Vol. 144, No. 2, 1987

April 29, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1069-]075

THE MOUSE HOMOLOG OF THE HUMAN AMYLOID B PROTEIN (AD-AP) GENE IS LOCATED ON THE DISTAL END OF MOUSE CHROMOSOME 16, FURTHER

EXTENSION OF THE HOMOLOGY BETWEEN HUMAN CHROMOSOME 21 AND MOUSE CHROMOSOME 16

Michael Lovett*, Dmitry Goldgaber§, Patricia Ashley*, David R. Cox*, D. Carleton Gajdusek§, and Charles 3. Epstein*

*Departments of Pediatrics, Biochemistry, and Biophysics, University of California, San Francisco, CA 94143

§Laboratory Of Central Nervous System Studies, National Institute of Neurological and Communicative Disorders and Stroke,

Bethesda, ~ 20892

Received March 24, 1987

SUGARY: The human amyloid B protein is the major constituent of the brain amyloid plaques found in Alzheimer disease. The gene that encodes this protein is located on chromosome 21, and individuals with Down syndrome (trisomy 21) also exhibit an early onset form of Alzheimer disease. We have used the cloned human amyloid B protein gene and a panel of somatic cell hybrids to map the location of the mouse homolog of this gene. We report here that the mouse gene is located on chromosome 16 within the region 16C3+ter, in common with three other genes which map within the Down syndrome region of human chromosome 21. ~ ~987 Ac~d~io Press, ~

Alzheimer Disease (AD) is a progressive neurodegenerative disorder in

humans that is characterized by the presence of neuritic plaques and other

neuropathological lesions in the brains of affected individuals (1,2).

Neuritic plaques are composed of an amyloid plaque core (APC) of lOnm fila-

ments surrounded by degenerating neurites (3,4). Similar filaments are

also found outside neuritic plaques in cerebrovascular amyloid (CVA) and

neurofibrillary tangles (5,6,7). A 4.2 kilodalton amyloid B protein subun-

it is a major constituent of both types of filaments, and analysis of both

APC and CVA amyloid B proteins has revealed an identical 28 amino acid

sequence (5,6,8). Recently, four groups (9-12) have isolated molecular

clones from brain cDNA libraries using synthetic oligonucleotide probes

that correspond to the APC amino acid sequence. These cDNA clones are

derived from a 3.4 kb transcript that is expressed in a variety of tissues,

the product of which is presumably cleaved to yield the short amyloid B

protein subunit. The gene for this transcript is on human chromosome 21

(9-12).

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0006-291X/87 $1.50 Copyright © 1987 by Academic Press, Inc.

All rights of reproduction in any form reserved.

Vol. 144, No. 2, 1987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A proportion of AD cases (familial Alzheimer disease) are caused by a

dominant genetic defect that has been mapped to human chromosome 21 (13),

and the amyloid B protein gene appears to be closely linked to this locus

(lO,13). This observation is particularly interesting because individuals

with Down syndrome (trisomy for human chromosome 21) develop an early onset

form of AD in the fourth decade of life (14). However , the region to which

the amyloid B protein (AD-AP) gene maps (21qll.2÷q21) (lO) may be more

centromere-proximal than the region that is considered to be the obligate

Down syndrome region (21q22.1÷qter) (15,16).

Comparative gene mapping has demonstrated that four genes within the

obligate Down syndrome region of human chromosome 21 are located on mouse

chromosome 16 (17-20). In addition, trisomy 16 mice and, to a lesser

extent, trisomy 16 ~ 2n chimeric mice share several phenotypic abnor-

malities with Down syndrome (21-23). On the basis of these observations,

mouse trisomy 16 has been identified as an animal model of Down syndrome.

In view of the potential role of the gene for amyloid B protein in one of

the Down syndrome phenotypic abnormalities, and in order to assess further

the degree of homology between human chromosome 21 and mouse chromosome 16,

we undertook to map the chromosomal location of this gene in the mouse. We

report here that the mouse homolog of the human AD-AP gene is located on

the distal end of mouse chromosome 16, within the region 16C3,*ter.

MATERIALS AND METHODS

Parental and hybrid cells: Chinese hamster x mouse somatic cell hybrids segrega-ting mouse chromo-

somes were made by polyethylene glycol fusion of mouse spleen cells or peritoneal macrophages with an established Chinese hamster cell line (380-6) deficient in hypoxanthine phosphoribosyltransferase (EC 2.4.2.8). The derivation and characterization of these hybrid clones have been pre- viously described (24). Ten hybrid cell lines, which together contain all the mouse chromosomes except for chromosomes 6, ll, 17 and the Y chromo- some, were used (Table 1). The genetic constitution of the hybrid cells was verified by electrophoretic analysis of mouse chromosome-specific gene products and/or karyotype analysis at the time the DNA was prepared.

Gel electrophoresis, Southern transfer, and hybridization: High molecular weight DNAs isolated from cell lines or tissues were

digested with a five-fold excess of the indicated restriction endonucleases (New England Biolabs). Samples of lOpg were separated by electrophoresis in 0.8% agarose gels. Gels were cast and run in 40 mM Tris, 1 mM EDTA, 50 mM sodium acetate, pH 7.6. DNAs were transferred to MSI Magna Nylon 66 membranes (Micron Separations Inc.) or nitrocellulose filters (25). Nylon filters were prehybridized and hybridized in 0.5 M sodium phosphate, pH 7.4, 7~ sodium dodecyl sulfate (SDS), l~ bovine serum albumin (BSA), and 1 mM EDTA at 50"C. Nitrocellulose filters were prehybridized and hybridized in 3x SSC (Ix SSC is 0.15 M NaCI, 0.015 M sodium citrate), 0.1% SDS, 0.2% polyvinylpyrrolidone, 0.2% BSA and 0.2% ficoll at 50"C. All filters were washed in O. lx SSC, 0.1% SDS at 60"C for 1 hour and were autoradiographed.

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DNA probes: The amyloid B protein was a human cDNA (kAm4) 1.1 kb in length that

corresponds to the 3° end of the gene and contains within it the amyloid core sequence (9)' The control pRI probe was a single copy mouse genomic DNA EcoRI fragment 1.1 kb in length that is located on chromosome 2 adja- cent to the agouti coat color locus (26). DNAs were 32P-radiolabeled to specific activities of >lO 8 cpm/~g by nick translation (27).

RESULTS

The human amyloid 6 protein cDNA clone lAm4 was assigned to a mouse

chromosome using a panel of ten Chinese hamster-mouse somatic cell hybrids

that segregate mouse chromosomes. Southern blots of mouse and Chinese

hamster genomic DNAs digested with PstI and hybridized with XAm4 revealed a

major cross-hybridizing mouse DNA fragment of 1.9 kb, clearly resolvable

from the hamster fragment of 2.5 kb (Fig. t). Analysis of the segregation

of the AD-AP gene and mouse chromosomes in the hybrid cell clones showed

concordant segregation with chromosome 15 and discordant segregation with

each of the other mouse chromosomes (Table l, Fig. l). In addition, a

number of lo~er homology mouse DNA fragments ~ere seen at the moderate

hybridization stringencies ~e employed in these experiments. However, the

signal intensities of these fragments were too lo~ to permit mapping of

their chromosomal location(s).

To confirm the chromosome 15 location independently~ ~e conducted an

analysis of the dosage of the amyloid B protein se%enees in the genomic

~ Detection of mouse DNA seQt~iqce{ bo-qo o.om t' \~¢.~:L ]~- somatic cell s. Genomic DNAs (tO pg per ]~,;,;:; ~rom ,T,c:,sc, ~":~t.r..~ -,rid the -'_,:ci-

ca ted h y b r i d c e l l l i n e s were d i g e s t e 3 w i t h ,~.%T electrc:~,,h:,,.,:~sed i n a 0 ,8~ agarose g e l , t r a n s f e r r e d to an MS[ n'~]~n memorane ant! h y b r i d i z e d ~,itk, r a d i o l a b e l e d XAM4. At the r i g h t a l e showq the ; ] -~iL.{cns a t ~:~hJch the major c r o s s - h y b r i d i z i n g hamster (H) and mouse (~f! DN.q ftagmer, i ; ' : , :g ra te . TV~ mouse chromosome c o n t e n t o f t he h y b r i d c e - i ~ ~s ts sr.,,,~':~ i n Table i ,

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Vol. 144, No. 2, 1987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table i. Mouse chromosome content of cell hybrids

Mouse chromosome

Hybrid i 2 5 4 5 6 7 8 9 i0 II 12 13 14 15 16 17 18 19 X 216 162 Gene*

F]

III-13 + + + + L+J + + I-3A-2 + + + + + + + + + + + VT-25 + [4 [÷] + + + VI-21 + [+i + + + + + + VI-6 + + AdCT-25 + + I-7B-4 + + + + + + + I I I - 2 3 (+ ) + [+ ] + + + [+ ] + (+ ) 1-8-5 + + + + + + I - 3 A - 3 + + + + + + +

* + indicates presence of the 1.9 kb PstI mouse DNA fragment (Fig. i) [ ) Indicates the presence of translocat-Ton chromosomes (25) [ ] Indicates the presence of part of the chromosome by isozyme data

DNA of mice trisomic for chromosome 16 compared to diploid control DNA.

After densitometry and normalization to an internal control probe that is

located on chromosome 2, the signal from trisomy 16 DNA compared to control

DNA shows a ratio of 1.57:1.0, very close to the expected ratio of 1.5:1.0,

thus confirming the chromosomal assignment (Table 2).

Three of the somatic cell hybrids used in this study were derived from

a mouse homozygous for the balanced reciprocal chromosomal translocation

T(2;16)28H and have retained a portion of chromosome 16 either proximal

(16 2) or distal (2 .6) to this translocation breakpoint. Therefore, we can

Table 2. Dosage analysis of AD-AP homologous locus in trisomy 16 (Tsl6) and diploid (2n) mouse DNA

Signal intensity XAm4 probe Control probe XAm4/control Tsl6/2n

EcoRI digest Tsl6 570 382 1.49 2n 235 243 0.97

PstI digest Ts16 705 455 1.62 2n 229 226 1.01

i. 54

i . 6 0

Genomic DNAs from trisomy 16 and diploid control mice were digested with EcoRI and RstI, electrophoresed in a 0.8% agarose gel, transferred to n-l-trocelluT~e and hybridized with XAm4 and with a control probe (pRI) for a sequence located on chromosome 2. Signal intensities were compared by scanning densitometry, and each value represents the integrated area beneath signal peak, expressed in arbitrary units. The normalized values, XAm4/control, were calculated to correct for the amounts of DNA loaded.

1 0 7 2

Vol. 144, No. 2, 1987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

regionally assign the mouse sequences homologous to the human AD-AP gene tr

a segment of mouse chromosome 16. One of these hybrids (VI-6) contains the

162 translocation chromosome, with the proximal portion of chromosome 16

linked to the distal end of chromosome 2. The other two (VI-25 and AdCT25)

contain the reciprocal (216) translocation chromosome ~.~., the proximal

portion of chromosome 2 linked to the distal end of chromosome 16 [with a

breakpoint in band C3 of chromosome 16 (28)]. Of these, one (AdCT25) con-

tains the 216 chromosome as the only mouse chromosome (18). Thus the pre-

sence of the 1.9 kb PstI fragment in this hybrid and in VI-25 and its

absence from VI-6 localizes the mouse homolog of the human AD-AP gene to

within the distal segment of mouse chromosome 16 that is shared in common

between these two hybrid cell lines.

DISCUSSION

We have shown that the mouse gene homologous to the human AD-AP gene

is located on the d i s ta l end of chromosome 16 wi th in the region 16C3+ter.

Three other human chromosome 21 genes, superoxide dismutase-1 [SOD~ (18) ,

phosphoribosylglycinamide synthetase ~RGS] (18), and the protooncogene

ETS2 (19), have also been local ized to th i s r e l a t i ve l y short segment of

mouse chromosome 16. A l l three of these are located wi th in the Down

syndrome region of human chromosome 21 (21q22.1÷qter) (17,18,20). The

human AD-AP gene may be located more proximal to the centromere wi th in

21qll.2+q21 (10), possibly outside the Down syndrome region. The data

reported here ind icate, therefore, that the region of homology between

human chromosome 21 and mouse chromosome 16 extends substant ia l ly far ther

than was previously known and may include the ent i re Down syndrome region.

Recently, the human AD-AP gene has been found to be duplicated in

three individuals with sporadic AD and in two karyotypically normal indivi-

duals with the Down syndrome phenotype (29). The latter also had three

copies of SO01 and ETS2. It is therefore possible that the region

necessary for all of the Down syndrome phenotypic features may extend out-

side the 21q22.1÷ter region and may include the AD-AP gene. The exact role

that the AD-AP gene plays in Down syndrome and AD is unknown, and a mouse

model for investigating this role would clearly be very useful.

Unfortunately, trisomy 16 mice do not survive to term and would therefore

probably not exhibit any of the late onset AD-related abnormalities.

However, it is possiblethat trisomy 16 ~ 2n chimeric mice (21-23) with a

high level of chimerism in the brain may be a valuable model system for

investigating the development and pathogenesis of both Down syndrome and AD.

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Vol. 144, No. 2, 1987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Human chromosome 21 comprises 1.6% by length of the haploid genome

(30), of which about one third is within 21q22.1÷qter. Mouse chromosome 16

is substantially larger, 3.8% of the haploid genome (31), and about 0.7% of

the haploid genome is contained within the region 16C3+ter that is linked

to chromosome 2 in th@ reciprocal chromosomal translocation T(2~16)28H.

Our data indicate that this short tran$1ocated segment contains a substan-

tial region of homology with the Down syndrome region of human chromosome

21 and may be a valuable source of material for a molecular genetic com-

parison between these two chromosomes.

ACKNOWLEDGEMENTS

This work was supported by a grant from the National Institute of Child Health and Human Development (HD-17001) and by the Weingart Program in Developmental Genetics.

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