Transposon Tagging of the Sulfur Gene of Tobacco Using Engineered Maize Ac/Ds Elements

Abstract The Sulfur gene of tobacco is nuclearly encoded. A Su allele at this locus acts as a dominant semilethal mutation and causes reduced accumulation of chlorophyll, resulting in a yellow color in the plant. An engineered transposon tagging system, based upon the maize element Ac/Ds, was used to mutate the gene. High frequency of transposon excision from the Su locus produced variegated sectors. Plants regenerated from the variegated sector exhibited a similar variegated phenotype. Genetic analyses showed that the variegation was always associated with the transposase construct and the transposon was linked to the Su locus. Sequences surrounding the transposon were isolated, and five revertant sectors possessed typical direct repeats following Ds excisions. These genetic and molecular data are consistent with the tagging of the Su allele by the transposon.

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Short direct repeats mediate spontaneous high-frequency deletions in DNA of minute virus of mice.

Molecular and Cellular Biology ◽

10.1128/mcb.4.10.2239 ◽

1984 ◽

Vol 4 (10) ◽

pp. 2239-2242 ◽

Cited By ~ 11

Author(s):

A Hogan ◽

E A Faust

Keyword(s):

Base Pair ◽

High Frequency ◽

Repeated Sequence ◽

Model Systems ◽

High Rate ◽

Direct Repeats ◽

Minute Virus Of Mice ◽

Minute Virus ◽

Novel Model ◽

Deletion Junction

Previous work (E. A. Faust and D. C. Ward, J. Virol. 32:276-292, 1979) revealed a remarkably high rate of spontaneous deletion in viral DNA during lytic infection of cultured murine cells with minute virus of mice (MVM), an autonomous parvovirus. In the present study, we have isolated plasmid and phage recombinants containing MVM DNA inserts bearing deletions and we have determined the DNA sequence spanning three deletion junctions. The deletions, which average 3 kilobases in length, occur between pairs of perfectly homologous 4- to 10-base-pair direct repeats, such that one copy of the repeated sequence is lost, whereas the other remains behind at the deletion junction. When compared, the three sets of direct repeats exhibit no apparent sequence homology and have an A + T content of between 50 and 80%. These results indicate that 4- to 10-base-pair homologies mediate spontaneous deletion formation in the MVM genome and highlight parvoviruses as novel model systems for studies of this ubiquitous pathway of genetic variation.

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The behavior of insertions near a site of mitotic gene conversion in yeast.

Genetics ◽

10.1093/genetics/119.3.535 ◽

1988 ◽

Vol 119 (3) ◽

pp. 535-540

Author(s):

J E Golin ◽

S C Falco

Keyword(s):

Gene Conversion ◽

High Frequency ◽

Direct Repeat ◽

Large Plasmid ◽

Direct Repeats ◽

Heteroduplex Dna ◽

Relative Stabilities ◽

Extensive Region ◽

Mitotic Gene Conversion ◽

A Site

Abstract In yeast, coincident gene conversion events involving the LEU1 and TRP5 loci (16 cM apart) occur at frequencies that are far greater than is expected for two independent acts of recombination. When a large plasmid (pJM53) is placed between these genes so that a direct repeat is produced, there is frequent loss of the insert among coincident convertants. Previous results strongly suggest that this is due to a separate, intrachromosomal exchange between the direct repeats rather than to excision from an extensive region of heteroduplex DNA. In this paper, we extend our genetic and molecular analysis to a plasmid insertion (pKSH) which replaces rather than duplicates the chromosomal material. The relative stabilities of pKSH and pJM53 are compared among coincident Leu+Trp+ convertants and convertants involving only one locus (LEU1). The pKSH insertion is significantly more stable in the latter which constitute a large majority of the selectable recombinants. In the former, both insertions are lost with high frequency. These results are used to argue that, while most mitotic conversion does not result from long intermediates, coincident convertants may arise from either multiple intermediates or extensive heteroduplex regions.

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Transposition pattern of the maize element Ds in Arabidopsis thaliana.

Genetics ◽

10.1093/genetics/134.4.1221 ◽

1993 ◽

Vol 134 (4) ◽

pp. 1221-1229 ◽

Cited By ~ 5

Author(s):

I Bancroft ◽

C Dean

Keyword(s):

Arabidopsis Thaliana ◽

Donor Site ◽

Transposon Tagging ◽

Selection For ◽

Tagging System

Abstract As part of establishing an efficient transposon tagging system in Arabidopsis using the maize elements Ac and Ds, we have analyzed the inheritance and pattern of Ds transposition in four independent Arabidopsis transformants. A low proportion (33%) of plants inheriting the marker used to monitor excision contained a transposed Ds. Selection for the transposed Ds increased this to at least 49%. Overall, 68% of Ds transpositions inherited with the excision marker were to genetically linked sites; however, the distribution of transposed elements varied around the different donor sites. Mapping of transposed Ds elements that were genetically unlinked to the donor site showed that a proportion (3 of 11 tested) integrated into sites which were still physically linked.

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Definition and characterization of an artificial En/Spm-based transposon tagging system in transgenic tobacco

Plant Molecular Biology ◽

10.1007/bf00021428 ◽

1993 ◽

Vol 23 (1) ◽

pp. 157-178 ◽

Cited By ~ 12

Author(s):

Guillermo H. Cardon ◽

Monika Frey ◽

Heinz Saedler ◽

Alfons Gierl

Keyword(s):

Transgenic Tobacco ◽

Transposon Tagging ◽

Tagging System

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Development of a TILLING resource in durum wheat for reverse- and forward-genetic analyses

Crop and Pasture Science ◽

10.1071/cp13226 ◽

2014 ◽

Vol 65 (1) ◽

pp. 112 ◽

Cited By ~ 18

Author(s):

R. Bovina ◽

A. Brunazzi ◽

G. Gasparini ◽

F. Sestili ◽

S. Palombieri ◽

...

Keyword(s):

Durum Wheat ◽

High Frequency ◽

Reverse Genetics ◽

Seed Morphology ◽

Wheat Genome ◽

Phenotypic Screening ◽

Starch Metabolism ◽

Genetic Analyses ◽

Morphological Alterations ◽

Local Lesions

A durum wheat TILLING (targeting induced local lesions in genomes) population of 2601 M3 families was developed from cv. Svevo using ethyl methanesulfonate as a chemical mutagen. The entire M3 population was field-grown for phenotypic evaluations. Despite the polyploid nature of the wheat genome, a preliminarily phenotypic screening showed a high frequency of morphological alterations (~22%); specific phenotyping for seed morphology was undertaken. Furthermore, a reverse-genetics experiment was performed on DNA collected from M2 leaves for the homoeologous genes SBEIIa-A and SBEIIa-B involved in starch metabolism. One non-sense mutation for both genes was identified; specific crosses are planned in order to pyramid the two mutations.

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The genetic basis of viral virulence

Philosophical Transactions of the Royal Society of London Series B Biological Sciences ◽

10.1098/rstb.1983.0094 ◽

1983 ◽

Vol 303 (1114) ◽

pp. 209-218 ◽

Cited By ~ 6

Keyword(s):

High Frequency ◽

Genetic Basis ◽

Rna Viruses ◽

Capsid Proteins ◽

Tissue Tropism ◽

Genetic Studies ◽

Genetic Analyses ◽

Viral Virulence ◽

Viral Components ◽

Outer Capsid

The precise genetic and molecular determ inants of viral virulence are poorly understood. Genetic studies with influenza and reovirus have indicated that virulence is multigenic. The high frequency of m utation of RNA viruses can complicate genetic analyses of virulence, resulting in phenotypes that are difficult to interpret. The ease with which the reoviruses reassort genome segments has made it possible to isolate reassortants from parental viruses causing different patterns of anim al disease. It has thus been feasible to show that each of the three outer capsid proteins plays a m ajor role in the pathogenesis of anim al infection: the viral haem agglutinin determines the specificity of the im mune response and cell and tissue tropism ; the p ic protein plays a central role in determ ining yield at portals of entry as well as in differentiated tissues; the δ3 protein inhibits host m acrom olecular synthesis. Thus virulence is clearly multigenic, with each of the viral components playing distinct roles.

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Prevalence of Cardinium Bacteria in Planthoppers and Spider Mites and Taxonomic Revision of “Candidatus Cardinium hertigii” Based on Detection of a New Cardinium Group from Biting Midges

Applied and Environmental Microbiology ◽

10.1128/aem.01583-09 ◽

2009 ◽

Vol 75 (21) ◽

pp. 6757-6763 ◽

Cited By ~ 77

Author(s):

Yuki Nakamura ◽

Sawako Kawai ◽

Fumiko Yukuhiro ◽

Saiko Ito ◽

Tetsuo Gotoh ◽

...

Keyword(s):

High Frequency ◽

Taxonomic Revision ◽

Single Species ◽

Molecular Data ◽

Spider Mites ◽

Parasitic Nematodes ◽

Biting Midges ◽

Group A ◽

Morphological And Molecular Data ◽

Group B

ABSTRACT Cardinium bacteria, members of the phylum Cytophaga-Flavobacterium-Bacteroides (CFB), are intracellular bacteria in arthropods that are capable of inducing reproductive abnormalities in their hosts, which include parasitic wasps, mites, and spiders. A high frequency of Cardinium infection was detected in planthoppers (27 out of 57 species were infected). A high frequency of Cardinium infection was also found in spider mites (9 out of 22 species were infected). Frequencies of double infection by Cardinium and Wolbachia bacteria (Alphaproteobacteria capable of manipulating reproduction of their hosts) were disproportionately high in planthoppers but not in spider mites. A new group of bacteria, phylogenetically closely related to but distinct from previously described Cardinium bacteria (based on 16S rRNA and gyrB genes) was found in 4 out of 25 species of Culicoides biting midges. These bacteria possessed a microfilament-like structure that is a morphological feature previously found in Cardinium and Paenicardinium. The bacteria close to the genus Cardinium consist of at least three groups, A, B, and C. Group A is present in various species of arthropods and was previously referred to as “Candidatus Cardinium hertigii,” group B is present in plant parasitic nematodes and was previously referred to as “Candidatus Paenicardinium endonii,” and group C is present in Culicoides biting midges. On the basis of morphological and molecular data, we propose that the nomenclature of these three groups be integrated into a single species, “Candidatus Cardinium hertigii.”

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Transposition of the Autonomous Fot1 Element in the Filamentous Fungus Fusarium oxysporum

Genetics ◽

10.1093/genetics/151.3.1005 ◽

1999 ◽

Vol 151 (3) ◽

pp. 1005-1013

Author(s):

Quirico Migheli ◽

Richard Laugé ◽

Jean-Michel Davière ◽

Catherine Gerlinger ◽

Fiona Kaper ◽

...

Keyword(s):

Nitrate Reductase ◽

Fusarium Oxysporum ◽

Dna Sequences ◽

Reductase Activity ◽

Transposon Tagging ◽

Reading Frame ◽

Frameshift Deletion ◽

Chromosome Sequence ◽

Autonomous Mobility ◽

Tagging System

Abstract Autonomous mobility of different copies of the Fot1 element was determined for several strains of the fungal plant pathogen Fusarium oxysporum to develop a transposon tagging system. Two Fot1 copies inserted into the third intron of the nitrate reductase structural gene (niaD) were separately introduced into two genetic backgrounds devoid of endogenous Fot1 elements. Mobility of these copies was observed through a phenotypic assay for excision based on the restoration of nitrate reductase activity. Inactivation of the Fot1 transposase open reading frame (frameshift, deletion, or disruption) prevented excision in strains free of Fot1 elements. Molecular analysis of the Nia+ revertant strains showed that the Fot1 element reintegrated frequently into new genomic sites after excision and that it can transpose from the introduced niaD gene into a different chromosome. Sequence analysis of several Fot1 excision sites revealed the socalled footprint left by this transposable element. Three reinserted Fot1 elements were cloned and the DNA sequences flanking the transposon were determined using inverse polymerase chain reaction. In all cases, the transposon was inserted into a TA dinucleotide and created the characteristic TA target site duplication. The availability of autonomous Fot1 copies will now permit the development of an efficient two-component transposon tagging system comprising a trans-activator element supplying transposase and a cis-responsive marked element.

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