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Vol. 10, Issue 2, 165-173, February 2000
REPORT
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
RESULTS
DISCUSSION
METHODS
REFERENCES
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Nephropathic cystinosis is an autosomal recessive disorder caused by the defective transport of cystine out of lysosomes. Recently, the causative gene (CTNS) was identified and presumed to encode an integral membrane protein called cystinosin. Many of the disease-associated mutations in CTNS are deletions, including one >55 kb in size that represents the most common cystinosis allele encountered to date. In an effort to determine the precise genomic organization of CTNS and to gain sequence-based insight about the DNA within and flanking cystinosis-associated deletions, we mapped and sequenced the region of human chromosome 17p13 encompassing CTNS. Specifically, a bacterial artificial chromosome (BAC)-based physical map spanning CTNS was constructed by sequence-tagged site (STS)-content mapping. The resulting BAC contig provided the relative order of 43 STSs. Two overlapping BACs, which together contain all of the CTNS exons as well as extensive amounts of flanking DNA, were selected and subjected to shotgun sequencing. A total of 200,237 bp of contiguous, high-accuracy sequence was generated. Analysis of the resulting data revealed a number of interesting features about this genomic region, including the long-range organization of CTNS, insight about the breakpoints and intervening DNA associated with the common cystinosis-causing deletion, and structural information about five genes neighboring CTNS (human ortholog of rat vanilloid receptor subtype 1 gene, CARKL, TIP-1, P2X5, and HUMINAE). In particular, sequence analysis detected the presence of a novel gene (CARKL) residing within the most common cystinosis-causing deletion. This gene encodes a previously unknown protein that is predicted to function as a carbohydrate kinase. Interestingly, both CTNS and CARKL are absent in nearly half of all cystinosis patients (i.e., those homozygous for the common deletion).
[The sequence data described in this paper have been submitted to the GenBank data library under accession nos. AF168787 and AF163573.]
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
METHODS
REFERENCES
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Nephropathic cystinosis is a rare autosomal recessive, lysosomal
storage disease with an incidence estimated at 1 per
100,000-200,000 live births (see http://www.ncbi.nlm.nih.gov/omim;
OMIM 219800). The classic disorder is characterized clinically by renal
tubular Fanconi syndrome in the first year of life, growth retardation in childhood, renal glomerular failure at ~10 years of age,
hypothyroidism, and a variety of other complications, including
photophobia and corneal crystal formation (Gahl 1986
; Gahl et al.
1995). After renal transplantation, cystine accumulation continues in
nonrenal organs, frequently causing a distal vacuolar myopathy (Charnas et al. 1994), swallowing difficulty (Sonies et al. 1990), or retinal dysfunction (Kaiser-Kupfer et al. 1986), and occasionally causing diabetes mellitus (Fivush et al. 1987), pancreatic exocrine
insufficiency (Fivush et al. 1988), or neurological deterioration
(Ehrich et al. 1979; Fink et al. 1989). These complications arise
because defective lysosomal transport of the disulfide cystine (Gahl et al. 1982a) causes this amino acid to accumulate within the lysosomes of
many different cell types, which then triggers cystine crystal formation (Gahl et al. 1982b). The cystine transporter is the first of
many lysosomal membrane carriers to be characterized biochemically
(Thoene 1992), and cystinosis is the most common of a group of
lysosomal transport disorders (Gahl et al. 1995).
The gene altered in patients with cystinosis (CTNS) was
recently identified by a positional cloning strategy (Town et al. 1998). CTNS is a 12-exon gene that is transcribed into a
~2.6-kb mRNA. The encoded protein, named cystinosin, consists of a
predicted 367 amino acids, appears to be an integral membrane protein,
and most likely functions as a cystine transporter. A number of
cystinosis-causing CTNS mutations have now been reported
(Shotelersuk et al. 1998a; Town et al. 1998). The most prevalent
mutation reported to date is a large (>55-kb) deletion, with
33%-44% of affected patients being homozygous for this deletion
(Town et al. 1998; Anikster et al. 1999). In addition, at least 11 other smaller disease-causing deletions have been reported (Shotelersuk
et al. 1998a; Forestier et al. 1999), suggesting that this genomic
region may be prone to rearrangement.
We sought to establish the long-range organization of the segment of chromosome 17p13 harboring CTNS and to determine the sequence of this clinically important gene and its surrounding DNA. Here we report the assembly of a detailed bacterial artificial chromosome (BAC)-based physical map encompassing CTNS. In addition, two BAC clones spanning >200 kb were sequenced to high accuracy, providing insight into the molecular architecture of the CTNS gene and the genomic segment commonly deleted in cystinosis patients.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
METHODS
REFERENCES
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Physical Mapping
Our goal was to construct a high-resolution, long-range physical map
of the region of chromosome 17p13 containing CTNS.
Specifically, we sought to isolate the region in overlapping BAC clones
(Shizuya et al. 1992; Birren et al. 1999) and to order a large set of
sequence-tagged sites (STSs) across the interval. Although this genomic
segment has been isolated in yeast artificial chromosomes (YACs)
(McDowell et al. 1996; Stec et al. 1996; Peters et al. 1997), few
markers were available for BAC isolation and mapping. Consequently, we generated new STSs across the region using several sources of DNA
sequence, including known genes (e.g., ASPA) and genetic
markers (e.g., D17S2167, D17S2054, D17S1828), a YAC spanning the
interval [CEPH YAC 767F9 (McDowell et al. 1996; Peters et al. 1997)],
and BAC insert ends. Available human BAC libraries were screened by PCR- and hybridization-based methods for the available STSs. Following STS-content analysis, nascent contigs were assembled, and clones residing at contig ends were selected and used to derive additional BAC
insert-end sequences. New STSs were developed from the latter and used
to screen the BAC libraries again. This scheme was repeated in an
iterative fashion, eventually allowing assembly of the contig map
depicted in Figure 1.
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The resulting BAC-based STS-content map contains 95 clones and provides
ordering information for 43 STSs. The contig is estimated to span >1
Mb based on previous YAC-based mapping of the interval (McDowell et al.
1996; Peters et al. 1997). The average redundancy of BACs per STS is
~14; such redundancy provides strong support for the indicated BAC
overlaps and deduced STS order.
Genomic Sequencing
Two overlapping BACs (RG147P12 and RG87B10; see Fig. 1), which
together contain the entire CTNS gene, were sequenced to an estimated accuracy of >99.99% by a shotgun sequencing strategy (Wilson and Mardis 1997). The clone inserts were found to be 68,220 and
138,720 bp in size, respectively, and to overlap by 6703 bp. Thus, a
total of 200,237 bp of nonredundant sequence was generated (GenBank
accession no. AF168787). Comparison of the sequence with a collection
of known human repetitive elements revealed that this genomic region is
relatively rich in repeats (constituting 42.6% of the total sequence),
in particular short interspersed repetitive elements (SINEs).
Alu repeats comprise nearly 30% of the sequence (Table
1).
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Genomic Organization of the CTNS Gene
Comparison of the CTNS cDNA sequence and the generated
genomic sequence allows the precise structure of the gene to be
deduced, including details about intron/exon organization (Table 2;
Fig. 2). The published
CTNS cDNA sequence (GenBank accession no. AJ222967) is
distributed across 24,816 bp of genomic DNA [positions 72,070-96,885 (GenBank accession no. AF168787)]. This cDNA sequence matches our
established genomic sequence throughout, except for a silent A:G
substitution at nucleotide position 843 in exon 8 (of the cDNA
sequence), the presence of an additional T residue at position 2273 in
the 3'-untranslated region (UTR), and a G:A substitution at
position 2594 in the 3' UTR. Furthermore, based on the genomic sequence, intron 1 is 276 bp in length, shorter than that described previously (Town et al. 1998).
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Deletion Breakpoint Mapping
The breakpoints of the most common cystinosis-causing deletion were
identified and sequenced in numerous cystinosis patients and reported
previously (Anikster et al. 1999; Forestier et al. 1999). The
availability of sequence data for the region encompassing CTNS
allowed precise characterization of this deletion. Aligning the
breakpoint sequences to the normal genomic sequence reveals that the
common deletion spans 57,257 bp, notably smaller than the ~65-kb
estimate reported previously (Town et al. 1998). The 5' (telomeric)
deletion breakpoint occurs after nucleotide position 36,253 (GenBank
accession no. AF168787). The 3' (centromeric) deletion breakpoint
occurs before nucleotide position 93,511 and interrupts exon 10 of the
CTNS gene. Note that it cannot be determined whether the C
nucleotide at the deletion junction originated from the 5' or
3' end of the deletion; thus, the breakpoint position at either end
may be plus or minus one nucleotide. Whereas the regions immediately
surrounding the deletion breakpoints are rich in Alu
repetitive elements, the breaks themselves do not occur within these repeats.
Detection of a Novel Gene (CARKL) in the Common Cystinosis-Causing Deletion
Toward the telomeric end of the 57-kb segment commonly deleted in cystinosis is a region matching a series of expressed-sequence tags (ESTs; GenBank accession nos. AA70014, AA553482, AA618422, AA340511, AA331298, AA313538, and AA355260; see Fig. 3), three of which comprise a UniGene cluster (UniGene Hs.190207). These ESTs were derived from various tissues (including colon, fetal kidney, fetal liver/spleen, human embryo, Jurkat T cells, and Schwannoma tumor) and matched the genomic sequence with nearly 100% identity. In addition, gene-prediction programs indicate the presence of a seven-exon gene between CTNS and the matching ESTs (with the 3' end of the predicted gene residing adjacent to the ESTs; see Fig. 3).
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In light of its apparent presence within the genomic interval commonly
deleted in cystinosis patients, we characterized this putative gene in
greater detail. PCR primers were designed from the predicted exons and
used in various combinations to amplify human fetal kidney cDNA. The
resulting PCR products were sequenced, eventually allowing the assembly
of 3838 bp of the mRNA (GenBank accession no. AF163573). Note that the
sequence of the most upstream portion of exon 1 has not been
determined. These results confirmed the presence of the gene [named
CARKL (carbohydrate kinase-like); see below], which contains a
1434-bp ORF encoding a predicted 478 amino-acid protein. Both GENSCAN
(Burge and Karlin 1997) and GRAIL2 (Xu et al. 1994) nicely predicted
the intron/exon organization of CARKL, the details of which
are now known based on the genomic and cDNA sequence data (Table 2).
Northern analysis (Fig. 4) of CARKL revealed
the strong expression of a ~3.9-kb transcript in liver, kidney, and
pancreas, weaker expression in heart and placenta, and very weak
expression in brain and lung. In addition to the ~3.9-kb mRNA, a
~2.7-kb transcript was also detected in liver and, to a lesser
extent, in heart.
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The predicted amino-acid sequence encoded by CARKL shows 30%
identity and 42% similarity over 321 amino acids to the hypothetical Caenorhabditis elegans protein T25C8.1 (GenBank accession
no. Z83241) and 24% identity and 37% similarity across 320 amino acids to a Streptomyces rubiginosus xylulose kinase protein
(GenBank accession no. P27156). CARKL has weak homology to
several other carbohydrate kinases from a variety of species (data not
shown). The predicted protein does not appear to contain a signal
sequence, suggesting that it localizes in the cytoplasm. A search for
protein motifs identified weak similarity to two domains of the FGGY
family of carbohydrate kinases (PROSITE PS00933 and PS00445).
Carbohydrate kinases are a class of proteins involved in the
phosphorylation of sugars as they enter a cell, inhibiting return
across the cell membrane (Worley et al. 1995). In light of the weak
similarity to the carbohydrate kinases and the absence of a known
substrate for the encoded protein, the gene was named CARKL.
Genes Neighboring CTNS and CARKL
By a combination of sequence database comparisons and computational
gene predictions, three additional genes were detected in the 200-kb
region immediately surrounding CTNS and CARKL (Fig. 2; Table 3). At the telomeric end of this interval is
the likely human ortholog of the rat vanilloid receptor subtype 1 gene
(Caterina et al. 1997). Most of the gene is contained within the
sequenced region. The encoded receptor, which is a cation channel whose ligands include capsaicin, functions as a transducer of pain stimuli. An alternative splicing variant of this gene, called the
stretch-inhibitable nonselective cation channel (SIC), has been
reported independently (Suzuki et al. 1999). At the centromeric end of
the region resides most of the gene encoding the integrin 
E
precursor (HUMINAE). The mRNA sequence of HUMINAE has
been established, with 3647 nucleotides (of 3927 total) identified in
the genomic sequence. The integrin E precursor is a component of a
cell adhesion protein complex expressed on a subclass of T lymphocytes
known as intraepithelial lymphocytes, which are interspersed among
mucosal epithelial cells (Shaw et al. 1994). Also present in the
sequenced interval are genes encoding the ionotropic ATP receptor
(P2X5), a developmentally regulated gene expressed as two
splicing variants (Le et al. 1997), and the Tax interaction protein 1 (TIP-1), a protein containing a PDZ domain that has been found
to interact with the HTLV-1 Tax oncoprotein (Rousset et al.
1998).
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EST T85505, reported previously to reside within the telomeric end of
the common 57-kb cystinosis-causing deletion (Town et al. 1998), was
analyzed in greater detail. This EST is part of a larger cluster
(UniGene Hs.193738). All of the cDNA clones in this cluster were
derived from fetal liver/spleen, and the sizes of the corresponding
inserts are nearly identical (744-746 bp), based on overlapping the
generated 5' and 3' ESTs with the genomic sequence. Searches
against the public databases failed to identify significant matches to
known genes or proteins. The 3' ESTs in this cluster begin at a
polyadenosine stretch located at the end of a partial Alu
sequence. No polyadenylation signal is found within the 3' ESTs,
and Northern analysis did not detect a transcript in multiple tissues
tested (data not shown). Furthermore, GRAIL2 and GENSCAN failed to
predict any exons or genes within the 15-kb interval surrounding these
ESTs. It seems, therefore, that this T85505-specific sequence in 17p13
likely represents a pseudogene or an artifact of cDNA cloning.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
METHODS
REFERENCES
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The systematic sequencing of large genomic segments represents a
powerful tool for revealing the long-range molecular architecture of
biologically important chromosomal regions. In the study reported here,
we have focused on the segment of human chromosome 17p harboring CTNS, the gene recently implicated in nephropathic cystinosis (Town et al. 1998). Specifically, following the construction of a
detailed BAC-based physical map of the region, we generated >200 kb
of high-accuracy genomic sequence from two overlapping clones that
together contain the entire CTNS gene.
Our sequence data reveal the molecular structure, size, and intron/exon
organization of CTNS, as well as insight about the size and
sequence context of cystinosis-causing deletions. Our findings reveal
that this genomic region is rich in Alu sequences. There is
direct and circumstantial evidence that such repetitive motifs may have
a role in other chromosomal rearrangements (Luzi et al. 1995; Harteveld
et al. 1997; Super et al. 1997; Jeffs et al. 1998; Strout et al. 1998).
One might speculate that such instability may have contributed to the
genetic event leading to the common 57-kb deletion as well as the other
described cystinosis-causing deletions, although a direct involvement
of Alu repeats in these deletions has certainly not been established.
The comprehensive sequence data now available for CTNS should
facilitate efforts to define the mutational spectrum associated with
cystinosis. Already, this sequence has been used to characterize the
breakpoints of the common deletion, allowing the development of a PCR
assay for diagnosing individuals that are heterozygous or homozygous
for that deletion (Anikster et al. 1999). This assay serves as the
primary diagnostic tool for cystinosis in the Western Hemisphere, as
nearly half of the known cystinosis alleles contain the 57-kb deletion.
It should now be straightforward to determine the precise breakpoints
in any cystinosis-associated deletion and to design suitable PCR assays
for detecting such deletions, such as the second large
cystinosis-causing deletion reported by Forestier et al. (1999). For
cystinosis patients with splice-site mutations, the intronic sequence
will permit the identification of cryptic or alternative splice sites
and allow the design of primers for PCR amplification and sequencing of
the intronic DNA flanking each exon.
Another use of the sequence has been demonstrated by the discovery of a
number of genes flanking CTNS (Fig. 2). In principle, deletions affecting CTNS and any of these flanking genes may
lead to more complex phenotypes than those encountered in conventional cystinosis patients; specifically, contiguous gene deletion syndromes may be recognized. In that regard, the most intriguing findings are
those associated with the novel gene CARKL, which presumably encodes a carbohydrate kinase. Strikingly, CARKL is fully
contained within the 57-kb region commonly deleted in cystinosis
patients. Because nearly half of all known cystinosis patients are
homozygous for this deletion, these individuals are devoid of both
cystinosin and the CARKL-encoded protein. Once the function of
the latter protein has been elucidated and its putative substrate(s)
identified, it will be important to study the clinical features of
cystinosis patients harboring different CTNS deletions (e.g.,
those with or without the common 57-kb deletion). It is possible that
the presence/absence of CARKL may account for the clinical
heterogeneity seen in cystinosis patients with respect to distal
vacuolar myopathy (Charnas et al. 1994), nephrocalcinosis
(Theodoropoulos et al. 1995), and other complications of the disease
(Gahl and Kaiser-Kupfer 1987; Gahl et al. 1995). In this regard, we
hypothesize that CARKL may be a modifier for the cystinosis phenotype.
The study of patients presumably lacking a carbohydrate kinase may also provide insight about the functional role of this putative enzyme and its associated biochemical pathway. Studies in human biochemical genetics often reveal pathways whose existence and function are elucidated by discovery of individuals lacking a key enzyme; the CARKL gene may provide the latest example.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
METHODS
REFERENCES
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STS Generation
STSs were developed from the following sources of DNA sequence: (1)
known genes and genetic markers; (2) plasmid subclones derived from
random restriction fragments of CEPH YAC 767F9 [which spans the entire
region harboring CTNS (McDowell et al. 1996; Peters et al.
1997)]; and (3) insert ends of isolated BACs. For generating the
latter, BAC DNA was purified using an Autogen 740 Automated Nucleic
Acid System (Integrated Separation Systems) and concentrated to 200 ng/µl using a Microcon-100 column (Millipore Corp., Bedford, MA).
Fluorescent DNA sequencing was performed with the -40M13 universal
primer (5'-GTTTTCCCAGTCACGAC-3') or -28M13 reverse primer
(5'-CAGGAAACAGCTATGACC-3') and BigDye-terminator chemistry
(Perkin Elmer/Applied Biosystems Division, Foster City, CA). The
20-µl sequencing reaction contained 11 µl of purified BAC DNA
(at 200 ng/µl), 1 µl of primer (at 10 µM), and 8 µl of BigDye-reaction mixture. Thermal cycling was performed as
suggested by the manufacturer. The products were then purified on a
Centrisep column (Princeton Separations, NJ), dried, suspended in 2 µl of formamide loading buffer, and analyzed on an Applied
Biosystems 377XL automated fluorescent sequencing instrument (Perkin
Elmer/Applied Biosystems Division, Foster City, CA). For developing
suitable STS-specific PCR assays, sequences were analyzed for
repetitive elements, and apparently unique sequences were then used to
design PCR primers using the computer program OSP (Hillier and Green 1991). PCR assays were optimized essentially as described (Green 1993).
Information about the STSs and their corresponding PCR assays is
available in GenBank and/or GDB.
BAC Contig Construction
BACs were isolated from the Research Genetics
(http://www.resgen.com) and Genome Systems
(http://www.genomesystems.com) human BAC libraries by PCR-based
screening (according to the suppliers' instructions) and from the
Roswell Park Cancer Institute human BAC library RPCI-11
(http://bacpac.med.buffalo.edu) by hybridization-based screening using
STS-specific "overgo" probes (Vollrath 1999; see http://genome.wustl.edu/gsc). Positive clones were colony purified, and
individual colonies were tested by PCR analysis. As nascent BAC contigs
were assembled based on the STS content of the BACs, new STSs were
developed from insert-end sequences derived from strategically selected
BACs and used to isolate additional clones. This process was repeated
in an iterative fashion. Our general strategy for constructing human
BAC contigs has been reported previously (Ellsworth et al. 1999).
Genomic Sequencing
BAC clones RG147P12 and RG87B10 were sequenced to high accuracy
using a shotgun sequencing strategy (Wilson and Mardis 1997). Briefly,
purified BAC DNA (http://genome.wustl.edu/gsc/Protocols/BAC.shtml) was
kinetically sheared with a nebulizer (CIS-US, Inc., Bedford, MA), and
the resulting fragments were end-repaired with T4 DNA polymerase and
Klenow and then subcloned into plasmid pBC (Stratagene, La Jolla, CA)
and M13mp18 vectors. Randomly selected subclones were sequenced from
one (M13mp18) or both (pBC) ends to a final estimated average
redundancy of 10-fold. Fluorescent sequencing reactions were performed
with BigDye-terminator (Perkin Elmer/Applied Biosystems Division,
Foster City, CA) and energy transfer (ET) dye-primer
(Amersham-Pharmacia Biotech, Piscataway, NJ) chemistries, and the
resulting products analyzed with Applied Biosystems 377XL automated
fluorescent sequencing instruments. Individual sequences were edited
and assembled using the Phred/Phrap/Consed suite of programs (Gordon et
al. 1998; Ewing and Green 1998; Ewing et al. 1998) to a final estimated
error frequency of <1 in 104 bp as determined by Phrap and
Consed. The validity of each sequence assembly was confirmed by the
concordance of forward and reverse sequencing reads from individual
plasmid subclones and by alignment with known cDNA sequences.
Sequence Analysis
Genomic sequence was analyzed for the presence of known human
repetitive elements using the program RepeatMasker
(http://ftp.genome.washington.edu/cgi-bin/RepeatMasker) and Crossmatch
(http://www.genome.washington.edu/UWGC/analysistools/swat.htm) (A.F.A.
Smit and P. Green, unpubl.). Sequence comparisons with public databases
were performed with PowerBLAST (Zhang and Madden 1997) using the
following parameters: BLASTN (M = 1, N = 
3, S = 40,
S2 = 40) and BLASTX (S = 90, S2 = 90, FILTER = SEG). The results from PowerBLAST were collated and viewed using Sequin (Benson
et al. 1997). As part of our sequence analysis process, the gene
prediction programs GRAIL2 (Xu et al. 1994) and GENSCAN (http://ccr-081.mit.edu/GENSCAN.html) (Burge and Karlin 1997) were used
to identify putative genes. Protein motifs were identified using the
MOTIF tools (http://www.genome.ad.jp/SIT/MOTIF.html), whereas
prediction of signal peptides was performed using the PSORT program
(http://psort.nibb.ac.jp).
cDNA Sequencing
Fragments of the CARKL cDNA were generated by PCR amplification of human fetal kidney cDNA (Clontech, Palo Alto, CA) using primers designed from GENSCAN-predicted exons (details available on request). The resulting DNA fragments were sequenced using BigDye-terminator chemistry as described above, eventually allowing assembly of the cDNA.
Northern Analysis
A 1072-bp CARKL-specific DNA probe was generated by PCR
from human fetal kidney cDNA (Clontech, Palo Alto, CA) with primers 5'-GAGTAGAATCCTCCAAGCCCTACAC-3' and
5'-GAAGCATGGAGTGCAGGTTCTG-3' (see GenBank accession no.
AF163573 for corresponding positions within the cDNA sequence). The
resulting PCR product was radiolabeled with [-32P]dCTP
(NEN Life Science Products, Boston, MA) and hybridized to a human
multiple tissue Northern blot (Clontech, Palo Alto, CA) as described
(Shotelersuk et al. 1998b).
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ACKNOWLEDGMENTS |
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Y.A. is a Howard Hughes Medical Institute Physician Postdoctoral fellow. We thank M. Furgusson, E. Sorbello, A. Cunningham, A. Gupta, R. Torkzadeh, C. Varner, and M. Walker for excellent technical assistance with DNA sequencing as well as John McPherson and the staff of the Washington University Genome Sequencing Center for assistance in BAC isolation. We also thank Drs. A. Baxevanis, L. Biesecker, L. Everett, W. Gan, and C. Jamison for general advice, assistance, and/or critical review of the manuscript.
The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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FOOTNOTES |
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4 Corresponding author.
E-MAIL egreen{at}nhgri.nih.gov; FAX (301) 402-4735.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
METHODS
REFERENCES
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Received October 21, 1999; accepted in revised form December 13, 1999.
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J. W. Touchman, A. Dehejia, O. Chiba-Falek, D. E. Cabin, J. R. Schwartz, B. M. Orrison, M. H. Polymeropoulos, and R. L. Nussbaum Human and Mouse alpha -Synuclein Genes: Comparative Genomic Sequence Analysis and Identification of a Novel Gene Regulatory Element Genome Res., January 1, 2001; 11(1): 78 - 86. [Abstract] [Full Text]
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) The STS was verified to be
present in that clone by PCR testing; (
) the STS was derived from
the insert end of that BAC. The two BACs subjected to complete
sequencing (see Fig. 
-actin-specific probe
(b). Autoradiography was performed for 24 and 2 hr,
respectively.