6. Recomendaciones y cuestiones abiertas
6.2 Cuestiones abiertas
4.2.3.1
S2-cells and polytene chromosomes
Whereas no specific nuclear signal for CHRAC14 or CHRAC16 was observed with any of the monoclonal rat antibodies on S2 cells (Figure 4.8 A), the 6G6 antibody (and to some extent the 1H2 antibody, not shown) stained some bands on Drosophila polytene chromosomes. Some of the bands co-localised with the ISWI-staining, whereas others did not (Figure 4.8 B). However, the signals were weak and the level of background staining was very high. Therefore, it cannot be excluded that the bands seen on polytene chromosomes, or at least some of them, are non-specific.
Figure 4.8: A: Immunostaining of S2-cells with the α-p14 antibody 1H2. B: Co-immunostaining of polytene
chromosomes with α-ISWI antibody (green) and the α-p16 antibody 6G6 (red). Some of the overlapping
4.2.3.2
Drosophila embryos
Also the immunostainings of Drosophila embryos with the monoclonal antibodies directed against p14 and p16 were hard to interpret. As already seen on polytene chromosomes, there was a high level of non-specific staining with most of the antibodies. Different developmental stages displayed different degrees of background staining, with a tendency of later stages (after cellularisation) being stained more intensively than earlier stages. In the early stages (until blastoderm), a shell at the embryo surface and the area surrounding the nuclei was stained particularly strong by the antibodies. Although the embryo surface staining is likely to be non- specific, it cannot be easily decided whether the staining around the nuclei contains a p14-p16- specific component.
Pre-incubation of the p16-specific antibody 6G6 with immobilised recombinant GSTp14- p16 suppresses the overall staining of the embryos (Figure 4.9, compare panels A and B). This can be seen in all developmental stages and suggests that the observed signal is indeed due to the 6G6 staining; however it does not necessarily mean that the staining is specific for p16. In fact, in some embryos, this antibody stains certain speckles that are clearly non-specific, since they co-localise neither with the ISWI staining nor the DNA staining (Figure 4.9 C). However, it cannot be ruled out that the antibody recognises p16 that is not associated with ISWI, but with another unknown factor. Whatever may be the case, it is very difficult to gain a credible p16 staining with the 6G6 antibody.
The only antibody that gave a definite signal was the anti-p14 antibody 5C7 (Figure 4.9 D). In very early developmental stages after only few nuclear divisions, it clearly stains the nuclei and co-localises with the ISWI staining. During mitosis, the antibody stains the centromeres (Figure 4.9 E), and the staining is very similar to the ACF1 staining on mitotic chromosomes (M. Chioda, unpublished observation). However, the signal intensity decreases with embryo age, whereas the intensity of the background staining increases, so that no distinct signal can be observed after the blastoderm stage. This suggests that CHRAC14-CHRAC16 are most abundant in the early developmental stages of the Drosophila embryo. Unfortunately, this monoclonal antibody recognises an additional band with a molecular weight of approximately 70 kDa on Western blots with embryo extracts (see Figure 4.5). Hence, although the signal co- localises with ISWI and shows a similar pattern than ACF1, it cannot be excluded that the immunostainings performed with this antibody do not solely reflect CHRAC14, but also another unknown protein.
Figure 4.9: Immunofluorescence on Drosophila embryos with the monoclonal antibodies directed against p14 and p16. The 6G6 antibody-staining (A) can be competed away by pre-incubation with recombinant p14-p16 (B), but gives non-specific signals in some embryos (C, see arrowheads). The signal 5C7 antibody signal co- localises with the ISWI-signal in early embryos (D) and stains the centromeres on mitotic chromosomes (E) (Panel E shows synchronous nuclear divisions during anaphase).
4.2.4
RT-PCR
For most applications, the monoclonal antibodies raised against CHRAC14 and CHRAC16 are not very reliable (see 4.2.1 to 4.2.3). Whereas the antibodies recognised CHRAC14 and CHRAC16 in Drosophila embryos, the detection in Drosophila cell lines was less successful (see 4.2.1 and 4.2.3.1). Therefore, the expression levels of the two small CHRAC subunits were checked by RT-PCR in KC and S2 cells. These cell lines are derived from Drosophila embryos, but possibly the expression pattern differs from the original embryonic tissues. It is imaginable that the expression of the small CHRAC subunits has been altered or downregulated.
Figure 4.10 shows the result of a typical RT-PCR reaction. Whereas there is a strong signal for both CHRAC14 and CHRAC16 from embryonic RNA (lanes 7 and 13), the signals from KC- cell RNA (lanes 9 and 15) and S2-cell RNA (lanes 11 and 17) are significantly weaker. The control RT-PCR with primers specific for the gene of the chromosomal kinase JIL-1 (lanes 1 to 6) indicates that this difference in mRNA levels is specific for the CHRAC14- and CHRAC16 subunits, because in the case of JIL-1, the difference in signal intensity between embryonic RNA and RNA from KC- and S2-cells is less pronounced (compare lanes 1, 3 and 5). The same result was obtained when primers specific for U6-RNA had been used for the control RT-PCR (not shown).
These experiments show that CHRAC14-CHRAC16 are expressed in KC and S2 cells, but – compared to Drosophila embryos – to a lesser extent. This could explain why it is difficult to detect the proteins in these cells with the weak monoclonal rat antibodies. The elevated mRNA levels in the embryos might not solely be due to higher expression, but also due to mRNA deposition by the mother (maternal contribution).
Figure 4.10: RT-PCR with primers specific for JIL-1 (lanes 1 to 6), CHRAC14 (lanes 7 to 12) and CHRAC16 (lanes 13 to 18). The CHRAC14 and CHRAC16 mRNA levels are significantly lower in KC- and S2 cells than in