EmtA, a rRNA methyltransferase conferring high-level evernimicin resistance.

Enterococcus faecium strain 9631355 was isolated from animal sources on the basis of its resistance to the growth promotant avilamycin. The strain also exhibited high-level resistance to evernimicin, a drug undergoing evaluation as a therapeutic agent in humans. Ribosomes from strain 9631355 exhibited a dramatic reduction in evernimicin binding, shown by both cell-free translation assays and direct-binding assays. The resistance determinant was cloned from strain 9631355; sequence alignments suggested it was a methyltransferase and therefore it was designated emtA for evernimicin methyltransferase. Evernimicin resistance was transmissible and emtA was localized to a plasmid-borne insertion element. Purified EmtA methylated 50S subunits from an evernimicin-sensitive strain 30-fold more efficiently than those from a resistant strain. Reverse transcription identified a pause site that was unique to the 23S rRNA extracted from resistant ribosomes. The pause corresponded to methylation of residue G2470 (Escherichia coli numbering). RNA footprinting revealed that G2470 is located within the evernimicin-binding site on the ribosome, thus providing an explanation for the reduced binding of the drug to methylated ribosomes.

Interaction of glutathione S-transferase M1 and T1 genotypes and malignant melanoma.

The mu and theta classes of glutathione S-transferases (GST) may affect the development of cutaneous malignant melanoma (CMM) by decreasing cellular oxidative stress in skin. These isozymes are absent in a large proportion of the population because of germ-line homozygous deletions in the genes encoding GSTM1 and GSTT1. To determine the association between GSTM1 and GSTT1 homozygous deletions (GSTM1 null and GSTT1 null, respectively) and CMM, we studied 212 patients with CMM, 150 patients with CMM and dysplastic nevi (DN), 147 patients with DN alone, and 124 healthy persons without CMM or DN. Comparing CMM cases (n = 362) to participants without CMM (n = 271), we found no association with GSTM1 null [odds ratio (OR), 1.2; 95% confidence interval (CI), 0.86-1.6] or GSTT1 null (OR, 0.82; 95% CI, 0.56-1.2), either independently or in combination (OR, 1.4; 95% CI, 0.81-2.2), after adjusting for age. However, among the subset of participants with red or blond hair, those with CMM were twice as likely to carry GSTM1 null (OR, 2.2; 95% CI, 1.2-4.2) and nearly 10-fold more likely to carry both GSTM1 null and GSTT1 null (OR, 9.5; 95% CI, 1.2-73) compared with those without CMM. These data suggest that among persons with hair colors traditionally associated with increased risk for melanoma, absence of both GSTM1 and GSTT1 may act to further elevate CMM risk.

Growth inhibition caused by overexpression of the structural gene for glutamate dehydrogenase (gdhA) from Klebsiella aerogenes.

Two linked mutations affecting glutamate dehydrogenase (GDH) formation (gdh-1 and rev-2) had been isolated at a locus near the trp cluster in Klebsiella aerogenes. The properties of these two mutations were consistent with those of a locus containing either a regulatory gene or a structural gene. The gdhA gene from K. aerogenes was cloned and sequenced, and an insertion mutation was generated and shown to be linked to trp. A region of gdhA from a strain bearing gdh-1 was sequenced and shown to have a single-base-pair change, confirming that the locus defined by gdh-1 is the structural gene for GDH. Mutants with the same phenotype as rev-2 were isolated, and their sequences showed that the mutations were located in the promoter region of the gdhA gene. The linkage of gdhA to trp in K. aerogenes was explained by postulating an inversion of the genetic map relative to other enteric bacteria. Strains that bore high-copy-number clones of gdhA displayed an auxotrophy that was interpreted as a limitation for alpha-ketoglutarate and consequently for succinyl-coenzyme A (CoA). Three lines of evidence supported this interpretation: high-copy-number clones of the enzymatically inactive gdhA1 allele showed no auxotrophy, repression of GDH expression by the nitrogen assimilation control protein (NAC) relieved the auxotrophy, and addition of compounds that could increase the alpha-ketoglutarate supply or reduce the succinyl-CoA requirement relieved the auxotrophy.

Effects of mutations in ribosomal protein L16 on susceptibility and accumulation of evernimicin.

Chemical mutagenesis of Staphylococcus aureus RN450 generated two strains that displayed a stable reduction (30- to 60-fold) in susceptibility to evernimicin. Cell-free translation reactions demonstrated that the resistance determinant was located in the ribosomal fraction. Compared to ribosomes isolated from a wild-type strain, ribosomes from the mutant strains displayed an 8- to 10-fold reduction in affinity for [(14)C]evernimicin. In contrast, the mutants displayed no alteration in either binding affinity or in vitro susceptibility to erythromycin. Exponential cultures of the mutant strains accumulated significantly less [(14)C]evernimicin than the wild-type strain, suggesting that accumulation is dependent on the high affinity that evernimicin displays for its binding site. Sequencing rplP (encodes ribosomal protein L16) in the mutant strains revealed a single base change in each strain, which resulted in a substitution of either cysteine or histidine for arginine at residue 51. Introduction of a multicopy plasmid carrying wild-type rplP into the mutant strains restored sensitivity to evernimicin, confirming that the alterations in rplP were responsible for the change in susceptibility. Overexpression of the mutant alleles in S. aureus RN450 had no effect on susceptibility to evernimicin, demonstrating that susceptibility is dominant over resistance.

Evernimicin binds exclusively to the 50S ribosomal subunit and inhibits translation in cell-free systems derived from both gram-positive and gram-negative bacteria.

Evernimicin (SCH 27899) is a new antibiotic with activity against a wide spectrum of gram-positive bacteria and activity against some gram-negative bacteria. Previous metabolic labeling studies indicated that evernimicin specifically inhibited protein synthesis in Staphylococcus aureus. Using a susceptible Escherichia coli strain, we demonstrated that evernimicin also inhibited protein synthesis in E. coli. In cell-free translation assays with extracts from either E. coli or S. aureus, evernimicin had a 50% inhibitory concentration of approximately 125 nM. In contrast, cell-free systems derived from wheat germ and rabbit reticulocytes were inhibited only by very high levels of evernimicin. Evernimicin did not promote transcript misreading. [(14)C]evernimicin specifically bound to the 50S subunit from E. coli. Nonlinear regression analysis of binding data generated with 70S ribosomes from E. coli and S. aureus and 50S subunits from E. coli returned dissociation constants of 84, 86, and 160 nM, respectively. In binding experiments, performed in the presence of excess quantities of a selection of antibiotics known to bind to the 50S subunit, only the structurally similar drug avilamycin blocked binding of [(14)C]evernimicin to ribosomes.

Olanzapine increases weight and serum triglyceride levels.

BACKGROUND: Previous studies have suggested that clozapine is associated with increases in both weight and serum triglyceride (but not cholesterol) levels. Because of the pharmacologic similarities between clozapine and olanzapine, we decided to evaluate if olanzapine use was associated with an increase in triglycerides. METHOD: Twenty-five inpatients (21 men, 4 women) were treated with olanzapine, and their outcomes were tracked prospectively in a medication utilization evaluation study. RESULTS: After 12 weeks on a mean +/- SD dose of 13.8+/-4.4 mg/day, weight increased a mean of 12 lb (5.4 kg; from 190+/-37 lb [85.5+/-16.7 kg] to 202+/-30 lb [90.9+/-13.5 kg]), while fasting triglycerides increased a mean of 60 mg/dL (from 162+/-121 mg/dL to 222+/-135 mg/dL). Both increases were significant at p < .05. Fasting total cholesterol did not increase. The triglyceride increase was even larger when we excluded 8 patients who received various interventions to lower lipid levels (e.g., pravastatin, low-fat diet) during the olanzapine trial. There was a strong association between weight change and triglyceride change (p < .02); after controlling for weight, analysis of covariance showed no independent increase in triglycerides. CONCLUSION: These results suggest olanzapine has significant effects on weight and serum triglyceride levels. Clinical implications are discussed.

Collection of genomic DNA by buccal swabs for polymerase chain reaction-based biomarker assays.

Studies in molecular and genetic epidemiology require a high-throughput, low cost, and reliable means of genomic DNA collection. Buccal (cheek) swabs have been proposed as a means of achieving these goals, but there is little information about the practical application of this approach. From January 1995 to December 1997, we processed 995 buccal swabs for use in polymerase chain reaction (PCR)-based genotype assays in the context of ongoing molecular epidemiologic studies. Six hundred forty-seven of these swabs were processed immediately after collection and 348 were received by mail. We were able to obtain at least one genotype from 99.7% (645 of 647) of fresh-processed and 97.4% (330 of 339) of mailed biosamples. A PCR success rate of 90.3% (2,546 genotypes from 2,819 assays) was achieved. Genotypes were obtained from 96.1% (1, 865 genotypes from 1,941 assays) of fresh-processed biosamples and 77.6% (681 genotypes from 878 assays) of mailed biosamples. PCR success rates at any single locus ranged from 92.6 to 98.8% (fresh-processed) and 75.5 to 79.6% (mailed). The PCR success rate among fresh-processed biosamples was significantly higher than among mailed biosamples (Fisher's exact test p < 0.0001), and more attempts were required to obtain a successful PCR result for mailed biosamples as compared to fresh-processed biosamples. For one locus (CYP3A4), a subset of mailed biosamples was purified if two or more PCR failures occurred. Additional genotypes were obtained in 58.3% of these previously failed biosamples. Time from biosample receipt to DNA extraction had no effect on PCR success. After storage of processed biosamples for as long as 3 years, there was no appreciable decrease in the rate of PCR success. These results suggest that adequate DNA for PCR-based applications can be obtained from buccal swabs, but sampling or processing considerations may be important in obtaining optimal results.

Separation and analysis of DNA sequence reaction products by capillary gel electrophoresis.

This paper demonstrates the potential of capillary gel electrophoresis with laser induced fluorescence detection as a tool for DNA sequence determination. Both synthetic oligonucleotides and single-stranded phage DNA were utilized as templates in the standard chain termination procedure. Primer molecules were tagged at the 5' end with the fluorescent dye, JOE. First, baseline resolution of a dA extended primer from 18 to 81 bases long, a total of 64 fragments, was observed. A second synthetic template was designed to yield alternating stretches of dA and dT extensions of the primer. Thirdly, the sequence reaction products from a synthetic oligonucleotide template containing all four bases was analyzed in four independent runs, one for each of the four base-specific reactions. In all cases, the expected number and patterns of peaks were observed by capillary gel electrophoretic analysis. Finally, separation of sequence reaction products generated with single-strand M13mp18 phage DNA as template exhibited baseline resolution of fragments differing in length by a single nucleotide and from 18 to greater than 330 bases total length.

Composite in situ vein bypass for upper extremity revascularization.

Chronic upper extremity arterial insufficiency is rare. Consequently, major reports specifically limited to the topic are scarce, and the clinical experience is small. In addition, symptomatology, diagnostic criteria, and guidelines for surgical management remain ill-defined. In the lower extremities, however, in situ vein bypass has been attempted for nearly three decades. This technique offers many advantages over traditional revascularization methods. Although the procedure has become popular for the lower extremity, no report of its use in the upper extremity is found in the literature. We report what may be the first case in which in situ bypass was used in the upper extremity for a threatened limb secondary to diabetic occlusive vascular disease complicated by a previous shunt used for hemodialysis. Revascularization of the upper extremity using the in situ vein bypass technique may offer a new alternative to traditional methods of revascularization.

Rapid separation and purification of oligonucleotides by high-performance capillary gel electrophoresis.

Picomole amounts of oligodeoxynucleotides [polydeoxyadenylic acids, (dA)40-60] were baseline resolved and analyzed in less than 8 min by high-performance capillary electrophoresis with polyacrylamide gels. In addition, fast analysis of a crude 70-mer oligodeoxynucleotide and a slab gel-purified 99-mer oligodeoxynucleotide was accomplished, demonstrating the ability of high-performance capillary electrophoresis to characterize rapidly synthesized oligonucleotides. Besides analytical separations, 800 ng of a primer (20-mer) was isolated in less than 20 min. The purified species was collected in water and subsequently used as a probe in a standard dot-blot analysis. The use of high-performance capillary electrophoresis for the analysis and purification of a variety of biopolymers is simple, rapid, and has the potential for automation.

Rapid separation of DNA restriction fragments using capillary electrophoresis.

Open-tube capillary electrophoresis has been applied to the separation of restriction fragments of DNA with a Tris-borate buffer containing 7 M urea and 0.1% sodium dodecyl sulfate. The importance of sample pretreatment and of the injection of heated samples has been demonstrated. In one separation, a DNA restriction fragment mixture from 72 to 23,130 base pairs (DRIgestTM III) (molecular weight range from 4.6.10(4) to 1.5.10(7] has been electrophoresed in 10 min on a column of 15 cm effective length. Over 600,000 plates have been obtained for individual peaks. Several of the peaks have been identified, by spiking slab gel electrophoretically purified components. Other examples of restriction fragment separations are illustrated in this paper. The results of this study when further validated with full characterization of individual species, open up the possibility of rapid restriction enzyme mapping.

DNA sequence and transcript mapping of MOD5: features of the 5' region which suggest two translational starts.

A mutation in the yeast nuclear gene MOD5 drastically reduces the biosynthesis of the modified base isopentenyladenosine in tRNAs located in different cellular compartments: the mitochondria and the nucleus or cytoplasm. Several lines of evidence strongly suggest that MOD5 is the structural gene encoding the tRNA-modifying enzyme delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase. DNA sequence analysis of MOD5 reveals an open reading frame of 428 amino acids. A set of mRNAs heterogeneous at both the 5' and 3' termini are transcribed from this gene. Although all of these transcripts initiate upstream of the first AUG codon of the open reading frame, a subset has an extremely short (greater than or equal to 1 base) 5' leader. Furthermore, in positions important for efficient initiation of translation and generally occupied by purines, this first AUG codon is flanked by a U (position -3) and a C (position +4). It is possible that two proteins, one with an amino-terminal extension of basic charge, could be generated from the MOD5 gene via differential translational starts.

Isolation and characterization of MOD5, a gene required for isopentenylation of cytoplasmic and mitochondrial tRNAs of Saccharomyces cerevisiae.

The mod5-1 mutation is a nuclear mutation in Saccharomyces cerevisiae that reduces the biosynthesis of N6-(delta 2-isopentenyl)adenosine in both cytoplasmic and mitochondrial tRNAs to less than 1.5% of wild-type levels. The tRNA modification enzyme, delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase, cannot be detected in vitro with extracts from mod5-1 cells. A characterization of the MOD5 gene would help to determine how the same enzyme activity in different cellular compartments can be abolished by a single nuclear mutation. To that end we have cloned the MOD5 gene and shown that it restores delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase activity and N6-(delta 2-isopentenyl)adenosine to tRNA in both the mitochondria and the nucleus/cytoplasm compartments of mod5-1 yeast cells. That MOD5 sequences are expressed in Escherichia coli and can complement an N6-(delta 2-isopentenyl)-2-methylthioadenosine-deficient E. coli mutant leads us to conclude that MOD5 is the structural gene for delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase.

Sequence and expression of four mutant aspartic acid tRNA genes from the mitochondria of Saccharomyces cerevisiae.

Expression of the mitochondrial tRNAAsp gene of Saccharomyces cerevisiae has been examined in five syn- mutants known to affect tRNAAsp function, and in a rho- mutant which accumulates precursor tRNAs. By comparison of wild-type versus mutant DNA sequence, the lesion in each syn- mutant has been identified as a single base change within the mitochondrial tRNAAsp structural gene. The mutant tRNAAsp genes are transcribed, and the transcripts can be processed to mature 4S-size tRNAAsp. The steady-state level of each mutant tRNAAsp is lower than that of wild-type tRNAAsp. The RNA from two of the syn- mutants contained a second, slow-migrating form of mitochondrial tRNAAsp which is correctly processed at the 5' end. We conclude that the lesions in the syn- mitochondrial tRNAAsp genes block neither transcription of these genes, nor 5'-end processing of the transcripts. The effect of each point mutation must be manifested at the level of 3'-end processing, or at a functional level.

In vitro transcription and promoter strength analysis of five mitochondrial tRNA promoters in yeast.

The in vitro transcriptional initiation sites of four yeast mitochondrial tRNA genes have been investigated in a run-off transcription assay. Precise initiation originating within the 9-nucleotide mitochondrial promoter sequence was detected for the phenylalanine, initiator formyl methionine, cysteine, and one of the two threonine tRNA genes. The relative promoter strength of each of these tRNA promoters as well as that for the previously described glutamate tRNA promoter was determined in a competition assay. This assay measured the utilization of a particular tRNA promoter relative to the amount of transcription arising from a control 14 S rRNA promoter present in the same reaction. The competition strength of the tRNAPhe, tRNAMetf, and tRNAGlu promoters is 20-fold greater than that for the tRNAThrACN and tRNACys promoters. Comparison of the nucleotide sequences at the +2 and +3 positions in the transcripts reveals a homology among the strong promoters not duplicated in the weak promoters.

A mutation in the tRNAAsp gene from yeast mitochondria. Effects on RNA and protein synthesis.

We have characterized a mutation that affects the tRNAAsp coded by yeast mitochondrial DNA. Comparison of the DNA sequences of the tRNAAsp gene from a wild type strain and the mutant demonstrates that the mutant differs by a C to U base change in position 72 of the tRNA. This mutation abolishes mitochondrial protein synthesis, presumably because the tRNAAsp made from this gene cannot be charged with aspartic acid (FAye, G., Bolotin-Fukuhara, M., and Fukuhara, H. (1976) in The Genetics and Biogenesis of Chloroplasts and Mitochondria (Bucher, C. T., Neupert, W., Sebalt, W., and Werner, S., eds) pp. 547-555, North Holland Publishing Co., Amsterdam). It also reduces the amount of tRNAAsp transcripts in the mutant as compared to the wild type.