scholarly journals Lox’d in translation: Contradictions in the nomenclature surrounding common lox site mutants and their implications in experiments

2020 ◽  
Author(s):  
Daniel Shaw ◽  
Luis Serrano ◽  
Maria Lluch-Senar
Keyword(s):  
Dna Repair ◽  
Genetic Engineering ◽  
Mycoplasma Pneumoniae ◽  
Dna Sequences ◽  
Double Mutant ◽  
Toxic Effects ◽  
Supplementary Data ◽  
Naming Convention ◽  
Data Files ◽  
Dna Recombinase

AbstractThe Cre-Lox system is a highly versatile and powerful DNA recombinase mechanism, mainly used in genetic engineering to insert or remove desired DNA sequences. It is widely utilised across multiple fields of biology, with applications ranging from plants, to mammals, to microbes. A key feature of this system is its ability to allow recombination between mutant lox sites, traditionally named lox66 and lox71, to create a functionally inactive double mutant lox72 site. However, a large portion of the published literature has incorrectly annotated these mutant lox sites, which in turn can lead to difficulties in replication of methods, design of proper vectors, and confusion over the proper nomenclature. Here, we demonstrate common errors in annotations, the impacts they can have on experimental viability, and a standardised naming convention. We also show an example of how this incorrect annotation can induce toxic effects in bacteria that lack optimal DNA repair systems, exemplified by Mycoplasma pneumoniae.Data SummaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

scholarly journals Lox’d in translation: contradictions in the nomenclature surrounding common lox-site mutants and their implications in experiments

Microbiology ◽  
2020 ◽  
Author(s):  
Daniel Shaw ◽  
Luis Serrano ◽  
Maria Lluch-Senar
Keyword(s):  
Dna Repair ◽  
Genetic Engineering ◽  
Mycoplasma Pneumoniae ◽  
Dna Sequences ◽  
Type Species ◽  
Double Mutant ◽  
Toxic Effects ◽  
Content Type ◽  
Naming Convention ◽  
Dna Recombinase

The Cre-Lox system is a highly versatile and powerful DNA recombinase mechanism, mainly used in genetic engineering to insert or remove desired DNA sequences. It is widely utilized across multiple fields of biology, with applications ranging from plants, to mammals, to microbes. A key feature of this system is its ability to allow recombination between mutant lox sites. Two of the most commonly used mutant sites are named lox66 and lox71, which recombine to create a functionally inactive double mutant lox72 site. However, a large portion of the published literature has incorrectly annotated these mutant lox sites, which in turn can lead to difficulties in replication of methods, design of proper vectors and confusion over the proper nomenclature. Here, we demonstrate common errors in annotations, the impacts they can have on experimental viability, and a standardized naming convention. We also show an example of how this incorrect annotation can induce toxic effects in bacteria that lack optimal DNA repair systems, exemplified by Mycoplasma pneumoniae .

scholarly journals Molecular cloning and chromosomal localization of DNA sequences associated with a human DNA repair gene.

1985 ◽  
Vol 5 (2) ◽  
pp. 398-405 ◽  
Author(s):  
J S Rubin ◽  
V R Prideaux ◽  
H F Willard ◽  
A M Dulhanty ◽  
G F Whitmore ◽  
...  
Keyword(s):  
Dna Repair ◽  
Dna Sequences ◽  
Chromosomal Localization ◽  
Excision Repair ◽  
Repair Process ◽  
Human Cells ◽  
Gene Products ◽  
Repair Gene ◽  
Human Dna ◽  
Dna Repair Gene

The genes and gene products involved in the mammalian DNA repair processes have yet to be identified. Toward this end we made use of a number of DNA repair-proficient transformants that were generated after transfection of DNA from repair-proficient human cells into a mutant hamster line that is defective in the initial incision step of the excision repair process. In this report, biochemical evidence is presented that demonstrates that these transformants are repair proficient. In addition, we describe the molecular identification and cloning of unique DNA sequences closely associated with the transfected human DNA repair gene and demonstrate the presence of homologous DNA sequences in human cells and in the repair-proficient DNA transformants. The chromosomal location of these sequences was determined by using a panel of rodent-human somatic cell hybrids. Both unique DNA sequences were found to be on human chromosome 19.

EMBRYO MANIPULATION AND GENE TRANSFER IN LIVESTOCK

1985 ◽  
Vol 65 (3) ◽  
pp. 527-538 ◽  
Author(s):  
R. B. CHURCH ◽  
F. J. SCHAUFELE ◽  
K. MECKLING
Keyword(s):  
Gene Transfer ◽  
Genetic Engineering ◽  
Embryo Transfer ◽  
Dna Sequences ◽  
Recombinant Dna ◽  
Fusion Gene ◽  
Bovine Genome ◽  
Monozygotic Twins ◽  
Genetically Engineered ◽  
Livestock Species

In the past few years significant progress has been made in manipulation of reproduction and in development of genetic engineering techniques which can be applied to animal species. Artificial insemination and embryo transfer are now used widely in the livestock industry. The advent of non-surgical embryo collection and transfer, embryo freezing and splitting along with estrus synchronization has allowed the industry to move from the laboratory to the farm. Embryo manipulation now involves embryo splitting to produce monozygotic twins, in vitro fertilization, cross-species fertilization, embryo sexing, and chimeric production of tetraparental animals among others. Advances in recombinant DNA, plasmid construction and embryo manipulation technologies allow the production of genetically engineered animals. The application of recombinant DNA technology involves the isolation and manipulation of desired genes which have potential for significant changes in productivity in genetically engineered livestock. Recombinant DNA constructs involve the coupling of promoter, enhancer, regulatory and structural DNA sequences to form a "fusion gene" which can then be multiplied, purified, assayed and expressed in cell culture prior to being introduced into an animal genome. Such DNA gene constructs are readily available for many human and mouse genes. However, they are not readily available for livestock species because the detailed molecular biology has not yet been established in these species. Gene transfer offers a powerful new tool in animal research. Transfer of genes into the bovine genome has been accomplished. However, successful directed expression of these incorporated genes has not been achieved to date. New combinations of fusion genes may be an effective way of producing transgenic domestic animals which show controlled expression of the desired genes. Embryo manipulation and genetic engineering in livestock species is moving rapidly. The problems being addressed at present in numerous laboratories will result in enhanced livestock production in the not too distant future. Key words: Embryo transfer, embryo manipulation, transgenic livestock, genetic engineering, gene transfer, monozygotic twins

Development of T7 phage and T7 phage containing apurinic sites in an exonuclease III, endonuclease IV double mutant of Escherichia coli

1992 ◽  
Vol 70 (7) ◽  
pp. 605-608 ◽  
Author(s):  
Giselle Sanchez ◽  
Margaret D. Mamet-Bratley
Keyword(s):  
Escherichia Coli ◽  
Dna Repair ◽  
Dna Synthesis ◽  
Double Mutant ◽  
Bacteriophage T7 ◽  
Bacterial Dna ◽  
Exonuclease Iii ◽  
Repair Enzymes ◽  
Phage Dna ◽  
T7 Phage

The development of bacteriophage T7 was examined in an Escherichia coli double mutant defective for the two major apurinic, apyrimidinic endonucleases (exonuclease III and endonuclease IV, xth nfo). In cells infected with phages containing apurinic sites, the defect in repair enzymes led to a decrease of phage survival and a total absence of bacterial DNA degradation and of phage DNA synthesis. These results directly demonstrate the toxic action of apurinic sites on bacteriophage T7 at the intracellular level and its alleviation by DNA repair. In addition, untreated T7 phage unexpectedly displayed reduced plating efficiency and decreased DNA synthesis in the xth nfo double mutant.Key words: apurinic sites, DNA repair, T7 phage.

ISOLATION AND GENETIC ANALYSIS OF MMS-SENSITIVE MUS MUTANTS OF NEUROSPORA

1980 ◽  
Vol 22 (4) ◽  
pp. 535-552 ◽  
Author(s):  
E. Käfer ◽  
E. Perlmutter
Keyword(s):  
Dna Repair ◽  
Linkage Group ◽  
Genetic Analysis ◽  
Double Mutant ◽  
Excision Repair ◽  
Methylmethane Sulfonate ◽  
New Genes ◽  
Mutant Strains ◽  
Error Prone Repair ◽  
Isogenic Strains

With the aim of obtaining mutants that affect DNA repair or recombination, mutants sensitive to methylmethane sulfonate (MMS) have been isolated in the ascomycete Neurospora crassa. Seven of these mutants were backcrossed repeatedly to produce isogenic strains for measurements of relative mutagen sensitivities and for analysis of recombination frequencies. The new mus (mutagen sensitives) were compared to four previously known radiation-sensitive mutants which were shown to be cross-sensitive to MMS. Tests for allelism assigned the mus mutants to five new genes, mus-7 to mus-11, each mapping in a different linkage group. In homozygous crosses all mutants were sterile, except the two alleles of gene mus-10 which occasionally produced some viable ascospores. Complementation tests on MMS-media identified double mutant strains from many intercrosses. Such strains can be used for analysis of interactions between mutant alleles from different genes and of possible epistatic groupings for repair-deficient mutants in Neurospora. Four of these double mutant strains, all containing mus-8 and previously known mutants, were checked for survival on MMS media and their sensitivities were compared to those of their parental single mutant strains. Results indicate that mus-8 may be epistatic to uvs-2 which is deficient in excision repair, but not to mutants like uvs-3 that appear to be deficient in error-prone repair.

Meiosis progression and donor age affect expression profile of DNA repair genes in bovine oocytes

Zygote ◽  
2013 ◽  
Vol 23 (1) ◽  
pp. 11-18 ◽  
Author(s):  
S. Bilotto ◽  
R. Boni ◽  
G.L. Russo ◽  
M.B. Lioi
Keyword(s):  
Dna Repair ◽  
Dna Sequences ◽  
Calf Serum ◽  
Internal Standard ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Gene Marker ◽  
Donor Age ◽  
Immature Oocytes

SummarySeveral genetic and physiological factors increase the risk of DNA damage in mammalian oocytes. Two critical events are: (i) meiosis progression, from maturation to fertilization, due to extensive chromatin remodelling during genome decondensation; and (ii) aging, which is associated with a progressive oxidative stress. In this work, we studied the transcriptional patterns of three genes, RAD51, APEX-1 and MLH1, involved in DNA repair mechanisms. The analyses were performed by real-time quantitative PCR (RT-qPCR) in immature and in vitro matured oocytes collected from 17 ± 3-month-old heifers and 94 ± 20-month-old cows. Batches of 30–50 oocytes for each group (three replicates) were collected from ovarian follicles of slaughtered animals. The oocytes were freed from cumulus cells at the time of follicle removal, or after in vitro maturation (IVM) carried out in M199 supplemented with 10% fetal calf serum, 10 IU luteinising hormone (LH)/ml, 0.1 IU follicle-stimulating hormone (FSH)/ml and 1 μg 17β-oestradiol/ml. Total RNA was extracted by Trizol method. The expression of bovine GAPDH gene was used as the internal standard, while primers for bovine RAD51, APEX-1 and MLH1 genes were designed from DNA sequences retrieved from GenBank. Results obtained indicate a clear up-regulation of RAD51, APEX-1 and MLH1 genes after IVM, ranging between two- and four-fold compared with germinal vesicle (GV) oocytes. However, only RAD51 showed a significant transcript increase between the immature oocytes collected from young or old individuals. This finding highlights RAD51 as a candidate gene marker for discriminating bovine immature oocytes in relation to the donor age.

scholarly journals Investigation of genes involved in somatic embryogenesis and plant-Agrobacterium interactions through transcriptional profiling

2021 ◽  
Author(s):  
◽  
Christopher Willig
Keyword(s):  
Somatic Embryogenesis ◽  
Genetic Engineering ◽  
Candidate Genes ◽  
Dna Sequences ◽  
Zygotic Embryo ◽  
Transcriptional Profiling ◽  
Differential Expression Analysis ◽  
Genetic Material ◽  
Plant Cells ◽  
Plant Genes

Plant genetic engineering relies on the ability to transmit and express cloned DNA sequences in plant cells (transformation) as well as the capacity for the cells carrying this DNA to undergo division and differentiation (regeneration), eventually giving rise to a mature whole plant. The breadth of application for genetic engineering is limited by constraints on one or both of these factors in many plant species and individual varieties. Uncovering plant genes which are involved in important aspects of either component can inform the development of technologies that serve to enable or improve the efficiency of genetic modification methods. The most commonly employed method of delivering exogenous genetic material into plant cells is via disarmed strains of the plant pathogen Agrobacterium tumefaciens. Somatic embryogenesis is a frequently applied mode of plant regeneration following DNA delivery, especially in major cereal crops such as maize, rice, and sorghum. In the work reported here, whole transcriptome sequencing (RNA-seq) was used in two different experiments to capture transcriptional dynamics throughout early somatic embryogenesis in immature zygotic embryo tissue of the major crop plant sorghum (Sorghum bicolor), and throughout early times following host plant inoculation with A. tumefaciens in seedlings of the model plant Arabidopsis thaliana (Chapters 2 and 3, respectively). In both cases, differential expression analysis revealed many genes which were induced either during somatic embryogenesis or in response to inoculation with either virulent or avirulent A. tumefaciens strains. Several of these genes were highlighted as candidates for future study into their potential role in the respective processes. Multiple candidate genes were functionally tested, using transgenic methods, for the possibility of having a role in the regulation of somatic embryogenesis in sorghum (Chapter 4). These experiments failed to confirm an influence over the process for all candidate genes that were evaluated. The experimental work documented herein contributes to a growing body of literature documenting plant genes which could serve as possible targets for techniques that work to enhance the utility of biotechnological methods for improving traits in plants.

scholarly journals CRISPR systems: what’s new, where next?

2021 ◽  
Author(s):  
Ashley Parkes ◽  
Fiona Kemm ◽  
Liu He ◽  
Tom Killelea
Keyword(s):  
Dna Repair ◽  
Immune System ◽  
Genetic Engineering ◽  
Gene Editing ◽  
Adaptive Immune System ◽  
Feature Article ◽  
Adaptive Immune ◽  
The Past ◽  
Domains Of Life ◽  
Made In

The genetic signature of natural CRISPR-Cas systems were first noted in a 1989 publication and were characterized in detail from 2002 to 2007, culminating in the first report of a prokaryotic adaptive immune system. Since then, CRISPR-Cas enzymes have been adapted into molecular biology tools that have transformed genetic engineering across domains of life. In this feature article, we describe origins, uses and futures of CRISPR-Cas enzymes in genetic engineering: we highlight advances made in the past 10 years. Central to these advances is appreciation of interplay between CRISPR engineering and DNA repair. We highlight how this relationship has been manipulated to create further advances in the development of gene editing.

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