I. Field of the Invention
The present invention relates generally to the field of molecular biology. More particularly, it concerns methods for isolating centromere DNA.
II. Description of Related Art
It is well documented that centromere function is crucial for stable chromosomal inheritance in almost all eukaryotic organisms, including essentially all plants (reviewed in Nicklas 1988) or animals. For example, broken chromosomes that lack a centromere (acentric chromosomes) are rapidly lost from cell lines, while fragments that have a centromere are faithfully segregated. The centromere accomplishes this by attaching, via centromere-associated proteins, to the spindle fibers during mitosis and meiosis, thus ensuring proper gene segregation during cell divisions.
To date, the most extensive and reliable characterization of centromere sequences has come from studies of lower eukaryotes such as S. cerevisiae and S. pombe, where the ability to analyze centromere functions has provided a clear picture of the desired DNA sequences. None of the essential components identified in unicellular organisms, however, function in higher eukaryotic systems. This has seriously hampered efforts to produce artificial chromosomes in higher organisms.
Genetic characterization of centromeres has relied primarily on segregation analysis of chromosome fragments, and in particular on analysis of trisomic strains that carry a genetically marked, telocentric fragment (for example, see Koornneef 1983). This approach is imprecise, however, because a limited set of fragments can be obtained, and because normal centromere function is influenced by surrounding chromosomal sequences (for example, see Koornneef, 1983).
A more precise method for mapping centromeres that can be used in intact chromosomes is tetrad analysis (Mortimer et al., 1981), which provides a functional definition of a centromere in its native chromosomal context. However, the technique is currently limited to a small number of organisms and is relatively labor intensive (Preuss 1994, Smyth 1994). To date, among higher plants, the technique has only been used successfully in Arabidopsis (Copenhaver, 1999).
Another avenue of investigation of centromeres has been study of the proteins that are associated with centromeres (Bloom 1993; Earnshaw 1991). Human autoantibodies that bind specifically in the vicinity of the centromere have facilitated the cloning of centromere-associated proteins (CENPs, Rattner 1991). Yeast centromere-associated proteins also have been identified, both through genetic and biochemical studies (Bloom 1993; Lechner et al., 1991).
Despite the aforementioned methods of analysis, the centromeres of most organisms remain poorly defined. Although repetitive DNA fragments mapping both cytologically and genetically to centromeric regions in plants and other higher eukaryotes have been identified, little is known regarding the functionality of these sequences (see Richards et al., 1991; Alfenito et al., 1993; and Maluszynska et al., 1991). Many of these sequences are tandemly-repeated satellite elements and dispersed repeated sequences in series of repeats ranging from 300 kB to 5000 kB in length (Willard 1990). Whether repeats themselves represent functional centromeres remains controversial, as other genomic DNA is required to confer inheritance upon a region of DNA (Willard, 1997).
One characteristic of centromeres which is not well understood is the methylation of cytosines at the carbon 5 position (Martinez-Zapater et al., 1986; Maluszynska and Heslop-Harrison, 1991; Vongs et al., 1993). Methylation is a characteristic feature of many eukaryotic genomes and has been shown to be correlated with heterochromatic regions including regions of repetitive DNA and centromeres (Martienssen and Richards, 1995; Ng and Bird, 1999).
The genomes of both animals and plants contain cytosine methylation, with overall levels of CpG modification often reaching 60 to 90% (Jones and Wolffe, 1999; Gruenbaum et al., 1981). In euchromatin, DNA methylation is concentrated in small regions such as CpG islands and provides epigenetic modifications that regulate genome imprinting, gene expression, and DNA repair (Robertson and Jones, 2000; Singer et al., 2001). In contrast, the role of the extensive DNA methylation found in repetitive, heterochromatic portions of the genome is less clear. In some cases, this methylation reduces recombination; in others, it may play a structural role (J. Bender, 1998; Vongs et al., 1993; Yoder et al., 1997).
A means that has been utilized to study the distribution of methylation in genomes is the use of methylation sensitive restriction endonucleases either alone or in combination with isoschizomeric restriction endonucleases lacking sensitivity to methylation (Jeddeloh and Richards, 1996). An example of such an isoschizomeric pair is HpaII and MspI, which both cut the sequence 5′-C/CGG-3′, but each enzyme differs in its sensitivity to cytosine methylation (Butkus et al., 1987; McClelland et al., 1994). Such analyses involving methylation have often been directed to the sparsely methylated portion of genomes, which comprises the majority of coding sequences.
While the above studies have been useful in helping to elucidate the structure and function of centromeres, they have failed to provide an efficient method for cloning centromere nucleic acid sequences. The development of such methods could allow the isolation of centromeres from a broad variety of organisms, potentially allowing the creation of artificial chromosome vectors tailored to numerous economically important species. Such a technique would avoid the need for costly methodologies described by the prior art and represent a significant advance in biotechnology research.