Gene 370 (2006) 34 – 42
www.elsevier.com/locate/gene

Human Endogenous Retrovirus (HERV)-R family in primates:
Chromosomal location, gene expression, and evolution
Heui-Soo Kim a,b,⁎, Joo-Mi Yi a , Hirohisa Hirai c , Jae-Won Huh a , Mi-Suk Jeong d , Se-Bok Jang d ,Choong-Gon Kim e , Naruya Saitou f , Byung-Hwa Hyun g , Won-Ho Lee aa

Division of Biological Sciences, College of Natural Sciences, Pusan National University, Busan 609-735, Koreab
PBBRC, Interdisciplinary Program of Bioinformatics, Pusan National University, Busan 609-735, Koreac
Department of Cellular and Molecular Biology, Primate Research Institute, Kyoto University, Inuyama, Aichi 484-8506, Japand
Korea Nanobiotechnology Center, Pusan National University, Busan 609-735, Korea
eMarine Living Resources Research Division, Korea Ocean Research and Development Institute, Ansan 426-744, Korea
f
Division of Population Genetics, National Institute of Genetics, Mishima 411-8540, Japang
National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Korea
Received 19 July 2005; received in revised form 31 October 2005; accepted 2 November 2005
Available online 27 January 2006
Received by T. Sekiya

Abstract
Hitherto, full-length endogenous retrovirus (HERV)-R has been located at human chromosome 7q11.2, and mRNA and envelope proteins havebeen detected in placenta and a variety of other cell types. In the present study, using a probe derived from the gorilla fosmid library, we detected
the paralogous locus (7q31.3) of the HERV-R env gene in human chromosome 7q11.2, and also determined the chromosomal location in apes and
Old World monkeys. The HERV-R gene was not detected in New World monkeys or prosimians with FISH and PCR analyses. We determined thesequences of the HERV-R env genes obtained from the genomic DNA of primates using PCR and sequencing tools. Except for a HERV-R env
sequence derived from gorilla DNA, the functional domains of putative envelope proteins are conserved, suggesting that those domains could
have a functional capacity in the primate genome. In addition, we investigated the env gene expression of HERV-R in various human tissues andcancer cells. An RT-PCR approach indicated that the env gene was expressed in several human tissues (brain, prostate, testis, kidney, placenta,
thymus, and uterus) and cancer cells (RT4, BT-474, MCF7, OVCAR-3, LOX-IMVI, and AZ521). Taken together, our data could be of great use
for understanding the evolutionary dynamics of HERV-R through primate radiation as well as the implications of its functional role in humantissues and cancers cells.
© 2005 Elsevier B.V. All rights reserved.
Keywords: HERV-R; Primates; FISH; Expression; Human tissues; Cancer cells

1. Introduction
Some of the human endogenous retroviruses (HERVs) havebeen fixed in the human population, and have also been inherited as stable genomic components, through generations in theevolution of species. Approximately 8% of the entire humanAbbreviations: HERV-R, human endogenous retrovirus R; FISH, Fluorescence in Situ Hybridization.
⁎ Corresponding author. Division of Biological Sciences, College of Natural
Sciences, Pusan National University, Busan 609-735, Korea. Tel.: +82 51 510
2259; fax: +82 51 581 2962.
E-mail address: khs307@pusan.ac.kr (H.-S. Kim).
0378-1119/$ - see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.gene.2005.11.008

genome consists of sequences of retroviral origin including
retrotransposons (International Human Genome Sequencing
Consortium, 2001). They are thought to be remnants of ancientgerm line infections by exogenous retroviruses. Most HERVs
seem to have entered the genome between 10 and 50 million
years ago, and they comprise over 200 distinct groups andsubgroups (Jurka, 2000). They can be classified into over 22
HERV families, containing various types from a single copy to
one thousand copies (Tristem, 2000). Previously, phylogenetic
significance and evolutionary studies have suggested that
HERVs inserted into the genome mostly early in primate evolution (Costas, 2001; Yi and Kim, 2004). Most of them wereH.-S. Kim et al. / Gene 370 (2006) 34–42

highly defective with large deletions, stop codons, and frameshifts in the open reading frames (ORFs). However, structuralgenes from some HERV families are expressed preferentially inhuman placenta (Mi et al., 2000; Venables et al., 1995) andseveral cancer cell lines (Armbruester et al., 2002; Yi et al.,2004). This is growing evidence that HERVs could be involved
in important cellular processes such as gene regulation, differentiation, immuno-modulation, cell fusion, and tumorigenesis(Krieg and Steinberg, 1990; Larsson and Andersson, 1998).
Expression of HERVs can influence the outcome of infections
in different ways that can be either beneficial or detrimental tothe host. The multiple-copy families scattered throughout the
genome have been reported to have regulatory functions on thegene expression of nearby located genes (Akopov et al., 1998).
Full-length retroviral sequences might interact with cellular
oncogenes (Varmus, 1982), and retroviral long terminal repeat(LTR) sequences have the capacity to exert a regulatory influence as promoters and enhancers of cellular genes (Leib-Moschet al., 1993). Some HERVs have been implicated in certain
autoimmune diseases, psychiatric diseases, and cancers. Furthermore, they might have a role in disease etiology and pathology (Nelson et al., 1999; Karlsson et al., 2001).
HERV-R (ERV3) was identified by low-stringency hybridization to be derived from the pol region of the chimpanzee
endogenous provirus CH2 together with the long terminal
repeat (LTR) of the baboon endogenous retrovirus (O'Connell
et al., 1984). HERV-R has inserted into the genomes of all great
apes and Old World monkeys during primate evolution (Shih etal., 1991). In addition, HERV-R is a provirus containing the poland env genes and a 3′ LTR. It is present as a single copy
located on chromosome 7, and conserves a full-length sequenceof Type C retrovirus containing a long ORF with mRNA
expression in the env region (O'Connell and Cohen, 1984;
O'Connell et al., 1984; Cohen et al., 1985). Based on the
HERV-R expression, a HERV-R proviral DNA clone was isolated from a human fetal liver library, and demonstrated that
HERV-R mRNAs of 9, 7.3, and 3.5 kb are expressed abundantly in the placental chorionic villi throughout gestation (O'Connell et al., 1984; Kato et al., 1987). Levels of HERV-R mRNA
expression for the env region differ among tissues and different
individuals (Kato et al., 1987; Cohen et al., 1988).
A large number of endogenous retroviral sequences arepresent in the human genome and some of them have beenshown to influence the regulation of normal cellular genes; theERV9 and ERV3 have altered the expression of Krüppel-related
zinc finger genes (Di Cristofano et al., 1995; Kato et al., 1990).
H-plk (Human-proviral-linked Krüppel) fused with HERV-R
was strongly expressed in the placenta. The activation of Hplk could be due to insertion of the HERV-R upstream of thegene (Abrink et al., 1998). Recently, tissue-specific expressionof two HERV transcripts (HERV-K cORF/rec and HERV-R
env) was found in human normal tissues (kidney, tongue,
heart, liver, central nervous system) during embryogenesis,suggesting a possible role in the development and differentiation of human tissues (Andersson et al., 2002).
In the present study, based on the chromosomal localizationof HERV-R on 7q11.2 by radiation hybrid mapping (Kim et al.,352000), we detected and identified the paralogous locus of
HERV-R on human 7q31.3 and that of various primate species.
To understand the molecular and evolutionary features of
HERV-R, its sequences and expression were analyzed in relation to functional capacity in various primate genomes, humantissues, and cancer cells.
2. Materials and methods
2.1. Isolation of genomic DNAs from primates and PCRamplification
Genomic DNA segments were isolated from heparinizedblood samples of the following species using a standard protocol: hominoid primates; human (Homo sapiens), chimpanzee(Pan troglodytes), bonobo (Pan paniscus), gorilla (Gorilla
gorilla), orangutan (Pongo pygmaeus), and gibbon (Hylobates
agilis); Old World monkey, Japanese monkey (Macaca fuscata), New World monkeys, night monkey (Aotus trivirgatus),
and common marmoset (Callithrix jacchus); prosimian, ringtailed lemur (Lemur catta). The genomic DNA samples were
subjected to PCR amplification. The env fragments of the
HERV-R were amplified by the primer pairs HS41 (5′GCCTTTACAGACCCAGTAGG-3′, bases 1741-1760) andDS10 (5′-AGGGCCATTAGTCGTGTGGT-3′ bases 26712690) from HERV-R (GenBank, accession no. M12140). The
PCR conditions were performed as previously described(Kim et al., 1996) with a modified annealing temperature of
56 °C.
2.2. Cell culture and total RNA isolation
Human cancer cells (RT4, PFSK-1, BT-474, HCT-116, TE1, UO-31, Jurkat, HepG2, A549, MCF7, OVCAR-3, MIAPaCa-2, PC3, LOX-IMVI, AZ521, 2F7, U-937, C-33A) weregrown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum, 2 mM
glutamine, 1 mM nonessential amino acids, 1 mM sodium
pyruvate, 100 U/ml penicillin, 0.1 mg/ml streptomycin at
37 °C, and 5% CO2 incubator. Total RNA from human cancer
cells was extracted by High Pure RNA isolation kit (Roche).
Total RNA from human tissues (brain, prostate, testis, heart,kidney, liver, lung, placenta, skeletal muscle, spleen, thymus,
uterus) was purchased from Roche company.
2.3. Molecular cloning of PCR products
The PCR products were separated on a 1.5% agarose gel,purified with the QIAEX II gel extraction kit (Qiagen) andcloned into the pGEM T-easy vector (Promega). The cloned
products were isolated by the alkali lysis method using the highpure plasmid isolation kit (Roche).
2.4. RT-PCR analysis for HERV-R Env gene
Using the pure 1 ug/ul mRNA only, expression patterns of
the HERV-R env gene were examined by the Titan One Tube

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H.-S. Kim et al. / Gene 370 (2006) 34–42

RT-PCR System (Roche). We performed PCR amplification
without reverse transcription reaction with pure mRNA samples, indicating that the prepared mRNA samples from thehuman tissues and cancer cells did not contain the genomic
DNA. Using the RT-PCR approach, the HERV-R env gene wasamplified by the same primer pairs HS41 and DS10. The RTPCR conditions followed were standard protocol of Titan One
Tube RT-PCR System with an annealing temperature of 56 °C.
As a standard control, G3PDH was amplified by the primers
GPH-S (5′-CAAAGTTGTCATGGATGACC-3′, bases 3172131740) and GPH-AS (5′-CCATGGAGAAGGCTGGGG-3′,
bases 31898-31915) from the human G3PDH (GenBank accession no. AC068657).
2.5. In vitro transcription and translation assay
The HERV-R gene encoding residues 270-834 was amplified
by PCR technique with forward (5′-CCGGAATTCATGACTAAAACCCTGTTGTATCAC-3′) and reverse (5′-CCGCTCGAGCTATCCTTTCCAAGTCTGAACTG-3′) primers fromhuman clone (R-HSenv37). The PCR product was purified,digested with EcoR I and Xho I and then ligated into thepGEX-4T1. E. coli strain DH5α was used for cloning andmaintaining the constructs. The resulting vector was introduced
into E. coli BL21(DE3) and BL21(DE3)RIL strain. The expressions of GST-HERV-R fusion proteins were introduced by
1 mM isopropyl-D-thiogalacto pyranoside (IPTG) at opticaldensities of 0.5–0.6 at 37 °C for 5 h and at 16 °C for 16 h.
Bacterial lysates were prepared by sonication in buffer A(50 mM Tris-HCl pH 7.5 and 200 mM NaCl). The supernatantand pellet were used in SDS-PAGE analyses to determine thesolubilities of the expressed proteins.
2.6. Fluorescence in situ hybridization (FISH) of HERV-R Env
gene in primates
Fosmid clone GGP42M6 derived from gorilla genomic
DNA (17.5 μg/μl) was denatured with boiling for 5 min, andlabeled with BioPrime DNA labeling system containing biotin14-dCTP as a hapten (Invitrogen). For DNA precipitation afterlabeling, a total 740 μl mixture of 50 μl the labeled probe and690 μl containing 465 μl 99.5% cold (− 20 °C) ethanol, 0.5 M
NaCl and 19 μg salmon testes DNA was centrifuged at 21,500 g
after keeping at 4 °C for 60 min. The labeled probe DNA pellet
was saturated with 50 μl formamide. Hybridization solution
was made with 1 μl probe DNA (350 ng), 8 μl formamide,
1 μl cot I DNA (10 μg), an 15 μl hybridization buffer (3 : 1 30%
dextran sulfate : 20 × SSC), then was denatured at 72 °C for
10 min before starting hybridization. The chromosome DNA
was denatured with 0.05 M NaOH (pH 12.5) in 2 × SSC for
4.5 min, followed by dehydration with 70% and 99.5% ethanol for 5 min each. After drying, the denatured probe DNA
was applied onto the denatured chromosomes slide preparation. The slide covered with parafilm (cut to size) was incubated in a moist chamber at 37 °C for 12–16 h. Signals weredetected with FITC-avidin DCS (Vector) after washing adequately and observed recorded under a fluorescence micro-

scope (Axioplan 2, Zeiss) mounted CCD camera connected toa personal computer (Apple G4) running the IPLab imaging
software (Scanalytics, Inc.). These procedures were modified apreviously described technique for BAC DNA clones (Hirai
and Hirai, 2004). To identify human chromosome 7, a painting
probe of biotin-labeled human chromosome 7 (Cambio) wasused according to a manufacture's protocol, and the signals
were detected with avidin-Rhodamin (Leinco Technologies,Inc.) or avidin-FITC (Vector).
2.7. DNA sequencing and data analyses
Individual plasmid DNA was digested by restriction enzyme
EcoRI. Positive samples were subjected to sequence analyses
on both strands with T7 and M13 reverse primers using an
automated DNA sequencer (Model 373A) and the DyeDeoxy
terminator kit (Applied Biosystems). Nucleotide sequence analyses were performed using the GAP, PILEUP, and PRETTY
from the GCG software (University of Wisconsin) and MEGA2
program (Kumar et al., 2001).
2.8. Nucleotide sequence accession numbers
The nucleotide sequence data reported in this paper appear in
the DDBJ/EMBL/GenBank nucleotide sequence databases withthe accession numbers: CH (AB198934), GO (AB198935), OR(AB198936), GI (AB198937), JM (AB198938).
3. Results
3.1. Structure and chromosomal location of HERV-R family
HERV-R (ERV3) is a provirus containing the pol, env genes
and a 3′ LTR (accession no. M12140). We determined thedetailed structure of the HERV-R using a protein domain searchby functional domain information from the Pfam HMM database (Fig. 1A). In our previous study, HERV-R (M12140) was
been mapped on the human chromosome 7q11.2 using radiation hybrid mapping (Fig. 1B; Kim et al., 2000). In order toconfirm the presence of the HERV-R env fragment, we performed PCR amplification using genomic DNAs from various
primates. The PCR products appeared in the samples of apes
and Old World monkeys, but not in those of New World
monkeys or a prosimian, indicating that the HERV-R family
was integrated into the primate genome before the divergenceof apes and Old World monkeys (Fig. 2A). Each of the PCR
products from the genomic DNA of the various primates wascloned and sequenced. They showed a high sequence similarity(95.7–98.6%) to that of HERV-R (M12140), except for thesequences derived from gorilla genomic DNA. These
sequences showed a low similarity (76.9%) to that of human
DNA (Table 1). The gorilla samples contained a 35 bp insertion, unlike those of the other primates. Likewise, five different
individuals among the gorilla samples exhibited such an insertion event (Fig. 2B). This information motivated us to investigate the evolutional implications of HERV-R for primate
evolution.

H.-S. Kim et al. / Gene 370 (2006) 34–42

37

Fig. 1. (A) Putative structure of the HERV-R family (M12140) and (B) chromosomal location of the HERV-R family.

Kim et al. (2003) have constructed and characterized afosmid clone library from gorilla DNA with a PCR screening
method, suggesting that it is useful for comparative genomics
of human and apes. Based on this system, one fosmid clone(GGP42M6) from gorilla genomic DNA was identified. We
performed a FISH analysis to determine the chromosomal
location of the HERV-R element in various primate species(chimpanzee, gorilla, orangutan, gibbon, Japanese monkey,

New World monkey, and prosimian), including humans. The
clone GGP42M6 (50 kb in length) containing the HERV-R
element was located in the bottom region of human chromosome 7q31.3 (Fig. 3a, b). A more exact location might be
7q31.32–33. This location of HERV-R, newly detected at chromosome 7q31.3, is the paralogous locus of human chromosome
7q11.2 mapped by radiation hybrid mapping (Kim et al., 2000).
The localization was limited to the orthologous locus in all
species used here, that is, the long arm of chromosome 6 ofchimpanzees (Fig. 3c, d, e) and gorillas (Fig. 3f, g), chromosome 10 of orangutans (Fig. 3h, i), chromosome 1 of gibbons(Fig. 3j, k, l, m), and chromosome 2 of macaques (Fig. 3n, o, p)(Yunis and Prakash, 1982; Weber et al., 1986; Jauch et al.,
1992; Wienberg et al., 1992). Because representatives of the
New World monkeys (night monkey, marmoset, and lemur) andprosimians were not hybridized with the probe, the HERV-R
element seemingly was inserted into the genomic site of theapes and Old World monkeys lineage approximately 30 million
years ago.
Table 1
Percentage similarity of nucleotide sequences of env fragments

Fig. 2. (A) PCR analysis of genomic DNAs for the presence of the env geneof the HERV-R family in primates and (B) five different individuals ofgorilla.

HERV-R

1

2

3

4

5

6

1. HU
2. CH
3. GO
4. OR
5. GI
6. JM

−
98.6
76.9
97.3
96.6
95.7

−
77.0
97.8
97.4
96.3

−
76.1
76.5
76.3

−
97.0
96.2

−
96.3

−

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H.-S. Kim et al. / Gene 370 (2006) 34–42

Fig. 3. FISH mapping with a fosmid clone GGP42M6 derived from gorilla genomic DNA on metaphase chromosomes of human (a, b), chimpanzee (c–e), gorilla(f, g), orangutan (h, i), gibbon (j–m), and Japanese monkey (n–p). Some species were checked homologous chromosome with human using a painting probe ofhuman chromosome 7 (pink or green) (e, m, p). Arrowheads indicate locations of signal. The location corresponds to human 7q31.3 (32–33). Scale is 10 μm. (Forinterpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.2. Identification and sequence analysis of HERV-R env genein primates
A full-length endogenous provirus was localized on humanchromosome 7q11.2 (O'Connell et al., 1984; Kim et al.,2000). We determined the nucleotide sequences of the
HERV-R env fragments in various primates (human, chimpanzee, gorilla, orangutan, gibbon, and Japanese monkey). The
env sequences showed a high degree of sequence similarity(95.7–98.6%) to that of human HERV-R (M12140) except for
the env sequences (76.9%) from gorilla DNA (Table 1).
Amino acid sequences of the env fragments of the HERV-R
from the various primates were aligned and analyzed (Fig. 4).
The putative amino acid sequences of the env fragments
indicated no translation interruptions by point mutations ordeletion/insertions in any of the clones identified in thisstudy with the exception of the env sequences from gorilla.
The percentage identity of the 246 amino acid sequences ofthe HERV-R env fragments ranged from 95.9% to 97.6%
among primates (Table 2A). In order to discover the evolutionary forces at work, we also analyzed synonymous andnon-synonymous substitutions within the env fragments of
HERV-R. As shown in Table 2B, the mean synonymous
substitution (Ks) ranged from 0.8% to 7.9%, whereas themean non-synonymous substitution (Ka) ranged from 1.6%
to 3.1%. In Ka / Ks ratio, most of the values in pairwise com-

parisons were b1. The data indicates that negative selection
has been acting on these sequences of the HERV-R family
during primate radiation.
3.3. HERV-R Env gene expression in human tissues and cancercells
The expression pattern of the HERV-R env gene in twelve
human normal tissues and eighteen human cancer cell lines was
examined using an RT-PCR approach. The HERV-R env gene
was expressed in brain, prostate, testis, kidney, placenta, thymus and uterus of the human normal tissues (Fig. 5A). In thecase of the cancer cells, the expression pattern was only
detected in the following cell lines: RT4, BT-474, MCF7,OVCAR-3, LOX-IMVI, and AZ521 (Fig. 5B). The RT-PCR
products were cloned and sequenced (data not shown), indicating that all of the sequences identified from human tissues andcancer cells originated from HERV-R (M12140) on humanchromosome 7q11.2 by primer specificity.
3.4. In vitro transcription and translation analysis of HERV-R
Env gene
Active HERVs can serve multiple functions including
early placental differentiation, fetal development, and immunosuppression. Along with these functions, a possible role forH.-S. Kim et al. / Gene 370 (2006) 34–42

39

Table 2B
Synonymous substitutions per site (Ks), nonsynonymous substitutions per site(Ka) and their ratio (Ka / Ks) in env fragments
HERV-R

Ks

Ka

Ka / Ks

HU and CH
HU and OR
HU and GI
HU and JM
CH and OR
CH and GI
CH and JM
OR and GI
OR and JM
GI and JM

0.8
5.2
4.6
6.2
4.4
3.8
5.4
6.2
7.9
5.8

1.6
2.0
3.1
3.0
2.0
2.6
3.0
2.6
2.5
2.8

2.00
0.39
0.67
0.48
0.46
0.69
0.55
0.42
0.32
0.48

fusion systems were high, but the fusion proteins were in
insoluble forms.
4. Discussion

Fig. 4. Amino acid sequence alignments of the HERV-R env fragments could
encode functional proteins without nonsense or frameshift mutations. Consensus sequences are shown on the top row. Dashes indicate residues identical tothe consensus sequences and dot indicates gap introduced to maximize thealignments. ⁎, ⁎⁎, and ⁎⁎⁎ indicate the SU/TM cleavage signal, immunosuppressive domain CKS-17 peptide, and cysteine loop, respectively.

HERV-R in the differentiation of syncytiotrophoblast and U937 cells has been proposed (Larsson et al., 1996). Therefore,
we examined in vitro transcription/translation assays using
HERV-R env derived from human clone (R-HSenv37),
which has only one transition mutation compared to theM12140 sequences. In order to compare the expression efficiencies, two kinds of E. coli strains (BL21(DE3) and BL21
(DE3)RIL) and a fusion system (glutathione S-transferase[GST]) were used. An SDS-PAGE analysis of the recombinant clones containing all of the two plasmids indicated highlevels of production of the recombinant proteins after induction with 1 mM IPTG at 37 °C for 5 h and at 16 °C for 16 h,
respectively. The recombinant proteins were found to be
insoluble, since they were detected in the insoluble fractionsof the E. coli sonicate (Fig. 6). Growth temperature and E.
coli strains did not affect the expression level or the solubilityof fusion proteins. As a result of a comparison of the expression level and the solubility, the expression rate for GST

Most HERVs entered into the genome between 10 and
50 million years ago during primate evolution (Jurka, 2000).
HERV-R, one of the HERV-ERI supergroup, was integrated
into the primate genomes before the divergence of apes and
Old World monkeys about 30 million years ago (O'Connell etal., 1984; Shih et al., 1991). Ten members of HERV-R including related sequences have been reported (Tristem, 2000). The
exact location, expression, and ORF sequences of the structuralgenes of the HERV-R provirus in primate species are notknown. In the present study, using the probe GGP42M6 derived
from the gorilla fosmid library (Kim et al., 2003), we determined the chromosomal location of HERV-R on 7q31.3 inhuman DNA. It seems to be proliferated by duplication event,suggesting that the HERV-R on 7q31.3 is paralogous to humanchromosome 7q11.2, mapped previously by radiation hybrid

Table 2A
Percentage identity of 246-amino-acid sequences of env fragments
HERV-R

1

2

4

5

6

1. HU
2. CH
3. OR
4. GI
5. JM

−
97.6
97.2
95.9
96.3

−
97.2
95.9
96.3

−
96.3
96.3

−
97.2

−

Fig. 5. Expression analysis of HERV-R env gene from human tissues (A) andcancer cells (B) by RT-PCR approach.

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H.-S. Kim et al. / Gene 370 (2006) 34–42

Fig. 6. SDS-PAGE analysis of the expressed proteins in E. coli. (A) Expressed GST-HERV-R fusion protein in E. coli BL21(DE3) at 37 °C. (B) Expressed GSTHERV-R fusion protein in E. coli BL21(DE3)RIL. (C) Expressed GST-HERV-R fusion protein in E. coli BL21(DE3) at 16 °C. Lane 1, molecular weight markers;
Lane 2, supernatant of lysed bacteria before induction by IPTG; Lane 3, supernatant after induction by IPTG; Lane 4, pellet of lysed bacteria before induction by
IPTG; Lane 5, pellet of after induction by IPTG.

mapping (Kim et al., 2000). HERV-R was clearly detected in
chimpanzee, gorilla, orangutan, gibbon, and Japanese monkey,
but not in New World monkeys or prosimian, by FISH approach. Taken together, HERV-R on the 7q31.3 and 7q11.2 lociof the human chromosome is conserved in the genomes of apes
and Old World monkeys, as shown in previous data (O'Connell
et al., 1984; Shih et al., 1991).
The HERV-R env sequences on the 7q11.2 locus were analyzed by PCR amplification and sequencing from genomic
DNA of apes and Old World monkeys, but not from New
World monkeys and prosimians, considering an integration
event more than approximately 30 million years ago with asubsequent changing in gorillas (Steiper et al., 2004; FukamiKobayashi et al., 2005; see also Figs. 2 and 3). We identified
and analyzed the HERV-R env sequences derived from chimpanzee, orangutan, gibbon, and Japanese monkey DNA. In thecase of gorilla DNA, the HERV-R env sequences showed a
76.9% similarity to 35 bp insertion compared to humans. This
fact implies that the change of HERV-R in gorilla DNA can be
explained by the rearrangement or translocation after duplication of the HERV-R family during primate evolution. Most of
the sequences derived from the various primates showed higher
values for synonymous substitutions than for non-synonymous
substitutions in each of the comparisons (Table 2B), suggestingthat the HERV-R env sequences might have an important biological function in primates. As shown in Fig. 4, the putative
amino acid sequences of the HERV-R env fragments arewell conserved during primate evolution. Interestingly, several functional regions of the protein showed high conservation in primates with only a few changes. These include theimmunosuppressive domain, the CKS-17 peptide of mammalian type C retroviruses (amino acids 190 to 206), the cysteinloop (amino acids 207 to 216), and the sequence related to the
SU/TM cleavage signal (amino acids 108 to 113). Taken together, these HERV-R env gene sequences could be associatedwith an active provirus in primates.
Several HERVs are also highly expressed in steroid-dependent organs such as the placenta, the adrenals, the fetal primitive adrenal cortex, the corpus luteum, the testis and thesebaceous glands. HERV-R is expressed in most human tissues,and its expression is usually found to be low by comparison tothat in human placenta, using Northern blot analysis. Elevatedexpression of HERV-R has recently been reported in sebaceous
glands (Andersson et al., 1996). In contrast, the level of HERVR expression in choriocarcinoma cell lines is extremely low(Kato et al., 1987, 1988). In a different case, the HERV-K env
gene was expressed in many breast cancer tissues and cell lines,
but no expression was detected in normal breast tissues, suggesting that HERV-K env expression could be a tumor marker(Wang-Johanning et al., 2003). In a comparative analysis between normal tissues and cancer cells, the HERV-R env gene
was expressed in brain, prostate, and kidney tissues (Fig. 5A),
whereas no expression appeared in PFSK-1, PC3, or UO-31
cancer cells derived from brain, prostate, or kidney tissues,
respectively (Fig. 5B). Tissue-specific expression of two
HERVs transcripts (HERV-K cORF/rec and HERV-R env)
was found in human normal tissues (kidney, tongue, heart,
liver, central nervous system) during embryogenesis, suggesting a possible role in the development and differentiation ofhuman tissues (Andersson et al., 2002).
Until now, there has been no clear evidence of the functionalroles of HERV-R. There is growing body of evidence that
HERV-R might be involved in normal cellular processes. For
instance, identification of specific demethylation of the ERV3H-plk gene in human cell lines and expression levels of thevarious H-plk transcripts have been reported (Abrink et al.,1998). It has been found, by investigation of the cellular mechanism, that HERV-R env expression affects the expression of βhCG and cell proliferation (Lin et al., 2000). It has beenstrongly suggested that enhancer and promoter elements inretroviral LTRs influence the transcription of neighboring
genes (Kowalski et al., 1999). This can result in transcriptional
activation or gene silencing as well as in changes of tissue
specificity expression (Ting et al., 1992; Schulte et al., 1996).
In addition, the biological role of HERV-R in vivo has beenstudied using established transgenic rats carrying a full sequence of HERV-R under control of its own LTR promoter(Tanaka et al., 2003).
In this paper, we describe dynamic evolutionary features ofthe HERV-R family. Their putative amino acids without any
mutations such as insertion or deletion in primates and in vitrotranscription and translation data, suggest the important biological role of the HERV-R family. Accordingly, the relation of the
HERV-R env gene to neighboring genes in the genome and toH.-S. Kim et al. / Gene 370 (2006) 34–42

gene function related to human diseases could be the focus offurther research.
Acknowledgments
We thank Yuriko Hirai for her technical assistance for FISH
and chromosome painting analyses. This research was supported by the KRIBB Research Initiative Program and by the
Grant for the Biodiversity Research of the 21st Century COE(A14).
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