Mouse cytosolic and mitochondrial deoxyribonucleotidases: cDNA cloning of the mitochondrial enzyme, gene structures, chromosomal mapping and comparison with the human orthologs

Abstract Two of the five known mammalian 5 0 -nucleotidases show a preference for the dephosphorylation of deoxynucleoside-5 0 -phosphates. One is a cytoplasmic enzyme (dNT-1), the other occurs in mitochondria (dNT-2). The human mitochondrial enzyme, recently discovered and cloned by us, is encoded by a nuclear gene located on chromosome 17 p11.2 in the critical region deleted in the Smith-Magenis syndrome (SMS), a genetic disease of unknown etiology. Looking for a model system to study the possible involvement of dNT-2 in the disease, we have cloned the cDNA of the mouse ortholog. The deduced protein sequence is 84% identical to the human ortholog, has a very basic NH 2 -terminus, a very high calculated probability of being imported into mitochondria and contains the DXDXT/V motif conserved among nucleotidases. Expression in Escherichia coli of the predicted processed form of the protein produced an active deoxyribonucleotidase. We also identified in genomic sequences present in the data base the structures of the murine genes for the cytosolic and mitochondrial deoxyribonucleotidases (Nt5c and Nt5m). PAC clones for the two loci were isolated from a library and used for chromosomal localization by fluorescent in situ hybridization. Both genes map on chromosome 11: Nt5c at 11E and Nt5m at 11B, demonstrating the presence of the dNT-2 locus in the mouse shaker-2 critical region, the murine counterpart of the human SMS region. We performed pair-wise dot-plot and PIP (percent identity plot) analyses of mouse and human deoxyribonucleotidase genes, and found a strong conservation that extends also to some intronic sequences of possible regulatory significance.

Comprehensive genetic and epigenetic analysis of sporadic meningioma for macro-mutations on 22q and micro-mutations within the NF2 locus

BACKGROUND: Meningiomas are the most common intracranial neoplasias, representing a clinically and histopathologically heterogeneous group of tumors. The neurofibromatosis type 2 (NF2) tumor suppressor is the only gene known to be frequently involved in early development of meningiomas. The objective of this study was to identify genetic and/or epigenetic factors contributing to the development of these tumors. A large set of sporadic meningiomas were analyzed for presence of 22q macro-mutations using array-CGH in order to identify tumors carrying gene dosage aberrations not encompassing NF2. The NF2 locus was also comprehensively studied for point mutations within coding and conserved non-coding sequences. Furthermore, CpG methylation within the NF2 promoter region was thoroughly analyzed. RESULTS: Monosomy 22 was the predominant finding, detected in 47% of meningiomas. Thirteen percent of the tumors contained interstitial/terminal deletions and gains, present singly or in combinations. We defined at least two minimal overlapping regions outside the NF2 locus that are small enough (~550 kb and ~250 kb) to allow analysis of a limited number of candidate genes. Bialleinactivationo the NF2 gne was detected in 36% of meningiomas. Among the monosomy 22 cases, no additional NF2 mutations could be identified in 35% (17 out of 49) of tumors. Furthermore, the majority of tumors (9 out of 12) with interstitial/terminal deletions did not have any detectable NF2 mutations. Methylation within the NF2 promoter region was only identified at a single CpG site in one tumor sample. CONCLUSION: We confirmed previous findings of pronounced differences in mutation frequency between different histopathological subtypes. There is a higher frequency of biallelic NF2 inactivation in fibroblastic (52%) compared to meningothelial (18%) tumors. The presence of macro-mutations on 22q also shows marked differences between fibroblastic (86%) and meningothelial (39%) subtypes. Thus, inactivation of NF2, often combined with the presence of macro-mutation on 22q, is likely not as important for the development of the meningothelial subtype, as opposed to the fibroblastic form. Analysis of 40 CpG sites distributed within 750 bp of the promoter region suggests that NF2 promoter methylation does not play a major role in meningioma development

Tissue-specific variation in DNA methylation levels along human chromosome 1

<p>Abstract</p> <p>Background</p> <p>DNA methylation is a major epigenetic modification important for regulating gene expression and suppressing spurious transcription. Most methods to scan the genome in different tissues for differentially methylated sites have focused on the methylation of CpGs in CpG islands, which are concentrations of CpGs often associated with gene promoters.</p> <p>Results</p> <p>Here, we use a methylation profiling strategy that is predominantly responsive to methylation differences outside of CpG islands. The method compares the yield from two samples of size-selected fragments generated by a methylation-sensitive restriction enzyme. We then profile nine different normal tissues from two human donors relative to spleen using a custom array of genomic clones covering the euchromatic portion of human chromosome 1 and representing 8% of the human genome. We observe gross regional differences in methylation states across chromosome 1 between tissues from the same individual, with the most striking differences detected in the comparison of cerebellum and spleen. Profiles of the same tissue from different donors are strikingly similar, as are the profiles of different lobes of the brain. Comparing our results with published gene expression levels, we find that clones exhibiting extreme ratios reflecting low relative methylation are statistically enriched for genes with high expression ratios, and <it>vice versa</it>, in most pairs of tissues examined.</p> <p>Conclusion</p> <p>The varied patterns of methylation differences detected between tissues by our methylation profiling method reinforce the potential functional significance of regional differences in methylation levels outside of CpG islands.</p

Hot Nano-particles in Polar or Paramagnetic Liquids Interact as Monopoles.

When neutral nano-particles are heated or cooled in a polar liquid, they will interact with each other as if they carry an electrostatic charge that is proportional to the temperature difference between the particle and the surrounding fluid. The same should hold for suspensions liquids of asymmetric ferromagnetic particles, in which case the heated nano-particles should behave as magnetic monopoles. However, the analogy with electrostatics/magnetostatics is not complete: heated/cooled nano-particles do not move under the influence of an applied homogeneous field. They should, however, interact as monopoles with each other and should move in inhomogeneous fields.This is the author accepted manuscript. The final version is available from the American Chemical Society via https://doi.org/10.1021/acs.jpcb.6b0184

Genetic predisposition to mosaic Y chromosome loss in blood.

Mosaic loss of chromosome Y (LOY) in circulating white blood cells is the most common form of clonal mosaicism1-5, yet our knowledge of the causes and consequences of this is limited. Here, using a computational approach, we estimate that 20% of the male population represented in the UK Biobank study (n = 205,011) has detectable LOY. We identify 156 autosomal genetic determinants of LOY, which we replicate in 757,114 men of European and Japanese ancestry. These loci highlight genes that are involved in cell-cycle regulation and cancer susceptibility, as well as somatic drivers of tumour growth and targets of cancer therapy. We demonstrate that genetic susceptibility to LOY is associated with non-haematological effects on health in both men and women, which supports the hypothesis that clonal haematopoiesis is a biomarker of genomic instability in other tissues. Single-cell RNA sequencing identifies dysregulated expression of autosomal genes in leukocytes with LOY and provides insights into why clonal expansion of these cells may occur. Collectively, these data highlight the value of studying clonal mosaicism to uncover fundamental mechanisms that underlie cancer and other ageing-related diseases.This research has been conducted using the UK Biobank Resource under application 9905 and 19808. This work was supported by the Medical Research Council [Unit Programme number MC_UU_12015/2]. Full study-specific and individual acknowledgements can be found in the supplementary information

Immune cells lacking Y chromosome show dysregulation of autosomal gene expression

Funder: Kjell och Märta Beijers Stiftelse (SE)Funder: Hjärnfonden; doi: http://dx.doi.org/10.13039/501100003792Funder: Cancerfonden; doi: http://dx.doi.org/10.13039/501100002794Funder: Vetenskapsrådet; doi: http://dx.doi.org/10.13039/501100004359Funder: Alzheimerfonden; doi: http://dx.doi.org/10.13039/501100008599Funder: Konung Gustaf V:s och Drottning Victorias Frimurarestiftelse (SE)Funder: Science for Life Laboratory (SE)Funder: Fundacja na rzecz Nauki Polskiej (PL)Funder: Uppsala UniversityAbstract: Epidemiological investigations show that mosaic loss of chromosome Y (LOY) in leukocytes is associated with earlier mortality and morbidity from many diseases in men. LOY is the most common acquired mutation and is associated with aberrant clonal expansion of cells, yet it remains unclear whether this mosaicism exerts a direct physiological effect. We studied DNA and RNA from leukocytes in sorted- and single-cells in vivo and in vitro. DNA analyses of sorted cells showed that men diagnosed with Alzheimer’s disease was primarily affected with LOY in NK cells whereas prostate cancer patients more frequently displayed LOY in CD4 + T cells and granulocytes. Moreover, bulk and single-cell RNA sequencing in leukocytes allowed scoring of LOY from mRNA data and confirmed considerable variation in the rate of LOY across individuals and cell types. LOY-associated transcriptional effect (LATE) was observed in ~ 500 autosomal genes showing dysregulation in leukocytes with LOY. The fraction of LATE genes within specific cell types was substantially larger than the fraction of LATE genes shared between different subsets of leukocytes, suggesting that LOY might have pleiotropic effects. LATE genes are involved in immune functions but also encode proteins with roles in other diverse biological processes. Our findings highlight a surprisingly broad role for chromosome Y, challenging the view of it as a “genetic wasteland”, and support the hypothesis that altered immune function in leukocytes could be a mechanism linking LOY to increased risk for disease

Genomic microarrays in the spotlight

Microarray-based comparative genomic hybridization (array-CGH) has emerged as a revolutionary platform, enabling the high-resolution detection of DNA copy number aberrations. In this article we outline the use and limitations of genomic clones, cDNA clones and PCR products as targets for genomic microarray construction. Furthermore, the applications and future aspects of these arrays for DNA copy number analysis in research and diagnostics, epigenetic profiling and gene annotation are discussed. These recent developments of genomic microarrays mark only the beginning of a new generation of high-resolution and high-throughput tools for genetic analysis

Development of NF2 gene specific, strictly sequence defined diagnostic microarray for deletion detection

Neurofibromatosis type 2 (NF2) is an autosomal dominant cancer syndrome caused by the biallelic inactivation of the neurofibromin 2 tumor suppressor gene ( NF2). Current molecular diagnostic methods for NF2 involve the detection of point mutations and/or microdeletions across the 100-kb locus from 22q12. Despite the fact that NF2 gene inactivating deletions occur in 25-30% of NF2 patients, the available approaches for high-resolution and high-throughput detection of deletions are underdeveloped. This need for improved methodology for gene copy number analysis is especially apparent when compared to a variety of methods available for accurate detection of point mutations. The microarray-based form of comparative genomic hybridization has been previously applied in the high-resolution analysis of gene copy number variation across large genomic regions. In this study we apply a PCR-based, strictly sequence-defined, repeat-free approach for the preparation of a diagnostic microarray for the detection of disease-causing deletions in the NF2 gene. The methodology is based on the preselection of target DNA by excluding redundant sequence within the NF2 locus using bioinformatics. This approach allows a significant increase in the resolution of deletion detection. The current average resolution of analysis across the NF2 locus is 23 kb. Therefore this NF2 gene-specific microarray is the first high-resolution tool for detection of diagnostically significant gene copy number aberrations. This microarray should now be applied in the analysis of an extensive series of NF2 patients, and hence we would like to call for such samples