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.     

Spatial Analysis of Slowly Oscillating Electric Activity in the Gut of Mice Using Low Impedance Arrayed Microelectrodes

Smooth and elaborate gut motility is based on cellular cooperation, including smooth muscle, enteric neurons and special interstitial cells acting as pacemaker cells. Therefore, spatial characterization of electric activity in tissues containing these electric excitable cells is required for a precise understanding of gut motility. Furthermore, tools to evaluate spatial electric activity in a small area would be useful for the investigation of model animals. We thus employed a microelectrode array (MEA) system to simultaneously measure a set of 8×8 field potentials in a square area of ∼1 mm². The size of each recording electrode was 50×50 µm², however the surface area was increased by fixing platinum black particles. The impedance of microelectrode was sufficiently low to apply a high-pass filter of 0.1 Hz. Mapping of spectral power, and auto-correlation and cross-correlation parameters characterized the spatial properties of spontaneous electric activity in the ileum of wild-type (WT) and W/W^v mice, the latter serving as a model of impaired network of pacemaking interstitial cells. Namely, electric activities measured varied in both size and cooperativity in W/W^v mice, despite the small area. In the ileum of WT mice, procedures suppressing the excitability of smooth muscle and neurons altered the propagation of spontaneous electric activity, but had little change in the period of oscillations. In conclusion, MEA with low impedance electrodes enables to measure slowly oscillating electric activity, and is useful to evaluate both histological and functional changes in the spatio-temporal property of gut electric activity.     

A 5-bp Insertion in Mip Causes Recessive Congenital Cataract in KFRS4/Kyo Rats

We discovered a new cataract mutation, kfrs4, in the Kyoto Fancy Rat Stock (KFRS) background. Within 1 month of birth, all kfrs4/kfrs4 homozygotes developed cataracts, with severe opacity in the nuclei of the lens. In contrast, no opacity was observed in the kfrs4/+ heterozygotes. We continued to observe these rats until they reached 1 year of age and found that cataractogenesis did not occur in kfrs4/+ rats. To define the histological defects in the lenses of kfrs4 rats, sections of the eyes of these rats were prepared. Although the lenses of kfrs4/kfrs4 homozygotes showed severely disorganised fibres and vacuolation, the lenses of kfrs4/+ heterozygotes appeared normal and similar to those of wild-type rats. We used positional cloning to identify the kfrs4 mutation. The mutation was mapped to an approximately 9.7-Mb region on chromosome 7, which contains the Mip gene. This gene is responsible for a dominant form of cataract in humans and mice. Sequence analysis of the mutant-derived Mip gene identified a 5-bp insertion. This insertion is predicted to inactivate the MIP protein, as it produces a frameshift that results in the synthesis of 6 novel amino acid residues and a truncated protein that lacks 136 amino acids in the C-terminal region, and no MIP immunoreactivity was observed in the lens fibre cells of kfrs4/kfrs4 homozygous rats using an antibody that recognises the C- and N-terminus of MIP. In addition, the kfrs4/+ heterozygotes showed reduced expression of Mip mRNA and MIP protein and the kfrs4/kfrs4 homozygotes showed no expression in the lens. These results indicate that the kfrs4 mutation conveys a loss-of-function, which leads to functional inactivation though the degradation of Mip mRNA by an mRNA decay mechanism. Therefore, the kfrs4 rat represents the first characterised rat model with a recessive mutation in the Mip gene.     

The APC/C activator Cdh1 regulates the G2/M transition during differentiation of placental trophoblast stem cells

Differentiation of placental trophoblast stem (TS) cells to trophoblast giant (TG) cells is accompanied by transition from a mitotic cell cycle to an endocycle. Here, we report that Cdh1, a regulator of the anaphase-promoting complex/cyclosome (APC/C), negatively regulates mitotic entry upon the mitotic/endocycle transition. TS cells derived from homozygous Cdh1 gene-trapped (Cdh1^GT/GT) murine embryos accumulated mitotic cyclins and precociously entered mitosis after induction of TS cell differentiation, indicating that Cdh1 is required for the switch from mitosis to the endocycle. Furthermore, the Cdh1^GT/GT TS cells and placenta showed aberrant expression of placental differentiation markers. These data highlight an important role of Cdh1 in the G2/M transition during placental differentiation.     

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.     

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.