The 5S rDNA of the bivalve Cerastoderma edule: nucleotide sequence of the repeat unit and chromosomal location relative to 18S-28S rDNA

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The 5S rDNA of the bivalve Cerastoderma edule:

nucleotide sequence of the repeat unit and chromosomal

location relative to 18S-28S rDNA

Ana Insua, Ruth Freire, Josefina Méndez

To cite this version:

Original

article

The

5S rDNA of the bivalve

Cerastoderma

edule:

nucleotide

_sequence

of

the

_repeat

unit

and

chromosomal

location

relative

to

18S-28S rDNA

Ana

Insua

Ruth

Freire

Josefina Méndez*

Departamento

de

Biologia

Celular _y

_Molecular,

Universidade da

_{Coruña,}

A

_Zapateira

_sin,

15071 A Coruña,

Spain

(Received

12

_February

_1999;

_accepted

2

_July

₁₉₉₉₎

Abstract - The whole 5S rDNA

repeated

unit of the bivalve Cerastoderma edule was

_{amplified by}

PCR and several clones were

_sequenced.

In

_addition,

the PCR

product

from several individuals was

_digested

with restriction enzymes. The results

obtained indicate that 5S rDNA is

_organized

in tandem _repeats of 544-546

_bp,

120

of which could represent the

coding region

and 424-426 the spacer

region.

Minimal intra- and inter-individual variation was

_detected,

_always

within the _spacer

_region.

In

_comparison

to the

_published

5S rRNA _sequences of three other

bivalves,

C. edule

displays

a maximum of four different nucleotide

positions.

A

specific probe

of C. edule 5S rDNA was

_{generated by}

PCR and used for FISH. Five chromosome

pairs

were identified that carried a cluster of 5S rDNA at the telomere of the

long

arm. After

performing

FISH with a

_heterologous

18S-28S rDNA

probe

and

C-banding,

absence of

_linkage

between 5S and 18S-28S rDNA was demonstrated.

_©

_{Inra/Elsevier,}

Paris

5S rRNA gene

/

non-transcribed spacer

/

Cerastoderma edule

/

FISH

Résumé - L’ADNr 5S chez le bivalve Cerastoderma edule : séquence nucléotidique

de l’unité de répétition et localisation chromosomique par rapport à l’ADNr 18S-28S. L’unité de

_{répétition complète}

de l’ADNr 5S a été

amplifiée

_par PCR chez le bivalve Cerastoderma edule et

_plusieurs

clones ont été

séquencés.

En outre, le

_produit

de PCR de

plusieurs

individus a été

_digéré

_par des _enzymes de restriction. Les résultats obtenus

_indiquent

que l’ADNr 5S est

_organisé

sous forme de

répétitions

en tandem dont l’unité mesure 544-546

_{pb, parmi lesquelles}

120

pourraient représenter

la

région

*

Correspondence

and

_reprints

codante et 424-426 la

region

de

1’espaceur

non codante. Des variations intra- et

interindividuelles minimes ont été

_{détectées, toujours}

dans la

_region

de

_{1’espaceur.}

Par _rapport aux trois autres

_sequences

d’ARNr 5S

_publi6es

chez les

_bivalves,

C. edule

présente

un maximum de _quatre

_{positions nucléotidiques}

differentes. D’autre _part, une sonde

spécifique

_pour 1’ADNr 5S de C. edule a été

_générée

par PCR et utilisée dans des essais de FISH.

_{Cinq paires chromosomiques}

_portent un _groupement d’ADNr 5S sur le telomere du bras

_{long. Apr6s}

avoir realise la FISH avec une sonde d’ADNr 18S-28S

_{hétérologue}

et le marquage des bandes

C,

1’absence de liaison entre 1’ADNr 5S et 18S-28S a été demontrée.

©

Inra/Elsevier,

Paris

genes d’ARNr 5S

_/

espaceur non transcrit

_/

Cerastoderma edule

_/

FISH

1. INTRODUCTION

The 5S ribosomal DNAs

_{(5S rDNA)}

of _many

_eukaryotes

has been cloned and characterized. In most _cases, it is

_organized

as clusters of tandem

_repeats

of several hundred base

_pairs

_(bp),

_consisting

of a

_{coding region}

and a non-transcribed spacer

region

!14!.

Accumulated data demonstrate

_that,

while the

coding region

is

_highly

conserved _among

_taxa,

both with

_respect

to

_length

and nucleotide _sequence, the _spacer

_region

evolves more

_rapidly

and can show variation both within and between

_species

_{(e.g. [7, 19!).}

In addition to the gene

encoding

the 5S

rRNA,

many

species

contain gene variants and

_pseudogenes,

_differing

from the _gene

_by

a variable number of sub-stitutions and deletions

_[5,

_18,

_24!.

_Moreover,

it has been well documented that

Xenopus

laevis has three

_types

of 5S rDNA sequences with

developmentally

regulated

expression

!32].

More than one

_type

of 5S rDNA _sequence with dif-ferential

_expression

was also seen in the chicken

_[13]

and some fish

[12].

As

is true for several other families of

_{tandemly repeated}

_genes, the 5S rDNA

repeats

evolve

_concertedly

_!3!,

i.e. in

_{intra-specific comparisons}

a

_{high degree}

of _sequence

_similarity

is

_usually

observed between

_independent

_repeats.

_Thus,

5S rDNA sequences are

_regarded

as

_potentially

useful in

_{revealing phylogenetic}

relationships.

In contrast to _genes

_encoding

_18S,

5.8S and 28S rRNA

_{(18S-28S rDNA),}

where chromosomal location can be determined

_by

selective

_staining

of nucleo-lus

_{organizer regions}

and in situ

_{hybridization,}

5S rDNA can

_only

be detected

by

in situ

_{hybridization.}

This could

_explain

the fact

_that,

in

_general,

there is

less information available on _{the chromosomal location of 5S rDNA. In bivalve}

molluscs,

very little attention has been

paid

to 5S rDNA. To

_date,

_only

the 5S rRNA of three

_species

_belonging

to different subclasses has been

_sequenced:

Solemya

velum

_{(Protobranchia),}

_{Calyptogena magnifica}

_{(Heterodonta) [25]}

and

Mytilus

edulis

_{(Pteriomorphia) !6!.}

The chromosomal location of 5S rDNA was determined in a

_pectinid

_species,

_Aequipecten

_opercularis

_!10!.

This work

_provides

for the first time the nucleotide _sequence of the whole 5S rDNA

repeated

unit of a bivalve

_species,

the cockle Cerastoderma edule

(Heterodonta, Cardiidae),

and an

analysis

of the intra- and inter-individual

variation. In

_addition,

it

_reports

the chromosomal location of 5S rDNA and its

2. MATERIALS AND METHODS

Specimens

of C. edule were collected from several locations

_(Pontevedra,

Vilanova de

_Arousa,

_Ponteceso,

Ria do

_Burgo

and

_Cedeira)

_along

the Galician coast

_{(NW Spain).}

Genomic DNA was extracted from muscle tissue

_according

to

_{Winnepenninckx}

et al.

_!31!.

2.1. PCR

_{amplification,}

_cloning

and

_sequencing

The

_{amplification}

mixture used for PCR

₍₅₀

_O

_L)

contained 500 ng of ge-nomic

_{template DNA,}

1

_vM

each

_primer,

250

_{vM dNTPs,}

1.25 U of

_Taq

poly-merase

(Boehringer Mannheim)

and the buffer recommended

_{by polymerase}

suppliers.

The

_primers

were 5’-CAACGTGATATGGTCGTAGAC-3’

_(A)

and 5’-AACACCGGTTCTCGTCCGATC-3’

_(B),

obtained from the 5S rRNA

se-quence of the mussel M. edulis

[6],

and 5’-CAAGCACAGAGGCAGGAG-3’

(C)

and 5’-CGATCCGCGGTTTACCTG-3’

_(D)

obtained from the C. edule 5S rDNA spacer

region.

Thirty

standard PCR

_{amplification cycles}

were per-formed at an

_annealing

_temperature

of 64 °C with

_primers

A and B and 56 °C with

_primers

C and D. The PCR

_product

_generated

with both sets of

primers

was

_{purified using}

Geneclean

_(BIO

_101,

_INC),

_ligated

into the

_plasmid

pGEM-T Easy, using pGEM-T Easy

Vector

_System

II

_(Promega),

and

subse-quently

transformed into E. coli JM109 cells. Recombinant clones were selected as white colonies on

_{ampicillin plates containing X-gal}

and IPTG. Plasmid DNA

_purification

of four clones

_(two

with insert obtained with

_primers

A and

B,

and the other two with insert obtained with

_primers

C and

_D)

was car-ried out as described

by

Sambrook et al.

_[23].

Both strands of each clone were

sequenced by

the

_{dideoxy-sequencing}

method with the AutoRead kit

(Phar-macia).

Automatic

_sequencing

was

performed

on an A.L.F. _{express sequencer}

(Pharmacia).

Sequences

were

aligned

_using

CLUSTAL V with both fixed and

floating

gap

penalties

of 10

_!9!.

The nucleotide _sequences have been

deposited

in the EMBL DNA data base under the accession numbers AJ132196-132199.

2.2. Chromosome

_preparation

and FISH

Metaphases

were obtained from

_gill

cells

_following

the

_procedure

described

by Thiriot-(!uievreux

and

_Ayraud

_!28!.

A

_{specific probe}

of C. edule 5S rDNA was

_{produced by}

PCR

using

the

_primers

A and B.

_Labelling

was obtained

using

the PCR

_procedure

described

_above,

but with a different dNTP con-centration

₍₁₀₀

_{vM dATP,}

100

wM dCTP,

100

_O

_M

_dGTP,

160

_R

_M

dTTP and 35

_vM

_{digoxigenin-11-dUTP).}

A recombinant

_plasmid

_{containing 185,}

5.8S and 28S genes

plus intergenic

spacers of

Drosophila melanogaster

was used as

probe

to localize 18S-28S rDNA. After extraction

_by

alkaline

_lysis

_(23],

the whole

plasmid

was labelled with

_{digoxigenin-11-dUTP employing}

the

_Boehringer

Mannheim nick translation kit. FISH was carried out as in Insua et al.

_!10!,

but the

_{post-hybridization washing}

was carried out with a 65

%

formamide solution in the case of 5S rDNA.

_C-banding

was

_performed

_according

to the method of Sumner

_[26]

but

slides

were stained with acridine _orange

_following

Martinez-Lage

et al.

_[15].

The examination of chromosome

_spreads

was per-formed with a Nikon fluorescence

_{photomicroscope}

_equipped

with

_appropriate

3. RESULTS

The 5S rDNA

repeat

unit of C. edule was

_amplified

_by

PCR

_{using primers}

A and

_B,

_designed

from the 5S rRNA sequence of M. edulis

(6!.

PCR

amplification

produced

a

single

band of

approximately

550

bp.

This

amplification product

was cloned and then two clones were

_sequenced

(Cel

and

Ce2).

Two additional

primers,

C and

_D,

derived from the spacer

region

of the

_just

_sequenced

C. edule 5S

_rDNA,

were also used to

produce

a new PCR

amplification

of the 5S rDNA

repeat

unit. A

_single

band of 550

bp

was also

_obtained,

and after

cloning

two

clones were

_sequenced

(Ce3

and

Ce4).

The

_complete

repeat

unit consists of 544-546

_bp

and the

_alignment

of

full-length

sequences of the four clones consists of 548

bp

(figure 1).

Comparison

with available bivalve sequences

[6, 25]

allows us to infer that the

_coding

_region

starts 5’ with GTC and ends with CTT to

_give

a 5S rRNA size of 120 nucleotides

(figure !).

However,

the 5S rRNA from mussel M. edulis ends with ACA and has a size of 119 nucleotides

(6!.

Therefore,

the

assignment

of the 3’ end and the 5S rRNA size must be considered tentative. The inferred

_coding

region

of the C. edule 5S rDNA is invariable between

_{clones, ignoring}

the sequence

corresponding

to

_primer

A in clones Cel and Ce2.

Several sequences of the

eukaryotic

5S rDNA involved in the

transcription

can be identified in the sequences obtained from C. edule: internal control

region

[22];

sequence elements related to

upstream

regulatory

regions

of the

coding region

as TATATA

[17];

and terminator _sequences

composed

of four

thymidine

residues

_[1]

located downstream of the

_coding

region

(figure

1).

The

G/C

content,

determined in the consensus _sequence of the four

clones,

is

higher

in the

_coding

_region

_{(54.2 %)}

than in the spacer

region

(45.8 %).

The _spacer

_region

showed some

variation,

_ranging

from 424 to 426

bp

in

length. Eight

variable sites were detected in the

alignment,

four of which

correspond

to _gaps and four to nucleotide substitutions

_(figure

_1).

Nevertheless,

three of the clones

_(Cel,

Ce2 and

_Ce3)

are almost

identical,

only

two _gaps associated with a run of

thymidine

residues at the 5’ end

_being

observed. To further examine the extent of the

_variation,

the 5S rDNA was

_amplified

with

primers

A and B from nine additional individuals collected

along

the Galician

coast. The

_product

obtained was

_digested

with the _enzymes Alu

I,

Hae

III,

Rsa I and

_Taq

I. No variation was found in the restriction

_pattern

generated by

these _enzymes,

_except

in one individual which showed intra-individual variation

concerning

the Rsa I restriction

pattern.

This had three bands as is to be

expected

from

the

sequences determined

here,

but also an additional

_band,

resulting

from the absence of one _enzyme

_target

in the _spacer

_region.

Comparison

of the 5S rDNA

_coding

sequence of C. edule with

previously

published

sequences of other bivalve

species

(figure

2)

reveals no differences with

_{Calyptogena ma}

_g

_nifica

_{(Heterodonta, Vesicomyidae),}

and four nucleotide differences with

_Solemya

velum

_{(Protobranchia, Solemyidae)}

and

_Mytilus

edulis

(Pteriomorphia, Mytilidae).

The chromosomal location of the 5S rDNA was determined

_{by FISH,}

_using

a

specific probe

obtained

by

PCR.

Forty-one metaphases, belonging

to four

of C. edule are submetacentric

_{(12 pairs),}

and the

_remaining

chromosomes are subtelocentric

_{(4 pairs)}

or telocentric

(3

pairs),

with small differences in size

[11],

identification of chromosome

_pairs

_carrying

5S rDNA cannot be

_accurately

determined.

To establish the relative

_position

of 5S rDNA and 18S-28S

_rDNA,

FISH was carried out with a

_heterologous

18S-28S rDNA

_{probe. Forty-seven metaphases}

from four individuals were examined.

_In

all cases,

hybridization signals

were found

_spread

_along

the short arms of one

_pair

of chromosomes characterized

in these heterochromatin

_regions

_(figure

_3a)

_indicating

that 5S and 18S-28S rDNA are not linked.

4. DISCUSSION

This is the first

report

on the characterization of the whole 5S rDNA

_repeat

unit in a bivalve

_species.

The results obtained

_suggest

that C. edule 5S rDNA exhibits the conventional tandem

_arrangement.

Evidence is

_{provided by}

the fact that the PCR

amplification

of the 5S rDNA unit was obtained

_{using contiguous}

primers

located in

_opposite

orientation. The

_length

of the inferred 5S _gene is 120

_bp

and the _spacer

_region

ranges between 424 and 426

bp. Totalling

544-546

_bp,

this size is intermediate between other 5S rDNA

repeat

units observed in invertebrates such as the 300-450

_bp

of some

_Diptera

[8]

and the 589

bp

of

a crustacean

_species

_!20!.

The four

_sequenced

clones of C. edule

display

an identical

coding region

and

_{potential regulatory}

elements are identifiable in all of them.

_Therefore,

the occurrence of _{gene variants} or

_pseudogenes

_among the

_repeat

units

analysed

here can be considered

_improbable.

In the _spacer

_region

of 5S

_rDNA,

three of the clones

_analysed

are almost identical as

only

two variable sites were found and these _{appear to} result from

reduction/expansion

of the

_{thymidine-rich}

_region

at the 5’ end.

Excluding

the variable sites associated with these

_thymidine

_regions,

the fourth clone

_displays

differences at five nucleotide

_positions.

As the clones

_analysed

were

obtained

by PCR,

it cannot be excluded that some of the nucleotide variations were due to

Taq

polymerase

misincorporation.

The occurrence of this minimal intra-individual variation and the identification of the same restriction

_patterns

after

enzyme

digestion

in the individuals examined

suggest

that 5S rDNA would be an

_{appropriate region}

for

_assessing

the

relationships

between C. edule and other bivalves.

Comparison

of the 5S rDNA

coding

sequence of C. edule and the three

available _sequences of bivalves shows little

_{interspecific}

variation. C. edule and

Calyptogena magnifica,

two

_species

whose

_{superfamilies}

are considered to have a

common ancestor in Late Paleozoic

_!16!,

share the same _sequence. On the other

hand,

C. edule differs from S. velum and from M. edulis at

_only

four

nucleotide

positions,

although

C. edule and S. velum share a common ancestor in Pre-cambrian and C. edule and M. edulis in

_Early

Paleozoic

_!16!.

This

_might

indicate that

_phylogenetic

information

_{provided by}

the

_coding

_{sequence may} be

_limited,

but a

_{sufficiently large}

species sample

and

_comparison

of the

supposedly

fast-evolving

spacer sequences could establish a

_utility

of 5S rDNA for

_phylogeny

estimations.

FISH revealed a total of nine

_{hybridization}

sites.

_Thus,

one of the

_pairs

seems

to bear 5S rDNA in

_{heterozygosity;}

that

_is,

it is absent in one chromosome or is

present

in such a low _copy number that it is not sufficient for detection

_by

FISH. Since chromosome

_pairs

cannot be

_{distinguished unambiguously}

after

propid-ium iodide

_{counterstaining}

it is not

_possible

to determine if it is

_always

the same

pair

that shows 5S rDNA in

heterozygosity.

Differences

between

heterochromatin

_regions

identified here in C. edule

_display

different sizes. Sister chromatid

_exchanges

and

_unequal

meiotic

_{crossing-over}

events could cause the

size of rDNA clusters to fluctuate

randomly

so that

heterozygosity

of the rDNA clusters is the

_rule,

and

_homozygosity

the

_exception.

The distribution of 5S rDNA in C. edule _{is very} different from that found in

the bivalve

_analysed

so

far,

A.

_opercudaris.

In this

_pectinid

_species,

5S rDNA was identified at two sites of one arm of a metacentric

_pair

_(10!.

The differences between these two

_species

_contrast,

for

_example,

with the

_tendency

of 5S rDNA

in mammals to be localized in the terminal

_region

of a

_pair

of chromosomes

(27!.

However,

they

are similar to those found in other _groups such as anuran

amphibians

where both number as well as

_position

can be _very different between

species

(30!.

Unlike the 5S

_rDNA,

the location of 18S-28S rDNA in C. edule is restricted to short arms of one chromosome

_pair,

as was

_already

observed in individuals of C.

_{glaucum populations using}

silver

_staining

_(29!.

Both

_types

of rDNA have been found to be linked in several animals and

_plants

_[2,

_4,

_21!,

_{nevertheless,}

the most common situation in

_{higher eukaryotes}

is the absence of

_linkage.

The results obtained in this work show that this also occurs in C. edule.

ACKNOWLEDGEMENT

This work has been

_{supported by project}

XUGA 10302B97 of the Galician Government.

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