Identification of a cAMP-responsive region in thyroglobulin gene promoter.

The DNA sequences involved in transcription control by a cAMP-dependent mechanism have been localized in the thyroglobulin gene promoter region by a functional assay. The proximal 5'-flanking sequences from the bovine thyroglobulin gene were linked to the bacterial chloramphenicol acetyl-transferase gene. Transient expression of this reporter gene was studied in dog thyrocytes in primary culture in the presence, or absence, of cAMP stimulation. Deletion analysis showed that the cAMP-responsive region is contained within the first 250 base-pairs of the promoter, and suggests that it could correspond to a sequence conserved between species. These DNA sequences do not bear significant homology with cAMP-responsive elements (CRE) described previously. By contrast, some similarities were found with the fat-specific element (FSE2) of genes under cAMP control in adipocytes and with DNA elements mediating cAMP-dependent regulation of expression of two different genes in the lower eukaryote Dictyostelium discoideum. This suggests that control of Tg gene transcription by cAMP could involve a mechanism different from the one mediated by a classical CRE.

[Studies on iodine exchange in thermal therapy with salsobromoiodic water]. / Osservazioni sul ricambio dello iodio nella terapia termale con acque salsobromoiodiche.

We have attempted a quantitative evaluation of the iodine taken in with the thermal waters from Salsomaggiore during therapeutic bathing, inhalation (dry and damp spray), or ingestion. For this purpose to 127I we have applied the metabolic parameters obtained through a 131I inhalation test and a 125I ingestion test. Of the iodine inhaled by aerosol 45% becomes exhaled; by 24 hours 2% is in the serum and in the extra-thyroid area of iodine distribution, 16% in the thyroid, 16% in the urine. By adding the amount of iodine exhaled to that found in the metabolic cycle of iodine, we find that about 21% of the inhaled iodine is still missing. This amount is trapped in the respiratory tract from where it disappears only very gradually. At the end of the 24 hours, therefore, in the metabolic cycle of the iodine we find 34% of that inhaled, whereas we find 87% of that ingested. The level of iodine in the serum reached in thermal therapeutic inhalation, never stays at a level which might alter the functioning of a normal thyroid. The amount of inhaled iodine which is excreted with the urine is usually eliminated during the first excretions. Experimental studies suggest that the iodine taken in during bathing in the thermal-pools mainly comes from iodine released from the water through the addition of hypochlorites, and is then inhaled through breathing the air just above the water.

Searching for non-B DNA-forming motifs using nBMST (non-B DNA motif search tool).

This unit describes basic protocols on using the non-B DNA Motif Search Tool (nBMST) to search for sequence motifs predicted to form alternative DNA conformations that differ from the canonical right-handed Watson-Crick double-helix, collectively known as non-B DNA, and on using the associated PolyBrowse, a GBrowse-based genomic browser. The nBMST is a Web-based resource that allows users to submit one or more DNA sequences to search for inverted repeats (cruciform DNA), mirror repeats (triplex DNA), direct/tandem repeats (slipped/hairpin structures), G4 motifs (tetraplex, G-quadruplex DNA), alternating purine-pyrimidine tracts (left-handed Z-DNA), and A-phased repeats (static bending). The nBMST is versatile, simple to use, does not require bioinformatics skills, and can be applied to any type of DNA sequences, including viral and bacterial genomes, up to an aggregate of 20 megabasepairs (Mbp).

Mung bean nuclease cleavage pattern at a polypurine.polypyrimidine sequence upstream from the mouse metallothionein-I gene.

Mung bean nuclease, an enzyme specific for single-stranded DNA, was used to probe a non-B DNA structure present in the mouse metallothionein-I gene. The region sensitive to the enzyme was constituted by a 128 base-pair long polypurine.polypyrimidine sequence located at 1.2-kb from the start of transcription. A detailed analysis of the mung bean nuclease cleavage pattern revealed that: (i) under conditions of supercoiling and low pH a triplex structure was formed, (ii) the triplex was flanked by a sequence with the potential of forming a Z-DNA structure, (iii) most of the enzymatic activity was localized at some of the junctions between double-stranded and triple-stranded DNA and at mismatches in the triplex, (iv) no unpaired bases were observed in the loop or outside the triplex, and (v) the triplex was present in more than one configuration.

Triplet repeat instability and DNA topology: an expansion model based on statistical mechanics.

The variance of writhe, the contribution of writhe to supercoiling, and the free energies of supercoiling were calculated for (CTG.CAG)n and (CGG.CCG)n triplet repeat sequences (TRS) by statistical mechanics from the bending and torsional moduli previously determined. Expansions of these sequences are inherited by non-mendelian transmission and are linked with several hereditary neuromuscular diseases. The variance of writhe was greater for the TRS than for random B-DNA. For random B-DNA, (CGG)n, and (CTG)n, the contribution of writhe to supercoiling was 70, 78, and 79%, whereas the free energy of supercoiling at a length of 10 kilobase pairs was 1040.RT, 760.RT, and 685.RT, respectively. These data indicate that the TRS are preferential sites for the partitioning of supercoiling. Calculations of the differences in free energy of supercoiling between the TRS and random B-DNA revealed a local minimum at approximately 520 base pairs. Human medical genetic studies have shown that individuals carrying up to 180-200 copies of TRS (540-600 base pairs, premutations) in the fragile X or myotonic dystrophy gene loci are usually asymptomatic, whereas large expansions (>200 repeats, full mutations), which lead to disease, are observed in their offspring. Therefore, the length corresponding to the local minimum in free energy of supercoiling correlates with the genetic breakpoint between premutation and full mutation. We propose that (a) TRS instability is mediated by DNA mispairing caused by the accumulation of supercoiling within the repeats, and (b) the expansions that take place at the premutation to full mutation threshold are associated with increased mispairing caused by the optimal partitioning of writhe within the TRS at this length.

Nucleotide excision repair affects the stability of long transcribed (CTG*CAG) tracts in an orientation-dependent manner in Escherichia coli.

The influence of nucleotide excision repair (NER), the principal in vivo repair system for DNA damages, was investigated in Escherichia coli with uvrA, uvrB and uvrAuvrB mutants with the triplet repeat sequences (TRS) involved in myotonic dystrophy, the fragile X syndrome and Friedreich's ataxia. (CTG*CAG)175was more stable when the (CTG) strand was transcribed than when the (CAG) strand was transcribed in the alternate orientation. A lack of the UvrA protein dramatically increases the instability of this TRS in vivo as compared with the stability of the same sequence in uvrB mutant, which produces an intact UvrA protein. We propose that transcription transiently dissociates the triplet repeat complementary strands enabling the non-transcribed strand to fold into a hairpin conformation which is then sufficiently stable that replication bypasses the hairpin to give large deletions. If the TRS was not transcribed, fewer deletions were observed. Alternatively, in the uvrA-mutant, the hairpins existing on the lagging strand will suffer bypass DNA synthesis to generate deleted molecules. Hence, NER, functionally similar in both prokaryotes and eukaryotes, is an important factor in the genetic instabilities of long transcribed TRS implicated in human hereditary neuro-logical diseases.

Pkd1 unusual DNA conformations are recognized by nucleotide excision repair.

The 2.5-kilobase pair poly(purine.pyrimidine) (poly(R.Y)) tract present in intron 21 of the polycystic kidney disease 1 (PKD1) gene has been proposed to contribute to the high mutation frequency of the gene. To evaluate this hypothesis, we investigated the growth rates of 11 Escherichia coli strains, with mutations in the nucleotide excision repair, SOS, and topoisomerase I and/or gyrase genes, harboring plasmids containing the full-length tract, six 5'-truncations of the tract, and a control plasmid (pSPL3). The full-length poly(R.Y) tract induced dramatic losses of cell viability during the first few hours of growth and lengthened the doubling times of the populations in strains with an inducible SOS response. The extent of cell loss was correlated with the length of the poly(R.Y) tract and the levels of negative supercoiling as modulated by the genotype of the strains or drugs that specifically inhibited DNA gyrase or bound to DNA directly, thereby affecting conformations at specific loci. We conclude that the unusual DNA conformations formed by the PKD1 poly(R.Y) tract under the influence of negative supercoiling induced the SOS response pathway, and they were recognized as lesions by the nucleotide excision repair system and were cleaved, causing delays in cell division and loss of the plasmid. These data support a role for this sequence in the mutation of the PKD1 gene by stimulating repair and/or recombination functions.

Recombinant human DNA (cytosine-5) methyltransferase. III. Allosteric control, reaction order, and influence of plasmid topology and triplet repeat length on methylation of the fragile X CGG.CCG sequence.

Steady-state kinetic analyses revealed that the methylation reaction of the human DNA (cytosine-5) methyltransferase 1 (DNMT1) is repressed by the N-terminal domain comprising the first 501 amino acids, and that repression is relieved when methylated DNA binds to this region. DNMT1 lacking the first 501 amino acids retains its preference for hemimethylated DNA. The methylation reaction proceeds by a sequential mechanism, and either substrate (S-adenosyl-l-methionine and unmethylated DNA) may be the first to bind to the active site. However, initial binding of S-adenosyl-l-methionine is preferred. The binding affinities of DNA for both the regulatory and the catalytic sites increase in the presence of methylated CpG dinucleotides and vary considerably (more than one hundred times) according to DNA sequence. DNA topology strongly influences the reaction rates, which increased with increasing negative superhelical tension. These kinetic data are consistent with the role of DNMT1 in maintaining the methylation patterns throughout development and suggest that the enzyme may be involved in the etiology of fragile X, a syndrome characterized by de novo methylation of a greatly expanded CGG.CCG triplet repeat sequence.

An intramolecular triplex in the human gamma-globin 5'-flanking region is altered by point mutations associated with hereditary persistence of fetal hemoglobin.

The properties of an intramolecular triplex formed in vitro at the 5'-flanking region of the human gamma-globin genes were studied by chemical and physical probes. Chemical modifications performed with osmium tetroxide, chloroacetaldehyde, and diethyl pyrocarbonate revealed the presence of non-paired nucleotides on the "coding strand" at positions -209 through -217. These reactivities were induced by negative supercoiling, low pH, and magnesium ions. Downstream point mutations associated with hereditary persistence of fetal hemoglobin (HPFH) altered the extent of the modifications and some of the patterns. Specifically, C-202-->G and C-202-->T significantly decreased the reactivities, whereas the patterns were increased and altered in the T-198-->C. C-196-->T and C-195-->G caused local decreases in reactivity. Modifications at the upstream flanking duplex were modulated by the composition of the vector sequence. In summary, our data indicates the formation of an intramolecular triplex between nucleotides -209 to -217 of the "non-coding strand" and the downstream sequence containing the HPFH mutations. All of the HPFH point mutations altered the structure. More than one sequence alignment is possible for each of the triplexes. In addition, a consequence of some of the point mutations may be to facilitate slippage of the third strand relative to the Watson-Crick duplex.

Recombinant human DNA (cytosine-5) methyltransferase. I. Expression, purification, and comparison of de novo and maintenance methylation.

A method is described to express and purify human DNA (cytosine-5) methyltransferase (human DNMT1) using a protein splicing (intein) fusion partner in a baculovirus expression vector. The system produces approximately 1 mg of intact recombinant enzyme >95% pure per 1.5 x 10(9) insect cells. The protein lacks any affinity tag and is identical to the native enzyme except for the two C-terminal amino acids, proline and glycine, that were substituted for lysine and aspartic acid for optimal cleavage from the intein affinity tag. Human DNMT1 was used for steady-state kinetic analysis with poly(dI-dC).poly(dI-dC) and unmethylated and hemimethylated 36- and 75-mer oligonucleotides. The turnover number (k(cat)) was 131-237 h(-1) on poly(dI-dC).poly(dI-dC), 1.2-2.3 h(-1) on unmethylated DNA, and 8.3-49 h(-1) on hemimethylated DNA. The Michaelis constants for DNA (K(m)(CG)) and S-adenosyl-L-methionine (AdoMet) (K(m)(AdoMet)) ranged from 0.33-1.32 and 2.6-7.2 microM, respectively, whereas the ratio of k(cat)/K(m)(CG) ranged from 3.9 to 44 (237-336 for poly(dI-dC).poly(dI-dC)) x 10(6) M(-1) h(-1). The preference of the enzyme for hemimethylated, over unmethylated, DNA was 7-21-fold. The values of k(cat) on hemimethylated DNAs showed a 2-3-fold difference, depending upon which strand was pre-methylated. Furthermore, human DNMT1 formed covalent complexes with substrates containing 5-fluoro-CNG, indicating that substrate specificity extended beyond the canonical CG dinucleotide. These results show that, in addition to maintenance methylation, human DNMT1 may also carry out de novo and non-CG methyltransferase activities in vivo.

Recombinant human DNA (cytosine-5) methyltransferase. II. Steady-state kinetics reveal allosteric activation by methylated dna.

Initial velocity determinations were conducted with human DNA (cytosine-5) methyltransferase (DNMT1) on unmethylated and hemimethylated DNA templates in order to assess the mechanism of the reaction. Initial velocity data with DNA and S-adenosylmethionine (AdoMet) as variable substrates and product inhibition studies with methylated DNA and S-adenosylhomocysteine (AdoHcy) were obtained and evaluated as double-reciprocal plots. These relationships were linear for plasmid DNA, exon-1 from the imprinted small nuclear ribonucleoprotein-associated polypeptide N, (CGG.CCG)(12), (m(5)CGG. CCG)(12), and (CGG.CCG)(73) but were not linear for (CGG. Cm(5)CG)(12). Inhibition by AdoHcy was apparently competitive versus AdoMet and uncompetitive/noncompetitive versus DNA at

Small slipped register genetic instabilities in Escherichia coli in triplet repeat sequences associated with hereditary neurological diseases.

Genetic instability investigations on three triplet repeat sequences (TRS) involved in human hereditary neurological diseases (CTG.CAG, CGG.CCG, and GAA.TTC) revealed a high frequency of small expansions or deletions in 3-base pair registers in Escherichia coli. The presence of G to A polymorphisms in the CTG.CAG sequences served as reporters for the size and location of these instabilities. For the other two repeat sequences, length determinations confirmed the conclusions found for CTG.CAG. These studies were conducted in strains deficient in methyl-directed mismatch repair or nucleotide excision repair in order to investigate the involvement of these postreplicative processes in the genetic instabilities of these TRS. The observation that small and large instabilities for (CTG.CAG)175 fall into distinct size classes (1-8 repeats and approximate multiples of 41 repeats, respectively) leads to the conclusion that more than one DNA instability process is involved. The slippage of the complementary strands of the TRS is probably responsible for the small deletions and expansions in methyl-directed mismatch repair-deficient and nucleotide excision repair-deficient cells. A model is proposed to explain the observed instabilities via strand misalignment, incision, or excision, followed by DNA synthesis and ligation. This slippage-repair mechanism may be responsible for the small expansions in type 1 hereditary neurological diseases involving polyglutamine expansions. Furthermore, these observations may relate to the high frequency of small deletions versus a lower frequency of large instabilities observed in lymphoblastoid cells from myotonic dystrophy patients.

An unusually long poly(purine)-poly(pyrimidine) sequence is located upstream from the human thyroglobulin gene.

A region of human genomic DNA encompassing the 5' end of the thyroglobulin gene has been sequenced and the position of the transcriptional start site has been determined. The 5' non-translated portion of the mRNA displays a quasi-palindromic sequence which could allow this region to adopt a hairpin structure. The first exon of the gene encodes a 19 amino-acids signal peptide and the 3 first amino acids of the mature protein. Apart from the canonical TATA-Box and from a CAAT-Box homology, the promoter region contains a 209 bp-long poly(purine)-poly (pyrimidine) sequence located between positions-512 and -304 relative to the transcription start. When contained in a supercoiled plasmid, this sequence exhibits sensitivity to S1 nuclease at two distinct positions. A precise mapping of the borders of the sensitive regions was achieved by extending primers from both ends of the sequence after digestion by the enzyme. The resulting data can be explained by a model involving the formation of a triple helix structure.

Chimeric human immunodeficiency virus type 1/type 2 reverse transcriptases display reversed sensitivity to nonnucleoside analog inhibitors.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), an important therapeutic target in the treatment of AIDS, is effectively inhibited by a class of nonnucleoside analog compounds that includes nevirapine (BI-RG-587) and tetrahydroimidazo[4,5,1-jk]-[1,4]benzodiazepin-2(1H)-one and -thione. We show that both tyrosine residues at positions 181 and 188 flanking the putative catalytic site of HIV-1 RT are required for sensitivity of the enzyme to these compounds. HIV-2 RT, which does not have tyrosines at these positions, is resistant to these nonnucleoside analog inhibitors. Substitution of the HIV-2 RT amino acid residues at position 181 or 188 into HIV-1 RT results in an enzyme that is resistant to these compounds while retaining sensitivity to 3'-azido-2',3'-dideoxythymidine triphosphate. HIV-2 RT substituted with amino acids 176-190 from HIV-1 RT acquires sensitivity to these nonnucleoside analog inhibitors.

Flexible DNA: genetically unstable CTG.CAG and CGG.CCG from human hereditary neuromuscular disease genes.

The properties of duplex CTG.CAG and CGG.CCG, which are involved in the etiology of several hereditary neurodegenerative diseases, were investigated by a variety of methods, including circularization kinetics, apparent helical repeat determination, and polyacrylamide gel electrophoresis. The bending moduli were 1.13 x 10(-19) erg.cm for CTG and 1.27 x 10(-19) erg.cm for CGG, approximately 40% less than for random B-DNA. Also, the persistence lengths of the triplet repeat sequences were approximately 60% the value for random B-DNA. However, the torsional moduli and the helical repeats were 2.3 x 10(-19) erg.cm and 10.4 base pairs (bp)/turn for CTG and 2.4 x 10(-19) erg.cm and 10.3 bp/turn for CGG, respectively, all within the range for random B-DNA. Determination of the apparent helical repeat by the band shift assay indicated that the writhe of the repeats was different from that of random B-DNA. In addition, molecules of 224-245 bp in length (64-71 triplet repeats) were able to form topological isomers upon cyclization. The low bending moduli are consistent with predictions from crystallographic variations in slide, roll, and tilt. No unpaired bases or non-B-DNA structures could be detected by chemical and enzymatic probe analyses, two-dimensional agarose gel electrophoresis, and immunological studies. Hence, CTG and CGG are more flexible and highly writhed than random B-DNA and thus would be expected to act as sinks for the accumulation of superhelical density.

Amino acid substitutions in HIV-1 reverse transcriptase with corresponding residues from HIV-2. Effect on kinetic constants and inhibition by non-nucleoside analogs.

Nevirapine is a highly potent and specific inhibitor of human immunodeficiency virus type 1 (HIV-1) polymerase, but is inactive against HIV-2 and other polymerase. Previous studies demonstrated that residues 176-190 of HIV-1 reverse transcriptase (RT) can confer nevirapine sensitivity to HIV-2 RT. To better characterize the role of this sequence in HIV-1 RT, we have progressively substituted residues 176-190 of HIV-2 RT for those of HIV-1 RT and monitored the impact on the kinetic properties; inhibitory activity of nevirapine (11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido[2,3-b:2',3'-e] [1,4]diazepin-6-one), E-BPU (5-ethyl-1-benzyloxymethyl-6-(phenylthio)-uracil), and TIBO-R82150 ((+)-S-4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)imidazo[4,5,1-j k] [1,4]benzodiazepin-2(1H)-thione); and inhibitor-induced fluorescence changes of the mutant enzymes. The study revealed that in addition to Try-181 and Tyr-188, a new amino acid residue (Gly-190) plays an important role in determining susceptibility to nevirapine and E-BPU, but not to TIBO-R82150. These data argue that these non-nucleoside inhibitors fit differently, even though they share a common binding pocket. Nevirapine was seen to exert inhibitory activity by altering the interaction of the enzyme with the template-primer. Kinetic parameters were modulated by the template (DNA versus RNA) as well as by some of the mutations.