scholarly journals Isolating an active and inactive CACTA transposon from lettuce color mutants and characterizing their family

2021 ◽  
Author(s):  
Csanad Gurdon ◽  
Alexander Kozik ◽  
Rong Tao ◽  
Alexander Poulev ◽  
Isabel Armas ◽  
...  
Keyword(s):  
Anthocyanin Biosynthesis ◽  
Mobile Element ◽  
Bioinformatic Analysis ◽  
Relative Expression Level ◽  
Gene Coding ◽  
Genes Encoding ◽  
Key Enzyme ◽  
Natural Derivative ◽  
Dark Green ◽  
Transposon Insertions

Abstract Dietary flavonoids play an important role in human nutrition and health. Flavonoid biosynthesis genes have recently been identified in lettuce (Lactuca sativa); however, few mutants have been characterized. We now report the causative mutations in Green Super Lettuce (GSL), a natural light green mutant derived from red cultivar NAR; and GSL-Dark Green (GSL-DG), an olive-green natural derivative of GSL. GSL harbors CACTA 1 (LsC1), a 3.9-kb active nonautonomous CACTA superfamily transposon inserted in the 5′ untranslated region of anthocyanidin synthase (ANS), a gene coding for a key enzyme in anthocyanin biosynthesis. Both terminal inverted repeats (TIRs) of this transposon were intact, enabling somatic excision of the mobile element, which led to the restoration of ANS expression and the accumulation of red anthocyanins in sectors on otherwise green leaves. GSL-DG harbors CACTA 2 (LsC2), a 1.1-kb truncated copy of LsC1 that lacks one of the TIRs, rendering the transposon inactive. RNA-sequencing and reverse transcription quantitative PCR of NAR, GSL, and GSL-DG indicated the relative expression level of ANS was strongly influenced by the transposon insertions. Analysis of flavonoid content indicated leaf cyanidin levels correlated positively with ANS expression. Bioinformatic analysis of the cv Salinas lettuce reference genome led to the discovery and characterization of an LsC1 transposon family with a putative transposon copy number greater than 1,700. Homologs of tnpA and tnpD, the genes encoding two proteins necessary for activation of transposition of CACTA elements, were also identified in the lettuce genome.

scholarly journals LoxTnSeq: random transposon insertions combined with cre/lox recombination and counterselection to generate large random genome reductions

2020 ◽  
Author(s):  
Daniel Shaw ◽  
Samuel Miravet‐Verde ◽  
Carlos Piñero‐Lambea ◽  
Luis Serrano ◽  
Maria Lluch‐Senar
Keyword(s):  
Transposon Insertions

scholarly journals Agrobacterium-Mediated Transformation of Aspergillus awamori in the Absence of Full-Length VirD2, VirC2, or VirE2 Leads to Insertion of Aberrant T-DNA Structures

2004 ◽  
Vol 186 (7) ◽  
pp. 2038-2045 ◽  
Author(s):  
Caroline B. Michielse ◽  
Arthur F. J. Ram ◽  
Paul J. J. Hooykaas ◽  
Cees A. M. J. J. van den Hondel
Keyword(s):  
Single Copy ◽  
Full Length ◽  
Aspergillus Awamori ◽  
Wild Type ◽  
Dna Structures ◽  
Dna Integration ◽  
Virulence Proteins ◽  
Type Transformation ◽  
The Right ◽  
Transposon Insertions

ABSTRACT Reductions to 2, 5, and 42% of the wild-type transformation efficiency were found when Agrobacterium mutants carrying transposon insertions in virD2, virC2, and virE2, respectively, were used to transform Aspergillus awamori. The structures of the T-DNAs integrated into the host genome by these mutants were analyzed by Southern and sequence analyses. The T-DNAs of transformants obtained with the virE2 mutant had left-border truncations, whereas those obtained with the virD2 mutant had truncated right ends. From this analysis, it was concluded that the virulence proteins VirD2 and VirE2 are required for full-length T-DNA integration and that these proteins play a role in protecting the right and left T-DNA borders, respectively. Multicopy and truncated T-DNA structures were detected in the majority of the transformants obtained with the virC2 mutant, indicating that VirC2 plays a role in correct T-DNA processing and is required for single-copy T-DNA integration.

scholarly journals GLC3 and GHA1 of Saccharomyces cerevisiae are allelic and encode the glycogen branching enzyme.

1992 ◽  
Vol 12 (1) ◽  
pp. 22-29 ◽  
Author(s):  
D W Rowen ◽  
M Meinke ◽  
D C LaPorte
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glycogen Metabolism ◽  
Striking Difference ◽  
Branching Enzyme ◽  
Yeast Saccharomyces Cerevisiae ◽  
Storage Carbohydrate ◽  
Functional Product ◽  
Cloned Gene ◽  
Transposon Insertions ◽  
Glycogen Branching Enzyme

In the yeast Saccharomyces cerevisiae, glycogen serves as a major storage carbohydrate. In a previous study, mutants with altered glycogen metabolism were isolated on the basis of the altered iodine-staining properties of colonies. We found that when glycogen produced by strains carrying the glc-1p (previously called gha1-1) mutation is stained with iodine, the absorption spectrum resembles that of starch rather than that of glycogen, suggesting that this mutation might reduce the level of branching in the glycogen particles. Indeed, glycogen branching activity was undetectable in extracts from a glc3-1p strain but was elevated in strains which expressed GLC3 from a high-copy-number plasmid. These observations suggest that GLC3 encodes the glycogen branching enzyme. In contrast to glc3-1p, the glc3-4 mutation greatly reduces the ability of yeast to accumulate glycogen. These mutations appear to be allelic despite the striking difference in the phenotypes which they produce. The GLC3 clone complemented both glc3-1p and glc3-4. Deletions and transposon insertions in this clone had parallel effects on its ability to complement glc3-1p and glc3-4. Finally, a fragment of the cloned gene was able to direct the repair of both glc3-1p and glc3-4. Disruption of GLC3 yielded the glycogen-deficient phenotype, indicating that glycogen deficiency is the null phenotype. The glc3-1p allele appears to encode a partially functional product, since it is dominant over glc3-4 but recessive to GLC3. These observations suggest that the ability to introduce branches into glycogen greatly increases the ability of the cell to accumulate that polysaccharide. Northern (RNA) blot analysis identified a single mRNA of 2,300 nucleotides that increased in abundance ca. 20-fold as the culture approached stationary phase. It thus appears that the expression of GLC3 is regulated, probably at the level of transcription.

scholarly journals Spermidine Inversely Influences Surface Interactions and Planktonic Growth in Agrobacterium tumefaciens

2016 ◽  
Vol 198 (19) ◽  
pp. 2682-2691 ◽  
Author(s):  
Yi Wang ◽  
Sok Ho Kim ◽  
Ramya Natarajan ◽  
Jason E. Heindl ◽  
Eric L. Bruger ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Biofilm Formation ◽  
Direct Synthesis ◽  
Wild Type ◽  
Cyclic Diguanylate ◽  
Content Type ◽  
Exogenous Addition ◽  
In The Wild ◽  
Growth Requirement ◽  
Transposon Insertions

ABSTRACTIn bacteria, the functions of polyamines, small linear polycations, are poorly defined, but these metabolites can influence biofilm formation in several systems. Transposon insertions in an ornithine decarboxylase (odc) gene inAgrobacterium tumefaciens, predicted to direct synthesis of the polyamine putrescine from ornithine, resulted in elevated cellulose. Null mutants forodcgrew somewhat slowly in a polyamine-free medium but exhibited increased biofilm formation that was dependent on cellulose production. Spermidine is an essential metabolite inA. tumefaciensand is synthesized from putrescine inA. tumefaciensvia the stepwise actions of carboxyspermidine dehydrogenase (CASDH) and carboxyspermidine decarboxylase (CASDC). Exogenous addition of either putrescine or spermidine to theodcmutant returned biofilm formation to wild-type levels. Low levels of exogenous spermidine restored growth to CASDH and CASDC mutants, facilitating weak biofilm formation, but this was dampened with increasing concentrations. Norspermidine rescued growth for theodc, CASDH, and CASDC mutants but did not significantly affect their biofilm phenotypes, whereas in the wild type, it stimulated biofilm formation and depressed spermidine levels. Theodcmutant produced elevated levels of cyclic diguanylate monophosphate (c-di-GMP), exogenous polyamines modulated these levels, and expression of a c-di-GMP phosphodiesterase reversed the enhanced biofilm formation. Prior work revealed accumulation of the precursors putrescine and carboxyspermidine in the CASDH and CASDC mutants, respectively, but unexpectedly, both mutants accumulated homospermidine; here, we show that this requires a homospermidine synthase (hss) homologue.IMPORTANCEPolyamines are small, positively charged metabolites that are nearly ubiquitous in cellular life. They are often essential in eukaryotes and more variably in bacteria. Polyamines have been reported to influence the surface-attached biofilm formation of several bacteria. InAgrobacterium tumefaciens, mutants with diminished levels of the polyamine spermidine are stimulated for biofilm formation, and exogenous provision of spermidine decreases biofilm formation. Spermidine is also essential forA. tumefaciensgrowth, but the related polyamine norspermidine exogenously rescues growth and does not diminish biofilm formation, revealing that the growth requirement and biofilm control are separable. Polyamine control of biofilm formation appears to function via effects on the cellular second messenger cyclic diguanylate monophosphate, regulating the transition from a free-living to a surface-attached lifestyle.

scholarly journals Identification of Genes Required for Synthesis of the Adhesive Holdfast in Caulobacter crescentus

2003 ◽  
Vol 185 (4) ◽  
pp. 1432-1442 ◽  
Author(s):  
Chris S. Smith ◽  
Aaron Hinz ◽  
Diane Bodenmiller ◽  
David E. Larson ◽  
Yves V. Brun
Keyword(s):  
Outer Membrane ◽  
Gene Cluster ◽  
Xanthomonas Campestris ◽  
Caulobacter Crescentus ◽  
Sequence Similarity ◽  
Gram Negative ◽  
Transmission Electron ◽  
Polar Development ◽  
Transposon Insertions ◽  
Polar Organelle

ABSTRACT Adhesion to both abiotic and biotic surfaces by the gram-negative prothescate bacterium Caulobacter crescentus is mediated by a polar organelle called the “holdfast,” which enables the bacterium to form stable monolayer biofilms. The holdfast, a complex polysaccharide composed in part of N-acetylglucosamine, localizes to the tip of the stalk (a thin cylindrical extension of the cell wall and membranes). We report here the isolation of adhesion mutants with transposon insertions in an uncharacterized gene cluster involved in holdfast biogenesis (hfs) as well as in previously identified polar development genes (podJ and pleC), and the holdfast attachment genes (hfa). Clean deletions of three of the four genes in the hfs gene cluster (hfsDAB) resulted in a severe holdfast biogenesis phenotype. These mutants do not bind to surfaces or to a fluorescently labeled lectin, specific for N-acetylglucosamine. Transmission electron microscopy indicated that the hfsDAB mutants fail to synthesize a holdfast at the stalk tip. The predicted hfs gene products have significant sequence similarity to proteins necessary for exopolysaccharide export in gram-negative bacteria. HfsA has sequence similarity to GumC from Xanthomonas campestris, which is involved in exopolysaccharide export in the periplasm. HfsD has sequence similarity to Wza from Escherichia coli, an outer membrane protein involved in secretion of polysaccharide through the outer membrane. HfsB is a novel protein involved in holdfast biogenesis. These data suggest that the hfs genes play an important role in holdfast export.

scholarly journals Novel Genomic Locus with Atypical G+C Content that Is Required for Extracellular Polysaccharide Production and Virulence in Xanthomonas oryzae pv. oryzae

2001 ◽  
Vol 14 (11) ◽  
pp. 1335-1339 ◽  
Author(s):  
Sridhar Dharmapuri ◽  
J. Yashitola ◽  
M. R. Vishnupriya ◽  
Ramesh V. Sonti
Keyword(s):  
Sequence Data ◽  
Extracellular Polysaccharide ◽  
Genomic Region ◽  
Xanthomonas Oryzae ◽  
Genomic Locus ◽  
Reading Frame ◽  
Glycosyl Transferase ◽  
Clone Sequence ◽  
Gum Gene Cluster ◽  
Transposon Insertions

Three exopolysaccharide (EPS)- and virulence-deficient mutants of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight of rice, were isolated by Tn5 mutagenesis. These insertions are not located within the gum gene cluster. A 40-kb cosmid clone that restored EPS production and virulence to all three mutants was isolated, and the three transposon insertions were localized to contiguous 4.3- and 3.5-kb EcoRI fragments that are included in this clone. Sequence data indicate that two of the transposon insertions are in genes that encode a putative sugar nucleotide epimerase and a putative glycosyl transferase, respectively; the third insertion is located between the glycosyl transferase gene and a novel open reading frame (ORF). A 5.5-kb genomic region in which these three ORFs are located has a G+C content of 51.7%, quite different from the G+C content of approximately 65.0% that is typical of X. oryzae pv. oryzae. Homologues of this locus have not yet been reported in any other xanthomonad.

scholarly journals Chromosome-scale, haplotype-resolved assembly of human genomes

2020 ◽  
Author(s):  
Shilpa Garg ◽  
Arkarachai Fungtammasan ◽  
Andrew Carroll ◽  
Mike Chou ◽  
Anthony Schmitt ◽  
...  
Keyword(s):  
Killer Cell ◽  
Human Leukocyte ◽  
Population Diversity ◽  
Pedigree Information ◽  
Structural Variants ◽  
High Quality ◽  
Leukocyte Antigen ◽  
Haplotype Variation ◽  
Human Genomes ◽  
Transposon Insertions

AbstractHaplotype-resolved or phased genome assembly provides a complete picture of genomes and their complex genetic variations. However, current algorithms for phased assembly either do not generate chromosome-scale phasing or require pedigree information, which limits their application. We present a method named diploid assembly (DipAsm) that uses long, accurate reads and long-range conformation data for single individuals to generate a chromosome-scale phased assembly within 1 day. Applied to four public human genomes, PGP1, HG002, NA12878 and HG00733, DipAsm produced haplotype-resolved assemblies with minimum contig length needed to cover 50% of the known genome (NG50) up to 25 Mb and phased ~99.5% of heterozygous sites at 98–99% accuracy, outperforming other approaches in terms of both contiguity and phasing completeness. We demonstrate the importance of chromosome-scale phased assemblies for the discovery of structural variants (SVs), including thousands of new transposon insertions, and of highly polymorphic and medically important regions such as the human leukocyte antigen (HLA) and killer cell immunoglobulin-like receptor (KIR) regions. DipAsm will facilitate high-quality precision medicine and studies of individual haplotype variation and population diversity.

Sign in / Sign up

Export Citation Format

Share Document