In vitro Packaging

In vitro Packaging

In vitro packaging of lambda DNA is possible and, indeed, is widely usedto package restructured DNA in genetic engineering operations. Such packaging is done by preparing and mixing two highly concentrated extracts of cells, each infected with lambda mutants incapable of form-ing complete lambda particles or synthesizing DNA. Together these extracts contain all the required proteins for packaging. In vitro packing proceeds well if one of the extracts contains a head precursor formed as a result of mutations in genes D or F. If such an extract is mixed with an extract prepared from bacteria growing lambda-defective in the E gene and lambda DNA is added, mature lambda are formed. This in vitro packaging requires ATP, as would be expected from the fact that DNA somehow is stuffed into the head.

In this chapter we have seen two extremes for biological assembly. The “unique” structure of the ribosome is generated with ribosomal RNA and, except for one duplicated protein, with single copies of each of the ribosomal proteins. On the other extreme, many phage coats are virtually crystalline in that they contain mainly one protein used over and over in a regular array. Although we know many facts about assembly, in reality we understand very little about the actual process. We cannot look at the structure of a protein like the lambda coat protein and predict that it is capable of forming a regular icosahedron. We are also quite in the dark about understanding how nucleic acid is inserted into phage coats. Far more remains to be learned in this area than is already known.