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The most widely studied “relaxed” mutant of the relAlocus, the relA1allele, is shown here to consist of an IS2 insertion between the 85th and 86th codons of the otherwise wild-type relAstructural gene, which normally encodes a 743-amino acid (84 kDa) protein. The RelA protein is a ribosome-dependent ATP:GTP (GDP) pyrophosphoryltransferase that is activated during the stringent response to amino acid starvation and thereby occasions the accumulation of guanosine 3′,5′-bispyrophosphate (ppGpp). We propose that the IS2 insertion functionally splits the RelA protein into two (α and β) peptide fragments which can complement each other in trans to yield residual ppGpp synthetic activity; neither fragment shows this activity when expressed alone.

During the acute phase response to inflammation the plasma concentration of some proteins, such as α(1-acid glycoprotein (AGP), increases dramatically. Since breakdown and remodeling of tissue is common to both nidation and inflammation we studied the tissue distribution and regulation of AGP mRNA levels during the embryonic development of the rat. High levels of mRNA coding for AGP were detected in the placenta during early fetal development. Expression of this mRNA was confined to the decidua and was first observed approximately 1 day after implantation when proliferation of the decidua is already well advanced. Maximum levels were attained about 5 days after implantation, after which the levels decreased rapidly. In contrast to the high levels of AGP mRNA in the decidua only very low levels were detected in fetal liver and visceral yolk sac, and there was only a small increase in the levels in maternal liver. Corticosteroid hormone responsiveness of AGP mRNA synthesis by hepatocytes appeared 3 days before birth. It is likely that the synthesis of AGP by the cells of the decidua is important in establishing the precisely controlled interaction between mother and embryo during nidation.

Levels of transferrin mRNA were measured by hybridization to transferrin cDNA in extracts from various areas of rat brain and other tissues. The highest concentrations of transferrin mRNA were found in the liver and the choroid plexus of the lateral and third ventricles. Lower concentrations were observed in the medulla and thalamus, choroid plexus of the fourth ventricle, cortex, hypothalamus, cerebellum, pituitary, testis, placenta, stomach, spleen, kidney, muscle, and heart. Yolk sac, small intestine, and adrenal glands did not contain detectable transferrin mRNA levels. The size of transferrin mRNA was the same in liver, brain, and testis. Upon incubation of choroid plexus pieces with [14C]leucine in vitro, about 4% of the radioactive protein secreted into the medium was found to be transferrin. Together with previous data (Dickson, P.W., Howlett, G.J., and Schreiber, G. (1985) J. Biol. Chem. 260, 8214-8219; Dickson, P.W., Aldred, A.R., Marley, P.D., Bannister, D., and Schreiber (1986) J. Biol. Chem. 261, 3475-3478) the obtained data suggest that the choroid plexus plays a role in maintenance of homeostasis in the microenvironment of the central nervous system by synthesizing and secreting plasma proteins.

The method of competition kinetics, which measures the binding kinetics of an unlabeled ligand through its effect on the binding kinetics of a labeled ligand, was employed to investigate the kinetics of muscarinic agonist binding to rat brain medulla pons homogenates. The agonists studied were acetylcholine, carbamylcholine, and oxotremorine, with N-methyl-4-[3H]piperidyl benzilate employed as the radiolabeled ligand. Our results suggested that the binding of muscarinic agonists to the high affinity sites is characterized by dissociation rate constants higher by 2 orders of magnitude than those of antagonists, with rather similar association rate constants. In contrast, the major differences between the kinetic binding parameters of agonists and antagonists to the low affinity agonist binding sites are in the association rate constants, which were 2-5 orders of magnitude lower for agonists. This demonstrates that there are basic differences in the interactions of agonists with the low and high affinity sites. Our findings also suggest that isomerization of the muscarinic receptors following ligand binding is significant in the case of antagonists, but not of agonists. Moreover, it is demonstrated that in the medulla pons preparation, agonist-induced interconversion between high and low affinity bindings sites does not occur to an appreciable extent.

We propose a method for obtaining kinetic parameters of unlabeled ligands which is based on analyzing the effect of their competition on the binding kinetics of a labeled ligand. In this method (competition kinetics), the binding kinetics of a labeled ligand are measured in the absence and in the presence of given concentrations of a competing unlabeled ligand. The rate equations appropriate to the particular kinetic model may be solved employing linear homogeneous differential equations. The rate constants appearing in the final integrated expressions can be evaluated by nonlinear regression. We have demonstrated the validity and applicability of the method to study receptor systems, using the muscarinic receptors in rat brain cortex as an experimental system. We have determined the various kinetic parameters of two muscarinic antagonists (N-methyl-4-[3H]piperidyl benzilate and (-)-N-[3H]methylscopolamine) by following directly their binding kinetics. These parameters were compared with those obtained for the same unlabeled ligands in homogeneous and heterogeneous competition studies. In all cases, the parameters obtained by the competition kinetics method were close to those extracted from direct binding kinetics, and in cases where both types of studies were performed on the same preparations, the parameters obtained by the two methods were essentially identical.

The relA gene product of Escherichia coli is known to be responsible for the synthesis of guanosine 3',5'-bispyrophosphate (ppGpp) during the stringent response to amino acid starvation. This report presents the sequence of the relA gene region and assignment of its 743-codon open reading frame by the following criteria: 1) genetic complementation of ppGpp synthesis in a relaxed (relA1) mutant during the stringent response; 2) changes in 3-aminotriazole resistance during growth to mimic a relA+ phenotype; 3) verification of the presence of an amber codon at the normal carboxyl terminus of the relA gene; and 4) immunological assays of expression of the RelA protein. The apparent molecular mass of the cloned relA gene product is calculated to be 83,856 daltons and as visualized by immunoblotting is identical to that of the previously characterized protein. A promoter has been identified that directs relA gene transcription towards the pyrG gene, in a counterclockwise direction on the E. coli chromosome. Genomic Southern blot analyses verify that the relA regions cloned and subjected to nucleotide sequence analysis correspond to homologous regions on the E. coli chromosome.

The rat transthyretin gene, 7.3 kilobase pairs (kb) long, with 14.5 kb of 5' flanking and 12.2 kb of 3' flanking region was cloned and characterized. The gene contained four exons. A “TATA box” sequence (5'-TATATAA-3') and a “CAAT box” sequence (5'-GTCAAT-3') were located 23 and 95 nucleotides upstream, respectively, from the major transcription start site. Nucleotides -51 to -189 were highly conserved (93% homology between rats and humans, 97% homology between rats and mice). Tandem repeats of sequences of 5'-AC-3' and 5'-ACACATGC-3' in the 5' flanking region, of 5'-GAAA-3' in the first intron, and of 5'-GT-3' in the third intron of the gene were observed. Using specific cDNA probes, tissue specificity and regulation of transthyretin mRNA biosynthesis during embryogenesis were analyzed. Transthyretin expression occurred first in the yolk sac, then decreased when expression increased in fetal liver. Presumptive choroid plexus cells in the inner lining of the neural tube expressed transthyretin early in gestation (11 days before birth) with a maximum immediately preceding the spurt of brain growth around birth. Partial hepatectomy of adult rats induced both an acute phase response and regenerative growth in liver. The decrease in transcription of the transthyretin gene in liver, which is characteristic for the acute phase response, was overridden by stimulation of gene expression after partial hepatectomy. This stimulation also affected transthyretin expression in choroid plexus.

Transthyretin cDNA was isolated from a rat liver cDNA library. Analysis of the nucleotide sequence revealed a signal peptide-like sequence preceding a section coding for a full length subunit and an untranslated sequence at the 3' end. The deduced primary structure of rat transthyretin was compared with that of human transthyretin. It was highly conserved at the binding sites for thyroxine and the interfaces and core regions of the subunits. The cDNA for transthyretin was used to measure mRNA levels by hybridization. During acute inflammation, the amount of transthyretin mRNA in liver decreased (reaching a minimum of 25% of the normal level 36 h after inducing inflammation), suggesting regulation of transthyretin synthesis at the mRNA level. Transthyretin mRNA was found only in the liver and in the choroid plexus, but not in other parts of the central nervous system nor in the adrenal glands, kidney, spleen, testes, heart, lung, intestine, and ovaries. One gram of choroid plexus contained about 25 times larger amounts of transthyretin mRNA than 1 g of liver. By synthesizing an important hormone carrier protein, the choroid plexus may be an important link in the chemical communication between the central nervous system and the bloodstream.

A rat ceruloplasmin cDNA clone was isolated from a rat liver cDNA library and identified by partial nucleotide sequence analysis. Rat liver ceruloplasmin mRNA levels were measured during the acute phase response to inflammation by cytoplasmic dot hybridization to ceruloplasmin cDNA. Regulation of ceruloplasmin synthesis appeared to be at the mRNA level, with the concentration of ceruloplasmin mRNA increasing significantly 12 h after induction of inflammation, reaching a maximum of 350% of normal at 36 h and returning to normal levels within 60 h. Using Northern blot analysis, extrahepatic ceruloplasmin gene expression was observed in choroid plexus, yolk sac, placenta, and testis. All these tissues are at the interface between, and possibly involved in maintaining homeostasis in, adjacent extracellular compartments. No ceruloplasmin mRNA was detected in RNA from stomach and small intestine.

The rat apolipoprotein E gene was isolated from a genomic library by screening with a cDNA probe. The nucleotide sequence of the gene plus a 5' flanking region of 623 nucleotides and a 3' flanking region of 234 nucleotides was determined. The gene contained three introns, which were located in the 5' nontranslated region and in the regions coding for the signal peptide and the mature protein. A "TATA box" sequence TATAATT was found beginning at nucleotide -32. Two copies of the sequences GGGCGG or CCGCCC, potential binding sites for the transcription protein factor Sp 1, were found adjacent to the TATA box, beginning at nucleotides -52 and -72. The 5' proximal flanking region, up to about 140 nucleotides, was found to be highly conserved (82% homology) in the gene for rat and human. This region was GC-rich (68% G + C) and contained self-complementary sequences with the potential to form hairpin loop structures, suggesting a regulatory role of this region in gene transcription. Two open reading frames with promoter sequences were found, one located in the first and the other in the second intron. Expression of rat apolipoprotein E mRNA in murine L cells transfected with the cloned gene was detected at levels comparable to that in rat liver. Rat apolipoprotein E expressed in transfected cells was secreted from the cells into the medium.

Two major acute phase alpha 1-protein (alpha 1-MAP, also called thiostatin) genes, one kininogen gene and one structurally related pseudogene, were isolated from a Buffalo rat genomic library and characterized. Comparison of the nucleotide sequence in the 5' proximal flanking region (1 kilobase) of a strongly inducible alpha 1-MAP gene with that in the non-inducible rat kininogen gene showed an overall homology of 90%, with a small number of randomly distributed base changes. In the intron downstream of the exon coding for Ile-Ser-bradykinin, the two alpha 1-MAP genes contained sequence sections similar to the section in the human kininogen gene which codes for the carboxyl-terminal chain of high molecular weight kininogen. Various features of the DNA related to gene expression such as initiation and termination sites of transcription, receptor binding sites, a Z-DNA section, and exon/intron splicing sites were identified. mRNAs expressed by alpha 1-MAP and kininogen genes in liver were studied by RNA blot hybridization using specific oligonucleotide probes. The kininogen gene expressed two mRNA species, one coding for high molecular weight kininogen, the other coding for low molecular weight kininogen. The levels of the two kininogen mRNAs in liver did not increase during acute inflammation. Only one type of mRNA, similar in size to low molecular weight kininogen RNA, was expressed by each of the two alpha 1-MAP genes. The levels of both alpha 1-MAP mRNAs increased strongly during acute inflammation.

The role of the choroid plexus in thyroid hormone transport between body and brain, suggested by strong synthesis and secretion of transthyretin in this tissue, was investigated in in vitro and in vivo systems. Rat choroid plexus pieces incubated in vitro were found to accumulate thyroid hormones from surrounding medium in a non-saturable process. At equilibrium, the ratio of thyroid hormone concentration in choroid plexus pieces to that in medium decreased upon increasing the concentration of transthyretin in the medium. Fluorescence quenching of fluorophores located at different depths in liposome membranes showed maximal hormone accumulation in the middle of the phospholipid bilayer. Partition coefficients of thyroxine and triiodothyronine between lipid and aqueous phase were about 20,000. After intravenous injection of 125I-labeled thyroid hormones, choroid plexus and parts of the brain steadily accumulated 125I-thyroxine, but not [125I]triiodothyronine, for many hours. The accumulation of 125I-thyroxine in choroid plexus preceded that in brain. The amount of 125I-thyroxine in non-brain tissues and the [125I]triiodothyronine content of all tissues decreased steadily beginning immediately after injection. A model is proposed for thyroxine transport from the bloodstream into cerebrospinal fluid based on partitioning of thyroxine between choroid plexus and surrounding fluids and binding of thyroxine to transthyretin newly synthesized and secreted by choroid plexus.

Synthesis of total protein and of transthyretin in rat choroid plexus was studied by measuring the incorporation of radioactive leucine into proteins in choroid plexus tissue incubated in vitro. About 20% of the protein newly synthesized in choroid plexus and about 50% of the newly synthesized protein secreted into the medium was transthyretin. Evidently, the choroid plexus is very active in the biosynthesis of this carrier protein for thyroid hormones and could be an important link in the chemical communication between the body and the central nervous system. Acute inflammation, which leads to a profound rearrangement of the pattern of plasma protein synthesis rates in the liver, produced distinct changes in the levels for plasma protein mRNAs in the liver. The levels of the mRNAs for alpha 1-acid glycoprotein and major acute phase alpha 1-protein increased more than 30-fold, those for transthyretin and albumin decreased to 27 and 57% of normal, respectively. The pattern of the observed changes in the levels of mRNAs for plasma proteins in the liver was independent of whether the acute inflammation was produced by subcutaneous injection of turpentine or intraperitoneal injection of a suspension of talcum. However, levels of transthyretin mRNA in choroid plexus were affected only very slightly, or not at all. Apparently, transthyretin synthesis in liver and choroid plexus is regulated independently during the acute phase response. No mRNA was detected in choroid plexus for albumin, alpha 1-acid glycoprotein, and major acute phase alpha 1-protein under any conditions.

The regulation of the synthesis of plasma proteins in rat liver during the acute phase response was studied by measuring gene transcription activities in a cell-free nuclear transcription system. The transcription activities for the genes of major acute phase alpha 1-protein, the beta-chain of fibrinogen, transferrin, alpha 1-acid glycoprotein, and alpha 2-macroglobulin increased, reaching a maximum level between 18 and 36 h after inducing an acute inflammation. The transcription activities for the genes of alpha 2u-globulin, albumin, and transthyretin (formerly called prealbumin) decreased, reaching a minimum level after 12 to 24 h. The extent of the relative changes in transcription activities was similar to that of the relative changes in mRNA levels for major acute phase alpha 1-protein, the beta-chain of fibrinogen, transferrin, alpha 2u-globulin, albumin, and transthyretin. This is consistent with the assumption that the principal mechanism of the regulation of the synthesis of these proteins operates at the level of transcription. In contrast, the relative changes in transcription activities for alpha 1-acid glycoprotein and alpha 2-macroglobulin were far smaller than the changes of their mRNA levels, suggesting that, in addition to transcriptional changes, other mechanism(s) are involved in the regulation of the synthesis of these proteins.