scholarly journals Inducible cell ablation in Drosophila by cold-sensitive ricin A chain

Development ◽  
1992 ◽  
Vol 114 (3) ◽  
pp. 681-687 ◽  
Author(s):  
K.G. Moffat ◽  
J.H. Gould ◽  
H.K. Smith ◽  
C.J. O'Kane
Keyword(s):  
Single Amino Acid ◽  
Temperature Sensitive ◽  
Temperature Dependent ◽  
Ricin Toxin ◽  
Cell Ablation ◽  
Active Site Cleft ◽  
Cold Sensitive ◽  
Low Toxicity ◽  
A Chain ◽  
Ricin A

We have developed a system for temperature-inducible killing of specific cells in the fruitfly Drosophila melanogaster. The system overcomes many of the limitations of existing cell ablation methods and is in principle applicable to any non-homeothermic eukaryote. Temperature-sensitive and cold-sensitive mutations in the ricin toxin A chain (RTA) of castor bean were generated in yeast. One cold-sensitive mutation, RAcs2, produced temperature-dependent ablation of eye cells in Drosophila when expressed under control of the eye-specific sev enhancer. At 29 degrees C, cell death was observed within 7 hours in the developing eye and no obvious toxic effects were observed elsewhere; at 18 degrees C, extremely low toxicity was observed. DNA sequencing of RAcs2 revealed a single amino acid substitution in the RTA active site cleft.

Selection and characterization of ricin toxin A-chain mutations in Saccharomyces cerevisiae

1989 ◽  
Vol 9 (2) ◽  
pp. 415-420
Author(s):  
A Frankel ◽  
D Schlossman ◽  
P Welsh ◽  
A Hertler ◽  
D Withers ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ricinus Communis ◽  
Single Amino Acid ◽  
Toxin A ◽  
Reactive Material ◽  
Ricin Toxin ◽  
Gal1 Promoter ◽  
Active Site Cleft ◽  
A Chain

A DNA sequence encoding the A chain of ricin toxin (RTA) from the castor bean plant, Ricinus communis, was placed under GAL1 promoter control and transformed into Saccharomyces cerevisiae. Induction of expression of RTA was lethal. This lethality was the basis for a selection of mutations in RTA which inactivated the toxin. A number of mutant alleles which encoded cross-reactive material were sequenced. Eight of the first nine mutant RTAs studied showed single-amino-acid changes involving residues located in the proposed active-site cleft.

scholarly journals Selection and characterization of ricin toxin A-chain mutations in Saccharomyces cerevisiae.

1989 ◽  
Vol 9 (2) ◽  
pp. 415-420 ◽  
Author(s):  
A Frankel ◽  
D Schlossman ◽  
P Welsh ◽  
A Hertler ◽  
D Withers ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ricinus Communis ◽  
Single Amino Acid ◽  
Toxin A ◽  
Reactive Material ◽  
Ricin Toxin ◽  
Gal1 Promoter ◽  
Active Site Cleft ◽  
A Chain

A DNA sequence encoding the A chain of ricin toxin (RTA) from the castor bean plant, Ricinus communis, was placed under GAL1 promoter control and transformed into Saccharomyces cerevisiae. Induction of expression of RTA was lethal. This lethality was the basis for a selection of mutations in RTA which inactivated the toxin. A number of mutant alleles which encoded cross-reactive material were sequenced. Eight of the first nine mutant RTAs studied showed single-amino-acid changes involving residues located in the proposed active-site cleft.

Cell ablation using wild-type and cold-sensitive ricin-a chain indrosophila embryonic mesoderm

genesis ◽  
2002 ◽  
Vol 34 (1-2) ◽  
pp. 132-134 ◽  
Author(s):  
Marcus J. Allen ◽  
Cahir J. O'Kane ◽  
Kevin G. Moffat
Keyword(s):  
Wild Type ◽  
Ricin A Chain ◽  
Cell Ablation ◽  
Cold Sensitive ◽  
A Chain ◽  
Ricin A

KEX2 mutations suppress RNA polymerase II mutants and alter the temperature range of yeast cell growth

1989 ◽  
Vol 9 (6) ◽  
pp. 2341-2349
Author(s):  
C Martin ◽  
R A Young
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Gene ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Temperature Dependent ◽  
Growth Properties ◽  
Cold Sensitive ◽  
Dependent Growth ◽  
Kex2 Protease

Suppressors of a temperature-sensitive RNA polymerase II mutation were isolated to identify proteins that interact with RNA polymerase II in yeast cells. Ten independently isolated extragenic mutations that suppressed the temperature-sensitive mutation rpb1-1 and produced a cold-sensitive phenotype were all found to be alleles of a single gene, SRB1. An SRB1 partial deletion mutant was further investigated and found to exhibit several pleiotropic phenotypes. These included suppression of numerous temperature-sensitive RNA polymerase II mutations, alteration of the temperature growth range of cells containing wild-type RNA polymerase, and sterility of cells of alpha mating type. The ability of SRB1 mutations to suppress the temperature-sensitive phenotype of RNA polymerase II mutants did not extend to other temperature-sensitive mutants investigated. Isolation of the SRB1 gene revealed that SRB1 is KEX2. These results indicate that the KEX2 protease, whose only known substrates are hormone precursors, can have an important influence on RNA polymerase II and the temperature-dependent growth properties of yeast cells.

scholarly journals The isolation and characterization of temperature-dependent ricin A chain molecules inSaccharomyces cerevisiae

FEBS Journal ◽  
2007 ◽  
Vol 274 (21) ◽  
pp. 5586-5599 ◽  
Author(s):  
Stuart C. H. Allen ◽  
Katherine A. H. Moore ◽  
Catherine J. Marsden ◽  
Vilmos Fülöp ◽  
Kevin G. Moffat ◽  
...  
Keyword(s):  
Temperature Dependent ◽  
Ricin A Chain ◽  
Chain Molecules ◽  
Isolation And Characterization ◽  
A Chain ◽  
Ricin A

scholarly journals Expression of soluble and fully functional ricin A chain in Escherichia coli is temperature-sensitive.

1988 ◽  
Vol 263 (10) ◽  
pp. 4837-4843 ◽  
Author(s):  
M Piatak ◽  
J A Lane ◽  
W Laird ◽  
M J Bjorn ◽  
A Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Temperature Sensitive ◽  
Ricin A Chain ◽  
A Chain ◽  
Ricin A

scholarly journals KEX2 mutations suppress RNA polymerase II mutants and alter the temperature range of yeast cell growth.

1989 ◽  
Vol 9 (6) ◽  
pp. 2341-2349 ◽  
Author(s):  
C Martin ◽  
R A Young
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Gene ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Temperature Dependent ◽  
Growth Properties ◽  
Cold Sensitive ◽  
Dependent Growth ◽  
Kex2 Protease

Suppressors of a temperature-sensitive RNA polymerase II mutation were isolated to identify proteins that interact with RNA polymerase II in yeast cells. Ten independently isolated extragenic mutations that suppressed the temperature-sensitive mutation rpb1-1 and produced a cold-sensitive phenotype were all found to be alleles of a single gene, SRB1. An SRB1 partial deletion mutant was further investigated and found to exhibit several pleiotropic phenotypes. These included suppression of numerous temperature-sensitive RNA polymerase II mutations, alteration of the temperature growth range of cells containing wild-type RNA polymerase, and sterility of cells of alpha mating type. The ability of SRB1 mutations to suppress the temperature-sensitive phenotype of RNA polymerase II mutants did not extend to other temperature-sensitive mutants investigated. Isolation of the SRB1 gene revealed that SRB1 is KEX2. These results indicate that the KEX2 protease, whose only known substrates are hormone precursors, can have an important influence on RNA polymerase II and the temperature-dependent growth properties of yeast cells.

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