Robert D. Ivarie

A rapid sensitive assay for specific protein synthesis in cells and in cell-free translations: Use of Staphylococcus aureus as an adsorbent for immune complexes

Abstract Formalin-fixed, heat-inactivated Staphylococcus aureus Cowen Type I (SAC) (Kessler, S. W. (1975) J. Immunol. 115 , 1617–1624) has been used to isolate antibody-bound tyrosine aminotransferase (EC 2.6.1.5) from radioactively labeled cell-free extracts and translation reactions of polysomes and poly(A)-containing messenger RNA. Under our conditions, SAC immunoprecipitation of cell-free extracts generated a radioactive background in control serum precipitations of 0.1%, an average of fivefold lower than backgrounds obtained by direct and indirect antibody precipitations. It is shown that the major source of the background, which limits sensitivity of the assay, comes from the amounts of antiserum and bacterial adsorbent in the assay. Procedures are presented to provide low backgrounds in more general applications of the precipitation method. From these experiments, SAC immunoprecipitation is shown to be a rapid, economical method to assay for specific protein synthesis under a variety of experimental conditions with noise levels much lower than with other standard methods.

Synthesis of growth hormone by bacteria

A hybrid gene was constructed between the β-lactamase gene of plasmid pBR322 and the cloned coding sequence for rat growth hormone. This gene is expressed in bacteria and growth hormone sequences are detectable by immunological methods.

Thyroid hormone receptors and responses

Publisher Summary This chapter discusses the responses of thyroid hormone receptors. The thyroid hormone receptor is unique in that it is an intrinsic chromosomal nonhistone protein known to be involved in the regulation of the expression of specific genes. These receptors are bound to chromatin in the absence or the presence of thyroid hormone. They can be solubilized from chromatin and can bind to DNA. The hormone (mainly T4 or T3) enters the cell where it can be metabolized. Several metabolites of thyroxine (T4), such as T3, 3, 3’-T2, reverse T3, triac, and tetrac, have biological and receptor-binding activity; however, quantitatively, T3 is the most important hormone in vivo. This model for thyroid hormone action contrasts with the case of steroid hormones. Whereas the chromatin stimulates the capability of the thyroid hormone–receptor to bind the hormone, steroids stimulate the binding of their receptors to chromatin. Shortly after the interaction of thyroid hormone with the receptor, there is a major increase in the chromatins capacity to bind bacterial RNA polymerase and the distribution of chromatin proteins is altered. These data suggest that the hormone rapidly modifies chromatin in some way. These influences nevertheless are transmitted into effects only on the expression of a small subset of the cellular genes.

Hormonal domains of response: actions of glucocorticoid and thyroid hormones in regulating pleiotropic responses in cultured cells.

Publisher Summary This chapter discusses the pleiotropic responses of thyroid and glucocorticoid hormones in cultured hepatoma cells (H35 and HTC) and cultured anterior pituitary cells (GH3). Both hormones influence a limited and highly specific domain in each cell type. Approximately 1% or less of the detected gene products are affected by each hormone. Evidence presents that there is only a very limited overlap in the glucocorticoid domains of HTC and H35 cells, as only tyrosine amino-transferase is induced by dexamethasone in both cell types. There can be heterogenity in glucocorticoid domains among parenchymal cells from livers of the same strain of rats. Alternatively, differences in the levels of expression of domain members or transformation-dependent influences on the glucocorticoid domains of different cell lines can account for these results. In addition, the glucocorticoid hormone domains between GH3 and either HTC or H35 cells are almost completely non-identical. Overlap between the glucocorticoid and thyroid hormone domains has been demonstrated in GH3 cells. The two hormones can regulate the same gene product in one of three ways: conduction, co-repression, or induction by one hormone and repression by the other. Both glucocorticoid and thyroid hormones are capable of inducing and repressing individual domain members.

Prolactin-deficient variants of GH3 rat pituitary tumor cells: linked expression of prolactin and another hormonally responsive protein in GH3 cells.

GH3 cells normally synthesize and secrete two pituitary polypeptide hormones, prolactin and growth hormone. From an ethyl methane sulfonate-mutagenized population, prolactin low-producing variants have been isolated at a frequency near 20%. Intracellular prolactin synthesis in the variants was reduced 40- to 100-fold compared to wild-type cells while growth hormone synthesis varied less than 2-fold. This decrease was paralleled by a decrease in intracellular preprolactin mRNA. Although reduced, prolactin synthesis was still repressible by glucocorticoids. There was a coordinate loss of expression of p21, a thyroid and glucocorticoid hormone-regulated protein, in GH3 cells, whereas the synthesis and regulation of other hormonally responsive proteins were unimpaired in the variants. Since p21 expression was coordinately regained in a high-producing prolactin revertant cell, expression of the two proteins is tightly coupled in GH3 cells. The stability of the low-producing phenotype differed among variants. One (B2) gave rise to revertants at about 20% frequency even after two rounds of subcloning, whereas another (B3) was more stable in that only 1 weak revertant was found in 47 subclones. The reversion frequency of B3 cells was also measured at less than 0.5%. Unmutagenized GH3 cells were phenotypically stable in that no prolactin-deficient variant was found among 57 subclones. Since variants were ony found after ethyl methane sulfonate mutagenesis, the DNA alkylating agent appears to have promoted an epigenetic change in pituitary gene expression.

Isolation of two different molecular weight polypeptides copurifying with rat liver tyrosine aminotransferase.

Abstract An affinity chromotography resin highly specific for rat liver tyrosine aminotransferase (EC 2.6.1.5) has been synthesized and used in the purification of this enzyme. The structure of the resin, N -(5′-phosphopyridoxyl)- l -tyrosyl-aminoocytl-Sepharose 4B, was designed to resemble the tyrosine-pyridoxal phosphate Schiffs base intermediate in the reaction pathway catalyzed by this enzyme. Use of this resin in combination with octyl-agarose chromatography on partially purified enzyme resulted in a tyrosine aminotransferase preparation with a specific activity of about 450 units/mg protein. When analyzed on one-dimensional polyacrylamide-sodium dodecyl sulfate slab gels, the highly purified enzyme was composed of two polypeptides with molecular weights of about 56,000 and 53,000. Radioiodinated tryptic peptides from each of these polypeptides were essentially identical following two-dimensional analysis. Although the two polypeptides could not be separated from each other in an active form, it was found that (i) both polypeptides have pyridoxal phosphate-binding sites, (ii) the coenzyme is probably bound to both polypeptides as a Schiffs base, (iii) both polypeptides have binding sites for l -tyrosine and l -glutamic acid, the two specific substrates for the enzyme, and (iv) both polypeptides can catalyze the formation of the initial amino acid-pyridoxal phosphate Schiffs base adduct in the overall reaction pathway. Since the ratios of these polypeptides differed from preparation to preparation of purified enzyme, the 53,000 M r species probably arises by proteolysis of tyrosine aminotransferase in crude liver extracts. These results imply that if tyrosine aminotransferase isozymes exist, they are not the result of translation products produced by different structural genes.