Статті в журналах: "Gene copy stability"

1

Saito, I., R. Groves, E. Giulotto, M. Rolfe, and G. R. Stark. "Evolution and stability of chromosomal DNA coamplified with the CAD gene." Molecular and Cellular Biology 9, no. 6 (June 1989): 2445–52. http://dx.doi.org/10.1128/mcb.9.6.2445-2452.1989.

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We have compared clones of Syrian hamster cells selected for the first amplification of the CAD gene with clones selected for further amplification. The large domain amplified initially was not reamplified as an intact unit. Instead, subregions were reamplified preferentially, and parts of the initial array were often lost. These events reduced the average amount of coamplified DNA accompanying each copy of the selected gene. The degree of amplification was small in the first step (about three extra copies of CAD per cell), but second-step amplifications to a high copy number (up to 60 extra copies per cell) occurred frequently. After several separate steps of amplification, highly condensed arrays that brought many CAD genes close together were formed. In striking contrast to the stability of these highly amplified arrays, the low-copy chromosomal arrays formed early were quite unstable and were often lost completely within 1 or 2 months of growth without selection. The results suggest that different mechanisms may be involved in the first step of amplification and in the later evolution of an already amplified array.

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2

Saito, I., R. Groves, E. Giulotto, M. Rolfe, and G. R. Stark. "Evolution and stability of chromosomal DNA coamplified with the CAD gene." Molecular and Cellular Biology 9, no. 6 (June 1989): 2445–52. http://dx.doi.org/10.1128/mcb.9.6.2445.

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We have compared clones of Syrian hamster cells selected for the first amplification of the CAD gene with clones selected for further amplification. The large domain amplified initially was not reamplified as an intact unit. Instead, subregions were reamplified preferentially, and parts of the initial array were often lost. These events reduced the average amount of coamplified DNA accompanying each copy of the selected gene. The degree of amplification was small in the first step (about three extra copies of CAD per cell), but second-step amplifications to a high copy number (up to 60 extra copies per cell) occurred frequently. After several separate steps of amplification, highly condensed arrays that brought many CAD genes close together were formed. In striking contrast to the stability of these highly amplified arrays, the low-copy chromosomal arrays formed early were quite unstable and were often lost completely within 1 or 2 months of growth without selection. The results suggest that different mechanisms may be involved in the first step of amplification and in the later evolution of an already amplified array.

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3

Banaszkiewicz, Sylwia, Ewa Wałecka-Zacharska, Justyna Schubert, Aleksandra Tabiś, Jarosław Król, Tadeusz Stefaniak, Ewelina Węsierska, and Jacek Bania. "Staphylococcal Enterotoxin Genes in Coagulase-Negative Staphylococci—Stability, Expression, and Genomic Context." International Journal of Molecular Sciences 23, no. 5 (February 25, 2022): 2560. http://dx.doi.org/10.3390/ijms23052560.

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In the current study, we screened a collection of coagulase-negative staphylococci (CoNS) isolates for orthologues of staphylococcal enterotoxins (SEs) involved in S. aureus-related staphylococcal food poisoning (SFP). The amplicons corresponding to SEs were detected in S. chromogenes, S. epidermidis, S. haemolyticus, S. borealis, S. pasteuri, S. saprophyticus, S. vitulinus, S. warneri, and S. xylosus. All amplicons were sequenced and identified as parts of known S. aureus or S. epidermidis SE genes. Quantitative real-time PCR allowed determining the relative copy number of each SE amplicon. A significant portion of the amplicons of the sea, seb, sec, and seh genes occurred at low copy numbers. Only the amplicons of the sec gene identified in three isolates of S. epidermidis displayed relative copy numbers comparable to sec in the reference enterotoxigenic S. aureus and S. epidermidis strains. Consecutive passages in microbiological media of selected CoNS isolates carrying low copy numbers of sea, seb, sec, and seh genes resulted in a decrease of gene copy number. S. epidermidis isolates harbored a high copy number of sec, which remained stable over the passages. We demonstrated that enterotoxin genes may occur at highly variable copy numbers in CoNS. However, we could identify enterotoxin genes only in whole-genome sequences of CoNS carrying them in a stable form at high copy numbers. Only those enterotoxins were expressed at the protein level. Our results indicate that PCR-based detection of enterotoxin genes in CoNS should always require an additional control, like analysis of their presence in the bacterial genome. We also demonstrate S. epidermidis as a CoNS species harboring SE genes in a stable form at a specific chromosome site and expressing them as a protein.

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4

Morlino, Giovanni B., Lorenza Tizzani, Reinhard Fleer, Laura Frontali, and Michele M. Bianchi. "Inducible Amplification of Gene Copy Number and Heterologous Protein Production in the Yeast Kluyveromyces lactis." Applied and Environmental Microbiology 65, no. 11 (November 1, 1999): 4808–13. http://dx.doi.org/10.1128/aem.65.11.4808-4813.1999.

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ABSTRACT Heterologous protein production can be doubled by increasing the copy number of the corresponding heterologous gene. We constructed a host-vector system in the yeast Kluyveromyces lactis that was able to induce copy number amplification of pKD1 plasmid-based vectors upon expression of an integrated copy of the plasmid recombinase gene. We increased the production and secretion of two heterologous proteins, glucoamylase from the yeast Arxula adeninivorans and mammalian interleukin-1β, following gene dosage amplification when the heterologous genes were carried by pKD1-based vectors. The choice of the promoters for expression of the integrated recombinase gene and of the episomal heterologous genes are critical for the mitotic stability of the host-vector system.

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5

Fong, Ryan, Zhihao Hu, C. Richard Hutchinson, Jianqiang Huang, Stanley Cohen, and Camilla Kao. "Characterization of a Large, Stable, High-Copy-Number Streptomyces Plasmid That Requires Stability and Transfer Functions for Heterologous Polyketide Overproduction." Applied and Environmental Microbiology 73, no. 4 (December 1, 2006): 1296–307. http://dx.doi.org/10.1128/aem.01888-06.

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ABSTRACT A major limitation to improving small-molecule pharmaceutical production in streptomycetes is the inability of high-copy-number plasmids to tolerate large biosynthetic gene cluster inserts. A recent finding has overcome this barrier. In 2003, Hu et al. discovered a stable, high-copy-number, 81-kb plasmid that significantly elevated production of the polyketide precursor to the antibiotic erythromycin in a heterologous Streptomyces host (J. Ind. Microbiol. Biotechnol. 30:516-522, 2003). Here, we have identified mechanisms by which this SCP2*-derived plasmid achieves increased levels of metabolite production and examined how the 45-bp deletion mutation in the plasmid replication origin increased plasmid copy number. A plasmid intramycelial transfer gene, spd, and a partition gene, parAB, enhance metabolite production by increasing the stable inheritance of large plasmids containing biosynthetic genes. Additionally, high product titers required both activator (actII-ORF4) and biosynthetic genes (eryA) at high copy numbers. DNA gel shift experiments revealed that the 45-bp deletion abolished replication protein (RepI) binding to a plasmid site which, in part, supports an iteron model for plasmid replication and copy number control. Using the new information, we constructed a large high-copy-number plasmid capable of overproducing the polyketide 6-deoxyerythronolide B. However, this plasmid was unstable over multiple culture generations, suggesting that other SCP2* genes may be required for long-term, stable plasmid inheritance.

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6

Kendall, Kevin, and John Cullum. "A vector for studying plasmid stability functions in Streptomyces." Genetical Research 51, no. 1 (February 1988): 71–74. http://dx.doi.org/10.1017/s0016672300023971.

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SummaryWe constructed a cloning vector (pMT603) based on the low copy number plasmid SCP2*. pMT6O3 is unstable because it lacks the SCP2* stability region and carries the selectable marker thiostrepton-resistance and a tyrosinase gene which results in melanin production. This allows easy testing of plasmid stability and we demonstrated its usefulness by cloning a plasmid stability function.

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7

Chen, Xiaoyun, Huiru Yu, Pengfei Wang, Cheng Peng, Xiaofu Wang, Xiaoli Xu, Junfeng Xu, Jingang Liang, and Liang Li. "Digital PCR-Based Characterization of a g10evo-epsps Gene-Specific Matrix Reference Material for Its Food and Feed Detection." Foods 11, no. 13 (June 25, 2022): 1888. http://dx.doi.org/10.3390/foods11131888.

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g10evo-epsps is a novel glyphosate herbicide-resistant gene that has been transferred to various crops such as soybean, corn, cotton, and rice. Here, we developed a gene-specific digital Polymerase Chain Reaction (dPCR) detection method for absolute quantitative analysis of g10evo-epsps, and characterized g10evo-epsps certified reference materials (CRM) using ZUTS-33 soybean powder as the candidate material. Stability tests of matrix CRMs demonstrate that these CRMs can be stored stably for 6 months and transported for 10 days at room temperature and withstand summer high temperatures (below 60 °C). CRM characterization is based on the copy number ratio of g10evo-epsps to lectin. Eight qualified laboratories independently validated the CRM using dPCR method, with a measurement of 0.98 (copy/copy) and an extended uncertainty of 0.08 (copy/copy). The g10evo-epsps matrix CRM described here may be used for qualitative and quantitative testing, method evaluation, laboratory quality control, and other related fields.

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8

Listanto, Edy, Eny Ida Riyanti, Tri Joko Santoso, Toto Hadiarto, and A. Dinar Ambarwati. "GENETIC STABILITY ANALYSIS OF RB GENE IN GENETICALLY MODIFIED POTATO LINES TOLERANT TO Phytophthora infestans." Indonesian Journal of Agricultural Science 16, no. 2 (February 19, 2016): 51. http://dx.doi.org/10.21082/ijas.v16n2.2015.p.51-58.

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Development of potato cultivars with high levels of broad spectrum resistance is a key long-term management strategy against late blight disease caused by Phytophthora infestans. Six progeny lines of hybridization between transgenic potato Katahdin SP951 with non-transgenic Granola and Atlantic were selected based on agronomical characteristics and resistance to late blight disease. The study aimed to analyze the number of insertions and stability of inserted RB gene in the transgenic potato lines. The research was carried out through plant DNA extraction, southern blot analysis and polymerase chain reaction (PCR). Southern blot analysis was used to detect the number of inserts integrated into potato genome, while PCR analysis was used to detect stability of RB gene from generation to generation. The results showed that the progenies obtained from hybridization between Atlantic and transgenic Katahdin SP951 (lines No. 20 and 27) and between Granola and transgenic Katahdin SP951 (line No. 69) contained one copy number of RB gene, according to the probing of nptII. The result is similar to that of inserted RB gene found in the parental transgenic Katahdin SP951. The presence of RB gene in four different generations (G0, G1, G2 and G3) showed stable integration of the gene into the plant genome. The single copy number of RB gene will repress the occurrence of silencing gene expression. The stability analysis of RB gene can determine that the gene is still present in plant genome after several generations.

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9

Listanto, Edy, Eny Ida Riyanti, Tri Joko Santoso, Toto Hadiarto, and A. Dinar Ambarwati. "GENETIC STABILITY ANALYSIS OF RB GENE IN GENETICALLY MODIFIED POTATO LINES TOLERANT TO Phytophthora infestans." Indonesian Journal of Agricultural Science 16, no. 2 (February 19, 2016): 51. http://dx.doi.org/10.21082/ijas.v16n2.2015.pp.51-58.

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Development of potato cultivars with high levels of broad spectrum resistance is a key long-term management strategy against late blight disease caused by Phytophthora infestans. Six progeny lines of hybridization between transgenic potato Katahdin SP951 with non-transgenic Granola and Atlantic were selected based on agronomical characteristics and resistance to late blight disease. The study aimed to analyze the number of insertions and stability of inserted RB gene in the transgenic potato lines. The research was carried out through plant DNA extraction, southern blot analysis and polymerase chain reaction (PCR). Southern blot analysis was used to detect the number of inserts integrated into potato genome, while PCR analysis was used to detect stability of RB gene from generation to generation. The results showed that the progenies obtained from hybridization between Atlantic and transgenic Katahdin SP951 (lines No. 20 and 27) and between Granola and transgenic Katahdin SP951 (line No. 69) contained one copy number of RB gene, according to the probing of nptII. The result is similar to that of inserted RB gene found in the parental transgenic Katahdin SP951. The presence of RB gene in four different generations (G0, G1, G2 and G3) showed stable integration of the gene into the plant genome. The single copy number of RB gene will repress the occurrence of silencing gene expression. The stability analysis of RB gene can determine that the gene is still present in plant genome after several generations.

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10

Wilson, J. A., R. A. Godke, and K. R. Bondioli. "437 DETERMINING GENE COPY NUMBER IN TRANSFECTED CAPRINE FIBROBLAST CELLS." Reproduction, Fertility and Development 22, no. 1 (2010): 375. http://dx.doi.org/10.1071/rdv22n1ab437.

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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.

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11

Dmowski, Michał, Izabela Sitkiewicz та Piotr Cegłowski. "Characterization of a Novel Partition System Encoded by the δ and ω Genes from the Streptococcal Plasmid pSM19035". Journal of Bacteriology 188, № 12 (15 червня 2006): 4362–72. http://dx.doi.org/10.1128/jb.01922-05.

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ABSTRACT High segregational stability of the streptococcal plasmid pSM19035 is achieved by the concerted action of systems involved in plasmid copy number control, multimer resolution, and postsegregational killing. In this study, we demonstrate the role of two genes, δ and ω, in plasmid stabilization by a partition mechanism. We show that these two genes can stabilize the native pSM19035 replicon as well as other θ- and σ-type plasmids in Bacillus subtilis. In contrast to other known partition systems, in this case the two genes are transcribed separately; however, they are coregulated by the product of the parB-like gene ω. Analysis of mutants of the parA-like gene δ showed that the Walker A ATPase motif is necessary for plasmid stabilization. The ParB-like product of the ω gene binds to three regions containing repeated WATCACW heptamers, localized in the copS (regulation of plasmid copy number), δ, and ω promoter regions. We demonstrate that all three of these regions can cause partition-mediated incompatibility. Moreover, our data suggest that each of these could play the role of a centromere-like sequence. We conclude that δ and ω constitute a novel type of plasmid stabilization system.

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12

Berkner, Silvia, and Georg Lipps. "Characterization of the Transcriptional Activity of the Cryptic Plasmid pRN1 from Sulfolobus islandicus REN1H1 and Regulation of Its Replication Operon." Journal of Bacteriology 189, no. 5 (December 15, 2006): 1711–21. http://dx.doi.org/10.1128/jb.01586-06.

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ABSTRACT The plasmid pRN1 from Sulfolobus islandicus REN1H1 belongs to the crenarchaeal plasmid family pRN. The plasmids in this family encode three conserved proteins that participate in plasmid replication and copy number regulation, as suggested by biochemical characterization of the recombinant proteins. In order to deepen our understanding of the molecular biology of these plasmids, we investigated the transcriptional activity of the model plasmid pRN1. We detected five major transcripts present at about 2 to 15 copies per cell. One long transcriptional unit comprises the genes for the plasmid-copy-number control protein Orf56/CopG and the replication protein Orf904. A second transcript with a long 3′-untranslated region codes for the DNA binding protein Orf80. For both transcripts, we identified countertranscripts which could play a regulatory role. The function of the fifth transcript is unclear. For the five transcripts, we determined the start site, the transcript end, the stability, and the abundance in different growth phases. Reporter gene experiments demonstrated that the copy number control protein Orf56 represses transcription of the orf56-orf904 cotranscript in vivo.

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13

Horsfall, Wendy H., and Ronald E. Pearlman. "Micronuclear DNA sequences from Tetrahymena do not confer mitotic stability on ARS plasmids in Saccharomyces." Genome 30, no. 5 (October 1, 1988): 690–96. http://dx.doi.org/10.1139/g88-116.

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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.

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14

Koprek, Thomas, Sergio Rangel, David McElroy, Jeanine D. Louwerse, Rosalind E. Williams-Carrier, and Peggy G. Lemaux. "Transposon-Mediated Single-Copy Gene Delivery Leads to Increased Transgene Expression Stability in Barley." Plant Physiology 125, no. 3 (March 1, 2001): 1354–62. http://dx.doi.org/10.1104/pp.125.3.1354.

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15

Markt, Rudolf, Markus Mayr, Evelyn Peer, Andreas O. Wagner, Nina Lackner, and Heribert Insam. "Detection and Stability of SARS-CoV-2 Fragments in Wastewater: Impact of Storage Temperature." Pathogens 10, no. 9 (September 18, 2021): 1215. http://dx.doi.org/10.3390/pathogens10091215.

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SARS-CoV-2 wastewater epidemiology suffers from uncertainties concerning sample storage. We show the effect of the storage of wastewater on the detectable SARS-CoV-2 load. Storage at 4 °C for up to 9 days had no significant effect, while storage at −20 °C led to a significant reduction in gene copy numbers.

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16

Altamura, Raffaele, Jiten Doshi, and Yaakov Benenson. "Rational design and construction of multi-copy biomanufacturing islands in mammalian cells." Nucleic Acids Research 50, no. 1 (December 10, 2021): 561–78. http://dx.doi.org/10.1093/nar/gkab1214.

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Abstract Cell line development is a critical step in the establishment of a biopharmaceutical manufacturing process. Current protocols rely on random transgene integration and amplification. Due to considerable variability in transgene integration profiles, this workflow results in laborious screening campaigns before stable producers can be identified. Alternative approaches for transgene dosage increase and integration are therefore highly desirable. In this study, we present a novel strategy for the rapid design, construction, and genomic integration of engineered multiple-copy gene constructs consisting of up to 10 gene expression cassettes. Key to this strategy is the diversification, at the sequence level, of the individual gene cassettes without altering their protein products. We show a computational workflow for coding and regulatory sequence diversification and optimization followed by experimental assembly of up to nine gene copies and a sentinel reporter on a contiguous scaffold. Transient transfections in CHO cells indicates that protein expression increases with the gene copy number on the scaffold. Further, we stably integrate these cassettes into a pre-validated genomic locus. Altogether, our findings point to the feasibility of engineering a fully mapped multi-copy recombinant protein ‘production island’ in a mammalian cell line with greatly reduced screening effort, improved stability, and predictable product titers.

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17

Kloos, Ralf, Edward Stevens, and Walter Oettmeier. "Complete Sequence of One Copy of the psbA Gene from the Thermophilic Cyanobacterium Synechococcus elongatus." Zeitschrift für Naturforschung C 48, no. 9-10 (October 1, 1993): 799–802. http://dx.doi.org/10.1515/znc-1993-9-1019.

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Abstract One copy of the psbA. gene which codes for the photosystem II reaction center D-1 protein from the thermophilic cyanobacterium Synechococcus elongatus has been sequenced. It is feasible that a disulfide bridge between D-1 Cys212 and D-2 Cys2I2 is reponsible for the thermo stability of the photosystem II reaction center from Synechococcus elongatus.

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18

Lopez, Francesca B., Antoine Fort, Luca Tadini, Aline V. Probst, Marcus McHale, James Friel, Peter Ryder, et al. "Gene dosage compensation of rRNA transcript levels in Arabidopsis thaliana lines with reduced ribosomal gene copy number." Plant Cell 33, no. 4 (February 2, 2021): 1135–50. http://dx.doi.org/10.1093/plcell/koab020.

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Abstract The 45S rRNA genes (rDNA) are among the largest repetitive elements in eukaryotic genomes. rDNA consists of tandem arrays of rRNA genes, many of which are transcriptionally silenced. Silent rDNA repeats may act as ‘back-up’ copies for ribosome biogenesis and have nuclear organization roles. Through Cas9-mediated genome editing in the Arabidopsis thaliana female gametophyte, we reduced 45S rDNA copy number (CN) to a plateau of ∼10%. Two independent lines had rDNA CNs reduced by up to 90% at the T7 generation, named low copy number (LCN) lines. Despite drastic reduction of rDNA copies, rRNA transcriptional rates, and steady-state levels remained the same as wild-type plants. Gene dosage compensation of rRNA transcript levels was associated with reduction of silencing histone marks at rDNA loci and altered Nucleolar Organiser Region 2 organization. Although overall genome integrity of LCN lines appears unaffected, a chromosome segmental duplication occurred in one of the lines. Transcriptome analysis of LCN seedlings identified several shared dysregulated genes and pathways in both independent lines. Cas9 genome editing of rRNA repeats to generate LCN lines provides a powerful technique to elucidate rDNA dosage compensation mechanisms and impacts of low rDNA CN on genome stability, development, and cellular processes.

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19

Carrier, Trent, Kristala L. Jones, and J. D. Keasling. "mRNA stability and plasmid copy number effects on gene expression from an inducible promoter system." Biotechnology and Bioengineering 59, no. 6 (September 20, 1998): 666–72. http://dx.doi.org/10.1002/(sici)1097-0290(19980920)59:6<666::aid-bit2>3.0.co;2-d.

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20

Slavcev, Roderick A., and Barbara E. Funnell. "Identification and Characterization of a Novel Allele of Escherichia coli dnaB Helicase That Compromises the Stability of Plasmid P1." Journal of Bacteriology 187, no. 4 (February 15, 2005): 1227–37. http://dx.doi.org/10.1128/jb.187.4.1227-1237.2005.

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ABSTRACT Bacteriophage P1 lysogenizes Escherichia coli cells as a plasmid with approximately the same copy number as the copy number of the host chromosome. Faithful inheritance of the plasmids relies upon proper DNA replication, as well as a partition system that actively segregates plasmids to new daughter cells. We genetically screened for E. coli chromosomal mutations that influenced P1 stability and identified a novel temperature-sensitive allele of the dnaB helicase gene (dnaB277) that replaces serine 277 with a leucine residue (DnaB S277L). This allele conferred a severe temperature-sensitive phenotype to the host; dnaB277 cells were not viable at temperatures above 34°C. Shifting dnaB277 cells to 42°C resulted in an immediate reduction in the rate of DNA synthesis and extensive cell filamentation. The dnaB277 allele destabilized P1 plasmids but had no significant influence on the stability of the F low-copy-number plasmid. This observation suggests that there is a specific requirement for DnaB in P1 plasmid maintenance in addition to the general requirement for DnaB as the replicative helicase during elongation.

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21

de Oliveira, Vanessa Cristina, Kelly Cristine Santos Roballo, Clésio Gomes Mariano Junior, Sarah Ingrid Pinto Santos, Fabiana Fernandes Bressan, Marcos Roberto Chiaratti, Elena J. Tucker, Erica E. Davis, Jean-Paul Concordet, and Carlos Eduardo Ambrósio. "HEK293T Cells with TFAM Disruption by CRISPR-Cas9 as a Model for Mitochondrial Regulation." Life 12, no. 1 (December 24, 2021): 22. http://dx.doi.org/10.3390/life12010022.

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The mitochondrial transcription factor A (TFAM) is considered a key factor in mitochondrial DNA (mtDNA) copy number. Given that the regulation of active copies of mtDNA is still not fully understood, we investigated the effects of CRISPR-Cas9 gene editing of TFAM in human embryonic kidney (HEK) 293T cells on mtDNA copy number. The aim of this study was to generate a new in vitro model by CRISPR-Cas9 system by editing the TFAM locus in HEK293T cells. Among the resulting single-cell clones, seven had high mutation rates (67–96%) and showed a decrease in mtDNA copy number compared to control. Cell staining with Mitotracker Red showed a reduction in fluorescence in the edited cells compared to the non-edited cells. Our findings suggest that the mtDNA copy number is directly related to TFAM control and its disruption results in interference with mitochondrial stability and maintenance.

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22

Fan, Hua-Ying, Robert J. Merker, and Hannah L. Klein. "High-Copy-Number Expression of Sub2p, a Member of the RNA Helicase Superfamily, Suppresses hpr1-Mediated Genomic Instability." Molecular and Cellular Biology 21, no. 16 (August 15, 2001): 5459–70. http://dx.doi.org/10.1128/mcb.21.16.5459-5470.2001.

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ABSTRACT We report on a novel role for a pre-mRNA splicing component in genome stability. The Hpr1 protein, a component of an RNA polymerase II complex and required for transcription elongation, is also required for genome stability. Deletion of HPR1 results in a 1,000-fold increase in genome instability, detected as direct-repeat instability. This instability can be suppressed by the high-copy-numberSUB2 gene, which is the Saccharomyces cerevisiae homologue of the human splicing factor hUAP56. Although SUB2 is essential, conditional alleles grown at the permissive temperature complement the essential function of SUB2 yet reveal nonessential phenotypes. These studies have uncovered a role for SUB2 in preventing genome instability. The genomic instability observed in sub2 mutants can be suppressed by high-copy-numberHPR1. A deletion mutant of CDC73, a component of a PolII complex, is also unstable for direct repeats. This too is suppressed by high-copy-number SUB2. Thus, defects in both the transcriptional machinery and the pre-mRNA splicing machinery can be sources of genome instability. The ability of a pre-mRNA splicing factor to suppress the hyperrecombination phenotype of a defective PolII complex raises the possibility of integrating transcription, RNA processing, and genome stability or a second role for SUB2.

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23

Wei, Junjie, Zhicheng Dong, and David W. Ow. "Spontaneous reactivation of a site-specifically placed transgene independent of copy number or DNA methylation." Journal of Experimental Botany 71, no. 4 (November 19, 2019): 1574–84. http://dx.doi.org/10.1093/jxb/erz514.

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Abstract As millions of seeds are produced from a breeding line, the long-term stability of transgene expression is vital for commercial-scale production of seeds with transgenic traits. Transgenes can be silenced by epigenetic mechanisms, but reactivation of expression can occur as a result of treatment with chromatin modification inhibitors such as 5-azacytidine, from stress such as heat or UV-B, or in mutants that have acquired a defect in gene silencing. Previously, we targeted a gfp reporter gene into the tobacco (Nicotiana tabacum) genome by site-specific recombination but still found some silenced lines among independent integration events. One such line also had a second random copy and both copies showed DNA hypermethylation. To test whether removing the second copy would reactivate gfp expression, two T1 plants were backcrossed to the wild type. Whereas the silenced status was maintained in the progenies from one backcross, spontaneous partial reactivation of gfp expression was found among progenies from a second backcross. However, this reactivation did not correlate with loss of the second random copy or with a significant change in the pattern or amount of DNA hypermethylation. This finding supports the suggestion that gene reactivation does not necessarily involve loss of DNA homology or methylation.

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24

Shukla, Ranti Dev, Ágnes Zvara, Ákos Avramucz, Alona Yu Biketova, Akos Nyerges, László G. Puskás, and Tamás Fehér. "inPOSE: A Flexible Toolbox for Chromosomal Cloning and Amplification of Bacterial Transgenes." Microorganisms 10, no. 2 (January 21, 2022): 236. http://dx.doi.org/10.3390/microorganisms10020236.

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Cloning the genes and operons encoding heterologous functions in bacterial hosts is now almost exclusively carried out using plasmid vectors. This has multiple drawbacks, including the need for constant selection and variation in copy numbers. The chromosomal integration of transgenes has always offered a viable alternative; however, to date, it has been of limited use due to its tedious nature and often being limited to a single copy. We introduce here a strategy that uses bacterial insertion sequences, which are the simplest autonomous transposable elements to insert and amplify genetic cargo into a bacterial chromosome. Transgene insertion can take place either as transposition or homologous recombination, and copy number amplification is achieved using controlled copy-paste transposition. We display the successful use of IS1 and IS3 for this purpose in Escherichia coli cells using various selection markers. We demonstrate the insertion of selectable genes, an unselectable gene and a five-gene operon in up to two copies in a single step. We continue with the amplification of the inserted cassette to double-digit copy numbers within two rounds of transposase induction and selection. Finally, we analyze the stability of the cloned genetic constructs in the lack of selection and find it to be superior to all investigated plasmid-based systems. Due to the ubiquitous nature of transposable elements, we believe that with proper design, this strategy can be adapted to numerous other bacterial species.

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25

Deshmukh, M., Y. F. Tsay, A. G. Paulovich, and J. L. Woolford. "Yeast ribosomal protein L1 is required for the stability of newly synthesized 5S rRNA and the assembly of 60S ribosomal subunits." Molecular and Cellular Biology 13, no. 5 (May 1993): 2835–45. http://dx.doi.org/10.1128/mcb.13.5.2835-2845.1993.

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Ribosomal protein L1 from Saccharomyces cerevisiae binds 5S rRNA and can be released from intact 60S ribosomal subunits as an L1-5S ribonucleoprotein (RNP) particle. To understand the nature of the interaction between L1 and 5S rRNA and to assess the role of L1 in ribosome assembly and function, we cloned the RPL1 gene encoding L1. We have shown that RPL1 is an essential single-copy gene. A conditional null mutant in which the only copy of RPL1 is under control of the repressible GAL1 promoter was constructed. Depletion of L1 causes instability of newly synthesized 5S rRNA in vivo. Cells depleted of L1 no longer assemble 60S ribosomal subunits, indicating that L1 is required for assembly of stable 60S ribosomal subunits but not 40S ribosomal subunits. An L1-5S RNP particle not associated with ribosomal particles was detected by coimmunoprecipitation of L1 and 5S rRNA. This pool of L1-5S RNP remained stable even upon cessation of 60S ribosomal subunit assembly by depletion of another ribosomal protein, L16. Preliminary results suggest that transcription of RPL1 is not autogenously regulated by L1.

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26

Deshmukh, M., Y. F. Tsay, A. G. Paulovich, and J. L. Woolford. "Yeast ribosomal protein L1 is required for the stability of newly synthesized 5S rRNA and the assembly of 60S ribosomal subunits." Molecular and Cellular Biology 13, no. 5 (May 1993): 2835–45. http://dx.doi.org/10.1128/mcb.13.5.2835.

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Анотація:

Ribosomal protein L1 from Saccharomyces cerevisiae binds 5S rRNA and can be released from intact 60S ribosomal subunits as an L1-5S ribonucleoprotein (RNP) particle. To understand the nature of the interaction between L1 and 5S rRNA and to assess the role of L1 in ribosome assembly and function, we cloned the RPL1 gene encoding L1. We have shown that RPL1 is an essential single-copy gene. A conditional null mutant in which the only copy of RPL1 is under control of the repressible GAL1 promoter was constructed. Depletion of L1 causes instability of newly synthesized 5S rRNA in vivo. Cells depleted of L1 no longer assemble 60S ribosomal subunits, indicating that L1 is required for assembly of stable 60S ribosomal subunits but not 40S ribosomal subunits. An L1-5S RNP particle not associated with ribosomal particles was detected by coimmunoprecipitation of L1 and 5S rRNA. This pool of L1-5S RNP remained stable even upon cessation of 60S ribosomal subunit assembly by depletion of another ribosomal protein, L16. Preliminary results suggest that transcription of RPL1 is not autogenously regulated by L1.

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27

Zeng, Chunhua, Tao Yang, Qinglin Han, Chun Zhang, Dong Tian, and Hua Wang. "Noises-induced toggle switch and stability in a gene regulation network." International Journal of Modern Physics B 28, no. 31 (December 8, 2014): 1450223. http://dx.doi.org/10.1142/s0217979214502233.

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It is well-known that noises are inevitable in gene regulatory networks due to the low-copy numbers of molecules and environmental fluctuations. In this paper, we investigate the stationary probability distribution (SPD) between both low (OFF state) and high (ON state) protein levels and mean first passage time (MFPT) in an abstract model of the Myc/E2F/miR-17-92 network presented by Aguda et al., PNAS 105, 19678 (2008), where the gene expression is assumed to be disturbed simultaneously by intrinsic and extrinsic noises that were correlated. Our results show that (i) the OFF state is enhanced by the extrinsic noise (D), while the ON state is enhanced by the intrinsic noise (Q) or cross-correlation between two noises (λ); (ii) for the cases of negative or no cross-correlation (λ⩽0.0), the increase of the noise intensity (D or Q) leads to a decline of the MFPT and enhances the probability of toggle switch to the OFF state; (iii) but for the case of positive cross-correlation (λ>0.0), the MFPT as a function of the noise intensity (D or Q) exhibits a maximum, this maximum for MFPT identifies the characteristic of noise enhanced stability of the ON state and (iv) the cross-correlation between two noises can enhance stability of the ON state.

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28

Kimmerly, W. J., and J. Rine. "Replication and segregation of plasmids containing cis-acting regulatory sites of silent mating-type genes in Saccharomyces cerevisiae are controlled by the SIR genes." Molecular and Cellular Biology 7, no. 12 (December 1987): 4225–37. http://dx.doi.org/10.1128/mcb.7.12.4225-4237.1987.

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In Saccharomyces cerevisiae, two cis-acting regulatory sites called E and I flank the silent mating-type gene, HMRa, and mediate SIR-dependent transcriptional repression of the a1-a2 promoters. It has been shown previously that the E and I sites have plasmid replicator (ARS) activity. We show in this report that the ARS activity of the E and I sites is governed by the SIR genotype of the cell. In wild-type cells, a plasmid carrying the E site from HMRa (HMR E) in the vector YIp5 exhibited very high mitotic stability at a copy number of approximately 25 per cell. However, in sir2, sir3, or sir4 mutants, plasmids with HMR E had the low mitotic stability characteristic of plasmids containing ARS1, a SIR-independent replicator. Elevated mitotic stability of plasmids that carry HMR E is due to a segregation mechanism provided by SIR and HMR E. In sir2 and sir4 mutants, the plasmid copy number was significantly lowered, suggesting that these gene products also participate in the replication of plasmids carrying HMR E. The phenotype of point mutations introduced at an 11-base-pair ARS consensus sequence present at HMR E indicated that this sequence is functional but not absolutely required for autonomous replication of the plasmid and that it is not required for SIR-dependent mitotic stabilization. A plasmid carrying both a centromere and HMR E exhibited reduced mitotic stability in wild-type cells. This destabilization appeared to be due to antagonism between the segregation functions provided by the centromere and by HMR E.

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29

Kimmerly, W. J., and J. Rine. "Replication and segregation of plasmids containing cis-acting regulatory sites of silent mating-type genes in Saccharomyces cerevisiae are controlled by the SIR genes." Molecular and Cellular Biology 7, no. 12 (December 1987): 4225–37. http://dx.doi.org/10.1128/mcb.7.12.4225.

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Анотація:

In Saccharomyces cerevisiae, two cis-acting regulatory sites called E and I flank the silent mating-type gene, HMRa, and mediate SIR-dependent transcriptional repression of the a1-a2 promoters. It has been shown previously that the E and I sites have plasmid replicator (ARS) activity. We show in this report that the ARS activity of the E and I sites is governed by the SIR genotype of the cell. In wild-type cells, a plasmid carrying the E site from HMRa (HMR E) in the vector YIp5 exhibited very high mitotic stability at a copy number of approximately 25 per cell. However, in sir2, sir3, or sir4 mutants, plasmids with HMR E had the low mitotic stability characteristic of plasmids containing ARS1, a SIR-independent replicator. Elevated mitotic stability of plasmids that carry HMR E is due to a segregation mechanism provided by SIR and HMR E. In sir2 and sir4 mutants, the plasmid copy number was significantly lowered, suggesting that these gene products also participate in the replication of plasmids carrying HMR E. The phenotype of point mutations introduced at an 11-base-pair ARS consensus sequence present at HMR E indicated that this sequence is functional but not absolutely required for autonomous replication of the plasmid and that it is not required for SIR-dependent mitotic stabilization. A plasmid carrying both a centromere and HMR E exhibited reduced mitotic stability in wild-type cells. This destabilization appeared to be due to antagonism between the segregation functions provided by the centromere and by HMR E.

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30

Wegierski, T., A. Dmochowska, A. Jabłonowska, A. Dziembowski, E. Bartnik, and P. P. Stepień. "Yeast nuclear PET127 gene can suppress deletions of the SUV3 or DSS1 genes: an indication of a functional interaction between 3' and 5' ends of mitochondrial mRNAs." Acta Biochimica Polonica 45, no. 4 (December 31, 1998): 935–40. http://dx.doi.org/10.18388/abp.1998_4352.

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Saccharomyces cerevisiae nuclear genes SUV3 and DSS1 encode putative RNA helicase and RNase II, respectively, which are subunits of the mitochondrial degradosome (mtEXO): a three-protein complex which has a 3' to 5' exoribonuclease activity and plays a major role in regulating stability of mitochondrial RNA. Lack of either of the two gene products results in a respiratory negative phenotype, while on the molecular level it causes a total block of mitochondrial translation, loss of the in vitro exoribonuclease activity and changes in stability and processing of many mtRNAs. We have found that the yeast nuclear gene PET127 present on a low or high copy number vector can effectively suppress the effects of the SUV3 or DSS1 gene disruptions. Since the product of the PET127 gene is involved in processing of the 5' ends of mitochondrial mRNAs, we suggest that there is a functional coupling between the 5' and 3' ends of mitochondrial mRNAs.

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31

Zhang, H., Y. Bian, X. Gou, Y. Dong, S. Rustgi, B. Zhang, C. Xu, et al. "Intrinsic karyotype stability and gene copy number variations may have laid the foundation for tetraploid wheat formation." Proceedings of the National Academy of Sciences 110, no. 48 (November 11, 2013): 19466–71. http://dx.doi.org/10.1073/pnas.1319598110.

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32

NEWELL, MARTINA, AIDEN J. McLOUGHLIN, and CAROLINE HUSSEY. "The effect of gene expression on copy number and heritable stability of recombinant plasmids in Bacillus subtilis." Biochemical Society Transactions 16, no. 2 (April 1, 1988): 192–93. http://dx.doi.org/10.1042/bst0160192a.

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33

Hoekema, A., R. A. Kastelein, M. Vasser, and H. A. de Boer. "Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression." Molecular and Cellular Biology 7, no. 8 (August 1987): 2914–24. http://dx.doi.org/10.1128/mcb.7.8.2914-2924.1987.

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The coding sequences of genes in the yeast Saccharomyces cerevisiae show a preference for 25 of the 61 possible coding triplets. The degree of this biased codon usage in each gene is positively correlated to its expression level. Highly expressed genes use these 25 major codons almost exclusively. As an experimental approach to studying biased codon usage and its possible role in modulating gene expression, systematic codon replacements were carried out in the highly expressed PGK1 gene. The expression of phosphoglycerate kinase (PGK) was studied both on a high-copy-number plasmid and as a single copy gene integrated into the chromosome. Replacing an increasing number (up to 39% of all codons) of major codons with synonymous minor ones at the 5' end of the coding sequence caused a dramatic decline of the expression level. The PGK protein levels dropped 10-fold. The steady-state mRNA levels also declined, but to a lesser extent (threefold). Our data indicate that this reduction in mRNA levels was due to destabilization caused by impaired translation elongation at the minor codons. By preventing translation of the PGK mRNAs by the introduction of a stop codon 3' and adjacent to the start codon, the steady-state mRNA levels decreased dramatically. We conclude that efficient mRNA translation is required for maintaining mRNA stability in S. cerevisiae. These findings have important implications for the study of the expression of heterologous genes in yeast cells.

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34

Hoekema, A., R. A. Kastelein, M. Vasser, and H. A. de Boer. "Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression." Molecular and Cellular Biology 7, no. 8 (August 1987): 2914–24. http://dx.doi.org/10.1128/mcb.7.8.2914.

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Анотація:

The coding sequences of genes in the yeast Saccharomyces cerevisiae show a preference for 25 of the 61 possible coding triplets. The degree of this biased codon usage in each gene is positively correlated to its expression level. Highly expressed genes use these 25 major codons almost exclusively. As an experimental approach to studying biased codon usage and its possible role in modulating gene expression, systematic codon replacements were carried out in the highly expressed PGK1 gene. The expression of phosphoglycerate kinase (PGK) was studied both on a high-copy-number plasmid and as a single copy gene integrated into the chromosome. Replacing an increasing number (up to 39% of all codons) of major codons with synonymous minor ones at the 5' end of the coding sequence caused a dramatic decline of the expression level. The PGK protein levels dropped 10-fold. The steady-state mRNA levels also declined, but to a lesser extent (threefold). Our data indicate that this reduction in mRNA levels was due to destabilization caused by impaired translation elongation at the minor codons. By preventing translation of the PGK mRNAs by the introduction of a stop codon 3' and adjacent to the start codon, the steady-state mRNA levels decreased dramatically. We conclude that efficient mRNA translation is required for maintaining mRNA stability in S. cerevisiae. These findings have important implications for the study of the expression of heterologous genes in yeast cells.

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35

MIYAMOTO, Suzanne, John A. CHIORINI, Elena URCELAY та Brian SAFER. "Regulation of gene expression for translation initiation factor eIF-2α: importance of the 3′ untranslated region". Biochemical Journal 315, № 3 (1 травня 1996): 791–98. http://dx.doi.org/10.1042/bj3150791.

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Gene expression of the α-subunit of eukaryotic initiation factor-2 (eIF-2α), involves transcriptional and post-transcriptional mechanisms. eIF-2α is a single-copy gene expressing two mRNAs, 1.6 and 4.2 kb in size. Cloning and sequencing of the cDNA for the 4.2 kb mRNA revealed that it is the result of alternative polyadenylation site selection. Four polyadenylation sites were identified within the 3´ untranslated region (UTR) of eIF-2α, only two of which are normally utilized in human and mouse tissues. A functional role for the extended 3´ UTR was assessed by comparing the translatability and stability of the 1.6 and 4.2 kb mRNAs. Both the 1.6 and 4.2 kb transcripts could be translated in vitro and were identified in vivo as being distributed on large polyribosomes. This indicates that both mRNAs are efficiently translated. Stability studies showed that in activated T-cells the 4.2 kb mRNA was more stable than the 1.6 kb mRNA. Polyadenylation site selection and mRNA stability differ for the two mRNAs of eIF-2α. These activities might be modulated by sequence elements contained within the untranslated regions of the eIF-2α gene.

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36

Blaukat, Andree, and Michael Kemme. "Effects of Gene Dosage on the Expression of a Functionally Active C-Terminal Domain of Human Mucus Proteinase Inhibitor in E. Coli." Protein & Peptide Letters 6, no. 1 (February 1999): 35–41. http://dx.doi.org/10.2174/092986650601221107155931.

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Abstract: A gene dosage strategy was developed for efficient production of the C-terminal domain of human mucus proteinase inhibitor (cMPI) in E. coli with polycistronic expression cassettes containing one, two and four tandemly arranged cMPI genes. Upon induction, the synthesis of soluble cMPI increased proportionally to the number of genes integrated due to amplified plasmid copy numbers and enhanced protein stability, reaching a maximum up to 20 mg/I culture. Purified recombinant cMPI retained the inhibitory activity of native MPI.

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37

Brown, Jennifer R., Megan Hanna, Bethany Tesar, Lillian Werner, Hazel Reynolds, Stacey M. Fernandes, Laura Macconaill, et al. "High Resolution Genomic Analysis In CLL Demonstrates Genomic Stability In Untreated Patients and Novel Markers of Progression In Treated Patients." Blood 116, no. 21 (November 19, 2010): 2426. http://dx.doi.org/10.1182/blood.v116.21.2426.2426.

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Abstract Abstract 2426 Chronic lymphocytic leukemia is the most common leukemia of adults but still incurable. Prognosis at diagnosis is widely variable, and the key cytogenetic abnormalities determined by FISH remain one of the best predictors of prognosis and treatment response. We therefore undertook very high resolution genomic analysis of 161 CLLs with matched germline samples using Affymetrix 6.0 SNP arrays, in an effort to identify additional predictors of prognosis, and have also performed gene expression profiling on most of this patient cohort. The median age at diagnosis for the cohort was 55 (31–79), and the median time to sampling was 4.6 months (0.5–291). 22% of the cohort was previously treated, with an additional 21% of patients receiving treatment during the follow-up period, for a total of 43% treated, with a median time from diagnosis to treatment of 41 months (0.4-161.2 months). The genomic data were analyzed both by GISTIC, which identifies significant deletions and amplifications based on analysis of the frequency and amplitude of each aberration in the tumor samples alone, as well as by paired copy number analysis of each tumor and its cognate germline, using Birdseed, PLINK and PennCNV. Our results show that the CLL genome is overall quite stable, with a median of only one acquired copy number aberration per sample, excluding rearrangements at the immunoglobulin gene loci. GISTIC analysis on the entire population identified the known common CLL abnormalities at frequencies that would be expected in a largely untreated cohort: 57% del 13q, 6.2% deletion 11q, 5.0% deletion 17p, and 12% trisomy 12. The presence of two or more acquired copy number aberrations (CNAs) of any type was associated with a significantly shorter time to first therapy (p<0.0001). A higher number of CNAs was strongly associated with deletions of 11q or 17p, but the predictive power of a higher number of CNAs was still present in those CLLs without deletions 11q or 17p. Detailed analysis of 13q deletion revealed no association of longer deletions or homozygous deletions with time to first therapy. However, any additional somatic copy number aberration in addition to 13q deletion significantly reduced the time to first therapy, making it comparable to non-13q patients. In order to identify genetic markers of progression, we compared treated to untreated patients using GISTIC. This analysis revealed that untreated patients showed peaks largely limited to deletion 13q and trisomy 12. Treated patients however showed three additional significant peaks: a deletion peak at 8p, as well as significant amplification peaks at 3q26.32 and 8q24.21. The deletion peak at 8p was observed in 8 of 161 samples tested (5.0%), and was large, with a common region of deletion spanning 11.0–29.6 Mb. Six of eight of these patients were untreated at the time of sampling but had a very short time to treatment thereafter, independent of whether they had coexistent deletions of 17p or 11q, suggesting that this deletion carries a very poor prognosis in itself. Another notable region of amplification was found on 3q26.32 in nine patients (9/161 or 5.6%). Although many of these amplified regions were large, three of the nine patients carrying this amplification demonstrated focal somatic amplification of the final exon of PIK3CA, the alpha catalytic subunit of PI3K. Finally the amplification on 8q24 was present in 6 of 161 CLLs (3.7%), two of which were focal and amplified only the gene desert region previously implicated in CLL risk by genome-wide association study and located approximately 335 kb centromeric to MYC. Analysis of gene expression profiling comparing patients with and without amplifications demonstrated upregulation of MYC mRNA expression and alteration of downstream targets of MYC in samples with amplification. We conclude that very high resolution copy number analysis with matched germline comparison in CLL reveals a quite stable genome in untreated patients, and identifies amplifications of 3q26 and MYC at 8q24 as progression events. Disclosures: No relevant conflicts of interest to declare.

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38

Deregowska, Anna, Marek Skoneczny, Jagoda Adamczyk, Aleksandra Kwiatkowska, Ewa Rawska, Adrianna Skoneczna, Anna Lewinska, and Maciej Wnuk. "Genome-wide array-CGH analysis reveals YRF1 gene copy number variation that modulates genetic stability in distillery yeasts." Oncotarget 6, no. 31 (September 10, 2015): 30650–63. http://dx.doi.org/10.18632/oncotarget.5594.

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39

Yu, Miao, Peiwu Wang, Yang Song, Yong Qi Feng, Jing Qu, Jie Rong, Mo Zhang, and Zhuo Zhang. "Genetic Stability and Disease Resistance Analysis of Hrpzpsta Gene in Transgenic Soybean Lines." Journal of Plant Studies 6, no. 2 (May 29, 2017): 45. http://dx.doi.org/10.5539/jps.v6n2p45.

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This experiment was carried out to evaluate genetic stability and disease resistance in transformed soybean lines with hrpZpsta gene using PCR analysis, southern blotting, real-time quantitative PCR (qRT-PCR) and to analyze the resistance against Phytophthora sojae (P. sojae) and Cercospora sojina (C. sojina) after inoculation. The results obtained using PCR and southern blotting analytical methods showed that exogenous gene functional elements were stably inherited in transgenic soybean and hrpZpsta gene was successfully integrated into the soybean genome in a single copy. Results at high-generation (T7, T8) transgenic lines of hrpZpsta revealed that their relative expression of hrpZpsta gene was the highest in leaves followed by roots, and much lower in stems, flowers, and seeds. Activity change rates of peroxidase (POD), polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL) showed that transgenic lines significantly enhanced receptor species. The resistance of transgenic strains T7 and T8 generations against P. sojae was significantly increased with artificial inoculation methods, and the resistance against C. sojina was increased from susceptibility to the level of resistance. Under natural conditions in the field, the response of T8 transgenic lines to C. sojina reached disease resistance level. There were no significant differences in transgenic lines and recipient variety in maturing stage, leaf shape, flower color, plant height, 100-grain weight and quality content, and the two years average yield of plots increased to 11.59% and 8.19%, which significantly higher than recipient cultivar. The current results provide data support for the release of transgenic lines.

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40

Gomes, Luciana, Gabriel Monteiro, and Filipe Mergulhão. "The Impact of IPTG Induction on Plasmid Stability and Heterologous Protein Expression by Escherichia coli Biofilms." International Journal of Molecular Sciences 21, no. 2 (January 16, 2020): 576. http://dx.doi.org/10.3390/ijms21020576.

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This work assesses the effect of chemical induction with isopropyl β-D-1-thiogalactopyranoside (IPTG) on the expression of enhanced green fluorescent protein (eGFP) by planktonic and biofilm cells of Escherichia coli JM109(DE3) transformed with a plasmid containing a T7 promoter. It was shown that induction negatively affected the growth and viability of planktonic cultures, and eGFP production did not increase. Heterologous protein production was not limited by gene dosage or by transcriptional activity. Results suggest that plasmid maintenance at high copy number imposes a metabolic burden that precludes high level expression of the heterologous protein. In biofilm cells, the inducer avoided the overall decrease in the amount of expressed eGFP, although this was not correlated with the gene dosage. Higher specific production levels were always attained with biofilm cells and it seems that while induction of biofilm cells shifts their metabolism towards the maintenance of heterologous protein concentration, in planktonic cells the cellular resources are directed towards plasmid replication and growth.

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41

Woodson, William R. "Phenotypic and Genetic Stability of Petunias Expressing Sense and Antisense RNAs of Endogenous Genes." HortScience 30, no. 4 (July 1995): 903F—903. http://dx.doi.org/10.21273/hortsci.30.4.903f.

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The target of many genetic engineering experiments is to inhibit the expression of an endogenous gene. For example, research in my laboratory attempts to suppress the expression of ethylene biosynthetic pathway genes to inhibit the production of ethylene and delay flower senescence. The silencing of endogenous genes is generally accomplished by engineering plants to express either antisense or sense RNAs homologous to the target sequence. The mechanism by which gene silencing occurs is not clearly understood. Genetic and molecular analyses of transgene-induced silencing has revealed both meiotically reversible and fully stable phenotypes resulting from the expression of the transgene. In several cases, the mechanisms potentially involved in the silencing of the transgene and concomitant reversion of phenotype have been studied. These include transgene copy number, configuration of the integrated DNA, level of transgene RNA, and environmental factors. In many cases the silencing of transgenes was correlated with DNA methylation. These phenomena and the implications for engineering horticultural crops to express transgenes will be discussed in this workshop.

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42

Udugama, Maheshi, Elaine Sanij, Hsiao P. J. Voon, Jinbae Son, Linda Hii, Jeremy D. Henson, F. Lyn Chan, et al. "Ribosomal DNA copy loss and repeat instability in ATRX-mutated cancers." Proceedings of the National Academy of Sciences 115, no. 18 (April 18, 2018): 4737–42. http://dx.doi.org/10.1073/pnas.1720391115.

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ATRX (alpha thalassemia/mental retardation X-linked) complexes with DAXX to deposit histone variant H3.3 into repetitive heterochromatin. Recent genome sequencing studies in cancers have revealed mutations in ATRX and their association with ALT (alternative lengthening of telomeres) activation. Here we report depletion of ATRX in mouse ES cells leads to selective loss in ribosomal RNA gene (rDNA) copy number. Supporting this, ATRX-mutated human ALT-positive tumors also show a substantially lower rDNA copy than ALT-negative tumors. Further investigation shows that the rDNA copy loss and repeat instability are caused by a disruption in H3.3 deposition and thus a failure in heterochromatin formation at rDNA repeats in the absence of ATRX. We also find that ATRX-depleted cells are reduced in ribosomal RNA transcription output and show increased sensitivity to RNA polymerase I (Pol I) transcription inhibitor CX5461. In addition, human ALT-positive cancer cell lines are also more sensitive to CX5461 treatment. Our study provides insights into the contribution of ATRX loss of function to tumorigenesis through the loss of rDNA stability and suggests the therapeutic potential of targeting Pol I transcription in ALT cancers.

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43

Mei, Han, Barbara Arbeithuber, Marzia A. Cremona, Michael DeGiorgio, and Anton Nekrutenko. "A High-Resolution View of Adaptive Event Dynamics in a Plasmid." Genome Biology and Evolution 11, no. 10 (September 18, 2019): 3022–34. http://dx.doi.org/10.1093/gbe/evz197.

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Abstract Coadaptation between bacterial hosts and plasmids frequently results in adaptive changes restricted exclusively to host genome leaving plasmids unchanged. To better understand this remarkable stability, we transformed naïve Escherichia coli cells with a plasmid carrying an antibiotic-resistance gene and forced them to adapt in a turbidostat environment. We then drew population samples at regular intervals and subjected them to duplex sequencing—a technique specifically designed for identification of low-frequency mutations. Variants at ten sites implicated in plasmid copy number control emerged almost immediately, tracked consistently across the experiment’s time points, and faded below detectable frequencies toward the end. This variation crash coincided with the emergence of mutations on the host chromosome. Mathematical modeling of trajectories for adaptive changes affecting plasmid copy number showed that such mutations cannot readily fix or even reach appreciable frequencies. We conclude that there is a strong selection against alterations of copy number even if it can provide a degree of growth advantage. This incentive is likely rooted in the complex interplay between mutated and wild-type plasmids constrained within a single cell and underscores the importance of understanding of intracellular plasmid variability.

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44

Burbank, Lindsey P., and Drake C. Stenger. "Plasmid Vectors for Xylella fastidiosa Utilizing a Toxin-Antitoxin System for Stability in the Absence of Antibiotic Selection." Phytopathology® 106, no. 8 (August 2016): 928–36. http://dx.doi.org/10.1094/phyto-02-16-0097-r.

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The phytopathogen Xylella fastidiosa causes disease in a variety of important crop and landscape plants. Functional genetic studies have led to a broader understanding of virulence mechanisms used by this pathogen in the grapevine host. Plasmid shuttle vectors are important tools in studies of bacterial genetics but there are only a limited number of plasmid vectors available that replicate in X. fastidiosa, and even fewer that are retained without antibiotic selection. Two plasmids are described here that show stable replication in X. fastidiosa and are effective for gene complementation both in vitro and in planta. Plasmid maintenance is facilitated by incorporation of the PemI/PemK plasmid addiction system, consisting of PemK, an endoribonuclease toxin, and its cognate antitoxin, PemI. Vector pXf20pemIK utilizes a native X. fastidiosa replication origin as well as a high-copy-number pUC origin for propagation in Escherichia coli cloning strains. Broad-host-range vector pBBR5pemIK is a medium- to low-copy-number plasmid based on the pBBR1 backbone. Both plasmids are maintained for extended periods of time in the absence of antibiotic selection, as well as up to 14 weeks in grapevine, without affecting bacterial fitness. These plasmids present an alternative to traditional complementation and expression vectors which rely on antibiotic selection for plasmid retention.

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45

Cahyani, Inswasti, Andrew G. Cridge, David R. Engelke, Austen R. D. Ganley, and Justin M. O'Sullivan. "A Sequence-Specific Interaction between the Saccharomyces cerevisiae rRNA Gene Repeats and a Locus Encoding an RNA Polymerase I Subunit Affects Ribosomal DNA Stability." Molecular and Cellular Biology 35, no. 3 (November 24, 2014): 544–54. http://dx.doi.org/10.1128/mcb.01249-14.

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The spatial organization of eukaryotic genomes is linked to their functions. However, how individual features of the global spatial structure contribute to nuclear function remains largely unknown. We previously identified a high-frequency interchromosomal interaction within theSaccharomyces cerevisiaegenome that occurs between the intergenic spacer of the ribosomal DNA (rDNA) repeats and the intergenic sequence between the locus encoding the second largest RNA polymerase I subunit and a lysine tRNA gene [i.e.,RPA135-tK(CUU)P]. Here, we used quantitative chromosome conformation capture in combination with replacement mapping to identify a 75-bp sequence within theRPA135-tK(CUU)Pintergenic region that is involved in the interaction. We demonstrate that theRPA135-IGS1 interaction is dependent on the rDNA copy number and the Msn2 protein. Surprisingly, we found that the interaction does not governRPA135transcription. Instead, replacement of a 605-bp region within theRPA135-tK(CUU)Pintergenic region results in a reduction in theRPA135-IGS1 interaction level and fluctuations in rDNA copy number. We conclude that the chromosomal interaction that occurs between theRPA135-tK(CUU)Pand rDNA IGS1 loci stabilizes rDNA repeat number and contributes to the maintenance of nucleolar stability. Our results provide evidence that the DNA loci involved in chromosomal interactions are composite elements, sections of which function in stabilizing the interaction or mediating a functional outcome.

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46

Kampmann, K., S. Ratering, I. Kramer, M. Schmidt, W. Zerr, and S. Schnell. "Unexpected Stability of Bacteroidetes and Firmicutes Communities in Laboratory Biogas Reactors Fed with Different Defined Substrates." Applied and Environmental Microbiology 78, no. 7 (January 13, 2012): 2106–19. http://dx.doi.org/10.1128/aem.06394-11.

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ABSTRACTIn the present study, bacterial communities in 200-liter biogas reactors containing liquid manure consecutively fed with casein, starch, and cream were investigated over a period of up to 33 days. A 16S rRNA gene clone library identifiedBacteroidetesandFirmicutesas the most abundant bacterial groups in the starting material, at 58.9% and 30.1% of sequences, respectively. The community development of both groups was monitored by real-time PCR and single-strand conformation polymorphism (SSCP) analysis. TheFirmicutesandBacteroidetescommunities were unexpectedly stable and hardly influenced by batch-feeding events. The continuous feeding of starch led to community shifts that nevertheless contributed to a stable reactor performance. A longer starving period and a change in the pH value resulted in further community shifts within theBacteroidetesbut did not influence theFirmicutes. Predominant DNA bands from SSCP gels were cloned and sequenced. Sequences related toPeptococcaceae,Cytophagales, andPetrimonas sulfuriphilawere found in all samples from all experiments. Real-time PCR demonstrated the abundance of members of the phylumBacteroidetesand also reflected changes in gene copy numbers in conjunction with a changing pH value and acetate accumulation.

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47

Hsu, Chin-Chen, and Carton W. Chen. "Linear Plasmid SLP2 Is Maintained by Partitioning, Intrahyphal Spread, and Conjugal Transfer in Streptomyces." Journal of Bacteriology 192, no. 1 (October 30, 2009): 307–15. http://dx.doi.org/10.1128/jb.01192-09.

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ABSTRACT Low-copy-number plasmids generally encode a partitioning system to ensure proper segregation after replication. Little is known about partitioning of linear plasmids in Streptomyces. SLP2 is a 50-kb low-copy-number linear plasmid in Streptomyces lividans, which contains a typical parAB partitioning operon. In S. lividans and Streptomyces coelicolor, a parAB deletion resulted in moderate plasmid loss and growth retardation of colonies. The latter was caused by conjugal transfer from plasmid-containing hyphae to plasmidless hyphae. Deletion of the transfer (traB) gene eliminated conjugal transfer, lessened the growth retardation of colonies, and increased plasmid loss through sporulation cycles. The additional deletion of an intrahyphal spread gene (spd1) caused almost complete plasmid loss in a sporulation cycle and eliminated all growth retardation. Moreover, deletion of spd1 alone severely reduced conjugal transfer and stability of SLP2 in S. coelicolor M145 but had no effect on S. lividans TK64. These results revealed the following three systems for SLP2 maintenance: partitioning and spread for moving the plasmid DNA along the hyphae and into spores and conjugal transfer for rescuing plasmidless hyphae. In S. lividans, both spread and partitioning appear to overlap functionally, but in S. coelicolor, spread appears to play the main role.

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48

Dong, Chongmei, Ryan Whitford, and Peter Langridge. "A DNA mismatch repair gene links to the Ph2 locus in wheat." Genome 45, no. 1 (February 1, 2002): 116–24. http://dx.doi.org/10.1139/g01-126.

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DNA mismatch repair is an essential system for maintaining genetic stability in bacteria and higher eukaryotes. Based on the conserved regions of the bacterial MutS gene and its homologues in yeast and human, a wheat cDNA homologue of MSH6, designated TaMSH7, was isolated by RTPCR. The deduced amino acid sequence of TaMSH7 shows conserved domains characteristic of other MSH6 genes, with highest similarity to maize MSH7 and Arabidopsis MSH7. TaMSH7 is expressed in meristem tissue associated with a high level of mitotic and meiotic activity, with maximum expression in the reproductive organs of young flower spikes. TaMSH7 is located on the short arms of chromosomes 3A, 3B, and 3D and has been mapped within barley chromosome 3HS. The copy on 3DS is located within the region deleted in the wheat mutant ph2a, which shows altered recombination frequency in the interspecific hybrids. The relationship between the ph2a mutant and TaMSH7 gene function is discussed.Key words: wheat, DNA mismatch repair gene, expression, chromosomal location, Ph2.

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49

Barbour, Leslie, Yu Zhu, and Wei Xiao. "Improving synthetic lethal screens by regulating the yeast centromere sequence." Genome 43, no. 5 (October 1, 2000): 910–17. http://dx.doi.org/10.1139/g00-050.

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The synthetic lethal screen is a useful method in identifying novel genes functioning in an alternative pathway to the gene of interest. The current synthetic lethal screen protocol in yeast is based on a colony-sectoring assay that allows direct visualization of mutant colonies among a large population by their inability to afford plasmid loss. This method demands an appropriate level of stability of the plasmid carrying the gene of interest. YRp-based plasmids are extremely unstable and complete plasmid loss occurs within a few generations. Consequently, YCp plasmids are the vector of choice for synthetic lethal screens. However, we found that the high-level stability of YCp plasmids resulted in a large number of false positives that must be further characterized. In this study, we attempt to improve the existing synthetic lethal screen protocol by regulating the plasmid stability and copy number. It was found that by placing a yeast centromere sequence under the control of either inducible or constitutive promoters, plasmid stability can be significantly decreased. Hence, altering the conditions under which yeast cells carrying the plasmid PGAL1-CEN4 were cultivated allowed us to develop a method that eliminated virtually 100% of false positives and drastically reduced the time required to carry out a synthetic lethal screen.Key words: synthetic lethal screen, yeast, centromere, inducible promoter, MRE11.

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50

Miller, Danny E. "Synaptonemal Complex-Deficient Drosophila melanogaster Females Exhibit Rare DSB Repair Events, Recurrent Copy-Number Variation, and an Increased Rate of de Novo Transposable Element Movement." G3: Genes|Genomes|Genetics 10, no. 2 (December 27, 2019): 525–37. http://dx.doi.org/10.1534/g3.119.400853.

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Genetic stability depends on the maintenance of a variety of chromosome structures and the precise repair of DNA breaks. During meiosis, programmed double-strand breaks (DSBs) made in prophase I are normally repaired as gene conversions or crossovers. DSBs can also be made by other mechanisms, such as the movement of transposable elements (TEs), which must also be resolved. Incorrect repair of these DNA lesions can lead to mutations, copy-number changes, translocations, and/or aneuploid gametes. In Drosophila melanogaster, as in most organisms, meiotic DSB repair occurs in the presence of a rapidly evolving multiprotein structure called the synaptonemal complex (SC). Here, whole-genome sequencing is used to investigate the fate of meiotic DSBs in D. melanogaster mutant females lacking functional SC, to assay for de novo CNV formation, and to examine the role of the SC in transposable element movement in flies. The data indicate that, in the absence of SC, copy-number variation still occurs and meiotic DSB repair by gene conversion occurs infrequently. Remarkably, an 856-kilobase de novo CNV was observed in two unrelated individuals of different genetic backgrounds and was identical to a CNV recovered in a previous wild-type study, suggesting that recurrent formation of large CNVs occurs in Drosophila. In addition, the rate of novel TE insertion was markedly higher than wild type in one of two SC mutants tested, suggesting that SC proteins may contribute to the regulation of TE movement and insertion in the genome. Overall, this study provides novel insight into the role that the SC plays in genome stability and provides clues as to why the sequence, but not structure, of SC proteins is rapidly evolving.

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