Jennifer Aparicio - Academia.edu (original) (raw)
Papers by Jennifer Aparicio
Fluorescent reporter pluripotent stem cell (PSC) derived retinal organoids are powerful tools to ... more Fluorescent reporter pluripotent stem cell (PSC) derived retinal organoids are powerful tools to investigate cell type-specific development and disease phenotypes. When combined with live imaging, they enable direct and repeated observation of cell behaviors within a developing retinal tissue. Here, we generated a human cone photoreceptor reporter line by CRISPR/Cas9 genome editing of WTC11-mTagRFPT-LMNB1 human induced pluripotent stem cells (iPSCs) by inserting enhanced green fluorescent protein (EGFP) coding sequences and a 2A self-cleaving peptide at the N-terminus ofGuanine Nucleotide-Binding Protein Subunit Alpha Transducin 2(GNAT2). In retinal organoids generated from these iPSCs, the GNAT2-EGFP allele robustly and exclusively labeled both immature and mature cones starting at culture day 34. Episodic confocal live imaging of hydrogel immobilized retinal organoids allowed tracking of morphological maturation of individual cones for >18 weeks and revealed inner segment accum...
Retinoblastomas form in response to biallelicRB1mutations orMYCNamplification and progress to mor... more Retinoblastomas form in response to biallelicRB1mutations orMYCNamplification and progress to more aggressive and therapy-resistant phenotypes through accumulation of secondary genomic changes. Progression-related changes include recurrent somatic copy number alterations and typically non-recurrent nucleotide variants, including synonymous and non-coding variants, whose significance has been unclear. To assess synonymous and non-coding variant contributions to recurrently altered processes, we identified altered genes and over-represented variant gene ontologies in 168 exome or whole-genome-sequenced retinoblastomas and 12 tumor-matched cell lines. In addition to initiatingRB1mutations,MYCNamplification, and established retinoblastoma SCNAs, the analyses revealed enrichment of variant genes related to diverse biological processes including histone monoubiquitination, mRNA processing (P) body assembly, and mitotic sister chromatid segregation and cytokinesis. Importantly, inclusion o...
Development, 2021
The development of the first synapse of the visual system between photoreceptors and bipolar cell... more The development of the first synapse of the visual system between photoreceptors and bipolar cells in the outer plexiform layer (OPL) of the human retina is crucial for visual processing but poorly understood. By studying the maturation state and spatial organization of photoreceptors, depolarizing bipolar cells and horizontal cells in the human fetal retina, we establish a pseudo-temporal staging system for OPL development that we term OPL-Stages 0 to 4. This was validated through quantification of increasingly precise subcellular localization of bassoon to the OPL with each stage (P<0.0001). By applying these OPL staging criteria to human retinal organoids (HROs) derived from human embryonic and induced pluripotent stem cells, we observed comparable maturation from OPL-Stage 0 at day 100 in culture up to OPL-Stage 3 by day 160. Quantification of presynaptic protein localization confirmed progression from OPL-Stage 0 to 3 (P<0.0001). Overall, this study defines stages of huma...
Cellular Therapies for Retinal Disease, 2017
Retinal cell replacement is a promising therapeutic approach to restore vision to millions of peo... more Retinal cell replacement is a promising therapeutic approach to restore vision to millions of people worldwide. Three-dimensional culture methods can produce large numbers of human embryonic retinal cells from pluripotent stem cells (PSCs), stimulating optimism for clinical use. These cells develop in a tissue environment that recapitulates many aspects of development in vivo. Most notably, the retinal progenitor cells are competent to give rise to all major retinal cells types, which segregate to rudimentary retinal layers. The cells are expected to mimic their natural counterparts at the molecular level, but that has yet to be demonstrated. These methodologies can produce impressive embryonic retinal structures, but will require optimization before meeting clinical needs. Using current methods PSC-derived retinal organoids do not fully mature, suggesting that the in vitro environment does not reproduce that of late fetal development. However, studies to date suggest that immature photoreceptor or retinal ganglion cells, roughly those of mid-gestation, function best for transplantation. This developmental stage appears to be well reproduced in vitro, and therefore the PSC-derived retinal cells may be appropriate for cell replacement approaches. Three-dimensional culture methods using PSC-derived cells offer hope to better understand and treat retinal disease.
Investigative ophthalmology & visual science, Jul 1, 2017
Human pluripotent stem cell (hPSC)-derived retinal organoids are a platform for investigating ret... more Human pluripotent stem cell (hPSC)-derived retinal organoids are a platform for investigating retinal development, pathophysiology, and cellular therapies. In contrast to histologic analysis in which multiple specimens fixed at different times are used to reconstruct developmental processes, repeated analysis of the same living organoids provides a more direct means to characterize changes. New live imaging modalities can provide insights into retinal organoid structure and metabolic function during in vitro growth. This study employed live tissue imaging to characterize retinal organoid development, including metabolic changes accompanying photoreceptor differentiation. Live hPSC-derived retinal organoids at different developmental stages were examined for microanatomic organization and metabolic function by phase contrast microscopy, optical coherence tomography (OCT), fluorescence lifetime imaging microscopy (FLIM), and hyperspectral imaging (HSpec). Features were compared to tho...
Experimental eye research, Jan 17, 2016
Human retinal ganglion cells (RGCs) derived from pluripotent stem cells (PSCs) have anticipated v... more Human retinal ganglion cells (RGCs) derived from pluripotent stem cells (PSCs) have anticipated value for human disease study, drug screening, and therapeutic applications; however, their full potential remains underdeveloped. To characterize RGCs in human embryonic stem cell (hESC) derived retinal organoids we examined RGC markers and surface antigen expression and made comparisons to human fetal retina. RGCs in both tissues exhibited CD184 and CD171 expression and distinct expression patterns of the RGC markers BRN3 and RBPMS. The retinal progenitor cells (RPCs) of retinal organoids expressed CD184, consistent with its expression in the neuroblastic layer in fetal retina. In retinal organoids CD184 expression was enhanced in RGC competent RPCs and high CD184 expression was retained on post-mitotic RGC precursors; CD171 was detected on maturing RGCs. The differential expression timing of CD184 and CD171 permits identification and enrichment of RGCs from retinal organoids at differi...
BMC genomics, Jan 26, 2006
Eukaryotic replication origins exhibit different initiation efficiencies and activation times wit... more Eukaryotic replication origins exhibit different initiation efficiencies and activation times within S-phase. Although local chromatin structure and function influences origin activity, the exact mechanisms remain poorly understood. A key to understanding the exact features of chromatin that impinge on replication origin function is to define the precise locations of the DNA sequences that control origin function. In S. cerevisiae, Autonomously Replicating Sequences (ARSs) contain a consensus sequence (ACS) that binds the Origin Recognition Complex (ORC) and is essential for origin function. However, an ACS is not sufficient for origin function and the majority of ACS matches do not function as ORC binding sites, complicating the specific identification of these sites. To identify essential origin sequences genome-wide, we utilized a tiled oligonucleotide array (NimbleGen) to map the ORC and Mcm2p binding sites at high resolution. These binding sites define a set of potential Autono...
Methods in Molecular Biology, 2009
Chromatin immunoprecipitation (ChIP) is a widely used method to study the interactions between pr... more Chromatin immunoprecipitation (ChIP) is a widely used method to study the interactions between proteins and discrete chromosomal loci in vivo. Originally, ChIP was developed for analysis of protein associations with DNA sequences known or suspected to bind the protein of interest. The advent of DNA microarrays has enabled the identification of all DNA sequences enriched by ChIP, providing a genomic view of protein binding. This powerful approach, termed ChIP-chip, is broadly applicable and has been particularly valuable in DNA replication studies to map replication origins in Saccharomyces cerevisiae based on the association of replication proteins with these chromosomal elements. We present a detailed ChIP-chip protocol for S. cerevisiae that uses oligonucleotide DNA microarrays printed on polylysine-coated glass slides and can also be easily adapted for commercially available high-density tiling microarrays from NimbleGen. We also outline general protocols for data analysis; however, microarray data analyses usually must be tailored specifically for individual studies, depending on experimental design, microarray format, and data quality.
Molecular and Cellular Biology, 2004
Cyclin-dependent kinase (CDK) is required for the initiation of chromosomal DNA replication in eu... more Cyclin-dependent kinase (CDK) is required for the initiation of chromosomal DNA replication in eukaryotes. In Saccharomyces cerevisiae , the Clb5 and Clb6 cyclins activate Cdk1 and drive replication origin firing. Deletion of CLB5 reduces initiation of DNA synthesis from late-firing origins. We have examined whether checkpoints are activated by loss of Clb5 function and whether checkpoints are responsible for the DNA replication defects associated with loss of Clb5 function. We present evidence for activation of Rad53 and Ddc2 functions with characteristics suggesting the presence of DNA damage. Deficient late origin firing in clb5 Δ cells is not due to checkpoint regulation, but instead, directly reflects the decreased abundance of S-phase CDK, as Clb6 activates late origins when its dosage is increased. Moreover, the viability of clb5 Δ cells depends on Rad53. Activation of Rad53 by either Mrc1 or Rad9 contributes to the survival of clb5 Δ cells, suggesting that both DNA replicati...
Molecular and Cellular Biology, 2004
The replication of eukaryotic genomes follows a temporally staged program, in which late origin f... more The replication of eukaryotic genomes follows a temporally staged program, in which late origin firing often occurs within domains of altered chromatin structure(s) and silenced genes. Histone deacetylation functions in gene silencing in some late-replicating regions, prompting an investigation of the role of histone deacetylation in replication timing control in Saccharomyces cerevisiae . Deletion of the histone deacetylase Rpd3 or its interacting partner Sin3 caused early activation of late origins at internal chromosomal loci but did not alter the initiation timing of early origins or a late-firing, telomere-proximal origin. By delaying initiation relative to the earliest origins, Rpd3 enables regulation of late origins by the intra-S replication checkpoint. RPD3 deletion suppresses the slow S phase of clb5 Δ cells by enabling late origins to fire earlier, suggesting that Rpd3 modulates the initiation timing of many origins throughout the genome. Examination of factors such as Um...
Journal of Cellular Biochemistry, 1990
The modB mutation eliminates specific carbohydrate epitopes from glycoproteins which are expresse... more The modB mutation eliminates specific carbohydrate epitopes from glycoproteins which are expressed primarily in prespore and spore cells of differentiating Dictyostelium discoideum. Spores formed by the mutant show several phenotypes. Whereas mutant spores germinate efficiently after heat activation, they germinate poorly after urea activation. Following germination, at least one glycosylation‐defective glycopro‐tein is cleaved, and the larger fragment is released in soluble form from the spore coat. However, an earlier difference in the spore coat can be traced back to the nongerminated spore coat, as detected by the elutability of protein from intact spores by chemical extraction. An altered character of the pregermination spore coat is also suggested by increased labeling by a fluorescent lectin which binds to its interior. The findings are consistent with a change in the character of certain molecular contacts leading to altered characteristics of the mutant spore coat, which ar...
Journal of Biological Chemistry, 1996
The photoreceptor membrane guanylate cyclase is a member of a family of proteins with a set of fo... more The photoreceptor membrane guanylate cyclase is a member of a family of proteins with a set of four structural motifs: an extracellular ligand binding domain, a transmembrane domain, an intracellular protein kinase-like domain, and an intracellular catalytic domain. Purified preparations of the photoreceptor guanylate cyclase have allowed us to explore the function of the protein kinase-like domain. ATP enhances the guanylate cyclase activity 2-fold in membranes stripped of peripheral proteins. The stimulation can be mimicked by ATP␥S (adenosine 5-O-(3-thiotriphosphate)), AMPPNP (5-adenylyl ,␥-imidodiphosphate), and ADP, but not AMP. While this effect is lost by solubilizing guanylate cyclase, ATP binds the purified, solubilized enzyme in a site distinct from the catalytic GTP site as shown by specific labeling with 8-N 3 [␣-32 P]ATP. The enzyme has a protein kinase activity that is Mg 2؉-dependent and autophosphorylates serine residues. Myelin basic protein serves as a substrate for the kinase and enables further characterization of the kinase properties. The K m for ATP is 81 M. The kinase activity is unaffected by calcium, cyclic nucleotides, and phorbol 12-myristate 13acetate/L-␣-phosphatidylserine/Ca 2؉ and is inhibited by high concentrations of staurosporine. These properties are distinct from other Ser/Thr kinases identified in rod outer segment preparations including protein kinase A, protein kinase C, and rhodopsin kinase. The observations offer the first biochemical evidence that a member of the receptor guanylate cyclase family has intrinsic protein kinase activity.
Genes & Development, 2008
Replication fork stalling at a DNA lesion generates a damage signal that activates the Rad53 kina... more Replication fork stalling at a DNA lesion generates a damage signal that activates the Rad53 kinase, which plays a vital role in survival by stabilizing stalled replication forks. However, evidence that Rad53 directly modulates the activity of replication forks has been lacking, and the nature of fork stabilization has remained unclear. Recently, cells lacking the Psy2–Pph3 phosphatase were shown to be defective in dephosphorylation of Rad53 as well as replication fork restart after DNA damage, suggesting a mechanistic link between Rad53 deactivation and fork restart. To test this possibility we examined the progression of replication forks in methyl-methanesulfonate (MMS)-damaged cells, under different conditions of Rad53 activity. Hyperactivity of Rad53 in pph3Δ cells slows fork progression in MMS, whereas deactivation of Rad53, through expression of dominant-negative Rad53-KD, is sufficient to allow fork restart during recovery. Furthermore, combined deletion of PPH3 and PTC2, a ...
Gene, 1997
The gene encoding the bovine guanylate cyclase isoform E (GC-E) was isolated as a single 18 kb ge... more The gene encoding the bovine guanylate cyclase isoform E (GC-E) was isolated as a single 18 kb genomic clone and shown to have 20 exons and 19 introns. Comparison of the structure of the GC-E gene with structures of other membrane guanylate cyclase genes indicates that the GC-E is most closely related to the subfamily of sensory guanylate cyclases. Comparison of the GC-E structure with that of the more distantly related guanylate cyclase isoform A (GC-A) gene shows the most divergence in the extracellular and C-terminal regions, but general conservation of introns and exons in the intracellular kinase-like and catalytic domains. RT-PCR from several bovine tissues shows that GC-E is expressed only in the retina. Consistent with this pattern of expression, elements for the retinal-specific transcription factors RET-1, RET-2 and Talpha-1 are located in the 5' flanking promoter region.
Methods in Enzymology, 2000
Fluorescent reporter pluripotent stem cell (PSC) derived retinal organoids are powerful tools to ... more Fluorescent reporter pluripotent stem cell (PSC) derived retinal organoids are powerful tools to investigate cell type-specific development and disease phenotypes. When combined with live imaging, they enable direct and repeated observation of cell behaviors within a developing retinal tissue. Here, we generated a human cone photoreceptor reporter line by CRISPR/Cas9 genome editing of WTC11-mTagRFPT-LMNB1 human induced pluripotent stem cells (iPSCs) by inserting enhanced green fluorescent protein (EGFP) coding sequences and a 2A self-cleaving peptide at the N-terminus ofGuanine Nucleotide-Binding Protein Subunit Alpha Transducin 2(GNAT2). In retinal organoids generated from these iPSCs, the GNAT2-EGFP allele robustly and exclusively labeled both immature and mature cones starting at culture day 34. Episodic confocal live imaging of hydrogel immobilized retinal organoids allowed tracking of morphological maturation of individual cones for >18 weeks and revealed inner segment accum...
Retinoblastomas form in response to biallelicRB1mutations orMYCNamplification and progress to mor... more Retinoblastomas form in response to biallelicRB1mutations orMYCNamplification and progress to more aggressive and therapy-resistant phenotypes through accumulation of secondary genomic changes. Progression-related changes include recurrent somatic copy number alterations and typically non-recurrent nucleotide variants, including synonymous and non-coding variants, whose significance has been unclear. To assess synonymous and non-coding variant contributions to recurrently altered processes, we identified altered genes and over-represented variant gene ontologies in 168 exome or whole-genome-sequenced retinoblastomas and 12 tumor-matched cell lines. In addition to initiatingRB1mutations,MYCNamplification, and established retinoblastoma SCNAs, the analyses revealed enrichment of variant genes related to diverse biological processes including histone monoubiquitination, mRNA processing (P) body assembly, and mitotic sister chromatid segregation and cytokinesis. Importantly, inclusion o...
Development, 2021
The development of the first synapse of the visual system between photoreceptors and bipolar cell... more The development of the first synapse of the visual system between photoreceptors and bipolar cells in the outer plexiform layer (OPL) of the human retina is crucial for visual processing but poorly understood. By studying the maturation state and spatial organization of photoreceptors, depolarizing bipolar cells and horizontal cells in the human fetal retina, we establish a pseudo-temporal staging system for OPL development that we term OPL-Stages 0 to 4. This was validated through quantification of increasingly precise subcellular localization of bassoon to the OPL with each stage (P<0.0001). By applying these OPL staging criteria to human retinal organoids (HROs) derived from human embryonic and induced pluripotent stem cells, we observed comparable maturation from OPL-Stage 0 at day 100 in culture up to OPL-Stage 3 by day 160. Quantification of presynaptic protein localization confirmed progression from OPL-Stage 0 to 3 (P<0.0001). Overall, this study defines stages of huma...
Cellular Therapies for Retinal Disease, 2017
Retinal cell replacement is a promising therapeutic approach to restore vision to millions of peo... more Retinal cell replacement is a promising therapeutic approach to restore vision to millions of people worldwide. Three-dimensional culture methods can produce large numbers of human embryonic retinal cells from pluripotent stem cells (PSCs), stimulating optimism for clinical use. These cells develop in a tissue environment that recapitulates many aspects of development in vivo. Most notably, the retinal progenitor cells are competent to give rise to all major retinal cells types, which segregate to rudimentary retinal layers. The cells are expected to mimic their natural counterparts at the molecular level, but that has yet to be demonstrated. These methodologies can produce impressive embryonic retinal structures, but will require optimization before meeting clinical needs. Using current methods PSC-derived retinal organoids do not fully mature, suggesting that the in vitro environment does not reproduce that of late fetal development. However, studies to date suggest that immature photoreceptor or retinal ganglion cells, roughly those of mid-gestation, function best for transplantation. This developmental stage appears to be well reproduced in vitro, and therefore the PSC-derived retinal cells may be appropriate for cell replacement approaches. Three-dimensional culture methods using PSC-derived cells offer hope to better understand and treat retinal disease.
Investigative ophthalmology & visual science, Jul 1, 2017
Human pluripotent stem cell (hPSC)-derived retinal organoids are a platform for investigating ret... more Human pluripotent stem cell (hPSC)-derived retinal organoids are a platform for investigating retinal development, pathophysiology, and cellular therapies. In contrast to histologic analysis in which multiple specimens fixed at different times are used to reconstruct developmental processes, repeated analysis of the same living organoids provides a more direct means to characterize changes. New live imaging modalities can provide insights into retinal organoid structure and metabolic function during in vitro growth. This study employed live tissue imaging to characterize retinal organoid development, including metabolic changes accompanying photoreceptor differentiation. Live hPSC-derived retinal organoids at different developmental stages were examined for microanatomic organization and metabolic function by phase contrast microscopy, optical coherence tomography (OCT), fluorescence lifetime imaging microscopy (FLIM), and hyperspectral imaging (HSpec). Features were compared to tho...
Experimental eye research, Jan 17, 2016
Human retinal ganglion cells (RGCs) derived from pluripotent stem cells (PSCs) have anticipated v... more Human retinal ganglion cells (RGCs) derived from pluripotent stem cells (PSCs) have anticipated value for human disease study, drug screening, and therapeutic applications; however, their full potential remains underdeveloped. To characterize RGCs in human embryonic stem cell (hESC) derived retinal organoids we examined RGC markers and surface antigen expression and made comparisons to human fetal retina. RGCs in both tissues exhibited CD184 and CD171 expression and distinct expression patterns of the RGC markers BRN3 and RBPMS. The retinal progenitor cells (RPCs) of retinal organoids expressed CD184, consistent with its expression in the neuroblastic layer in fetal retina. In retinal organoids CD184 expression was enhanced in RGC competent RPCs and high CD184 expression was retained on post-mitotic RGC precursors; CD171 was detected on maturing RGCs. The differential expression timing of CD184 and CD171 permits identification and enrichment of RGCs from retinal organoids at differi...
BMC genomics, Jan 26, 2006
Eukaryotic replication origins exhibit different initiation efficiencies and activation times wit... more Eukaryotic replication origins exhibit different initiation efficiencies and activation times within S-phase. Although local chromatin structure and function influences origin activity, the exact mechanisms remain poorly understood. A key to understanding the exact features of chromatin that impinge on replication origin function is to define the precise locations of the DNA sequences that control origin function. In S. cerevisiae, Autonomously Replicating Sequences (ARSs) contain a consensus sequence (ACS) that binds the Origin Recognition Complex (ORC) and is essential for origin function. However, an ACS is not sufficient for origin function and the majority of ACS matches do not function as ORC binding sites, complicating the specific identification of these sites. To identify essential origin sequences genome-wide, we utilized a tiled oligonucleotide array (NimbleGen) to map the ORC and Mcm2p binding sites at high resolution. These binding sites define a set of potential Autono...
Methods in Molecular Biology, 2009
Chromatin immunoprecipitation (ChIP) is a widely used method to study the interactions between pr... more Chromatin immunoprecipitation (ChIP) is a widely used method to study the interactions between proteins and discrete chromosomal loci in vivo. Originally, ChIP was developed for analysis of protein associations with DNA sequences known or suspected to bind the protein of interest. The advent of DNA microarrays has enabled the identification of all DNA sequences enriched by ChIP, providing a genomic view of protein binding. This powerful approach, termed ChIP-chip, is broadly applicable and has been particularly valuable in DNA replication studies to map replication origins in Saccharomyces cerevisiae based on the association of replication proteins with these chromosomal elements. We present a detailed ChIP-chip protocol for S. cerevisiae that uses oligonucleotide DNA microarrays printed on polylysine-coated glass slides and can also be easily adapted for commercially available high-density tiling microarrays from NimbleGen. We also outline general protocols for data analysis; however, microarray data analyses usually must be tailored specifically for individual studies, depending on experimental design, microarray format, and data quality.
Molecular and Cellular Biology, 2004
Cyclin-dependent kinase (CDK) is required for the initiation of chromosomal DNA replication in eu... more Cyclin-dependent kinase (CDK) is required for the initiation of chromosomal DNA replication in eukaryotes. In Saccharomyces cerevisiae , the Clb5 and Clb6 cyclins activate Cdk1 and drive replication origin firing. Deletion of CLB5 reduces initiation of DNA synthesis from late-firing origins. We have examined whether checkpoints are activated by loss of Clb5 function and whether checkpoints are responsible for the DNA replication defects associated with loss of Clb5 function. We present evidence for activation of Rad53 and Ddc2 functions with characteristics suggesting the presence of DNA damage. Deficient late origin firing in clb5 Δ cells is not due to checkpoint regulation, but instead, directly reflects the decreased abundance of S-phase CDK, as Clb6 activates late origins when its dosage is increased. Moreover, the viability of clb5 Δ cells depends on Rad53. Activation of Rad53 by either Mrc1 or Rad9 contributes to the survival of clb5 Δ cells, suggesting that both DNA replicati...
Molecular and Cellular Biology, 2004
The replication of eukaryotic genomes follows a temporally staged program, in which late origin f... more The replication of eukaryotic genomes follows a temporally staged program, in which late origin firing often occurs within domains of altered chromatin structure(s) and silenced genes. Histone deacetylation functions in gene silencing in some late-replicating regions, prompting an investigation of the role of histone deacetylation in replication timing control in Saccharomyces cerevisiae . Deletion of the histone deacetylase Rpd3 or its interacting partner Sin3 caused early activation of late origins at internal chromosomal loci but did not alter the initiation timing of early origins or a late-firing, telomere-proximal origin. By delaying initiation relative to the earliest origins, Rpd3 enables regulation of late origins by the intra-S replication checkpoint. RPD3 deletion suppresses the slow S phase of clb5 Δ cells by enabling late origins to fire earlier, suggesting that Rpd3 modulates the initiation timing of many origins throughout the genome. Examination of factors such as Um...
Journal of Cellular Biochemistry, 1990
The modB mutation eliminates specific carbohydrate epitopes from glycoproteins which are expresse... more The modB mutation eliminates specific carbohydrate epitopes from glycoproteins which are expressed primarily in prespore and spore cells of differentiating Dictyostelium discoideum. Spores formed by the mutant show several phenotypes. Whereas mutant spores germinate efficiently after heat activation, they germinate poorly after urea activation. Following germination, at least one glycosylation‐defective glycopro‐tein is cleaved, and the larger fragment is released in soluble form from the spore coat. However, an earlier difference in the spore coat can be traced back to the nongerminated spore coat, as detected by the elutability of protein from intact spores by chemical extraction. An altered character of the pregermination spore coat is also suggested by increased labeling by a fluorescent lectin which binds to its interior. The findings are consistent with a change in the character of certain molecular contacts leading to altered characteristics of the mutant spore coat, which ar...
Journal of Biological Chemistry, 1996
The photoreceptor membrane guanylate cyclase is a member of a family of proteins with a set of fo... more The photoreceptor membrane guanylate cyclase is a member of a family of proteins with a set of four structural motifs: an extracellular ligand binding domain, a transmembrane domain, an intracellular protein kinase-like domain, and an intracellular catalytic domain. Purified preparations of the photoreceptor guanylate cyclase have allowed us to explore the function of the protein kinase-like domain. ATP enhances the guanylate cyclase activity 2-fold in membranes stripped of peripheral proteins. The stimulation can be mimicked by ATP␥S (adenosine 5-O-(3-thiotriphosphate)), AMPPNP (5-adenylyl ,␥-imidodiphosphate), and ADP, but not AMP. While this effect is lost by solubilizing guanylate cyclase, ATP binds the purified, solubilized enzyme in a site distinct from the catalytic GTP site as shown by specific labeling with 8-N 3 [␣-32 P]ATP. The enzyme has a protein kinase activity that is Mg 2؉-dependent and autophosphorylates serine residues. Myelin basic protein serves as a substrate for the kinase and enables further characterization of the kinase properties. The K m for ATP is 81 M. The kinase activity is unaffected by calcium, cyclic nucleotides, and phorbol 12-myristate 13acetate/L-␣-phosphatidylserine/Ca 2؉ and is inhibited by high concentrations of staurosporine. These properties are distinct from other Ser/Thr kinases identified in rod outer segment preparations including protein kinase A, protein kinase C, and rhodopsin kinase. The observations offer the first biochemical evidence that a member of the receptor guanylate cyclase family has intrinsic protein kinase activity.
Genes & Development, 2008
Replication fork stalling at a DNA lesion generates a damage signal that activates the Rad53 kina... more Replication fork stalling at a DNA lesion generates a damage signal that activates the Rad53 kinase, which plays a vital role in survival by stabilizing stalled replication forks. However, evidence that Rad53 directly modulates the activity of replication forks has been lacking, and the nature of fork stabilization has remained unclear. Recently, cells lacking the Psy2–Pph3 phosphatase were shown to be defective in dephosphorylation of Rad53 as well as replication fork restart after DNA damage, suggesting a mechanistic link between Rad53 deactivation and fork restart. To test this possibility we examined the progression of replication forks in methyl-methanesulfonate (MMS)-damaged cells, under different conditions of Rad53 activity. Hyperactivity of Rad53 in pph3Δ cells slows fork progression in MMS, whereas deactivation of Rad53, through expression of dominant-negative Rad53-KD, is sufficient to allow fork restart during recovery. Furthermore, combined deletion of PPH3 and PTC2, a ...
Gene, 1997
The gene encoding the bovine guanylate cyclase isoform E (GC-E) was isolated as a single 18 kb ge... more The gene encoding the bovine guanylate cyclase isoform E (GC-E) was isolated as a single 18 kb genomic clone and shown to have 20 exons and 19 introns. Comparison of the structure of the GC-E gene with structures of other membrane guanylate cyclase genes indicates that the GC-E is most closely related to the subfamily of sensory guanylate cyclases. Comparison of the GC-E structure with that of the more distantly related guanylate cyclase isoform A (GC-A) gene shows the most divergence in the extracellular and C-terminal regions, but general conservation of introns and exons in the intracellular kinase-like and catalytic domains. RT-PCR from several bovine tissues shows that GC-E is expressed only in the retina. Consistent with this pattern of expression, elements for the retinal-specific transcription factors RET-1, RET-2 and Talpha-1 are located in the 5' flanking promoter region.
Methods in Enzymology, 2000