Genetic Analysis of the Saccharomyces Cerevisiae Centromere-Binding Protein CP1: a Thesis

CP1 is a sequence specific DNA-binding protein of the yeast Saccharomyces cerevisiae which recognizes the highly conserved centromere DNA element I (CDEI) of yeast centromeres. The gene encoding CP1, which was designated CEP1 for centromere protein 1, was cloned and sequenced. CEP1 encodes a highly acidic protein of molecular weight 39,400. CEP1 was mapped to a position 4.6 centiMorgans centromere distal to SUP4 on the right arm of chromosome X. Phenotypic analysis of cep1 mutants demonstrated that yeast strains lacking CP1 are viable but have a 35% increase in cell doubling time, a ninefold increase in the rate of mitotic chromosome loss, and are methionine auxotrophs. Detailed analysis of the mitotic chromosome-loss phenotype showed that the loss is primarily due to chromosome nondisjunction (2:0 segregation). During meiosis cep1 null mutants exhibited aberrant segregation of centromere containing plasmids, chromosome fragments, and chromosomes. The predominant missegregation event observed was precocious sister segregation. The mutants also displayed a nonrandom 20% decrease in spore viability. Missegregation of chromosomes accounted for some but not all of this decreased spore viability, the remainder of which is presumed to be related to the pleiotropic consequences of the cep1 mutation. Together with the observed mitotic missegregation phenotype the results are interpreted as suggesting that CP1 promotes sister chromatid-kinetochore adhesion. The following conclusions are based on my mutational analysis of CP1: (1) CP1 is normally present in functional excess, (2) the C-terminal 143 amino acids are sufficient for full CP1 function in chromosome segregation and methionine metabolism, and (3) while DNA binding is apparently necessary for function, DNA binding per se is not sufficient. All of the mutations which caused an observable phenotype affected both centromere function and methionine metabolism. In addition, a direct correlation was observed in the degree to which both phenotypes were affected by different mutations. None of the mutant proteins displayed trans-dominant effects in a wild type background; however, two nonfunctional DNA binding-competent mutants exerted a dominant negative effect on the ability of PHO4 to suppress cep1 methionine auxotrophy. The data are consistent with a model in which CP1 performs a similar function at centromeres and promoters

Human Immunodeficiency Virus Type-1 Infection of Human Myeloid Cells

Infection with human immunodeficiency virus type 1 (HIV-1) results in a wide range of immunologic and hematopoietic abnormalities. The overall goal of this dissertation was directed toward obtaining a better understanding of the interactions of HIV-1 and myeloid cells in relation to the pathogenesis of AIDS. The human myelomonocytic cell line, HL-60, was used as a model system to determine if HIV-1 infects myeloid progenitor cells and subsequently, if infection affects their differentiation. HL-60 cells and the human prototypic T cell line, H9 were infected with three different HIV-l isolates (IIIB, PM213, and NL4-3) which are known to infect T cells. All three isolates productively infected both H9 and HL-60 cells; however, HIV-1 antigen expression and cytopathicity was delayed by approximately 15 days in infected HL-60 cells compared H9 cells. To examine the effect of HIV-l infection on myeloid differentiation, chronically infected HL-60 cells and clonal lines derived from them were induced to differentiate into either granulocytes by treatment with dimethyl formamide (DMF) or into monocytes by treatment with phorbol l2-myristate 13 acetate (PMA). By both cellular morphology and function, approximately the same percentage of treated, HIV-infected HL-60 cells differentiated into either granulocytes or monocytes as treated, control HL-60 cells. Taken together, these results indicate that HIV-1 infection does not affect the morphological or functional differentiation of HL-60 cells. In an effort to understand the differences in the regulation of HIV-l infection in myeloid versus T cells, the life cycle of NL4-3 was examined in HL-60 cells and H9 cells. Initially, NL4-3 replication was restricted in HL-60 cells compared to H9 cells. This restriction was overcome 15 days after infection by the generation of a viral isolate, NL4-3(M). NL4-3(M), harvested during the lytic phase of NL4-3 infection of HL-60 cells, caused cell death approximately 8 days after infection in both H9 and HL-60 cells. Although measurements of viral entry kinetics demonstrated that the timing of entry of NL4-3 and NL4-3(M) in HL-60 cells and NL4-3 in H9 cells was similar, a quantitative polymerase chain reaction (PCR) analysis of newly reverse transcribed NL4-3 DNA in H9 and HL-60 cells revealed that NL4-3 infected H9 cells and NL4-3(M) infected HL-60 cells contain consistently higher amounts of newly reverse transcribed DNA than NL4-3 infected HL-60 cells. The delay in NL4-3 replication in HL-60 cells was further amplified by inefficient spread of the virus throughout the HL-60 culture as measured by RNA production and DNA integration suggesting that another step in the viral life cycle after reverse transcription was also restricted. These results suggest that the efficiency of NL43 replication in HL-60 cells is restricted at several steps in the viral life cycle. Further, these restrictions are overcome by the generation of a viral variant, NL4-3(M), which efficiently replicates in myeloid cells. The tropism of NL4-3(M) was further characterized by testing its growth in monocyte-derived macrophages (MDM). Unlike NL4-3, NL4-3(M) productively infected MDM cultures. The ability of NL4-3(M) to infect macrophages was conferred by the envelope gene. This was demonstrated by the ability of the recombinant virus, NL4-3envA, which contains the envelope of NL4-3(M) in the context of the NL4-3 genome, to infect and replicate in MDM cultures. The envelope gene of NL4-3(M), however, did not confer ability to rapidly kill HL-60 cells. Together, these findings demonstrate that viral determinants controlling entry into MDM are different trom the determinants controlling the cytopathic phenotype in HL-60 cells

Gem-induced cytoskeleton remodeling increases cellular migration of HTLV-1-infected cells, formation of infected-to-target T-cell conjugates and viral transmission

Efficient HTLV-1 viral transmission occurs through cell-to-cell contacts. The Tax viral transcriptional activator protein facilitates this process. Using a comparative transcriptomic analysis, we recently identified a series of genes up-regulated in HTLV-1 Tax expressing T-lymphocytes. We focused our attention towards genes that are important for cytoskeleton dynamic and thus may possibly modulate cell-to-cell contacts. We first demonstrate that Gem, a member of the small GTP-binding proteins within the Ras superfamily, is expressed both at the RNA and protein levels in Tax-expressing cells and in HTLV-1-infected cell lines. Using a series of ChIP assays, we show that Tax recruits CREB and CREB Binding Protein (CBP) onto a c-AMP Responsive Element (CRE) present in the gem promoter. This CRE sequence is required to drive Tax-activated gem transcription. Since Gem is involved in cytoskeleton remodeling, we investigated its role in infected cells motility. We show that Gem co-localizes with F-actin and is involved both in T-cell spontaneous cell migration as well as chemotaxis in the presence of SDF-1/CXCL12. Importantly, gem knock-down in HTLV-1-infected cells decreases cell migration and conjugate formation. Finally, we demonstrate that Gem plays an important role in cell-to-cell viral transmission

Memories of John N. Brady: scientist, mentor and friend

Friends and colleagues remember John N. Brady, Ph.D., Chief of the Virus Tumor Biology Section of the Laboratory of Cellular Oncology, who died much too young at the age of 57 on April 27, 2009 of colon cancer. John grew up in Illinois and received his Ph.D. with Dr. Richard Consigli at Kansas State University studying the molecular structure of polyomavirus. In 1984 John came to the National Institutes of Health as a Staff Fellow in the laboratory of Dr. Norman Salzman, Laboratory of Biology of Viruses NIAID, where he was among the first to analyze SV40 transcription using in vitro transcription systems and to analyze regulatory sequences for SV40 late transcription. He then trained with Dr. George Khoury in the Laboratory of Molecular Virology NCI, where he identified SV40 T-antigen as a transcriptional activator protein. His research interests grew to focus on the human retroviruses: human T-cell lymphotropic virus type I (HTLV-I) and human immunodeficiency virus (HIV), analyzing how interactions between these viruses and the host cell influence viral gene regulation, viral pathogenesis and viral transformation. His research also impacted the fields of eukaryotic gene regulation and tumor suppressor proteins. John is survived by his wife, Laraine, and two sons, Matt and Kevin

Transcriptomic analyses reveal that the cellular Gem protein promotes HTLV-1 infected cell migration and viral transmission

International audienc

Differences in the Curing of [PSI+] Prion by Various Methods of Hsp104 Inactivation

[PSI+] yeast, containing the misfolded amyloid conformation of Sup35 prion, is cured by inactivation of Hsp104. There has been controversy as to whether inactivation of Hsp104 by guanidine treatment or by overexpression of the dominant negative Hsp104 mutant, Hsp104-2KT, cures [PSI+] by the same mechanism– inhibition of the severing of the prion seeds. Using live cell imaging of Sup35-GFP, overexpression of Hsp104-2KT caused the foci to increase in size, then decrease in number, and finally disappear when the cells were cured, similar to that observed in cells cured by depletion of Hsp104. In contrast, guanidine initially caused an increase in foci size but then the foci disappeared before the cells were cured. By starving the yeast to make the foci visible in cells grown with guanidine, the number of cells with foci was found to correlate exactly with the number of [PSI+] cells, regardless of the curing method. Therefore, the fluorescent foci are the prion seeds required for maintenance of [PSI+] and inactivation of Hsp104 cures [PSI+] by preventing severing of the prion seeds. During curing with guanidine, the reduction in seed size is an Hsp104-dependent effect that cannot be explained by limited severing of the seeds. Instead, in the presence of guanidine, Hsp104 retains an activity that trims or reduces the size of the prion seeds by releasing Sup35 molecules that are unable to form new prion seeds. This Hsp104 activity may also occur in propagating yeast

Gem-Induced Cytoskeleton Remodeling Increases Cellular Migration of HTLV-1-Infected Cells, Formation of Infected-to-Target T-Cell Conjugates and Viral Transmission

International audienceEfficient HTLV-1 viral transmission occurs through cell-to-cell contacts. The Tax viral transcriptional activator protein facilitates this process. Using a comparative transcriptomic analysis, we recently identified a series of genes up-regulated in HTLV-1 Tax expressing T-lymphocytes. We focused our attention towards genes that are important for cytoskeleton dynamic and thus may possibly modulate cell-to-cell contacts. We first demonstrate that Gem, a member of the small GTP-binding proteins within the Ras superfamily, is expressed both at the RNA and protein levels in Tax-expressing cells and in HTLV-1-infected cell lines. Using a series of ChIP assays, we show that Tax recruits CREB and CREB Binding Protein (CBP) onto a cAMP Responsive Element (CRE) present in the gem promoter. This CRE sequence is required to drive Tax-activated gem transcription. Since Gem is involved in cytoskeleton remodeling, we investigated its role in infected cells motility. We show that Gem co-localizes with F-actin and is involved both in T-cell spontaneous cell migration as well as chemotaxis in the presence of SDF-1/CXCL12. Importantly, gem knock-down in HTLV-1-infected cells decreases cell migration and conjugate formation. Finally, we demonstrate that Gem plays an important role in cell-to-cell viral transmission

Expression Of Mir-34a In T-cells Infected By Human T-lymphotropic Virus 1

Human T-lymphotropic virus 1 (HTLV-1) immortalizes T-cells and is the causative agent of adult T-cell leukemia/lymphoma (ATLL). HTLV-1 replication and transformation are governed by multiple interactions between viral regulatory proteins and host cell factors that remain to be fully elucidated. The present study investigated the impact of HTLV-1 infection on the expression of miR-34a, a microRNA whose expression is downregulated in many types of cancer. Results of RT-PCR assays showed that five out of six HTLV-1-positive cell lines expressed higher levels of miR-34a compared to normal PBMC or purified CD4+ T-cells. ATLL cell line ED, which did not express miR-34a, showed methylation of the miR-34a promoter. Newly infected PBMC and samples from 10 ATLL patients also showed a prominent increase in miR-34a expression compared to PBMC controls. The primary miR-34a transcript expressed in infected cell line C91PL contained binding motifs for NF-kappa B and p53. Pharmacological inhibition of NF-kappa B with Bay 11-7082 indicated that this pathway contributes to sustain miR-34a levels in infected cells. Treatment of infected cell lines with the p53 activator nutlin-3a resulted in a further increase in miR-34a levels, thus confirming it as a transcriptional target of p53. Nutlin-3a-treated cells showed downregulation of known miR-34a targets including the deacetylase SIRT1, which was accompanied by increased acetylation of p53, a substrate of SIRT1. Transfection of C91PL cells with a miR-34a mimic also led to downregulation of mRNA targets including SIRT1 as well as the pro-apoptotic factor BAX. Unlike nutlin-3a, the miR-34a mimic did not cause cell cycle arrest or reduce cell viability. On the other hand, sequestration of miR-34a with a sponge construct resulted in an increase in death of C91PL cells. These findings provide evidence for a functional role for miR-34a in fine-tuning the expression of target genes that influence the turnover of HTLV-1-infected cells