scholarly journals The mouse c-rel protein has an N-terminal regulatory domain and a C-terminal transcriptional transactivation domain.

1990 ◽  
Vol 10 (10) ◽  
pp. 5473-5485 ◽  
Author(s):  
P Bull ◽  
K L Morley ◽  
M F Hoekstra ◽  
T Hunter ◽  
I M Verma
Keyword(s):  
Amino Acids ◽  
Fusion Proteins ◽  
Mammalian Cells ◽  
Dna Binding Domain ◽  
Transactivation Domain ◽  
Regulatory Domain ◽  
Amino Acid Residues ◽  
Terminal Portion ◽  
In Cis ◽  
Transcriptional Transactivator

We have shown that the murine c-rel protein can act as a transcriptional transactivator in both yeast and mammalian cells. Fusion proteins generated by linking rel sequences to the DNA-binding domain of the yeast transcriptional activator GAL4 activate transcription from a reporter gene linked in cis to a GAL4 binding site. The full-length mouse c-rel protein (588 amino acids long) is a poor transactivator; however, the C-terminal portion of the protein between amino acid residues 403 to 568 is a potent transcriptional transactivator. Deletion of the N-terminal half of the c-rel protein augments its transactivation function. We propose that c-rel protein has an N-terminal regulatory domain and a C-terminal transactivation domain which together modulate its function as a transcriptional transactivator.

The mouse c-rel protein has an N-terminal regulatory domain and a C-terminal transcriptional transactivation domain

1990 ◽  
Vol 10 (10) ◽  
pp. 5473-5485
Author(s):  
P Bull ◽  
K L Morley ◽  
M F Hoekstra ◽  
T Hunter ◽  
I M Verma
Keyword(s):  
Amino Acids ◽  
Fusion Proteins ◽  
Mammalian Cells ◽  
Dna Binding Domain ◽  
Transactivation Domain ◽  
Regulatory Domain ◽  
Amino Acid Residues ◽  
Terminal Portion ◽  
In Cis ◽  
Transcriptional Transactivator

We have shown that the murine c-rel protein can act as a transcriptional transactivator in both yeast and mammalian cells. Fusion proteins generated by linking rel sequences to the DNA-binding domain of the yeast transcriptional activator GAL4 activate transcription from a reporter gene linked in cis to a GAL4 binding site. The full-length mouse c-rel protein (588 amino acids long) is a poor transactivator; however, the C-terminal portion of the protein between amino acid residues 403 to 568 is a potent transcriptional transactivator. Deletion of the N-terminal half of the c-rel protein augments its transactivation function. We propose that c-rel protein has an N-terminal regulatory domain and a C-terminal transactivation domain which together modulate its function as a transcriptional transactivator.

scholarly journals Sir3p Domains Involved in the Initiation of Telomeric Silencing in Saccharomyces cerevisiae

Genetics ◽  
1998 ◽  
Vol 150 (3) ◽  
pp. 977-986 ◽  
Author(s):  
Yangsuk Park ◽  
John Hanish ◽  
Arthur J Lustig
Keyword(s):  
Amino Acids ◽  
Active Site ◽  
Fusion Proteins ◽  
Initiation Site ◽  
Negative Control ◽  
Model System ◽  
Mutant Protein ◽  
Regulatory Domain ◽  
C Terminus ◽  
Necessary And Sufficient

Abstract Previous studies from our laboratory have demonstrated that tethering of Sir3p at the subtelomeric/telomeric junction restores silencing in strains containing Rap1-17p, a mutant protein unable to recruit Sir3p. This tethered silencing assay serves as a model system for the early events that follow recruitment of silencing factors, a process we term initiation. A series of LexA fusion proteins in-frame with various Sir3p fragments were constructed and tested for their ability to support tethered silencing. Interestingly, a region comprising only the C-terminal 144 amino acids, termed the C-terminal domain (CTD), is both necessary and sufficient for restoration of silencing. Curiously, the LexA-Sir3N205 mutant protein overcomes the requirement for the CTD, possibly by unmasking a cryptic initiation site. A second domain spanning amino acids 481-835, termed the nonessential for initiation domain (NID), is dispensable for the Sir3p function in initiation, but is required for the recruitment of the Sir4p C terminus. In addition, in the absence of the N-terminal 481 amino acids, the NID negatively influences CTD activity. This suggests the presence of a third region, consisting of the N-terminal half (1-481) of Sir3p, termed the positive regulatory domain (PRD), which is required to initiate silencing in the presence of the NID. These data suggest that the CTD “active” site is under both positive and negative control mediated by multiple Sir3p domains.

Targeting motifs and functional parameters governing the assembly of connexins into gap junctions

2000 ◽  
Vol 349 (1) ◽  
pp. 281-287 ◽  
Author(s):  
Patricia E. M. MARTIN ◽  
James STEGGLES ◽  
Claire WILSON ◽  
Shoeb AHMAD ◽  
W. Howard EVANS
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gap Junctions ◽  
Gap Junction ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Lucifer Yellow ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Green Fluorescent

To study the assembly of gap junctions, connexin-green-fluorescent-protein (Cx-GFP) chimeras were expressed in COS-7 and HeLa cells. Cx26- and Cx32-GFP were targeted to gap junctions where they formed functional channels that transferred Lucifer Yellow. A series of Cx32-GFP chimeras, truncated from the C-terminal cytoplasmic tail, were studied to identify amino acid sequences governing targeting from intracellular assembly sites to the gap junction. Extensive truncation of Cx32 resulted in failure to integrate into membranes. Truncation of Cx32 to residue 207, corresponding to removal of most of the 78 amino acids on the cytoplasmic C-terminal tail, led to arrest in the endoplasmic reticulum and incomplete oligomerization. However, truncation to amino acid 219 did not impair Cx oligomerization and connexon hemichannels were targeted to the plasma membrane. It was concluded that a crucial gap-junction targeting sequence resides between amino acid residues 207 and 219 on the cytoplasmic C-terminal tail of Cx32. Studies of a Cx32E208K mutation identified this as one of the key amino acids dictating targeting to the gap junction, although oligomerization of this site-specific mutation into hexameric hemichannels was relatively unimpaired. The studies show that expression of these Cx-GFP constructs in mammalian cells allowed an analysis of amino acid residues involved in gap-junction assembly.

Mapping of the p53 and mdm-2 interaction domains

1993 ◽  
Vol 13 (7) ◽  
pp. 4107-4114 ◽  
Author(s):  
J Chen ◽  
V Marechal ◽  
A J Levine
Keyword(s):  
P53 Protein ◽  
Cellular Protein ◽  
Deletion Mutants ◽  
Cdna Clones ◽  
Transactivation Domain ◽  
Amino Acid Residues ◽  
Terminal Portion ◽  
Interaction Domains ◽  
General Transcription Machinery

The 90-kDa cellular protein encoded by the mouse mdm-2 oncogene binds to the p53 protein in vivo and inhibits its transactivation function (J. Momand, G. P. Zambetti, D. C. Olson, D. George, and A. J. Levine, Cell 69:1237-1245, 1992). cDNA clones encoding the human homolog of the mdm-2 protein (also called hdm-2) were isolated from a HeLa cell cDNA library. A series of monoclonal antibodies have been generated against human mdm-2 protein, and the epitopes recognized by these antibodies have been mapped. By construction of a series of deletion mutants, the region of the mdm-2 protein that is critical for complex formation with the p53 protein has been mapped to the N-terminal portion of the human mdm-2 protein. Interestingly, a monoclonal antibody with an epitope located in this same region failed to immunoprecipitate the mdm-2-p53 complex and appeared to recognize only free mdm-2 protein. The domain of the p53 protein that is sufficient for interaction with human mdm-2 protein has been mapped to the N-terminal 52 amino acid residues of the p53 protein. This region contains the transactivation domain of p53, suggesting that mdm-2 may inhibit p53 function by disrupting its interaction with the general transcription machinery.

scholarly journals Amino acids 473V and 598P of PB1 from an avian-origin influenza A virus contribute to polymerase activity, especially in mammalian cells

2012 ◽  
Vol 93 (3) ◽  
pp. 531-540 ◽  
Author(s):  
Chen Xu ◽  
Wei-Bin Hu ◽  
Ke Xu ◽  
Yun-Xia He ◽  
Tong-Yan Wang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Influenza A Virus ◽  
Mammalian Cells ◽  
Influenza A ◽  
Polymerase Activity ◽  
Amino Acid Residues ◽  
Lower Growth ◽  
Efficient Replication ◽  
Avian Origin

It has been reported that the avian-origin influenza A virus PB1 protein (avian PB1) enhances influenza A virus polymerase activity in mammalian cells when it replaces the human-origin PB1 protein (human PB1). Characterization of the amino acid residues that contribute to this enhancement is needed. In this study, it was found that PB1 from an avian-origin influenza A virus [A/Cambodia/P0322095/2005, H5N1 (Cam)] could enhance the polymerase activity of an attenuated human isolated virus, A/WSN/33, carrying the PB2 K627E mutation (WSN627E) in vitro. Furthermore, 473V and 598P in the Cam PB1 were identified as the residues responsible for this enhanced activity. The results from recombinant virus experiments demonstrated the contribution of PB1 amino acids 473V and 598P to polymerase activity in mammalian cells and in mice. Interestingly, 473V is conserved in pH1N1 viruses from the 2009 pandemic. Substitution of 473V by leucine in pH1N1 PB1 led to a decreased viral polymerase activity and a lower growth rate in mammalian cells, suggesting that the PB1 473V also plays a role in maintaining efficient virus replication of the pH1N1 virus. Thus, it was concluded that two amino acids in avian-origin PB1, 473V and 598P, contribute to the polymerase activity of the H5N1 virus, especially in mammalian cells, and that 473V in PB1 also contributes to efficient replication of the pH1N1 strain.

scholarly journals Cohesin-Dockerin Interactions of Cellulosomal Subunits of Clostridium cellulovorans

2001 ◽  
Vol 183 (18) ◽  
pp. 5431-5435 ◽  
Author(s):  
Jae-Seon Park ◽  
Yutaka Matano ◽  
Roy H. Doi
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Fusion Proteins ◽  
Sequence Similarity ◽  
Binding Specificity ◽  
Scaffolding Protein ◽  
Amino Acid Residues ◽  
Clostridium Cellulovorans ◽  
Level Sequence

ABSTRACT The cellulosome of Clostridium cellulovorans consists of three major subunits: CbpA, EngE, and ExgS. The C. cellulovorans scaffolding protein (CbpA) contains nine hydrophobic repeated domains (cohesins) for the binding of enzymatic subunits. Cohesin domains are quite homologous, but there are some questions regarding their binding specificity because some of the domains have regions of low-level sequence similarity. Two cohesins which exhibit 60% sequence similarity were investigated for their ability to bind cellulosomal enzymes. Cohesin 1 (Coh1) was found to contain amino acid residues corresponding to amino acids 312 to 453 of CbpA, which contains a total of 1,848 amino acid residues. Coh6 was determined to contain amino acid residues corresponding to residues 1113 to 1254 of CbpA. By genetic construction, these two cohesins were each fused to MalE, producing MalE-Coh1 and MalE-Coh6. The abilities of two fusion proteins to bind to EngE, ExgS, and CbpA were compared. Although MalE-Coh6 could bind EngE and ExgS, little or no binding of the enzymatic subunits was observed with MalE-Coh1. Significantly, the abilities of the two fusion proteins to bind CbpA were similar. The binding of dockerin-containing enzymes to cohesin-containing proteins was suggested as a model for assembly of cellulosomes. In our examination of the role of dockerins, it was also shown that the binding of endoglucanase B (EngB) to CbpA was dependent on the presence of EngB's dockerin. These results suggest that different cohesins may function with differing efficiency and specificity, that cohesins may play some role in the formation of polycellulosomes through Coh-CbpA interactions, and that dockerins play an important role during the interaction of cellulosomal enzymes and cohesins present in CbpA.

scholarly journals Mapping of the p53 and mdm-2 interaction domains.

1993 ◽  
Vol 13 (7) ◽  
pp. 4107-4114 ◽  
Author(s):  
J Chen ◽  
V Marechal ◽  
A J Levine
Keyword(s):  
P53 Protein ◽  
Cellular Protein ◽  
Deletion Mutants ◽  
Cdna Clones ◽  
Transactivation Domain ◽  
Amino Acid Residues ◽  
Terminal Portion ◽  
Interaction Domains ◽  
General Transcription Machinery

The 90-kDa cellular protein encoded by the mouse mdm-2 oncogene binds to the p53 protein in vivo and inhibits its transactivation function (J. Momand, G. P. Zambetti, D. C. Olson, D. George, and A. J. Levine, Cell 69:1237-1245, 1992). cDNA clones encoding the human homolog of the mdm-2 protein (also called hdm-2) were isolated from a HeLa cell cDNA library. A series of monoclonal antibodies have been generated against human mdm-2 protein, and the epitopes recognized by these antibodies have been mapped. By construction of a series of deletion mutants, the region of the mdm-2 protein that is critical for complex formation with the p53 protein has been mapped to the N-terminal portion of the human mdm-2 protein. Interestingly, a monoclonal antibody with an epitope located in this same region failed to immunoprecipitate the mdm-2-p53 complex and appeared to recognize only free mdm-2 protein. The domain of the p53 protein that is sufficient for interaction with human mdm-2 protein has been mapped to the N-terminal 52 amino acid residues of the p53 protein. This region contains the transactivation domain of p53, suggesting that mdm-2 may inhibit p53 function by disrupting its interaction with the general transcription machinery.

Critical Amino Acid Residues Within the ɸC31 Integrase DNA-Binding Domain Affect Recombination Activities in Mammalian Cells

2010 ◽  
Vol 21 (9) ◽  
pp. 1104-1118 ◽  
Author(s):  
Raphael Liesner ◽  
Wenli Zhang ◽  
Nadja Noske ◽  
Anja Ehrhardt
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Mammalian Cells ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Amino Acid Residues ◽  
Critical Amino Acid

scholarly journals Mutational Analysis of the N-Terminal DNA-Binding Domain of Sleeping Beauty Transposase: Critical Residues for DNA Binding and Hyperactivity in Mammalian Cells

2004 ◽  
Vol 24 (20) ◽  
pp. 9239-9247 ◽  
Author(s):  
Stephen R. Yant ◽  
Julie Park ◽  
Yong Huang ◽  
Jacob Giehm Mikkelsen ◽  
Mark A. Kay
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Dna Binding ◽  
Mammalian Cells ◽  
Nuclear Translocation ◽  
Mutational Analysis ◽  
Sleeping Beauty ◽  
Binding Domain ◽  
Single Amino Acid ◽  
Dna Binding Domain

ABSTRACT The N-terminal domain of the Sleeping Beauty (SB) transposase mediates transposon DNA binding, subunit multimerization, and nuclear translocation in vertebrate cells. For this report, we studied the relative contributions of 95 different residues within this multifunctional domain by large-scale mutational analysis. We found that each of four amino acids (leucine 25, arginine 36, isoleucine 42, and glycine 59) contributes to DNA binding in the context of the N-terminal 123 amino acids of SB transposase, as indicated by electrophoretic mobility shift analysis, and to functional activity of the full-length transposase, as determined by a quantitative HeLa cell-based transposition assay. Moreover, we show that amino acid substitutions within either the putative oligomerization domain (L11A, L18A, L25A, and L32A) or the nuclear localization signal (K104A and R105A) severely impair its ability to mediate DNA transposition in mammalian cells. In contrast, each of 10 single amino acid changes within the bipartite DNA-binding domain is shown to greatly enhance SB's transpositional activity in mammalian cells. These hyperactive mutations functioned synergistically when combined and are shown to significantly improve transposase affinity for transposon end sequences. Finally, we show that enhanced DNA-binding activity results in improved cleavage kinetics, increased SB element mobilization from host cell chromosomes, and dramatically improved gene transfer capabilities of SB in vivo in mice. These studies provide important insights into vertebrate transposon biology and indicate that Sleeping Beauty can be readily improved for enhanced genetic research applications in mammals.

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