Linked Life Data

7651337

Source:http://linkedlifedata.com/resource/pubmed/id/7651337

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
1995-9-22
pubmed:abstractText
To characterize homologous recombination of transforming DNA in the filamentous fungus Alternaria alternata, we have compared the frequencies of gene targeting by circular and linear DNA fragments in the fungus. The A. alternata BRM1 gene, which is an essential gene for melanin biosynthesis, was selected as a target locus. BRM1 targeting events are easily identified because loss of function leads to a change in mycelial color from black to light brown. We constructed targeting vectors by inserting 0.6 to 3.1 kb internal BRM1 segments into a plasmid containing the hygromycin B phosphotransferase gene. When circular plasmids were used, melanin-deficient (Mel-) transformants accounted for 30 to 80% of hygromycin B-resistant (HyR) transformants, correlating closely with the size of the BRM1 segment in the transforming DNA. Restriction enzyme digestion within the BRM1 region greatly enhanced the frequency of gene targeting: integration of the linear plasmids was almost completely attributable to homologous recombination, regardless of the size of the BRM1 segments. Plasmids carrying both BRM1 segments and rDNA segments were transformed into the fungus to examine the effect of the number of target copies on homologous recombination. Using the circular plasmids, Mel- transformants accounted for only 5% of HyR transformants. In contrast, when the linear plasmid produced by restriction enzyme digestion within the BRM1 segment was used, almost all transformants were Mel-. These results indicate that homologous integration of circular molecules in A. alternata is sensitive to the length of homology and the number of targets, and that double-strand breaks in transforming DNA greatly enhance homologous recombination.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jul
pubmed:issn
0026-8925
pubmed:author
pubmed:issnType
Print
pubmed:day
28
pubmed:volume
248
pubmed:geneSymbol
BRM1
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
142-50
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
1995
pubmed:articleTitle
Efficient gene targeting in the filamentous fungus Alternaria alternata.
pubmed:affiliation
School of Agricultural Sciences, Nagoya University, Japan.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't