scholarly journals Assembly of Multiple Full‐Size Genes or Genomic DNA Fragments on Human Artificial Chromosomes Using the Iterative Integration System

2021 ◽  
Vol 1 (12) ◽  
Author(s):  
Nicholas C. O. Lee ◽  
Nikolai S. Petrov ◽  
Vladimir Larionov ◽  
Natalay Kouprina
Keyword(s):  
Genomic Dna ◽  
Dna Fragments ◽  
Integration System ◽  
Full Size ◽  
Artificial Chromosomes ◽  
Iterative Integration

Enzymatic amplification and characterization of large DNA fragments from genomic DNA

Gene ◽  
1988 ◽  
Vol 71 (1) ◽  
pp. 211-216 ◽  
Author(s):  
Phouthone Keohavong ◽  
Cindy C. Wang ◽  
Rita S. Cha ◽  
William G. Thilly
Keyword(s):  
Genomic Dna ◽  
Dna Fragments ◽  
Enzymatic Amplification

Development of a fungal transformation system based on selection of sequences with promoter activity

1987 ◽  
Vol 7 (9) ◽  
pp. 3297-3305
Author(s):  
B G Turgeon ◽  
R C Garber ◽  
O C Yoder
Keyword(s):  
Genomic Dna ◽  
Pathogenic Fungus ◽  
Low Frequency ◽  
Hygromycin B ◽  
Fungal Transformation ◽  
Dna Fragments ◽  
Transformation System ◽  
Chromosomal Dna ◽  
General Utility ◽  
Novel Strategy

A novel strategy was used to develop a transformation system for the plant pathogenic fungus Cochliobolus heterostrophus. Sequences capable of driving the expression of a gene conferring resistance to the antibiotic hygromycin B in C. heterostrophus were selected from a library of genomic DNA fragments and used, with the selectable marker, as the basis for transformation. The library of random 0.5- to 2.0-kilobase-pair fragments of C. heterostrophus genomic DNA was inserted at the 5' end of a truncated, promoterless Escherichia coli hygromycin B phosphotransferase gene (hygB) whose product confers resistance to hygromycin B. C. heterostrophus protoplasts were transformed with the library and selected for resistance. Resistant colonies arose at low frequency. Each colony contained a transformation vector stably integrated into chromosomal DNA. When the transforming DNA was recovered from the genome and introduced into C. heterostrophus, resistant colonies appeared at higher frequency. We determined the sequences of two of the C. heterostrophus DNA fragments which had been inserted at the 5' end of hygB in the promoter library and found that both made translational fusions with hygB. One of the two fusions apparently adds 65 and the other at least 86 amino acids to the N-terminus of the hygB product. Plasmids containing hygB-C. heterostrophus promoter fusions can be used unaltered to drive hygB expression in several other filamentous ascomycetes. This approach to achieving transformation may have general utility, especially for organisms with relatively undeveloped genetics.

Identification of developmental regulatory genes in Aspergillus nidulans by overexpression.

Genetics ◽  
1995 ◽  
Vol 139 (2) ◽  
pp. 537-547 ◽  
Author(s):  
J F Marhoul ◽  
T H Adams
Keyword(s):  
Growth Inhibition ◽  
Aspergillus Nidulans ◽  
Genomic Dna ◽  
Regulatory Gene ◽  
Regulatory Genes ◽  
Dna Fragments ◽  
Expression Library ◽  
Phenotypic Changes ◽  
Inhibition Of Growth ◽  
Induction Sequence

Abstract Overexpression of several Aspergillus nidulans developmental regulatory genes has been shown to cause growth inhibition and development at inappropriate times. We set out to identify previously unknown developmental regulators by constructing a nutritionally inducible A. nidulans expression library containing small, random genomic DNA fragments inserted next to the alcA promoter [alcA(p)] in an A. nidulans transformation vector. Among 20,000 transformants containing random alcA(p) genomic DNA fusion constructs, we identified 66 distinct mutant strains in which alcA(p) induction resulted in growth inhibition as well as causing other detectable phenotypic changes. These growth inhibited mutants were divided into 52 FIG (Forced expression Inhibition of Growth) and 14 FAB (Forced expression Activation of brlA) mutants based on whether or not alcA(p) induction resulted in accumulation of mRNA for the developmental regulatory gene brlA. In four FAB mutants, alcA(p) induction not only activated brlA expression but also caused hyphae to differentiate into reduced conidiophores that produced viable spores from the tips as is observed after alcA(p)::brlA induction. Sequence analyses of the DNA fragments under alcA(p) control in three of these four sporulating strains showed that in two cases developmental activation resulted from overexpression of previously uncharacterized genes, whereas in the third strain, the alcA(p) was fused to brlA. The potential uses for this strategy in identifying genes whose overexpression results in specific phenotypic changes like developmental induction are discussed.

Isolation of large terminal sequences of BAC inserts based on double-restriction-enzyme digestion followed by anchored PCR

Genome ◽  
2000 ◽  
Vol 43 (2) ◽  
pp. 412-415 ◽  
Author(s):  
Zhong-Nan Yang ◽  
T Erik Mirkov
Keyword(s):  
Restriction Enzyme ◽  
Genomic Dna ◽  
Enzyme Digestion ◽  
Restriction Enzyme Digestion ◽  
Artificial Chromosomes ◽  
Bac Clones ◽  
Anchored Pcr ◽  
Pcr Products ◽  
Left And Right ◽  
The Right

Isolation of the terminal portions of genomic DNA cloned in bacterial artificial chromosomes (BACs) is an important step in map-based cloning, and several methods have been developed. Here, we present a new method based on double-restriction-enzyme digestion followed by anchored PCR. BAC DNA was digested with two enzymes: NotI and one of four enzymes (EcoRV, HpaI, StuI, or XmnI) that produce blunt termini. After dephosphorylation, these digestions were ligated to NotI- and EcoRV-digested pMSK, a new cloning vector developed in this work that is derived from pBluescript SK(+). PCR products representing the left- and right-terminal sequences of BAC inserts were obtained using a primer complementary to pMSK and a primer complementary to sequences in either the left arm or the right arm of the BAC vector pBeloBAC11. We have tested this method with 15 different BAC clones, and PCR products representing both the left- and right-terminal sequences have been obtained from all 15 BAC clones. This method is simple, fast, reproducible, and uses the same set of primers for any restriction enzyme used. With some modifications, it can also be used for isolating the terminal portions of genomic DNA cloned in yeast artificial chromosomes and P1-derived artificial chromosomes. Key words: BAC, anchored PCR, terminal sequence isolation, chromosome walk.

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