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Breakage of the human Y-chromosome short arm between two blocks of tandemly repeated DNA sequences
GENOMICS
6,153-156
(1989)
SHORT COMMUNICATION Breakage of the Human Y-Chromosome Short Arm between Blocks of Tandemly Repeated DNA Sequences ULRICH MOLLER,*‘t *Division
MARC
LALANDE,*+*
TIMOTHY
A. DONLON,~
AND MICHAEL
Two
W. tiEARTLEIN*+$
of Genetics and Mental Retardation Center, The Children’s Hospital; tDepartment of Pediatrics, Harvard Medical School; and *Howard Hughes Medical Institute, Boston, Massachusetts 02115; and Klinical Cytogenetics, Stanford University Hospital, Stanford, California 94305 Received
November
25, 1988;
revised
February
7, 1989
Recently we detected a cluster of Y-specific moderately repeated DNA sequences.These sequencesare defined by the three DNA probes Y-156, Y-190, and Y-223a specific for a proximal region of Yp (Miiller et al., 1986a,b,c). The Y-specific DNA fragments hybridize to a 20-kb tandem repeat (Tyler-Smith et al., 1988) and are organized into two blocks. A major block is variable in size, comprises between 20 and 40 copies of the basic 20-kb unit, and is polymorphic. A smaller block consists of approximately 3 copies and was not found to be polymorphic (Tyler-Smith et al., 1988). The present investigation was undertaken to study the relationship of these Yp-specific repeats to Y-chromosomal rearrangements. The 17 46,Xx males studied were cases 460, 462, 548, 756, 775, 693, 547, 11, GM2626, GM2670, 102, 510, GM1189, 481, and 385 of Miiller et al. (1986a,c, 1987), many of which were kindly supplied by Prof. Dr. U. Wolf, Freiburg. A single patient with a cytogenetically detectable translocation (t(X;Y)(p22.23;p11.2)) was from Magenis et al. (1982). CHl was from The Children’s Hospital, Boston. The 46,XY females were cases 651, 620, and 740 of Miiller et al. (1986c), 1 and 2 of Miiller et al. (1986b) and Disteche et al. (1986), and three additional cases, two from The Children’s Hospital, Boston (CH2 and CH3), and one from the Cytogenetics Laboratory of Stanford University (Stan 1). A iytogenetic deletion of Yp was detected in 46,XY females 1 and 2. In 46,XY females 651, 620, 740, CH2, CH3, and Stan 1, a deletion of Yp was not detected cytologically. A 49,XXXXY lymphoblastoid line (GM1202) was purchased from the Human Genetic Cell Repository, Institute for Medical Research (Camden, NJ). Two types of pulsed-field gel electrophoresis systems were employed: orthogonal pulsed-field gel electrophoresis (OFAGE) (Schwartz and Cantor, 1984), with modifications as described in Hermann et al. (1987),
Y-chromosomal rearrangements, a common cause of sex reversal in man, frequently occur between two blocks of repeated DNA. Both blocks are composed of 20-kb tandemly repeated Y-chromosome-specific DNA sequences. They are located in the proximal portion of the Y short arm on a Not1 restriction fragment of approximately 6.3 Mb and on an MuI fragment of approximately 5.6 Mb. Chromosome breaks positioned between the two blocks were detected in two of three 46,XY females with deletions of Yp and in five of six 46,Xx males positive for the repeat sequences. The rearranged Not1 fragments in the 46,Xx males were 4.4 Mb and the MZuI fragments were 2.0 Mh in length. This indicates that breaks occur within a small region of Yp defined by the two blocks of specific repeated DNA sequences. The region between the two blocks thus appears to be a focus of structural lability in the human Y chromosome. o less Academic PXW, I~C.
Y -chromosomal rearrangements are a frequent cause of sex reversal in man. Aberrant X/Y interchanges are the most frequent underlying cause of XX maleness (Ferguson-Smith, 1966; Magenis et al., 1982; Andersson et al., 1986; Affara et al., 1986; Miiller et al., 1987; Weissenbach et al., 1987; Petit et al., 1987; Page et al., 1987), while XY females may arise from deletions of Yp (Rosenfeld et al., 1979; Miiller et al., 1986a,b,c; Disteche et al., 1986). Little is known about the DNA sequences involved in Y-chromosomal rearrangements. So far there is only one report demonstrating that Alu repeat sequencesmay be involved in X/Y interchanges in XX males (Rouyer et al, 1987). More information is required for a better understanding of Y-chromosomal rearrangements at the molecular level. It is especially important to learn whether breaks of the Y chromosome occur randomly or at preferential sites. 153
Copyright 0 1989 All rights of reproduction
osss-7543/89 $3.00 by Academic Press, Inc. in any form reserved.
154
SHORT
COMMUNICATION
and field-inversion gel electrophoresis (FIGE) (Carle et al., 1986), with modifications as described by Lalande et al. (1987) and Turmel and Lalande (1988). All DNA probes used in this study were derived from a recombinant phage library constructed from DNA enriched by flow sorting for the human Y chromosome (Miiller et al., 198613). Probe sizes and regions of their Y -chromosomal localization are given in Miiller (1987). DNA extraction, restriction enzyme cleavage, blotting (Southern, 1975), determination of DNA content, and hybridization conditions were those described in Miiller et al. (1986c). Eight 46,XY females were investigated for Y-chromosomal DNA content. Deletions of the distal sequences of Yp (Y-228, Y-280, Y-286; Table 1) were detected in three of these individuals (1, 2, and 651; Table 1). Sequences hybridizing with Y-190, Y-156, and Y-223a were reduced in copy number in two of these patients (2 and 651; Table 1) but not in the third (1 (Miiller et aZ., 1986; Disteche et al., 1986)). In the third 46,XY female (l), the repeated DNA sequences detected by Y-190, Y-156, and Y-223a were present, but the more distal DNA sequences hybridizing with probes Y-228, Y-280, and Y-286 were absent as in the other two 46,XY females. The results are summarized in Table 1. Hybridization with Y-chromosomal DNA probes to DNA from the five remaining 46,XY females (group VI of Table 1) did not show any deletions and thus did not reveal differences from Y chromosomes of normal males. TABLE
1
Hybridization of Y-Specific DNA Probes with from 46,Xx Males and 46,XY Females
DNA
I
II
III
IV
102
v
1
VI
Y-286 Y-280 Y-228 Y-190 Y-156 Y-223a Y-198 Y-253
+ + + + + + -
+ t + -
+ t -
-
+ t +
Red. Red. Red.
+ + t
+
-
t t +
t
t -
Y-216a
-
-
-
-
-
+
y-202
y-221
-
-
-
-
-
-
-
1
+
+
t t +
t t + t t
t t + t t
Note. “I” comprises 46,Xx males 460,462,548,756,775,693,547, 11, and CHl and the t(X,Y)(p22.23;p11.2) patient. “II” comprises 46,Xx males GM2626 and GM2670. “III” comprises 46,Xx male 510. “IV” comprises 46,Xx males GM1189,481, and 385. “V” comprises 46,XY females 2 and 651. “VI” comprises 46,XY females 620, 740, CH2, CH3, Stan 1; “1” is XY female 1. Note that Y-chromosomal rearrangements more complex than linear aberrant X/Y interchanges have occurred in the paternal ancestry of 46,Xx male 102. The Yspecific repeat cluster is absent in this patient. “Red.“: reduced copy number of Y-specific repeated DNA sequences. Probes Y-198, Y253, Y-202, Y216a, and Y-221 have been mapped to Yq (14).
Hybridization of various Y DNA probes to Southern blots of HindHI-cleaved DNA from 17 46,Xx males revealed the presence of Y-chromosomal DNA sequences in 14 cases (Table 1). Yp-specific repeated DNA sequences such as those hybridizing with probes Y-190, Y-156, Y-223a were detected in 10 cases. The intensity of hybridization of the 3.5,4.4-, and 0.75kb Hind111 fragments detected by probes Y-190, Y-156, and Y-223a was approximately the same in the XX males as that in controls (not shown). The Y-156/Y-190/Y-223a repeat cluster was analyzed by OFAGE in several of the above 46,XY females and 46,Xx males. In agreement with earlier findings (Tyler-Smith et al, 1988), digestion of normal male control DNA with XbaI revealed two major repeated blocks upon hybridization with Y-190 (Fig. 1). The larger block is polymorphic in size and ranges from 400 to 800 kb. The smaller block appears not to be polymorphic in normal males and has an approximate size of 60 kb. Both blocks also hybridize with Y-156 and Y-223a (not shown). In the two 46,XY females (2 and 651) with an apparent reduction in copy number of the repeated sequences, the major block of the repeat was missing and only the smaller block was present (Fig. 1A and Tyler-Smith et al., 1988). Conversely, in five of six 46,Xx males positive for Y-190, the smaller block was missing (cases 547, 462, 469, 756, 775 of Table 1 and Figs. 1B and 1C). In the sixth 46,Xx male studied, however, both blocks were present (case 693 of Fig. 1C). To determine the relative distance of the two repeat clusters, we searched for restriction enzymes that cleave outside the two blocks. Not1 and MZuI were found to meet this criterion. The FIGE experiment of Fig. 2 indicates that Not1 cleavage-of normal male DNA results in an approximately 5.3-Mb restriction fragment (Fig. 2A) and that MluI results in a fragment of approximately 5.5 Mb (Fig. 2B). Both the Not1 and the &flu1 fragments hybridize with Y-190. Restriction fragments of uniform sizes (4.4-Mb Not1 and 2.0-Mb MZuI fragments) were detected in the DNA from 46,Xx males 547, 462, 469, 756, and 775 (Figs. 2A and 2B). NotI-cleaved DNA from 46,XY females 2 and 651 resulted in Y-190-positive restriction fragments of 4.4 and 4.9 Mbp, respectively. The experiments demonstrate that breaks of the Ychromosome short arm frequently occur between the two blocks of Yp-specific repeated DNA sequences. The breaks are confined to a small region between the blocks, as was demonstrated by the similar sizes of rearranged Not1 (4.4 Mb) and i&flu1 fragments (2.0 Mb) in the five XX males tested (Fig. 2). This is consistent with hybridization data using a battery of Yp-specific DNA probes which suggested that chromosome breaks may occur at preferential sites of Yp (Miiller et al., 1986a; Vergnaud et al., 1986; Affara et al., 1987). The
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FIG. 1. Hybridization of Y-190 with XbaI-digested DNA from two 46,XY females (651: A, lane 3; 2: A, lane 4), from 46,xX males (462: B, lane 3; 469: B, lane 4; 756: B, lane 5; 775: B, lane 6; 547: C, lane 2; 693: C, lane 3) and from normal male and female controls. DNA was separated by OFAGE on 1% agarose gels in 0.5% Tris-borate (TBE) buffer (89 n&f Tris base, pH 8.0/89 m&f boric acid/2 m&f EDTA). The pulse time was 50 s at 330 V at 1l’C. “Std” indicates A concatamer size markers; “yeast” indicates the sizes of Saccharomyces cercuisioc chromosomes.
rearranged Not1 fragments described in 46,XY females 2 and 651 also favor the notion of a comparatively small region on Yp- defined by the two repeat blocks in which the breaks occur. Unlike the breaks in 46,xX males, however, the breaks in the two XY females did not occur at similar sites.
At present, little is known about the composition of the region between the two blocks. Further studies may reveal whether the breaks occur at or near the boundaries of the tandemly repeated DNA sequences. ACKNOWLEDGMENTS
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We are indebted to Dr. S. Latt for stimulating discussions and most useful comments on the manuscript. Drs. H. Cooke, T. Monaco, and L. M. Kunkel are gratefully acknowledged for critically reading the manuscript, Dr. C. Tyler-Smith for stimulating discussions and invaluable suggestions, Dr. M. Gessler for his advice on pulsed-field gel electrophoresis, and Prof. Dr. U. Wolf for cell lines. This work was supported by NIH Grants HD18658, GM33579, and HD24381 and by funds from the Howard Hughes Medical Institute.
REFERENCES
B)
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FIG. 2. Hybridization of probe Y-190 with Not1 (A)- and MZuI (B)-digested DNA from normal females (A, lane 1; B, lane l), from normal males (A, lanes 2 and 3; B, lane 2), from 46,Xx males (462, 469,547, 756, and 775: in A, lanes 4 to 8; in B, lanes 3 to 7), and from 46,XY females (2: A, lane 9; 651: A, lane 10). FIGE was carried out as described previously under pulse conditions that resolve DNA molecules in the size range 3000 to 6000 kb (21).
AFFARA, N. A., FERGUSON-SMITH, M. A., TOLMIE, J., KWOK, K., MITCHELL, M., JAMIESON, D., COOKE, A., AND FLORENTIN, L. (1986). Variable transfer of Y specific sequences in XX males. Nucleic Acids Res. 14: 5375-5387. AFFARA, N. A., FERGUSON-SMITH, M. A., MAGENIS, R. E., TOLMIE, J. L., BOYD, E., COOKE, A., JAMIESON, D., KWOK, K., MITCHELL, M., AND SNADDEN, L. (1987). Mapping the testis determinants by an analysis of Y-specific sequences in males with apparent XX and X0 karyotypes and females with XY karyotypes. Nuc.?eic Acids Res. 16: 7325-7342. ANDERSSON, M., PAGE, D. C., AND DE LA CHAPELLE, A. (1986). Chromosome Y-specific DNA is transferred to the short arm of X chromosome in human XX males. Science 233: 786-788. CARLE, G. F., FRANK, M., AND OLSON, M. V. (1986). Electro-
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phoretic separations of large DNA molecules by periodic inversion of the electric field. Science 232: 65-68. DISTECHE, C. M., CASANOVA, M., SAAL, H., FRIEDMAN, C., SYBERT, V., GRAHAM, J., THULINE, H., PAGE, D. C., AND FELLOUS, M. (1986). Small deletions of the short arm of the Y chromosome in 46,XY females. Proc. Natl. Acad. Sci. USA 83: 784-7844. FERGUSON-SMITH, M. A. (1966). X-Y chromosomal interchange in the aetiology of true hermaphroditism and of XX Klinefelter’s syndrome. Lancet 2: 475-476. HERMANN, B. G., BARLOW, D. P., AND LEHRACH, H. (1987). A large inverted duplication allows homologous recombination between chromosomes heteroxygous for the proximal t complex inversion. Cell 48: 813-825. LALANDE, M., NOOLANDI, J., TURMEL, C., ROUSSEAU, J., AND SLATJZR,G. W. (1987). Pulsed-field electrophoresis: Application of a computer model to the separation of large DNA molecules. Proc. Natl. Acad. Sci. USA 84: 8011-8015. MAGENIS, R. E., WEBB, M. J., MCKEAN, R. S., TOMAR, D., ALLEN, L. J., KAMMER, H., VAN DYKE, D. L., AND LOVRIEN, E. (1982). Translocation (X,Y)(p.22.23:~11.2) in XX males: Etiology of male phenotype. Hum, Genet. 62: 271-276. MOLLER, U., DONLON, T., SCHMID, M., FITCH, N., RICHER, C. L., LALANDE, M., AND LAW, S. A. (1986a). Deletion mapping of the testis determining locus with DNA probes in 46,xX males and in 46,XY and 46,X&(Y) females. Nucleic Acids Res. 14: 6489-6505. MULLER, U., LALANDE, M., DISTECHE, C. M., AND LATT, S. A. (1986b). Construction, analysis, and application to 46,XY gonadal dysgenesis of a recombinant phage DNA library from flow-sorted human Y chromosomes. Cytometry 7: 418-424. MULLER, U., LALANDE, M., DONLON, T., AND LATT, S. A. (1986c). Moderately repeated DNA sequences specific for the short arm of the human Y chromosome are present in XX males and reduced in copy number in an XY female. Nucleic Acids Res. 14: 1325-1340. MILLER, U. (1987). Mapping of testis-determining locus on Yp by the molecular genetic analysis of XX males and XY females. Development (Supplement) 101: 51-58. MOLLER, U., LATT, S. A., AND DONLON, T. (1987). Y-specific
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DNA sequences in male patients with 46,Xx and 47,XxX karyotypes. Amer. J. Med. Genet. 28: 393-401. PAGE, D. C., BROWN, L. G., AND DE LA CHAPELLE, A. (1987). Exchange of terminal portions of X- and Y-chromosomal short arms in human XX males. Nature (London) 328: 437-440. PETIT, C., DE LA CHAPELLE, A., LEVILLIERS, J., CASTILLO, S., NOEL, B., AND WEISSENBACH, J. (1987). An abnormal terminal X-Y interchange accounts for most but not all cases of human XX maleness. Cell 49: 595-602. ROSENFELD, R. G., LUZ~ATTI, L., HINTZ, R. L., MILLER, 0. J., Koo, G. C., AND WACHTEL, S. S. (1979). Sexual and somatic determinants of the human Y chromosome: Studies in a 46,XYp- phenotypic female. Amer. J. Hum. Genet. 31: 458468. ROUYER, F., SIMMLER, M. C., PAGE, D. C., AND WEISSENBACH, J. (1987). A sex chromosome rearrangement in a human XX male caused by Alu-Alu recombination. Cell 51: 417-425. SCHWARTZ, D. C., AND CANTOR, C. R. (1984). Separation of yeast chromosome-sized DNAs by pulsed field gel electrophoresis. Cell 37: 67-75. SOUTHERN, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. MOE. Biol. 98:503-517. TURMEL, C., AND LALANDE, M. (1988). Resolution of Schizosaccharomyces pombe chromosomes by field inversion gel electrophoresis. Nucleic Acids Res. 16: 4727. TYLER-SMITH, C., TAYLOR, L., AND MOLLER, U. (1988). Structure of hypervariable tandemly repeated DNA sequence on the short arm of the human Y chromosome. J. Mol. Biol. 203: 837848. VERGNAUD, G., PAGE, D. C., SIMMLER, M.-C., BROWN, L., ROUYER,F., NOEL, B., BOTSTEIN, D., DE LA CHAPELLE, A., AND WEISSENBACH, J. (1986). A deletion map of the human Y chromosome based on DNA hybridization. Amer. J. Hum. Genet. 38: 109-124. WEISSENBACH, J., LEVILLIERS, J., PETIT, C., ROUYER, F., AND SIMMLER, M.-C. (1987). Normal and abnormal interchanges between the human X and Y chromosomes. Development (Sup@.) 101:67-74.
6,153-156
(1989)
SHORT COMMUNICATION Breakage of the Human Y-Chromosome Short Arm between Blocks of Tandemly Repeated DNA Sequences ULRICH MOLLER,*‘t *Division
MARC
LALANDE,*+*
TIMOTHY
A. DONLON,~
AND MICHAEL
Two
W. tiEARTLEIN*+$
of Genetics and Mental Retardation Center, The Children’s Hospital; tDepartment of Pediatrics, Harvard Medical School; and *Howard Hughes Medical Institute, Boston, Massachusetts 02115; and Klinical Cytogenetics, Stanford University Hospital, Stanford, California 94305 Received
November
25, 1988;
revised
February
7, 1989
Recently we detected a cluster of Y-specific moderately repeated DNA sequences.These sequencesare defined by the three DNA probes Y-156, Y-190, and Y-223a specific for a proximal region of Yp (Miiller et al., 1986a,b,c). The Y-specific DNA fragments hybridize to a 20-kb tandem repeat (Tyler-Smith et al., 1988) and are organized into two blocks. A major block is variable in size, comprises between 20 and 40 copies of the basic 20-kb unit, and is polymorphic. A smaller block consists of approximately 3 copies and was not found to be polymorphic (Tyler-Smith et al., 1988). The present investigation was undertaken to study the relationship of these Yp-specific repeats to Y-chromosomal rearrangements. The 17 46,Xx males studied were cases 460, 462, 548, 756, 775, 693, 547, 11, GM2626, GM2670, 102, 510, GM1189, 481, and 385 of Miiller et al. (1986a,c, 1987), many of which were kindly supplied by Prof. Dr. U. Wolf, Freiburg. A single patient with a cytogenetically detectable translocation (t(X;Y)(p22.23;p11.2)) was from Magenis et al. (1982). CHl was from The Children’s Hospital, Boston. The 46,XY females were cases 651, 620, and 740 of Miiller et al. (1986c), 1 and 2 of Miiller et al. (1986b) and Disteche et al. (1986), and three additional cases, two from The Children’s Hospital, Boston (CH2 and CH3), and one from the Cytogenetics Laboratory of Stanford University (Stan 1). A iytogenetic deletion of Yp was detected in 46,XY females 1 and 2. In 46,XY females 651, 620, 740, CH2, CH3, and Stan 1, a deletion of Yp was not detected cytologically. A 49,XXXXY lymphoblastoid line (GM1202) was purchased from the Human Genetic Cell Repository, Institute for Medical Research (Camden, NJ). Two types of pulsed-field gel electrophoresis systems were employed: orthogonal pulsed-field gel electrophoresis (OFAGE) (Schwartz and Cantor, 1984), with modifications as described in Hermann et al. (1987),
Y-chromosomal rearrangements, a common cause of sex reversal in man, frequently occur between two blocks of repeated DNA. Both blocks are composed of 20-kb tandemly repeated Y-chromosome-specific DNA sequences. They are located in the proximal portion of the Y short arm on a Not1 restriction fragment of approximately 6.3 Mb and on an MuI fragment of approximately 5.6 Mb. Chromosome breaks positioned between the two blocks were detected in two of three 46,XY females with deletions of Yp and in five of six 46,Xx males positive for the repeat sequences. The rearranged Not1 fragments in the 46,Xx males were 4.4 Mb and the MZuI fragments were 2.0 Mh in length. This indicates that breaks occur within a small region of Yp defined by the two blocks of specific repeated DNA sequences. The region between the two blocks thus appears to be a focus of structural lability in the human Y chromosome. o less Academic PXW, I~C.
Y -chromosomal rearrangements are a frequent cause of sex reversal in man. Aberrant X/Y interchanges are the most frequent underlying cause of XX maleness (Ferguson-Smith, 1966; Magenis et al., 1982; Andersson et al., 1986; Affara et al., 1986; Miiller et al., 1987; Weissenbach et al., 1987; Petit et al., 1987; Page et al., 1987), while XY females may arise from deletions of Yp (Rosenfeld et al., 1979; Miiller et al., 1986a,b,c; Disteche et al., 1986). Little is known about the DNA sequences involved in Y-chromosomal rearrangements. So far there is only one report demonstrating that Alu repeat sequencesmay be involved in X/Y interchanges in XX males (Rouyer et al, 1987). More information is required for a better understanding of Y-chromosomal rearrangements at the molecular level. It is especially important to learn whether breaks of the Y chromosome occur randomly or at preferential sites. 153
Copyright 0 1989 All rights of reproduction
osss-7543/89 $3.00 by Academic Press, Inc. in any form reserved.
154
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COMMUNICATION
and field-inversion gel electrophoresis (FIGE) (Carle et al., 1986), with modifications as described by Lalande et al. (1987) and Turmel and Lalande (1988). All DNA probes used in this study were derived from a recombinant phage library constructed from DNA enriched by flow sorting for the human Y chromosome (Miiller et al., 198613). Probe sizes and regions of their Y -chromosomal localization are given in Miiller (1987). DNA extraction, restriction enzyme cleavage, blotting (Southern, 1975), determination of DNA content, and hybridization conditions were those described in Miiller et al. (1986c). Eight 46,XY females were investigated for Y-chromosomal DNA content. Deletions of the distal sequences of Yp (Y-228, Y-280, Y-286; Table 1) were detected in three of these individuals (1, 2, and 651; Table 1). Sequences hybridizing with Y-190, Y-156, and Y-223a were reduced in copy number in two of these patients (2 and 651; Table 1) but not in the third (1 (Miiller et aZ., 1986; Disteche et al., 1986)). In the third 46,XY female (l), the repeated DNA sequences detected by Y-190, Y-156, and Y-223a were present, but the more distal DNA sequences hybridizing with probes Y-228, Y-280, and Y-286 were absent as in the other two 46,XY females. The results are summarized in Table 1. Hybridization with Y-chromosomal DNA probes to DNA from the five remaining 46,XY females (group VI of Table 1) did not show any deletions and thus did not reveal differences from Y chromosomes of normal males. TABLE
1
Hybridization of Y-Specific DNA Probes with from 46,Xx Males and 46,XY Females
DNA
I
II
III
IV
102
v
1
VI
Y-286 Y-280 Y-228 Y-190 Y-156 Y-223a Y-198 Y-253
+ + + + + + -
+ t + -
+ t -
-
+ t +
Red. Red. Red.
+ + t
+
-
t t +
t
t -
Y-216a
-
-
-
-
-
+
y-202
y-221
-
-
-
-
-
-
-
1
+
+
t t +
t t + t t
t t + t t
Note. “I” comprises 46,Xx males 460,462,548,756,775,693,547, 11, and CHl and the t(X,Y)(p22.23;p11.2) patient. “II” comprises 46,Xx males GM2626 and GM2670. “III” comprises 46,Xx male 510. “IV” comprises 46,Xx males GM1189,481, and 385. “V” comprises 46,XY females 2 and 651. “VI” comprises 46,XY females 620, 740, CH2, CH3, Stan 1; “1” is XY female 1. Note that Y-chromosomal rearrangements more complex than linear aberrant X/Y interchanges have occurred in the paternal ancestry of 46,Xx male 102. The Yspecific repeat cluster is absent in this patient. “Red.“: reduced copy number of Y-specific repeated DNA sequences. Probes Y-198, Y253, Y-202, Y216a, and Y-221 have been mapped to Yq (14).
Hybridization of various Y DNA probes to Southern blots of HindHI-cleaved DNA from 17 46,Xx males revealed the presence of Y-chromosomal DNA sequences in 14 cases (Table 1). Yp-specific repeated DNA sequences such as those hybridizing with probes Y-190, Y-156, Y-223a were detected in 10 cases. The intensity of hybridization of the 3.5,4.4-, and 0.75kb Hind111 fragments detected by probes Y-190, Y-156, and Y-223a was approximately the same in the XX males as that in controls (not shown). The Y-156/Y-190/Y-223a repeat cluster was analyzed by OFAGE in several of the above 46,XY females and 46,Xx males. In agreement with earlier findings (Tyler-Smith et al, 1988), digestion of normal male control DNA with XbaI revealed two major repeated blocks upon hybridization with Y-190 (Fig. 1). The larger block is polymorphic in size and ranges from 400 to 800 kb. The smaller block appears not to be polymorphic in normal males and has an approximate size of 60 kb. Both blocks also hybridize with Y-156 and Y-223a (not shown). In the two 46,XY females (2 and 651) with an apparent reduction in copy number of the repeated sequences, the major block of the repeat was missing and only the smaller block was present (Fig. 1A and Tyler-Smith et al., 1988). Conversely, in five of six 46,Xx males positive for Y-190, the smaller block was missing (cases 547, 462, 469, 756, 775 of Table 1 and Figs. 1B and 1C). In the sixth 46,Xx male studied, however, both blocks were present (case 693 of Fig. 1C). To determine the relative distance of the two repeat clusters, we searched for restriction enzymes that cleave outside the two blocks. Not1 and MZuI were found to meet this criterion. The FIGE experiment of Fig. 2 indicates that Not1 cleavage-of normal male DNA results in an approximately 5.3-Mb restriction fragment (Fig. 2A) and that MluI results in a fragment of approximately 5.5 Mb (Fig. 2B). Both the Not1 and the &flu1 fragments hybridize with Y-190. Restriction fragments of uniform sizes (4.4-Mb Not1 and 2.0-Mb MZuI fragments) were detected in the DNA from 46,Xx males 547, 462, 469, 756, and 775 (Figs. 2A and 2B). NotI-cleaved DNA from 46,XY females 2 and 651 resulted in Y-190-positive restriction fragments of 4.4 and 4.9 Mbp, respectively. The experiments demonstrate that breaks of the Ychromosome short arm frequently occur between the two blocks of Yp-specific repeated DNA sequences. The breaks are confined to a small region between the blocks, as was demonstrated by the similar sizes of rearranged Not1 (4.4 Mb) and i&flu1 fragments (2.0 Mb) in the five XX males tested (Fig. 2). This is consistent with hybridization data using a battery of Yp-specific DNA probes which suggested that chromosome breaks may occur at preferential sites of Yp (Miiller et al., 1986a; Vergnaud et al., 1986; Affara et al., 1987). The
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FIG. 1. Hybridization of Y-190 with XbaI-digested DNA from two 46,XY females (651: A, lane 3; 2: A, lane 4), from 46,xX males (462: B, lane 3; 469: B, lane 4; 756: B, lane 5; 775: B, lane 6; 547: C, lane 2; 693: C, lane 3) and from normal male and female controls. DNA was separated by OFAGE on 1% agarose gels in 0.5% Tris-borate (TBE) buffer (89 n&f Tris base, pH 8.0/89 m&f boric acid/2 m&f EDTA). The pulse time was 50 s at 330 V at 1l’C. “Std” indicates A concatamer size markers; “yeast” indicates the sizes of Saccharomyces cercuisioc chromosomes.
rearranged Not1 fragments described in 46,XY females 2 and 651 also favor the notion of a comparatively small region on Yp- defined by the two repeat blocks in which the breaks occur. Unlike the breaks in 46,xX males, however, the breaks in the two XY females did not occur at similar sites.
At present, little is known about the composition of the region between the two blocks. Further studies may reveal whether the breaks occur at or near the boundaries of the tandemly repeated DNA sequences. ACKNOWLEDGMENTS
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We are indebted to Dr. S. Latt for stimulating discussions and most useful comments on the manuscript. Drs. H. Cooke, T. Monaco, and L. M. Kunkel are gratefully acknowledged for critically reading the manuscript, Dr. C. Tyler-Smith for stimulating discussions and invaluable suggestions, Dr. M. Gessler for his advice on pulsed-field gel electrophoresis, and Prof. Dr. U. Wolf for cell lines. This work was supported by NIH Grants HD18658, GM33579, and HD24381 and by funds from the Howard Hughes Medical Institute.
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FIG. 2. Hybridization of probe Y-190 with Not1 (A)- and MZuI (B)-digested DNA from normal females (A, lane 1; B, lane l), from normal males (A, lanes 2 and 3; B, lane 2), from 46,Xx males (462, 469,547, 756, and 775: in A, lanes 4 to 8; in B, lanes 3 to 7), and from 46,XY females (2: A, lane 9; 651: A, lane 10). FIGE was carried out as described previously under pulse conditions that resolve DNA molecules in the size range 3000 to 6000 kb (21).
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