Microsatellite Organization in the Grasshopper Abracris flavolineata (Orthoptera: Acrididae) Revealed by FISH Mapping: Remarkable Spreading in the A and B Chromosomes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Delimiting the Origin of a B Chromosome by FISH Mapping, Chromosome Painting and DNA Sequence Analysis in Astyanax paranae (Teleostei, Characiformes)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

FISH mapping of 10 canine BAC clones harbouring genes and microsatellites in the arctic fox and the Chinese raccoon dog genomes

Cytogenetic mapping of the arctic fox and the Chinese raccoon dog were performed using a set of canine probes derived from the Bacterial Artificial Chromosome (BAC) library. Altogether, 10 BAC clones containing sequences of selected genes (PAX3, HBB, ATP2A2, TECTA, PIT1, ABCA4, ESR2, TPH1, HTR2A, MAOA) and microsatellites were mapped by fluorescence in situ hybridization (FISH) experiments to chromosomes of the canids studied. At present, the cytogenetic map on the arctic fox and Chinese raccoon dog consists of 45 loci each. Chromosomal localization of the BAC clones was in agreement with data obtained by earlier independent comparative chromosome painting. However, two events of telomere-to-centromere inversions were tentatively identified while compared with assignments in the dog karyotype.

Assignment of chromosome rearrangements between X chromosomes of human and cattle by laser microdissection and Zoo-FISH

Cross-species fluorescence in-situ hybridization (Zoo-FISH) was performed on cattle metaphase spreads using Homo sapiens X chromosome (HSAX) painting probes specific for the p- and q-arms to identify the cytogenetic location of a chromosome breakpoint between HSAX and the Bos taurus X chromosome (BTAX). The existence of a breakpoint is strongly suggested by recent radiation hybrid and FISH mapping results. Hybridization probes were generated by microdissection of HSAX p- and q-arms using the contact-free technology of Laser Microdissection and Pressure Catapulting (LMPC), amplification of the isolated chromosome material by DOP-PCR, and labelling of the PCR products with digoxigenin in a secondary PCR. Independent Zoo-FISH of the two painting probes on bovine metaphase chromosomes (detected by antidigoxigenin-fluorescein) resulted in clear hybridization signals on BTAX. A breakpoint was identified between HSAXp and HSAXq on BTAX, and narrowed down between the G-bands BTAXq25 and BTAXq26. The assumed centromere transposition between HSAX and BTAX associated with the rearranged chromosome segments is supported by cytogenetic assignments of the genes BGN and G6PD to BTAX.

Tracking the evolution of sex chromosome systems in Melanoplinae grasshoppers through chromosomal mapping of repetitive DNA sequences

Background: The accumulation of repetitive DNA during sex chromosome differentiation is a common feature of many eukaryotes and becomes more evident after recombination has been restricted or abolished. The accumulated repetitive sequences include multigene families, microsatellites, satellite DNAs and mobile elements, all of which are important for the structural remodeling of heterochromatin. In grasshoppers, derived sex chromosome systems, such as neo-XY♂/XX♀ and neo-X1X2Y♂/X 1X1X2X2♀, are frequently observed in the Melanoplinae subfamily. However, no studies concerning the evolution of sex chromosomes in Melanoplinae have addressed the role of the repetitive DNA sequences. To further investigate the evolution of sex chromosomes in grasshoppers, we used classical cytogenetic and FISH analyses to examine the repetitive DNA sequences in six phylogenetically related Melanoplinae species with X0♂/XX♀, neo-XY♂/XX♀ and neo-X1X2Y♂/X1X1X 2X2♀ sex chromosome systems. Results: Our data indicate a non-spreading of heterochromatic blocks and pool of repetitive DNAs (C 0 t-1 DNA) in the sex chromosomes; however, the spreading of multigene families among the neo-sex chromosomes of Eurotettix and Dichromatos was remarkable, particularly for 5S rDNA. In autosomes, FISH mapping of multigene families revealed distinct patterns of chromosomal organization at the intra- and intergenomic levels. Conclusions: These results suggest a common origin and subsequent differential accumulation of repetitive DNAs in the sex chromosomes of Dichromatos and an independent origin of the sex chromosomes of the neo-XY and neo-X1X2Y systems. Our data indicate a possible role for repetitive DNAs in the diversification of sex chromosome systems in grasshoppers. © 2013Palacios-Gimenez et al.; licensee BioMed Central Ltd.

Consolidation of the genetic and cytogenetic maps of turbot (Scophthalmus maximus) using FISH with BAC clones

Bacterial artificial chromosomes (BAC) have been widely used for fluorescence in situ hybridization (FISH) mapping of chromosome landmarks in different organisms, including a few in teleosts. In this study, we used BAC-FISH to consolidate the previous genetic and cytogenetic maps of the turbot (Scophthalmus maximus), a commercially important pleuronectiform. The maps consisted of 24 linkage groups (LGs) but only 22 chromosomes. All turbot LGs were assigned to specific chromosomes using BAC probes obtained from a turbot 5x genomic BAC library. It consisted of 46,080 clones with inserts of at least 100 kb and < 5 % empty vectors. These BAC probes contained gene-derived or anonymous markers, most of them linked to quantitative trait loci (QTL) related to productive traits. BAC clones were mapped by FISH to unique marker-specific chromosomal positions, which showed a notable concordance with previous genetic mapping data. The two metacentric pairs were cytogenetically assigned to LG2 and LG16, and the nucleolar organizer region (NOR)-bearing pair was assigned to LG15. Double-color FISH assays enabled the consolidation of the turbot genetic map into 22 linkage groups by merging LG8 with LG18 and LG21 with LG24. In this work, a first-generation probe panel of BAC clones anchored to the turbot linkage and cytogenetical map was developed. It is a useful tool for chromosome traceability in turbot, but also relevant in the context of pleuronectiform karyotypes, which often show small hardly identifiable chromosomes. This panel will also be valuable for further integrative genomics of turbot within Pleuronectiformes and teleosts, especially for fine QTL mapping for aquaculture traits, comparative genomics, and whole-genome assembly.

黑麂和费氏麂四种卫星DNA的克隆特征和染色体定位

近年来,分子细胞遗传学研究已基本证实了染色体的串联融合(端粒一着丝粒融合)是麂属动物核型演化的主要重排方式.尽管染色体串联融合的分子机制还不清楚,但通过染色体的非同源重组,着丝粒区域的卫星DNA被认为可能介导了染色体的融合.以前的研究发现在赤麂和小麂染色体的大部分假定的串联融合位点处存在着非随机分布的卫星DNA.然而在麂属的其他物种中,这些卫星DNA的组成以及在基因组中的分布情况尚未被研究.本研究从黑麂和费氏麂基因组中成功地克隆了4种卫星DNA (BMC5、BM700、BM1.1k和FM700),并分析了这些卫星克隆的特征以及在小麂、黑麂、贡山麂和费氏麂染色体上的定位情况.结果表明,卫星Ⅰ和Ⅱ DNA (BMC5,BM700和FM700)的信号除了分布在这些麂属动物染色体的着丝粒区域外,也间隔地分布在这些物种的染色体臂上.其研究结果为黑麂、费氏麂和贡山麂的染色体核型也是从一个2n=70的共同祖先核型通过一系列的串联融合进化而来的假说提供了直接的证据.

麂属动物以及六带犰狳DNA重复序列家族的克隆、序列分析和染色体定位

1．黑麂和费氏麂卫星DNA的克隆、序列分析和染色体定位 麂属动物在很短的时间内经历了快速的物种辐射，并且种间染色体数目存在巨大差异，是研究动物核型进化和物种起源的理想模型。近二十年来的分子细胞遗传学研究已基本上证实染色体串联融合(端粒－着丝粒融合)是麂属动物核型演化的主要染色体重排方式。尽管染色体串联融合的分子机制仍不清楚，但研究提示着丝粒区域的卫星DNA可能介导染色体的非同源重组。因此，着丝粒卫星DNA的克隆、分析序列以及染色体定位研究不仅有助于阐明麂属染色体核型演化规律，还可能揭示染色体串联融合的分子机制。迄今为止，上述研究工作已经在赤麂、小麂和小麂台湾亚种开展过。但是，尚无有关黑麂、费氏麂和贡山麂卫星 DNA 克隆、序列分析以及染色体定位研究的报道。 在本研究中，我成功地克隆了黑麂的卫星DNA I、II和IV，分别命名为BMC5、BM700和BM1.1k，并且从费氏麂中克隆了卫星DNA II，命名为FM700。对这些卫星DNA克隆进行序列分析，并将这些克隆探针分别与黑麂、费氏麂、贡山麂和小麂的染色体杂交。研究结果表明： 1)黑麂的卫星DNA I(BMC5)与小麂卫星DNA I(C5)序列高度相似，并且在小麂、黑麂、费氏麂和贡山麂染色体上的大部分串联融合位点处均有分布，因此卫星DNA I可能代表着染色体发生串联融合后保存下来，来源于麂属动物祖先染色体着丝粒的一种卫星DNA。卫星DNA I在这四种麂属动物染色体上的分布也表明黑麂、费氏麂和贡山麂与赤麂的核型演化过程相似，很可能从一个2n ＝ 70的共同祖先通过一系列的串联易位进化而来。 2) 将卫星DNA II（BM700和FM700）克隆探针分别杂交到黑麂和费氏麂的染色体上，只检测到几对间隔分布的信号。这提示在核型进化过程中不同卫星DNA间可能发生了广泛的重组，从而导致卫星DNA II大量丢失。大部分重组断裂位点可能位于卫星DNA I 与卫星DNA II之间，或者在卫星DNA II 区域内。 2．六带犰狳重复序列家族的克隆、序列分析和染色体定位 六带犰狳属于犰狳科、贫齿目，是六带犰狳属中唯一的一个代表物种。系统发育研究认为贫齿目与非洲兽总目是有胎盘哺乳动物中最原始的两个类群。C显带结果揭示六带犰狳30%的基因组是由组成性异染色质构成的，并且C带分布的位置也较复杂，提示在六带犰狳基因组中存在多种重复序列元件。 为了研究六带犰狳异染色质的组成，我从六带犰狳的基因组中克隆了七种位点特异性的重复序列。根据测序结果以及它们在染色体上的分布，将这些重复序列分为五个重复序列家族。其中AMD-EcoRI 837与AMD-BglII 811的序列相似，都是由大小约116 bp的单位组成，分布在大多数染色体的着丝粒区域，同时在一些染色体臂也有分布。AMD-EcoRI 832，AMD-EcoRI 836和AMD-EcoRI 934是特定染色体的重复序列，并且都分布于着丝粒区域。另外，AMD-BglII 634，AMD-EcoRI 731两个克隆都属于长散在分布重复序列(L1)，倾向于分布在G带阳性、富含AT碱基的区域，并且这两种重复序列在染色体上的定位与C带阳性的非着丝粒的异染色质区域很相似。本研究提供了六带犰狳异染色质区域的部分基因组信息，并且这些重复序列家族也可以用于研究六带犰狳及其近缘物种的系统发育关系。

Heterochromatin, Sex Chromosomes and rRNA Gene Clusters in Coprophanaeus Beetles (Coleoptera, Scarabaeidae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

B chromosome in the beetle Coprophanaeus cyanescens (Scarabaeidae): emphasis in the organization of repetitive DNA sequences

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Dinâmica evolutiva dos clusters de pequenos RNAs nucleares (RNAsn) nos cariótipos de espécies de gafanhotos com ênfase em Acrididae

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Breakpoint analysis of human chromosome 3 inversions during hominoid evolution

Comparative fluorescence in situ hybridization (FISH) mapping revealed four large DNA segments which have been conserved in their entirety between human chromosome 3 and Bornean orangutan chromosome 2 as well as three evolutionary breakpoints which distinguish between the human and Bornean orangutan chromosome forms. Examination of the structural and functional features of evolutionary breakpoints provides new insights into the possible effects of evolutionary rearrangements on genome function and the relationship between human chromosome pathology and evolution. FISH of human BAC clones which were assesssed in human genomic sequence to primate chromosomes, combined with precise breakpoint localizations by polymerase chain reaction (PCR) analysis of flow-sorted chromosomes and in silico analysis, were used to characterize the evolutionary breakpoints. None of the three breakpoints studied disrupts a validated gene(s), however they are all associated with segmental duplications. At least eleven DNA segments (&a

Chromosomal assignment of the canine melanophilin gene (MLPH): a candidate gene for coat color dilution in Pinschers

Pinschers affected by coat color dilution show a specific pigmentation phenotype. The dilute pigmentation phenotype leads to a silver-blue appearance of the eumelanin-containing fur and a pale sandy color of pheomelanin-containing fur. In Pinscher breeding, dilute black-and-tan dogs are called "blue," and dilute red or brown animals are termed "fawn" or "Isabella fawn." Coat color dilution in Pinschers is sometimes accompanied by hair loss and a recurrent infection of the hair follicles. In human and mice, several well-characterized genes are responsible for similar pigment variations. To investigate the genetic cause of the coat color dilution in Pinschers, we isolated BAC clones containing the canine ortholog of the known murine color dilution gene Mlph. RH mapping of the canine MLPH gene was performed using an STS marker derived from BAC sequences. Additionally, one MLPH BAC clone was used as probe for FISH mapping, and the canine MLPH gene was assigned to CFA25q24.

Unlocking pathology archives for molecular genetic studies: a reliable method to generate probes for chromogenic and fluorescent in situ hybridization

Chromogenic (CISH) and fluorescent ( FISH) in situ hybridization have emerged as reliable techniques to identify amplifications and chromosomal translocations. CISH provides a spatial distribution of gene copy number changes in tumour tissue and allows a direct correlation between copy number changes and the morphological features of neoplastic cells. However, the limited number of commercially available gene probes has hindered the use of this technique. We have devised a protocol to generate probes for CISH that can be applied to formalin-fixed, paraffin-embedded tissue sections (FFPETS). Bacterial artificial chromosomes ( BACs) containing fragments of human DNA which map to specific genomic regions of interest are amplified with phi 29 polymerase and random primer labelled with biotin. The genomic location of these can be readily confirmed by BAC end pair sequencing and FISH mapping on normal lymphocyte metaphase spreads. To demonstrate the reliability of the probes generated with this protocol, four strategies were employed: (i) probes mapping to cyclin D1 (CCND1) were generated and their performance was compared with that of a commercially available probe for the same gene in a series of 10 FFPETS of breast cancer samples of which five harboured CCND1 amplification; (ii) probes targeting cyclin-dependent kinase 4 were used to validate an amplification identified by microarray-based comparative genomic hybridization (aCGH) in a pleomorphic adenoma; (iii) probes targeting fibroblast growth factor receptor 1 and CCND1 were used to validate amplifications mapping to these regions, as defined by aCGH, in an invasive lobular breast carcinoma with FISH and CISH; and (iv) gene-specific probes for ETV6 and NTRK3 were used to demonstrate the presence of t(12; 15)(p12; q25) translocation in a case of breast secretory carcinoma with dual colour FISH. In summary, this protocol enables the generation of probes mapping to any gene of interest that can be applied to FFPETS, allowing correlation of morphological features with gene copy number.