Construction, characterization and chromosomal mapping of bacterial
arti¢cial chromosome (BAC) library of Yunnan snub-nosed monkey
Huai-liang Xu1,3,4, Ya-ping Qian2, Wen-hui Nie1, Jian-xiang Chi1,3, Feng-tang Yang1& Bing Su1,2*
1Key Laboratory of Cellular and Molecular Evolution, Kunming Institute of Zoology,
The Chinese Academy of Sciences, Kunming, China; E-mail: firstname.lastname@example.org;
2Center for Genome Information, Department of Environmental Health, University of Cincinnati, OH, USA;
Tel: (þ1)513-558-6678; Fax: (þ1)513-558-4397; E-mail: email@example.com;
3Graduate School of the Chinese Academy of Sciences, Beijing, China;4College of Animal Science and
Technology, Sichuan Agricultural University, Yaan, China
Received 18 December 2003. Accepted for publication by Adrian Sumner 21 January 2004
Key words: bacterial artificial chromosome (BAC), chromosome mapping, fluorescence in-situ
hydridization, Rhinopithecus bieti, Yunnan snub-nosed monkey
We constructed a high redundancy bacterial artificial chromosome library of a seriously endangered Old
World Monkey, the Yunnan snub-nosed monkey (Rhinopithecus bieti) from China. This library contains
a total of 136320 BAC clones. The average insert size of BAC clones was estimated to be 148kb. The per-
centage of small inserts (50–100kb) is 2.74%, and only 2.67% non-recombinant clones were observed.
Assuming a similar genome size with closely related primate species, the Yunnan snub-nosed monkey
BAC library has at least six times the genome coverage. By end sequencing of randomly selected BAC clones,
we generated 201 sequence tags for the library. A total of 139 end-sequenced BAC clones were mapped onto
the chromosomes of Yunnan snub-nosed monkey by fluorescence in-situ hybridization, demonstrating a high
degree of synteny conservation between humans and Yunnan snub-nosed monkeys. Blast search against
human genome showed a good correlation between the number of hit clones and the size of the chromosomes,
an indication of unbiased chromosomal distribution of the BAC library. This library and the mapped BAC
clones will serve as a valuable resource in comparative genomics studies and large-scale genome sequencing
of nonhuman primates. The DNA sequence data reported in this paper were deposited in GenBank and
assigned the accession number CG891489-CG891703.
Comparison between mammalian genomes pro-
vides insight into the common features of biologic
mechanisms and helps to identify experimental
models forstudying complex
unique details of gene structure and function
(Collins et al. 1998). It also has been considered
experimentation that helps to identify functional
elements and evolutionary constraints within the
genomes (Sidow 2002). With the completion of
whole genome sequencing of human and a series
of model species (Lander et al. 2001, Waterston
Chromosome Research 12: 251–262, 2004.
# 2004 Kluwer Academic Publishers. Printed in the Netherlands
et al. 2002, Larkin et al. 2003), there has been an
ever-increasing need for large insert DNA librar-
ies of other species that are phylogenetically
informative and biomedically significant. Cur-
rently, the bacterial artificial chromosome (BAC)
library is the common choice of resource for
research. It provides an easy access to stable
material for DNA manipulation such as exon
trapping, cDNA selection, direct sequencing,
microsatellite marker isolation, fluorescence in-
situ hybridization (FISH) and physical mapping
(Beck 2001, Martins-Wess 2002, Li 1999).
In particular, sharing a close behavioral and
genetic kinship with humans, nonhuman primates
are the most suitable biomedical models for com-
parative genomic studies. However, most of the
primate species are seriously endangered and may
face extinction (Mittermeier et al. 1999). Hence,
construction of nonhuman primate libraries would
provide the most practical and cost-e¡ective means
to interrogate the genetic bases for primates traits
relevant to evolution and human disease, and also
resources which can be utilized persistently (Eich-
ler and de Jong, 2002). To date, a number of non-
human primates libraries have been constructed
(Pieter de Jong’s laboratory at Children’s Hospital
Oakland Research Institute, hhttp://www.chori.
org/bacpaci; Qian et al. 2003), and more BAC
libraries are certainly necessary to cover the
diverse evolutionary lineages in primates.
The Yunnan snub-nosed monkey (Rhinopithecus
bieti) diverged from the human lineage about
25 million years ago (Goodman 1998, 1999), and
is one of the rarest and most endangered primates
in the world (Long et al. 1994). Morphological
study suggests it occupies an intermediate position
between the Old World monkeys and the lesser
apes (Peng et al. 1985, 1988), and is, therefore, a
keystone species in understanding primate evolu-
tion. Unfortunately, the low genetic diversity,
small population size and fragmented distribution
all act to increase the endangered status of this
rare species with less than 2000 individuals in the
wild (Long et al. 1994, Lan et al. 1995, Su & Shi
In the Old World monkeys, genome-wide
comparative chromosome maps between human
and three species of leaf monkeys have been estab-
lished by cross-species chromosome painting with
human chromosome-speci¢c probes (Bigoni et al.
1997a, Nie et al. 1998). These comparative maps
showed a high degree of conservation of chromoso-
mal syntenies, and
chromosomal rearrangements (involving human
chromosomes 1, 2, 6, 14, 15, 16, 19, 21 and 22) dif-
ferentiate the karyotypes of the leaf monkeys and
humans. This observation suggests that human 14
and 15 homologues as well as 21 and 22 homo-
logues have been involved in Robertsonian translo-
cation during the evolution of Asian colobine
monkeys (Bigoni et al. 1997a, 1997b, Nie et al.
1998, Wu et al. manuscript in preparation). The
leaf monkeys are closely related to the snub-nosed
monkeys with the same diploid chromosome
number (2n¼44) like most of the Asian colobine
monkeys (Bigoni et al. 2003). In this study, we con-
structed a high-redundancy BAC library of the
Yunnan snub-nosed monkey using pBACe3.6 vec-
tor (Frengen et al. 1999). With insert size testing,
BAC clone end sequencing, we showed that this
library has an average insert size of 148kb, and at
least six times the genome coverage with unbiased
chromosomal distribution. Using FISH, we map-
ped the chromosomal locations of 139 BAC
clones. Our results demonstrated a high degree of
synteny conservation between humans and Yun-
nan snub-nosed monkeys.
Materials and methods
A. BAC library construction
(1) Preparation of high-molecular-weight
The lymphoblast cell line (KCB-96009) of a male
Yunnan snub-nosed monkey with a normal
2n¼44 karyotype was obtained from Kunming
Cell Bank and the cell concentration was adjus-
ted to 5?108cells/ml. An equal volume of pre-
warmed 1% InCert agarose (BioWhittaker Mole-
cular Applications) was added. The agarose/cell
mixture was briefly stirred and transferred into a
disposable plug mold (Bio-Rad) placed on ice.
The plugs were then incubated twice (3h and
overnight with change of the buffer) at 50?C
0.01mol/L Tris-Cl pH 7.6, 0.02mol/L NaCl and
1% N-lauroylsarcosine) containing 0.1mg/ml of
252Huai-liang Xu et al.
proteinase K. The plugs were washed five successive
times at 4?C in TE buffer (pH 7.6, 10mmol/L
Tris-Cl, 1mmol/L EDTA) and incubated for 30
min at 50?C in TE buffer containing 40mg/ml
Sigma, 20mg/ml in isopropanol stored at –20?C),
followed by three successive 30-min dialyses
against TE (pH 7.6). The DNA plugs were stored
at 4?C in 20% NDS (2mmol/L Tris, 6.8mmol/L
N-lauroylsarcosine and 127mmol/L EDTA pH
(2) Preparation of insert DNA
Five agarose plugs were equilibrated twice in
sterile 0.5?TE (pH 8.0) for 1h at 4?C and then
in the EcoRI digestion buffer (2mmol/L MgCl2)
for 30min. The optimal amount of enzyme com-
bination was determined by digesting the DNAs
with 1 U EcoR I and varying amounts of EcoR I
methylase. Partial digestion of each plug was per-
formed with 10ml of EcoRI restriction endonu-
clease (0.1U/ml) and 1ml of EcoRI methylase
1.25ml SAM (final concentration: 80mmol/L) and
5ml 100?BSA (final concentration: 100ng/ml) at
37?C overnight. Reactions were stopped by add-
ing 20% NDS. The partial digested DNAs were
size-selected twice by pulsed-field gel electrophor-
esis (PFGE) on 1% agarose gel (Fisher Biotech)
using the CHEF-DRIII system (Bio-Rad). The
agarose gel containing 100–250kb DNAs was cut
out and sliced into two equal parts.A second
PFGE was then performed to remove small
DNA fragments trapped within the gel slices
using the same conditions.
(3) Recovery of high-molecular-weight EcoRI
The DNA size-selected agarose slices were recov-
ered by electroelution. After electroelution, the
DNA solution was dialyzed against 0.5?TE
(pH 8.0) at 4?C overnight. DNA concentration
was estimated by gel electrophoresis with l DNA
of known concentration.
(4) Ligation and transformation
Using a 1:10 molar ratio of insert over vector
fragments were incubated with the pBACe3.6
vectors in a 50-ml total volume containing 0.5
unit of T4 DNA ligase (Invitrogen) at 15?C
overnight. The ligation mixture was dialyzed
using microdialysis filters (0.025-mm, Millipore)
in sterile 0.5?TE (pH 8.0) buffer for 1.5h.
Two microliters of ligation product was used to
transform 20ml of ElectroMAX DH10B compe-
tent E. coli cells (Invitrogen). Electroporation
was carried out using a Cell-Porator E. coli
Electroporation System (Gibco BRL). After ele-
troporation, the cells were incubated in 1ml
S.O.C. medium at 37?C for 1h. The cells were
12.5mg/ml chloramphenicol and incubated at
B. BAC library characterization
(1) Size estimation of BAC clones
A total of 225 BAC clones were randomly picked
from the Yunnan snub-nosed monkey library.
These clones were inoculated in 2ml LB medium
containing 12.5mg/ml chloramphenicol at 37?C
for 16h. The insert DNAs were extracted using a
modified alkaline-lysis method (Osoegawa et al.
2002). The insert DNAs were digested with NotI
(New England Biolabs) and then subjected to
PFGE. The molecular weights of the BAC inserts
were calculated using the image analysis program
TotalLab 1D gel analysis (http://www.totallab.
com/home.asp) with the Low-Range size marker
(New England BioLabs).
(2) BAC end sequencing
A total of 206 BAC clones were randomly picked
performed using plasmid BAC mini-prep kit
(Oligo-Chem). A 20-mer T7 primer: 50TAA-
end sequencing. Fifteen clones were also
sequenced at the 30ends using the SP6 primer: 50
ATTTAGGTGACACTATAGAAGGATC 30. The
ABI 3100 Genetic Analyzer (Applied Biosystem)
was used for
C. Chromosomal mapping using FISH
The metaphase preparation and G-banding fol-
lowed the procedures described in Nie et al. (1998).
BAC library construction of Yunnan snub-nosed monkey253
A total of 139 BAC clones of the end-sequenced
clones were mapped using FISH. Mini-preps of
BAC DNAs were conducted using the standard
alkaline lysis method (Sambrook et al. 2001). The
DNA samples were subject to further purification
by phenol/chloroform extraction and isopropanol
precipitation. A 1–2mg aliquot of DNA was used
to prepare probes labeled with biotin-14-dCTP
by nick-translation in 25ml mixtures containing
2.5ml 10?nick-translation buffer, 1.9ml ATG
(0.5mmol/L dATP, dTTP, GTP), 1ml DNase I
(1mU), 1.8ml biotin-14-d CTP (0.4mmol/L), 2ml
DNA polymerase/DNase I (0.5U DNA poly-
merase I – 0.4mU DNase I/ml) and 0.5ml DNA
polymerase I (10 U/ml) (Invitrogen). The mixtures
were incubated for 1–1.5h at 16?C and stopped by
adding 1/10 volume of 0.5mol/L EDTA (pH 8.0)
and incubated for 10min at 65?C. About 2ml
(about 100ng) of labeled BAC DNA and 1mg of
human Cot-1 DNA were mixed with 12ml hybridi-
zation buffer (50% deionized formamide, 10% dex-
tran sulfate, 2?SSC, 0.5mol/L phosphate buffer,
pH 7.3 and 1?Denhardt’s solution). The probes
were denatured for 10min at 70?C, directly trans-
ferred to 37?C, and allowed to pre-anneal for at
least 30min. The metaphase slides were treated
with 0.01% pepsin in 10mmol/L HCl for 7min at
room temperature and stopped in 2?SSC solu-
tion, and then subject to dehydration in an ethanol
series (70%, 90% and 100%) for 2 min. The slides
were incubated for 1–3h at 65?C before hybridiza-
tion. The slides were denatured in 70% formamide-
2?SSC for 1–1.5min at 65?C, quenched in
ice-cold 70% ethanol, and dehydrated in an ethanol
series. After air-drying, slides were hybridized to
probes at 37?C for 15–18h. Following hybridiza-
tion, the coverslips were removed and the slides
were washed in 50% formamide-2?SSC twice and
2?SSC twice for 5min at 43?C. The hybridization
was checked with Cy3-avidin (1:1000 dilution), and
then were stained in 2?SSC containing 0.6mg/ml
DAPI (40,6-diamidino-2-phenylindole). The fluor-
escent images were captured using the Genus sys-
tem (Applied Imaging) with a Cohu CCD camera
mounted on a Zeiss microscope (Axioplan 2). The
chromosomes of the Yunnan snub-nosed monkey
were identified based on the DAPI-bandings which
are similar to G-bandings following the nomen-
clature described by Wu et al. (manuscript in
Results and discussion
To generate enough BAC clones for sufficient
genome coverage, genomic DNAs were prepared
and partially digested with EcoRI, size fraction-
ized, and then ligated with pBACe3.6 vectors
(Frengen 1999). A total of 136320 BAC clones
were produced and arrayed into 355 384-well
Insert size testing
To estimate the average insert size of the
library, a total of 225 clones were randomly
selected and tested through pulse field gel elec-
trophoresis (PFGE) (Figure 1 and Figure 2).
By subtracting the vector size (8.7kb), the
insert size was calculated using the TotalLab
1D gel analysis program. Our result showed
that the average insert size is 148kb. Six non-
insert clones were observed in the 225 clones
tested (2.67%).The insert size distribution of the
BAC clones is shown in Figure 2. Most of the
clones in the library fall in the range of 120kb
to 170kb (80.4%). The ratio of small inserts
(<100kb) is only 2.74% (6/219), indicating a
high ratio of large insert clones in this library.
According to the published data on haploid C
values (CV) of the primate genomes (http://
a similar genome size with the average size in
subfamily Colobinae, the Yunnan snub-nosed
monkey BAC library has at least six times
coverage of its genome.
BAC clone end-sequencing and blast search analysis
In order to generate sequence tags and evaluate
the chromosomal distribution of the BAC clones,
we sequenced a total of 206 randomly picked
primer.There were five clones showing no inserts
(2.43%), consistent with the test using PFGE.
The BAC end sequences were subjected to blast
search against the human genome and dbEST
databases. A summary of the blast search result
is shown in Table 1. The blast search result
254 Huai-liang Xu et al.
Figure 2. The insert size distribution of the Yunnan snub-nosed monkey BAC library. Insert sizes were determined for 225 BAC
clones. The horizontal axis refers to the size ranges in kb while the vertical axis indicates the number of clones.
Figure 1. The electromorph of PFGE for insert size testing. There are 28 BAC clones tested. The low-range molecular weight marker
was used as size standard in the two flanking lanes. The vector band is 8.7kb.
BAC library construction of Yunnan snub-nosed monkey255
sequences and 95 (47.3%) repetitive sequences,
and 4 (2%) sequences without obvious homo-
logies in the human genome. This observation is
lower than the ratio of repetitive sequences (58%)
observed in the human genome (Zhao et al.
2000), which is probably due to lack of EcoRI
recognition sites in some of the repetitive regions
in the Yunnan snub-nosed monkey. Based on the
analysisof the 102
sequence similarity between humans and Yunnan
snub-nosed monkeys was estimated as 92.2% on
average (Table 1).The blast search result also
indicated a fairly good correlation between the
number of hit clones and the size of the human
chromosomes (Figure 3), hence, an implication
of unbiased chromosomal distribution of the
BAC clones of the library. There are 24 (11.9%)
thatthereare 102 (50.7%) unique
end sequences showing hits in the dbEST data-
base (score?300, according to Zhao et al. 2000).
In addition, we also sequenced 15 of the 201
clones at the 30ends using the SP6 primer, the
blast results indicated the same chromosome hits
as the 50end sequences. The average read length
of the BAC ends after trimming the vector
sequences is 484bp and the total length of all the
BAC end reads is 104.8kb. The 201 sequence
tags were deposited in GenBank under accession
Mini-prepared DNAs from the BAC clones were
used in the FISH experiments to evaluate the
chimerism of the library and to map the chromo-
somal locations of the clones. A total 139
Figure 3. Human chromosomal distribution of the 100 unique BAC end sequences of the Yunnan snub-nosed monkey. The data was
generated by blast search against the human genome database.
Table 1. Summary of the blast search result of the 201 BAC end sequences.
Repet Uniq UnknownEST
No. of hits
Note: Repet, repetitive sequences, Uniq, unique sequences, Unknown, no sequence matches in
the human genome, EST, sequences with hits on human ESTs (score5300). The average
trimmed read length of the 201 BAC-end sequences is 484bp, and the average sequence
similarity between human and Yunnan snub-nosed monkey is 92.2%.
256 Huai-liang Xu et al.
end-sequenced BAC clones were mapped using
Yunnan snub-nosed monkey. The FISH examples
were shown in Figure 5 and the chromosomal
assignment of all BAC clones were summarized
on a G-banded karyotype of the Yunnan snub-
nosed monkey (Figure 6 and Table 2). Among the
139 BAC clones tested, 10 clones had hybridiza-
tion signals at the centromeric regions of all the
chromosomes except for the Y chromosome (Figure
5a), an implication of repetitive sequences in those
BAC clones. All the other 129 BAC clones had
unique chromosomal locations and no chimeric
hybridization signals were observed. We were able
to confidently determine the approximate chromo-
somal locations of 100 BAC clones on well-spread
chromosomes (Table 2 and Figure 6). The chro-
mosomal distribution of the mapped clones is
illustrated in Figure 4, demonstrating a good cor-
relation between the number of clones and the
size of the chromosomes of the Yunnan snub-
nosed monkey, which is consistent with the blast
search result against human genome (Figure 3).
Based on the chromosomal homology between
human and Yunnan snub-nosed monkey estab-
chromosomes of the
lished by chromosome painting (Wu et al. 2003,
manuscript in preparation), most of the BAC
clones hitting individual human chromosomes
were hybridized onto the corresponding monkey
chromosomes, indicating conserved chromosomal
syntenies between human and Yunnan snub-nosed
monkey. There are five clones (243A5, 183E2,
243A3, 330D1 and 330N3) hitting sequences on
multiple human chromosomes. This is probably
caused by segment duplications that frequently
occurred in the human genome (Johnson et al.
2001). Interestingly, the clones hitting human
chromosome 14/15 were mapped onto chromo-
some 5 of the Yunnan snub-nosed monkey, and
the clones hitting human chromosome 21/22 were
mapped onto chromosome 15 of the Yunnan
snub-nosed monkey. This observation is con-
sistent with the proposed HAS 14/15, 21/22
Robertsonian translocation by chromosome paint-
ing studies (Wienberg et al. 1990, 1992, Bigoni
et al. 1997a, 1997b, Nie et al. 1998, Wu et al.
2003, manuscript in preparation). Our result also
suggested that human chromosome 1 has four
syntenic regions in the Yunnan snub-nosed monkey
genome, which was confirmed by the chromosome
Figure 4. Chromosomal distribution of the 100 BAC clones mapped onto the chromosomes of Yunnan snub-nosed monkey using
BAC library construction of Yunnan snub-nosed monkey257
painting study (Wu et al. 2003, manuscript in
preparation). Seventeen clones with a single hit on
both human and monkey chromosomes were found
to be located at different arms of the homologous
chromosomes (Table 2), which implies that these
chromosomes have probably undergone intra-
chromosomal rearrangements during evolution.
In summary, we have constructed a Yunnan
136320 clones. Based on the estimated insert size,
the number of clones and the putative genome
size, this library should represent at least 6-fold
coverage of the R.bieti genome. We have
demonstrated that this library has no obvious
chromosome-coverage bias. As a preliminary
e¡ort, we have generated 201 BAC end sequences
and 139 of them were mapped onto the chromo-
somes of the Yunnan snub-nosed monkey. There-
library would serve as a valuable resource for
Figure 5. The examples of FISH mapping results of four BAC clones. (a) BAC clone 243M5 with signals on the centromeric regions
of all chromosomes except for the Y chromosome (arrowed). (b) BAC clone 243J2 hybridized onto the middle region of the long arm
of chromosome 15. (c) BAC clone 243N6 hybridized onto the distal regions of the short arm of chromosome 2 (d) BAC clone 243J6
hybridized onto the distal regions of the long arms of chromosome 7. No chimerism was observed for all the BAC clones tested.
258Huai-liang Xu et al.
Figure 6. The G-banding karyotype of a male Yunnan snub-nosed monkey and the idiogram with the mapped BAC clones.
The clone ID of each mapped BAC can be found in Table 2.
BAC library construction of Yunnan snub-nosed monkey 259
Table 2. The blast search and FISH mapping results of the
100 BAC clones.
Clone ID. Human chr. Monkey chr.
Table 2. (Continued).
Clone ID.Human chr. Monkey chr.
Note: The numbers in parenthesis indicate the serial numbers of
the approximate chromosomal locations in Figure 6. The
chromosomal locations of the five BAC clones with multiple
hits on human chromosomes are:
c243A3: 3q21.1, 2q37, 7q36, 9p23–22.1, 17q25;
9q22, 7p13–12, 3p14, Xp22.2–22.11;
a243A5: 11p13, 15q23–24,
b183E2: 10p12, 13q12, 9q32–34.11, 7q11–22;
e330D1: 19q12, 12q13,
260Huai-liang Xu et al.
The ¢lters and BAC clones of the Yunnan snub-
nosed monkey BAC library is available upon
Laboratory of Cellular and Molecular Evolution,
Kunming Institute of Zoology, CAS.
We are grateful to the technical help of Jin-Huan
Wang and Xiao-Na
supported by grants from the Chinese Academy
of Sciences (CAS) and the National Science
Foundation of China (NSFC).
Beck TW, Menninger J, Voigt G et al. (2001) Comparative
feline genomics: a BAC/PAC contig map of the major
histocompatibility complex class II region. Genomics 71:
Bigoni F, Koehler U, Stanyon R, Ishida T, Wienberg J (1997a)
Fluorescence in situ hybridization establishes homology
between human and silvered leaf monkey chromosomes,
reveals reciprocal translocations between chromosomes
homologous to human Y/5, 1/19 and 6/16 and delineates
an X1X2Y1Y2/X1X1X2X2 sex-chromosome system. Am J
Phys Anthropol 98: 315–328.
Bigoni F, Stanyon R, Koehler U, Morescalchi AM, Wienberg J
(1997b) Mapping homology between human and black and
white colobine monkey chromosomes by fluorescence in situ
hybridization. Am J Primatol 42: 289–98.
Bigoni F, Stanyon R, Wimmer R, Schempp W (2003)
Chromosome painting shows that the proboscis monkey
(Nasalis larvatus) has a derived karyotype and is phylo-
genetically nested within Asian colobines. Am J Primatol 60:
Collins FS, Patrinos A, Jordan E et al. (1998) New goals for
the U.S. human genome project: 1998–2003. Science 282:
Eichler EE, de Jong PJ (2002) Biomedical applications and
studies of molecular evolution: a proposal for a primate
genomic library resource. Genome Res 12: 673–678.
Frengen E, Weichenhan D, Zhao B et al. (1999) A modular,
positive selection bacterial artificial chromosome vector with
multiple cloning sites. Genomics 58: 250–253.
Goodman M, Porter CA, Czelusiak J et al. (1998) Toward a
phylogenetic classification of primates based on DNA
evidence complemented by fossil evidence. Mol Phylogenet
Evol 9: 585–598.
Goodman M (1999) Molecular evolution 99: the genomic
record of humankind’s evolutionary roots. Am J Hum Genet
Johnson ME, Viggiano L, Bailey JA et al. (2001) Positive
selection of a gene family during the emergence of humans
and African apes. Nature 413: 514–519.
Lander ES, Linton LM, Birren B et al. (2001) Initial
sequencing and analysis of the human genome. Nature 409:
Lan H, Zhang WY, Wang W, Su B, Shi LM (1995) Genetic
diversity in the snub-nosed monkey (Rhinopithecus bieti)
based on random amplified polymorphic DNA. Folia
Primatol 65: 154–158.
Larkin DM, Everts-van der Wind A, Rebeiz M et al. (2003) A
cattle–human comparative map built with cattle BAC-ends
and human genome sequence. Genome Res 13: 1966–1972.
Li R, Mignot E, Faraco J et al. (1999) Construction
genomic bacterial artificial chromosome library. Genomics
Long YC, Kirkpatrick RC (1994) Report on the distribution,
population and ecology of Yunnan snub-nosed monkey
(Rhinopithecus bieti). Primates 35: 241–250.
Martins-Wess F, Voß-Nemitz R, Dro ¨gemu ¨ller C, Brenig B,
Leeb T (2002) Construction of a 1.2-Mb BAC/PAC contig of
the porcine gene RYR1 region on SSC 6q1.2 and compa-
rative analysis with HAS 19Q13.13. Genomics 80: 416–422.
Mittermeier RA, Rylands AB, Konstant WR (1999) Primates
of the world: an introduction. In: Nowak R, ed. Primates of
the world, Baltimore and London: Johns Hopkins Uni-
versity Press, pp 31–41.
Nie W, Liu R, Chen Y, Wang J, Yang F (1998) Mapping
chromosomal homologies between humans and two langurs
(Semnopithecus francoisi and S. phayrei) by chromosome
painting. Chromosome Res 6: 447–453.
Osoegawa K, de Jong P, Frengen E, Ioannou PA (2002)
Construction of bacterial artificial chromosome (BAC/PAC)
libraries. In: Ausubel FM, Kingston RE et al., eds., Current
protocol in molecular biology on CD-ROM, John Wiley &
Sons, Inc., unit 5.9.
Peng YZ, Ye ZZ, Zhang YP, Liu RL (1985) Observations on
the position of genus Rhinopethecus in phylogeny. Zool Res
5: 174–181 [in Chinese with English abstract].
Peng YZ, Ye ZZ, Zhang YP, Pan RL (1988) The classification
and phylogeny of snub-nosed monkey (Rhinopithecus spp.)
based on gross morphological characters. Zool Res 9:
239–248 [in Chinese with English abstract].
Qian YP, Jin L, Su B (2003) Construction and characterization
of bacterial artificial chromosome library of black-handed
spider monkey (Ateles geoffroyi). Genome (in press).
SambrookJ, RussellD (2001)
Laboratory Manual, 3rd edn. Cold Spring Harbor, NY:
Cold Spring Harbor Laboratory Press.
Sidow A (2002) Sequence first, ask question later. Cell 111:
Su B, Shi LM (1995) Genetic diversity in the snub-nosed
monkey (Rhinopithecus bieti) as estimated by protein
electrophoresis. Conserv Biol 9: 947–951.
Wienberg J, Jauch A, Stanyon R, Cremer T (1990) Molecular
cytotaxonomy of primates by chromosomal in situ sup-
pression hybridization. Genomics 8: 347–350.
Wienberg J, Stanyon R, Jauch A, Cremer T (1992) Homologies
in human and Maca fuscata chromosomes revealed by in situ
suppression hybridization with human chromosome specific
DNA libraries. Chromosoma 101: 265–270.
BAC library construction of Yunnan snub-nosed monkey261
Waterston RH, Lindblad-Toh K, Birney E et al. (2002) Initial Download full-text
sequencing and comparative analysis of the mouse genome.
Nature 420: 520–562.
Zhao SY, Malek J, Mahairas G et al. (2000) Human BAC ends
quality assessment and sequence analyses. Genomics 63:
262 Huai-liang Xu et al.