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At least 631 records · Page 35Linked to original sources
PubMed1994

Oligo(A) sequences of human respiratory syncytial virus G protein gene: assessment of their genetic stability in frameshift mutants.

We have described previously antibody-resistant mutants of the human respiratory syncytial virus Long strain that contained frameshift changes generated by deletions or insertions of a single adenosine in oligo(A) tracts (mRNA sense) of the G protein gene. Since these mutations introduced drastic structural and antigenic changes in the G protein C-terminal third, we decided to test the mutant stability by passaging the viruses in either the presence or the absence of selective antibody. Two such mutants (R63/1/2/3 and R63/2/4/8), with a single reading frame shift, reverted after a few passages in the absence of antibody to the wild-type genotype, by insertion of an A at the same homopolymeric tract as in the original deletion. In contrast, a double frameshift mutant (R63/2/4/1), generated by deletion of an A after nucleotide 623 and insertion of another A seven triplets later, was stably maintained after passage in either the absence or the presence of antibody. The stability of this mutant was manifested in its capacity to gradually displace the Long strain from mixed infections and by the fact that mutant R63/2/4/8 acquired the genotype of R63/2/4/1 after several passages in the presence of antibody. These results were indicative of genetic instability in the oligo(A) tract length of certain G protein mutants, which resulted in frameshift changes. The frequency of such errors among the viral RNA population obtained from a single infectious cycle was estimated to be lower than 1%. The relevance of these results for respiratory syncytial virus evolution is discussed.

Antibodies, Monoclonal↗
PubMed2004

Triplex mediated delivery of a platinum complex to a specific DNA target site.

Tethering an ethylene diamine linker to the 5' terminus of an oligothymidine sequence provides a site for complexation with K(2)PtCl(4). Due to the low reactivity of dT toward a platinum source, we chose dT(8) and dT(15) as our initial synthetic targets for platination. Post-synthetic reaction of the platinum reagent with the diamino oligothymidine generates the diamino dichloro platinum-DNA conjugate that can be used for DNA duplex targeting by oligodeoxyncleotide-mediated triplex formation. The dT(8) sequence is not sufficiently long to facilitate triplex formation and Pt-cross-linking, whereas with a dT(15) sequence cross-linking between the third strand and the duplex occurs exclusively with the duplex target strand directly involved in triplex formation. No examples of cross-linking to the complementary target strand, or of cross-linking to both target strands are observed. Most efficient cross-linking occurs when the dinucleotide d(GpG) is present in the target strand and no cross-linking occurs with the corresponding 7-deazaG dinucleotide target. Cross-linking is also observed when dC or dA residues are present in the target strand, or even with a single dG residue, but it is not observed in any cases to dT residues. Triplex formation provides the ability to target specific sequences of double-stranded DNA and the orientational control arising from triplex formation is sufficient to alter the binding preferences of platinum. Conjugates of the type described here offer the potential of delivering a platinum complex to a specific DNA site.

Base Sequence↗
PubMed2003

Correction of an enzyme trafficking defect in hereditary kidney stone disease in vitro.

In normal human hepatocytes, the intermediary-metabolic enzyme alanine:glyoxylate aminotransferase (AGT) is located within the peroxisomes. However, in approx. one-third of patients suffering from the hereditary kidney stone disease primary hyperoxaluria type 1, AGT is mistargeted to the mitochondria. AGT mistargeting results from the synergistic interaction between a common P11L (Pro11-->Leu) polymorphism and a disease-specific G170R mutation. The polymorphism generates a functionally weak mitochondrial targeting sequence, the efficiency of which is increased by the mutation. The two substitutions together, but not in isolation, inhibit AGT dimerization, highlighting the different structural requirements of the peroxisomal and mitochondrial protein-import machineries. In the present study, we show that treatments known to increase the stability of proteins non-specifically (i.e. lowering the temperature from 37 to 30 degrees C or by the addition of glycerol) completely normalize the intracellular targeting of mutant AGT expressed in transfected COS cells. On the other hand, treatments known to decrease protein stability (e.g. increasing the temperature from 37 to 42 degrees C) exacerbate the targeting defect. Neither of the treatments affects the relative efficiencies of the peroxisomal and mitochondrial protein-import pathways intrinsically. Results are discussed in the light of the known structural requirements of the two protein trafficking pathways and the formulation of possible treatment strategies for primary hyperoxaluria type 1.

Alleles↗
PubMed2004

A third base pair for the polymerase chain reaction: inserting isoC and isoG.

Two additional bases (isoguanosine and isocytosine), generating a third base pair, have been implemented in PCR. Enzyme fidelity for the third base pair is demonstrated using molecular thermodynamic melting, chemical cleavage and molecular beacons. When amplifying as few as 15 targets containing multiple non-natural base pairs with 40 cycles of amplification, our results confirm sequence conservation. The additional sequence space provided by three base pairs allows for the construction of molecular tools that achieve higher complexity and better discrimination than those possible with natural DNA alone.

Adenosine↗
PubMed1998

Structure and genetic stability of mitochondrial genomes vary among yeasts of the genus Saccharomyces.

Several yeast species/isolates belonging to the genus Saccharomyces were examined for the organization of their mtDNAs and ability to generate petite mutants. A general characteristic for all of the mtDNAs tested was that they were very A+T-rich. However, restriction patterns and inducibility of petite mutations revealed a great diversity in the organization and genetic behaviour of mtDNAs. One group of yeasts, Saccharomyces sensu stricto, contains mtDNA ranging in size from 64 to 85 kb. mtDNAs form these yeasts contain a high number of restriction sites that are recognized by the enzymes Haelll and Mspl, which cut specifically in G+C clusters. There are three to nine ori/rep sequences per genome. These yeasts spontaneously generate respiration deficient mutants. Ethidium bromide (Et-Br), at low concentrations, induces a majority of cells to give rise to petites. A second group of yeasts, Saccharomyces sensu lato, contains smaller mtDNAs, ranging in size from 23 to 48 kb, and probably only a few intergenic G+C clusters and no ori/rep sequences. These yeasts also generate petite clones spontaneously. but Et-Br, even when present at high concentrations, does not substantially increase the frequency of petites. In most petite clones from these yeasts only a small fragment of the wild-type molecule is retained and apparently multiplied. A third group, represented by Saccharomyces kluyveri, does not give rise to petite mutants either spontaneously or after induction.

Base Composition↗
PubMed1995

Factor XIIIA Calgary: a candidate missense mutation (Leu667Pro) in the beta barrel 2 domain of the factor XIIIA subunit.

Molecular analysis performed on a Canadian family with congenital factor XIII deficiency revealed a homozygous missense mutation (Leu667Pro) in exon 14 of the A subunit gene in three affected siblings. The mutation results from a T-to-C transition at nucleotide position 2087 and generates a new Msp1 restriction site. Digestion of an amplified fragment containing exon 14 with this restriction enzyme enabled the heterozygous allele to be identified in both parents (who were third cousins) and three other family members. SSCP analysis detected no additional mutations in the coding or consensus splice sequences of the A subunit gene. The mutant nucleotide substitution was absent in 60 normal alleles and 10 unrelated patients with XIIIA deficiency. Leu667 is located in the carboxyl terminal beta barrel 2 domain of the A subunit molecule. Computer modelling based on 3D crystallographic data predicts that the mutant protein has aberrant folding and is likely to be rapidly degraded following translation.

Factor VIIIa↗
PubMed2000

Molecular changes in the D-bifunctional protein cDNA sequence in Australasian patients belonging to the bifunctional protein complementation group.

The cDNA sequence for the human D-bifunctional protein (D-BP: 17 beta-hydroxysteroid dehydrogenase IV) was investigated in patients with peroxisomal disorders belonging to the BP complementation group (CG). In three cases, analysis of polymerase chain reaction products generated from the patients' cDNA indicated the presence of a deletion within the region corresponding to nucleotides 209-537 of the normal cDNA sequence. Subsequent sequencing revealed that, in two of the patients, 47 base pairs were missing, with the deletion corresponding to nucleotides 302/3-349/50 of the normal sequence. In the third patient, a smaller deletion of 22 bp (nucleotides 280/1-302/3) was characterized. Only the mutant sequence was detected in each of these cases, consistent with parental consanguinity. Both deletions cause a frameshift, and would lead to premature termination of the BP. Available family members were also investigated, and the findings conformed with expectations for an autosomal recessive disorder. In addition to the deletions, a number of other base changes have been identified in this series of patients. In particular, one patient, whose parents were also consanguineous, was homozygous for a base change, which results in a nonconservative substitution of serine 177 with a phenylalanine residue. The functional significance of this amino acid substitution, as well as the other identified changes, is still to be determined. Nevertheless, our data provide strong support for the hypothesis that defects in the gene for the D-BP are responsible for the beta-oxidation defect in patients belonging to the BP CG.

17-Hydroxysteroid Dehydrogenases↗
PubMed2001

Molecular mechanisms of microheterogeneity-induced defect formation in ferritin crystallization.

We apply in situ atomic force microscopy to the crystallization of ferritins from solutions containing approximately 5% (w/w) of their inherent molecular dimers. Molecular resolution imaging shows that the dimers consist of two bound monomers. The constituent monomers are likely partially denatured, resulting in increased hydrophobicity of the dimer surface. Correspondingly, the dimers strongly adsorb on the crystal surface. The adsorbed dimers hinder step growth and on incorporation by the crystal initiate stacks of up to 10 triple and single vacancies in the subsequent crystal layers. The molecules around the vacancies are shifted by approximately 0.1 molecular dimensions from their crystallographic positions. The shifts strain the lattice and, as a consequence, at crystal sizes > 200 microm, the accumulated strain is resolved by a plastic deformation whereupon the crystal breaks into mosaic blocks 20-50 microm in size. The critical size for the onset of mosaicity is similar for ferritin and apoferritin and close to the value for a third protein, lysozyme; it also agrees with theoretical predictions. Trapped microcrystals in ferritin and apoferritin induce strain with a characteristic length scale equal to that of a single point defect, and, as a consequence, trapping does not contribute to the mosaicity. The sequence of undesired phenomena that include heterogeneity generation, adsorption, incorporation, and the resulting lattice strain and mosaicity in this and other proteins systems, could be avoided by improved methods to separate similar proteins species (microheterogeneity) or by increasing the biochemical stability of the macromolecules against oligomerization.

Adsorption↗
PubMed2003

Decision-making strategies by panic disorder subjects are more sensitive to errors.

BACKGROUND: Decision-making is a complex process, which can be assessed experimentally by the two-choice prediction task. Error-rate, i.e. the frequency of incorrect predictions during this task, is an important factor for the response selection during decision-making. This investigation examined whether the frequency of incorrect predictions has an augmented effect on the number of different strategies underlying decision-making in patients with panic disorder. METHODS: Patients with a DSM-IV diagnosis of panic disorder (PD; N=18), unipolar major depressive disorder (MDD; N=18) and normal comparison subjects (C; N=35) were tested with the two-choice prediction task using three error-rate conditions (20, 50, or 80%). The dynamical entropy of the response sequences was used to quantify the number of different strategies generated during the different error-rates. RESULTS: At 20% error-rates, panic disorder subjects when compared to MDD and C subjects generated more strategies and switched more frequently between strategies as measured by the dynamical entropy and the range of local dynamical entropies. Response bias measures during the two-choice prediction task and post-test self-assessment did not differ between panic disorder subjects and MDD or C subjects. LIMITATIONS: First, panic disorder subjects were medicated. Second, the frequency and intensity of panic attacks and the degree of avoidance behaviors, was not assessed. Third, subjects were tested once only. CONCLUSIONS: Panic disorder subjects show uniformly high response sequence unpredictability in the presence of low error-rates, which is consistent with continued search for an optimal response strategy even when the error-rate is low.

Adult↗
PubMed1988

Base sequence damage in DNA from X-irradiated monkey CV-1 cells.

Two kinds of 3'-ends were detected in DNA scission fragments of highly repetitive primate component alpha DNA which were isolated from irradiated monkey CV-1 cells. The fragments' 3'-ends were characterized by 5'-32P-end labelling the DNA, followed by examination in high-resolution polyacrylamide gels under denaturing conditions. Hydrolysis of the labelled fragments' termini with exonuclease III of E. coli or by the 3'-phosphatase activity of T4 polynucleotide kinase generated a third, slowest migrating species in each mobility size class. Reference to mobility size class standards makes it highly probable that the fragment ends generated by X-rays in cells are 3'-phosphoryl and 3'-phosphoglycolate, and that they are converted to slower migrating fragments with 3'-OH ends, similar to results obtained with DNA irradiated in water (Henner et al. 1982, 1983 a, b). Densitometer measurements of gel autoradiograms showed that X-ray induction of DNA fragments with 3'-phosphoryl and 3'-phosphoglycolate ends was dose-dependent over a range 100-900 Gy. In CV-1 cells the frequency of single-strand breaks in alpha DNA was 8.6 x 10(-7) breaks/nt/Gy. The two kinds of ends disappeared in post-radiation incubation with a half-time of 1.6 h. These results provide a new means to study X-ray damage and repair of specific sequences in animal cells.

Animals↗
PubMed2003

Microarray analysis in the clinical management of cancer.

Because of the many genetic and epigenetic alterations that define a given tumor cell, and given the heterogeneity of these changes, between, as well as within a tumor class, progress in the understanding and treatment of cancer has been slow. The sequencing of the human genome, in combination with advances in robotics, computing, and imaging technologies, has resulted in rapid advances in the development of microarray methodology. This technology now places us in a position to simultaneously consider the consequence of all of these genetic changes through measurement of a large proportion of the complement of genes expressed in a given tissue at a given time. The power of this methodology for (1) the classification and identification of tumor classes, (2) gene discovery, (3) determining mechanisms of drug action, and (4) predicting drug response has now clearly been demonstrated: however, several challenges remain. First, there is a growing need for a standardization of the methodology, such that different datasets may be compared directly and meaningfully. The complete sequencing and annotation of the human genome may be the first step toward this attainable goal. Also, the databases generated should be made publicly available to facilitate further analysis by other researchers. To this end, the results of our studies are available at http://sequence.aecom.yu.edu/bioinf/Augenlicht/default.html. Second, a number of reported findings on tumor classification require further validation in independent patient data sets. Third, extensive clinical studies with appropriate patient follow-up are required to determine the validity of this method for the prediction of patient response to chemotherapy. Finally, the possibility needs to be considered that gene expression profiling may need to be combined with other global approaches such as proteomics and mutation screening analyses, for optimization of its potential. The advent of methodologies that enable gene expression profiling provides an opportunity to gain insights into the genetic makeup of a cancer cell on a global scale. Given the heterogeneity of this disease, such a global approach is likely to enhance significantly our understanding and management of this disease.

Gene Expression Profiling↗
PubMed1990

Mouse V lambda x gene sequence generates no junctional diversity and is conserved in mammalian species.

The lambda x, a new mouse Ig lambda L chain, is produced by rearrangement of the V lambda x, J lambda 2, and C lambda 2 gene segments. The V lambda x amino acid sequence is as divergent to other V lambda as to Vk gene sequences. Additionally, its third hypervariable region (CDR3) is four amino acids longer than those of all other variable gene segments of murine L chain. We have cloned and sequenced the germ-line V lambda x gene and found that the unexpected CDR3 length is encoded by the V lambda x gene. Junctional diversity is prevented by a TAA termination codon localized at the V lambda x 3' extremity. Moreover, we show a striking conservation of the V lambda x sequence in various mammalian species. Portions of the V lambda x sequence display more than 70% of nucleotide sequence identity with rabbit and human variable regions. These results suggest that V lambda x predated the divergence of mammalian species.

Amino Acid Sequence↗
PubMed1985

Nucleotide sequence of the birA gene encoding the biotin operon repressor and biotin holoenzyme synthetase functions of Escherichia coli.

A 2.2-kb region of DNA containing the birA gene of Escherichia coli has been sequenced. The birA gene sequence predicts a 35.3-kDal [321 amino acids (aa)] bifunctional protein containing biotin-operon-repressor and biotin-holoenzyme-synthetase activities. Mutations, generated by random insertion of XhoI linkers, defined the extent of the gene. Mutations affecting one or more of five discernable properties of birA [Barker, D. and Campbell, A., J. Bacteriol., 143 (1980) 789-800] were mapped. Three mutations that result in temperature-sensitive (ts) growth, birA85, birA215, and birA879 mapped in the N-terminal two-thirds of the protein. The birA352 mutation, which partially complements birA215 and birA879, maps in the N-terminal third of the protein. Finally, birA361 maps closest to the amino terminus.

Amino Acid Sequence↗
PubMed1996

Cloning and characterization of cDNAs encoding the epsilon-subunit of eukaryotic initiation factor-2B from rabbit and human.

A rabbit reticulocyte lysate cDNA library was screened with a polyclonal antiserum directed against eukaryotic initiation factor eIF-2B (eIF-2B). A 2508 base pair cDNA (pA1) was isolated and determined to encode the epsilon-subunit of eIF-2B based on the immunoreactivity of the fusion protein expressed from the cDNA in Escherichia coli and the presence of two peptide sequences obtained from two V8 fragments of purified nonrecombinant eIF-2B epsilon in the deduced amino acid sequence of the cDNA. The open reading frame of the cDNA began with the third nucleotide of the cDNA with the first AUG codon at nucleotide 522. Mutational analysis of pA1 indicated that the cDNA did not code for full-length eIF-2B epsilon. Seven missing codons of the reading-frame and the 71 nucleotide 5' non-coding region of the eIF-2B epsilon mRNA were obtained by 5' RACE. A human eIF-2B epsilon cDNA fragment, which corresponded to a similar 2.3 kb fragment generated by digestion of the rabbit pA1 cDNA with EcoRI, was isolated from a human histiocytic lymphoma (U-937) cell cDNA library constructed in lambda gt10. The nucleotide and amino acid sequences were highly conserved between the rabbit and human cDNAs, showing approx. 90% sequence identity within the open reading frame. Northern and Western blot analyses of reticulocyte lysate and other rabbit tissue extracts indicated that the eIF-2B epsilon polypeptide has a similar apparent molecular weight in all tissues examined, and is coded for by a single approximately 2.8 kilobase mRNA species which is ubiquitously expressed.

Amino Acid Sequence↗
PubMedDate not supplied

Conserved, erythropoietin-responsive VL30 promoters isolated from erythroid progenitor cells.

Virus-like 30S (VL30) elements are endogenous retro-elements of the mouse retrotransposon family. These elements are transcriptionally responsive in a temporal and tissue-specific manner due to the U3 promoter region of the elements' long terminal repeat (LTR). We have analyzed VL30 promoters from erythroid progenitor cell lines (MEL 585S and ELM-I-1) that contrasted in their response to erythropoietin (epo). Through RT-PCR-generated cDNAs, VL30 promoters were identified and showed homology to the third and fourth U3 subgroups, with GATA-1, Jak2/STAT5, and B10 RRE sites. One clone (ELM5) showed 97% homology to BVL-1, a putative epo-responsive VL30 element. In addition, a novel U3 promoter (MEL/ELM CONSTIT) showed complete sequence homology between both cell lines. Ribonuclease protection confirmed that epo-induced VL30 promoters were activated in ELM-I-1 cells, whereas the conserved VL30 MEL-ELM CONSTIT VL30 promoter showed no enhanced expression in the epo-unresponsive MEL cells. Identification of these U3 promoters suggests that VL30s are conserved and can be transcriptionally activated in an epo-specific manner.

Animals↗
PubMed2005

Transferable DHA-1 cephalosporinase in Escherichia coli.

Three Escherichia coli isolates resistant to third-generation cephalosporins but negative for extended-spectrum beta-lactamase production were isolated from hospitalised patients in Zagreb, Croatia, during June 2003 to February 2004. Resistance was due to the inducible production of a DHA-1 cephalosporinase. Each isolate contained an integron-associated bla(DHA-1)-ampR sequence carried by similar-sized plasmids, of which one was self-transferable. Serotyping and polymerase chain reaction typing using ERIC2 primer indicated that the isolates were distinct. This is the first description of DHA beta-lactamase production in E. coli.

Bacterial Proteins↗
PubMed2001

What do implantable cardioverter/defibrillators teach us about the mechanisms of sudden cardiac death?

Almost two decades of experience with the ICD have resulted in a better understanding of the nature of life-threatening ventricular tachyarrhythmias. The most pertinent achievements in this respect stem from clinical follow-up observations in patients with important clinical entities such as dilated cardiomyopathy or the Brugada syndrome. For instance, patients with dilated cardiomyopathy presenting with syncope and a negative electrophysiological study have been shown to have a high incidence of appropriate usage of their ICD. On the other hand, relative little has been gained from the extensive electrogram storage capabilities of third and fourth generation ICDs. Basically, careful evaluation of these electrograms has confirmed previous anecdotal data obtained from analysis of Holter recordings of patients who died suddenly while wearing the ECG recorder. In most instances, VT/VF episodes are triggered by relatively late-coupled premature beats whereas short--long--short sequences have been observed only in few patients. One future research avenue concerns the more detailed analysis of changes in cardiac autonomic tone preceding the occurrence of sustained ventricular tachyarrhythmias which can be assessed from the electrogram storage of the ICD.

Arrhythmias, Cardiac↗
PubMed1987

Primary structure of Paim I, an alpha-amylase inhibitor from Streptomyces corchorushii, determined by the combination of Edman degradation and fast atom bombardment mass spectrometry.

Paim I, a protein alpha-amylase inhibitor, inhibits animal alpha-amylases from pig, dog, cow, horse, etc. but has no activity against human salivary and pancreatic amylases. The primary structure of Paim I has been determined by Edman degradation and fast atom bombardment mass spectrometry (FABMS). This protein is a single-chain polypeptide of 73 amino acid residues with a calculated molecular weight from the sequence data of 7415.3 (monoisotopic molecular weight) and 7420.2 (average molecular weight). The sequencing strategy chosen for Paim I consists of four steps. First, the accurate molecular weights of the intact and tetra-S-carboxymethylated Paim I are determined by fast atom bombardment mass spectrometry. Second, the primary fragments generated by Staphylococcus aureus V8 protease are isolated by reversed-phase high-performance liquid chromatography. The molecular weights of these subpeptides are determined by FABMS. The peptides that must be sequenced are selected by the molecular weights of these subpeptides and the tetra-S-carboxymethylated Paim I. Third, these subpeptides and the whole protein are sequenced by automated Edman degradation. Finally, the primary structure of tetra-S-carboxymethylated Paim I is confirmed by the combination of tryptic, chymotryptic, and S. aureus V8 protease digestion and FABMS. The sequence of Paim I is compared with those of Haim II, Hoe-467A, Z-2685, and AI-3688 because they have different alpha-amylase inhibition spectra against mammalian alpha-amylases but belong to a family of related proteins.

Amino Acid Sequence↗