Exercise 13.8 - Prestalk vs Prespore Effect

LEVEL III

Figure 13.7 Prestalk/prespore immunofluoresence

Materials

• Agar cultures of D. discoideum
  
• 20 mM Na-K Phosphate buffer, pH 7.0
  
• 20 mM Na-K Phosphate buffer plus 20 mM EDTA, pH 7.0
  
• Hypodermic syringe with 16 gauge needle
  
• Nylon mesh
  
• 70% v/v Percoll in 20 mM EDTA, 5 mM MES, pH 7.0
  
• Preparative centrifuge, rotor and tubes

Procedure 28

1. Collect migrating slugs by washing them from the agar with cold 20 mM Na-K Phosphate Buffer, pH 7.0. A bent glass rod may be used to assist in dislodging the slugs.

2. Wash the slugs once with the same buffer and resuspend in 20 mM Na-K Phosphate Buffer containing 20 mM EDTA.

3. Pipette the slugs up and down through a 16 gauge needle for about 5 minutes to dissociate the cells. 29

4. Pass the suspension of cells through a fine nylon mesh to remove clumps, wash with the EDTA buffer and resuspend in 2-3 ml of fresh EDTA buffer. Collect the cells by centrifuging for 2 minutes at 300 xg. Resuspend the cells to make a final concentration of 4 X 10 cells per ml of EDTA buffer.

5. Form two Percoll gradients by placing 12 ml of Percoll into two centrifuge tubes and centrifuging at 21,000 xg for 40 minutes at 4° C.

6. Layer 250 µl of the cell suspension onto a preformed Percoll gradient. Either distribute the cells between the two gradients, or use one tube as a balance in the centrifuge.

7. Centrifuge the amoeba in the preformed Percoll gradient at 13,000 xg at 15° C for 3 minutes.

8. Collect and identify the bands of cells. Wash the cells free of Percoll with EDTA buffer.

Notes

Once separated on Percoll, the cells can be analyzed for any number of activities. One of the principal analyses is for the production of specific gene products which would indicate DNA control of differentiation. The effect of differential gene activity can be studied by extracting RNA from the prestalk and prespore cells with subsequent analysis of the RNA.
Since the ultimate gene product is a protein, several investigators have identified and isolated proteins characteristic of prestalk and prespore differentiation. By producing antibodies to those proteins, immunofluorescent techniques may be applied to the detection of early differentiation. Figure 13.7 demonstrates such an analysis applied to prestalk/prespore cells.

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