Analyse von dominant-negativ wirkenden Lamin-Deletionsproteinen und Identifizierung eines Spermien-spezifischen Lamins in Xenopus laevis

Abstract

Lamins are type V intermediate filament proteins. They form the lamina, a filamentous meshwork underlying the inner nuclear membrane. The nuclear lamina provides structural support to maintain nuclear shape and stability. Furthermore, lamins are involved in replication, transcription, and gene regulation. Conserved regions at the N- and C- terminus of the central alpha-helical rod domain affect the assembly of lamins into higher order structures. Within the course of this work three Xenopus lamin LIII mutants (Flag DN rod LIII, Flag DC rod, Flag DNDC rod LIII) lacking these conserved regions were generated. To analyse the function of the deletion proteins, they were expressed in Xenopus oocytes and embryos as well as in Cos-7 cells. In oocytes the three lamin mutants induce intranuclear membranes. The appearance of these membranes is different for all three deletion proteins and also differs from those induced by wildtype lamin LIII. The expression of the mutant proteins in Xenopus embryos leads to dominant-negative phenotypes. In the second part of this thesis, the sperm specific lamin LIV was identified. This lamin is a splice variant of the LIIIb-gene. It contains a 40 amino acid residues insertion in the coil 2 of the rod domain which interrupts the heptade repeats characteristic for the coiled-coil structures of lamins. RT-PCR- and Western blot- analyses of different Xenopus tissues have shown that expression of lamin LIV is restricted to testis. Moreover, database analyses of species expressing lamin LIII showed that the presence of lamin LIV is restricted to the amphibian lineage.