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Biomedical subjects

R K Wilson

Publications and source records attributed to R K Wilson.

At least 19 recordsLinked to original sources
PubMed1996

Preferential RNA editing at specific sites within transcripts of two plant mitochondrial genes does not depend on transcriptional context or nuclear genotype.

Transcripts of most plant mitochondrial protein-coding genes exhibit C-to-U RNA editing events. In Petunia, two co-transcribed genes, nad3 and rps12, exhibit transcripts which are not fully edited at all potential editing sites. We investigated the nad3/rps12 transcript population in four different genotypes. In one pair of genotypes, the nuclear genome is identical but the nad3/rps12 genes are in different transcriptional contexts. Both the nad3/ rps12 genes and the plant mitochondrial genomes are identical in a second pair of genotypes, but the nuclear background is derived from two different Petunia species. We found that the overall extent of editing varied greatly between genotypes and is affected by nuclear genotype but not by the global transcriptional context. Local sequence context around a particular site does affect editing frequency. In all genotypes, certain sites exhibit high editing frequency, but these sites do not share obvious primary sequence characteristics. In all genotypes examined, editing sites which do not affect the encoded amino acid are less frequently edited than sites which alter codons to non-synonymous forms. All these data indicate that an unidentified property of the sequences immediately surrounding a cytosine affect its selection as a target in the editing process.

Amino Acid Sequence↗
PubMed1996

Protein polymorphism generated by differential RNA editing of a plant mitochondrial rps12 gene.

The rps12 gene transcripts encoding mitochondrial ribosomal protein S12 are partially edited in petunia mitochondria. Different petunia lines were found vary in the extent of rps12 transcript editing. To test whether multiple forms of RPS12 proteins are produced in petunia mitochondria as a result of partial editing, we probed mitochondrial proteins with specific antibodies against edited and unedited forms of a 13-amino-acid RPS12 peptide spanning two amino acids affected by RNA editing. Both antibodies reacted with mitochondrial proteins at the expected size for RPS12 proteins. The amounts of unedited RPS12 protein in different petunia lines correlate with the abundance of unedited transcripts in these plants. Unedited rps12 translation products are also detected in other plant species, indicating that polymorphism in mitochondrial rps12 expression is widespread. Moreover, we show that RPS12 proteins recognized by both edited-specific and unedited-specific antibodies are present in a petunia mitochondrial ribosome fraction. These results demonstrate that partially edited transcripts can be translated and that the protein product can accumulate to detectable levels. Therefore, genes exhibiting incompletely edited transcripts can encode more than one gene product in plant mitochondria.

Amino Acid Sequence↗
PubMed1995

The construction and analysis of M13 libraries prepared from YAC DNA.

Yeast artificial chromosomes (YACs) provide a powerful way to isolate and map large regions of genomic DNA and their use in genome analysis is now extensive. We modified a series of procedures to produce high quality shotgun libraries from small amounts of YAC DNA. Clones from several different libraries have been sequenced and analyzed for distribution, sequence integrity and degree of contamination from yeast DNA. We describe these procedures and analyses and show that sequencing at about 1-fold coverage, followed by database comparison (survey sequencing) offers a relatively quick method to determine the nature of previously uncharacterized cosmid or YAC clones.

Bacteriophage M13↗
PubMed1995

Genomic structure of a cytoplasmic dynein heavy chain gene from the nematode Caenorhabditis elegans.

We report the cloning and sequencing of genomic DNA encoding a cytoplasmic dynein heavy chain from the nematode Caenorhabditis elegans. In a contiguous stretch of 35,103 bp of DNA from the left arm of linkage group I, we have found a gene that is predicted to encode a protein of 4,568 amino acids. This gene is composed of 15 exons and 14 relatively short introns, and it has significant homology to the other dynein heavy chains in the databases. The deduced molecular mass of the derived polypeptide is 512,624 Da. As with other dynein heavy chains that have been sequenced to date, it contains four GXXGXGK(S/T) motifs that form part of a consensus sequence for the nucleotide triphosphate-binding domains. Comparison of the axonemal and cytoplasmic dynein heavy chains shows that regions of homology among all dyneins are clustered in the carboxyl terminal two-thirds of the polypeptide, whereas the amino terminal one-third of the heavy chains may contain domains that specify functions that differ between the axonemal and cytoplasmic forms of the dynein heavy chain.

Animals↗
PubMed1995

Genomic DNA sequencing methods.

Sequence analysis of cosmids from C. elegans and other organisms currently is best done using the random or "shotgun" strategy (Wilson et al., 1994). After shearing by sonication, DNA is used to prepare M13 subclone libraries which provide good coverage and high-quality sequence data. The subclones are assembled and the data edited using software tools developed especially for C. elegans genomic sequencing. These same tools facilitate much of the subsequent work to complete both strands of the sequence and resolve any remaining ambiguities. Analysis of the finished sequence is then accomplished using several additional computer tools including Genefinder and ACeDB. Taken together, these methods and tools provide a powerful means for genome analysis in the nematode.

Animals↗
PubMed1995

An infrared fluorescent dATP for labeling DNA.

Near-infrared fluorescence provides a nonradioactive method of detection with high sensitivity and low background. An infrared fluorophore has been attached covalently to the nucleotide deoxyadenosine triphosphate (dATP) to provide a reagent for enzymatic labeling of various types of DNA molecules and for facilitating their detection with an automated DNA sequencing and analysis system. DNA sequencing reaction products can be labeled internally by performing limited polymerization utilizing infrared-labeled dATP (IR-dATP) as the sole source of adenine deoxynucleotide prior to a dideoxy-specific termination reaction. PCR products can be labeled fluorescently by the addition of limited quantities of IR-dATP to the amplification reaction. This latter strategy has been utilized for detection of short tandem repeat polymorphisms (STRPs) which are useful for gene mapping, genetic diagnostics, forensic analysis, and paternity testing. Restriction fragments can be labeled also by fill-in reactions of appropriate 5' overhangs. Diminutive amounts of such fluorescently labeled DNA molecules can be visualized rapidly and conveniently using infrared detection technology.

Adenosine Triphosphate↗
PubMed1995

Large-scale complementary DNA sequencing methods.

Complementary DNA libraries are useful tools for uncovering genes of interest in C. elegans and finding specific homologies to genes in other organisms (Waterston et al., 1992; McCombie et al., 1992). When working with existing cDNA libraries, be sure to carefully choose which libraries would be most beneficial to the type of research being done. Some libraries may be specific for genes that are present in lower copy numbers, whereas others may be of a more general nature. It is important to fully understand the source and construction of the library you will be working with. Once an appropriate library has been chosen, work may begin to isolate a specific cDNA and sequence it completely or to survey many cDNAs by single-pass DNA sequencing. Whatever the project, it is important to develop a specific strategy for both the sequencing and the organization of the clones being characterized. The strategies and procedures we have outlined in this chapter have proven effective for rapid and comprehensive cDNA characterization.

Animals↗
PubMed1994

Using the PDSA cycle to standardize a quality assurance program in a quality improvement-driven environment.

BACKGROUND: At Parkview Episcopal Medical Center (Pueblo, Colorado), QA means not only quality assurance but also quality alarms-statistical monitoring and analysis of key indicators that lead to the discovery of opportunities for continuous improvement. Data are monitored using statistical process control. Continuous improvement supports quality assurance (QA) just as it supports all other functions at Parkview. METHODS: A PDSA (Plan-Do-Study-Act) analysis form was created for use in conjunction with the data collection tool selected by the user. The data collected and analyzed are made meaningful through the standardization of formats and methods. RESULTS: Successes with this form demonstrate that QA and quality improvement (QI) can be integrated, allowing processes and outcomes to be improved. Since standardization occurred successfully in 1993, QA has been integrated into the strategic-planning process for 1994. Expectations are that duplicate reports and data gathering will be eliminated.

Clinical Competence↗
PubMed1994

Sequencing, processing, and localization of the petunia CMS-associated mitochondrial protein.

The petunia mitochondrial fused gene (pcf), which is associated with cytoplasmic male sterility (CMS), is composed of sequences derived from atp9, coxII, and an unidentified reading frame termed urfS. Pcf transcripts are modified by editing at 11 sites. Codon usage and nearest neighbor analysis suggest that the urfS region is not derived originally from a plant mitochondrial coding region. Although the gene contains an open reading frame coding for a 43 kDa protein, a 25 kDa gene product has previously been identified (Nivison and Hanson, 1989). N-terminal sequencing revealed that the 25 kDa protein is encoded within the urfS portion of pcf and that its actual molecular mass is 19.5 kDa. Through pulse-chase labeling of protein in isolated mitochondria, the 25 kDa protein was found to be processed from a 43 kDa precursor protein representing the entire pcf gene sequence. Antibodies to synthetic peptides encoded by the atp9 and coxII portions of pcf recognized petunia ATP9 or COXII but no other mitochondrial proteins on immunoblots. Controlled proteolysis experiments showed that both the 43 kDa precursor and the 25 kDa protein are soluble or loosely associated with membranes. Thus, the 25 kDa protein appears to be the only pcf-encoded protein that accumulates in mitochondria.

Amino Acid Sequence↗
PubMed1994

Variations on cycle sequencing.

Linear amplification or cycle sequencing is an advance that has made the use of automated fluorescent DNA sequencing instruments truly practical for large-scale genome analysis. We have investigated several variations of our standard cycle sequencing method. First, we have reduced the number of amplification cycles, which resulted in improved data quality and faster sequencing. Second, we have used different thermostable enzymes, which again improved data quality. Lastly, we have devised a method for precipitating reaction products in 96-tube trays. Together, these variations have allowed us to develop an improved cycle sequencing method that significantly increases sequencing throughput, improves efficiency and data quality, and reduces the cost of sequencing reactions.

Gene Amplification↗
PubMed1993

Automated fluorescent DNA sequencing of polymerase chain reaction products.

The methods described in this chapter provide some useful approaches for DNA sequencing of templates produced by PCR. These procedures have been employed successfully for large-scale DNA sequencing of cosmid fragments subcloned in plasmid or M13 vectors, and for sequence analysis of cDNAs cloned in bacteriophage lambda vectors. In addition, the method describing direct sequencing from PEG-precipitated PCR product has been used successfully for analysis of Caenorhabditis elegans genomic and cDNA sequences. It is important to reiterate that for every combination of amplification primer pair and target DNA, there is an optimal method for PCR amplification; the ability to sequence the products of any PCR experiment directly will also vary. A coupled PCR/DNA sequencing method that works well for one experimental system may work quite poorly with others. Hence, a few days or hours spent optimizing PCR amplification conditions and selecting the best DNA sequencing method for the target DNA of interest will be time well spent.

Animals↗
PubMed1993

High-throughput purification of M13 templates for DNA sequencing.

Two variants of methods for high-throughput preparation of single-stranded M13 DNA are described. Both variants are derived from previously described chemistry and are appropriate for purification of M13 templates in 96-deep well plates. In both variants, phenol extraction is replaced by treatment with sodium iodide to disrupt phage proteins prior to ethanol precipitation of M13 DNA. In one of the variants, nonderivatized paramagnetic particles are employed to collect aggregated M13 phage particles and DNA, thereby replacing the need for centrifugation. The other variant omits the magnetic particles and utilizes a centrifuge that can accommodate the 96-deep well plates. Although the purification scheme that uses magnetic separation results in a decreased yield of M13 DNA, it is amenable to robotic automation strategies and thus will be useful for genomic sequencing projects. Performed manually, either method can easily produce 192 templates in a few hours. Although both variants produce DNA of sufficient quantity for automated fluorescent DNA sequencing, the procedure that utilizes magnetic separation provides template DNA of higher quality.

Autoanalysis↗
PubMed1992

Duck liver 'malic' enzyme. Expression in Escherichia coli and characterization of the wild-type enzyme and site-directed mutants.

A cDNA for duck liver 'malic' enzyme (EC 1.1.1.40) was subcloned into pUC-8, and the active enzyme was expressed in Escherichia coli TG-2 cells as a fusion protein including a 15-residue N-terminal leader from beta-galactosidase coded by the lacZ' gene. C99S and R70Q mutants of the enzyme were generated by the M13 mismatch technique. The recombinant enzymes were purified to near homogeneity by a simple two-step procedure and characterized relative to the enzyme isolated from duck liver. The natural duck enzyme has a subunit molecular mass of approx. 65 kDa, and the following kinetic parameters for oxidative decarboxylation of L-malate at pH 7.0: Km NADP+ (4.6 microM); Km L-malate (73 microM); kcat (160 s-1); Ka (2.4 microM) and Ka' (270 microM), dissociation constants of Mn2+ at 'tight' (activating) and 'weak' metal sites; and substrate inhibition (51% of kcat. at 8 mM-L-malate). Properties of the E. coli-derived recombinant wild-type enzyme are indistinguishable from those of the natural duck enzyme. Kinetic parameters of the R70Q mutant are relatively unaltered, indicating that Arg-70 is not required for the reaction. The C99S mutant has unchanged Km for NADP+ and parameters for the 'weak' sites (i.e. inhibition by L-malate, Ka'); however, kcat. decreased 3-fold and Km for L-malate and Ka each increased 4-fold, resulting in a catalytic efficiency [kcat./(Km NADP+ x Km L-malate x Ka)] equal to 3.7% of the natural duck enzyme. These results suggest that the positioning of Cys-99 in the sequence is important for proper binding of L-malate and bivalent metal ions.

Amino Acid Sequence↗
PubMed1992

DNA sequencing with dye-labeled terminators and T7 DNA polymerase: effect of dyes and dNTPs on incorporation of dye-terminators and probability analysis of termination fragments.

The incorporation of fluorescently labeled dideoxynucleotides by T7 DNA polymerase is optimized by the use of Mn2+, fluorescein analogs and four 2'-deoxyribonucleoside 5'-O-(1-thiotriphosphates) (dNTP alpha S's). The one-tube extension protocol was tested on single-stranded templates, as well as PCR fragments which were made single-stranded by digestion with T7 gene 6 exonuclease. Dye primer sequencing using four dNTP alpha S's was shown to give uniform termination patterns which were comparable to four dNTPs. Efficiency of the polymerase also appeared to improve with the dNTP alpha S's. A mathematical model was developed to predict the pattern of termination based on enzyme activity and ratios of ddNTP/dNTPs. This method can be used to optimize sequencing reactions and to estimate enzyme discrimination constants of chain terminators.

Base Sequence↗
PubMed1992

Fluorescence chemistries for automated primer-directed DNA sequencing.

A major limitation in the applicability of automated DNA sequencing instruments has been the difficulty in using user-defined oligonucleotide primers which allow sequencing reactions to start at any specific point in a region of interest. Recently, new chemistries have become available for fluorescent labeling which will begin to facilitate the use of any oligonucleotide primer with automated DNA sequencers. In this report, we describe several methods for automated primer-directed DNA sequencing, and compare and discuss the relative merits and limitations of these methods.

Automation↗