Construction of artificial cyclic amide amidohydrolases using molecular imprinting technique

A general molecular imprinting approach is proposed to synthesize artificial enzymes to mimic the family of cyclic amide amidohydrolases which share similar active site and catalytic mechanism. The artificial enzymes were constructed by co-polymerizing 4(5)-vinylimidazole-Co²⁺-methacrylic acid clusters with divinylbenzene micro-spheres in the presence of corresponding substrates. The artificial enzymes mimicked creatininase and hydantoinase by showing specific affinity towards the corresponding substrates in buffer. The artificial hydantoinase also showed specific affinity towards corresponding substrate in organic solvent, and catalyzed the hydrolysis of hydantoin.     

Conditions Favoring Differentiation and Stabilization of the Life Cycle of the Yeast <Emphasis Type="Italic">Pachysolen tannophilus</Emphasis>

Conditions favoring differentiation and stabilization of the life cycle of the yeast Pachysolen tannophilus have been studied. When concentrations of the carbon source in the medium were lower than 100 g/l, it was found to be favorable to the mating of vegetative cells, both haploid and diploid. The addition of nitrogen and sulfur sources to the medium influenced the life phases of haploid cells and partially stabilized the vegetative growth of diploid cells. Enrichment of the nutrient medium with potassium, vitamins, and microelements was shown to be necessary for the formation and maturation of conjugated ascospores. Microelements, vitamins, and phosphorus in excessive amounts activated conjugation but did not provide for the distinct phases of formation of unconjugated asci and spores in the diploid cells. Possible reasons for the unstable diplophase in the yeast P. tannophilus have been discussed.__________Translated from Mikrobiologiya, Vol. 74, No. 4, 2005, pp. 483–488.Original Russian Text Copyright © 2005 by Bolotnikova, Mikhailova, Shabalina, Bodunova, Ginak.     

Intergeneric somatic hybridization and its application to crop genetic improvement

Related or distant species of cultivated cs are a large pool of many desirable genes. Gene transfer from these species through conventional breeding is difficult owing to post- and pre-zygotic sexual incompatibilities. Somatic hybridization via protoplast fusion is a possible alternative for gene transfer from these species to cultivated crops. Since the early days of somatic hybridization many intergeneric somatic hybrids have been developed through symmetric fusion, asymmetric fusion and microfusion. Somatic hybrids are mainly selected by using markers such as specific media or fusion parents with special features, biochemical mutants, antibiotic resistance and complementation strategy. The hybridity of the regenerants is determined based on morphological, cytological and molecular analysis. The inheritance patterns of nuclear and cytoplasmic genomes in the somatic hybrids are diverse. Nuclear DNA from both fusion parents co-exists congruously in some hybrids with translocation and rearrangement of chromosomes, but spontaneous elimination of chromosomes from either or both fusion parents has been observed very often. In asymmetric fusion, chromosome elimination is an important issue that is a complicated process influenced by many factors, such as irradiation dose, phylogenetic relatedness, ploidy level of fusion parent and regenerants. As for chloroplast genome, uniparental segregation is mainly detected, though co-existence is also reported in some cases. The mitochondrial genome, in contrast to chloroplast, undergoes recombination and very frequent rearrangements. Somatic cell fusion has potential applications for crop genetic improvement by overcoming sexual incompatibility or reproductive barriers, and by realizing novel combinations of nuclear and/or cytoplasmic genomes.     

Indirect consequences of artificial selection on plasticity to light quality in Arabidopsis thaliana

Covariation between light quality- and photoperiod-mediated phenotypic plasticity was investigated using Arabidopsis thaliana. Three episodes of artificial selection were imposed on an index that quantified the plastic response to reduced red to far-red ratios (R:FR), with higher index values indicating greater plasticity. Relative to control lines, two high plasticity (HP) lines showed 1.6- and 2.4-fold increases in the index; low plasticity (LP) lines showed 1.4- and 1.1-fold decreases. A factorial experiment combining high and low R:FR conditions with long and short photoperiods assessed indirect consequences of selection on plasticity. Despite divergent R:FR-mediated plasticities in HP vs. LP lines, all four lines showed increases in photoperiod-mediated responses and decreases in mean leaf number. Complex relationships among trait means, plasticities and underlying mechanisms caution against generalizing about the genetic architecture of plastic traits. Partially independent developmental and evolutionary responses to R:FR and photoperiod are somewhat unsurprising, given this species' cosmopolitan nature.     

Comparing artificial and natural selection in rate of adaptation to genetic stress in Aspergillus nidulans

In an experimental study of adaptation to negative pleiotropic effects of a major fungicide resistance mutation in the filamentous fungus Aspergillus nidulans we have investigated the relative effectiveness of artificial selection vs. natural selection on the rate of compensatory evolution. Using mycelial growth rate as a fitness measure, artificial selection involved the weekly transfer of the fastest growing sector onto a fresh plate. Natural selection was approximated by transferring random samples of all the spores produced by the mycelium. Fungicide resistant and fungicide sensitive haploid and diploid strains were used in an evolution experiment over 10 weekly transfers, which is equivalent to 1200 cell cycles. Two different environmental conditions were applied: a constant fungicide-free environment and a weekly alternation between presence and absence of fungicide. Results show that for all strains and conditions used the transfer of a random sample of all spores leads to more rapid adaptation than the transfer of the visually 'fittest' sector. The rates of compensatory evolution in the constant and the alternating environment did not differ. Moreover, haploid strains tend to have a higher rate of adaptation than isogenic diploid strains.     

Male sterility at extreme temperatures: a significant but neglected phenomenon for understanding Drosophila climatic adaptations

The thermal range for viability is quite variable among Drosophila species and it has long been known that these variations are correlated with geographic distribution: temperate species are on average more cold tolerant but more heat sensitive than tropical species. At both ends of their viability range, sterile males have been observed in all species investigated so far. This symmetrical phenomenon restricts the temperature limits within which permanent cultures can be kept in the laboratory. Thermal heat sterility thresholds are very variable across species from 23 degrees C in heat sensitive species up to 31 degrees C in heat tolerant species. In Drosophila melanogaster, genetic variations are observed among geographic populations. Tropical populations are more tolerant to heat induced sterility and recover more rapidly than temperate ones. A genetic analysis revealed that about 50% of the difference observed between natural populations was due to the Y chromosome. Natural populations have not reached a selection limit, however: thermal tolerance was still increased by keeping strains at a high temperature, close to the sterility threshold. On the low temperature side, a symmetrical reverse phenomenon seems to exist: temperate populations are more tolerant to cold than tropical ones. Compared to Mammals, drosophilids exhibit two major differences: first, male sterility occurs not only at high temperature, but also at a low temperature; second, sterility thresholds are not evolutionarily constrained, but highly variable. Altogether, significant and sometimes major genetic variations have been observed between species, between geographic races of the same species, and even between strains kept in the laboratory under different thermal regimes. In each case, it is easily argued that the observed variations correspond to adaptations to climatic conditions, and that male sterility is a significant component of fitness and a target of natural selection.     

Experimental Visualization of Chromoneme as a Higher Level of Chromatin Compactization in the Mitotic Chromosome

We succeeded to visualize the chromoneme or a filamentous chromatin structure, with the mean thickness 0.1–0.2 μm, as a higher level of chromatin compactization in animal and plant cells at different stages of chromosome condensation at mitotic prophase and during chromatid decondensation at telophase. Under the natural conditions, chromoneme elements are not detected in the most condensed chromatin of metaphase chromosomes on ultrathin sections. We studied the ultrastructure and behavior of the chromatin of mitotic chromosomes in situ in cultured mouse L-197 cells under the conditions selectively demonstrating the chromoneme structure of the mitotic chromosomes in the presence of Ca²⁺. Loosely packaged dense chromatin bands, ca. 100 nm in diameter, chromonemes, were detected in chromosome arms in a solution containing 3 mM CaCl₂. When transferred in a hypotonic solution containing 10 mM tris-HCl, these chromosomes swelled, lost the chromoneme level of structure, and rapidly transformed in loose aggregates of elementary DNP fibrils, 30 nm in diameter. After this decondensation in the low ionic strength solution, the chromoneme structure of mitotic chromosomes was restored when they were transferred in a Ca₂₊ containing solution. The morphological characteristics of the chromoneme and pattern of its packaging in the chromosome were preserved. However, when the mitotic cells with chromosomes, in which the chromoneme structure was visualized with the help of 3 mM CaCl₂, were treated with a photosensitizer, ethidium bromide, and illuminate with a light with the wavelength 460 nm, chromatic decondensation under the hypotonic solution was not observed. The chromoneme elements in a stabilized chromatin of the mitotic chromosome preserved specific interconnection and the general pattern of their packaging in the chromatid was also preserved. The chromoneme elements in the chromosomes stabilized by light preserved their density and diameter even in a 0.6 M NaCl solution, which normally leads to chromoneme destruction. An even more rigid treatment of the stabilized chromosomes with a 2 M NaCl solution, which normally fully decondenses the chromosomes, made it possible to detect a 3D reticular skeleton devoid of any axial structures. __________ Translated from Ontogenez, Vol. 36, No. 5, 2005, pp. 323–332. Original Russian Text Copyright ? 2005 by Burakov, Tvorogova, Chentsov.     

Design of improved membrane protein production experiments: quantitation of the host response

Eukaryotic membrane proteins cannot be produced in a reliable manner for structural analysis. Consequently, researchers still rely on trial-and-error approaches, which most often yield insufficient amounts. This means that membrane protein production is recognized by biologists as the primary bottleneck in contemporary structural genomics programs. Here, we describe a study to examine the reasons for successes and failures in recombinant membrane protein production in yeast, at the level of the host cell, by systematically quantifying cultures in high-performance bioreactors under tightly-defined growth regimes. Our data show that the most rapid growth conditions of those chosen are not the optimal production conditions. Furthermore, the growth phase at which the cells are harvested is critical: We show that it is crucial to grow cells under tightly-controlled conditions and to harvest them prior to glucose exhaustion, just before the diauxic shift. The differences in membrane protein yields that we observe under different culture conditions are not reflected in corresponding changes in mRNA levels of FPS1, but rather can be related to the differential expression of genes involved in membrane protein secretion and yeast cellular physiology.     

Identifying natural substrates for chaperonins using a sequence-based approach

The Escherichia coli chaperonin machinery, GroEL, assists the folding of a number of proteins. We describe a sequence-based approach to identify the natural substrate proteins (SPs) for GroEL. Our method is based on the hypothesis that natural SPs are those that contain patterns of residues similar to those found in either GroES mobile loop and/or strongly binding peptide in complex with GroEL. The method is validated by comparing the predicted results with experimentally determined natural SPs for GroEL. We have searched for such patterns in five genomes. In the E. coli genome, we identify 1422 (about one-third) sequences that are putative natural SPs. In Saccharomyces cerevisiae, 2885 (32%) of sequences can be natural substrates for Hsp60, which is the analog of GroEL. The precise number of natural SPs is shown to be a function of the number of contacts an SP makes with the apical domain (N(C)) and the number of binding sites (N(B)) in the oligomer with which it interacts. For known SPs for GroEL, we find approximately 4 < N(C) < 5 and 2 相似文献