Vitellogenin gene transcription is not under strict estrogen control in winter flounder.

Although it is almost axiomatic that vitellogenin gene expression is under exclusive control of estrogen in oviparous animals, our results with winter flounder demonstrate that vitellogenin gene transcription in females can continue independent of estradiol. Winter flounder were hypophysectomized in January, i.e. several months after the onset of vitellogenesis. Thirty or more days after hypophysectomy, all fish had negligible levels of estradiol in the serum, and yet vitellogenin gene transcription was quite active in the liver. Our results also suggest that a pituitary factor may be involved in the normal repression of the vitellogenin gene.

Antifreeze protein gene transcription in winter flounder is not responsive to temperature.

Although the endogenous rhythm of antifreeze protein gene expression in winter flounder is primarily regulated through the pituitary, the effect of water temperature on the annual cycle is poorly understood. In order to determine the specific effects of temperature on antifreeze gene transcription we did a series of experiments with intact and hypophysectomized winter flounder kept at various temperature regimes. Our results demonstrate that temperature shifts do not induce or suppress antifreeze gene transcription as determined by "run-on" transcription assays or by Northern blot analysis of liver mRNA in hypophysectomized flounder. However, warm temperature reduces the amount of antifreeze protein in the plasma, and apparently reduces the half-life of antifreeze protein mRNA.

Hormonal regulation of antifreeze protein gene expression in winter flounder.

The essential features of experiments carried out over the past fifteen years are brought together with new data to formulate a model describing the hormonal regulation of the annual plasma antifreeze polypeptide (AFP) cycle in winter flounder (Pseudopleuronectes americanus). The precise time of onset of antifreeze synthesis in the fall appears to be regulated by photoperiod acting through the central nervous system (CNS) on the pituitary gland. During the summer, growth hormone (GH) blocks transcription of the AFP genes. With the loss of long daylengths in the fall, the CNS inhibits the release of GH allowing AFP gene transcription in the liver to proceed. In the spring GH is again released from the pituitary and AFP gene transcription is blocked.

A soluble biotinyl-alpha-amanitin affinity probe for the isolation of RNA polymerase B.

Utilizing the 6'-hydroxyindole moiety of alpha-amanitin for substitution, biotinyl-alpha-amanitin has been synthesized to use as a soluble affinity probe for the isolation of RNA polymerase B from mammalian cell culture. The synthetic biotinyl-alpha-amanitin remains a potent inhibitor of RNA polymerase B having a Ki of 4.1 X 10(-8) M as compared with a Ki of 5 X 10(-9) M for natural alpha-amanitin. RNA polymerase B complexed with biotinyl-alpha-amanitin can be isolated on Bio-Gel P300 polyacrylamide gel beads to which avidin has been attached. RNA polymerase B may then be released from the complex by treatment with sodium dodecyl sulfate or by monochromatic irradiation at 314 nm which destroys the anatoxin moiety. We have used this affinity probe to analyze the subunit composition of RNA polymerase B from various mouse myeloma cell lines. We believe that the biotinyl-alpha-amanitin may be very useful for the isolation of factors which associate with RNA polymerase B; e.g., we have substantiated that actin can be associated with RNA polymerase.

Membrane damage of liposomes by the mushroom toxin phallolysin.

We have investigated the membrane-damaging effect of phallolysin on liposomes varying in phospholipid composition, net charge and physical constitution. Liposomes were prepared from lipids extracted from bovine or human erythrocyte ghosts. The liposomes composed of bovine lipids (the intact cell showing little sensitivity to phallolysin) were found comparably sensitive to those prepared from lipids of human red cells (these cells being of high sensitivity). In addition, artificial mixtures of lipids were used for the preparation of liposomes, consisting of (a) negatively charged phospholipids such as dicetyl phosphate or phosphatidylserine, (b) cholesterol, and (c) either sphingomyelin (as the major component of erythrocytes from ruminants) or phosphatidylcholine (as the major component of erythrocytes from non-ruminants). Again, we found only little difference in the susceptibilities of sphingomyelin- and phosphatidylcholine-containing liposomes. On the other hand, the susceptibility depended on the presence of phospholipids with negative net charges. Omittance of phosphatidylcholine or dicetyl phosphate, or replacement by the positively charged stearylamine, decreased the susceptibility by a factor of more than 20. Finally, we prepared liposomes from dicetyl phosphate, cholesterol and phosphatidylcholine in two physical states: large unilamellar and smaller multilamellar liposomes. The unilamellar liposomes were about 10-times more sensitive to phallolysin. We conclude: (1) Phallolysin damages phospholipid-membranes in the absence of receptor proteins, but high concentrations of the toxin are required. (2) Membrane damage takes place with liposomes containing phosphatidylcholine as well as those containing sphingomyelin. (3) Phallolysin damages only liposomes containing phospholipids with a negative net charge.

Formation of a single phosphodiester bond by RNA polymerase B from calf thymus is not inhibited by alpha-amanitin.

The template-directed synthesis of a single phosphodiester bond by highly purified calf thymus RNA polymerase B is not inhibited by high concentrations of alpha-amanitin (10(-6) M). However, a subsequent internucleotide bond is not synthesized in the presence of alpha-amanitin. These results suggest that translocation of the nascent RNA and RNA polymerase B along the DNA template is the enzymatic process inhibited by alpha-amanitin. It is also shown that the formation of a single phosphodiester bond by RNA polymerase B results in a stable ternary transcription complex, i.e., between the enzyme, the DNA, and the nascent RNA. Under reaction conditions which normally favor the elongation of RNA, the transcriptional process is arrested at initiation by alpha-amanitin. Such ternary initiation complexes have been isolated by agarose gel electrophoresis.

The effect of the chemical nature of the side chains of amatoxins in the inhibition of eukaryotic RNA polymerase B.

The inhibition constants (Ki) of DNA-dependent RNA polymerase B (or II) from calf thymus were measured for eight synthetically obtained bicyclic amanitin-like thioethers, two R-sulfoxides, and two S-sulfoxides. These Ki values were compared with those of alpha-amanitin, its 6'-O-methylether Ia (an R-sulfoxide), the S-sulfoxide, the sulfone, the S-deoxo derivative (Id) of Ia, and several previously described amatoxins. The necessity of a beta-methyl side chain in position 3 and a hydroxy group in proline-2 was confirmed. Additionally, the presence of an isoleucine side chain in position 6 and the absence of a side chain in position 5 was recognized as important for binding to the enzyme. In the three sulfoxide samples examined, the R-diastereomer was found to be a stronger inhibitor than the S-form. The contribution of every structural element to biological activity has been discussed.

Purification and characterization of RNA polymerase II resistant to alpha-amanitin from the mushroom Agaricus bisporus.

The DNA-dependent RNA polymerases II or B (ribonucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) from the mushroom Agaricus bisporus has been purified to apparent homogeneity. The purification procedures involve precipitation with polyethylenimine, selective elution of RNA polymerase II from the polyethylenimine precipitate, ammonium sulfate fractionation, DEAE-cellulose chromatography, CM-cellulose chromatography, and exclusion chromatography on Bio-Gel A-1.5M. With this procedure 11 mg of RNA polymerase II is recovered from 1.5 kg of mushroom tissue. RNA polymerase II from Agaricus bisporus has 12 subunits with the following molecular weights: 182,000, 140,000, 89,000, 69,000, 53,000, 41,000, 37,000, 31,000, 29,000, 25,000, 19,000, and 16,500. Purified RNA polymerase II from Agaricus bisporous was half-maximally inhibited by the mushroom toxin alpha-amanitin at a concentration of 6.5 microgram/mL (7 X 10(-6) M), which is 650-fold more resistant than mammalian RNA polymerases II. The apparent Ki for the alpha-amanitin-RNA polymerase complex was estimated to be 12 X 10(-6) M. The activity of purified RNA polymerase II from the mushroom was quite typical of other eukaryotic RNA polymerase II with regard to template preference, salt optima, and divalent metal cation optima.

The insensitivity of mushroom nuclear RNA polymerase activity to inhibition by amatoxins.

Nuclear fractions isolated from Amanita phalloides, Amanita muscaria and Agaricus bisporus were subjected to in vitro RNA synthesis assays in the presence of various concentrations of amatoxins. The mushroom nuclei were highly insensitive to inhibition by amatoxin when compared to assays of nuclear fractions isolated from the Oömycete fungus, Achlya ambisexualis and from rabbit brain.