Isolation and characterization of further cis- and trans-acting regulatory elements involved in the synthesis of glucose-repressible alcohol dehydrogenase (ADHII) in Saccharomyces cerevisiae.

Summary Starting with yeast cells lacking the constitutive alcohol dehydrogenase activity (ADHI), mutants with partially glucose-insensitive formation of ADHII were isolated. Genetic analysis showed that four mutants (designated ADR3 ^c) were linked to the ADHII-structural gene, ADR2, and were cis-dominant. On derepression, two of them produced elevated ADHII-levels, indicating a promotor function of the altered controlling site. The other ADR3 ^c-mutant alleles affected the ADHII-subunit association in diploids carrying two electrophoretically distinct alleles of the structural gene ADR2. Twelve semidominant constitutive mutants could be attributed to gene ADR1 (ADR1 ^c-alleles) previously identified by recessive mutants with blocked derepression. This suggested a positive regulatory role of the ADR1 gene product on the expression of the ADHII-structural gene. A pleiotropic mutation ccr1 (Ciriacy, 1977) was epistatic over glucose-resistant ADHII-formation caused by ADR1 ^c-alleles. From this it was concluded that CCR1 specifies for a product co-activating the structural gene or modifying the ADR1-gene product. A further regulatory element (gene designation ADR4) not linked to the structural gene could be identified upon isolation of recessive constitutive mutants adr4 from a ccr1 ADR1 ^c-double mutant.     

Identification and Characterization of Three Genes That Affect Expression of Adh2 in Saccharomyces Cerevisiae

Using a new selection protocol we have identified and preliminarily characterized three new loci (ADR7, ADR8 and ADR9) which affect ADH2 (alcohol dehydrogenase isozyme II) expression. Mutants were selected which activate ADH2 expression in the presence of an over-expressed, normally inactive ADR1 allele. The mutants had very similar phenotypes with the exception that one was temperature sensitive for growth. In the absence of any ADR1 allele, the mutants allowed ADH2 to partially escape glucose repression. However, unlike wildtype strains deleted for ADR1, the mutants were able to efficiently derepress ADH2. The mutations allowed a small escape from glucose repression for secreted invertase, but had no effect on the glucose repression of isocitrate lyase or malate dehydrogenase. The mutations were shown to be nonallelic to a wide variety of previously characterized mutations, including mutations that affect other glucose-repressed enzymes.     

A mutation outside the two zinc fingers of ADR1 can suppress defects in either finger.

A second-site mutation that restored DNA binding to ADR1 mutants altered at different positions in the two zinc fingers was identified. This mutation (called IS1) was a conservative change of arginine 91 to lysine in a region amino terminal to the two zinc fingers and known from previous experiments to be necessary for DNA binding. IS1 increased binding to the UAS1 sequence two- to sevenfold for various ADR1 mutants and twofold for wild-type ADR1. The change of arginine 91 to glycine decreased binding twofold, suggesting that this arginine is involved in DNA binding in the wild-type protein. The increase in binding by IS1 did not involve protein-protein interactions between the two ADR1 monomers, nor did it require the presence of the sequences flanking UAS1. However, the effect of IS1 was influenced by the sequence of the first finger, suggesting that interactions between the region amino terminal to the fingers and the fingers themselves could exist. A model for the role of the amino-terminal region based on these results and sequence homologies with other DNA-binding motifs is proposed.     

Mutagenesis directed by phosphotriester analogs of oligonucleotides

20-mer oligodeoxyribonucleotides d-ACGACGG (R') CCAG (R') TGATCCGTA, where R' = R' = H (20), F' = Et, R' = H (20-Et), or R' = R' = Et (20-Et2) were synthesized by modified triester method. Ethylated dinucleotide blocks were prepared by transesterification method from chlorophenyl derivatives. Structures of oligonucleotides were confirmed by Maxam - Gilbert method. Mutagenesis induced by oligonucleotides was studied on DNA of M13mpB phage. Oligonucleotides were not totally complementary to this DNA in the region of 4-11 codons of Z'-gene. They all were shown to direct the formation of the designed deletion mutants, phosphotriester analogues (20-Et) and (20-Et2) being more effective mutagens. The specificity of oligonucleotides: DNA binding and mutant DNA structure were shown by Sanger method.     

An efficient method for the sequence analysis of oligodeoxyribonucleotides

Modifications of the chemical method of DNA sequence analysis that permit rapid and reliable sequence determination of single-stranded oligodeoxyribonucleotides as short as 4 nucleotides in length are reported. The principal changes made were increasing the level of chemical modification and optimizing the conditions for recovery of the chemically modified oligodeoxyribonucleotides. This method includes two approaches to the removal of [γ-³²P]ATP from ³²P-labeled oligodeoxyribonucleotides and is especially useful in the determination of the sequence of chemically synthesized oligodeoxyribonucleotides, which are generally between 4 and 20 nucleotides in length.     

Cis-dominant regulatory mutations affecting the formation of glucose-repressible alcohol dehydrogenase (ADHII) in Saccharomyces cerevisiae

Summary The formation of ADHII in Saccharomyces cerevisiae is regulated by carbon catabolite repression. There are two genes involved in the formation of ADHII: ADR2, the structural gene as identified by electrophoretic variants and ADR1, possibly a regulatory gene. A new genetic element involved in the regulation of ADHII was identified by three allelic mutants insensitive to strong glucose repression. They were called ADR3 ^c (wild type designation ADR3) and found to be tightly linked to the structural gene, ADR2. The alcohol dehydrogenase found in ADR3 ^c mutants could not be distinguished electrophoretically from the ADHII of the glucose-sensitive wild type, ADR3.Dominance relations between ADR3 ^c and ADR3 were established in diploids heterozygous for ADR3 and the two alleles of ADR2 (ADR2-S: slow ADHII, ADR2-F: fast ADHII). During growth on 10% glucose, an ADR3 ^c ADR2-F/ADR3 ADR2-S heterozygous diploid formed only the fast ADHII variant whereas an ADR3 ^c ADR2-S/ADR3 ADR2-F heterozygote produced only the slow form. This was taken as evidence of the cis-dominance of all ADR3 ^c alleles. The cis-effect of ADR3 ^c was also demonstrated in glucose-derepressed diploids. The ADR3 ^c mutations do not only cause glucose-insensitive ADHII formation, but also reduce the activity of the adjacent structural gene during derepression. Thus ADR3 ^c alleles were considered to be controlling site mutations. No pleiotropic effects were observed on the formation of enzymes related to the function of ADHII.An adr1 ADR2 ADR3 single mutant did not form ADHII. In contrast to this, an adr1 ADR2 ADR3 ^c double mutant formed ADHII at a similar level as an ADR1 ADR2 ADR3 ^c mutant. This showed that ADR3 ^c was epistatic over adr1 (previously suggested as a positive regulatory gene). From this it was concluded that ADR1 is in fact a positive regulatory gene the function of which is required for the expression of the structural gene for ADHII, ADR2. ADR3 is the controlling site for the structural gene ADR2. Mutations at this site, ADR3 ^c , alleviate the requirement for the ADR2 gene product. ADR3 ^c is discussed as a promotor or operator site.     

Convenient modification of the method for oligonucleotide-directed in vitro mutagenesis of cloned DNA

A new modification of the oligonucleotide-mediated mutagenesis technique has been developed. The proposed methodology has been used to produce specific base changes in the double-stranded plasmid DNA. For this purpose, special cloning vectors have been constructed using the synthetic oligodeoxyribonucleotides. The developed method allows the production of mutant DNA from those of the wild-type with a yield of 10-20%.     

Drought tolerance established by enhanced expression of the CC-NBS-LRR gene, ADR1, requires salicylic acid, EDS1 and ABI1

An activation-tagged allele of activated disease resistance 1 (ADR1) has previously been shown to convey broad spectrum disease resistance. ADR1 was found to encode a coiled-coil (CC)-nucleotide-binding site (NBS)-leucine-rich repeat (LRR) protein, which possessed domains of homology with serine/threonine protein kinases. Here, we show that either constitutive or conditional enhanced expression of ADR1 conferred significant drought tolerance. This was not a general feature of defence-related mutants because cir (constitutive induced resistance)1, cir2 and cpr (constitutive expressor of PR genes)1, which constitutively express systemic acquired resistance (SAR), failed to exhibit this phenotype. Cross-tolerance was not a characteristic of adr1 plants, rather they showed increased sensitivity to thermal and salinity stress. Hence, adr1-activated signalling may antagonise some stress responses. Northern analysis of abiotic marker genes revealed that dehydration-responsive element (DRE)B2A but not DREB1A, RD (response to dehydration)29A or RD22 was expressed in adr1 plant lines. Furthermore, DREB2A expression was salicylic acid (SA) dependent but NPR (non-expressor of PR genes)1 independent. In adr1/ADR1 nahG (naphthalene hydroxylase G), adr1/ADR1 eds (enhanced disease susceptibility)1 and adr1/ADR1 abi1 double mutants, drought tolerance was significantly reduced. Microarray analyses of plants containing a conditional adr1 allele demonstrated that a significant number of the upregulated genes had been previously implicated in responses to dehydration. Therefore, biotic and abiotic signalling pathways may share multiple nodes and their outputs may have significant functional overlap.     

Analysis of mutations affecting Ty-mediated gene expression in Saccharomyces cerevisiae

Summary Yeast translocatable, Ty, elements can cause constitutive synthesis of the glucose-repressible alcohol dehydrogenase (ADHII) when inserted upstream from the 5 end of the structural gene, ADR2. These insertion mutations, ADR3 ^c, are unstable and give rise to secondary ADHII^– mutations. The majority of such mutants, adr3, can be attributed to excision of the insertion sequence, leaving behind a single copy of the -sequence which occurs as a direct repeat at the ends of the Ty elements. A few adr3 mutants appear to be generated by DNA-rearrangements in the vicinity of the Ty insertion. The occurrence of recessive mutants, tye, which are unlinked to ADR2 indicates that the constitutive expression of ADR2 caused by the Ty insertions requires the function of trans-acting genes. These results support the idea that regulation of Ty-linked ADR2 is actively mediated by the insertion sequence and is probably not due to a mere disruption of the wild-type controlling site.     

Targeted activation tagging of the Arabidopsis NBS-LRR gene,ADR1, conveys resistance to virulent pathogens

A transgenic Arabidopsis line containing a chimeric PR-1::luciferase (LUC) reporter gene was subjected to mutagenesis with activation tags. Screening of lines via high-throughput LUC imaging identified a number of dominant Arabidopsis mutants that exhibited enhanced PR-1 gene expression. Here, we report the characterization of one of these mutants, designated activated disease resistance (adr) 1. This line showed constitutive expression of a number of key defense marker genes and accumulated salicylic acid but not ethylene or jasmonic acid. Furthermore, adr1 plants exhibited resistance against the biotrophic pathogens Peronospora parasitica and Erysiphe cichoracearum but not the necrotrophic fungus Botrytis cinerea. Analysis of a series of adr1 double mutants suggested that adr1-mediated resistance against P. parasitica was salicylic acid (SA)-dependent, while resistance against E. cichoracearum was both SA-dependent and partially NPR1-dependent. The ADR1 gene encoded a protein possessing a number of key features, including homology to subdomains of protein kinases, a nucleotide binding domain, and leucine-rich repeats. The controlled, transient expression of ADR1 conveyed striking disease resistance in the absence of yield penalty, highlighting the potential utility of this gene in crop protection.     

The isolation and genetic analysis of V79-derived etoposide sensitive Chinese hamster cell mutants: two new complementation groups of etoposide sensitive mutants

Using a replica plating microwell method, three Chinese hamster V79-derived cell lines, designated ETO1, ETO2 and ETO3, which exhibit hypersensitivity to the non-intercalating topoisomerase II inhibitor etoposide have been isolated. Mutant lines ETO2 and ETO3 are cross-sensitive to the topoisomerase II inhibitors adriamycin and streptonigrin; however, neither mutant is sensitive to the topoisomerase I inhibitor camptothecin, the bifunctional alkylating agent mitomycin C, nor hydrogen peroxide. In contrast, ETO1 is cross-sensitive to camptothecin but displays only slight sensitivity to adriamycin, streptonigrin and hydrogen peroxide, and is not sensitive to mitomycin C. It has been established through extensive cell fusion studies that all three mutants are genetically distinct, and that ETO2 and ETO3 genetically complement all other known etoposide-sensitive Chinese hamster cell mutants (i.e., irs1, XR-1, xrs1, V3, BLM2, ADR1, ADR3, ADR4 and ADR5) thus defining two new complementation groups of etoposide sensitive mutants. Interestingly, the hybrids created by the fusion irs2TOR (thioguanine and ouabain resistant)xETO1 and the reciprocal cross ETO1TORxirs2 both exhibited a response to camptothecin intermediate with respect to V79 and ETO1. It has been hypothesised that this partial complementation may be the result of intragenic complementation and that both ETO1 and irs2 result from mutations in the gene XRCC8. This study indicates that cellular responses to topoisomerase II inhibitors are complex and hypersensitivity may result from mutations in many different genes.     

Mechanism of DNA binding by the ADR1 zinc finger transcription factor as determined by SPR

The ADR1 protein recognizes a six base-pair consensus DNA sequence using two zinc fingers and an adjacent accessory motif. Kinetic measurements were performed on the DNA-binding domain of ADR1 using surface plasmon resonance. Binding by ADR1 was characterized to two known native binding sequences from the ADH2 and CTA1 promoter regions, which differ in two of the six consensus positions. In addition, non-specific binding by ADR1 to a random DNA sequence was measured. ADR1 binds the native sites with nanomolar affinities. Remarkably, ADR1 binds non-specific DNA with affinities only approximately tenfold lower than the native sequences. The specific and non-specific binding affinities are conferred mainly by differences in the association phase of DNA binding. The association rate for the complex is strongly influenced by the proximal accessory region, while the dissociation reaction and specificity of binding are controlled by the two zinc fingers. Binding kinetics of two ADR1 mutants was also examined. ADR1 containing an R91K mutation in the accessory region bound with similar affinity to wild-type, but with slightly less sequence specificity. The R91K mutation was observed to increase binding affinity to a suboptimal sequence by decreasing the complex dissociation rate. L146H, a change-of-specificity mutation at the +3 position of the second zinc finger, bound its preferred sequence with a slightly higher affinity than wild-type. The L146H mutant indicates that beneficial protein-DNA contacts provide similar levels of stabilization to the complex, whether they are hydrogen-bonding or van der Waals interactions.     

Improvements in the phosphoramidite procedure for the synthesis of oligodeoxyribonucleotides.

The paper describes an improved method for the synthesis of oligodeoxyribonucleotides using phosphoramidite chemistry. Our procedure relies on novel phosphoramidite intermediates, the deoxyribonucleoside-3'-morpholine-methoxyphosphins. These compounds are extremely stable and can be purified readily. Condensation reactions during solid-phase synthesis can thus be performed with high efficiency and result in a high yield synthesis of long chain oligodeoxyribonucleotides.