Mitonuclear mismatch alters performance and reproductive success in naturally introgressed populations of a montane leaf beetle*

Coordination between nuclear and mitochondrial genomes is critical to metabolic processes underlying animals' ability to adapt to local environments, yet consequences of mitonuclear interactions have rarely been investigated in populations where individuals with divergent mitochondrial and nuclear genomes naturally interbreed. Genetic variation in the leaf beetle Chrysomela aeneicollis was assessed along a latitudinal thermal gradient in California's Sierra Nevada. Variation at mitochondrial cytochrome oxidase II (COII) and the nuclear gene phosphoglucose isomerase (PGI) shows concordance and was significantly greater along a 65 km transect than 10 other loci. STRUCTURE analyses using neutral loci identified a southern and northern subpopulation, which interbreed in the central drainage Bishop Creek. COII and PGI were used as indicators of mitochondrial and nuclear genetic variation in field and laboratory experiments conducted on beetles from this admixed population. Fecundity, larval development rate, running speed and male mating frequency were higher for beetles with geographically “matched” than “mismatched” mitonuclear genotypes. Effects of mitonuclear mismatch were largest for individuals with northern nuclear genotypes possessing southern mitochondria and were most pronounced after heat treatment or at high elevation. These findings suggest that mitonuclear incompatibility diminishes performance and reproductive success in nature, effects that could intensify at environmental extremes.     

Xenopus as a model to study alternative splicing in vivo

An increasing number of genes are being identified for which the corresponding mRNAs contain different combinations of the encoded exons. This highly regulated exon choice, or alternative splicing, is often tissue-specific and potentially could differentially affect cellular functions. Alternative splicing is therefore not only a means to increase the coding capacity of the genome, but also to regulate gene expression during differentiation or development. To both evaluate the importance for cellular functions and define the regulatory pathways of alternative splicing, it is necessary to progress from the in vitro or ex vivo experimental models actually used towards in vivo whole-animal studies. We present here the amphibian, Xenopus, as an experimental model highly amenable for such studies. The various experimental approaches that can be used with Xenopus oocytes and embryos to characterize regulatory sequence elements and factors are presented and the advantages and drawbacks of these approaches are discussed. Finally, the real possibilities for large-scale identification of mRNAs containing alternatively spliced exons, the tissue-specific patterns of exon usage and the way in which these patterns are modified by perturbing the relative amount of splicing factors are discussed.     

Historical male‐mediated introgression in horseshoe bats revealed by multilocus DNA sequence data

Instances of hybridization between mammalian taxa in the wild are rarely documented. To test for introgression between sibling species of horseshoe bat (Rhinolophus yunanensis and R. pearsoni) and two subspecies of the latter (R. p. pearsoni and R. p. chinensis), we sequenced two mtDNA and two ncDNA markers in individuals sampled from multiple localities within their overlapping ranges. The interspecific mtDNA gene tree corresponded to the expected taxonomic divisions, and coalescent‐based analyses suggested divergence occurred around 4 MYA. However, these relationships strongly conflicted with those recovered from two independent nuclear gene trees, in which R. yunanensis clustered with R. p. pearsoni to the exclusion of R. p. chinensis. This geographically widespread discordance is best explained by large‐scale historical introgression of ncDNA from R. yunanensis to R. pearsoni by male‐mediated exchange in mixed species colonies during Pleistocene glacial periods, when ranges may have contracted and overlapped more than at present. Further species tree–gene tree conflicts were detected between R. p. pearsoni and R. p. chinensis, also indicating past and/or current introgression in their overlapping regions. However, here the patterns point to asymmetric mtDNA introgression without ncDNA introgression. Analyses of coalescence times indicate this exchange has occurred subsequent to the divergence of these subspecies from their common ancestor. Our work highlights the importance of using multiple data sets for reconstructing phylogeographic histories and resolving taxonomic relationships.     

Differential Gene Expression Analysis by DNA Microarray Technology and Its Application in Molecular Oncology

Accumulation of genetic and epigenetic aberrations leads to malignant transformation of normal cells. Functional studies of cancer using genomic and proteomic tools aim to reveal the true complexity of the processes leading to cancer development in humans. Until recently, diagnosis and prognosis of cancer was based on conventional pathologic criteria and epidemiological evidence. Certain tumors were divided only into relatively broad histological and morphological subcategories. Rapidly developing methods of differential gene expression analysis promote the search for clinically relevant genes changing their expression levels during malignant transformation. DNA microarrays offer a unique possibility to rapidly assess the global expression picture of thousands genes in any given time point and compare the results of detailed combinatory analysis of global expression profiles for normal and malignant cells at various functional stages or separate experimental conditions. Acquisition of such genetic portraits allows searching for regularity and difference in expression patterns of certain genes, understanding their function and pathological importance, and ultimately developing the molecular nosology of cancer. This review describes the basis of DNA microarray technology and methodology, and focuses on their application in molecular classification of tumors, drug sensitivity and resistance studies, and identification of biological markers of cancer.     

Adaptive auditory risk assessment in the dogbane tiger moth when pursued by bats

Moths and butterflies flying in search of mates risk detection by numerous aerial predators; under the cover of night, the greatest threat will often be from insectivorous bats. During such encounters, the toxic dogbane tiger moth, Cycnia tenera uses the received intensity, duration and emission pattern of the bat''s echolocation calls to determine when, and how many, defensive ultrasonic clicks to produce in return. These clicks, which constitute an acoustic startle response, act as warning signals against bats in flight. Using an integrated test of stimulus generalization and dishabituation, here we show that C. tenera is able to discriminate between the echolocation calls characteristic of a bat that has only just detected it versus those of a bat actively in pursuit of it. We also show that C. tenera habituates more profoundly to the former stimulus train (‘early attack’) than to the latter (‘late attack’), even though it was initially equally responsive to both stimuli. Matched sensory and behavioural data indicate that reduced responsiveness reflects habituation and is not merely attributable to sensory adaptation or motor fatigue. In search of mates in the face of bats, C. tenera''s ability to discriminate between attacking bats representing different levels of risk, and to habituate less so to those most dangerous, should function as an adaptive cost–benefit trade-off mechanism in nature.     

Regulation of alternative RNA splicing by exon definition and exon sequences in viral and mammalian gene expression

Intron removal from a pre-mRNA by RNA splicing was once thought to be controlled mainly by intron splicing signals. However, viral and other eukaryotic RNA exon sequences have recently been found to regulate RNA splicing, polyadenylation, export, and nonsense-mediated RNA decay in addition to their coding function. Regulation of alternative RNA splicing by exon sequences is largely attributable to the presence of two majorcis-acting elements in the regulated exons, the exonic splicing enhancer (ESE) and the suppressor or silencer (ESS). Two types of ESEs have been verified from more than 50 genes or exons: purine-rich ESEs, which are the more common, and non-purine-rich ESEs. In contrast, the sequences of ESSs identified in approximately 20 genes or exons are highly diverse and show little similarity to each other. Through interactions with cellular splicing factors, an ESE or ESS determines whether or not a regulated splice site, usually an upstream 3 splice site, will be used for RNA splicing. However, how these elements function precisely in selecting a regulated splice site is only partially understood. The balance between positive and negative regulation of splice site selection likely depends on thecis-element's identity and changes in cellular splicing factors under physiological or pathological conditions.     

利用抑制性扣除杂交技术克隆水稻磷饥饿诱导基因

磷素是植物生长所必需的重要元素.在缺磷环境中,植物能够调节自身的形态、生理生化和基因表达水平来适应环境的变化.为研究水稻(Oryza sativa L.)耐低磷胁迫的分子机理,采用抑制性扣除杂交技术(SSH)构建磷饥饿诱导的水稻根系扣除cDNA文库.通过文库筛选和测序获得18个已知基因和47个功能未知基因.这些基因参与了不同的代谢过程,包括磷吸收和转运、信号传导、蛋白质合成和降解、碳水化合物代谢和胁迫反应.Northern杂交结果表明,在磷饥饿胁迫下这些基因呈现不同的表达模式,并且不同代谢过程中的基因对磷饥饿有着不同的反应.     

扣除杂交法筛选与非小细胞性肺癌转移相关的基因

非小细胞性肺癌病人的术后死亡率很高 ,其原因是该病易发生转移。收集了肺癌早期患者的样品 ,并根据患者资料分成转移型 (n =4)和非转移型 (n =5 ) ,采用扣除杂交法筛选与非小细胞性肺癌转移相关的基因。扣除后的cDNA文库中得到了 2 2 5个有效克隆。对这些克隆进行了测序 ,在基因文库中比较了核苷酸同源性 ,初步确定了这些克隆对应的基因 ,并根据基因可能涉及的功能加以分类。通过实时 (realtime)RT PCR鉴定 ,发现 10种基因在用于扣除试验的转移病人样品中的平均表达量比非转移病人高。进一步对 70位患非小细胞性肺癌病人 (I至IIIA期 )的样品进行了检测。根据统计分析 ,在I和II期病人中 ,2种基因MALA1和EIF4A1在发生转移的病人样品中的表达与在非转移的病人中有显著差异性。这些结果将有助于分析非小细胞性肺癌病变发生转移的可能性     

利用抑制性扣除杂交技术克隆水稻磷饥饿诱导基因

磷素是植物生长所必需的重要元素。在缺磷环境中,植物能够调节自身的形态、生理生化和基因表达水平来适应环境的变化。为研究水稻(Oryzn sativa L.)耐低磷胁迫的分子机理,采用抑制性扣除杂交技术(SSH)构建磷饥饿诱导的水稻根系扣除cDNA文库。通过文库筛选和测序获得18个已知基因和47个功能未知基因。这些基因参与了不同的代谢过程,包括磷吸收和转运、信号传导、蛋白质合成和降解、碳水化合物代谢和胁迫反应。Northern杂交结果表明,在磷饥饿胁迫下这些基因呈现不同的表达模式,并且不同代谢过程中的基因对磷饥饿有着不同的反应。     

Cellular localization of a pollen-specific mRNA by in situ hybridization and confocal laser scanning microscopy

Summary The application of confocal laser scanning microscopy together with in situ hybridization experiments in tobacco pollen enabled a detailed localization of a pollen-specific mRNA. The three-dimensional distribution of this specific mRNA over the whole pollen grain was reconstructed by means of optical sections of one specimen.