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R G Rutledge

Publications and source records attributed to R G Rutledge.

16 recordsLinked to original sources
PubMed2004

Sigmoidal curve-fitting redefines quantitative real-time PCR with the prospective of developing automated high-throughput applications.

Quantitative real-time PCR has revolutionized many aspects of genetic research, biomedical diagnostics and pathogen detection. Nevertheless, the full potential of this technology has yet to be realized, primarily due to the limitations of the threshold-based methodologies that are currently used for quantitative analysis. Prone to errors caused by variations in reaction preparation and amplification conditions, these approaches necessitate construction of standard curves for each target sequence, significantly limiting the development of high-throughput applications that demand substantive levels of reliability and automation. In this study, an alternative approach based upon fitting of fluorescence data to a four-parametric sigmoid function is shown to dramatically increase both the utility and reliability of quantitative real-time PCR. By mathematically modeling individual amplification reactions, quantification can be achieved without the use of standard curves and without prior knowledge of amplification efficiency. Combined with provision of quantitative scale via optical calibration, sigmoidal curve-fitting could confer the capability for fully automated quantification of nucleic acids with unparalleled accuracy and reliability.

Calibration↗
PubMed2003

Characterization of a MYBR2R3 gene from black spruce (Picea mariana) that shares functional conservation with maize C1.

PCR amplification with degenerate primers targeted to highly conserved amino acid motifs within the MYB domain was used to demonstrate that black spruce (Picea mariana) possesses a diverse MYB gene family. Amino acid sequence comparisons revealed three broad MYB subfamilies, one of which shares extensive similarity with maize C1, a central regulator of anthocyanin biosynthesis. A cDNA clone encoding a MYBR2R3 protein from P. mariana with high levels of sequence homology to maize C1 was shown to transactivate the Bz2 promoter in combination with maize R in embryonal tissues of both black spruce and larch. Functional dependence on the maize R protein, and the presence of a conserved C-terminal GIDPxTH motif, support the conservation of MYBR2R3 function in conifers, and demonstrate that the basic components of MYBR2R3-dependent transcriptional regulation have been conserved between angiosperms and gymnosperms.

Amino Acid Sequence↗
PubMed2003

Mathematics of quantitative kinetic PCR and the application of standard curves.

Fluorescent monitoring of DNA amplification is the basis of real-time PCR, from which target DNA concentration can be determined from the fractional cycle at which a threshold amount of amplicon DNA is produced. Absolute quantification can be achieved using a standard curve constructed by amplifying known amounts of target DNA. In this study, the mathematics of quantitative PCR are examined in detail, from which several fundamental aspects of the threshold method and the application of standard curves are illustrated. The construction of five replicate standard curves for two pairs of nested primers was used to examine the reproducibility and degree of quantitative variation using SYBER Green I fluorescence. Based upon this analysis the application of a single, well- constructed standard curve could provide an estimated precision of +/-6-21%, depending on the number of cycles required to reach threshold. A simplified method for absolute quantification is also proposed, in which quantitative scale is determined by DNA mass at threshold.

DNA↗
PubMed2003

Transgene integration patterns and expression levels in transgenic tissue lines of Picea mariana, P glauca and P abies.

Seventy transgenic tissue lines (translines) of three spruce species ( Picea mariana, P. glauca and P. abies) were characterized with respect to the integration pattern of the gus (beta-glucuronidase) gene, and the level of GUS activity was determined in 81 lines. The majority of the P. mariana translines (18 lines of 22) integrated multicopies of the transgene, whereas mostly single integrations were detected in the other two species. The activity levels of GUS varied widely among the individual translines of P. mariana, and there was a strong indication that the logarithm of GUS activity increased with the number of gus copies ( P=0.0003) in lines with one to five known insertions (uncensored). The average level of GUS activity, in lines that integrated one gene copy, was the highest in white spruce followed by black spruce and Norway spruce (22.7, 16.5 and 6.3 nmol 4-methylumbelliferone min(-1 )mg(-1 )protein, respectively).

Culture Techniques↗
PubMed2002

An improved MUG fluorescent assay for the determination of GUS activity within transgenic tissue of woody plants.

Despite the success of the MUG fluorometric assay for quantitative analysis of beta-glucuronidase (GUS) activity within a vast array of transgenic plant species and tissues, attempts to apply this protocol for analysis of woody plants has been found to be problematic, primarily due to the interfering effects of phenolics and other secondary metabolites. Our analysis of transgenic spruce needles and poplar leaves illustrates that low tissue mass to extract volume, along with the inclusion of polyvinylpolypyrolidone and either beta-mercaptoethanol or metabisulphite, are essential for producing reliable results. The primary action of these additives was found to involve increased GUS extractability and the preservation of GUS activity during extract manipulation, but they were not completely effective in eliminating GUS enzymatic inhibitors. Normalization of GUS activity upon DNA concentration was also found to be an effective alternative to protein concentration, providing the ability to make cross-species and inter-tissue comparisons of gusA transgene activity.

Fluorescence↗
PubMed2000

Induction of chalcone synthase expression in white spruce by wounding and jasmonate.

The phenylpropanoid pathway has important functions in angiospermous plants following exposure to environmental stresses, such as wounding and pathogen attack, that lead to production of compounds including lignin, flavonoids and phytoalexins. Chalcone synthase (CHS) is a key enzyme in this pathway, catalyzing the first step in flavonoid biosynthesis, whose expression can be induced in response to environmental stress. To explore the response of conifers to environmental stress, expression of spruce CHS and its inducibility were investigated. A partial spruce CHS cDNA clone was isolated using PCR. Examination of the expression patterns of the CHS gene family in white spruce revealed accumulation of CHS mRNA in needle tissue following mechanical wounding, or application of signal molecules like jasmonic acid or methyl jasmonate. Repeated mechanical wounding or jasmonate applications had an enhancing effect on transcript accumulation in needles. A systemic accumulation of CHS mRNAs following wounding was also observed. Conifers thus appear to possess a general wound response similar to that found for angiosperms, which includes CHS induction as well as its inducibility by jasmonic acid and airborne methyl jasmonate.

Acyltransferases↗
PubMed1999

Synthesis and oxidative insolubilization of cell-wall proteins during osmotic stress.

The cell walls in the new white roots of jack pine (Pinus banksiana Lamb.) were observed to constrict around the shrinking protoplast of osmotically stressed roots, and pressure was maintained via an apparent adjustment of cell-wall size and elasticity. These elastic alterations of the cell wall permitted the root cells to maintain full turgor despite the loss of most of the water in the tissue. The constriction of the root cell wall around the dehydrating protoplasts to maintain turgor may reflect changes in cell wall structure. We found that these shrinking root cells synthesize and secrete into the intercellular fluid a set of proteins. These proteins become tightly associated (i.e. guanidine HCl- and sodium dodecyl sulfate-insoluble) with the cell wall but can be released from the matrix, after briefly boiling in 0.1% sodium dodecyl sulfate, by the combination of guanidine HCl, CaCl2 and dithiothreitol. However, these cell-wall proteins became insoluble with time. The proteins could subsequently be destructively extracted from the wall with acid NaClO2 treatments. After these proteins were incorporated into the cell walls, the roots adopted a new, smaller maximal tissue volume and elastic coefficients returned to normal levels.

Amino Acid Sequence↗
PubMed1992

Members of the acetohydroxyacid synthase multigene family of Brassica napus have divergent patterns of expression.

Brassica species possess the most complex acetohydroxyacid synthase (AHAS) multigene family reported for plants. The AHAS genes code for an essential enzyme in branched-chain amino acid biosynthesis. In the allotetraploid species, B. napus, four (AHAS1-4) of the five AHAS genes have been cloned and sequenced. The transcripts were examined by RNase protection assays using gene-specific, antisense RNA probes. Only AHAS1, AHAS2 and AHAS3 were shown to be expressed in B. napus and one of the diploid progenitor species B. campestris or B. oleracea. AHAS1 and AHAS3 are highly conserved genes that presumably code for the essential AHAS housekeeping functions. They were expressed as low abundance mRNA in all somatic and reproductive tissues examined. AHAS2, which is structurally distinct from all other plant AHAS genes, was only expressed in mature ovules and extraembryonic tissues of immature seeds. This study provides direct evidence for multiple AHAS isoforms in plants and for an AHAS gene which is developmentally regulated in a tissue-specific manner. The discovery raises questions concerning the functional significance of AHAS in seed development.

Acetolactate Synthase↗
PubMed1991

Molecular characterization and genetic origin of the Brassica napus acetohydroxyacid synthase multigene family.

The Brassica napus rapeseed cultivar Topas contains an acetohydroxyacid synthase (AHAS) multigene family consisting of five members (AHAS 1-5). DNA sequence analysis indicate that AHAS1 and AHAS3 share extensive homology. They probably encode the AHAS enzymes essential for plant growth and development. AHAS2 has diverged significantly from AHAS1 and AHAS3 and has unique features in the coding region of the mature polypeptide, transit peptide and upstream non-coding DNA, which raises the possibility that it has a distinct function. AHAS4 and AHAS5 have interrupted coding regions and may be defective. The complexity of the AHAS multigene family in the allotetraploid species B. napus is much greater than reported for Arabidopsis thaliana and Nicotiana tabacum. Analysis of the presumptive progenitor diploid species B. campestris and B. oleracea indicated that AHAS2, AHAS3 and AHAS4 originate from the A genome, whereas AHAS1 and AHAS5 originate from the C genome. Further variation within each of the AHAS genes in these species was found.

Acetolactate Synthase↗
PubMed1988

Isolation and expression of cDNA clones coding for two sequence variants of Xenopus laevis histone H5.

We have cloned and characterized cDNAs coding for two variants of Xenopus laevis H5 histone protein (previously called H1s). cDNA was synthesized from RNA of immature erythrocytes in a single reaction using a modification of the method of Gubler and Hoffman [Gene 25 (1983) 263-269], and blunt-end ligated into the HincII site of the phage vector M13mp9. Immunological screening with a polyclonal antibody yielded two clones expressing H5 peptide. Sequence characterization revealed that both clones contained partial cDNA inserts and that the smaller 340-bp clone initiated reverse transcription within the coding region, at a site rich in adenine. Rescreening of the cDNA bank by nucleic acid hybridization produced eleven additional H5 clones, one of which coded for a second variant of H5. These two variants, called XLH5A and XLH5B, are very similar in sequence and code for proteins of 195 and 193 amino acids, respectively, which may be the H1D and H1E variants observed previously. XLH5, avian H5 and human H1O share identity at both nucleotide and amino-acid sequence levels. Further, the XLH5-coding mRNA is likely polyadenylated and lacks the highly conserved, 23-nucleotide dyad symmetry element found within the 3' untranslated regions of most histone-coding mRNAs.

Amino Acid Sequence↗
PubMed1988

Rapid synthesis and cloning of complementary DNA from any RNA molecule into plasmid and phage M13 vectors.

We describe several modifications of the Gubler and Hoffman procedure [Gene 25 (1983) 263-269] for complementary DNA (cDNA) synthesis that expand the versatility of this method for the rapid synthesis and cloning of double-stranded (ds) cDNA. These modifications include: (1) The combination of first and second strand synthesis into a single two-step reaction, which reduces the time for synthesis of blunt-ended ds-cDNA to less than 4 h. (2) The use of random hexadeoxyribonucleotide primers (RP) for the synthesis of ds-cDNA, which allows the synthesis of cDNA from any RNA template. (3) The combined use of random primers and DNA ligase treatment of cDNA/RNA hybrids prior to second-strand synthesis, which promotes the production of nearly full length ds-cDNA molecules. (4) The use of gel filtration to size-fractionate ds-cDNA, which allows the selection of specific size classes of ds-cDNA for cloning. (5) The use of blunt-end ligation to insert the ds-cDNA into the vector, which reduces the total time required for the construction of cDNA libraries to less than 24 h.

Autoradiography↗
PubMed1987

A complete complementary DNA for the oncodevelopmental calcium-binding protein, oncomodulin.

RNA from a rat liver tumor (Morris hepatoma 5123tc) was used to construct cDNAs together comprising the complete coding sequence of rat oncomodulin mRNA. Information obtained from these cDNAs as well as from primer extension analysis gave a deduced length for the complete oncomodulin mRNA of approximately 680 nucleotides (excluding the poly(A) tail) including a 5'-untranslated region of 97 +/- 2 nucleotides, a 324-nucleotide-coding sequence and a 259-nucleotide 3'-noncoding region. Comparison of the oncomodulin cDNA sequence with those coding for other members of the calcium-binding protein family shows little homology with the exception of a recently reported parvalbumin cDNA where the oncomodulin and parvalbumin nucleotide sequences are 59% identical in the protein-coding region. RNA blot analysis of poly(A+) RNA from normal adult rat liver gave no evidence of oncomodulin expression in this tissue. A single RNA species was detected, however, in RNA extracts from the hepatoma and from rat and human placentas. A probe prepared from one of the rat oncomodulin cDNAs hybridized with a single DNA species in restriction digests of hepatoma and normal DNA from rat and sequences in DNA of humans and other mammals. A 38-nucleotide sequence spanning the 5'-untranslated region and the first seven codons of the oncomodulin cDNA, was far less homologous than was the same region of a parvalbumin cDNA, to a chicken calmodulin cDNA sequence coding for the first calcium-binding domain. The oncomodulin gene appears to have diverged more from that of calmodulin than has the parvalbumin gene.

Amino Acid Sequence↗
PubMed1984

Uncoupled synthesis of H1o-like histone H1s during late erythropoiesis in Xenopus laevis.

This study investigated the synthesis of Xenopus histones during erythropoiesis. Although cessation of DNA replication in the mid-stages of erythroid maturation is accompanied by arrested synthesis of histone H1 and core histones, synthesis of H1o (an H1o-like histone) was found to continue into late stages of erythropoiesis, as has been reported for avian erythrocyte histone H5. This was accompanied by a threefold increase in the relative amount of Xenopus H1s, similar to the accumulation reported for H5 during avian erythropoiesis and for H1o in some differentiated mammalian cells. The structural and metabolic homologies of avian H5, mammalian H1o, and Xenopus H1s imply that these lysine-rich histones have closely related functions distinct from those of H1, and thus represent a subclass of lysine-rich histones.

Anemia↗
PubMed1981

Surgical intervention in chronic (Hashimoto's) thyroiditis.

The incidence of chronic (Hashimoto's) thyroiditis in surgical specimens is relatively high, i.e., 13% in collected studies, for a disease with clinical and laboratory characteristics that are sufficiently specific, that thyroidectomy should rarely be required for diagnosis or treatment. This incidence is presumably related to the difficulty in distinguishing between thyroiditis and a thyroid neoplasm. Experience with 260 thyroidectomies at the North Carolina Memorial Hospital performed between 1875 and 1980 for a dominant thyroid mass was reviewed to determine the reliability of criteria for diagnosis and the indications for surgical treatment. Using the criteria of clinical findings, complemented by laboratory studies, e.g., free thyroxine index, thyroid autoantibodies, TSH level, thyroid scan, in addition to the judicious use of the cutting (core) needle biopsy procedure, the incidence of Hashimoto's thyroiditis in this series was 3% and cancer-27%. Four patients had Hashimoto's thyroiditis coincidental to another disease for which thyroidectomy was performed. In seven patients Hashimoto's thyroiditis alone constituted the indications for operation. The indications for operation in these patients were: autonomous function with mild hyperthyroidism (2 patients); associated cold nodule (2 patients); thyromegaly unresponsive to suppressive therapy (2 patients); and rapidly enlarging mass simulating a neoplasm (1 patient). Only one of 71 patients with well differentiated carcinoma had Hashimoto's thyroiditis. One patient with Hashimoto's thyroiditis had associated lymphoma. In most patients, Hashimoto's thyroiditis can be identified using appropriate clinical and laboratory criteria without resorting to thyroidectomy to differentiate between thyroiditis and a neoplasm. Operations are indicated in patients with suspected or established chronic thyroiditis for: 1) the presence of a dominant mass with incomplete regression on suppressive therapy. 2) Progression of thyromegaly despite suppressive therapy. 3) Historic or physical findings suggest a malignancy, e.g., irradiation, multiple endocrine adenomatosis (MEA) syndrome, nerve paralysis, pain, tracheal compression, stipple calcification and cervical lymph node enlargement. 4) Indeterminant findings on cutting needle biopsy, e.g., lymphoma versus thyroiditis. Rarely, an operation is required for an oppressive goiter or associated hyperthyroidism.

Adult↗
PubMed1981

The similarity of histones from turtle erythrocytes and liver.

Using the fresh-water "red-eared turtle" Pseudomys scriptans elegans, we have confirmed the existence of a minor component H1s among the lysine-rich histones of turtle erythrocytes by three forms of gel electrophoresis and two forms of chromatography. It was separated from the major components by both cation-exchange chromatography and molecular-exclusion chromatography and shown to differ slightly but significantly in content of several amino acids as well as being shorter than the other lysine-rich histones. Although its composition is close to that of the "tissue-specific" F1b of sea turtle erythrocytes, it is present in even greater relative amount in red-eared turtle livers. In most respects it resembles the satellite histone H10 of mammalian tissues. No unusual histones were observed among the perchloric acid insoluble histones of turtle erythrocytes.

Amino Acids↗