Correlación de las variables de estudio y dimensiones de investigación

The levels o f NrcA protein expressed during the growth o f vegetative cells were examined.

The Dictyostelium cyclin B and Cdc2 protein levels are both constant and high during

vegetative growth but decline markedly upon entry into stationary phase (Luo et a l , 1995), a pattern o f expression consistent with a cell cycle role with strong expression during

exponential growth and low expression when cells are not actively dividing.

Vegetative Dictyostelium A x-2 cells were grown in axenic liquid culture and equal numbers o f cells were removed at various time points, shown in figure 7.5A. Samples o f

1 xlO^ cells were analysed by SDS-PAGE and immunoblotted with the anti-NrcA antibody serum. I initially analysed the results o f film that had been exposed for a normal period o f time to the ECL reaction, but inconsistencies described below caused me to return to this experiment and analyse the results o f a much longer exposure, shown in figure 7.5B. The levels o f the single strong band previously recognised in a Dictyostelium cell extract (figure 7.4 lane 14) remained virtually constant throughout vegetative growth (figure 7.5B, lanes 5 to

11). These bands were analysed by scanning densitometry o f less overexposed film which confirmed that the protein levels fluctuated very little during exponential growth (time points A to E) and early stationary phase (time point F) and there was only a small drop o f about 25% in level after the cells had been in stationary phase for over a day (time point G).

Antigen IVT Antigen IVT k-Da 116 93 66 58 29 1 2 3 4 5 6 7 Pre-im m une 8 9 10 11 12 13 14 Immune

Figure 7.4 The immune serum from a rabbit injected with the truncated NrcA polypeptide antigen recognises NrcA protein

Each sam ple w as analysed by S D S -P A G E and im m unoblotted with either the rabbit pre-im m une or im m une serum before d etection by ECL. The sam ples included 10-fold serial d ilutions o f the p ellet fraction, from lysed ce lls that had expressed the original polypeptide antigen, cou p led in vitro transcription / translations (IV T ) with either no D N A or nrcA D N A added and a sam ple o f

D ictyo steliu m vegetative ce ll extract.

A

,

.

B

Aniigen ^ Growth curve ^ time points V ^ A B C D E F G

• r t

Weak hand ot similar s i / e to IVT nrcA

Strong endogenous band

29

1 2 3 4 5 6 7 8 9 10 11

Figure 7.5 The levels of NrcA protein expression during growth

(A) C ell counts o ï D ictyo ste liu m vegetative am oebae grow n in axenic liquid culture were taken over a period ol 4 days using a Neubauer cham ber.

(B ) Equal num bers o f ce lls were harvested from this culture at the time points show n and analysed by S D S -P A G E before im m unoblotting with the anti-N rcA antibody and detection by ECL. A lso analysed were 10-fold serial dilutions o f the p ellet fraction, from lysed c e lls that had expressed the original polypeptide antigen, and a coupled in vitro transcription / translation (IV T ) o f nrcA D N A .

The long exposure also revealed a number o f much weaker additional bands which were undetectable with a normal length exposure (lanes 5 to 11). One o f these bands corresponded in size to that obtained by coupled in vitro transcription / translation o f nrcA D N A (lane 4). This band w as present at nearly constant levels throughout exponential growth (lanes 5 to 9, time points A to E) but disappeared when cells entered stationary phase (lanes 10 and 11, time points F and G). The only other, slightly larger, band corresponding approximately in size to in vitro translated nrcA varied significantly in level during exponential growth with higher levels at a later stage o f growth which persisted into early stationary phase (time point F). Thus, the weak band o f the same size as in vitro translated nrcA bore the closest relation to a cell cycle protein with higher, constant expression during exponential growth and a rapid reduction in levels as cells entered stationary phase.

Because a polyclonal antibody was used, only approximate comparisons o f the band intensities with those o f known quantities o f the smaller NrcA polypeptide antigen (figure 7.5B, lanes 1 to 3) can be made. These indicate that about 3 ng /lO^ cells o f the strong band was present during exponential growth, corresponding to 30 000 copies/cell and about 0.1 ng /lO^ cells o f the weak band o f the same size as in vitro translated nrcA was present, equivalent to 1000 copies/cell.

Attempts were then made to synchronise Dictyostelium vegetative cell growth to determine if the levels o f these protein bands oscillated during the cell cycle, which would have given a much better indication o f whether one o f the bands exhibited cyclin-like behaviour. I tried to synchronise cells by the well established D ictyostelium method o f release from stationary phase (Weijer et a l , 1984). Good synchrony should have resulted in a period o f low increase in cell number after release followed by a rapid cell doubling. Unfortunately, my preliminary attempts at synchrony gave a relatively constant rise in cell number after release and I did not have sufficient time to repeat the experiment.