Karen Lech

DNA-bound Fos proteins activate transcription in yeast.

We constructed genes encoding the DNA binding region of the bacterial LexA repressor fused to the v-fos and c-fos oncogene products. The resulting LexA-Fos fusion proteins activated transcription in yeast. Transcription activation by these proteins was as strong as transcription activation by proteins native to yeast. LexA-Fos fusion proteins only activated transcription of genes when they were bound to LexA binding sites inserted upstream of those genes. Transcription was activated less strongly by similar proteins in which the DNA binding region of LexA was fused to vMyc and cMyc. Transcription was not activated by native LexA or by proteins containing the DNA binding domain of LexA fused to bacteriophage 434 repressor or yeast MAT alpha 2 protein. These results demonstrate that Fos proteins activate eukaryotic gene expression when they are bound to promoter DNA, and thus suggest that Fos proteins exert some of their effects because they stimulate transcription of cellular genes. Regulation of transcription by Fos and Myc proteins in yeast provides a phenotype that may facilitate genetic analysis of the function of these proteins in higher organisms.

Preparing Lambda DNA from Phage Lysates

DNA extracted from lambda‐derived vectors is typically subcloned into plasmid or filamentous phage vectors. The first two protocols describe methods for isolating phage DNA from large‐ and medium‐scale liquid lysates. These two methods use either density‐gradient centrifugation or ion‐exchange chromatography to purify the phage particles. The third protocol describes a rapid procedure for isolating phage DNA, suitable for small‐scale liquid lysates.

Growing Lambda‐Derived Vectors

It is often necessary to grow large quantities of lambda‐derived vectors, so that DNA can be made from them. This unit describes a procedure for making a phage stock by plate lysis, and an gives details on how to make a liquid lysate. Storage of phage lysates is described in a .

Plating Lambda Phage to Generate Plaques

This unit provides detailed protocols for isolating a single plaque by titering serial dilutions or streaking on a lawn of cells. A discussion of phage transfection and in vitro packaging is also included.

Lambda as a Cloning Vector

Advantages of lambda as a cloning vector are discussed along with considerations for the insert DNA (i.e., size, spi and hfl state). Maps of lambda‐derived cloning vectors are provided in addition to a discussion and map of a cosmid (a lambda‐derived plasmid vector).

Techniques for Bacterial Cell Culture: Media Preparation and Bacteriological Tools

Basic information for the culture of bacteria is presented in this appendix, using Escherichia coli as a representative organism. The techniques and recipes should be readily transferrable. Specifics are given for liquid and solid media, rich and minimal media, stab agar, as well as a description of the tools required to inoculate and culture bacteria.

Growing Bacteria in Liquid Media

The procedure for inoculating overnight (starter) cultures of E. coli from a single colony is described along with considerations for growing larger cultures. Also included are two methods for monitoring cell growth using a spectrophotometer or a hemacytometer.

Growing Bacteria on Solid Media

Detailed protocols are provided for titering and isolating bacterial colonies by serial dilutions, or alternatively by streaking or spreading a plate. Support protocols describe replica plating as well as methods for storing strains as agar stabs or frozen glycerol stocks.

Introduction to Lambda Phages

The biology of lambda‐derived vectors is extremely well understood. This introduction to the biology of lambda and related phages is included to help readers use the lambda vectors that are employed in the manual and to help readers understand new lambda vectors that are being developed. Specific topics include a discussion of lytic growth (i.e., requirements for DNA replication, packaging and lysis) and lysogenic growth (i.e., requirements for lysogenization and induction of a lysogen).