GLAM assay

The GLAM assay is a simple reporter assay used for detection of mutations and agents influencing mRNA biogenesis in a gene length dependent manner in yeast (1).

It consists in the transformation of every strain to be tested with three different plasmids (see below), and the comparison of the expression from each plasmid, either by an enzymatic assay (Acid Phosphatase) or by Northern Blot.

A detailed protocol is as follows:

Yeast transformation

1.Transform your favourite strain and an isogenic WT with the following plasmids:

yCPlac33 _ An empty vector used to calculate the endogenous Acid Phosphatase activity.

pSCh202 _ “Short” transcription unit, comprising the PHO5 ORF under a GAL1 promoter.

pSCh212 _ “Long” transcription unit, containing a lacZ gene transcriptionally fused to the 3´end of the “short” transcription unit.

2.Select transformants in minimal media minus uracil.

Acid phosphatase

Based on (2).

3.Collect 3 ml of culture at 0,8-1 O.D.600nm. Wash with H2Od. Freeze the pellet at – 20 ºC.

4.Resuspend cells in 1 ml of H2Od. Measure O.D.600nm.

5.Take 30 µl of cells, add 400 µl of 70 mM Na-acetate  pH4.

6.Start the reaction adding 50 µl of pNPP.

7.Incubate 10 min. at 37ºC.

8.Stop the reaction adding:

•120 µl of 25% TCA.

•600 µl of saturated Na2CO3.

9.Measure O.D.405nm.(Dilute if it is too yellow).

10. Apply the next equation to obtain the Acid Phosphatase activity:

                   O.D.405nm*0,066

                   O.D.600nm*time(min=10)*volume (ml=0,03)

11. Subtract the value of yCPlac33-transformed yeast to both short and long transcript-transformed yeast.

12. If your mutant (or treatment) has a defect in transcription elongation (or gene length dependent mRNA biogenesis) you expect a decrease in the ratio long/short transcript (GLAM ratio) in comparison with the WT.

Northern Blot

13. To check the ratio by northern blot, we use the 900 bp fragment you obtain with a digestion of pSCh202 with EcoRV as a probe.

Solutions

70mM NaAc pH4: 0,57 g NaAc

         2,5 ml of Glacial acetic acid.

                              To 100 ml with H2Od.

pNPPs (4-nitrophenyl-phosphate): Prepare 50 mg/ml in buffer NaAc.

Saturated Na2CO3: 172 g in 400 ml of H2Od.

1.Morillo-Huesca, M., Vanti, M. and Chavez, S. (2006) A simple in vivo assay for measuring the efficiency of gene length-dependent processes in yeast mRNA biogenesis. FEBS J, 273, 756-769.

2.Haguenauer-Tsapis, R. and Hinnen, A. (1984) A deletion that includes the signal peptidase cleavage site impairs processing, glycosylation, and secretion of cell surface yeast acid phosphatase. Molecular and cellular biology, 4, 2668-2675.