Microgram Quantities Of Tissue Research Articles

Next generation sequencing and comparative analyses of Xenopus mitogenomes

BackgroundMitochondrial genomes comprise a small but critical component of the total DNA in eukaryotic organisms. They encode several key proteins for the cell’s major energy producing apparatus, the mitochondrial respiratory chain. Additonally, their nucleotide and amino acid sequences are of great utility as markers for systematics, molecular ecology and forensics. Their characterization through nucleotide sequencing is a fundamental starting point in mitogenomics. Methods to amplify complete mitochondrial genomes rapidly and efficiently from microgram quantities of tissue of single individuals are, however, not always available. Here we validate two approaches, which combine long-PCR with Roche 454 pyrosequencing technology, to obtain two complete mitochondrial genomes from individual amphibian species.ResultsWe obtained two new xenopus frogs (Xenopus borealis and X. victorianus) complete mitochondrial genome sequences by means of long-PCR followed by 454 of individual genomes (approach 1) or of multiple pooled genomes (approach 2), the mean depth of coverage per nucleotide was 9823 and 186, respectively. We also characterised and compared the new mitogenomes against their sister taxa; X. laevis and Silurana tropicalis, two of the most intensely studied amphibians. Our results demonstrate how our approaches can be used to obtain complete amphibian mitogenomes with depths of coverage that far surpass traditional primer-walking strategies, at either the same cost or less. Our results also demonstrate: that the size, gene content and order are the same among xenopus mitogenomes and that S. tropicalis form a separate clade to the other xenopus, among which X. laevis and X. victorianus were most closely related. Nucleotide and amino acid diversity was found to vary across the xenopus mitogenomes, with the greatest diversity observed in the Complex 1 gene nad4l and the least diversity observed in Complex 4 genes (cox1-3). All protein-coding genes were shown to be under strong negative (purifying selection), with genes under the strongest pressure (Complex 4) also being the most highly expressed, highlighting their potentially crucial functions in the mitochondrial respiratory chain.ConclusionsNext generation sequencing of long-PCR amplicons using single taxon or multi-taxon approaches enabled two new species of Xenopus mtDNA to be fully characterized. We anticipate our complete mitochondrial genome amplification methods to be applicable to other amphibians, helpful for identifying the most appropriate markers for differentiating species, populations and resolving phylogenies, a pressing need since amphibians are undergoing drastic global decline. Our mtDNAs also provide templates for conserved primer design and the assembly of RNA and DNA reads following high throughput “omic” techniques such as RNA- and ChIP-seq. These could help us better understand how processes such mitochondrial replication and gene expression influence xenopus growth and development, as well as how they evolved and are regulated.

Separate enzymatic microassays for aspartate aminotransferase isoenzymes

The properties of the cytosolic and mitochondrial isoenzymes of aspartate aminotransferase were studied using a commercial preparation of the cytosolic isoenzyme, a mitochondrial preparation, and whole brain homogenate. Based on these properties, microassays were developed and shown to be highly specific and quantitatively accurate for measuring the activity of either the cytosolic or mitochondrial isoenzyme in microgram quantities of tissue. The assays have been successfully applied to homogenates of a wide variety of tissues. They can be used to measure the activities of aspartate aminotransferase isoenzymes in sub-microgram samples of freeze-dried tissue.

Radiotracer assay of pyruvate kinase

A radiotracer enzyme assay for the determination of pyruvate kinase activity is described. The rate of the enzyme catalyzed reaction is determined by radioassay of either ATP- 3H formed or unreacted ADP- 3H, or both after their separations from the assay reaction mixture by TLC. The method is extremely sensitive, permitting the use of microgram quantities of tissue to assay for the enzymic formation of μμmole quantities of product.

An assay for organic phosphorus fractions in microgram quantities of tissue

A fluorometric technique for the determination of acid-soluble phosphorus, lipid phosphorus, and nucleic acid phosphorus in 0.5 to 1.0 μg quantities of tissue is described. The assay is based on the stoichiometric generation of NADPH from P i by the use of purified crystalline enzymes, a feature which confers the advantage of extension to even greater sensitivity by application of the “NADPH cycling” system. Assays of mouse brain and mouse brain tumor were performed at both the microgram and milligram level and compared. The fluorometric assay offers about fivefold increase in sensitivity with a sacrifice of analytical accuracy of approximately 6% (4% vs. 10%) when compared to an established spectrophotometric procedure.

Spectrophotometric Determination of Nanogram Amounts of Total Cholesterol in Microgram Quantities of Tissue or Microliter Volumes of Serum.

Spectrophotometric Determination of Nanogram Amounts of Total Cholesterol in Microgram Quantities of Tissue or Microliter Volumes of Serum.

Studies in histochemistry. LVI. Microbiological determination of biotin in microgram amounts of tissue

A microbiological procedure employing Lactobacillus arabinosus for the determination of biotin in microgram quantities of tissue is described. Data pertaining to effects of various conditions of acid hydrolysis on the liberation and destruction of biotin in rat liver are presented and compared with data obtained by using Saccharomyces cerevisiae. An analysis of the reproducibility of the method is given, and data for biotin content of liver are included.

Studies in histochemistry. LII. Quantitative determination of coenzyme A in microgram amounts of tissue by a microbiological technique

A procedure for the determination of coenzyme A in microgram quantities of tissue has been developed by an adaptation of the method of Novelli and Schmetz. Assay conditions suitable for measurement on the micro scale have been elaborated. An analysis of the range and reliability of the method has been presented. A micro adaptation of the method of Moat and Delwiche for the determination of coenzyme A without hydrolysis has been developed and examined; its usefulness for assay of pure samples has been discussed.

Studies in histochemistry. XXXIII. Distribution of phenolsulfatase in the adrenal of various species. A correction

Errors in a previously published report on the quantitative distribution of phenolsulfatase in the adrenal of the rat, rabbit, hog, and cow have been corrected. High activities which had been thought characteristic of the medulla in these species were shown to be low or negligible when corrections were made for color-contributing substances in this region of the adrenal. Most of the activity was found in the fascicular and reticular zones of the rat, rabbit, and hog adrenals but no activity was demonstrated in any region of the cow adrenal. The adrenals of the monkey and dog were also studied, and the enzyme activities in the various histological zones of the monkey adrenal were found to be negligible. In the dog organ relatively high activities were observed in the fascicular region, and presumably also in the medulla although the latter is less sure. An improved procedure for the measurement of phenolsulfatase activity in microgram quantities of tissue has been given.

Flame photometric determination of potassium in microgram quantities of tissue and the distribution of potassium and lipid in the adrenal of the monkey and guinea pig. Studies in histochemistry. XXXII.

A flame photometric method for the determination of potassium in microgram quantities of tissue was described. A simple water extraction of the potassium from microtome sections of adrenal tissue was shown to be preferable to either digestion or dry-ashing in the preparation of the sample for analysis. The quantitative histological distributions of total dry matter, total lipid, and potassium in the adrenal glands of M. rhesus and cynomolgus monkeys and guinea pigs were determined. No significant strain or species differences were observed among the data collected. The total lipid in the fascicular zone reached maximum concentrations of 55-70% of the total dry-weight. The potassium concentration remained within the approximate range of 1.4-1.7% of the fat-free dry-weight throughout all of the regions of the adrenals of the animals used.

Studies in histochemistry: XXVII. The determination of L-ascorbic acid, and dehydro-L-ascorbic acid plus diketo-L-gulonic acid in microgram quantities of tissue.

The determination of l-ascorbic, and dehydro-l-ascorbic plus diketo-l-gulonic acids in microgram quantities of tissue was described. The procedure used is an adaptation to the micro scale of macro methods based on spectrophotometric measurements of reactions involving 2,6-dichlorophenol indophenol and 2,4-dinitrophenylhydrazine, and it is well suited to serial analyses on large numbers of samples because of its relatively great simplicity and speed. The standard deviation in the ascorbic acid method is about 2% corresponding to about 3 mµg. of ascorbic acid for the equipment used. With pipettes and cuvettes of smaller capacity a reduction in the latter value by about one-third can be obtained without inconvenience.

Determination of beta-glucuronidase in microgram quantities of tissue and its distribution in the cow adrenal.

The procedure of Talalay, Fishman and Huggins was adapted to the determination of β-glucuronidase activity in microgram quantities of tissue. The quantitative histological distribution of β-glucuronidase activity in the cow adrenal was studied, and it was found that the activity is high in the glomerulosa and outer fasciculata but comparatively low in other regions.

Determination of Cholesterol in Microgram Quantities of Tissue

Determination of Cholesterol in Microgram Quantities of Tissue

Studies in histochemistry. XXV. Determination of phenolsulfatase in microgram quantities of tissue and its distribution in the adrenal of several species

1. 1. A quantitative method for the determination of phenolsulfatase activity in microgram quantities of tissue has been described. 2. 2. The quantitative distribution of this enzyme in the adrenal gland of hog, cow, rat, and rabbit was determined. The same general distribution was found in all four species. In the cortex, the highest activity was demonstrated in the fascicular zone. The concentration in the medulla was the same as, or higher than, that in the fasciculata. 3. 3. Per unit dry weight, the rat adrenal had about twice the activity of the glands of the other species, which showed activities that varied little from one another.