Am. J. Hum. Genet. 51:263-272, 1992

Isolation, Characterization, and Regional Mapping of Microclones from a Human Chromosome 21 Microdissection Library Jingwei Yu,* Judith Hartz,* Yisheng Xu,* Robert M. Gemmill,*t Julie R. Korenberg,t David Patterson, *t and Fa-Ten Kao *T *Eleanor Roosevelt Institute for Cancer Research and TDepartment of Biochemistry, Biophysics, and Genetics, and Cancer Center, University of Colorado Health Sciences Center, Denver; and tAhmanson Department of Pediatrics, Cedars-Sinai Medical Center, University of California, Los Angeles

Summary Thirty-four unique-sequence microclones were isolated from a previously described microdissection library of human chromosome 21 and were regionally mapped using a cell hybrid mapping panel which consists of six cell hybrids and divides chromosome 21 into eight regions. The mapping results showed that the microclones were unevenly distributed along chromosome 21, with the majority of microclones located in the distal half portion of the long arm, between 21q21.3 and 21qter. The number of unique-sequence clones began to decrease significantly from 21q21.2 to centromere and extending to the short arm. This finding is consistent with those reported in other chromosome 21 libraries. Thus, it may be inferred that the proximal portion of the long arm of chromosome 21 contains higher proportions of repetitive sequences, rather than unique sequences or genes. The microclones were also characterized for insert size and were used to identify the corresponding genomic fragments generated by HindIII. In addition, we demonstrated that the microclones with short inserts can be efficiently used to identify YAC (yeast artificial chromosome) clones with large inserts, for increased genomic coverage for high-resolution physical mapping. We also used 200 uniquesequence microclones to screen a human liver cDNA library and identified two cDNA clones which were regionally assigned to the 21q21.3-q22.1 region. Thus, generation of unique-sequence microclones from chromosome 21 appears to be useful to isolate and regionally map many cDNA clones, among which will be candidate genes for important diseases on chromosome 21, including Down syndrome, Alzheimer disease, amyotrophic lateral sclerosis, and one form of epilepsy.

Introduction Chromosome 21 is the location of genes important for Alzheimer disease (AD) (St George-Hyslop et al. 1987), amyotrophic lateral sclerosis (ALS) (Siddique et al. 1991), leukemia (Rowley 1981), at least one form of epilepsy (Lehesjoki et al. 1991), and mental retardation (Munke et al. 1988). It is the chromosome which, when present in three copies, is responsible for Down syndrome (DS). Recent work shows that Received January 22, 1992; revision received March 11, 1992. Address for correspondence and reprints: Dr. Fa-Ten Kao, Eleanor Roosevelt Institute, 1899 Gaylord Street, Denver, CO 80206.

o 1992 by The American Society of Human Genetics. All rights reserved. 0002-9297/92/5102-OOO5$02.00

specific regions of chromosome 21 can be associated with specific phenotypes (Epstein et al. 1991; Korenberg et al. 1990, 1992). Molecular genetic analysis, including physical mapping, of chromosome 21 is essential for understanding the role of genes on chromosome 21 in human development and disease. This analysis requires the availability of large numbers of unique-sequence probes for the construction of highresolution physical maps of chromosome 21 and for the identification of many expressed sequences encoded by chromosome 21 as candidate genes, for analyzing these diseases. In spite of considerable effort expended thus far, the number of chromosome 21specific DNA clones available is still insufficient, especially for certain regions of the chromosome. Thus, 263

264

techniques such as chromosome microdissection and the construction of region-specific libraries (Scalenghe et al. 1981; Rohme et al. 1984; Ludecke et al. 1989) may be important in overcoming these difficulties. Elsewhere we have described a procedure for microdissection of human metaphase chromosomes and for the subsequent cloning of the minute quantities of DNA into plasmid vectors to construct large genomic libraries, by means of a microcloning technique assisted by PCR (Kao and Yu 1991). This technology provides direct access to important chromosomal regions, for isolating large numbers of probes for molecular genetic analysis, as first described by Scalenghe et al. (1981) using Drosophila polytene chromosomes and as later applied to mouse chromosomes (Rohme et al. 1984). The technology has become more efficient with the incorporation of PCR to amplify the dissected DNA to construct large libraries from banded and stained human chromosomes (Ludecke et al. 1989). This added feature offers additional advantages in that (a) only 20-30 chromosome fragments are needed for microdissection (Ludecke et al. 1989) and (b) the resulting libraries are large, for a more complete representation of the dissected chromosomal region (Saunders et al. 1989). Because of the increased precision of microdissection, the resulting microclones in the microdissection library were generally derived from the dissected region once they could be confirmed as being of human origin and chromosome specific (Buiting et al. 1990; Kao and Yu 1991). This technology has gained wide applicability in the construction of region-specific libraries for studying chromosomal regions associated with genetic diseases, e.g., the Prader-Willi syndrome region (Buiting et al. 1990), the fragile-X region (MacKinnon et al. 1990), the Langer-Giedion syndrome region (Ludecke et al. 1991), the neurofibromatosis-2 region (Fiedler et al. 1991), and the adenomatous polyposis coli region (Hampton et al. 1991). We previously used this technology to construct a microdissection library for whole human chromosome 21, for general studies of DS and other disease loci on this chromosome (Kao and Yu 1991). This library consists of 700,000 recombinant microclones with a mean insert size of 416 bp. Individual clones from the library were isolated and screened for unique-sequence inserts by using total human DNA as labeled probe in colony hybridization. About 60% of the clones in the library contained unique or very-lowcopy-number sequences. A subset of microclones isolated from the library were confirmed to be of human

Yu et al. origin and chromosome 21 specific. Two microclones were regionally mapped on chromosome 21 by using four cell hybrids from a mapping panel. In this paper, we report both isolation of additional unique-sequence microclones from the library and regional mapping of a total of 34 unique-sequence microclones, 2 of which have been described elsewhere, by using a smaller mapping panel (Kao and Yu 1991). In the present study, a larger mapping panel, consisting of six cell hybrids and dividing chromosome 21 into eight regions, was used, thus providing a better assessment of the general distribution of the uniquesequence microclones on the chromosome. The microclones have been characterized for insert size, and their corresponding genomic fragments generated by HindIII cleavage have been detected. In addition, for microclones to be most useful for molecular analysis of chromosome 21, they need to be usable for screening genomic and cDNA libraries. In this paper, we demonstrate the efficient screening of a human YAC (yeast artificial chromosome) library by using the unique-sequence microclones as labeled probes in Southern hybridization, and we identify corresponding YAC clones with inserts several hundred times larger than those in the microclones. We also show in this paper that the microclones with uniquesequence inserts can be pooled for screening cDNA libraries to isolate cDNA clones derived from chromosome 21. Since only a limited number of genes (fewer than 20) have been cloned for chromosome 21 (Solomon and Rawlings 1991), it seems desirable to isolate as many cDNA clones as possible from this chromosome, to serve as candidate genes for studying DS, AD, ALS, and other diseases and phenotypes associated with chromosome 21. The general approach described here, using microdissection and microcloning techniques to construct region-specific libraries and using unique-sequence microclones to isolate regionspecific YAC clones and cDNA clones, appears valuable for high-resolution genome analysis of chromosome 21 and for an efficient search of crucial genes underlying many genetic diseases and specific forms of cancer that have known regional positions on the chromosome. Material and Methods Isolation and Insert-Size Determination of Unique-Sequence Microclones of Chromosome 21 The procedures for isolation of unique-sequence microclones from the chromosome 21 microdissection

Chromosome 21 Microdissection Clones

library and for the determination of insert size were essentially the same as those described elsewhere (Kao and Yu 1991). In brief, DNA samples of the PCR products from the library were cleaved with Mbol and were cloned into pUC19. Recombinant microclones were individually isolated and screened for unique-sequence clones by using labeled total human DNA in colony hybridization. The insert size in the microclones was determined by PCR using the following primers flanking the multiple cloning site of pUC19: 5'-ACAGGAAACAGCTATGACCA-3' and 5'-CGTTGTAAAACGACGGCCAG-3'. Bacterial colonies containing recombinant pUC1 9 were used directly for PCR, and the insert size was determined by gel electrophoresis (2% NuSieve + 1% agarose). In the microdissection library, over 80% of the unique-sequence microclones were usable clones which contained inserts greater than 50 bp and were shown to be of human origin and derived from chromosome 21. Hybrid Mapping Panel of Chromosome 21 Figure 1 presents a diagram of the hybrid mapping panel. The following six cell hybrids containing partial chromosome 21 and dividing the chromosome

into eight regions were used for regional mapping: 153E7b (containing 21pl1.1 -qter, including the centromere and the entire long arm), R50-3 (containing 21q1 1.2-qter and defined by the translocation breakpoint in t[6;21]), 21q + (containing 21pter-q22.1 and defined by t[8;21]), GA9-3 (containing 21q21.1-qter and defined by t[4;21]), 1881C-13b (containing 21q21.2-qter and defined by t[1;21]), and JC-6 (containing 21pter-ql1.2 and 21q21.3-qter). Detailed

analysis of these hybrids and the karyotype have been described elsewhere (Van Keuren et al. 1986; Gardiner et al. 1988, 1990; Patterson et al. 1988). In addition, DNAs from Chinese hamster ovary (CHO), human (a lymphoblastoid cell line), and cell hybrid 38-14 containing an intact chromosome 21 (Kao and Yu 1991) were used as controls. Southern Blot Hybridization of Microclones by the Hybrid Mapping Panel The insert from each microclone was amplified by PCR using primers specific for pUC19, as described above. The PCR products from each clone were puri-

fied by low-melting-point agarose-gel electrophoresis and were labeled with [32P]dCTP by the primer extension procedure, as described elsewhere (Kao and Yu 1991). The DNAs from the mapping panel were cleaved with HindIII, were separated by gel electro-

265 13

P

11.2 IS.I 11.1

11.2

6'21

q 21.1 21.2 21.3

4;21 1;21 J34

22.1 121

22.2 22.3

21

IW.MF.h R5O. 1(6;21

21q+

t(;21U

GA9-3 t(4:21)

IUI(.-

JC.

13b t(121)

Figure I Regional mapping panel of chromosome 21, comprising six cell hybrids and dividing the chromosome into eight regions, each of which is defined by a breakpoint identified by cytogenetic analysis as described in the text. The vertical lines represent the portions of chromosome 21 that are retained in the respective hybrids.

phoresis, and were transferred to a Hybond-N + nylon membrane (Amersham) for 2-3 h by the alkali-blotting procedure (transfer buffer 0.4 NaOH), according to the manufacturer's protocol. The filters were prehybridized at 65°C for 2 h before the probes were added at a concentration of 1-3 x 106 cpm/ml. Hybridization was done at 65°C for 16-20 h, and washing included a final wash in 1 x SSPE + 0.1% SDS at 650C for 10-20 min. The X-ray film was exposed for 4-5 d. YAC Library Screening Using Pooled YAC Clones and Pooled Unique-Sequence Microclones

A new YAC library screening method described by Mendez et al. (1991) was used. In brief, the human genomic YAC library of Washington University (Brownstein et al. 1989), containing approximately 60,000 clones (5 x genomic equivalent) was arrayed onto three gels, with 50 lanes/gel. Each lane contained DNAs from 384 pooled YAC clones. After pulsed-field gel electrophoresis (PFGE) and Southern transfer, the filters were hybridized to the pooled microclones. A pool of three unique-sequence microclones was used in the screening. The inserts in the microclones were individually amplified by PCR, were purified by low-melting-point gel electrophoresis, were pooled to prepare labeled probes by primer extension, and hybridized to filters at 420C overnight at a concentration of 2 x 106 cpm /ml, in a hybridization

Yu et al.

266

buffer containing 50% formamide, 5 x SSC, 1 x Denhardt, 20 mM NaPO4 pH 6.7, 100 ,ug denatured salmon sperm DNA/ml, and 10% dextran sulfate. The filters were washed three times, including a final wash in 0.1 x SSC + 0.1% SDS at 650C for 30 min. The X-ray film was exposed for 2-9 d at - 700C. Screening of a cDNA Library, Using Pooled Unique-Sequence Microclones

Inserts from unique-sequence microclones were individually amplified and pooled, 10 or 20 clones in a group, to prepare labeled probes. A human liver Xgtl 1 cDNA library, provided by Dr. Savio Woo, was used. Phages from the library were inoculated at a density of 50,000 pfu/150-mm dish. One or two dishes were screened for each group ofpooled microclones. Duplicate filters were prepared for each dish and were used in hybridization to confirm positive signals. Labeled probes at a concentration of 2 x 106 cpm/ml were added to each bag and hybridized at 420C for 16 h in a hybridization buffer containing 50% formamide, 2.5 x Denhardt, 1 M NaCl, 1% SDS, 25 mM phosphate buffer pH 6.8, and 100 gg denatured salmon sperm DNA/ml. After hybridization, the filters were washed two times, for 5 min each, at room temperature in 2 x SSC + 0.1% SDS, and then in 0.2 x SSC + 0.1% SDS at 550C for 30 min. The X-ray film was exposed for 3-5 d. Positive areas detected in both duplicate filters in the primary screening were isolated, and the plaques were purified by secondary and tertiary screening. Results Regional Mapping of 34 Unique-Sequence Microclones, Using the Hybrid Mapping Panel The chromosome 21 regional mapping panel used in the study comprised six cell hybrids which divided the chromosome into eight regions as shown in figure 1. The precision of the division in each region was based on cytogenetic resolution analyzed on the translocation chromosomes in the hybrids and also on physical mapping, as reported elsewhere (Gardiner et al. 1988, 1990). Figure 2 presents the assignment of 34 microclones to the eight regions on chromosome 21. The insert sizes and the hybridizing genomic HindIII bands of each microclone are summarized in table 1.

Typical Southern blot hybridization of the microclones by use of the hybrid mapping panel is shown in figure 3.

13

12 P 11.2 11.1

MM5Eh 21I-201

11.2

JC-6

q 21.1

4;21

21.2 21.3

121 JC4

22.1 22.2 22.3

21E-210 21E-313 21 E-.W 21 E-222 21 E-226 21 E:-L. 21 E-22 21 F-353

21E-24 21E-35 21E-56 21F-101 21E-1" 21E-2l1 21E-253 2F1200

21E-312 21E-327 21E-352 '1F-356

8;1.:

21 E-362 21 E479 21E-30

21MA4I 21E-209

21E-215 21F-239 21F-246 21E-2S1 21E.256 211-333 21 .-335 21 F.339

21 Regional assignment of 34 unique-sequence miFigure 2 croclones from a microdissection library of chromosome 21. For details of these microclones, refer to table 1.

From the regional mapping results shown in figure 2, it is clear that the unique-sequence microclones are largely distributed in the distal half of the long arm, starting from the Giemsa dark band 21q21.3 and proceeding toward 21qter. The number of uniquesequence microclones was drastically decreased from 21q21.2 toward the proximal part of the long arm and extending to the short arm. Thus, nearly 90% (30 of 34) of the clones were clustered in approximately 60% of the long arm. However, the dark-band region 21q21 is not completely devoid of unique-sequence clones. The distal portion of 21q21, including 21q21.3, contains as many (if not more) uniquesequence microclones per unit length as does the Giemsa light-band region 21q22. Only the proximal portion of the dark band 21q21 appears to have significantly fewer unique-sequence clones. As shown in table 1 and figure 3, several microclones, including 21E-210 (fig. 3F), 21E-209 (fig. 3E), and 21E-251, also showed cross-hybridization with CHO DNA. More information on species conservation in other microclones and with other species should be available when a "zoo" blot with more species is used. Although the present study used only a limited number of individuals and a few restriction enzymes, some information on RFLP was evident in some microclones, as described in table 1 and figure 3. A more extensive survey, using additional enzymes and individuals, should reveal more information on RFLP in these microclones.

Chromosome 21 Microdissection Clones

267

Table I Regional Location, Insert Size, and Hybridizing Hindill Genomic Bands of 34 Chromosome 21 Microdissection Clones

Chromosomal Region 21pter-pl1.1 ............. 21pll.l-ql1.2 .......... 21ql1.2 ................... 21q21.l ................... 21q21.1-q21.2 ........... 21q21.2-q21.3 ...........

21q21.3-q22.1 ...........

21q22.2-qter .............

Microclone

Insert Size

Hindill Bands

(bp)

(kb)

21E-201

740

5.8 and 6.2

21E-210 21E-313 21E-340 21E-222 21E-226 21E-238 21E-282 21E-353 21E-24 21E-35 21E-56 21E-181 21E-199 21E-231 21E-253 21E-280 21E-312 21E-327 21E-352 21E-356 21E-362 21E-479 21E-30 21E-41 21E-209 21E-215 21E-239 21E-246 21E-251 21E-286 21E-333 21E-335 21E-339

390 400 550 170 490 270 360 870 250 300 280 850 380 740 220 370 480 380 1,210 384 210 1,300 500 560 740 380 270 220 720 360 280 630 690

9.4 1.2 11.0 7.7 1.95 and 4.5 2.4 3.3 10.5 9.2 7.8 14.1

13.8 13.2 6.8 5.1 and 3.9 4.1 .95 and .85

Remarks

Contiguous HindIlI fragments Cross-hybridizing to CHO at 3.6 and 1.38 kb (fig. 3F)

Fig 3B Fig 3A Polymorphic for HindIlI (fig. 3D)

Polymorphic for EcoRI Fig. 3C; identifying cDNA 21E-C1 Seven human bands; 6.8 kb unique to chromosome 21 Polymorphic for HindIII Two contiguous HindIII fragments

3.6

9.1 and 2.8 6.6 1.6 5.1 and 2.7 5.7 and 3.5 13.8 13.0 2.4 5.9 6.0 6.2 2.4 2.6 7.6 and 12.3 14.7

Use of Unique-Sequence Microclones in Screening of a YAC Library The human genomic YAC library was screened with unique-sequence microclones by using the pooled YAC screening procedure (Mendez et al. 1991) as described in Material and Methods. Three microclones were used as pooled probes: 21E-56 (280 bp), 21E181 (850 bp), and 21E-199 (380 bp) (table 1). After two-thirds of the library (two filters) was screened, five positive YAC clones were identified. Three of the

Polymorphic for HindIll Fig. 3E Two contiguous HindlIl fragments; identifying cDNA 21E-C2 Two contiguous HindIlI fragments

Cross-hybridizing to CHO at 3.9 and 1.35 kb (fig. 3E)

Cross-hybridizing to CHO BamHI band at 4.7 kb

Polymorphic for HindIll

five positive YACs were detected in one filter and are shown in figure 4. One positive YAC clone was found in lane B66-B69, with an insert of approximately 250 kb. Another positive clone was in lane B101-B105, with an insert of approximately 230 kb. One YAC clone with a small insert of about 50 kb was found in lane B169-B172. Individual YAC clones corresponding to each microclone can thus be isolated from the 384 pooled YACs by hybridization of the microclones to colony filters prepared from the four microtiter

Yu et al.

268 A

kb

kb

11.0 7.7

1 23 45 6

C.-,,

7 8

D 4

I

4

a a

I

13.8

a

1

Z

3

4 5

78

E

4

.49.4

13.0

43.6 .4

3.9 7

m

1.35 *i

1

2 3

4

5

6

1.3

7 8

,

r-T.. ..h I;S 3S"

..

1 2 3 4 5 6 7 8

Southern blot analysis for regional assignment of microclones by using a cell hybrid mapping panel. Lanes 1, CHO-K1. Figure 3 Lanes 2, Human. Lanes 3, lS3E7b. Lane 4, R50-3. LaneS5,21q + (8;21). Lane 6, GA9-3 (4;21). Lane 7,1881-Cl3b (1;21). Lane 8,JC-6. A, 21E-222, negative for hybrid JC-6 (lane 8) and mapped to 21q21.2-21.3. B, 21E-340, negative for hybrids 1881C-13b (lane 7) and JC-6 (lane 8) and mapped to 21q21.1-q21.2. C, 21E-181, positive for all hybrids and mapped to 21q21.3-q22.1; this microclone also identifies the cDNA clone 21E-C1. D, 21E-226, polymorphic for HindIll sites and yielding two fragments, at 4.5 and 1.95 kb, and negative for JC-6 (lane 8) and mapped to 21q21.2-q21.3. E, 21E-209, cross-hybridizing to CHO (lane 1) to produce two fragments, at 3.9 and 1.35 kb; the human band at 13.0 kb was absent in hybrid 21q + (lane 5) and mapped to 21q22.2-qter. F, 21E-210, cross-hybridizing to CHO (lane 1) at 3.6 and 1.38 kb; the human band of 9.4 kb was absent in hybrids GA9-3 (lane 6), 1881C-13b (lane 7), and JC-6 (lane 8) and mapped to 21q21.1.

plates constituting the pool. Thus, it appears that the pooled YAC screening method can be used in screening YAC libraries with unique-sequence microclones, to expand the genomic coverage in the dissected region.

Isolation and Preliminary Characterization of cDNA Clones from Chromosome 21

Table 2 presents the results of screening a human liver cDNA library by pooling 10 or 20 uniquesequence microclones from the chromosome 21 li-

269

Chromosome 21 Microdissection Clones CD

B66 B69

B101 I

X:

B105

>-

V

V

kb

V

580_ 440_.. 360 280 230 t

ItI I

A

8169 1 B172

Figure 4 Screening of the human YAC library by a rapid procedure using pooled YAC clones and pooled unique-sequence microclones. The gel shown in the left photo contained SO lanes, with 384 YAC clones/lane, separated by PFGE. DNA was transferred to filter for Southern hybridization using three pooled microclones as mixed probes. The photo on the right shows an autoradiogram of one of the filters containing three positive YAC clones, indicated by arrowheads. The corresponding lanes on the pulsed-field gel are indicated by the arrowheads in the photo on the left. YPH149 is a yeast chromosome size standard which was included in one well per row.

Table 2 Screening of Human Liver Xgtl I cDNA Library by Using Pools of Microclones from Human Chromosome 21 Microdissection Library

No. of Clones Pooled

No. of Positive Clones per Plate

10 ............ 10 oa............

Oa

10 ...... 10 oa............ 10 oa............ 10 oa............

lb

10 10 10 10

............

Oa

............

Oa

............ ............

20 ...... 20 ...... 20 ....... 20 ...... 20 ...... Total .... 200

cDNA Clone Identified

21E-C1

0.

Oa

0' Oc

1

21E-C2

0' 0' 2

Two plates screened. Two plates screened, one of which had one positive clone. ' One plate screened. a

b

brary that were used as labeled probes. Two cDNA clones, 21E-C1 and 21E-C2, were isolated after screening the library with 200 microclones. Preliminary characterization of the cDNA clone 21E-C1 showed that it contains an insert of 1.5 kb. This cDNA clone was isolated from a pool of 10 microclones and was identified as derived from microclone 21E-181. Regional mapping assigned 21E-181 to 21q21.3q22. 1 (figs. 2 and 3C and table 1). This mapping position was also confirmed by using cDNA clone 21E-C1 as labeled probe (data not shown). Partial sequencing of this cDNA clone revealed that the sequence bears no homology with any of the previously reported DNA sequences in the GenBank (IntelliGenetics, Inc.), an indication of a possible new cDNA clone from chromosome 21. Complete sequencing of the cDNA clone in progress. The other cDNA clone, 21E-C2, contains an insert of approximately 1 kb. This cDNA clone was isolated from a pool of 20 microclones and was subsequently identified as derived from microclone 21E-479, which was mapped to the same region as 21E-C1, i.e., is

21 q21.3-q22.1

(fig.

2 and table

1). This mapping posi-

270 tion was confirmed by using 21E-C2 as probe. Partial sequencing of 21E-C2 also failed to reveal any homology with reported DNA sequences in the GenBank; thus it is also a possible new cDNA clone from chromosome 21. Discussion

Regional mapping of 34 microdissection clones containing unique-sequence inserts established local assignment of the clones on human chromosome 21. The microdissection library described here is particularly suitable for the isolation of large numbers of unique-sequence probes by a simple screening procedure. The microclones with short inserts are convenient for sequencing, to prepare sequence-tagged sites (STS) at strategic locations as genomic landmarks for chromosome 21 (Olson et al. 1989). In addition, the microclones can be conveniently used as probes to screen YAC libraries for isolating corresponding YAC clones with large inserts. Thus, if a chromosomal region of 10 Mb is microdissected and cloned, it appears feasible to use unique-sequence microclones from the library to isolate 20-35 corresponding YACs with an average insert size of 500 kb, to construct contigs and high-resolution physical maps for the entire dissected region.

Yu

et

al.

should be helpful to isolate more cDNA clones as candidate genes for DS and other disease loci on this chromosome. The present approach appears worthy of being developed further for an efficient isolation of cDNA clones from region-specific libraries. The regional mapping of microclones from the microdissection library shows uneven distribution on the chromosome (fig. 2). This finding is consistent with several other studies in which uneven distribution of unique-sequence probes on chromosome 21 has been reported; in most of these studies, the majority of unique sequences have clustered in the distal half of the chromosome (Korenberg et al. 1987; Gardiner et al. 1988, 1990; Tantravahi et al. 1988; Gao et al. 1991), and relatively fewer genes have clustered in the proximal half of the chromosomes (Gardiner et al. 1988, 1990). Thus, these findings may suggest that the proximal portion of the long arm of chromosome 21, including the large Giemsa dark band 21q21, contains higher proportions of repetitive sequences than of unique sequences and genes. It should also be pointed out that it is not the entire Giemsa dark band 21q21 that is less represented by unique sequences. A significant portion of this dark band, including 21 q21.3, appears to contain unique sequences as abundant as those in the Giemsa light-band region 21q22 (fig. 2). In fact, the gene coding for the 0-amyloid precursor protein is located in this dark-band region, at 21q21.2-q21.3 (Patterson et al. 1988). Thus, one should not be tempted to generalize that any Giemsa dark band in the human genome is a region with abundant repetitive sequences. If there is uneven distribution of unique sequences isolated from chromosome 21 libraries, it appears that one of the most efficient ways to isolate unique-sequence probes from the proximal portion of the long arm of chromosome 21 would be by direct microdissection and cloning of this region. We are currently constructing microdissection libraries specifically for the 21q21 region. Such directed efforts should yield unique-sequence probes from any underrepresented region, so that sufficient YAC clones can be isolated for contig construction and physical mapping and so that cDNA clones can be isolated for studying genotype/phenotype correlations (Korenberg et al. 1990, 1992; Epstein et al. 1991).

The microdissection library is also valuable in providing large numbers of unique-sequence probes for direct screening of cDNA libraries, without the problem of repetitive sequences commonly present not only in most genomic clones with larger inserts but also in some cDNA clones. In our pooled probes of 10 or 20 unique-sequence microclones as a group, corresponding cDNA clones can be confidently identified without interference due to repetitive sequences. It is important, however, that the procedure and criteria used for isolating unique-sequence clones must be carefully exercised, to avoid possible inclusion of some lowrepetitive-sequence clones. Occasionally, microclones containing very low repeats or members of a gene family may be selected, and corresponding cDNA clones may be isolated. These clones can be identified by subsequent Southern and northern analyses. The results for the two cDNA clones isolated in the present study, although preliminary, suggest that they could be new cDNA clones from chromosome 21. Complete sequencing and detailed characterization of these cDNA clones should provide more definitive evi-

Acknowledgments

dence about transcribed gene sequences and may reveal their possible functions. Since the number of cloned genes on chromosome 21 is still limited, it

We thank Michael Mendez, Suhong Tong and Jinna Shu for excellent technical assistance. This work was supported by NIH grants HD17749 (to D.P. and F.-T.K.), CA41183,

Chromosome 21 Microdissection Clones and HG00353 (both to R.M.G.), Department of Energy grant DE-FG02-91ER61139 (to F.-T.K.), The Council for Tobacco Research USA grant CTR2321 (to F.-T.K.), and by grants from The American Health Assistance Foundation (to J.R.K.), The Alzheimer's Association (to J.R.K. and D.P.), The March of Dimes (to J.R.K.), and The Lucille P. Markey Charitable Trust (to R.M.G.).

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