gb-2002-3-12-research0084 GBJ Research <p>The transposable elements of the <it>Drosophila melanogaster </it> euchromatin: a genomics perspective</p> Kaminker S Joshua Bergman M Casey Kronmiller Brent Carlson Joseph Svirskas Robert Patel Sandeep Frise Erwin Wheeler A David Lewis E Suzanna Rubin M Gerald Ashburner Michael ma11@gen.cam.ac.uk Celniker E Susan

Department of Molecular and Cellular Biology, University of California, Berkeley, CA 94720, USA

Drosophila Genome Project, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA

Amersham Biosciences, 2100 East Elliot Rd, Tempe, AZ 85284, USA

Howard Hughes Medical Institute

Human Genome Sequencing Center and Department of Molecular and Cell Biology, Baylor College of Medicine, Houston, TX 77030, USA

Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK

Current address: Department of Bioinformatics and Computational Biology, Iowa State University, Ames, IA 50011, USA

These authors contributed equally to this work

 Genome Biology 1465-6906 2002 3 12 research0084.1 0084.20 http://genomebiology.com/2002/3/12/research/0084 This article is part of a series of refereed research articles from Berkeley Drosophila Genome Project, FlyBase and colleagues, describing Release 3 of the Drosophila genome, which are freely available at http://genomebiology.com/drosophila/. 10.1186/gb-2002-3-12-research0084 12537573 7 10 2002 11 11 2002 25 11 2002 23 12 2002 2002 Kaminker et al., licensee BioMed Central Ltd

The transposable elements of the Drosophila melanogaster euchromatin: a genomics perspective

Using Release 3 of the euchromatic genomic sequence of Drosophila melanogaster, 85 known and eight novel families of transposable element have been identified, varying in copy number from one to 146. A total of 1,572 full and partial transposable elements were identified, comprising 3.86% of the sequence.

Abstract

Background

Transposable elements are found in the genomes of nearly all eukaryotes. The recent completion of the Release 3 euchromatic genomic sequence of Drosophila melanogaster by the Berkeley Drosophila Genome Project has provided precise sequence for the repetitive elements in the Drosophila euchromatin. We have used this genomic sequence to describe the euchromatic transposable elements in the sequenced strain of this species.

Results

We identified 85 known and eight novel families of transposable element varying in copy number from one to 146. A total of 1,572 full and partial transposable elements were identified, comprising 3.86% of the sequence. More than two-thirds of the transposable elements are partial. The density of transposable elements increases an average of 4.7 times in the centromere-proximal regions of each of the major chromosome arms. We found that transposable elements are preferentially found outside genes; only 436 of 1,572 transposable elements are contained within the 61.4 Mb of sequence that is annotated as being transcribed. A large proportion of transposable elements is found nested within other elements of the same or different classes. Lastly, an analysis of structural variation from different families reveals distinct patterns of deletion for elements belonging to different classes.

Conclusions

This analysis represents an initial characterization of the transposable elements in the Release 3 euchromatic genomic sequence of D. melanogaster for which comparison to the transposable elements of other organisms can begin to be made. These data have been made available on the Berkeley Drosophila Genome Project website for future analyses.

 Molecular biology Genome studies Model organisms Bioinformatics

Background

Transposable element sequences are abundant yet poorly understood components of almost all eukaryotic genomes [1]. As a result, many biologists have an interest in the description of transposable elements in completely sequenced eukaryotic genomes. The evolutionary biologist wants to understand the origin of transposable elements, how they are lost and gained by a species and the role they play in the processes of genome evolution; the population geneticist wants to know the factors that determine the frequency and distribution of elements within and between populations; the developmental geneticist wants to know what roles these elements may play in either normal developmental processes or in the response of the organism to external conditions; finally, the molecular geneticist wants to know the mechanisms that regulate the transposition cycle of these elements and how they interact with the cellular machinery of the host. It is for all of these reasons and more that a description of the transposable elements in the recently completed Release 3 genomic sequence of D. melanogaster is desirable.

Our understanding of transposable elements owes much to research on Drosophila. Over 75 years ago, Milislav Demerec discovered highly mutable alleles of two genes in D. virilis, miniature and magenta ([2,3,4], reviewed in [5]). Both genes were mutable in soma and germline and, for the miniature-3α alleles, dominant enhancers of mutability were also isolated by Demerec. In retrospect, it seems clear that the mutability of these alleles was the result of transposition of mobile elements. The dominant enhancers may have been particularly active elements or mutations in host genes that affect transposability (see below). There matters stood until McClintock's analysis of the Ac and Ds factors in maize, which led to the discovery of transposition [6] and the discovery of insertion elements in the gal operon of Escherichia coli (see [7]).

Green [8] synthesized the available evidence to make a strong case for insertion as a mechanism of mutagenesis in Drosophila. Concurrently, Hogness' group had begun a molecular characterization of two elements in D. melanogaster, 412 and copia [9,10] and provided evidence that they were transposable [11,12,13]. Glover [14] unknowingly characterized the first eukaryotic transposable element at the molecular level, the insertion sequences of 28 S rRNA genes. The discovery of male recombination [15], and two systems of hybrid dysgenesis in D. melanogaster (see [16,17]) bridged the gap between genetic and molecular analyses. The discovery of the transposable elements that cause hybrid dysgenesis, the P element [18] and the I element [19], led to the first genomic analyses of transposable elements in a eukaryote.

The publication of the Release 1 genomic sequence in March 2000 [20] and the Release 2 genomic sequence in October 2000 encouraged several studies on the genomic distribution and abundance of transposable elements in D. melanogaster [21,22,23,24,25]. Unfortunately, neither release was suitable for rigorous analysis of its transposable elements. In the whole-genome shotgun assembly process, repetitive sequences (including transposable elements) were masked by the SCREENER algorithm and remained as gaps between unitigs [26]. During the repeat-resolution phase of the whole-genome assembly, an attempt was made to fill these gaps. However, comparisons of small regions sequenced by the clone-by-clone approach versus the whole-genome shotgun method show that this process did not produce accurate sequences for transposable elements [26,27]. These results demonstrate that rigorous analyses of the transposable elements, or any other repetitive sequence, requires a sequence of higher quality, now publicly available as Release 3 [28]. For the first time, the nature, number and location of the transposable elements can reliably be analyzed in the euchromatin of D. melanogaster.

Results and discussion

Identification of known and novel transposable elements

Eukaryotic transposable elements are divided into those that transpose via an RNA intermediate, the retrotransposons (class I elements), and those that transpose by DNA excision and repair, the transposons (class II elements [1]). Within the retrotransposons, the major division is between those that possess long terminal repeats (LTR elements) and those that do not (LINE and SINE elements [29]). Among the transposons, the majority transpose via a DNA intermediate, encode their own transposase and are flanked by relatively short terminally inverted repeat structures (TIR elements). Foldback (FB) elements, which are characterized by their property of reannealing after denaturation with zero-order kinetics, are quite distinct from prototypical class I or II elements, and have been included in our analyses [30]. Other classes of repetitive elements, such as DINE-1 [31,32,33], which are structurally distinct from all other classes, have not been included in this study.

We used a criterion of greater than 90% identity over more than 50 base-pairs (bp) of sequence to assign individual elements to families (see Materials and methods for details; a classification is shown in the additional data available (see Additional data files)). Subsequently, in order to ensure proper inclusion of elements in appropriate families, we generated multiple alignments for all families of transposable element represented by multiple copies. This allowed us to identify and remove spurious hits to highly repetitive regions of the genome, and it also enabled us to distinguish sequences of closely related families that share extensive regions of similarity.

A summary by class of the total number of complete and partial transposable elements in the Release 3 Drosophila euchromatic sequence is presented in Table 1, and detailed results for individual families of transposable element are listed in Table 2. Including those described here, there are 96 known families of transposable elements in D. melanogaster: 49 LTR families, 27 LINE-like families, 19 TIR families and the FB family. We have identified 1,572 full or partial elements from 93 of these 96 families (Table 1). In total, 3.86% (4.5 Mb) of the Release 3 sequence is composed of transposable elements. Previous analysis of both the euchromatic and heterochromatic sequences has suggested that 9% of the Drosophila genome is composed of repetitive elements [34]. One reason for this difference may be that the proportion of transposable element sequences in heterochromatic regions is higher than the genomic average [22,35].

<p>Table 1</p>

An overview of the numbers of transposable elements in the euchromatic genome of D. melanogaster

Class

Arm

Total transposable element sequence (in bp)

% of arm

Total number of transposable elements

Number full length

% Full length

Number of transposable elements perMb in genome

Number of transposable elements perMb in proximal 2 Mb

All families

X

828,370

3.80

276

83

30.43

12.67

50

2L

878,471

3.95

305

100

32.79

13.73

58.5

2R

870,914

4.29

313

84

26.84

15.42

89

3L

938,947

4.02

288

100

34.72

12.33

66.5

3R

866,971

3.11

288

102

35.76

10.33

24.5

4

127,874

10.33

102

9

8.82

82.40

-

Total

4,511,547

1,572

478

Average

3.86

30.53

13.46

57.70

LTR

X

628,924

2.89

134

54

41.04

6.15

19.00

2L

603,536

2.72

127

67

52.76

5.72

20.00

2R

573,034

2.82

140

54

38.57

6.90

30.50

3L

618,441

2.65

117

58

49.57

5.01

24.00

3R

621,272

2.23

154

67

44.16

5.52

7.50

4

44,121

3.56

10

4

40.00

8.08

-

Total

3,089,328

682

304

Average

2.65

44.87

5.84

20.20

LINE-like

X

136,348

0.63

71

18

25.35

3.26

14.50

2L

185,499

0.83

98

20

20.41

4.41

18.50

2R

225,984

1.11

109

18

16.51

5.37

37.50

3L

251,077

1.08

106

27

25.47

4.54

24.00

3R

176,355

0.63

70

19

27.14

2.51

4.50

4

37,399

3.02

32

1

3.12

25.85

-

Total

1,012,662

486

103

Average

0.87

21.19

4.16

19.80

TIR

X

45,324

0.21

59

7

11.86

2.71

14.50

2L

82,761

0.37

76

11

14.47

3.42

18.00

2R

69,291

0.34

62

11

17.74

3.05

20.50

3L

52,743

0.23

57

12

21.05

2.44

16.50

3R

63,359

0.23

60

14

23.33

2.15

12.50

4

44,195

3.57

58

3

5.17

46.85

-

Total

357,673

372

58

Average

0.31

15.59

3.19

16.40

FB

X

17,774

0.08

12

4

33.33

0.55

2.00

2L

6,675

0.03

4

2

50.00

0.18

2.00

2R

2,605

0.01

2

1

50.00

0.1

0.50

3L

16,686

0.07

8

3

37.50

0.34

2.00

3R

5,985

0.02

4

2

50.00

0.14

0.00

4

2,159

0.17

2

1

50.00

1.62

-

Total

51,884

32

13

Average

0.04

40.62

0.27

1.30

For each class, the total numbers of each family of element, together with the numbers (and percentage of elements) that are full length is given for each chromosome arm. Column 3 gives the total base pairs contained within transposable elements, column 4 the percentage of each chromosome arm composed of transposable element sequences, column 8 the number of elements perMb, and column 9 the numbers of elements within the most proximal 2 Mb of each of the five major chromosome arms. Differences in density and amount of transposable element sequence were tested by binning major chromosomal arms into 50-kb windows and testing significance by Mann-Whitney U tests. The only significant difference (p < 0.05) observed, either including or excluding the proximal 2 Mb, was an increase in density and amount of sequence on the X chromosome relative to 3R.

 <p>Table 2</p>

The transposable elements of D. melanogaster

Class

Family

Canonical length

X

2L

2R

3L

3R

4

Total number

Number full length

Number partial

Number in proximal 2 Mb

Average pairwise distance

LTR

17.6

7439

2

0

3

5

2

0

12

7

5

4

0.006

1731

4648

1

0

0

0

1

0

2

1

1

1

0.000

297

6995

22

12

6

7

10

0

57

18

39

12

0.032

3S18

6126

4

0

1

1

0

0

6

4

2

3

0.075

412

7566

8

0

7

11

5

0

31

24

7

6

0.024

accord

7404

0

0

1

0

0

0

1

0

1

0

-

aurora

4263

0

0

2

1

0

0

3

1

2

3

0.074

blastopia

5034

5

2

7

1

2

0

17

13

4

4

0.016

blood

7410

1

11

2

3

5

0

22

22

0

6

0.001

Burdock

6411

2

4

4

0

3

0

13

7

6

4

0.002

Circe

6356

0

0

2

0

0

0

2

0

2

2

0.057

copia

5143

4

13

4

5

4

0

30

26

4

3

0.002

diver

6112

1

1

3

1

3

0

9

9

0

1

0.002

diver2

4917

0

4

3

2

0

0

9

0

9

9

0.032

Dm88

4558

0

0

2

0

30

0

32

0

32

2

0.015

frogger

2483

0

1

0

0

0

0

1

1

0

1

-

GATE

8507

1

0

16

0

0

3

20

0

20

17

0.077

gtwin

7411

2

2

0

2

0

0

6

2

4

3

0.038

gypsy

7469

0

1

1

0

0

0

2

1

1

1

0.000

gypsy2

6841

1

0

0

2

0

0

3

1

2

2

0.067

gypsy3

6973

0

0

2

0

0

0

2

1

1

2

0.038

gypsy4

6852

0

1

0

0

1

0

2

1

1

2

0.041

gypsy5

7369

1

0

0

1

0

0

2

1

1

0

0.005

gypsy6

7826

0

1

0

0

0

0

1

0

1

1

-

HMS-Beagle

7062

4

5

1

0

3

0

13

9

4

3

0.054

Idefix

7411

1

2

0

3

1

0

7

2

5

5

0.022

invader1

4032

0

0

4

3

18

1

26

1

25

7

0.023

invader2

5124

1

4

3

2

0

0

10

3

7

10

0.053

invader3

5484

2

5

2

2

5

0

16

3

13

5

0.044

invader4

3105

0

4

2

1

1

1

9

2

7

2

0.068

invader5

4038

1

4

0

1

0

0

6

0

6

6

0.068

McClintock

6450

0

0

0

1

0

1

2

2

0

1

0.002

mdg1

7480

5

2

9

6

3

0

25

13

12

2

0.012

mdg3

5519

3

5

2

2

4

0

16

8

8

8

0.009

micropia

5457

1

0

0

0

4

0

5

2

3

1

0.010

opus

7521

3

6

6

6

3

0

24

16

8

5

0.003

qbert

7650

0

0

1

0

0

0

1

1

0

1

-

Quasimodo

7387

2

7

0

4

1

0

14

5

9

5

0.016

roo

9092

35

22

31

31

27

0

146

58

88

22

0.012

rooA

7621

1

0

0

1

1

2

5

0

5

3

0.045

rover

7318

3

0

1

0

2

0

6

3

3

3

0.035

springer

7546

2

1

4

1

3

0

11

5

6

5

0.061

Stalker

7256

3

1

0

5

3

0

12

3

9

7

0.014

Stalker2

8119

4

0

5

2

1

1</