THE GRANA OF STARCH-FREE CHLOROPLASTS OF NICOTIANA RUSTICA

The grana of chloroplasts of starch-free leaves of Nicotiana rustica are described in detail. Leaf sections were fixed in 2.5 per cent KMnO₄ and embedded in mixtures of butyl and ethyl methacrylate. Chain length of the polymer was modified by use of a transfer agent. The grana are composed of compartments consisting of electron-scattering partitions and electron-transparent loculi. Compartments are not open to the stroma so that the grana are distinct subplastid organelles. Adjacent grana are connected by an anastomosing fretwork system composed of flexuous channels bordered by electron-scattering membranes. Ten different kinds of granum margins are described. These marginal variations depend upon grana-fretwork connections and internal marginal connections between adjacent loculi. A study of serial sections suggests, at least in some plastids, the occurrence of a possible orderly spiral arrangement of compartment-fretwork connections. Adjacent grana may have common compartments. Grana may branch. Variations in shape may depend upon the angle the section bears to the axis of the cylinder. This should also influence the relative thickness and sharpness of the partitions. Since all shapes and variations in partition thickness and sharpness cannot be accounted for on the basis of the orientation of the grana, such variations probably occur naturally. Grana vary in size, ranging from those few which have a single partition to those having 50 or more compartments which extend completely through the width of a plastid. Relationships between grana of different sizes and between compartments and frets indicate the possibility of growth of grana from union or extension of compartments and formation of compartments from the union of frets.     

A degenerate alpha satellite probe, detecting a centromeric deletion on chromosome 21 in an apparently normal human male, shows limitations of the use of satellite DNA probes for interphase ploidy analysis.

A degenerate alpha satellite DNA probe specific for a repeated sequence on human chromosomes 13 and 21 was synthesized using the polymerase chain reaction (PCR). Fluorescence in situ hybridization (FISH) with this probe to normal metaphase spreads revealed strong probe binding to the centromeric regions of human chromosomes 13 and 21 with negligible cross-hybridization with other chromosomes. FISH to normal interphase cell nuclei showed four distinct domains of probe binding. However, hybridization with probe to interphase and metaphase preparations from one apparently normal human male resulted in only three major binding domains. Metaphase chromosome analysis revealed a centromeric deletion on one chromosome 21 that caused greatly reduced probe binding. The result suggest caution in the interpretation of interphase ploidy studies performed with chromosome-specific alphoid DNA probes.     

Improved gold chloride staining method for anatomical analysis of sensory nerve endings in the shoulder capsule and labrum as examples of loose and dense fibrous tissues

Consistency in gold chloride staining is essential for anatomical analysis of sensory nerve endings. The gold chloride stain for this purpose has been modified by many investigators, but often yields inconsistent staining, which makes it difficult to differentiate structures and to determine nerve ending distribution in large tissue samples. We introduce additional steps and major changes to the modified Gairns’ protocol. We controlled the temperature and mixing rate during tissue staining to achieve consistent staining and complete solution penetration. We subjected samples to sucrose dehydration to improve cutting efficiency. We then exposed samples to a solution containing lemon juice, formic acid and paraformaldehyde to produce optimal tissue transparency with minimal tissue deformity. We extended the time for gold chloride impregnation 1.5 fold. Gold chloride was reduced in the labrum using 25% formic acid in water for 18 h and in the capsule using 25% formic acid in citrate phosphate buffer for 2 h. Citrate binds gold nanoparticles, which minimizes aggregation in the tissue. We stored samples in fresh ultrapure water at 4° C to slow reduction and to maintain color contrast in the tissue. Tissue samples were embedded in Tissue Tek and sectioned at 80 and 100 μm instead of using glycerin and teasing the tissue apart as in Gairns’ modified gold chloride method. We attached sections directly to gelatin subbed slides after sectioning with a cryostat. The slides then were processed and coverslipped with Permount. Staining consistency was demonstrated throughout the tissue sections and neural structures were clearly identifiable.     

Molecular cytogenetic characterization of chromosome 9-derived material in a human thyroid cancer cell line

The incidence of papillary thyroid carcinoma (PTC) increases significantly after exposure of the head and neck region to ionizing radiation, yet we know neither the steps involved in malignant transformation of thyroid epithelium nor the specific carcinogenic mode of action of radiation. Such increased tumor frequency became most evident in children after the 1986 nuclear accident in Chernobyl, Ukraine. In the eight years following the accident, the average incidence of childhood PTCs (chPTC) increased 70-fold in Belarus, 200-fold in Gomel, 10-fold in the Ukraine and 50-fold in Tschnigov, Kiev, Rovno, Shitomyr and Tscherkassy compared to the rate of about 1 tumor incidence per 106 children per year prior to 1986 (Likhtarev et al., 1995; Sobolev et al., 1997; Jacob et al., 1998). To study the etiology of radiation-induced thyroid cancer, we formed an international consortium to investigate chromosomal changes and altered gene expression in cases of post-Chernobyl chPTC. Our approach is based on karyotyping of primary cultures established from chPTC specimens, establishment of cell lines and studies of genotype-phenotype relationships through high resolution chromosome analysis, DNA/cDNA micro-array studies, and mouse xenografts that test for tumorigenicity. Here, we report the application of fluorescence in situ hybridization (FISH)-based techniques for the molecular cytogenetic characterization of a highly tumorigenic chPTC cell line, S48TK, and its subclones. Using chromosome 9 rearrangements as an example, we describe a new approach termed 'BAC-FISH' to rapidly delineate chromosomal breakpoints, an important step towards a better understanding of the formation of translocations and their functional consequences.     

Rejecting strictly allopatric speciation on a continental island: prolonged postdivergence gene flow between Taiwan (Leucodioptron taewanus,Passeriformes Timaliidae) and Chinese (L. canorum canorum) hwameis

Allopatry is conventionally considered the geographical mode of speciation for continental island organisms. However, strictly allopatric speciation models that assume the lack of postdivergence gene flow seem oversimplified given the recurrence of land bridges during glacial periods since the late Pliocene. Here, to evaluate whether a continental island endemic, the Taiwan hwamei (Leucodioptron taewanus, Passeriformes Timaliidae) speciated in strict allopatry, we used weighted‐regression‐based approximate Bayesian computation (ABC) to analyse the genetic polymorphism of 18 neutral nuclear loci (total length: 8500 bp) in Taiwan hwamei and its continental sister species, the Chinese hwamei (L. canorum canorum). The nonallopatry model was found to fit better with observed genetic polymorphism of the two hwamei species (posterior possibility = 0.82). We also recovered unambiguous signals of nontrivial bidirectional postdivergence gene flow (N_em » 1) between Chinese hwamei and Taiwan hwamei until 0.5 Ma. Divergence time was estimated to be 3.5 to 2 million years earlier than that estimated from mitochondrial cytochrome b sequences. Finally, using the inferred nonallopatry model to simulate genetic variation at 24 nuclear genes examined showed that the adiponectin receptor 1 gene may be under divergent adaptation. Our findings imply that the role of geographical barrier may be less prominent for the speciation of continental island endemics, and suggest a shift in speciation studies from simply correlating geographical barrier and genetic divergence to examining factors that facilitate and maintain divergence, e.g. differential selection and sexual selection, especially in the face of interpopulation gene flow.