Ornithine aminotransferase and 4-aminobutyrate aminotransferase are related pyridoxal phosphate-dependent enzymes having different substrate specificities. The atomic structures of these enzymes have shown (i) that active site differences are limited to the steric positions occupied by two tyrosine residues in ornithine aminotransferase and (ii) that, uniquely among related, structurally characterized aminotransferases, the conserved arginine that binds the ␣-carboxylate of ␣-amino acids interacts tightly with a glutamate residue. To determine the contribution of these residues to the specificities of the enzymes, we analyzed site-directed mutants of ornithine aminotransferase by rapid reaction kinetics, x-ray crystallography, and 13 C NMR spectroscopy. Mutation of one tyrosine (Tyr-85) to isoleucine, as found in aminobutyrate aminotransferase, decreased the rate of the reaction of the enzyme with ornithine 1000-fold and increased that with 4-aminobutyrate 16-fold, indicating that Tyr-85 is a major determinant of specificity toward ornithine. Unexpectedly, the limiting rate of the second half of the reaction, conversion of ketoglutarate to glutamate, was greatly increased, although the kinetics of the reverse reaction were unaffected. A mutant in which the glutamate (Glu-235) that interacts with the conserved arginine was replaced by alanine retained its regiospecificity for the ␦-amino group of ornithine, but the glutamate reaction was enhanced 650-fold, whereas only a 5-fold enhancement of the ketoglutarate reaction rate resulted. A model is proposed in which conversion of the enzyme to its pyridoxamine phosphate form disrupts the internal glutamate-arginine interaction, thus enabling ketoglutarate but not glutamate to be a good substrate.Ornithine aminotransferase (Orn-AT) 3 and 4-aminobutyrate aminotransferase (GABA-AT) are members of a large family of pyridoxal 5Ј-phosphate (PLP)-dependent enzymes that catalyze a wide range of reactions on amino acids (1). Each enzyme operates by a mechanism, common to all aminotransferases ( Fig. 1), in which the cofactor shuttles between pyridoxaldimine and pyridoxamine forms by means of two coupled half-reactions (2, 3). The half-reaction converting ketoglutarate to glutamate is the same for both enzymes as well as for the majority of other aminotransferases. In this half-reaction, the chemical changes occur at the ␣-carbon. The substrate specificity of the enzymes thus arises from the other half-reaction that transfers an amino group distant from the ␣-carbon. For this reason, the enzymes are known as "-aminotransferases" (4). In the case of GABA-AT, the amino group transferred is the only amino group in the substrate molecule, whereas Orn-AT specifically selects the -amino group of ornithine despite the presence of a second amino group on C␣ with the same configuration as that in glutamate (3).The three-dimensional structures of both enzymes have been solved in the unliganded form as well as in complex with various substrate analogues (5-7). The enzymes have the same b...
Holocentric chromosomes possess multiple kinetochores along their length rather than the single centromere typical of other chromosomes [1]. They have been described for the first time in cytogenetic experiments dating from 1935 and, since this first observation, the term holocentric chromosome has referred to chromosomes that: i. lack the primary constriction corresponding to centromere observed in monocentric chromosomes [2]; ii. possess multiple kinetochores dispersed along the chromosomal axis so that microtubules bind to chromosomes along their entire length and move broadside to the pole from the metaphase plate [3]. These chromosomes are also termed holokinetic, because, during cell division, chromatids move apart in parallel and do not form the classical V-shaped figures typical of monocentric chromosomes [4][5][6]. Holocentric chromosomes evolved several times during both animal and plant evolution and are currently reported in about eight hundred diverse species, including plants, insects, arachnids and nematodes [7,8]. As a consequence of their diffuse kinetochores, holocentric chromosomes may stabilize chromosomal fragments favouring karyotype rearrangements [9,10]. However, holocentric chromosome may also present limitations to crossing over causing a restriction of the number of chiasma in bivalents [11] and may cause a restructuring of meiotic divisions resulting in an inverted meiosis [12]. Evolution and structure of holocentric chromosomes Evolution of holocentric chromosomesHolocentric chromosomes were described for the first time in 1935 to identify chromosomes with a diffuse kinetochore (or with a diffuse kinetochore activity) making these chromosomes able to bind to microtubules along their entire length. In the last decades, several studies assessed that the same behaviour during mitosis can be observed not only for holocentric/ holokinetic chromosomes, but also for polykinetic chromosomes that contain numerous (but discrete) microtubule-binding sites, but the term "holocentric/holokinetic" is still used for both [1,5,7].Before molecular methods became available, the presence of holocentric chromosomes was evaluated mostly using cytology and, considering that many species are difficult to study cytologically, it can be surmised that the true presence of holocentrism may be underestimated. In addition, there are several taxa, whose chromosomes are still uncharacterized, but their
This review summarizes conventional and recent applications of genomic in situ hybridization (GISH). GISH is a well recognized technique, but its modifications and applications have not been widely used. Here, we show how modifications to the GISH technique can be used as tools to ‘paint’ plant chromosomes. In addition, we describe novel applications, e.g. how GISH banding could be used for karyotyping plant chromosomes. We further discuss recent phylogenetic applications of GISH that allow a semiquantitative signal analysis and the possibility of comparing and combining this cytogenetic technique with DNA sequence-based phylogenetic trees.
The dioecious plant species Silene latifolia has a sex determination mechanism based on an active Y chromosome. Here, we used inter-specific hybrids in the genus Silene to study the effects of gene complexes on the Y chromosome. If the function of Y-linked genes has been maintained in the same state as in the hermaphrodite progenitor species, it should be possible to substitute such genes by genes coming from a related hermaphrodite species. In the inter-specific hybrid, S. latifolia x S. viscosa, anthers indeed develop far beyond the early bilobal stage characteristic of XX S. latifolia female plants. The S. viscosa genome can thus replace the key sex determination gene whose absence abolishes early stamen development in females (loss of the stamen-promoting function, SPF), so that hybrid plants are morphologically hermaphrodite. However, the hybrids have two anther development defects, loss of adhesion of the tapetum to the endothecium, and precocious endothecium maturation. Both these defects were also found in independent Y-chromosome deletion mutants of S. latifolia. The data support the hypothesis that the evolution of complete gender dimorphism from hermaphroditism involved a major largely recessive male-sterility factor that created females, and the appearance of new, dominant genes on the Y chromosome, including both the well-documented gynoecium-suppressing factor, and two other Y specific genes promoting anther development.
Summary• Stabilizing selection is a key evolutionary mechanism for which there is relatively little experimental evidence. To date, stabilizing selection has never been observed at the whole-genome level.• We tested the effect of selection on genome size in a field experiment using seeds collected in a population of Festuca pallens with a highly variable genome size. Using flow cytometry, we measured the genome size in germinating seedlings and juvenile plants grown with or without high intraspecific competition (908 individuals). Above-ground biomass and leaf number were used as measurements of individual vegetative performance. The possible confounding effect of seed weight was controlled for in a separate experiment.• Growth under high competition had a significant stabilizing effect on genome size. Because no relationship was observed between genome size and vegetative performance, we assume that the elimination of plants with extreme genome sizes was the result of decreased survival as a consequence of some unrecognized stress.• Our results indicate that genome size may be under direct selection. The equal disadvantaging of either large or small genomes indicates that the selection for optimum genome size in species may be fully context dependent. This study demonstrates the power of competition experiments for the detection of weak selection processes.
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