Abstract: Excess amount of modified subtracter DNA from control cells is generated by carboxylating the base structures of its certain nucleotides with chemical agents in order to introduce covalent affinity between the modified subtracter and a non-modified tester DNA. Hybridization of the control subtracter and the experimental tester DNA is performed with a heat-melting and then cool-reassociation technique. While the desired different (heterologous) sequences remain in the form of hydrogen-binding, common (homologous) sequences of the hybridized DNA are covalently bonded to each other. Since the covalent bonding of the common sequences can not be broken during a polymerase chain reaction, resulting in no amplification of the common sequences but great amplification of the desired different sequences. The desired DNA sequences present after such covalent homologue subtraction and selective amplification represent those DNA sequences which only exist in the tester but not in the subtracter DNA library.

Abstract: The present invention provides a method for fast, simple, and reliable isolation of desired different sequences from two DNA libraries. Excess amount of nucleotide analog-containing DNA subtracter from control cells is generated by incorporating nucleotide analog with a template-dependent extension reaction to introduce susceptible-sites for subsequent enzymatic digestion. Hybridization of the control subtracter and experimental DNA is performed with a heat-melting and then cool-reassociation technique. The hybridized DNAs are subtracted with nucleotide analog-removing enzyme first, resulting in nicking or gapping all nucleotide analog-containing hybrid duplexes which are further digested by single-strand-specific nuclease. Desired DNA sequences from the experimental cells, but not the control ones stay intact throughout the digestion procedure and can be selectively amplified at the end.

Abstract: Two follistatin proteins with inhibin-like activity were isolated from porcine follicular fluid using heparin-Sepharose affinity chromatography, followed by gel filtration on Sephacryl S-200 and then six steps of high-performance liquid chromatography. The larger protein has 315 residues and is believed to be glycosylated. The smaller protein is a 288-residue, C-terminally shortened version thereof. These proteins specifically inhibit basal secretion of FSH, but not of LH, in a rat anterior pituitary monolayer culture system. The half-maximal effective dose for both is 2.5-6.0 ng/ml. Human and rat follistatins exhibit very high homology with the porcine protein, with the human differing from porcine in only 4 residues out of 315 and with the rat differing from porcine in only 8 residues out of 315. Using the porcine amino acid sequence information, cDNA clones encoding these proteins were identified from a porcine ovarian cDNA library.

Abstract: Two follistatin proteins with inhibin-like activity were isolated from porcine follicular fluid using heparin-Sepharose affinity chromatography, followed by gel filtration on Sephacryl S-200 and then six steps of high-performance liquid chromatography. Each isolated molecule is a monomer having a molecular weight of at least about 32,000 daltons. Microsequencing revealed the NH.sub.2 -terminal portions both to be Gly-Asn-Cys-Trp-Leu-Arg-Gln-Ala-Lys-Asn-Gly-Arg-Cys-Gln-Val-Leu. The larger protein has 315 residues and is believed to be glycosylated. The smaller protein is a 288-residue, C-terminally shortened version thereof. These proteins specifically inhibit basal secretion of FSH, but not of LH, in a rat anterior pituitary monolayer culture system. The Half-maximal effective dose for both is 2.5-6.0 ng/ml.

Abstract: A 28,000-dalton protein with FSH-releasing activity is isolated from porcine follicular fluid in a multi-step procedure including gel filtration, ion exchange chromatography and several reverse-phase high-performance liquid chromatography steps. The 28 kD protein promotes secretion of FSH, but not of LH, in a rat anterior pituitary monolayer culture system, exhibiting an EC.sub.50 of 0.5 ng/ml. These peptides are dimers of inhibin .beta.-chains of mammals, and homodimers or heterodimers of the .beta..sub.A and .beta..sub.B chains are biologically active to regulate fertility of various mammalian species, including humans.

Abstract: Two 32,000-dalton proteins with inhibin activity were isolated from porcine follicular fluid using heparin-Sepharose affinity chromatography, followed by gel filtration on Sephacryl S-200 and then four steps of reverse-phase high-performance liquid chromatography. Each isolated molecular is composed of two chains having molecular weights of about 18,000 and about 14,000 daltons, respectively, which are bound together by disulfide bonding. Microsequencing revealed the NH.sub.2 -terminal portion of the 18K chain of both to be Ser-Thr-Ala-Pro-Leu-Pro-Trp-Pro-Trp-Ser-Pro-Ala-Ala-Leu-Arg-Leu-Leu-Gln-Ar g-Pro-Pro-Glu-Glu-Pro-Ala-Val, of one of the 14K chains to be Gly-Leu-Glu-Cys-Asp-Gly-Arg-Thr-Asn-Leu-Cys-Cys-Arg-Gln-Gln-Phe-Phe-Ile-As p-Phe-Arg-Leu-Ile-Gly-Trp, and of the other 14K chain to be Gly-Leu-Glu-Cys-Asp-Gly-Lys-Val-Asn-Ile-Cys-Cys-Lys-Lys-Gln-Phe-Phe-Val-Se r-Phe-Lys-Asp-Ile-Gly-Trp-Asn-Asp-Trp-Ile-Ile-Ala-Pro.

Abstract: A substantially purified substance has been isolated from bovine gonadal secretions which mediates pituitary hormone secretion, using an extraction and purification sequence that includes gel filtration, partition chromatography and HPLC in that order. The substance, designated gonadostatin, is a moderate-size peptide which below a threshold level selectively inhibits LRF-stimulated pituitary secretion of LH and FSH. Above a threshold level, gonadostatin generally stimulates pituitary hormone secretion.