A rate-independent technique for analysis of nucleic acid sequences: evolutionary parsimony

The method of evolutionary parsimony--or operator invariants--is a technique of nucleic acid sequence analysis related to parsimony analysis and explicitly designed for determining evolutionary relationships among four distantly related taxa. The method is independent of substitution rates because it is derived from consideration of the group properties of substitution operators rather than from an analysis of the probabilities of substitution in branches of a tree. In both parsimony and evolutionary parsimony, three patterns of nucleotide substitution are associated one-to-one with the three topologically linked trees for four taxa. In evolutionary parsimony, the three quantities are operator invariants. These invariants are the remnants of substitutions that have occurred in the interior branch of the tree and are analogous to the substitutions assigned to the central branch by parsimony. The two invariants associated with the incorrect trees must equal zero (statistically), whereas only the correct tree can have a nonzero invariant. The chi 2-test is used to ascertain the nonzero invariant and the statistically favored tree. Examples, obtained using data calculated with evolutionary rates and branchings designed to camouflage the true tree, show that the method accurately predicts the tree, even when substitution rates differ greatly in neighboring peripheral branches (conditions under which parsimony will consistently fail). As the number of substitutions in peripheral branches becomes fewer, the parsimony and the evolutionary-parsimony solutions converge. The method is robust and easy to use.      

Phylogenetic analysis of the South American electric fishes (order Gymnotiformes) and the evolution of their electrogenic system: a synthesis based on morphology, electrophysiology, and mitochondrial sequence data

The order Gymnotiformes (South American electric fishes) is a fascinating assemblage of freshwater fishes that share the unusual ability to produce and sense electric fields used for electrolocation and social communication. In the last few decades, the electrogenic and electrosensory systems (EES) of these fish have served as an excellent model to study motor and sensory physiology in vertebrates. In an attempt to the evolution of characters associated with the EES in the group, we applied maximum-parsimony (MP), minimum-evolution (ME), and maximum-likelihood (ML) methods to analyze 302 aligned bases of the mitochondrial 12S rRNA and 416 bases of the mitochondrial 16S rRNA of 19 gymnotiform genera representing all six recognized families. Six catfish genera (order Siluriformes) were also sequenced and used as outgroups. The phylogenetic hypothesis resultant from molecular data analysis differs in some respects from previous hypotheses based on morphological studies. Our results were most informative within the family level, as we were unable to elucidate the relationships among deeper branches in this order with sufficient confidence by using molecular data alone. The phylogenetic information of both mitochondrial DNA segments appears to be affected by functional constraints, and the resultant topologies were sensitive to different weighting schemes and the algorithm used. Nonetheless, we found unanimous support for the following phylogenetic relationships: (1) the family Sternopygidae is an unnatural group, and Sternopygus is the sole representative of a unique lineage within the order; (2) the family Hypopomidae is not monophyletic; and (3) the order Gymnotiformes is composed of at least six natural clades: Sternopygus, family Apteronotidae, a new clade consisting of the remaining sternopygids, families Hypopomidae + Rhamphicthyidae, family Electrophoridae, and family Gymnotidae. By combining molecular, morphological, and physiological information, we propose a new hypothesis for the phylogeny of this group and suggest a new family Eigenmanniidae n. (order Gymnotiformes).      

Formation of enlarged mitochondria in a liver cell line in response to a synthetic glucocorticoid

For a number of years it has been recognized that glucocorticoids cause alterations in liver cell morphology (6, 9). Several investigators have shown that in liver in vivo mitochondria can be enlarged to many times their normal volume by treatment with cortisone (13, 15). There is a concomitant decrease in mitochondrial number, and the results of Kimberg and Loeb suggest that this is due to mitochondrial fusion (7). However, the exact mechanism whereby mitochondrial volume is altered and whether in fact cortisone is the direct causal agent are not known due to the complexity of studying these questions in a whole animal system. We have found that dexamethasone sodium phosphate (dex), a synthetic glucocorticoid, causes the formation of enlarged mitochondria in a liver cell line RLC-GAI, which grows in defined medium. In this paper we present our observations on the amount of enlargement that occurs after 5 days of treatment. The formation of enlarged mitochondria is reversible upon removal of the hormone from the medium, and we have attempted to determine whether "mitochondrial" or "nonmitochondrial" inhibitors are more effective in blocking the return of mitochondria to their normal size when the hormone is removed.     

Does progressive nuclear staining with hemalum (alum hematoxylin) involve DNA,and what is the nature of the dye-chromatin complex?

Previous investigators have disagreed about whether hemalum stains DNA or its associated nucleoproteins. I review here the literature and describe new experiments in an attempt to resolve the controversy. Hemalum solutions, which contain aluminum ions and hematein, are routinely used to stain nuclei. A solution containing 16 Al³⁺ ions for each hematein molecule, at pH 2.0–2.5, provides selective progressive staining of chromatin without cytoplasmic or extracellular “background color.” Such solutions contain a red cationic dye-metal complex and an excess of Al³⁺ ions. The red complex is converted to an insoluble blue compound, assumed to be polymeric, but of undetermined composition, when stained sections are blued in water at pH 5.5–8.5. Staining experiments with DNA, histone and DNA + histone mixtures support the theory that DNA, not histone, is progressively colored by hemalum. Extraction of nucleic acids, by either a strong acid or nucleases at near neutral pH, prevented chromatin staining by a simple cationic dye, thionine, pH 4, and by hemalum, with pH adjustments in the range, 2.0–3.5. Staining by hemalum at pH 2.0–3.5 was not inhibited by methylation, which completely prevented staining by thionine at pH 4. Staining by hemalum and other dye-metal complexes at pH ≤ 2 may be due to the high acidity of DNA-phosphodiester (pK_a ~ 1). This argument does not explain the requirement for a much higher pH to stain DNA with those dyes and fluorochromes not used as dye-metal complexes. Sequential treatment of sections with Al₂(SO₄)₃ followed by hematein provides nuclear staining that is weaker than that attainable with hemalum. Stronger staining is seen if the pH is raised to 3.0–3.5, but there is also coloration of cytoplasm and other materials. These observations do not support the theory that Al³⁺ forms bridges between chromatin and hematein. When staining with hematein is followed by an Al₂(SO₄)₃ solution, there is no significant staining. Taken together, the results of my study indicate that the red hemalum cation is electrostatically attracted to the phosphate anion of DNA. The bulky complex cation is too large to intercalate between base pairs of DNA and is unlikely to fit into the minor groove. The short range van der Waals forces that bind planar dye cations to DNA probably do not contribute to the stability of progressive hemalum staining. The red cation is precipitated in situ as a blue compound, insoluble in water, ethanol and water-ethanol mixtures, when a stained preparation is blued at pH > 5.5.     

Phylogenetic analysis of carbamoylphosphate synthetase genes: complex evolutionary history includes an internal duplication within a gene which can root the tree of life

Carbamoylphosphate synthetase (CPS) catalyzes the first committed step in pyrimidine biosynthesis, arginine biosynthesis, or the urea cycle. Organisms may contain either one generalized or two specific CPS enzymes, and these enzymes may be heterodimeric (encoded by linked or unlinked genes), monomeric, or part of a multifunctional protein. In order to help elucidate the evolution of CPS, we have performed a comprehensive phylogenetic analysis using the 21 available complete CPS sequences, including a sequence from Sulfolobus solfataricus P2 which we report in this paper. This is the first report of a complete CPS gene sequence from an archaeon, and sequence analysis suggests that it encodes an enzyme similar to heterodimeric CPSII. We confirm that internal similarity within the synthetase domain of CPS is the result of an ancient gene duplication that preceded the divergence of the Bacteria, Archaea, and Eukarya, and use this internal duplication in phylogenetic tree construction to root the tree of life. Our analysis indicates with high confidence that this archaeal sequence is more closely related to those of Eukarya than to those of Bacteria. In addition to this ancient duplication which created the synthetase domain, our phylogenetic analysis reveals a complex history of further gene duplications, fusions, and other events which have played an integral part in the evolution of CPS.      

Trichoderma spp. and Bacillus spp. as growth promoters in maize (Zea mays L.)

Microbes that are beneficial to plants are used to enhance the crop growth, yield and are alternatives to chemical fertilizers. Trichoderma and Bacillus are the predominant plant growth-promoting fungi and bacteria. The objective of this study was select, characterize, and evaluate isolates of Trichoderma spp. and Bacillus spp. native from the northern region of Sinaloa, Mexico, and assess their effect on growth promotion in maize (Zea mays L.). In greenhouse conditions, four Trichoderma isolates and twenty Bacillus isolates, as well as two controls, were tested in a completely randomized design with three replicates. We selected the two best strains of Trichoderma and Bacillus: TB = Trichoderma asperellum, TF = Trichoderma virens, B14 = Bacillus cereus sensu lato and B17 = Bacillus cereus, which were evaluated in the field in a completely randomized blocks in factorial arrangement design with three replicates applying different rates of nitrogen fertilizer (0, 150 kg N/ha, and 300 kg N/ha). Treatments 5 (B17 = B. cereus) and 11 (TF = T. virens) both fertilized with 150 kg N/ha showed similar yields and they did not reveal significant differences from the treatments fertilized with 300 kg N/ha. This indicated that treatment 5 (B17= B. cereus with 150 kg N/ha) and treatment 11 (TF= T. virens with 150 kg N/ha) were efficient as growth promoters, by not showing significant differences in root volume and dry weight of foliage. The results indicated a reduction of 50% in the rate of nitrogen to fertilizer required for maize (Zea mays L.) crops. These microorganisms Trichoderma and Bacillus could be an alternative to reduce the use of chemical fertilizers in maize.     

Identification of a novel allosteric GLP-1R antagonist HTL26119 using structure-based drug design

A series of novel allosteric antagonists of the GLP-1 receptor (GLP-1R), exemplified by HTL26119, are described. SBDD approaches were employed to identify HTL26119, exploiting structural understanding of the allosteric binding site of the closely related Glucagon receptor (GCGR) (Jazayeri et al., 2016) and the homology relationships between GCGR and GLP-1R. The region around residue C347^6.36b of the GLP-1R receptor represents a key difference from GCGR and was targeted for selectivity for GLP-1R.