Long telomeres in the polytene chromosomes of Drosophila melanogaster are associated with amplification of subtelomeric repeat sequences

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Long telomeres in the polytene chromosomes of

Drosophila melanogaster are associated with

amplification of subtelomeric repeat sequences

On Danilevskaya, Ge Lapta

To cite this version:

Original

article

Long

telomeres in the

_polytene

chromosomes of

_Drosophila

_melanogaster

are

associated with

amplification

of subtelomeric

_repeat

_sequences

ON

_Danilevskaya

GE

_Lapta

1

USSR

_{Academy of Sciences,}

Institute

_of

Molecular

_Genetics,

123182

Moscow;

2

Kharkov State

_{University, Kharkov,}

USSR

(Received

23

_{April 1990; accepted}

7 November

₁₉₉₀₎

Summary - The T-32 strain of

_{Drosophila melanogaster,}

which was

_originally

selected for heat

_resistance,

is characterized

_{by having exceptionally long tips}

in chromosomes X and 2L in the

_{polytene salivary gland}

chromosomes. Six sublines

_differing

in telomere

length

in different chromosomes were selected at an elevated temperature

(32°C).

The

morphological

characteristics of the telomeres in each subline are stable for a number

of

_generations

at 23°C. The additional chromatin in the

_long

telomeres

_effectively

hybridizes

with a DNA _sequence

_designated

_Dm665, which is related to the He-T

_family

of heterochromatin- and telomere-associated sequences. The short telomeres show weak

or no

_{hybridization}

with the Dm665

_fragment.

This result

_implies

that at least _part of the

morphological changes

in telornere

_length

in

_polytene

chromosomes results from different

degrees

of

amplification

of the telomere-associated sequences.

D _melanogaster

_/

_polytene chromosome

_/

telomere _length

_/

_repeat sequence

/

amplification

Résumé — Les télomères _longs des chromosomes _polytènes de _{Drosophila melanogaster}

sont associés à _{l’amplification} de séquences répétées subtélomériques. La souche 7°-32 de D

_{melanogaster,}

sélectionnée à

_l’origine

_pour sa résistance à la

_chaleur,

se

caractérise par des extrémités

exce

P

tionnellement allongées

de ses chromosomes X et

2L

_(chromosomes

_polytènes

des

_glandes

_salivaires).

Six

_{sous-lignées, différant}

par la

longueur

des télomères de

_différents

_chromosomes, ont été sélectionnées à

_température

élevée

_(32°

_C).

Dans chacune des

_{sous-lignées,}

les

_{caractéristiques}

des télomères demeurent

stables

_{pendant plusieurs générations}

à 23° C. La chromatine additionnelle des

_longs

télomères

_s’hybride

avec une

_séquence

d’ADN

(Dm 665)

_apparentée

à la

_famille

He-T de

séquences

associées à l’hétérochromatine et aux lélomères. Les télomères courts

préserctent

une

_hybridation

_faible

ou nulle avec le

_fragment

Dm 665. Ces résultats

indiquent

qu’au

moins une

_partie

des

_{modifications morphologiques}

de la

_longueur

des télomères des

chromosomes

polytènes

peut être attribuée à des variations du

degré

d’amplification

des

séquences

associées aux télomères.

D

_melanogaster

_{/ chromosome polytène}

_{/ longueur}

de

_télomère

_{/ séquence}

répétée

/ amplification

*

INTRODUCTION

Polytene

chromosomes from different

_wildtype

strains of

_{Drosophila melanogaster}

can differ

_by

the amount of chromatin in the telomeres of different chromosomes.

Morphologically,

some telomeres _appear

_long

and others short

(Roberts,

1979).

The additional chromatin in the

_long

telomeres has no well defined band structure

but can _vary in size and

_{general morphology.}

The

_{morphological peculiarities}

of telomeres can

_persist

for a number of

_generations

and serve as

_cytological

markers in certain

_wildtype

stocks

_(Lapta

and

Shakhbazov,

1986).

It seemed

_possible

that the

_long

and short telomeres

_might

result from differential

amplification

of one or more subtelomeric

_repeat

_sequences found in the

_Drosophila

genorne. This

hypothesis

was examined

_using

the cloned DNA

fragment

Dm665

and the 10.5-kb insert of the

_{phage A 17,}

which contains Dm665 and 2

_copies

of the

transposable

element

_hoppel

_{(Danilevskaya}

et

_al,

_1984).

The Dm665

_fragment

is an AT-rich

repeated

_sequence found at the

_tips

of chromosomes and in

hetero-chromatin,

particularly

in the Y chromosome. It is related to the He-T

_family

of heterochromatic-telomeric _sequences

_(Young

et

_al,

_1983;

Traverse and

_Pardue,

1989).

The

_Drosophila

strains used in the

_analysis

were derived from a stock

des-ignated T-32,

which is characterized

by

the _presence of additional chromatin in

the telomeres of the chromosome arms X and 2L. T-32

_originates

from flies col-lected in

_N’Djamena

_(Chad,

Central

_Africa)

that were

_subjected

to artificial

selec-tion for increased heat tolerance

_(Tikhomirova

and

_Belyatskaya,

_1980).

Flies of the

T-32 strain are

_exceptional

in that

_they

can

_develop

from _egg to

_imago

at 32°C

without

_losing

their

_{fertility. Furthermore,}

when T-32 is reared at

_high

tempera-tures, sublines can be obtained that have additional chromatin at the

_tips

of one or more chromosome arms in the

_{salivary gland}

chromosomes. The mechanism of

ori-gin

of these telomeric variants is

_uncertain,

but the telomere

_morphology

is stable

in successive

_generations

when the sublines are reared at 23°C. In this _way we

ob-tained 6 sublines of T-32 that exhibited different telomere

_{morphologies, including}

sublines that differ from one another

_{by long}

and short telomere variants of the

same chromosome arm.

We have found that the additional chromatin of the

_long

telomeres

_intensively

hybridizes

with labeled Dm665 _sequences. The short telomeres show a much weaker

hybridization

(or

none at

_all)

with the

_probe

sequences. We conclude that the

morphological changes

in the telomeres of

_polytene

chromosomes are caused

_by

different

_degrees

of

_{amplification}

of at least Dm665 and

_perhaps

other telomere-associated _sequences.

MATERIALS AND METHODS

Drosophila

stocks

The heat-resistant strain

_T-32,

_{kindly provided}

_by

MM

_Tikhomirova,

served as

starting

material. One

_generation

of flies was reared at

_32°C,

and a number

was examined in

polytene

chromosome

squashes prepared by

standard

procedure

(Atherton

and

_Gall,

₁₉₇₂₎

with minor modifications.

In situ

_{hybridization}

Polytene

chromosomes of

_salivary

_glands

of

_D

₇

_-osophila

larvae were

_prepared

ac-cording

to Gall and Pardue

_(1971).

The DNA

_probes

were labeled with tritiated

thymidine by

nick translation. The 2.4-kb Dm665

_fragment

was obtained

by

sub-cloning

the Dm665 HindIII

_fragment

from

plasmid p665

into

_pBR322

_{(Danilevskaya}

et

_al,

_1984).

A

_{larger fragment homologous}

to Dm665 was obtained from a 10.5-kb

insert in

_{bacteriophage}

A 17. This

_fragment

includes not

_only

_sequences

homolo-gous to Dm665 but also 2

_copies

of a novel

transposable

element

designated hoppel

(Kurenova, personal

communication).

Hybridization

sites of the

_probes

were

deter-mined in 5-10 individuals in each subline.

_Although

the

_figures

in this _paper were

prepared

from material

_hybridized

with A17 for the sake of

_signal

_intensity,

the same

results were obtained when the material was

_hybridized

with Dm665. Hence we

at-tribute the

_pattern

of

_{hybridization}

_signals

obtained with A17 as

_coming

at least from the Dm665 _sequences it contains and

perhaps

also from the

_hoppel

elements.

RESULTS

Selection of T-32 sublines with different telomere

_morphology

The

_polytene

chromosomes of strain T-32 have additional blocks of chromatin in

the telomeres of the chromosome arms I and 2L. When T-32 is reared at

_high

temperatures

(32°C),

sublines can be isolated that differ in telomere

_morphology,

and these variants are

_stably

maintained at 23°C. The

_generation

of these stable sublines allowed us to

_study

sublines with additional telomeric chromatin in

different

chromosomes,

and we were able to obtain different combinations of chromosomes with

_long

and short telomeres in the same

_{genetic background.}

The

morphology

of the

_long

and short telomeres found at the

_tips

of the

_{X, 2L,}

and 3R is

illustrated in

_figure

1. Six sublines

_differing

in telomere

_morphology

with

_particular

combinations of

_long,

intermediate,

and short telomeres are

diagrammed

in

figure

2

(lines

A-F).

The telomeres in some lines are not as

_long

as the

_{long telomeres,}

and not as short as the short

_telomeres,

and we call them &dquo;intermediate&dquo;

_only

to indicate this

_ambiguity.

Flies with short telomeres in all the chromosomes

_(exemplified

_by

subline

_E)

occurred with the lowest

_frequency.

Such sublines are somewhat unstable

and

_{quickly acquire long}

telomeres in some

_chromosomes,

most often in the

_right

arm of chromosome 3

_(eg,

subline

_F).

In situ

_{hybridization}

of larval

_{salivary gland polytene}

chromosomes from different T-32 sublines with telomere-associated

_probes

Polytene

chromosomes from the 6 sublines with

_{morphologically}

different telomeres

were

_subjected

to in situ

_{hybridization}

with 2

_probes

_containing

_sequences

homol-ogous to the subtelomeric

_repeat

_designated

Dm665

_{(Danilevskaya}

et

_al,

_1984).

glands

from the same larva. More intense

_{hybridizations}

of the same telomeres were obtained with a 10.5-kb _sequence cloned in

_{bacteriophage A17,}

but the

pres-ence of the mobile element

_hoppel

in the A

_phage

also results in

_{hybridization}

with

the chromocenter and several sites internal to the chromosomes arms.

A

_diagrammatic

_summary of the

_labelling

data with the telomere-associated

probes

is

_presented

in

_figur0

2. This

_composite

is based on results like those

illustrated in

_{figures 3-6,}

which show larval

_{salivary gland polytene}

chromosomes from sublines

_A,

B,

C and

_D,

_{respectively,}

with and without

_{hybridization. Figures}

3

(subline A)

and 5

_{(subline C)}

show the telomeric

_{regions only,}

while

_figures

4

(subline B)

and 6

_{(subline D)}

show the entire euchromatic

complement.

The arrows

indicate the additional chromatin found in the

_long

telomeres.

In all the sublines the

_long

X and 2L telomeres

_carrying

additional chromatin can

be seen to

_{hybridize effectively}

with the telomere-associated

_probes.

The _appearance of additional chromatin in the 3R telomere

_(sublines

D and

_F)

leads to enhanced

hybridization

of this chromosome

_tip

_(figs

_1,

_6).

Note that these sublines also exhibit

All the short-telomere variants of the X chromosome also

_hybridize

with

_a17,

but less

_intensely

than X chromosomes with the

_long

telomeres. The short 2L telomeres in sublines

_B,

E and F do not

_hybridize

to a detectable level with A17

(fig 4),

nor do the short 3L telomeres in sublines

A, B,

C and E

(figs

4,

5).

The

pattern

of

_{hybridization}

is

_especially

clear in

_{heterozygotes produced by crossing}

the

_sublines,

in which

_only

one of the

_homologues

has additional chromatin in

the 2L telemore.

_Figure

7 shows the intensive

_labelling

of the

_long

2L

_homologue

as

_compared

with the

_hardly

noticeable label in the

_homologue

with the short

telomere. This result demonstrates that the short telomeres do contain a small

number

of

copies

of telomere-associated sequences

homologous

to a17. These are

revealed

by

an

_{appropriately}

labeled

_probe,

even

though they

are not associated with

_{morphologically}

visible additional chromatin.

Overall,

our results

_imply

that

the

_{labeling intensity}

of telomeres when

hybridized

with the telomere-associated

probe

Dm665 or A1

directly

correlates with the _presence of additional chromatin.

DISCUSSION

Morphological

differences _{among the telomeric}

_regions

of

_polytene

chromosomes in

different

Drosophila

strains have been known for some time

_{(Roberts, 1979).}

The

chromosomes.

However,

most of the differences have been observed in strains of

very diverse

genetic backgrounds.

In our

study

the sublines

differing

in telomere

length

all derive from T-32 and so have a common

_{genetic background.}

The obvious next _step in the

analysis

of

_{morphological}

variation in telomeres is

to determine what DNA _sequences

_comprise

the additional chromatin. The molec-ular structure of telomeres is under intensive

_{investigation}

in a number of

sys-tems, and a

_general

_pattern

seems to have

_emerged

_(Blackburn

and

_Szostak,

_1984).

The ends of chromosomal DYA consist of

_{simple, tandemly}

satellite-like _sequences

that are

species specific. Adjoining

these are telomere-associated

_repeats

of

_varying

length,

often

_organized

in tandem _arrays. The sequences at the chromosome

_tips

in

Droso

P

hila

have not been

_established,

but telomere-associated

_fragments

have been cloned in several laboratories

_(Rubin,

1978;

Young

et

_{al, 1983;}

Renkawitz-Pohl and

_Bialojan,

_1984).

_{They belong}

to the He-T

_family

of sequences found in the

telomeric and heterochromatic

_regions

_{of the genome}

_(Young

et

_{al, 1983;}

Traverse and

_{Pardue, 1989;}

Biessmann et

_al

_n

_{1990; Levis,}

_personal

_{communication).}

The

fragment

Dm665

_{(Danilevskaya}

et

_al,

_1984),

which is a member of the He-T

_family

of

_repetitive

_sequences, is located in the Y chromosome and shares

_homology

with

unpub-lished

_{observations).}

The telomere-associated sequences also

frequently

contain

mo-bile elements of various kinds

_{(Danilevskaya,}

_unpublished

_{observations).}

One of these mobile

_{elements, designated hoppel,}

is

_frequently

associated with the He-T

The

_{Drosophila melanogaster}

strain

_{T-32, originally}

selected for increased heat

tolerance,

has

_provided

an excellent

experimental

model for

obtaining

sublines with

altered telomere

_{morphology. Rearing}

T-32 at 32°C results in a

_{high frequency}

of alterations in telomere

_morphology,

and sublines

_differing

in telomere

_morphology

can be

_stably

maintained at 23° C.

Hybridization

of labeled telomere-associated

_probes

with

_salivary

_gland

_polytene

chromosomes from different sublines of T-32 demonstrated that sequences homolo-gous to Dm665 are

amplified

in the

long

telomeres. The short telomeres show a far

less intense

_{hybridization,}

and in some cases no detectable

_{hybridization.}

Hence,

the

_{morphologically}

visible additional chromatin in the

_long

telomeres results from

the

_{amplification}

of at least Dm665 and

perhaps

other telomere-associated DNA

sequences.

Roberts

₍₁₉₇₉₎

proposed

that the

_long

and short telomeres result from a

heritable difference in the location of a transition zone between

polytene

and

non-polytene

chromatin. Our results

_suggest

that the

_long

telomeres _may result from the

_{amplification}

of telomere-associated sequences. This may occur in the

_germline

and

_merely

be observed in the

polytene chromosomes,

or the

amplification

_may occur

specifically

in the

_{salivary gland}

and

_perhaps

other

_polytene

chromosomes.

A critical test will

_require

direct in situ

_comparison

of

_salivary

_gland

chromosomes and

_metaphase

chromosomes from other

_tissues,

in order to allow an estimation of the number of

_copies

of Dm665 that occur in the

_tips

of

_metaphase

chromosomes

in

_diploid

tissues.

The effect of

_temperature

on the

_morphology

and

_pattern

of

_{hybridization}

of

telomeres in sublines selected from T-32 is

_highly

unusual. The result

suggests

that elevated

_temperature

_may stimulate

_{deletion, amplification,}

or

_{rearrangement}

of the

telomere-associated _gene

_{family. Except}

for the effect of elevated

_temperature,

the

processes involved may be

entirely conventional,

for

example, unequal

crossing

over or _gene conversion.

_However,

the He-T _gene

family

_may be

subject

to

_{unprecedented}

processes. For

example,

Traverse and Pardue

₍₁₉₈₈₎

and Biessmann et al

₍₁₉₉₀₎

have described the de novo addition of He-T

family

_sequences to the

_tips

of broken

chromosomes. These additions serve the role of telomeres in

stabilizing

the

_tips

against

the loss of DNA

_{during replication. Moreover,}

the addition of the He-T

sequences to the ends of broken chromosomes does not

_require

_sequence

_homology

to the ends. What _processes are involved have not

_yet

been

_identified,

but it is

possible

that the _process is

_{promoted by}

elevated

_temperatures

in the T-32 strain.

ACKNOWLEDGMENTS

The authors thank VG Nikiforov and VG Shakhbazov for support and _{encouragement,}

MM Tikhomirova for strain T-32

stock,

ES

Belyaeva

and BA Leibovich for their aid and

advice,

and DL Hartl for his

_help

and encouragement.

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