D C Tiemeier

In vitro packaging of a λ Dam vector containing EcoRI DNA fragments of Escherichia coli and phage P1

Abstract In this report we describe a coliphage λ vector system for cloning endo R. Eco RI DNA fragments. This system differs significantly from those previously described in two ways. First, restricted and ligated DNA is encapsidated in vitro. Second, with increasing λ DNA size in the range 78 to 100% that of wild-type, the efficiency of DNA encapsidation into infectious phage particles markedly increases. For λ wild-type DNA the efficiency of in vitro packaging (10 6 to 10 7 plaques produced per μg of added DNA) is equal to, or better than, the standard CaCl 2 transfection method. The use of a D am mutation to facilitate recognition of size classes of inserted fragments is described. Using this vector and in vitro packaging, several E. coli and phage P1 endo R. Eco RI fragments were cloned.

A comparison of two cloned mouse β-globin genes and their surrounding and intervening sequences

The BALC/c mouse has two nonallelic beta-globin genes that appear to reside on two different Eco R1 fragments of genomic DNA. We have already cloned one of these fragments and shown that the gene encoded within it is interrupted by at least one large intervening sequence of DNA. We have now cloned and characterized the second beta-globin gene-containing fragment. The coding sequence of its gene is also interrupted by an intervening sequence of DNA that occurs in about the same position, relative to the coding sequence, as does the first. Because some shared features of the structure of these two genes might be responsible for their coordinate expression and the elimination of their intervening sequences, we have compared their surrounding, coding and intervening sequences by restriction endonuclease analysis and by visualization of the heteroduplex structures formed between them. Of the 7000 bp of sequence compared in this way, we find only a few hundred base pairs of homology in addition to the coding sequence. These shared sequences flank the coding sequence and appear to include only those portions of the intervening sequence immediately adjacent to the interrupted structural gene.

Use of an EK-2 vector for the cloning of DNA from higher organisms.

A certified EK2 bacteriophage lambda vector, which is useful for cloning fragments of DNA from higher organisms, is described.