Identification of the Gene Whose Mutation Leads to the Appearance of Recessive Lethal Germlings in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The data are presented on genetic and molecular-genetic analysis of a mutant from the collection of morphological insertion mutants of Arabidopsis thaliana we obtained earlier, which belongs to the phenotypic class of recessive lethal germlings. A nucleotide DNA sequence, 147 bp in size, was identified, which adheres to the left border area of T-DNA insertion. The site of localization of the insertion was determined using computer analysis.__________Translated from Ontogenez, Vol. 36, No. 3, 2005, pp. 222–224.Original Russian Text Copyright © 2005 by Ogarkova, Tomilov, Tomilova, Tarasov.     

SNP-Ratio Mapping (SRM): Identifying Lethal Alleles and Mutations in Complex Genetic Backgrounds by Next-Generation Sequencing

We present a generally applicable method allowing rapid identification of causal alleles in mutagenized genomes by next-generation sequencing. Currently used approaches rely on recovering homozygotes or extensive backcrossing. In contrast, SNP-ratio mapping allows rapid cloning of lethal and/or poorly transmitted mutations and second-site modifiers, which are often in complex genetic/transgenic backgrounds.     

Genetic Analysis of Recessive Lethal Mutations Induced by the Influenza Virus in the X Chromosome of Drosophila melanogaster

Mutagenic potential of the influenza virus was evaluated. Based on its capacity of inducing recessive lethal mutations in the X chromosome of Drosophila melanogaster, the influenza virus can be classified as a moderate-activity mutagen. Its mutagenicity does not depend on ability to reproduce in the cell system. This virus was shown to disrupt formation of the wing, particularly wing vein M_{1 + 2}. Cytogenetic examination of polytene X chromosomes bearing recessive lethal mutations in Drosophilasalivary glands did not reveal chromosome rearrangements. These lethals are assumed to be small deletions or point mutations. The determination of the lethal activity stage of these mutations showed that they disrupt the expression of genes functioning at various developmental stages of Drosophila.Two of them were conditionally lethal (temperature-sensitive). Two of 15 mutations analyzed were mapped to region 2B9-10–3C10-11.     

PKHD1基因缺陷与常染色体隐性遗传性多囊肾病的研究进展

PKHD1是目前所知人类常染色体隐性遗传多囊肾病(autosomal recessive polycystic kidney disease,ARPKD)的惟一致病基因。ARPKD临床病变以双肾多发性进行性充液囊泡为主要特征。目前对PKHDl基因在ARPKD发病中的作用了解并不多。该文对ARPKD的发病机制和PKHD1基因功能最新研究进展进行综述。     

Accumulation of Spontaneous Mutations in the Ciliate Tetrahymena thermophila

Knowledge of the rate and fitness effects of mutations is essential for understanding the process of evolution. Mutations are inherently difficult to study because they are rare and are frequently eliminated by natural selection. In the ciliate Tetrahymena thermophila, mutations can accumulate in the germline genome without being exposed to selection. We have conducted a mutation accumulation (MA) experiment in this species. Assuming that all mutations are deleterious and have the same effect, we estimate that the deleterious mutation rate per haploid germline genome per generation is U = 0.0047 (95% credible interval: 0.0015, 0.0125), and that germline mutations decrease fitness by s = 11% when expressed in a homozygous state (95% CI: 4.4%, 27%). We also estimate that deleterious mutations are partially recessive on average (h = 0.26; 95% CI: –0.022, 0.62) and that the rate of lethal mutations is <10% of the deleterious mutation rate. Comparisons between the observed evolutionary responses in the germline and somatic genomes and the results from individual-based simulations of MA suggest that the two genomes have similar mutational parameters. These are the first estimates of the deleterious mutation rate and fitness effects from the eukaryotic supergroup Chromalveolata and are within the range of those of other eukaryotes.     

Deletion of one of the duplicated Hsp70 genes causes hereditary myopathy of diaphragmatic muscles in Holstein-Friesian cattle

Heat-shock protein 70 (Hsp70) is a major chaperone that folds protein and prevents aggregation. The Hsp70 family contains both constitutive and stress-inducible forms. In humans, two of the inducible Hsp70 genes are located within the human major histocompatibility complex (MHC) on 6p21.3, as a duplicated locus, 12 kb apart from each other. We report that loss of one of the duplicated Hsp70 genes, the bovine homologue within the bovine MHC, is responsible for hereditary myopathy of diaphragmatic muscles (HMDM) in Holstein-Friesian cattle. Although the remaining Hsp70 gene is intact, Hsp70 protein levels are dramatically decreased in affected cattle. In normal diaphragmatic muscle, Hsp70 binds several proteins involved in energy metabolism including glycogen phosphorylase (PYGM). Immunohistochemical staining indicated that PYGM accumulated in the HMDM-specific core-like structures in affected cattle. Misfolding of energy-related proteins due to Hsp70 deficiency might lead to protein aggregation and muscle fibre degeneration.     

Parkin mediates the degradation-independent ubiquitination of Hsp70

Mutations in the parkin gene cause autosomal recessive, juvenile-onset parkinsonism. Parkin is an E3 ubiquitin ligase that mediates the ubiquitination of protein substrates. Disease-associated mutations cause a loss-of-function of parkin which may compromise the poly-ubiquitination and proteasomal degradation of specific protein substrates, potentially leading to their deleterious accumulation. Here, we identify the molecular chaperones, Hsp70 and Hsc70, as substrates for parkin. Parkin mediates the ubiquitination of Hsp70 both in vitro and in cultured cells. Parkin interacts with Hsp70 via its second RING finger domain and mutations in/near this domain compromise Hsp70 ubiquitination. Ubiquitination of Hsp70 fails to alter its steady-state levels or turnover, nor does it promote its proteasomal degradation. Consistent with this observation, Hsp70 levels remain unaltered in brains from parkin-deficient autosomal recessive, juvenile-onset parkinsonism subjects, whereas alternatively, Hsp70 levels are elevated in the detergent-insoluble fraction of sporadic Parkinson's disease/dementia with Lewy bodies brains. Parkin mediates the multiple mono-ubiquitination of Hsp70/Hsc70 consistent with a degradation-independent role for this ubiquitin modification. Our observations support a novel functional relationship between parkin and Hsc/Hsp70 and support the notion that parkin is a multi-purpose E3 ubiquitin ligase capable of modifying proteins either via attachment of alternatively linked poly-ubiquitin chains or through multiple mono-ubiquitination to achieve alternate biological outcomes.     

Towards an understanding of the genetics of human male infertility: lessons from flies

It has been argued that about 4–5% of male adults suffer from infertility due to a genetic causation. From studies in the fruitfly Drosophila, there is evidence that up to 1500 recessive genes contribute to male fertility in that species. Here we suggest that the control of human male fertility is of at least comparable genetic complexity. However, because of small family size, conventional positional cloning methods for identifying human genes will have little impact on the dissection of male infertility. A critical selection of well-defined infertility phenotypes in model organisms, combined with identification of the genes involved and their orthologues in man, might reveal the genes that contribute to human male infertility.     

Population Growth Inflates the Per-Individual Number of Deleterious Mutations and Reduces Their Mean Effect

This study addresses the question of how purifying selection operates during recent rapid population growth such as has been experienced by human populations. This is not a straightforward problem because the human population is not at equilibrium: population genetics predicts that, on the one hand, the efficacy of natural selection increases as population size increases, eliminating ever more weakly deleterious variants; on the other hand, a larger number of deleterious mutations will be introduced into the population and will be more likely to increase in their number of copies as the population grows. To understand how patterns of human genetic variation have been shaped by the interaction of natural selection and population growth, we examined the trajectories of mutations with varying selection coefficients, using computer simulations. We observed that while population growth dramatically increases the number of deleterious segregating sites in the population, it only mildly increases the number carried by each individual. Our simulations also show an increased efficacy of natural selection, reflected in a higher fraction of deleterious mutations eliminated at each generation and a more efficient elimination of the most deleterious ones. As a consequence, while each individual carries a larger number of deleterious alleles than expected in the absence of growth, the average selection coefficient of each segregating allele is less deleterious. Combined, our results suggest that the genetic risk of complex diseases in growing populations might be distributed across a larger number of more weakly deleterious rare variants.     

An Assessment of the Carcinogenic Potential of Trichloroethylene in Humans

Controversy surrounds the assessments of carcinogenic potential associated with human exposure to trichloroethylene (TCE). The American Conference of Governmental Industrial Hygienists states that TCE is “not suspected to be a human carcinogen.” In contrast, the International Agency for Research on Cancer has classified TCE as a probable human carcinogen, based primarily on the results of animal toxicity studies. Chronic high-dose TCE exposures cause hepatic and pulmonary tumors in mice and renal tumors in rats. Human epidemiology studies, however, do not support a causal association between exposure to TCE at environmentally relevant levels and cancers of the lung, liver, or kidney. The apparent discrepancy between the animal data and the human data can be explained by (1) differences in TCE exposure levels between laboratory animals and humans, (2) species-specific differences in TCE metabolism, and (3) other species-specific mechanisms involved in the development of cancer in rodents. This paper critically assesses the experimental and epidemiological data relevant to the carcinogenic potential of TCE. From the analysis, we conclude that TCE exposure at concentrations likely to be encountered in most environmental media is not likely to cause liver, lung, or kidney cancers in humans.     

卫星搭载鸡冠花种子SP2代的生长、发育与性状变异研究

对卫星搭载处理的鸡冠花SP2代种子萌发、植株生长、开花以及主要性状在群体中的变异进行了观察.与对照比较,SP2种子发芽势提高,发芽率有所下降,萌发延迟.群体茎粗与对照相比显著增加;选择系在株幅、茎粗间出现了较大差异.在SP2株系间开花时间和花期出现了明显分离,株系群体中有花期明显提前和延迟的单株出现.20个株系中有16个株系的单株种子量比对照显著减少.在SP2株系中株高、株幅呈非正态分布,茎粗和花期呈偏正态分布.在处理的株系群体中有植株显著高大、多分枝、叶色、叶型、冠型、冠色及花期等显著性状变异出现,产生性状变异的单株频率为0.07％-1.26％,表明空间诱变所产生的变异性状开始在SP2代出现.     

DCC Is Required for the Development of Nociceptive Topognosis in Mice and Humans

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Recessive hyperekplexia mutations of the glycine receptor α1 subunit affect cell surface integration and stability

The human neurological disorder hyperekplexia is frequently caused by recessive and dominant mutations of the glycine receptor α1 subunit gene, GLRA1 . Dominant forms are mostly attributed to amino acid substitutions within the ion pore or adjacent loops, resulting in altered channel properties. Here, the biogenesis of glycine receptor α1 subunit mutants underlying recessive forms of hyperekplexia was analyzed following recombinant expression in HEK293 cells. The α1 mutant S231R resulted in a decrease of surface integrated protein, consistent with reduced maximal current values. Decreased maximal currents shown for the recessive α1 mutant I244N were associated with protein instability, rather than decreased surface integration. The recessive mutants R252H and R392H encode exchanges of arginine residues delineating the intracellular faces of transmembrane domains. After expression, the mutant R252H was virtually absent from the cell surface, consistent with non-functionality and the importance of the positive charge for membrane integration. Surface expression of R392H was highly reduced, resulting in residual chloride conductance. Independent of the site of the mutation within the α1 polypeptide, metabolic radiolabelling and pulse chase studies revealed a shorter half-life of the full-length α1 protein for all recessive mutants as compared to the wild-type. Treatment with the proteasome blocker, lactacystin, significantly increased the accumulation of α1 mutants in intracellular membranes. These observations indicated that the recessive α1 mutants are recognized by the endoplasmatic reticulum control system, and degraded via the proteasome pathway. Thus, the lack of glycinergic inhibition associated with recessive hyperekplexia may be attributed to sequestration of mutant subunits within the endoplasmatic reticulum quality control system.     

Mutations in the human homologue of the Drosophila segment polarity gene patched in oral squamous cell carcinoma cell lines

In the present study, we have analyzed tumor deoxyribonucleic acid from oral squamous cell carcinoma (OSCC) cells for patched mutations using an exon-by-exon single strand conformation polymorphism assay and direct sequencing. We found two missense mutations which affected the conserved residue in the transmembrane domains of the gene product and in the intracellular loop at the C-terminal residue implicated in regulating the smoothened molecule. In addition, we demonstrated that the N-terminal fragment of sonic hedgehog (Shh-N) stimulates the growth of normal epithelial cells, the OSCC cell line, NA, and the salivary gland adenocarcinoma cell lines, HSG and HSY, which have no detectable mutation in patched. On the other hand, Shh has no effect on human SCC cells (UE, KA, KO, NI, A431 cells) that have mutations in patched. These results strongly suggest that an Shh-patched signaling is involved in the cell growth of oral epithelial cells and in the tumorigenesis of OSCCs.     

Tailoring the Peptide-Binding Specificity Of An RNA by Combinations of Specificity-Altering Mutations

In this study, the ability to tailor the peptide-binding specificity of an RNA was investigated. First, variants of the Rev-response element (RRE) RNA with different specificities toward the natural binding partner, Rev, and two RRE-binding aptamers, the RSG-1.2 and the Kl peptides, were identified. Next, hybrid RRE mutants with combinations of two sets of specificity-altering substitutions were tested for peptide-binding specificity. It was shown that in most cases the results of the combination of individual mutations were of an additive nature, therefore providing a way to manipulate the peptide-binding specificity of an RNA in a predictable manner.     

酮色林对人类ether-a-go-go相关基因钾通道的阻断作用

采用双电极电压钳技术,研究酮色林对表达在非洲爪蟾卵母细胞上的野生型和Y652突变型人类ether-a-go-go相关基因(human ether-a-go-go-related gene,HERG)钾通道的阻断效应,观测HERG通道的分子位点特性改变对其阻断效应的影响.结果显示,酮色林以电压依赖性和浓度依赖性的方式阻断野生型的HERG钾通道电流.尾电流包裹程序记录电流显示酮色林对HERG钾通道微小的张力性阻断.阻断特征符合对开放状态通道的阻断特征.酮色林也能调节失活状态的HERG钾通道.位于孔道S6区的氨基酸位点突变Y652A和Y652R可显著减弱酮色林对HERG通道的阻断作用.同野生犁HERG钾通道的阻断相比,Y652A突变使阻断的IC50提高72倍,而Y652R突变使阻断的IC50提高53倍.Y652A和Y652R的阴断效应之间没有明显的差别.以上结果提示,酮色林优先阻断开放状态的HERG钾通道,而Y652是酮色林与通道结合的关键位点之一.     

Role of the Protective Antigen Octamer in the Molecular Mechanism of Anthrax Lethal Toxin Stabilization in Plasma

Anthrax is caused by strains of Bacillus anthracis that produce two key virulence factors, anthrax toxin (Atx) and a poly-γ-D-glutamic acid capsule. Atx is comprised of three proteins: protective antigen (PA) and two enzymes, lethal factor (LF) and edema factor (EF). To disrupt cell function, these components must assemble into holotoxin complexes, which contain either a ring-shaped homooctameric or homoheptameric PA oligomer bound to multiple copies of LF and/or EF, producing lethal toxin (LT), edema toxin, or mixtures thereof. Once a host cell endocytoses these complexes, PA converts into a membrane-inserted channel that translocates LF and EF into the cytosol. LT can assemble on host cell surfaces or extracellularly in plasma. We show that, under physiological conditions in bovine plasma, LT complexes containing heptameric PA aggregate and inactivate more readily than LT complexes containing octameric PA. LT complexes containing octameric PA possess enhanced stability, channel-forming activity, and macrophage cytotoxicity relative to those containing heptameric PA. Under physiological conditions, multiple biophysical probes reveal that heptameric PA can prematurely adopt the channel conformation, but octameric PA complexes remain in their soluble prechannel configuration, which allows them to resist aggregation and inactivation. We conclude that PA may form an octameric oligomeric state as a means to produce a more stable and active LT complex that could circulate freely in the blood.