Department of Biological Sciences, University of Notre Dame
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 2000
) has given rise to neosex chromosomes that still reflect their common ancestry (Steinemann and Steinemann 1992, 1998
; Steinemann, Steinemann, and Lottspeich 1993
). 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 1993
; Carvalho, Lazzaro, and Clark 2000
). 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. 1996
; Kalmykova et al. 1997
; Carvalho, Lazzaro, and Clark 2000
). The human Y also has a class of genes without X homologs, derived from autosomal genes by (retro)transposition (e.g., Saxena et al. 1996
; Lahn and Page 1997, 1999
). 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, 1999
).
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 1993
), there are also regions devoid of satellite DNA that harbor moderately repetitive transposable element families, dominated by retrotransposons (Dimitri 1997
). Both the Drosophila and human Y accumulate retrotransposons preferentially (Steinemann and Steinemann 1992
; Dimitri 1997
; Erlandsson, Wilson, and Paabo 2000
). 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 1993
; Steinemann and Steinemann 1998
). 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 1994
; Chalvet et al. 1998
).
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-LTRcontaining 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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 digoxigenin11-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 DASH II (Stratagene) genomic library, prepared from Sau3AI partially digested A. gambiae SUA DNA from both sexes (Salazar et al. 1994
), 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)
. 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 1999
). 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. 1997
) 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 1993
).
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 1999
). 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.
|
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.
|
Estimation of Copy Number
Copy number was estimated as described previously (Besansky 1990
). Briefly, the genomic DNA of males from A. gambiae PEST (Mukabayire and Besansky 1996
) 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 2024 h after bloodmeal and prepared for in situ hybridization and detection according to Kumar and Collins (1994)
. 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In reverse Southern analysis, two contiguous 4 kb XbaI subclones and an overlapping 1.4 kb EcoRI subclone (E1C) of
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 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 1996
), the frameshift may occur in a mononucleotide hexamer (A)6 (positions 14601465), as described for the human retrovirus HIV-1 and postulated for Drosophila retrotransposon HeT-A (Pardue et al. 1996
). 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)
, 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.
|
|
|
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).
|
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 1992
), 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
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 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 (2.34.2) 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 AF387850AF387861). 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.
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 1995
; Charlesworth and Charlesworth 2000
). 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 1992
) 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 1997
; Stuart-Rogers and Flavell 2001
). Stuart-Rogers and Flavell (2001)
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 1.9more 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 1997
; Stuart-Rogers and Flavell 2001
). 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. 1998
). 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. 1999
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. 1994
; Song et al. 1994
). 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. 1995
). 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. 1998
). 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 1997
). 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. 1997
) and the DAZ gene cluster on the human Y (Saxena et al. 1996
) 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
Present address: Parasite and Vector Biology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, UK.
Keywords: Anopheles gambiae
malaria vector
male-specific transcripts
retrotransposon
Y chromosome
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
.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bachtrog D., B. Charlesworth, 2000 Reduced levels of microsatellite variability on the neo-Y chromosome of Drosophila miranda Curr. Biol 10:1025-1031[ISI][Medline]
Baker R. H., R. K. Sakai, 1979 Triploids and male determination in the mosquito, Anopheles culicifacies J. Hered 70:345-346[ISI][Medline]
Besansky N. J., 1990 A retrotransposable element from the mosquito Anopheles gambiae Mol. Cell. Biol 10:863-871[ISI][Medline]
Besansky N. J., J. R. Powell, 1992 Reassociation kinetics of Anopheles gambiae (Diptera: Culicidae) DNA J. Med. Entomol 29:125-128[ISI][Medline]
Bonaccorsi S., G. Santini, M. Gatti, S. Pimpinelli, M. Colluzzi, 1980 Intraspecific polymorphism of sex chromosome heterochromatin in two species of the Anopheles gambiae complex Chromosoma 76:57-64[ISI][Medline]
Camirand A., N. Brisson, 1990 The complete nucleotide sequence of the Tst1 retrotransposon of potato Nucleic Acids Res 18:4929[ISI][Medline]
Capy P., C. Bazin, D. Higuet, T. Langin, 1998 Dynamics and evolution of transposable elements Landes Bioscience, Georgetown, Tex.
Carvalho A. B., B. P. Lazzaro, A. G. Clark, 2000 Y chromosomal fertility factors kl-2 and kl-3 of Drosophila melanogaster encode dynein heavy chain polypeptides Proc. Natl. Acad. Sci. USA 97:13239-13244
Chalvet F., C. di Franco, A. Terrinoni, A. Pelisson, N. Junakovic, A. Bucheton, 1998 Potentially active copies of the gypsy retroelement are confined to the Y chromosome of some strains of Drosophila melanogaster possibly as the result of the female-specific effect of the flamenco gene J. Mol. Evol 46:437-441[ISI][Medline]
Charlesworth B., D. Charlesworth, 2000 The degeneration of Y chromosomes Philos. Trans. R. Soc. Lond. B Biol. Sci 355:1563-1572[ISI][Medline]
Clare J., P. Farabaugh, 1985 Nucleotide sequence of a yeast Ty element: evidence for an unusual mechanism of gene expression Proc. Natl. Acad. Sci. USA 82:2829-2833[Abstract]
Clements A. N., 1992 The biology of mosquitoes Chapman & Hall, London.
Collins F. H., M. A. Mendez, M. O. Rasmussen, P. C. Mehaffey, N. J. Besansky, V. Finnerty, 1987 A ribosomal RNA gene probe differentiates member species of the Anopheles gambiae complex Am. J. Trop. Med. Hyg 37:37-41[ISI][Medline]
Dimitri P., 1997 Constitutive heterochromatin and transposable elements in Drosophila melanogaster Genetica 100:85-93[ISI][Medline]
Erlandsson R., J. F. Wilson, S. Paabo, 2000 Sex chromosomal transposable element accumulation and male-driven substitutional evolution in humans Mol. Biol. Evol 17:804-812
Farabaugh P. J., 1996 Programmed translational frameshifting Annu. Rev. Genet 30:507-528[ISI][Medline]
Felsenstein J., 1993 PHYLIP (Phylogeny Inference Package) Version 3.5c. Department of Genetics, University of Washington, Seattle. (Distributed by the author)
Flavell A. J., 1984 Role of reverse transcription in the generation of extrachromosomal copia mobile genetic elements Nature 310:514-516[ISI][Medline]
Flavell A. J., S. R. Pearce, P. Heslop-Harrison, A. Kumar, 1997 The evolution of Ty1-copia group retrotransposons in eukaryote genomes Genetica 100:185-195[ISI][Medline]
Fourcade-Peronnet F., L. d'Auriol, J. Becker, F. Galibert, M. Best-Belpomme, 1988 Primary structure and functional organization of Drosophila 1731 retrotransposon Nucleic Acids Res 16:6113-6125[Abstract]
Gatti M., S. Bonaccorsi, S. Pimpinelli, M. Coluzzi, 1982 Polymorphism of sex chromosome heterochromatin in the Anopheles gambiae complex Pp. 3248 in W. W. M. Steiner, W. J. Tabachnick, K. S. Rai, and S. Narang, eds. Recent developments in the genetics of insect disease vectors. Stipes Publishing Co., Champaign, Ill
Genetics Computer Group. 1999 Wisconsin package Version 10.0. Genetics Computer Group (GCG), Madison, Wis
Grandbastien M. A., A. Spielman, M. Caboche, 1989 Tnt1, a mobile retroviral-like transposable element of tobacco isolated by plant cell genetics Nature 337:376-380[ISI][Medline]
Graves J. A. M., 1995 The origin and function of the mammalian Y chromosome and Y-borne genesan evolving understanding Bioessays 17:311-321[ISI][Medline]
Hackstein J. H., R. Hochstenbach, E. Hauschteck-Jungen, L. W. Beukeboom, 1996 Is the Y chromosome of Drosophila an evolved supernumerary chromosome? Bioessays 18:317-323[ISI][Medline]
Hammer M. F., S. L. Zegura, 1996 The role of the Y chromosome in human evolutionary studies Evol. Anthropol 5:116-134
Hirochika H., H. Otsuki, 1995 Extrachromosomal circular forms of the tobacco retrotransposon Tto1 Gene 165:229-232[ISI][Medline]
Jobling M. A., C. Tyler-Smith, 1995 Fathers and sons: the Y chromosome and human evolution Trends Genet 11:449-456[ISI][Medline]
Junakovic N., A. Terrinoni, C. Di Franco, C. Vieira, C. Loevenbruck, 1998 Accumulation of transposable elements in the heterochromatin and on the Y chromosome of Drosophila simulans and Drosophila melanogaster J. Mol. Evol 46:661-668[ISI][Medline]
Kalmykova A. I., Y. Y. Shevelyov, A. A. Dobritsa, V. A. Gvozdev, 1997 Acquisition and amplification of a testis-expressed autosomal gene, SSL, by the Drosophila Y chromosome Proc. Natl. Acad. Sci. USA 94:6297-6302
Kim A., C. Terzian, P. Santamaria, A. Pelisson, N. Purd'homme, A. Bucheton, 1994 Retroviruses in invertebrates: the gypsy retrotransposon is apparently an infectious retrovirus of Drosophila melanogaster Proc. Natl. Acad. Sci. USA 91:1285-1289[Abstract]
Kumar V., F. H. Collins, 1994 A technique for nucleic acid in situ hybridization to polytene chromosomes of mosquitoes in the Anopheles gambiae complex Insect Mol. Biol 3:41-47[Medline]
Lahn B. T., D. C. Page, 1997 Functional coherence of the human Y chromosome Science 278:675-680
. 1999 Retroposition of autosomal mRNA yielded testis-specific gene family on human Y chromosome Nat. Genet 21:429-433[ISI][Medline]
Lankenau S., V. G. Corces, D. H. Lankenau, 1994 The Drosophila micropia retrotransposon encodes a testis-specific antisense RNA complementary to reverse transcriptase Mol. Cell. Biol 14:1764-1775[Abstract]
Lohe A. R., A. J. Hilliker, P. A. Roberts, 1993 Mapping simple repeated DNA sequences in heterochromatin of Drosophila melanogaster Genetics 134:1149-1174
Marchi A., R. Mezzanotte, 1990 Inter- and intraspecific heterochromatin variation detected by restriction endonuclease digestion in two sibling species of the Anopheles maculipennis complex Heredity 65:135-142[ISI][Medline]
McAllister B. F., J. H. Werren, 1997 Phylogenetic analysis of a retrotransposon with implications for strong evolutionary constraints on reverse transcriptase Mol. Biol. Evol 14:69-80[Abstract]
McLain D. K., F. H. Collins, A. D. Brandling-Bennett, J. B. Were, 1989 Microgeographic variation in rDNA intergenic spacers of Anopheles gambiae in western Kenya Heredity (Edinburgh) 62:257-264[Medline]
Mitchell S. E., J. A. Seawright, 1989 Recombination between the X and Y chromosomes in Anopheles quadrimaculatus species A J. Hered 80:496-499[ISI]
Mossie K. G., M. W. Young, H. E. Varmus, 1985 Extrachromosomal DNA forms of copia-like transposable elements, F elements and middle repetitive DNA sequences in Drosophila melanogaster. Variation in cultured cells and embryos. J. Mol. Biol 182:31-43[ISI][Medline]
Mount S. M., G. M. Rubin, 1985 Complete nucleotide sequence of the Drosophila transposable element copia: homology between copia and retroviral proteins Mol. Cell Biol 5:1630-1638[ISI][Medline]
Mukabayire O., N. J. Besansky, 1996 Distribution of T1, Q, Pegasus and mariner transposable elements on the polytene chromosomes of PEST, a standard strain of Anopheles gambiae Chromosoma 104:585-595[ISI][Medline]
Mukabayire O., A. J. Cornel, E. M. Dotson, F. H. Collins, N. J. Besansky, 1996 The tryptophan oxygenase gene of Anopheles gambiae Insect Biochem. Mol. Biol 26:525-528[ISI][Medline]
Pardue M.-L., O. N. Danilevskaya, K. Lowenhaupt, J. Wong, K. Erby, 1996 The gag coding region of the Drosophila telomeric retrotransposon, HeT-A, has an internal frame shift and a length polymorphic region J. Mol. Evol 43:572-583[ISI][Medline]
Powell J. R., V. Petrarca, A. della Torre, A. Caccone, M. Coluzzi, 1999 Population structure, speciation, and introgression in the Anopheles gambiae complex Parasitologia 41:101-113[Medline]
Prud'homme N., M. Gans, M. Masson, C. Terzian, A. Bucheton, 1995 Flamenco, a gene controlling the gypsy retrovirus of Drosophila melanogaster Genetics 139:697-711
Salazar C. E., D. Mills-Hamm, V. Kumar, F. H. Collins, 1993 Sequence of a cDNA from the mosquito Anopheles gambiae encoding a homologue of human ribosomal protein S7 Nucleic Acids Res 21:4147[ISI][Medline]
Salazar C. E., D. Mills-Hamm, D. M. Wesson, C. B. Beard, V. Kumar, F. H. Collins, 1994 A cytoskeletal actin gene in the mosquito Anopheles gambiae Insect Mol. Biol 3:1-13[Medline]
SanMiguel P., B. S. Gaut, A. Tikhonov, Y. Nakajima, J. L. Bennetzen, 1998 The paleontology of intergene retrotransposons of maize Nat. Genet 20:43-45[ISI][Medline]
Saxena R., L. G. Brown, T. Hawkins, et al. (8 co-authors) 1996 The DAZ gene cluster on the human Y chromosome arose from an autosomal gene that was transposed, repeatedly amplified and pruned Nat. Genet 14:292-299[ISI][Medline]
Shen P., F. Wang, P. A. Underhill, et al. (10 co-authors) 2000 Population genetic implications from sequence variation in four Y chromosome genes Proc. Natl. Acad. Sci. USA 97:7354-7359
Song S. U., T. Gerasimova, M. Kurkulos, J. D. Boeke, V. G. Corces, 1994 An env-like protein encoded by a Drosophila retroelement: evidence that gypsy is an infectious retrovirus Genes Dev 8:2046-2057[Abstract]
Steinemann M., S. Steinemann, 1992 Degenerating Y chromosome of Drosophila miranda: a trap for retrotransposons Proc. Natl. Acad. Sci. USA 89:7591-7595[Abstract]
. 1998 Enigma of Y chromosome degeneration: neo-Y and neo-X chromosomes of Drosophila miranda a model for sex chromosome evolution Genetica 102103:409-420
Steinemann M., S. Steinemann, F. Lottspeich, 1993 How Y chromosomes become genetically inert Proc. Natl. Acad. Sci. USA 90:5737-5741[Abstract]
Stuart-Rogers C., A. J. Flavell, 2001 The evolution of Ty1-copia group retrotransposons in gymnosperms Mol. Biol. Evol 18:155-163
Swofford D. L., 1999 PAUP*: phylogeny analysis using parsimony (and other methods) Version 4.0b2. Sinauer, Sunderland, Mass
Thompson J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, D. G. Higgins, 1997 The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools Nucleic Acids Res 24:4876-4882
Underhill P. A., L. Jin, A. A. Lin, S. Q. Mehdi, T. Jenkins, D. Vollrath, R. W. Davis, L. L. Cavalli-Sforza, P. J. Oefner, 1997 Detection of numerous Y chromosome biallelic polymorphisms by denaturing high-performance liquid chromatography Genome Res 7:996-1005
Underhill P. A., P. Shen, A. A. Lin, et al. (18 co-authors) 2000 Y chromosome sequence variation and the history of human populations Nat. Genet 26:358-361[ISI][Medline]
Varmus H., P. Brown, 1989 Retroviruses Pp 53108 in D. Berg and M. Howe, eds. Mobile DNA. American Society for Microbiology, Washington, D.C
Voytas D. F., F. M. Ausubel, 1988 A copia-like transposable element family in Arabidopsis thaliana Nature 336:242-244[ISI][Medline]
Warren A. M., M. A. Hughes, J. M. Crampton, 1997 Zebedee: a novel copia-Ty1 family of transposable elements in the genome of the medically important mosquito Aedes aegypti Mol. Gen. Genet 254:505-513[ISI][Medline]