DNA Inversion Regulates Outer Membrane Vesicle Production in Bacteroides fragilis

Phase changes in Bacteroides fragilis, a member of the human colonic microbiota, mediate variations in a vast array of cell surface molecules, such as capsular polysaccharides and outer membrane proteins through DNA inversion. The results of the present study show that outer membrane vesicle (OMV) formation in this anaerobe is also controlled by DNA inversions at two distantly localized promoters, IVp-I and IVp-II that are associated with extracellular polysaccharide biosynthesis and the expression of outer membrane proteins. These promoter inversions are mediated by a single tyrosine recombinase encoded by BF2766 (orthologous to tsr19 in strain NCTC9343) in B. fragilis YCH46, which is located near IVp-I. A series of BF2766 mutants were constructed in which the two promoters were locked in different configurations (IVp-I/IVp-II = ON/ON, OFF/OFF, ON/OFF or OFF/ON). ON/ON B. fragilis mutants exhibited hypervesiculating, whereas the other mutants formed only a trace amount of OMVs. The hypervesiculating ON/ON mutants showed higher resistance to treatment with bile, LL-37, and human β-defensin 2. Incubation of wild-type cells with 5% bile increased the population of cells with the ON/ON genotype. These results indicate that B. fragilis regulates the formation of OMVs through DNA inversions at two distantly related promoter regions in response to membrane stress, although the mechanism underlying the interplay between the two regions controlled by the invertible promoters remains unknown.     

Genomic analysis of the appearance of testicular oocytes in MRL/MpJ mice

Mammals produce sperm or oocytes depending on their sex; however, newborn MRL/MpJ (MRL) male mice produce oocytes within their testes. We previously reported that one of the genes responsible for this phenotype is present on the MRL-type Y chromosome (Y^MRL), and that multiple genes, probably autosomal, are also required for the development of this phenotype. In this study we focused on the autosomal genes and examined their relationship with this phenotype by analyzing the progeny from crosses between MRL mice and other strains. We first observed the male F1 progeny from the crosses between female A/J, C57BL/6 (B6), BALB/c, C3H/He, or DBA/2 mice and male MRL mice, and two consomic strains, male B6-Y^MRL and MRL-Y^B6. Testicular oocytes that were morphologically similar to those of MRL mice were detected in all mouse strains except BALBMRLF1; however, the incidence of testicular oocytes was significantly lower than that in MRL mice. The appearance of testicular oocytes in MRL-Y^B6 mice indicates that this phenotype is strongly affected by genomic factors present on autosomes, and that there is at least one other causative gene on the MRL-type autosomes (MRL testicular oocyte production, mtop) other than that on Y^MRL. Furthermore, a quantitative trait locus (QTL) analysis using N2 backcross progeny from crosses between female MRLB6F1 and male MRL mice revealed the presence of susceptibility loci for the appearance of testicular oocytes at 8–17 cM on Chr 15. These findings demonstrate that the appearance of testicular oocytes is regulated by the genetic factors on Chr 15 and on Y^MRL.

     

High Free-Energy Barrier of 1D Diffusion Along DNA by Architectural DNA-Binding Proteins

Architectural DNA-binding proteins function to regulate diverse DNA reactions and have the defining property of significantly changing DNA conformation. Although the 1D movement along DNA by other types of DNA-binding proteins has been visualized, the mobility of architectural DNA-binding proteins on DNA remains unknown. Here, we applied single-molecule fluorescence imaging on arrays of extended DNA molecules to probe the binding dynamics of three structurally distinct architectural DNA-binding proteins: Nhp6A, HU, and Fis. Each of these proteins was observed to move along DNA, and the salt concentration independence of the 1D diffusion implies sliding with continuous contact to DNA. Nhp6A and HU exhibit a single sliding mode, whereas Fis exhibits two sliding modes. Based on comparison of the diffusion coefficients and sizes of many DNA binding proteins, the architectural proteins are categorized into a new group distinguished by an unusually high free-energy barrier for 1D diffusion. The higher free-energy barrier for 1D diffusion by architectural proteins can be attributed to the large DNA conformational changes that accompany binding and impede rotation-coupled movement along the DNA grooves.     

Physical interactions among flavonoid enzymes in snapdragon and torenia reveal the diversity in the flavonoid metabolon organization of different plant species

Flavonoid metabolons (weakly‐bound multi‐enzyme complexes of flavonoid enzymes) are believed to occur in diverse plant species. However, how flavonoid enzymes are organized to form a metabolon is unknown for most plant species. We analyzed the physical interaction partnerships of the flavonoid enzymes from two lamiales plants (snapdragon and torenia) that produce flavones and anthocyanins. In snapdragon, protein–protein interaction assays using yeast and plant systems revealed the following binary interactions: flavone synthase II (FNSII)/chalcone synthase (CHS); FNSII/chalcone isomerase (CHI); FNSII/dihydroflavonol 4‐reductase (DFR); CHS/CHI; CHI/DFR; and flavonoid 3′‐hydroxylase/CHI. These results along with the subcellular localizations and membrane associations of snapdragon flavonoid enzymes suggested that FNSII serves as a component of the flavonoid metabolon tethered to the endoplasmic reticulum (ER). The observed interaction partnerships and temporal gene expression patterns of flavonoid enzymes in red snapdragon petal cells suggested the flower stage‐dependent formation of the flavonoid metabolon, which accounted for the sequential flavone and anthocyanin accumulation patterns therein. We also identified interactions between FNSII and other flavonoid enzymes in torenia, in which the co‐suppression of FNSII expression was previously reported to diminish petal anthocyanin contents. The observed physical interactions among flavonoid enzymes of these plant species provided further evidence supporting the long‐suspected organization of flavonoid metabolons as enzyme complexes tethered to the ER via cytochrome P450, and illustrated how flavonoid metabolons mediate flower coloration. Moreover, the observed interaction partnerships were distinct from those previously identified in other plant species (Arabidopsis thaliana and soybean), suggesting that the organization of flavonoid metabolons may differ among plant species.     

Podocalyxin is crucial for the growth of oral squamous cell carcinoma cell line HSC-2

Oral cancers constitute approximately 2% of all cancers, with the most common histological type being oral squamous cell carcinoma (OSCC), representing 90% of oral cancers. Although diagnostic technologies and therapeutic techniques have progressed, the survival rate of patients with OSCC is still 60%, whereas the incidence rate has increased. Podocalyxin (PODXL) is a highly glycosylated type I transmembrane protein that is detected in normal tissues such as heart, breast, and pancreas as well as in many cancers, including lung, renal, breast, colorectal, and oral cancers. This glycoprotein is associated with the progression, metastasis, and poor outcomes of oral cancers. PODXL overexpression was strongly detected using our previously established anti-PODXL monoclonal antibody (mAb), PcMab-47, and its mouse IgG_2a-type, 47-mG_2a. In previous studies, we also generated PODXL-knock out (PODXL-KO) cell lines using SAS OSCC cell lines, in order to investigate the function of PODXL in the proliferation of oral cancer cells. The growth of SAS/PODXL-KO cell lines was observed to be lower than that of parental SAS cells. For this study, PODXL-KO OSCC cell lines were generated using HSC-2 cells, and the role of PODXL in the growth of OSCC cell lines in vitro was assessed. Decreased growth was observed for HSC-2/PODXL-KO cells compared with HSC-2 parental cells. The influence of PODXL on tumor growth of OSCC was also investigated in vivo, and both the tumor volume and the tumor weight were observed to be significantly lower for HSC-2/PODXL-KO than that for HSC-2 parental cells. These results, taken together, indicate that PODXL plays an important role in tumor growth, both in vitro and in vivo.     

The Regulatory Functions of the rolB and rolC Genes of Agrobacterium rhizogenes are Conserved in the Homologous Genes (Ngrol) of Nicotiana glauca in Tobacco Genetic Tumors

To compare patterns of expression between the Ngrol genes ofN. glauca and the Rirol genes of Agrobacterium rhizogenes, weperformed fluorometric and histochemical analysis of transgenicgenetic tumors on the hybrid of Nicotiana glauca x N. langsdorffü(Fl) that harbored a rß- glucuronidase (GUS) reportergene fused to the promoter of NgrolB, NgrolC, RirolB or RirolC The promoters of NgrolB and NgrolCNgrolC had 2- to 3-fold loweractivity than those of RirolB and RirolC However, the changesin patterns of GUS activity caused by deletion of NgrolB andNgrolCpromoters were similar to those of RirolB and RirolC promoters.This result suggests that the cis-acting sequences that regulatethe level of expression of RirolB and RirolC are conserved inthe NgrolB and NgrolC promoters. Furthermore, an auxin dependent(NAA-dependent) increase in GUS activity was observed in thecase of NgrolB-GUS and RirolB-GUS. Histochemical analysis showedGUS activity encoded by both NgrolB-GUS and RirolB-GUS in normal-typeFl transgenic plants was located in meristematic zones, whilethat encoded by NgrolC-GUS and RirolC-GUS was detected mainlyin vascular systems of various organs. Thus, the patterns ofexpression of the Ngrol genes were the same as those of theRirol genes in terms of promotion by auxin and tissue-specificity,indicating that regulatory mechanisms for both sets of geneshave been conserved during the evolution of the genus Nicotianaafter transfer from a progenitor of Agrobacterium to that ofNicotiana. (Received May 2, 1995; Accepted June 13, 1995)     

The TraB protein, which mediates the intermycelial transfer of the Streptomyces plasmid pSN22, has functional NTP-binding motifs and is localized to the cytoplasmic membrane

The traB gene on the Streptomyces conjugative plasmid pSN22 is required for intermycelial plasmid transfer and the mobilization of chromosomal markers (Cma). The predicted amino acid sequence of TraB contains one Walker type-A and two type-B NTP-binding motifs. Site-directed mutagenesis revealed that the type-A motif and one of the type-B motifs, 109 amino acid residues downstream of the type-A motif, were essential for both plasmid transfer and Cma. The second type-B sequence could be changed without any phenotypic effect. A modified traB gene was constructed, resulting in the production of a functional protein with an amino-terminal c-Myc epitope tag for immunological analysis. This protein was associated with the cytoplasmic membrane, suggesting that TraB is a membrane protein that uses energy from ATP hydrolysis to transport DNA between mycelia. The c-Myc tagging of TraB decreased the efficiency of intramycelial plasmid spread, suggesting that TraB is involved in both inter- and intramycelial transfer processes.     

Production of a T-Cell Clone Which Reacts with Membrane Proteins of Acholeplasma laidlawii

The role of cellular immunity in mycoplasma infection is not completely understood. In this study, we established mycoplasma-specific T-cell clones to evaluate cellular immunity in mycoplasma infection. We developed a T-cell clone (G-10) which was stimulated with Acholeplasma laidlawii. The T-cell clone G-10, CD4⁺ and T-cell receptor (TCR) αβ⁺ recognized the 42- and 65-kilodalton (kDa) membrane proteins of A. laidlawii and responded to A. hippikon. Hence, the application of mycoplasma-specific T cells such as G-10 in animal models may allow the assessment of cellular immune response to mycoplasma infection.