Chromosomal Acid Research Articles

PCR-based detection of nucleic acids in chromosomes, cells, and tissues: technical considerations on PRINS and in situ PCR and comparison with in situ hybridization.

The polymerase chain reaction (PCR) is an extremely sensitive technique allowing the detection of rare and low copy nucleic acid sequences (up to 1–10 copies in DNA or mRNA extracts from 1 million cells) by solution-phase amplification using specific primer sets and Taq DNA polymerase and visualization of the resulting PCR products by gel electrophoresis and blotting techniques (see chapters in this book). However, the obligatory cell and tissue destruction required for nucleic acid extraction does not permit the correlation of results with histopathological features or the localization of targets in specific cell types. This may now be overcome by combining recently developed micromanipulation systems, such as laser-assisted microdissection, to isolate the cells of interest (up to the level of a single cell) from a large population of cells or from the tissue, and to apply single-cell PCR on the extracted nucleic acids (for a review, see ref. ). Alternatively, and in case it is unknown in which cell a certain target nucleic acid, for example, a virus particle, may be present, cellular localization of DNA and RNA can be accomplished by in situ hybridization (ISH). This procedure has a history of more than 30 years and has been improved continuously. In particular, the development of nonradioactive approaches and the recent implementation of tyramide signal amplification have made ISH a powerful technique for use in many applications (). Some 10 to 15 years ago, however, ISH detection limits were only in the range of 10 to 20 copies of mRNA or viral DNA per cell, and probe detection periods could be very long when using radioactive procedures (, , ).

Consequences of phosphate imbalance.

Phosphorus is the sixth most abundant element in the body after oxygen, hydrogen, carbon, nitrogen, and calcium. It comprises about 1% of the total body weight of humans. Eighty-five percent of it is stored in the bone in the form of hydroxyapatite crystal; 14% is in the soft tissues in the form of energy-storing bonds with nucleotides (ATP, GTP), nucleic acids in chromosomes and ribosomes, 2,3-DPG in the red blood cells, and phospholipids in the cells' membranes. Less than 1% is in the extracellular fluids. Phosphate balance is maintained by multiple systems. The gut is responsible for the absorption of two thirds of the 4-30 mg/kg/day of phosphate intake. Absorption sites are all along the gut; in humans the most active site is the jejunum. The kidney filters 90% of the plasma phosphate and reabsorbs it in the tubuli. In states of hypophosphatemia the kidney can reabsorb the filtered phosphates very efficiently, reducing the amount excreted in the urine virtually to zero. The healthy kidney can excrete high loads of phosphate and rid the body of phosphate overload. Through the vitamin D-PTH axis the endocrine system regulates the phosphate balance by influencing the kidney, gut, and bone. Other hormones, including thyroid, insulin, glucagon, glucocorticosteroid, and thyrocalcitonin, play a lesser role in regulation of phosphate metabolism. Because of the complex control of phosphate homeostasis, various clinical conditions may lead to hypophosphatemia. These include nutritional repletion, gastrointestinal malabsorption, use of phosphate binders, starvation, diabetes mellitus, and increased urinary losses due to tubular dysfunction. The clinical picture of phosphate depletion is manifested in different organs and is due mainly to the fall in intracellular levels of ATP and decreased availability of oxygen to the tissues, secondary to 2,3-DPG depletion. The various manifestations of phosphate depletion are listed in Table 2. The treatment of hypophosphatemia consists of administering enteral or parenteral phosphate salts. An important aspect of dealing with the potentially serious effects of phosphate depletion is to prevent the depletion from happening in the first place. Hyperphosphatemia can occur in renal failure, hemolysis, tumor lysis syndrome, and rhabdomyolysis. The treatment of hyperphosphatemia usually consists of fluid administration (in the absence of kidney failure). In chronic hyperphosphatemia, phosphate binders such as aluminum and magnesium salts can reduce the phosphate load. The use of these phosphate binders is limited by their potential side effects.

Nucleolar-derived ribonucleic acid in chromosomes, nuclear sap, and cytoplasm of Chironomus tentans salivary gland cells.

The distribution of monodisperse high molecular weight RNA (38, 30, 28, 23, and 18S RNA) was studied in the salivary gland cells of Chironomus tentans. RNA labeled in vitro and in vivo with tritiated cytidine and uridine was isolated from microdissected nucleoli, chromosomes, nuclear sap, and cytoplasm and analyzed by electrophoresis on agarose-acrylamide composite gels. As shown earlier, the nucleoli contain labeled 38, 30, and 23S RNA. In the chromosomes, labeled 18S RNA was found in addition to the 30 and 23S RNA previously reported. The nuclear sap contains labeled 30 and 18S RNA, and the cytoplasm labeled 28 and 18S RNA. On the basis of the present and earlier analyses, it was concluded that the chromosomal monodisperse high molecular weight RNA fractions (a) show a genuine chromosomal localization and are not due to unspecific contamination, (b) are not artefacts caused by in vitro conditions, but are present also in vivo, and (c) are very likely related to nucleolar and cytoplasmic (pre)ribosomal RNA. The 30 and 23S RNA components are likely to be precursors to 28 and 18S ribosomal RNA. The order of appearance of the monodisperse high molecular weight RNA fractions in the nucleus is in turn and order: (a) nucleolus, (b) chromosomes, and (c) nuclear sap. Since both 23 and 18S RNA are present in the chromosomes, the conversion to 18S RNA may take place there. On the other hand, 30S RNA is only found in the nucleus while 28S RNA can only be detected in the cytoplasm, suggesting that this conversion takes place in connection with the exit of the molecule from the nucleus.

Effect of colchicine on the utilization of thymidine labelled with tritium during chromosomal reproduction.

EXPERIMENTS1 designed to verify the findings of Taylor, Woods and Hughes2 on the incorporation of thymidine labelled with tritium in the deoxyribonucleic acid in chromosomes of growing root meristems of seedlings of Vicia faba have produced evidence that colchicine influences either the synthesis of the acid or its subsequent distribution among sister chromatids at metaphase. This evidence was derived from two types of experiment.