Rabbit sucrase-isomaltase contains a functional intestinal receptor for Clostridium difficile toxin A.

The intestinal effects of Clostridium difficile toxin A are inidated by toxin binding to luminal enterocyte receptors. We reported previously that the rabbit ileal brush border (BB) receptor is a glycoprotein with an alpha-d-galactose containing trisaccharide in the toxin-binding domain (1991. J. Clin. Invest. 88:119-125). In this study we characterized the rabbit ileal BB receptor for this toxin. Purified toxin receptor peptides of 19 and 24 amino acids showed 100% homology with rabbit sucrase-isomaltase (SI). Guinea pig receptor antiserum reacted in Western blots with rabbit SI and with the purified toxin receptor. Antireceptor IgG blocked in vitro binding of toxin A to rabbit ileal villus cell BB. Furthermore, anti-SI IgG inhibited toxin A-induced secretion (by 78.1%, P < 0.01), intestinal permeability (by 80.8%, P < 0.01), and histologic injury (P < 0.01) in rabbit ileal loops in vivo. Chinese hamster ovary cells transfected with SI cDNA showed increased intracellular calcium increase in response to native toxin (holotoxin) or to a recombinant 873-amino acid peptide representing the receptor binding domain of toxin A. These data suggest that toxin A binds specifically to carbohydrate domains on rabbit ileal SI, and that such binding is relevant to signal transduction mechanisms that mediate in vitro and in vivo toxicity.

Characterization of the promoter elements required for hepatic and intestinal transcription of the human apoB gene: definition of the DNA-binding site of a tissue-specific transcriptional factor.

The promoter elements important for intestinal and hepatic transcription of the human apoB gene have been localized downstream of nucleotide -150. Footprinting analysis using hepatic nuclear extracts identified four protected regions, -124 to -100, -97 to -93, -86 to -33, and +33 to +52. Gel electrophoretic mobility shift assays showed that multiple factors interact with the apoB sequence -86 to -33, while the region -88 to -61 binds a single nuclear factor. Methylation interference analysis and nucleotide substitution mutagenesis identified the binding site of the factor between residues -78 and -68. Binding competition experiments indicate that this factor recognizes the regulatory elements of other liver-specific genes.

DNA sequence of the early E3 transcription unit of adenovirus 5.

The DNA sequence of the early E3 transcription unit of adenovirus 5 (Ad5) has been determined and it has been compared to Ad2, as published previously [J. Hérissé, G. Courtois, and F. Galibert (1980), Nucl. Acids Res. 8, 2173-2192; J. Hérissé and F. Galibert (1981), Nucl. Acids Res. 9, 1229-1240]. The E3 regions of Ad5 and Ad2 are quite homologous despite being nonessential for Ad growth in cultured cells. The major differences are "gaps" that exist either in Ad5 or Ad2 in intergenic regions. The conservation of sequences suggests that E3 plays a beneficial role in natural infection of humans. E3 appears to encode about seven to nine proteins; based on sequence, seven of these may be membrane proteins. Thus, E3 may be a transcription unit devoted to the synthesis of membrane proteins. The E3 genes lie essentially one after the other along the genome, and which gene is expressed from a given primary transcript is determined by the choice of the 3' end site and the 5' and 3' splice sites. Almost all E3 mRNAs contain nonfunctional AUGs that are 5' to the initiation codon. Codon usage is nonrandom. Although the CG dinucleotide frequency is low, CG clusters exist in the promoter and other regions.

Dietary alteration of translatable mRNA sequences coding for rat liver pyruvate kinase.

Poly(A+) RNA (RNA containing a polyadenylic acid sequence) was isolated from individual livers of rats fed standard lab chow, fasted, or fasted and refed a high carbohydrate diet. The level of functional mRNA coding for pyruvate kinase was assayed using a rabbit reticulocyte in vitro translation system. The total 35S incorporation into newly synthesized liver pyruvate kinase was measured and compared to 35S incorporation into albumin, total trichloroacetic acid-precipitated proteins, and released polypeptide chains. The relative level of mRNA coding for liver pyruvate kinase decreases almost 60% upon fasting and increases approximately 15-fold upon refeeding with a high carbohydrate diet for 24 h. These observed changes in the amount of mRNA coding for liver pyruvate kinase agree with the previously reported changes in the relative rates of liver pyruvate kinase synthesis measured in vivo during these dietary stresses. Thus, it is suggested that the alterations in the amount of pyruvate kinase in liver in response to these dietary stresses primarily result from alteration in the amount of functional mRNA coding for the enzyme.

Purification and characterization of the nuclear factor BA1. A transcriptional activator of the human apoB gene.

The promoter region of human apoB -79 to -63 is recognized by a sequence-specific DNA binding protein, designated NF-BA1, which is essential for the transcriptional activation of the apoB gene in hepatic and intestinal cells. This protein has been purified to apparent homogeneity from rat liver nuclear extracts. The purification steps involve Q-Sepharose, Bio-Rex 70, S-Sepharose, and DNA-specific affinity chromatography. The purified protein was identified as a polypeptide of 60 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and photoaffinity cross-linking. In vitro transcription complementation experiments using normal and mutated -268 to +8 apoB promoter sequences as templates indicated that the purified protein retained the ability to stimulate apoB transcription when added to hepatic nuclear extracts. The stimulation of transcription is associated with the binding of the factor to its recognition sequence. A synthetic promoter consisting of five contiguous -79 to -63 elements in front of the core apoB promoter region -38 to +8 is strongly activated by NF-BA1 protein in hepatic extracts in vitro. Footprinting analysis showed that purified NF-BA1 binds to the regulatory regions of apoCIII (-87 to -63), apoAII (-740 to -719), and apoAI (-212 to -191) genes and may be involved in the regulation of their expression.

Organization of the regulatory elements and nuclear activities participating in the transcription of the human apolipoprotein B gene.

The human apolipoprotein B (apoB) promoter region (-150 to +124) is transcriptionally active in HepG2 and CaCo-2 but not in HeLa cells. In the present study, we demonstrate that transcription of the human apoB gene in hepatic cells is achieved through the interaction of proteins that bind to five regulatory elements, I (-118 to -98), II (-112 to -94), III (-86 to -62), IV (-72 to -53), and V (-53 to -33). Elements I, IV, and V can bind to bacterially expressed enhancer-binding protein (C/EBP) although with different affinities. In addition, elements I, IV, and V interact with at least two other heat-stable nuclear factors, designated NF-BA2 and NF-BA3, that are distinct from C/EBP. These activities have been separated by ion exchange chromatography and shown to produce footprints in elements I, IV, and V that are similar to those obtained with C/EBP. Element II is recognized by three activities designated NF-BCB1, NF-BCB2, and NF-BCB3, which have been separated by heparin-Sepharose chromatography. Saturation mutagenesis of the region established that they belong to a family of proteins recognizing the same motif, 5'-AAAAGCAAACAG-3'. Substitution mutations in element II that abolish binding of all three activities in vitro also reduce hepatic transcription in vivo to 10% of control, indicating that the binding of NF-BCB1, NF-BCB2, and NF-BCB3 activities is required for efficient transcription of the apoB gene. Furthermore, element III is recognized by the previously characterized nuclear factor NF-BA1, the binding of which is essential for the transcriptional activation of the apoB gene in hepatic and intestinal cells.

Mapping the 5' ends, 3' ends, and splice sites of mRNAs from the early E3 transcription unit of adenovirus 5.

Using nuclease gel analyses, the sites in the approximately 4000 nucleotide (nt) E3 transcription unit of adenovirus 5 (Ad5) that encode the 5' ends, 3' ends, and 5' and 3' splice sites of the approximately 10 E3 mRNAs were determined. Transcription initiation of all mRNAs occurs at two major (nt 1 and 8) and approximately two minor sites, situated 20-30 nt 3' to a TATA box. There are two major 3' end sites (nt 2227 and 3308), located approximately 20 nt downstream from ATTAAA and an AATAAA sequences, respectively. Thus, ATTAAA, as well as the usual AATAAA, apparently can function as a 3' end signal. There are two 5' splice sites (nt 372 and 923), both with GT at the intron boundary. There are four 3' splice sites (nt 766, 1817, 2201, and 2880), all with AG at the intron boundary. The nt 1817, 2201, and 2880 3' splice sites are located immediately upstream from open reading frames, such that splicing at the different sites allows synthesis of completely different proteins.

Regulation of the human ApoA-II gene by the synergistic action of factors binding to the proximal and distal regulatory elements.

We have recently shown that the human apoA-II promoter contains a set of 11 distal regulatory elements between nucleotides -903 and -255 and three proximal regulatory elements between nucleotides -126 and -33 that are essential for hepatic and intestinal transcription of the apoA-II gene (Chambaz, J., Cardot, P., Pastier, D., Zannis, V. I., and Cladaras, C. (1991) J. Biol. Chem. 266, 11676-11685). Deletion or nucleotide substitution analysis has shown that alterations in elements L (nucleotides -803 to -773) and K (nucleotides -760 to -743) reduced hepatic transcription to 25 and 20% and intestinal transcription to 8 and 4% of control, respectively, as measured by chloramphenicol acetyltransferase assays, indicating that these elements play an important regulatory role. Nucleotide substitutions in element AB (nucleotides -65 to -33) reduced hepatic and intestinal transcription to 60 and 36% of control, respectively. The factors that recognize regulatory regions L, K, and AB were analyzed by DNA binding gel electrophoretic and competition assays. This analysis has shown that elements AB, K, and L bind with different affinities to a newly characterized heat-stable factor, CIIIB1, which is a transcriptional activator of the human apoC-III gene (Ogami, K., Kardassis, D., Cladaras, C., and Zannis, V. I. (1991) J. Biol. Chem. 266, 9640-9646). In addition, elements AB and K bind a heat-labile activity, designated AIIAB1, and element L binds to several CCAAT box binding activities. Mutations in domain L that prevented the binding of CCAAT box binding activities reduced both hepatic and intestinal transcription to 30% of control, indicating the importance of these factors in transcription. Simultaneous nucleotide substitutions that prevented the binding of CIIIB1 activity in elements AB, K, and L reduced hepatic and intestinal transcription to 7 and 6% of control, respectively, suggesting that the synergistic interaction of CIIIB1 (bound to the proximal and distal regulatory elements) with CCAAT box proteins (bound to element L) can modulate the level of transcription of the human apoA-II gene.

Purification and characterization of a heat stable nuclear factor CIIIB1 involved in the regulation of the human ApoC-III gene.

The apoC-III promoter region -86 to -74 is recognized by two nuclear factors designated CIIIB1 and CIIIB2 (NF-BA1) which are both activators of apoC-III gene transcription (Ogami, K., Hadzopoulou-Cladaras, M., Cladaras, C., and Zannis, V. I. (1990) J. Biol. Chem. 265, 9808-9815). In this communication we report the purification of factor CIIIB1 from rat liver nuclear extracts. The purification procedure included anion- and cation-exchange chromatography, DNA sequence-specific affinity chromatography, and heat treatment at 85 degrees C for 5 min. The ligand used for affinity chromatography was a mutated apoC-III -90 to -73 promoter sequence which binds only the CIIIB1 factor. The purified protein was identified as a poly-peptide of Mr 41,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and photoaffinity cross-linking. The binding site of CIIIB1, defined by DNase I footprinting and methylation interference assays, contains the octameric motif CAGGTGAC. Nucleotide substitutions within this sequence abolished the binding of the purified factor. DNase I footprinting analysis showed that purified CIIIB1 protein protects the apoA-II promoter region -65 to -48 which contains an identical octameric CAGGTGAC motif in the antisense strand suggesting that CIIIB1 may also play a role in apoA-II gene regulation.

rev protein of human immunodeficiency virus type 1 affects the stability and transport of the viral mRNA.

rev (trs/art) is an essential human immunodeficiency virus type 1 (HIV-1) regulatory protein. rev increases the levels of the gag- and env-producing mRNAs via a cis-acting element in the env region of HIV-1, named rev-responsive element. Our results show that rev increases the stability of the unspliced viral mRNA, while it does not affect the stability of the multiply spliced viral mRNAs that do not contain the rev-responsive element. The study of mutated proviral constructs producing mRNA that cannot be spliced revealed that the effect of rev on stability is independent of splicing. Our experiments also indicate that rev promotes the transport of the viral mRNA containing the rev-responsive element from the nucleus to the cytoplasm. The proposed functions of rev are consistent with its nuclear localization as shown by immunofluorescence. The selective effects of rev on the levels of the viral mRNA suggest a model for feedback regulation by rev leading to a steady state of viral expression.

Mapping a new gene that encodes an 11,600-molecular-weight protein in the E3 transcription unit of adenovirus 2.

The DNA sequence of the early E3 transcription unit of adenovirus 2 (Ad2) (J. Hérissé et al., Nucleic Acids Res. 8:2173-2192, 1980), indicates that an open reading frame exists between nucleotides 1860 and 2163 that could encode a protein of Mr 11,600 (11.6K). We have determined the DNA sequence of the corresponding region in Ad5 (closely related to Ad2) and have established that this putative gene is conserved in Ad5 (a 10.5K protein). To determine whether this protein is expressed, we prepared an antiserum in rabbits against a synthetic peptide corresponding to amino acids 66 to 74 in the 11.6K protein of Ad2. The peptide antiserum immunoprecipitated a ca. 13K-14K protein doublet, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, from [35S]methionine-labeled Ad2- or Ad5-early-infected KB cells. The antiserum also immunoprecipitated a 13K-14K protein doublet translated in vitro from Ad2 or Ad5 early E3-specific mRNA purified by hybridization to Ad2 EcoRI-D (nucleotides -236 to 2437). The synthetic peptide successfully competed with the 13K-14K protein doublet in immunoprecipitation experiments, thereby confirming the specificity of the antiserum. As deduced from the DNA sequence, the 11.6K protein (and the corresponding 10.5K Ad5 protein) has a conserved 22-amino-acid hydrophobic domain, suggesting that the protein may be associated with membranes. We conclude that a gene located at nucleotides 1860 to 2143 in the Ad2 E3 transcription unit (nucleotides 1924 to 2203) in the Ad5 E3 transcription unit) encodes an 11.6K protein (10.5K in Ad5).

cyt gene of adenoviruses 2 and 5 is an oncogene for transforming function in early region E1B and encodes the E1B 19,000-molecular-weight polypeptide.

A total of 59 cytocidal (cyt) mutants were isolated from adenovirus 2 (Ad2) and Ad5. In contrast to the small plaques and adenovirus type of cytopathic effects produced by wild-type cyt+ viruses, the cyt mutants produced large plaques, and the cytopathic effect was characterized by marked cellular destruction. cyt mutants were transformation defective in established rat 3Y1 cells. cyt+ revertants and cyt+ intragenic recombinants recovered fully the transforming ability of wild-type viruses. Thus, the cyt gene is an oncogene responsible for the transforming function of Ad2 and Ad5. Genetic mapping in which we used three Ad5 deletion mutants (dl312, dl313, and dl314) as reference deletions located the cyt gene between the 3' ends of the dl314 deletion (nucleotide 1,679) and the dl313 deletion (nucleotide 3,625) in region E1B. Restriction endonuclease mapping of these recombinants suggested that the cyt gene encodes the region E1B 19,000-molecular-weight (175R) polypeptide (nucleotides 1,711 to 2,236). This was confirmed by DNA sequencing of eight different cyt mutants. One of these mutants has a single missense mutant, two mutants have double missense mutations, and five mutants have nonsense mutations. Except for one mutant, these point mutations are not located in any other known region E1B gene. We conclude that the cyt gene codes for the E1B 19,000-molecular-weight (175R) polypeptide, that this polypeptide is required for morphological transformation of rat 3Y1 cells, and that simple amino acid substitutions in the protein can be sufficient to produce the cyt phenotype.

Novel deletion mutants that enhance a distant upstream 5' splice in the E3 transcription unit of adenovirus 2.

Region E3 of adenovirus is a "complex" transcription unit: i.e. different mRNAs and proteins arise by differential RNA 3' end selection and differential splicing of the primary transcript. We are using viable virus mutants to understand the controls that dictate the specificity and efficiency of the RNA processing signals. We describe a novel class of deletion mutations that enhance a natural 5' splice site located approximately 740 nucleotides (nt) upstream. In particular, deletions within nt 1691-2044 in the E3 transcription unit result in a 5-fold enhancement of the 5' splice site at nt 951 (as reflected in steady-state mRNA). The effect is specific, because the deletions do not affect the 5' splice site at nt 372, and because deletions within nt 2044-2214 do not affect either the 951 or the 372 5' splice sites. As a consequence of the enhanced splicing at the 951 5' site, synthesis of the major E3 mRNA and the major E3 protein (gp19K) are dramatically reduced. At least one of the natural 3' splice sites, located at nt 2157, is the recipient of the enhanced splicing at the 951 5' splice site. We conclude that sequences located within nt 1691-2044 affect (probably in cis) splicing at the 951 5' splice site. We speculate that nt 1691-2044 includes a splicing control region which functions to suppress splicing at the 951 5' splice site.

The 19-kDa glycoprotein coded by region E3 of adenovirus. Purification, characterization, and structural analysis.

The 19-kDa glycoprotein (gp 19K) coded by early region E3 of adenovirus is of interest as a model for glycoprotein processing and sorting, as well as for the interaction between viral antigens and class I transplantation antigens. In this paper, we show that gp 19K is a major protein synthesized during early stages of infection of human KB cells. We report the purification of gp 19K to near homogeneity, the preparation of a gp 19K antiserum, and structural analyses on the protein. We have determined the DNA sequence of the gp 19K gene in adenovirus type 5 (Ad5) for comparison with the published sequence (Hérissé, J., Courtois, G., and Galibert, F. (1980) Nucleic Acids Res. 8, 2173-2192) of adenovirus type 2 (Ad2). Fragments produced by cyanogen bromide cleavage of Ad2 gp 19K are in accord with the DNA sequence, as are synthetic peptide antibodies targeted to the NH2 terminus of Ad2 gp 19K and the COOH terminus of Ad5 gp 19K. The Ad2 and Ad5 proteins are quite homologous. Conserved features include an NH2-terminal signal sequence, two potential Asn-linked glycosylation sites, and a 20-residue putative transmembrane hydrophobic domain followed by a 15-residue polar domain at the COOH terminus. We show that cleavage of the signal peptide occurs between the 17th and 18th amino acids on both the Ad2 and Ad5 versions of gp 19K and that both potential sites are glycosylated with exclusively high-mannose (as opposed to complex) oligosaccharides. Secondary structure predictions suggest six alpha-helix regions including the signal peptide and transmembrane domain, two or three beta-sheet regions, and about eight beta-turns including the two glycosylation sites and the regions flanking the transmembrane domain.

Functional domains of the nuclear receptor hepatocyte nuclear factor 4.

The hepatocyte nuclear factor 4 (HNF-4) is a member of the nuclear receptor superfamily and participates in the regulation of several genes involved in diverse metabolic pathways and developmental processes. To date, the functional domains of this nuclear receptor have not been identified, and it is not known whether its transcriptional activity is regulated by a ligand or other signals. In this report, we show that HNF-4 contains two transactivation domains, designated AF-1 and AF-2, which activate transcription in a cell type-independent manner. AF-1 consists of the extreme N-terminal 24 amino acids and functions as a constitutive autonomous activator of transcription. This short transactivator belongs to the class of acidic activators, and it is predicted to adopt an amphipathic alpha-helical structure. In contrast, the AF-2 transactivator is complex, spanning the 128-366 region of HNF-4, and it cannot be further dissected without impairing activity. The 360-366 region of HNF-4 contains a motif that is highly conserved among transcriptionally active nuclear receptors, and it is essential for AF-2 activity, but it is not necessary for dimerization and DNA binding of HNF-4. Thus, HNF-4 deletion mutants lacking the 361-465 region bind efficiently to DNA as homo- and heterodimers and behave as dominant negative mutants. Remarkably, the full transactivation potential of AF-2 is inhibited by the region spanning residues 371-465 (region F). The inhibitory effect of region F on the HNF-4 AF-2 activity is a unique feature among members of the nuclear receptor superfamily, and we propose that it defines a distinct regulatory mechanism of transcriptional activation by HNF-4.

Promoter elements and factors required for hepatic and intestinal transcription of the human ApoCIII gene.

We have mapped the promoter elements required for hepatic and intestinal transcription of the human apoCIII gene by deletion, nucleotide substitution, and DNase I footprinting analyses of the promoter region (nucleotides -1411 to +24). Deletion of the region -1020 to -871 increased 2-fold intestinal transcription without affecting hepatic transcription. Deletion of the region -890 to -686 decreased hepatic and intestinal transcription 34- and 13-fold respectively. Internal deletions of the -686 to -553 region increased intestinal transcription 2-fold and decreased hepatic transcription 9-fold. Finally, internal deletions in the region -408 to -163 decreased hepatic transcription 2- to 4-fold without affecting the intestinal transcription. Footprinting analysis using rat liver nuclear extracts identified 10 protected regions as follows: A, -32 to -25; B, -87 to -72; C, -138 to -119; D, -160 to -142; E, -414 to -403; F, -611 to -592; G, -669 to -648; H, -705 to -690; I, -766 to -726; and J, -792 to -779. The findings indicate that the region -890 to -686 is recognized by nuclear factors which promote both intestinal and hepatic transcription, whereas the region -686 to -553 is recognized by factors which promote only hepatic transcription. DNA binding and methylation interference assays indicated that the region -86 to -74 is recognized by two mutually exclusive nuclear factors with overlapping domains. One factor (CIIIB1) is unique to apoCIII and the other (CIIIB2) recognizes the regulatory elements of other apolipoprotein promoters. Binding of factor CIIIB2 is associated with normal transcription. In contrast, binding of factor CIIIB1 is associated with reduced transcription indicating that this factor may act as a modulator of transcription.

Promoter elements and factors required for hepatic transcription of the human ApoA-II gene.

The apolipoprotein A-II (apoA-II) gene regulatory region -911 to +29 strongly promotes the transcription of the promotorless chloramphenicol acetyltransferase (CAT) gene in cells of hepatic (HepG2) and intestinal (CaCo2) origin but not in HeLa cells. Deletion of nucleotides -911 to -860 decreased the hepatic and intestinal transcription to 7% and 18% of control, respectively. Further progressive deletions extending to nucleotides -614, -440, -230, and -80 abolished both hepatic and intestinal transcription, indicating that the distal promoter region -911 to -614 contains regulatory elements that are essential for intestinal and hepatic transcription. An internal deletion of the -614 to -230 region decreased hepatic transcription 60% while it increased intestinal transcription 140% of control indicating that the elements which control hepatic and intestinal transcription of the apoA-II gene may be different within this regulatory region. DNase I footprinting analysis with rat liver nuclear extracts identified 14 protected regions: A, -40 to -33; B, -65 to -42; C, -126 to -110; D, -276 to -255; E, -377 to -364; F, -404 to -384; G, -468 to -455; H, -573 to -554; I, -706 to -680; J, -734 to -716; K, -760 to -743; L, -803 to -773; M, -853 to -829, and N, -903 to -879, as the DNA binding sites for nuclear factors. Five of the regions (B, C, G, H, and K) bind to heat-stable factors. DNA binding gel electrophoretic assays indicated that region N (-903 to -879), which is essential for efficient transcription, binds predominantly a nuclear activity designated AIIN3. This activity is present in cells of hepatic and intestinal origin but absent in HeLa cells. Similar analysis showed that region H (-573 to -554) binds to the liver-specific factor HNF1/LFB1. Deletion of this region decreased hepatic and intestinal transcription 80 and 64% of control, respectively, suggesting that HNF1/LFB1 or a related activity contributes to optimal transcription but is not essential for the tissue-specific expression of the human apoA-II gene.

The complete sequence and structural analysis of human apolipoprotein B-100: relationship between apoB-100 and apoB-48 forms.

We have isolated and sequenced overlapping cDNA clones covering the entire sequence of human apolipoprotein B-100 (apoB-100). DNA sequence analysis and determination of the mRNA transcription initiation site by S1 nuclease mapping showed that the apoB mRNA consists of 14,112 nucleotides including the 5' and 3' untranslated regions which are 128 and 301 nucleotides respectively. The DNA-derived protein sequence shows that apoB-100 is 513,000 daltons and contains 4560 amino acids including a 24-amino-acid-long signal peptide. The mol. wt of apoB-100 implies that there is one apoB molecule per LDL particle. Computer analysis of the predicted secondary structure of the protein showed that some of the potential alpha helical and beta sheet structures are amphipathic, whereas others have non-amphipathic neutral to apolar character. These latter regions may contribute to the formation of the lipid-binding domains of apoB-100. The protein contains 25 cysteines and 20 potential N-glycosylation sites. The majority of cysteines are distributed in the amino terminal portion of the protein. Four of the potential glycosylation sites are in predicted beta turn structures and may represent true glycosylation positions. ApoB lacks the tandem repeats which are characteristic of other apolipoproteins. The mean hydrophobicity the mean value of H1 and helical hydrophobic moment the mean value of microH profiles of apoB showed the presence of several potential helical regions with strong polar character and high hydrophobic moment. The region with the highest hydrophobic moment, between amino acid residues 3352 and 3369, contains five closely spaced, positively charged residues, and has sequence homology to the LDL receptor binding site of apoE. This region is flanked by three neighbouring regions with positively charged amino acids and high hydrophobic moment that are located between residues 3174 and 3681. One or more of these closely spaced apoB sequences may be involved in the formation of the LDL receptor-binding domain of apoB-100. Blotting analysis of intestinal RNA and hybridization of the blots with carboxy apoB cDNA probes produced a single 15-kb hybridization band whereas hybridization with amino terminal probes produced two hybridization bands of 15 and 8 kb. Our data indicate that both forms of apoB mRNA contain common sequences which extend from the amino terminal of apoB-100 to the vicinity of nucleotide residue 6300. These two messages may have resulted from differential splicing of the same primary apoB mRNA transcript.