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At least 19 recordsLinked to original sources
PubMed1989

A system for on-line detection and resolution of radiolabeled DNA molecules and its application to automated DNA sequence analysis.

We describe a system for the real-time detection of radioactively labeled DNA molecules in gel matrix, and we demonstrate the application of this system to DNA sequence analysis. DNA sequencing reactions prepared by the Sanger chain termination method are resolved by electrophoresis on 8% polyacrylamide gels. During electrophoresis the 32P-labeled DNA fragments are detected by solid state detectors positioned 22 cm from the top surface of the gel. This system is able to resolve a DNA sequence of 300 bases or greater. Optimized protocols that allow sequence information to be obtained from single stranded and double-stranded templates are described. A linear relationship exists between the input dpm and the integrated peak values over a 20-fold range indicating that accurate DNA quantitation is also possible using this system.

Automation↗
PubMedDate not supplied

DNA sequence analysis and structural relationships among the cytoskeletal actin genes of the sea urchin Strongylocentrotus purpuratus.

The general organization and primary amino acid sequences of the S. purpuratus cytoskeletal actin genes CyIIb and CyIIIb have been determined from restriction enzyme analysis, DNA sequencing, and RNA mapping studies. As is the case with the other sea urchin cytoskeletal actin genes previously studied, the CyIIb and CyIIIb genes contain two introns that interrupt the coding DNA following codon 121 and within codon 204. An intron ending 26-27 nucleotides (nt) upstream of the initiation codon has also been localized in the 5'-flanking region of both genes. The CyIIb gene, which is part of a cluster of three genes linked in the order CyI-CyIIa-CyIIb, encodes a protein that differs from CyI by a single residue and from CyIIa by three residues. The substitutions observed within this linkage group are relatively conservative changes, and pairwise comparisons between genes indicate less than 5% mismatch in nucleotide sequence within the coding region. Nucleotide sequence comparisons of 5'-flanking region and intron DNA, however, indicate greater similarity between the CyI and CyIIb genes than the CyIIa gene that separates them, suggestive of a potential gene conversion event between the flanking genes in the CyI-CyIIa-CyIIb linkage. The CyIIIb gene, part of a separate cluster of two functional genes ordered CyIIIa-CyIIIb, shares little similarity outside of coding DNA with genes of the other linkage group. Although CyIIIb exhibits strong nucleotide sequence similarity outside of coding DNA with the neighboring CyIIIa gene, it differs from that gene at six codons. The CyIIIb gene encodes a protein considerably different from all cytoskeletal actins previously reported, with changes clustered in the latter 40% of the coding sequence. An 81-nt tandem duplication of the C-terminal coding region is located adjacent to the termination codon of the CyIIIb gene, a potential relic of a slipped mispairing and replication event.

Actins↗
PubMed1992

Cloning and DNA sequence analysis of the mercury resistance genes of Streptomyces lividans.

A broad-spectrum mercury resistance locus (mer) from a spontaneous chloramphenicol-sensitive (Cms), arginine auxotrophic (Arg-) mutant of Streptomyces lividans 1326 was isolated on a 6 kb DNA fragment by shotgun cloning into the mercury-sensitive derivative S. lividans TK64 using the vector pIJ702. The mer genes form part of a very large amplifiable DNA sequence present in S. lividans 1326. This element was amplified to about 20 copies per chromosome in the Cms Arg- mutant and was missing from strains like S. lividans TK64, cured for the plasmid SLP3. DNA sequence analysis of a 5 kb region encompassing the whole region required for broad-spectrum mercury resistance revealed six open reading frames (ORFs) transcribed in opposite directions from a common intercistronic region. The protein sequences predicted from the two ORFs transcribed in one direction showed a high degree of similarity to mercuric reductase and organomercurial lyase from other gram-negative and gram-positive sources. Few, if any, similarities were found between the predicted polypeptide sequences of the other four ORFs and other known proteins.

Amino Acid Sequence↗
PubMed1979

A rapid screening for the specific DNA sequence: analysis of transforming DNA segments in adenovirus-transformed cells.

The viral DNA sequences in cells transformed by adenovirus type-12 (Ad12) DNA fragments were investigated by spot hybridization, a detection by autoradiography of nucleic acid hybrids formed between cell DNAs spotted on a membrane filter and various nick-translated Ad12 DNA fragments. In CY1 cell line, a rat cell line transformed by the EcoRI-C fragment (left hand 16%), all of the HindIII fragments included in the EcoRI-C fragment are shown to be present. In GY1 cell line, a rat cell line transformed by the HindIII-G fragment (left hand 7%), both of the BpaI-H and a part of the BpaI-J, two components consisting of the HindIII-G fragment, were found. A dominant presence of the Ad12 BpaI-H fragment (left hand end 4.5%) of the Ad12 DNA molecule, approximately 60% of the Ad12 transforming DNA sequences (Ad12 HindIII-G), was also shown in GY1. Spot hybridization used in the experiment will be of general use for detecting viral nucleic acid sequences in cells and provides a simple and useful screening method for investigating viral etiology of tumors and transformed cells.

Adenoviruses, Human↗
PubMed1990

DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Effect of pyrophosphorolysis and metal ions.

Pyrophosphorolysis by bacteriophage T7 DNA polymerase leads to the degradation of specific dideoxynucleotide-terminated fragments on DNA sequencing gels. This reaction can be prevented by pyrophosphatase. It is also inhibited by a high concentration of dNTPs; only the dNTP complementary to the next base in the template is an effective inhibitor, suggesting the formation of a stable polymerase-primer-template-nucleotide complex despite the absence of a 3' hydroxyl group on the primer. The use of pyrophosphatase, a genetically modified T7 DNA polymerase that lacks exonuclease activity, and Mn2+ rather than Mg2+ to eliminate discrimination between dideoxynucleotides and deoxynucleotides (Tabor, S., and Richardson, C. C. (1989) Proc. Nat. Acad. Sci. U. S. A. 86, 4076-4080) generates bands of uniform intensity on a DNA sequencing gel. Uniform band intensities simplify the analysis of a DNA sequence, particularly with automated procedures. For example, when genomic DNA is sequenced directly, heterozygotic sequences are readily detected because their bands have half the intensity of homozygotic sequences. A procedure for automated DNA sequencing is described that exploits the uniformity. A single reaction with a single labeled primer is carried out using four different ratios of dideoxynucleotides to deoxynucleotides; after gel electrophoresis in a single lane, the sequence is determined by the relative intensity of each band.

Animals↗
PubMedDate not supplied

Fluorescence detection in automated DNA sequence analysis.

We have developed a method for the partial automation of DNA sequence analysis. Fluorescence detection of the DNA fragments is accomplished by means of a fluorophore covalently attached to the oligonucleotide primer used in enzymatic DNA sequence analysis. A different coloured fluorophore is used for each of the reactions specific for the bases A, C, G and T. The reaction mixtures are combined and co-electrophoresed down a single polyacrylamide gel tube, the separated fluorescent bands of DNA are detected near the bottom of the tube, and the sequence information is acquired directly by computer.

Automation↗
PubMed1985

The synthesis of oligonucleotides containing an aliphatic amino group at the 5' terminus: synthesis of fluorescent DNA primers for use in DNA sequence analysis.

A rapid and versatile method has been developed for the synthesis of oligonucleotides which contain an aliphatic amino group at their 5' terminus. This amino group reacts specifically with a variety of electrophiles, thereby allowing other chemical species to be attached to the oligonucleotide. This chemistry has been utilized to synthesize several fluorescent derivatives of an oligonucleotide primer used in DNA sequence analysis by the dideoxy (enzymatic) method. The modified primers are highly fluorescent and retain their ability to specifically prime DNA synthesis. The use of these fluorescent primers in DNA sequence analysis will enable DNA sequence analysis to be automated.

Base Sequence↗
PubMed1986

Localization and DNA sequence analysis of the C gene of bacteriophage Mu, the positive regulator of Mu late transcription.

The C gene of bacteriophage Mu, required for transcription of the phage late genes, was localized by construction and analysis of a series of deleted derivatives of pKN50, a plasmid containing a 9.4 kb Mu DNA fragment which complements Mu C amber mutant phages for growth. One such deleted derivative, pWM10, containing only 0.5 kb of Mu DNA, complements C amber phages and transactivates the mom gene, one of the Mu late genes dependent on C for activation. The DNA sequence of the 0.5 kb fragment predicts a single long open reading frame coding for a 140 amino acid protein. Sequence analysis of DNA containing a C amber mutation located the base change to the second codon of this reading frame. Generation of a frameshift mutation by filling in a BglII site spanning codon 114 of this reading frame resulted in the loss of C complementation and transactivation activity. These results indicate that this open reading frame encodes the Mu C gene product. Comparison of the predicted amino acid sequence of the C protein with those of other transcriptional regulatory proteins revealed some similarity to a region highly conserved among bacterial sigma factors.

Amino Acid Sequence↗
PubMed1987

DNA sequence analysis of the mutational specificity of u.v. light in the SUP4-o gene of yeast.

We have characterized mutations induced in the SUP4-o gene of Saccharomyces cerevisiae by u.v. irradiation. Mutants were selected following treatment with 60 J/m2 u.v. light which reduced cell survival to 10% and increased the SUP4-o mutation frequency 100-fold above background. DNA sequence analysis of 120 mutants revealed that u.v. induced all types of base substitutions, although transitions, in particular G:C----A:T events predominated. In addition, a small number of single base pair deletions and double mutations, occurring in tandem or separated by a few base pairs, were recovered. The base pair substitutions were not distributed randomly in the SUP4-o gene and, with one exception, were all located at sites of adjacent pyrimidines, suggesting that they were targeted by u.v. photolesions. A substantial fraction of the mutations were detected at hotspots for u.v. mutagenesis. The majority of changes occurred at the 3' base of dipyrimidine sequences where both cyclobutane dimers and [6-4]-photoproducts could form. Approximately one-third of the induced base substitutions were found at potential pyrimidine dimer sites where [6-4]-photoproducts would be expected to occur rarely. The possible origins of the induced mutations and the role of cyclobutane dimers as premutational u.v. lesions in yeast are considered.

Base Sequence↗
PubMed1992

Cloning and DNA sequence analysis of an aac(3)-Vb gene from Serratia marcescens.

The AAC(3)-V resistance mechanism is characterized by high-level resistance to the aminoglycosides gentamicin, netilmicin, 2'-N-ethylnetilmicin, and 6'-N-ethylnetilmicin and moderate resistance levels to tobramycin. Serratia marcescens 82041944 contains an AA(3)-V resistance mechanism as determined from aminoglycoside resistance profiles. This strain, however, does not exhibit hybridization with a probe derived from the previously cloned aac(3)-Va gene, (R. Allmansberger, B. Bräu, and W. Piepersberg, Mol. Gen. Genet. 198:514-520, 1985). High-pressure liquid chromatography analysis of the acetylation products of sisomicin carried out by extracts of S. marcescens 82041944 have demonstrated the presence of an AAC(3) enzyme. We have cloned the gene encoding this acetyltransferase and have designated it aac(3)-Vb. Nucleotide sequence comparisons show that the aac(3)-Va and aac(3)-Vb genes are 72% identical. The predicted AAC(3)-Vb protein is 28,782 Da. Comparisons of the deduced amino acid sequences show 75% identity and 84% similarity between the AAC(3)-Va and AAC(3)-Vb proteins. The use of a DNA fragment internal to the aac(3)-Vb as a hybridization probe demonstrated that the aac(3)-Vb gene is very rare in clinical isolates possessing an AAC(3)-V mechanism.

Amino Acid Sequence↗
PubMed1990

Identification of phosphate starvation-inducible genes in Escherichia coli K-12 by DNA sequence analysis of psi::lacZ(Mu d1) transcriptional fusions.

Twenty-four independent phosphate starvation-inducible (psi) transcriptional fusions made with Mu d1(lacZbla) were analyzed by sequencing the psi::lacZ(Mu d1) chromosomal junctions by using DNAs amplified with the polymerase chain reaction or mini-Mu cloning. Our DNA sequence analysis showed that the MuR DNA in Mu d1 has an unexpected structure that is comprised of 104 bases of MuR DNA in the form of a large inverted repeat, which we denoted Mu d1-R. Also, Mu d1s in the phoA and phn (psiD) loci of the phosphate regulon showed regional specificities for the insertion sites despite the randomness of Mu d1 insertions into the genome as a whole. Gene products or open reading frames were identified for seven unknown psi::lacZ(Mu d1) transcriptional fusions by searching DNA data bases with the sequences adjacent and upstream of the Mu d1s. One psiC::lacZ(Mu d1) lies in the ugpB gene of the ugpBAEC operon, which encodes a periplasmic sn-glycerol-3-phosphate-binding protein; two psiQ::lacZ(Mu d1)s lie in the gltB gene, and one psiQ::lacZ(Mu d1) lies in the gltD gene of the gltBDF operon, encoding the large and small subunits of glutamate synthase, respectively; and the psi-51::lacZ(Mu d1) lies in the glpB gene of the glpABC operon, which codes for the anaerobically regulated glycerol-3-phosphate dehydrogenase. psiE and psiF::lacZ(Mu d1)s lie in uncharacterized open reading frames near the xylE and phoA genes, respectively. Six other psi::lacZ(Mu d1)s lie in yet unreported Escherichia coli sequences.

Bacteriophage mu↗
PubMed1991

DNA sequence analysis of X-ray induced Adh null mutations in Drosophila melanogaster.

The mutational spectrum for 28 X-ray induced mutations and 2 spontaneous mutations, previously determined by genetic and cytogenetic methods, consisted of 20 multilocus deficiencies (19 induced and 1 spontaneous) and 10 intragenic mutations (9 induced and 1 spontaneous). One of the X-ray induced intragenic mutations was lost, and another was determined to be a recombinant with the allele used in the recovery scheme. The DNA sequence of two X-ray induced intragenic mutations has been published. This paper reports the results of DNA sequence analysis of the remaining intragenic mutations and a summary of the X-ray induced mutational spectrum. Only one of the X-ray induced mutations is a single base change, a C to G transversion (AdhnLA80). Therefore, the mutational spectrum of X-ray induced mutations consists predominantly of deletions that are observed to range in size from two base pairs to deletions of a large number of loci as determined by genetic complementation analysis. The combination of DNA sequence analysis with genetic complementation analysis shows a continuous distribution in size of deletions rather than two different types of mutations consisting of deletions and "point mutations." Sequencing is shown to be essential for detecting intragenic deletions. Of particular importance for future studies is the observation that all of the intragenic deletions consist of a direct repeat adjacent to the break-point with one of the repeats deleted.

Alcohol Dehydrogenase↗
PubMed1987

DNA sequence analysis with a modified bacteriophage T7 DNA polymerase.

A chemically modified phage T7 DNA polymerase has three properties that make it ideal for DNA sequencing by the chain-termination method. The enzyme is highly processive, catalyzing the polymerization of thousands of nucleotides without dissociating. By virtue of the modification the 3' to 5' exonuclease activity is eliminated. The modified polymerase efficiently uses nucleotide analogs that increase the electrophoretic resolution of bands in gels. Consequently, dideoxynucleotide-terminated fragments have highly uniform radioactive intensity throughout the range of a few to thousands of nucleotides in length. There is virtually no background due to terminations at pause sites or secondary-structure impediments. Processive synthesis with dITP in place of dGTP eliminates band compressions, making possible the unambiguous determination of sequences from a single orientation.

Base Sequence↗
PubMed1987

DNA sequence analysis of host range mutants of the promiscuous IncP-1 plasmids R18 and R68 with Tn7 insertions in oriV.

Transposon Tn7 insertions in the origin of vegetative replication (oriV) result in host range mutants of the promiscuous IncP-1 plasmids R18 and R68 which affect plasmid replication in Escherichia coli but not in Pseudomonas aeruginosa. The sites of these insertions have been analyzed by DNA sequence analysis. In two mutants, the insertions generated direct duplications of 5'GTATT3' at the target site which included the first base at the 5' end of the fourth 17-bp direct repeat in oriV. In a third mutant the duplication of 5'GACAC3' also involved the same direct repeat also at the 5' end but contiguous with the previous duplication. DNA sequence analysis of another Tn7-induced host range mutant of R18, characterized by reduced conjugational transmissibility into P. stutzeri while retaining normal transmissibility within P. aeruginosa, showed that the insertion generated a 474-bp deletion which brought the insertion 20 bp 5' to the 17-bp direct repeat between oriV and the oxytetracycline hydrochloride-resistant gene. The analysis of the DNA sequence data at the site of the Tn7 insertions shows that particular segments of the DNA sequence in oriV are differentially required for the replication of these plasmids in different bacterial hosts and thus of importance to the promiscuity of these plasmids.

Base Sequence↗
PubMed1985

Genetic mapping and DNA sequence analysis of mutations in the polA gene of Escherichia coli.

DNA polymerase I of Escherichia coli provides an excellent model for the study of template-directed enzymatic synthesis of DNA because it is a single subunit enzyme, it can be obtained in large quantities and the three-dimensional structure of the polymerizing domain (the Klenow fragment) has recently been determined (Ollis et al., 1985). One approach to assigning functions to particular portions of the structure is to correlate the altered enzymatic behavior of mutant forms of DNA polymerase I with the change in the primary sequence of the protein. Towards this end we have developed a rapid procedure for mapping any polA mutation to a region no larger than 300 base-pairs within the polA gene. Two series of polA deletion mutants with defined end-points were constructed in vitro and cloned into bacteriophage lambda. These phages can then be used to map precisely E. coli polA mutants. Twelve polA- alleles have been mapped in this way and for nine of them the nature of the mutational change has been determined by DNA sequence analysis. Two of the mutations, polA5 and polA6, which affect the enzyme-DNA interaction, provide evidence for the location of the DNA binding region on the polymerase three-dimensional structure.

Base Sequence↗
PubMed1987

DNA sequence analysis of I-Ak beta mutants reveals serologically immunodominant region.

We have produced a series of in vitro serologically selected cell lines that express mutant I-Ak molecules. In this report we describe the DNA sequence analysis of the Ak beta gene of four cell lines that express serologically altered Ak beta polypeptides in association with wild-type Ak alpha polypeptides. Each of the major serologic epitopes on the Ak beta polypeptide has been altered in one or more of the four mutants. In addition, the four mutants exhibit a broad spectrum of functional defects when used to stimulate a panel of T hybridomas of various specificities. The DNA sequence analysis revealed that each mutant had sustained a single nucleotide substitution resulting in a single amino acid substitution. All four independent substitutions occurred within or near the third of the four variable regions defined in the beta 1 domain of the A beta polypeptide by allelic comparisons. These data strongly suggest that the third variable region is the major determinant of alloantigenicity on the Ak beta polypeptide.

Animals↗
PubMed1992

DNA sequence analysis of 1-nitrosopyrene-induced mutations in the hprt gene of Chinese hamster ovary cells.

DNA sequence was determined in 21 mutants induced at the hprt locus of Chinese hamster ovary (CHO) cells by 1-nitrosopyrene, a metabolite of the tumorigenic environmental pollutant 1-nitropyrene. Following cDNA synthesis using RNA from each of the mutants, the hprt protein-coding region was amplified by the polymerase chain reaction (PCR) and subjected to direct DNA sequence analysis. Sixteen primary mutations were found: seven were G:C----T:A transversions, five were G:C----A:T transitions, two were single basepair insertions, one was a single basepair deletion, and one was a complex mutation involving substitutions at two A:T basepairs. The simple basepair substitution mutations preferentially occurred with one or two purines 3' to the mutated dG, and mutations in exons 1-4 disproportionately occurred with the mutated dG on the nontranscribed DNA strand. In addition, 12 of the mutants produced one or more cDNA PCR products with partial or complete exon deletions. Seven mutants with multiple PCR products had point mutations in one of the products; exon deletions in the other product(s) removed these point mutations. A group of solvent control mutants had a different distribution of basepair substitution mutations and a lower proportion of cDNAs with exon deletions than that found for the 1-nitrosopyrene-induced mutants. The results indicate a specificity for the induction of mutations in the hprt gene of CHO cells by 1-nitrosopyrene with respect to both the types of mutations produced and their location in the hprt gene. Also, the elimination of point mutations in many of the cDNA PCR products with exon deletions suggests that mutations in the protein-coding sequence affect hprt mRNA processing.

Amino Acid Sequence↗