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Michael Snyder

Publications and source records attributed to Michael Snyder.

At least 37 records · Page 2Linked to original sources
PubMed2006

ProCAT: a data analysis approach for protein microarrays.

Protein microarrays provide a versatile method for the analysis of many protein biochemical activities. Existing DNA microarray analytical methods do not translate to protein microarrays due to differences between the technologies. Here we report a new approach, ProCAT, which corrects for background bias and spatial artifacts, identifies significant signals, filters nonspecific spots, and normalizes the resulting signal to protein abundance. ProCAT provides a powerful and flexible new approach for analyzing many types of protein microarrays.

Cyclic AMP-Dependent Protein Kinases↗
PubMed2006

High-throughput methods of regulatory element discovery.

With the number of organisms whose genomes have been sequenced, a vast amount of information concerning the genetic structure of an organism's genome has been collected. However, effective experiment means to study how this information is accessed have only recently been developed. In this review, three basic methods for identifying regions of protein-DNA interaction will be introduced. The first two, chromatin immunoprecipitation (ChIP)-chip and ChIP-PET (for paired-end ditag), rely on the enrichment provided by chromosomal immunoprecipitation to interrogate the genomic sequence for the interaction sites of a protein of interest. In contrast, protein microarrays allow the identification of DNA binding protein that interacts with a DNA sequence of interest. These complementary methods of exploring protein-DNA interactions will increase our fundamental knowledge of how the information contained within the genome sequence is accessed and processed.

Chromatin Immunoprecipitation↗
PubMed2005

Design optimization methods for genomic DNA tiling arrays.

A recent development in microarray research entails the unbiased coverage, or tiling, of genomic DNA for the large-scale identification of transcribed sequences and regulatory elements. A central issue in designing tiling arrays is that of arriving at a single-copy tile path, as significant sequence cross-hybridization can result from the presence of non-unique probes on the array. Due to the fragmentation of genomic DNA caused by the widespread distribution of repetitive elements, the problem of obtaining adequate sequence coverage increases with the sizes of subsequence tiles that are to be included in the design. This becomes increasingly problematic when considering complex eukaryotic genomes that contain many thousands of interspersed repeats. The general problem of sequence tiling can be framed as finding an optimal partitioning of non-repetitive subsequences over a prescribed range of tile sizes, on a DNA sequence comprising repetitive and non-repetitive regions. Exact solutions to the tiling problem become computationally infeasible when applied to large genomes, but successive optimizations are developed that allow their practical implementation. These include an efficient method for determining the degree of similarity of many oligonucleotide sequences over large genomes, and two algorithms for finding an optimal tile path composed of longer sequence tiles. The first algorithm, a dynamic programming approach, finds an optimal tiling in linear time and space; the second applies a heuristic search to reduce the space complexity to a constant requirement. A Web resource has also been developed, accessible at http://tiling.gersteinlab.org, to generate optimal tile paths from user-provided DNA sequences.

Algorithms↗
PubMed2005

Global analysis of protein phosphorylation in yeast.

Protein phosphorylation is estimated to affect 30% of the proteome and is a major regulatory mechanism that controls many basic cellular processes. Until recently, our biochemical understanding of protein phosphorylation on a global scale has been extremely limited; only one half of the yeast kinases have known in vivo substrates and the phosphorylating kinase is known for less than 160 phosphoproteins. Here we describe, with the use of proteome chip technology, the in vitro substrates recognized by most yeast protein kinases: we identified over 4,000 phosphorylation events involving 1,325 different proteins. These substrates represent a broad spectrum of different biochemical functions and cellular roles. Distinct sets of substrates were recognized by each protein kinase, including closely related kinases of the protein kinase A family and four cyclin-dependent kinases that vary only in their cyclin subunits. Although many substrates reside in the same cellular compartment or belong to the same functional category as their phosphorylating kinase, many others do not, indicating possible new roles for several kinases. Furthermore, integration of the phosphorylation results with protein-protein interaction and transcription factor binding data revealed novel regulatory modules. Our phosphorylation results have been assembled into a first-generation phosphorylation map for yeast. Because many yeast proteins and pathways are conserved, these results will provide insights into the mechanisms and roles of protein phosphorylation in many eukaryotes.

Eukaryotic Cells↗
PubMed2005

Biochemical and genetic analysis of the yeast proteome with a movable ORF collection.

Functional analysis of the proteome is an essential part of genomic research. To facilitate different proteomic approaches, a MORF (moveable ORF) library of 5854 yeast expression plasmids was constructed, each expressing a sequence-verified ORF as a C-terminal ORF fusion protein, under regulated control. Analysis of 5573 MORFs demonstrates that nearly all verified ORFs are expressed, suggests the authenticity of 48 ORFs characterized as dubious, and implicates specific processes including cytoskeletal organization and transcriptional control in growth inhibition caused by overexpression. Global analysis of glycosylated proteins identifies 109 new confirmed N-linked and 345 candidate glycoproteins, nearly doubling the known yeast glycome.

Cell Growth Processes↗
PubMed2005

Global changes in STAT target selection and transcription regulation upon interferon treatments.

The STAT (signal transducer and activator of transcription) proteins play a crucial role in the regulation of gene expression, but their targets and the manner in which they select them remain largely unknown. Using chromatin immunoprecipitation and DNA microarray analysis (ChIP-chip), we have identified the regions of human chromosome 22 bound by STAT1 and STAT2 in interferon-treated cells. Analysis of the genomic loci proximal to these binding sites introduced new candidate STAT1 and STAT2 target genes, several of which are affiliated with proliferation and apoptosis. The genes on chromosome 22 that exhibited interferon-induced up- or down-regulated expression were determined and correlated with the STAT-binding site information, revealing the potential regulatory effects of STAT1 and STAT2 on their target genes. Importantly, the comparison of STAT1-binding sites upon interferon (IFN)-gamma and IFN-alpha treatments revealed dramatic changes in binding locations between the two treatments. The IFN-alpha induction revealed nonconserved STAT1 occupancy at IFN-gamma-induced sites, as well as novel sites of STAT1 binding not evident in IFN-gamma-treated cells. Many of these correlated with binding by STAT2, but others were STAT2 independent, suggesting that multiple mechanisms direct STAT1 binding to its targets under different activation conditions. Overall, our results reveal a wealth of new information regarding IFN/STAT-binding targets and also fundamental insights into mechanisms of regulation of gene expression in different cell states.

Antineoplastic Agents↗
PubMed2005

Substrate specificity analysis of protein kinase complex Dbf2-Mob1 by peptide library and proteome array screening.

BACKGROUND: The mitotic exit network (MEN) is a group of proteins that form a signaling cascade that is essential for cells to exit mitosis in Saccharomyces cerevisiae. The MEN has also been implicated in playing a role in cytokinesis. Two components of this signaling pathway are the protein kinase Dbf2 and its binding partner essential for its kinase activity, Mob1. The components of MEN that act upstream of Dbf2-Mob1 have been characterized, but physiological substrates for Dbf2-Mob1 have yet to be identified. RESULTS: Using a combination of peptide library selection, phosphorylation of optimal peptide variants, and screening of a phosphosite array, we found that Dbf2-Mob1 preferentially phosphorylated serine over threonine and required an arginine three residues upstream of the phosphorylated serine in its substrate. This requirement for arginine in peptide substrates could not be substituted with the similarly charged lysine. This specificity determined for peptide substrates was also evident in many of the proteins phosphorylated by Dbf2-Mob1 in a proteome chip analysis. CONCLUSION: We have determined by peptide library selection and phosphosite array screening that the protein kinase Dbf2-Mob1 preferentially phosphorylated substrates that contain an RXXS motif. A subsequent proteome microarray screen revealed proteins that can be phosphorylated by Dbf2-Mob1 in vitro. These proteins are enriched for RXXS motifs, and may include substrates that mediate the function of Dbf2-Mob1 in mitotic exit and cytokinesis. The relatively low degree of sequence restriction at the site of phosphorylation suggests that Dbf2 achieves specificity by docking its substrates at a site that is distinct from the phosphorylation site.

Amino Acid Sequence↗
PubMed2005

Spindle checkpoint maintenance requires Ame1 and Okp1.

Kinetochore proteins are required for high fidelity chromosome segregation and as a platform for checkpoint signaling. Ame1 is an essential component of the COMA (Ctf19, Okp1, Mcm21, Ame1) sub-complex of the central kinetochore of budding yeast. In this study, we describe the isolation and characterization of an Ame1 conditional mutant, ame1-4. ame1-4 cells exhibit chromosome segregation defects and Mad2-dependent cell cycle delay similar to okp1-5 cells. However, the viability of ame1-4 cells is markedly reduced relative to wild type and okp1-5 cells after three hours at restrictive temperature. To determine if ame1-4 cells enter anaphase with mis-segregated chromosomes, we monitored the localization of Bub3:VFP as a marker for anaphase onset. ame1-4 cells containing mis-segregated sister chromatids initially accumulate Bub3:VFP at kinetochores, indicating checkpoint activation and a metaphase arrest. Subsequently, Bub3:VFP de-localizes and cells reinitiate DNA duplication and budding without cytokinesis in the presence of un-segregated chromosomes. Overexpression of OKP1 in ame1-4 cells restores ame1-4 protein localization and a stable arrest. Based on our results, we propose that Ame1 and Okp1 are required for a sustained checkpoint arrest in the presence of mis-segregated chromosomes. Our results suggest that checkpoint response might be controlled not only at the level of activation but also via signals that ensure maintenance of the response.

Cell Cycle↗
PubMed2005

Calixarene derivative as a tool for highly sensitive detection and oriented immobilization of proteins in a microarray format through noncovalent molecular interaction.

One important factor in fabricating protein microarray is to immobilize proteins without losing their activity on a solid phase. To keep them functional, it is necessary to immobilize proteins in a way that preserve their folded structural integrity. In a previous study, we developed novel Calixarene derivatives for the immobilization of proteins on the surface of a glass slide (1). In this study, we compared the sensitivity and the specificity of the linker molecules with those of five other protein attachment agents on glass slides using a prostate-specific antigen and its antibodies as a model system. The Calixcrown-coated protein chip showed a superior sensitivity and a much lower detection limit than those chips prepared by other methods. When we tested the capability of Calixcrown to immobilize antibody molecules, it appeared that Calixcrown makes arrangement of antibody be more regular with the vertical orientation than the covalent-bond agent. We also observed that the Calixcrown chip could be used for the diagnostic application with clinical samples from prostate cancer and HIV patients. Finally, we applied the Calixcrown chip using an antibody microarray to identify up- or down-regulated proteins in specific tissue and detected several up- or down-regulated proteins from a rat liver by administering toxin. Thus, the Calixcrown chip can be used as a powerful tool with a wide range of applications, including protein-protein interaction, protein-DNA interaction, and an enzyme activity assay.

Animals↗
PubMed2005

Applications of DNA tiling arrays to experimental genome annotation and regulatory pathway discovery.

Microarrays have become a popular and important technology for surveying global patterns in gene expression and regulation. A number of innovative experiments have extended microarray applications beyond the measurement of mRNA expression levels, in order to uncover aspects of large-scale chromosome function and dynamics. This has been made possible due to the recent development of tiling arrays, where all non-repetitive DNA comprising a chromosome or locus is represented at various sequence resolutions. Since tiling arrays are designed to contain the entire DNA sequence without prior consultation of existing gene annotation, they enable the discovery of novel transcribed sequences and regulatory elements through the unbiased interrogation of genomic loci. The implementation of such methods for the global analysis of large eukaryotic genomes presents significant technical challenges. Nonetheless, tiling arrays are expected to become instrumental for the genome-wide identification and characterization of functional elements. Combined with computational methods to relate these data and map the complex interactions of transcriptional regulators, tiling array experiments can provide insight toward a more comprehensive understanding of fundamental molecular and cellular processes.

Gene Expression Profiling↗
PubMed2005

A pilot study of transcription unit analysis in rice using oligonucleotide tiling-path microarray.

As the international efforts to sequence the rice genome are completed, an immediate challenge and opportunity is to comprehensively and accurately define all transcription units in the rice genome. Here we describe a strategy of using high-density oligonucleotide tiling-path microarrays to map transcription of the japonica rice genome. In a pilot experiment to test this approach, one array representing the reverse strand of the last 11.2 Mb sequence of chromosome 10 was analyzed in detail based on a mathematical model developed in this study. Analysis of the array data detected 77% of the reference gene models in a mixture of four RNA populations. Moreover, significant transcriptional activities were found in many of the previously annotated intergenic regions. These preliminary results demonstrate the utility of genome tiling microarrays in evaluating annotated rice gene models and in identifying novel transcription units that will facilitate rice genome annotation.

Carbocyanines↗
PubMed2005

Global analysis of protein function using protein microarrays.

Protein microarrays containing thousands of proteins arrayed at high density can be prepared and probed for a wide variety of activities, thereby allowing the large scale analysis of many proteins simultaneously. In addition to identifying the activities of many previously uncharacterized proteins, protein microarrays can reveal new activities of well-characterized proteins, thus providing new insights about the functions of these proteins. Below, we describe the construction and use of protein microarrays and their applications using yeast as a model system.

Animals↗
PubMed2005

Sexual dimorphism in mammalian gene expression.

Males and females have obvious phenotypic differences; they also exhibit differences related to health, life span, cognitive abilities and have different responses to diseases such as anemia, coronary heart disease, hypertension and renal dysfunction. Although the anatomical, hormonal and chemical differences between the sexes are well known, there are few molecular descriptors for gender-specific physiological traits and health risks. Recent studies using microarrays and other methods have made significant progress towards elucidating the molecular differences between mammalian sexes in a variety of tissues and towards identifying the transcription factors that regulate sex-biased gene expression. These findings are providing new insights into the molecular and genetic differences that dictate the different behaviors and physiologies of mammalian sexes.

Animals↗
PubMed2005

Issues in the analysis of oligonucleotide tiling microarrays for transcript mapping.

Traditional microarrays use probes complementary to known genes to quantitate the differential gene expression between two or more conditions. Genomic tiling microarray experiments differ in that probes that span a genomic region at regular intervals are used to detect the presence or absence of transcription. This difference means the same sets of biases and the methods for addressing them are unlikely to be relevant to both types of experiment. We introduce the informatics challenges arising in the analysis of tiling microarray experiments as open problems to the scientific community and present initial approaches for the analysis of this nascent technology.

Algorithms↗
PubMed2005

Advances in functional protein microarray technology.

Numerous innovations in high-throughput protein production and microarray surface technologies have enabled the development of addressable formats for proteins ordered at high spatial density. Protein array implementations have largely focused on antibody arrays for high-throughput protein profiling. However, it is also possible to construct arrays of full-length, functional proteins from a library of expression clones. The advent of protein-based microarrays allows the global observation of biochemical activities on an unprecedented scale, where hundreds or thousands of proteins can be simultaneously screened for protein-protein, protein-nucleic acid, and small molecule interactions. This technology holds great potential for basic molecular biology research, disease marker identification, toxicological response profiling and pharmaceutical target screening.

Animals↗
PubMed2004

DNA replication-timing analysis of human chromosome 22 at high resolution and different developmental states.

Duplication of the genome during the S phase of the cell cycle does not occur simultaneously; rather, different sequences are replicated at different times. The replication timing of specific sequences can change during development; however, the determinants of this dynamic process are poorly understood. To gain insights into the contribution of developmental state, genomic sequence, and transcriptional activity to replication timing, we investigated the timing of DNA replication at high resolution along an entire human chromosome (chromosome 22) in two different cell types. The pattern of replication timing was correlated with respect to annotated genes, gene expression, novel transcribed regions of unknown function, sequence composition, and cytological features. We observed that chromosome 22 contains regions of early- and late-replicating domains of 100 kb to 2 Mb, many (but not all) of which are associated with previously described chromosomal bands. In both cell types, expressed sequences are replicated earlier than nontranscribed regions. However, several highly transcribed regions replicate late. Overall, the DNA replication-timing profiles of the two different cell types are remarkably similar, with only nine regions of difference observed. In one case, this difference reflects the differential expression of an annotated gene that resides in this region. Novel transcribed regions with low coding potential exhibit a strong propensity for early DNA replication. Although the cellular function of such transcripts is poorly understood, our results suggest that their activity is linked to the replication-timing program.

Cell Differentiation↗
PubMed2004

Rapid analysis of the DNA-binding specificities of transcription factors with DNA microarrays.

We developed a new DNA microarray-based technology, called protein binding microarrays (PBMs), that allows rapid, high-throughput characterization of the in vitro DNA binding-site sequence specificities of transcription factors in a single day. Using PBMs, we identified the DNA binding-site sequence specificities of the yeast transcription factors Abf1, Rap1 and Mig1. Comparison of these proteins' in vitro binding sites with their in vivo binding sites indicates that PBM-derived sequence specificities can accurately reflect in vivo DNA sequence specificities. In addition to previously identified targets, Abf1, Rap1 and Mig1 bound to 107, 90 and 75 putative new target intergenic regions, respectively, many of which were upstream of previously uncharacterized open reading frames. Comparative sequence analysis indicated that many of these newly identified sites are highly conserved across five sequenced sensu stricto yeast species and, therefore, are probably functional in vivo binding sites that may be used in a condition-specific manner. Similar PBM experiments should be useful in identifying new cis regulatory elements and transcriptional regulatory networks in various genomes.

Base Sequence↗