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MOLECULAR GENETIC ANALYSIS OF THE CENTROMERE OF HIGHER EUKARYOTES

Objective


A structural analysis has been performed of the human Y chromosome centromeric deoxyribonucleic acid (DNA) sequences. At the centromere are major arrays of satellite DNAs: an alphoid satellite (170 base pairs (bp) repeat) which is variable in size and ranges from 250 kb to 1400 kb, and a 5 bp satellite which is about 400 kb in size. On the short arm side are a small block of the 48 bp satellite and a small block of alphoid DNA (about 18 kb). On the long arm side are small blocks of the 48 bp satellite (21 kb), the 5 bp satellite (17 kb) and the 68 bp satellite (27 kb). Between these tandemly repeated arrays are many novel repeated sequences which do not appear to be tandem repeats.

In situ hybridization techniques have been developed for mapping cloned centromeric sequences onto wild type and rearranged Drosophila chromosomes, in particular the Drosophila melanogaster dodecasatellite sequence. It is located at a unique site, within the pericentric heterocromatin of the right arm of the third chromosome of D melanogaster, closely linked to the primary constriction. In polytene chromosomes, dodecasatellite is found as one or a few dots, in the central region of the chromocentre. In untreated diploid cells, dodecasatellite sequences are found as 1 or 2 dots through the cell cycle. In interphase cells, hypotonic shock promotes the decondensation of the genomic region containing this satellite, resulting in a string like structure. Some of the precociously separated sister chromatids produced by colchicine treatment show dodecasatellite within the intervening space connecting the main dodecasatellite signals of each chromatid. The distribution of dodecasatellite seems to be constant between individuals of the same species but the distribution of dodecasatellite showed a remarkable degree of variation amongst closely related species of the melanogaster subgroup ranging from nondetectable signal in D. yakuba and D. teissieri, to staining in the X, second and third chromosome of D. mauritiana.

Dodecasatellite has a repeat unit length of 11-12 bp and these repeats have an asymetric distribution of guanine (G) and cytosine resulting in one strand being relatively guanine rich in comparison with the other. The G-strand oligonucleotides can associate to form stable non-Watson-Crick foldback structures containing guanine guanine and guanine adenine base pairs.
The objective of this project is to understand the structure and function of the centromeres of higher eukaryotic chromosomes. This requires analysis at the molecuar and at the genetic level, and both human and Drosophila chromosomes will be studied. In humans, a partial physical map of the DNA of one centromeric region is available and a functional test will be developed for the centromeric activity of YAC clones from this region. In Drosophila, DNA probes from the centromeric regions will be obtained by microdisection of the chromocenter of polytene chromosomes and used to construct physical maps of normal and rearranged chromosomes, thus localizing functionally important areas. Proteins required for centromere function in Drosophila will be identified and studied by genetic and immunological methods. The project will make it possible to construct minichromosome vectors for higher eukaryotic cells, thus facilitating their genetic manipulation.

Coordinator

CONSEJO SUPERIOR DE INVESTIGACIONES CIENTIFICAS
Address
Calle Serrano 117
Madrid
Spain

Participants (2)

THE CHANCELLOR, MASTERS AND SCHOLARS OF THE UNIVERSITY OF OXFORD
United Kingdom
Address
University Offices, Wellington Square
Oxford
University of Dundee
United Kingdom
Address
The Nethergate
DD1 4HN Dundee