Tetracycline-regulatable factors with distinct dimerization domains allow reversible growth inhibition by p16.

Continuous regulation is required to maintain a given cell state or to allow it to change in response to the environment. Studies of the mechanisms underlying such regulation have often been hindered by the inability to control gene expression at will. Among the inducible systems available for regulating gene expression in eukaryotes, the tetracycline (tet) regulatable system has distinct advantages. It is highly specific, non-toxic and non-eukaryotic, and consequently does not have pleiotropic effects on host cell genes. Previously this system also had drawbacks, as it did not extinguish gene expression completely, precluding the study of toxic or growth-inhibitory gene products. We report here the development of a facile reversible tetracycline-inducible retroviral system (designated RetroTet-ART) in which activators and repressors together are expressed in cells. Gene expression can now be actively repressed in the absence of tet and induced in the presence of tet, as we have engineered distinct dimerization domains that allow co-expression of homodimeric tet-regulated transactivators and transrepressors in the same cells, without the formation of non-functional heterodimers. Using this system, we show that growth arrest by the cell cycle inhibitor p16 is reversible and dependent on its continuous expression.

Highly conserved RNA sequences that are sensors of environmental stress.

The putative function of highly conserved regions (HCRs) within 3' untranslated regions (3'UTRs) as regulatory RNA sequences was efficiently and quantitatively assessed by using modular retroviral vectors. This strategy led to the identification of HCRs that alter gene expression in response to oxidative or mitogenic stress. Databases were screened for UTR sequences of >100 nucleotides that had retained 70% identity over more than 300 million years of evolution. The effects of 10 such HCRs on a standard reporter mRNA or protein were studied. To this end, we developed a modular retroviral vector that can allow for a direct comparison of the effects of different HCRs on gene expression independent of their gene-intrinsic 5'UTR, promoter, protein coding region, or poly(A) sequence. Five of the HCRs tested decreased mRNA steady-state levels 2- to 10-fold relative to controls, presumably by altering mRNA stability. One HCR increased translation, and one decreased translation. Elevated mitogen levels caused four HCRs to increase protein levels twofold. One HCR increased protein levels fourfold in response to hypoxia. Although nonconserved UTR sequences may also have a role, these results provide evidence that sequences that are highly conserved during evolution are good candidates for RNA motifs with posttranscriptional regulatory functions in gene expression.

Adenylosuccinate synthetase: a dominant amplifiable genetic marker in mammalian cells.

Adenylosuccinate synthetase (AdSS) functions at the branchpoint of purine nucleotide metabolism leading to the synthesis of AMP. The enzyme is inhibited by a metabolite of alanosine, an aspartic acid analog that is highly cytotoxic for most cells. We show here that it is possible to use alanosine selection to isolate from a population of transformants those cells having the highest levels of AdSS activity resulting from uptake and expression of AdSS minigenes. Transformants isolated in this way were selected for resistance to even higher concentrations of alanosine and resulted in the isolation of cells with highly amplified copies of the transfected AdSS minigenes. We demonstrated that nonselectable genes can be cotransferred and coamplified with AdSS minigenes. These findings indicate that AdSS minigenes can be used as dominant amplifiable genetic markers in mammalian cells.

The highest levels of purine catabolic enzymes in mice are present in the proximal small intestine.

Recent studies on the tissue distribution and developmental regulation of adenosine deaminase (ADA) activity in mice show that very high ADA levels exist in the murine alimentary tract (tongue, esophagus, forestomach, proximal small intestine) and at the fetal-maternal interface. To understand the role of ADA in these tissues, we measured the levels of three other enzymes involved in purine catabolism, purine nucleoside phosphorylase (PNP), guanine deaminase (GDA), and xanthine dehydrogenase (XDH), to see how their levels correlated with ADA activity. Our results show that the highest level of PNP, GDA, and XDH is present in the proximal small intestine. Levels of these purine catabolic enzymes are much lower in the tongue, esophagus, forestomach, and fetal-maternal interface in marked contrast to ADA distribution. We also determined mRNA levels encoding PNP, XDH, and ADA in a variety of tissues. Tissue-specific differences in PNP, XDH, and ADA activity correlated with RNA abundance, indicating that the regulation of gene expression is at the level of mRNA production. Thus, ADA is part of a purine catabolic pathway leading to the production of uric acid that is present at the highest known level in the proximal small intestine. ADA may have additional roles in other tissues.

Molecular cloning and expression of a mouse muscle cDNA encoding adenylosuccinate synthetase.

Adenylosuccinate synthetase (EC 6.3.4.4) catalyzes the first step in formation of AMP from IMP. At least two isozymes exist in vertebrate tissue. An acidic form, present in most tissues, has been suggested to be involved in de novo biosynthesis while a basic isozyme, which predominates in muscle, appears to function in the purine nucleotide cycle. Antibodies specific for the basic isozyme detect a single protein in mouse tissues with highest levels in skeletal muscle, tongue, esophagus, and heart tissue consistent with a role for the enzyme in muscle metabolism. A series of degenerate oligonucleotides were constructed based on peptide sequences from purified rat muscle enzyme and then used to clone a mouse muscle cDNA encoding the basic isozyme. The clone contains a open reading frame of 1356 bases with 452 amino acids. Northern analysis of RNA from mouse tissues showed a tissue distribution similar to that of the protein, indicating a high level of gene expression in muscle. Transfection of COS cells with the mouse muscle cDNA allows expression of a functional protein with a molecular mass of approximately 50 kDa, consistent with the open reading frame and the size of the isolated rat enzyme. The deduced amino acid sequence of the mouse synthetase is 47 and 37% identical to the synthetase sequences from Dictyostelium discoideum and Escherichia coli, respectively. The availability of antibodies and cDNA clones specific for the basic isozyme of adenylosuccinate synthetase from muscle will facilitate future genetic and biochemical analysis of this protein and its role in muscle physiology.

Amplification of an adenylosuccinate synthetase gene in alanosine-resistant murine T-lymphoma cells. Molecular cloning of a cDNA encoding the "non-muscle" isozyme.

Adenylosuccinate synthetase (EC 6.3.4.4) catalyzes the initial step in the conversion of IMP to AMP. Two isoforms of this enzyme have been observed in vertebrates. A muscle isozyme is highly abundant in cardiac and skeletal muscle tissue and is thought to play a role in muscle energy metabolism. The non-muscle isozyme, which is present at low levels in most tissues, likely functions in de novo AMP biosynthesis. The analysis of the non-muscle isozyme has been hampered by its low abundance and instability during purification. In this study a genetic selection scheme was used to generate a murine T-lymphoma cell line which was at least 100-fold enriched for the non-muscle isozyme, as a result of amplification of the non-muscle synthetase gene. This cell line made possible the purification of the non-muscle isozyme, and the subsequent isolation of isozyme-specific peptides. Based on peptide sequence information a degenerate oligonucleotide probe was designed and used to screen a mouse kidney cDNA library. A 1.5-kilobase cDNA encoding the non-muscle isozyme was cloned and found to contain an open reading frame of 1368 base pairs encoding 456 amino acids. Gene transfer experiments showed that the cDNA encoded a 50-kDa protein, the size expected for mammalian synthetases, that correlated with the presence of high levels of synthetase activity. The deduced amino acid sequence of the mouse non-muscle synthetase is approximately 75% identical to the previously reported mouse muscle synthetase. Southern blot analysis of mouse genomic DNA with the isozyme-specific cDNA probes revealed that the synthetase isozymes are encoded by separate genes. The non-muscle gene is expressed in most tissues but is virtually undetectable in striated muscle tissues. Three different transcripts (1.7, 2.8, and 3.4 kilobases) are detected for the non-muscle isozyme which show a similar tissue distribution. The availability of a cDNA for the non-muscle isozyme of adenylosuccinate synthetase will facilitate further comparative analyses with the previously cloned muscle isozyme.

Transcriptional control: rheostat converted to on/off switch.

Individual cells translate concentration gradients of extracellular factors into all-or-none threshold responses leading to discrete patterns of gene expression. Signaling cascades account for some but not all such threshold responses, suggesting the existence of additional mechanisms. Here we show that all-or-none responses can be generated at a transcriptional level. A graded rheostat mechanism obtained when either transactivators or transrepressors are present is converted to an on/off switch when these factors compete for the same DNA regulatory element. Hill coefficients of dose-response curves confirm that the synergistic responses generated by each factor alone are additive, obviating the need for feedback loops. We postulate that regulatory networks of competing transcription factors prevalent in cells and organisms are crucial for establishing true molecular on/off switches.

Structure and expression of the murine muscle adenylosuccinate synthetase gene.

A muscle-specific isoform of adenylosuccinate synthetase (AdSS1, EC) is one of three enzymes that constitute the purine nucleotide cycle, a muscle-specific metabolic cycle. Previously, we showed that the muscle Adss1 gene was highly expressed in both skeletal muscle and heart of the adult mouse. Here we have shown that the Adss1 gene is initially activated early in embryonic development in skeletal muscle and heart precursors and is subsequently up-regulated perinatally. The earliest detectable gene expression corresponds with the establishment of the first myogenic and cardiac lineages. To allow identification of the genetic signals controlling this developmental pattern of expression, the Adss1 gene was cloned and its structure determined. Transgenic analysis has shown that 1.9 kilobase pairs of 5' flank can activate expression in skeletal muscle progenitors and direct enhanced expression to adult cardiac muscle. Sequence analysis of the promoter and 5' flanking region revealed the presence of numerous potential muscle-specific cis-regulatory elements.