DNA replication stress restricts ribosomal DNA copy number

Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100–200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how “normal” copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a “normal” rDNA copy number

RNAseq Analyses Identify Tumor Necrosis Factor-Mediated Inflammation as a Major Abnormality in ALS Spinal Cord

ALS is a rapidly progressive, devastating neurodegenerative illness of adults that produces disabling weakness and spasticity arising from death of lower and upper motor neurons. No meaningful therapies exist to slow ALS progression, and molecular insights into pathogenesis and progression are sorely needed. In that context, we used high-depth, next generation RNA sequencing (RNAseq, Illumina) to define gene network abnormalities in RNA samples depleted of rRNA and isolated from cervical spinal cord sections of 7 ALS and 8 CTL samples. We aligned \u3e50 million 2X150 bp paired-end sequences/sample to the hg19 human genome and applied three different algorithms (Cuffdiff2, DEseq2, EdgeR) for identification of differentially expressed genes (DEG’s). Ingenuity Pathways Analysis (IPA) and Weighted Gene Co-expression Network Analysis (WGCNA) identified inflammatory processes as significantly elevated in our ALS samples, with tumor necrosis factor (TNF) found to be a major pathway regulator (IPA) and TNFα-induced protein 2 (TNFAIP2) as a major network “hub” gene (WGCNA). Using the oPOSSUM algorithm, we analyzed transcription factors (TF) controlling expression of the nine DEG/hub genes in the ALS samples and identified TF’s involved in inflammation (NFkB, REL, NFkB1) and macrophage function (NR1H2::RXRA heterodimer). Transient expression in human iPSC-derived motor neurons of TNFAIP2 (also a DEG identified by all three algorithms) reduced cell viability and induced caspase 3/7 activation. Using high-density RNAseq, multiple algorithms for DEG identification, and an unsupervised gene co-expression network approach, we identified significant elevation of inflammatory processes in ALS spinal cord with TNF as a major regulatory molecule. Overexpression of the DEG TNFAIP2 in human motor neurons, the population most vulnerable to die in ALS, increased cell death and caspase 3/7 activation. We propose that therapies targeted to reduce inflammatory TNFα signaling may be helpful in ALS patients

Applications of microarray technology in breast cancer research

Microarrays provide a versatile platform for utilizing information from the Human Genome Project to benefit human health. This article reviews the ways in which microarray technology may be used in breast cancer research. Its diverse applications include monitoring chromosome gains and losses, tumour classification, drug discovery and development, DNA resequencing, mutation detection and investigating the mechanism of tumour development

An integrated genetic map of the pearl locus of mouse chromosome 13.

We have used a Mus domesticus/spretus congenic animal and two interspecific backcross panels to map genetically 30 sequence-tagged sites (STSs) and 13 genes to the vicinity of the pearl locus on mouse chromosome 13. The STSs defining the mapped region are from D13Mit9 to D13Mit37, spanning 10.6 cM. Genes mapped to this region include Versican (Cspg2), GTPase activating protein (Rasa), dihydrofolate reductase (Dhfr), arylsulfatase (As-1), thrombin receptor (Cf2r), hexosaminidase b(Hexb), 3-hydroxy-3-methylglutaryl coenzyme A reductase (Hmgcr), microtubule associated protein 5/1b (Mtap5), phosphodiesterase (Pde), phosphatidylinositol 3' kinase (Pik3rl), rat integrin a1-subunit (Itga1), collagen receptor a2-subunit (Itga2), and 5-hydroxytryptamine 1a receptor (Htr1a). This high resolution genetic map of the pearl region of chromosome 13 establishes the order of multiple markers, including genes whose human homologs are located within a limited region of human chromosome 5, with respect to the phenotypic anchor marker pearl

An integrated genetic map of the pearl locus of mouse chromosome 13.

We have used a Mus domesticus/spretus congenic animal and two interspecific backcross panels to map genetically 30 sequence-tagged sites (STSs) and 13 genes to the vicinity of the pearl locus on mouse chromosome 13. The STSs defining the mapped region are from D13Mit9 to D13Mit37, spanning 10.6 cM. Genes mapped to this region include Versican (Cspg2), GTPase activating protein (Rasa), dihydrofolate reductase (Dhfr), arylsulfatase (As-1), thrombin receptor (Cf2r), hexosaminidase b(Hexb), 3-hydroxy-3-methylglutaryl coenzyme A reductase (Hmgcr), microtubule associated protein 5/1b (Mtap5), phosphodiesterase (Pde), phosphatidylinositol 3' kinase (Pik3rl), rat integrin a1-subunit (Itga1), collagen receptor a2-subunit (Itga2), and 5-hydroxytryptamine 1a receptor (Htr1a). This high resolution genetic map of the pearl region of chromosome 13 establishes the order of multiple markers, including genes whose human homologs are located within a limited region of human chromosome 5, with respect to the phenotypic anchor marker pearl