Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1997-06-09
2001-09-11
Saoud, Christine J. (Department: 1647)
Chemistry: molecular biology and microbiology
Vector, per se
C435S243000, C435S252330, C530S350000
Reexamination Certificate
active
06287853
ABSTRACT:
FIELD OF THE INVENTION
This invention relates (a) to a 540 kb YAC which encodes the necessary species-specific factor(s) and is able to substitute for human Chromosome 21 to reconstitute the Hu-IFN-gamma receptor-mediated induction of class I HLA antigens; (b) to the construction of a plasmid to integrate the selective marker for antibiotic G418 resistance into YACs and to delete some of the human DNA fragments from YACs in order to facilitate the manipulation of human genomic DNA in yeast artificial chromosome (YAC) clones; (c) to two fragmentation vectors, pSE1 and pSE2, which contain a neomycin resistance and URA3 gene, developed for targeting yeast artificial chromosomes (YACs) containing human genomic DNA; (d) to a chromosomal fragmentation procedure employed to produce a deletion set of yeast artificial chromosomes (YACs) from a parental YAC (GART D142H8) known to map to Chromosome 21q and to encode the human interferon-gamma receptor (Hu-IFN-gamma R) accessory factor gene as well as the phosphoribosylglycinamide formyltranisferase (GART) gene; and (e) to the isolation of cDNA clones that encode the necessary species-specific factor and that are able to substitute for human Chromosome 21 to reconstitute the Hu-IFN-gamma receptor-mediated induction of class I HLA antigens.
BACKGROUND OF THE INVENTION
The disclosures referred to herein to illustrate the background of the invention and to provide additional detail with respect to its practice are incorporated herein by reference and, for convenience, are numerically referenced in the following text and respectively grouped in the appended bibliography.
YAC Clone Encoding an Accessory Factor
Human interferon gamma (Hu-IFN-gamma) induces a variety of biological responses such as antiviral, antiproliferative, and immunomodulatory activities in sensitive cells (1-5). Immunoregulatory functions induced by Hu-IFN-gamma such as induction of class I and class II human HLA antigens, activation of macrophages, regulation of Ig class switching, and up-regulation of Fc receptor expression are involved in modulating a variety of other host defense mechanisms (5-10). The first event in inducing these responses is the specific binding of IFN-gamma to its cell surface receptor encoded on human Chromosome 6 (11) or mouse Chromosome 10 (12,64). However, human Chromosomes 6 and 21 (14,15) and mouse Chromosomes 10 and 16 (16) are required for sensitivity to IFN-gamma as measured by the induction of class I MHC antigens, which indicates that the binding of IFN-gamma to the receptor is necessary, but not sufficient to induce these antigens. Interaction between the extracellular domain of the Hu-IFN-gamma receptor and the species-specific accessory factor (AF-1) which leads to class I HLA antigen induction upon treatment with Hu-IFN-gamma was suggested by experiments with chimeric receptors (17-19). A five-amino acid sequence (YDKPH) SEQ ID NO. 17 of the intracellular domain of the Hu-IFN-gamma receptor is required for this activity (20,21). Moreover, at least two additional accessory factors (AF-2 and AF-3) are involved in EMCV and VSV antiviral activity (20,65). However, little is known about how the binding of Hu-IFN-gamma to its receptor initiates this diverse array of functional changes and the nature of the accessory factors.
The region of Chromosome 21 which is necessary for sensitivity to Hu-IFN-gamma as assayed by the expression of class I HLA antigens was localized within a region of about 1-3 mb of Chromosome 21q with irradiation-reduced hamster/human somatic hybrid cells (22). The cloned Hu-IFN-gamma receptor cDNA (23,24) was expressed in hamster/human hybrid cells which have human Chromosome 21q as the sole human Chromosome to reconstitute a biologically-active Hu-IFN-gamma receptor for HLA class I antigen induction (25), and EMCV antiviral protection activity (20). Thus, it appeared that human Chromosome 21q encodes all necessary factors for both activities. This was confirmed by another study with mouse/human hybrid cells transfected with the Hu-IFN-gamma receptor cDNA that showed that human Chromosome 21 is sufficient for induction of class I MHC antigens, EMCV antiviral activity and induction of 2′,5′-oligoadenylate synthetase in response to Hu-IFN-gamma (26).
The cloning of DNA into yeast artificial chromosomes (YACs) has allowed isolation of much larger DNA fragments than was previously possible (27). Also, development of polymerase chain reaction (PCR)-based YAC screening methods has facilitated the identification of specific YAC clones (28). These large cloned segments have potential applications for the isolation of functional domains from chromosomes (29). To assay for the biological functions of genes included in YACs, yeast cells can be fused to cultured mammalian cells. Experiments to demonstrate the functional expression of genes carried on YACs have been carried out with G6PD (glucose-6-phosphate dehydrogenase), HPRT (hypoxanthine phosphoribosyltransferase) and GART YAC clones (30-32). To allow selection for fused cells in these cases, a gene cassette that confers resistance to antibiotic G418 on mammalian cells can be introduced into the YAC by homologous recombination targeted to human genomic sequences (33,34).
Alu-targeting YAC Deletion Plasmids
Yeast artificial chromosomes (YACs) provide a system for the cloning of up to one megabase of contiguous DNA (55,56). While the large size of the YAC insert facilitates the isolation of specific large segments of DNA (59), identification and localization of genes within the YAC requires conventional yeast genetic techniques to manipulate the YAC insert (39).
Chromosome fragmentation has been developed for
Saccharomyces cerevisiae
YPH252 with a fragmentation plasmid (48,60). Fragmentation takes advantage of homologous recombination between Alu family sequences or LINE human repetitive DNA elements in the fragmentation plasmid and the YAC insert. The recombination event causes chromosome breakage at the site where homologous integration occurs and deletion of all portions of the YAC distal to the targeted site. Similarly another fragmentation plasid system has been developed for
S. cerevisiae
AB1380 (58). The plasmid developed by Cook et al. contained the gene for neomycin phosphotransferase as a eukaryotic selectable marker. A second type of YAC modification plasmid, an integrating plasmid, has been described (33) for
S. cerevisiae
YPH252. In contrast to the fragmentation plasmid, the integrating plasmid contains two recombinogenic sequences resulting in the insertion of the plasmid into the YAC. The integrating plasmid has been used mainly to insert a neomycin-resistance cassette to be used as a selectable marker following fusion of YAC-containing yeast spheroplasts with mammalian cells (33). Another integrating plasmid containing a DNA fragment derived from the DNA of the YAC insert, not Alu family sequences, has been described (34).
Yeast Artificial Chromosome Fragmentation Vectors
Yeast artificial chromosome vectors have enabled the cloning of up to 1 megabase of DNA (27). In order to reduce YACs to smaller sizes for mapping and manipulation, Reeves et al. (62) and Pavan et al. (48) have developed fragmentation vectors which are capable of producing a set of deletions in a parental YAC containing human genomic DNA. The fragmented YACs obtained by this procedure are useful not only for mapping studies but also for determining the location of genes encoding certain biochemical functions. The vectors which have been reported utilize transformation to a His
+
phenotype to select for clones containing fragmented YACs (48,60). While this procedure is applicable to YACs carried in his3
−
YPH252 yeast cells, it cannot be used with the AB1380 yeast strain which is the host for many YAC libraries.
Localization of the Human Interferon-gamma Receptor Accessory Factor Gene
The human and murine interferon gamma receptors (IFN-gamma R) have been shown to be homologous to a considerable degree (94). Nevertheless, these receptors are very specific in terms
Cook Jeffry R.
Donnelly Robert J.
Emanuel Stuart
Kotenko Serguei
Mariano Thomas M.
Muccino Richard R.
Saoud Christine J.
University of Medicine & Dentistry of New Jersey
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