Identification of the coding region for the putidaredoxin reductase gene from the plasmid of Pseudomonas putida

The first 12 NH₂-terminal amino acids of the Pseudomonas putida putidaredoxin reductase were shown to be Met-Asn-Ala-Asn-Asp-Asn-Val-Val-Ile-Val-Gly-Thr. Comparison of these data with the DNA sequence of the BamHI-HindIII 197-base fragment derived from the PstI 2.2-kb fragment obtained from the P. putida plasmid showed that the putidaredoxin reductase gene was downstream from the cytochrome P-450 gene and the intergenic region had the 24-nucleotide sequence TAAACACATGGGAGTGCGTGCTAA. The Shine-Dalgarno sequence GGAG was detected in this region. The initiating triplet for the reductase gene was GTG, which normally codes for valine, but in the initiating codon position codes for methionine. From the amino acid sequence and X-ray data comparisons with other flavoproteins, what appears to be the AMP binding region of the FAD can be recognized in the NH₂-terminal portion of the reductase involving residues 5–35.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Solution Radioactivated by Hadron Radiation Can Increase Sister Chromatid Exchanges

When energetic particles irradiate matter, it becomes activated by nuclear reactions. Radioactivation induced cellular effects are not clearly understood, but it could be a part of bystander effects. This investigation is aimed at understanding the biological effects from radioactivation in solution induced by hadron radiation. Water or phosphate buffered saline was activated by being exposed to hadron radiation including protons, carbon- and iron-ions. 1 mL of radioactivated solution was transferred to flasks with Chinese hamster ovary (CHO) cells cultured in 5 mL of complete media. The induction of sister chromatid exchanges (SCE) was used to observe any increase in DNA damage responses. The energy spectrum and the half-lives of the radioactivation were analyzed by NaI scintillation detector in order to identify generated radionuclides. In the radioactivated solution, 511 keV gamma-rays were observed, and their half-lives were approximately 2 min, 10 min, and 20 min. They respectively correspond to the beta+ decay of ¹⁵O, ¹³N, and ¹¹C. The SCE frequencies in CHO cells increased depending on the amount of radioactivation in the solution. These were suppressed with a 2-hour delayed solution transfer or pretreatment with dimethyl sulfoxide (DMSO). Our results suggest that the SCE induction by radioactivated solution was mediated by free radicals produced by the annihilated gamma-rays. Since the SCE induction and DMSO modulation are also reported in radiation-induced bystander effects, our results imply that radioactivation of the solution may have some contribution to the bystander effects from hadron radiation. Further investigations are required to assess if radioactivation effects would attribute an additional level of cancer risk of the hadron radiation therapy itself.     

Evolutionary relationships between laboratory mice and subspecies of Mus musculus based on the genetic study of pancreatic proteinase loci,Prt-1, Prt-2, Prt-3, and Prt-6

Various patterns of mouse pancreatic proteinase activity bands were observed on agarose gel electrophoresis. Prt-1 ^a and Prt-1 ^b genes control the positive (PRT-1A) and negative (PRT-1B) expression of tryptic band V, respectively; Prt-2 ^a and Prt-2 ^b correspond to chymotryptic bands II (PRT-2A) and III (PRT-2B); Prt-3 ^a and Prt-3 ^b control the low (PRT-3A) and high (PRT-3B) tryptic activities of band IV; the Prt-1 and Prt-3 loci are closely linked on the same chromosome; Prt-6 ^a and Prt-6 ^b correspond to tryptic bands I (PRT-6A) and I (PRT-6B). Twenty-four laboratory strains from the United States showed the phenotype PRT-1A, PRT-3A, and PRT-2A. Of laboratory strains established in Europe, 6 showed PRT-1A, PRT-3A, and PRT-2A, and 10 had PRT-1B, PRT-3A, and PRT-2A bands. Most wild mice around the world and their descendants showed the phenotype PRT-1B, PRT-3B, and PRT-2A. Only the phenotype of M. m. brevirostris was PRT-1A, PRT-3A, and PRT-2A, which was the same as most laboratory inbred strains. PRT-2B was observed mainly in Japanese (M. m. molossinus) and Korean (M. m. yamashinai) wild mice. PRT-6B was detected only in Mus spicilegus and Mus caroli, but all other mice including wild populations and laboratory strains showed PRT-6A. New biochemical phenotypes such as PRT-2C and PRT-3C were also found in this study.     

Polymorphism of T-cell receptor genes among laboratory and wild mice: Diverse origins of laboratory mice

Southern blots of genomic DNA from 23 strains of laboratory mice and 19 individual wild mice were examined for restriction fragment length polymorphisms in their loci encoding the T-cell receptors (Tcr): the constant regions of the α, β, and γ chains (C _α,C _β, andC _γ) and a variable region family of the β chain (V _β8). Only a few polymorphisms were observed for each locus in the laboratory mice after using three restriction enzymes,Bam HI,Eco RI, andHind III. All the laboratory mice examined fall into one of two types for theC _α,C _β andV _β8 loci and one of three types for theC _γ. These types are found in some of the wild mice studied, indicating that they were already present in the founder mice of laboratory mouse strains. In contrast, theTcr genes are highly polymorphic among wild mice. Analysis of the polymorphisms in these loci suggests that laboratory mice have inherited their genes not only fromMus musculus domesticus, but also from other subspecies, and much more than previously believed from Asian subspecies.     

The locus controlling liver GM1(NeuGc) expression is mapped 1 cM centromeric to H-2K

The polymorphic variation of liver GM1 (NeuGc) ganglioside was found in inbred strains of the mouse. The genetic analysis using C57BL/10 (GM1-negative) and SWR (GM1-positive) mice revealed that a single autosomal gene (Ggm-1) was involved in the expression of liver GM1(NeuGc) and that C57BL/10 mice lacking GM1(NeuGc) expression carried a defective gene on Ggm-1. Since our previous study on H-2 congenic mice indicated that Ggm-1 was linked to the H-2 complex, in this study we measured recombination frequencies among Ggm-1, Go-1 and H-2K in the backcross progeny between (C57BL/10 × SWR)F₁ and C57BL/10. Ggm-1 was mapped 1 cM centromeric to H-2K on chromosome 17.Abbreviations used in this paper GM1(NeuGc) Gal1-3GalNAc1-4 (NeuGc2-3)Gal1-4Glc1-ceramide - GM2(NeuGc) Gal1-4(Neu Gc2-3)Gal1-4Glc1-ceramide - GM3(NeuGc) NeuGc2-3Gal1-4 Glc1-ceramide - GD1a(NeuGc) NeuGc2-3Gal1-3GalNAc1-4 (NeuGc2-3)Gal1-4Glc1-ceramide     

Effects of dietary retinoid and triglyceride on the lipid composition of rat liver stellate cells and stellate cell lipid droplets

Hepatic stellate cells store the majority of the liver's retinoid (vitamin A) reserves as retinyl esters in stellate cell lipid droplets. A study was conducted to explore the effects of differences in dietary retinoid and triglyceride intake on the composition of the stellate cell lipid droplets. Weanling rats were placed on one of five diets that differed in retinoid or triglyceride contents. The dietary groups were: 1) control (2.4 mg retinol (as retinyl acetate)/kg diet and 20.5% of the calories supplied by triglyceride (as peanut oil]; 2) low retinol (0.6 mg retinol/kg diet and control triglyceride levels); 3) high retinol (24 mg retinol/kg diet and control triglyceride levels); 4) low triglyceride (2.4 mg retinol/kg diet and 5% of the calories supplied by triglyceride); and 5) high triglyceride (2.4 mg retinol/kg diet and 45% of the calories supplied by triglyceride). Stellate cells were isolated using the pronase-collagenase method and stellate cell lipid droplets were isolated by differential centrifugation. The levels of retinoids and other lipids were measured by high performance liquid chromatography. The stellate cells from control rats contained 113 micrograms total lipid/10(6) cells. Control stellate cell lipid droplets had the following mean percent lipid composition: 39.5% retinyl ester; 31.7% triglyceride; 15.4% cholesteryl ester; 4.7% cholesterol; 6.3% phospholipids; and 2.4% free fatty acids. Both the concentration of stellate cell lipids and the composition of stellate cell lipid droplets were markedly altered by changes in dietary retinoid. The low and high retinol groups contained, respectively, 82 and 566 micrograms total lipid/10(6) cells, with retinyl ester representing, respectively, 13.6% and 65.4% of the lipid present in the stellate cell lipid droplets. Low and high triglyceride groups were similar to controls in both stellate cell lipid content and the composition of the stellate cell lipid droplets. These findings indicate that the composition of stellate cell lipid droplets is strongly regulated by dietary retinoid status but not by dietary triglyceride intake.     

Molecular cloning of mouse beta 2-glycoprotein I and mapping of the gene to chromosome 11.

beta 2-Glycoprotein I (beta 2 GPI), a plasma protein that binds to anionic phospholipids, is composed of five repeating units called a short consensus repeat (SCR), which is found mostly in the regulatory proteins of the complement system. Recently the human beta 2 GPI gene has been assigned to chromosome 17, not to chromosome 1 where most of the genes of the SCR-containing proteins are clustered. In this report, we have isolated a full-length cDNA clone of mouse beta 2 GPI and determined the chromosomal localization of the gene. The amino acid sequence deduced from the nucleotide sequence of mouse beta 2 GPI revealed 76.1% identity with that of human beta 2 GPI. A genetic mapping by in situ hybridization and linkage analysis using 50 backcross mice has shown that the mouse beta 2 GPI gene (designated B2gp1) is located on the terminal portion of the D region of chromosome 11, closely linked to Gfap, and is 18 cM distal to Acrb, extending a conserved linkage group between mouse chromosome 11 and human chromosome 17. On the basis of these results, the evolutionary relationships among the SCR-containing proteins are discussed.     

The amplified long genomic sequence (ALGS) located in the centromeric regions of mouse chromosomes.

We reported previously that the haploid genome of standard strains of laboratory mice contains approximately 70 copies of an amplified long genomic sequence, designated ALGS, that includes a retroposon of the gene for elongation factor 2 (MER). The length of each repeating unit is more than 60 kb, and the sequence of the unit is highly conserved among the repeats. In the present study, Southern blot analysis of the genomes of wild rodents demonstrated that the ALGS is present in all subspecies of Mus musculus and is abundant in M. spicilegus, whereas it is absent in M. spretus as well as in Rattus and other closely related genera. This result indicates that the amplification occurred after the species differentiation with the genus Mus and at least prior to the differentiation of subspecies of M. musculus. To locate chromosomal positions of the ALGS, in situ hybridization was carried out with laboratory strains and wild mice. It appears that the ALGS is located in the centromeric regions of most chromosomes in laboratory mice, M. musculus and M. spicilegus, whereas no positive signals were observed with M. spretus, in accordance with the results from the Southern blotting analysis.     

Gamma-MSH and its related peptides do not augment ACTH-induced steroidogenesis in isolated rat adrenal cells

In a sensitive ACTH bioassay system using isolated rat adrenal cells, we tested the effect of gamma-MSH related peptides on ACTH-induced steroidogenesis. Peptides, including synthetic gamma1-, gamma2-, gamma3- and Lys-gamma3-MSH, exerted no effect in augmenting ACTH-induced steroidogenesis. None of the 16 kilodalton fragment of ACTH/beta-lipotropin precursor and its cleaved fragment had such an activity. The results are in contrast with previous reports concerning ACTH-potentiating activity of gamma-MSH related peptides and, therefore, indicate the necessity of further investigation of the principle involved in this unique biological activity.