The Influence of Copy-Number of Targeted Extrachromosomal Genetic Elements on the Outcome of CRISPR-Cas Defense

In agriculture, various chemicals are used to control the weeds. Out of which, glyphosate is an important herbicide invariably used in the cultivation of glyphosate-resistant crops to control weeds. Overuse of glyphosate results in the evolution of glyphosate-resistant weeds. Evolution of glyphosate resistance (GR) in Amaranthus palmeri (AP) is a serious concern in the USA. Investigation of the mechanism of GR in AP identified different resistance mechanisms of which 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene amplification is predominant. Molecular analysis of GR AP identified the presence of a 5- to >160-fold increase in copies of the EPSPS gene than in a glyphosate-susceptible (GS) population. This increased copy number of the EPSPS gene increased the genome size ranging from 3.5 to 11.8%, depending on the copy number compared to the genome size of GS AP. FISH analysis using a 399-kb EPSPS cassette derived from bacterial artificial chromosomes (BACs) as probes identified that amplified EPSPS copies in GR AP exist in extrachromosomal circular DNA (eccDNA) in addition to the native copy in the chromosome. The EPSPS gene-containing eccDNA having a size of ∼400 kb is termed EPSPS-eccDNA and showed somatic mosacism in size and copy number. EPSPS-eccDNA has a genetic mechanism to tether randomly to mitotic or meiotic chromosomes during cell division or gamete formation and is inherited to daughter cells or progeny generating copy number variation. These eccDNAs are stable genetic elements that can replicate and exist independently. The genomic characterization of the EPSPS locus, along with the flanking regions, identified the presence of a complex array of repeats and mobile genetic elements. The cytogenomics approach in understanding the biology of EPSPS-eccDNA sheds light on various characteristics of EPSPS-eccDNA that favor GR in AP.

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The copy-number of plasmids and other genetic elements can be determined by SYBR-Green-based quantitative real-time PCR

Journal of Microbiological Methods ◽

10.1016/j.mimet.2005.09.007 ◽

2006 ◽

Vol 65 (3) ◽

pp. 476-487 ◽

Cited By ~ 44

Author(s):

Miguel A. Providenti ◽

Jason M. O'Brien ◽

Robyn J. Ewing ◽

E. Suzanne Paterson ◽

Myron L. Smith

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Copy Number ◽

Sybr Green ◽

Quantitative Real Time Pcr ◽

Genetic Elements

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Transposition of the I element and copia in a natural population of Drosophila melanogaster

Genetics Research ◽

10.1017/s0016672300026914 ◽

1987 ◽

Vol 49 (2) ◽

pp. 121-128 ◽

Cited By ~ 36

Author(s):

Andrew J. Leigh Brown ◽

Julie E. Moss

Keyword(s):

Drosophila Melanogaster ◽

Natural Population ◽

Copy Number ◽

Evolutionary Dynamics ◽

Data Sets ◽

X Chromosomes ◽

Genetic Elements ◽

Population Structuring ◽

Transposable Genetic Elements

SummaryIn order to increase our understanding of the evolutionary dynamics of transposable genetic elements we have studied the chromosal location of copies of 2 element families in 20 X chromosomes extracted from a natural population of Drosophila melanogaster from Spain. The I element was localized at a total of 64 chromosomal sites and copia at 45 sites in this sample with a mean copy number of 3·2 and 2·3 elements/chromosome respectively. Both elements were highly variable in location, with no site reaching a higher frequency than 4/20 in either case. Comparisons with other data sets suggest that insertion frequencies can be used to detect population structuring.

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Transgenerational dynamics of rDNA copy number in Drosophila male germline stem cells

10.1101/199679 ◽

2017 ◽

Author(s):

Kevin L. Lu ◽

Jonathan O. Nelson ◽

Natalie Warsinger-Pepe ◽

Yukiko M. Yamashita

Keyword(s):

Copy Number ◽

Replicative Senescence ◽

Yeast Cells ◽

Rrna Genes ◽

Germline Stem Cell ◽

Genetic Elements ◽

Male Germline ◽

Rdna Loci ◽

Rdna Copy ◽

Rdna Copy Number

AbstractrDNA loci, composed of hundreds of tandemly duplicated arrays of rRNA genes, are known to be among the most unstable genetic elements due to their repetitive nature. rDNA instability underlies aging (replicative senescence) in yeast cells, however, its contribution to the aging of multicellular organisms is poorly understood. In this study, we investigate the dynamics of rDNA loci during aging in the Drosophila male germline stem cell (GSC) lineage, and show that rDNA copy number decreases during aging. Our study further reveals that this age-dependent decrease in rDNA copy number is heritable from generation to generation, yet GSCs in animals that inherit reduced rDNA copy number are capable of recovering normal rDNA copy number. Based on these findings, we propose that rDNA loci are dynamic genetic elements, where rDNA copy number changes dynamically yet is maintained through a recovery mechanism in the germline.

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Transgenerational dynamics of rDNA copy number in Drosophila male germline stem cells

eLife ◽

10.7554/elife.32421 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 20

Author(s):

Kevin L Lu ◽

Jonathan O Nelson ◽

George J Watase ◽

Natalie Warsinger-Pepe ◽

Yukiko M Yamashita

Keyword(s):

Copy Number ◽

Replicative Senescence ◽

Yeast Cells ◽

Rrna Genes ◽

Germline Stem Cell ◽

Genetic Elements ◽

Male Germline ◽

Rdna Loci ◽

Rdna Copy ◽

Rdna Copy Number

rDNA loci, composed of hundreds of tandemly duplicated arrays of rRNA genes, are known to be among the most unstable genetic elements due to their repetitive nature. rDNA instability underlies aging (replicative senescence) in yeast cells, however, its contribution to the aging of multicellular organisms is poorly understood. In this study, we investigate the dynamics of rDNA loci during aging in the Drosophila male germline stem cell (GSC) lineage, and show that rDNA copy number decreases during aging. Our study further reveals that this age-dependent decrease in rDNA copy number is heritable from generation to generation, yet GSCs in young animals that inherited reduced rDNA copy number are capable of recovering normal rDNA copy number. Based on these findings, we propose that rDNA loci are dynamic genetic elements, where rDNA copy number changes dynamically yet is maintained through a recovery mechanism in the germline.

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Identification, Characterization, and Application of the Replicon Region of the Halophilic Temperate Sphaerolipovirus SNJ1

Journal of Bacteriology ◽

10.1128/jb.00131-16 ◽

2016 ◽

Vol 198 (14) ◽

pp. 1952-1964 ◽

Cited By ~ 10

Author(s):

Yuchen Wang ◽

Linshan Sima ◽

Jie Lv ◽

Suiyuan Huang ◽

Ying Liu ◽

...

Keyword(s):

Copy Number ◽

Life Cycles ◽

Replication Initiation ◽

Lytic Cycle ◽

Negative Regulator ◽

Content Type ◽

Genetic Tools ◽

Shuttle Vectors ◽

Genetic Elements ◽

Protein Expression And Purification

ABSTRACTThe temperate haloarchaeal virus SNJ1 displays lytic and lysogenic life cycles. During the lysogenic cycle, the virus resides in its host,Natrinemasp. strain J7-1, in the form of an extrachromosomal circular plasmid, pHH205. In this study, a 3.9-kb region containing seven predicted genes organized in two operons was identified as the minimal replicon of SNJ1. Only RepA, encoded by open reading frame 11-12 (ORF11-12), was found to be essential for replication, and its expression increased during the lytic cycle. Sequence analysis suggested that RepA is a distant homolog of HUH endonucleases, a superfamily that includes rolling-circle replication initiation proteins from various viruses and plasmids. In addition to RepA, two genetic elements located within both termini of the 3.9-kb replicon were also required for SNJ1 replication. SNJ1 genome and SNJ1 replicon-based shuttle vectors were present at 1 to 3 copies per chromosome. However, the deletion of ORF4 significantly increased the SNJ1 copy number, suggesting that the product of ORF4 is a negative regulator of SNJ1 abundance. Shuttle vectors based on the SNJ1 replicon were constructed and validated for stable expression of heterologous proteins, both in J7 derivatives and inNatrinemapallidumJCM 8980T, suggesting their broad applicability as genetic tools forNatrinemaspecies.IMPORTANCEArchaeal viruses exhibit striking morphological diversity and unique gene content. In this study, the minimal replicon of the temperate haloarchaeal virus SNJ1 was identified. A number of ORFs and genetic elements controlling virus genome replication, maintenance, and copy number were characterized. In addition, based on the replicon, a novel expression shuttle vector has been constructed and validated for protein expression and purification inNatrinemasp. CJ7 andNatrinema pallidumJCM 8980T. This study not only provided mechanistic and functional insights into SNJ1 replication but also led to the development of useful genetic tools to investigate SNJ1 and other viruses infectingNatrinemaspecies as well as their hosts.

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Different classes of retrotransposons in coniferous spruce species

Genome ◽

10.1139/g00-077 ◽

2000 ◽

Vol 43 (6) ◽

pp. 1084-1089 ◽

Cited By ~ 10

Author(s):

Yvan L'Homme ◽

Armand Séguin ◽

Francine M Tremblay

Keyword(s):

Reverse Transcriptase ◽

Picea Glauca ◽

Copy Number ◽

Direct Repeat ◽

Mobile Genetic Elements ◽

High Copy Number ◽

Ltr Retrotransposons ◽

Genetic Elements ◽

Black And White ◽

Gypsy Group

We have used the conservation of reverse transcriptase and integrase domains among retroelements to PCR-amplify three well-known types of these mobile genetic elements. Reverse transcriptase sequences from Ty1-copia were identified in spruce in this way, as well as integrase sequences from the Ty3-gypsy group. Using these sequences as probes against a Picea glauca genomic bank, individual members from the LTR (long terminal direct repeat) groups were obtained. A partial Ty1-copia-type element named Spcl was isolated along with a Ty3-gypsy-type element named Spdl. Genomic Southern hybridizations revealed the complexity and high copy number of LTR retrotransposons in black and white spruce.Key words: copia, gypsy, Picea, PCR.

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Homeostatic responses regulate selfish mitochondrial genome dynamics in C. elegans

10.1101/050930 ◽

2016 ◽

Cited By ~ 3

Author(s):

Bryan L. Gitschlag ◽

Cait S. Kirby ◽

David C. Samuels ◽

Rama D. Gangula ◽

Simon A. Mallal ◽

...

Keyword(s):

Mitochondrial Genome ◽

Copy Number ◽

Digital Pcr ◽

Mtdna Copy Number ◽

Genetic Elements ◽

C Elegans ◽

Selfish Genetic Elements ◽

Copy Number Control ◽

Genome Dynamics ◽

Bona Fide

SummarySelfish genetic elements have profound biological and evolutionary consequences. Mutant mitochondrial genomes (mtDNA) can be viewed as selfish genetic elements that persist in a state of heteroplasmy despite having potentially deleterious consequences to the organism. We sought to investigate mechanisms that allow selfish mtDNA to achieve and sustain high levels. Here, we establish a large 3.1kb deletion bearing mtDNA variant uaDf5 as a bona fide selfish genome in the nematode Caenorhabditis elegans. Next, using droplet digital PCR to quantify mtDNA copy number, we show that uaDf5 mutant mtDNA replicates in addition to, not at the expense of, wildtype mtDNA. These data suggest existence of homeostatic copy number control for wildtype mtDNA that is exploited by uaDf5 to ‘hitchhike’ to high frequency. We also observe activation of the mitochondrial unfolded protein response (UPRmt) in animals with uaDf5. Loss of UPRmt results in a decrease in uaDf5 frequency whereas constitutive activation of UPRmt increases uaDf5 levels. These data suggest that UPRmt allows uaDf5 levels to increase. Interestingly, the decreased uaDf5 levels in absence of UPRmt recover in parkin mutants lacking mitophagy, suggesting that UPRmt protects uaDf5 from mitophagy. We propose that cells activate two homeostatic responses, mtDNA copy number control and UPRmt, in uaDf5 heteroplasmic animals. Inadvertently, these homeostatic responses allow uaDf5 levels to be higher than they would be otherwise. In conclusion, our data suggest that homeostatic stress response mechanisms play an important role in regulating selfish mitochondrial genome dynamics.

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