Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1995-06-07
2003-09-02
Gambel, Phillip (Department: 1644)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S024300, C536S024310, C530S350000, C435S069100, C435S252300, C435S320100, C435S455000, C435S471000
Reexamination Certificate
active
06613891
ABSTRACT:
TECHNICAL FIELD
This invention relates, inter alia, to the construction of cloning vectors that contain deoxyribonucleic acid (DNA) sequences which code for part or all of the hormone inhibin, and of host cells such as bacterial strains containing such vectors, and of host cells such as bacterial strains which produce part, all or precursors of inhibin. In addition it relates to the production and uses of expression products of said vectors and strains and to the production and uses of fragments of the expression products and vectors, be they natural or synthetic in origin.
BACKGROUND ART
It was suggested in 1932 that the gonads produce a non-steroidal factor called inhibin which is involved in feedback regulation of pituitary function (McCullagh, D. R. (1932) Science 76, 19-20). Since that time, it has been shown that the anterior pituitary produces at least two gonadotrophins, follicle stimulating hormone or follitropin (FSH) and luteinising hormone or lutropin (LH) which together regulate the development and functioning of the gonads. Sensitive radioimmunoassays have permitted accurate independent monitoring of each hormone and have shown feedback regulation of these hormones by the gonads. The feedback regulation of LH appears to be predominantly via steroids whereas that of FSH is via the protein or glycoprotein factor, inhibin, in addition to steroids.
Inhibin can now be defined as a protein or glycoprotein hormone secreted from the granulosa cells in the ovary or Sertoli cells in the testis. It is secreted in response to FSH and acts on the pituitary as a feedback inhibitor of FSH synthesis and secretion but which leaves the basal synthesis and secretion of LH largely intact. Whether inhibin, its mRNA or precursors are formed in other cells and tissues is not yet known.
Since the early 1970's a number of attempts have been made to purify inhibin from a variety of gonadal sources, both testicular and ovarian, and with conflicting results (de Jong, F. H. (1979) Mol. Cell. Endocrinol. 13, 1-10) due in part to the use of a variety of bioassay systems where any suppression of FSH in pituitary cells in vitro or in vivo was assumed to be due to inhibin and checks were not always made for non-specific toxic effects of the test substances (Baker, H. W. G. et al. (1981) in Intragonadal Regulation of Reproduction (Franchimont, P. and Channing, C. P. Eds.), Academic Press, London pp. 193-228; Baker H. W. G. et al., (1982) Ann. New York Acad. Sci. 383, 329-342).
Recently, inhibin from bovine follicular fluid (bFF) has been purified to homogeneity. (International Patent Application PCT/AU85/00119; Robertson, D. M. et al., (1985) Biochem. Biophys. Res. Commun. 126, 220-226). This achievement was aided by the use of a rigorous cultured rat pituitary cell assay (Scott, R. S. et al., (1980) Endocrinol. 107, 1536-1542) which incorporates a means of assessing the cytotoxic effects of the substances under test (Robertson, D. M. et al., (1982) Mol. Cell. Endocrinol. 26, 119-127) so that non-specific toxic effects that lowered the FSH content of the cells under measurement could be identified as distinct from the effects of inhibin. The standard employed was a bovine follicular fluid preparation with an inhibit activity of 20 U/ml in terms of a previously described ovine testicular lymph standard assigned an arbitary activity of 1 U/mg (Scott, R. S. et al., (1980) Endocrinol. 107, 1536-1542)
The inhibin from bFF was purified about 3,500-fold to a specific activity of 200,000 units/mg protein and is a protein of 58 kD composed of two disulphide-linked subunits A and B of approximately 43 kD and 15 kD respectively as evidenced from electrophoresis in polyacrylamide gels in the presence of sodium dodecylsulphate (SDS-PAGE). The amino acid sequences for the NH
2
-terminus of each subunit have been determined. This purified substance has the in vitro physiological properties defined earlier as those of inhibin, namely inhibiting the synthesis and release of endogenous FSH from rat pituitary cells whilst leaving intact the synthesis and release of LH prolactin and thyroid stimulating hormone.
Since FSH is important for determining the incidence and rate of ovulation in females and spermatogenesis in males, it follows that the main potential applications of inhibin, analogues, or homologues of inhibin or antibodies against inhibin will be to inhibit or to stimulate gonadal function in man and domestic animals and as diagnostic tools for analysis of gonadal function. Many experiments have been performed in vivo using crude or partially fractionated gonadal extracts or secretions in attempts to analyse the physiological effects of inhibin. Effects attributed to be due to the inhibin or antibodies against inhibin in these experiments include
1. Inhibition of gonadal function (Moudgal, N. R. et al. (1985) in Gonadal Proteins and Peptides and their Biological Significance (Sairam, M. R. and Atkinson, L. E., Eds.) World Scientific Publishing Co., Singapore, pp 21-37).
2. An increase in ovulation rate (O'Shea, T. et al., (1982) Proc. Aust. Soc. Rep. Biol. 14, 85; O'Shea, T. et al. (1983) Proc. Aust. Soc. Rep. Biol. 15, 22; Henderson, K. M. et al., (1984) J. Endocrinol. 102, 305-309).
3. An advancement of the onset of puberty (Al-Obaidi, F. A. R. et al., (1983) Proc. Aust. Soc. Rep. Biol. 15, 80).
The commercial exploitation of these properties and further physiological studies in live animals require large quantities of pure inhibin or fragments thereof or inhibin agonists and antagonists whether of natural or synthetic origin. Such quantities cannot be obtained solely by the present methods of purification since source material may be limited (e.g. human follicular fluid) and a typical extraction starting with 50 ml bFF yields only 5-10 &mgr;g of purified material.
The present invention seeks, inter alia, to overcome these limitations by the identification and characterisation of the genes which code inhibin so as to allow the molecular cloning of genes or parts of genes coding for inhibin into a host such as
Escherichia coli
and by manipulating the cloned genes or parts thereof to create hosts, such as bacterial strains, which can synthesise all, part or precursors of the inhibin molecule, including subunits thereof.
DESCRIPTION OF THE INVENTION
In a first embodiment the present invention provides a first DNA sequence which acts as a coding sequence for amino acid sequences of all, part of precursors of inhibin, or a DNA sequence which hybridises to said first DNA sequence, said sequences being from whatever source obtained, including natural, synthetic or semi-synthetic sources, said sequences including sequences related by mutation, including single or multiple base substitutions, deletions, insertions, and inversions and including DNA sequences which on expression code for all, part or precursors or homologues and analogues of a polypeptide which is inhibin or which displays similar immunological or biological activity as that of inhibin.
Preferred sequences of the invention are those coding for a polypeptide corresponding to the 43 kD and 15 kD (A and B) subunits of bovine and human inhibin as described hereinafter in more detail and depicted in
FIGS. 5
,
6
,
7
and
8
of the accompanying drawings.
In another preferred embodiment, the DNA codes for the 20 kD (A
C
) subunit of inhibin described hereinafter.
The DNA sequences embraced by the present invention can be prepared for example from vertibrate cells by extracting total DNA therefrom and isolating the sequences by standard techniques. Alternatively the DNA may be prepared in vitro, synthetically or biosynthetically, such as by the use of an mRNA terplate.
Also, within the scope of the present invention, is a process for selecting a DNA or RNA sequence coding for all, part or precursors of a polypeptide which is inhibin or which displays an immunological or biological activity similar to inhibin, which process comprises providing one or more DNA or RNA sequences, and determining which of said sequences hybridises with a DNA
de Kretser David Moritz
Findlay John Kerr
Forage Robert Gregory
Milne-Robertson David Mark
Stewart Andrew George
Gambel Phillip
Inhibin Pty. Limited
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