Open AccessRestriction‐Enzyme‐Cleavage Maps of Bactenophage M13
Author(s) -
HONDEL Cees A.,
PENNINGS Leo,
SCHOENMAKERS John G. G.
Publication year - 1976
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1976.tb10764.x
Subject(s) - restriction enzyme , intergenic region , biology , gene , genome , restriction map , genetics , dna , microbiology and biotechnology , bacteriophage , restriction fragment , plasmid , escherichia coli
Replicative form DNA of bacteriophage M13 was cleaved into specific fragments by an endonuclease isolated from Hemophilus aegyptius (endoR.HaeII) and an endonuclease from Arthrobacter luteus (endoR.AluI). The fragments were ordered so as to construct a circular map of the phage M13 genome by: (a) using each fragment as a primer for the synthesis in vitro of its respective neighbour and (b) digesting the isolated fragments with the Hemophilus aegyptius enzyme endoR.HaeIII or the Hemophilus aphirophilus enzyme endoR.HapII and subsequent analysis of the overlapping sets of fragments. The resulting physical map was correlated with the M13 genetic map by marker rescue experiments with amber mutant phage DNAs and purified wild‐type fragments. From the results of these analyses it has been concluded that gene II and gene V are contiguous on the genetic map. Evidence is provided that there is an internal start of RNA synthesis within the C‐terminal region of gene II which then ultimately leads to the synthesis of X protein. Furthermore, we conclude that there is an intergenic space of considerable length (450‐500 base pairs) which is located between gene II and gene IV on the M13 genome. The function of this intergenic region as the origin site for phase‐ DNA replication is discussed.