A contiguous 3-Mb sequence-ready map in the S3-MX region on 21q22.2 based on high- throughput nonisotopic library screenings

Abstract

Progress in complete genomic sequencing of human chromosome 21 relies on the construction of high-quality bacterial clone maps spanning large chromosomal regions. To achieve this goal, we have applied a strategy based on nonradioactive hybridizations to contig building. A contiguous sequence-ready map was constructed in the Down syndrome congenital heart disease (DS-CHD) region in 21q22.2, as a framework for large-scale genomic sequencing and positional candidate gene approach. Contig assembly was performed essentially by high throughput nonisotopic screenings of genomic libraries, prior to clone validation by (1) restriction digest fingerprinting, (2) STS analysis, (3) Southern hybridizations, and (4) FISH analysis. The contig contains a total of 50 STSs, of which 13 were newly isolated. A minimum tiling path (MTP) was subsequently defined that consists of 20 PACs, 2 BACs, and 5 cosmids covering 3 Mb between D21S3 and MX1. Gene distribution in the region includes 9 known genes (c21-LRP, WRB, SH3BGR, HMG14, PCP4, DSCAM, MX2, MX1, and TMPRSS2) and 14 new additional gene signatures consisting of cDNA selection products and ESTs. Forthcoming genomic sequence information will unravel the structural organization of potential candidate genes involved in specific features of Down syndrome pathogenesis.

Figure 1

Example of nonradioactive hybridization (DIG labeled) of a PAC filter containing 27,648 clones with riboprobe 54E24-SP6. Filter is bar-coded (at left). Image was captured by a CCD camera and analyzed with the Xdigitize program. A grid was automatically superimposed on the filter, that is, 48 × 48 squares placed around guide dots (black ink spotted on the filter). Each of the squares contains 12 different clones, spotted in duplicate around the central guide dot, according to the pattern shown in the Classification’s window. Each pair of numbers represents a clone pair (guide dot is noted by −9). Levels of relative intensities are indicated as follows: 3 = high, 2 = medium, and 1 = faint. Two positive clones were scored on this filter; the arrow indicates clone 228A2.

Figure 2

Contig and gene map between D21S3 and TMPRSS2. The sequenced MTP consists of two BACs, 20 PACs, and five cosmids depicted in red. Sizes of clones are indicated in parentheses. Markers are shown above the clone map: STSs are displayed in the lower layer; EST markers are in blue. NotI linking clones are marked with a dot. Riboprobes are shown in the upper layer; riboprobes in magenta correspond to the initial cosmid anchors (pockets are prefixed by ICRF and Soeda’s bins by Q). Riboprobes used for Southern hybridizations to confirm the contig are shown as follows: DIG riboprobes are marked by a green dot and ³²P-labeled riboprobes by a red square at the end of corresponding clones. Known genes are depicted by blue lines below the clones. Transcription units (TUs) are indicated below the PACs in magenta. The raw primary data are shown below the actual MTP and gene map. Positive clone names are given for probes showing <20 hits. Color boxes correspond to probes identifying >20 clones for which only numbers are given (cosmids are indicated by a C and PACs by a P). Box color shadings are as follows: 20–50 hits are light yellow, 50–100 hits are yellow, 100–200 hits are dark yellow, and >200 hits are brown. Each probe is indicated above the clone stack.

Figure 2

Contig and gene map between D21S3 and TMPRSS2. The sequenced MTP consists of two BACs, 20 PACs, and five cosmids depicted in red. Sizes of clones are indicated in parentheses. Markers are shown above the clone map: STSs are displayed in the lower layer; EST markers are in blue. NotI linking clones are marked with a dot. Riboprobes are shown in the upper layer; riboprobes in magenta correspond to the initial cosmid anchors (pockets are prefixed by ICRF and Soeda’s bins by Q). Riboprobes used for Southern hybridizations to confirm the contig are shown as follows: DIG riboprobes are marked by a green dot and ³²P-labeled riboprobes by a red square at the end of corresponding clones. Known genes are depicted by blue lines below the clones. Transcription units (TUs) are indicated below the PACs in magenta. The raw primary data are shown below the actual MTP and gene map. Positive clone names are given for probes showing <20 hits. Color boxes correspond to probes identifying >20 clones for which only numbers are given (cosmids are indicated by a C and PACs by a P). Box color shadings are as follows: 20–50 hits are light yellow, 50–100 hits are yellow, 100–200 hits are dark yellow, and >200 hits are brown. Each probe is indicated above the clone stack.

Figure 3

(A) Vistra green-stained agarose gel of HindIII digest patterns of nine PACs. Marker lanes correspond to λ HindIII. Arrows in lanes 39C17 and 247E2 indicate the position of the restriction fragments identified by 247E2-SP6 riboprobe in B. (B) Autoradiograph from a Southern hybridization of the gel shown in A confirming the overlap between PACs 247E2 and 39C17. Radiolabeled 247E2-SP6 riboprobe identified a single HindIII fragment in PACs 39C17 and 247E2.

Figure 3

(A) Vistra green-stained agarose gel of HindIII digest patterns of nine PACs. Marker lanes correspond to λ HindIII. Arrows in lanes 39C17 and 247E2 indicate the position of the restriction fragments identified by 247E2-SP6 riboprobe in B. (B) Autoradiograph from a Southern hybridization of the gel shown in A confirming the overlap between PACs 247E2 and 39C17. Radiolabeled 247E2-SP6 riboprobe identified a single HindIII fragment in PACs 39C17 and 247E2.