In light of present progress in the understanding of how messenger RNA is constructed in eukaryotic cells, the levels of gene control are discussed. Transcriptional control, assessed by the rate of synthesis of specific nuclear RNA, is strongly indicated to be the most frequent level of control. Definition of eukaryotic transcription units as simple (encoding one protein} and complex (encoding two or more proteins) affords a framework in which to discuss control at the level of RNA processing for which several examples also are known. Finally, differential stability of cytoplasmic mRNAs and differential translation, both well established, are briefly described.

Variety in the level of gene control in eukaryotic cells

Publisher Summary This chapter describes the different aspects of vitellogenesis and oocyte assembly The major components of fish oocytes are derived from the blood-borne high-molecular-weight compound, vitellogenin, which is synthesized in the liver of oviparous vertebrates The classification of vitellogenin as a phospholipoglycoprotein indicates the crucial functional groups that are carried on the protein backbone of the molecule—namely, lipids, some carbohydrates, and phosphate groups In the rainbow trout, estrone administration leads to the induction of vitellogenin synthesis in the liver and its release into the bloodstream, but estrone displays only 5%–12% of the potency of estradiol The biochemical information concerning vitellogenin clearly indicates that a great deal of posttranslational modification must occur in the liver cell to reach the finished product seen in the serum An integral part of estradiol action is the observed hypercalcemia in vitellogenic fish, which can largely be ascribed to the calcium-binding properties of phosphorylated and highly charged, components of the native vitellogenin molecule

5 Vitellogenesis and Oocyte Assembly

Liver-specific mRNA sequences were examined in primary cultures of mouse hepatocytes. After cell disaggregation by collagenase treatment and for at least 24 h in culture, little change in liver-specific mRNA concentrations was noted. Gradually over a period of 140 h, liver-specific mRNAs declined. In contrast, transcriptional assays in which liver cell nuclei were used to produce 32P-labeled nuclear RNA showed that liver-specific gene transcription was greatly diminished within 24 h, while polymerase II transcription of "common" genes and transcription of tRNA and rRNA did not decline. Thus, a prompt differential transcriptional effect seems to underlie the gradual loss of tissue specificity of the primary cultures.

Changes in liver-specific compared to common gene transcription during primary culture of mouse hepatocytes.

The evolution of egg-yolk proteins

https://www.cell.com/cell/pdf/0092-8674(83)90335-5.pdf

Temporal order of chromatin structural changes associated with activation of the major chicken vitellogenin gene

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.

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

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.

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

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.

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

Publisher Summary This chapter focuses on the regulation at the level of gene expression. The structural genes for what the transcription frequency can be regulated have been the subject of intensive investigation over the past two decades. There are many steps in the process of transcription of a given gene that finally results in a change, in a specific cellular function; some of these steps that are capable of being regulated are defined. Binding of ribonucleic acid (RNA) polymerase to the promoter involves a number of different proteins that modify the activity and specificity of the polymerase, allowing gene expression to be regulated. The effects of the regulatory proteins may be negative, restricting transcription, or positive, facilitating transcription. The activities of the regulatory proteins are themselves regulated by specific small molecules that communicate the metabolic needs of the cell to the sites at which gene expression is controlled. Gene expression could be modulated by alterations in the stability of the mRNA, in the frequency with what specific portions of a polycistronic mRNA are translated, in the availability of the various species of tRNA or their synthesis, or in any of the many catalytic events involved in the translation process. The activity of a protein may be modified by specific small molecules through noncovalent interactions, such as those involved in “feedback” inhibition, or through covalent changes, such as adenylylation. By describing the mechanisms of modulating gene expression, the basic principles involved and to indicate how complex is the total story of gene regulation, and how extensively interlocking are its parts are elucidated.

Regulation of Gene Expression in Prokaryotic Organisms

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.

Roger G. Deeley

Papers

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

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

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

Regulation of Gene Expression in Prokaryotic Organisms

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