B~ECHNICAL B'~IPS Nonisotopic SSCP detection in PCR products by ethidium bromide staining Single-stranded conformation polymorphism (SSCP) is a sensitive method for detecting single base differences in polymerase chain reaction (PCR) productsL Detection by autoradiography~ or silver staining2 is time-consuming, costly and inconvenient. We discuss here optimized conditions for performing SSCP nonisotopically with routinely available gel oo D...... . (b) ~o,.,__up apparatus and ethidium bromide staining; the method is (a) ~ ~ t g I m I simple and rapid (2 h). Mineral oil. if used in PCR, interferes with sample loading, and is extracted with chloroform. Then, 0.5-10 ~tl of PCR product (at least 40 ng of amplified DNA per lane, the sensitivity limit), brought to a total volume of 10 ~1 with water, is denatured with alkali at 42°(2 for 5 min after the addition of 1 ~1 0.5 M NaOH, 10 mM EDTA: heat denaturation gives a poorer yield of single-stranded DNA. Just before loading, 1 lal ss G ds I~~ of formamide containing 0.5% bromophenol blue and 0.5% ssl~ xylene cyanol is added. Formamide lowers surface tension, ss allowing at least 15 lal to be added to each 20 I.tl well, and disds solves any chloroform inadvertently pipetted. Nondenaturing gels (0.4-1.5 mm thick, 5-10% polyacrylamide), with and without 5% glycerol, are made in a standard vertical gel FIGgll apparatus (e.g. Biorad Protean II). While thick gels are easier Nonisotopic SSCP of an alkali-denatured 185 bp fragment of to handle and more DNA can be loaded, thin .:gels are the p53 gene, which has a single base pair polymorphism in preferred as they can be mn at a higher voltage and staining codon 72. DNA is amplified from a plasmid ('C' allele), MDA is faster. Use of 0.5xTBE gives adequate buffering capacity cell DNA ('G' allele) and HeLa cell DNA (heterozygous 'CG') and also a lower running current. Gels, run typically at on 10% acrylamide gels (a) with and (b) without 5% glycerol. 20-30 V cmq for 1.5 h and maintained at 22°C by circulating UD, undenatured DNA controls; MW, 1 kb DNA ladder; cold water, are neutralized and stained in 0.5xTBE containing ss, single-stranded DNA; ds, double-stranded DNA. 0.5 I.tgmlq ethidium bromide for 10 min. Double-stranded DNA, which fluoresces more intensely with ethidium, gives an indication of product size, while the differential mobilities of one or both single strands reflect sequence differences (Fig. 1). Acridine orange staining (0.3 ~tg mlq ) is less sensitive and requires a destaining step. REFERENCES

1 0 r i t a , M. et al. (1989) Genomics 5, 874-879 2 Dockhorn-Dwomiczak, B. et al. (1991) Nucleic Acids Res. 19, 2500

Contributed by Eric P.H. Yap and James O'D. McGee (to whom correspondence should be addressed), University of Oxford, Nuffi'eld Department of Pathology and Bacteriology, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK.

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Signal transduction in yeast mating: Receptors, transcription

factors,and the kinaseconnection By GeorgeE Sprague,Jr (T/G7 no. 11/12, 393-398) In Figure 3 of this review, the labels for the genes FUS3 and FAR1 were accidentally transposed during preparation of the manuscript in the T/G Editorial office. The correct Figure and its legend are s h o w n here: the product of the gene FUS3 inhibits cyclin CLN3, and the product of FAR1 inhibits CLN2. We apologize to Dr Sprague for this error. Differentiation and cell cycle arrest in response to pheromone. Binding of pheromone (hatched circle) alters the conformation of the receptor. As a result of an attendant conformational change in Gc,, GTP replaces GDP, and GD is released. Activated GD then initiates a signal that passes throughSTE5 to four protein kinases (STE11, STE7, FUS3 and KSS1). The transcriptional activator STE12 is rapidly phosphorylated (asterisk) and transcription of target genes is stimulated. Some target genes are directly involved in differentiation (FUS1, KAR3), others in cell cycle arrest (FUS3, FAR1). As a result of the action of FUS3, FAR1, and another, hypothetical protein(s) (X), CLN products do not accumulate and cell cycle arrest in G1 ensues. Lines with arrowheads indicate stimulation; lines with terminal bars indicate inhibition. Other symbols are as in Fig. 2. See the text for a discussion of possible relationships among the kinases.

STE5

STE11, STE7, FUS3, KSS1

STE12 k

,, "-. uction

CLN1

TIG FEBRUARY1992 VOL. 8 NO. 2

:)9

STE12*

G1

CLN2 CDC28

CLN3

.~- ~ S

Cell cycle arrest

Differentiation