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V I Zannis

Publications and source records attributed to V I Zannis.

At least 19 recordsLinked to original sources
PubMed1995

Complex interactions between SP1 bound to multiple distal regulatory sites and HNF-4 bound to the proximal promoter lead to transcriptional activation of liver-specific human APOCIII gene.

Footprinting analysis of the human apoCIII promoter identified a set of four proximal (A-D) and six distal (E-J) regulatory elements between nucleotides -792 and -25 [Ogami, K., et al. (1990) J. Biol. Chem. 265, 9808-9815]. The distal regulatory elements of the apoCIII gene increase by 10-fold the strength of the homologous as well as of heterologous proximal promoters. Required for such transcriptional enhancement is the presence of an intact hormone response element (HRE) on the proximal promoter which binds a variety of nuclear hormone receptors. To understand the mechanism of this transcriptional activation, we identified the nature and the importance of the factors which bind to the upstream regulatory elements of the apoCIII promoter by DNA binding, competition, supershift, and transient transfection assays. These analyses showed that the upstream apoCIII promoter contains multiple binding sites for the ubiquitous transcription factor SP1, which recognizes the regulatory elements F, H, and I. The regulatory element G represents a specialized HRE which is recognized by the orphan receptors ARP-1 and EAR-3 but not by HNF-4. A single activity designated CIII J1 binds to the regulatory element J. The same or a similar activity binds as a minor component to the regulatory elements F and I where SP1 is the predominant binding activity. Finally, a minor activity designated CIII 15 binds to the regulatory element I.(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoprotein C-III↗
PubMed1994

Transcriptional regulation of the apolipoprotein A-IV gene involves synergism between a proximal orphan receptor response element and a distant enhancer located in the upstream promoter region of the apolipoprotein C-III gene.

Apolipoprotein A-IV expression is limited to intestinal and hepatic cells, suggesting a tissue specific transcriptional regulation of its gene. To investigate the mechanism controlling apo A-IV transcription we have analysed its promoter region by in vitro DNA binding and transient transfection experiments. DNase I footprinting analysis of the proximal promoter with rat liver nuclear extracts revealed four protected regions: AIVA (-32 to -22), AIVB (-84 to -42), AIVC (-148 to -92) and AIVD (-274 to -250). Element AIVC which is necessary for maximal promoter activity, binds HNF-4, Arp-1 and Ear-3 with similar affinity in a mutually exclusive manner. HNF-4 transactivated chimeric constructs containing intact AIVC site in the context of either the apo A-IV promoter or the heterologous thymidine kinase minimal promoter, while Arp-1 and Ear-3 repressed this activation. Increasing amounts of HNF-4 alleviated Arp-1 or Ear-3 mediated repression, suggesting that the observed opposing effects is a result of direct competition of these factors for the same recognition site. In transient transfection assays the apo A-IV promoter region (-700 to +10) had a very low activity in cells of hepatic (HepG2) and intestinal (CaCo2) origin. This activity was increased 13 to 18-fold when the upstream elements of the distantly linked apo C-III gene were fused to the proximal promoter. Results obtained with different 5' and 3' deletion constructs indicated that the cis-acting elements F to J between the nucleotides -500 and -890 of the apo C-III promoter were absolutely necessary to drive maximal enhancement in HepG2 and CaCo2 cells. The apo C-III upstream elements enhanced the activity of the minimal AdML promoter or the apo A-IV site C mutant less efficiently than the intact apo A-IV or AdML promoter constructs containing single HNF-4 sites. The findings suggest that the enhancer effect is mediated by synergistic interactions between the trans-acting factors which recognize the apo C-III regulatory elements and HNF-4 which binds to the proximal apo A-IV promoter.

Animals↗
PubMed1994

Purification and characterization of nuclear factors binding to the negative regulatory element D of human apolipoprotein A-II promoter: a negative regulatory effect is reversed by GABP, an Ets-related protein.

We have previously shown that transcription of the human apolipoprotein A-II (apoA-II) gene is controlled by a complex set of regulatory elements [Cardot et al. (1993) Biochemistry 32, 9080-9093]. We have also identified previously described, as well as new activities which bind to these elements and influence to varying degrees the transcription of the apoA-II gene. DNA binding and competition assays indicated that element D binds three new activities, designated AIID1, AIID2, and AIID4, as well as an activity related to C/EBP. Activities AIID1, AIID2, and AIID4 were purified and characterized further in order to determine their function on the transcriptional regulation of human apoA-II gene. SDS-PAGE analysis as well as photoaffinity cross-linking of the affinity-purified AIID2 showed that it consists of three proteins with molecular masses ranging between 54 and 63 kDa. The amino acid sequence of tryptic peptides obtained from AIID2 protein bands revealed that it is homologous to GABP, an Ets-related protein. Similar analysis showed that affinity-purified AIID4 has an apparent molecular mass of 130 kDa. AIID1 activity was purified partially; in addition to binding to the apoA-II promoter, AIID1 also binds to the regulatory element C of apoCIII and may play a role in the transcriptional regulation of both genes. Methylation interference of G residues and permanganate modification of T residues indicated that the binding sites of AIID2 and AIID4 were contiguous on element D. However, the binding site of AIID1 overlaps with the binding sites of both AIID2 and AIID4. This suggests that the binding of AIID1 and AIID2 or of AIID1 and AIID4 may be mutually exclusive, whereas AIID2 and AIID4 may bind simultaneously. Transcription from a minimal promoter containing elements AB, C, and D of apoA-II increased 1.5- to 1.6-fold when element D is deleted, as well as by promoter mutations which eliminated the binding of both AIID1 and/or AIID4 to element D, but permitted the binding of AIID2/GABP. The findings suggest that element D has a negative regulatory role on apoA-II gene transcription when it is occupied by protein AIID1 and/or AIID4. This negative effect is reversed when element D is occupied only by the regulatory factor AIID2/GABP.

Amino Acid Sequence↗
PubMed1994

Intracellular early and late modifications of human apolipoprotein A-II. Effect of glutamine- +1 to leucine substitution.

It has been shown previously that apoA-II undergoes several intracellular modifications in HepG2 cells (Hussain & Zannis, 1990). In the present study, we have generated permanent cell lines in mouse C127 cells which express the normal apoA-II gene and a mutated form in which Gln+1 was substituted with Leu (Leu+1). This modification was designed to prevent cyclization of the N-terminal glutamine of apoA-II and thus identify the isoproteins which are precursors and products of the N-terminal cyclization reaction. The C127-expression cells were also utilized to study the cellular compartments where the apoA-II modifications occur as well as the importance of the modifications for apoA-II trafficking and secretion. We have found that apoA-II (Gln+1) synthesized by C127 and HepG2 cells had similar isoproteins. In both cell types, unmodified pro-apoA-II, designated isoprotein 3, had a similar isoelectric point as the cell-free translation product of apoA-II mRNA, suggesting that isoprotein 3 results from cleavage of the signal peptide. Isoprotein 3 represents an unmodified apoA-II isoprotein and undergoes an early modification into a more acidic isoprotein 1, which differs from isoprotein 3 by two negative charges. Brefeldin A treatment of the cells did not prevent the formation of isoprotein 1, suggesting that this modification occurs in a pre-Golgi compartment. Neuraminidase treatment of secreted apoA-II isoproteins did not affect isoprotein 1, indicating that it is not sialylated isoprotein. Isoprotein 1 undergoes further modifications which are consistent with cleavage of the propeptide, N-terminal cyclization and sialylation most likely resulting from O-glycosylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoprotein A-II↗
PubMed1993

An indirect negative autoregulatory mechanism involved in hepatocyte nuclear factor-1 gene expression.

Recent studies have revealed that hepatocyte nuclear factor 4 (HNF-4) is an essential positive regulator of another liver enriched transcription factor HNF-1, defining a transcriptional hierarchy between the two factors operating in hepatocytes. To assess the possible autoregulation of the HNF-1 gene we have examined the effect of HNF-1 on its own transcription. In transient transfection assays, HNF-1 strongly down-regulated transcription driven by its own promoter in HepG2 cells. In addition HNF-1 also repressed the activity of HNF-4 dependent ApoCIII and ApoAI promoters. The same effect was observed using vHNF-1, a distinct but highly related protein to HNF-1. Both HNF-1 and vHNF-1 downregulated HNF-4 activated transcription from intact and chimeric promoter constructs carrying various HNF-4 binding sites implying that they act by impeding HNF-4 binding or activity. DNA binding and cell free transcription experiments however failed to demonstrate any direct or indirect interaction of HNF-1 and vHNF-1 with the above regulatory regions. Both factors repressed HNF-4 induced transcription of the ApoCIII and HNF-1 genes in HeLa cells, arguing against the requirement of a hepatocyte specific function. These findings define an indirect negative autoregulatory mechanism involved in HNF-1 gene expression, which in turn may affect HNF-4 dependent transcription of other liver specific genes.

Animals↗
PubMed1993

Factors participating in the liver-specific expression of the human apolipoprotein A-II gene and their significance for transcription.

We have shown previously that the hepatic and intestinal transcription of the human apolipoprotein A-II (apoA-II) gene in cell cultures is controlled by a complex set of regulatory elements A-N [Chambaz, et al. (1991) J. Biol. Chem. 266, 11676-11685; Cardot, et al. (1991) J. Biol. Chem. 266, 24460-24470]. In the present communication, we have assessed the functional importance of each of the regulatory elements. In addition, we have used DNA binding and competition assays and protein fractionation to identify the hepatic nuclear activities which are involved in the regulation of the human apoA-II gene. Such activities may be of general importance for the regulation of liver-specific genes. The DNA binding and competition analysis showed that the regulatory elements M, D, and F bind new activities which have not been identified in apolipoprotein or other liver-specific promoters. These activities have been designated AIIM1 and AIIM2 for element M, AIID1 and AIID2 for element D, and AIIF2 for element F. The activity AIIM2 is present in liver, but absent in CaCo-2 cells. A set of regulatory elements binds activities which resemble liver-enriched or ubiquitous factors previously shown to play important roles in the regulation of their target genes. Thus, element I binds to activities related to NF1, and elements L, C, D, G, AB, and F bind to C/EBP alpha as well as other heat-stable activities. The affinity of the bacterially expressed C/EBP alpha for the various apoA-II regulatory regions follows the order: AIIL approximately AIIC > AIID > AIIF > AIIG > AIIAB. Protein fractionation showed that element J binds at least three hepatic nuclear activities and is also recognized by members of the nuclear receptor family, HNF4, EAR2, EAR3, and ARP1. Another liver-enriched factor, HNF1, was shown previously to bind to element H. Despite the importance of HNF1, HNF4, NF1, and C/EBP alpha in the regulation of numerous other target genes, deletion of the HNF1, NF1, and HNF4 and several C/EBP binding sites did not drastically affect the hepatic transcription of the apoA-II gene. Rather, the hepatic and intestinal transcription is affected severely by deletion of elements A, B, K, L, and N. In addition, the intestinal transcription is affected by deletion of elements C, J, and M. The in vivo physiological importance of these elements will require analysis of their function in transgenic animals. This analysis establishes the organization of several nuclear activities on the human apoA-II promoter.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Site-directed mutagenesis and structure-function analysis of the human apolipoprotein A-I. Relation between lecithin-cholesterol acyltransferase activation and lipid binding.

We have mutagenized the human apoA-I gene and have generated cell lines which express normal and mutant apoA-I forms. Point mutations were introduced which changed Gln-1, Gln-2 to Arg,Arg, Pro99 to His, and Pro121 to His. In addition, the following amino acid deletions (delta) were generated: delta 113-124, delta 148-186, delta 212-233, and delta 213-243. The apoA-I form isolated from the culture medium of C127 cells was analyzed for its ability to activate lecithin-cholesterol acyltransferase (LCAT) and to bind to phospholipid vesicles and high density lipoprotein (HDL). Compared with the wild type (WT) apoA-I, the relative activation of LCAT achieved by the point mutations Gln-1, Gln-2----Arg,Arg, Pro99----His, and Pro121----His were 106 +/- 7, 92 +/- 6, and 77 +/- 9%, respectively. Kinetic analysis of one mutant apoA-I form showed similar Vmax but a 15-fold increase in the Km of the mutant apoA-I form. Furthermore, the activation achieved by the internal deletion mutants delta 113-124, delta 148-186, delta 212-233, and delta 213-243 was 47 +/- 3, 0.5 +/- 0.4, 28 +/- 4 and 13 +/- 5%, respectively. Mutants deficient in their ability to activate LCAT displayed alterations in liposome and HDL binding, compared with WT as determined by density gradient ultracentrifugation analysis of the culture medium. Thus, the peak recovery (approximately 50%) of apoA-I bound to HDL was at density 1.14 g/ml for the WT apoA-I, at 1.18 g/ml for the mutants delta 113-124 and delta 148-186, and at d greater than 1.21 g/ml for the delta 212-233 and delta 213-243. Electron microscopy of the proteoliposome LCAT substrate generated by WT and mutant apoA-I forms showed that the carboxyl-terminal deletion mutants which displayed aberrant binding to HDL also displayed reduced ability to convert the spherical lecithin-cholesterol vesicles into discs compared with WT. The findings suggest that (a) the importance of the carboxyl terminus of apoA-I for LCAT activation is related to its ability to bind to lipid and/or to form discoidal substrate for LCAT, and (b) the interaction of several domains of apoA-I are required for the activation of LCAT.

Algorithms↗
PubMed1992

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.

Animals↗
PubMed1992

Expression of human apolipoprotein E but not that of apolipoprotein A-I by mouse C127 cells is associated with increased secretion of lipids in the form of vesicles and discs.

Transfected mouse mammary-derived cells (C127) expressing human apolipoprotein (apo) E (C127E) were used a) to determine whether the lipid-binding character of apoE is sufficient to promote its assembly with lipid to form lipoprotein-like particles when expressed by cells not normally expressing apolipoproteins; b) to characterize the secreted complexes in terms of morphology, size, and composition; and finally c) to determine whether apoE or apoA-I gene expression by these transfected cells has any effect on the levels and the profiles of the synthesized and secreted lipids. The findings of the present study demonstrate that: a) as determined by density gradient ultracentrifugation and gel filtration chromatography, about 20% of the secreted [35S]methionine-labeled apoE expressed by C127E cells is lipid-associated. b) Negative-stain electron microscopic analysis of the lipid-protein complexes recovered in the lipoprotein fractions (d less than 1.21 g/ml) revealed that approximately 13% of the total population of particles were discs (16 +/- 5 nm mean diameter and 4-6 nm thick), resembling nascent high density lipoproteins (HDL). The majority of the particles however (greater than 82%) appeared vesicular with varying diameters (48 +/- 40 nm mean diameter). The discoidal and the vesicular appearance of the particles secreted by C127E cells is consistent with the composition of lipids. These consisted mostly of surface lipids, phospholipids (45 +/- 18%), diacylglycerols (36 +/- 17%), and free cholesterol (17 +/- 7%) (by weight). c) Expression of apoE by C127E cells was associated with an increased release of [35S]methionine-labeled protein and [3H]glycerol-labeled lipid (3- to 5- and 4- to 8-fold, respectively) compared to nontransfected C127 cells. Expression of mutant apoE or normal apoA-I, however, was not associated with increased release of the major lipid classes compared to the parent C127 cells, strongly suggesting that this character of C127E cells is specific to apoE expression. The release of lipids by C127E cells could be reduced considerably by the addition of the metabolic inhibitors, colchicine or cycloheximide (10 and 1 microM, respectively), suggesting that lipid release by C127E cells is an active process requiring both protein synthesis and functional secretory mechanisms. Taken together the findings suggest that apoE expression by C127 cells promotes the formation of nascent discoidal lipoprotein-like particles and enhances the release of vesicular lipids, possibly by promoting shedding of cell plasma membrane fragments.

Animals↗
PubMed1991

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.

Animals↗
PubMed1991

Expression, secretion, and lipid-binding characterization of the N-terminal 17% of apolipoprotein B.

The N-terminal 17% of human apolipoprotein B (apoB-17) was expressed in murine C127 cells following transfection with a bovine papilloma virus-based expression vector. A permanent cell line overexpressing the expected 89-kDa protein was selected and characterized. Pulse-chase experiments showed that the depletion of intracellular apoB-17 follows an apparent first-order kinetics with t1/2 = 51 min. Under conditions of continuous labeling, greater than 60% of the total synthesized apoB-17 was secreted in a soluble form, approximately 98% lipid-poor and approximately 2% lipid-bound. Inclusion of 1.2 mM oleate resulted in 5- and 2.5-fold increases in the amount of labeled apoB-17 in the p less than 1.063 g/ml and 1.063 less than p less than 1.21 g/ml fractions, respectively, which was coordinated with increased secretion of radiolabeled core lipids, triacylglycerols, and cholesteryl esters. Thus under conditions in which lipid pools are enriched a greater fraction of apoB-17 may be secreted on lipoprotein-like particles. The lipid-poor apoB-17 present in p greater than 1.21 g/ml readily associates with exogenously added dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles to form discoidal particles. Discs formed with DMPC/apoB-17, 7:1 (wt/wt), are 239 +/- 43 A in diameter and 61 +/- 4 A thick and contain approximately 2 molecules of apoB-17 and 2250 molecules of DMPC per disc. Based on volume calculations we conclude that apoB-17 forms an annulus about one bilayer high and 10 A thick surrounding the DMPC disc. Circular dichroic spectra of apoB-17 on DMPC discs showed apoB-17 to contain 39% alpha-helix, 36% beta-sheet, 9% beta-turn, and 16% random coil. To be consistent with this model greater than 70% of apoB-17 on DMPC discs must bind to lipid. These data suggest that the N-terminal 17% of apoB-100 can bind lipid and may contribute to some extent to the stabilization of triglyceride-rich lipoproteins.

Amino Acid Sequence↗
PubMed1991

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.

Apolipoprotein A-II↗
PubMed1991

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.

Animals↗
PubMed1991

Promoter elements and factors involved in hepatic transcription of the human ApoA-I gene positive and negative regulators bind to overlapping sites.

DNase I footprinting analysis of the proximal apoA-I promoter sequences with rat liver nuclear extracts identified four protected regions: A, -22 to +17; B, -128 to -77; C, -175 to -148; and D, -220 to -190. Region D (-220 to -190) binds at least two distinct activities, designated AID1 and AID2, respectively, which can be separated by ion exchange chromatography. Region C (-175 to -148) forms five DNA protein complexes. Three of the complexes (2, 4, and 5) originate from the binding of more than one heat-stable nuclear factor, and two (1 and 3), from the binding of two heat-labile factors. The heat-stable factors bind in the -175 to -148 region and can be distinguished from C/EBP, which recognizes the same region, with DNA binding gel electrophoretic assays. Both factors 1 and 3 bind in the -168 to -148 apoA-I region. Despite the lack of a CCAAT motif in this region, the binding of factor 1 is competed out by oligonucleotides containing the binding sites of NFY and NFY*. Mutagenesis of the promoter region showed that mutations in the -171 to -166 and -158 to -153 regions diminished the binding of the heat-stable factors and reduced hepatic transcription to 14 and 8% of control, respectively. In contrast, a mutation in the -164 to -159 region abolished the binding of factor 1 and was associated with a 4.6-fold increase in hepatic transcription. These findings suggest that the heat-stable factors act as positive regulators, whereas factor 1 acts as a negative regulator in apoA-I gene transcription.

Animals↗
PubMed1991

Cloning and expression of a rat brain alpha 2B-adrenergic receptor.

We have isolated a cDNA clone (RB alpha 2B) and its homologous gene (GR alpha 2B) encoding an alpha 2B-adrenergic receptor subtype by screening a rat brain cDNA and a rat genomic library. Nucleotide sequence analysis showed that both clones code for a protein of 458 amino acids, which is 87% homologous to the human kidney glycosylated adrenergic receptor (alpha 2-C4) and divergent from the rat kidney nonglycosylated alpha 2B subtype (RNG alpha 2). Transient expression of RB alpha 2B in COS-7 cells resulted in high-affinity saturable binding (Kd = 0.25 nM) for [3H]rauwolscine and a high receptor number (Bmax = 7.7 pmol/mg of protein) in the membranes of transfected COS-7 cells. Pharmacological analysis demonstrated that the expressed receptor bound adrenergic ligands with the following order of potency: rauwolscine greater than yohimbine greater than prazosin greater than oxymetazoline, with a prazosin-to-oxymetazoline Ki ratio of 0.34. This profile is characteristic of the alpha 2B-adrenergic receptor subtype. Blotting analysis of rat brain mRNA gave one major (3.0-kilobase) and two minor (4.6- and 2.3-kilobase) mRNA species, and hybridization with strand-specific probes showed that both DNA strands of GR alpha 2B may be transcriptionally active. These findings show that rat brain expresses an alpha 2B-adrenergic receptor subtype that is structurally different from the rat kidney nonglycosylated alpha 2B subtype. Thus the rat expresses at least two divergent alpha 2B-adrenergic receptors.

Amino Acid Sequence↗
PubMed1991

Secretion of lipid-poor nascent human apolipoprotein apoAI, apoCIII, and apoE by cell clones expressing the corresponding genes.

The human apolipoprotein apoAI, apoCIII, and apoE genes were placed under the control of the mouse metallothionein 1 promoter in a bovine papilloma virus vector that also contained the human metallothionein 1A gene. Following transfection of mouse C127 cells with the expression vector, cell clones resistant to Cd2+ were selected and found to express in high abundance specific apolipoprotein genes. Individual cell clones expressing apoAI, apoCIII, or apoE genes were used further to study the isoprotein composition and the flotation properties of the corresponding nascent apolipoproteins. It was found that the lipoproteins secreted by cell clones expressing the apoAI, apoCIII, and apoE genes consisted of the proapoAI disialylated form of apoCIII (apoCIIIS2) and mainly sialylated forms of apoE. Separation of the secreted apolipoproteins by density gradient ultracentrifugation resulted in limited flotation of nascent apoAI, apoE and apoCIII in the high density lipoprotein (HDL) fraction. Similar analysis in the presence of human serum increased the flotation of apoAI, apoE, and apoCIII to 6.5-, 4.5-, and 5.5-fold, respectively, and resulted in their redistribution to various lipoprotein fractions. HDL increased the flotation of apoAI to 12-fold and very low density lipoprotein (VLDL) increased the flotation of apoCIII and apoE to 6.5- and 5.5-fold, respectively. These findings suggest that in the cell system used, the majority of nascent apoAI, apoCIII and apoE is secreted in the lipid-poor form, which then associates extracellularly with preexisting lipoproteins.

Animals↗