Biomedical subjects

R G Deeley

Publications and source records attributed to R G Deeley.

At least 109 records · Page 6Linked to original sources

PubMed1990

Developmental regulation of specific protein interactions with an enhancerlike binding site far upstream from the avian very-low-density apolipoprotein II gene.

Expression of the avian very-low-density apolipoprotein II (apoVLDLII) gene is completely dependent on estrogen and restricted to the liver. We have identified binding sites for nonhistone nuclear proteins located between -1.96 and -2.61 kilobases. One of these sites, located at -2.6 kilobases (designated site 1), was found to span an MspI site that becomes demethylated between days 7 and 9 of embryogenesis, the stage of development at which competence to express the apoVLDLII gene begins to be acquired. Levels of the factor(s) involved were high at day 7 of embryogenesis, decreased two- to threefold by days 9 to 11, and continued to decline more slowly until hatching. Furthermore, the mobility of the complex formed underwent a well-defined shift between days 11 to 13 embryogenesis. Methylation interference studies showed that modification of the outer guanosines of the MspI site resulted in marked inhibition of the formation of the protein-DNA complex. Competition studies, fractionation of nuclear extracts, and tissue distribution indicated that the factor was not the avian homolog of hepatocyte nuclear factor 1, nuclear factor 1, or CCAAT/enhancer-binding protein (C/EBP). However, site 1 could complete for binding to an oligonucleotide, previously shown to be recognized by C/EBP, in a nonreciprocal fashion. These studies demonstrate that the sequence recognized by the protein includes a C/EBP consensus sequence but that elements in addition to the core enhancer motif are essential for binding.

Age Factors↗

PubMed1989

Detection and characterization of degradative intermediates of avian apo very low density lipoprotein II mRNA present in estrogen-treated birds and following destabilization by hormone withdrawal.

The apo very low density lipoprotein II (apoVLDLII) gene is dormant in embryos, chicks, and roosters but can be activated by estrogen. ApoVLDLII mRNA is relatively stable in estrogen-treated birds. However, its stability decreases 4-5-fold following withdrawal of hormone. We have characterized degradative intermediates of apoVLDLII mRNA detected in liver total RNA from estrogen-treated birds and searched for alterations in the pattern of intermediates that occur upon hormone-withdrawal. Primer extension and S1 nuclease analyses have demonstrated that these intermediates consist of fragments of the molecule with intact 5' ends but which lack various 3' regions. Estrogen withdrawal results in a decrease in the steady state levels of several of these intermediates and the detection of two new species. The end points of the major fragments present in RNA from both estrogen-treated and withdrawn birds all map in, or within four nucleotides of, the tetranucleotide, GAUG. The two fragments detected only in RNA from withdrawn birds have 3' ends that immediately precede the sequence, CAGU. Based on secondary structures predicted by a global folding program, the end points also appear to be preferentially located in, or at the base of, internal "bulge-loops".

Animals↗

PubMed1988

Estrogen-dependent activation of the avian very low density apolipoprotein II and vitellogenin genes. Transient alterations in mRNA polyadenylation and stability early during induction.

Administration of estrogen to egg-laying vertebrates activates unscheduled, hepatic expression of major, egg-yolk protein genes in immature animals and mature males. Two avian yolk protein genes, encoding very low density apolipoprotein II (apoVLDLII) and vitellogenin II, are dormant prior to stimulation with estrogen, but within three days their cognate mRNAs accumulate to become two of the most abundant species in the liver. Accumulation of these mRNAs has been attributed to both induction of transcription and selective, estrogen-dependent mRNA stabilization. We have detected alterations in the size of apoVLDLII mRNA that occur during the first 24 hours that are attributable to a shift in the extent of polyadenylation as steady-state is approached. In vitro transcription assays indicate that primary activation of both genes takes place relatively slowly and that maximal rates of mRNA accumulation occur when the apoVLDLII and vitellogenin II genes are expressed at only 30% and 10% of their fully induced levels, respectively. Transcription data combined with the structural alteration of apoVLDLII mRNA suggest that stability of the two mRNAs may change as steady-state is approached. We have assessed the compatibility of this suggestion with earlier estimates of the kinetics of accumulation of both mRNAs by developing a generally useful algorithm that predicts approach to steady-state kinetics under conditions where both the rate of synthesis and mRNA stability change throughout the accumulation phase of the response. The results predict that the stability of both mRNAs decreases by at least two- to threefold during the approach to steady-state and that, although an additional destabilization of apoVLDLII mRNA may occur following withdrawal of estrogen, the steady-state stability of vitellogenin mRNA is not significantly decreased upon removal of hormone.

Animals↗

PubMed1988

Organization, sequence and nuclease hypersensitivity of repetitive elements flanking the chicken apoVLDLII gene: extended sequence similarity to elements flanking the chicken vitellogenin gene.

Analysis of nuclease hypersensitivity of regions flanking the estrogen-dependent, chicken apoVLDLII gene has revealed an hepatic, DNaseI hypersensitive site whose sensitivity is influenced by both the developmental stage and sex of the bird. The site is located 3.0kb upstream from the gene, in a block of middle repetitive elements. Contact hybridization studies indicate that the block consists of contiguous copies of two elements with reiteration frequencies of 500-1000 and 10,000-30,000 copies per haploid genome. Sequencing of 1.8kb spanning the repeats has revealed that the higher frequency element is a member of the CR1 family. The adjacent lower frequency repeat can also be found next to another member of the CR1 family located in the 3' flanking region of the vitellogenin gene. The hypersensitive site has been mapped to one of the two most highly conserved regions of the CR1 element. This region displays homology with a silencer sequence recently identified in a CR1 element flanking the chicken lysozyme gene.

Animals↗

PubMed1988

25-Hydroxylation of vitamin D3 in the human hepatoma cell lines Hep G2 and Hep 3B.

Two human hepatoma cell lines, Hep G2 and Hep 3B, were screened for vitamin D3-25-hydroxylase enzyme activity by incubation with radioactive vitamin D3. A compound co-chromatographing with 25-OH-D3 was synthesized in both cell lines but its rate of synthesis was tenfold greater in Hep 3B than in Hep G2 cells. The identity of the compound was confirmed by comparing its chromatographic properties with authentic 25-OH-D3 on three different high pressure liquid chromatography systems. Its production was suppressed by adding fetal calf serum (10%), lipoprotein-deficient fetal calf serum, or pure vitamin D-binding globulin to the medium. The mechanism of action of these plasma proteins appears to involve retardation of uptake of the substrate. These two cell lines offer considerable potential as defined in vitro models for studying the effects of physiological factors on the 25-hydroxylation of vitamin D3.

Animals↗

PubMed1987

Structure and evolution of primate cytochrome c oxidase subunit II gene.

The sequence of cytochrome oxidase subunit II (COII) mRNA from the cynomolgus macaque has been determined. Availability of the sequence from a non-human primate has allowed examination of the evolution of the COII gene and protein along the primate lineage. Comparison with existing protein and DNA sequences, combined with estimates of divergence derived from calculations designed to compensate for multiple mutation and reversion events, indicates that although the rate of fixation of nucleotide substitutions at silent sites is somewhat lower in primates than non-primates, the rate of fixation at replacement sites is 4-5-fold higher. The data also suggest that the rate of divergence at replacement sites along the primate lineage has not been uniform, but has decreased 2-2.5-fold since the higher primate branch point, in the absence of a comparable change in the rate substitution at silent sites. Both primate mRNAs differ from their non-primate homologues in having 3'-untranslated regions of 20-25 nucleotides. Examination of the monkey and human untranslated sequences suggests that these regions have evolved by duplication events occurring in both cases within 2-3 nucleotides following the translational stop codon. The primate mRNAs are also exceptional in that both can form stable stem and loop structures immediately preceding the postulated duplication site that may have played a role in facilitating the mutational events involved. Comparison of the human and monkey protein sequences has revealed regions conserved in primates that are significantly more hydrophobic than their non-primate counterparts. The possible effects of these alterations on the interaction between COII and cytochrome c are discussed.

Amino Acid Sequence↗

PubMed1987

Long-term effects of estrogen on avian liver: estrogen-inducible switch in expression of nuclear, hormone-binding proteins.

The stimulation of chicks or embryos with estrogen results in transient, hepatic expression of the vitellogenin gene, as well as long-term, propagatable alterations in the rapidity with which the gene can be reactivated. We examined the possibility that nuclear, type II estrogen-binding sites are involved in this long-term change in response characteristics. We demonstrate that the primary induction kinetics of type II sites in embryos and chicks correlated with the expression of the vitellogenin gene and that once their induction was triggered by estrogen, they accumulated, were propagated, and persisted for months after withdrawal of the hormone. We also show that their accumulation in the embryo was accompanied by prolonged expression of both the vitellogenin and very low-density apolipoprotein II genes, in the absence of elevated levels of type I receptor, and that the type II sites, like the classical receptor, appear to be preferentially associated with active or potentially active chromatin. Finally, we describe a regulatory mechanism, tested by computer modelling, that simulated the behavioral characteristics of these nuclear estrogen-binding sites and which may explain their role in mediating the long-term effects of estrogen.

Age Factors↗

PubMed1986

Estrogen memory effect in human hepatocytes during repeated cell division without hormone.

Transient stimulation of target tissues by sex steroids can cause long-lasting changes that may facilitate or alter responses to subsequent hormonal treatment. How these altered characteristics are propagated during cell division in the absence of the stimulating hormone is unknown. The human hepatocarcinoma cell line HepG2 was used as a model to examine the effects of estrogen on the synthesis of serum apolipoproteins in vitro. Treatment with low concentrations of estrogen for 24 to 48 hours resulted in long-lasting alterations in the kinetics with which the cells responded to subsequent stimulation with estrogen. Manifestation of this memory effect was correlated quantitatively with the induction and propagation of a moderate-affinity, nuclear, estrogen-binding protein with the characteristics of a type II estrogen receptor. The data indicate that transient exposure of these cells to estrogen can induce changes in their response characteristics and composition of nuclear proteins that are inherited by daughter cells grown in the absence of hormone for more than ten generations.

Animals↗

PubMed1986

Kinetics of estrogen-dependent modulation of apolipoprotein A-I synthesis in human hepatoma cells.

Apolipoprotein (apo) A-I is the principal protein component of high density lipoproteins and the major in vivo activator of lecithin-cholesterol acyltransferase. We have used the human hepatoma cell line, HepG2, as a model to examine the ability of estrogen to modulate hepatic synthesis of apo-A-I. Primer extension studies have demonstrated that the major transcriptional initiation site of the apo-A-I gene utilized in the hepatoma cells is the same as that used in human liver. The kinetics of induction of high- and low-affinity estrogen-binding sites, rates of secretion of apolipoproteins, and apo-A-I mRNA levels were examined following treatment of the cells with estrogen. Initial concentrations of 20 nM 17 beta-estradiol resulted in a 14-15-fold increase in the levels of high-affinity nuclear estrogen-binding sites within 8 h, while the level of low-affinity sites increased by only 10%. During the same period, the levels of apo-A-I mRNA and the rate of accumulation of the secreted protein increased by 55 and 50%, respectively. New steady state levels of apo-A-I mRNA and rates of accumulation of protein, approximately twice those in control cultures, were established within 24-48 h of exposure to hormone. Experiments with a 50-fold higher concentration of estrogen resulted in only an additional 10% increase in mRNA levels. The increase in mRNA levels following estrogen treatment was adequate to account for 85-90% of the elevation observed in the rate of accumulation of secreted apo-A-I. Comparison of the apo-A-I mRNA levels in HepG2 cells with those present in human liver revealed that the concentration of the mRNA was approximately 3-fold lower than that found in vivo.

Apolipoprotein A-I↗

PubMed1986

Biphasic effects of estrogen on apolipoprotein synthesis in human hepatoma cells: mechanism of antagonism by testosterone.

Treatment of HepG2 cells with various concentrations of 17 beta-estradiol has revealed two distinct thresholds for induction of different apolipoproteins. Maximal increases in apolipoprotein AI and CII (apoAI and apoCII) secretion can be obtained with initial concentrations of hormone of 20 nM or greater, while a similar induction of apoB and apoE requires in excess of 500 nM. Both responses involve alterations in the concentrations of apolipoprotein mRNAs. Analyses of the kinetics of accumulation of the apolipoproteins in response to high concentrations of hormone indicate that induction of apoB and apoE occurs coordinately, but it lags behind that of apoAI and apoCII by 5-6 hr. This lag can be eliminated by preexposing the cells to low concentrations of hormone. The ability to induce apoAI and apoCII and the kinetics with which they respond to low levels of estrogen correlate with levels of nuclear type I estrogen binding sites, while increases in apoE and apoB synthesis in response to high concentrations of hormone correlate with the induction of type II sites. Testosterone alone has no effect on the rates of apolipoprotein secretion, but it does increase the concentration of estrogen required to maximally induce apoCII and apoAI by a mechanism that involves high-affinity androgen receptors. This effect may be attributable to the testosterone-dependent induction of a cytoplasmic moderate-affinity estrogen-binding component.

Animals↗

PubMed1985

Effects of estrogen on apolipoprotein secretion by the human hepatocarcinoma cell line, HepG2.

We have examined the effect of estrogen on the rate of accumulation of apolipoproteins secreted by the human hepatocarcinoma cell line, HepG2. Prior to exposure to hormone, we detected less than 300 high-affinity, nuclear, estrogen-binding sites/cell. Within 48 h of growth in the presence of 20 nM 17 beta-estradiol this number rose to 3000-3500 sites/cell. Rates of accumulation of two of the major apolipoproteins, apo-C-II and apo-A-I increased 2.5- and 2.0-fold, respectively, in response to estrogen treatment. Other major apolipoproteins were not affected at this concentration of hormone. Induction of both proteins was completely antagonized by 20 nM testosterone. The density distribution of apolipoproteins secreted by the hepatocytes was similar to that reported using perfused liver systems. The consequences of estrogen treatment were to increase the apo-C-II/apo-C-III ratio in very low density lipoproteins as well as to decrease the overall very low density lipoprotein:high density lipoprotein ratio.

Apolipoprotein A-I↗

PubMed1985

Apolipoprotein C-II mRNA levels in primate liver. Induction by estrogen in the human hepatocarcinoma cell line, HepG2.

We have demonstrated that physiological concentrations of 17 beta-estradiol increase nuclear estrogen-specific binding sites in the human hepatoma cell line HepG2 7- to 10-fold and the rate of accumulation of secreted apolipoprotein C-II (apo-C-II), 2.5-fold (Tam, S-P., Archer, T. K., and Deeley, R. G. (1985) J. Biol. Chem. 260, 1670-1675). Apo-C-II is the major activator of lipoprotein lipase, an enzyme which plays a key role in lipoprotein catabolism. In order to define more precisely the mechanism by which estrogen influences apo-C-II production, we have synthesized a triacontanucleotide DNA probe that is complementary to apo-C-II mRNA. We have used the probe both in Northern hybridization experiments and in DNA excess titrations to quantify apo-C-II mRNA in hormonally treated HepG2 cells and various primate tissues. These studies revealed that: 1) the concentration of apo-C-II mRNA in HepG2 cells is comparable with that present in human liver; 2) treatment of the cells with low levels of estrogen results in a doubling of the apo-C-II mRNA concentration; 3) the apo-C-II mRNA concentration in monkey liver is 60- to 70-fold greater than in the intestine and 2.5-fold higher than in human liver.

Animals↗

PubMed1985

The effects of estrogen on apolipoprotein synthesis.

Sex steroids are important regulators of apolipoprotein synthesis and metabolism in a wide variety of species. In man, epidemiological studies have established strong correlations between sex steroid levels, circulating apolipoprotein profiles, and atherosclerotic risk. In this review, we compare data obtained from studies on two model systems that are being used to examine molecular mechanisms by which estrogens and androgens may influence hepatic lipoprotein synthesis. The first involves estrogen-dependent induction of lipoprotein synthesis in avian liver and the second involves estrogen-dependent modulation of lipoprotein synthesis in the human hepatocarcinoma cell line HepG2.

Animals↗

PubMed1984

Estrogen-dependent modification of ribosomal proteins. Effects of estrogen withdrawal on the distribution of constitutive and hormonally regulated mRNAs.

Estrogen-dependent modification of ribosomal proteins during induction of egg-yolk protein synthesis in avian liver was examined in vivo and in cultured hepatocytes. Modification of two proteins of the 40S ribosomal subunit was detected in vivo, within 40 min of injection of hormone. One of the proteins was identified as S6 and the other, tentatively, as S3a. Estrogen treatment resulted in the appearance of multiple, phosphorylated forms of S6 and a shift in electrophoretic mobility of the other protein that was consistent with its dephosphorylation. The steady state achieved within 2 h of injection could be maintained for up to 2 weeks when the hormone was administered from silastic implants. Removal of the implants resulted in a return to the preinduction state within 20-40 min. Similar modifications were induced in hepatocytes maintained in defined medium, with 17 beta-estradiol as the only hormonal supplement. In order to check on the possibility that the modifications observed could selectively influence mRNA utilization, the cytoplasmic distributions of two abundant mRNAs were monitored during the first few hours following withdrawal. One of these was serum albumin mRNA, the levels of which are unaffected by estrogen. The other was very-low-density apolipoprotein II mRNA which specifies a major egg-yolk protein. The synthesis of this mRNA is absolutely dependent on estrogen and its half-life is also markedly affected by the hormone.

Animals↗

PubMed1983

Selective block of albumin gene expression in chick embryo hepatocytes cultured without hormones and its partial reversal by insulin.

Rates of synthesis of albumin and transferrin were compared with the levels of their cognate mRNAs in primary monolayer cultures of chick embryo hepatocytes over a period of 72 h. These liver cells were exposed, from the onset of culture, only to a chemically defined medium devoid of hormonal or macromolecular supplement. Synthesis of transferrin was constant whereas synthesis of albumin diminished to near 0 in 3 days. RNA prepared from hepatocyte monolayers, maintained for various lengths of time in culture, was translated in wheat germ extracts and Xenopus oocytes. Translation products were immunoprecipitated and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. In addition, total RNA from the cultured cells was hybridized to labeled cDNA probes to determine the number of sequences specific for each protein. Albumin mRNA was found to decrease with time in culture in all three assay systems, while transferrin mRNA remained constant. Analysis of the kinetics suggests a selective decay, with a half-life of 7 h, of the amount of albumin-specific RNA present at the beginning of culture. Apparently, in the hormone-deprived hepatocytes, there is little or no further transcription of the albumin gene. After addition of insulin to the cultures, albumin mRNA levels increased, suggesting an effect of this hormone on albumin gene utilization.

Albumins↗

PubMed1983

The 5' noncoding and flanking regions of the avian very low density apolipoprotein II and serum albumin genes. Homologies with the egg white protein genes.

We have isolated and sequenced the 5' proximal exons and flanking regions of the chicken very low density apolipoprotein II (apo-VLDLII) and serum albumin genes. These genes specify the most abundant mRNA species present in livers of hens or estrogen-treated roosters. Sequencing revealed that the promoter region of the estrogen-dependent apo-VLDLII gene contained at least two potential transcriptional initiation sites, preceded by appropriately positioned "ATA" sequences. One is homologous with the cap site of the ovalbumin gene, and the other exhibits a match of 10 out of 12 nucleotides with the cap site of the serum albumin gene. S1 nuclease mapping indicates that both sites are active in vivo, although the "ovalbumin"-like site is by far the most efficient at all stages of the estrogenic response. The relative efficiencies of these two sites are maintained during in vitro transcription of truncated templates. A third site, that was not anticipated from sequencing data, is active in vivo but inactive in vitro. A conserved 5' flanking element, initially identified during studies on egg white protein genes, is also present upstream from the apo-VLDLII and serum albumin genes. Its removal does not affect initiation site selection in vitro. Regions of the apo-VLDLII and ovalbumin genes extending 100 nucleotides downstream from the "TATA box" contain several striking homologies that suggest a common mode of evolution.

Amino Acid Sequence↗

PubMed1981

Cloning and structural characterization of an estrogen-dependent apolipoprotein gene.

ApoVLDLII is a major polypeptide component of avian very low density lipoprotein. Its production in the liver of the maturing female chick is developmentally synchronized with vitellogenesis. The apoVLDLII gene is normally dormant in males but can be activated by the administration of estrogen. In these studies, we describe the isolation of three recombinant bacteriophages that contain apoVLDLII genes. The genes appear to be identical from preliminary restriction analyses, although heterogeneity in flanking sequences is evident. The structure of the gene has been characterized and its organization correlated with the structure of the apoVLDLII polypeptide.

Animals↗

PubMed1980

Could poly(A) align the splicing sites of messenger RNA precursors?

In general, poly(A)-mRNA appears to be derived from larger nuclear RNA precursors. The maturation of these precursors involves excision of sequences of variable length from within the molecule and splicing of the remaining structural and coding sequences. The mechanism by which this process occurs is not known. It does not appear to operate solely through the recognition of a defined primary sequence or through the formation of a consistent secondary structure. We propose an alternative model in which poly(A) facilitates the splicing event by promoting the formation of triple-stranded structures within the mRNA precursor.

Animals↗