Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 30.344
Filtrar
1.
Methods Mol Biol ; 2392: 161-171, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34773622

RESUMO

Transgenic events are defined as exogenous DNA insertion in the genome through genetic transformation. It is a powerful means for the improvement of crop plants and to understand the gene function. Multiple DNA insertion events may occur at one or several chromosomal locations. One of the important tasks, after validation of the transformation of transgenic plants, is the identification of single copy in the transgenic. This means the insertion of exogenous DNA fragment only in a single locus in the genome. Southern blot hybridization is a convincing and reliable method, for estimation of copy number in transgenic lines but it is cumbersome and time-consuming process. One of the other well-known methods is quantitative polymerase chain reactions (qPCR), a simple and rapid method to identify copy number from a population of independent transgenic lines. In comparison to the Southern hybridization method, qPCR is simpler to perform, requires less DNA, lesser time and does not require any labeled probes. This method utilizes specific primers to amplify target transgenes and endogenous reference genes. Designing an appropriate and specific primer pair is a very crucial part of the estimation of the gene copy number. In this chapter, we have illustrated a detailed methodology for identification of the gene copy of the transgenic plants.


Assuntos
Dosagem de Genes , Southern Blotting , Plantas Geneticamente Modificadas/genética , Reação em Cadeia da Polimerase em Tempo Real , Transgenes
2.
Nat Commun ; 12(1): 5784, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34599161

RESUMO

Cardiac regeneration involves the generation of new cardiomyocytes from cycling cardiomyocytes. Understanding cell-cycle activity of pre-existing cardiomyocytes provides valuable information to heart repair and regeneration. However, the anatomical locations and in situ dynamics of cycling cardiomyocytes remain unclear. Here we develop a genetic approach for a temporally seamless recording of cardiomyocyte-specific cell-cycle activity in vivo. We find that the majority of cycling cardiomyocytes are positioned in the subendocardial muscle of the left ventricle, especially in the papillary muscles. Clonal analysis revealed that a subset of cycling cardiomyocytes have undergone cell division. Myocardial infarction and cardiac pressure overload induce regional patterns of cycling cardiomyocytes. Mechanistically, cardiomyocyte cell cycle activity requires the Hippo pathway effector YAP. These genetic fate-mapping studies advance our basic understanding of cardiomyocyte cell cycle activity and generation in cardiac homeostasis, repair, and regeneration.


Assuntos
Miócitos Cardíacos/citologia , Animais , Southern Blotting , Ciclo Celular/fisiologia , Proliferação de Células/fisiologia , Feminino , Citometria de Fluxo , Coração/fisiologia , Masculino , Camundongos , Microscopia de Fluorescência , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo
3.
Sci Rep ; 11(1): 10543, 2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34007007

RESUMO

Pigeon pea, a grain legume of the semiarid tropics, is a rich source of high-quality protein. The productivity of this pulse is seriously affected by lepidopteron insect pests. To generate a sustainable insect-resistant plant, synthetically prepared bioactive key constituents of a crystal protein (Syn Cry1Ab) of Bacillus thuringiensis were expressed in pigeon pea under the guidance of a tissue-specific promoter of the RuBP carboxylase/oxygenase small subunit (rbcS) gene. Regenerated transgenic plants with the cry1Ab expression cassette (cry1Ab-lox-bar-lox) showed the optimum insect motility rate (90%) in an in vitro insect bioassay with second instar larvae, signifying the insecticidal potency of Syn Cry1Ab. In parallel, another plant line was also generated with a chimaeric vector harbouring a cre recombinase gene under the control of the CaMV 2 × 35S promoter. Crossing between T1 plants with a single insertion of cry1Ab-lox-bar-lox T-DNA and T1 plants with moderate expression of a cre gene with a linked hygromycin resistance (hptII) gene was performed to exclude the bialaphos resistance (bar) marker gene. Excision of the bar gene was achieved in T1F1 hybrids, with up to 35.71% recombination frequency. Insect-resistant pigeon pea plants devoid of selectable marker genes (syn Cry1Ab- bar and cre-hptII) were established in a consecutive generation (T1F2) through genetic segregation.


Assuntos
Proteínas de Bactérias/metabolismo , Cajanus/metabolismo , Inseticidas/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Animais , Toxinas de Bacillus thuringiensis/genética , Southern Blotting , Endotoxinas/genética , Proteínas Hemolisinas/genética
4.
Methods Mol Biol ; 2267: 191-205, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33786793

RESUMO

Damage to DNA elicits both checkpoint and repair responses. These are complex events that involve many genes whose products assemble at lesions and form signaling cascades to recruit additional factors and regulate the cell cycle. The fission yeast Schizosaccharomyces pombe has proven to be an excellent model to study these events, and has led gene and pathway discovery efforts. Recent progress has involved a more detailed analysis of the earliest events at lesions, particularly double-stranded DNA breaks (DSBs). Here we describe several methods for the analysis of events at DSBs, both on the DNA and the recruitment of proteins to these lesions, using S. pombe as a model. However, each of these methods is easily applicable to any experimental system with minor modifications to the protocols.


Assuntos
Sequenciamento de Cromatina por Imunoprecipitação/métodos , Quebras de DNA de Cadeia Dupla , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reparo de DNA por Recombinação , Schizosaccharomyces/genética , Southern Blotting/métodos , Western Blotting/métodos , Ciclo Celular , Técnicas Microbiológicas/métodos , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo
5.
Ann Lab Med ; 41(4): 394-400, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-33536358

RESUMO

Background: Conventional diagnosis of fragile X syndrome (FXS) is based on a combination of fragment analysis (FA) and Southern blotting (SB); however, this diagnostic approach is time- and labor-intensive and has pitfalls such as the possibility of missing large number alleles. Triplet repeat primed PCR (TP-PCR) is a current alternative used to overcome these limitations. We evaluated the diagnostic usefulness of TP-PCR compared with the conventional diagnostic protocol consisting of FA and/or SB in terms of allele categorization, repeat number correlation, and zygosity concordance in female genetic carriers. Methods: From November 2013 to March 2018, 458 patients (326 males, 132 females) were simultaneously examined using FA and/or SB and TP-PCR by detecting CGG repeat numbers in FMR1 gene and diagnosed as per American College of Medical Genetics guidelines. Results: The TP-PCR results showed high concordance with the FA and/or SB results for all three aspects (allele categorization, repeat number correlation, and zygosity concordance in female genetic carriers). TP-PCR detected CGG expansions ≥200 in all full mutation (FM) allele cases in male patients, as well as both the normal allele (NL) and FM allele in female carriers. In premutation (PM) allele carriers, the TP-PCR results were consistent with the FA and/or SB results. In terms of zygosity concordance in female genetic carriers, 12 NL cases detected by TP-PCR showed a merged peak consisting of two close heterozygous peaks; however, this issue was resolved using a 10-fold dilution. Conclusions: TP-PCR may serve as a reliable alternative method for FXS diagnosis.


Assuntos
Síndrome do Cromossomo X Frágil , Alelos , Southern Blotting , Feminino , Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/genética , Humanos , Masculino , Mutação , Reação em Cadeia da Polimerase , Repetições de Trinucleotídeos
6.
Am J Epidemiol ; 190(7): 1406-1413, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33564874

RESUMO

Researchers increasingly wish to test hypotheses concerning the impact of environmental or disease exposures on telomere length (TL), and they use longitudinal study designs to do so. In population studies, TL is usually measured with a quantitative polymerase chain reaction (qPCR)-based method. This method has been validated by calculating its correlation with a gold standard method such as Southern blotting (SB) in cross-sectional data sets. However, in a cross-section, the range of true variation in TL is large, and measurement error is introduced only once. In a longitudinal study, the target variation of interest is small, and measurement error is introduced at both baseline and follow-up. In this paper, we present results from a small data set (n = 20) in which leukocyte TL was measured twice 6.6 years apart by means of both qPCR and SB. The cross-sectional correlations between qPCR and SB were high at both baseline (r = 0.90) and follow-up (r = 0.85), yet their correlation for TL change was poor (r = 0.48). Moreover, the qPCR data but not the SB data showed strong signatures of measurement error. Through simulation, we show that the statistical power gain from performing a longitudinal analysis is much greater for SB than for qPCR. We discuss implications for optimal study design and analysis.


Assuntos
Southern Blotting/estatística & dados numéricos , Correlação de Dados , Leucócitos/ultraestrutura , Reação em Cadeia da Polimerase em Tempo Real/estatística & dados numéricos , Telômero , Estudos Transversais , Humanos , Estudos Longitudinais , Reprodutibilidade dos Testes , Projetos de Pesquisa
7.
Invest Ophthalmol Vis Sci ; 62(1): 17, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33444430

RESUMO

Purpose: To characterize inheritance, penetrance, and trinucleotide repeat expansion stability in Fuchs endothelial corneal dystrophy (FECD). Methods: One thousand unrelated and related subjects with and without FECD were prospectively recruited. CTG18.1 repeat length (CTG18.1L) was determined via short tandem repeat assay and Southern blotting of leukocyte DNA. Multivariable logistic regression and generalized estimating equation models were employed. Results: There were 546 unrelated FECD cases (67.6% female; 70 ± 10 years) and 235 controls (63.8% female; 73 ± 8 years; all ≥ 50 years). CTG18.1 expansion (CTG18.1exp+) was observed in 424 (77.7%) cases and 18 (7.7%) controls (P = 2.48 × 10-44). CTG18.1 expansion was associated with FECD severity (P = 5.62 × 10-7). The family arm of the study included 331 members from 112 FECD-affected families; 87 families were CTG18.1exp+. Autosomal dominant inheritance with variable expression of FECD was observed, regardless of expansion status. FECD penetrance of CTG18.1 expansion increased with age, ranging from 44.4% in the youngest (19-46 years) to 86.2% in the oldest (64-91 years) age quartiles. Among 62 parent-offspring transmissions of CTG18.1exp+, 48 (77.4%) had a change in CTG18.1L ≤ 10 repeats, and eight (12.9%) were ≥50 repeats, including five large expansions (∼1000-2000 repeats) that contracted. Among 44 offspring who did not inherit the CTG18.1exp+ allele, eight (18.2%) exhibited FECD. Conclusions: CTG18.1 expansion was highly associated with FECD but demonstrated incomplete penetrance. CTG18.1L instability occurred in a minority of parent-offspring transmissions, with large expansions exhibiting contraction. The observation of FECD without CTG18.1 expansion among family members in CTG18.1exp+ families highlights the complexity of the relationship between the FECD phenotype and CTG18.1 expansion.


Assuntos
Distrofia Endotelial de Fuchs/genética , Fator de Transcrição 4/genética , Expansão das Repetições de Trinucleotídeos/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Southern Blotting , DNA/genética , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Padrões de Herança , Masculino , Repetições de Microssatélites , Pessoa de Meia-Idade , Linhagem , Penetrância , Reação em Cadeia da Polimerase , Polimorfismo Genético , Estudos Prospectivos , Adulto Jovem
8.
FASEB J ; 35(2): e21308, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33481304

RESUMO

Peroxisome proliferator-activated receptor gamma (PPARγ) is a master regulator of adipogenesis and lipogenesis. To understand its roles in fiber formation and fat deposition in skeletal muscle, we successfully generated muscle-specific overexpression of PPARγ in two pig models by random insertion and CRISPR/Cas9 transgenic cloning procedures. The content of intramuscular fat was significantly increased in PPARγ pigs while had no changes on lean meat ratio. PPARγ could promote adipocyte differentiation by activating adipocyte differentiating regulators such as FABP4 and CCAAT/enhancer-binding protein (C/EBP), along with enhanced expression of LPL, FABP4, and PLIN1 to proceed fat deposition. Proteomics analyses demonstrated that oxidative metabolism of fatty acids and respiratory chain were activated in PPARγ pigs, thus, gathered more Ca2+ in PPARγ pigs. Raising of Ca2+ could result in increased phosphorylation of CAMKII and p38 MAPK in PPARγ pigs, which can stimulate MEF2 and PGC1α to affect fiber type and oxidative capacity. These results support that skeletal muscle-specific overexpression of PPARγ can promote oxidative fiber formation and intramuscular fat deposition in pigs.


Assuntos
DNA Mitocondrial/metabolismo , Músculo Esquelético/metabolismo , PPAR gama/metabolismo , Adipócitos/metabolismo , Adipogenia/genética , Adipogenia/fisiologia , Animais , Southern Blotting , Western Blotting , Proteína alfa Estimuladora de Ligação a CCAAT , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Variações do Número de Cópias de DNA/genética , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Metabolismo dos Lipídeos/genética , Metabolismo dos Lipídeos/fisiologia , Oxirredução , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Perilipina-1/genética , Perilipina-1/metabolismo , Proteômica , Reação em Cadeia da Polimerase em Tempo Real , Suínos
9.
Jpn J Infect Dis ; 74(1): 48-53, 2021 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-32741932

RESUMO

JC polyomavirus (JCPyV) causes progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the central nervous system affecting immunocompromised patients. The study of PML-type JCPyV in vitro has been limited owing to the inefficient propagation of the virus in cultured cells. In this study, we carried out long-term culture of COS-7 cells (designated as COS-IMRb cells) transfected with PML-type M1-IMRb, an adapted viral DNA with a rearranged non-coding control region (NCCR). The JCPyV derived from COS-IMRb cells were characterized by analyzing the viral replication, amount of virus by hemagglutination (HA), production of viral protein 1 (VP1), and structure of the NCCR. HA assays indicated the presence of high amounts of PML-type JCPyV in COS-IMRb cells. Immunostaining showed only a small population of JCPyV carrying COS-IMRb cells to be VP1-positive. Sequencing analysis of the NCCR of JCPyV after long-term culture revealed that the NCCR of M1-IMRb was conserved in COS-IMRb cells without any point mutation. The JCPyV genomic DNA derived from a clone of COS-IMRb-3 cells was detected, via Southern blotting, as a single band of approximately 5.1 kbp without deletion. These findings suggest the potential of using COS-IMRb-3 cells as a useful tool for screening anti-JCPyV drugs.


Assuntos
Vírus JC/crescimento & desenvolvimento , Vírus JC/genética , Leucoencefalopatia Multifocal Progressiva/virologia , Cultura de Vírus/métodos , Animais , Southern Blotting/métodos , Células COS , Chlorocebus aethiops , Replicação do DNA , DNA Viral/isolamento & purificação , Hemaglutinação , Humanos , Transfecção , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral
10.
Plant Sci ; 302: 110671, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33288000

RESUMO

Repeated sequences and polyploidy play a central role in plant genome dynamics. Here, we analyze the evolutionary dynamics of repeats in tetraploid and hexaploid Spartina species that diverged during the last 10 million years within the Chloridoideae, one of the poorest investigated grass lineages. From high-throughput genome sequencing, we annotated Spartina repeats and determined what sequence types account for the genome size variation among species. We examined whether differential genome size evolution correlated with ploidy levels and phylogenetic relationships. We also examined the tempo of repeat sequence dynamics associated with allopatric speciation over the last 3-6 million years between hexaploid species that diverged on the American and European Atlantic coasts and tetraploid species from North and South America. The tetraploid S. spartinae, whose phylogenetic placement has been debated, exhibits a similar repeat content as hexaploid species, suggesting common ancestry. Genome expansion or contraction resulting from repeat dynamics seems to be explained mostly by the contrasting divergence times between species, rather than by genome changes triggered by ploidy level change per se. One 370 bp satellite may be exhibiting 'meiotic drive' and driving chromosome evolution in S. alterniflora. Our results provide crucial insights for investigating the genetic and epigenetic consequences of such differential repeat dynamics on the ecology and distribution of the meso- and neopolyploid Spartina species.


Assuntos
Elementos de DNA Transponíveis/genética , DNA Satélite/genética , Evolução Molecular , Poaceae/genética , Poliploidia , Southern Blotting , Genoma de Planta/genética , Sequenciamento de Nucleotídeos em Larga Escala , Filogenia
11.
Mol Plant Pathol ; 22(2): 216-230, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33231927

RESUMO

Badnaviruses are double-stranded DNA pararetroviruses of the family Caulimoviridae. Badnaviral sequences found in banana are distributed over three main clades of the genus Badnavirus and exhibit wide genetic diversity. Interestingly, the nuclear genome of many plants, including banana, is invaded by numerous badnaviral sequences although badnaviruses do not require an integration step to replicate, unlike animal retroviruses. Here, we confirm that banana streak viruses (BSVs) are restricted to clades 1 and 3. We also show that only BSVs from clade 3 encompassing East African viral species are not integrated into Musa genomes, unlike BSVs from clade 1. Finally, we demonstrate that sequences from clade 2 are definitively integrated into Musa genomes with no evidence of episomal counterparts; all are phylogenetically distant from BSVs known to date. Using different molecular approaches, we dissected the coevolution between badnaviral sequences of clade 2 and banana by comparing badnavirus integration patterns across a banana sampling representing major Musa speciation events. Our data suggest that primary viral integrations occurred millions of years ago in banana genomes under different possible scenarios. Endogenous badnaviral sequences can be used as powerful markers to better characterize the Musa phylogeny, narrowing down the likely geographical origin of the Musa ancestor.


Assuntos
Badnavirus/genética , Musa/virologia , Badnavirus/classificação , Coevolução Biológica , Southern Blotting , DNA Viral/análise , Genoma de Planta , Musa/genética , Filogenia , Reação em Cadeia da Polimerase , Uganda , Integração Viral
12.
Plant Cell Environ ; 44(3): 842-855, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33377200

RESUMO

The phase transition from vegetative to reproductive growth is triggered by internal and external signals that participate in circadian clock in plants. We identified a rice floral inhibitor OsPRR73 encoding a CONSTANS protein. Overexpression of OsPRR73 resulted in late heading under both long-day (LD) and short-day (SD) conditions. Knockout mutants led to early heading under LD conditions but no change under SD. OsPRR73 mRNA accumulated at noon and exhibited a robust oscillation under constant light (LL) and constant darkness (DD) conditions. OsPRR73 overexpression exerted negative feedback on endogenous OsPRR73 expression and altered diurnal expressions of key flowering genes and circadian clock genes. OsPRR73 bound to the promoters of the floral gene Ehd1 and the circadian gene OsLHY, and significantly suppressed their expression at dawn. In LL and DD, the oscillatory patterns of the circadian genes OsLHY, OsTOC1, OsGI and OsELF3 were varied in OsPRR73OX and osprr73 mutants. OsPRR73 expression was decreased in osphyb mutants, and overexpression of OsPRR73 complemented the early heading date phenotype of osphyb, indicating OsPRR73 works downstream of OsPhyB. Therefore, OsPRR73 is involved in a feedback loop of the rice clock and connects the photoperiod flowering pathway by binding to the Ehd1 promoter in rice.


Assuntos
Relógios Circadianos , Oryza/metabolismo , Fotoperíodo , Proteínas de Plantas/metabolismo , Proteínas Repressoras/metabolismo , Southern Blotting , Relógios Circadianos/fisiologia , Ensaio de Desvio de Mobilidade Eletroforética , Regulação da Expressão Gênica de Plantas , Oryza/fisiologia , Proteínas de Plantas/fisiologia , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/fisiologia , Técnicas do Sistema de Duplo-Híbrido
13.
J Cell Mol Med ; 25(2): 840-854, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33263949

RESUMO

Hepatitis B virus (HBV) infection is a major public health problem. The high levels of HBV DNA and HBsAg are positively associated with the development of secondary liver diseases, including hepatocellular carcinoma (HCC). Current treatment with nucleos(t)ide analogues mainly reduces viral DNA, but has minimal, if any, inhibitory effect on the viral antigen. Although IFN reduces both HBV DNA and HBsAg, the serious associated side effects limit its use in clinic. Thus, there is an urgent demanding for novel anti-HBV therapy. In our study, viral parameters were determined in the supernatant of HepG2.2.15 cells, HBV-expressing Huh7 and HepG2 cells which transfected with HBV plasmids and in the serum of HBV mouse models with hydrodynamic injection of pAAV-HBV1.2 plasmid. RT-qPCR and Southern blot were performed to detect 35kb mRNA and cccDNA. RT-qPCR, Luciferase assay and Western blot were used to determine anti-HBV effects of MLN4924 and the underlying mechanisms. We found that treatment with MLN4924, the first-in-class neddylation inhibitor currently in several phase II clinical trials for anti-cancer application, effectively suppressed production of HBV DNA, HBsAg, 3.5kb HBV RNA as well as cccDNA. Mechanistically, MLN4924 blocks cullin neddylation and activates ERK to suppress the expression of several transcription factors required for HBV replication, including HNF1α, C/EBPα and HNF4α, leading to an effective blockage in the production of cccDNA and HBV antigen. Our study revealed that neddylation inhibitor MLN4924 has impressive anti-HBV activity by inhibiting HBV replication, thus providing sound rationale for future MLN4924 clinical trial as a novel anti-HBV therapy.


Assuntos
Ciclopentanos/farmacologia , Vírus da Hepatite B/metabolismo , Vírus da Hepatite B/patogenicidade , Fator 4 Nuclear de Hepatócito/metabolismo , Pirimidinas/farmacologia , Fatores de Transcrição/metabolismo , Animais , Southern Blotting , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Ciclopentanos/uso terapêutico , Células Hep G2 , Vírus da Hepatite B/efeitos dos fármacos , Fator 4 Nuclear de Hepatócito/genética , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Camundongos , Pirimidinas/uso terapêutico , RNA Mensageiro/metabolismo , Fatores de Transcrição/genética
14.
Methods Mol Biol ; 2153: 33-45, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32840770

RESUMO

Generation of 3' single-stranded DNA (ssDNA) at the ends of a double-strand break (DSB) is essential to initiate repair by homology-directed mechanisms. Here we describe a Southern blot-based method to visualize the generation of ssDNA at the ends of site-specific DSBs generated in the Saccharomyces cerevisiae genome.


Assuntos
DNA de Cadeia Simples/metabolismo , Reparo de DNA por Recombinação , Saccharomyces cerevisiae/genética , Southern Blotting , Quebras de DNA de Cadeia Dupla , DNA Fúngico/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Eletroforese , Proteínas de Saccharomyces cerevisiae/metabolismo
15.
Methods Mol Biol ; 2153: 47-57, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32840771

RESUMO

DNA double-strand break (DSB) end resection is an essential step for homologous recombination. It generates 3' single-stranded DNA needed for the loading of the strand exchange proteins and DNA damage checkpoint proteins. To study the mechanism of end resection in fission yeast, we apply a robust, quantitative and inducible assay. Resection is followed at a single per genome DSB synchronously generated by the tet-inducible I-PpoI endonuclease. An additional assay to follow resection involves recombination between two direct repeats by single-strand annealing (SSA), since SSA requires extensive resection to expose two single-strand repeats for annealing. The kinetics of resection and SSA repair are then measured using Southern blots.


Assuntos
DNA de Cadeia Simples/metabolismo , Reparo de DNA por Recombinação , Schizosaccharomyces/genética , Southern Blotting , Quebras de DNA de Cadeia Dupla , DNA Fúngico/metabolismo , Endodesoxirribonucleases/metabolismo
16.
Methods Mol Biol ; 2153: 71-86, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32840773

RESUMO

DNA repeats capable of adopting stable secondary structures are hotspots for double-strand break (DSB) formation and, hence, for homologous recombination and gross chromosomal rearrangements (GCR) in many prokaryotic and eukaryotic organisms, including humans. Here, we provide protocols for studying chromosomal instability triggered by hairpin- and cruciform-forming palindromic sequences in the budding yeast, Saccharomyces cerevisiae. First, we describe two sensitive genetic assays aimed to determine the recombinogenic potential of inverted repeats and their ability to induce GCRs. Then, we detail an approach to monitor chromosomal DSBs by Southern blot hybridization. Finally, we describe how to define the molecular structure of DSBs. We provide, as an example, the analysis of chromosomal fragility at a reporter system containing unstable Alu-inverted repeats. By using these approaches, any DNA sequence motif can be assessed for its breakage potential and ability to drive genome instability.


Assuntos
Quebra Cromossômica , Cromossomos Fúngicos/metabolismo , Saccharomyces cerevisiae/genética , Elementos Alu , Southern Blotting , Cromossomos Fúngicos/química , DNA Cruciforme/metabolismo , Sequências Repetidas Invertidas , Conformação de Ácido Nucleico
17.
Methods Mol Biol ; 2153: 221-238, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32840783

RESUMO

By using an inducible site-specific double-strand break (DSB) in budding yeast, it is possible to monitor-in real time-the repair of the break by homologous recombination. A method is described using an ectopic homologous donor sequence to repair an HO endonuclease-induced DSB. These gene conversion events can occur with or without crossing-over, the products of which are distinguished as different-sized restriction endonuclease fragments. The method of Southern blotting is described in detail.


Assuntos
DNA Fúngico/genética , Conversão Gênica , Saccharomyces cerevisiae/genética , Southern Blotting , DNA , Enzimas de Restrição do DNA/metabolismo
18.
Methods Mol Biol ; 2153: 395-402, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32840794

RESUMO

Upon telomerase inactivation telomeres are getting shorter at each round of DNA replication and progressively lose capping functions and hence protection against homologous recombination. In addition, telomerase-minus cells undergo a round of stochastic DNA damage before the bulk of telomeres become critically short because telomeres are difficult regions to replicate. Although most of the cells will enter finally replicative senescence, those that unleash recombination can eventually recover functional telomeres and growth capacity. Formation of these survivors in yeast depends on various recombination mechanisms. Here, we present assays that we developed to analyze and quantify recombination at telomeres.


Assuntos
Saccharomyces cerevisiae/crescimento & desenvolvimento , Telomerase/genética , Telômero/metabolismo , Southern Blotting , Replicação do DNA , Recombinação Homóloga , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
19.
Methods Mol Biol ; 2153: 403-425, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32840795

RESUMO

The ribosomal RNA (rDNA) sequence is the most abundant repetitive element in the budding yeast genome and forms a tandem cluster of ~100-200 copies. Cells frequently change their rDNA copy number, making rDNA the most unstable region in the budding yeast genome. The rDNA region experiences programmed replication fork arrest and subsequent formation of DNA double-strand breaks (DSBs), which are the main drivers of rDNA instability. The rDNA region offers a unique system to understand the mechanisms that respond to replication fork arrest as well as the mechanisms that regulate repeat instability. This chapter describes three methods to assess rDNA instability.


Assuntos
DNA Ribossômico/metabolismo , Eletroforese em Gel de Campo Pulsado/métodos , Saccharomyces cerevisiae/genética , Southern Blotting , Cromossomos Fúngicos/genética , Quebras de DNA de Cadeia Dupla , Replicação do DNA , DNA Circular/química , DNA Circular/metabolismo , DNA Fúngico/química , DNA Fúngico/metabolismo , DNA Ribossômico/química
20.
Methods Mol Biol ; 2196: 245-255, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32889727

RESUMO

DNA double-strand break (DSB) is one of the most deleterious types of DNA lesions threatening genome integrity. Cells have evolved several exquisite pathways to repair these breaks. Homologous recombination (HR) is an essential DSB repair mechanism that utilizes an intact homologous sequence as a template to repair DSBs with high fidelity. To initiate the HR repair, the 5'-ends of DSBs have to be nucleolytically cleaved by nucleases to generate 3'-single-strand DNA (ssDNA). Exposed 3'-ssDNA recruits the ssDNA binding protein complex RPA to activate the DNA damage checkpoint. RPA is subsequently replaced by Rad51 recombinase to form Rad51 nucleoprotein filament that catalyzes strand invasion and formation of the D-loop. Processing of 5'-ends (called resection) is a crucial step that determines the choice of repair pathways. Here we introduce an assay for monitoring the dynamics of resection at different locations from a site-specific DSB in yeast.


Assuntos
Southern Blotting/métodos , Quebras de DNA de Cadeia Dupla , Reparo de DNA por Recombinação , Genoma Fúngico , Rad51 Recombinase/metabolismo , Leveduras/genética , Leveduras/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...