Biomedical subjects

Elizabeth A Amiott

Publications and source records attributed to Elizabeth A Amiott.

3 recordsLinked to original sources

PubMed2006

Sensitivity of the yeast mitochondrial RNA polymerase to +1 and +2 initiating nucleotides.

Despite a simple consensus sequence, there is considerable variation of promoter strengths, transcription rates, and the kinetics of initiating nucleotide incorporation among the promoters found in the Saccharomyces cerevisiae mitochondrial genome. We asked how changes in the initiating (+1 and +2) nucleotides, conformation of the promoter DNA template, and mutation of the mitochondrial RNA polymerase (mtRNAP) affect the kinetics of nucleotide (NTP) utilization. Using a highly purified in vitro mitochondrial transcription system, we found that 1) the mtRNAP requires the highest concentrations of the +1 and +2 initiating NTPs, intermediate concentrations of NTPs at positions 5 to 11, and low concentrations of elongating NTPs; 2) the mtRNAP requires a higher concentration of the +2 NTP than the +1 NTP for initiation; 3) the kinetics of +2 NTP utilization are altered by a point mutation in the mtRNAP subunit Mtf1; and 4) a supercoiled or pre-melted promoter DNA template restores normal +2 NTP utilization by the Mtf1 mutant. Based on comparisons to the structural and biochemical properties of the bacterial RNAP and the closely related T7 RNAP, we propose that initiating nucleotides, particularly the +2 NTP, are required at high concentrations to drive mitochondrial promoter opening or to stabilize a productive open complex.

DNA-Directed RNA Polymerases↗

PubMed2006

Mitochondrial transcription is regulated via an ATP "sensing" mechanism that couples RNA abundance to respiration.

The information encoded in both the nuclear and mitochondrial genomes must be coordinately regulated to respond to changes in cellular growth and energy states. Despite identification of the mitochondrial RNA polymerase (mtRNAP) from several organisms, little is known about mitochondrial transcriptional regulation. Studying the shift from fermentation to respiration in Saccharomyces cerevisiae, we have demonstrated a direct correlation between in vivo changes in mitochondrial transcript abundance and in vitro sensitivity of mitochondrial promoters to ATP concentration (K(m)ATP). Consistent with the idea that the mtRNAP itself senses in vivo ATP levels, we found that transcript abundance correlates with respiration, but only when coupled to mitochondrial ATP synthesis. In addition, we characterized mutations in the mitochondrial promoter and the mtRNAP accessory factor Mtf1 that alter both in vitro K(m)ATP and in vivo transcription in response to respiratory changes. We propose that shifting cellular pools of ATP coordinately control nuclear and mitochondrial transcription.

Adenosine Triphosphate↗

PubMed2002

Validation of a 16-locus fluorescent multiplex system.

STR multiplexes have been indispensable for the efficient genotyping of forensic samples. The PowerPlex 16 System contains the coreCODIS loci, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, CSF1PO, FGA, THOI, TPOX, vWA, the sex determinant locus, amelogenin, and two pentanucleotide STR loci, Penta D and Penta E. This multiplex satisfies the locus requirements for most national databases and is the most efficient currently available system due to its single PCR amplification. To provide the groundwork for judicial acceptance, including the publication of primer sequences, and to evaluate laboratory-to-laboratory variation, a developmental validation for casework on this commercially available system was performed in 24 laboratories and produced the following conclusions. Amplification was reliable on a variety of thermal cyclers and product could be analyzed on either an ABI PRISM 310 Genetic Analyzer or an ABI PRISM 377 DNA Sequencer. Genotyping using single source samples was consistent between 0.25 and 2 ng of input DNA template with a few laboratories obtaining complete genotypes at 0.0625 ng. However, heterozygote allele imbalance (<60% peak height balance) caused by stochastic effects was observed at a rate of 13% with 0.125 ng DNA and 22% at 0.0625 ng DNA. Mixture analyses were done using a total of 1 ng of DNA template. Most alleles were detected in mixtures of 4 to 1 and some minor alleles were detected in mixtures of 19 to 1. Optimum amplification cycle number was dependent on the sensitivity of the detection instrument used and could also be adjusted to accommodate larger amounts of DNA on solid supports such as FTA paper. Reaction conditions including volume, annealing temperature, and concentrations of primer, AmpliTaq Gold, and magnesium were shown to be optimal yet robust enough to withstand moderate variations without affecting genotype analysis. Environmental, matrix and standard source analyses revealed an ability to obtain complete genotypes in all sample types except those exposed to 80 degrees C for 12-48 days. Finally, comparison of genotype results from the PowerPlex 16 System with other commercially available systems on non-probative reference and forensic samples showed consistent results.

Alleles↗