Genetic Selection Strategies Research Articles

Flagellar Motor-switch Binding Face of CheY and the Biochemical Basis of Suppression by CheY Mutants That Compensate for Motor-switch Defects in Escherichia coli

CheY is a response regulator protein of Escherichia coli that interacts with the flagellar motor-switch complex to modulate flagellar rotation during chemotaxis. The switch complex is composed of three proteins, FliG, FliM, and FliN. Recent biochemical data suggest a direct interaction of CheY with FliM. In order to determine the FliM binding face of CheY, we isolated dominant suppressors of fliM mutations in cheY with limited allele specificity. The protein products of suppressor cheY alleles were purified and assayed for FliM binding. Six out of nine CheY mutants were defective in FliM binding. Suppressor amino acid substitutions were mapped on the crystal structure of CheY showing clustering of reduced binding mutations on a solvent-accessible face of CheY, thus revealing a FliM binding face of CheY. To examine the basis of genetic suppression, we cloned, purified, and tested FliM mutants for CheY binding. Like the wild-type FliM, the mutants were also defective in binding to various CheY suppressor mutants. This was not expected if CheY suppressors were compensatory conformational suppressors. Furthermore, a comparison of flagellar rotation patterns indicated that the cheY suppressors had readjusted the clockwise bias of the fliM strains. However, a chemotaxis assay revealed that the readjustment of the clockwise bias was not sufficient to make cells chemotactic. Although the suppressors did not restore chemotaxis, they did increase swarming on motility plates by a process called "pseudotaxis." Therefore, our genetic selection scheme generated suppressors of pseudotaxis or switch bias adjustment. The binding results suggest that the mechanism for this adjustment is the reduction in binding affinity of activated CheY. Therefore, these suppressors identified the switch-binding surface of CheY by loss-of-function defects rather than gain-of-function compensatory conformational changes.

Genetic selection strategies: computer modeling

There are four primary factors to consider in genetic selection strategies: 1) accuracy of selection, 2) selection intensity, 3) effective population size, and 4) mating system. Current theory indicates that optimum response to selection is achieved by maximizing the first three factors and using a mating systems that allows optimization of reproductive characteristics in dam lines and production characteristics in sire lines. However, with limited resources, compromises among the first three factors are needed. Simulations are useful for examining those compromises. Unrealistic simplifying assumptions are necessary for analytic theoretical results and thus do not address real world breeding problems. Using simulations, the relationship between selection accuracy, which is increased by use of family selection indices or Best Linear Unbiased Prediction (BLUP), and response to selection was examined. Results show that those procedures place a great restriction on effective population size, which offsets most of their advantage, i.e., there is too little emphasis on effective population size. A revision of the methodology and a reappraisal of the results of selection theory for optimization of genetic response is required. Another relationship that is of fundamental importance in breeding programs is that between selection intensity and effective population size. Analytical results for the additive case have been developed but have never been extended to heterotic traits. A gene level simulation program was developed to examine that relationship. Results show that the optimal selection strategy depends on the trait being selected. For additive traits and in the short term (20 generations), one should maximize selection intensity. For heterotic traits, an intermediate proportion (25% of each sex) gives optimal response. In all breeding strategies, primary attention must be given to the rate of inbreeding, which is increased by increasing either accuracy of selection or selection intensity. Inbreeding reduces response to selection in two ways. First, for both additive and nonadditive traits, inbreeding is a measure of the amount of random genetic drift that has occurred. Genetic drift causes loss of favorable alleles. Once lost, those alleles can never be recovered and thus genetic drift lowers the selection limit. Second, for heterotic traits, inbreeding results in a depression of the mean caused by directional dominance.

Genetic selection strategies–population genetics

This paper provides an overview of the association between population genetics and selection strategies in poultry. Relationships between artificial and natural selection and among causes contributing to limits to artificial selection are discussed. Homeostasis and resource allocations at the individual and at the population level are reviewed. Examples from poultry demonstrate where human intervention has circumvented biological limits. Lastly, this paper considers the role of population genetics in future breeding strategies for poultry.

Gene isolation in Arabidopsis thaliana by conditional overexpression of cDNAs toxic to Saccharomyces cerevisiae: identification of a novel early response zinc-finger gene.

In an effort to identify novel regulatory plant genes, conditional overexpression of toxic Arabidopsis thaliana gene products in Saccharomyces cerevisiae was evaluated as a genetic selection scheme. The screening method was tested on a fraction of a cDNA expression library and led to the identification of two Arabidopsis cDNA clones that were toxic to yeast; one corresponded to histone H1 and the other to a previously unidentified gene. This new gene, named ATL2, combines a RING-like zinc-binding motif and a putative signal anchor sequence for membrane insertion in the same molecule. Furthermore, inspection of the 3' untranslated region reveals two types of sequences which appear to be key determinants in rapid transcript decay. Indeed, rapid and transient accumulation of transcript occurs in the presence of a protein synthesis inhibitor and of the growth regulator auxin. These features provide evidence that ATL2 is an early-response gene. Thus, ATL2 represents one of the first early-response plant genes to be described which possesses a distinct regulatory domain; the fact that ATL2 mRNA is induced by auxin suggests that it might have a role during the response of plants to this growth regulator.

The reverse two-hybrid system: a genetic scheme for selection against specific protein/protein interactions

The yeast two-hybrid system is a powerful experimental approach for the characterization of protein/ protein interactions. A unique strength of the yeast two-hybrid system is the provision for genetic selection techniques that enable the identification of specific protein/protein interactions. We now report the development of a modified yeast two-hybrid system which enables genetic selection against a specific protein/protein interaction. This reverse two-hybrid system utilizes a yeast strain which is resistant to cycloheximide due to the presence of a mutant cyh2 gene. This strain also contains the wild-type CYH2 allele under the transcriptional control of the Gal1 promoter. Expression of the wild-type Gal4 protein is sufficient to restore growth sensitivity to cycloheximide. Growth sensitivity towards cycloheximide is also restored by the coexpression of the avian c-Rel protein and its I kappa B alpha counterpart, p40, as Gal4 fusion proteins. Restoration of growth sensitivity towards cycloheximide requires the association of c-Rel and p40 at the Gal1 promoter and correlates with the ability of the c-Rel/p40 interaction to activate expression from the Gal1 promoter. A genetic selection scheme against specific protein/protein interactions may be a valuable tool for the analysis of protein/protein interactions.

Genetic selection strategies for generating and characterizing catalysts

Abstract

Dynamics of Bacteriophage T4 DNA Polymerase Function: Identification of Amino Acid Residues that Affect Switching between Polymerase and 3′ → 5′ Exonuclease Activities

Many DNA polymerases are multifunctional with the ability to replicate DNA as well as to proofread misincorporated nucleotides. Since polymerase and 3′ → 5′ exonuclease activities appear to reside in spatially distinct active centers, there must be some mechanism for coordinating replication with proofreading and for transferring DNA between the two active centers. We have designed a genetic selection scheme to isolate bacteriophage T4 mutant DNA polymerases that are defective in "switching" between polymerase and exonuclease activities. Amino acid residues that affected active-site-switching were identified in four regions of the T4 DNA polymerase: two regions in the proposed polymerase domain and two regions in the proposed exonuclease domain. Representative mutant DNA polymerases from each region were purified for biochemical studies. We propose that amino acid substitutions identified by mutational analysis affect critical contacts between T4 DNA polymerase and DNA that are required for transfer of DNA between the polymerase and exonuclease active centers.

In vitro integration of retrotransposon Ty1: a direct physical assay.

Retrotransposon Ty1 of Saccharomyces cerevisiae inserts a double-stranded Ty1 cDNA into the yeast genome by a reaction analogous to the integration mechanism used by retroviruses. A quantitative in vitro integration assay that directly detects integrative recombination products was developed for Ty1. Blunt-ended artificial radioactive substrates bearing Ty1 termini integrate into circular or linear target DNAs. The reaction is specific for native integrase isolated in the form of virus-like particles; virus-like particles prepared from integrase mutants were completely inactive in this assay. The products are radioactive, allowing direct detection after gel electrophoresis by autoradiography. Using this simple and amenable system, we characterized the biochemical requirements of the system and the structures of the major integration products. Two classes of products were detected: those that were the result of bona fide complete integration events (concerted reactions) and single-end joinings of substrate to target (half-reactions). Additionally, we used a genetic selection scheme to identify and characterize target sites of complete integration events into a circular target plasmid; a 5-bp target site duplication flanking the inserted DNA resembling the duplication characteristic of in vivo integration was observed.

In vitro integration of retrotransposon Ty1: a direct physical assay.

Retrotransposon Ty1 of Saccharomyces cerevisiae inserts a double-stranded Ty1 cDNA into the yeast genome by a reaction analogous to the integration mechanism used by retroviruses. A quantitative in vitro integration assay that directly detects integrative recombination products was developed for Ty1. Blunt-ended artificial radioactive substrates bearing Ty1 termini integrate into circular or linear target DNAs. The reaction is specific for native integrase isolated in the form of virus-like particles; virus-like particles prepared from integrase mutants were completely inactive in this assay. The products are radioactive, allowing direct detection after gel electrophoresis by autoradiography. Using this simple and amenable system, we characterized the biochemical requirements of the system and the structures of the major integration products. Two classes of products were detected: those that were the result of bona fide complete integration events (concerted reactions) and single-end joinings of substrate to target (half-reactions). Additionally, we used a genetic selection scheme to identify and characterize target sites of complete integration events into a circular target plasmid; a 5-bp target site duplication flanking the inserted DNA resembling the duplication characteristic of in vivo integration was observed.

1005 GENETIC AND MORPHOLOGICAL BASES FOR ROOT-BASED SEEDLING STRESS RESISTANCE

Different root types have different temporal and morphological patterns of development, functionality and adaptation. Root based adaptation to stresses can not be assessed on a root system basis, but must be employed on an individual root type basis. Three different types of root are developed by most seedlings: tap root, basal roots, and lateral roots. These have been demonstrated to have temporally and spatially different developmental and functional patterns. If a stress occurs prior to the onset of an especially sensitive type of root or after that type has shut down functionally, the seedling will demonstrate resistance, when it is not correctly resistant. Timing of screening treatments and scoring of the results is, therefore, extremely critical. Different genotypes within a species demonstrate strong differences in numbers and timing of root initiation and functional maturity for each of the root typos. In addition, different types of root demonstrate sensitivity/tolerance to different chemicals, suggesting that functionality and, therefore, resistance/tolerance mechanisms may differ. All root types present on a seedling must be scored for resistance/sensitivity to the stress, even if morphological/physiological symptoms are not readily apparent. The technology (knowledge, software and equipment) necessary to detect tolerance/resistance and to establish genetic selection schemes is available. Root type differences and the potential for genetic selection will be discussed.

Amplification of an adenylosuccinate synthetase gene in alanosine-resistant murine T-lymphoma cells. Molecular cloning of a cDNA encoding the “non-muscle” isozyme.

Adenylosuccinate synthetase (EC 6.3.4.4) catalyzes the initial step in the conversion of IMP to AMP. Two isoforms of this enzyme have been observed in vertebrates. A muscle isozyme is highly abundant in cardiac and skeletal muscle tissue and is thought to play a role in muscle energy metabolism. The non-muscle isozyme, which is present at low levels in most tissues, likely functions in de novo AMP biosynthesis. The analysis of the non-muscle isozyme has been hampered by its low abundance and instability during purification. In this study a genetic selection scheme was used to generate a murine T-lymphoma cell line which was at least 100-fold enriched for the non-muscle isozyme, as a result of amplification of the non-muscle synthetase gene. This cell line made possible the purification of the non-muscle isozyme, and the subsequent isolation of isozyme-specific peptides. Based on peptide sequence information a degenerate oligonucleotide probe was designed and used to screen a mouse kidney cDNA library. A 1.5-kilobase cDNA encoding the non-muscle isozyme was cloned and found to contain an open reading frame of 1368 base pairs encoding 456 amino acids. Gene transfer experiments showed that the cDNA encoded a 50-kDa protein, the size expected for mammalian synthetases, that correlated with the presence of high levels of synthetase activity. The deduced amino acid sequence of the mouse non-muscle synthetase is approximately 75% identical to the previously reported mouse muscle synthetase. Southern blot analysis of mouse genomic DNA with the isozyme-specific cDNA probes revealed that the synthetase isozymes are encoded by separate genes. The non-muscle gene is expressed in most tissues but is virtually undetectable in striated muscle tissues. Three different transcripts (1.7, 2.8, and 3.4 kilobases) are detected for the non-muscle isozyme which show a similar tissue distribution. The availability of a cDNA for the non-muscle isozyme of adenylosuccinate synthetase will facilitate further comparative analyses with the previously cloned muscle isozyme.

Identification and characterization of multiple A/T-rich cis-acting elements that control expression from Dictyostelium actin promoters: the Dictyostelium actin upstream activating sequence confers growth phase expression and has enhancer-like properties.

The promoter elements in the Dictyostelium actin 15 and actin 6 genes required for full growth phase expression were identified by assaying promoter/luciferase reporter constructs. We find that these promoters contain common cis-acting elements, an actin upstream activating sequence (UAS) and sequences proximal to the transcription start site that overlap with a poly(dT) region. The actin 15 promoter has two additional cis-acting elements not present in the actin 6 promoter that may account for the higher level of expression from the actin 15 promoter. All of the identified promoter elements are unusual for Dictyostelium in that they are all A/T-rich. Two cis-acting elements, the actin UAS and the poly(dT) domain were studied in greater detail. The actin UAS was tested on a heterologous promoter from the prespore-specific gene SP60 and shown to have the ability to confer growth phase expression. The actin UAS also exhibited the ability to function in a distance- and orientation-independent manner and activate expression synergistically when present in two copies. The poly(dT) domain of the actin 15 promoter was studied in greater detail by using a genetic selection scheme to define parameters that effect the strength of this element. This element is comprised of 45 consecutive dT residues immediately upstream of the putative TATA box. We show that the length of the homopolymer dT region correlates with the expression level of the promoter. The poly(dT) element is also shown to function to promote wild-type levels of expression with small deviations in the sequence, indicating that the element is not required to be homopolymeric to function.

Genetic selection schemes — an important part of sustainable livestock improvement?

Genetic selection schemes — an important part of sustainable livestock improvement?

Conversion of the lac Repressor into an Allosterically Regulated Transcriptional Activator for Mammalian Cells

A novel mammalian regulatory system was created by using the Escherichia coli lac repressor. The lac repressor was converted into a mammalian transcriptional activator by modifying the lac repressor coding region to include a nuclear localization signal from the simian virus 40 (SV40) large tumor antigen and the transcription activation domain from the herpes simplex virus type 1 virion protein 16. The lac activator protein (LAP) fusions were potent activators of several promoters containing lac operator sequences positioned either upstream or downstream of the transcription unit. A single lac operator allowed for transactivation, whereas multiple operators acted synergistically when separated by a small distance. Promoters containing 14 or 21 operator sequences were induced at least 1,000-fold in response to LAP, reaching levels of activity 20 to 30 times greater than that of the SV40 early promoter in HeLa cells. Activation was strongly inhibited by isopropyl-beta-D-thiogalactoside (IPTG), indicating that LAP retained the functions needed for allosteric regulation. LAP was bifunctional, also acting as a repressor of expression of an SV40 promoter containing an operator immediately downstream of the TATA box. Finally, genetic selection schemes were developed such that LAP-expressing cell lines can be generated at high frequency from either established or primary cells in culture.

Conversion of the lac repressor into an allosterically regulated transcriptional activator for mammalian cells.

A novel mammalian regulatory system was created by using the Escherichia coli lac repressor. The lac repressor was converted into a mammalian transcriptional activator by modifying the lac repressor coding region to include a nuclear localization signal from the simian virus 40 (SV40) large tumor antigen and the transcription activation domain from the herpes simplex virus type 1 virion protein 16. The lac activator protein (LAP) fusions were potent activators of several promoters containing lac operator sequences positioned either upstream or downstream of the transcription unit. A single lac operator allowed for transactivation, whereas multiple operators acted synergistically when separated by a small distance. Promoters containing 14 or 21 operator sequences were induced at least 1,000-fold in response to LAP, reaching levels of activity 20 to 30 times greater than that of the SV40 early promoter in HeLa cells. Activation was strongly inhibited by isopropyl-beta-D-thiogalactoside (IPTG), indicating that LAP retained the functions needed for allosteric regulation. LAP was bifunctional, also acting as a repressor of expression of an SV40 promoter containing an operator immediately downstream of the TATA box. Finally, genetic selection schemes were developed such that LAP-expressing cell lines can be generated at high frequency from either established or primary cells in culture.