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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.

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.

The first enzyme for histidine biosynthesis, encoded in the hisG gene, is involved in regulation of expression of the histidine operon in Salmonella typhimurium. The studies reported here concern the question of how expression of the histidine operon is affected by a mutation in the hisG gene that alters the allosteric site of the first enzyme for histidine biosynthesis, rendering the enzyme completely resistant to inhibition by histidine. The intracellular concentrations of the enzymes encoded in the histidine operon in a strain carrying such a mutation on an episome and missing the chromosomal hisG gene are three- to fourfold higher than in a strain carrying a wild-type hisG gene on the episome. The histidine operon on such a strain fails to derepress in response to histidine limitation and fails to repress in response to excess histidine. Furthermore, utilizing other merodiploid strains, we demonstrate that the wild-type hisG gene is trans dominant to the mutant allele with respect to this regulatory phenomenon. Examination of the regulation of the histidine operon in strains carrying the feedback-resistant mutation in an episome and hisT and hisW mutations in the chromosome showed that the hisG regulatory mutation is epistatic to the hisT and hisW mutations. These data provide additional evidence that the first enzyme for histidine biosynthesis is involved in autogenous regulation of expression of the histidine operon.

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.

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.

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.

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.

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.

A new method is described for assaying the ATP phosphoribosyltransferase (EC 2.4.2.17) that catalyzes the first step of histidine biosynthesis in bacterial cells. This is a highly sensitive radioassay, capable of detecting fewer than 10 9 molecules of enzyme. In contrast to the previously available assay, it can be carried out in the presence of other molecules that absorb uv light. The assay may find useful application in studies on the in vitro synthesis of the enzymes for histidine biosynthesis and in studies on the various aggregation states of the enzyme.

A new term, autogenous regulation, is used to describe a phenomenon that is not a new discovery but rather is newly appreciated as a mechanism common to a number of systems in both prokaryotic and eukaryotic organisms. In this mechanism the product of a structural gene regulates expression of the operon in which that structural gene resides. In many (perhaps all) cases, the regulatory gene product has several functions, since it may act not only as a regulatory protein but also as an enzyme, structural protein, or antibody, for example. In a few cases, this protein is the multimeric allosteric enzyme that catalyzes the first step of a metabolic pathway, gearing together the two most important mechanisms for controlling the biosynthesis of metabolites in bacterial cells—feedback inhibition and repression. Autogenous regulation may provide a mechanism for amplification of gene expression (84); for severe and prolonged inactivation of gene expression (85); for buffering the response of structural genes to changes in the environment (45, 52); and for maintaining a constant intracellular concentration of a protein, independent of cell size or growth rate (86). Thus, autogenous regulation provides the cell with means for accomplishing a number of different regulatory tasks, each suited to better satisfying the needs of the organism for its survival.

Prior studies to this article by Anfinsen, Epstein, and Goldberger established that the time required for synthesizing a chain of protein such as ribonuclease in the tissues of higher organisms would be approximately two minutes. Here, the three researchers noted that an enzyme system in the endoplasmic reticulum of cells that catalyzed the disulfide interchange reaction and which, when added to solutions of reduced ribonuclease, catalyzed the rapid formation of the native disulfide pairing in a period less than two minutes. Subsequent studies would find that such motile proteins as staphylococcal nuclease or myoglobin could undergo virtually complete renaturation in only a few seconds or less.

Previous studies showed that the enzyme (phosphoribosyltransferase) which catalyzes the first step of the histidine pathway in Salmonella typhimurium plays a role in regulation of the histidine operon. Since histidyl transfer ribonucleic acid (His-tRNA) is required for repression of the histidine operon, we considered the possibility that the role of phosphoribosyltransferase might be realized through an interaction with His-tRNA. One prediction inherent in this idea is that the enzyme should interact with His-tRNA in vitro. Evidence is presented for such an interaction. Binding of 3 H-His-tRNA to purified phosphoribosyltransferase was tested on Sephadex columns and on nitrocellulose filters. The enzyme was found to have a high affinity for tRNA. Comparing the binding of 3 H-His-tRNA with that of tRNA aminoacylated with other 3 H-amino acids disclosed that the binding of the histidyl species of tRNA is favored over that of other species and is dependent upon magnesium-ion concentration.

An enzyme which catalyzes sulfhydryl-disulfide interchange in proteins containing "incorrect" disulfide bonds has been isolated from beef liver microsomes. The purified form of this enzyme shows two bands upon electrophoresis in starch gels and polyacrylamide gels. The proteins represented by these bands are considered to be variants of the same protein because both are enzymically active after elution from starch gels and because they appear as a single band after reduction and alkylation. A contaminant of higher molecular weight was identified in the purified enzyme preparation both as a faint third band in starch and polyacrylamide gels and as a faster sedimenting component in the analytical ultracentrifuge. By the latter technique the quantity of the contaminant was estimated to be 8 to 10% that of the microsomal enzyme. The molecular weight of the enzyme was found to be 42,000 by analytical ultracentrifugation. Amino acid analyses of the enzyme disclosed the presence of 3 half-cystine residues, and 44 arginine plus lysine residues, per molecule. Peptide maps, prepared from tryptic digests of the enzyme, contained 48 ninhydrin-positive spots. The visible and ultraviolet absorption spectra of the enzyme showed no peaks other than one with a maximum at 278 mµ.

Recent studies on repression of the enzymes for histidine bio-synthesis in Salmonella typhimurium demonstrated that the kinetic pattern in which the enzymes become repressed is influenced by the state of the feedback-sensitive site of the first enzyme of the pathway (Kovach et al., J. Bacteriol., 97, 1283 (1969)). In the present study we demonstrate that under certain conditions alteration of the feedback-sensitive site of the first enzyme prevents repression of the histidine operon. We conclude that the first enzyme plays a previously unrecognized role in regulation of the histidine system.

The specificity of the interaction between phosphoribosyltransferase and partially purified preparations of various species of transfer ribonucleic acid (tRNA) was investigated with the use of a filter binding assay. The enzyme showed a higher affinity for histidyl-tRNA than for arginyl- or glutamyl-tRNA. Competition experiments revealed that the enzyme does not distinguish between the aminoacylated and deacylated forms of arginine tRNA or glutamic acid tRNA, since all the binding of the aminoacylated tRNA could be inhibited by deacylated tRNA. The enzyme does, however, distinguish between the aminoacylated and deacylated forms of histidine tRNA. Approximately 70% of the binding of aminoacylated histidine tRNA is specific, since only 30% of the binding could be inhibited by deacylated tRNA. The possibility that the regulatory role of phosphoribosyltransferase is carried out as a complex with histidyl-tRNA is consistent with these data.

Imidazolylacetolphosphate aminotransferase, which catalyzes the eighth step in the biosynthesis of histidine in Salmonella typhimurium, has been purified from derepressed histidine auxotrophs. The procedure involves fractionation of cell extracts with protamine sulfate and ammonium sulfate, chromatography on diethylaminoethyl cellulose columns, and gel filtration on Sephadex G-150. By a variety of techniques, the enzyme preparation was shown to be homogeneous and to have a molecular weight of 59,000 daltons. One pyridoxal phosphate group was found to be associated with each molecule of the enzyme. The enzyme displays a marked tendency to polymerize upon aging. This polymerization can be reversed by incubation of the enzyme with β-mercaptoethanol.

A method is presented for the purification of the isomerase catalyzing the fourth step of histidine biosynthesis in Salmonella typhimurium. The enzyme was isolated from extracts of histidine auxotrophs which (when derepressed) produced 25 to 30 times the wild type levels of enzyme. The purification procedure involved fractionation with protamine sulfate and ammonium sulfate, gel filtration on Sephadex G-100, ion exchange chromatography on diethylaminoethyl Sephadex, and continuous flow high voltage electrophoresis. The final product is homogeneous with respect to size, with a weight average molecular weight of 29,000, but is composed of three enzymically active forms (in the ratio 90:9:1) which may be distinguished by their differing electrophoretic mobilities in alkaline polyacrylamide gels.

The pathway for histidinle biosyinthesis in Salmonella typhimurium, elucidated largely through the efforts o:f Dr. B. N. Ames and co-workers, 1-5 is shown in Figure 1. The figure also shows that portioin of the pathway for puriine biosynthesis which involves the further metabolism of 4-amino-5-imidazole carboxamide riboinucleotide (phosphoribosyl-AIC).6 Dr. Phillip Hartmain and his colleagues have shown that the structural genes for the enzymes catalyziing the reactioins of the histidine pathway are localized in a small segment of the bacterial chromosome, the histidine operon.7' 8 The phenomenoni of coordiniate derepressioin of the histidine enzymes was originally described by Ames and Garry, 9 who found that the histidine operon functions as a single unit in response to the level of histidiine available to the organism. Evidence presented by Martin10 inidicates that the histidinie operon is transcribed into a polycistroiiic messenger-RNA, anid studies oIn polarity in the histidine operon7' 11,12 suggest that this messenger-RNA is trainslated from the end which

Kinetic studies on the derepression of 5 of the 10 enzymes for histidine biosynthesis in Salmonnella typhimurium reveal that derepression may proceed by two different modes, depending upon the site of mutation in the various histidine auxotrophs examined. Simultaneous derepression of the enzymes is observed in those mutants incapable of producing the intermediate, 4-amino-5-imidazolecarboxamide ribonucleotide, through the histidine pathway. Derepression of the enzymes in a temporal sequence corresponding to the positional sequence of the genes in the histidine operon is observed in those mutants which do produce this intermediate. The addition of 4-amino-5-imidazolecarboxamide ribonucleoside to cultures of mutants ordinarily characterized by the simultaneous mode of derepression served to shift the mode from simultaneous to sequential, but did not influence the derepressed rate of enzyme synthesis. In organisms in which the sequential mode is ordinarily observed, adenine effected a shift to the simultaneous mode, causing all the enzymes to derepress at the same time, but did not influence the derepressed rate of enzyme synthesis. Chloramphenicol, at low concentrations, reduced the growth rate of derepressed cells by a factor of 2 to 3 and decreased the rate of derepression of the histidine enzymes in all organisms tested. In the histidine auxotroph, hisE11, and in the wild type, LT-2 (derepressed by the addition of thiazole alanine), chloramphenicol prolonged the intervals between the times of derepression of the histidine enzymes. On the other hand, in hisG52, in which the enzymes for histidine biosynthesis derepress simultaneously and not sequentially, chloramphenicol did not alter the time of derepression of the enzymes. In the presence of chloramphenicol, adenine shifted the mode of derepression of hisE11 and LT-2 from sequential to simultaneous, just as it did in the absence of chloramphenicol. However, adenine did not eliminate the effect of chloramphenicol on the rate of depression. The data presented here suggest that the histidine operon is transcribed into a polycistronic message which, under conditions in which sequential derepression is observed, is translated from one end only. Under conditions in which simultaneous derepression is observed, the translation of this message is initiated at multiple sites.

An in vitro system was developed for transcription of the histidine operon of Esherichia coli carried in the genome of a defective ϕ80 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 ϕ80 and ϕ80d his 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 ϕ80d his DNA template. The RNA complementary to the histidine operon was synthesized on the “sense” strand (the R strand) of ϕ80d his 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.

In this follow-up to an earlier article, Anfinsen, Goldberger, and Epstein reported "the solubilization and partial purification of the active microsomal protein," and described some of the properties of the soluble system. This and other studies seemed to make clear that the integrity of microsomes as organized particles was not required for catalyzing the reactivation of the reduced forms of ribonuclease and lysozyme. Their findings suggested that the microsomal system might function to facilitate the conversion of at least two different polypeptide chains to the corresponding native proteins during the synthesis of most proteins containing disulfide bonds.

This article was published after Anfinsen's laboratory had delineated and demonstrated the major principles of protein folding and during a period when research at the lab turned to the disulfide interchange enzyme that accelerates the reactivation of the reduced form of ribonuclease. In the lab, Givol and Goldberger had worked to achieve in vitro the reactivation rates that would be compatible with the rate of protein biosynthesis. Purifying the enzyme proved difficult, so the team decided to utilize disulfide interchange activity to demonstrate other aspects related to protein folding and structure. The results of these experiments were consistent with the idea that the disulfide bonds in chymtrypsinogen were formed according to the information present in the single-chain protein, which is subsequently converted by peptide bond cleavage to the metastable three-chain chymotrypsin. Based on this, Anfinsen's group reasoned that the inactivation of a multi-chain protein by disulfide interchange indicated its origin as a single-chain protein during biosynthesis.

Kinetic studies on repression of the enzymes for histidine biosynthesis in Salmonella typhimurium showed that, upon addition of histidine to a derepressed culture, the enzymes became repressed in a temporal sequence which corresponds with the positional sequence of the genes in the histidine operon. This serial pattern of repression occurred under conditions in which the feedback site of the first enzyme for histidine biosynthesis is intact. When this site was rendered nonfunctional the pattern of repression was changed so that all of the enzymes became repressed concomitantly. These results suggest that the first enzyme for histidine biosynthesis plays a hitherto unrecognized role in control of the histidine system.

We previously proposed that the first enzyme for histidine biosynthesis in Salmonellatyphimurium plays a role in regulating expression of the histidine operon and that in order to play this role the enzyme must form a complex with histidyl-tRNA. Among the many observations that led to these conclusions were 1) that regulation of the histidine operon is defective in strains carrying a mutation in the gene for the first enzyme that renders the enzyme resistant to inhibition by histidine; and 2) that the enzyme purified from the wild type strain interacts specifically, and with high affinity, with histidyl-tRNA. The present study was carried out to test the prediction that the enzyme purified from the mutant strain described above would display a defect in its interaction with histidyl-tRNA. This prediction was fulfilled by the finding that purified histidine-insensitive enzyme does not bind histidyl-tRNA. Our results therefore suggest that the capacity of the enzyme to bind histidyl-tRNA invitro is a reflection of its regulatory function invivo.

Previous studies suggested that phosphoribosyltransferase, which catalyzes the first step of the pathway for histidine biosynthesis in Salmonella typhimurium and which is sensitive to inhibition by histidine, plays a role in repression of the histidine operon. Recently, we showed that the enzyme has a high affinity for histidyl transfer ribonucleic acid (His-tRNA), which is known to participate in the repression process. In the present study, we have investigated further the interaction between the enzyme and His-tRNA. We found that His-tRNA binds at a site on phosphoribosyltransferase distinct from the catalytic site and the histidine-sensitive site; that the substrates of the enzyme inhibit the binding of His-tRNA, whereas histidine does not do so; that, once a complex has been formed between phosphoribosyltransferase and His-tRNA, the substrates of the enzyme decrease the stability of the complex, whereas histidine is without effect; and that purified phosphoribosyltransferase which has a defect in its inhibition by histidine (produced by mutation) displays an altered ability to bind His-tRNA, a finding which may be a reflection of the fact that mutants producing such a defective enzyme display an alteration of the repression process.

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.

Administration of a single dose of estrogen to a rooster results in de novo synthesis of vitellogenin mRNA and vitellogenin in the liver. When the estrogen response has apparently ceased and vitellogenin is no longer detectable in the liver or in the plasma (post-estrogen-serum-negative state) vitellogenin mRNA persists in appreciable amounts in the liver of the rooster. This mRNA has the same size as vitellogenin mRNA from stimulated roosters. However, this vitellogenin mRNA is no longer associated with polysomes. The possible role of this persisting vitellogenin mRNA in the greater rapidity and magnitude of the response to a subsequent estrogen stimulation will be discussed.

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.

About four years ago, we turned our attention from regulation of gene expression in prokaryotes and began to search for a system that would tell us something about regulation in eukaryotic cells. We were guided in this search by the following considerations. First, we did not want to work with a system in which cellular differentiation was itself an integral part of the regulatory response. We felt that we could obtain more clear-cut information if we could study a response in a tissue that was already fully differentiated and in which the regulatory response did not require DNA synthesis. We recognized that a hormonally responsive system would be the easiest to manipulate, and for practical reasons we wanted a system in which the hormonal response was of very great magnitude. And as long as were asking, we thought we might as well put in a request that the tissue we study also contain a whole group of genes that are hormonally responsive, so that we would be able to study not only the effect of hormone on a specific gene but also the mechanisms that coordinate the expression of the hormone-responsive domain of the tissue. In addition, we wanted our hypothetical tissue to have a gene expressed at a high level that is not responsive to hormonal stimulation. Such a gene would provide an ideal control for in vitro transcription studies, where one must show that the specificity of hormonal responsiveness reflects that of the tissue in vivo.

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.

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.

One of the most striking characteristics of living systems is that they function in an orderly manner despite their high degree of complexity. One workable definition of regulation, in fact, is the set of mechanisms that allows organisms to maintain this orderly functioning. It is important to realize, however, that regulation was not superimposed upon living systems; orderly processes are simply more successful than are disorderly ones, and therefore tend to be preserved through the evolutionary process by conferring advantages upon organisms that possess them. The thousands of chemical reactions occurring in cells are controlled by regulatory mechanisms that operate at many different levels. This introductory chapter focuses on those that operate at the level of gene expression and will introduce some of the strategies of genetic regulation that have evolved in prokaryotic organisms. Scanning the table of contents of this brief essay should suffice to tell the reader that a very general overview is in store for him. The renaissance in biological research that occurred in the last 25 years has been due mostly to the exciting studies concerning genetic regulation in prokaryotes. I have tried to abstract from those studies the most important basic principles they illustrate and to organize into a few generalizations the enormous body of data they have produced. I believe it is these principles and generalizations with which the reader will need to arm himself before proceeding further into this volume. It is to be hoped that the necessarily simplistic view of regulation they provide will be preferable to the bewilderment that so often results from exhaustive reviews that include the details of many specific regulated systems.

Selbst die kleinsten Zellen enthalten noch Billionen von Atomen und Molekulen, die nach Zusammensetzung und Funktion eine Reihe von Fragen aufwerten

Aus dem 16. Jahrhundert stammen unsere ersten Kenntnisse zur Anatomie des menschlichen Korpers, ohne das davon die medizinische Praxis wesentlich beeinflust wurde. Der Arzt verordnete Medikamente und Kuren, die haufig auf Aberglauben oder empirischen Erfahrungen beruhten. Zwar befinden sich unter den Arzneien eine Reihe von wirksamen Heilmitteln, die Mehrzahl war aber bar jeder pharmakologischen Aktivitat. In den folgenden Jahrhunderten nahmen die Kenntnisse auf dem Gebiet der Chemie und Physik zu, Probleme der biologischen Funktionen wurden jedoch nur selten untersucht. Erst zu Beginn des 19. Jahrhunderts nahm die Physiologie ihren grosen Aufschwung, in Deutschland mit Muller, in Frankreich mit Magendie, Bernard und Pasteur. Nachdem sich die wissenschaftliche Methodik bei der Untersuchung lebender Systeme bewahrt hatte, erkannte man, das sich damit ein auserordentlich fruchtbarer Wissenzweig eroffnete, der die Grundlagen zum Verstandnis der Funktionen des lebenden Korpers und seiner Krankheiten legte.