GST-LAMIN FUSION PROTEINS ACT AS DOMINANT-NEGATIVE MUTANTS IN XENOPUSEGG EXTRACT AND REVEAL THE FUNCTION OF THE LAMINA IN DNA-REPLICATION

A cDNA encoding Xlamin B-1 was cloned from a whole ovary mRNA by RT-PC R, GST-lamin fusion constructs were generated from this cDNA by first creating convenient restriction sites within the Xlamin B-1 coding seq uence, using PCR directed mutagenesis, and then sub-cloning relevant s equences into pGEX-4T-3. Two expression constructs were made, the firs t, termed Delta 2+ lacked sequences encoding the amino-terminal 'head domain' of lamin B1 but included sequences encoding the nuclear locali zation signal sequence (NLS), The second expression construct, termed Delta 2-, lacked sequences encoding the amino-terminal 'head domain' a s well as sequences encoding the NLS, Purified fusion proteins express ed from these constructs, when added to egg extracts prior to sperm pr onuclear assembly, formed hetero-oligomers with the endogenous lamin B -3. The Delta 2+ fusion protein prevented nuclear lamina assembly but not nuclear membrane assembly, The resulting nuclei were small (simila r to 10 mu m in diameter), did not assemble replication centers and fa iled to initiate DNA replication, When the Delta 2-fusion protein was added to egg extracts prior to sperm pronuclear assembly, lamina assem bly was delayed but not prevented, The resulting nuclei although small (similar to 12 mu m), did form replication centers and initiated DNA replication, When added to egg extracts after sperm pronuclear assembl y was completed Delta 2+, but not Delta 2-, entered the pre-formed nuc lei causing lamina disassembly. However, the disassembly of the lamina by Delta 2+ did not result in the disruption of replication centers a nd indeed these centres remained functional. These results are consist ent with the hypothesis that lamina assembly precedes and is required for the formation of replication centers but does not support those ce nters directly.