Chromosoma (1992) 102:32-40

CHROMOSOMA 9 Springer-Verlag1992

A repetitive DNA element, associated with telomeric sequences in Drosophila melanogaster, contains open reading frames* Olga N. Danilevskaya 1,2,**, Dmitri A. Petrov 1,***, Maria N. Pavlova 2, Akihiko Koga 1,****, Elena V. Kurenova 2, and Daniel L. Hartl 1,*** Department of Genetics, WashingtonUniversitySchool of Medicine, St. Louis, MO 63110-8232, USA 2 Institute of MolecularGenetics, USSR Academyof Sciences, Moscow 123182, USSR Received June ll, 1992 Accepted July 4, 1992 by W. Hennig

Abstract. He-T sequences are a complex repetitive family of DNA sequences in Drosophila that are associated with telomeric regions, pericentromeric heterochromatin, and the Y chromosome. A component of the He-T family containing open reading frames (ORFs) is described. These ORF-containing elements within the He-T family are designated T-dements, since hybridization in situ with the polytene salivary gland chromosomes results in detectable signal exclusively at the chromosome tips. One T-element that has been sequenced includes ORFs of 1,428 and 1,614 bp. The ORFs are overlapping but one nucleotide out of frame with respect to each other. The longer ORF contains cysteine-histidine motifs strongly resembling nucleic acid binding domains of gaglike proteins, and the overall organization of the T-element ORFs is reminiscent of LINE elements. The Telements are transcribed and appear to be conserved in Drosophila species related to D. melanogaster. The results suggest that T-elements may play a role in the structure and/or function of telomeres.

Introduction The Drosophila genome contains a complex repetitive family of DNA sequences designated the He-T family, which is associated with telomeric regions, the pericentromeric heterochromatin, and the Y chromosome * The sequences reported in this paper have been deposited in the Gen Bank data base under accessionnumbers 68806 and 68816. ** Present address: Department of Biology, Massachusetts Institute of Technology,Boston, MA 02139, USA *** Present address: Department of Organismic and Evolutionary Biology, The Biological Laboratories, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA **** Present address: Department of Biology,Universityof Hiroshima, Hiroshima, Japan Correspondence to." D.L. Hartl

(Young et al. 1983; Traverse and Pardue 1989; Danilevskaya et al. 1991). The number of copies of He-T sequences associated with telomeres differs among chromosomes and varies within (Renkawitz-Pohl and Bialojan 1984) and between (Traverse and Pardue 1989) Drosophila stocks. Genetic rearrangements appear to occur at high frequency in the He-T family. For example, the He-T repeats are amplified in certain stocks resulting in the formation of " l o n g " telomeres (Danilevskaya and Lapta 1991). Furthermore, a portion of the He-T sequence known as the HeT-A box can undergo translocation to the ends of broken chromosomes and appears to participate in chromosome "healing" by which the chromosome ends become genetically stable (Traverse and Pardue 1988; Biessmann et al. 1990). These results suggest an important role for the He-T family in the structure of Drosophila telomeres. Although DNA sequences from the He-T family studied to date appear to be noncoding (Biessmann et al. 1990; Valgeirsdottir et al. 1990; Danilevskaya et al. t991), we have found a novel repetitive sequence within the He-T family that contains open reading frames (ORFs). We refer to this class of He-T sequences as T-elements, since hybridization in situ with the polytene salivary gland chromosomes results in detectable signal exclusively at the chromosome tips. One copy of the T-element that has been sequenced includes two ORFs of/,428 and 1,6/4 bp. The ORFs are overlapping but one nucleotide out of frame with respect to each other. The longer ORF contains the gag-like cysteine-histidine motifs strongly resembling nucleic acid binding domains. The structure of the T-elements is reminiscent of the LINE type of transposable element (Hutchison et al. 1989), but detailed sequence analysis also reveals many differences. Hence, if the T-elements do undergo transposition, they may represent a novel class of transposable elements. Furthermore, although T-elements are specifically associated with the He-T family, their sequence and position in the genome are conserved between species of Drosophila, whereas other He-T family sequences are more divergent.

33

M a t e r i a l s and m e t h o d s Drosophila strains. The plasmid, phage 2 and YAC (yeast artificial chromosome) clones originate from Drosophila strain Oregon RC. The bacteriophage P1 library of clones originated from an isogenic strain of genotype y; cn bw sp. Origin of cloned DNA. Plasmids Dm4568, Dm665 and Dm2103, containing DNA fragments from the Drosophila strain Oregon RC, have been described previously (Danilevskaya et al. 1984, 1991). Strains DH5:~ and LE392 of Escherichia coli were used for plasmid and 2 phage propagation, respectively. The 2 library was prepared and generously provided by N. Churikov (Institute of Molecular Biology, USSR Academy of Science) using Oregon RC DNA in 247.1 with E. coli host strain LE392, essentially as described in Sambrook et al. (1989). The YAC clones, containing DNA fragments averaging about 200 kb, were generated from Oregon RC DNA as described in Garza et al. (1989) for randomly sheared DNA and in Danilevskaya et al. (1991) for Notl fragments. YAC library screening was carried out as described in Garza et al. (1989). The bacteriophage Pl library, containing DNA fragments in the size range 75-100 kb, originated from Sau3A partial digests of DNA from an isogenic strain of genotype y; cn bw sp, and its construction and methods for screening are described in Smoller et al. (1991). Cytological analysis. Polytene chromosomes of salivary gland nuclei from third instar larvae of Oregon RC were prepared as described by Johnson-Schlitz and Lira (1987). Probe DNA was labeled with biotin-dCTP (Enzo Biochemicals) by random oligonucleotide primer extension (Feinberg and Vogelstein 1984) and in situ hybridization was carried out at 37~ C for 16-18 h. DNA hybridization and sequencing. Genomic DNA was prepared as described in Lis et al. (1983). Restriction fragments were separated in 0.7% agarose gels and transferred to Hybond N (Amersham) nylon membranes (Southern 1975). Filters were hybridized with probe labeled to high specific activity with random oligonuc-

BamHI

leotide primer extension (Feinberg and Vogelstein 1984). Hybridizations were carried out overnight at 65~ in high phosphate buffer (100raM Na2HPO4, 500mM NaC1, 5.2mM EDTA, pH 7.0, containing 1% Sarcosyl and 100 mg/ml sonicated denatured salmon sperm DNA). Washes were performed under conditions of moderate stringency (five 20 min washes at 25~ C in I mM Tris-HC1, pH 7.5, containing 0.1% sodium lauryl sulfate). DNA sequences were obtained from clone Dm2103 and from the 5.7 kb EcoRI fragment from 223 by the method of Sanger et al. (1977). All other methods for DNA manipulation were as described in Sambrook et al. (1989).

Results Origin o f clones containing T-elements

In order to identify Drosophila clones originating from the chromosome tips, approximately 200 in situ hybridizations with Drosophila salivary gland chromosomes were carried out using randomly selected pBR322 clones containing inserts of Oregon RC DNA (Danilevskaya et al. 1984). One of these clones, designated Dm4568 in Fig. 1 A, hybridized in situ only to the chromosome tips. Detailed analysis of Dm4568 subclones indicated that the BamHI insert in the clone contained two types of sequence. A subclone consisting of the right-hand PstI-BamHI fragment in Fig. 1A hybridized strongly with the chromocenter as well as the chromosome tips; this subclone contains noncoding He-T family sequences homologous to those in the previously described clone Dm665 (Danilevskaya et al. 1984, 1991). A subclone of

BamHI

Dm4568 Dm2103

B k23

24~ 4

8

"

"

>,

I,'//////////////J

I

I

TTT' "

...................

I

~ V ECORI

9 HindIII

9 BglII

i kb

Fig. 1. A Restriction map of Drosophila DNA in the plasmid clone Dm4568. The filled region designates the Din2103 subfragment. The shaded region designates homology with He-T sequences previously defined by clone Dm665 (Danilevskaya et al. 1991). B Restriction maps of 2 clones with homology to Dm2103 and/or DM665. The arrows indicate the positions of T-elements and the orientation of the open reading frames. The arrows with solid outline indicate copies of T-elements that were sequenced (T-2103 from Dm2103 and T-23 from 223); the other T-elements were identified by DNA hybridization

Fig. 2, Polytene chromosomes of Oregon RC stained with Giemsa and hybridized with biotin-labeled Dm2103. Bar represents 12 ~tm

34 1

~tga~t~gta~ct~a~agCaaagttaa~a~tc~gt~tCCata~tCca~Ca~aaQtaaaaaaaaaaaaaaaaaaaataaataaataaaataa

I01

attaaacaattaactaaataattaaataactaaaattaataatataatcgcgctt•g•g••aaagactCtcacgcgcataactaattaaaatcgttttca

201

agttga•aaataaatggtttaaaattgtc•t•aggct•caaagaaaagccgcggcaacaataaacattta•tgaca•gcgaaaagcgacatttgattagt

301

gtaatacttgtgcaaa••gacaagctgccg•cataacaaaacggagacgaagaatcataaagaacaaaagctaaatcaccag•atagcaaaaataaatta

401

acaaataaaataaaagcaaatttaaataacataataaattaaa•ttatttaataaacaccataatttaattatcataacgctaatctacatata•tccac

501

gcgcaaattaatt~aaatcgtCtttctagttaataaat~aaaagtttaaaaat~gtctccggccgcaaaatttgaaccgCgacgataaaaacatttaatt

601

gacaaacaaaaagcgaacaattattcagtgaactatttgtgcaaaattgacaagcagacg•cataattaaaaggagaagaagccaaaagacgaagagaag

701

aaagcaacCagaagaact•aaagaagaaaaggag•aaagc•CaattaaagaaagccagggtatttataccttaCa•ttat•gtttaatataacaaaaac•

801

M S M S D N L F S D D E V L S I S S S P E Q R S S P F Y L N I S ~aacATGTCCATGTCCGAC~ccTTTTTTCTGACGATGAGGTACTTTC~TTTCCTC~GCCCAG~CAGCGATCTTCTCCGTTcTACCTC~TATATCG

901

P M S H G S D N S Q I N T V I I N S K K L P S N Q A D I S L K N S ~CCATGTC~CACGGATCAGAc~TTCTCAGATT~TACAGTcATCATTAATTcG~G~ATTGCCCTCA~Tc~GCAGACAT~GTTT~cTCTT

S G A A I K I V N S L S H K K K E N T N V N N A Q K D P L S L T N T i001 C T G G G G c T G C T A T A A ~ T T G T T ~ T T C C C T T T C A c A C ~ G ~ G A ~ G A G A A c A c ~ A C G T T ~ T ~ T G c c C A ~ G A C C C C C T C T C A c T C A C C ~ T A C T A S T C G A K S S I S E G K L S S P P S T S H T Y E G K L L T K ii01 TACTGC~GCACTTGTGGcGCCAAAAGCAGCATCTCAGAGGGG~ATTGTCTTCTCCTCCGTCcACCTCACACACATATGAGGGGAAATTACTCACA~A L T H T H T D F R G A K T S D A M G S F P S L S H S D N S I E K N 1201 C T T A C T c A C A C A c A C A C A G A C T T T A G A G G C G C c ~ C G A G C G A T G C ~ T G G G ~ G T T T c C c C T C T C T c T C G C A C A G C G A C ~ T A G C A T A G A G A A A A A T C L S S S T K I G P N A S S P P S H A H T H T S K S T D I S L E S R S 1301 TGAGTT~TTCCACcAAAATTGGACC~ACGCTTCTTCCCCTCCTTCTCATGCAcACACTcACACTAGCA~TCCACTGATAT~GCTTAGA~GCCGCTC K H P A L A N f D A R S I K A N A N D N G E I F S S L I Q I D E R 1401 ~ C A T C C C G C G C T T G C C ~ T A C G G A C G C A C G C T c T A T ~ G C C ~ T G c T A A T G A C A A T G G G G ~ A T T T T C T c C T c A c T T A T A c A A A T T G A C G ~ C G C K Q E E R P C T T I N A F W S I F K P K P D V T K L S L K R K P T 1501 ~ G C ~ G A G G ~ A G G C C T T G C A C ~ C T A T C ~ C G C T T T T T G G T C T A T T T T T ~ A C C C ~ G C C G G A C G T T A C T A ~ C T ~ G T C T ~ A G A G G A ~ C C C A C C A N P T K N T G K K C I S P H K K S A Y L C P S A Q D D L N L N L N P 1601 A T C c C A C T ~ A C A C T G G G A A A A A A T G C A T C T C C C C T C A T ~ A G A G C G C T T A T T T A T G C C C T T c C G C T c A G G A T G A T T T A ~ T T T A ~ T T T A A A c C C K S S A K P T V V N L P A A R I L S R P A A K R D L F K S S S S R 1701 cA~TCTAGCGCCAAGCCCACTGTGGTGAATTTACCAGcTGCCCGCATCcT~GCcGGCCTGcAGCC~GcGGGATTTATTTA~TCATCATCCTCcCGA S P D E Q P M S F S E V V A G T G S I F A A P C V P A P L T K T P 1801 AGCcCAGACGAGCAGCCTATGAGTTTTTCGG~GTGGTCGCTGGCACGGGTTC~TTTTTGCGGcACCCTGTGTCCCGGCACCTTT~CGAAAACTCCAG K I M R D F Q L C N S Q H F S A G K R T N D D L D C S N F K T P N K K L C A T S N F V T P S I F P P 1901 G C ~ G C G G A C ~ A C G A C G A T C T G G A C T G C T C C A A ~ T T T ~ G A c G c C C ~ T A ~ A A A T T A T G C G c G A C T T C C ~ C T T T G T ~ c T C C C A G C A T T T T T C C G C C A

H H S R F Q E Q G S S I C L R G I Q S Q K W T P P A G P R L Q H L I T P V F K S K A A Q S V Y E E S K A R N G P P P P A L A C S I 2001 G C T c A T C A C T C C C G T T T T c ~ G A G C ~ G G C A G C T C ~ T c T G T T T A C G A G G A A T C C ~ A G C C A G ~ A T ~ G A C C C C C C C C G C C G G C C C T C G C C T ~ C A G C A T c Q

C L C S Q R T A P P G I A P L P P H N T D A E L P P W K I V P Q S N A S A R S A R R H P G S P P Y P L I I Q M Q S C L H G K S C P R 2101 ~TGCcTCTGCTCGCAGCGcACGGcGCCAcccGGGATCGCCCcCCTACCCCcTCATAATAcAGATGCAGAGCTGCCTCCATGG~TCGTGCCCCAGAG R R A P P I L V N D V K E I V P L L E K L N Y T A G V S S Y T T R A V E H L L Y S S M M E n d 2201 ccGTAGAGCACCTCCTATACTCGTc~TGATGT~AGGAAATTGTAcCTCTACTGGA~AGcTGAAcTAcACAGcAGGAGTCTCcAGCTATACTACTAGG A I E G N G V R I Q A K D M T A ~ K I K E V L ~ A N G ~ P L F T 2301 G C T A T A G ~ G A ~ C G G G G T C A G G A T A C A G G C A ~ G G A C A T G A c C G C C T A T ~ C A A A A T T A ~ G A A G T C C T G G T G G C C ~ C G G A C T T c C T T T A T T C A C C A 1

GGATCCAGGCC~GGACATGACCGCCTTCCACAAAATTA~GAAGTTCTTACAGCAA-~CGGCTTCCCTCTTTTTACTA

~g. 3. DNA sequence and in~rred amino acid sequence of T-23 (the T-element sequenced from clone 223). The sequence of T-2103 (from plasmid Dm2103) is also shown beneath T-23 nucleotides 2323-3567. Dots indicate identical nucleotides in the sequences.

Amino acids that di~r between T-23 and T-2103 are un~rlmed. The cysteine-histidine moti~ are enclosed in r e c t a ~ . The HeT-A box upstream of ORFI is un~rlmed

the left-hand BamHI-PstI fragment in Fig. 1 A, designated Dm2103, did not hybridize with Dm665. This observation stimulated additional analysis since it suggested that Dm2103 might represent a novel family o f telomere-associated sequences.

In situ hybridization o f Dm2103 with the polytene c h r o m o s o m e s o f Oregon RC yielded no detectable signal in the chromocenter, which consists largely o f relatively underreplicated pericentromeric heterochromatin and includes the entire Y c h r o m o s o m e . Strong hybridization

35

2401

N Q P K S E R G F R V I I R H L H H S T P C S W I V E E L L K L G F A C C A G C C C A A G T CC G A G A G A G G C T T CC G A G T C A T C A T C A G A C A T C T C C A C C A C T C C A C A C C A T G C TC G T G G A T A G T C G A G G A A C T G C T G A A G C T C G G A T T

79

A-CAGCCCAAGT CCGAAAG-GGC TTCCGAGTAG TCATCAGACATCTTCACCAT

2501

O A R F V R N M T N P A T G G P M R M F E V E I V M A K D G S H D C C A A G C G C G A T T C G T C A G A A A T A T G A C G A A T C C G G C T A C A G G T G G C C C C A T G C G A A T G T T T G A A G TG G A G A T C G T C A T G G C C A A A G A C G G C A G TCAT G A C

177

CCATGCGCGC TTCG TCAGAAATATGACGAATCC

2601

]i I L S L K Q I G G Q R V D I E R K N R T R E P V AA3~ATACTCTCACTCA/~ACAAATCGGTGGGCAAAGGGTGGACATTGA~GGAAAAACAGGACACGGGAGCCAGTCCA(

277

AGAATTATC TCACTCAAACAAATCGGTGGGCAAAGGGTTGATATAGAAAGGAA~TAGGACACGGGAGCCGG

2701

R H A K N S C M R P P R C M K S A G E H L S S C C T K P R T T P A T G G C A T G C C A A A A A C T C T T G C A T G A G G C C G C C A A G A T G C A T G A A A T C G G C TG G C G A A C A C C T G T C T TC C T G T T G C A C C A A A C C A A G A A C C A C CC C C G C C A C

377

GGCAT T CAAAAAAC TCTTGCATGAGGCCGCCAAAATGCATGAAAT C

~

N

C

S

~

I

S

A

Y

K

TCCACACCATGT TCGT GGATAGTTGAGGAACTGCTGAAGC

TCGGAT T

G G C C A C A G G T GG C C C C A T G A G A A T G T TT G A A G T G G A G A T C G T C A T G G C C A A A G A C G G C A G T C A C G A C

G

C

I

P

Q

C Y R C Q G F ~GCTACAGATGCCAAGGCTTCA

TC C A G ~GC T A C A G A T G C C A G G G C T T C A

GCGC TGGCGATCACCTGTCATCCTGTTGCACCAAAC ~

Y

K

A

E

K

Q

K

L

A

A

N

N

C A A G A T C C A C CC C C G C C A C ~

D

I

N

477

cTGcATc~CTGCTCTGAAGAcGACATTAGTGCTTAc~GGGATG~cCGTTTAT~GGCCGA~CAAA~GCTTGCAGc~AC~CATTGACATA~T

2901

K I R T I K D A T N N ~ Y N R Q G P P L R N N ~ P R L P H S S A I AA-AATAAG~CAATCA/~AGA~Gc~CA~T~CTTTTAT~ACGTc~GGCCCcCCTCTACGC~cAACACccCTCGGCTACCGcACAGcTCAGc~TCC

577

~TACGGACAATTAAAGAC~C~CAACT~CATATATAGACGTC~GGTccTCCTCCTCGC~TAACGcCCCTCGGCTGCCACACAGCTCAGC~TTC

3001

L S K S I A E A ~ Q E A A R K S M L N P F R ~ N I N D ~ R P R F S S TGAGcA~Tc~TTGCCGAAGcTcGCCAGGAGGCAGCCAGA~GTCGATGTTAAATccATTCcGACA~ATAT~ACGAcAGAAGACCACGATTCTCCTC

677

TGAGcA~TC~TCGccGAAGcTGcccAGGAGGcAGCCAGA~GTCTATGcTA~ccTTTCCGGcC~ATATGAACGACAT~GGCcAcGTTTTTcCTc

3101

H D T A I Q K R L N K W R R N T N K I P K K G R I A L K ~ N ~ K P CCAcGAcACGGcCATTcAG~GcGTcTGAAT~ATGGCGCCGA~cAcC~cAAAATACccAAAAAGGGTAGGATAGccTT~AGGATAATGcAAAGCcA

777

CcATGAcACTGCCATCCAG~GCGGCTGAAA/~AATGGCGcCGA~CAcT~TAAGATACCCAAAAAGGGTAGAACAGTcCC~AG~CAATGTAAAACCG

3201

R P ~ T S N P A Q R H L Z ~ Y Q D ~ L R ~ E R S E [ N ~ Q E I CGACCGGCACATAGGACAAGT~cccAGCGC~AGACATCTGGAGGACTACCAGGACATGCTCCG~GGGAAAGGAGTG~GA~AcGACCAGG~TCTG

877

cGACCGAT~CC~GACAAGC~TCcAGcGC~AGACATCTGG~cTACCAGGAc~G~TcCG~GAAAGGAGTG~AC~AcGGCcAGG~cCTG

E K G T P N T K Q ~ G N D S P P T T S R A A R A S F K P R I I ~ 3301 A G ~ G G G c A C C C C C ~ T A C C A A G C A G G T C G G C A A T G A C A G C C C T c C G A C C A C G A G C A G A G C A G C c A G A G C C A G c T T T ~ G C C A A G ~ T C A T T G A C G A T A C 977

~AGGTACCcCA~CCCCATGCAACTCGGCAATGACAGCcCTCCGACcACCAGCAGAGCCGCCAGAGcTAGCTTCAAACCAAGAGTCATTG~GA~A

3401

T P ~ P ~ N P ~ Q K G ~ D D P T T ~ L A N R V ~ N L E ~ cACGCCATCGcc~AAATCTGc~TCCCAACTCAc~AAAGGccTCTTGGAcGACCCcACAAC~GCTTAGcT~TAGAGTCGAC~TTTAGAA-~GAA

K

1077 C A c G c C A T T G C C ~ G A A A C T C C ~ C C C A T A C C T A c ~ A A ~ G C T C C T C G G A C G A C C C C A C C A C ~ A C C T A G C A ~ T A G A G T T G T T ~ T T T A G A G T C A ~ A

3500

I D I L M A L I I ~ G R N N N ~ D M ~ T S ~ E n d ~TTGAcATTTT~TGGCCTT~TCATAC~GG~GA~TAAC~TcTTGACATGGATACATCCAATTAAtctta~aactactta-tatattcttta--a

1177 ~ T T G A C A T T T T ~ T G G C G T T ~ T T A T A G ~ G G ~ G G ~ T A A C ~ C A T T G A T A T G G A T A C A T C C A C T T A A a t c t a c a t t t g c t t a t t c ~ a t t t t t g a c t a 3597

taaatatatccaatagaaaagcgcacgtcggtctgcttttaaaatccttcaccgtcatcac•ttcctcgacg--gagcctaatttatt-ggaaaaataaa

1277 t a t c t a t a t c c g a t - g a a t a g t g c a c c t c c g t c t g g t c t c a a a t t c c t t t c t t g a c a t c a t • c t c c t c e a a g c a a a g c c t a a t t t a t t g g g g a a a a t a a a 3694

tcaattatatgttggcacaaaaatg~aaacacacactcacctaaacg~acccggacgaacaagcctatgacaacgeactccagctgatctgtaagaaaca

1376 tcaattagatgatggcataaaagttttataatacactcaccttaacgcaaccggatgaacaagcctatgacaacgcactccagctgatccggacgaaaca 3794

aaaaatatgaatagatagatcgatatgaaagga-Zagtgcggcag-aacatgatg--gcaaaggcgactcgctgcagcaacttatgcacaacgtcactta

1476 taaaagatgaatcgatagatagaaataaaaggartagtgcggcagaaacatgatgaaEttaaggcgactcgctgcagcaatttatgcacaacgtcactta 3890

cctgaaatttcaaatttcttgccgtacgat-ctcctgtagtat-ccttatcaacagctgcaatttcta•ttgcaatgctgcactgcaataaacgtactac

1576

cctgaatctt ....... cttgccatgcgttccttttgaaatatccct•atca-c•gctgcaatc--ta•atacattgatgcagcg-aaaagacggtcaac

3988

aaagctgcatac-ttttgarc-ggacacct•gtgccagtgctaaaaatttgctttctgctg•tcttattgagtccaaactt

1666

catgtcgtctccgagtcgatcaggacacct--tcctgctctcaaaaacctccagcctgacgagcgccaacagcagttgacg

Fig, 3 (continued)

36 was observed at the tips of chromosomes X, 2L, 2R, and 4, as illustrated in Fig. 2. Weaker hybridization was also observed at the tips of chromosomes 3L and 3R in some preparations. The in situ hybridizations in Fig. 2 were carried out with biotin-labeled probe, which is generally sensitive enough to identify sites with imperfect homology. For example, the Drosophila sequences in the He-T-related clone Dm665 share only 600 bp of imperfect homology with the Stellate gene and yet hybridize with the euchromatic site at 12E~-2 in which the Stellate gene is located (Danilevskaya et al. 1991). Among a collection of about 20 2 clones bearing Drosophila DNA inserts with homology to the He-T related clone Dm665, 4 also contained regions of homology with Dm2103. Restriction maps of the inserts in these 4 clones are shown in Fig. 1 B. The sequences homologous to Din2103 are interspersed with sequences homologous to Dm665. The Dm2103 sequences are found as single sequences (248), tandem repeats (233), or as several repeats interrupted by sequences homologous to Dm665 (223, 244). These data indicate that Dm2103 represents a family of repeated sequences that are associated with chromosome tips, and we designate the sequences as T-elements.

The 5.7 kb fragment from 223 actually contains two ORFs defining T-23. The first ORF (here designated ORF1) is 1,428 bp long. ORF1 overlaps for 288 bp, and is one nuclcotide out of frame with, the second ORF (designated ORF2) of 1,614 bp. The final 1,245 bp in the 3' portion of ORF2 (nucleotides 2323-3567) corresponds to the ORF in T-2103, and across this region of homology the two sequences are 84.5% identical. ORF1 begins with an ATG codon at position 805, and the context of this codon matches the consensus Drosophila translational start site at 14/16 nucleotides (Cavener and Ray 1991). We searched GenBank and EMBL data bases for sequences sharing homology with T-element sequences. No significant homologies were detected with ORF1, at either the nucleotide or the amino acid level. However, ORF2 shares homology in a region of cysteine-histidine motif that is highly conserved in the Drosophila LINEtype retroposons G (Di Nocera 1988) and Jockey (Priimagi et al. 1988). In particular, nucleotides 2520-2966 (amino acids 188-336) in ORF2 code for a complex cysteine histidine motif, which is present in T-23 as well as T-2103. The corresponding region of the G element is a 150 amino acid domain that has 28% identity (45% similarity) with the T-23 amino acid sequence (Fig. 4A). The corresponding region of the Jockey element is a 95 amino acid domain with 42% identity and 55% amino acid similarity (Fig. 4 B). The cysteine-histidine motif (Cys-X2-Cys-X4-His-X4-Cys) present in the T-element ORF2 at amino acids 241-245 and 260-272 (Fig. 4A) is highly conserved among retroviral gag proteins (Covey 1986), and it is also found in a number of retroposons of the non-LTR class (Schwarz-Sommer et al. 1987; Jakubczak et al. 1990). The number of Cys X2Cys-Xg-His-X4-Cys motifs, and the spacing of 5-6 amino acids between them, is conserved in Drosophila LINE-type retroposons and the gag proteins of retroviruses. This same organization is found in ORF2 in T-23 and T-2103 (Fig. 5). Note, however, that in T-23 the Ser263 underlined in Fig. 5 is expected to be Cys263; and in T-2103 the Asp289 underlined in Fig. 5 is expected to be His289. These differences may mean that

The T-elements contain two ORFs with gag-like cysteine -histidine motifs In order to investigate the nature of the T-elements we sequenced the Dm2103 fragment. The size of the insert in Din2103 is 1,556 bp. The insert included an ORF of 1,179 bp (putatively coding for 393 amino acids) oriented from the BamHI to the PstI site in Fig. 1 A. The sequence suggested that the ORF detected in Dm2103 could extend further upstream in larger clones. In an attempt to find the start of the ORF, we sequenced 4.1 kb from the 5.7 kb EcoRI fragment possessing homology with Dm2103 present in clone 223 (Fig. 1 B). The results of the sequencing are summarized in Fig. 3. The particular T-elements sequenced from clones Dm2103 and 223 are denoted T-2103 and T-23, respectively.

A G

26

DLHNPIGKKSKEPLGIFFVNLEPASNNTDIYKLKRICRSVVTVEPPLKFN

II

I

l

II

I

I

T-23

188

NMTNPATGGPMRMFEVEIV.MAKDGSHDKILSLKQIGGQRVDIERKNRTR

G

76

DVPQCFRCQGFGHTQRYCFLEFRCVKCGGLHDSRACEKKEDEKACCLHCQ

II

llllJ

I

I

II

I

I I I

l I

236

I

237

EPVQCYRCQGFRHAKNSCMRPPRCMKSAGEHLSSCCTKPRTTPATCVNCS

G

126

ADHPASFKGCPAYKKAKAQQAPKPKARSMESNNKPSFELPNITNGMSYRD

T-23

287

IIIIIII

I

I

125

I l

T-23

I

75

I

I

286 175

I

I

GQHISAYKGCPAYKAEKQKLAANNVDINKIRTIKDATNNFYKRQGPPLRN

336

B J-I

301

348

SLKITRLGRYRVTVERATRRKELLQCQRCQIFGHSKNYCAQDPICG

III

I

II

II

J

I

II

III

1 I II

I

I I

263

T-23

218

SLK..QIGGQRVDIERKNRTREPVQCYRCQGFRHAKNSCMRPFRCM

J-I

349

K C S G P H M T G F A L C .... I S D V C L C I N C G G D H V S T D K S C F V R A E K A K K L K

E T-23

273

I I

I

I II

I I I

I II

l rl

KSAGEHLSS...CCTKPRTTPATCVNCSGQHISAYKGCP..AYKAEKQK

I I

395

315

Fig. 4. A Alignment of amino acid sequence of T-23 with the Drosophila G element in the region of the cysteine histidine motif (data from Di Nocera 1988). B Alignment of amino acid sequenceof T-23 with the Drosophila Jockey (J-l) element in the region of the cysteine-histidine motif (data from Priimagi et al. 1988). Conserved amino acids in the cysteine-histidine motif are in boldface; identical amino acids are indicatedby vertical lines

37 RIDm RIBm F G T-23 T-2103 Jockey I RSV HIV-I HTLV-I

CHRCVGFDHKVSEC CNKCQQYGHPEKFC CINCQEYGHTRSYC CFRCQGFGHTQRYC CYRCQGFRHAKNSC CYRCQGFRHSKNSC CQRCQIFGHSKNYC CKKCLRFGHPTPIC CYTCGSPGHYQAQC CFNCGKEGHIARNC CFRCGKAGHWSRDC

6aa 6aa 5aa 5aa 5aa 5aa 5aa 5aa 5aa 5aa 5aa

CRQCGQQGHTAAKC CGRCGEDGHRMEAC CVVCGDL-HDSKQC CVKCGGL-HDSRAC CMK~AGE-HLSSCC CMKCAGE-HLSSCC CGKCSGP-HMTGFALC CZNCSETKHTNDGEKC CWKCGKEGHQMKDC CWKCGKEGHQMKDC CPLCQDPTHWKRDC

5aa 6aa llaa 9aa 9aa 9aa 6aa 5aa

CRNCRHRGQPSG-HYMLSNAC CATCRRFRREAM-HPTASRDC CNNCGGN ...... HTANYRGC CLHCQAD ...... HPASFKGC CVNCSGQ ...... HISAYKGC CINCSED ...... ~ISAYKGC CINCGGD ...... HVSTDKSC CLNCRNNPELDHQHSPIDRKC

both T-23 and T-2103 represent nonfunctional copies of the authentic T-element family. The 223 clone also includes a copy of the HeT-A box described by Biessmann et al. (1990), which begins 774 bp upstream from the putative translational initiation codon in ORF1. The HeT-A sequence is underlined in Fig. 3; it has only two mismatches with the HeT-A consensus sequence and includes a 20 bp region of poly(A). Comparison of the T-element sequence in Fig. 3 with the sequence from the B fragment of the 2T-A clone (Valgeirsdottir et al. 1990) revealed 89% identity across nucleotides 1579-2875. However, the B fragment has three small deletions in comparison with the T-element sequence that disrupt the ORF.

Fig. 5. Comparison of amino acid sequences in the gag-like cysteine motifs found in the T-elements T-23 and T-2103 with various retroposons (data from Jakubczak et al. 1990). The number of amino acid residues (aa) separating the multiple cysteine motifs is indicated

two clones containing sequences homologous to Dm2103, and both of these had also hybridized with Dm665. The Dm2103 probe was also used to screen a bacteriophage PI library containing approximately 2.2 genome equivalents of Sau3A inserts of D. melanogaster D N A in the size range 75-100 kb (Smoller et al. 1991). Six P1 clones were identified, and each of these also contained sequences homologous to Dm665. The co-occurrence of Dm2103 and Dm665 sequences in plasmid, 2, Pl and YAC libraries strongly suggests that the Telements regularly occur in association with other He-T sequences in the Drosophila genome.

Presence of T-element sequences in the Y chromosome The T-elements are included in the He-T family of sequences Further analysis of the relation between Dm2103 (containing a portion of the T-element) and Dm665 (containing one noncoding subfamily of He-T sequences) was encouraged by the presence of Dm2103 sequences in association with Dm665 in plasmid Dm4568, as well as their co-occurrence in several 2 clones isolated from a genomic library screened with Dm665 (Fig. 1). The analysis was carried out by screening YACs (Garza et al. 1989; Danilevskaya et al. 1991) and bacteriophage Pl libraries (Smoller etal. 1991) containing Drosophila DNA, using probes derived from Dm2103 and Dm665. Clones yielding positive signals from colony hybridizations were confirmed by D N A blotting and in situ hybridization with salivary gland chromosomes. The results are summarized in Table 1. A m o n g 3,456 Y A C clones in two different libraries (containing an estimated 4.5 haploid genome equivalents of D. melanogaster DNA), we isolated 27 clones containing sequences homologous to Dm665. The same libraries yielded only

Restriction fragment length polymorphisms in T-elements unique to the Y chromosome suggest that the T-element copies in 223 and in the YACs N03-89 (containing a NotI fragment) and DY698 (containing a ran-

130 O" co ,o C~ >'-

Type of genomic library YAC (sheared) YAC (NotI) PI (Sau3A)

Number of clones screened 768 2,688 3,840

Number of clones hybridizing with Dm665

Number of clones hybridizing with Dm2103

3 24 a

1 1 6

a P1 library was screened only with Dm2103. All clones identified with Dm2103 also hybridized with Dm665

COL

,< s

2' w

4.23,5-

2.]

-

2.0-

Table 1. Co-occurrence of Dm2103 and Dm665 homologous se-

quences in genomic libraries of Drosophila meIanogaster

a- Oregon oo RC

1.3-

].0-

0.9-

6

Fig. 6. Evidence that copies of T-elements are present in the Y chromosome, An equivalent amount of DNA fi'om Oregon RC males and females, YAC clones (DY698 and N03-89) and 223 were digested with HindIII, fractionated by electrophoresis, blotted, and hybridized with 32p-labeled DNA from Din2103

38 domly sheared fragment) originate from the Y chromosome. Figure 6 shows a DNA blot of Oregon RC DNA from males and females digested with HindlII and probed with Dm2103. While the differences between DNA of males and females are not as dramatic as observed with Dm665 (Danilevskaya et al. 1991), there are nevertheless some differences. The most intense band in both sexes is approximately 2.1 kb, which corresponds to the HindIII fragment in ORF2 of T-23 in Fig. 1 (the HindIII sites are at nucleotides 1383 and 3463 in Fig. 3, and so the actual size of the band is 2,080 bp). In Fig. 6, HindIII fragments smaller than 0.9 kb may originate from genomic sequences similar to those in phages 233 or 244 in Fig. 1. Among the bands specific for the Y chromosome, the most intense is about 3.0 kb. YAC clone DY698 contains this band, which strongly suggests that this YAC originates from the Y chromosome. In YAC N03-89, two faint bands of 2.0 and 3.2 kb comigrate with bands in DY698 and with minor bands in DNA of males; hence, N03-89 may also come from the Y chromosome and partly overlap DY698. In 223 there is also a band of about 4.0 kb, which comigrates with a band in DY698 and a male-specific band. (This band corresponds to the region between the middle HindIII sites in Fig. 1. A band of the same size is also present in N03-98, but it is too faint to be seen clearly in the reproduction in Fig. 6.) These results suggest that the genomic fragment in 223 may also have originated from the Y chromosome. T-elements are conserved in the Drosophila melanogaster species subgroup To determine whether T-element sequences are conserved among related species, we carried out DNA blots, using a probe derived from Dm2103, against genomic DNA from species closely related to D. melanogaster. The species D. simulans, D. mauritiana, and D. sechellia are all very closely related to D. melanogaster (estimated divergence about I million years), but D. yakuba is somewhat more distant (estimated divergence about 4 million years). All species exhibited one or more restriction fragments that hybridized with Dm2103 under conditions of moderate stringency (Fig. 7). To determine whether the euchromatic localization of T-elements is similar among related species, we also carried out in situ hybridization of Dm2103 with the polytene chromosomes of D. simulans and D. yakuba. In neither species did we observe hybridization to euchromatic sites other than chromosome tips (data not shown). With D. simuIans, the probe from Dm2103 hybridized in situ with the tips of all chromosomes except chromosome 4, and with D. yakuba, hybridization occurred only with the tips of chromosomes Jr" and 4 (data not shown). Transcription of T-elements RNA blots hybridized with probe from Dm2103 yielded weakly hybridizing bands with poly(A) + RNA isolated

13 >-

23.19.4-

6.64.4-

2.3-

2.0-

.~i!~~

ii ~

Fig. 7. Occurrence of T-element sequences among Drosophila species closely related to D. melanogaster. DNA samples from D. melanogaster (Mel), D. sechellia (Sech), D. simulans (Sim), D. mauritiana (Maur) and D. yakuba (Yak) were digested with HindIII + BglII, fractionated by electrophoresis, blotted and hybridized with 32p-labeled DNA from Dm2103

from pupae or adult flies but no detectable hybridization with poly(A) § RNA isolated from embryos or larvae (data not shown). Libraries of cDNAs in the vector 2gtl0 that were prepared from embryos, larvae, pupae or adults were also screened with the 5.7 kb EcoRI fragment from phage 223 (Fig. 1). The pupal cDNA library yielded nine positive clones, and the adult cDNA library yielded four positive clones, whereas the embryonic and larval libraries gave none (data not shown). Hybridization of individual cDNA clones with portions of the 5.7 kb EcoRI fragment homologous to Dm665 and Dm2103 indicated that all cDNA clones contained sequences homologous to Dm665 but only four contained sequences homologous to Din2103. Hence, the Dm2103related T-elements are apparently cotranscribed with the Dm665-1ike HeT sequences.

Discussion

Drosophila telomeres are associated with a heterogeneous collection of repetitive sequences designated the He-T family (Traverse and Pardue 1988), which is also present in/~-heterochromatin (Young et al. 1983; Pardue and Hennig 1990) and in extensive regions of the Y chromosome (Traverse and Pardue 1989; Danilevskaya et al. 1991). The most intriguing feature of the He-T family is its involvement in the healing of broken chromosome ends. Traverse and Pardue (1989) have demonstrated that the newly acquired telomeres of broken ring chromosomes contain He-T sequences, and Biessmann et al.

39 (1990) have shown that unstable terminal deletions of the X chromosome can be stabilized by spontaneous addition of He-T sequences. In the cases of chromosome healing analyzed in detail, a sequence with a consensus known as the HeT-A box was found at the junction between the broken chromosome and new sequences transposed to the end (Biessmann et al. 1990). These data suggest an important role for He-T family sequences in telomere structure and/or function. The nucleotide sequences of two fragments corresponding to I-Ie-T sequences have been reported: (1) the 4,826 bp )~T-A fragment, which most likely originates from near the tip of a wild-type X chromosome (Valgeirsdottir et al. 1990), and (2) the 2,443 bp sequence Dm665, which originates from an internal segment of the Y chromosome designated the " C " segment (Danilevskaya et al. 1991). The organization of 2T-A and Dm665 sequences in the genome suggests that the He-T family of sequences consists of a scrambled mosaic of different types of elements, at least some of which are transposable. However, no ORF is present in the 2T-A sequences, nor in Dm665, nor in the HeT-A box. While studying the telomere-specific clone Dm4568 described previously (Danilevskaya et al. 1984, 1991), we found an ORF in the subfragment designated Dm2103. This clone included part of an ORF in a repetitive family of sequences that we designate the T-elements. Screening a set of He-T-containing 2 clones with Dm2103 resulted in a number of clones containing longer T-element sequences. One T-element (T-23, from clone 223), was analyzed in detail and found to include two overlapping ORFs of 1,428 bp (ORF1) and 1,614 bp (ORF2) that were one nucleotide out of frame. The ORF2 contains a cysteine-histidine motif that is highly conserved in the Drosophila LINE-type retroposons G (Di Nocera 1988) and Jockey (Priimagi et al. 1988), as well as among retroviral gag proteins (Covey 1986). A very similar cysteine-histidine motif is also found in some other genes. For example, Rajavashisth et al. (1989) have described a protein that contains a virtually identical cysteine-histidine motif, which forms a zincfinger DNA binding domain that participates in the sterol-mediated repression of gene transcription in humans. In regard to the possibility that the T-elements are related to retroviruses or retroposons, it is noteworthy that we have not found any recognizable reverse transcriptase domain in the deduced amino acid sequence of T-23, even though this domain is the most conserved structure shared by all known retroviruses and retroposons. We also did not find a poly(A) tract at the 3' end of T-23, which is one distinguishing feature of LINE elements (Hutchison et al. 1989). It is, however, possible that T-23 comprises only part of a longer transposable element. Furthermore, there are several reasons for suggesting that T-23 and T-2103 (the portion of the T-element in plasmid Dm2103) may both be nonfunctional. First, both sequences contain an important deviation from the conserved cysteine-histidine motif: in particular, amino acid residue 263 in T-23 is Ser instead of Cys, and amino acid residue 289 in T-2103 is Asp instead of His. Secondly, in the 1,245 bp in the 3' portion of ORF2 in which

T-23 and T-2103 overlap, there are 83 nucleotide differences that are synonymous and 82 nucleotide differences that result in amino acid replacements; such a high ratio of synonymous to replacement differences suggests relaxation or elimination of selective constraints on amino acid sequence. Third, the clone 223 derives from the Y chromosome, which appears to contain many apparently degenerate copies of He-T sequences (Young et al. 1983; Danilevskaya et al. 1991). Of considerable interest is the apparent obligatory association between T-elements and the Dm665 class of He-T repeats. Sequences homologous to Din2103 occur along with sequences homologous to Dm665 in all relevant clones so far examined, including YAC, bacteriophage PI, phage 2 and plasmid clones. On the other hand, numerous clones have been found in each type of library that contain Dm665 sequences but not Din2103 sequences. Furthermore, cDNAs containing Telement sequences also contain Dm665 sequences, hence the sequences are co-transcribed. These observations suggest that T-elements are always, or almost always, embedded in blocks of other types of He-T sequences, although blocks of He-T sequences also exist that do not contain T-elements. Sequence families consisting of complex mosaics of various repeats are found adjacent to the telomeres of many eukaryotic chromosomes. One of the best studied examples is the family of Y' repeats in Saccharomyces cerevisiae. The Y' repeats are located just proximal to the functioning telomere, which is composed of (G1- 3T)n repeats. The complete sequence of Y' has been published only recently (Louis and Haber 1992) and shows some interesting similarities to the overall structure of the T-elements. For example, the reported sequence contains two overlapping ORFs of 1709 bp (ORF1) and 3914 bp (ORF2) that are one nucleotide out of frame. There is a potential transcription start site within 100 bp of the first ATG in ORF1, and RNAs homologous to ORF1 and ORF2 have been detected (Louis and Haber 1992). There are also potential splice sites in the sequences that, if used, would bring the two ORFs into frame, but hybridization using probes spanning the putative splice site does not reveal the lower molecular weight RNA fragments expected for spliced mRNA. The consensus sequence for the - 1 translational frameshifting that occurs in many viruses (Jacks et al. 1988; Icho and Wickner 1989) has not been found. The putative proteins show no strong homologies to known proteins, except for a region in ORF2 that has 27% similarity with RNA helicases (Louis and Haber 1992). In particular, neither ORF shows homology with reverse transcriptase or gag protein. The data are not inconsistent with Y' being a mobile genetic element, although all of the movements of the Y' sequences documented so far can be explained by (and sometimes require) recombination. The general similarity of the Y' repeats and the Telements raises the possibility of a common function in the genome. It has been shown that all the survivors of the mutation E S T I in S. cerevisiae, which results in defective telomere elongation (Lundblad and Szostak

40 1989), h a v e a c q u i r e d Y ' r e p e a t s a t the ends o f m a n y , if n o t all, c h r o m o s o m e s (see L o u i s a n d H a b e r 1992). T h e a d d e d Y ' sequences m a y p r o v i d e a s o r t o f b u f f e r p a r t i a l l y to offset the h a r m f u l effects o f p r o g r e s s i v e loss o f t e r m i n a l sequences d u r i n g each r o u n d o f r e p l i c a t i o n . This s i t u a t i o n is v e r y a n a l o g o u s to the b e h a v i o r o f the H e T sequences except that, in Drosophila, n o c o n v e n t i o n a l t e l o m e r a s e a c t i v i t y has y e t b e e n d e t e c t e d , a n d so p e r h a p s t h e H e - T sequences in Drosophila a r e even m o r e i m p o r t a n t as buffers n e a r the tips o f the c h r o m o s o m e s . In a n y event, the p o s s i b i l i t y t h a t T - e l e m e n t s m i g h t p l a y a role in the s t r u c t u r e a n d / o r f u n c t i o n o f t e l o m e r e s in Drosophila is s u g g e s t e d b y the f o l l o w i n g lines o f evid e n c e : (1) the l o c a t i o n o f T - e l e m e n t s at the tips o f c h r o m o s o m e s in D. melanogaster a n d r e l a t e d species; (2) the p r e s e n c e o f O R F s in the T - e l e m e n t s ; (3) the o c c u r r e n c e o f t r a n s c r i p t i o n o f T - e l e m e n t s ; a n d (4) the a p p a r e n t l y o b l i g a t o r y a s s o c i a t i o n o f T - e l e m e n t s w i t h H e T - A sequences.

Acknowledgements. We express our appreciation to the following individuals: V.G. Nikiforov for his interest; R. DuBose and J. Lawrence for computer analysis and stimulating discussions; D.V. Bebikhov, A.G. Veselkov and A.E.C. Vellek for technical help; and M.L. Pardue and V.G. Nikiforov for suggestions on the manuscript.

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Howe MM (eds) Mobile DNA. American Society for Microbiology, Washington, DC, pp 593-617 Icho T, Wickner RB (1989) The double-stranded RNA genome of yeast virus L-A encodes its own putative RNA polymerase by fusing two open reading frames. J Biol Chem 264:6716-6723 Jacks T, Madhani HD, Masiarz FR, Varmus HE (1988) Signals for ribosomal frameshifting in the Rous sarcoma virus gag-pol region. Cell 55:447-458 Jakubczak JL, Xiong Y, Eickbush TH (1990) Type I (RI) and type II (R2) ribosomal DNA insertion of Drosophila melanogaster are retrotransposable elements closely related to those of Bombyx mori. J Mol Biol 212:37-52 Johnson-Schlitz D, Lim JK (1987) Cytogenetics of Notch mutations arising in the unstable X chromosome Uc of Drosophila melanogaster. Genetics 115: 701-709 Lis JT, Simon JA, Sutton CA (1983) New heat shock puffs and beta-galactosidase activity resulting from transformation of Drosophila with an hsp70-lacZ hybrid gene. Cell 35:403-410 Louis E J, Haber JE (1992) The structure and evolution of subtelomeric Y' repeats in Saccharomyces cerevisiae. Genetics 131:559-574 Lundblad V, Szostak JW (1989) A mutant witt~ a defect in telomere elongation leads to senescence in yeast. Cell 57:633-643 Pardue ML, Hennig W (1990) Heterochromatin: Junk or collectors item? Chromosoma 100:3-7 Priimagi AF, Mizrokhi LJ, Ilyin JV (1988) The Drosophila mobile element Jockey belongs to LINEs and contains coding sequences homologous to some retroviral proteins. Gene 70:253262 Rajavashisth TB, Taylor AK, Andalibi A, Svenson KL, Lusis AJ (1989) Identification of a zinc finger protein that binds to the sterol regulatory element. Science 241 : 640-643 Renkawitz-Pohl R, Bialojan S (1984) A DNA sequence of Drosophila melanogaster with a different telomeric distribution. Chromosoma 89:206-211 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-termination inhibitors. Proc Natl Acad Sci USA 74: 5463-5467 Schwarz-Sommer Z, Leclercq L, Gobel E, Saedler H (1987) Cin4, an insert altering the structure of the A1 gene in Zea mays, exhibits properties of nonvirat retrotransposons. EMBO J 6:3873-3880 Smoller DA, Petrov D, Hartl DL (1991) Characterization of bacteriophage PI library contain inserts of Drosophila DNA of 75100 kilobase pairs. Chromosoma 100:487-494 Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mot Biol 98 : 503517 Traverse KL, Pardue ML (1988) A spontaneously opened ring chromosome of Drosophila metanogaster has acquired He-T DNA sequences at both new telomeres. Proc Natl Acad Sci USA 85:8116~8120 Traverse KL, Pardue ML (1989) Studies of He-T DNA sequences in the pericentric regions of Drosophila chromosomes. Chromosoma 97 : 261-271 Valgeirsdottir K, Traverse KL, Pardue ML (1990) He-T DNA: A family of mosaic repeated sequences specific for heterochromatin in Drosophila melanogaster. Proc Nat1 Acad Sci USA 87 : 7998-8002 Young BS, Pession A, Traverse KL, French C, Pardue ML (1983) Telomere regions in Drosophila share complex DNA sequences with pericentric heterochromatin. Cell 34:85 94

A repetitive DNA element, associated with telomeric sequences in Drosophila melanogaster, contains open reading frames.

He-T sequences are a complex repetitive family of DNA sequences in Drosophila that are associated with telomeric regions, pericentromeric heterochroma...
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