Selective inhibition of uracil tRNA methylases of E. coli by ethionine.

L-ethionine has been found to inhibit uracil tRNA methylating enzymes in vitro under conditions where methylation of other tRNA bases is unaffected. No selective inhibitor for uracil tRNA methylases has been identified previously. 15 mM L-ethionine or 30 mM D,L-ethionine caused about 40% inhibition of tRNA methylation catalyzed by enzyme extracts from E. coli B or E. coli M3S (mixtures of methylases for uracil, guanine, cytosine, and adenine) but did not inhibit the activity of preparations from an E. coli mutant that lacks uracil tRNA methylase. Analysis of the 14CH3 bases in methyl-deficient E. coli tRNA after its in vitro methylation with E. coli B3 enzymes in the presence or absence of ethionine showed that ethionine inhibited 14CH3 transfer to uracil in tRNA, but did not diminish significantly the 14CH3 transfer to other tRNA bases. Under similar conditions 0.6 mM S-adenosylethionine and 0.2 mM ethylthioadenosine inhibited the overall tRNA base methylating activity of E. coli B preparations about 50% but neither of these ethionine metabolites preferentially inhibited uracil methylation. Ethionine was not competitive with S-adenosyl methionine. Uracil methylation was not inhibited by alanine, valine, or ethionine sulfoxide. It is suggested that the thymine deficiency that we found earlier in tRNA from ethionine-treated E. coli B cells, resulted from base specific inhibition by the amino acid, ethionine, of uracil tRNA methylation in vivo.     

A rapid assay technique for RNA ribose methylases.

A rapid technique for quantitative separation of ribose-methylated nucleosides from base-methylated and non-methylated nucleosides by chromatography on DEAE-cellulose paper in the presence of borate is described. The method has been used as an assay for tRNA ribose methylases from yeast, using under methylated Escherichia coli tRNA as substrate. The main product formed with a partly purified yeast enzyme was characterized as 2'-O-methylcytidine.     

Modified assay procedures for the phosphotransferase system in enteric bacteria.

Conditions for the assay of individual components of the bacterial phosphotransferase system (PTS) are presented wich offer two important improvements over earlier methods. First, a lactate dehydrogenase-coupled assay for phosphocarrier proteins (HPr, FPr, and Factor III^Gle) which permits their measurement in either pure or partially pure form was developed. Quantitation by this assay does not rely on the level of activity of the enzymes used. Second, conditions under which Enzyme I activity was proportional to enzyme concentration are given. With these methods levels of PTS components have been measured that are 2-to 20-fold higher than those previously reported. These levels can now account for various PTS functions measured in vivo. Further, we have shown that the phosphocarrier proteins HPr and Factor III^Gle are substrates for their respective enzymes which show typical Michaelis-Menten kineties. In addition, a method for the partial purification of Enzyme II-B^Gle essentially free of Enzyme II^Man activity is presented.     

A propionate-inducible expression system for enteric bacteria

A series of new expression vectors (pPro) have been constructed for the regulated expression of genes in Escherichia coli. The pPro vectors contain the prpBCDE promoter (P(prpB)) responsible for expression of the propionate catabolic genes (prpBCDE) and prpR encoding the positive regulator of this promoter. The efficiency and regulatory properties of the prpR-P(prpB) system were measured by placing the gene encoding the green fluorescent protein (gfp) under the control of the inducible P(prpB) of E. coli. This system provides homogenous expression in individual cells, highly regulatable expression over a wide range of propionate concentrations, and strong expression (maximal 1,500-fold induction) at high propionate concentrations. Since the prpBCDE promoter has CAP-dependent activation, the prpR-P(prpB) system exhibited negligible basal expression by addition of glucose to the medium.     

A prevalence survey for zoonotic enteric bacteria in a research monkey colony with specific emphasis on the occurrence of enteric Yersinia

Transmissible pathogenic and opportunistic zoonotic enteric bacteria comprise a recognized occupational health threat to exposed humans from non-human primates (NHPs). In an effort to evaluate the occurrence of selected enteric organisms with zoonotic and biohazard potential in a research colony setting, we performed a prevalence study examining 61 juvenile and young adult rhesus macaques participating in a transplant immunology project. Primary emphasis was directed specifically to detection of pathogenic enteric Yersinia, less well-documented and reported NHP pathogens possessing recognized significant human disease potential. NHPs were surveyed by rectal culture during routine health monitoring on three separate occasions, and samples incubated using appropriate media and specific selective culture methods. Enteric organisms potentially transmissible to humans were subcultured and identified to genus and species. Significant human pathogens of the Salmonella/Shigella, Campylobacter, and enteric Yersinia groups were not isolated throughout the survey, suggesting prevalence of these organisms may generally be quite low.     

Escherichia coli formylmethionine tRNA: methylation of specific guanine and adenine residues catalyzed by HeLa cells tRNA methylases and the effect of these methylations on its biological properties

Purified HeLa cell tRNA methylases have been used for site-specific methylations of Escherichia coli formylmethionine transfer ribonucleic acid (tRNA^fMet). Guanine-N²-methylase catalyzed the methylation of a specific guanine residue (G₂₇) and adenine-1-methylase that of a specific adenine residue (A₅₉). The combined action of both of these enzymes leads to a total incorporation of two methyl groups and results in the methylation of both G₂₇ and A₅₉.The effect of introducing additional methyl groups on the function of tRNA has been studied by a comparison in vitro of the biological properties of tRNA^fMet and enzymically methylated tRNA^fMet. It was found that none of the following properties of E. coli tRNA^fMet are altered to any significant extent by methylation: (a) rate, extent, and specificity of aminoacylation, (b) ability of methionyl-tRNA to be enzymically formylated, and (c) ability of formylmethionyl-tRNA to initiate protein synthesis in cell-free extracts of E. coli in the presence of f2 RNA as messenger. Also, the temperature versus absorbance profile of the doubly methylated tRNA^fmet was virtually identical to that of the E. coli tRNA^fMet, and enzymically methylated tRNA^fmet resembled tRNA^fMet in that both were resistant to deacylation by E. coli, N-acylaminoacyl-tRNA hydrolase.     

Studies of odd bases in yeast mitochondrial tRNA: III. Characterization of the tRNA methylases associated with the mitochondria

Whereas m¹G, m²G, m₂²G, m⁷G, T, m¹A, m⁵C and Cm methylase activities were found in total cell enzyme of $\text{Saccharomyces cerevisiae}$ using under-methylated $\text{E. coli}$ tRNA and $\text{E. coli}$ B tRNA in reaction with or without Mg⁺⁺, only m¹G, m²G, m₂²G and T methylases occurred in mitochondria. Mitochondrial and cytoplasmic tRNA cannot be methylated by their homologous enzymes; only mitochondrial tRNA can be methylated in a heterologous reaction by total cell enzyme with formation of T, m⁵C, m¹A and low amounts of m²G and m₂²G.     

A simple micro-growth assay for enumerating bacteria

A simple method for nonspecific determination of bacteria concentrations in a variety of liquid samples was developed. The assay was based on the time required for a sample grown in liquid media to reach a threshold turbidity. Samples were combined with media in a covered 96-well microwell plate and the turbidity was monitored in real time as the bacteria grew in a temperature-controlled plate reader. A significant problem with growth in microwells was condensation on the cover, which prevented accurate turbidity measurement. This problem was overcome by coating the cover with a small amount of surface-active agent. Salmonella and E. coli concentrations could be determined with a relative error of approximately 20% at levels from 10 to 10(6) cells/ml (eight replicates). An assay of 10 samples with standards required 10 min to set up and 20 min for data processing using a computer spreadsheet program. Growth time at 37 degrees C ranged from 4 h for samples at 10(7) cells/ml to 16 h for samples at 10 cells/ml.     

The use of cloned tRNA genes for the purification and measurement of specific tRNAs

We have previously reported the ability of a cloned tRNAMeti gene (pt145) to bind tRNAMeti specifically [5]. In this paper, we show that a pBR322 plasmid containing the tRNAAsn gene of Xenopus (pt38 - donated by Stuart Clarkson) will specifically bind to mouse tRNAAsn when total mouse tRNA, extracted from uninduced Friend erythroleukemia cells, is hybridized to the gene probe. One-dimensional electrophoresis of the hybridizing tRNA in 20% polyacrylamide reveals one major band and several small-molecular-weight minor bands. The hybridizing tRNA has been identified as tRNAAsn by partial RNA sequencing and the detection of both the Q base and t6A. The steady-state concentration of tRNAAsn in the uninduced Friend cell was determined by hybridizing tRNA labeled in vitro to pt38. 1% of the total tRNA hybridized, representing 0.017 pg tRNAAsn/cell. The fraction of newly synthesized tRNA representing tRNAAsn or tRNAMeti was also determined by hybridizing tRNA labeled in vivo to either pt38 or pt145, respectively. 0.96% and 0.85% of the tRNA hybridized to pt38 and pt145, respectively.     

Penetration of fimbriate enteric bacteria through basement membranes: A hypothesis

A mechanism for penetration of basement membranes by Escherichia coli is presented. The mechanism is based on the ability of the S fimbriae of meningitis-associated E. coli to bind to vascular endothelium and choroid plexuses in brain and to basement membranes. On the other hand, the S and the type 1 fimbriae of E. coli immobilize plasminogen and tissue-type plasminogen activator; this process generates proteolytic plasmin activity on the surface of fimbriate cells. Our hypothesis is that bacterium-bound plasma activity, directed to basement membranes through fimbrial binding, promotes bacterial penetration through basement membranes.     

The mechanism of action of tRNA methylases studied with immobilized tRNAs.

Each of the individual tRNAs immobilized on aminohydroxybutyl-cellulose (ABC) through their oxidized 3'-terminal binds affinitively all methylases present in the enzyme extract irrespective of whether this tRNA will be involved in the following step of methylation or not. These data allow to suggest that (a) the formation of a methylase-tRNA complex and the catalytic act of methylation are indeed autonomous processes and (b) the first step of interaction between tRNAs and tRNA methylases is rather unspecific and consists in the recognition of the whole class of tRNA molecules.     

A specific kinetic assay for phenylalanine hydroxylase

An assay procedure is given which is speedy, accurate, and specific, permitting direct recording of velocities, and obviating the use of reagents other than those necessary for the enzymatic reaction itself. The method is suitable for the study of enzyme mechanism and inhibition and also offers distinct advantages when used for other purposes, e.g., assay during purification of enzymes or for measurement of phenylalanine hydroxylase activity in the liver of hyperphenylalaninemics.The method is based on the phenylalanine-dependent change in absorbance of the tetrahydropteridine cofactor as it is oxidized to the dihydro form. The reaction rate measured by this procedure is linear over a wide range of enzyme concentration. The K_m and V for both tetrahydropteridine and for phenylalanine were the same as the values determined by the old procedure. Measurement of the stoichiometry of the reaction showed that one dihydropteridine is formed per tyrosine formed, or per DPNH consumed. The rate of reaction was identical to that measured by a coupled assay using DPNH and purified dihydropteridine reductase.     

A sensitive specific enzymatic-fluorometric assay for homocarnosine

We have developed a sensitive and specific assay for homocarnosine in tissues. Homocarnosine is separated from GABA by ion exchange chromatography. After hydrolysis of homocarnosine with swine kidney carnosinase, the evolved GABA is measured by an enzymatic-fluorometric procedure. As little as 0.1 nmol of tissue homocarnosine can be detected by this procedure. Homoanserine, which would be detected by this assay, can be separated from homocarnosine by thin layer chromatography. No homoanserine could be detected in any tissues examined. There are marked regional variations in levels of homocarnosine in guinea-pig brain that do not correspond to regional differences in GABA levels.     

A quick and specific assay for hydroxyproline

Hydroxyproline is oxidized by periodic acid and various periodates in strong acid medium, while proline is oxidized only in neutral medium (1). This phenomenon led us to elaborating an assay method for quantitative determination of hydroxyproline in the presence of proline. The procedure and its application for quantitative assay of hydroxyproline in isolated collagen and in skin and urine are reported below.     

Eukaryotic DNA methylases and their use for in vitro methylation

DNA methylases from mouse and pea have been purified and characterized. Both are high molecular mass enzymes that show greater activity with hemimethylated than unmethylated substrate DNA. Both methylate cytosines in CpG preferentially, but not exclusively and show similar kinetics of methylation, which makes it difficult to saturate all possible sites on the DNA, but procedures are described that circumvent this problem.