Characterization of Caenorhabditis elegans Homologs of the Down Syndrome Candidate Gene DYRK1A
a Department of Biochemistry and Molecular Biophysics, Center for Neurobiology and Behavior, Columbia University College of Physicians and Surgeons, New York, New York 10032,b Howard Hughes Medical Institute, Columbia University College of Physicians and Surgeons, New York, New York 10032f$sh, 百拇医药
c Hubrecht Laboratory, 3584 CT Utrecht, The Netherlandsf$sh, 百拇医药
ABSTRACTf$sh, 百拇医药
The pathology of trisomy 21/Down syndrome includes cognitive and memory deficits. Increased expression of the dual-specificity protein kinase DYRK1A kinase (DYRK1A) appears to play a significant role in the neuropathology of Down syndrome. To shed light on the cellular role of DYRK1A and related genes we identified three DYRK/minibrain-like genes in the genome sequence of Caenorhabditis elegans, termed mbk-1, mbk-2, and hpk-1. We found these genes to be widely expressed and to localize to distinct subcellular compartments. We isolated deletion alleles in all three genes and show that loss of mbk-1, the gene most closely related to DYRK1A, causes no obvious defects, while another gene, mbk-2, is essential for viability. The overexpression of DYRK1A in Down syndrome led us to examine the effects of overexpression of its C. elegans ortholog mbk-1. We found that animals containing additional copies of the mbk-1 gene display behavioral defects in chemotaxis toward volatile chemoattractants and that the extent of these defects correlates with mbk-1 gene dosage. Using tissue-specific and inducible promoters, we show that additional copies of mbk-1 can impair olfaction cell-autonomously in mature, fully differentiated neurons and that this impairment is reversible. Our results suggest that increased gene dosage of human DYRK1A in trisomy 21 may disrupt the function of fully differentiated neurons and that this disruption is reversible.
TRISOMY of chromosome 21, or Down syndrome, is the most frequent chromosomal abnormality in human infants that come to term. Besides manifesting a characteristic set of facial and physical features, heart defects, and abnormalities in the immune and endocrine systems, patients with Down syndrome have deficits in spatial memory and difficulty in converting short-term to long-term memories (JOHANNSEN et al. 1996 ; TAKASHIMA 1997 ). Although the cognitive defects of Down syndrome are likely to arise from increased dosage of many genes, several lines of evidence suggest that increased expression of the dual-specificity protein kinase DYRK1A plays a significant role. First, the DYRK1A locus maps to the Down syndrome candidate region (DSCR), a region of 70–100 genes (SHINDOH et al. 1996 ). Rare patients with a partial trisomy of the DSCR display defects in cognition, suggesting that the genes in this region are sufficient to produce the cognitive defects of Down syndrome. Second, a 180-kb region on human chromosome 21 containing the DYRK1A locus is sufficient to produce defects in learning and memory in transgenic mice (SMITH et al. 1997 ). Third, transgenic mice overexpressing the full-length Dyrk1A cDNA exhibit impairment in spatial learning and cognitive flexibility (ALTAFAJ et al. 2001 ). The cellular and molecular consequences of these DYRK1A perturbations are unknown.
The first vertebrate member of the DYRK family was originally identified in a PCR screen for protein kinases (KENTRUP et al. 1996 ) and subsequently shown to belong to a larger family of related dual-specificity kinases (BECKER and JOOST 1999 ) to which we refer here as the DYRK/minibrain family. Members of this family are localized to distinct subcellular compartments and phosphorylate various substrates in vitro (BECKER and JOOST 1999 ). They share the unusual property of tyrosine-directed autophosphorylation as well as phosphorylation of serine/threonine residues in exogenous substrates (BECKER and JOOST 1999 ). Their precise cellular role, however, remains elusive. Insights into their physiological relevance were provided by the finding that the Drosophila homolog of DYRK1A, termed minibrain, is involved in neuroblast proliferation in the fly brain (TEJEDOR et al. 1995 ). A similar reduction in brain size has been recently observed in DYRK1A knockout mice (FOTAKI et al. 2002 ). The sole Saccharomyces cerevisiae representative of the DYRK/minibrain family, Yak1p, acts in parallel to a protein kinase A pathway to negatively regulate cell-cycle progression (GARRETT and BROACH 1989 ; GARRETT et al. 1991 ).
In metazoan animals, the only DYRK/minibrain family member for which mutant alleles exist is the Drosophila minibrain gene. Here, we describe the expression pattern and loss-of-function alleles of all three DYRK/minibrain family members in the nematode Caenorhabditis elegans. In contrast to Drosophila minibrain, we observe no obvious morphological defects in mbk-1 mutants, but find that another DYRK/minibrain family member, mbk-2, is required for viability. In an attempt to model the cellular consequences of DYRK1A overexpression that are observed in Down syndrome patients, we analyzed the effects of providing extra copies of the worm ortholog of DYRK1A and describe dosage-sensitive and reversible defects in the processing of acute sensory information.9, 百拇医药
MATERIALS AND METHODS9, 百拇医药
cDNAs:9, 百拇医药
A full-length mbk-1 cDNA was amplified with SL1 and gene-specific primers. The structure of the cDNA is similar to the predicted T04C10.1 gene, with the exception of an incorrect first exon predicted in T04C10.1. The mbk-2 locus generates two messages, a long form (mbk-2L) and a short form (mbk-2S) that uses an internal start site from an alternatively spliced exon. Both splice forms are represented in expressed sequence tag (EST) clones (a gift from Y. Kohara) that have been completely sequenced. Similarly, hpk-1 full-length cDNA clones are represented in Y. Kohara's EST collection.
DNA constructs:g@y, 百拇医药
Constructs are shown schematically in 1.g@y, 百拇医药
fig.ommittedg@y, 百拇医药
Figure 1. Reporter gene constructs and deletion alleles used to study minibrain-like genes. Boxes denote exons of the respective genes. Dark shaded bars above the genomic locus denote the location of the deletion in the respective mutant alleles (see MATERIALS AND METHODS for precise location). Kinase domains are lightly shaded. The kinase domain was predicted using SMART. The large size of the mbk-2 genomic locus prevented us from constructing a full-length, translational gfp fusion construct. To monitor subcellular localization of mbk-2 we thus fused the cDNA of the mbk-2L splice form to a heterologous promoter, phsp16-2 (FIRE et al. 1990 ). The two alternatively spliced mbk-2 isoforms produced by the mbk-2 locus were deduced by cDNA analysis (see MATERIALS AND METHODS).g@y, 百拇医药
mbk-1: To build mbk-1::gfp, the mbk-1 genomic locus, including 7 kb of 5' noncoding sequence and all exons and introns, was amplified by Expand long-template PCR (Boehringer Mannheim, Indianapolis). The PCR product was cloned in frame with gfp in the promoterless vector pPD95.75 (from A. Fire), generating pBR104. To build mbk-1(YA)::gfp, the QuickChange kit (Stratagene, La Jolla, CA) was used to modify pBR104 with primers YAs (5'-ctggacaccgaatcgcccaggccattcagtcgagattctatcg) and YAas (5'-cgatagaatctcgactgaatggcctgggcgattcggtgtccag), generating pBR113. To build mbk-1(pk1389)::gfp, the mbk-1 locus was amplified from homozygous mbk-1(pk1389) animals by Expand long-template PCR (Boehringer Mannheim). Following the cloning and sequencing of this PCR product, a 4.0-kb BglII-Asp718 fragment was subcloned into pBR104, generating pBR177. To build pgcy-10::mbk-1, the gcy-10 promoter (YU et al. 1997 ) was amplified from N2 genomic DNA by Expand long-template PCR (Boehringer Mannheim) and cloned in front of the full-length mbk-1 cDNA, generating pBR145. To build pgcy-10::mbk-1(YA), the QuickChange kit (Stratagene) was used to modify pBR145 with primers YAs and YAas, generating pBR147. To build phsp16-2::mbk-1::gfp, the full-length mbk-1 cDNA was cloned into the heat-shock vector pPD49.78 (from A. Fire), generating pBR144. To generate a fusion with gfp, the promoter and cDNA were subsequently fused in frame with pBR104, generating pBR144.
mbk-2: To build pmbk-2L::gfp, 8 kb of 5' noncoding sequence was amplified by Expand long-template PCR (Boehringer Mannheim) and cloned into pPD95.77 (from A. Fire), generating pBR126. To build mbk-2S::gfp, Expand long-template PCR (Boehringer Mannheim) was used to amplify exons 7–11 of the mbk-2 locus and fused in frame with gfp in pPD95.77 (from A. Fire), generating pBR138. This construct uses intron 6 as the 5' noncoding sequence. pmbk-2L::gfp and mbk-2S do not contain the full mbk-2 genomic locus, and this may result in an artifactual or incomplete expression pattern. To build phsp16-2::mbk-2L::gfp, full-length mbk-2L cDNA was fused with gfp and inserted into the heat-shock vector pPD49.79 (from A. Fire), generating pBR169.0'9du.{, http://www.100md.com
hpk-1: To build a translational fusion between HPK-1 and green fluorescent protein (GFP), the full-length hpk-1 genomic locus, including 4 kb of 5' noncoding sequence and all exons and introns, was amplified by Expand long-template PCR (Boehringer Mannheim) and cloned into the promoterless vector pPD95.77 (from A. Fire), generating pBR132.
Transgenic and mutant strains:-4, 百拇医药
The strains are as follows:-4, 百拇医药
EK224 cmIs6 [pBR104, pNC4.21] I; 6x outcrossed-4, 百拇医药
EK270 cmIs8 [pBR113, pNC4.21]; 2x outcrossed-4, 百拇医药
EK176 cmEx20 [pBR145, unc-122::gfp]-4, 百拇医药
EK179 cmEx21 [pBR147, unc-122::gfp]-4, 百拇医药
EK173 unc-4(e120) II; cmEx19 [pBR144, pNC4.21]-4, 百拇医药
EK234 unc-4(e120) II; cmEx31 [pBR169, pNC4.21]-4, 百拇医药
EK123 cmEx6 [pBR126, pRF4]-4, 百拇医药
EK251 cmEx16 [pBR138, pRF4]-4, 百拇医药
EK135 cmEx11 [pBR132, pRF4]-4, 百拇医药
EK228 mbk-1(pk1389) X; 6x outcrossed-4, 百拇医药
EK264 cmEx36 [pBR177, pRF4]-4, 百拇医药
EK273 hpk-1(pk1393) X; 6x outcrossed-4, 百拇医药
EK275 mbk-2(pk1427)/mgIs18 IV; 3x outcrossed-4, 百拇医药
All expression constructs were injected at 50 ng/µl. pNC4.21 [unc-4(+)] and unc-122::gfp were injected at 50 ng/µl. pRF4 [rol-6(d)] was injected at 100 ng/µl. Integrated lines were obtained using a protocol described at .
Isolation of deletions in the mbk-1, mbk-2, and hpk-1 loci:5?, 百拇医药
PCR screening of a chemically induced deletion library was done as previously described (JANSEN et al. 1997 ). The relative position within the genomic loci of all three mutant alleles is schematically indicated in 1. A 1658-bp deletion mutant of mbk-1, pk1389, was isolated using primers MBK1A (5'-gcagacgtgcctgacaatcttc) and MBK1D (5'-tgtaggtatggcggtatccgtc) and nested primers MBK1B (5'-ctcaaatacccagcaacactcac) and MBK1C (5'-caatagtagatcccatcctcag). The deleted PCR product from pk1389 was sequenced and the deletion was confirmed by Southern analysis. The following sequence in capitals is deleted by pk1389: 5'-ataaaGCTTT-TTACGactat. The deletion extends from the first intron into the sixth exon. If in this context exon 1 is spliced to exon 7 (the beginning of the kinase domain), a frameshift and a premature stop would result; splicing of exon 1 to more downstream exons would also create frameshifts and/or delete the kinase domain. The mbk-2 deletion, pk1427, spans 3398 bp in the genomic region represented in cosmid F49E11 and deletes the capitalized sequence 5'-tcgtcGTCCG-AAAACttgta. The hpk-1 deletion, pk1393, spans 1457 bp in the genomic region represented in cosmid F20B6 and deletes the capitalized sequence 5'-cacacATGCC-TGACAtaatg. All deletion alleles lead to a disruption of the majority of the respective kinase domains and are predicted to act as null alleles.
RNAi:$(rjr, http://www.100md.com
mbk-2 dsRNA was delivered by bacterial feeding as previously described (TIMMONS et al. 2001 ). The effectiveness of RNAi could be assessed by monitoring the decrease in fluorescence of transgenic animals that expressed gfp-tagged mbk-2.$(rjr, http://www.100md.com
Behavioral assays:$(rjr, http://www.100md.com
Chemotaxis assays toward volatile odorants were performed as described (BARGMANN et al. 1993 ). Chemotaxis index = (the number of animals attracted to the odorant) - (the number of animals at the opposite end of the plate)/(the total number of animals at both ends of the plate). When transgenic animals carrying extrachromsomal arrays were scored, only those animals that carry the injection marker were scored.$(rjr, http://www.100md.com
Scoring neuroanatomy:$(rjr, http://www.100md.com
AWC morphology was observed by crossing kyIs140, a chromosomally integrated str-2::gfp construct (TROEMEL et al. 1999 ), into the respective mutant background. The generation, proliferation, and anatomy of several sensory neuron classes (ASK, ADL, ASI, AWB, ASH, ASJ, PHA, and PHB) were visualized by filling exposed sensory neurons with the lipophilic dye DiI, as previously described (HEDGECOCK et al. 1985 ). Briefly, a mixed population of well-fed worms was washed with M9 buffer, incubated at room temperature with 10 µg/ml DiI for 1 hr, washed several times with M9, and then mounted on a compound fluorescence microscope.
DAPI staining:z$, 百拇医药
4',6-Diamidino-2-phenylindole (DAPI) staining on gfp-expressing transgenic lines was done by placing animals in a drop of water on a coverslip, letting the water evaporate, adding a drop of acetone, letting the drop evaporate, drying for 20 min, and then adding 1 µg/ml DAPI in M9 medium.z$, 百拇医药
RESULTSz$, 百拇医药
Identification of DYRK/minibrain-like genes in C. elegans:z$, 百拇医药
Through sequence homology searching of the complete C. elegans genome sequence, we identified two C. elegans genes with close homology to the DYRK/mini-brain family, termed mbk-1 and mbk-2 (for minibrain- kinase), and one gene, hpk-1 (named after its vertebrate homologs, homeodomain-interacting protein kinase 1-3), with a more distant homology (2A). mbk-1 and mbk-2 both carry the characteristic DYRK family signature motifs in their kinase domain, as well as a DH-box, a conserved sequence motif preceding the catalytic domain (BECKER and JOOST 1999 ). mbk-1 is unique among the C. elegans DYRK/minibrain family members in clustering in the DYRK1A subgroup (2A). In addition to highest overall sequence similarity, MBK-1 and DYRK1A can also be distinguished from other DYRK family members by their sharing of an N-terminal nuclear localization sequence, a leucine zipper in the kinase domain, and stretches of homopolymeric amino acids at their C termini (2B). We thus consider MBK-1 the C. elegans ortholog of human DYRK1A.
fig.ommittedk, http://www.100md.com
Figure 2. The DYRK/minibrain-like gene family in C. elegans. (A) Classification of the DYRK subfamily of protein kinases. On the basis of BLAST- and PFAM-based similarity searches of the complete genome sequence, C. elegans has two members of the DYRK subfamily of protein kinases, MBK-1 (T04C10.1; LGX; GenBank accession no. AY064464) and MBK-2 (F49E11.1; LGIV; GenBank accession no. AY090019), as well as a related kinase called HPK-1 (F20B6.8; LGX). The gene structures of mbk-1 and mbk-2 were confirmed by sequence analysis of EST clones. The alignment and dendrogram were generated from the predicted kinase domains using ClustalX and Align with default parameters. The DYRK1A group is boxed by dark shading, the DYRK subfamily is boxed by light shading, C. elegans proteins are in boldface type and underlined, and the percentage identity between each kinase domain and that of DYRK1A is listed to the right. (B) MBK-1 is the closest homolog to DYRK1A, whose overexpression is implicated in Down syndrome. The relative positions of the N-terminal nuclear localization sequence [NLS; amino acids (aa) 120–127], the DH box, the kinase domain (aa 317–649), leucine zipper (L-L; aa 443–464; contains four leucines each spaced by 6 aa), and C-terminal homo-polymeric stretch of amino acids (histidines in DYRK1A, glutamines in MBK-1) sites are shown. DYRK kinases are regulated through the phosphorylation of two DYRK-specific tyrosines within the activation loop (Y-Y; aa 488 and 490). For a more detailed alignment and more sequence features of DYRK kinases, see BECKER and JOOST 1999 .
Expression and subcellular localization of DYRK/minibrain-like genes in C. elegans:8, 百拇医药
To determine the sites of expression and subcellular localization of DYRK/minibrain-like genes, we fused gfp in frame with the respective genomic loci (1). The mbk-1::gfp construct included 7 kb of 5' noncoding sequence and all of the exons and introns present in the endogenous mbk-1 locus. The mbk-1::gfp gene product is expressed in all somatic cells and primarily localizes to nuclei (3), similar to the reported expression and subcellular localization of human DYRK1A (SONG et al. 1997 ; BECKER et al. 1998 ). During development, mbk-1::gfp expression can first be observed around the 300-min stage, when cell division ceases and morphogenesis begins (3A). Expression levels increase during later embryonic stages and remain at comparable levels throughout larval and adult stages (3).8, 百拇医药
fig.ommitted8, 百拇医药
Figure 3. Expression of the DYRK1A/minibrain ortholog mbk-1 gene as assayed by reporter gene analysis. MBK-1::GFP is expressed in all somatic tissues and primarily localizes to nuclei in embryos (A), larvae (B), and adults (C). Nuclear localization can be observed in all cells and is particularly obvious in the enlarged nuclei of hypodermal cells (D, MBK-1::GFP; E, DAPI staining; the dark center is the nucleolus). Expression is monitored from a chromosomally integrated array, cmIs6. (A) Embryos at different stages; the embryo at the top is roughly at 300 min of development entering morphogenesis and showing the first signs of mbk-1::gfp expression; the embryo on the lower left is at the comma stage with slightly increased mbk-1::gfp expression. Maximal levels of mbk-1::gfp expression are then observed around threefold stages (embryos on left).8, 百拇医药
In contrast to mbk-1, mbk-2::gfp reporter constructs were expressed in subsets of tissues, including the nervous system, body wall muscle, and the pharynx (4, A–C). To determine the subcellular localization of MBK-2 protein, we fused a cDNA—encoding the longer splice form of mbk-2 (see 1 and MATERIALS AND METHODS)—to gfp and expressed it under control of a ubiquitously expressed promoter (1). Unlike MBK-1::GFP, but like its vertebrate ortholog DYRK2 (BECKER et al. 1998(William B. Raich Celine Moorman Clay O. Lacefield Jonah Lehrer Dusan Bartsch Ronald H. A. Plasterk E)
c Hubrecht Laboratory, 3584 CT Utrecht, The Netherlandsf$sh, 百拇医药
ABSTRACTf$sh, 百拇医药
The pathology of trisomy 21/Down syndrome includes cognitive and memory deficits. Increased expression of the dual-specificity protein kinase DYRK1A kinase (DYRK1A) appears to play a significant role in the neuropathology of Down syndrome. To shed light on the cellular role of DYRK1A and related genes we identified three DYRK/minibrain-like genes in the genome sequence of Caenorhabditis elegans, termed mbk-1, mbk-2, and hpk-1. We found these genes to be widely expressed and to localize to distinct subcellular compartments. We isolated deletion alleles in all three genes and show that loss of mbk-1, the gene most closely related to DYRK1A, causes no obvious defects, while another gene, mbk-2, is essential for viability. The overexpression of DYRK1A in Down syndrome led us to examine the effects of overexpression of its C. elegans ortholog mbk-1. We found that animals containing additional copies of the mbk-1 gene display behavioral defects in chemotaxis toward volatile chemoattractants and that the extent of these defects correlates with mbk-1 gene dosage. Using tissue-specific and inducible promoters, we show that additional copies of mbk-1 can impair olfaction cell-autonomously in mature, fully differentiated neurons and that this impairment is reversible. Our results suggest that increased gene dosage of human DYRK1A in trisomy 21 may disrupt the function of fully differentiated neurons and that this disruption is reversible.
TRISOMY of chromosome 21, or Down syndrome, is the most frequent chromosomal abnormality in human infants that come to term. Besides manifesting a characteristic set of facial and physical features, heart defects, and abnormalities in the immune and endocrine systems, patients with Down syndrome have deficits in spatial memory and difficulty in converting short-term to long-term memories (JOHANNSEN et al. 1996 ; TAKASHIMA 1997 ). Although the cognitive defects of Down syndrome are likely to arise from increased dosage of many genes, several lines of evidence suggest that increased expression of the dual-specificity protein kinase DYRK1A plays a significant role. First, the DYRK1A locus maps to the Down syndrome candidate region (DSCR), a region of 70–100 genes (SHINDOH et al. 1996 ). Rare patients with a partial trisomy of the DSCR display defects in cognition, suggesting that the genes in this region are sufficient to produce the cognitive defects of Down syndrome. Second, a 180-kb region on human chromosome 21 containing the DYRK1A locus is sufficient to produce defects in learning and memory in transgenic mice (SMITH et al. 1997 ). Third, transgenic mice overexpressing the full-length Dyrk1A cDNA exhibit impairment in spatial learning and cognitive flexibility (ALTAFAJ et al. 2001 ). The cellular and molecular consequences of these DYRK1A perturbations are unknown.
The first vertebrate member of the DYRK family was originally identified in a PCR screen for protein kinases (KENTRUP et al. 1996 ) and subsequently shown to belong to a larger family of related dual-specificity kinases (BECKER and JOOST 1999 ) to which we refer here as the DYRK/minibrain family. Members of this family are localized to distinct subcellular compartments and phosphorylate various substrates in vitro (BECKER and JOOST 1999 ). They share the unusual property of tyrosine-directed autophosphorylation as well as phosphorylation of serine/threonine residues in exogenous substrates (BECKER and JOOST 1999 ). Their precise cellular role, however, remains elusive. Insights into their physiological relevance were provided by the finding that the Drosophila homolog of DYRK1A, termed minibrain, is involved in neuroblast proliferation in the fly brain (TEJEDOR et al. 1995 ). A similar reduction in brain size has been recently observed in DYRK1A knockout mice (FOTAKI et al. 2002 ). The sole Saccharomyces cerevisiae representative of the DYRK/minibrain family, Yak1p, acts in parallel to a protein kinase A pathway to negatively regulate cell-cycle progression (GARRETT and BROACH 1989 ; GARRETT et al. 1991 ).
In metazoan animals, the only DYRK/minibrain family member for which mutant alleles exist is the Drosophila minibrain gene. Here, we describe the expression pattern and loss-of-function alleles of all three DYRK/minibrain family members in the nematode Caenorhabditis elegans. In contrast to Drosophila minibrain, we observe no obvious morphological defects in mbk-1 mutants, but find that another DYRK/minibrain family member, mbk-2, is required for viability. In an attempt to model the cellular consequences of DYRK1A overexpression that are observed in Down syndrome patients, we analyzed the effects of providing extra copies of the worm ortholog of DYRK1A and describe dosage-sensitive and reversible defects in the processing of acute sensory information.9, 百拇医药
MATERIALS AND METHODS9, 百拇医药
cDNAs:9, 百拇医药
A full-length mbk-1 cDNA was amplified with SL1 and gene-specific primers. The structure of the cDNA is similar to the predicted T04C10.1 gene, with the exception of an incorrect first exon predicted in T04C10.1. The mbk-2 locus generates two messages, a long form (mbk-2L) and a short form (mbk-2S) that uses an internal start site from an alternatively spliced exon. Both splice forms are represented in expressed sequence tag (EST) clones (a gift from Y. Kohara) that have been completely sequenced. Similarly, hpk-1 full-length cDNA clones are represented in Y. Kohara's EST collection.
DNA constructs:g@y, 百拇医药
Constructs are shown schematically in 1.g@y, 百拇医药
fig.ommittedg@y, 百拇医药
Figure 1. Reporter gene constructs and deletion alleles used to study minibrain-like genes. Boxes denote exons of the respective genes. Dark shaded bars above the genomic locus denote the location of the deletion in the respective mutant alleles (see MATERIALS AND METHODS for precise location). Kinase domains are lightly shaded. The kinase domain was predicted using SMART. The large size of the mbk-2 genomic locus prevented us from constructing a full-length, translational gfp fusion construct. To monitor subcellular localization of mbk-2 we thus fused the cDNA of the mbk-2L splice form to a heterologous promoter, phsp16-2 (FIRE et al. 1990 ). The two alternatively spliced mbk-2 isoforms produced by the mbk-2 locus were deduced by cDNA analysis (see MATERIALS AND METHODS).g@y, 百拇医药
mbk-1: To build mbk-1::gfp, the mbk-1 genomic locus, including 7 kb of 5' noncoding sequence and all exons and introns, was amplified by Expand long-template PCR (Boehringer Mannheim, Indianapolis). The PCR product was cloned in frame with gfp in the promoterless vector pPD95.75 (from A. Fire), generating pBR104. To build mbk-1(YA)::gfp, the QuickChange kit (Stratagene, La Jolla, CA) was used to modify pBR104 with primers YAs (5'-ctggacaccgaatcgcccaggccattcagtcgagattctatcg) and YAas (5'-cgatagaatctcgactgaatggcctgggcgattcggtgtccag), generating pBR113. To build mbk-1(pk1389)::gfp, the mbk-1 locus was amplified from homozygous mbk-1(pk1389) animals by Expand long-template PCR (Boehringer Mannheim). Following the cloning and sequencing of this PCR product, a 4.0-kb BglII-Asp718 fragment was subcloned into pBR104, generating pBR177. To build pgcy-10::mbk-1, the gcy-10 promoter (YU et al. 1997 ) was amplified from N2 genomic DNA by Expand long-template PCR (Boehringer Mannheim) and cloned in front of the full-length mbk-1 cDNA, generating pBR145. To build pgcy-10::mbk-1(YA), the QuickChange kit (Stratagene) was used to modify pBR145 with primers YAs and YAas, generating pBR147. To build phsp16-2::mbk-1::gfp, the full-length mbk-1 cDNA was cloned into the heat-shock vector pPD49.78 (from A. Fire), generating pBR144. To generate a fusion with gfp, the promoter and cDNA were subsequently fused in frame with pBR104, generating pBR144.
mbk-2: To build pmbk-2L::gfp, 8 kb of 5' noncoding sequence was amplified by Expand long-template PCR (Boehringer Mannheim) and cloned into pPD95.77 (from A. Fire), generating pBR126. To build mbk-2S::gfp, Expand long-template PCR (Boehringer Mannheim) was used to amplify exons 7–11 of the mbk-2 locus and fused in frame with gfp in pPD95.77 (from A. Fire), generating pBR138. This construct uses intron 6 as the 5' noncoding sequence. pmbk-2L::gfp and mbk-2S do not contain the full mbk-2 genomic locus, and this may result in an artifactual or incomplete expression pattern. To build phsp16-2::mbk-2L::gfp, full-length mbk-2L cDNA was fused with gfp and inserted into the heat-shock vector pPD49.79 (from A. Fire), generating pBR169.0'9du.{, http://www.100md.com
hpk-1: To build a translational fusion between HPK-1 and green fluorescent protein (GFP), the full-length hpk-1 genomic locus, including 4 kb of 5' noncoding sequence and all exons and introns, was amplified by Expand long-template PCR (Boehringer Mannheim) and cloned into the promoterless vector pPD95.77 (from A. Fire), generating pBR132.
Transgenic and mutant strains:-4, 百拇医药
The strains are as follows:-4, 百拇医药
EK224 cmIs6 [pBR104, pNC4.21] I; 6x outcrossed-4, 百拇医药
EK270 cmIs8 [pBR113, pNC4.21]; 2x outcrossed-4, 百拇医药
EK176 cmEx20 [pBR145, unc-122::gfp]-4, 百拇医药
EK179 cmEx21 [pBR147, unc-122::gfp]-4, 百拇医药
EK173 unc-4(e120) II; cmEx19 [pBR144, pNC4.21]-4, 百拇医药
EK234 unc-4(e120) II; cmEx31 [pBR169, pNC4.21]-4, 百拇医药
EK123 cmEx6 [pBR126, pRF4]-4, 百拇医药
EK251 cmEx16 [pBR138, pRF4]-4, 百拇医药
EK135 cmEx11 [pBR132, pRF4]-4, 百拇医药
EK228 mbk-1(pk1389) X; 6x outcrossed-4, 百拇医药
EK264 cmEx36 [pBR177, pRF4]-4, 百拇医药
EK273 hpk-1(pk1393) X; 6x outcrossed-4, 百拇医药
EK275 mbk-2(pk1427)/mgIs18 IV; 3x outcrossed-4, 百拇医药
All expression constructs were injected at 50 ng/µl. pNC4.21 [unc-4(+)] and unc-122::gfp were injected at 50 ng/µl. pRF4 [rol-6(d)] was injected at 100 ng/µl. Integrated lines were obtained using a protocol described at .
Isolation of deletions in the mbk-1, mbk-2, and hpk-1 loci:5?, 百拇医药
PCR screening of a chemically induced deletion library was done as previously described (JANSEN et al. 1997 ). The relative position within the genomic loci of all three mutant alleles is schematically indicated in 1. A 1658-bp deletion mutant of mbk-1, pk1389, was isolated using primers MBK1A (5'-gcagacgtgcctgacaatcttc) and MBK1D (5'-tgtaggtatggcggtatccgtc) and nested primers MBK1B (5'-ctcaaatacccagcaacactcac) and MBK1C (5'-caatagtagatcccatcctcag). The deleted PCR product from pk1389 was sequenced and the deletion was confirmed by Southern analysis. The following sequence in capitals is deleted by pk1389: 5'-ataaaGCTTT-TTACGactat. The deletion extends from the first intron into the sixth exon. If in this context exon 1 is spliced to exon 7 (the beginning of the kinase domain), a frameshift and a premature stop would result; splicing of exon 1 to more downstream exons would also create frameshifts and/or delete the kinase domain. The mbk-2 deletion, pk1427, spans 3398 bp in the genomic region represented in cosmid F49E11 and deletes the capitalized sequence 5'-tcgtcGTCCG-AAAACttgta. The hpk-1 deletion, pk1393, spans 1457 bp in the genomic region represented in cosmid F20B6 and deletes the capitalized sequence 5'-cacacATGCC-TGACAtaatg. All deletion alleles lead to a disruption of the majority of the respective kinase domains and are predicted to act as null alleles.
RNAi:$(rjr, http://www.100md.com
mbk-2 dsRNA was delivered by bacterial feeding as previously described (TIMMONS et al. 2001 ). The effectiveness of RNAi could be assessed by monitoring the decrease in fluorescence of transgenic animals that expressed gfp-tagged mbk-2.$(rjr, http://www.100md.com
Behavioral assays:$(rjr, http://www.100md.com
Chemotaxis assays toward volatile odorants were performed as described (BARGMANN et al. 1993 ). Chemotaxis index = (the number of animals attracted to the odorant) - (the number of animals at the opposite end of the plate)/(the total number of animals at both ends of the plate). When transgenic animals carrying extrachromsomal arrays were scored, only those animals that carry the injection marker were scored.$(rjr, http://www.100md.com
Scoring neuroanatomy:$(rjr, http://www.100md.com
AWC morphology was observed by crossing kyIs140, a chromosomally integrated str-2::gfp construct (TROEMEL et al. 1999 ), into the respective mutant background. The generation, proliferation, and anatomy of several sensory neuron classes (ASK, ADL, ASI, AWB, ASH, ASJ, PHA, and PHB) were visualized by filling exposed sensory neurons with the lipophilic dye DiI, as previously described (HEDGECOCK et al. 1985 ). Briefly, a mixed population of well-fed worms was washed with M9 buffer, incubated at room temperature with 10 µg/ml DiI for 1 hr, washed several times with M9, and then mounted on a compound fluorescence microscope.
DAPI staining:z$, 百拇医药
4',6-Diamidino-2-phenylindole (DAPI) staining on gfp-expressing transgenic lines was done by placing animals in a drop of water on a coverslip, letting the water evaporate, adding a drop of acetone, letting the drop evaporate, drying for 20 min, and then adding 1 µg/ml DAPI in M9 medium.z$, 百拇医药
RESULTSz$, 百拇医药
Identification of DYRK/minibrain-like genes in C. elegans:z$, 百拇医药
Through sequence homology searching of the complete C. elegans genome sequence, we identified two C. elegans genes with close homology to the DYRK/mini-brain family, termed mbk-1 and mbk-2 (for minibrain- kinase), and one gene, hpk-1 (named after its vertebrate homologs, homeodomain-interacting protein kinase 1-3), with a more distant homology (2A). mbk-1 and mbk-2 both carry the characteristic DYRK family signature motifs in their kinase domain, as well as a DH-box, a conserved sequence motif preceding the catalytic domain (BECKER and JOOST 1999 ). mbk-1 is unique among the C. elegans DYRK/minibrain family members in clustering in the DYRK1A subgroup (2A). In addition to highest overall sequence similarity, MBK-1 and DYRK1A can also be distinguished from other DYRK family members by their sharing of an N-terminal nuclear localization sequence, a leucine zipper in the kinase domain, and stretches of homopolymeric amino acids at their C termini (2B). We thus consider MBK-1 the C. elegans ortholog of human DYRK1A.
fig.ommittedk, http://www.100md.com
Figure 2. The DYRK/minibrain-like gene family in C. elegans. (A) Classification of the DYRK subfamily of protein kinases. On the basis of BLAST- and PFAM-based similarity searches of the complete genome sequence, C. elegans has two members of the DYRK subfamily of protein kinases, MBK-1 (T04C10.1; LGX; GenBank accession no. AY064464) and MBK-2 (F49E11.1; LGIV; GenBank accession no. AY090019), as well as a related kinase called HPK-1 (F20B6.8; LGX). The gene structures of mbk-1 and mbk-2 were confirmed by sequence analysis of EST clones. The alignment and dendrogram were generated from the predicted kinase domains using ClustalX and Align with default parameters. The DYRK1A group is boxed by dark shading, the DYRK subfamily is boxed by light shading, C. elegans proteins are in boldface type and underlined, and the percentage identity between each kinase domain and that of DYRK1A is listed to the right. (B) MBK-1 is the closest homolog to DYRK1A, whose overexpression is implicated in Down syndrome. The relative positions of the N-terminal nuclear localization sequence [NLS; amino acids (aa) 120–127], the DH box, the kinase domain (aa 317–649), leucine zipper (L-L; aa 443–464; contains four leucines each spaced by 6 aa), and C-terminal homo-polymeric stretch of amino acids (histidines in DYRK1A, glutamines in MBK-1) sites are shown. DYRK kinases are regulated through the phosphorylation of two DYRK-specific tyrosines within the activation loop (Y-Y; aa 488 and 490). For a more detailed alignment and more sequence features of DYRK kinases, see BECKER and JOOST 1999 .
Expression and subcellular localization of DYRK/minibrain-like genes in C. elegans:8, 百拇医药
To determine the sites of expression and subcellular localization of DYRK/minibrain-like genes, we fused gfp in frame with the respective genomic loci (1). The mbk-1::gfp construct included 7 kb of 5' noncoding sequence and all of the exons and introns present in the endogenous mbk-1 locus. The mbk-1::gfp gene product is expressed in all somatic cells and primarily localizes to nuclei (3), similar to the reported expression and subcellular localization of human DYRK1A (SONG et al. 1997 ; BECKER et al. 1998 ). During development, mbk-1::gfp expression can first be observed around the 300-min stage, when cell division ceases and morphogenesis begins (3A). Expression levels increase during later embryonic stages and remain at comparable levels throughout larval and adult stages (3).8, 百拇医药
fig.ommitted8, 百拇医药
Figure 3. Expression of the DYRK1A/minibrain ortholog mbk-1 gene as assayed by reporter gene analysis. MBK-1::GFP is expressed in all somatic tissues and primarily localizes to nuclei in embryos (A), larvae (B), and adults (C). Nuclear localization can be observed in all cells and is particularly obvious in the enlarged nuclei of hypodermal cells (D, MBK-1::GFP; E, DAPI staining; the dark center is the nucleolus). Expression is monitored from a chromosomally integrated array, cmIs6. (A) Embryos at different stages; the embryo at the top is roughly at 300 min of development entering morphogenesis and showing the first signs of mbk-1::gfp expression; the embryo on the lower left is at the comma stage with slightly increased mbk-1::gfp expression. Maximal levels of mbk-1::gfp expression are then observed around threefold stages (embryos on left).8, 百拇医药
In contrast to mbk-1, mbk-2::gfp reporter constructs were expressed in subsets of tissues, including the nervous system, body wall muscle, and the pharynx (4, A–C). To determine the subcellular localization of MBK-2 protein, we fused a cDNA—encoding the longer splice form of mbk-2 (see 1 and MATERIALS AND METHODS)—to gfp and expressed it under control of a ubiquitously expressed promoter (1). Unlike MBK-1::GFP, but like its vertebrate ortholog DYRK2 (BECKER et al. 1998(William B. Raich Celine Moorman Clay O. Lacefield Jonah Lehrer Dusan Bartsch Ronald H. A. Plasterk E)