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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.

The primary structure of rat cellular retinol-binding protein (CRBP) II has been determined from a cloned cDNA. Alignment of this 134-amino acid, 15,580-Da polypeptide with rat CRBP revealed that 75 of 133 comparable residues are identical. Both proteins contain four tryptophan residues, which occupy identical relative positions in the two primary structures, providing a structural explanation for their similar fluorescence spectra when complexed to retinol. Two of the three cysteines in each single-chain protein are comparably positioned. Both polypeptides contain reactive thiol groups, but the rate of disruption of CRBP II-retinol complexes by p-chloromercuribenzoate is greater than that of CRBP-retinol. The small intestine contains the highest concentrations of CRBP II mRNA in adult rats. CRBP II mRNA is first detectable in intestinal RNA during the 19th day of gestation, a time that corresponds to the appearance of an absorptive columnar epithelium. Unlike in intestine, a dramatic fall in liver CRBP II mRNA concentration occurs immediately after birth. The CRBP II gene remains quiescent in the liver during subsequent postnatal development. These data suggest that ligand-protein interactions may be somewhat different for the two rat CRBPs. They also support the concept that CRBP II plays a role in the intestinal absorption or esterification of retinol and suggest that changes in hepatic metabolism of vitamin A occur during development.

Mice homozygous for the piebald lethal (sl) mutation, which have a complete deletion of endothelin receptor-B, fail to form ganglion cells in the distal large intestine and are nearly devoid of cutaneous melanocytes. These phenotypic features stem from incomplete colonization of the hindgut and skin by neural crest-derived neuroblasts and melanoblasts, respectively. We have used expression of a transgene, dopamine-beta-hydroxylase-nlacZ, to study colonization of the enteric nervous system in sl/sl embryos and sl/sl <--> wild-type chimeric mice. Enteric neuroblasts derived from the vagal neural crest colonize the developing foregut, midgut and distal small intestine of sl/sl embryos in a cranial-to-caudal manner indistinguishable from sl/+ or +/+ embryos. However, colonization of the large intestine is retarded and the distal large intestine is never colonized, a developmental defect identical to that observed in lethal spotted (endothelin-3 deficient) embryos. The coat pigmentation and relative distributions of mutant and wild-type ganglion cells in sl/sl <--> wild-type chimeras indicate that the defect associated with endothelin receptor-B gene deletion is not strictly neuroblast autonomous (independent of environmental factors). Instead, intercellular interactions downstream of the endothelin receptor-B mediate complete colonization of the skin and gut by neural crest cells.

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 Fabplgene 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.

The amino acid sequences of the 65-kilodalton antigens of Mycobacterium leprae, Mycobacterium tuberculosis, and Mycobacterium bovis BCG display greater than 95% homology.

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.

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.

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)

A recombinant DNA expression strategy has been used to deduce the amino acid sequences of six different antigenic determinants in a single protein of Mycobacterium leprae, the etiologic agent of leprosy. The gene encoding the M. leprae 65-kDa antigen was sequenced and a lambda gt11 gene sublibrary was constructed with fragments of the gene. Recombinant DNA clones producing specific antigenic determinants were isolated by screening with monoclonal antibodies, and the sequences of their insert DNAs were determined with a rapid primer-extension method. The amino acid sequence of each determinant was deduced from the minimum overlap of insert DNAs from multiple antibody-positive DNA clones. Amino acid sequences for six different epitopes were elucidated. A peptide containing sequences for one of these epitopes was synthesized and shown to bind the appropriate monoclonal antibody; this antigenic determinant is unique to M. leprae. The approach described here can be used to rapidly elucidate protein epitopes that are recognized by antibodies or T cells.

Eukaryotic RNA polymerases are complex aggregates whose component subunits are functionally ill-defined. The gene that encodes the 140,000-dalton subunit of Saccharomyces cerevisiae RNA polymerase II was isolated and studied in detail to obtain clues to the protein's function. This gene, RPB2, exists in a single copy in the haploid genome. Disruption of the gene is lethal to the yeast cell. RPB2 encodes a protein of 138,750 daltons, which contains sequences implicated in binding purine nucleotides and zinc ions and exhibits striking sequence homology with the beta subunit of Escherichia coli RNA polymerase. These observations suggest that the yeast and the E. coli subunit have similar roles in RNA synthesis, as the beta subunit contains binding sites for nucleotide substrates and a portion of the catalytic site for RNA synthesis. The subunit homologies reported here, and those observed previously with the largest RNA polymerase subunit, indicate that components of the prokaryotic RNA polymerase "core" enzyme have counterparts in eukaryotic RNA polymerases.

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.

To understand the immune response to infection by tuberculosis and leprosy bacilli and to develop improved vaccines, the nature of antigens that are involved in humoral and cell-mediated immunity was investigated. We have determined that five immunodominant protein antigens under study are homologues of stress proteins. This finding and observations with other pathogens suggest that infectious agents may respond to the host environment by producing stress proteins and that these proteins can be important immune targets. We postulate that abundant and highly conserved stress proteins may have "immunoprophylactic" potential for a broad spectrum of human pathogens.