mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species

Reconstructions of the human-African great ape phylogeny by using mitochondrial DNA (mtDNA) have been subject to considerable debate. One confounding factor may be the lack of data on intraspecific variation. To test this hypothesis, we examined the effect of intraspecific mtDNA diversity on the phylogenetic reconstruction of another Plio- Pleistocene radiation of higher primates, the fascicularis group of macaque (Macaca) monkey species. Fifteen endonucleases were used to identify 10 haplotypes of 40-47 restriction sites in M. mulatta, which were compared with similar data for the other members of this species group. Interpopulational, intraspecific mtDNA diversity was large (0.5%- 4.5%), and estimates of divergence time and branching order incorporating this variation were substantially different from those based on single representatives of each species. We conclude that intraspecific mtDNA diversity is substantial in at least some primate species. Consequently, without prior information on the extent of genetic diversity within a particular species, intraspecific variation must be assessed and accounted for when reconstructing primate phylogenies. Further, we question the reliability of hominoid mtDNA phylogenies, based as they are on one or a few representatives of each species, in an already depauperate superfamily of primates.      

Isoformes of Malate Dehydrogenase from <Emphasis Type="Italic">Rhodovulum Steppense</Emphasis> A-20s Grown Chemotrophically under Aerobic Conditions

Three malate dehydrogenase isoforms (65-, 60-, and 71-fold purifications) with specific activities of 4.23, 3.88, and 4.56 U/mg protein were obtained in an electrophoretically homogenous state from Rhodоvulum steppense bacteria strain A-20s chemotrophically grown under aerobic conditions. The physicochemical and kinetic properties of malate dehydrogenase isoforms were determined. The molecular weight and the Michaelis constants were determined; the effect of hydrogen ions on the forward and reverse MDH reaction was studied. The results of the study demonstrated that the enzyme consists of subunits; the molecular weight of subunits was determined by SDS-PAGE.     

Express hybridization of molecular colonies with fluorescent probes

DNA colonies formed during PCR in a polyacrylamide gel and RNA colonies grown in an agarose gel containing Qβ replicase can be identified using the procedure of transfer of molecular colonies onto a nylon membrane followed by membrane hybridization with fluorescent oligonucleotide probes. The suggested improvements significantly simplify and shorten the procedure. By the example of a chimeric AML1-ETO sequence, a marker of frequently occurring leukemia, the express hybridization method was shown to allow the rapid identification of single molecules and the determination of titers of DNA and RNA targets. Hybridization with a mixture of two oligonucleotide probes labeled with different fluorophores complementary to components of the chimeric molecule ensures the identification of molecular colonies containing both parts of the chimeric sequence and improves the specificity of diagnostics.     

The Role of Malate Dehydrogenase Isoforms in the Regulation of Anabolic and Catabolic Processes in the Colorless Sulfur Bacterium Beggiatoa leptomitiformis D-402

The functional role of tetrameric and dimeric isoforms of malate dehydrogenase in the carbon metabolism of the colorless sulfur bacterium Beggiatoa leptomitiformis, strain D-402, was studied. This strain can grow both lithotrophically and organotrophically. By use of inhibition analysis, the tetrameric isoenzyme was shown to operate in the glyoxylate cycle and the dimeric form was found to be involved in the TCA cycle. The dynamics of the dimeric isoenzyme conversion to the tetrameric isoform was found to be determined by the rate of thiosulfate oxidation. The regulation of the carbon metabolism in Beggiatoa leptomitiformis is supposed to be accomplished by means of structural and functional changes in the protein molecule of malate dehydrogenase.     

Isolation and properties of malate dehydrogenase from Meso-and thermophilic bacteria

A scheme of purification of malate dehydrogenase from Macromonas bipunctata strain D-405 and Vulcanithermus medioatlanticus DSM 14978^T was developed. This scheme was used to obtain electrophoretically homogeneous enzyme preparations of the mesophilic bacterium M. bipunctata (specific activity, 26.9 ± 0.8 U/mg protein; yield, 10.9%) and the thermophilic bacterium V. medioatlanticus (specific activity, 5.0 ± 0.2 U/mg protein; yield, 19.2%). Using these high-purity enzymatic preparations, the physicochemical and regulatory properties of malate dehydrogenase were studied and the differences in kinetic characteristics and thermal stability of the preparations were determined.     

Purification and Investigation of Physicochemical and Regulatory Properties of Homogeneous L-Lactate: Cytochrom <Emphasis Type="Italic">c</Emphasis> Oxidoreductase Obtained from the Nonsulfur Purple Bacterium <Emphasis Type="Italic">Rhodovulum steppense</Emphasis>

L-Lactate: cytochrome c oxidoreductase activity was detected in cells of strain A-20s of the nonsulfur haloalkalophilic purple bacterium Rhodovulum steppense. An electrophoretically homogeneous preparation of the enzyme was obtained by purification. The enzyme had a specific activity of 4.75 U/mg protein, a 81.9-fold purification degree, and a 2.2% yield. The kinetic and physicochemical characteristics were determined. The value of the Michaelis constant with lactate was 15 μM. The temperature optimum for the studied enzyme was 31°C; optimum of pH was 8.2. It was found that the enzyme was a homodimer with a molecular weight of ~140 kDa; the mass of individual subunit was 68 kDa.     

Isolation, Purification, and Properties of Malate Dehydrogenase from Sulfur Bacterium Beggiatoa leptomitiformis

Malate dehydrogenase (E.C. 1.1.1.37) from the bacterium Beggiatoa leptomitiformis was isolated and purified 123-fold using a five-step purification procedure including the enzyme extraction, ammonium sulfate protein fractionation, gel filtration, ion exchange chromatography, and gel chromatography. The enzyme was homogenous according to the electrophoresis data; its activity was 20.43 U/mg protein. This malate dehydrogenase is a homotetramer (Mr = 172 kDa). The catalytic and thermodynamic properties, as well as the analysis of the published data suggest that the tetrameric structure of the enzyme allows it to participate in constructive metabolism supplying the cell with organic acids as a source of carbon.     

Oligomeric forms of bacterial malate dehydrogenase: a study of the enzyme from the phototrophic non-sulfur bacterium Rhodovulum steppense A-20s

Malate dehydrogenase (EC 1.1.1.37) was purified to homogeneity from the phototrophic purple non-sulfur bacterium Rhodovulum steppense A-20s. According to gel-chromatography and electrophoretic studies, malate dehydrogenase is present as a dimer, tetramer and octamer depending on cultivation conditions. In phototrophic aerobic conditions only the tetrameric form was present, in chemotrophic aerobic conditions all three forms were detected, while in the absence of oxygen the octameric form disappeared. The malate dehydrogenase oligomers are encoded by a single gene and composed of the same 35 kDa polypeptide but differ in pH and temperature optimum, in affinities to malate, oxaloacetate, NADH and NAD⁺ and in regulation by cations and citrate. By modulating the cultivation conditions, it has been established that the dimer participates in the glyoxylate cycle; the tetramer operates in the tricarboxylic acid cycle, and the octamer may be involved in the adaptation to oxidative stress.