The male sex lethal proteins (MSLs) first associate with the male X chromosome as early as blastoderm stage, slightly earlier than the Histone H4 isoform acetylated at lysine 16 is detected on the X chromosome. MSL binding to the male X chromosome is observed in all somatic tissues of embryos and larvae. Binding of the MSLs to the X chromosome is also interdependent in male embryos and prevented in female embryos by the expression of Sex-lethal (Sxl). A delayed onset of binding of the MSLs in male progeny of homozygous mutant msl-1 or mle mothers coupled with the previous finding that such males have an earlier lethal phase supports the idea that msl-mediated dosage compensation begins early in embryogenesis. Other results show that the Maleless protein on embryo and larval chromosomes differs in its reactivity with antibodies. The functional significance of this finding remains to be explored (Franke, 1996).


When salivary gland chromosomes are spread to reveal the association of Mle with the chromosomes, a striking difference in the distribution of the Mle protein in males and females is found. In males, Mle protein virtually covers the X chromosome, binding to hundreds of sites and giving the male X chromosome a heavily banded appearance. Females lack predominant staining of any one chromosome arm. Rather, the female X and the autosomes each show weak anti-Mle staining (Kuroda, 1991).

Effects of Mutation or Deletion

Mle protein, overexpressed and purified from Sf9 cells infected with recombinant baculovirus, possesses RNA/DNA helicase, adenosine triphosphatase (ATPase) and single-stranded (ss) RNA/ssDNA binding activities, properties identical to RNA helicase A. The helicase activity demonstrates a degree of substrate specificity. Mle displaces substrates containing single stranded RNA regions, i.e. RNA:RNA and DNA:RNA hybrids, 2.5-fold more efficiently than substrates containing single stranded DNA regions. In keeping with the higher helicase activity observed with duplex substrates containing single stranded RNA, complexes of polynucleotides with Mle are formed more efficiently (3- to 4-fold) with single stranded RNA than with single stranded DNA. A mutant of Mle (mle-GET) was created that contains a glutamic acid in place of lysine in the conserved ATP binding site A. This mutation abolishes both NTPase and helicase activities of Mle but affects the ability of Mle to bind to ssRNA, ssDNA and guanosine triphosphate (GTP) less severely. In vivo, mle-GET protein can still localize to the male X chromosome coincidentally with the male-specific lethal-1 protein, Msl-1, but fails to complement mle1 mutant males. These results indicate that the NTPase/helicase activities are essential functions of Mle for dosage compensation, perhaps utilized for chromatin remodeling of X-linked genes (Lee, 1997).

The mutational effect of the maleless (mle) gene in Drosophila has been reexamined. Earlier work had suggested that mle along with other male-lethal genes was responsible for hypertranscription of the X chromosome in males to bring about dosage compensation. Prompted by studies on dosage sensitive regulatory genes, a test was made for effects of mle temperature sensitive mutant on the phenotypes of 16 X chromosome or autosomal mutations in adult escapers of lethality. In third instar larvae, prior to the major lethal phase of mle, activities of 6 X-linked or autosomally encoded enzymes were examined, as well as steady state mRNA levels of 15 X-linked or autosomal genes and transcripts from two large genomic segments derived from either the X or from chromosome 2 and present in yeast artificial chromosomes. Pronounced effects due to mle are found on the expression of both X-linked and autosomal loci such that a large proportion of the tested genes are increased in expression, while only two X-linked loci are reduced. The most prevalent consequence is an increase of autosomal gene expression, which can explain previously observed reduced X:autosome transcription ratios. These observations suggest that if mle plays a role in the discrimination of the X and the autosomes, it may do so by modification of the effects of dosage sensitive regulatory genes (Hiebert, 1994).

A male-specific lethal gene, maleless kills males but not females in homozygous condition, regardless of whether female parents are heterozygous or homozygous for mle. Many, if not most, homozygous males survive up to the third instar larval stage, but cannot pupate and die eventually as larvae. No interactions with sex-transforming genes transformer and doublesex are observed. It is proposed that mle interacts with a gene(s) on the X chromosome, which is not dosage compensated (Fukunaga, 1975).

Although no maternal effect is seen with respect to the male-specific lethality, the lethal stage is influenced by whether parental females were homozygous or heterozygous for mle. Thus, mle/mle males from mle/mle mothers die mostly in the late third instar larval stage, while practically all males with heterozygous mothers survived to the pupal stage. In the dying mle/mle males, pupae often complete differentiation of adult external head and thorax structures but abdominal structures are usually incomplete, forming at most only a few segments in the majority of cases. Imaginal discs from third instar mle/mle male larvae (from mle/mle mothers) destined to die as larvae, are able to differentiate into adult structures upon transplantation into normal third instar larval hosts (Tanaka, 1976).

To investigate the possibility that kinesin transports vesicles bearing proteins essential for ion channel activity, the effects of kinesin (Khc) and ion channel mutations were compared in Drosophila using established tests. Khc mutations produce defects and genetic interactions characteristic of paralytic (para) and maleless (mle) mutations that cause reduced expression or function of the alpha-subunit of voltage-gated sodium channels. mle is thought to reduce the dosage of sodium channels by reducing the expression of the para gene. Like para and mle mutations, Khc mutations cause temperature-sensitive (TS) paralysis. When combined with para or mle mutations, Khc mutations cause synthetic lethality and a synergistic enhancement of TS-paralysis. Khc mutations suppress Shaker and ether-a-go-go mutations that disrupt potassium channel activity. In light of previous physiological tests that show that Khc mutations inhibit compound action potential propagation in segmental nerves, these data indicate that kinesin activity is required for normal inward sodium currents during neuronal action potentials. Tests for phenotypic similarities and genetic interactions between kinesin and sodium/potassium ATPase mutations suggest that impaired kinesin function does not affect the driving force on sodium ions. It is hypothesize that a loss of kinesin function inhibits the anterograde axonal transport of vesicles bearing sodium channels (Hurd, 1996).

maleless (mle) is essential in Drosophila melanogaster males both in somatic cells and in germ cells. In somatic cells mle is necessary for X-chromosome dosage compensation. The role of mle in the germline is unknown. The expression pattern and localization of MLE, the other MSLs and acetylated isoforms of histone H4 in male germ cells have been examined to address whether dosage compensation and/or X inactivation occur in the Drosophila germline. MLE is the only MSL expressed in the male germ cells and is not localized to the X chromosome, nor with any other chromosomal cluster. Weak Mle expression is detectable in spermatogonia and early spermatocytes. Mle is very abundant in the nuclei of fully developed primary spermatocytes; it continues to be detectable in round stage spermatids and elongated spermatides. Mle protein is not detectable during later stages of spermatid development. Studies using an mle temperature sensitive allele reveal that, genetically, the amount of mle activity required for fertility is higher than that required for viability. The analysis of mle mutant gonads reveals that complete loss of mle in the germline and in the soma does not affect the development of male germ cells. Spermatogeneisis can proceed to the final stages of differentiation in the apparent absence of Mle protein. In male germ cells, loss of mle has no detectable effect on the expression or localization of Histone H4 acetylated on amino acid 16. The lack of specific X chromosome localization of H4Ac16 in the transcriptionally active stages of spermatogenesis argues against a role of H4Ac16 in dosage compensation and favors a more general role in transcriptional activation. It is concluded that in the germline mle is not involved in chromosomal dosage compensation but, in its requirement for male fertility, it may be involved in post-transcriptional gene regulation, perhaps mediated by the Mle helicase function. The acetylation pattern of histone H4 is very dynamic during spermatogenesis. While the pattern is not compatible with dosage compensation or X inactivation, it is consistent with all premeiotic chromosomes being in an active configuration that is replaced in post-meiotic stages with an inactive chromatin constitution (Rastelli, 1998).

Dosage compensation works to heighten the activity of the single X chromosome in males. This heightened expression of the X chromosome in males is accomplished through the action of male-specific lethal (MSL) proteins. Immunostaining of chromosomes shows that the MSL proteins are associated with all female chromosomes at a low level but are sequestered to the X chromosome in males. Histone-4 Lys-16 acetylation follows a similar pattern in normal males and females, being higher on the X and lower on the autosomes in males than in females. However, the staining pattern of acetylation and the mof gene product, a putative histone acetylase, returns to a uniform genome-wide distribution as found in females and in males that are mutant for the msl gene. Gene expression on the autosomes correlates with the level of histone-4 acetylation. With minor exceptions, the expression levels of X-linked genes are maintained with either an increase or decrease of acetylation, suggesting that the MSL complex renders gene activity unresponsive to H4Lys16 acetylation. Evidence has also been found for the presence of nucleation sites for the association of the MSL proteins with the X chromosome rather than individual gene binding sequences (Bhadra, 1999).

Three different approaches (chromosomal morphology, specific RNA quantitation, and binding of MSL proteins) were taken to investigate the role of the MSL complex on dosage compensation. Both chromosomal morphology and the measurement of specific transcripts reveal that the lack of MSL binding and histone H4 modification in the mle mutant males neither reduces X-chromosomal size specifically nor eliminates dosage compensation of most X-linked transcripts. Two X-derived transgenes, white and yellow, which are normally partially compensated on the autosomes, are elevated in expression by the homozygous mle mutation. Thus, transgenes do not lose dosage compensation in the mle::mle males; rather, they become fully compensated on the autosomes in the absence of the Mle protein. In addition, the presence of Msl-2 in H83M2 females (H83M2 is a stock in which Msl-2 is ectopically expressed in females) does not promote hypertranscription of the X-derived mini-white and yellow transgenes inserted into the autosomes. Rather the strong association of the MSL proteins and H4Ac16 enrichment with the two X chromosomes and concomitant reduction of H4Ac16 residues on the autosomes is correlated with reduced autosomal transgene expression. Sequestration of chromatin proteins from one location in the genome to another as a means to affect gene expression has also been described in the case of Sir silencing proteins in yeast (Marcand, 1996). In contrast, changes of the acetylation level on the X chromosome appear to have minimal consequences. MSL proteins do not associate with the X-derived transgenes or with a small X segment (>179 kb) in the autosomes. This result indicates that MSL proteins do not initiate binding to every gene on the X chromosome because the lack of binding within the >179-kb segment suggests the absence of potential nucleation sites in that region, while the same cytological bands are associated with MSL proteins when residing on a larger segment. Apparently, the MSL proteins associate with nucleation sites on the X that allow initial recognition followed by polymerization. It has been suggested that the presence of MSL proteins on the X is sufficient evidence to conclude they condition dosage compensation. However, it is now thought that measures of gene expression are necessary to understand the consequences of the msl mutations. Without such experiments, one can come to no conclusion about an involvement of male sex lethal proteins in any process (Bhadra, 1999 and references).

With regard to dosage compensation there are five levels of gene expression to be explained. The most commonly discussed is the twofold increase of the X in males, as compared to females. However, dosage compensation also occurs in females that have three X chromosomes (metafemales 3X;2A). In order for this to occur, the expression of each of the gene copies present must be reduced to two-thirds so that the total amount from the three X's is equivalent to a normal female. Autosomal expression in these flies is also reduced to approximately two-thirds of normal. Dosage compensation in males and metafemales is related as shown by the fact that several white alleles that fail to compensate in males also fail in metafemales. Triploid females with three sets of all chromosomes have the same per gene expression as diploid females. Reduction in the dosage of the X chromosome to two results in triploid intersexes (2X;3A), while further reduction to one copy is referred to as triploid metamales (X;3A). Both exhibit dosage compensation of their X chromosomes. The increases in per gene expression for compensation in these cases are 1.5- and 3-fold, respectively (Bhadra, 1999 and references).

The magnitude of the inverse dosage effect, commonly observed in aneuploids, can account for the different levels of gene expression. When the dosage of large chromosomal segments is reduced from 2 to 1, the expression of unlinked genes goes from 1 to 2. When the dosage of the same segment is increased from 2 to 3, gene expression is reduced to 2/3. Genes present on the varied segment that are similarly affected become dosage compensated because the structural gene dosage effect is canceled by this simultaneously produced inverse dosage effect. The magnitude observed in triploid situations follows closely an inverse correlation between the dosage and the effect on gene expression. Because the inverse effect is the most prevalent type of dosage response, it is likely to be the basis of the five levels of gene expression that occur with the different doses of the X. Indeed, when the white gene is present on the autosomes in constant copy number and a dosage series of the X is produced in the form of males (1X), females (2X), and metafemales (3X), an inverse effect on w expression is found (Bhadra, 1999 and references).

Invoking an inverse dosage effect alone as the basis of X-chromosomal dosage compensation is inadequate on two counts: (1) although large aneuploids exhibit dosage compensation for the majority of linked genes, this fraction of compensated genes is not as great as that for the X chromosome, and (2) this hypothesis predicts an increased autosomal gene expression in males, as compared to females, concomitant with dosage compensation of the X. In general, this is not the case. However, the results of the present study resolve these two issues. The increased acetylation of the X in normal males might intensify the response of some X-linked genes to the inverse dosage effect by increasing the fraction of X-linked genes exhibiting full compensation. An example in the present study is Adh and from previous work, Salivary gland secretion protein 4 (Sgs4), both of which show lower expression on the X in mle mutant males. However, X-linked gene expression is lowered in only a minority of mle males, when the histone acetylation drops on the X to female levels. Indeed, in the absence of MOF activity, the majority of the genes resident in their normal locations on the X retain compensation. The product of the mof gene is predicted to have histone acetylase activity and has been postulated to be responsible for the H4 acetylation that is correlated with the presence of the MSL complex (Hilfiker 1997). The failure of most X-linked genes to respond to lowered acetylation in mof1 mutants or, in the example of y+, to increased levels in H83M2/+ females, suggests that at least some component of the MSL complex nullifies a response of most X-linked genes to their acetylation levels. These results could also suggest that the presence of the MSL complex on the X in normal males prevents genes from responding to the high levels of H4Ac16. The consequence of this action would be that the hyperactivation of these genes could remain at the twofold level because of the inverse dosage effect of the X, rather than a greater overexpression brought on by extremely high levels of acetylation (Bhadra, 1999 and references).

The lowered acetylation of the autosomes in normal males appears to reduce the strength of the inverse dosage effect there. In general, histone acetylation is thought to result in a more open chromatin configuration and is associated with higher levels of gene expression. In the mle mutant males, the acetylation is increased on the autosomes and the inverse dosage effect is intensified. The reduced acetylation of the autosomes in normal males, when compared to females, provides a basis for the nearly equal expression of the autosomes between the sexes despite the 'monosomic' condition of nearly 20% of the genome, which would be expected to increase gene expression throughout all chromosomes under other circumstances. It is suggested that autosomal genes inserted on the X become compensated, because they are moved from a weakly acetylated region to one where the dosage of the X on them can be effective. This acquisition of dosage compensation of autosomal genes is consistent with the conclusion that the MSL complex spreads from nucleation sites and that individual genes on the X do not have MSL binding sites. On the other hand, the partial compensation of white on the autosomes as well as other genes can be explained because they are moved from a chromosome where the dosage of the X can be effective on them to one where it cannot. Thus, the MSL sequestration would maximize equality of expression between males and females for the whole genome (Bhadra, 1999 and references).

In this article, evidence is presented that chromosomal proteins are sequestered from the autosomes to the X in normal males. Therefore, when any of the msl loci are mutated, there is no sequestration. The X remains basically compensated and the autosomal expression is increased in general because MOF becomes uniformly distributed in the nucleus, resulting in a return of acetylation levels to those of females. The msl mutations have little effect in females because they are chromosomally balanced and therefore have no dosage effects operating. Also, there is a similar distribution of acetylation in mutants and normals. In contrast, in H83M2/+ females, the product of Msl-2 promotes sequestration of the acetylase to the X, which lowers the level of autosomal acetylation. As a result, the expression of transgenes inserted in the autosomes is reduced (Bhadra, 1999).

There is much to be learned before a full comprehension of the evolution of this situation can be formulated. However, one speculation might be that the degeneration of one member of a homologous pair of chromosomes that begins the formation of the Y would leave a small segment of the opposing homolog in the haplo state. A selective advantage would be afforded to individuals carrying any change, such as elevated acetylation, that would result in increased expression of haplo-insufficient genes in this region. Although a focus has been placed on the situation in which acetylation or lack thereof modifies the effect of sex-chromosomal dosage, it is also the case, as exemplified by the ectopic expression of Msl-2, that changes in acetylation will modify gene expression without involving a dosage effect. As the haplo region expands, the dosage in dosage-dependent regulatory genes would be altered. It is interesting to note that dosage-dependent regulatory genes are more likely to have an effect in the haplo condition than genes encoding metabolic functions. Their acetylation might be selected to eliminate their global effects on target genes throughout the genome, but some fraction must remain dosage dependent in normal males to produce compensation and the observed autosomal effects (Bhadra, 1999 and references).

Because an inverse effect is more common than a positive dosage response, there would be a tendency to increase gene expression throughout the genome, including rendering the haplo regions at least partially dosage compensated. To date there are over twenty genes identified that produce a trans-acting dosage effect on white. By analogy with modifiers of position-effect variegation, many of which have been identified as transcriptional regulators, there are scores of dosage-dependent modifiers of a single process. These genes appear to operate in a dosage-dependent cascade such that any one or several produce much the same effect on the target locus. Thus, statistically speaking, any region involving only a few percent of the genome will potentially carry a dosage-dependent modifier of any one target gene. Despite the large number of such modifiers, varying multiples does not in general generate cumulative effects beyond the inverse limits. This conclusion is derived from two different types of observations: (1) combinations of mutations do not exceed this limit and (2) large aneuploids produce trans-acting dosage effects that remain within this range. Thus, the combination of acetylation and the inverse dosage effect could provide for a synergistic expansion of the haplo regions. This combination would produce dosage compensation whether the affected target genes are haplo insufficient or not. The inverse dosage effect provides a numerical explanation of how the process of dosage compensation doubles the expression of many genes transcribed at widely differing rates, although the mechanism remains to be elucidated. The sequestration of acetylase to the X in increasingly larger quantities would lower the autosomal acetylation and thus mute the increased expression of the autosomes that would be expected to result from a lowered dosage of the evolving X. On the X, some component of the MSL complex or associated proteins would eventually render most genes unresponsive to the acetylation level. Indeed, there may be a selective advantage to maintaining transcriptional regulators as dosage dependent in order to maintain compensation (Bhadra, 1999 and references).

Such a situation might evolve if continued increases in acetylation caused a tendency for extensive overexpression of the X. Inactivation of a response to acetylation would hold the hyperactivation at the twofold level because of the effect of the X dosage. Moreover, there is a strong skewing in favor of TATA-less promoters on the X chromosome. Therefore, it is also possible that many individual X-linked genes have evolved features that render them more sensitive to the X dosage effects or less sensitive to the acetylation level. Consequently, with the available information, it is envisioned that as the heteromorphic sex chromosome situation evolved in Drosophila by degeneration of one member of a homologous pair of chromosomes to form the Y, dosage effects would come into play and produce compensation of most X-linked genes together with a tendency for the doubling of the expression of the autosomes in males. The sequestration of a histone acetylase to the X chromosome would mute the effect on the autosomes. The MSL complex on the X would inactivate the response to H4Lys16 acetylation for most genes, to maintain the hyperactivation at the twofold level. Thus it is suggested that sequestration of the MSL proteins occurs in males to nullify on the autosomes and maintain on the X (an inverse effect produced by negatively acting dosage-dependent regulatory genes) as a consequence of the evolution of the X/Y sex chromosomal system. Thus, this model proposes a single mechanism that explains the five levels of transcription involved with the various cases of dosage compensation, the MSL binding on the X in males, and the gene expression pattern in the msl mutants (Bhadra, 1999).

The mlenapts mutation in the maleless RNA helicase gene causes temperature-dependent blockade of action potentials resulting from decreased abundance of para-encoded Na+ channels. The napts allele (for no action potential) of mle was originally isolated on the basis of its temperature-sensitive (ts) paralytic phenotype, which is associated with a temperature-dependent block in nerve conduction at restrictive temperatures (Wu, 1978). The behavioral and electrophysiological phenotypes of the mlenapts mutation are nearly indistinguishable from those of parats mutations, which are conditional mutations in the gene encoding the primary type of Na+ channel expressed in the Drosophila nervous system. Numerous genetic experiments strongly suggest that the phenotypic defects of mlenapts result from an unconditional decrease in the expression of para-encoded Na+ channels and that this deficit in wild-type Na+ channel expression confers a ts paralytic phenotype on mutant flies. The most compelling genetic evidence is that the paralytic phenotype of mlenapts mutants can be completely rescued by the addition of a single extra dose of para+ (Reenan, 2000 and references therein).

Although maleless encodes a double-stranded RNA (dsRNA) helicase, exactly how mlenapts affects para expression has remained uncertain. para transcripts undergo adenosine-to-inosine (A-to-I) RNA editing via a mechanism that apparently requires dsRNA secondary structure formation encompassing the edited exon and the downstream intron. In an mlenapts background, >80% of para transcripts are aberrant, owing to internal deletions that include the edited exon. It is proposed that the Mle helicase is required to resolve the dsRNA structure and that failure to do so in an mlenapts background causes exon skipping because the normal splice donor is occluded. These results explain how mlenapts affects Na+ channel expression and provide new insights into the mechanism of RNA editing (Reenan, 1999).

What is known about the mechanism of this type of editing in mammalian glutamate receptor subunits (GluRs) highlights the possibility that the mlenapts defect is exerted through an involvement in RNA editing. RNA editing of GluRs and serotonin 5-hydroxytryptamine 2C receptor (5-HT2CR) in the mammalian brain introduces changes in the coding potential of messages via hydrolytic deamination of adenosine (A) residues to inosine (I). Altered coding is due to the base-pairing properties of inosine, which resemble those of guanosine (G). Editing of two distinct sites (the Q/R and R/G sites) in transcripts from several GluR genes has profound functional consequences. For instance, the Q/R site controls Ca2+ permeability, while the R/G site affects rates of receptor desensitization. As elucidated for GluR transcript editing sites, the mechanism of A-to-I editing requires a cis region of the primary transcript that extends into the intron downstream of the exonic editing site. This intronic region contains an editing-site complementary sequence (ECS) that base pairs with the exonic sequences surrounding the edited adenosine to form a dsRNA substrate for the editing enzyme, an adenosine deaminase that acts on RNA, or ADAR. Furthermore, the region between the ECS and the exonic editing site may contain extended inverted repeat hairpins or more extensive secondary structures with significant stretches of duplex dsRNA. Formation of this large scale dsRNA structure brings the ECS and the editing site sequences into appropriate juxtaposition for efficient editing. It is presumed that subsequent to editing, the dsRNA structure must be resolved before splicing can take place (Reenan, 1999).

If the mechanism of editing of para transcripts is similar to that of mammalian GluR transcripts, then the downstream intron should be capable of forming an extended dsRNA secondary structure that juxtaposes the para editing site and the ECS. RNA secondary structure prediction programs were used to obtain hypothetical RNA secondary structures using the entire edited exon and downstream intronic sequences of D. melanogaster as input sequence. The program predicted a complex, highly base-paired RNA secondary structure for this sequence. The salient feature of this structure is that it brings the putative ECS into register with the edited exonic sequences. Although the sequence of the downstream intron of D. virilis differs substantially from that of D. melanogaster outside of the ECS, the computer program also predicted an extensive dsRNA secondary structure for the D. virilis sequence that aligns the editing site with the corresponding ECS. The D. melanogaster and D. virilis RNA structures also share a high level of 'double-strandedness' (64% and 67% base pairing, respectively), although the overall secondary structures bear little resemblance to one another. The key structural feature in common is the alignment of editing sites with intronic ECSs. The D. melanogaster and D. virilis structures are nearly identical near the editing sites; edited adenosine residues are in identical positions. Moreover, the edited adenosine residues are contained in regions of dsRNA duplex that bear striking similarity to the proposed structures for the 5-HT2CR and GluR-B,5,6 Q/R editing sites (Reenan, 1999).

The mlenapts mutation causes ts paralysis and action potential failure in both males and females, demonstrating that its effect is independent of dosage compensation. Null mutations of mle are male lethal because of a failure in dosage compensation leading to inadequate expression of X-linked genes. However, homozygous mle females are viable and do not manifest any behavioral or electrophysiological defects. These results imply that although mlenapts is fully recessive to mle+, it encodes an altered protein whose effect on Na+ channels is more severe than the complete absence of the protein. To investigate whether this is also true at the molecular level, several other mle genotypes were examined for their effects on para transcripts. Examination of para RT-PCR products derived from females homozygous for a null allele of mle, reveals no detectable deletions caused by exon skipping. Analysis of para transcripts from mlenapts/mle+ heterozygotes yields similar results. Thus, there is complete concordance between the behavioral phenotype of mlenapts and the occurrence of the splicing catastrophe of the para transcript. Furthermore, with respect to anomalous splicing of the para transcript, the effect of the protein encoded by the mlenapts allele is recessive to the wild-type protein and more severe than complete absence of the protein. A model is presented to explain the results. According to this model, the Mle helicase is required to resolve the dsRNA editing structure prior to splicing. The mutant protein encoded by mlenapts binds to the dsRNA substrate but fails to resolve the structure triggering the occurrence of aberrant splicing and exon skipping. In addition, binding of the mutant protein prevents other functionally redundant helicases from acting. In the complete absence of the Mle helicase, these other helicases, which may be part of the splicing machinery itself, could compensate for the loss of Mle (Reenan, 1999).

The roX1 and roX2 genes of Drosophila produce non-coding transcripts that localize to the X-chromosome. In spite of their lack of sequence similarity, they are redundant components of an RNA/protein complex that up-regulates the male X-chromosome, contributing to the equalization of X-linked gene expression between males and females. roX1 is detected at 2 h AEL, prior to formation of the complex, and is present in both sexes. Maternally provided MLE (Maleless) is required for roX1 stability. By contrast, roX2 is male-specific and is first observed at 6 h. Either roX transcript can support X-localization of the complex, but localization is delayed in roX1 mutants until roX2 expression. These results support a model for the ordered assembly of the complex in embryos (Meller, 2003).

During larval and adult stages, roX1 is highly unstable in the absence of an intact dosage compensation complex. The embryonic transcription of roX1 precedes expression of MSL2 and formation of the complex, yet roX1 appears stable in embryos. In addition, until 10 h AEL roX1 transcripts are detected in female embryos, which lack MSL2. With the exception of MSL2, the MSL proteins are maternally provisioned and support the formation of the initial dosage compensation complexes. To test the hypothesis that one or more of these maternal proteins is responsible for roX1 stability, roX1 was examined in embryos from mothers homozygous for mutations in each of the msl genes. As anticipated, the maternal genotype with respect to the missense msl21 mutation has no effect. Early roX1 expression is also unchanged in embryos from females homozygous for a null allele of male-specific lethal 1 (msl1L60, a 2 kb deletion removing most of the coding region), male-specific lethal 3 (msl32), and males absent on first (mof1 and mof2, missense and nonsense mutations, respectively). By contrast, although an initial burst of roX1 expression is detected in blastoderm stage embryos produced by mothers homozygous for the nonsense mle1 mutation, roX1 disappears upon gastrulation. Between 4 and 5 h, roX1 can once more be detected. MLE is a member of the DExH family of helicases, and has RNA and DNA helicase activity in vitro. Interestingly, a similar lack of roX1 stability was detected in embryos expressing the mutated MLEDQIH protein that lacks helicase activity, indicating that this activity is required for roX1 stability. MLE has also been linked to the ability of the roX transcripts to travel from their sites of synthesis on the polytene X-chromosome. In embryos from mle1 mothers, spots of transcription within blastoderm nuclei are particularly apparent, and embryos displaying either one or two sites of synthesis per nucleus are readily observed. This suggests that MLE is also required to move roX1 from its site of synthesis in embryos. These results demonstrate that none of the MSL proteins are required for initial roX1 transcription, but maternal stores of MLE contribute to roX1 RNA stability in early embryos (Meller, 2003).

The occasional observation of weak foci of MSL2 staining in older male embryos carrying X-chromosomes mutated for both of the roX genes suggests either that roX RNA is not absolutely essential for localization of the dosage compensation complex, or that the roX1ex6 allele, a partial excision, retains some ability to produce functional transcripts, or that other genes produce transcripts which can partially support complex assembly and localization. All of these theories have been previously raised to account for a low level of developmentally delayed adult male escapers that are roX1ex6 roX2- (Meller, 2003 and references therein).

The findings of this study will support a model for the assembly of dosage compensation complexes during embryogenesis. roX1, highly expressed in all blastoderm stage embryos, is transcribed in advance of MSL2 translation and is likely to form an initial complex with MLE. It is possible that several of the MSL proteins must contact roX1 during assembly of the complex. In all, three members of the dosage compensation complex, MLE, MSL3 and MOF, have been reported to have RNA binding activity in vitro, or to be removed from the X-chromosome by RNase A digestion. With the exception of MSL2, all of the MSL proteins are present upon initial transcription of roX1 at 2 h AEL. The onset of dosage compensation has been linked to the male-limited production of MSL2 about 3 h AEL. This sequence of events suggests that MSL2 may complete a complex that is already organized by several RNA-binding proteins and the roX1 transcript. The proposed primary association between roX1 and MLE could reflect a need for the MLE helicase activity to disrupt incorrect base pairing or RNA/protein interactions preventing the large roX1 transcripts from correctly assembling with the other RNA-binding proteins of the dosage compensation complex (Meller, 2003).

In Drosophila, dosage compensation requires assembly of the Male Specific Lethal (MSL) protein complex for doubling transcription of most X-linked genes in males. The recognition of the X chromosome by the MSL complex has been suggested to include initial assembly at ~35 chromatin entry sites and subsequent spreading of mature complexes in cis to numerous additional sites along the chromosome. To understand this process further, MSL patterns were examined in a range of wild-type and mutant backgrounds producing different amounts of MSL components. The data support a model in which MSL complex binding to the X is directed by a hierarchy of target sites that display different affinities for the MSL proteins. Chromatin entry sites differ in their ability to provide local intensive binding of complexes to adjacent regions, and need high MSL complex titers to achieve this. A set of definite autosomal regions (~70), competent to associate with the functional MSL complex in wild-type males, was mapped. Overexpression of both MSL1 and MSL2 stabilizes this binding and results in inappropriate MSL binding to the chromocenter and the 4th chromosome. Thus, wild-type MSL complex titers are critical for correct targeting to the X chromosome (Demakova, 2003).

A functional dosage compensation complex required for male killing in Drosophila

Bacteria that selectively kill males ('male-killers') were first characterized more than 50 years ago in Drosophila and have proved to be common in insects. However, the mechanism by which sex specificity of virulence is achieved has remained unknown. This study tested the ability of Spiroplasma poulsonii to kill Drosophila melanogaster males carrying mutations in genes that encode the dosage compensation complex. The bacterium failed to kill males lacking any of the five protein components of the complex (Veneti, 2005).

Certain isofemale lines of Drosophila only give rise to daughters following the death of male embryos. Male death is due to the presence of intracellular bacteria that pass from a female to her progeny and that selectively kill males during embryogenesis. These male-killing bacteria are found in a wide range of other insect species, and many different bacteria have evolved male-killing phenotypes. In some host species, male-killers drive the host population sex ratio to levels as high as 100 females per male and alter the pattern of mate competition. However, the underlying processes that produce male-limited mortality are unclear. This study examined the interaction between the male-killing bacterium Spiroplasma poulsonii and the sex determination pathway of D. melanogaster (Veneti, 2005).

The primary signal of sex in Drosophila is the X-to-autosome ratio. This signal is permanently established in expression of Sex-lethal (Sxl) in females and its absence in males. This, in turn, effects three processes: germline sexual identity, somatic sexual differentiation, and dosage compensation, the process by which the gene expression titer on the X chromosome is equalized between two sexes despite their difference in X chromosome number. Mutations in the gene tra that convert XX individuals to male somatic sex do not induce female death. These observations indicate germline formation and migration happen correctly in male embryos and that dying male embryos do not express Sxl. Therefore, the requirement of the Spiroplasma for genes within the system of dosage compensation was examined (Veneti, 2005).

In Drosophila, the single X of males is hypertranscribed. This process of hypertranscription requires the formation of the dosage compensation complex (DCC) and its binding to (and modification of) the X chromosome. SXL in female Drosophila inhibits the production of MSL-2 protein, which is thus only present in male Drosophila. MSL-2 forms a complex with four other proteins, MSL-1, MSL-3, MLE, and MOF, which collectively form the DCC. MSL-1, MSL-3, MLE, and MOF are constitutively present in both males and females and are also supplied maternally. The complete DCC binds, with JIL-1, to the male X chromosome at various entry points, and, with the products of two noncoding RNAs, RoX1 and RoX2, it affects the modification of the single X chromosome and its hypertranscription (Veneti, 2005).

The effect was examined of mutations within the host DCC on the ability of the male-killer to function. The survival of male progeny beyond embryogenesis to L2/L3 (and in one case adult) was scored in the presence of different loss-of-function mutations within the dosage compensation system (normal male-killing occurs during embryogenesis), in the presence and absence of infection. Because many genes within this group additionally show strong maternal effects, the effect of mutations was in each case tested by using both mothers that were heterozygous for the loss-of-function mutation and mothers homozygous for it (Veneti, 2005).

Uninfected males homozygous for loss-of-function mutations within the dosage compensation system generally survive to the third larval instar. Survival to the third larval instar was studied for loss-of-function alleles of msl-1 (alleles msl-11 and msl-1b), msl-2 (msl-2g227 and msl-2g134), msl-3 (msl-3132), mle (mle9, mle1), and mof (mof1), and survival to adult for mle1/mle6 transheterozygotes. In the case of all alleles of msl-1, msl-3, mle, and mof, a similar pattern is observed: Males homozygous or hemizygous for the loss-of-function mutation have appreciable survival in the presence of infection when their mother is also homozygous for the loss-of-function mutation. In contrast, heterozygous male embryos that were siblings of the above (that will have a wild-type DCC) were always killed, as were all male embryos in the case where the mother was heterozygous (where maternal supply of these proteins enables dosage compensation to be initiated, although not maintained). In the case of mle1/mle6 transheterozygotes, male survival to adult was observed. For the case of mof, male-killing was restored to full efficiency when 18H1, a transgenic copy of mof, was added to the mof1 loss-of-function background. Within the above crosses, three observations make sure the observation that Spiroplasma was fully operational. First, crosses involving heterozygous mothers, where male-killing was complete, were conducted concurrently with those using homozygous mothers, and the females in these crosses were siblings from the same vials. Second, in each cross and vial where homozygous males survived, heterozygous males (with wild-type function) still died. Finally, F1 females derived from these crosses, when mated to wild-type males, produced a full, female-biased sex ratio (Veneti, 2005).

In the case of msl-2, where there is no maternal supply of MSL-2, survival of homozygous sons was observed for both homozygous and heterozygous mothers for the case of the mutation msl-2g134. For the case of msl-2 g227, no male progeny were observed from infected females (table S5). This mutation does not rescue males in the assay, probably because the two mutations have different effects on msl-2 expression. The msl-2g134 allele prevents formation of MSL-2 protein, whereas msl-2 g227 potentially encodes the RING-finger element of a truncated MSL-2 protein (Veneti, 2005).

Thus, absence or reduced function of any of the proteins of the DCC can reduce the efficiency of male killing, and a functional DCC is required for male killing by S. poulsonii. The fact that the genes mediating this process in Drosophila have been well studied can be exploited to yield further insights into the mechanism of male killing (Veneti, 2005).

maleless: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | References

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