Faculty Opinions recommendation of A competitive PCR-based method to detect a single copy of T-DNA insertion in transformants.

Abstract Photosynthesis, an indispensable biological process of plants, produces organic substances for plant growth, during which photorespiration occurs to oxidize carbohydrates to achieve homeostasis. Although the molecular mechanism underlying photosynthesis and photorespiration has been widely explored, the crosstalk between the two processes remains largely unknown. In this study, we isolated and characterized a T-DNA insertion mutant of tomato (Solanum lycopersicum) named yellow leaf (yl) with yellowish leaves, retarded growth, and chloroplast collapse that hampered both photosynthesis and photorespiration. Genetic and expression analyses demonstrated that the phenotype of yl was caused by a loss-of-function mutation resulting from a single-copy T-DNA insertion in chaperonin 60α1 (SlCPN60α1). SlCPN60α1 showed high expression levels in leaves and was located in both chloroplasts and mitochondria. Silencing of SlCPN60α1using virus-induced gene silencing and RNA interference mimicked the phenotype of yl. Results of two-dimensional electrophoresis and yeast two-hybrid assays suggest that SlCPN60α1 potentially interacts with proteins that are involved in chlorophyll synthesis, photosynthetic electron transport, and the Calvin cycle, and further affect photosynthesis. Moreover, SlCPN60α1 directly interacted with serine hydroxymethyltransferase (SlSHMT1) in mitochondria, thereby regulating photorespiration in tomato. This study outlines the importance of SlCPN60α1 for both photosynthesis and photorespiration, and provides molecular insights towards plant genetic improvement.

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High-resolution mapping of the origin of DNA replication in the hamster dihydrofolate reductase gene domain by competitive PCR.

Molecular and Cellular Biology ◽

10.1128/mcb.16.10.5358 ◽

1996 ◽

Vol 16 (10) ◽

pp. 5358-5364 ◽

Cited By ~ 51

Author(s):

C Pelizon ◽

S Diviacco ◽

A Falaschi ◽

M Giacca

Keyword(s):

Dna Replication ◽

Dihydrofolate Reductase ◽

Cultured Cells ◽

Chinese Hamster ◽

Single Copy ◽

Genomic Region ◽

Competitive Pcr ◽

Dhfr Gene ◽

Mapping Procedure ◽

Dna Replication Origin

By the use of a highly sensitive mapping procedure allowing the identification of the start sites of DNA replication in single-copy genomic regions of untreated, exponentially growing cultured cells (M. Giacca, L. Zentilin, P. Norio, S. Diviacco, D. Dimitrova, G. Contreas, G. Biamonti, G. Perini, F. Weighardt, S. Riva, and A. Falaschi, Proc. Natl. Acad. Sci. USA 91:7119-7123, 1994), the pattern of DNA replication of the Chinese hamster dihydrofolate reductase (DHFR) gene domain was investigated. The method entails the purification of short stretches of nascent DNA issuing from DNA replication origin regions and quantification, within this sample, of the abundance of different adjacent segments by competitive PCR. Distribution of marker abundance peaks around the site from which newly synthesized DNA had emanated. The results obtained by analysis of the genomic region downstream of the DHFR single-copy gene in asynchronous cultures of hamster CHO K1 cells are consistent with the presence of a single start site for DNA replication, located approximately 17 kb downstream of the gene. This site is coincident with the one detected by other studies using different techniques in CHO cell lines containing an amplified DHFR gene domain.

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JOINT DISTRIBUTION OF INSERTION ELEMENTS IS4 AND IS5 IN NATURAL ISOLATES OF ESCHERICHIA COLI

Genetics ◽

10.1093/genetics/111.2.219 ◽

1985 ◽

Vol 111 (2) ◽

pp. 219-231

Author(s):

Daniel E Dykhuizen ◽

Stanley A Sawyer ◽

Louis Green ◽

Raymond D Miller ◽

Daniel L Hartl

Keyword(s):

Escherichia Coli ◽

Dna Sequences ◽

Significant Negative Correlation ◽

Single Copy ◽

Common Mechanism ◽

Reference Collection ◽

Insertion Elements ◽

Natural Isolates ◽

Dna Insertion ◽

Flanking Sequences

ABSTRACT A reference collection of natural isolates of Escherichia coli has been studied in order to determine the distribution, abundance and joint occurence of DNA insertion elements IS4 and IS5. Among these isolates, 36% were found to contain IS4 and 30% were found to contain IS5. Among strains containing IS4 the mean number of copies per strain was 4.4 ± 0.8; the comparable figure for IS5 was 3.7 ± 1.0. Although the presence of the elements among the isolates was independent, among those isolates containing both IS4 and IS5, there was a significant negative correlation in the number of copies of the elements.— The reference collection was also studied for the presence of the DNA sequences flanking the single copy of IS4 in the chromosome of E. coli K12. Homologous sequences were found in only 26% of the isolates. The sequences flanking the IS4 invariably occur together, and their presence is significantly correlated with the presence of IS4. In eight of the strains that carry these flanking sequences, an IS4 is located between them, and the sequences are present at the homologous position as in the K12 strain. We suggest that IS4 and its flanking sequences share a common mechanism of dissemination, such as plasmids, and we present evidence that they are included in a much larger transposable element.

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Establishment of Agrobacterium tumefaciens-Mediated Transformation of Cladonia macilenta, a Model Lichen-Forming Fungus

Journal of Fungi ◽

10.3390/jof7040252 ◽

2021 ◽

Vol 7 (4) ◽

pp. 252

Author(s):

Rundong Liu ◽

Wonyong Kim ◽

Jaycee Augusto Paguirigan ◽

Min-Hye Jeong ◽

Jae-Seoun Hur

Keyword(s):

Agrobacterium Tumefaciens ◽

Incubation Temperature ◽

Single Copy ◽

Insertion Mutant ◽

Symbiotic Association ◽

Slow Growth Rate ◽

Cultivation Period ◽

C Terminus ◽

Genetic Mechanisms ◽

Dna Insertion

Despite the fascinating biology of lichens, such as the symbiotic association of lichen-forming fungi (mycobiont) with their photosynthetic partners and their ability to grow in harsh habitats, lack of genetic tools manipulating mycobiont has hindered studies on genetic mechanisms underpinning lichen biology. Thus, we established an Agrobacterium tumefaciens-mediated transformation (ATMT) system for genetic transformation of a mycobiont isolated from Cladonia macilenta. A set of combinations of ATMT conditions, such as input biomass of mycobiont, co-cultivation period with Agrobacterium cells, and incubation temperature, were tested to identify an optimized ATMT condition for the C. macilenta mycobiont. As a result, more than 10 days of co-cultivation period and at least 2 mg of input biomass of the mycobiont were recommended for an efficient ATMT, owing to extremely slow growth rate of mycobionts in general. Moreover, we examined T-DNA copy number variation in a total of 180 transformants and found that 88% of the transformants had a single copy T-DNA insertion. To identify precise T-DNA insertion sites that interrupt gene function in C. macilenta, we performed TAIL-PCR analyses for selected transformants. A hypothetical gene encoding ankyrin repeats at its C-terminus was interrupted by T-DNA insertion in a transformant producing dark-brown colored pigment. Although the identification of the pigment awaits further investigation, this proof-of-concept study demonstrated the feasibility of use of ATMT in construction of a random T-DNA insertion mutant library in mycobionts for studying genetic mechanisms behind the lichen symbiosis, stress tolerance, and secondary metabolite biosynthesis.

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Activity of the c-myc Replicator at an Ectopic Chromosomal Location

Molecular and Cellular Biology ◽

10.1128/mcb.19.8.5685 ◽

1999 ◽

Vol 19 (8) ◽

pp. 5685-5695 ◽

Cited By ~ 48

Author(s):

Michelle Malott ◽

Michael Leffak

Keyword(s):

Single Copy ◽

Acceptor Site ◽

Competitive Pcr ◽

Donor Plasmid ◽

Autonomously Replicating Sequence ◽

Flp Recombinase ◽

Before And After ◽

Dna Strands

ABSTRACT DNA replication starts at multiple discrete sites across the human chromosomal c-myc region, including two or more sites within 2.4 kb upstream of the c-myc gene. The corresponding 2.4-kb c-myc origin fragment confers autonomously replicating sequence (ARS) activity on plasmids, which specifically initiate replication in the origin fragment in vitro and in vivo. To test whether the region that displays plasmid replicator activity also acts as a chromosomal replicator, HeLa cell sublines that each contain a single copy of the Saccharomyces cerevisiae FLP recombinase target (FRT) sequence flanked by selectable markers were constructed. A clonal line containing a single unrearranged copy of the transduced c-myc origin was produced by cotransfecting a donor plasmid containing the 2.4-kb c-myc origin fragment and FRT, along with a plasmid expressing the yeast FLP recombinase, into cells containing a chromosomal FRT acceptor site. The amount of short nascent DNA strands at the chromosomal acceptor site was quantitated before and after targeted integration of the origin fragment. Competitive PCR quantitation showed that the c-myc origin construct substantially increased the amount of nascent DNA relative to that at the unoccupied acceptor site and to that after the insertion of non-myc DNA. The abundance of nascent strands was greatest close to the c-myc insert of the integrated donor plasmid, and significant increases in nascent strand abundance were observed at sites flanking the insertion. These results provide biochemical and genetic evidence for the existence of chromosomal replicators in metazoan cells and are consistent with the presence of chromosomal replicator activity in the 2.4-kb region of c-myc origin DNA.

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