Analysis of the CYP21A2 Gene with Intergenic Recombination and Multiple Gene Deletions in the RCCX Module

2011 ◽  
Vol 15 (1-2) ◽  
pp. 35-42 ◽  
Author(s):  
Shwu-Fen Chang ◽  
Hsien-Hsiung Lee
Keyword(s):  
Cyp21a2 Gene ◽  
Gene Deletions ◽  
Multiple Gene ◽  
Intergenic Recombination

scholarly journals Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining

2019 ◽  
Author(s):  
Gabriel A. Suárez ◽  
Kyle R. Dugan ◽  
Brian A. Renda ◽  
Sean P. Leonard ◽  
Lakshmi S. Gangavarapu ◽  
...  
Keyword(s):  
Gene Knockout ◽  
Single Gene ◽  
Golden Gate ◽  
Acinetobacter Baylyi ◽  
Gene Deletions ◽  
Multiple Gene ◽  
A Genome ◽  
Strain Collection ◽  
Acinetobacter Baylyi Adp1 ◽  
Improved Methods

ABSTRACTOne goal of synthetic biology is to improve the efficiency and predictability of living cells by removing extraneous genes from their genomes. We demonstrate improved methods for engineering the genome of the metabolically versatile and naturally transformable bacterium Acinetobacter baylyi ADP1 and apply them to a genome streamlining project. In Golden Transformation, linear DNA fragments constructed by Golden Gate Assembly are directly added to cells to create targeted deletions, edits, or additions to the chromosome. We tested the dispensability of 55 regions of the ADP1 chromosome using Golden Transformation. The 19 successful multiple-gene deletions ranged in size from 21 to 183 kilobases and collectively accounted for 24.6% of its genome. Deletion success could only be partially predicted on the basis of a single-gene knockout strain collection and a new Tn-Seq experiment. We further show that ADP1’s native CRISPR/Cas locus is active and can be retargeted using Golden Transformation. We reprogrammed it to create a CRISPR-Lock, which validates that a gene has been successfully removed from the chromosome and prevents it from being reacquired. These methods can be used together to implement combinatorial routes to further genome streamlining and for more rapid and assured metabolic engineering of this versatile chassis organism.

scholarly journals Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining

2020 ◽  
Vol 48 (8) ◽  
pp. 4585-4600
Author(s):  
Gabriel A Suárez ◽  
Kyle R Dugan ◽  
Brian A Renda ◽  
Sean P Leonard ◽  
Lakshmi Suryateja Gangavarapu ◽  
...  
Keyword(s):  
Gene Deletion ◽  
Single Gene ◽  
Acinetobacter Baylyi ◽  
Gene Deletions ◽  
Multiple Gene ◽  
Transposon Insertion ◽  
A Genome ◽  
Acinetobacter Baylyi Adp1 ◽  
Transposon Insertion Sequencing ◽  
Improved Methods

Abstract One goal of synthetic biology is to improve the efficiency and predictability of living cells by removing extraneous genes from their genomes. We demonstrate improved methods for engineering the genome of the metabolically versatile and naturally transformable bacterium Acinetobacter baylyi ADP1 and apply them to a genome streamlining project. In Golden Transformation, linear DNA fragments constructed by Golden Gate Assembly are directly added to cells to create targeted deletions, edits, or additions to the chromosome. We tested the dispensability of 55 regions of the ADP1 chromosome using Golden Transformation. The 18 successful multiple-gene deletions ranged in size from 21 to 183 kb and collectively accounted for 23.4% of its genome. The success of each multiple-gene deletion attempt could only be partially predicted on the basis of an existing collection of viable ADP1 single-gene deletion strains and a new transposon insertion sequencing (Tn-Seq) dataset that we generated. We further show that ADP1’s native CRISPR/Cas locus is active and can be retargeted using Golden Transformation. We reprogrammed it to create a CRISPR-Lock, which validates that a gene has been successfully removed from the chromosome and prevents it from being reacquired. These methods can be used together to implement combinatorial routes to further genome streamlining and for more rapid and assured metabolic engineering of this versatile chassis organism.

scholarly journals Characterization of Sigma Factor Genes in Streptomyces lividans TK24 Using a Genomic Library-Based Approach for Multiple Gene Deletions

2018 ◽  
Vol 9 ◽  
Author(s):  
Yuriy Rebets ◽  
Konstantinos C. Tsolis ◽  
Elísabet Eik Guðmundsdóttir ◽  
Joachim Koepff ◽  
Beata Wawiernia ◽  
...  
Keyword(s):  
Sigma Factor ◽  
Genomic Library ◽  
Streptomyces Lividans ◽  
Gene Deletions ◽  
Multiple Gene

scholarly journals Markerless Multiple-Gene-Deletion System for Streptococcus mutans

2008 ◽  
Vol 74 (7) ◽  
pp. 2037-2042 ◽  
Author(s):  
Anirban Banerjee ◽  
Indranil Biswas
Keyword(s):  
Dental Caries ◽  
Streptococcus Mutans ◽  
Genetic Background ◽  
Double Mutant ◽  
Gene Deletion ◽  
Low Ph ◽  
Gene Deletions ◽  
Multiple Gene ◽  
Modified Method ◽  
Oxidative Agents

ABSTRACT Inactivation or selective modification is essential to elucidate the putative function of a gene. The present study describes an improved Cre-loxP-based method for markerless multiple gene deletion in Streptococcus mutans, the principal etiological agent of dental caries. This modified method uses two mutant loxP sites, which after recombination creates a double-mutant loxP site that is poorly recognized by Cre recombinase, facilitating multiple gene deletions in a single genetic background. The effectiveness of this modified strategy was demonstrated by the construction of both single and double gene deletions at the htrA and clpP loci on the chromosome of Streptococcus mutans. HtrA and ClpP play key roles in the processing and maturation of several important proteins, including many virulence factors. Deletion of these genes resulted in reducing the organism's ability to withstand exposure to low pH and oxidative agents. The method described here is simple and efficient and can be easily implemented for multiple gene deletions with S. mutans and other streptococci.

scholarly journals Molecular Analysis of CYP21A2 Gene Mutations among Iraqi Patients with Congenital Adrenal Hyperplasia

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Ruqayah G. Y. Al-Obaidi ◽  
Bassam M. S. Al-Musawi ◽  
Munib Ahmed K. Al-Zubaidi ◽  
Christian Oberkanins ◽  
Stefan Németh ◽  
...  
Keyword(s):  
Congenital Adrenal Hyperplasia ◽  
Molecular Analysis ◽  
Point Mutations ◽  
Gene Mutations ◽  
Adrenal Hyperplasia ◽  
Hydroxylase Deficiency ◽  
Cyp21a2 Gene ◽  
Gene Deletions ◽  
Large Gene ◽  
21 Hydroxylase Deficiency

Congenital adrenal hyperplasia is a group of autosomal recessive disorders. The most frequent one is 21-hydroxylase deficiency. Analyzing CYP21A2 gene mutations was so far not reported in Iraq. This work aims to analyze the spectrum and frequency of CYP21A2 mutations among Iraqi CAH patients. Sixty-two children were recruited from the Pediatric Endocrine Consultation Clinic, Children Welfare Teaching Hospital, Baghdad, Iraq, from September 2014 till June 2015. Their ages ranged between one day and 15 years. They presented with salt wasting, simple virilization, or pseudoprecocious puberty. Cytogenetic study was performed for cases with ambiguous genitalia. Molecular analysis of CYP21A2 gene was done using the CAH StripAssay (ViennaLab Diagnostics) for detection of 11 point mutations and >50% of large gene deletions/conversions. Mutations were found in 42 (67.7%) patients; 31 (50%) patients were homozygotes, 9 (14.5%) were heterozygotes, and 2 (3.2%) were compound heterozygotes with 3 mutations, while 20 (32.3%) patients had none of the tested mutations. The most frequently detected mutations were large gene deletions/conversions found in 12 (19.4%) patients, followed by I2Splice and Q318X in 8 (12.9%) patients each, I172N in 5 (8.1%) patients, and V281L in 4 (6.5%) patients. Del 8 bp, P453S, and R483P were each found in one (1.6%) and complex alleles were found in 2 (3.2%). Four point mutations (P30L, Cluster E6, L307 frameshift, and R356W) were not identified in any patient. In conclusion, gene deletions/conversions and 7 point mutations were recorded in varying proportions, the former being the commonest, generally similar to what was reported in regional countries.

Self-excising Cre/mutant lox marker recycling system for multiple gene integrations and consecutive gene deletions in Aspergillus oryzae

2017 ◽  
Vol 123 (4) ◽  
pp. 403-411 ◽  
Author(s):  
Silai Zhang ◽  
Akihiko Ban ◽  
Naoki Ebara ◽  
Osamu Mizutani ◽  
Mizuki Tanaka ◽  
...  
Keyword(s):  
Aspergillus Oryzae ◽  
Gene Deletions ◽  
Multiple Gene ◽  
Recycling System ◽  
Marker Recycling

scholarly journals Multiple gene deletions within the human immunoglobulin heavy-chain cluster.

1984 ◽  
Vol 81 (18) ◽  
pp. 5811-5815 ◽  
Author(s):  
N. Migone ◽  
S. Oliviero ◽  
G. de Lange ◽  
D. L. Delacroix ◽  
D. Boschis ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Immunoglobulin Heavy Chain ◽  
Human Immunoglobulin ◽  
Gene Deletions ◽  
Multiple Gene

scholarly journals Cre-lox-Based System for Multiple Gene Deletions and Selectable-Marker Removal in Lactobacillus plantarum

2006 ◽  
Vol 73 (4) ◽  
pp. 1126-1135 ◽  
Author(s):  
Jolanda M. Lambert ◽  
Roger S. Bongers ◽  
Michiel Kleerebezem
Keyword(s):  
Lactobacillus Plantarum ◽  
Model Organism ◽  
Gene Replacement ◽  
Gram Positive ◽  
Gene Deletions ◽  
Chloramphenicol Resistance ◽  
Multiple Gene ◽  
Double Crossover ◽  
Pcr Products ◽  
Eukaryotic Organisms

ABSTRACT The classic strategy to achieve gene deletion variants is based on double-crossover integration of nonreplicating vectors into the genome. In addition, recombination systems such as Cre-lox have been used extensively, mainly for eukaryotic organisms. This study presents the construction of a Cre-lox-based system for multiple gene deletions in Lactobacillus plantarum that could be adapted for use on gram-positive bacteria. First, an effective mutagenesis vector (pNZ5319) was constructed that allows direct cloning of blunt-end PCR products representing homologous recombination target regions. Using this mutagenesis vector, double-crossover gene replacement mutants could be readily selected based on their antibiotic resistance phenotype. In the resulting mutants, the target gene is replaced by a lox66-P32-cat-lox71 cassette, where lox66 and lox71 are mutant variants of loxP and P32-cat is a chloramphenicol resistance cassette. The lox sites serve as recognition sites for the Cre enzyme, a protein that belongs to the integrase family of site-specific recombinases. Thus, transient Cre recombinase expression in double-crossover mutants leads to recombination of the lox66-P32-cat-lox71 cassette into a double-mutant loxP site, called lox72, which displays strongly reduced recognition by Cre. The effectiveness of the Cre-lox-based strategy for multiple gene deletions was demonstrated by construction of both single and double gene deletions at the melA and bsh1 loci on the chromosome of the gram-positive model organism Lactobacillus plantarum WCFS1. Furthermore, the efficiency of the Cre-lox-based system in multiple gene replacements was determined by successive mutagenesis of the genetically closely linked loci melA and lacS2 in L. plantarum WCFS1. The fact that 99.4% of the clones that were analyzed had undergone correct Cre-lox resolution emphasizes the suitability of the system described here for multiple gene replacement and deletion strategies in a single genetic background.

scholarly journals The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

10.3791/4072 ◽  
2012 ◽  
Author(s):  
Yo Suzuki ◽  
Jason Stam ◽  
Mark Novotny ◽  
Nozomu Yachie ◽  
Roger S. Lasken ◽  
...  
Keyword(s):  
Gene Deletions ◽  
Multiple Gene ◽  
Yeast Strains
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