Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-11-30
2002-10-22
Nguyen, Dave T. (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S069100, C435S455000, C435S471000, C435S325000, C536S023100, C536S023500, C536S023510
Reexamination Certificate
active
06468770
ABSTRACT:
1. INTRODUCTION
The present invention relates to Drosophila insulin-like genes and methods for identifying insulin-like genes. The invention provides nucleotide sequences of Drosophila insulin-like genes, amino acid sequences of their encoded proteins (including peptide or polypeptide), and derivatives (e.g., fragments) and analogs thereof. The invention further relates to fragments (and derivatives and analogs thereof) of insulin-like proteins which comprise one or more domains of an insulin-like protein. Antibodies to an insulin-like protein, and derivatives and analogs thereof, are provided. Methods of production of an insulin-like protein (e.g., by recombinant means), and derivatives and analogs thereof, are provided. Methods to identify the biological function of a Drosophila insulin-like gene are provided, including various methods for the functional modification (e.g., overexpression, underexpression, mutation, knock-out) of one gene, or of two or more genes simultaneously. Methods to identify a Drosophila gene which modifies the function of, and/or functions in a downstream pathway from, an insulin-like gene are provided. The invention further provides for use of Drosophila insulin-like proteins as a media additive or pesticide.
2. BACKGROUND OF THE INVENTION
Citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention.
2.1. THE INSULIN SUPERFAMILY
Insulin-like proteins are a large and widely-distributed group of structurally-related peptide hormones that have pivotal roles in controlling animal growth, development, reproduction, and metabolism (Blundell and Humbel, 1980, Nature 287:781-787). Consequently, the insulin superfamily has become one of the most intensively investigated classes of peptide hormones. Such hormones have a vast array of uses including, for example, clinical applications in human disease, management of fish and livestock, and the control of agriculturally-important animal pests. At least five different subfamilies of insulin-like proteins have been identified in vertebrates, represented by insulin (Steiner et al., 1989, in
Endocrinology
, DeGroot, ed., Philadelphia, Saunders, pp. 1263-1289), insulin-like growth factor (IGF, previously termed somatomedin) (Humbel, 1990, Eur. J. Biochem. 190:445-462), relaxin (Schwabe and Bullesback, 1994, FASEB J. 8:1152-1160), relaxin-like factor (RLF, previously called Leydig cell-specific insulin-like peptide) (Adham et al., 1993, J. Biol. Chem. 268:26668-72; Ivell, 1997, Reviews of Reproduction 2:133-138), and placentin (also known as early placenta insulin-like peptide, or ELIP) (Chassin et al., 1995, Genomics 29:465-470).
Insulin superfamily members in invertebrates have been less extensively analyzed than in vertebrates, but a number of different subgroups have been defined. Such subgroups include molluscan insulin-related peptides (MIP-I to MIP-VII) (Smit et al., 1988, Nature 331:535-538; Smit et al., 1995, Neuroscience 70:589-596), the bombyxins of lepidoptera (originally referred to as prothoracicotropic hormone or PTTH) (Kondo et al., 1996, J Mol. Biol. 259:926-937), and the locust insulin-related peptide (LIRP) (Lagueux et al., 1990, Eur. J. Biochem. 187:249-254). Most recently, there have been descriptions of an exceptionally large insulin-like gene family in the nematode
C. elegans
(U.S. patent application Ser. No. 09/062,580, filed Apr. 17, 1998 (Attorney Docket No. 7326-059) entitled “NUCLEIC ACIDS AND PROTEINS OF
C. ELEGANS
INSULIN-LIKE GENES AND USES THEREOF” by Homburger et al; U.S. patent application Ser. No. 09/074,984, filed May 8, 1998 (Attorney Docket No. 7326-068) entitled “NUCLEIC ACIDS AND PROTEINS OF
C. ELEGANS
INSULIN-LIKE GENES AND USES THEREOF” by Buchman et al; U.S. patent application Ser. No. 09/084,303, filed May 26, 1998 (Attorney Docket No. 7326-069) entitled “NUCLEIC ACIDS AND PROTEINS OF
C. ELEGANS
INSULIN-LIKE GENES AND USES THEREOF” by Ferguson et al; Duret, et al., 1998, Genome Res. 8:348-353; Brousseau, et al., 1998, Early 1998 East Coast Worm Meeting, abstract 20; Kawano, et al., 1998, Worm Breeder's Gazette 15(2):47; Pierce and Ruvkun, 1998, Early 1998 East Coast Worm Meeting, abstract 150; Wisotzkey and Liu, 1998, Early 1998 East Coast Worm Meeting, abstract 206). Also, putative orthologs of both vertebrate insulin and IGF have been identified in a tunicate (McRory and Sherwood, 1997, DNA and Cell Biology 116:939-949). Tunicates are thought to be the closest living invertebrate relative to the progenitor from which vertebrates evolved (McRory and Sherwood, 1997, DNA and Cell Biology 16:939-949).
Comparison of the primary sequence of insulin superfamily peptides, cDNAs, and genes, as well as the overall conservation of functional and structural domains of insulin-like genes and proteins, lead to the conclusion that existing members of the insulin superfamily evolved from a common ancestral gene (Blundell and Humbel, 1980, Nature 287:781-787; LeRoith, et al., 1986, Recent Prog. Horm. Res. 42:549-87; Murray-Rust, et al., 1992, BioEssays 14:325-331; LeRoith, et al., 1993, Mol. Reprod. Dev. 35(4):332-8). From the extensive sequence divergence evident among known subfamilies of insulin-like proteins, it is assumed that this is an ancient family of regulatory hormones that evolved to control growth, reproduction and metabolism in early metazoans. However, the precise evolutionary origins of this important family remain unclear.
2.1.1. COMMON STRUCTURAL THEMES
There are common structural themes that unite the insulin superfamily of proteins. Insulin-like peptide hormones are synthesized in vivo as precursor proteins having structures that are variations of the structure schematically represented in FIG.
1
. Most precursor forms of the insulin superfamily can be divided into four domains, termed Pre, B, C, and A domains, extending in order from the N-terminus to the C-terminus of a precursor polypeptide (see FIG.
1
). Precursors of the IGF subfamily are distinguished by having two additional domains at the C-terminal end, termed D and E domains. The precursors of the locust LIRP protein and some
C. elegans
insulin-like proteins are distinctive in that they possess another domain, here designated as the F domain, positioned between the Pre domain and the B peptide. The N-terminal Pre domain typically contains a hydrophobic signal sequence which directs secretion of the hormone from cells and is removed by the enzymatic action of a signal peptidase during transit into the endoplasmic reticulum (see the asterisk in FIG.
1
). Upon folding, the prohormone undergoes additional processing which, in most cases, involves proteolytic cleavage at two sites that excise the C peptide from the mature hormone (see the two middle arrows illustrated in FIG.
1
). These processing steps are mediated by prohormone convertases that cleave at specific positions next to basic residues in the C peptide sequence. As a result, most forms of mature insulin superfamily hormones consist of two polypeptide chains, the A and B peptides, which are covalently joined by disulfide linkages (S—S) between Cys residues (see S—S linkages illustrated in FIG.
1
). The precise arrangement of Cys residues and disulfide linkages, both between the A and B peptides and within the A peptide, is highly characteristic of the insulin superfamily of hormones. The vast majority of known insulin superfamily members contain six precisely-positioned Cys residues, two in the B chain and four in the A chain, which participate in the formation of three disulfide bonds. Two of these disulfide linkages covalently join the B and A chains (i.e., they form inter-chain bonds), whereas the third disulfide linkage occurs within the A peptide (i.e., as an intra-chain bond) and appears to stabilize a bend in the A chain fold.
The IGF subfamily of hormones has a unique processing pathway. In this subfamily, the connecting C peptide is not removed by processing of the prohormone. Instead, a single proteolytic cleavage event removes the C-terminal E domain (see
Buchman Andrew Roy
Doberstein Stephen Kohl
Keyes Linda Nolan
Reddy Bindu Priya
Ruddy David Andrew
Exelixis Pharmaceuticals, Inc.
Nguyen Dave T.
Nguyen Quang
Pennie & Edmonds LLP
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