Typically genes reside in chromosomal regions characterized by low chromatin condensation while highly condensed chromosomal loci such as centromeres and telomeres are thought to be genetically inactive, Exceptions to this have been reported for both yeast and Drosophilia, because they posses genes that function in highly condensed centromeres. On the other hand, there are no reports of such heterochromatic genes that code for mRNA in vertebrates. If there are gen es in vertebrate centromeres, they must reside in heterochromatin for their function and mutation of such genes could be responsible for hereditary genetic defects. The ability to microdissect specific segments of mitotic chromosomes using the laser scissors and then amplify the DNA of a small number of pieces using the polymerase chain reaction (PCR) represents a unique approach to the analysis of vertebrate chromosomal regions which are refractile to standard molecular cloning approaches. We are using this methodology to investigate the possibility that the centromere region of a vertebrate chromosome contains genes. Mouse chromosome centromeres were microdissected with laser, centromeres were collected with the glass needles, DNA was amplified by PCR with specific a-satellite DNA primers and amplified DNA was used as a probe to screen mouse embryonic cDNA library. Several clones have been recovered, one clone was randomly selected for the detailed analysis. The sequence analysi s revealed that one third of it had 96% homology to the retrotransposable elements, while the rest of the sequence was unique. Restrictional analysis of the genomic DNA suggested that the sequence is represented as a single copy. The unique part of the sequence was used as a probe to screen mouse genomic library in order to identify the gene that codes for this mRNA. Characterization of the gene is in progress. To simplify the recovery of microdissected chromosome region as more efficient procedure has been designed which exploits micro electrol mechanical devices (MEMS).
Showing the most recent 10 out of 663 publications
