Structure and Evolution of mtanga, a Retrotransposon Actively Expressed on the Y Chromosome of the African Malaria Vector Anopheles gambiae

Cherise J. B. Rohr, Hilary Ranson1, Xuelan Wang and Nora J. Besansky

Department of Biological Sciences, University of Notre Dame


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Here we report the discovery of a novel family of long terminal repeat (LTR)-retrotransposons designated mtanga-Y, specific to the Y chromosome of the African malaria vector, Anopheles gambiae. mtanga-Y elements represent the first Y-linked sequences and the first members of the Ty1-copia superfamily of retrotransposons described from this mosquito. Analysis of a full-length 4,284-bp element revealed the presence of two intact overlapping open reading frames bounded by LTRs of 119 bp. Evidence suggests that the elements are capable of retrotransposition, as transcripts and potential replication intermediates (one-LTR circles) were detected. However, the ~12 copies of mtanga-Y appear to be clustered rather than dispersed on the Y chromosome. Absent from the Y chromosome of four sibling species (A. arabiensis, A. quadriannulatus, A. melas, and A. merus), similar, but often defective, mtanga elements are present elsewhere in these genomes, as well as in A. gambiae. These data are consistent with a relatively recent invasion of the A. gambiae Y chromosome by an intact element. The presence of functional mtanga-Y elements suggests that the Y chromosome may be a source, not just a sink, for retrotransposons.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
The diploid karyotype of most mosquitoes (Diptera: Culicidae) consists of two pairs of autosomes and a pair of morphologically indistinguishable chromosomes bearing a sex-determining locus (Clements 1992Citation ). Only members of the subfamily Anophelinae have heteromorphic sex chromosomes and classical sex linkage. Sparse genetic and cytogenetic data indicate that the anopheline Y chromosome (hereafter, the Y) is male determining, heterochromatic, polymorphic in banding pattern, and nonrecombining over all or most of its length (Baker and Sakai 1979Citation ; Bonaccorsi et al. 1980Citation ; Gatti et al. 1982Citation ; Mitchell and Seawright 1989Citation ; Marchi and Mezzanotte 1990Citation ). A longstanding view of the Y as a gene-depauperate sink for junk repetitive DNA and the exclusion of males from disease transmission have conspired against detailed studies on this chromosome. Yet, molecular characterization of the Y in other model systems has uncovered a wealth of informative genetic markers for inference of population and species histories (e.g., Jobling and Tyler-Smith 1995Citation ; Hammer and Zegura 1996Citation ; Underhill et al. 1997, 2000Citation ; Shen et al. 2000Citation ). In addition, molecular analysis of the Y has yielded insights into the processes that shaped and continue to reshape the evolution and structure of this chromosome (e.g., Graves 1995Citation ; Kalmykova et al. 1997Citation ; Lahn and Page 1997, 1999Citation ; Carvalho, Lazzaro, and Clark 2000Citation ). This study was undertaken as a first step in characterizing the organization and evolution of the Y chromosome at the molecular level in a medically important insect and emerging model system, the African malaria vector Anopheles gambiae.

The evolution of the heteromorphic sex chromosomes from an indistinguishable pair of autosomes is triggered by the suppression of recombination. Whereas gene content and function is preserved on the X, the Y degenerates through point mutations, structural rearrangements, heterochromatization, and accumulation of repetitive DNA. This process has been studied in Drosophila miranda, where an autosome:Y translocation that occurred within the last 1 Myr (Bachtrog and Charlesworth 2000Citation ) has given rise to neo–sex chromosomes that still reflect their common ancestry (Steinemann and Steinemann 1992, 1998Citation ; Steinemann, Steinemann, and Lottspeich 1993Citation ). A common ancestry between the sex chromosomes is no longer apparent in Drosophila hydei or Drosophila melanogaster, where Y-linked satellite DNAs and most known genes lack recognizable X homologs (e.g., Lohe, Hilliker, and Roberts 1993Citation ; Carvalho, Lazzaro, and Clark 2000Citation ). Instead, evidence suggests that many Y-linked genes were acquired through (retro)transposition of autosomal genes, fueling the hypothesis that much of the extant Drosophila Y has arisen de novo (Hackstein et al. 1996Citation ; Kalmykova et al. 1997Citation ; Carvalho, Lazzaro, and Clark 2000Citation ). The human Y also has a class of genes without X homologs, derived from autosomal genes by (retro)transposition (e.g., Saxena et al. 1996Citation ; Lahn and Page 1997, 1999Citation ). Together, these data emphasize that content of the Y has been shaped at least as much by the flow of genetic information from the autosomes as by the preservation of X homologs (Lahn and Page 1997, 1999Citation ).

Retrotransposons are likely mediators in the retroposition of autosomal genes and are mobile elements that rely on reverse transcriptase (RT) to replicate through an RNA intermediate. They may perform both constructive and destructive roles in shaping gene content and expression on the Y. Although most of the D. melanogaster Y consists of blocks of highly repeated satellite DNA (Lohe, Hilliker, and Roberts 1993Citation ), there are also regions devoid of satellite DNA that harbor moderately repetitive transposable element families, dominated by retrotransposons (Dimitri 1997Citation ). Both the Drosophila and human Y accumulate retrotransposons preferentially (Steinemann and Steinemann 1992Citation ; Dimitri 1997Citation ; Erlandsson, Wilson, and Paabo 2000Citation ). On the neo-Y of D. miranda, the rapid accumulation of retrotransposons led to the transcriptional silencing of the larval cuticle protein genes and heterochromatization along the entire chromosome (Steinemann, Steinemann, and Lottspeich 1993Citation ; Steinemann and Steinemann 1998Citation ). These Y-linked retrotransposons are often silenced themselves, being defective in both sequence and structure. Important exceptions, such as micropia and gypsy, may be intact and actively expressed within the introns of Y-linked fertility genes (Lankenau, Corces, and Lankenau 1994Citation ; Chalvet et al. 1998Citation ).

Here, we report the discovery of a family of intact copia-like retrotransposons on the A. gambiae Y, named mtanga-Y. These elements were identified during the course of a screen for Y-specific sequences in A. gambiae. Evidence suggests that mtanga-Y elements are functional. Not only are copies specifically transcribed in males but we also identify one-LTR–containing circular elements that we interpret as intermediates in the retrotransposition process. More diverse and often defective mtanga elements are present on non-Y chromosomes in A. gambiae and closely related sibling species (A. arabiensis, A. quadriannulatus, A. melas, and A. merus), consistent with a relatively recent invasion of the A. gambiae Y by an active source.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Mosquitoes
Specimens of A. gambiae and four sibling species in the A. gambiae complex were obtained from laboratory colonies maintained at the University of Notre Dame or from archived (frozen) colonies (table 1 ). Additional field specimens of A. gambiae were the F1 progeny of wild females captured in May of 1987 (B6-Q) from Asembo Bay, Kenya (McLain et al. 1989Citation ) or in July of 1997 (BAN) from Banambani, Mali. Only one male and one female offspring from each female was used for the genetic analysis.


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Table 1 Colonies of Anopheles gambiae and Sibling Species Used in This Study

 
Isolation of Nucleic Acids
Genomic DNA was prepared from individual adult males and females as described (Collins et al. 1987Citation ) and resuspended in TE buffer pH 8.0. Bulk genomic DNA was prepared using Genomic-tips (Qiagen). RNA was isolated from single adult specimens (ZAN/U and RSP strains) using TRI REAGENT (Sigma Chemical Co.). To remove any contaminating DNA, RNA samples were treated with 1 U DNAse I, as instructed by the supplier (GIBCO BRL).

Southern Hybridization
For experiments performed using radioactively labeled probes, DNA was transferred from 0.8% agarose gels to Duralon membranes (Stratagene) in 10x standard saline citrate (SSC) transfer buffer. Southern blots were hybridized overnight at 65°C in 6x SSC, 5x Denhardt's reagent, 0.5% sodium dodecyl sulfate (SDS), and 100 µg/ml denatured salmon sperm DNA. Three posthybridization washes at high stringency were performed in 0.1x SSC/0.1% SDS at 65°C for 15 min each. For experiments performed using digoxigenin–11-dUTP (DIG)–labeled probes, blots were prehybridized in DIG EasyHyb solution (Boehringer-Mannheim) for 4 h at 42°C, then hybridized overnight with probes labeled with DIG by random priming (DIG HI PRIME, Boehringer-Mannheim). High-stringency washes were: Wash I (2x SSC, 0.1% SDS), 2 times for 10 min at room temperature; Wash II (0.1x SSC, 0.1% SDS), 3 times for 15 min at 68°C. Low-stringency washes were reduced to 10 min each and the third replication of Wash II was omitted. Detection protocols were as provided by the supplier.

Isolation of Y Chromosome Sequences
A differential library screening approach was adopted to identify male-specific sequences. A {lambda}DASH II (Stratagene) genomic library, prepared from Sau3AI partially digested A. gambiae SUA DNA from both sexes (Salazar et al. 1994Citation ), was plated at ~33,000 pfu on each of the 12 (150 mm) plates. Filter lifts using Duralose membranes (Stratagene) were performed in quadruplicate. Probes were prepared from equivalent amounts of A. gambiae WE (see table 1) total genomic DNA from each sex, by random prime labeling. Two sets of filters each were hybridized with male or female probes and washed at high stringency. Phage that reproducibly hybridized only with the male total genomic DNA were plugged into 1 ml of phage-dilution buffer, plaque purified, and rescreened as before. DNA from 11 putative male-specific phage inserts was harvested from liquid lysates, following Salazar et al. (1994)Citation . Phage inserts were subcloned into pBluescript SK+ (Stratagene) and amplified in Esherichia coli XL1 Blue (Stratagene).

DNA Sequencing and Analysis
Plasmid and PCR templates were purified using QIAprep Spin Miniprep and QIAquick Spin Purification Kits, respectively (Qiagen). Cycle sequencing was performed using PE BigDye Terminator Ready Reaction Kit (PE Applied Biosystems) and M13 universal/reverse or synthetic primers (GIBCO BRL) spaced at ~400-bp intervals on both the strands. Sequences were analyzed on an ABI 377 automated sequencer. Verification and contig assembly was accomplished with ABI AutoAssembler software. GenBank accession numbers are AF387850AF387862.

Further analyses were conducted using the GCG Sequence Analysis Package (Genetics Computer Group 1999Citation ). Database searches were implemented on the NCBI server using BLAST. The amino acid alignment encompassing the deduced RT regions of copia/TY1-related retrotransposons was created with ClustalX (Thompson et al. 1997Citation ) using the default options and displayed with Boxshade 3.21 (http://www.ch.embnet.org/software/BOX_form.html). Phylogenetic inference based on this alignment was performed using the PHYLIP 3.5c programs SEQBOOT (500 replicates), PROTPARS (with jumble option = 10), and CONSENSE (Felsenstein 1993Citation ).

Following alignment with the default options of Pileup (GCG), phylogenetic analysis of mtanga integrase (INT) sequences using maximum parsimony was performed using the branch and bound search option in PAUP* 4.0b4a (Swofford 1999Citation ). Gaps were treated as missing data, but three informative gaps were also coded as single events (regardless of length). In a few instances where a nucleotide could not be unambiguously determined by direct sequencing, it was coded using International Union of Biochemistry symbols and interpreted by PAUP* as polymorphic. Bootstrapping was performed using 1,000 pseudoreplicates.

mtanga PCR
PCR reactions consisted of 1 µl template DNA (1/100 of the DNA extracted from a single mosquito), 1.5 mM MgCl2, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 0.2 mM dNTPs, 30 pM of each primer, and 1 U Taq polymerase, in a total volume of 25 µl. All PCR reactions, performed in a Perkin-Elmer 9600, were initiated with a 5-min denaturation step at 94°C followed by 35 cycles (parameters varied; see below) and terminated with a 10-min extension at 72°C. An ~800-bp region spanning the INT domain was amplified using MTA1 (5'-CTCCAAAACATTTCGTCATC-3', fig. 1A ) and MTA2 (5'-TTAAAAACCCACTTACTGCCC-3', fig. 1A ). Cycling at 94°C for 20 s, 65°C for 20 s, and 72°C for 5 s achieved A. gambiae male-specific amplification.



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Fig. 1.—Organization of mtanga. A, Structure of the mtanga element found in {lambda}1. Open triangles indicate the long terminal repeats, which are flanked by 5-bp target site duplications (filled triangles). Open rectangles indicate long ORFs (ORF1, ORF2) overlapping in the -1 frame. Conserved amino acid domains within them are indicated by: NC, nucleocapsid; PRO, protease; INT, integrase; RT, reverse transcriptase; and RH, RnaseH. PBS-/+ represent putative minus and plus strand PBSs, respectively. Vertical arrows represent restriction sites: X, XbaI; E, EcoRI. Horizontal bar represents the EcoRI fragment E1C used as a probe in Southern analysis. Horizontal arrows indicate the position and orientation of primer pairs referred to in the text. B, Sequence similarity between the termini of mtanga and copia LTRs and sequence complementarity between the PBS- of these elements and the 3'-end of tRNAMet from Drosophila melanogaster (K00461). C, Alignment of nucleotide sequence flanking mtanga-Y in A. gambiae {lambda}1 and corresponding empty site sequences from the Y chromosome of two strains (ARMOR, AHERO) of A. arabiensis (ARA-Y), one strain each of A. melas (MEL-Y) and A. quadriannulatus (QUA-Y), and from BAC clone 32P11. Only nucleotide positions different from the mtanga-Y reference are indicated; dots represent identical positions and dashes represent missing sequence. M = A or C and Y = C or T. Target site duplications are underlined

 
The unique Y chromosomal location of mtanga in A. gambiae was demonstrated by PCR using two pairs of primers at the 5' and 3' boundaries of the element, respectively. For each pair, one primer was designed to anneal within the element pointing outward toward flanking DNA, the other annealed to flanking DNA and pointed toward the element. The 5' pair, MTA3 (flanking; 5'-CCACACCATAGCAACATACC-3'; 377-bp upstream of LTR, fig. 1A ) and MTA4 (5'-AAACTTTCCGCCATCC-3', fig. 1A ), amplified the predicted 559-bp product exclusively from males after cycling at 94°C for 20 s, 65°C for 20 s, and 72°C for 30 s. The 3' pair, MTA5 (5'-GAAACTTGGTGA AGA-3', fig. 1A ) and MTA6 (flanking; 5'-CACATTGGGATAGATTCTCACAAC-3'; 53-bp downstream of LTR, fig. 1A ), amplified male-specific products of the expected 671 bp after cycling at 94°C for 20 s, 50°C for 20 s, and 72°C for 1 min. Finally, employing only the flanking pair of primers MTA3 and MTA6, the empty mtanga insertion site (empty site) was amplified from males of other sibling species after cycling at 94°C for 20 s, 55°C for 20 s, and 72°C for 30 s.

Circular mtanga molecules were amplified with a pair of PCR primers pointing toward the LTRs (see fig. 9 ): MTAR2 (5'-GCAGTCATCCACAAGAAGAG-3') and EHF7 (5'-ATCCGTGAAGATAACCAG-3'). Cycling conditions were 94°C for 20 s, 50°C for 20 s, and 72°C for 45 s.



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Fig. 9.—Evidence for an extrachromosomal one-LTR circular form of mtanga. Top: Proposed derivation of circular mtanga DNA with a single LTR, by homologous recombination between the LTRs of linear DNA. Primers EHF7 and MTAR2 were used for PCR amplification across the LTR circle junction. Bottom: DNA sequence of the polypurine tract (PPT), LTR, and primer-binding site (PBS) determined from 18 cloned PCR products. Only partial sequences from the 119-bp LTR are shown: the first and last two positions and polymorphic positions (numbered above). Sequence shown in the first line, identical in 12 independent clones, is used as a reference. Sequences from the remaining six clones are shown only if they differ from the reference. LTR position 101, indicated by an asterisk, is polymorphic for C/A between the 5' and 3' LTRs in the cloned copy of mtanga-Y derived from {lambda}1

 
mtanga RT-PCR
First strand cDNA was synthesized from 1 to 5 µg of RNA isolated from individual specimens (pretreated with DNAse I) and 0.5 µg oligo(dT) adaptor using the Superscript kit (GIBCO BRL) as recommended by the supplier. The resulting cDNA was used in two separate mtanga PCR amplifications, using either MTA1 and MTA2 from ORF2, as described above, or MTA7 (5'-CCTGAATCATTGACCGAAACC-3', fig. 1A ) and MTA8 (5'-CAGCTTACTTTTCACCAATTCC-3', fig. 1A ) primers that span the nucleocapsid (NC) domain of ORF1. Male-specific amplification was achieved with the latter primer pair by cycling at 94°C for 20 s, 56°C for 20 s, and 72°C for 20 s. As positive controls for RNA quality, separate PCR reactions were carried out on each RNA sample using primers designed to amplify the housekeeping gene encoding S7 ribosomal protein (Salazar et al. 1993Citation , GenBank accession number L20837): SPC (5'-GTGCCGGTGCCGAAACAGAA-3') and SPD (5'-AGCACAAACACTCCAATAATCAAG-3'). To test for DNA contamination, negative control PCR reactions were also performed on each sample of RNA, using an aliquot removed before treatment with RT, and the SPC + SPD primers. Only RNA samples that did not amplify the S7 gene in the negative control reactions, but did so in the positive control reactions, were used for mtanga RT-PCR.

Estimation of Copy Number
Copy number was estimated as described previously (Besansky 1990Citation ). Briefly, the genomic DNA of males from A. gambiae PEST (Mukabayire and Besansky 1996Citation ) was serially diluted, in parallel with serial dilutions of mtanga subclone E1C, a ~1.4-kb EcoRI fragment encompassing the RT domain of the element (fig. 1A ). Each dilution series was applied to a Duralon membrane in a dot blot apparatus. The membrane was probed with the purified E1C insert, washed at high stringency, and the extent of hybridization measured by direct liquid scintillation counting of dots cut from the membrane.

In situ Hybridization
Ovaries from A. gambiae PEST were cropped 20–24 h after bloodmeal and prepared for in situ hybridization and detection according to Kumar and Collins (1994)Citation . Probes were prepared from plasmid or BAC DNA and labeled using the BioNick DNA Labeling System (GIBCO BRL). Sites of hybridization were localized according to the map of M. Coluzzi (personal communication).


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Identification of a Y-linked Retrotransposon
As a part of the ongoing research on A. gambiae Y chromosome organization and evolution, sequences specific to, or enriched on, the Y were identified through differential screening of a dual-sex A. gambiae genomic library with single-sex total genomic DNA probes. Eleven recombinant phage that produced signals only with the male probe were isolated, and among them a phage was designated {lambda}1.

In reverse Southern analysis, two contiguous ~4 kb XbaI subclones and an overlapping 1.4 kb EcoRI subclone (E1C) of {lambda}1 hybridized only faintly with the male total genomic DNA probe, indicating moderate to low copy number. BLASTX searches of GenBank based on partial sequences of these subclones revealed significant amino acid similarity to a number of different LTR retrotransposons, most notably copia from Drosophila.

Complete DNA sequence analysis of the {lambda}1 subclones revealed the presence of an intact 4,284-bp retrotransposon, bounded by 119-bp LTRs and flanked by a 5-bp target site duplication (fig. 1A and C ; GenBank accession number AF387862). The terminal sequences of the LTRs, 5'-TG...CA-3', are shared by retroviruses and many retrotransposons; the sequence similarity to copia extends for nine or eight nucleotides at the 5' and 3' ends, respectively (fig. 1B ). No canonical transcription initiation signal was identified in the LTRs. However, a putative termination signal (AATAAA) was located at position 4223. Moreover, immediately adjacent to the 5' LTR, is a 14-bp sequence identical to the minus strand primer binding site (PBS) of copia and complementary to the tRNAMet genes from various organisms (fig. 1B ). Adjacent to the 3' LTR is a short polypurine tract (GAGGAGGAG), which may be a plus strand PBS.

The bulk of the element comprises two long overlapping open reading frames (ORFs) of 1,446 and 2,826 bp, respectively (fig. 1A ). From the first start methionine, ORF1 potentially encodes a protein of 476 amino acids with conserved motifs characteristic of retroviral NC and protease proteins. Overlapping ORF1 by 253 bp in the -1 frame, ORF2 potentially encodes a protein of 858 amino acids from the first methionine, which overlaps the stop codon of ORF1. Given that retroviruses and retrotransposons normally express overlapping genes by ribosomal frameshifting, programmed frameshifting is likely to occur in this element as well. Despite the absence of a typical frameshift heptanucleotide signal (Farabaugh 1996Citation ), the frameshift may occur in a mononucleotide hexamer (A)6 (positions 1460–1465), as described for the human retrovirus HIV-1 and postulated for Drosophila retrotransposon HeT-A (Pardue et al. 1996Citation ). The predicted product of ORF2 includes motifs characteristic of retroviral INT, RT, and RNAseH proteins, in that order.

The presence of two complete LTRs and two intact ORFs potentially encoding the gag and pol polyproteins suggests that this sequence represents a complete and possibly active retrotransposable element in A. gambiae. It has been designated mtanga, Swahili for wanderer.

mtanga is in the Ty1-copia Superfamily
According to the transposable element classification scheme elaborated by Capy et al. (1998)Citation , mtanga belongs in Class I with other RNA-mediated elements, Subclass I with LTR-retrotransposons, and Superfamily Ty1-copia on the basis of the lack of an envelope glycoprotein gene and the order of domains within the pol gene: the INT domain lies 5' to the RT domain. Figure 2 shows an alignment of RT amino acid sequences from mtanga and seven other Ty1-copia elements. A phylogenetic analysis on the basis of this alignment placed mtanga within the copia clade (fig. 3 ). mtanga and copia are 40% identical (80/202) at the amino acid level in the RT region.



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Fig. 2.—Amino acid sequence alignment of RT sequences from mtanga and other Ty1-copia retrotransposons. Shading is according to identity (black) or similarity (grey) to a column consensus. Sources for each element are: Zebedee1, (Warren et al. 1997); Tnt1, (Grandbastien et al. 1989); Ta1, (Voytas and Ausubel 1988); Tst1, (Camirand and Brisson 1990); 1731, (Fourcade-Peronnet et al. 1988); copia, (Mount and Rubin 1985); Ty1, (Clare and Farabaugh 1985)

 


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Fig. 3.—Maximum parsimony tree based on the amino acid alignment of RT sequences in figure 2 . Shown are bootstrap values exceeding 50% of 500 replicates

 
A Subfamily of mtanga is Associated with the Y Chromosome of A. gambiae
To assess the distribution and sequence conservation among mtanga copies in A. gambiae and sibling species, Southern analysis was conducted. Blots of EcoRI-digested male and female genomic DNA isolated from single specimens of five members of the A. gambiae species complex were probed with a ~1.4-kb EcoRI fragment of mtanga encompassing the RT domain of ORF2 (fig. 1A ). At high stringency, hybridization of mtanga was limited to males of A. gambiae (fig. 4A ), represented by strains (table 1 ) from both West Africa (SUA, GASEL) and East Africa (ZAN/U). Neither A. gambiae females nor males and females of the other species contain sequences that hybridize at high stringency with mtanga, implying that the homolog to the probe was located on the Y in A. gambiae. As a control, the same blot was hybridized at high stringency with a probe prepared from the single copy tryptophan oxygenase (tox) gene (Mukabayire et al. 1996Citation ). A band of roughly equal intensity was detected in all lanes of both sexes, much fainter than the band corresponding to mtanga sequences in A. gambiae males (data not shown). Hereafter we will refer to these Y-specific copies of mtanga in A. gambiae as the mtanga-Y subfamily to distinguish them from less conserved mtanga copies in this and other species.



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Fig. 4.—Southern blot analysis of EcoRI-digested genomic DNA extracted from single specimens of species in the A. gambiae complex, probed with the EcoRI fragment E1C of mtanga. The same blot was washed at high stringency (A) and reduced stringency (B). Note that panel B was deliberately overexposed to enhance faint bands. In both panels, DNA from females and males from the same source was segregated in the left and right set of eight lanes, respectively. For each set, lanes 1–3: A. gambiae ZAN, SUA, and GASEL; lanes 4–5: A. arabiensis AHERO and ARMOR; lane 6: A. quadriannulatus CHIL; lane 7: A. melas BAL; lane 8: A. merus V12. Positions of the {lambda}/HindIII size marker are indicated

 
PCR experiments gave results consistent with the Y-linkage of mtanga from {lambda}1. Primers were designed to amplify a product extending from within the 5' or 3' LTR out into flanking DNA regions present in the clone {lambda}1 (5': MTA3 + MTA4; 3': MTA5 + MTA6; fig. 1A ). PCR products of the expected size were obtained exclusively from A. gambiae males, despite repeated attempts to amplify the fragment from A. gambiae females and both sexes of the other species (data not shown). Conversely, using only the flanking PCR primers oriented across the site of insertion (MTA3 + MTA6), PCR products were obtained only from males of A. arabiensis, A. melas, and A. quadriannulatus, respectively. (Amplification across the ~4.3-kb mtanga element in A. gambiae exceeded the limits of PCR under the conditions employed). The sequence determined from these products revealed an empty site devoid of an mtanga element. In addition, the 5-bp target was not duplicated in these sequences (fig. 1C ).

The male-specificity of the mtanga-Y subfamily contrasts with the distribution of other mtanga copies. At lowered stringency, hybridization of mtanga was apparent in both males and females of A. gambiae and all four sibling species examined (fig. 4B ). This suggests that in addition to mtanga-Y, there are more distantly related copies located on the Y, X, and autosomes (or all the three) of this and other species. Indeed, in situ hybridization of the ovarian nurse cell polytene chromosomes of A. gambiae PEST revealed four sites of mtanga hybridization on the autosomes: 19A and 19D on chromosome 2R, 21A on chromosome 2L, and 29A on chromosome 3R (fig. 5 ). Hybridization was detected neither to the centromeric or pericentromeric heterochromatin nor to the X chromosome. (As the Y is heterochromatic and does not polytenize, no hybridization would have been expected on the Y, even if the polytene chromosomes from larval salivary glands of males had been used).



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Fig. 5.—Ovarian polytene chromosomes of A. gambiae PEST hybridized in situ with biotin-labeled E1C from mtanga-Y. Arrows indicate sites of hybridization

 
Of interest was our discovery of a BAC clone, 32P11, containing sequences nearly identical (92% or 142 of 155 aligned positions excluding insertion and deletions) to sequences flanking the mtanga element from {lambda}1 but devoid of mtanga itself (fig. 1C ). As the source of this clone was a library prepared only from female A. gambiae PEST DNA (F. Collins, personal communication), Y chromosome sequences were not present. In situ hybridization of A. gambiae PEST polytene chromosomes using biotin-labeled 32P11 as a probe produced one signal on the X chromosome in subdivision 5A (data not shown). This result suggests that the insertion site on the Y is homologous to the sequences on the X chromosome.

Subfamily mtanga-Y Consists of ~12 Homogeneous Copies
Copy number of mtanga-Y was estimated by a dot blot experiment in which defined amounts of DNA from A. gambiae PEST males and mtanga-Y subclone E1C were hybridized at high stringency with the 1.4-kb E1C insert. Radioactive counts hybridized per dot of E1C were plotted against the amount of plasmid DNA per dot (data not shown). Assuming a haploid genome size of 0.27 pg (Besansky and Powell 1992Citation ), the number of counts hybridized per 100 ng dot of PEST DNA indicated that mtanga-Y consists of ~12 conserved copies on the Y. These results do not preclude the presence of more diverged mtanga copies on the Y (see below).

Southern analysis suggested that, unlike other mtanga copies, mtanga-Y copies are structurally conserved, as judged by the ORF2 region. At high stringency, one canonical band of hybridization was present in males, at the expected size of 1.4 kb (fig. 4A ). By contrast, reduced stringency revealed additional noncanonical EcoRI bands in females (and males), each presumably representing a copy in which one or both EcoRI sites was altered by mutation (fig. 4B ). The size and number of noncanonical EcoRI bands varied not only between species but also among strains of A. gambiae. Moreover, a comparison of the profile of bands between male and female lanes from the same inbred laboratory colony revealed that whereas all female bands were shared with males, the converse was not always true. Although within-colony polymorphism cannot be ruled out as an explanation, it seems more likely that additional bands in males represent more divergent Y-linked copies.

To further evaluate the polymorphism within and divergence between mtanga-Y and other copies of mtanga, DNA sequence comparisons were made in a segment encompassing the INT domain, located upstream of the RT domain in E1C (see fig. 1A ). PCR primers MTA1 and MTA2 (fig. 1A ) were used to amplify genomic DNA extracted from four A. gambiae laboratory strains (PEST, RSP, ZAN/U, SUA) and two field-derived specimens (B6-Q, BAN) of both sexes from East and West Africa. At an annealing temperature of 65°C, only A. gambiae males gave the expected ~800-bp PCR product. Direct sequencing of purified products from each of the six male A. gambiae specimens revealed unambiguous electropherograms. This result cannot be taken as evidence that all element copies within an individual are identical in sequence, but it suggests that the majority of amplified sequences were identical at each nucleotide position. As these sequences are effectively a consensus of all amplified copies of mtanga, we refer to them as majority-rule sequences. All majority-rule sequences from each of the six specimens were identical to one another and differed from the cloned mtanga-Y copy in {lambda}1 by only two transitions at positions 2520 and 2966. Both substitutions potentially result in an amino acid replacement from Ile -> Met in the conserved INT motif and from Leu -> Ser.

When annealing temperature during the PCR was lowered by 6°C (to 59°C), a fragment of the expected size amplified in corresponding females of five A. gambiae colonies or isofemale families (RSP was not included). At all but a few (<5) nucleotide positions, direct sequencing resulted in an unambiguous A. gambiae female majority-rule sequence for each female. Sequence diversity was much higher among A. gambiae females, with an average of 13 nt differences (~2.1% per site) in pairwise comparisons (complete sequence alignment available in PopSet database of GenBank, accession numbers AF387850AF387861). In addition, all except PEST contained deletions causing frameshifts and premature termination of ORF2. One 14-bp deletion was shared by all females except PEST, and a second 8-bp deletion was unique to SUA. The female PEST sequence contained the only intact ORF, distinct from mtanga-Y because of an in-frame insertion (a tandem duplication) of 18 bp. Between mtanga-Y and female-derived mtanga sequences from A. gambiae there was an average of ~19 nt differences (~3.2% per site excluding alignment gaps), three of which were fixed.

Under the standard assumptions of neutrality, including equal sex ratio and no strong asymmetry in male reproductive success, nucleotide diversity on the Y is expected to be 1/4 of that for autosomes. Given that a minimum of 30 segregating sites were observed in the female mtanga INT consensus sequences determined from each of the five A. gambiae isolates, between seven and eight segregating sites (30÷4) would have been expected from the Y chromosome consensus sequence of the same isolates. Yet, none was observed. The likelihood of obtaining this outcome by chance alone was approximated through coalescent simulation of over 10,000 replicates conditioned on seven segregating sites and no recombination. The 99% confidence interval for expected nucleotide diversity (2.3–4.2) did not include zero, indicating that some unaccounted factor was responsible for lowering the effective population size of mtanga-Y copies below neutral expectation.

mtanga elements from females of other sibling species were also directly sequenced and compared (table 2 ; for alignment, see PopSet AF387850–AF387861). As seen for most A. gambiae sequences, sequences from A. arabiensis and A. merus contained frameshifting deletions. In the latter species, direct sequencing resulted in ambiguous electropherograms. Thus, PCR products were cloned, and a consensus sequence was constructed from six individual clones chosen at random. Both A. arabiensis, AHERO and ARMOR, shared the 14-bp deletion found in A. gambiae females, and a second 8-bp deletion was common to ARMOR and A. gambiae SUA. Two private deletions of 22 and 15 bp were detected in A. merus. Only A. melas and A. quadriannulatus consensus sequences were intact, although the latter contained the 18-bp in-frame insertion, also found in A. gambiae PEST.


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Table 2 Average Pairwise Nucleotide Distances (%, per site) Between INT Consensus Sequences of Males () and Females ()

 
Parsimony analysis of these mtanga sequences revealed the unrooted tree shown in figure 6 . Most striking is the placement of the mtanga-Y clade as a sister group not only to the female-derived mtanga sequences of A. gambiae but also to the female-derived mtanga sequences from all species examined. It is also noteworthy that the female A. gambiae sequences are polyphyletic, with monophyletic A. arabiensis sequences embedded in one well-supported clade and the PEST sequence (the only intact copy from A. gambiae females) basal to all other female sequences.



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Fig. 6.—Maximum parsimony tree of mtanga relationships based sequences encompassing the INT domain of ORF2 (for sequence alignment, see PopSet AF387850–AF387861). Bootstrap estimates derived from 500 replicates (values <50% not shown) are given below; branch lengths are given above branches. GAM-Y represents the sequences from all six males of A. gambiae strains PEST, RSP, ZAN/U, SUA, B6-Q, and BAN, as they were identical. MTA1 represents the sequence determined from the mtanga-Y clone from {lambda}1. Abbreviations are defined in table 1

 
mtanga-Y Copies are Clustered
The genomic organization of mtanga-Y was investigated by Southern analysis, using the E1C probe. Genomic DNA from single specimens were digested with PstI, whose recognition sequence was absent from the {lambda}1 mtanga element. If dispersed on the Y as it is on the autosomes, mtanga should have hybridized to an irregular profile of bands >=4.3 kb in length, with each band representing a different copy of the element. For mtanga copies detected at reduced stringency, this expected pattern of hybridization was confirmed (fig. 7B ). Several bands were detected in females (and males) of each species, indicating a consistently low copy number on the X or autosomes (or both). Surprisingly, this pattern was not observed for mtanga-Y. Instead, at high stringency, a single band at ~9 kb was detected in male A. gambiae (fig. 7A ).



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Fig. 7.—Southern blot analysis of PstI-digested genomic DNA extracted from single specimens of species in the A. gambiae complex, probed with the EcoRI fragment E1C of mtanga. The same blot was washed at high stringency (panel A) and reduced stringency (panel B). The latter was deliberately overexposed to enhance faint bands. In both panels, DNA from females and males from the same source was segregated in the left and right set of eight lanes, respectively. For each set, lanes 1–3: A. gambiae ZAN, SUA, and GASEL; lanes 4–5: A. arabiensis AHERO and ARMOR; lane 6: A. quadriannulatus CHIL; lane 7: A. melas BAL; lane 8: A. merus V12. Positions of the {lambda}/HindIII size marker are indicated

 
mtanga-Y is Expressed in Males
To determine whether mtanga-Y is transcribed, total RNA was isolated from single adult specimens of two strains of A. gambiae (RSP and ZAN/U) and treated with DNAse I to remove contaminating DNA. RT-PCR was performed using pairs of primers that targeted ORF1 and ORF2 of mtanga (MTA7/8 and MTA1/2, fig. 1A ). At 56° annealing temperature, the expected product from each ORF amplified only in males. Figure 8 shows the results from ORF2, using MTA1/2 as primers. Results were similar for ORF1 (data not shown). Sequences determined directly from each of these cDNAs were identical to the genomic consensus from mtanga-Y, indicating that mtanga-Y is actively transcribed on the Y of A. gambiae males. Reduction of the annealing temperature by 6° allowed amplification of ORF2 cDNAs from five of six females, as assessed by Southern blotting and probing with E1C (necessary because PCR products were not visible by EtBr fluorescence; data not shown). Direct sequencing of one ZAN female-derived mtanga cDNA revealed two deletions, each causing frameshifts and premature termination of ORF2 (GenBank accession number AY039510). Thus, although there is some expression of mtanga apart from the Y, the transcripts may not encode functional proteins.



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Fig. 8.—RT-PCR products obtained at high stringency from mtanga (panel A) and ribosomal S7 gene (panel B) transcripts isolated from the same adult specimen. In both panels, PCR products from females and males from the same source were segregated in the left and right set of six lanes, respectively. For each set, lanes 1–3: A. gambiae RSP; lanes 4–6: A. gambiae ZAN/U. Lane 7: negative control reaction. M: panel A, 100-bp ladder (GIBCO/BRL); panel B, 1-kb ladder (GIBCO/BRL)

 
In hosts of replicating retrotransposons, it is sometimes possible to detect replication intermediates: unintegrated linear and circular molecules with one or two LTRs (Flavell 1984Citation ; Mossie, Young, and Varmus 1985Citation ; Hirochika and Otsuki 1995Citation ). A preliminary screen for these extrachromosomal molecules was based on probing a Southern blot of undigested male A. gambiae PEST total genomic DNA (30 µg) with mtanga-Y probe E1C. A strong band of hybridization was detected at ~5.5 kb, with two fainter bands at ~4.3 kb and ~3 kb. As the DNA was undigested and did not appear degraded, these bands were interpreted as possible unintegrated linear (4.3 kb) and monomeric or oligomeric circular forms (3 kb, 5.5 kb) of mtanga (data not shown). Stronger evidence for the presence of one-LTR circular forms of mtanga was obtained by PCR. Primers were designed to anneal within mtanga, oriented toward the LTRs (fig. 9 ). Successful amplification using these PCR primers should be limited to either unintegrated circular forms of mtanga or to adjacent mtanga elements that had integrated in a head-to-tail manner. Using genomic DNA extracted from single specimens of A. gambiae SUA and PEST strains, PCR products of the size predicted for a one-LTR circle (603 bp) were obtained from both strains and cloned. Sequence determined from 18 of 26 individual clones exactly matched the pattern expected for circular mtanga DNA with a single LTR (fig. 9 ). No evidence of two-LTR circles was found.


    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
During the course of screening for male-specific sequences on the Y chromosome of A. gambiae, we discovered a novel family of LTR retrotransposons designated mtanga-Y. mtanga-Y elements represent the first Y-linked sequences and the first members of the Ty1-copia superfamily of retrotransposons described from this mosquito. The mere presence of a copia-like element in the A. gambiae genome is not unexpected as these retrotransposons are distributed widely among eukaryotes (Flavell et al. 1997Citation ). The location on the Y is also not surprising, given the tendency for transposable elements, and retrotransposons in particular, to accumulate preferentially on this chromosome (Dimitri 1997Citation ; Junakovic et al. 1998Citation ; Steinemann and Steinemann 1998Citation ; Erlandsson, Wilson, and Paabo 2000Citation ). What is unusual is that despite their Y-linkage, mtanga-Y elements are apparently intact, actively expressed in males, and potentially functional (i.e., competent to transpose through an RNA intermediate), in contrast to autosomal copies that are more diverse. Their functionality is suggested by evidence of intermediates in the retroviral-like life cycle (Varmus and Brown 1989Citation ): full-length linear mtanga DNA molecules detected on Southern blots of undigested host genomic DNA and circular mtanga DNA with a single LTR, detected by PCR and confirmed by sequencing. The precision with which the sequences matched the pattern expected of one-LTR circles, without intervening DNA at the LTR junctions and only a single LTR present, is inconsistent with amplification across adjacent mtanga elements integrated head-to-tail. In light of the clustered arrangement of mtanga-Y elements, a model of concatameric elements sharing 5' and 3' LTRs cannot be ruled out. However, the {lambda}1 phage containing an mtanga-Y element shows no evidence of this type of organization. Linear and circular extrachromosomal forms of retrotransposons also have been observed in Drosophila and plants. In principle, these molecules can be explained by processes other than RT-mediated replication, such as LTR-LTR recombinative excision, although homologous recombination between LTRs is relatively rare (Varmus and Brown 1989Citation ). Detailed analysis has suggested that at least some proportion is likely replication intermediates (Flavell 1984Citation ; Mossie, Young, and Varmus 1985Citation ; Hirochika and Otsuki 1995Citation ).

Models of Y chromosome evolution suggest that the cessation of crossing over initiates an inexorable erosion of gene function mediated by various population genetic processes (Charlesworth and Charlesworth 2000 and references therein). Genes remaining active either represent new arrivals destined for extinction, or old survivors, having escaped extinction by conferring a selective advantage in the heterogametic sex (Graves 1995Citation ; Charlesworth and Charlesworth 2000Citation ). It has been suggested that retrotransposons play important and possibly beneficial roles of insertional mutagenesis or downregulation of deleterious alleles arising on the neo-Y (Steinemann and Steinemann 1992Citation ) but they ultimately should be subject to the same forces of deterioration as the genes themselves. What, then, is the significance of an active mtanga-Y? There are at least three possibilities, none of which is mutually exclusive. First, mtanga may have been evolving under purifying selection throughout its history. Second, it may have transposed onto the Y quite recently and has yet to degrade. Third, the Y may be the only location in which mtanga activity is not detrimental to the host genome. Each of these possibilities is discussed below.

There is evidence for strong evolutionary constraints on the RT domain of retrotransposons (McAllister and Werren 1997Citation ; Stuart-Rogers and Flavell 2001Citation ). Stuart-Rogers and Flavell (2001)Citation reported 80%–85% sequence conservation in the RT region of Ty1-copia group retrotransposons from gymnosperm lineages separated by over 260 Myr. As would be predicted if selective constraints were responsible, high ratios (>10) of synonymous (Ks) to nonsynonymous (Ka) substitutions were found. The lineages leading to D. melanogaster and A. gambiae have probably been separated for a comparable length of time, but RT sequence comparison between copia (white-apricot) and mtanga-Y showed only 50% nucleotide sequence conservation, a Ka rate of 0.42 per site, and a Ks/Ka ratio of 1.9—more typical of the level of sequence heterogeneity generally observed among retrotransposons. An explanation that could reconcile these discrepant data sets is the unintended comparison of paralogous rather than orthologous sequences in the case of copia and mtanga, which would be a consequence of multiple retrotransposon lineages in a primordial genome, each subject to stochastic loss of replication ability (McAllister and Werren 1997Citation ; Stuart-Rogers and Flavell 2001Citation ). Thus, although convincing evidence is lacking for selective constraint on mtanga-Y, this possibility should not be dismissed.

On the other hand, at least three lines of evidence support the conclusion that mtanga-Y was acquired relatively recently by the Y and has not had time to degrade. First, as dictated by the process of retrotransposition, the two LTRs should be identical at the time of integration. Afterward, successive nucleotide substitutions cause the LTR sequences to diverge. mtanga-Y LTRs are mismatched at only two positions (~1.7%), indicating relatively recent retrotransposition (see SanMiguel et al. 1998Citation ). Second, no X chromosome homolog of mtanga-Y was detected in A. gambiae, at least not in the PEST strain. Barring recombinative excision from the X chromosome, the absence of mtanga from the X suggests that this element invaded the Y after X:Y recombination was halted. Third, mtanga sequences are present in the genomes of four sibling species closely related to A. gambiae. A maximum parsimony tree of mtanga consensus sequences from females largely recapitulated the inferred evolutionary relationships among species in the A. gambiae complex, including genetic introgression between A. gambiae and A. arabiensis (see fig. 6 , Powell et al. 1999Citation and references therein). This implies that mtanga was present in the common ancestor of the A. gambiae complex prior to lineage splitting and diversification into its descendant species. Nevertheless, Southern analysis demonstrated that mtanga-Y occurs exclusively in A. gambiae males. Successful PCR amplification and sequencing across the mtanga-Y empty site on the Y chromosomes of three sibling species revealed the precise absence of this element from intact flanking regions. We discount the possibility that mtanga-Y was lost from the Y of all sibling species except A. gambiae because it is less parsimonious and because the empty sites show no evidence of imprecise excision events. Therefore, it is likely that retrotransposition onto the Y occurred following speciation of A. gambiae.

Perhaps mtanga-Y remains active on the Y chromosome because it is the only location in which mtanga activity is not detrimental to the host genome. This idea has received some circumstantial support in the studies of another retrotransposon, gypsy, an infectious retrovirus-like member of the gypsy/Ty3 group (Kim et al. 1994Citation ; Song et al. 1994Citation ). Retrotransposition of gypsy is under maternal control as it not only requires active gypsy copies but also requires mothers that are homozygous for permissive alleles of the X-linked host regulatory gene flamenco (flam) (Prud'homme et al. 1995Citation ). A study of multiple Drosophila strains found that in a background of restrictive flam alleles, potentially active (but silenced) gypsy copies could be found on any chromosome; in a permissive background, active gypsy copies were often restricted to the Y, where transmission presumably would have no deleterious effects (Chalvet et al. 1998Citation ). Further investigation will be required to understand if there is a relationship between enrichment or confinement of active mtanga copies on the Y and host regulation.

The mtanga phylogeny gives some insight into the evolutionary dynamics of mtanga in the larger context of the A. gambiae complex. The phylogeny was built mainly from sequences determined directly from primary PCR products, without a cloning step, a strategy that generates a consensus expected to reflect the ancestral sequence of the replicating source element (assuming that copies were sampled in an unbiased manner). It is noteworthy that among six of the seven different geographic isolates of A. gambiae, mtanga consensus sequences from females shared the same frameshifting 14-bp deletion. Thus, the majority of mtanga copies within each genome share the potentially inactivating deletion, a situation made possible only if these defective copies co-opt the RT machinery encoded by functional elements. A similar observation was made for a retrotransposon found in a group of parasitoid wasps (McAllister and Werren 1997Citation ). That defective elements can nonetheless transpose suggests that at least one functional element must reside somewhere in the genome, even though it is not in the majority. Could this source element be present on the Y in A. gambiae? The mtanga phylogeny is consistent with this proposal. The putatively active copies on the Y form one clade. The next clades to branch off contain female consensus sequences that, because they lack frameshifts or in-frame stop codons, also are potentially functional. More distant from mtanga-Y are branches leading to mtanga sequences from A. gambiae, A. arabiensis, and A. merus. The recombinational isolation of the Y may be reflected in the distant relationship of the A. gambiae mtanga-Y clade and the A. gambiae autosomal mtanga clade. As for the disjunct placement of the A. gambiae PEST clade, more closely allied with mtanga-Y than to all other A. gambiae autosomal clades, it is tempting to speculate that the PEST elements retrotransposed relatively recently from an active source on the Y, although retrotransposition in the reverse direction cannot be ruled out. In either case, the presence of replication-competent mtanga elements on the Y suggests that the Y chromosome can be a source, not just a sink, for retrotransposons.

Why is sequence diversity of mtanga-Y significantly reduced below neutral expectation? Must selective factors operating to further reduce the effective population size of the Y chromosome (e.g., high variance in male reproductive success, selective sweeps, Muller's ratchet, background selection) be invoked? Not necessarily. mtanga-Y is not dispersed randomly on the Y, as might be expected if the transposition events were independent. Rather, Southern analysis of PstI digests suggests that the ~12 copies are clustered together. This clustering may be the result of a transposition-independent process of DNA amplification that occurred subsequent to, or possibly concurrent with, a single transposition event. The mechanism(s) responsible for DNA amplification on the Y are poorly understood, but the propensity of the Y to undergo such rearrangements is well known. The Su(Ste) repeats on the Drosophila Y (Kalmykova et al. 1997Citation ) and the DAZ gene cluster on the human Y (Saxena et al. 1996Citation ) are but two examples. If a copy of mtanga transposed to the Y and amplified, this alone would be sufficient to explain the low DNA variability within the mtanga-Y repeats, without having to invoke other evolutionary forces. Ultimately, a convincing explanation for the reduced variation in mtanga-Y will require estimates of variation from several other regions of the A. gambiae Y, an effort currently underway in our laboratory.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank Cristina Salazar Rafferty for assistance with library screening and F. Collins, J. Feder, D. Severson, and J. Krzywinski for constructive discussions and for critical comments on the manuscript. This work received financial support from the National Institutes of Health Predoctoral Fellowship grant GM17263-04 to C.R. and NIH grant R01 AI44003 to N.J.B.


    Footnotes
 
Pierre Capy, Reviewing Editor

Present address: Parasite and Vector Biology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, UK. Back

Keywords: Anopheles gambiae malaria vector male-specific transcripts retrotransposon Y chromosome Back

Address for correspondence and reprints: Nora J. Besansky, Department of Biological Sciences, P.O. Box 369, 317 Galvin Life Sciences Building, University of Notre Dame, Notre Dame, Indiana 46556. besansky.1{at}nd.edu . Back


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 Materials and Methods
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Accepted for publication September 20, 2001.