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Analysis of chimeric UmuC proteins: identification of regions inSalmonella typhimurium UmuC important for mutagenic activity (1996)

Koch, W. H. ; Kopsidas, G. ; Meffle, B. ; [et al.]

Springer

Molecular genetics and genomics 251 (1996), S. 121-129

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ISSN: 1617-4623

Keywords: Escherichia coli ; Salmonella typhimurium ; SOS mutagenesis ; Chimeric proteins ; UmuC

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Abstract UnlikeEscherichia coli, the closely related bacteriumSalmonella typhimurium is relatively unresponsive to the mutagenic effects of DNA-damaging agents. Previous experiments have suggested that these phenotypic differences might result from reduced activity of theS. typhimurium UmuC protein. To investigate this possibility, we have taken advantage of the high degree of homology between the UmuC proteins ofE. coli andS. typhimurium and have constructed a series of plasmid-encoded chimeric proteins. The possibility that the phenotypic differences might be due to differential expression of the respective UmuC proteins was eliminated by constructing chimeric proteins that retained the first 25 N-terminal amino acids of either of the UmuC proteins (and presumably the same translational signals), but substituting the remaining 397 C-terminal amino acids with the corresponding segments from the reciprocal operon. Constructs expressing mostlyE. coli UmuC were moderately proficient for mutagenesis whereas those expressing mostlyS. typhimurium UmuC exhibited much lower frequencies of mutation, indicating that the activity of the UmuC protein ofS. typhimurium is indeed curtailed. The regions responsible for this phenotype were more precisely localized by introducing smaller segments of theS. typhimurium UmuC protein into the UmuC protein ofE. coli. While some regions could be interchanged with few or no phenotypic effects, substitution of residues 212–395 and 396–422 ofE. coli UmuC with those fromS. typhimurium resulted in reduced mutability, while substitution of residues 26–59 caused a dramatic loss of activity. We suggest, therefore, that the primary cause for the poor mutability ofS. typhimurium can be attributed to mutations located within residues 26–59 of theS. typhimurium UmuC protein.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF02172909

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Cells transformed by a wide variety of agents express higher abundance levels of some cellular RNA species (1982)

Schutzbank, T. ; Levine, A. J.

New York, N.Y. : Wiley-Blackwell

Journal of Cellular Biochemistry 19 (1982), S. 259-265

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ISSN: 0730-2312

Keywords: cytoplasmic RNA ; messenger RNA ; 3T3 cells ; C3HEF ; SV40 ; Life and Medical Sciences ; Cell & Developmental Biology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Chemistry and Pharmacology , Medicine

Notes: A cDNA-cloned library was prepared from mRNA synthesized by SV40-transformed mouse cells. Eleven cDNA clones were selected based on their ability to hybridize higher levels of mRNA in SV40-transformed 3T3 cells than in 3T3 cells. These cDNA clones were employed to screen the steady-state levels of cytoplasmic RNAs in a wide variety of viral (SV40, polyoma, adenovirus, and Rous sarcoma virus) and nonviral (methylcholanthrene, embryonal carcinoma) transformed cell lines. Two of the cDNA clones - A17 and 104 - detected greater than 40-100-fold higher levels of mRNA in all the transformed cell lines tested when compared to nontransformed cells (3T3, C3HEF). The levels of mRNA complementary to these two cDNAs were regulated in a temperature-sensitive fashion (87-100-fold) in both SV40tsA- and RSV ts-src-transformed murine cell lines. These two cDNA clones detected greater than 100-fold, higher levels of complementary RNA derived from SV40 tumor tissue than in normal mouse liver. RNA species complementary to cDNA clones A17 or 104 were not detected in either actively growing nontransformed cells or in serum-stimulated 3T3 cells. The abundance levels of mRNAs detected by these two cDNA clones appear to be regulated 100-fold or greater by the transformed state, independent of the transforming agent. The higher levels of these RNA species detected in transformed mouse cells appear not to be solely regulated by the state of growth of nontransformed cells.

Additional Material: 3 Tab.