WorldCat Identities

Fire, Andrew Zachary

Works: 36 works in 36 publications in 1 language and 43 library holdings
Roles: Thesis advisor, Author
Publication Timeline
Most widely held works by Andrew Zachary Fire
A role for the let-7 primary microRNA in target gene recognition and repression by Robin Deis Trujillo( )

1 edition published in 2010 in English and held by 2 WorldCat member libraries worldwide

MicroRNA (miRNA) genes produce three noncoding RNA products: the long primary transcript (pri-miRNA), the ~70 nucleotide pre-miRNA, and the ~22-nt mature miRNA. Only the mature miRNA is considered to be the functional species of a miRNA gene in recognizing cognate target mRNAs and modulating their expression. However, mature miRNAs are processed from the primary transcript through sequential endonucleolytic steps. As a result, the mature miRNA sequence is present in all three RNA products of a miRNA gene. It has thus been intrinsically difficult to determine the contribution of each miRNA gene product to target repression. In fact, direct functional roles for pri- and pre-miRNAs have never been ruled out. Here we show that pri- and pre-miRNAs may not be mere transitory intermediates of mature miRNA biogenesis. We found that ectopic expression of the C. elegans miRNA gene let-7 (cel-let-7) in human culture cells results in the production of truncated pre- and mature miRNAs that lack the first two 5' nucleotides, one of which is the first nucleotide of the miRNA seed region (nucleotide SD1). We found this nucleotide to be required for repression of target reporters by cel-let-7 in these cells, demonstrating that pri-let-7 may have a direct role in target repression. Further, we show that the nucleotide sequence and structure of both the pri-/pre-let-7 loop and stem regions play a key role in miRNA gene function in reporter assays. In vitro and in vivo analyses indicated the significance of these regions may be in the mediation of a physical interaction between pri-let-7 and target RNAs. These observations suggest that regulatory information encoded in the structured pri-miRNAs, but absent from mature miRNAs, could be directly interpreted for target recognition and repression through RNA:RNA interaction. Intriguingly, some mutations in the loop nucleotide sequence also restored processing of the 5' ends of C. elegans pre- and mature let-7 in culture cells, demonstrating that the pri-/pre-miRNA loop region can also regulate the precision of mature miRNA biogenesis. Importantly, in the presence of functional pre- and mature let-7, cel-let-7 activity in target repression consists of both SD1-independent and SD1-dependent components, implying potential contributions by both pri- and mature let-7. Finally, we interrogated the effects of pri-/pre-let-7 loop mutations on their ability to rescue a let-7 loss-of-function mutant phenotype in C. elegans. Our results indicate decreased significance of these parameters in the control of worm vulval development, although context-dependent differences in mature miRNA biogenesis between heterologous culture and live animals may partially explain this discrepancy. Taken together the work presented here reveals a novel layer of regulatory complexity encoded in long primary miRNAs that may have broad implications in understanding the mechanisms by which miRNA genes control target expression
Genetics and genomics of mammalian pigment patterns by Zuocheng Lewis Hong( )

1 edition published in 2011 in English and held by 2 WorldCat member libraries worldwide

Hundreds of coat color loci have been cloned and studied in the laboratory mouse, and most aspects of the mouse pigmentary system are conserved in other mammals. However, comparative zoologic studies suggest that some components of mammalian pigmentation are not represented as coat color mutations in laboratory mice. This dissertation aims to advance our molecular understanding of mammalian pigmentation, and strives to identify novel components of the genetic toolkit involved in generating the diverse array of coat color patterns found in nature. In the Syrian hamster, X-linked inheritance of the Sex-linked yellow (Sly) mutation results in irregular patches of black and yellow hair in heterozygous females. Femalespecific variegation of black and yellow coat color is also present in the domestic cat, but curiously absent from other vertebrate species. Using a combination of comparative genomics and linkage mapping, I made key contributions towards defining a candidate interval for the Sly mutation and discovering that the hamster and cat mutations lie in non-homologous regions of the X chromosome. Periodic color patterns on animal skin, such as cheetah spots and zebra stripes, are evident across the mammalian phylogeny, but their molecular basis remains a mystery. Taking advantage of improvements in ultra high-throughput sequencing technologies, I developed a methodology called EDGE, or EcoP15I-tagged Digital Gene Expression, that is well suited for gene expression profiling in non-model organisms. Applying EDGE to black spot and yellow background regions of cheetah skin, I discovered that localized differences in Endothelin-3 expression is likely to be responsible for maintaining black spotting through a pigment-type switching mechanism in hair follicle melanocytes. The use of EDGE and other genomic approaches to generate comparative molecular profiles of mammalian skin patterns is likely to open new avenues of research into mammalian pigment patterning and initiate a molecular renaissance for a set of questions that has mostly resided in the area of theoretical biology for the past few decades
Systematic identification of mirna targets and the steps in gene expression regulated by mirnas by David Gillis Hendrickson( )

1 edition published in 2009 in English and held by 2 WorldCat member libraries worldwide

In the last decade, RNA interference (RNAi), the process by which small RNAs direct the post-transcriptional silencing of cognate mRNA transcripts has revolutionized longstanding paradigms about RNA function. In addition, researchers have harnessed this pathway for experimentally induced gene silencing in what is arguably one of the most important technological advances in modern biology. That disruption of the RNAi pathway results in aberrant development, cancer, and embryonic death suggesting that RNAi is an integral component of eukaryotic gene expression programming. Although RNAi is a vast and diverse pathway with marked distinctions between species, the basic organization has been established largely from work carried out in worms, flies, humans and mice. Double stranded RNA (dsRNA) inputs are "diced" by the class III ribonuclease Dicer into small dsRNA intermediates of ~21-22nt in length which are transferred to the RNA induced silencing complex (RISC) wherein the guide strand is selected and bound to an Argonaute (Ago) family protein. Target mRNAs are then recruited to RISC through Watson-Crick base pairing to the guide strand. Silencing of target transcripts can be directed by Ago mediated cleavage, or through Ago mediated recruitment of factors that induce translational inhibition and mRNA degradation. MicroRNAs (miRNAs) are the most common class of endogenous small silencing RNAs. Despite these advances, many of molecular details of RISC mediated gene silencing are poorly understood as current models are based on only a few miRNA:mRNA target pairs. Here, we present a method for systematic identification of specific miRNA targets. We demonstrate that immuno-affinity purification (IP) of Argonaute proteins is a viable method for isolating RISC associated miRNAs and mRNAs for identification using DNA microarrays. The strong enrichment of mRNAs with binding sites to the experimentally introduced miR-1 and miR-124 in Ago IPs from human embryonic kidney 293T cells (HEK293T) validates the utility of this method. Furthermore, mRNAs classified as targets of miR-1 and miR-124 using this approach behave like bona fide targets in that they exhibit significant down-regulation at the mRNA level. To learn about the steps in gene expression regulated by miRNAs, we simultaneously measured miR-124 mediated changes in Ago enrichment, mRNA abundance, and ribosome occupancy and ribosome density for ~8,000 genes. The translational parameters were used to estimate apparent changes in translational rate and were collected using standard polysome profiling in tandem with DNA microarrays and a novel gradient encoding scheme. We found that for the majority of the miR-124 targets, changes in mRNA concentration and apparent translation rate are concordant and that ~75% of the estimated change in protein levels could be accounted for by changes in mRNA abundance. Our data is most consistent with models of miRNA inhibition of translation initiation. To rule out miRNA mediated repressive mechanisms that would not be visible to our translational profiling (concordant reductions in translational initiation and elongation, co-translational proteolysis) we tested the protein levels for 13 targets by Western blot and found that our estimated changes in protein were nearly identical to the actual changes for 12/13 of the proteins measured. In addition, we observed a large dynamic range for miR-124 mediated down-regulation of mRNA abundance and apparent translation rate, and estimated protein abundance demonstrating the versatility of miRNA mediated regulation. The concordance between miR-124 specific changes in mRNA level and translation supports a model wherein these two regulatory outcomes are functionally linked in a sequential process or regulated by the same cis factors. We have also sought to learn about the RNAi pathway from a Dicer-centric perspective. We generated a library of Dicer truncations to test the contribution of Dicer's conserved protein domains to in vitro dicing reactions to learn about potentially interesting in vivo function as well as for increasing the efficacy of in vitro dicing as a gene silencing tool. We found that the domain of unknown function 283 (DUF283) may be important for proper spacing in dicing reactions and is part of Dicer's "molecular ruler". In addition we found that the ATPase/Helicase domains may inhibit Dicer activity and are dispensable for in vitro dicing, but may play a role in non-canonical substrate recognition
COSA-1, a meiotic crossover site associated protein by Rayka Yokoo( )

1 edition published in 2011 in English and held by 2 WorldCat member libraries worldwide

The formation of crossovers during meiosis is important both for the reassortment of genetic traits, and for creating a physical connection between homologous chromosomes to ensure faithful segregation. This thesis explores the role of a novel protein, COSA-1 (crossover site associated-1), in forming crossovers in the nematode C. elegans. cosa-1 was isolated in a screen for dead embryos, signaling missegregation of the autosomes, and a high incidence of males, signaling missegregation of the X chromosome. Interestingly, obvious orthologs of COSA-1 are only present in metazoans with the notable exception of Drosophilids. Thus no obvious orthologs of COSA-1 exist in the common model organisms, S. cerevisiae, S. pombe, D. melanogaster, and A. thaliana, making C. elegans one of the few organisms in which COSA-1 could have been isolated and studied. Yet, COSA-1 is conserved into humans suggesting the study of COSA-1 will have implications for human reproduction. Not only does C. elegans cosa-1 play a crucial role in promoting the formation of crossovers, evidenced by the lack of chiasmata in the cosa-1 mutant, GFP::COSA-1 exhibits an interesting localization pattern to presumptive crossover sites. In particular, unlike other crossover promoting proteins MSH-5 and ZHP-3, GFP::COSA-1 does not exhibit an initial broader localization but instead localizes to just 6 foci, 1 focus for the 1 crossover on each of the 6 chromosomes. This localization of GFP::COSA-1 to 6 foci was robust under various conditions. Mathematical modeling of the relationship between irradiation dose and COSA-1 foci revealed a relationship between obligate crossover, the phenomenon in which at least one crossover is formed per chromosome, and crossover interference, the phenomenon in which the formation of a crossover inhibits the formation of other crossovers nearby. In addition, analysis of GFP::COSA-1 foci in mutants that form extra crossovers, in particular rtel-1 and dpy-28, revealed a separation between the number of GFP::COSA-1 foci, 6, and the number of COs made, more than 6, suggesting that interference is still operating on GFP::COSA-1foci. Thus GFP::COSA-1 may serve as a better readout for crossover interference than crossovers per se. The ability to visualize GFP::COSA-1 in live worms holds great potential for elucidating the mechanisms involved in crossover interference, crossover distribution, and crossover designation
Regulatory controls of microRNA genes beyond the mature microRNAs by Gwen Liu( )

1 edition published in 2010 in English and held by 2 WorldCat member libraries worldwide

MicroRNA (miRNA) genes, which encode an abundant class of ~22-nucleotide (nt), evolutionarily conserved small RNAs, control a fundamental layer of genetic programs at the post-transcriptional level. However, little is known about how the activity of miRNA genes is regulated and how the regulatory information controlling their activity is encoded. Interestingly, mature miRNAs can often be classified into large families consisting of members with identical seeds (nucleotides 2 through 8 of the mature miRNAs) and highly homologous ~22-nt mature miRNA sequences, but with divergent sequences and structural elements beyond their mature miRNAs. Here we investigated whether members of a miRNA gene family that encode identical or nearly identical mature miRNAs are functionally interchangeable in vivo and if not, why? We compare the activities of the mir-181 gene family in promoting double positive T cell development and show that miRNA genes that encode identical to nearly identical mature miRNAs can have distinct activities. The differences in activity between mir-181a-1 and mir-181c are largely determined by their unique primary/precursor-miRNA (pri/pre-miRNA) loop nucleotides, and the differences in activity between mir-181a-1 and mir-181b-1 are determined by both the pri/pre-miRNA loop and stem. Furthermore, the organization of mir-181a-1/b-1 in a cluster is important for its full activity. We also show that mir-181 family members can differentially regulate target genes quantitatively, and that some target genes can be upregulated. Taken together, we have demonstrated that regulatory information encoded in a miRNA gene beyond the mature miRNA plays a critical role in controlling the activity of the miRNA gene, suggesting that miRNA family members could have evolved different functions through their divergent miRNA gene sequences and structural elements beyond their mature miRNAs. Although, proteins may have evolved to recognize the structural and sequence elements of the pri/pre-miRNAs, we suggest that the regulatory information encoded in the structured pri/pre-miRNA may be directly interpreted through target and pri/pre-miRNA interactions
Molecular signatures of virome-host interactions by Poornima Parameswaran( )

1 edition published in 2010 in English and held by 2 WorldCat member libraries worldwide

Small RNAs that are 19-30 nucleotides in length use the genetic information encoded in their sequence to effect gene regulation in a sequence-directed manner. Infections with RNA viruses in plants, worms and flies generate short viral-derived RNAs ("vsRNAs") that map to the genome of the infecting virus. The regulated production of these vsRNAs and their engagement by the immune apparatus is essential for inhibiting viral growth, making vsRNAs important components of antiviral immunity in these organisms. RNA virus-derived vsRNA-mediated gene silencing is yet to be demonstrated in mammalian systems. We investigated diverse RNA virus-mammalian systems, and characterized changes in small RNA populations that occur during viral infection in animal cells using high-throughput sequencing. Due to the large number of samples to be analyzed, we designed DNA barcodes to 'tag' RNA samples from individual experiments, which facilitated sequencing in parallel from multiple samples. Our work demonstrated the generality of RNA virus-derived vsRNA production, and the ability of the cellular short RNA apparatus to engage these vsRNAs in worms during Flock House Virus replication, and in mammalian cells during infections with Hepatitis C, Polio, Dengue, Vesicular Stomatitis or West Nile virus. In addition to the appearance of vsRNAs during infection, we saw a number of specific changes in host-encoded small RNA (miRNA) profiles. For several infection models investigated in more detail, the RNAi and Interferon pathways modulated the abundance of vsRNAs. We found evidence for populations of vsRNAs that exist as duplexed small interfering RNAs ("siRNAs; " effectors of gene silencing) with zero to three nucleotide 3' overhangs. We also observed strand-selective loading of siRNAs onto Argonaute complexes, which are mediators of gene silencing. We quantitated the capacity of these HCVrep-derived vsRNAs to down-regulate target mRNAs in a sequence-specific manner in mammalian systems. We found that abundant HCVrep vsRNAs are not capable of mediating robust silencing (i.e. 2-fold or higher) of Luciferase reporters that have been engineered with vsRNA targets. Additionally, over-expression of siRNAs corresponding to five abundant vsRNAs failed to enhance silencing of Hepatitis C Virus mRNA. These results complement each other and suggest that in mammalian hosts, the virus may coexist with an abundant population of vsRNAs. Questions still remain as to whether robust gene silencing may be achieved by the cooperative action of abundant vsRNAs, or if abundant vsRNAs specifically inhibited from functioning in gene silencing, or alternatively, if they have novel roles in pathways distinct from gene silencing
Identification of RNA regulatory information in the Saccharomyces cerevisiae transcriptome by Daniel Patrick Riordan( )

1 edition published in 2011 in English and held by 2 WorldCat member libraries worldwide

The unique post-transcriptional behavior of each mRNA is thought to be largely determined by features present in its molecular sequence, representing a type of RNA regulatory code. However, the details by which distinct regulatory outcomes are programmed into the sequences of different transcripts are mostly unknown. We set out to identify features of yeast mRNAs that influence their post-transcriptional fates. Using bioinformatic and in vitro selection approaches, we characterized several RNA recognition elements involved in mediating specific interactions with individual yeast RNA-binding proteins (RBPs). Most of the RNA elements we uncovered were associated with significant mRNA expression changes and were phylogenetically conserved in related yeasts, providing insights into the function and evolution of the corresponding interactions. We also analyzed RNA-protein interaction sites for the yeast Puf3 RBP by high-throughput sequencing under different growth conditions. These results provided high-resolution experimental evidence for Puf3 binding at consensus RNA elements in the transcriptome, and enabled detailed comparisons of individual interaction sites. Finally, we developed complementary methods for transcriptome-wide mapping of potential sites of RNA 2'-O-methylation. Application of these methods to the yeast transcriptome successfully recovered known sites of RNA modification and suggested that ribose methylation of functionally-related transcripts may occur and influence the regulation of endogenous yeast mRNAs. Overall, these results contribute to understanding of how RNA sequence features help to specify global differences in gene expression characteristics
Priming by streptococcus pneumoniae causes changes in gene expression in Drosophila melanogaster by Junaid Ziauddin( )

1 edition published in 2013 in English and held by 1 WorldCat member library worldwide

Insects are typically described as having no memory yet they can be primed by past exposure to survive a specific pathogenic challenge. To determine how a primed immune response might work in the context of a transcriptional response to a pathogen, we performed a microarray analysis that identified genes modulated by priming during a S. pneumoniae infection in Drosophila melanogaster. We followed 22 transcripts at a high level of resolution using qRT-PCR of tight timelines of infected primed and naïve flies. We knocked down expression of 17 of these transcripts/genes and observed changes in survival and bacterial growth dynamics. We found that priming affected the transcriptional response in three ways. First, the basal levels of some transcripts changed permanently upon priming. Second, the induced maximal expression level of genes was altered between the first and second infection. Third, there were changes in sensitivity where genes were induced at earlier times in the infection in the primed flies. We hypothesized that these changes are evolved to adapt, where the fly's immunity changes in a fashion that allows it to respond more effectively to that infection in subsequent exposures. We highlight 5 genes that follow our gene induction model and show that when knocked down, primed flies exhibit survival and bacterial growth phenotypes that demonstrate their important role in the primed response. These data show how a simple signaling network can possess memory which allows it to respond more effectively to threats based upon past experiences. We also added Serratia marcescens to the repertoire of pathogens that induce the primed immune response. We showed that this memory lasts for the life of the fly and the Toll pathway is necessary for the response. We demonstrated that Serratia is not cleared in primed flies despite longer survival times, indicating a tolerance effect. We found the highest protection to be species specific and nutrient deprivation to negatively impact priming. It is the first time gram-negative bacteria have been characterized in the primed response and the first time tolerance-based priming against any pathogen has been demonstrated in Drosophila
The role of the 5'-3' exoribonuclease Xrn2 in RNA virus infection by Cecilia D Sedano( )

1 edition published in 2014 in English and held by 1 WorldCat member library worldwide

Hepatitis C virus (HCV) is a small, hepatotropic, RNA virus that can establish persistent infections leading to chronic hepatitis and hepatocellular carcinoma (HCC). With an estimated 2% of the world's population infected, HCV is a serious global health problem. Curiously, this virus has the unique feature of depending on the liver-specific, microRNA miR-122. In the current model, miR-122 interacts with the 5'-end of the HCV RNA genome, protecting it from hitherto unknown degradation machinery. The work described in this dissertation highlights a novel cytoplasmic antiviral function for the mammalian 5'-3' exoribonuclease Xrn2, and enzyme best known for its role in RNA polymerase II transcription termination. We discovered that Xrn2 modulates HCV RNA abundance by destabilizing the viral RNA genome. Importantly, during sequestration of miR-122, Xrn2 depletion restored HCV RNA abundance, suggesting that Xrn2 depletion eliminates the miR-122 requirement for viral RNA stability. Thus, Xrn2 is an antiviral effector so potent that HCV has evolved a mechanism to evade it, through the subversion of miR-122 to form a protective oligomeric complex at the 5' end of the viral genome. To determine if Xrn2's antiviral function is specific to HCV, we studied its role in poliovirus (PV) infection. Our experiments showed that Xrn2 interacts with PV RNA early during infection, and that Xrn2 activity may be suppressed by proteolytic cleavage that is mediated by the PV protease 2A. This work revealed an unprecedented role for Xrn2 and highlighted the broad function of this novel antiviral factor in RNA virus infection. In this dissertation, we also investigated the regulation of noncoding RNAs in HCV infection. Sucrose-gradient analysis of miR-122 during HCV infection uncovered that this highly conserved miRNA and its isomiR variants form unique complexes, which are specifically modulated during infection. This highlights the ability of HCV to modulate the engagement of specific miR-122-complexes, and the distinct functions in gene regulation these complexes may play. We also examined the role of Xrn2 in mammalian long noncoding RNA (lncRNA) degradation, and their regulation during HCV infection, by performing lncRNA microarrays. Our analyses will provide important insights into the biological functions of lncRNAs and help us identify novel therapeutic targets for HCV infection and HCC. Taken together, the work described here has uncovered a novel cytoplasmic antiviral function for Xrn2 in RNA virus infection. Our research highlights the potential of studying lncRNAs and miRNA variants during viral infection, which can lead the way for the discovery of new biomarkers and therapeutics
An in vivo characterization of chromatin state and its relationship to the expression of foreign, non-integrating transgenic DNA in mouse liver by Lia Gracey( )

1 edition published in 2012 in English and held by 1 WorldCat member library worldwide

The pursuit of gene therapy to treat a wide-spectrum of diseases holds great promise, but its application in the clinic is still blocked by several barriers. Gene therapy approaches that rely on a virus to carry genetic material are plagued by problems with a host immune response and difficulty reaching therapeutic doses. Nonviral approaches have the advantage that delivering naked DNA does not illicit the same significant immune response and therapeutic levels are often initially easily attained. But, one prominent barrier to nonviral gene therapy is the lack of in vivo sustained expression from a foreign transgene. In the early stages after delivery, robust expression can be achieved from transgenic DNA, but this expression is very quickly and efficiently silenced, with loss of the desired effect of gene replacement. Our work took a two-step approach to further our understanding of how to create a better expression vector, both for gene therapy purposes as well as basic scientific goals of sustaining expression from delivered transgenic DNA. First, we studied whether we could manipulate and control the chromatin structure that a delivered gene adopts in vivo via nucleosome positioning signals on plasmid DNA in mouse liver. We developed a selective hybridization assay that allowed us to use a high-throughput sequencing approach to more rapidly screen the nucleosome occupancy and positioning of numerous constructs with varied DNA elements. We were able to transiently control the initial chromatin structure, but other forces in the cell soon overcame the thermodynamic preferences of nucleosome formation. We now believe that the use of nucleosome eviction elements may be a more promising approach: it will likely be more feasible to exclude nucleosome formation using rigid DNA sequences instead of permanently positioning a nucleosome using DNA sequence alone. Secondly, we gained a more fundamental understanding of the mechanism(s) responsible for the silencing of episomal (non-integrating) DNA constructs in mouse liver. We studied the differences between plasmid DNA and minicircle DNA, which was derived from a parental plasmid but lacked bacterial backbone sequences. At 6 weeks after delivery, plasmid DNA was effectively silenced but there was still active expression from the minicircle. We used high-throughput sequencing approaches to quantitate the levels of transcription and the enrichment of various histone modifications and RNA polymerase II (Pol II) on plasmid and minicircle. We found that Pol II appeared to stall at the transcription start site on the plasmid but was present at the 5' and 3' ends of the gene on the minicircle. This observation may explain the 28-fold higher level of transcript produced by the minicircle versus the plasmid. Though we saw enrichment of activating and silencing histone modifications on both plasmid and minicircle (likely due to our analysis of a population of cells which at any given point could have a construct that is actively expressed or silenced) we observed a striking abundance of a well-characterized histone modification associated with silencing on the plasmid and not on the minicircle. These results represent a contribution to the fields of gene therapy and chromatin biology that will help us move in the direction of achieving safe, therapeutic, and sustained expression of transgenic DNA
Bayesian assembly of reads from high throughput sequencing by Jonathan Daniel Laserson( )

1 edition published in 2012 in English and held by 1 WorldCat member library worldwide

The high-throughput sequencing revolution allows us to take millions of noisy short reads from the DNA in a sample, essentially taking a snapshot of the genomic material in the sample. To recover the true genomes, these reads are assembled by algorithms exploiting their high coverage and overlap. I focus on two scenarios for sequence assembly. The first is de novo assembly, where the reads come from an unknown and diverse population of genomes. The second is variant assembly, where the reads come from short but clonally related genomes, only slightly mutated from each other. In both cases I use the same principled Bayesian approach to design an algorithm that uncovers the composition of the genomic sequences that produced the reads. I will demonstrate the algorithms' performance on real data taken from various metagenomic environments, as well as the immune system B cells. On that latter dataset, collected from 10 organ donors each providing 4 tissue samples, the results show evidence of clone migration between tissues and provide new insights on the organization of the immune system
Regulation of meiotic recombination DNA double-strand break formation and repair in C. elegans by Simona Rosu( )

1 edition published in 2012 in English and held by 1 WorldCat member library worldwide

Sexually reproducing organisms undergo meiotic recombination to increase genetic diversity and to ensure correct segregation of chromosomes at the first meiotic division. During meiotic recombination, DNA double-strand breaks (DSBs) are deliberately induced, and a subset of the breaks are repaired as inter-homolog crossovers (COs). Inter-homolog COs provide physical connections between homologous chromosomes that enable them to segregate away from each other. Despite reliance on COs for chromosome segregation, few COs are made per chromosome pair, implying mechanisms for robust CO control. In addition, DNA breaks are potentially dangerous lesions, therefore DSB formation and repair must be tightly regulated, both to ensure the formation of COs, but also to protect against deleterious effects. This thesis provides new insights into mechanisms that regulate DSB formation and repair, using the nematode Caenorhabditis elegans as a model organism. CO regulation mechanisms were probed by monitoring the repair outcome of a DSB induced at a defined site at different stages of meiotic progression, in WT and mutant situations. This analysis uncovered a previously unappreciated level of control in restricting CO number, which is to limit the duration of access to the homolog as a repair template. In addition, this work showed that when no competing breaks are present, a single induced DSB is converted to a CO with high efficiency. This feature helps achieve CO assurance. Insights into regulation of DSB formation were uncovered from the identification and characterization of novel protein DSB-2. DSB-2 is required for break formation, but is dispensable for later steps of meiotic recombination. DSB-2 localizes to chromatin during the time of break formation, suggesting it acts to promote competence for DSB formation. Both approaches presented in this thesis uncovered evidence for negative feedback regulatory mechanisms, suggesting a model wherein formation of CO-eligible recombination intermediates signals shutdown of DSB formation as well as shutdown of inter-homolog access. The proposed regulatory networks provide a mechanism to both ensure sufficient breaks and COs are made, and to shut down these processes to prevent deleterious effects as meiosis progresses
In the right place at the right time understanding basic microrna biology through the control of developmental timing by lin-4 and let-7 in caenorhabditis elegans by Huibin Zhang( )

1 edition published in 2012 in English and held by 1 WorldCat member library worldwide

MicroRNAs are an important class of short RNA molecules that regulate gene expression in diverse organisms. Despite their short history, extensive research on microRNAs has revealed how microRNAs are made, their regulatory mechanisms and functions in different biological processes. In addition, novel experimental techniques and computational algorithms have been developed to study microRNAs. Our knowledge of microRNAs is constantly evolving as newer studies turn up exceptions to canonical models established by early studies. This highlights how our understanding of microRNAs is far from complete and much work is still needed to investigate unexplored aspects of the microRNA world. The founding microRNAs, C. elegans lin-4 and let-7, have well-characterized heterochronic defects in mutant animals and their target genes have been genetically validated. Combined with powerful experimental tools available in C. elegans, the microRNA function of lin-4 and let-7 in developmental timing of C. elegans is an ideal in vivo model system for testing microRNA-related hypotheses. Using functional assays to evaluate lin-4 and let-7 rescue activities in transgenic animals, we have investigated several poorly understood aspects of microRNAs, from biogenesis to functional mechanism. In this work, we first demonstrated the cell autonomous nature of lin-4 in C. elegans temporal development. Through tissue/cell-specific expression of lin-4 and the use of tissue/cell-specific reporters and microRNA sponges, we have shown that lin-4 rescue activities were limited to sites of lin-4 expression, despite its broad endogenous expression and function in many tissues. Next, we showed that intron-containing microRNAs ("inc-miRs") are functional in C. elegans, raising the possibility that mature microRNAs need not be encoded as contiguous units in the genome. This result also highlights a blindspot in our current novel microRNA discovery methods that assume inc-miRs do not exist. Lastly, we investigated the partnership between microRNA "seed" and "non-seed" sequences in providing microRNA function during C. elegans temporal development. By assessing microRNA function in transgenic mutant animals expressing lin-4 or let-7 mutant microRNA, we found contrasting results between lin-4 and let-7. While the results suggest a functional requirement for lin-4 seed sequence, mutations in either let-7 seed or non-seed sequences provided similar levels of functional activities. Using C. elegans lin-4 and let-7 as an experimental model, this work has furthered our understanding on microRNA autonomy, microRNA biogenesis and functional partnership between microRNA seed and non-seed sequence
Inducing and mapping cellular reprogramming by transcription factor overexpression by Ernesto George Lujan( )

1 edition published in 2015 in English and held by 1 WorldCat member library worldwide

The capability to directly convert one cell type into another by transcription factor overexpression is a modern marvel of biology. Recent advances have suggested that this process can principally convert any cell type to any cell type without restriction to germline origin or tissue. In this thesis, I demonstrate that FoxG1, Sox2 and Brn2 can directly convert fibroblasts into neural precursor cells. These cells can self-renew, differentiate into functional neurons, astrocytes and oligodendrocytes and integrate into the mouse brain. Additionally, this thesis demonstrates my work in induced pluripotent stem cell reprogramming where I identified a series of early intermediates that allowed us to interrogate the mechanism involved during early reprogramming induction

1 edition published in 2013 in English and held by 1 WorldCat member library worldwide

Intrinsic retroviral reactivation in human preimplantation embryos and pluripotent cells by Edward J Grow( )

1 edition published in 2015 in English and held by 1 WorldCat member library worldwide

Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections, which comprise nearly 8% of the human genome. The most recently acquired human ERV is HERV-K (HML-2), which repeatedly infected the primate lineage both before and after the divergence of humans and chimpanzees. Unlike most other human ERVs, HERV-K retained multiple copies of intact open reading frames (ORFs) encoding retroviral proteins. However, HERV-K is transcriptionally silenced by the host with exception of certain pathological contexts, such as germ cell tumors, melanoma, or HIV infection. Here we demonstrate that DNA hypomethylation at LTR elements representing the most recent genomic integrations, together with transactivation by OCT4, synergistically facilitate HERV-K expression. Consequently, HERV-K is transcribed during normal human embryogenesis beginning with embryonic genome activation (EGA) at the 8-cell stage, continuing through the emergence of epiblast cells in pre-implantation blastocysts, and ceasing during hESC derivation from blastocyst outgrowths. Remarkably, HERV-K viral-like particles and Gag proteins are detected in human blastocysts, indicating that early human development proceeds in the presence of retroviral products. We further show that overexpression of one such product, HERV-K accessory protein Rec, in a pluripotent cell line is sufficient to increase IFITM1 levels on the cell surface and inhibit viral infection, suggesting at least one mechanism through which HERV-K can induce viral restriction pathways in early embryonic cells. Moreover, Rec directly binds a subset of cellular RNAs and modulates their ribosome occupancy, arguing that complex interactions between retroviral proteins and host factors can fine-tune regulatory properties of early human development
In vitro transcription studies of adenovirus by Andrew Zachary Fire( )

1 edition published in 1983 in English and held by 1 WorldCat member library worldwide

Modulation of hepatitis C virus RNA abundance by the liver-specific microRNA miR-122 by Erica Machlin Cox( )

1 edition published in 2012 in English and held by 1 WorldCat member library worldwide

Hepatitis C virus (HCV) is a global health problem, infecting approximately 2% of the world's population. The virus is hepatotropic, replicating in liver cells, and its only known hosts are humans and chimpanzees. HCV is an unusual virus in that it requires the liver-specific host microRNA (miRNA) miR-122 for HCV RNA accumulation. Though the precise mechanism by which miR-122 upregulates HCV RNA is still under investigation, it is known that miR-122 must bind to two adjacent sites in the 5' end of the HCV genome. In this dissertation, a stepwise mutational analysis of the entire sequence of miR-122 was performed to identify residues important for HCV RNA accumulation. All mutant miRNAs were tested in canonical miRNA reporter assays and in HCV RNA accumulation assays. The identities of two nucleotides within miR-122, at positions 15 and 16, were shown to be dispensable for canonical miRNA and siRNA activity but required for HCV RNA accumulation. Compensatory mutations in the HCV genome upstream of the original binding sites uncovered supplementary binding sites for nucleotides 15 and 16 of miR-122. This analysis led to a new model for miR-122-HCV RNA interactions. To further define the requirements of HCV for miR-122, we investigated whether the predecessor of mature miR-122, a long hairpin precursor designated pre-miR-122, was also able to mediate HCV RNA accumulation. The function of pre-miR-122 was tested in miRNA, siRNA, and HCV RNA accumulation assays. Inhibition of pre-miR-122 processing was achieved by substituting deoxyribonucleotides into the loop of pre-miR-122 to prevent Dicer-mediated cleavage. Full-length pre-miR-122 was demonstrated to be functional in miRNA and siRNA assays and to be sufficient for HCV RNA accumulation. Pre-miR-122 also required traditional components of the RNA-induced silencing complex (RISC) for activity. Taken together, this research has uncovered novel requirements of miR-122 for HCV RNA accumulation. Components shown to be dispensable for canonical miRNA interactions were necessary for this unusual microRNA-target RNA interaction. Uncovering hepatitis C virus's stringent requirements for the mature and precursor forms of miR-122 will pave the way for new antiviral therapies targeting a host factor
Characterization of an RNase III protein and its potential roles in the RNA interference pathway of the protozoan parasite, Entamoeba histolytica by Justine Michelle Pompey( )

1 edition published in 2012 in English and held by 1 WorldCat member library worldwide

Entamoeba histolytica, a human intestinal parasite and a leading cause of death worldwide, contains a complex repertoire of endogenous small RNAs. Core elements of the RNAi machinery have been identified in the E. histolytica genome including three Argonaute proteins (EhAgo2-1, EhAgo2-2, and EhAgo2-3) and two genes with RNA-directed RNA polymerase (RdRP) domains. However, to date no canonical Dicer enzyme, an RNaseIII endonuclease responsible for cleaving double-stranded RNA (dsRNA) to 20-30nt small RNAs and a critical player in the RNAi pathway, has been identified in the E. histolytica genome. We conducted bioinformatics searches of the genome which revealed only one gene with an RNaseIII domain, EhRNaseIII. Interestingly, this candidate lacks the canonical Dicer structure - it is substantially smaller than other Dicers, contains only a single RNaseIII domain, and lacks PAZ or double-stranded RNA binding domain (dsRBD). Most eukaryotic Dicer proteins identified to date contain two RNaseIII domains, which form a heterodimer required for cleavage of the dsRNA, and contain PAZ or dsRBDs. We determined that EhRNaseIII exists as a homodimer in E. histolytica trophozoites. We sought to probe the contributions of EhRNaseIII, EhAgo2-2, and EhRdRP1 to the RNAi pathway in E. histolytica. Attempts to downregulate these genes using RNA-based methods including antisense mediated silencing and the production of gene-specific secondary small RNAs were unsuccessful. Expression of EhRNaseIII dominant-negative mutants also failed to impair production of small RNAs. In order to determine if EhRNaseIII was capable of generating small RNAs of the size observed in E. histolytica trophozoites, we conducted in vitro cleavage assays of dsRNA. Despite using multiple protein sources (E. histolytica whole cell lysate, immunoprecipitated EhRNaseIII, and recombinant EhRNaseIII protein) and two structurally distinct dsRNA substrates, we were unable to detect small RNA cleavage products. We further investigated whether EhRNaseIII was sufficient to act as a Dicer enzyme by co-expressing it with a dsRNA substrate in Saccharomyces cerevisiae, which lacks an RNAi pathway. However, no small RNAs were detected. We also assessed whether EhAgo2-2, which associates with 5'-polyphosphate small RNAs in E. histolytica, could mediate silencing using 5'-monophosphated small RNAs in S. cerevisiae. No downregulation in reporter transcript or protein was observed. These data indicate that EhRNaseIII is not a minimal Dicer enzyme and suggest that small RNA biogenesis may occur solely through a Dicer-independent small pathway in E. histolytica. These data also suggest that EhAgo2-2 may be specific for 5'-polyphosphate small RNAs. Future studies will continue to investigate the mechanism of small RNA-induced silencing in E. histolytica
Statistical analyses on high-thoughput sequencing of B cell and T cell receptors by Yi Liu( )

1 edition published in 2014 in English and held by 1 WorldCat member library worldwide

The immune system is typically described as having two major branches: the adaptive branch, and the innate branch. In the adaptive immune system, T cells and B cells learn to discern foreign pathogens from self. B cell repertoires mediate the host's ability to mount appropriately pathogen-specific humoral responses against immunological challenges. VDJ somatic recombination of the immunoglobulin chains, affinity maturation, and B cell clonal selection all contribute to the generation of a healthy B cell repertoire. Despite starting from only a limited number of genomic segments, these processes lead to diverse and optimized repertoires of clones, where each clone has its own distinct binding specificity for foreign antigens. High depth sequencing data on the repertoire of B cell receptors has enabled us to examine the diverse receptors in great detail. My thesis will showcase three aspects of the B cell repertoire in terms of how they relate to ageing and/or pathology: clonal convergence, clonal expansion, and phylogenetic hypermutation. In a separate but related work, we apply a novel combination of sequencing method and statistical techniques to examine T cell receptors. We raised the lower bounds on T cell receptor repertoires beta chain richness in healthy humans, and examined how T cell receptor diversity relates to age
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Audience level: 0.79 (from 0.68 for In vitro t ... to 0.81 for An in vivo ...)

Alternative Names
Andrew Fire Amerikaans bioloog

Andrew Fire nhà sinh học đoạt giải thưởng Nobel

Andrew Z. Fire

Andrew Z. Fire US-amerikanischer Mediziner

Andrew Zachary Fire

Andrew Zachary Fire biologo statunitense

Ендрю Фаєр

Файер, Эндрю

Эндру Фаер

Эндрю Файер

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English (20)