 1996 Oxford University Press

Human Molecular Genetics, 1996, Vol. 5, No. 1

23–32

ARTICLE

Human pseudoautosomal boundary-like sequences:
expression and involvement in evolutionary
formation of the present-day pseudoautosomal
boundary of human sex chromosomes
Tatsuo Fukagawa, Yasukazu Nakamura, Katsuzumi Okumura1, Masahiro Nogami1,
Asako Ando2, Hidetoshi Inoko2, Naruya Saitou and Toshimichi Ikemura*
Department of Evolutionary Genetics, National Institute of Genetics, and The Graduate University for Advanced
Studies, Mishima, Shizuoka-ken 411, Japan, 1Faculty of Bioresources, Mie University, Tsu, Mie-ken 514, Japanand 2School of Medicine, Tokai University, Bohseidai, Isehara, Kanagawa-ken 259-11, Japan
Received August 23, 1995; Revised and Accepted October 10, 1995

The human genome is composed of long-range mosaic structures of G+C% (GC%), which are thought to berelated to chromosome bands. We previously identified a boundary of Mb-level domains of GC% mosaic
structures in the human major histocompatibility complex (MHC) and found in the domain boundary a sequence
very similar to pseudoautosomal boundary (PAB) sequences of human sex chromosomes. We designated it‘PABL’ and found many PABLs in the human genome. By analysis of six genomic and six transcribed PABLs, acore and consensus sequence of about 650 nt was defined; the 3′- and 5′-edges of the PABLs were strictlyconserved. Northern blot analysis showed sizes of PABL transcripts to be 5–10 kb in length. Divergence time of
PABLs was estimated to be 60–120 million years ago by analysis of human PABLs and PABXY1 of seven primates,
and the evolutionary rates deduced showed PABLs to have been under selective constraints. A model for
evolutionary formation of the present pseudoautosomal boundary was proposed by postulation of illegitimate
recombination between two PABLs.

INTRODUCTION
The human genome, like the genomes of warm-blooded vertebrates in general, has long-range G+C% (GC%) mosaic structures related to chromosome bands: Giemsa-dark G bands arecomposed mainly of AT-rich sequences, T bands (a subgroup of
Giemsa-pale R bands) are of GC-rich sequences, and ordinary R
bands are heterogeneous and appear to be intermediate (1–6).
Bernardi et al. called the GC% mosaic domains ‘isochores’ (1).
DNA replication timing, gene density, CpG island density, codon
usage, chromosome condensation, repeat sequence density, andchromosome behavior such as recombination are related tochromosome bands and to long-range GC% mosaic domains(5–12). Because chromosome bands are structures observed by
microscopes, precise location of their boundaries may seem
meaningless. However, considering the various genome beha-viors connected with chromosome bands, we may precisely
locate their boundaries by using informative landmarks on
genomic DNA. Boundaries may be structurally assigned where
there are clear GC% transition points, and characteristic signals
may be found that punctuate and/or differentiate distinct functions, such as a signal that switches from early to late DNA
replication. We previously reported a boundary of long-range
GC% mosaic domains in the human major histocompatibility
complex (MHC) disclosing a sharp GC% transition, and found in
the boundary region high sequence similarity with the pseudoautosomal boundary (PAB) sequence in the short arms of humansex chromosomes (13).
Human sex chromosomes have two functionally distinct
regions, sex-specific sequences and pseudoautosomal regions(PARs). X and Y chromosomes exchange DNA sequences
through homologous recombination in PARs during each male

*To whom correspondence should be addressed
Sequence data from this article have been deposited with the GenBank/EMBL/DDBJ Data Libraries under Accession Nos. D55638 (Bc4), D55639 (Mo1),
D55640 (Mo2), D55641 (Sk13), D55642 (PABLSp2), D55643 (Sp2), D55644 (Sp3), D63517 (PABL consensus sequence)24

Human Molecular Genetics, 1996, Vol. 5, No. 1

meiosis, and PAR sequences in the two chromosomes arepractically identical because of the obligatory recombination(14–17). There are two PARs for human sex chromosomes: PAR1
is at the distal ends of the short arms of the X and Y chromosomes
and PAR2 is at the ends of the long arms (17,18). The interface
between the PAR1 of 2.6 Mb and the sex-specific region is thepseudoautosomal boundary, PAB1, and therefore PAB1 is thecentromeric limit to recombination in PAR1. Ellis and Goodfellow (16) and Ellis et al. (19,20) reported sequences around theinterface, PABX1 and PABY1 sequences (abbreviated
PABXY1). A sequence found in the boundary of long-range
GC% mosaic domains in the MHC is very similar to PABXY1
being the functional interface in sex chromosomes. We designated the sequence found in the MHC ‘pseudoautosomal
boundary-like sequence 1’, abbreviated PABL1 (13). With the
PABL1 segment as a probe, many copies of pseudoautosomal
boundary-like sequences (PABLs) were detected by Southernblot hybridization against genomic DNAs. In the present study wecharacterized both genomic and transcribed PABLs.
RESULTS
Core sequences of genomic PABLs
Using the PABL1 segment found in the human MHC as a probe,
we isolated about 150 independent cosmid clones having PABLs.
Figure 1 shows alignment of genomic PABLs sequenced thus far
and PABXY1 plus their flanking sequences; the sequenceorientation for PABXY1 is from telomere to centromere (i.e.,from PAR1 to the sex-specific region). Similarity for all pairs of
PABLs and PABXY1 were high (∼80% nucleotide identity), andthe 3′-terminus of the similar region among all PABLs corresponds closely to the PABXY1 homology terminus reported by
Ellis et al. (19,20) as where the X and Y chromosome sequences
diverge completely (sex-specific in Fig. 2).
In the case of the 5′-terminus of PABXY1 sequences there wasno reason to assign a specific site because the upstream 2.6 Mb
sequence of PAR1 should be practically identical in sex
chromosomes. In fact, the 5′-terminus of PABXY1 sequences
reported (19) was an EcoRI site chosen arbitrarily in PAR1.
Fukagawa et al. (13) noted that, even if PABXY1 and PABLs
correspond to a certain functional signal, the 5′-end of therespective signal cannot be elucidated by comparison of the X andY sequences, and that only comparison with or among PABLs
will define the 5′-terminus of the hypothesized signal. Recently
Dr N. A. Ellis kindly supplied us with his unpublished PAR1
sequence upstream of the EcoRI site. PABXY1 in Figure 1
includes this sequence. Multiple alignments of Figure 1 showedthat sequence similarity among all PABLs and PABXY1 actuallyextended 200 nt upstream of the EcoRI site of PABXY1. We
propose that a total of 650 nt including the 200 nt extended is thecore form of PABLs. Figure 2 is a diagram of their structure. Strict
conservation of both termini for all PABLs and PABXY1 may be
due to their function.
The transcripts of PABLs
Although multiple copies of PABLs were found in the humangenome, genomic sequences in the recent GenBank (Release 87
including update data; 1995) exhibiting significant similarity
with the PABLs were confined to PABXY1. In the case of
expressed sequence tag (EST) sequences, however, eleven ESTs

were similar (∼80% nucleotide identity) to separate portions of
PABLs (Fig. 2), suggesting that some, if not all, PABLs aretranscribable and presumably have functions. For identificationof the characteristics of the predicted PABL transcripts, six
human cDNA libraries of different tissues and cells were screened
with the PABL1 segment as a probe. Clones were obtained fromall six libraries, and inserts of all 20 cDNA clones analyzed werelonger than the PABL size. We then sequenced the following six
cDNA clones: Mo1 and Mo2 from the monocyte library; Bc4
from the B-cell library; Sk13 from the skin library; and Sp2 andSp3 from the spleen library. Alignment around the 5′-terminal
portion of all available PABLs found in the cDNA clones
including ESTs (T92306 and R12279) is shown in Figure 3A. The
similarity terminus of the 5′-portion of these transcribed PABLs
corresponds closely to that defined for genomic PABLs. It should
first be noted that a portion of the Mo2 cDNA sequence waspractically identical to one of the ESTs mentioned above,
HSAAAAWAH. The identity portion spanned not only the PABL
sequence but also the entire flanking region of 266 nt
HSAAAAWAH, predicting the EST to correspond to a portion of
the Mo2 transcript and the transcript to be fairly abundant.
Sequences of three recently reported ESTs (R12279, R07404, andT99392, derived from at least two independent cDNA libraries)
were almost identical with each other, indicating the transcripts
are also abundant.
Figure 3B shows alignment around the 3′ portion of thetranscribed PABLs and one EST (T47905). Again, the similarity
terminus of these transcribed PABLs closely corresponds to thatdefined for genomic PABLs and thus to the homology terminus
between PABX1 and PABY1. Therefore, the results of Figures 1
and 3 showed conservation of both 5′- and 3′-termini of genomic
and transcribed PABLs. Open reading frames (ORFs) withsignificant sizes could not be found for the obtained cDNAs, forthe core sequences or their flanking sequences. We searched thesecDNA sequences, using the BLASTX program, against theprotein sequence database compiled by the Human Genome
Center of Japan. Little if any similarity with known proteins was
detected. GRAIL, a computer program that predicts protein-coding ORFs in human DNA (21), did not detect reliable proteincoding capacity. For identification of the genomic structures of
the transcribed PABLs, a λ-phage library of human genomic
DNA was screened with a probe of the Sp2 cDNA fragment
deprived of the PABL core. The genomic PABLSp2 sequence
shown in Figure 1 is derived from the phage clone thus isolated.
The sequences of the 5′-flank of about 50 nt and the 3′-flank ofabout 300 nt so far known for Sp2 cDNA were identical to thoseof the genomic PABLSp2 clone, showing that there were no
intron/exon structures around this PABL core.
Northern hybridization analysis
The six cDNAs of PABLs analyzed were longer than the PABL
size. For estimation of the intact sizes of PABL transcripts,
northern blot analysis of human total or polyA+ RNA fraction was
done with the PABL1 probe. Figure 4A shows the results for the
total RNA extracted from B-cell line GM01416D. Broad bands
mainly with mobility slower than that of 28S rRNA and estimatedto be 5–10 kb in length were detected. The results for the polyA+
RNA fractions were practically the same for total RNA (Fig. 4B).
All samples shown in Figure 4 gave signals, although expression
levels seemed to differ. The broad bands shown are likely to25
Human Acids
Molecular
Genetics,
Vol.No.
5, No.
Nucleic
Research,
1994,1996,
Vol. 22,
1 1

25

Figure 1. Comparison of genomic sequences of PABXY1 and PABLs. Multiple alignment of nucleotide sequences was performed with the MALIGN program. An
Alu sequence present in PABY1 was omitted in the alignment. Identical nucleotides are marked with asterisks, and positions where five of six nucleotides are identicalare marked with dots. PABXY1 sequences upstream of the EcoRI site were supplied by Dr Nathan A. Ellis. Arrows show termini of the proposed core unit of PABLs.

26

Human Molecular Genetics, 1996, Vol. 5, No. 1

Figure 2. Genomic structure of PABXY1 and PABLs. For both PABXY1 and PABLs, regions before and after the Alu-insertion site are marked separately. The coreunit of PABL (∼650 nt) is present in all sequences, and their flanking sequences are divergent. In the case of PABXY1, the 3′ flank constitutes the sex specific region.
EST sequences showing evident similarity with PABLs are shown by horizontal lines: GenBank loci HSAAAAWAH (accession no. Z19872), hbc671 (T11103),
HSGS04158 (D25790), and GenBank loci and accession nos. R07404, R12279, T99392, T47905, T92306, R38816, and R43643.

correspond to many transcripts hybridized with PABL sequences,and their length being long was consistent with the finding that the
PABL cDNAs were longer than the PABL size. For detection ofa single transcript corresponding to a unique cDNA, a Sp2 cDNA
fragment deprived of its PABL core was used as the hybridization
probe, and in this case a single sharp band of 7.5 kb was detected(Fig. 4C).
Phylogenetic relationships among PABLs and PABXY1
Obligatory pairing and crossover in the PAR ensure accurate
segregation of the sex chromosomes during male meiosis. An
unusually high rate of homologous recombination in PAR1
(20-fold the genome average) seems to arise from a mechanismthat promotes obligatory physical association and the successive
crossover between the sex chromosomes. The strict limit toterminate the high frequency recombination in PAR1 is thepseudoautosomal boundary 1 (PAB1). An Alu element is situatedin PAB1 of the human Y chromosome, PABY1, but not in PABX1(19). Ellis et al. defined first the Alu element as the strict boundaryof PAR1. A later study of the boundaries of PAR1 in Old World
monkeys, however, found no Alu repeats on the PABY1 (20). In
spite of the lack of the Alu element, they proposed the
Alu-insertion site itself as the strict boundary of PAR1 for thefollowing reasons. In pair-wise comparisons of the X and Y
boundary sequences within each species of several hominoids
and Old World monkeys, they found sequences about 220 nt long
downstream of the Alu insertion site to be more divergent between
sex chromosomes than the sequences of the Alu-upstream region(∼78% vs. ∼97% nucleotide identity; see Fig. 2 for human
PABXY1), whether or not the Alu element was present. By
extensive analysis of the nucleotide substitution patterns, they
concluded that the Alu-insertion site, which corresponds to theabrupt transition between the high- and reduced homology

regions, is the strict limit for high-frequency recombination inPAR1 and thus the exact boundary of PAR1 (20). The position of
the boundary in Old World monkeys and hominoids waspractically the same. This shows that the limit of PAR1
recombination was at the Alu-insertion site before divergence of
lineages of Old World monkeys and great apes, and that an Alu
element was inserted into the preexisting boundary of the Y
chromosome in the great ape lineage. As did Ellis et al., this site
is called the ‘Alu-insertion site’ in this paper whether or not the
Alu element is present; the high- and reduced-homology regions
are thus called the Alu-upstream and Alu-downstream regions,
respectively. Downstream of the Alu-downstream region, there isno similarity between X and Y sequences, which are called thesex-specific regions in Figure 2.
Sequences around the Alu-insertion site being the exact
interface that divides sex chromosomes into two functionallydistinct regions are known to be strictly conserved in species ofhominoids and Old World monkeys (20). At the insertion site
there is a 10 nt sequence strictly conserved even among PABLs,
although the Alu element is absent (see Fig. 1). Strict sequenceconservation around this Alu-insertion site, as well as strict
conservation of the 5′- and 3′-termini, during evolution may berelated to possible functions of PABXY1 and PABLs. In ourprevious paper, we reported a rather unexpected finding that thesequence similarity of the Alu-downstream portion of PABX1 or
PABY1 with the PABL2 (82.2 or 80.5% nucleotide identity) is
higher than that between PABX1 and PABY1 (77.8% identity)
and the similarity among PABX1, PABY1, and PABL1 isequivalent (13). For clarifying evolutionary processes thatformed the present PABXY1 and PABLs and investigating
functional constraints on the sequences, phylogenetic relationships and evolutionary rates were examined for all available
PABLs. The reported PABXY1 sequences of great apes and Old
World monkeys (20) were included for estimation of the27
Human Acids
Molecular
Genetics,
Vol.No.
5, No.
Nucleic
Research,
1994,1996,
Vol. 22,
1 1

27

Figure 4. Northern blot analysis of human total or polyA+ RNA. (A) Total RNA
was extracted from GM01416D cells by the AGPC method (39). Twenty-fiveµg of the total RNA was electrophoresed on a 1% agarose gel in a buffer
containing 6% formaldehyde, 20 mM MOPS (pH 7.0), 1 mM EDTA, and 5 mM
sodium acetate, and then transferred to a Hybond-N+ membrane. The probe
used for hybridization was a 598 nt PCR fragment of PABL1. Hybridization
was done in solution containing 5 × SSPE, 10 × Denhardt’s solution, 100 µg/ml
freshly denatured salmon sperm DNA, and 2% SDS. The final washing wasdone at 60C in 0.1 × SSC containing 0.1% SDS for 15 min. (B) Two µg of
polyA+ RNA fractions derived from different tissues (lane 1, spleen; lane 2,
thymus; lane 3, prostate gland; lane 4, testis; lane 5, ovary; lane 6, small
intestine; lane 7, colon; lane 8, peripheral blood leukocytes) were tested. The
RNAs were obtained from Clontech (Palo Alto, CA), and the probe was the
PABL1 segment described above. (C) The polyA+ RNA filter in (B) was used
after removal of the PABL1 probe. The probe used for this hybridization wasa unique 800 nt fragment of Sp2 cDNA deprived of the PABL sequence.

evolutionary rates. Divergence between great apes and Old World
monkeys is postulated to have occurred 25 million years ago.
Because of the PAR characteristic, PABX1 and PABY1
sequences upstream of the Alu-insertion site are practically
identical within a single species. For avoidance of complications
arising from this peculiar characteristic, individual PABXY1 and
PABLs were divided into two regions and unrooted phylogenetic
trees were separately constructed by the neighbor-joining method(Fig. 5). Trees obtained from evolutionary distances on the basisof one- and two-parameter methods were identical in theirbranching patterns or topologies, and branch lengths were nearly
the same.
The downstream portion, and thus the reduced-homology
portion for sex chromosomes, was analyzed first. PABX1 andPABY1 of all species examined were separated into two distinct

Figure 3. (A) Comparison of the 5′-region of PABLs of cDNA clones with thatof PABL1 and PABX1. Positions of identical nucleotides are marked withasterisks, and positions where four of five nucleotides are identical are marked
with dots. Arrow shows the 5′-edge of the proposed core unit of PABLs. Near
the 5′-edge, two EST sequences, T92306 and R12279, were added to thealignment. The multiple alignment analyzing only cDNA and EST sequences
showed the 5′-terminus of similarity to be practically identical to that for the
genomic PABLs. (B) Comparison of the 3′-region of PABLs of cDNA clones
with that of PABL1 and PABX1. Near the 3′-edge, one EST, T47905, was added
to the alignment. The multiple alignment analyzing only cDNA and EST
sequences showed that the 3′ terminus of similarity approximately corresponds
to that for the genomic PABLs. One edge of cDNAs and ESTs were often within
the PABL core. This may be due to the stable secondary structure predicted for
PABLs which presumably inhibits cDNA extension by reverse transcriptase:the most stable secondary structure for PABLs was calculated with the
GCGFOLD program managed by the University of Wisconsin Genetics
Computer Group (UWGCG), and energy levels found for individual PABLs
were equivalent to or lower than those of RNAs known to form stable secondary
structures such as mitochondrial rRNAs and 7SL RNA (data not shown).

28

Human Molecular Genetics, 1996, Vol. 5, No. 1
groups and the topology within each group reflects the known
phylogenetic relationships of the species (Fig. 5A). The divergence between PABX1 and PABY1 sequences was similar inextent to the divergence of PABX1 (or PABY1) from PABLs.
This is consistent with our previous finding obtained analyzing
PABL1 and 2, as well as with the suggestion of Ellis et al. (20)that genetic contact resulting in sequence homogenization
between sex chromosomes did not occur in the Alu-downstream
portion after the divergence of great ape and Old World monkey
lineages and the strict limit of PAR1 recombination was at the
Alu-insertion site in the ancestral species of all extant higher
primates. From the evolutionary distance between human
PABX1 and Old World monkey PABX1, as well as that for
PABY1, the divergence times of PABXY1 and thus of PABLs
was estimated to be 60–120 million years. This estimate isconsistent with the finding that PABLs are present in the bovine
genome (13). Based on the divergence time, evolutionary rates ofindividual PABLs were estimated (Table 1).
Table 1. Evolutionary rates of PABLs and PABXY1
Rates
Upstream

Downstream

PABL1

1.2 ± 0.4

1.5 ± 0.5PABL2

0.6 ± 0.2

0.4 ± 0.1PABL3

0.7 ± 0.3

1.3 ± 0.4Sp2

0.9 ± 0.3

0.8 ± 0.3Sp3

0.1 ± 0.05

1.5 ± 0.5PABX1

0.9 ± 0.3

1.0 ± 0.4PABY1

0.9 ± 0.3

1.5 ± 0.5

All rates are in units of 10–9 substitutions per site per year. Divergence time of
PABLs is assumed to be 60–120 million years.

Figure 5. Phylogenetic trees of PABXY1 and PABL sequences, based on thedownstream (A) and upstream (B) portions to the Alu-insertion site. Trees wereconstructed for PABXY1 of four hominoids and three Old World monkeys andfive human PABLs by the neighbor-joining method. Mainly because of limited
availability of PABXY1 sequences of great apes and Old World monkeys, theportions analyzed (151 nt for downstream and 177 nt for upstream) were shorter
than those of the PABL core. Abbreviations for the species are as follows:Homosapiens (human; Hum), Pan troglodytes (chimpanzee; Chi), Gorilla gorilla(gorilla; Gor), Pongo pygmaeus (orangutan; Ora), Theropithecus gelada(gelada baboon; Gel), Macaca sylvanus (barbary macaque; Bar), and Cercopithecus cephus (moustached guenon; Gue). The root of trees was predicted by
the UPGMA method (40). Branch lengths were proportional to the estimatednumber of nucleotide substitutions. Evolutionary distances (number ofnucleotide substitutions) were estimated by Kimura’s two parameter method(41), and these distances were used to construct neighbor-joining trees.
Bootstrap probabilities, based on 1000 resamplings, were calculated for eachinternal branch of neighbor-joining trees with the NJBOOT2 program (kindly
provided by Dr K. Tamura, Tokyo Metropolitan University).

We then analyzed the upstream portion of the Alu-insertion site
(Fig. 5B). As expected from the PAR characteristic, PABX1 andPABY1 do not separate into two groups. Evolutionary distancesof several PABLs such as PABL2, PABL3, and PABLSp3 (PABL
portion of Sp3 sequence) differ significantly between the
upstream and downstream portions of the Alu-insertion site (Fig.
5A vs. B). This difference suggests that the Alu-insertion site
corresponds to recombinational and/or functional interface
within a PABL as observed for PABXY1 although the Alu
element itself is absent. Evolutionary rates for the upstream
portion estimated from the PABL divergence time obtained forthe downstream portion are also listed in Table 1. The evolutionary rates of some PABLs were estimated to be far smaller than 1
× 10–9 substitutions per site per year (e.g. 0.1 × 10–9 substitutions
per site per year for the Alu-upstream portion of PABLSp3). Thisfinding indicates that the sequences evolved more slowly thantypical noncoding regions, since the averages of evolutionary
rates for mammalian pseudogenes and introns of transcribedgenes have been estimated to be 4.9 × 10–9 and 3.7 × 10–9
substitutions per site per year, respectively (22). These results
suggest that PABLs and PABXY1 have been under selective
constraints and presumably have functions. We also analyzed a
300 nt X-specific sequence immediately downstream of PABX1(sex-specific in Fig. 2), which has been reported in hominoids and
Old World monkeys (20). Pairwise-comparison of the sequence29
Human Acids
Molecular
Genetics,
Vol.No.
5, No.
Nucleic
Research,
1994,1996,
Vol. 22,
1 1
between species and the same comparisons for the Alu-upstreamor -downstream PABX1 sequence were conducted for estimationof the nucleotide divergence in the regions. In most cases,
nucleotide divergence in the PABX1 sequences was lower thanthat of the X-specific sequence, presenting evidence for evolutionary and functionary constraints on the PABX1 sequence(Table 2).
Table 2. Percentage of divergence in pairwise comparison of sequences
wthin or around pseudoautosomal boundary 1 of X chromosome
PABL sequenceSex-specific

Upstreamof Alu insertion

Downstreamof Alu insertion

1.5

1.2

1.0

HumX/GorX

1.0

0.6

2.1

ChiX/GorX

0.5

0.6

1.7

HumX/OraX

4.1

1.2

5.5

ChiX/OraX

4.6

1.2

5.1

GorX/OraX

4.1

0.6

6.2

HumX/BarX

10.7

8.1

11.6

HumX/GelX

9.6

6.9

10.8

HumX/GueX

11.2

9.4

14.2

ChiX/BarX

10.2

8.1

11.2

ChiX/GelX

9.1

6.9

10.5

HumX/ChiX

ChiX/GueX

9.6

9.4

13.9

GorX/BarX

10.7

7.5

12.2

GorX/GelX

9.6

6.2

11.5

GorX/GueX

10.2

8.8

14.5

OraX/BarX

9.6

8.1

10.8

OraX/GelX

8.6

6.9

10.6

OraX/GueX

10.2

9.4

12.8

BarX/GelX

2.0

4.4

3.8

BarX/GueX

4.6

6.2

5.1

GelX/GueX

3.6

5.6

5.8

Abbreviations for species are listed in Figure 5 legend.

More than ten PABL sequences (genomic PABLs, PABXY1,
and transcribed PABLs) have been studied, and their sequences
are about 80% identical or more. This high similarity allowed
deduction of a consensus sequence of 646 nt which we deposited
with GenBank/EMBL/DDBJ (accession no. D63517); a uniquebase could be assigned to 626 nucleotide positions in the 646 nt
PABL core. Tree topology in Figure 5 indicates the consensus
sequence is most likely related to their ancestor sequence. Whenthe consensus sequence was aligned with PABLSp3, divergencefound for the Alu downstream portion (11.9% nucleotide
difference) was higher than that for the upstream portion (5.7%
difference). This difference and results shown in Figure 5 andTable 1 support the model that the Alu insertion site is arecombinational and/or functional interface within a PABL as
with PABXY1.

29

DISCUSSION
Boundaries of long-range GC% mosaic domains
At a standard 850 band level, the human MHC harboring PABL1
is on a wide R band, 6p21.3, which has been assigned to a T-type
R band (T bands); T bands are an evidently heat-stable subgroupof R bands and mainly composed of GC-rich sequences. By
high-resolution banding, a thin G positive subband, 6p21.32, wasfound within the MHC (23). On the basis of detailed basecomposition analysis, Fukagawa et al. (13) predicted that the
genome portion harboring the evidently AT-rich 200 kb aroundthe junction between classes II and III constitutes a portion of thethin G positive subband. PABL1 was within the AT-rich domain
but close to the domain edge, suggesting PABL1 is located neara chromosome band boundary. Considering the wide range offunctional behaviors of chromosome bands and GC% mosaic
domains, their boundaries are most probably composed ofmultiple signals and structures related with multiple functions.
Near the GC% mosaic boundary in the MHC the following
characteristic structures have been found: on the GC-rich side adense 20 kb Alu cluster and on the GC-poor side a dense 30 kb
LINE-l cluster and PABL1 (13). If these characteristics are foundfor other boundaries, it will add to our comprehensive understanding of both DNA sequences and chromosome structures. We
focus here on GC% distribution and band structures around
PABXY1. PAR1 has been assigned to an R band (Xp22.33 andYp11.32), and judging from the high density of CpG islands amajor portion of 2.6 Mb of PAR1 is thought GC-rich (17). The
boundaries with the neighboring G-positive subbands, Xp22.32
and Yp11.31, appear rather close to PABXY1 (17,24) (Fig. 6).
PABXY1 and their neighboring sex-specific sequences, including SRY, RPS4Y, and ZFY located downstream of PABY1, areAT-rich (20). We therefore previously predicted that PABXY1 isnear a boundary of the long-range GC% mosaic domains and ofchromosome bands in the sex chromosomes (13). Very recently,
analyzing a contiguous 41 kb sequence harboring PABY1 (22.5
kb of PARY1 and 18.5 kb of Y-specific sequence), Whitfield etal. (25) showed that the PARY1 and Y-specific portions differ
dramatically in GC%. It thus becomes clear that not only PABL1
in the MHC but also PABY1 are located in the GC% mosaic
boundaries and therefore genome characteristics in regions
harboring these elements are similar.
To further study genome features of regions with PABLs, werecently characterized a 3 Mb portion harboring PABL2 byanalyzing YAC and cosmid clones in the respective regions. The
3 Mb region was found to be composed of long-range GC%
mosaic domains and PABL2 was in the domain boundary region(our unpublished data). Therefore, genome characteristics around
PABL2 are also analogous to those around PABL1 and PABY1.
For identification of chromosome locations of other PABLs, weused a standard fluorescence in situ hybridization (FISH) onto
metaphase chromosomes. Most of the ten PABLs mapped alreadywere on T-type and/or terminal R bands in which there are internal
narrow G subbands (our unpublished data). Characteristics of the
genome portions harboring PABLs appear again to resemble eachother.
Possible functions of PABLs and PABXY1
Functionally important molecules evolve more slowly than lessimportant molecules (26). PABLs probably have biological30

Human Molecular Genetics, 1996, Vol. 5, No. 1
here that intact size of PABL transcripts ranges from 5 to 10 kb
but the length of cDNA sequences obtained in this study is 2 kb
or less. Therefore we can not exclude the possibility that
protein-coding regions are present in the unanalyzed portions.
Alternatively, these results may suggest that the functional formof the transcripts is RNA molecules. PABL1 and PABXY1 wereboth found in experiments that searched for molecular structuresand signals differentiating global characteristics in the humangenome. Large PABL transcripts may be necessary for recognition or differentiation of global genome characteristics. XISTRNA which is 17 kb long has been thought important in suchdetermination and differentiation in the inactivated X chromosome (33).

Figure 6. Chromosome bands and long-range GC% mosaic structures aroundthe pseudoautosomal boundary of the short arms of the human sex chromosomes. Pseudoautosomal regions (PAR1) have been assigned to R bands(Xp22.33 and Yp11.32) and, judging from the high density of CpG islands, amajor portion of PAR1 is thought to be GC-rich. The boundaries with theneighboring Giemsa-positive subbands, Xp22.32 and Yp11.31, appear rather
close to PABXY1 and tentatively placed near PAB1 according to Mandel et al.
(24). PABXY1 and their neighboring sex-specific sequences, including SRY,RPS4Y, and ZFY are known to be AT-rich. These observations suggest the
PABXY1 to be in a boundary of the long-range GC% mosaic domains.

functions because they are transcribable and their evolutionary
rates were slower than nonfunctional regions. Concerning thefunctions of boundaries of chromosome bands and GC%-mosaic
domains, the switching of DNA replication timing is an importantcandidate (11,13). Multi-color FISH of interphase nuclei (27), in
which contiguous λ phage and cosmid clones in the MHC (13,28)
were used as probes, showed that PABL1 is located in a switchingregion of DNA replication timing (Okumura et al., in preparation). Major portions of the methylated and transcriptionallyinactive X chromosome replicate very late in S phase, but PAR1
escaping from the X-inactivation is thought to replicate muchearlier (29,30). Therefore, a switch in DNA-replication timing, atleast in the inactivated X chromosome, presumably occurs near
PAB1. PABLs including PABXY1 may be related to possible
signals for DNA replication timing such as a pause signal for the
replication. Considering that high frequency-recombination inPAR1 between sex chromosomes terminates abruptly at PAB1,
PABLs are expected to be involved in a certain process ofrecombination. A model for evolutionary formation of the present
pseudoautosomal boundaries is proposed below by postulation ofillegitimate recombination between two PABLs.
Characteristics of PABL transcripts
PABX1 was found within an intron of PBDX (pseudoautosomal
boundary divided on the X chromosome), which encodes the Xga
antigen and was recently renamed XG (31,32). When RT–PCR
analyses were done on human total RNA from a B-cell line,
primers designed for the present cDNAs reproducibly gave PCR
products with the expected sizes under standard conditions (datanot shown). However, primers designed for the PABX1 region
showed only occasionally a faint band of the expected size under
the same or modified conditions. RNA molecules corresponding
to the present cDNAs may be more abundant or have longer
life-span than the RNA of the XG intron.
Significant ORFs were not found for the present cDNA
sequences, for either PABLs or their flanks. Computer searches
could assign no protein-coding capacity. It should also be noted

Evolutionary processes in formation of sex-chromosome PABs
Recently Ellis et al. (31) proposed a model for the evolutionary
formation of the present-day pseudoautosomal boundary of theshort arms, PABXY1. They hypothesized a pericentric inversionof the Y chromosome with one break point in ancestral XG and
the other break point 5 kb distal to the ancestral SRY believed to
be on the earlier long arm (see legend of Fig. 7). Here we propose
a model in which the hypothesized inversion occurred byillegitimate recombination between the two PABL elements, one
PABL in the ancestral XG and the other near the ancestral SRY;
before recombination, the two elements on the earlier Y
chromosome were ordinary PABLs (Fig. 7A). After recombination, the characteristics of a pseudoautosomal boundary was
acquired and the recombinant PABL became PABY1 (Fig. 7C).
Ellis et al. (20) mentioned two kinds of models for formationof a strict boundary of PAR. One is based on genome rearrangement such as insertion, deletion, inversion, and translocation; themodel proposed above postulating illegitimate recombination
belongs to this category. The other model is based on thefollowing ‘attrition’ process, and our model is different from the‘attrition’ model. High-frequency recombination acts to maintainsequence similarity in the PAR between the sex chromosomes,
but this event is infrequent in sequences close to the PAB. The
attrition model predicts that when enough sex-specific differences accumulate immediately distal to the boundary, theprobability of recombination in the region with mismatches isreduced. Once recombination is limited, divergence between the
X and Y chromosomes accumulates more rapidly until recombination events no longer include the region of mismatches,
resulting in formation of a new strict boundary. In the attrition
model, the region of reduced homology in PABXY1 (i.e.,
downstream of the Alu insertion site) is taken to correspond to the
portion derived from a single PABL after attrition occurred.
Therefore, when based on this model, the most probableexplanation for why the 3′ homology terminus between PABX1and PABY1 is almost identical to the termini among PABLs is astrong functional constraint that preserves the 3′-terminal position during course of evolution. Furthermore, an extra mechanism
may act to put an evolutionally stable interface between theregions of high- and reduced-homology (i.e., the Alu-insertionsite) during attrition. Our explanation for the reduced-homology
supposed in Figure 7 is different from this attrition process, andis based mainly on the finding that the diversity between PABX1and PABY1 is similar to the diversity of either PABX1 or PABY1
from PABLs in the respective region (Fig. 5A). In this connection,
it is worthwhile to consider the sequence organization of the

31
Human Acids
Molecular
Genetics,
Vol.No.
5, No.
Nucleic
Research,
1994,1996,
Vol. 22,
1 1

31

Figure 7. Model of the evolutionary formation of the PABY1, in a modification of the model proposed by Ellis et al. (31). As the molecular process in the pericentric
inversion hypothesized by them, an illegitimate recombination between two PABLs is postulated. (A) According to Ellis et al., in early primates, PAB1 was somewhereproximal to the present-day amelogenin gene on the X chromosome (AMGX). We suggest that, in the old Y chromosome, one PABL was within XG and another was
5 kb distal to SRY. (B) After the divergence of lineages to higher primates and prosimians, a new boundary is considered to have been formed by a pericentric inversion
on the Y chromosome initiated by break points inside XG and 5 kb distal to SRY. As the molecular process of this inversion, we propose illegitimate recombination
between the two PABLs mentioned above. (C) An inverted Y chromosome with the present PABY1 was thus formed. Further evolutionary events such as pericentric
inversion transferring AMGY to its present location were described by Ellis et al. (31).

boundary of PAR2, the pseudoautosomal regions of the long armsof the human sex chromosomes. At the breakpoint of the X-Y
sequence homology in PAR2, Kvaløy et al. (18) found a portionof LINE-l sequence (about 780 nt), and they proposed thatillegitimate recombination between two independent LINEs onearlier X and Y chromosomes were involved in the formation of
the present PAR2 boundary, PABXY2. The similarity between
the reported LINE-1 sequences in PABX2 and PABY2 is 92%
nucleotide identity. One explanation for this reduced homology
may be due to attrition derived from a single LINE-1 sequence.
To test this possibility, we used 780 nt of the LINE-1 sequencesof X and Y chromosomes in a search of GenBank sequences.
LINE-1 of the X chromosome had 98% identity with LINE-1 of
HSL1G and LINE-1 of the Y chromosome had 96% identity with
LINE-1 of HSRETBLAS, indicating separate origins for theLINE-1 sequences in PABX2 and PABY2. This finding is notconsistent with the attrition model postulating these two LINE-1
sequences with 92% identity have been derived from a single
LINE-1, but consistent with the model supposing the illegitimate
recombination between two LINEs. Boundaries of both PAR1 andPAR2 therefore were probably produced by analogous processes,that is, by illegitimate recombination between repetitive elements:
PABLs for PABXY1 and LINEs for PABXY2. Analogous
processes may have been involved in forming the present-day
human genome, which is composed of GC% mosaic structures.

MATERIALS AND METHODS
Cloning and sequencing
A cosmid library constructed from the total human DNA of an
HLA homogeneous B cell line on vector pWE15 by Inoko et al.
(34) and a λ-EMBL3 library constructed from the human DNAof peripheral blood cells (35) were used. Six human cDNA
libraries cloned on vector λgt10 or λgt11 were obtained from
Clontech (Palo Alto, CA): placenta 5′-stretch plus (λgt11),
placenta (λgt11), monocyte (λgt11), B cell (λgt10), spleen(λgt10), and skin fibroblast (λgt10). Cloning of PABLs, theirsubcloning into pUC118, sequencing, and database searches of
GenBank/EMBL/DDBJ, as well as of PIR and Swiss-Prot protein
databases, were done as described previously (13,28).
Sequence alignment and construction of phylogenetic trees
Alignments and calculations of sequence identity were done with the
MALIGN program available on DDBJ. For identification of the coresequence of PABLs, all pairs of sequences with PABLs or PABXY1
were first aligned, and multiple alignments of the similar portions
thus found were done as described by Hein (36). Phylogenetic trees
were constructed by using the neighbor-joining method (37).
Evolutionary rates were estimated as described by Nei (38).

32

Human Molecular Genetics, 1996, Vol. 5, No. 1

ACKNOWLEDGEMENTS
The authors are very grateful to Dr N. Ellis for kindly providing
us with his unpublished sequence and valuable comments; to Drs
K. Sugaya, T. Tenzen and K. Matsumoto for discussion; to Mrs
Y. Miyauchi for technical assistance. The computers at the DDBJ
and the Human Genome Center of Japan were used. This work
was supported by a JSPS Fellowship for Japanese Junior
Scientists to T.F., Grant-in-Aid for Creative Basic Research(Human Genome Project) to T.I. and for Scientific Research on
Priority Areas (Genome Informatics) to T.I., and by Grants-inAid of Scientific Research from the Ministry of Education,
Science and Culture of Japan to T.F. and T.I.
ABBREVIATIONS
MHC, major histocompatibility complex; PAB, pseudoautosomal boundary; PABL, pseudoautosomal boundary-like sequence; PABX1, pseudoautosomal boundary sequence of shortarm of X chromosome; PABY1, pseudoautosomal boundary
sequence of short arm of Y chromosome; PABXY1, PABX1 andPABY1 sequences; PAR1, pseudoautosomal region of short armof sex chromosomes; PAR2, pseudoautosomal region of long armof sex chromosomes; EST, expressed sequence tag.

REFERENCES
1. Bernardi, G., Olofsson, B., Filipski, J., Zerial, M., Salinas, J., Cuny, G.,
Meunier-Rotival, M. and Rodier, F. (1985) The mosaic genome ofwarm-blooded vertebrates. Science, 228, 953–958.
2. Ikemura, T. (1985) Codon usage and tRNA content in unicellular andmulticellular organisms. Mol. Biol. Evol., 2 ,13–34.
3. Aota, S. and Ikemura, T. (1986) Diversity in G+C content at the third position ofcodons in vertebrate genes and its cause. Nucleic Acids Res., 14, 6345–6355.
4. Bernardi, G. (1989) The isochores organization of the human genome. Annu.
Rev. Genet., 23, 637–661.
5. Bernardi, G. (1993) The isochores organization of the human genome and its
evolutionary history—A review. Gene, 135, 57–66.
6. Ikemura, T. and Aota, S. (1988) Global variation in G+C content alongvertebrate genome DNA; possible correlation with chromosome band
structures. J. Mol. Biol., 203, 1–13.
7. Korenberg, J. R. and Rykowski, M. C. (1988) Human genome organization:Alu, Lines and the molecular structure of metaphase chromosome bands.
Cell, 53, 391–400.
8. Gardiner, K., Aissani, B. and Bernardi, G. (1990) A compositional map ofhuman chromosome 21. EMBO J., 9,1853–1858.
9. Ikemura, T., Wada, K. and Aota, S. (1990) Giant G+C% mosaic structures of
the human genome found by arrangement of GenBank human DNA
sequences according to genetic positions. Genomics, 8, 207–216.
10. Ikemura, T. and Wada, K. (1991) Evident diversity of codon usage patterns ofhuman genes with respect to chromosome banding patterns and chromosome
numbers; relation between nucleotide sequence data and cytogenetic data.
Nucleic Acids Res., 19, 4333–4339.
11. Holmquist, G. P. (1992) Chromosome bands, their chromatin flavors, andtheir functional features. Am. J. Hum. Genet., 51, 17–37.
12. Craig, J. M. and Bickmore, W. A. (1994) The distribution of CpG islands inmammalian chromosomes. Nature Genet., 7, 376–382.
13. Fukagawa, T., Sugaya, K., Matsumoto, K., Okumura, K., Ando, A., Inoko, H.
and Ikemura, T. (1995) A boundary of long-range G+C% mosaic domains in
the human MHC locus: pseudoautosomal boundary-like sequence exists near
the boundary. Genomics, 25, 184–191.
14. Cooke, H. J., Brown, W. R. and Rappold, G. A. (1985) Hypervariable
telomeric sequences from the human sex chromosomes are pseudoautosomal. Nature, 317, 687–692.
15. Simmler, M.- C., Rouyer, F., Vergnaud, G., Nystrom-Lahti, M., Ngo, K. Y., dela Chapelle, A. and Weissenbach, J. (1985) Pseudoautosomal DNA sequencesin the pairing region of the human sex chromosomes. Nature, 317, 692–697.

16. Ellis, N. and Goodfellow, P. N. (1989) The mammalian pseudoautosomal
region. Trends Genet., 5, 406–410.
17. Rappold, G. A. (1993) The pseudoautosomal regions of the human sex
chromosomes. Hum. Genet., 92, 315–324.
18. Kvaløy, K., Galvagni, F. and Brown, W. R. A. (1994) The sequenceorganization of the long arm pseudoautosomal region of the human sex
chromosomes. Hum. Mol. Genet., 3, 771–778.
19. Ellis, N. A., Goodfellow, P. J., Pym, B., Smith, M., Palmer, M., Frischauf,
A.-M. and Goodfellow, P. N. (1989) The pseudoautosomal boundary in man
is defined by an Alu repeat sequence inserted on the Y chromosome. Nature,
337, 81–84.
20. Ellis, N., Yen, P., Neiswanger, K., Shapiro, L. J. and Goodfellow, P. N. (1990)
Evolution of the pseudoautosomal boundary in Old World monkeys and great
apes. Cell, 63, 977–986.
21. Uberbacher, E. C. and Mural, R. J. (1991) Locating protein-coding regions inhuman DNA sequences by a multiple sensor-neural network approach. Proc.
Natl. Acad. Sci. USA, 88, 11262–11265.
22. Li, W.-H., Luo, C.-C. and Wu, C.-I. (1985) In MacIntyre, R. J. (ed.),
Molecular Evolutionary Genetics. Plenum Press, New York, pp. 1–94.
23. Senger, G., Ragoussis, J., Trowsdale, J. and Sheer, D. (1993) Fine mapping of
the human MHC class II region within chromosome band 6p21 andevaluation of probe ordering using interphase fluorescence in situ hybridization. Cytogenet. Cell Genet., 64, 49–53.
24. Mandel, J.-L., Monaco, A. P., Nelson, D. L., Schlessinger, D. and Willard, H.
(1992) Genome Map III. Science, 258, 87–102.
25. Whitfield, L. S., Hawkins, T. L., Goodfellow, P. N. and Sulston, J. (1995) 41
kilobases of analyzed sequence from the pseudoautosomal and sex-determining region of short arm of human Y chromosome. Genomics, 27, 306–311.
26. Kimura, M. (1983) The Neutral Theory of Molecular Evolution, Cambridge
Univ. Press, Cambridge, UK.
27. Selig, S., Okumura, K., Ward, D. C. and Cedar, H. (1992) Delineation of DNA
replication time zones by fluorescence in situ hybridization. EMBO J., 11,1217–1225.
28. Sugaya, K., Fukagawa, T., Matsumoto, K., Mita, K., Takahashi, E., Ando, A.,
Inoko, H. and Ikemura, T. (1994) Three genes in the human MHC class m region
near the junction with the class III: gene for receptor of advanced glycosylation
end products, PBX2 homeobox gene and a Notch-homolog, human counterpartof mouse mammary tumor gene int-3. Genomics, 23, 408–419.
29. Goodfellow, P., Pym, B., Mohandas, T. and Shapiro, L. J. (1984) The cellsurface antigen locus, MIC2X, escapes X-inactivation. Am. J. Hum. Genet.,
36, 777–782.
30. Schiebel, K., Weiss, B., Wohrle, D. and Rappold, G. (1993) A humanpseudoautosomal gene, ADP/ATP translocase, escapes X-inactivation whereas a homologue on Xq is subject to X inactivation. Nature Genet., 3, 82–87.
31. Ellis, N. A., Ye, T.-Z., Patton, S., German, J., Goodfellow, P. N. and Weller, P.
(1994) Cloning of PBDX, an MIC2-related gene that spans the pseudoautosomal boundary on chromosome Xp. Nature Genet., 6, 394–400.
32. Ellis, N. A., Tippett, P., Petty, A., Reid, M., Weller, P. A., Ye, T. Z., German, J.,
Goodfellow, P. N., Thomas, S. and Banting, G. (1994) PBDX is the XG bloodgroup gene. Nature Genet., 8, 285–290.
33. Brown, C. J., Hendrich, B. D., Rupert, J. L., Lafreniere, R. G., Xing, Y.,
Lawrence, J. and Willard, H. (1992) The human XIST gene: Analysis of 17 kb
inactive X-specific RNA that contains conserved repeats and is highly
localized within the nucleus. Cell, 71, 527–542.
34. Inoko, H., Ando, A., Kimura, M. and Tsuji, K. (1985) Isolation andcharacterization of the cDNA clone and genomic clones of a new HLA class
II antigen heavy chain, DO alpha. J. Immunol., 135, 2156–2159.
35. Tomatsu, S., Kobayashi, Y., Fukumaki, T., Yubisui, T., Orii, T. and Sakaki, Y.
(1989) The organization and the complete nucleotide sequence of human
NADH-cytochrome b5 reductase gene. Gene, 80, 353–361.
36. Hein, J. (1990) Unified approach to alignment and phylogenies. Methods
Enzymol., 183, 626–645.
37. Saitou, N. and Nei, M. (1987) The neighbor-joining method: A new method
for reconstructing phylogenetic trees. Mol. Biol. Evol., 4, 406–425.
38. Nei, M. (1987) Molecular Evolutionary Genetics, Columbia Univ. Press,
New York.
39. Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolationby acid guanidinium thiocyanate-PhOH-chloroform extraction. Anal. Biochem., 162, 156–159.
40. Sneath, P. H. A. and Sokal, R. R. (1973) Numerical taxonomy. Freeman, San
Francisco.
41. Kimura, M. (1980) A simple method for estimating evolutionary rate of basesubstitutions through comparative studies of nucleotide sequences. J. Mol.
Evol., 16, 111–120.