Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-12-03
2002-01-29
Zitomer, Stephanie W. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S320100, C435S325000, C435S091100, C435S091200, C435S069100, C435S173600, C435S455000, C536S023100, C536S024330, C536S024300, C436S504000
Reexamination Certificate
active
06342354
ABSTRACT:
BACKGROUND OF THE INVENTION
(a) Field of the Invention
The invention relates to a method for mapping and isolating regions of chromosomes that interact together or associate functionally in vivo. The method of the present invention is also referred to as recombination access mapping (RAM).
(b) Description of Prior Art
Currently in the field of molecular biology, it is becoming quite evident that gene regulation occurs through a complex network of processes. Transcription, replication and recombination of DNA must occur in a timely and appropriate manner or the outcome may be disastrous. Extreme control over these processes is required during development and differentiation of tissues in multicellular organisms. In contrast, disorder of these processes occurs during oncogenesis. Cancer cells often exhibit aberrant expression of genes as well as general genomic instability, a hallmark of which is an increase in recombination rates.
Activation or repression of gene expression by transcription factors or repressors respectively has been studied in great detail both in vitro and in vivo. Changes in chromatin structure are intimately linked to the activation or inactivation of a gene and can affect the replication or recombination of DNA. Mostly in vitro studies and limited in vivo data has been used to determine such changes in chromatin as they relate to DNA transcription, replication and recombination. From this data it is apparent that chromatin is organized in DNA loop domains that are organized through interaction with a nuclear protein matrix. These domains average in size from 60-100 kb and are assumed to be flanked by matrix attachment regions (MARS). MARS have been associated with functional domains of transcription and replication. Indirect methods such as DnaseI sensitivity and DNA cleavage assays involving topoisomerase inhibitors, have provided further evidence of higher order chromatin domains which may correspond to single loops (60-100 kb) and loop arrays (300 kb). Functional chromatin domains seem to exist for transcription and replication (reviewed by in Jackson, D. A.,
Bioessays
17:587-591, 1995). Strong evidence exists which suggests chromatin structure also plays a role in recombination as in VDJ recombination, formation and repair of double strand breaks in irradiated cells, and during meiosis; differences in meiotic recombination between imprinted domains.
Therefore, the central question is raised as to the interaction between chromatin domains and their accessibility to biological molecules are involved in gene regulation. Many studies of chromatin focus on in vitro data at the nucleosomal level (Wolffe, A. Chromatin: Structure and Function, Second Ed.
Academic Press Inc
. San Diego 1995). Unfortunately, it is most likely that large scale changes in chromatin packaging beyond the nucleosomal level are primarily responsible for the maintenance of certain chromatin states, such as early or late replication and hetero vs. euchromatin. Very few techniques exist to analyze DNA interaction in vivo in a global fashion over the entire genome.
Ectopic gene targeting is an alternative outcome of the gene targeting process in which a targeting vector acquires sequences from a genomic target but proceeds to integrate elsewhere in the genome. More specifically, ectopic gene targeting is a process by which an extra chromosomal molecule (recipient) obtains DNA sequence from a target locus via one-end invasion and gene conversion followed by release of the recipient molecule and integration, complete with the newly acquired sequence from the target locus, Aelsewhere in the genome. Such events were first observed in gene targeting experiments involving the adenine phosphoribosyl transferase (APRT) locus in CHO cells (Adair, G. M., et al.,
Proc. Natl. Acad. Sci. USA
86:4574-4578, 1989) and in experiments involving retroviral transfection of rat cells (Ellis, J. et al.,
Mol. Cell. Biol
. 9:1621-1627, 1989). Consequently, a model has been proposed for the mechanism of ectopic gene targeting (Belmaaza, A., et al.,
Nucl. Acids Res
. 18:6385-6391, 1990; and Belmaaza, A. et al.,
Mut. Res
. 314:199-208, 1994). Instances of ectopic gene targeting and/or ectopic gene conversion have been seen in Drosophila (roo element, p and hobo elements), plants, yeast (between dispersed repeated genes or Ty 1 repeat elements), fungi (in Ustilago maydis, chickens (Ig rearrangement)), rabbit (generation of antibody repertoire), mice (germline ectopic gene conversion in spermatids, gene conversion between Line-1 elements, and humans (gene conversion between Line-1 elements and pseudo autosomal region on X and Y chromosome).
Although the phenomenon of ectopic gene targeting is well documented, the question of where the recipient molecule integrates, with respect to the target locus, has not been determined. It is apparent from Southern analysis that the recipient integrates in most cases at a distinct site from the target but Southern analysis does not permit the determination of the relative position of the ectopic sites with regard to the target locus.
It would be highly desirable to be provided with a method allowing the identification of interactions between chromatin domains within or between chromosomes which may be involved in gene regulation. With such a method, the functional organization of the genome could be mapped. The understanding the three-dimensional (3-D) in vivo interactions between chromosome could help to better understand complex gene regulation during cancer or other disease states.
SUMMARY OF THE INVENTION
One aim of the present invention is to provide a method allowing to define functional organization of chromatin in vivo with respect to interchromosomal and interchromatin domain interactions involved in gene regulation, including but not limited to replication, transcription and recombination.
Another aim of the present invention is to provide a method allowing to mark domains of chromatin that interact functionally in vivo with a given gene locus for the purpose of cloning such domains or their visualization in 3-D using confocal fluorescent microscopy.
Another aim of the present invention is to provide a method allowing to define points of interaction between chromosomes involved in translocation or ectopic gene conversion within or between chromosomes.
Another aim of the present invention is to provide a method allowing to define chromatin domain interactions between chromosomes involved in epigenetic phenomenon such as imprinting, position effect variegation and transvection.
Another aim of the present invention is to provide a method allowing to produce diagnostic ectopic gene targeting distribution profiles, such as fingerprints, for a given gene locus.
Another aim of the present invention is to provide a method allowing to determine changes in genomic organization associated with various disease states as a means of monitoring disease progression or onset.
Another aim of the present invention is to provide a method allowing to study developmental changes in multicellular organisms such as during tissue development.
Another aim of the present invention is to provide a method allowing for the placement of DNA elements or recognition sites for enzymes for the purpose of chromosomal engineering.
Another aim of the present invention is to provide a means for mapping the distribution of double strand breaks in DNA, which are natural breaks or induced by any means, over a given region of a chromosome with respect to a chromosomal DNA sequence to be studied, which in turn allows for the definition, characterization and cloning of structural and/or functional genomic domain(s) containing the chromosomal sequence being studied.
A further aim of the present invention is to provide a method allowing to assess the affects of a given drug or chemical on genomic organization and stability such as for defining oncogenic potential of a substance.
A still further aim of the present invention is to provide a method allowing to define at what time in a cell cycle chromatin domai
Chartrand Pierre
Dellaire Graham
Crowell & Moring LLP
Universite de Montreal
Wilder Cynthia B
Zitomer Stephanie W.
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