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R G Deeley

Publications and source records attributed to R G Deeley.

123 records · Page 7Linked to original sources
PubMed1980

Coordinate regulation of two estrogen-dependent genes in avian liver.

Livers of egg-laying species contain abundant mRNAs encoded by both estrogen-responsive and constitutively expressed genes. We have recently constructed cDNA clones from three members of the abundant mRNA class of hen liver. One of these mRNA species was identified as serum albumin mRNA, and another as vitellogenin mRNA. In this study we have identified the third member of the group as apo VLDLII mRNA. Hybridization analyses using cloned cDNA probes indicate that expression of the apo VLDLII gene in rooster liver, like that of the vitellogenin gene, is completely dependent upon the administration of estrogen. The apo VLDLII and vitellogenin genes appear to be the only genes capable of high rates of expression in the liver that exhibit such an exceptional response to the hormone. Administration of estrogen resulted in the appearance of both mRNA species within 30 min, followed by a rapid accumulation to several thousand copies per cell. Removal of the hormone caused a marked destabilization of both vitellogenin mRNA and apo VLDLII mRNA. In contrast, the absolute levels of serum albumin mRNA were unaffected by the hormone. Comparative studies on the structure and organization of these three genes may reveal elements involved in determining their rates of expression in the presence and absence of estrogen.

Animals↗
PubMed1979

Specific transcription in chicken liver chromatin by endogenous RNA polymerase II. Comparison of an estrogen-inducible gene with a constitutively expressed gene.

We have developed a system for the in vitro transcription of specific genes in rooster liver chromatin by endogenous RNA polymerase II that maintains the specificity of transcription in vivo. Radioactive transcripts synthesized in vitro were identified and quantitated by hybridization to a vast excess of cloned cDNA. The cDNA preparations employed corresponded to vitellogenin mRNA, the synthesis of which is responsive to estrogen stimulation in vivo, and chicken serum albumin mRNA, the synthesis of which is not significantly affected by estrogen stimulation in vivo. Comparing the pattern of transcription of the albumin and vitellogenin genes in chromatin from the liver of the normal rooster with the pattern in chromatin from the liver of the estrogen-stimulated rooster, we found that prior estrogen treatment of the rooster is attended by a slight decrease in the differential rate of transcription of the albumin gene and approximately a 10-fold increase in the differential rate of transcription of the vitellogenin gene. Because this pattern of transcription reflects the estrogen-induced changes in transcription observed in vivo, chromatin preparations from the livers of normal and estrogen-stimulated roosters can be used to investigate regulation of specific gene transcription at the molecular level in vitro.

Amanitins↗
PubMed1978

Cloning of a double-stranded cDNA that codes for a portion of chicken preproalbumin. A general method for isolating a specific DNA sequence from partially purified mRNA.

A scheme is presented for cloning a double-stranded cDNA molecule that codes for a portion of chicken preproalbumin. This method, which does not require pure mRNA or cDNA, has widespread applicability. Chicken preproalbumin was identified as a Mr = 72,000 polypeptide by immunoprecipitation of proteins synehesized in a wheat germ cell-free translation system from total, guanidine.HCl-extracted, rooster liver RNA. After removal of the bulk of the ribosomal RNA by poly(U)-Sephadex G-10 chromatography, albumin mRNA was enriched approximately 2-fold by centrifugation through low salt, isokinetic sucrose gradients, until it represented about 30% of the mRNA sequences present. Double-stranded cDNA prepared from this mRNA was then inserted into the Pst 1 site of the plasmid PBR322 by the "G-C tailing" technique and the recombinant DNA was used to transform Echerichia coli stran X1776. Transformants containing putative albumin DNA sequences were identified by colony hybridization with a cDNA probe that was highly enriched for albumin cDNA sequences. This probe was isolated by hybridizing the partially purified RNA preparation to its cDNA, under conditions of RNA excess, to a R0t value such that only the most abundant cDNA sequences had hybridized. Unhybridized, less abundant, sequences were destroyed by subsequent S1 nuclease digestion. The identity of clones that hybridized to this abundant class cDNA was established by DNA-mRNA hybrid-arrested cell-free translation. Hybridization of nick-translated, albumin-containing, plasmid DNA to total liver poly(A)+ RNA, that had been separated on methyl mercury agarose gels and transferred to diazobenzyloxymethyl paper, established that avian albumin mRNA has a molecular weight of 850,000. This molecular weight corresponds to approximately 2,600 nucleotides, or 600 nucleotides longer than the size required to code for the preproalbumin polypeptide.

Animals↗
PubMed1978

Primary induction of vitellogenin mRNA in the rooster by 17beta-estradiol.

We have studied the kinetics of vitellogenin mRNA accumulation in rooster liver after a primary injection of 17beta-estradiol. The levels of vitellogenin mRNA have been determined both by hybridization of total cellular RNA to vitellogenin cDNA and by translation of vitellogenin mRNA in a wheat germ cell-free system. The results obtained by both methods of analysis are in good agreement and indicate that vitellogenin mRNA is present in the liver of normal roosters at a level of 0-5 molecules per liver cell and increases in amount during the 3 days following injection of estrogen, reaching a level of almost 6000 molecules per cell at the peak of the response. The level of vitellogenin mRNA declined exponentially during the next 14 days with a half-life of 29 hr, reaching a level of less than 10 molecules per cell at 17 days after injection of the hormone. The levels of vitellogenin mRNA after stimulation with estrogen have been correlated with the in vivo rate of synthesis of the vitellogenin polypeptide. The results indicate that the rate of vitellogenin synthesis is closely correlated with the level of vitellogenin mRNA. On the basis of these findings, we conclude that vitellogenin mRNA does not exist in the liver in an untranslated form after withdrawal from estrogen.

Animals↗
PubMed1977

Kinetics of avian vitellogenin messenger RNA induction. Comparison between primary and secondary response to estrogen.

Following either primary or secondary stimulation of cockerels with 17beta-estradiol, vitellogenin mRNA begins to accumulate in the liver after about 30 min, reaches a maximum in 3 days, and decays thereafter with a half-life of 30 h. During primary induction, accumulation of vitellogenin mRNA begins at a low rate (50 nucleotides/s/nuclear equivalent of DNA) and after 4 h, shifts to a higher rate (340 nucleotides/s/nuclear equivalent of DNA). In contrast, during secondary induction (administration of estrogen several weeks after the primary response has ceased), accumulation of vitellogenin mRNA begins at the rate of 350 nucleotides/s/nuclear equivalent of DNA and subsequently increases by about 40%. These accumulation rates result in a maximal level of vitellogenin mRNA that is approximately 1.5 times higher during secondary stimulation than that found during primary stimulation. This difference is sufficient to explain the anamnestic response to secondary hormonal stimulation that results in higher levels of circulating vitellogenin in the plasma of the rooster.

Animals↗
PubMed1977

In vitro translation of avian vitellogenin messenger RNA.

Administration of 17beta-estradiol to roosters induced the synthesis of vitellogenin in the liver. The mRNA that specifies this protein has been purified from the livers of estrogen-treated roosters and has been shown to have a molecular weight of 2.3 X 10(6) (Deeley, R.G., Gordon, J.I., Burns, A.T.H., Mullinix, K.P., Bina-Stein, M., and Goldberger R.F. (1977) J. Biol. Chem. 252, 8310-8319). In order to rigorously establish the identity of the polypeptide specified by this mRNA, we used a staphylococcal nuclease-treated, mRNA-dependent wheat germ cell-free translation system capable of synthesizing polypeptides as large as vitellogenin (monomer Mr = 240,000). Vitellogenin mRNA directs the in vitro synthesis of a polypeptide with the following features: (a) it co-migrates with authentic vitellogenin in SDS-polyacrylamide gels; (b) it is highly enriched for serine but is not phosphorylated; (c) it is immunoprecipitated by purified, monospecific, anti-vitellogenin antibody; and (d) it has an unusual cyanogen bromide cleavage pattern characteristic of vitellogenin. The most striking characteristic of the cyanogen bromide cleavage products is an extremely large polypeptide (Mr = 90,000) that contains two phosvitins. The kinetics of incorporation of serine and methionine into vitellogenin synthesized in the wheat germ cell-free translation system indicates that the phosvitins are located near the COOH-terminal portion of the molecule.

Animals↗
PubMed1976

Induction of vitellogenin synthesis by estrogen in avian liver: relationship between level of vitellogenin mRNA and vitellogenin synthesis.

We have investigated the estrogen-mediated induction of vitellogenin synthesis in rooster liver. We compared the concentrations of vitellogenin messenger RNA (mRNA) in the liver with the concentrations of vitellogenin in the sera of roosters that had recieved various treatments with estrogen. We found no vitellogenin mRNA in the livers of the unstimulated roosters. An initial injection of estrogen was attended by de novo synthesis of vitellogenin mRNA in the liver and accumulation of vitellogenin in the serum. When vitellogenin was no longer present in the serum or liver (the "post-estrogen-serum-negative" state), the liver was found to contain appreciable amounts of vitellogenin mRNA. This mRNA was of the same size as that found in the liver of the rooster actively synthesizing vitellogenin in response to estrogen. Whereas vitellogenin mRNA was in large polysomes in the livers of the roosters actively synthesizing vitellogenin, the vitellogenin mRNA in the liver of the post-estrogen-serum-negative rooster was not associated with polysomes. The possible relevance of these findings to the fact that the rooster responds differently to a primary stimulation with estrogen than to subsequent stimulations is discussed.

Animals↗
PubMed1975

Vitellogenin synthesis in the avian liver. Vitellogenin is the precursor of the egg yolk phosphoproteins.

Administration of estrogen to roosters induces the synthesis of egg yolk phosphoproteins in the liver. We have demonstrated that these proteins are synthesized in the form of a large precursor, vitellogenin, and that vitellogenin is the only phosphoprotein found in the plasma of the estrogen-treated rooster. Vitellogenin is cleaved to form the egg yolk phosphoproteins, lipovitellin, and phosvitin. We have purified vitellogenin to hemogeneity by two methods: chromatography on diethylaminoethyl-52-cellulose and affinity chromatography on an antibody-Sepharose column. Antibodies were elicited in rabbits and sheep by immunization with vitellogenin and lipovitellin, and these antibodies were purified by affinity chromatography on antigen-Sepharose columns. We found that phosvitin was not immunogenic in its native form or in any of the large variety of modified forms we have tested. We have determined the molecular weights of native and denatured vitellogenin and have examined the immunological relationship between vitellogenin and lipovitellin. On the basis of these studies, together with data from phosphate analyses, we suggest that avian vitellogenin is composed of two polypeptides, each of which has a molecular weight of approximately 240,000 and contains within it lipovitellin and two phosvitins.

Animals↗
PubMed1975

Effect of estrogen on gene expression: purification of vitellogenin messenger RNA.

We report initial studies on estrogen-mediated regulation of egg yolk protein synthesis in the rooster. Egg yolk proteins are normally synthesized as a large precursor, vitellogenin, in the liver of the laying hen; roosters synthesize vitellogenin only when treated with estrogen. Polysomal RNA from the liver of estrogen-treated roosters was translated in a reticulocyte cell-free system, and the newly synthesized proteins were identified by a highly specific and sensitive indirect immunoprecipitation reaction. The messenger RNA that specifies vitellogenin has been purified more than 800-fold from rooster liver polysomal RNA by a combination of methods, including immunoprecipitation of polysomes and chromatography of RNA on poly(U)-Sepharose.

Animals↗
PubMed1975

Specific binding of the first enzyme for histidine biosynthesis to the DNA of histidine operon.

Studies were done to examine direct binding of the first enzyme of the histidine biosynthetic pathway (phosphoribosyltransferase) to 32P-labeled phi80dhis DNA and competition of this binding by unlabeled homologous DNA and by various preparations of unlabeled heterologous DNA, including that from a defective phi80 bacteriophage carrying the histidine operon with a deletion of part of its operator region. Our findings show that phosphoribosyltransferase binds specifically to site in or near the regulatory region of the histidine operon. The stability of the complex formed by interaction of the enzyme with the DNA was markedly decreased by the substrates of the enzyme and was slightly increased by the allosteric inhibitor, histidine. These findings are consistent with previous data that indicate that phosphoribosyltransferase plays a role in regulating expression of the histidine operon.

ATP Phosphoribosyltransferase↗
PubMed1975

Interaction between phosphoribosyltransferase and purified histidine tRNA from wild type Salmonella typhimurium and a derepressed hisT mutant strain.

We have examined the interaction between phosphoribosyltransferase and purified tRNA-His from the wild type strain of Salmonella typhimurium, LT-2, and the histidine regulatory mutant hisTl504. Histidyl-tRNA from the mutant strain functions normally in protein synthesis but is defective in its role in the repression mechanism of the histidine operon. Phosphoribosyltransferase has been suggested as a possible aporegulator for this operon and as such might be expected to interact abnormally with tRNA-His from hisT1504. In these studies we have been unable to detect any difference between the affinities of phosphoribosyltransferase for tRNA-His from LT-2 or hisT1504, and thus we conclude that if the complex between phosphoribosyltransferase and histidyl-tRNA does function in regulation, the defect in the hisT1504 mutant must influence the interaction of the complex with some other regulatory element.

Adenosine Triphosphate↗
PubMed1973

Inhibition of transcription of the histidine operon in vitro by the first enzyme of the histidine pathway.

An in vitro system was developed for transcription of the histidine operon of Esherichia coli carried in the genome of a defective varphi80 transducing phage. The messenger RNA (mRNA) of the histidine operon synthesized in the in vitro system was detected by hybridization to single strands of both varphi80 and varphi80dhis DNA, and by competition of this hybridization with unlabeled histidine mRNA that had been synthesized in vivo (RNA extracted from cells in which the histidine operon had been derepressed). Under the conditions used, RNA complementary to the histidine operon was about 15% of the total RNA that was synthesized in vitro from the varphi80dhis DNA template. The RNA complementary to the histidine operon was synthesized on the "sense" strand (the R strand) of varphi80dhis in the form of a polycistronic message with a sedimentation coefficient (about 38 S) very close to that observed for the histidine mRNA synthesized in vivo. Synthesis of the histidine operon RNA appears to be subject to control in vitro. Addition of the first enzyme of the pathway for histidine biosynthesis blocked transcription of the histidine operon specifically, strongly suggesting that this enzyme acts as a regulatory protein for the histidine operon.

Cell-Free System↗