Dataset Information

Informatic selection of a neural crest-melanocyte cDNA set for microarray analysis.

ABSTRACT: With cDNA microarrays, it is now possible to compare the expression of many genes simultaneously. To maximize the likelihood of finding genes whose expression is altered under the experimental conditions, it would be advantageous to be able to select clones for tissue-appropriate cDNA sets. We have taken advantage of the extensive sequence information in the dbEST expressed sequence tag (EST) database to identify a neural crest-derived melanocyte cDNA set for microarray analysis. Analysis of characterized genes with dbEST identified one library that contained ESTs representing 21 neural crest-expressed genes (library 198). The distribution of the ESTs corresponding to these genes was biased toward being derived from library 198. This is in contrast to the EST distribution profile for a set of control genes, characterized to be more ubiquitously expressed in multiple tissues (P < 1 x 10(-9)). From library 198, a subset of 852 clustered ESTs were selected that have a library distribution profile similar to that of the 21 neural crest-expressed genes. Microarray analysis demonstrated the majority of the neural crest-selected 852 ESTs (Mel1 array) were differentially expressed in melanoma cell lines compared with a non-neural crest kidney epithelial cell line (P < 1 x 10(-8)). This was not observed with an array of 1,238 ESTs that was selected without library origin bias (P = 0.204). This study presents an approach for selecting tissue-appropriate cDNAs that can be used to examine the expression profiles of developmental processes and diseases.

SUBMITTER: Loftus SK

PROVIDER: S-EPMC17770 | biostudies-literature | 1999 Aug

REPOSITORIES: biostudies-literature

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Informatic selection of a neural crest-melanocyte cDNA set for microarray analysis.

Loftus S K SK Chen Y Y Gooden G G Ryan J F JF Birznieks G G Hilliard M M Baxevanis A D AD Bittner M M Meltzer P P Trent J J Pavan W W

Proceedings of the National Academy of Sciences of the United States of America 19990801 16

With cDNA microarrays, it is now possible to compare the expression of many genes simultaneously. To maximize the likelihood of finding genes whose expression is altered under the experimental conditions, it would be advantageous to be able to select clones for tissue-appropriate cDNA sets. We have taken advantage of the extensive sequence information in the dbEST expressed sequence tag (EST) database to identify a neural crest-derived melanocyte cDNA set for microarray analysis. Analysis of cha ...[more]

PMID: 10430933

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Project description:Phosphatidylinositides are important signaling molecules that interact with a myriad of cellular proteins, many of which remain unidentified. We previously screened a yeast surface displayed human proteome library to identify protein fragments with affinity for the phosphatidylinositides, phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate. Much of the diversity in the screened selection outputs was represented by clones present at low frequencies, suggesting that a significant number of additional phosphatidylinositide-binding protein fragments might be present in the selection outputs. In the studies described in this report, we developed a novel cDNA library analysis method and comprehensively analyzed the polyclonal selection outputs from the phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate selections using a high-density exon microarray. In addition to the nine previously reported phosphatidylinositide-binding protein fragments, we identified 37 new phosphatidylinositide-binding candidates. Nine of 37 contain known phosphatidylinositide-binding domains, whereas the remaining 28 contain no known phosphatidylinositide-binding domain. We cloned and confirmed phosphatidylinositide binding by fluorescence-activated cell sorting for 17 of these novel candidate protein fragments. Our experiments suggest that phosphatidylinositide binding by these 17 novel protein fragments is dependent on both the inositol phosphate "headgroup" and the lipid "tail." This is in contrast with the PH domain containing fragments we tested, for which the inositol phosphate headgroup was sufficient for binding. The novel PtdIns-binding fragments come from a wide variety of proteins, including splicing factors, transcription factors, a kinase, and a polymerase. Intriguingly, 11 of the phosphatidylinositide-binding protein fragments are from nuclear proteins, including four containing homeobox domains. We found that phosphatidylinositides and double-stranded DNA oligonucleotides derived from homeobox domain target sequences compete for binding to homeobox domain-containing protein fragments, suggesting a possible mechanism for phospholipid-dependent transcriptional regulation. FACS enrichment of target-binding clones in yeast human cDNA display libraries coupled with comprehensive analysis of the selection output by DNA microarray analysis is an effective method for investigating common as well as rare protein interactions. In particular, this method is well suited for the study of small molecule/protein and drug/protein interactions.

Dataset Information

Informatic selection of a neural crest-melanocyte cDNA set for microarray analysis.

Publications

Informatic selection of a neural crest-melanocyte cDNA set for microarray analysis.

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