Katsuhide Miyake

Primary structure of AfsR, a global regulatory protein for secondary metabolite formation in Streptomyces coelicolor A3(2)

The afsR gene of Streptomyces coelicolor A3(2) complements afsB mutations affecting production of pigmented antibiotics. It also directs pigment production in Streptomyces lividans when carried on a plasmid vector. Nucleotide sequencing of the afsR gene revealed that it codes for a 993-amino acid protein (Mr 105,600) with A- and B-type ATP-binding consensus sequences at its N-terminal portion and two DNA-binding consensus sequences with a helix-turn-helix motif at its C-terminal portion. Each of the N- and C-terminal halves was capable of conferring pigment production, to some extent, in S. lividans, when carried separately on a multicopy plasmid. In addition, expression in trans of the two regions on the same plasmid conferred pigment production to almost the same extent as did the intact afsR gene. Mutations at the two ATP-binding consensus sequences, that were generated by in vitro site-directed mutagenesis, revealed their functional importance. Disruption of the S. coelicolor A3(2) chromosomal afsR gene in either the N- or C-terminal half using phage phi C31 KC515 resulted in significant, but not complete, loss of pigment production. These data suggest that the AfsR protein comprises two domains, viz., an ATP-binding and a DNA-binding domain, each of which could function as a positive regulator for pigment production. These afsR mutants sporulate normally. In addition to an internal promoter, which we previously detected in the middle of the AfsR coding region, S1 nuclease mapping revealed two tandem transcriptional start points, separated by 64 bp, upstream from a putative ATG start codon of the AfsR product.

BRG1 chromatin remodeling activity is required for efficient chromatin binding by repressor element 1-silencing transcription factor (REST) and facilitates REST-mediated repression

Chromatin remodeling enzymes such as SWI/SNF use the hydrolysis of ATP to power the movement of nucleosomes with respect to DNA. BRG1, one of the ATPases of the SWI/SNF complex, can be recruited by both activators and repressors, although the precise role of BRG1 in mechanisms of repression has thus far remained unclear. One transcription factor that recruits BRG1 as a corepressor is the repressor element 1-silencing transcription factor (REST). Here we address for the first time the mechanism of BRG1 activity in gene repression. We found that BRG1 enhanced REST-mediated repression at some REST target genes by increasing the interaction of REST with the local chromatin at its binding sites. Furthermore, REST-chromatin interactions, mediated by BRG1, were enhanced following an increase in histone acetylation in a manner dependent on the BRG1 bromodomain. Our data suggest that BRG1 facilitates REST repression by increasing the interaction between REST and chromatin. Such a mechanism may be applicable to other transcriptional repressors that utilize BRG1.

Sumoylation of CCAAT/Enhancer-binding Protein α and Its Functional Roles in Hepatocyte Differentiation

The sumoylation of CCAAT/enhancer-binding proteins (C/EBPs) by small ubiquitin-related modifier-1 (SUMO-1) has been reported recently. In this study, we investigated the functional role of the sumoylation of C/EBPα in the differentiation of hepatocytes. The amount of sumoylated C/EBPα gradually decreased during the differentiation, which suggests that the sumoylation is important for the control of growth/differentiation especially in the fetal liver. To analyze the function of the sumoylation of C/EBPα in liver-specific gene expression, we studied its effects on the expression of the albumin gene. The C/EBPα-mediated transactivation of the albumin gene was reduced by sumoylation of C/EBPα in primary fetal hepatocytes. The enhancement of C/EBPα-mediated transactivation by BRG1, a core subunit of the SWI/SNF chromatin remodeling complex, was hampered by sumoylation in a luciferase reporter assay. In addition, we discovered that sumoylation of C/EBPα blocked its inhibitory effect on cell proliferation by leading to the disruption of a proliferation-inhibitory complex because of a failure of the sumoylated C/EBPα to interact with BRG1. BRG1 was recruited to the dihydrofolate reductase promoter in nonproliferating C33a cells but was not detected in proliferating cells where C/EBPα, BRG1, and SUMO-1 were overexpressed. This result suggests that BRG1 down-regulates the expression of the dihydrofolate reductase gene. These findings provide the insight that SUMO acts as a space regulator, which affects protein-protein interactions.

Screening of bacteriophages producing endo-N-acetylneuraminidase

Seven different bacteriophages were isolated from sewage with Escherichia coli K1 strains as host bacteria. These phages showed specific degrading activity for the host capsular polysaccharide, α2,8-linked polysialic acid. The bacteriophage-associated endo-N-acetylneuraminidases degraded only a homopolymer of α2,8-linked sialic acid and were not able to degrade other sialylated carbohydrates such as sialyl lactose. With respect to the minimum substrate size, each enzyme had its own specificity. Among the seven phages isolated, three enzymes exhibited novel endo-N-acetylneuraminidase activity in that they produced sialyl dimer as a main product and two of them degraded sialyl trimer or tetramer.

Organization and nucleotide sequence of the secE-nusG region of Streptomyces griseus

The nusG genes of Streptomyces griseus and Streptomyces coelicolor A3(2) were cloned by the DNA-probing method with synthetic oligonucleotides designed on the basis of the nucleotide sequence of the nusG gene of Streptomyces virginiae. The amino acid sequences of the NusG proteins deduced from the nucleotide sequences showed significant homologies to those from a variety of microorganisms. Nucleotide sequence analysis of the region upstream of the nusG gene of S. griseus revealed the presence of the secE gene, suggesting that secE and nusG are organized as an operon as is found in other microorganisms.

Molecular cloning and characterization of a novel bacteriophage-associated sialidase

Bacteriophage 63D, previously isolated from sewage, is associated with alpha-2,8-linked polysialic acid degrading activity. We cloned a DNA fragment containing the sialidase gene from a 63D phage genomic library and the enzyme was functionally expressed in Escherichia coli. Determination of the nucleotide sequence of the fragment revealed that it contained one open reading frame (ORF) coding for a 108-kDa polypeptide consisting of 984 amino acid residues. The fragment had promoter sequences similar to the E. coli consensus promoters for sigma70. The deduced amino acid sequence of the central region of the ORF showed homology to those of phages K1F (51.6% identity) and PK1E (51.7% identity) endosialidases. Two Asp-box motifs that are widely found in sialidases were conserved. Purification of the soluble enzyme from lysed culture broth of infected E. coli yielded a 90-kDa protein upon SDS polyacrylamide gel electrophoresis, suggesting that the primary translational product is processed to the mature 90-kDa protein. The molecular mass of the enzyme was determined as 360 kDa by gel filtration, indicating that the native enzyme was probably a tetramer of identical 90-kDa subunits.

Simple assay method for endocrine disrupters by in vitro quail embryo culture: Nonylphenol acts as a weak estrogen in quail embryos

Many chemicals that are not structurally related to estrogen have estrogen-like activity. In this study, we tried to apply a quail embryo culture system for assessing the in vivo effects of such chemicals on the development of quail embryos. Beta-estradiol induced feminization of the gonads of genetically male embryos, which was confirmed by the increase in the size of the left gonad and female-specific aromatase expression, while male-specific SOX9 expression was not affected. Nonylphenol, which has a weak estrogenic activity, reduced the viability and body weight of embryos. Simultaneously, several genetically male embryos were feminized in terms of gonadal size and aromatase expression. These results indicate that the avian embryo culture system was useful for evaluating endocrine disrupters.

Blocking adhesion of cancer cells to endothelial cell types by S. agalactiae type-specific polysaccharides.

By using immortalized and normal endothelial cells, we were able to detect inhibitory effects of type specific polysaccharides from Streptococcus agalactiae on adhesion of cancer cells to endothelial cells, which is an essential step of cancer metastasis. The inhibition was probably due to specific structures of the bacterial polysaccharides, since the structures of the saccharides are very similar to those of cancer specific sialyl Lewis carbohydrates (sialyl Lea and Lex) which bind to ELAM-1 of endothelial cells. This result indicated that the bacterial polysaccharides from S. agalactiae could be very useful and hopeful as cancer metastasis inhibitors.

Genetic modification of a chicken expression system for the galactosylation of therapeutic proteins produced in egg white

As a tool for large scale production of recombinant proteins, chickens have advantages such as high productivity and low breeding costs compared to other animals. We previously reported the production of erythropoietin, the tumor necrosis factor receptor fused to an Fc fragment, and an Fc-fused single-chain Fv antibody in eggs laid by genetically manipulated chickens. In egg white, however, the incomplete addition of terminal sugars such as sialic acid and galactose was found on N-linked glycans of exogenously expressed proteins. This could be a draw back to the use of transgenic chickens since the loss of these terminal sugars may affect the functions and stability of recombinant proteins purified from chicken egg white for pharmaceutical usage. To overcome this problem, we studied galactosyltransferase (GalT) activity in the magnum where the majority of egg-white proteins are secreted. In the magnum, lower β1,4-GalT1 expression and poor galactose-transfer activity were observed. Thus, we supposed that the lack of GalT1 activity may partly cause the incomplete glycosylation of egg-white proteins, and generated genetically manipulated chickens expressing GalT1 by retrovirus-mediated gene transfer. In a Golgi fraction prepared from magnum cells of the genetically manipulated chickens, significant GalT activity was detected. The series of analyses revealed a considerable improvement in the galactosylation of native egg-white proteins as well as an exogenously expressed single-chain Fv antibody fused to an Fc fragment. We conclude that chickens with genetically modified GalT activity in the magnum could be an attractive platform for producing galactosylated therapeutics.

Chicken oviduct-specific expression of transgene by a hybrid ovalbumin enhancer and the Tet expression system.

We generated genetically manipulated chickens and quail by infecting them with a retroviral vector expressing the human growth hormone under the control of chicken ovalbumin promoter/enhancer up to -3861 bp from the transcriptional start site. The growth hormone was expressed in an oviduct-specific manner and was found in egg white, although its level was low. The DNA sequence of the integrated form of the viral vector in the packaging cells was shown to be truncated and contained only the sequence spanning -3861 to -1569 bp. This represented only the DNase I hypersensitive site (DHS) III of the 4 DHSs and lacked the proximal promoter of the ovalbumin control region. We found several TATA-like and other promoter motifs of approximately -1800 bp and considered that these promoter motifs and DHS III may cause weak but oviduct-specific expression of the growth hormone. To prove this hypothesis and apply this system to oviduct-specific expression of the transgene, the truncated regulatory sequence was fused to an artificial transactivator-promoter system. In this system, initial weak but oviduct-specific expression of the Tet activator from the promoter element in the ovalbumin control sequence triggered a self-amplifying cycle of expression. DsRed was specifically expressed in oviduct cells of genetically manipulated chickens using this system. Furthermore, deletion of a short region possibly containing the promoter elements (-2112 to -1569 bp) completely abrogated oviduct-specific expression. Taken together, these results suggest that weak expression of this putative promoter causes oviduct-specific expression of the transgene.