Thomas C. Spelsberg

Chromatin of the developing chick oviduct: Changes in the acidic proteins

Abstract 1. The chromatin of the oviducts of immature chicks undergoing estrogen-induced differentiation was studied. Both the species and quantitative level of the histones remained relatively unaltered throughout development. In contrast, the level of the acidic proteins displayed a biphasic pattern, increasing during the first few days of differentiation followed by a decrease during the final stages of maturation. The level of chromatin-associated RNA and the capacities of chromatin to serve as template in in vitro DNA-dependent RNA synthesis showed a similar biphasic pattern. 2. Fractionation of the acidic chromatin proteins into four groups followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated a marked heterogeneity of the acidic chromatin proteins. The majority of protein bands were associated with two of the four groups (AP1 and AP2). Quantitative analysis revealed that variations primarily in the level of one fraction (AP2) were responsible for the changes observed in the total chromatin acidic proteins. Amino acid composition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunochemical analysis demonstrated that alterations in the molecular species of acidic proteins occurred during development. Using circular dichroism to probe the secondary structure of whole and dehistonized chromatin, a gradual conformational change was observed in both the DNA and protein components during oviduct development. 3. The observed changes in chromatin structure and composition are suspected to be involved in the changing pattern of gene expression during development of the chick oviduct.

Rapid isolation of total acidic proteins from chromatin of various chick tissues

Abstract A method is described for the rapid isolation of the nuclear acidic proteins of chromatin. Bovine pancreatic deoxyribonuclease I is used to hydrolyze DNA of dehistonized chromatin into acid-soluble fragments. The acidic proteins are recovered with more than 95% yield. Characterization of the isolated acidic proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis reveals reproducible heterogeneous banding patterns which are specific for the following chick tissues: liver, spleen, erythrocyte and heart, as well as oviduct at various stages of development. [3H]Ovalbumin added to dehistonized chromatin is not degraded throughout the procedure demonstrating that the multiple bands are not due to proteolytic activity.

Alterations in liver chromatin during perinatal development of the rat

Abstract Chromatins were isolated from liver nuclei of 19-day fetuses, 2-, 5-, 21-day old and adult rats. Very little variation was observed in the mass ratio of total histones to DNA or in the spectrum of histones as determined by polyacrylamide gel electrophoresis. On the other hand, the amount and banding pattern of acidic proteins indicated pronounced changes during liver development. The composition of acidic proteins may be specific for the stage of development as evidenced immunochemically. Antibody against acidic protein-DNA complexes from adult rat liver were produced in rabbits. Whereas adult liver acidic protein-DNA complexes interacted strongly with the antibody, fetal liver preparations showed very little affinity. Complexes from 2-day-old animals reacted more strongly than fetal complexes while preparations from 5-day-old and 21-day-old displayed further increases in affinity. The results support the idea that chromatin acidic proteins play an important role in genetic expression during the ontogeny.

Mammalian RNA polymerases I and II: Independent diurnal variations in activity

Abstract Diurnal variations in the activities of nucleolar (I) and non-nucleolar (II) DNA-dependent RNA polymerases of rat liver were observed. Both polymerase I and II displayed diphasic shifts in activity levels. However, the patterns of the individual shifts were not synchronous. Polymerase I activity was highest between 0100–1100 hours and lowest between 1700–2000 hours while polymerase II peaks between 2400 and 0500 hours with a low point at 1000 hours. The daily fluctuations and the dissimilar patterns of polymerase I and II activity may be of such significance as to require reevaluation and redesign of studies dealing with regulation of enzyme activity.

Changes in chromatin composition and hormone binding during chick oviduct development

Abstract 1. 1. Previous studies have demonstrated that progesterone, complexed with the oviduct receptor protein, is extensively bound by the deoxyribonucleoproteins (chromatin) of the mature chick oviduct. The chromatin of other tissues of the chick did not display this extensive binding. Results of studies presented here show that the progesterone receptor complex is extensively bound by the chromatin of the chick oviduct at all stages of development. 2. 2. Analyses of the chromatins indicate that the levels of nonhistone (acidic) protein and RNA as well as the ability of the chromatins to serve as a template for DNA-dependent RNA synthesis are altered in a specific pattern during differentiation of the chick oviduct. Differences in the levels of histones were less and showed no specific pattern. The capacity of the oviduct chromatins to bind the progesterone-receptor complex also varies throughout the development of the oviduct. The extent of binding the hormone receptor complex appears to be correlated with the levels of acidic proteins and RNA of the oviduct chromatins. 3. 3. Since chromatin of the undifferentiated oviduct binds the progesterone-receptor complex more extensively than the chromatins of other tissues, e.g., spleen or erythrocyte, it is speculated that the hormone binding capacity is not established during the estrogen induced maturation of the oviduct but must be established earlier at some stage of organogenesis of the chick oviduct.

Tissue specific binding invitro of progesterone-receptor to the chromatins of chick tissues

Summary When H 3 -progesterone is incubated with chick oviduct cytosol, it binds to a specific receptor protein. The subsequent binding of this complex to oviduct chromatin is greater than that of free progesterone alone. This specific affinity for the progesterone-receptor complex is not observed with spleen, heart, liver or erythrocyte chromatin. Progesterone incubated with the cytosols of non-target tissues such as liver or spleen shows no specific binding to chromatin from any source. Artificial complexes of chick histone and DNA display a low degree of non-specific binding. The results suggest that the chromatin of target tissue (oviduct) may contain “acceptor” sites for the hormone-receptor complex contained in the cytoplasm of the cell. The genome may thus be preprogrammed to receive the hormone receptor complex as it enters the nucleus of the target cell.

Hormone-Receptor Interactions with the Genome of Eucaryotic Target Cells

Progesterone induces de novo synthesis of the specific protein avidin in estrogen-primed chick oviducts. The specificity and kinetics of induction of this protein has been the subject of a series of past publications from our laboratory (1–6). We have previously shown that progesterone causes major quantitative and qualitative alterations in oviduct gene transcription. Induction of avidin was blocked by actinomycin D. Changes in rapidly-labeled nuclear RNA and nuclear RNA polymerase activities supported a transcriptional effect of the hormone (4–7).

Structural alterations of acidic proteins by acid treatment of chromatin

SummaryRecent studies into the properties and biological function of the acidic (non-histone) chromatin proteins have utilized inorganic or organic acids to first remove the histones prior to analysis of the acidic proteins. Examination of the effects of the acid treatment on the DNA and acidic proteins by immunochemistry, circular dichroism, and the ability of the DNA to serve as a template in thein vitro DNA-dependent RNA synthesis, has demonstrated a marked structural change (denaturation) in the proteins and DNA after the acid treatment. Other methods of removing histones, e.g., by high salt or salt and urea, are recommended for studies, especially for those of the biological functions, of the DNA and acidic proteins.

A specific oviduct target-tissue receptor for progesterone: identification characterization partial purification inter-compartmental transfer kinetics and specific interaction with the genome.

Researchers at the Vanderbilt University School of Medicine have used the chick oviduct in many of their efforts to understand the mechanism of steroid hormone action. In this paper they describe their efforts to establish the existence and characteristics of a specific receptor for progesterone in target cells and to relate the initial hormone-receptor interactions to subsequent alterations in RNA synthesis induced in the oviduct cell nucleus. Areas treated include progesterone binding to macromolecules following in vivo administration characterization of macromolecular-progesterone complex formed by oviduct cytosol in vitro partial purification of cytoplasmic complex evidence that binding components mediate progesterone action transfer of steroid-receptor complex from cytoplasm to nucleus and receptor interaction with the genome. OMalley leads a discussion further considering the nature of the progesterone receptor.