Marshall H. Edgell

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.

High capacity gel preparative electrophoresis for purification of fragments of genomic DNA

Abstract We have designed a high-capacity gel electrophoretic device for the purification of large amounts of restriction endonuclease fragments of genomic DNA. This device exploits the high resolution of gel electrophoresis in conjunction with an electronic system permitting discontinuous sample elution over a large gel surface area. This feature preserves resolution and greatly increases capacity and yield. The resulting DNA fractions may be used in restriction endonuclease, ligation, transfection, and transformation reactions without further extensive manipulation. Furthermore, DNA fragments of a broad size range are recovered with high efficiency from the gel.

Evaluations of wasted mouse fibroblasts and SV-40 transformed human fibroblasts as models of ataxia telangiectasia in vitro.

Fibroblast cultures from wasted mice have been derived and the responses of these cultures to bleomycin treatment or gamma-irradiation have been examined. No differences were observed between wasted fibroblasts and littermate controls in the post-treatment inhibition of DNA replication. In contrast, a human SV-40 transformed ataxia telangiectasia fibroblast line mimicked the abnormal response of primary ataxia telangiectasia fibroblasts to gamma-rays or bleomycin and thus appears to be a useful in vitro model of ataxia telangiectasia.

[40] Use of preparative gel electrophoresis for DNA fragment isolation

Publisher Summary This chapter discusses the use of preparative gel electrophoresis for DNA fragment isolation. Large amounts of DNA may be fractionated with high resolution by means of preparative electrophoresis utilizing discontinuous collection. A very sharp band of DNA when electrophoresed into a collection chamber, which is well mixed and through, to which there is a continuous flow of collection buffer, will emerge from the chamber with a sharp leading edge but with an exponential tail due to continuous dilution in the collection chamber. To avoid this problem, the electrophoresis is limited to a suitable period of time and then the contents of the collection chamber are drained into a fraction collector, refill the chamber, and resume electrophoresis. This cycle can be repeated under control of a programmable timer that switches the power to the electrophoresis power supply and to the fill and drain pumps. The high resolution attained with this collection system does not reduce significantly by going to very large gel electrophoresis slabs. The chapter presents an alternate simpler apparatus. The new apparatus retains the high capacity and resolution but requires less than half the shop time to construct and appears even more trouble-free to operate.