Purification of dihydrofolate reductase amplified in Escherichia coli K-12

The Escherichia coli strain carrying pTP 6-10 which was constructed in our previous work (Iwakura, M., et al. (1983) J. Biochem. 93, 927-930) produces more than 400-fold dihydrofolate reductase as compared with the strain without the plasmid. Dihydrofolate reductase was highly purified from the cell-free extract of the plasmid strain simply by two steps; ammonium sulfate fractionation and ion-exchange chromatography. By 10-fold purification, the enzyme was essentially homogeneous as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The restriction map of pTP 6-10 was also determined and the plasmid was shown to have an Ava I, an EcoR I, a Pst I, a Pvu I, and a Pvu II site. Our results indicate that the plasmid strain is suitable as a source of the enzyme and that plasmid pTP 6-10 is promising as a versatile plasmid vector for efficiently yielding the product of the cloned gene.     

Starch phosphorus content in potato (Solanum tuberosum L.) cultivars and its effect on other starch properties

The research presented herein provides valuable data with respect to the phosphorus content of starches from many potato (Solanum tuberosum L.) cultivars using an energy-dispersive X-ray fluorescence technique. In all starches examined, the phosphorus content ranged from 308 to 1244 ppm. Furthermore, the estimation of the starch characteristics of representative samples differing manifestly in their phosphorus content indicated that enhancing the starch phosphate resulted in significant increases in the swelling power, peak viscosity, and breakdown and significant but small increases in the onset and peak temperatures of gelatinization. Other starch quality parameters, such as the amylose content, median granule size, and the gelatinization enthalpy, did not change significantly due to the degree of phosphate substitution of starch.     

MAP kinases function downstream of HSP90 and upstream of mitochondria in TMV resistance gene N-mediated hypersensitive cell death

Although the involvement of heat shock protein 90 (HSP90), mitogen-activated protein kinase (MAPK) cascades and organelle dysfunction in plant hypersensitive cell death has been suggested, the mutual relationship among them has not been elucidated. Here, we show the molecular network of HSP90, the wound-induced protein kinase (WIPK)/salicylic acid-induced protein kinase (SIPK)-mediated MAPK cascade and mitochondrial dysfunction in tobacco mosaic virus (TMV) resistance gene N-dependent cell death. p50, the Avr component for N, NtMEK2(DD), a constitutively active form of a MAPK kinase of WIPK/SIPK, and a mammalian pro-apoptotic factor Bax were used for cell death induction. Suppression of HSP90 and treatment with geldanamycin, a specific inhibitor of HSP90, compromised p50- but not NtMEK2(DD)- or Bax-mediated cell death accompanying the reduction of NtMEK2, WIPK and SIPK activation. In WIPK/SIPK-double knockdown plants, p50- and NtMEK2(DD)- but not Bax-mediated cell death was suppressed. All three types of cell death induced mitochondrial dysfunction, but they were similarly suppressed by Bcl-xL, which is a mammalian anti-apoptotic factor, and prevents mitochondrial dysfunction in plants as it does in animals in the cell death signal pathway. Taken together with the expression profile of hypersensitive reaction marker genes, it was indicated that the MAPK cascade functions downstream of HSP90 and transduces the cell death signal to mitochondria for N gene-dependent cell death. Furthermore, we found that WIPK and SIPK are functionally redundant in cell death signaling using WIPK/SIPK single or double knockdown plants.     

Region-specific insertion of transposons in combination with selection for high plasmid transferability and stability accounts for the structural similarity of IncP-1 plasmids

The overall architecture of IncP-1 plasmids is very conserved in that the accessory genes are typically located in one or two specific regions: between oriV and trfA and between the tra and trb operons. Various hypotheses have been formulated to explain this, but none have been tested experimentally. We investigated whether this structural similarity is due to region-specific transposition alone or also is reliant on selection for plasmids with insertions limited to these two regions. We first examined the transposition of Tn21Km into IncP-1beta plasmid pBP136 and found that most Tn21Km insertions (67%) were located around oriV. A similar experiment using the oriV region of IncP-1beta plasmid pUO1 confirmed these results. We then tested the transferability, stability, and fitness cost of different pBP136 derivatives to determine if impairment of these key plasmid characters explained the conserved plasmid architecture. Most of the pBP136 derivatives with insertions in transfer genes were no longer transferable. The plasmids with insertions in the oriV-trfA and tra-trb regions were more stable than other plasmid variants, and one of these also showed a significantly lower fitness cost. In addition, our detailed sequence analysis of IncP-1 plasmids showed that Tn402/5053-like transposons are situated predominantly between the tra and trb operons and close to the putative resolution site for the ParA resolvase, a potential hot spot for those transposons. Our study presents the first empirical evidence that region-specific insertion of transposons in combination with selection for transferable and stable plasmids explains the structural similarity of IncP-1 plasmids.     

Effect of luteinizing hormone (LH), pregnancy-specific protein B (PSPB), or arachidonic acid (AA) on secretion of progesterone and prostaglandins (PG) E (PGE; PGE(1) and PGE(2)) and F(2alpha) (PGF(2alpha)) by ovine corpora lutea of the estrous cycle or pregnancy in vitro

LH regulates luteal progesterone secretion during the estrous cycle in ewes and cows. However, PGE, not LH, stimulated ovine luteal progesterone secretion in vitro at day 90 of pregnancy and at day 200 in cows. The hypophysis is not obligatory after day 50 nor the ovaries after day 55 to maintain pregnancy in ewes. LH has been reported to regulate ovine placental PGE secretion up to day 50 of pregnancy and by pregnancy-specific protein B (PSPB) after day 50 of pregnancy. The objective of this experiment was to determine if and when a switch from LH to PGE occurred as the luteotropin regulating luteal progesterone secretion during pregnancy in ewes. Ovine luteal tissue slices of the estrous cycle (days 8, 11, 13, and 15) or pregnancy (days 8, 11, 13, 15, 20, 30, 40, 50, 60, and 90) were incubated in vitro with vehicle, LH, AA (precursor to PGE(2) and PGF(2alpha) synthesis), or PSPB in M199 for 4 h and 8 h. Concentrations of progesterone in jugular venous plasma of bred ewes increased (P< or =0.05) after day 50 and continued to increase through day 90. Secretion of progesterone by luteal tissue of non-bred ewes on days 8, 11, 13 and 15 and by bred ewes on days 8, 11, 13, 15, 20, 30, 40, and 50 was increased (P< or =0.05) by LH, but not by luteal tissue from pregnant ewes after day 50 (P> or =0.05). LH-stimulated progesterone secretion by luteal tissue from day 15 bred ewes was greater (P< or =0.05) than day 15 luteal tissue from non-bred ewes. Concentrations of progesterone in media were increased (P< or =0.05) when luteal tissue from pregnant ewes on day 50, 60, or 90 were incubated with AA or PSPB. Concentrations of PGE in media of non-bred ewes on days 8, 11, 13, or 15 and bred ewes on days 8 and 11 did not differ (P> or =0.05). Concentrations of PGE were increased (P< or =0.05) in media by luteal slices from bred ewes on days 13, 15, 20, 30, 40, 50, 60, and 90 of vehicle, LH, AA or PSPB-treated ewes. In addition, PSPB increased (P< or =0.05) PGE in media by luteal slices from pregnant ewes only on days 40, 50, 60, and 90. Concentrations of PGF(2alpha) were increased in media (P<0.05) of vehicle, AA, LH, or PSPB-treated luteal tissue from non-bred ewes and bred ewes on day 15 and by luteal tissue from bred ewes on days 20 and 30 after which concentrations of PGF(2alpha) in media declined (P< or =0.05) and did not differ (P> or =0.05) from non-bred or bred ewes on days 8, 11, or 13. It is concluded that LH regulates luteal progesterone secretion during the estrous cycle of non-bred ewes and up to day 50 of pregnancy, while only PGE regulates luteal progresterone secretion by ovine corpora lutea from days 50 to 90 of pregnancy. In addition, PSPB appears to regulate luteal secretion of progesterone from days 50 to 90 of pregnancy through stimulation of PGE secretion by ovine luteal tissue.     

The identification of catalytic pentad in the haloalkane dehalogenase DhmA from Mycobacterium avium N85: reaction mechanism and molecular evolution

Haloalkane dehalogenase DhmA from Mycobacterium avium N85 showed poor expression and low stability when produced in Escherichia coli. Here, we present expression DhmA in newly constructed pK4RP rhodococcal expression system in a soluble and stable form. Site-directed mutagenesis was used for the identification of a catalytic pentad, which makes up the reaction machinery of all currently known haloalkane dehalogenases. The putative catalytic triad Asp123, His279, Asp250 and the first halide-stabilizing residue Trp124 were deduced from sequence comparisons. The second stabilizing residue Trp164 was predicted from a homology model. Five point mutants in the catalytic pentad were constructed, tested for activity and were found inactive. A two-step reaction mechanism was proposed for DhmA. Evolution of different types of catalytic pentads and molecular adaptation towards the synthetic substrate 1,2-dichloroethane within the protein family is discussed.     

Risk of accidental invasion and expansion of allochthonous mice in Tokyo metropolitan coastal areas in Japan

House mouse (Mus musculus) is one of the perilous animal vectors for imported zoonosis such as a lymphocytic choriomeningitis (LCMV) infectious disease, and probably unknown emerging and/or re-emerging infectious diseases as well. It is necessary to prevent such diseases by regular surveys for behavioral trends of these allochthonous mice. However, such a trial has never been attempted in Japan. From 1998 to 2002, we analyzed partial sequences of the D-loop region in mtDNA, which provides powerful diagnostic SNPs for subspecies identification in the Mus musculus species, from 301 individuals of mice collected in 23 international bays or airports in Japan. We found that invasion of many allochthonous mice, which were identified as European subspecies, Mus musculus domesticus, occurred in Tokyo metropolitan coastal area. Based on the evidence, we warn that extensive invasion of allochthonous mice has occurred recently and, therefore, the risk of emerging and/or re-emerging infectious diseases invasion might be high in Tokyo metropolitan area.     

Different origins of bird and reptile sex chromosomes inferred from comparative mapping of chicken Z-linked genes

Recent progress of chicken genome projects has revealed that bird ZW and mammalian XY sex chromosomes were derived from different autosomal pairs of the common ancestor; however, the evolutionary relationship between bird and reptilian sex chromosomes is still unclear. The Chinese soft-shelled turtle (Pelodiscus sinensis) exhibits genetic sex determination, but no distinguishable (heteromorphic) sex chromosomes have been identified. In order to investigate this further, we performed molecular cytogenetic analyses of this species, and thereby identified ZZ/ZW-type micro-sex chromosomes. In addition, we cloned reptile homologues of chicken Z-linked genes from three reptilian species, the Chinese soft-shelled turtle and the Japanese four-striped rat snake (Elaphe quadrivirgata), which have heteromorphic sex chromosomes, and the Siam crocodile (Crocodylus siamensis), which exhibits temperature-dependent sex determination and lacks sex chromosomes. We then mapped them to chromosomes of each species using FISH. The linkage of the genes has been highly conserved in all species: the chicken Z chromosome corresponded to the turtle chromosome 6q, snake chromosome 2p and crocodile chromosome 3. The order of the genes was identical among the three species. The absence of homology between the bird Z chromosome and the snake and turtle Z sex chromosomes suggests that the origin of the sex chromosomes and the causative genes of sex determination are different between birds and reptiles.