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Gene Structure and Glial Expression of the Glycine Transporter Glytl in Embryonic and Adult Rodents

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Gene Structure and Glial Expression of the Glycine Transporter Glytl in Embryonic and Adult Rodents The Journal of Neuroscience, March 1995, 1.5(3): 2524-2532 Gene Structure and Glial Expression of the Glycine Transporter GlyTl in Embryonic and Adult Rodents Ralf H. Adams,’ Kohji Sato,ls2 Shoichi Shimada, Masaya Tohyama,3 Andreas W. Piischel,’ and Heinrich Betzl ‘Abteilung Neurochemie, Max-Planck-lnstitut fijr Hirnforschung, D-60528 Frankfurt/Main, Germany and 2Department of Neuroanatomy, Biomedical Research Center, and 3Department of Anatomy and Neuroscience, Osaka University Medical School, Osaka, Japan Na+/CI--dependent glycine transporters are crucial for the and on surrounding glial cells (for a recent review, see Schloss termination of neurotransmission at glycinergic synapses. et al., 1994) and are crucial for the rapid removal of neurotrans- Two different glycine transporter genes, GlyTl and GlyT2, mitters from the synaptic cleft. This reuptake terminatessynaptic have been described. Several isoforms differing in their 5’ transmissionand helps to replenishtransmitter pools in the pre- ends originate from the GlyTl gene. We have determined synaptic nerve terminal. the genomic structure of the murine Glyfl gene to eluci- Cloning of the transportersfor GABA (Guastellaet al., 1990) date the genetic basis underlying the different isoforms. and norepinephrine(Pacholczyck et al., 1991) allowed the sub- Analysis of cDNA 5’-ends revealed that the GlyTla and 1 b/ sequentisolation of a number of cDNAs encoding homologous lc mRNAs are transcribed from two different promoters. Na+/Cll-dependent transporters,including those for dopamine During murine embryonic development GlyTl mRNAs were (Giros et al., 1991; Kilty et al., 1991; Shimada et al., 1991; detectable by RNase protection assays as early as embry- Usdin et al., 1991), 5-HT (Blakely et al., 1991; Hoffman et al., onic day E9 and reached maximal levels between El3 and 1991), and glycine (Guastella et al., 1992; Liu et al., 1992b; E15. In situ hybridization revealed GlyTl expression in the Smith et al., 1992). All these transporters share a postulated developing spinal cord mainly in the ventral part of the ven- structure of 12 transmembranedomains deduced from hydrop- tricular zone at E12. At later stages (E15) transcripts were athy plots (Guastella et al., 1992; Smith et al., 1992). Several also found in the lateral half of the basal and intermediate neurotransmitteruptake systemsdisplay an unexpected molec- gray matter. In contrast, the second glycine transporter ular heterogeneity as shown by the cloning of four different gene GlyTP displayed a completely different expression GABA (Lopez-Corcuera et al., 1992; Liu et al., 1993a) and gly- pattern. At El1 it is expressed in the mantle zone, and at tine (Borowsky et al., 1993; Liu et al, 1993b; Kim et al., 1994) later stages throughout the ventral horns. In the adult rat transporter cDNAs. Three of the latter (GlyTla, lb, and lc) are brain and spinal cord, GlyTl hybridization signals were encodedby a single gene and differ only in their amino-terminal found exclusively in glial cells. Our data indicate that GlyTl region (Kim et al., 1994). The fourth transporter GlyT2 is gen- is an early marker of neural development and encodes glia- erated from a separategene with 48% amino acid sequenceiden- specific transporter proteins. tity to GlyTl and is pharmacological distinguishablefrom the [Key words: g/ycine transporter, gene structure, alternate different GlyTl isoforms (Liu et al., 1993b). promoters, g/y&e receptor, in situ hybridization, embry- The expressionof GlyTla and GlyTlb in adult rats and mice onic development, spinal cord] has been studied by in situ hybridization (Guastellaet al., 1992; Liu et al., 1992b; Smith et al., 1992; Borowsky et al., 1993). Tremendousprogress has been made in understandingthe mo- Both variants are expressedin many regions of the CNS includ- lecular mechanismsof synaptic transmission,and an increasing ing hypothalamus, mesencephalon,brainstem, and spinal cord. number of both pre- and postsynaptic proteins has been studied GlyTl a mRNA is presentin regions with a high density of neu- at the molecular level. With the recent cloning of several mem- ronal cell bodies and was proposedto colocalize with inhibitory bers of the Na+-/Cl--dependent neurotransmitter transporter glycine receptor subunit mRNA (Borowsky et al., 1993). In ad- family another important synaptic component, neurotransmitter dition GlyTla is expressedin several peripheral tissues,for ex- uptake, has become amenableto functional studies.Neurotrans- ample, liver, lung, and stomach.In contrast, in brain the GlyTlb mitter transportersare located both in the presynaptic membrane mRNA was suggestedto colocalize with the NMDA subtype of glutamate receptors (Smith et al., 1992). Glycine transporters Received July 13, 1994; revised Sept. 23, 1994; accepted Sept. 27, 1994. might modulate the concentration of glycine at its coagonist We thank Birgit Pilz for expert technical assistence and M. Ehms-Sommer binding site on the NMDA receptor (Johnsonet al., 1987; Ku- for help with photography. Part of this work was supported by the Deutsche ryatow et al., 1994) by uptake into neighboring glial cells or by Forschungsgemeinschaft (Leihnia-Programm and SFB 269), by the Ministry of Education, Science and Culture of Japan, and by a postdoctoral fellowship of reverse transporter-mediatedrelease (Attwell et al., 1992, 1993), the Alexander-vowHumboldt Foundation to K.S. and thereby affect the efficacy of glutaminergic synapses.The Correspondence should be addressed to H. Betz, Abteilung Neurochemie, GlyT2 mRNA was found to be restricted to the central nervous Max-Plan&Institut ftir Hirnforschung, Deutschordenstrasse 46, D-60528 Frankfurt/Main, Germany. system and to be mainly localized in spinal cord and brainstem Copyright 0 1495 Society for Neuroscience 0270-6474/95/152524-09$05.00/O (Liu et al., 1993b), which correlates well with the distribution The Journal of Neuroscience, March 1995, E(3) 2525 Table 1. Origin of GlyTl-specific probes used in this article Probe Reference Genomic screening 460 bp PCR-fragment derived from genomic DNA Present results with primers from exon 3 (sense) and 4 (antisense), includes 0.4 kb intronic sequence cDNA screening GlyT740: PCR-fragment including nucleotides Guastella et al., 1992 409-I 148 of the rat GlyTl cDNA RNase protection assay antisense RNA derived from a 450 bp BglII-BsaAI Liu et al., 1992b fragment corresponding to nucleotides 2 lo-660 of the mouse GlyTla cDNA Northern blot 0.9 kb BglII fragment of the Glytla cDNA Liu et al., 1992b nucleotides 210-l 121 In situ hybridization of GlyTla/b: 1.9 kb NcoI-BsaBI fragment of the Liu et al., 1992b mouse embryo mouse GlyTl a cDNA, nucleotides 239-2092 sections GlyTla: 209 bp EcoRI-BglII fragment of the Liu et al., 1992b mouse GlyTla cDNA, nucleotides l-210 GlyTIb: 253 bp PCR fragment derived from Present results: mouse genomic DNA including coding sequence Fig. 2 of exon 1b and 208 bp upstream from the start codon In situ hybridization of Antisense oligonucleotides complementary to bases Smith et al., 1992 rat brain sections 536580 or 1813-1857 of the rat GlyTlb cDNA Application and designations of the probes (see Materials and Methods) are listed as well as the sequences to which they correspond. of inhibitory, strychnine-sensitive glycine receptors (Betz, quenced after subcloning into Bluescript using a T7 DNA sequencing 1992). kit (Pharmacia, Freiburg, Germany) and synthetic oligonucleotide prim- ers. Computer analysis was done using the HUSAR 3.0 software To further understand the roles of the different GlyTl iso- (Deutsches Krebsforschungszentrum, Heidelberg, Germany). forms in glycinergic and glutaminergic neurotransmission we Polymerase chain reaction and recombinant DNA. Probes for cDNA analyzed both the genomic structure and the developmentaland screening and in situ hybridization were generated by amplifying DNA regional expressionof the murine GlyTl gene. Our data show fragments from rat brain cDNA (GLYTl: Glyt740 probe, sense 5’- that two isoforms, GlyTla and lb, originate from transcription GGTATGATGGTGGTGTCCACGTAC-3’, antisense 5’-GACACA- TCCACACCCAGGTGATTG-3’) by 30 cycles of denaturation (45 set, initiated at alternate promoters,whereas GlyTlc is a splice vari- 94”C), annealing (45 set, 65°C) and extension (1 min, 72”(Z), 50 ng of ant of the 1b transcript. In situ hybridization revealed that in the mouse genomic DNA (300 hp GlyRal: sense 5’-GGAGAATTCTT- CNS GlyTl transcripts are expressedin glial cells. CAGGATGATGAGGGTGG-3’, antisense 5 ‘-GGGGATCCAGCClT- CACTTGTTGTGGAC-3’: 300 bo GlvRa2: sense: 5’-CCCTTTGCA- Materials and Methods TGGTGATGcicG-3', antisense:‘5 r-GITCACTAGCTGCCGGTACA- 3’; 340 bp GlyRB: sense 5’-GCGGATCCCATAATTGCTGATCTGTG- Isolation of the GLYTI cDNA. We screened 1.6 X lo6 pfu of a mouse 3’, antisense: 5’-GGGATTCGATACTGCATACATGGAC-3’) by 30 cy- brain cDNA library (Stratagene, Heidelberg, Germany) with a 740 bp cles of denaturation (1 min, 94”C), annealing (I 5 min, 60°C) and PCR fragment (Glyt740 probe; see Table l), and a single hybridization- positive clone was isolated. Partial sequencing showed that the cDNA extension (1.5 min, 72”C), or mouse brain cDNA (300 bp GlyT2: sense sequence was identical to that published by Liu et al. (1992b). 5’-GGGGATCCCACAGGGTGGAATCTACATG-7, antisense 5 ‘- Full-length cDNA 5’.ends were isolated by using the 5’-RACE Ready GGGAATTCGCATCAGCCATCCAAGCACC-3’) by 30 cycles of de- cDNA kit (Clontech, Palo Alto, CA). Two subsequent PCR amplifica- naturation (1 min, 94”C), annealing (1.5 min, 55°C) and
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