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Vol. 9, Issue 5, 428-436, May 1999
LETTER
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ABSTRACT |
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 Top
 Abstract
 Introduction
RESULTS
DISCUSSION
METHODS
References
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Williams syndrome (WS) is a complex developmental disorder resulting from the deletion of a large (~1.5-2 Mb) segment of human chromosome 7q11.23. Physical mapping studies have revealed that this deleted region, which contains a number of known genes, is flanked by several large, nearly identical blocks of DNA. The presence of such highly related DNA segments in close physical proximity to one another has hampered efforts to elucidate the precise long-range organization of this segment of chromosome 7. To gain insight about the structure and evolutionary origins of this important and complex genomic region, we have constructed a fully contiguous bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) contig map encompassing the corresponding region on mouse chromosome 5. In contrast to the difficulties encountered in constructing a clone-based physical map of the human WS region, the BAC/PAC-based map of the mouse WS region was straightforward to construct, with no evidence of large duplicated segments, such as those encountered in the human WS region. To confirm this difference, representative human and mouse BACs were used as probes for performing fluorescence in situ hybridization (FISH) to metaphase and interphase chromosomes. Human BACs derived from the nonunique portion of the WS region hybridized to multiple, closely spaced regions on human chromosome 7q11.23. In contrast, corresponding mouse BACs hybridized to a single site on mouse chromosome 5. Furthermore, FISH analysis revealed the presence of duplicated segments within the WS region of various nonhuman primates (chimpanzee, gorilla, orangutan, and gibbon). Hybridization was also noted at the genomic locations corresponding to human chromosome 7p22 and 7q22 in human, chimpanzee, and gorilla, but not in the other animal species examined. Together, these results indicate that the WS region is associated with large, duplicated blocks of DNA on human chromosome 7q11.23 as well as the corresponding genomic regions of other nonhuman primates. However, such duplications are not present in the mouse.
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INTRODUCTION |
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Top
Abstract
Introduction
RESULTS
DISCUSSION
METHODS
References
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Williams syndrome (WS; also known as Williams-Beuran syndrome) is a
well-studied disorder with features involving multiple physiological
systems [Online Mendelian Inheritance in Man (OMIM) 194050;
http://www.ncbi.nlm.nih.gov/Omim]. The syndrome is
characterized by congenital heart and vascular disease, dysmorphic
facial features, infantile hypercalcemia, and a unique cognitive and
personality profile (Burn 1986
; Morris et al. 1988; Bellugi et al.
1990; Keating 1997). Major strides have been made in recent years to
elucidate the genetic basis of WS. Following the landmark finding that
WS is caused by hemizygous microdeletions within human chromosome 7q11.23 that include the elastin gene (ELN) (Ewart et al.
1993), numerous other genes have been found to reside within the
commonly deleted interval. These include LIMK1 (encoding
LIM kinase-1) (Frangiskakis et al.
1996; Tassabehji et al. 1996), RFC2 (encoding replication factor C subunit
2) (Peoples et al. 1996), FZD3 (encoding a homolog
of the Drosophila frizzled wnt receptor) (Wang et al. 1997),
CYLN2 (encoding cytoplasmic
linker-2) (Hoogenraad et al. 1998),
STX1A (encoding syntaxin
1A) (Osborne et al. 1997b; Nakayama et al. 1998),
FKBP6 (Meng et al. 1998b), WSTF (Lu et al. 1998), GTF2I (encoding general transcription
factor II-1) (Perez Jurado et al. 1998),
BCL7B (Meng et al. 1998a; Jadayel et al. 1998), WS-
TRP (Meng et al. 1998a),
WS-bHLH (Meng et al. 1998a), CPETR1 (Paperna et al.
1998), CPETR2 (Paperna et al. 1998), and several others
(Osborne et al. 1996). The wide spectrum of phenotypic features
observed in WS is likely a consequence of haploinsufficiency of some of
the above genes and/or yet-to-be-identified genes in the deleted region.
Various studies suggest that the microdeletions associated with WS span
~1.5-2 Mb of DNA within 7q11.23 (Osborne et al. 1996; Perez Jurado
et al. 1996; Robinson et al. 1996; Urban et al. 1996; Wang et al. 1997;
Meng et al. 1998a; Wu et al. 1998). Although difficult to isolate in
yeast artificial chromosome (YAC) clones, most of the deleted region
(e.g., the segment immediately flanking ELN) has been
relatively straightforward to map using bacterial-based clones
[bacterial artificial chromosomes (BACs) (Shizuya et al. 1992) and
P1-derived artificial chromosomes (PACs) (Ioannou et al. 1994)].
However, detailed physical mapping efforts of the broader genomic
region encompassing the commonly deleted segment (i.e., the WS region)
have been hindered greatly by the presence of large, duplicated
segments of DNA. Our laboratory (Gorlach et al. 1997; E.D. Green and
B.J. Trask, unpubl.) and others (Osborne et al. 1997a) have generated
data suggesting the likely presence of three such duplicated blocks of
DNA that reside close together in 7q11.23. These duplicated segments
contain gene and pseudogene sequences of the p47-phox
(NCF1) (Francke et al. 1990; Gorlach et al. 1997) and
GTF2I (Perez Jurado et al. 1998) genes as well as members of
the PMS2 mismatch repair gene family (Osborne et al. 1997a).
Establishing the precise long-range organization of these duplicated
regions relative to the WS-associated deletions has proven particularly
difficult, although evidence emerging from our (E.D. Green and B.J.
Trask, unpubl.) and several other (Osborne et al. 1996, 1997a,b;
Perez Jurado et al. 1996, 1998; Robinson et al. 1996; Wang et al. 1997;
Lu et al. 1998; Meng et al. 1998a) laboratories is converging towards a
reasonably consistent model for the physical map of this region (see
Fig. 1).
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Human chromosome 7q11.23 has conserved synteny to a segment on distal
mouse chromosome 5 (DeBry and Seldin 1996). Several mouse homologs
of the above-mentioned genes, including Eln (Wydner et
al. 1994), Limk1 (Mao et al. 1996), Cyln2 (Hoogenraad
et al. 1998), Cpetr1 (Paperna et al. 1998), Cpetr2
(Paperna et al. 1998), Gtf2i (Wang et al. 1998), and
p47-phox (Jackson et al. 1994) (also see
http://www.informatics.jax.org and
http://www.ncbi.nlm.nih.gov/Homology), have been genetically or
cytogenetically mapped to this region of the mouse genome. In light of
the complex organization of the human WS region, we sought to gain
insight into the structure of the WS region in a distantly related
mammal. Here we report the results of comparative physical mapping of
the human and mouse WS regions as well as fluorescence in situ
hybridization (FISH) studies that demonstrate evolutionary differences
in the presence of the duplicated segments residing within this complex
genomic region.
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RESULTS |
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Top
Abstract
Introduction
RESULTS
DISCUSSION
METHODS
References
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Construction of a BAC/PAC Contig Map of the Mouse WS Region
The collective data generated by studies from several groups
(Osborne et al. 1996, 1997a,b; Perez Jurado et al. 1996, 1998; Robinson
et al. 1996; Wang et al. 1997; Lu et al. 1998; Meng et al. 1998a; E.D.
Green and B.J. Trask, unpubl.) indicate that the WS region of human
chromosome 7q11.23 consists of a unique segment harboring a number of
known single-copy genes flanked by several large, closely related
genomic segments (Fig. 1; see Introduction for specific references).
The latter contain a number of known genes or related pseudogene
sequences [e.g., p47-phox (Gorlach et al. 1997),
GTF2I (Perez Jurado et al. 1998), PMS2 family members (Osborne et al. 1997a)]. Our interest in human-mouse comparative genome analysis prompted us to construct a BAC/PAC-based physical map
of the region of mouse chromosome 5 corresponding to the human WS region.
Little pre-existing physical mapping information or DNA sequence was
available for this region of the mouse genome. Using partial sequence
data for the mouse Limk1 (GenBank accession no. X86569) and
p47-phox (S.J. Chanock, pers. comm.) genes
and information about an intronic polymorphism in the mouse
Eln gene (Wydner et al. 1994; also see
http://www.informatics.jax.org), sequence-tagged site
(STS)-specific PCR assays were developed and used to screen mouse BAC
and PAC libraries. Sequences were then derived from the insert ends of
positive clones and used to develop new STS-specific PCR assays, which
in turn were used to isolate additional clones. This process was
repeated for several rounds, which in some cases also included
hybridization-based screening of BAC/PAC libraries and the use of STSs
derived from other sources. Eventually, this process and the resulting
STS-content data allowed assembly of the BAC/PAC contig depicted in
Figure 2.
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The assembled contig of the mouse WS region contains 28 clones and
provides ordering information for 44 STSs. Of the 44 STSs, 39 correspond to BAC insert ends, 4 to known genes, and 1 to a conserved
human sequence. The average number of clones identified per STS is 4.9, whereas the average number of STSs within each clone is 7.7. Importantly, the clone overlaps depicted in the contig shown in Figure
2 were confirmed by restriction enzyme digest-based fingerprint
analysis (Marra et al. 1997; data not shown). The latter analysis also
revealed that the total length of the contig is ~680 kb, as
assessed by summing the size of all unique restriction fragments, and
extends >100-200 kb on either side of the interval defined by
Eln and p47-phox. Thus, with the mapping of 44 STSs across ~680 kb, the average STS spacing within the contig
map is ~15 kb. Typical variability in the depth of clone coverage
(ranging from two to seven positive clones per STS) was encountered
across the mouse WS region, with no evidence of a particular region(s)
with strikingly higher representation. This pattern is in sharp
contrast to the mapping of the human WS region, where the ratio of
p47-phox-containing to ELN-containing clones
(e.g., YAC, BAC, P1) is typically ~3:1 (Gorlach et al. 1997;
E.D. Green and B.J. Trask, unpubl.) Preliminary sequence analysis of several mouse BACs has revealed the expected
presence of known genes residing between Eln and
p47-phox (e.g., Limk1, Wscr1,
Rfc2, Cyln2, Gtf2i; compare the bottoms of
Figs. 2 with 1), providing additional support for the colinearity of
these regions in human and mouse. To date, we have not detected
Pms2-related sequences within the mouse WS region.
FISH-Based Comparative Analysis of the WS Region
To gain additional insight into the structure of the human and mouse WS regions, comparative FISH analysis was performed using representative BAC clones. Markedly different results were obtained in each case. Hybridization of human metaphase and interphase chromosomes with a BAC containing ELN (RG030E19) yields results routinely encountered with single-copy genomic regions (Fig. 3). In contrast, FISH analysis with a BAC containing p47-phox (RG350L10) reveals the presence of multiple, closely spaced hybridizing segments in human 7q11.23 as well as additional weaker hybridizing segments in 7p22 and 7q22 (Fig. 3). The number of signals observed in interphase nuclei is consistent with the presence of three to four copies of the genomic region containing p47-phox sequences within 7q11.23 and confirms the single-copy nature of the ELN-containing region (Fig. 3; Table 1). In contrast, FISH analyses of mouse chromosomes using BACs containing Eln (42J20) or p47-phox (391O16) yield virtually indistinguishable results (Fig. 3). No evidence is seen with mouse metaphase or interphase chromosomes for the presence of duplicated p47-phox-containing segments (Fig. 3; Table 1).
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Sequences separated by >100 kb can be resolved in interphase
chromatin (Trask et al. 1989; van den Engh et al. 1992). Note that the
similarity of FISH patterns obtained with the Eln- and p47-phox-containing BACs in interphase nuclei, both with
respect to the signal intensity (Fig. 3) and number of resolvable dots (Table 1), argue against the possibility of even closely spaced duplicated segments in mouse. Three other BACs, which together with the
Eln- and p47-phox-containing clones provide a tiling path across the ~680-kb mouse WS region, gave similar results, consistent with the single-copy nature of the WS region in mouse (Table
1; data not shown).
To establish if duplications of the WS region are present in other
primates, the chromosomes of chimpanzee, gorilla, orangutan, and gibbon
were analyzed by FISH using human ELN- and
p47-phox-containing BACs. The FISH patterns obtained with the
ELN BAC are consistent with the presence of a single-copy
segment in all these animal species (data not shown). The ELN
sequence maps to the expected genomic locations corresponding to human
chromosome 7q11.23, based on comparison of the banding patterns of
chromosome-7 orthologs (Yunis and Prakash 1982; Jauch et al. 1992)
(Fig. 4a,b). These chromosomes are referred to as
PTR6, GGO6, PPY6, and HLA1, respectively (note that the locations are
denoted henceforth using nomenclature for the human karyotype). The
gorilla and orangutan chromosomes differ from the human and chimpanzee
chromosomes by the presence of one and two inversions, respectively
(Yunis and Prakash 1982). One breakpoint of the paracentric inversion
that differentiates the gorilla and human chromosomes lies in or near
7q11.23. However, the position of the ELN-associated signal is
unaffected, and thus it lies proximal to this evolutionary breakpoint.
Relative to the human and chimpanzee 7, orangutan shows the same
paracentric inversion seen in gorilla, plus a large pericentric
inversion. The ELN-associated signal resides within the latter
inverted unit, resulting in its location on the small short arm of the
orangutan chromosome.
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The FISH pattern obtained with the p47-phox-containing BAC is strikingly different than that with the ELN-containing BAC in the nonhuman primates. As in human, a very large FISH signal is seen in the region corresponding to 7q11.23 in metaphase chromosomes from the four nonhuman primates tested (Fig. 4). Multiple duplicated segments are also evident in interphase nuclei. In addition, signals are observed on chimpanzee and gorilla chromosomes at locations corresponding to 7p22 and 7q22. These additional copies of p47-phox-related sequence are not observed in either orangutan or gibbon (Fig. 4).
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DISCUSSION |
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Top
Abstract
Introduction
RESULTS
DISCUSSION
METHODS
References
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Elucidating the genetic bases of WS represents an important area of
ongoing investigation. The general region of human chromosome 7q11.23
deleted in WS is complex in structure, which may in part account for
its propensity to undergo interstitial deletions. As a complement to
the intensive studies of the human WS region (Osborne et al. 1996,
1997a,b; Perez Jurado et al. 1996, 1989; Robinson et al. 1996; Wang et
al. 1997; Lu et al. 1998; Meng et al. 1998a), we are pursuing the
detailed characterization of the mouse WS region for several reasons.
First, the resulting information should facilitate the
identification of genes deleted in WS, thereby enhancing studies aiming
to define the basis for each of the phenotypic features of this
contiguous gene deletion syndrome. Whereas a number of deleted genes
have been identified already [e.g., ELN (Ewart et al. 1993),
LIMK1 (Frangiskakis et al. 1996; Tassabehji et al. 1996),
RFC2 (Peoples et al. 1996), FZD3 (Wang et al. 1997), STX1A (Osborne et al. 1997b; Nakayama et al. 1998b),
CYLN2 (Hoogenraad et al. 1998), FKBP6 (Meng et al.
1998a), WSTF (Lu et al. 1998), WSCR1 (Osborne et al.
1996), GTF2I (Perez Jurado et al. 1998), BCL7B
(Meng et al. 1998a; Jadayel et al. 1998),
WS-TRP (Meng et al. 1998), WS-bHLH
(Meng et al. 1998a), CPETR1 (Paperna et al. 1998a),
CPETR2 (Paperna et al. 1998)], comparative mouse-human sequence analysis should provide a more robust approach for identifying all the encoded genes and their regulatory elements. Systematic sequencing of both the human and mouse WS regions is underway (U. DeSilva and E.D. Green unpubl.; see bottom of Fig. 2). Second, the
resulting mapping and sequencing data generated from the mouse WS
region should aid the generation of mouse models of WS, for example,
the creation of mice lacking one or more of the genes located within
the commonly deleted interval. Finally, comparative studies of the
human and mouse WS regions should provide insight into the
evolutionary origins of this structurally complex genomic region.
The findings reported here reveal that the human and mouse WS regions
are strikingly different. Numerous genetic (Dutly and Schinzel 1996;
Perez Jurado et al. 1996; Robinson et al. 1996; Urban et al. 1996;
Baumer et al. 1998) and physical (Osborne et al. 1997a; Perez Jurado et
al. 1998; E.D. Green and B.J. Trask, unpubl.) mapping studies have
revealed the presence of large, duplicated blocks of DNA that flank the
region deleted commonly in WS. Some studies also indicate that both
inter- and intrachromosomal rearrangements are responsible for WS
deletions (Dutly and Schinzel 1996; Perez Jurado et al. 1996, 1998;
Robinson et al. 1996; Urban et al. 1996; Baumer et al. 1998).
Presumably, the duplicated segments on chromosome 7 homologs or
chromatids misalign and mediate meiotic or mitotic rearrangements.
In contrast, our physical (Fig. 2) and cytogenetic (Fig. 3) mapping
studies indicate the absence of such duplicated segments in the mouse
WS region. These results are consistent with studies showing that
Gtf2I (Wang et al. 1998) and p47-phox (S.J. Chanock,
pers. comm.) sequences are present in a single-copy fashion in the
mouse genome [whereas these gene sequences reside within the
duplicated segments in the human WS region (see Fig. 1)]. The absence
of such duplicated segments in the mouse genome indicates that the
duplication event(s) that created the complex structure of the human WS
region must be of recent evolutionary origin.
Our comparative FISH analyses of the WS region suggest that the
duplication of the p47-phox sequences occurred sometime after the split of the ancestors of rodents and hominoids [~65 million years ago (Mya)] and before the diversification of the hominoids (~15-20 Mya) (see Kumar and Hedges 1998). Local duplication of this
segment within the WS region on 7q11.23 (or its equivalent position)
was observed in all animal species tested, except the mouse. Additional
copies of p47-phox sequences appear to have spread to two
locations elsewhere on chromosome 7 (7p22 and 7q22) in the ancestor
common to human, chimpanzee, and gorilla.
The association of evolutionary breakpoints in the orthologs of human
chromosome 7 and the locations of genomic segments containing p47-phox sequences is striking. The gorilla chromosome
differs from the human and chimpanzee chromosomes by a paracentric
inversion with breakpoints at 7q11.23-q21 and 7q22, and
p47-phox sequences are present in both regions. The
organization of chromosome 7 material in orangutan differs from that in
the other hominoids by a pericentric inversion, with breakpoints in
7p22 and 7q11.23-q21. Again, p47-phox sequences are found in
the vicinity of both breakpoints. This inversion event may explain the
single cluster of p47-phox sequences on the short arm of the
orangutan chromosome. It is, however, surprising that no
p47-phox sequences are detected at the 7q22 site in orangutan
as they are in gorilla, given that the banding pattern of the orangutan
chromosome (Yunis and Prakash 1982) reveals the same paracentric
inversion involving 7q11.23-7q22 as that seen in gorilla.
Our comparative analyses could be explained by a scenario in which a p47-phox-containing segment first duplicated locally and then some copies were distributed to three locations on chromosome 7 by inversion events. The presence of similar sequences at these various locations may lead to homologous recombination and subsequent intrachromosomal rearrangements. It is therefore possible that the inversions that occurred during primate evolution and the relatively frequent deletions that lead to WS in humans are mediated by the same duplicated sequences.
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METHODS |
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Top
Abstract
Introduction
RESULTS
DISCUSSION
METHODS
References
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Isolation of BAC/PAC Clones
BAC (Shizuya et al. 1992) and PAC (Ioannou et al. 1994) clones were
isolated by PCR- and/or hybridization-based screening of the following
libraries: (1) Research Genetics CITB-CJ7-B mouse BAC library
(http://www.resgen.com); (2) Genome Systems C57BL/6 mouse BAC library
(http://www.genomesystems.com); and (3) Roswell Park Cancer Institute
RPCI-21 129SV mouse PAC library (http://bacpac.med.buffalo.edu). PCR
screening was performed as recommended by the library supplier. For
hybridization-based screening, the STS-specific PCR assay was used to
generate an appropriate product, which was then isolated by gel
electrophoresis, purified using a Geneclean II kit (BIO 101), and
radiolabeled with [
-32P]dCTP using Ready-To-Go DNA
labeling beads (Pharmacia). The resulting radiolabeled probe was then
separated from unincorporated nucleotides by passage over a
Sephadex-G50 column (Pharmacia). The purified probe was then hybridized
with membrane filters containing the arrayed clones in 50% formamide
buffer, essentially as described (Sambrook et al. 1989). Filters were
then washed two times in 2× SSC at room temperature and two times in
0.1× SSC/0.2% SDS at 65°C prior to overnight exposure to Kodak
X-Omat film. Candidate positive clones were then evaluated by
additional hybridization and/or PCR analyses.
Sequencing of BAC Insert Ends
A freshly grown BAC colony was inoculated into 2 ml of 2× TY
broth (Sambrook et al. 1989) containing 12.5 µg/ml of
chloramphenicol and incubated shaking at 37°C for 4-5 hr. The
culture was then added to 30 ml of 2× TY broth containing 12.5 µg/ml of chloramphenicol in a 50-ml tube and incubated shaking at
37°C for 16-22 hr. A total of 20 ml of the final culture was used
to isolate BAC DNA with an Autogen 740 Plasmid Isolation System using
the manufacturer's protocol (v. 1.3). The resulting DNA from the 20 ml
of culture was resuspended in 400 µl of water, and 5 µl of
RNase cocktail (500 U/ml of RNase A and 20,000 U/ml of RNase T1;
Ambion) was added. Following incubation at 37°C for 30 min, samples
were transferred onto a Microcon-100 column (Amicon) and centrifuged at
800g for 15 min. The columns were then inverted into the
collection tubes and centrifuged at top speed in a microcentrifuge for
5-10 sec. The resulting DNA was adjusted to a final concentration of
~200-250 ng/µl.
DNA sequencing was performed using DYEnamic Energy Transfer Primers
(Amersham) and an adaptation of the described protocol (Marra et al.
1996). Briefly, each sequencing reaction contained 2 µl of
ThermoSequenase reagent mix (Amersham), 1 µl of DYEnamic Energy
Transfer Primer (
40 universal or 28 reverse), and 5 µl of
the above-purified BAC DNA. Thermal cycling was performed in either a
Catalyst Molecular Biology Workstation (Perkin-Elmer) or an MJ Research
PTC-200 DNA Engine using the following conditions: 95°C for 2 min
followed by 20 cycles of 95°C for 10 sec, 50°C (28 reverse
primer) or 55°C (40 universal primer) for 20 sec, and 72°C
for 60 sec. Reactions corresponding to the four dideoxynucleotides were
pooled, and the DNA precipitated by addition of 5 µl of glycogen and 132 µl of 100% ethanol. Samples were centrifuged at 3200 rpm for 30 min in a Jouan GR422 centrifuge. Following removal of the supernatant, the DNA pellets were dried and then resuspended in 5 µl (for analysis on an Applied Biosystems 373 sequencer) or 3 µl (for analysis on an Applied Biosystems 377 sequencer) of the
supplied loading buffer (Amersham), heated to 60°C for 10 min, and
analyzed on either an Applied Biosystems 373 or 377 sequencer.
Development of BAC Insert End-Specific STSs
Sequences derived from BAC insert ends were imported into MacVector DNA analysis program (Kodak), and BAC vector sequences were removed manually. The resulting sequence was analyzed for murine repeats using the Repeat Masker 2 Web server (http://ftp.genome.washington.edu/cgi-bin/RepeatMasker), and PCR primers were designed from nonrepetitive sequences using the PCR primer design module of MacVector. Oligonucleotide primers were then synthesized commercially (Life Technologies). Information about the individual STS-specific PCR assays is available in GenBank (http://www.ncbi.nlm.nih.gov).
Determination of BAC/PAC Insert Sizes
BAC and PAC DNA was purified using the Autogen 740 Plasmid
Isolation System as described above and digested to completion with
NotI (New England Biolabs). The digested sample was then loaded onto a 1% SeaPlaque (FMC Bioproducts) agarose gel in 0.5× TBE and electrophoresed for 14 hr in a CHEF DRII pulsed-field gel
apparatus (Bio-Rad) (5-12 sec ramped switching, 120 V, 12°C). Concatemerized 
DNA samples (New England Biolabs) were included on
all gels to facilitate establishing the size(s) of the resulting BAC-derived band(s) following ethidium bromide staining (Riethman et
al. 1997).
BAC/PAC Fingerprint Analysis
BACs and PACs were subjected to restriction enzyme digest-based
fingerprint analysis, essentially as described (Marra et al. 1997).
Specifically, purified BAC/PAC DNA was digested with HindIII, electrophoresed in a 1% SeaKem LE agarose (FMC Bioproducts) gel at 45 V for 16 hr, and stained with SYBR green (FMC Bioproducts). The gel was
imaged using a Molecular Dynamics FluorImager. The resulting image was
then transferred to a UNIX workstation for band calling and contig
assembly. Restriction fragment bands were identified using the program
Image 3.3, and contigs were constructed using FPC.
FISH Analysis
Purified BAC DNA was biotinylated by nick translation and
hybridized (20 ng/slide in a 10-µl volume of hybridization
solution) to immobilized mouse and human interphase and metaphase
chromosomes. Mouse interphase cells were prepared from uncultured mouse
splenocytes. Mouse metaphase spreads were prepared from mouse
splenocytes after culture in lipopolysaccharide. Human
G0/G1 interphase cells were prepared from
growth-inhibited fibroblast cultures. Human metaphase spreads were
prepared from PHA-stimulated peripheral blood lymphocyte cultures.
Metaphase and interphase cells were harvested from actively growing
cultures of chimpanzee (Pan troglodytes, CRL-1847), gorilla (Gorilla gorilla, CRL-1854), orangutan (Pongo
pygmaeus, CRL-1850), and gibbon (Hylobates lar, TIB-201)
cell lines obtained from the American Type Culture Collection (ATCC)
(http://www.atcc.org). The culture and preparation of cells is
described elsewhere (Trask 1999). All cells were incubated in hypotonic
75 mM KCl, fixed several times in 3:1 methanol:acetic
acid, and dropped on clean slides. The same hybridization and wash
stringencies were used in all experiments (hybridization solution: 50%
formamide, 2× SSC, 10% dextran sulfate at 37°C, with washes in
50% formamide, 2× SSC at 42°C). Mouse or human Cot1
DNA was included in the hybridization solution to suppress labeling of
highly repetitive sequences. Hybridization was detected by incubation
of the slides in avidin-FITC, biotinylated goat anti-avidin, and
avidin-FITC, with wash steps between each labeling step. In some
experiments, two probes, one labeled with digoxigenin and one with
biotin, were hybridized simultaneously and detected with different
fluorochromes (Trask 1999). The nuclei were counterstained with DAPI.
Images of the FITC and DAPI fluorescence were collected separately
using Signal Analytics IPLab Spectrum software, a Princeton CCD camera,
and a Zeiss Axiophot microscope equipped with separate excitation filters in a Ludl filter wheel mounted between the 100W lamp and the
sample and a triple-band multichroic mirror and emission filter mounted
between the sample and the detector (known as the Pinkel arrangement).
The FITC image was pseudocolored in red and then overlaid on a
gray-scale DAPI image. In some experiments, the number of hybridization
dots was counted within the clusters of FISH signals observed in 100 interphase chromosomes.
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ACKNOWLEDGMENTS |
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We thank Johannah Doyle, Curt Jamison, Jackie Idol, Danny Wangsa, and Thomas Ried for various contributions to these studies. This work was supported in part by grants from the Department of Energy (DE-FG03-96ER62173) and the National Institutes of Health (GM57070).
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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3 Corresponding authors.
E-MAIL btrask{at}u.washington.edu; FAX (206) 685-7354.
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 February 2, 1999; accepted in revised form March 16, 1999.
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,
) The STS is
confirmed to be present in that clone by PCR testing. When an STS
corresponds to a clone insert end, a red square is present at the end
of the clone from which it was derived. PAC clones are indicated with a
P at the beginning of their names. Four of the BAC clones were isolated
from the Genome Systems C57BL/6 mouse library (111E07, 285A04, 155F12,
and 159L24), with the rest of the BACs coming from the Research
Genetics CITB-CJ7-B mouse library. The size of each BAC/PAC, as
assessed by pulsed-field gel analysis, is also provided. The indicated
BAC/PAC overlaps were confirmed by restriction enzyme digest-based
fingerprint analysis (Marra et al. 1997
