Performance comparison between bootstrap and multiscale bootstrap for assessing phylogenetic tree for RNA polymerase

Phylogenetic inference refers to the reconstruction of evolutionary relationships among various species that is usually presented in the form of a tree. This study constructs the phylogenetic tree by using a novel distance-based method known as Modified one step M-estimator (MOM) method. The branches of the phylogenetic tree constructed were then evaluated to see their reliability. The performance of the reliability was then compared between the p-value of multiscale bootstrap (AU value) and bootstrap p-value (BP value). The aim of this study was to compare the performance between the AU value and BP value for assessing phylogenetic tree of RNA polymerase. The results have shown that multiscale bootstrap analysis can detect high sampling errors but not in bootstrap analysis. To overcome this problem, the multiscale bootstrap analysis has reduced the sampling error by increasing the number of replications. The clusters were indicated as significant if AU values or BP values were 95% or higher. From the analysis, the results showed that the BP and AU values differ at 11th and 15th branch of the phylogenetic tree. The BP values at both branches were 72 and 85%, respectively, thereby making the cluster not significant but by looking at the AU values, the two branches were more than 95% and the clusters were significant. This was due to the biasness in calculation of the probability of bootstrap analysis, therefore, the multiscale bootstrap analysis has improved the calculation of the probability value compared to the bootstrap analysis

The complete mitochondrial genome of a basal teleost, the Asian arowana (Scleropages formosus, Osteoglossidae)

BACKGROUND: Mitochondrial DNA-derived sequences have become popular markers for evolutionary studies, as their comparison may yield significant insights into the evolution of both the organisms and their genomes. From the more than 24,000 teleost species, only 254 complete mtDNA sequences are available (GenBank status on 06 Sep 2006). In this paper, we report the complete mitochondrial genome sequence of Asian arowana, a basal bonytongue fish species, which belongs to the order of Osteoglossiformes. RESULTS: The complete mitochondrial genomic sequence (mtDNA) of Asian arowana (Scleropages formosus) was determined by using shotgun sequencing method. The length of Asian arowana mtDNA is ca. 16,650 bp (its variation is due to polymorphic repeats in the control region), containing 13 protein-coding genes, 22 tRNA and 2 rRNA genes. Twelve of the thirteen protein coding genes were found to be encoded by the heavy strand in the order typically observed for vertebrate mitochondrial genomes, whereas only nad6 was located on the light strand. An interesting feature of Asian arowana mitogenome is that two different repeat arrays were identified in the control region: a 37 bp tandem repeat at the 5' end and an AT-type dinucleotide microsatellite at the 3' end. Both repeats show polymorphism among the six individuals tested; moreover the former one is present in the mitochondrial genomes of several other teleost groups. The TACAT motif described earlier only from mammals and lungfish was found in the tandem repeat of several osteoglossid and eel species. Phylogenetic analysis of fish species representing Actinopterygii and Sarcopterygii taxa has shown that the Asian arowana is located near the baseline of the teleost tree, confirming its status among the ancestral teleost lineages. CONCLUSION: The mitogenome of Asian arowana is very similar to the typical vertebrate mitochondrial genome in terms of gene arrangements, codon usage and base composition. However its control region contains two different types of repeat units at both ends, an interesting feature that to our knowledge has never been reported before for other vertebrate mitochondrial control regions. Phylogenetic analysis using the complete mtDNA sequence of Asian arowana confirmed that it belongs to an ancestral teleost lineage

THE DETECTION AND CHARACTERIZATION OF SOME VIRUSES INFECTING BLACKBERRY AND CHERRY IN SOUTH CAROLINA

Three separate virus research projects were conducted. Blackberry yellow vein disease (BYVD), a disorder caused by virus complexes, has become a major threat to blackberry production in the United States, especially in the southeastern part of the country where blackberries are grown for the fresh market. More than 30 viruses have been found to be associated with the disease. Most of these induce no symptoms when infecting the plant alone. However, when more than a single virus is present in the host visible symptoms are displayed. The incidence of 6 different viruses (Blackberry yellow vein-associated virus, Blackberry virus Y, Blackberry chlorotic ringspot virus, Blackberry virus E, Blackberry virus Ω, and Tobacco ringspot virus) that have been commonly found in BYVD-infected plants was studied using sentinel plants dispersed in plantings of blackberry in the field. Experiments were completed at the two largest commercial blackberry farms in South Carolina using more than 1200 sentinel plants over the course of three years. The sentinel plants were tested for the presence of the 6 viruses before they were exposed in the field and were again tested for the presence of the 6 viruses after the plants had been recovered from the field and allowed to overwinter in the greenhouse. Both Blackberry virus E, and Blackberry virus Ω were found infecting blackberry in South Carolina for the first time. A potential new ilarvirus was identified in blackberry and veronica. Partial sequence information for the 3 genomic molecules has been obtained. The virus shows closest homology to the members of subgroup 1 of the genus Ilarvirus, but is unique. This subgroup includes BCRV, one of the viruses previously associated with the BYVD complex. Symptoms typical of virus infection were observed in the suckers/watersprouts growing from the ‘Mazzard’ rootstock of a flowering cherry tree growing at Musser Farm, Clemson University in 2011. However, the scion of the tree, Prunus serrulata cv. Shirofugen, displayed no symptoms. Double-stranded RNA was isolated from the symptomatic tissues of the rootstock and used to provide templates for cDNA cloning and for nucleotide sequencing. Sequence data showed the virus to be most closely related to Cherry rusty mottle-associated virus

Functional identification of a Ligase in the Red Sea Atlantis II deepest Layer

Red sea, described as one of the unique marine ecosystems, incorporates up to 25 deep-sea brine pools. These pools posses multiple extreme conditions influencing the evolution and survival of their inhabiting microbial community. The combination of maximum depth (2194 m), high temperature (68C), anoxia, high salinity (26%), high pressure and high concentrations of heavy metals in the lower convective layer (LCL) of the Atlantis II brine pool makes it an ideal environment for identification of novel enzymes with unique characteristics and potential biotechnological applications. Here we describe the identification and the preliminary in vivo functional investigation of the ligase domain of an ATP-dependent DNA ligase from the DNA of the prokaryotic community extracted from water samples of the LCL of Atlantis II brine pool. Previously, these water samples were serially filtered on different membranes and the DNA isolated from the 0.1­m filter was subjected to 454 pyrosequencing. A metagenomic dataset was initiated and used in this study to mine for genes encoding DNA ligases through Pfam search of conserved domains. The search and subsequent bioinformatic analysis resulted in the identification of a contig harboring an ORF of 915 bp (305 amino acids) that encodes a putative DNA ligase (LigATII). Homology search of the putative DNA ligase showed highest similarity to Erysiopelotrichaceae Bacterium (39% identity, 54% positive). LigATII displays modular architecture that is similar to two distinct domains-(the adenylation domain of LigD and the oligonucleotide binding (OB) fold domain)-that are conserved in ATP-dependent DNA ligases. Functional annotation of the LigATII ORF, identification of the functional conserved amino acids by the Consurf tool, 3D modeling and comprehensive phylogenetic analysis were conducted. These analyses have revealed the relatedness of LigATII to the family of ATP-dependent DNA ligases that has been recently identified through computational studies to exist in prokaryotes. This family is expected to be involved in the specialized form of genomic DNA repair through the non-homologous end joining pathway which acts to join double-stranded breaks (DSBs) or to promote genetic diversity under conditions of selection pressures. Accordingly, the putative LigATII was amplified from the whole genome DNA amplification of LCL. Sanger sequencing confirmed the sequence of the gene before cloning into pET100 Topo directional expression vector. The cloned LigATII was transformed into a temperature sensitive mutant strain of Escherichia coli; strain GR501, with mutation in the DNA ligase gene. LigATII complemented the temperature sensitive strain at the non-permissive temperature (43â—¦C) verifying the in vivo functional activity. The biochemical characteristics of the novel LigATII protein will be described

Filogenetska analiza i molekularna karakterizacija virusa humane imunodeficijencije u Srbiji

Human immunodeficiency virus (HIV) is a retrovirus, the causative agent of Acquired immunodeficiency syndrome (AIDS). Since the beginning of the epidemic over 35 years ago, more than 78 million people have been infected so far and over 30 million have died. The high genetic variability and rapid evolution of HIV have been critical to its persistence and spread throughout the world. HIV-1 and HIV-2 comprise two distinct types of HIV. HIV-1 has diversified extensively into numerous genetic forms, including four groups (M, N, O, P), of which group M is causing the pandemic of HIV infection and AIDS. Group M viruses are further classified in multiple phylogenetically distinct subtypes (A-D, F, G, H, J and K), sub-subtypes (A1, A2, F1 and F2) and numerous recombinant forms. The global distribution of HIV-1 is complex and dynamic with regional epidemics representing only a subset of the global diversity. Molecular phylogenetic analysis, a method of reconstructing evolutionary relationships between nucleotide sequences, is one of the strategies for studying viral diversity and transmission dynamics. It is estimated that around half of HIV infected people are undiagnosed, making identification of transmission networks important for targeted public health intervention programs...Virus humane imunodeficijencije (HIV) je retrovirus koji uzrokuje sindrom stečene imunodeficijencije. Od početka epidemije pre 35 godina, ovim virusom je inficirano više od 78 miliona ljudi a preko 30 miliona je umrlo. Visoka genetička varijabilnost i brza evolucija HIV-a su ključni uzroci opstanka i globalnog širenjaepidemije. HIV je filogenetski klasifikovan u dva tipa: HIV-1 i HIV-2. Visoki diverzitet HIV-1 ogleda u postojanju četiri grupe (M, N, O, P) od kojih su virusi grupe M uzročnici globalne HIV-1 pandemije. Grupa M virusa je podeljena u više filogenetski različitih podtipova (A-D, F-H, J i K), pod-podtipove (A1, A2, F1 i F2) i cirkulišuće rekombinantne forme. Distribucija podtipova u svetu je složena i dinamična sa regionalnim HIV-1 epidemijama unutar globalnog diverziteta. Molekularna filogenetska analiza, metod za rekonstrukciju evolutivnih odnosa između nukleotidnih sekvenci, je tehnika za proučavanje varijabilnosti virusa i dinamike transmisije unutar regionalnih populacija. Procenjuje se da kod blizu polovine inficiranih osoba HIV infekcija nije dijagnostikovana, zbog čega je identifikacija puteva transmisije izuzetno značajna u cilju javno zdravstvenog nadzora. U ovom istraživanju primenjene su savremene filogenetske metode u analizi HIV-1 sekvenci izolata iz Srbije u cilju karakterizacije molekularne epidemiologije i dinamike transmisije, što je ključno za bolje razumevanje karakteristika aktuelne HIV-1 epidemije u Srbiji..

Phylogenetic Structure of Two Central Mexican Centruroides Species Complexes

Central Mexico is home to numerous species of highly toxic Centruroides scorpions. Two species complexes C. infamatus, (C.L. Koch, 1844), and C. limpidus (Karsch, 1879) typify the complex relationships that exist between and within the complexes. Their existing taxonomic status is based on morphological features such as coloration and morphosculpture. A complete and modern study of these scorpions does not exist, and is needed. In an attempt to clarify the status and relationship between these complexes we initiated a molecular based approach applying mitochondrial gene markers (16S and CO1). This study confirms two divergent clades within C. infamatus; divergence rate estimates their common ancestor’s age as 2-4 Ma for HKY+G+I divergence rate (11.7 ± 0.9 %) and 3-5 Ma for uncorrected p (7.2 ± 0.4 %). Further study is necessary with sampling all over the range of both taxa, to confirm existence of two independent study also suggests that more than one ancient monophyletic lineage (possibly, more than one species) exist within currently accepted Centruroides limpidus limpidus. The type locality of Centruroides limpidus is Puebla, which lies in the same geographic area as Guerrero. Thus, we might assume that the Querétaro/Guerrero lineage corresponds to ‘true’ C. limpidus, and that the Balsas Depression populations could belong to another, ‘cryptic’, or ‘sibling’ species. Further, detailed investigations should be done to test these preliminary conclusions: the need for many more populations from the entire range of C. limpidus is needed. Several data sets (mitochondrial and nuclear genes, allozymes, morphology, toxin structure/activity, etc.) could be analyzed to establish the true taxonomic and genetic structure of the populations and species of Centruroides

Characterization of genes involved in sumoylation during embryogenesis in rainbow trout (Oncorhynchus mykiss)

SUMOylation is the post-translational modification of proteins by the addition of the small ubiquitin-like modifier (SUMO), which plays an important role in various cellular processes. It has been reported that SUMO and its related proteins are important in diverse reproductive functions such as ovulation, gametogenesis, and embryogenesis. Modification of target proteins by SUMO is an ATP-dependent enzymatic cascade involving three key enzymes: E1 activating enzyme (the heterodimer SAE1-SAE2), E2 conjugating enzyme (UBC9) and several E3 ligating enzymes (PIAS, RanBP2/Nup358 and Pc2). The objectives of this study were to characterize the genes involved in SUMOylation and determine their expression profiles during embryogenesis in rainbow trout. Through database analysis, ten Sumo related genes, which include Sumo1, Sumo2, Sumo3, Sae1, Sae2, Ubc9, Pias1, Pias4, Cbx4, and Nse2, were identified. Analysis of protein sequences of SUMO1 and UBC9, the key components in the pathway, revealed that they are highly conserved among human, mice, rat, cattle, pig, chicken, Xenopus, zebrafish and rainbow trout species. The expression profiles of the Sumo related genes during embryonic development in rainbow trout were analyzed by quantitative real time PCR using cDNAs derived from unfertilized eggs and embryos of 17 different developmental stages (0h, 3h, 7.5h, 11.5h, 13.5h, 18h, 27h, 34h, 2d, 3d, 4d, 5d, 6d, 8d, 10d, 12d, 16d and 25d post fertilization). The expression of Sumo3, Ubc9, Pias4, and Nse2 genes showed similar patterns, being low in unfertilized eggs and increasing gradually in early embryos until 18 h post fertilization followed by a gradual decrease in embryos after 18 h post fertilization; both Sumo1 and Sumo2 genes were highly expressed during maternal to zygotic transition (3d-5d post fertilization); while Sae1, Sae2, Pias1, and Cbx4 were expressed constitutively at steady-state levels throughout embryogenesis. The data indicate that the expression of Sumo related genes are dynamically regulated during the embryonic development in rainbow trout. To better understand how SUMO modification regulates embryonic development, two oocyte specific factors, FIGLA and LHX8, were studied for their interactions with SUMO. Under the experimental conditions used in the study, no apparent interactions of FIGLA or LHX8 with SUMO were detected. The study represents the first attempt to characterize genes involved in SUMOylation in rainbow trout. Further studies to understand the role of SUMOylation in controlling early embryogenesis may ultimately lead to the development of molecular markers for egg quality and embryonic development potential in rainbow trout

Discover And Analysis Of Grapevine Vein-Clearing Virus In Ampelopsis Cordata

A recent threat to the sustainability of grape production is Grapevine vein-clearing virus (GVCV), the first DNA virus discovered in grapevines. Infection with GVCV leads to vine decline, lower quality berries, and eventual death of the grapevine. Since GVCV was discovered in cultivated grapevines, research has been dedicated to investigating its range and origin. The entire genome of the first GVCV isolate from a grape cultivar ‘Chardonel\u27 has been deposited in GenBank and is used as a reference genome. More recently, two GVCV isolates were found in native Vitis rupestris in Missouri. In this thesis project, I applied polymerase chain reaction (PCR) assays to screen for GVCV in native Ampelopsis cordata, which is also in the Vitaceae family. I found GVCV in two accessions of this wild plant species. The entire genomes of the two GVCV isolates, GVCV-AMP1 and GVCV-AMP2, from A. cordata were sequenced. The GVCV-AMP1 genome is composed of double-stranded DNA, 7,749 bp long, while GVCV-AMP2 is 7,765 bp long. Genomic analysis indicated that they are new isolates with signature 9base pair inserts in open reading frame II. A survey of GVCV in seventeen A. cordata plants around the Springfield area found that five were infected with GVCV, suggesting high incidence of GVCV among these native plants. These results demonstrated that GVCV spreads among species across genera in native habitats, and yielded crucial clues on origin and epidemics of GVCV. These findings will aid in developing new strategies for the management of GVCV-associated disease

Molecular approach to the identification and phylogenetic relationship among actinopterygii using mitochondrial DNA 12S rRNA, 16S rRNA and cytochrome B genes in Peninsular Malaysia

The morphological based method has been utilised to identify and classify fish species. However, this method is lacking genetic information which makes it less useful in phylogenetic studies. The present study utilises mtDNA markers namely 12S rRNA, 16S rRNA and cytochrome b genes to characterize, identify the polymorphism and genetic relationship among marine fish species. The genetic relationship among the selected marine fish species was inferred through phylogenetic trees using Neighbor-joining (NJ), Maximum parsimony (MP) and Maximum likelihood (ML) methods. The result showed that 12S rRNA gene was more conserved than 16S rRNA and cytb genes based on the polymorphisms reported. The finding also revealed that Nemipterus nemurus exhibits the highest number of polymorphisms indicating that this species might have experienced the most changed events and thus possessing the most genetic deviation from the common shared ancestry. All the phylogenetic trees that were constructed based on 12S rRNA, 16S rRNA and cytochrome b genes showed a similar clustering with slight differences in their bootstrap values. Some species with greatly morphological resemblance such as Liza macrolepis and Liza vaigiensis however, showed a genetically distant relationship. Nevertheless, another morphological resemblance species namely Pampus argenteus / Pampus chinensis and Drepane longimana / Drepane punctata were consistently observed in all topologies with high bootstrap support (97-100%). Having a distinctive morphological, the Megalaspis cordyla and Caranx sexfasciatus occupied a highly similar nucleotide sequences and showed a consistent clustering in all topologies. This study also revealed that the combined genes provide a better resolution for all phylogenetic trees. Overall, these findings showed that the molecular based method using mtDNA genes were successfully applied to study the diverse Malaysia marine fish species in class Actinopterygii