<i>In vivo</i> system for the detection of low level activity barnase mutants

Jucovic, Milan; Hartley, Robert W.

doi:10.1093/protein/8.5.497

Cited by 10 publications

(10 citation statements)

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“…It has been previously reported that E. coli cells are able to tolerate low levels of barnase activity and have been used for in vivo assays of barnase mutant activities (Jucovic and Hartley, 1995;Axe et al, 1996;Yazynin et al, 1999). We demonstrated that the in vivo system reported in this paper was able to detect low levels of RNase activity of barnase mutants, and this system helped us identify mutants with sufficient activity to achieve high and consistent pollen ablation efficacy while minimizing any vegetative damage.…”

Section: Discussionsupporting

confidence: 51%

“…However, the in vivo comparison of barnase mutant activities in this study revealed that barnaseH102E had no detectable activity in the E. coli colony assay. Many single amino acid substitutions of the His at position 102 of the barnase protein have been reported, including substitution with Ala, Gln, Tyr, Asn, Asp, Arg, Lys, Gly, Leu, and Pro (Mossakowska et al, 1989;Meiering et al, 1992;Jucovic and Hartley, 1995;Axe et al, 1998). Among all the single substitution mutants listed above, only mutants carrying a substitution with Leu or Pro had detectable activities, while all others were found to render the enzyme inactive, including barnaseH102D.…”

Section: Discussionmentioning

confidence: 99%

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Control of Pollen-Mediated Gene Flow in Transgenic Trees

Zhang¹,

Norris-Caneda²,

Rottmann³

et al. 2012

Plant Physiology

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Pollen elimination provides an effective containment method to reduce direct gene flow from transgenic trees to their wild relatives. Until now, only limited success has been achieved in controlling pollen production in trees. A pine (Pinus radiata) male cone-specific promoter, PrMC2, was used to drive modified barnase coding sequences (barnaseH102E, barnaseK27A, and barnaseE73G) in order to determine their effectiveness in pollen ablation. The expression cassette PrMC2-barnaseH102E was found to efficiently ablate pollen in tobacco (Nicotiana tabacum), pine, and Eucalyptus (spp.). Large-scale and multiple-year field tests demonstrated that complete prevention of pollen production was achieved in greater than 95% of independently transformed lines of pine and Eucalyptus (spp.) that contained the PrMC2-barnaseH102E expression cassette. A complete pollen control phenotype was achieved in transgenic lines and expressed stably over multiple years, multiple test locations, and when the PrMC2-barnaseH102E cassette was flanked by different genes. The PrMC2-barnaseH102E transgenic pine and Eucalyptus (spp.) trees grew similarly to control trees in all observed attributes except the pollenless phenotype. The ability to achieve the complete control of pollen production in field-grown trees is likely the result of a unique combination of three factors: the male cone/anther specificity of the PrMC2 promoter, the reduced RNase activity of barnaseH102E, and unique features associated with a polyploid tapetum. The field performance of the PrMC2-barnaseH102E in representative angiosperm and gymnosperm trees indicates that this gene can be used to mitigate pollen-mediated gene flow associated with large-scale deployment of transgenic trees.

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Section: Discussionsupporting

confidence: 51%

Section: Discussionmentioning

confidence: 99%

Control of Pollen-Mediated Gene Flow in Transgenic Trees

Zhang¹,

Norris-Caneda²,

Rottmann³

et al. 2012

Plant Physiology

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“…The promoterless barnase gene is inserted with the complete barstar gene in the expression plasmid pMI43a in the antisense orientation to prevent a basal level of toxic barnase expression. When heat-induced, this vector will invert the barnase-barstar cassette and barnase will be transcribed by the tandem Ptac-lac promoters (13). Inversion is mediated by interaction between attP and attB sites derived from A phage (15).…”

Section: Introductionmentioning

confidence: 99%

“…Inversion is mediated by interaction between attP and attB sites derived from A phage (15). Since (13), that this mutation produces a barnase with residual RNase activity that is not properly inhibited by barstar and thereby limits growth. In the two-stage barnase expression system pMI43a-b (Fig.…”

Section: Introductionmentioning

confidence: 99%

Protein-protein interaction: a genetic selection for compensating mutations at the barnase-barstar interface.

Jucovic

Hartley

1996

Proc. Natl. Acad. Sci. U.S.A.

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Barnase and barstar are trivial names of the extracellular RNase and its intracellular inhibitor produced by Bacillus amyloliquefaciens. Inhibition involves the formation of a very tight one-to-one complex of the two proteins. With the crystallographic solution of the structure of the barnasebarstar complex and the development of methods for measuring the free energy of binding, the pair can be used to study protein-protein recognition in detail. In this report, we describe the isolation of suppressor mutations in barstar that compensate for the loss in interaction energy caused by a mutation in barnase. Our suppressor search is based on in vivo selection for barstar variants that are able to protect host cells against the RNase activity of those barnase mutants not properly inhibited by wild-type barstar. This approach utilizes a plasmid system in which barnase expression is tightly controlled to keep the mutant barnase gene silent. When expression of barnase is turned on, failure to form a complex between the mutant barnase and barstar has a lethal effect on host cells unless overcome by substitution of the wild-type barstar by a functional suppressor derivative. A set of barstar suppressors has been identified for barnase mutants with substitutions in two amino acid positions (residues 102 and 59), which are critically involved in both RNase activity and barstar binding. The mutations selected as suppressors could not have been predicted on the basis of the known protein structures. The single barstar mutation with the highest information content for inhibition of barnase (H102K) has the substitution Y30W. The reduction in binding caused by the R59E mutation in barnase can be partly reversed by changing Glu-76 of barstar, which forms a salt bridge with the Arg-59 in the wild-type complex, to arginine, thus completing an interchange of the two charges.Much of biological existence is determined by unique interactions at the molecular level. A vast array of molecular recognitions are specific protein-protein interactions between enzymes and their substrates, enzymes and their inhibitors, the assembly of identical or different subunits into larger structures, etc. What is the nature of the interactions involved in protein-protein recognition? Which forces stabilize protein complexes? How plastic are the interfaces? One approach to this problem is to determine which changes in one partner will compensate for weakened binding caused by mutation in the other.We have developed a system to do just this, using in vivo selection to find such complementing mutations. The gene for barnase, the extracellular RNase of Bacillus amyloliquefaciens, is lethal when expressed in Escherichia coli without concurrent expression of its intracellular polypeptide inhibitor barstar. The genes for both proteins have been cloned in E. coli plasmids and both can be produced in quantity as plasmidThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertis...

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“…5 Recently, we introduced a strategy for producing soluble ICK proteins in Escherichia coli. 6 To this end, various ICK peptides were fused to the C terminus of the catalytically inactive H102A mutant of the RNase barnase from Bacillus amyloliquefaciens 7,8 separated by the four residue linker SSSM. Chemical cleavage with CNBr or enzymatic cleavage by thrombin allows isolation of the ICK peptide from the carrier protein.…”

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confidence: 99%