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W R Pearson

Publications and source records attributed to W R Pearson.

At least 55 records · Page 3Linked to original sources
PubMed1988

Tissue-specific induction of murine glutathione transferase mRNAs by butylated hydroxyanisole.

Three murine liver glutathione transferase (GT, EC 2.5.1.18) have been cloned and sequenced. Two of the cDNA clones, pGT875 and pGT55, encode the murine class-mu GT isoenzymes, GT8.7 and GT9.3, respectively. These two cDNA clones share 85% DNA sequence identity with one another, and the GT8.7 sub-unit encoded by pGT875 shares 92% protein sequence identity with the class-mu rat-3 (Yb1) GT subunit. The third cDNA clone, pGT41, encodes a class-alpha GT subunit that shares 96% protein sequence identity with a mouse Ya gene, 95% identity with a rat-1 (Ya) GT subunit, and 70% identity with the rat-2 (Yc) subunit. These cDNA clones and an oligonucleotide derived from the sequence of a rat class-pi cDNA clone were used to measure the induction of the mu, alpha, and pi classes of GT mRNA in different tissues of mice that were fed the dietary antioxidant 2(3)-tert-butyl hydroxyanisole (BHA). These tissues included liver, intestinal mucosa, kidney, lung, spleen, and brain. Class-mu GT mRNAs that hybridize with pGT875 are most abundant in liver and intestinal mucosa but are also found in kidney and lung, and at low levels in brain and spleen. Class-alpha GT mRNAs are most abundant in BHA-induced and uninduced intestinal mucosa, kidney, and induced liver, and were not found in spleen and brain. Class-mu and -alpha GT mRNA levels increased 15- and 50-fold, respectively, in the liver and 15- and 100-fold in intestinal mucosa in response to BHA induction. BHA increases class-mu mRNAs less than 5-fold in the kidney and lung. Class-pi mRNAs were found in all the tissues examined but were much less responsive to BHA induction. The expression of two cytochrome P-450 mRNAs increased 3-5-fold in liver and intestine after BHA induction. Oligonucleotides from divergent portions of the pGT875 and pGT55 cDNA clones have been used to examine the expression of specific mRNAs from individual class-mu GT genes; these experiments suggest that the GT mRNAs expressed in BHA-induced tissues are also expressed in the uninduced tissue. Measurements of transcription rates in isolated nuclei showed that increased GT mRNA levels are due to increased rates of transcription.

Amino Acid Sequence↗
PubMed1988

Hereditary differences in the expression of the human glutathione transferase active on trans-stilbene oxide are due to a gene deletion.

Glutathione transferase (GT; EC 2.5.1.18) mRNA levels were measured in human liver samples by using mouse and human cDNA clones that encode class-mu and class-alpha GT. Although all the RNA samples examined contained class-alpha GT mRNA, class-mu GT mRNA was found only in individuals whose peripheral leukocytes expressed GT activity on the substrate trans-stilbene oxide. The mouse class-mu cDNA clone was used to identify a human class-mu GT cDNA clone, lambda GTH411. The amino acid sequence of the GT encoded by lambda GTH411 is identical with the 23 residues determined for the human liver GT-mu isoenzyme and shares 76-81% identity with mouse and rat class-mu GT isoenzymes. The mouse and human class-mu GT cDNA inserts hybridize with multiple BamHI and EcoRI restriction fragments in the human genome. One of these hybridizing fragments is missing in the DNA of individuals who lack GT activity on trans-stilbene oxide. Hybridizations with nonoverlapping subfragments of lambda GTH411 suggest that there are at least three class-mu genes in the human genome. One of these genes appears to be deleted in individuals lacking GT activity on trans-stilbene oxide.

Amino Acid Sequence↗
PubMed1988

Improved tools for biological sequence comparison.

We have developed three computer programs for comparisons of protein and DNA sequences. They can be used to search sequence data bases, evaluate similarity scores, and identify periodic structures based on local sequence similarity. The FASTA program is a more sensitive derivative of the FASTP program, which can be used to search protein or DNA sequence data bases and can compare a protein sequence to a DNA sequence data base by translating the DNA data base as it is searched. FASTA includes an additional step in the calculation of the initial pairwise similarity score that allows multiple regions of similarity to be joined to increase the score of related sequences. The RDF2 program can be used to evaluate the significance of similarity scores using a shuffling method that preserves local sequence composition. The LFASTA program can display all the regions of local similarity between two sequences with scores greater than a threshold, using the same scoring parameters and a similar alignment algorithm; these local similarities can be displayed as a "graphic matrix" plot or as individual alignments. In addition, these programs have been generalized to allow comparison of DNA or protein sequences based on a variety of alternative scoring matrices.

Amino Acid Sequence↗
PubMed1985

Rapid and sensitive protein similarity searches.

An algorithm was developed which facilitates the search for similarities between newly determined amino acid sequences and sequences already available in databases. Because of the algorithm's efficiency on many microcomputers, sensitive protein database searches may now become a routine procedure for molecular biologists. The method efficiently identifies regions of similar sequence and then scores the aligned identical and differing residues in those regions by means of an amino acid replacability matrix. This matrix increases sensitivity by giving high scores to those amino acid replacements which occur frequently in evolution. The algorithm has been implemented in a computer program designed to search protein databases very rapidly. For example, comparison of a 200-amino-acid sequence to the 500,000 residues in the National Biomedical Research Foundation library would take less than 2 minutes on a minicomputer, and less than 10 minutes on a microcomputer (IBM PC).

Amino Acid Sequence↗
PubMed1983

Increased synthesis of glutathione S-transferases in response to anticarcinogenic antioxidants. Cloning and measurement of messenger RNA.

Glutathione S-transferase activities in mouse hepatic cytosols are elevated as much as 11-fold following the administration of BHA (2(3)-tert-butyl-4-hydroxyanisole), a widely used antioxidant food additive. Ethoxyquin (1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline) and disulfiram [bis(diethyldithiocarbamyl)disulfide] also enhance the activities of glutathione S-transferases and certain other enzymes. Each of these compounds protects rodents against mutagenic and carcinogenic metabolites. A major (pI 8.7) and a minor (pI 9.3) component of the family of mouse hepatic glutathione S-transferases have been purified to homogeneity. These transferases are immunologically cross-reactive, and have a high degree of NH2-terminal sequence homology (but are not identical). The enzymes differ in a number of molecular and catalytic properties. The transferases are 12-fold elevated by dietary BHA as demonstrated by immunotitration. The mRNA for the major glutathione S-transferase is increased more than 20-fold in the liver RNA of BHA-fed mice, as determined by translation of total liver mRNA and characterization of the products by immunoprecipitation and sodium dodecyl sulfate-gel electrophoresis or by two-dimensional gel electrophoresis. A cDNA plasmid complementary to glutathione S-transferase mRNA was constructed. Translation of liver mRNA selected by hybridization with this plasmid gave products similar to or identical with glutathione S-transferase polypeptides. The cDNA insert has been partially sequenced and its orientation has been determined. Its sequence corresponds to the NH2-terminal region (beginning at residue 9) of the amino acid sequence of the glutathione S-transferase with pI 9.3. Hybridization of the 32P-labeled cDNA plasmid with total liver RNA indicates a 26-fold increase in homologous mRNA in response to the feeding of BHA.

Amino Acid Sequence↗
PubMed1982

Automatic construction of restriction site maps.

A computer program is described which constructs maps of restriction endonuclease cleavage sites in DNA molecules, given only the fragment lengths. The program utilizes fragment length data from single and double restriction enzyme digests to generate maps for linear or circular molecules. The search for a map can be limited to the unknown (insert) region of a recombinant phage or plasmid. Typical restriction maps with four or five enzymes which cut at three to five unknown sites can be calculated in a few minutes.

Autoanalysis↗
PubMed1982

Regulation of synthesis of amyloid A-related protein.

The concentration of serum amyloid A polypeptide (SAAL) increases greatly during the acute phase responses to infection or inflammation. We find that SAAL synthesis comprises 2.5% of murine hepatic protein synthesis after lipopolysaccharide (LPS) administration, but much less in normal liver. SAAL messenger RNA (mRNA) in liver increases at least 500-fold above the normal level. A recombinant plasmid homologous to SAAL mRNA has been isolated, as has most of the mouse genome DNA encoding the plasmid's nucleotide sequence. This gene is transcribed into RNA much more frequently after LPS administration than it is in normal liver. In a number of other mammalian genes, cytosine methylation is inversely related to the rate of transcription. Methylation of CCGG sequences in hepatic DNA homologous to the recombinant plasmid has been examined. Little or no change is found after LPS administration. This suggests that other factors are responsible for the increase in SAAL mRNA in the acute phase response.

Amino Acid Sequence↗
PubMed1981

Repeated DNA sequences near the 5'-end of the silk fibroin gene.

Four different repeated sequence elements have been found in cloned silk moth DNA, extending from 5700 base pairs upstream from the silk fibroin gene to 600 base pairs within its intervening sequence. Only 2 of the 12 overlapping restriction fragments examined, ranging from 164 to 1800 base pairs in length, do not contain a repeated sequence. The 830 base pairs adjoining the 5'-end of the fibroin gene hybridize with only one sequence in the Bombyx mori genome. Fibroin gene transcription initiates within this single-copy DNA at least 500 base pairs downstream from any repeated DNA sequence. The data show that repeated sequences comprise a major portion of the DNA flanking this developmentally regulated gene but not the DNA immediately 5' to it; that an intervening sequence can contain a repeated element; and that repeated sequences can be interspersed with one another as well as with single-copy DNA in a typical animal genome. In addition, filter transfer hybridization can be used to quantitate repetition frequency and sequence divergence and to distinguish patterns of repeated sequence organization.

Animals↗
PubMed1981

Long and short repeats of sea urchin DNA and their evolution.

Repeated sequences cloned from the DNA of the sea urchin S. purpuratus were used as probes to measure the lengths of individual families of repeats. Some probes reassociated much more rapidly with preparations of long repeats than with short repeats while others reassociated more rapidly with short repeats than with long repeats. In this way two of five cloned repeats were shown to represent families with a great majority of sequences in the long class. One represented a family with similar number of long and short class members. Two were members of predominantly short class families - The cloned repeats representing long class families, formed more precise duplexes than those representing short class families. Thermal stability measurements using S. purpuratus or S. franciscanus driver DNA showed that precise repetitive sequences have as great an interspecies sequence difference as the less precise repeats. Thus the precision of many families may result from recent multiplication rather than from selective pressure on the DNA sequences. Measurements of evolutionary frequency change show a clear correlation between the frequency change and the size of families of repeats in S. purpuratus. Comparison with S. franciscanus indicates that many of the large size families in S. purpuratus are those that have grown in size since these two species diverged.

Animals↗
PubMed1981

Discrete-length repeated sequences in eukaryotic genomes.

Two of the four repeated DNA sequences near the 5' end of the silk fibroin gene hybridize with discrete-length families of repeated DNA. These two families comprise 0.5% of the animal's genome. A repeated sequence with a conserved length has also been found in the short class of moderately repeated sequences in the sea urchin. The discrete length, interspersion, and sequence fidelity of these moderately repeated sequences suggests that each has been multiplied as a discrete unit. Thus, transposition mechanisms may be responsible for the multiplication and dispersion of a large class of repeated sequences in phylogenetically diverse eukaryotic genomes. The repeat we have studied in most detail differs from previously described eukaryotic transposable elements: it is much shorter (1300 base pairs) and does not have terminal repetitions detectable by DNA hybridization. A simple technique for identifying such discrete-length repeated sequences is described.

Animals↗
PubMed1979

Computer programs for analysis of nucleic acid hybridization, thermal denaturation, and gel electrophoresis data.

Computer programs for the analysis of data from techniques frequently used in nucleic acids research are described. In addition to calculating non-linear, least-squares solutions to equations describing these systems, the programs allow for data editing, normalization, plotting and storage, and are flexible and simple to use. Typical applications of the programs are described.

Computers↗
PubMed1978

Analysis of rat repetitive DNA sequences.

Parameters of repetitive sequence organization have been measured in the rat genome. Experiments using melting, hydroxylapatite binding, and single strand specific nuclease digestion have been used to measure the number, length, and arrangement of repeated DNA sequences. Renaturation and melting or S1 nuclease digestion of 1.0 kbp DNA fragment show about 20% of rat DNA sequences are 3000-fold repeated. Renatured duplexes from 4.0 kbp DNA fragments display two repetitive size fractions after nuclease digestion. About 60% of the repeated sequences are 0.2-0.4 kbp long while the remainder are longer than 1.5 kbp. The arrangement of the repeated sequences has been measured by hydroxylapatite fractionation of DNA fragments of varying lengths bearing a repeated sequence. Repeated DNA sequences are interspersed among 2.5 kbp long nonrepeated sequences throughout more than 70% of the rat genome. There are approximately 350 different 3000-fold short repeated sequences in the rat interspersed among 600,000 nonrepeated DNA sequences.

Animals↗
PubMed1978

Sequence organization of the rat genome by electron microscopy.

The size and arrangement of repetitive and inverted repeat (foldback) sequences in rat DNA were studied by visualization of hybrid and heteroduplex structures in the electron microscope. The self-reassociation of repetitive sequence-bearing DNA strands often results in the formation of four-ended "H" structures, whose duplex regions equal the repetitive sequence length and can be measured in the electron microscope. In this way, it was determined that the average size of the class of numerous short repetitive sequences is 0.40 +/- 0.15 kbp. Heteroduplex structures were prepared between long whole DNA single strands and short repeat-sequence-bearing strands. The analysis of these structures confirms that the size of the repetitive sequences in 0.4 kbp on average. Length measurements between adjacent duplexes show that the average spacing between two interspersed repeats is at least 1.5-1.8 kbp. By examining 29.4-kbp single strands after brief renaturation, the size and distribution of foldback sequences were determined. There are 1.9 X 10(5) foldback apirs per rat genome, spaced an average of 9.7 kbp apart according to our measurement. Repetitive, inverted repeat and unique sequences are interspersed with each other in at least half the genome.

Animals↗
PubMed1978

Kinetic determination of the genome size of the pea.

Renaturation of pea (Pisum sativum) DNA has been used to estimate the size of the pea genome and the fraction of pea DNA containing repeated DNA sequences. Pea DNA renaturation and single copy tracer renaturation indicate that the size of the pea genome is 0.5 picograms. More than 70% of pea DNA sequences are repeated from 100 to 5,000 times.

Journal Article↗
PubMed1977

Sequence relationship between long and short repetitive DNA of the rat: a preliminary report.

Long and short repetitive sequences of rat DNA can be isolated and characterized. Long [greater than 1.5 kilobases (kb)] sequences can be separated from short (0.2-0.4 kb) sequences by exclusion chromatography after renaturation of 4-kb DNA fragments to a repetitive Cot and digestion with the single-strand-specific S1 nuclease. (Cot is the initial concentration of DNA in mol of nucleotides/liter multiplied by time in sec.) Long repetitive DNA can be driven by an excess of whole rat DNA can also be used to drive tracer quantities of either long (self-renaturation) or short repetitive DNA. Both the extent and the rate of the renaturations are found to be similar, suggesting that long and short DNA fragments share sequences. When long repetitive DNA is used to drive whole DNA tracers of various lengths, a 3.2-kb interspersion period is found. These data are consistent with the concept that short repetitive sequences are present within long repetitive DNA sequences in the rat genome.

Animals↗
PubMed1977

A program for least squares analysis of reassociation and hybridization data.

A computer program is described for the rapid calculation of least squares solutions for data fitted to different functions normally used in reassociation and hybridization kinetic measurements. The equations for the fraction not reacted as a function of Cot follow: First order, exp(-kCot); second order, (1+kCot)-1; variable order, (1+kCot)-n; approximate fraction of DNA sequence remaining single stranded, (1+kCot)-.44; and a function describing the pairing of tracer when the rate constant for the tracer (k) is distinct from the driver rate constant (kd): (formula: see text). Several components may be used for most of these functional forms. The standard deviations of the individual parameters at the solutions are calculated.

Computers↗
PubMed1976

Absence of short period interspersion of repetitive and non-repetitive sequences in the DNA of Drosophila melanogaster.

A sensitive search has been made in Drosophila melanogaster DNA for short repetitive sequences interspersed with single copy sequences. Five kinds of measurements all yield the conclusion that there are few short repetitive sequences in this genome: () Comparison of the kinetics of reassociation of short (360 nucleotide) and long (1,830 nucleotide) fragments of DNA; 2) reassociation kinetics of long fragments (2,200 nucleotide) with an excess of short (390 short nucleotide) fragments; 3) measurement of the size of S1 nuclease resistant reassociated repeated sequences; 4) measurement of the hyperchromicity of reassociated repetive fragments as a function of length; 5) direct assay by kinetics of reassociation of the amount of single copy sequence present on 1,200 nucletodie long fragments which also contain repetitive sequences.

Animals↗