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The rat intestinal fatty acid binding protein (I-FABP) gene exhibits cell-specific as well as regional differences in its expression within the continuously regenerating small intestinal epithelium. To investigate the underlying mechanisms, we linked portions of its 5' nontranscribed domain to the human growth hormone (hGH) gene and analyzed expression of the hGH reporter in transgenic mice by RNA blot, solution hybridization, and immunocytochemical techniques. Sequences located within 277 nucleotides of the start site of I-FABP transcription are sufficient to limit hGH expression to the intestine. Although the absolute levels of hGH mRNA in the duodenum and proximal jejunum of these transgenic mice were similar to those of I-FABP mRNA, steady-state hGH mRNA concentrations were approximately 100 times lower in their distal small intestine. Addition of nucleotides -278 to -1178 of the I-FABP gene "restored" hGH mRNA concentrations in the distal jejunum and ileum to levels comparable to murine I-FABP mRNA. Serum hGH levels were 1000 times lower in the "short promoter" transgenic mice compared to animals with the "long promoter" transgene, indicating that efficient distal small intestinal hGH expression is required to produce elevated hGH concentrations in serum. The distribution of hGH in villus-associated enterocytes and goblet cells and its lack of expression in the crypts of Lieberkuhn mimicked that of the endogenous I-FABP gene product in all transgenic pedigrees. However, bands of hGH-negative cells extending from the base to the tips of villi were frequently observed in mice that were heterozygous for the short promoter transgene. This mosaic staining was not observed for I-FABP. These data suggest that (i) different cis-acting sequences may be required for complete expression of proximal-distal I-FABP gradients than for recapitulation of its normal crypt-villus tip distribution; (ii) differences may exist in the export pathways of secreted proteins within enterocytes located in various regions of the small intestine; and (iii) there may be subtle genetic differences among various crypt stem cells that can be detected in vivo by observing mosaic patterns of transgene expression along the villus epithelium.

Regional as well as cell-specific differences in gene expression are established and maintained in the perpetually regenerating intestinal epithelium. We have recently linked regions of the 5'-nontranscribed domain of the rat "liver" fatty acid binding protein (L-FABP) gene which is normally expressed in both liver and intestine, to a reporter, the human growth hormone (hGH) gene, and examined hGH expression in adult transgenic mice (Sweetser, D. A., Birkenmeier, E. H., Hoppe, P. C., McKeel, D. W., and Gordon, J. I. (1988) Genes Dev. 2, 1318-1332). Our results indicated that cis-acting elements, including an orientation-independent suppressor, could produce a pattern of cellular and geographic expression of hGH which mimics that of the intact, endogenous murine Fabpl gene in both organs. We now show that nucleotides -4000 to +21 of the rat L-FABP gene can direct "appropriate" cell-specific, regional, and temporal expression of the hGH reporter during a period of remarkable cellular expansion, cytodifferentiation, and morphologic transformation of the fetal gut epithelium. These studies also indicate that the polyclonal stem cell population located in the intervillous regions of the late fetal intestine exhibits a different pattern of transgene regulation than does the monoclonally derived crypt stem cell population in adult transgenic mice. Nucleotides -4000 to +21 are not sufficient to reproduce the normal temporal pattern of L-FABP gene activation in liver. Precocious expression of growth hormone in this pedigree of transgenic mice results in early induction of insulin-like growth factor I mRNA accumulation in liver but has no effect on insulin-like growth factor II mRNA levels. In contrast, local synthesis of growth hormone in the small intestine does not influence its insulin-like growth factor I or II mRNA levels.

The intestine is lined by a continuously regenerating epithelium that maintains gradients in 'liver' fatty acid binding protein (L-FABP) gene expression along its horizontal and vertical axes, i.e., from duodenum to colon and from crypt to villus tip. To identify cis-acting DNA sequences responsible for these regional differences, we linked portions of the L-FABP gene's 5' nontranscribed region to the human growth hormone (hGH) gene and examined hGH expression in transgenic mice. Nucleotides -596 to +21 of the rat L-FABP gene correctly directed hGH expression to enterocytes and hepatocytes. However, anomalous expression was observed in small intestinal crypts, colon, and renal proximal tubular epithelial cells. Addition of nucleotides -4000 to -597 of the L-FABP gene, in either orientation, suppressed renal hGH expression and restored a nearly normal horizontal, but not a vertical, hGH gradient in the intestine. Thus, horizontal gradients of gene expression within the intestine can be maintained by orientation-independent, cis-acting suppressor elements.

We have determined the primary structure of human liver fatty acid binding protein from an analysis of a full length cDNA. This 127-residue 14,178-Da protein exhibits a high degree of sequence conservation when compared to its orthologous homologue, rat liver fatty acid binding protein. It appears likely that this polypeptide arose from two intragenic duplication events. Using a variety of computational techniques, we were unable to find any evidence of amphipathic alpha helical domains in this protein nor any sequence similarities to apolipoproteins and serum albumins. A family of paralogous proteins was defined, whose members share a remarkable degree of sequence homology with share a remarkable degree of sequence homology with human liver fatty acid binding protein. These include rat intestinal fatty acid binding protein, the cellular the P2 protein of myelin. It appears that the small cytosolic fatty acid binding proteins have evolved structural features necessary for lipid-protein interaction which are different from those present in some familiar and better studied extracellular sequences.

Intestinal fatty acid binding protein (I-FABP) is believed to participate in the uptake, intracellular metabolism, and/or transport of long chain fatty acids within enterocytes. The 15.1-kDA rodent proteins is a member of a family of low Mr cytoplasmic proteins that have evolved to bind different ligands. We have now determined the nucleotide sequence of the gene encoding human I-FABP and defined the primary structure of its protein product. The human I-FABP gene spans 3382 nucleotides and contains 4 exons (103 or 128, 173, 108, and 312 base pairs). interrupted by 3 introns (1194, 1023, and 444 base pairs). The 132-residue rat and human I-FABPs have 82% amino acid sequence identity. Blot hybridization studies of RNAs prepared from a variety of adult rhesus monkey tissues as well as human intestine and liver indicate that I-FABP mRNA is confined to the intestine. I-FABP mRNA was not detectable in a number of cultured human enterocyte-like cell lines, suggesting it may be a sensitive marker for differentiated, villus-associated, small intestinal lining cells. Given the similar patterns of tissue-specific expression exhibited by the rat and human genes, we compared their 5' nontranscribed regions. Optimal alignments of the two sequences disclosed 64% identity among the 260 nucleotides immediately 5' to the start site of transcription. Matrix plots revealed a 14-nucleotide long repeated sequence (5'-TGAACTTTGAACTT-3') in the 5' nontranscribed region of both genes as well as in a comparable region of another family member that is expressed in enterocytes, cellular retinol binding protein II. The linkage relationships between I-FABP and the homologous liver FABP (L-FABP) gene were defined in mice and humans. The mouse genes were mapped using restriction fragment length polymorphisms and recombinant inbred strains. The I-FABP gene is located on mouse chromosome 3 between the amylase 1,2 (Amy 1,2) and alcohol dehydrogenase 3 (Adh-3) loci while the L-FABP gene is on mouse chromosome 6 within 3 centimorgans of the lymphocyte antigen-2 (Ly-2) locus. Mouse L-FABP may be identical to the major liver protein-1 (Lvp-1) which is encoded by a gene situated within a centimorgan of Ly-2. Human gene mapping studies were carried out using a panel of mouse-human somatic cell hybrid clones as well as in situ hybridization to metaphase chromosomes.(ABSTRACT TRUNCATED AT 400 WORDS)

We have determined the nucleotide sequence of the gene encoding rat liver fatty acid-binding protein (L-FABP). Previous structural studies have shown that L-FABP belongs to a family of low molecular weight cytosolic proteins which bind hydrophobic ligands. Rat L-FABP is the first member of this family whose genomic organization has been defined. The L-FABP transcription unit spans 3790 nucleotides and contains four exons (115, 173, 93, and 121 base pairs) interrupted by three introns (1454, 1224, and 610 base pairs). No other abundant mRNAs appear to be transcribed from sequences located within 4 kilobases 5' or 6.5 kilobases 3' of this gene. Sequence analyses have detected the presence of several related amino acid sequence blocks within L-FABP which may serve similar structural roles. A variety of computational techniques were used to compare the oligopeptides specified by each exon with other known members of the protein family. The results indicate that only the amino acid sequence present in the first exon is conserved among the homologous proteins. This suggests that the first exon may encode a shared structural and functional domain important in the interaction of these proteins with their ligands.