A gene cluster responsible for alkylaldoxime metabolism coexisting with nitrile hydratase and amidase in Rhodococcus globerulus A-4

An enzyme "alkylaldoxime dehydratase (OxdRG)" was purified and characterized from Rhodococcus globerulus A-4, in which nitrile hydratase (NHase) and amidase coexisted with the enzyme. The enzyme contains heme b as a prosthetic group, requires reducing reagents for the reaction, and is most active at a neutral pH and at around 30 degrees C, similar to the phenylacetaldoxime dehydratase from Bacillus sp. OxB-1 (OxdB). However, some differences were seen in subunit structure, substrate specificity, and effects of activators and inhibitors. The corresponding gene, oxd, encoding a 1059-base pair ORF consisting of 353 codons, was cloned, sequenced, and overexpressed in Escherichia coli. The predicted polypeptide showed 30.3% identity to OxdB. The gene is mapped just upstream of the gene cluster encoding the enzymes involved in the metabolism of aliphatic nitriles, i.e., NHase and amidase, and their regulatory and activator proteins. We report here the existence of an aldoxime dehydratase genetically linked with NHase and amidase, and responsible for the metabolism of alkylaldoxime in R. globerulus.     

Construction of protease-deficient Candida boidinii strains useful for recombinant protein production: cloning and disruption of proteinase A gene (PEP4) and proteinase B gene (PRBI)

The yeast Candida boidinii PEP4 and PRB1 genes, encoding proteinase A (PrA) and proteinase B (PrB), respectively, have been cloned and their primary structures were analyzed. The open reading frames of the PEP4 gene (1263 bp encoding a protein of 420 amino acids) and the PRBI gene (1683 bp encoding a protein of 560 amino acids) were found. The deduced amino acid sequences of PrA and PrB are very similar to Saccharomyces cerevisiae PrA and PrB (64% and 61% identities, respectively). Both PEP4 and PRBI genes were disrupted in the C. boidinii genome by one-step gene disruption. The resultant pep4delta and the pep4delta prb1delta strains lost protease activity when compared with the wild-type original strain. The constructed C. boidinii strains are expected to be useful hosts for heterologous protein production.     

Kinetic study of azole-bridged dinuclear platinum(II) complexes reacting with a hairpin-stabilized double-stranded oligonucleotide

The cytotoxic dinuclear platinum(II) complexes [[cis-Pt(NH(3))(2)](2)(mu-OH)(mu-pz)](NO(3))(2) (pz=pyrazolate) (1) and [[cis-Pt(NH(3))(2)](2)(mu-OH)(mu-1,2,3-ta-N1,N2)](NO(3))(2) (1,2,3-ta=1,2,3-triazolate) (2), were allowed to react with the hairpin-stabilized double-stranded oligonucleotide d(TATGGCATT(4)ATGCCATA), to determine the amounts of intrastrand and interstrand DNA adducts. The reaction kinetics was investigated by reversed-phase HPLC, and the resulting products were analyzed using mass spectroscopy combined with enzymatic digestion, and Maxam-Gilbert sequencing. The reaction of 1 results in the formation of the 1,2-intrastrand d(GG) adduct as the major final product. The two most abundant products of 2 were identified as isomeric 1,2-intrastrand d(GG) adducts differing probably in platinum coordination to the triazole ring. No GG-interstrand crosslinks were detected with either compound. d(GGC)-d(GCC) sequences of DNA do thus not appear to represent significant targets for forming interstrand crosslinks with either 1 or 2.     

Chromosomal instability and human hepatocarcinogenesis

Recently, many studies have identified losses and gains of several chromosomal loci in human hepatocellular carcinoma (HCC) with fine microsatellite analysis and comparative genomic hybridization. Although distribution of aberrant chromosomal arms differs among HCCs, loss of 1p, 4q, 6q, 8p, 9p, 10q, 13q, 16q and 17p, and gain of 1q, 6p, 8q, 17q and 20q have been recurrently reported, and loss of 4q and 16q seems to occur preferentially in hepatitis B virus-related HCCs. Accumulation of these aberrant chromosomal regions is associated with tumor progression, and some chromosomal aberrations, such as loss of 1p, are frequently identified in well-differentiated HCCs and also detected even in dysplastic nodule and cirrhotic nodule. This evidence suggests that chromosomal instability (CIN) emerges at an early stage during hepatocarcinogenesis and is successively inherent to tumor cells, resulting in acquisition of malignant phenotype. The molecular basis of CIN is beginning to be explored; however, several mechanisms may be involved for CIN of HCC.     

Regulation of shoot epidermal cell differentiation by a pair of homeodomain proteins in Arabidopsis

In higher plants, the outermost cell layer (L1) of the shoot apex gives rise to the epidermis of shoot organs. Our previous study demonstrated that an 8-bp motif named the L1 box functions as a cis-regulatory element for L1-specific gene expression in the shoot system of ARABIDOPSIS: We show here that PROTODERMAL FACTOR2 (PDF2), a member of the HD-GL2 class of homeobox genes, is expressed exclusively in the L1 of shoot meristems and that recombinant PDF2 protein specifically binds to the L1 box in vitro. Although knockout mutants of PDF2 and ATML1, another L1-specific HD-GL2 class gene sharing the highest homology with PDF2, display normal shoot development, the double mutant results in severe defects in shoot epidermal cell differentiation. This suggests that PDF2 and ATML1 are functionally interchangeable and play a critical role in maintaining the identity of L1 cells, possibly by interacting with their L1 box and those of downstream target-gene promoters.     

ACAULIS5, an Arabidopsis gene required for stem elongation, encodes a spermine synthase

Polyamines have been implicated in a wide range of biological processes, including growth and development in bacteria and animals, but their function in higher plants is unclear. Here we show that the Arabidopsis: ACAULIS5 (ACL5) gene, whose inactivation causes a defect in the elongation of stem internodes by reducing cell expansion, encodes a protein that shares sequence similarity with the polyamine biosynthetic enzymes spermidine synthase and spermine synthase. Expression of the recombinant ACL5 protein in Escherichia coli showed that ACL5 possesses spermine synthase activity. Restoration of the acl5 mutant phenotype by somatic reversion of a transposon-induced allele suggests a non-cell-autonomous function for the ACL5 gene product. We also found that expression of the ACL5 cDNA under the control of a heat shock gene promoter in acl5 mutant plants restores the phenotype in a heat shock-dependent manner. The results of the experiments showed that polyamines play an essential role in promotion of internode elongation through cell expansion in Arabidopsis: We discuss the relationships to plant growth regulators such as auxin and gibberellins that have related functions.