Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-09-28
2001-10-02
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S455000, C435S463000, C435S254210, C435S354000, C435S366000, C435S320100
Reexamination Certificate
active
06297029
ABSTRACT:
TECHNICAL FIELD
This invention relates to mammalian artificial chromosomes (MACs) that can replicate autonomously, can be stably maintained extra-chromosomally and can be transmitted efficiently in mammalian cells. The invention includes methods to construct, modify and stably maintain yeast artifical chromosomes (YACs) in yeast cells which have the potential ability to form MACs when introduced into mammalian cells.
BACKGROUND ART
Constructing a mammalian artificial chromosome (MAC) carrying the known functional elements required for chromosome replication, stable extra-chromosomal maintenance and segregation in a manipulatable form will be of great value not only for basic studies concerning the organization and function of mammalian chromosomes but also as a vector to introduce DNA segments (genes) of interest to test their functions in mammalian cells or bodies, since the genes carried by MACs will neither be subject to variable expression due to integration position effects nor cause unpredictable insertion mutation in the host chromosomes. Furthermore, a MAC will have the capacity to accommodate a DNA segment having a size in the megabase range, wherein an entire large gene or group of genes and regulatory elements could be included. For these reasons, MACs will offer exciting alternative vectors to currently existing vectors for somatic gene therapy, because frequently used infectious vectors derived from viruses are either integrated randomly into host chromosomes or exist extra-chromosomally but only transiently. Besides, these vectors are able to carry only short DNA segments. A new way to generate transgenic mice will be provided by the invention of MACs, if the stability of MACs during meiosis is established in mammalian development. However, the construction of a MAC has not yet been achieved due to technical difficulties (Willard, Proc. Natl. Acad. Sci. 93, 6874-6850,1996).
Yeast artificial chromosomes (YACs) has been constructed (Burke et al., Science, 236, 806-812,1987) with three essential DNA elements from the budding yeast,
Saccharomyces cerevisiae;
namely, an origin of replication or autonomously replicating sequence (ORI or ARS) is required for initiation of DNA replication, telomere sequences (TEL) are required to stabilize and facilitate complete replication of chromosomal ends, and a centromere (CEN) is required for faithful segregation of the sister chromatids after replication. As a result, YACs have become a major tool for cloning large gene segments of complex genomes. Similar to YACs, MACs are believed to be constructable with the three essential elements derived from mammalian genomes. Among the three, telomeres have been isolated from mammalian chromosomes and used for the mammalian chromosome manipulation (Brown et al., Hum. Mol. Genet., 27-1237,1994; Farr et al., EMBO J., 14, 5444-5454,1995), but centromeres and the origins of replication for mammalian chromosomes were found to be difficult to isolate because of the unavailability of activity assays.
SUMMARY OF THE INVENTION
The present investigators have analyzed the specific structure of the mammalian centromere locus in order to obtain information concerning the essential functional structure of mammalian centromeres. They have found that centromere protein B (CENP-B), which is one of the antigens recognized by anti-centromere antibodies (Moroi et al., Proc. Natl. Acad. Sci. USA, 77,1627-1631,1980) at centromeres of various mammalian chromosomes, specifically recognizes and binds the 17 bp sequence (CENP-B box) of the centromere satellite DNA (alphoid DNA) in the human genome (Masumoto et al., J. Cell Biol., 109, 1963-1973, 1989; Muro et al., J. Cell Biol., 116, 585-596, 1992). The recognition sequences of CENP-B were found in centromeric satellite DNA of mouse species (Masumoto et al., J. Cell Biol., 109, 1963-1973, 1989; Kipling et al., Mol. Cell. Biol., 15, 4009-4020, 1995) and the consensus sequence of the CENP-B box was established to be 5′-NTTCGNNNNANNCGGGN-3′ (Masumoto et al., NATO ASI Series, vol.H72, Springer-Verlag, 31-43, 1993; Yoda et al., Mol. Cell. Biol.,16, 5169-5177, 1996). They have also demonstrated that a pair of CENP-B sequences form a dimer at the C-terminal and bind to a CENP-B box located in the centromeric satellite DNA at the N-terminal of each CENP-B polypeptide, so that a stable complex, which consists essentially of the dimer protein and the two regions (or strands) of the DNA, was formed (Muro et al., J. Cell Biol.,116,585-596,1992; Yoda et al., J. Cell Biol., 119, 1413-1427,1992; Kitagawa et al., Mol. Cell. Biol. 15, 1602-1612,1995). Further, the investigators studied the location of the CENP-B box in human chromosome 21 (Ikeno et al., Hum. Mol. Genet., 3, 1245-1257,1994), and found that there are two distinct regions in the alphoid DNA array of human chromosome 21 with regard to the CENP-B box. Specifically, one (&agr;21-I) contains a regular series of CENP-B box sequences, and the other (&agr;21-II) scarcely contains any CENP-B box sequences, the two regions extend side by side for a stretch of about several megabases, the former is located at a position in the chromosome where the centromere proteins are located and the latter is located at a position slightly shifted towards the short arm of the chromosome (Ikeno et al., Hum. Mol. Genet., 3, 1245-1257,1994).
The present investigation relates to the DNA region within the CENP-B box. It is an object of the investigation to provide artificial chromosomes derived from the above region that can be stably maintained in the extra-chromosomal region of mammalian cells, especially in human cells, and can be safely transmitted to cells of succeeding generations. The present investigation includes development of methods to construct, modify and stably maintain the precursors of such artificial chromosomes in yeast cells as YACs, which have the potential ability to form mammalian artificial chromosomes when introduced into mammalian cells.
This invention has been achieved by using homologous recombination in yeast cells to determine a method for constructing a yeast artificial chromosome construct comprising a DNA sequence including CENP-B box sequences from the alphoid DNA region of human chromosome 21 that can interact with CENP-B or the centromere protein B, and a segment of the human telomere sequence. Further, the invention involves the determination that when the construct is introduced into a human cell, the construct is replicated autonomously in a human cell and is stably maintained in the cell lineage.
This invention provides a DNA construct comprising a mammalian telomere and a centromere, wherein the centromere has a DNA sequence containing a plurality of copies of the CENP-B box sequence consisting of:
5′-NTTCGNNNNANNCGGGN-3′,
wherein N is any one of A,T,C and G.
In a preferred embodiment of this invention, a DNA construct comprises a mammalian telomere and a centromere, wherein the centromere has a DNA sequence containing a plurality of copies of the CENP-B box sequence designated herein as sequence No. 1.
Preferably, the centromere contains spaced repeats of the CENP-B box sequence.
This invention provides a DNA construct comprising a mammalian telomere and a centromere, wherein the centromere has a DNA sequence containing a plurality of copies of the sequence designated as sequence No. 2 or a sequence derived from sequence No.2 in which one or more nucleotides are added, deleted and/or replaced.
In a preferred embodiment of this invention, the centromere is derived from a human chromosome. The DNA construct further comprises one or more sequences necessary for the DNA construct to multiply in yeast cells. The DNA construct further comprises a sequence encoding a selectable marker gene. The DNA construct is capable of being maintained as a chromosome in a transformed cell with the DNA construct. Preferably, the DNA construct is capable of being maintained as a chromosome in a human cell. Further, the DNA construct is capable of being maintained as a chromosome in a mouse cell.
T
Cooke Howard J.
Grimes Brenda Rose
Ikeno Masashi
Masumoto Hiroshi
Okazaki Tsuneko
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