Nonsense mutations in many operons have polar effects, that is, they reduce the expression of downstream genes. In efforts to study these effects more carefully, in the late 1960s Malamy, Shapiro, and Starlinger each isolated and characterized strongly polar mutations in the lac and gal operons. In addition to the expected classes of nonsense mutations, another type was also found. This new type was highly polar but not suppressible by known nonsense suppressors. Since the mutations reverted, albeit at very low rates, it seemed likely that they were insertions and not deletions. Therefore it became of great interest to find out just what these mutations were. Nowadays this question would probably be answered by cloning a portion of the DNA containing the mutation and sequencing it to determine the exact nature of the mutation. Such a procedure was impossible at the time and less direct experiments were performed.

One likely cause of the polarity was the insertion of extraneous nucleotides into the lac and gal genes. Fortunately, this possibility could be tested by the techniques then available. If the insertions were larger than a few hundred nucleotides, they could be detected directly because they would increase the density of lac or gal transducing phage. There fore the mutations were crossed onto λdgal and φ80plac phage and the densities of phage both with and without the mutations were carefully compared by equilibrium gradient centrifugation (Fig. 1). The results showed that transducing phage carrying the polar mutations were denser than the corresponding transducing phage not carrying the mutations. Therefore the mutations were caused by the insertion of DNA elements. These later came to be called IS, for insertion sequences.

Fig1. The positions of Lac+ and strongly polar Lac- phage particles in the centrifuge tube after CsCl equilibrium gradient centrifugation. The polar mutation makes the phage more dense and it bands closer to the bottom of the tube.

At first, the insertions were suspected of being merely random sequences that somehow had been inserted into the genes being studied. The mechanism of their generation is not so easy to imagine, however. Point mutations, insertions and deletions of a nucleotide or two, and sizable deletions may all be created by simple reactions likely to occur in cells. Not so for sizable insertions.

Additional study of the IS sequences began to clarify their origin. One particularly valuable technique for their study was the formation of DNA heteroduplexes (Fig.2). DNA isolated from the gal and lac transducing phage with and without the IS sequences was used in these experiments. When necessary, the strands of these phage could even be separated and purified. The lengths of IS sequences fell into discrete size classes, and any particular IS sequence was found to insert into a gene in either of the two possible orientations. The existence of the length classes suggested that a small number of IS sequences might be responsible for all the insertions. Indeed, fewer than ten different IS sequences have been found in Escherichia coli (Table1). The demonstration that IS elements were specific sequences did not explain how they were inserted into different sites in the cell’s chromosome, but by analogy to lambda phage, the elements were expected to code for one or more proteins involved in the process. By now the IS elements have been obtained on phage and plasmids and they have been sequenced and characterized using in vitro mutagenesis as described below. From these studies a comprehensive picture of their structure and function has emerged.

Fig2. (a) DNA heteroduplex between lambda and lambda containing IS2. The single stranded bubble is the DNA of IS2. (b) DNA heteroduplex formed between two r strands of lambda, each containing IS2 but in opposite orientations.

Table 1.  Sizes of Various Bacterial IS Sequences and the Number of Base Pairs of Host DNA they Duplicate Upon Insertion.

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مواضيع ذات صلة

Discovery of Tn Elements

Structure and Properties of IS Elements

Holliday Structures and Branch Migration in Integration

Structure of the Intasome

In vitro Assay of Integration and Excision

Inhibition By a Downstream Element

Demonstrating Xis is Unstable

The Protein Kinase Domain

Posttranslational Modifications

The Double att Phage, att2

Transducing Phage Carrying Genes Other than gal and bio

Incorrect Excision and gal and bio Transducing Phage