[Monitoring of patients with a cardiac pacemaker].
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Electrocardiography↗
Biomedical subjects
R Frank
Publications and source records attributed to R Frank.
The complete primary structure of GTP:AMP phosphotransferase from beef heart mitochondria.
To complete the amino acid sequence of GTP:AMP phosphotransferase (MgGTP + AMP in equilibrium with MgGDP + ADP) from beef heart mitochondria it was necessary to sequence an intermediate region of about 33 residues after position 102 [(1984) Eur. J. Biochem. 143, 331-339] and find a suitable overlap with the rest of the protein. The required peptides were obtained by cleaving the enzyme with endoproteinase Lys-C. One peptide, covering the region from residue 79 to 144, was sequenced up to residue 124. Another peptide, extending from residue 79 to 169, was subcleaved with Staphylococcus aureus V8 protease and provided the fragment from residue 99 to 139 which was sequenced. Several other peptides from endoproteinase Lys-C cleavage were used to check large sections of the previously published sequence work. The complete sequence contains 225 amino acids and has an Mr of 25 469.
Amino Acid Sequence↗
Role of thymidine residues in DNA recognition by the EcoRI and EcoRV restriction endonucleases.
We have synthesized a series of oligonucleotides containing the EcoRI (GAATTC) or EcoRV (GATATC) recognition site within which or adjacent to which thymidine was substituted by uridine or derivatives of uridine. The effects of these substitutions on the rate of the EcoRI and EcoRV catalyzed cleavage reaction were investigated. Our results show that most of the substitutions within the site are quite well tolerated by EcoRI, not, however, by EcoRV. We conclude that the thymin residues most likely are not directly involved in the recognition process of the EcoRI reaction. In contrast, they are major points of contact, between substrate and enzyme in the EcoRV reaction. The effects of substitutions in the position adjacent to the recognition site is also markedly different for EcoRI and EcoRV. Here, EcoRI seems to be considerably more selective than EcoRV.
Base Sequence↗
RNA-protein cross-linking in Escherichia coli ribosomal subunits: localization of sites on 16S RNA which are cross-linked to proteins S17 and S21 by treatment with 2-iminothiolane.
Treatment of E. coli ribosomal subunits with 2-iminothiolane coupled with mild ultraviolet irradiation leads to the formation of a large number of RNA-protein cross-links. In the case of the 30S subunit, a number of sites on 16S RNA that are cross-linked to proteins S7 and S8 by this procedure have already been identified (see ref. 6). Here, by using new or modified techniques for the partial digestion of the RNA and the subsequent isolation of the cross-linked RNA-protein complexes, three new iminothiolane cross-links have been localized: Protein S17 is cross-linked to the 16S RNA within an oligonucleotide encompassing positions 629-633, and protein S21 is cross-linked to two sites within oligonucleotides encompassing positions 723-724 and positions 1531-1542 (the 3'-end of the 16S RNA).
Cross-Linking Reagents↗
Translation arrest by oligodeoxynucleotides complementary to mRNA coding sequences yields polypeptides of predetermined length.
We investigated the arrest of mRNA translation at predetermined sites by oligodeoxynucleotides complementary to defined coding sequences within a mRNA. An in vitro transcription and a wheat germ cell-free translation system were used for the synthesis of mRNA and protein, respectively. Oligodeoxynucleotides (10-, 15- and 20-mer) arrested polypeptide synthesis in a concentration-dependent manner at the site of their hybridization to the mRNA, as judged by the size of the translation products. A 5-mer oligodeoxynucleotide did not prevent synthesis of the full length protein. Ribosomes arrested by an oligodeoxynucleotide transiently stacked up and eventually disassembled. Upon dissociation of the ribosomes from the blocked site, nascent chains were released as peptidyl-tRNAs which in turn became rapidly converted to free polypeptide chains. None of these results was affected by the position within the reading frame to which the 3' end of the oligodeoxynucleotide hybridized. The general applicability of translation arrest by oligodeoxynucleotides was demonstrated for different mRNAs. Only partial arrest of translation was obtained when oligodeoxynucleotides were used to arrest translation in the reticulocyte cell-free system.
Animals↗
Structure of gene and pseudogenes of human apoferritin H.
Ferritin is composed of two subunits, H and L. cDNA's coding for these proteins from human liver (1,2,3), lymphocytes (4) and from the monocyte-like cell line U937 (5) have been cloned and sequenced. Southern blot analysis on total human DNA reveals that there are many DNA segments hybridizing to the apoferritin H and L cDNA probes (1,2,4,6). In view of the tissue heterogeneity of ferritin molecules (7,8), it appeared possible that apoferritin molecules could be coded by a family of genes differentially expressed in various tissues (1,2). In this paper we describe the cloning and sequencing of the gene coding for human apoferritin H. This gene has three introns; the exon sequence is identical to that of cDNA's isolated from human liver, lymphocytes, HeLa cells and endothelial cells. In addition we show that at least 15 intronless pseudogenes exist, with features suggesting that they were originated by reverse transcription and insertion. On the basis of these results we conclude that only one gene is responsible for the synthesis of the majority of apoferritin H mRNA in various tissues examined, and that probably all the other DNA segments hybridizing with apoferritin cDNA are pseudogenes.
Apoferritins↗
Mitochondrial adenylate kinase (AK2) from bovine heart. The complete primary structure.
The sequence analysis of adenylate kinase isoenzyme 2 (AK2) was completed using a gas-phase sequencer constructed in our laboratory. The enzyme contains 238 amino acid residues in the following order: (sequence; see text) The four cysteine residues of AK2 were reinvestigated. Cys-41 and Cys-233 contain free thiols, which can be carboxymethylated in the intact protein without loss of enzymic activity. Chemical and model-building studies suggest that the pair Cys-43/Cys-93 forms a disulfide in native AK2. The relative molecular mass of AK2, as deduced from the sequence, is 26104. Other methods, including titration of -SH groups, sedimentation equilibrium ultracentrifugation and gel filtration yielded Mr values in the range from 26 000 to 31 500, each value depending on the respective method of determination. Bovine heart AK2 contains 44 residues more than the homologous isoenzyme AK1 (myokinase). As all but one single insertions and deletions cancel, the higher Mr of AK2 is due to 9 residues preceding the N terminus of AK1, a stretch of 30 residues in the middle of the molecule and 6 residues at the end. AK2 and AK1 are similar in their active-site geometry. In contrast, AK2 does not possess any of the three antigenic sites of AK1, which is consistent with the lack of immunological cross-reactivity between AK1 and AK2.
Adenylate Kinase↗
The primary structure of bovine chromogranin A: a representative of a class of acidic secretory proteins common to a variety of peptidergic cells.
We have determined the primary structure of bovine chromogranin A as a first step in the elucidation of the function of this widespread protein. After oligonucleotide screening of a cDNA library of bovine adrenal medulla, a clone (insert length 1.9 kb) containing the entire coding region for chromogranin A was isolated and sequenced. The authenticity of the sequence was verified by comparison with N-terminal, several internal, and C-terminal amino acid sequences as well as the amino acid composition of chromogranin A. The cDNA clone hybridized to an mRNA of 2.1 kb and, after in vitro transcription-translation, yielded a polypeptide with a similar electrophoretic mobility in SDS gels to chromogranin A. The polypeptide chain of chromogranin A comprises 431 amino acid residues, corresponding to an unmodified protein of 48 kd, and is preceded by a cleaved signal peptide of 18 amino acid residues. Interesting features of the chromogranin A structure include repeated clusters of glutamic acid residues, the occurrence of eight potential dibasic cleavage sites, six of which are located in the C-terminal domain, and the presence, in the N-terminal domain, of -Arg-Gly-Asp- (RGD), a three amino acid sequence involved in the binding of several constitutively secreted proteins to cell membranes.
Adrenal Medulla↗
The mom gene of bacteriophage Mu: the mechanism of methylation-dependent expression.
Transcription of the DNA modification gene (mom) of bacteriophage Mu requires methylation of three GATC sites upstream of the mom promoter by the Escherichia coli deoxyadenosine methylation function (Dam). The three sites map within a 40-bp segment termed region I. Small deletions, inversions, duplications and specific point mutations have been introduced in region I. Their effect on mom expression has been studied in dam+ and dam strains. Dam-dependent expression of the mom gene requires a specific arrangement of the three GATC sites and the presence of the methylated base in at least two of the three sites. We show that mom specific modification is regulated by a host protein. The Mom function is expressed in dam strains if they are defective in one component of the methylation-instructed mismatch correction system, mutH. We suggest that the product of mutH functions as a transcriptional repressor by binding to region I.
Base Sequence↗
Formation of the 3' end on U snRNAs requires at least three sequence elements.
The structural requirements for 3' end formation on the Xenopus laevis U1B snRNA gene have been studied. Three sequence elements are shown to be required. The first is a conserved sequence element found immediately 3' of all vertebrate U snRNA genes studied so far. The second is a gene internal sequence potentially capable of forming a stem-loop structure close to the 3' end of the RNA. The third element lies upstream of these, and may be part of the gene promoter. Experiments designed to investigate the mechanism of 3' end formation on primary U1B snRNA transcripts failed to find evidence for a processing event.
Acetyltransferases↗
The economic burden of depression.
This article provides estimates of direct treatment costs and indirect costs from lost productivity associated with the morbidity and mortality of depression. Data are based on epidemiologic estimates of the prevalence of major depressive illness and on the number of suicides assumed to be secondary to depression. The number of hospitalizations, hospital days, physician and mental health provider visits, home/nursing home costs, and pharmaceutical costs are estimated. The direct and indirect costs are estimated to be approximately $16.3 billion per year. These economic figures provide a lower-bound estimate of the full economic burden of major depression and further emphasize the need for timely recognition and treatment to potentially minimize the negative impact of the illness on society.
Absenteeism↗
The timing of paradoxical wall motion in ventricular aneurysms and in asynergic ventricles.
The timing of paradoxical wall motion was investigated in the left ventricular cineangiograms of 15 patients in whom ventricular aneurysm was diagnosed or excluded at surgery. Eight had aneurysm and 7 had asynergic ventricles without aneurysm. Areas of paradoxical motion and inward motion were planimetered in each quarter of ejection. In both aneurysmal and asynergic ventricles paradoxical motion occurred within large akinetic areas. The extent of paradoxical motion was small, constituting only 3.4 and 2.6% of end-systolic areas. Over 80% of the maximal paradoxical motion occurred in the first half of ejection. In the latter half of ejection, further changes in paradoxical motion were small and inconsistent. There were no significant differences in the extent of quarterly paradoxical wall motion between the two patient groups. These data suggest that in the presence of marked left ventricular asynergy, the extent and timing of paradoxical wall motion from cineangiograms may not be useful in detection ventricular aneurysm.
Cineangiography↗
Molecular cloning of the plasmid RP4 primase region in a multi-host-range tacP expression vector.
Plasmid RP4 primase was overproduced by utilizing autoregulated high-level expression vector systems in Escherichia coli and in four other Gram-negative bacterial species. Analysis of the products in E. coli revealed that in addition to the two primase polypeptides of 118 and 80 kDa the pri region of RP4 encodes two smaller proteins of 16.5 and 8.6 kDa. The transcript for the four RP4-specified products is polycistronic. The vector system used in E. coli is based on the plasmid pKK223-3 (Brosius and Holy, 1984), a ColE1-type replicon which contains a polylinker sequence flanked on one side by the controllable tac promoter and on the other side by two strong transcriptional terminators. The gene for the lac repressor (lacIQ) was inserted to render the use of the plasmid independent from repressor-overproducing strains. The gene cartridge essential for high-level expression and selection was combined with the RSF1010 replicon to generate a vector plasmid functioning in a wide variety of Gram-negative hosts. The versatility of the vector family was extended by constructing derivatives that contain the polylinker in inverted orientation relative to the tac promoter. Therefore, the orientation of the cloned fragment can be chosen by 'forced cloning' into the appropriately selected vector.
Cloning, Molecular↗
Overall and regional lung function in dogs exposed acutely to ozone.
Regional distribution of ventilation and overall function of the lungs were compared in anesthetized, paralyzed, mechanically ventilated dogs exposed to ozone. Three animals were exposed at each concentration (0,0.13,0.22, and 0.45 ppm ozone) for 3 hr. There were no significant changes in pulmonary flow resistance, dynamic compliance, or flow rates derived from maximum forced expiratory maneuvers. The distribution of inspired gas was determined using air containing 13N and a positron camera to follow wash-in in each of 80 or more regions of the lung. The mean wash-in time (T) and its standard deviation did not change in sham-treated dogs, but T increased by 40 to 100% in exposed animals. Dogs showing increases in T also had increased frequency dependence of compliance (correlation significant at P less than 0.05). The changes in 13N wash-in reflected less uniform distribution of ventilation, with the greatest disparity occurring between central and marginal regions. Altered regional mechanical function, in the absence of overall changes, suggested that the initial effect of ozone was on the small airways. The measurement may be a sensitive indicator of acute irritation in the lung periphery.
Animals↗
Predicting DNA duplex stability from the base sequence.
We report the complete thermodynamic library of all 10 Watson-Crick DNA nearest-neighbor interactions. We obtained the relevant thermodynamic data from calorimetric studies on 19 DNA oligomers and 9 DNA polymers. We show how these thermodynamic data can be used to calculate the stability and predict the temperature-dependent behavior of any DNA duplex structure from knowledge of its base sequence. We illustrate our method of calculation by using the nearest-neighbor data to predict transition enthalpies and free energies for a series of DNA oligomers. These predicted values are in excellent agreement with the corresponding values determined experimentally. This agreement demonstrates that a DNA duplex structure thermodynamically can be considered to be the sum of its nearest-neighbor interactions. Armed with this knowledge and the nearest-neighbor thermodynamic data reported here, scientists now will be able to predict the stability (delta G degree) and the melting behavior (delta H degree) of any DNA duplex structure from inspection of its primary sequence. This capability should prove valuable in numerous applications, such as predicting the stability of a probe-gene complex; selecting optimal conditions for a hybridization experiment; deciding on the minimum length of a probe; predicting the influence of a specific transversion or transition on the stability of an affected DNA region; and predicting the relative stabilities of local domains within a DNA duplex.
Base Composition↗
Localization of the exposed N-terminal region of the B800-850 alpha and beta light-harvesting polypeptides on the cytoplasmic surface of Rhodopseudomonas capsulata chromatophores.
Proteinase K and trypsin were used to determine the orientation of the light-harvesting B800-850 alpha and beta polypeptides within the chromatophores (inside-out membrane vesicles) of the mutant strain Y5 of Rhodopseudomonas capsulata. With proteinase K 7 amino acid residues of the B800-850 alpha polypeptide were cleaved off up to position Trp-7--Thr-8 of the N terminus, and 11 residues were cleaved off up to position Leu-11-Ser-12 of the beta chain N terminus. The C termini of the B800-850 alpha and beta polypeptides, including the hydrophobic transmembrane portions, remained intact. It is proposed that the N termini of the alpha and beta subunits, each containing one transmembrane alpha-helical span, are exposed on the cytoplasmic membrane surface and the C termini are exposed to or directed toward the periplasm.
Amino Acids↗
Structure and transforming function of transduced mutant alleles of the chicken c-myc gene.
A small retroviral vector carrying an oncogenic myc allele was isolated as a spontaneous variant (MH2E21) of avian oncovirus MH2. The MH2E21 genome, measuring only 2.3 kilobases, can be replicated like larger retroviral genomes and hence contains all cis-acting sequence elements essential for encapsidation and reverse transcription of retroviral RNA or for integration and transcription of proviral DNA. The MH2E21 genome contains 5' and 3' noncoding retroviral vector elements and a coding region comprising the first six codons of the viral gag gene and 417 v-myc codons. The gag-myc junction corresponds precisely to the presumed splice junction on subgenomic MH2 v-myc mRNA, the possible origin of MH2E21. Among the v-myc codons, the first 5 are derived from the noncoding 5' terminus of the second c-myc exon, and 412 codons correspond to the c-myc coding region. The predicted sequence of the MH2E21 protein product differs from that of the chicken c-myc protein by 11 additional amino-terminal residues and by 25 amino acid substitutions and a deletion of 4 residues within the shared domains. To investigate the functional significance of these structural changes, the MH2E21 genome was modified in vitro. The gag translational initiation codon was inactivated by oligonucleotide-directed mutagenesis. Furthermore, all but two of the missense mutations were reverted, and the deleted sequences were restored by replacing most of the MH2E21 v-myc allele by the corresponding segment of the CMII v-myc allele which is isogenic to c-myc in that region. The remaining two mutations have not been found in the v-myc alleles of avian oncoviruses MC29, CMII, and OK10. Like MH2 and MH2E21, modified MH2E21 (MH2E21m1c1) transforms avian embryo cells. Like c-myc, it encodes a 416-amino-acid protein initiated at the myc translational initiation codon. We conclude that neither major structural changes, such as in-frame fusion with virion genes or internal deletions, nor specific, if any, missense mutations of the c-myc coding region are necessary for activation of the basic oncogenic function of transduced myc alleles.
Alleles↗
A new covalently modified support for gas-liquid phase sequencing.
A new method for activation of glass fiber supports for immobilisation of proteins and peptides in gas-liquid phase sequencing is described. The new support offers several advantages over the presently used carrier polybrene: no precycling is required, initial yields are improved and background contamination is lower. This leads to an overall increase in detection sensitivity. The derivatisation method includes acid activation and subsequent covalent coating of glass fibers with quaternary ammonium groups thereby giving the glass surface a high binding capacity for both proteins and peptides. The activated glass has been successfully used for sequencing proteins and peptides isolated by HPLC as well as by electroelution from polyacrylamide gels.
Chromatography, Gas↗