Ethanol-hypersensitive and ethanol-dependent cdc − mutants in Schizosaccharomyces pombe

Ethanol-hypersensitive strains (ets mutants), unable to grow on media containing 6% ethanol, were isolated from a sample of mutagenized Schizosaccharomyces pombe wild-type cells. Genetic analysis of these ets strains demonstrated that the ets phenotype is associated with mutations in a large set of genes, including cell division cycle (cdc) genes, largely non-overlapping with the set represented by the temperature conditional method; accordingly, we isolated some ets non-ts cdc ^? mutants, which may identify novel essential genes required for regulation of the S. pombe cell cycle. Conversely, seven well characterized ts cdc ^? mutants were tested for their ethanol sensitivity; among them, cdc1–7 and cdc13–117 exhibited a tight ets phenotype. Ethanol sensitivity was also tested in strains bearing different alleles of the cdc2 gene, and we found that some of them were ets, but others were non-ets; thus, ethanol hypersensitivity is an allele-specific phenotype. Based on the single base changes found in each particular allele of the cdc2 gene, it is shown that a single amino acid substitution in the p34^cdc2 gene product can produce this ets phenotype, and that ethanol hypersensitivity is probably due to the influence of this alcohol on the secondary and/or tertiary structure of the target protein. Ethanol-dependent (etd) mutants were also identified as mutants that can only be propagated on ethanol-containing media. This novel type of conditional phenotype also covers many unrelated genes. One of these etd mutants, etd1-1, was further characterized because of the lethal cdc ^? phenotype of the mutant cells under restrictive conditions (absence of ethanol). The isolation of extragenic suppressors of etd1-1, and the complementation cloning of a DNA fragment encompassing the etd1 ⁺ wild-type gene (or an extragenic multicopy suppressor) demonstrate that current genetic techniques may be applied to mutants isolated by using ethanol as a selective agent.     

Does a cdc2 Kinase-Like Recognition Motif on the Core Protein of Hepadnaviruses Regulate Assembly and Disintegration of Capsids?

Hepadnaviruses are enveloped viruses, each with a DNA genome packaged in an icosahedral nucleocapsid, which is the site of viral DNA synthesis. In the presence of envelope proteins, DNA-containing nucleocapsids are assembled into virions and secreted, but in the absence of these proteins, nucleocapsids deliver viral DNA into the cell nucleus. Presumably, this step is identical to the delivery of viral DNA during the initiation of an infection. Unfortunately, the mechanisms triggering the disintegration of subviral core particles and delivery of viral DNA into the nucleus are not yet understood. We now report the identification of a sequence motif resembling a serine- or threonine-proline kinase recognition site in the core protein at a location that is required for the assembly of core polypeptides into capsids. Using duck hepatitis B virus, we demonstrated that mutations at this sequence motif can have profound consequences for RNA packaging, DNA replication, and core protein stability. Furthermore, we found a mutant with a conditional phenotype that depended on the cell type used for virus replication. Our results support the hypothesis predicting that this motif plays a role in assembly and disassembly of viral capsids.     

p34cdc2: the S and M kinase?

In the yeast cell cycle, the induction of two very different processes, DNA synthesis (S-phase) and mitosis (M-phase), requires the same serine/threonine-specific protein kinase p34cdc2, which has been highly conserved through evolution. On the basis of work conducted largely in multicellular eukaryotes, it has recently been suggested that p34cdc2 is able to perform these two mutually exclusive roles by phosphorylating different sets of substrates through a cell cycle-dependent association with other proteins that dictate the substrate specificity of the protein kinase. To recognize its mitotic substrates, p34cdc2 associates with one of the cyclins--a family of proteins of two distinct but related types (A and B) characterized by their periodic destruction at each mitosis. In interphase, the formation of a complex between p34cdc2 and another protein (or proteins) would allow the phosphorylation of a different set of proteins involved in the G1 to S transition. This review focuses on the evidence for this appealing simple model and the nature of the putative substrates proposed.     

The Schizosaccharomyces pombe cdc6 gene encodes the catalytic subunit of DNA polymerase δ

The cdc6 mutants of Schizosaccharomyces pombe have been classified as being defective in progression through the G2 phase of the cell cycle. We cloned an S. pombe gene that could complement the temperature-sensitive growth of the cdc6-23 mutant. Unexpectedly, the cloned gene was allelic to pol3, which encodes the catalytic subunit of DNA polymerase δ. Integration mapping confirmed that cdc6 and pol3 are identical. The cdc6-23 mutant carries one amino acid substitution in the conserved N3 region of Pol3.     

小鼠受精卵早期发育过程中PKC对cdc2和cdc25C活性的影响

为研究小鼠受精卵细胞早期发育过程中PKC对cdc2和cdc2 5C活性的影响 ,采用免疫印迹和电泳迁移率差异分析的方法 ,观察PKC的激活剂TPA及其抑制剂星形孢子素对小鼠受精卵一细胞期cdc2和cdc2 5C活性的影响 .10nmol L的TPA作用 10min后 ,小鼠受精卵一细胞期卵裂率明显大于对照组 (P <0 0 5 ) ,而星形孢子素作用后卵裂率显著下降 (P <0 0 1) .TPA处理后 ,受精卵中呈去磷酸化状态的活性cdc2明显增加 ,没有活性呈磷酸化状态的cdc2 5C明显减少 ;而星形孢子素处理的受精卵中没有活性的cdc2明显增加 ,有活性的cdc2 5C明显减少 .结果表明 ,TPA短时间作用可以促进小鼠一细胞期受精卵分裂 ,星形孢子素抑制受精卵的分裂 ;TPA可以促进cdc2的去磷酸化以及cdc2 5C的磷酸化 ,从而促进G2 M转换 ,星形孢子素则抑制cdc2和cdc2 5C的活性 ,阻止受精卵由G2 期进入M期     

Synthesis and Cytotoxicity of О- and N-Acyl Derivatives of Azepanobetulin

Russian Journal of Bioorganic Chemistry - A five-stage synthesis of azepanobetulin from betulin with a total yield of 47% has been carried out. The acylation of azepanobetulin with anhydrides or...     

Cloning and sequencing of inulinase and β-fructofuranosidase genes of a deep-sea Microbulbifer species and properties of recombinant enzymes

An inulinase-producing Microbulbifer sp. strain, JAM-3301, was isolated from a deep-sea sediment. An inulin operon that contained three open reading frames was cloned and sequenced. Two of the three genes were expressed. One product was an endo-inulinase, and the other was a β-fructofuranosidase. Both enzymes worked together to effectively degrade inulin.     

Identification and Characterisation of the α and β Subunits of Succinyl CoA Ligase of Tomato

Despite the central importance of the TCA cycle in plant metabolism not all of the genes encoding its constituent enzymes have been functionally identified. In yeast, the heterodimeric protein succinyl CoA ligase is encoded for by two single-copy genes. Here we report the isolation of two tomato cDNAs coding for α- and one coding for the β-subunit of succinyl CoA ligase. These three cDNAs were used to complement the respective Saccharomyces cerevisiae mutants deficient in the α- and β-subunit, demonstrating that they encode functionally active polypeptides. The genes encoding for the subunits were expressed in all tissues, but most strongly in floral and leaf tissues, with equivalent expression of the two α-subunit genes being expressed to equivalent levels in all tissues. In all instances GFP fusion expression studies confirmed an expected mitochondrial location of the proteins encoded. Following the development of a novel assay to measure succinyl CoA ligase activity, in the direction of succinate formation, the evaluation of the maximal catalytic activities of the enzyme in a range of tissues revealed that these paralleled those of mRNA levels. We also utilized this assay to perform a preliminary characterisation of the regulatory properties of the enzyme suggesting allosteric control of this enzyme which may regulate flux through the TCA cycle in a manner consistent with its position therein.     

Preparation and Characterization of Oligonucleotides of D- and L-2’ Deoxyuridine

Abstract

The synthesis of oligonucleotides of 2'deoxyuridine containing both the natural D-2'deoxyribose and the unnatural L-2'deoxyribose is described. Units up to the 18-mer have been made via a modified triester procedure and characterized by HPLC.     

Rate of utilization of glucose and `compartmentation'' of α-oxoglutarate and glutamate in rat brain

1. The rate of incorporation of ¹⁴C into pyruvate, α-oxoglutarate, lactate and glucose of rat tissues was measured after the subcutaneous injection of uniformly labelled glucose. 2. In rat brain the specific radioactivities of lactate and glucose were similar to that of alanine. In liver the specific radioactivity of glucose was considerably higher than that of lactate or alanine. 3. The specific radioactivities of α-oxo acids of rat brain were lower than those of corresponding amino acids, alanine and glutamate. These findings have been explained in relation to metabolic compartments in vivo. 4. The approximate estimated rate of glucose utilization in rat brain in vivo is 0·96μmole/g. of brain/min.     

裂殖壶菌EST文库cdc2基因的筛选与分析

利用已建立的裂殖壹菌(Schizochytrium sp FJU-512)EST文库,通过序列比对分析发现p34cdc2相似基因的3′端序列,分离含该EST序列的质粒ROCHA2004,以T7引物进行反向测序,获得p34cdc2相似基因的全长序列,结合NCBI的ORF finder以及Blastx程序获得p34cdc2蛋白的氨基酸序列;利用同源模建方法构建了该蛋白的三维空间结构,并预测其功能结构域;对该蛋白进行系统发育分析,构建进化树,结果显示该蛋白同小麦(Triticum aestivum)的p34cdc2序列亲缘关系最近。     

分裂中期促进因子和cdc蛋白调控着细胞有丝分裂

当细胞进入M相,细胞核和细胞质的结构发生着戏剧性重组。一般来说,有三条途径可供研究这类重组的调控机制;其一是选用细胞分裂周期有缺陷的突变种(cdc突变种)进行遗传分析;其二是挑选活性随细胞周期波动的蛋白激酶和其它酶进行生化分析;其三对分裂细胞中的有丝分裂诱导物进行生物活性测试。研究的结果均表明,所有的真核细胞,由G_2相转入M相的调控机制相似。     

Summer-autumn ichthyoplankton of the Sea of Okhotsk and the Sea of Japan and special traits of feeding of fish larvae and fry in 2003–2004

In summer-autumn of 2003–2004, the ichthyoplankton of the Sea of Okhotsk comprised 35 species. In this period the most widely distributed and numerous were larvae of the lord Hemilepidotus gilberti, the Pacific stout sand lance Ammodytes hexapterus, and the Sakhalin dab Limanda sakhalinensis. The maximum catches of fish larvae were attributed to coastal waters off eastern Sakhalin and to the shelf of the northern part of the Sea of Okhotsk. In November of 2003, the ichthyoplankton of the Sea of Japan was represented by fish larvae belonging mainly to the boreal ichthyocomplex. The catches consisted predominantly of larvae of the arabesque greenling Pleurogrammus azonus, the ronquil Bathymaster derjugini, and the rockfish Sebastes owstoni. Fish larvae and fry in the northwestern part of the Sea of Japan were caught principally within 43°–45° N and 137°–139° E above the depth 1500–2000 m. The food spectrum of fish larvae in the Sea of Okhotsk and the Sea of Japan comprised over 20 plankters of various size belonging to seven taxa. Irrespective of fish species, the food items common of all fish were copepods Pseudocalanus minutus and Oithona similis. The daily rations were calculated for mass species (Hemilepidotus gilberti, Ammodytes hexapterus, Hexagrammos stelleri, Pleurogrammus azonus, Bathymaster derjugini, and Sebastes owstoni). The larvae of all considered species in the Sea of Japan and in the Sea of Okhotsk fed predominantly in the light period of the day.     

α-Lactalbumin binding and membrane integrity—effect of charge and degree of unsaturation of glycerophospholipids

Several studies have shown that the physical state of the phospholipid membrane has an important role in protein-membrane interactions, involving both electrostatic and hydrophobic forces. We have investigated the influence of the interaction of the calcium-depleted, (apo)-conformation of bovine α-lactalbumin (BLA) on the integrity of anionic glycerophospholipid vesicles by leakage experiments using fluorescence spectroscopy. The stability of the membranes was also studied by measuring surface tension/molecular area relationships with phospholipid monolayers. We show that the degree of unsaturation of the acyl chains and the proportion of charged phospholipid species in the membranes made of neutral and acidic glycerophospholipids are determinants for the association of BLA with liposomes and for the impermeability of the bilayer. Particularly, tighter packing counteracted interaction with BLA, while unsaturation—leading to looser packing—promoted interaction and leakage of contents. Equimolar mixtures of neutral and acidic glycerophospholipids were more permeable upon protein binding than pure acidic lipids. The effect of lipid structure on BLA-membrane interaction and bilayer integrity may throw new light on the membrane disrupting mechanism of a conformer of human α-lactalbumin (HAMLET) that induces death of tumour cells but not of normal cells.     

Hydrophilicity and antigenicity of proteins — A case study of myoglobin and hemoglobin

Hydrophilicity index is used to locate antigenic determinants on two related groups of proteins-myoglobin and hemoglobin. The data on 41 species (including 34 mammals) of myoglobin show that average hydrophilicity for the complete myoglobin molecules as well as the average hydrophilicity for all hydrophilic regions put together seem to remain constant; the variation in the size and location of the antigenic determinants in these species is very small indicating that the antigenic sites are not shifted during evolution. In the case of both the proteins there is a good agreement between the antigenic sites picked up by using hydrophilicity index and the experimentally determined antigenic sites. The data on 56 species of hemoglobin α-chains and 44 species of hemoglobinβ-chains showed that although there are few sites on hemoglobin which have remained invariant during evolution, there is a significant variation in other sites in terms of either a splitting of a site, or a drastic change in the hydrophilicity values and/or a length of the site. Comparison of the hydrophilicity data on these two groups of proteins suggests that hemoglobins which perform a variety of functions as compared to myoglobins are evolving faster than myoglobins supporting the contention of earlier workers.     

Analysis and modeling of time-variant amplitude–frequency couplings of and between oscillations of EEG bursts

Low-frequency (0.5-2.5 Hz) and individually defined high-frequency (7-11 or 8-12 Hz; 11-15 or 14-18 Hz) oscillatory components of the electroencephalogram (EEG) burst activity derived from thiopental-induced burst-suppression patterns (BSP) were investigated in seven sedated patients (17-26 years old) with severe head injury. The predominant high-frequency burst oscillations (>7 Hz) were detected for each patient by means of time-variant amplitude spectrum analysis. Thereafter, the instantaneous envelope (IE) and the instantaneous frequency (IF) were computed for these low- and high-frequency bands to quantify amplitude-frequency dependencies (envelope-envelope, envelope-frequency, and frequency-frequency correlations). Time-variant phase-locking, phase synchronization, and quadratic phase couplings are associated with the observed amplitude-frequency characteristics. Additionally, these time-variant analyses were carried out for modeled burst patterns. Coupled Duffing oscillators were adapted to each EEG burst and by means of these models data-based burst simulations were generated. Results are: (1) strong envelope-envelope correlations (IE courses) can be demonstrated; (2) it can be shown that a rise of the IE is associated with an increase of the IF (only for the frequency bands 0.5-2.5 and 7-11 or 8-12 Hz); (3) the rise characteristics of all individually averaged envelope-frequency courses (IE-IF) are strongly correlated; (4) for the 7-11 or 8-12 Hz oscillation these associations are weaker and the variation between the time courses of the patients is higher; (5) for both frequency ranges a quantitative amplitude-frequency dependency can be shown because higher IE peak maxima are accompanied by stronger IF changes; (6) the time range of significant phase-locking within the 7-11 or 8-12 Hz frequency bands and of the strongest quadratic phase couplings (between 0.5-2.5 and 7-11 or 8-12 Hz) is between 0 and 1,000 ms; (7) all phase coupling characteristics of the modeled bursts accord well with the corresponding characteristics of the measured EEG burst data. All amplitude-frequency dependencies and phase locking/coupling properties described here are known from and can be discussed using coupled Duffing oscillators which are characterized by autoresonance properties.     

Journal of Genetics and Genomics

(Started in 1974, Monthly)WWW.jgenetgenomics.org (Free electronic papers published in 1974-2005)Journal of Genetics and Genomics (JGG) is one of China's leading journals in the life science. JGG covers all areas of