gbb12046-sup-0001-figures1.docWord document3758KFigure S1: Hypothetical models of NRX-1 proteins of C. elegans. Three-dimensional structure models of the wild-type NRX-1 (a), NRX-1-ok1649 (b) and NRX-1-tm1961 (c) proteins are shown. Conformational changes in the proteins coded by both mutant alleles are observed. Models were powered by Swiss-Model Proteomic Serve (Arnold et al. 2006; Guex & Peitsch 1997; Schwede et al. 2003). (D) Structure of C. elegans nrx-1 locus on chromosome V and identification of the nrx-1 (ok1649) and nrx-1 (tm1961) deletion alleles. Exons of the transcript are shown as numbered shaded boxes connected by lines representing the introns. The inset shows a PCR amplification of N2 wild type, CRR3 (tm1961) and CRR1 (ok1649) genomic DNA using the following primers flanking the deletion: nrx-1(ok1649) forward 5′-CGGAAGCAAAGAAACCAAAG-3′, nrx-1(ok1649) reverse 5′-GTTGAGCATTTGCAATCGAA-3′, nrx-1(tm1961) forward 5′-ATCTGGCCGATCAAAGTTAC-3′ and nrx-1(tm1961) reverse 5′-TCTAACCTCCCGTTGAGCAT-3′. The expected sizes were as follows: 2459 bp (ok1649 allele), 3320 bp (wild type control); 1398 bp (tm1961 allele), 1826 bp (wild type control).
gbb12046-sup-0002-figures2.docWord document3758KFigure S2: Exonic organization and protein domains of C. elegans NRX-1β isoform. (a) nrx-1α transcript isoform (coding transcript C29A12.4b) and nrx-1β isoform. The exons are indicated with yellow boxes and exon numbers are labeled above or below each exon. (b) Domains organization of the C. elegans neurexin proteins. The α-neurexins contain a N-terminal signal peptide (SP) that is removed from the final form functional protein, followed by three repeats units consisting of two laminin-G domains (LNS) flanking an EGF domains, a transmembrane domain (TM) and a short cytoplasmatic domain (not shown). The putative worm NRX-1β protein is also shown.
gbb12046-sup-0003-figures3.docWord document3758KFigure S3: Caenorhabditis elegans nrx-1β isoform sequences. (a) nrx-1β isoform-coding sequence corresponding to isolated cDNA from Bristol N2 wild-type strain (see Materials and Methods section). (b) Putative NRX-1β full-length protein sequence translated.
gbb12046-sup-0004-figures4.docWord document3758KFigure S4: GFP or CFP fluorescence in transgenic strains CRR114 expressing human NRXN-1β cDNAs in neurexin deficient mutants of C. elegans. Expression of GFP in pharynx muscles (a), and expression of CFP in neurons of head ganglia (b) or embryo (f). The transgenic strain contains the extra-array {crrEx14 [pDD04 NeoR (pmyo-2::GFP); pPD95.75 (Pnrx-1::NRXN-1β::CFP)]}. The Bristol N2 wild-type strain was used as a control for unspecific fluorescence signals (d, h). Asterisks indicate autofluorescence signal. The inset in (b) shows a detail of extranuclear expression patterns in body cell of head neurons (arrows). The images (c), (e), (g) and (i) correspond to DIC. (j) Translational construction used for human NRXN-1β expression.
gbb12046-sup-0005-figures5.docWord document3758KFigure S5: Comparative amino acid sequences of C. elegans NRX-1β and human NRXN-1β isoforms, showing a full length protein sequence (a), extracellular (b) and intracellular domains (c) alignments. Accession numbers for nucleotide and protein sequences are as follow: Homo sapiens, NM_1387535.2 and NP_620072.1, consensus CDS: CCDS1845.1. The extra- and intracellular regions of C. elegans and human protein isoforms were predicted using TMHMM server v. 2.0 (Center for Biological Sequence Analysis, Technical University of Denmark, DTU). The nrx-1β-coding transcript sequence was translated and alignment of protein sequences was performed using the Clustal W method (BLOSUM62 Similarity Matrix). Identity (I) and similarity (S) percentages: I = 11.73/S = 23.25 with full-length proteins; I = 8.19/S = 16.93 among extracellular domains; I = 17.89/S = 32.63 among intracellular domains.

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