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.

Comparative mapping of the cri du chat and DiGeorge syndrome regions in the great apes.

Structural variations between great ape and human chromosomes due to pericentric inversions and translocations have created at apparent controversy during the reconstruction of hominoid phylogeny. One such variation involves human chromosome 5, which is equivalent to chromosome 4 in chimpanzee and orangutan but equivalent to segments of chromosomes 4 and 19 in gorilla. Obviously, neither banding patterns nor centromeric indecies in these chromosomes match. The pathological condition of cri du chat syndrome is due to the cytogenetic deletion of band p15.2 of chromosome 5. Is this region involved during pericentric inversion of apes chromosome 4? We used a human cosmid probe for cri du chat syndrome as a phylogenetic marker in search of the aforementioned question. The genomic sequences for cri du chat syndrome region were conserved in chimpanzee (PTR4) and orangutan (PPY4) but displayed a positional divergence in gorilla on chromosome 19(GG019). In addition, we used a human cosmid DNA probe for DiGeorge syndrome which is located on chromosome 22 band q11.2 and was conserved within band 23q11.2 in apes. The loci specific human genomic probes may help to describe the inversions and translocations for other chromosomes.

Graded transcriptional response to different concentrations of a single transactivator.

Threshold mechanisms of transcriptional activation are thought to be critical for translating continuous gradients of extracellular signals into discrete all-or-none cellular responses, such as mitogenesis and differentiation. Indeed, unequivocal evidence for a graded transcriptional response in which the concentration of inducer directly correlates with the level of gene expression in individual eukaryotic cells is lacking. By using a novel binary tetracycline regulatable retroviral vector system, we observed a graded rather than a threshold mechanism of transcriptional activation in two different model systems. When polyclonal populations of cells were analyzed at the single cell level, a dose-dependent, stepwise increase in expression of the reporter gene, green fluorescent protein (GFP), was observed by fluorescence-activated cell sorting. These data provide evidence that, in addition to the generally observed all-or-none switch, the basal transcription machinery also can respond proportionally to changes in concentration of extracellular inducers and trancriptional activators.

A unique genomic sequence in the Wolf-Hirschhorn syndrome [WHS] region of humans is conserved in the great apes.

The Wolf-Hirschhorn syndrome (WHS) is caused by a partial deletion in the short arm of chromosome 4 band 16.3 (4p 16.3). A unique-sequence human DNA probe (39 kb) localized within this region has been used to search for sequence homology in the apes' equivalent chromosome 3 by FISH-technique. The WHS loci are conserved in higher primates at the expected position. Nevertheless, a control probe, which detects alphoid sequences of the pericentromeric region of humans, is diverged in chimpanzee, gorilla, and orangutan. The conservation of WHS loci and divergence of DNA alphoid sequences have further added to the controversy concerning human descent.

Unique genomic sequences in human chromosome 16p are conserved in the great apes.

In humans, acute myelomonocytic leukemia (AMML) with abnormal bone marrow eosinophilia is diagnosed by the presence of a pericentric inversion in chromosome 16, involving breakpoints p13;q23 [i.e., inv(16)(p13;q23)]. A pericentric inversion involves breaks that have occurred on the p and q arms and the segment in between is rotated 180 degrees and reattaches. The recent development of a "human micro-coatasome" painting probe for 16p contains unique DNA sequences that fluorescently label only the short arm of chromosome 16, which facilitates the identification of such inversions and represents an ideal tool for analyzing the "divergence/convergence" of the equivalent human chromosome 16 (PTR 18, GGO 17 and PPY 19) in the great apes, chimpanzee, gorilla and orangutan. When the probe is used on the type of pericentric inversion characteristic of AMML, signals are observed on the proximal portions (the regions closest to the centromere) of the long and short arms of chromosome 16. The probe hybridized to only the short arm of all three ape chromosomes and signals were not observed on the long arms, suggesting that a pericentric inversion similar to that seen in AMML has not occurred in any of these great apes.

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.