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Molecular Biology and Genetics |
Department of Surgery, Section of General Thoracic Surgery [L. L., S. A., M. B. O., D. G. B.] and Departments of Human Genetics [T. W. G.] and Pediatrics [T. W. G., S. H.], University of Michigan Medical School, Ann Arbor, Michigan 48109
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Gene amplification is one mechanism of oncogene activation leading to tumorigenesis. Barrett’s esophagus and esophageal adenocarcinoma provide an ideal tumor progression model to examine gene amplification because a well-defined metaplasia-dysplasia-carcinoma sequence is observed (8) . Several proto-oncogenes demonstrate gene amplification in esophageal adenocarcinomas, including the erbB2 gene which is amplified in 22%, the EGFR gene in 13%, and the K-ras gene in 10% (9) .3 Additional chromosomal amplicons have been identified using CGH4 as well as other techniques including the amplicon at 8p22–23 (10) . Candidate genes for these amplicons, however, remain largely unknown because narrowing the minimal critical amplified region using CGH alone still leaves relatively large chromosomal intervals involving many uncharacterized genes (reviewed in Ref. 11 ).
We have identified recently a novel amplicon at 8p22–23 in esophageal adenocarcinomas using the RLGS 2D-gel technique (10) . The CTSB gene was proposed as a potential candidate gene for the 8p22–23 amplicon, based on the fact that it was found to be amplified in 13% and overexpressed in >25% of esophageal adenocarcinomas examined, and that CTSB is overexpressed in many other human cancers (12, 13, 14) . The 8p22–23 amplicon, however, spans 12 cM and includes many genes in this region. To better characterize the minimal critical region of this amplicon, in the present study, we implemented a novel application, which we have termed STS-amplification mapping. This approach uses integrated physical maps and databases to search highly resolved microsatellite markers (STS) mapped within the chromosomal region of the amplicon. The selected STS markers are then applied to screen a large number of normal-tumor pairs of genomic DNA by a QG-PCR assay. The minimal critical area is determined by mapping the minimal overlapping amplified region between tumors. This application allowed us to construct an STS-amplification map and narrow the 12-cM amplicon to a <5-cM minimal amplified region in the present series of 66 esophageal adenocarcinomas. A positional candidate approach was then applied to this region, and the minimal critical region was further reduced to <2 cM. A YAC clone (725-c-12), which contained a 420-kb insert including CTSB, was analyzed previously using FISH to determine its chromosomal location (10) . This YAC was localized within the minimal amplified region in the present study. The GATA-4 gene was also verified to be included within YAC 725-c-12. GATA-4 was observed to demonstrate gene amplification as well as mRNA and protein overexpression in the esophageal tumors in the present study, as was the case for CTSB in the previous study (10) . GATA-4 is a member of zinc finger transcription factor family that play developmental roles including gut endodermal differentiation (15) . The function of the GATA-4 gene in adult tissue, however, remains unknown. Furthermore, by using QG-PCR assay, we were able to demonstrate genomic amplification of 8p22–23 in both esophageal and gastric cardia adenocarcinomas but not in esophageal squamous cell carcinomas or lung adenocarcinomas.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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STS-Amplification Mapping Using QG-PCR.
Two BAC clones (393H12 and 538D6), representing two individual
NotI-HinfI two-dimensional gel spots found to be
amplified in the esophageal adenocarcinomas and mapped by FISH
(10)
, were used as anchors to localize the telomeric and
centromeric sites of the 8p22–23 amplicon. By STS database
analyses,5
DNA microsatellite (STS) markers in the 8p22–23 amplicon were
selected. PCR primers for each selected DNA fragment were carefully
designed (slightly modified from the primers chosen using DNASTAR
software) to ensure that the melting temperature (Tm) of the
STS PCR fragments matched the Tm of the internal control
(GAPDH), which was coamplified in each PCR reaction. Genomic DNA from
tumor and normal tissues was quantified by fluorometry (TKO100; Hoefer
Scientific Instruments, San Francisco, CA). The forward primers of the
control and test fragments were end-labeled with
[
-32P[ATP (NEN Life Science Products,
Boston, MA) using T4 polynucleotide kinase (New England Biolabs,
Beverly, MA). PCR was conducted with incorporation of a 50-ng template
in 25 µl of total reaction volume using Taq polymerase (Promega
Corp., Madison, WI). The template was amplified for 18–22 cycles to
maintain linearity of the PCR products. The PCR conditions included an
initial denaturing step at 94°C for 2 min and 18–25 cycles each
consisted of 95°C for 40 s, 58–63°C (depending on the
Tm of individual primers of the tested STS markers) for
40 s, and 70–72°C for 1 min using the PTC100 thermal cycler (M
& J Research, Watertown, MA). The PCR products were then resolved on
8% denaturing polyacrylamide gels. Vacuum-dried gels were exposed to
PhosphorImager screens (Molecular Dynamics, Sunnyvale, CA). The signal
ratios (Ts/c:Ns/c) for both the tumor (Ts/c, tumor STS fragment/tumor
GAPDH fragment) and normal DNA samples (Ns/c, normal STS
fragment/normal GAPDH fragment) were determined after quantitation
using ImageQuant software (Molecular Dynamics). Esophageal tumors
showing recurrent increased DNA copy number were further analyzed, and
the minimal amplified region was determined.
Positional Candidate Analysis Using Quantitative RT-PCR.
Several
databases6
were searched to select available ESTs and known genes within the
defined minimal amplified region of the 8p22–23 amplicon. Total RNAs
from all samples were treated with DNase I (Promega) prior to
performing reverse transcription. Two µg of total RNA were reverse
transcribed using reverse transcriptase (Life Technologies) and primed
by both (dT)18 and random hexamers in a total 40
µl of reaction volume. Two µl of the cDNA were then subjected to
RT-PCR with GAPDH coamplified as the internal control. The PCR products
were resolved on 8% PAGE gels, and gel data analyses were performed
using ImageQuant software as in QG-PCR analysis.
cDNA Library Screening.
Human testis cDNA library (Clontech, Palo Alto, CA) was plated onto
150-mm LB agar plates at a high cell titer. The individual clone DNAs
were lifted onto nylon membranes and then hybridized to EST
stSG1554, according to the supplied protocol from the
manufacturer (NEN Life Science Products, Boston, MA). The hybridization
probe used was a 296-bp RT-PCR fragment of stSG1554 labeled
with [
-32P]dCTP by direct incorporation. A
Sephadex G-50 column (Boehringer Mannheim, Indianapolis, IN) was used
to remove unincorporated [-32P]dCTP. After
hybridization and washing, the membranes were subjected to
autoradiography using Hyperfilm (Amersham). The positive clones were
selected for secondary screening, and the isolated clones were then
picked. Mini-preps of isolating clone DNA were conducted by the
alkaline method as described by Sambrook et al.
(17)
.
Northern Blot Analysis.
Ten µg of total RNA from tumor samples, Barrett’s mucosa, and
surrounding normal esophagus were size-fractionated in 1.2%
formaldehyde/formamide agarose gels and vacuum-transferred to nylon
membranes. The same 296-bp RT-PCR fragment of EST stSG1554
used for cDNA library screening was
[-32P]dCTP labeled and hybridized to the RNA
blotting membranes following the conditions described previously
(18)
. Hybridization signals and analysis were obtained
after PhosphorImager scanning.
Sequencing of Cloned DNA and RT-PCR Products.
The sequences of the cloned DNA were obtained manually according to the
protocol of fmol DNA Cycle Sequencing System (Promega) and through the
University of Michigan DNA Sequencing Core using an ABI PRISM Model
377XL DNA Sequencer (PE Applied Biosystems, Foster City, CA). A 0.7-kb
RT-PCR fragment was amplified from an esophageal tumor that contained
the 8p22–23 amplicon. One primer of the RT-PCR products overlapped the
3' end of the published GATA-4 gene sequence (GATA4-s2,
5'-CTGCTGCCGGCCTTTGCTC-3'), and another primer was located within the
cloned DNA sequence (SG554-rf1, 5'-TCCAGCATCAGGGGCAGAAAC-3'). The
purified RT-PCR products were sequenced through the University of
Michigan DNA Sequencing Core from both the 5' and 3' ends,
respectively, primed with the sense primer GATA4-s2 and an antisense
primer that overlaps the 5' end of the cloned DNA sequence
(SG554–75', 5'-ACAGACAGCAGGTGGGCCAGC-3').
Immunohistochemical and Western Blot Analyses.
Frozen specimens were sectioned at 5 µm, placed on 0.1%
poly-L-lysine-coated slides, and fixed in 100% acetone at
-20°C for 10 min. Endogenous peroxidase activity was quenched with
five changes of 1.2% hydrogen peroxide for 30 min each. Nonspecific
binding was blocked using a 1:20 dilution of rabbit serum in PBS-1%
BSA. The GATA-4 protein was detected by using the anti-GATA-4 antibody
(Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:500 dilution in
PBS-1% BSA. The antibody for GATA-4, although demonstrating
cross-reactivity with rat, mouse, and human GATA-4, does not recognize
other GATA family members (e.g., GATA-1, GATA-2, GATA-3,
GATA-5, and GATA-6). Adult mouse heart, which normally expresses the
nuclear GATA-4 gene product, was used as positive control. A
section of every tissue was incubated without the primary antibody as a
negative control. Immunoreactivity was detected by using the Vectastain
avidin/biotin complex kit (Vector Laboratories, Burlingame, CA) with
3,3'-diaminobenzidine as a substrate. The slides were lightly
counterstained with Harris-modified hematoxylin and permanently mounted
as described previously (18)
.
For Western blot analysis, tissue protein was extracted in a buffer containing NP40 as described (18) . Ten µg of total protein extract were fractionated by 10% SDS/PAGE and transferred to nylon membranes. The GATA-4 protein was detected with the same anti-GATA-4 polyclonal antibody used in immunohistochemical study.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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12 cM within the 8p22–23
amplicon. STS markers, spaced 1 cM apart, were carefully chosen from
the highly resolved maps integrated from several STS databases, as
described in "Materials and Methods." A housekeeping gene,
GAPDH, was incorporated as an internal control and
coamplified with the genomic target (STS or genes) fragments in each
QG-PCR reaction. The primer labeling and reaction cycles were
controlled such that neither the internal control nor the test fragment
ever reached amplification saturation or reaction plateau, maintaining
product linearity. The PCR products were quantified, and the ratio of
(Ts/c):(Ns/c) was computed. Fig. 1
shows the results of the PCR-amplified normal-tumor pairs using four
different STS or gene markers by the QG-PCR assay. As shown, some
tumors demonstrate genomic amplification at only one marker, whereas
some tumors show recurrent increased DNA copy number at all markers
(Fig. 1)
. The data using the QG-PCR assay in the present study were
found to be consistent with previous data obtained by Southern blot
analysis of genes in the 8p22–23 amplicon in this series of esophageal
adenocarcinomas (10)
. Fig. 2
shows the tumor STS-amplification map of the 8p22–23 amplicon based on
the QG-PCR data by screening the 66 normal-tumor pairs of genomic DNA
with 11 STS markers, one EST, and one known gene inside the amplicon.
By analyzing the individual tumor patterns for the recurrent increased
DNA copy number using these STS and gene markers, the minimal amplified
region was mapped to the area between the markers D8S552 and
D8S1759, 5 cM. The minimal critical region was further
narrowed and determined to be <2 cM, based on the tumor overexpression
patterns of the ESTs selected from the RH physical maps within the
defined 5-cM area (Table 1A)
. YAC 725-c-12, containing 420 kb of the insert and the
STS/EST marker WI-8953, was mapped within the minimal amplified region
(Fig. 2)
. The sequence of WI-8953 is homologous to CTSB and
was confirmed in our previous study (10)
. EST
stSG1554 was analyzed using PCR and confirmed to be located
inside this YAC (data not shown), indicating that the distance between
the two genes are within 0.5 cM. The map order is based on the regional
amplification patterns of our 66 normal-tumor pairs but closely follows
the published genetic, YAC contig, and radiation hybrid maps from the
NCBI, WICGR, and SHGC databases.
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, RT-PCR analysis for this gene failed to
show elevation of mRNA expression in the amplified tumors, suggesting
that BLK was not likely to be an important candidate gene
for the amplicon (data not shown).
Nine available ESTs within the minimal amplified
region7
and four potential cancer-related genes in the 8p22–23
amplicon8
were selected for quantitative RT-PCR analysis (Table 1)
. Quantitative
RT-PCR of normal-tumor pairs was directly chosen as the testing method,
rather than testing DNA amplification first, because of the lack of
genomic information for these ESTs. Theoretically, all ESTs within the
minimal amplified area should show amplification in tumors bearing the
amplicon. Moreover, the ultimate goal of the study is to identify
overexpressed gene(s) within this amplified genomic DNA segment. All
total RNAs were treated with DNase prior to performing reverse
transcription to avoid any DNA contamination. Quantitative RT-PCR was
performed with the normal-tumor pairs containing genomic amplification
as well as with pairs not demonstrating increased DNA copy numbers.
Among the nine ESTs and four known genes examined in the present study,
EST stSG1554 was the only one demonstrating RNA
overexpression in the amplified tumors relative to their corresponding
normal tissues. Table 1
lists the expression status of all 12 ESTs
within the minimal amplified region and four known genes examined from
the present study as well as from our previous study (10)
.
The four selected known genes are cytogenetically close to the minimal
amplified region; however, their precise positions in terms of location
within the genetic map are not currently available.
Cloning and Sequencing a Novel Sequence Leading to the
Identification of Amplification and Overexpression of the
GATA-4 Gene in Esophageal Adenocarcinomas.
The EST, stSG1554, was found to show elevated mRNA
expression in all tumors containing the 8p22–23 amplicon using
quantitative RT-PCR but not in nonamplified tumors (Figs. 1D
and Fig. 3, A and B
). Northern blot hybridization using
stSG1554 as the probe confirmed the RT-PCR results (Fig. 4)
. Overexpression of this EST was observed in primary and metastatic
adenocarcinomas but not in Barrett’s metaplasia (Fig. 4)
. BLAST search
of this EST (407 bp, gi = 689325) indicated >82%
sequence homology with the rat GATA-GT2 gene (gb = L22761) in the 3'UTR region. The human GATA-4 gene is
the homologue of rat GATA-GT2 gene and has been mapped
previously to chromosome 8p22 (19)
. The sequence retrieved
for human GATA-4 from GenBank (gb = L34357) is 2226 bp
in length. The coding sequence of the human GATA-4 mRNA
starts at 241 bp and ends at 1569 bp. To further examine the
relationship between the EST stSG1554 and the
GATA-4 gene, RT-PCR was applied to the coding region of the
GATA-4 gene from 890 to 1188 bp (Fig. 3C)
. A
similar mRNA expression pattern was observed between EST
stSG1554 and human GATA-4 gene in esophageal
adenocarcinomas (Fig. 3)
. To obtain and fill the sequence gap between
the EST stSG1554 and human GATA-4 mRNA, a human
testis cDNA library was screened using EST stSG1554 as a
probe. Two clones were obtained and yielded a 577-bp-read sequence but
still did not reach the sequence at the 3' end of the published human
GATA-4 sequence. A 0.7-kb RT-PCR fragment was then obtained
from tumor cDNA using two primers. One primer overlapped the 3' end of
the published GATA-4 sequence, and the other primer was derived from
the clone sequence. An 865-bp sequence of the human GATA-4
gene at 3'UTR region was identified, and this novel sequence is now
accessible in the GenBank with the accession number of AF180736.
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. Positive nuclear staining was also observed in two other tumors, M47
and D15. The genomic amplifications of these two tumors were not known
because of lack of tumor DNA sources. Western blot analysis were
consistent with immunohistochemical staining of the GATA-4 amplified
esophageal tumors (data not shown).
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and 2)
. Genomic amplification
was not detected in any of the 24 esophageal squamous carcinomas nor
the 39 lung adenocarcinomas (Table 2)
. However, DNA amplification was observed for both CTSB and
D8S1695 in one gastric cardia adenocarcinoma of eight
(12.5%) tumors examined (Table 2)
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. Analysis revealed a genomic amplification in only one
tumor (D06) but not in any other esophageal adenocarcinomas containing
the 8p22–23 amplicon. Expression status of MASL1 in the
tumors was also tested by RT-PCR analysis, and no overexpression was
observed (Fig. 6B)
. Tumor D06 possessed the largest amplicon
(>12 cM) at 8p22–23 of all 66 esophageal adenocarcinomas examined
(Figs. 2
and 6)
. Because D06 was the only tumor showing amplification
of MASL1 (Fig. 6A)
, the MASL1 gene
might either represent a different amplicon less frequently amplified
in esophageal adenocarcinomas, or more likely, lie within the same
amplicon but not within the minimal amplified region and thus is less
likely to represent a critical cancer-related gene within this amplicon
(Fig. 6C)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because many known genes, ESTs, and genomic sequences are now accessible using extensive databases, the positional candidate approach used in the present study is an effective way to identify new genes of interest (27 , 28) . Twelve ESTs and four known genes were analyzed as potential candidate genes within the minimal amplified region in the 8p22–23 amplicon. The location of the ESTs were determined using GeneMap’99.9 Known genes were selected on their potential relationship to tumor development and progression. All four selected known genes did not demonstrate mRNA overexpression in this series of esophageal adenocarcinomas, suggesting that these genes are unlikely to be critical candidate genes for the 8p22–23 amplicon.
8p22–23 amplification was identified recently as a novel amplicon in esophageal adenocarcinomas using RLGS 2D-gel technique (10) . Genomic amplification of 8p21–22 was also observed in gastric adenocarcinomas by CGH technology (29) . Gene amplification of erbB2 was observed in 21–22% of esophageal adenocarcinomas, EGFR in 13%, and K-ras in 10% (9) .3 Comparable with the frequencies of amplification for these oncogenes, genomic amplification at 8p22–23 is detected in 13.6% in esophageal adenocarcinomas and 12.5% in gastric cardia adenocarcinomas in the present study. These results suggest a nonrandom event favoring tumorigenesis or tumor progression of the cancer types examined and indicate that an oncogenic gene(s) may reside in this region. Interestingly, genomic amplification of 8p22–23 was not observed in lung adenocarcinomas or in esophageal squamous carcinomas examined in the present study, suggesting that the oncogenic event of the 8p22–23 amplicon may be tumor type specific, favoring the development or progression of esophageal and gastric cardia adenocarcinomas.
EST stSG1554 was the only one among 12 ESTs examined that
showed elevated mRNA expression in the tumors with 8p22–23
amplification. Further analysis of this EST led to the identification
of novel sequences and the determination of these sequences as part of
the human GATA-4 gene. The GATA-4 gene has been
mapped previously to 8p22 (19)
. GATA genes are
a family of zinc finger transcription factors, recognizing a consensus
WGATAR motif of target genes through a conserved multifunctional
DNA-binding domain (30)
. Six GATA genes have
been identified, among which GATA-1, GATA-2, and GATA-3 function in the
hematopoietic cell lineages (31)
and GATA-4, GATA-5, and
GATA-6 regulate gene expression in developing tissues, including heart
and gut (32)
. Several target genes in the cardiac system
regulated by GATA-4 have been studied, including the brain
natriuretic peptide gene (33)
, the -myosin
heavy-chain gene (34)
, and a recently identified
gene, FOG2 (35
, 36)
. Rat GATA-GT2, the
homologue of human GATA-4, was identified in the gastric epithelium and
found to bind to the upstream sequence of the
H+/K+-ATPase ß gene
containing the GATA motif (37)
. The intestinal fatty
acid-binding protein was also proposed as the downstream target of
GATA-4 (38)
. Murakami et al., (39)
recently reported that FGF3 oncogene may be a target of the
GATA-4 transcription factor in the undifferentiated tumor cells. The
function and downstream target gene(s) for GATA-4 in adult tissues,
however, are unclear and will require further study.
Our primary characterization of the 8p22–23 amplicon revealed that CTSB was both amplified and overexpressed in esophageal adenocarcinomas (10) . Overexpression of CTSB has been observed in many other tumor types, including lung, colon, breast, prostate, and gastric (12, 13, 14) . The data from the present study demonstrate that amplification of the GATA-4 gene results in GATA-4 overexpression in the same esophageal tumors that have also been found to have CTSB amplification. STS-amplification mapping and YAC 725-c-12 analyses indicate that CTSB and GATA-4 are located within the minimal amplified region and closely linked to each other (<0.5 Mb). Overexpression of GATA-4 was only observed in the tumors containing the 8p22–23 amplicon. Abundant nuclear staining was also observed in small portions of two additional tumors by immunohistochemical analysis; however, gene amplification analysis of these two tumors was not possible because of lack of tumor DNA. Gene amplification of CTSB was observed in 13% of the tumors examined, but overexpression of the mRNA was seen in 25% of the tumors, including those without gene amplification (10) . Increased CTSB protein staining was detected even in 75% of the tumors examined, suggesting that overexpression of CTSB could result from other mechanisms in addition to gene amplification. It is possible that amplifications of GATA-4 and CTSB are attributable to their cosegregation with an additional cancer-related gene located within the minimal critical region of the 8p22–23 amplicon. Further functional characterization is required to clarify the roles of CTSB, GATA-4, and other unknown genes within the minimal critical region in the development or progression of esophageal and gastric cardia adenocarcinomas.
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FOOTNOTES |
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1 Supported by NCI Grant CA71606. 
2 To whom requests for reprints should be
addressed, at Department of Surgery, Section of General Thoracic
Surgery, MSRB II B560, Box 0686, University of Michigan Medical School,
Ann Arbor, MI 48109. Phone: (734) 763-0325: Fax: (734) 763-0323;
E-mail: dgbeer{at}umich.edu
3 D. G. Beer et al.,
unpublished data.
4 The abbreviations used are: CGH, comparative
genomic hybridization; CTSB, cathepsin B; STS, sequence tagged site;
QG-PCR, quantitative genomic-PCR; YAC, yeast artificial chromosome;
EST, expressed sequence tag; FISH, fluorescence in situ
hybridization; RLGS, restriction landmark genomic scanning; Ts/c and
Ns/c, the intensity ratio of tumor (Ts/c) or normal (Ns/c) sample
versus GAPDH control from QG-PCR; NCBI, National Center
of Biotechnology Information; WICGR, Whitehead Institute Center for
Genome Research; SHGC, Stanford Human Genome Center; UTR, untranslated
region.
5 Internet addresses: NCBI,
http://www.ncbi.nlm.nih.gov; WICGR, http://www.genome. wi.mit.edu;
SHGC, http://www.shgc.stanford.edu.
6 NCBI, WICGR, and SHGC, and the Human Genome
Research Center (http://www.genethon.fr).
7 NCBI,
http://www.ncbi.nlm.nih.gov/genome/guide.
8 NCBI, http://www.ncbi.nlm.nih.gov/Omim/.
9 Internet address: http://www.ncbi.
nlm.nih.gov/genemap.
Received 8/27/99. Accepted 1/ 5/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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L. Lin, M. S. Prescott, Z. Zhu, P. Singh, S. Y. Chun, R. D. Kuick, S. M. Hanash, M. B. Orringer, T. W. Glover, and D. G. Beer Identification and Characterization of a 19q12 Amplicon in Esophageal Adenocarcinomas Reveals Cyclin E as the Best Candidate Gene for this Amplicon Cancer Res., December 1, 2000; 60(24): 7021 - 7027. [Abstract] [Full Text]
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 I. Ketola, V. Pentikäinen, T. Vaskivuo, V. Ilvesmäki, R. Herva, L. Dunkel, J. S. Tapanainen, J. Toppari, and M. Heikinheimo Expression of Transcription Factor GATA-4 during Human Testicular Development and Disease J. Clin. Endocrinol. Metab., October 1, 2000; 85(10): 3925 - 3931. [Abstract] [Full Text]
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M. P. E. Laitinen, M. Anttonen, I. Ketola, D. B. Wilson, O. Ritvos, R. Butzow, and M. Heikinheimo Transcription Factors GATA-4 and GATA-6 and a GATA Family Cofactor, FOG-2, Are Expressed in Human Ovary and Sex Cord-Derived Ovarian Tumors J. Clin. Endocrinol. Metab., September 1, 2000; 85(9): 3476 - 3483. [Abstract] [Full Text]
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, the DNA amplification is not detected at the loci applied.
The map order is based on the DNA amplification analysis of 66
normal-tumor pairs but closely follows the published genetic, YAC
contigs, and radiation hybrid maps from the NCBI, WICGR, and SHGC
databases. As shown in the map, the minimal amplified area is confined
to an approximate 5 cM region between markers D8S1759
and D8S552. The minimal amplified region was further
limited to a <2-cM region, including the YAC 725-c-12, by analyzing
the ESTs selected that spanned 3–5 cR in the GeneMap’99 physical map.
This YAC was confirmed to contain CTSB and was hybridized to metaphase
chromosomes to determine the chromosomal location using FISH analysis
