Practicable approaches to facilitate rapid and accurate molecular cytogenetic mapping in birds and mammals.

Molecular cytogenetic mapping by FISH is a common feature of most genome projects as it provides a global, low-resolution overview of the genome and facilitates comparative genomics. An essential prerequisite for cytogenetic mapping is the ability to identify accurately the chromosome on which the clone (e.g. BAC) resides. This is not usually a barrier to human mapping as knowledge of the human karyotype is commonplace. For other species however accurate assignment can be problematic either because, as in birds, the karyotype is too complex to analyze by standard means or because of the paucity of individuals skilled to perform the karyotyping. Using chicken as a model we have developed a reproducible approach for accurate cytogenetic mapping that involves: a single colour FISH, measurement of the ratio of the size of the signal bearing chromosome to that of chromosome 8, and final assignment through a small series of dual colour experiments. Reference values for size ratios were established using base pair estimate information from the Ensembl browser. By this method cytogenetic mapping to highly complex karyotypes can be achieved in a small number of simple steps. We have also developed and tested a karyotyping tutorial programme adapted from one previously reported in this journal. That is, we have used pig as an example of a model species with a relatively tractable karyotype and demonstrated that scientists and students, even after only one hour using our tutorial, can readily identify pig chromosomes and thus make appropriate assignments using FISH. Simple, practicable means often provide preferable solutions than complex alternatives (e.g. m-FISH) to the solution of scientific problems. Such is the case for the approaches described here.

Subcellular fractionation of liver organelles from phenobarbital-treated rats by counter-current partition and sucrose gradient centrifugation.

Counter-current partition, sucrose density gradient centrifugation and enzymic analysis were used to explore the changes in rat liver organelles induced by phenobarbital. There was a small increase in partition coefficient and marker enzyme activity of the endoplasmic reticulum. The modal density of the marker enzyme showed a significant decrease reflecting the proliferation of the smooth endoplasmic reticulum. The mitochondria showed a bimodal distribution with a small reduction of marker enzyme activity. In contrast, lysosomes and peroxisomes were relatively unaffected by phenobarbital treatment. Phenobarbital caused a small but statistically insignificant increase in gamma-glutamyl transferase activity: density gradient centrifugation studies indicated that the increased activity was predominantly in the biliary canalicular elements. In contrast, cytosolic gamma-glutamyl hydrolase activity was strikingly reduced by phenobarbital treatment.

Subcellular distribution of rat pituitary homogenates by poly(ethylene glycol)-dextran countercurrent partitioning.

Rat pituitary homogenates were subjected to two phase countercurrent partition in a poly(ethylene glycol)-dextran mixture using a simple apparatus with enhanced gravity to facilitate the phase separations. Assay of the fractions for organelle marker enzymes and prolactin after 17 transfers showed similar distributions for endoplasmic reticulum, lysosomes, prolactin granules and plasma membrane at the lowest dextran concentrations. Increasing the dextran concentrations had a differential effect on the various organelles. Excellent resolution of endoplasmic reticulum from the other organelles was obtained and marked organelle heterogeneity was demonstrated. Two-phase countercurrent partition thus offers an alternative approach to the subcellular fractionation of pituitary homogenates and should prove useful in separating endoplasmic reticulum from plasma membrane and other cell components.

Subcellular fractionation of rat liver homogenates using two-polymer phase systems in a toroidal-coil centrifuge.

The principal organelles of rat liver homogenates were fractionated by two-phase partition chromatography using toroidal-coil centrifugation with a mixture of dextran T 500 and poly(ethylene glycol) 6000 in 0.26 M-sucrose containing 10 mM-sodium phosphate/phosphoric acid buffer, pH 7.4. The effects of varying the following parameters on organelle elution profiles, as reflected by their marker-enzyme activities, were studied: centrifuge speed; the composition and relative proportion of dextran-rich and poly(ethylene glycol)-rich phases in the eluent; flow rate; sample volume; homogenate concentration; helix diameter; tubing bore and the number of loops in the coil. Optimal resolution of the organelles was achieved with a toroidal coil of internal diameter 1.07 mm with a 4.55 mm helix diameter on a 0.42 m-diameter rotor running at 1000 rev./min. The eluent was prepared by combining, in a ratio of 93:7 (v/v), the poly(ethylene glycol)-rich upper phase and dextran-rich lower phase obtained from a phase mixture containing 3.3% (w/w) dextran and 5.4% (w/w) poly(ethylene glycol). The flow rate of the eluent was 14ml/h. Optimal conditions for separation of the organelles were evaluated. Resolution of plasma membrane and lysosomes was achieved. Separation of endoplasmic reticulum, which showed marked heterogeneity, from plasma membrane was also demonstrated. DNA and marker enzymes for peroxisomes, mitochondria and cytosol showed distinct elution profiles.

Microanalytical partition of rat-liver homogenates by poly(ethylene glycol)-dextran counter-current distribution.

1. The principal organelles from rat liver homogenates were fractionated by counter-current partition between solutions of dextran T50 and poly(ethylene glycol) 6000 in 0.25 M sucrose containing 10 mM sodium phosphate/phosphoric acid buffer, pH 7.4. 2. A small-volume (1.3-ml sample well)counter-current partition apparatus capable of 17 transfers, in which separation of the phases was enhanced by low-speed centrifugation, was used. 3. Variations in distribution of organelles was observed with increasing concentration of dextran and/or poly(ethylene glycol). At a constant polymer composition of 5.0% (w/w) dextran and 4.0% (w/w) poly(ethylene glycol), increasing concentrations of sodium chloride (2.5 mM, 5 mM and 10 mM) also altered the partition of the organelles. 4. Useful analytical resolution of the various organelles was achieved. In particular, separation of endoplasmic reticulum from plasma membrane components was obtained.