecific hybrids and MAALs of <Emphasis Type="Italic">Oryza </Emphasis><Emphasis Type="Italic">officinalis</Emphasis> and <Emphasis Type="Italic">Oryza sativa</Emphasis>

Wild rice is a valuable resource for the genetic improvement of cultivated rice (Oryza sativa L., AA genome). Molecular markers are important tools for monitoring gene introgression from wild rice into cultivated rice. In this study, Simple sequence repeat (SSR) markers were used to analyze interspecific hybrids of O. sativa-O. officinalis (CC genome), the backcrossing progenies and the parent plants. Results showed that most of the SSR primers (335 out of 396, 84.6%) developed in cultivated rice successfully amplified products from DNA samples of wild rice O. officinalis. The polymorphism ratio of SSR bands between O. sativa and O. officinalis was as high as 93.9%, indicating differences between the two species with respect to SSRs. When the SSR markers were applied in the interspecific hybrids, only a portion of SSR primers amplified O. officinalis-specific bands in the F(1) hybrid (52.5%), BC(1) (52.5%), and MAALs (37.0%); a number of the bands disappeared. Of the 124 SSR loci that detected officinalis-specific bands in MAAL plants, 96 (77.4%) showed synteny between the A and C-genomes, and 20 (16.1%) showed duplication in the C-genome. Sequencing analysis revealed that indels, substitution and duplication contribute to the diversity of SSR loci between the genomes of O. sativa and O. officinalis.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.