Christine Ko

Electrophoretic characterization of shuttling and nonshuttling small nuclear RNAs

Abstract snRNAs that shuttle nonrandomly between nucleus and cytoplasm, and snRNAs that do not leave the interphase nucleus have been characterized by transplanting 32 P-RNA nuclei into unlabeled amebae followed by electrophoresis of RNA extracted from grafted nuclei, host cell nuclei, and cytoplasms. Amebae were found to possess snRNAs designated: 3.5S, 4S, 5S, 5.5S, U1, U2, U3, and 8S, which range from about 80 to about 200 nucleotides long. Because they migrate from grafted to host nucleus, 5S, U1, U3, and 8S are recognized as the shuttling RNAs. The 3.5S, 5.5S, and U2 RNAs, which do not migrate, clearly are nonshuttling. The nuclear and cytoplasmic concentrations of 4S RNA are approximately equal, presumably reflecting a lack of a nuclear envelope barrier to its free intracellular movement. 5S RNA is about 250 times and 8S RNA about 750 times more concentrated in nucleus than cytoplasm, and the other snRNAs are substantially greater than 750 times more concentrated in the nucleus. The shuttling RNAs thus migrate into the nucleus from the cytoplasm against steep concentration gradients. The shuttling 5S RNA appears to be unrelated to ribosomal 5S RNA, although they are electrophoretically indistinguishable. In the presence of actinomycin D the shuttling RNAs continue to shuttle but nonshuttling and shuttling snRNAs appear in the cytoplasm in significant amounts, suggesting that some snRNAs at least are normally bound to chromatin. This finding also demonstrates that the observed shuttling behavior does not reflect reutilization, in new transcription events, of labeled RNA breakdown products. How snRNAs may be related to gene function is considered briefly.

Chapter 12 A Method for the Mass Culturing of Large Free-Living Amebas

Publisher Summary This chapter presents a method for mass culturing of large free-living amebas. The cultures are grown in Pyrex glass-baking dishes with inside dimensions of 33 × 21 × 4.5 cm; on the bottom of each dish is a plastic platform (30 × 20 cm) on small plastic feet of 2–3 mm high. This platform provides an added surface area on which the amebas can grow. Each dish normally contains ameba medium to a depth of 1–2 cm. The ameba medium is made up of 50 mg CaHPO4, 60 mg KC1, 40 mg MgSO4, and 1 liter glass-distilled H2O. Each culture dish normally contains 0.5–1.5 × 106 amebas. All estimations of ameba number are based on the finding that 4–5 × 105 living amebas pack into a volume of 1 ml when centrifuged for 1 minute at 200 g. The method described yields 20–25 × 106 amebas (or 40–50 ml packed cells) per week. Given sufficient resources, the system can be scaled up to provide considerably greater yields if desired.

Nuclear actin: an apparent association with condensed chromatin.

In Amoeba proteus the concentration of actin is the same in the nucleus and cytoplasm. Certain characteristics, e.g., the ready loss of actin from isolated nuclei, suggest that the association with nuclei is normally not a tight one. Here we report, however, that when nuclei are isolated several hours after mitotic amebas are placed in actinomycin D (which allows normal progression of mitosis), the nuclei retain substantial amounts of actin. Since this finding correlates with other observations that the chromatin of such cells is extensively condensed, we suggest that a relationship may exist between actin and chromatin condensation.

The characteristics of shuttling RNAs confirmed

Shuttling RNAs are recognized as molecules that migrate against steep concentration gradients from one nucleus (through the cytoplasm) into another nucleus in the same cell. In previous work these molecules were identified through experiments involving the separation of two kinds of nuclei utilizing differences in the nuclei that may have produced misleading results. In the experiments reported here normal, randomly-chosen ameba (A. proteus) nuclei containing [32P]RNA were implanted into unlabeled normal, randomly-chosen cells and, after suitable incubation, the labeled RNAs present in each kind of nucleus were characterized by gel electrophoresis. The previously obtained results were confirmed: i.e. (a) the recipient cell nuclei acquired the same four small, distinct RNAs, which are recognized as shuttling ones because they migrate from one nucleus to the other; (b) the grafted nuclei possess, in addition to the four shuttling RNAs, three small, distinct RNAs, which are recognized as non-shuttling RNAs. New evidence also is presented to show that the acquisition by a nucleus of labeled RNAs in the above kind of experiment is not a result of new synthesis of RNAs from the labeled turnover products emanating from the transplanted nucleus.

Identification of the small nuclear RNAs associated with the mitotic chromosomes of Amoeba proteus

Amebas contain 7 electrophoretically distinct species of small nuclear RNAs (snRNAs), some of which are known to associate in a striking manner with mitotic chromosomes. These RNAs can be divided into 2 classes, one consisting of 4 snRNA species that shuttle in a non-random way between nucleus and cytoplasm during interphase and one consisting of 3 snRNA species that do not leave the nucleus at all during interphase. In the work reported here we sought to determine which class is associated with mitotic chromosomes. Through a series of micromanipulative procedures we arranged for the shuttling snRNAs to be the only radioactive molecules in the cell. Such cells were allowed to enter mitosis, whereupon they were fixed and subjected to autoradiography. In those cells no radioactive snRNAs were found associated with mitotic chromosomes. It is concluded, therefore, that those snRNAs that do associate with mitotic chromosomes must be one or more of the non-shuttling species. — In the Discussion, how the non-shuttling snRNAs may function in cell activities is considered.

Manipulation of the amount of RNA in post-mitotic nuclei.

Abstract Because all (or almost all) nuclear RNAs are liberated to the cytoplasm during mitosis and then return to the post-mitotic nuclei, we expected that if cytoplasm were amputated from mitotic cells the post-division nuclei would possess less than normal amounts of RNA. Experiments performed with amebae ( A. proteus ) show that this is in fact what happens. Furthermore, since the enucleate fragment cut from a mitotic cell possesses an “excess” of returnable nuclear RNAs, a normal interphase nucleus implanted into such mitotic cytoplasm might be expected to acquire above-normal amounts of RNA. Experiments reported here show that this expectation also is realized. Thus, the regulation of the normal nuclear concentration of these RNAs involves mechanisms other than a limited number of intranuclear “binding” sites and most likely is restricted by the rate of synthesis of these RNAs. The demonstration that nuclei can be depleted or enriched for RNAs, many of which are unique to nuclei, makes it possible to determine the consequences for cell metabolism of altered amounts of nuclear RNA. Hopefully, such studies will reveal the function(s) of these RNAs.