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Biomedical subjects

S E Harris

Publications and source records attributed to S E Harris.

At least 127 records · Page 7Linked to original sources
PubMed1976

Effects of estrogen on gene expression in the chick oviduct. Control of ovalbumin gene expression by non-histone proteins.

Chromatin was isolated from the oviducts of chicks which had either received diethylstilbestrol stimulation for 14 days (stimulated chromatin) or been treated and then withdrawn from hormone administration (withdrawn chromatin). These chromatin preparations were dissociated and then reconstituted in the presence of various non-histone protein fractions. cDNAov was used as a probe for measuring the level of mRNAov sequences synthesized in vitro from various reconstituted chromatins. Our results indicate that extractable non-histone proteins from stimulated chromatin are capable of activating the in vitro transcription of the ovalbumin gene when included in the reconstitution of withdrawn chromatin. On the other hand, addition of extractable non-histone proteins from withdrawn chromatin to stimulated chromatin does not affect the synthesis of mRNAov sequences. Therefore, the extractable non-histone proteins from stimulated chromatin contain a positive regulator(s) which controls the in vitro expression of the ovalbumin gene.

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PubMed1976

Effects of estrogen on gene expression in chick oviduct. The role of chromatin proteins in regulating transcription of the ovalbumin gene.

Histones, extractable non-histone proteins, and a tightly bound non-histone protein DNA complex were fractionated from chromatins which were isolated from estrogen-stimulated and hormone-withdrawn chick oviducts. Reconstitution of homologous constituents was performed and RNA was transcribed from these reconstituted chromatins using Escherichia coli RNA polymerase. DNA complementary to ovalbumin mRNA was then used as a hybridization probe to estimate the concentration of ovalbumin messenger RNA sequences in these in vitro transcripts. Our results demonstrated that 0.011% of the in vitro RNA transcripts produced from reconstituted estrogen-stimulated chromatin was ovalbumin mRNA sequences as compared to 0.0015% obtained for reconstituted hormone-withdrawn chromatin. Thus, the ratio of ovalbumin mRNA sequences in the in vitro transcripts of reconstituted stimulated and withdrawn chromatins was 8 to 1, identical with the value obtained from native estrogen-stimulated and hormone-withdrawn chromatins. Furthermore, the number of initiation sites for RNA synthesis on reconstituted and native chromatins are indistinguishable. Thus, the specificity of transcription of isolated chromatins appears to be conserved upon dissociation and fractionation of the chromatin proteins followed by reconstitution of these constituents to DNA. The effect of chromatin proteins on gene expression was further examined by reconstituting components from different development stages. Reconstitution of extractable non-histone proteins from estrogen-stimulated chromatins to tightly bound non-histone protein. DNA complexes from hormone-withdrawn chromatins resulted in the synthesis of a substantial amount of ovalbumin mRNA sequences. Conversely, when extractable non-histone proteins from withdrawn chromatins were reconstituted to tightly bound non-histone protein DNA complexes from estrogen-stimulated chromatins, very low levels of mRNAov sequences were detected. In contrast, interchange of the histones and tightly bound non-histone protein DNA complexes from hormone-withdrawn and estrogen-stimulated chromatins during reconstitution did not affect the level of mRNAOV sequences produced. Therefore, the extractable non-histone proteins of chromatin appear to be extremely important in regulating specific gene expression.

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PubMed1976

Effect of estrogen on gene expression in the chick oviduct. Effect of estrogen on the sequence and population complexity of chick oviduct poly(A)-containing RNA.

Total cellular RNA preparations were isolated from chicken oviducts at three different development stages: (a) immature chicks which were chronically stimulated with estrogen; (b) estrogen-stimulated chicks which were then withdrawn from hormone for 12 days; and (c) laying hens. Total cellular RNA containing 3'-poly(A) sequences (poly(A)-RNA) were than isolated from these preparations using oligo(dT)-cellulose chromatography. The number average nucleotide length of the poly(A)-RNA preparations in each case was approximately 2000 nucleotides. The number average nucleotide length of the poly(A) residues at the 3'-terminal end of each RNA preparation was approximately 70 adenylate residues. Complementary DNA (cDNA) copies to each preparation of poly(A)-RNA were synthesized using avian myeloblastosis virus RNA-directed DNA polymerase. The cDNApoly(A) preparations were then utilized in DNA excess hybridization experiments to analyze the complexity of the DNA sequences from which these RNAs were transcribed. Approximately 22% of each of the total cellular poly(A)-RNAs were transcribed from repeated DNA sequences (average repeat frequency of 35 copies/genome) while the remaining majority were transcribed from single copy or unique sequence DNA. It was possible to estimate the number of different poly(A)-RNA sequences per cell by analyzing the kinetics of hybridization of these cDNApoly(A) preparations to total cellular poly(A)-RNA extracts under conditions of RNA excess. The results revealed that 41% of the poly(A)-RNA from laying hen oviduct consisted of, on the average, three different sequences/cell, each of which was present in approximately 25,000 copies/cell. The remainder of the poly(A)-RNA in this tissue consisted of approximately 25,000 different sequences/cell, which were present largely in only two or three copies/cell. A somewhat similar sequence complexity was found for oviduct cells prepared from estrogen-stimulated chicks. We estimated that there were approximately 20,000 different poly(A)-RNA sequences/cell, each represented in only one to two copies/cell. However, there were five sequences which were present, on the average, in a concentration of 5600 copies/cell. The poly(A)-RNAs from hormone-wtihdrawn tissue, on the other hand, had a lower sequence complexity. There were only approximately 10,000 different poly(A)-RNA sequences/cell, each present in about three copies/cell. Furthermore, the few sequences present in a great abundance in hen and hormone-stimulated tissues were apparently absent in oviduct tissue from hormone-wtihdrawn chicks, suggesting that the intracellular concentrations of these high frequency RNA sequences are dependent on estrogen.

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PubMed1976

Effect of estrogen on gene expression in the chick oviduct.

Hen oviduct RNA polymerase II and Escherichia coli RNA polymerase holoenzyme and core enzyme were used to study the initiation of RNA synthesis on chromatin. In either the presence or absence of estrogenic stimulation, changes in the level of oviduct chromatin initiation sites as measured in the presence of either homologous or heterologous polymerases followed a similar pattern. Comparison of the initiation sited utilized by these enzymes on chick oviduct chromatin indicated that these enzymes compete with each other for the same initiation regions. In contrast to chromatin, however, the majority of the initiation sites on DNA which are utilized by the oviduct RNA polymerase II are different from those utilized by E. coli holoenzyem. These results suggest that chromatin proteins are involved in the selection of initiation sites on chromatin for RNA polymerases. The in vitro transcripts of these RNA polymerases on stimulated chick oviduct chromatin were analyzed by hybridization to a cDNA probe transcribed from ovalbumin mRNA. The relative concentration of ovalbumin sequences transcribed by these three polymerases was 4:1.5:1 for oviduct RNA polymerase II, E. coli core enzyme, and holoenzyme respectively. Therefore, the efficiency of transcribing a specific gene appears to depend on the interaction between RNA polymerase and chromosomal elements in the initiation region.

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PubMed1976

Effect of estrogen on gene expression in the chick oviduct. In vitro transcription of the ovalbumin gene in chromatin.

RNA was transcribed from chromatin isolated from chick oviduct and spleen by using RNA polymerase from Escherichia coli. RNA was also transcribed from whole chick DNA using E. coli RNA polymerase. DNA complementary to ovalbumin messenger RNA (cDNAov) was then used as a hybridization probe to estimate the concentration of ovalbumin messenger RNA sequences (mRNAov) in these in vitro transcripts. Although chromatin from unstimulated chick oviduct was capable of substantial RNA synthesis, no detectable mRNAov sequences could be found in the transcript. Likewise, mRNAov sequences could not be found in RNA synthesized from spleen chromatin using E. coli RNA polymerase. However, chromatin from estrogen-stimulated chick oviducts was capable of supporting synthesis of ovalbumin mRNA. We estimate that approximately 0.01% of the RNA synthesized from estrogen-stimulated chromatin was mRNAov sequences. When RNA synthesized from chick DNA was tested with the cDNAov probe, mRNAov sequences could be detected in a concentration of approximately 10% that found in the RNA transcript from estrogen-stimulated chromatin. This was as expected if the ovalbumin gene is considered to be in the "open or derepressed" region of the estrogen-stimulated oviduct chromatin. Chromatin isolated from chicks withdrawn from hormone for 12 days was only capable of supporting mRNAov synthesis in vitro at a level of 5 to 10% of that observed in chromatin prepared from estrogen-stimulated chicks, thus indicating the requirement for estrogen to maintain the ovalbumin gene in the available or "open" state in the majority of oviduct cells. These data militate against post-transcriptional control as the primary mechanism of steroid hormone regulation of specific mRNA synthesis in the chick oviduct system, and favor primary gene derepression as the most likely mechanism for estrogen induction of ovalbumin synthesis.

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PubMed1976

The synthesis and properties of the complete complementary DNA transcript of ovalbumin mRNA.

The synthesis of a complementary DNA copy (cDNA) of hen ovalbumin mRNA using AMV RNA-directed DNA polymerase was studied under different conditions of salt, deoxyribonucleotide concentrations, temperature, and time. It was observed that in the absence of monovalent cation at 46 degrees C a complete transcript of ovalbumin mRNA could be effected by the enzyme. The minimum deoxyribonucleotide requirement for complete synthesis was 35 muM for dATP, dGTP, and dCTP and 200 muM dTTP. By a number of different experimental criteria which included sedimentation on alkaline sucrose gradients and electrophoresis in polyacrylamide gels containing 98% formamide, direct electron microscope visualization, and protection of ovalbumin [25I]mRNA from nuclease digestion it could be demonstrated that a considerable fraction of a complete mRNA transcript was indeed synthesized. The cDNA/ovalbumin mRNA hybrid had a Tm on hydroxylapatite of 92 degrees C, indicating the synthesis of a RNA transcript with a high fidelity. When such a complete ovalbumin [3H]cDNA was synthesized with a specific activity of 10(8) cpm/mug and hyfridized to an excess of chick DNA, the kinetics of hybridization indicated that the cDNA was comprised of a nonrepetitive sequence.

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PubMed1976

Molecular biology and biochemistry of differentiation.

Recent work in the field of molecular biology and differentiation has been directed towards an assessment of the number of different genes involved in the development and differentiation process. By the techniques of RNA-DNA hybridization to single copy DNA, it appears that some 60,000-200,000 different RNA sequences are expressed during embryonic development in the mouse. The differentiating brain shows the highest degree RNA transcription diversity. The new technique of cDNA-poly(A)-containing RNA hybridization is described. The nuclear poly(A)-containing RNA appears to reflect the high complexity of sequences as determined in RNA-DNA experiments described above. The cytoplasmic poly(A)-containing RNA or messenger RNA appears to represent a subset of approximately 10-20% of the information in the nuclear poly (A)-RNA. Also, the major portion of frequency class of messenger RNA in different organs such as kidney, spleen, and liver are common to these different organs. However, brain, although containing many of the cytoplasmic messenger RNA sequences found in liver, kidney, and spleen, has a large class of messenger RNA sequences which are specific to the nervous system.

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PubMed1975

Use of a specific probe for ovalbumin messenger RNA to quantitate estrogen-induced gene transcripts.

DNA complementary to purified ovalbumin messenger RNA (cDNA ov) was synthesized in vitro using RNA-directed DNA olymerase from avian myeloblastosis virus. This cDNAov was then employed in hybridization assays to determine the effect of estrogen on the number of ovalbumin mRNA (MRNAov) molecules per tubular gland cell of the chick oviduct. The changes in mRNAov were measured in immature chicks during primary stimulation, after hormone withdrawal and again following secondary stimulation of the chick oviduct with estrogen. The number of mRNAov per tubular gland cell was also determined for egg-laving hen. Daily estrogen administration to the immature chick resulted in growth of the oviduct, differentiation of epithelial cells to tubular glands, and a corresponding increase in the concentration of mRNAov in the tubular gland cell from essentially zero before estrogen administration to 48,000 molecules per cell after 18 days of estrogen treatment. Upon withdrawal of estrogen from the chick, the mRNAov concentration decreased to a level of 0-10 molecules/tubular gland cell after 12 days. Readministration of a single dose of estrogen to these chicks resulted in a dramatic and rapid increase in the concentration of mRNAov. Within 30 min, the mRNAov concentration approximately doubled and by 29 hr the tubular gland cell concentration had reached 17,000 molecules. The initial transcription rate for the ovalbumin gene was 12 mRNAov molecules/min. With these data, we have calculated that the half-life of the ovalbumin messinger RNA should be on the order of 40-60 hr and that the steady-state concentration of mRNAov per tubular gland cell was 50,000 molecules. Similarly, each messenger RNA molecule was translated approximately 50,000 times during its lifetime in order to effect the necessary quantity of ovalbumin required for egg production. These data substantiate the hypothisis that estrogen exerts its primary action at the level of transcription to effect the synthesis of nascene mRNA molecules which in turn code for synthesis of hormone-induced proteins.

Animals↗
PubMed1975

Estrogen induction of ovalbumin mRNA: evidence for transcription control.

Estrogen induces the synthesis and accumulation of the specific messenger RNA for the egg white protein ovalbumin. The messenger RNA has been purified to apparent homogeneity on a preparative scale and utilized to synthesize a radioactive complementary DNA copy. This complementary DNA probe was first used to reveal that although ovalbumin constitutes 60% of the total protein of chick oviduct the gene which codes for the ovalbumin nRNA is represented only once in each haploid genome: The induction of genetranscription and subsequent accumulation of ovalbumin mRNA during estrogen-mediated tissue differentiation was also investigated. Ovalbumin mRNA sequences were quantifiedusing the complementary DNA probe and by an in vitro heterologous translation system. Similar experiments were performed using chicks which were withdrawn from hormone treatment and then given a single injection of estrogen. The data suggest pure transcriptional control for the mechanism by which estrogen regulates the synthesis of the tissuespecific protein ovalbumin. Finally, several in vitro translation systems are comparedwith respect to their usefullness to assess the effects of hormones on mRNA production. It is concluded that the protein synthesis system derived from wheat germ offers thegreatest advantages for initial studies.

Animals↗
PubMed1975

Steroid hormone regulation of specific messenger RNA and protein synthesis in eucaryotic cells.

Evidence is presented that the induction of specific proteins in the chick oviduct by the steroid hormones estrogen and progesterone, involves a primary effect at the level of gene transcription. The intracellular levels of mRNA's which code for the synthesis of the egg-white proteins, ovalbumin and avidin, have been quantitated in a heterologous protein synthesizing system. It is demonstrated that these levels are directly dependent upon the inducing steroid, estrogen or progesterone, respectively. Ovalbumin mRNA has been purified to apparent homogeneity. This ovalbumin mRNA was then used as a template for the synthesis of a complementary DNA copy catalyzed by the enzyme reverse transcriptase which was isolated from avian myeloblastosis virus. This radioactively labeled complementary DNA was used to demonstrate, by means of DNA excess hybridization, that the ovalbumin gene is represented only once in each haploid genome of the chick cell. Next the complementary DNA copy of the ovalbumin mRNA was used as a genetic probe to determine the precise number of sequences of ovalbumin mRNA present at any one time after the administration of estrogen. It was demonstrated that the unstimulated chick contained no sequences of ovalbumin mRNA. Within a very short period of time after estrogen is administered the ovalbumin sequences begin to appear and reach a steady state level of 140,000 molecules per tubular gland cell. It could also be calculated that each ovalbumin molecule is probably translated some 50,000 times during its life which explains why ovalbumin comprises some 60% of the total protein in the oviduct cell. Following withdrawal of the oviduct from estrogen treatment, ovalbumin mRNA sequences again drop to undetectable levels. However, following a single injection of estrogen to these withdrawn animals, new ovalbumin mRNA sequences could be detected within 30 minutes. These data suggest that estrogen controls the activity of the ovalbumin gene via a pure transcriptional control mechanism. It is also demonstrated that the efficiency of the complementary DNA as a means of quantitating specific mRNA sequences is some 1,000 times more sensitive than the best available in vitro translation system. Finally, the efficacy of four popular translation systems is compared. It is suggested that for initial studies involving hormonal control of mRNA levels, the translation system derived from wheat germ is the simplest and most sensitive.

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PubMed1973

Synthesis of (3H)DNA complementary to ovalbumin messenger RNA: evidence for limited copies of the ovalbumin gene in chick oviduct.

Accumulation of ovalbumin messenger RNA in chick oviduct is absolutely dependent upon estrogen. After estrogen treatment, ovalbumin comprises 60-65% of the total oviduct protein. We used maximally stimulated animals to extract and partially purify the ovalbumin messenger RNA. The final product was enriched about 100-fold in activity with respect to this specific messenger RNA. This ovalbumin messenger RNA fraction was used to direct the synthesis of a complementary [(3)H]DNA in the presence of RNA-dependent DNA polymerase isolated from avian myeloblastosis virus. The complementary [(3)H]DNA (specific radioactivity, 8 x 10(7) cpm/mug) was a faithful transcript since about 90% would hybridize back to the original messenger RNA template. Ovalbumin complementary [(3)H]DNA was reannealed with an excess of chick-oviduct total DNA. The kinetics of this reaction indicate that only one copy of the ovalbumin gene exists in each haploid genome. These data suggest that estrogen may affect the oviduct genome to stimulate production of large numbers of ovalbumin messenger RNA molecules from a single copy of the ovalbumin gene.

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