ADP-ribosylation factor-like proteins

Details ADP-ribosylation factor-like proteins ADP-ribosylation factor-like proteins

2001-05-04

2003-05-13

Yucel, Remy (Department: 1636)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Fungi

C435S255200, C435S471000

Reexamination Certificate

active

06562614

ABSTRACT:

BACKGROUND OF THE INVENTION
ADP-ribosylation factors (ARFs) are a family of proteins, each about 20 kDa in size and having the ability to bind and hydrolyze GTP. ARFs are also characterized by their ability to enhance the ADP-ribosyltransferase activity of cholera toxin (Kahn et al., J Biol Chem 259:6228-6234, 1984; and Tsai et al., J Biol Chem 263:1768-1772, 198). Some members of the ARF protein family are involved in regulating vesicle transport in cells as diverse as yeast and human cells.
The ARF-like protein (ARL) family is related to ARFs by amino acid sequence homology and, like ARFs, are characterized by their ability to bind and hydrolyze GTP. However, ARLs can be distinguished from ARFs as they do not enhance the ADP-ribosyltransferase activity of cholera toxin.
SUMMARY OF THE INVENTION
The invention features an antibody which specifically binds the ARL3 polypeptide having the amino acid sequence of SEQ ID NO:2, which can be encoded by a DNA molecule having the sequence of SEQ ID NO:1. By “specifically binds” is meant that the antibody binds the ARL3 polypeptide having the sequence of SEQ ID NO:2 but not specifically bind other molecules in that sample. For example, the antibody of the invention will not bind to other members of the yeast ARF and ARL families.
The invention also features a transgenic knockout yeast (e.g.,
Saccharomyces cerevisiae
) having a homozygous disruption in its endogenous ARL3 gene, where the disruption prevents the expression of a functional ARL3 protein and the phenotype of the knockout yeast relative to a yeast having a wild type ARL3 gene includes impaired growth at about 15° C. The impaired growth can represent 50, 10, 5, or 1% of the growth of wild type yeast at that temperature. The disruption can include an insertion of a nucleic acid sequence into a wild type ARL3 gene in the genome of a parent yeast. Alternatively, the disruption can include an insertion into a mutated but functional ARL3 gene. In some embodiments, the nucleic acid sequence encodes a polypeptide (e.g., one that confers a selectable phenotype on the transgenic knockout yeast). For example, the parent yeast can be incapable of growth in a medium free of uracil, and the selectable phenotype can be the ability to grow in a medium free of uracil.
The antibody of the invention can be used to isolate and clone genes expressing polypeptides homologous to SEQ ID NO:2. Such an antibody is also useful for quantifying the amount of ARL3 in a sample. The transgenic knockout yeast of the invention is useful for identifying genes which are involved in vesicle transport. Such genes can be identified by their ability to complement the growth defect conferred by disruption of the ARL3 sequence.
Other features or advantages of the present invention will be apparent from the following drawings and detailed description, and also from the claims.
DETAILED DESCRIPTION
The invention relates to the identification of an expressed yeast ARL3 polypeptide and a nucleic acid which encodes it. The polypeptide and nucleic acid were then used to produce antibodies which specifically bind the ARL3 polypeptide and transgenic knockout yeast with a disruption in the ARL3 gene, respectively.
I. Antibodies
Both polyclonal and monoclonal anti-ARL3 antibodies are within the scope of the invention. Polyclonal anti-ARL3 antibodies can be prepared by immunizing a suitable animal, e.g., a rabbit, with an ARL3 immunogen. The anti-ARL3 antibody titer in the immunized animal can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized ARL3. The antibody molecules directed against ARL3 can be isolated from a mammal (e.g., from the blood of the mammal) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the anti-ARL3 antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler et al., Nature 256:495-497, 1975; Kozbor et al. (1983) Immunol Today 4:72, 1983; and Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985. The technology for producing various monoclonal antibody hybridomas is well known (see, e.g., Coligan et al. eds., Current Protocols in Immunology, John Wiley & Sons, Inc., New York, N.Y., 1994). Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with an ARL3 immunogen, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds ARL3.
Any of the many well known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating an anti-ARL3 monoclonal antibody (see, e.g., Current Protocols in Immunology, supra; Galfre et al. (1977) Nature 266:55052; R. H. Kenneth, in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, N.Y. (1980); and Lerner (1981) Yale J. Biol. Med., 54:387-402. Moreover, the ordinary skilled worker will appreciate that there are many variations of such methods which also would be useful. Typically, the immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian species as the lymphocytes. For example, murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line, e.g., a myeloma cell line that is sensitive to culture medium containing hypoxanthine, aminopterin and thymidine (HAT medium). Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. These myeloma lines are available from ATCC. Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol (PEG). Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed). Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind ARL3, e.g., using a standard ELISA assay.
As an alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal anti-ARL3 antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with ARL3 to thereby isolate immunoglobulin library members that bind ARL3. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAPJ Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S. Pat. No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCT Publication WO 92/20791; PCT Publication No. WO 92/15679; PCT Publication WO 93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs et al., Bio/Technology 9:1370-1372, 1991; Hay et al., Hum Antibod Hybridomas 3:81-85, 1992; Huse et al., Science 246:1275-1281, 1989; and Griffiths et al. EMBO J 12:725-734, 1993.
Additionally, recombinant anti-ARL3 antibodies, such as chimeric and humanized monoclonal antibodies, including both human and non-human portions, which can be made using standard recombinant DNA techniques, are also within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods describ

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