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Molecular organization of heterochromatin in malaria mosquitoes of the Anopheles maculipennis subgroup
Gene 448 (2009) 192–197
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Gene j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / g e n e
Molecular organization of heterochromatin in malaria mosquitoes of the Anopheles maculipennis subgroup Olga G. Grushko a,b, Maria V. Sharakhova c, Vladimir N. Stegnii a, Igor V. Sharakhov c,⁎ a b c
Research Institute of Biology and Biophysics, Tomsk State University, Lenin prospect 36, Tomsk, Russia Life Sciences Institute, University of Michigan, 210 Washtenaw Avenue, Ann Arbor, MI 48109-2216, USA Department of Entomology, 203 Fralin Life Science Institute, West Campus Drive, Virginia Tech, Blacksburg, VA 24061, USA
a r t i c l e
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Article history: Received 29 April 2009 Received in revised form 17 July 2009 Accepted 24 July 2009 Available online 5 August 2009 Received by I. King Jordan Keywords: Transposable elements Heterochromatic genes Repetitive DNA DNA turnover Polytene chromosomes Pericentric heterochromatin Anopheles maculipennis subgroup Malaria mosquito evolution
a b s t r a c t Although heterochromatin makes up a significant portion of the malaria mosquito genome, its organization, function, and evolution are poorly understood. Sibling species of the Anopheles maculipennis subgroup, the European malaria mosquitoes, are characterized by striking differences in the morphology of pericentric heterochromatin; however, the molecular basis for the rapid evolutionary transformation of heterochromatin is not known. This study reports an initial survey of the molecular organization of the pericentric heterochromatin in nonmodel species from the A. maculipennis subgroup. Molecular identity and chromosomal localization were established for short DNA fragments obtained by microdissection from the pericentric diffuse β-heterochromatin of A. atroparvus. Among 102 sequenced clones of the Atr2R library, twenty had sequence similarity to transposable elements (TEs) from the Anopheles gambiae and Aedes aegypti genomes. At least six protein-coding single-copy genes from A. gambiae and four single-copy genes from Drosophila melanogaster were homologous to eight clones from the library. Most of these conserved genes were heterochromatic in A. gambiae but euchromatic in D. melanogaster. The remaining 74 clones were characterized as noncoding repetitive DNA. Comparative chromosome mapping of twelve clones in the sibling species A. atroparvus and A. messeae demonstrated that the noncoding repetitive sequences and the TEs have undergone independent chromosome-specific and species-specific gains and losses in the morphologically different pericentric heterochromatic regions, in accordance with the “library model.” © 2009 Elsevier B.V. All rights reserved.
1. Introduction Мalaria mosquitoes, Anopheles, are genetically diverse and rapidly adapting infectious disease vectors; hence, any progress in understanding the structure and function of their genome is extremely important. Approximately 33% of the Anopheles genome consists of heterochromatin (Sharakhova et al., 2007). As in Drosophila, two morphological types of pericentric heterochromatin in polytene chromosomes have been described in Anopheles: condensed α- and diffuse β-heterochromatin (Stegnii and Sharakhova, 1991; Sharakhov et al., 2001). In Drosophila, the compact central part of the chromocenter (α-type) is enriched with satellite DNA, while the distal diffuse area (β-type) contains mostly transposable elements (TEs) (Miklos et al., 1988; Vaury et al., 1989). In addition,
Abbreviations: TEs, transposable elements; Atr2R, the library of DNA fragments from the pericentric β-heterochromatin of the 2R arm of A. atroparvus; DOP-PCR, degenerated oligonucleotide primed PCR; FISH, fluorescent in situ hybridization; GIRI, Genetic Information Research Institute; LTR, long terminal repeat; EST, expressed sequence tag. ⁎ Corresponding author. Tel.: +1 540 231 7316; fax: +1 540 231 7126. E-mail address: [email protected] (I.V. Sharakhov). 0378-1119/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2009.07.020
Drosophila melanogaster heterochromatin includes approximately 450 protein-coding genes, many of which are essential for viability (Hoskins et al., 2002; Smith et al., 2007). However, in Anopheles, the molecular content of heterochromatin is not well known, not to mention that the link between DNA content and heterochromatin structure has not been established. Malaria mosquito species of the Anopheles maculipennis subgroup are a good model system to study the molecular content of heterochromatin because pericentromeric regions of ovarian nurse cell polytene chromosomes do not form the chromocenter and have unambiguous interchromosomal and interspecific morphological differences. The shape of heterochromatin (amount of diffuse β-type and condensed α-type), as well as the relationship of the pericentric regions to the nuclear envelope (“strong” permanent attachment, “weak” contact by the β-heterochromatin fibers, or no visible connections), differs even among closely related homosequential species (Stegnii, 1987; Sharakhova et al., 1997). Heterochromatin is the most quickly evolving part of a eukaryotic genome because of the predominance of repetitive DNA sequences, which are capable of rapid changes. Species-specificity of the heterochromatin structure of mitotic chromosomes is associated with the species-specific composition of repetitive elements in
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mammals (Modi et al., 2003), insects (Ross et al., 1997), and plants (Vershinin et al., 1996). Therefore, structural rearrangements of heterochromatin of polytene chromosomes during diversification of the A. maculipennis subgroup most likely have been facilitated by rapid turnover of repetitive DNA sequences. To test this hypothesis, the library of DNA fragments from the pericentric β-heterochromatin of the 2R arm of A. atroparvus (Atr2R) was generated by chromosome microdissection, a cytogenetic technique that physically cuts a large section of a chromosome (Grushko et al., 2004). The microdissected DNA fragments were amplified by degenerated oligonucleotide primed PCR (DOP-PCR) and cloned in a plasmid vector. Comparative in situ hybridization of the region-specific DOP-PCR-based probes to the chromosomes of A. atroparvus and of the two other members of the A. maculipennis group, A. messeae and A. beklemishevi, detected interspecies and interchromosomal molecular homogeneity among morphologically different pericentric regions. It was unclear whether the structural rearrangement of heterochromatin within the A. maculipennis subgroup is a sequence-independent process, as the hybridization of the whole region-specific DOP-PCR-based probe with Anopheles chromosomes was not informative. Also, it was intriguing to determine if any of the Atr2R clones represent TEs and heterochromatic protein-coding genes. To address these questions, we attempted to map twelve individual clones of the polytene chromosomes of A. atroparvus and A. messeae — sibling species of the A. maculipennis subgroup. Although a partial in silico analysis of the library was performed (Grushko et al., 2004), it was limited to the comparative analysis with the euchromatic part of Anopheles gambiae and D. melanogaster genomes. In 2007, the A. gambiae heterochromatin assembly (Sharakhova et al., 2007), the annotated D. melanogaster heterochromatin assembly (Smith et al., 2007), and the Aedes aegypti genome sequence assembly became available (Nene et al., 2007). Therefore, we performed in silico analysis of all 102 sequenced DNA fragments from the Atr2R library, conducting searches of TE databases as well as TBLASTX against the A. gambiae and D. melanogaster genomes and search of tandem repeats within the fragments.
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2. Materials and methods 2.1. Mosquito collection and chromosome preparation Chromosomal spreads were prepared from ovaries, at the stage of active maturation of nurse cells, fixed in Carnoy fixative (96% ethanol and glacial acetic acid in a 3:1 ratio). Ovaries were isolated from females of the laboratory strain of A. atroparvus and the wild strain of A. messeae collected in the settlement of Krivosheino in the Tomsk region. Genomic DNA was extracted from larvae fixed in 96% ethanol (Bender et al., 1983). Polytene chromosome spreads were prepared according to the standard method (Sharakhov et al., 2002). 2.2. Fluorescent in situ hybridization (FISH) To map DNA clones to the polytene chromosomes of A. atroparvus and A. messeae, FISH was performed. Labeling and in situ hybridization procedures were conducted according to protocols described earlier (Grushko et al., 2004; Grushko et al., 2006). Detection was performed by use of Rhodamin-Avidin and Biotinylated Anti-Avidin (Vector Laboratories) kits. Chromosomes were stained with YOYO-1 (Sigma) and embedded into DABCO (Sigma) solution. Data analysis and recording were carried out by use of a Bio-Rad MRC 1024 Scanning Confocal (2 channel/LaserSharp 3.2 program/networked) System confocal microscope. 2.3. In silico analysis of the DNA sequences The GenBank accession numbers for all sequences analyzed in this paper are: DQ072281–DQ072378, FJ972616–FJ972618. A search for homology between the cloned fragments and the D. melanogaster genome was carried out using the TBLASTX program at FlyBase (FB2009_03, March 20, 2009, http://www.fruitfly.org). A search for homologous sequences in the A. gambiae genome was performed using the VectorBase TBLASTX program (AgamP3, Feb. 2006, genebuild: AgamP3.4, July, 2007, http://www.vectorbase.org/Tools/
Table 1 Homologies of DNA clones isolated from 2R heterochromatin of A. atroparvus to known mosquito transposable elements. Clone name
TE name [organism]
TE class
Similarity (GIRI) and/or E-value (TEfam)
Atr2R-50aa Atr2R-53 Atr2R-25b Atr2R-46a Atr2R-39
GYPSY4-I_AG [Anopheles gambiae] GYPSY42-I_AG [Anopheles gambiae] GYPSY61-I_AG [Anopheles gambiae] GYPSY7-I_AG [Anopheles gambiae] TF000083|Outcast_Ele6 [Anopheles gambiae] TF000172|Outcast_Ele7 [Aedes aegypti] TF000438|Pao_Bel_Ele195 [Aedes aegypti]
LTR/Gypsy LTR/Gypsy LTR/Gypsy LTR/Gypsy Non-LTR/Outcast Non-LTR/Outcast Bel/Pao-like LTR
0.7383 0.7455 0.6952 0.7381 9e− 13 1e− 12 3e− 24
TF000079|Jockey_Ele1 [Anopheles gambiae]
Non-LTR/Jockey
8e− 24
TF000083|Outcast_Ele6 [Anopheles gambiae] GYPSY59-I_AG [Anopheles gambiae] Mariner2_AG [Anopheles gambiae]
Non-LTR/Outcast LTR/Gypsy Tc1-like/Mariner
2e− 06 0.7215 0.4595
TF000555|Tc1_Ele7|MsqTc3 [Aedes aegypti] TF001005|Tc1_Ele8 [Aedes aegypti] TF000083|Outcast_Ele6 [Anopheles gambiae]
Tc1 “cut and paste DNA transposon” Tc1 “cut and paste DNA transposon” Non-LTR/Outcast
8e− 09 1e− 08 0.55
BEL9-I_AG [Anopheles gambiae] TF000393|Pao_Bel_Ele40 [Aedes aegypti] Mosqul_Aa2 [Aedes aegypti] TF000083|Outcast_Ele6 [Anopheles gambiae] AARA8_AG [Anopheles gambiae] I_ele33 [Aedes aegypti] I_Ele2 [Anopheles gambiae] AgaP8 [Anopheles gambiae] Pao_Bel_Ele214 [Aedes aegypti]
LTR/Bel Bel/Pao-like LTR Non-LTR/MosquI-Aa2 Non-LTR/Outcast Non-LTR Non-LTR/I Non-LTR/I P-like transposon/AgaP8 Bel/Pao-like LTR
0.4128 2e− 15 0.4167 5e− 19 0.6866 5e− 28 9e− 28 0.7283 4e− 08
Atr2R-5a Atr2R-85ab Atr2R-109a Atr2R-23 Atr2R-70b Atr2R-70aa Atr2R-90 Atr2R-64ba
Atr2R-118 Atr2R-28aa Atr2R-43a Atr2R-35 Atr2R-133
Atr2R-69b Atr2R-57bca a b
Clones determined in the previous study by Southern blot hybridization as conserved repetitive DNA (Grushko et al., 2004). Clones located in the same row share sequence similarity.
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BLAST/#) (Lawson et al., 2009). Homology to known mosquito TEs was identified by a CENSOR software tool (GIRI) (Kohany et al., 2006) and a TEfam program (http://tefam.biochem.vt.edu/tefam/index. php). A search for tandem repeats within the clones of the library was carried out using the Tandem Repeats Finder program (http:// tandem.bu.edu/trf/trf.html) (Benson, 1999). 3. Results and discussion
has members of TE classes that are common in the genome of the African malaria vector A. gambiae. We also analyzed the entire library for the presence of tandem repeats. Only two copies of a 43 bp tandem repeat were found within the Atr2R-57a clone. Therefore, we concluded that micro- and minisatellites are not common in the microdissected region of A. atroparvus and that the molecular content of β-heterochromatin is similar between a mosquito and a fruit fly (Miklos et al., 1988; Vaury et al., 1989; Smith et al., 2007).
3.1. Transposable elements (TE) in the A. atroparvus heterochromatin Sequencing and annotation of the A. gambiae and A. aegypti genomes (Holt et al., 2002; Nene et al., 2007) provided us with an opportunity to identify TEs in the Atr2R library. Similarities with known mosquito TEs were determined using the CENSOR software tool (Kohany et al., 2006) and the TEfam program (http://tefam. biochem.vt.edu/tefam/index.php) (Table 1). The majority of TEs identified in the library belong to long terminal repeat (LTR) retrotransposons of the Gypsy and Bel/Pao-like families. Their sequences were ~ 70% similar to those of the A. gambiae TEs. In addition, sequences homologous to Outcast, Jockey, MosquI-Aa2, and I Non-LTR retrotransposons were found among the clones. Two fragments (Atr2R-90 and Atr2R-64b) had 45% identity with the A. aegypti Tc1_Ele8 TE of the Tc1 “cut and paste” DNA transposon family, and another fragment (Atr2R-69b) had 73% identity with the P-like transposon AgaP8 of A. gambiae. Thus, the β-heterochromatin of the European malaria mosquito A. atroparvus
3.2. Evolutionary turnover of repetitive DNA sequences in the A. maculipennis subgroup To test our hypothesis of sequence-dependent heterochromatin formation, we mapped individual DNA fragments from the Atr2R heterochromatin library to the polytene chromosomes of A. atroparvus and A. messeae by FISH. Comparison between these two species was especially interesting because the pericentromeric regions of 2R in A. atroparvus consist of only diffuse β-heterochromatin, while pericentromeric regions of 2R in A. messeae have large blocks of compact α-heterochromatin surrounded by euchromatin. Other pericentromeric regions in A. messeae have more α-heterochromatin than pericentromeric regions in A. atroparvus. Twelve clones were selected based on their nonrelatedness by primary sequences and on their repetitive and conserved nature within the A. maculipennis subgroup, which was detected by Southern blot hybridizations of the library with labeled genomic DNA from A. atroparvus and A. messeae
Fig. 1. Localization of repetitive DNA sequences of clones Atr2R-73 and Atr2R-25a in pericentromeric heterochromatin of polytene chromosomes of A. atroparvus and A. messeae. A) Clone Atr2R-73 on A. atroparvus chromosomes; B) clone Atr2R-73 on A. messeae chromosomes; C) clone Atr2R-25a on A. atroparvus chromosomes; D) clone Atr2R-25a on A. messeae chromosomes. When the red signal overlaps with the green color in the highly condensed heterochromatin, it gives a yellow color. Arrows—labeled regions of chromosomes. X, 2L, 2R and 3R—chromosomal arms. Scale—10 μm.
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14C–15D). Сlone Atr2R-22 was found only in condensed αheterochromatin of chromosome 2 (subdivision 14C) in A. messeae; 3) Selective distribution (chromosome 2 and some other regions): clones Atr2R-25a and Atr2R-90 were localized in the heterochromatin regions of chromosomes X and 2 of A. atroparvus but not in 3. Clone Atr2R-118 was located in the pericentric heterochromatin of all three chromosomes of A. atroparvus, except heterochromatin of the 3L arm (subdivision 33C). Clone Atr2R-25a was localized in chromosomes 2 and 3 but not in chromosome X of A. messeae.
Fig. 2. Differences in structure and molecular composition among regions of pericentromeric heterochromatin of sibling species A. atroparvus and A. messeae. Black bars—compact α-heterochromatin blocks; grey shading—diffuse β-heterochromatin; zigzag structures—the nuclear envelope attachment regions.
(Grushko et al., 2004). FISH revealed several possible patterns of chromosomal localization (Figs. 1 and 2):
1) Universal distribution (all three chromosomes): clone Atr2R-136 was detected in all pericentric regions of each chromosome in A. atroparvus. Clone Atr2R-73 was detected in condensed α-heterochromatin of each chromosome in A. messeae. 2) Chromosome-specific distribution (chromosome 2 оnly): in A. atroparvus, clones Atr2R-46a, Atr2R-73, and Atr2R-85a were found only in pericentric β-heterochromatin chromosome 2 (subdivision
No obvious correlation was detected between the type of labeled heterochromatin and the molecular nature of the hybridizing clones: the same clones hybridized to both α- and β-types within the same species (Atr2R-136 in A. atroparvus) or to only one type in one and different type(s) in another species. For example, clone Atr2R-73 was localized in pericentric β-heterochromatin of chromosome 2 in A. atroparvus and in condensed α-heterochromatin of each chromosome in A. messeae (Fig. 1A, B). Clone Atr2R-25a was localized in both types of heterochromatin of chromosomes X and 2 in A. atroparvus, but only in β-heterochromatin in the 3R arm and in euchromatin of chromosome 2 in A. messeae (subdivision 14C) (Fig. 1C, D). Overall, hybridization with A. atroparvus chromosomes was stronger than hybridization with A. messeae chromosomes. Nevertheless, the comparative physical mapping identified interspecies differences between A. messeae and A. atroparvus in the distribution of repetitive sequences in the pericentric heterochromatin (Figs. 1 and 2). Clone Atr2R-22 hybridized to α-type heterochromatin of chromosome 2 in A. messeae but did not hybridize to A. atroparvus chromosomes, where its presence was confirmed by Southern blot hybridization (Grushko et al, 2004). Clones Atr2R-68, Atr2R-43, and Atr2R-42 did not give any signal in any species, while a positive control in the same experiment (Atr2R-73) did. Obviously, factors such as copy number and representation in polytene chromosome are also important for detecting homologous sequences by FISH.
Table 2 Homologies of DNA clones isolated from 2R heterochromatin of A. atroparvus to the A. gambiae and D. melanogaster genes. TBLASTX of the A. gambiae genome (VectorBase assembly: AgamP3, Feb. 2006, genebuild: AgamP3.4, July, 2007)
TBLASTX of the D. melanogaster genome (FB2009_03, March 20, 2009)
Ensembl Gene ID E-value
Chromosome location Coordinates Chromatin type
FlyBase ID Gene name E-value
Chromosome location Coordinates Chromatin type
GO terms
Atr2R-6a Atr2R-4
AGAP004696 3e− 55
2L:20C 1,926,520–1,965,505 Pericentric heterochromatin
FBgn0000611 Extradenticle 5.3e− 42
X:14A5 15,886,520–15,890,040 Euchromatin
Atr2R-71a
AGAP004672 2e− 10
2R:19D 61,246,251–61,247,458 Pericentric heterochromatin
FBgn0030205 CG17255 5.8e− 10
X:9C 10,357,851–10,367,618 Euchromatin
Atr2R-107
AGAP004707 7e− 13
2L:20C 2,358,158–2,431,617 Pericentric heterochromatin-proximal euchromatin boundary
FBgn0003036 paralytic 4.1e− 06
X:14C 16,357,638–16,421,448 Euchromatin
Atr2R-33 Atr2R-138
AGAP004733 2e− 35
2L:20C 2,917,655–2,919,864 Proximal euchromatin
FBgn0030417 CG15725-PA 3.6e− 25
X:11B 12,600,231–12,602,603 Euchromatin
Atr2R-64a
AGAP003510 6e− 11
2R:15D 38,955,964–38,958,261 Euchromatin
No similarity
No similarity
Atr2R-147b
AGAP009302 7e− 04
3R:34A 30,920,086–30,923,139 Euchromatin
No similarity
No similarity
Molecular function: DNA binding, protein binding, sequence-specific DNA binding, transcription factor activity Biological process: brain development, oenocyte development, DNA-dependent regulation of transcription Cellular component: nucleus Molecular function: protein binding, ATP binding Biological process: protein folding Cellular component: cytoplasm Molecular function: voltage-gated sodium channel activity Biological process: response to DDT, response to pyrethroid, sodium ion transport Cellular component: voltage-gated sodium channel complex Molecular function: protein binding, ATP binding, zinc ion binding Biological process: protein folding Cellular component: intracellular Molecular function: not known Biological process: not known Cellular component: not known Molecular function: DNA binding, sequence-specific DNA binding, transcription factor activity Biological process: DNA-dependent regulation of transcription Cellular component: nucleus
Clone name
a
Clones located in the same row share sequence similarity.
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Our data strongly indicate that heterochromatic regions contain species- and chromosome-specific pools of conserved repetitive DNA. The rapid evolutionary turnover of repetitive DNA sequences between the two sibling species of the A. maculipennis subgroup suggests that these elements are still active. We conclude that repetitive elements underwent independent amplification and loss in the pericentric heterochromatin regions of the A. maculipennis species subgroup according to the “library model” (Ugarkovic and Plohl, 2002). This model states that different satellite or TE families can be preferentially amplified or lost in the derived chromosomes, therefore, changing the relative contribution of each family to the pericentromeric heterochromatin. Thus, the availability of polytene chromosomes in anopheline ovarian nurse cells provide an opportunity to link the molecular organization of heterochromatin to dramatic variations in its structure among sibling species of the A. maculipennis subgroup. More sequencing data are needed to link specific molecular content with a certain heterochromatin type.
and demonstrates rapid heterochromatin remodeling on a short evolutionary scale. Because heterochromatin is predominantly formed by various repetitive DNA sequences, which are difficult to clone and to map, deciphering its DNA content requires utilizing nontraditional approaches, such as microdissection. In this study, microdissection proved to be a useful tool for obtaining and characterizing DNA sequences from heterochromatin of polytene chromosomes and for revealing evolutionary dynamics of homologous sequences. Thus, this approach can be applied for initial evaluation of DNA content in cytologically interesting chromosomal regions of nonmodel organisms. Our analysis determined that heterochromatin of a European malarial mosquito, A. atroparvus, contains noncoding repetitive DNA, TEs, and sequences with high similarity to protein-coding genes. Noncoding repetitive sequences and TEs underwent chromosome-specific and species-specific gains and losses in the morphologically different pericentric heterochromatic regions of sibling species, in accordance with the “library model.”
3.3. Conserved protein-coding genes in the mosquito heterochromatin A search for similarity between the Atr2R cloned fragments and genomes of A. gambiae and D. melanogaster was carried out by TBLASTX. Clones Atr2R-4, Atr2R-6, Atr2R-33, Atr2R-138, Atr2R-107, Atr2R-71a, Atr2R-147b, and Atr2R-64a were similar to protein-coding DNA sequences. Similarity to sequences of A. gambiae was higher than to the corresponding sequences of D. melanogaster (Table 2). Five clones (Atr2R-4, Atr2R-6, Atr2R-33, Atr2R-138, and Atr2R-107) were similar to three single-copy genes in pericentromeric subdivision 20C of chromosome 2L of A. gambiae, suggesting the preservation of a microsynteny. In contrast, the orthologous genes in D. melanogaster were found in different subdivisions of the X chromosome euchromatin. Fragments Atr2R-4 and Atr2R-6 were similar to a single-copy A. gambiae gene, AGAP004696, and to a single-copy D. melanogaster gene, extradenticle. In D. melanogaster, this gene encodes a homeobox-containing transcription factor involved in developmental regulation. Clones Atr2R-33 and Atr2R-138 had similarity to a single-copy gene important for protein folding in A. gambiae and D. melanogaster. Fragment Atr2R-107 was similar to the voltage-gated sodium channel gene in A. gambiae and to the paralytic gene in D. melanogaster. This mosquito heterochromatic gene has implications for development of insecticide resistance and, thus, for vector control. Clone Atr2R-71a had similarity to a single-copy A. gambiae heterochromatic gene, AGAP004672, and to the single-copy D. melanogaster euchromatic gene, CG1725, which encodes a product important for protein folding. A single-copy A. gambiae euchromatic gene, AGAP009302, which does not have an orthologue in Drosophila, encodes for a transcription factor and had similarity to heterochromatic fragment Atr2R-147b. Another A. gambiae euchromatic gene, AGAP003510, was similar to heterochromatic clone Atr2R-64a. Four of six A. gambiae genes homologous to the Atr2R clones are single-copy and located in heterochromatic regions. All their known Drosophila orthologues are also single-copy and vitally important; however, they reside in euchromatin rather than heterochromatin. According to Vector Base, all A. gambiae heterochromatic genes and one euchromatic gene AGAP003510 have expressed sequence tag (EST) evidence. Interestingly, heterochromatic localization of the light gene cluster remains conserved only among species of the D. melanogaster subgroup but not in D. virilis, D. pseudoobscura, or D. ananassae (Yasuhara et al., 2005), suggesting frequent gene transpositions between euchromatin and heterochromatin. Location of these single-copy genes, especially genes coding for transcription factors, in the heterochromatin urges further investigation of their regulation in a repressive environment. 4. Conclusions This paper provides the first description of the molecular content in the heterochromatin of the European malaria mosquito
Acknowledgements This work was supported by the Agricultural Experimental Station at Virginia Tech, by a Grant of the President of the Russian Federation supporting the leading scientific schools (grant no. NSh-15.2003.4), the Russian State Foundation for Basic Research (grant nos. 01-0449712 and 02-04-4817), and by the Russian State Program “Integration” (grant no. E0371+E0206). We thank Prof. Nora Besansky for providing us with access to the confocal microscope and with FISH reagents. We also thank Raquel Assis, Melissa Wade, and Janet B. Webster, Ph.D., for correction of the English text.
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O.G. Grushko et al. / Gene 448 (2009) 192–197 Smith, C.D., Shu, S., Mungall, C.J., Karpen, G.H., 2007. The Release 5.1 annotation of Drosophila melanogaster heterochromatin. Science 316, 1586–1591. Stegnii, V.N., 1987. Systemic reorganization of the architectonics of polytene chromosomes in the onto- and phylogenesis of malaria mosquitoes. II. Species specificity in the pattern of chromosome relations with the nuclear envelope of nutrient ovarian cells. Genetika 23, 1194–1199. Stegnii, V.N., Sharakhova, M.V., 1991. Systemic reorganization of the architechtonics of polytene chromosomes in onto- and phylogenesis of malaria mosquitoes. Structural features regional of chromosomal adhesion to the nuclear membrane. Genetika 27, 828–835.
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Ugarkovic, D., Plohl, M., 2002. Variation in satellite DNA profiles—causes and effects. EMBO J. 21, 5955–5959. Vaury, C., Bucheton, A., Pelisson, A., 1989. The beta heterochromatic sequences flanking the I elements are themselves defective transposable elements. Chromosoma 98, 215–224. Vershinin, A.V., Alkhimova, E.G., Heslop-Harrison, J.S., 1996. Molecular diversification of tandemly organized DNA sequences and heterochromatic chromosome regions in some Triticeae species. Chromosome Res. 4, 517–525. Yasuhara, J.C., DeCrease, C.H., Wakimoto, B.T., 2005. Evolution of heterochromatic genes of Drosophila. Proc. Natl. Acad. Sci. U. S. A. 102, 10958–10963.
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Gene j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / g e n e
Molecular organization of heterochromatin in malaria mosquitoes of the Anopheles maculipennis subgroup Olga G. Grushko a,b, Maria V. Sharakhova c, Vladimir N. Stegnii a, Igor V. Sharakhov c,⁎ a b c
Research Institute of Biology and Biophysics, Tomsk State University, Lenin prospect 36, Tomsk, Russia Life Sciences Institute, University of Michigan, 210 Washtenaw Avenue, Ann Arbor, MI 48109-2216, USA Department of Entomology, 203 Fralin Life Science Institute, West Campus Drive, Virginia Tech, Blacksburg, VA 24061, USA
a r t i c l e
i n f o
Article history: Received 29 April 2009 Received in revised form 17 July 2009 Accepted 24 July 2009 Available online 5 August 2009 Received by I. King Jordan Keywords: Transposable elements Heterochromatic genes Repetitive DNA DNA turnover Polytene chromosomes Pericentric heterochromatin Anopheles maculipennis subgroup Malaria mosquito evolution
a b s t r a c t Although heterochromatin makes up a significant portion of the malaria mosquito genome, its organization, function, and evolution are poorly understood. Sibling species of the Anopheles maculipennis subgroup, the European malaria mosquitoes, are characterized by striking differences in the morphology of pericentric heterochromatin; however, the molecular basis for the rapid evolutionary transformation of heterochromatin is not known. This study reports an initial survey of the molecular organization of the pericentric heterochromatin in nonmodel species from the A. maculipennis subgroup. Molecular identity and chromosomal localization were established for short DNA fragments obtained by microdissection from the pericentric diffuse β-heterochromatin of A. atroparvus. Among 102 sequenced clones of the Atr2R library, twenty had sequence similarity to transposable elements (TEs) from the Anopheles gambiae and Aedes aegypti genomes. At least six protein-coding single-copy genes from A. gambiae and four single-copy genes from Drosophila melanogaster were homologous to eight clones from the library. Most of these conserved genes were heterochromatic in A. gambiae but euchromatic in D. melanogaster. The remaining 74 clones were characterized as noncoding repetitive DNA. Comparative chromosome mapping of twelve clones in the sibling species A. atroparvus and A. messeae demonstrated that the noncoding repetitive sequences and the TEs have undergone independent chromosome-specific and species-specific gains and losses in the morphologically different pericentric heterochromatic regions, in accordance with the “library model.” © 2009 Elsevier B.V. All rights reserved.
1. Introduction Мalaria mosquitoes, Anopheles, are genetically diverse and rapidly adapting infectious disease vectors; hence, any progress in understanding the structure and function of their genome is extremely important. Approximately 33% of the Anopheles genome consists of heterochromatin (Sharakhova et al., 2007). As in Drosophila, two morphological types of pericentric heterochromatin in polytene chromosomes have been described in Anopheles: condensed α- and diffuse β-heterochromatin (Stegnii and Sharakhova, 1991; Sharakhov et al., 2001). In Drosophila, the compact central part of the chromocenter (α-type) is enriched with satellite DNA, while the distal diffuse area (β-type) contains mostly transposable elements (TEs) (Miklos et al., 1988; Vaury et al., 1989). In addition,
Abbreviations: TEs, transposable elements; Atr2R, the library of DNA fragments from the pericentric β-heterochromatin of the 2R arm of A. atroparvus; DOP-PCR, degenerated oligonucleotide primed PCR; FISH, fluorescent in situ hybridization; GIRI, Genetic Information Research Institute; LTR, long terminal repeat; EST, expressed sequence tag. ⁎ Corresponding author. Tel.: +1 540 231 7316; fax: +1 540 231 7126. E-mail address: [email protected] (I.V. Sharakhov). 0378-1119/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2009.07.020
Drosophila melanogaster heterochromatin includes approximately 450 protein-coding genes, many of which are essential for viability (Hoskins et al., 2002; Smith et al., 2007). However, in Anopheles, the molecular content of heterochromatin is not well known, not to mention that the link between DNA content and heterochromatin structure has not been established. Malaria mosquito species of the Anopheles maculipennis subgroup are a good model system to study the molecular content of heterochromatin because pericentromeric regions of ovarian nurse cell polytene chromosomes do not form the chromocenter and have unambiguous interchromosomal and interspecific morphological differences. The shape of heterochromatin (amount of diffuse β-type and condensed α-type), as well as the relationship of the pericentric regions to the nuclear envelope (“strong” permanent attachment, “weak” contact by the β-heterochromatin fibers, or no visible connections), differs even among closely related homosequential species (Stegnii, 1987; Sharakhova et al., 1997). Heterochromatin is the most quickly evolving part of a eukaryotic genome because of the predominance of repetitive DNA sequences, which are capable of rapid changes. Species-specificity of the heterochromatin structure of mitotic chromosomes is associated with the species-specific composition of repetitive elements in
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mammals (Modi et al., 2003), insects (Ross et al., 1997), and plants (Vershinin et al., 1996). Therefore, structural rearrangements of heterochromatin of polytene chromosomes during diversification of the A. maculipennis subgroup most likely have been facilitated by rapid turnover of repetitive DNA sequences. To test this hypothesis, the library of DNA fragments from the pericentric β-heterochromatin of the 2R arm of A. atroparvus (Atr2R) was generated by chromosome microdissection, a cytogenetic technique that physically cuts a large section of a chromosome (Grushko et al., 2004). The microdissected DNA fragments were amplified by degenerated oligonucleotide primed PCR (DOP-PCR) and cloned in a plasmid vector. Comparative in situ hybridization of the region-specific DOP-PCR-based probes to the chromosomes of A. atroparvus and of the two other members of the A. maculipennis group, A. messeae and A. beklemishevi, detected interspecies and interchromosomal molecular homogeneity among morphologically different pericentric regions. It was unclear whether the structural rearrangement of heterochromatin within the A. maculipennis subgroup is a sequence-independent process, as the hybridization of the whole region-specific DOP-PCR-based probe with Anopheles chromosomes was not informative. Also, it was intriguing to determine if any of the Atr2R clones represent TEs and heterochromatic protein-coding genes. To address these questions, we attempted to map twelve individual clones of the polytene chromosomes of A. atroparvus and A. messeae — sibling species of the A. maculipennis subgroup. Although a partial in silico analysis of the library was performed (Grushko et al., 2004), it was limited to the comparative analysis with the euchromatic part of Anopheles gambiae and D. melanogaster genomes. In 2007, the A. gambiae heterochromatin assembly (Sharakhova et al., 2007), the annotated D. melanogaster heterochromatin assembly (Smith et al., 2007), and the Aedes aegypti genome sequence assembly became available (Nene et al., 2007). Therefore, we performed in silico analysis of all 102 sequenced DNA fragments from the Atr2R library, conducting searches of TE databases as well as TBLASTX against the A. gambiae and D. melanogaster genomes and search of tandem repeats within the fragments.
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2. Materials and methods 2.1. Mosquito collection and chromosome preparation Chromosomal spreads were prepared from ovaries, at the stage of active maturation of nurse cells, fixed in Carnoy fixative (96% ethanol and glacial acetic acid in a 3:1 ratio). Ovaries were isolated from females of the laboratory strain of A. atroparvus and the wild strain of A. messeae collected in the settlement of Krivosheino in the Tomsk region. Genomic DNA was extracted from larvae fixed in 96% ethanol (Bender et al., 1983). Polytene chromosome spreads were prepared according to the standard method (Sharakhov et al., 2002). 2.2. Fluorescent in situ hybridization (FISH) To map DNA clones to the polytene chromosomes of A. atroparvus and A. messeae, FISH was performed. Labeling and in situ hybridization procedures were conducted according to protocols described earlier (Grushko et al., 2004; Grushko et al., 2006). Detection was performed by use of Rhodamin-Avidin and Biotinylated Anti-Avidin (Vector Laboratories) kits. Chromosomes were stained with YOYO-1 (Sigma) and embedded into DABCO (Sigma) solution. Data analysis and recording were carried out by use of a Bio-Rad MRC 1024 Scanning Confocal (2 channel/LaserSharp 3.2 program/networked) System confocal microscope. 2.3. In silico analysis of the DNA sequences The GenBank accession numbers for all sequences analyzed in this paper are: DQ072281–DQ072378, FJ972616–FJ972618. A search for homology between the cloned fragments and the D. melanogaster genome was carried out using the TBLASTX program at FlyBase (FB2009_03, March 20, 2009, http://www.fruitfly.org). A search for homologous sequences in the A. gambiae genome was performed using the VectorBase TBLASTX program (AgamP3, Feb. 2006, genebuild: AgamP3.4, July, 2007, http://www.vectorbase.org/Tools/
Table 1 Homologies of DNA clones isolated from 2R heterochromatin of A. atroparvus to known mosquito transposable elements. Clone name
TE name [organism]
TE class
Similarity (GIRI) and/or E-value (TEfam)
Atr2R-50aa Atr2R-53 Atr2R-25b Atr2R-46a Atr2R-39
GYPSY4-I_AG [Anopheles gambiae] GYPSY42-I_AG [Anopheles gambiae] GYPSY61-I_AG [Anopheles gambiae] GYPSY7-I_AG [Anopheles gambiae] TF000083|Outcast_Ele6 [Anopheles gambiae] TF000172|Outcast_Ele7 [Aedes aegypti] TF000438|Pao_Bel_Ele195 [Aedes aegypti]
LTR/Gypsy LTR/Gypsy LTR/Gypsy LTR/Gypsy Non-LTR/Outcast Non-LTR/Outcast Bel/Pao-like LTR
0.7383 0.7455 0.6952 0.7381 9e− 13 1e− 12 3e− 24
TF000079|Jockey_Ele1 [Anopheles gambiae]
Non-LTR/Jockey
8e− 24
TF000083|Outcast_Ele6 [Anopheles gambiae] GYPSY59-I_AG [Anopheles gambiae] Mariner2_AG [Anopheles gambiae]
Non-LTR/Outcast LTR/Gypsy Tc1-like/Mariner
2e− 06 0.7215 0.4595
TF000555|Tc1_Ele7|MsqTc3 [Aedes aegypti] TF001005|Tc1_Ele8 [Aedes aegypti] TF000083|Outcast_Ele6 [Anopheles gambiae]
Tc1 “cut and paste DNA transposon” Tc1 “cut and paste DNA transposon” Non-LTR/Outcast
8e− 09 1e− 08 0.55
BEL9-I_AG [Anopheles gambiae] TF000393|Pao_Bel_Ele40 [Aedes aegypti] Mosqul_Aa2 [Aedes aegypti] TF000083|Outcast_Ele6 [Anopheles gambiae] AARA8_AG [Anopheles gambiae] I_ele33 [Aedes aegypti] I_Ele2 [Anopheles gambiae] AgaP8 [Anopheles gambiae] Pao_Bel_Ele214 [Aedes aegypti]
LTR/Bel Bel/Pao-like LTR Non-LTR/MosquI-Aa2 Non-LTR/Outcast Non-LTR Non-LTR/I Non-LTR/I P-like transposon/AgaP8 Bel/Pao-like LTR
0.4128 2e− 15 0.4167 5e− 19 0.6866 5e− 28 9e− 28 0.7283 4e− 08
Atr2R-5a Atr2R-85ab Atr2R-109a Atr2R-23 Atr2R-70b Atr2R-70aa Atr2R-90 Atr2R-64ba
Atr2R-118 Atr2R-28aa Atr2R-43a Atr2R-35 Atr2R-133
Atr2R-69b Atr2R-57bca a b
Clones determined in the previous study by Southern blot hybridization as conserved repetitive DNA (Grushko et al., 2004). Clones located in the same row share sequence similarity.
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BLAST/#) (Lawson et al., 2009). Homology to known mosquito TEs was identified by a CENSOR software tool (GIRI) (Kohany et al., 2006) and a TEfam program (http://tefam.biochem.vt.edu/tefam/index. php). A search for tandem repeats within the clones of the library was carried out using the Tandem Repeats Finder program (http:// tandem.bu.edu/trf/trf.html) (Benson, 1999). 3. Results and discussion
has members of TE classes that are common in the genome of the African malaria vector A. gambiae. We also analyzed the entire library for the presence of tandem repeats. Only two copies of a 43 bp tandem repeat were found within the Atr2R-57a clone. Therefore, we concluded that micro- and minisatellites are not common in the microdissected region of A. atroparvus and that the molecular content of β-heterochromatin is similar between a mosquito and a fruit fly (Miklos et al., 1988; Vaury et al., 1989; Smith et al., 2007).
3.1. Transposable elements (TE) in the A. atroparvus heterochromatin Sequencing and annotation of the A. gambiae and A. aegypti genomes (Holt et al., 2002; Nene et al., 2007) provided us with an opportunity to identify TEs in the Atr2R library. Similarities with known mosquito TEs were determined using the CENSOR software tool (Kohany et al., 2006) and the TEfam program (http://tefam. biochem.vt.edu/tefam/index.php) (Table 1). The majority of TEs identified in the library belong to long terminal repeat (LTR) retrotransposons of the Gypsy and Bel/Pao-like families. Their sequences were ~ 70% similar to those of the A. gambiae TEs. In addition, sequences homologous to Outcast, Jockey, MosquI-Aa2, and I Non-LTR retrotransposons were found among the clones. Two fragments (Atr2R-90 and Atr2R-64b) had 45% identity with the A. aegypti Tc1_Ele8 TE of the Tc1 “cut and paste” DNA transposon family, and another fragment (Atr2R-69b) had 73% identity with the P-like transposon AgaP8 of A. gambiae. Thus, the β-heterochromatin of the European malaria mosquito A. atroparvus
3.2. Evolutionary turnover of repetitive DNA sequences in the A. maculipennis subgroup To test our hypothesis of sequence-dependent heterochromatin formation, we mapped individual DNA fragments from the Atr2R heterochromatin library to the polytene chromosomes of A. atroparvus and A. messeae by FISH. Comparison between these two species was especially interesting because the pericentromeric regions of 2R in A. atroparvus consist of only diffuse β-heterochromatin, while pericentromeric regions of 2R in A. messeae have large blocks of compact α-heterochromatin surrounded by euchromatin. Other pericentromeric regions in A. messeae have more α-heterochromatin than pericentromeric regions in A. atroparvus. Twelve clones were selected based on their nonrelatedness by primary sequences and on their repetitive and conserved nature within the A. maculipennis subgroup, which was detected by Southern blot hybridizations of the library with labeled genomic DNA from A. atroparvus and A. messeae
Fig. 1. Localization of repetitive DNA sequences of clones Atr2R-73 and Atr2R-25a in pericentromeric heterochromatin of polytene chromosomes of A. atroparvus and A. messeae. A) Clone Atr2R-73 on A. atroparvus chromosomes; B) clone Atr2R-73 on A. messeae chromosomes; C) clone Atr2R-25a on A. atroparvus chromosomes; D) clone Atr2R-25a on A. messeae chromosomes. When the red signal overlaps with the green color in the highly condensed heterochromatin, it gives a yellow color. Arrows—labeled regions of chromosomes. X, 2L, 2R and 3R—chromosomal arms. Scale—10 μm.
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14C–15D). Сlone Atr2R-22 was found only in condensed αheterochromatin of chromosome 2 (subdivision 14C) in A. messeae; 3) Selective distribution (chromosome 2 and some other regions): clones Atr2R-25a and Atr2R-90 were localized in the heterochromatin regions of chromosomes X and 2 of A. atroparvus but not in 3. Clone Atr2R-118 was located in the pericentric heterochromatin of all three chromosomes of A. atroparvus, except heterochromatin of the 3L arm (subdivision 33C). Clone Atr2R-25a was localized in chromosomes 2 and 3 but not in chromosome X of A. messeae.
Fig. 2. Differences in structure and molecular composition among regions of pericentromeric heterochromatin of sibling species A. atroparvus and A. messeae. Black bars—compact α-heterochromatin blocks; grey shading—diffuse β-heterochromatin; zigzag structures—the nuclear envelope attachment regions.
(Grushko et al., 2004). FISH revealed several possible patterns of chromosomal localization (Figs. 1 and 2):
1) Universal distribution (all three chromosomes): clone Atr2R-136 was detected in all pericentric regions of each chromosome in A. atroparvus. Clone Atr2R-73 was detected in condensed α-heterochromatin of each chromosome in A. messeae. 2) Chromosome-specific distribution (chromosome 2 оnly): in A. atroparvus, clones Atr2R-46a, Atr2R-73, and Atr2R-85a were found only in pericentric β-heterochromatin chromosome 2 (subdivision
No obvious correlation was detected between the type of labeled heterochromatin and the molecular nature of the hybridizing clones: the same clones hybridized to both α- and β-types within the same species (Atr2R-136 in A. atroparvus) or to only one type in one and different type(s) in another species. For example, clone Atr2R-73 was localized in pericentric β-heterochromatin of chromosome 2 in A. atroparvus and in condensed α-heterochromatin of each chromosome in A. messeae (Fig. 1A, B). Clone Atr2R-25a was localized in both types of heterochromatin of chromosomes X and 2 in A. atroparvus, but only in β-heterochromatin in the 3R arm and in euchromatin of chromosome 2 in A. messeae (subdivision 14C) (Fig. 1C, D). Overall, hybridization with A. atroparvus chromosomes was stronger than hybridization with A. messeae chromosomes. Nevertheless, the comparative physical mapping identified interspecies differences between A. messeae and A. atroparvus in the distribution of repetitive sequences in the pericentric heterochromatin (Figs. 1 and 2). Clone Atr2R-22 hybridized to α-type heterochromatin of chromosome 2 in A. messeae but did not hybridize to A. atroparvus chromosomes, where its presence was confirmed by Southern blot hybridization (Grushko et al, 2004). Clones Atr2R-68, Atr2R-43, and Atr2R-42 did not give any signal in any species, while a positive control in the same experiment (Atr2R-73) did. Obviously, factors such as copy number and representation in polytene chromosome are also important for detecting homologous sequences by FISH.
Table 2 Homologies of DNA clones isolated from 2R heterochromatin of A. atroparvus to the A. gambiae and D. melanogaster genes. TBLASTX of the A. gambiae genome (VectorBase assembly: AgamP3, Feb. 2006, genebuild: AgamP3.4, July, 2007)
TBLASTX of the D. melanogaster genome (FB2009_03, March 20, 2009)
Ensembl Gene ID E-value
Chromosome location Coordinates Chromatin type
FlyBase ID Gene name E-value
Chromosome location Coordinates Chromatin type
GO terms
Atr2R-6a Atr2R-4
AGAP004696 3e− 55
2L:20C 1,926,520–1,965,505 Pericentric heterochromatin
FBgn0000611 Extradenticle 5.3e− 42
X:14A5 15,886,520–15,890,040 Euchromatin
Atr2R-71a
AGAP004672 2e− 10
2R:19D 61,246,251–61,247,458 Pericentric heterochromatin
FBgn0030205 CG17255 5.8e− 10
X:9C 10,357,851–10,367,618 Euchromatin
Atr2R-107
AGAP004707 7e− 13
2L:20C 2,358,158–2,431,617 Pericentric heterochromatin-proximal euchromatin boundary
FBgn0003036 paralytic 4.1e− 06
X:14C 16,357,638–16,421,448 Euchromatin
Atr2R-33 Atr2R-138
AGAP004733 2e− 35
2L:20C 2,917,655–2,919,864 Proximal euchromatin
FBgn0030417 CG15725-PA 3.6e− 25
X:11B 12,600,231–12,602,603 Euchromatin
Atr2R-64a
AGAP003510 6e− 11
2R:15D 38,955,964–38,958,261 Euchromatin
No similarity
No similarity
Atr2R-147b
AGAP009302 7e− 04
3R:34A 30,920,086–30,923,139 Euchromatin
No similarity
No similarity
Molecular function: DNA binding, protein binding, sequence-specific DNA binding, transcription factor activity Biological process: brain development, oenocyte development, DNA-dependent regulation of transcription Cellular component: nucleus Molecular function: protein binding, ATP binding Biological process: protein folding Cellular component: cytoplasm Molecular function: voltage-gated sodium channel activity Biological process: response to DDT, response to pyrethroid, sodium ion transport Cellular component: voltage-gated sodium channel complex Molecular function: protein binding, ATP binding, zinc ion binding Biological process: protein folding Cellular component: intracellular Molecular function: not known Biological process: not known Cellular component: not known Molecular function: DNA binding, sequence-specific DNA binding, transcription factor activity Biological process: DNA-dependent regulation of transcription Cellular component: nucleus
Clone name
a
Clones located in the same row share sequence similarity.
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Our data strongly indicate that heterochromatic regions contain species- and chromosome-specific pools of conserved repetitive DNA. The rapid evolutionary turnover of repetitive DNA sequences between the two sibling species of the A. maculipennis subgroup suggests that these elements are still active. We conclude that repetitive elements underwent independent amplification and loss in the pericentric heterochromatin regions of the A. maculipennis species subgroup according to the “library model” (Ugarkovic and Plohl, 2002). This model states that different satellite or TE families can be preferentially amplified or lost in the derived chromosomes, therefore, changing the relative contribution of each family to the pericentromeric heterochromatin. Thus, the availability of polytene chromosomes in anopheline ovarian nurse cells provide an opportunity to link the molecular organization of heterochromatin to dramatic variations in its structure among sibling species of the A. maculipennis subgroup. More sequencing data are needed to link specific molecular content with a certain heterochromatin type.
and demonstrates rapid heterochromatin remodeling on a short evolutionary scale. Because heterochromatin is predominantly formed by various repetitive DNA sequences, which are difficult to clone and to map, deciphering its DNA content requires utilizing nontraditional approaches, such as microdissection. In this study, microdissection proved to be a useful tool for obtaining and characterizing DNA sequences from heterochromatin of polytene chromosomes and for revealing evolutionary dynamics of homologous sequences. Thus, this approach can be applied for initial evaluation of DNA content in cytologically interesting chromosomal regions of nonmodel organisms. Our analysis determined that heterochromatin of a European malarial mosquito, A. atroparvus, contains noncoding repetitive DNA, TEs, and sequences with high similarity to protein-coding genes. Noncoding repetitive sequences and TEs underwent chromosome-specific and species-specific gains and losses in the morphologically different pericentric heterochromatic regions of sibling species, in accordance with the “library model.”
3.3. Conserved protein-coding genes in the mosquito heterochromatin A search for similarity between the Atr2R cloned fragments and genomes of A. gambiae and D. melanogaster was carried out by TBLASTX. Clones Atr2R-4, Atr2R-6, Atr2R-33, Atr2R-138, Atr2R-107, Atr2R-71a, Atr2R-147b, and Atr2R-64a were similar to protein-coding DNA sequences. Similarity to sequences of A. gambiae was higher than to the corresponding sequences of D. melanogaster (Table 2). Five clones (Atr2R-4, Atr2R-6, Atr2R-33, Atr2R-138, and Atr2R-107) were similar to three single-copy genes in pericentromeric subdivision 20C of chromosome 2L of A. gambiae, suggesting the preservation of a microsynteny. In contrast, the orthologous genes in D. melanogaster were found in different subdivisions of the X chromosome euchromatin. Fragments Atr2R-4 and Atr2R-6 were similar to a single-copy A. gambiae gene, AGAP004696, and to a single-copy D. melanogaster gene, extradenticle. In D. melanogaster, this gene encodes a homeobox-containing transcription factor involved in developmental regulation. Clones Atr2R-33 and Atr2R-138 had similarity to a single-copy gene important for protein folding in A. gambiae and D. melanogaster. Fragment Atr2R-107 was similar to the voltage-gated sodium channel gene in A. gambiae and to the paralytic gene in D. melanogaster. This mosquito heterochromatic gene has implications for development of insecticide resistance and, thus, for vector control. Clone Atr2R-71a had similarity to a single-copy A. gambiae heterochromatic gene, AGAP004672, and to the single-copy D. melanogaster euchromatic gene, CG1725, which encodes a product important for protein folding. A single-copy A. gambiae euchromatic gene, AGAP009302, which does not have an orthologue in Drosophila, encodes for a transcription factor and had similarity to heterochromatic fragment Atr2R-147b. Another A. gambiae euchromatic gene, AGAP003510, was similar to heterochromatic clone Atr2R-64a. Four of six A. gambiae genes homologous to the Atr2R clones are single-copy and located in heterochromatic regions. All their known Drosophila orthologues are also single-copy and vitally important; however, they reside in euchromatin rather than heterochromatin. According to Vector Base, all A. gambiae heterochromatic genes and one euchromatic gene AGAP003510 have expressed sequence tag (EST) evidence. Interestingly, heterochromatic localization of the light gene cluster remains conserved only among species of the D. melanogaster subgroup but not in D. virilis, D. pseudoobscura, or D. ananassae (Yasuhara et al., 2005), suggesting frequent gene transpositions between euchromatin and heterochromatin. Location of these single-copy genes, especially genes coding for transcription factors, in the heterochromatin urges further investigation of their regulation in a repressive environment. 4. Conclusions This paper provides the first description of the molecular content in the heterochromatin of the European malaria mosquito
Acknowledgements This work was supported by the Agricultural Experimental Station at Virginia Tech, by a Grant of the President of the Russian Federation supporting the leading scientific schools (grant no. NSh-15.2003.4), the Russian State Foundation for Basic Research (grant nos. 01-0449712 and 02-04-4817), and by the Russian State Program “Integration” (grant no. E0371+E0206). We thank Prof. Nora Besansky for providing us with access to the confocal microscope and with FISH reagents. We also thank Raquel Assis, Melissa Wade, and Janet B. Webster, Ph.D., for correction of the English text.
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