The KetelD Dominant-Negative Mutations Identify Maternal Function

Copyright 2000 by the Genetics Society of America

The KetelD Dominant-Negative Mutations Identify Maternal Function of theDrosophila Importin-b Gene Required for Cleavage Nuclei Formation

Laszlo Tirian,* Jaakko Puro,† Miklos Erdelyi,‡ Imre Boros,‡ Bernadett Papp,‡

Monika Lippai* and Janos Szabad*

*Faculty of General Medicine, Department of Biology, University of Szeged, H-6720 Szeged, Hungary, ‡Biological Research Center of theHungarian Academy of Sciences, H-6701, Szeged, Hungary and †Department of Biology, University of Turku, SF-20500 Turku, Finland

Manuscript received January 27, 2000Accepted for publication September 6, 2000

ABSTRACTThe KetelD dominant female-sterile mutations and their ketelr revertant alleles identify the Ketel gene,

which encodes the importin-b (karyopherin-b) homologue of Drosophila melanogaster. Embryogenesis doesnot commence in the KetelD eggs deposited by the KetelD/1 females due to failure of cleavage nucleiformation. When injected into wild-type cleavage embryos, cytoplasm of the KetelD eggs does not inhibitnuclear protein import but prevents cleavage nuclei formation following mitosis. The Ketel1 transgenesslightly reduce effects of the KetelD mutations. The paternally derived KetelD alleles act as recessive zygoticlethal mutations: the KetelD/2 hemizygotes, like the ketel r/ketel r and the ketel r/2 zygotes, perish duringsecond larval instar. The Ketel maternal dowry supports their short life. The KetelD-related defects originatemost likely following association of the KetelD-encoded mutant molecules with a maternally provided partner.As in the KetelD eggs, embryogenesis does not commence in eggs of germline chimeras with ketel r/2 germlinecells and normal soma, underlining the dominant-negative nature of the KetelD mutations. The ketel r homozygousclones are fully viable in the follicle epithelium in wings and tergites. The Ketel gene is not expressed in mostlarval tissues, as revealed by the expression pattern of a Ketel promoter-lacZ reporter gene.

THE commencement of embryogenesis is one of many 2000), we cloned the Ketel gene and learned that itencodes the Drosophila homologue of importin-b, aintriguing biological phenomena and raises ques-protein believed to be essential for import of some typestions about the origin and function of factors requiredof nuclear proteins.for the initiation of a new life. What are those factors?

Biochemical studies have clarified a number of nu-Where and how are they made? What are their func-clear import and export processes and several of thetions? It has long been known that most of the factorscomponents (for recent reviews see Mattaj and Engl-that are required during early embryogenesis are depos-meier 1998; Weis 1998; Wozniak et al. 1998; Gorlichited into the egg cytoplasm during oogenesis and areand Kutay 1999). However, the developmental rolematernally provided. The importance of maternal con-of proteins involved in nuclear transport and genetictribution is emphasized by the fact that there is verycomplexity of the process have been poorly studied. Tolittle, if any, zygotic gene expression during the initialunderstand Ketel gene requirement during develop-cleavage divisions in Drosophila.ment and reveal unknown aspects of nuclear proteinGenetic dissection and the use of female-sterile muta-import, we analyzed the mutant phenotypes associatedtions have been an efficient approach to identifyingwith the KetelD gain-of-function and the ketel r loss-of-genes coding for the maternally provided factors re-function alleles, constructed different types of geneticquired during Drosophila embryogenesis (Wieschausmosaics, and studied the expression pattern of a Ketel1996). During genetic dissection of maternal effects inpromoter-operated lacZ reporter gene. The present arti-Drosophila melanogaster, we isolated 75 dominant female-cle describes that function of Drosophila importin-bsterile mutations including the four KetelD alleles (Erde-is required—in association with a maternally providedlyi and Szabad 1989; Szabad et al. 1989; Erdelyi et al.component—for the formation of cleavage nuclei. We1997). Since embryogenesis is terminated at the verypropose that several of the dominant female-sterile (Fs)beginning in the so-called KetelD eggs deposited by themutations identify maternal function of essential zygoticKetelD/1 females, we assumed that the normal Ketel genegenes and the functions become manifested followingproduct is maternally provided and performs essentialassociation of the Fs-encoded mutant gene productsfunctions during the commencement of embryogenesis.with a maternally provided partner “designed” for theAs described in the accompanying article (Lippai et al.initial stages of embryogenesis.

We also report that clones homozygous for ketel null

alleles are viable in the female germline, the follicleCorresponding author: Janos Szabad, Faculty of General Medicine,

epithelium, and wing and tergite cells referring to theDepartment of Biology, University of Szeged, H-6720 Szeged, SomogyiB. u. 4, Hungary. E-mail: [email protected] existence of nuclear protein import mechanisms that

Genetics 156: 1901–1912 (December 2000)

1902 L. Tirian et al.

females was monitored. The extra Ketel gene copies (1) werecompensate lost Ketel gene functions. With few excep-introduced through the Tp(2;Y)G chromosome or throughtions the Drosophila importin-b encoding the Ketel geneone of the Ketel1 (K1) transgenes described in the accompa-

is expressed in mitotically active cells and is not ex- nying article (Lippai et al. 2000).pressed in those larval and adult cells that are mitotically The Tp(2;Y)G chromosome: For production of XXTp(2;Y)G;

Ketel D/1 females, XXTp(2;Y)G; Df(2L)Sd68, pr/CyO, pr femalesinactive.were mated with XY; Ketel D/CyO, pr males. [In the Tp(2;Y)Gchromosome the 36B5-C1 to 40F segment of the second chro-mosome—including the Ketel and the purple (pr) loci—wasMATERIALS AND METHODStransposed onto a Y chromosome. The Df(2L)Sd68 deficiencyremoves both the Ketel and the pr loci (Lindsley and ZimmThe Ketel alleles: The four Ketel D alleles were isolated in a1992).] The XXTp(2;Y)G; Ketel D/CyO, pr females were matedscreen for dominant female-sterile mutations. The 27 recessivewith XTp(2;Y)G; pr/pr males. As determined in a cross betweenketel r alleles were generated through second mutagenesis ofXXTp(2;Y)G; Df(2L)Sd68, pr/CyO, pr females and XY; pr/prthe Ketel D mutations (Szabad et al. 1989; Erdelyi et al. 1997).males, 39% of the progeny females are XXTp(2;Y)G.The ketel r/ketel r homozygotes and the ketel r/2 hemizygotes

The Ketel1 (K1) transgenes: To study the effects of the K1were produced by crossing y/y; ketel r/y1CyO or y/y; ketel rX32/transgenes, we generated females that were heterozygous fory1CyO females with y/Y; ketel r/y1CyO males. [The ketel rX32 alleleone of the Ketel D alleles and carried one or two additionalis a small deficiency that removes the Ketel and a few neigh-Ketel gene copies in one of the three different types of K1boring loci (Erdelyi et al. 1997).] The y1CyO balancer chro-transgenes. The K1 transgenes were labeled with the mini-mosome carries a y1 transgene (Timmons et al. 1993). Headwhite marker gene that ensured, on white background, lightskeleton and ventral setae of the descending y/y (or y/Y);yellow and orange eyes in one and two copies, respectively.ketel r/ketel r (or ketel r/2) zygotes are yellow and thus the homo-One group of the K1; Ketel D/1 and the K1/K1; Ketel D/1and the hemizygous larvae can be separated from the heterozy-females was mated with wild type (1/1), the other groupgous nonyellow (y1CyO) sibling larvae in which the chitinwith K1/Y; 1/1 males (see Table 1).structures are dark. The Ketel D/2 hemizygotes were produced

Germline chimeras: We constructed germline chimerasby crossing y/y; ketel rX32/y1CyO females with y/Y; Ketel D/y1CyOthrough the transplantation of pole cells, ancestors of themales. For explanation of the genetic symbols throughoutgermline (Illmensee 1973). In the chimeras functionally nor-this article see Lindsley and Zimm (1992). The Drosophilamal soma surrounded the germline cells that were hemizygouscultures were kept at 258.for ketel rX13, a ketel r null allele. (In ketel rX13 z 9/10 of the 39 endThe Ketel D mutant phenotypes: The Ketel D dominant pheno-of the open reading frame was deleted.) In practice, pole cellstype was established by cytological analysis of Ketel D eggs.of the embryos from a cross between ketel rX13/Bc Gla femalesSquashes were prepared from eggs newly deposited by Ketel D/

1 females. The egg squashes were stained by Feulgen and and ketel rX32/CyO males were transplanted into host embryosGiemsa, a procedure appropriate for the staining of chromo- that derived from wild-type (1/1) females and Fs(1)KI237/Ysomes, centrosomes, and the spindle apparatus (Puro and males. Fs(1)K1237 (5 ovoD1) is a dominant female-sterile muta-Nokkala 1977; Puro 1991). tion that disrupts function of the germline cells without affect-

Cytoplasm injections: Two types of cytoplasm injections ing the soma (Busson et al. 1983; Komitopoulou et al. 1983).were carried out: The eclosing K1237/1 females were mated with lt bw/lt bw

males. [The ketel rX13 and ketel rX32-carrying chromosomes are la-1. A sample of z40 pl Ketel D1 egg cytoplasm (z0.4% total egg beled with the lt and the bw recessive marker mutations (Lind-

volume) was injected on one side into the presumptive sley and Zimm 1992; Erdelyi et al. 1997).]head region at 70% egg length and 70% egg diameter of Follicle cell mosaics: Follicle cell clones homozygous forwild-type embryos. The antennal and the maxillary sense ketel rX13 were generated through mitotic recombination. Inorgans, characteristic landmark structures, derive from the practice, ketel rX13 pr/Bc Gla adult females were mated withchosen blastoderm region ( Jurgens et al. 1986). The donor Fs(2)Ugra/Bc Gla males. [Three independent ketel rX13 pr linesand the recipient embryos were ,30 min old. In another were recovered following meiotic recombination to removeset of cytoplasm injections the wild-type embryos received possible second-site lethal mutations that were induced duringz300 pl Ketel D1 egg cytoplasm. Cuticles of the developing (i) the EMS treatment to induce the Ketel D1 mutation andembryos and larvae were analyzed ( Jurgens et al. 1986; (ii) the X-ray treatment when the ketel rX13 revertant allele wasWieschaus and Nusslein-Volhard 1986). As a control, generated (Szabad et al. 1989; Erdelyi et al. 1997). The threecytoplasm samples of ,30-min-old wild-type embryos were lines gave identical results in the clonal analysis experimentsinjected. and hence pooled data are presented in Tables 3 and 4. Fs(2)2. A small sample (20 mg/ml) of the red fluorescent classic Ugra (5 Ugra) is a dominant female-sterile mutation thatnuclear localization signal-phycoerythrin (cNLS-PE; Cser-

disrupts follicle cell function without affecting the germlinepan and Udvardy 1995) was first injected into Ketel D1 eggs.cells (Szabad et al. 1989, 1991).] Early third instar larvae wereA sample of z200 pl cNLS-PE containing Ketel D1 cytoplasmX-irradiated for the induction of mitotic recombination bywas subsequently injected into wild-type cleavage embryos.1500 R of X rays (150 kV; 0.5 mm Al filter, 1000 R/min). TheIn the control the cNLS-PE substrate solution was first in-eclosing ketel rX13/Ugra and the control lt bw/Ugra adult femalesjected into newly deposited wild-type eggs and the cNLS-were tested for offspring production. Whether the ketel rX13/PE containing wild-type egg cytoplasm was subsequentlyketel rX13 homozygous follicle cells can support egg developmentinjected into wild-type cleavage embryos. Import of thewas decided by comparing the frequencies of follicle cell mosa-cNLS-PE substrate into the cleavage nuclei was followed inicism in the ketel rX13/Ugra and the lt bw/Ugra control femalesa Zeiss (Thornwood, NY) LSM410 confocal microscope.(Szabad et al. 1989).The injections were done at 208.

Wing and tergite mosaics: Clones homozygous for the ket-el rX13 allele were induced through mitotic recombination inThe Ketel D/1/1 and the Ketel D/1/1/1 females: We con-f 36a/Y; ketel rX13/f 1 ck pwn larvae. The f 1 symbol stands for astructed Ketel D/1/1 and Ketel D/1/1/1 females which, inforked1 transgene in the 30B cytological region. It compensatesaddition to the Ketel D allele, carried two and three normal

Ketel gene (1) copies. Egg and progeny production of the effects of the f 36a mutation (P. Martın and A. Garcıa-Bel-

1903Drosophila Importin-b Mutations

lido, personal communication). The f 36a, ck, and pwn symbols F; Foe et al. 1993; Callaini and Riparbelli 1996). Tworepresent cell marker mutations that allow recognition of the nuclei form shortly in wild-type eggs and cleavage divi-different types of clones. Mitotic recombination was induced

sions commence and follow with a cycle of 8–10 minin young third instar larvae 72–80 hr after egg deposition(Figure 1H; Foe et al. 1993). In 6- to 7-min-old fertilized(1500 R, 150 kV, 0.5 mm Al filter, 1000 R/min). Following

mitotic recombination, the majority of the forked (f ) clones KetelD eggs the female and the male pronuclei are poorlyare homozygous for ketel rX13. The ck pwn twin clones served as contoured and suggest nuclear envelope (NE) defectsreference in analysis of the f clones. Wings and abdomens of (Figure 1, C and E). As a rule, disorganized masses ofthe f 36a/Y; ketel rX13/f 1 ck pwn males were mounted and analyzed

MTs form instead of the gonomeric spindle. The MTfor clones in a compound microscope. Types, frequencies,mass appears as a prominent sperm aster and persistsand clone sizes were recorded.

The Ketel-lacZ reporter gene: We constructed a reporter for several minutes (Figure 1, E and G). The centrosomegene in which a 1378-bp upstream segment of the Ketel gene replicates; however, the daughter centrosomes cannotbetween positions 21336 and 142 was combined with the separate in absence of intact NE (Figure 1G). The chro-Escherichia coli lacZ gene. (See the corresponding sequence

mosomes fail to segregate in absence of the gonomericunder the accession no. AJ002729 in the EMBL nucleotidespindle and disintegrate within minutes (Figure 1, Gsequence database.) The 1378-bp fragment contains the entire

Ketel promoter with the transcription start site (Lippai et al. and I). The centrosomes may replicate two to three2000). The 1378-bp segment was cloned into the pP(CaSpeR- times but instead of separation they organize rudimen-AUG-gal) P-element transformation vector (Thummel et al. tary asters of MTs along with a general decay of the egg1988). Four germline transformant lines of the reporter gene

cytoplasm. (Behavior of the polar body nuclei was notwere generated using the CaSpeR vector with the mini-whitedifferent in wild-type and in KetelD eggs and served as amarker gene. Two of the transgenes were inserted into the X

and two into the 3rd chromosome. Flies homozygous for any reference for timing the initial events of embryogene-of the transgenes were viable and fertile. b-Galactosidase activi- sis.) The KetelD mutant phenotype is identical for theties of transgene homozygotes were studied in adult, embry- four KetelD alleles.onic, and larval stages of development according to standard

The KetelD egg cytoplasm prevents cleavage nucleiprocedures.formation: Since the KetelD mutations have been knownto be gain-of-function type (Szabad et al. 1989), theabove-described defects are most likely brought aboutRESULTSby KetelD-encoded mutant gene products. To elaborate

The KetelD mutant phenotype suggests NE-related the above possibility we carried out egg cytoplasm injec-function of the normal Ketel gene product: The four tions:KetelD dominant female-sterile mutations of D. melanogas-ter emerged following EMS mutagenesis (Szabad et al. 1. First a sample of z40 pl KetelD egg cytoplasm was

injected into one side of the presumptive head region1989). The KetelD/1 females and the males are fully vi-able and male fertility is normal. However, the KetelD/1 of each of 59 wild-type cleavage embryos. (In the

control, where wild-type cytoplasm was injected intofemales are sterile. Development of the egg primordiais normal in all the KetelD/1 females and the meiotic wild-type embryos, only 5 of the 72 larvae showed

minor head defects.) The corresponding head struc-divisions are indistinguishable from wild type (J. Puro,unpublished data). The KetelD/1 females deposit nor- tures were invariably missing at the site of injection

(Figure 2). The anterior structures were entirely miss-mal numbers of normal-looking so-called KetelD eggs.All the KetelD eggs are normally fertilized as revealed by ing following the injection of z300 pl KetelD egg

cytoplasm per wild-type embryo (44 embryos).the presence of sperm tail in the egg cytoplasm. As inwild type, the nuclei are well contoured in the newly 2. In the second set of experiments, a small sample of

cNLS-PE solution was first injected into newly depos-deposited KetelD eggs, suggesting compact nuclei (Fig-ure 1, A and B). The four haploid nuclei, products of ited KetelD1 and (as control) into wild-type eggs. A

sample of the cNLS-PE containing egg cytoplasm wasthe meiotic divisions, appear compact in the unfertilizedKetelD eggs deposited by virgin KetelD/1 females. The subsequently injected into wild-type cleavage em-

bryos and the fate of the red fluorescent cNLS-PEbundle of microtubules (MTs) between the two innerhaploid nuclei is most likely the central spindle pole substrate was followed in a laser scanning micro-

scope. Whether the cNLS-PE was introduced in wild-body that persists following the second meiotic division,a phenomenon never seen in wild-type eggs, whether type or in KetelD egg cytoplasm, the cNLS-PE substrate

readily entered the cleavage nuclei, implying thatfertilized or not (Figure 1B; Puro 1991; Riparbelli andCallaini 1996). Severe KetelD-related defects appear 6–7 the KetelD-encoded molecules do not prevent nuclear

import of the cNLS-PE substrate (Figure 3). In themin after fertilization, during the commencement ofembryogenesis when the female and the male pronuclei control the cNLS-PE substrate was essentially homo-

geneously distributed during mitosis in the egg cyto-become juxtaposed (Figure 1, C–E). In wild type, thedaughter centrosomes separate and move around the plasm (Figure 3B). The cNLS-PE substrate high-

lighted the nuclei—which doubled in numbers—uponperimeter of the male pronucleus to the opposite poleand organize the first mitotic spindle (Figure 1, D and onset of the next interphase (Figure 3C). Following


Ketel D1 egg cytoplasm injections some nuclei ap- The KetelD alleles are very strong antimorph muta-tions: To find out whether the KetelD-encoded productspeared normal; however, many small nuclei ap-

peared (Figure 3D). Both the small and the normal- participate in the same process as the wild-type counter-part (i.e., the KetelD alleles are antimorph mutations;sized nuclei entered mitosis (with some delay as com-

pared to control); however, nuclei did not form at Muller 1932) or whether they disrupt a process inwhich the normal Ketel gene products are not involvedthe end of mitosis as indicated by the homogeneously

distributed cNLS-PE substrate in the egg cytoplasm (i.e., the KetelD alleles are neomorphs), we constructedfemales that, in addition to a KetelD allele, carried two(Figure 3F).


Figure 2.—Phase-contrast photomicrographs of the headof first instar larvae as seen in cuticle preparations. About 40pl wild-type (A) or KetelD egg cytoplasm (B) was injected intothe presumptive head region of wild-type cleavage embryos.Blastoderm cells that originate from the injected region giverise to the antennal (AntSO) and the maxillary sense organs Figure 3.—Import of the cNLS-PE red fluorescent substrate(MxSO; Jurgens et al. 1986). Note the absence of sense organs into nuclei of wild-type cleavage Drosophila embryos. Theon the side injected with KetelD egg cytoplasm (B). Bar, 50 mm. cNLS-PE substrate was coinjected with wild-type (A–C) or with

KetelD1 (D–F) egg cytoplasm at positions shown by the arrows.Import of the cNLS-PE molecules into the nuclei was followed

or three normal Ketel gene copies and studied whether in a laser scanner microscope. A and D, B and E, and Cand F photographs were taken 15, 22, and 29 min followingdominant female sterility can be overcome in thecytoplasm injections, respectively. Bar, 100 mm.KetelD/1/1 and in the KetelD/1/1/1 females. Two

systems were analyzed:

1. In the XXTp(2;Y)G; KetelD/CyO females the Tp(2;Y)G tion was 5.6 3 1023 offspring/(female 3 day)]. Ofthe 56 offspring, 13 females carried the KetelD2 allelechromosome carried a normal Ketel gene as part of

the second chromosome transposed onto a Y chro- and were sterile. Analysis of the KetelD2/1/1 femalesshowed the strong antimorph nature of KetelD2 andmosome. The females were mated with XTp(2;Y)G; 1/

CyO males. About 1% of the eggs of the 446 implies involvement of the KetelD2-encoded and thenormal Ketel gene products in the same process. TheXXTp(2;Y)G; KetelD2/CyO females turned brown, indi-

cating the progression of embryogenesis to the stage Tp(2;Y)G chromosome did not reduce sterility im-posed by the other three KetelD alleles: every eggof embryonic cuticle formation. [An analysis of the

cuticles of the deceased embryos revealed gross cell remained white and not a single offspring descendedfrom the 1196, 1341, and 945 XXTp(2;Y)G; KetelD/death apparently without any preference to the dif-

ferent body regions (J. Szabad, unpublished re- CyO females that carried the KetelD1, the KetelD3, andthe KetelD4 alleles, respectively.sults).] In addition, 56 offspring descended from the

total of 466 XXTp(2;Y)G; KetelD2/CyO females during 2. In the second set of experiments we made use ofthree types of seven Ketel1 (K1) transgenes linked tothe 3-wk test period. [The rate of offspring produc-

Figure 1.—The first embryonic division cycle in Feulgen-Giemsa-stained egg squashes of wild type (A, D, F, and H) and KetelD1/1females (B, C, E, G, and I). The left and right panels correspond to roughly identical stages as inferred from appearance of thepolar body nuclei. Thin arrows point to centrosomes. (A) Female and male pronuclei come to lie side by side following fertilization.The centrosome is attached to the NE of the male pronucleus. The three polar body nuclei are shown on the right. The well-defined nuclei imply the presence of intact NE. (D) In wild type one of the daughter centrosomes migrates to the opposite polealong the male pronuclear envelope; the other remains attached to the sperm tail (arrows). (Three polar body nuclei are shownon the right.) (F) The first mitotic spindle (that is gonomeric in Drosophila) in wild type. The maternal (right) and paternal(left) sets of chromosomes are separated. (Chromosomes of the polar body nuclei are shown on the right.) (H) Two zygoticnuclei form in wild type following completion of the first cleavage division. The centrosomes have already replicated and areabout to become separated. (B) Four well-contoured meiotic products in an egg of a virgin KetelD1/1 female. The dark structuremost likely represents the central spindle pole body, a remainder of the meiotic divisions. (C) The pronuclear stage in a fertilizedKetelD1/1-derived egg. The fuzzy nuclei suggest decomposing NE. (Only one polar body nucleus is shown on the right.) Thesperm-tail-associated sperm aster, which disassembles in wild type, persists. (E) NE of the pronuclei seems to be missing. Thesperm aster persists. (G) Centrosome migration does not take place in the KetelD1/1-derived eggs and bipolar spindles fail toform. The chromosomes embedded in the microtubular aggregate disintegrate. Enlarged polar body chromosomes are shownin insert on the right. Arrowheads point to sister chromatids and refer to chromosome replication during the pronuclear stage.(I) NE and nuclei never form in the KetelD1/1-derived eggs. While the chromatin (thick arrows) disintegrates the centrosomespass through two to three rounds of replication but fail to separate and are surrounded by asters of microtubules. Bars, 10 mm.


TABLE 1

Effects of an X-linked Ketel1 (K1) transgene on KetelD-related dominant female sterility

Genotype of tested females and the features of offspring production

K1/X; KetelD/1 females K1/K1; KetelD/1 females

Rate of Rate ofKetelD Male Test offspring Test offspringallele partner Tested Offspring perioda productionb Tested Offspring perioda productionb

KetelD1 XY; 1/1 233 0 25.1 — 250 0 19.6 —K1Y; 1/1 201 0 26.0 — 217 21 14.4 6.7 3 1023

KetelD2 XY; 1/1 237 2 17.8 0.5 3 1023 261 13 12.7 3.9 3 1023

K1Y; 1/1 172 11 19.6 3.3 3 1023 169 15 16.0 5.5 3 1023

KetelD3 XY; 1/1 188 0 19.5 — 179 0 22.8 —K1Y; 1/1 167 0 17.8 — 163 15 11.1 8.3 3 1023

KetelD4 XY; 1/1 168 0 27.2 — 433 0 27.5 —K1Y; 1/1 365 0 26.9 — 449 0 25.6 —

a Average test period per female (days).b Offspring/(female 3 day).

the X and to the third chromosomes (Lippai et al. Furthermore, the paternal rescue of embryonic lethality2000). Since effects of the transgenes were very simi- shows expression and function of the zygotic Ketel genelar, results related to only one of the X-linked K1 during embryogenesis.transgenes are presented. The transgene carries a The ketel r alleles reveal the zygotic requirement of22-kb genomic fragment including the promoter and the Ketel gene: We generated, through second mutagen-the structural parts of the Ketel gene. Flies homozy- esis of the KetelD alleles, 27 loss-of-function ketel r alleles.gous for the transgene are fully viable and fertile. Twenty-five of the 27 ketel r alleles are recessive zygoticWe constructed both K1/X; KetelD/1 and K1/K1; lethal mutations. The most severe ketel r/ketel r homozy-KetelD/1 females in which the wild-type:KetelD ratios gous and the ketel r/2 hemizygous combinations, includ-were 2:1 and 3:1, respectively. One group of the ing ketelrX13/2, bring about death during the secondfemales was mated with wild-type (X/Y; 1/1), the larval instar. [The 2 symbol stands for ketel rX32, a smallother group with K1/Y; 1/1 males (Table 1). As deficiency that removes the Ketel and a few adjacentin the case of the Tp(2;Y)G chromosome, a slight loci (Erdelyi et al. 1997).] The mutant larvae becomereduction of female sterility was apparent in the sluggish and decease within a day without any apparentK1/X; KetelD2/1 females: cuticle developed in z1% morphological defect. Since second larval instar deathof the eggs and a few offspring descended (Table is the most severe defect associated with the ketel r alleles,1). Apparently offspring production of the K1/X; it most likely represents the complete loss-of-functionKetelD2/1 females significantly increased when they mutant phenotype. The zygotic lethal nature of the ketel r

were mated with K1/Y; 1/1 males showing contribu- alleles clearly shows the zygotic requirement of the Keteltion of paternal rescue of the mutant phenotype. gene.Female sterility was further reduced in the K1/K1; When paternally derived the KetelD mutations behaveKetelD2/1 females, especially when they were mated as the zygotic lethal ketel r alleles, the KetelD/ketel r andwith K1/Y; 1/1 males (Table 1). the KetelD/2 larvae also perish during second larval in-

star and cannot be distinguished from the ketel r/2 ones.As expected, one copy of the K1 transgene did notGermline chimeras without a functional Ketel geneovercome female sterility brought about by the other

revealed important features of Ketel gene function: Tothree KetelD alleles, whether or not the K1/X; KetelD/1decide whether the short life of the ketel r/2 hemizygotesfemales were mated with wild-type or with K1/Y; 1/1is made possible by the normal Ketel gene products pro-males (Table 1). The K1/K1; KetelD/1 females werevided by the 1/2 mothers, we constructed germlinealso sterile when mated with wild-type males (Table 1).chimeras with normal soma and ketel r/2 female germ-However, when mated with K1/Y; 1/1 males, the K1/line cells that lacked a functional Ketel gene (Table 2).K1; KetelD1/1 and the K1/K1; KetelD3/1 females yieldedThe chimeras deposited normal-looking eggs. Althougha few offspring (Table 1). About 50% of the progenythe eggs were fertilized, embryogenesis did not com-females carried the KetelD allele. Results of the transgenemence inside them due to the lack of cleavage nucleiexperiments confirmed (i) the highly toxic nature offormation following fertilization, and the defects werethe KetelD-encoded gene products and (ii) that three of

the KetelD alleles are very strong antimorph mutations. indistinguishable from those described for the KetelD


TABLE 2

Features of the germline chimeras with normal soma and mutant germline

ketel rX13/2 germline chimeras KetelD1/2 germline chimeras

Genotype of the No. of Rate of egg Genotype of the No. of Rate of egggermline cells chimeras productiona germline cells chimeras productiona

Bc Gla/Cy Roi 3 Bc Gla/Cy Roi 2Ketel rX13/Bc Gla 4 9.3 6 6.7b Ketel D1/Bc Gla 2 7.7 6 5.3b

Ketel rX32/Cy Roi 3 Ketel rX32/Cy Roi 2Ketel rX13/ketelrX32 2 6.8 6 4.5 Ketel D1/ketelrX32 3 5.9 6 3.8

The donor embryos derived from a cross between ketelrX32/Bc Gla females and ketelrX13/Cy Roi males in theketelrX13/2 chimeras and ketelrX32/Bc Gla females and KetelD1/Cy Roi males in the KetelD1/2 chimeras. The ketelrX32

allele is a small deficiency that removes the Ketel and a few adjacent loci (Erdelyi et al. 1997).a Average daily egg production (and standard deviation) over a 10-day test period.b For the chimeras with Bc Gla and/or Cy Roi chromosomes.

eggs. Analysis of the germline chimeras revealed three mal follicle cells may develop because (i) function ofthe Ketel gene is not required in the germline or (ii)features of Ketel gene requirement: (1) Function of the

Ketel gene is not required in the female germline since the follicle cells compensate Ketel gene function absentin the germline. To distinguish between the above possi-the ketel r/2 cells are viable and are sources of normal-

looking eggs; (2) since embryogenesis does not com- bilities, we generated mosaic egg primordia in whichsome or all the enveloping follicle cells lacked Ketel genemence in eggs of the above chimeras, development of

the ketel r/2 larvae to the second larval instar must be function and the germline cells were normal. To pro-duce the latter type of mosaic egg primordia, we X-irradi-supported by the Ketel maternal dowry present in the

egg cytoplasm [maternal support of embryogenesis is a ated ketel rX13/Ugra larvae for the generation, throughmitotic recombination, of ketel rX13/ketel rX13 follicle cellrather general phenomenon in Drosophila (for a recent

review see Szabad 1998).], and (3) the identical pheno- clones. Egg and progeny production of the eclosingfemales were monitored. Results of the experiment cantype seen in eggs of the above germline chimeras and

in the KetelD eggs shows that the KetelD-encoded mutant be summarized as follows (Table 3): (1) Similar frequen-cies of the ketel rX13/Ugra and the 1/Ugra control femalesgene products impede function of the normal Ketel gene

products; i.e., the KetelD alleles are dominant-negative were mosaic and (2) the two types of mosaic femalesproduced eggs with similar rates. However, the larvaemutations.

To further clarify the function of the KetelD alleles, hatched from eggs of the ketel rX13/Ugra females witha reduced rate as compared to the control femaleswe constructed germline chimeras with normal soma

and KetelD1/2 germline cells (Table 2). As in the ketel r/2 (Table 3).The ketel null homozygous clones are fully viable ongermline chimeras, the KetelD1/2 germline cells allowed

proliferation of the female germline cells and were the wings and the tergites: To further characterize therequirement of the Ketel gene in cell types of the soma,sources of normal-looking eggs. However, embryogene-

sis did not commence in their eggs due to the lack of we analyzed clones of ketel rX13 homozygous wing andtergite cells. The ketel rX13 homozygous clones were in-cleavage nuclei formation.

Function of the Ketel gene is not required in the folli- duced through mitotic recombination in f 36a/Y; ketel rX13/f1 ck pwn young third instar larvae. Most of the forkedcle cells: The normal-looking eggs that derive from mo-

saic egg primordia with ketel r/2 germline cells and nor- homozygous clones were homozygous for ketel rX13. As

TABLE 3

Characteristics of the ketel rX13 homozygous follicle cell clones

Females Egg production

Frequency of Eggs Egg/(mosaic Egg hatchSpecimena Tested Mosaic mosaicism (%) deposited female 3 day) Hatched rate (%)

Control 132 16 12.1 37 0.29 18 48.6ketel rX13 379 43 11.3 80 0.23 23 28.8

a lt bw/Fs(2)Ugra (as control) and ketel rX13/Fs(2)Ugra females were irradiated as third instar larvae and the developing adultfemales were tested.


summarized in Table 4, frequencies and sizes of thedifferent types of clones were similar in the ketel rX13 andin the control experiments in both the wings and thetergites. Several of the f clones included as many as70–100 cells. More than seven rounds of cell divisionsare required following mitotic recombination to reachclones of that size. Features of the f clones revealed thatKetel gene function is not required for life and functionof the wing imaginal disk and the abdominal histoblastcells.

Expression pattern of the Ketel gene as revealed bya reporter gene: To study the expression pattern of theKetel gene, we generated four transgenes in which theKetel promoter regulated expression of a lacZ reportergene and analyzed b-gal activities in embryos as well asin different larval and adult tissues. The reporter geneis expressed in the ovaries and its expression pattern inthe ovaries and during embryogenesis is identical withthat detected by RNA in situ hybridizations and by theanti-Ketel antibody. [For details on Ketel gene expressionduring oogenesis and embryogenesis see Lippai et al.(2000).] The reporter gene is also expressed in thetestes and sperm pump. However, the reporter gene isnot expressed in the bulk of the adult tissues. (Someb-gal activities appeared in the central nervous system.The blood-producing organ could not be included inthe reporter gene analysis due to its intrinsic b-gal ex-pression.) In third instar larvae the reporter gene isintensively expressed in all the imaginal discs, in thelarval gonads, in the imaginal ring cells of salivaryglands, and in the ring gland (Figure 4). There was nob-gal activity present in most larval cells, including thesalivary glands, fat body, larval epidermis, larval muscu-lature, and Malpighian tubules (Figure 4).

DISCUSSION

The KetelD-encoded importin-b molecules preventcleavage nuclei formation: Embryogenesis fails to com-mence in the KetelD eggs deposited by the KetelD/1 fe-males: the KetelD-encoded mutant gene products preventformation of the first zygotic nuclei. Instead of cleavagenuclei formation MT bundles and asters persist. As re-sults of cytoplasm injections revealed, the defects arebrought about by “toxic” components in the KetelD eggcytoplasm that “poison” wild-type cleavage embryosthrough the blocking of cleavage nuclei formation fol-lowing cleavage mitoses.

In higher eukaryotes the nuclei disassemble upon theinitiation of mitosis. The process is under the controlof p34cdc2, the cyclin-B-dependent mitotic kinase (Gantand Wilson 1997; Marshall and Wilson 1997; Collas1998). Phosphorylation of the lamins, the nucleoporins,and the integral nuclear membrane proteins are impor-tant events of NE disassembly. Some of the disassemblednuclear membrane complexes disperse in the cyto-

TA

BL

E4

Cha

ract

eris

tics

ofth

eke

telrX

13ho

moz

ygou

sw

ing

and

terg

ite

clon

es

Win

gsT

ergi

tes

Typ

esof

clon

esC

lon

esi

zea

Typ

esof

clon

esC

lon

esi

zeb

Spec

imen

Scre

ened

f/ck

pwn

fck

pwn

fck

pwn

Scre

ened

f/ck

pwn

fck

pwn

fck

pwn

Con

trol

2052

1118

6.6

61.

76.

46

1.9

2042

1216

4.4

62.

64.

06

1.9

kete

lrX13

2045

1310

6.2

61.

56.

66

2.2

2052

129

4.2

62.

04.

26

2.0

aT

he

aver

age

num

ber

ofce

lldi

visi

ons

requ

ired

tore

ach

the

obse

rved

clon

esi

ze.

bT

he

aver

age

num

ber

ofbr

istl

esin

clud

edin

acl

one.

plasm; others are stored in vesicles (Marshall and Wil-son 1997). In Drosophila NE disassembly is complete


Figure 4.—Expression pat-tern of a Ketel promoter-regu-lated lacZ reporter gene in dif-ferent tissues of late third in-star larvae. Blue staining corre-sponds to expression of theKetel gene as revealed throughactivities of b-galactosidase mo-lecules. The reporter gene isexpressed in all the imaginaldiscs (id), in the larval ovaries(lo) and testes (lt), in imaginalrings of the salivary glands (ir),and in the ring gland (rg). Thereporter gene is not expressedin the larval epidermis and theoverlying muscles (lem), the fatbody (fb), the salivary glands(sg), the central nervous sys-tem (cns), the gut (gt), and the

Malpighian tubules (mt). (The slight staining in different sections of the digestive tract is due to endogenous b-galactosidaseactivities.) Bar, 1 mm on A–C and 100 mm on D–H.

only opposite the spindle poles. The nuclear pore com- and Ran-GDP is cytoplasmic. Ran carries out its GTPaseplexes (NPCs) and the nuclear lamina dissociate from cycle to supply energy for the nucleocytoplasmic trans-the rest of the nuclear membrane and so-called spindle port of macromolecules. During this cycle it shuttlesenvelopes form (Foe et al. 1993). between the nucleus and cytoplasm (Melchior and

Formation of nuclei, as studied mostly in Xenopus Gerace 1998; Azuma and Dasso 2000).egg extracts, begins with reassembly of the NE, a process Two possible mechanisms seem feasible to explainproposed to occur in the following major steps (Sutov- the KetelD-related defects, i.e., the failure of cleavagesky et al. 1998; Zhang and Clarke 2000). The process nuclei formation following mitosis:begins during telophase when Ran-GDP associates with

1. Since the Ketel gene encodes the Drosophila homo-chromatin (Zhang et al. 1999). [Ran is a Ras-related Glogue of importin-b, a component of nuclear proteinprotein without membrane anchoring site (for a recentimport (Lippai et al. 2000), the KetelD-encoded pro-review see Azuma and Dasso 2000).] The chromatin-tein molecules may simply block the import of mole-associated Ran-GDP promotes (i) the binding of mem-cules required for the formation of cleavage nucleibrane vesicles to chromatin; (ii) the recruitment offollowing cleavage mitoses. This possibility is sup-RCC1 (regulator of chromatin condensation), a chro-ported by two observations: (i) KetelD egg cytoplasmmatin-associated nuclear protein that is the only knownand ovary extracts of KetelD/1 females (Lippai et al.guanine nucleotide exchange factor for Ran; and (iii)2000) do not disrupt already established NEs andthe association of nucleoporins (Goldberg et al. 1997;(ii) the formation of miniature nuclei besides theGant et al. 1998). RCC1 generates Ran-GTP from Ran-normal-sized ones (Figure 3D). The cNLS-PE experi-GDP, and Ran-GTP causes fusion of the vesicles andments do not rule out the above possibility. Theformation of a double nuclear membrane (Gant et al.cNLS-PE molecules may be imported into the nuclei1998; Zhang and Clarke 2000). Formation of the NEthrough an alternative pathway (Mattaj and Engl-with NPCs establishes conditions for resumed nuclearmeier 1998; Pemberton et al. 1998; Gorlich andprotein import and the formation of functional nuclei.Kutay 1999). The possible existence of alternativeThe process proceeds in vitro where NE forms from eggnuclear protein import pathway(s) is supported bycytoplasm extract components over the demembra-two findings described in this article: (a) Cells innated sperm chromatin (Burke and Gerace 1986) inwhich the Ketel gene is expressed function normallya process similar to NE assembly around the spermwithout Ketel gene product and (b) the Ketel gene ischromatin during male pronucleus formation followingnot expressed in most of the terminally differentiatedfertilization (Sutovsky et al. 1998). As described re-and nondividing cell types.cently by Zhang and Clarke (2000), functional NEs

2. Importin-b (the Ketel protein) has been known toform over Sepharose beads loaded with Ran-GDP ininteract with importin-a, Ran, and a number ofXenopus egg extract in the absence of DNA or chro-nucleoporins during nuclear protein import, and thematin.regions of interactions have been determined (KutayThe intrinsic GTPase activity or Ran is activated byet al. 1997; Wozniak et al. 1998). In the case of humanRanGAPs, the RanGTPase activating proteins. The Ran-

GAPs are cytoplasmic and hence Ran-GTP is nuclear importin-b, as in other members of the importin-b,


the Ran binding domain resides at the N-terminal tant gene products in the 1/1 cells (Szabad et al.1989); (3) the gain-of-function features of the KetelDregion. Truncated importin-b molecules lack the

N-terminal sections and thus cannot bind Ran, can alleles can be eliminated during second mutagenesisand loss-of-function ketel r recessive alleles are generatedassociate with NPCs, and have a dominant-negative

effect on nuclear protein import as determined in the (Szabad et al. 1989; Erdelyi et al. 1997); and (4) thedeleterious effects of three of the four KetelD alleles candigitonin-permeabilized HeLa assay system (Kutay

et al. 1997); the NPC binding domain slightly overlaps be reduced by the addition of normal (1) Ketel genecopies: a few offspring derive from the KetelD2/1/1/1the Ran binding domain and resides toward central

regions of the protein. The importin-a binding do- females. Interestingly, mating of the KetelD2/1/1/1females with males that provided two wild-type Ketelmain is located toward the C terminus.genes significantly increased the rate of offspring pro-

Abnormal interactions between the KetelD-encoded duction (as compared to the wild-type males that pro-molecules and components of the nuclear transport vided only one Ketel gene). In fact, the KetelD1/1/1/1process may lead to the failure of cleavage nuclei forma- and the KetelD3/1/1/1 females yielded a few offspringtion. In fact, dominant-negative mutations in both Ran only when their male partners provided two normaland RCC1 have been known to disrupt NE formation. Ketel gene copies during fertilization. The paternal res-The RanT24N (substitution of Thr at position 24 by cue effect on KetelD-associated maternal-effect lethalityAsn) dominant-negative mutant allele of Ran encodes clearly shows expression of the Ketel gene during em-a protein that is defective in nucleotide binding and bryogenesis.profoundly disrupts NE assembly in the Xenopus laevis Analysis of the KetelD/1/1/1 females revealed theegg extract system (Dasso et al. 1994; Zhang and strong antimorph nature of the KetelD mutations andClarke 2000). Similarly to RanT24N, the nonhydrolyz- imply involvement of the normal and the KetelD-encodedable guanosine 59-triphosphate analogues have also mutant gene products in the same process (Mullerbeen known to inhibit NE assembly through disrupted 1932). Since products of the antimorph mutations im-function of Ran, which has been known to play a key pede action of the normal gene products they are alsorole in NE integrity and exit from mitosis (Demeter et called dominant-negative mutations. The gain-of-func-al. 1995; Macaulay and Forbes 1996). The pim1-d1ts tion and loss-of-function mutant phenotypes are ex-mutant allele of RCC1 of Schizosaccharomyces pombe pro- pected to be identical in the case of strong antimorphhibits the reestablishment of the NE following mitosis mutations. [The TomajD mutations, which identify the(Kornblut et al. 1994). Mutations in a number of Tubulin67C gene of Drosophila, represent a typical ex-nucleoporins have also been reported to lead to nuclear ample (Mathe et al. 1998).] Although the KetelD allelesfragmentation in yeast (reviewed in Corbett and Sil- are very strong dominant-negative mutations, the gain-ver 1997). of-function and loss-of-function mutant phenotypes are

The disturbing effects on NE organization of the Ket- not identical: failure of the commencement of em-el D-encoded molecules is also supported by persistence bryogenesis in the KetelD eggs vs. death during secondof the MT bundles and asters seen in the KetelD eggs. larval instar of the ketel r homo- and hemizygotes. When,We interpret the persistence of the MT bundles and however, the Ketel gene product is removed from theasters by leakage of Ran-GTP from the nuclei through eggs (in eggs of the germline chimeras with normalthe inappropriately assembled NE. Ran-GTP has re- soma and ketel r/2 hemizygous germline cells) the gain-cently been reported to stabilize microtubule asters and of-function and the loss-of-function Ketel mutant pheno-promote spindle assembly (for reviews see Kahana and types become identical: they lack cleavage nuclei forma-Cleveland 1999; Nishimoto 1999; Azuma and Dasso tion. Obviously, the normal Ketel gene products, as part2000). Evidently several types of molecules participate of the maternal dowry provided by the 1/2 mothers,in NE disassembly and reassembly upon the onset and support life of the ketel r/2 hemizygotes up to secondtoward the end of mitosis and the KetelD mutations larval instar when they perish.should help in understanding the role of importin-b in The KetelD-related defects are brought about follow-the process. ing interaction of the KetelD-encoded molecules with

The KetelD alleles are dominant-negative mutations: maternally provided partner(s): In understanding theFour observations show the gain-of-function nature of mode of KetelD action, the following facts should bethe KetelD mutations and that mutant gene products considered:bring about the KetelD-related defects: (1) The KetelD eggcytoplasm is toxic: when injected into wild-type embryos 1. The Ketel maternal dowry and the “toxic” nature

of the KetelD egg cytoplasm imply deposition by theit prevents formation of cleavage nuclei; (2) followingthe induction of 1/1 clones (through mitotic recombi- KetelD/1 mothers of both the normal and the KetelD-

encoded mutant gene products into the egg cyto-nation) in the germline of KetelD/1 females, the 1/1clones appear with reduced frequencies and with several plasm.

2. Lethality of the ketel r homo- and hemizygotes impliesdays delay due to perdurance of the KetelD-encoded mu-


expression of the normal Ketel and the KetelD alleles tute lost Ketel gene function and proteins normallyduring development. Yet the KetelD-encoded mutant imported through the importin-b route are importedgene products exert their deleterious effects only on into the nucleus through another system. The hu-cleavage embryos. man ribosomal protein L23a, for example, can be

3. When paternally derived, the KetelD alleles behave as imported through at least four routes (Jakel andthe ketel r loss-of-function alleles: they do not disturb Gorlich 1998). Similarly, transportin, a member ofbut also cannot support cell functions. the importin-b superfamily, recognizes and assists

import of rather different types of proteins into theWe interpret the deleterious effects of the KetelD muta-nucleus (reviewed in Gorlich and Kutay 1999).tions on cleavage embryos by interaction of the KetelD-However, function of the Ketel gene must be essentialencoded molecules with a maternally provided partnerin some cell type(s) since the ketel rX13/2 hemizygotespresent in the egg cytoplasm but absent from the so-die during second larval instar. “Focus,” i.e., the cellmatic cells. The complexes not only inhibit cleavagetype in which function of the Ketel gene is essential,nuclei formation but also impede activities of the com-remains to be elucidated.plexes composed from normal Ketel molecule(s) and

4. It is also possible that the Ketel gene is not expressedthe maternally provided partner. During cellular stagesin the studied cell types. To clarify this possibility,of development, when the maternally provided partnerwe constructed a reporter gene in which the Ketelis absent, the KetelD-encoded gene products may well bepromoter regulated expression of the lacZ reporterpresent but cannot exert their deleterious activities ingene. In adult females b-gal activities appeared inthe absence of an appropriate partner and consequentlythe ovaries, including the female germline and thethe cells survive and function normally. Thus the KetelD

follicle cells. Expression patterns of the reporter andmutations identify maternal function of a zygoticallythe Ketel genes—as determined by in situ hybridiza-essential gene and the identified function, if manifested,tions and Western blot analysis—were in harmonyfollowing interaction with a maternally provided part-(see also the accompanying article by Lippai et al.ner. The maternally provided component that interacts2000). In harmony with the genetic data, the eggwith the Ketel and the KetelD-encoded molecules re-cytoplasm was loaded with b-gal and there was b-galmains to be identified.activity present in virtually every cell of the cleavageRequirement and expression patterns of the Keteland cellular embryos. However, except for minorgene indicate parallel nuclear protein import pathways:b-gal activities in the brain, no other cells in the adultWe assumed that Ketel gene function was required infemales possessed b-gal activities. In late third instarevery cell for the import of cNLS-containing nuclearlarvae b-gal activity appeared in imaginal discs, larvalproteins. Surprisingly, cells in the female germline, folli-gonads, imaginal rings of the salivary glands, and incle epithelium, and wing and abdominal histoblasts are

viable and function normally without the Ketel gene. the ring gland. However, the other larval tissues didThe following possible explanations may be considered not possess b-gal activity. It thus appears that theto account for viability of the above cells: Ketel gene is expressed, with exception of the ring

gland cells, in certain types of the mitotically active1. Perdurance of the normal Ketel gene products incells. (The neuroblast cells in the central nervousclones of cells without a functional Ketel gene is notsystem and the histoblast cells, for example, do notlikely the reason for survival of the cells for the follow-possess b-gal activity.) The role of the Ketel proteining reasons. In female germline chimeras the pole-in cell cycle progression remains to be elaborated.cell-derived normal Ketel gene products are expectedInterestingly, the Drosophila importin-b codingto decay and/or dilute between pole cell formation(Ketel) gene is not expressed in the mitotically quies-and egg production. Even if they survived it is un-cent cells. This observation raises questions aboutlikely that they can support the immense biochemicalimport of cNLS-containing nuclear proteins in, e.g.,activities associated with egg cytoplasm production.the larval cells.It is also rather improbable that perdurance of the

We thank Revesz Kati and Kissne Ani for excellent technical helpnormal Ketel gene products supports life of the wingand our colleagues Drs. I. Belecz, E. Mathe, J. Mihaly, Z. Laszlo, anddisc cells over more than seven rounds of cell divi-A. Udvardy for stimulating discussions. We are grateful to Drs. A.

sions following the induction of mitotic recombina- Garcia-Bellido for the f 1/ck pwn system and A. Shearn for the y1CyOtion. chromosome. We express our gratitude to Dr. David Glover, who

2. Nonautonomy of the lack of Ketel gene function (i.e., organized support through the Preadhesion pour les Dix Pays d’Eu-rope Centrale et Orientale No. CEC ERB CIPD CT 94 0049 EC Cellthe supply with normal Ketel gene products of theCycle Network program. Support for the “Ketel project” came frommutant cells by the normal cells) is also unlikelythe following additional sources: OTKA 922, OTKA T5537, OTKAsince the 94-kD Ketel protein lacks signal sequencesT32540 from the Hungarian National Science Foundation, FKFP grant

required for excretion and uptake from and into 1348/1997 from the Hungarian Education and Science Foundationcells. and the Poland and Hungary: Action for the Restructuring of the

Economy-ACCORD Program No. H-9112-0528.3. Most likely parallel nuclear import pathways substi-


Lindsley, D. L., and G. G. Zimm, 1992 The Genome of DrosophilaLITERATURE CITEDmelanogaster. Academic Press, San Diego and London.

Lippai, M., L. Tirian, I. Boros, J. Mihaly, M. Erdelyi et al., 2000Azuma, Y., and M. Dasso, 2000 The role of Ran in nuclear function.The Ketel gene encodes the Drosophila homologue of Importin-b.Curr. Opin. Cell Biol. 12: 302–307.Genetics 156: 1889–1900.Burke, B., and L. Gerace, 1986 A cell free system to study reassembly

Macaulay, C., and D. J. Forbes, 1996 Assembly of the nuclearof the nuclear envelope at the end of mitosis. Cell 44: 639–652.pore—biochemically distinct steps revealed with NEM, GTPgS,Busson, D., M. Gans, K. Komitopoulou and M. Masson, 1983 Ge-and BAPTA. J. Cell Biol. 132: 5–20.netic analysis of three dominant female sterile mutations located

Marshall, I. B., and K. L. Wilson, 1997 Nuclear envelope assemblyon the X chromosome of Drosophila melanogaster. Genetics 105:after mitosis. Trends Cell Biol. 7: 69–74.309–325.

Mathe, E., I. Boros, K. Josvay, K. Li, J. Puro et al., 1998 TheCallaini, G., and M. G. Riparbelli, 1996 Fertilization in DrosophilaTomaj mutant alleles of Tubulin67C reveal a requirement for themelanogaster: centrosome inheritance and organization of the firstencoded maternal specific tubulin isoform in the sperm aster, themitotic spindle. Dev. Biol. 176: 199–208.cleavage spindle apparatus and neurogenesis during embryonicCollas, P., 1998 Nuclear envelope disassembly in mitotic extractsdevelopment in Drosophila. J. Cell Sci. 111: 887–896.require functional nuclear pores and a nuclear lamina. J. Cell

Mattaj, I. W., and L. Englmeier, 1998 Nucleocytoplasmic trans-Sci. 111: 1293–1303.port: the soluble phase. Annu. Rev. Biochem. 67: 265–306.Corbett, A. H., and P. A. Silver, 1997 Nucleocytoplasmic transport

Melchior, F., and L. Gerace, 1998 Two-trafficking with Ran.of macromolecules. Microbiol. Mol. Biol. Rev. 61: 193–211.Trends Cell Biol. 8: 175–179.Cserpan, I., and A. Udvardy, 1995 The mechanism of nuclear

Muller, H. J., 1932 Further studies on the nature and causes oftransport of natural or artificial transport substances in digitoningene mutations. Proc. 6th Int. Cong. Genetics, Brooklyn Botanicpermeabilized cell. J. Cell Sci. 108: 1849–1861.Gardens USA 1: 213–255.Dasso, M., T. Seki, Y. Azuma, T. Ohba and T. Nishimoto, 1994

Nishimoto, T., 1999 A new role of Ran GTPase. Biochem. Biophys.A mutant form of the Ran/TC4 protein disrupts nuclear functionRes. Commun. 262: 571–574.in Xenopus laevis egg extracts by inhibiting the RCC1 protein, a

Pemberton, L. F., G. Blobel and J. Rosenblum, 1998 Transportregulator of chromosome condensation. EMBO J. 13: 5732–5744.through the nuclear pore complex. Curr. Opin. Cell Biol. 10:Demeter, J., M. Morphew and S. Sazer, 1995 A mutation in the392–399.RCC1-related protein pim1 results in nuclear envelope fragmen-

Puro, J., 1991 Differential mechanism governing segregation of andtation in fission yeast. Proc. Natl. Acad. Sci. USA 28: 1436–1440.univalent in oocytes and spermatocytes of Drosophila melanogaster.Erdelyi, M., and J. Szabad, 1989 Isolation and characterization ofChromosoma 100: 205–314.dominant female sterile mutations of Drosophila melanogaster.

Puro, J., and S. Nokkala, 1977 Meiotic segregation of chromosomesI. Mutations on the third chromosome. Genetics 122: 111–127.in Drosophila melanogaster oocytes: a cytological approach. Chro-Erdelyi, M., E. Mathe and J. Szabad, 1997 Genetic and develop-mosoma 63: 273–286.mental analysis of mutant Ketel alleles that identify the Drosoph-

Riparbelli, M. G., and G. Callaini, 1996 Meiotic spindle organiza-ila importin-b homologue. Acta Biol. Hung. 48: 323–338.tion in fertilized Drosophila oocyte: presence of centrosomalFoe, V. E., G. M. Odell and B. A. Edgar, 1993 Mitosis and morpho-components in the meiotic apparatus. J. Cell Sci. 109: 911–918.genesis in the Drosophila embryo: point and counterpoint, pp.

Sutovsky, P., C. Simerly, L. Hewitson and G. Schatten, 1998149–300 in The Development of Drosophila melanogaster, edited byAssembly of nuclear pore complexes and annulate lamellae pro-M. Bate and A. Martinez-Arias. Cold Spring Harbor Laboratorymotes normal pronuclear development in fertilized mammalianPress, Cold Spring Harbor, NY.oocytes. J. Cell Sci. 111: 2841–2854.Gant, T. M., and K. L. Wilson, 1997 Nuclear assembly. Annu. Rev.

Szabad, J., 1998 Genetic requirement of epidermal and female germCell Dev. Biol. 13: 669–695.line cells in Drosophila in the light of clonal analysis. Int. J. Dev.Gant, T. M., M. W. Goldberg and T. D. Allen, 1998 NuclearBiol. 42: 257–262.envelope and nuclear pore assembly: analysis of assembly interme-

Szabad, J., M. Erdelyi, G. Hoffmann, J. Szidonya and T. R. F.diates by electron microscopy. Curr. Opin. Cell Biol. 10: 409–415.Wright, 1989 Isolation and characterization of dominant fe-Goldberg, M. W., C. Wiese, T. D. Allen and K. L. Wilson, 1997male sterile mutations of Drosophila melanogaster. II. Mutations onDimples, pores, star-rings, and thin rings on growing nuclearthe second chromosome. Genetics 122: 823–835.envelopes: evidence for structural intermediates in nuclear pore

Szabad, J., V. A. Jursnich and P. J. Bryant, 1991 Requirement forassembly. J. Cell Sci. 110: 409–420. cell-proliferation control genes in Drosophila oogenesis. GeneticsGorlich, D., and U. Kutay, 1999 Transport between the cell nu- 127: 525–533.cleus and the cytoplasm. Annu. Rev. Cell Dev. Biol. 15: 607–660. Thummel, K. S., A. M. Boulet and H. D. Lipshitz, 1988 VectorsIllmensee, K., 1973 The potentialities of early gastrula nuclei of D. for Drosophila P-element-mediated transformation and tissue cul-

melanogaster: production of their imago descendants by germ line ture transfection. Gene 74: 445–456.transplantation. Wilhelm Roux Archiv. 171: 331–343. Timmons, L., E. Hersperger, E. Woodhouse, J. Xu, L. Z. Liu et al.,

Jakel, S., and D. Gorlich, 1998 Importin beta, transportin, RanBP5 1993 The expression of the Drosophila awd gene during normaland RanBP7 mediate nuclear import of ribosomal proteins in development and in neoplastic brain tumors caused by lgl muta-mammalian cells. EMBO J. 17: 4491–4502. tions. Dev. Biol. 158: 364–379.

Jurgens, G., R. Lehmann, M. Schardin and C. Nusslein-Volhard, Weis, K., 1998 Importins and exportins. Trends Biol. Sci. 23: 185–1986 Segmental organisation of the head in the embryo of 189.Drosophila melanogaster. Roux’s Arch. Dev. Biol. 195: 359–377. Wieschaus, E., 1996 Embryonic transcription and the control of

Kahana, J. A., and D. W. Cleveland, 1999 Beyond nuclear trans- developmental pathways. Genetics 142: 5–10.port: Ran-GTP as a determinant of spindle assembly. J. Cell Biol. Wieschaus, E., and C. Nusslein-Volhard, 1986 Looking at em-146: 1205–1209. bryos, pp. 199–227 in Drosophila: A Practical Approach, edited by

Komitopoulou, K., M. Gans, L. M. Margaritis, F. Kafatos and D. B. Roberts. I.R.L. Press, Oxford.M. Masson, 1983 Isolation and characterization of sex-linked Wozniak, R. W., M. P. Rout and J. D. Aitchison, 1998 Karyopherinsfemale-sterile mutants in Drosophila melanogaster. Genetics 105: and kissing cousins. Trends Cell Biol. 8: 184–188.897–920. Zhang, C., M. Hughes and P. R. Clarke, 1999 Ran-GTP stabilizes

Kornblut, S., M. Dasso and J. Newport, 1994 Evidence for a dual microtubule asters and inhibits nuclear assembly in Xenopus eggrole for TC4 protein in regulating nuclear structure and cell extracts. J. Cell Sci. 112: 2453–2461.cycle progression. J. Cell Biol. 125: 705–719. Zhang, C., and P. R. Clarke, 2000 Chromatin-independent nuclear

Kutay, U., E. Izaurralde, F. R. Bischoff, I. W. Mattaj and D. envelope assembly induced by Ran GTPase Xenopus egg extracts.Gorlich, 1997 Dominant-negative mutants of importin-b block Science 288: 1429–1432.multiple pathways of import and export through the nuclearpore complex. EMBO J. 16: 1153–1163. Communicating editor: T. C. Kaufman

Documents

The KetelD Dominant-Negative Mutations Identify Maternal Function