Hybridization between diploid Centaurea pseudophrygia and tetraploid C. jacea (Asteraceae): the role of mixed pollination,unreduced gametes,and mentor effects

We studied hybridization between the diploid Centaurea pseudophrygia and the tetraploid C. jacea by performing crossing experiments and screening natural populations using flow cytometry. The experiments confirm that the studied species exhibit strong reproductive isolation. Interspecific hybrids were formed at a low frequency, including triploids (originating from reduced gametes) and tetraploids (involving unreduced gametes of the diploids). In contrast, hybrids were almost absent among seeds and adult plants of natural mixed populations and among the offspring from experimental pollinations with a mixture of pollen of both ploidy levels. We found that mixed pollination is an important mechanism for preventing hybridization between plants of different ploidy levels and sustaining the reproduction of the tetraploids. A mentor effect (induced selfing in the presence of pollen of different ploidy levels) was observed in both diploids and tetraploids, reinforcing the reproductive isolation between cytotypes. Higher ploidy levels (pentaploid, hexaploid) involving unreduced gametes of the tetraploid species were identified. Notably, pentaploids were discovered for the first time in Centaurea sect. Jacea. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 104 , 93–106.     

马卵母细胞体外成熟的初步研究

采用抽吸法和切割法两种采卵方法收集马卵母细胞,显示采用切割法的卵母细胞回收率为83%,高于抽吸法的回收率46.5%(P＜0.05),在卵母细胞的成熟上,使用M199和DMEM/F12两种培养液为基础液的成熟体系,紧凑型(Cp)COCs和扩展型(Ex)COCs在以M199为基础液和以DMEM/F12为基础液的培养液中的卵母细胞成熟率分别为41.7%和64.7%、46.7%和66.7%,差异不显著(P＞0.05).两种培养体系中成熟的Cp COCs采用Ionomycin与6-DMAP和CHX联合激活,在以M199和以DMEM/F12为基础液的培养液中成熟的卵母细胞的卵裂率分别为45%和57.1%,差异不明显(P＞0.05).     

FATE MAP OF THE ORGANIZING SHOOT APEX IN GOSSYPIUM

Chimeral seedlings from a “semigametic” strain of cotton were used as the basis for a clonal analysis of events in the progressive organization of the nascent shoot apex. At the time the proembryo becomes a globular stage embryo with a distinct dermatogen, the surface of the embryo contains an eight-celled compartment for each cotyledon, a two-celled compartment for the first leaf, a one-celled compartment for the second leaf, and a three-celled compartment for the apical initials and all subsequent leaves and aerial structures. The developmental history of the first two leaves differs in a fundamental way from that of all the other leaves.     

CHARACTERIZATION OF THE NUCLEAR DNA OF HORDEUM VULGARE ROOT HAIRS: AMPLIFICATION DISAPPEARS UNDER SALT STRESS

In Hordeum vulgare L., the nucleus of differentiating root hairs contains amplified, extrachromosomal DNA sequences. Cytophotometry shows that the nuclei of trichoblasts and root hairs grown under normal conditions contain up to 50% more DNA than those grown in 200 mM salt. Although the root hairs develop and differentiate under salt stress, amplification of their nuclear DNA is suppressed. From this, we conclude that amplification is not necessary for differentiation at the cellular level. Characterization of the amplified nuclear DNA of the root hair is based on the physical/chemical nature of the DNA. The amplified sequences separate as a satellite band when total nucleic acids are centrifuged on CsCl gradients. Enzyme restriction of the satellite and main bands with Msp I and Hpa II followed by agarose gel electrophoresis shows that the satellite band is not more highly methylated than the main band. Restriction of the root hair DNA with EcoRI reveals repetitive DNA sequences not seen in similarly restricted whole root, leaf or salt-stressed root hair preparations. While these unique, repetitive sequences in the 2–6 kb region of the gel do not hybridize with ribosomal, chloroplast, or mitochondrial DNAs, RNA hybridization shows that some of them are transcribed. We believe that the amplified sequences are extrachromosomal based on their selective degradation during root hair senescence, their separation as a satellite band and their restriction patterns.     

The structure of L-tyrosine 2,3-aminomutase from the C-1027 enediyne antitumor antibiotic biosynthetic pathway

The SgcC4 l-tyrosine 2,3-aminomutase (SgTAM) catalyzes the formation of (S)-beta-tyrosine in the biosynthetic pathway of the enediyne antitumor antibiotic C-1027. SgTAM is homologous to the histidine ammonia lyase family of enzymes whose activity is dependent on the methylideneimidazole-5-one (MIO) cofactor. Unlike the lyase enzymes, SgTAM catalyzes additional chemical transformations resulting in an overall stereospecific 1,2-amino shift in the substrate l-tyrosine to generate (S)-beta-tyrosine. Previously, we provided kinetic, spectroscopic, and mutagenesis data supporting the presence of MIO in the active site of SgTAM [Christenson, S. D.; Wu, W.; Spies, A.; Shen, B.; and Toney, M. D. (2003) Biochemistry 42, 12708-12718]. Here we report the first X-ray crystal structure of an MIO-containing aminomutase, SgTAM, and confirm the structural homology of SgTAM to ammonia lyases. Comparison of the structure of SgTAM to the l-tyrosine ammonia lyase from Rhodobacter sphaeroides provides insight into the structural basis for aminomutase activity. The results show that SgTAM has a closed active site well suited to retain ammonia and minimize the formation of lyase elimination products. The amino acid determinants for substrate recognition and catalysis can be predicted from the structure, setting the framework for detailed mechanistic investigations.     

X-ray crystal structure of aristolochene synthase from Aspergillus terreus and evolution of templates for the cyclization of farnesyl diphosphate

Aristolochene synthase from Aspergillus terreus catalyzes the cyclization of the universal sesquiterpene precursor, farnesyl diphosphate, to form the bicyclic hydrocarbon aristolochene. The 2.2 A resolution X-ray crystal structure of aristolochene synthase reveals a tetrameric quaternary structure in which each subunit adopts the alpha-helical class I terpene synthase fold with the active site in the "open", solvent-exposed conformation. Intriguingly, the 2.15 A resolution crystal structure of the complex with Mg2+3-pyrophosphate reveals ligand binding only to tetramer subunit D, which is stabilized in the "closed" conformation required for catalysis. Tetramer assembly may hinder conformational changes required for the transition from the inactive open conformation to the active closed conformation, thereby accounting for the attenuation of catalytic activity with an increase in enzyme concentration. In both conformations, but especially in the closed conformation, the active site contour is highly complementary in shape to that of aristolochene, and a catalytic function is proposed for the pyrophosphate anion based on its orientation with regard to the presumed binding mode of aristolochene. A similar active site contour is conserved in aristolochene synthase from Penicillium roqueforti despite the substantial divergent evolution of these two enzymes, while strikingly different active site contours are found in the sesquiterpene cyclases 5-epi-aristolochene synthase and trichodiene synthase. Thus, the terpenoid cyclase active site plays a critical role as a template in binding the flexible polyisoprenoid substrate in the proper conformation for catalysis. Across the greater family of terpenoid cyclases, this template is highly evolvable within a conserved alpha-helical fold for the synthesis of terpene natural products of diverse structure and stereochemistry.     

Stress-triggered Activation of the Metalloprotease Oma1 Involves Its C-terminal Region and Is Important for Mitochondrial Stress Protection in Yeast

Functional integrity of mitochondria is critical for optimal cellular physiology. A suite of conserved mitochondrial proteases known as intramitochondrial quality control represents one of the mechanisms assuring normal mitochondrial function. We previously demonstrated that ATP-independent metalloprotease Oma1 mediates degradation of hypohemylated Cox1 subunit of cytochrome c oxidase and is active in cytochrome c oxidase-deficient mitochondria. Here we show that Oma1 is important for adaptive responses to various homeostatic insults and preservation of normal mitochondrial function under damage-eliciting conditions. Changes in membrane potential, oxidative stress, or chronic hyperpolarization lead to increased Oma1-mediated proteolysis. The stress-triggered induction of Oma1 proteolytic activity appears to be associated with conformational changes within the Oma1 homo-oligomeric complex, and these alterations likely involve C-terminal residues of the protease. Substitutions in the conserved C-terminal region of Oma1 impair its ability to form a labile proteolytically active complex in response to stress stimuli. We demonstrate that Oma1 genetically interacts with other inner membrane-bound quality control proteases. These findings indicate that yeast Oma1 is an important player in IM protein homeostasis and integrity by acting in concert with other intramitochondrial quality control components.     

Conversion of human steroid 5β-reductase (AKR1D1) into 3β-hydroxysteroid dehydrogenase by single point mutation E120H: example of perfect enzyme engineering

Human aldo-keto reductase 1D1 (AKR1D1) and AKR1C enzymes are essential for bile acid biosynthesis and steroid hormone metabolism. AKR1D1 catalyzes the 5β-reduction of Δ(4)-3-ketosteroids, whereas AKR1C enzymes are hydroxysteroid dehydrogenases (HSDs). These enzymes share high sequence identity and catalyze 4-pro-(R)-hydride transfer from NADPH to an electrophilic carbon but differ in that one residue in the conserved AKR catalytic tetrad, His(120) (AKR1D1 numbering), is substituted by a glutamate in AKR1D1. We find that the AKR1D1 E120H mutant abolishes 5β-reductase activity and introduces HSD activity. However, the E120H mutant unexpectedly favors dihydrosteroids with the 5α-configuration and, unlike most of the AKR1C enzymes, shows a dominant stereochemical preference to act as a 3β-HSD as opposed to a 3α-HSD. The catalytic efficiency achieved for 3β-HSD activity is higher than that observed for any AKR to date. High resolution crystal structures of the E120H mutant in complex with epiandrosterone, 5β-dihydrotestosterone, and Δ(4)-androstene-3,17-dione elucidated the structural basis for this functional change. The glutamate-histidine substitution prevents a 3-ketosteroid from penetrating the active site so that hydride transfer is directed toward the C3 carbonyl group rather than the Δ(4)-double bond and confers 3β-HSD activity on the 5β-reductase. Structures indicate that stereospecificity of HSD activity is achieved because the steroid flips over to present its α-face to the A-face of NADPH. This is in contrast to the AKR1C enzymes, which can invert stereochemistry when the steroid swings across the binding pocket. These studies show how a single point mutation in AKR1D1 can introduce HSD activity with unexpected configurational and stereochemical preference.     

Structural and mechanistic analysis of trichodiene synthase using site-directed mutagenesis: probing the catalytic function of tyrosine-295 and the asparagine-225/serine-229/glutamate-233-Mg2+B motif

Trichodiene synthase from Fusarium sporotrichioides contains two metal ion-binding motifs required for the cyclization of farnesyl diphosphate: the "aspartate-rich" motif D(100)DXX(D/E) that coordinates to Mg2+A and Mg2+C, and the "NSE/DTE" motif N(225)DXXSXXXE that chelates Mg2+B (boldface indicates metal ion ligands). Here, we report steady-state kinetic parameters, product array analyses, and X-ray crystal structures of trichodiene synthase mutants in which the fungal NSE motif is progressively converted into a plant-like DDXXTXXXE motif, resulting in a degradation in both steady-state kinetic parameters and product specificity. Each catalytically active mutant generates a different distribution of sesquiterpene products, and three newly detected sesquiterpenes are identified. In addition, the kinetic and structural properties of the Y295F mutant of trichodiene synthase were found to be similar to those of the wild-type enzyme, thereby ruling out a proposed role for Y295 in catalysis.