Functional cloning, sorting, and expression profiling of nucleic acid-binding proteins.
A major challenge in the post-sequencing era is to elucidate the activity and biological function of genes that reside in the human genome. An important subset includes genes that encode proteins that regulate gene expression or maintain the structural integrity of the genome. Using a novel oligonucleotide-binding substrate as bait, we show the feasibility of a modified functional expression-cloning strategy to identify human cDNAs that encode a spectrum of nucleic acid-binding proteins (NBPs). Approximately 170 cDNAs were identified from screening phage libraries derived from a human colorectal adenocarcinoma cell line and from noncancerous fetal lung tissue. Sequence analysis confirmed that virtually every clone contained a known DNA- or RNA-binding motif. We also report on a complementary sorting strategy that, in the absence of subcloning and protein purification, can distinguish different classes of NBPs according to their particular binding properties. To extend our functional annotation of NBPs, we have used GeneChip expression profiling of 14 different breast-derived cell lines to examine the relative transcriptional activity of genes identified in our screen and cluster analysis to discover other genes that have similar expression patterns. Finally, we present strategies to analyze the upstream regulatory region of each gene within a cluster group and select unique combinations of transcription factor binding sites that may be responsible for dictating the observed synexpression.
Ramanathan
Y
Y
Center for Applied Genomics, Public Health Research Institute, International Center for Public Health W420M, Newark, New Jersey 07103, USA.
Zhang
Haibo
H
Aris
Virginie
V
Soteropoulos
Patricia
P
Aaronson
Stuart A
SA
Tolias
Peter P
PP
D000230:Adenocarcinoma / Q000235:genetics / Q000473:pathology; D003001:Cloning, Molecular / Q000379:methods*; D015179:Colorectal Neoplasms / Q000235:genetics / Q000473:pathology; D019295:Computational Biology; D018076:DNA, Complementary / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology; D005333:Fetus / Q000737:chemistry / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D015894:Genome, Human; D006801:Humans; D008168:Lung / Q000737:chemistry / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D015345:Oligonucleotide Probes; D016601:RNA-Binding Proteins