Single-Molecule LATE-PCR Analysis of Human Mitochondrial Genomic Sequence Variations

It is thought that changes in mitochondrial DNA are associated with many degenerative diseases, including Alzheimer's and diabetes. Much of the evidence, however, depends on correlating disease states with changing levels of heteroplasmy within populations of mitochondrial genomes, rather than individual mitochondrial genomes. Thus these measurements are likely to either overestimate the extent of heteroplasmy due to technical artifacts, or underestimate the actual level of heteroplasmy because only the most abundant changes are observable. In contrast, Single Molecule (SM) LATE-PCR analysis achieves efficient amplification of single-stranded amplicons from single target molecules. The product molecules, in turn, can be accurately sequenced using a convenient Dilute-‘N’-Go protocol, as shown here. Using these novel technologies we have rigorously analyzed levels of mitochondrial genome heteroplasmy found in single hair shafts of healthy adult individuals. Two of the single molecule sequences (7% of the samples) were found to contain mutations. Most of the mtDNA sequence changes, however, were due to the presence of laboratory contaminants. Amplification and sequencing errors did not result in mis-identification of mutations. We conclude that SM-LATE-PCR in combination with Dilute-‘N’-Go Sequencing are convenient technologies for detecting infrequent mutations in mitochondrial genomes, provided great care is taken to control and document contamination. We plan to use these technologies in the future to look for age, drug, and disease related mitochondrial genome changes in model systems and clinical samples

Compromised Mitochondrial Fatty Acid Synthesis in Transgenic Mice Results in Defective Protein Lipoylation and Energy Disequilibrium

A mouse model with compromised mitochondrial fatty acid synthesis has been engineered in order to assess the role of this pathway in mitochondrial function and overall health. Reduction in the expression of mitochondrial malonyl CoA-acyl carrier protein transacylase, a key enzyme in the pathway encoded by the nuclear Mcat gene, was achieved to varying extents in all examined tissues employing tamoxifen-inducible Cre-lox technology. Although affected mice consumed more food than control animals, they failed to gain weight, were less physically active, suffered from loss of white adipose tissue, reduced muscle strength, kyphosis, alopecia, hypothermia and shortened lifespan. The Mcat-deficient phenotype is attributed primarily to reduced synthesis, in several tissues, of the octanoyl precursors required for the posttranslational lipoylation of pyruvate and a-ketoglutarate dehydrogenase complexes, resulting in diminished capacity of the citric acid cycle and disruption of energy metabolism. The presence of an alternative lipoylation pathway that utilizes exogenous free lipoate appears restricted to liver and alone is insufficient for preservation of normal energy metabolism. Thus, de novo synthesis of precursors for the protein lipoylation pathway plays a vital role in maintenance of mitochondrial function and overall vigo

Trademarks, Certification Marks and Technical Standards

The names of many technical standards such as Wi-Fi, Bluetooth and DVD have become household terms known throughout the developed world. This chapter describes different approaches that have been taken with respect to the naming and legal protection of technical standards, ranging from those that are wholly unregulated to those that are administered under strict certification and compliance regimes. It concludes by questioning the need for aggressive protection of marks that exist largely to inform consumers about technical product features rather than the source of standards themselves

Table of sequences from the NTC amplifications.

<p>NTC-1, NTC-2, and NTC-3 are the first, second, and third NTC amplification runs. All changes are based off of the Anderson sequence.</p

List of results for hair shaft mitochondria experiments.

<p>Samples with an a or b following mean that they were two sequences in the same tube. (–) There were no mutations.</p

Table of sequences from the re-amplification of the M2 sample.

<p>All changes are based off of the M2 sequences. Heteroplasmy is indicated by a/.</p

Amplification plots of (a) dilution series and (b) single molecules using blood lymphocyte mtDNA.

<p>(a) (*) equal NTC, (▪) 10,000 copies, (♦) 1,000, (▴) 100, and (•) 10 copies. (b) Single copy amplification has scattered Ct values. (▪) equal single copy mtDNA, (*) equal NTCs. (c) SYBR Green melts of HV1 samples. The predicted melt of the amplicon is 86.5. The samples do melt with a sharp peak at that temperature showing that the sample has been amplified not only correctly but also cleanly.</p

Compromised mitochondrial fatty acid synthesis in transgenic mice results in defective protein lipoylation and energy disequilibrium.

A mouse model with compromised mitochondrial fatty acid synthesis has been engineered in order to assess the role of this pathway in mitochondrial function and overall health. Reduction in the expression of mitochondrial malonyl CoA-acyl carrier protein transacylase, a key enzyme in the pathway encoded by the nuclear Mcat gene, was achieved to varying extents in all examined tissues employing tamoxifen-inducible Cre-lox technology. Although affected mice consumed more food than control animals, they failed to gain weight, were less physically active, suffered from loss of white adipose tissue, reduced muscle strength, kyphosis, alopecia, hypothermia and shortened lifespan. The Mcat-deficient phenotype is attributed primarily to reduced synthesis, in several tissues, of the octanoyl precursors required for the posttranslational lipoylation of pyruvate and α-ketoglutarate dehydrogenase complexes, resulting in diminished capacity of the citric acid cycle and disruption of energy metabolism. The presence of an alternative lipoylation pathway that utilizes exogenous free lipoate appears restricted to liver and alone is insufficient for preservation of normal energy metabolism. Thus, de novo synthesis of precursors for the protein lipoylation pathway plays a vital role in maintenance of mitochondrial function and overall vigor