Human histone genes are interspersed with members of the Alu family and with other transcribed sequences

Vol. 104, No. 2, 1982 January

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

29, 1982

Pages 785-792

HUMAN HISTONE GENES ARE INTERSPERSED WITH MEMBERS OF THE ALU FAMILY AND WITH OTHER TRANSCRIBED SEQUENCES F.

Sierral,

A.

Leza*,

F.

S.

Clark3,

J,

Department Department University

of of

Marashil,

M.

Plumb’,

R,

Ricklesl,

Wells3,

G.S,

Stein’

and

J.L,

Biochemistry immunology of Florida,

T.

Van

Dyke’,

Stein’

and Molecular Biology’ and Medical Microbiology2 Gainesvi I le, FL 32610 and

University

ReceiTred

Department Adelaide,

of

r)ecemSer

1,

of Biochemistry3 Adelaide, South

Australia

5001

1981

SUMMARY.

We have isolated a series of recombinant ACh4A phages containing human histone genes. Histone H2A, H2B, H3 and H4 genes have been found to be clustered, but are not present in any simple repeat pattern. Hybridization of a blot containing phage DNA with S phase polysomal cDNA indicates the presence of additional sequences complementary to HeLa polysomal RNA sequences. Northern blot analysis using these clones as probes has also shown the presence of sequences complementary to non-histone-coding RNAs, some of which accumulate differentially in different stages of the cell cycle. We have also found, by hybridization with appropriate probes, that histone genes are interspersed with several copies of the Alu DNA family; however, not all of the histone genes are associated with an Alu DNA sequence,

We have

isolated

human histone

genes

to heterologous

in w&o genes

a series (1).

histone

but

was readily

evident genes,

From these

clustered,

%onopkiea that

our

detectable

clones

to 7-11s

we have shown

that

H3 and H4 coding

HeLa cell

apparent.

regions,

northern studies

the

a sequence

that

analysis

HeLa cells, contains

hybridizes

histone

of sea urchin studies,

other

than

it

In a previous in

the

addition

and became

histone

as by hybridization

strongly

during

selection-

in humans,

of these

as well

predominantly

and hybrid

that

sequences

genomic

by hybridization

to those

course

xHHG 41,

containing

located

comparable

analysis,

clones,

present

blot

transcribed

RNA from

were

we concluded

repeat

blot

polysomal

xCh4A phages

regions

During

contained

one of our

330 nucleotides,

coding

no tandem

by northern

cDNA prepared

of about

Histone DNA probes,

translation. are

of recombinant

to report

(2),

to histone

to an RNA species Gl phase

of the

cycle. 0006-291X/82/020785-08$01.00/0 185

Cop.vright 0 1982 b-v Academic Press, Inc. A I1 rights of reproduction m anv form reserved.

Vol. 104, No. 2. 1982 Tashima

e2 c& have

a human genomic DNA sequences

long

these

the

genome

single

copy sequences,

(4).

Although

concerning

Alu

that into

over

suggesting

its

their

that

repeated

interspersed

DNA sequences

histone

genes,

human histone

sequences

present

copy

DNA family

is

predominant

300,000

have been

found

to be interspersed

In this genes

also

per

families

(6),

of clustered,

communication, are

300

sequences

being

among families

Alu

approximately

single

multigene

in

containing

number

as dispersed

presence

recombinants

to a probe

with

the Alu

repetition

RESEARCH COMMUNICATIONS

95% of the

hCh4A hybridize

In humans,

as well

such as the

of the Alu

found

sequences,

(5).

evidence

AND BIOPHYSICAL

In most eukaryotes,

genome

sequences,

cloned

have been

reiterated

available

reported

library (3).

nucleotides throughout

BIOCHEMICAL

among

haploid with

no data

are

repeated we present

interspersed

with

members

obtained

by screening

DNA family.

Microorganisms human genomic

MATERIALS AND METHODS and DNA Isolation: Histone clones were

a

DNA library containing fetal liver DNA cloned into the Eco RI The I ibrary was generously provided by Dr, T, sites of the vector hCh4A (7). Maniatis. Bacteriophage were grown in E. co& strain DP~OAU~F (obtained from Dr. F. Blattner) in NZCYM.DT broth as suggested by Blattner ti & (8), Phage DNA was isolated by a modification of the method described by Blattner (81, The Alu DNA containing clone pCDF’2 (9) was a gift from Dr. S. Weissman and was grown in L broth in the presence of 50 ug/ml of ampicillin. Plasmid DNA was isolated by the cleared lysate procedure (IO), followed by CsCIethidium bromide density gradient centrifugation. DNAs were digested with restriction endonucteases, electrophoresed on 0,8% or I .2 % (w/v) agarose gels, and transferred to nitrocellutose essentially as described by Southern ( I I ). Nick-translated probes were prepared as described by Maniatis ti c& (121, At1 experiments involving viable bacteriophage and bacteria containing recombinant DNA were performed under conditions specified by the NIH Guidelines for Research Involving Recombinant DNA Molecules. Hybrid Selection-& w&?L~ Translation: 30-120 pg of each AHHG phage DNA were immobilized on nitrocellulose filters and hybridized at 43O for 6 hours in 50% formamide, 20 mM Hepes pH 6.8, 005 M NaCt, i0 mM EDTA and 0,5% SDS. Fitters were washed with buffers of decreasing ionic strength in the absence of formami de, followed by several washes in IO mM Hepes, pH 6.8 to remove SDS. Hybridized RNAs were eluted in 90% formamide, 20 mM Hepes, pH 6.8, 0.5 M NaCl and IO mM EDTA. RNAs were precipitated by ethanol and transt ated as described (13). In U&O translated proteins were analyzed on a slab acid-urea gel to display h;stone polypeptides (14). Preparation of cDNA: 7-11s RNA from S phase HeLa 53 cetts was polyadenylated using ATP-polynucleotidyl exotransferase from maize (15) in a reaction containing 70 mM Tris*HCI pH 8,8, I mM ATP, IO mM dithiothreitol, I mM MnC12. Pol yadenyl ated RNA was reverse transcribed by AMV reverse transcriptase (kindly provided by Dr, J. Beard) in the presence of [a-32P]dCTP in a reaction containing 40 ug/ml RNA, 50 mM Tris*HCl pH 8,3, 20 mM f3-mercaptoethanol, IO mM MgCI2, 30 mM NaCI, 20 pg/ml oligo (dT), 50 ug/ml actinomycin 0, I mM each of dATP, dGTP and dTTP, 30 mM dCTP, and 200 units/ml reverse transcriptase, Hybridization of Southern blots to [ 32P]cDNA was carried out as described Hybridization of Southern blots to nick-translated ptasmid by Lawn e/t UR (7). DNA was done as described by Southern (II),

786

Vol. 104, No. 2, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

1 =Eco RI t =Hittd A =Bam

III HI

Figure I. Maps of AHHG recombinant phage. Eco RI, Hind III restriction sites were determined by single and double digestions; Clone coding regions were assigned as described in the text. in an orientation (with respect to A) opposite to that of the Boxes indicate restriction fragments to which histone coding been assigned; full lines above each clone indicate restriction hybridize to an Alu DNA probe; broken lines above each clone restriction fragments that hybridize to cDNA to HeLa 7-115 S RNA. The two fragments denoted with an asterisk (*I miqrate therefore the hybFidizr ,ion results with respect to these two inconclusive.

and

Barn HI histone AHHG 22 is shown other hHHG clones. sequences have fragments that indicate phase polysomal as a doublet, and fragments are

RESULTS AND DISCUSSION Isolation

of Clones

cloned from

into ~2.6,

a plasmid Fifteen

further

cleases these

clones

H4 coding obtained

containing clones

characterized

Eco RI,

restricted

ning

xCh4A was screened

sequences. were

Conta

Hind

were

III

from

~2.6

(not

histone

chicken

(designated

located several

determined shown).

Genes: genes

genomic

A human genomic

by using histone isolated

mapping,

using

1).

Histone

by hybridization specific

H2B was determined 787

and seven restriction coding

of Southern

heterologous

by hybridization

as a probe

library the

insert

H3 and H4 coding

hHHG) were

and Barn HI (Figure

initially

were

for

by restriction

hHHG DNAs with regions

Human Histone

with

of these endonu-

regions

within

blots

of

histone

probes.

defined

fragments

by hybridization

with

H3 and

yet

Vol. 104, No. 2. 1982

8lOCHEMlCAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

-Ill -Ii3 -Ii29 -li2A

-lM

Figure filter-bound

another

Hybrid AHHG

2.

chicken

genomic

fragment

derived

shown).,

The presence

by hybrid shows

selection-h DNAs and

from

cloned

and in

the polypeptides

in clones

obtained

v&a

Taken

have

pattern

has been found. while

in our

Hl histone

Only

Hl has not

under

coding

regions, is

study

fall

as well

apparently

41 and 55 have shown

to heterologous

into

with

on the structure

the

been localized

either

Preliminary

data

of microheterogeneity 788

Assignment on hybrid

of the

human

repeat

have been detected clones,

blot

analysis,

results).

sites; obtained

techniques of an

respect seventh

from clones with

by

Six of the

with the

so far

either

the presence

(unpublished

endonuclease

histone

no simple

one of two arrangements

as restriction

some degree

but

used to detect

clone

the

of the xHHG inserts.

in any of our

or by Northern

genomic

unique.

genes

2

to be present

all

and organization

histone

Figure

by hybridization

was based

subclones

to be clustered,

core

RNA,

probes.

fragments

(not

was determined

H2A genes

us to confirm

an H2B

genes

RNA selected

showed

obtained

genes

histone

of HeLa polysomal using

by using Methods.

with

the hHHG clones

allowed

have been successfully

gene in a chicken

in

restriction data

were selected Materials and

puh)l)w~~Xti)

experiments

V,ULIJ translation

hands

clones

v&w

and also

shown these

selection-in

(S,

by hybridization

studies

genes

xHHG 39,

These

translation

histone

in clusters

&

RNAs in

as by hybridization

regions

to specific

together,

as well

translation

translated

regions

selection-&

hybrid

C&LO

xHHG 5 and xHHG 55,

of H2A coding

~4.8,

translation. as described

sea urchin

xHHG DNAs.

gene assignments

which

clone,

of H2A coding

selection

to filter-bound

C&LO analyzed

was

respect

to clone, xHHG 5,

to other

BIOCHEMICAL

Vol. 104, No. 2, 1982

5

AND BIOPHYSICAL

6

17

39

22

RESEARCH COMMUNICATIONS

41

55 _'

P -23.7

v

A armA arm-

Figure 3. Hybridization of AHHG clones to HeLa cDNA. C3’PlcDNA to 7-115 polysomal RNA from S phase HeLa cells was prepared as described in Materials and’Methods and hybridized for 20 hrs to a’southern blot containing Eco RINumbers on the right indicate the size (in Kbp) of A digested hHHG DNAs. Hind III markers electrophoresed in the same gel. Black dots at the left of hybridizing bands indicate Eco RI fragments to which histone genes have been assigned by other methods (see text).

restriction clones

endonucleases. may have arisen

Human Histone digested

histone

7-11s genes

specific, contain

with

or alternatively, in

the

broken

in the

suggest

areas

with

hybridized

that

RNA (Figure

Other

3),

Sequences:

obtained

with

These weaker

sequences

that

under-represented

are

partial

homology

Hybridizing

maps shown

in

with

fragments

Figure 789

1,

polyadenylated

known

signals;

sequences,

phages,

When Eco RI-

Eco RI fragments

hybridization were

overlapping

of the genome,

coding

only

seemingly

to a cDNA made to .in v&Jxo

signals

bear

recombinant lines

different

gave the strongest

any histone

results

Interspersed

polysomal

hybridization

hybridization

from

Genes Are

hHHG DNAs were

HeLa cell

These

to contain

weaker,

although

Eco RI fragments signals

could

in the the

genomic

are

indicated

that

do not

be due to

cDNA preparation, sequences by parallel

present

Vol. 104, No. 2. 1982

A

5

6

BIOCHEMICAL

17

39

22

AND BIOPHYSICAL

41

RESEARCH COMMUNICATIONS

5 617 2239

55

41 55

23.7-

23.79

W

m

w

0.60 Figure 4. Hybridization of XHHG clones to of a Southern blot containing Eco RI-digested nick-translated pCDF2, a plasmid containing ethidium bromide staining pattern of a gel at the left of each panel indicate the size electrophoresed in the same gel.

In view recombinant were

of this phages

selected

sequences

(3),

experiments

of the Alu sequence

present

and the

in the

have been found

DNA sequences

inserted

observation

it

in our

histone

genomic

pBR322

family, found

(9).

with

some flanking

in the human s-globin

that

most,

but

not

all,

from which

the

xHHG clones

family

of DNA

members of the Alu directly

gene containing plasmid

Most of this

sequences. A. Hybridization DNA with [32Pl-labeled, DNA sequence. 6. The Numbers to that shown in A. of AHind III markers

library

to look

was pCDF2, a recombinant into

DNA XHHG an Afu similar (in Kbp)

fact

to contain

was of interest

Alu

clones,

(300

790

of Alu

used

in these

a 482 bp sequence bp)

corresponds

corresponding

gene cluster.

representation

The probe

containing

insert regions

at the

of the

Figure

to a member

to a single

copy

4 shows the

hybrid-

Vol. 104. No. 2. 1982 ization

pattern

BIOCHEMICAL

obtained

DNAs was hybridized indicated

to note

three

histone

Comparison those

are not

Alu

This

On the other

which

330 nucleotides during

the

unrelated

used in our clone

the

the cell

cycle.

Taken histone other

DNA sequences

together,

transcribed

are

is

observation

cytoplasm

in poly

in the

with

members

blots

sequences. 791

RNA. (or

to be

other

clones the

have indicated

conditions that

In this

of HeLa cells

the moderately Alu

preferentially

RNA under

sequence.

of the

A+ polysomal

appears

since

cytoplasm

that

(16).

to an RNA of approximately

to this

a non-histone-coding

polysomes

hHHG 41 of a sequence

of DNA sequences,

northern

the

in CCRF-CEM,

sequence

to hybridize

of HeLa

although

with

that

analysis

This

other

of human K562 cells,

associated

in clone

sets

are

polysomes

that,

cycle

indicate

both

results

in the

represented

(2),

DNA sequences

may be due to sequences

of HeLa cells

fail

results

both

have

at least

cDNA show that

in the cytoplasm

to be present

interspersed

to the Alu

have reported

blot

in which

that

hybridize

that

inter-

sequences

suggest

and present

presence

family

contain

our

xHHG are

It

DNA sequences.

possible

are

Additionally,

RNA appears

genes

the

Alu

experiments.

however,

sequences

of the cell

XHHG 39 may also

1.

Alu

by the

by northern

to the repeated Alu

is

u$ (17)

present

results

have been found

ti

Alu

is

Figure

but no Alu

HeLa polysomal

supported

sequneces

hybridizes

Gl phase

containing

7-11s

in HnRNA and in the

reported

that

that

transcribed

is

line,

We have already that

with

cDNA hybridization

Calabretta

human cell

sequences)

associated

It

are

present

hand,

fragments

in

detected, These

not

to the

possibility are

Eco RI-digested

Hybridizing

to pCDF2,

Eco RI fragments

or none of these

another

have been

superimposable.

or histone,

sequences

little

is

and some of the

S3 cells. Alu

regions

hybridize

completely

containing

xHHG 5 and XHHG 55 have an Eco RI fragment

genes

of the

that

independent, than

clones

RESEARCH COMMUNICATIONS

in the maps shown

by hybridization

one of these

blot pCDF2.

lines

mRNA coding

been identified

with

solid

that

histone

when a Southern

to nick-translated

by parallel

esting

AND BIOPHYSICAL

case, throughout

reiterated

DNA family

and with

human

Vol. 104, No. 2. 1982

8lOCHEMlCAL

AND BIOPHYSICAL

ACKNOWLEDGEMENT.

RESEARCH COMMUNICATIONS

Sherman Weissman and Julian We thank Drs. Richard Boyce for a gift of A DNA. These studies and Dr. grant PCM 80-18075 from the National Science Foundation,

Pan for pCDF2 DNA were supported by

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Sierra, F,, Lichtler, A., Marashi, F., Rickles, R., Van Dyke, T., Clark, S., Wells, J., Stein, G. and Stein, J. (1982) Proc. Natl. Acad. Sci., in press. 2. Rickles, R., Marashi, F., Sierra, F., Clark, S,, Wells, J., Stein, J. and Stein, G. (1981) Proc. Natl. Acad. Sci., in press. 3. Tashima, M,, Calabretta, B., Torelli, G., Scofield, M., Maizel, A. and Saunders, G.F. (1981) Proc. Natl. Acad. Sci. 78, 1508-1512. E.H., Galau, G.A., Angerer, R.C. and Britten, R.J. (1975) 4. Davidson, Chromosoma 51, 253-259. 5. Houck, C-M., Rinehart, F.P. and Schmid, C.W. (1979) J. Mol. Biol. 132, 289-306. W.R., Toomey, T.P., Leinwand, L,, Duncan, C.H., Biro, P.A., 6. Jelinek, Choudary, P-V., Weissman, S.M., Rubin, C.M., Houch, C.M., Deininger, P.L. and Schmid, C.W, (1980) Proc. Natl. Acad. Sci. 77, 1398-1402. 7, Lawn, R.M., Fritsch, E.F,, Parker, R.C., Blake, G. and Maniatis, T. (1978) Cell 15, 1157-1174, F.R., Williams, B.G,, Blechl, A.E., Denniston-Thompson, K,, 8, Blattner, Faber, H.E., Furlong, L.A., Grunwald, D.J., Kieter, D,O., Moore, D.D., Schuman, J.W., Sheldon, E.L. and Smithies, 0. (1977) Science 196, 161-169. 9, Duncan, C.H., Jagadeeswaran, P., Wang, R.R.C, and Weissman, S.M. (1981) Gene 13, 185-196. 10. Clewell, D.B. and Helinski, D.R. (1970) Biochemistry 9, 4428-4440. 11. Southern, E.M. (1975) J. Mol. Biol. 98, 503-517. A, and Kleid, D.G. (1975) Proc. Natl, Acad. Sci. 12. Maniatis, T., Jeffrey, 72, 1184-1188. Marashi, F., Rickles, R., Stein, G. and Stein, J,, manuscript submitted. 1143: Panyim, S. and Chalkley, R. (1969) Arch, Biochem. Biophys. 130, 337-353. Chem. 250, 3672-3678. 15. Mans, R.J. and Huff, N,J. (1975) J, Biol. Elder, J.T., Pan, J,, Duncan, C.H, and Weissman, S. M. (1981) Nucl. Acids 16. Res. 9, 1171-1189. Calabretta, B,, Robberson, D.L., Maizel, A,L. and Saunders, G.F. (1981) 17. Proc. Natl. Acad. Sci. 78, 6003-6007,

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