Tomomasa Watanabe

AMP deaminase isozymes in human tissues

In human, there are four AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) isozymes: E1, E2, M and L. Chromatographic, electrophoretic and immunological studies showed the existence of isozymes E1 and E2 in erythrocytes, isozyme M in muscle and isozyme L in liver and brain. The tissues such as heart, kidney and spleen contained isozymes E1, E2 and L. Isozymes E1, M and L were isolated as apparently homogeneous preparations. The three isozymes were all tetramers composed of identical subunits, but differing slightly in molecular weight; isozyme E1 showed a subunit molecular weight of 80,000, isozyme M 72,000 and isozyme L 68,000. They were immunologically different from one another. The antisera precipitated only the corresponding enzyme and did not precipitate any other isozyme. The three isozymes were also different in kinetic and regulatory properties. Isozyme E2 was very similar to isozyme E1 in immunological an kinetic properties, although isozyme E2 could be separated from isozyme E1 by phosphocellulose chromatography, and zonal electrophoresis.

A molecular genetic linkage map of mouse chromosome 19, including thelpr, Ly-44, andTdt genes

The mouselpr gene, which is an autosomal recessive gene causing autoimmune disease with features of human systemic lupus erythematosus and eventually death from severe immune-complex glomerulonephritis, has been mapped on chromosome 19. To determine its exact chromosomal location, a three-point backcross was carried out by mating (MRL/MpJ-lpr/lpr × MOL-MIT)F1 × MRL/MpJ-lpr/lpr using the genesLy-44 (lymphocyte differentiation antigen-44) andTdt (terminal deoxynucleotidyl transferase) as markers. The following order of genes is proposed, with the distances between genes given in parentheses: centromere-Ly-44 (19.3 cM)-lpr (6.1 cM)-Tdt-telomere. TheLy-44a andTdta alleles are found in all laboratory strains and in the wild Western European subspecies,domesticus andbrevirostris. In contrast, theLy-44b andTdtb alleles are found in some Asian subspecies, Chinese mice of wild origin,yamashinai andmolossinus. Furthermore the thirdTdt allele,Tdtc, is detected incastaneus.

Polymorphism of mitochondrial DNA in pigs based on restriction endonuclease cleavage patterns.

Restriction endonuclease cleavage patterns of mitochondrial DNA (mtDNA) of pigs and Japanese wild boars were analyzed using 17 enzymes which recognize six nucleotides. The map of cleavage sites was made by double-digestion methods. Polymophism of mtDNA was detected in the digestion by BglII, EcoRV, ScaI, and StuI. The restriction cleavage patterns were identical among the breeds of Landrace, Hampshire, Duroc I, and Large White I (A type). The patterns of Large White II were the same as those of Japanese wild boars (B type). A difference between the A type and the B type of mtDNA was found in the case of three restriction enzymes, BglII, ScaI, and StuI, and the nucleotide alterations between them were estimated as more than six. On the other hand, a difference between mtDNA from almost all pigs and mtDNA from Duroc II was detected using EcoRV. We suggest that the difference of mtDNA between the A type and the B type of mtDNA could result from the different origin of boars, that is, whether they were of European or Asian origin.

Mouse Mx2 protein inhibits hantavirus but not influenza virus replication.

Summary. The antiviral potential of Mx2 protein remains unknown, because the Mx2 gene in commonly used strains of laboratory mice is nonfunctional. Our previous study showed that functional Mx2 protein in some feral-origin strains was induced upon interferon treatment, was localized in the cytoplasm, and inhibited vesicular stomatitis virus replication. In the present study, we have demonstrated that the embryonic fibroblastic cells from a feral-origin strain (SPR) expressed 74 kDa Mx2 protein, which prevented the accumulation of viral transcripts and proteins of hantaviruses when the Mx2 gene was constitutively expressed in transfected Vero cells. Furthermore, the cells showed significantly lower titers of the virus than control cells. In contrast, influenza virus replication was not affected by the expression of Mx2 protein in the Vero cells.

Characterization and Expression of the Mx1 Gene in Wild Mouse Species

The mouse Mx1 gene encodes an interferon(IFN)-inducible nuclear protein and confers resistanceto influenza virus infection. The standard laboratorymouse strains all carry the Mx1- allele andare susceptible to influenza virus. In this study, severalmouse strains established from wild mice were tested todetermine their Mx1+ or Mx1-allele status with polymerase chain reaction-restrictionfragment length variation (PCR-RFLV), sequence analysis, reversetranscription (RT)-PCR, and immunofluorescence staining.All of the mouse strains originating from wild mice werefound uniformly to carry the Mx1+ allele.Therefore, it is conceivable that the Mx1+allele in wild populations serves a function againstsome pathogens related to orthomyxoviruses. The PCR-RFLVand sequence analysis allowed us to classify theMx1+ alleles of the laboratory and wild-origin mouse strainsinto distinct classes. RT-PCR and immunofluorescencestaining demonstrated that the Mx1 transcripts andproteins were induced by IFN-alpha/beta in macrophages from wild mouse species.

A Naturally Occurring Variant of Porcine Mx1 Associated with Increased Susceptibility to Influenza Virus In Vitro

Mx1 has been implicated in resistance to the influenza virus. We have now identified four alleles of the Mxl gene in domesticated breeds of pigs. Two of the alleles encode deletion variants (a 3-bp deletion in exon 13 and an 11-bp deletion in exon 14), which might be expected to interfere with Mx activity. The porcine Mxl genes corresponding to wild type, the 3-bp deletion mutant, and the 11-bp deletion mutant were cloned and expressed in NIH3T3 cells, and the antiviral activity for influenza virus was assayed. Virus yield was observed to be 10–100-fold greater with the 11-bp deletion allele than that for wild type and the 3-bp deletion alleles. The results suggest that the 11-bp deletion type is lacking antiviral activity able to contribute to the interference of influenza virus replication.

Pig mitochondrial DNA: polymorphism, restriction map orientation, and sequence data

Restriction endonuclease cleavage patterns of mitochondrial DNA (mtDNA) in pigs were analyzed using 18 enzymes which recognize six nucleotides and 1 four-nucleotide-recognizing enzyme. Pigs including Taiwan native breeds and miniature strains maintained in Japan were examined in this study; four commercial breeds of pigs and Japanese wild boars have been investigated earlier [Watanabe, T., et al. (1985). Biochem. Genet.23:105]. mtDNA polymorphisms were observed in the cleavage patterns of five restriction enzymes, Bg1II, EcoRV, ScaI, StuI, and TaqI. The results support the previous hypothesis that pigs must be derived from two different maternal origins, European and Asian wild boars, and that a breed, Large White, arises from both European and Asian pigs. Two HindIII cleavage fragments were cloned into the HindIII site of M13mp10 and were partially sequenced by the dideoxynucleotide-chain termination method. Furthermore, DraI and StuI cleavage sites were newly determined on the restriction endonuclease map. On the basis of these results, the restriction endonuclease cleavage map of pig mtDNA was rewritten. Comparing sequence data of pig mtDNA at 237 positions with those of cow, human, mouse, and rat mtDNA, the sequence difference, silent and replacement changes, and transitions and transversions among mammalian species were estimated. The relationships among them are discussed.

Mouse Mx2 gene: organization, mRNA expression and the role of the interferon-response promoter in its regulation

Mx is an interferon (IFN)-inducible intracellular protein found in various vertebrates that mediates resistance against negative-strand RNA viruses. We have demonstrated previously that feral mouse strains are able to express a functional Mx2 mRNA, while that of the inbred laboratory strains was non-functional because of a single nucleotide insertion in the open reading frame, and under the detectable level. In the present study, we examined the regulation of Mx2 expression in vivo using a congenic mouse carrying Mx1 and Mx2 genes derived from feral strain SPR. Mx2 mRNA was induced strongly in the spleen, ovary and white adipose tissue after the treatment with IFNalpha/beta. Furthermore, we identified the structure of the Mx2 gene. It consists of 14 exons, greatly homologous to the Mx1 gene. The promoter region of Mx2 contained two putative IFN-stimulated response elements (ISREs). We found that the proximal ISRE site positioned between -69 and -55 was essential to IFNalpha/beta-induced transcription by transient transfection assay using reporter gene constructs with mutants of the Mx2 promoter. These results indicate the similarity of the mechanisms of mRNA induction among Mx genes.

cDNA sequence analysis of CP94: Rat lens fiber cell beaded-filament structural protein shows homology to cytokeratins

To study the molecular structure of the gene responsible for a lens fiber cell beaded-filament structural protein of 94kDa (CP94), we isolated its specific cDNA from a rat lens cDNA library by use of anti-mouse CP94 antiserum. The expressed fusion protein kept the epitopes specific against anti-chick CP97 as well as anti-mouse CP94 antibody, and the size was estimated as 190-200kDa, indicating that the cDNA insert of the clone seemed to encode a polypeptide with 80-90kDa in appearance. Northern analysis indicated that CP94 mRNA is expressed only in the lens, and not in the brain, skin, heart, kidney, lung, and liver, and the size was estimated to 2.1-2.3kb. In a lens of inherited microphthalmic mouse, Elo, a trace amount of mRNA with the size closely similar to that of rat mRNA was observed. The entire compiled sequence (1,873bp) showed an open reading frame covering the sequence of 533 amino acids totalling 58,857Da. No sequence homologous to the entire CP94 was found among the entries of any nucleotide and amino acid sequence databases; but with respect to a limited amino acid sequence of N-side region of CP94, a significant homology with cytokeratins was found.

Bovine mitochondrial DNA polymorphism in restriction endonuclease cleavage patterns and the location of the polymorphic sites

Cleavage patterns of mitochondrial DNA (mtDNA) by restriction endonuclease analysis were examined in four Japanese Black cows, three Japanese Shorthorn cows, and six Holstein cows. Seventeen restriction enzymes which recognize six base pairs and two restriction enzymes which recognize four base pairs were used in this study. Polymorphism was observed with three restriction enzymes, HindIII, TaqI, and MspI, and was detected within the breeds. Nucleotide substitution was determined in the HindIII polymorphic site by DNA cloning and sequencing; this is C ↔ T at position 10126 of the URF-3 region. Furthermore, the MspI and TaqI polymorphic sites were located on the physical map.