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PubMed2001

Transcription factor AP-2 functions as a repressor that contributes to the liver-specific expression of serum amyloid A1 gene.

We previously identified transcription factor AP-2 as the nuclear factor that interacts with the tissue-specific repressor element in the rat serum amyloid A1 (SAA1) promoter. In this report, we provide evidence for a second AP-2-binding site and show that both AP-2 sites participate in mediating the transcription repression of SAA1 promoter. This proximal AP-2 site overlaps with the NFkappaB-binding site known to be essential for SAA1 promoter activity. Protein binding competition experiments demonstrated that AP-2 and NFkappaB binding to these overlapping sites were mutually exclusive. Furthermore, the addition of AP-2 easily displaced prebound NFkappaB, whereas NFkappaB could not displace AP-2. These results thus suggest that one mechanism by which AP-2 negatively regulates SAA1 promoter activity may be by antagonizing the function of NFkappaB. Consistent with a repression function, transient expression of AP-2 in HepG2 cells inhibited conditioned medium-induced SAA1 promoter activation. This inhibition was dependent on functional AP-2-binding sites, since mutation of AP-2-binding sites abolished inhibitory effects of AP-2 in HepG2 cells as well as resulted in derepression of the SAA1 promoter in HeLa cells. In addition to SAA1, we found that several other liver gene promoters also contain putative AP-2-binding sites. Some of these sequences could specifically inhibit AP-2.DNA complex formation, and for the human complement C3 promoter, overexpression of AP-2 also could repress its cytokine-mediated activation. Finally, stable expression of AP-2 in hepatoma cells significantly reduced the expression of endogenous SAA, albumin, and alpha-fetoprotein genes. Taken together, our results suggest that AP-2 may function as a transcription repressor to inhibit the expression of not only SAA1 gene but also other liver genes in nonhepatic cells.

Cell Differentiation↗
PubMed1993

Natural killer cells and the MHC class I pathway of peptide presentation.

Natural killer (NK) cells are TcR-Ig- lymphocytes that can mediate resistance to tumor growth, virus infections and bone marrow transplants. The receptors and specificity of NK cells are not clear, although it has been suggested that cytolytic triggering may be facilitated by lack of host matched MHC expression in the target or the graft. This article reviews and analyzes the role of the MHC class I pathway of peptide presentation in regulation of NK cells. This subject is introduced by brief overviews on NK cells, the 'missing self hypothesis' and studies establishing that MHC class I molecules of the target and the host can influence NK reactivity against certain normal and malignant cells. The main focus is then set on recent studies indicating that this influence occurs through interactions involving the antigen binding groove or its bound peptides. This includes discussion of modulation of NK sensitivity by peptides, beta 2 microglobulin, allelic differences or point mutations in MHC class I heavy chains, virus infection and peptide loading deficiency. While it is clear that alterations affecting the groove and its contents can change NK sensitivity, the evidence should be interpreted with caution, as the structural integrity of the whole class I molecule is dependent on the conformation and the stability of the antigen binding groove. Two models for recognition, 'target interference' and 'effector inhibition', are discussed. Each of these (mutually non-exclusive) models can accommodate a role for the groove and its peptide.

Alleles↗
PubMed1998

Structural organization of the human reduced folate carrier gene: evidence for 5' heterogeneity in lymphoblast mRNA.

The reduced folate carrier (rfc1) gene encodes a protein that is involved in the intracellular accumulation of folates. Point mutations in this gene and alterations resulting in the down regulation of its message are major factors involved in the resistance to antifolate chemotherapeutic compounds. As a framework for understanding the significance of such changes in relation to gene expression and function, in this report we describe the organization of the rfc gene from human lymphoblasts. The gene contains 5 exons (2 to 6) coding for protein. At least four 5' exons, used in a mutually exclusive manner in the production of the rfc message from lymphoblast cells, are spliced to exon 2, which contains the translational start site. "Semi-quantitative" PCR indicates that exon 1 is preferentially used. The major transcriptional start site has been mapped by RACE and RNase protection to a region 109 to 135 base pairs 5' to the start of exon 1. The 5' region of the gene has no TATA box-like sequence but contains several consensus binding sites for transcriptional factors such as SP-1, MZF1, CREB, AP-1, ETS, GATA-1 and GATA-2. The overall organization of the human gene is similar to that of the hamster and mouse genes.

Alternative Splicing↗
PubMed2005

Polycystin-2--an intracellular or plasma membrane channel?

The intracellular location of polycystin-2 is a hotly debated topic in the field of polycystic kidney disease. Two not necessarily mutually exclusive hypotheses state that polycystin-2 is located in the endoplasmic reticulum or in the plasma membrane, respectively. Although a variety of techniques have been employed to prove one or the other location, no definite consensus has been reached yet. It is generally acknowledged, however, that the COOH-terminus of polycystin-2 contains a retention signal for the endoplasmic reticulum. Another facet has been added to the discussion due to the fact that many genes mutated in patients with cystic kidney diseases, among them PKD2, encode proteins which have been detected in primary cilia. Since there is no evidence that the endoplasmic reticulum extends into the primary cilium, polycystin-2 has to reach the plasma membrane at least in this case. An unbiased approach towards elucidating the physiological location of polycystin-2 would involve the characterization of its intracellular trafficking. Using the COOH-terminus of polycystin-2 in a two-hybrid screen, my group has identified a novel coiled-coil protein which we call PIGEA-14 (polycystin-2 interactor, Golgi- and endoplasmic reticulum-associated protein with a molecular weight of 14 kDa). PIGEA-14 also interacts with GM130, a protein associated with the Golgi matrix, and may therefore represent one important component of the trafficking machinery for polycystin-2.

Animals↗
PubMed2001

Two alternating structures of the HIV-1 leader RNA.

In this study we demonstrate that the HIV-1 leader RNA exists in two alternative conformations, a branched structure consisting of several well-known hairpin motifs and a more stable structure that is formed by extensive long-distance base pairing. The latter conformation was first identified as a compactly folded RNA that migrates unusually fast in nondenaturing gels. The minimally required domains for formation of this conformer were determined by mutational analysis. The poly(A) and DIS regions of the leader are the major determinants of this RNA conformation. Further biochemical characterization of this conformer revealed that both hairpins are disrupted to allow extensive long-distance base pairing. As the DIS hairpin is known to be instrumental for formation of the HIV-1 RNA dimer, the interplay between formation of the conformer and dimerization was addressed. Formation of the conformer and the RNA dimer are mutually exclusive. Consequently, the conformer must rearrange into a branched structure that exposes the dimer initiation signal (DIS) hairpin, thus triggering formation of the RNA dimer. This structural rearrangement is facilitated by the viral nucleocapsid protein NC. We propose that this structural polymorphism of the HIV-1 leader RNA acts as a molecular switch in the viral replication cycle.

Alternative Splicing↗
PubMed1997

NMR studies of the E140Q mutant of the carboxy-terminal domain of calmodulin reveal global conformational exchange in the Ca2+-saturated state.

In the present investigation, the Ca2+ activation of the C-terminal domain of bovine calmodulin and the effects of replacing the bidentate Ca2+-coordinating glutamic acid residue in the 12th and last position of loop IV with a glutamine are studied by NMR spectroscopy. The mutation E140Q results in sequential Ca2+ binding in this domain and has far-reaching effects on the structure of (Ca2+)2 TR2C, thereby providing further evidence for the critical role of this glutamic acid residue for the Ca2+-induced conformational change of regulatory EF-hand proteins. Analyses of the NOESY spectra of the mutant under Ca2+-saturated conditions, such that 97% of the protein is in the (Ca2+)2 form, revealed two sets of mutually exclusive NOEs. One set of NOEs is found to be consistent with the closed structure observed in the apo state of the C-terminal domain of the wild-type protein, while the other set supports the open structure observed in the Ca2+-saturated state. In addition, several residues in the hydrophobic core exhibit broadened resonances. We conclude that the (Ca2+)2 form of the mutant experiences a global conformational exchange between states similar to the closed and open conformations of the C-terminal domain of wild-type calmodulin. A population of 65 +/- 15% of the open conformation and an exchange rate of (1-7) x 10(4) s(-1) were estimated from the NMR data and the chemical shifts of the wild-type protein. From a Ca2+ titration of the 15N-labeled mutant, the macroscopic binding constants [log(K1) = 4.9 +/- 0.3 and log(K2) = 3.15 +/- 0.10] and the inherent chemical shifts of the intermediate (Ca2+)1 form of the mutant were determined using NMR. Valuable information was also provided on the mechanism of the Ca2+ activation and the roles of the structural elements in the two Ca2+-binding events. Comparison with the wild-type protein indicates that the (Ca2+)1 conformation of the mutant is essentially closed but that some rearrangement of the empty loop IV toward the Ca2+-bound form has occurred.

Amino Acid Sequence↗
PubMed1998

Directed evolution of a thermostable esterase.

We have used in vitro evolution to probe the relationship between stability and activity in a mesophilic esterase. Previous studies of these properties in homologous enzymes evolved for function at different temperatures have suggested that stability at high temperatures is incompatible with high catalytic activity at low temperatures through mutually exclusive demands on enzyme flexibility. Six generations of random mutagenesis, recombination, and screening stabilized Bacillus subtilis p-nitrobenzyl esterase significantly (>14 degreesC increase in Tm) without compromising its catalytic activity at lower temperatures. Furthermore, analysis of the stabilities and activities of large numbers of random mutants indicates that these properties are not inversely correlated. Although enhanced thermostability does not necessarily come at the cost of activity, the process by which the molecule adapts is important. Mutations that increase thermostability while maintaining low-temperature activity are very rare. Unless both properties are constrained (by natural selection or screening) the evolution of one by the accumulation of single amino acid substitutions typically comes at the cost of the other, regardless of whether the two properties are inversely correlated or not correlated at all.

Amino Acid Substitution↗
PubMed2001

Mapping of calmodulin and Gbetagamma binding domains within the C-terminal region of the metabotropic glutamate receptor 7A.

Ca(2+)/calmodulin (Ca(2+)/CaM) and the betagamma subunits of heterotrimeric G-proteins (Gbetagamma) have recently been shown to interact in a mutually exclusive fashion with the intracellular C terminus of the presynaptic metabotropic glutamate receptor 7 (mGluR 7). Here, we further characterized the core CaM and Gbetagamma binding sequences. In contrast to a previous report, we find that the CaM binding motif localized in the N-terminal region of the cytoplasmic tail domain of mGluR 7 is conserved in the related group III mGluRs 4A and 8 and allows these receptors to also bind Ca(2+)/CaM. Mutational analysis of the Ca(2+)/CaM binding motif is consistent with group III receptors containing a conventional CaM binding site formed by an amphipathic alpha-helix. Substitutions adjacent to the core CaM target sequence selectively prevent Gbetagamma binding, suggesting that the CaM-dependent regulation of signal transduction involves determinants that overlap with but are different from those mediating Gbetagamma recruitment. In addition, we present evidence that Gbetagamma uses distinct nonoverlapping interfaces for interaction with the mGluR 7 C-terminal tail and the effector enzyme adenylyl cyclase II, respectively. Although Gbetagamma-mediated signaling is abolished in receptors lacking the core CaM binding sequence, alpha subunit activation, as assayed by agonist-dependent GTPgammaS binding, was not affected. This suggests that Ca(2+)/CaM may alter the mode of group III mGluR signaling from mono- (alpha) to bidirectional (alpha and betagamma) activation of downstream effector cascades.

Amino Acid Sequence↗
PubMed1992

During negative regulation of the human papillomavirus-16 E6 promoter, the viral E2 protein can displace Sp1 from a proximal promoter element.

The principal early promoter of human papillomaviruses (HPVs), designated P97 in the case of HPV-16, contains four characteristically aligned cis-responsive elements, namely one binding site for Sp1, two for the viral E2 proteins, and the TATA box. The Sp1 binding site is needed to mediate activation of P97 by the remote epithelial-specific enhancer, and the two E2 binding sites contribute to a negative feedback-loop of viral gene expression. The Sp1 consensus motif and the TATA-box distal E2 binding site are spaced in all genital papillomaviruses by a single nucleotide. We show here that at physiological concentrations, the binding of E2 proteins and Sp1 are mutually exclusive events, since a bandshift analysis with nuclear extracts from ID13, a mouse cell line transformed by BPV-1, showed only the E2 or the Sp1 bandshift, but no complex indicative of the concomitant binding of both factors. Increasing concentrations of in vitro translated E2 protein compete efficiently with the Sp1 factor for binding to an oligonucleotide containing both binding sites. Interference between Sp1 and E2 protein binding is apparently relevant for P97 repression in vivo, since a mutational analysis revealed that both E2 binding sites are necessary for negative transcriptional regulation: Alone, neither the distal site, where E2 protein can induce Sp1 displacement, nor the proximal site, where E2 protein interferes with formation and function of the pre-initiation complex, have a significant effect, but two functional E2 binding sites lead to repression of P97.

Animals↗
PubMed1998

Genetic interactions and cell behaviour in blistered mutants during proliferation and differentiation of the Drosophila wing.

In this work, we analyse the blistered function in wing vein development by studying genetic mosaics of mutant cells, genetic interactions with other genes affecting vein development and blistered expression in several mutant backgrounds. blistered encodes for a nuclear protein homologous to the mammalian Serum Response Factor and is expressed in presumptive intervein cells of third larval instar and pupal wing discs. Clones of blistered mutant cells proliferate normally but tend to grow along veins and always differentiate as vein tissue. These observations indicate that vein-determined wing cells show a particular behaviour that is responsible for their allocation to vein regions. We observe strong genetic interactions between blistered, veinlet and genes of the Ras signaling cascade. During disc proliferation, blistered expression is under the control of the Ras signal transduction pathway, but its expression is independent of veinlet. During the pupal period, blistered and veinlet expression become interdependent and mutually exclusive. These results link the activity of the Ras pathway to the process of early determination of intervein cells, by the transcriptional control of the blistered nuclear factor.

Animals↗
PubMed2003

Pathogenesis of selective expansion of PNH clones.

Hemolysis, a characteristic of paroxysmal nocturnal hemoglobinuria (PNH), is caused by the expansion of an affected stem cell with a mutation of the PIG-A gene. Increasing evidence has shown that the presence of the PIG-A mutation alone does not induce the expansion. Two theories have been proposed. One, the growth advantage hypothesis, is supported by current data indicating the presence of several intrinsic alterations that might confer a proliferative advantage to PNH clones over normal cells. Alternatively, the PIG-A mutation might confer a relative survival advantage to PNH clones. This theory is supported by clinical observation indicating that PIG-A mutant cells survive immune-mediated bone marrow injury in patients with aplastic anemia, PNH, and myelodysplastic syndromes. The latter theory is also supported by current experimental data indicating that PIG-A mutant cells are relatively resistant to cytotoxic attack by natural killer cells and cytotoxic T-lymphocytes. The 2 theories appear complementary rather than mutually exclusive. Rapid progress in this field can be expected in the near future.

Cell Division↗
PubMed1993

Functional stoichiometry of Shaker potassium channel inactivation.

Shaker potassium channels from Drosophila are composed of four identical subunits. The contribution of a single subunit to the inactivation gating transition was investigated. Channels carrying a specific mutation in a single subunit can be labeled in a heterogeneous population and studied quantitatively with scorpion toxin sensitivity as a selection tag. Linkage within a single subunit of a mutation that removes the inactivation gate to a second mutation that affects scorpion toxin sensitivity demonstrates that only a single gate is necessary to produce inactivation. The inactivation rate constant for channels with a single gate was one-fourth that of channels with four gates. In contrast, the rate of recovery from inactivation was independent of the number of gates. It appears that each of the four open inactivation gates in a Shaker potassium channel is independent, but only one of the four gates closes in a mutually exclusive manner.

Animals↗
PubMed2005

The genesis of Barrett esophagus: has a histologic transition from gastroesophageal reflux disease-damaged epithelium to columnar metaplasia ever been seen in humans?

Has a histologic transition from gastroesophageal reflux disease-damaged epithelium to columnar metaplasia ever been seen in humans? The answer to this question seems to be that it has but that we either do not readily recognize it or it is not readily recognizable with regular light microscopy. There are at least 3 possible mechanisms for the genesis of Barrett esophagus. The first is ulceration at the gastroesophageal junction with subsequent repair by an epithelium that differentiates into Barrett epithelium. The second is metaplasia through multilayered epithelium. The third is creeping columnar metaplasia at the Z-line proximally followed by intestinalization. These 3 hypotheses may not be mutually exclusive, and all may be operative, depending on the local circumstances, amount of inflammation, erosion, ulcers, healing, acid and alkaline reflux, and use of proton pump inhibitors. Any of the epithelial types involved could be stable and not progress. They might even be reversible, which may also in part explain the mosaic of epithelial types that typify Barrett esophagus, and may be modified by any of the molecular mechanisms that turn protein transcription on and off (eg, promoter methylation, mutations). These mechanisms ultimately may also be involved in the genesis of neoplastic transformation.

Barrett Esophagus↗
PubMed1997

Transposable elements as sources of variation in animals and plants.

A tremendous wealth of data is accumulating on the variety and distribution of transposable elements (TEs) in natural populations. There is little doubt that TEs provide new genetic variation on a scale, and with a degree of sophistication, previously unimagined. There are many examples of mutations and other types of genetic variation associated with the activity of mobile elements. Mutant phenotypes range from subtle changes in tissue specificity to dramatic alterations in the development and organization of tissues and organs. Such changes can occur because of insertions in coding regions, but the more sophisticated TE-mediated changes are more often the result of insertions into 5' flanking regions and introns. Here, TE-induced variation is viewed from three evolutionary perspectives that are not mutually exclusive. First, variation resulting from the intrinsic parasitic nature of TE activity is examined. Second, we describe possible coadaptations between elements and their hosts that appear to have evolved because of selection to reduce the deleterious effects of new insertions on host fitness. Finally, some possible cases are explored in which the capacity of TEs to generate variation has been exploited by their hosts. The number of well documented cases in which element sequences appear to confer useful traits on the host, although small, is growing rapidly.

Adaptation, Physiological↗
PubMed1997

What is a bona fide mating-type gene? Internuclear complementation of mat mutants in Podospora anserina.

In the heterothallic ascomycete Podospora anserina, the mating-type locus is occupied by two mutually exclusive sequences termed mat+ and mat-. The mat+ sequence contains only one gene, FPR1, while the mat- sequence contains three genes: FMR1, SMR1 and SMR2. Previous studies have demonstrated that FPR1 and FMR1 are required for fertilization. Further analyses have led to the hypothesis that mat+ and mat- genes establish a mat+ and mat- nuclear identity, allowing recognition between nuclei of opposite mating type within the syncytial cells formed after fertilization. This hypothesis was based on the phenotypes of strains bearing mutations in ectopic mat genes. Here we present an analysis of mutations in resident mat- genes which suggests that, unlike FMR1 and SMR2, SMR1 is not involved in establishing nuclear identity. In fact, mutations in these two genes impair nuclear recognition, leading to uniparental progeny, while mutations in SMR1 block the sexual process, probably at a step after nuclear recognition. The nuclear identity hypothesis has also been tested through internuclear complementation tests. In these experiments, the mat- mutants were crossed with a mat+ strain carrying the wild-type mat- genes. Our rationale was that internuclear complementation should not be possible for nuclear identity genes: the relevant genes should show nucleus-restricted expression, and diffusion of their products to other nuclei should not occur. This test confirmed that SMR1 is not a bona fide mat gene since it can fulfill its function whatever its location, in either a mat- or a mat+ nucleus, and even when present in both nuclei. SMR2, but not FMR1, behaves like a nuclear identity gene with respect to internuclear complementation tests. A model is proposed that tentatively explains the ambiguous behaviour of the FMR1 gene and clarifies the respective functions of the three mat- proteins.

Alleles↗
PubMed2002

The molecular determinants of ionic regulatory differences between brain and kidney Na+/Ca2+ exchanger (NCX1) isoforms.

The Na(+)/Ca(2+) exchanger gene NCX1 undergoes alternative splicing leading to several isoforms that differ in a small portion of the large cytoplasmic loop. This loop is involved in many regulatory processes of NCX1, including ionic regulation by the transported substrates Na(+) and Ca(2+). High intracellular Ca(2+) can alleviate intracellular Na(+)-dependent inactivation in exon A (NCX1.4)-containing isoforms but not in those containing the mutually exclusive exon B (NCX1.3). Giant excised patches from Xenopus oocytes expressing various NCX1 constructs were used to examine the specific amino acids responsible for these observed regulatory differences. Using a chimeric approach, the region responsible was narrowed down to the small central part of exon A (IDDEEYEKNKTF). Replacing the second aspartic acid of this sequence with arginine (the corresponding amino acid in exon B) in an exon A background completely prevented the effect of Ca(2+) on intracellular Na(+)-dependent inactivation. Mutating the second lysine to cysteine (exon B) had a similar, but only partial, effect. The converse double mutant, but neither single mutation alone, introduced into an exon B background (arginine to aspartic acid and cysteine to lysine) was able to restore the NCX1.4 regulatory phenotype. These data demonstrate that aspartic acid 610 and lysine 617 (using the rat NCX1.4 numbering scheme) are critical molecular determinants of the unique Ca(2+) regulatory properties of NCX1.4.

Amino Acid Sequence↗
PubMed2005

Analysis of Paramecium tetraurelia A-51 surface antigen gene mutants reveals positive-feedback mechanisms for maintenance of expression and temperature-induced activation.

In Paramecium tetraurelia, variable surface antigen loci show mutually exclusive expression which is controlled primarily at the transcriptional level. Clonally stable expression of a single antigen has attracted models involving self-regulation by their gene products. However, direct demonstration of self-feedback at the molecular level has been complicated due to the inability to separate the functional gene from its product as well as copy number effects associated with injected extrachromosomal DNA in the polygenomic somatic nucleus. In this study, we exploited several germ line termination and frameshift mutations in the A-51 surface antigen gene to analyze variable surface antigen expression. These mutant alleles have the same copy number as the wild-type allele and therefore eliminate possible copy number effects. The mutant alleles were not transcribed at 27 degrees C, consistent with positive-feedback models for gene expression. However, further analysis showed that high temperatures (34 degrees C) induced transcription of the mutant A genes even in the presence of a different antigen on the cell surface. Thus, transcription was temperature dependent. Unlike wild-type cells, transcription of the mutant A genes at high temperatures was not maintained after temperature shift back to 27 degrees C in homozygous mutant cells. Importantly, transcription of the mutant allele was maintained at 27 degrees C in heterozygous cells with one copy of the wild-type allele. These results indicate that expression of the wild-type gene is required to stabilize its own transcriptional state at 27 degrees C.

Amino Acid Sequence↗
PubMed1991

In vitro analysis of the rat liver-type arginase promoter.

Genes for urea cycle enzymes including liver-type arginase are expressed mainly in the liver and are regulated developmentally, nutritionally, and hormonally in a coordinated manner. The promoter region of the rat arginase gene was investigated with an in vitro transcription system using nuclear extracts prepared from rat tissues. Accurate initiation of the transcription in liver nuclear extracts was confirmed by runoff analysis and S1 nuclease mapping. The arginase promoter was transcribed more efficiently in liver nuclear extracts than in brain extracts, reproducing the in vivo tissue specificity qualitatively. Analysis of deletion mutants of the 5'-flanking region in liver nuclear extracts revealed a positive regulatory region spanning nucleotides -90 to -51 relative to the transcription start site. Overlapping this region, two protected areas were detected by DNase I footprinting. Competition analysis with synthetic oligonucleotides showed that the more downstream protected area was occupied, in a mutually exclusive manner, by two factors each related to CTF/NF-1 and Sp1. The other more upstream protected area was recognized by a factor related to the liver-enriched transcription factor C/EBP, which was recently shown to interact with regulatory regions of two other urea cycle enzyme genes.

Animals↗