Showing papers in "Nucleic Acids Research in 1990"
TL;DR: A new DNA polymorphism assay based on the amplification of random DNA segments with single primers of arbitrary nucleotide sequence is described, suggesting that these polymorphisms be called RAPD markers, after Random Amplified Polymorphic DNA.
Abstract: Molecular genetic maps are commonly constructed by analyzing the segregation of restriction fragment length polymorphisms (RFLPs) among the progeny of a sexual cross. Here we describe a new DNA polymorphism assay based on the amplification of random DNA segments with single primers of arbitrary nucleotide sequence. These polymorphisms, simply detected as DNA segments which amplify from one parent but not the other, are inherited in a Mendelian fashion and can be used to construct genetic maps in a variety of species. We suggest that these polymorphisms be called RAPD markers, after Random Amplified Polymorphic DNA.
13,764 citations
TL;DR: The generality of the arbitrarily primed PCR method is demonstrated by application to twenty four strains from five species of Staphylococcus, eleven strains of Streptococcus pyogenes and three varieties of Oryza sativa.
Abstract: Simple and reproducible fingerprints of complex genomes can be generated using single arbitrarily chosen primers and the polymerase chain reaction (PCR). No prior sequence information is required. The method, arbitrarily primed PCR (AP-PCR), involves two cycles of low stringency amplification followed by PCR at higher stringency. We show that strains can be distinguished by comparing polymorphisms in genomic fingerprints. The generality of the method is demonstrated by application to twenty four strains from five species of Staphylococcus, eleven strains of Streptococcus pyogenes and three varieties of Oryza sativa (rice).
5,472 citations
TL;DR: From these 'sequence logos', one can determine not only the consensus sequence but also the relative frequency of bases and the information content at every position in a site or sequence.
Abstract: A graphical method is presented for displaying the patterns in a set of aligned sequences. The characters representing the sequence are stacked on top of each other for each position in the aligned sequences. The height of each letter is made proportional to its frequency, and the letters are sorted so the most common one is on top. The height of the entire stack is then adjusted to signify the information content of the sequences at that position. From these 'sequence logos', one can determine not only the consensus sequence but also the relative frequency of bases and the information content (measured in bits) at every position in a site or sequence. The logo displays both significant residues and subtle sequence patterns.
3,232 citations
TL;DR: Together, the pBabe vectors and omega E cell line should prove useful in experiments where highest frequencies of gene transfer, or concomitant expression of several different genes within a single cell are required with minimal risk of helper virus contamination.
Abstract: We report the development of an advanced system for transfer and expression of exogenous genes in mammalian cells based on Moloney murine leukemia virus (Mo MuLV). Extensive deletion/mutagenesis analysis to identify cis-acting signals involved in virus transmission has led to the design of a family of novel, highly efficient retroviral vectors and a partner helper-free packaging cell line. The pBabe retroviral vector constructs transmit inserted genes at high titres and express them from the Mo MuLV Long Terminal Repeat (LTR). Each of these vectors has been constructed with one of four different dominantly acting selectable markers, allowing the growth of infected mammalian cells in the presence of G418, hygromycin B, bleomycin/phleomycin or puromycin, respectively. The high titre ecotropic helper free packaging cell line, omega E, was designed in conjunction with the pBabe vectors to reduce the risk of generation of wild type Mo MuLV via homologous recombination events. The omega E cell line was generated with separate gagpol and ecotropic env expression constructs with minimal sequence overlap and decreased sequence homology achieved by 'codon wobbling'. Homologous env coding sequences were deleted from the pBabe vectors without diminishing recombinant vector titre. Together, the pBabe vectors and omega E cell line should prove useful in experiments where highest frequencies of gene transfer, or concomitant expression of several different genes within a single cell are required with minimal risk of helper virus contamination.
2,246 citations
TL;DR: The pEFBOS vector will be used to produce a large amount of growth factors and proteins in mammalian cells, to express a high level of anti-sense RNA, and will be an ideal positive control for CAT assay in various cell types.
Abstract: Polypeptide chain elongation factor l a (EF-la) is an eukaryotic counterpart of E. coli EF-Tu which promotes the GTP-dependent binding of an aminoacyl-tRNA to ribosomes. EF-la is one of the most abundant proteins in eukaryotic cells, and expressed in almost all kinds of mammalian cells. Recently, we have isolated human chromosomal gene coding for EF-la, and shown that the promoter of EF-la chromosomal gene very efficiently stimulates the in vitro transcription (1). In this report, we have constructed a powerful mammalian expression vector, pEF-BOS, using the promoter of human EF-la chromosomal gene. As shown in Fig. 1, pEF-BOS carries the SV40 replication origin (311 bp of EcoRH G fragment), the promoter region of human EF-la chromosomal gene (1.2 kb), the stuffer fragment (450 bp) from CDM8 vector (2) and poly(A) adenylation signal from human G-CSF cDNA (700 bp EcoSll ~ EcoRl DNA fragment) (3) in HindUL-EcoRl site of pUC119. The promoter region of EF-la gene is from nucleotide position 373 to 1561 (1) which includes 203 bp 5' flanking region, 33 bp first exon, 943 bp first intron and 10 bp of the part of the second exon located at 20 bp upstream of the ATG initiation codon. The size of pEFBOS is 5.8 kb, and the cDNA to be expressed can be inserted at BstXl site using BstXl adapter, or Xbal site using Xbal linker. Human G-CSF cDNA (4) was inserted into BstXl site of pEFBOS or CDM8, or into BamhU site of pKCR vector containing SV40 early promoter (5). As shown in Table 1, when these plasmids were transfected into COS cells by DEAEdextran/chloroquine method, the construct in pEF-BOS has directed the synthesis of human G-CSF about 20 times more efficiently than the construct in CDM8, and 50 ~ 200 times more efficiently than the construct in pKCR. In addition, when E. coli chloramphenicol acetyltransferase (CAT) gene was inserted into pEF-BOS, the CAT activities observed with pEF-BOS-CAT were 1.5 ~ 50 times higher than that of pSV2-CAT or pRSV-CAT after transfection into various cell lines including murine L929, human HeLa, CHU-2 and simian COS cells (Table 2). The pEFBOS vector, therefore, will be used to produce a large amount of growth factors and proteins in mammalian cells, to express a high level of anti-sense RNA. Furthermore, the pEF-BOS-CAT will be an ideal positive control for CAT assay in various cell types.
1,743 citations
1,362 citations
TL;DR: A new procedure has been developed for the efficient cloning of complex PCR mixtures, resulting in libraries exclusively consisting of recombinant clones, and the procedure is applied for the cloning of inter-ALU fragments from hybrid cell-lines and human cosmid clones.
Abstract: A new procedure has been developed for the efficient cloning of complex PCR mixtures, resulting in libraries exclusively consisting of recombinant clones. Recombinants are generated between PCR products and a PCR-amplified plasmid vector. The procedure does not require the use of restriction enzymes, T4 DNA ligase or alkaline phosphatase. The 5'-ends of the primers used to generate the cloneable PCR fragments contain an additional 12 nucleotide (nt) sequence lacking dCMP. As a result, the amplification products include 12-nt sequences lacking dGMP at their 3'-ends. The 3'-terminal sequence can be removed by the action of the (3'----5') exonuclease activity of T4 DNA polymerase in the presence of dGTP, leading to fragments with 5'-extending single-stranded (ss) tails of a defined sequence and length. Similarly, the entire plasmid vector is amplified with primers homologous to sequences in the multiple cloning site. The vector oligos have additional 12-nt tails complementary to the tails used for fragment amplification, permitting the creation of ss-ends with T4 DNA polymerase in the presence of dCTP. Circularization can occur between vector molecules and PCR fragments as mediated by the 12-nt cohesive ends, but not in mixtures lacking insert fragments. The resulting circular recombinant molecules do not require in vitro ligation for efficient bacterial transformation. We have applied the procedure for the cloning of inter-ALU fragments from hybrid cell-lines and human cosmid clones.
1,185 citations
TL;DR: The effects of various primer-template mismatches on DNA amplification of an HIV-1 gag region by the polymerase chain reaction (PCR) were investigated, although the G:G mismatches appeared to be more sensitive to sequence context and dNTP concentrations than other mismatches.
Abstract: We investigated the effects of various primer-template mismatches on DNA amplification of an HIV-1 gag region by the polymerase chain reaction (PCR). Single internal mismatches had no significant effect on PCR product yield while those at the 3'-terminal base had varied effects. A:G, G:A, and C:C mismatches reduced overall PCR product yield about 100-fold, A:A mismatches about 20-fold. All other 3'-terminal mismatches were efficiently amplified, although the G:G mismatches appeared to be more sensitive to sequence context and dNTP concentrations than other mismatches. It should be noted that mismatches of T with either G, C, or T had a minimal effect on PCR product yield. Double mismatches within the last four bases of a primer-template duplex where one of the mismatches is at the 3' terminal nucleotide, in general, reduced PCR product yield dramatically. The presence of a mismatched T at the 3'-terminus, however, allowed significant amplification even when coupled with an adjacent mismatch. Furthermore, even two mismatched Ts at the 3'-terminus allowed efficient amplification.
1,060 citations
TL;DR: Using PCR, it is found that normal heart muscle and brain from adult human individuals contain low levels of a specific mitochondrial DNA deletion, previously found only in patients affected with certain types of neuromuscular disease.
Abstract: Using PCR, we found that normal heart muscle and brain from adult human individuals contain low levels of a specific mitochondrial DNA deletion, previously found only in patients affected with certain types of neuromuscular disease. This deletion was not observed in fetal heart or brain. Experimental tests support the idea that the deletion exists in vivo in adult mitochondria and is not an in vitro artifact of PCR. Our data provide direct experimental support for the idea that accumulation of mitochondrial DNA deletions may be important in aging.
754 citations
TL;DR: The optimal annealing temperature (TaOPT) values for several primer-template pairs are experimentally determined and a method for its calculation is developed and found to be a function of the melting temperatures of the less stable primer- template pair and of the product.
Abstract: In the polymerase chain reaction (PCR) technique, DNA is amplified in vitro by a series of polymerization cycles consisting of three temperature-dependent steps: DNA denaturation, primer-template annealing, and DNA synthesis by a thermostable DNA polymerase. The purity and yield of the reaction products depend on several parameters, one of which is the annealing temperature (Ta). At both sub- and super-optimal Ta values, non-specific products may be formed, and the yield of products is reduced. Optimizing the Ta is especially critical when long products are synthesized or when total genomic DNA is the substrate for PCR. In this article we experimentally determine the optimal annealing temperature (TaOPT) values for several primer-template pairs and develop a method for its calculation. The TaOPT is found to be a function of the melting temperatures of the less stable primer-template pair and of the product. The fact that experimental and calculated TaOPT values agree to within 0.7 degree C eliminates the need for determining TaOPT experimentally. Synthesis of DNA fragments shorter than 1 kb is more efficient if a variable Ta is used, such that the Ta is higher in each consecutive cycle.
679 citations
TL;DR: A method for the rapid isolation of terminal sequences from YAC clones is developed, which provides a convenient method of generating Sequence Tagged Sites for the mapping of complex genomes.
Abstract: The recent development of yeast artificial chromosome (YAC) vectors has provided a system for cloning fragments that are over ten times larger than those that can be cloned in more established systems. We have developed a method for the rapid isolation of terminal sequences from YAC clones. The YAC clone is digested with a range of restriction enzymes, a common linker is ligated to the DNA fragments and terminal sequences are amplified using a vector specific primer and a linker specific primer. Sequence data derived from these terminal specific products can be used to design primers for a further round of screening to isolate overlapping clones. The method also provides a convenient method of generating Sequence Tagged Sites for the mapping of complex genomes.
TL;DR: Chaos Game Representation permits the representation and investigation of patterns in sequences, visually revealing previously unknown structures, and is a new tool for investigating gene structure.
Abstract: This paper presents a new method for representing DNA sequences. It permits the representation and investigation of patterns in sequences, visually revealing previously unknown structures. Based on a technique from chaotic dynamics, the method produces a picture of a gene sequence which displays both local and global patterns. The pictures have a complex structure which varies depending on the sequence. The method is termed Chaos Game Representation (CGR). CGR raises a new set of questions about the structure of DNA sequences, and is a new tool for investigating gene structure.
TL;DR: PCR co-amplification of two distinct HIV1 tat gene sequences lead to the formation of recombinant DNA molecules, and crossover sites mapped essentially to three discrete regions suggesting specific Taq DNA polymerase pause or termination sites.
Abstract: PCR co-amplification of two distinct HIV1 tat gene sequences lead to the formation of recombinant DNA molecules. The frequency of such recombinants, up to 5.4% of all amplified molecules, could be decreased 2.7 fold by a 6 fold increase in Taq DNA polymerase elongation time. Crossover sites mapped essentially to three discrete regions suggesting specific Taq DNA polymerase pause or termination sites. PCR mediated recombination may be a problem when studying heterogeneous genetic material such as RNA viruses, multigene families, or repetitive sequences. This phenomenon can be exploited to create chimeric molecules from related sequences.
TL;DR: The new method is considerably more powerful, detecting approximately 50% more likely helix-turn-helix sequences without an increase in false predictions.
Abstract: We present an update of our method for systematic detection and evaluation of potential helix-turn-helix DNA-binding motifs in protein sequences [Dodd, I. and Egan, J. B. (1987) J. Mol. Biol. 194, 557-564]. The new method is considerably more powerful, detecting approximately 50% more likely helix-turn-helix sequences without an increase in false predictions. This improvement is due almost entirely to the use of a much larger reference set of 91 presumed helix-turn-helix sequences. The scoring matrix derived from this reference set has been calibrated against a large protein sequence database so that the score obtained by a sequence can be used to give a practical estimation of the probability that the sequence is a helix-turn-helix motif.
TL;DR: Three sequences in the vicinity of poly (A) addition sites are conserved among vertebrate mRNAs and point mutations at the conserved adenosine shift the 3' end of the most abundant 5' half-molecule downstream by a single nucleotide.
Abstract: Three sequences in the vicinity of poly (A) addition sites are conserved among vertebrate mRNAs. We analyze the effects of single base changes in each position of AAUAAA and in the nucleotide to which poly (A) is added on 3' end formation in vitro. All 18 possible single base changes of the AAUAAA sequence greatly reduce addition of poly (A) to RNAs that end at the poly (A) addition site, and prevent cleavage of RNAs that extend beyond. The magnitude of reduction varies greatly with the position changed and the base introduced. For any given mutation, cleavage and polyadenylation are reduced to similar extents, strongly suggesting that the same factor interacts with AAUAAA in both reactions. Mutations at and near the conserved adenosine to which poly (A) is added disturb the accuracy, but not the efficiency, of 3' end formation. For example, point mutations at the conserved adenosine shift the 3' end of the most abundant 5' half-molecule downstream by a single nucleotide. The mechanism by which these mutations might exert their effects on the precision of 3' end formation are discussed.
TL;DR: Mobile genetic elements are useful genetic tools and have been found in most organisms which have been examined and will be used as probe for the identification of the M. tuberculosis complex.
Abstract: Mobile genetic elements are useful genetic tools. They have been found in most organisms which have been examined (for recent reviews see 1). /S900 was isolated from M. paratuberculosis (2) and IS6100 from a M. fortuitum strain (our unpublished results). IS elements have been used as taxonomic markers useful for diagnostic purposes, (2, 3). From a IA. tuberculosis cosmid library constructed in pHC79 (4), an IS-like element, 1S6110, was identified as a repeated sequence, by screening the library with labelled M. tuberculosis total DNA. This sequence (1361 nt) possesses characteristics of IS elements, i.e., inverted (28bp with 3 mismatched bp) and direct (3bp) repeats of the target sequence at its extremities. A search in the EMBL data bank has revealed homologies with IS3411, an insertion element from E. coli (5). Cross-hybridization was observed between 1S6110 and a repeated sequence previously isolated from M. tuberculosis (6). IS6110 has been found in M. tuberculosis and M. bovis but not in any of the other mycobacteria tested (our unpublished data). Therefore, IS6110 will be used as probe for the identification of the M. tuberculosis complex. ACKNOWLEDGEMENTS
TL;DR: Fifty sequences from the mouse genome database containing simple sequence repeats or microsatellites have been analysed for size variation using the polymerase chain reaction and gel electrophoresis to facilitate construction of high resolution maps of both the mouse and human genomes.
Abstract: Fifty sequences from the mouse genome database containing simple sequence repeats or microsatellites have been analysed for size variation using the polymerase chain reaction and gel electrophoresis. 88% of the sequences, most of which contain the dinucleotide repeat, CA/GT, showed size variations between different inbred strains of mice and the wild mouse, Mus spretus. 62% of sequences had 3 or more alleles. GA/CT and AT/TA-containing sequences were also variable. About half of these size variants were detectable by agarose gel electrophoresis. This simple approach is extremely useful in linkage and genome mapping studies and will facilitate construction of high resolution maps of both the mouse and human genomes.
TL;DR: The antiviral activity depended on the sequence of the oligodeoxynucleotide, but some compounds having little or no base complementarity to the viral target were also effective and were not toxic to the cells.
Abstract: Triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (2) was coupled to the 5' terminus of oligodeoxynucleotides via hydrogen phosphonate solid support DNA synthesis methodology. Duplex DNA oligomers with a single 5'-phospholipid melted at lower temperatures than the corresponding unmodified duplex, but duplexes bearing lipids at each 5' end had higher Tms. In uptake experiments with L929 cells, 8-10 times more lipid-DNA became cell-associated than did unmodified DNA. Unmodified antisense diesters were inactive in a VSV antiviral assay in L929 cells (at up to 200 microM). Attachment of a lipid to the oligomer, however, led to a greater than 90% at 150 microM (greater than 80% at 100 microM) reduction in viral protein synthesis. The antiviral activity depended on the sequence of the oligodeoxynucleotide, but some compounds having little or no base complementarity to the viral target were also effective. Phosphorothioate derivatives reduced viral protein synthesis by 20-30% at 100 microM in the VSV assay. The lipid-DNA compounds were not toxic to the cells at up to 100 microM.
TL;DR: A computer program which rapidly scans nucleic acid sequences to select all possible pairs of oligonucleotides suitable for use as primers to direct efficient DNA amplification by the polymerase chain reaction is designed.
Abstract: We have designed a computer program which rapidly scans nucleic acid sequences to select all possible pairs of oligonucleotides suitable for use as primers to direct efficient DNA amplification by the polymerase chain reaction. This program is based on a set of rules which define in generic terms both the sequence composition of the primers and the amplified region of DNA. These rules (1) enhance primer-to-target sequence hybridization avidity at critical 3'-end extension initiation sites, (2) facilitate attainment of full length extension during the 72 degrees C phase, by minimizing generation of incomplete or nonspecific product and (3) limit primer losses occurring from primer-self or primer-primer homologies. Three examples of primer sets chosen by the program that correctly amplified the target regions starting from RNA are shown. This program should facilitate the rapid selection of effective and specific primers from long gene sequences while providing a flexible choice of various primers to focus study on particular regions of interest.
TL;DR: The results suggest that CpG methylation inhibits proenkephalin gene expression by directly interfering with the binding of a positively acting transcription factor previously shown to be essential for maximal basal, cAMP, and TPA inducible transcription.
Abstract: DNA methylation at HpaII (CmCGG) sites inhibits expression of a human proenkephalin-CAT fusion gene when it is transiently expressed in CV-1 cells or stably expressed in C6-glioma cells. The inhibitory effects of HpaII methylation have been mapped to a site within the human proenkephalin promoter located at position -72 relative to the start site of transcription. This region spans a cAMP and phorbol ester inducible enhancer and methylation at this position inhibits both basal transcription and transcription induced by either cAMP or TPA. The HpaII site is located within an element which binds the transcription factor AP-2. In vitro methylation at this HpaII site inhibits the binding of AP-2. These results suggest that CpG methylation inhibits proenkephalin gene expression by directly interfering with the binding of a positively acting transcription factor previously shown to be essential for maximal basal, cAMP, and TPA inducible transcription.
TL;DR: It is demonstrated that despite lacking a 3'----5' proofreading exonuclease, the Thermus aquaticus (Taq) DNA polymerase can catalyze highly accurate DNA synthesis in vitro.
Abstract: We demonstrate that despite lacking a 3'----5' proofreading exonuclease, the Thermus aquaticus (Taq) DNA polymerase can catalyze highly accurate DNA synthesis in vitro. Under defined reaction conditions, the error rate per nucleotide polymerized at 70 degrees C can be as low as 10(-5) for base substitution errors and 10(-6) for frameshift errors. The frequency of mutations produced during a single round of DNA synthesis of the lac Z alpha gene by Taq polymerase responds to changes in dNTP concentration, pH, and the concentration of MgCl2 relative to the total concentration of deoxynucleotide triphosphates present in the reaction. Both base substitution and frameshift error rates of less than 1/100,000 were observed at pH 5-6 (70 degrees C) or when MgCl2 and deoxynucleotide triphosphates were present at equimolar concentrations. These high fidelity reaction conditions for DNA synthesis by the Taq polymerase may be useful for specialized uses of DNA amplified by the polymerase chain reaction.
TL;DR: The high-efficiency alkali cation method circumvents many of the shortcomings of the spheroplast method generally used for Schiz.
Abstract: We describe a highly efficient alkali cation method and library transducing vectors for cloning mammalian cDNAs by trans-complementation of fission yeast Schizosaccharomyces pombe mutants. cDNA libraries constructed with the pcD or pcD2 vector are transduced into yeast by cotransfection with a linearized vector, which allows an enhanced homologous recombination between the yeast vector and the library plasmid leading to the efficient formation of concatemers containing pcD molecules. The transformation frequencies obtained by the method are 10(6) colonies per 10(8) cells transfected with 2 micrograms of library and 1 microgram of vector, 50-60% of which contain pcD molecules. The high-efficiency alkali cation method circumvents many of the shortcomings of the spheroplast method generally used for Schiz. pombe transfection. The vectors are maximized for the efficiency of library transduction and minimized for the rearrangements of pcD molecules during propagation in yeast. This system allows rapid screening of multi-million cDNA clone libraries for rare cDNAs in a routine scale of experiments. Using this system, various mammalian cDNAs that are extremely difficult, time-consuming, or unclonable to clone by other methods have been cloned.
TL;DR: The isolation of a recombinant baculovirus expression vector from any of the AcMNPV transfer vectors currently in use can be facilitated by linearization of the viral DNA at the appropriate location.
Abstract: Engineered derivatives of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV) possessing a unique restriction site provide a source of viral DNA that can be linearized by digestion with a specific endonuclease. Circular or linearized DNA from two such viruses were compared in terms of their infectivity and recombinogenic activities. The linear forms were 15- to 150-fold less infectious than the corresponding circular forms, when transfected into Spodoptera frugiperda cells using the calcium phosphate method. Linear viral DNA was, however, proficient at recombination on co-transfection with an appropriate transfer vector. Up to 30% of the progeny viruses were recombinant, a 10-fold higher fraction of recombinants than was obtained from co-transfections with circular AcMNPV DNA. The isolation of a recombinant baculovirus expression vector from any of the AcMNPV transfer vectors currently in use can thus be facilitated by linearization of the viral DNA at the appropriate location.
TL;DR: Capillary gel electrophoresis has been demonstrated for the separation and detection of DNA sequencing samples and for genomic sequencing, and agreement of measured values for velocity, resolution and separation efficiency with theory predicts further improvements will result from increased electric field strengths.
Abstract: Capillary gel electrophoresis has been demonstrated for the separation and detection of DNA sequencing samples. Enzymatic dideoxy nucleotide chain termination was employed, using fluorescently tagged oligonucleotide primers and laser based on-column detection (limit of detection is 6,000 molecules per peak). Capillary gel separations were shown to be three times faster, with better resolution (2.4 x), and higher separation efficiency (5.4 x) than a conventional automated slab gel DNA sequencing instrument. Agreement of measured values for velocity, resolution and separation efficiency with theory, predicts further improvements will result from increased electric field strengths (higher voltages and shorter capillaries). Advantages of capillary gel electrophoresis for automatic DNA sequencing instruments and for genomic sequencing are discussed.
TL;DR: An optimal set of conditions is determined, which consists in: a temperature T of — 70°C, a time course L between 15 and 45 seconds, a ratio R around 20, and several PCR products of different lengths were sequenced on both strands by several investigators.
Abstract: The use of the dideoxy chain termination procedure (1) to sequence linear double-stranded PCR products is hampered by the fast renaturation of the template. To minimize renaturation, the template is usually heat-denatured in the presence of the primer and the reaction is initiated after the shortest possible annealing phase. The recommended times and temperatures of incubation as well as DNA concentrations in the reaction mixture differ widely from author to author (2, 3, 4, 5, 6, 7). We have systematically varied three parameters: 1. the annealing temperature (T), to which the sample is transferred after denaturation by boiling, 2. the time course (L) of the labeling reaction, which is carried out at room temperature, 3. the primentemplate ratio (R) in the reaction mixture. We have determined an optimal set of conditions, which consists in: 1. a temperature T of — 70°C, 2. a time course L between 15 and 45 seconds, 3. a ratio R around 20. Under these conditions, several PCR products of different lengths were sequenced on both strands by several investigators.
TL;DR: Two expression vectors were constructed that differ only in the presence of an intervening sequence (IVS) in their 5'-untranslated leaders, and results imply that splicing is coupled to a polyadenylation/transport pathway.
Abstract: Two expression vectors were constructed that differ only in the presence (+) or absence (-) of an intervening sequence (IVS) in their 5'-untranslated leaders. Transient transfection into four mammalian cell lines resulted in higher levels of the indicator protein (CAT) from the IVS(+) vector (6 to 50-fold). Cytoplasmic RNA concentrations in 293s and HeLa cell lines corresponded directly to resultant protein levels; measurements in 293s cells of transcription initiation and elongation, steady-state total nuclear RNA, and cytoplasmic RNA stability, were equivalent for the two vectors. Surprisingly, the amount of poly(A)+ nuclear RNA was greater from the IVS(+) vector. Since this difference matches the ratio seen with polyadenylated cytoplasmic RNA, our results imply that splicing is coupled to a polyadenylation/transport pathway.
TL;DR: Computer analysis showed a correlation between the location of the deletion breakpoints and sequences in human mtDNA similar to the target sequence for Drosophila topoisomerase II.
Abstract: Large-scale deletions of mitochondrial DNA (mtDNA) have been described in patients with progressive external ophthalmoplegia (PEO) and ragged red fibers. We have determined the exact deletion breakpoint in 28 cases with PEO, including 12 patients already shown to harbor an identical deletion; the other patients had 16 different deletions. The deletions fell into two classes. In Class I (9 deletions; 71% of the patients), the deletion was flanked by perfect direct repeats, located (in normal mtDNA) at the edges of the deletion. In Class II (8 deletions; 29% of patients), the deletions were not flanked by any obviously unique repeat element, or they were flanked by repeat elements which were located imprecisely relative to the breakpoints. Computer analysis showed a correlation between the location of the deletion breakpoints and sequences in human mtDNA similar to the target sequence for Drosophila topoisomerase II. It is not known how these deletions originate, but both slipped mispairing and legitimate recombination could be mechanisms playing a major role in the generation of the large mtDNA deletions found in PEO.
TL;DR: It is shown that epitope tagging can be used to allow recovery of sites when specific antibodies are unavailable, and that specific sites bound by the Fos protein are easily recovered by this technique.
Abstract: We describe a sensitive and rapid method for determination of the sequence specificity of DNA binding proteins. The method allows recovery of specific sites using the small amounts of protein present in crude cell extracts or produced by cell-free translation reactions. Extract proteins are incubated with a pool of random sequence oligonucleotides, complexes purified by immunoprecipitation, and bound DNA amplified by the Polymerase Chain Reaction (PCR). This DNA is then used in further rounds of binding, immunoprecipitation, and amplification, until specific binding is detectable. With the transcription factor SRF as a model system, we demonstrate that authentic high affinity binding sites are recovered, and show that epitope tagging can be used to allow recovery of sites when specific antibodies are unavailable. We also show that specific sites bound by the Fos protein, which binds DNA with high affinity only when complexed with other polypeptides, are easily recovered by this technique.
TL;DR: The catalytic domain within the sequence of the negative strand of the satellite RNA of tobacco ringspot virus is identified and an appropriately engineered 'hairpin' catalyst is capable of cleaving heterologous RNA.
Abstract: We have identified the catalytic domain within the sequence of the negative strand of the satellite RNA of tobacco ringspot virus. Minimum energy RNA folding calculations predict a two dimensional model with four major helical regions which are supported by mutagenesis experiments. This model for the catalytic complex consists of a 50 base catalytic RNA and a 14 base substrate RNA folded together in a type of hairpin two dimensional structure. Part of the recognition region between the catalyst and substrate is two helices of 6 bases and 4 bases respectively. Catalytic activity remains when the bases in these two helices are changed but base pairing is maintained. Thus an appropriately engineered 'hairpin' catalyst is capable of cleaving heterologous RNA.