Mutational analysis of a ras catalytic domain

1986 ◽  
Vol 6 (7) ◽  
pp. 2646-2654
Author(s):  
B M Willumsen ◽  
A G Papageorge ◽  
H F Kung ◽  
E Bekesi ◽  
T Robins ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Mutational Analysis ◽  
Nucleotide Binding ◽  
Membrane Localization ◽  
Wide Range ◽  
Murine Sarcoma Virus ◽  
Sarcoma Virus ◽  
Transforming Activity ◽  
Type V

We used linker insertion-deletion mutagenesis to study the catalytic domain of the Harvey murine sarcoma virus v-rasH transforming protein, which is closely related to the cellular rasH protein. The mutants displayed a wide range of in vitro biological activity, from those that induced focal transformation of NIH 3T3 cells with approximately the same efficiency as the wild-type v-rasH gene to those that failed to induce any detectable morphologic changes. Correlation of transforming activity with the location of the mutations enabled us to identify three nonoverlapping segments within the catalytic domain that were dispensable for transformation and six other segments that were required for transformation. Segments that were necessary for guanosine nucleotide (GDP) binding corresponded to three of the segments that were essential for transformation; two of the three segments share strong sequence homology with other purine nucleotide-binding proteins. Loss of GDP binding was associated with apparent instability of the protein. Lesions in two of the three other required regions significantly reduced GDP binding, while small lesions in the last required region did not impair GDP binding or membrane localization. We speculate that this latter region interacts with the putative cellular target of ras. The results suggest that transforming ras proteins require membrane localization, guanosine nucleotide binding, and an additional undefined function that may represent interaction with their target.

scholarly journals Mutational analysis of a ras catalytic domain.

1986 ◽  
Vol 6 (7) ◽  
pp. 2646-2654 ◽  
Author(s):  
B M Willumsen ◽  
A G Papageorge ◽  
H F Kung ◽  
E Bekesi ◽  
T Robins ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Mutational Analysis ◽  
Nucleotide Binding ◽  
Membrane Localization ◽  
Wide Range ◽  
Murine Sarcoma Virus ◽  
Sarcoma Virus ◽  
Transforming Activity ◽  
Type V

We used linker insertion-deletion mutagenesis to study the catalytic domain of the Harvey murine sarcoma virus v-rasH transforming protein, which is closely related to the cellular rasH protein. The mutants displayed a wide range of in vitro biological activity, from those that induced focal transformation of NIH 3T3 cells with approximately the same efficiency as the wild-type v-rasH gene to those that failed to induce any detectable morphologic changes. Correlation of transforming activity with the location of the mutations enabled us to identify three nonoverlapping segments within the catalytic domain that were dispensable for transformation and six other segments that were required for transformation. Segments that were necessary for guanosine nucleotide (GDP) binding corresponded to three of the segments that were essential for transformation; two of the three segments share strong sequence homology with other purine nucleotide-binding proteins. Loss of GDP binding was associated with apparent instability of the protein. Lesions in two of the three other required regions significantly reduced GDP binding, while small lesions in the last required region did not impair GDP binding or membrane localization. We speculate that this latter region interacts with the putative cellular target of ras. The results suggest that transforming ras proteins require membrane localization, guanosine nucleotide binding, and an additional undefined function that may represent interaction with their target.

scholarly journals Mutational Analysis and Homology-Based Modeling of the IntDOT Core-Binding Domain

2009 ◽  
Vol 191 (7) ◽  
pp. 2330-2339 ◽  
Author(s):  
Karolina Malanowska ◽  
Joel Cioni ◽  
Brian M. Swalla ◽  
Abigail Salyers ◽  
Jeffrey F. Gardner
Keyword(s):  
Amino Acid ◽  
Dna Cleavage ◽  
Catalytic Domain ◽  
Mutational Analysis ◽  
Dimensional Structure ◽  
Common Amino Acid ◽  
Mutant Proteins ◽  
Tyrosine Recombinases ◽  
Wide Range

ABSTRACT Tyrosine recombinases mediate a wide range of important genetic rearrangement reactions. Models for tyrosine recombinases have been based largely on work done on the integrase of phage lambda and recombinases like Cre, Flp, and XerC/D. All of these recombinases share a common amino acid signature that is important for catalysis. Several conjugative transposons (CTns) encode recombinases that are also members of the tyrosine recombinase family, but the reaction that they catalyze differs in that recombination does not require homology in the attachment sites. In this study, we examine the role of the core-binding (CB) domain of the CTnDOT integrase (IntDOT) that is located adjacent to the catalytic domain of the protein. Since there is no crystal structure for any of the CTn integrases, we began with a predicted three-dimensional structure produced by homology-based modeling. Amino acid substitutions were made at positions predicted by the model to be close to the DNA. Mutant proteins were tested for the ability to mediate integration in vivo and for in vitro DNA-binding, cleavage, and ligation activities. We identified for the first time nonconserved amino acid residues in the CB domain that are important for catalytic activity. Mutant proteins with substitutions at three positions in the CB domain are defective for DNA cleavage but still proficient in ligation. The positions of the residues in the complex suggest that the mutant residues affect the positioning of the cleaved phosphodiester bond in the active site without disruption of the ligation step.

Mouse cells contain two distinct ras gene mRNA species that can be translated into a p21 onc protein

1982 ◽  
Vol 2 (11) ◽  
pp. 1339-1345
Author(s):  
R W Ellis ◽  
D DeFeo ◽  
M E Furth ◽  
E M Scolnick
Keyword(s):  
Normal Mouse ◽  
Velocity Sedimentation ◽  
Ras Gene ◽  
Mrna Species ◽  
P21 Ras ◽  
Murine Sarcoma Virus ◽  
Sarcoma Virus ◽  
Mouse Cells ◽  
Murine Sarcoma

The Kirsten (Ki) and Harvey (Ha) strains of murine sarcoma virus encode a 21,000-dalton protein (p21 ras) which is the product of the transforming gene of these viruses. Normal cells express low levels of p21 ras encoded by cellular genes (Ki-ras and Ha-ras) homologous to the Ki and Ha murine sarcoma virus transformation genes. A bone marrow-derived mouse cell line, 416B, has been shown to express unusually high levels of p21 ras. In this manuscript, we investigated the molecular biology of p21 ras gene expression in 416B and other normal mouse cells. We identified four distinct polyadenylated and polysome-associated RNAs, two related to Ki-ras and two to Ha-ras. The levels in 416B cells of the two Ki-ras RNAs, sized 5.2 and 2.0 kilobases, were both elevated approximately 25-fold over levels found in normal mouse cells; there was no corresponding change in 416B cells in the levels of the two Ha-ras RNAs. We partially purified the two Ki-ras mRNAs and separated them by velocity sedimentation in sucrose density gradients. Both the 5.2- and 2.0-kilobase mRNAs could be translated in vitro into p21 ras. These results show that a cellular onc protein can be translated from two distinct cellular mRNA species.

scholarly journals A SCANNING ELECTRON MICROSCOPE STUDY OF SURFACE FEATURES OF VIRAL AND SPONTANEOUS TRANSFORMANTS OF MOUSE BALB/3T3 CELLS

1973 ◽  
Vol 59 (3) ◽  
pp. 633-642 ◽  
Author(s):  
Keith R. Porter ◽  
George J. Todaro ◽  
Virginia Fonte
Keyword(s):  
Parent Cell ◽  
Cell Of Origin ◽  
3T3 Cells ◽  
Scanning Electron Microscope Study ◽  
Murine Sarcoma Virus ◽  
Sarcoma Virus ◽  
Surface Morphologies ◽  
Murine Sarcoma ◽  
Scanning Electron

Cells of the mouse line Balb/3T3 as well as three virus-induced transformants and two spontaneous transformants grown in vitro have been studied for their topography by scanning electron microscopy. The parent cell in confluent culture closely resembles an endothelial cell in its form and in the structure of its association with adjacent cells. The tumorigenic transformants produced by SV40, murine sarcoma virus, or polyoma viruses are fusiform to pleomorphic and distinctly different from the cell of origin. They show relatively smooth surfaces except for blebs and marginal microvilli. Perhaps most surprising is the similarity they bear to one another. This is made the more singular by the very different form shown by the tumorigenic transformants of spontaneous origin. One of these, S2-4, possesses a thickened rather than the lamellar form of the parent A31 cell and is covered by long microvilli and many spherical blebs. The other, TuT3, more closely resembles the cell of origin but shows extensive ruffling at its margins. All transformants grow without evidence of contact inhibition. The significance of the surface morphologies and the factors influencing cell form are discussed.

In Vivo and In Vitro Studies on the Morphogenesis of Tumors Induced by Murine Sarcoma Virus (Harvey) 2

1973 ◽  
Vol 51 (5) ◽  
pp. 1541-1549 ◽  
Author(s):  
W. R. Thomas ◽  
E. J. Aw ◽  
J. M. Papadimitriou ◽  
P. J. Simons
Keyword(s):  
In Vitro Studies ◽  
Murine Sarcoma Virus ◽  
Sarcoma Virus ◽  
Murine Sarcoma
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