Specific mammalian genes functionally and dynamically associate together within the nucleus. Yet, how an array of many genes along the chromosome sequence can be spatially organized and folded together is unknown. We investigated the 3D structure of a well-annotated, highly conserved 4.3-Mb region on mouse chromosome 14 that contains four clusters of genes separated by gene “deserts.” In nuclei, this region forms multiple, nonrandom “higher order” structures. These structures are based on the gene distribution pattern in primary sequence and are marked by preferential associations among multiple gene clusters. Associating gene clusters represent expressed chromatin, but their aggregation is not simply dependent on ongoing transcription. In chromosomes with aggregated gene clusters, gene deserts preferentially align with the nuclear periphery, providing evidence for chromosomal region architecture by specific associations with functional nuclear domains. Together, these data suggest dynamic, probabilistic 3D folding states for a contiguous megabase-scale chromosomal region, supporting the diverse activities of multiple genes and their conserved primary sequence organization.
Fluorescence emission after two-photon absorption of coumarins and xanthenes in an alcoholic solution was measured in the tuning range of a femtosecond-pulsed titanium-sapphire laser (750-840 nm). Xanthenes, which have a low one-photon absorption in the near UV, show a higher fluorescence signal after two-photon absorption than the UV-excitable coumarins. When fluxes of 10(28) photons/(cm(2) s) are used, the two-photon absorption cross sections for xanthenes are 1 order of magnitude higher than the two-photon absorption cross sections of the coumarins. Absolute cross sections have been estimated for three coumarins and three xanthenes. For the xanthenes a significant wavelength-dependent departure from the expected fluorescence intensity square law was observed. The coumarins follow the square-law dependence. The consequences of the findings are discussed for analytic and diagnostic methods. An especially important result is that the resolution in two-photon microscopy of xanthenes is worse than expected.
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