Unusual RNA polymerase content of Trypanosomabrucei nuclei

Vol.

13 1, No. 2, 1985

September

16,

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1985

Pages

844-848

UNUSUAL RNA POLYMERASE CONTENT OF TRYPANOSCMA BRUCE1 NUCLEI David

L. Earnsha&,

*Department **Department

Received

Trevor

of biochemistry, Brighton

of biochemical

June

25,

J.C.

Beebee*

and Win E. Gutteridge**

University of Sussex, BNl SQG.,U.K.

parasitology, Wellcome Beckenham, Kent, U.K.

Falmer,

Research

Laboratories,

1985

SUMMARY: Nuclei were isolated from bloodstream forms of Trypanosoma brucei by nitrogen cavitation and sedimentation through percoll density gra=. Transcription studfes with these nuclei demonstrated features not --in vitro seen with other eukaryotes: RNA synthesis was much greater in the presence of Mn2+ than with Mg2+ and was sensitive to high concentrations (lo-100 pg/ml) of (Y-amanitin at all salt concentrations tested (25-300 mM ammonium sulphate). RNA polymerase extracted from nuclei by sonication at high ionic strength to highC&amanitin concentrations, chromatographed as a single peak, sensitive on DEAE-sephadex under conditions which resolved the classic three RNA polymerase forms when rat liver nuclear extracts were used. @1985 Academic Press, 1~.

It

has become clear

in Trypanosoma yet

studied.

variable

reported

concentrations

intermediate

is

similar

with

of transcription sensitivity

isolated

isolated

concentrations

chromatographic

properties

0006-291X/85 $1.50 Copyright 0 1985 by Academic Press. Inc. AN rights of reproduction in any form reserved.

In this with

of cr-amanitin to that

844

methods

isolated

been

from whole peak,

sensitive

of DEAE-sephadex established

from 2. brucei

resistant,

associated

not

also

has

as a single

enzyme was however

(4).

It

elution

the

physical

(1,2).

by standard

eukaryotes

in expressing

may include

chromatographs

nuclei

in other

involved

on gradient

to a-amanitin

to high

which

of gene expression

observed

in the chromosomes

reactions

activity

not

the processes

activity

of this

assays

of RNA polymerase

sensitive

are

or 1. cruzi

origin

the mechanisms

features

of'(Y-amanitin,

work , and "run-on" the existence

sites

2. brucei

that

(VSG) genes,

RNA polymerase

The intracellular

bulk

among these

to telomeric that

years

unique

glycoprotein

of either

to high

include

Foremost

surface

translocation

cells

brucei

in recent

have

in this indicated

highly

sensitive

paper

we report

that

2. brucei

nuclei

isolated and after from

extraction

whole

cells;

(3).

and with the

has

Vol.

131,

No. 2, 1985

multiplicity

BIOCHEMICAL

of trypanosome

We have

therefore

RNA polymerase

BIOPHYSICAL

RNA polymerases

identified activity

AND

the

of r.

RESEARCH

was not evident

intracellular

location

COMMUNICATIONS

using

this

of the major

approach. cellular

brucei. MATERIALS

AND METHODS

Blood trypomastigote forms of 2. brucei were maintained by syringe passage in mice every 2-3 days and grown in rats for large scale preparations of nuclei. Cells were purified by DEAE-cellulose chromatography (5) and nuclei isolated according to Shapiro and Doxsey (6) but with 0.1 mM dithiothreitol in all buffers After removal of percoll particles washed nuclei could be stored in 50 mM Tris-HCl pH 7.9, 12.5 mM ammonium sulphate, 5 mM MgC12, 0.1 mM EDTA, 0.1 mM dithiothreitol, 50% (v/v) glycerol at -196' without loss of transcriptional activity. RNA polymerase activity was solubilised from nuclei by sonication at high ionic strength essentially as described for whole cells by Kitchin et al (3). Chromatography was carried out using columns packed with 10 ml DEAE-sephadex A-25 and eluting with 70 ml gradients of 25-400 mM ammonium sulphate. RNA polymerase assays were in duplicate at 37O, normally in final volumes of 100 ~1. Each contained 50 mM Tris-HCl pH 7.9, l-l.5 mM MnC12, 1 mM dithiothreitol, 0.3 mM each of ATP, GTP and CTP, c3H)-UTP as described in figure legends, salt as in figure legends or 40 mM ammonium sulphate for solubilised enzymes (which also had 10 /kg denatured calf thymus DNA), and aliquots of nuclear suspension or soluble polymerase. After incubation assays were terminated by addition of 50 pg carrier DNA and 10% trichloroacetic acid, followed by filtration on GF/C filter discs and liquid scintillation counting. Nuclei assayed at high salt concentrations were briefly sonicated prior to filtration to disrupt chromatin aggregates. RESULTS Yields microscopy RNA synthesis around

of nuclei

were

usually

and DNA measurements over

a broad

200 mM annnonium

range

sulphate

in the region (data

not

of salt (figure

of 20-25%

shown).

Such nuclei

concentrations 1).

However,

0.1

0.2 (NHq)2S04

with at all

as judged were

by active

an optimum ionic

in at

strengths

0.3

(11)

w. Salt optimisation for nuclear transcription. Nuclei were isolated from trypanosomes as described in methods. 20 /Ll of guclear suspension containing 1.3 ~8 DNA were used in each assay with 2 pCi ( H)-UTP and incubations were for 15 minutes. 0, with 1.5 mM Mn2+; 0, with 8 &I Mg2+. 845

Vol.

131,

BIOCHEMICAL

No. 2, 1985

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

I

r T

h

(I

1

1

kl

m

w. Characterisation of transcription in isolated nuclei. Assays contained 15 ~1 nuclear suspension and 2 /Ki (3H)-UTP and were incubated for 10 minutes. (a) control, uninhibited; (b),(c),(d) with 10, 50 and 100 fig/ml a-amanitin and 75 mM ammonium sulphate; (e),(f),(g) as (b),(c),(d) but with 200 mM ammonium sulphate; (h),(i) with 5 or 25 pg/ml DNase I; (j),with 5 pg/ml RNase A; (k),(l) with 5 or 50 ,cg/ml actinomycin D; (m), with 100 pg/ml rifamycin AF/O-13. Assays (h) to (m) inclusive were at 75 mu ammonium sulphate.

tested

transcription

by factors these

was much greater

ranging

between

experiments

was clearly

was that

a poor

highly

had no measurable solubilised of transcription (not

shown)

cells

latter

was occuring.

This

conditions

(7).

was supported

were

Conversely,

by 50-100

isolated

unusual. of this

in nuclei

of activity

pg/mla-amanitin. 846

or no initiation studies from

eukaryotic

I and II.

and therefore

the bulk

the

by time-course

At low salt drug

was seen at 200 mM ammonium

II

of

AF/O-13

inhibited

little

of nuclei

however

to DNase I and

Rifamycin

totally that

to RNA polymerases

most RNA synthesis

was inhibited

indicating

cation

common features

templates.

which

by low concentrations

by RNA polymerase

Cryamanitin

2),

common feature

respect

of RNA synthesis

catalysed

tested

shown)

of (Y-amanitin

inhibition

to RNase A (figure chromatin

used in

nuclei.

sensitive

with

not

with

The effects

reduction

(data

in --T. brucei

Mg 2+

with

and this

was partially

at concentrations

and is another

at least

negligible

--in vitro

effect

enzyme

nuclei

sensitive

reactions

shown to be optimal,

RNA synthesis

in isolated

D but

RNA synthesis

for

of Mn 2+ than

presence

The Mg2+ concentration

and four.

previously

cofactor

Transcription actinomycin

three

in the

from

and only sulphate.

higher

is abolished at both

there about Under

eukaryotes by 10 pg/ml ionic

was

strengths

20% the is

Vol.

131,

No. 2, 1985

BIOCHEMICAL

OL

AND

BIOPHYSICAL

50

RESEARCH

COMMUNICATIONS

100

ELUTION VOL.

(ml)

m. DFAE-sephadex chromatography of RNA polymerases. 2 ml of crude solubilised RNA polymeraess from rat liver nuclei (A) or 2. brucei nuclei (B) were chromatographed on identical DEAE-sephadex columns. 30-50 ~1 aliquots of column frac ions were assayed for RNA polymerase activity in the presence of 1 ,%Ci ( 3 H)-UTP. Trypanosome polymerases were extracted from nuclei isolated from 2-3 x 1010 cells. RNA

single

solubilised

polymerase

from

peak on DEAE-sephadex

observed liver

by Kitchin

nuclear

the usual

higher

peaks

nuclear

inhibitors

in the salt

(3)

polymerases

three

trypanosome

et al

eluting using

of activity extracts

(data

not

like

most

was,

to high

concentrations

of a-amanitin.

(data

not

whole

which

RNA polymerase

with

same low

cell

0.1

an apparent

M salt

3). that

might

Mixing

as a

concentration of --T. brucei.Rat conditions

experiments

were

revealed with

no general

The single

activity

weight

and

eluting

at

peak of trypanosome

in intact

also

rat

polymerase

have masked activities

nuclei,

chromatographed

on CM-sephadex

molecular

salt

identical

there

shown).

It

chromatographed

extracts

under

(figure

nuclear

gradients

at the

confirmed

trypanosomes

at about

nuclei

chromatographed

concentrations

peak eluting

isolated

as a single

and sedimented

in the region

sensitive

in glycerol

of 450-500,000

shown). DISCUSSION

We have sufficiently

isolated large

transcriptionally scale

as to permit

active

nuclei

characterisation 847

from 2. brucei of total

on a

RNA synthesis

Vol.

131,

No. 2, 1985

--in vitro

and demonstrated

RNA polymerase

polymerase

with

to high

by Kitchin

et al

and unlikely

and Borst

totally

resistant

concentrations of a-amanitin

from

in isolated

gradient

include: that

only

though

small

similar

amounts to those

inappropriate

polymerases

than

is

not be detected

to this

used,

notably

type

with

to low

to high

concentrations

nitrogen for

I and II

detectable cavitation

RNA polymerases

(B)

assay

virtually

all

other

in much lower

relative

amounts

of specific

resolve

possibilities.

genes.

type

eukaryotes.

assays

which

Work is currently

in and

such

procedures,

present

by "run-on"

of

the disparities

nuclei;

other

those

sensitive

(C) type

in most

enzyme

such as the kinetoplast.

enzymes;

the case

isolated

RNA and VSG transcripts

explanations

for

nuclear

the VSG RNAs were

in isolated adequate

species

observations

sensitive

of trypanosome

major

atypical

RNA

the polymerase

source

except

highly

of their

trypanosome

transcription these

All

to solubilise

some of the

are actually

trypanosomes would

for

in respect

transcripts

Possible

found

were

riboscmal

we have

survive

organelles

these

transcripts

nuclei.

lability

possible

identical

large

centrifugation.

(A) High

not

COMMUNICATIONS

is

Clearly

tubulin

'mini-exon'

it

an extranuclear

who detected

RESEARCH

of a single

is to reconcile

made by the same method

density

are

if

to (Y-amanitin, and

These

concentrations.

problem (4)

that

to consist

was similar

outstanding

time

eukaryotes

appears

to have arisen

Kooter

other

a-amanitin (3)

BIOPHYSICAL

such nuclei.

from

which

AND

the first

from

those

content,

sensitive

A major

for

activity

by comparison

nuclei

BIOCHEMICAL

eukaryotes, I and II in

Such a discrepancy selectively

in hand

measure

to try

and

ACKNOWLEDGEMENTS We thank

the SERC for

a CASE studentship

in support

of D.L.Earnshaw.

REFERENCES 1. De Lange, T., Berkvens, T.M., Veerman, H.J.G., Frasch, A.C.C., Barry, and Borst, P. (1984) Nut. Acids Res. ll, 4431-4443. D.A. and Boothroyd, J.C. (1984) Nature 311, 2. Campbell, D.A., Thornton, 350-355. 3. Kitchin, P.A., Ryley, J.F. and Gutteridge, W.E. (1984) Comp. Biochem. Physiol. z, 223-231. 4. Kooter, J.M. and Borst, P. (1984) Nut. Acids Res. l2, 9457-9472. 5. Lanham, S.M. (1968) --Nature 218, 1273-1274. 6. Shapiro, S.Z. and Doxsey, S.J. (1982) Anal. Bioch. 127, 112-115. 7. Beebee, T.J.C. (1978) Biochem. J. 176, 715-725. 848

J.D.