Platelet-derived growth factor-stimulated expression of the MCP-1 immediate-early gene involves an inhibitory multiprotein complex.

We have demonstrated previously that the seven-nucleotide (nt) motif TTTTGTA (the heptamer) that is present within the proximal 3' untranslated sequences of numerous immediate-early genes is essential for platelet-derived growth factor (PDGF)-stimulated induction of the MCP-1 immediate-early gene. On this basis, the heptamer was suggested to be a conserved regulatory element involved in immediate-early gene expression, although its mechanism of action was unknown. Herein, we demonstrate that the heptamer functions to remove an inhibition of PDGF induction of MCP-1 maintained by two independently acting inhibitory elements present in the MCP-1 5' flanking sequences (designated I* elements). PDGF treatment relieves the I*-mediated inhibition of MCP-1 expression only if the heptamer is also present. One inhibitory element is contained within a 59-nt portion of MCP-1 5' flanking sequences and functions in an orientation-independent and heptamer-regulated manner. Significantly, proteins binding to two DNA sequences contribute to the formation of a single multiprotein complex on the 59-nt I* element. The I*-binding complex contains Sp3, an Sp1-like protein, and a novel DNA-binding protein. Moreover, the complex does not form on two 59-nt sequences containing mutations that reverse the inhibition of PDGF induction maintained by the wild-type I* element. We propose to call the multiprotein I*-binding complex a repressosome and suggest that it acts to repress PDGF-stimulated transcription of MCP-1 in the absence of the heptamer TTTTGTA.

Platelet-derived growth factor induction of the immediate-early gene MCP-1 is mediated by NF-kappaB and a 90-kDa phosphoprotein coactivator.

A broad panel of agents including serum, interleukin-1, double-stranded RNA, and platelet-derived growth factor (PDGF) stimulate transcription of the "slow" immediate-early gene MCP-1. These disparate inducers act through a tight cluster of regulatory elements in the distal 5'-flanking sequences of the MCP-1 gene. We describe a 22-base element in this cluster which, in single copy, confers PDGF-inducibility to a tagged MCP-1 reporter gene. In mobility shift assays, the element binds a PDGF-activated form of NF-kappaB, and a 90-kDa protein (p90) which binds constitutively. Antibody supershift and UV cross-linking experiments indicate that the PDGF-activated NF-kappaB species is a Rel A homodimer. The DNA binding form of p90 is a nuclear-restricted serine/threonine phosphoprotein. Mutagenesis of the 22-base element shows that the NF-kappaB and p90 binding sites overlap, but binding of the two species is mutually independent. Both sites, however, are required for optimum PDGF induction of MCP-1. Therefore, p90 appears to be a coactivator with NF-kappaB in PDGF-mediated induction of MCP-1.

A new platelet-derived growth factor-regulated genomic element which binds a serine/threonine phosphoprotein mediates induction of the slow immediate-early gene MCP-1.

The MCP-1 chemokine gene belongs to a cohort of immediate-early genes that are induced with slower kinetics than c-fos. In this study, we identified a cluster of four platelet-derived growth factor (PDGF)-responsive elements within a 240-bp enhancer found in the distal 5' flanking MCP-1 sequences. Two of the elements bind one or more forms of the transcription factor NF-kappa B. We focused on the other two elements which are hitherto unreported, PDGF-regulated genomic motifs. One of these novel elements, detected as a 28-mer by DNase I footprinting, restores PDGF inducibility when added in two copies to a 5' truncated MCP-1 gene. A single copy of the second novel element, a 27-mer, restores PDGF inducibility to a 5' truncated MCP-1 gene. The 27-base element interacts with a PDGF-activated serine/threonine phosphoprotein that is detected only within the nucleus of PDGF-treated 3T3 cells. DNA binding of this phosphoprotein is activated by PDGF treatment with slow kinetics that match the time course of MCP-1 gene expression, and activation is not inhibited by cycloheximide. PDGF-activated binding to the 27-mer is shown to involve a single 30-kDa protein by UV-cross-linking analysis.

A novel 7-nucleotide motif located in 3' untranslated sequences of the immediate-early gene set mediates platelet-derived growth factor induction of the JE gene.

A cohort of the serum and growth factor regulated immediate-early gene set is induced with slower kinetics than c-fos. Two of the first immediate-early genes characterized as such, c-myc and JE, are contained within this subset. cis-acting genomic elements mediating induction of the slower responding subset of immediate-early genes have never been characterized. Herein we characterize two widely separated genomic elements which are together essential for induction of the murine JE gene by platelet-derived growth factor, serum, interleukin-1, and double-stranded RNA. One of these elements is novel in several regards. It is a 7-mer, TTTTGTA, found in the proximal 3' sequences downstream of the JE stop codon. The 3' element is position dependent and orientation independent. It does not function in polyadenylation, splicing, or destabilization of the JE transcript. Copies of the 7-mer or its inverse are found at comparable 3' sites in 25 immediate-early genes that encode transcription factors or cytokines. Given its general occurrence, the 7-mer may be a required cis-acting control element mediating induction of the immediate-early gene set.

Regulation of murine oocyte meiosis: evidence for a gonadotropin-induced, cAMP-dependent reduction in a maturation inhibitor.

We have developed an assay that can detect relative changes in the amount of a non-cAMP inhibitor of maturation present in cumulus cells (Eppig et al., 1983, Dev. Biol., 100:39-49). Using this assay in which accelerated maturation of a group of treated cumulus cell-oocyte complexes relative to untreated complexes indicates a decrease in the amount of inhibitor, results of the experiments described here suggest a possible relationship between elevation of cAMP levels and subsequent decreased amounts of a non-cAMP inhibitor. Mouse oocytes obtained from cumulus cell-oocyte complexes treated with luteinizing hormone (LH) resumed meiosis prior to oocytes obtained from untreated complexes; the degree of acceleration of maturation was dependent on LH concentration. A similar result was obtained with follicle-stimulating hormone (FSH). Correlated with LH- or FSH-acceleration of maturation was an LH- or FSH-induced elevation of cumulus cell cAMP levels. Inhibiting LH-induced elevation of cumulus cell cAMP levels inhibited LH-induced acceleration of maturation. An initial incubation of complexes in medium containing dibutyryl cAMP (dbcAMP) also promoted acceleration of maturation. In contrast, maturation of denuded oocytes was not altered by treatment with either LH, FSH, or dbcAMP. Complexes initially incubated in dbcAMP-containing medium still demonstrated acceleration of maturation after a subsequent 2 h incubation in dbcAMP-free medium. Relative to untreated complexes, none of these treatments disrupted intercellular communication between cumulus cells and the oocyte. Elevating follicle cAMP levels with cholera toxin induced maturation of follicle-enclosed oocytes when cumulus cell-oocyte coupling was still fully maintained. These results are interpreted to indicate that gonadotropin-mediated acceleration of maturation is via a cAMP-dependent reduction in the level of a maturation inhibitor present in granulosa/cumulus cells.

Inhibition of oocyte maturation in the mouse: participation of cAMP, steroid hormones, and a putative maturation-inhibitory factor.

The hypothesis that cumulus cells inhibit oocyte maturation by a cAMP-dependent process was tested (R.M. Schultz, R. Montgomery, P.F. Ward-Bailey, and J.J. Eppig (1983). Dev. Biol. 95, 294-304.). Treatment of isolated cumulus cell-oocyte complexes with follicle-stimulating hormone (FSH) resulted in a dose-dependent increase in both cumulus cell cAMP levels and in the extent of inhibition of germinal vesicle breakdown (GVBD), the first morphological manifestation of oocyte maturation. Furthermore, it was found that concentrations of a membrane-permeable analog of cAMP, dibutyryl cAMP (dbcAMP), that were below those required for complete meiotic inhibition had a greater inhibitory effect on cumulus cell-enclosed oocytes than on denuded oocytes. Cumulus cell-enclosed and denuded oocytes matured at the same time in the absence of dbcAMP. Ablation of the gap junctions that couple cumulus cells to the oocyte abolished the maturation-inhibitory action of cumulus cells that was promoted either by FSH or low concentrations of dbcAMP. These results are consistent with the hypothesis that inhibition of oocyte maturation is mediated by a factor of granulosa/cumulus cell origin, other than cAMP, which requires cAMP for its activity and/or generation, and an intact intercellular coupling pathway between cumulus cells and the oocyte. A variety of steroid hormones potentiated the FSH-induced inhibition of maturation in cumulus cell-enclosed oocytes. In addition, steroid hormones inhibited maturation in denuded oocytes, but only when oocyte cAMP levels were elevated by cAMP analogs or forskolin. Steroids alone did not inhibit maturation of either cumulus cell-enclosed or denuded oocytes. Moreover, the steroids alone or in combination with FSH did not affect metabolic coupling between the cumulus cells and oocytes, nor did testosterone affect the forskolin-induced level of cAMP in denuded oocytes. Therefore, it is proposed that the oocyte is a site for the synergistic activity of steroid hormones with a cAMP-dependent process in inhibiting maturation. Results of these studies are discussed in terms of the roles of intercellular communication, cAMP, a putative maturation-inhibiting factor, and steroid hormones in the inhibition of maturation of mouse oocytes.

Experimental and mathematical models of Escherichia coli plasmid transfer in vitro and in vivo.

Little is known about the factors that govern plasmid transfers in natural ecosystems such as the gut. The consistent finding by earlier workers that plasmid transfer in the normal gut can be detected only at very low rates, if at all, has given rise to numerous speculations concerning the presence in vivo of various inhibitors of plasmid transfer. Plasmids R1, R1drd-19, and pBR322 were studied in Escherichia coli K-12 and wild-type E. coli hosts in two experimental systems: (i) gnotobiotic mice carrying a synthetic indigenous microflora (F-strains) which resemble in their function the normal indigenous microflora of the mouse large intestine, and (ii) anaerobic continuous-flow cultures of indigenous large intestinal microflora of the mouse, which can simulate bacterial interactions observed in the mouse gut. Mathematical models were developed to estimate plasmid transfer rates as a measure of the "fertility," i.e., of the intrinsic ability to transfer the plasmid under the environmental conditions of the gut. The models also evaluate the effects of plasmid segregation, reduction of the growth rates of plasmid-bearing bacterial hosts, repression of transfer functions, competition for nutrients, and bacterial attachment to the wall of the gut or culture vessel. Some confidence in the validity of these mathematical models was gained because they were able to reproduce a number of known phenomena such as the repression of fertility of the R1 plasmid, as well as known differences in the transmission and mobilization of the plasmids studied. Interpretation of the data obtained permitted a number of conclusions, some of which were rather unexpected. (i) Fertility of plasmid-bearing E. coli in the normal intestine was not impaired. The observed low rates of plasmid transfer in the normal gut can be explained on quantitative grounds alone and do not require hypothetical inhibitory mechanisms. (ii) Conditions for long-term spread and maintenance throughout human or animal populations of a diversity of conjugative and nonconjugative plasmids may be optimal among E. coli strains of low fertility, as are found among wild-type strains. (iii) E. coli strains carrying plasmid pBR322 plus R1drd-19 were impaired in their ability to transfer R1drd-19, but strains carrying pBR322 were significantly better recipients of R1drd-19 than a plasmid-free recipient E. coli. (iv) Long-term coexistence of plasmid-bearing and plasmid-free E. coli, in spite of undiminished fertility, appeared to be due to a detrimental effect of the plasmid on the growth rate of its host bacterium, rather than due to high rates of plasmid segregation. (v) Mathematical analysis of experimental data published by earlier investigators is consistent with the conclusion that plasmid transfer occurs consistently in the human gut, but that the resulting transconjugant E. coli populations are too small to be detected regularly with the culture methods used by earlier investigators. It is concluded that the long-term interactions observed were often the consequences of minor differences in parameters such as growth rates, fertility, rates of segregation, etc., which were too small to be detected except by precise mathematical analysis of long-term experiments, but which were nevertheless decisive determinants of the ultimate fates of the plasmids and their hosts.

On the evolution of accuracy and cost of proofreading tRNA aminoacylation.

Aminoacylation of tRNA occurs with a high degree of accuracy in many cases because misacylated molecules are effectively proofread on the aminoacyl-tRNA synthetase by preferential hydrolysis. This hydrolysis releases an ATP equivalent of energy. An explicit relationship between cost of proofreading and the resulting degree of accuracy is presented. Experimental data from Escherichia coli for isoleucyl-tRNA synthetase proofreading valyl-tRNAIle are examined by means of this relationship, and a conjecture concerning the natural selection of accuracy and proofreading costs is put forth and tested. We have found the energy cost of accurate proofreading to be high. The minimum error, derived in previous theoretical studies, is never actually reached. Instead, higher values, determined by the balance between energy wasted in the cell as a consequence of error and the energy cost of proofreading, appear to be selected. The total cost of proofreading all types of tRNA aminoacylations is estimated to be approximately 2% of the energy required to synthesize a bacterial cell.

Energy cost of proofreading to increase fidelity of transfer ribonucleic acid aminoacylation.

The paradox of relatively error free function in biological systems composed of relatively error prone components has recently come under intensive investigation. In the case of tRNA aminoacylation, aminoacyl-tRNA synthetases were discovered to have a separate function that allows misacylated molecules to be hydrolyzed more rapidly than correctly acylated molecules. This additional function of the synthetases provides a proofreading or verification mechanism that is believed to improve significantly the overall accuracy of tRNA aminoacylation. In this paper we provide an explicit relationship between the accuracy achieved by proofreading and the energy cost. Experimental data available in the literature are examined in light of this relationship. The following are the principal conclusions from our study: (1) high-accuracy proofreading of tRNA aminoacylation has a high energy cost, as much as 100 times greater than indications from early experimental work; (2) the minimum net error derived in previous theoretical studies is never actually reached; (3) mechanisms in which misacylation and subsequent proofreading occur on the surface of the same synthetase molecule achieve a much higher accuracy than mechanisms in which these functions occur on the surface of different synthetase molecules.