Production of lampbrush chromosome

Details Production of lampbrush chromosome Production of lampbrush chromosome

1998-11-16

2001-03-27

Fredman, Jeffrey (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091500, C435S806000, C435S810000, C424S520000

Reexamination Certificate

active

06207382

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a process for inducing a transcriptionally active chromosome (i.e., a “lampbrush” chromosome) from condensed chromatin or nuclei.
2. Description of the Related Art
More than 100 years ago, Flemming described giant chromosomes in the oocyte nucleus or germinal vesicle (GV) of the salamander
Ambystoma mexicanum.
His initial brief observations were soon followed by detailed investigations that established the existence of giant chromosomes in GVs of many animals, both vertebrate and invertebrate. Rückert named them lampbrush chromosomes (LBCs) because of their fancied resemblance to the brushes used for cleaning kerosene lamp chimneys.
Lampbrush chromosomes of amphibian oocytes are the largest known chromosomes, rivaled only by the giant polytene chromosomes of Drosophila and other flies. They are characterized by the presence of hundreds or thousands of transcriptionally-active regions that loop out laterally from the main axis of the chromosome. Each loop consists of one or a small number of active genes. Striking variations in the morphology and/or molecular composition of individual loops and of other landmarks along the main axis (e.g., centromeres, telomeres, axial granules, and the like) make it possible to map these chromosomes at the gene level by conventional light microscopy, using combinations of morphology, antibody binding, and in situ nucleic acid hybridization.
Although lampbrush chromosomes are found in a variety of organisms, they are conspicuously absent from many others or are not analyzable in some cases because of the small size of germinal vesicles in some organisms. For instance, it is still debatable whether typical lampbrush chromosomes occur in mammalian oocytes, including human oocytes, but even if they do, the germinal vesicle is too small to analyze such chromosomes by the techniques of the prior art.
The overall organization and functional significance of lampbrush chromosomes have been the subject of extensive experimentation and speculation (reviewed in Callan, 1986; Davidson, 1986). Because LBCs occur in oocytes during the growth period, they are in an arrested diplotene phase of meiosis I with homologous chromosomes held together at one or more chiasmata. Each homologue has a DAPI-positive axis of chromomeres, which correspond to transcriptionally-inactive regions where sisters are intimately associated. Numerous pairs of loops extend laterally from the chromomere axis; these consist of transcriptionally-active regions where sisters are completely independent of one another. Even at the light microscope level of analysis, loops are not of uniform thickness, but contain of one or more “thin-to-thick” regions, which correspond to transcription units. The electron microscope shows that the “thin-to-thick” morphology reflects the increasing length of nascent RNP fibrils along the transcription unit in the direction of transcription. This structure of LBCs reflects their transcriptionally active state.
Despite a wealth of detailed morphological and molecular information on lampbrush chromosomes, fundamental questions remain about their structure and especially about the significance of their highly active transcription. At one time their structure was regarded as unusual, but the discovery of looped chromatin domains in somatic nuclei (Paulson and Laemmli, 1977; Saitoh and Laemmli, 1993; Yokota et al., 1995) suggests that the lampbrush condition is a good model for chromosomes in general. However, the cis and trans factors which cause a condensed chromosome to assume the lampbrush condition had not been identified.
These and other questions about LBCs would be easier to address in a system in which the lampbrush state could be artificially induced and manipulated. In the present application, we disclose that condensed chromatin injected into a heterologous germinal vesicle gives rise within hours to distinctive transcriptionally-active LBCs. This system will be useful in assessing the relative importance of cis and trans acting factors in establishing the morphological and molecular attributes of LBCs. It should also permit analysis of transcriptionally-active chromosomes from organisms whose oocytes cannot be handled by current techniques or do not go through a typical lampbrush stage. Furthermore, LBCs from interphase nuclei exhibit a pattern of loops useful for cytogenetic analysis with higher resolution than interphase chromosomes painted by fluorescent in situ hybridization. Such loop patterns allow mapping, essentially at the single gene level.
SUMMARY OF THE INVENTION
In one embodiment of the invention, a process is provided of contacting a chromosome from sperm or other nuclei with the contents of a germinal vesicle of an oocyte to produce a transcriptionally active chromosome (i.e., a lampbrush chromosome). Examples of such are microinjection into an intact germinal vesicle, exposure to an extract of germinal vesicles or fractionated oocytes in a cell-free system, and incubation with isolated fractions of germinal vesicle contents. Preferably, the chromosome and the germinal vesicle are derived from organisms of different species (i.e., a heterologous process).
The chromosome-containing nuclei may be derived from sperm, other totipotent cells, interphase cells, somatic or differentiated cells, hematopoietic cells, benign or metastatic tumors, normal or transformed solid tissues, non-proliferating cells, and non-replicating cells. The source of the nuclei may be a vertebrate, such as a fish, mammal, hamster, mouse, rat, or human.
Germinal vesicles and oocytes are preferably from an amphibian (e.g., frog, newt, salamander, toad) but are not a preferred source of chromosomes. In the heterologous process, the chromosome produced has a morphology which resembles lampbrush chromosomes of the source of the germinal vesicle.
Disruption or removal of membranes, especially the nuclear envelope, surrounding the chromosome is preferred prior to contact with the germinal vesicle contents. Detergents, enzymes, mechanical disruption, or a combination thereof, may be used for removal of the membrane.
Another embodiment of the invention is providing the transcriptionally active chromosome (i.e., a lampbrush chromosome) produced by the invention on a solid substrate. Preferably, the chromosome produced is attached to a glass microscope slide or electron microscope grid.
The advantages of the invention include the ability to manipulate the conditions under which lampbrush chromosomes are produced, to provide chromosomes from interphase nuclei with distinctive loop patterns, and to produce such looped chromosomes from organisms or tissues which do not naturally contain lampbrush chromosomes.


REFERENCES:
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patent: 95/21860 (1995-08-01), None
Montag et al., “In vitro Decondensation of Mammalian Sperm and Subsequent Formation of Pronuclei-Like Structures for Micromanipulation”, Molecular Reproduction and Development, vol. 33, 1992, pp. 338-346, XP-00209706.
Gurdon, Injected Nuclei in Frog Oocytes: Fate, Enlargement, and Chromatin Dispersal, Journal of Embryology and Experimental Morphology, vol. 36, 1976, pp. 523-540, XP-002097751.
Gall et al., “Assembly of Lampbrush Chromosomes from Sperm Chromatin”, Molecular Biology of the Cell, vol. 9, Apr. 1998, pp. 733-747, XP-002097707.
Gall et al., “Lampbrush Chromosomes from Xenopus Sperm Chromatin”, Molecular Biology of the Cell (Abstracts), vol. 8, No. Suppl, 13-17 Dec. 1997, p. 4A, XP-002097705.
Lohka et al., “Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components,” Science, 1983, 220:719-721.
Lohka et al., “Roles of cytosol and cytoplasmic particles in nuclear envelope assembly and sperm pronuclear formation in cell-free preparations from amphibian eggs,” Journal of Cell Biology, 1984, 98:1222-1230.
Kirschner et al., “The timing of early developmental events in Xenopus,” Trends in Genetics, 1995, 1:41-47.
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