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...