Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-06-09
2002-06-25
McKelvey, Terry (Department: 1636)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
Reexamination Certificate
active
06410722
ABSTRACT:
BACKGROUND OF THE INVENTION
(a) Field of the Invention
The invention relates to human and mammalian DNA replication origin consensus sequences; the use of consensus sequences for control of initiation of mammalian DNA replication; small sequences which will allow the maintenance of circular plasmid constructs which are capable of being replicated semiconservatively in proliferating mammalian cells; to small sequences suitable for use in human gene therapy; to small sequences suitable for inclusion in mammalian and human artificial chromosome vectors; to a protein binding to double-stranded DNA; to an anti-gene to DNA replication; and to a method of inhibiting DNA replication in vitro or in vivo.
(b) Description of Prior Art
Building a human artificial chromosome would not only provide a valuable tool for addressing difficult questions about chromosome biology, but would also create an all-human transfection vector with the capacity to carry large chromosomal regions including complete transcriptional units, from even the largest genes, for the purpose of complementation mapping, or for gene therapy (Huxley, C. (1994)
Gene Ther.
1:7-12).
Artificial chromosomes require three cis-acting functional components: replication origins, telomeres, and a centromere.
S. cerevisiae
origin-containing yeast ARS (autonomously replicating sequence) plasmids provided the basis for the addition of telomeres, TEL, and centromere, CEN elements to complete the construction of stable yeast artificial chromosomes, or YACs (Murray, A. W. & Szostak J. W. (1983)
Nature,
305, 189-193). A similar strategy proved successful for artificial chromosome assembly in the fission yeast
Schizosaccharomyces pombe
, in spite of the far more complicated structure of its centromeres (Hahnenberger, K. M. et al. (1989)
Proc. Nat. Acad. Sci. USA,
86:577-581).
The first component required in such a “ground up” strategy for the assembly of a prototype human artificial chromosome is a functional human replication origin. Different techniques have permitted the identification of a limited but rapidly-increasing number of putative and proven mammalian origins of DNA replication (DePamphilis, M. L. (1993)
Annu. Rev. Biochem.
62:29-63).
Our group has been able to isolate large numbers of putative origins using such techniques as nascent strand extrusion (Kaufmann, G. et al. (1985) Mol. Cell. Biol., 5:721-727) and anti-cruciform immunoaffinity purification (Bell, D. et al. (1991)
Biochim. Biophys. Acta,
1089:299-308); these sequences permit short-term autonomous replication of plasmids transfected into human cells, and can act as replication origins in their native chromosomal position (Wu, C. et al. (1993a)
Biochim. Biophys. Acta,
1174:241-257).
To use such isolated origin sequences for the construction of a first stage human artificial chromosome, they must be cloned into a circular vector which permits transfection into human cells and selection of transfected clonal subpopulations, and has the capacity for further modification to carry human-functional telomeres and putative centromere elements which could be hundreds of kilobases. In addition, methods are adapted and applied which demonstrate that these constructs are maintained in long-term culture as independent episomal elements, not integrated into a host chromosome.
We have identified CDNA clones which contain ARS (Wu, C. et al. (1993a)
Biochim. Biophys. Acta,
5 1174:241-257) and demonstrated that clone 343, located on chromosome 6q (Shihab-El-Deen, A. et al. (1993)
Somat. Cell Mol. Genet.
19:103-109), can be mapped as a chromosomal origin in vivo (Wu, C. et al. (1993b)
Biochim. Biophys. Acta,
1174:258-266). We have also demonstrated that the bi-directional origin (ori&bgr;) of the dihydrofolate reductase, DHFR, locus from Chinese hamster), within small or large fragments, has ARS activity in vivo in DpnI resistance assays after transfection into HeLa cells, and in vitro with our mammalian in vitro DNA replication system (Zannis-Hadjopoulos, M. et al. (1994)
Gene,
151:273-277). (We also showed that the in vivo and in vitro replication assays of ARS and origin function give the same results, and initiation of replication in the plasmid constructs occurs within the ori&bgr; containing insert).
We have also conducted the identification and characterization of an ors (origin enriched sequence) binding activity (now also referred to as origin binding activity) (OBA) partially purified from HeLa cell extracts that are used in our in vitro DNA replication system, co-purifying with known replication proteins (Ruiz, M. T. et al. (1995)
J. Cell. Biochem.
58:221-236). The regulation of eukaryotic DNA replication is one of the most important biologic mechanisms. We have demonstrated the long-term maintenance as episomes of origin containing fragments (S3 (Nielsen, T. et al. (1994)
Mol. Gen. Genet.
242:280-288) and 343) under selection in a YACneo plasmid, further demonstrating the functionality of small fragments containing ARS as origins.
Although previous sequence analysis failed to reveal specific nucleotide consensus sequences, we have now had the opportunity to examine larger numbers of monkey and human ARS and to group small DNA fragments containing ARS activity in order to derive two putative minimal core ARS consensus sequences (36 bp and 91 bp). Preliminary analysis by in vivo and in vitro replication assays indicates that both are capable of functioning as origins in these assays of episomal replication in mammalian cells. Furthermore, one specific sequence has been shown to bind to OBA and to be as effective of a competitor for binding as the fragments of the 186 bp minimal ARS of the ORS8 origin (Todd, A. et al. (1995)
J. Cell. Biochem.
58:221-236).
Because of the collective weight of our original work and resources, we are confident in the likelihood of identification of a mammalian minimal core ARS consensus sequence, which like the identification of the yeast minimal core ARS consensus should rapidly advance discoveries of the mechanism of DNA replication in mammalian cells.
It would be highly desirable to be provided with a minimal consensus sequence from which versions of human core ARS could be used to create shuttle vector constructs for use in definition of essential mammalian chromosomal elements that are required for the maintenance of chromosome function, and for use in gene therapy.
SUMMARY OF THE INVENTION
One aim of the present invention is to provide consensus sequences for control of initiation of mammalian DNA replication.
Another aim of the present invention is to provide small sequences which will allow the maintenance of circular plasmid constructs which are capable of being replicated semiconservatively in proliferating mammalian cells.
Another aim of the present invention is to provide small sequences suitable for inclusion in mammalian and human artificial chromosome vectors.
In accordance with one embodiment of the present invention there is provided a specific 36-bp consensus sequence called ‘alphaconsensus’ and a specific 91-bp consensus sequence called ‘uniorsconsensus’.
In accordance with another embodiment of the present invention the alphaconsensus comprises the nucleotide sequence set forth as follows:
CCTMDAWKSGBYTSMAAWYWBCMYTTRSCAAATTCC (SEQ ID NO:1).
The alphaconsensus sequence (36 bp) was derived from autonomously replicating sequences associated with alpha-satellite sequences from African Green Monkey CV-1 cells (ORS14 & ORS23 Landry, S. & Zannis-Hadjopoulos, M. (1991)
Biochim. Biophys. Acta,
1088:234-244) and associated with alpha-satellite sequences from normal human skin fibroblasts (F5 and F20 Nielsen, T. et al. (1994)
Mol. Gen. Genet.
242:280-288).
The alphaconsensus is able to replicate DNA in BVDR incorporation assay using murine fibroblasts NIH 3T3 and murine embryonic carcinomas cells P19.
In accordance with another embodiment of the present invention the consensus is a functional variant thereof having a sequence with at least 70% homology with the alphaconsensus, such as Y.343 w
Cossons Nandini H.
Nielsen Torsten O.
Price Gerald B.
Zannis-Hadjopoulos Maria
McGill University
McKelvey Terry
Nixon & Peabody LLP
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