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In human beings, serum transferrin levels increase during iron deficiency and decrease with iron overload. Yet, whether or not iron levels actually affect the synthesis of transferrin in human liver cells is not known. In previous studies, iron was shown to suppress the expression of chimeric human transferrin genes in livers of transgenic mice. The goal of this study was to determine if iron suppresses intact endogenous human transferrin synthesis by testing the effects of changes in iron levels on synthesis of transferrin in a human hepatoma cell line HepG2. In HepG2 cells, normalized(35)S-metabolically labeled transferrin synthesis was consistently less following iron treatment with hemin or ferric citrate, than following treatment with an iron-chelator deferroxamine. Thus, this study provides new evidence that iron can regulate synthesis of intact endogenous human transferrin., (Copyright 2000 Academic Press.)

Transcription of the autosomal phosphoglycerate kinase gene, Pgk-2, is initiated at the onset of meiosis during spermatogenesis in mammals. However, in the mouse, the 5' portion of the endogenous Pgk-2 coding sequence undergoes a specific demethylation event that precedes transcriptional activation by 10-12 days. Here we show that transgenes consisting of the Pgk-2 core promoter ligated to the CAT reporter gene undergo a similar tissue-, stage-, and cell type-specific demethylation in the 5' portion of the CAT coding sequence, whereas transgenes consisting of the CAT reporter sequence alone, or of the CAT sequence ligated to the CpG island-containing transferrin gene promoter, demonstrate different patterns of demethylation. These results indicate that specific promoter sequences can influence the pattern of tissue-specific demethylation within different genes and that a signal for spermatogenic cell-specific demethylation resides within the core promoter of the mammalian Pgk-2 gene.

Human transferrin mRNA contains a 5'-untranslated region that (1) has homology to an iron responsive element and (2) is implicated in translational iron regulation of human transferrin transgenes in transgenic mice. Ferritin mRNA contains a 5'-untranslated region iron-responsive element, but iron regulation of ferritin differs from that of human transferrin transgenes in both magnitude and direction. Structural differences between the ferritin iron-responsive element and the human transferrin putative iron-responsive element may influence their iron-regulatory protein interactions and direct the differing translational responses. This study examines human transferrin RNA nucleotide sequence requirements for binding of cytoplasmic proteins and purified iron-regulatory protein. Mutations of the putative transferrin iron-responsive element similarly affected binding of purified iron-regulatory protein and liver cytoplasmic proteins, providing evidence that the IRP is one of the liver cytoplasmic proteins that binds the human transferrin iron-regulatory element and suggesting that it may be involved in iron-regulation of transferrin.

Four independent lines of transgenic mice were produced carrying integrated copies of a chimeric gene composed of 3.3 kb of the human haptoglobin 5' regulatory region fused to the CAT (chloramphenicol acetyl transferase) reporter gene. Although the endogenous mouse haptoglobin (Hp) and human haptoglobin (HP) genes express mainly in liver and lung, expression of the human 3.3-kb HP-CAT transgene was not detected until after induction of inflammation and then only in lungs. The results indicated that the transgene maintained the regulatory DNA elements required for lung specific responsiveness to inflammation in vivo but lacked the DNA sequence required for robust expression in liver. The DNA sequence(s) responsible for the normally high level of HP expression in liver either reside outside the 3.3-kb regulatory region of the HP chimeric gene or this region contains a suppressor sequence affecting tissue specific expression in the liver.

O6-Methylguanine DNA methyltransferase (MGMT; EC 2.1.1.63) is an unusual DNA repair protein in that it directly and specifically repairs a premutagenic DNA lesion without involving other proteins. MGMT removes the alkyl group from O6-alkylguanine in DNA in a unique stoichiometric reaction by accepting the alkyl group on a cysteine residue. The intracellular level of MGMT varies among tissues and appears to be inversely correlated to tissue-specific tumorigenesis induced by monofunctional alkylating agents. Because MGMT acts in solo, genetic manipulation of its expression may provide valuable insight into its contribution to cellular resistance to alkylation toxicity and to tumor induction. The human MGMT full length cDNA has been fused with a portion of the human transferrin (TF) 5'-flanking region (TF/MGMT). Transgenic founder mice were produced carrying the TF/MGMT transgene and then bred to establish stable transgenic lines. Human MGMT transcripts were specifically expressed in abundance in transgenic brain and liver tissues. In vitro MGMT assays revealed approximately 150-fold and approximately 25-fold increases in MGMT activity in transgenic brain and liver extracts respectively. Western blot analysis confirmed that human MGMT protein is specifically synthesized in transgenic brain and liver tissues.

The major iron-transport protein in serum is transferrin (TF) which also has the capacity to transport other metals. This report presents evidence that synthesis of human TF can be regulated by the metal lead. Transgenic mice carrying chimeric human TF-chloramphenicol acetyl transferase (CAT) genes received lead or sodium salts by intraperitoneal injections or in drinking water. Transgene expression in liver was suppressed 31 to 50% by the lead treatment. Lead regulates human TF transgenes at the mRNA level since liver CAT enzyme activity, CAT protein, and TF-CAT mRNA levels were all suppressed. The dosages of lead did not alter synthesis of the other liver proteins, mouse TF and albumin, as measured by Northern blot analysis of total liver RNA and rocket immunoelectrophoresis of mouse sera. Moderate levels of lead exposure were sufficient to evoke the human TF transgene response; blood lead levels in mice that received lead acetate in drinking water ranged from 30 micrograms/dl to 56 micrograms/dl. In addition to suppressing expression of TF-CAT genes in transgenic mice, lead also suppressed synthesis of TF protein in cultured human hepatoma HepG2 cells. The regulation of human TF apparently differs from the regulation of mouse TF which is unresponsive to lead exposure.

Transferrin, the transferrin receptor, and ferritin are integral to the body's management of iron, an element required for life but highly toxic when present in excess. The transferrin receptor and ferritin are regulated posttranscriptionally by iron: the transferrin receptor by mRNA stability and ferritin by mRNA translation. Results described here indicate that transferrin, like ferritin, is regulated by iron at the level of translation. Chimeric genes introduced into the mouse genome were composed of the human transferrin 5' regulatory region fused to the chloramphenicol acetyl transferase (CAT) reporter gene. Iron administration to transgenic mice resulted in a significant decrease of transferrin-directed CAT enzyme activity and CAT protein in liver, but no significant decrease in human transferrin-CAT mRNA levels. Binding of specific RNA iron regulatory elements by proteins in cytoplasmic extracts have been shown to regulate ferritin and transferrin receptor synthesis. Similar results have been obtained with transferrin mRNA. A decreased binding of human transferrin 5'-untranslated region RNA by factors in cytoplasmic extracts of livers from mice receiving iron was found when compared to extracts from control mice. A human transferrin RNA-protein complex migrated electrophoretically with the same mobility as a ferritin iron responsive element RNA-iron responsive element binding protein complex. The ferritin iron responsive element RNA also competed with the human transferrin 5'-untranslated region RNA-protein complexes formed and vice versa. Therefore, iron modulation of human transferrin may share a factor common or similar to that observed in ferritin and transferrin receptor iron modulation.

Transferrin (TF) gene expression is tissue specific and is regulated during development. Transgenic mice have been developed which carry 1.2 or 0.67 kb of the TF 5' flanking region of the human TF gene fused to the bacterial chloramphenicol acetyltransferase (CAT) gene. The onset of expression of the chimeric human TF-CAT transgenes in liver and brain during development has been studied in these transgenic mice. In brain, the TF(0.67)CAT transgene began to express between 5 and 10 days after birth; in liver, the TF(0.67)CAT transgene was turned on between 10 and 20 days after birth. Endogenous mouse TF mRNA levels in liver and brain have also been measured during development by Northern analysis. In brain, the developmental expression pattern of the TF(0.67)CAT transgene is the same as the mouse endogenous TF gene; in liver, the transgene is turned on later than the endogenous mouse TF gene. DNA-protein mobility shift assays and DNase I footprinting analyses were conducted in the region of -621 to -409 bp of the human TF gene by using TF-CAT expressing liver nuclear extract from 27-day-old mice and nonexpressing liver nuclear extract from 7-day-old mice. The level of protein-DNA complex formation is several times higher in the expressing extracts, and the region from -481 to -463 bp of human TF gene is protected by the expressing extract but not the nonexpressing extracts. As demonstrated by this and other studies, the transgenic mouse model furnishes a unique opportunity to analyze developmental regulation of human transgenes.

In order to determine whether all the extrinsic salivary glands synthesize transferrin mRNA, the polyadenylated ribonucleic acids [poly(A)+ RNAs] from parotid, submandibular, and sublingual glands, liver, midbrain, testis, spleen, heart, kidney, and the mucosae of oesophagus and stomach from adult male baboons were analysed, using oligo(dT)-cellulose chromatography, agarose gel electrophoresis, followed by transfer of the mRNAs to nitrocellulose filters and identification with transferrin and tubulin cDNA probes. Transferrin and tubulin mRNAs were visualized by autoradiography and analysed by measuring specific activity from beta emitting nuclides following transfer to nitrocellulose filters and hybridizing with [alpha-32P]-labelled human transferrin and tubulin cDNA probes. The results indicate that transferrin mRNA is present in all the extrinsic salivary glands (submandibular, sublingual, parotid) of baboons.

Transgenic mice provide a means to study human gene expression in vivo throughout the aging process. A DNA sequence containing 668 bp of the 5' regulatory region of the human transferrin gene was fused to the bacterial reporter gene chloramphenicol acetyl transferase (TF-CAT) and introduced into the mouse genome. Expression of the human chimeric transferrin gene was similar to the tissue patterns of mouse and human transferrin. In aging transgenic mice, expression of the human chimeric transferrin gene was found to diminish 40% in livers between 18 and 26 months of age. Transferrin levels and serum iron levels in aging humans also diminish, as observed from measurements of total iron binding capacity and percent iron saturation in sera from 701 individuals ranging from 0 to 99 years of age. In contrast, in transgenic mice and nontransgenic mice, the mouse endogenous plasma transferrin and endogenous Tf mRNA increase significantly during aging. Neither the decrease of human TF-CAT nor the increase of mouse transferrin during aging appears to be part of a typical inflammatory reaction. Although the 5' regions of the human transferrin and mouse transferrin genes are homologous, sequence diversities exist which could account for the different responses to inflammation and aging observed.

Introduction of human plasma protein genes into the mouse genome to produce transgenic mice furnishes an in vivo model for correlating chromosomal DNA sequences with developmental and tissue-specific expression. The liver produces an array of plasma proteins that circulate throughout the body contributing to homeostasis. Non-hepatic tissue sites of synthesis have been identified where a local provision of plasma proteins is needed. Analysis of expression of human plasma protein genes in ageing transgenic mice appears especially promising in identifying DNA sequences that respond to environmental adversities such as inflammatory factors, hormonal changes and metal toxicity. The results indicate that human genes encoding and controlling liver plasma proteins serve as useful models for studying genetic regulation in the background of development and ageing.

Expression of genes encoding transferrin and the vitamin D-binding protein is described in a cell line, U-2 OS, derived from a human osteogenic sarcoma. The mRNA transcripts of transferrin and vitamin D-binding protein were shown to be the lengths of those found in normal human liver. The cells synthesize and secrete the transferrin and vitamin D-binding proteins, in addition to human albumin and ceruloplasmin. The U-2 OS cells were successfully transfected with chimeric genes carrying 670 bp of the 5' regulatory sequence of the human transferrin gene fused to a reporter chloramphenicol acetyltransferase gene. These data indicate that the appropriate transcriptional factors required for expression of four plasma proteins are produced by U-2 OS nuclei and that the U-2 OS cell line will be useful for studies analyzing regulation of these genes.

The iron-binding plasma protein transferrin (TF) is essential for supplying iron to cells and the prevention of iron toxicity. Our laboratory has cloned and characterized the human TF gene. Comparison of promoter regions of TF genes from human, chicken, and mouse reveals a strong nucleotide sequence conservation. This study demonstrates that 5' flanking regions of the TF gene are sufficient for directing expression of a heterologous gene in transgenic mice and transfected cells. For cell-specific expression, more than 150 base pairs appear to be required.

Characterization of the 5'-flanking region of the DBP genomic sequence is reported. Nucleotide sequences that serve as cis-regulatory elements for transcription in other genes have been found and include metal regulatory elements, viral enhancers, adipocyte and mitotic signals. The promoter region of DBP is not homologous to the 5'-flanking region of the albumin and alpha-fetoprotein genes despite the strong protein homology and evolutionary relationship among the three proteins.

Using both in vitro translation and hybridization to an adenosine deaminase (ADA) cDNA probe, ADA mRNA has been characterized in B lymphoblast lines established from seven ADA-deficient children, two parents of an ADA-deficient child, and three normal people. All ADA-deficient lines except GM-2825A, including those with less than 1% of normal catalytic activity, had normal or greater amounts of hybridizable, 1.6 kilobase in size, ADA mRNA. Immunoreactive ADA protein of normal size was produced by in vitro translation of the mRNAs. Deficiency of ADA activity in these lines appears secondary to synthesis of structurally altered proteins rather than to a quantitative deficiency in ADA mRNA. The GM-2825A line contains electrophoretically abnormal species of RNA which hybridize to the cDNA probe. Deficiency of ADA activity in this line appears at least in part secondary to a structural defect in the ADA mRNA or its precursors.

Tetrahymena pyriformis contains an enzyme which hydrolyzed dolichyl phosphate. This activity was solubilized from lyophilized samples of this organism and was relatively stable when stored frozen. The soluble enzyme preparation had an acid pH optimum and hydrolyzed both dolichyl and phytanyl phosphates at equivalent rates. The polyprenylphosphate phosphatase activity was compared with the acid phosphatases which hydrolyzed p-nitrophenyl phosphate and marked differences were found. Dolichyl phosphate hydrolysis required Mg2+ for maximum activity while the bulk of the phosphatase activity was not effected by the absence of this ion. Other differences were that the polyprenylphosphate phosphatase was relatively insensitive to inhibitors such as tartrate and vanadium oxide sulfate which had a pronounced effect on the rate of p-nitrophenyl phosphate hydrolysis. The two activities also appeared to have different subcellular distributions. The polyprenylphosphate phosphatase was markedly inhibited by ethoxy formic anhydride, a reagent which is active against enzymes containing a histidine residue at their active site, while p-nitrophenyl phosphate hydrolysis was unaffected. The polyprenylphosphate phosphatase may be important in regulating the level of dolichyl phosphate in T. pyriformis and thus the rate of glycoprotein synthesis. It is also a useful tool which is capable of liberating dolichol from dolichyl phosphate under mild conditions which will permit the further characterization of the polyprenols.

Dolichols of Tetrahymena pyriformis were isolated and characterized by TLC, HPLC and mass spectrometry. Four strains of Tetrahymena were studied and found to have relatively small amounts of dolichol, from 0.26 to 2.60 mg dolichol/kg wet weight. All four strains had approximately the same relative proportions of isoprenologs, dolichol-13 (2%), dolichol-14 (74%), dolichol-15 (23%), and dolichol-16 (less than 1%). Tetrahymena dolichols were found mainly in the mitochondrial subcellular fraction (86%). The pellicle fraction contained 9% and the microsomal fraction, 5% of the remaining dolichol. Free dolichol has also been found in the mitochondrial fraction of four other organisms. We were not able to demonstrate dolichyl esters in these organisms, but their presence is inferred, because reduced yields of dolichol were obtained if the lipid extracts were not saponified prior to HPLC assay.

The nucleotide sequence for an unusual, cloned human adenosine deaminase cDNA has been determined. Contained within a sequence of 1535 nucleotides is a coding sequence of 1089 nucleotides that encodes a protein of 40,762 daltons. The coding sequence is interrupted by a non-coding region containing 76 nucleotides. Both the 3' and 5' ends of this region have consensus sequences generally associated with splice sites. The 3' untranslated sequence contained 308 nucleotides, including a polyadenylation signal sequence 20 nucleotides from the end. The cloned cDNA appears to correspond to a nuclear mRNA precursor which contains a small intron.

A dolichyl palmitate esterase was found in cell-free extracts of both pancreas and intestinal mucosa. The substrate for the reaction was dolichyl palmitate that was synthesized with labeled fatty acid. The reaction was monitored by the liberation of the free fatty acid and HPLC. All polyprenol esters studied were hydrolyzed despite differences in chain length. The role of this enzyme might be to promote the absorption of dolichol from the diet.

Transferrin is a major plasma protein that transports iron to proliferating cells throughout the body. A clone containing the 5' region of the human transferrin gene has been isolated and characterized. A 14 kb EcoRI fragment was identified that contained the first 8 exons of the transferrin gene and 3.6 kb of its 5' flanking region. Conserved sequences identical or homologous to regulatory elements responding to heavy metals, glucocorticoid receptor and a putative acute phase reaction signal were identified in the 5'flanking region and intron 1. Also, the regulatory region of the transferrin gene contains a 14-bp sequence which closely matches sequences found in the interleukin-2 and gamma-interferon genes. All three genes are expressed by T lymphocytes before proliferation. A secondary loop structure similar to that proposed for the ovotransferrin gene can be formed by sequences in the 5' untranslated region of the transferrin mRNA.