Influence of insertions on packaging of host sequences covalently linked to bacteriophage Mu DNA.

Abstract

Insertions in bacteriophage Mu DNA have been identified. These insertions are responsible for at least seven X mutations, all of which eliminate essential Mu functions. The insertions are about 800 base pairs long and are located to the left of the cleavage site of restriction endonuclease EcoRI, near the immunity end of Mu DNA. We have found that such-insertions cause a reduction in the length of nonhomologous terminal sequences which are seen as split ends in denatured and renatured Mu DNA molecules. These heterogeneous sequences apparently arise from packaging of host DNA from maturation precursors in which Mu and host DNA are covalently linked. We infer that a single Mu genome length is too short to be cut during morphogenesis, and thus some host DNA is packaged into mature virions. Since the insertions increase the length of Mu DNA, they decrease the amount of host DNA needed for packaging.The temperate bacteriophage Mu is characterized by the highly promiscuous integration of its DNA into the genome of its host bacterium Escherichia coli (1-3). The prophage Mu DNA can be excised precisely from the different integration sites, and as a result wild-type function of the gene into which Mu was inserted can be restored (4). This reversal of Mu integration can be seen if the prophages contain the X mutations, which block the lethal and essential phage functions.Mu X mutants revert to wild type at a frequency of about 10-8. However, no X amber mutants have been found. Thus, we proposed that the X mutations are insertions (4). We have investigated the nature of the X mutations by agarose-gel electrophoresis of Mu DNA fragments produced by specific endonucleases, and by electron microscopic examination of Mu DNA heteroduplexes. These studies have shown that the X mutations are insertions near the immunity (c) end of Mu DNA. In one X mutant, the insertion is demonstrated to be of about 720 base pairs, located 4 kilobases (kb) away from the c end.The isolation of insertions in Mu DNA has allowed us to analyze further some aspects of Mu DNA structure. Mu DNA, a double-stranded linear duplex of about 37 kb (5), has some interesting features. When it is denatured and reannealed, two types of homoduplex molecules are obtained (Fig. 1). All the molecules show single-stranded tails or split ends, measuring about 4% of the molecular length. The split ends, which apparently represent sequences picked up randomly from the, E. coli chromosome, are always found at the S gene end, called here the SE end, of Mu DNA (6-8). Some molecules show another nonrenaturable region, termed the G bubble, near the SE end (9). Recently, we observed that the c end of Mu DNA is also not fixed, and probably varies in length by about 100 base pairs (10, 11).Abbreviation: kb, kilobase. t To whom correspondence should be directed.One plausible model for the origin of the heterogeneity of the SE end is that Mu DNA is packaged into phage heads from maturation precursors which contain both Mu DNA and host sequences. This hypothesis implies that...

Determining DNA Packaging Strategy by Analysis of the Termini of the Chromosomes in Tailed-Bacteriophage Virions

“…A headful type packaging event then extends from this initial cleavage, across the integrated phage DNA and beyond, to include about 1,800 bp of host DNA at the other side (Fig. 7.1) (54). Again the ends are thought to be blunt by their ability to be ligated to other blunt ends (e.g., EcoRV ends in ref.…”

My life with Mu

“…Mu DNA was thus matured by a full head packaging process, most likely starting at the left end of Mu replicas still integrated at the time of packaging. 12 The 1976 CSHL " DNA Insertion Elements, Plasmids and Episomes" Meeting and Jim Shapiro's concluding remarks "Mu uses transposition as a life style," made it obvious, to me at least, that chromosomal rearrangements and Mu replication were intimately linked and that Mu did not excise prior to replication.…”

Regulation of bacteriophage Mu transposition

“…This prevents killing of the bacteria following excision either by the induced prophage or by superinfection by the phages produced by the surrounding bacteria. These Mucts62X mutants carry an insertion in their B gene (Bukhari & Taylor, 1975). As a result, they do not replicate (Pato & Waggoner, 1987) nor do they express the phage kil gene, the two means by which Mu kills its host.…”