Data decisiveness,data quality,and incongruence in phylogenetic analysis: an example from the monocotyledons using mitochondrial atp A sequences

We examined three parallel data sets with respect to qualities relevant to phylogenetic analysis of 20 exemplar monocotyledons and related dicotyledons. The three data sets represent restriction-site variation in the inverted repeat region of the chloroplast genome, and nucleotide sequence variation in the chloroplast-encoded gene rbcL and in the mitochondrion-encoded gene atpA, the latter of which encodes the alpha-subunit of mitochondrial ATP synthase. The plant mitochondrial genome has been little used in plant systematics, in part because nucleotide sequence evolution in enzyme-encoding genes of this genome is relatively slow. The three data sets were examined in separate and combined analyses, with a focus on patterns of congruence, homoplasy, and data decisiveness. Data decisiveness (described by P. Goloboff) is a measure of robustness of support for most parsimonious trees by a data set in terms of the degree to which those trees are shorter than the average length of all possible trees. Because indecisive data sets require relatively fewer additional steps than decisive ones to be optimized on nonparsimonious trees, they will have a lesser tendency to be incongruent with other data sets. One consequence of this relationship between decisiveness and character incongruence is that if incongruence is used as a criterion of noncombinability, decisive data sets, which provide robust support for relationships, are more likely to be assessed as noncombinable with other data sets than are indecisive data sets, which provide weak support for relationships. For the sampling of taxa in this study, the atpA data set has about half as many cladistically informative nucleotides as the rbcL data set per site examined, and is less homoplastic and more decisive. The rbcL data set, which is the least decisive of the three, exhibits the lowest levels of character incongruence. Whatever the molecular evolutionary cause of this phenomenon, it seems likely that the poorer performance of rbcL than atpA, in terms of data decisiveness, is due to both its higher overall level of homoplasy and the fact that it is performing especially poorly at nonsynonymous sites.     

Biogeography and floral evolution of baobabs (Adansonia,Bombacaceae) as inferred from multiple data sets

The phylogeny of baobab trees was analyzed using four data sets: chloroplast DNA restriction sites, sequences of the chloroplast rpl16 intron, sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA, and morphology. We sampled each of the eight species of Adansonia plus three outgroup taxa from tribe Adansonieae. These data were analyzed singly and in combination using parsimony. ITS and morphology provided the greatest resolution and were largely concordant. The two chloroplast data sets showed concordance with one another but showed significant conflict with ITS and morphology. A possible explanation for the conflict is genealogical discordance within the Malagasy Longitubae, perhaps due to introgression events. A maximum-likelihood analysis of branching times shows that the dispersal between Africa and Australia occurred well after the fragmentation of Gondwana and therefore involved overwater dispersal. The phylogeny does not permit unambiguous reconstruction of floral evolution but suggests the plausible hypothesis that hawkmoth pollination was ancestral in Adansonia and that there were two parallel switches to pollination by mammals in the genus.     

Duplicated plastid and triplicated cytosolic isozymes of triosephosphate isomerase in maize (Zea mays L.)

We studied electrophoretic variation and inheritance of triosephosphate isomerase (TPI) isozymes in maize (Zea mays L.). In contrast to most diploid plants, in maize, TPI exists as multiple isozymes in both the plastid and cytosolic subcellular compartments. Phenotypes result from the overlay of two independent sets of isozymes and allozymes, representing the plastid (encoded by the nuclear genes Tpi1 and Tpi2) and cytosolic (encoded by Tpi3, Tpi4, and Tpi5) systems. All possible intragenic and intergenic dimeric enzymes are formed between polypeptides within each subcellular compartment. No heterodimers are formed between plastid and cytosolic polypeptides. Extensive surveys of accessions of land races and inbred lines revealed 22 allelic variants for the five loci. Most alleles have been formally validated by segregation analysis. We describe two null alleles at Tpi4, distinguished by their relative abilities to form intergenic heterodimers with polypeptides specified by Tpi3 and Tpi5. Linkage analyses and crosses with B-A translocation stocks were effective in determining the chromosome locations of all five loci. Duplicated genes for both the plastid and cytosolic isozymes were localized to genomic regions that possess numerous other redundant sequences. We placed Tpi1 on the long arm of chromosome 7, approximately 23 centimorgans (cM) distal to g11; we localized its duplicate--Tpi2--17 cM distal to v4 on the long arm of chromosome 2. The triplicate loci encoding cytosolic TPIs reside on chromosomes 3 and 8. Tpi4 is approximately equidistant (11 cM) from d1 and Lg3, near the centromere of chromosome 3. Tpi3 and Tpi5 are located on distal ends of the most poorly marked maize chromosome; Tpi3 is 29 cM distal to Idh 1 on 8L, and Tpi5 is on 8S or near the centromere on 8L. In contrast to most duplicated maize sequences, which often occur in parallel linkages on different chromosomes, Tpi3 and Tpi5 provide an example of intrachromosomal gene duplication. Several of the Tpi loci are located in sparsely mapped regions of the genome, and Tpi1 is the first isozyme marker for chromosome 7.     

5-Oxoprolinase from rat kidney, I. Assay, purification, and determination of kinetic parameters.

A new assay for the determination of 5-oxoprolinase activity is described. The enzyme 5-oxoprolinase was purified from rat kidney 285-fold to apparent homogeneity, as judged by analytical disc electrophoresis and discontinous polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. The specific activity of the preparation was 122 mU/mg of protein. A complete initial rate kinetic analysis of the forward reaction catalyzed by 5-oxoprolinase was carried out using 5-oxo-L-proline and MgATP2theta as substrates. The computer-fitted double reciprocal plots showed intersecting patterns indicating a sequential mechanism. The data were fitted by weighted linear regression analysis using the complete equation for bisubstrate reactions. The limiting Michaelis constants for 5-oxoproline and MgATP2theta were calculated to be 31.6 +/- 2.3 muM and 172.7 +/- 11.5muM, respectively. The maximum forward rate is 1.2 +/- 0.02 mumol X min-1; the turnover number 7.0 min-1.     

5-Oxoprolinase from rat kidney, II. Molecular weight determinations.

The molecular weight of 5-oxoprolinase from rat kidney was estimated by gel filtration on Sephadex G-200 and G-150 to be 460 000 +/- 30 000. A value of 230 000 +/- 10 000 was obtained by zonal sedimentation in a sucrose gradient. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate yielded a molecular weightof 115 000 +/- 6 000. It is concluded that 5-oxoprolinase consists of four subunits of 115 000 daltons each. The dissociation or aggregation behavior of the enzyme seems to be influenced neither by the presence of the substrates 5-oxo-L-proline and MgATP2theta nor by the presence of the stabilizing compounds glutathione mercaptoethanol or dithioerythritol.     

15-Hydroxyprostaglandin dehydrogenase from human placenta. 1. Isolation and characterization]

15-Hydroxyprostaglandin dehydrogenase was isolated from human term placenta up to a final purification of 380-fold. A spec. act. of 2000 mU/mg of protein was reached. The preparation was not homogeneous as judged by analytical disc electrophoresis. The enzyme could be stored in the presence of 50% glycerol and 10mM 2-mercaptoethanol without any loss of activity for at least one year. A distinct single protein band stained after discontinuous polyacrylamide gel electrophoresis was shown by enzymatic activity staining to correspond to 15-hydroxyprostaglandin dehydrogenase activity. Thus no evidence for the exitstence of isoenzymes was obtained. The protein in the final preparation steps showed neither alcohol dehydrogenase, NAD reductase, nor NADH oxidase activity, nor enzymatic conversion of prostaglandin or 15-oxoprostaglandin in the absence of NAD and NADH. No spontaneous reactions between NAD and prostaglandin or NADH and 15-oxoprostaglandin were detectable in the absence of the enzyme. Ethanol and glycerol slightly inhibited the reaction. Various buffers (Tris/HC1, potassium phosphate, HEPES, and triethanolamine) and salts (ammonium chloride, ammonium sulfate, potassium chloride, and sodium chloride) had different effects on the reaction rate. The pH profile of the reaction shows a plateau between pH 7.0 and 7.8 and a steep maximum at pH 9.5. A linear Arrhenius plot was obtained for the temperature dependence of the reaction from 20 to 37 degrees C. The molar activation enthalpy of the reaction was calculated to be 13.1 kcal/mole. The molecular weight of 15-hydroxyprostaglandin dehydrogenase was estimated to be 32000 -/+ 3000 by gel filtration on Sephadex G-150 in the presence of 10mM mercaptoethanol.     

Separation and quantitative determination of 2-acetyl-aminofluorene and its hydroxylated metabolites by high pressure liquid chromatography

A method utilizing high pressure liquid chromatography has been developed for the separation and quantitative estimation of all the major metabolites of the carcinogen 2-acetylaminofluorene in a single chromatographic determination. The method was used to separate 7-hydroxy-2-acetylaminofluorene, 5-hydroxy-2-acetylaminofluorene, 3-hydroxy-2-acetylaminofluorene, 1-hydroxy-2-acetyl-aminofluorene, 2-aminofluorene, N-hydroxy-2-acetylaminofluorene, and 2-acetylaminofluorene when 2-acetylaminofluorene was incubated with mouse liver microsomes and NADPH.This new high pressure liquid chromatography method for separating the metabolites arising from hydroxylations of 2-acetylaminofluorene should also prove useful in the isolation and quantitative analysis of metabolites from other N-acetylarylamines.     

The fate of extracellular glutathione in the rat.

When intravenously administered to rats, [U-14C]glycine-labelled GSSG, GSH and its analogue ophthalmic acid were rapidly removed from the blood. In perfusion studies with isolated liver, however, the compounds did not enter the liver tissue. Thus, uptake by this tissue is obviously not responsible for the removal of gamma-glutamyl tripeptides from the blood. Instead, rapid hydrolysis of the tripeptides was observed. The undegraded tripeptides were only detected in the blood immediately after administration. Within tissue the degradation product glycine accounted for all the radioactivity. After intravenous injection of the labelled tripeptides the radioactivity accumulated first in the kidney, as shown by autoradiographic studies and chemical analysis of different tissues. The hydrolysis of the gamma-glutamyl tripeptides decreased markedly after the renal arteries were clamped. These observations strongly suggest that renal tissue is the principal site of the degradation of the tripeptides. Inhibition studies and experiments with isolated renal tubules revealed that gamma-glutamyl transpeptidase catalyses the fast hydrolysis of the extracellular peptides. The results indicate that, when entering the extracellular space, glutathione and its analogues are completely hydrolysed and must be resynthesized after reuptake of the constituent amino acids. It is concluded that the degradation occurs mainly on the luminal surface of the renal brush-border membrane and that gamma-glutamyl transpeptidase is a glutathionase acting on extracellular glutathione.     

Novel patterns of gene expression in polyploid plants

Genome doubling, or polyploidy, is a major factor accounting for duplicate genes found in most eukaryotic genomes. Polyploidy has considerable effects on duplicate gene expression, including silencing and up- or downregulation of one of the duplicated genes. These changes can arise with the onset of polyploidization or within several generations after polyploid formation and they can have epigenetic causal factors. Many expression alterations are organ-specific. Specific genes can be independently and repeatedly silenced during polyploidization, whereas patterns for other genes appear to be more stochastic. Three recent reports have provided intriguing new insights into the patterns, timing and mechanisms of gene expression changes that accompany polyploidy in plants.