An assay was designed to measure the sensitivity of Escherichia coli mutants to bacteriophage infection via growth curves, using a Tecan temperature controlled plate reader. I screened 3985 single-gene deletion strains in Escherichia coli K12 from the Keio collection and identified 43 strains displaying varying degrees of resistance to four different phages, three non-contractile tailed phages (λcI857, HK97, and HK243) and the myoviridae T6, including 20 genes not previously implicated in phage infection. Additional assays, such as adsorption and tests of DNA-injection, were designed to further characterize resistant strains. The use of these assays helped identify varying sensitivities to LPS structure and LamB receptor concentration in the three non-contractile tailed phages, showing HK97 is the most sensitive to changes and HK243 the least. I also found that the periplasmic chaperone, FkpA, is required for HK97 DNA-injection.

An assay was designed to measure the sensitivity of Escherichia coli mutants to bacteriophage infection via growth curves, using a Tecan temperature controlled plate reader. I screened 3985 single-gene deletion strains in Escherichia coli K12 from the Keio collection and identified 43 strains displaying varying degrees of resistance to four different phages, three non-contractile tailed phages (λcI857, HK97, and HK243) and the myoviridae T6, including 20 genes not previously implicated in phage infection. Additional assays, such as adsorption and tests of DNA-injection, were designed to further characterize resistant strains. The use of these assays helped identify varying sensitivities to LPS structure and LamB receptor concentration in the three non-contractile tailed phages, showing HK97 is the most sensitive to changes and HK243 the least. I also found that the periplasmic chaperone, FkpA, is required for HK97 DNA-injection.

Seed dormancy is a mechanism with great importance in plant fitness and it inhibits seed germination until is broken and seeds can germinate under optimal environmental conditions favorable for successful reproduction. Primary dormancy is contingent to the environment that seeds and the mother plant experience. Temperature is a major factor participating in the regulation of this complex trait. High and low levels of dormancy are induced during seed maturation by cold and warm temperatures respectively but the mechanism at the basis of temperature signaling in seeds is not well understood. Climate change and increased weather variability threaten the constant supply of high quality seeds into the market hence agriculture productivity. Therefore, understanding and taking control of the molecular mechanism behind the regulation of seed dormancy and germination will help to control and predict seed behavior in the field. Here I describe and discuss a forward genetic screen for the selection of mutant seed lines with altered seed dormancy in response to cool temperature during seed set. Putative mutant seed lines designated awake1 to awake52, were preliminarily characterized. Eleven awake lines were selected for further analysis and one was investigated in more detail. It was revealed that awake1 seeds shares common phenotype with seeds of a suberin deficient mutant which were previously reported to display increased dormancy but, here, I show they also display a reduction of seed dormancy. Segregation analysis suggests that the reduced dormancy phenotype is maternally inherited as the suberin deficient mutants. Also, transcriptomic analysis shows that many suberin associated genes are temperature-regulated. I conclude that control of suberin deposition may play a role in the regulation of dormancy in response to cool temperature.

DAWDLE (DDL) is one of the eighteen genes in Arabidopsis thaliana that encodes a protein with a Fork-Head Associated domain, a phospho-threonine binding domain providing a role in DNA repair and cell cycle regulation. DDL also contains an arginine-rich N terminal domain with putative Nuclear Localization Signals and a region for RNA binding. Two ddl knockout alleles in the WS-2 ecotype exhibit a strong pleiotropic phenotype with developmental defects such as short root and hypocotyl, reduced fertility, and distorted organs. This developmentally delayed phenotype is due to reduced accumulation of microRNAs and the phenotype is similar to those displayed by mutants involved in microRNA biogenesis pathway, suggesting a function for DDL in the microRNA biogenesis. One of the goals of my research was to characterize DDL protein through a structureunction study. Twelve point mutants were isolated in a mutagenesis screen and a comparative phenotypic and molecular characterization of each mutant with wildtype (WT) plants was performed. This revealed a few functionally significant amino acid residues of DDL. Traits for comparison included hypocotyl and root length, plant height, fertility, and microRNA accumulation. While all the mutants displayed reduced fertility, some of them had significantly varying stem height, hypocotyl and root length, and microRNA accumulation compared to the WT. Another objective of my research was to identify components involved in the DDL pathway, which in-turn would contribute to discovering additional components in microRNA biogenesis pathway. One such component, ddl suppressor1 (dds1), was identified through a second site mutagenesis screen of ddl-2. Phenotypic and molecular characterization revealed that dds1 is a strong suppressor of ddl that was mapped on chromosome 3 of Arabidopsis. Another component identified was MODIFIER OF DDL (MDL), a natural variation between Col and WS-2 ecotype of Arabidopsis. The variation has been mapped to an interval consisting of 37 genes on chromosome 5. MDLCol is the dominant allele and imparts a weak phenotype to ddl knockouts, whereas the recessive MDLWS-2 does not modify a strong ddl knockout phenotype. ddl MDLCol does not display abnormality in microRNA accumulation unlike ddl MDLWS-2 indicating that MDL has a function related to microRNA biogenesis.

Embryonic stem (ES) cells can be maintained indefinitely in culture while retaining the ability to give rise to cellular derivatives from the three germ layers. These unique characteristics hold great promise for regenerative medicine and underscore the importance of understanding the molecular mechanisms behind ES cell maintenance. The embryonic stem cell state is supported by a delicate equilibrium of mechanisms that maintain pluripotency, prevent differentiation, and promote proliferation and survival. We sought to find genes that could contribute to one or more of these processes in human ES cells by using a gain-of-function screen of over 8000 human open reading frames (ORFs). We identify Vestigial-like 4 (Vgll4), a co-transcriptional regulator with no previously known function in ES cells, as a positive regulator for survival of human ES cells. Specifically, Vgll4 protects human ES cells from dissociation stress, and enhances colony formation from single cells. These effects may be attributable in part to the ability of Vgll4 to decrease the activity of initiator and effector caspases. Based on global transcriptional analysis, we hypothesize that Vgll4 enhances survival of hES cells at clonal densities by regulating changes in the cytoskeleton, which may in turn regulate pathways known to result in hES cell death. This dissertation introduces a novel approach for studying hES cell survival in the context of cell dissociation and presents Vgll4 as a novel regulator of this process. We also propose that Vgll4 could have multiple functions in hES cells including possible roles in pluripotency, cell cycle dynamics, Hippo pathway regulation, and \(TGF\beta\) signaling. A direct regulator of survival in human embryonic stem cells could have important implications for facilitating the generation of transgenic cell lines and reporters, thus harnessing the therapeutic application of these cells.

Plant growth and development depends on controlled cell expansion. This, in itself, is determined by the plant cell wall, a structural matrix of polysaccharides encasing the plant cell. One line of investigation that has proven particularly successful in elucidating the components of the plant cell wall machinery has been the forward genetic screens of cell wall mutants. In this study, the molecular and cellular characterisation of sloughy, a cell separation mutant in Arabidopsis thaliana, was commenced. This mutant has a striking phenotype, with files of elongating epidermal cells snaking away from the adjacent epidermal cells and from the underlying cortex, loosing contact from the side walls while remaining attached at the cell ends, in a manner reminiscent of border-like cells in the root cap of arabidopsis. The sloughy mutation was fine mapped to a short region on chromosome I using high resolution melt point analysis. On sequencing all five genes in this region, a single nucleotide mutation, introducing a stop codon, was detected in exon 2 in the previously-described heat shock transcription factor SCHIZORIZA that results in a truncated protein missing several conserved domains essential for activity. SCHIZORIZA acts as a cell fate determinate in the root meristem to promote cortex fate, while suppressing epidermal and root cap fate in the mature ground tissue. Although the literature on schizoriza mutants has focused on the developing root meristem, with little documentation on the cell separation phenotype further up in the roots, the investigation of a collection of schizoriza TILLING mutants revealed that aberrant cell separation was ubiquitous to schizoriza mutants with a severely truncated protein. To investigate cell identity in the mature roots, sloughy was crossed to GAL4-GFP enhancer trap lines that act as cell-specific markers. Epidermal identity lines revealed that sloughy possessed a supernumerary ground tissue layer with epidermal identity. A cortex and endodermal line revealed that these two identities are restricted to the endodermal layer and the next ground tissue layer out. There was no indication of root cap identity in the mature root with any of the root cap lines used, although partial lateral root cap identity has been previously described in the epidermal and subepidermal cell layers in the meristem of schizoriza mutants expressing SOMBRERO-GFP, a lateral root cap-specific transcription factor. Immunolabelling of cell wall epitopes revealed that the JIM13 antibody, which specifically labels arabinogalactan-proteins in wild-type root caps, often labelled the epidermal cells and surrounding mucilage further up the in the roots of sloughy. The aberrant cell separation present in sloughy is thought to be a consequence of epidermal cells possessing partial lateral root cap identity. The data on sloughy/schizoriza is sufficient to generate a model on how a meristem developmental gene can generate a cell separation phenotype in the mature roots. Loss of SCHIZORIZA causes confused cell identity in the root meristem that results in an epidermal and subepidermal layer possessing mixed epidermal and lateral root cap identity. The distinctive properties of border-like cells in the root cap of arabidopsis have been linked to unique cell wall maturation and developmental processes, implicating the cellulases CEL3 and CEL5, the pectin glycosyltransferase QUA1, the pectin methyltransferase QUA2 and other pectolytic enzymes. The ectopic expression of these cell wall enzymes in the epidermal and subepidermal layers of sloughy roots result in reduced adhesion along the sides of the cell, while the ends remain attached, causing the observed cell separation phenotype.

Pattern formation is the process by which undifferentiated cells divide and differentiate to generate complex tissues and organs. In plants, pattern formation begins in embryogenesis and continues post-embryonically with the function of the meristems. microRNAs (miRNAs), which are small regulatory RNAs that repress gene expression, are involved in a variety of patterning processes in plants, including the formation and function of the meristems and establishment of polarity. For example, regulation of the class III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP III) transcription factors by miR165/6 is not only involved in the formation and function of the meristems, but also in polarity establishment in the leaf, and in axial patterning during embryogenesis. To gain a better understanding of the role of miRNAs in embryonic patterning, I investigated the tissue-specific functions of the miRNA biogenesis protein SERRATE (SE), which is required for the regulation of the HD-ZIP IIIs via miR165/6. By expressing SE in various domains in se-5 null mutant embryos, I revealed that although SE is expressed throughout the embryonic body, tissue-specific expression of SE from either the upper or lower tier of the embryo is sufficient for correct patterning. This observation suggests a SE-dependent non-cell autonomous and bi-directional mechanism that influences patterning in Arabidopsis embryos. Furthermore, through a suppressor screen of a se-3 loss-of-function mutant allele, I identified mutants in genes that likely function upstream of SE, and downstream or in parallel of the HD-ZIP IIIs. One of those se-3 suppressors is likely to be a mutant in the BELL homeobox gene POUND-FOOLISH (PNF).

No description available.

No description available.

Mutations in the telomere length maintenance pathway can lead to a spectrum of diseases called telomere syndromes, however, the pathway is not fully understood and there may still be unknown components. We designed a forward genetic screen to identify new genes involved in telomere length maintenance. Of the top ranked genes, ZNF827, a zinc finger protein, is the most promising candidate gene. The possible discovery of a new component involved in telomere length maintenance increases our understanding of the pathway and opens new avenues of research. Recent advances in molecular biology techniques, such as the use of RNA-guided nuclease CRISPR associated protein 9 (Cas9), have made screens like this possible. Cas9 is a nuclease that uses a guide RNA(gRNA) to direct its endonuclease activity. The use of Cas9 has revolutionized the field of genome engineering, providing scientists with more efficient methods to knockout and modify genomes. We sought to optimize CRISPR-Cas9 genome editing to make it as widely accessible as possible. We compared plasmid, ribonucleoprotein (RNP), and RNA only lipid-mediated transfection of CRISPR-Cas9 into cell lines using a novel reporter system to measure genome editing efficiency. All methods were successful to some extent, however, RNP lipofection was the most efficient and has many advantages over other methods. We also found that short homology arms of 30-35bp on donor templates was able to mediate site specific editing. These methods should broaden the accessibility of CRISPR-Cas9 genome editing.