©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cyclin-dependent Kinase Site-regulated Signal-dependent Nuclear Localization of the SWI5 Yeast Transcription Factor in Mammalian Cells (*)

David A. Jans (1) (2)(§), Thomas Moll (3), Kim Nasmyth (4), Patricia Jans (1) (2)

From the  (1)Max-Planck-Institut für Biophysik, Frankfurt am Main, Federal Republic of Germany, the (2)Nuclear Signalling Laboratory, Division for Biochemistry and Molecular Biology, John Curtin School of Medical Research, Canberra City, Australian Capital Territory 2601, Australia, (3)Vienna International Research Cooperation Center, Vienna, Austria, and the (4)Institut für Molekulare Pathologie, Vienna, Austria

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Control over the nuclear transport of transcription factors (TFs) represents a level of gene regulation integral to cellular processes such as differentiation, transformation and signal transduction. The Saccharomyces cerevisiae TF SWI5 is excluded from the nucleus in a cell cycle-dependent fashion, mediated by phosphorylation by the cyclin-dependent kinase (cdk) CDC28. Nuclear entry occurs in G. -galactosidase fusion proteins carrying SWI5 amino acids 633-682, including the nuclear localization sequence (NLS: Lys-Lys-Tyr-Glu-Asn-ValVal-Ile-Lys-Arg-Ser-Pro-Arg-Lys-Arg-Gly-Arg-ProArg-Lys) were analyzed for subcellular localization in appropriate temperature-sensitive yeast strains blocked in G or G/M using indirect immunofluorescence, and for nuclear import kinetics in living rat hepatoma or Vero African green monkey kidney cells microinjected with fluorescently labeled bacterially expressed protein and quantitative confocal laser microscopy. Cell cycle-dependent nuclear localization in yeast was both NLS and cdk site-dependent, whereby mutation of the cdk site serines (Ser and Ser) to alanine resulted in constitutive nuclear localization. In mammalian cells, the SWI5 fusion proteins were similarly transported to the nucleus in an NLS-dependent fashion, while the mutation to Ala of the cdk site serines increased the maximal level of nuclear accumulation from about 1- to over 8-fold. We suggest that phosphorylation at the cdk sites inhibits nuclear transport of SWI5, consistent with our previous observations for the inhibition of SV40 large tumor antigen nuclear transport by phosphorylation by the cdk cdc2. The results indicate for the first time that a yeast NLS and, fascinatingly, its regulatory mechanisms are functional in higher eukaryotes, implying the universal nature of regulatory signals for protein transport to the nucleus.


INTRODUCTION

Precisely scheduled nuclear import of transcription factors (TFs)() is a key factor in the regulation of gene expression and signal transduction. While proteins such as histones appear to be constitutively targeted to the nucleus, others are only translocated to the nucleus under specific conditions, otherwise being cytoplasmic(1, 2, 3) . TFs regulating nuclear gene expression are no different from other proteins in terms of their being synthesized in the cytoplasm and thereby subject to specific mechanisms regulating nuclear protein import. The advantages of a conditionally cytoplasmic location for a TF include the potential to control its activity by regulating its nuclear uptake, and its direct accessibility to cytoplasmic signal-transducing systems. The nuclear translocation of various TFs (2, 4, 5, 6, 7) and oncogene products (8, 9, 10, 11) has been shown to accompany changes in the differentiation or metabolic state of eukaryotic cells precisely, indicating that nuclear protein import is a key control point in the regulation of gene expression and signal transduction. TFs able to undergo inducible nuclear import include the glucocorticoid receptor(12) , the -interferon-regulated factor ISGF-3(13) , the nuclear v-jun oncogenic counterpart of the AP-1 transcription complex member c-jun(14) , the Saccharomyces cerevisiae TF SWI5(6) , the Drosophila melanogaster morphogen dorsal(7) , and NF-B(2, 4, 5) .

Proteins larger than 45 kDa require a nuclear localization sequence (NLS) (1) in order to be targeted to the nucleus. Little is known, however, concerning the regulation of import kinetics, which is likely to be critical in cuing the aforementioned cell cycle transitions, morphogenesis and transformation. In addition to the NLS, specific signals carried by the transported proteins appear to function in a regulatory fashion, whereby covalent modifications such as phosphorylation may play a role(3, 15, 16, 17, 18) . We have demonstrated that the nuclear localization of hybrid proteins in which fragments of the SV40 large T-antigen (T-Ag) are fused to the Escherichia coli -galactosidase enzyme is completely dependent on the presence of the T-Ag NLS (amino acids 126-132)(15, 16) . However, the kinetics of import are markedly enhanced by the presence of the N-terminal sequence (amino acids 111-125) adjacent to the NLS(15, 16) . The rate of nuclear import is regulated by the casein kinase II (CKII) phosphorylation site (Ser)(16, 18) , while phosphorylation at the cyclin-dependent kinase (cdk) cdc2 site (Thr) adjacent to the NLS determines the maximal extent of nuclear accumulation(17) . The CKII and cdk sites, together with the NLS, constitute the CcN motif, responsible for phosphorylation-regulated T-Ag nuclear transport(17) . CKII also appears to enhance nuclear import of the Xenopus laevis nuclear phosphoprotein nucleoplasmin(19) .

The yeast TF SWI5 is involved in mating switch determination through regulating transcription of the HO endonuclease(6) . It exhibits cell cycle-dependent nuclear exclusion, entering the nucleus specifically in G(6, 20) . This nuclear exclusion is effected by phosphorylation by the cdk CDC28, the yeast equivalent of cdc2(6, 20) , where three cdk sites, one of which is within the spacer of the SWI5 bipartite NLS, are proposed to prevent nuclear localization by inactivating NLS function through charge or conformational effects (6) .

We were interested in measuring the kinetics of cdk site-dependent regulation of SWI5 nuclear transport and decided to apply our previous approach of measuring the nuclear uptake of bacterially expressed -galactosidase fusion proteins microinjected into living mammalian cells using confocal laser microscopy(16, 17, 18) . Fusion proteins carrying SWI5 amino acids 633-682, including the NLS (Lys-Lys-Tyr-Glu-Asn-Val-ValIle-Lys-Arg-Ser-Pro-Arg-Lys-Arg-Gly-Arg-Pro-Arg-Lys), were found to be transported to the nucleus in an NLS-dependent fashion, whereby the mutation to Ala of the CDC28 site serines (Ser and Ser) in the vicinity of the SWI5 NLS increased the maximal level of nuclear accumulation from about 1- to over 8-fold. These results were consistent with qualitative findings for subcellular localization of the same SWI5-galactosidase fusion proteins in appropriate yeast strains. The results indicate that a yeast NLS together with its regulatory mechanisms are functional in higher eukaryotes, implying the universal nature of constitutive and regulatory signals for protein transport to the nucleus. A cell cycle-dependent NLS (``CDN''), able to function in diverse eukaryotic cell types, is thus functionally defined here for the first time.


MATERIALS AND METHODS

Chemicals and Reagents

Isopropyl-1-thio--Dgalactopyranoside, -galactosidase (EC 3.2.1.23.37), and polyethylene glycol 1500 were from Boehringer Mannheim, and 5-iodacetamidofluorescein from Molecular Probes. Other reagents were from the sources described previously(16, 17, 18) .

Yeast Strains

The S. cerevisiae strains used were ``wild type'' K1258 (Mata/Mata, can1-100, ura3, ho) and the temperature-sensitive cell cycle mutants K1509 (Mat, cdc13-1, ura3, ho) and K1550 (Mata, cdc28-4, can1-100, ura3, ho)(6, 21) .

Cell Culture

HTC rat hepatoma tissue culture (a derivative of Morris hepatoma 7288C) and Vero (African green monkey kidney) cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum(16, 17, 18) .

SWI5/-Galactosidase Fusion Proteins

To prepare the SWI5--galactosidase fusion proteins, PCR was used to generate a fragment of SWI5 encoding amino acids residues Ser to Leu. The oligonucleotides employed were used to design an initiation (methionine) codon immediately 5` of the SWI5 sequence and to produce an EcoRI-HindIII fragment, the sequence of which was verified by DNA sequencing. The fragment was used to replace the EcoRI-HindIII fragment of YCpGAL/SWI5 (22) and the E. coli -galactosidase gene was inserted as a HindIII fragment from plasmid pMC1871(23) . The SWI5 (633-682: CDC28) and SWI5 (633-682: NLS) -galactosidase fusion proteins are expressed from the GAL1-10 promoter.

E. coli expression plasmids for SWI5--galactosidase fusion proteins, exactly comparable to those for expression in yeast, were derived by inserting the SWI5 sequences as EcoRI (blunt-ended)-HindIII fragments into the polylinker of the T7-expression vector pMWT7 restricted with NdeI (blunt-ended)/HindIII. The lacZ gene from plasmid pMC1871 was inserted C-terminal to the SWI5 sequences as a HindIII fragment. E. coli expression plasmids for fusion proteins in which SV40 T-Ag amino acids 111-135 are fused to the N terminus of E. coli -galactosidase (amino acids 9-1023) have been described(15, 16) .

Immunofluorescence

SWI5--galactosidase fusion proteins were localized in fixed yeast cells as described previously(6, 21) using mouse monoclonal anti--galactosidase antibody, followed by a rhodamine-conjugated goat anti-mouse secondary antibody. Nuclei were visualized by mounting the cells in the DNA-specific dye diamidinophenyl indole (DAPI).

Fusion Protein Expression

1 mM isopropyl-1-thio--Dgalactopyranoside was used in media to induce fusion protein expression. Proteins were purified by affinity chromatography and labeled with 5-iodacetamidofluorescein as described(15) .

Microinjection and Laser Microscopy

HTC cells were fused with polyethylene glycol about 1 h prior to microinjection to produce polykaryons(15) . Microinjection and single-cell microscopic fluorescence measurements on HTC and Vero cells were performed as previously(15, 16, 17, 18) . Quantitation of fluorescence using confocal microscopy and microphotolysis has been described previously in detail (see (24, 25, 26, 27) for applications). Histochemical staining of fixed cells for -galactosidase activity after microinjection was performed as described(15) .


RESULTS AND DISCUSSION

The Cell Cycle-dependent NLS of SWI5 Can Target a Heterologous Protein to the Nucleus in Yeast in an NLS- and cdk Site-dependent Fashion

The bipartite NLS of SWI5 residing within amino acids 633 and 682 (which also includes the cdk/CDC28 sites at Ser and Ser) has been shown previously to be sufficient and necessary to confer cell cycle-dependent nuclear localization on both SWI5 itself and the heterologous E. coli protein -galactosidase in yeast(6) . We were interested in investigating the sequences responsible for this cell cycle-dependent regulation, as well as examining the nuclear import kinetics. We made SWI5--galactosidase fusion protein derivatives (amino acid sequences are shown in Fig. 3) for expression both in yeast under the control of the GAL1-10 promoter, and in bacteria, to enable the examination of nuclear import of microinjected protein in mammalian cells.


Figure 3: Time course of nuclear import of SWI5 -galactosidase fusion proteins in vivo in microinjected HTC cells. Results represent the average of at least 8 single-cell measurements (S.D. not greater than 28% the value of the mean) for Fn/c (fluorescence quantitated in the nucleus, relative to that in the cytoplasm, as determined by fluorescence microphotolysis; see ``Materials and Methods'')(16, 17, 18) . The results for nuclear transport rates determined by curve-fitting (17) are shown in Table 1(see legend).





SWI5 amino acids 633-682 are capable of conferring cell cycle-dependent nuclear localization on the heterologous protein -galactosidase, the protein being nuclear in G (Fig. 1A) and cytoplasmic during the rest of the cell cycle (e.g. in G; see Fig. 1B)(6) . This nuclear localization was dependent on the SWI5 bipartite NLS (amino acids 636-655) (6) since the SWI5-NLS construct exhibited strictly cytoplasmic localization, even in cells arrested in G (Fig. 1A). When the cdk/CDC28 site serines 646 and 664 were replaced by alanine residues, the SWI5--galactosidase protein was constitutively nuclear in contrast to wild type, even in G (Fig. 1B). The CDN of SWI5 could thus be defined as the bipartite NLS together with the cdk sites (Ser/Ser), able to confer cell cycle-dependent nuclear localization on a heterologous protein.


Figure 1: The CDC28-k sites within and C-terminal to the bipartite NLS of SWI5 inhibit NLS-dependent nuclear transport of SWI5--galactosidase fusion proteins. A, cdc28-4 cells expressing the SWI5 -galactosidase constructs as indicated were arrested in G by incubation at the restrictive temperature (36 °C) and the distribution of fusion proteins revealed by staining with a mouse monoclonal anti--galactosidase antibody, followed by a rhodamine-conjugated goat anti-mouse secondary antibody. The leftpanels show rhodamine fluorescence, and the rightpanels the same fields stained with DAPI. B, cdc13-1 cells expressing the SWI5 -galactosidase constructs indicated were treated as in A above to effect arrest in G/mitosis. Left-hand panels show rhodamine fluorescence, and right-handpanels DAPI fluorescence.



The CDN of SWI5 Confers NLS- and cdk Site-dependent Nuclear Transport in Mammalian Cells

In vivo nuclear import kinetics of the fluorescently labeled SWI5 -galactosidase fusion proteins were measured after microinjection in HTC polykaryons or Vero cells ( Fig. 2and 3, Table 1). Results for both cell lines were completely comparable (Fig. 2, and not shown) in that the wild type SWI5 construct was only weakly transported to the nucleus in interphase cells, in contrast to the CDC28 site-mutated derivative, which localized in the nucleus very strongly and rapidly. This nuclear localization was clearly NLS-dependent, since the SWI5-NLS derivative was exclusively cytoplasmic even 2 h after microinjection (Fig. 2).


Figure 2: Visualization of nuclear import of SWI5--galactosidase fusion proteins in vivo in microinjected HTC polykaryons (A) and Vero (B) cells. In the case of panel A, fluorescent visualization is shown 15 (D) and 30 (A and G) min after microinjection. Other panels show histochemical staining for -galactosidase activity, where cells were fixed and stained as described under ``Materials and Methods'' (15) 15 (E), 30 (B and H) and 120 (C, F, and I) min, respectively, after microinjection. In the case of panel B, fluorescent visualization is shown 10 min (A), and staining for -galactosidase activity 15 (B), 30 (C), and 120 (D) min after microinjection, respectively.



Quantitative results (Fig. 3, Table 1) for the SWI5--galactosidase fusion proteins confirmed these observations, whereby the CDC28 site-mutated derivative was accumulated to levels about 8 times that in the cytoplasm within about 40 min after microinjection. As shown in Table 1, these transport properties are comparable to those for T-Ag--galactosidase fusion proteins containing the T-Ag CcN motif (the NLS together with the requisite phosphorylation sites)(15, 16, 17, 18) . Similar results were obtained using the same SWI5 fusion proteins in our mechanically perforated HTC cell in vitro nuclear transport system (see (17) ) (not shown). Nuclear transport kinetics were examined mostly in interphase cells principally for reasons of convenience with respect to both microinjection and HTC cell perforation, but the CDC28 SWI5 fusion protein derivative appeared to be transported constitutively to the nucleus, irrespective of the stage of the cell cycle (not shown).

The cdk/CDC28 sites of the SWI5 CDN thus function to inhibit nuclear entry of SWI5, determining the end point (Fn/c) of nuclear import (see Table 1). This is consistent with our observations concerning cdk/cdc2 site-mediated inhibition of nuclear transport of T-Ag(17) , where phosphorylation adjacent to the T-Ag NLS reduces the maximal extent of nuclear accumulation of T-Ag fusion proteins. The mechanisms of the cdk site-mediated effects appear to be different; whereas cdc2 inhibition of T-Ag transport appears to be through phosphorylation increasing the affinity of T-Ag for a putative cytoplasmic retention factor(17) , the CDC28 sites in SWI5 appear to inhibit nuclear localization by inactivating or masking the function of the NLS (see (6) ). NLS masking by phosphorylation has been reported to regulate nuclear transport of both lamin B2 (inhibited by phosphorylation at two protein kinase C sites N-terminally adjacent to the NLS)(28) , and the actin-binding protein cofilin (whose nuclear translocation upon heat shock is accompanied by dephosphorylation at a multifunctional calmodulin-dependent protein kinase site adjacent to the putative NLS)(29, 30) .

CDN/cdk-mediated Regulation of Nuclear Protein Import

A number of examples of cell cycle-dependent phosphorylation (cdk)-mediated regulation of nuclear entry are known, the best examples being those of T-Ag(17) , SWI5 ( (6) and see above), and the ``retinoblastoma susceptibility factor'' tumor-suppressor gene p110, whose tightness of association with the nucleus (``nuclear tethering'') appears to be reduced by cell cycle-dependent phosphorylation(31, 32) . Table 2lists proteins whose nuclear localization is specifically regulated during the cell cycle or known to be regulated by cdks and/or CDNs. Although their respective activities themselves may be cdk-regulated, kinases other than cdks (e.g. CKII in the case of the Drosophila lodestar protein; see Table 2) (34) also appear to be capable of regulating cell cycle-dependent nuclear entry. CDNs may well be a general mechanism of controlling protein import with respect to the cell cycle, enabling precise timing of nuclear entry as required.



In conclusion, this study shows that the SWI5 CDN can confer NLS- and cdk site-dependent nuclear transport on a heterologous protein in mammalian cells. Importantly, the results show that a bipartite yeast NLS and, fascinatingly, its regulatory mechanisms are functional in higher eukaryotes, implying the universal nature of constitutive and regulatory signals for protein transport to the nucleus (see Refs. 3, 41, and 42). The regulation of nuclear protein import through cell cycle-dependent or other phosphorylation may be as universal as NLSs themselves(3, 17) .


FOOTNOTES

*
This work was supported by the Clive and Vera Ramaciotti Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Nuclear Signalling Laboratory, Division for Biochemistry and Molecular Biology, John Curtin School of Medical Research, Australian National University, P. O. Box 334, Canberra City, A.C.T. 2601, Australia. Tel.: 616-2494188; Fax: 616-2490415; daj224{at}huxley.anu.edu.au; Telex: curtmed 62033.

The abbreviations used are: TF, transcription factor; NLS, nuclear localization sequence; CKII, casein kinase II; cdk, cyclin-dependent kinase; CDN, cell cycle-dependent NLS; DAPI, diamidinophenyl indole.


ACKNOWLEDGEMENTS

We thank Reiner Peters (Institut für Medizinische Physik, Münster, Germany) for support in preliminary studies.


REFERENCES
  1. Miller, M., Park, M. Y. & Hanover, J. A.(1991)Physiol. Rev. 71, 909-949 [Free Full Text]
  2. Schmitz, M. L., Henkel, T. & Baeuerle, P. A.(1991)Trends Cell Biol. 1, 130-137
  3. Jans, D. A. (1995) Biochem. J., in press
  4. Shirakawa, F. & Mizel, S. B.(1989)Mol. Cell. Biol. 9, 2424-2430 [Medline] [Order article via Infotrieve]
  5. Lenardo, M. J. & Baltimore, D.(1989)Cell 58, 227-229 [Medline] [Order article via Infotrieve]
  6. Moll, T., Tebb, G., Surana, U., Robitsch, H. & Nasmyth, K.(1991)Cell 66, 743-758 [Medline] [Order article via Infotrieve]
  7. Govind, S. & Steward, R.(1991)Trends Genet. 7, 119-125 [Medline] [Order article via Infotrieve]
  8. Roux, P., Blanchard, J.-M., Fernandez, A., Lamb, N., Jeanteur, Ph. & Piechaczyk, M. (1990)Cell 63, 341-351 [Medline] [Order article via Infotrieve]
  9. Gusse, M., Ghysdael, J., Evan, G., Soussi, T. & Mechali, M.(1989)Mol. Cell. Biol. 9, 5395-5403 [Medline] [Order article via Infotrieve]
  10. Van Etten, R. A., Jackson, P. & Baltimore, D.(1989)Cell 58, 669-678 [Medline] [Order article via Infotrieve]
  11. Capobianco, A. J., Simmons, D. L. & Gilmore, T. D.(1990)Oncogene 5, 584-591
  12. Picard, D., Salser, S. J. & Yamamoto, K. R.(1988)Cell 54, 1073-1080 [Medline] [Order article via Infotrieve]
  13. Schindler, C., Shuai, K., Prezioso, V. R. & Darnell, J. E., Jr.(1992) Science 257, 809-813 [Medline] [Order article via Infotrieve]
  14. Chida, K. & Vogt, P. K.(1992)Proc. Natl. Acad. Sci. U. S. A. 89, 4290-4294 [Abstract]
  15. Rihs, H.-P. & Peters, R.(1989)EMBO J. 8, 1479-1484 [Abstract]
  16. Rihs, H.-P., Jans, D. A., Fan, H. & Peters, R.(1991)EMBO J. 10, 633-639 [Abstract]
  17. Jans, D. A., Ackermann, M., Bischoff, J. R., Beach, D. H. & Peters, R.(1991) J. Cell Biol. 115, 1203-1212 [Abstract]
  18. Jans, D. A. & Jans, P.(1994)Oncogene 9, 2961-2968 [Medline] [Order article via Infotrieve]
  19. Vancurova, I., Paine, T. M., Lou, W. & Paine, P. L.(1995)J. Cell Sci. 108, 779-787 [Abstract/Free Full Text]
  20. Nasmyth, K., Adolf, G., Lydall, D. & Seddon, A.(1990)Cell 62, 631-647 [Medline] [Order article via Infotrieve]
  21. Nasmyth, K. & Shore, D.(1987)Science 237, 1162-1170 [Medline] [Order article via Infotrieve]
  22. Stillman, D. J., Bankier, A. T., Seddon, A., Groenhout, G. & Nasmyth, K. A. (1988)EMBO J. 7, 485-494 [Abstract]
  23. Casadaban, M. J., Martinez-Arias, A., Shapira, S. K. & Chou, J.(1983) Methods Enzymol. 100, 293-308 [Medline] [Order article via Infotrieve]
  24. Jans, D. A., Peters, R., Zsigo, J. & Fahrenholz, F.(1989)EMBO J. 8, 2481-2488 [Abstract]
  25. Jans, D. A., Peters, R. & Fahrenholz, F.(1990)EMBO J. 9, 2693-2699 [Abstract]
  26. Jans, D. A., Peters, R., Jans, P. & Fahrenholz, F.(1991)J. Cell Biol. 114, 53-60 [Abstract]
  27. Jans, D. A.(1992) Biochim. Biophys. Acta 1113, 271-276 [Medline] [Order article via Infotrieve]
  28. Hennekes, H., Peter, M., Weber, K. & Nigg, E. A.(1993)J. Cell Biol. 120, 1293-1304 [Abstract]
  29. Nishida, E., Iida, K., Yonezawa, N., Koyasu, S., Yahara, I. & Sakai, H.(1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5262-5266 [Abstract]
  30. Ohta, Y., Nishida, E., Sakai, H. & Miyamoto, E.(1989)J. Biol. Chem. 264, 16143-16148 [Abstract/Free Full Text]
  31. Templeton D. J., Park, S. H., Lanier, L. & Weinberg, R. A.(1991)Proc. Natl. Acad. Sci. U. S. A. 88, 3833-3837
  32. Templeton, D. J. (1992)Mol. Cell. Biol. 12, 435-443 [Abstract]
  33. Zacksenhaus, E., Bremner, R., Phillips, R. A. & Gallie, B. L.(1993) Mol. Cell. Biol. 13, 4588-4599 [Abstract]
  34. Girdham, C. H. & Glover, D. M.(1991)Genes & Dev. 5, 1786-1799
  35. Deleted in proof
  36. Pines, J. & Hunter, T.(1991)J. Cell Biol. 115, 1-17 [Abstract]
  37. Pines, J. & Hunter, T.(1994)EMBO J. 13, 3772-3781 [Abstract]
  38. Shaulsky, G., Goldfinger, N., Tosky, M. S., Levine, A. J. & Rotter, V. (1990)Oncogene 6, 2055-2065
  39. Ryan, J. J., Prochownik, E., Gottlieb, C. A., Apel, I. J., Merino, R., Nunez, G. & Clarke, M. F.(1994)Proc. Natl. Acad. Sci. U. S. A. 91, 5878-5882 [Abstract]
  40. Shaulsky, G., Goldfinger, N., Ben Zeev, A. & Rotter, V.(1990) Mol. Cell. Biol. 10, 6565-6577 [Medline] [Order article via Infotrieve]
  41. Shiozaki, K. & Yanagida, M.(1992)J. Cell Biol. 119, 1023-1036 [Abstract]
  42. Tinland, B., Koukolikova-Nicola, Z., Hall, M. N. & Hohn, B.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7442-7446 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.