Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1999-01-06
2004-04-13
Wilson, Michael (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S022000
Reexamination Certificate
active
06720472
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention pertains to a method for treating obesity in a mammal which comprises reducing the biological activity of HMGI genes in the mammal. In another embodiment, the invention pertains to a method for treating a tumor in a patient by reducing the biological activity of normal HMGI genes which comprises administering to the patient a therapeutically effective amount of an inhibitor compound active against normal HMGI-C or HMGI(Y) genes. In another embodiment, the invention pertains to a method of producing a transgenic non-human mammal, the germ cells and somatic cells of which contain an inactivated HMGI gene sequence introduced into the mammal, or an ancestor of the mammal, at an embryonic stage. In another embodiment, the invention pertains to a method for screening candidate compounds capable of inhibiting the biological activity of normal HMGI proteins, or a fragment thereof, which comprises the steps of (a) incubating a HMGI protein, or a fragment thereof, with a candidate compound under conditions which promote optimal interaction; and (b) measuring the binding affinity of the candidate compound to the HMGI protein, or a fragment thereof; and (c) determining from the binding affinity which candidate compounds inhibit the biological activity of HMGI proteins, or a fragment thereof. In another embodiment, the invention pertains to a method for screening candidate compounds capable of inhibiting the biological activity of normal HMGI genes which comprises the steps of (a) transfecting into a cell a DNA construct which contains a reporter gene under control of a normal HMGI protein-regulated promoter; (b) administering to the cell a candidate compound; (c) measuring the levels of reporter gene expression; and (d) determining from the levels of reporter gene expression which candidate compounds inhibit the HMGI biological activity. In another embodiment, the invention pertains to a method for detecting normal HMGI proteins as a diagnostic marker for a tumor using a probe that recognizes normal HMGI proteins, which comprises the steps of (a) contacting normal HMGI proteins from a sample from a patient with a probe which binds to HMGI proteins; and (b) analyzing for normal HMGI proteins by detecting levels of the probe bound to the normal HMGI proteins, wherein the presence of normal HMGI proteins in the sample is positive for a tumor. In another embodiment, the invention pertains to a method for detecting antibodies to normal HMGI proteins using a probe that recognizes antibodies to HMGI normal proteins, which comprises the steps of (a) treating a sample from a patient with a probe which binds to antibodies to normal HMGI proteins; and (b) analyzing for antibodies to HMGI proteins by detecting levels of the probe bound to the antibodies to HMGI proteins, wherein the presence of antibodies to normal HMGI proteins in the sample is positive for a tumor. In another embodiment, the invention pertains to HMGI genes and proteins for use as a starting point to isolate downstream target genes regulated by the HMGI genes and proteins.
2. Description of the Background
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 referenced in the following text and respectively grouped in the appended bibliography.
HMGI Proteins in Adipogenesis and Mesenchyme Differentiation
Understanding various genes and pathways underlying development of multicellular organisms provide insights into the molecular basis of the highly regulated processes of cellular proliferation and differentiation. In turn, genetic aberrations in control of cell growth lead to a variety of developmental abnormalities and, most prominently, cancer (Aaronson, 1991). To pursue identification of genes involved in these fundamental biological processes, the viable pygmy mutation (MacArthur, 1944) was investigated because it gives rise to mice of small stature due to a disruption in overall growth and development of the mouse. An insertional transgenic mutant facilitated cloning of the locus (Xiang et al., 1990) and subsequently it was shown that expression of the HMGI-C gene was abrogated in three pygmy alleles (unpublished results).
HMGI-C belongs to the HMG (high mobility group) family of DNA-binding proteins which are abundant, heterogeneous, non-histone components of chromatin (Grosschedl et al., 1994). HMG proteins are divided into three distinct families, the HMG box-containing HMG1/2, the active chromatin associated HMG14/17 and the HMGI proteins (Grosschedl et al., 1994). At present, the last family consists of two genes, HMGI(Y) (Johnson et al., 1988; Friedmann et al., 1993) which produces two proteins via alternative splicing (Johnson et al., 1989) and HMGI-C (Manfioletti et al., 1991; Patel et al., 1994). A prominent feature of HMGI proteins is the presence of DNA-binding domains which bind to the narrow minor groove of A-T rich DNA (Reeves and Nissen, 1990) and are therefore referred to as A-T hooks. Recently, valuable insights have been gained into their mechanism and role in transcription (Thanos and Maniatis, 1992; Du et al., 1993). The HMGI proteins have no transcriptional activity per se (Wolffe, 1994), but through protein-protein and protein-DNA interactions organize the framework of the nucleoprotein-DNA transcriptional complex. This framework is attained by their ability to change the conformation of DNA and these proteins are therefore termed architectural factors (Wolffe, 1994). In the well-studied case of HMGI(Y) and the interferon B promoter, HMGI(Y) stimulates binding of NF-KB and ATF-2 to appropriate sequences and alters the DNA structure which allows the two factors to interact with each other and presumably with the basal transcription machinery (Thanos and Maniatis, 1992; Du et al., 1993).
A number of studies have revealed an association between increased expression levels of HMGI proteins and transformation (Giancotti et al., 1987, 1989, 1993). For example, in chemically, virally or spontaneously derived tumors, appreciable expression of HMGI-C was found in contrast to no detectable expression in normal tissues or untransformed cells (Giancotti et al., 1989). A recent study has demonstrated a more direct role for HMGI-C in transformation (Berlingieri et al., 1995). Cells infected with oncogenic retroviruses failed to exhibit various phenotypic markers of transformation if HMGI-C protein synthesis was specifically inhibited.
DNA probes adjacent to HMGI-C were mapped to the distal portion of mouse chromosome 10 in a region syntenic to the long arm of human chromosome 12 including and distal to band q13 (Justice et al., 1990). This genomic region is under intensive investigation because it is the location of consistent rearrangements in a number of neoplasms, mainly of mesenchymal origin (Schoenberg Fejzo et al., 1995). Lipomas, tumors mainly composed of mature fat cells, are one of the most common mesenchymal neoplasms that occur in humans (Sreekantaiah et al., 1991). Approximately 50% of lipomas are characterized by cytogenetic rearrangements and the predominant alteration is a presumably balanced translocation involving 12q14-15 with a large variety of chromosomal partners including 1, 2, 3, 4, 5, 6, 7, 10, 11, 13, 15, 17, 21, and X (Sreekantaiah et al., 1991; Fletcher et al., 1993). This variability in reciprocal translocations along with duplications, inversions, and deletions of 12q14-15 in these tumors, strongly indicates a primary role of a gene on chromosome 12 in lipomas. Furthermore, this gene may play a key role in normal differentiation of primitive mesenchyme as not only lipomas, but also uterine leiomyomas (smooth muscle tumors), lipoleiomyomas (smooth muscle and adipose components), and pulmonary chondroid hamartomas (primitive mesenchyme, smooth muscle, adipose, and mature cartilage components) are all clonal proliferations that are characterized by rearrangements of 12
Ashar Hena
Chada Kiran K.
Tkachenko Alex
Zhou Xianjin
Perkins Coie LLP
University of Medicine and Dentistry of New Jersey
Wilson Michael
LandOfFree
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