[Avian cytogenetics goes functional] Third report on chicken genes and chromosomes 2015

High-density gridded libraries of large-insert clones using bacterial artificial chromosome (BAC) and other vectors are essential tools for genetic and genomic research in chicken and other avian species... Taken together, these studies demonstrate that applications of large-insert clones and BAC libraries derived from birds are, and will continue to be, effective tools to aid high-throughput and state-of-the-art genomic efforts and the important biological insight that arises from them

Characterization of candidate disease genes from human chromosomes 11g13 and 22q

Two regions of the human genome, 11q13 and 22q, have been shown to harbor numerous disease-related genes, among them tumor suppressor genes (TSGs). The aim of this project was to construct transcription maps within these chromosomal segments, and thus provide a basis for further analysis of some of the genes for their involvement in disease-related processes. The main strategy used in identification of candidate genes was large-scale genomic sequencing combined with "in silico" cloning. In total 27 genes were cloned, including human paralogs and orthologs in other organisms. The following diseases have been assigned to 11q13: Bardet-Biedl syndrome; spino-cerebellar ataxia type 5; familial paraganglioma type 2; insulin dependent diabetes mellitus; the translocation t(11;17) in B-cell non-Hodgkin's lymphoma; and multiple endocrine neo-plasia type 1 (MEN 1). Our aim was to identify genes in the vicinity of PYGM and FAU loci using a comparative genomic sequencing approach. We sequenced a PAC around the PYGM locus from human 11q13 and a cosmid from Fugu rubripes containing the Pygm gene. The region surrounding Pygm locus in F. Rubripes was not syntenic with the region neighboring the human PYGM gene. We established a transcriptional map around the human PYGM gene which includes: i) two genes previously localized to 11q13, PYGM and a zinc-finger protein (ZFM1) gene; ii) germinal center kinase (GCK) gene; iii) a novel human CDC25-like (HCDC25L) gene; iv) a dystrophia myotonica protein kinase-like (DMPKL) gene; and v) a novel ubiquitously expressed gene of unknown function (germinal center kinase neighboring gene, GCKNG). The GCKNG gene was also cloned by other groups and confirmed to be the MEN1 tumor suppressor gene. Deletions on 22q have been described in multiple tumors, which implies that this chromosome harbors TSGs. Previous deletion mapping studies in meningiomas have defined four candidate regions on 22q. From one of these the neurofibromatosis type 2 gene (NF2) was cloned and shown to be inactivated in a subset of meningiomas. However, further studies indicate that additional 22q-located gene(s) may be important in development of these and other tumors. We analyzed six genes from meningioma-deleted regions on 22q. These were: clathrin heavy chain 2 gene (CLH-22); beta-prime adaptin gene (BAM22); human homologue of C. elegans NIPSNAP gene 1 (NIPSNAP1); synaptogyrin 1 gene (SYNGR1a, SYNGR1b, SYNGR1c); human homolog of chicken TOM1 gene (TOM1); and SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily B, member 1 (SMARCB1a, SMARCB1b). For the majority of these human genes, human paralogs and mouse orthologs were also investigated. This resulted in the characterization of a novel family of genes called synaptogyrins (SYNGR1-4), as well as NIPSNAP2 and TOM1L genes in humans. The human SMARCB1 gene is a tumor suppressor gene located on 22q11.2 and it is inactivated in malignant rhabdoid tumors. We performed mutational analysis of the SMARCB1 gene as well as CLH22, BAM22, and TOM1 genes in meningiomas and schwannomas with negative results. The closest paralogs of three genes (CLH-22, BAM22, and SYNGR1) studied in this thesis are located on human chromosome 17. Furthermore, the available literature and searches of databases show that 13 pairs of paralogs are located on chromosome 17 and 22. We propose that this is a resuit of an ancient chromosomal duplication event

Protein identification and quantification by two-dimensional infrared spectroscopy: Implications for an all-optical proteomic platform

Electron-vibration-vibration two-dimensional coherent spectroscopy, a variant of 2DIR, is shown to be a useful tool to differentiate a set of 10 proteins based on their amino acid content. Two-dimensional vibrational signatures of amino acid side chains are identified and the corresponding signal strengths used to quantify their levels by using a methyl vibrational feature as an internal reference. With the current apparatus, effective differentiation can be achieved in four to five minutes per protein, and our results suggest that this can be reduced to <1 min per protein by using the same technology. Finally, we show that absolute quantification of protein levels is relatively straightforward to achieve and discuss the potential of an all-optical high-throughput proteomic platform based on two-dimensional infrared spectroscopic measurements

Haplotype selection as an adaptive mechanism in the protozoan pathogen Leishmania donovani

International audienceThe parasite Leishmania donovani causes a fatal disease termed visceral leishmaniasis. The process through which the parasite adapts to environmental change remains largely unknown. Here we show that aneuploidy is integral for parasite adaptation and that karyotypic fluctuations allow for selection of beneficial haplotypes, which impact transcriptomic output and correlate with phenotypic variations in proliferation and infectivity. To avoid loss of diversity following karyotype and haplotype selection, L. donovani utilizes two mechanisms: polyclonal selection of beneficial haplotypes to create coexisting subpopulations that preserve the original diversity, and generation of new diversity as aneuploidy-prone chromosomes tolerate higher mutation rates. Our results reveal high aneuploidy turnover and haplotype selection as a unique evolutionary adaptation mechanism that L. donovani uses to preserve genetic diversity under strong selection. This unexplored process may function in other human diseases, including fungal infection and cancer, and stimulate innovative treatment options

Global and single-nucleotide resolution detection of 7-methylguanosine in RNA

RNA modifications, including N-7-methylguanosine (m7G), are pivotal in governing RNA stability and gene expression regulation. The accurate detection of internal m7G modifications is of paramount significance, given recent associations between altered m7G deposition and elevated expression of the methyltransferase METTL1 in various human cancers. The development of robust m7G detection techniques has posed a significant challenge in the field of epitranscriptomics. In this study, we introduce two methodologies for the global and accurate identification of m7G modifications in human RNA. We introduce borohydride reduction sequencing (Bo-Seq), which provides base resolution mapping of m7G modifications. Bo-Seq achieves exceptional performance through the optimization of RNA depurination and scission, involving the strategic use of high concentrations of NaBH4, neutral pH and the addition of 7-methylguanosine monophosphate (m7GMP) during the reducing reaction. Notably, compared to NaBH4-based methods, Bo-Seq enhances the m7G detection performance, and simplifies the detection process, eliminating the necessity for intricate chemical steps and reducing the protocol duration. In addition, we present an antibody-based approach, which enables the assessment of m7G relative levels across RNA molecules and biological samples, however it should be used with caution due to limitations associated with variations in antibody quality between batches. In summary, our novel approaches address the pressing need for reliable and accessible methods to detect RNA m7G methylation in human cells. These advancements hold the potential to catalyse future investigations in the critical field of epitranscriptomics, shedding light on the complex regulatory roles of m7G in gene expression and its implications in cancer biology.</p

Variable degree of mosaicism for tetrasomy 18p in phenotypically discordant monozygotic twins-Diagnostic implications

Background: Phenotypically discordant monozygotic twins (PDMZTs) offer a unique opportunity to study post-zygotic genetic variation and provide insights into the linkage between genotype and phenotype. We report a comprehensive analysis of a pair of PDMZTs. Methods: Dysmorphic features and delayed neuro-motor development were observed in the proband, whereas her twin sister was phenotypically normal. Four tissues (blood, skin, hair follicles, and buccal mucosa) from both twins were studied using four complementary methods, including whole-exome sequencing, karyotyping, array CGH, and SNP array. Results: In the proband, tetrasomy 18p affecting all studied tissues except for blood was identified. Karyotyping of fibroblasts revealed isochromosome 18p [i(18p)] in all metaphases. The corresponding analysis of the phenotypically normal sister surprisingly revealed low-level mosaicism (5.4%) for i(18p) in fibroblasts. Conclusion: We emphasize that when mosaicism is suspected, multiple tissues should be studied and we highlight the usefulness of non-invasive sampling of hair follicles and buccal mucosa as a convenient source of non-mesoderm-derived DNA, which complements the analysis of mesoderm using blood. Moreover, low-level mosaic tetrasomy 18p is well tolerated and such low-level mosaicism, readily detected by karyotyping, can be missed by other methods. Finally, mosaicism for low-level tetrasomy 18p might be more common in the general population than it is currently recognized, due to detection limitations

Genome and transcriptome analysis of the Mesoamerican common bean and the role of gene duplications in establishing tissue and temporal specialization of genes

Background: Legumes are the third largest family of angiosperms and the second most important crop class. Legume genomes have been shaped by extensive large-scale gene duplications, including an approximately 58 million year old whole genome duplication shared by most crop legumes. Results: We report the genome and the transcription atlas of coding and non-coding genes of a Mesoamerican genotype of common bean (Phaseolus vulgaris L., BAT93). Using a comprehensive phylogenomics analysis, we assessed the past and recent evolution of common bean, and traced the diversification of patterns of gene expression following duplication. We find that successive rounds of gene duplications in legumes have shaped tissue and developmental expression, leading to increased levels of specialization in larger gene families. We also find that many long non-coding RNAs are preferentially expressed in germ-line-related tissues (pods and seeds), suggesting that they play a significant role in fruit development. Our results also suggest that most bean-specific gene family expansions, including resistance gene clusters, predate the split of the Mesoamerican and Andean gene pools. Conclusions: The genome and transcriptome data herein generated for a Mesoamerican genotype represent a counterpart to the genomic resources already available for the Andean gene pool. Altogether, this information will allow the genetic dissection of the characters involved in the domestication and adaptation of the crop, and their further implementation in breeding strategies for this important crop.This work was supported by Ibero-American Programme for Science, Technology and Development - CYTED (PhasIbeAm project); Spanish Government - Ministry of Economy and Competitiveness (EUI2009-04052, BIO2011-26205); Brazilian Government — National Council for Scientific and Technological Development - CNPq/Prosul (490725/2010-4) and Brazilian Agricultural Research Corporation - Embrapa (MP2-0212000050000); Ministerio de Ciencia, Tecnología e Innovación Productiva de la República Argentina; the European Molecular Biology Laboratory; Consejo Nacional de Ciencia y Tecnología - Conacyt, Mexico (J010-214-2009) for financial support to undertake parts of research presented in this study. We acknowledge support of the Spanish Ministry of Economy and Competitiveness, ‘Centro de Excelencia Severo Ochoa 2013-2017’, SEV-2012-0208 and Instituto Nacional de Bioinformatica (INB, Project PT13/0001/0021, ISCIII — Subdirección General de Evaluación y Fomento de la Investigación/FEDER “Una Manera de hacer Europa”)