Flipping a genetic switch by subunit exchange

Correction to: The EMBO Journal (2001) 20, 7149–7159. doi:10.1093/emboj/20.24.7149Two different structures of the anti-σ factor T4 AsiA in its dimeric state have been reported, one by us and a second by Urbauer et al (2002). The principal distinction between these structures was in the monomer fold, which displayed an approximate mirror image relationship to one another. This difference prompted a re-examination of our AsiA structure in solution, leading us to conclude that our structure of the T4 AsiA dimer was incorrectly determined in the original report. The resolution of this discrepancy was driven by the solution of the AsiA structure in two new states: a free monomer and a monomer bound to conserved region 4 from Escherichia coli σ⁷⁰ (EcSR4) (see report by Lambert et al in this issue (The EMBO Journal (2004) 23, 2952–2962). In each of these states, the chemical shifts for all atoms were similar to each other and very different from those observed in AsiA dimer, enabling a completely independent effort at the solution of the AsiA structure. The fold of AsiA determined in each of these new states was found to be very similar to that reported by Urbauer et al. Most convincing in this analysis were the 220 intermolecular NOEs observed between AsiA and EcSR4, which were only consistent with the fold reported by Urbauer et al. Both monomer states were further refined by residual dipolar coupling (RDC) analysis. We subsequently reanalyzed our original AsiA dimer NMR spectra and collected RDCs on the original dimer. No errors in chemical shift assignment were found and less than 4% of all NOEs needed to be reassigned for the fold of AsiA dimer to conform to the fold reported by Urbauer et al. This represented less than 3% of those NOEs between nonsequential residues in the polypeptide chain (17 NOEs in all); these NOEs were not previously appreciated to be ambiguous with respect to a mirror image fold. Figure 1 displays the revised structure and compares it to that of Urbauer et al. The coordinates of our structure with PDB accession code 1KA3 have been replaced. The PDB ID for the corrected AsiA dimer is 1TKV.Open in a separate windowFigure 1Comparison of recalculated structure of AsiA dimer to the structure of Urbauer et al (PDB accession code 1JR5). On the left is the recalculated structure family with a ribbon representation of the monomer shown at the bottom. On the right is the C_α trace of one model from 1JR5 and a ribbon representation at the bottom. The two structures are very similar to one another, with the exception of the position of helix A6. In 1JR5, the C-terminus is tucked against the back face of helices A1 and A4, while in our revised structures these restraints are not observed. Helix A6 in 1JR5 is also one turn shorter than that observed by us. Despite these differences, the two structures are in overall agreement.     

Introduction of the negative selection marker into replacement vectors by a single ligation step.

Gene targeting is a powerful method for introducing mutations into the genome of embryonic stem cells. The most widely used approach is the positive-negative selection method in which a gene encoding a negative selection marker is cloned into the replacement vector to obtain an enrichment of properly targeted clones. Here, we present an alternative means to introduce any given negative selection marker at the ends of a replacement vector using a single ligation step, thereby avoiding laborious cloning procedures. Our results demonstrate that this fast and simple method consistently provides a high level of enrichment of appropriately targeted clones.     

Successful genetic transformation of Chinese cabbage using phosphomannose isomerase as a selection marker

A mannose selection system was adapted for use in the Agrobacterium-mediated transformation of Chinese cabbage. This system makes use of the pmi gene that encodes phosphomannose isomerase, which converts mannose-6-phosphate to fructose-6-phosphate. Hypocotyl explants from 4–5-day-old seedlings of Chinese cabbage inbred lines were pre-cultured for 2–3 days and then infected with Agrobacterium. Two genes (l-guluno-γ-lactone oxidase, GLOase, and jasmonic methyl transferase, JMT) were transformed into Chinese cabbage using the transformation procedure developed in this study. We found that supplementing the media with 7 g l⁻¹ mannose and 2% sucrose provides the necessary conditions for the selection of transformed plants from nontransformed plants. The transformation rates were 1.4% for GLOase and 3.0% for JMT, respectively. The Southern blot analysis revealed that several independent transformants (T ₀) were obtained from each transgene. Three different inbred lines were transformed, and most of the T ₁ plants had normal phenotypes. The transformation method presented here for Chinese cabbage using mannose selection is efficient and reproducible, and it can be useful to introduce a desirable gene(s) into commercially useful inbred lines of Chinese cabbage.     

Toward genetic transformation of mitochondria in mammalian cells using a recoded drug-resistant selection marker

Due to technical difficulties, the genetic transformation of mitochondria in mammalian cells is still a challenge. In this report, we described our attempts to transform mammalian mitochondria with an engineered mitochondrial genome based on selection using a drug resistance gene. Because the standard drug-resistant neomycin phosphotransferase confers resistance to high concentrations of G418 when targeted to the mitochondria, we generated a recoded neomycin resistance gene that uses the mammalian mitochondrial genetic code to direct the synthesis of this protein in the mitochondria, but not in the nucleus (mitochondrial version). We also generated a universal version of the recoded neomycin resistance gene that allows synthesis of the drug-resistant proteins both in the mitochondria and nucleus. When we transfected these recoded neomycin resistance genes that were incorporated into the mouse mitochondrial genome clones into mouse tissue culture cells by electroporation, no DNA constructs were delivered into the mitochondria. We found that the universal version of the recoded neomycin resistance gene was expressed in the nucleus and thus conferred drug resistance to G418 selection, while the synthetic mitochondrial version of the gene produced no background drug-resistant cells from nuclear transformation. These recoded synthetic drug-resistant genes could be a useful tool for selecting mitochondrial genetic transformants as a precise technology for mitochondrial transformation is developed.     

Generation of double gene disruptions in Dictyostelium discoideum using a single antibiotic marker selection

Gene targeting is a powerful molecular genetic technique that has been widely used to understand specific gene function in vivo. This technique allows the ablation of an endogenous gene by recombination between an introduced DNA fragment and the homologous target gene. However, when multiple gene disruptions are needed, the availability of only a limited number of marker genes becomes a complication. Here we describe a new approach to perform double gene disruptions in Dictyostelium discoideum by simultaneous transfection of two gene targeting cassettes followed by performing clonal selection against only one marker gene. The subsequent PCR-based screens of blasticidin-resistant clones revealed the integration of both the selected and the nonselected targeting cassettes at their original respective loci creating complete gene disruptions. For the genes we have tested in these studies (myosin heavy chain kinases B and C), the efficiency of the double gene targeting event is found in the range of 2%-5% of all blasticidin-resistant colonies following the transfection step. This approach for the simultaneous disruptions of multiple genes should prove to be a valuable tool for other laboratories interested in creating multiple gene disruptants in Dictyostelium or other organisms where a limited number of selectable markers are available.     

Objective evaluation measures of genetic marker selection in large-scale SNP genotyping

High-throughput single nucleotide polymorphism (SNP) genotyping systems provide two kinds of fluorescent signals detected from different alleles. In current technologies, the process of genotype discrimination requires subjective judgments by expert operators, even when using clustering algorithms. Here, we propose two evaluation measures to manage fluorescent scatter data with nonclear plot aggregation. The first is the marker ranking measure, which provides a ranking system for the SNP markers based on the distance between the scatter plot distribution and a user-defined ideal distribution. The second measure, called individual genotype membership, uses the membership probability of each genotype related to an individual plot in the scatter data. In verification experiments, the marker ranking measure determined the ranking of SNP markers correlated with the subjective order of SNP markers judged by an expert operator. The experiment using the individual genotype membership measure clarified that the total number of unclassified individuals was remarkably reduced compared to that of manually unclassified ones. These two evaluation measures were implemented as the GTAssist software. GTAssist provides objective standards and avoids subjective biases in SNP genotyping workflows.     

Natural selection and the origin and maintenance of standard genetic marker systems

Natural selection has always been assumed to be the major force of evolution, but its presence has been difficult to demonstrate. A review of the evidence for selective differences among genotypes for most human genetic polymorphisms indicates there is little of a direct nature. Indirect theoretical evidence, however, seems to support a major role for natural selection, and it does not seem to support the hypothesis that most amino acid substitutions within the human species are neutral. Among small isolates, most of the gene frequency differences are most likely due to genetic drift or the founder effect, and the principal counterbalancing force is gene flow or migration. But genetic differences among the major human subdivisions do not seem to be due to the same interacting forces. One reason for the inability to detect selection has been an oversimplified view of its operation, which assigns genotypes a constant fitness in every generation. Many recent theoretical developments of more complicated kinds of selection may lead to a resolution of the problem and suggest better interpretations of the enormous amount of data on human genetic variation that is rapidly accumulating.     

Accuracy of marker-assisted selection with single markers and marker haplotypes in cattle

A key question for the implementation of marker-assisted selection (MAS) using markers in linkage disequilibrium with quantitative trait loci (QTLs) is how many markers surrounding each QTL should be used to ensure the marker or marker haplotypes are in sufficient linkage disequilibrium (LD) with the QTL. In this paper we compare the accuracy of MAS using either single markers or marker haplotypes in an Angus cattle data set consisting of 9323 genome-wide single nucleotide polymorphisms (SNPs) genotyped in 379 Angus cattle. The extent of LD in the data set was such that the average marker-marker r2 was 0.2 at 200 kb. The accuracy of MAS increased as the number of markers in the haplotype surrounding the QTL increased, although only when the number of markers in the haplotype was 4 or greater did the accuracy exceed that achieved when the SNP in the highest LD with the QTL was used. A large number of phenotypic records (>1000) were required to accurately estimate the effects of the haplotypes.     

Modeling the evolution of a classic genetic switch

Background  

The regulatory network underlying the yeast galactose-use pathway has emerged as a model system for the study of regulatory network evolution. Evidence has recently been provided for adaptive evolution in this network following a whole genome duplication event. An ancestral gene encoding a bi-functional galactokinase and co-inducer protein molecule has become subfunctionalized as paralogous genes (GAL1 and GAL3) in Saccharomyces cerevisiae, with most fitness gains being attributable to changes in cis-regulatory elements. However, the quantitative functional implications of the evolutionary changes in this regulatory network remain unexplored.     

recA-dependent genetic switch generated by transposon Tn10

We describe a new type of regulatory switch generated in bacteriophage lambda by transposon Tn10. By this switch, phage genes alternate reversibly between expressed and non-expressed states as the direct consequence of a reversible DNA rearrangement. The switch itself has arisen via Tn10-promoted recombination. The subsequent “flip-flop” in gene expression occurs by general recombination between two IS10 elements serving as “portable regions of homology”.     

Prediction of sample size to maintain genetic variation in doubled-haploid populations following marker selection

Marker selection (MS) and doubled-haploid (DH) technologies have the potential to reduce the time taken to breed new cereal cultivars. However, a limiting factor is the potential increased genetic drift. The aim of this study was to design and test a genetic model for predicting the sample sizes needed to maintain genetic variation among DH plants following marker selection. The model estimates the amount of the genome that is fixed during the production of DH populations of a given size using a given number of markers. To test the model, doubled-haploids were produced from wheat plants selected for three PCR-based markers. When the genetic variation of the DH population (108 plants), produced from 15 selected F₂ plants homozygous at three loci, was compared to the genetic variation of an unselected F₃ population (200 plants), five of the six measured quantitative traits were identical and normally distributed. This model should prove to be a valid breeding tool, allowing a breeder to apply MS to a breeding programme and estimate the minimum DH population sizes required for minimal loss of genetic variation through genetic drift. Received: 16 October 2000 / Accepted: 20 March 2001     

Haptoglobin polymorphism--not only a genetic marker]

The biological activities of the haptoglobin polymorphism are controlled by continuous DNA sequences coding for the HP alpha and Hp beta polypeptide chains and forming with a linked Hp related gene the haptoglobin gene complex on chromosome 16. Probably, this DNA domain originates from the gene family of the serine proteases after having lost the informations for the proteolytic functions. Instead of this, the haptoglobins have acquired other qualities, among them the hemoglobin binding capacity, inserted into the Hp beta chain. The Hp polymorphism is constituted by the evolutionary progressive DNA sequences for the Hp alpha chains, which probably have activation functions. The haptoglobins display immunoregulative abilities, which can be immunosuppressive by inhibition of the lymphocyte reactivity or immunoinductive by influencing the IgM biosynthesis, adapted to the functional requirements. In this field, Hp 2-2 has a stronger effect than the two other Hp types. Moreover, the haptoglobins inhibit the prostaglandin synthesis and protect against harmful oxidation processes. These qualities are based on the hemoglobin binding ability and can be realized by Hp 1-1 with the comparatively highest efficacy. Further on, the haptoglobins are protease inhibitors. Finally, Hp 2-2 is associated with higher albumin and ceruloplasmin serum levels than Hp 2-1 and Hp 1-1. Evidently, the haptoglobins are inserted into a widely ramified network of biological functions. The selective advantages and disadvantages of the Hp polymorphism are noticeable under pathological conditions in case of malignant tumors, inflammations, autoimmune diseases, allergic illness, affective psychoses and affective lability favouring addiction.     

Systems for the removal of a selection marker and their combination with a positive marker

Many systems have been developed for the removal of a selection marker in order to generate marker-free transgenic plants. These systems consist of (1) a site-specific recombination system (Cre/lox) or a phage-attachment region (attP) to remove the selectable marker gene and (2) a transposable element system (Ac) or a co-transformation system to segregate the gene of interest from the selectable marker gene. Overall, the process is more time-consuming than conventional transformation methods because two rounds of transformation - two steps of regeneration or sexual crossings - are required to obtain the desired transgenic plants. Recently, removal systems combined with a positive marker, denoted as MAT vectors, have been developed to save time and effort by generating marker-free transgenic plants through a single-step transformation. We summarize here the transformation procedures using these systems and discuss their feasibility for practical use.     

Linking genetic mechanisms of heterozygosity-fitness correlations to footprints of selection at single loci

Investigations of heterozygosity-fitness correlations (HFCs) are central to the understanding how genetic diversity is maintained in natural populations. Advanced genome-wide approaches will enrich the number of functional loci to be tested. We argue that a combined analysis of the genetic mechanisms of HFCs and selection signals at single loci will allow researchers to better understand the micro-evolutionary basis of HFCs. Different dominance relationships among the alleles at the locus can lead to positive, negative or null HFCs depending on the allele frequency distribution. These scenarios differ in the temporal stability of the HFCs and in the patterns of allele frequency changes over time. Here, we describe a simple theoretical framework that links the analyses of heterozygosity-fitness associations (ecological timescale) with tests for selection signals (evolutionary timescale). Different genomic footprints of selection can be expected for the different underlying genetic mechanisms of HFCs, and this information can be independently used for the classification of HFCs. We suggest that in addition to inbreeding and single-locus overdominant effects also loci under directional selection could play a significant role in the development of heterozygosity-fitness effects in large natural populations under recent or fluctuating ecological changes.     

A model for marker-assisted selection among single crosses with multiple genetic markers

 Trait means of marker genotypes are often inconsistent across experiments, thereby hindering the use of regression techniques in marker-assisted selection. Best linear unbiased prediction based on trait and marker data (TM-BLUP) does not require prior information on the mean effects associated with specific marker genotypes and, consequently, may be useful in applied breeding programs. The objective of this paper is to present a flanking-marker, TM-BLUP model that is applicable to interpopulation single crosses that characterize maize (Zea mays L.) breeding programs. The performance of a single cross is modeled as the sum of testcross additive and dominance effects at unmarked quantitative trait loci (QTL) and at marked QTL (MQTL). The TM-BLUP model requires information on the recombination frequencies between flanking markers and the MQTL and on MQTL variances. A tabular method is presented for calculating the conditional probability that MQTL alleles in two inbreds are identical by descent given the observed marker genotypes (G ^k _obs) at the kth MQTL. Information on identity by descent of MQTL alleles can then be used to calculate the conditional covariance of MQTL effects between single crosses given G ^k _obs. The inverse of the covariance matrix for dominance effects at unmarked QTL and MQTL can be written directly from the inverse of the covariance matrices of the corresponding testcross additive effects. In practice, the computations required in TM-BLUP may be prohibitive. The computational requirements may be reduced with simplified TM-BLUP models wherein dominance effects at MQTL are excluded, only the single crosses that have been tested are included, or information is pooled across several MQTL. Received: 22 June 1997 / Accepted: 25 February 1998     

Quantification of a genetic message in selection

The genetic communication system includes the following components: the parent, which represents the information source and which emits messages; the gametes, which are the messenger carriers; and the offspring, which results from the decoding of two of these messages and can, in turn, become an information source.In a diploid species, a pair of heterozygous homologous loci may emit two equally probable messages, the quantity of genetic information (Q) produced being equivalent to: Q=log₂ 2=1 bit. For n independent pairs of heterozygous homologous loci, Q=n.log₂ 2=n bits. The evolution of Q is examined whenever the parent is used in inbreeding or crossbreeding. In the case of inbreeding, the initial Q is depleted as the loci become homozygous; for hybridization the evolution of Q is unpredictable.In the case of pairs of linked heterozygous homologous loci, Q is represented by an equation similar to that used to describe entropy. The value of entropy is lower when linkage between loci is tighter, the freedom of choice in selection is reduced.