In order to clone the mouse homologue of MCOLN1, the human amino acid sequence was compared to the high throughput genomic sequence (HTGS) database using TBLASTN, which identified the mouse BAC clone RPCI-23_387H4 (GB No. AC079544.1). Correspondence with the Joint Genome Institute and the Lawrence Livermore National Laboratory (LLNL) Human Genome Center confirmed the location of this BAC to mouse chromosome 8 and allowed us to construct a physical map of this region 14 (Fig. 1A). The BAC sequence was then compared to the mouse EST database using BLASTN, and multiple ESTs and their corresponding I.M.A.G.E. clones were identified.

Three clones (ID Nos. 604971, 1228665, 1247566) from the UniGene cluster Mm. 8356 were obtained, sequenced, and assembled into a 2 kb transcript with an open reading frame of 580 amino acids designated Mcoln1 (GB No. AF302010). Comparison of the cDNA sequence to the BAC clone showed that the gene consists of 14 exons. We then created a transcript map of the Mcoln1 region of mouse chromosome 8 (Fig. 1B), noting the presence of the UniGene Cluster Mm. 39099, a homologue of the human zinc finger gene (GB No. AC001252) that terminates approximately 1.8 kb before the start of Mcoln1; and Nte, the mouse homologue of the human neuropathy target esterase gene (NTE) beginning 130 base pairs after the polyadenylation signal for Mcoln1. The transcript map of the region surrounding Mcoln1 is similar to the corresponding region of the homologous human MCOLN1 region 15, and the presence of the zinc finger gene and Nte confirms and extends the region of synteny between human chromosome 19 and mouse chromosome 8.

Comparison of the mouse and human peptide sequences showed 91% identity (Fig. 2). The C. elegans homologue cup-5 shows 34% identity with Mcoln1 and BLASTP analysis of Mcoln1 identified a putative Drosophila melanogaster homologue that shows 38% identity (Fig. 2). Interestingly, two MCOLN1 amino acid substitutions that result in MLIV occur at conserved amino acids. TMPred analysis http://www.ch.embnet.org/software/TMPRED_form.html predicts a protein structure that is nearly identical to MCOLN1, containing 6 transmembrane domains with the N- and C-termini residing in the cytoplasm (Fig. 2) 9.

Mouse adult multiple tissue and embryonic Northern blots were hybridized using a probe generated from mouse exon 2 (probe 1, Fig. 1B), yielding a band of approximately 2.4 kb (isoform 1), as expected, and a less abundant and unexpected 4.4 kb band (isoform 2) (Figs. 3A &3B). The 2.4 kb band shows ubiquitous but variable expression, with the highest expression in brain, liver and kidney. The fetal tissue blot shows decreasing levels of the 2.4 kb message with increasing gestational age. Since the human MCOLN1 gene encodes a single transcript 789, we carried out additional hybridizations with probes generated from various regions of the coding sequence and 3' UTR, and all probes identified the same two transcripts in the mouse (data not shown). In order to verify the presence of a single mouse locus, we hybridized a mouse Southern blot with the exon 2 probe. Four different restriction enzymes were used, and only the expected size bands for the chromosome 8 locus were detected (data not shown).

In order to determine the coding sequence for the larger transcript, we searched the mouse EST database using each intron as well as the genomic sequence flanking the Mcoln1 gene. Two ESTs were identified that contained sequence from intron 12 (GB No. AI430291 and AA874645), and the corresponding clones were sequenced. Clone 408619 (ESTs: GB No. AI430291, AI429558) begins approximately 1.1 kb before exon 13 and continues through the exon and splices correctly to exon 14. Clone 1281641 (EST:GB No. AA874645) begins 175 bp before exon 13 and also splices correctly to exon 14. A mouse multiple tissue Northern was hybridized using a probe generated from the putative intron sequence in clone 408619 (Probe 2, Fig. 1B), which detected only the 4.4 kb band (Fig. 3C).

In order to determine the sequence of the entire transcript, RT-PCR using primers in exons 10 and 11 paired with a primer in intron 12 was performed using BALB/c mouse brain total RNA and the resulting products sequenced. These products show that the larger transcript is due to an alternative splice event that results in an expanded exon 13. Specifically, exon 12 splices at bp 436 of intron 12, creating a large 1614 bp exon 13 that splices correctly to exon 14. The open reading frame of this alternatively spliced transcript is 611 amino acids, 28 amino acids longer than the message encoded by the 2.4 kb transcript.

TMPred analysis predicts that isoform 2 encodes a protein identical in structure to Mcoln1, possessing 6 transmembrane domains and a channel pore, however the protein sequences diverge at amino acid 526. The 55 amino acid C-terminal cytoplasmic tail encoded by the 2.4 kb transcript is completely different from the 86 amino acid tail encoded by the murine specific 4.4 kb transcript (Fig. 4). Clontech Mouse RNA Master Blots were hybridized with the exon 2 and intron 12 probes mentioned above in an attempt to determine if these two transcripts showed differences in expression patterns, however, there was no significant difference in the 22 tissues represented (data not shown).

Next, we directly compared the nucleotide and amino acid sequence of the alternatively spliced mouse transcript to the entire human MCOLN1 genomic sequence and found no significant similarity. As mentioned previously, Northern blots performed with human MCOLN1 probes show only one 2.4 kb transcript. In addition, we hybridized a human multiple tissue Northern and human Southern with a probe in human intron 12 that is adjacent to exon 13. The probe was located in the region syntenic to that which encodes the alternate mouse transcript. Only the expected bands were detected on the Southern and no bands were detected on the Northern, confirming that this alternative transcript is specific to murine Mcoln1. Recent BLASTP analysis of the alternate Mcoln1 transcript yields a match to a putative 145 amino acid anonymous protein (GB No. BAB25862) predicted from a RIKEN clone. It is obvious from our results, however, that the identification of this sequence as a full-length protein is incorrect since probes unique to the clone, as well as probes containing the Mcoln1 coding sequence, identify the same transcripts.

We conducted database searches using BLAST http://www.ncbi.nlm.nih.gov/blast and Draft Human Genome Browser http://genome.ucsc.edu/. Sequences from UniGene http://www.ncbi.nlm.nih.gov/UniGene were used to confirm the Mcoln1 sequence. We performed motif searches using ProfileScan http://www.isrec.isb-sib.ch/software/PFSCAN_form.html and TMPred http://www.ch.embnet.org/software/TMPRED_form.html and alignment of protein sequences using Pileup (GCG) and Boxshade http://www.ch.embnet.org/softward/BOX_form.html.

I.M.A.G.E. Clones were purchased from Research Genetics (Huntsville, AL). Sequencing was performed using the AmpliCycle sequencing kit from Applied Biosystems (Foster City, CA) on a Genomyx LR programmable DNA sequencer.

BALB/c mouse brain total RNA was purchased from Clontech Laboratories (Palo Alto, CA) and made to a 1 μg / ml concentration. This RNA was used as a template to create cDNA via RT-PCR using random hexamers and oligo dT primer. The RT product was used to confirm the alternatively spliced form using primers: 5'-CATCTACCTGGGCTATTGC-3' (forward) and 5'-GCTCTCAGGTGGTGGACAC-3' (reverse) in a PCR reaction with an annealing temperature of 61°C.

Total mouse genomic DNA was digested using EcoRI, BamHI, PstI, and XbaI. The digests were electrophoresed on a 1% agarose gel at 60V overnight and were transferred onto a Hybond N+ membrane from Amersham Pharmacia Biotech (Piscataway, NJ). A ³²P-dATP labeled PCR fragment of the Mcoln1 coding region corresponding to exon 2 was used as a probe (primers 5'-CCCCACAGAAGAGGAAGAC-3' (forward) and 5'-AGATCTTGACCACCTGCAG-3' (reverse) with an annealing temperature of 59°C). Hybridization and washes were carried out in standard conditions 16.

Mouse embryo multiple-tissue northern blot and mouse adult multiple-tissue northern blot filters were purchased from Clontech Laboratories (Palo Alto, CA). The filters were hybridized with the ³²P-dATP labeled DNA fragment of Mcoln1 coding region corresponding to exon 2 (see above). For the alternative transcript, a probe was generated in the region between exons 12 and 13 with primers 5'-GTGTCCACCACCTGAGAG-3' (forward) and 5'-GAAGTAGCATTCCTGCAGGC-3' (reverse) with an annealing temperature of 62°C. The filters were then hybridized with β-actin probes. Hybridizations and washes were carried out in standard conditions, with the stripping of previously bound probes in between 16.