Biochemical and genetic analysis of a mutant with altered alkaline phosphatase activity in Dictyostelium discoideum

Alkaline phosphatase is one of several enzymes that accumulate in a temporally regulated sequence during the development of Dictyostelium discoideum. These enzymes can be used to monitor specific gene expression; moreover, isolation and analysis of mutations in the structural gene(s) can serve to indicate some of the essential steps in programmed synthesis and morphogenesis. A mutation (alpA) which affects the activity and substrate affinity of alkaline phosphatase was isolated in D discoideum using a procedure for screening large numbers of clones. Alkaline phosphatase activity at all stages of vegetative growth and development was altered by the mutation. Several physical properties of the enzyme from growing cells and developed cells were compared and found to be indistinguishable. It is likely that a single enzyme is responsible for the majority of alkaline phosphatase activity in growth and development. The mutation is coexpressed in diploids heterozygous for alpA and maps to linkage group III. One of the haploid segregants isolated from these diploids carries convenient markers on each of the six defined linkage groups and can be used for linkage analysis of other genetic loci.     

Correlation of phenotypes of the apterous mutation in Drosophila melanogaster

The apterous (ap) mutant in Drosophila melanogaster exhibits phenotypes of wing deficiency, precocious adult death, and nonvitellogenic oocyte development. The latter phenotype previously has been shown to result from juvenile hormone (JH) deficiency in the adult stage. To explore the relationship between the hormone deficiency and the other phenotypes, the expression of each phenotype was measured in five alleles of ap (including a new, chemically-induced allele, ap^77f) as wing length, survival five days after eclosion, and initiation and progress of vitellogenic oocyte development. No correlation could be found between severity of wing phenotype and that of precocious adult death or nonvitellogenesis. However, the latter phenotypes were correlated in both ap homozygotes and allelic heterozygotes, since adults that survive have wild-type vitellogenesis, and those fated for precocious death fail to develop vitellogenic oocytes. These results indicate that no relationship exists between wing and JH deficiencies, but that precocious adult death is related to hormone deficiency — probably through pleiotropy, rather than through causality.     

β-Carotene biosynthesis by extracts of the C115 mutant of Phycomyces blakesleeanus

A cell extract of the yellow C115 car-42 mad-107(?) mutant of Phycomyces blakesleeanus, capable of converting MVA-[2-¹⁴C] into isoprenoids, was used to investigate the formation of β-carotene. The incorporation of radioactivity into β-carotene was reduced by the addition of unlabelled carotenes, solubilised using detergent, to the incubation mixtures. On reisolation of these carotenes after anaerobic incubations, they were found to carry radioactivity. The relative efficiencies of these carotenes as trapping agents are discussed in relation to the pathways of carotene cyclisation and to the apparent operation of a system for the negative feedback control of carotene biosynthesis.     

Control of membrane morphogenesis in bacteriophage PM2

The regulation of membrane formation in bacteriophage PM2 serves as a simple model for changes in membrane structure in eukaryotic cells. Prior to Pseudomonas host lysis, wild-type virions mature to an icosahedral morphology at the inner face of the cytoplasmic membrane. The proliminary charcterization of two temperature-sensitive mutants of PM2 is described. In cells infected at the restrictive temperature with ts 1, an abundance of “empty” virus-size membrane vesicles are seen. Synthesis of DNA is also reduced in ts 1 infected cells. The preponderance of vesicles is not sen in cells infected with wil-type virus or with ts 1 at the permissive temperature. The “empty” appearance of the viral membranes suggests that viral DNA is not encapsulated. The major viral capsid protein (MW 26,000) is located just out side the viral membrane and normallyl sediments with host and virus membranes; insted, large amounts of capsid protein can be precipitated from the supernatant with TCA. Compared to cells infected with wild type virus, cells infected with is 5 at th restrictive temperature produce inside the cell an aboundance of virus-soze membrane vesicles. Taken Together, These results with viral mutants suggest that formation of a viral membrane of the proper size does not require a DNA core around which to form, or an outer scaffolding of coat protein against which to form a spherical bilayer.     

What limits meiotic crossovers?

Comment on: Crismani W, et al. Science 2012; 336:1588-90.     

Cometabolic turnover of aniline,phenol and some of their monochlorinated derivatives by the Rhodococcus mutant strain AM 144

One-step conversion of aniline, phenol and some of their monochlorinated derivatives into the corresponding catechols by resting pre-adapted cells of the Rhodococcus mutant strain AM 144 (defective in synthesis of catechol 1,2-dioxygenase) was shown to depend on the availability of an additional metabolizable carbon substrate, e.g. glucose or acetate. A stoichiometric relation existed between the amount of the latter compounds added and the amount of aniline (or phenol, respectively) converted into catechol suggesting that the primary function of the cosubstrates was to provide reducing power to the oxygenative transformation reaction. The observed cosubstrate-dependence generally parallels that seen in previous studies on turnover of different monochloroaromatic non-growth substrates by aromatics-utilizing Rhodococcus wildtype-strains. Cell cultures of strain AM 144 growing at the expense of acetate also proved able to convert aniline into catechol. Typically, growth of the cells was retarded during the phase of aniline transformation as compared to the respective control cultures. Based on the results of these model experiments, it was concluded that (i) in natural microbial communities cometabolically active bacteria would hardly enrich under cometabolic conditions over fast-growing non-cometabolizing bacteria if the latter organisms will tolerate the particular non-growth substrate, and (ii) cometabolizing bacteria would have a selective advantage only if the non-growth substrate to be transformed is a toxic one or if it can serve as a potential nutrient source (e.g., of nitrogen or sulfur).Abbreviations MCA monochloroaniline - MCP monochlorophenol - MCC monochlorocatechol - TLC thin-layer chromatography - MS mass spectrometry - GLC gas-liquid chromatography - UV ultraviolet (range of the spectrum)     

Development of near-infrared firefly luciferin analogue reacted with wild-type and mutant luciferases

Interestingly, only the D-form of firefly luciferin produces light by luciferin–luciferase (L–L) reaction. Certain firefly luciferin analogues with modified structures maintain bioluminescence (BL) activity; however, all L-form luciferin analogues show no BL activity. To this date, our group has developed luciferin analogues with moderate BL activity that produce light of various wavelengths. For in vivo bioluminescence imaging, one of the important factors for detection sensitivity is tissue permeability of the number of photons emitted by L–L reaction, and the wavelengths of light in the near-infrared (NIR) range (700–900 nm) are most appropriate for the purpose. Some NIR luciferin analogues by us had performance for in vivo experiments to make it possible to detect photons from deep target tissues in mice with high sensitivity, whereas only a few of them can produce NIR light by the L–L reactions with wild-type luciferase and/or mutant luciferase. Based on the structure–activity relationships, we designed and synthesized here a luciferin analogue with the 5-allyl-6-dimethylamino-2-naphthylethenyl moiety. This analogue exhibited NIR BL emissions with wild-type luciferase (λ_max = 705 nm) and mutant luciferase AlaLuc (λ_max = 655 nm).     

‘培矮64S’与其eui突变体‘长选3S’穗颈节间蛋白质组差异分析

为从蛋白质表达水平了解长穗颈温敏核不育水稻穗颈节间伸长机理,该研究以长穗颈(EUI)温敏核不育水稻‘长选3S’为材料,温敏核不育水稻‘培矮64S’为对照,采用固相pH梯度双向凝胶电泳和质谱分析方法,对2个水稻材料抽穗前2 d的穗颈节间蛋白质进行分离,并进行差异蛋白质组学的比较研究。结果表明:(1)获得了分辨率和重复性较好的双向凝胶电泳图谱。(2)对40个差异蛋白质点进行MALDI-TOF-TOF-MS肽质谱指纹图谱分析,成功鉴定其中27个差异蛋白质点;与‘培矮64S’相比,‘长选3S’中有17个上调表达和10个下调表达的蛋白质。(3)差异蛋白质按照其功能可分为6类,其中主要是与细胞代谢相关蛋白,其次是与细胞壁重建相关蛋白;并且这些差异蛋白质可能与‘长选3S’抽穗期穗颈节间剧烈伸长生长有关,尤其是细胞壁重建相关蛋白与细胞的伸长密切相关。(4)实时荧光定量PCR对随机挑选的蛋白点2、7、8、24、35和36所对应的基因在两个材料最上节间的表达结果显示,‘长选3S’的2(Os10g08550)、7(Os12g42876)、8(Os01g55830)基因的表达量较‘培矮64S’明显下调,而24(Os06g48760)、35(Os05g25850)、36(Os07g42300)基因的表达量较‘培矮64S’显著上调,表明q-PCR的结果与蛋白凝胶图分析结果一致。研究认为,水稻eui基因可能是通过调节抽穗期穗颈节间这些蛋白质的表达,从而促进穗颈节间细胞分裂,尤其是细胞的伸长生长。     

A new unique form of microRNA from human heart,microRNA-499c,promotes myofibril formation and rescues cardiac development in mutant axolotl embryos

Background

A recessive mutation “c” in the Mexican axolotl, Ambystoma mexicanum, results in the failure of normal heart development. In homozygous recessive embryos, the hearts do not have organized myofibrils and fail to beat. In our previous studies, we identified a noncoding Myofibril-Inducing RNA (MIR) from axolotls which promotes myofibril formation and rescues heart development.

Results

We randomly cloned RNAs from fetal human heart. RNA from clone #291 promoted myofibril formation and induced heart development of mutant axolotls in organ culture. This RNA induced expression of cardiac markers in mutant hearts: tropomyosin, troponin and α-syntrophin. This cloned RNA matches in partial sequence alignment to human microRNA-499a and b, although it differs in length. We have concluded that this cloned RNA is unique in its length, but is still related to the microRNA-499 family. We have named this unique RNA, microRNA-499c. Thus, we will refer to this RNA derived from clone #291 as microRNA-499c throughout the rest of the paper.

Conclusions

This new form, microRNA-499c, plays an important role in cardiac development.