Combining Multicolor FISH with Fluorescence Lifetime Imaging for Chromosomal Identification and Chromosomal Sub Structure Investigation

Understanding the structure of chromatin in chromosomes during normal and diseased state of cells is still one of the key challenges in structural biology. Using DAPI staining alone together with Fluorescence lifetime imaging (FLIM), the environment of chromatin in chromosomes can be explored. Fluorescence lifetime can be used to probe the environment of a fluorophore such as energy transfer, pH and viscosity. Multicolor FISH (M-FISH) is a technique that allows individual chromosome identification, classification as well as assessment of the entire genome. Here we describe a combined approach using DAPI as a DNA environment sensor together with FLIM and M-FISH to understand the nanometer structure of all 46 chromosomes in the nucleus covering the entire human genome at the single cell level. Upon DAPI binding to DNA minor groove followed by fluorescence lifetime measurement and imaging by multiphoton excitation, structural differences in the chromosomes can be studied and observed. This manuscript provides a blow by blow account of the protocol required to perform M-FISH-FLIM of whole chromosomes.

The value of the larval head pattern for differentiating Simulium exiguum s.l. and S. gonzalezi (Diptera: Simuliidae) in the onchocerciasis focus of Ecuador

In the Esmeraldas focus of onchocerciasis in Ecuador, the larvae of the primary vector Simulium exiguum Roubaud are found in sympatry with the non-vector S. gonzalezi Vargas & Díaz Nájera. Distinguishing the pre-ultimate stage larvae of these two species usually relies on the pattern of markings on the cephalic apotome. These patterns appear either as dark spots against a pale cuticle (positive head pattern) or as pale spots within a darkened area of the cuticle (negative head pattern). Larvae of S. exiguum s.l. usually exhibit positive or no head patterns whilst those of S. gonzalezi usually have negative head patterns. The purpose of this paper was to investigate head pattern variation (scored in one of six categories) in (polytene) chromosomally identified larvae of the two species and cytotypes of S. exiguum from three collection sites in Ecuador (including one in the onchocerciasis focus). The head patterns of a total of 1183 larvae were studied. Chromosomal identification of penultimate instar larvae revealed only the presence of the Cayapa and Quevedo cytotypes of S. exiguum (n=208). Both cytotypes exhibited a wide range of head patterns from positive to negative. The results indicate that negative head patterns cannot be used to reliably distinguish between larvae of S. exiguum s.l. and S. gonzalezi. All except one larva with negative head patterns were females (n=53), suggesting that head pattern in this species complex might be a sex-linked character.

THE CHROMOSOMAL BASIS OF VIABILITY IN INTERSPECIFIC HYBRIDS BETWEEN DROSOPHILA ARIZONENSIS AND DROSOPHILA MOJAVENSIS

The chromosomal effects on differences in viability among progeny from interspecific crosses was studied in the interfertile pair Drosophila arizonensis and Drosophila mojavensis. Interspecific crossing-over was avoided by crossing hybrid males to pure-species females, and chromosomal identification in backcross progeny was possible by means of electrophoretic markers. The main findings are as follows. One chromosome supresses viability when in the heterospecific state, this being mainly so when the rest of the genotype is predominantly of mojavensis type; the other chromosomes show occasional interspecific heterosis, but are neutral in the majority of cases; interactions are not significant, except in one pair of chromosomes within a mojavensis background; there is no correlation between numbers of heterospecific chromosomes and viability scores. It is concluded that hybrid and backcross progeny inviability is not a very potent mechanism for keeping these species apart. Should etiological and ecological barriers break down, introgressive hybridization is a real possibility. The findings are compared with those from studies concerning hybrid sterility and mating behavior in these two species. It is concluded that there is no common chromosomal basis for these phenomena. In particular, the role of interspecific inversions to speciation remains obscure.