A 21-base pair DNA fragment directs transcription attenuation within the simian virus 40 late leader.
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Transcription through the late simian virus 40 (SV40) sequences have been examined in vivo and in vitro for identifying attenuation signals involved in regulating late RNA transcription. In addition to the previously identified and characterized attenuator 1 situated 93 nucleotides downstream from the major late transcription start site, a second attenuator, attenuator 2, situated 55 nucleotides downstream from it, has been identified. Attenuated transcripts mapping to this site have been observed in vivo as well as in several different in vitro transcription systems. The signal inducing transcription attenuation has been localized to a 21-base pair DNA fragment and has been found to function independently of the promoter directing transcription or its distance from the transcription start site. Furthermore, this attenuator, unlike that preceding it, does not include a region of dyad symmetry or A+T-rich sequences. Also, similar to the adenovirus 2 attenuator, but in contrast to SV40 attenuator 1, the block to elongation at the SV40 attenuator 2 appears to be modulated by the general transcription factors. It is concluded that in SV40 there are at least two types of attenuators: one that is dependent on RNA secondary structure and a second that is sequence specific and is modulated, at least in part, by the general transcription factors. (+info)
Functional analysis of the Pseudomonas putida regulatory protein CatR: transcriptional studies and determination of the CatR DNA-binding site by hydroxyl-radical footprinting.
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CatR, a LysR family protein, positively regulates the Pseudomonas putida catBC operon, which is required for growth on benzoate as a sole carbon source. Transcriptional studies show that the catR and catBC promoters are divergent and overlapping by 2 bp. A beta-galactosidase promoter probe vector was constructed to analyze expression from the catR and catBC promoters under induced and uninduced conditions. As predicted, the catBC promoter is expressed only under induced conditions, while the catR promoter is constitutive. CatR has been shown to specifically bind the catRBC promoter region, and this property was used to devise a purification protocol for CatR. Linear M13 DNA containing the catRBC control region was covalently bound to cyanogen bromide-activated Sepharose in order to construct a DNA affinity column. Crude extracts containing hyperproduced CatR protein were then incubated with the affinity resin under binding conditions, and the CatR protein was eluted with 1 M NaCl. CatR was also purified by heparin-agarose chromatography. This highly purified protein was used for gel retardation and hydroxyl-radical footprinting studies. From this analysis, it was shown that CatR binds upstream of the catBC promoter within the transcribed region of catR. (+info)
MicroRNA fingerprints during human megakaryocytopoiesis.
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microRNAs are a highly conserved class of noncoding RNAs with important regulatory functions in proliferation, apoptosis, development, and differentiation. To discover novel regulatory pathways during megakaryocytic differentiation, we performed microRNA expression profiling of in vitro-differentiated megakaryocytes derived from CD34(+) hematopoietic progenitors. The main finding was down-regulation of miR-10a, miR-126, miR-106, miR-10b, miR-17 and miR-20. Hypothetically, the down-regulation of microRNAs unblocks target genes involved in differentiation. We confirmed in vitro and in vivo that miR-130a targets the transcription factor MAFB, which is involved in the activation of the GPIIB promoter, a key protein for platelet physiology. In addition, we found that miR-10a expression in differentiated megakaryocytes is inverse to that of HOXA1, and we showed that HOXA1 is a direct target of miR-10a. Finally, we compared the microRNA expression of megakaryoblastic leukemic cell lines with that of in vitro differentiated megakaryocytes and CD34(+) progenitors. This analysis revealed up-regulation of miR-101, miR-126, miR-99a, miR-135, and miR-20. Our data delineate the expression of microRNAs during megakaryocytopoiesis and suggest a regulatory role of microRNAs in this process by targeting megakaryocytic transcription factors. (+info)
DNase protection analysis of the stable synaptic complexes involved in Mu transposition.
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Several critical steps in phage Mu transposition involve specialized protein-DNA complexes. Cleavage of Mu donor DNA by MuA protein leads to the formation of the stable cleaved donor complex (CDC), in which the two Mu DNA ends are held together by MuA. In the subsequent strand-transfer reaction the CDC attacks a target DNA to generate the strand-transfer complex, in which the donor and the target DNAs are covalently joined. We have carried out DNase I protection experiments on these protein-DNA complexes and found that only three MuA binding sites (L1, R1, and R2 of the six total) at the two Mu ends are stably bound by MuA to maintain the paired Mu end structure. The protection extends beyond the ends of the Mu sequence for different lengths (7-20 nucleotides) depending on the strand and the type of complex. After formation of the CDC, the other MuA binding sites (L2, L3, and R3) and internal activation sequence become dispensable for the subsequent strand-transfer reaction. (+info)
Histone H1 inhibits eukaryotic DNA topoisomerase I.
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Histone H1 inhibits the catalytic activity of topoisomerase I in vitro. The relaxation activity of the enzyme is partially inhibited at a molar ratio of one histone H1 molecule per 40 base pairs (bp) of DNA and completely inhibited at a molar ratio of one histone H1 molecule per 10 base pairs of DNA. Increasing the amount of enzyme at a constant histone H1 to DNA ratio antagonizes the inhibition. This indicates that topoisomerase I and histone H1 compete for binding sites on the substrate DNA molecules. Consistent with this we show on the sequence level that histone H1 inhibits the cleavage reaction of topoisomerase I on linear DNA fragments. (+info)
Different allele-distribution of mthfr 677 C -> T and mthfr -393 C -> a in patients classified according to subtypes of Lesch's typology.
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AIMS: The typology by Lesch distinguishes between four subtypes: type 1 (model of allergy), type 2 (model of anxiety or conflict), type 3 (alcohol as an antidepressant), and type 4 (alcohol as adaptation). Taking into account that alcohol dependence is associated with elevated homocysteine levels, this study was undertaken to investigate different MTHFR (methylenetetrahydrofolate reductase) genotypes related to homocysteine metabolism in patients with alcohol dependence who were classified according to Lesch's typology (LT). SUBJECTS AND METHODS: 134 non-abstinent chronic alcoholics (112 males, 22 females; mean age 44.2 (SD 8.9) years) were classified according to LT and divided into four groups: LT 1 (n = 26), LT 2 (n = 65), LT 3 (n = 58), and LT 4 (n = 18). Total plasma homocysteine levels and MTHFR genotypes -393, 677, and 1,793 were determined. RESULTS: We observed a significantly higher frequency of the thermolabile MTHFR 677 C-->T variant (TT) in patients classified as subtype LT4 when compared with subtypes LT2 and LT3 (P = 0.005). Furthermore, for the MTHFR -393 C --> A-polymorphism, significantly more AC/AA variants were found in subtype LT4 (P = 0.034). No differences in allele-distribution were detected for MTHFR 1793. CONCLUSION: To our knowledge, this is the first study evaluating MTHFR genotypes in patients who were classified according to LT. Significantly different distributions of MTHFR 677 and -393 variants within Lesch Type 4 as compared with Types 2 and 3 hint at genetic determination of Lesch subtypes. (+info)
Mapping noncovalent ligand binding to stemloop domains of the HIV-1 packaging signal by tandem mass spectrometry.
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The binding modes and structural determinants of the noncovalent complexes formed by aminoglycoside antibiotics with conserved domains of the HIV-1 packaging signal (Psi-RNA) were investigated using electrospray ionization (ESI) Fourier transform mass spectrometry (FTMS). The location of the aminoglycoside binding sites on the different stemloop structures was revealed by characteristic coverage gaps in the ion series obtained by sustained off-resonance irradiation collision induced dissociation (SORI-CID) of the antibiotic-RNA assemblies. The site positions were confirmed using mutants that eliminated salient structural features of the Psi-RNA domains. The effects of the mutations on the binding properties of the different substrates served to validate the position of the aminoglycoside site on the wild-type structures. Additional information was provided by docking experiments performed on the different aminoglycoside-stemloop complexes. The results have shown that, in the absence of features disrupting the regular A-helix of the double-stranded stem, aminoglycosides tend to bind in an area situated between the upper stem and the loop regions, as demonstrated for stemloop SL3. The presence of a tandem wobbles motif in SL4 modifies the regular geometry of the upper stem, which does not affect the general site location, but greatly increases its solution binding affinity compared with SL3. The platform motif in SL2 locates the binding site in the stem midsection and confers upon this stemloop an intermediate affinity toward aminoglycosides. In SL3 and SL4, the extensive overlap of the antibiotic site with the region used to bind the nucleocapsid (NC) protein provides the basis for a competition mechanism that could explain the aminoglycoside inhibition of the NC.SL3 and NC.SL4 assemblies. In contrast, the minimal overlap between the aminoglycoside and the NC sites in SL2 accounts for the absence of inhibition of the NC.SL2 complex. (+info)
MicroRNA-cancer connection: the beginning of a new tale.
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Cancer initiation and progression can involve microRNAs (miRNA), which are small noncoding RNAs that can regulate gene expression. Their expression profiles can be used for the classification, diagnosis, and prognosis of human malignancies. Loss or amplification of miRNA genes has been reported in a variety of cancers, and altered patterns of miRNA expression may affect cell cycle and survival programs. Germ-line and somatic mutations in miRNAs or polymorphisms in the mRNAs targeted by miRNAs may also contribute to cancer predisposition and progression. We propose that alterations in miRNA genes play a critical role in the pathophysiology of many, perhaps all, human cancers. (+info)