Drosophila melanogaster - Genes & Developmentgenesdev.cshlp.org/content/8/10/1160.full.pdfThe Doa...

The Doa locus encodes a member of a new protein kinase family and is essential for eye and embryonic development in Drosophila melanogaster B o k y o u n g Yun, Robert Farkas, 1 Kun Lee, and Leonard Rabinow

Waksman Institute, Rutgers University, Piscataway, New Jersey 08855-0759 USA

Mutations at the Darkener of apricot (Doa) locus of Drosophila cause roughened eyes and increase transcript accumulation from the retrotransposon copia up to fourfold. Cloning of the gene and sequencing of cDNAs reveals that it encodes a putative serine/threonine protein kinase. Sequence data base searches identify it is a member of a novel highly conserved protein kinase family, with homologs in humans, mice, and Saccharomyces cerevisiae, not related to each other previously. Family members are characterized by a peptide motif reading EHLAMMERILG at kinase subdomain X, which is virtually 100% identical in all homologs. We therefore refer to this new family as the LAMMER protein kinases. As predicted from its primary sequence, Doa protein possesses intrinsic protein kinase activity when expressed in bacteria, as assayed via autophosphorylation. The gene is expressed throughout development, and both stage and tissue-specific RNAs are found. Its function is essential, because maternally deposited or zygotically transcribed mRNA is required for development to larval stages, and defects in segmentation and development of the nervous system are observed in embryos derived from heteroaUelic mothers. Doa function is also critical to Drosophila eye development, because the organization and development of pigment cells, bristles, and photoreceptors are affected in various mutant classes. In the most extreme cases that survive to adulthood, retinal photoreceptors degenerate prior to eclosion. These results demonstrate that the kinase encoded by Doa is required at multiple stages of development, for both differentiation and maintenance of specific cell types.

[Key Words: Drosophila; Doa; protein kinase; LAMMER; eye development]

Received February 14, 1994; revised version accepted March 31, 1994.

Families of conserved protein kinases, with homologs in eukaryotic organisms ranging from yeast to man, regulate crucial cellular processes such as the cell cycle, differentiation, and signal transduction (e.g. Neigebom and Mitchell 1991; Woodgett 1991; Meyerson et al. 1992; Pelech and Sanghera 1992; Crews and Erikson 1993; Ko- sako et al. 1991; Neiman 1993; Puziss et al. 19941. In several instances, kinases with high levels of amino acid conservation have been proven to possess functional as well as structural homology, by complementation of mutations in yeast (Jimenez et al. 1990; Lehner and O'Farell 1990; Leopold and O'Farrell 1991; Ninomiya- Tsuji et al. 1991; Hughes et al. 1993) or Drosophila (Sieg- fried et al. 1992).

Protein kinases are grouped into phylogenetically related families, based upon their amino acid sequences (Hanks et al. 1988; Hanks and Quinn 1991). These families are broadly classified into those that phosphorylate

1Present address: Institute of Experimental Endocrinology, Bratislava, Slovakia.

tyrosine residues or those that phosphorylate serine/ threonine residues. Kinases in these two classes possess specific amino acid residues within their catalytic domains, allowing the classification of untested members as either tyrosine or serine/threonine kinases. Recently, however, a small number of protein kinases, classified as serine/threonine kinases, based on sequence, autophos- phorylate on both serine/threonine and tyrosine residues (for review, see Lindberg et al. 19921. Two members of this "dual-specificity" kinase class, wee1 (Featherstone and Russell 1991; Parker et al. 1992} and MAPK-K (Na- kielny et al. 1992), also phosphorylate exogenous substrates on serines/threonines and tyrosines, in addition to autophosphorylating on these residues. However, niml protein kinase, which autophosphorylates in vitro with dual specificity, apparently does not phosphorylate its substrate, wee1, on tyrosines (Parker et al. 1993; Wu and Russell 19931. Thus, further studies on additional dual-specificity kinases are required to understand their function and the significance of their unusual specificity.

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LAMMERprotein kinases

We are investigating the function of the Darkener of apricot (Doa) locus of Drosophila. Doa mutants were isolated in screens for dosage-sensitive modifiers of white ~p~c°t (w a) (Rabinow and Birchler 1989). Because this allele is caused by the insertion of the copia retrotransposon into the second intron of the white (w) gene (Bingham and Judd 1981), many loci isolated in screens for modifiers of w ~ interact specifically with the copia element (Birchler et al. 1989; Rabinow and Birchler 1990). Doa is one of these (Rabinow and Birchler 1989).

Unlike the majority of modifiers of retrotransposon- induced mutations in Drosophila, Doa alleles are dominant in their suppression of w ~ (Rabinow and Birchler 1989) and at least two other copia-induced mutations (Rabinow and Birchler 1990; Rabinow et al. 1993). Mu- tant Doa alleles elevate copia transcript accumulation, probably because of increased rates of copia-specific transcription (Rabinow et al. 1993). The product of Doa has a vital role in the fly, because most mutant alleles are recessive lethal. The requirement for Doa function was characterized previously as occurring during early larval stages. However, complex complementation patterns among alleles allow occassional trans-heterozygotes to escape lethality; their survival rate depends on the direction of the cross producing them, demonstrating that Doa function is maternally contributed to the oocyte (Rabinow et al. 1993). Adult trans-heterozygotes display roughened eye morphology and are also frequently missing bristles on the scutellum and head capsule, suggesting a role for Doa in the development of these structures.

These intriguing phenotypes prompted us to examine the role of Doa in Drosophila development more closely

and to characterize the gene molecularly. We report here that segmentation and nervous system defects are observed in homozygous mutant embryos derived from heteroallelic mothers. In rare homozygous adult mutants escaping lethality, photoreceptor cells degenerate prior to eclosion. Molecular cloning of Doa reveals that it is expressed in developmentally regulated patterns, including stage- and tissue-specific transcripts. The gene encodes a protein kinase, with highly conserved homologs m humans, mice, Arabidopsis thaliana, and the yeast Saccharomyces cerevisiae, which were not recognized previously as being related to each other. The murine homolog is a dual-specifity protein kinase, suggesting that other family members may also possess this property. Finally, when expressed as a fusion in Esche- richia coli, Doa protein autophosphorylates, demonstrating that it possesses intrinsic protein kinase activity.

Results

Retinal photoreceptors are missing in rare Doa homozygotes

We had observed previously that the highly ordered ommatidial pattern of wild-type Drosophila (Fig. 1A, B), is disorganized in Doa heteroallelic individuals (Fig. 1C, D; Rabinow and Birchler 1989). Detailed observations of these defects reveal ommatidia of varying size, as well as disorganization of the interommatidial bristles (Fig. 1D). These defects are drastically increased in rare adults escaping lethality in homozygotes of two unusual alleles,

A ~'7/~ r C E

B

( ;-5;-" ?- . ' : / . k - - /.-/7,-. ' ':,. ",.,,

D Figure 1. Scanning electron microscopy of wild-type and Doa mutants. (A,B) Wild- type; {C,D) DoanD/Doa l°s heteroallelic fly; (E,F) Doa Msue homozygote. Female flies are shown, (A,C,E) 80x magnification; {B,D,F) 800x magnification. Note the ommatidia of varying sizes and disor- dered patterns of interommatidial bristles in the mutants, relative to wild type. In- creasing disorder of the normal wild-type ommatidial array corresponds with increasing severity of the lesion in Doa.

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Yun et al.

Doa Msu2 and Doa vA {Fig. 1E, F). These alleles are almost certainly not null, and we presume that the rare homozygotes possess low levels of Doa function, allowing their survival.

The superficial ommatidial disorganization observed in Doa mutants suggested that the gene might be involved in the development of one or more specific cell types. We therefore examined saggital and transverse sections from flies of the genotypes shown in Figure 1. These reveal defects in both photoreceptors and pigment cells (Fig. 2). Saggital eye sections of heteroallelic Doa adults reveal random interruption of the normal lattice of pigment cells (Fig. 2C). Random vacuolization of the retina is seen in the corresponding transverse sections of these flies. Rhabdomeres, the light-gathering organelle of the photoreceptor cells, are not affected in either number or identity in this genotype. Consistent with the effects of Doa mutations as suppressors of w ~, pigment granules are restored to at least normal levels, as seen in both saggital and transverse sections of Doa heteroallelic flies, compared to w ~ (Fig. 2B, C).

The most severe effects of Doa mutations on retinal organization are found in homozygotes and in one heteroallelic combination examined. In these flies, photoreceptors are completely lacking in the adult retina, as seen in both saggital and transverse sections (Fig. 2D). Photoreceptors in the ocelli (the simple eyes) of Doa homozygotes are also affected, although not as dramati- cally; occassional homozygotes are completely missing one or more ocelli. Pigment granules are randomly distributed in the pigment cell lattice, as might be expected given the variegating phenotype of these flies. The lattice of pigment cells is also randomly disrupted, as in the heteroallelic flies. Transverse sections of the homozygotes further reveal that the sizes of the lamina and optic lobe are reduced, perhaps because of loss of proliferative cues from the photoreceptors.

Photoreceptors degenerate late in deve lopmen t in Doa m u t a n t s

Differentiation of pigment and other accessory cells in the Drosophila eye is an inductive process, directed by the photoreceptor precursors. The photoreceptors them- selves develop through a series of sequential inductive steps, initiated by photoreceptor precursor 8 (for review, see Cagan and Zipursky 1992). The dramatic effects of homozygosity for Doa mutations on photoreceptor differentiation could thus be explained either by failure of the photoreceptors to undergo terminal differentiation or by their differentiation and subsequent degeneration prior to eclosion of the adults. We determined when aberrant development occurred by immunohistochemical staining of developing eye-antennal imaginal discs from mutant third-instar larvae and from pupal retinas at 60 hr of pupal development. The results demonstrate that photoreceptor precursors differentiate normally and then degenerate prior to eclosion of adults.

Antigens common to all neuronal species are expressed normally in the eye-antennal imaginal discs of

A

B

G

D

Figure 2. Tissue-sections of wild-type, w ~ and Doa mutants. {A) Wild type; (B) w~; + / + ; (C) w a, Doan~/Doal°5; (D) w a, DoaMSU2/Doa Msu2. (Left) 1.5 ~m. Transverse sections through the eye and brain, magnified 100x. (Right) 1.0 ~m. Saggital sections magnified 315x. Note the normal morphology, but loss of pigment granules in B (w ~) compared with A (wild type). Heteroallelism for DoaHD/Doa I°s (C) restores pigment accumulation in pigment cells to at least wild-type levels, consistent with the phenotype of Doa as a suppressor of wC Random dis- ruption in the pigment cell lattice is observed in both saggital and transverse sections; it is visible as random vacuolization in the latter. In Doa homozygotes (D), defects in addition to those found in heteroallelic flies include the complete loss of photoreceptor cells, a disrupted lamina, and random accumulation of pigment granules, consistent with the variegating pigment phenotype of these flies. This phenotype is also observed in the heteroallelic combination of Doal°S/Doa M~u (not shown).

Doa homozygotes (Fig. 3A,B), as revealed with antibodies to the neuronal nuclear protein ELAV (Robinow and

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LAMMERptotein kinases

White 1988) and to horseradish peroxidase (HRP, not shown), which specifically label neurons in insects (Jan and Jan 1982). Expression of chaoptin, a photoreceptor- specific antigen (Zipursky et al. 1984, 1985), is also normal in eye-antennal imaginal discs derived from the rare Doa homozygotes (not shown). Progression of the mor- phogenetic furrow, behind which ommat id ia l organization occurs, also appears to be normal. We also examined expression of glass, which encodes a zinc finger DNA- binding protein required for differentiation of photoreceptors (Moses et al. 1989; Moses and Rubin 1991). Mu- tants at glass lack photoreceptors, s imilar ly to Doa homozygotes, and it was thus possible that the lack of photoreceptors in Doa homozygotes was attributable to altered glass expression. Immunohis tochemica l staining of eye- imaginal discs from Doa heteroallelic and homozygous mutants using an anti-Glass monoclonal an-

tibody (Ellis et al. 1993) revealed normal levels and dis- tr ibution of Glass in all genotypes (Fig. 3C, D).

Because larval development of the eye- imagina l discs was normal, pupal retinas were examined at 60 hr of postpupariation development. By this t ime the determination of the various cell types in the ommat id ia is complete (Cagan and Zipursky 1992}. No differences were found between pupal retinas derived from wild-type and Doa heteroallelic or homozygous individuals, when stained wi th antibodies against HRP (which recognizes a general neuronal antigen) or chaoptin (photoreceptor- specific). Because both nonspecific neuronal and photoreceptor-specific gene expression occurs normal ly in eye discs obtained from Doa homozygotes, the photoreceptor cells mus t degenerate after differentiation but prior to the eclosion of adults.

Although immunohis tochemica l staining of eye-an-

A B

(3 D

E

J

F

G H

Figure 3. Immunohistochemical staining of eye-antennal imaginal discs and embryos, and embryonic cuticle preparations. (Leftl Wild-type samples; (right) Doa mutants. All samples were photographed at 122 x. IA,B,C,DI Differentiation of photoreceptor precursors as photoreceptors occurs normally in A {wild-type) and B (Doa homozygotes escaping lethality), as revealed with antisera to the nonspecific neuronal antigens ELAV and HRP (not shown). Differentiation of the neurons as photoreceptors also appears normal in wild-type and homozygous mutants, as revealed by immunohistochemical staining with mAb 24Bt0, directed against chaoptin {not shown). Normal expression of GLASS, a zinc finger transcription factor required for differentiation of photoreceptors, is also oberved in C (wild- type) and D (Doa homozygotes). However, in all cases, the imaginal discs derived from Doa homozygous mutant larvae are smaller than those of wild type. Posterior is to the left; anterior is to the right. (E,F,G,H) Embryonic phenotypes in homozygous embryos derived from Doa heteroallelic females. The Doa mutant embryos result from a cross between a DoaHD/Doa 1°5 mutant female and a Doa~3B/TM3 male. Mutant embryos show a wide range of defects, and those shown are among those developing the furthest, probably because of perdurance of maternally contributed Doa function. Staining of neurons in the embryonic central nervous system, as revealed with anti-HRP is normal in wild-type embryos (E). Doa mutant embryos from the above cross show substantially aberrant neuronal development (F). Al- though some neuronal cells form, the longitudi- nal axons and commissures are completely disrupted in the mutant embyro, relative to the wild type. Embryonic cuticles, mounted in Hoyer's medium and viewed under dark-field illumina- tion, reveal normal segmentation and cuticular structures in wild-type embyros (G). Extensive disorganization and malformation of anterior structures, as well as disorganization of the segmentation pattern, is observed in Doa mutants (H). Anterior is to the left; posterior is to the right.





Yun et al.

tennal imaginal discs from Doa heteroallelic or homozygous larvae shows normal differentation of all cell types, one nonspecific abnormality is readily observed. Eye- imaginal discs derived from homozygous mutants are significantly smaller than wild type (Fig. 3). This obser- vation also extends to at least the wing and leg imaginal discs, although the size differences are less dramatic. These size differences may reflect a nonspecific effect of Doa mutations, rather than a specific role for the gene in cellular proliferation, because homozygous larvae, pupae, and adults are noticeably smaller than wild type.

Segmentation and nervous system defects m homozygous embryos derived from heteroallelic Doa mothers

Recessive lethality of Doa alleles was initially characterized as occurring at early larval stages (Rabinow and Birchler 1989). However, because the direction of recip- rocal crosses influenced the survival rate of Doa trans- heterozygotes, a maternal contribution of Doa function to embryos was suggested (Rabinow et al. 1993). This in turn raised the possibility that defects would be found in Doa mutant embryos derived from mutant mothers. To deplete mutant embyros of as much of the maternal Doa contribution as possible, large numbers of female trans- heterozygotes were produced. These semifertile females were crossed with male heterozygotes of an allele that does not produce any heteroallelic flies. Nonhatching embryos from this cross were scored (for details, see Ma- terials and methods).

Although only 50% of the embyros from this cross were trans-heterozygotes of an inviable heteroallelic combination, only -20% of them hatched. Thus, maternal contribution of Doa product, its mRNA or modified substrates is essential to embryonic development, although rescue by zygotic transcription of the paternal allele occurs occasionally.

Embryos failing to develop from the above crosses were characterized by examination of cuticles, and immunohistochemical staining of the nervous system (Fig. 3E, F,G,H). Defects in both were observed. Both the stage at which embryonic development arrested and the observed defects were variable among individuals, probably because of the residual maternal contribution of Doa and its perdurance in the embryos.

Molecular cloning of Doa

We cloned Doa, benefiting from the r e v e r t a n t Dfd RX1, a transposition of the Dfd locus into 98F1-2 (Hazelrigg and Kaufman 1983), the established location of Doa. Genetic tests demonstrated that the Dfd gxl insertion had generated a Doa allele (for details, see Materials and methods). A wild-type Dfd clone, kindly provided by W. McGinnis (Yale University, New Haven, CT), was used to cross the breakpoint into Doa, and phage clones distal to to it were isolated from a wild-type genomic library. A subclone of one of these hybridized to polytene chromosome bands 98F1-2 (Fig. 4). Restriction mapping of DNA from four

mutant Doa alleles and two independent revertants demonstrated conclusively that we had cloned the locus, delimiting a minimal region of -35 kb encoding the locus (Fig. 4; for details, see Materials and methods).

Doa mutations alter expression of a 2.7-kb transcript

The 7-kb EcoRI-SalI genomic fragment encompassing the closely linked Dfd Rxl and Doa ~B breakpoints (approximate coordinates - 4 to + 4, Fig. 4) was used as a probe to isolate phage from a 3- to 12-hr embryonic cDNA library (Poole et al. 1985). The four cDNA clones obtained hybridize to genomic clones covering 37 kb and encompass the breakpoints and insertions described above (Fig. 4}. The large size of Doa is consistent with the large number of mutant alleles recovered with several different mutagens (Rabinow and Birchler 1989; Peng and Mount 1990; Kurkulos et al. 1991; Rabinow et al. 1993). Two of the cDNA clones derive from putative introns, based on the lack of open reading frames within them and also because they detect no RNA on Northern blots (not shown). The other two eDNA clones substantially overlap on the basis of restriction maps and DNA sequence, comprising a composite eDNA of 2567 bp.

When the composite eDNA was used to probe North- ern transfers, a prominent 2.7-kb RNA was observed throughout development (Fig. 5, lanes 1-13). Because of (1) the nearly ubiquitous expression of the 2.7-kb transcript; (2} the fact that it matches closely in size the composite cDNA encoding a long open reading frame (below); and (3) the fact that several different Doa mutations specifically reduce its accumulation (below), we believe that this transcript encodes the primary form of functional Doa mRNA.

Final confimation that the 2.7-kb eDNA encodes Doa was provided by examination of RNA isolated from both heteroallelic and heterozygous mutants (Fig. 5, lanes 14-- 20). RNA from pupal trans-heterozygotes escaping lethality examined on Northern blots shows virtual elim- ination of the 2.7-kb transcript, compared with heterozygous siblings or wild-type strains (Fig. 5, lanes 14-16). Interestingly, RNA from heteroallelic adults does not show a strong reduction in this mRNA species (not shown), suggesting a developmentally specific defect in expression in one or both of the alleles (HD and 105) used to produce these animals. Doa adult heterozygotes of two of three additional alleles examined also show no- ticeable reductions in the 2.7-kb RNA relative to wild type (Fig. 5, lanes 17,18,19,20).

Developmental analysis of Doa transcription

Analysis of the transcripts hybridizing to the composite eDNA reveal that the transcript pattern of the locus var- ies significantly during development (Fig. 5). In addition to the 2.7-kb RNA described above, major transcripts of 3.8, 4.0, and 5.5 kb are expressed with precise developmental specificity (Fig. 5). A minor pupal-specific transcript >7 kb is also observed. The higher molecular weight transcripts in Figure 5 may represent splicing in-





A

LAMMER protein kinases

B 36 34 32 30 28 26 24 22 20 18 16 14 12 i0 8 6 4 2 0 -2 -4 -6 I I I I I I I I I I , I I 1 I I I I I I I I I

kDoall k, Doa8

kDoa7

kDoal

-8 -i0 -12 I I I

~Doa2

y3B

BBB B B xs E BE .~BBS ~ESX E B S i i ~1 i i ] , i r

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Figure 4. Physical organization of the Doa locus. (A) In situ hybridization to polytene chromosomes using a cloned genomic fragment. The 7-kb EcoRI-SalI subclone of phage Doal (approximate coordinates - 4 to + 41 was used as a probe to salivary gland polytene chromosomes of the wild-type strain Canton-S. The clone hybridizes to 98F1-2, the location of Doa. (B) The Doa genomic region: restriction map, genomic phage clones, structure of the sequenced eDNA, and lesions in four mutant alleles. Restriction sites shown are BamHI (B), SalI (S), EcoRI (E), and XhoI (X). The scale shows distances in kilobases; the breakpoint of Dfd Rxl, which was used as the entry point to the Doa locus, is the arbitrary origin, 0.0. Phage from the chromosome walk are shown above the genomic restriction map. The vertical arrows indicate the Doa v3B and Dfd nxl breakpoints. The insertion sites of the two insertion elements in Doa HD and Doa D~m are also shown. An exact intron-exon map of the sequenced eDNA, as determined by sequencing genomic fragments hybridizing to it, is shown below the genomic restriction map. The hatched region in the most 5' exon denotes the uncertainty in the site of transcriptional initiation. The 3' hatched region indicates the long untranslated region.

termediates, because genomic DNA subclones that do not contain exons present in our composite cDNA (introns 3 and 4: Fig. 4} detect the 4.0- and 5.5-kb RNAs when used as probes on Northern transfers (not shown). Alternatively, these RNAs may encode differentially spliced, initiated, or terminated functional messengers.

Tissue-specific transcripts were also observed. One, of 1.9 kb, is restricted to the first 4 hr of embyronic development (Fig. 5, lane 1). Because Doa alleles have maternal effects, and zygotic transcription does not commence until - 2 hr of development, we determined whether the 1.9-kb transcript was of maternal origin. Analysis of RNA prepared from dissected ovaries of adult females reveals the 1.9-kb and ubiquitous 2.7-kb transcripts (not shown), confirming that the former is maternally contributed to the developing oocyte. The structural basis for its reduced size is unknown.

A second tissue-specific transcript is of particular in- terest, because of the effects of D o a mutations on retinal development. RNA prepared from total adults shows predominant transcripts of 4.0, 3.8, and 2.7 kb. (Fig. 5, lane 10). Minor transcripts of 5.5 and 2.9 kb are also found. Adult Drosoph i la were fractionated into head and body samples, from which RNA was prepared. The ubiquitous 2.7-kb transcript is reduced in quantity in the head fraction, and a novel 2.9-kb transcript is found (Fig. 5, lane 11, overexposed in lane 13). The 4.0-kb transcript is found in both head and body fractions, whereas the 3.8-kb transcript is found only in samples from adult bodies (Fig. 5, lane 12). It is possible that the 2.9-kb transcript is actually present throughout the nervous system and is observed only in the head samples because neural cells are highly enriched by this fractionation.

To identify other possible transcription units in the





Yun et al.

1 2 3 4 5 6 7 8 9 10 11 12 13

2.7-

1.9-

rp49 -

region, genomic DNA subclones covering the entire 50- kb cloned region were used to probe Northern transfers of RNA samples derived from 0- to 4- and 16- to 20-hr- old embryos, pupae, and adults. With one exception, these probes detected only the transcripts hybridizing to the cDNAs described above. The exception, a 1.8-kb transcript, is expressed only in pupae and adults. It is detected only with genomic subclones from beyond the BamHI site approximately at coordinate +32 (Fig. 4), which is beyond the region any genomic clones hybridized to Doa transcripts. The 1.8-kb transcript cannot be related to Doa, because of both the locations of lesions in mutants and its restricted developmental expression.

4.0-

2.7-

14 15 16 17 18 19 20

. " ' - o o I o e Q ! Figure 5. Northern analysis of Doa expression during development and in mutants. A 1.8-kb cDNA clone containing the Doa open reading frame was used as the probe. The same transfers were reprobed with a probe for RNA from the ribosomal rp49 gene as a loading control. (Lanes 1-13) RNA from developmentally staged organisms. ILanes 1-6) Four hour windows during embryonic development. (Lane 1) 0-4 hr; (lane 2) 4--8 hr; (lane 3: 8-12 hr; (lane 4} 12-16 hr; (lane 5) 16-20 hr; {lane 6) 20-24 hr; Ilane 7) first-instar larvae; (lane 8) late third-instar larvae; (lane 9) 0- to 24-hr-old pupae; (lane 10) total adult (mixed sex; no differences were observed between total RNA derived from sexed flies; not shown); (lane 11) adult head; (lane 12) adult body; (lane 13) the same adult head sample as lane I 1, overexposed to show the 2.9-kb head-specific transcript. The 2.7-kb transcript is expressed throughout development. The 1.9-kb transcript restricted to 0- to 4-hr-old embryos is maternal in origin, because zygotic transcription does not commence until - 3 hr of development, and this transcript can also be detected in RNA derived from dissected ovaries (not shown). The RNAs >2.7 kb may represent unspliced precursors to the 2.7-kb RNA (see text for details). (Lanes 14--20) RNA from Doa mutants shows a reduction in the ubiquitous 2.7-kb transcript. (Lanes 14-16) RNA from Doa mutant pupae. (Lane 14) Canton-S; (lane 15) Doa heterozygous mutants (alleles 105 or HD, mixed); (lane 16) Doa heteroallelic mutants (105/HD). Samples in lanes 15 and 16 are derived from siblings originating from a cross between balanced stocks of Doa l°s and Doa HD (for details, see

Materials and methods). (Lanes 17-20) RNA from adults heterozygous for Doa mutations. (Lane 17) Canton-S; (lane 18) Doa~3B/TM3; (lane 19) DoaHD/TM6; (lane 20) DfdRX~/TM3. Note that heteroallelism for Doa mutations virtually elimi- nates expression of the 2.7-kb transcript in pupal samples, whereas in adult heterozygotes, expression of the same transcript is also reduced in two of the three mutant strains examined, relative to wild type (Canton-S).

Doa encodes a protein kinase

To further our understanding of Doa function, we sequenced the two cDNAs yielding a single long composite clone. The deduced amino acid sequence of the 1.5-kb open reading frame contained in the 2567-bp composite cDNA sequence (Fig. 6) reveals perfect matches wi th the conserved residues and domains of a ser ine- threonine protein kinase (Hanks et al. 1988; Hanks and Qu inn 1991). A 3'-untranslated region of 1035 bp terminates in a poly(A) tail of 20 nucleotides, indicating that the entire 3' end of an m R N A was cloned. Each genomic fragment hybridizing to the cDNA was also sequenced to determine the exact exon- int ron junctions. The composite cDNA comprises 10 exons (Figs. 4 and 6).

The composite cDNA of 2567 bases nearly matches the size of the highly expressed ubiquitous 2.7-kb RNA and, thus, is nearly full length. However, the conceptual translation product of the cDNA began wi th a his t idine residue (tailed arrow, Fig. 6), indicating that the initiat- ing methionine residue and the 5' end of the m R N A were missing. To identify the probable ini t iat ing methionine residue, we used a probe from the 5' end of the cDNA to identify the genomic sequences hybridizing to it. The sequence of this fragment revealed an in-frame methionine codon with no open reading frame 5' to it, 13 amino acids 5' to the end of the composite cDNA (Fig. 6). A second methionine codon is found 7 residues down- stream of the first; either might be uti l ized in vivo. The DNA sequence adjacent to the upstream potential initi- ating methionine (GACA ATG) shows two of four matches wi th the Drosophila consensus translat ion start (C/A AA C/A ATG; Cavener 1987), although the down- stream potential init iator (TCAG ATG) shows fewer matches, favoring the upstream meth ionine as the translational initiator. Although a l ikely candidate for the ini- tiating methionine was found in an appropriate context and location, this analysis does not e l iminate the possi- bil i ty that yet another intron separates the sequenced cDNA from its actual translation start. The point of Doa transcriptional ini t iat ion also may lie nearby, because the composite eDNA is nearly full length compared wi th the 2.7-kb transcript, and RNA transfers probed wi th re-

s t r i c t i o n fragments 5' to the SalI site at +30 (Fig. 5) do not reveal Doa transcripts.

The putative protein kinase catalytic domain lies to-






-% ATGTGTGTACGTTTTCAGATGCCCAGAACAAGGAGACTCCACCACTCAAGGGATCGCTCCTCGGcGGGcACACGAGACAAGCGCCGACGACACGATACTGCCGATCATTCGCCACCCCTA 120 M C V R F Q M P R T R R L H H S R D R S S A G T R D K R R R H D T A D H S P P L 40

GCTGAGGCCCCATCGCCGCCGCGCATCACCAACACACATCACACAAGATCGGCGGCcAAGCGAAGGCGACAcGAGCTCGATGCCAAGAAGGCCCAGATATCCAAGGAACCCACTTTCGAT 240 A E A P S P P R I T N T H H T R S A A K R R R H E L D A K K A Q I S K E P T F D 80

GACAGcATCTCAACCCGCCGACGCAAGGAGCGTTCAAAGCGTTCGCACCGCAAGTCGCCCGCCGCCAGTCGACGGCAGCACAAATACCG~TACAGGGACGAGACGTCCCATTCGAGCTCC 360 D S I S T R R R K E R S K R S H R K S P A A S R R Q H K Y R Y R D E T S H S S S 120

CGCCGGCGGCACCGTGATCGCGCCAAGGACGAACGCGACAGCCK~ACGCAACAACCGCCAGTCG GGCTAA'GAC AAA CG TC A CA AGC GCC TCAT AAG TGATGCTGATGGTCACTTAATTTAC 480 R R R H R D R A K D E R D S G R N N R Q S Q A K T A K P V I Q D D A D G H L I Y 160

CACACCGGAGACATTCTCCATCACAGATATAAGATCATGGCCACACTGGGCG T AGCK~AACTTTTGGACGTGTGGTCAAGGTCAAAGATATGGAGCGTGATTACTGCATGGCTTTAAAGATT 600 H T G D I L H H R Y K I M A T L G E G T F G R V V K V K D M E R D Y C M A L K I 200

ATTAAGAACGTGGAAAAGTA•CGCGAAGCTGCCAAGCTGGAAATAAATGCTTTGGAAAAGATTGCCCAAAAGGATCCGCATTGTGATCATTTGTGCGTCAAAATGATTGACTGGTTTGAT 720 I K N V E K Y R E A A K L E I N A L E K I A Q K D P H C D H L C V K M I D W F D 240

TATCATGGACACATGTGTATAGTTTTTGAAATGTT~TCAGTGTTTTCGATTTTTTGCGAGAGAACAACTATGAGCCATACCCGCTGGACCAAGTGCGCCATATGGCCTATCAATTA 840 Y H G H M C I V F E M L G L S V F D F L R E N N Y E P Y P L D Q V R H M A Y Q L 280

TGCTACTCTGTGAAATTTCTACATGACAATCGTTTAACGCACACAGATCTCAAGCCGGAGAACATACTCTTCGTCGACTCCK~ATTATACTTCTCACTATAATCATAAGATTAACCGCGAG 960 C Y S V K F L H D N R L T H T D L K P E N I L F V D S D Y T S H Y N H K I N R E 320

GTGCGCCGCGTCAAGAATACGGACGTTCGCCTAATCGACTTCGGGTCGGCCACCTTCGACCACGAGCACCACAGCACAATTGTCTCGACGCGACATTACCGCGCGCCAGAGGTCATACTG 1080 V R R V K N T D V R L I D F G S A T F D H E H H S T I V S T R H Y R A P E V I L 360

T GAGCT~TGGTCACAGCCGTGTGACGTTTGGTCCATTGGGTGCATCTTGTTCGAACTGTA TCTGGGAATCACGCTC TTCCAAACGCA CGACAA TCGCGAACACTTGGCCATGA TGGAG 1200 E L G W S Q p C D V W S I G C I L F E L Y L G I T L F Q T H D N R E H L A M M E 400

CGAATCTTGGGACAAATACCATATCG CATGGCACGCAAAACCAAAACAAAGTACTTCTATCATGGTAAGTTAGATTGGGATGAGAAGTCCAGTGCGGGTCGCTACGTCCGTGAT•ACTGC 1320 R I L G Q I P Y R M A R K T K T K Y F Y H G K L D W D E K S S A G R Y V R D H C 440

T AAGCCGTTGTTCCTGTGCCA~GAGCGACAGCGAGGACCACTGTGAGCTCTTCAGTCTGATCAAGAAGATGCTGGAGTACGAGCCGTCGTCACGCATCACGTTAGGrGAGC~2CCT~CAT 1440 K P L F L C Q L S D S E D H C E L F S L I K K M L E Y E P S S R I T L G E A L H 480

CATCcGTTCTT'I~ATAGGCTGCCAcCACACCATCGAGTAC'G'~X~AGGTCAGCAACAAGCAGCCcCT~I~CGTCGGGCAG CAGCAG CCGCG~CGATCGCATAGCCTCTC CAGA ~ A G ~ T T A 15 6 0 H P F F D R L P P H H R V G E V S N K Q P L S S G S S S R E R S H S L S R * 517

CAC, CGATTTGGTTATTGTTGACAGACAGCGCTAATTAAGCACTCACTCA TGCAAACATACACTCAAGGCAGTCG TCCAGCAACGTCTTGCACCCGACTCGTAGAGTAGACCAGCAACTTT 1680

ACGACGAACCCTATTTTAAGAGCAAAAGTCTTATTTTAAATTTTTGAACTTGACTTGTATAGATGTTTATTATACAAAAATCCA TTGATCCCTTTTGATCCTTTGTTTCCAAATACTTCG 1800

AGCACATGTTGCTGTGCATTATATTTAGAGCTGTGTAGCGCCATCGTGCGCTTGTGCATTGTACCATACCCGCCAA•AGTGTTTGTTCTGTAATGATAACCAAATCGTGTATATTATTGA 1920

AAGTGCGGTGCGT•TTCCCGACACCAGAACCTAATTGTAAATGTCTTGCATTGTGCACACCCCAAGGTGGAGGAGACTAATTGAA•CGACTGCAAACCGATAATTGGAATATACAAATGT 2040

AGTTGTAAAATAAGAAAGGTTCGCTTAAGCGCTAGTTGGAATGCATCCCCATGTCTACACCTGTCACTGAACCGGGAGCCGACTAATTTGTAATTAATGCACCATTTCGAGAGCGTGAAC 2160

TGAAAGCTAATGCAAGCCATTATGTACACCGCCCACGAATGTACGGATTATTGTAAATGTAAAGAGCAATTGTTATGCCACCATGTGTA CACTTCAGCGATCGCAGCGCATTTTGTGTAA 2280

ATTCAGTAGCAGCCCCAGAGTCATCTCTCTTAGAATACGTTTTCAAATGTGTAGAACCGTTGCCGGCCTGTTTTGTTCGTTTT•GTTTACAACCGAATATcGAAAGATCAAcCCATTTAC 2400

A~aT~^~A~a~AaA~TaT~T~TAT~TTaT~eaTa~TT~aTT~ATT~GA~TTaGTTT~T~TAcAT~TATT~TTAT~TTa~TTaTTT~TTT~ 2520 T a T T ~ G T Q ~ T ~ T C ~ T ~ 2606

Figure 6. The nucleotide and deduced amino acid sequence of a Doa-encoding composite cDNA. Sequence of 2.6 kb is shown. The predicted amino acids are shown below the eDNA sequence. Translation terminates with the codon TGA (nucleotides 1552-1554), yielding a 1055-bp 3'-untranslated region (including a 20-bp poly(A) tract). The downward arrowheads indicate the positions of introns. The downward-pointing tailed arrow indicates the start of the sequenced cDNA clones. The sequence yielding the initial 13 amino acids is derived from genomic clones hybridizing to the most 5' cDNA fragment. The open reading frame shown commences with the initial methionine residue shown, although we cannot confirm that this is the actual site of translation initiation. The protein kinase catalytic domain is delimited with upward-pointing tailed arrows.

ward the caroboxyl terminus of the deduced 517-amino- acid Doa product, beginning at Y170 and ending at F484 (Fig. 6}, assuming translat ion begins at the AUG 39 nucleotides 5' to the end of the composite eDNA-- the lysine residue crucial to Mg-ATP binding that is invari- ant in all protein kinases is K199. The kinase catalytic domain is del imited by upward-pointing tailed arrows in Figure 6.

As expected, Doa protein (DOA) possesses intr insic protein kinase activity. When expressed in E. coli, a purified fusion protein containing the entire catalytic domain autophosphorylates in a f i l ter-renaturat ion assay (Fig. 7; for details, see Materials and methods). The purified fusion protein, detected wi th Coomassie blue, is recognized by antisera directed against the maltose-bind-

ing domain used in the fusion, is the 84 kD predicted for the fusion construct, and all three properties (stained protein, immunodetec ted material, and protein kinase activity) comigrate on SDS gels.

Highly conserved protein kinases homologous to Doa in widely diverged species define a new protein kinase family

Searches of computer data bases wi th the conceptual Doa translation product and DNA sequence revealed a number of highly conserved homologs. The first identified was a mur ine kinase known as cdc2-1ike kinase (elk, Ben-David et al. 1991), or STY (Howell et al. 1991)(Fig. 8; Table 1). Clk/STY was independent ly identified in two





Yun et al.

1 0 7 - -

7 6 - - .

1 2 3 4

: . .. . :

human T-cell c D N A library with degenerate oligonucleotides designed to identify serine/threonine protein kinases related to p34 cat2 (Johnson and Smith 1991). A second human homolog was identified in the catalytic domain sequence of pSK-G1 {Hanks and Quinn 1991), which was isolated from a HeLa cell c D N A library, also via use of degenerate oligonucleotides (S. Hanks, pets.

Figure 7. DOA protein possesses intrinsic protein kinase activity in vitro. A cDNA subclone encompassing the entire Doa catalytic domain was expresssed in E. coli as a fusion with maltose-binding protein {mBP) and purified on an amylose column. Following gel electrophoresis and transfer to nitrocellulose membrane, the samples were denatured in guanidine and rena- tured, before being incubated in kinase assay conditions. (Lane 1) IPTG- induced crude lysate from E. coli harboring the vector used for expression (without Doa); (lane 2) IPTG-induced crude lysate from the MBP-Doa fusion strain; (lane 3) purified MBP- DOA fusion protein; (lane 4) crude lysate from a TrpE-clk fusion strain, as a positive control. Approximately 3 ~g of amylose column-purified material was loaded in lane 3, compared with 30 ~g of crude lysates. DOA and Clk fusion proteins autophos- phorylate, demonstrating that they possess intrinsic protein kinase activity. The trpE-Clk fusion protein coincidentally has the same molecular mass (84 kD1 as the MBP-DOA fusion.

laboratories using using anti-phosphotyrosine in screens of c D N A expression libraries derived, respectively, from Friend erythroleukemia cells, and the PC19 embyronal carcinoma cell line. Clk/STY autophosphorylates on serine, threonine, and tyrosine residues, as shown via reaction with anti-phosphotyrosine, as well as phos- phoamino acid analysis of autophosphorylated bacteri- ally expressed fusion protein. Clk/STY also phosphorylates an artificial substrate peptide, poly(Glu, Tyr) on tyrosines in vitro (Ben-David et al. 19911, suggesting that this specificity extends to exogenous substrates. Clk/ STY is expressed in a wide variety of tissues. Its transcription patterns change following stimulation of em- bryonal stem cells to differentiate (Howell et al. 1991). These RNAs exist in at least two forms in several mu- fine tissues and are altered in size in several malignant cell lines (Ben-David et al. 1991; Howell et al. 19911.

Relatively restricted amino acid identities in a few kinase catalytic domains place clk in the phylogenetic cdc2 kinase family (Hanks and Quinn 1991 ), although no conserved functions are implied by this grouping. No evidence exists either for or against clk being a cyclin- dependent kinase. Aside from the homologs described here, the catalytic domain of clk is most closely related to the MAP kinase FUS3 from S. cerevisiae (Ben-David et al. 1991).

Further searches of the literature on protein kinases and of the GenBank and EMBL D N A sequence libraries revealed three additional protein kinases highly homologous to murine clk and to Doa that had not been recognized previously as being related. A human homolog, also named cdc2-1ike kinase, was isolated in screens of a

1 70

Doa ......................................................................

psk-G1 ......................................................................

Humanclk ......................................................................

Musclk ......................................................................

KNSI MSQNIQIGTR KRSRANMNNS T~fGPANNTS SNKT?LDNFE ETRTNKLLDE MFARQNSFLT DNLRNSLDLN

71 140

Doa ......................................................................

psk -GI ......................................................................

Humanclk ......................................................................

Musclk ...................................................................... KNSI QADNPLRPRQ HQHQLFLDNE NAIELDEEPR IIN'I~INNSN NHNSSRVDED ADDDIIFIKE QPlQFSSPLI

141 210

Doa ............. MCVRFQM PRTRRLHHSR DRSSAGTRDK RRRHDTADHS PPLAEAPSPP RITN'rHHTRS

psk-G1 ......................................................................

Humanclk ......................... MR--K RTYCPDWD-- DWDYGKWRS- SSHKRRKRSH SSAQENKRCK

Musclk ......................... MR--K RTYCPDWDER DWDYG~WRS- SSHKRKKRSH SSAREQKRCR

KNS1 LPSSSSINNN NNIVTSNNPG CGTAATSN-T YIT~PKKFK- Q-TISLPQLP LSKLSYQSNY FNVpDQTNAI

Doa

psk-Gl

Humanclk

Musclk

KNSI

211 280

AAKRRRHELD AKKAQISKEP T?DDSIST~R RKERSKRSHR KSPAASRRQH KYRYRDETSH SSSRR...RH

......................................................................

YNHSKMCDSH YLESRSIN-K DYHSRRYIDE YRNDYT~GCE PGHRQRDHES R-QNHSSK-S GR-G-SSYKS

YDHSKTIDSY YLESRSIN-K AYHSRRYVDE YRNDY.MGYE PGHPYGEPGS R-QMHSSK-S GR-G-SSYKS

VPRV'I~TENE LLHLTG-CAK TLEGNKAVNL TIAHSTSPFS NPPAQIASLP QSNLKKQIGS -LRKFTSNGS

281 350

Doa RDRAKDERDS GRNNRQSQAK TAKPVIQDDA DGHLIYHTGD IL..HHRYKI MATLGEGTFG RVVKVKDMER

psk-Gl ............................................... -E- VS ........... QSV-HR-

Humanclk KH-IHHSTSH R-SHGD ................................ E- VD ..... A-- K--ECI-HKA

Musclk KH-SRHHTSQ HHSHGK-HRR KRSREVE--E E .... CQS-- V-..SA--E- VD ..... A-- K--ECI-HKV

KNSI SESASSNKSN ............... FKT-K ---YVYQEN- -FGSGG-~/V KDL--Q .... K-LKCI-NKY

I

351

Doa DYC.MALKII KNVEKYREAA KLEINALEKI

psk-Gl GGARV ........... K--- R .... V ....

Humanclk GGRHV-V--V ---DR-C--- RS--QV--HL

Musclk GGRRV-V--V ---DR-C--- QS--QV--HL

KNSI EPNYV-V-V- RA-DR ...... T-LRILQT-

420

AQKDPHCDHL CVKMIDWFDY HGHMCIVFEM LGLSVFDFLR

NE---DNKN- --Q-F ........... S--L .... T .... K

N~--NSTFR --Q-LE--EH ---I ..... L .... TY--IK

NTT---STFR --Q-LE--EH R--I ..... L .... TY--IK

LNN--QGQFQ -LLLREC--- KN-I-L-TDL y-R-IY--MC

II

421

Doa ENNYEPYPLD

psk-Gl D-N-L---IH

Humanclk --GFL-FR--

Musclk --SFL-FRM-

KNS% S-GIAq~-.A

Ikm

psk-Gl

Humanclk

Musclk

KNSI

Doa

psk-Gl

Humanclk

Musclk

KNS 1

• . • -S--S-A

• . , -TLI-P-

• . . -TIV- P-

GKRKIL-- P~

561

' i l t ~ l . m ~ t . . . . . . . . .

I-E-I/--M--

I I I IV

490

QVRHMAYQLC YSVKFLHDNR LTHT6LKPEN ILFVDSDY . . . . . . . . TSHY NHKINREV,,

. . . . . . F - - - QA . . . . . . . K . . . . . . . . . . . . . . N - - - . . . . . . . . ELT- -LEKK-DE..

H I -K . . . . I - K - -N - - -S -K . . . . . . . . . . . . . . Q - - - . . . . . . . . -EA- -P - -K -DE . .

H I -K . . . . I - K - -N - - -S -K . . . . . . . . . . . . . . K - - - . . . . . . . . -EA- -P-MK-DE..

LISGHCR-~I R--C .... ~ II .......... IC-ETHIA QKLPLK-VQS L~-RR--ASK

Via VIb

560

VRLIDFGSAT FDHEHHSTIV STRHYRAP~'V ILELGWSQPC ~VWSIGCILF ELYLGITLFQ

A-W ............................................ I- -Y-V-F ....

IKVV ...... Y-D ..... L ............. A ................ I -y---F-V-P

IKVV ...... Y-D ..... L ............. A ................ I -Y---F-V-P

IKI ...... I -HY-Y-PPVI ......... I V-G .... F-- -I---A-V-V --VI-ES-YP

VII VIII IX

630

MERILG~QIP YRMARK . . . . . . . . . . . . . . . . . T,KTKYF YHG..KLDWD EKSSAG . . . .

........ FP S--I-- , ................ -R-Q .... R-. ,R ..... NT---, . ..

...... IPL- KH-IQ-. ................ -R-R--- H-D. ,R ..... H .... ,. ,.

...... PL- KH-IQ-. ................ -R-RR-- H-D..R ..... H .... ....

-Q--N. PF- TDIID-MFYK SKHKLGNSPS DLNSTVI-H- DRKTLS-Q-P --NKR-DTIT

Doa

psk-Gl

Humanelk

Musclk

KNSI

Doa

psk-Gl

Humanclk

Musclk

KNSI

x

6 3 1 6 8 0

. . . . . RYVRD H C K P L F L C Q . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L S D S E D H C

. . . . . - - - - E N . . . . RRYL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T - E A - E - H

. . . . . ---SR A .... KEFM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . --QDVE-E

. . . . . - - - S R R . . . . KEFM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - Q D A E - E

TEKSMKR-LQ S-DR-DIYIS KVLKQDYGDS LSINWNLPPE KNWSLINSKL AWKRQTHSSS SSTT-ELDKE

XI

701 768

ELF . . . . . . S LIKKMLEYEP SSRITLGEAL HHPFFDRLPP HHRVGEVSNK QDLESGSSSR ERSHSLSR

Q--. .... D --ES ...... AK-L ...... Q .... . ................................

R--. ..... D --Q ..... D- AK .... R--- M ..... L-KK SI ..........................

F--. ..... D -VG-I---D- AK .... K--- K .... YP-KK -T ..........................

TFLFWYWFID -LR--F-FD- TK---AKD-- D-EW-NLGIL DDGIATYN-T QG ................

Figure 8. The LAMMER protein kinase family. Alignment of Doa with human clk, psk-G1 [human), mouse clk, and KNS1 {yeast}. Amino acids identical between Doa and any of the other proteins are indicated by hyphens. Gaps in alignment are indicated by periods. The open box represents the LAMMER motif, found only in members of this protein kinase family, which all family members possess in an identical location relative to kinase catalytic domains, as marked.





LAMMERprotein kinases

Table 1. Comparative values of amino acid identity among LAMMER kinases

Doa H u m a n clk pSK-G1 Mouse clk AFC1 KNS1

100 62 75 62 48 38 Doa (100) (46) (56) (46) (36) (27)

100 67 92 62 40 Human clk (100) (55) (87) (46) (29) 100 64 45 35

pSK-G1 (100) (54) (35) (26) 100 77 40

Mouse clk (100) (56) (30) 100 41

AFC1 (100) (32) 100

KNS1 (100)

The top number in each case represents amino acid identity in the kinase catalytic domain; the number in parentheses compares the overall amino acid identity between the proteins. The species of origin of the homologs are Doa, Drosophila melanogaster; clk, cdc2-1ike kinase (human and mouse); pSK-G1, human; AFC1, Arabidopsis thaliana; KNS1, S. cerevisiae. The sequence comparisons with AFC1 and the overall comparison with pSK-G1 were made possible by Judith Bender and Gerry Fink, and Steve Hanks, respectively, who generously provided unpublished data.

comm.), pSK-G1 is probably encoded at a locus distinct from that of human Clk, based on the numerous differences in amino acid sequence between the two putative kinases distributed throughout the protein (Fig. 8), essentially ruling out alternative splicing as the origin of the RNA that yielded these cDNAs.

A third homolog, Kinase Nex t to Spa2 (KNS1), was identified in S. cerevisiae (Padmanabha et al. 1991 ). This putative kinase gene was identified in sequencing a region 3' to the SPA2 locus. The gene has no identified function nor a mutant phenotype when deleted or overexpressed, indicating that it is either nonessential or that a redundant function is encoded in the yeast ge- nome.

Alignments of the deduced amino acid sequences are shown in Figure 8; the extensive amino acid identity among these homologs is summarized in Table 1, which also includes a comparative summary of sequence data of the entire pSK-G1 cDNA, as well as that of AFC1, an A. thaliana homolog (J. Bender and G. Fink, pers. comm.). Unpublished data on pSK-G1 and AFC-1 was generously made available by S. Hanks {Vanderbilt Uni- versity, Nashville, TN) and J. Bender (Whitehead Insti- tute, Cambridge, MA).

These kinases all display similar structure and high amino acid identity. Their catalytic domains are located near the carboxyl terminus; noncatalytic domains of varying length lie at their amino termini. Spacing among the catalytic domains is highly conserved, with the exception of the S. cerevisiae homolog KNS1, which possesses several small inserts between catalytic domains relative to the other four family members. Amino acid residue coordinates referring to the family are based on the relationships displayed in Figure 8, which are num- bered from the first residue in the substantially longer amino terminus of KNS1.

All family members contain potential nuclear localization signals, as recognized during the characterization of STY (Howell et al. 1991). Their significance requires

further analysis, although their presence in all family members suggests that they may be utilized.

More striking than the amino acid identity values of catalytic domains among these protein kinase homologs are the long runs of 100% amino acid identity in domains critical to protein kinase function and that are implicated in interactions with substrates. Examples are found in domain VIb, and the 50 amino acid residues encompassing domains VII, VIII, and part of domain IX (amino acid residues 501-549). In addition to playing a role in the transfer of the ~/-phosphate from ATP to the peptide substrate, these regions may be involved in substrate recognition (Taylor et al. 1992).

In addition to extremely high amino acid sequence identity of kinase-related motifs, virtual 100% identity of a motif not related to any known protein kinase or other function is found at the amino terminus of kinase domain X. This motif reads EHLAMMERILG in all homologs except in S. cerevisiae, in which three substitu- tions occur (Fig. 8, box). A similar sequence containing a single conserved substitution is also found in AFC1 (J. Bender and G. Fink, pers. comm.). Correlations of the location of this motif with the known three-dimensional structure of other protein kinases (Taylor et al. 1992; DeBondt et al. 1993) places it in an a-helix below the substrate-binding cleft, where it might be reasonably expected to make contact with substrates. Combined with identical spacing and high amino acid identity levels throughout catalytic domains, the LAMMER motif de- fines a new protein kinase family, and we suggest the name LAMMER kinases to describe it. The LAMMER kinases must play a role in the regulation of a basic cellular function, because they are highly conserved in widely diverged eukaryotes, including fungi, green plants, and animals.

Discussion

Mutants at the Doa locus were characterized previously





Yun et al.

as increasing transcript levels of the copia retrotransposon, probably through altering transcription rates (Rab- inow et al. 1993). Additionally, Doa mutat ions decrease levels of transcripts t runcated in the copia element inserted in w ~ (Rabinow and Birchler 1989). Here, we demonstrate an essential role for Doa in Drosophila embryonic development and maintenance of photoreceptor cells. The product of the locus is a protein kinase that possesses the potential for dual specificity, with homologs in widely diverged eukaryotes defining a novel family.

Aside from the analyses presented here, little is known about the function of the LAMMER kinases. The phenotypes of Doa mutan ts suggest that the kinase is required during both embryonic and imaginal development. Dif- ferentiation, maintenance, and proliferation of specific cell types are affected, because photoreceptors degenerate in the retina of Doa mutants , whereas the imaginal discs of mutan t s are smaller because of a reduction in cell number. Because Doa regulates copia transcript levels, it is particularly intriguing that potential nuclear localization signals are found in all LAMMER kinases, raising the possibility that they may phosphorylate nuclear proteins directly.

Defects in the adult eye are not restricted to photoreceptors and are also observed in pigment cells and in the organization of interommatidial bristles. These defects are only observable in animals that possess residual Doa activity; thus, these phenotypes may be less severe than those that would be observed in a complete null back- ground. Defects in both embyronic segmentat ion and differentiation of the nervous system are found in zy- gotes substantial ly deprived of both maternal and zygotic Doa function. The variability in the development of these embryos suggests that there must be continued contribution of Doa function from their heteroallelic mutan t mothers. Small amounts of residual function may allow cell differentiation and survival past the stage when Doa is actually required, during both imaginal and embryonic development. Further analyses to determine the role Doa plays during embryogenesis and photoreceptor development will require el imination of residual activity using genetic means.

The highly conserved sequence identity and structure of the LAMMER protein kinases suggest that they regulate one or more conserved cellular processes. Further analysis of the biological consequences of Doa mutations and the identification of molecules wi th which it interacts will help to elucidate the role of the LAMMER protein kinases throughout eukaryotes.

M a t e r i a l s a n d m e t h o d s

Drosophila culture and genetic crosses

Stocks and crosses were grown on standard medium (cornmeal, molasses, agar). All crosses were performed at 25°C. Crosses with Dfd Rxl were performed with Doa alleles HD, 15, and CC, the former two of which have normal cytology. Each allele was crossed as females with two different balanced stocks carrying DfdUX~: Df(3R)30c76 Tp(3:3)Dfd Rx~, ri pv/TM3, kindly provided

by E. Stephenson (University of Alabama, Tuscaloosa), and Tp(3:3)DfdRXI pV M(3)$31/TM3, kindly provided by W. McGin- his. The Df(3R)30c76 Tp(3:3)Dfd Rxl, ri pp/TM3 stock was found to suppress w ~, a phenotype typical of Doa. Crosses to generate heteroallelic DoaHO/Doa l°s individuals were performed as described (Rabinow et al. 1993). The two alleles yielding extremely rare homozygotes, Msu2 and 7A, were induced by ethylmethane sulfonate (EMS) mutagenesis and gamma-irradi- ation, respectively. Neither is homozygous viable, except at extremely low rates when balanced with TM6, for unknown rea- sons. Msu2 and 7.4 are probably attributable to position effects, based on their variegating phenotype when homozygous, the existence of cytologically visible breakpoints in or near the gene, and their ability to complement the recessive lethality of some but not all other Doa alleles (Csink et al. 1994; B. Yun and L. Rabinow, unpubl.). Additionally, in combination with Doa alleles allowing survival of trans-heterozygotes at or near Men- delian ratios, both Msu2 and 7A often produce eye-roughening and complete suppression of w ~ to wild-type pigment levels, both phenotypes found in trans-heterozygotes of mutant Doa alleles (B. Yun and L. Rabinow, unpubl.). The induced revertant Doa HD2R2 was induced during crosses of HD with the A2-3 P-element at 99B (Robertson et al. 1988). The spontaneous revertant, Doa HD1R~, was recovered as a homozygote and occurred on a e Doa riD1 chromosome in a stock balanced with TM3.

A Deformed revertant is a Doa allele

We tested whether a Doa allele had been induced by the Dfd RX~ transposition by performing complementation tests based on the recessive lethality of Doa. Two different stocks of Dfd Rxl were crossed independently with three Doa alleles, two which have normal cytology, to rule out the possible induction of closely linked secondary recessive lethal mutations in both the Doa and Dfd Rx~ chromosomes. No Dfd Rxt segregants from either stock survived as trans-heterozygotes with any Doa allele tested. Additionally, Dfd axl suppresses w ~ (one stock tested), a phenotype characteristic of Doa.

Embryonic phenotypes

To obtain embryos as depleted of the maternal Doa contribution as possible, DoaHD~/Doa ~°5 females were generated and crossed with DoaV3B/TM3 males. The latter allele does not produce heteroallelic escapers in any combination, causes a dramatic reduction in the 2.7-kb Doa RNA (this report), and does not complement several different classes of Doa alleles, including Msu2 and RemTA (B. Yun and L. Rabinow, unpubl.). Em- bryos obtained from this cross were collected on fine nylon mesh and treated for antibody staining or cuticle preps, as described (Ashbumer 1989).

Scanning electron microscopy

Samples for scanning electron microscopy were processed as described by Kretzschmar et al. (1992). Fixed adult flies were dehydrated through an ascending ethanol series (30%, 50%, 70%, 96%, and 3x 100% ), followed by critical point drying and spatter-coating with gold/paladium. Flies were viewed and photographed in a Jeol JSM 35-C scanning electron microscope op- erating at 10, 15, or 25 kV.

Histology and immunocytochemistry

Plastic sections were prepared as described by Tomlinson and Ready (1987), as modified by Kimmel et al. (1990). Adult heads






were dissected in Drosophila saline, and holes were made between the eyes to aid penetration of fixatives and embedding components. Heads were fixed in 2% glutaraldehyde in phosphate buffer, postfixed in 1% osmium tetroxide, and dehydrated in ethanol. After infiltration in propylene oxide-Durcupan they were embedded in Durcupan ACM plastic resin (Fluka AG, Buchs, Switzerland). Plastic sections of 1.5-2 ~m were stained with toluidine blue (Basler and Hafen 1988), and mounted in DPX (Fluka AG).

Immunohistochemical staining of imaginal discs and pupal retinas was performed with anti-HRP (Cappel), anti-chaoptin (mAb 24b10), kindly provided by S. Benzer (California Institute of Technology, Pasadena), and anti-ELAV and anti-Glass, kindly provided by E. O'Neill and G. Rubin (University of California, Berkeley). Dissected discs or pupal retinas were fixed in 4% paraformaldehyde in PBS for 3 hr at room temperature and washed 3 x in PBS. The peripodial membrane was removed using heptane-dimethylsulfoxide, and discs were permeabilized with PBS +0.1% Triton X-100 (PBT). After 4 hr of blocking in PBT+ 2% BSA and 1% normal rabbit serum, discs were incubated with primary antibodies overnight. After washing with PBT, secondary biotinylated anti-goat antibody (Vector Labora- tories) was added for another 3 hr at room temperature. Staining was performed with strepavidin-conjugated-HRP, followed by extensive washing and incubation with 0.1% diaminobenzidine and 0.02% hydrogen peroxide in 0.1 M Tris-HC1 (pH 7.6).

Clones from 98F1-2 detect DNA polymorphisms in Doa alleles

The generation of a Doa allele by the Dfd Rxl reversion allowed us to "jump" into the Doa locus, because the precise breakpoint in Dfd axl had been mapped (Regulski et al. 1987). A clone from transposed DNA near the break was used to isolate genomic phage from a Dfd RxI genomic library, crossing the breakpoint between the two loci. Because Dfd Rx~ is maintained as a balanced stock, only one-half the cloned phage should reveal the breakpoint. Of four phage isolated, one had a restriction map differing from the wild-type Dfd locus, suggesting that it de- fined the Dfd-Doa breakpoint. To confirm this, a restriction fragment distal to the putative Dfd Rxl breakpoint was used as a probe to screen a wild-type genomic library, from which four overlapping phage were isolated (Fig. 4). A subclone of phage Doal (approximate coordinates - 4 to + 4, Fig. 5) was used as a probe to salivary gland polytene chromosomes of the Canton-S wild-type strain. Chromosomal walking from our entry point isolated additional phage from the 98F 1-2 region, using the ends of both genomic and cDNA (see below) clones to probe k libraries from wild-type Drosophila. Six of those characterized span 50 kb at 98F1-2 (Fig. 4). Restriction maps from five Doa mutant alleles were then constructed, based on data from Southern transfers using the wild-type genomic clones as probes, to identify lesions in the gene and aid in defining its structure (Fig. 4). We particularly wished to locate aberrations associated with three-hybrid dysgenically induced alleles. This desire stemmed in part from the existence of two independent revertants, one of which occurred spontaneously (B. Yun and L. Rabinow, unpubl.). A second was induced during crosses to the A2-3 element tRobertson et al. 1988}. The finding of DNA polymorphisms coincidentally induced with a Doa mutant allele, which were subsequently altered during reversions, would provide un- ambiguous evidence that we had cloned the gene.

An insertion of - 5 kb was found in the identical location in both the HD1 and HD2 alleles (Fig. 41. This is not surprising, as both potentially originated from a single event during mass crosses {Rabinow and Birchler 1989). Furthermore, this insertion is missing in both revertants described above, presumably

through excision of the uncharacterized 5-kb transposable element. A 2-kb insertion was found at a different location in a third dysgenically induced allele, Dem (Fig. 4). We also found a breakpoint in Doa ~3B, which closely flanks the Dfd Rxl insertion. This allele is associated with a small chromosomal abnormality at 98F1-2, probably a localized inversion, an interpreta- tion supported by our restriction maps.

Molecular biology

Genomic library construction, plaque isolation, and subcloning, Southern blots, probe preparation, and so forth, all made use of standard protocols, (e.g. Ausubel et al. 1989}. The Dfd Rxl genomic library was constructed by Sau3A1 partial digestion and cloning of genomic fragments into phage EMBL3 at the BamHI site. DNA sequencing was performed by double-stranded sequencing of plasmids using the dideoxy method (Sanger et al. 1977). All restriction sites were crossed, and both strands of cDNA and genomic clones were sequenced. Computerized searches of sequence data bases and alignments were generated using GCG software. Polytene chromosome squashes and in situ hybridization were performed using biotinylated DNA probes and alkaline phosphatase detection, essentially as described (Ashbumer 1989). RNA isolation, Northern blots, and RNA probes were performed as described (Rabinow et al. 1993). Briefly, total RNA was extracted from frozen organisms by guanidine-HC1 extraction (Cox 1968). Five micrograms per lane of total RNA was separated on 1.5% formaldehyde-agarose gels {Lehrach et al. 1977), after which the RNA was transferred to Biotrans filters. The RNA was fixed to the filter by both UV cross-linking and baking under vacuum at 80°C for 2 hr. An- tisense RNA probes were synthesized with T3 or T7 P, NA poly- merase. Loading controls were performed by rehybridizing the blots with a probe for rp49 (O'Connell and Rosbash 1984). Blots were hybridized at 60°C, under described conditions {Dorsett et al. 1989), at a probe concentration of >2x 10 6 cpm/ml. Filters were washed with 0.1 x SSC, 0.4% SDS, at 75°C after overnight hybridization.

Expression of Doa protein in E. coli and autophosphorylation assays

A subclone of the composite Doa cDNA encoding the entire catalytic domain, starting at K129 to a StyI site in the 3'-untranslated region, was inserted into the XbaI site of pMAL-C2 of New England Biolabs. Expression of the fusion protein in E. coli was induced with IPTG, and the fusion was recovered on an amylose column, analyzed for purity on an SDS gel, and transferred to a nitrocellulose membrane. Kinase-renaturation was performed as described for routine Clk (Ben-David et al. 1991).

A c k n o w l e d g m e n t s

Many individuals provided us with stocks, reagents, and unpublished data, greatly facilitating this work. Excellent assistance was provided by Christophe Hitte and Jean Hyde for the embyronic cuticle preparations and the maltose binding protein (MBP)-Doa fusion construct, respectively. We are grateful to Ed Stephenson for informing us about the Dfd Rxl breakpoint and for providing a stock of itl Bill McGinnis for a Dfd clone and a second Dfd Rxl stock; Judith Bender for communicating unpublished DNA sequence data on AFC1 and suggesting that we examine the KNS1 sequence; Steve Hanks for communicating the unpublished noncatalytic domain sequence of pSK-G1; Sey- mour Benzer for the gift of mAb 24b10 (anti-chaoptin); Tony





Yun et ai.

Pawson and Karen Colwill for the TrpE-clk fusion construct; Elizabeth O'Neill and Gerry Rubin for the generous gifts of anti-Glass and anti-ELAV antibodies and cell lines; Rick Padgett for an aliquot of a wild-type genomic library; and last, but not least, Lee Simon for generous donation of his time for the scanning electron microscopy. We particularly thank Ross Cagan, Karen Colwill, Michael Greenberg, Tony Pawson, and Marie-Laure Samson for critical comments on the manuscript. This work was supported by grants to L.R. from the New Jersey Cancer Commission and the National Science Foundation (MCB92-19371).

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

N o t e added i n proof

The sequence data described in this paper have been deposited into the EMBL/GenBank data library under accession number X78715.

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