Restriction mapping of the rRNA genes from Artemia larvae

BIOCHEMICAL

Vol. 98, No. 2,198l January

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30, 1981

MAPPING OF TEE rREA GENES FROM Artemia LARVAE

RESTRICTION Jesiis

Cruces,

Instituto

Jesiis

de Enzimologia Universidad

Received

November

Sebasti&

and Jaime

de1 CSIC,

Facultad

AutSnoma,

Renart de Medicina,

Madrid-34,

Spain

21, 1980

SUMMARY A restriction endonuclease analysis of the genes coding for the ribosomal RNA from Artemia larvae has shown that these genes consist of a repeat unit of 16.2 kilobase pairs (10.7 Mdaltons) and that the repeat unit seems to be homogeneous in size. INTRODUCTION The knowledge rification

are

Likewise,

of the molecular

important

a current

the molecular

used

identification order

is

well

field

of specific

genes

regulation is

The repeated

two problems

and their

pu-

of gene expression.

of evolution

genes.

these

the rRNA genes

anostraca) suited

opmental

program

of view,

since

the

gene

comparison families

due to their

of are

relatively

the

simple

system

for

the

this

type

is very

little

of their

of study

interesting

data

Artemia (subclass

crustacean

the regulation

Arternia is also

(l-3). there

from

to study

due to its from

bran-

Artemia

expression. the

peculiar

devel-

evolutionary

on the rRNA gene structure

point

of crusta-

(4). The overall

a repeat inner

in the

in studying

We have chosen

higher

of specific the

and isolation.

chiopoda,

tea

structure

in studying

approach

structure

most widely

a very

tools

unit

structure that

eukaryotes: transcribed

spacer

One way to approach

tion

endonuclease

MATERIALS

here

rRNA genes

in all

5' - non-transcribed

3'.

We present

of the

is mantained - "28 the

analysis, the

physical

region

S" RNA coding

study whereby map for

is well

organisms

of the

- "18 region

structure

a physical

known

studied

(4).

It

consists

from bacteria

S" RNA coding

region

- non-transcribed of these

genes

restric-

from Artemia.

AND METHODS

Artemia cysts

were obtained from San Francisco Bay Brand Inc., Division of Metaframe Co., Menlo Park, CA 94025 (batches % 2018 and 1808). a-amylase, Abbreviations: SDS, sodium dodecyl sulfate; SSC, 0.15 M NaCl, 0.015 M trisodium citrate; kb, kilobase pairs. 0006-291X/81/020404-06$01.00/0 Copyrighr 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

-

region is

map of them can be obtained.

the rRNA genes

in

to -

Vol. 98, No. 2,1981

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pancreatic ribonuclease and ribonuclease Tl were from Calbiochem. was from Merk. Restriction endonucleases Hpa I, Pst I, Sal I, Sma were from BioLabs and kindly supplied by Dr. E. Tabards and Dr. V. RI, Hind 111,Bam HI and polynucleotide kinase were from Boehringer All of them were used according to the manufacturer's instructions. (,2,000 Ci/mmol) was from the Radiochemical Center, Amersham. Wild was the generous gift of Dr. V. Rubio. Obtention of Artemia larvae. Artemia cysts were treated (5) and incubated in 0.25 M NaCl in a shaking incubator Naiplii were harvested in a separation funnel, filtered weighed and used immediately. with distilled water,

Proteinase K I and Kba I Rubio; Eco Mannheim. (Y-~~P)ATP type X DNA

as previously described at 30°C for 20 hr. through a cloth, washed

Preparation of high molecular weight DNA. New born nauplii were homogenized in 5 vol of 10 mM Tris-HCI, pH 8.0, 3 mM MgC12, 400 mM sucrose with 8 strokes in a glass hand-homogenizer. The crude extract was filtered through 8 layers of gauze and the filtrate was centrifuged at 5OOxg for 10 min at 4'C. The nuclear pellet was resuspended in 3 vol of 10 mM Tris-HCl, pH 8.0, 10 mM NaCl, 10 mM EDTA by vortexing and SDS was added to a final concentration of 0.25%. Proteinase K was added at 50 pg/ml and incubated with very gentle agitation for 30 min at 37'C. SDS concentration was raised to 2% and the incubation followed another 30 min at room temperature. This nuclear homogenate was chilled and extracted immediately with 1 vol of phenol:chloroform:isoamil alcohol (25:24:1) saturated with 500 mM Tris-HCl, pH 8.0, 10 mM NaCl, 10 mM EDTA for 30 min at 4'C with gentle agitation. The two phases were separated by centrifugation at 10,000 rpm for 15 min at 4°C in the HB-4 rotor of a Sorvall centrifuge. After the aqueous phase was dialyzed against another phenol-chloroform extraction, 500 vol of 50 mM Tris-HCl, pH 8.0, 10 mM NaCl, 10 mM EDTA for 24-36 hr at 4°C until the phenol was eliminated. The solution was then treated inside the dialysis bag with 50 pg/ml pancreatic ribonuclease and 100 U/ml ribonuclease Tl (both heated at 80°C for 30 min) for 3.5 hr at 37'C. a-amylase was added (25 pg/ml) and incubated 1.5 hr at 37°C. Finally, SDS was added at 0.25% and the solution digested with 50 pg/ml of Proteinase K for 3 hr at 37°C. After the the solution was extracted at least 3 times with phenol-chlolast digestion, roform and dialyzed against 10 mM Tris-HCl, pH 8.0, 10 mM NaCl, 10 mM EDTA. DNA was stored at 4'C in this buffer. When needed, DNA was dialyzed against 10 mM Tris-HCl, pH 8.0. The molecular weight of the DNA preparations was analyzed by electrophoresis in 0.25% or 0.15% agarose gels (6) and was higher than 200 Mdaltons. Preparation of rRNA. Riboeomes traction and sucrose gradient formed as described by Cruces labeled in their 5' ends with except that the hydrolysis of fragments. of 100 bases.

were obtained from new born nauplii. rRNA exseparation of the 17 S and 25 S species were peret al. (manuscript in preparation). rRNAs were polynucleotide kinase according to Maize1 (7), both rRNAs was for 30 min at 90°C, to obtain

Transfer of DNA to nitrocellulose filters and hybridization. 5 pg aliquots of DNA were cleaved with different restriction endonucleases (or mixtures of them) at 37°C for 12 hr with enough enzyme to give complete digestion. Restricted DNA was electrophoresed in 0.5% agarose gels (23x14.5x0.3 cm) in slots of 24 mm2 at 1 V/cm in Tris-borate buffer(6) until the marker dye was 15 cm from the origin. After staining with ethidium bromide and photographing, DNA was transferred to nitrocellulose filters (Millipore) as indicated by Southern (8). Hybridization was carried out in plastic bags (Seal-N-Save, Sears) at 42°C for 36 hr with SO-100 pl/cm2 of hybridization buffer (50% formamide, 4xSSC, 0.1% SDS, 25 mM EDTA, 20 mM sodium phosphate, pH 7.0) plus 2x105 cpm/slot of (32P)rRNA (specific activity, lo7 cpm/pg) and lo-20 pg of the non-labeled rRNA. After hybridization the filters were washed twice at 42'C for 30 min with hy-

405

Vol. 98, No. 2,198l

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BamH/

Ban1 H I

Xba

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Xba I EC0 R I

/

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Eco R I

Hpa

!

w-80

f-30

w-23

.--IO

to.5

Figure

1. Pattern of hybridization or rRNA with restriction --withdifferent endonucleases on Artmia DNA. In each digest, the left lane representshybridization with lane with 25 S rRNAs. Numbers representing the sizes of the pressed in kb.

fragments

obtained

17 S and fragments

the right are ex-

bridization buffer (100 ml), twice with 2xSSC, 0.5% SDS (250 ml) and twice with O.lxSSC, 0.5% SDS (250 ml). After air-drying, filters were exposed to sensitized X-ray film (Kodak X-omat XS-5) with an intensifying screen (DuPont Cronex Lightning-plus XL) at -7O'C (9). In each gel (32P)5' end-labeled A DNA digested with Hind III was included as an internal molecular weight marker for hybridization.

RESULTS AND DISCUSSION Figure

1 shows

the results

rRNAs of the DNA fragments and mixtures tions ences from

to which

performed.

the fragment

Sal

lengths

can be accounted the

gels,

obtained

of them on total

and the probe

they I,

of the hybridization

hybridize

Pst for

16.8

different

restriction of the

are

and 16.9

by the difficulty steepness

17 S and 25 S (32P)

DNA. The size

I and Hpa I cut

of 17,

due to the

with

Artemia

with

of the

406

shown

in Table

only

I for

all

once per repeat

kb respectively. in assessing log(MW)

endonucleases different

vs.

These true mobility

fragments the digesunit,

being

small

differ-

molecular curve

weights in this

Vol. 98, No. 2,198l

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Table I. Summary of restriction

fragments obtained complementary to rRNA.

Endonuclease

from Artemia

DNA

Fragments

Sal Sal Pst Pst Sma Sma

I I + Pst I I I + sma I I I + Hpa I

Hpa Hpa Hind Hind Eco Eco

I I + Hind III III III + Eco RI RI RI + Xba I

17.0 11.1 16.8 14.6 14.7 13.5 1.3 16.9 7.4 7.4 4.6 13.4 10.9 1.1 12.4 8.0 0.7 8.0 0.7 7.9 0.7

Xba I Xba I + Barn HI Barn HI Barn HI + Sal I

(25, (25); (25, (25, (25, (25, (17) (25, (25); (25); (25); (25, (25) (25) (25) (25); (17); (25); (17); (25); (17);

17) 5.2 17) 17); 17); 17);

Total

17.0 16.3 16.8 15.6 16.3

(17) 1.0 (25) 1.6 (25) 1.5 (25)

16.3 16.9 10.4 10.4 10.3 16.2

17) I.8 3.0 3.0 17); ; 2.5

(17); 1.2 (17) (17) (17); 2.7 (25) 2.8 (25) (17); 1.7 (25);

; 3.8 2.3 0.55 3.4 0.55 3.4 0.55

(25, 17) (25, 17); 1.0 (25); (25); 0.35 (25) (25, 17); 3.1 (17); (25); 0.35 (25) (25, 17); 3.1 (17); (25); 0.35 (25)

16.2 16.2 12.9 16.1 16.0

Fragment size is expressed in kb. Numbers between brackets which rRNA the fragment hybridizes.

range

(6).

To calculate

to use enzymes for

the size

that

kb for

10.4

We believe

kb.

probe

cut more

of the repeat

and 16.1 within

a more accurate

Barn HI. that

once per a value

high

molecular

weight

given

digest.

The only

unit

I and Hpa I digests

for

of 16.2

is because

Hind

the

other would

kb for III

in

same hybridization

possible be if

way to get these

three

the

one obtains

Eco RI and Xba I,

since

cut

either

of the repeat

no double are

detected

the

for

of

fragment

with

same size

enzymes

a size

a "silent"

be detected

pattern

is better

gives

the size

size,

to

it

case,

which

generates

that

unit

Sma I,

III,

cannot

we estimate

seems to be homogeneous with

In this

is Hind

and therefore

refer

the repeat

repeat.

exception

In conclusion,

kb.

Pst

this region

16.2

The repeat

unit

The cnly

a non-transcribed (see below).

than

size

length

the

repeat

is

bands

of

in a Sal very

I,

Vol. 98, No. 2,1981

BIOCHEMICAL

Sal I Pst

Hind

I

Pst

Pst I Sma I

I

Hind 111 Eco R I

III

AND

Em

Sma

BIOPHYSICAL

Sma I Hpa I

I

Eco R I Xba I

R I

RESEARCH

Xbe

Hpa

Xba Barn

I

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Hpa Hind

I

I H I

Barn

I III

Hnd

Bang Sal

H I

III

H I I

Sel

;g;$y

“7’c

0.7 0.55 0.35

Figure

close

2. Generation and lengths from ArtemiarDNA ---*

to each other

cribed

region,

enzymes

and on both

and this

give

is not

different

(in kb) of the restriction ----

ends of an extra the case,

patterns

0.1* -I-

DNA fragment

as different

(compare,

for

fragments

in

a non-trans-

double

digests

Sal

I + Pst

instance,

obtained

with

these

I and Pst

I

+ sma I). Figure

2 shows a representation

relationship. (10).

This

An asterisk

type

on a given

the data

presented

has been

constructed

in Figure

3. It

tions

with

III

different gion,

Hind regions

1.9 kb;

I and Fig.

also

explains

the production

repeat

25 S coding

have been 3.9

region,

spacer

regions

the transcribed

tensively,

et al.,

17 S coding

are within

all

recognition is

both

1 kb and the distance

almost

it

kb,

noteworthy

giving

very

the cuts

of about produced

regions. sequences that small

This based

Hpa II fragments

"silent".

obtained. digest.

fragments

in diges-

The sizes

of the

with

in preparation). between A close

the restriction

the

408

re-

the molecular The inner

trans-

3' end of 25 S and

examination

of the map

endonucleases

the enzymes

used

used have hexa-

on A and T residues. shown).

unit

the map: 17 S coding

in agreement

not

all

The map is

(CCGG) and Hha I (GCGC) cut (data

With

map of the repeat

from

is because mainly

and their and Gall

of silent

9 kb.

with

obtained from Wild is

fragment

deduced

is of about

shows that

spect,

every

and in the Xba I t Ram HI double of the

it

2, a physical

for

of 17 S and 25 S rRNAs (Cruces

nucleotide

taken

that

accounts

cribed

5' end of

indicates

which

weight the

the fragments has been

fragment

in Table

shown

of all

of representation

the

In this

re-

repeat

ex-

I

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Vol. 98, No. 2,1981

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RESEARCH

------Figure

characteristics

to be solved,

define

/I

the

including

size

rRNA coding

region,

usually

(8,

been conserved confirmed the

in

it

fragment ference

the characterization

of the pre-rRNA

molecule

is

the

hybridizes

repeat

large only

is a general

spacer, in the

that

the divergence (12): fragment

of Eco RI in Artmia

fragments,

in Artmia

found

18 S rRNA and the small

pattern

the

since the avian

feature

the

and the localization

inner the

transcribed

spacer. point

of amphibians the

large

with

both

hybridizes

25 S rRNA.

It

in

to

of

5.8 S

view.

the rRNA genes

and mammals, fragment

re-

of the

evolutionary

two Eco RI sites

rDNA is different. one which

with

Pst I.

of the rRNA genes

can be made from

11) has suggested

(I):

of the structure

of the non-transcribed

Some considerations Southern

-----

3. Physical= of the rRNA repeat --unit from Artemia. --(t): Barn HI. (7): Eco RI. (7): Hind III. (7): Hpa I. (7): Sal I. (7): Sma I. (9): Xba I.

Several main

COMMUNICATIONS

and this

hybridizes

have

has been

only

with

18 S and 28 S. The restriction Although with

is not

in the rDNA of crustacea

there

both yet

are also

rRNAs,

clear and in

while

whether

two the

small

this

dif-

the evolution

of

rRNA genes.

ACKNOWLEDGEMENTS This rrollo

work

was supported

de la Investigaci6n

by grants

from

the Fondo National

para

el Desa-

Cientifica.

REFERENCES 1. Hentschel, C.C. and Tata, J.R. (1976) Trends Biochem. Sci. 1, 97-100. 2. Bagshaw, J.C. and Warner, A.H., eds. (1979) Biochemistry of Artemia development. University Microfilms International, Ann Arbor. 3. Persoone, G., Sorgeloos, P., Roels, 0. and Jaspers, E., eds.(1980) The Brine Shrimp Artemia. Universal Press, Wetteren, Belgium, in press. 4. Long, E.O. and Dawid, I.B. (1980) Ann. Rev. Biochem. 49, 727-764. 5. Osuna, C. and Sebastign, J. (1980) Eur. J. Biochem. 109, 383-389. 6. Fangman, W.L. (1978) Nucl. Acids Res. 5, 653-665. 7. Maizel, N. (1976) Cell 9, 431-438. 8. Southern, E.M. (1975) J. Mol. Biol. 98, 503-517. 9. Laskey, R.A. and Mills, A.D. (1977) FEBS Lett. 82, 314-316. 10. Wild, M.A. and Gall, J.G. (1979) Cell 16, 565-573. 11. Arnheim, N. and Southern, E.M. (1977) Cell 11, 363-370. 12. McClements, W. and Skalka, A.M. (1977) Science 196, 195-197.

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