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At least 217 records · Page 12Linked to original sources
PubMed1981

Restriction and modification in Bacillus subtilis: two DNA methyltransferases with BsuRI specificity. II. Catalytic properties, substrate specificity, and mode of action.

The properties of two DNA methyltransferases, termed M. BsuRIa and M. BsuRIb, whose isolation was described in the preceding paper (Günthert, U., Freund, M., and Trautner, T. A. (1981) J. Biol. Chem. 256, 9340-9345) were compared. Both enzymes recognize the same target sequence in double-stranded DNA, leading to methylation of the internal cytosine: 5'GGCC. The enzymes have identical reaction constants with their substrates, DNA (km = 2.7 nM for the 5' GGCC sequence), and S-adenosyl-L-methionine (km = 0.7 microM). Initial rates of methyl group transfer were proportional to enzyme concentration over a range of 50-fold, indicating absence of aggregation. The enzymes are different in their ionic strength requirements using Tris-HCl, pH 8.4. M. BsuRIa is most active at 100 mM, M. BsuRIb at 440 mM. As measured by incorporation kinetics and heat inactivation, M. BsuRIa is the more stable enzyme of the two. Equilibrium dialysis was used to study the mode of methyl group transfer to the DNA with either enzyme. The data indicate that initially S-adenosyl-L-methionine binds to methyltransferase. This complex attaches to either modified or nonmodified DNA. The methyl group will then be transferred to a nonmodified target sequence, leading to the dissociation of enzyme and S-adenosyl-L-homocysteine from the DNA.

Bacillus subtilis↗
PubMed1989

Coordinate action of a proximal homeoprotein binding site and a distal sequence confers the Ultrabithorax expression pattern in the visceral mesoderm.

Spatially regulated expression of the homeotic gene Ultrabithorax (Ubx) in the visceral mesoderm can be mimicked in transformed Drosophila embryos by expression of a Ubx--beta-galactosidase fusion gene. Here we show that a proximal homeoprotien binding sequence downstream of the Ubx transcription start site, the B element, is required for this pattern. A distal upstream Ubx sequence, but not the B element, is sufficient to confer the pattern if linked to an hsp70 TATA box in a heterologous construct. The pattern in this case requires Ubx function, like endogenous Ubx expression in the visceral mesoderm, suggesting that the distal upstream sequence contains an important target sequence for autoregulation. We propose that the B element, in the context of the Ubx promoter, functions to mediate enhancer function of the distal sequence. Thus, the visceral mesoderm pattern requires coordinate action of a proximal and a distal regulatory element.

Animals↗
PubMed1988

N-(2-hydroxyethyl)phenazinium derivatives of oligonucleotides as effectors of the sequence-specific modification of nucleic acids with reactive oligonucleotide derivatives.

It has been found that mono- and especially diphenazinium derivatives of oligonucleotides complementary to the DNA sequence adjacent to the target sequence of the addressed alkylation of DNA, significantly enhance the extent and specificity of alkylation with p-(N-2-chloroethyl-N-methylamino)benzylamide derivatives of the addressing oligonucleotides, thus playing the role of effector of the sequence-specific (complementary addressed) modification.

Alkylation↗
PubMed1988

Pure exogenous singlet oxygen: nonmutagenicity in bacteria.

Singlet oxygen (1 delta gO2) is the lowest energy-excited state of molecular oxygen, and more reactive than the triplet ground-state molecule. Although singlet oxygen has been implicated in a variety of biological effects, including reactions with DNA or some of its components, evidence for mutagenesis by singlet oxygen has remained unclear. We have previously described a system for bacterial exposure to pure exogenous singlet oxygen that eliminates ambiguity regarding the identity of the reactive species responsible for observed results. Despite the potent toxicity of pure singlet oxygen for several different strains of bacteria, we have found no evidence for mutagenicity of singlet oxygen in 26 Salmonella typhimurium histidine-auxotrophic strains killed to 35% survival. These strains included a variety of base-pair substitution or frameshift target sequences for reversion, including targets responsive to oxidative damage and targets rich in GC base pairs. Some strains combined histidine mutations with one or more mutations affecting DNA-repair capacity. 4 strains possessing the hisG46 mutation also were not mutated when exposed to dose ranges killing less than 28% and up to 99% of the bacteria. The relative frequency of small inphase deletions was assayed in hisG428 bacteria exposed to single oxygen and found to be the same as the spontaneous level. In addition to lack of induction of mutation in these strains, the 8-azaguanine forward mutation assay yielded no evidence of mutagenesis by singlet oxygen in strains killed to 15% survival. No induction of genetic changes by singlet oxygen was seen in an assay for duplication of approximately 1/3 of the bacterial chromosome. Tests for the ability of singlet oxygen to induce lambda prophage in E. coli K12 also proved negative. These studies collectively indicate that pure singlet oxygen generated outside the bacterial cell does not react significantly with the bacterial chromosome in ways leading to base-pair substitutions, frameshift mutations, small or large deletions, large duplications, or damage that interferes with DNA replication and induces the SOS system.

Azaguanine↗
PubMed1990

MyoD binds cooperatively to two sites in a target enhancer sequence: occupancy of two sites is required for activation.

MyoD is a master regulatory gene for myogenesis. Its product, the MyoD protein, appears to act by binding to muscle-specific enhancer sequences. We show that MyoD binds cooperatively to two sites in the muscle-specific creatine kinase enhancer; this is dramatically reflected in dissociation-rate measurements. A deletion of the acidic N terminus (residues 3-56) results in a protein that binds normally to single sites but fails to bind cooperatively to two adjacent sites, suggesting a role of the N terminus in cooperative interactions. In transfection assays, a reporter gene flanked by a single MyoD binding site fails to be activated by cotransfected MyoD expression vectors. In contrast, a reporter with two or more MyoD binding sites is activated by wild-type MyoD but not by N-terminally deleted MyoD. A reporter gene with a single binding site, although not activated by MyoD, can nonetheless compete for expression with a reporter gene containing three sites. Thus, in vivo, a single site can bind MyoD, but occupancy of two or more sites is required for subsequent transcriptional activation.

Animals↗
PubMed1992

Tn10 insertion specificity is strongly dependent upon sequences immediately adjacent to the target-site consensus sequence.

Transposon Tn10 inserts preferentially into particular "hotspots" that have been shown by sequence analysis to contain the symmetrical consensus sequence 5'-GCTNAGC-3'. This consensus is necessary but not sufficient to determine insertion specificity. We have mutagenized a known hotspot to identify other determinants for insertion into this site. This genetic dissection of the sequence context of a protein binding site shows that a second major determinant for Tn10 insertion specificity is contributed by the 6-9 base pairs that flank each end of the consensus sequence. Variations in these context base pairs can confer variations of at least 1000-fold in insertion frequency. There is no discernible consensus sequence for the context determinant, suggesting that sequence-specific protein-DNA contacts are not playing a major role. Taken together with previous work, the observations presented suggest a model for the interaction of transposase with the insertion site: symmetrically disposed subunits bind with specific contacts to the major groove of consensus-sequence base pairs, while flanking sequences influence the interaction through effects on DNA helix structure. We also show that the determinants important for insertion into a site are not important for transposition out of that site.

Base Sequence↗
PubMed1990

Structural requirements in the herpes simplex virus type 1 transactivator Vmw65 for interaction with the cellular octamer-binding protein and target TAATGARAT sequences.

Herpes simplex virus type 1 virion protein Vmw65 forms a complex (TRF.C) with TAATGARAT sequences and the cellular transcription factor oct-1, which has been implicated as an intermediate in the activation of gene expression by Vmw65. To examine structural requirements within Vmw65 for this interaction, we analyzed extracts of transfected cells that express mutant Vmw65 proteins by gel retardation assay and identified two regions in the primary sequence of Vmw65 which are necessary for in vitro assembly of TRF.C. The amino-terminal boundary for complex assembly and trans activation mapped between residues 49 and 75. At the carboxyl terminus, deletion as far as residue 388 did not affect in vitro TRF.C assembly, although trans-activating activity was abolished. Deletion beyond residue 388 rapidly impaired the ability of the protein to participate in the TRF.C complex, such that a truncated mutant of 380 residues was completely inactive. These requirements towards the carboxyl terminus overlap a region of strong local sequence similarity between Vmw65 and terminal protein p3 of bacteriophage phi 29. Although substitution of corresponding p3 residues into Vmw65 failed to produce a functional chimera, site-directed mutagenesis within the region of similarity identified a number of single-point mutant proteins which were completely deficient for TRF.C formation. These mutant proteins were also unable to trans activate expression from immediate-early promoters, despite the integrity of the acidic carboxyl terminus. The extreme sensitivity of both TRF.C formation and trans activation to single-residue substitutions within this region of Vmw65 suggests that it is directly involved in the protein-protein or protein-DNA interactions required for assembly of a transcriptional complex containing oct-1.

Amino Acid Sequence↗
PubMed1987

Rapid prenatal diagnosis of sickle cell anemia by a new method of DNA analysis.

We have used a new method of DNA analysis for the rapid prenatal diagnosis of sickle cell anemia in two fetuses at risk for this disease. This method of detecting the sickle gene is a modification of standard restriction-enzyme techniques and requires only a small amount of DNA. The first step involves a 200,000-fold enzymatic amplification of the specific beta-globin DNA sequences that may carry the sickle mutation. This provides a sufficient quantity of DNA for the analysis. Next, a short radiolabeled synthetic DNA sequence homologous to normal beta A-globin gene sequences is hybridized to the amplified target sequences. The hybrid "duplexes" are then digested sequentially with two restriction endonucleases. The presence of beta A- or beta S-globin gene sequences in the amplified target DNA from the patient determines whether the beta A-hybridization probe anneals perfectly or with a single nucleotide mismatch. This difference affects the restriction-enzyme digestion of the DNA and the size of the resulting radiolabeled digestion products, which can be distinguished by electrophoresis followed by autoradiography. This method is sufficiently sensitive and rapid that the prenatal diagnosis of sickle cell anemia can be made on the same day that the fetal DNA is made available. It can also be applied to the diagnosis of hemoglobin C disease.

Anemia, Sickle Cell↗
PubMed1987

Profile analysis: detection of distantly related proteins.

Profile analysis is a method for detecting distantly related proteins by sequence comparison. The basis for comparison is not only the customary Dayhoff mutational-distance matrix but also the results of structural studies and information implicit in the alignments of the sequences of families of similar proteins. This information is expressed in a position-specific scoring table (profile), which is created from a group of sequences previously aligned by structural or sequence similarity. The similarity of any other sequence (target) to the group of aligned sequences (probe) can be tested by comparing the target to the profile using dynamic programming algorithms. The profile method differs in two major respects from methods of sequence comparison in common use: (i) Any number of known sequences can be used to construct the profile, allowing more information to be used in the testing of the target than is possible with pairwise alignment methods. (ii) The profile includes the penalties for insertion or deletion at each position, which allow one to include the probe secondary structure in the testing scheme. Tests with globin and immunoglobulin sequences show that profile analysis can distinguish all members of these families from all other sequences in a database containing 3800 protein sequences.

Amino Acid Sequence↗
PubMed2024

Sex differences in MAGEL2 gene promoter methylation in high functioning autism - trends from a pilot study using nanopore Cas9 targeted long read sequencing.

BACKGROUND: MAGEL2 is an autism susceptibility gene whose deficiency has been associated with autism-related behaviors in animal models and in syndromic human autism spectrum disorders (ASDs) such as Schaaf-Yang syndrome, but has not been studied in the broader autism spectrum. Given the capabilities of long-read sequencing technologies, this pilot study used a targeted nanopore sequencing approach to simultaneously examine MAGEL2 DNA sequence and methylation in adults with high-functioning autism (HFA) compared to neurotypical controls (NC). METHODS: Using DNA extracted from peripheral blood, Cas9-targeted nanopore DNA sequencing was used to analyze MAGEL2, including its entire regulatory construct (chr15:23639316-23651466), for sequence variation and 5-methyl-cytosine (5mC) modification in a cohort of adults with HFA compared to sex- and age-matched NC. Given the known sex differences in ASD and MAGEL2 KO animal models, results were further analyzed by sex. RESULTS: 20 adults with HFA (10 males, 10 females) and 20 NC were included. While there were no overall differences in MAGEL2 DNA sequence and 5mC modification between HFA and NC, we found a significant difference in MAGEL2 gene promoter methylation between males and females with HFA and NC of both sexes, with HFA males tending to show hypomethylation in a 300 bp long differentially methylated region (chr15:23647640-23647939) around the MAGEL2 transcription start site. CONCLUSIONS: In this pilot study utilizing nanopore Cas9 targeted DNA sequencing, significant sex-specific differences in MAGEL2 gene promoter methylation were identified in male adults with HFA in comparison to control groups, suggesting the potential for sex-specific epigenetic differences. However, further replication in larger cohorts is required to validate these findings.

Humans↗
PubMed1992

Multiple copies of SUC4 regulatory regions may cause partial de-repression of invertase synthesis in Saccharomyces cerevisiae.

Transformation to generate multiple copies of regulatory DNA sequences has been used to study the interactions between regulatory proteins and their target sequences, since a high copy number of these sequences may titrate trans-acting regulatory proteins. We have analyzed the synthesis of invertase in yeast strains carrying different SUC genes transformed with the multiple-copy plasmid pSH143, a derivative of pJDB207 containing the promoter and upstream regulatory sequences of SUC4. The results obtained seem to be strain dependent. Under repressing conditions a high copy number of SUC4 promoter regions may cause increased expression of the invertase genes resulting in the synthesis of external glycosylated protein. A similar result was obtained under de-repressing conditions since transformants from some strains showed higher levels of activity. These results suggest that transcriptional regulatory (negative) factors may become limiting when the copy number of their target DNA sequences is increased. This effect may depend on the amount of active repressor molecules as well as on their affinity for SUC4 upstream sequences. This is discussed on the basis of the nucleotide sequences of SUC promoters.

Base Sequence↗
PubMed1992

Age differences in short-term memory: organization or internal noise?

Older and young adults' letter search performances were examined on a short-term memory (STM) task where subjects compared a five-letter target sequence stored in short-term memory to a subsequently presented five-letter probe sequence. The same five letters were always presented on target and probe portions of a given trial, but on half of the trials, two letters were transposed in the first chunk, second chunk, or between the first and second chunks of the probe sequence ("No" trials). On the remaining half of the trials, the target and probe sequences were identical ("Yes" trials). Both young and older adults showed increases in reaction time (RT) when the chunk boundary for "yes" trials was different for the target and probe sequences of a given trial. This finding indicated that both age groups were organizing the sequences in STM in the same qualitative manner. However, older adults showed a relatively greater increase in RT and errors than young adults for second-chunk transpositions than for first-chunk or between-chunk transpositions, and this finding suggested that an age difference in task complexity could not account for this effect. We propose, though, that these data are consistent with an internal noise model.

Adolescent↗
PubMed1989

Studies on the biogenesis of the mammalian ATP synthase complex: isolation and characterization of a full-length cDNA encoding the rat F1-beta-subunit.

Screening a rat cDNA library with a fragment of the gene encoding the corresponding bovine protein, we have isolated cDNA clones encoding the complete precursor to the rat F1-beta-subunit. Comparison with other mammalian beta-subunit sequences reveals a 5' untranslated sequence of 27 bp, a targeting sequence of 48 amino acid residues, a 480 amino acid long mature polypeptide and a 3' untranslated region of approximately 150 bp. In vitro translation of F1-beta mRNA reveals the synthesis of multiple fragments of the protein. Preliminary characterization of intron-containing cDNA isolates indicates that the gene encoding the rat F1-beta subunit contains at least two introns.

Amino Acid Sequence↗
PubMed1991

Transposon-facilitated DNA sequencing.

We describe here a transposon-based DNA sequencing strategy that allows the introduction of sequencing priming sites throughout a target sequence by bacterial mating. A miniplasmid was designed to select against transposon insertions into the vector. Sites of transposon insertion are mapped by the polymerase chain reaction with bacterial overnight cultures providing the templates. A small set of plasmids with transposons spaced several hundred base pairs apart can then be sequenced. Sequencing primers corresponding to the transposon ends allow sequencing in both directions. Thus, the entire sequence of both strands can be easily determined.

Base Sequence↗
PubMed1990

Site-specific transposition of insertion sequence IS630.

IS630 is a 1.15-kilobase sequence in Shigella sonnei that, unlike many mobile elements, seems not to mediate cointegration between different replicons. To assess its transposition, we constructed composite elements containing inverted copies of IS630 flanking a drug resistance gene. We found that these composite elements transposed to plasmid ColE1 in Escherichia coli. DNA sequencing showed that transposition was, in all cases, to the dinucleotide sequence 5'-TA-3'. There were two preferred insertion sites which corresponded to the TA sequences in the inverted repeats of a 13-base-pair stem region of the [rho]-dependent transcription terminator. IS630 is flanked by TA, and nucleotide substitution by in vitro mutagenesis at these ends did not affect transposition activity of a composite element or its ability to insert preferentially into TA within the 13-base-pair inverted repeat sequences or to duplicate the target sequence.

Base Composition↗