Thymidylate synthase (TS; 5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) is essential for the de novo synthesis of thymidylate, a precursor of DNA. Previous studies have shown that the cellular level of this protein is regulated at both the transcriptional and posttranscriptional levels. The regulation of human TS mRNA translation was studied in vitro with a rabbit reticulocyte lysate system. The addition of purified human recombinant TS protein to in vitro translation reactions inhibited translation of TS mRNA. This inhibition was specific in that recombinant TS protein had no effect on the in vitro translation of mRNA for human chromogranin A, human folate receptor, preplacental lactogen, or total yeast RNA. The inclusion of dUMP, 5-fluorodUMP, or 5,10-methylene-tetrahydrofolate in in vitro translation reactions completely relieved the inhibition of TS mRNA translation by TS protein. Gel retardation assays confirmed a specific interaction between TS protein and its corresponding mRNA but not with unrelated mRNAs, including human placenta, human .8-actin, and yeast tRNA. These studies suggest that translation of TS mRNA is controlled by its own protein end product, TS, in an autoregulatory manner.Thymidylate synthase (TS; 5,10-methylenetetrahydrofolate: dUMP C-methyltransferase, EC 2.1.1.45) catalyzes the conversion of 2'-deoxyuridine 5'-monophosphate (dUMP) and 5,10-methylenetetrahydrofolate (5,10-methylene-H4PteGlu, where H4PteGlu is tetrahydropteroylglutamic acid) to thymidine monophosphate (dTMP) and dihydrofolate (H2PteGlu) (1). This enzymatic reaction provides the sole intracellular de novo source of dTMP, and because of its central role in the synthesis of DNA precursors, TS remains an important target enzyme in cancer chemotherapy (2).Both the cDNA and corresponding mRNA clones for mouse (3) and human (4) TS have been isolated and sequenced, and these probes have facilitated the analysis of TS structure and expression and the study of the molecular basis of TS regulation. This enzyme has been purified and well characterized from various sources, including bacteria, bacteriophage, yeast, viruses, parasites, and mammals (5-9). TS is a dimeric protein with identical subunits, each =35 kDa, and comparison of the predicted primary amino acid sequence ofthe protein from eight different sources reveals that it is one of the most highly conserved proteins.Previous studies examining regulation of TS expression have concentrated on cell-cycle-directed events. Various investigators have shown that maximal TS activity occurs during periods of active DNA synthesis (10-12). Moreover, this increase in TS enzyme levels that arises as cells enter S phase appears to be regulated at both the transcriptional and posttranscriptional levels (13-15). Takeishi et al. (4) also suggested the possibility of translational regulation of TS expression given the theoretical potential of three interconvertible secondary structures, each containing a stem-loop structure in the 5' untranslated region (5' U...

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During spermatogenesis, several genes are expressed in a germ cell-specific manner. Previous studies have demonstrated that rat and mouse spermatogenic cells produce a 1,700-nucleotide proenkephalin RNA, while somatic cells that express the proenkephalin gene contain a 1,450-nucleotide transcript. Using cDNA cloning, RNA protection, and primer extension analyses, we showed that transcription of the rat and mouse spermatogenic-cell RNAs is initiated downstream from the proenkephalin somatic promoter in the first somatic intron (intron As). In both species, the germ cell cap site region consists of multiple start sites distributed over a length of approximately 30 base pairs. Within rat and mouse intron As, the region upstream of the germ cell cap sites is GC rich and lacks TATA sequences. A consensus binding site for the transcription factor SP1 was identified in intron As downstream of the proenkephalin germ cell cap site region. These features are characteristic of several previously described promoters that lack TATA sequences. Homologies were also identified between the proenkephalin and rat cytochrome c spermatogenic-cell promoters, including the absence of a TATA box, a multiple start site region, and several common sequences. This promoter motif thus may be shared with other genes expressed in male germ cells.Spermatogenesis is a complex program of cellular differentiation that results in the formation of haploid spermatozoa. While this developmental sequence of events has been well characterized morphologically, a description of the molecular mechanisms regulating spermatogenic cell differentiation has only recently been initiated (18). Germ cell gene expression is highly stage specific, with different gene products being expressed at distinct phases of development. Both stage-specific transcriptional and translational regulation of early-transcribed mRNAs contribute to these differentiational changes (18,19). Another characteristic of spermatogenic-cell gene expression is the presence of germ cell-specific transcripts not produced in somatic cells. These unique RNAs may be generated by multiple mechanisms, including transcription of genes selectively expressed in germ cells (5,37,49), utilization of distinct transcriptional initiation or termination sites (12, 39), and alternative RNA splicing.The gene for the opioid precursor proenkephalin is expressed in both spermatogenic and somatic cells (13,(28)(29)(30) MATERIALS AND METHODSIsolation of proenkephalin cDNA from mouse testis. A mouse testis cDNA library constructed in the EcoRI site of AgtlO (kindly provided by Ken Kleene, Biology Department, University of Massachusetts, Boston) was screened for proenkephalin-containing phage by hybridization to a PvuII fragment from rat brain proenkephalin cDNA [pRPE-1(165-600) (21)]. The largest insert from the positive bacteriophage clones was isolated by EcoRI digestion and subcloned into the EcoRI site of pBluescript SK (Stratagene, San Diego, Calif.). Standard procedures were used throughout for the growth and...

Steady-state levels of phosphatidyl inositol (PtdIns) turnover are examined in MCF-7 human breast cancer cells in response to estradiol treatment. Elevated levels of PtdIns are observed 12-24 h after estradiol treatment, occur at estradiol concentrations as low as 10(-12) M, and are competitively blocked by the antiestrogen LY117018. MCF-7 cells secrete a transforming growth factor (TGF) alpha-like material which can partly replace estradiol in conferring tumorgenicity in nude mice. We show that acute or chronic treatment of MCF-7 cells with TGF alpha results in elevated PtdIns turnover and that chronic treatment increases growth rate. In contrast TGF beta is growth inhibitory and blocks estradiol-induced increases in PtdIns turnover. A phosphatidyl inositol 4,5-bisphosphate specific phospholipase-C activity has been identified and is elevated in association with estradiol treatment. These data are consistent with estradiol-induced autocrine growth factors, including TGF alpha, acting through the PtdIns turnover pathway as part of their mechanism of action.

During spermatogenesis, several genes are expressed in a germ cell-specific manner. Previous studies have demonstrated that rat and mouse spermatogenic cells produce a 1,700-nucleotide proenkephalin RNA, while somatic cells that express the proenkephalin gene contain a 1,450-nucleotide transcript. Using cDNA cloning, RNA protection, and primer extension analyses, we showed that transcription of the rat and mouse spermatogenic-cell RNAs is initiated downstream from the proenkephalin somatic promoter in the first somatic intron (intron As). In both species, the germ cell cap site region consists of multiple start sites distributed over a length of approximately 30 base pairs. Within rat and mouse intron As, the region upstream of the germ cell cap sites is GC rich and lacks TATA sequences. A consensus binding site for the transcription factor SP1 was identified in intron As downstream of the proenkephalin germ cell cap site region. These features are characteristic of several previously described promoters that lack TATA sequences. Homologies were also identified between the proenkephalin and rat cytochrome c spermatogenic-cell promoters, including the absence of a TATA box, a multiple start site region, and several common sequences. This promoter motif thus may be shared with other genes expressed in male germ cells.

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Two distinct forms of proenkephalin messenger RNA (mRNA) are present in the murine testis, a family of 1.7 kilobases (kb), germ cell-specific transcripts and a 1.45-kb form that is also found in somatic tissues. In situ hybridization and molecular analysis of purified spermatogenic cell types were used to characterize the cellular localization of these different transcripts during development of the mouse testis. Both forms of proenkephalin mRNA were observed in isolated germ cells by RNA gel-blot analysis, but in distinct developmental patterns; the 1.7-kb transcripts were present in cells undergoing meiosis and spermiogenesis, whereas the 1.45-kb mRNA was detected primarily in type B spermatogonia. In contrast, in situ hybridization analysis did not detect significant amounts of the 1.45-kb transcript in any spermatogenic cell type. Using transcript-specific probes, distinct patterns of developmental expression were evident for the two mRNAs. The 1.45-kb transcript was the only form detected in the prepubertal testis, where it was localized mainly in interstitial cells. In contrast, the 1.7-kb transcripts were the major mRNAs observed in the adult testis and were localized to spermatogenic cells. A transition from the prepubertal to the adult pattern occurred on or about postnatal day 21, when proenkephalin-expressing pachytene spermatocytes begin to populate the seminiferous tubules. In situ hybridization analysis further demonstrated that proenkephalin gene expression in mutant (at/at) mice, which lack germ cells, was identical to that observed in the early prepubertal testis. These results suggest that the 1.45-kb proenkephalin mRNA is developmentally down-regulated in mouse interstitial cells and that this process requires ongoing spermatogenesis.

Experiments were performed to assess the capacity of lectin (Con A), ionomycin, phorbol ester (PMA), and recombinant IL 2 to mediate proliferation as well as the expression of cell surface IL 2 receptors, two lymphokine genes, IL 2 and IFN-gamma, and the c-myc proto-oncogene in cloned T cell populations. Stimulation of T cell clones with recombinant IL 2 resulted in proliferation and sustained expression of the c-myc cellular proto-oncogene, but did not induce the expression of mRNA for the lymphokines IFN-gamma and IL 2. In contrast, stimulation of cloned T cells with lectin alone induced significant IFN-gamma and IL 2 mRNA expression, up-regulation of the number of cell surface IL 2 receptors, and transient c-myc expression. Ionomycin alone was not a sufficient signal for lymphokine mRNA induction. The phorbol ester PMA alone induced neither proliferation nor lymphokine gene expression but potentiated lectin and ionomycin-mediated signals. We also performed experiments to examine whether the T cell response to extracellular stimuli was a function of the activation state of the cell. Reexposure of 48-hr antigen-activated cloned cells to identical stimuli revealed several differences. Low but significant levels of IFN-gamma mRNA were now also reinduced in activated clones cells in response to IL 2 or PMA alone. Activated cells were refractory to reinduction of IL 2 mRNA by any stimulus, which may reflect a physiologic mechanism to limit clonal expansion after antigenic stimulation. This could be partially reversed by restimulation with lectin in the presence of cycloheximide, suggesting a role for a labile protein repressor in the down-regulation of IL 2 mRNA expression. PMA alone induced an IL 2-independent proliferative response. We demonstrate that distinct signals are required for lymphokine gene expression vs cellular proliferation in cloned T lymphocyte populations, and that the capacity of extracellular stimuli to reinduce expression of lymphokine genes or to mediate cell proliferation is altered by prior activation.