Biomaterial Database

Isolation and characterization of a differentiation-dependent gene in the human colonic cell line HT29-18. 1993

M M Oliva, and T C Wu, and V W Yang

Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.

The human colonic epithelial cell line HT29, and its clonal derivatives HT29-18 and HT-29-18-C1, differentiate in vitro. Differential screening of a subtraction cDNA library enriched for sequences unique to HT29-18-C1, a highly differentiated subclone of HT29-18, resulted in the isolation of a differentiation-dependent cDNA clone, A4. A full-length clone encoding A4 was obtained and sequenced to its entirety. It is 945 bp in length and contains an open reading frame (ORF) of 456 bp. The amino acid sequence deduced from the ORF reveals a polypeptide of 152 amino acids with a predicted molecular mass of 17,000 Da, a size confirmed by coupled in vitro transcription and translation directed by the full-length A4 cDNA. This polypeptide contains four potential membrane-spanning domains and consensus sequences for N-linked glycosylation as well as phosphorylation sites for protein kinase C and casein kinase II. Comparison of A4 to published DNA and protein sequences revealed no significant homology. Genomic Southern blot analysis suggests that the gene is present in a single copy within the human genome and is conserved in the rat. Northern blot analysis of RNA obtained from various rat tissues shows that the expression of the A4 gene is tissue-selective and is enriched in colonic mucosa. In situ hybridization using human intestinal tissues indicates that the expression of A4 follows a gradient along the crypt-to-villus axis with the most abundant message occurring in the lower half of the crypt. Furthermore, nuclear run-on assays suggest that the induction of the A4 gene during differentiation of HT29-18 is regulated at a transcriptional level. A clone was isolated from a human genomic library and found to contain all five exons of A4. S1 nuclease analysis localized the start site of transcription to an adenosine residue 91 nucleotides upstream from the ATG translation initiation codon. Examination of the immediate sequence 5' to the mRNA start site reveals no TATA box and multiple known enhancer sequences. A4 is also noted to share certain features with the gene encoding the cystic fibrosis transmembrane conductance regulator protein. They include a similar vertical distribution of expression along the intestinal epithelium, enhanced transcription upon differentiation of HT29-18, and multiple shared putative regulatory sequences in the promoter regions. Further characterization of the mechanisms regulating expression of the A4 gene could contribute to the understanding of mammalian intestinal differentiation.

UI	MeSH Term	Description	Entries
D008565	Membrane Proteins	Proteins which are found in membranes including cellular and intracellular membranes. They consist of two types, peripheral and integral proteins. They include most membrane-associated enzymes, antigenic proteins, transport proteins, and drug, hormone, and lectin receptors.	Cell Membrane Protein,Cell Membrane Proteins,Cell Surface Protein,Cell Surface Proteins,Integral Membrane Proteins,Membrane-Associated Protein,Surface Protein,Surface Proteins,Integral Membrane Protein,Membrane Protein,Membrane-Associated Proteins,Membrane Associated Protein,Membrane Associated Proteins,Membrane Protein, Cell,Membrane Protein, Integral,Membrane Proteins, Integral,Protein, Cell Membrane,Protein, Cell Surface,Protein, Integral Membrane,Protein, Membrane,Protein, Membrane-Associated,Protein, Surface,Proteins, Cell Membrane,Proteins, Cell Surface,Proteins, Integral Membrane,Proteins, Membrane,Proteins, Membrane-Associated,Proteins, Surface,Surface Protein, Cell
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D009693	Nucleic Acid Hybridization	Widely used technique which exploits the ability of complementary sequences in single-stranded DNAs or RNAs to pair with each other to form a double helix. Hybridization can take place between two complimentary DNA sequences, between a single-stranded DNA and a complementary RNA, or between two RNA sequences. The technique is used to detect and isolate specific sequences, measure homology, or define other characteristics of one or both strands. (Kendrew, Encyclopedia of Molecular Biology, 1994, p503)	Genomic Hybridization,Acid Hybridization, Nucleic,Acid Hybridizations, Nucleic,Genomic Hybridizations,Hybridization, Genomic,Hybridization, Nucleic Acid,Hybridizations, Genomic,Hybridizations, Nucleic Acid,Nucleic Acid Hybridizations
D011510	Proteolipids	Protein-lipid combinations abundant in brain tissue, but also present in a wide variety of animal and plant tissues. In contrast to lipoproteins, they are insoluble in water, but soluble in a chloroform-methanol mixture. The protein moiety has a high content of hydrophobic amino acids. The associated lipids consist of a mixture of GLYCEROPHOSPHATES; CEREBROSIDES; and SULFOGLYCOSPHINGOLIPIDS; while lipoproteins contain PHOSPHOLIPIDS; CHOLESTEROL; and TRIGLYCERIDES.
D002454	Cell Differentiation	Progressive restriction of the developmental potential and increasing specialization of function that leads to the formation of specialized cells, tissues, and organs.	Differentiation, Cell,Cell Differentiations,Differentiations, Cell
D003106	Colon	The segment of LARGE INTESTINE between the CECUM and the RECTUM. It includes the ASCENDING COLON; the TRANSVERSE COLON; the DESCENDING COLON; and the SIGMOID COLON.	Appendix Epiploica,Taenia Coli,Omental Appendices,Omental Appendix,Appendices, Omental,Appendix, Omental
D003110	Colonic Neoplasms	Tumors or cancer of the COLON.	Cancer of Colon,Colon Adenocarcinoma,Colon Cancer,Cancer of the Colon,Colon Neoplasms,Colonic Cancer,Neoplasms, Colonic,Adenocarcinoma, Colon,Adenocarcinomas, Colon,Cancer, Colon,Cancer, Colonic,Cancers, Colon,Cancers, Colonic,Colon Adenocarcinomas,Colon Cancers,Colon Neoplasm,Colonic Cancers,Colonic Neoplasm,Neoplasm, Colon,Neoplasm, Colonic,Neoplasms, Colon
D004247	DNA	A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine).	DNA, Double-Stranded,Deoxyribonucleic Acid,ds-DNA,DNA, Double Stranded,Double-Stranded DNA,ds DNA
D004848	Epithelium	The layers of EPITHELIAL CELLS which cover the inner and outer surfaces of the cutaneous, mucus, and serous tissues and glands of the body.	Mesothelium,Epithelial Tissue,Mesothelial Tissue,Epithelial Tissues,Mesothelial Tissues,Tissue, Epithelial,Tissue, Mesothelial,Tissues, Epithelial,Tissues, Mesothelial
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man