scholarly journals Essentiality of the Early Transcript in the Replication Origin of the Lactococcal Prolate Phage c2

2004 ◽  
Vol 186 (23) ◽  
pp. 8010-8017 ◽  
Author(s):  
Anja H. Schiemann ◽  
Jasna Rakonjac ◽  
Michael Callanan ◽  
James Gordon ◽  
Kayla Polzin ◽  
...  
Keyword(s):  
Plasmid Vector ◽  
Model System ◽  
Internal Deletion ◽  
Specific Sequence ◽  
Template Sequence ◽  
Unrelated Sequence ◽  
Origin Of Dna Replication ◽  
Sequence Encoding ◽  
Origin Function

ABSTRACT The genome of the prolate-headed lytic lactococcal bacteriophage c2 is organized into two divergently oriented blocks consisting of the early genes and the late genes. These blocks are separated by the noncoding origin of DNA replication. We examined the functional role of transcription of the origin in a plasmid model system. Deletion of the early promoter PE1 abolished origin function. Introduction of mutations into PE1 which did not eliminate promoter activity or replacement of PE1 with an unrelated but functional promoter did not abolish replication. The A-T-rich region upstream of PE1, which is conserved in prolate phages, was not required for plasmid replication. Replacement of the PE1 transcript template sequence with an unrelated sequence with a similar G+C content abolished replication, showing that the sequence encoding the transcript is essential for origin function. Truncated transcript and internal deletion constructs did not support replication except when the deletion was at the very 3′ end of the DNA sequence coding for the transcript. The PE1 transcript could be detected for all replication-proficient constructs. Recloning in a plasmid vector allowed detection of PE1 transcripts from some fragments that did not support replication, indicating that stability of the transcript alone was not sufficient for replication. The data suggest that production of a transcript of a specific length and with a specific sequence or structure is essential for the function of the phage c2 origin in this model system.

scholarly journals Serine and Threonine Phosphorylation of the Paxillin LIM Domains Regulates Paxillin Focal Adhesion Localization and Cell Adhesion to Fibronectin

1998 ◽  
Vol 9 (7) ◽  
pp. 1803-1816 ◽  
Author(s):  
Michael C. Brown ◽  
Joseph A. Perrotta ◽  
Christopher E. Turner
Keyword(s):  
Cell Adhesion ◽  
Focal Adhesion ◽  
Binding Sites ◽  
Protein Localization ◽  
Model System ◽  
Lim Domain ◽  
Amino Terminal ◽  
Lim Domains ◽  
Inside Out

We have previously shown that the LIM domains of paxillin operate as the focal adhesion (FA)-targeting motif of this protein. In the current study, we have identified the capacity of paxillin LIM2 and LIM3 to serve as binding sites for, and substrates of serine/threonine kinases. The activities of the LIM2- and LIM3-associated kinases were stimulated after adhesion of CHO.K1 cells to fibronectin; consequently, a role for LIM domain phosphorylation in regulating the subcellular localization of paxillin after adhesion to fibronectin was investigated. An avian paxillin-CHO.K1 model system was used to explore the role of paxillin phosphorylation in paxillin localization to FAs. We found that mutations of paxillin that mimicked LIM domain phosphorylation accelerated fibronectin-induced localization of paxillin to focal contacts. Further, blocking phosphorylation of the LIM domains reduced cell adhesion to fibronectin, whereas constitutive LIM domain phosphorylation significantly increased the capacity of cells to adhere to fibronectin. The potentiation of FA targeting and cell adhesion to fibronectin was specific to LIM domain phosphorylation as mutation of the amino-terminal tyrosine and serine residues of paxillin that are phosphorylated in response to fibronectin adhesion had no effect on the rate of FA localization or cell adhesion. This represents the first demonstration of the regulation of protein localization through LIM domain phosphorylation and suggests a novel mechanism of regulating LIM domain function. Additionally, these results provide the first evidence that paxillin contributes to “inside-out” integrin-mediated signal transduction.

scholarly journals Role of Papillomavirus E1 Initiator Dimerization in DNA Replication

2005 ◽  
Vol 79 (13) ◽  
pp. 8661-8664 ◽  
Author(s):  
Stephen Schuck ◽  
Arne Stenlund
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Severe Effect ◽  
Origin Of Dna Replication ◽  
Initiation Of Dna Replication

ABSTRACT Viral initiator proteins are polypeptides that form oligomeric complexes on the origin of DNA replication (ori). These complexes carry out a multitude of functions related to initiation of DNA replication, and although many of these functions have been characterized biochemically, little is understood about how the complexes are assembled. Here we demonstrate that loss of one particular interaction, the dimerization between E1 DNA binding domains, has a severe effect on DNA replication in vivo but has surprisingly modest effects on most individual biochemical activities in vitro. We conclude that the dimer interaction is primarily required for initial recognition of ori.

The Arabidopsis leaf as a model system for investigating the role of cell cycle regulation in organ growth

2005 ◽  
Vol 119 (1) ◽  
pp. 43-50 ◽  
Author(s):  
Gerrit T. S. Beemster ◽  
Steven Vercruysse ◽  
Lieven De Veylder ◽  
Martin Kuiper ◽  
Dirk Inzé
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Regulation ◽  
Model System ◽  
Organ Growth ◽  
Cycle Regulation

On the role of surface composition and curvature on biointerface formation and colloidal stability of nanoparticles in a protein-rich model system

2013 ◽  
Vol 108 ◽  
pp. 110-119 ◽  
Author(s):  
Guillermo Orts-Gil ◽  
Kishore Natte ◽  
Raphael Thiermann ◽  
Matthias Girod ◽  
Steffi Rades ◽  
...  
Keyword(s):  
Colloidal Stability ◽  
Surface Composition ◽  
Model System ◽  
Protein Rich

Expression of human recombinant plasminogen activators enhances invasion and experimental metastasis of H-ras-transformed NIH 3T3 cells

1989 ◽  
Vol 9 (5) ◽  
pp. 2133-2141
Author(s):  
J H Axelrod ◽  
R Reich ◽  
R Miskin
Keyword(s):  
Serine Proteinase ◽  
Tissue Type ◽  
Metastatic Lesions ◽  
Metastatic Behavior ◽  
Sequence Encoding ◽  
Direct Genetic Evidence ◽  
Nih 3T3 ◽  
Gene Transfer Technique

The gene transfer technique was used to examine the role of plasminogen activator (PA) in the invasive and metastatic behavior of tumorigenic cells. H-ras-transformed NIH 3T3 clonal cells producing a very low level of PA were generated and further transfected with an expression plasmid containing a cDNA sequence encoding either the urokinase-type or the tissue-type human PA. Compared with the parental transformed cells, clonal cells expressing high levels of both types of recombinant PA invaded more rapidly through a basement membrane reconstituted in vitro. Furthermore, cells expressing high levels of recombinant urokinase-type PA also caused a higher incidence of pulmonary metastatic lesions after intravenous injection into nude mice. Both activities were reduced by the serine proteinase inhibitor EACA; invasion was also suppressed by antibodies blocking the activity of human PAs and by the synthetic collagenase inhibitor SC-44463. These findings provide direct genetic evidence for a causal role of PA in invasive and metastatic activities.

Tissue inhibitor of metalloproteinases (TIMP) regulates extracellular type I collagen degradation by chondrocytes and endothelial cells

1987 ◽  
Vol 87 (2) ◽  
pp. 357-362
Author(s):  
J. Gavrilovic ◽  
R.M. Hembry ◽  
J.J. Reynolds ◽  
G. Murphy
Keyword(s):  
Endothelial Cells ◽  
Type I Collagen ◽  
Model System ◽  
Collagen Degradation ◽  
Type I ◽  
Tissue Inhibitor Of Metalloproteinases ◽  
Regulatory Factor ◽  
Tissue Inhibitor ◽  
Cells Cultured

A specific antiserum to purified rabbit tissue inhibitor of metalloproteinases (TIMP) was raised in sheep, characterized and used to investigate the role of TIMP in a model system. Chondrocytes and endothelial cells cultured on 14C-labelled type I collagen films and stimulated to produce collagenase were unable to degrade the films unless the anti-TIMP antibody was added. The degradation induced was inhibited by a specific anti-rabbit collagenase antibody. It was concluded that TIMP is a major regulatory factor in cell-mediated collagen degradation.

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