Ds-Induced Alleles at the OPAQUE-2 Locus of Maize

Transposon mutagenesis has been used to isolate mutable alleles at the Opaque-2 (O2) locus of maize. Plants with the Activator-Dissociation (Ac-Ds) system of transposable elements and O2 were crossed as males to a stable o2 tester line. Among a population of 200,000 kernels, 198 exceptional kernels with somatic instability were recovered. In four cases, designated O2-m1, o2-m2, O2-m3 and O2-m4, variegated phenotypes appeared in F₂ and subsequent generations. Genetic analyses indicated that the presence of Ds near or within the O2 gene was responsible for the observed somatic instability at the O2 locus. The phenotypes of the newly induced alleles were of two types. Alleles O2-m1, O2-m3 and O2-m4, in the absence of Ac, were characterized by kernel phenotypes indistinguishable from the wild type; in the presence of Ac they generated kernels with opaque sectors interspersed within a vitreous background. In contrast, the mutable allele o2-m2, in the absence of Ac, was characterized by kernels with a recessive phenotype similar to o2 recessive mutants. In the presence of Ac, it reverted somatically to wild-type-producing kernels with vitreous spots in an o2 background. The association of the Ds element with the O2 locus may prove a valuable tool directed to the isolation of DNA fragments bearing the O2 gene.     

Mu Transposon Insertion Sites and Meiotic Recombination Events Co-Localize with Epigenetic Marks for Open Chromatin across the Maize Genome

The Mu transposon system of maize is highly active, with each of the ∼50–100 copies transposing on average once each generation. The approximately one dozen distinct Mu transposons contain highly similar ∼215 bp terminal inverted repeats (TIRs) and generate 9-bp target site duplications (TSDs) upon insertion. Using a novel genome walking strategy that uses these conserved TIRs as primer binding sites, Mu insertion sites were amplified from Mu stocks and sequenced via 454 technology. 94% of ∼965,000 reads carried Mu TIRs, demonstrating the specificity of this strategy. Among these TIRs, 21 novel Mu TIRs were discovered, revealing additional complexity of the Mu transposon system. The distribution of >40,000 non-redundant Mu insertion sites was strikingly non-uniform, such that rates increased in proportion to distance from the centromere. An identified putative Mu transposase binding consensus site does not explain this non-uniformity. An integrated genetic map containing more than 10,000 genetic markers was constructed and aligned to the sequence of the maize reference genome. Recombination rates (cM/Mb) are also strikingly non-uniform, with rates increasing in proportion to distance from the centromere. Mu insertion site frequencies are strongly correlated with recombination rates. Gene density does not fully explain the chromosomal distribution of Mu insertion and recombination sites, because pronounced preferences for the distal portion of chromosome are still observed even after accounting for gene density. The similarity of the distributions of Mu insertions and meiotic recombination sites suggests that common features, such as chromatin structure, are involved in site selection for both Mu insertion and meiotic recombination. The finding that Mu insertions and meiotic recombination sites both concentrate in genomic regions marked with epigenetic marks of open chromatin provides support for the hypothesis that open chromatin enhances rates of both Mu insertion and meiotic recombination.     

Inositol Mutants of SACCHAROMYCES CEREVISIAE: Mapping the ino1 Locus and Characterizing Alleles of the ino1, ino2 and ino4 Loci

An extensive genetic analysis of inositol auxotrophic mutants of yeast is reported. The analysis includes newly isolated mutants, as well as those previously reported (Culbertson and Henry 1975). Approximately 70% of all inositol auxotrophs isolated are shown to be alleles of the ino1 locus, the structural gene for inositol-1-phosphate synthase, the major enzyme involved in inositol biosynthesis. Alleles of two other loci, ino2 and ino4, comprise 9% of total mutants, with the remainder representing unique loci or complementation groups. The ino1 locus was mapped by trisomic analysis with an n + 1 disomic strain constructed with complementing alleles at this locus. The ino1 locus is shown to be located between ura2 (11.1 cm) and cdc6 (21.8 cm) on chromosome X. An extended map of chromosome X of yeast is presented. Unlike most yeast loci, but similar to the his1 locus, the ino1 locus lacks allelic representatives that are suppressible by known suppressors. This finding suggests that premature termination of translation of the ino1 gene product may be incompatible with cell viability.     

Transposon Mutagenesis of Azospirillum brasilense and Azospirillum lipoferum: Physical Analysis of Tn5 and Tn5-Mob Insertion Mutants

Tn5-induced insertion mutants were generated in Azospirillum brasilense Sp7 and A. lipoferum SpBr17 by mating with Escherichia coli strains carrying suicide plasmid vectors. The sources of Tn5 were the suicide plasmids pGS9 and pSUP2021. Kanamycin-resistant Azospirillum colonies appeared from crosses with E. coli at maximum frequencies of 10⁻⁷ per recipient cell. Transposon Tn5 also conferred streptomycin resistance on Azospirillum colonies as was observed earlier for Rhizobium sp. Eight Tn5-induced Km^r Sm^rA. brasilense Sp7 mutants with reduced nitrogen-fixing capacity were isolated. The potential use of Tn5-Mob for labeling and mobilization of Azospirillum-indigenous plasmids was demonstrated by isolating Tn5-Mob insertions in the megaplasmids of A. brasilense Sp7.     

Germinal Excisions of the Maize Transposon Activator Do Not Stimulate Meiotic Recombination or Homology-Dependent Repair at the Bz Locus

Double-strand breaks have been implicated both in the initiation of meiotic recombination in yeast and as intermediates in the transposition process of nonreplicative transposons. Some transposons of this class, notably P of Drosophila and Tc1 of Caenorhabditis elegans, promote a form of homology-dependent premeiotic gene conversion upon excision. In this work, we have looked for evidence of an interaction between Ac transposition and meiotic recombination at the bz locus in maize. We find that the frequency of meiotic recombination between homologues is not enhanced by the presence of Ac in one of the bz heteroalleles and, conversely, that the presence of a homologous sequence in either trans (homologous chromosome) or cis (tandem duplication) does not promote conversion of the Ac insertion site. However, a tandem duplication of the bz locus may be destabilized by the insertion of Ac. We discuss possible reasons for the lack of interaction between Ac excision and homologous meiotic recombination in maize.     

ESSENTIALS: Software for Rapid Analysis of High Throughput Transposon Insertion Sequencing Data

High-throughput analysis of genome-wide random transposon mutant libraries is a powerful tool for (conditional) essential gene discovery. Recently, several next-generation sequencing approaches, e.g. Tn-seq/INseq, HITS and TraDIS, have been developed that accurately map the site of transposon insertions by mutant-specific amplification and sequence readout of DNA flanking the transposon insertions site, assigning a measure of essentiality based on the number of reads per insertion site flanking sequence or per gene. However, analysis of these large and complex datasets is hampered by the lack of an easy to use and automated tool for transposon insertion sequencing data. To fill this gap, we developed ESSENTIALS, an open source, web-based software tool for researchers in the genomics field utilizing transposon insertion sequencing analysis. It accurately predicts (conditionally) essential genes and offers the flexibility of using different sample normalization methods, genomic location bias correction, data preprocessing steps, appropriate statistical tests and various visualizations to examine the results, while requiring only a minimum of input and hands-on work from the researcher. We successfully applied ESSENTIALS to in-house and published Tn-seq, TraDIS and HITS datasets and we show that the various pre- and post-processing steps on the sequence reads and count data with ESSENTIALS considerably improve the sensitivity and specificity of predicted gene essentiality.     

Maize Mu transposons are targeted to the 5' untranslated region of the gl8 gene and sequences flanking Mu target-site duplications exhibit nonrandom nucleotide composition throughout the genome

The widespread use of the maize Mutator (Mu) system to generate mutants exploits the preference of Mu transposons to insert into genic regions. However, little is known about the specificity of Mu insertions within genes. Analysis of 79 independently isolated Mu-induced alleles at the gl8 locus established that at least 75 contain Mu insertions. Analysis of the terminal inverted repeats (TIRs) of the inserted transposons defined three new Mu transposons: Mu10, Mu 11, and Mu12. A large percentage (>80%) of the insertions are located in the 5' untranslated region (UTR) of the gl8 gene. Ten positions within the 5' UTR experienced multiple independent Mu insertions. Analyses of the nucleotide composition of the 9-bp TSD and the sequences directly flanking the TSD reveals that the nucleotide composition of Mu insertion sites differs dramatically from that of random DNA. In particular, the frequencies at which C's and G's are observed at positions -2 and +2 (relative to the TSD) are substantially higher than expected. Insertion sites of 315 RescueMu insertions displayed the same nonrandom nucleotide composition observed for the gl8-Mu alleles. Hence, this study provides strong evidence for the involvement of sequences flanking the TSD in Mu insertion-site selection.     

Cloning of the y1 Locus of Maize,a Gene Involved in the Biosynthesis of Carotenoids

The y1 gene is one of the genes responsible for the production of [beta]-carotene in the endosperm and leaves of maize. We have cloned a Robertson's Mutator-tagged allele of the y1 gene (y1-mum) by using a Mu3 element as a hybridization probe. We substantiate that the cloned sequence is a portion of the y1 gene by molecular analyses of a revertant of a putative Mutator-induced y1 allele and the incidence of insertions within the cloned y1 sequence from several independently derived Mutator-induced y1 mutant stocks. The y1-mum sequence was used to isolate the standard Y1 allele, which conditions the presence of [beta]-carotene in the endosperm of the maize kernel.     

The Mutation Bronze-Mutable 4 Derivative 6856 in Maize Is Caused by the Insertion of a Novel 6.7-Kilobase Pair Transposon in the Untranslated Leader Region of the Bronze-1 Gene

The Ds-controlled allele, bz-m4 Derivative 6856 [bz-m4 D6856], is reported to have an altered temporal- and tissue-specific pattern of gene expression. We have cloned this allele and have characterized it at the molecular level. The mutation was caused by the insertion of a complex transposon-like structure 36 base pairs downstream from the Bz mRNA cap site. The insert is 6.7-kbp long. Ds elements, each approximately 2 kbp in length, are at both ends of the insert. The sequence between the Ds elements is a partial duplication of flanking sequences from the 3' end of the Bz gene. These data suggest that Ds initially inserted near the 3' end of the gene and mobilized adjacent sequences as it transposed.     

Control of the Frequency of Reversion of the Mutable Alleles at the opaque2 Locus by the Bg-rbg System of Transposable Elements in Maize

Maize lines differing in the frequency of reversion of the opaque2 (o2) mutable alleles controlled by the Bg-rbg system of transposable elements were studied. In the presence of the Bg regulatory element, these alleles can revert to normal. When reversion occurs prior to the first division of the primary endosperm nucleus, phenotypically normal kernels or whole endosperm revertants (WER) develop. It was shown that the low frequency of formation of whole endosperm revertant may be produced by different genetic mechanisms. The frequency of WER formation was shown to nonlinearly depend on the dose of the Bg-hf regulatory element. A dose increase from two to three failed to cause an essential increase in the number of revertants. The regulatory elements Bg-lf andBg-hfdiffered in their ability to induce excision of the receptor element at the same dose. The frequency of reversion of the receptive alleles was shown to be regulated by epigenetic mechanisms so that high frequency of reversion of receptive alleles requires preliminary premeiotic association between the regulatory and receptor elements. The inheritance of the maize alleles o2-hfand o2-lf proved to be similar to that of an3 mutable alleles in petunia.     

Complex Study of Drosophila Mutants in the agnostic Locus: A Model for Coupling Chromosomal Architecture and Cognitive Functions

After the devastation of genetics in our country, Academician Leon A. Orbeli has provided an opportunity for the studies on evolutionary conservatism of genes controlling the main properties of the higher nervous activity and conditioning. For the last few years, determination and bioinformatic analysis of genome sequences in the plant, worm, Drosophila, and human genome have revealed, indeed, a high interspecies homology of genes. Studies on Drosophila mutants have shown that components of intracellular signalization systems regulating neuronal functions and gene expression are organized in supramolecular complexes. It has become evident that the chromosomal architecture predetermines the appearance of deletions, duplications, insertions, and translocations and, therefore, plays an important role not only in evolution but also in generating human pathological syndromes with multiple manifestations, including cognitive dysfunctions. There appeared a new approach, comparative genomics, that allows revealing functions of human disease genes on the basis of their sequence homology to the known Drosophila gene with various well-studied mutant phenotypes. For this reason, the Drosophila genes should be saturated with mutant phenotypes, and these are to be studied in comparison with the chromosomal architecture. Our complex behavioral and molecular-genetic study of spontaneous, induced, and P-insertional mutations in the Drosophila agnostic locus and the bioinformatic analyses of genomic sequences has allowed us to assign the locus to the Drosophila genomic scaffold AE003489 from the 11AB X-chromosomal region that contains the CG1848 gene coding for LIM-kinase 1. Mutations, insertions, and deletions in the agnostic locus lead to an increased activity of Ca²⁺/calmodulin-dependent PDE1, resistance to ether, an inactivator of synaptic transmission, impairments of the brain structures, learning and memory defects in conditioned courtship suppression paradigm, alterations in sound production and in structural-functional chromosomal organization. Therefore, the agnostic locus represents a model to study the human Williams syndrome with multiple dysfunctions due to a contiguous deletion in the 7q11.23 spanning 17 genes, among them the gene for LIM-kinase 1 presumed to be responsible for cognitive defects. The Williams syndrome is considered to be a most efficient model to study human cognition, human genome organization, and evolution.     

Identification of a Maize Locus That Modulates the Hypersensitive Defense Response,Using Mutant-Assisted Gene Identification and Characterization

Potentially useful naturally occurring genetic variation is often difficult to identify as the effects of individual genes are subtle and difficult to observe. In this study, a novel genetic technique called Mutant-Assisted Gene Identification and Characterization is used to identify naturally occurring loci modulating the hypersensitive defense response (HR) in maize. Mutant-Assisted Gene Identification and Characterization facilitates the identification of naturally occurring alleles underlying phenotypic variation from diverse germplasm, using a mutant phenotype as a “reporter.” In this study the reporter phenotype was caused by a partially dominant autoactive disease resistance gene, Rp1-D21, which caused HR lesions to form spontaneously all over the plant. Here it is demonstrated that the Rp1-D21 phenotype is profoundly affected by genetic background. By crossing the Rp1-D21 gene into the IBM mapping population, it was possible to map and identify Hrml1 on chromosome 10, a locus responsible for modulating the HR phenotype conferred by Rp1-D21. Other loci with smaller effects were identified on chromosomes 1 and 9. These results demonstrate that Mutant-Assisted Gene Identification and Characterization is a viable approach for identifying naturally occurring useful genetic variation.POTENTIALLY useful naturally occurring genetic variation is often difficult to identify as the effects of individual genes are subtle and difficult to observe. Furthermore, so many different alleles are available that it is a major challenge just to sift through the enormous diversity available. To this end, we recently conceptualized a simple yet effective method to discover and characterize variation present naturally in plant germplasm (Johal et al. 2008). This method, Mutant-Assisted Gene Identification and Characterization, makes use of a mutant phenotype for a gene affecting the trait of interest as a reporter to discover and analyze relevant, interacting genes present naturally in diverse germplasm. Mutant-Assisted Gene Identification and Characterization involves crossing a mutant to diverse germplasm and then evaluating the mutant progeny for transgressive changes (both suppressed and severe) in the mutant phenotype(s). If the mutation is recessive, the population needs to be advanced to the F₂ generation to be able to detect and analyze such variation. However, for a dominant or partially dominant mutant, evaluations can be made immediately in the F₁ to discover lines that contain suppressors or enhancers of the trait (mutation) under study. Mutant F₁ progenies from such crosses can then be propagated further to identify, map, and clone genes/QTL that affect the trait positively or negatively. In the case of maize and other species for which genetically characterized mapping populations are available, modifying loci can be rapidly mapped by crossing a mutant line to each member of a mapping population and evaluating the resulting F₁ families. In this study we provide a proof-of-concept for the Mutant-Assisted Gene Identification and Characterization technique, using it to identify loci involved in the defense response of maize.Plants are constantly exposed to numerous potential pathogens with diverse modes of attack. Nevertheless, it is rather rare to see plants succumbing to disease. One key reason for this is the presence of a highly effective and inducible defense system, a major component of which is the hypersensitive response (HR). HR is usually associated with a specific recognition event and is activated after other nonspecific resistance mechanisms have been overcome or evaded (see Bent and Mackey 2007). Although it was initially coined to refer to the rapid collapse of cells at the site of infection, over the years the term HR has been used to refer to both cell death and the associated induction of a number of other defense responses, including the accumulation of phytoalexins and pathogenesis-related (PR) proteins at the site of infection, to name a few (Mur et al. 2007). Reactive oxygen species such as superoxide and H₂O₂ appear to be causally involved in cell death underlying the HR response (Jones and Dangl 2006).HR is under the control of a subset of disease-resistance genes, commonly referred to as R genes. These R genes specifically recognize matching avirulence (Avr) effectors from the pathogen. Many R genes encode products containing a nucleotide-binding site (NBS) domain in the middle of the protein and a leucine-rich repeat (LRR) domain at the C-terminal end (Bent and Mackey 2007). R proteins are involved both in the recognition of the pathogen and the subsequent induction of the HR response. How R proteins remain in a quiescent but “vigilant” state remains to be established. Certain mutations in R genes have been found that abolish their dependence on AVR proteins for activation. Such aberrant R genes mostly behave as dominant or partially dominant alleles and trigger the HR constitutively in the absence of the pathogen (Hu et al. 1996; Zhang et al. 2003; Dodds et al. 2006). Two consequences of such “autoactive” or “ectopically active” R genes are a massive induction of cell death and the consequential stunting of the organism (Dodds et al. 2006). Although autoactive R genes have been found to exist in many plant species, the first few examples came from the maize Rp1 locus, which confers race-specific resistance to common rust, caused by Puccinia sorghi (Hu et al. 1996). Such autoactive R genes can be used to investigate HR genetics and etiology in the absence of confounding effects from the pathogen and constitute an excellent candidate for analysis using Mutant-Assisted Gene Identification and Characterization.The details of the HR cell death reaction as well as the pathway(s) that link R gene activation with the HR remain unclear (Mur et al. 2007). Despite considerable research over the past decade, only a few components have been found thus far. Some of these, Ndr1, Eds1, Pad4, Rar1, and Sgt1, were identified in mutagenesis screens conducted to identify mutants that failed to undergo an HR reaction in response to infection by an avirulent pathogen (reviewed in Bent and Mackey 2007). A few others, RIN4, for example, were identified in yeast two-hybrid assays using an NBS–LRR protein as bait (Mackey et al. 2003). Recently, an Arabidopsis gain-of-function mutant that carries a point mutation in an R gene analog (a gene with the structure of an R gene but not known to be involved in resistance to any pathogen) was used to isolate a few more potential genes in the HR pathway in a second site suppressor approach following mutagenesis with ethane methyl sulfonate (EMS) (Palma et al. 2005; Zhang and Li 2005; Goritschnig et al. 2007). A problem with approaches based on intentional mutagenesis is that they fail to uncover genes that have either redundant or essential functions. One way to avoid this problem would be to seek naturally occurring allelic variants affecting HR. Such natural variation is pervasive in all species, being generated and selected for over millions of years of evolution.Although natural variation has served as a constant provider of the R genes in all plant species, natural variability has not been tapped as a tool for understanding other aspects of the disease-resistance response (Holub 2007). The Rp1-D21 gene is an autoactive allele from the maize Rp1 disease-resistance locus that initiates HR randomly all over the plant (Pryor 1993; Collins et al. 1999; Sun et al. 2001). Our objective for this study was to use the Rp1-D21 gene phenotype as a test case for the Mutant-Assisted Gene Identification and Characterization approach. We show here that enormous variation exists in the maize germplasm that is capable of affecting the HR response positively or negatively and we identify loci that modulate expression of the HR phenotype segregating in the well-known Intermated B73 × Mo17 (IBM) advanced intercross line (AIL) population (Coe et al. 2002; Lee et al. 2002). This constitutes the first demonstration of the utility of the Mutant-Assisted Gene Identification and Characterization approach—an approach that is likely to prove widely applicable.     

P-Element Insertion Alleles of Essential Genes on the Third Chromosome of Drosophila Melanogaster: Mutations Affecting Embryonic Pns Development

To identify novel genes and to isolate tagged mutations in known genes that are required for the development of the peripheral nervous system (PNS), we have screened a novel collection of 2460 strains carrying lethal or semilethal P-element insertions on the third chromosome. Monoclonal antibody 22C10 was used as a marker to visualize the embryonic PNS. We identified 109 mutant strains that exhibited reproducible phenotypes in the PNS. Cytological and genetic analyses of these strains indicated that 87 mutations affect previously identified genes: tramtrack (n = 18 alleles), string (n = 15), cyclin A (n = 13), single-minded (n = 13), Delta (n = 9), neuralized (n = 4), pointed (n = 4), extra macrochaetae (n = 4), prospero (n = 3), tartan (n = 2), and pebble (n = 2). In addition, 13 mutations affect genes that we identified recently in a chemical mutagenesis screen designed to isolate similar mutants: hearty (n = 3), dorsotonals (n = 2), pavarotti (n = 2), sanpodo (n = 2), dalmatian (n = 1), missensed (n = 1), senseless (n = 1), and sticky ch1 (n = 1). The remaining nine mutations define seven novel complementation groups. The data presented here demonstrate that this collection of P elements will be useful for the identification and cloning of novel genes on the third chromosome, since >70% of mutations identified in the screen are caused by the insertion of a P element. A comparison between this screen and a chemical mutagenesis screen undertaken earlier highlights the complementarity of the two types of genetic screens.