(unc-44) Is Necessary for Proper Axonal Guidance in ...

An Ankyrin-related Gene (unc-44) Is Necessary for Proper Axonal Guidance in Caenorhabditis elegans Anthony J. Otsuka,*¢ Rodr igo Franco,~ Bin Yang,* K y u - H w a n Shim,* Lan Z h a o Tang,* Yu Yong Zhang,* P ra tumt ip Boontrakulpoontawee,* Ayyamperumal Jeyaprakash,~ Edward Hedgecock,§ Virginia I. Wheaton,~ and Alan Sobery*

* Department of Biological Sciences, 4120 Illinois State University, Normal, Illinois 61790-4120; ~ Department of Genetics, University of California, Berkeley, California 94720; and § Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218

Abstract. Caenorhabditis elegans unc-44 mutations result in aberrant axon guidance and fasciculation with inappropriate partners. The unc-44 gene was cloned by transposon tagging, and verified by genetic and molecular analyses of six transposon-induced alleles and their revertants. Nucleotide sequence analyses demonstrated that unc-44 encodes a series of putative ankyrin-related proteins, including AO49 ankyrin (1815 aa, 198.8 kD), AO66 ankyrin (1867 aa, 204 kD), and

AOI3 ankyrin (x<4700 aa, ~<517 kD). In addition to the major set of •6 kb alternatively spliced transcripts, minor transcripts were observed at -,3, 5, 7, and 14 kb. Evidence is provided that mutations in the *14-kb AO13 ankyrin transcript are responsible for the neuronal defects. These molecular studies provide the first evidence that ankyrin-related molecules are required for axonal guidance.

TrIOUGH the molecular basis of neural development has been the object of intense study in recent years, the detailed mechanisms of axon guidance remain

unknown (for general reviews see Dodd and Jessell, 1988; Jessell, 1988; Takeichi, 1988; Sanes, 1989; Takeichi, 1991; Rathjen et al., 1992; Gumbiner, 1993; for C. elegans reviews see Hedgecock et al., 1987; Wadsworth and Hedgecock, 1992).

Mutations in the unc-44 gene affect the direction of axonal outgrowth for many axons (Hedgecock et al., 1985; Sid- diqui, 1990; Siddiqui and Culotti, 1991; Mclntire et al., 1992), including the postdeirid (PDE) ~ axon, which nor- mally extends from the postdeirid sensillum on the lateral surface of the nematode to the ventral nerve cord (White et al., 1986). In unc-44 mutants, the initial direction of PDE axon outgrowth along the basement membrane is apparently random, and the misdirected PDE axon fasciculates with inappropriate partners (Hedgecock et al., 1985).

Address all correspondence to A. Otsuka, Department of Biological Sci- ences, 4120 Illinois State University, Normal, IL 61790-4120. Tel.: (309) 438-5220. Fax: (309) 438-3722.

The current address of Dr. Franco is Wyeth-Ayerst Research, CNS000, Princeton, NJ 08543-8000.

The current address of Dr. Jeyaprakash is Entomology and Nematology Department, Institute of Food and Agricultural Sciences, University of Florida, Bldg. 970, Hull Road, P. O. Box 110620, Gainesville, FL 32611- 0620.

1. Abbreviations used in this paper: AE1, anion exchanger 1; PDE, postdeirid; RBC, red blood cell.

The discovery that the C. elegans unc-6 gene encodes a laminin B chain-related product provided evidence that directed axonal outgrowth and cell migration require interactions with the extracellular matrix (Hedgecock et al., 1990), and that these interactions use laminin or related proteins in both invertebrates and vertebrates (Jessell, 1988; Sanes, 1989; Hedgecock et al., 1990; Serafini et al., 1994). The product of the unc-5 gene, which affects dorsalward cell migrations and axon outgrowth, has been proposed to be a cell surface protein which may interact with the extracellular matrix (Leung-Hagesteijn et al., 1992). Thus, it was likely that other mutations affecting axonal outgrowth and guidance were defects in cytoskeletal or extracellular matrix structures. The actin/a-actinin framework of growth cone filopodia or the spectrin/ankyrin network underlying the cytoplasmic surface of the plasma membrane could be the targets for mutations affecting axon outgrowth and growth cone adhesion. In this study, we have discovered that the wild-type unc-44 gene, which is required for proper axonal guidance, encodes a series of putative ankyrin-related proteins.

Ankyrin (or bands 2.1 and 2.2) has been most thoroughly studied in erythrocyte "ghosts" (for reviews see Lazarides and Woods, 1989; Bennett, 1990; Bennett, 1992; Michaely and Bennett, 1992; Lambert and Bennett, 1993; Peters and Lux, 1993). In erythrocytes, ankyrin monomers anchor the spectrin network to the transmembrane anion exchanger (AE1 or band 3). The AEl-binding domain can also bind to tubulin, microtubules, and intermediate filaments (Geor- gatos et al., 1987). Ankyrin is composed of three domains: (/) a membrane protein-binding domain containing 23

© The Rockefeller University Press, 0021-9525/95/05/1081/12 $2.00 The Journal of Cell Biology, Volume 129, Number 4, May 1995 1081-1092 1081

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,~33-amino acid repeats (ankyrin repeats), (2) a spectrin- binding domain, and (3) a regulatory domain (Wallin et al., 1984; Weaver and Marchesi, 1984; Weaver et al., 1984; Hail and Bennett, 1987; Davis and Bennett, 1990; Lambert et al., 1990; Lux et al., 1990). In this report, the sequence heterogeneity between the vertebrate ankyrins and the unc- 44 products defines an additional "linker" domain between the ankyrin repeat and spectrin-binding domains (see Fig. 1 a; see also Plattet al., 1993).

Ankyrin is found in a number of tissues, including the brain (Drenckhahn and Bennett, 1987). Molecular analysis of human brain ankyrin cDNA clones reveals several alternatively spliced RNAs (Otto et al., 1991). The predicted products of two of the RNAs are brain ankyrin 2 (202 kD) with a gross structural similarity to erythrocyte ankyrin, and brain ankyrin 1 ("~440 kD) which contains the typical ankyrin repeat, linker, and spectrin-binding domains, but has an extensive carboxyl-terminal domain (Otto et al., 1991; Chan et al., 1993). Brain and erythrocyte ankyrins bind to distinct sites on kidney membranes, suggesting different transmembrane protein targets for the different ankyrins (Davis et al., 1989). Recently, it was demonstrated that ankyrin binds a protein related to the neurofascin cell adhesion molecule in the rat brain (Davis et al., 1993). Thus, ankyrin may interact with other proteins, including cell surface receptors, localiz- ing them on the cell surface.

A minor form of ankyrin (band 2.2), which results from an alternative splicing event that removes a 16-kD polypep- tide region from within the regulatory domain, is perma- nently "activated" to allow enhanced binding to membrane- associated sites (Hall and Bennett, 1987; Davis et al., 1992). In addition, calpain proteolysis of the AEl-binding domain decreases ankyrin binding to AE1 (Hall and Bennett, 1987). Altering the binding affinity of ankyrin for its substrates may allow cytoskeletal remodeling during cell growth and loco- motion (Hall and Bennett, 1987).

In previous studies, unc-44 mutations were isolated in a high level transposition background in order to tag the gene

Figure 1. (a) The proposed structures of ankyrin-related proteins. Multiple proposed forms of human erythrocyte ankyrin (Lambert et al., 1990; Lux et al., 1990), human brain ankyrin 2, a partial structure of brain ankyrin 1 (Otto et al., 1991), and partial structures of the putative nematode unc-44 ankyrin-related products are shown. Brain ankyrins 1 and 2 contain different carboxyl termini produced by alternative splicing at aa position 1444 (Otto et al., 1991). In brain ankyrin 1, alternative splicing results in insertion of 2085 aa, including 15 repeats of a 12-aa sequence, into the carboxyl-terminal domain to produce a 440-kD product (Kunimoto et al., 1991, Chan et al., 1993). For unc-44, the amino acids are numbered from the start of the partial cDNAs, while those expected in the fuU-length protein are noted in parentheses. In the uric-44 products, alternative splicing modifies the carboxyl terminus (at cDNA aa positions 983 and 910) in a manner similar to that found in erythrocyte ankyrin. In the linker domain, between the ankyrin repeats and the spectrin-binding domain, the DIM/PAO49 product contains a 6-aa alternatively spliced microexon relative to DD #PAO66. An inversion of the nucleic acid sequence (boxed arrow) occurs in the linker domain in the DD#AO66 cDNA clone. The ankyrin repeat domains shown as dashed lines are those expected on the basis of Northern blot analysis. (b) Map of the unc-44 re-

gion. The positions and extents of the cosmid (BO350 and C44A), phage (DD#LRFI), genomic plasmid subclones (DD#PRF6, DD #PRF7, DD#PPB40, and DD#PSLRS), and cDNA clones (DD#PAO 13, DD#PAO49, and DD#PAO66) are displayed. The positions of unc-44 mutations are shown above the genomic map. The ankyrin domains present on the ll-kb BamHI fragment in DD #PPB40 are shown along with the exon map (filled block.s) above the genomic map. Relevant restriction sites for BamHI (B) and SalI (S) are noted. The dashed lines extending from the cDNA clones estimate the full extent of the RNAs as determined by Northern blot analysis. The cDNA clones were obtained by screening cDNA libraries with probes corresponding to DD#PPB40 (11 kb) and DD#PSLR8 (3.7 kb). The dashed lines extending from cosmid clone C44A represent the uncertainty of nematode DNA junctions in the clone. (c) The genomic organization of the 11-kb BamHI fragment. The 1 l-kb region from DD#PPB40 was sequenced by the exonuclease RI deletion method using the subelones DD#PRF6 and DD#PRF7. The exons are represented by boxes and the introns by a line. The ankyrin repeats subdivide the boxes and the strong spectrin-binding domain similarity is shown in black. The primed numbers represent breaks within the individual ankyrin repeats.

The Journal of Cell Biology, Volume 129, 1995 1082

with transposons (Otsuka et al., 1987). In this paper, we report the molecular cloning and characterization of the unc- 44 gene. The DNA sequence analysis, Southern and North- ern blot analysis, and genetic complementation tests demonstrate that the unc-44 gene has been cloned. Analysis of six spontaneous uric-44 alleles ascertained that all were due to DNA insertions (see Fig. 1 b). Reversions of these six alleles result in in-frame deletions of the transposons or secondary insertions of transposons at RNA splicing junctions.

The composite structures of the unc-44 ankyrins have been obtained from a combination of cloned genomic and cDNA sequences. These studies demonstrate that alternative splicing produces several transcripts from a single ankyrin- related gene. There is a major set of 6 kb transcripts and several minor transcripts. Paralleling the human ANK2 gene, unc-44 encodes "conventional" ankyrin isoforms (AO49 and AO66 ankyrins) with gross similarities to erythrocyte ankyrin and brain ankyrin 2, as well as a much larger form of ankyrin (AO13 ankyrin). Although AO13 ankyrin is predicted to be similar in size to vertebrate brain ankyrin 1, its carboxyl-terminal domain sequence is highly acidic and distinct from that reported from brain ankyrin 1 (Chan et al., 1993).

Materials and Methods

Cloned DNAs and Nematode Strains

Nematode strains and recombinant DNA clones are listed in Table I. The insertion mutations define a single complementation group because they fail to complement in all combinations tested, i.e., the rhlOl3 allele failed to complement both q331 and rh1042 mutations, while the q331 allele failed to complement the rh1042 allele.

Preparation of DNA

Nematodes were cultured and DNA prepared as described previously (Brenner, 1974; Sulston and Brenner, 1974). Plasmid and phage DNAs were prepared by standard methods (Maniatis et al., 1982).

Southern Blot Hybridization

3 ttg of each restriction enzyme-digested DNA were fractionated on Tris- borate 0.7 % agarose gels containing ethidium bromide, and Southern blots were prepared (Maniatis et al., 1982). After prehybridizing the nitrocellu- lose filters (Schleicher and Schuell, Inc., Keene, NH), the hybridization was performed in 6 x S SC, 0.01 M EDTA, 5 x Denhardt's solution, 0.5 % SDS, 100/~g of denatured herring sperm DNA/ml, and 1 ttg 32p-labeled probe (107 cpm//~g), for 12-16 h at 68°C. The blots were washed exten- sively in 2 x SSC and 0.5 % SDS at 68°C. The Tel probe was the plasmid pCe2003 (Emmons and Yesner, 1984).

Northern Blot Analysis

RNA was prepared from a mixed-stage population of N2 worms by French pressure cell disruption, lysis with a Polytron homogenizer, or grinding in liquid nitrogen in the presence of guanidinium chloride, followed by selective ethanol precipitation (MacI.ex~ et al., 1981). Messenger RNA was purified on an oligodeoxythymidylic acid-cellulose column (Aviv and Leder, 1972). 5 #g of poly A selected or 20 #g of total nematode RNA were separated on denaturing formaldehyde agarose gels (Lehrach et al., 1977) and analyzed by Northern blot hybridization using Zetaprobe charged nylon membranes (BioRad Laboratories, Richmond, CA) and [u.32p] UTP-labeled riboprobes (5 × 107 cpm, 200 Ci/mmol of nucleotide) transcribed from eDNA clones (DD#PAO13, DD#PAO49, and DD#PAO66) or ankyrin repeat (DD#PLTI840) plasmids using a T3/T7 RNA polymerase transcription kit (Stratagene, Inc., La Julia, CA). The hybridization conditions (manufacturer recommended buffer including 5× SSC, 42°C) and washing conditions (0.1× SSC plus 0.1% SDS, 68°C) were those recommended by Stratagene.

Screening Clone Banks

Six genomic clones were obtained from a phage bank probed by standard methods (Maniatis et al., 1982). Five independent eDNA clones were obtained from ",,1.4 million phage of the Barstead-Waterston bank without additional amplification (Barstead and Waterston, 1989).

DNA Sequencing and Computer Analysis

DNA was subcloned into pTZ18R, pTZ19R (Pharmacia LKB Biotechnol- ogy, Piscataway, NJ), or pBhlescript SK(-) (Stratagene) and sequenced by the dideoxynucleotide method (Sanger et al., 1977). Appropriate subclones were obtained by using suitable restriction fragments or by the exonuclease III deletion method (Henikoff, 1984). DNA sequence was obtained from both DNA strands except in some of the introns. Sequences determined from a single strand were done at least four times. Additional analysis was done using the Pustell and MacVector DNA Sequence Analysis Programs (Eastman Kodak Co., New Haven, CT) and our own programs.

Resul ts

Cloning the unc-44 Gene

To identify a restriction fragment length polymorphism associated with the unc-44 gene, a set of recombinants was constructed in the unc-44 region (Table I). Linkage of a Tc/ transposon to the unc-44 gene was demonstrated by the presence of a 12.6-kb Tc/-containing EcoRI fragment in the rh1042 mutant and in recombinants retaining the unc-44 (rh1042) allele (Fig. 2, lanes 2 and 4). The wild-type N2 strain and recombinants that have lost the rid042 mutant phenotype do not contain this fragment (Fig. 2, lanes 1, 5, and 6). Because the wild-type DNA does not contain a transposon insertion in unc-44, no unc-44-specific band appears in Fig. 2, lane 1. However, wild-type DNA did contain an 11-kb fragment when probed with an unc-44-specific probe (data not shown). In the rh1042 revertant, the characteristic 12.6-kb mutant fragment is missing and is replaced by a 14.2-kb fragment (Fig. 2, lane 3), due to the insertion of a second Tc/element. Reversion of transposon-induced mutations by insertion of additional transposons has been described previously (Mount et al., 1988).

EcoRI-cleaved rh1042 DNA fragments in the 12.6-kb size range were cloned into the EMBL3 bacteriophage lambda vector, and the resulting library was screened with a Tc/ probe to yield clone DD#LRF7 (Table I). The region flanking the Tc/element was subcloned into a plasmid vector and used to screen a wild-type nematode genomic library in EMBL3. Six unc-44 clones were obtained (Table I) and used to identify cosmid clones (Table I and Fig. 1 b).

Southern Blot Analysis of unc-44 DNA Insertion Mutations

To unambiguously demonstrate that the unc-44 gene had been cloned, six putative transposon-induced mutations and their revertants (Table I) were analyzed by Southern blot hybridization with unc-44-specific probes and the results are summarized in Fig. 1 b. By Southern analysis with BamHI, EcoRI, PstI, or SalI, and by polymerase chain reaction amplification, the q331 and rh1013 mutations were found to be Tc/insertions toward the 5'-end of the gene, and their rever- rants were Tc/ excisions which restore the unc-44 reading frame. The rh1042, mn259, mn339, and st200 mutations were DNA insertions toward the 3'-end of the gene. The

Otsuka et al. Ankyrin and Axonal Guidance 1083

rh1042 allele is a Ted insertion within the DD#PAO13 open reading frame. The six insertion mutations and their in- frame excision in mn259, q331, and rh1013 revertants provide proof that the unc-44 gene has been cloned. The revertants of rh1042 and st200 are secondary Ted insertions at RNA splicing junctions and presumably restore gene activity by altering RNA splicing.

Cloning of cDNAs

To clone cDNAs corresponding to the regions surrounding the unc-44 DNA insertions, two genomic DNA fragments were used to probe nematode eDNA libraries: (/) the ll-kb genomic BamHI fragment flanking the Ted insertions in q331

Table 1. Nematode Strains and Cloned DNAs

and rh1013 toward the 5'-end of the gene (corresponding to clone DD#PPB40 in Fig. 1 b), and (2) the 3.7-kb SalI fragment flanking the Ted insertion in rh1042 toward the Y-end of the gene (corresponding to DD#PSLR8 in Fig. 1 b). DNA sequencing was performed on two clones, DD#PAO49 and DD#PAO66, obtained with the 11-kb probe, and one (DD #PAO13) of the three independent clones with identical restriction patterns obtained with the 3.7-kb probe.

The Ankyrin Repeats Are Grouped into Six Clusters

The overall structure of the AO49 and AO66 ankyrin isoforms (Fig. 3) can be inferred from the domains present on the l l-kb BamHI genomic (DD#PPB40) and the cDNA

Strain Genotype Relevant properties Source of reference

N2 wild-type Bristol strain Brenner, 1974 NJ82 wild-type Bristol/Bergerac hybrid otsuka et al., 1987 RW7097 mut-6 (stT02) High transposition Mori et al., 1988 TR679 mut-2 (r459) High transposition Collins et al., 1987 uric-44 (rh1013) derivatives NJ94 uric-44 (rh1013) Sp. mut. from NJ82 Otsuka et al., 1987 NJ280 unc-44 (rh1013"10), bli-6 (sc16) Double mut. This work DD196 uric-44 (rh1013"10), bli-6 (+) Unc-44 from NJ280 This work NJ101 unc-44 (rh1013"1) rev-1 Sp. rev. This work NJI05 unc-44 (rh1013) rev-2 Sp. rev. This work unc-44 (rh1042) derivatives NJ401 unc-44 (rh1042"4) Sp. mut. from RW7097 This work NJ441 uric-44 (rh1042"8) Outcrossed 8x This work NJ416 uric-44 (rh1042"3) bli-6 (sc16) Unc-44 Bli This work NJ417 unc-44 (+), bli-6 (sc16) Non-Unc Bli This work NJ418 unc-44 (+), bli-6 (sc16) Non-Unc Bli This work NJ413 unc-44 (rh1042) rev-l*2 Sp. rev. This work Other unc-44 mutants and revertants CB362 unc-44 (e362) Canonical allele Brenner, 1974 SP1161 dpy-13 (e184) unc-44 (q331) Sp. mut. T. Schedl* JK1040 unc-44 (q331, q332 rev) q331 rev. T. Schedl* RW7220 unc-44 (st200) Sp. mut. D. Moerman* SP1167 unc-44 (st200, mn343 rev) st200 rev. W. Li* SP1162 unc-44 (mn259) Sp. mut. from TR679 W. Li* SP1164 unc-44 (mn259, mn340 rev) mn259 rev. W. Li* SPl163 unc-44 (mn339) Sp. mut. from TR679 W. Li* SP1166 unc-44 (mn339, mn342 rev) mn339 rev. W. Li* Plasmids and bacteriophages DD#LRF7 kEMBL3 unc-44 (rh1042): Tcl 12.6 kb EcoRI frag. This work DD#LRF1 kEMBL3 unc-44 N2 partial Sau3aI This work

to DD#LRF6 kEMBL3 unc-44 N2 partial Sau3aI This work BO350 pJB8 uric-44 AmpR N2 partial Sau3aI cosmid This work:~ C44A pJB8 unc-44 AmpR N2 partial Sau3aI cosmid This work:~ DD#PPB40 pTZ18R unc-44 AmpR 11 kb BO350 BamHI This work DD#PLT1840 pTZ18R unr-44 AmpR 6 kb BO350 BamI-II/SalI This work DD#PRF6 pTZ18R unc-44 AmpR 5 kb BO350 BamHI/SalI This work DD#PRF7 pTZ18R unc-44 AmpR 6 kb BO350 BamHI/SalI This work DD#PSLR8 pTZ18R unc-44 AmpR 3.7 kb BO350 SalI This work DD#PAO13 pBluescript unc-44 AmpR N2 partial site B eDNA This work§ DD#PAO49 pBluescript unc-44 AmpR N2 partial site A cDNA This work§ DD#PAO66 pBluescript unc-44 AmpR N2 partial site A eDNA This work§

The number of crosses to N2 is indicated after the allele number. For example, crossing of a strain containing the rhlO13 allele ten times to N2 would be indicated by rhlO13*lO. * These mutations were isolated in the laboratories of J. Kimble, J. Shaw, R. Waterston, and R. Herman, and kindly provided by C. Kari and R. Herman. ~: BO350 and C44A were kindly provided by A. Coulson and J. Sulston from their library (Coulson et al., 1986). § These eDNA clones were isolated from a bank provided by R. Barstead and R. Waterston (1989). Mut., mutation; rev., revertant; sp., spontaneous.


Figure 2. Southern blot analysis of the unc- 44 (rh1042) allele. A Southern blot was prepared from EcoRI-digested DNA samples and probed with a Te/plasmid. The sam- pies are as follows: (lane 1 ) wild-type N2; (lane 2) NJ401 unc-44 (rh1042); (lane 3) NJ413 unc-44 (rh1042) rev-1 revertant; (lane 4) NJ416 unc-44 (rh1042) b li-6 (st6) recombinant; (lane 5) NJ417 unc-44 (+) bli-6 (sc16) non-Unc, Blister recombinant; and (lane 6) NJ418 uric-44 (+) bli-6(sc-16). Note the 12.6-kb fragment in the unc-44 mutant strains (arrows in lanes 2 and 4) and absence of this band in the other strains. In the case ofa revertant, the 12.6-kb band is converted to a 14.2-kb band (arrow in lane 3).

clones (DD#PAO49 and DD#PAO66) (Fig. 1 b). The carboxyl-terminal end of the large ankyrin isoform was obtained from eDNA clone DD#PAO13 (Fig. 1 b).

The ankyrin repeat, spectrin binding, and most of the conventional regulatory domains are arranged in 12 exons on clone DD#PPIM0 (Figs. 1 c and 4). The 23 ankyrin repeats are grouped into six exons containing essentially 1, 5, 8½, 6, 1½, and 1 repeats (Figs. 1 c and 4). Introns cleanly sepa- rate exons that encode the first ankyrin repeat and the last ankyrin repeat, as is the case in vertebrate ankyrin (Tse, 1990). The clustering of repeat elements and the bifurcation of individual repeats is in contrast to the large number of single ankyrin repeat exons in the human ANK/ gene (Tse, 1990).

Each individual repeat is more closely related to the corresponding repeat from other organisms than to the other nematode repeats (Fig. 5). The conservation of individual repeat sequences in organisms as divergent as humans and nematodes suggests a functional or structural role for each repeat. With the exception of a 7-aa insertion in repeat 5 of human brain ankyrin, the lengths of the corresponding nematode, mouse erythrocyte, human erythrocyte, and brain ankyrin repeats are identical. The constancy of the individual repeat lengths suggests stringent limits on the three- dimensional structure. The 7-aa insertion in the human brain ankyrin repeat 5 is not present in the unc-44 genomic DNA, and therefore could not be obtained by simple alternative RNA splicing in the nematode. Repeats 2 through 6 and 8 through 12 contain the greatest number of identical residues (18 or more). There is 52% identity (396 of 755 residues) in the ankyrin repeat domains of the various ankyrins. The unc-44 ankyrin repeat domain is more closely related to brain ankyrin (13 % or 96 brain-specific residues, noted by asterisks in Fig. 5) than to erythrocyte ankyrin (5 % or 39 erythrocyte-specific residues, noted by equal signs in Fig. 5). A number of residues are unique to the vertebrate ankyrin (13 % or 101 residues, noted by periods in Fig. 5). The UNC- 44 amino-terminal domain preceding the ankyrin repeats is also more similar in size and sequence to human brain ankyrin than to erythrocyte ankyrin (Fig. 5). These results sug- gest that the unc-44 products are more closely related to the vertebrate brain ankyrin than to erythrocyte ankyrin.

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z 2 3 1-) Linker Domain 641 E SEDE G~AAEE~GGGABEKD FSDNLTQGLQD8 TGV~ HTG~QLLQRS QE LENGGAI pK i 9 0 1 NSGGB4S PEKEFAE IAPVAT88 P ZAT SNSQ 8 FG IAPRAGS Z S GQ ~QQ pLB GAGPEDNLEE 961 L~PINAGNYDNGGVAMLENGBADb~rp I GREVT~PSKL~ERTFL 18 FLVDARGGA

8~=tri~-Bin~ing Domain 1021 ~D%GC~H 8 GVRI I VPPRKKS~P I RVTCR¥ LRKDKLA~ P Pp LSE G~E LASR113DSAPAGAK F

1081 LGPVI LEVPH FAS LRDREREIVI LI%SDDGQHW~EH~LEATEDAVQEVLNE 8 FDAED LAQ L 1141 DDL~TPRI TRI LTSDFPMY FAVVTRVR~EVECVGPEGGVI Z 3 SVVPRVQAI FPD G8 LT~T

1201 I KVSV~AQ~GN~VAV8 P IVTVEPRRRKFBEP I TLC I PLI~ S ~ T Q ¥ 1261 $ G~G~E PPTLRLLC S 1 TGGBAPA~WED I TGTTQLTFTGDEV8 FTTTVSAR~4L)®D CQTP

1321 RDAARI GQEVYNEAI S I pY~U%KFAVFARRTFPVEGQLRYFCA~fDDKEDKTLEK~EB FKL I 1381 AKSRDVEVLKG~QFLE FS GNLVP I TK8 GDQ L8 LFFLPFQENRIJLFMVKTRSNDDNEAAT

Regulatory Do~a£n 1441 EGRI GFMAE 1~I RSDALP~P I C TLAI $ LPEY TG~ I KTAPP~ LT pFEQR¥ GSALEK 1501 DLPE FVEQMVLKG I GADWPRLGRAIJ~/PHRD I QH I RQNY PG~ECENTLK I WI H LKKEDAN

1561 ~ D ~ I G~DD IVRS IA¥ G~ PDAL INY S~AD 8 P8 QKRDAI~ FEEVPAAT LVKREV 1621 RTED LVTREPV~PT~BVE~ATTVVQEP 8¥ 8APVB~5 PVPEEPEb~J~A~VA~TVVRTE

1681 PJ~V~D SEDGPVVEERTI TTTYEDDVAV~D~ IVDRTVP LNEDEQQKWEELNR/J~DE 5 S p S p 1741 A~RSTIVAESTSE~FEDVE~SVESESHREDDGTIVTTTVTTS BI 8ESPSGSPTRRSVE p

/GST 1815 A089 1601 EEERESQEEDBE

\A~GAEQH 5 G~D $ GTTQKKETAEE GFTNEDGSVVVSKEMTRVVTTTRT TLpG~G~J~PSR~GE 1867 AO66

Figure 3. Predicted amino acid sequences of AO49 and AO66 ankyrin isoforms. The predicted protein sequences of AO49 and AO66 ankyrins are shown with the ankyrin repeats (rl-r23), linker, spectrin-binding, and regulatory domains noted. The alternatively spliced micmexon present in AO49 ankyrin is underlined. At the bottom of the figure, the alternative carboxyl termini are noted. Amino acid residue positions are noted at the left margin.

unc-44 cDNA Fragments Encode Spectrin-binding and Regulatory Domains

DNA sequence analysis of DD#PAO49 and DD#PAO66 partial eDNA clones reveals linker, spectrin-binding, and regulatory domains (Fig. 6). The eDNA sequences are essentially identical in the central coding region with divergence at the 5' and 3' ends. The predicted protein fragments are hydrophilic and contain dispersed cysteine and proline residues. The open reading frame of DD#PAO49 includes a 6-aa alternatively spliced microexon adjacent to the strong spectrin-binding domain sequence similarity (Fig. 6). The divergence at the 5' end of the DD#PAO66 eDNA is due to an inversion which is not detectable in the genome. The divergence at the 3' end is due to alternative splicing, and leads to predicted full-length isoforms of 1815 aa (AO49 ankyrin, 198.8 kD) and 1867 aa (AO66 ankyrin, 204 kD) (Figs. 3 and 6).

The linker domain in C. elegans is larger (187 aa) than in human erythrocyte (113 aa) or brain (141 aa) ankyrins (Fig. 5). The linker domain contains the acidic portion of the spectrin-binding domain as defined by Davis and Bennett (1990), but has been separated in this report because the nematode sequence has considerably diverged from it, and


1 GGATCCCCCCTT¢C~,TAO/~ATUGG4d~CAC-AaC, C~CCCAACGASCTCCS S a ~ C , G G C G T C ~ T T T T ~ A / t ~ C C C r A A ~ r T C T C C T C ~ A A T T A r C C A c r ^ r r ~ c ~ T o m r r a o ~ m ¢ c r z c . c , ~ c c c r c c r ^ c c c c r r c r c c c c l ~ * CAACGCCO~rAC^CACTrC-CCATTU~}T/~C, A h C C m ~ h ~ , ~ ¢ ~ ~ u u ~ r . c ' ~ c r T r r r ~ c r ^ t r r T r c l ~ r C ~ ¢ T C r C ^ T C ^ C G T C T ~ T ^ C r e T e r C A r C C . C C T C r C - - T C C X C r C T ^ T r C C r C r r G C r C C G 301 A G A G C T C C ~ A ~ T C C T C T C C C G T C C . A I ~ d ~ . ~ C C A / ~ I G G T T G C G G T T C, GTT~GmT, C A A I O ~ G G G G T C , . W ~ A C A J ~ T T T t ; 1 T G A T T T G I ~ T ^ T ~ T C I T G T ~ C T C C C T C ~ T U C r c r c C C T T T X C ~ " r c ~ c ~ i b i GGA.*J~AGC,$*GCA$*GTGASGCTCATTCGTATTT~.~GAM*.AC,~VV~*G A C . C A C A T T T ~ C T T C C r ~ X X r T r r T C / t C T T T C C I T T Y r r / * T G T C C T I Y I T C T T C C A t } T A C T I I ' G T T T C y A ~ X I ~ C , . ~ ^ C A C G A a ~ C C A C C T A G T C C T C T 60~ ^ c ^ r C T C ^ T ¢ * T T T T T m ~ C , C r T C C ~ T C C C ' T T T T T T T C C C C C T C ~ A O A GerT~TC, C~T^Tt ,~ATC, c r c r T ~ T T T ' m T C - ~ r r r ~ T A C , A A C r T T ^ T ~c, c C r r T C C ~ r r A A r r o c ' i r ^ T C C ~ C C ~ r T T T C r ~ A m r x x z ~ 1 C C m C T T ~ T C r S T ' ~ T T T ' ~ e C ' T r C - X ~ A C A T I T T ~ I T T T T r T ~ T T ~ A m Z C C ~ O t ~ C A ~ m ~ T ^ C ^ T r ~ I T C C ^ T A ~ ' : r ~ A ~ C A T A T T T C ' T T T T C T T C C C . m e C A A T T T ~ O V ~ A r ~ r C 4 0 ~ C r G r C A C r T C A T C A m ^ T T 901 r rTGTrAATC, C r ~ ¢ T A A ~ T T ' ~ d ~ A T T I T A T A A A T O T T T T ~ K r C T ~ T A T C T G r T T G A C A ' ~ . ~ T I ~ f ~ T T A ~ C T T T ~ T G A ~ * ¢ A ~ T A A T T T T T T C A A A ~ A T T A A G ~ C / ~ C r l C C ' I ¢ C ~ C ¢ C T O ~ T C , eCTTGACC;¢

1,0~1 TTTCTCCAA#J~CTTTCCTC4~TTTCYrgAT~ATTGAATTCATGI~CTTOG ^ T ^ T C ~ k G G $ O ~ T . * d O ~ T ~ I ~ 7 , A A r r T G A ~ * C A ~ T T T r A T T G ~ T T T T O ~ G AACTATTh~GJdkTAC~V~CAACATA~TATTCTTGT$*AATTAATTT^GT^ 1 , 2 ~ 1 A G A A r r T ~ A A ~ C T O T ~ T r ~ T C ~ * X T m O U ~ A T ~ T ~ T O ~ C C ~ C r G T C C C T ~ T m C T C C ^ T c r m A T T e C m C a ' ~ a C r T T T C T C T ^ C ^ T T C U T T T C C A T T T T m T C r e c c r ~ T m A A C . ~ T ~ T C r T C T T C C r a . ~ ~ l r C T T C T 1 C X C T ^ T T C C m C r T C r T S C C C ~ r T T ~ A C , C C C C ^ C r T C C C C C C G ^ T G G r C ~ C A T C ~ . ~ T T I C C r C r T T T e C r T C C T C C T r T T T C C r m T T T C r T T ^ C r T C r e A / V , m ~ C m ~ T m m ^ ' m T T T C A m r T T ^ ~ G m ~ T ~ : , 50~ TTK~XTTTTCTTTTTTTTCTITC.CAGGACT~V, CATCTM~CCC;^CCATGTC GAACGÂGGC~ATCCACCACAACCTCAACAACAACAC'Cr-SC4~TC^CA~G ~ T T C ~ . C T C ~ V * C C C C , GACGTCCA(;.V,C~TTCCC-CA

~6 S F L g A A g A G D L g K V L g L L R ~ G T D I N ? S N A W G L N S L H L A S K g G H $ E V V g E L -1

801 A T C ~ C G A C A # G C A C ~ * T ? G A T G C ~ G C C A C ~ G T T m T T T T T T A T ^ TATATTTTTG^TCACAT~d~ACTG~TGTMk~CTC~A~ZS~M~Z; C T ^ T Z T T ~ A T A C r T G ^ C ^ C A G ~ T ~ G ? ~ G I O ~ I ~ T Z Z A T ~ ' G T T

951 I C T ~ I T G A ~ ^ T A G T K ~ T A T G ~ T T T T T T T G A A G A G T T T G T T A A T A A A I r G m C T T I I T T T T T T T ^ A T C C r , I T A / U ~ . C A A Z C C ~ T T G C T ^ T Y ~ ^ T r ^ C C ~ , a ~ C ^ A T T C ~ U ~ T * / ~ C T ^ ^ ~ A C I T ~ C X C A C r m ~ A C ^ ? C A m ~ t O , A 10 t ~ G T T G ~ T T T m I G T r r C T G C ~ ¢ ¢ m r T G ~ G A m ~ , ~ , A m r m r C T S T ^ T ^ r r ^ . r ~ r A r T ~ r G x x G G c c A ~ r r r r r G T ~ r ^ r c ~ G x r ~ a T C ~ . ~ C C r r ~ m r T C - c m ~ o ~ C ~ C ^ C C C C C C ~ C C m m C C C T T C

~2 : . : ~ T T T G G C T ~ T C A m C ¢ C T T ~ T ~ T C A C ~ T T C T m T C ~ A . V ~ r ~ X C ~ C ^ ^ C G T O A A C X ~ Z T C A A T C A G Z T ~ r ~ C T T C m C C C ^ C X C T m ~ T ~ , : X G C T c ~ G G m ~ T C ^ ~ * ~ r r n r ~ Z ^ r c r c z r G ~ a ~ ¢ ~ ¢ A ~

i c 8 L A G Q S L I V T I L V E N O A n V S V ¢ S V . G r r ~ L Y 8 ~, ^ • Q g N H S S V V g y L L K a C A N

2 . ~C l C C A C ~ C G C I T T ¢ ¢ m A G , ~ G m ~ m T C m T ¢ C ^ T T C ~ C ~ r r ~ ¢ T C T T ¢ ~ C ~ . ~ G G ^ C ^ Z ~ T ~ T G T T G r r G c ~ r ¢ ¢ T T C T C G ~ T G ~ T T C ;AAG G G h ' . ~ A ~ T ¢ O G T C T T C C m C A C T T C A T m r G C T C , C ~ m G m ^ C 158 Q A L S T £ 0 G F T g L A V A L 0 Q G H D R V V A V L L g N D S r G g V R L P A L H I A A g g O D T

2 , ~ 5 1 ~ACAGffmCGACACTTTTACTTC~TG~CAC~TCCAGATGTGACAT ~AAATCTGGAYTCACTCCACTTCATATTC.CAGCCCATTATGGACATGAA mTCTTGGACAGCTGTTGTT~*~A.~*GGA~CA&ATGTCh~TTATCAAGC 198 T A h T L L L Q N E H N F D V T S K S G F T P L H I A A H y G H ~ N V n Q L L L E K G A N V N Y Q A

2. ~ 01 TA~CACÂTATA:..GGTGAGACTT.V~GTGTT'f'f'f'tTCAC~TACA:..T.V.. ACGTTTT~T~AT~AT~CT~CGTG~J~CAGT~a~TAATAAA~K~ APdo~ACCTATTTTG~A~AGTTCATTTC~A~TTT~J~TTTT~P~TTT

2.@51 CAAGCC~ATGTT~T~GTA~V~TGTTAJU~C~TTAATATTTT~T~U~ ~A/~TGyTTTTCGCATTI~CTC~T~ATGTGTTTT^C.PCh~TT/~T T P w ~ A T ~ A A C T T T A ~ T / ~ T T T C S d k R A ~ / ~ T ~ T & T C G ~.001 A/V~A~TGTTCAC?TCh&CCTA/~TTCTTAC~YTCCA/~CTGATATTTAT TGT/Ot~TGTTGCATGTT~T~TT~AC~dgT~GCTCC~TI~GTT Tt'AC'gTT~TTT~'¢GT~TAT.I~%T/~.TCCATTACG~A~CTJt~C~A 3, JSl C~TATC~TACATTTC~TTTGT~A¢~TTATG~C ~¢~TAT~TT~T~TTTT~TTTTTTTT~TTT~T~TTTT~ ~ T T T t A y ~ T ~ T ~ T T T ~ A y ~ T ~ T T ~ , 3 0 1 ATTTGTTTCCCC.CAAAATCGTTTCATTTCTATTTCCC47CATCTAGAATCC KTAACCAC~TGGCACACCAGTTCTCAGCTGTCTATTTTGT&TAATC£AAT ~TAT~TA/~CCT~'CTC~TCTT&T/U~IAA J , t51 GTCTCTT~ACA~TCT~T~CC~TGT~A~T~TC~TTGTGT C ~ C A C ~ A ~ T ~ T T ~ T T ~ T ~ C A T C A A ~ T ~ n T T T ~ T ~ A C ~ T ~ A ~ T

2 ~ ~ L ~ v ^ T X w a ~ ~ . + 0 ~ A C ~ . ~ r ^ r c , c , C ~ T C r C T T C C T T I C C C G T m X c - c r ~ x c m ~ ^ ~ c ~ C O r ^ C A ~ O ~ C ~ r C r C r I ~ A C r C C A C T T C X C X C . C C C C C C C C ~ r T C ~ C ~ A C ^ I G ^ C C ~ T T G T X ~ . ~ / c r T e r T S T ~ r r c ~ a ~ m , c ^ c e ~ * c r c r e ~ A ~ A

2~3 r N M A N L L L S a G A I i D $ R T X O L ~ T ~ Z . C ^ ^ n S G , n o v v o ~ ~ v v o z ^ v i s , r r$

~ , ~s 1 ^CGAAh~ACC~ATIC, CCACC^C~CXTmC, GC'ZC.CCC~C~maIC^C'GI a C ~ T ¢ , C T C ~ C m A A C T C T t ~ T ~ T ^ C C A T C G T C ~ T C C ~ G T ~ G ^ r G ~ ¢ G T C ~ CTOrrG^~T~TT~G~tC~CC~C'ZCCXC'G~C,C-C~C~tCarTGTGC,~cArGTT 323 T K N G I. A ~ L H M A A O G D I( V D A A R r L L Y H a A p V D D V ? V D y L T P L 8 V A A R C G H V

~, 9 o l ~TT~T~GT~AT~ATCT~ATCC~CTCC~ T~CBT~TTTCA~CCATTACACATT~GT~BT~CATB ~T~TTGB~TCTT~G~TAC~T~@T~BCB~

zn 4,0 S ~ G~rcc~.~c~s^crcosrrsc^c~TC~Ce~¢crrcmc~Gmo.~Tc*a T^TTSr~^rCrXCrr~CrZC*aC~W/~CC~TCCm~G~^Cm TCCa~C~XCaCTCCrCrTC^rrrC~.Cm-CrCSrGCC;aTC~CCS~C

~23 ~ S S L Z r L H V A A r H G h I N I V 1 y L L Q Q ~ A N ~ n V E T V R O £ T p t . L A A ~ A N O r I) ~12 ~Z~

I , 201 GTTGTT~TGTG~CATC~TBT~TTfi~T~AC~A~ T~TT~CACCTCTACACATTGCC~T~TTT~BqA~GACA TTGTGATT~TTTGTT{~BTT~BT~@C~A ~73 V V ~ V L I R N G A r V U A Q A g Z L Q T P L H I A $ R t G , T D I V I L L L Q A S h g S N A T r ~

~, ~ 1 G A T ~ T T A C T C G C C A T T G C A T A T T G ~ T ~ A T T T T G ~ ~ T G ~ C C ~ T ~ T T ~ T T G T G ~ T G T ~ T T T ~ C A T A T G ~ T T T ~ T A T ~ C C ~ T T ~ C A T T T A T ~

~. 501 ~ C ~ T T T T T T T ~ T ~ C C ~ C C ~ ATGT~TTTC~TT~T~TT~C~TTTTTC ~ C A T ~ T ~ T ~ T T A T ~ A C ~ T T T T T ~ , 65 x T~ACAC¢GA,IOk.~A~AAAACACTT~CTmT/OO~O~ACTACACCTGCTTTGA ~ V * ~ T T C T T ~ C A ~ T A A A A T T T ' g T T C A I T T C C A G G A C A ~ G A . ~ , A A ~ T T ~ C ~ G C A I I C T ~ C C A C ~ A A ~ A C T C T I C T C h

5 ~ 6 Q g S V k O I L L D H ~ & D r ~ L L ~,801 CC~G~TCACACCAC~CCAT~ATCC~TAT~TCTT G~T~TT~TCTT~T~TA~CC~TT~TAT~ ~CC~TGACACCA~CCA~CA~AC~C~CA

~, ~Sl ~C~TC~C~ar~crTcrScrc~m~*re~ac.ccrcc~c;a~c~-cccrscc ~a~*Ct~T^T^CrCC^TT~^T^TCSC~CC~;aS~TC~.~TSS^ ~TT~C^TC~^CmTXrT~C~rTC~UW~crG^TCC~rC~r~Ich~ 60¢K V A M L L L E W G A S A K ^ A ~. g N ~ y T P L a I A ^ K I ¢ a Q g B I A $ r L L Q F X A D p N A X $

rZ~ 5,1ol G~C~IITCACAC~CCAT~TTC~C~AC~B ATTT~CTT~TATTG~B~TT~GATGTT~C~B CBT~CTTACgBT~ATCTTIGT~AC~ATCATGTTCC~

6S*g A o r T e Z H I. S ^ Q g S , K E I S G L L I g N O S D V O ~. K A N N G L T A M B L C A Q E D ]q V P n ~ r 2 0

5,251 TTGCTC~ATT~TTAT~C~T~G~TC~T~T~A~T ~TTACACTCC~TC~TT~TGTCA~TTT~AC~CTC~T~T ~TC~TT~T~TTG~T~TGTT~T~TTTTTA 7 0 ~ V A c.l 1 ~ ¥ N , G A g I N S K T N A G Y T P L M V A C I 4 F G Q L l q M V g r L V i , O A D V O

r 2 t S . 1 0 1 C ~ V ~ A T r A T C A T C T G T T T C A C T A A ~ A I O T T ~ T T G ~ C T G r G ^ T r ? G ^ I OAATITTC-ATTAATTS.W.CTGICAIGT~^TSÂT~ITGATGAA??rTC, G ¢ G a r r r o m o A ~ r r r c r A A r ^ o a r r c r a c c c c ~ A r c ~ c T r r T a ~ A r r T r ~ s.ssl ^TIT^CAC~AC, O,A.L~.,C~IC,¢CTC^TACA¢CCCICrZCACCm}C, CC~ CCCAACASC,~TCACAACAACTC.C~TCC~XT~,CCrrCIOG~*.AArC~AC, C X rCC-CC~A~C~mCAAACTC.CU~TA/*C~CCACTAAC-CTTT1X~e~CACATIC

~ 5 0 g g T R A $ ¥ T P L N Q A A Q O G I( N N C V R Y L L $ N G A S ~ N g Q r A

I C ~ r C z22 5 701 A ATCATACTTCCAC~BC~CTTCTC~TTTC~TTTC GTGT~TCATTT~TTTT~RCTBT~TCAT~T~TTTTTATG~ CTTTTC~T~TTCTCBTTCBT~TATTTTC~AC~

rn 5. 851 CACCI~CTCTCCATTGCCCAACSCCTTC, GATA¢GTGTCA~TCGTTC4WAACT c r r ~ . ~ c r o t c A c c ~ c A ~ c m r c m c ^ c m m A c c * c ~ c T o r c ~ ^ c~As, m ^ r ^ c ~ c c m A x ~ A r c c r ~ A A r c . o t ~ m m T ~ r l c r

7 9 0 T P L S I A cd R b G Y V $ V V E r L R T V l E T T V I r E T r r v D g a y K r Q S r g h M N | r N F

6. 001 CCGAATCAGAAGAI~AAC, G ^ C A A C , ¢ I G C ~ A S C A T G M , G~C'C~TGGCC, CT CAC~IV~AC~TTTCTCCX~kCAATTTGAC3*CA/*C.GATIG~A/,~ATTCG^C T G ~ C a I I C A T A T 6 A T T C A T A C T ~ I A C - A C A T I C A C ~ I C C ^ C ^ ~1o s g s g D ~ G ~ A A g N l G O G A a g K D ~ S O , L T Q O L Q O s T G V R M I N T G g Q

~. 151 T A A T A T T A T T T T G G & T A A A T T ~ T G T O ~ T G T C T T C ~ q T G A C A T A C T ~ A T T C ~ T T T A ~ T T A T T ~ A T B T T ~ T A T T I T T C , CT~ACTG~C, CAAA/t~C AATTA~TTACCGA2utA;~CAATCGATTATAATTTTTATTAATGACTASTT s. ~ 0 l ^ ~ T C , ~ / , 6 A ~ A T A ~ M ' - - , A T I t ~ C . C ¢ C T ^ C T I ' T T C r T A A / ~ ^ T A C ~ A A I ~ Q ~ A ~ ' A T ' ~ & ~ T ~ T C T T T C T 3 0 ~ q A T T T ~ A C T G T T T C / ~ C A C A T T T T T T A T T A C A J ~ T ( ~ T T T T ~ I ~ I ~ t T A T T ~ A A T C G G C A T A T T G ~ - T C C I T 6 , I S t T C C ~ O U W . C n C ~ T ~ I t C C m C m } C ~ T I ~ C , A / t ~ a ~ & X ~ m U * X r , C T ~ C ~ r C e ^ T T ~ C A ~ e r C ' r ~ r T T T C ~ A t ~ , C r ~ T ~ T ~ - C ~ I ~ T ~ C m , CCAe-CCC, C.C 6. 601 TCGTGI~JtC, I ~ T C A I G G A I C ~ T ~ O ~ T ~ O ~ J * ~ T ~ I ~ I C C A T r I A I r r T o A I V , ATO~^TCCTrITC'rCA ATGTG~CATTICACTmTCACTTTCTTCTTCTTCTT~TT^T'I~TC&TTC 6 , 7 5 1 G C % ~ T T T C T C A T C . * * I C A . ~ t ~ T T C C T ~ T T I C T T ~ . ~ . ~ C a ~ T A T C ~ * T ~ * T C ~ J t ~ 7 ~ T ~ O ~ C C ~ I ~ T / * X T I A C A C C ~ T T . I ~ C C A I ~ A A ~ C ~ A I G T ~ T T C G T ~ C A C I T ~ T C A / ~ , A T T & T G A ~ T I O t ~ * G A s, ~o I A m ~ O ~ T C m ~ T C A C ~ C , ¢ I X ~ G C l ~ ' m C ~ . ~ A T O l a ~ C ~ O ~ O ~ q ~ C , ~ C ~ . . C ~ 4 t ¢ ~ C ' r O S r C I ~ W A T ~ I A T T T ~ T T T T C T T A I I C / ~ - ~ ' T T T I T I I T O ~ / O ~ A T A T A C T G I T I C ' r G A / O t C A T T T T T T T ~ T C T O C m T W T G

88$ s Q £ L K N G G A I P K l ~ S G G M 5 P g K S F A K I A p V k T S s P I A T S N S Q S F G I A ~ ~ A 7,20~ CGG^TCTATTTCOGGACAATTCCAACAACAACCACTTCACGGTC.CAC, GAC C~EA~EACAACTT~C,A~C, AACTTSTT~ACAGAATCATCCGATC A A C G C T G 4 A A A C T A C G A T ~ A T ~ G A ~ A G T T G O C A T G T T ~ A G ~ A T G G A C A

938 G S I 5 G Q F O Q 0 p L H G A S ~ ~ D N L £ S L V ~ ~ A Q N H P I N A G N Y D N G G V A M L g N G H ? . ) 5~ TGCGGATAATGTCCCAATTGGACATCATGICACACAC47C/O~GGTTC~TGG A T / ~ A ~ m ~ - - . . , I ~ W ~ A T T T T A ~ T A T I C A A K ~ . T ~ Y G A T G C C C G T CG^TGGCAYGT$~I~TACCAT$Ok~.TGTTCTCAAT/*~AT.~ATCÂCAT~G

9B~ ^ D N V ~ I G H H V r Q p $

7, 50~ TG~C~T~TT~CA~TTT~TCCATATATTTCA~TC ~TTGT~CC~TTATCB~TT~TAC@TTTTTTGATAT @C~BC~TC~TTAT~TATTTT~CABTTT~T ] . 651 T T T T T A ~ I ~ T T ~ T y ~ A ~ T ~ T ~ T ~ T ~ T T T ~ T ~ T r A ~ T T C A O T T C A T A T T T T ~ T ~ A ~ ~ ~ T ~ 0 0 I T T T q G A T T / / C ~ C T G A J ~ N . I ~ T ~ T ( ~ a & A C L k ~ T C G / O ~ I ~ C - ~ m e C T T C - J t ~ I A I ~ T ~ T C A T I G A T A T T T T T ~ % T T A J ~ T . * * A ~ - . A T m T C , A J O O k I ~ T C ~ / O ~ I * T ~ T T T T ~ T 7 , 9 ~ 1 ~ T T T ~ I ~ m I C ~ T ~ C ~ T ~ T A T ~ T ~ T T ~ A T ~ Y A T T ~ A I ~ I ~ T m T C ~ T ~ A ~ y r r ~ r T r ~ i ~ i 6 i ~ l ~ B. 101 ~ T T C ~ A I T ~ T T T ~ T A T ~ A ~ T T A T T A T T T T ~ T T C ~ T ~ C ~ T T ~ T ~ A T C ~ T A T I ~ T T ~ T ~ Y ~ B, 251 A ~ T A J O k C ' / I C A I t ~ A ~ . r ~ C G T . M ~ r I Y ~ T T e ~ , . C T T T T ~ I ~ J ~ , ~ A C C~AJ~tTT~mIT~TC~T~TTG*ICATTTCTC~T~Ty&~I tCG~T' i 'C ' ICTATC T C T T T t ~ T A T ~ T T C A . I ~ d t T A T ~ . A ~ T T T C A ~ I ~ T A I W T y T T T ~ I ~ T T I I ~,00~ r L o . n r S, IoI T ~ T T T C ~ C A ~ T T C ~ T ~ ~ ~ T C ~ T C A T ~ T T C ~ T C ~ C ~ T T ~ T T A ~ T T A T ~ T A ~ ] T T T ~ A 1 . 0 0 8 F L I $ t L V D A R G ~ A N K o c R N $ o v ~ i i v t ~ R X K S Q t I g V T C ~ V L It g

i ~ S p * ¢ t r i n - b i n d t n g D t ~ a i n

D X ~ N '~ e p L $ s a S g I~ $ g I L i Iq A I~ A O A g I ~ @ V X L II V t II

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l . t 91 F p D G L T I( T I IC V V 0 & Q e V ~ Q t N V I L I I ~ N t l V A V S p I V T V S I~ It i~ i c t M K ~ . Z~S c c ~ c x c r c r r ~ ¢ ~ r ~ c c ~ c r T C C ~ c o a u T ~ a C ~ m C ~ c ~ G ~ ~ ^ C m ~ T ~ C r ~ C a ~ W . ~ O ~ C a C ~ W , h ~ c c r c c ~ a c ~ c r ~ c ~ m ~ ^ Z T ^ T C - C ~ C G m * ~ C r ~ T G ~ ^ r c r c ~ c c m , c ~ c ~ a a ~ ¢ ~ a ~ C ^ T C ~ C r 1,24~ I T L C P L P Q s N i~ G M L T Q ¥ $ G Q Q G Q P P T L II L L C $ I T G G $ A A Q W | C I T 9. 3 0 1 G G ~ C ~ C A C ~ C . C T C A C T T T T & C T I ~ A I G A A ~ T I } T C A T T C A C G A C ~ C ~,~TATCTGCCAC, A T T ~ A t ~ 4 ~ T T G C C ^ G ^ C ~ C C T O ~ T G A T G C C G C C * a ~ C ' ~ C ~ a ~ a T C - r ^ C A A T C , M~CAXTTTC ' I / t~TCC^r~ tCAT~ X , 2 9 ~ r T Q L F T G D S V $ ~ T r V S A I t F W I . I ~ D Q ~ ~ I~ O A A I I I G O I V y W A I $ I e ¥ N 9,4~I GCCAAGTTTCC;~GTATTTGCACGAAC, AA~TTTCCCAGTT~ACAACT TOSTGTATTCTGTAT3ACTGATGACAAC, GA~GATAAGACACT~AG~&f,C A~AG~ATTTCAAGT~ATTC, C/OO~G~T~T~TTCTTAAA 1 . ~ { i K F A V & K I~ l F ~ V G Q L ~ v F C N ~ D D X O K ~ L S X Q i H ; K L I & K I 0 V I V L K 9 , 6 0 1 ~ N / ~ A C A T C A t ~ T T C C T T C - A A ' r t C ' T ~ C C T C ~ T ~ t ~ T C ^ A ~ C A C T A ^ A T C T G ~ A G A T C A A C T T T C c C r A T T C r T C C T C W T C A Z t C C J ~ S A a T C ~ T C T T C , ~ A r T T A t C . G t G ~ I * C ~ C G ~ C . ~ I ~ A T C , ~ .CA 1 , 3 9 ~ K H Q F E F S G N L V £ T K s G D Q L S L I I P p Q | N B L A F M V K T R S D D N | A A T

i ~ ~ * g u X * t o r y ~ £ n ~ , 7S~ ~A~C, G A ~ G A ~ T C ~ . , A T r r ^ r C , C, C A ~ C ~ C ~ r C m A r C m ^ r C . C A C ' T T C C ~ A ~ C ~ T ~ T ~ A T T ~ A T ~ T ~ T a T A ~ T C ~ A C ~ C C A ~ C ~ T ¢ ~ T ~ C 1 , 4 1 1 g G R I G F M A I P K I ~ $ D A L P P O Q P I C T L A I S L P I Y T G K I K T A e P p K X D L T r ~ 9 , 9 o l G ~ C ~ T A T ~ A T ~ A ~ T ~ T ~ T C C ~ T T T G T C C A T C ~ T ~ G ~ C ~ T ~ T ~ T G C A ~ T C C C A C A C ~ T A T T C ~ C A C A T T ~ T T ~ 1.~9~ E Q ~ y G $ & L S K D L p g F V H Q N V L K ~ I G A D W p II L G R A L E V p H • 0 I Q M I I 0 N Y ~

10 , 0 ~ C ~ a C ~ r C ~ h ~ C X C A C Z C ~ ^ r c r c ~ r T C m T T G ~ C ~ C ~ ^ T C C ~ a c c ~ c a c s a ~ c r r c a r c ~ a C ^ C T C X C a C ~ ^ r r G c a ; ~ x c c ~ c ^ r ~ G r ~ o ~ ^ ~ c c ~ r c ~ c c r ^ r ~ c ~ c a c , c c m ~ r t ~ c ~ c a T c ~ a c I . S t I G Q S C K N Z L K I W I ½ L K K K I) A N Q D N I. 0 Q A L ~ Q I G R D D I V It $ I A y G E V D A L I W

1 0 , 2 0 1 TAC~TC~GATT~CC~TC~A~BTT~TCA~T T ~ T T C C ~ C A ~ T A ~ B C ~ A ~ T ~ T B C ~ C C ~ T T C ~ A C A C ~ T C A ~ A l , s ~ l Y $ o A o s p s Q g a D ^ i ~ . r s • v e ^ ~ t L v x ~ ~ v ~ T K D I. V T ~ S r V Q V I H 14 V ~ Q &

1 0 , 3%1 ACCA~T~TTC~CAT~TATTC~AC~TTCATCAT~TCC TGTTC~CC~T~TC~T~ATAC@~ATTTT~ ~TTCCATTGAT~TAT~T~@T~TTTATT~ 1 . 6 ~ I T T V V Q E ~ S Y S A ~ V H H S ~ v P s K p E , s E

1o , 5 o ] ~CATG~TT~TTCT~CT~TTTTATTTC~CCC~T ~T~TA~TTGTT~ATGTTCATGAC~TG ~ A T ~ A C C ~ T T G T ~ A T T ~ C ~ C A T R ~ T 1 , 6 6 7 E A p V ^ ~ M ~ ~ v v B T s R , v H O $ E 0 G ~ V V g K m T I T T T Y E D

~0, 6SI G A ~ T ~ T C ~ C A T T G T T G A C ~ A ~ T T C C A T T G ~ A ~ A ~ C ~ T ~ T ~ T A C ~ C ~ C A T ~ A T ~ T T T C ~ T T T ~ T A T T T T T T ~ A T ~ T T ~ T C ~ 1.705 D V A V N g N I V D n T V P L N £ D S 0 Q K W E g L N R L A D K S

1 0 , s 0 1 C A C C m C C C C A ~ n C A A C C - C r C A A c A ~ r r G T ~ ' T C a ~ 4 ~ C A C C T C C ~ m C ~ T T C C ~ T ~ T ~ T ~ C A T ~ ~ T G A ~ C ~ T ~ T C A ~ C ~ T ~ C C ~ A T C ~

1 , 7 3 7 5 ~ $ e A Q R $ T I V A £ $ T $ g Q V P £ V V E Q $ V E $ E $ H K g D D O T I V T T T V T T $ ~( 1 $ ~ X0, ~51 SrAAC, GAAAA~TZT~'C,C~^Tm~rZrC, C.arAACT(~C~-~ATCC

Figure 4. The nucleotide sequence of the ll-kb BamHI genomic DNA fragment. The nucleotide (nt) sequence of the 11-kb BamI-II genomic fragment including the ankyrin repeat, linker, spectrin binding, anda portion of the regulatory domains is shown. The ankyrin repeats are noted below the protein sequence. The point of overlap with the DD#PAO49 eDNA clone is noted. Amino acid residue positions are noted at the left margin. These sequence data are available from EMBL/GenBank/DDBJ under accession number U21734.

it is not known whether the spectrin interaction with this region is specific (Davis and Bennett, 1990; Platt et al., 1993). The presence of the three introns in the linker domain could provide sequences for additional alternatively spliced exons.

Searches of the intron sequences for vertebrate-like sequences failed to reveal cryptic exons.

A 47 % protein sequence identity (198 residues) was found between brain, erythrocyte, and nematode ankyrins in the


Figure 5. Comparison of the ankyrin repeat, linker, and spectrin-binding domains. The ankyrin repeat, linker, and spectrin-binding domains of nematode (n), mouse erythrocyte (mr) (White et al., 1992), human RBC (hr) (Lambert et al., 1990; Lux et al., 1990), and human brain (hb) (Otto et al., 1991) are compared. Identical residues are shown as inverse text. The consensus (c) sequence is shown below the various ankyrin sequences with brain-specific (*), erythrocyte-specific (=), and vertebrate-specific (.) residues noted. Ankyrin repeat numbers are listed at the left and the positions of introns in the nematode ankyrin are shown by the vertical arrows.

424-aa region of greatest spectrin-binding domain similarity (Fig. 5). The spectrin-binding domain similarity is intermediate between vertebrate brain and erythrocyte isoforms, suggesting that a single nematode gene may substitute for the

multiple ankyrin genes in vertebrates. As with the ankyrin repeat domain, the spectdn-binding domain contains more brain-specific residues (12% or 50 residues) than erythrocyte-specific sequences (6% or 24 residues), and 18% (75

Otsuka et al, Ankyrin and Axonal Guidance 1087

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Figure 6. The AO49 and AO66 eDNA and predicted protein sequences. The DD#PAO49 sequence has been broken into thr~ parts and aligned with the DD#PAO66 sequence. The 5' end of DD#PAO49 joins the DD#PAO66 sequence at nt 97 ( ~ 31)just to the Nght of the DNA recombination site. The 18-nt insertion due

residues) were vertebrate-specific. The longest nematode spectrin-binding domain sequences that matched the vertebrate sequences were VDARGGAMRGCRH, SPIVTVEP- RRRKFHKPITL, PTLRLLCSITGG, and PAQWEDITGTT.

The A049 and,4066 Ankyrin Carboxyl-terminal Domains As is the case among the vertebrate conventional ankyrins (White et al., 1992), the regulatory domains of the AO49 and AO66 ankyrins are less well conserved than the ankyrin repeat and spectrin-binding domains. For this reason, the carboxyl-terminal assignments of sequence similarity are tentative. The nematode ankyrin regulatory domains are smaller than those in vertebrates. Using the regulatory binding domain boundary defined by Lux et al. (1990), the sizes of the regulatory domains are as follows: AO66, 381 aa; AO49, 324 aa; human red blood cell (RBC) 2.1 ankyrin, 497 aa; 2.2 ankyrin, 336 aa; and human brain ankyrin 2,401 aa. Although weak, the amino acid sequence identities are greatest at the extremities of the regulatory domain (Fig. 7). Starting at aa position 1432 of full-length AO49 ankyrin, which marks the beginning of the predicted regulatory domain, a region of '~160 aa can be aligned with the vertebrate ankyrins. Beyond this point, neither AO49 nor AO66 anky- ,ins shows an obvious version of the band 2.1 exon that is spliced out in band 2.2 ankyrin. At position 1655, there is a short sequence in the vertebrate erythroid and nematode ankyrins that resembles a single copy of the 12-aa repeat found in brain ankyrin 1 (Otto et al., 1991). At position 1702, there is a poor similarity to a small region at the 3' end of the "2.1 exonY Downstream of this sequence at position 1755 are two copies of a sequence that shares a similarity with brain ankyrin 1. The alternative splice in AO49 ankyrin near position 1810 provides an ending that is similar to the alternative splice at the very 3' end of erythrocyte ankyrin (Lambert et al., 1990). Present at position 1815 of the AO66 ankyrin carboxyl terminus, but not in AO49 ankyrin, is a similarity to the carboxyl termini of the conventional vertebrate ankyrins.

DNA Sequence Analysis of the AOI3 Ankyrin Carboxyl Terminus The DNA sequence of DD#PAO13 reveals a potential protein fragment that is quite different from that putatively encoded by the 6-kb transcripts (Fig. 8). Northern blot analysis has shown that the DD#PAO13 cDNA clone corresponds to the 3' end of a ~14-kb RNA. The deduced protein fragment is

to alternative splicing is shown below the DD#PAO66 sequence. The 3' end of DD#PAO49 follows nt 2728 of DD#PAO66. The inverted repeat sequences at the breakpoints of the inversion near the 5' end of the cDNAs (starting at nt positions 100 and 275 in DD#PAO49 and at position 74 in DD#PAO66) and a potential polyadenylation site (AATAAA) at the 3' end of DD#PAO49 are underlined. A single nt difference at position 731 results in arginine 244 (AGA) being substituted by lysine (AAA) in DD#PAO49 and is as- sumed to be a cloning artifact. Potential hairpin structures near the 3' ends of the cDNAs are noted by arrows. These sequence data are available from EMBL/GenBank/DDBJ under accession numbers U21731 and U21732.


ter of the deduced protein fragment, the most notable feature is a repeat of the sequence S(L/V) (T/S) SL (Q/A) EFERL- EKE in the central portion (repeat A in Fig. 8). The transposon insertion in the rh1042 allele is located between these repeats (Fig. 8). A shorter set of repeats containing the sequence TDSL occurs near the carboxyl terminus (repeat B in Fig. 8).

In the 3' untranslated region of the cDNA, there are four inverted repeat sequences which may fold into hairpin structures in the mRNA. Three of the structures contain the sequence GCCCCAA in the loop of the hairpin.

Figure 7. Comparison of ankyrin carboxyl-terminal domains. The carboxyl-terminal domains of nematode AO49, AO66, mouse erythrocyte (mr), human RBC (hr), human brain 1 (hbl), and human brain 2 (hb2) ankyrins are compared. Identical residues are presented as inverse text. The solid vertical arrows represent the positions of introns while the open arrow indicates the spectrin- binding/regulatory domain boundary (aa 1383) defined by Lux et al. (1990).

very acidic (predicted pI of 4.0). For this reason, searches of Entrez (release 11.0, June 1994) yielded the highest scores for acidic proteins, for example, the intermediate filament proteins. However, the structural characteristics found in the intermediate filament family of proteins (Steinert and Roop, 1988) are not obvious in the predicted protein product. The predicted AO13 ankyrin fragment contains a central 612-aa portion that is devoid of cysteine residues. The lack of cys- teines (and therefore the possibility of disulfide cross-links in the central region), the hydrophilic character of the protein fragment, and the high predicted u-helical content sug- gest a filamentous structure. In addition to the acidic charac-

Multiple Transcripts Encode Conventional and Large Ankyrins

Northern blot analysis revealed a major 6-kb band and several minor transcripts, including a '~14-kb transcript(s). To analyze the transcripts that arise from the 5'-half of the gene, probes from the AO49 spectrin-binding and regulatory domains were used (Fig. 9 a, lanes 1-6). When blots of wild- type poly A-selected RNA (Fig. 9 a, lane 1 ) or total RNA (Fig. 9 a, lane 2) were probed, a major band at 5.95 + 0.26 kb (n=79) was observed along with several less prevalent bands, including 3.19 + 0.13 kb (n=45), 5.07 5= 0.10 kb (n=20), 6.91 + 0.32 kb (n=32), and 13.86 + 1.25 kb (n=8). Of particular interest is the wild-type ,~14-kb minor transcript, near the limit of detection in Fig. 9 a, lanes 1 and 2, and Fig. 9 b, lanes 1 and 2. As detailed below, the '~14-kb transcript is the only transcript affected by all the insertion mutations, and therefore alterations in this transcript are responsible for the uncoordinated phenotype.

Besides the 5.95-kb, 6.91-kb, and 13.86-kb bands that were present in wild-type RNA, additional bands differing by the 1.6-kb size of Tc/ were detected at 7.64 + 0.28 kb (n=7), 8.55 + 0.10 kb (n=4), and 14.79 5= 0.79 kb (n=9) in rh1013 mutant RNAs (Fig. 9 a, lanes 3 and 4). It appears that the transposon is transcribed into RNA, and somatic excision of the transposon gives rise to the normal sized RNAs in the mutants (Emmons and Yesner, 1984). In the rh1042 mutant, the 5.07-, 5.95-, and 6.91-kb transcripts were unaffected by the downstream Tc/element (Fig. 9 a, lanes 5 and 6). How- ever, as in the rh1013 mutant, the rh1042 mutant RNA revealed the ~14-kb and '~15-kb bands. The '~15-kb band ac- cumulates in the mutants to greater levels than the '~14-kb band, making it more readily detectable.

Transcripts overlapping the 3'-end of the gene were analyzed with DD#PAO13 riboprobe (Fig. 9 a, lanes 7-10). The '~14-kb transcript was observed in wild-type RNA (Fig. 9 a, lanes 7and 8), and both ,'~14- and '~15-kb bands were found in the rh1013 mutant (Fig. 9 b, lanes 9 and 10).

The spanning of the entire gene by the ~14-kb RNA was demonstrated by probing blots of total RNA with the ankyrin repeat probe, stripping the blots, and reprobing the same blots with the DD#PAO13 riboprobe. Probing wild-type RNA with an ankyrin repeat probe (Fig. 9 b, lane 1 ) revealed a weakly hybridizing '~14-kb band at exactly the same position as that obtained by reprobing with a DD#PAO13 probe (Fig. 9 b, lane 2). Further evidence for a transcript that spans the entire gene is provided by analysis of the rh1013 and rh1042 mutant RNAs. Common bands at '~14 and '~15 kb were found in rhlO13 RNA with both probes (Fig. 9 b, lanes


i GAA TTT TCT ACT CAA CTC ACA GAT °AT GTC ATT CAA G~A GCT GAA ¢GA GAC GCC ACA

1 E F s T Q L T D D V I Q E k E C D A T

5 8 ACT CAT ATA ATG A T ° ACT CAA ° C T GAA TAT TCA CCA AGA AGA TCC AAA TTC TTA AAA

20 T ~ I ~ ~ T ~ ~ • Y s ~ ~ ~ s z r L

115 CAA GAG AGC TGC CA° GAA ATT TCC AAT °~ °CA OAA GTC °AT TAT TAT TCG GAT CTT

39 Q E S C Q E I S N E P E V D ¥ ¥ S D L

172 CKA GAA A~A CT° AAT ATC TTG GCT GGA GAG AAG AAC AAT CTT CAT °CT CTC GTG GAA

58 ~ Z K L N ! L A U E Z N N L a A L v s

229 GAG GAA CCT TC° AGC AGT GCG TCT GAT CAA CTT °AT GTA ATC CAT GAA TCT °AT CA°

7 7 E E P s s $ A s D O L D V I H z s v Q

286 GAA °CA CAC CTT GAA CAA GAG CAT CA° CA° GAA °CA °CA GCT CAG AAA GAG CA° GAC

96 E A H L E Q E H Q Q E A A A Q K E Q O

343 GAG AAC GAA GAA GCT GCT GAA TAC ACT GCC ACA CTG TT° GTA °AT °AT GTA CTT CAA

1 1 5 E K E ~ A A E ~ T A T L L v D D v L

400 CAC GTT GTA CAA GAA ATT AAC GAA GAA GAC GAC °AT AAG AAA ACT ATG ACT TCA ACT

134 ~ V V Q E I N E E D D D K K T M T S T

457 TCT TAT CTA ACG GCA ACA GAA AAA GAC °AT CAA GAG TAT GAC ACT TGT GTA ACA TCT 153 S y L T A T E K D D Q E Y D T C V T S

514 CA° °AC GAC AC° TAT GAA TCC °CC CAA GGC TC~ ACT TCT CAP. GAT TCA GRA TAT ACT 1 7 2 Q D D T ¥ E $ A Q ° W r $ Q D $ "E Y T

571 ACA ¢CA ACC AGT CAA GCA CCA AGC CGC CTT AGC GAC TCT °AT GCT GAG CTG ACT GCT

191 T A r s ~ A P s a L S O s O A • L r A

628 CGC °AT CAC OAT CAA GAA CGA C~d% GAG ACG TCG ACT °CA CAA GCG ATC CTT TCG CCT

210 R D H D Q E R Q E T S T P Q A I L S P

685 GTG °AT TCT °AC CGA CAT TTT ACA GTT CAA GAG GAC TTC GAA AT° CCT °TA ATT CG°

229 V D S D R H F T V Q Q D F E M p v I R

742 GCT TTC GAT CCT GAC °AT TTC AT° CAt ACT ACA GCT AGA TCC ACA CCA GAT GTT GCT

248 A F D P D D F M Q T T a R s T P D V A

799 CTT CAG GTA ACT AT° GAG GAA GAG GAT GAG AGT °AT GAC AAG CTG °CA ATC AGT CCA

267 L Q V T I E E E D E S D D K L P I S P

85~ AOT GGA ATT CTT CTT CCA CCA AAA CAC GAT °CA ~A CGT °CA ATT TCC CCA °TG °CA 28~ s U I 5 L • ~ ~ H O ~ O ~ • 1 s • v p

305 e A x s o T e ~ O P v ~ v e

970 GAA GAG GAC ACA ATT GCT GAA CCA ACA CCA CCA °CA CAA CCC °CA GAA CCA GAA CAA

324 E E D T I A E P T P P P Q P A- E P E Q

1027 CTT GCC GAA TAT GAA GCC ACA A~ °AA TCA GA~ CAT CA~ CAA ATC ACA GAA GGA GAC

343 L A E Y E A T K E S E H Q Q I T E G D

1084 GTG CCG CAA GAG GKA GAA GAA AC° GAG AT° AGG CGA CAG GAG ACC GAA CGC ATC CAT

3fi2 V P Q E E E E T E M" R R Q E T E R I H

1041 AGT TTA GCC ATG GAG GC° TCG TCC GAT CTC C~A AAT AGC GAG TCT TCA CGT TAC TCG

~81 s L A ~ Z A s s D L G N s z s s ~ ~ s

i~98 CGA CAG CTA TCG GAC CTG TCG TCG TC° GCG G~A AGC CAT GCG GAC ACT GTG ATC CGT

400 R Q L S O L S S $ A ~ S H A D T V I R 1 2 5 5 GTT GAA AGT GAA CGA AGT GGA TCA ~GT GAT TCA TTG GAA GTT GTT AGT GTT ATC AGT

419 V g S E R S G S S O $ L E V V S V I S 1312 °CA GGG AAG CAT GCA CAA AT° TCT GAA /tAG TCC CTG ACT °CA CA/& °AT CCT GAG AdKA

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1369 °CA ACT °AT TeA a T ° CAA GAG CAT CAA CAA AAA °AT CAG GAA CAA GCT GAA GCA GAA

457 P T O S V Q E H Q Q K O Q E Q A E A z

1426 GAT CAA AGG GAA °CA GAA GAT CTT GGT TTT GAK GTC TAC GAT GCT OAT ACG GAA GRA

4 7 6 D ~ ~ E A e D L U r z v Y P A O T Z Z

. . . . . ~' ~ . . . . . . . . . . Y T T T . . . . . . . . . . . . . . . . . . . . . . . . . . . 495 Q Q L T v E E e P g D S D

1540 TCA CTG AAC GAA GGA GGA AAT CAT TCA TCT C, GA CAT TCC TCT GTC GGA OTT CCA GCT 514 s L s z G G ~ H ~ s ° H s s v G v p A

G t y c

1597 GAT ACT CTT GCC AT° ATT GGC ~G TAT CGC CAT C~A TCA AGT GAC AAT CTC TCA TTA 533 D r L A ~t I u K • ~ H ~ s s ~ ~ k s L

°lye uric-44 (rhZO42;Tcl)

1654 ACA TCA GTT CAA GAG TTT GAA AGG CTT GAA AAA GAA GTA GGA GCT CUT GGA GAT GGT

5 5 2 T s L o • F E K L Z K Z V G A ~= ~ D G ~ * p e a t A I t 'D | | * q u * n c *

1711 TCA CTA ACA AGA TCT CA]& ATT GAA TTG CTT CTT GCC GGA AGA TTG AAC AAA TCA GGA

5 7 1 S L T a s ~ I • L L v k C ~ L ~ S ~ U

G l y c i~68 GAA GGA TCA GTC AGC TCA CTT GCT GAG TTC GAA CGC CTC GAA AAA GAG AT° ACC GAG

590 E O S V S S L A R~pel 8tF A E R ~ ~__L~ H T E

1825 AAT CAA TCC °CA CCA GAA GAT GTT AT° AT° CTT TCT GAT ATT ~T GAG GAA TCA GAA 609 ~ ~I ~ P p E D V M M L S D I R E E S E

G t y c

1882GCAG~GATA~AGCATT~AGATGATGATGAGG~GATG~G~AAGTGATACA

~ 2 8 & E D M $ I R D D D E E D A E G S D T

1939G~T~GAGC~ACCAGTA~GG~G~GATCTCAGAG~ATCACACCAATTGCT 6 4 7 E L K S R P V Q E E D L R V I T P I A

1996 CCA TCA °CA A~ GAT TCT CTT °AT ~T G~ GTT GAT GCC GTT CA° ATG C~ TAT CTC 666 P ~ H A V V A V Q M Q Y L

Re~*t S 2053 GAG ACA AGe ACC GAC TCT TTG GAG CCA ACG TTC CAA GAA ATT GAA GTT GAG CAA CGT

665 Z 2 . _ _ l . _ _ 2 . _ _ D . _ _ ~ l ~ T F Q Z I E v z Q

R e p * * t n

211¢ CCA GAA AGT GTT GAA GAC ACA TCA CTA ACT GAG TAT GAG GTC ATT CCA CGC GTC ATG

704 P E ~ T E Y E V I P R V M R * ~ a t n

2167 GAG ¢CC ~0C ACC ACC GAC TCU CTA CAT GGA ACT OC~ ACT ATT ¢A0 ~ G CAC TCT a t e 7 2 3 ~ A ~ ¢ T ^ T Z • K O s v

742 L P A N L H M D S s p

2281 GTG ACA CAA T~ OAR, CA° T~ TCA CTG AT° AT° ATe GAG ATT CTT TGG AT° GAG AAA 7 6 1 v r 0 w • ~ W S L ~ ~ Z Z I ~ W ~ ~ •

2138 TGT CGA °CA TGC TTC AAT CCT ATC CAP* CTA CA° TTA CCA CAT TCC AAA CAA GAG TTG 780 C a A c ~ N ~ l ~ L ~ L P H s x ~ ~ L

2395 TCG GAC C&G ATG GCT CTC TTC AGA °CA TTT CAA GAC GTG TTG AGA CAA AGT CA° CGA

7 9 ~ s D 0 M A L r ~ ~ ~ 0 ~ v L ~ 0 s H R 2452 CCC &TT AGT GTC °CA CGT TAC ATT TAC TGG AAC AGA /tAG °CA &GA ACG °CT TGACCAAC

818 ~ ! $ V P R Y I ¥ W H g K P ~ T A ~nd

2511 TACCOGATOA TGA~CA~TTT GAGACTGT'GG ATACCU~G¢ ~A~TOT~ACA AG~CCaC'~T TCCATCOGG^ 2SSl GCACO~CCaA C~¢C~ACCAC ACTTTr~U~A GTTC~TC~CC ~ C A T C A e ~ CCCACTTCA~ ^ T A T ~ c ~ ' r c 2~SX T C ~ T ~ T ~ T TCCACTC~AC AC~C~TTTa OTTCTCTTAT ~TTCT~AT^C ~TTCCATATT TTTTGATCCC 2 ~ 2 1 ACAATTTACC AGTTTTCACO CCCTAATCCA GTGACTTGAG CTTACAGTAT TTTTGTTTTT TTTCCATGAG

1 279~. CTTTTAATTT ATTTTTTATT TTTCA~ GaTCCAATCA GTAAAT&CAA TTTCTTCGCC CCAAAGKATT

. . . . . . . 2 . . . . . . . . . . . . . • - • . - . . . . . . . . . . . . . . 2861 TGTATTTTTT TAT~GAAATT TGAAATGATA TTCAAGATGC CCCAACTTGA ATATCATTCA ATAGCTTTCC

3 . . . . * . . . . . . . . . * • • • . . . . . . . . . • . . . .

2 9 3 1 &TC&TTTAAT TTTCTGAAAA TCGCCCCAAT T T T C A G A C ~ TTTCAGAAAT GTTTCAAGCT TTATCTTTAA

3001 CTTTTCATGT TTTAAOCTTT CATCTGCCCA ACTTCTACCA ATATTTCAAT GATTTTCTCA GAGTTGTTAG 3071 CCCC~CAT CTTCTGRATT ATTATAGAAA TTTTTTTCAG TAATTAACTT CTACCATTTC CCATCATCTT

4 . . . . . . . . . . . . . • . . . . . . . • . . . . 3141 T~TTGTATTT CGTTTTTAGT CTACGCGGTT TTCCTTTTTT TTTAATTTTA GAGCTCTAAA TGTAAAAGTT

3211 CTTTACA~ A/%C~CTI&GT CACTCTAAAA ATGCC, CTCCC CATTTCCATC TTTTCATGTT TTTTTTTCTC 3281 ATTTTTTCGT TCAATTATTG ATTTGAATTA TTCATTTOCT AAT&&,n~ ...........

Figure 8. The predicted AO13 ankyrin carboxyl domain sequence. The nt and predicted aa sequences are presented as in Fig. 5. The uncertainty in the position of the Tc/element in the rh1042 allele is due to the two-base-pair duplication created during To/ insertion. The repeated motifs are noted by double underlines. Potential glycosylation sites (Glyc) and an RGD sequence (Singer et al.,

Figure 9. Northern blot analysis of uric-44 RNAs. In panel a, Northern blots were prepared from poly A-selected RNA (odd lanes) or total RNA (even lanes) separated on a 1% agarose- formaldehyde gel. The blots were probed with an DD#PAO49 riboprobe (lanes 1-6) or DD#PAO13 riboprobe (lane 7-10). RNAs are as follows: N2 wild-type, lanes 1, 2, 7, and 8; unc-44 (rh1013), lanes 3, 4, 9, and 10; and uric-44 (rh1042), lanes 5 and 6. The sizes of the RNAs were determined relative to 0.24-9.49-kb RNA mark- ers (GIBCO-BRL, Gaithersburg, MD). The sizes of the RNAs larger than 9.49 kb were determined by extrapolation from the size marker curve (Otto et al., 1991). In panel b, common bands in the '~14-16-kb region (arrow) were detected by probing blots with the ankyrin repeat probe (odd lanes), and then reprobing the stripped blot with the DD#PAO13 riboprobe (even lanes). Total RNA sam- pies are as follows: N2 wild-type, lanes 1 and 2; uric-44 (rh1013), lanes 3 and 4; and unc-44 (rh1042), lanes 5 and 6. In panel c, the '~14-16-kb region of the Northern blot has been expanded by run- ning the gel for an extended period of time. A '~14-kb band is present in wild-type RNA (lane 1), while '~14- and '~15-kb bands (arrows) are present in RNAs from uric-44 (q331) (lane 2) and unc-44 (rh1013) (lane 3) when probed with the ankyrin repeat probe. In a manner similar to rh1013, the q331 mutation affects several of the smaller mRNAs (data not shown).

3 and 4), and also in rh1042 mutant RNA (Fig. 9 b, lanes 5 and 6).

To clearly distinguish the '~14- and '~15-kb RNAs (Fig. 9 b, arrow), wild-type and mutant RNAs were separated by extensive electrophoresis (Fig. 9 c). As can be clearly seen, wild-type RNA contains a '~14-kb band (Fig. 9 c, lane 1 ), while both the '~14- and '~15-kb bands are present in the q331 and rh1013 mutants (Fig. 9 c, lanes 2 and 3).

D i s c u s s i o n

In this paper, we report the molecular cloning of unc-44, a gene that is required for the correct targeting of axons to appropriate partners, and the identification of the putative gene

1987) are noted by underlines. Potential hairpin structures are noted by arrows, and numbered, above the DNA sequences. Al- though the hairpin stems differ, three of the four loops contain the sequence GCCCCAA. Potential polyadenylation sequences, AAT- AAA, are underlined near the start of the polyadenylic acid tract at position 3340. These sequence data are available from EMBL/ GenBanL/DDBJ under accession number U21733.


products as ankyrin-related proteins. Although the presence of ankyrin in the brain has been known for some time (Drenkhahn and Bennett, 1987), this paper provides the first evidence that ankyrins are required, directly or indirectly, for axonal guidance.

The following facts demonstrate that the unc-44 gene has been definitively cIoned. First, six unc-44 mutations are due to DNA insertions. Four alleles (mn259, q331, rhlO13, and rh1042) are Tc/ insertions. The remaining two alleles (mn3~39 and st200) are insertions that are larger than Tc/. Second, four revertants of q331, rhlO13, and mn259 are in- frame excisions of Tc/. Because in-frame excisions of Tc/are unusual, these results demonstrate that the restoration of the reading frame is critical for the function of the unc-44 ankyrins. Third, complementation tests show that the DNA insertion mutations define a single complementation group, and that this complementation group corresponds to unc-44. Fourth, Northern blot and cDNA sequence analysis demon- strates that multiple transcripts are generated from uric-44, but that only the ~14-kb transcript(s) is affected by all the insertion alleles tested.

The isolation of several different eDNA clones demonstrated that the uric-44 gene produces several alternatively spliced transcripts, with the most abundant RNA being --6 kb. The ~6-kb RNA is smaller than the major vertebrate erythrocyte mRNAs which range from 6.8 to 9.5 kb (Lam- bert et al., 1990; Lux et al., 1990). However, the uric-44 conventional ankyrins have smaller regulatory domains than the vertebrate proteins, which might account for part of the difference. The pattern of multiple mRNAs generated from unc-44 (~3, 5, 6, 7, and 14 kb) is similar to the pattern of 4, 7, 9, and 13 kb RNAs from the neuronal ANK-2 gene (Otto et al., 1991). The presence of alternatively spliced unc-44 RNAs suggests that the products of this gene may play varied roles in the organism.

From the unc-44 mutant phenotype, the complementation data, the RNA analysis, and the positions of the uric-44 mutations, we propose that the large AO13 ankyrin is required for proper axonal guidance in C. elegans. The lack of cys- teines in the central region of the AO13 ankyrin fragment and the predicted highly u-helical character of the domain sug- gest that the carboxyl-terminal domain has an extended structure. Exact repeats of the sequence EFERLEKE may indicate a functional role for these sequences.

This is the first demonstration that ankyrin plays a functional role in neural development. A role for AO13 ankyrin in neural development is reinforced by the finding that the 440-kD ANK-2 product is present in the developing rat brain and is localized to neuronal processes (Kunimoto et al., 1991; Otto ct al., 1991; Chan et al., 1993). Consequently, the predominant 6-kb unc-44 messenger RNAs and the AO49 and AO66 ankyrins may not be critical for axon guidance.

Because the nematode unc-44 ankyrin gene represents an evolutionarily primitive form, the conserved amino acid residues provide a starting point for structure-function analysis by site-directed mutagenesis and DNA transformation. The cloning and partial sequencing of the uric-44 gene provides the foundation for molecular genetic analyses which may reveal specific roles for the various unc-44 products in neurons and other cell types.

We thank the group of R. Herman for the kind gifts of the unc-44 (mn259), mn339, q331, and st200 mutants and revertants isolated in the laboratories of R. Herman, J. Kimble, J. Shaw, and R. Waterston; K. Nishiwaki and J. Miwa for the nematode genomic bank; R. Barstead, R. Waterston, I. Schauer, and W. Wood for cDNA banks; A. Coulson and J. Sulston for iden- tifying and providing the cosmid clones; S. Emmons for the Tc/clone; J. Matsuzaki and S. Weldon for discussions; H. T. Cheung for his continued encouragement; W. Li for communication of unpublished results; H. T. Cheung and H. Brockman for critically reading the manuscript; and the Caenorhabditis Genetics Center for strains.

R. Franco was a National Institutes of Health postdoctoral trainee (grant T32-HDO-7299) for a one year period. This work was funded by gifts from TONEN, Inc., Saitama, Japan, a Biomedical Research Support Grant, Il- linois State University Research Grants, and grants from the American Heart Association Illinois Affiliate and the National Science Foundation to A. J. Otsuka.

Portions of this work were previously presented in abstract form (Otsuka, A. J., R. Franco, B. Yang, K.-H. Shim, L. Z. Tang, A. Jeyaprakash, and E Boontrakulpoontawee. 1991. Z Cell Biol. 115:465a. Abstr. 2706; Ot- suka, A. J., E Boontrakulpoontawee, Y.-Y. Zhang, L. Z. Tang, and A. Sobery. 1992. Mol. Biol. Cell 3:196a. Abstr. 1137; Boontrakulpoontawee, E, and A. J. Otsuka. 1993. FASEB (Fed. Am. Soc. Exp. Biol.) Z 7:A1079. Abstr. 159; Boontrakulpoontawe, c, E, and A. J. Otsuka. 1993. Mol. Biol. Cell. 4:571a. Abstr. 326; Boontrakulpoontawee, E, and A. J. Otsuka. 1994. Mol. Biol. Cell. 5:44a. Abstr. 255).

Received for publication 17 November 1994 and in revised form 28 February 1995.

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