Isolation and regional localization by in situ hybridization of a unique gene segment to chromosome 21

Vol. 121, No. 1, 1984 May 31, 1984

BIOCHEMICAL

Isolation

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 380-385

and Regional Localization by j,~ &t--u Hybridization of a Unique Gene Segment to Chromosome21

Evelyn A. Devine', Sarah L. Nolin I, George E. Houck, Jr.', EdmundC. Jenkins', David L. Miller' and W. Ted Brown' 1Departments of HumanGenetics and 2Molecular Biology, New York State Office of Mental Retardation and Developmental Disabilities, Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314 Received April

9, 1984

A chromosome21 (ch. 21) flow-sorted library was screened for the presence of unique DNA segments which are specific for the 21st chromosome. By combining the techniques of somatic cell genetics and j& z&& hybridization, we have identified several of these recombinant probes and have regionally mapped one of them to the distal half of the long .arm of This represents the first report of chromosome21 (q22.1->qter). the sublocalization of a unique DNA segment to chromosome21 by ti & hybridization. SUMMARY:

Down Syndrome (DS) is a chromosomal disorder caused by the presence of an extra copy of all

or part (21q22->qter)

of ch. 21

To date, accurate diagnosis of DS can only be made by

(1,2).

chromosomal analysis,

a time consuming and expensive process.

Since the incidence of DS in the general population

is fairly

high, - 1 in 1000 live

of a more

efficient

births

(l),

the availability

assay would be both advantageous and economical.

Recombinant DNA techniques have allowed the identification isolation

and

of unique DNAsegments which can be mappedto

chromosomal regions by Southern blot analysis using somatic cell hybrids

and in gj&

hybridization.

Several of these unique

segments have been used to quantitate

chromosomedosage and would

be useful in the diagnosis of aneuploid conditions We have identified tentatively Abbreviations: 0006-291X/84 Copyright All rights

(3,4).

a series of unique probes which have been

assigned to humanch. 21 by Southern blot DS, DownSyndrome; ch., chromosome.

$1.50

0 1984 by Academic Press, Inc. of reproduction in any form reserved.

380

analysis of

Vol.

121,

No.

1, 1984

DNA from

BIOCHEMICAL

normal

diploid

mouse-human hybrid assignment confirmed

only

of one of these situ

BIOPHYSICAL

fibroblasts,

whose

by in

AND

RESEARCH

COMMUNICATIONS

mouse fibroblasts

human ch. is

probes

21 (WA17d).

(DS21Dl)

hybridization

and a

to

ch. 21 has

and regionally

The been

mapped to

21q22.1->qter.

-IALS

AND METHODS

Cell Lines: Total genomic DNA was prepared as described (5) from normal diploid cell lines, the mouse-human somatic cell hybrid WA17 (6) and from the murine parent of the hybrid cell line, Ag. Isolation of DNA Probm An EcoRI-generated chromosome21 flow-sorted 1fPrary (7) was screened with nick-translated total human DNA (10 cpm/ugm) by the Benton-Davis technique (8) to identify ch. 21 specific sequences which lacked repetitive DNA. The sequences were then hybridized to Southern blots (9) containing 20 mcg each of total human, WA17and A9 DNAto confirm that they contained unique humanDNA. Jn situ Hvbridi&&ion and Autoradioaranhv: The hybridization protocol of Harper and Saunders (10) as modified by Zabel et al. (11) was used. Slides were stained immediately after developing using the method of Zabel et al. (11). The chromosomespreads were analyzed microscopically and grain locations were indicated on a chromosomeidiogram (12).

Isolation

of aniaue

DNASegment: Of the 5,000 recombinant phage

examined, approximately labelled

total

170 (3.4%) showed no hybridization

humanDNAand were selected for a second screen.

Several of these were chosen randomly, amplified restricted

with

EcoRI, blotted

sequences by hybridization approximately

to

individually,

and rescreened for

against total

repetitive

humanDNA.

Of these

70% did not show any homology to repetitive

DNA. In the final and hybridized

screen, the

human

recombinant clones were labelled

to Southern blots containing

20 mcg each of total

human, WA17and A9 DNA. Clones which showed unique bands after hybridization tentatively

and autoradiography assigned to ch. 21.

to total The first

DS21D1, carried

a 7.0 kb insert

characterization

by in situ hybridization.

humanand WA17DNAwere probe to be identified,

and was chosen for

381

further

BIOCHEMICAL

Vol. 121, No. 1, 1984 Jn situ

Hybridizam.

hybridized normal from

overnight 46,Xx,

The major

shown

in

grains of label,

the

to metaphase The

spreads

site

are

metaphase

spread spread

approximately

19

7%

20

clone,

chromosome composite

Fig.

were

was

preparations

in the

was the in

DS21D1,

from

hybridization

illustrated

of hybridization

per metaphase

FIGURE 1:

recombinant

individual.

80 metaphase

1.

The

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

2.

observed

results

idiogram

long

in Fig.

arm of ch.

On the

average,

to the

long

21 as 5.9

and of the total

was localized

amount

arm of 21.

3

4

5

6

15

16

17

18

21

22

Idiogram of high resolution Obanded mid-metaphaee chromosomes showing the composite hybridization data of recombinant probe DSZlDl from 80 metaphase spreads. As is shown, the major area of hybridization is to the long arm of ch.21. Over 53% of the label on this chromosome hybridizes

to q22.1->qter.

382

a

Vol. 121, No. 1, 1984

FIGURE2:

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

U u hybridization of probe DSElDl to a G-banded human metaphase spread. The major area of hybridization to the terminus of ch. 21 is indicated by an arrow.

Of the grains visualized 2lq22.1 ->qter

on ch.

21,

535 were localized

to the

region of the chromosome. In addition,

metaphase spreads which contained a labelled

ch. 21 were examined

in order to obtain more precise mapping data. these grains were also localized

30 more

The majority

to the region q22.1->qter.

of Probe

DS21Dl also appeared to have somehomoiogy to the centromere of ch. 15. However, over 50% of the grains which localized

to this

region were obtained from one of the three slides used in analysis; labelled

whereas the ch. 21 region q22.1->qter on all

of the slides.

These le u

the

was consistently

results

corroborate

the assignment of DS21Dl to chromosome21 and have delineated the mapping

to

q22.1->qter.

383

Vol. 121, No. 1, 1984

In

BIOCHEMICAL

situ

hybridization

be a powerful

of unique

method

We have

genome .

for

screened

precise

a ch.

21 library

of unique

gene fragments

to oh.

21 on the

basis

the

arm

using

q22.1->qter,

The homology

ti

for

the morphological hybridization

are

Southern

blots,

the

Southern

blot

genomic

located

concentrated had

in

the

Alternatively, some degree

may allow

area

probe

order

chromosome,

the

the is&J&

Since

present the

this

techniques. 384

in

unique

probe

DNA families If

be are

the probe

this

could

of oh.

on ch.

15.

21 which

had

on 15.

b is

in a trisomic diagnosis

used to

centromere

a gene

than

may or may not

DNA element,

encode

first

this

which

to the

of

homology

of DNA (13).

to a pseudogene

to be the

more

repetitive

repetitive

could

conditions

conditions

that

regions

to preserve

experiments

stringency

indicating

of

background

in

for

hybridization

for

the

may represent

many low

to a low

is

some evidence

DNA segments

the centromeric

this

regionally

showed

we observed

other

of homology

uhich

the

the

DS21D1,

21 DNA segment.

chromosome DS21Dl

stringent

Second,

the

We believe unique

of

apparent

to total

has been

First,

hybridization,

some homology

explain

integrity

21.

a

assigned

to

this

reasons.

for

human

identified

specifically

15 also

several

DNA with

on ch.

oh.

By lonering

has some affinity

the

patterns

DS21D1,

21,

We believe

less

for

and have

hybridization

ch.

along

to

hybridization.

of

DS21Dl.

hybridization

total

u

centromere to

of

has proved

we have tentatively

One of these,

long

segments mapping

which

of their

human and kTAl7 DNA. to

gene

their

series

mapped

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of

&.Q also

localization the

amount DS using

area

of a of

the

in DS, the recombinant

probe

Vol. 121, No. 1, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

-NT We thank Doctors Krumlauf and Young for kindly their

ch. 21 flow-sorted

Massari

for helpful

library,

providing

Doctors Nick Robakis

us with and Ron

discussions and Marie Cappola for preparation

of the manuscript.

1.

2. 3. 4. 5.

de la Cruz, F.F. and Gerald, P.S. (1981) Trisomy 21 (Down Syndrome) pp. 3-67, University Park Press, Baltimore. Jenkins, E.C., Duncan, C.J., Wright, C.E., Giordano, F.M., Wilbur, L., Wisniewski, K., Sklower, S.L., French, J.H., Jones, C., and Brown, W.T. (1983) Clin. Genet. 2&, 97-102. Junien, C., Huerre, C., and Rethore, M.-O. (1983) Amer. J. Hum. Genet. 35, 584-591. Camerino, G., Grzeschik, K.H., Jaye, M., DeLaSalle, H., Tolstoshev, P., Lecocq, P., Heilig, R., and Wandel, J.L. (1984) Proc. Nat'l. Acad. Sci. 81, 498-502. Blin, N. and Stafford, D.W. (1976) Nucleic Acids Res. 3, 2303-2310.

6. 7. 8. 9. 10.

Dutkowski, R.T., Brown, W.T., and Darlington, G.J. (1984) In Vitro, submitted. Krumlauf, R., J8anpierre, M., and Young, B. (1982) Proc. Nat'l. Acad. Sci. B, 2971-2975. Benton, W.D., and Davis, R.W. (1977) Science 1pd, 180-182. Southern, E.M. (1975) J. Mol. Biol. 98, 503-517. Harper, M.E., and Saunders, G.F. (1982) Chromosoma 83, 431-439.

11.

12. 13.

Zabel, B.U., Naylor, S.L,, Sakaguchi, A.Y., Bell, G.I., and Shows, T.B. (1983) Proc. Nat’l. Acad. Sci. Bp, 6932-6936. Yunis, J.J., Sawyer, J.R., and Ball, D.W. (1978) Cytog. Cell. Genet. 22, 679-683. Manuelidis, L. (1978) Chromosoma 44, 23-32.

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