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PubMed1993

An alternative Pit-1 RNA splicing product reveals modular binding and nonmodular transcriptional activities of the POU-specific domain.

We have identified a form of the pituitary-specific POU protein Pit-1 that results from deletion of the POU-specific (POUs) domain by alternative RNA splicing. This natural variant of Pit-1 (called delta 4Pit-1) has revealed several aspects of the function of the POUs domain. The delta 4Pit-1 protein was characterized using a delta 4Pit-1-specific antiserum. Further, selection assays of random oligonucleotide pools identified binding site preferences for both wild type and delta 4Pit-1. Methylation interference, copper phenanthrolene, and missing contact analyses were used to compare the binding characteristics of the two forms of Pit-1 on a selected site. DNA binding affinity assays on several DNA elements revealed that the POUs domain contains a modular DNA binding activity affecting the DNA binding affinity of the entire POU domain on some, but not on other, DNA sites. Functional analysis on such DNA elements has revealed that the POUs domain is an essential, but nonmodular, component of the Pit-1 trans-activation domain dependent on its natural context within the Pit-1 protein.

Amino Acid Sequence↗
PubMedDate not supplied

[A kinetic model of the cleavage of permutational variants of the antigenomic HDV-ribozyme].

The kinetic characteristics have been studied for noncircularly permuted variants of the human hepatitis delta virus (HDV) antigenomic ribozyme to find out the cause of the two-phase kinetics of the self-cleavage reaction. Different ways of reaction initiation, suboptimal conditions, and jumpwise changes of reaction conditions have been used, and the temperature dependences have been studied. A correlation has been shown between the apparent kinetic constant of the first reaction phase and the portion of the ribozyme molecules that self-cleaved during the first phase. Partial restoration of the initial reaction characteristics has been shown by the reinitiation of reaction being stopped after completing the first phase. On the basis of all the data obtained, a scheme of the self-cleavage reaction has been proposed including: (i) activation of the ribozyme with energy of 40-50 kcal/mol and a characteristic time of several deciminutes under optimal reaction conditions; (ii) fast and reversible reaction of the phosphodiester bond cleavage; (iii) reaction leading to isomerization of the 3',5'-phosphodiester bond to the 2',5' bond in the self-cleavage site with a characteristic activation time of tens of minutes; and (iv) practically irreversible conformational change leading to fixation of the cleavage by immobilization of the 5'-terminal nucleotide of the product in the center of the formed structure and displacement of the 3'-terminal nucleotide to the periphery. The latter process has a characteristic time of tens of minutes and a low activation energy.

Base Sequence↗
PubMed1994

Repression of AP-1-stimulated transcription by c-Ets-1.

The transcriptional activities of c-Ets-1 and v-Ets and their functional interaction with the AP-1 factor c-Jun were investigated. Several recombinant Ets proteins were produced and purified either from bacteria or from insect cells. Plasmid DNAs that contained the polyoma virus enhancer Ets/AP-1 element were used as templates for in vitro transcription assays in the presence of HeLa nuclear extract and various combinations of the Jun and Ets proteins. Under these conditions full-length c-Ets-1 on its own does not markedly influence transcription but abolishes the strong transcriptional stimulation normally elicited by Jun. This repression depends on the Ets-binding site and on specific features of c-Ets-1 structure, as both v-Ets and a natural splicing variant c-Ets-1 (delta VII) fail to inhibit Jun activity. These findings suggest that c-Ets may act both as a transcriptional repressor or activator depending on promoter context and splicing pattern.

Alternative Splicing↗
PubMed1999

Mutational analysis of the antigenomic trans-acting delta ribozyme: the alterations of the middle nucleotides located on the P1 stem.

Our previous report on delta ribozyme cleavage using a trans -acting antigenomic delta ribozyme and a collection of short substrates showed that the middle nucleotides of the P1 stem, the substrate binding site, are essential for the cleavage activity. Here we have further investigated the effect of alterations in the P1 stem on the kinetic and thermodynamic parameters of delta ribozyme cleavage using various ribozyme variants carrying single base mutations at putative positions reported. The kinetic and thermodynamic values obtained in mutational studies of the two middle nucleotides of the P1 stem suggest that the binding and active sites of the delta ribozyme are uniquely formed. Firstly, the substrate and the ribozyme are engaged in the formation of a helix, known as the P1 stem, which may contain a weak hydrogen bond(s) or a bulge. Secondly, a tertiary interaction involving the base moieties in the middle of the P1 stem likely plays a role in defining the chemical environment. As a con-sequence, the active site might form simultaneously or subsequently to the binding site during later steps of the pathway.

Binding Sites↗
PubMed1994

Biochemical and molecular characterization of a new sporadic glucose-6-phosphate dehydrogenase variant described in Italy: G6PD Modena.

A new glucose-6-phosphate dehydrogenase variant detected in an Italian man from the Po delta is described and designated as G6PD Modena. Biochemical characterization of the variant enzyme revealed an activity 21% of normal, a slow electrophoretic mobility, increased Km value for NADP, decreased Km value for G6P and a complete absence of NADPH inhibition, which could account for the apparently nonhaemolytic feature of this variant. The cloning and sequencing of the G6PD Modena allele showed a G-->C transition at nucleotide 844 in exon VIII causing a Asp-->His amino acid substitution. On the basis of biochemical characterization, G6PD Modena is classified as a genuine variant but it has the same mutation as G6PD Seattle-like.

Adult↗
PubMed1995

A C-terminal domain, which prevents secretion of the neuroendocrine protein 7B2 in Saccharomyces cerevisiae, inhibits Kex2 yet is processed by the Yap3 protease.

Recent reports reveal that the C-terminal half of the neuroendocrine polypeptide 7B2 selectively inhibits and binds PC2, a mammalian prohormone converting enzyme that is homologous to the yeast pro-alpha-factor processing protease Kex2. During attempted secretion of the 185 amino-acid human 7B2 in Saccharomyces cerevisiae, we observe that the protein is mostly retained inside the cell. However a mutant polypeptide (7B2 delta 1), where the C-terminal 48 amino acids of 7B2 are deleted, is efficiently secreted. Two shorter C-terminal truncations either permit poor secretion or no secretion at all. Surprisingly, full-length 7B2 but not 7B2 delta 1 abolishes the catalytic activity of Kex2, indicating that C-terminal residues of 7B2 might also be important for inhibition of the yeast protease. When the KEX2 gene is disrupted, yeast cells unexpectedly secrete a 7B2 variant similar in size to 7B2 delta 1, suggesting involvement of the alternate yeast prohormone convertase Yap3 in processing. Secretion is enhanced by overexpression of Yap3 and by the presence of a Lys-Arg residue at the processing site of precursor 7B2. These results purport that, in neuroendocrine cells too, secretion of 7B2 could be mediated by a homologue of Yap3.

Aspartic Acid Endopeptidases↗
PubMed2004

A mutational study in the transmembrane domain of Ccc2p, the yeast Cu(I)-ATPase, shows different roles for each Cys-Pro-Cys cysteine.

Ccc2p is homologous to the human Menkes and Wilson copper ATPases and is herein studied as a model for human copper transport. Most studies to date have sought to understand how mutations in the human Menkes or Wilson genes impair copper homeostasis and induce disease. Here we analyze whether eight conserved amino acids of the transmembrane domain are important for copper transport. Wild-type Ccc2p and variants were expressed in a ccc2-Delta yeast strain to check whether they were able to restore copper transport by complementation. Wild-type Ccc2p and variants were also expressed in Sf9 cells using baculovirus to study their enzymatic properties on membrane preparations. The latter system allowed us to measure a copper-activated ATPase activity of about 20 nmol/mg/min for the wild-type Ccc2p at 37 degrees C. None of the variants was as efficient as the wild type in restoring copper homeostasis. The mutation of each cysteine of the (583)CPC(585) motif into a serine resulted in nonfunctional proteins that could not restore copper homeostasis in yeast and had no ATPase activity. Phosphorylation by ATP was still possible with the C583S variant, although it was not possible with the C585S variant, suggesting that the cysteines of the CPC motif have a different role in copper transport. Cys(583) would be necessary for copper dissociation and/or enzyme dephosphorylation and Cys(585) would be necessary for ATP phosphorylation, suggesting a role in copper binding.

Adenosine Triphosphatases↗
PubMedDate not supplied

[The study of orthopoxvirus genes for Kelch-like proteins. II. Construction of cowpox virus variants with targeted gene deletions].

Integrative plasmids p delta C, p delta D, and p delta G were designed to contain a selective marker beyond the region of homology to virus DNA and to allow construction of recombinant cowpox viruses (CPV) that lack C18L, D11L, or G3L coding for kelch-like proteins. CPV mutants lacking one (C18L, D11L, or G3L), two (D11L/G3L or C18L/D11L), or three (D11L/G3L/C18L, that is, all) kelch-like protein genes of the left variable region of the virus genome were obtained. Impaired reproduction was observed for the triple mutant. Pocks produced by the triple mutant and the original virus differed in size and morphology. In addition, the two CPV variants differed in destructive changes caused in the chorioallantoic membrane of chicken embryos.

Animals↗
PubMed1995

3-D models of the antigenomic ribozyme of the hepatitis delta agent with eight new contacts suggested by sequence analysis of 188 cDNA clones.

We mapped 359 mutations at 25 positions in synthetic variants of the antigenomic ribozyme of the hepatitis delta agent by analyzing the sequences of 188 cDNA clones. These data were used to identify three features of the ribozyme: highly conserved nucleotides, positions with restricted nucleotide substitutions and three-dimensional relationships between nucleotides. The distribution of mutations at the 25 positions was as follows: G-11 (the eleventh nucleotide from the cleavage site) was mutated in 56 clones; G-12 in 36; U-15 in 33; C-13 in 26; G-28 in 23; C-27 in 21; C-29 in 19; U-26 in 17; C-18 in 14; A-14 in 13; C-16 in 13; C-19 in 12; U-17 in 11; A-20 in 10; G-42 in 9; G-40 in 7; G-41 in 7; C-24 in 6; U-32 in 6; U-23 in 5; C-25 in 4; C-21 in 3; G-30 in 3; G-31 in 3; C-22 in 1. All clones containing a mutation at C-25 had an A at this position, suggesting that the extra cyclic amino group present in adenine and cytosine may function during the cleavage event. Mutations at certain positions were common in simple clones (containing only one or two mutations), while mutations at other positions were over-represented in more complex clones. Both compensatory base changes and co-mutational frequencies were used to identify eight pairs of nucleotides which may interact with each other: G-11 and C-18, G-12 and C-27, C-13 and G-28, C-21 and U-23/C-24, C-21 and G-30, U-23 and G-31/U-32, C24 and G-30, C-27 and G-42. These pairs, which involve some of the most conserved positions in the molecule, suggest interactions among nucleotides previously depicted in open-loop structures. The newly proposed points of contact between pairs of nucleotides are compatible with both the axehead and pseudoknot secondary structural models and were combined with previously proposed Watson-Crick base paired helices to produce two three dimensional models. In both of these, C-25 and C-76 are placed near the cleavage site.

Base Sequence↗
PubMed1995

Structural analysis of the Bacillus subtilis delta factor: a protein polyanion which displaces RNA from RNA polymerase.

RNA polymerase from Bacillus subtilis is a complex mixture comprising a common core (beta beta' alpha 2), the 20.4 kDa delta (delta) protein, and of one of several sigma (sigma) specificity factors. The delta protein, together with several truncated variants, has been overproduced and purified from Escherichia coli. It is highly acidic (pI = 3.6) and contains two distinct regions, a 13 kDa amino-terminal domain with fairly uniform charge distribution and a glutamate and aspartate residue-rich carboxyl-terminal region. The purified amino-terminal domain (delta N) contains 32% alpha-helix and 16% beta-sheet, as judged by circular dichroism analysis. In contrast, an 8.5 kDa tryptic fragment containing the carboxyl-terminal region (delta C) is largely unstructured and highly charged (net charge of -47). RNA polymerase purified from a B. subtilis mutant with an insertion in the delta gene (rpoE::cat) contains a truncated delta protein, indicating that the amino-terminal domain is stable in vivo and contains a core-binding function. Addition of delta, but not sigma A or delta N, displaces RNA bound to RNA polymerase in a binary complex. The ability of delta to displace RNA efficiently requires the activities of both the amino-terminal core-binding domain and the polyanionic carboxyl-terminal region. Although delta C can also displace nucleic acids from RNA polymerase, this activity requires the addition of a large molar excess of protein and is relatively non specific in that both DNA and RNA are displaced. This suggests that the function of the amino-terminal domain is to bind and orient the carboxyl-terminal region on the surface of RNA polymerase.

Amino Acid Sequence↗
PubMed2006

CFTR 5T variant has a low penetrance in females that is partially attributable to its haplotype.

PURPOSE: The study's purpose was to understand the molecular basis for different clinical phenotypes of the 5T variant, a tract of 5 thymidines in intron 8 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which disrupts processing of CFTR mRNA and reduces synthesis from the corresponding CFTR alleles. METHOD: We analyzed the polymorphic TG dinucleotide repeat adjacent to the 5T variant in intron 8 and the codon 470 in exon 10. Patients selected for this study were positive for both the 5T variant and the major cystic fibrosis mutation, Delta F508. Almost all Delta F508 mutation alleles occur in a 10TG-9T-470M haplotype. Therefore, it is possible to determine the haplotype of the 5T variant in trans. RESULTS: Of the 74 samples analyzed, 41 (55%) were 11TG-5T-470M, 31 (42%) were 12TG-5T-470V, and 2 (3%) were 13TG-5T-470M. Of the 49 cases for which we had clinical information, 17.6% of females (6/34) and 66.7% of males (10/15) showed symptoms resembling atypical cystic fibrosis. The haplotype with the highest penetrance in females (42% or 5/12) and more than 80% (5/6) in males is 12TG-5T-470V. We also evaluated 12 males affected with congenital bilateral absence of vas deferens and positive for the 5T variant; 10 of 12 had the 12TG-5T-470V haplotype. CONCLUSION: Overall, the 5T variant has a milder clinical consequence than previously estimated in females. The clinical presentations of the 5T variant are associated with the 5T-12TG-470M haplotype.

Alleles↗
PubMed2005

delta2 opioid receptor agonist facilitates mean arterial pressure recovery after hemorrhage in conscious rats.

delta opioid receptor agonists exert potent hemodynamic effects under ischemic conditions. This study was designed to assess the cardiovascular effects of Deltorphin-D(variant) (Delt-D(var)), a selective delta(2) opioid receptor agonist, in conscious, freely moving male rats during the posthemorrhage, recompensatory phase of a hemorrhagic trauma. Rats were fitted with femoral arterial and venous catheters for measurements of mean arterial pressure (MAP), heart rate (HR), and intravenous (i.v.) injections of isotonic saline, 1 mg/kg Delt-D(var), or 2 mg/kg Delt-D(var). During hemorrhaging, 30% (approximately 5 mL) of total blood volume was collected from the arterial catheter. MAP-HR was fitted to a logistic equation to determine baroreceptor reflex properties. Hemorrhaged rats progressed through three distinct phases: compensation, decompensation, and recompensation. Saline and 1 mg/kg Delt-D(var) rats treated posthemorrhage had similar MAP and HR after hemorrhage. In contrast, 2 mg/kg Delt-D(var) administered after hemorrhaging led to a faster and more complete recovery of MAP than compared with the other groups. In hemorrhaged rats, the average HR gain (bpm/mmHg) after 2 mg/kg Delt-D(var) treatment was greater and the BP(50) (BP at one-half the HR range) was significantly lower than after saline treatment. The results show that after hemorrhage, during the recompensatory period, stimulation of delta(2) opioid receptors leads to improved MAP, and this recovery may involve a change in baroreflex sensitivity.

Amino Acid Sequence↗
PubMed1997

Hepatitis viruses: genetic variants and clinical significance.

Variants of hepatitis B, C, and delta virus have been identified in patients both with acute and chronic infections. In the hepatitis B virus genome, naturally occurring mutations have been found in all viral genes, most notably in the genes coding for the structural envelope and nucleocapsid proteins. In the hepatitis C virus genome, the regions coding for the structural envelope proteins E1 and E2, as well as the 3'-contiguous non-structural region NS1, were found to be hypervariable. Viral variants may be associated with a specific clinical course of the infection, e.g., acute, fulminant or chronic hepatitis. Specific mutations may reduce viral clearance by immune mechanisms ('vaccine escape' and 'immune escape'), response to antiviral therapy ('therapy escape'), as well as detection ('diagnosis escape'). The exact contribution, however, of specific mutations to the pathogenesis and natural course of hepatitis B, C, or delta virus infection, including hepatocellular carcinoma development, and the response to antiviral treatment remains to be established.

Antiviral Agents↗
PubMed1996

Fatty acid interactions with a helix-less variant of intestinal fatty acid-binding protein.

Intestinal fatty acid-binding protein (I-FABP) binds a single molecule of long-chain fatty acid in an enclosed cavity surrounded by two antiparallel beta-sheets. The structure also contains two short alpha-helices which form a cap over one end of the binding cavity adjacent to the methyl terminus of the fatty acid. In this study, we employed a helix-less variant of I-FABP known as delta 17-SG [Kim, K., Cistola, D.P.,& Frieden, C. (1996) Biochemistry 35, 7553-7558] to investigate the role of the helical region in maintaining the integrity of the binding cavity and mediating the acquisition of ligand. Fluorescence and NMR experiments were used to characterize the energetic, structural, and kinetic properties of fatty acid binding to this variant, and the results were compared and contrasted with those of wild-type I-FABP and a single-site mutant, R106T. Remarkably, oleate bound to delta 17-SG with a dissociation constant of 4.5 microM, a value comparable to that for R106T and approximately 20-100-fold higher than that for wild-type I-FABP. Heteronuclear two-dimensional NMR spectra for [2-13C]palmitate complexed with delta 17-SG revealed a pattern nearly identical to that observed for the wild-type protein, but distinct from that for R106T. In addition, the ionization behavior of bound [1-13C]palmitate and the nearest neighbor patterns for [2-13C]palmitate derived from 13C-filtered NOESY experiments were very similar for delta 17-SG and the wild-type protein. These results implied that the fatty acid-protein interactions characteristic of the carboxyl end of the fatty acid binding cavity in the wild-type protein were essentially intact in the helix-less variant. In contrast, 13C-filtered NOESY spectra of [16-13C]palmitate bound to delta 17-SG indicated that the fatty acid-protein interactions at the methyl end of the binding cavity were disrupted. As determined by stopped-flow fluorescence, the observed ligand association rates for both delta 17-SG and wild-type I-FABP increased with increasing oleate concentration, but only the wild-type protein exhibited a limiting value of 1000 s-1. This rate-limiting process was interpreted as a conformational change involving the helical region that allows the ligand access to the internal cavity. Simulation and fitting of the kinetic results yielded ligand association rates for delta 17-SG and wild-type I-FABP that were comparable. However, the dissociation rate for wild-type protein was 16-fold lower than that for delta 17-SG. We conclude that the alpha-helices of I_FABP are not required to maintain the integrity of the fatty acid binding cavity but may serve to regulate the affinity of fatty acid binding by selectively altering the dissociation rate constant. In this manner, conformational changes involving the alpha-helical domain may help control the transfer of fatty acids within the cell.

Amino Acid Sequence↗
PubMed2003

Angelman syndrome: difficulties in EEG pattern recognition and possible misinterpretations.

PURPOSE: This study aimed to evaluate the sensitivity of the EEG in Angelman syndrome (AS), to verify the age at onset of suggestive EEGs and to study EEG patterns, analyzing variations and comparing our findings with nomenclature previously used. METHODS: Seventy EEG and 15 V-EEGs of 26 patients were analyzed. Suggestive EEG patterns of AS were classified in delta pattern (DP), theta pattern (TP), and posterior discharges (PDs). Generic terms were used to simplify the analysis. RESULTS: Suggestive EEGs were observed in 25 (96.2%) patients. DP occurred in 22 patients with four variants-hypsarrhythmic-like: irregular, high-amplitude, generalized delta activity (DA) with multifocal epileptiform discharges (EDs); slow variant: regular, high-amplitude, generalized DA with rare EDs; ill-defined slow spike-and-wave: regular, high-amplitude, generalized DA with superimposed EDs characterizing a slow wave, with notched appearance; triphasic-like: rhythmic, moderate-amplitude DA over anterior regions with superimposed EDs. TP was observed in eight patients, as generalized or over the posterior regions. PDs were seen in 19 patients as runs of sharp waves or runs of high-amplitude slow waves with superimposed EDs. TP was the only age-related pattern (younger than 8 years) and observed only in patients with deletion. In 15 patients who had an EEG before the clinical diagnosis, 60% had a suggestive tracing. CONCLUSIONS: Although some EEG descriptions are not very detailed, and every author describes findings in a slightly different manner, obviously a common denominator must exist. In this context, EEG seems to be a very sensitive method for the diagnosis of AS, offering an opportunity to corroborate this etiologic diagnosis. Conversely, we do not believe that these patterns may be accounted as specific, except for the delta pattern, which seems to be extremely unusual in other syndromes. Other EEG patterns observed in AS, such as theta activity and PDs, occur in a wide variety of disorders. Nonetheless, their importance for the EEG diagnosis of AS is supported by the fact that they are associated with other features and may be helpful in a proper clinical setting.

Adolescent↗
PubMed2000

The specific expression of three novel splice variant forms of human metalloprotease-like disintegrin-like cysteine-rich protein 2 gene inBrain tissues and gliomas.

We have previously identified 67 exons on a yeast artificial chromosome contig spanning 1.5 Mb around the multidrug resistance 1 gene region of human chromosome 7q21.1. In this study, we identified three novel cytoplasmic variants (MDC2-gamma, MDC2-delta, and MDC2-epsilon) of the human metalloprotease-like disintegrin-like cysteine-rich protein 2 (MDC2) among these exons by screening a human brain cDNA library and also by using a reverse transcription polymerase chain reaction. Genomic sequence analysis strongly supported the idea that the variations in the cytoplasmic domain were generated by alternative splicing. The expression of MDC2 variant forms in human brain tissue and gliomas was examined by reverse transcription polymerase chain reaction and RNase protection assay. MDC2-epsilon was expressed only in the cortical and hippocampal regions in human brain, but not in gliomas. In contrast, MDC2-gamma was a major form expressed in human gliomas. Specific expression of these cytoplasmic variants of MDC2 in human brain and its malignancies is discussed.

ADAM Proteins↗
PubMed2001

Isolation of stable hemolysin and catalase variants of Staphylococcus aureus S6C includes one with an exoprotein-deficient phenotype.

Previous studies suggested that the exoprotein-deficient phenotype of a Delta 1058::Tn551 insertion/deletion mutant of Staphylococcus aureus S6C was not owing to the insertion/deletion event, but instead was owing to the inherent instability of the agrC gene during transduction of the Delta 1058::Tn551 region into S6C. The purpose of the following study was to examine S6C as a potential source of exoprotein-deficient mutants that would account for their appearance after transposition and transduction. Four stable variants of S6C were isolated that differed in their hemolysin and catalase activities. Surprisingly, the agr regulatory molecule, RNAIII, was undetectable in one of these variants, which most likely accounted for the exoprotein-deficient phenotype of this variant. When the original Delta 1058::Tn551 mutation was transduced into the hemolytic, catalase-positive variant of S6C, none of the transductants exhibited an exoprotein-deficient phenotype. These data suggest that, while the exoprotein-deficient phenotype of the S6C variant is most likely due to mutations in the agr regulatory system, these mutations are not caused by the transduction of the Delta 1058::Tn551 region into S6C, but instead already exist in an exoprotein-deficient variant of S6C.

Blotting, Northern↗
PubMed1996

Selection in vitro of trans-acting genomic human hepatitis delta virus (HDV) ribozymes.

In an effort to identify the functional structure as well as new active variants of the trans-acting genomic ribozyme of human hepatitis delta virus (HDV), we applied an in vitro selection procedure. A total of 14 rounds of selection and amplification was repeated and various mutant ribozymes in G10 and G14 pools analyzed. Active ribozymes which were isolated in the present study (from G10 and G14) all possessed conserved bases (that were identified earlier) in the cis-acting molecule. A dominant clone G10-68 variant was accumulated in generation 14. Interestingly, when base substitutions were analyzed in G10-68 variant, we found that this variant appears to be close to antigenome-like HDV ribozyme molecule. Further investigations of G10-68 confirmed that each mutated base was the most appropriate nucleotide at every position of the HDV ribozyme.

Base Sequence↗