Variable copy number DNA sequences in rice

1987 ◽  
Vol 210 (3) ◽  
pp. 373-380 ◽  
Author(s):  
Shoshi Kikuchi ◽  
Fumio Takaiwa ◽  
Kiyoharu Oono
Keyword(s):  
Dna Sequences ◽  
Copy Number

Micronuclear DNA sequences from Tetrahymena do not confer mitotic stability on ARS plasmids in Saccharomyces

Genome ◽  
1988 ◽  
Vol 30 (5) ◽  
pp. 690-696 ◽  
Author(s):  
Wendy H. Horsfall ◽  
Ronald E. Pearlman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequences ◽  
Copy Number ◽  
Tetrahymena Thermophila ◽  
Assay System ◽  
Mitotic Stability ◽  
Yeast Saccharomyces Cerevisiae ◽  
Genomic Libraries ◽  
Ade2 Gene

Genomic libraries containing micronuclear DNA sequences from Tetrahymena thermophila have been constructed in a vector containing ARS1, SUP11, and ura3 sequences from the yeast Saccharomyces cerevisiae. When transformed into a strain of S. cerevisiae carrying a suppressible ochre mutation in the ade2 gene, viable transformants are obtained only if the transforming plasmid is maintained at a copy number of one or two per cell. Mitotic segregation of the plasmid is easily assessed in a colour assay of transformants. Using this assay system, we showed that micronuclear DNA from Tetrahymena does not contain sequences that confer mitotic stability on yeast ARS-containing plasmids; i.e., sequences that function analogously to yeast centromere sequences. One transformant was analyzed that carries Tetrahymena sequences that maintain the copy number of the ARS plasmid at one or two per cell. However, these sequences do not confer mitotic stability on the transformants and they confer a phenotype in this assay similar to that of the REP3 gene of the yeast 2 μm plasmid.Key words: mitotic stability, centromere, Tetrahymena, Saccharomyces.

Distribution, expression and germ line transmission of exogenous DNA sequences following microinjection into Xenopus laevis eggs

Development ◽  
1987 ◽  
Vol 99 (1) ◽  
pp. 15-23
Author(s):  
L.D. Etkin ◽  
B. Pearman
Keyword(s):  
Xenopus Laevis ◽  
Dna Sequences ◽  
Copy Number ◽  
Germ Line ◽  
Gene Promoter ◽  
Early Gene ◽  
Mosaic Pattern ◽  
Exogenous Dna ◽  
Gene Coding ◽  
Germ Line Transmission

We analysed the fate, expression and germ line transmission of exogenous DNA which was microinjected into fertilized eggs of Xenopus laevis. DNA was injected into fertilized eggs within 1 h following fertilization. The injected DNA was dispersed around the site of injection and became localized to cleavage nuclei by stage 6. Injected DNA persisted in the tissues of 6- to 8-month-old frogs and exhibited a mosaic pattern of distribution with regard to the presence or absence and copy number between different tissues. We detected the exogenous DNA sequences in 60% of injected frogs. Restriction digestion analysis of this DNA suggested that it is not rearranged and was organized as head-to-tail multimers. The copy number varied from 2 to 30 copies/cell in various tissues of the same frog. Plasmid pSV2CAT which contains the prokaryotic gene coding for chloramphenicol acetyl transferase (CAT) enzyme linked to the SV40 early gene promoter was expressed in 50% of the animals containing the gene. The pattern of expression, however, varied between different animals and could not be correlated with copy number. We also showed that the exogenous DNA sequences were transmitted through the male germ line and that each offspring contained the gene integrated into a different region of the genome.

scholarly journals Optimization of Droplet Digital Polymerase Chain Reaction (Ddpcr) For Dna Copy Number Variation Analysis of Cnv Esv27061 in Young Adults with Higher Blood Pressure

2017 ◽  
Vol 16 (1) ◽  
Author(s):  
Siti Radziah Shaik Alaudeen ◽  
Aszrin Abdullah ◽  
Azarisman Shah Mohd Shah ◽  
Norlelawati Abdul Talib
Keyword(s):  
Blood Pressure ◽  
Young Adults ◽  
Copy Number Variation ◽  
High Blood Pressure ◽  
Dna Sequences ◽  
Copy Number ◽  
Healthy Controls ◽  
Cross Sectional ◽  
Dna Concentration ◽  
Number Variation

Introduction: Copy number variation (CNV) caused by changes in DNA sequences of 1000 or more bases is implicated with susceptibility to common diseases. A study on CNV esv27061 among hypertensive Australian adults reported association with high blood pressure (BP). In Malaysia, no study on CNV among hypertensive young adults is available. Thus, this investigation aimed to assess the CNV esv27061 of young Malaysian adults with high blood pressure using optimized ddPCR. Materials and method: Ten samples each from hypertensive and healthy controls were randomly selected from samples collected for an on-going comparative cross-sectional research project among young adults living in Kuantan. The DNAs were purified using Maxwell RSC Buffy Coat DNA Kit and the concentration was measured using SimpliNano spectrophotometer. Subsequently, restriction digestion of DNAs by EcoRV was performed prior to ddPCR. The products were later subjected to droplet generation (QX100 Droplet Generator), PCR amplification and finally CNV was read by QX100 Droplet reader. Unfortunately, the above method did not yield any result. Thus, an alternative method in which purified DNA concentration was determined by QuantiFluor ONE dsDNA System (Quantus fluorometer). The DNAs (60 ng) and Alu1 were added in master mix during ddPCR and CNV esv27061 analysis was performed as stated above. Results: Optimization of method in this study showed that the detection of CNV esv27061 was possible by the use of more sensitive measurement of DNA concentration, Alu1 restriction enzyme instead of EcoRV and digestion in ddPCR reaction method rather than prior digestion. The finalized protocol run on selected hypertensive and healthy controls has shown to be reproducible and easily interpretable discrimination of gene's copy numbers. Conclusion: This optimized protocol for CNV esv27061 analysis proved useful in identifying CNV and will allow a reproducible assay evaluation and the application of this method to a bigger sample size.

BK virus-transformed inbred hamster brain cells: status of viral DNA in subclones

1982 ◽  
Vol 2 (7) ◽  
pp. 837-844
Author(s):  
M M Pater ◽  
A Pater ◽  
G di Mayorca ◽  
E Beth ◽  
G Giraldo
Keyword(s):  
Cell Line ◽  
Dna Sequences ◽  
Copy Number ◽  
Tumor Antigens ◽  
Blot Hybridization ◽  
Virus Production ◽  
Bk Virus ◽  
Free Form ◽  
Brain Cells ◽  
Viral Dna

We have recently reported that viral DNA sequences in inbred LSH hamster brain cells transformed by the GS variant of BK virus (LSH-BR-BK) are present predominantly in a free form (Beth et al., J. Virol. 40:276-284, 1981). In this report, we confirm that the presence of viral DNA sequences in these cells is not due to virus production, since viral capsid proteins were not detected by immunoprecipitation. Furthermore, we examined the status of viral DNA in 15 subclones of this cell line and detected free and integrated viral DNA sequences in only 5 of the subclones. The other 10 subclones contained exclusively integrated viral DNA sequences, as shown by the blot hybridization of high-molecular-weight cell DNA which was uncleaved or digested with HincII, for which there are no sites in viral DNA. The arrangement of viral DNA in these clones was further analyzed by cleavage of cellular DNA with HpaII and HindIII. Mitomycin (0.03 microgram/ml) treatment of subclones containing only integrated sequences resulted in the appearance of free viral DNA sequences in some of these cells. This result supports the postulation that free viral DNA in LSH-BR-BK cells is made up of excision products of observed tandemly repeated integrated sequences. In addition to the large T- and small t-antigens, LSH-BR-BK and all of its 15 subclones contained two antigen species which were larger than large T and one species which was smaller than small t. The number of tumor antigens in the LSH- BR-BK cell line and its subclones with a large copy number in a free form was not more than in the subclones with low copy number and integrated DNA. This suggests that free viral DNA is not a template for tumor antigen production in transformed cells.

scholarly journals Introduction of extra telomeric DNA sequences into Saccharomyces cerevisiae results in telomere elongation.

1989 ◽  
Vol 9 (4) ◽  
pp. 1488-1497 ◽  
Author(s):  
K W Runge ◽  
V A Zakian
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequences ◽  
Copy Number ◽  
Plasmid Copy Number ◽  
High Copy Number ◽  
Limiting Factor ◽  
Telomeric Dna ◽  
Base Pairs ◽  
Plasmid Copy ◽  
Telomere Elongation

The termini of Saccharomyces cerevisiae chromosomes consist of tracts of C1-3A (one to three cytosine and one adenine residue) sequences of approximately 450 base pairs in length. To gain insights into trans-acting factors at telomeres, high-copy-number linear and circular plasmids containing tracts of C1-3A sequences were introduced into S. cerevisiae. We devised a novel system to distinguish by color colonies that maintained the vector at 1 to 5, 20 to 50, and 100 to 400 copies per cell and used it to change the amount of telomeric DNA sequences per cell. An increase in the number of C1-3A sequences caused an increase in the length of telomeric C1-3A repeats that was proportional to plasmid copy number. Our data suggest that telomere growth is inhibited by a limiting factor(s) that specifically recognizes C1-3A sequences and that this factor can be effectively competed for by long tracts of C1-3A sequences at telomeres or on circular plasmids. Telomeres without this factor are exposed to processes that serve to lengthen chromosome ends.

PstI repeat: a family of short interspersed nucleotide element (SINE)-like sequences in the genomes of cattle, goat, and buffalo

Genome ◽  
2002 ◽  
Vol 45 (1) ◽  
pp. 44-50 ◽  
Author(s):  
Faruk G Sheikh ◽  
Sudit S Mukhopadhyay ◽  
Prabhakar Gupta
Keyword(s):  
Nucleotide Sequence ◽  
Dna Sequences ◽  
Sequence Homology ◽  
Copy Number ◽  
Southern Hybridization ◽  
Repetitive Dna Sequences ◽  
Type I ◽  
Type Ii ◽  
Repeat Type ◽  
Highly Repetitive Dna

The PstI family of elements are short, highly repetitive DNA sequences interspersed throughout the genome of the Bovidae. We have cloned and sequenced some members of the PstI family from cattle, goat, and buffalo. These elements are approximately 500 bp, have a copy number of 2 × 105 – 4 × 105, and comprise about 4% of the haploid genome. Studies of nucleotide sequence homology indicate that the buffalo and goat PstI repeats (type II) are similar types of short interspersed nucleotide element (SINE) sequences, but the cattle PstI repeat (type I) is considerably more divergent. Additionally, the goat PstI sequence showed significant sequence homology with bovine serine tRNA, and is therefore likely derived from serine tRNA. Interestingly, Southern hybridization suggests that both types of SINEs (I and II) are present in all the species of Bovidae. Dendrogram analysis indicates that cattle PstI SINE is similar to bovine Alu-like SINEs. Goat and buffalo SINEs formed a separate cluster, suggesting that these two types of SINEs evolved separately in the genome of the Bovidae.Key words: repeat, SINE, Bovidae, 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.

Evolution of low-copy number and major satellite DNA sequences coexisting in two Pimelia species-groups (Coleoptera)

Gene ◽  
2003 ◽  
Vol 312 ◽  
pp. 85-94 ◽  
Author(s):  
Branka Bruvo ◽  
Joan Pons ◽  
Đurđica Ugarković ◽  
Carlos Juan ◽  
Eduard Petitpierre ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Satellite Dna ◽  
Copy Number ◽  
Major Satellite ◽  
Low Copy Number ◽  
Species Groups

A minimal c-fes cassette directs myeloid-specific expression in transgenic mice

Blood ◽  
2000 ◽  
Vol 96 (9) ◽  
pp. 3040-3048
Author(s):  
Ahlke Heydemann ◽  
Soren Warming ◽  
Cynthia Clendenin ◽  
Kirsten Sigrist ◽  
J. Peter Hjorth ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Dna Sequences ◽  
Copy Number ◽  
Messenger Rna ◽  
Transgene Expression ◽  
Fluorescent Protein ◽  
Control Function ◽  
Integration Site ◽  
Myeloid Cells ◽  
Independent Copy

The c-fes proto-oncogene encodes a 92-kd protein tyrosine kinase whose expression is restricted largely to myeloid and endothelial cells in adult mammals. A 13.2-kilobase (kb) humanc-fes genomic fragment was previously shown to containcis-acting element(s) sufficient for a locus control function in bone marrow macrophages. Locus control regions (LCRs) confer transgene expression in mice that is integration site independent, copy number dependent, and similar to endogenous murine messenger RNA levels. To identify sequences required for this LCR,c-fes transgenes were analyzed in mice. Myeloid-cell–specific, deoxyribonuclease-I–hypersensitive sites localized to the 3′ boundary of exon 1 and intron 3 are required to confer high-level transgene expression comparable to endogenous c-fes, independent of integration site. We define a minimal LCR element as DNA sequences (nucleotides +28 to +2523 relative to the transcription start site) located within intron 1 to intron 3 of the human locus. When this 2.5-kb DNA fragment was linked to a c-fes complementary DNA regulated by its own 446–base-pair promoter, integration-site–independent, copy-number–dependent transcription was observed in myeloid cells in transgenic mice. Furthermore, this 2.5-kb cassette directed expression of a heterologous gene (enhanced green fluorescent protein) exclusively in myeloid cells. The c-fes regulatory unit represents a novel reagent for targeting gene expression to macrophages and neutrophils in transgenic mice.

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