A genetic element that increases the frequency of gene amplification.

A repetitive mammalian genetic element, HSAG-1, has been shown to promote the amplification of the vector, pSV2-DHFR, containing the functional cDNA for dihydrofolate reductase (DHFR). LR-73 cells, a line of Chinese hamster ovary cells, were transfected with this recombinant construct or with the parent vector, then subjected to culture in selective medium containing steadily increasing concentrations of methotrexate, a drug which specifically inhibits DHFR. Cultures transfected with the HSAG-1-containing construct acquired drug resistance faster than those transfected with the parent vector. This acceleration of acquisition of drug resistance was due to an increased probability of the generation and subsequent selection of cellular variants with increased copy numbers of the vector. The effect has also been observed in CHO(DHFR-) and HeLa cell lines. Possible mechanisms for the effect of the HSAG-1 element on gene amplification are discussed.     

The scute (T4) gene acts as a numerator element of the X:a signal that determines the state of activity of sex-lethal in Drosophila.

The ratio of X chromosomes to sets of autosomes (X:A) is the primary genetic signal that determines sex and dosage compensation in Drosophila. The gene Sex-lethal (Sxl) receives this signal and is responsible for the execution of the alternative developmental programmes of males and females. We have found that the scute (T4) gene, which is involved in neurogenesis, also plays a role in the activation of Sxl. The following results suggest that scute (T4) may be a numerator element of the X:A signal: scute (T4) mutations show female-specific lethality. There are female-specific lethal synergistic interactions between sis-a, a previously described numerator element, and mutants for T4. The female lethality is suppressed by SxlM1, a constitutive allele which expresses an active Sxl product independently of the X:A ratio. The Hw685 mutation, which overexpresses T4, is lethal to males with a duplication of sis-a. This lethality is suppressed by either Sxlf1, or the T4 point mutation sc10-1. There are female-specific lethal interactions between sc10-1 and daughter-less (da), a gene needed maternally for Sxl to become active. The sc10-1 mutation masculinizes triploid intersexes.     

A distinct thyroid hormone response element mediates repression of the human cholesterol 7alpha-hydroxylase (CYP7A1) gene promoter.

We examined the molecular basis by which T3 regulates the human cholesterol 7alpha-hydroxylase gene (CYP7A1) promoter. L-T3 decreased chloramphenicol acetyltransferase activity in hepatoma cells cotransfected with a plasmid encoding the T3 receptor (TR) alpha [NR1a1] and a chimeric gene containing nucleotides -372 to +61 of the human CYP7A1 gene fused to the chloramphenicol acetyltransferase structural gene. Deoxyribonuclease I footprinting revealed that recombinant TRalpha protected two regions in this segment of the human CYP7A1 gene promoter. In EMSAs, TRalpha bound to both regions. The binding was competed by oligonucleotides bearing an idealized TRalpha binding motif and abolished by mutation of these elements. In assays of promoter function, mutation of only one of the TRalpha binding sites blocked repression by T3. The results indicate that T3-dependent repression of human CYP7A1 gene expression is mediated via a novel site in the human CYP7A1 gene promoter.     

A cyclic AMP response element mediates repression of tyrosine aminotransferase gene transcription by the tissue-specific extinguisher locus Tse-1

Tyrosine aminotransferase (TAT) gene expression is liver specific and inducible by glucocorticoids and via the cAMP signaling pathway. In fibroblasts and other nonliver cells the gene is subject to negative control by the trans-dominant tissue-specific extinguisher locus Tse-1. We identified a hepatocyte-specific enhancer that is repressed by Tse-1. Two distinct sequence motifs are absolutely essential for function of this enhancer: a cAMP response element (CRE), which is the target for repression by Tse-1, and a hepatocyte-specific element. The specificity of the enhancer is generated by the combination of these two essential elements, which are fully interdependent. In vivo footprinting indicates that Tse-1 acts by affecting protein binding at the CRE. A direct antagonism between Tse-1 and the cAMP signaling pathway suggests that Tse-1 plays a role in control of developmental activation of the TAT gene.     

A new cAMP response element in the transcribed region of the human c-fos gene.

In NIH 3T3 cells the c-fos gene is induced rapidly and transiently by cAMP. As shown by the analysis of 3T3 cells stably transfected with promoter mutants of the human c-fos gene this induction does not depend on the dyad symmetry element (position -320 to -300), but involves at least two other non-related sites: an element located around position -60 resembling the cAMP response element of the fibronectin and somatostatin genes (which has been described before), and an element located between positions +18 and +38. Destruction of one or the other element in the c-fos gene reduces cAMP inducibility. The cAMP response of c-fos promoter CAT gene constructs also depends on these elements in transient transfection assays. When cloned in front of the albumin TATA box, both elements independently mediate cAMP inducibility. These elements do not bind the same protein as shown in gel retardation analyses, suggesting that two different cAMP inducible factors mediate the activation of the c-fos gene by cAMP.     

Molecular mechanisms of pattern formation by the BRE enhancer of the Ubx gene.

The core activity of the Ubx gene enhancer BRE (bx region enhancer) is encoded within a 500 bp module. bx DNA outside this active module increases the level of expression, expands the expression into ventro-lateral ectoderm and partially stabilizes the late expression pattern. The products of the gap genes hb and tll and of the pair-rule gene ftz bind to the 500 bp BRE module and control directly its initial pattern of expression. ftz enhances expression in even-numbered parasegments within the correct spatial domain whose boundaries are set by hb and tll. In addition, en and twi products activate the enhancer, probably directly. en broadens the parasegmental stripe while twi cooperates with ftz to enhance expression in the mesoderm. Binding sites for the five regulators are closely clustered, often overlapping extensively with one another. In vitro, hb blocks the binding of ftz and can also displace ftz protein pre-bound to an overlapping site, suggesting that competitive binding and/or interference by hb sets the initial boundaries of the domain of expression. Our results also suggest that this interaction is short-range and the long distance interactions among different enhancers may depend on each enhancer's ability to complex with the promoter.     

A gene from Xanthomonas campestris pv. vesicatoria that determines avirulence in tomato is related to avrBs3.

Strains of Xanthomonas campestris pv. vesicatoria that were avirulent in tomato leaves but virulent in pepper leaves were identified. A cloned gene, avrBsP, from one of the strains, Xv 87-7, converted a virulent strain in tomato to avirulent in tomato. A 1.7-kb subclone containing the avirulence gene cross-hybridized with the avirulence gene, which determines race 1 within the pepper group of strains (avrBs3). However, the two avirulence genes differ in their biological activity. The base sequences of the two avirulence genes were almost identical through the 1.7-kb segment of avrBsP, with significant differences only in some bases in the repeat region.     

Magnesium spin state determines the energetics of an adenosinetriphosphate molecule

The molecular dynamics method (density functional theory) DFT:B3LYP (6-3IG** basis set, t = 310 K) was used to study interactions between a molecule of adenosinetriphosphate (ATP) (ATP subsystem) and the [Mg(H₂O)₆]²⁺ magnesium cofactor (Mg subsystem) in an aqueous medium simulated by 78 water molecules in the singlet (S) and triplet (T) states. Potential energy surfaces (PESs) for the S (lowest in energy) and T states (highest in energy) are significantly separated in space. Motion along them directs the Mg complex either to oxygen atoms of the γ-β-phosphate groups (O1–O2) (S state of PES) or to oxygen atoms of the β-α-phosphate groups (O2–O3) (T state of PES). Chelation of the γ-β- and β-α-phosphates leads to formation of a stable low-energy ([Mg(H₂O)₄-(OI-O2)ATP]^2?) complex or a metastable high-energy ([Mg(H₂O)₂-(O2–O3)ATP]^2?) complex, respectively, which differ in number of water molecules surrounding the Mg atom. Intersection of two T PESs is accompanied by formation of an unstable state characterized by redistribution of spins between the Mg and ATP subsystems. This state, being sensitive to interaction with the Mg nuclear spin (²⁵Mg), induces an unpaired electron spin, which initiates the ATP cleavage by the ion-radical mechanism, yielding a reactive ion radical of adenosinemonophosphate (·AMP^?), which was earlier found experimentally by the method of chemically induced dynamic nuclear polarization (CIDNP). Biological aspects of the results obtained are discussed.