Simulation of dephosphorylation curves by an ensemble of enzyme molecules with different rate constants.
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Kinetics↗
Biomedical subjects
D W Martin
Publications and source records attributed to D W Martin.
Mucoid Pseudomonas aeruginosa in cystic fibrosis: signal transduction and histone-like elements in the regulation of bacterial virulence.
The profuse production of the exopolysaccharide alginate results in mucoidy, a critical virulence factor expressed by Pseudomonas aeruginosa during chronic respiratory tract infections in cystic fibrosis. Studies of the regulation of this pathogenic determinant have unravelled at least two levels of control, including bacterial signal transduction systems and histone-like elements. Although only in its initial phase, an understanding of the dual control of mucoidy may help to illuminate adaptive processes that depend on the combination of these regulatory factors. Integration of specific signals transduced by the two-component systems with inputs generated by the general state of bacterial nucleoids may govern the expression of certain virulence determinants and provide a framework facilitating selection of phenotypes successful under particular environmental conditions and selective pressures.
Alginates↗
Analysis of the herpes simplex virus type 1 OriS sequence: mapping of functional domains.
The herpes simplex virus type 1 (HSV-1) OriS region resides within a 90-bp sequence that contains two binding sites for the origin-binding protein (OBP), designated sites I and II. A third presumptive OBP-binding site (III) within OriS has strong sequence similarity to sites I and II, but no sequence-specific OBP binding has yet been demonstrated at this site. We have generated mutations in sites I, II, and III and determined their replication efficiencies in a transient in vivo assay in the presence of a helper virus. Mutations in any one of the sites reduced DNA replication significantly. To study the role of OriS sequence elements in site I and the presumptive site III in DNA replication, we have also generated a series of mutations that span from site I across the presumptive binding site III. These mutants were tested for their ability to replicate and for the ability to bind OBP by using gel shift analyses. The results indicate that mutations across site I drastically reduce DNA replication. Triple-base-pair substitution mutations that fall within the crucial OBP-binding domain, 5'-YGYTCGCACT-3' (where Y represents C or T), show a reduced level of OBP binding and DNA replication. Substitution mutations in site I that are outside this crucial binding sequence show a more detrimental effect on DNA replication than on OBP binding. This suggests that these sequences are required for initiation of DNA replication but are not critical for OBP binding. Mutations across the presumptive OBP-binding site III also resulted in a loss in efficiency of DNA replication. These mutations influenced OBP binding to OriS in gel shift assays, even though the mutated sequences are not contained within known OBP-binding sites. Replacement of the wild-type site III with a perfect OBP-binding site I results in a drastic reduction of DNA replication. Thus, our DNA replication assays and in vitro DNA-binding studies suggest that the binding of the origin sequence by OBP is not the only determining factor for initiation of DNA replication in vivo.
Animals↗
High efficiency Ca2+ transport by the sarcoplasmic reticulum Ca2(+)-ATPase in the absence of the 53-kilodalton glycoprotein.
Although the Ca2(+)-ATPase is the predominant protein species of the skeletal sarcoplasmic reticulum membrane, the functional significance of other minor protein species remains unresolved. The proposition has been tested that the membrane-bound 53-kDa glycoprotein (GP-53) may be required or significantly involved in regulating the coupling of ATP hydrolysis to Ca2+ transport by the Ca2(+)-ATPase. Ca2(+)-ATPases originating from preparations with and without GP-53 were reconstituted into phosphatidylcholine liposomes, and Ca2+ uptake and pumping efficiency were determined. The reconstituted Ca2+ pump from all preparations transported Ca2+ with high efficiency (Ca2+:ATP greater than 1.5). The results demonstrate that GP-53 is not required to couple ATP hydrolysis to Ca2+ transport. Additionally, the observed high coupling efficiency is inconsistent with GP-53 functioning as a substantial positive regulator of coupling.
Adenosine Triphosphate↗
Mucoid Pseudomonas aeruginosa in cystic fibrosis: mutations in the muc loci affect transcription of the algR and algD genes in response to environmental stimuli.
Increased levels of alginate biosynthesis cause mucoidy in Pseudomonas aeruginosa, a virulence factor of particular importance in cystic fibrosis. The algR gene product, which controls transcription of a key alginate biosynthetic gene, algD, is homologous to the activator members of the two-component, environmentally responsive systems (NtrC, OmpR, PhoB, ArcA, etc). In this report, we show that mutations in the muc loci, (muc-2, muc-22, and muc-23, in the standard genetic P. aeruginosa strain PAO, as well as a mapped muc allele in an isolate from a cystic fibrosis patient) affect transcription of algD and algR. This influence was strongly dependent on environmental factors. Regulation by nitrogen was observed in all strains examined, but the absolute transcriptional levels, determining the mucoid or nonmucoid status, were strain (muc allele)-dependent. Increased concentrations of NaCl in the medium, an osmolyte which is elevated in cystic fibrosis lung secretions, resulted in an increased algD transcription and mucoid phenotype in a muc-2 strain; the same conditions, however, produced a nonmucoid phenotype in the muc-23 background and abolished algD transcription. Mutations in the muc loci may cause mucoidy by deregulating the normal response of the alginate system to environmental stimuli.
Alginates↗
Role of the far-upstream sites of the algD promoter and the algR and rpoN genes in environmental modulation of mucoidy in Pseudomonas aeruginosa.
The role of several regulatory elements in environmental modulation of mucoidy in Pseudomonas aeruginosa was studied. Transcriptional activation of algD, necessary for the mucoid phenotype, was found to depend on FUS, the newly identified far-upstream sites of the algD promoter. The FUS were delimited to a region spanning nucleotides -432 to -332 relative to the algD mRNA start site. Insertional inactivation of algR in PAO568 abolished the algD promoter response to nitrogen availability and greatly diminished but did not completely eliminate reactivity to changes in salt concentration. Insertional inactivation of rpoN (ntrA) in PAO568 did not affect algR and algD transcription.
Base Sequence↗
Insights into dolphin sonar discrimination capabilities from human listening experiments.
A variety of dolphin sonar discrimination experiments have been conducted, yet little is known about the cues utilized by dolphins in making fine target discriminations. In order to gain insights on cues available to echolocating dolphins, sonar discrimination experiments were conducted with human subjects using the same targets employed in dolphin experiments. When digital recordings of echoes from targets ensonified with a dolphinlike signal were played back at a slower rate to human subjects, they could also make fine target discriminations under controlled laboratory conditions about as well as dolphins under less controlled conditions. Subjects reported that time-separation-pitch and duration cues were important. They also reported that low-amplitude echo components 32 dB below the maximum echo component were usable. The signal-to-noise ratio had to be greater than 10 dB above the detection threshold for simple discrimination and 30 dB for difficult discrimination. Except for two cases in which spectral cues in the form of "click pitch" were important, subjects indicated that time-domain rather than frequency-domain processing seemed to be more relevant in analyzing the echoes.
Animals↗
Tau consists of a set of proteins with repeated C-terminal microtubule-binding domains and variable N-terminal domains.
Tau proteins consist of a family of proteins, heterogeneous in size, which associate with microtubules in vivo and are induced during neurite outgrowth. In humans, tau is one of the major components of the pathognomonic neurofibrillary tangles in Alzheimer's disease brain. Screening of a cDNA library prepared from bovine brain led to the isolation of several cDNA clones encoding tau proteins with different N termini and differing by insertions or deletions, suggesting differential splicing of the tau transcripts. One of the N-terminal domains and the repeated C-terminal domain of the encoded tau proteins are recognized by polyclonal antibodies to bovine tau. The bovine tau proteins are highly homologous to murine and human tau, especially within the repeated C-terminal domain. Compared with murine and human tau, bovine tau contains the insertion of three longer segments, one of which is an additional characteristic repeat. Portions of tau proteins generated by in vitro translation were used to show that these repeats represent tubulin-binding domains, two of which are sufficient to bind to microtubules assembled from purified tubulin in the presence of taxol.
Amino Acid Sequence↗
Molecular cloning of the cDNA for a mutant mouse ribonucleotide reductase M1 that produces a dominant mutator phenotype in mammalian cells.
Mammalian ribonucleotide reductase is regulated by the binding of dATP and other nucleotide effectors to allosteric sites on subunit M1. Using mRNA from a mutant mouse T-lymphoma (S49) cell line, we have isolated a cDNA which encodes an altered, dATP feedback-resistant subunit M1. The mutant cDNA contains a single point mutation (a G-to-A transition) at codon 57, converting aspartic acid to asparagine. Proof that this mutation is responsible for the phenotype of dATP feedback resistance is provided by the following evidence. (i) The mutation was detected only in mutant S49 cells containing dATP feedback-resistant ribonucleotide reductase and not in wild-type or other mutant S49 cells. (ii) Transfection of Chinese hamster ovary cells with an expression plasmid containing the mutant M1 cDNA resulted in the production of dATP feedback-resistant ribonucleotide reductase. Transfected CHO cells expressing the mutant M1 cDNA exhibited a 15- to 25-fold increase in the frequency of spontaneous mutation to 6-thioguanine resistance, confirming that dATP feedback-resistant ribonucleotide reductase produces a mutator phenotype in mammalian cells. The availability of a cDNA which encodes dATP feedback-resistant subunit M1 thus provides a means of manipulating by transfection the frequency of spontaneous mutation in mammalian cells.
Animals↗
Signal for attachment of a phospholipid membrane anchor in decay accelerating factor.
Decay accelerating factor (DAF) belongs to a novel group of membrane proteins anchored to the cell surface by a glycophospholipid membrane anchor that is covalently attached to the carboxyl terminus of the protein. The last 37 amino acids of membrane DAF, when fused to the carboxyl terminus of a secreted protein, are sufficient to target the fusion protein to the plasma membrane by means of a glycophospholipid anchor. This approach provides a novel means of targeting proteins to the cell-surface membrane.
Animals↗
A human purine nucleoside phosphorylase deficiency caused by a single base change.
Purine nucleoside phosphorylase (PNP) deficiency in humans is associated with a severe defect in T-lymphocyte function. The mutant gene was cloned from one PNP-deficient patient who was the offspring of a consanguineous mating. The exons and intron/exon boundaries of the mutant PNP gene were sequenced and compared with the wild-type cDNA sequence. A single base difference was found in the coding region of the mutant gene, a G to A transition in the third exon. This single base mutation alters the codon at position 89 from Glu to Lys, a result which is consistent with previously published peptide mapping data. The patient was demonstrated to be autozygous for the single base mutation on the basis of hybridization of synthetic oligomers to genomic DNA digests. A mammalian expression vector was constructed containing the entire mutant gene under the transcriptional regulation of its own promoter. In another construction, the single base mutation was reverted to the wild-type sequence by in vitro mutagenesis. An isoelectric focusing gel containing extracts of the cells transfected with the mutant and reverted PNP gene was stained histochemically for PNP activity. The proteins from a similar gel were blotted on a nitrocellulose membrane, and immunoreactive human PNP protein was visualized. Cells transfected with the mutant gene contained no human PNP activity, but expressed immunoreactive PNP which focused at an abnormally alkaline pI. Cells transfected with the reverted gene expressed human PNP activity which co-focused with human PNP from a HeLa cell control, proving that the observed single base change was responsible for the loss of catalytic function.
Amino Acid Sequence↗
Human purine nucleoside phosphorylase and adenosine deaminase: gene transfer into cultured cells and murine hematopoietic stem cells by using recombinant amphotropic retroviruses.
Cell lines were established which produced high titers (approximately 10(6) infectious units per ml) of amphotropic, replication-defective recombinant retroviruses which transduced sequences encoding either human purine nucleoside phosphorylase (PNP) or adenosine deaminase (ADA). These viruses also contained a human hypoxanthine phosphoribosyltransferase gene as a selectable marker and a mouse metallothionein promoter (MMP) sequence just upstream from the PNP or ADA genes. Virus structure was maintained through the replication cycle if a short (216-base pair) MMP sequence was used. However, the use of a longer (1,834-base pair) MMP sequence resulted in the deletion of a significant portion of the recombinant virus genome, including the transcriptional regulatory elements of the MMP sequence. Northern analysis indicated a predominance of genome length transcripts in cells infected with deleted virus. The demonstration of substantial human PNP or ADA activity in virus-infected mouse fibroblasts by isozyme analysis suggested that active gene product was translated from either spliced or bicistronic message. The deleted ADA and PNP viruses were introduced into mouse hematopoietic stem cells by cocultivating freshly explanted bone marrow with virus producer cells. The infected marrow cells were injected into irradiated, syngeneic recipient mice, and the presence of integrated ADA or PNP proviral sequences was demonstrated in the DNA of spleen colonies by Southern analysis. Failure of these integrated proviral sequences to express active, human isozyme in spleen colony tissue indicated the existence of some regulatory constraint not active in cultured mouse cells.
Adenosine Deaminase↗
Molecular basis of a human purine nucleoside phosphorylase deficiency.
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Amino Acid Sequence↗
Cloned mouse ribonucleotide reductase subunit M1 cDNA reveals amino acid sequence homology with Escherichia coli and herpesvirus ribonucleotide reductases.
We have isolated and sequenced overlapping cDNA clones containing the entire coding region of mouse ribonucleotide reductase subunit M1. The coding region comprises 2.4 kilobases and predicts a polypeptide of 792 amino acids (Mr 90,234) which shows striking homology with ribonucleotide reductases from Escherichia coli and the herpesviruses, Epstein-Barr virus and herpes simplex virus. The homologies reveal three domains: an N-terminal domain common to the mammalian and bacterial enzymes, a C-terminal domain common to the mammalian and viral ribonucleotide reductases, and a central domain common to all three. We speculate on the functional basis of this conservation.
Amino Acid Sequence↗
The effects of exogenous thymidine on endogenous deoxynucleotides and mutagenesis in mammalian cells.
The intracellular deoxyribonucleoside triphosphate pools in mammalian cells affect diverse biological functions including the spontaneous or induced mutability. We have isolated from murine T-lymphosarcoma S49 cells, a mutant that is unable to convert dCMP to dUMP, contains deranged intracellular dNTP pools, and exhibits a mutator phenotype. The enzymatic defect in araC-6-1 cells is a deficiency of deoxycytidylate deaminase, which accounts for the high dCTP and low TTP intracellular pools. The addition of increasing concentrations of exogenous thymidine to araC-6-1 cells alters these dNTP pools in a predictable manner: increasing the TTP and diminishing the dCTP. Concomitant with this reversal of the dCTP:TTP ratio is a marked decrease in the mutation rate followed by an increase in the mutation rates at higher exogenous thymidine concentrations. This response of the mutation rate is in contrast to that seen in the control cell line containing normal deoxycytidylate deaminase. In the latter case, increasing thymidine concentration induces an enhanced mutation rate that parallels the later phase of the thymidine-induced mutation rate in araC-6-1 cells. The deficiency of deoxycytidylate deaminase, the endogeneous dNTP pool alterations, and the mutator phenotype of araC-6-1 cells are all recessive traits in cell-cell hybrids. These observations allow one to predict whether exogenous thymidine will be mutagenic, antimutagenic, or both for a given cell line and provide a basis for understanding conflicting reports in the literature concerning the effects of the thymidine on genomic stability.
Animals↗
Mammalian expression of cloned cDNA sequences encoding human purine nucleoside phosphorylase and adenosine deaminase.
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Adenosine Deaminase↗
Expression of a cDNA sequence encoding human purine nucleoside phosphorylase in rodent and human cells.
A cDNA sequence which contains the entire coding region for human purine nucleoside phosphorylase (PNP) was recombined for selection and expression in mammalian cells. Plasmids containing either the simian virus 40 early promoter or the mouse metallothionein promoter positioned just upstream of the PNP coding sequence were constructed. These plasmids also contained the gene for a methotrexate-resistant dihydrofolate reductase, allowing for selection and amplification of positive transferrents after transfection of cells by the DNA-calcium phosphate coprecipitation technique. Expression of human PNP activity was readily detected in both mouse (L) and CHO cells by isoelectric focusing of cell extracts followed by histochemical staining for PNP activity. The simian virus 40 early promoter directed considerable expression of human PNP activity in CHO cells but only scant activity in mouse cells. The mouse metallothionein promoter was not successful in effecting human PNP expression in CHO cells but provided substantial human PNP activity in mouse cells and was inducible by incubation with zinc. HeLa cell transferrents were isolated and screened for the presence of transferred PNP cDNA sequences by Southern hybridization analysis. RNA transcripts derived from the transferred PNP cDNA were identified in one of these cell lines.
Animals↗
Solubilized monomeric sarcoplasmic reticulum Ca pump protein. Phosphorylation by inorganic phosphate.
Phosphorylation (by inorganic phosphate) of sarcoplasmic reticulum Ca pump protein has been studied in a detergent solution in which the protein has been previously shown to exist as a monomer. The course of the reaction is qualitatively similar to that observed for membrane-bound (possibly oligomeric) protein. In particular, the results indicate that alternation between the two principal conformational states of the Ca pump protein persists in the monomeric state, which suggests that the machinery for coupling of ATP hydrolysis to Ca2+ transport is intact. There are quantitative differences between monomeric and membrane-bound protein with respect to phosphorylation, but they are not necessarily related to the state of association.
Animals↗