Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1996-12-10
2001-02-13
Patterson, Jr., Charles L. (Department: 1652)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C530S350000
Reexamination Certificate
active
06187749
ABSTRACT:
The present invention relates to methods for decondensation and/or condensation of chromatin or chromosomes through the addition of substances to condensed or decondensed chromatin/chromosomes respectively. In particular, the invention relates to a method for decondensing and subsequently optionally recondensing chromatin in cells at the interphase stage of the cell cycle and to a method for condensing chromatin/chromosomes following disruption of sperm. The present invention also relates to substances for the decondensation and/or condensation of chromatin/chromosomes and to kits comprising said substances, as well as to the use of these substances in chromatin/chromosome analysis, reproduction in particular fertility assessment, the diagnosis and treatment of infertility, assisted conception either in the clinic or naturally and contraception.
Chromatin is a DNA protein substance of which chromosomes are made. The expression ‘chromatin/chromosome’ used herein means that both terms can be applied.
The analysis of chromatin/chromosomes is of fundamental importance in assessing the genetic well-being of an organism. In man, analysis of chromosomes is undertaken routinely on foetal cells isolated, for example, by amniocentesis and this allows the prenatal diagnosis of such disabling genetic diseases as Down Syndrome usually caused by the presence of an extra copy of chromosome 21. In several cases, the peripheral blood cells are analysed in relation to the suspected presence of a genetic disease in the individual. For many cancers, chromosomes are analysed in order to identify abnormalities which either correlate with the occurrence of a particular tumour type or which predict the subsequent rate of progression of the cancer and thus the well-being of the patient. In addition to these routine analyses of chromosomes, there has arisen in recent years a desire to undertake the same type of chromosome analysis on human sperm especially with suggestions that exposure to ionising radiation and chemicals acting in the same way as ionising radiation (so-called radiomimetic agents) might lead to inheritable chromosome defects in the germ-line.
Despite these examples of the analysis of chromosomes undertaken at present, it is clear that the more widespread analysis of chromatin/chromosomes in medical diagnosis is limited by the technical difficulties of undertaking such a analyses. Chromatin/chromosomes undertake changes in gross structure during cell division whereby chromatin/chromosomes at the metaphase (mitotic phase) of the cell cycle are condensed and usually readily visible upon cell lysis with hypotonic solutions but whereby at the interphase stages of the cell cycle, the chromatin/chromosomes are decondensed and not readily visible upon cell lysis. Chromatin/chromosome analysis most often relies upon culture of cells in vitro to accumulate actively dividing cells with condensed chromosomes in the metaphase stage of the cell cycle. The in vitro cell culture required may take days or weeks to accomplish, and some cells, especially those which are “terminally” differentiated such as nerve cells or sperm cells, are not at all amenable to cell culture in vitro.
As even in actively dividing cell populations, metaphase cells are rare, several methods have been developed to allow analysis of chromosomes in mammalian cell preparations. Commonly, dividing cells are treated with chemicals such as colchicine and vincristine which block dividing mammalian cells in metaphase producing an increased number of cells with condensed visible chromosomes.
More recently, fluorescently-labelled nucleic acid probes specific for the centromeric regions of individual chromosomes have been used to detect chromatin/chromosomes directly in interphase cells whereby fluorescent probes hybridise to very small segments of the decondensed chromatin/chromosomes in order to produce a well defined fluorescent spot indicating the presence of a chromosome.
Both of these widely used methods have drawbacks. Metaphase inhibitors rely on actively growing cell populations to be effective and many cell samples isolated from blood or tissues are usually slowly growing thus precluding an abundance of cells trapped in metaphase. Whilst fluorescent probes which recognise small regions on individual chromatin/chromosomes can give well defined fluorescent spots on interphase cells, probes which recognise a larger region such as a whole chromosome or large fragment of chromatin/chromosome may give rise to a diffuse fluorescent smear which may preclude, for example, the counting of individual chromatin/chromosomes or chromatin/chromosome fragments recognised by the probes. The use of nucleic acid probes can present another problem in relation to chromatin/chromosome decondensation whereby, in order for a nucleic acid probe to hybridise to a chromatin/chromosome, the DNA in the chromatin/chromosome has to be denatured, a process which itself can disrupt the structure of the chromatin/chromosome to make identification by the probe difficult.
A further technique which is applicable in certain circumstances is known as premature chromosome condensation (PCC). It was first described by Hittelman and Rao, (1978) Cancer Res. 38:416-423. This technique results in the condensation of chromatin/chromasomes in interphase cells. It may be achieved in vitro using CHO or HeLa cells, or inactivated Sendai virus. Alternatively non-physiological agents such as polyethylene glycol (PEG) may be involved as well as synthetic acidic proteins such as poly L-glutamic acid and extracts from non-mammalian cells such as Xenopus egg extracts from germ-line cells such as hamster oocytes. The technique has been utilised many times in the art, for example in studies of acute lymphblastic leukaemia (Macleod et al., Genes, Chromosomes & Cancer, (1989) 1, 135-138). It is however a very difficult technique to apply in the laboratory and only a limited number of research groups utilise it.
For sperm cells, current techniques for genetic analysis are difficult. This is because of the unusually tight compactment of nucleic acids in sperm heads thus precluding analysis. Often the microinjection of individual sperm into hamster eggs in order to achieve visible chromosomes from human sperm cells is required (“pseudofertilisation”). This is a peculiarly difficult technique to operate and only a few research groups undertake it. Another technique utilised is fluorescence in situ hybridisation (FISH) which employs fluorescently-labelled nucleic acid probes specific for the centromeric region of the chromosome as discussed above. The application of this technique is described by for example Hultén and Goldman in Chromosomal Alterations ed. obe and Natarajan (1994), Springer-Verlag.
A number of in vitro systems, which mimic the events at fertilisation, have been developed in order to enable the changes in chromatin structure and the mechanisms of chromatin remodelling to be studied. These are reviewed by Leno et al.,in John Innes Review, The Chromosome, Ed. J S Heslop-Harrison, (1992) R. B Flavell Bros. Scientific Publishers. p135-147. The principal component found to be effective in the decondensation and remodelling of sperm is nucleoplasmin, a protein isolated from the eggs of
Xenopus laevis.
For many cell types, such as foetal cells in amniotic fluid, the quality of chromosome preparation is highly variable between different samples even after several weeks of in vitro cell culture. Precise identification of chromosomes is often undertaken by “banding” whereby chromosomes stained with various dyes show a characteristic banding pattern across the length of the chromosome. This is particularly important in identifying fragments of chromosomes which have translocated onto another chromosome and also in scoring less gross lesions in chromosomes such as small deletions of chromosome material which might have little visible effect on chromosome length but which might result in the visible loss of a stained band. Critical in the achievement of a good interpretable banding pa
Banerjee Subhasis
Hulten Maj
Larson & Taylor PLC
Patterson Jr. Charles L.
Simeg Ltd.
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