Formation of an inverted duplication can be an initial step in gene amplification

We have developed a gene transfer approach to facilitate the identification and isolation of chromosomal regions which are prone to high-frequency gene amplification. Such regions are identified by assaying for transformants which show high-frequency resistance to PALA and/or methotrexate by amplification of a vector containing the genes which encode the enzyme targets of these antiproliferative agents. We identified 2 of 47 transformants which displayed high-frequency amplification of the transfected genes, and in this report we describe the analysis of one of them (L46). Molecular analysis of the integration site in transformant L46 revealed that the donated genes were at the center of an inverted duplication which spanned more than 70 kilobase pairs and consisted largely of host DNA. The data suggest that integration of the transfected sequences generates a submicroscopic molecule containing the inverted duplication and at least 750 kilobases of additional sequences. The donated sequences and the host sequences were readily amplified and lost in exponentially growing cultures in the absence of drug selection, which suggests that the extrachromosomal elements are acentric. In contrast to the instability of this region following gene insertion, the preinsertion site was maintained at single copy level under growth conditions which produced copy number heterogeneity in L46. The implications of our results for mechanisms of genetic instability and mammalian gene amplification are discussed.

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Advances in Agrobacterium transformation and vector design result in high frequency targeted gene insertion in maize

Plant Biotechnology Journal ◽

10.1111/pbi.13613 ◽

2021 ◽

Author(s):

Dave Peterson ◽

Pierluigi Barone ◽

Brian Lenderts ◽

Chris Schwartz ◽

Lanie Feigenbutz ◽

...

Keyword(s):

High Frequency ◽

Agrobacterium Transformation ◽

Gene Insertion ◽

Design Result

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High frequency repeat-induced point mutation (RIP) is not associated with efficient recombination in Neurospora.

Genetics ◽

10.1093/genetics/138.4.1093 ◽

1994 ◽

Vol 138 (4) ◽

pp. 1093-1103 ◽

Cited By ~ 3

Author(s):

J T Irelan ◽

A T Hagemann ◽

E U Selker

Keyword(s):

Point Mutation ◽

Dna Sequences ◽

High Frequency ◽

Recombination Frequency ◽

Single Copy ◽

Sexual Cycle ◽

Base Pairs ◽

Copy Gene ◽

The Relationship ◽

Repeat Induced Point Mutation

Abstract Duplicated DNA sequences in Neurospora crassa are efficiently detected and mutated during the sexual cycle by a process named repeat-induced point mutation (RIP). Linked, direct duplications have previously been shown to undergo both RIP and deletion at high frequency during premeiosis, suggesting a relationship between RIP and homologous recombination. We have investigated the relationship between RIP and recombination for an unlinked duplication and for both inverted and direct, linked duplications. RIP occurred at high frequency (42-100%) with all three types of duplications used in this study, yet recombination was infrequent. For both inverted and direct, linked duplications, recombination was observed, but at frequencies one to two orders of magnitude lower than RIP. For the unlinked duplication, no recombinants were seen in 900 progeny, indicating, at most, a recombination frequency nearly three orders of magnitude lower than the frequency of RIP. In a direct duplication, RIP and recombination were correlated, suggesting that these two processes are mechanistically associated or that one process provokes the other. Mutations due to RIP have previously been shown to occur outside the boundary of a linked, direct duplication, indicating that RIP might be able to inactivate genes located in single-copy sequences adjacent to a duplicated sequence. In this study, a single-copy gene located between elements of linked duplications was inactivated at moderate frequencies (12-14%). Sequence analysis demonstrated that RIP mutations had spread into these single-copy sequences at least 930 base pairs from the boundary of the duplication, and Southern analysis indicated that mutations had occurred at least 4 kilobases from the duplication boundary.

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Unstable amplification of two extrachromosomal elements in alpha-difluoromethylornithine-resistant Leishmania donovani.

Molecular and Cellular Biology ◽

10.1128/mcb.12.12.5499 ◽

1992 ◽

Vol 12 (12) ◽

pp. 5499-5507 ◽

Cited By ~ 21

Author(s):

S Hanson ◽

S M Beverley ◽

W Wagner ◽

B Ullman

Keyword(s):

Gene Amplification ◽

Leishmania Donovani ◽

Molecular Mechanisms ◽

Gene Copy Number ◽

Gene Copy ◽

Unstable Gene ◽

Extrachromosomal Elements ◽

Circular Dnas ◽

Amplification Mechanism

We describe the first example of unstable gene amplification consisting of linear extrachromosomal DNAs in drug-resistant eukaryotic cells. alpha-Difluoromethylornithine (DFMO)-resistant Leishmania donovani with an amplified ornithine decarboxylase (ODC) gene copy number contained two new extrachromosomal DNAs, both present in 10 to 20 copies. One of these was a 140-kb linear DNA (ODC140-L) on which all of the amplified copies of the odc gene were located. The second was a 70-kb circular DNA (ODC70-C) containing an inverted repeat but lacking the odc gene. Both ODC140-L and ODC70-C were derived from a preexisting wild-type chromosome, probably by a conservative amplification mechanism. Both elements were unstable in the absence of DFMO, and their disappearance coincided with a decrease in ODC activity and an increase in DFMO growth sensitivity. These results suggest the possibility that ODC70-C may play a role in DFMO resistance. These data expand the diversity of known amplification mechanisms in eukaryotes to include the simultaneous unstable amplification of both linear and circular DNAs. Further characterization of these molecules will provide insights into the molecular mechanisms underlying gene amplification, including the ability of linear amplified DNAs to acquire telomeres and the determinants of chromosomal stability.

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The multicopy appearance of a large inverted duplication and the sequence at the inversion joint suggest a new model for gene amplification.

The EMBO Journal ◽

10.1002/j.1460-2075.1988.tb02828.x ◽

1988 ◽

Vol 7 (2) ◽

pp. 407-417 ◽

Cited By ~ 68

Author(s):

O. Hyrien ◽

M. Debatisse ◽

G. Buttin ◽

B. R. de Saint Vincent

Keyword(s):

Gene Amplification ◽

New Model ◽

Inverted Duplication

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Bacteriophage Lambda: a Paradigm Revisited

Journal of Virology ◽

10.1128/jvi.02177-09 ◽

2010 ◽

Vol 84 (13) ◽

pp. 6876-6879 ◽

Cited By ~ 23

Author(s):

Paul C. M. Fogg ◽

Heather E. Allison ◽

Jon R. Saunders ◽

Alan J. McCarthy

Keyword(s):

Escherichia Coli ◽

Sequence Analysis ◽

High Frequency ◽

Southern Hybridization ◽

Integration Site ◽

Bacteriophage Lambda ◽

Rare Event ◽

Low Frequency ◽

E Coli ◽

Very Low Frequency

ABSTRACT Bacteriophage lambda has an archetypal immunity system, which prevents the superinfection of its Escherichia coli lysogens. It is now known that superinfection can occur with toxigenic lambda-like phages at a high frequency, and here we demonstrate that the superinfection of a lambda lysogen can lead to the acquisition of additional lambda genomes, which was confirmed by Southern hybridization and quantitative PCR. As many as eight integration events were observed but at a very low frequency (6.4 × 10−4) and always as multiple insertions at the established primary integration site in E. coli. Sequence analysis of the complete immunity region demonstrated that these multiply infected lysogens were not immunity mutants. In conclusion, although lambda superinfection immunity can be confounded, it is a rare event.

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Excision and transposition of Tn5 upon insertion in the hha gene of Escherichia coli

Canadian Journal of Microbiology ◽

10.1139/m94-095 ◽

1994 ◽

Vol 40 (7) ◽

pp. 597-601 ◽

Cited By ~ 1

Author(s):

C. Badenas ◽

C. Madrid ◽

A. Juárez

Keyword(s):

Escherichia Coli ◽

High Frequency ◽

Chromosomal Location ◽

Single Copy

We report the occurrence of Tn5 secondary transpositions in Escherichia coli HB101 hha::Tn5(pANN202-312). Tn5-induced hha mutants (hemolysin oveiproducers) segregated at high frequency either hemolysin-negative clones or clones showing the parental hemolytic phenotype (reduced hemolysin production). Secondary transpositions of Tn5 appeared to be responsible for the phenotypes of both types of derivatives. The hemolysin-negative clones no longer harboured Tn5 in the hha gene, but rather Tn5 was in the hly genes of the hemolytic plasmid pANN202-312. The derivatives that recovered the parental hemolytic phenotype contained a single copy of Tn5 in a chromosomal location different from that of hha. Both kinds of transpositional events appeared to restore the function of the hha gene.Key words: Tn5, transposition, Escherichia coli, excision.

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MOLECULAR DOCKING STUDIES OF CURCUMA LONGA AND ALOE VERA FOR THEIR POTENTIAL ANTICANCER EFFECTS

Asian Journal of Pharmaceutical and Clinical Research ◽

10.22159/ajpcr.2018.v11i4.23995 ◽

2018 ◽

Vol 11 (4) ◽

pp. 314 ◽

Cited By ~ 3

Author(s):

Parul Tripathi ◽

Saad Sabir Siddiqui ◽

Anju Sharma ◽

Parul Johri ◽

Aditi Singh

Keyword(s):

Topoisomerase I ◽

Active Sites ◽

Rational Design ◽

Curcuma Longa ◽

Aloe Vera ◽

Initial Step ◽

Docking Study ◽

Single Copy ◽

Docking Studies ◽

Anticancer Effects

Objective: In this paper, docking study is presented to use these phytocompounds for their prospective role in various types of cancers.Methods: A group of the different set of phytocompounds (aloesin, barbaloin, curcumin, and emodin) were taken and docked into the active sites of Topoisomerase I, a 91-kDa monomer (having 765 amino acids), is encoded by a single copy gene (Top 1) located on chromosome 20q12–13.2 using Autodock4 Software. The docking studies of the selected proteins were also docked to study the anticancerous property of the selected phytocompounds.Result: These studies were based on binding energy, docking energy and other relevant scores that revealed emodin could be the potential lead molecule for the inhibition of signal potent for different types of cancer. Furthermore, the important residues for potential drug target were identified.Conclusion: This paper is an initial step toward a rational design of novel selective and potent phytocompounds inhibitors for the treatment of deadly disease cancer.

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Spontaneous germ line virus infection and retroviral insertional mutagenesis in eighteen transgenic Srev lines of mice.

Molecular and Cellular Biology ◽

10.1128/mcb.9.1.177 ◽

1989 ◽

Vol 9 (1) ◽

pp. 177-184 ◽

Cited By ~ 17

Author(s):

S E Spence ◽

D J Gilbert ◽

D A Swing ◽

N G Copeland ◽

N A Jenkins

Keyword(s):

Dna Replication ◽

High Frequency ◽

Insertional Mutagenesis ◽

Germ Line ◽

Integration Site ◽

Viral Integration ◽

Mammalian Development ◽

Copy Numbers ◽

Cell Embryo ◽

Dna Copy Numbers

SWR/J-RF/J hybrid mice spontaneously acquire new germ line ecotropic proviruses at high frequency. In the studies described here, we used these hybrids to produce 18 transgenic mouse lines, each carrying a single newly acquired Srev locus (SWR/J-RF/J ecotropic proviral locus). All of the newly acquired proviruses identified in mosaic founder SWR/J-RF/J mice that could be transmitted through the germ line were also present in somatic tissues, demonstrating that viral integration occurred before the germ line was set aside from the somatic lineages. Quantitative analysis of proviral DNA copy numbers in somatic and germinal tissues of mosaic founder parents combined with structural analysis of Srev loci indicated that these proviruses are acquired after multiple rounds of somatic viral reinfection and that most of these viral integration events occurred after DNA replication in the zygote and before DNA replication in the four-cell embryo. The frequency of provirus acquisition in Srev lines that expressed the infectious ecotropic virus was similar to that in SWR.RF mice carrying Emv-16 and Emv-17, suggesting that the chromosomal integration site of the parental locus is not an important determinant for high-frequency provirus acquisition. The frequency of recessive lethal mutations induced by spontaneous viral integration was 5%, which was similar to that induced by preimplantation embryo infection. This approach represents a simple and viable strategy for inducing and studying mutations that affect mammalian development.

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