In 1901 a physician, Archibald Garrod, observed a patient with black urine. He used this simple observation to demonstrate that a single mutant gene can produce a discrete block in a biochemical pathway, which he called an “inborn error of metabolism”. Garrod’s brilliant insight anticipated by 40 years the one gene-one enzyme concept of Beadle and Tatum. In similar fashion the chemist Linus Pauling and the biochemist Vernon Ingram, through study of patients with sickle cell anemia, showed that mutant genes alter the amino acid sequences of proteins. Clearly, many fundamental advances in biology were spawned by perceptive studies of human genetic diseases (1). We began our work in 1972 in an attempt to understand a human genetic disease, familial hypercholesterolemia or FH. In these patients the concentration of cholesterol in blood is elevated many fold above normal and heart attacks occur early in life. We postulated that this dominantly inherited disease results from a failure of end-product repression of cholesterol synthesis. The possibility fascinated us because genetic defects in feedback regulation had not been observed previously in humans or animals, and we hoped that study of this disease might throw light on fundamental regulatory mechanisms. Our approach was to apply the techniques of cell culture to unravel the postulated regulatory defect in FH. These studies led to the discovery of a cell surface receptor for a plasma cholesterol transport protein called low density lipoprotein (LDL) and to the elucidation of the mechanism by which this receptor mediates feedback control of cholesterol synthesis (2,3). FH was shown to be caused by inherited defects in the gene encoding the LDL receptor, which disrupt the normal control of cholesterol metabolism. Study of the LDL receptor in turn led to the understanding of receptor-mediated endocytosis, a genera! process by which cells communicate with each other through internalization of regulatory and nutritional molecules (4). Receptor-mediated endocytosis differs from previously described biochemical pathways because it depends upon the continuous and highly controlled movement of membraneembedded proteins from one cell organelle to another in a process termed

https://science.sciencemag.org/content/sci/232/4746/34.full.pdf

A receptor-mediated pathway for cholesterol homeostasis.

https://www.cell.com/cell/pdf/S0092-8674(05)80085-6.pdf

Role of Insulin-like Growth Factors in Embryonic and Postnatal Growth

All scientific investigations begin with distinct objectives: first is the hypothesis upon which studies are undertaken to disprove, and second is the overall aim of obtaining further information, from which future and more precise hypotheses may be drawn Studies focusing on the generation and use of gene-targeted animal models also apply these goals and may be loosely categorized into sequential phases that become apparent as the use of the model progresses Initial studies of knockout models often focus on the plausibility of the model based on prior knowledge and whether the generation of an animal lacking the particular gene will prove lethal or not Upon the successful generation of a knockout, confirmatory studies are undertaken to corroborate previously established hypotheses of the function of the disrupted gene product As these studies continue, observations of unpredicted phenotypes or, more likely, the lack of a phenotype that was expected based on models put forth from past investigations are noted Often the surprising phenotype is due to the loss of a gene product that is downstream from the functions of the disrupted gene, whereas the lack of an expected phenotype may be due to compensatory roles filled by alternate mechanisms As the descriptive studies of the knockout continue, use of the model is often shifted to the role as a unique research reagent, to be used in studies that 1) were not previously possible in a wild-type model; 2) aimed at finding related proteins or pathways whose existence or functions were previously masked; or 3) the subsequent effects of the gene disruption on related physiological and biochemical systems The alpha ERKO mice continue to satisfy the confirmatory role of a knockout quite well As summarized in Table 4, the phenotypes observed in the alpha ERKO due to estrogen insensitivity have definitively illustrated several roles that were previously believed to be dependent on functional ER alpha, including 1) the proliferative and differentiative actions critical to the function of the adult female reproductive tract and mammary gland; 2) as an obligatory component in growth factor signaling in the uterus and mammary gland; 3) as the principal steroid involved in negative regulation of gonadotropin gene transcription and LH levels in the hypothalamic-pituitary axis; 4) as a positive regulator of PR expression in several tissues; 5) in the positive regulation of PRL synthesis and secretion from the pituitary; 6) as a promotional factor in oncogene-induced mammary neoplasia; and 7) as a crucial component in the differentiation and activation of several behaviors in both the female and male The list of unpredictable phenotypes in the alpha ERKO must begin with the observation that generation of an animal lacking a functional ER alpha gene was successful and produced animals of both sexes that exhibit a life span comparable to wild-type The successful generation of beta ERKO mice suggests that this receptor is also not essential to survival and was most likely not a compensatory factor in the survival of the alpha ERKO In support of this is our recent successful generation of double knockout, or alpha beta ERKO mice of both sexes The precise defects in certain components of male reproduction, including the production of abnormal sperm and the loss of intromission and ejaculatory responses that were observed in the alpha ERKO, were quite surprising In turn, certain estrogen pathways in the alpha ERKO female appear intact or unaffected, such as the ability of the uterus to successfully exhibit a progesterone-induced decidualization response, and the possible maintenance of an LH surge system in the hypothalamus [ABSTRACT TRUNCATED]

Estrogen receptor null mice: what have we learned and where will they lead us?

There is currently widespread interest in the IGFs (IGF-I and IGF-II) and their roles in the regulation of growth and differentiation of an ever increasing number of tissues are being reported. This selective review focused on the current state of our knowledge about the structure of mammalian IGFs and the multiple forms of mRNAs which arise from alternative splicing and promoter sites which arise from gene transcription. Current progress in the immunological measurement of the IGF is reviewed including different strategies for avoiding binding protein interference. The results of measurements of serum IGF-I and IGF-II in fetus and mother and at various stages of postnatal life are described. Existing knowledge of the concentration of these peptides in body fluids and tissues are considered. Last, an attempt is made to indicate circumstances in which the IGFs are exerting their actions in an autocrine/paracrine mode and when endocrine actions predominate. In the latter context it was concluded that an important role for GH action on skeletal tissues via hepatic production of IGF-I and endocrine action of IGF-I on growth cartilage is likely.

Insulin-Like Growth Factors I and II. Peptide, Messenger Ribonucleic Acid and Gene Structures, Serum, and Tissue Concentrations

PATHWAYS OF RECEPTOR-MEDIATED ENDOCYTOSIS 3 Entry Into Coated Pits 3 Intracellular Routes 4

Receptor-Mediated Endocytosis: Concepts Emerging from the LDL Receptor System

The insulin-like growth factors (IGFs) have both metabolic and growth-promoting activities in many cell and tissue types. Although the IGFs are present in serum, they are also thought to have important autocrine and paracrine functions. Using complementary DNA (cDNA) probes for rat IGF-I and mouse IGF-II, we have investigated the tissue distribution of messenger RNAs (mRNAs) for these growth factors in adult rats. IGF-I cDNA hybridized with three groups of transcripts, 7.0, 1.8 and 0.7-1.1 kilobases, which were detectable in all tissues examined, with liver demonstrating the highest level of expression. IGF-II cDNA also hybridized to a number of mRNAs, the most abundant of which was 4.0 kilobases. Of the tissues examined, IGF-II expression was highest in the brain, barely detectable in the liver, and undetectable under the conditions used, in lung, ovary, testes, and mammary gland. These studies support the notion of paracrine or autocrine function for IGF-I and demonstrate tissue-specific IGF-II expression in the adult rat.

Tissue distribution of insulin-like growth factor I and II messenger ribonucleic acid in the adult rat.

Estrogens stimulate the in vivo proliferation of epithelial cells of the mouse uterus. The cumulative evidence from several earlier studies suggests that the mitogenic effect of estrogens is mediated indirectly through a polypeptide growth factor. The primary focus of the present investigation was to determine whether an epidermal growth factor (EGF) -related polypeptide originates in the uterus of the immature or adult mouse under normal or altered estrogen status. Hybridization experiments revealed the presence of the 4.7- kilobase prepro-EGF mRNA in uteri of immature CD-I mice. The level of this mRNA was augmented at least 2-fold in immature mice treated for 4 days with estrogen, but levels remained markedly low compared to those in submaxillary gland or kidney. Two preparations of pooled uterine luminal fluid from estrogen-treated immature mice contained EGF immunoreactivity (1.2 and 1.7 ng/ml) that was stable in response to acid (50 mM acetic acid) and heat. Negligible EGF (<20 pg/uterus) was detecte...

Influence of Estrogens on Mouse Uterine Epidermal Growth Factor Precursor Protein and Messenger Ribonucleic Acid

The amino acid sequences of the human low-density lipoprotein (LDL) receptor and the human precursor for epidermal growth factor (EGF) show 33 percent identity over a stretch of 400 residues. This region of homologous is encoded by eight contiguous exons in each respective gene. Of the nine introns that separate these exons, five are located in identical positions in the two protein sequences. This finding suggests that the homologous region may have resulted from a duplication of an ancestral gene and that the two genes evolved further by recruitment of exons from other genes, which provided the specific functional domains of the LDL receptor and the EGF precursor.

Cassette of Eight Exons Shared by Genes for LDL Receptor and EGF Precursor

The effect of GH administration to hypophysectomized rats on expression of the c-myc proto-oncogene and insulin-like growth factor I (IGF-I) in the liver and kidney was examined. In both tissues maximal expression of c-myc occurred by 1 h after a single injection of human GH (100 micrograms/100 g bw). In the liver the maximal c-myc mRNA level was increased 12 +/- 3.9-fold (mean +/- SEM; n = 4), while the maximal c-myc level in the kidney was increased 3.4-fold (n = 2) compared to levels in basal hypophysectomized rats. In both the liver and kidney the IGF-I cDNA hybridized under stringent conditions to three transcripts with apparent sizes of 7.0, 1.8, and diffuse group of transcripts of 0.7-1.1 kilobases. Each of these transcripts demonstrated some degree of GH dependence. Under the hybridization condition used, the 7.0-kilobase IGF-I mRNA was virtually undetectable in the hypophysectomized control rat liver, while the smaller transcripts were easily detectable. Peak expression of each of the IGF-I transcripts occurred 6-12 h after GH administration. Maximal IGF-I expression in the kidney occurred 9 h after the GH injection. To determine whether the increase in c-myc expression following GH could result from a small but undetectable increase in IGF-I expression in these tissues, we administered human recombinant IGF-I to hypophysectomized rats (50 micrograms/100 g bw, ip). Despite a significant increase in serum IGF-I concentrations to levels greater than those present in the first 3 h after GH administration, no increase in c-myc expression was apparent in either the liver or kidney. These observations suggest that GH itself, rather than IGF-I, initiates the mitogenic response in the liver and kidney that follows GH administration to hypophysectomized rats.

Growth hormone stimulates sequential induction of c-myc and insulin-like growth factor I expression in vivo.

T h e n on sen se cod on , U G A , h as for th e first tim e recen tly b eensh ow n to en cod e selen ocystein e in tw o p rotein s, m ou seglu tath ion e p eroxid ase (G S H -P x) (E C 1.11.1.9) an d b acterialform ate d eh yd rogen ase. A co-tran slation al rath er th an p ost-tran slation al selen iu m -in corp oration m ech an ism h as b eenim p licated . F u rth erm ore, h igh exp ression levels of G S H -P xh ave su ggested th at su p p ression of term in ation is efficient andsp ecific. W e h ave isolated an d ch aracterized p itu itary, kidneyan d p lacen ta cD N A s for bovine , h u m an an d m ou se G S H -P xresp ectively. It is d em on strated th at th is n ovel su p p ressioneven t occu rs in d iverse tissu es, in at least th ree m am m aliansp ecies an d at th e tran slation al step . S u rp risin gly, G S H -P xis sh ow n to b e extram itoch on d rially en cod ed , in d icatin g acytosolic su p p ression even t rath er th an on e u tilizin g th em itoch on d ria's w ell-d ocu m en ted exten d ed cod on -read in gab ility. S eq u en ce an alysis reveals th at a sim p le p roxim alcon textu al p attern resp on sib le for read th rou gh d oes not exist.A n alysis of p red icted secon d ary stru cu tres of m R N A s,h ow ever, h as revealed a con form ation w h ich m ay b e u n iq u eto selen ocystein e p rotein s an d m ay p rove u sefu l as a tool forartificial in corp oration of selen ocystein es. A h u m an n itronfor G S H -P x from an u n sp lk ed m R N A h as b een isolated w hosep osition indicate s an an cien t, d ivergen t evolu tion aryrelation sh ip w ith thk>redoxin-S2, rath er th an an in d ep en d en tcon vergen t on e.Key words: codon usage/glutathione peroxidase/isom orphousreplacem ent/ selenocysteine/suppressionIn trod u ctionB ecause the selenocysteine residue (-C H

Graeme I. Bell

Papers

Tissue distribution of insulin-like growth factor I and II messenger ribonucleic acid in the adult rat.

Influence of Estrogens on Mouse Uterine Epidermal Growth Factor Precursor Protein and Messenger Ribonucleic Acid

Cassette of Eight Exons Shared by Genes for LDL Receptor and EGF Precursor

Growth hormone stimulates sequential induction of c-myc and insulin-like growth factor I expression in vivo.

Selenocysteine's mechanism of incorporation and evolution revealed in cDNAs of three glutathione peroxidases.