Organization of the biosynthetic gene cluster for the polyketide macrolide mycinamicin in Micromonospora griseorubida

Mycinamicin, composed of a branched lactone and two sugars, desosamine and mycinose, at the C-5 and C-21 positions, is a 16-membered macrolide antibiotic produced by Micromonospora griseorubida A11725, which shows strong antimicrobial activity against Gram-positive bacteria. The nucleotide sequence (62 kb) of the mycinamicin biosynthetic gene cluster, in which there were 22 open reading frames (ORFs), was completely determined. All of the products from the 22 ORFs are responsible for the biosynthesis of mycinamicin II and self-protection against the compounds synthesized. Central to the cluster is a polyketide synthase locus (mycA), which encodes a seven-module system comprised of five multifunctional proteins. Immediately downstream of mycA, there is a set of genes for desosamine biosynthesis (mydA-G and mycB). Moreover, mydH, whose product is responsible for the biosynthesis of mycinose, lies between mydA and B. On the other hand, eight ORFs were detected upstream of the mycinamicin PKS gene. The myrB, mycG, and mycF genes had already been characterized by Inouye et al. The other five ORFs (mycCI, mycCII, mydI, mycE, and mycD) lie between mycA1 and mycF, and these five genes and mycF are responsible for the biosynthesis of mycinose. In the PKS gene, four regions of KS and AT domains in modules 1, 4, 5, and 6 indicated that it does not show the high GC content typical for Streptomyces genes, nor the unusual frame plot patterns for Streptomyces genes. Methylmalonyl-CoA was used as substrate in the functional units of those four modules. The relationship between the substrate and the unusual frame plot pattern of the KS and AT domains was observed in the other PKS genes, and it is suggested that the KS-AT original region was horizontally transferred into the PKS genes on the chromosomal DNA of several actinomycetes strains.     

Genetic polymorphism of the swine major histocompatibility complex (SLA) class I genes, SLA-1, -2 and -3

In order to identify and characterize genetic polymorphism of the swine major histocompatibility complex (Mhc: SLA) class I genes, RT-PCR products of the second and third exons of the three SLA classical class I genes, SLA-1, SLA-2 and SLA-3 were subjected to nucleotide determination. These analyses allowed the identification of four, eight and seven alleles at the SLA-1, SLA-2 and SLA-3 loci, respectively, from three different breeds of miniature swine and one mixed breed. Among them, 12 alleles were novel. Construction of a phylogenetic tree using the nucleotide sequences of those 19 alleles indicated that the SLA-1 and -2 genes are more closely related to each other than to SLA-3. Selective forces operating at single amino acid sites of the SLA class I molecules were analyzed by the Adaptsite Package program. Ten positive selection sites were found at the putative antigen recognition sites (ARSs). Among the 14 positively selected sites observed in the human MHC (HLA) classical class I molecules, eight corresponding positions in the SLA class I molecules were inferred as positively selected. On the other hand, four amino acids at the putative ARSs were identified as negatively selected in the SLA class I molecules. These results suggest that selective forces operating in the SLA class I molecules are almost similar to those of the HLA class I molecules, although several functional sites for antigen and cytotoxic T-lymphocyte recognition by the SLA class I molecules may be different from those of the HLA class I molecules.The DNA sequence data reported in this paper have been submitted to the DDBJ, EMBL and GenBank nucleotide databases and have been assigned the accession numbers, AB105379, AB105380, AB105381, AB105382, AB105383, AB105384, AB105385, AB105386, AB105388, AB105389, AB105390 and AB105391     

Impaired differentiation of fetal hepatocytes in homozygous jumonji mice

Homozygous jumonji (jmj(-)/jmj(-)) mice were previously shown to exhibit hepatic hypoplasia and defective hematopoiesis in the liver and die at around embryonic day 15.5 (E15.5), suggesting that jmj is essential for liver development. In order to gain insight into the mechanism of liver development, we analyzed the expression and function of jmj in fetal hepatocytes. The number of hepatocytes in jmj(-)/jmj(-) mice was markedly reduced in comparison with control mice and the expression of jmj in hepatocytes increased along with development. As jmj(-)/jmj(-) embryos die by E15.5, we employed an in vitro culture system in which fetal hepatocytes differentiate in response to oncostatin M. The proliferation potential of jmj(-)/jmj(-) hepatocytes was comparable to that of wild type cells in vitro, however maturation of hepatocytes as evidenced by the expression of liver enzymes such as tyrosine amino transferase was severely impaired by the jmj gene inactivation. These results suggested that jmj plays a pivotal role in the development of mid-fetal hepatocytes to the neonatal stage.     

N-linked glycan structures of human lactoferrin produced by transgenic rice

Human lactoferrin was produced in genetically engineered rice. N-linked glycan structures of recombinant human lactoferrin were determined. The oligosaccharides liberated by hydrazinolysis were labeled with 2-aminopyridine (PA). The PA-labeled glycans were purified by reverse-phase and size-fractionation HPLCs. The structures of these glycans were identified by HPLC, exoglycosidase digestion, and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The glycan structures determined were ManFucXylGlcNAc(2) (3.4%), Man(2)FucGlcNAc(2) (2.1%), Man(3)FucGlcNAc(2) (2.5%), Man(3)FucXylGlcNAc(2) (42.5%), two isomers of Man(2)FucXylGlcNAc(2) (39.1%), Man(3)XylGlcNAc(2) (6.5%), and Man(2)XylGlcNAc(2) (3.9%).     

Simple and rapid PCR method for identification of streptococcal species relevant to animal infections based on 23S rDNA sequence

A PCR identification system targeting 23S rDNA sequences for the identification of eight streptococcal species relevant to animal infections (Streptococcus agalactiae, S. bovis, S. canis, S. dysgalactiae, S. equi, S. porcinus, S. suis and S. uberis) was developed. This system consists of two PCR reactions, A and B, in which seven and eight primers, respectively, are used simultaneously, and was designed so that each amplification product indicates a species by its size. A total of 111 cultures, including the type strain of eight species, could be successfully identified and differentiated as individual species, except for the cross reactivity between S. bovis and S. equinus. The developed PCR system can complete the identification procedure for eight streptococcal species through two tube reactions per isolate, and, therefore, might provide a rapid, simple and accurate diagnostic tool for veterinary laboratories.     

Identification of mRNA/protein (mRNP) complexes containing Puralpha,mStaufen, fragile X protein,and myosin Va and their association with rough endoplasmic reticulum equipped with a kinesin motor

Puralpha, which is involved in diverse aspects of cellular functions, is strongly expressed in neuronal cytoplasm. Previously, we have reported that this protein controls BC1 RNA expression and its subsequent distribution within dendrites and that Puralpha is associated with polyribosomes. Here, we report that, following treatment with EDTA, Puralpha was released from polyribosomes in mRNA/protein complexes (mRNPs), which also contained mStaufen, Fragile X Mental Retardation Protein (FMRP), myosin Va, and other proteins with unknown functions. As the coimmunoprecipitation of these proteins by an anti-Puralpha antibody was abolished by RNase treatment, Puralpha may assist mRNP assembly in an RNA-dependent manner and be involved in targeting mRNPs to polyribosomes in cooperation with other RNA-binding proteins. The immunoprecipitation of mStaufen- and FMRP-containing mRNPs provided additional evidence that the anti-Puralpha detected structurally or functionally related mRNA subsets, which are distributed in the somatodendritic compartment. Furthermore, mRNPs appear to reside on rough endoplasmic reticulum equipped with a kinesin motor. Based on our present findings, we propose that this rough endoplasmic reticulum structure may form the molecular machinery that mediates and regulates multistep transport of polyribosomes along microtubules and actin filaments, as well as localized translation in the somatodendritic compartment.     

Transgenic tobacco cultivars resistant to Pseudomonas syringae pv. tabaci

 Six oriental cultivars of tobacco (Nicotiana tabacum L.) were evaluated for transformation and foreign gene expression. Leaf-disc explant tissue was transformed with Agrobacterium tumefaciens strain LBA4404 carrying the plasmid pARK21, which contains NPTII gene and ttr (tabtoxin resistance) gene conferring the resistance to Pseudomonas syringae pv. tabaci. The disease resistance of regenerated plants and segregation of this trait up to R₇ progeny were investigated in a greenhouse and under field conditions. Our results indicated that the resistance to Pseudomonas syringae pv. tabaci introduced by transformation is heritable. Received: 10 June 1997 / Accepted: 31 March 1998     

Mixed monolayers of fatty acids with distearoylglycerophosphocholine

Mixed monolayer states of long normal-chain fatty acids with distearoylglycerophosphocholine (DSPC) have been studied by a previously described thermodynamic treatment. The surface pressures of mixed monolayers of tetradecanoic, pentadecanoic and octadecanoic acids with DSPC were measured at various compositions and temperatures. The two-dimensional phase diagrams of both the tetradecanoic acid-DSPC and pentadecanoic acid-DSPC systems were determined. They both exhibited eutectic type phase diagrams. In the octadecanoic acid-DSPC system the two components were immiscible both in the liquid-expanded and liquid-condensed states. The apparent molar entropy and energy changes for the tetradecanoic acid-DSPC system were calculated. The values for tetradecanoic acid were negative, as is the usual case. However, the values of DSPC were positive, and their absolute values wer about $\text{1}\text{4}$ of that of tetradecanoic acid.