A cDNA Encoding the 57 kDa Subunit of a Cytokinin-Binding Protein Complex from Tobacco: the Subunit Has High Homology to S-Adenosyl-L-Homocysteine Hydrolase

130 kDa cytokinin-binding protein complex (CBP130) is a majorcytokinin binding entity in tobacco leaves (Nicotiana sylvestris)and contains two protein species of 57 and 36 kDa [CBP57 andCBP36, Mitsui and Sugiura (1993) Plant Cell Physiol. 34: 543].We have determined partial amino acid sequences of CBP57 andisolated cDNAs encoding the protein from a tobacco cDNA libraryusing an oligonucleotide probe. Sequence analysis revealed significanthomology between CBP57 and S-adenosyl-L-homocysteine hydrolasefrom other organisms, which catalyzes the reversible hydrolysisof S-adenosyl-L-homocysteine, a methyltransferase inhibitor.CBP57 contains an additional sequence of 41 amino acids whichis not present in animal and slime mold Sadenosyl-L-homocysteinehydrolases. This additional sequence is also found in the parsleyand Rhodobacter enzymes, suggesting that it is unique to photosyntheticorganisms. CBP57 is encoded by more than one nuclear genes intobacco. Northern and western blot analyses revealed that thelevel of expression of the genes is high in roots and low inleaves. They are also expressed in cultured tobacco cells. Wediscuss the possibility that at least some of the physiologicaleffects of cytokinin are mediated through the control of methylation/demethylationby regulating the intracellular concentration of S-adenosyl-L-homocysteinevia the hydrolase. (Received June 24, 1993; Accepted August 14, 1993)     

Inhibition of fibronectin binding of some bacterial cells by subtle pH increase within the physiological range

The fibronectin (Fn)-binding ability of microorganisms is considered to be involved in their pathogenicities. Granulicatella adiacens, a member of the oral flora and a causative agent of culture-negative infective endocarditis, showed nearly maximum binding to immobilized Fn at pH 7.2 but greatly reduced binding at a slightly higher pH 7.4 and almost no binding at pH 7.6 in the presence of physiological concentration of NaCl (0.15 M). A similar pH-sensitive Fn-binding property was noted with Escherichia coli and Abiotrophia defectiva, but not with Streptococcus pyogenes nor Staphylococcus aureus. In contrast, bindings to laminin and fibrinogen observed for some of these strains were unaffected by the same pH changes. This fastidious pH-dependency of Fn-binding abilities of some bacteria warns that the pH condition must be seriously considered in the in vitro assay of bacterial adherence to fibronectin.     

Detection of quantitative trait loci causing abnormal spermatogenesis and reduced testis weight in the small testis (Smt) mutant mouse.

The small testis (Smt) mutant mouse is characterized by a small testis of one third to one half the size of a normal testis, and its spermatogenesis is mostly arrested at early stages of meiosis, although a small number of spermatocytes at the late prophase of meiosis and a few spermatids can sometimes be seen. We performed quantitative trait locus (QTL) analysis of these spermatogenic traits and testis weight using 221 F2 males obtained from a cross between Smt and MOM (Mus musculus molossinus) mice. At the genome-wide 5% level, we detected two QTLs affecting meiosis on chromosomes 4 and 13, and two QTLs for paired testis weight as a percentage of body weight on chromosomes 4 and X. In addition, we found several QTLs for degenerated germ cells and multinuclear giant cells on chromosomes 4, 7 and 13. Interestingly, for cell degeneration, the QTL on chromosome 13 interacted epistatically with the QTL on chromosome 4. These results reveal polygenic participation in the abnormal spermatogenesis and small testis size in the Smt mutant.     

Nucleotide Sequence of a 28-kbp Portion of Rice Mitochondrial DNA: the Existence of Many Sequences That Correspond to Parts of Mitochondrial Genes in Intergenic Regions

The nucleotide sequence of a 27,588-bp region of rice mitochondrialDNA was determined. This sequence contains putative genes thatencode initiator methionine tRNA (trnfM), subunits III (nad3)and IV (nad4) of the NADH dehydrogenase complex, and ribosomalproteins S3 (rps3), S12 (rps12) and L16 (rp116). An open readingframe that contains sequences homologous to parts of rps2 andatpA is also present. In addition to these regions, there aremany short sequences with homology to fragments of mitochondrialDNAs from rice or other plants. These sequences may be remnantsof multiple rearrangements of the genome and their presenceseems to explain, in part, the large sizes of the mitochondrialgenomes of higher plants. (Received July 15, 1994; Accepted September 26, 1994)     

Redox-induced protein structural changes in cytochrome bo revealed by Fourier transform infrared spectroscopy and [13C]Tyr labeling

Cytochrome bo is a heme-copper terminal ubiquinol oxidase of Escherichia coli under highly aerated growth conditions. Tyr-288 present at the end of the K-channel forms a Cepsilon-Nepsilon covalent bond with one of the Cu(B) ligand histidines and has been proposed to be an acid-base catalyst essential for the O-O bond cleavage at the Oxy-to-P transition of the dioxygen reduction cycle (Uchida, T., Mogi, T., and Kitagawa, T. (2000) Biochemistry 39, 6669-6678). To probe structural changes at tyrosine residues, we examined redox difference Fourier transform infrared difference spectra of the wild-type enzyme in which either L-[1-13C]Tyr or L-[4-13C]Tyr has been biosynthetically incorporated in the tyrosine auxotroph. Spectral comparison between [1-13C]Tyr-labeled and unlabeled proteins indicated that substitution of the main chain carbonyl of a Tyr residue(s) significantly affected changes in the amide-I (approximately 1620-1680 cm(-1)) and -II ( approximately 1540-1560 cm(-1)) regions. In contrast, spectral comparison between [4-13C]Tyr-labeled and unlabeled proteins showed only negligible changes, which was the case for both the pulsed and the resting forms. Thus, protonation of an OH group of tyrosines including Tyr-288 in the vicinity of the heme o-Cu(B) binuclear center was not detected at pH 7.4 upon full reduction of cytochrome bo. Redox-induced main chain changes at a Tyr residue(s) are associated with structural changes at Glu-286 near the binuclear metal centers and may be related to switching of the K-channel operative at the reductive phase to D-channel at the oxidative phase of the dioxygen reduction cycle via conformational changes in the middle of helix VI.     

Structure of the E.coli aspartate transcarbamoylase trapped in the middle of the catalytic cycle

Snapshots of the catalytic cycle of the allosteric enzyme aspartate transcarbamoylase have been obtained via X-ray crystallography. The enzyme in the high-activity high-affinity R state contains two catalytic chains in the asymmetric unit that are different. The active site in one chain is empty, while the active site in the other chain contains an analog of the first substrate to bind in the ordered mechanism of the reaction. Small angle X-ray scattering shows that once the enzyme is converted to the R state, by substrate binding, the enzyme remains in the R state until substrates are exhausted. Thus, this structure represents the active form of the enzyme trapped at two different stages in the catalytic cycle, before the substrates bind (or after the products are released), and after the first substrate binds. Opening and closing of the catalytic chain domains explains how the catalytic cycle occurs while the enzyme remains globally in the R-quaternary structure.