Biomedical subjects

Kitai Kim

Publications and source records attributed to Kitai Kim.

7 recordsLinked to original sources

PubMed2006

Histocompatible embryonic stem cells by parthenogenesis.

Genetically matched pluripotent embryonic stem (ES) cells generated via nuclear transfer or parthenogenesis (pES cells) are a potential source of histocompatible cells and tissues for transplantation. After parthenogenetic activation of murine oocytes and interruption of meiosis I or II, we isolated and genotyped pES cells and characterized those that carried the full complement of major histocompatibility complex (MHC) antigens of the oocyte donor. Differentiated tissues from these pES cells engrafted in immunocompetent MHC-matched mouse recipients, demonstrating that selected pES cells can serve as a source of histocompatible tissues for transplantation.

Animals↗

PubMed2005

Identification of cis-acting elements that mediate the replication and maintenance of human papillomavirus type 16 genomes in Saccharomyces cerevisiae.

Papillomaviruses contain small double-stranded DNA genomes that are maintained in persistently infected mammalian host epithelia as nuclear plasmids and rely upon the host replication machinery for replication. Papillomaviruses encode a DNA helicase, E1, which can specifically bind to the viral genome and support DNA synthesis. Under some conditions in mammalian cells, E1 is not required for viral DNA synthesis, leading to the hypothesis that papillomavirus DNA can be replicated solely by the host replication machinery. This machinery is highly conserved among eukaryotes. We and others found that papillomavirus DNA could replicate in a simple eukaryote, Saccharomyces cerevisiae. Specifically, papillomavirus DNA could substitute for the function of the autonomously replicating sequence (ARS) and centromere (CEN) elements that are normally both required for the stable replication of extrachromosomal DNAs in yeast. Furthermore, this form of replication in yeast was E1 independent. In this study, we map the elements in the human papillomavirus type 16 (HPV16) genome that can substitute for yeast ARS and CEN elements. A single element, termed rep, was identified that can substitute for ARS, and multiple elements, termed mtc, could substitute for CEN. The location of one of these mtc elements overlaps the location of rep, and this approximately 1,000-bp region of HPV16 was sufficient to support stable replication of a bacterial-yeast shuttle plasmid deleted of both ARS and CEN elements.

Genome, Viral↗

PubMed2004

Characterization of altered regulation variants of dinitrogenase reductase-activating glycohydrolase from Rhodospirillum rubrum.

In Rhodospirillum rubrum, nitrogenase activity is subject to posttranslational regulation through the adenosine diphosphate (ADP)-ribosylation of dinitrogenase reductase by dinitrogenase reductase ADP-ribosyltransferase (DRAT) and dinitrogenase reductase-activating glycohydrolase (DRAG). To study the posttranslational regulation of DRAG, its gene was mutagenized and colonies screened for altered DRAG regulation. Three different mutants were found and the DRAG variants displayed different biochemical properties including an altered affinity for divalent metal ions. Taken together, the results suggest that the site involved in regulation is physically near the metal binding site of DRAG.

ADP Ribose Transferases↗

PubMed2003

Derivation of embryonic germ cells and male gametes from embryonic stem cells.

Egg and sperm cells (gametes) of the mouse are derived from a founder population of primordial germ cells that are set aside early in embryogenesis. Primordial germ cells arise from the proximal epiblast, a region of the early mouse embryo that also contributes to the first blood lineages of the embryonic yolk sac. Embryonic stem cells differentiate in vitro into cystic structures called embryoid bodies consisting of tissue lineages typical of the early mouse embryo. Because embryoid bodies sustain blood development, we reasoned that they might also support primordial germ cell formation. Here we isolate primordial germ cells from embryoid bodies, and derive continuously growing lines of embryonic germ cells. Embryonic germ cells show erasure of the methylation markers (imprints) of the Igf2r and H19 genes, a property characteristic of the germ lineage. We show that embryoid bodies support maturation of the primordial germ cells into haploid male gametes, which when injected into oocytes restore the somatic diploid chromosome complement and develop into blastocysts. Our ability to derive germ cells from embryonic stem cells provides an accessible in vitro model system for studies of germline epigenetic modification and mammalian gametogenesis.

Animals↗

PubMed2003

Methylation patterns of papillomavirus DNA, its influence on E2 function, and implications in viral infection.

The biological activities of the papillomavirus E2 protein in transcription, replication, and maintenance of the papillomavirus genome rely on the E2 protein's ability to bind that genome specifically. The E2 binding sites (E2BSs), located within the long control region (LCR) of human papillomavirus (HPV) genomes, contain potential sites for 5'methylation at cytosine (CpG) residues. The E2 protein's capacity to bind E2BS in vitro is inhibited by methylation of these cytosines (59). Herein, we describe experiments to assess the influence of methylation on E2 function in cells. E2's ability to activate transcription was inhibited by the global methylation of CpG dinucleotides in E2-responsive transcriptional templates or when only the CpG dinucleotides within the E2BSs of a transcriptional template were methylated. Thus at least one biological activity of E2 that is dependent on its ability to bind DNA in a site-specific manner is influenced by the methylation status of its cognate binding site. The activity of DNA methylases is influenced by the differentiation status of mammalian cells. The life cycle of HPVs is tied to the differentiation of its host cells within stratified squamous epithelia. To investigate whether methylation of the papillomavirus genomes is influenced by the differentiation status of host epithelial cells, we analyzed HPV16 DNA harvested from a cervical epithelial cell line that was isolated from an HPV16-infected patient. We found, using bisulfite treatment to discriminate between methylated and unmethylated cytosines, that the HPV16 LCR was selectively hypomethylated in highly differentiated cell populations. In contrast, the HPV16 LCR from poorly differentiated, basal cell-like cells contained multiple methylated cytosines and were often methylated at E2BSs, particularly E2BS(2). These experiments indicate that the methylation state of the viral genome, and particular that of E2BSs, may vary during the viral life cycle, providing a novel means for modulating E2 function. These studies also uncovered an extensive pattern of methylation at non-CpG dinucleotides indicative of de novo methylation. The potential implications of this de novo methylation pattern are discussed.

Cell Line↗

PubMed2002

E1 protein of bovine papillomavirus 1 is not required for the maintenance of viral plasmid DNA replication.

Derivatives of bovine papillomavirus 1 (BPV1) with temperature-sensitive and dominant-negative mutation the E1 gene were used to determine the requirement for E1 in the maintenance of viral plasmid DNA replication. The abilities of these mutant BPV1 genomes to replicate as nuclear plasmids were monitored at permissive (32 degrees C) and nonpermissive (37 degrees C) temperatures in mouse C127 cells. We found that the temperature-sensitive E1 mutant BPV1 genomes replicate as nuclear plasmids as efficiently as does wild-type BPV1 in C127 cells after shifting to the nonpermissive temperature. These findings indicate that BPV1 does not require E1 for the maintenance of viral plasmids.

Animals↗

PubMed2002

Stable replication of papillomavirus genomes in Saccharomyces cerevisiae.

Papillomaviruses normally replicate in stratified squamous epithelial tissues of their mammalian hosts, in which the viral genome is found as a nuclear plasmid. Two viral proteins, E1, a helicase, and E2, a transcriptional activator and plasmid maintenance factor, are known to contribute to the episomal replication of the viral genome. Recently, our laboratory discovered that papillomaviruses can also replicate in an E1-independent manner in mammalian cells (K. Kim and P. F. Lambert, Virology, in press; K. Kim and P. F. Lambert, submitted for publication). In this study, we describe experiments investigating the capacity of the human papillomavirus type 16 (HPV16) genome to replicate in yeast (Saccharomyces cerevisiae). The full-length HPV16 genome, when linked in cis to a selectable yeast marker gene, either TRP1 or URA3, could replicate stably as an episome in yeast. The replication of papillomavirus genomes in yeast is not limited to HPV16. Bovine papillomavirus type 1 and HPV6b, -11, -16, -18, and -31 were all capable of replicating in short-term assays over a period of 20 cell doublings. The long-term persistence of viral episomes did not require any one viral gene, as mutant genomes defective in single genes also replicated episomally. These results indicate that the viral episome can replicate in the absence of the E1 DNA helicase. Similarly, E2 was also not required for replication in yeast, and E2 mutant viral genomes were stably maintained in the absence of selection, indicating the existence of an E2-independent mechanism for plasmid maintenance. The episomal replication of papillomavirus genomes in yeast provides a genetically manipulatable system in which to investigate cellular factors required for episomal replication and may provide a novel means for generating infectious papillomavirus.

DNA Helicases↗