male-specific lethal 3
DEVELOPMENTAL BIOLOGY

Effects of Mutation or Deletion

Investigations of the regulation of dosage compensation in Drosophila

In Drosophila, dosage compensation occurs by transcribing the single male X chromosome at twice the rate of each of the two female X chromosomes. This hypertranscription requires four autosomal male-specific lethal (msl) genes and is negatively regulated by the Sxl gene in females. Two of the msls, the mle and msl-1 genes, encode proteins that are associated with hundreds of specific sites along the length of the male X chromosome. MLE and MSL-1 X chromosome binding are negatively regulated by Sxl in females and require the functions of the other msls in males. To investigate further the regulation of dosage compensation and the role of the msls in this process, another msl, the msl-3 gene, has been cloned and molecularly characterized. MSL-3 was also found to be associated with the male X chromosome. It was further investigated whether Sxl negatively regulates MSL-3 X-chromosome binding in females and whether MSL-3 X-chromosome binding requires the other msls. The results suggest that the MLE, MSL-1 and MSL-3 proteins may associate with one another in a male-specific heteromeric complex on the X chromosome to achieve its hypertranscription (Gorman, 1995).

To investigate the role of the MSL-3 protein in the hypertranscription of the male X chromosome, polyclonal rat antibodies were raised against two separate parts of the MSL-3 protein. The first antibodies (ab1 antibodies) were raised against a MSL-3(aa324-500)-TRPE fusion protein; the second antibodies (ab2 antibodies) were raised against a beta-GALACTOSIDASE- MSL-3(aa141-323) fusion protein. Both of these antibodies were affinity purified. The predicted size of the MSL-3 protein is approximately 57,103 Mr. Consistent with this prediction, in Western analyses, ab2 antibodies detect an approximately 58,103 Mr protein that is present in wild-type male and female larvae, but is severely reduced in amount in females. This protein is not detectable in two different msl-3 mutants but protein reappears in msl-3 mutants that are transgenic for a construct that contains the entire msl-3 gene. Furthermore, in msl-3 mutants that are transgenic for construct E, which contains a genomic fragment that is predicted to produce a MSL-3 protein that is missing the carboxyl 23 amino acids, a smaller protein is detected. Similar results are obtained with ab1 antibodies (Gorman, 1995).

To investigate the intracellular localization of the MSL-3 protein, the salivary gland polytene chromosomes of male and female third instar larvae were stained with anti-MSL-3 antibodies. It was found that the MSL-3 protein, like the MLE and MSL-1 proteins, is associated with hundreds of specific sites along the length of the male X chromosome, suggesting that MSL-3 may also be a direct mediator of dosage compensation. No anti-MSL-3 staining is detected in females. Also, 10-20 autosomal sites of MSL-3 association was detected in males. These sites are in general more difficult to detect than the X-linked sites of MSL- 3 association, particularly at lower magnification. Additionally, they are highly variable in intensity, with a given band being sometimes quite bright but sometimes not detectable. This variability is seen among different cells in the same salivary gland. Of the 10-20 sites, three sites that were seen relatively consistently were mapped to 21B, 30B and 56A. The autosomal sites of MSL-3 association are detected by both ab1 and ab2 anti-MSL-3 antibodies and are not detected in msl-3P homozygotes (Gorman, 1995).

The association of the MLE, MSL-1 and MSL-3 proteins with hundreds of sites along the length of the male X chromosome, raises the question whether these proteins are associated with the same sites. The MLE and MSL-1 proteins are co-localized on the male X chromosome. To investigate whether MSL-3 is co-localized with MLE and MSL-1, double-labeling experiments were performed with either anti-MSL-3 and anti-MSL-1 antibodies or anti-MSL-3 and anti-MLE antibodies. It was found that both the sites of MSL-3 and MSL-1 association and the sites of MSL-3 and MLE association are indistinguishable. Furthermore, the intensities of the MSL-3 and MSL-1 or MSL-3 and MLE bands appear to be correlated. These data suggest that the MSL proteins function at the same X-linked sites and are present at these sites in similar relative amounts. MSL-1 is also associated with 10-20 autosomal sites in males and MLE is associated with 30-40 autosomal sites in both sexes. In males, all the autosomal sites of MSL-1 association are also sites of MLE association. Similarly, MSL-3 and MSL-1 are co-localized at autosomal sites. These data suggest that the MSL proteins may function similarly at X-linked sites and the subset of autosomal sites where all of them are found (Gorman, 1995).

Dosage compensation is under the control of the Sxl gene, which prevents both the MLE and MSL-1 proteins from associating with the female X chromosomes. To determine whether Sxl also prevents the MSL-3 protein from binding to the female X chromosomes, the salivary gland chromosomes were stained of females that were heteroallelic for a null mutation in Sxl, Sxlf1 and a partial loss-of-function mutation in Sxl, Sxlfhv1. Although females homozygous for Sxl null mutations die as embryos, some heteroallelic Sxlf1/Sxlfhv1 females live long enough and are healthy enough, to yield chromosome squashes. The salivary glands of these females are mosaic with respect to Sxl expression. When they are stained with an anti-SXL antibody, which recognize 40-50 sites in the chromosomes of wild-type females, but does not stain the chromosomes of wild-type males, some cells show anti-SXL staining, whereas others do not. When the salivary gland chromosomes of Sxlf1/Sxlfhv1 females were stained with both anti-SXL and anti-MSL-3 antibodies, it was found that the cells that did not have Sxl function, as assayed by the lack of anti-SXL staining, did indeed have MSL-3 associated with the two X chromosomes. These results indicate that Sxl functions in females to prevent not only the MLE and MSL-1, but also the MSL-3 protein from associating with the female X chromosomes (Gorman, 1995).

The co-localization of the MLE, MSL-1 and MSL-3 proteins on the male X chromosome suggests that these three proteins function at the same sites on the X chromosome. The MLE and MSL-1 proteins require the functions of all the other msls for wild-type X-chromosome binding. It has been asked whether or not the MSL-3 protein also requires the functions of all the other msls for X-chromosome binding. Although msl mutant males die as late larvae or early pupae, mle and msl-3 mutant males sometimes live long enough and are healthy enough to yield chromosome squashes. To determine whether MSL-3 X-chromosome binding requires mle, the salivary gland chromosomes of mlepml8 mutant male larvae were squashed and stained with anti-MSL-3 antibodies. No staining was seen at most X-linked sites, although around 20 weak bands were detected. Similar results were obtained in Sxlf1/Sxlfhv1 females. Thus, the stable association of MSL-3 with most X-linked sites requires the function of the mle gene. msl-1 and msl-2 mutant males are not as healthy as mle mutant males. Therefore, the same approach was used that was used to determine whether MLE and MSL-1 X-chromosome binding requires the other msls to determine whether MSL-3 X-chromosome binding requires msl-1 and msl-2. Because MSL-3 binds to the X chromosomes in the cells that lack Sxl function in Sxlf1/Sxlfhv1 females, it was asked whether msl-1 and msl-2 are required for this female X-chromosome binding. When anti-SXL and anti-MSL-3 antibodies were used to stain the chromosomes of Sxlf1/Sxlfhv1; msl-1b/msl-1b or Sxlf1/Sxlfhv1; msl-2/msl-2 females, it was found that no MSL-3 X-chromosome binding was detected in the cells that lacked anti-SXL staining. Thus, the stable association of the MSL-3 protein with the X chromosome requires the wild-type functions of the mle, msl-1 and msl-2 genes, although MSL-3 appears to be able to associate weakly with around 20 X-linked sites in the absence of mle function (Gorman, 1995).

The requirements for MSL-1 X chromosome association were tested. No sites of MSL-1 association were detected in a msl-2 mutant background, but 20-40 sites of weak MSL-1 association are detected in a mle mutant background and approximately 40 sites of stronger MSL-1 association are detected in a msl-3 mutant. In double labeling experiments, the sites of MSL-1 and MSL-3 association in mle mutants are equivalent. The association of MSL-1 with approximately 40 sites in the non-Sxl-expressing cells in Sxlf1/Sxlfhv1; msl-3P/msl-3P females raised the possibility that there was also a small amount of MLE bound to the Xs in such cells. To investigate this possibility, rat antibodies were raised against the same portion of the MLE protein against which earlier antibodies had been raised. In stains of wild-type males, these rat antibodies detected X-chromosome sites relative to autosomal sites much more strongly than had the rabbit antibody with which previous analyses were performed. Rat anti-MLE antibodies revealed MLE associated with around 40 sites in the chromosomes of the non-Sxl-expressing cells in these females. Double labeling experiments further demonstrated that the sites of MLE and MSL-1 association in Sxlf1/Sxlfhv1; msl- 3P/msl-3P females are coincident. No MLE X-chromosome staining was detected in the non- Sxl-expressing cells in Sxlf1/Sxlfhv1; msl-1b/msl-1b or Sxlf1/Sxlfhv1; msl-2/msl-2 females (Gorman, 1995).

In summary, these results indicate that each of the MSL proteins requires all the other msls for wild-type X-chromosome binding. However, while no anti-MSL staining is seen in a msl-1 or msl-2 background, a small number of sites of MSL association can be detected in a mle or msl-3 mutant background. The sites of MSL-1 and MSL-3 association detected in mle mutants are much more variable, and on the whole much weaker, than the sites of MLE and MSL-1 association detected in msl-3 mutants. Additionally, in msl-3 mutants, the MLE sites are more variable, and weaker, than the MSL-1-binding sites (Gorman, 1995).

The mle mutation used in these experiments results in a truncated MLE protein that is almost certainly nonfunctional. Therefore, it is likely that MSL-1 and MSL-3 can weakly associate with some X-chromosome sites in the absence of functional Mle protein. The molecular nature of the msl-3 mutation, msl-3P, that was used in these experiments, is not known. Although no protein was detectable by Western analyses and no anti-MSL-3 staining was seen in the non-Sxl expressing cells in Sxlf1/Sxlfhv1; msl-3P/msl-3P females, it remains possible that a mutant MSL-3 protein that retains some msl-3 function is produced (Gorman, 1995).

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).


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male-specific lethal 3: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 30 December 2015

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