A collection of 11 800 single-copy Ds transposon insertion lines in Arabidopsis

More than 10 000 transposon-tagged lines were constructed by using the Activator (Ac)/Dissociation (Ds) system in order to collect insertional mutants as a useful resource for functional genomics of Arabidopsis. The flanking sequences of the Ds element in the 11 800 independent lines were determined by high-throughput analysis using a semi-automated method. The sequence data allowed us to map the unique insertion site on the Arabidopsis genome in each line. The Ds element of 7566 lines is inserted in or close to coding regions, potentially affecting the function of 5031 of 25 000 Arabidopsis genes. Half of the lines have Ds insertions on chromosome 1 (Chr. 1), in which donor lines have a donor site. In the other half, the Ds insertions are distributed throughout the other four chromosomes. The intrachromosomal distribution of Ds insertions varies with the donor lines. We found that there are hot spots for Ds transposition near the ends of every chromosome, and we found some statistical preference for Ds insertion targets at the nucleotide level. On the basis of systematic analysis of the Ds insertion sites in the 11 800 lines, we propose the use of Ds-tagged lines with a single insertion in annotated genes for systematic analysis of phenotypes (phenome analysis) in functional genomics. We have opened a searchable database of the insertion-site sequences and mutated genes (http://rarge.gsc.riken.go.jp/) and are depositing these lines in the RIKEN BioResource Center as available resources (http://www.brc.riken.go.jp/Eng/).     

A collection of sequenced and mapped Ds transposon insertion sites in Arabidopsis thaliana

Insertional mutagenesis is a powerful tool for generating knockout mutations that facilitate associating biological functions with as yet uncharacterized open reading frames (ORFs) identified by genomic sequencing or represented in EST databases. We have generated a collection of Dissociation (Ds) transposon lines with insertions on all 5 Arabidopsis chromosomes. Here we report the insertion sites in 260 independent single-transposon lines, derived from four different Ds donor sites. We amplified and determined the genomic sequence flanking each transposon, then mapped its insertion site by identity of the flanking sequences to the corresponding sequence in the Arabidopsis genome database. This constitutes the largest collection of sequence-mapped Ds insertion sites unbiased by selection against the donor site. Insertion site clusters have been identified around three of the four donor sites on chromosomes 1 and 5, as well as near the nucleolus organizers on chromosomes 2 and 4. The distribution of insertions between ORFs and intergenic sequences is roughly proportional to the ratio of genic to intergenic sequence. Within ORFs, insertions cluster near the translational start codon, although we have not detected insertion site selectivity at the nucleotide sequence level. A searchable database of insertion site sequences for the 260 transposon insertion sites is available at http://sgio2.biotec.psu.edu/sr. This and other collections of Arabidopsis lines with sequence-identified transposon insertion sites are a valuable genetic resource for functional genomics studies because the transposon location is precisely known, the transposon can be remobilized to generate revertants, and the Ds insertion can be used to initiate further local mutagenesis.     

A collection of sequenced and mapped Ds transposon insertion sites in Arabidopsis thaliana

Insertional mutagenesis is a powerful tool for generating knockout mutations that facilitate associating biological functions with as yet uncharacterized open reading frames (ORFs) identified by genomic sequencing or represented in EST databases. We have generated a collection of Dissociation(Ds) transposon lines with insertions on all 5 Arabidopsischromosomes. Here we report the insertion sites in 260 independent single-transposon lines, derived from four different Ds donor sites. We amplified and determined the genomic sequence flanking each transposon, then mapped its insertion site by identity of the flanking sequences to the corresponding sequence in the Arabidopsisgenome database. This constitutes the largest collection of sequence-mapped Ds insertion sites unbiased by selection against the donor site. Insertion site clusters have been identified around three of the four donor sites on chromosomes 1 and 5, as well as near the nucleolus organizers on chromosomes 2 and 4. The distribution of insertions between ORFs and intergenic sequences is roughly proportional to the ratio of genic to intergenic sequence. Within ORFs, insertions cluster near the translational start codon, although we have not detected insertion site selectivity at the nucleotide sequence level. A searchable database of insertion site sequences for the 260 transposon insertion sites is available at http://sgio2.biotec.psu.edu/sr. This and other collections of Arabidopsislines with sequence-identified transposon insertion sites are a valuable genetic resource for functional genomics studies because the transposon location is precisely known, the transposon can be remobilized to generate revertants, and the Ds insertion can be used to initiate further local mutagenesis.     

A dominant mutation in the COL1A1 gene that substitutes glycine for valine causes recurrent lethal osteogenesis imperfecta

Summary Type I collagen chains of a proband from a family with recurrent lethal osteogenesis imperfecta (OI) migrated as a doublet when submitted to gel electrophoresis. Cyanogen bromide (CNBr) peptide mapping demonstrated that the post-translational over-modifications were initiated in 1ICB7. Chemical cleavage of cDNA-RNA heteroduplexes identified a mismatch in the 1I cDNA; this mismatch was subsequently confirmed by sequencing a 249-bp fragment amplified by the polymerase chain reaction. A G to T transition in the second base of the first codon of exon 41 resulted in the substitution of glycine 802 by valine. This mutation impaired collagen secretion by dermal fibroblasts. The over-modified chains were retained intracellularly and melted at a lower temperature than normal chains. Collagen molecules synthesized by parental fibroblasts had a normal electrophoretic mobility, but hybridization of genomic DNA with allele-specific oligonucleotides revealed the presence of the mutant allele in the mother's leukocytes. The mutation was not detected in her fibroblasts consistent with the protein data. These results support the hypothesis that somatic and germ-line mosaicism in the phenotypically normal mother explain the recurrence of OI.     

Identification of the gene whose mutation leads to the appearance of recessive lethal germlings in Arabidopsis thaliana

The data are presented on genetic and molecular-genetic analysis of a mutant from the collection of morphological insertion mutants of Arabidopsis thaliana we obtained earlier, which belongs to the phenotypic class of recessive lethal germlings. A nucleotide DNA sequence, 147 bp in size, was identified, which adheres to the left border area of T-DNA insertion. The site of localization of the insertion was determined using computer analysis.     

A large MSH2 Alu insertion mutation causes HNPCC in a German kindred

Hereditary non-polyposis colorectal cancer (HNPCC) syndrome is an autosomal, dominantly inherited disease accounting for about 1%–5% of all colorectal cancer cases. HNPCC predisposition is caused by germline mutations in at least five genes coding for DNA mismatch repair (MMR) proteins. More than 400 MMR gene mutations have been identified in HNPCC patients. About 90% of mutations affect the MLH1 and MSH2 genes. The mutational spectrum mainly includes point mutations and small deletions or insertions. Here, we report a large 184 base-pair Alu insertion mutation in exon 6 of the MSH2 gene in a German HNPCC family. The inserted sequence contains repetitive Alu sequence elements that present the highest homology with the old Alu J subfamily. The Alu J insertion was most likely derived from Alu-mediated recombination, since Alu J elements have been found close to the insertion site in adjacent introns, and since elements pivotal for Alu retrotransposition are missing. Our results suggest that the recombination event occurred at least one generation ago. This is the first report of an Alu insertion in the coding sequence of a MMR gene as the cause of HNPCC. Our data thus further extend the spectrum of MMR gene mutations causative for HNPCC.M. Kloor and C. Sutter contributed equally to this work     

A benchmark and an algorithm for detecting germline transposon insertions and measuring de novo transposon insertion frequencies

Transposons are genomic parasites, and their new insertions can cause instability and spur the evolution of their host genomes. Rapid accumulation of short-read whole-genome sequencing data provides a great opportunity for studying new transposon insertions and their impacts on the host genome. Although many algorithms are available for detecting transposon insertions, the task remains challenging and existing tools are not designed for identifying de novo insertions. Here, we present a new benchmark fly dataset based on PacBio long-read sequencing and a new method TEMP2 for detecting germline insertions and measuring de novo ‘singleton’ insertion frequencies in eukaryotic genomes. TEMP2 achieves high sensitivity and precision for detecting germline insertions when compared with existing tools using both simulated data in fly and experimental data in fly and human. Furthermore, TEMP2 can accurately assess the frequencies of de novo transposon insertions even with high levels of chimeric reads in simulated datasets; such chimeric reads often occur during the construction of short-read sequencing libraries. By applying TEMP2 to published data on hybrid dysgenic flies inflicted by de-repressed P-elements, we confirmed the continuous new insertions of P-elements in dysgenic offspring before they regain piRNAs for P-element repression. TEMP2 is freely available at Github: https://github.com/weng-lab/TEMP2.     

A novel Arabidopsis gene causes Bax-like lethality in Saccharomyces cerevisiae

Overexpression of the mammalian proapoptotic protein Bax induces cell death in plant and yeast cells. The Bax inihibitor-1 (BI-1) gene rescues yeast and plant from Bax-mediated lethality. Using the Arabidopsis BI-1 (AtBI-1) gene controlled by the GAL1 promoter as a cell death suppressor in yeast, Cdf1 (cell growth defect factor-1) was isolated from Arabidopsis cDNA library. Overexpression of Cdf1 caused cell death in yeast, whereas such an effect was suppressed by co-expression of AtBI-1. The Cdf1 protein fused with a green fluorescent protein was localized in the mitochondria and resulted in the loss of mitochondrial membrane potential in yeast. The Bax-resistant mutant BRM1 demonstrated tolerance against Cdf1-mediated lethality, whereas the Deltaatp4 strain was sensitive to Cdf1. Our results suggest that Cdf1 and Bax cause mitochondria-mediated yeast lethality through partially overlapped pathways.     

Analysis of transposon insertion mutants highlights the diversity of mechanisms underlying male progamic development in Arabidopsis

To identify genes with essential roles in male gametophytic development, including postpollination (progamic) events, we have undertaken a genetic screen based on segregation ratio distortion of a transposon-borne kanamycin-resistance marker. In a population of 3359 Arabidopsis Ds transposon insertion lines, we identified 20 mutants with stably reduced segregation ratios arising from reduced gametophytic transmission. All 20 mutants showed strict cosegregation of Ds and the reduced gametophytic transmission phenotype. Among these, 10 mutants affected both male and female transmission and 10 mutants showed male-specific transmission defects. Four male and female (ungud) mutants and 1 male-specific mutant showed cellular defects in microspores and/or in developing pollen. The 6 remaining ungud mutants and 9 male-specific (seth) mutants affected pollen functions during progamic development. In vitro and in vivo analyses are reported for 5 seth mutants. seth6 completely blocked pollen germination, while seth7 strongly reduced pollen germination efficiency and tube growth. In contrast, seth8, seth9, or seth10 pollen showed reduced competitive ability that was linked to slower rates of pollen tube growth. Gene sequences disrupted in seth insertions suggest essential functions for putative SETH proteins in diverse processes including protein anchoring, cell wall biosynthesis, signaling, and metabolism.     

Cis-by-Trans regulatory divergence causes the asymmetric lethal effects of an ancestral hybrid incompatibility gene

The Dobzhansky and Muller (D-M) model explains the evolution of hybrid incompatibility (HI) through the interaction between lineage-specific derived alleles at two or more loci. In agreement with the expectation that HI results from functional divergence, many protein-coding genes that contribute to incompatibilities between species show signatures of adaptive evolution, including Lhr, which encodes a heterochromatin protein whose amino acid sequence has diverged extensively between Drosophila melanogaster and D. simulans by natural selection. The lethality of D. melanogaster/D. simulans F1 hybrid sons is rescued by removing D. simulans Lhr, but not D. melanogaster Lhr, suggesting that the lethal effect results from adaptive evolution in the D. simulans lineage. It has been proposed that adaptive protein divergence in Lhr reflects antagonistic coevolution with species-specific heterochromatin sequences and that defects in LHR protein localization cause hybrid lethality. Here we present surprising results that are inconsistent with this coding-sequence-based model. Using Lhr transgenes expressed under native conditions, we find no evidence that LHR localization differs between D. melanogaster and D. simulans, nor do we find evidence that it mislocalizes in their interspecific hybrids. Rather, we demonstrate that Lhr orthologs are differentially expressed in the hybrid background, with the levels of D. simulans Lhr double that of D. melanogaster Lhr. We further show that this asymmetric expression is caused by cis-by-trans regulatory divergence of Lhr. Therefore, the non-equivalent hybrid lethal effects of Lhr orthologs can be explained by asymmetric expression of a molecular function that is shared by both orthologs and thus was presumably inherited from the ancestral allele of Lhr. We present a model whereby hybrid lethality occurs by the interaction between evolutionarily ancestral and derived alleles.     

A two-element Enhancer-Inhibitor transposon system in Arabidopsis thaliana

The Enhancer-Inhibitor (En-I), also known as Suppressor-mutator (Spm-dSpm), transposable element system of maize was modified and introduced into Arabidopsis by Agrobacterium tumefaciens transformation. A stable En/Spm transposase source under control of the CaMV 35S promoter mediated frequent transposition of I/dSpm elements. Transposition occurred continuously throughout plant development over at least seven consecutive plant generations after transformation. New insertions were found at both linked and unlinked positions relative to a transposon donor site. The independent transposition frequency was defined as a transposition parameter, which quantified the rate of unique insertion events and ranged from 7.8% to 29.2% in different populations. An increase as well as a decrease in I/dSpm element copy number was seen at the individual plant level, but not at the population level after several plant generations. The continuous, frequent transposition observed for this transposon system makes it an attractive tool for use in gene tagging in Arabidopsis.     

Phenotypic manifestations of yeast transposon insertion in the LYS2 gene and deletions resulting from imprecise excision of the transposon

The lys2-32 mutant allele resulted from Ty1 element insertion was identified and cloned. The expression and reversions of lys2-32 localized in an autonomous plasmid were studied. The insertion was shown to inactivate LYS2 gene incompletely. Spontaneous reversions to complete or almost complete prototrophy were also obtained. About 50% of revertants retained the insertion. Others arise as a result of imprecise excision events leading to deletions of adjacent LYS2 sequences.     

A semidominant mutation in an Arabidopsis mitogen-activated protein kinase phosphatase-like gene compromises cortical microtubule organization

Reversible protein phosphorylation regulates many cellular processes, including the dynamics and organization of the microtubule cytoskeleton, but the events mediating it are poorly understood. A semidominant phs1-1 allele of the Arabidopsis thaliana PROPYZAMIDE-HYPERSENSITIVE 1 locus exhibits phenotypes indicative of compromised cortical microtubule functions, such as left-handed helical growth of seedling roots, defective anisotropic growth at low doses of microtubule-destabilizing drugs, enhancement of the temperature-sensitive microtubule organization1-1 phenotype, and less ordered and more fragmented cortical microtubule arrays compared with the wild type. PHS1 encodes a novel protein similar to mitogen-activated protein kinase (MAPK) phosphatases. In phs1-1, a conserved Arg residue in the noncatalytic N-terminal region is exchanged with Cys, and the mutant PHS1 retained considerable phosphatase activity in vitro. In mammalian MAPK phosphatases, the corresponding region serves as a docking motif for MAPKs, and analogous Arg substitutions severely inhibit the kinase-phosphatase association. Transgenic studies indicate that the phs1-1 mutation acts dominant negatively, whereas the null phs1-2 allele is recessive embryonic lethal. We propose that the PHS1 phosphatase regulates more than one MAPK and that a subset of its target kinases is involved in the organization of cortical microtubules.     

Site-specific insertion of three structural gene cassettes in transposon Tn7.

Transposon Tn7 has been known to carry genes for resistance to trimethoprim and spectinomycin. A poorly expressed streptothricin resistance gene, identical to the sat gene found in transposons Tn1825 and Tn1826, was localized between the two mentioned genes in Tn7. The surroundings of all three resistance genes indicated site-specific insertion of genetic cassettes.     

A novel mutation in the mitochondrial tRNA Asn gene associated with a lethal disease

We describe a lethal mitochondrial disease in a 10-month-old child who presented with encephalomyopathy. Histochemical and electron microscopy examinations of skeletal muscle biopsy revealed abnormal mitochondria associated with a combined deficiency of complexes I and IV. After excluding mitochondrial DNA deletions and depletion, direct sequencing was used to screen for mutation in all transfer RNA (tRNA) genes. A T-to-C substitution at position 5693 in the tRNA(Asn) gene was found in blood and muscle. Microdissection of muscle biopsy and its analysis revealed the highest level of this mutation in cytochrome c oxidase (COX)-negative fibres. We suggest that this novel mutation would affect the anticodon loop structure of the tRNA(Asn) and cause a fatal mitochondrial disease.     

Disruption of plE2, the gene for the E2 subunit of the plastid pyruvate dehydrogenase complex,in Arabidopsis causes an early embryo lethal phenotype

The pyruvate dehydrogenase multi-enzyme complex is the main source of acetyl-CoA formation in the plastids of plants and is composed of multiple copies of four different subunits, E1, E1, E2, and E3. A T-DNA insertion into the gene for the plastidic E2 (dihydrolipoyl acetyltransferase) subunit, plE2, of the complex in Arabidopsis destroys the expression of that gene. The resulting mutation has no apparent phenotype in the heterozygous state, but the homozygous mutation is lethal. Haploid sperm and eggs that contain only the disrupted plE2 gene function normally resulting in the formation of an embryo that is homozygous for the mutation. This embryo only develops to an early stage before the development arrests resulting in an early embryo-lethal phenotype. While the mutation could not be complemented with the cDNA for the plE2 gene under control of the 35S, the AtSERK1, or the napin promoter, it could be complemented using the endogenous plE2 promoter to drive expression of the plE2 cDNA. This verifies the essential nature of the plastidic pyruvate dehydrogenase complex and its role in embryo formation.     

A mutation in the MATP gene causes the cream coat colour in the horse

In horses, basic colours such as bay or chestnut may be partially diluted to buckskin and palomino, or extremely diluted to cream, a nearly white colour with pink skin and blue eyes. This dilution is expected to be controlled by one gene and we used both candidate gene and positional cloning strategies to identify the "cream mutation". A horse panel including reference colours was established and typed for different markers within or in the neighbourhood of two candidate genes. Our data suggest that the causal mutation, a G to A transition, is localised in exon 2 of the MATP gene leading to an aspartic acid to asparagine substitution in the encoded protein. This conserved mutation was also described in mice and humans, but not in medaka.     

A murine gene with circadian expression revealed by transposon insertion: self-sustained rhythmicity in the liver and the photoreceptors

We have previously identified in some mouse strains (e.g. BALB/c, DBA/2) a murine Intracisternal A-particle (IAP) transposable element specifically expressed in the liver. This IAP sequence is inserted within a gene, mCCR4/m. nocturnin, the sequence of which is related to the circadian Xenopus nocturnin gene. Here we show, using real-time quantitative RT-PCR, that both the IAP sequence and the m. nocturnin gene display strong circadian expression in the liver, with peak abundance after dusk. Circadian oscillations of m. nocturnin RNA are maintained in mice without the IAP insertion (e.g. CBA/J, 129/sv), are free-running under constant light and dark conditions, and persist upon food and water privation, demonstrating that m. nocturnin is a circadian gene. In situ hybridization analyses (in 129/sv mice) further show circadian expression of m. nocturnin also in the retina, precisely at the level of the photoreceptors, a result consistent with the previously described circadian expression of the Xenopus gene. These results strengthen the strong conservation of the nocturnin gene with the identification of a functional mouse ortholog of the Xenopus gene, and demonstrate the reciprocal influence of nearby genes on the expression of transposable elements via "position effects".