DNA sequence alteration resulting from a mutation impairing promoter function in the lac repressor gene

1980 ◽  
Vol 178 (1) ◽  
pp. 225-227 ◽  
Author(s):  
Michèle P. Calos ◽  
Jeffrey H. Miller
Keyword(s):  
Dna Sequence ◽  
Lac Repressor ◽  
Repressor Gene ◽  
Promoter Function ◽  
Sequence Alteration

scholarly journals Novel method for identifying sequence-specific DNA-binding proteins.

1985 ◽  
Vol 5 (9) ◽  
pp. 2307-2315 ◽  
Author(s):  
D Levens ◽  
P M Howley
Keyword(s):  
Dna Binding ◽  
Fusion Protein ◽  
Dna Sequence ◽  
Binding Proteins ◽  
Binding Protein ◽  
Protein S ◽  
Dna Binding Proteins ◽  
Lac Repressor ◽  
Lac Operator ◽  
Beta Galactosidase

We developed a general method for the enrichment and identification of sequence-specific DNA-binding proteins. A well-characterized protein-DNA interaction is used to isolate from crude cellular extracts or fractions thereof proteins which bind to specific DNA sequences; the method is based solely on this binding property of the proteins. The DNA sequence of interest, cloned adjacent to the lac operator DNA segment is incubated with a lac repressor-beta-galactosidase fusion protein which retains full operator and inducer binding properties. The DNA fragment bound to the lac repressor-beta-galactosidase fusion protein is precipitated by the addition of affinity-purified anti-beta-galactosidase immobilized on beads. This forms an affinity matrix for any proteins which might interact specifically with the DNA sequence cloned adjacent to the lac operator. When incubated with cellular extracts in the presence of excess competitor DNA, any protein(s) which specifically binds to the cloned DNA sequence of interest can be cleanly precipitated. When isopropyl-beta-D-thiogalactopyranoside is added, the lac repressor releases the bound DNA, and thus the protein-DNA complex consisting of the specific restriction fragment and any specific binding protein(s) is released, permitting the identification of the protein by standard biochemical techniques. We demonstrate the utility of this method with the lambda repressor, another well-characterized DNA-binding protein, as a model. In addition, with crude preparations of the yeast mitochondrial RNA polymerase, we identified a 70,000-molecular-weight peptide which binds specifically to the promoter region of the yeast mitochondrial 14S rRNA gene.

Novel method for identifying sequence-specific DNA-binding proteins

1985 ◽  
Vol 5 (9) ◽  
pp. 2307-2315
Author(s):  
D Levens ◽  
P M Howley
Keyword(s):  
Dna Binding ◽  
Fusion Protein ◽  
Dna Sequence ◽  
Binding Proteins ◽  
Binding Protein ◽  
Protein S ◽  
Dna Binding Proteins ◽  
Lac Repressor ◽  
Lac Operator ◽  
Beta Galactosidase

We developed a general method for the enrichment and identification of sequence-specific DNA-binding proteins. A well-characterized protein-DNA interaction is used to isolate from crude cellular extracts or fractions thereof proteins which bind to specific DNA sequences; the method is based solely on this binding property of the proteins. The DNA sequence of interest, cloned adjacent to the lac operator DNA segment is incubated with a lac repressor-beta-galactosidase fusion protein which retains full operator and inducer binding properties. The DNA fragment bound to the lac repressor-beta-galactosidase fusion protein is precipitated by the addition of affinity-purified anti-beta-galactosidase immobilized on beads. This forms an affinity matrix for any proteins which might interact specifically with the DNA sequence cloned adjacent to the lac operator. When incubated with cellular extracts in the presence of excess competitor DNA, any protein(s) which specifically binds to the cloned DNA sequence of interest can be cleanly precipitated. When isopropyl-beta-D-thiogalactopyranoside is added, the lac repressor releases the bound DNA, and thus the protein-DNA complex consisting of the specific restriction fragment and any specific binding protein(s) is released, permitting the identification of the protein by standard biochemical techniques. We demonstrate the utility of this method with the lambda repressor, another well-characterized DNA-binding protein, as a model. In addition, with crude preparations of the yeast mitochondrial RNA polymerase, we identified a 70,000-molecular-weight peptide which binds specifically to the promoter region of the yeast mitochondrial 14S rRNA gene.

scholarly journals Sliding of a single lac repressor protein along DNA is tuned by DNA sequence and molecular switching

2018 ◽  
Vol 46 (10) ◽  
pp. 5001-5011 ◽  
Author(s):  
Alessia Tempestini ◽  
Carina Monico ◽  
Lucia Gardini ◽  
Francesco Vanzi ◽  
Francesco S Pavone ◽  
...  
Keyword(s):  
Dna Sequence ◽  
Lac Repressor ◽  
Repressor Protein ◽  
Molecular Switching

DNA sequence mapping in interphase and metaphase chromosomes by fluorescence in situ hybridization

1992 ◽  
Vol 50 (1) ◽  
pp. 496-497
Author(s):  
Barbara Trask ◽  
Susan Allen ◽  
Anne Bergmann ◽  
Mari Christensen ◽  
Anne Fertitta ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Dual Band ◽  
Nick Translation ◽  
Metaphase Chromosomes ◽  
Band Pass ◽  
Texas Red ◽  
Fluorescent Spot

Using fluorescence in situ hybridization (FISH), the positions of DNA sequences can be discretely marked with a fluorescent spot. The efficiency of marking DNA sequences of the size cloned in cosmids is 90-95%, and the fluorescent spots produced after FISH are ≈0.3 μm in diameter. Sites of two sequences can be distinguished using two-color FISH. Different reporter molecules, such as biotin or digoxigenin, are incorporated into DNA sequence probes by nick translation. These reporter molecules are labeled after hybridization with different fluorochromes, e.g., FITC and Texas Red. The development of dual band pass filters (Chromatechnology) allows these fluorochromes to be photographed simultaneously without registration shift.

Phylogenetic relationships ofSalvia(Lamiaceae) in China: Evidence from DNA sequence datasets

2012 ◽  
pp. n/a-n/a
Author(s):  
Qian-Quan Li ◽  
Min-Hui Li ◽  
Qing-Jun Yuan ◽  
Zhan-Hu Cui ◽  
Lu-Qi Huang ◽  
...  
Keyword(s):  
Dna Sequence ◽  
Phylogenetic Relationships

Corrected DNA Sequence of the Platelet Glycoprotein IX Gene

1997 ◽  
Vol 77 (05) ◽  
pp. 1034-1035 ◽  
Author(s):  
Tomohiro Hayashi ◽  
Keijiroh Suzuki ◽  
Akito Yahagi ◽  
Jiroh Akiba ◽  
Katsushi Tajima ◽  
...  
Keyword(s):  
Dna Sequence ◽  
Platelet Glycoprotein
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