Métodos para formular raciones alimenticias para animales de especie menor

A Tunen DIG labelled antisense RNA probe was synthesised by digesting CDN16.6/pBS.3 with Asp718 and transcribing with T3 RNA polymerase. This was used to stain embryos from overnight yellow white (yw) collections. During very early stages of embryo development Tunen transcript expression can occasionally be seen in stripes (Figure 21a and b). Tunen is first expressed at high levels in the hindgut primordium and a segmental repeat is found in the ectoderm during germ band extension (Figure 21c). In later stages of germ band extension it is strongly expressed on the dorsal side of the posterior midgut (Figure 2 Id). Expression can also be seen in a medial ectoderm stripe and head region during germ band extension (Figure 2 le) In a germ band shortened embryo it is found expressed in the gut, nervous system and ectoderm (Figure 211).

Figure 21: Expression of Tunen transcript in developing Drosophila embryos

A lateral view of Drosophila embryos expressing Tunen RNA (blue) is shown in (a) - (d). Occasional stripes are seen in very early stages of development when germ cells are forming at the pole end (arrow head), (a) and the beginning o f germ band extension (b).Tunen transcript can be seen in the hindgut and a segmental repeat in the ectoderm (c) and (d). In the later stages of germ band extension Tunen is strongly expressed on the dorsal side of the posterior midgut (d).

Dorsal views of germ band extended (e) and ventral view of germ-band-shortened (f) embryos show Tunen RNA expression in a medial ectodermal stripe and head region (e) gut, nervous system and ectoderm (f). (Anterior to the left in all examples shown).

Tunen transcript expression was compared to Wunen using a DIG labelled antisense probe to Wunen generated by digesting pBSclS (Wunen cDNA cloned into pBlueScript, a gift Ifom Ken Howard) with BarnWl and transcribing with T7 polymerase. Figure 22 shows Wunen transcript expression in comparison to Tunen. These transcripts appear to be expressed in identical patterns.

Wunen Tunen

%

#

Figure 22: Comparison of Wunen and Tunen RNA expression in Drosophila

embryos.

Wunen (a), (c), (e) and Tunen (b), (d), (e), show very similar patterns of RNA expression (blue) during embryo development, (a), (b) (c) and (d) = lateral views, (e) and (f) = ventral view. (Anterior to the left in all examples shown).

Due to the nucleotide homology between Tunen and Wunen and similarity in expression pattern, cross-hybridisation was a concern. To determine the extent of cross­ hybridisation, unlabelled sense RNA for Tunen was made by digesting CDN16.6pBS.3 with Xba\ and transcribing with T7 polymerase. Wunen unlabeled sense RNA was made by digesting pBS c l 8 with Hindlll and transcribing with T3 polymerase. The transcribed sense RNA was checked on an agarose gel (Figure 23a). The concentration o f Tunen sense RNA was approximately 8 fold higher than Wunen, therefore, a 1/8 dilution in water was performed to allow equimolar concentrations of both RNAs to be tested. Using the diluted Tunen probe, ten fold serial dilutions were prepared in water for each RNA and Ipl of each dilution spotted onto a nylon membrane, cross linked by UV and the membranes placed in 1.5 ml ependorf tubes. The membranes were

prehybridised and then probed with antisense DIG labelled probes to Tunen and Wunen in standard in situ hybridisation conditions.

Weak cross hybridisation signals are only detected at the two highest RNA concentrations tested (Figure 23b and c). These cross-hybridisation signals should not be a concern for in situ hybridisation experiment as the level of RNA used at which they are seen is likely to far exceed that expressed in the embryo tissue.

(a)

2 3

Wunen

4 5 6

Tunen

(b ) Probed w ith W unen a n tisen se R N A

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neat 10 1 102 10^ l o ' '

(d ilu tion factor) (c ) Probed w ith Tunen an tisen se R N A

W unen se n se R N A Tunen se n se R N A ^ . < C L neat 10 * ,0^ 10^ W u nen se n se R N A Tunen se n se R N A

(dilu tion factor)

Figure 23: Analysis of cross-hybridisation between Wunen and Tunen RNAs

(see next page for details)

Figure 23: Analysis of cross-hybridisation between Wunen and Tunen RNAs

(a) Transcribed RNA for Wunen and Tunen. Lanes 1,2 and 3 were loaded with 0.5, 1, and 2|l i1of product from the Wunen in vitro transcription reaction. Lanes 4,5, and 6 are

loaded with 0.5, 1, and 2pi o f product from the Tunen in vitro transcription reaction. (b)The top row has Wunen sense RNA and the second row has Tunen sense RNA bound at different concentrations as shown. This was then probed with digoxygenin labelled anti-sense RNA for Wunen by in situ hybridisation

(c) Sense RNA was bound in the same way as in (b) but this filter was probed with digoxygenin labelled anti-sense RNA for Tunen.

3.1.6 Summary

A second member of the Drosophila melanogaster LPP family was identified and cloned. This protein which was called Tunen shares 34% amino acid homology with Wunen. Tunen also has the three phosphatase domains which are described[Stukey, 1997 #201;(Neuwald, 1997)]. These domains are almost identical to those in Wunen.The least homologous regions noted are at the amino and carboxy termini of these two proteins. The amino and carboxy termini are predicted to be cytoplasmic. This suggests that these regions are important in defining isoform specificity possibly through interactions with intracellular proteins. As yet, no obvious binding motifs have been identified in these regions of Drosophila or mammalian LPPs.

The pattern of expression of these two RNAs was studied in Drosophila embryos and showed that both transcripts are expressed in very similar patterns. As for Wunen, Tunen RNA is first expressed at high levels in the hindgut primordium and later on the dorsal side o f the gut. Strong expression is also seen in the developing nervous system.

To ensure that the similarity in expression pattern was not due to probe cross hybridisation, the ability of antisense Tunen to bind to Wunen sense RNA and vice versa was tested. Results demonstrated that the transcript pattern is specific for each RNA.These data confirm that there are at least two LPPs in Drosophila. The most homologous regions appear to be those areas neccessary for catalytic activity. These two proteins also share a very similar expression pattern. Because of the similarity in

expression, a role for Tunen in germ cell migration will be investigated. Tunen, like Wunen, is also expressed in the developing nervous system, thus a role for this protein in guiding neurons will be examined.