Supplementary Note 4:

Six sequence species in three heterochromatic regions

We found that the MSY heterochromatin encompasses at least six distinct
sequence species (Supplementary Table 10), each forming long, rather
homogenous tandem arrays, and two of which are located in the centromeric
region.  One centromeric array (DYZ3), based on a 171-bp alphoid primary
unit with a 5.9-kb secondary periodicity, gradually merges, across a
distance of 50 kb, with the euchromatic sequences of Yp (Supplementary
Fig. 7f).  On the opposite side of the centromere, an unrelated
heterochromatic array (DYZ17), based on a GGAAT repeat unit, abruptly
adjoins the euchromatic sequences of Yq (Supplementary Fig. 7h).  Within
the centromeric region, the alphoid (DYZ3) and GGAAT (DYZ17) arrays meet
cleanly and without intermixing (Supplementary Fig. 7g).

Another species of MSY heterochromatic sequences, the newly identified
DYZ19 repeats, form a sharply demarcated island of tandemly repeated
125-bp units in the midst of the euchromatic sequences of proximal Yq
(Supplementary Fig. 7i,j).  This DYZ19 island, comprised of >3,000 repeat
units, is roughly 400 kb in length and is remarkably homogeneous, with
no Alu, LINE-1, or other insertions identified.  When human metaphase
chromosomes are stained with DAPI, the euchromatic region of Yq exhibits
a single, thin pale band (Yq11.22), which we suspected might correspond
to the DYZ19 island. This hypothesis was confirmed by hybridizing a 
fluorescent DYZ19 probe to DAPI-stained metaphase chromosomes in situ
(FISH analysis; Supplementary Fig. 3a).  The DYZ19 island can now serve
as a cross-referenced landmark linking the light microscopic (cytogenetic)
and sequence-based maps of the Y chromosome.

The massive heterochromatic region of distal Yq, also known as Yq12, is
extraordinarily polymorphic in length (1-3) and typically spans 40 Mb, 
or more than 1% of the haploid genome (4).  We found that the Yq12 
heterochromatin is comprised of at least three distinct sequence species 
(Supplementary Table 10).  Immediately distal to the euchromatic sequences 
of Yq is a relatively homogenous array (DYZ18), not previously described, 
that is based on a GGAAT primary unit with a 2.9-kb secondary periodicity 
(Supplementary Fig. 7L).  Chromosomal FISH analysis indicates that the 
DYZ18 sequences are restricted to this interface with Yq euchromatin and 
are not found elsewhere in the Yq12 heterochromatin (Supplementary Fig. 
2b).  The DYZ1 arrays, based on a GGAAT primary unit with a remarkably 
regular 3.6-kb secondary periodicity (Supplementary Fig. 7m), may comprise 
the bulk of the Yq12 heterochromatin (FISH studies not shown).  The DYZ1 
repeats were the first male-specific DNA sequences to be identified in 
the human genome (5, 6).  We identified no BACs containing DYZ2, a third 
sequence species in Yq12 (Supplementary Table 10), and here only limited 
sequence information is available.  Our FISH studies suggest the existence 
of at least two DYZ2 arrays that are separated by a large mass of DYZ1 
repeats (not shown).

We have not sequenced the entirety of any of the three heterochromatic 
regions, and thus additional sequence species may reside within one or 
more of these regions.  In particular, our efforts to identify the distal 
boundary of the Yq12 heterochromatin were unsuccessful (Supplementary 
Fig. 1).  In the course of these efforts, however, we discovered 65 kb 
of euchromatic, male-specific DNA that is located distal to the Yq12 
heterochromatin but proximal to the long-arm pseudoautosomal region 
(Fig. 2).  These most distal MSY sequences exhibit little similarity to 
the X chromosome or to other regions of the Y chromosome, but they are 
remarkably similar (as much as 98.5% identical) to sequences in human 
chromosomes 1, 2, 7, 10, and 16.  We found no evidence of functional genes 
among these most distal MSY sequences.

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