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Depletion of any of the essential Lsm proteins, Lsm2-5p or Lsm8p, delayed pre-rRNA processing and led to the accumulation of many aberrant processing intermediates, indicating that an Lsm complex is required to maintain the normally strict order of processing events. In addition, high levels of degradation products derived from both precursors and mature rRNAs accumulated in Lsm-depleted strains. Depletion of the essential Lsm proteins reduced the apparent processivity of both 5' and 3' exonuclease activities involved in 5.8S rRNA processing, and the degradation intermediates that accumulated were consistent with inefficient 5' and 3' degradation. Many, but not all, pre-rRNA species could be coprecipitated with tagged Lsm3p, but not with tagged Lsm1p or non-tagged control strains, suggesting their direct interaction with an Lsm2-8p complex. We propose that Lsm proteins facilitate RNA protein interactions and structural changes required during ribosomal subunit assembly.

The henny-feathering trait is an autosomal dominant mutation that causes the expression of aromatase activity and accumulation of aromatase mRNA in extragonadal tissues of chickens. The current studies establish that the aromatase gene is not amplified and is organized similarly in control (Leghorn) and henny-feathered (Sebright and Campine) birds. Nucleotide sequence analysis of the nine coding exons of the aromatase gene reveals that the predicted amino acid sequence is identical in all three strains. We, therefore, characterized the genomic DNA segments flanking the coding segment of the Sebright, Leghorn, and Campine aromatase genes. The site of transcription initiation utilized in the ovary of all three strains is located approximately 147 nucleotides upstream of the initiator methionine. In addition to aromatase mRNA derived from this common ovarian promoter, another species of aromatase mRNA is present in Sebright and Campine ovary and is the only type detected in Sebright fibroblasts. cDNA copies of this second species of aromatase mRNA contain a unique 5' terminus, suggesting that a second promoter controls extragonadal aromatase expression in birds that carry the henny-feathering trait. Nucleotide sequence analysis of this 5' terminus indicates that this segment is derived from a retroviral long terminal repeat.

The activity of 5alpha-reductase, the enzyme that converts testosterone to dihydrotestosterone, has been assessed in cell-free extracts of fibroblasts grown from foreskin, labia majora, scrotum, and nongenital skin from control subjects, from patients with developmental defects of the urogenital system, drom two subjects with the type 2 form of familial incomplete male pseudohermaphroditism and from individuals with other forms of hereditary male pseudohermaphoditism. Enzyme activity was shown to be maximal in the pH range of 5 to 6. Substrate specificity studies indicated that the enzyme so assayed is the 5alpha-reductase previously characterized in human foreskin. The activity of the enzyme was low in normal fibroblasts grown from nongenital skin and high in most fibroblasts grown from genital skin. 5alpha-Reductase activity in extracts of foreskin fibroblasts from two subjects with the type 2 disorder was undetectable at pH 5.5. Activity in comparable fibroblast extracts from most patients with other forms of hereditary male pseudohermaphroditism was easily measurable.

Regulation of the in vitro synthesis of the serum amyloid proteins A and P has been studied with hepatocyte cultures from CBA/J and C3H/HeJ mice. Liver cells were isolated by the collagenase perfusion technique and established for 48 h in the presence of fetal calf serum. Viable cells could then be maintained in the absence of serum for at least 72 h and in the presence of serum for up to 2 weeks. Serum amyloid A synthesis differed from serum amyloid P synthesis in three significant ways. 1) Serum amyloid A production was stimulated in the presence of fetal calf serum, whereas serum amyloid P was not; 2) serum amyloid A synthesis was increased 200% by monokine-rich macrophage supernatants while serum amyloid P was increased only 10 to 20%; 3) serum amyloid A synthesis was strikingly resistant to cycloheximide inhibition as compared with serum amyloid P and other liver proteins. It is concluded that the in vivo asynchrony of the acute phase serum amyloid A and serum amyloid P responses results at least in part from differences in regulatory mechanisms for their synthesis by hepatocytes.

A complementary DNA (cDNA) copy of the aromatase P-450 has been isolated from a chicken ovary library using as probe a partial cDNA believed to encode the human placental aromatase. The predicted amino acid sequence of the chicken aromatase cDNA possesses regions of homology to that of its human counterpart, but only limited homology to other cytochrome P-450 enzymes. The introduction of the cDNA clone into COS-1 cells results in the production of high levels of aromatase activity. The chicken enzyme is targeted to the appropriate subcellular fraction in the transfected COS cells, and the apparent Km of the chicken aromatase activity, measured in microsomes prepared from the transfected cells, is similar to that of the enzyme prepared from chicken ovary microsomes. These findings establish that the cDNA clone encodes chicken ovarian aromatase and demonstrate that this protein can catalyze the three successive oxidation reactions necessary to form estrogen from androgen.

The henny feathering trait in chickens leads to a marked increase in the conversion of androgen to estrogen in skin and other peripheral tissues with the result that feathers of affected males are feminized. To gain insight into the mechanisms responsible for this increased estrogen synthesis, we studied the conversion of testosterone to estrogen in fibroblasts cultured from the skin of control chickens and from two breeds carrying the henny feathering trait, the Sebright bantam and the Campine. Estrogen synthesis was measured in suspensions of intact fibroblasts and in cell-free fibroblast extracts by two assays: 1) direct measurement of 17 beta-estradiol formation from [1,2,6,7-3H]testosterone, and 2) assessment of 3H2O release from [1 beta-3H]testosterone. Both assays gave comparable results. Estrogen formation was as much as several hundred-fold higher in fibroblasts cultured from skin of chickens carrying the henny feathering trait compared to that observed in fibroblasts from skin of control chickens. The current data indicate that increased estrogen formation in skin of chickens with the henny feathering trait is due to an enhanced activity of the aromatase complex of enzymes responsible for estrogen synthesis. The molecular basis for this increased activity is unclear.

Steroid 5 alpha-reductase in skin fibroblasts varies in two ways: 1) mean activity in uncloned fibroblasts corresponds to the activity in skin from which fibroblasts are derived (high in genital skin and low in nongenital skin); 2) activity in normal genital skin fibroblasts varies over a wide range (more than 200-fold). Studies of activity in fibroblast clones provides an explanation for this variability. Activity in clones of genital skin fibroblasts varies from the limits of detectability to very high levels, whereas activity in fibroblasts cloned from nongenital skin is uniformly low. The mean value of 5 alpha-reductase in genital skin clones is similar to the value in uncloned fibroblasts from the same explant. This suggests that the numbers of high and low activity cells in a given explant influence the overall enzyme activity. Variability among normal genital skin strains is also due to inherent differences in the range of enzyme activities. Furthermore, high activity genital skin clones give rise to a mixture of both high and low-activity subclones, whereas low activity genital skin and nongenital skin clones give rise to populations with uniformly low activity. Thus, with time genital skin gives rise to high activity fibroblasts that continually undergo shift in phenotypic expression to low activity.

The metabolism of progesterone-1,2-3H has been studied in the estrogen-primed pullet. Following the intravenous administration of the hormone, radioactivity was recovered within 5 min in oviduct magnum, and at all time intervals up to 1 hour approximately one-fourth of the magnum radioactivity was localized within the nuclei of this tissue. Although the metabolism of progesterone-3H to a variety of metabolites is rapid in all tissues studied, progesterone itself and its 5α-reduced derivative allopregnanedione are the principal compounds identified in magnum cytoplasm and the only labeled steroids recovered from oviduct nuclei. The conversion of progesterone to allopregnanedione was shown to take place in magnum nuclei, and this metabolite of progesterone was recovered in significant amounts only in the magnum and shell gland of the oviduct, the comb, and the brain following the intravenous administration of progesterone-1,2-3H. At all times studied progesterone itself is the predominant species (80% or greater) bound to nuclei obtained from the magnum, whereas allopregnanedione is the principal radioactive steroid bound to nuclei in the shell gland of the oviduct.

Membranes have been isolated from fresh rectal glands of the dogfish shark, Squalus acanthias, in which the specific activity of the (sodium + potassium)-activated adenosine triphosphatase (NaK ATPase) is as high as 400 µmoles of Pi per mg of protein per hour. Membranes isolated from frozen rectal glands have only one-fourth to one-third this specific activity. Solubilization of membranes from frozen glands with the nonionic detergent Lubrol WX activates the NaK ATPase 2-fold, but there is no activation of the enzyme with membranes from fresh glands. Purification of Lubrol extracts by zonal centrifugation and a novel ammonium sulfate fractionation gives an enzyme preparation with a specific activity of 1,500 µmoles of Pi per mg of protein per hour. The same specific activity and gel pattern is obtained with enzyme purified from membranes from either fresh or frozen glands. The loss of Lubrol activation of the enzyme from frozen glands occurs on zonal centrifugation, where free Lubrol is removed. The over-all yield of enzyme from the membrane stage is 70%. A mince of 10 rectal glands weighing about 10 to 15 g fresh weight yields about 20 to 30 mg of purified enzyme. The enzyme is completely stable at 0° for 10 days in either the membranous form or in the most highly purified form. It is stable on freezing at -70°. Disc gel electrophoresis shows a gradual elimination of proprotein bands on purification. At the final stage of purification, scanning of Coomassie blue-stained gels gives 72% of the protein as the 97,000 molecular weight catalytic subunit, 19% as the 55,000 molecular weight glycoprotein, and 8.5% as the protein running with the tracking dye. The catalytic subunit and the glycoprotein can be isolated in milligram quantities by solubilization and chromatography on Sephadex G-150. About 90% of the protein applied to the column can be recovered. Sephadex chromatography gives a protein composition of 66% for the catalytic subunit, 28% for the glycoprotein, and 5% for the protein running with the tracking dye. The catalytic subunit and the glycoprotein enrich more or less in parallel as purification proceeds. At the last stage of purification, both proteins are found in the form of vesicles, which bear a striking resemblance on negative staining to purified cytochrome oxidase. Electron-translucent rods and rings which are approximately 80 A in diameter are also formed with projections of about 35 to 55 A at regular intervals. These rods and rings resemble reconstituted oligomycin-sensitive mitochondrial ATPase. The projections appear to be more hydrophilic, and this, coupled with their size, suggests that they are the glycoprotein. Incubation of the enzyme with [γ-32P]ATP, magnesium, and sodium gives 4,080 pmoles of acyl phosphate per mg of protein, which is 2 to 3 times higher than the highest levels of phosphorylation previously reported, thus attesting to the high purity of the enzyme preparation (the level of phosphorylation is generally accepted as being proportional to the number of NaK ATPase molecules in the preparation). The turnover number of the preparation is 6,300 min-1, which is within the range found for most NaK ATPase preparations from different organs and species. Arguments are presented for and against the view that the glycoprotein is a subunit of the NaK ATPase. If the catalytic subunit of molecular weight of 97,000 and the glycoprotein of molecular weight of 55,000 are both integral components of the NaK ATPase, the enzyme is 90 to 95% pure. If the 97,000 molecular weight subunit is the only subunit in the enzyme, the present preparation is 66 to 72% pure.

Utilizing density gradient centrifugation, polyacrylamide gel electrophoresis, and equilibrium dialysis, the in vitro binding of [1,2-3H]dihydrotestosterone by cytoplasmic extracts of submandibular glands from normal mice was compared to that of mice carrying the X-linked gene for testicular feminization (Tfm). By these techniques it was shown that supernatant from Tfm animals (Tfm/Y) exhibit greater capacity and higher affinity for androgen binding than does supernatant from normal male mice. Since this high affinity binding was also demonstrable in preparations from heterozygous female (Tfm/X) but not from normal female mice (X/X), the abnormality appeared to be a valid and useful biochemical marker for the Tfm gene. Although the exact genetic mechanism that leads to this phenomenon is not clear, it is possible that the enhanced binding of androgen by submandibular gland cytoplasm may be involved in the pathogenesis of the male pseudohermaphroditism and the androgen resistance in the Tfm mouse.

The intranuclear binding of radioactive hormone in prostate after the intravenous administration of testosterone-1,2-3H to rats has been studied. Nuclei obtained by sucrose density gradient centrifugation were extracted with buffer containing 0.6 m NaCl, and the soluble radioactivity was separated into bound and free fractions by gel filtration on Sephadex G-25 or G-200. As early as 15 min after testosterone administration, the radioactivity recovered in the bound form was predominantly dihydrotestosterone. By gel filtration of nuclear extracts on Sephadex G-200 and by the use of digestive enzymes, it was shown that dihydrotestosterone was bound to an acidic nuclear protein. Finally, several characteristics of this binding phenomenon were defined; the binding was stable to freezing for as long as 8 days, stable to short term incubation at 20° but not at 37°, and partially stable to repeated gel filtration on Sephadex.

l-Ribulose 5-phosphate 4-epimerase from Aerobacter aerogenes was inactivated by treatment with EDTA and was reactivated to varying extents by the addition of divalent metal ions in the order: Mn++ g Co++ g Ni++ g Ca++ g Zn++ g Mg++. When optimal Mn++ was present, the homogeneous enzyme had a specific activity of 70 µmoles-1 min-1 mg of protein at 28° and pH 7.2. This value is about five times greater than that displayed by the crystalline enzyme as isolated and assayed in the absence of added metal ion. In other mechanistic studies, l-ribulose 5-phosphate 4-epimerase was found to be stable to treatment with sodium sulfite and arsenite in the presence of a thiol compound. It was also stable to sodium borohydride in the presence or absence of substrate. Further, a reaction of tetranitromethane with the enzyme-substrate complex could not be detected. Possible mechanisms for l-ribulose 5-phosphate 4-epimerase are discussed.

l-Ribulose 5-phosphate 4-epimerase (EC 5.1.3._.) from Aerobacter aerogenes was purified to homogeneity as determined by a constant specific activity with recrystallization, a single protein band and coincident activity peak in a polyacrylamide gel electrophoresis, and a constant molecular weight across the cell in high speed equilibrium ultracentrifugation. The latter method gave an approximate molecular weight of 1.14 x 105. No bound NAD+ was detected when (a) the purified enzyme was tested for NAD+ by microbiological assay, (b) the epimerase was purified from a nicotinic acid auxotroph grown on 14C-nicotinic acid, or (c) fluorescence and absorption spectra were measured. Activity measurements indicated that the enzyme does not require NAD+ for activity nor is the rate of epimerization enhanced by the presence of NAD+, NADH, NADP+, or NADPH. These results show that the mechanism of 4-epimerization for l-ribulose-5-phosphate and d-xylulose-5-phosphate differs from that for UDP-glucose and UDP-galactose. It is evident that the possibilities now include oxidation-reduction utilizing an as yet unrecognized acceptor or an entirely different mechanism.

The intranuclear localization of the 5α-reductase that converts testosterone to dihydrotestosterone in rat ventral prostate has been investigated in nuclei purified by sedimentation through 2.2 m sucrose. First, as the result of chemical analyses of the isolated nuclei, light microscopy studies, and an investigation of the subcellular distribution of several marker enzymes in this tissue, it was concluded that the nuclei prepared in this manner were free from major contamination with other cytoplasmic constituents. In keeping with previous studies of the enzyme approximately half the enzyme activity in rat prostate was found in the nucleus. Second, the nuclei were fragmented by exposure to sonic oscillation and subjected to centrifugation in previously formed cesium chloride density gradients. Under these conditions, all the enzyme activity was found to coincide with a visible band of turbidity at approximately d 1.23 to 1.27. On the basis of the buoyant density, the chemical composition of the fraction, and similar flotation patterns of NADH-cytochrome c reductase, a known enzyme of the nuclear membrane, it was concluded that the 5α-reductase in prostatic nuclei is located in a nuclear membrane. In the isolation of this nuclear membrane fraction a 90-fold purification of the enzyme has been achieved.

The binding of [1,2-3H]dihydrotestosterone by cytoplasmic extracts of submandibular glands from normal male mice and from mice carrying the X-linked gene for testicular feminization (Tfm/Y) has been studied by density gradient centrifugation and gel filtration. Androgen binding in this tissue was found exclusively in the portion of the gland that is known to contain androgen-dependent elements. No binding was demonstrated in the glands of either genotype immediately after birth, but at 2 weeks of age and after high affinity binding of the hormone was found in the glands of both male and Tfm animals. At all ages studied, the apparent dissociation constant for this binding was similar in the two genotypes (around 10 nm), but the number of binding sites in Tfm animals at 6 and 12 weeks was 3-fold higher. This difference did not appear to be the result of differential degradation during the preparation of the extracts. The observation that normal and Tfm animals have identical binding parameters at 2 weeks of age suggests: (a) that the postnatal development of androgen binding in submandibular gland is not androgen-dependent; (b) that androgen binding develops prior to the onset of sexual dimorphism of the gland; and (c) that the later development of enhanced binding of androgen by submandibular gland cytoplasm of the adult Tfm mouse is not caused by the appearance of an altered androgen binding protein but is the result of the accumulation of a physiologically normal binding protein.