Forensic DNA typing.
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DNA Fingerprinting↗
Biomedical subjects
L R Yarbrough
Publications and source records attributed to L R Yarbrough.
Conserved beta-tubulin binding domain for the microtubule-associated motors underlying sperm motility and fast axonal transport.
An antiserum against tubulin, NS20, has been previously shown to inhibit anterograde and retrograde axonal transport by 50% in vivo and in vitro. We report here that Protein A purified NS20 antibodies also attenuate sperm motility by 50% in demembranated sea urchin sperm. This inhibition is absorbed out by preincubating the NS20 antibodies with a biochemically purified porcine microtubule preparation, with recombinant Trypanosoma beta- (but not alpha-) tubulin and most specifically, with a 37 amino acid (a.a.) synthetic peptide corresponding to a domain near (but not including) the porcine beta-tubulin C terminus. Furthermore, addition of this beta-tubulin peptide alone is sufficient to attenuate motility by 50% in demembranated sperm, indicating that this critical 37a.a. NS20 antigen is a motor binding domain. Together, the results suggest that at least two phenotypically distinct forms of microtubule-based motility, axonal transport and flagellar beating, are homologous at the fundamental level of the microtubule domains (the beta-tubulin peptide and we suggest a distinct but similarly located alpha-tubulin domain) mediating the attachment of tubulin-associated motors.
Amino Acid Sequence↗
Effects of age on myosin and creatine kinase isoforms in left ventricles of Fischer 344 rats.
Left ventricles of hearts from male Fischer 344 rats of 2, 8 and 23 months of age were analyzed to determine if aging results in significant alterations in the isoform distribution of myosin and creatine kinase protein and mRNAs. Left ventricles of maturing (2-month) rats contained almost exclusively alpha-myosin heavy chain (MHC) mRNA and protein. In adults (8 months) there was a 5-fold increase in beta-MHC (fetal isoform) and an approximate 10% decrease in alpha-MHC mRNA levels, relative to 2 months. By 23 months (senescence), beta-MHC mRNA levels had increased by 11-fold and alpha-MHC mRNA levels had decreased by about 30%. These changes corresponded to an increase in the relative proportion of beta-MHC protein, from undetectable levels at 2 months, to about 40% by 8 months and to about 60% by 23 months. Increased levels of beta-MHC and its mRNA in older rats correlated with decreased serum thyroid hormone levels. The specific activity of creatine kinase in crude homogenates decreased with age, as has been reported previously. Relative to 2-month controls, the specific activity of creatine kinase had decreased by 21% at 8 months and by 37% at 23 months. Analysis of creatine kinase activity showed no large increase in levels of the fetal (B) isoform with age, as was found for myosin. Levels of mRNAs encoding the B and M isoforms of creatine kinase were significantly reduced in senescent rats. Thus, the decreased levels of creatine kinase in aging rats is correlated with decreased levels of mRNA encoding the BCK and MCK isoforms but not an isoform shift.
Aging↗
Comparison of myosin and creatine kinase isoforms in left ventricles of young and senescent Fischer 344 rats after treatment with triiodothyronine.
The effects of perturbation of thyroid hormone levels on expression of myosin and creatine kinase isoforms were examined in maturing and senescent rats. Whereas LV of maturing rats contain only alpha-MHC, LV of senescent rats contain nearly equal amounts of both alpha-MHC and beta-MHC. When maturing rats were made hypothyroid by treatment for 14 days with the antithyroid agent, propylthiouracil (PTU), beta-MHC and beta-MHC mRNA levels increased significantly. Administration of T3 to senescent rats, or maturing rats made hypothyroid by PTU treatment produced similar decreases in levels of both beta-MHC and beta-MHC mRNA. In contrast, treatment with T3 produced little change in creatine kinase isoform distribution. Thus, thyroid hormone appears to play a critical role in regulating expression of the isoforms of myosin but not of creatine kinase.
Aging↗
Structure and regulation of genes encoding phycocyanin and allophycocyanin from Anabaena variabilis ATCC 29413.
Gene clones encoding phycocyanin and allophycocyanin were isolated from an Anabaena variabilis ATCC 29413-Charon 30 library by using the phycocyanin (cpc) genes of Agmenellum quadruplicatum and the allophycocyanin (apc) genes of Cyanophora paradoxa as heterologous probes. The A. variabilis cpcA and cpcB genes occur together in the genome, as do the apcA and apcB genes; the two sets of genes are not closely linked, however. The cpc and apc genes appear to be present in only one copy per genome. DNA-RNA hybridization analysis showed that expression of the cpc and apc genes is greatly decreased during nitrogen starvation; within 1 h no cpc or apc mRNA could be detected. The source of nitrogen for growth did not influence expression of the genes; vegetative cells from nitrogen-fixing and ammonia-grown cultures had approximately the same levels of cpc and apc mRNAs. Heterocysts had less than 5% as much cpc mRNA as vegetative cells from nitrogen-fixing cultures. Northern hybridization (RNA blot) analysis showed that the cpc genes are transcribed to give an abundant 1.4-kilobase (kb) RNA as well as two less prominent 3.8- and 2.6-kb species. The apc genes gave rise to two transcripts, a 1.4-kb predominant RNA and a minor 1.75-kb form.
Cyanobacteria↗
Spectroscopic techniques for study of phosphodiester bond formation by Escherichia coli RNA polymerase.
Nucleotides containing the fluorophore 1-aminonaphthalene-5-sulfonate attached to the gamma phosphoryl group via a phosphoamidate bond are excellent substrates for Escherichia coli DNA-dependent RNA polymerase. Cleavage of the alpha-beta-phosphoryl bond produces significant changes in both absorption and fluorescence spectra. These alterations provide a sensitive and precise means for continuous monitoring of transcription. Under appropriate conditions one can detect the utilization of less than 1 nmol of nucleotide. Since the spectroscopic techniques measure nucleotide utilization they can be used in conjunction with measurements of incorporation of radiolabeled precursor such as [3H]UTP into acid-insoluble material to determine whether significant amounts of acid-soluble oligonucleotides are formed.
DNA-Directed RNA Polymerases↗
Synthesis and properties of fluorescent nucleotide substrates for DNA-dependent RNA polymerases.
A new class of fluorescent nucleotide analogs which contain the fluorophore 1-aminonaphthalene-5-sulfonate attached via a gamma-phosphoamidate bond has been synthesized. Both the purine and pyrimidine analogs have fluorescence emission maxima at 460 nm. Cleavage of the alpha-beta-phosphoryl bond produces change in both the absorption and fluorescence emission spectra. The fluorescence of the pyrimidine analogs is quenched; cleavage of the alpha-beta-phosphoryl bond of the UTP analog produces about a 14-fold increase in fluorescence intensity at 500 nm. Under the same conditions the fluorescence of the CTP analog increases about 8-fold, whereas the fluorescence of the purine analogs shows only a slight change. These derivatives are good substrates for Escherichia coli RNA polymerase with only slightly increased Km values and with Vmax values about 50 to 70% that of the normal nucleotides. They are used less efficiently by wheat germ RNA polymerase II. The ATP analog can be used by E. coli RNA polymerase to initiate RNA chains.
DNA-Directed RNA Polymerases↗
Synthesis and properties of a new fluorescent analog of ATP: adenosine-5'-triphosphoro-gamma-1-(5-sulfonic acid) napthylamidate.
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Adenosine Triphosphate↗
Conformational transition of Escherichia coli RNA polymerase induced by the interaction of sigma subunit with core enzyme.
The isolated sigma subunit of Escherichia coli RNA polymerase has been labeled covalently with a fluorescent probe, N-(1-pyrene)maleimide. The labeled sigma subunit (PM-sigma) still retained its biological activity in stimulating transcription of T7 DNA by core enzyme. When a stoichiometric amount of core enzyme was added to a solution of PM-sigma, there was a decrease in fluorescence intensity without shifts in emission maxima of PM-sigma. The kinetics of the interaction between the sigma subunit and core enzyme was investigated with the stopped-flow technique by monitoring the fluorescence quenching. A biphasic change of fluorescence intensity with respect to time was observed when PM-sigma was rapidly mixed with an excess of core enzyme. The kinetic data can be analyzed in terms of a mechanism in which a fast bimolecular binding of sigma to core enzyme is followed by a relatively slow isomerization of the holoenzyme formed. From the best-fit kinetic parameters, an overall binding constant of less than or equal to 3X10(-10)M was estimated for the PM-sigma core complex, in agreement with that obtained by the fluorimetric titration. In addition, we have studied the effect of temperature on the rate constant associated with the conformational change of the holoenzyme, which shows a temperature transition around 20 degrees C. The nonlinear Arrhenius plot obtained implies that the conformational transition is complex and may be composed of several processes. The activation energy for the "overall" conformational change was estimated to be 6.7 kcal/mol. The kinetic evidence for the conformational transition of holoenzyme induced by the interactions of sigma subunit with core enzyme presented here further supports the proposition that the sigma subunit acts on core enzyme to trap a unique conformation of RNA polymerase which recognizes the proper promoters and initiates the synthesis of specific RNA chains.
DNA-Directed RNA Polymerases↗
N-(1-pyrene)maleimide: a fluorescent cross-linking reagent.
N-(1-Pyrene)maleimide is nonfluorescent in aqueous solution but forms strongly fluorescent adducts with sulfhydryl groups of organic compounds or proteins. The conjugation reactions of N-(1-pyrene)maleimide are relatively fast and can be monitored by the increase in fluorescence intensity of the pyrene chromophore. In cases where primary amino groups are also present in the system, we have observed a red shift of the emission spectra of the fluorescent adducts subsequent to the initial conjugation, as characterized by the disappearance of three emission peaks at 376, 396, and 416 nm, and the appearance of two new peaks at 386 and 405 nm. Model studies with N-(1-pyrene)maleimide adducts of L-cysteine and cysteamine indicate that the spectral shift is the result of an intramolecular aminolysis of the succinimido ring in the adducts. Evidence from both chemical analysis and nuclear magnetic resonance studies of the addition products supports this reaction scheme. N-(1-Pyrene)maleimide adducts of N-acetyl-L-cysteine and beta-mercaptoethanol, which have no free amino group, do not exhibit a spectral shift. Among several protein conjugates only the N-(1-pyrene)maleimide adduct of bovine serum albumin (PM-BSA) shows the spectral shift resembling that of PM-cysteine. N-(1-Pyrene)maleimide reacts with the sulfhydryl group of the single cysteine residue at position 34 in BSA. The finding that the alpha-amino group of the N-terminus in PM-BSA is blocked after the spectral shift is completed strongly suggests that N-(1-pyrene)maleimide cross-links the N-terminus and the cysteine residue in BSA. The relative proximity of the sulfhydryl and amino groups is very critical in the cross-linking as demonstrated by the observation that the spectral shift observed with PM-BSA can be prevented by addition of denaturing reagents such as 1% sodium dodecyl sulfate immediately after labeling, and by the failure of PM-glutathione to undergo the intramolecular aminolysis. Since the intramolecular rearrangement of PM adducts is associated with characteristic fluorescence changes, N-(1-pyrene)maleimide can serve as a fluorescent cross-linking reagent which provides information about the spatial proximity of sulfhydryl and amino groups in proteins.
Binding Sites↗
Molecular mechanism of the rifampicin -RNA polymerase interaction.
Equilibrium and kinetic studies of the interaction of rifampicin with RNA polymerase of Escherichia coli were performed by exploiting the quenching of intrinsic fluorescence of the protein by the drug. Fluorimetric titrations show that rifampicin binds stoichiometrically to the core and holoenzyme with an apparent Kd of less than or equal to 3 x 10(-9) M. Neither the addition of template nor the formation of the initiation complex in the presence of dinucleotide and nucleoside triphosphate prevents the rifampicin-enzyme interaction. Although the equilibrium binding constant for the rifampicin-RNA polymerase complex is about the same for the core and holoenzyme and the holoenzyme-T7 DNA complex, stopped-flow studies indicate that the rates at which rifampicin interacts with these enzyme forms are different. In all three cases, the kinetic data can be interpreted in terms of a mechanism in which the rapid bimolecular binding of rifampicin to RNA polymerase is followed by a relatively slow isomerization of the drug enzyme complex: (See article). While the values of dissociation constant K1 = (k-1/k1), for the first binary complex (ER) are similar, the rate constant for the forward isomerization, k2, decrease in the order of core enzyme greater than holoenzyme greater than the holoenzyme-T7 DNA complex. The fact that this order is parallel to the relative rates of inactivation of the enzymes and the enzyme-DNA complex suggests that the inactivation may be due to the rifampicin-induced isomerization (conformational change) of the enzyme. This is supported by our observations that an enzyme complex which is in the process of elongating RNA chains can still bind rifampicin, although the enzyme activity is not inhibited by such binding. The values of overall binding constants calculated from the kinetic parameters, 1-2 x 10(-9) M, are in good agreement with the values of the apparent Kd obtained from fluorimetric titrations and Ki determined by enzymatic assays. In addition, the observations that the formation of an initiation complex leads to a significant but not complete rifampicin-resistant RNA synthesis and the recent finding that rifampicin only partly inhibits the formation of the first phosphodiester bond in an abortive initiation of RNA chains are consistent with our kinetic mechansim, i.e., the existence of two forms of the rifampicin-RNA polymerase complex, only one of which is able to initiate the RNA chains.
Binding Sites↗
Spatial relationship of the sigma subunit and the rifampicin binding site in RNA polymerase of Escherichia coli.
sigma subunit of Escherichia coli RNA polymerase is known to stimulate specific RNA chain initiation. Rifampicin, an inhibitor of RNA chain initiation, binds to a single site on the beta subunit of RNA polymerase. We have used the fluorescence energy transfer technique to deduce proximity relationships of sigma subunit and rifampicin binding site on the enzyme. Isolated sigma subunit was covalently labeled with fluorescent donors in two ways: specific labeling of a single sulfhydryl residue with N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonate (1,5-I-AENS) and nonspecific labeling on the surface of the protein with dansyl chloride (Dns-Cl) adsorbed on Celite. The labeled sigma subunits were biologically active and formed a stoichiometric complex with core polymerase. The efficiency of energy transfer was obtained from the fluorescence intensity and the excited-state lifetime of the sigma-labeled holoenzyme in the presence and absence of rifampicin, which served as an energy acceptor. The transfer efficiency (2%) from AENS to rifampicin placed AENS somewhere between 42 and 85 A away from the rifampicin binding site. The rotational mobility of the donor was determined by nanosecond fluorescence depolarization spectroscopy, while the acceptor orientation was assumed to be fixed at some unknown angle. The efficiency measured for energy transfer from Dns to rifampicin was 10% in the presence of 0.2 M KCl. The distance from the surface of sigma subunit to the rifampicin binding site was calculated to be 27--38 A for a model having a randomly distributed and oriented array of donors on the surface of a spherical sigma subunit of 31-A radius. Our results indicate that rifampicin does not inhibit the initiation of transcription by RNA polymerase through a direct interaction with sigma subunit. In addition, energy transfer measurements under low salt conditions suggest that in RNA polymerase dimer the two rifampicin binding sites are symmetric with respect to each sigma subunit.
Binding Sites↗
Sigma cycle during in vitro transcription: demonstration by nanosecond fluorescence depolarization spectroscopy.
Studies of RNA chain initiation have suggested that the sigma subunit of Escherichia coli RNA polymerase (RNA nucleotidyltransferase; nucleosidetriphosphate: RNA nucleotidyltransferase; EC 2.7.7.6) is released from the enzyme-template complex during transcription and may be reused by another core polymerase. Nanosecond fluorescence depolarization spectroscopy was used to follow the sigma cycle. Isolated sigma subunit labeled with the fluorescent probe dansyl (DNS) chloride bound stoichiometrically to core polymerase and stimulated transcription of phage T7 DNA to the same extent as did unlabeled sigma. DNS-sigma showed an exponential fluorescence anisotropy decay corresponding to a rotational correlation time of about 100 nsec. This value was unaffected by addition of T7 DNA, but increased about 6-fold when core polymerase was added, and increased further when T7 DNA was added. Such increases are expected for the formation of molecular complexes. Using the anisotropy decays for free DNS-sigma and DNS-sigma-core enzyme bound to T7 DNA, we calculated theoretical decay curves for various mixtures of free and bound sigma. Comparison of the observed anisotropy decay with the calculated curves indicated that about 55% of DNA-sigma was released from the enzyme-T7 DNA complex in the presence of four nucleoside triphosphates under low salt conditions. Sigma release did not occur if rifampicin was added prior to addition of four nucleoside triphosphates or if only three nucleoside triphosphates were present. After sigma was released, addition of core polymerase with rifampicin reduced the free sigma to less than 15%, indicating that the released sigma was accessible to the added core enzyme. Thus these studies have provided physical evidence for the sigma cycle during in vitro transcription.
Binding Sites↗
Changes in myosin and creatine kinase mRNA levels with cardiac hypertrophy and hypothyroidism.
Rats were treated with three methods which produce alterations in the expression of myosin isozymes: coarctation of the abdominal aorta, treatment with low doses of isoproterenol, and administration of propylthiouracil. The steady-state levels of the left ventricle mRNAs for alpha myosin heavy chain (alpha-MHC), beta myosin heavy chain (beta-MHC), M creatine kinase (MCK), and B creatine kinase (BCK) were then determined using Northern and slot blot hybridizations. Cardiac hypertrophy was induced by an acute systolic pressure overload, or beta adrenergic stimulation. At 7 days following systolic pressure overload, the induced cardiac hypertrophy was accompanied by alterations in the levels of MHC mRNAs, as has been previously reported. In RNA from left ventricles of treated animals alpha-MHC mRNA levels decreased by 15% by day 3 and 20% by day 7. In contrast, beta-MHC mRNA levels increased to 250% of control levels by day 3 and then declined to a value 150% of controls by day 7. Levels of MCK and BCK mRNAs showed little or no changes by day 3; at day 7 both MCK and BCK mRNAs showed decreases of 20% relative to controls. Cardiac hypertrophy induced by low doses of isoproterenol produced decreases of alpha-MHC mRNA levels to 70% of control values at day 3 and 50% at day 7. Over the same time periods there was an increase in the levels of the fetal mRNA isoform (beta-MHC) to 190%, then 130% of control values, respectively. At 3 days, both BCK and MCK mRNA levels had declined by approximately 20-25%. By 7 days, MCK mRNA levels had decreased by approximately 50% and BCK mRNA levels by 30%. Hypothyroidism induced by PTU treatment led to a 50% decrease in alpha-MHC mRNA levels by day 3, which then further decreased to 10% of control levels at day 7. beta-MHC mRNA levels increased to 350% of control levels at day 3 and then decreased to 275% of control levels at day 7. For creatine kinase mRNAs the level of the M isoform was increased by 30% at day 3, whereas there appeared to be no significant change in levels of B isoform mRNA at this time. At day 7 neither BCK nor MCK mRNA levels were significantly different from controls. These results show three treatments which produce an alteration in myosin mRNA isoforms produce little or no change in creatine kinase isoform mRNAs. Thus, the MHC and CK genes respond differently to either cardiac hypertrophy or a reduction in thyroid hormone levels.
Animals↗