Activin from secondary follicles causes small preantral follicles to remain dormant at the resting stage

The sequences controlling the expression of the Rhodobacter capsulatus recA and uvrA genes belonging to the SOS DNA repair system have been identified by PCR mutagenesis. Data obtained demonstrated that the GTTCN7GTAC and GAACN7GAAC motifs present upstream of the recA gene and the GTTCN7GTTC motif found upstream of the uvrA gene are required for their respective DNA damage-mediated induction. Alignment of recA promoters of R. capsulatus, Rhodobacter sphaeroides and Rhodopseudomonas viridis with the uvrA promoters of R. capsulatus and R. sphaeroides has identified the consensus sequence GTTCVYVYTWTGTTC as the SOS operator site of the Rhodospirillaceae family.     

Identification of functional elements in the promoter region of the human gene for thymidylate synthase and nuclear factors that regulate the expression of the gene

To identify the essential motifs of the promoter of the human gene for thymidylate synthase (TS), we constructed a set of deletion mutants from the 5'-terminal region of the human TS gene. From the results of assays of the expression of chloramphenicol acetyltransferase (CAT), we identified two functional elements with positive effects on the promoter activity: a CACCC box (CCACACCC) and an Sp1-binding motif (GAGGCGGA) that was homologous to the Sp1-binding site in the mouse TS gene. In addition, negative regulatory sequences were identified between the two positive elements and in the region upstream of the CACCC box. The results of gel mobility shift analyses suggested that Sp1 binds to the Sp1-binding motif of the human TS gene promoter and that multiple nuclear factors that are related to Sp1 bind to the CACCC box. Furthermore, the binding of Sp1 to mutated Sp1-binding motifs in the promoter region of the human TS gene was correlated with the promoter activity, as measured by the CAT assay. Therefore, the Sp1 motif seems to be a major contributor to the basic promoter activity of the human TS gene, although multiple positive and negative regulatory elements are involved in the regulated expression of this gene.     

Regulation of embryonic/fetal globin genes by nuclear hormone receptors: a novel perspective on hemoglobin switching

The CCAAT box is one of the conserved motifs found in globin promoters. It binds the CP1 protein. We noticed that the CCAAT-box region of embryonic/fetal, but not adult, globin promoters also contains one or two direct repeats of a short motif analogous to DR-1 binding sites for non-steroid nuclear hormone receptors. We show that a complex previously named NF-E3 binds to these repeats. In transgenic mice, destruction of the CCAAT motif within the human epsilon-globin promoter leads to substantial reduction in epsilon expression in embryonic erythroid cells, indicating that CP1 activates epsilon expression; in contrast, destruction of the DR-1 elements yields striking epsilon expression in definitive erythropoiesis, indicating that the NF-E3 complex acts as a developmental repressor of the epsilon gene. We also show that NF-E3 is immunologically related to COUP-TF orphan nuclear receptors. One of these, COUP-TF II, is expressed in embryonic/fetal erythroid cell lines, murine yolk sac, intra-embryonic splanchnopleura and fetal liver. In addition, the structure and abundance of NF-E3/COUP-TF complexes vary during fetal liver development. These results elucidate the structure as well as the role of NF-E3 in globin gene expression and provide evidence that nuclear hormone receptors are involved in the control of globin gene switching.     

Evidence that a combined activator-repressor protein regulates Dictyostelium stalk cell differentiation

The ecmA gene is expressed in Dictyostelium prestalk cells and is inducible by differentiation-inducing factor (DIF), a low-molecular-weight lipophilic substance. The ecmB gene is expressed in stalk cells and is under negative control by two repressor elements. Each repressor element contains two copies of the sequence TTGA in an inverted relative orientation. There are activator elements in the ecmA promoter that also contain two TTGA sequences, but in the same relative orientation. Gel retardation assays suggest that the same protein binds to the ecmB repressor and the ecmA activator. We propose that DIF induces prestalk cell differentiation by activating this protein and that the protein also binds to the promoters of stalk-specific genes, acting as a repressor that holds cells in the prestalk state until culmination is triggered.     

Role of the CCAAT-binding protein CBF/NF-Y in transcription

The CCAAT motif is one of the common promoter elements present in the proximal promoter of numerous mammalian genes transcribed by RNA polymerase II. CBF (also called NF-Y and CP1) consists of three different subunits and interacts specifically with the CCAAT motif. In each CBF subunit, the segment needed for formation of the CBF-DNA complex is conserved from yeast to human and, interestingly, the conserved segment of two CBF subunits, CBF-A and CBF-C, are homologous to the histone-fold motif of eukaryotic histones and archaebacterial histone-like protein HMf-2. The histone fold motifs of CBF-A and CBF-C interact with each other to form a heterodimer that associates with CBF-B to form a heterotrimeric CBF molecule, which then binds to DNA.     

An essential E box in the promoter of the gene encoding the mRNA cap-binding protein (eukaryotic initiation factor 4E) is a target for activation by c-myc

The mRNA cap-binding protein (eukaryotic initiation factor 4E [eIF4E]) binds the m7 GpppN cap on mRNA, thereby initiating translation. eIF4E is essential and rate limiting for protein synthesis. Overexpression of eIF4E transforms cells, and mutations in eIF4E arrest cells in G, in cdc33 mutants. In this work, we identified the promoter region of the gene encoding eIF4E, because we previously identified eIF4E as a potential myc-regulated gene. In support of our previous data, a minimal, functional, 403-nucleotide promoter region of eIF4E was found to contain CACGTG E box repeats, and this core eIF4E promoter was myc responsive in cotransfections with c-myc. A direct role for myc in activating the eIF4E promoter was demonstrated by cotransfections with two dominant negative mutants of c-myc (MycdeltaTAD and MycdeltaBR) which equally suppressed promoter function. Furthermore, electrophoretic mobility shift assays demonstrated quantitative binding to the E box motifs that correlated with myc levels in the electrophoretic mobility shift assay extracts; supershift assays demonstrated max and USF binding to the same motif. cis mutations in the core or flank of the eIF4E E box simultaneously altered myc-max and USF binding and inactivated the promoter. Indeed, mutations of this E box inactivated the promoter in all cells tested, suggesting it is essential for expression of eIF4E. Furthermore, the GGCCACGTG(A/T)C(C/G) sequence is shared with other in vivo targets for c-myc, but unlike other targets, it is located in the immediate promoter region. Its critical function in the eIF4E promoter coupled with the known functional significance of eIF4E in growth regulation makes it a particularly interesting target for c-myc regulation.     

An ethanolic-aqueous extract of Curcuma longa decreases the susceptibility of liver microsomes and mitochondria to lipid peroxidation in atherosclerotic rabbits

Escherichia coli K12 assay-system is designed in order to detect bioantimutagens, agents preventing mutagenesis by modulation of DNA repair and replication. The assay is composed of four tests aimed at the detection of inhibition of spontaneous and induced mutations (Tests A and B) and at the estimation whether the anti-mutagenic agent acts by increasing the fidelity of DNA replication (Test B), by inhibition of SOS error prone repair (Test C), or by favoring error-free recombinational repair (Test D). In Test A, repair proficient strain and its uvrA counterpart are used for detection of spontaneous and UV-induced mutations, while in Test B mismatch repair deficient strains (mutH, mutS, mutL and uvrD) are used for amplified detection of spontaneous mutations caused by replication errors. In Test C, repair proficient strain carrying sfiA::lacZ fusion is used for measuring the level of SOS induction by monitoring the level of beta-galactosidase. In Test D, the strains carrying different recA alleles (recA+, recA730 and DeltarecA) are used for measuring intrachromosomal recombination between nonoverlapping deletions in duplicated lac operon, by monitoring Lac+ recombinants. The assay-system is validated with model bioantimutagens and used for detection of anti-mutagenic potential of different terpenoid fractions from sage (Salvia officinalis L.). Extract E1/3 of cultivated sage, distinguished from others by its high content of monoterpenoid camphor, reduces UV-induced mutagenesis in Test A, while it has no effect in Tests B and C. In Test D, it enhances intrachromosomal recombination in untreated and UV-irradiated recA+ and recA730 strains. The results suggest that the protective effect is due to stimulation of recombinational repair, similarly to coumarin. We speculate that monoterpenoids from sage enhance genetic recombination by intervening in a formation of RecA-DNA complex and channeling it into recombination reaction.     

Characterization of a mutant RecA protein that facilitates homologous genetic recombination but not recombinational DNA repair: RecA423

A recA mutant (recA423; Arg169-->His), with properties that should help clarify the relationship between the biochemical properties of RecA protein and its two major functions, homologous genetic recombination and recombinational DNA repair, has been isolated. The mutant has been characterized in vivo and the purified RecA423 protein has been studied in vitro. The recA423 cells are nearly as proficient in conjugational recombination, transductional recombination, and recombination of lambda red- gam- phage as wild-type cells. At the same time, the mutant cells are deficient for intra-chromosomal recombination and nearly as sensitive to UV irradiation as a recA deletion strain. The cells are proficient in SOS induction, and results indicate the defect involves the capacity of RecA protein to participate directly in recombinational DNA repair. In vitro, the RecA423 protein binds to single-stranded DNA slowly, with an associated decline in the ATP hydrolytic activity. The RecA423 protein promoted a limited DNA strand exchange reaction when the DNA substrates were homologous, but no bypass of a short heterologous insert in the duplex DNA substrate was observed. These results indicate that poor binding to DNA and low ATP hydrolysis activity can selectively compromise certain functions of RecA protein. The RecA423 protein can promote recombination between homologous DNAs during Hfr crosses, indicating that the biochemical requirements for such genetic exchanges are minimal. However, the deficiencies in recombinational DNA repair suggest that the biochemical requirements for this function are more exacting.     

Binding of the recA protein of Escherichia coli to single- and double-stranded DNA

The recA protein of Escherichia coli binds both single- (SS) and double-stranded (DS) DNA; however, the optimal conditions differ for interaction with these DNA substrates. Binding of DS DNA by recA protein is pH dependent (optimum near pH 6.2) and requires a nucleoside triphosphate (ATP) and divalent cation. Substitution of the 5'-O-3'-thiotriphosphate (ATP(gamma S)) for ATP leads to formation of stable complexes of recA protein and DNA that dissociate very slowly. Formation of these complexes is extremely sensitive to ionic strength and pH. However, once formed, the complexes resist changes in pH and high salt concentrations. SS DNA binds to recA protein in the absence of a nucleoside triphosphate, but recA protein-SS DNA complexes are stabilized by ATP(gamma S). At high recA protein/DNA ratios (1 recA protein monomer/30 nucleotides), these complexes sediment in sucrose gradients as large protein-DNA aggregates. Although ATP(gamma S) blocks dissociation of recA protein from DNA, ATP stimulates the release of recA protein from SS DNA. Hydrolysis of the ATP is not required for dissociation since it is also enhanced by ADP and certain nucleoside triphosphates that are not hydrolyzed by recA protein. recA protein binds with different affinities to ribohomopolymers and deoxyhomopolymers. It preferentially binds polydeoxythymidylate and polydeoxycytidylate but does not bind short oligonucleotides, indicating that there is a minimum size requirement for the binding step. The recA protein exists as a heterogeneous aggregate at pH 7.5 and at low ionic strength. At pH 6.2 in the presence of Mg2+, the protein sediments homogeneously as a dimer. At pH 6.2, ATP or ATP(gamma S) promotes an oligomerization of the recA protein which can be observed as filamentous structures by electron microscopy. Oligomerization is not induced by UTP, a nucleoside triphosphate that is efficiently hydrolyzed by the recA protein, but fails to stimulate efficiently recA protein-promoted annealing and assimilation of single-stranded DNA.