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
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