Publication Date:
1990-10-12
Description:
The 15-megabase pair Saccharomyces cerevisiae and the 4.7-megabase pair Escherichia coli genomes were completely cleaved at a single predetermined site by means of the Achilles' heel cleavage (AC) procedure. The symmetric lac operator (lacOs) was introduced into the circular Escherichia coli genome and into one of the 16 yeast chromosomes. Intact chromosomes from the resulting strains were prepared in agarose microbeads and methylated with Hha I (5'-GCGC) methyltransferase (M.Hha I) in the presence of lac repressor (LacI). All Hae II sites (5'-[sequence: see text]) with the exception of the one in lacOs, which was protected by LacI, were modified and thus no longer recognized by Hae II. After inactivation of M.Hha I and LacI, Hae II was used to completely cleave the chromosomes specifically at the inserted lacOs. These experiments demonstrate the feasibility of using the AC approach to efficiently extend the specificity of naturally occurring restriction enzymes and create new tools for the mapping and precise molecular dissection of multimegabase genomes.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Koob, M -- Szybalski, W -- 5-P30-CA-07175/CA/NCI NIH HHS/ -- GM 39715/GM/NIGMS NIH HHS/ -- New York, N.Y. -- Science. 1990 Oct 12;250(4978):271-3.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/2218529" target="_blank"〉PubMed〈/a〉
Keywords:
Base Sequence
;
DNA, Bacterial/genetics/*metabolism
;
DNA, Fungal/genetics/*metabolism
;
Deoxyribonucleases, Type II Site-Specific/*metabolism
;
Escherichia coli/*genetics
;
*Genes, Bacterial
;
*Genes, Fungal
;
Hydrolysis
;
Molecular Sequence Data
;
Operon
;
Repressor Proteins/*metabolism
;
Saccharomyces cerevisiae/*genetics
;
Substrate Specificity
Print ISSN:
0036-8075
Electronic ISSN:
1095-9203
Topics:
Biology
,
Chemistry and Pharmacology
,
Computer Science
,
Medicine
,
Natural Sciences in General
,
Physics
