Gretchen Kiser - Academia.edu (original) (raw)
Papers by Gretchen Kiser
20. Cells were grown overnight in rich medium at 23°C to early log phase. Cells were diluted to a... more 20. Cells were grown overnight in rich medium at 23°C to early log phase. Cells were diluted to an absor-bance at 600 (A600) of 0.1 and incubated at 37°C. Samples were tested every hour for 9 hours. Cell viability was calculated as the number of viable col-onies formed after plating at 23°C at each time point divided by the number of colonies that formed after plating at 23°C at the 0 hour time point. 21. Supplementary material is available at www. sciencemag.org/feature/data/974048.shl. 22. A. Dillin and J. Rine, data not shown. 23. U. Surana et al., Cell 65, 145 (1991). 24. Cdc28p was isolated and assayed essentially as de-scribed (33). The orc5-1 cultures were grown to A600! 0.1 and either arrested with "-factor, hydroxyurea, or nocodazole for 3 hours at 23°C or shifted to 37°C.
Archives of Biochemistry and Biophysics, 2001
Many cystic fibrosis disease-associated mutations cause a defect in the biosynthetic processing a... more Many cystic fibrosis disease-associated mutations cause a defect in the biosynthetic processing and trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Yeast mutants, defective at various steps of the secretory pathway, have been used to dissect the mechanisms of biosynthetic processing and intracellular transport of several proteins. To exploit these yeast mutants, we have employed an expression system in which the CFTR gene is driven by the promoter of a structurally related yeast ABC protein, Pdr5p. Pulse-chase experiments revealed a turnover rate similar to that of nascent CFTR in mammalian cells. Immunofluorescence microscopy showed that most CFTR colocalized with the endoplasmic reticulum (ER) marker protein Kar2p and not with a vacuolar marker. Degradation was not influenced by the vacuolar protease mutants Pep4p and Prb1p but was sensitive to the proteasome inhibitor lactacystin -lactone. Blocking ER-to-Golgi transit with the sec18-1 mutant had little influence on turnover indicating that it occurred primarily in the ER compartment. Degradation was slowed in cells deficient in the ER degradation protein Der3p as well as the ubiquitin-conjugating enzymes Ubc6p and Ubc7p. Finally a mutation (sec61-2) in the translocon protein Sec61p that prevents retrotranslocation across the ER membrane also blocked degradation. These results indicate that whereas approximately 75% of nascent wild-type CFTR is degraded at the ER of mammalian cells virtually all of the protein meets this fate on heterologous expression in Saccharomyces cerevisiae.
We have carried out an analysis of the serological and molecular diversity of a panel of monoclon... more We have carried out an analysis of the serological and molecular diversity of a panel of monoclonal anti-DNA autoantibodies and serum autoantibodies from NZB and (NZB X NZW) F1 mice, in an attempt to obtain insights into the mechanisms responsible for the development of systemic autoimmune disease. Our data show that the autoantibodies are quite diverse. A dominant, binding-site idiotope on one of our monoclonal autoantibodies is expressed at variable levels in anti-DNA binding antibodies in the sera of both NZB and (NZB X NZW) F1 mice, but on none of the other monoclonal autoantibodies in our panel. We have cloned and sequenced the heavy chain variable region (VH) gene of one anti-DNA hybridoma and by hybridization have determined the VH and V kappa gene segments expressed by 14 others. All of the autoantibodies express members of known V gene subfamilies. A total of four different VH and at least six V kappa subfamilies are expressed by the hybridomas. Thus, a broad spectrum of th...
Yeast (Chichester, England), 1995
While sequencing a region of chromosome IV adjacent to the checkpoint gene MEC3, we identified a ... more While sequencing a region of chromosome IV adjacent to the checkpoint gene MEC3, we identified a gene we call GUF1 (GTPase of Unknown Function), which predicts a 586 amino acid GTPase of the elongation factor-type class. The predicted Guf1p protein bears striking sequence similarity to both LepA from Escherichia coli (43% identical) and LK1236.1 from Caenorhabditis elegans (42% identical). Analysis of both a guf1 delta deletion and a putative constitutive-activating mutant (GUF1HG) revealed that GUF1 is not essential nor did mutant cells reveal any marked phenotype.
Immunogenetics, 1989
We have studied the restriction fragment length polymorphisms (RFLPs) found in the germline T-cel... more We have studied the restriction fragment length polymorphisms (RFLPs) found in the germline T-cell receptor genes of 25 inbred Mus musculus strains and 8 wild Mus species. Included in the inbred mice tested were several strains which spontaneously develop systemic autoimmune disease. Extensive polymorphism was evident for the variable (V) gene segments of the a gene family for both the
Cell cycle (Georgetown, Tex.), 2005
The use of stable cell lines expressing fusions with green fluorescent protein (GFP) has increase... more The use of stable cell lines expressing fusions with green fluorescent protein (GFP) has increased significantly in recent years. In this study we have used a range of complimentary analytical techniques to examine the characteristics of a cell line stably expressing a EGFP cell cycle sensor relative to parental U2OS cells. Analysis of cell cycle duration and cell cycle phase distribution by cell growth assays and flow cytometry revealed that the two cell lines had identical doubling times and cell cycle distributions. Measurement of EGFP fusion protein mRNA by quantitative RT-PCR indicated a EGFP sensor expression level equivalent to endogenous Cyclin B1 (7000 copies/cell in G2). Microarray analysis showed a 0.9% (>2 fold at p<0.001 across 20,000 genes) difference in global gene expression levels between parental and EGFP expressing U2OS cells, with no significant differences in expression of A, B, C, D, E, F, G, H, I, K, L, M or T type Cyclins between the two cell types. The...
Methods in Enzymology, 1998
ABSTRACT This chapter describes the special needs for, limitations to, and requirements of vector... more ABSTRACT This chapter describes the special needs for, limitations to, and requirements of vectors and hosts used for the expression of the cystic fibrosis transmembrane conductance regulator (CFTR). While heterologous expression is an integral part of the study of the protein product of virtually all cloned genes, it is also essential for the investigations of the structure, function, and biosynthesis of CFTR. This is not the only reason that the CFTR gene identified and characterized before the protein is detected but also because it is present endogenously at very low copy number. Hence, no natural rich source of the protein exists. Initial attempts to express CFTR heterologously are thwarted by difficulties in propagating plasmids containing the full-length CFTR cDNA in bacteria. No single heterologous expression system is sufficient for every avenue of CFTR investigation required to gain an understanding of its structure, function, and biosynthetic processing. The use of promoters that direct the expression to lung epithelial cells show that the overexpression is without detrimental effects and intestinal epithelial cells is capable of restoring chloride and fluid secretion to animals in which the endogenous CFTR gene is inactivated.
Neurobiology of disease, 2006
Large-scale genomics approaches are now widely utilized to study a myriad of human diseases. Thes... more Large-scale genomics approaches are now widely utilized to study a myriad of human diseases. These powerful techniques, when combined with data analysis tools, detect changes in transcript abundance in diseased tissue relative to control. We hypothesize that specific differential gene expression underlies important pathogenic processes in Parkinson's disease, which is characterized by the gradual loss of dopaminergic neurons in the substantia nigra and consequent loss of dopamine in the striatum. We have therefore examined gene expression levels in the human parkinsonian nigrostriatal pathway, and compared them with those of neurologically normal controls. Using unsupervised clustering methods, we demonstrate that relatively few genes' expression levels can effectively distinguish between disease and control brains. Further, we identify several interesting patterns of gene expression that illuminate pathogenic cascades in Parkinson's disease. In particular is the robust ...
Nucleic Acids Research, 2005
The comparability and reliability of data generated using microarray technology would be enhanced... more The comparability and reliability of data generated using microarray technology would be enhanced by use of a common set of standards that allow accuracy, reproducibility and dynamic range assessments on multiple formats. We designed and tested a complex biological reagent for performance measurements on three commercial oligonucleotide array formats that differ in probe design and signal measurement methodology. The reagent is a set of two mixtures with different proportions of RNA for each of four rat tissues (brain, liver, kidney and testes). The design provides four known ratio measurements of .200 reference probes, which were chosen for their tissue-selectivity, dynamic range coverage and alignment to the same exemplar transcript sequence across all three platforms. The data generated from testing three biological replicates of the reagent at eight laboratories on three array formats provides a benchmark set for both laboratory and data processing performance assessments. Close agreement with target ratios adjusted for sample complexity was achieved on all platforms and low variance was observed among platforms, replicates and sites. The mixed tissue design produces a reagent with known gene expression changes within a complex sample and can serve as a paradigm for performance standards for microarrays that target other species.
Neurobiology of Aging, 2005
The gradual loss of striatal dopamine and dopaminergic neurons residing in the substantia nigra (... more The gradual loss of striatal dopamine and dopaminergic neurons residing in the substantia nigra (SN) causes parkinsonism characterized by slow, halting movements, rigidity, and resting tremor when neuronal loss exceeds a threshold of approximately 80%. It is estimated that there is extensive compensation for several years prior to symptom onset, during which vulnerable neurons asynchronously die. Recent evidence would argue that much of the compensatory response of the nigrostriatal system is multimodal including both pre-synaptic and striatal mechanisms. Although parkinsonism may have multiple causes, the classic syndrome, Parkinson's disease (PD), is frequently modeled in small animals by repeated administration of the selective neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Because the MPTP model of PD recapitulates many of the known behavioral and pathological features of human PD, we asked whether the striatal cells of mice treated with MPTP in a semi-chronic paradigm enact a transcriptional program that would help elucidate the response to dopamine denervation. Our findings reveal a time-dependent dysregulation in the striatum of a set of genes whose products may impact both the viability and ability to communicate of dopamine neurons in the SN.
Molecular Biology of the Cell, 1996
In eukaryotic cells, checkpoint genes cause arrest of cell division when DNA is damaged or when D... more In eukaryotic cells, checkpoint genes cause arrest of cell division when DNA is damaged or when DNA replication is blocked. In this study of budding yeast checkpoint genes, we identify and characterize another role for these checkpoint genes after DNA damagetranscriptional induction of genes. We found that three checkpoint genes (of six genes tested) have strong and distinct roles in transcriptional induction in four distinct pathways of regulation (each defined by induction of specific genes). MECI mediates the response in three transcriptional pathways, RAD53 mediates two of these pathways, and RAD17 mediates but a single pathway. The three other checkpoint genes (including RAD9) have small (twofold) but significant roles in transcriptional induction in all pathways. One of the pathways that we identify here leads to induction of MECI and RAD53 checkpoint genes themselves. This suggests a positive feedback circuit that may increase the cell's ability to respond to DNA damage. We make two primary conclusions from these studies. First, MECI appears to be the key regulator because it is required for all responses (both transcriptional and cell cycle arrest), while other genes serve only a subset of these responses. Second, the two types of responses, transcriptional induction and cell cycle arrest, appear distinct because both require MEC1 yet only cell cycle arrest requires RAD9. These and other results were used to formulate a working model of checkpoint gene function that accounts for roles of different checkpoint genes in different responses and after different types of damage. The conclusion that the yeast MECI gene is a key regulator also has implications for the role of a putative human homologue, the ATM gene.
Genes & Development, 1994
In eukaryotes a cell-cycle control termed a checkpoint causes arrest in the S or G2 phases when c... more In eukaryotes a cell-cycle control termed a checkpoint causes arrest in the S or G2 phases when chromosomes are incompletely replicated or damaged. Previously, we showed in budding yeast that RAD9 and RAD17 are checkpoint genes required for arrest in the G2 phase after DNA damage. Here, we describe a genetic strategy that identified four additional checkpoint genes that act in two pathways. Both classes of genes are required for arrest in the G2 phase after DNA damage, and one class of genes is also required for arrest in S phase when DNA replication is incomplete. The Gz-specific genes include MEC3 (for mitosis entry checkpoint), RAD9, RAD17, and RAD24. The genes common to both S phase and G2 phase pathways are MECl and MEC2. The MEC2 gene proves to be identical to the RAD53 gene. Checkpoint mutants were identified by their interactions with a temperature-sensitive allele of the cell division cycle gene CDC13-, cdcl3 mutants arrested in G2 and survived at the restrictive temperature, whereas all cdcl3 checkpoint double mutants failed to arrest in G2 and died rapidly at the restrictive temperature. The cell-cycle roles of the RAD and MEC genes were examined by combination of rad and mec mutant alleles with 10 cdc mutant alleles that arrest in different stages of the cell cycle at the restrictive temperature and by the response of rad and mec mutant alleles to DNA damaging agents and to hydroxyurea, a drug that inhibits DNA replication. We conclude that the checkpoint in budding yeast consists of overlapping S-phase and G2-phase pathways that respond to incomplete DNA replication and/or DNA damage and cause arrest of cells before mitosis.
FEBS Letters, 1997
Cystic fibrosis is characterized by an impaired cyclic adenosine 3,5-monophosphate (cAMP) activat... more Cystic fibrosis is characterized by an impaired cyclic adenosine 3,5-monophosphate (cAMP) activated CI-conductance in parallel with an enhanced amiloride sensitive Na+ conductance (ENaC) of the respiratory epithelium. Very recently, acute downregulation of ENaC by the cystic fibrosis transmembrane conductance regulator (CFTR) was demonstrated in several studies. The mechanism, however, by which CFTR exerts its inhibitory effect on ENaC remains obscure. We demonstrate that cytosolic domains of human CFTR are sufficient to induce inhibition of rat epithelial Na + currents (rENaC) when coexpressed in Xenopus oocytes and stimulated with 3-isobutyl-l-methylxanthine (IBMX). Moreover, mutations of CFTR, which occur in cystic fibrosis, abolish CFTRdependent downregulation of rENaC. Yeast two hybrid analysis of CFTR domains and rENaC subunits suggest direct interaction between the proteins. Enhanced Na + transport as found in the airways of cystic fibrosis patients is probably due to a lack of CFTR dependent downregulation of ENaC.
Experimental Neurology, 2007
The pathophysiological processes that cause Parkinson's disease (PD) affect dopamine neurons resi... more The pathophysiological processes that cause Parkinson's disease (PD) affect dopamine neurons residing in the substantia nigra with devastating consequences for normal movement. One important gene involved in both familial and sporadic PD is α-synuclein. We have generated three strains of α-synuclein transgenic mice to study the pathologic consequences of the targeted expression of mutant or wild-type human α-synuclein in a model system. We have analyzed gene expression patterns in these mice using high throughput microarrays in anatomical regions implicated in disease (substantia nigra and brainstem). Our study reveals gene dosage-dependent dysregulation of several genes important for the dopaminergic phenotype in mice over-expressing wild-type human α-synuclein in the substantia nigra at time points preceding neuronal cell death. Analysis of mutant α-synuclein mice at a time point when pathology is advanced reveals several new candidate genes that may play a role in neuronal demise and/or protein accumulation.
Cell Cycle, 2005
ABSTRACT The use of stable cell lines expressing fusions with green fluorescent protein(GFP) has ... more ABSTRACT The use of stable cell lines expressing fusions with green fluorescent protein(GFP) has increased significantly in recent years. In this study we have useda range of complimentary analytical techniques to examine the characteristicsof a cell line stably expressing a EGFP cell cycle sensor relative to parentalU2OS cells. Analysis of cell cycle duration and cell cycle phase distribution bycell growth assays and flow cytometry revealed that the two cell lines hadidentical doubling times and cell cycle distributions. Measurement of EGFPfusion protein mRNA by quantitative RT-PCR indicated a EGFP sensorexpression level equivalent to endogenous Cyclin B1 (7000 copies/cell in G2).Microarray analysis showed a 0.9% (>2 fold at p
20. Cells were grown overnight in rich medium at 23°C to early log phase. Cells were diluted to a... more 20. Cells were grown overnight in rich medium at 23°C to early log phase. Cells were diluted to an absor-bance at 600 (A600) of 0.1 and incubated at 37°C. Samples were tested every hour for 9 hours. Cell viability was calculated as the number of viable col-onies formed after plating at 23°C at each time point divided by the number of colonies that formed after plating at 23°C at the 0 hour time point. 21. Supplementary material is available at www. sciencemag.org/feature/data/974048.shl. 22. A. Dillin and J. Rine, data not shown. 23. U. Surana et al., Cell 65, 145 (1991). 24. Cdc28p was isolated and assayed essentially as de-scribed (33). The orc5-1 cultures were grown to A600! 0.1 and either arrested with "-factor, hydroxyurea, or nocodazole for 3 hours at 23°C or shifted to 37°C.
Archives of Biochemistry and Biophysics, 2001
Many cystic fibrosis disease-associated mutations cause a defect in the biosynthetic processing a... more Many cystic fibrosis disease-associated mutations cause a defect in the biosynthetic processing and trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Yeast mutants, defective at various steps of the secretory pathway, have been used to dissect the mechanisms of biosynthetic processing and intracellular transport of several proteins. To exploit these yeast mutants, we have employed an expression system in which the CFTR gene is driven by the promoter of a structurally related yeast ABC protein, Pdr5p. Pulse-chase experiments revealed a turnover rate similar to that of nascent CFTR in mammalian cells. Immunofluorescence microscopy showed that most CFTR colocalized with the endoplasmic reticulum (ER) marker protein Kar2p and not with a vacuolar marker. Degradation was not influenced by the vacuolar protease mutants Pep4p and Prb1p but was sensitive to the proteasome inhibitor lactacystin -lactone. Blocking ER-to-Golgi transit with the sec18-1 mutant had little influence on turnover indicating that it occurred primarily in the ER compartment. Degradation was slowed in cells deficient in the ER degradation protein Der3p as well as the ubiquitin-conjugating enzymes Ubc6p and Ubc7p. Finally a mutation (sec61-2) in the translocon protein Sec61p that prevents retrotranslocation across the ER membrane also blocked degradation. These results indicate that whereas approximately 75% of nascent wild-type CFTR is degraded at the ER of mammalian cells virtually all of the protein meets this fate on heterologous expression in Saccharomyces cerevisiae.
We have carried out an analysis of the serological and molecular diversity of a panel of monoclon... more We have carried out an analysis of the serological and molecular diversity of a panel of monoclonal anti-DNA autoantibodies and serum autoantibodies from NZB and (NZB X NZW) F1 mice, in an attempt to obtain insights into the mechanisms responsible for the development of systemic autoimmune disease. Our data show that the autoantibodies are quite diverse. A dominant, binding-site idiotope on one of our monoclonal autoantibodies is expressed at variable levels in anti-DNA binding antibodies in the sera of both NZB and (NZB X NZW) F1 mice, but on none of the other monoclonal autoantibodies in our panel. We have cloned and sequenced the heavy chain variable region (VH) gene of one anti-DNA hybridoma and by hybridization have determined the VH and V kappa gene segments expressed by 14 others. All of the autoantibodies express members of known V gene subfamilies. A total of four different VH and at least six V kappa subfamilies are expressed by the hybridomas. Thus, a broad spectrum of th...
Yeast (Chichester, England), 1995
While sequencing a region of chromosome IV adjacent to the checkpoint gene MEC3, we identified a ... more While sequencing a region of chromosome IV adjacent to the checkpoint gene MEC3, we identified a gene we call GUF1 (GTPase of Unknown Function), which predicts a 586 amino acid GTPase of the elongation factor-type class. The predicted Guf1p protein bears striking sequence similarity to both LepA from Escherichia coli (43% identical) and LK1236.1 from Caenorhabditis elegans (42% identical). Analysis of both a guf1 delta deletion and a putative constitutive-activating mutant (GUF1HG) revealed that GUF1 is not essential nor did mutant cells reveal any marked phenotype.
Immunogenetics, 1989
We have studied the restriction fragment length polymorphisms (RFLPs) found in the germline T-cel... more We have studied the restriction fragment length polymorphisms (RFLPs) found in the germline T-cell receptor genes of 25 inbred Mus musculus strains and 8 wild Mus species. Included in the inbred mice tested were several strains which spontaneously develop systemic autoimmune disease. Extensive polymorphism was evident for the variable (V) gene segments of the a gene family for both the
Cell cycle (Georgetown, Tex.), 2005
The use of stable cell lines expressing fusions with green fluorescent protein (GFP) has increase... more The use of stable cell lines expressing fusions with green fluorescent protein (GFP) has increased significantly in recent years. In this study we have used a range of complimentary analytical techniques to examine the characteristics of a cell line stably expressing a EGFP cell cycle sensor relative to parental U2OS cells. Analysis of cell cycle duration and cell cycle phase distribution by cell growth assays and flow cytometry revealed that the two cell lines had identical doubling times and cell cycle distributions. Measurement of EGFP fusion protein mRNA by quantitative RT-PCR indicated a EGFP sensor expression level equivalent to endogenous Cyclin B1 (7000 copies/cell in G2). Microarray analysis showed a 0.9% (>2 fold at p<0.001 across 20,000 genes) difference in global gene expression levels between parental and EGFP expressing U2OS cells, with no significant differences in expression of A, B, C, D, E, F, G, H, I, K, L, M or T type Cyclins between the two cell types. The...
Methods in Enzymology, 1998
ABSTRACT This chapter describes the special needs for, limitations to, and requirements of vector... more ABSTRACT This chapter describes the special needs for, limitations to, and requirements of vectors and hosts used for the expression of the cystic fibrosis transmembrane conductance regulator (CFTR). While heterologous expression is an integral part of the study of the protein product of virtually all cloned genes, it is also essential for the investigations of the structure, function, and biosynthesis of CFTR. This is not the only reason that the CFTR gene identified and characterized before the protein is detected but also because it is present endogenously at very low copy number. Hence, no natural rich source of the protein exists. Initial attempts to express CFTR heterologously are thwarted by difficulties in propagating plasmids containing the full-length CFTR cDNA in bacteria. No single heterologous expression system is sufficient for every avenue of CFTR investigation required to gain an understanding of its structure, function, and biosynthetic processing. The use of promoters that direct the expression to lung epithelial cells show that the overexpression is without detrimental effects and intestinal epithelial cells is capable of restoring chloride and fluid secretion to animals in which the endogenous CFTR gene is inactivated.
Neurobiology of disease, 2006
Large-scale genomics approaches are now widely utilized to study a myriad of human diseases. Thes... more Large-scale genomics approaches are now widely utilized to study a myriad of human diseases. These powerful techniques, when combined with data analysis tools, detect changes in transcript abundance in diseased tissue relative to control. We hypothesize that specific differential gene expression underlies important pathogenic processes in Parkinson's disease, which is characterized by the gradual loss of dopaminergic neurons in the substantia nigra and consequent loss of dopamine in the striatum. We have therefore examined gene expression levels in the human parkinsonian nigrostriatal pathway, and compared them with those of neurologically normal controls. Using unsupervised clustering methods, we demonstrate that relatively few genes' expression levels can effectively distinguish between disease and control brains. Further, we identify several interesting patterns of gene expression that illuminate pathogenic cascades in Parkinson's disease. In particular is the robust ...
Nucleic Acids Research, 2005
The comparability and reliability of data generated using microarray technology would be enhanced... more The comparability and reliability of data generated using microarray technology would be enhanced by use of a common set of standards that allow accuracy, reproducibility and dynamic range assessments on multiple formats. We designed and tested a complex biological reagent for performance measurements on three commercial oligonucleotide array formats that differ in probe design and signal measurement methodology. The reagent is a set of two mixtures with different proportions of RNA for each of four rat tissues (brain, liver, kidney and testes). The design provides four known ratio measurements of .200 reference probes, which were chosen for their tissue-selectivity, dynamic range coverage and alignment to the same exemplar transcript sequence across all three platforms. The data generated from testing three biological replicates of the reagent at eight laboratories on three array formats provides a benchmark set for both laboratory and data processing performance assessments. Close agreement with target ratios adjusted for sample complexity was achieved on all platforms and low variance was observed among platforms, replicates and sites. The mixed tissue design produces a reagent with known gene expression changes within a complex sample and can serve as a paradigm for performance standards for microarrays that target other species.
Neurobiology of Aging, 2005
The gradual loss of striatal dopamine and dopaminergic neurons residing in the substantia nigra (... more The gradual loss of striatal dopamine and dopaminergic neurons residing in the substantia nigra (SN) causes parkinsonism characterized by slow, halting movements, rigidity, and resting tremor when neuronal loss exceeds a threshold of approximately 80%. It is estimated that there is extensive compensation for several years prior to symptom onset, during which vulnerable neurons asynchronously die. Recent evidence would argue that much of the compensatory response of the nigrostriatal system is multimodal including both pre-synaptic and striatal mechanisms. Although parkinsonism may have multiple causes, the classic syndrome, Parkinson's disease (PD), is frequently modeled in small animals by repeated administration of the selective neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Because the MPTP model of PD recapitulates many of the known behavioral and pathological features of human PD, we asked whether the striatal cells of mice treated with MPTP in a semi-chronic paradigm enact a transcriptional program that would help elucidate the response to dopamine denervation. Our findings reveal a time-dependent dysregulation in the striatum of a set of genes whose products may impact both the viability and ability to communicate of dopamine neurons in the SN.
Molecular Biology of the Cell, 1996
In eukaryotic cells, checkpoint genes cause arrest of cell division when DNA is damaged or when D... more In eukaryotic cells, checkpoint genes cause arrest of cell division when DNA is damaged or when DNA replication is blocked. In this study of budding yeast checkpoint genes, we identify and characterize another role for these checkpoint genes after DNA damagetranscriptional induction of genes. We found that three checkpoint genes (of six genes tested) have strong and distinct roles in transcriptional induction in four distinct pathways of regulation (each defined by induction of specific genes). MECI mediates the response in three transcriptional pathways, RAD53 mediates two of these pathways, and RAD17 mediates but a single pathway. The three other checkpoint genes (including RAD9) have small (twofold) but significant roles in transcriptional induction in all pathways. One of the pathways that we identify here leads to induction of MECI and RAD53 checkpoint genes themselves. This suggests a positive feedback circuit that may increase the cell's ability to respond to DNA damage. We make two primary conclusions from these studies. First, MECI appears to be the key regulator because it is required for all responses (both transcriptional and cell cycle arrest), while other genes serve only a subset of these responses. Second, the two types of responses, transcriptional induction and cell cycle arrest, appear distinct because both require MEC1 yet only cell cycle arrest requires RAD9. These and other results were used to formulate a working model of checkpoint gene function that accounts for roles of different checkpoint genes in different responses and after different types of damage. The conclusion that the yeast MECI gene is a key regulator also has implications for the role of a putative human homologue, the ATM gene.
Genes & Development, 1994
In eukaryotes a cell-cycle control termed a checkpoint causes arrest in the S or G2 phases when c... more In eukaryotes a cell-cycle control termed a checkpoint causes arrest in the S or G2 phases when chromosomes are incompletely replicated or damaged. Previously, we showed in budding yeast that RAD9 and RAD17 are checkpoint genes required for arrest in the G2 phase after DNA damage. Here, we describe a genetic strategy that identified four additional checkpoint genes that act in two pathways. Both classes of genes are required for arrest in the G2 phase after DNA damage, and one class of genes is also required for arrest in S phase when DNA replication is incomplete. The Gz-specific genes include MEC3 (for mitosis entry checkpoint), RAD9, RAD17, and RAD24. The genes common to both S phase and G2 phase pathways are MECl and MEC2. The MEC2 gene proves to be identical to the RAD53 gene. Checkpoint mutants were identified by their interactions with a temperature-sensitive allele of the cell division cycle gene CDC13-, cdcl3 mutants arrested in G2 and survived at the restrictive temperature, whereas all cdcl3 checkpoint double mutants failed to arrest in G2 and died rapidly at the restrictive temperature. The cell-cycle roles of the RAD and MEC genes were examined by combination of rad and mec mutant alleles with 10 cdc mutant alleles that arrest in different stages of the cell cycle at the restrictive temperature and by the response of rad and mec mutant alleles to DNA damaging agents and to hydroxyurea, a drug that inhibits DNA replication. We conclude that the checkpoint in budding yeast consists of overlapping S-phase and G2-phase pathways that respond to incomplete DNA replication and/or DNA damage and cause arrest of cells before mitosis.
FEBS Letters, 1997
Cystic fibrosis is characterized by an impaired cyclic adenosine 3,5-monophosphate (cAMP) activat... more Cystic fibrosis is characterized by an impaired cyclic adenosine 3,5-monophosphate (cAMP) activated CI-conductance in parallel with an enhanced amiloride sensitive Na+ conductance (ENaC) of the respiratory epithelium. Very recently, acute downregulation of ENaC by the cystic fibrosis transmembrane conductance regulator (CFTR) was demonstrated in several studies. The mechanism, however, by which CFTR exerts its inhibitory effect on ENaC remains obscure. We demonstrate that cytosolic domains of human CFTR are sufficient to induce inhibition of rat epithelial Na + currents (rENaC) when coexpressed in Xenopus oocytes and stimulated with 3-isobutyl-l-methylxanthine (IBMX). Moreover, mutations of CFTR, which occur in cystic fibrosis, abolish CFTRdependent downregulation of rENaC. Yeast two hybrid analysis of CFTR domains and rENaC subunits suggest direct interaction between the proteins. Enhanced Na + transport as found in the airways of cystic fibrosis patients is probably due to a lack of CFTR dependent downregulation of ENaC.
Experimental Neurology, 2007
The pathophysiological processes that cause Parkinson's disease (PD) affect dopamine neurons resi... more The pathophysiological processes that cause Parkinson's disease (PD) affect dopamine neurons residing in the substantia nigra with devastating consequences for normal movement. One important gene involved in both familial and sporadic PD is α-synuclein. We have generated three strains of α-synuclein transgenic mice to study the pathologic consequences of the targeted expression of mutant or wild-type human α-synuclein in a model system. We have analyzed gene expression patterns in these mice using high throughput microarrays in anatomical regions implicated in disease (substantia nigra and brainstem). Our study reveals gene dosage-dependent dysregulation of several genes important for the dopaminergic phenotype in mice over-expressing wild-type human α-synuclein in the substantia nigra at time points preceding neuronal cell death. Analysis of mutant α-synuclein mice at a time point when pathology is advanced reveals several new candidate genes that may play a role in neuronal demise and/or protein accumulation.
Cell Cycle, 2005
ABSTRACT The use of stable cell lines expressing fusions with green fluorescent protein(GFP) has ... more ABSTRACT The use of stable cell lines expressing fusions with green fluorescent protein(GFP) has increased significantly in recent years. In this study we have useda range of complimentary analytical techniques to examine the characteristicsof a cell line stably expressing a EGFP cell cycle sensor relative to parentalU2OS cells. Analysis of cell cycle duration and cell cycle phase distribution bycell growth assays and flow cytometry revealed that the two cell lines hadidentical doubling times and cell cycle distributions. Measurement of EGFPfusion protein mRNA by quantitative RT-PCR indicated a EGFP sensorexpression level equivalent to endogenous Cyclin B1 (7000 copies/cell in G2).Microarray analysis showed a 0.9% (>2 fold at p