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Genomics 82 (2003) 571–574

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Short Communication

Construction of a gorilla fosmid library and its PCR screening system
Choong-Gon Kim,a Asao Fujiyama,b and Naruya Saitoua,*abDivision of Population Genetics, National Institute of Genetics, Mishima 411-8540, Japan
National Institute of Informatics, Hitotsubashi 2-1-2, Chiyoda-ku, Tokyo 101-8430, Japan
Received 14 March 2003; accepted 29 May 2003

Abstract
A gorilla fosmid library of 261,120 independent clones was constructed and characterized. The fosmid vector is similar to the cosmid
in average insert size of ca. 40 kb but contains the F factor for replication, and it is more resistant to recombination. This clone library
represents about 3.7 times coverage of the gorilla genome. A simple screening system by PCR was established, and we successfully found
9 clones that cover the entire Hox A gene cluster of the gorilla genome. This gorilla fosmid DNA library is a useful resource for comparative
genomics of human and apes.
© 2003 Elsevier Inc. All rights reserved.
Keywords: Gorilla; Ape; Hominoid; Fosmid library; Comparative genomics; HOX; Evolution

Gorilla, chimpanzee, bonobo, and orangutan are great
apes, and they are the closest organisms to human. Despite
the clear morphological differences such as cranial shapeamong them, their DNA sequences differ only 1–3% [1,2].
Recently, the draft human genome sequences were reported[3,4] and they made it possible to conduct comparative
genomics of human and great ape genomes.
Genetic changes responsible for human-specific characters occurred in the human lineage during the past several
million years after it separated from the common ancestor ofhuman and chimpanzee, the closest organism to human[5,6]. This is why the chimpanzee genome is now the targetof human comparative genomic study [2]. However, we alsoneed genome sequence information of the outgroup speciesof human and chimpanzee to identify which differences are
specific to human. Therefore, we decided to construct a
fosmid library of gorilla. Although BAC libraries are often
used for mammalian genome studies [e.g., 7–9], fosmid
libraries have some advantages. First, a fosmid library iseasier to handle than a BAC library due to its small insert
size (ca. 40 kb). In many comparative genomic studies, the* Corresponding author: Fax: ⫹81-55-981-6789.
E-mail address: nsaitou@genes.nig.ac.jp (N. Saitou).
0888-7543/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved.
doi:10.1016/S0888-7543(03)00174-5

region of interest is not so large that one fosmid clone cancover it. Second, because of its relatively small insert size,
DNA extracted from a dead animal can be used. In fact, weused a liver sample stored in ethanol in room temperature inthis study.
Fosmid clones are similar in size to cosmids due to theirreliance on phage packaging for their introduction into bacteria, but they contain replicons derived from the F factor
for DNA replication and segregation. Because of this, they
are more stable than cosmids [10,11] and are suitable for
rapidly creating genomic or chromosome-specific libraries[12,13]. We report here the construction of a gorilla genome
fosmid DNA library and establishment of an efficient PCRscreening system.
The genomic DNA was prepared from liver tissue of adead female gorilla (“Taiko”), donated from the Ueno Zoo(Tokyo). The liver tissue was immediately put into 99%
ethanol and stored until use to prevent degradation of DNA
at room temperature. The genomic DNA was extracted from500 mg liver tissue using the QIAamp Tissue Kit (Qiagen).
The pKS143 fosmid vector was prepared in a manner similar to that of the pFOS1 vector [11]. This vector wasdeveloped by one of us (A.F.) and has been used for humanchromosome 21 genomic sequencing [14] as well as for
chimpanzee genome study [2]. Construction of this fosmid

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vector was performed according to the procedure for double
cos cosmids [10,15]. To generate two arms, the plasmids
were successively treated with BamHI, alkaline phosphatase, and HpaI. DNA molecules were then fragmented andblunt-ended before the ligation with pKS143 vector arms.
Fresh cells (Escherichia coli XL1-Blue) were cultured ina 50-ml flask containing 10 ml medium (NZY⫹ maltose⫹
Mg2⫹) at 37°C with shaking until the OD600 reached 0.7.
This took approximately 3–5 h at 150 rpm. Five millilitersof the cell suspension was transferred to a 15-ml tube, andwe performed centrifugation for harvesting cells for 15 min
at 3000 rpm, 30°C. The supernatant was discarded and 5 mlof 10 mM MgSO4 and 250 ␮l of the in vitro packaging
reaction mixture were added. The solution was carefully
mixed by tapping the tube bottom with a finger and thenincubated for 30 min at 25°C. It was transferred to a 50-ml
tube and suspended in 20 ml SOC, followed by a 35-min
incubation at 37°C with shaking at 150 rpm. We thenperformed centrifugation for 15 min at 3000 rpm, 30°C, andthe cells were resuspended in 6.4 ml SOC. Eight hundred
microliters of the cells was spread on 22 ⫻ 22-cm LB plates
containing 25 ␮g/ml ampicillin, and they were incubatedovernight at 37°C.
Well-separated colonies were picked and transferred into
individual wells of 384 microtiter plates containing 60 ␮l/well LB supplemented with 7.5% glycerol and 25 ␮g/ml
ampicillin using a Flexys colony picker (PBA Technology)
and then incubated overnight at 37°C. The plates werestored at ⫺80°C.
All 384-well plates containing gorilla fosmid clones
were replicated using MicroGrid (BioRobotics) and incubated overnight at 37°C. Each 384-well plate was reversed(using a U-bottom polypropylene plate (Iwaki) instead of
the 384-well plate cover) and centrifuged for 1 min at 1000
rpm, 4°C. These combined cells were carefully mixed andpurified using an automatic plasmid isolation system PI-100(Kurabo).
A total of 261,120 independent clones were isolated andarrayed to 680 ⫻ 384-well microtiter plates containing
LB–7.5% glycerol plus ampicillin. The plates were incubated overnight at 37°C, replicated, and stored at ⫺80°C.
To evaluate the average insert size in the library, 100 clones
were randomly selected from the gorilla fosmid library.
Fosmid clone DNA was then completely digested using
NotI, and the insert size was estimated by pulsed-field gelelectrophoresis. The insert size was more or less constant at
ca. 40 kb, as expected (data not shown). Therefore, the total
size of this gorilla fosmid library is estimated to be 10,445
Mb (⫽ 261,120 ⫻ 40 kb). The size of the gorilla genome
was estimated to be 102% of the human genome [16], which
was estimated to be 2800 Mb [3]. Our gorilla fosmid librarythus represents about 3.66 (10,445/(2800 ⫻ 1.02)) coverageof the gorilla genome.
We named this fosmid clone library GGFP, after “Gorilla gorilla genomic library constructed by using fosmid
vector pKS143.” This is the first gorilla fosmid library andit will become a useful resource for human and ape genomic
studies. This successful construction of a fosmid library also
paves the way for constructing clone libraries for endangered species, whose DNA is not easy to obtain.
We introduced a two-step PCR screening system for our
GGFP library. The first step is to use 384 fosmid clones in
one plate as the unit. DNA is extracted from the pool of all
384 clones and used as template. Several 96-well plate
PCRs are necessary for this step (Fig. 1A). The PCR is
performed for 35 cycles consisting of 94°C for 1 min, 60°C
for 1 min, and 72°C for 2 min.
It may be possible to pool further DNA of 10 or moreplates in a future modification. When a plate with positive
signal is found, the search is restricted to that plate, and thesecond step is to use DNA pooled from clones located in all
24 columns of the same row for the PCR template. At thesame time, DNA of this plate is pooled from clones locatedin all 16 rows of the same column for the PCR template. A
total of only 40 PCR reactions are necessary for this second
step (Fig. 1B). We can then identify positive clones efficiently. This kind of hierarchical screening system is also
used by various clone libraries [e.g., 10,17]. One advantage
of our system is that the unit of pooled DNA is always aclear physical entity (plate, row, and column), and they areeasy to handle.
The Hox A gene cluster is one of four Hox clusters inmammals and is known to establish the proper body patterning along the anterior–posterior axis [18]. It is of generalinterest to investigate the gorilla Hox A cluster as a comparative target with the human HOX A cluster. We thus
screened the Hox A gene cluster region to test the quality ofthis library. Five PCR primer pairs used in this study arelisted in Table 1. These primer sequences were based on thehuman BAC clone sequence (DDBJ/EMBL/GenBank International Nucleotide Sequence Database Accession No.
AC004080) containing the HOX A gene cluster genes located at human chromosome 7. Those primers were firsttested to obtain amplicons from the gorilla genomic DNA
used as template.
Table 1 shows the summary of the PCR screening results
for the five primer pairs. Because we were originally interested in obtaining any gorilla fosmid clones that cover the
Hox A region, the number of screened plates (hence clones)
varies for each primer pair. We calculated “the number offound clones/genome,” where 1 genome corresponds to2856 Mb or 71,400 fosmid clones. The average number of
positive clones found per gorilla genome was 0.89. Thisvalue is close to the expected number of clones detectable
per genome, namely 1.0. This shows that our gorilla fosmid
library GGFP well represents the 3.7⫻ coverage of thegorilla genome.
Using this screening method, a total of nine positive
clones were found, and they completely cover the gorilla
Hox A gene cluster region (Fig. 2). These nine positive
clones are GGFP-562J15, GGFP-367A20, GGFP-347D05,

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573

Fig. 1. A schematic illustration of our two-step PCR screening system. (A) The first step. The plates containing 384 independent fosmid clones were pooled
and purified, and then PCRs were performed in 96-well plates using target primers. (B) The second step. After a plate containing a positive clone(s) is found,row-by-row and column-by-column screenings follow by pooling clones from the same row and same column, respectively.

GGFP-175G7, GGFP-452O13, GGFP-012E07, GGFP210K06, GGFP-470N12, and GGFP-473C20. The tiling
map of positive clones shown in Fig. 2 was constructedusing fosmid-end sequencing of each clone. The end se-quencing procedures were the same as published [2]. The
seven clones that constitute a minimum tiling path are now
being used for Hox A region nucleotide sequence determination (Kitano et al., manuscript in preparation).

Table 1
PCR screening result of the Hox A gene cluster from the gorilla fosmid library GGFP
Primer pair (forward/reverse)

No. positive
clonesNo. screened plates(No. screened clones)

No. found clones/
genome(1) GATATAGCGAAAGGCGAGTC/CAGAAACTCAGTGTGGTACTT(2) TTTATTCTTAGCACATGGCT/TTCTCTTCTCCTGCTCCGTT(3) GAGCTGACTGTAACTGACTC/AATTCGCCTTTGTCTCCTGCGG(4) TTAACTAGTGGTTTTCAGTTTG/ACGTGGTCTCCCATCCCTCGG(5) ATCGCAGTTTTCTGTCTGGG/GAGCCACCCTCAGGAATGTA
Total

1
2
3
4
3
13a

672 (258,048)384 (147,456)
672 (258,048)576 (221,184)
384 (147,456)
2688 (1,032,192)

0.28
0.97
0.83
1.29
1.45
0.89

a

Some fosmid clones were found more than once by using different PCR primers (see Fig. 2).

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C.-G. Kim et al. / Genomics 82 (2003) 571–575

Fig. 2. The genomic map of the nine gorilla fosmid clones that cover the Hox A cluster region. The information on the human BAC clone was from theEnsembl genome server (http://www.ensembl.org/Homo_sapiens/). The position of each clone was determined by fosmid-end sequencing using the T3 andT7 primer sets. The clone names are represented by library name, plate number, and well position. For example, GGFP-562J15 stands for GGFP library plate
562, well J15. The seven shaded fosmid clones constitute the minimum tiling path. The triangles with numbers indicate primer pairs used for screening listed
in Table 1.

We additionally screened four genes to test the genomewide coverage of this gorilla fosmid library. The names ofthese genes and their chromosomal locations in the humangenome are the ABO blood group gene at chromosome 9 (twoclones using one PCR primer pair), the Rh blood group gene at
chromosome 1 (nine clones using two PCR primer pairs), thealcohol dehydrogenase gene at chromosome 4 (three clones
using two PCR primer pairs), and HERV at various chromosomes (seven clones using two PCR primer pairs). We foundmultiple positive clones in all cases. This is more evidence that
our fosmid library GGFP covers the gorilla genome quite well.
We hope this newly constructed gorilla fosmid librarywill become a useful resource for human and ape comparative genomic studies. Our library GGFP is available for
PCR screening upon request.
Acknowledgments
We are grateful to Ueno Zoo, Tokyo, for providing us withdead gorilla tissue. We appreciate Makoto Shimada, Akiko
Aoshima, Atsuko Ide, Yoshimi Noaki, Masayo Kondo, andReiko Noda of Saitou Lab and Yong-Sik Bong and Ayuko
Motoyama of Fujiyama Lab, for their skillful technical assistance. We also thank Atsushi Toyoda of RIKEN Genome
Science Center for providing us the fosmid-end sequencing
protocol and Aya Takahashi, Takashi Kitano, and Kenta Sumiyama of Saitou Lab for comments on the manuscript. This
work was supported by Grants-in-Aid for Scientific Research
on Priority Areas from the Ministry of Education, Culture,
Sport, Science, and Technology of Japan to N.S. and A.F. anda National Institute of Genetics COE Fellowship for visiting
foreign researcher to C.K.
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