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K B Jacobson

Publications and source records attributed to K B Jacobson.

At least 37 records · Page 2Linked to original sources
PubMed1991

An approach to the use of stable isotopes for DNA sequencing.

The sequencing of DNA by current procedures involves the use of radioisotopic or fluorescent labels. We propose that stable isotopes can be used as such labels and that the large number of stable isotopes available would allow multiplexing so that many DNA segments could be sequenced simultaneously. We have developed methods to use 57Fe2O3 to synthesize ferrocene and to attach the ferrocene to the 5' end of oligonucleotides. The 57Fe-labeled M13 universal primer functioned normally in a Sanger sequencing procedure. When a 57Fe-labeled oligonucleotide had migrated on a polyacrylamide gel it was readily located on the dried gel by scanning with resonance ionization spectroscopy (RIS) coupled with mass spectrometry. Using a 57Fe-labeled primer in a PCR reaction a 2000-bp DNA was produced that was detected by RIS on nylon membrane after agarose electrophoresis. The rapid analysis features of RIS coupled with the multispectral multiplexing possibilities of stable isotopes should significantly increase the rate of determination of DNA sequences.

Base Sequence↗
PubMed1991

Applications of mass spectrometry to DNA sequencing.

The ability of the mass spectrometer to analyze collectively the masses of DNA fragments that are produced in the Sanger procedure for sequencing may allow the gel electrophoresis step to be eliminated. On the other hand, if gel electrophoresis is required, the use of resonance ionization spectroscopy coupled to a mass spectrometer may enable much faster analysis of DNA bands labeled with stable isotopes. Other combinations of labeling of the DNA and its mass spectrometric analysis with or without gel electrophoresis are also considered. Recent advances in these areas of mass spectrometry are reviewed.

Base Sequence↗
PubMed1991

Resonance ionization spectroscopy for multiplex sequencing of tin-labeled DNA.

A method is described for synthesis of a tin reagent, triethylstannylpropanoic acid (TESPA), and its attachment to oligonucleotide primers. Except for the expected mobility retardation, the presence of [116Sn]-TESPA did not affect the sequencing ladder on electrophoresis gels. By using [120Sn]-TESPA and [35S]-dTTP simultaneously in the Sanger procedure, DNA bands on an electrophoresis gel were first located by autoradiography and then by resonance ionization spectroscopy to demonstrate the coincidence of the signals. Previous results using stable isotopes as labels on model compounds are now confirmed by their use in actual DNA sequencing products.

Autoradiography↗
PubMed1991

Queuine metabolism and cadmium toxicity in Drosophila melanogaster.

Queuine can replace guanine in the anticodon of certain tRNAs and is a hypermodified guanine derivative that can be synthesized by bacteria but not by mice. The study demonstrates that Drosophila can incorporate dietary queuine into tRNA but cannot synthesize it de novo for this purpose. Since an earlier study had shown that dietary CdCl2 caused Drosophila to increase greatly the proportion of queuine-containing tRNA over non-queuine tRNA the ability of dietary queuine to counteract cadmium toxicity was evaluated. When queuine was present in the cadmium-containing medium more pupae matured into adults than when queuine was absent. Other studies had demonstrated that the transglycosylase enzyme, that catalyzes the replacement of guanine in the anticodon of tRNA by queuine, is present in Drosophila larvae but the tRNA is virtually devoid of queuine. This study shows that in the presence of dietary queuine the larval tRNA contains abundant amounts of queuine. Therefore, we postulate a significant role for bacteria in supplying queuine to Drosophila for its incorporation into tRNA and that the control of this process by Drosophila is passive, i.e. is not an essential feature in differentiation.

Animals↗
PubMed1990

Purification and characterization of 6-pyruvoyl-tetrahydropterin synthase from Drosophila melanogaster.

The enzyme 6-pyruvoyl-tetrahydropterin synthase (PTP synthase), which catalyzes the conversion of 7,8-dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin, has been purified approx. 230-fold to apparent homogeneity from head extracts of Drosophila melanogaster. A partially purified 6-pyruvoyl-tetrahydropterin reductase (PTP reductase) was also prepared and in its presence, along with Mg2+ and NADPH, the purified PTP synthase converted 7,8-dihydroneopterin triphosphate to metastable 6-lactoyltetrahydropterin, which was autoxidized to sepiapterin under aerobic conditions. Purified PTP synthase had a specific activity of 3792 units per mg protein and migrated as a single protein band on both nondenaturing polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified active enzyme consisted of at least two identical subunits which had a molecular mass of 37.5 kDa on SDS-PAGE and NH2-Asx-Pro- as N-terminal amino acids. The native enzyme in crude extract was shown to be more complex, existing as higher multimeric forms.

Alcohol Oxidoreductases↗
PubMed1990

Selenocysteine tRNA[Ser]Sec gene is ubiquitous within the animal kingdom.

Recently, a mammalian tRNA which was previously designated as an opal suppressor seryl-tRNA and phosphoseryl-tRNA was shown to be a selenocysteyl-tRNA (B. J. Lee, P. J. Worland, J. N. Davis, T. C. Stadtman, and D. Hatfield, J. Biol. Chem. 264:9724-9727, 1989). Hence, this tRNA is now designated as selenocysteyl-tRNA[Ser]Sec, and its function is twofold, to serve as (i) a carrier molecule upon which selenocysteine is biosynthesized and (ii) as a donor of selenocysteine, which is the 21st naturally occurring amino acid of protein, to the nascent polypeptide chain in response to specific UGA codons. In the present study, the selenocysteine tRNA gene was sequenced from Xenopus laevis, Drosophila melanogaster, and Caenorhabditis elegans. The tRNA product of this gene was also identified within the seryl-tRNA population of a number of higher and lower animals, and the human tRNA[Ser]Sec gene was used as a probe to identify homologous sequences within genomic DNAs of organisms throughout the animal kingdom. The studies showed that the tRNA[Ser]Sec gene has undergone evolutionary change and that it is ubiquitous in the animal kingdom. Further, we conclude that selenocysteine-containing proteins, as well as the use of UGA as a codon for selenocysteine, are far more widespread in nature than previously thought.

Animals↗
PubMed1990

Scanning tunneling microscopy and spectroscopy of plasmid DNA.

We present scanning tunneling microscope (STM) images of uncoated deoxyribonucleic acid (DNA) electrochemically mounted on highly-oriented pyrolytic graphite (HOPG) and imaged in air. Images of linear abnormalities inherent to HOPG surfaces that can be confused with DNA are also presented. Scanning tunneling spectroscopic (STS) images generated by superimposing a small, high frequency ac bias onto the dc tunnel bias and recording the ac current signal were taken simultaneously with the topographic images. These spectroscopic images reveal contrast due to local conductivity variations and can be used to differentiate DNA molecules from graphite artifacts.

Carbon↗
PubMed1990

Monte Carlo calculations of free ammonia production in deoxygenated solutions of glycylglycine irradiated by X rays and 60Co gamma rays.

Detailed-history Monte Carlo computer codes were used to simulate the formation, diffusion, and chemical reaction of free-radical species within deoxygenated aqueous solutions of glycylglycine irradiated by 250-kVp X rays and by 60Co gamma rays. In one reaction, hydrated electrons react with the glycylglycine solute to produce unbound, or free, ammonia. This reaction is complete by 10(-6) s within individual electron tracks for glycylglycine concentrations greater than or equal to 0.025 M. For solute concentrations from 0.025 to 1.2 M, calculated G values of free ammonia are in excellent agreement with measured values. In addition, the computer model predicts a statistically significant difference between the G value of free ammonia produced under X irradiation and that produced under 60Co gamma irradiation.

Ammonia↗
PubMed1990

Measurement of free ammonia produced by X irradiation of glycylglycine in aqueous solution.

This research was initiated to test the validity of predictions based on Monte Carlo calculations of the effect of ionizing radiation on a simple dipeptide. The mechanism for the formation of ammonia, proposed by Garrison, Sokol, and Bennett-Corniea (Radiat. Res. 53, 376-384, 1973), was reevaluated by measuring the yields under deoxygenated and oxygenated conditions. Although free ammonia was formed under both conditions, the yields were different, depending on the concentrations of solute and molecular oxygen. The reaction probabilities of the specific interactions of free radicals formed in pure water with solute and oxygen are discussed to account for the observed difference. Our results obtained after low-dose-rate X irradiation are compared with those obtained by Garrison et al. after high-dose-rate 60Co gamma irradiation.

Ammonia↗
PubMed1989

Resistance of Drosophila to cadmium: biochemical factors in resistant and sensitive strains.

With regard to cadmium toxicity, Drosophila strains v;bw and Austin represent extremes in resistance and sensitivity, respectively. Both strains produced metallothionein (MT) in response to Cd ions in their diet. Austin produced more metallothionein than v;bw at Cd ion levels below 0.2 mM, when both strains were allowed lifetime development on Cd2+-containing media. When the rate of MT appearance was measured for 4 days in young adults the results showed no clear trend with time within a strain or between strains. The plot of LC50 vs. MT levels for identical developmental conditions revealed that for v;bw small increases in MT corresponded to large increases in resistance whereas for the sensitive Austin even large increases in MT had comparatively little effect on increasing LC50. Results given here suggest that differences in total MT content do not explain the genetically demonstrable difference in Cd2+-resistance between v;bw and Austin. However, since two MT genes are identified in Drosophila, differences in resistance could be reflective of greater relative amounts of one "more important" MT in the resistant fly.

Animals↗
PubMed1989

Effects of sepiapterin and 6-acetyldihydrohomopterin on the guanosine triphosphate cyclohydrolase I of mouse, rat and the fruit-fly Drosophila.

The regulation of GTP cyclohydrolase I would lead to the regulation of tetrahydrobiopterin, an important cofactor for synthesis of neurotransmitters. In an attempt to extend a previous finding [Bellahsene, Dhondt, & Farriaux (1984) Biochem. J. 217, 59-65] that GTP cyclohydrolase I of rat liver is inhibited by subnanomolar concentrations of reduced biopterin and sepiapterin, we found that this could not be verified with the enzyme from mouse liver, fruit-fly (Drosophila) heads or, indeed, from rat liver. It was shown, however, that 12 microM-sepiapterin inhibited mouse liver GTP cyclohydrolase I. Another compound, namely 6-acetyldihydrohomopterin, was also employed in the present study to explore its effect on enzymes that lead to its synthesis in Drosophila and for effects on mammalian systems; at 2-5 microM this compound was shown to stimulate one form of mouse liver GTP cyclohydrolase I and then to inhibit at higher concentrations (40 microM). Neither sepiapterin nor 6-acetyldihydrohomopterin caused any effect on the Drosophila head enzyme. On the other hand, the sigmoid GTP concentration curve for the Drosophila enzyme may indicate a regulatory characteristic of this enzyme. Another report, on the lower level of GTP cyclohydrolase I in mutant mouse liver [McDonald, Cotton, Jennings, Ledley, Woo & Bode (1988) J. Neurochem. 50, 655-657], was confirmed and extended. Instead of having 10% activity, we find that the hph-1 mouse mutant has less than 2% activity in the liver. These studies demonstrate that micromolar levels of reduced pterins may have regulatory effects on GTP cyclohydrolase I and that a mouse mutant is available that has low enough activity to be considered as a model for human atypical phenylketonuria.

Aminohydrolases↗
PubMed1989

Repetitive recycling of guanosine triphosphate cyclohydrolase I for synthesis of dihydroneopterin triphosphate.

A procedure for enzymatic production of dihydroneopterin triphosphate is described that allows GTP cyclohydrolase I to be reused repetitively. The reaction takes place in an ultrafiltration cell, and the product is collected in the filtrate, whereas the enzyme remains in the cell to be reused with additional substrate. This is repeated until the enzyme activity drops below a desirable level. The purity of the dihydroneopterin triphosphate is satisfactory for utilization of this compound for studies on enzymes involved in the synthesis of tetrahydrobiopterin and drosopterin. A procedure for purification of dihydroneopterin triphosphate is described that uses C18-silica and silica cartridges.

Aminohydrolases↗
PubMed1989

Ordering of metal-ion toxicities in different species--extrapolation to man.

Our previous attempts to predict the toxicities of 24 metal ions for a given species, using physicochemical parameters associated with the ions, are summarized. In our current attempt we have chosen indicators of toxicity for biological systems of increasing levels of complexity--starting with individual biological molecules and ascending to mice as representative of higher-order animals. The numerical values for these indicators have been normalized to a scale of 100 for Mg2+ (essentially nontoxic) and 0 for Cd2+ (very toxic). To give predicted toxicities to humans, extrapolations across biological species have been made for each of the metal ions considered. The predicted values are then compared with threshold limit values (TLV) from the literature. Both methods for predicting toxicities have their advantages and disadvantages, and both have limited success for metal ions. However, the second approach suggests that the TLV for Cu2+ should be lower than that currently recommended.

Animals↗
PubMed1988

Effect of zinc ions on tRNA structure: imino proton NMR spectroscopy.

The structure of tRNA in solution was explored by NMR spectroscopy to evaluate the effect of divalent cations, especially zinc, which has a profound effect on the chromatographic behaviour of tRNAs in certain systems. The divalent ions Mg2+ and Zn2+ have specific effects on the imino proton region of the 1H NMR spectrum of valine transfer RNA (tRNA(Val] of Escherichia coli and of phenylalanine transfer RNA (tRNA(Phe] of yeast. The dependence of the imino proton spectra of the two tRNAs was examined as a function of Zn2+ concentration. In both tRNAs the tertiary base pair (G-15).(C-48) was markedly affected by Zn2+ (shifted downfield possibly by as much as 0.4 ppm); this is the terminal base pair in the augmented dihydrouridine helix (D-helix). Base pair (U-8).(A-14) in yeast tRNA(Phe) or (s4U-8).(A-14) in tRNA1(Val), which are stacked on (G-15).(C-48), was not affected by Zn2+, except when 1-2 Mg2+ ions per tRNA were also present. Another imino proton that may be affected by Zn2+ in both tRNAs is that of the tertiary base pair (G-19).(C-46). The assignment of this resonance in yeast tRNA(Phe) is tentative since it is located in the region of highly overlapping resonances between 12.6 and 12.3 ppm. This base pair helps to anchor the D-loop to the T psi C loop.(ABSTRACT TRUNCATED AT 250 WORDS)

Cations, Divalent↗
PubMed1987

Effect of zinc ions on tRNA structure. I. Reversed-phase chromatography.

The effect of zinc on the chromatographic behavior of four tRNAs was examined on RPC-5 and Aminex A-28 columns. RPC-5 contains dichlorodifluoroethylene beads coated with a quaternary ammonium compound where the substituents are: R1 = methyl, and R2-4 = C8-10 hydrocarbons. Aminex A-28 contains quaternary ammonium covalently attached to styrene-divinylbenzene copolymer lattice and R1-3 are methyl groups. The retentions of tRNAVal, tRNAIle, and tRNALys of E. coli and yeast tRNAPhe on RPC-5 were all markedly increased by Zn2+ ions. In contrast, no increased retention due to Zn2+ was observed when tRNAPhe was chromatographed on Aminex A-28. A model for chromatography on RPC-5 is developed which treats the elution behavior of tRNAs from this matrix as the sum of ion-exchange and hydrophobic interactions. The chromatography of tRNA in the presence and absence of Zn2+ is interpreted in terms of this model and the effects of sodium chloride concentration, temperature, and pH were explored as the experimental variables. These experiments suggest that in the absence of Zn2+ tRNA does not interact appreciably with the hydrophobic surface of the column. The addition of Zn2+ has three effects on chromatography: a decrease in the number of anionic sites on the tRNA which interact with the positively charged ammonium ion, an increase in affinity of the tRNA for these ionic sites, and an increase in affinity of tRNA for hydrophobic sites on the column. All three effects were fully reversed by the addition of Cd2+ (10 mM) or Mg2+ (35 mM), but only partially reversed at lower concentrations of these competing ions. These results show that chromatography on RCP-5 can be a sensitive physical chemical technique for examination of the structure of tRNA, and probably for other nucleic acids as well.

Cadmium↗
PubMed1987

Cadmium resistance in Drosophila: a small cadmium binding substance.

A small cadmium-binding substance (CdBS) has been observed in adult Drosophila melanogaster that were raised for their entire growth cycle on a diet that contained 0.15 mM CdCl2. Induction of CdBS was observed in strains that differed widely in their sensitivity to CdCl2. This report describes the induction of CdBS and some of its characteristics.

Animals↗
PubMed1987

Mechanism of suppression in Drosophila: regulation of tryptophan oxygenase by the su(s)+ allele.

The suppressor gene, su(s)2, in Drosophila melanogaster restores the production of red and brown eye pigments for some purple and vermilion mutant alleles, respectively. We showed previously that the product of the su(s)+ allele caused inhibition of the sepiapterin synthase A produced by the purple mutant but did not affect the wild-type enzyme. Suppression was accomplished by removing su(s)+ from the genome. We now report that the tryptophan oxygenase, produced by suppressible vermilion alleles, is also inhibited by extracts from su(s)+ flies. The inhibition of the vermilion enzyme can be reduced or eliminated, respectively, by prior storage of the extract at 4 or -20 degrees C or by boiling, whereas the wild-type enzyme is not affected by extracts of su(s)+ flies. Also, when the suppressible vermilion strain is raised on certain diets, brown eye pigment production occurs. This epigenetic suppression was reduced by the presence of an extra copy of su(s)+ in the genome. These data support a posttranslational mechanism for regulation of enzyme activity in which the activity of the mutant enzyme is reduced by the product of the su(s)+ allele. How the su(s)+ gene product can distinguish between the normal and the mutant forms of these two enzymes is discussed, along with other mechanisms for suppression that are currently under investigation.

Alleles↗