Construction of a high-throughput rat genetic mapping system with 466 arbitrarily primed-representational difference analysis markers.

Linkage mapping of quantitative trait loci (QTLs) requires genetic markers that can be efficiently genotyped for a large number of individuals. To isolate genetic markers suitable for this purpose, we previously established the arbitrarily primed RDA (AP-RDA) method. Dot-blotting AP-PCR products (AP-amplicons) onto filters at a high density and hybridization of the filters with the AP-RDA markers made it possible to genotype a large number of individuals simultaneously for multiple loci. In this study, by using 25 primers or primer combinations, we isolated a total of 419 AP-RDA markers by subtracting the AP-amplicon of BUF rats from that of ACI rats, and vice versa. By combining 47 previously isolated markers, a rat genetic map was drawn with 466 AP-RDA markers. Between two given strains of rats other than ACI and BUF, the average informativeness of the markers was 38%. As for the intercross of ACI and BUF rats, 12 selected primers served to genotype 259 loci. In addition, the amounts and quality of genomic DNA to be used for AP-PCR were examined to guarantee reliable genotyping. Now, initial genome scanning of the rat for linkage analysis can be performed efficiently using this mapping system with AP-RDA markers.

Detection of mutagenicity in Ames test using a metalloporphyrin/oxidant model system for cytochrome P450.

A chemical model system for cytochrome P450, a porphyrin and an oxidant, was used in Ames assay as a substitute for S9 mix. In the presence of tetrakis(pentafluorophenyl)porphyrinatoiron(III) chloride [Fe(F5P)Cl] and tert-butyl hydroperoxide (t-BuOOH), mutagenicity of N-nitrosodibutylamine (NDB) in Salmonella typhimurium TA1535 was detected. The mutagenicity depended on the pre-incubation period, and also on the concentration of an oxidant and of bacteria. In the chemical model system, pH affected the mutagenicity of NDB, which suggested that as observed in an enzymatic activating system, the mutagenicity was due to the labile alkylating species which was derived from NDB activated in the chemical activation system and was sensitive to pH. Under the optimum conditions; a higher concentration of an oxidant, a higher concentration of bacterial culture, and a weakly acidic medium, mutagenicity of N-nitrosodipropylamine in S. typhimurium TA1535 was also detected. Besides N-nitrosodialkylamines, 2-aminofluorene (2-AF) and benzo[a]pyrene (BaP) were also used as mutagens. Mutagenicity of 2-AF and BaP in S. typhimurium TA1538 were both detected in the same system as used in detecting the mutagenicity of N-nitrosodialkylamines. Ames test using a metalloporphyrin/oxidant model system makes it possible to detect mutagenicity derived from both base pair substitution mutagens and frameshift mutagens without using enzymatic activating system. These results demonstrate that the assay with the chemical model system is useful in detecting unstable unknown active mutagens or investigating the mechanisms of the metabolic pathway of mutagens or carcinogens in a protein-free medium.

Interaction of 5SrRNA-L5 protein complex, methionyl-tRNA, and methionyl-tRNA synthetase in the macromolecular ARS complex.

Rat liver cytosol was incubated with a trace amount of rat liver 5SrRNA which was highly labeled at the 3'-end with cytidine 3',5'-[5'-32P]biphosphate, and with [35S]methionine in the presence of ATP mixture, and then with an antibody against ribosomal protein L5. The mixture was analyzed by protein A-Sepharose chromatography. The following results were obtained. (i) The eluate with glycine-HCl buffer (pH 3.0) from the protein A-Sepharose column contained an overlapping peak of 32P- and 35S-radioactivities. In a control experiment using the same amount of 32P-labeled Escherichia coli 5SrRNA with the same specific activity, no fraction of the eluate contained 32P-radioactivity. (ii) The fractions containing both 32P- and 35S-radioactivities from the protein A-Sepharose column were crosslinked by UV irradiation. The products was subjected to PAGE, and RNA in each gel slice was eluted and purified. The fraction containing both 32P- and 35S-radioactivities was present in a region of somewhat higher molecular weight than that of 5SRNP, whereas very low 32P- and 35S-radioactivities were present in this region in the control experiment without UV irradiation. This finding suggested that [35S]methionyl-tRNA interacted with 32P-labeled 5SRNP. (iii) The fraction containing overlapping 32P- and 35S-radioactivities described above was subjected to Sephadex G-150 chromatography. The component containing both radioactivities was distributed in the region corresponding to molecular weights of 10,000 to 250,000 with a peak at about 200,000, suggesting the presence of a complex containing Met-RS (Mr 108,000), 5SRNP (Mr 74,000), and methionyl-tRNA (Mr 25,000). Furthermore, this fraction showed definite Met-RS activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Presence of role of the 5SrRNA-L5 protein complex (5SRNP) in the threonyl- and histidyl-tRNA synthetase complex in rat liver cytosol.

A complex containing Thr-RS and His-RS was purified about 1000 to 2000-fold from rat liver cytosol by successive column chromatographies on Sephadex G-200, Phenyl-Sepharose CL-4B, and tRNA-Sepharose. The ratio of the specific activity of Thr-RS and His-RS was relatively constant throughout the purification steps, suggesting that the two synthetases were co-purified as a complex. Chromatographic analyses of the tRNA-Sepharose fraction by Sephadex G-150 column chromatography showed the presence of a hybrid form of the Thr-RS monomer and the His-RS monomer in addition to dimer forms of both enzymes from the pattern of activity of both enzymes. The monomer form of Thr-RS showed high activity comparable to the dimer form and the monomer form of His-RS showed definite activity. An association form of Thr-RS and His-RS dimers was detected by Sephadex G-200 chromatography of rat liver cytosol. Northern blot analysis of RNA prepared from the tRNA-Sepharose fraction showed the presence of 55SrRNA blot analysis of the tRNA-Sepharose fraction using an antibody against ribosomal protein L5, showed the presence of ribosomal protein L5 in this fraction. These findings suggest that the presence of a 5SRNA-L5 protein complex (5SRNP) in the Thr-RS and His-RS complex. 5SRNP enhanced the activity of Thr-RS in a freshly prepared tRNA-Sepharose fraction. It also enhanced the activity of the rat liver cytosol for the attachment of [3H]threonine to endogenous tRNA. This activity was inhibited by an antibody against protein L5, and the inhibition was reversed by addition of 5SRNP. These results indicate that 5SRNP plays a role as a positive effector of Thr-RS in the complex.

Occurrence of 5SrRNA in high molecular weight complexes of aminoacyl-tRNA synthetases in a rat liver supernatant.

The 5SrRNA in the rat liver postmicrosomal supernatant was investigated. Acrylamide gel electrophoresis and Northern blot analysis showed that most of the 5SrRNA was present in the fractions obtained on high molecular weight regions separated by Sephadex G-200 column chromatography of the supernatant, which contained the bulk of the methionyl-tRNA synthetase (Fraction I) and tyrosyl-tRNA synthetase (Fraction II). A high molecular weight complex containing nine aminoacyl-tRNA synthetases [Mirande, M., LeCorre, D., & Waller, J.-P. (1985) Eur. J. Biochem. 147, 281-289] was purified by fractional precipitation with polyethylene glycol 6000, gel filtration on Bio-Gel A-1.5m, and finally tRNA-Sepharose column chromatography, which gave two fractions. Fraction B showed the activities of nine aminoacyl-tRNA synthetases and gave protein bands corresponding to eight previously identified enzymes on SDS-PAGE. Fraction A, eluted with a lower KCl concentration than Fraction B, showed lower activities than fraction B of eight of the aminoacyl-tRNA synthetases, the exception being prolyl-tRNA synthetase. The staining patterns with ethidium bromide of the RNAs after PAGE showed 5SrRNA bands for Fraction A but not for Fraction B. However, Northern blot analysis indicated that 5SrRNA was present in both Fractions A and B. The staining pattern after SDS-PAGE of Fraction A with Coomassie Brilliant Blue showed several protein bands in addition to those observed for Fraction B, one of which, with a staining intensity comparable with those of other bands, was located at the same position as ribosomal protein L5, which is the protein moiety of the 5SrRNA-L5 protein complex of ribosomal 60S subunits.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of 5SrRNA as a positive effector of some aminoacyl-tRNA synthetases in macromolecular complexes, with specific reference to methionyl-tRNA synthetase.

1) Rat liver 5SrRNA enhanced the activity of methionyl-tRNA synthetase in the macromolecular aminoacyl-tRNA synthetase complex (Fraction B) purified from a rat liver supernatant. 5SrRNA-L5 protein complexes (5SrRNP) had similar effects, whereas other ribosomal RNAs and E. coli 5SrRNA had no effect. 2) 5SrRNA increased the activity of the complex for methionine-dependent ATP-PPi exchange. 3) 5SrRNA increased the activities of methionyl-, arginyl-, and isoleucyl-tRNA synthetases in the complex, but scarcely affected its leucyl-, lysyl-, and glutamyl-tRNA synthetase activities. 4) 5SrRNA increased the activities of the rat liver supernatant for the attachment of [35S]methionine, [3H]isoleucine, [3H]lysine, [3H]proline, [3H]threonine, [3H]tyrosine, and [3H]phenylalanine to endogenous tRNA markedly, and those for [3H]leucine, [3H]arginine, [3H]aspartic acid, and [3H]histidine slightly, but did not affect those for [3H]glutamic acid, [3H]glycine, [3H]valine, [3H]alanine, and [3H]tryptophan. 5) Preincubation of the rat liver supernatant with an antibody against Artemia salina ribosomal protein L5, that cross-reacted with the rat liver ribosomal protein L5, decreased the attachment of [35S]methionine and [3H]isoleucine to endogenous tRNA, and 5SrRNA and 5SRNP enhanced these activities of the supernatant preincubated with antibody. On the other hand, the antibody did not affect that for [3H]alanine. Immune dot blot analysis using the antibody against L5 showed the presence of immunologically the same protein as L5 in the liver supernatant. Northern blot analysis of RNA in the immunoprecipitate prepared from the liver supernatant incubated with the antibody against L5 indicated that 5SrRNA was complexed with L5.(ABSTRACT TRUNCATED AT 250 WORDS)

Two types of autoantibodies to adrenal medullary cells in type 1 (insulin-dependent) diabetic patients: prevalence, properties and implications.

Complement-fixing adrenal medullary antibodies were examined in sera from 170 (114 Type 1 and 56 Type 2) diabetic patients and normal subjects by indirect immunofluorescence methods. Two types of antibodies were detected; one showed a homogeneous immunofluorescence pattern (homogeneous-type) and the other a spotty pattern (spotty-type) in the cytoplasm of adrenal medullary cells. Both antibodies were IgG class and adrenal medulla-specific. The prevalence of the homogeneous-type was significantly higher in Type 1 diabetic patients with disease duration under 1 year (36%) than in those with duration of 1 year or more (1.1%), in Type 2 diabetic patients (1.8%) or in normal subjects (0%; P less than 0.01). Conversely, the prevalence of the spotty-type was not significantly different among all subjects examined (3.6-4.5%). The epitope for the homogeneous-type is likely to be a glycoconjugate since binding of this antibody was abolished after periodate oxidation. The epitope for spotty-type antibody is considered to be a peptide since it was trypsin sensitive. Patients who were positive for the homogeneous-type were also positive for islet cell antibodies, although their antibody titers were not correlated. We conclude that (1) adrenal medullary antibodies are of homogeneous-type or spotty-type and the antigenic determinants of these antibodies are different, and (2) the prevalence of the homogeneous-type is significantly higher in newly diagnosed Type 1 diabetic patients and its presence is associated with that of islet cell antibodies.

Effects of gastrectomy on insulin secretion in rats.

To examine the relative importance of the stomach for the enteroinsular axis, the portal insulin secretion was measured in the rats with gastrectomy, after intraduodenal (I.D.) infusion of 0.5 g/kg glucose and after intravenous (I.V.) infusion of the same deal of glucose, respectively. Portal plasma gastrin was decreased from 100 pg/ml to 5 pg/ml by gastrectomy. Blood sugar level after I.D. and I.V. infusion of glucose in the rats with gastrectomy was similar to controls. Insulin release to I.D. infusion of glucose was slightly lower at 30 minutes in the gastrectomized rats compared with controls, while insulin secretion to I.V. infusion of glucose was similar to controls. These results suggest that endogenous gastrin has no insulinotropic effect and that the stomach has no direct effect on glucose-induced insulin release.