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Approach to the physical and functional mapping of human genome in metaphaseand interphase chromosomes by in situ hybridisation and 3-dimensional confocal laser scan microscope imaging

Objective

The human genome is organized as a 3-dimensional entity packed inside the nucleus during interphase and as highly condensed chromosomes during mitosis. The relative position of specific parts of the genome and genes is likely to be of primary importance for both their regulation and possible rearrangements. The aim of this project is to develop approaches for the physical mapping of DNA sequences on metaphase and interphase chromosomes, and vice versa to project chromosomes' remarkable points onto the DNA sequence as it becomes available.
Approaches for the physical mapping of deoxyribonucleic acid (DNA) sequences on metaphase and interphase chromosomes have been developed. The development of fluorescent procedures for the detection of in situ hybridization (ISH) DNA probes was combined with the development of systems for 2-dimensional digital imaging and 3-dimensional digital imaging of the fluorescence signals. These systems wee optimized for maximal spatial resolution, sensitivity and optimal discrimination of multiple fluorescent probes.

New methodologies and technologies involved development of:
a new approach for the construction of analytical chromosomal bar codes adaptable to the particular needs of cytogenetic investigations and automated image analysis;
a new approach foer the rapid detection of amplifications and losses of genetic material in tumor cells, based on the simultaneous hybridization of tumoural and normal DNA to metaphase chromosomes, and on the measurement of fluorescence intensities ratios;
a new method for simulation of the imaging pathway in the confocal laser scanning microscope (a water immersion lens was designed which can be optimized for a spectral range of 400 to 700 nm and has correction for refractive index differences);
a special software version for the new laser scanning microscope LSM4, dedicated to the measurement of distances in space (this functionality gives the possibility to measure distances between different ISH fluorescent spots in interphase nuclei);
a software package (cytoFISH) on a Silicon Graphics workstation for 2-dimensional quantitative ISH probe imaging and 2-dimensional mapping;
an image preprocessing and processing software environment called EXPLORER (this was designed with the aim of providing an open environment able to be applied to a variety of processing tasks in the field of fluorescence imaging);
3-dimensional quantitative ISH probe imaging and mapping (this was applied to the study of the size and shape of chromosome territories in the cell nucleus and the results obtained strongly support a model which proposes that the genetic activity of chromosome territories in the cell nucleus is correlated with their 3-dimensional shape and surface, but not with their volume).
To reach these goals, the development of fluorescent procedures for the detection of in situ hybridization (ISH) DNA probes has to be combined with the development of systems for 2-dimensional and 3-dimensional digital imaging of the fluorescence signals. To bridge the gap between cytogenetics and molecular biology, these systems have to be optimized for maximal spatial resolution, sensitivity and discrimination of multiple fluorescent probes. These improvements will make it possible to order DNA sequences close to one another and thus contribute to increasing the precision of cytogenetic diagnosis of chromosomal rearrangements.

The research activities to be carried out will converge on :
the development of a routine fluorescent multicolour reproducible ISH approach to the location of probes and to the orientation of chromosome specific contigs on selected chromosomes (X,Y,17,19);
the development of a specific high resolution digital imaging system for the detection of fluorescent ISH signals, by improving the confocal laser scan microscope to interface with the image processing and analysis dedicated workstation;
the development of a database concept for information storage and knowledge representation of 2-dimensional and 3-dimensional probe locations compatible with the low level DNA sequence representation and the high level conventional physical and genetic maps of chromosomes.

Owing to the strong involvement of two industrial companies, the project will contribute to the provision for molecular biologists and cytogeneticists of a tool to locate and interpret chromosomal anomalies by reference to known DNA sequences. This, in turn, bridges the gap between molecular genetic knowledge and that of pathologies of genetic origin in order to more quickly collect new knowledge on human genome structure and function.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

UNIVERSITE JOSEPH FOURIER - GRENOBLE 1
Address
Avenue Centrale 621
38041 Grenoble
France

Participants (4)

ALCATEL TITN Answare
France
Address
34 Avenue Du General De Gaulle
38100 Grenoble
CARL ZEISS GMBH
Germany
Address
Carl Zeiss Straße
7082 Oberkochen
Ruprecht-Karls-Universität Heidelberg
Germany
Address
Im Neuenheimer Feld 328
69120 Heidelberg
Université de Grenoble I (Université Joseph Fourier)
France
Address
Domaine De La Merci
38700 La Tronche