Monophyly of the order Rodentia inferred from mitochondrial DNA sequences of the genes for 12S rRNA, 16S rRNA, and tRNA-valine

A recent analysis of amino acid sequence data (Graur et al.) suggested that the mammalian order Rodentia is polyphyletic, in contrast to most morphological data, which support rodent monophyly. At issue is whether the hystricognath rodents, such as the guinea pig, represent an independent evolutionary lineage within mammals, separate from the sciurognath rodents. To resolve this problem, we sequenced a region (2,645 bp) of the mitochondrial genome of the guinea pig containing the complete 12S ribosomal RNA, 16S ribosomal RNA, and transfer RNA(VAL) genes for comparison with the available sciurognath and other mammalian sequences. Several methods of analysis and statistical tests of the data all show strong support for rodent monophyly (91%-98% bootstrap probability, or BP). Calibration with the mammalian fossil record suggests a Cretaceous date (107 mya) for the divergence of sciurognaths and hystricognaths. An older date (38 mya) for the controversial Mus- Rattus divergence also is supported by these data. Our neighbor-joining analyses of all available sequence data (25 genes) confirm that some individual genes support rodent polyphyly but that tandem analysis of all data does not. We propose that the conflicting results are due to several compounding factors. The unique biochemical properties of some hystricognath metabolic proteins, largely responsible for generating this controversy, may have a single explanation: a cascade effect resulting from inactivation of the zinc-binding abilities of insulin. After excluding six genes possibly affected by insulin inactivation, analyses of all available sequence data (7,117 nucleotide sites, 3,099 amino acid sites) resulted in strong support for rodent monophyly (94% BP for DNA sequences, 90% for protein sequences), which lends support to the insulin-cascade hypothesis.      

Adh 1 first intron polymorphisms in Argentinean populations of Ceratitis capitata

DNA size polymorphisms were utilized in a study of 24 natural populations of Ceratitis capitata Wiedemann (Diptera: Tephritidae) from Argentina. The first intron of alcohol dehydrogenase 1 gene (Adh₁) was amplified using exon priming intron crossing‐polymerase chain reaction. Three size variants were detected among the 307 samples analyzed. To better differentiate the size variants, further digestion of PCR products with the EcoRI restriction enzyme was carried out. Complete nucleotide sequences of the three‐allele variants were obtained and single changes, insertions, deletions, and EcoRI recognition sites were located. Population allele frequencies were analyzed and a global mean heterozygosity (He) of 0.33 was obtained. In most populations, observed allelic frequencies conformed to Hardy–Weinberg expectations. Significant differences between provinces and sampling sites within these provinces, and among some populations were found. The average number of insects exchanged among populations (Nm) was estimated and high values were observed between Argentina and populations from two African countries (Morocco and Kenya), Australia, and Hawaii (Kauai). Pest introduction sources and dispersion patterns in Argentina are discussed based on these results as well as on available bibliographical data.     

High level expression of Acidothermus cellulolyticus β-1, 4-endoglucanase in transgenic rice enhances the hydrolysis of its straw by cultured cow gastric fluid

Background

In the hydrolysis of lignocellulosic materials, thermostable enzymes decrease the amount of enzyme needed due to higher specific activity and elongate the hydrolysis time due to improved stability. For cost-efficient use of enzymes in large-scale industrial applications, high-level expression of enzymes in recombinant hosts is usually a prerequisite. The main aim of the present study was to compare the biochemical and hydrolytic properties of two thermostable recombinant glycosyl hydrolase families 10 and 11 (GH10 and GH11, respectively) xylanases with respect to their potential application in the hydrolysis of lignocellulosic substrates.

Results

The xylanases from Nonomuraea flexuosa (Nf Xyn11A) and from Thermoascus aurantiacus (Ta Xyn10A) were purified by heat treatment and gel permeation chromatography. Ta Xyn10A exhibited higher hydrolytic efficiency than Nf Xyn11A toward birchwood glucuronoxylan, insoluble oat spelt arabinoxylan and hydrothermally pretreated wheat straw, and it produced more reducing sugars. Oligosaccharides from xylobiose to xylopentaose as well as higher degree of polymerization (DP) xylooligosaccharides (XOSs), but not xylose, were released during the initial hydrolysis of xylans by Nf Xyn11A, indicating its potential for the production of XOS. The mode of action of Nf Xyn11A and Ta Xyn10A on glucuronoxylan and arabinoxylan showed typical production patterns of endoxylanases belonging to GH11 and GH10, respectively.

Conclusions

Because of its high catalytic activity and good thermostability, T. aurantiacus xylanase shows great potential for applications aimed at total hydrolysis of lignocellulosic materials for platform sugars, whereas N. flexuosa xylanase shows more significant potential for the production of XOSs.     

Efficient Mutagenesis by Cas9 Protein-Mediated Oligonucleotide Insertion and Large-Scale Assessment of Single-Guide RNAs

The CRISPR/Cas9 system has been implemented in a variety of model organisms to mediate site-directed mutagenesis. A wide range of mutation rates has been reported, but at a limited number of genomic target sites. To uncover the rules that govern effective Cas9-mediated mutagenesis in zebrafish, we targeted over a hundred genomic loci for mutagenesis using a streamlined and cloning-free method. We generated mutations in 85% of target genes with mutation rates varying across several orders of magnitude, and identified sequence composition rules that influence mutagenesis. We increased rates of mutagenesis by implementing several novel approaches. The activities of poor or unsuccessful single-guide RNAs (sgRNAs) initiating with a 5′ adenine were improved by rescuing 5′ end homogeneity of the sgRNA. In some cases, direct injection of Cas9 protein/sgRNA complex further increased mutagenic activity. We also observed that low diversity of mutant alleles led to repeated failure to obtain frame-shift mutations. This limitation was overcome by knock-in of a stop codon cassette that ensured coding frame truncation. Our improved methods and detailed protocols make Cas9-mediated mutagenesis an attractive approach for labs of all sizes.     

Degradation of Phthalate and Di-(2-Ethylhexyl)phthalate by Indigenous and Inoculated Microorganisms in Sludge-Amended Soil

The metabolism of phthalic acid (PA) and di-(2-ethylhexyl)phthalate (DEHP) in sludge-amended agricultural soil was studied with radiotracer techniques. The initial rates of metabolism of PA and DEHP (4.1 nmol/g [dry weight]) were estimated to be 731.8 and 25.6 pmol/g (dry weight) per day, respectively. Indigenous microorganisms assimilated 28 and 17% of the carbon in [¹⁴C]PA and [¹⁴C]DEHP, respectively, into microbial biomass. The rates of DEHP metabolism were much greater in sludge assays without soil than in assays with sludge-amended soil. Mineralization of [¹⁴C]DEHP to ¹⁴CO₂ increased fourfold after inoculation of sludge and soil samples with DEHP-degrading strain SDE 2. The elevated mineralization potential was maintained for more than 27 days. Experiments performed with strain SDE 2 suggested that the bioavailability and mineralization of DEHP decreased substantially in the presence of soil and sludge components. The microorganisms metabolizing PA and DEHP in sludge and sludge-amended soil were characterized by substrate-specific radiolabelling, followed by analysis of ¹⁴C-labelled phospholipid ester-linked fatty acids (¹⁴C-PLFAs). This assay provided a radioactive fingerprint of the organisms actively metabolizing [¹⁴C]PA and [¹⁴C]DEHP. The ¹⁴C-PLFA fingerprints showed that organisms with different PLFA compositions metabolized PA and DEHP in sludge-amended soil. In contrast, microorganisms with comparable ¹⁴C-PLFA fingerprints were found to dominate DEHP metabolism in sludge and sludge-amended soil. Our results suggested that indigenous sludge microorganisms dominated DEHP degradation in sludge-amended soil. Mineralization of DEHP and PA followed complex kinetics that could not be described by simple first-order equations. The initial mineralization activity was described by an exponential function; this was followed by a second phase that was described best by a fractional power function. In the initial phase, the half times for PA and DEHP in sludge-amended soil were 2 and 58 days, respectively. In the late phase of incubation, the apparent half times for PA and DEHP increased to 15 and 147 days, respectively. In the second phase (after more than 28 days), the half time for DEHP was 2.9 times longer in sludge-amended soil assays than in sludge assays without soil. Experiments with radiolabelled DEHP degraders suggested that a significant fraction of the ¹⁴CO₂ produced in long-term degradation assays may have originated from turnover of labelled microbial biomass rather than mineralization of [¹⁴C]PA or [¹⁴C]DEHP. It was estimated that a significant amount of DEHP with poor biodegradability and extractability remains in sludge-amended soil for extended periods of time despite the presence of microorganisms capable of degrading the compound (e.g., more than 40% of the DEHP added is not mineralized after 1 year).     

Higher-level snake phylogeny inferred from mitochondrial DNA sequences of 12S rRNA and 16S rRNA genes

Portions of two mitochondrial genes (12S and 16S ribosomal RNA) were sequenced to determine the phylogenetic relationships among the major clades of snakes. Thirty-six species, representing nearly all extant families, were examined and compared with sequences of a tuatara and three families of lizards. Snakes were found to constitute a monophyletic group (confidence probability [CP] = 96%), with the scolecophidians (blind snakes) as the most basal lineages (CP = 99%). This finding supports the hypothesis that snakes underwent a subterranean period early in their evolution. Caenophidians (advanced snakes), excluding Acrochordus, were found to be monophyletic (CP = 99%). Among the caenophidians, viperids were monophyletic (CP = 98%) and formed the sister group to the elapids plus colubrids (CP = 94%). Within the viperids, two monophyletic groups were identified: true vipers (CP = 98%) and pit vipers plus Azemiops (CP = 99%). The elapids plus Atractaspis formed a monophyletic clade (CP = 99%). Within the paraphyletic Colubridae, the largely Holarctic Colubrinae was found to be a monophyletic assemblage (CP = 98%), and the Xenodontinae was found to be polyphyletic (CP = 91%). Monophyly of the henophidians (primitive snakes) was neither supported nor rejected because of the weak resolution of relationships among those taxa, except for the clustering of Calabaria with a uropeltid, Rhinophis (CP = 94%).      

Workflow management systems for gene sequence analysis and evolutionary studies – A Review

Post ‘omic’ era has resulted in the development of many primary, secondary and derived databases. Many analytical and visualization bioinformatics tools have been developed to manage and analyze the data available through large sequencing projects. Availability of heterogeneous databases and tools make it difficult for researchers to access information from varied sources and run different bioinformatics tools to get desired analysis done. Building integrated bioinformatics platforms is one of the most challenging tasks that bioinformatics community is facing. Integration of various databases, tools and algorithm is a challenging problem to deal with. This article describes the bioinformatics analysis workflow management systems that are developed in the area of gene sequence analysis and phylogeny. This article will be useful for biotechnologists, molecular biologists, computer scientists and statisticians engaged in computational biology and bioinformatics research.     

Taxol binds to polymerized tubulin in vitro

Taxol, a natural plant product that enhances the rate and extent of microtubule assembly in vitro and stabilizes microtubules in vitro and in cells, was labeled with tritium by catalytic exchange with (3)H(2)O. The binding of [(3)H]taxol to microtubule protein was studied by a sedimentation assay. Microtubules assembled in the presence of [(3)H]taxol bind drug specifically with an apparent binding constant, K(app), of 8.7 x 19(-7) M and binding saturates with a calculated maximal binding ration, B(max), of 0.6 mol taxol bound/mol tubulin dimer. [(3)H]Taxol also binds and assembles phosphocellulose-purified tubulin, and we suggest that taxol stabilizes interactions between dimers that lead to microtubule polymer formation. With both microtubule protein and phosphocellulose- purified tubulin, binding saturation occurs at approximate stoichiometry with the tubulin dimmer concentration. Under assembly conditions, podophyllotoxin and vinblastine inhibit the binding of [(3)H]taxol to microtubule protein in a complex manner which we believe reflects a competition between these drugs, not for a single binding site, but for different forms (dimer and polymer) of tubulin. Steady-state microtubules assembled with GTP or with 5’-guanylyl-α,β-methylene diphosphonate (GPCPP), a GTP analog reported to inhibit microtubule treadmilling (I.V. Sandoval and K. Weber. 1980. J. Biol. Chem. 255:6966-6974), bind [(3)H]taxol with approximately the same stoichiometry as microtubules assembled in the presence of [(3)H]taxol. Such data indicate that a taxol binding site exists on the intact microtubule. Unlabeled taxol competitively displaces [(3)H]taxol from microtubules, while podophyllotoxin, vinblastine, and CaCl(2) do not. Podophyllotoxin and vinblastine, however, reduce the mass of sedimented taxol-stabilized microtubules, but the specific activity of bound [(3)H]taxol in the pellet remains constant. We conclude that taxol binds specifically and reversibly to a polymerized form of tubulin with a stoichiometry approaching unity.     

Phosphorylation and biosynthesis of high molecular weight proteins of tumor nuclear matrix

INTRODUCTIONAsearlyasin1948wehavefr8CtionatedisolatednucleifromnormalandtumorcellsbyextractionwithiMNaCIanddilutealkali[1].Thenuclearresiduewasthenstudiedmorethoroughly[2,3].Lateron,sillillarproteinousnuclearresidueswereisolatedbyotherworkers[46]andasstud…