Structure and organization of a human EcoRI satellite II DNA family

HAL Id: hal-01501516

https://hal.archives-ouvertes.fr/hal-01501516

Submitted on 4 Apr 2017

HAL is a multi-disciplinary open access

archive for the deposit and dissemination of

sci-entific research documents, whether they are

pub-lished or not. The documents may come from

teaching and research institutions in France or

abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, est

destinée au dépôt et à la diffusion de documents

scientifiques de niveau recherche, publiés ou non,

émanant des établissements d’enseignement et de

recherche français ou étrangers, des laboratoires

publics ou privés.

Structure and organization of a human EcoRI satellite II

DNA family

Katia Sol, Michael Dubow

To cite this version:

Structure and organization of

a

human EcoRI satellite

II

DNA

family

Katia Sotl and Michael S. DuBow2

Department of Microbiology and Immunology, McGill-University, 3775 University St., Montreal, Quebec H3A 284, Canada

AE§TE4ET

Flighiy-repetitive (;.teliite

l)NA's

are maini' cûncentrated u,ithin ceniromeric heterochomatirr.'fhe organization of these DNA's within this chrornosomai region has been hampereci

by a

lack

of

fuii-length clones

of

these tandem arrays. We have previousl'' reported the cloning and sequencing

of a

i.797 Kô,

EcoRl setellile

Ii

DNÂ present in clone oKS36. tn

ti:i:

report, we show pKS3d-relateci sateililô

ii

DNAs It)

represent up tc 2Ûlo of the genomes of HeLri and Mell'a

human ceil lines. and to be organizeci mosily in tanderc arrays

oi

i .8 F'l b Kon

i

and Sau-34 DNA fragme nts and as 1.65

Kb,

1.95 Kb and 3.6

Kb

EcoRl eiements. A cell-specific organization

of

satellite DNAs, possibly

the

resuit

of'

chromosomal aberraîions inherent to

cultured cells, was observed in the t.wo human

(I/ela

and MeWo)

cell

lines.

The analysis,

by

southern hybridization, of satellite DNAs using lield inversion

gel electrophoresis revealed the presence, in HeLa cells, of F{indIIi satellite DNA clusters ranging from 150 Kb to 500 Kb in length. Using a panel of rodent-human hybrid cell DNAs, the members of the pKS36 satellite

lI

DNA family were found to reside mainly on human chromosomes 7, 12, 14, 15, 16, and 22.

INTRODUCTION

Molecular analysis

of

the human genome has revealed two types

of

tandemly orgânized repeated

DNA

sequences

that are

characteristic

of

heterochromatin: the alphoid satellite DNA and the so-called classical satenite

DNAs.

The term "classical"

satellite DNAs refer loosely to the collection of DNA

sequences detected by buoyant density sedimentation gradients as distinct components

of

nuclear DNA (Corneo

et al. l97l).

Satellite peaks consist

of

a mixture of sequences, some of which are related as in the case

of

satellite

II

and Iitr DNAs (Mitchell et al.

I _{Curent address: DuPont-Mercl Phermaceuticrls, DuPonl Experimeltal}

Rcsearch Slation Ë3281151, Wilmington, Deiaware 19898, USA

*Aulhor lo wbom correspondence should be rddressed

i q 19).

Satellite

Ii end IIi

peaks consist

oi

a

h,, ,;:rogeneous ccllection

of

repeatec

DNI'

sequences

thet

apparently

have

evolved

from a

common p+:nfameric ancestor 5'TT'CCA 3',(Frommer et al. i9&2: Pr.lsser ef al. 1986). Large blocks of satellite

II

and

iii

'-i\.r

h:ve been rdentifieci ùr, numan autosoma!

an ':

chromosomes. The K-d,;r*ain, described by

Bur.

'.r'

ai, (1985) anC observed

b)

oihers (i{olden et ai"

llr;'i.

Soi et â1. 1986), refers to the tandem organization

r,

1., Kb and 3.6 Kb Ko-fi,l satellite DNA usits. A mer,r3.r.'

of this K on I {amilv iD i 57. !.7 has been touno assoctet{

with the

nucleolar

organizer

and

centromeri:, heterochromatin

in

homogeneously staining region l (HSR) of chromosome l5 (Higgins et al. 1985; Hoide:r

et al. 1985). D- and

R-domains

specific

tc chromosomes

l6 and I,

respectively,

were

aiso identified (Burck et al. 1985). These domains represent the autosomal homologues of the male specific 3.4 Kb FIaeIII (or EçqRI) satellite DNA that wâs previously identified by Cooke ( 1976). Recently, a I .4 Kb EcoR 1

satellite

III

DNA, located on chromosome 14, has aiso

been reported (Choo et al. 1990). This satellite DNA was shown, by

in

situ hybridization, to not

otly

be

located in the heterochromatic regions of chromosome 14, but also on chromosomes

l,

9, 22 and

Y.

Using

pulse field gel

electrophoresis,

a

remarkable polymorphism in the organization of this satellite DNA

on

different chromosomes was demonstrated. on

chromosome 14, the 1.4

Kb

EsqRl satellite DNA is found clustered

in

a large tandem array

of

150 Kb,'

whereas

on

chromosome 22, shorter arrays

of

the satellite DNA, ranging from 20 to 150 Kb, are detected.

r60 Katia Sol & Michael S. DuBow related, but non-identical, sequences to recognize each

other during

hybridization experiments

can be

controlled by the stringency of the conditions imposed lor hybrid formation (Southern 1975; Beltz et al. 1983). lVe have previously isolated a 1,797 bp EçqRl satellite II DNA (clone pKS36) that was shown to be interrupted

in its tandem pentameric array by a region

of

49 bp (called the 49-mer) devoid

of

the sâtellite consensus sequence 5'TTCCA 3'(Sol et al. 1986). By varying the

concentrâtion

of

formamide

in the

hybridization reaction,

we

''vere able

to

distinguish between the genomic organization of the family

ol

related satellite II and III DNA sequences, and the specific organization

of

the

sub-group

of

pKS36-like satellite

II

DNA

sequences.

Using

the

technique

of field

inversion gel

electrophoresis (FIGE), the long range organization of

satellite

DNA

was explored (Schwartz and Cantor 1984). Although

it

is not clear to date

if

large blocs of

sateilite

DNA are

interspersed

with

unrelated sequences, we were able to identify, via FIGE, discrete blocks (ranging

from

150

Kb to

500

Kb in

size)

containing satellite II DNA sequences closely related to pKS36.

Using the DNA

of

a panel

of

rodent-human hybrid cell lines, we examined the copy number and

chromosomal location

of two

families

of

related satellite

DNA,

represented, respectively,

by clones

pKS36 and a previously cloned

l.8Kb

KpnI satellite

IIIIII

DNA in plasmid pBK1.8 [20] (Shâfit-Zâgardo et

al. 1982). The family

of

sequences related to pKS36 and pBKl.8 [20] was found to comprise 2 to 3% of the total genomic DNA of IleZa cells, and to reside mainly on chromosomes 7, I 2, I 4, I 5, I 6 and 22.

MATERIALS AND METHODS Muerials

Media and culture conditions, genomic and plasmid DNA isolation. and nick-translation of satellite DNA's are as described

in

Sol et al. (1986). Plasmid

pBKl.8 _{[20], a}

gift

from Dr. J. Maio (Albert Einstein College, NY), contains a 1.8

Kb

Konl

satellite DNA (Shafit-Zagardo

et al. 1982).

Recipes

for

PBS (phosphate buffered saline),

TE

(tris-EDTA), TBE (tris-borate-EDTA) and SSC (sodium citrate) are as per Maniatis et al. (1982).

Enzvmes and Conditions

All

restriction endonucleases were purchased

from Boerhinger-Mannheim Canada (BMC),

Gibco-Bethesda Research Labs (BRL), or Pharmacia Canada

(Montreal, Que.). Routinely, DNA was hydrolyzed in 6 _{mM Tris-HCl [pH} 7.5], 6 mM MgClr, 75 mM NaCl,

6 mM 2-mercaptoethanol and 125 pg/ml bovine serum albumin (BSA, Pentex fraction Y, Miles, Elkhart, IN).

DNA hydrolysis was conducted

for

4 hours at 37"C (unless otherwise indicated), using

I

unit of restriction

endonuclease per microgram of plasmid or phage DNA, or 5 units of enzyme per microgram of genomic DNA.

After hydrolysis, the samples were placed at 65"C for l0 minutes to stop the reaction, and completely separâte the cleâved fragments before electrophoresis. 0 I i çonucleotide owi f icalion

A crude oligonucleotide mix ol a synthesized 49 nucleotide long non-satellite sequence present in pKS36 (Sol et al. 1986), was generously provided by Dr. D. Garfinkel (National Cancer Institute, Frederick, MD.). The premature termination products were separated

from the 49-mer on a I2% polyacrylamide ge1 (Maniatis et al. 1982) and the full-length product was purified using the "crush and soak" procedure

ol

Maxam and

Gilbert

(1980).

The purified

oligonucleolide was

labelled

with

"l-32p-ATP (Amersham)

using

T4

polynucleotide kinase (Maniatis et al. 1982).

A p _{arose sel} e leclro ohore sis

Horizontal agarose gel electrophoresis of DNA was carried out on slab gels

in

I

x E buffer (Maniatis et al. 1982) at room temperature. The DNA samples

were loaded, along with a tracking dye consisting of 25%

_lw/vl

sucrose,

I x E buffer,

0.05% [w/v]

bromophenol blue and 0.05%

_[w/v]

xylene-cyanol. Large DNA fragments were routinely separated on a 0.75%

[w/v]

agarose gel, whereas smaller fragments

were

separated

_{in l% [w/v]}

agarose gels.

Electrophoresis was performed either

at 20

volts overnight or at 50 volts

for

I

to 5 hours, depending on the size of the gel.

Field Inversion eel electroohoresis

HeLa and MeWo (Holdelr et al. 1985) cells were

grown

to

confluency, trypsinized, harvested by centrifugation and resuspended

in

PBS

to I x

107 cells/ml (final concentration) at 37'C. An equal volume of cells and l% low gelling temperature agarose (LGT Sigma type VII, made in PBS) were mixed at 37'C and aspirated into sillicon tubing (3/32 inch inner diameter, Cole-Parmer). The tubing was then placed at 4"C for

l0

minutes

to

allow

the

agarose

to

harden.

The polymerized agarose containing the cells was extruded onto a sheet of saran-wrap (Fisher Scientific, Montreal, Que.), and cut into

I

cm "plugs". Each plug contained approximately 4 x 105 cells. The plugs (50 on average)

containing the embedded cells were placed in separate 50 ml conical tubes and incubated at 50'C for 24 hours

in 25 ml of a lysis buffer consisting of 0.5 M EDTA

(final concentration), l% [w/v] sarkosyl and 2 mg/ml proteinase

K.

The plugs were subsequently transferred to new conical tubes and washed with ice-cold

I

x TE

x TE.

The

washes were thereafter resumed as described above and the plugs were stored

in

0.5 M EDTA at 15"C. Just before use, the required number

of

plugs were washed extensively

in

I

x

TE,

and preincubated overnight (one plug per l.5 ml centrifuge tube)

ât

l5"C

in

0.5

ml

of

a

bulfer

consisting of

autoclaved gelatin (0.2 mglml), spermidine (5 mM), and the appropriate digestion buffer as recommended by the manufacturers. The next day, the.plugs were transferred to new tubes containing 0.2 ml of freshly made butter.

Fifty

units of the appropriate restriction

endonuclease were added, and the reaction was allowed

to

incubate

for 5

hours

at

the

temperature recommended

by

the manufacturers. The reactions were then stopped by placing each plug in a new tube containing 0.5 ml of 25 mM

EDTA.

The plugs to be

analyzed were soaked

for

30 minutes

in

a solution consisting of 0.5 x TBE plus 0.05% [w/v] bromophenol

blue.

The stained plugs were cut in half and placed, one per well, in the slots of a 0.75% _[w/v] agarose gel (GTG agarose, Sigma) made in 0.5 x TBE. The gel was

submerged

in

0.5

x

TBE and

field

inversion gel electrophoresis was conducted at 4"C for 96 hours at 80

volts using

a

PPI-100 device

(MJ

Research Inc.,

Cambridge, MA). The program used was number 9 and had the following characteristics:

A:

reverse time at beginning of ramp = 2 sec.

B:

amount added to reverse time at each step = 2 sec'

C:

forward time at beginning of ramp = 6 sec.

D:

âmount added to forward time ztt each step = 6 sec'

E:

number

of

complete reverse and forward runs before starting over with

initial values ₌ 22

F:

added to reverse increment at each step

=

-0'l

G:

added to forward increment at each step

= -0.6

Before starting the reverse/forward field flow, the gel was run for l0 minutes in the forward direction at 80 volts to allow migration of the DNA out

of

the

wells

and into the

gel.

Southern

blotting

and hybridization was performed under high stringencv conditions as described previously (Sol et al. 1986).

Dot-blot oreoaration

Fluman-rodent DNAs

(a

gift

from Dr.

M. Hansen, Ludwig Cancer Institute, Montreal, Canada), total genomic DNA, and plasmid DNA. were placed on nylon membranes using a BioRad dot blot filtration

unit.

Serial dilutions of the DNA were made in 200 pl

of

I

x

TE.

To each tube, 40

pl

of

I

M

NaOH was

added

to

denature

the

DNA.

After

l0

minutes incubation at room temperature,40 pl of I M Tris-HCl

[pH 7.5] and 40

pl

of

I

M

HCI were added

to

the

reactions and the tubes were kept on ice until needed.

A Genescreen nylon membrane was cut to the size of

the filtration unit, and soaked for l0 minutes in water, and thert in 6 x SSC.

It

was assembled on the dot blot filtration unit as specificied by the manufacturers. The membrane was washed under vacuum with 6

x

SSC,

and the samples were applied, vacuum,off, in the slots made by the apparatus. The samples were filtered by vacuum and the slots were washed twice with 6 x ssc.

After

filtration

was complete,

the

membrane was

released from the dot blot apparatus and washed in 2 x Denhardt's solution (100 x Denhardt's solution is 20,6

[w/v]

polyvinyl pyrollidone, 20Âlw/vl BSA, 2% _[w/v]

ficoll 400).

It

was then baked at 80'C under vacuum, and stored at room temperature for future use. When

using the 49-mer as

a

probe, the membranes were soaked for l0 minutes in 3 x SSC and prehybridized for

2 hours at 42"C

in

6 x SSC, 1

x

Denhardt's solution, 0.5%

_[w/v]

sodium dodecyl sulfate SDS, 0.05% _[w/v] sodium pyrophosphate, and 50 pg/ml of E. coli DNA (Woods, 1984). The hybridization was performed for

24 hours at 42'C in 6 x SSC,

I

x Denhardt's solution, 25 pg/ml E. coli DNA and 4 x 106 cpm of probe, followed by t',vo washes

of

l5 minutes at room temperature and one

lor

I0 minutes at 42C in 6 x SSC and 0.05% [w/v] sodium pyrophosphate.

The

autoradiograms were scanned using an

LKB

Broma 2202 Ultrascan laser

densitomer. The results were therl analyzed and plotted on an Apple II computer using the program GELSCAN by P. Heilmann

of

LKB.

When the EcoRl or Kpni

satellite DNA insert of plasmids pKS36 or pK1.8[20], respectively, were used as probes, the

filters

were

hybridized

for 24

hours

at

42"C

in

50% (v/v)

formamide, 5

x

SSC, 5

x

Denhardt's solution and 50

pg/ml Ë. coli DNA, plus 2

x

106 cpm

of

the nick-translated

DNA

fragments (Sol

et al.

1986).

The unbound probe was removed via two successive washes

àt 42"C for 30 minutes in 0.2% (w/v) SDS, 0.5 x SSC. RESULTS

Short ranse Satellite

II

DNA orsanizuion in HeLa cells In order to define the genomic organization

ol

the satellite DNA related to the cloned 1.8 Kb EcoRi satellite

DNA

of

pKS36,

a

series

of

southern blot

analyses of restriction enzyme hydrolyzed

llelc

DNA was probed, under increasing stringency conditions,

with the insert

of

pKS36 (Fig. 1, panels

|,2

and 4). The stringency was controlled, from low to high, by the percent of formamide added to the hybridization mix. Low stringency was defined by the presence

_of

tr5o/o

formamide

(Fig.

I,

panels

I

and

2),

whereas high stringency was obtained

in

the

presence

of

50ÿo formamide (Fig. I panel 4). Panel 2, a shorter exposure

of panel

i,

is presented to facilitate the identification

of the _EsqRl satellite DNA species positioned around 1.8Kb.

162 Katia Sol & Michael S. DuBow re*Ist'§

§Ê

P*r!§l §{K§J Pôn*l I

Fig.

i:

Southern hybridization

with

satetlite DNA, under increasing stringency, to cleaved HeLa ger,omtc

Ill.ia.

ûenomic DNÀ was hydroiyzed with restriction

ei.rtionucleases

Kpnl

(K),

EcoRl (E), or

HhdtII

(H).

U is

uncut

DNA.

Panels

i

and

2

present the autoradiograms of Southern hybridizations performed under low stringency (i5% formamide), whereas panel

4 presents the autoradiogram ol Southern hybridization performed under high stringency (50% formarnide), using the t .797 Kb EcoR

I

DNA f,ragment ol pKS36 as

a probe. Note that panel 2 is a lighter exposure of the panel I autoradiogram. Panel 3 is the autoradiogram of the Southern blot hybridization performed using the 49-mer as a probe. 'The positions of the l.B Kb and 3.6

Kb DNA bands are indicated. Arrowheads indicate the positions

_ol

the 1.8

Kb

rnullimeric DNA Fragments.

Dots indicare

the

positions

of

the

2.35

Kb

D|{A

fragments.

intense bands located

at

approximately

1.8

Kb

(monomers) and 3.6 Kb (dimers) were detected, along with faint bands (indicated by arrowheads; migrating

as trimers (5.4 Kb), tetramers (7.2 Kb) and pentamers (9 Kb), thus suggesting a regular tandem organization

for

Kpnl

satellite DNAS.

In contrast, the pattern of hybridization to the EcoRl-cleaved

I/ela

DNAs appeared more complex.

Under

low

stringency

(Fig. l,

panel

2,

lane E)"

prominent EcoRl bands are lormed at approxinlatel,v 1.8 Kb and 2"35

Kb

(itdieated by a

dot).

The other detectable bands (otrservecX in Fig.

l.

panei

l.

lane F),

iormed under

these permissire ;onditions,'irÿûie approximately 1.5 F-b, 1.65 Kb, 1.95 Kb,2.75 F.b, 2-.ç5

Kb,3.05

Kb,3.6 Kb,5.4 Kb,7.2 Kb,

and 9

Kb

in

length, along

with

many diverse sized fragments.

Although the presence of multimeric forms (3.6 Kb, 5.4

Kb,

'1

.2 Kb,

and

9 Kb),

indicative

of a

_tandem organization, could have been generated

by

point mutations at the EcoRl site between adjacent 1.8 Kb

E§gR I repeat units, the presence of the no'n-multimeric sized bands found

in

the EcoRl-cleaved DNA lanes suggest a non-contiguous organization lor some of the

genomic sequences relateC

to

the cloned satellite II

DNA.

Under high stringency conditions (Fig.

l,

panel

4, lane E),

hybridization

was found

to

occur predominantly with a 1.95 Kb EcoRl DNA fragment. In addition, major bands ,*'ere observe,J ât 1.65 Kb aû;

3.6Kb. It

thus appear that pK:;36-iike satellite

ii

sequences define a sub-class of the satellite

II

farnily whose members are likely to be cluslered on i.65 Kb.

1.95 Kb, and 3.6 Kb EcoRl DI{A fragrnents.

In addition, the distribution of satellile DNAs containing the 49-mer region specific to pK§36 was

investigated (Fig. 1, panel 3). The 49-mer, hybridized under

mild

condition as indicated

in

Materials and

Methods, annealed

with

EcoRl-cleaved

llela

DNA fragments in a pattern similar to that observed under iow stringency

for

the complete EcoRl element. bul with a very high background (Fig"

l,

panel 3, iane E). Though no bands with the 49-mer could be detected at

positions

ol

1.8 Kb or 3.6 Kb charâcterizirg the Kpni

satell;te DNAs,, a band was detectable af approximately 2.35 Kb (Fig. 1, panel 3, lane K).

Under the electrophoresis conditions used in

these experirnents to separate the hydrolyzed DNAs, no

bands were detected

with

restriction endonuclease

HindIII (Fig.

l,

panel 4, lane FI). Polvrnor phisms between cells lines

Fig. 2 presents the autoradiogram ol a Southern blot al HeLa aad MeWo DN.Às hydroiyzed with EcoRl

(E),

Kpnl (Ki,

Sar:34 (S), EcoRt plus

Konl

(EK),

EcoRl plus Sau3A {ES), and

Kpnl

plus Sau3A (KS).

As

seen

in

the previous section, HeLa pKS36-bke satellite

II

DNAs are exclusively organized in tandem arrâys as Kpnl fragments (1.8

Kb

and 3.6 Kb repeat

units), and

their EcoRl

pattern reveals

a

complex

distribution (Fig. 2,lanes

K

and E, respectively). In addition to a tandem array distribution

of 1.8

Kb and

3.6Kb Sau3A DNA fragments, pKS36-like sequences are present in abundence on 3.25 Kb Sau3A lragments (Fig. 2,lane S).

ln MeWo cells, this class of sâtellite II DNA was found mainly in tandem arrays

ol 1.8

Kb

iong Konl

and Sau3A Dl§A fragments (Fig. 2, lanes tr( and S,

respectiveiy).

Wircfl

MeV/o genomic

Dl.{A

was lrydlolyzed witl'r EcoRi

_iFig.

2, luleWc" \ane

Ë),

the majorit,v-

t;f

ihe satellite

ïI

Di'{As were iound as 2.?5

kKb,

2.Ç5

iib," aric

3.6

[ab

iong

DhiÂ

iiagments. However, minor bands were detected at

i.5 Kb, Ldi

A, lane pBKl.8[20] and 4-8, lanes

I

to

3).

However,

the

Konl

element

did

not

contain

sequences homologous to the 49-mer found in pKS36, as it failed to cross-hybridize to this unique region of pKS36 (Fig. 4-B, lane 6).

-

The oligonucleotide probe (the 49-mer) failed to hybridize to E. coli DNA (Fig. 4-C, lane 2), but a hybridization signal was detected with Rat DNA (Fie.

4-C,

lane

3).

The significance

of

this observation remains, at the moment, unclear. The intensities of the hybridization signals

of

the

_@Rl

probe to known amounts

of

its homoiogous DNA, pKS36 (Fig. 4-A), were recorded by laser densitometry, and the ratio of

copy number/signal intensity determined. This signai

ratio was then used to determine, by recording the hydridization signal

of

the ECqRI probe

to

known amounts

of

Hef-a genomic

DNA,

the genomic copy number of this satellite DNA element. Assuming the content

of

DNA

per nucleus

to

be approximately 6

billion base pairs,

this

1.797

Kb

satellite DNA (and

other closely related sequences) was estimated to

represent approximately 2%

of

the genome

of

HeLa cells. Though similar results were obtained with the 1.8

Kb

Konl

satellite probe, less than t% of the genomes

of HeLa and human

AKl43

(Goring et al. 1987) cells could hybridize to the oligonucleotide probo (Fig. a-C, lanes 4 and

5).

These results suggest that the actuâl copy number

of

the pKS36 family

of

satellite DNA (containg one copy

of

the 49-mer) is less than l% of

the human genome.

Chrcmosomal location of Satellite trI DNA

The human chromosome composition

_of

the hamster/human cell-line panel used

in

this study is presented in Table

l.

The distributioû of satellite DNAs was analysed according to Waye et al. ( I 988). For each of the human chromosomes,

the

degrees

of

discordance

(D)

and concordance (C) were determined, and the percent discordance (D/D+C) calculated.

In

this equation, D equals the number

of

positive hybrids

in

which the chromosome

is

absent plus the number

of

negative hybrids in which the chromosome is present, and C equals the number

of

positive hybrids

in

which the chromosome is present plus the number

of

negative hybrids

in

which the chromosome is absent. In this

manner, sequeûces related

to

both

pKS36 and

pBKl.8[20] were found

to

reside

mainly

on chromosomes 7, 12, 14, 15, 16, aad 22.

Equal quantities of the

l0

hybrid DNA's (wcl to wcl0), along with the control hamster DNA sample, were denatured, applied onto a nylon membrane in a BioRad dot blot apparatus at different concentrations, anrl _{hybridized under high stringency to the t.797 Kb}

EçoRi

insert

contained

in

clone

pKS36"

After

autoradiography, the probe was removed

from

the membrane by treatment with a basic solution (0.5 M

TABLE I

Rodent-Human Hybrid

Human Chrornosorne content

wcr

wc2 6,

I,

ll, x

wc3

i., 3, 4, 5, 8, 12, 13, 14, 16, 20,

2t,

Y

ivc4

1,2,5,7,8,

12, 13, 14,:.5, 17, 18, r9,21,22,

x

wc5

l,

3, 4.,5, 6,7, 8, 12, 14, 15, t6, \7

_,

\9, 21, 22, X

rvc6

3,4, 8, 9, tÛ, t5, 17. i9, 20. 22"

x,Y

wc7

3,4,8,9,

10, 15, r7, r9"2a,27,

x,v

wcE

6, 12, 13

wc9

3, 4, 5, 6, 9,

tt,

14, 17,22

wclo

2,3,6,7,8,

ll,

12, 13,

t4,

15, 17,20, 21,

x,

Y

KOH).

Subsequently, the stripped membrane was

hybridized, as per Materials and Methods, to the 49-mer specific to clone pKS36. The process

of

probe removal was repeated, and the membrane was finally

hybridized, under

high

stringency,

to the

nick-translated 1.8

Kb Konl

satellite

DNA

of

clone pBKl.E[20]. The complete removal of the probes by KOH treatment was assessed by autoradiography of the stripped membrane (not shown). Fig. 5 presents the autoradiograms

of

these hybridization experiments,

along

with

the

corresponding laser densitometer tracings. In all cases, no hybridization signal to wcl

166 Katia Sol & Michael S. DuBow

lllr

^^^A_l

aa

!8K 1.8[201

'aOO:sr!'

(!Al Sal.

Fig.

5:

Analysis of a rodent-human hvbrid cell panel'

Dai biot autoradiogram and densilcmeter profiles

cf

haIrlster-human chrcrrosomai bar:ks iianes

wcl0 tt

§,e -J.l hybridir:eri, as per bLâteriê.15 àrd Methods, with: pKS36 i,?!;? bp

I':roRt DNA,

pKS36 49-mer

non-satellite sequeTlee,

and

pEK1.8[20i

i.8 Kb

Konl

sareltrire

DNÀs. 'îhe

preeks ;ôrrespond

to

the

hybridization intensity reccrrJed lo:' each dr:t blot using a* LFi B laser densitometer scanner as per Jv{ateriâi§ aûd

,r,iethüCs.

DI§ÜL]§§ION

The human satellite

II

and

III

DNAs consist of

lamilies

of

evolutionarily related members that were found to be highly polymorphic in sequence (Beridze 1986). The poiymorphism of satellite DNAS extends to the level of their organization into diverse domains, as depicted

in the

series

ol

Southern

blot

analyses

presented

here.

Southern

blot

analysis, perlormed under low stringency, has revealed that, though the members of the satellite DNA

II

and

III

are organized as 1.8 Kb and 3.6 Ktr KonI tanden repeat units, a large

fraction

of

its

members are found as diverse sized EcoR.i fragments. Alternatively, these diverse sized lragments may indicate

that

there

is

considerable heterogeneity

in

EcoRl site distribution

in

tandemly

repeated satetrlite elernents. ln HeLa cells, clone pKS36 appeared

to

be

a

minor

member

of a

sub-family characterized by clusters

ol 1.65

Kb,

1.95 Kb and 3.6

Kb EcoRl satellite DNAs.

If

the 1.65 Kb and 1.95 Kb

units are organized

in

consecutive tandems ("'1.65-1.95-[ 1.65- 1.95]- 1.65-"'),

a

mutation

at

the EcoRl

site between two consecutive units (indicated within brackets) would result in the formation of the observed 3.6

Kb

EcoRl

composite dimer ("'1.65-[3.6]-1.65--.).

Examination

of

the sequence

of

the cloned l.797 Kb pKS36 satellite DNA revealed the,presence of

a

one base mismatch

EcoRl

site

(5'

GCATTC 3)

located I 50 bp from the 3' end of the elernent (Sol et al.

1986). Satetlite DNA repeats are characterized by the hypervariability

of

cytosine residues (Fowler

et

al.

1988).

A

single C->A point mutation within the 5' GCATTC 3' sequence would generate an EcoRl site

(G _IAATTC)

in

the original 1.8

Kb

monomeric unit,

and a new I .65 Kb fragment would appear upon EcoR I hydrolysis. Moreover, the same mutation affecting one

of the sub-repeats of a dimerized 1.8 Kb repeat would generate two new EcoRl fragments

of

1.65

Kb

and 1.95 Kb in length. Further amplification, in tandem,

of

these

two

fragments couid generate the type

ol

EcoRl organization that

is

observed

in

HeLa cells.

Furthermore, point mutations in other single mismatch E19-R.l sites scattered within the cloned element, may result

in

the apparent crganization of EçoRl satellite lllNAs observed in Érel-a cells.

Using the "49-mer" as a probe,

it

was observeri

that, under the permissive hybridization conditions

utilizeil, This element h.-vbridized in rnuch the same wâv

as the i.?9?

Kb

LçaRl sâtelliie elernent did under low stringeircy. 'I'hor-rgh the 4Ç-rn*r did ûot anneal to any

cf

the nrajor

Kpnl

saiellite ÜNA fragments,

it

wâs found to be present as a 2.35 Kb

Kpnl

DN.A species.

Thè identity

of

this

Kpnl

DNA

fragment remains

unknown, but the intetsity

ol

the hybridization signal suggests thar

it

may be repelitive.

In addition to the cell line HeLa, we examined

a

second

cell line

(MeWo) derived

lrom a

human melanoma (Holden et

aI..1985).

MeWo was chosen because

it

exhibits a chromosome

I5

homogeneously staining region containing amplilied copies of

Dl5Zl,

a satellite

II

DNA (Simmons et al. 1984; Higgins et al. 1985; Holden et â1. 1986). The sub-family of pK536-like satellite II DNA appea.s to be characteristic of the genome of HeLa cells, as it is absent (as 1"65 Kb and 1.95 Kb EçoRl DNA lragments) from the genome of the huûran cell line MeWo. Restriction analysis of these

two

human

cell

lines

for

satellile

II

DNAs closely related

to

pKS36 revealed

the

presence

of

diverse domains, potrymorphic in organization.

kt HeLa cells, the ;.65 Kb and 1.95 Kb EcoRt

DNA fragments, characterizing members

of

the sub-family of pK536-like satellile trI DNAs. are interrupted

with

Kpnl

sites. Though a fraction

of

the 1.95 Kb EcoRl fragments contains Sau3A sites, the bulk of the members of the pKS36-like sateilite family are devoid

of such sites.

in

contrast, tite members

ol

the Meÿl'o

pKS36-like satellite

II

DNA lamily are characterized

by

2.95

Kb

and 3.6Kb

EcoRl DNA

fragments.

Furthermore, the units

ol

repetition

of

these satellite

DNAs, as

Konl

and Sau3A fragments, was largely found to be 1.8 Kb in length, (as opposed to the equal distribution

of 1.8

Kb and 3.6

Kb

long

wits

in HeLa

cells). In Mel{o cells, the characteristic EcoRl DNA fragments appear

to

be interrupted by Sau3A sites, whereas only a fraction contain

Kpnl

sites.

Higgins et al. (1986) determined the genomic

distribution

of a

pKS36-related satellite

II

DNA

(Dl52,l), by hybridization to Melf o DNA and to male

and

female placental

DNA. The Dl52l

probe

hybridized to placental male DNA and MeWo (a male

cell line) DNA

in

a pattern similar to that observed with pKS36 in Mel{o. A low level of EcoRI restriction lragments with a

i.8 Kb

periodicity wurs detected in male DNA, but detectable hybridization was displayed

with

1.8

Kb

and

l.0.xU

EcqRl fragments

in

female piacental

DNA.

The

Konl,

Msol,

Sau3A and Rsal hybridization patterns

of Dl5Zl

were similar to the ones obtained with pKS36.

It

is not known, to date,

if

Dl52'l

contains a region homologous to the 49-mer of

pKS36.

However,

from

the

similar hybridization patterns

_of

the

two

cloned satellite DNAs and the presence

ol

numerous Taol sites in

Dl5Zl,

it

is likely that this satellite DNA belong to the sâtellite

II

family rather thân To the satellite

III

DNA family (Prosser et

a1. t986.t.

Lrsing FIGE to separate very iarge restriction

iragments, xve analyzed, by Southern

blot hybridization" the macro-organizâtion of satellite DNAs

in

ileLa eells. The restriction endonucleases EcoRl,

Clal, and BamÈIi do not appear to define large units of

amplified satellite

DNAs, as we

did not

observe

discrete

sized

hybridization bands

under

the electrophoretic conditions utilized. However, we were able to identify HindIII fragments, ranging from 150 Kb to 500 Kb, that contain satellite

II

DNAs. To date,

it

is unclear

if

these large biocks contain tandem or interspersed arrays of satellite DNAs. The intensity

ol

the 150 Kb and 500 Kb HindIII fragments suggest that

they

may

be

repetitive.

Alternatively,

they

rnay

contain numerous copies

of

satellite DNAs and be

present

in

single copy

in

HeLa

cells.

The

faint

but

discernabie

series

ol

fragments,

ranging

fmm

approximately 20C

Kb to

400

Kb,

could represent

singie oûpy DiliA

lragments containing

low concentrâtions

,;l

satellite

DNAs. The

Civerse

ciistribution

ol

satellite DNAs on

HlndIII

fragments

ma-v rellect their ireteromorphic distribution on hurnan chromosomes. The two rnajor

t{indlii

blocks might represent The sâteliite DNA organization common to â

subset of hurnan chromosomes. {n contrast, each of the

minor

blocks

might

represent

the

satellite DNA

organization

that is specific lor a

particular

chromosome.

The sâiellite II DNA family analyzed represents approximately 2oh

of

the genome of HeLa cells and its members are f,ound mainly clustered on chromosomes

7,12,14,15,

16 and

22.

\Yhile we did not see any significant hybridization to chromosomes 9 and Y, the major region of satellite

II

DNA has been previously mapped, by in situ hybridization and Taol restriction endonuclease banding analysis, to chromosomes

l,

9, 15, t6 and Y (Gosden et al. 1975; Tagarro et al. l99l).

The _{EçgRl sub-family of} satellire II DNA, represented by clone pKS36, appeared to represent no more than

l% of

the human genome. Thus, there

is

growing evidence that simple sequence, highly-repetitive human satellite DNA's are, in fact, a complex collection and organization

of

families of related, yet distinct, DNA

sequerces. The elucidation

of

their

structure and

evolution

will

provide clues

to

their

presence and function in the human genome.

ACKNOWLEDGEMENI-S

The authors are grateful to Dr. J. Maio for his generous

gift of

pKi.8[20] and

Dr.

Brian Sauer for

interesting discussions. 3he hamster-human hybrid cell

DNA panel was a

gilt

from Dr. M. ÉIansen _{Ludwig Cancer Institute, Montreali. KS was the recipient of a Fellowship

from

the

World University Service of

Canada (WUSC). MSD is a Chercheur-tsoursier de

Mérite Exceptionnel of lhe Fonds de la Rechorche en Santé _{du Québec} (FRSQi. Tltis work w.as supported by grant (OGP 0003222) from the Natural Sciences and

Engineering Research Councili

*l

Canada (NSERC). REFERENCES

Beltz, G..A., Jacots, K..A., Eiekbush, T"Fi., Cherbas, P.T., Kafatos, F.C. 1983. Methods Enzvrngl. i00: 266-285

Beridze,

T.

I986. Satellite

DNA,

Springer-Verlag, Berlin FRG

Burk, R.D., Szabo, P., O'Brien, S., Nash, tÿ.G., Yu, L., Smith, K.D. l9S5rChromosoma, 92: 225-233 Choo,

K.H.,

Earle, E., McQuillan,

C.

1990. Nucleic Acids Res., 1Ê: 5641 -5648

Cooke, F{..I., Flindley,

l.

1979. Nucleic Acids Res., §: 3177 -3197

Cooke, Fi.J. 1976. Nature, 262: 182-186

Corneo,

G.,

Gineiti,

E.,

Polli,

E.

1971. Biochir4. Bioohvs. Acta, _217: 528-534

Fowler, C., Drinl<rvater. Ë.., §kinner"

i.,

Burgoyne, L. !988. F{uman Genet., l9:2û5-272

Frommer. N{., Prosser, J., Tkachuk, ü., R.eisner, A.Ftr". Vincent, P.C. 1982. Nucieic .{c',eis 8-çs., lQ: 547-563 Gardiner,

K.,

Laas. W^, Patterson.

D.

1986. Somatic Cell Mol. Genet., l2: 185-i95

Gosden, J.R., Mitchelt,,{.R.., Buckland, R.4., Clyton,

R..P., Evans.

Il.J.

1975. Exp. Cell. Bes., 92:148-158 Goring, D.R", Gupta,

K.,

DuBow, M.S. Somatic Celi Molec. Genet., 13: 47 -56

Higgins, M.J., Wang,

H.,

Shtromas,

I.,

Haliotis, 'T'.,

168 Kalia Sol & Michael S.DuBow

Holden, J'J'A., R'eimer, D.L., Higgins, M.J.,

Roder,

shafit-Zagardo'

8.,

Maio,

J'J''

Brown'

F'L'

1982'

J.C., White,

B.N.

1985. Cancer Genet.

Cvtosenet.,

Nucleic AcidsBgS., l0:31?5-3193

14:13l-146

Simmons, M.C., Maxrvell,

J.,

tlatiotis,

T.,

Higgins, Holden, J.J.A., Reimer, D.L., Higgins, M.J.,

Roder,

M.J., Roder, J.C., White, 8.N., Holden, J.A. 1984.

_l.

J.C., \Yhite, B.N. t986. Cancer Genet. Cytoqenet.,

_2l:

Natl. Cancer I!s!., ZZ 801-808

221-217

Southern, E.M. 1975. J. À4s1. Eisl-, 98: 503-507

Maniatis,

T.,

Fritsch,

E'F.,

Sambrook,

J. 1982.

Sol,

K.,

Lapointe'

M-'

Macleod,

M',

Nadeau, C',

Molecular cloning, a laboratory manual, Cold

Spring

DuBow, M.S. 1986. Biochim. tsioohvs. Acta, E§E:

128-Harbor Laboratory Press, Cold Spring Harbor, N.

Y'

135

Mitchell, 4.R., Beauchamp, R.S., Bostock,C.l. 1979.

J.

Tagarro,I.,Gonzalez-Aguilera,J.J.,Fernandez-Peralta, Mol. Biol., 135l-

127-149

A.M. 1991. Genome, 34:251-254

Prosser, J., Frommer, M., Paul, C., Vincent, P.C.

1986.

Waye, J.S., Mitchell, A.R., lffillard, É{.F. 1988. Hum" J. Mol. Biol., 187:

145-155

Genet., 78:27-32