Biosynthetic Pathway of Thiamine Pyrophosphate: a Special Reference to the Thiamine Monophosphate-Requiring Mutant and the Thiamine Pyrophosphate-Requiring Mutant of Escherichia coli

alpha-Isopropylmalate synthase, the first specific enzyme in leucine biosynthesis, was purified approximately 100-fold from extracts of Saccharomyces sp. (strain 60615), the most effective step being specific elution with the feedback inhibitor leucine from a hydroxyapatite column. In the early steps of purification, special care was taken to protect the synthase against proteolytic activities. The apparent molecular weight of the enzyme as determined from gel filtration on a calibrated column was 137,000 in the absence and 121,000 in the presence of leucine. Inhibition by leucine was specific and strongly pH-dependent, with the leucine concentration necessary for half-maximal inhibition increasing about 10-fold as the pH increased from 7.5 to 8.5. Within this pH range, catalytic activity remained almost unchanged. The apparent K(m) values for the two substrates were found to be 16 mum for alpha-ketoisovalerate and 9 mum for acetyl-coenzyme A. K(+) was required for full activity, the apparent K(a) value being 2 mm. Leucine inhibition was of the mixed type, resulting in decreased V(max) and increased apparent K(m) values forboth substrates. Whereas no cooperative effects were observed with either substrate, positive cooperativity was seen with leucine in the presence of saturating substrate concentrations. Leucine and, to a lesser extent, alpha-ketoisovalerate stabilized the purified enzyme against heat-inactivation. The presence of acetyl-coenzyme A, on the other hand, accelerated the inactivation. In subsequent experiments, coenzyme A was recognized as the actual inactivating ligand, being effective even at lower temperatures and in concentrations which were estimated to be in the range of the enzyme concentration.     

Chromosomal rearrangements in Plantago insularis Eastw.

Seeds of Plantago insularisEastw. which were irradiated with gamma rays yielded 37–67% semi-sterile plants. Twenty-four out of sixty-four of these plants were heterozygous for one or more chromosomal rearrangements. Twothirds of these were translocations, and one-third were inversions. Homozygous lines for four translocations were established. The karyotypes of these provide chromosome markers either at pachynema or in mitotic divisions, or both.Breakage positions were usually located within hetrochromatic segments or at the ends of heterochromatic regions (72.6% of all breaks), and half of all breaks occurred at the juncture of the centromere with the proximal heterochromatin. The consequences of proximal breakage were non-random, in that 93% of such breaks resulted in translocations and only 7% in inversions, whereas more than half of breaks in non-centromeric regions became involved in inversions.The individual chromosomes differed in the types of breakage and of aberrations produced, and these differences appeared correlated with length ratios of heterochromatic segments flanking the centromeres.The research for this paper was supported by National Science Foundation Grant Number GB 5713X.     

Biosynthesis of Thiamine Pyrophosphate in Escherichia coli

In Escherichia coli, thiamine pyrophosphate is synthesized from thiamine monophosphate. Free thiamine is not involved as an intermediate in de novo synthesis of thiamine pyrophosphate.     

Inhibition of Thiamine Pyrophosphate Utilization by Thiamine or Its Monophosphate in Escherichia coli

The growth of a thiamine pyrophosphate auxotroph of Escherichi coli was inhibited by either thiamine or thiamine monophosphate, and the growth of a thiamine monophosphate auxotroph was inhibited by thiamine. The thiamine pyrophosphate-dependent oxidation of pyruvate was inhibited by thiamine with whole cells of the thiamine pyrophosphate auxotroph but not with cell extracts prepared from the same organism. In addition, the thiamine pyrophosphate uptake of the thiamine pyrophosphate auxotroph was inhibited by either thiamine or thiamine monophosphate. Although the thiamine pyrophosphate uptake of a revertant, selected for prototrophy from the thiamine monophosphate auxotroph, was inhibited by thiamine to an extent comparable to that observed with the thiamine monophosphate auxotroph, its growth was no longer inhibited by thiamine. A possible mechanism for the inhibition by thiamine and thiamine monophosphate in the utilization of thiamine pyrophosphate is discussed.     

水稻(Oryza sativa L.)苗期低温白化突变体al12的超微结构与基因定位

水稻苗期低温白化突变是水稻在发育早期对低温胁迫的一种适应性,是一种受发育和温度控制的条件表达,它与其他水稻白化突变有本质的不同.本研究利用便携式叶绿素测量仪测定了白化时期植株的叶绿素含量和用透射电镜观察了叶绿体的结构变化.结果发现叶绿素平均含量仅为1.2(SPAD),而叶绿体也不能正常发育仅有囊泡状结构.通过与9311的正反交实验及子代的分离表现证明该性状受一个隐性核基因的控制.另外利用SSR分子标记技术将该基因定位在第8染色体上,两侧最近的SSR标记RM5068和RM3702分别距基因0.5～1.1 cM和4.9 cM,基因被定位在约6个cM的区间内.我们将该基因暂时命名为al12.     

黑线仓鼠及其白化突变系毛囊中黑色素细胞的组织学分析

目的黑线仓鼠白化突变系是一种新的实验动物模型,本研究旨在组织水平上观察黑线仓鼠与白化突变系皮肤组织中黑色素细胞的分布与定位,以揭示黑线仓鼠白化突变系发生的细胞学机制。方法本实验选择黑线仓鼠和白化突变系各6只,取背部皮肤,制备石蜡切片,分别采用多巴染色、多巴-甲苯胺蓝复染,光镜观察、拍照。结果 Dopa染色显示,在黑线仓鼠与白化突变系皮肤组织中均有成熟黑色素细胞的分布,主要存在于毛囊毛乳头中,且黑线仓鼠黑色素阳性区要明显高于白化突变系。结论黑线仓鼠的酪氨酸酶活性要高于白化突变系。     

Inactivation of ccmO in Synechococcus sp. Strain PCC 7942 Results in a Mutant Requiring High Levels of CO2

Inactivation of ccmO in Synechococcus sp. strain PCC 7942 resulted in a mutant which possesses aberrant carboxysomes and a normal inorganic carbon uptake capability but a reduced ability to photosynthetically utilize the internal inorganic carbon pool. Consequently, it exhibits low apparent photosynthetic affinity for extracellular inorganic carbon and demands high levels of CO₂ for growth.     

Unusual Leucophore‐Like Cells Specifically Appear in the Lineage of Melanophores in the Periodic Albino Mutant of Xenopus laevis

In the periodic albino mutant (a^p/a^p) of Xenopus laevis, peculiar leucophore‐like cells appear in the skins of tadpoles and froglets, whereas no such cells are observed in the wild‐type (+/+). These leucophore‐like cells are unusual in (1) appearing white, but not iridescent, under incident light, (2) emitting green fluorescence under blue light, (3) exhibiting pigment dispersion in the presence of α‐melanocyte stimulating hormone (αMSH), and (4) containing an abundance of bizarre‐shaped, reflecting platelet‐like organelles. In this study, the developmental and ultrastructural characteristics of these leucophore‐like cells were compared with melanophores, iridophores and xanthophores, utilizing fluorescence stereomicroscopy, and light and electron microscopy. Staining with methylene blue, exposure to αMSH, and culture of neural crest cells were also performed to clarify the pigment cell type. The results obtained clearly indicate that: (1) the leucophore‐like cells in the mutant are different from melanophores, iridophores and xanthophores, (2) the leucophore‐like cells are essentially similar to melanophores of the wild‐type with respect to their localization in the skin and manner of response to αMSH, (3) the leucophore‐like cells contain many premelanosomes that are observed in developing melanophores, and (4) mosaic pigment cells containing both melanosomes specific to mutant melanophores and peculiar reflecting platelet‐like organelles are observed in the mutant tadpoles. These findings strongly suggest that the leucophore‐like cells in the periodic albino mutant are derived from the melanophore lineage, which provides some insight into the origin of brightly colored pigment cells in lower vertebrates.     

Inactivation of ccmO in Synechococcus sp. Strain PCC 7942 Results in a Mutant Requiring High Levels of CO(2)

Inactivation of ccmO in Synechococcus sp. strain PCC 7942 resulted in a mutant which possesses aberrant carboxysomes and a normal inorganic carbon uptake capability but a reduced ability to photosynthetically utilize the internal inorganic carbon pool. Consequently, it exhibits low apparent photosynthetic affinity for extracellular inorganic carbon and demands high levels of CO(2) for growth.     

Ultrastructure and Gene Mapping of the Albino Mutant al12 in Rice (Oryza sativa L.)

Seedling albino mutation resistant to low temperature is an adaptability of rice (Oryza sativa L.) to cold. The mutant, a conditional expression controlled by development and temperature, differs from other albino mutants. The chlorophyll content of the mutant was measured using a portable chlorophyll meter, and the ultrastructure of the chloroplast was observed using a transmission electron microscope. Chlorophyll content was 1.2 SPAD, and the chloroplast did not develop, with only small vesicle-like structures. A segregation analysis of the reciprocal crosses between the albino mutation line with the rice line 9311 demonstrated that the albino trait was controlled by a single recessive gene, which was flanked by SSR markers RM5068 and RM3702 on the short arm of chromosome 8 with a distance of 0.5-1.1 cM and 4.9 cM, respectively. This gene was mapped within a 6 cM interval region and was tentatively referred to as al12.     

An Ethylmethane Sulfonate Mutant Resource in Pre-Green Revolution Hexaploid Wheat

Mutagenesis is a powerful tool used for studying gene function as well as for crop improvement. It is regaining popularity because of the development of effective and cost efficient methods for high-throughput mutation detection. Selection for semi-dwarf phenotype during green revolution has reduced genetic diversity including that for agronomically desirable traits. Most of the available mutant populations in wheat (Triticum aestivum L.) were developed in post-green revolution cultivars. Besides the identification and isolation of agronomically important alleles in the mutant population of pre-green revolution cultivar, this population can be a vital resource for expanding the genetic diversity for wheat breeding. Here we report an Ethylmethane Sulfonate (EMS) generated mutant population consisting of 4,180 unique mutant plants in a pre-green revolution spring wheat cultivar ‘Indian’. Released in early 1900s, ‘Indian’ is devoid of any known height-reducing mutations. Unique mutations were captured by proceeding with single M₂ seed from each of the 4,180 M₁ plants. Mutants for various phenotypic traits were identified by detailed phenotyping for altered morphological and agronomic traits on M₂ plants in the greenhouse and M₃ plants in the field. Of the 86 identified mutants, 75 (87%) were phenotypically stable at the M₄ generation. Among the observed phenotypes, variation in plant height was the most frequent followed by the leaf morphology. Several mutant phenotypes including looped peduncle, crooked plant morphology, ‘gritty’ coleoptiles, looped lower internodes, and burnt leaf tips are not reported in other plant species. Considering the extent and diversity of the observed mutant phenotypes, this population appears to be a useful resource for the forward and reverse genetic studies. This resource is available to the scientific community.     

转绿型白化突变系W_ (25)转绿过程中幼苗叶片内碳水化合物和氨基酸组分的变化

水稻转绿型白化突变系W25与亲本2177s苗期叶片内碳水化合物总量及蛋白质含量差异明显。亲本2177s幼苗叶片内碳水化合物和蛋白质代谢在两种温度下基本稳定。但W25却有明显不同,25℃下W25叶片内蔗糖和淀粉的含量比20℃下的低,但还原糖含量明显地高,蛋白质含量较高,丙酮酸激酶和谷氨酸胺合成酶活性强,游离氨基酸中Ser、Glu、Val的含量明显较低,Arg、Pro、Thr的含量明显较高,尤其是Thr含量接近了亲本水平。     

An Analysis of Interactions between Fluorescently-Tagged Mutant and Wild-Type SOD1 in Intracellular Inclusions

Background

By mechanisms yet to be discerned, the co-expression of high levels of wild-type human superoxide dismutase 1 (hSOD1) with variants of hSOD1 encoding mutations linked familial amyotrophic lateral sclerosis (fALS) hastens the onset of motor neuron degeneration in transgenic mice. Although it is known that spinal cords of paralyzed mice accumulate detergent insoluble forms of WT hSOD1 along with mutant hSOD1, it has been difficult to determine whether there is co-deposition of the proteins in inclusion structures.

Methodology/Principal Findings

In the present study, we use cell culture models of mutant SOD1 aggregation, focusing on the A4V, G37R, and G85R variants, to examine interactions between WT-hSOD1 and misfolded mutant SOD1. In these studies, we fuse WT and mutant proteins to either yellow or red fluorescent protein so that the two proteins can be distinguished within inclusions structures.

Conclusions/Significance

Although the interpretation of the data is not entirely straightforward because we have strong evidence that the nature of the fused fluorophores affects the organization of the inclusions that form, our data are most consistent with the idea that normal dimeric WT-hSOD1 does not readily interact with misfolded forms of mutant hSOD1. We also demonstrate the monomerization of WT-hSOD1 by experimental mutation does induce the protein to aggregate, although such monomerization may enable interactions with misfolded mutant SOD1. Our data suggest that WT-hSOD1 is not prone to become intimately associated with misfolded mutant hSOD1 within intracellular inclusions that can be generated in cultured cells.     

Evidence for the Existence in Arabidopsis thaliana of the Proteasome Proteolytic Pathway: ACTIVATION IN RESPONSE TO CADMIUM*

Heavy metals are known to generate reactive oxygen species that lead to the oxidation and fragmentation of proteins, which become toxic when accumulated in the cell. In this study, we investigated the role of the proteasome during cadmium stress in the leaves of Arabidopsis thaliana plants. Using biochemical and proteomics approaches, we present the first evidence of an active proteasome pathway in plants. We identified and characterized the peptidases acting sequentially downstream from the proteasome in animal cells as follows: tripeptidyl-peptidase II, thimet oligopeptidase, and leucine aminopeptidase. We investigated the proteasome proteolytic pathway response in the leaves of 6-week-old A. thaliana plants grown hydroponically for 24, 48, and 144 h in the presence or absence of 50 μm cadmium. The gene expression and proteolytic activity of the proteasome and the different proteases of the pathway were found to be up-regulated in response to cadmium. In an in vitro assay, oxidized bovine serum albumin and lysozyme were more readily degraded in the presence of 20 S proteasome and tripeptidyl-peptidase II than their nonoxidized form, suggesting that oxidized proteins are preferentially degraded by the Arabidopsis 20 S proteasome pathway. These results show that, in response to cadmium, the 20 S proteasome proteolytic pathway is up-regulated at both RNA and activity levels in Arabidopsis leaves and may play a role in degrading oxidized proteins generated by the stress.