Molecular differentiation of sex chromosomes probed by comparative genomic hybridization

Comparative genomic hybridization (CGH) was used to identify and probe sex chromosomes in several XY and WZ systems. Chromosomes were hybridized simultaneously with FluorX-labelled DNA of females and Cy3-labelled DNA of males in the presence of an excess of Cot-1 DNA or unlabelled DNA of the homogametic sex. CGH visualized the molecular differentiation of the X and Y in the house mouse, Mus musculus, and in Drosophila melanogaster: while autosomes were stained equally by both probes, the X and Y chromosomes were stained preferentially by the female-derived or the male-derived probe, respectively. There was no differential staining of the X and Y chromosomes in the fly Megaselia scalaris, indicating an early stage of sex chromosome differentiation in this species. In the human and the house mouse, labelled DNA of males in the presence of unlabelled DNA of females was sufficient to highlight Y chromosomes in mitosis and interphase. In WZ sex chromosome systems, the silkworm Bombyx mori, the flour moth Ephestia kuehniella, and the wax moth Galleria mellonella, the W chromosomes were identified by CGH in mitosis and meiosis. They were conspicuously stained by both female- and male-derived probes, unlike the Z chromosomes, which were preferentially stained by the male-derived probe in E. kuehniella only but were otherwise inconspicuous. The ratio of female:male staining and the pattern of staining along the W chromosomes was species specific. CGH shows that W chromosomes in these species are molecularly well differentiated from the Z chromosomes. The conspicuous binding of the male-derived probe to the W chromosomes is presumably due to an accumulation of common interspersed repetitive sequences. Received: 6 January 1999; in revised form: 28 January 1999 / Accepted: 11 February 1999     

Cytological characterization of sex chromosomes and ribosomal DNA location in Anastrepha species (Diptera, Tephritidae)

This paper reports a comparative analysis of heterochromatin organization in the sex chromosomes of the fruit fly Anastrepha. Mitotic chromosomes of eight Anastrepha species from different taxonomic groups were stained with DAPI and chromomycin A3 fluorochromes followed by C-banding. A specific sex-chromosome banding pattern was obtained for each of the analyzed species. Fluorescence in situ hybridization (FISH) was performed to investigate the chromosomal location of rDNA loci. In all cases the rDNA sequences were found to localize exclusively to the sex chromosomes. The results further extend the chromosomal knowledge of Anastrepha and allow a precise species identification.     

Subpopulation differentiation of Anthoxanthum alpinum (Poaceae) along an altitudinal gradient detected by random amplified polymorphic DNA

Random amplified polymorphic DNA (RAPD) phenotypes generated by 15 primers were scored in 5 subpopulatious of Anthoxanthum alpinum along an altitudinal gradient. Although few subpopulation-specific markers were found, a significant population differentiation was revealed by the use of principal factor analysis and Ward' s cluster analysis. The result was also confirmed by the significant positive correlation when comparing beth the matrix of similarity between the individuals within subpopulations and the matrix among subpopulations, as well as the matrix of distances and the differences of altitude between the subpopulations (Mantel test). This study suggests that the differences of altitude between subpopulation sites may result in the differences of phenologicalperiod for flowering and growth which restrict gene flow. Decontaminated thianthrene disproportion. Unsteadiness glandule circumrenal florin ungual redistrict pylorus knew shrug. 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应用RAPD技术研究阿尔卑斯山黄花茅居群内的遗传分化

应用RAPD技术,沿一个海拔梯度研究了阿尔卑斯山黄花茅自然居群的遗传变异和分化。实验 表明,虽然在亚居群间有很少的亚居群独有遗传标记的存在,但通过RAPD资料的聚类分析、主因子分 析以及相关分析观察到遗传分化沿海拔梯度发生,而且亚居群间的遗传分化和它们的海拔高度(地理距 离)呈有意义的正相关。研究结果暗示,在阿尔卑斯山的高山-亚高山过渡区,亚居群间的海拔高度差别 可能导致黄花茅开花和生长物候期的变化,而后者限制了亚居群间的基因流,从而引起居群内的遗传分化。     

Homologies between X and Y chromosomes detected by DNA probes: localisation and evolution.

We have isolated and characterized DNA probes that detect homologies between the X and Y chromosomes. Clone St25 is derived from the q13-q22 region of the X chromosome and recognizes a 98% homologous sequence on the Y chromosome. Y specific fragments were present in DNAs from 5 Yq-individuals and from 4 out of 7 XX males analysed. An X linked TaqI RFLP is detected with the St25 probe (33% heterozygosity) which should allow one to establish a linkage map including other polymorphic X-Y homologous sequences in this region and to compare it to a Y chromosome deletion map. Probe DXS31 located in Xp223-pter detects a 80% homologous sequence in the Y chromosome. The latter can be assigned to Yq11-qter outside the region which contains the Y specific satellite sequences. ACT1 and ACT2, the actin sequences present on the X and Y chromosomes respectively, have been cloned. No homology was detected between the X and Y derived fragments outside from the actin sequence. ACT2 and the Y specific sequence corresponding to DXS31 segregate together in a panel of Y chromosomes aberrations, and might be useful markers for the region important for spermatogenesis in Yq. Various primate species were analysed for the presence of sequences homologous to the three probes. Sequences detected by St25 and DXS31 are found only on the X chromosome in cercopithecoidae. The sequences which flank ACT2 detect in the same species autosomal fragments but no male specific fragments. It is suggested that the Y chromosome acquired genetic material from the X chromosome and from autosomes at various times during primate evolution.     

Karyotypes ofMegaselia scalaris (Diptera) wild-type and translocation strains

Making use of somatic pairing of homologous chromosome arms and of balanced translocations as cytogenetic markers, the three chromosome pairs of the phorid flyMegaselia scalaris have been identified and described. From measurements of the compliments a standard karyotype was constructed. Identification of the chromosomes allows cytogenetic, phenotypic and molecular markers to be assigned to specific chromosomes. Sex linkage of t(1;2) and t(2;3) translocations define chromosome 2 as the normal sex determining chromosome pair in our translocation strains, and therefore also, probably, in the wild-type strain from which they were derived. No differences between X and Y with respect to size of arms or C-bands were detected.     

Molecular differentiation of two sibling species of the black fly Simulium vittatum (Diptera: Simuliidae) based on random amplified polymorphic DNA.

Larvae of the black fly morphospecies Simulium vittatum from Colorado, Montana, Nebraska, and New Hampshire were cytologically identified as either the IS-7 or the IIIL-1 cytospecies. DNA was PCR amplified from cytotyped larvae using eight 10-mer primers, labeled with 33P, and run on polyacrylamide gels. The entire data set of 96 amplicons produced incomplete separation of the two cytospecies when subjected to neighbor-joining and maximum parsimony analyses. However, when analyzed within geographical regions, separate species status was supported. Bootstrap support for distinctness of the two cytospecies was best in Colorado where they were collected in true sympatry. The IS-7 cytospecies was more polymorphic in the western states, where it differed most from IIIL-1, which was most variable in the eastern states. The frequencies of the 17 most common amplicons in the two cytospecies were inversely correlated. A model of speciation derived from the molecular evidence suggests that IS-7 evolved in the west and spread eastward, whereas IIIL-1 later originated in the east and spread westward.     

Surface ultrastructure of third-instar Megaselia scalaris (Diptera: Phoridae)

We describe some ultrastructure of the third-instar Megaselia scalaris (Diptera: Phoridae) using scanning electron microscopy, with the cephalic segment, anterior spiracle and posterior spiracle being emphasized. This study provides the taxonomic information of this larval species, which may be useful to differentiate from other closely-related species.     

蛆症异蚤蝇减数分裂染色体观察

以蛹精巢和卵巢组织为材料,采用空气干燥法制备染色体标本,Giemsa和硝酸银分别染色,对蛆症异蚤蝇Megaselia scalaris减数分裂染色体行为进行研究。结果表明:蛆症异蚤蝇的染色体数目n=3,由2条中着丝粒染色体和1条端着丝粒染色体组成;粗线期,第2条二价体具有较强的嗜银性,可能为性染色体;晚粗线期,第1条二价体的同源染色体之间出现一条细线,类似于联会复合体;终变期,第2条二价体形成环状结构;晚终变期,在3条二价体染色体臂上均产生条带,根据二价体着丝粒处是否成环可以将3条二价体分开。     

Chromosome assignment of two cloned DNA probes hybridizing predominantly to human sex chromosomes

Summary In situ hybridization experiments were carried out with two clones, YACG 35 and 2.8, which had been selected from two genomic libraries strongly enriched for the human Y chromosome. Besides the human Y chromosome, both sequences strongly hybridized to the human X chromosome, with few minor binding sites on autosomes. In particular, on the X chromosome DNA from clone YACG 35 hybridized to the centromeric region and the distal part of the short arm (Xp2.2). On the Y chromosome, the sequence was assigned to one site situated in the border region between Yq1.1 and Yq1.2. DNA from clone 2.8 also hybridized to the centromeric region of the X and the distal part of the short arm (Xq2.2). On the Y, however, two binding sites were observed (Yp1.1 and Yq1.2). The findings indicate that sex chromosomal sequences may be localized in homologous regions (as suggested from meiotic pairing) but also at ectopic sites.     

Molecular differentiation of the Old World Culicoides imicola species complex (Diptera, Ceratopogonidae), inferred using random amplified polymorphic DNA markers

Samples of seven of the 10 morphological species of midges of the Culicoides imicola complex were considered. The importance of this species complex is connected to its vectorial capacity for African horse sickness virus (AHSV) and bluetongue virus (BTV). Consequently, the risk of transmission may vary dramatically, depending upon the particular cryptic species present in a given area. The species complex is confined to the Old World and our samples were collected in Southern Africa, Madagascar and the Ivory Coast. Genomic DNA of 350 randomly sampled individual midges from 19 populations was amplified using four 20-mer primers by the random amplified polymorphic DNA (RAPD) technique. One hundred and ninety-six interpretable polymorphic bands were obtained. Species-specific RAPD profiles were defined and for five species diagnostic RAPD fragments were identified. A high degree of polymorphism was detected in the species complex, most of which was observed within populations (from 64 to 76%). Principal coordinate analysis (PCO) and cluster analysis provided an estimate of the degree of variation between and within populations and species. There was substantial concordance between the taxonomies derived from morphological and molecular data. The amount and the different distributions of genetic (RAPD) variation among the taxa can be associated to their life histories, i.e. the abundance and distribution of the larval breeding sites and their seasonality.     

华山新麦草自然居群沿海拔梯度的遗传分化

华山新麦草为我国特有种 ,仅分布于陕西华山 ,是国家一级珍稀濒危植物和急需保护的农作物野生亲缘种。应用 RAPD技术 ,选取 8条引物对华山新麦草自然分布区的 3个山峪(种群 ) 1 1个样方 (亚居群 ) 79个华山新麦草个体的总 DNA进行随机扩增 ,共得到 65个RAPD位点。统计分析表明 ,在华山的 3个山峪 (居群 )中 ,黄埔峪 (居群 )与其它 2个山峪 (居群 )发生较显著的遗传分化 ;华山峪 6个亚居群的个体平均 RAPD位点数有随海拔的升高而下降的趋势 ;6个亚居群间的相似性系数也有随海拔的升高而下降的趋势 ;华山峪的高海拔亚居群和低海拔亚居群间表现出明显差异。主成分分析结果进一步证明了黄埔峪居群与华山峪居群和仙峪居群间、以及华山峪的高海拔亚居群与低海拔亚居群间已发生一定程度的分化。研究结果暗示海拔差异是导致华山新麦草自然居群遗传分化的主要因素 ,海拔差异造成的有限的基因流可能才是影响居群和亚居群遗传分化的主要因子 ,而亚居群内遗传变异程度则与该亚居群的所处的特定生境有关     

Gene rearrangements detected by nonradioactive digoxigenin-labeled DNA probes.

The configurations of immunoglobulin, T-cell receptor beta chain and bcl-2 genes were analyzed in lympho-proliferative disorders using nonradioactive digoxigenin-labeled probes. The studies demonstrated the reliability of digoxigenin-labeled probes for the detection of single-copy genes after Southern blotting of genomic DNA: 1 microgram and sometimes even 0.2 microgram of restriction endonuclease-digested DNA could be detected either by JH, C beta or bcl-2 probes. The intensity of the signal was consistently satisfactory, and there was no background problem. Control experiments showed neither false-positive nor false-negative results caused by the use of the nonradioactive detection system.     

Ultrastructure of the larval salivary glands of Megaselia scalaris Loew (Diptera,Phoridae)

Each of the paired salivary glands of third instar larvae of the humpbacked fly Megaselia scalaris is a bag-like structure with a short neck region from which a single duct emerges. The two ducts form a common duct that empties into the ventral region of the pharynx near the mouthparts. The wall of the glands and ducts consists of a simple squamous epithelium that rests upon a connective tissue layer. Cells in the neck are less flattened than those found elsewhere. The basal surfaces of the cells are infolded most deeply in the neck and the least in the duct. The apical surfaces of the cells possess microvilli except in the duct where the apices of the cells are covered by a complex extracellular layer. This layer displays circularly arranged folds that accommodate a thread-like supportive structure resembling taenidial threads of tracheae. Elaborate junctional complexes are associated with the lateral surfaces of the cells. Elements of these complexes include a zonula adherens, a series of pleated septate desmosomes, and conventional desmosomes. The cytoplasm of the glandular cells is filled with RER and other organelles normally seen in cells that export proteins and mucosubstances. Secretory material found in the lumens of the glands reacts only moderately with the PAS procedure but more strongly with alcian blue and methods that demonstrate proteins. The nuclei of the glandular cells contain single large nucleoli and polytene chromosomes whose banding is rather indistinct. Treatment with EDTA produces detrimental effects on all of the foregoing ultrastructural features of the glands and ducts.     

Use of random amplified polymorphic DNA (RAPD) for generating specific DNA probes for microorganisms

We report the rapid generation of DNA probes for several Azospirillum strains. This method does not require any knowledge of the genetics and/or the molecular biology of the organism (genome) to be investigated. The procedure is based on the generation of random amplified polymorphic DNA (RAPD) fingerprints using primers with an embedded restriction site. The amplification product(s) peculiar to one strain or common to two or more strains can be purified, cloned, sequenced and used as molecular probes in hybridization experiments for the detection and identification of microorganisms. We have tested this methodology in the nitrogen-fixing bacterium Azospirillum by amplyfing the total DNA extracted from several Azospirillum strains. We have used amplification bands with different specificity as molecular probes in hybridization experiments performed on amplified DNA. Results obtained have demonstrated the usefulness of this methodology for Azospirillum. Its use in microbial ecology studies as a general strategy to generate specific DNA probes is also discussed.     

DNA probes for the identification of members of the Simulium damnosum complex (Diptera: Simuliidae)

Genomic libraries in plasmid have been constructed from various sibling species of blackflies of the Simulium damnosum complex from West Africa. Three cloned repetitive sequences, which show variation in copy number between sibling species, have been isolated. These clones can be used as probes for the dot-blot identification of larvae, pupae or adults into the three main West African subcomplexes, i.e. damnosum, squamosum and sanctipauli subcomplexes. The sequences also show some intraspecific variation in copy number.     

Molecular aspects of sex differentiation

Mammalian sex differentiation involves the action of a cascade of genes. Discovery of the sex-determining region of the Y chromosome (SRY) marked the beginning of the delineation of the genes in the cascade. Studies of the genetics of mammalian sex reversal and the embryogenesis of the mice are essential in this endeavor. A number of genes involved in the pathway have been identified and all except one of these genes have a putative role in male sex differentiation. Besides SRY being the master switch in male sex differentiation the hierarchical relationship of the genes identified are far from being understood. Similarly, our knowledge of the genetic regulation of female sex differentiation is minimal. Differential screening and gene expression profiling bring a new dimension to the pursuit with the identification of a number of genes previously unknown to be involved in sex differentiation. Wider application of functional genomic techniques and introduction of proteomic analyses are expected to shed light to our understanding of this complicated developmental process.     

Origin and molecular organization of supernumerary chromosomes of Prochilodus lineatus (Characiformes, Prochilodontidae) obtained by DNA probes

In Prochilodus lineatus B-chromosomes are visualized as reduced size extra elements identified as microchromosomes and are variable in morphology and number. We describe the specific total probe (B-chromosome probe) in P. lineatus obtained by chromosome microdissection and a whole genomic probe (genomic probe) from an individual without B-chromosome. The specific B-chromosome was scraped and processed to obtain DNA with amplification by DOP-PCR, and so did the genomic probe DNA. Fluorescence in situ hybridization using the B-chromosome probe labeled with dUTP-Tetramethyl-rhodamine and the genomic probe labeled with digoxigenin-FITC permitted to establish that in this species supernumerary chromosomes with varying number and morphology had different structure of chromatin when compared to that of the regular chromosomes or A complement, since only these extra elements were labeled in the metaphases. The present findings suggest that modifications in the chromatin structure of B-chromosomes to differentiate them from the A chromosomes could occur along their dispersion in the individuals of the population.     

Estimation of population profiles of two strains of the fly Megaselia scalaris (Diptera: Phoridae) by bootstrap simulation

Based on experimental population profiles of strains of the fly Megaselia scalaris (Phoridae), the minimal number of sample profiles was determined that should be repeated by bootstrap simulation process in order to obtain a confident estimation of the mean population profile and present estimations of the standard error as a precise measure of the simulations made. The original data are from experimental populations founded with SR and R4 strains, with three replicates, which were kept for 33 weeks by serial transfer technique in a constant temperature room (25 +/- 1.0 degrees C). The variable used was population size and the model adopted for each profile was a stationary stochastic process. By these simulations, the three experimental population profiles were enlarged so as to determine minimum sample size. After sample size was determined, bootstrap simulations were made in order to calculate confidence intervals and to compare the mean population profiles of these two strains. The results show that with a minimum sample size of 50, stabilization of means begins.