M&c-trlur

atrd Biorhemirul

Purosrtokyq~.

CI99 I I I 2 I - I30

45

I?1

Elsevter hlOLBI0

01478

Chromatin organization in Entamoeba histolytica Haydee Torres-Guerrero ‘. Debra A. Peattie’.” and Isaura Meza’ ‘Depurrumettro Hutwrd

de Biokyru

Crltdur.

School of Publrc Healrh.

C/M

ESTAl ‘-IPN. Me.uico Cig.

Mtwico:

‘Depurmrenr

Bos~nn. M.4. U.S..-\ .: .‘I trte.v Pharmucetrticulr

of Tropical

Incorporated.

Ptrblic Health.

Cumbrid,qr.

M.4. L’S A

(Received I August 1990: accepted I October 19901

The chromatin structure of Enrumocbu hisro/yrica ICBS investigated. It was found that this protozoan organizes its chromatin in nucleosome-like particles IO nm in diameter, but digestion of the chromatin with micrococcal nuclease did not render a regularly spaced DNA ladder in agarose gels. Southern blot analysis of the products of Enwnoehu chromatin digestion using total amebic DNA and a non-transcribed repetitive sequence produced a banding pattern characteristic of eukaryotic chromatin with a repetitive size of approximately I30 hp. Conversely. hybridization with two active gene probes. actin and ribosomal RNA. showed that these sequences are not part of the chromatin organized in nucleosomes. It was also found that the basic nuclear proteins differ from histones of higher eukaryotes in electrophoretic mobility. Screening of an E. hisro/ytica HMI-IMSS genomic library with Sucrhuromwes cerelisiue H3 and H4 genes and attempts to amplify E histol:ricu sequences. homologous to these yeast histone genes. gave negative results suggesting that the Entumoebu proteins involved tn chromatin organization are not typical htstones. Key words: Ettrumoebu

Chromatin: Nuclear proteins

hisro!\/ic-o:

Introduction

evolutionary

step in the strategy

otes to organize

Chromatin

in

most

in nucleosomes. complex H2A.

H2B.

wrapped

and

by electron [l].

with

nucleoprotein 160 bp of DNA particles

microscopy mild

varying

On the other

with

renders a lad-

from hand,

the can

in the so-

digestion 160-250 bacteria

bp have

small amounts of basic proteins associated their DNA [3], and some eukaryotes such

as dinoflagellates of these

in DNA

lack basic

compaction

histones

proteins could

[4,S]. and

represent

their

The

pres-

function

an important

Isaura Meza, Departamento de Biologia Celular. CINVESTAV-IPN. Apattado Postal 14-740. Mexico. D.F. 07000. Mexico. Correspondtvt~ c uddress:

PMSF, phenylmethylsulfonyl fluoride: NEM. N-ethyl maleimide: PHMB. p-Hydroxymercuribenzoate; PAGE. polyacry lamide gel electrophoresis.

Abbrt~\*rutions:

0166-68Sl/91/$03.S0

studies tion

of the histones

the chromatin

fragments

[I].

organized

core [I .2]. When

Upon

nuclease,

der of DNA

ence

H4

the protein

IO nm fiber

micrococcal

only with

nucleosomal

is spread. the nucleosomal

be visualized

in length

is

by an octamer

H3.

around

chromatin called

The

is formed

eukaryotes

their genomic

of chromatin

in primitive

provide

compaction

composition

forms

information

used by eukary-

material.

such as protozoa

about

of DNA

Therefore.

and organiza-

the transition

without

should from

histones

the

to the one

present in complex eukaryotes. The Protoctista kingdom [6] includes

very

versified

features:

organisms

with

(a) they

are unicellular

defined

nuclei.

In

two

common

and (b)

they

Mastigophora

have

such

diwell

as Tg-

panosoma cruzi [7]. Tyypanosoma brucei [8] and Chlamydomonas reinhardfii [9]. it is possible to extract

basic

acrylamide

urea gels and in polyacrylamide-SDS

gels

with

proteins

a profile

tones of higher

mena piriformis only stones ogous

similar

eukaryotes.

that

migrate

to that

in poly-

of

the macronucleus

(HI. H2A. H2B, H3. and to their counterparts from

the

his-

as Terruhy-

In ciliates

H4) calf

has 5 hihomolthymus

Digestion of U.lytricha nova (another cilmacronuclear chromatin [ 1 I] and T. brucei chromatin [8] with microccocal enzyme gen-

[IO].

iate)

erates codina

the characteristic DNA ladder. such as Enramoeba histolyfica,

18 1991 Elsevier Science Publishers B.V. (Biomedical Division,

In Sarlittle is

knot! n The

about life

the

cycle

composition

phases: (a) the trophozoite. hally

in the intestine

nal wall: causes

features

commenthe intesti-

is the form

that

possesses a single

of ancient reticulum

lar cgtoskeleton. out dissolution erwise

inside

mitotic

spindle

nucleus

have only where

chromatin

membrane.

is observed

Although

densed metaphasic chromatin-like photungstic

close

chromosomes have

been re-

to the nuclear

been reported has

of con-

I-l

composition

and

trophozoites.

We present

that

nucleosome-like

basic DNA-binding histones.

proteins

and that actively

Etr~unrochu

srmitrs.

buffer

mM

MgC12/0.6

mM

NaHS0,/0.3

added

(IO mM

suspension

and DNA the in

suggest by

from known

transcribed

genes

are

der the light

of the nuclei. were layered

at 6000

animals

cell un-

of E. hislolyfin TY I -S-

33 [ 181 at 36°C.

cell lysis and

as reported

with

filtered

and centrifuged

The nuclear

mM KCI/IO

The nuclear

pelbuffer

or chromatin

from

mouse

[?I].

Livers

liver

cells

from three

in 0.32 M sucrose containing

the tissue was minced this

cushion

and centrifuged

estraction

Nuclei

enized

sus-

IO ml of the nuclear

for protein

were pooled

i\as

and observed

in 2 ml of isolation

M&l?;

crose cushion

NP-IO

X&.

to monitor

I5 min at 4°C.

digestion.

KCI/IO NEM/IO The

spermidine)

3 mM

I”‘C.

and

mM Tris pH 8.0/3-l mM

x ,q for

prepared

mM mM

on a IO ml glycerol

let was resuspended nuclease

in 10 ml of isola-

of

agitated

microscope

MgC1?/0.6

here

PMSFA

by Ed-

moditications.

pH 8.0/3-l

concentration

ini-

performed

ah reported

solution. pellet

The

and homog-

homogenate

was

at 700 x in. Basic proteins were extracted from tropho-

trifuged

N-lauroylsarcosine

clone

added

The filters

We confirmed

exposing

bp The

were prepared

and exposed

DNA

I35

from a genomic

10h cpm ml-’

I3 h at 42°C.

Lvith intermittent

bromide.

Tris-HCI.

x

three

of 5 mM

nuclei

of I3

probes

and were

ml-’

by adding

and

concentration U

[27]

for

stopped

treated.

0.03

priming

from a cDNA

bated

concentration

digested

with

nuclease

times

NaCI/O. IS

mM CaCI:/lS

pH 8) and digested

per 2Az6(, of micrococcal of nuclear

mM

actin insert was derived

size of

in the trophozoite.

Micrrx-coca1 ttuclease digestion of Enraatoeba und nmtse liwr chromatic. Isolated nuclei of E. hisroi!ricu and mouse liver were resuspended in mibuffer

(b) pE.h. I2 in-

t I.5 kb); (dj ribo-

insert

IR

NaCI/ (O.l%, bovine

SDS/l 00

/lg ml-’ DNA at 42°C. Filters were hybridized with the following radioactive [“P]dATP-labeled

analyzed gel

gel using

Laemmli

electrophoresis

ter electrophoresis stained with 0.1% methanol/water/acetic ivith

ale~lr~tp/tot.t~sis. Proteins

the S:4:1

the

gel

SDS-polyacryl-

(PAGE)

[38].

Af-

was

fixed

and

Coomassie Blue acid (S:1: I ) and

mixture.

Ptmeitt blorrity und DNA bittding. solved lulose

dye in washed

Proteins

re-

by PAGE were transferred onto nitrocel[29] and incubated in IO mM Hepes. pH

8.0/50 mM NaCI/IO mM MgCIJO. I mM EDTA/I mM DlT/O.2% low fat milk with I x I@ cpm of purified E. hisrolwica DNA labeled with “P [30].

I

Kbp

1.07 0.87

0.60

0.31 0.28 0.23 0.19

IllCUbiltiWl

\\a

done for 3 h

.AtIer incubation IO0

iNKI

750

Illhl

i11

room teniperalture.

the paper> \\ere i\xhed NaCl

3t rooni

\\fith 50.

tZlllpr3riltLlre.

Results of E. ItiJtolwictr

Sptwiitr~

clttwtt~~titt.

Spreads

cated lq Rattner et al. [Ii I]. Llndrr these conditions

a particular portion of chromatin completeI! de\ oid of particles but rather beaded and non-beaded

a partial disassembly of chrornatin took place. and

regions here heen alternstin?

from \\ hole cells t\ere

different (Fig.

prepared

le\,el> of it5 organization

in PBS as indi-

could he seen

I ).

Estended nucleosomal chains chi~racteris;tic of masimullv dispersed St;lte> of chrornatin Lvere obsewed in-man\ regions of the hprend. The averqe diameter of the Ett~~tttwdxt nucleosome-like pnrticlex in these regions \\as IO nm as found in most sukaFotic nucleosomes. Hotkever. in contrast to osornal lengths rnce of

most eukngotes studied. the intemuclelinker DNA in Ettttrttrocbn had \ sriable (lwver magnification and inset I. The presnon-beaded stmcfure:s \%as not confined to

Di~persrtl pared in the

in the

11lwse

chromatin

of

same

sho\\ed

WHJ

5;ulie

tiber.

liver cells pre;1 regular appear-

ance (data not sho\vn 1.

Entctttroh~

hisrolyttcu

lrtd

tttouw

li\xlt. chortrutitr

tligcstiott \litit tttict.ocx.ocul tttudtww. Etmttrrwbu chromatin digested M ith the nuclease for different times did not render the regular intewal banding pattern of the nucleosomal structure (Fig. 2. lanes 2-7 1. The high-molecular-Height chrornutin Ht time 0 cFi9. 2. lane I ) is a broad band that diminished in

!

1234567

34567

2

I

1

234567

234567

Kbp

IO7 0 87 0.60 -

0 31 028$:;I

E

A Fig. 3. Different hybridization

patterns of chromatm

was carried

organization.

out with random-primed

probe: tCt actin gene: IDI

rDN.4

probe. Lanes

Enrornochu labeled

l-7

time. After

10 min (Fig. 2.

of DNA

was observed. Mouse liver conditions

of different

chromatin

showed

size

digested

the banding formed

under identical

pattern

character-

by the 3 core histones

HZB. H3. H4) at incubation

times

of both

10 min (Fig. 2. lane 8) and 30 min (Fig. 2. lane 91. The size of the nucleosomal chromatin tween

was determined

adjacent

standards.

to digestion

as in Fig. 2 \\as

The

oligomers average

unit of approximately

repeat in mouse liver by the difference relative

repeat

to known corresponds

l-IS bp. a size which

result from the loss of histone term digestions.

HI

during

beDNA to a could long-

oj- microccocal

tamoeha

cht~onu~titt.

with

micrococcal

mclease

times of 0, 0.5.

The

nuclease

nuclei for

itr Ett-

were

digested

different

lengths

I. 1. 1.

(B,

and

pE.h. 12

IO and 30 min. Molecular

The pattern generated by hybridization to total genomic E. hisrolyica DNA is shown in Fig. 3A. Even after short periods of digestion. 0.5 min (lane 2). a periodic banding strong background

pattern

(lane 7) the faint banding due to the increased average like

number

repeat

is smaller matin.

hybridization showed

At 30 min

was less apparent

background

smearins.

of the Etmnroeba repeat

the hybridization

nucleosomeof mouse

with

total

with the pE.h. 12 fragment

a clear

banding

The

to be 130 bp. which

than the DNA

Unlike

signal.

pattern

was calculated

e.g.. DNA that resem-

was seen in spite of a

hybridization

pattern

with

chroDNA.

(Fig.

little

3B)

smear-

ing. Bands appeared at the early times of incubation. e.g.. 0.5 and I min (Fig. 3B. lanes 2 and 3): at 30 min (Fig.

digesriott

to nylon

digested Cp X I74 DNA.

bands were more Parrems

blotted

with: 1.41 E~rran~oeha total DNA:

bled the nucleosomal

istic of nucleosomes (H?A.

fragments

clectrophorescd

Hybridtzation

to Hazlll

lane 6) a smear appeared and reached a maximum at 30 min (Fig. 3. lane 7) where a continuous distribution

DN.4

DNA.

correspond

weight marks correspond

size with the incubation

D

Hybridization probes with

did total

not DN.4

3B. lane 7) the corresponding intense

with reveal

and clearly

the

actin

the band

observed.

and

ribosomal

periodicity

and the pE.h. I? sequence

seen (Fig.

of time as shown in Fig. 2. and the resultant DNA fragments were electrophoresed, transferred

3C and D). Up to 3 min of digestion with micrococcal nuclease (Fig. 3C and D. lanes 1-S). actin

onto

and ribosomal molecular-weight

nylon

membrane.

and hybridized

individu-

ally with: (A) total Ettrantoeba DNA; (B) pE.h.12; (C) actin gene: (D) rRNA cluster (Fig. 3).

molecular-weight

sequences hybridized Ettruntoeha DNA. DNA

is

with This

transformed

highhighinto

a

I2

3

4

5

6

7

8

9

lo

123456-a

0.60

0 31 028

C

I3

smear ~kith longer incubation

time5 (Fig. K

and

D. lanes 6 and 7).

with

and pE.h. 12 (Fig.

DNA

(data not hho\in)

-lB. lane4 I-3,

Ptrrrt~rtrs prrt$td DNA

cf tttic~tnc~occtrl Ett~untod~t~ DN.4.

di~t~sriotr ot Purified Etuattudxr

trtrcletrse

~vas digested under the same conditions

the chromatin.

The times of digestion

IO. 30 and -IO min at ?7’C. \vcre slectrophorcsed

The DN.4

in a I.55

-IA,

of pure DNA

2 min of digestion DNA

UBS converted to a smear stretching from ‘3 to 0.5 kb (Fig. 1.4. lane I I: at -I min the molecuof the smear \vas between

DNA

degradation

E. Irisloluictr

digested in parallel tvith micrococcal

the broad band of hi_gh-moleculnr-weight

lar \\eight

tified due to complete

fragments

( lanes I -.S I \\ as faster compared \s ith that of chromatin tlanes 7 and 81. Within

a smear that decreased

lanes J-5).

agarose gel and

that the rate of digestion

generated

in size \\ith the progress of incubation. After 30 and -IO niin of incubation no signal could be iden-

were 7. -I,

blotted to a nylon membrane (Fig. 1). The ethidium bromide stained gel (Fig. shwed

as

1.37 and 0.7

kb. At IO min of incubation a smear of very small hize (< 150 bp I could be seen ( Fig. AA. lane 3 1. and at 30 min the DNA \~a4 completely degraded (Fig. -IA. lane -I ). The purified DNA \\as blotted onto a nylon membrane and hybridized kvith the same probes

total E. his-

ubed in Fig. 3. Hybridization

fo/uic.c7

hybridized

Conversely.

L$ith pE.h. I? revealed

(Fis. AB. chromatin

nuclease and periodicit!

of

the DNA bands (Fig. -IB. lanes 7-8). Hybridization Lvith the actin probe sho\vn in Fig. 1C. and ribosomal

genes (data

not bho\\n,.

produced

mearing pattern in both puritird DNA lanes I-5 I and chromatin I Fig. K. lanes bands Lvere evident

a

(Fig. -K. 7-X 1. No

in these h> bridizations.

SDS-pol~clc.t~ILInriLle ,yci t?lec,rtut~thot.tIsi~ c$ hrsic pt-ott~itts. The analysis in PAGE-SDS of the basic proteins obtained

from trophozoites

and from

amoeba and mouse liver nuclei can be seen in Fig. 5. In the acid cell extract. proteins of 97. 68. 15. -II+. and 10 kDa Lvere enriched. An enrichment of acid-soluble proteins from trophozoites that resemble histones in molecular tveight \vas

I23

Kd 200

45

kd

97

53-

IL

I

-

68

343025-

43

l925

16-

1E

tems uere uith

I x

nuclear

basic protems.

Trophozoites

and

isolared nuclei were incubated with 0.25 M HCI. The extracted soluble proteins were precipitated tionated by 10% SDS-P.rZGE.

Lane I. molecular

trophozoites:

tielght

IO’ cpm ml-’

from ameba nuclei: lane 5. acid-soluble

pro-

proteins

proteins from rat liver

(Fig. extracts

5. lane 3). Moreover,

mouse

liver

nuclei

of the proteins Acid

histones (Fig.

nuclear

However, from

cell extract cal histones.

and again

of nuclear

from

represent

most

in

binding calf

lane 2: and 2.50 mh.1 NaCI.

proteins

thymus,

of molecular

proteins

are dif-

in the acid weights

proteins

whole to typi-

of the most

are 34. 30. 23.

basic DNA-binding

and SO0 mM

NaCI.

was observed

phage

proteins

tern of nuclear staining

After lane I:

lane 3. Lanes -I te nuclei

respzcu\el).

Enrumoebu basic nuclear proteins were transferred to nitrocellulose paper and hybridized with “Plabeled calf thymus DNA, /\ phage DNA or total Enfumoeba DNA. The same pattern of DNA-

of Etltumoebu is shown.

From

basic proteins

in Fig.

5. only

DNA

with

DNAs. binding

the complex

In to pat-

seen by Coomassie

the proteins

to the S3-. 30- and 2S-kDa

creased slightly

(Fig.

of 53, 33.

ionic strength

was increased completely

(Fig. band mM

and to

I!+kDa

(Fig.

As a positive soluble

de-

to 100 mM. the DNA

from

6. lane 2). The DNA NaCl

proteins

6. lanes 2 and 3). When the

was removed

used acid proreins.

on hybridizing

X and Enrumoeba

30. 25. I9 and I6 kDa bound to DNA (Fig. 6. lane I). With 100 mM and 250 mM of NaCI. the binding

Entumoehu

do not correspond

The molecular

prominent basic nuclear 19. I7 and 16 kDa. Analysis

extract

range

the enriched

the ones enriched

pattern

5, lane 5).

extracts

5, lane 4) have a wide

weights. ferent

to a similar

where

extracted

soluble

acid sol-

gave a very complex

(Fig. 5, lane 4) compared

NaCI.

washed Nirh 30

nuclear uble nuclear

of labeled Iota1 amoeha DNA.

the hlters were washed with SO mhl NaCI.

Fig. 6. the pattern

cell nuclei.

not observed

Acid soluble pro-

paper and incubated

mark-

lane 3. acid-soluble

lane -I. acid soluble

proteins.

IO nitrocellulose

and 5 are acid soluble proteins from chicken eryhroc!

with 95% ethanol and frac-

ers: lane 2. E~rramoehm total extract: reins from whole

100 mhl

DNA-binding

transferred

incubation. Fig. 5. Ewunroeba

;-

Q

Fig. 6. Enromot+u

14

(Fig.

0

the I6-kDa

binding

proteins

protein

to the %I-kDa

was stable

at 250

6. lane 3). control

for the DNA

nuclear

proteins

binding from

we

chicken

erythrocytes (Fig. 6, lanes 4 and 5). The binding to these proteins was not modified with 250 mM NaCI, but at SO0 mM almost disappeared.

NaCl

the binding

to HS

I’S

Discussion

>uscrptihle

to digehtion

tribution Electron sho\red

microxop!

different

of the chromatin

levels

of

tion.

In the most extended

ture

corresponding

tihers

organiza-

a headed

to nucleosome-like

10 nm in diameter zones totally

\\as obsewed.

devoid

There

of these structures.

in some region?, the intemucleosomal variable.

resulting

pattern.

Analogous

in

an

plied

is variable

liver

gressive

same

chromatin

nuclease

der of

DNA

rendered

particles

spreadings.

digestion

coccal

technique

not

of pro-

with

micro-

a regular

as \+as observed conditions

ladunder

for mouse

liver

chromatin. The

of a regular

Etrrc~ttwch~ chromatin of proteins

banding

since purified

faster than the chromatin.

that Etrrtrttrodx~ chromatin

in modilied

pattern

nucleosomal

of chromatin

structure

ing micrococcal genes

of discoidin

structures.

The analysis organisms

to study

has led to different

active results

gene analyzed.

which

actively

mal banding

1 gene of Dicyysreliwn

significant express

is lost. This

gene of Dtmophila the ovalbumin

[.!?I.

and independ-

chromatin

ladder.

transcribed

with

sequence.

actin

the transcriptional

times

RNA

bands Lvere \ery

u ith total

in cells

region

gene [32]

c-oiticwnr [36].

[32-373.

histone

secretory

pro-

and the pE.h. I3

This Has tested lvith

Etrratnoehu

DNA.

structure

and

at A+T-rich

the digestion

H here no bands

of

were

support the conclusion that has a small proportion of

organized

in nucleosome-like

the DNA

ladder

pattern.

to the nuclease

Eukaqotes

strucbut that

action.

chromatin

as the result of transcriptional sequences.

pattern

after hybridiza-

cleavage

Not all the nuclease-sensitive essential

the period-

banding

DNA

nuclease

most of it is sensiti\e

genes

tein gene of Clritmot7zous [37]. The hybridization of Etrtatt~odxt digested chromatin with different probes corresponding to nontranscribed as knell as actively transcribed genes revealed that some DNA regions are organized in regularly repeating units while others are more

found

The

chromatin

Etrruttrodw

tures that gives

explained

cell embryos [M]. the and ribosomal genes of D. tlis-

and the hyperactive

[Ml.

DNA and the

When the actin and

was due to the chromatin

its chromatin

of

strong

for actively

not to preferential regions

hharp

\\a> un-

as has been

genes

Lvith total

h)

by a smear

observed. Our results Etuunwchtr chromatin

nucleoof other

and the nucleoso-

gene [3-l]. n-subtype

tion

This

Etrrcwwt4m

was vr~

by the digested

sequence

\ug-

and it \c’as replaced

tliscoidtwnt

is the case for the /zs/)70 the 5’ flanking

t Fig. 3C and DI transcribed

a

When pE.h. 12 L\HS hy-

genes \vere used ah probes.

disappeared

shown

cluster. is affected

of incubation.

like the h> bridization

icitg

that renders

structure

activit!.

M here the background ribosomal

is

and

pE.h. 13. and \%ith two active

the nucleosomal

even at longer

and

action.

an E. Irisrd~ric~c~ non-

and a ribosomal

gested that the chromatin bridized.

,tructurs

to nuclease

organization

Probing

purified

moditications

them.

us-

of Psc~n~tttt~c~ltitr~tsli\idrrs pCZ22

DNA

btructures

that is not pro-

nucleosoms

sensitive

In the case

[I+?]. the active genes eshlblt a typical somal pattern. HoLvever. the chromatin genes undergoes

DNA

hut rather is organized

in diverse

nuclease

ing on the particular

for

was not due to the absence

bound to the DNA.

\ias digested

active

more

band-

qgestins

chromatin

w-ucture

bands \vere not \‘ery apparent.

absence

suggests

bg a typical

therefore

genes.

the products

produce

fragments.

the same esperimental

spreads

an active

cor-

olxened

this regular

~%asapparent.

of at least tbo

in Etrrtrttrocbtr:

an inactive ap-

\iere clearI>

of the chromatin did

the presence tected

reported

regions.

nucleosome-like

seen in chromatin

nucleosomal

the intrmucleosomal

intemucleosomal

Although

H’;\s

>ho\rsd

bize could

particles

. Behind

microscop!,

of to-

chromatin

whose

to the nucleosome-like

ing. a strong background

where The

pattern

dis-

HJ bridization

to digested

by electron

no

although

been

respond

DNA banding

were

length

irregular

tal amoeba a regular

and give a continuous

fragments.

particles

have

[Jl].

to mouse

u ith regular

wuc-

results

for Least chromatin. linker

spreads

chromatin

of DNA

can be

activity

have more

of

DNA

than is necessary to encode essential functions [39.-W]. In most higher eukaryotes studied 1391. fewer than 30% of the single copy sequences are transcribed into polysomal mRNA diris c1t~gtrtr.s [-I 1] and Dtmophilu

In Ctwtwt-lrtrhttrt~lunoyctsret~

[43]. genetic studies have shown that there is one essential gene per 30 kb DNA. and in yeast [33] it has been found that onI> 12% of the yeast genome is essential. The E. Irisrolyricu genome

has O.-l-l.5

pg DNA/trophozoite

0.3 pg DNA/cell. bp. Taking

[U].

this corresponds

an average

chain

Assuming

to 3.96

x

Ifly

gene size of 2.0 kb based on

reaction

for H.

using different

although

thymus.

herring.

These amplified

sequences

were found in the actin gene) [2-l]. at least 191000

H4 gene (data

not shown).

proteins

actin cDNA

could

be encoded.

of Etzrumwhu

genome

be 38400

genes.

times

the

number

with

5000

genes

is not feasible a small One

for

DNA

D. tt?elotro,qclsir’t. then perhaps

onl) corre-

could

chromatin

the

organized

that render

nuclease.

non-transcribed

This

proteins

induced hibitors

histones since

bisulfite proteases fumoehu

extracts

in molecular

mass.

the protein

extraction

PMSF.

Five

do

to protease

of protease

pHMB

to purify

[16].

plement zation

in-

and sodium histones

proteins

from

of the amebic

This

from

that supports in the

matin

in an Earamoeba

genomic

library.

biochemical

organization

nu-

char-

proteins

would

in-

also com-

of nucleosome

organi-

in eukaqotes.

was

supported

for

by

Health.

the ameba by the UME

H.T.G.

and a MacArthur agreement

with Har-

We are very grateful

for her teaching

technique

CONACyT.

Foundation.

by CONACyT

of Public

were provided

acid-

of the chro-

and to E. France cultures.

EM

and

facilities

at CINVESTAV.

References

struc-

3

tures in this protozoan are different from known histones was our inability to identify H3 and HJ sequences

eukary

but lack

nuclear

student exchange

spreading

A. Boyzo

the idea that the

nucleosome-like

complete

and the MacArthur

to Dr. B. Hamkalo

2

involved

lower

Etr-

in the range of histones.

evidence

research

Foundation

to DNA.

Other

metaphasic

[17].

the understanding

vard School

have molecular

proteins

and

typical Other

of E. hisroiyic-u

was supported

I

weights

part

structures

Acknowledgements

soluble nuclear proteins could be related to histones since they are enriched in nuclear extracts. and bind

to protect

chromosomes

and evolution

and cilpossesses

(2)

not due

used to preserve

[-IS] and

although

and more

in chromatin

Mexico

in Etrrumoehu

at 4”‘C in presence

routinely

volved

yeast

the ability

are not visible.

acterization

but most of it could

identified

such as NEM.

and

proportion

sequences.

is probably

degradation

was performed

a small

as was

the tropho-

be in the cyst. where

to non-coding typical

during

Further

periodic chromatin

sequences

actively

be due to gene activity

difference

chromatin.

or histones

but could

not resemble The

with

for

pro-

or have

Ettrumor~bu

in nucleosome-like

cleosomes

chromatin.

The nuclear

DNA

condensed

transcribed

correspond

proteins

the

otes show

with the pE.h. I? sequence or genes that are phase.

specific

hand.

histone

modified

as reported

chromosomes

shown zoite

greatly.

On the other

orga-

bulk

DNAs.

to the yeast

consemed

are either

struc-

contain

the sensitive

iates.

condense

should

not being

diverged

of

the most

in evolution.

protozoan

this

particles

with

chromatin

sequences.

is that

in nucleosome-like

such a number

sequences.

at least in two different

( 1 ) the typical bands

teins

of the sensitive

possibility

DNA

H,

to almost

inferred

nizes its chromatin tures:

there should 8

ettrietii

hybridized

These results suggest that in Etttumoc~hu H3 and

ZOc;lr,

corresponds

[-IO]. Assuming

to expressed

that

is expressed.

in Entamoeba.

portion

sponds

This

Assuming

primers

to do so from calf

and L&~hmunim

of I .S kb (no introns

the E. hisrolvricu

degenerate

it was possible

We

screened the genomic library with the Suc-chutwrtwes c-ere\*isiue genes for Hj and H, and with primers designed from consensus sequences of those genes, and based on the Ettkawehu codon usage [30]. It was also impossible to amplify histone Emamoeba sequences by the polymerase

4

5

6 7

Komberg. R.D. I 1977) Strucrure of chromalln. Annu. Rev. B&hem. 46. 93 I-054. Thoma. F. and Keller. T. I 1981 I Unravelled nucleosomes. nuclcosomal heads and higher order strucmres of chromatin: inHuence of non-hlstone components and HI. J. Mol. Biol. 149. 709-733. Diirenhergrr. hl.. Bjomsti. h1.A.. Uetz. T.. Hobot. J.A. and Kellenberger. E. I 1088) Intracellular location of the histone-like protein HU in Esclrtwc~hiti w/r. J. Bacrrriol. 170. 47574768. Rizzo. P.J. and Moms. R.L. I 19841Some properties of the hislone-like prowin from C,.!prlre~otlinrrtm chrir I HCc I. Bioustems 16. 21 I-216. Rizzo. P.J. I 19871 Biochemistry of the dinoHagellate nucleus. In: The Biolog) of Dinoflagellares (Taylor. C.F.R.. ed. I, pp. 113-l 7.1.Blackwell. Osiord. hlargulis. L. and Schwanr. K.V. I 19x21 Five Kingdoms. 1V.H. Freeman. San Francisco. Ruhio. J.. Rosado. ‘u’. and Castaneda. hl. I l9X01 Suh-

krenner. S. 77 f t_ 7 I-94. Judd.

an;ltom\

pmmi~i

Harbor L&rato~. Cold Church. G.hl. and Gilbert.

Sprtng Harbor. N\I’. \\‘. I 198-11 Grnomic

_ wquencing.

; i474,

The genetik

oi (‘

&y~s.

Gcnrttcr

B.H.. Shrn. hl.\V. and Kauimm. T.C. t IYi2 I The 3rd I‘unction oi :I epmcnt of the N chromowme

mpm_-e~ are not e*wntidl ior cell prwth and divl\lon. Cell 46. YX3-YY?. B!crs. T.J. I 10861 hlnkular biolog! of DNA m %tirr. r/rc~~irkd. YY. ?I I-3X3 \on Halt. B.. Brad. H .F.. Cirq I~ng. H.J.. Lmdq. G.G.. Retiet. J.D.. Rodrigues. J..\.. Srhungcr. S. and Serell. B T. I IYXY I Iwdnticln and charactertrntion oi hiwxw. hlcthod~ Enq mol. 170. 13 I-M. hlszu. I.. Sdbanzro. hl.. Cnrnres. F. and Bryan. J. I lY8.31 Iwlatlon :~nd shuractctiratlon oi xttn from Em~~rtuwlw lI/t/t~/!/i~rl. J. Bid. Chem. 258. 3Y?h-?Y-lI. RIZZO. P.J. I lY851 Hi\tane\ in prottam c\olutron BIO\!~tern\\ 18. 24Y-32.

Chromatin organization in Entamoeba histolytica.

The chromatin structure of Entamoeba histolytica was investigated. It was found that this protozoan organizes its chromatin in nucleosome-like particl...
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