Showing papers on "Base pair published in 1988"
TL;DR: An assay for the presence of given DNA sequences has been developed, based on the ability of two oligonucleotides to anneal immediately adjacent to each other on a complementary target DNA molecule, which permits the rapid and standardized identification of single-copy gene sequences in genomic DNA.
Abstract: An assay for the presence of given DNA sequences has been developed, based on the ability of two oligonucleotides to anneal immediately adjacent to each other on a complementary target DNA molecule. The two oligonucleotides are then joined covalently by the action of a DNA ligase, provided that the nucleotides at the junction are correctly base-paired. Thus single nucleotide substitutions can be distinguished. This strategy permits the rapid and standardized identification of single-copy gene sequences in genomic DNA.
1,320 citations
TL;DR: It is discussed that triplex formation (site-specific RNA binding to a DNA duplex) could serve as the basis for an alternative program of gene control in vivo.
Abstract: A 27-base-long DNA oligonucleotide was designed that binds to duplex DNA at a single site within the 5' end of the human c-myc gene, 115 base pairs upstream from the transcription origin P1. On the basis of the physical properties of its bound complex, it was concluded that the oligonucleotide forms a colinear triplex with the duplex binding site. By means of an in vitro assay system, it was possible to show a correlation between triplex formation at -115 base pairs and repression of c-myc transcription. The possibility is discussed that triplex formation (site-specific RNA binding to a DNA duplex) could serve as the basis for an alternative program of gene control in vivo.
762 citations
TL;DR: By using end-labeled DNA probes containing T or C single-base-pair mismatches and conditions for limited cleavage, it was shown that cleavage was at the base predicted by sequence analysis and that mismatches in a length of DNA could be readily detected by such an approach.
Abstract: The chemical reactivity of thymine (T), when mismatched with the bases cytosine, guanine, and thymine, and of cytosine (C), when mismatched with thymine, adenine, and cytosine, has been examined. Heteroduplex DNAs containing such mismatched base pairs were first incubated with osmium tetroxide (for T and C mismatches) or hydroxylamine (for C mismatches) and then incubated with piperidine to cleave the DNA at the modified mismatched base. This cleavage was studied with an internally labeled strand containing the mismatched T or C, such that DNA cleavage and thus reactivity could be detected by gel electrophoresis. Cleavage at a total of 13 T and 21 C mismatches isolated (by at least three properly paired bases on both sides) single-base-pair mismatches was identified. All T or C mismatches studied were cleaved. By using end-labeled DNA probes containing T or C single-base-pair mismatches and conditions for limited cleavage, we were able to show that cleavage was at the base predicted by sequence analysis and that mismatches in a length of DNA could be readily detected by such an approach. This procedure may enable detection of all single-base-pair mismatches by use of sense and antisense probes and thus may be used to identify the mutated base and its position in a heteroduplex.
753 citations
TL;DR: It is demonstrated that during the reaction many, but not all, of the adenosine residues are converted to inosine residues, and it is proposed that the covalent modification is responsible for the irreversible change in base pairing properties.
657 citations
TL;DR: Analysis of a series of mutants of an Escherichia coli alanine transfer RNA shows that substitution of a single G-U base pair in the acceptor helix eliminates aminoacylation withAlanine in vivo and in vitro.
Abstract: Analysis of a series of mutants of an Escherichia coli alanine transfer RNA shows that substitution of a single G-U base pair in the acceptor helix eliminates aminoacylation with alanine in vivo and in vitro. Introduction of that base pair into the analogous position of a cysteine and a phenylalanine transfer RNA confers upon each the ability to be aminoacylated with alanine. Thus, as little as a single base pair can direct an amino acid to a specific transfer RNA.
541 citations
TL;DR: High-resolution x-ray crystallography was used to study the DNA-binding domain of phage 434 repressor in complex with a synthetic DNA operator, showing recognition of the operator by direct interactions with base pairs in the major groove, combined with the sequence-dependent ability of DNA to adopt the required conformation on binding repressor.
Abstract: The repressors of temperate bacteriophages such as 434 and lambda control transcription by binding to a set of DNA operator sites. The different affinity of repressor for each of these sites ensures efficient regulation. High-resolution x-ray crystallography was used to study the DNA-binding domain of phage 434 repressor in complex with a synthetic DNA operator. The structure shows recognition of the operator by direct interactions with base pairs in the major groove, combined with the sequence-dependent ability of DNA to adopt the required conformation on binding repressor. In particular, a network of three-centered bifurcated hydrogen bonds among base pairs in the operator helps explain why 434 repressor prefers certain sites over others. These bonds, which stabilize the conformation of the bound DNA, can form only with certain sequences.
481 citations
TL;DR: The structure of a presumptive DNA intermediate in the integration of retroviral DNA was studied in a cell-free reaction with exogenously added target DNA and demonstrates that the linear form of Mo-MLV DNA can integrate directly without prior circularization.
389 citations
TL;DR: The technique involves the ligation of large genomic fragments into a vector that provides centromeric, telomeric and selective functions; the constructs are then introduced into Saccharomyces cerevisiae, and replicate in the same manner as the host chromosomes.
Abstract: The haploid genome of Caenorhabditis elegans consists of some 80 x 10(6) base pairs of DNA contained in six chromosomes. The large number of interesting loci that have been recognized by mutation, and the accuracy of the genetic map, mean that a physical map of the genome is highly desirable, because it will facilitate the molecular cloning of chosen loci. The first steps towards such a map used a fingerprinting method to link cosmid clones together. This approach reached its practical limit last year, when 90-95% of the genome had been cloned into 17,500 cosmids assembled into some 700 clusters (contigs), but the linking clones needed were either non-existent or extremely rare. Anticipating this, we had planned to link by physical means--probably by hybridization to NotI fragments separated by pulse field gel electrophoresis. NotI recognizes an eight base sequence of GC pairs; thus the fragments should be large enough to bridge regions that clone poorly in cosmids, and, with no selective step involved, would necessarily be fully representative. However, with the availability of a yeast artificial chromosome (YAC) vector, we decided to use this alternative source of large DNA fragments to obtain linkage. The technique involves the ligation of large (50-1,000 kilobase) genomic fragments into a vector that provides centromeric, telomeric and selective functions; the constructs are then introduced into Saccharomyces cerevisiae, and replicate in the same manner as the host chromosomes.
387 citations
TL;DR: The present account reviews this area of research by classifying structures and mechanisms of action, with comments on stereochemistry, and describes various classes of pendant groups that can be attached to increase the efficacy of oligonucleotides.
Abstract: Oligonucleotides specifically bind to complementary sequences of either genomic DNA or genomic RNA through hydrogen bonding of base pairs. In principle, relatively short oligomers (<20 bases) can specifically hybridize with DNA or RNA and thus be used for novel drug design strategies involving targeted interference of genetic expression at the level of transcription or translation. Conceivable chemotherapeutic applications predicated on sequence-specific hybridization (“antisense” inhibition) require oligonucleotide analogues that are resistant to in vivo degradation by enzymes such as nucleases. Nuclease-resistant analogues having modified internucleoside linkages (e.g., methylphosphonates or phosphorothioates) or modified nucleosides (e.g., 2′-0-methylribose or α-anomers) are now readily available by means of automated synthesis, and there are various classes of pendant groups (e.g., alkylating or intercalating agents) that can be attached to increase the efficacy of these analogues. The present account reviews this area of research by classifying structures and mechanisms of action, with comments on stereochemistry. Biological studies are briefly summarized, and pharmaceutically related topics of interest are noted.
340 citations
TL;DR: Two divalent metal ions interacting with the phosphodiester to be hydrolyzed are proposed to catalyze the exonuclease reaction by a mechanism that may be related to mechanisms of other enzymes that catalyze phospho-group transfer including RNA enzymes.
Abstract: High-resolution crystal structures of editing complexes of both duplex and single-stranded DNA bound to Escherichia coli DNA polymerase I large fragment (Klenow fragment) show four nucleotides of single-stranded DNA bound to the 3'-5' exonuclease active site and extending toward the polymerase active site. Melting of the duplex DNA by the protein is stabilized by hydrophobic interactions between Phe-473, Leu-361, and His-666 and the last three bases at the 3' terminus. Two divalent metal ions interacting with the phosphodiester to be hydrolyzed are proposed to catalyze the exonuclease reaction by a mechanism that may be related to mechanisms of other enzymes that catalyze phospho-group transfer including RNA enzymes. We suggest that the editing active site competes with the polymerase active site some 30 A away for the newly formed 3' terminus. Since a 3' terminal mismatched base pair favors the melting of duplex DNA, its binding and excision at the editing exonuclease site that binds single-stranded DNA is enhanced.
317 citations
TL;DR: The idea that a binding cleft that snugly fits correct base pairs and excludes water at the active site may amplify base-pair free energy differences by reducing entropy differences and increasing enthalpy differences sufficiently to account for nucleotide insertion and extension fidelity is explored.
Abstract: The relation between DNA polymerase fidelity and base pairing stability is investigated by using DNA primer-template duplexes that contain a common 9-base template sequence but have either correct (A.T) or incorrect (G.T, C.T, T.T) base pairs at the primer 3' terminus. Thermal melting and enzyme kinetic measurements are compared for each kind of terminus. Analysis of melting temperatures finds that differences between the free energy changes upon dissociation (delta delta Go) are only 0.2, 0.3, and 0.4 kcal.mol-1 (1 cal = 4.18 J) for terminal A.T compared to G.T, C.T, and T.T mispairs, respectively, at 37 degrees C. We show that enthalpy changes are directly correlated with entropy changes for normal and abnormal base pairs in DNA in aqueous solution and that delta delta Go values are small because of near cancellation of corresponding enthalpy and entropy components. The kinetics of elongating primer termini are measured with purified Drosophila DNA polymerase alpha. The matched A.T terminus is found to be extended approximately 200 times faster than a G.T mismatch and 1400 and 2500 times faster than C.T and T.T mismatches, respectively. Enzymatic discrimination against elongating mismatched termini is based mainly on Km rather than Vmax differences. From Km at 37 degrees C, we find delta delta Go values of 2.6-3.7 kcal.mol-1, about an order of magnitude greater than indicated by melting data. A similar measurement of nucleotide insertion kinetics has previously found rates of forming A.T base pairs to be 500 times greater than G.T mispairs and 20,000 times greater than C.T and T.T mispairs. Here also, Km differences are mainly responsible for discrimination and indicate even larger delta delta Go values (4.3-4.9 kcal.mol-1). Thus, free energy differences between correct and incorrect base pairs in the active site cleft of polymerase appear to be greater than 10 times as large as in aqueous medium. We explore the idea that a binding cleft that snugly fits correct base pairs and excludes water at the active site may amplify base-pair free energy differences by reducing entropy differences and increasing enthalpy differences sufficiently to account for nucleotide insertion and extension fidelity.
TL;DR: The crystal structure of the complex between the dodecamer d(CGCGAATTCGCG) and a synthetic dye molecule Hoechst 33258 was solved by X-ray diffraction analysis and refined to an R-factor of 15.7% at 2.25 A resolution.
Abstract: The crystal structure of the complex between the dodecamer d(CGCGAATTCGCG) and a synthetic dye molecule Hoechst 33258 was solved by X-ray diffraction analysis and refined to an R-factor of 15.7% at 2.25 A resolution. The crescent-shaped Hoechst compound is found to bind to the central four AATT base pairs in the narrow minor groove of the B-DNA double helix. The piperazine ring of the drug has its flat face almost parallel to the aromatic bisbenzimidazole ring and lies sideways in the minor groove. No evidence of disordered structure of the drug is seen in the complex. The binding of Hoechst to DNA is stabilized by a combination of hydrogen bonding, van der Waals interaction and electrostatic interactions. The binding preference for AT base pairs by the drug is the result of the close contact between the Hoechst molecule and the C2 hydrogen atoms of adenine. The nature of these contacts precludes the binding of the drug to G-C base pairs due to the presence of N2 amino groups of guanines. The present crystal structural information agrees well with the data obtained from chemical footprinting experiments.
TL;DR: The pathway for base pair opening within a B-DNA duplex is investigated by theoretical molecular modeling and it is found that the opening process is greatly facilitated by DNA bending and that, conversely, once a base pair is disrupted, DNA can bend very easily.
Abstract: The pathway for base pair opening within a B-DNA duplex is investigated by theoretical molecular modeling. The results show that the disruption of a single base pair is energetically compatible with the deductions made from hydrogen exchange measurements. In addition, it is found that the opening process is greatly facilitated by DNA bending and that, conversely, once a base pair is disrupted, DNA can bend very easily. It appears that the energetic coupling between these two processes may play an important role in many biological reactions involving nucleic acid distortion.
TL;DR: A high degree of homology between the two gene products involved in iron-molybdenum cofactor biosynthesis and the two nitrogenase component I structural proteins (NifD and NifK) was found.
TL;DR: In this paper, a set of cDNA clones encoding human topoisomerase I were isolated from an expression vector library (lambda gt11) screened with autoimmune anti-topoisomerases I serum.
Abstract: cDNA clones encoding human topoisomerase I were isolated from an expression vector library (lambda gt11) screened with autoimmune anti-topoisomerase I serum. One of these clones has been expressed as a fusion protein comprised of a 32-kDa fragment of the bacterial TrpE protein linked to 67.7 kDa of protein encoded by the cDNA. Three lines of evidence indicate that the cloned cDNA encodes topoisomerase I. (i) Proteolysis maps of the fusion protein and human nuclear topoisomerase I are essentially identical. (ii) The fusion protein relaxes supercoiled DNA, an activity that can be immunoprecipitated by anti-topoisomerase I serum. (iii) Sequence analysis has revealed that the longest cDNA clone (3645 base pairs) encodes a protein of 765 amino acids that shares 42% identity with Saccharomyces cerevisiae topoisomerase I. The sequence data also show that the catalytically active 67.7-kDa fragment is comprised of the carboxyl terminus.
TL;DR: The results indicate that very few nucleotide or amino acid changes differentiate the antigenic and host range specificity of FPV and CPV.
Abstract: The genome of a canine parvovirus isolate strain (CPV-N) was cloned, and the DNA sequence was determined. The entire genome, including ends, was 5,323 nucleotides in length. The terminal repeat at the 3' end of the genome shared similar structural characteristics but limited homology with the rodent parvoviruses. The 5' terminal repeat was not detected in any of the clones. Instead, a region of DNA starting near the capsid gene stop codon and extending 248 base pairs into the coding region had been duplicated and inserted 75 base pairs downstream from the poly(A) addition site. Consensus sequences for the 5' donor and 3' acceptor sites as well as promotors and poly(A) addition sites were identified and compared with the available information on related parvoviruses. The genomic organization of CPV-N is similar to that of feline parvovirus (FPV) in that there are two major open reading frames (668 and 722 amino acids) in the plus strand (mRNA polarity). Both coding domains are in the same frame, and no significant open reading frames were apparent in any of the other frames of both minus and plus DNA strands. The nucleotide and amino acid homologies of the capsid genes between CPV-N and FPV were 98 and 99%, respectively. In contrast, the nucleotide and amino acid homologies of the capsid genes for CPV-N and CPV-b (S. Rhode III, J. Virol. 54:630-633, 1985) were 95 and 98%, respectively. These results indicate that very few nucleotide or amino acid changes differentiate the antigenic and host range specificity of FPV and CPV.
TL;DR: In this article, the authors co-crystallized a complex of DNase I with a self-complementary octanucleotide and refined the crystal structure at 2 A resolution.
Abstract: The cutting rates of bovine pancreatic deoxyribonuclease I (DNase I) vary along a given DNA sequence, indicating that the enzyme recognizes sequence-dependent structural variations of the DNA double-helix. In an attempt to define the helical parameters determining this sequence-dependence, we have co-crystallized a complex of DNase I with a self-complementary octanucleotide and refined the crystal structure at 2 A resolution. This structure confirms the basic features of an early model, namely that an exposed loop of DNase I binds in the minor groove of B-type DNA and that interactions do occur with the backbone of both strands. Nicked octamer duplexes that have lost a dinucleotide from the 3'-end of one strand are hydrogen-bonded across a two-fold axis in the crystal to form a quasi-continuous double helix of 14 base pairs. The DNA 14-mer has a B-type conformation and shows substantial distortion of both local and overall helix parameters, induced mainly by the tight interaction of Y73 and R38 in the unusually wide minor groove. Directly coupled to the widening of the groove by approximately 3A is a 21.5 degree bend of the DNA away from the bound enzyme towards the major groove, suggesting that both DNA stiffness and groove width are important in determining the sequence-dependence of the enzyme cutting rate. A second cut of the DNA which is induced by diffusion of Mn2+ into the co-crystals suggests that there are two active sites in DNase I separated by more than 15A.
TL;DR: RNA transfer (Northern) blot analysis was used to perform the physical characterization of the transcript expressed in murine sensory nerve ganglia latently infected with herpes simplex virus type 1 and its possible function in the latent phase of infection.
Abstract: RNA transfer (Northern) blot analysis was used to perform the physical characterization of the transcript expressed in murine sensory nerve ganglia latently infected with herpes simplex virus type 1. Most of this latency-associated transcript (LAT) was isolated in the poly(A)- fraction from ganglia. A smaller RNA species was also detected at less than 10% the abundance of the major one. LAT was not detected with probes from DNA outside the limits of the larger species. In situ hybridization data correlated well with Northern blot analysis; however, low levels of hybridization were seen with probes immediately outside the region of viral DNA giving positive Northern blot signals. S1 nuclease and primer extension mapping were used to locate the 5' end of the LAT 510 bases to the left of a KpnI site at 0.783 map units. The 3' end of the major latency-associated species was mapped to just within a 310-base-pair SmaI fragment located 660 to 970 base pairs to the right of the SalI site at 0.790 map units. These data were correlated with an analysis of the sequence of the DNA encoding this transcript and its possible function in the latent phase of infection.
TL;DR: New specificities indicate that the hydroxyl group of Thr81 makes a specific contact with one of the four critical base pairs in a trp operator half-site, and the methyl group of Thr81 determines specificity at a second, critical base pair.
Abstract: Oligonucleotide-directed mutagenesis of the codons for glutamine-68 (Gln68), lysine-72 (Lys72), isoleucine-79 (Ile79), alanine-80 (Ala80), and threonine-81 (Thr81) of the Escherichia coli trpR (tryptophan aporepressor) gene was used to make mutant repressors with each of 36 different amino acid changes Mutant repressors were tested for binding to each member of a set of 28 different operators closely related to the consensus trp operator Of the 36 mutant repressors, 11 bind a subset of the 28 operators; 5 of these have new binding specificities These new specificities indicate that the hydroxyl group of Thr81 makes a specific contact with one of the four critical base pairs in a trp operator half-site, and the methyl group of Thr81 determines specificity at a second, critical base pair The Trp repressor does not use the first two amino acids of its "recognition alpha-helix," Ile79 and Ala80, to make sequence-specific DNA contacts, and interacts with its operator in vivo in a way fundamentally different from the way that phage lambda repressor, lambda Cro protein, and coliphage 434 repressor contact their respective binding sites
TL;DR: Three of the lambda clones contained sequences that were not only expressed as fusion proteins with beta-galactosidase but were recognized by human hepatitis delta virus-specific antibody and indicated an exceptional ability for intramolecular base pairing.
Abstract: cDNA prepared from the single-stranded circular RNA genome of hepatitis delta virus was cloned in lambda gt11 by using RNA from the liver of an infected woodchuck. From the sequence of overlapping clones, we assembled the full sequence of 1,679 nucleotides. The sequence indicated an exceptional ability for intramolecular base pairing, yielding a rod structure with at least 70% of the bases paired and a predicted free energy of -805 kcal (-3,368 kJ)/mol. Three of the lambda clones contained sequences that were not only expressed as fusion proteins with beta-galactosidase but were recognized by human hepatitis delta virus-specific antibody. These clones were sequenced so as to establish the reading frame of the delta antigen on the antigenomic strand. The fusion protein produced by one clone was purified by immunoaffinity chromatography and then was used to raise rabbit antibodies specific for the delta antigen.
TL;DR: In vivo "footprints" of the glnA regulatory region under activating conditions demonstrate that the three most upstream activator sequences bind the protein NRI in the cell, placing interesting restrictions on models describing the mechanism by which NRI activates transcription from glnAp2 at a distance.
Abstract: In vivo "footprints" of the glnA regulatory region under activating conditions demonstrate that the three most upstream activator sequences bind the protein NRI in the cell. Together, protections at these sites span six of seven consecutive major grooves and lie on the same helix face. E sigma 54 protects two major grooves of DNA approximately 60 base pairs downstream at the glnAp2 promoter and primarily on the opposite helix face. Experiments using potassium permanganate to probe open complex formation in vivo demonstrate that NRI is absolutely required for E sigma 54 to open the promoter DNA. Together, the dimethyl sulfate and permanganate studies verify [Reitzer, L. J., Bueno, R., Cheng, W. D., Abrams, S. A., Rothstein, D. M., Hunt, T. P., Tyler, B. & Magasanik, B. (1987) J. Bacteriol. 169, 4279-4284] that E sigma 54 occupies the glnAp2 promoter in a closed complex in vivo even in the presence of excess nitrogen and the absence of NRI. Furthermore, the slow step in transcriptional activation is shown to be an NRI-dependent conformational change in the downstream promoter DNA, which results in DNA melting. These observations place interesting restrictions on models describing the mechanism by which NRI activates transcription from glnAp2 at a distance.
TL;DR: The crystal structure of the synthetic DNA dodecamer CGCATATATGCG has been solved at 2.2-A resolution and three general principles emerge from x-ray analyses of B-DNA oligonucleotides are emerged.
Abstract: The crystal structure of the synthetic DNA dodecamer CGCATATATGCG has been solved at 2.2-A resolution. Its central 6 base pairs adopt the alternating-B-DNA helix structure proposed nearly a decade ago. This alternating poly(AT) structure contrasts with the four known examples of what can be termed a poly(A) subfamily of B-DNA structures: CGCAAAAAAGCG, CGCAAATTTGCG, CGCGAATTCGCG, and CGCGAATTbrCGCG, their defining characteristic being a succession of two or more adenines along one strand, in a region of 4 or more A.T base pairs. All five helices show a characteristically narrow minor groove in their AT centers, but the mean propeller twist at A.T base pairs is lower in the alternating poly(AT) helix than in the poly(A) subfamily of helices. Three general principles emerge from x-ray analyses of B-DNA oligonucleotides: (i) GC and mixed-sequence B-DNA have a wide minor groove, whereas the minor groove is narrow in heteropolymer or homopolymer AT sequences. (ii) G.C base pairs have low propeller twist; A.T pairs can adopt a high propeller twist but need not do so. A high propeller twist can be stabilized by cross-strand hydrogen bonds in the major or minor groove, examples being the minor groove bonds seen in CCAAGATTGG and the major groove bonds that can accompany AA sequences in the poly(A) family. (iii) Homopolymer poly(A) tracts may be stiffer than are alternating AT or general-sequence DNA because of these cross-strand major groove hydrogen bonds. Poly(A) tracts appear internally unbent, but bends may occur at junctions with mixed-sequence DNA because of differences in propeller twist, base pair inclination, and base stacking on the two sides of the junction. Bending occurs most easily via base roll, favoring compression of the broad major groove.
TL;DR: It is shown that disruption of the postulated base pairs by single nucleotide substitutions, either in the S‐D sequence required for shifting or in nucleotide 1538 of 16S rRNA, decrease the amount of shifting, and that this defect is corrected by restoring complementary base pairing.
Abstract: Watson-Crick base pairing is shown to occur between the mRNA and nucleotides near the 3' end of 16S rRNA during the elongation phase of protein synthesis in Escherichia coli. This base-pairing is similar to the mRNA-rRNA interaction formed during initiation of protein synthesis between the Shine and Dalgarno (S-D) nucleotides of ribosome binding sites and their complements in the 1540-1535 region of 16S rRNA. mRNA-rRNA hybrid formation during elongation had been postulated to explain the dependence of an efficient ribosomal frameshift on S-D nucleotides precisely spaced 5' on the mRNA from the frameshift site. Here we show that disruption of the postulated base pairs by single nucleotide substitutions, either in the S-D sequence required for shifting or in nucleotide 1538 of 16S rRNA, decrease the amount of shifting, and that this defect is corrected by restoring complementary base pairing. This result implies that the 3' end of 16S rRNA scans the mRNA very close to the decoding sites during elongation.
TL;DR: Results indicate that the first strand transfer step in reverse transcription is an intermolecular event while that of the second transfer is intramolecular, and retroviruses contain two functionally active RNA's, and both may be required for the generation of a single linear DNA molecule.
Abstract: Retroviruses contain two copies of the plus stranded viral RNA genome. As a means of determining whether both of these RNA's are used in the reverse transcription reaction, cells were infected with heterozygous virus particles that varied in nucleotide sequence at two separate locations at the RNA termini. The DNA proviruses formed from a single cycle of reverse transcription were then examined. Of the 12 proviruses that were characterized, all exhibited long terminal repeats (LTR's) that would be expected to arise only if both RNA templates were used for the generation of minus strand DNA. In contrast, only a single minus strand DNA appeared to be used as template for the plus strand DNA in the generation of fully double-stranded viral DNA. These results indicate that the first strand transfer step in reverse transcription is an intermolecular event while that of the second transfer is intramolecular. Thus, retroviruses contain two functionally active RNA's, and both may be required for the generation of a single linear DNA molecule. Formation of heterozygotes during retrovirus infection would be expected to result in the efficient generation of LTR recombinants.
TL;DR: All three of theADH2 alleles and both of the ADH3 alleles are identified, including a new MaeIII site created in the third exon by the single base pair alteration in ADH2(2).
TL;DR: Mature enterotoxin A has 82, 72, 74, and 34 amino acid residues in common with staphylococcal enterotoxins B and C1, type A streptococcal exotoxin, and toxic shock syndrome toxin 1, respectively, and hybridization results indicate that considerable sequence divergence has occurred within this family of exotoxin.
Abstract: We determined the nucleotide sequence of the gene encoding staphylococcal enterotoxin A (entA). The gene, composed of 771 base pairs, encodes an enterotoxin A precursor of 257 amino acid residues. A 24-residue N-terminal hydrophobic leader sequence is apparently processed, yielding the mature form of staphylococcal enterotoxin A (Mr, 27,100). Mature enterotoxin A has 82, 72, 74, and 34 amino acid residues in common with staphylococcal enterotoxins B and C1, type A streptococcal exotoxin, and toxic shock syndrome toxin 1, respectively. This level of homology was determined to be significant based on the results of computer analysis and biological considerations. DNA sequence homology between the entA gene and genes encoding other types of staphylococcal enterotoxins was examined by DNA-DNA hybridization analysis with probes derived from the entA gene. A 624-base-pair DNA probe that represented an internal fragment of the entA gene hybridized well to DNA isolated from EntE+ strains and some EntA+ strains. In contrast, a 17-base oligonucleotide probe that encoded a peptide conserved among staphylococcal enterotoxins A, B, and C1 hybridized well to DNA isolated from EntA+, EntB+, EntC1+, and EntD+ strains. These hybridization results indicate that considerable sequence divergence has occurred within this family of exotoxins.
TL;DR: A mechanism is proposed in which folding of the untranslated region brings together codons separated by the interruption so that the elongating ribosome may skip the 50 nucleotides during translation.
Abstract: A 50-nucleotide untranslated region is shown to be present within the coding sequence of Escherichia coli bacteriophage T4 gene 60, which encodes one of the subunits for its type II DNA topoisomerase. This interruption is part of the transcribed messenger RNA and appears not to be removed before translation. Thus, the usual colinearity between messenger RNA and the encoded protein sequence apparently does not exist in this case. The interruption is bracketed by a direct repeat of five base pairs. A mechanism is proposed in which folding of the untranslated region brings together codons separated by the interruption so that the elongating ribosome may skip the 50 nucleotides during translation. The alternative possibility, that the protein is efficiently translated from a very minor and undetectable form of processed messenger RNA, seems unlikely, but has not been completely ruled out.
TL;DR: The nucleotide sequence of the xynZ gene, encoding the extracellular xylanase Z of Clostridium thermocellum, was determined and it was shown that the active site was located in the C-terminal half of the protein and that the conserved region was not involved in catalysis.
Abstract: The nucleotide sequence of the xynZ gene, encoding the extracellular xylanase Z of Clostridium thermocellum, was determined. The putative xynZ gene was 2,511 base pairs long and encoded a polypeptide of 837 amino acids. A region of 60 amino acids containing a duplicated segment of 24 amino acids was found between residues 429 and 488 of xylanase Z. This region was strongly similar to the conserved domain found at the carboxy-terminal ends of C. thermocellum endoglucanases A, B, and D. Deletions removing up to 508 codons from the 59 end of the gene did not affect the activity of the encoded polypeptide, showing that the active site was located in the C-terminal half of the protein and that the conserved region was not involved in catalysis. Expression of xylanase activity in Escherichia coli was increased up to 220-fold by fusing fragments containing the 39 end of the gene with the start of lacZ present in pUC19. An internal translational initiation site which was efficiently recognized in E. coli was tentatively identified 470 codons downstream from the actual start codon. Images
TL;DR: The cDNA clone for strictosidine synthase, the enzyme which catalyzes the stereospecific condensation of tryptamine with secologanin to form the key intermediate in indole alkaloid biosynthesis, strictosalid, has been identified with a synthetic oligodeoxynucleotide hybridization probe in a λgt11 cDNA library of cultured cells of Rauvolfia serpentina.
TL;DR: The DNA helicase activity associated with purified simian virus 40 (SV40) large tumor (T) antigen has been examined and it was found that under specified conditions, only duplex DNA fragments containing an intact SV40 core origin were unwound.