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Site-directed mutagenesis of cis-regulatory elements in the 5' flanking region of the rat pyruvate kinase M gene reveakd that two out of the three GC boxes (-133/-124 and -48/-39) are involved in the stimulation of a core promoter (-3511-46). These two regions were also protected in DNaseI footprinting assays. Spl and Sp3 were identified as binding proteins to all three GC boxes by supershift experiments.Cotransfections in Drosophilu SL2 cells revealed a strong stimulatory function of Spl and a synergistic effect of Sp3 to Spl in the activation of the pyruvate kinase M promoter. No inhibitory effect of Sp3 was detected.These data indicate that binding of Spl at two GC boxes is required for full promoter activity of the pymvate kinase M gene and thus contributes to the observed cell-cycle-dependent expression of this enzyme in proliferating rat thymocytes.Keywords: pymvate kinase M ; rat thymocytes; stimulatory protein 1 ; stimulatory protein 3.The four isozyme types of the glycolytic enzyme pyruvate kinase in rat are encoded by two genes. The M gene codes for both the My and the M, type. Tissue-specific synthesis of the two enzymes is due to differential splicing [ l , 21. L-type and Rtype isoforms are also encoded by a single gene through the use of two distinct promoters [3-51. The 5' flanking region of the M gene was cloned and sequenced by Takenaka et al. [6]. This region contains no TATA box and no CAAT box but putative Spl-binding sites. The reported assays of chloramphenicol acetyltransferase (CAT) revealed that a 0.5-kb fragment (Fig. 1) upstream to the transcription start site is sufficient for expression of the gene in dRLh-84 hepatoma cells, whereas no CAT activity in transfected primary hepatocytes could be detected. The authors discussed a role of Spl for the transcriptional regulation of the pyruvate kinase M (PKM) gene deduced from their sequence data without presenting functional evidence. From transient expression analyses of the promoter in dRLh-84 hepatoma cells, Wang et al. [7] suggest the presence of a cluster of three positive cis-acting regions (A, B, C in Fig. 1) within the sequence -279 to -216 which have a role in the regulation of the PKM gene. The authors, however, concluded that the potential Spl-binding sites in the region between -133/-39 identified by DNA sequencing [6] are not involved in the regulation of the gene.In previous studies [8], we analyzed the cell cycle of rat thymocytes by determining biochemical parameters associated with cell cycle progression. These studies revealed a coincidence of the time courses of the activity profiles of the glycolytic enzymes and the rates of DNA, RNA and protein synthesis. The activity and M,-type-specific mRNA levels of pyruvate kinase increased about 10-fold and !+fold, respectively, in the S-phase of stimulated cells compared to resting cells [9, 101. We could show further that glucose is essential for the induction of glycolytic enzymes that provoke a transition to glycolytic energy production in proliferating rat thymocytes [Ill. It...

The complete nucleotide sequence of a 14 kb segment of A. nidulans mtDNA reveals a rather compact organization of genes transcribed from the same strand and coding for two functionally known proteins, seven unidentified polypeptides (URFs), 24 tRNAs and two rRNAs. One of the URFs is located in the intron of the L-rRNA gene and codes for a basic protein of 410 residues. The other URFs are in spacer regions and code for hydrophobic proteins. URFa is homologous to human URF4, and URFb produces a polypeptide of 48 residues resembling the human URF6L product (hydrophobic N-terminus, basic C-terminus). The ATPase subunit 6 genes from mitochondria and E. coli appear to share a common ancestor. The codon frequencies of identified genes and URFs are similar, and codons ending with G or C are rarely used. The structures of tRNAs specific for arginine, asparagine, tyrosine and histidine are deduced from gene sequences.

To elucidate the role of polyamine metabolism in the regulation of mesangial cell growth, we examined the involvement of ornithine decarboxylase (ODC), the rate limiting enzyme for polyamine synthesis, in the mitogenesis of cultured rat mesangial cells (MCs). Resting MCs, stimulated with fetal calf serum (FCS 10%), showed an induction of ODC activity from undetectable values in resting cells to mean = 5035 nmol CO2/10(10) cells.hr (range 3157 to 7154, N = 5), which is 25-fold above the detection limit. We found a single peak of ODC activity eight to ten hours after stimulation, declining to 22 to 34% of peak levels after 24 hours. 3H-thymidine (TdR) uptake, an S-phase marker of MC replication, peaked at 24 hours, reaching 10.7-fold values of resting MCs. ODC mRNA levels were low in resting cells. After serum stimulation there was a two- to 10-fold increase in ODC mRNA with a maximum after six hours. ODC activity with similar kinetics but lower peak levels was also induced by incubating MCs with mitogens, such as platelet-derived growth factor (PDGF-AB 20 ng/ml), arginine vasopressin (AVP 10(-7) M), phorbol myristate acetate (PMA 10(-7) M), interleukin 1 alpha and beta (IL-1 alpha 10 U/ml, IL-1 beta 10 U/ml). In the presence of alpha-difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ODC, the growth rate of MCs, assessed by cell counts and by 3H-TdR uptake, was markedly reduced by 62 to 100%. This antiproliferative effect of DFMO could be reversed by addition of putrescine, the reaction product of ODC.(ABSTRACT TRUNCATED AT 250 WORDS)

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Thymosin p4 belongs to a family of ubiquitous peptides present at a high cellular content but still with an unknown intracellular function. The expression of this peptide was studied in concanavalin-A-stimulated, proliferating rat thymocytes during cell cycle progression. An early, transient 10-fold increase of the peptide occurred 1 h after stimulation without elevation of the corresponding mRNA level. This increase coincided with that of thymosin p4 biosynthesis. The sharp decline of the thymosin p4 content was not due to a secretion of the peptide into the medium. During S phase and mitosis, the biosynthetic rates as well as mRNA content, but not the cellular thymosin p4 concentration, increased again.

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