Karyoskeletal protein-enriched fraction

Isolation and Characterization of Karyoskeletal Protein-Enriched Fractions from Vertebrate Livers... 

Many methods have been developed for the isolation and subfractionation of vertebrate liver nuclei to produce relatively insoluble karyoskeletal protein-enriched fractions [for reviews of the early literature, please see Franke (1974), Harris and Agutter (1976), Fry (1977), Harris (1977, 1978), and Agutter and Richardson (1980)]. Morphologically, these fractions contain nuclear pore complexes attached to the lamina (Aaronson and Blobel, 1975 Dwyer and Blobel, 1976) and, in several instances, a meshwork of filaments apparently derived from the nuclear interior (see, e.g., P. A. Fisher et al, 1982). This chapter describes... 

Fig. 6 Nuclease treatment and subfractionation of nuclei isolated from vertebrate livers. Flow chart showing nuclease digestion of isolated nuclei as well as fractionation steps leading to a karyoskeletal protein-enriched fraction.

Preparation of D. melanogaster Karyoskeletal Protein-Enriched Fractions... 

Unless otherwise noted, all procedures should be performed at 4°C or on ice. Nuclear purification is the first step in preparation of karyoskeletal protein-enriched fractions from Drosophila cells and tissues. Two techniques have been used extensively in our laboratory. The first, as detailed previously in Fisher et al. (1982), is particularly useful for preparation of undegraded karyoskeletal proteins from embryos without regard for their biological activity. 

Fig. 2 Transmission electron micrograph of karyoskeletal protein-enriched fraction prepared from Drosophila embryos. The main panel shows a nucleuslike structure, bounded by a peripheral lamina (L) Inset Higher magnification showing tangential section through the periphery of karyoskeletal protein structure as shown in the main panel. Nuclear pore complex remnants can be readily appreciated as small ringlike structures.

Fig. 3 SDS-gradient PAGE and Coomassie blue staining. Lane 1, Final karyoskeletal protein-enriched fraction after nuclease digestion at 37°C lane 2,1 Af NaCl extract after nuclease digestion at 23°C. Migration positions of various Drosophila marker proteins are indicated to the right of the figure.

Figure 2 is a transmission electron micrograph of a nuclease-treated, Triton X-lOO-extracted, twice NaCl-extracted karyoskeletal protein-enriched fraction derived from Drosophila embryos. Nuclease treatment was at 37°C. Identifiable karyoskeletal elements are labeled. The SDS-PAGE profiles of the final karyoskeletal protein-enriched pellet fraction generated after subfractionation of nuclei treated with nucleases at 37°C are shown in Fig. 3 (lane 1) as well as the first 1 M NaCl extract generated after subfractionation of nuclei treated with nucleases at 23°C (also highly enriched for Drosophila karyoskeletal proteins but in soluble form) (lane 2). 

Fractions are as follows 1, filtered crude homogenate 2, postnuclear supernatant 3, supernatant from the first wash of the nuclei 4, supernatant from the second wash of the nuclei 5, resuspended nuclei 6, resuspended nuclei after treatment with nucleases 7, supernatant from resupended nuclei after treatment with nucleases and centrifugation 8, supernatant from nuclease-treated nuclear pellet after extraction with Triton X-lOO and centrifugation 9, supernatant from nuclease-treated, Triton X-lOO-treated nuclear pellet after extraction with NaCI and centrifugation 10, supernatant from nuclease-treated, Triton X-lOO-treated, NaCl-treated nuclear pellet after repeat extraction with NaO and centrifugation 11, final karyoskeletal protein-enriched fraction after purified nuclei were subjected to nuclease treatment, Triton X-100 treatment, and two NaCI treatments, all performed sequentially. 

Numbers in parentheses indicate the relative percent compositions of each of the final karyoskeletal protein-enriched fraction components. 

Berrios, S., and Fisher, P. A. (1988). Thermal stabilization of putative karyoskeletal protein-enriched fractions from Saccharomyces cerevisiae. Mol. Cell BioL 8,4573-4575. 

Fisher, P. A., Lin, L., McConneU, M., Greenleaf, A., Lee, J.-M., and Smith, D. E. (1989). Heat shock-induced appearance of RNA polymerase II in karyoskeletal protein-enriched (nuclear matrix ) fractions correlates with transcriptional shutdown in Drosophila melanogaster. J. Biol. Chem. 264, 3464-3469,... 

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