scholarly journals Endogenous RNA interference is driven by copy number

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Cristina Cruz ◽  
Jonathan Houseley
Keyword(s):  
Rna Interference ◽  
Transposable Elements ◽  
Copy Number ◽  
Defense Mechanism ◽  
Gene Copy Number ◽  
Single Copy ◽  
Gene Copy ◽  
Protein Coding ◽  
Endogenous Loci ◽  
Pervasive Transcription

A plethora of non-protein coding RNAs are produced throughout eukaryotic genomes, many of which are transcribed antisense to protein-coding genes and could potentially instigate RNA interference (RNAi) responses. Here we have used a synthetic RNAi system to show that gene copy number is a key factor controlling RNAi for transcripts from endogenous loci, since transcripts from multi-copy loci form double stranded RNA more efficiently than transcripts from equivalently expressed single-copy loci. Selectivity towards transcripts from high-copy DNA is therefore an emergent property of a minimal RNAi system. The ability of RNAi to selectively degrade transcripts from high-copy loci would allow suppression of newly emerging transposable elements, but such a surveillance system requires transcription. We show that low-level genome-wide pervasive transcription is sufficient to instigate RNAi, and propose that pervasive transcription is part of a defense mechanism capable of directing a sequence-independent RNAi response against transposable elements amplifying within the genome.

scholarly journals 437 DETERMINING GENE COPY NUMBER IN TRANSFECTED CAPRINE FIBROBLAST CELLS

2010 ◽  
Vol 22 (1) ◽  
pp. 375
Author(s):  
J. A. Wilson ◽  
R. A. Godke ◽  
K. R. Bondioli
Keyword(s):  
Dna Sequences ◽  
Copy Number ◽  
Genomic Dna ◽  
Gene Copy Number ◽  
Single Copy ◽  
Gene Copy ◽  
Transgene Copy Number ◽  
Transgenic Organisms ◽  
Plasmid Construct ◽  
The Mean

Transgene expression in stably transgenic organisms is affected by many factors, including the copy number of the transgene in the genome and by interactions between the transgene and flanking DNA sequences. Very high transgene copy number has also been shown to affect genetic stability in transgenic plants and animals. Two commonly used methods for transfecting cells prior to their use in nuclear transfer (NT) are liposome-mediated transfection and electroporation. Little is known about the transgene copy number or variability of the copy number with these techniques. The objective of this study was to determine transgene copy number after liposome-mediated transfection and electroporation. The mean transgene copy number and variability between individual integration events have been determined. Q-PCR conditions were optimized for primer annealing temperature and concentration when amplifying a region of a plasmid expressing green fluorescent protein (GFP) under the control of the human elongation factor (hEF) promoter (hEFGFP) used for transfection. The quantitative nature of the Q-PCR reaction was confirmed by amplifying 10-fold dilutions of the plasmid and plotting the threshold cycle (CT) value against the log of the plasmid concentration. A correlation coefficient of 1.00 and a calculated PCR efficiency of 93.3% were obtained from this analysis. Caprine fibroblasts were transfected by electroporation with 20 μg of DNA or FuGENE® HD (Roche, Nutley, NJ, USA) reagent with 6 μg of DNA using either a circular or linearized hEFGFP plasmid. Transformed cells were plated at low density in medium containing Geneticin® (Gibco, Grand Island, NY USA). After 10 days of culture, single-cell colonies were isolated and expanded. When cultures reached 1 to 2 million cells, genomic DNA was isolated. Transgene copy number was determined by amplifying genomic DNA from individual clones representing 1 × 105 cells with Q-PCR. Transgene copy number was calculated from a standard curve of the transgene plasmid. The mean transgene copy number for electroporation circular was 2.7 ± 0.75 (n = 32 colonies) and 1.3 ± 0.65 (n = 19) when using a linear DNA construct. FuGENE HD using a circular plasmid construct generated a mean gene copy number of 0.5 ± 0.11 (n = 14) and 0.64 ± 0.13 (n = 16) for the linear plasmid construct. One-way ANOVA followed by multiple pair-wise comparisons using Tukey’s method showed significant differences when comparing electroporation circular to all other treatments. However, there were no differences when comparing electroporation linear, FuGENE HD circular, and FuGENE HD linear to each other. Because the calculated mean copy number for transfection with FuGENE HD was consistently less than 1, it is assumed that these colonies consisted predominantly of single-copy integrations. Our results indicate that the transfection method can affect gene copy number. Electroporation resulted in multiple but few copies whereas Fugene HD resulted in predominantly single-copy integrations. The probability of transgene mutation with single-copy integration suggests that electroporation is preferable forproducing transgenic animals by NT.

scholarly journals Transcription-induced formation of extrachromosomal DNA during yeast ageing

2019 ◽  
Author(s):  
Ryan Hull ◽  
Michelle King ◽  
Grazia Pizza ◽  
Felix Krueger ◽  
Xabier Vergara ◽  
...  
Keyword(s):  
Copy Number ◽  
De Novo ◽  
Gene Copy Number ◽  
Phenotypic Selection ◽  
Copper Resistance ◽  
Gene Copy ◽  
Dna Double Strand Breaks ◽  
Protein Coding ◽  
Gene Copy Number Variation ◽  
Number Variation

Extrachromosomal circular DNA (eccDNA) facilitates adaptive evolution by allowing rapid and extensive gene copy number variation, and is implicated in the pathology of cancer and ageing. Here, we demonstrate that yeast aged under environmental copper accumulate high levels of eccDNA containing the copper resistance gene CUP1. Transcription of CUP1 causes CUP1 eccDNA accumulation, which occurs in the absence of phenotypic selection. We have developed a sensitive and quantitative eccDNA sequencing pipeline that reveals CUP1 eccDNA accumulation on copper exposure to be exquisitely site specific, with no other detectable changes across the eccDNA complement. eccDNA forms de novo from the CUP1 locus through processing of DNA double-strand breaks (DSBs) by Sae2 / Mre11 and Mus81, and genome-wide analyses show that other protein coding eccDNA species in aged yeast share a similar biogenesis pathway. Although abundant we find that CUP1 eccDNA does not replicate efficiently, and high copy numbers in aged cells arise through frequent formation events combined with asymmetric DNA segregation. The transcriptional stimulation of CUP1 eccDNA formation shows that age-linked genetic change varies with transcription pattern, resulting in gene copy number profiles tailored by environment.

scholarly journals Messenger RNAs encoding the beta subunits of guinea pig (Cavia porcellus) luteinizing hormone (gpLH) and putative chorionic gonadotropin (gpCG) are transcribed from a single-copy gpLH/CGbeta gene

2001 ◽  
Vol 26 (3) ◽  
pp. 267-280 ◽  
Author(s):  
GB Sherman ◽  
DF Heilman ◽  
AJ Hoss ◽  
D Bunick ◽  
LA Lund
Keyword(s):  
Guinea Pig ◽  
Chorionic Gonadotropin ◽  
Copy Number ◽  
Genomic Dna ◽  
Gene Locus ◽  
Gene Copy Number ◽  
Single Copy ◽  
Gene Copy ◽  
Beta Subunits ◽  
Coding Sequence

Neither gene locus nor gene sequence characterizations have been reported for the beta subunits of guinea pig (gp) LH and putative gp chorionic gonadotropin (CG). Descriptions of this locus would allow comparison with functionally relevant molecular genetic features of other species' homologous loci including the single-copy equid LH/CGbeta gene and the primate LHbeta-CGbeta gene cluster locus. Contiguous cDNA and genomic DNA fragments spanning the entire mature coding sequence of gpLHbeta mRNA, gpCGbeta mRNA and a homologous gpLH/CGbeta gene were amplified using PCR methodologies. With the exception of one silent mutation, the two cDNA and the genomic sequences were identical where they overlapped. Comparison of guinea pig coding sequence with LHbeta, CGbeta and LH/CGbeta sequences of other vertebrate species revealed the following order of similarity expressed as per cent coding sequence identity: rhinoceros LHbeta (83.6%)>pig LHbeta (81.8%)>donkey LH/CGbeta=bovine LHbeta (81.5%)> horse LH/CGbeta (80.6%)>dog LHbeta (79.7%)>human LHbeta (78.2%)>rat LHbeta (77.9%)>human CGbeta (75.8%)>turkey LHbeta (52.7%); values that are generally consistent with recently postulated phylogenetic relationships. Like the consensus mammalian LHbeta gene, the 5'-flanking region of the gpLH/CGbeta gene contains a single TATA sequence 37 bp upstream of the translation start codon. The first in-frame stop codon occurred at codon position +122 which is consistent with the 121 amino acid residue length of the consensus mammalian mature LHbeta peptide. To estimate gene copy number, full-length gpLHbeta cDNA was radiolabeled and hybridized to Southern blots of guinea pig genomic DNA digested with a panel of six restriction endonucleases. The resulting simple hybridization pattern strongly suggested that there is a single-copy gpLH/CGbeta gene. Northern analysis of total pituitary RNA using the same probe indicated that gpLHbeta transcript size is indistinguishable from that of consensus mammalian pituitary LHbeta mRNAs ( approximately 750 nucleotides). Despite amplifying gpCGbeta from placental RNA, positive signal was not detected in Northern blot lanes containing guinea pig total RNA prepared from placentae collected at three gestational ages (17.3 days, 24.3 days and 68 days (term)). Other data suggest that inability to detect Northern blot signal could have been due to low relative tissue concentrations of gpCGbeta transcript and/or sampling at gestational time-points that missed peak periods of mRNA expression. We conclude that, with respect to gene copy number, coding sequence and pituitary mRNA size, the gpLH/CGbeta gene locus reflects the CTP-less consensus mammalian LHbeta condition. However, based on the capacity of this single-copy gene to express in both pituitary and placental tissues, gpLH/CGbeta also exhibits functional similarities with the single-copy equine LH/CGbeta locus.

scholarly journals Digital PCR (dPCR) and qPCR Mediated Determination of Transgene Copy Number in the Forage Legume White Clover (Trifolium Repens).

2021 ◽  
Author(s):  
Rafael Narancio ◽  
Ulrik John ◽  
John Mason ◽  
Paula Giraldo ◽  
German Spangenberg
Keyword(s):  
White Clover ◽  
Reference Genes ◽  
Copy Number ◽  
Gene Copy Number ◽  
Single Copy ◽  
Digital Pcr ◽  
Gene Copy ◽  
Relative Quantification ◽  
Transgene Copy Number ◽  
Copy Number Estimation

Abstract Obtaining data on transgene copy number is an integral step in the generation of transgenic plants. Techniques such as Southern blot, segregation analysis, and quantitative PCR (qPCR) have routinely been used for this task, in a range of species. More recently, use of Digital PCR (dPCR) has become prevalent, with a measurement accuracy higher than qPCR reported. Here, the relative merits of qPCR and dPCR for transgene copy number estimation in white clover were investigated. Furthermore, given that single copy reference genes are desirable for estimating gene copy number by relative quantification, and that no single-copy genes have been reported in this species, a search and evaluation of suitable reference genes in white clover was undertaken. Results demonstrated a higher accuracy of dPCR relative to qPCR for copy number estimation in white clover. Two genes, Pyruvate dehydrogenase (PDH), and an ATP-dependent protease, identified as single-copy genes, were used as references for copy number estimation by relative quantification. Identification of single-copy genes in white clover will enable the application of relative quantification for copy number estimation of other genes or transgenes in the species. The results generated here validate the use of dPCR as a reliable strategy for transgene copy number estimation in white clover, and provide resources for future copy number studies in this species.

scholarly journals Why haploinsufficiency persists

2019 ◽  
pp. 201900437 ◽  
Author(s):  
Summer A. Morrill ◽  
Angelika Amon
Keyword(s):  
Gene Expression ◽  
Copy Number ◽  
Gene Copy Number ◽  
Single Copy ◽  
Gene Copy ◽  
Data Set ◽  
Cellular Processes ◽  
Sensitivity Data ◽  
Cell To Cell Variability ◽  
Decrease Gene Expression

Haploinsufficiency describes the decrease in organismal fitness observed when a single copy of a gene is deleted in diploids. We investigated the origin of haploinsufficiency by creating a comprehensive dosage sensitivity data set for genes under their native promoters. We demonstrate that the expression of haploinsufficient genes is limited by the toxicity of their overexpression. We further show that the fitness penalty associated with excess gene copy number is not the only determinant of haploinsufficiency. Haploinsufficient genes represent a unique subset of genes sensitive to copy number increases, as they are also limiting for important cellular processes when present in one copy instead of two. The selective pressure to decrease gene expression due to the toxicity of overexpression, combined with the pressure to increase expression due to their fitness-limiting nature, has made haploinsufficient genes extremely sensitive to changes in gene expression. As a consequence, haploinsufficient genes are dosage stabilized, showing much more narrow ranges in cell-to-cell variability of expression compared with other genes in the genome. We propose a dosage-stabilizing hypothesis of haploinsufficiency to explain its persistence over evolutionary time.

scholarly journals Associations of <i>ORMDL1</i> gene copy number variations with growth traits in four Chinese sheep breeds

2019 ◽  
Vol 62 (2) ◽  
pp. 571-578 ◽  
Author(s):  
Xiaogang Wang ◽  
Xiukai Cao ◽  
Yifan Wen ◽  
Yilei Ma ◽  
Ibrahim Elsaeid Elnour ◽  
...  
Keyword(s):  
Copy Number ◽  
Genomic Sequence ◽  
Growth Traits ◽  
Phenotypic Diversity ◽  
Body Height ◽  
Gene Copy Number ◽  
Copy Number Variations ◽  
Gene Copy ◽  
Protein Coding ◽  
Sheep Breeds

Abstract. Copy number variations (CNVs) are gains and losses of genomic sequence of more than 50 bp between two individuals of a species. Also, CNV is considered to be one of the main elements affecting the phenotypic diversity and evolutionary adaptation of animals. ORMDL sphingolipid biosynthesis regulator 1 (ORMDL1) is a protein-coding gene associated with diseases and development. In our study, the polymorphism of ORMDL1 gene copy numbers in four Chinese sheep breeds (abbreviated CK, HU, STH, and LTH) was detected. In addition, we analyzed the transcriptional expression level of ORMDL1 gene in different tissues of sheep and examined the association of ORMDL1 CNV with growth traits. The statistical analysis revealed that ORMDL1 CNV was remarkably correlated with body height, heart girth, and circumference of cannon bone in HU sheep (P<0.05), and there are significant effects on body weight, body height, body length, chest depth, and height of hip cross in STH sheep (P<0.05). In conclusion, our results provide a basis for the relationship between CNV of ORMDL1 gene and sheep growth traits, suggesting that ORMDL1 CNV may be considered a promising marker for the molecular breeding of Chinese sheep.

scholarly journals Estimating Copy-Number Proportions: The Comeback of Sanger Sequencing

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 283
Author(s):  
Eyal Seroussi
Keyword(s):  
Copy Number ◽  
Sanger Sequencing ◽  
Cytosine Methylation ◽  
Direct Sequencing ◽  
Information Source ◽  
Gene Copy Number ◽  
Cost Effective ◽  
Gene Copy ◽  
Base Editing ◽  
Recent Developments

Determination of the relative copy numbers of mixed molecular species in nucleic acid samples is often the objective of biological experiments, including Single-Nucleotide Polymorphism (SNP), indel and gene copy-number characterization, and quantification of CRISPR-Cas9 base editing, cytosine methylation, and RNA editing. Standard dye-terminator chromatograms are a widely accessible, cost-effective information source from which copy-number proportions can be inferred. However, the rate of incorporation of dye terminators is dependent on the dye type, the adjacent sequence string, and the secondary structure of the sequenced strand. These variable rates complicate inferences and have driven scientists to resort to complex and costly quantification methods. Because these complex methods introduce their own biases, researchers are rethinking whether rectifying distortions in sequencing trace files and using direct sequencing for quantification will enable comparable accurate assessment. Indeed, recent developments in software tools (e.g., TIDE, ICE, EditR, BEEP and BEAT) indicate that quantification based on direct Sanger sequencing is gaining in scientific acceptance. This commentary reviews the common obstacles in quantification and the latest insights and developments relevant to estimating copy-number proportions based on direct Sanger sequencing, concluding that bidirectional sequencing and sophisticated base calling are the keys to identifying and avoiding sequence distortions.

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