Mouse Ribosomal RNA Genes Contain Multiple Differentially Regulated Variants

Previous cytogenetic studies suggest that various rDNA chromosomal loci are not equally active in different cell types. Consistent with this variability, rDNA polymorphism is well documented in human and mouse. However, attempts to identify molecularly rDNA variant types, which are regulated individually (i.e., independent of other rDNA variants) and tissue-specifically, have not been successful. We report here the molecular cloning and characterization of seven mouse rDNA variants (v-rDNA). The identification of these v-rDNAs was based on restriction fragment length polymorphisms (RFLPs), which are conserved among individuals and mouse strains. The total copy number of the identified variants is less than 100 and the copy number of each individual variant ranges from 4 to 15. Sequence analysis of the cloned v-rDNA identified variant-specific single nucleotide polymorphisms (SNPs) in the transcribed region. These SNPs were used to develop a set of variant-specific PCR assays, which permitted analysis of the v-rDNAs' expression profiles in various tissues. These profiles show that three v-rDNAs are expressed in all tissues (constitutively active), two are expressed in some tissues (selectively active), and two are not expressed (silent). These expression profiles were observed in six individuals from three mouse strains, suggesting the pattern is not randomly determined. Thus, the mouse rDNA array likely consists of genetically distinct variants, and some are regulated tissue-specifically. Our results provide the first molecular evidence for cell-type-specific regulation of a subset of rDNA

Bacterial pathogen adaptation during human infections

Like all organisms on earth, bacteria must adapt to changes in their environment to survive. Thus, discovering bacterial adaptations reveals the tools bacteria use to be successful. Identifying how pathogenic bacteria adapt during infections can consequently identify the tools bacteria use to cause disease, and therapy design can then consider inhibiting these tools to treat or prevent infections. Here, the ways in which two worldwide human intestinal pathogens, Campylobacter jejuni and Enterotoxigenic E. coli (ETEC), adapt to the human host during infections are explored. Bacteria were studied directly in infected samples from controlled human infection models. In C. jejuni, genetic adaptations that were selected for during acute and persistent human infections identified the role of a previously uncharacterized flagellar modification gene during persistence. In ETEC, the bacteria’s ability to sense oxygen was linked to global virulence gene expression in human infection samples as well as biofilm formation. As environmental ETEC biofilms are associated with seasonal ETEC epidemics, oxygen sensing likely contributes to human infection inside and outside of the host. Together, these data demonstrate the scope of pathogen adaptation during infections, identified new targetable virulence factors, and can thus aid the design of new therapiesMicrobiolog

Genetic regulation of the tissue specific expression of Pyruvate kinase

SIGLEAvailable from British Library Document Supply Centre- DSC:D82962 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

Run-Off Replication of Host-Adaptability Genes Is Associated with Gene Transfer Agents in the Genome of Mouse-Infecting Bartonella grahamii

The genus Bartonella comprises facultative intracellular bacteria adapted to mammals, including previously recognized and emerging human pathogens. We report the 2,341,328 bp genome sequence of Bartonella grahamii, one of the most prevalent Bartonella species in wild rodents. Comparative genomics revealed that rodent-associated Bartonella species have higher copy numbers of genes for putative host-adaptability factors than the related human-specific pathogens. Many of these gene clusters are located in a highly dynamic region of 461 kb. Using hybridization to a microarray designed for the B. grahamii genome, we observed a massive, putatively phage-derived run-off replication of this region. We also identified a novel gene transfer agent, which packages the bacterial genome, with an over-representation of the amplified DNA, in 14 kb pieces. This is the first observation associating the products of run-off replication with a gene transfer agent. Because of the high concentration of gene clusters for host-adaptation proteins in the amplified region, and since the genes encoding the gene transfer agent and the phage origin are well conserved in Bartonella, we hypothesize that these systems are driven by selection. We propose that the coupling of run-off replication with gene transfer agents promotes diversification and rapid spread of host-adaptability factors, facilitating host shifts in Bartonella

Paternal Effects on Metabolism in Mammals: A Dissertation

The following work demonstrates that paternal diet controls medically important metabolic phenotypes in offspring. We observe transmission of dietary information to the zygote via sperm, and this information evades reprogramming that typically occurs after fertilization. Cytosine methylation is implicated as a major contributor to meiotic epigenetic inheritance in several transgenerational phenomena. Our extensive characterization of the sperm methylome reveals that diet does not significantly affect methylation patterns. However, we find that extensive epivariability in the sperm epigenome makes important contributions to offspring variation. Importantly, coordinate cytosine methylation and copy number changes over the ribosomal DNA locus contributes to variation in offspring metabolism. Thus, rDNA variability acts independently of postadolescent paternal diet to influence offspring metabolism. Therefore, at least two mechanisms exist for epigenetically controlling offspring metabolism: stochastic epivariation and diet acting by an unknown mechanism to further modulate metabolism. This work argues that an offspring\u27s phenotype can no longer be viewed solely as the result of genetic interactions with the developmental environment - the additional influences of paternal environment and inherited epigenetic variability must also be considered. These findings reveal novel contributions to metabolism that could revolutionize how we think about the risk factors for human health and disease

Novel Diagnostic and Therapeutic Approaches for Mitochondrial Disorders

Mitochondrial disorders are among the most common inherited genetic disorders, with a combined prevalence of 1:5,000. These are genetically, biochemically, and clinically heterogeneous disorders affecting any organ or tissue in the body. A poor understanding of gene-to-phenotype relationships and pathophysiological mechanisms has resulted in sometimes years-long diagnostic odysseys and a lack of curative therapies. Consequently, outcomes are often poor with most patients dying in early childhood. The aim of this project is to improve patient outlooks by using novel tools to address both the diagnostic and therapeutic challenges associated with mitochondrial disease. The diagnostic aspect of the study involved the creation of four interactive diagnostic resources which can complement next generation sequencing (NGS) technologies to achieve more rapid diagnoses for patients. MitoEpilepsy Map, MitoCardio Map, MitoLiver Map, and MitoMedicine Map were created to aid in the diagnosis of mitochondrial epilepsy, cardiomyopathy, liver disease, and the entirety of mitochondrial disease, respectively. These, maps were accurate in identifying candidate genes from clinical vignettes of genetically confirmed cases of mitochondrial disease in 69-100% of cases. These maps will be valuable resources for interpreting NGS results, hopefully facilitating quicker and more accurate genetic diagnoses for affected patients. The therapeutic aspect of the project aimed to develop a new treatment strategy for mitochondrial disease caused by nonsense mutations. Translational read-through therapy involves pharmacological incorporation of a near-cognate amino acid in place of a premature stop codon during translation. A systematic in vitro proof-of-principle study was performed in patient fibroblasts harbouring bi-allelic nonsense mutations in ten different mitochondrial disease genes. In five patient cell cultures, translational read-through therapy was able to restore transcript, protein, and mitochondrial function, thus demonstrating in vitro efficacy and paving the way for future clinical development. Together, these approaches help improve outcomes for patients suffering from mitochondrial disease

Bacteria-Mediated Delivery of mazF mRNA into Cancer Cells for Induction of Apoptosis

Programmed cell death (PCD), an active process that leads to cell suicide, is a critical mechanism in eukaryotes and prokaryotes. In eukaryotes, apoptosis, a well-characterized form of PCD, is a gene-directed program that can be inhibited by some oncogenic mutations, resulting in the development of cancer. Toxin-antitoxin (TA) is one example of an intracellular death program that is present in almost all bacteria and comprises a stable toxin and a labile antitoxin that blocks the lethal function of the toxin. MazEF, one of the best studied bacterial PCD systems, includes a toxin, namely, MazF that cleaves mRNAs at ACA sequences leading to the inhibition of protein synthesis. Although the exact mechanism remains unknown, it seems the MazF-mediated apoptosis depends on the presence of BAK, a pro-apoptotic protein in mammalian cells. Since cancer is a heterogeneous group of diseases with various sensitivities to different treatments, we examined the impact of MazF proteins on the growth and viability of three cancer cell lines: MCF7, HT29, and AGS. These cells lines were transfected with ACA-less mazF mRNAs, and the cells were evaluated for development of MazF-mediated cell death. Our data illustrated that expression of MazF proteins in the aforementioned cells leads to significant reductions in cell viability. In order to confirm the occurrence of apoptosis, the activation of caspase-3 and -7 and the presence of caspase-3’s substrate were evaluated in transfected cells. Our observations suggested that in the presence of MazF, the levels of activated caspase-3 and -7 were significantly elevated in transfected cells. In addition, the inhibition of protein synthesis by MazF in MCF7 and AGS cell lines were examined via quantifying mRNA translation on a single-cell basis. Our data confirmed that the expression of MazF causes a significant drop in the levels of protein translation in the examined cell lines. Recently, it has been reported that the DNA delivery system does not seem to be a suitable approach to transfer mazF in tumors due to the lack of continuous expression of MazF, degradation, and the incidence of mutations in mazF gene. To address these challenges, we developed an mRNA delivery system to transfer ACA-less mazF mRNA exclusively into cancer cells. To examine the expression and stability of mazF mRNA in cells, we synthesized mazF and GFP mRNA for IRES- or cap- dependent translation. Our results suggest that cap/IRES-dependency, poly-A tails, and modified/unmodified nucleotides played important roles in efficient expression of MazF proteins in transfected cell lines but is dependent on the cell line. MazF is a nonspecific ribonuclease that can induce death in both normal and cancer cells. Thus, the application of MazF is dependent on efficient expression and delivery system to transfer MazF into tumors. We developed a listerial bi-vector expression and delivery system to transfer mazF mRNA into specific HER2-positive SKBR3 human breast cancer cells. Our results showed that following the infection of cells by functionalized bacterial vectors, caspase activities elevated in SKBR3 cells 48 hours post-transfection. In this research, this expression and delivery system for the delivery of mazF mRNA as a potential therapeutic mRNA in cancer cells was reported for the first time