Abstract: A method for the preparation of a 3' end functionalized polynucleotide is disclosed. An amine-functionalized solid phase support is treated sequentially with an anhydride, then with an .omega.-hydroxylamine. A polynucleotide is chemically synthesized on the treated support and is subsequently cleaved therefrom by hydrolysis of the amide bonds. A polynucleotide having a 3' free primary amine is recovered for use in hybridization assays and other uses.

Abstract: Disclosed are novel circular DNA plasmids useful as vectors in recombinant methods to secure high levels of E.coli expression of exogenous genes. Plasmids of the invention comprise discrete DNA sequences operative to: (1) confer upon the plasmid the capacity for autonomous replication in a host cell; (2) control autonomous plasmid replication in relation to the temperature at which host cell cultures are maintained; (3) stabilize maintenance of the plasmid in host cell populations; (4) direct synthesis of a protein product indicative of plasmid maintenance in a host cell population; (5) provide, in series, a plurality of restriction endonuclease recognition sites, unique to the plasmid and facilitative of exogenous gene DNA sequence insertion; and (6) terminate mRNA transcription of adjacent DNA sequences and situated so as to terminate transcription of exogenous gene sequences inserted within the plasmid at said unique restriction endonuclease restriction sites.

Abstract: Disclosed are novel polypeptides possessing part or all of the primary structural conformation and one or more of the biological properties of mammalian erythropoietin ("EPO") which are characterized in preferred forms by being the product of procaryotic or eucaryotic host expression of an exogenous DNA sequence. Illustratively, genomic DNA, cDNA and manufactured DNA sequences coding for part or all of the sequence of amino acid residues of EPO or for analogs thereof are incorporated into autonomously replicating plasmid or viral vectors employed to transform or transfect suitable procaryotic or eucaryotic host cells such as bacteria, yeast or vertebrate cells in culture. Upon isolation from culture media or cellular lysates or fragments, products of expression of the DNA sequences display, e.g., the immunological properties and in vitro and in vivo biological activities of EPO of human or monkey species origins.

Abstract: Disclosed are novel synthetic peptides having primary structural homology to a continuous sequence of amino acid residues of human growth hormone in a region spanning positions thirty-two to forty-six ("hGH.sub.32-46 ", or "deletion peptide"). In preferred forms, peptides of the invention comprehend: duplicate portions (i.e., sequence fragments) of hGH.sub.32-46 ; stereochemical analogs and fragment analogs of hGH.sub.32-46 including one or more amino acid residues in D-isomeric configuration; and, "interspecies" analogs and fragment analogs of hGH.sub.32-46 including one or more non-homologous amino acid residues duplicating variant residues present in corresponding positions in corresponding regions of heterologous species growth hormones. Peptides of the invention are administered to mammals contemporaneously with exogenous insulin to generate hypoglycemic effects greater than available through administration of insulin alone. A presently preferred heptapeptide has the sequence, NH.sub.

Abstract: Described are rapid and highly efficient procedures for the total synthesis of linear, double stranded DNA sequences in excess of about 200 base pairs in length, which sequences may comprise entire structural genes. Novel sequences are prepared from two or more DNA subunits provided with terminal regions comprising restriction endonuclease cleavage sites facilitating insertion of subunits into a selected vector for purposes of amplification during the course of the total assembly process. The total, finally-assembled assembled sequences include at least one, and preferably two or more, unique restriction endonuclease cleavage site(s) at intermediate positions along the sequence, allowing for easy excision and replacement of subunits and the correspondingly facile preparation of multiple structural analogs of polypeptides coded for by the sequences.

Abstract: Disclosed are novel DNA sequences operative as promoters of microbial transcription of structural genes and comprising synthetic E.coli bacteriophage T5 "early" promoter replica DNA sequences. Disclosed also are novel promoter/operator DNA sequences comprising a synthetic T5 early promoter replica DNA sequence associated with a regulatable operator DNA sequence providing for selectively regulatable transcription of structural genes in, e.g., E.coli cells transformed with a DNA vector including same.

Abstract: Disclosed are methods for selective modification of double-stranded DNA sequences facilitating their storage and incorporation into expression vectors. Manufactured partially double-stranded DNA primers are hybridized to complementary regions of single-stranded forms of DNA sequences to be altered. Desired, selectively modified double-stranded DNA sequences are then formed by DNA polymerization and specific nuclease digestion of undesired double- and single-stranded regions. Illustratively provided are DNA sequences useful in the microbial expression of bovine prolactin and other valuable polypeptides such as avian growth hormone.

Abstract: Chromatographic procedures are individually and jointly applied to the rapid and efficient isolation of biologically active proteins and especially glycoproteins such as recombinant erythropoietin present in the medium of growth of genetically transformed mammalian host cells. Illustratively, recombinant EPO is isolated from culture fluids by reverse phase chromatography employing a C.sub.4 or C.sub.6 column and elution with ethanol. Recombinant erythropoietin may also be purified by anion exchange chromatography employing, e.g., a DEAE resin, with preliminary selective elution of contaminant materials having a lower pKa than erythropoietin from the resin under conditions mitigating against acid activated protease degradation. Practiced serially, the two chromatographic procedures allow for high yields of biologically active recombinant erythropoietin from mammalian cell culture media.

Abstract: Described are rapid and highly efficient procedures for the total synthesis of linear, double stranded DNA sequences of up to about 200 base pairs, which sequences may comprise entire structural genes. Illustratively disclosed is the preparation and expression of manufactured genes, including fusion genes, capable of directing synthesis of human .beta.-endorphin and of proteins which differ from human .beta.-endorphin in terms of the identity or relative position of one or more amino acids. Manufactured genes preferably include codons selected from among alternative codons specifying the same amino acid on the basis of preferential expression in a projected host microorganism (e.g., E. coli) to be transformed.

Abstract: Disclosed are monoclonal antibody substances specifically immunologically reactive with native and recombinant human immune interferon ("IFN-.gamma.") and with polypeptides having amino acid sequences substantially duplicative of sequences extant in IFN-.gamma.. In a presently preferred embodiment, antibody substances are produced by new mouse-mouse hybridoma tumor cell line A.T.C.C. HB 8291 and are immunoreactive with native IFN-.gamma., with recombinant IFN-.gamma. and polypeptide analogs thereof, and with a nonadecapeptide whose amino acid sequence duplicates that of the final nineteen amino acid residues of the carboxyl terminal of IFN-.gamma.. These preferred antibody substances, while displaying high affinity for IFN-.gamma., do not neutralize antiviral biological activity of the interferon. They are usefully employed in the detection, quantification and affinity purification of IFN-.gamma. and IFN-.gamma. analogs as well as in investigations relating to the mode of biological action of IFN-.gamma..

Abstract: Disclosed are novel methods and materials for developing hypoglycemic effects in mammals, including human. A pentadecapeptide ("deletion peptide") having the sequence, NH.sub.2 -Glu-Glu-Ala-Tyr-Ile-Pro-Lys-Glu-Gln-Lys-Tyr-Ser-Phe-Leu-Gln-COOH, is administered to the mammal contemporaneously with exogenous insulin to generate hypoglycemic effects greater than available through administration of insulin alone. The pentadecapeptide sequence is duplicative of the sequence of amino acid residues in human growth hormone in the region spanning positions thirty-two through forty-six.

Abstract: Disclosed is a new mouse-mouse hybridoma tumor cell line A.T.C.C. No. HB8209. A monoclonal antibody produced by said cell line is specifically immunologically reactive with erythropoietin and with a polypeptide whose amino acid sequence is substantially duplicative of a sequence extant is erythropoietin. Disclosed also are procedures for isolation of erythropoietin by affinity purification and for quantitative detection of erythropoietin in fluid samples.

Abstract: Microbial synthesis of indigo dyestuff in indole-free media is disclosed. Indigo production is preferably accomplished by genetic transformation of selected host cells having the capacity to produce and accumulate indole (either as a result of endogenous genomic capacity or genetic transformation) to incorporate the capacity for synthesis of an aromatic dioxygenase enzyme. Growth of transformed cells under suitable conditions facilitates aromatic dioxygenase enzyme catalyzed oxidative transformation of cellular indole, with consequent formation of indigo from the oxidized reaction products. In a highly preferred embodiment, E. coli cells having endogenous indole production capacity are transformed with a DNA expression vector comprising the structural gene for naphthalene dioxygenase, resulting in the microbial synthesis of isolatable quantities of indigo.

Abstract: Murine-derived hybridoma tumor cell lines and monoclonal anti-Colony Stimulating Factor Subclass Number 1 antibody substances produced by these cell lines. Use of said monoclonal antibody substances, alone or in combination, in immunological procedures for isolation of natural Colony Stimulating Factor Subclass Number 1 and for quantitative detection of colony Stimulating Factor Subclass Number 1 in fluid samples.