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1.
Hirokazu Fukuda & Eiichi Ohtsubo 《Genes to cells : devoted to molecular & cellular mechanisms》1997,2(12):735-751
Background:
The plasmid R100 encodes the TraI protein, which is required for conjugal DNA transfer. TraI has the activity of site- and strand-specific nicking of the supercoiled plasmid DNA. The molecular mechanism of this specific nicking, which is supposed to be the initiation reaction of DNA transfer, is not understood.Results:
We have demonstrated that TraI has the ability to cleave the single-stranded DNA at the same site as the nicking site (nic) in a region, which we here refer to as sbi. The product contained the TraI protein which was covalently linked to the newly generated 5′ end of the nicking reaction. Both the cleaving and nicking reactions took place under almost the same conditions and required the presence of the sbi region. DNase I-footprinting analysis revealed that the TraI bound to the single-stranded DNA of the sbi region. TraI did not cleave the double-stranded DNA fragment, but it did cleave the double-stranded DNA with a single-stranded DNA portion in the sbi region. KMnO4 mapping analysis revealed that TraI can melt the sbi region in the supercoiled DNA to generate a single-stranded portion. We have also demonstrated that TraI was able to rejoin the cleaved products. The rejoining reaction required the 5′ end of one cleaved product with the TraI covalently attached and the 3′ end of the other product containing the sbi region.Conclusions:
Our results demonstrate that the nicking reaction—the initiation reaction of DNA transfer—is actually the cleaving reaction of the single-stranded DNA. TraI, which has both cleaving and rejoining activities, is thought to be involved in the termination of DNA transfer, to give a copy of the conjugative plasmid by joining the 5′ end, which is generated by the initiation reaction, with the 3′ end, which will be generated upon cleavage of the sbi region appearing after one round of the rolling circle replication of the plasmid.2.
Replication profile of Saccharomyces cerevisiae chromosome VI 总被引:2,自引:0,他引:2
Katherine L. Friedman Bonita J. Brewer & Walton L. Fangman 《Genes to cells : devoted to molecular & cellular mechanisms》1997,2(11):667-678
Background:
An understanding of the replication programme at the genome level will require the identification and characterization of origins of replication through large, contiguous regions of DNA. As a step toward this goal, origin efficiencies and replication times were determined for 10 ARSs spanning most of the 270 kilobase (kb) chromosome VI of Saccharomyces cerevisiae.Results:
Chromosome VI shows a wide variation in the percentage of cell cycles in which different replication origins are utilized. Most of the origins are activated in only a fraction of cells, suggesting that the pattern of origin usage on chromosome VI varies greatly within the cell population. The replication times of fragments containing chromosome VI origins show a temporal pattern that has been recognized on other chromosomes—the telomeres replicate late in S phase, while the central region of the chromosome replicates early.Conclusions:
As demonstrated here for chromosome VI, analysis of the direction of replication fork movement along a chromosome and determination of replication time by measuring a period of hemimethylation may provide an efficient means of surveying origin activity over large regions of the genome.3.
4.
Tohru Yamamoto & Masami Horikoshi 《Genes to cells : devoted to molecular & cellular mechanisms》1998,3(6):347-355
Background:
TBP-associated factors contain a variety of structural motifs and their related in vivo significance has remained unclear. We have attempted to identify specific biological phenomena linked to a particular domain of a TAF by analysing domain-exchanged chimeric mutants between Schizosaccharomyces pombe (Sp) and Saccharomyces cerevisiae (Sc) counterparts.Results:
Contrary to the case of TBP, Sp TAF containing the WD40 repeat cannot be exchanged for its Sc counterpart, despite their highly conserved primary structures. This ‘species-specific’ function locates in the N-terminal region. The C-terminal region, largely consisting of the WD40 repeat, is exchangeable for the corresponding region of its Sc counterpart. Growth of the strain harbouring this C-terminal chimeric mutant is temperature-sensitive. The chimeric gene product did not disappear at a restrictive temperature, a finding which strongly suggests that the growth defect is caused by an aberration in the interactions through the WD40 repeat structural motif. With temperature elevation, the chimeric mutants underwent drastic morphological changes due to a defect in cytokinesis.Conclusions:
The WD40 repeat of TAF is primarily involved in reactions which might regulate cytokinesis in Sp.5.
6.
7.
Grb10/GrbIR as an in vivo substrate of Tec tyrosine kinase 总被引:2,自引:0,他引:2
Hiroyuki Mano Ken-ichi Ohya Akira Miyazato Yoshihiro Yamashita Wataru Ogawa Johji Inazawa Uichi Ikeda Kazuyuki Shimada Kiyohiko Hatake Masato Kasuga Keiya Ozawa & Sachiko Kajigaya 《Genes to cells : devoted to molecular & cellular mechanisms》1998,3(7):431-441
Background
Tec is a member of the recently emerging subfamily among nonreceptor protein-tyrosine kinases (PTKs). Although many members of this family have been shown to be involved in a wide range of cytokine-mediated signalling systems, the molecular mechanism by which they exert in vivo effects remains obscure. To gain insights into the downstream pathways of Tec, we here looked for Tec-interacting proteins (TIPs) by using the yeast two-hybrid screening.Results
One of TIPs turned out to be Grb10/GrbIR, which carries one pleckstrin homology domain and one Src homology 2 domain. Grb10/GrbIR was known to bind receptor PTKs in a ligand-dependent fashion, but not to be phosphorylated on tyrosine residues. In a transient expression system in human kidney 293 cells, however, Grb10/GrbIR becomes profoundly tyrosine-phosphorylated by Tec, but not by Syk, Jak2 or insulin receptor. We also reveal that expression of Grb10/GrbIR suppresses the cytokine-driven and Tec-driven activation of the c-fos promoter.Conclusion
Our results indicate a novel role of Grb10/GrbIR as an effector molecule to a subset of nonreceptor PTKs.8.
9.
Myosin II activation promotes neurite retraction during the action of Rho and Rho-kinase 总被引:8,自引:0,他引:8
Mutsuki Amano Kazuyasu Chihara Nao Nakamura Yuko Fukata Takeo Yano Masao Shibata Mitsuo Ikebe & Kozo Kaibuchi 《Genes to cells : devoted to molecular & cellular mechanisms》1998,3(3):177-188
Background
The Rho small GTPase regulates myosin II activity through the phosphorylation of the myosin light chain (MLC) by activating Rho-kinase, which is a target of Rho. Several lines of evidence point to an important role of Rho in the action of lysophosphatidic acid (LPA) and thrombin in provoking neurite retraction in N1E-115 neuroblastoma cells.Results
Here we examined whether Rho-kinase and myosin II are involved in neurite retraction in N1E-115 cells. We showed that the expression of constitutively active forms of Rho-kinase induced neurite retraction in N1E-115 cells and MLC phosphorylation in NIH 3T3 cells, whereas the expression of dominant negative forms of Rho-kinase inhibited the LPA-induced neurite retraction in N1E-115 cells and the serum-induced MLC phosphorylation in NIH 3T3 cells. The expression of mutant MLCT18D,S19D (substitution of Thr and Ser by Asp), which is known to lead to the activation of myosin ATPase and a conformational change of myosin II when reconstituted with myosin heavy chains in vitro, also promoted neurite retraction.Conclusion
These results indicate that Rho-kinase is involved in the LPA-induced neurite retraction downstream of Rho, and that myosin II activation promotes neurite retraction downstream of Rho and Rho-kinase.10.
Takashi Oku Soichiro Ikeda Hisashi Sasaki Kotaro Fukuda Hiroshi Morioka Eiko Ohtsuka Hiroshi Yoshikawa & Toshiki Tsurimoto 《Genes to cells : devoted to molecular & cellular mechanisms》1998,3(6):357-369
Background:
PCNA, an eukaryotic DNA sliding clamp interacts with replication factors and the cell cycle protein, p21(Cip1/Waf1) and functions as a molecular switch for DNA elongation. To understand how DNA replication is regulated through PCNA, elucidation of the precise mechanisms of these protein interactions is necessary.Results:
Loop-region mutants in which human PCNA sequences were substituted with the corresponding Saccharomyces cerevisiae PCNA regions were prepared. Analysis of their functions, along with previously prepared alanine scanning mutants, demonstrated that some loops interact with DNA polymerase δ (pol δ) and replication factor C (RFC). The p21 binding sites of PCNA, mapped by affinity measurement of the mutant forms, found to be located within a distinct structure of the PCNA monomer, overlap with RFC- and pol δ-interaction sites. Competition between p21 and pol δ or RFC for binding to PCNA results in efficient inhibition of its stimulation of pol δ DNA synthesis and RFC ATPase but not of PCNA loading on DNA by RFC.Conclusions:
Semi-saturated amounts of p21 selectively block formation of the active pol δ complex but not the RFC–PCNA complex at 3′-ends of DNA primers. This differential effect may explain the specific inhibition of DNA replication by p21.11.
Tsutomu Nakamura Fumihiko Hamada Takao Ishidate Ken-ichi Anai Kohichi Kawahara Kumao Toyoshima & Tetsu Akiyama 《Genes to cells : devoted to molecular & cellular mechanisms》1998,3(6):395-403
Background:
The Wnt/Wingless signalling pathway plays an important role in both embryonic development and tumorigenesis. β-Catenin and Axin are positive and negative effectors of the Wnt signalling pathway, respectively.Results:
We found that Axin interacts with β-catenin and glycogen synthase kinase-3β (GSK-3β). Furthermore, the regulation of the G-protein signalling (RGS) domain of Axin is associated with the colorectal tumour suppressor adenomatous polyposis coli (APC). Overexpression of Axin in the human colorectal cancer cell line SW480 induced a drastic reduction in the level of β-catenin. Interaction with β-catenin and GSK-3β was required for the Axin-mediated β-catenin reduction.Conclusion:
Axin interacts with β-catenin, GSK-3β and APC, and negatively regulates the Wnt signalling pathway, presumably by regulating the level of β-catenin.12.
Donna M. Winham 《American journal of human biology》2012,24(5):690-695
Objectives:
Childhood obesity remains a problem among Latino children in the United States. Acculturation to an American diet and sedentary lifestyle may be causative factors. The research purpose was to assess child growth status, including sitting height, in relation to acculturation among Mexican and Mexican‐American children.Methods:
Anthropometric measures of weight, height, and sitting height were taken in a cross‐sectional survey of Mexican and Mexican‐American elementary school children (N = 484) in Phoenix, Arizona. Height‐for‐age (HAZ), weight‐for‐age (WAZ), and body mass index (BMI) Z‐scores were calculated based on the Centers for Disease Control 2000 growth reference. Sitting height Z‐scores (SHZ) were determined from the NHANES III reference values. Questions about language usage were asked of the children as a proxy for acculturation. Differences in growth measures and acculturation between those born in the United States or Mexico were evaluated by chi‐square or t‐tests.Results:
The mean HAZ value (?0.23) was close to the reference median. There were no significant differences in HAZ or SHZ by birth country or gender. WAZ values for boys were significantly higher than for girls. More girls (64%) than boys (54%) had normal BMIs. More Mexican‐born boys (28%) were obese than Mexican‐born girls (17%; P = 0.026) in comparison to the US‐born boys (31%) and girls (24%; P = n.s.). Acculturation scale score and male gender predicted a small percentage of the variation in BMIZ.Conclusions:
Environmental and cultural factors that promote obesity among low‐income Mexican and Mexican‐American children are similar regardless of birth country but boys may be at greater risk of obesity than girls. Am. J. Hum. Biol. 2012. © 2012 The Authors. American Journal of Human Biology published by Wiley Periodicals LLC.13.
14.
Nuclear accumulation of Saccharomyces cerevisiae Mcm3 is dependent on its nuclear localization sequence 总被引:1,自引:0,他引:1
Michael R. Young Katsunori Suzuki Hong Yan Susan Gibson & Bik K. Tye 《Genes to cells : devoted to molecular & cellular mechanisms》1997,2(10):631-643
Background:
The proteins of the Mcm2-7 family are required for the initiation of DNA replication. In Saccharomyces cerevisiae the nuclear envelope does not break down during the mitotic phase of the cell cycle. Large nuclear proteins, such as the Mcm proteins, which accumulate in the nucleus during specific portions of the cell cycle, must have regulated mechanisms to direct their entry into the nucleus.Results:
We have identified a nuclear localization sequence (NLS) in Mcm3, and demonstrated that it is necessary for the translocation of Mcm3 into the nucleus and sufficient for directing Escherichia coliβ-galactosidase to the nucleus. Immediately adjacent to the nuclear localization sequence are four potential sites for phosphorylation by Cdc28. Mutagenesis of all four sites has no immediate phenotypic effect on cell growth or viability, nor does it affect nuclear accumulation of Mcm3, although two-dimensional protein gel analysis has shown that at least some of these sites are normally phosphorylated in vivo. Substitution of the Mcm3 NLS by the SV40 large T-antigen NLS also directs the nuclear accumulation of the Mcm3-T-antigen protein, although cell growth is compromised. Replication activity in cells bearing either the Mcm3-Cdc28 phosphorylation site mutations or the Mcm3 T-antigen NLS substitution, as measured by plasmid stability assays, is comparable to activity in wild-type cells.Conclusions:
The Mcm3 protein is imported into the nucleus by a specific NLS. The cell cycle specific nuclear accumulation of Mcm3 appears to be a result of nuclear retention or nuclear targeting, rather than nuclear import regulated through the NLS.15.
Large scale isolation of osteoclast-specific genes by an improved method involving the preparation of a subtracted cDNA library 总被引:7,自引:0,他引:7
Masato Kobori Yasushi Ikeda Hiromi Nara Miyuki Kato Masayoshi Kumegawa Hiroshi Nojima & Hiroyuki Kawashima 《Genes to cells : devoted to molecular & cellular mechanisms》1998,3(7):459-475
16.
Post-transcriptional control of the level of mRNA by hepatitis B virus X gene in the transient expression system using human hepatic cells 总被引:4,自引:0,他引:4
Tsutomu Shimazu Shinako Takada Yoshio Ueno Yasuyuki Hayashi & Katsuro Koike 《Genes to cells : devoted to molecular & cellular mechanisms》1998,3(7):477-484
17.
Kimikazu Kurozumi Michiru Nishita Kyoko Yamaguchi Takashi Fujita Naoto Ueno & Hiroshi Shibuya 《Genes to cells : devoted to molecular & cellular mechanisms》1998,3(4):257-264
Background
TGF-β superfamily members elicit signals through the stimulation of serine/threonine-kinase receptors. Recently, molecules associated with several TGF-β family receptors have been cloned. One such molecule, the immunophilin FKBP12, has been reported to interact with TGF-β family type I receptors. However, the identity of signalling specific molecules interacting with the receptor was unknown.Results
To clarify the factors mediating bone morphogenetic protein (BMP) receptor signalling, a cytoplasmic molecule associated with the BMP type IA receptor (BMPR-IA) was isolated using the yeast two-hybrid system. We designated the molecule BMP receptor associated molecule 1 (BRAM1). BRAM1 is an alternatively spliced form of BS69, a factor previously identified as an adenovirus E1A-associated protein. BRAM1 was localized to the cytoplasmic region in mammalian cells, whereas BS69 is localized to the nucleus. BRAM1 bound specifically to BMPR-IA in mammalian cells. The C-terminal half of BRAM1 was found to be sufficient for binding to BMPR-IA.Conclusions
BRAM1, a BMPR-IA associated molecule, was isolated using the yeast two-hybrid system, and found to associate specifically with BMPR-IA. BRAM1 may thus serve as an interacting protein in the BMP signal pathway.18.
19.
Chikara Meno Yuzuru Ito Yukio Saijoh Youichi Matsuda Kosuke Tashiro Satoru Kuhara & Hiroshi Hamada 《Genes to cells : devoted to molecular & cellular mechanisms》1997,2(8):513-524