GADD34 is a protein that is induced by stresses such as DNA damage. The function of mammalian GADD34 has been proposed by in vitro transfection, but its function in vivo has not yet been elucidated. Here we generated and analyzed GADD34 knockout mice. Despite their embryonic stage- and tissue-specific expressions, GADD34 knockout mice showed no abnormalities at fetal development and in early adult life. However, in GADD34-/- mouse embryonic fibroblasts (MEFs), recovery from a shutoff of protein synthesis was delayed when MEFs were exposed to endoplasmic reticulum (ER) stress. The phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2alpha) at Ser51 induced by thapsigargin or DTT was prolonged in GADD34-/- MEF, although following treatment with tunicamycin, the eIF2alpha phosphorylation level did not change in either GADD34+/+ or GADD34-/- cells. ER stress stimuli induced expressions of Bip (binding Ig protein) and CHOP (C/EBP homologous protein) in MEF of wild-type mice. These expressions were strongly reduced in GADD34-/- MEF, which suggests that GADD34 up-regulates Bip and CHOP. These results indicate that GADD34 works as a sensor of ER stress stimuli and recovers cells from shutoff of protein synthesis.

Tetracycline-resistant (Tet r ) bacteria were isolated from fishes collected at three different fish farms in the southern part of Japan in August and September 2000. Of the 66 Tet r gram-negative strains, 29 were identified as carrying tetB only. Four carried tetY, and another four carried tetD. Three strains carried tetC, two strains carried tetB and tetY, and one strain carried tetC and tetG. Sequence analyses indicated the identity in Tet r genes between the fish farm bacteria and clinical bacteria: 99.3 to 99.9% for tetB, 98.2 to 100% for tetC, 99.7 to 100% for tetD, 92.0 to 96.2% for tetG, and 97.1 to 100% for tetY. Eleven of the Tet r strains transferred Tet r genes by conjugation to Escherichia coli HB-101. All transconjugants were resistant to tetracycline, oxycycline, doxycycline, and minocycline. The donors included strains of Photobacterium, Vibrio, Pseudomonas, Alteromonas, Citrobacter, and Salmonella spp., and they transferred tetB, tetY, or tetD to the recipients. Because NaCl enhanced their growth, these Tet r strains, except for the Pseudomonas, Citrobacter, and Salmonella strains, were recognized as marine bacteria. Our results suggest that tet genes from fish farm bacteria have the same origins as those from clinical strains.Many different kinds of antibiotics have been used as therapeutic agents in aquaculture in Japan. Intensive work was done until the 1980s to develop guidelines for antibiotic usage in fish farms. The guidelines regulated doses and required a period of drug-free rearing before sale of fish and succeeded in keeping the residual antibiotics in cultured fish to nondetectable levels. However, Samuelsen et al. (35) found that antibiotic-resistant bacteria persisted in fish farm sediments for at least 18 months after chemotherapy. Since the products of aquaculture are consumed by humans and since many antibiotic resistance determinants are encoded by transferable plasmids, cultured fish may serve as a vehicle for transmission of antibiotic resistance to bacteria that are commensal or pathogenic to humans (34).Tetracyclines are among the therapeutic agents most commonly used in human and veterinary treatment. Oxytetracycline is permitted to be mixed with feed for fish, and food sanitation law in Japan permits certain residual levels in fish. Because of the widespread use of tetracycline, resistance to it has been disseminated to many species of marine bacteria (4,6,18,42,46).More than 30 different kinds of tetracycline resistance determinants have been published. Resistance genes have been mainly categorized into two major groups, those responsible for proton-dependent efflux of tetracycline (24) and those conferring ribosomal protection by cytoplasmic proteins (9). Dissemination of the proton-dependent tetracycline efflux protein in aquaculture environments has been reported (5,12,13,14,22,34,37). Previous work has identified the relevant genes by using DNA hybridization or PCR methods (4,12,13,14,15,16,27,28,34,37), but the nucleotide sequences of these determinants remain unkn...

We have reported that histone acetylation induced by trichostatin A (TSA) promotes p21 waf1/cip1 (p21) expression, the GC-box located just upstream of TATA box was responsible for TSA-induced promoter activation, and both Sp1 and Sp3 were the working activator of this GC-box. To understand the molecular pathway from histone acetylation to this Sp1 family factors-mediated promoter activation, we investigated the function of p300, one of the histone acetyltransferase, in the present work. The evidence supporting the linkage between p300 and TSA-induced p21 promoter activation were realized from the following findings: 1) cotransfection of p300 elevated p21 promoter activity, and this elevation was dependent on TSA-responsive GC-box; 2) TSA-induced promoter activation was blocked by the introduction of p300 dominant-negative mutant into cells; and 3) Sp1-or Sp3-mediated activation was also suppressed by this p300 dominant-negative mutant. Our data also suggested that p300 collaborates with Sp1 in a way which is different from that when p300 collaborates with p53 in p21 transcription. p21waf1/cip1 (p21) 1 is a gene functioning as a cell cycle blocker, and its expression is usually regulated at transcription level. p21 was first cloned and characterized as an important effector that acts to inhibit cyclin-dependent kinase activity in p53-mediated cell cycle arrest induced by DNA damage (1-4). Further studies indicated that p21 is also regulated by other transcription factors during cell differentiation and growth arrest (5-8). During the study of cellular senescence, we found that the inhibitors of histone deacetylase, either sodium butyrate or TSA, can promote p21 transcription and induce growth arrest and senescence-like state in NIH 3T3 cells (9).2 The minimal region of the mouse p21 promoter, containing from Ϫ60 to ϩ40 bp relative to the TATA box, is essential and sufficient for the induction of p21 promoter by TSA. We reported also that a GC-box in this region is critical for both basal and TSA-induced promoter activity and that Sp1 and Sp3 are the functional activators of this GC-box (10). However, one question remained, how does histone acetylation affect Sp1-mediated transcription?Recently, the study of transcriptional regulation has been moving its focus to chromatin level. The molecules involved in chromatin transcription include DNA (promoter, enhancer, or silencer), histones, and non-histone proteins. It has become increasingly apparent that the equilibrium of histone acetylation and deacetylation plays an important role in transcriptional regulation (11,12). Several mammalian histone acetyltransferases and histone deacetylases have been cloned in recent years (13)(14)(15)(16)(17)(18)(19). p300 was first cloned as an E1A associated protein with properties of a transcriptional adapter (20). This protein was found later to possess intrinsic histone acetyltransferase activity and works as a coactivator in MyoD-, p53-, and SRC-1-mediated transcription (21-23). Indirect evidence has implicated p300 in cell cyc...

Twelve strains (the largest number ever reported) of group C and G 1 streptococci (GCS and GGS, respectively) that caused streptococcal toxic shock syndrome (STSS) were collected and characterized. Eleven strains were identified as Streptococcus dysgalactiae subsp. equisimilis, and one strain was identified as Streptococcus equi subsp. zooepidemicus. We found that it was the first reported case of STSS caused by S. equi subsp. zooepidemicus. Cluster analysis according to the 16S rRNA gene (rDNA) sequences revealed that the S. dysgalactiae strains belonged to clusters I and II, both of which were closely related. The emm types and the restriction patterns of chromosomal DNA measured by pulsed-field gel electrophoresis were highly variable in these strains except BL2719 and N1434. The 16S rDNA sequences and other characteristics of these two strains were indistinguishable, suggesting the clonal dissemination of this particular S. dysgalactiae strain in Japan. As the involvement of superantigens in the pathogenesis of group A streptococcus-related STSS has been suggested, we tried to detect known streptococcal superantigens in GCS and GGS strains. However, only the spegg gene was detected in seven S. dysgalactiae strains, with none of the other superantigen genes being detected in any of the strains. However, the sagA gene was detected in all of the strains except Tokyo1291. In the present study no apparent factor(s) responsible for the pathogenesis of STSS was identified, although close genetic relationships of GCS and GGS strains involved in this disease were suggested.

We have used the yeast one-hybrid system to clone transcription factors that bind to specific sequences in the proximal promoters of the type I collagen genes. We utilized as bait the sequence between ؊180 and ؊136 in the pro-␣2(I) collagen promoter because it acts as a functional promoter element and binds several DNA-binding proteins. Three cDNA clones were isolated that encoded portions of the mouse SPR2 transcription factor, whereas a fourth cDNA contained a potential open reading frame for a polypeptide of 775 amino acids and was designated BFCOL1. Recombinant BFCOL1 was shown to bind to the ؊180 to ؊152 segment of the mouse pro-␣2(I) collagen proximal promoter and to two discrete sites in the proximal promoter of the mouse pro-␣1(I) gene. The N-terminal portion of BFCOL1 contains its DNA-binding domain. DNA transfection experiments using fusion polypeptides with the yeast GAL4 DNA-binding segment indicated that the C-terminal part of BFCOL1 contained a potential transcriptional activation domain. We speculate that BFCOL1 participates in the transcriptional control of the two type I collagen genes.Type I collagen is a protein that is abundantly synthesized by a discrete number of cell types including osteoblasts, odontoblasts, fibroblasts, smooth muscle cells, and mesenchymal cells. It is composed of two ␣1 chains and one ␣2 chain forming a characteristic triple helix. Expression of the genes for these polypeptides is coordinately regulated in a variety of physiological and pathological situations (1). Changes in the synthesis of type I collagen occur not only during embryonic development in specific tissues but changes also take place in disease states, for example during wound healing as well as in fibrotic diseases such as lung fibrosis, cirrhosis, and scleroderma. In many of these instances it is likely that the control of expression of the two type I collagen genes is mainly exerted at the level of transcription, but the precise mechanisms that control transcription of these genes are still poorly understood. Our long term goal is to identify the critical cis-acting elements in these two genes and both the cell-specific and ubiquitous transcription factors that presumably control their expression.Recently, transgenic mouse studies have identified strong tissue-specific enhancer elements in the 5Ј-flanking regions of both type I collagen genes (2-5). These elements are located further upstream than the proximal promoter elements. For instance, in the mouse pro-␣1(I) gene, a potent enhancer element for osteoblast and odontoblast expression was localized about 1.6 kilobases (kb) 1 upstream of the start of transcription, whereas another strong element for expression in tendon and fascia fibroblasts was found between Ϫ2.3 and Ϫ3.2 kb (2). Similar experiments from other laboratories have produced analogous results (3, 4). These experiments strongly suggested that separate elements control the expression of this gene in different type I collagen-producing cells. In the pro-␣2(I) gene, an element that strongly...