Gerardo Perez

The UCSC Genome Browser (https://genome.ucsc.edu) is a widely utilized web-based tool for visualization and analysis of genomic data, encompassing over 4000 assemblies from diverse organisms. Since its release in 2001, it has become an essential resource for genomics and bioinformatics research. Annotation data available on Genome Browser includes both internally created and maintained tracks as well as custom tracks and track hubs provided by the research community. This last year's updates include over 25 new annotation tracks such as the gnomAD 4.1 track on the human GRCh38/hg38 assembly, the addition of three new public hubs, and significant expansions to the Genome Archive[GenArk) system for interacting with the enormous variety of assemblies. We have also made improvements to our interface, including updates to the browser graphic page, such as a new popup dialog feature that now displays item details without requiring navigation away from the main Genome Browser page. GenePred tracks have been upgraded with right-click options for zooming and precise navigation, along with enhanced mouseOver functions. Additional improvements include a new grouping feature for track hubs and hub description info links. A new tutorial focusing on Clinical Genetics has also been added to the UCSC Genome Browser.

Transduction is the process in which bacterial DNA is transferred from one bacterial cell to another by means of a phage particle. There are two types of transduction, generalized transduction and specialized transduction. In this chapter two of the best-studied systems - Escherichia coli-phage P1, and Salmonella enterica-phage P22 - are discussed from theoretical and practical perspectives.

Now in its 25th year of operation, the UCSC Genome Browser (https://genome.ucsc.edu) provides a central location for researchers around the world to display and compare annotations on assembled genomes. Highlighted updates include a positional heatmap display, used to show data on functional consequences of mutation from MaveDB; QuickLift, a tool to copy annotation data seen on one assembly for display on another related assembly; and HubSpace, an initiative to simplify the process of creating and using track hubs by providing each user account with dedicated storage on UCSC's infrastructure.

Although a great deal is known about the life cycle of bacteriophage P22, the mechanism of phage DNA transport into Salmonella is poorly understood. P22 DNA is initially ejected into the periplasmic space and subsequently transported into the host cytoplasm. Three phage-encoded proteins (gp16, gp20, and gp7) are coejected with the DNA. To test the hypothesis that one or more of these proteins mediate transport of the DNA across the cytoplasmic membrane, we purified gp16, gp20, and gp7 and analyzed their ability to associate with membranes and to facilitate DNA uptake into membrane vesicles in vitro. Membrane association experiments revealed that gp16 partitioned into the membrane fraction, while gp20 and gp7 remained in the soluble fraction. Moreover, the addition of gp16, but not gp7 or gp20, to liposomes preloaded with a fluorescent dye promoted release of the dye. Transport of (32)P-labeled DNA into liposomes occurred only in the presence of gp16 and an artificially created membrane potential. Taken together, these results suggest that gp16 partitions into the cytoplasmic membrane and mediates the active transport of P22 DNA across the cytoplasmic membrane of Salmonella.

The short noncontractile tail of P22 allows it to eject its DNA together with three phage-encoded proteins (gp7, gp16, and gp20) into the periplasm of the host. However, the mechanism of phage P22 DNA transport across the cytoplasmic membrane of Salmonella enterica sv. Typhimurium is not known. This process could be mediated by phage-encoded or host-encoded proteins, or a combination of both. Genetic and biochemical approaches failed to identify host factors that are essential for P22 DNA uptake, suggesting that phage-encoded proteins are sufficient to catalyze P22 DNA uptake. This hypothesis was tested by studying the protein-protein interactions, the DNA-binding properties and the membrane partitioning characteristics of the P22 ejected proteins. The energy requirements for P22 DNA transport was also tested by studying the effects of inhibitors of the ATP synthase and uncouplers of the proton motive force. The pull-down assays revealed that the P22 ejected proteins interacted with each other. The gel retardation assays showed that these proteins not only bound DNA nonspecifically but also protected the DNA from degradation by DNase I. The protein gp16 partitioned into the detergent phase of Triton X-114 and into the liposomal fraction. Gp16 was also found to disrupt membranes by possibly forming a channel across the liposomal membrane. When ³²P-labeled DNA was used to detect the transport of DNA inside liposomes, transport of radiolabeled DNA into liposomes was possible only in the presence of gp16 and an artificially-created membrane potential. The requirement for the membrane potential in the transport of phage P22 DNA into the cytoplasm of Salmonella was shown when valinomycin plus potassium chloride reduced the transduction efficiency of the host cells by 63%. The findings reported in this thesis suggest that the transport of phage P22 DNA across the cytoplasmic membrane of the Salmonella host is mediated by the phage- encoded protein gp16, and that transport is driven by the membrane potential of the host. The primary role of the other ejected proteins, gp7 and gp20, might be to protect the ejected DNA from periplasmic nucleases