IDENTIFICATION OF PORTO-1, A NEW REPEATED SEQUENCE THAT LOCALIZES CLOSE TO THE CENTROMERE OF CHROMOSOME-2 OF DROSOPHILA-MELANOGASTER

We have used the polymerase chain reaction (PCR) technique to search t he Drosophila melanogaster genome for the presence of sequences with h omology to mammalian and yeast centromeric DNA. Using primers based on the human CENP-B box present in alpha-satellite DNA and part of the S accharomyces cerevisiae CDEIII centromeric sequence, a number of speci fic DNA fragments were amplified from total genomic DNA. In situ hybri dization to polytene and mitotic chromosomes showed these fragments to localise to centromeric and pericentromeric regions. Direct cloning o f the amplified fragments into conventional plasmids proved unsuccessf ul. However, a recombinant P1 clone containing D. melanogaster genomic DNA that supports PCR amplification by the primers was identified. Mo lecular characterisation of this clone revealed a DNA fragment that lo cal ises primarily to the centromere of chromosome 2. Sequence analysi s indicated that this fragment contains at least four different repeat s, including Rsp, transposable elements, Bari-1 and a new AT-rich repe ated sequence that we have designated Porto-1. Detailed fluorescence i n situ hybridization analysis shows that Porto-1 is localised very clo se to the primary constriction of chromosome 2. Sequence analysis sugg ests that this repeat was specifically amplified by our primers, altho ugh limited homology to the CENP-B box or CDEIII elements was found. I n situ hybridization to a number of Drosophila species shows Porto-1 t o be present only in D. melanogaster.