RIP60 nucleic acid and polypeptide sequences and uses therefor

Details RIP60 nucleic acid and polypeptide sequences and uses therefor RIP60 nucleic acid and polypeptide sequences and uses therefor

2000-01-04

2004-08-24

Smith, Lynette (Department: 1645)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S023100, C435S069100, C435S071100, C435S091400, C435S455000

Reexamination Certificate

active

06780986

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to nucleic acids and encoded polypeptides of the human zinc finger protein RIP60. The invention also relates to isolated nucleic acid molecules, expression vectors containing those molecules and host cells transfected with those molecules.
BACKGROUND OF THE INVENTION
The ability to transfer nucleic acids into cells has vast experimental and therapeutic implications. Many different chemical, electrochemical and biological approaches have been used for this purpose. In vitro chemical methods include osmotic shock transformation of prokaryotic cells and calcium phosphate transfection and liposome-mediated transfer for eukaryotic cells. Nucleic acids, namely DNA, have also been delivered to cells by electroporation. While this latter approach is amenable to nucleic acid transfer in vitro, it is inherently unsuitable for in vivo use. Biological approaches have focused on viral strategies which include retroviral and most recently adenoviral mediated gene transfer into cells in culture and, in some instances, cells in vivo. A common disadvantage of the above-mentioned strategies is their inability to specifically target cells for nucleic acid delivery. Targeting of cell subsets usually requires the selective harvesting of cells followed by in vitro delivery and re-introduction in vivo.
Viral mediated gene transfer requires the in vitro production of defective viral particles which encapsulate a nucleic acid of a finite size. The encapsulated nucleic acid, usually referred to as a viral vector, is a recombinant nucleic acid which contains a gene(s) of interest cloned between 5′ and 3′ flanking viral cis elements. The cis elements are required for integration into the host genome yet they are also capable of transcriptional regulation. As a result, these elements have the potential to interfere with the transcriptional activity of the cloned gene(s). Another limitation of viral mediated gene transfer is the need for and the difficulty in achieving high titre viral stocks. In vivo infection with viruses, when applicable, is generally not effective given the in vivo dilution of viral particles. Additionally, although both retroviral and adenoviral methods employ replication-defective viral particles, the possibility of producing replication-competent viruses and thereby causing active infection in vivo is an inherent danger of both systems.
For retroviral mediated gene transfer to occur, target cells whether in vitro or in vivo must be in a cycling status. Since retroviruses package nucleic acid in the form of RNA, reverse transcription of the RNA to DNA is required for integration into the host genome from where the gene exerts its effects. Cells which divide infrequently or never at all, such as some classes of stem cells or terminally differentiated end cells, are usually less amenable to gene transfer via retroviral infection as compared to rapidly dividing cells. Thus diseases for which a long-term cure is dependent upon stem cell or end cell manipulation are poor candidates for gene therapy treatment using retroviral transfection. Retroviral use is also limited to the restricted range of host infectivity specific to each strain of virus. In contrast adenoviruses which contain double stranded DNA do not require target cells to be cycling for infection, integration and propagation.
DNA has also been delivered to cells using receptor-mediated endocytosis. In this approach, DNA is initially complexed with polycations such as polylysine for condensation and charge neutralization purposes. Ligands for cell surface receptors, such as transferrin, are then coupled either biochemically or enzymatically to the polylysine moieties. In a further modification, the transferrin molecules are coupled to the outer surface of inactivated adenoviral particles. The adenoviral particles can effect the release of the DNA/polylysine/transferrin complex from endosomes prior to lysosome mediated degradation. The transfer of up to 48 kilobases (kb) of DNA has been reported using this approach. Cotten et al., PNAS v. 89, p.6094-6098 (1992).
In contrast to the use of polycations for complexing DNA, other approaches have incorporated specific DNA binding domains which recognize and bind distinct nucleic acid consensus sequences. An example of this is the use of the GAL4 DNA binding domain of yeast which selectively binds to a 17 bp sequence. Thus a nucleic acid to be delivered must usually be modified to incorporate artificial GAL4 binding sites. Likewise, other approaches which rely on a consensus sequence dependent DNA binding domain will similarly require modification of the transferred nucleic acid.
SUMMARY OF THE INVENTION
The invention also relates to the molecular cloning and characterization of RIP60, a zinc finger protein involved in cell division and nucleic acid replication.
The invention provides isolated RIP60 nucleic acid molecules, unique fragments of those molecules, expression vectors containing the foregoing, and host cells transfected with those molecules. The invention also provides isolated RIP60 polypeptides, and agents which bind RIP60 polypeptides, including antibodies.
According to one aspect of the invention, isolated nucleic acid molecules are provided that comprise: (a) nucleic acid molecules which hybridize under stringent conditions to a molecule consisting of a nucleic acid of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:50 and which code for a polypeptide having RIP60 activity, (b) deletions, additions and substitutions of (a) which code for a polypeptide having RIP60 activity, (c) nucleic acid molecules that differ from the nucleic acid molecules of (a) or (b) in codon sequence due to the degeneracy of the genetic code, and (d) complements of (a), (b) or (c). In certain embodiments, the isolated nucleic acid molecule comprises SEQ ID NO: 1. In other embodiments, the isolated nucleic acid molecule comprises SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:50. In some embodiments, the isolated nucleic acid molecules are those that code for a polypeptide comprising SEQ ID NO:2. In some embodiments, the isolated nucleic acid molecules are those that code for a polypeptide comprising SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:51. In an important embodiment, the nucleic acid molecules code for a native RIP60 polypeptide, including homologs and alleles. A native RIP60 polypeptide is one which possesses a native RIP60 function or activity, such as but not limited to DNA binding or protein multimerization. Another function or activity of a native RIP60 polypeptide is the ability to bind to either itself or to other proline rich region containing proteins, specifically through its proline rich region.
The invention in another aspect provides an isolated nucleic acid molecule selected from the group consisting of (a) a unique fragment of nucleic acid molecule of SEQ ID NO:1 of sufficient length to represent a sequence unique within the human genome, and (b) complements of (a), provided that the unique fragment includes a sequence of contiguous nucleotides which is not identical to a sequence selected from the sequence group consisting of (1) sequences having the GenBank and EMBL database accession numbers of Table 1, (2) complements of (1), and (3) fragments of (1) and (2).
In one embodiment, the sequence of contiguous nucleotides is selected from the group consisting of (1) at least two contiguous nucleotides nonidentical to the sequence group, (2) at least three contiguous nucleotides nonidentical to the sequence group, (3) at least four contiguous nucleotides nonidentical to the sequence group, (4) at least five contiguous nucleotides nonidentical to the sequence group, (5) at least six contiguous nucleotides nonidentical to the sequence group, and (6) at least seven contiguous nucleotides nonidentical to the sequence group.
In another embodiment, the fragment has a size selected from the group consisting of at least: 8 nucleotides, 10 nucleotides, 12 nucleotides, 14 nucleotides, 16 nucleotides, 18 nucleotides, 20, nucleotides, 22 nucl

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