Introduction

Polymorphisms of alleles residing in the nonrecombining portion of the human Y chromosome have been used to trace male-specific gene flow and human evolution (Spurdle et al 1994; Hammer and Horai 1995). In particular, the 47z/StuI polymorphism (Nakahori et al 1989) that is known to be specific to Asian populations is found in Japan, Korea, and Taiwan but is absent in other populations (Nakagome et al 1992; Hammer and Horai 1995; Shin et al 1998; Oh et al 2000). A simple PCR–RFLP method has been used to investigate the polymorphism, which is represented by two alleles, Y1 and Y2. Until now, the Y1 allele frequency has been shown to be higher than that of the Y2 allele in Asian populations (Shin et al 1998; Shinka et al 1999; Kim et al 2000). Chinese and Philippino populations are 100% in the Y1 allele, whereas the frequency of Y1 in Japan is relatively lower (83%) than other Asian populations.

In this study, we investigated the allelic variation in 47z/StuI polymorphism using the 47z/StuI PCR–RFLP method in 267 individual males in a Korean population. Here we report a novel allele of 47z/StuI polymorphism in addition to the two previously known alleles (Y1 and Y2), and discuss its consequences.

Materials and methods

47z/StuI PCR–RFLP

Using the standard method (Sambrook et al 1989), genomic DNAs were prepared from whole blood of 267 individual Korean males. Two copies of DXYS5 DNA at the loci of Xq21 (DXYS5X) and Yp (DXYS5Y) were amplified by PCR using AmpliTaq DNA polymerase (Perkin-Elmer, Boston, MA, USA), subsequently cut with StuI enzyme (Shin et al 1998) and separated on 3% agarose gel and 7% polyacrylamide gel. Primer sequences used were NS2F: 5′-TGAGTCAATGTCAATGAATC-3′ and NS2R: 5′-TAGTTACGCCTTGGCATAAC-3′. DNA amplification was performed. To determine the DNA sequences, the amplified DNAs were directly cloned into pGEM-T easy Vector (Promega), and sequenced with universal sequencing primers (F40 and R21) via BigDye Terminator Cycle Sequencing Kit (Applied Biosystems Ver 3.1. Foster City, CA, USA). Individual sequences were assembled into contigs using the Sequencher 4.1.4 (Gene Codes Corp., Ann Arbor, MI, USA).

Results and discussion

The most popular method used to detect the polymorphism of the DXYS5Y locus on the Y chromosome is the 47z/StuI PCR–RFLP system (Shin et al 1998; Shinka et al 1999). In this study, we investigated 47z (DXYS5) polymorphism using 267 individual Korean males using this system. Unexpectedly, we found a new band pattern which was distinct from the two previously known types, Y1 and Y2, and clearly demonstrated it by 7% polyacrylamide gel electrophoresis (Fig. 1). The new variant showed two bands, 280 and 100 bp, which suggests that DNAs to be amplified from both DXYS5Y and DXYS5X loci were clearly cut by StuI enzyme. Moreover, we analyzed the nucleotide sequence and confirmed that individuals showing the novel allele have StuI restriction sites (AGGCCT) on not only the DXYS5Y but also on the DXYS5X locus (Fig. 2). Consequently, our result implies that the DXYS5X locus is a polymorphic site although all previous studies have only considered Y-linked polymorphism (DXYS5Y), disregarding X-linked polymorphism (Shin et al 1998; Oh et al 2000).

Fig. 1
figure 1

Detection of the 47z/StuI polymorphism using the primers NS2-F and NS2-R. DNAs amplified from three different male individuals (Y1, Y2, and Novel) and subsequently digested by StuI enzyme (Y1*, Y2*, and Novel*) were separated using a 7% polyacrylamide gel electrophoresis method. All of the alleles were reformulated in this study as follows: X1–Y1, X2–Y1, X1–Y2, and X2–Y2. Expected sizes of the allelic variants are 382, 289, and 93 bp, respectively, and these depend on the sequence variation of the StuI enzyme site. The numbers on the left hand side indicate a 100 bp ladder size marker (M)

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Fig. 2
figure 2

Alignment result for novel allele sequences for 47z/StuI polymorphism. Two consensus sequences derived from X and Y chromosomes were aligned by the CLUSTALW program, and a reliable region discriminated the X sequence from the Y (gray region). Two sequences have the sequences of the StuI enzyme (black rectangle), and the cutting site of the StuI enzyme is represented by an arrow

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Based on these results, we reformulated allele types derived by the 47z/StuI PCR–RFLP method on the 47z (DXYS5) locus, as follows: X1–Y1, X1–Y2, X2–Y1, and X2–Y2. X1–Y2 and X2–Y1 correspond to the Y2 allele in previous reports, and X1–Y1 and X2–Y2 correspond to the Y1 allele of the previous and the new variant from this study, respectively (Fig. 1).

We further investigated the allele frequencies for 47z/StuI polymorphism using 267 individual Korean males, which showed 95.8% (X1–Y1), 3.4% (X1–Y2 and X2–Y1), and 0.8% (X2–Y2) or 2 out of the 267 individuals (Table 1). The frequency was similar to previous results seen in Korean populations. However, since the two bi-alleles of X1–Y2 and X2–Y1 cannot be classified by the method, previous results (Shin et al 1998; Shinka et al 1999; Oh et al 2000) may have overestimated the Y2 allele due to the misjudgment about the X and Y polymorphism. Therefore, our reformulated allele types on the 47z (DXYS5) locus should provide important clues for understanding not only the gene flow of the Y chromosome but also that of the X chromosome.

Table 1 Allele frequencies of 47z/StuI in eight East Asian populations
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A recent effort highlighted that the X-transposed region (XTR) on the Y chromosome resulted from the transposition of a chromosome segment from the X chromosome to the Y chromosome, which occurred after the speciation event that lead to human and chimpanzee lineages (Ross et al 2005). Our finding suggests that the translocated fragment including DXYS5 did not have the StuI site, and after translocation the base change on the Y chromosome resulted in the StuI site. This means that the same base change at the StuI site happened on the X chromosome independently without changing any of the surrounding sequence, as gene conversion. Therefore, our finding supplies an important clue to not only the migration route of human lineage but also the molecular evolution mechanism.