On the Genetic Control of Genes Located in the Sex-Chromosome Heterochromatin of DROSOPHILA MELANOGASTER

The genetic properties of a recessive autosomal point mutant, "daughterless" (symbol: da), are described. They are: (1) da maps in the euchromatin of 2L at about position 39 on the genetic map and between 27D and 31E on the salivary map; (2) when homozygous in females, da causes the production of unisexual male progenies owing to sex differential zygote mortality in the egg stage; (3) da has no effect on the progeny of mutant males; (4) female zygotes die, while at least some male zygotes survive, irrespective of the number of Y chromosomes or the amount of X-chromosome heterochromatin carried by either the mutant female or her progeny; (5) homozygous da progeny of heterozygous parents also show sex differential survival favoring males indicating a da effect in development as well as in oogenesis, but with the developmental effect much less pronounced; (6) extra heterochromatin from either the X or Y chromosome in either the mother or her progeny can reduce the mortality caused by da in development (and, therefore, conceivably can also reduce the abnormality in oogenesis, although this is obscured by the severity of the maternal effect.)—From these results, it is suggested that (1) da regulates either the activity of structural genes in the sex chromosome heterochromatin or the activity or stability of their products, and (2) it is a defective product of sex chromosome heterochromatic genes that causes the abnormalities resulting in the observed mortality of heterozygous progeny of homozygous mutant mothers and of homozygous mutant progeny of heterozygous mothers.—The striking parallels with the properties of the gene, "abnormal oocyte" are noted as is the close linkage between the two loci. The possibility of a special sex-chromosome-heterochromatin-regulator region on chromosome 2 is considered.     

On Biological Functions Mapping to the Heterochromatin of DROSOPHILA MELANOGASTER

We examined the behavior of an autosomal recessive maternal-effect mutation, abnormal-oocyte (abo), that is located in the euchromatin of the left arm of chromosome 2. When homozygous in females, abo results in a marked reduction in the probability that an egg produced by a mutant mother will develop into an adult. However, this probability is increased if the fertilizing sperm delivers to the egg either a normal allele of the maternal-effect gene or a specific type of heterochromatin (called ABO) that is located in small regions of the X and Y chromosome constitutive heterochromatin as well as in some autosomal heterochromatin. These regions, moreover, all react to Hoechst 33258 fluorescent dye identically and specifically. The amelioration of the maternal effect produced by this heterochromatin differs temporally from that caused by the normal allele of the euchromatic gene: the heterochromatin reduces only precellular blastoderm mortality, whereas the normal allele of the euchromatic gene reduces only postblastoderm mortality. Thus, although the genome of the preblastoderm Drosophila embryo is apparently mostly silent, the ABO-containing heterochromatin functions at this early time. Finally, preliminary data indicate that abo is but one member of a cluster of linked genes, each of which interacts with its own normal allele and with a different, locus-specific, heterochromatic factor. From these observations, it appears that Drosophila heterochromatin contains developmentally important genetic elements, and that a functional concomitant of heterochromatic location is gene action at a developmental stage during which the activity of the euchromatic genome is as yet undetectable. Some general implications of these inferences are considered.     

Analysis of the Autosomal Mutation abo and Its Interaction with the Ribosomal DNA of DROSOPHILA MELANOGASTER: The Role of X-Chromosome Heterochromatin

The autosomal recessive, maternal-effect mutation abnormal oocyte (abo: 2-38) preferentially lowers the viability os XO progeny. The severity of the sex-ratio distortion is reduced by duplications of maternal or zygotic heterochromatin (SANDLER 1970, 1977; PARRY and SANDLER 1974). Utilizing X-chromosome inversions that contain modifications in the quantity and arrangement of the heterochromatic functions, Xhabo and cr+, wer have extended our investigations of abo's influence on XO male recovery and rDNA redundancy (KRIDER, YEDVOBNICK and LEVINE 1979).--XO males bearing In(1)SCS1LSC4R or In(1)Wm4LSC4R are recovered twice as frequently as X chromosomes containing a single Xh region, implying that these inversions possess a duplication of Xhabo. abo mutant females heterozygous for In(1)SCS1LSC4R and wild-type X chromosomes generate XO progeny that do not contain elevated rDNA redundancies. XO males containing In(1)Wm4 exhibit male recoveries and rDNA elevations similar to those of males bearing a wild-type X chromosome, when both derive from a common abo/abo mother. Reciprocal crosses baetween In(1)Wm4 and Canton-S males to attached-X abo females show significant, though reuduced, sex ratios in the absence of an rDNA effect. The observation that abo can elevate the rDNA redundancy of In(1)Wm4, a chromosome that does not compensate, suggests that abo and cr+ functions are not directly related.     

Rescue from the Abnormal Oocyte Maternal-Effect Lethality by Abo Heterochromatin in Drosophila Melanogaster

The euchromatic maternal-effect mutation abnormal oocyte (abo), of Drosophila melanogaster interacts with regions of heterochromatin known as ABO, which reside on the X, Y and second chromosomes. Here, we show that survival of progeny from abo females depends in part upon the maternal dosage of ABO heterochromatin. A comparison was made of the recovery of genotypically identical progeny from abo mothers bearing sex chromosomes of various ABO contents. The results show that the recovery of daughters was decreased if mothers were ABO-/ABO-. However, no decrease was observed if mothers were ABO+/ABO-. In addition, the survival of daughters was greater when they received an ABO-X chromosome from an ABO-/ABO+ mother rather than the father. We suggest that these results reflect a complementation or interaction between the ABO-deficient X and the ABO heterochromatin in the maternal genome. This proposed interaction could occur early in oogenesis in the mother or prior to completion of meiosis I in the fertilized egg. To determine if zygotic dosage of ABO heterochromatin might also be important at very early stages of embryogenesis, we examined the timing of zygotic rescue by paternally donated ABO heterochromatin using a second mutation, paternal loss (pal). Homozygous pal males produce progeny which lose paternally derived chromosomes during the early zygotic divisions. Zygotes that have lost a paternal sex chromosome in a fraction of their nuclei will be mosaic for the amount of ABO heterochromatin. By monitoring the recovery of pal-induced mosaics from abo and abo+ females, we could determine the temporal and spatial requirements for ABO function. Results show that the survival of progeny from the abo maternal-effect lethality was increased if ABO heterochromatin was present prior to the pal-induced loss event. Analysis of mosaic patterns did not reveal a specific lethal focus. We conclude from these results that ABO heterochromatin serves its vital function prior to completion of the early cleavage divisions in progeny of abo mothers.     

Heterochromatic ABO locus of the Drosophila melanogaster X chromosome overlaps with the region of localization of repeats, including mobile elements and Stellate genes

A loss of certain heterochromatic regions (ABO loci) of various chromosomes dramatically distorts the early embryo development in the progeny of females mutant for the abnormal oocyte (abo) gene, which is located in euchromatin of chromosome 2. One ABO locus (X-ABO) is in X-chromosomal heterochromatin distal of the nucleolus organizer. A cluster of the Stellate repeats is located in the same heterochromatin block. Deletions of various fragments from distal heterochromatin were tested for the effect on expression of the abo mutation. The X-ABO locus was assigned to X-chromosomal heterochromatin segment h26 and shown to include repeats consisting mostly of mobile elements and defective Stellate copies. A major part of the regular Stellate tandem repeats proved to be distal of the X-ABO locus.     

The Abnormal Oocyte Phenotype Is Correlated with the Presence of Blood Transposon in Drosophila Melanogaster

The abnormal oocyte mutation (2;44) originates in the wild: it confers no visible phenotype on homozygous abo males or females, but homozygous abo females produce defective eggs and the probability of their developing into adults is much lower than that of heterozygous sister females. We isolated by chromosome walking 200 kb of DNA from region 32. This paper reports that a restriction enzyme site polymorphism analysis in wild type and mutant stocks allowed us to identify a DNA rearrangement present only in stocks carrying the abo mutation. The rearrangement is caused by a DNA insert on the abo chromosome in region 32E which, by restriction map and sequence analysis, was identified as copia-like blood transposon. The transposon, in strains that had remained in abo homozygous conditions for several generations and had lost the abo maternal-effect, was no longer present in region 32E. Certain features of the abo mutation, discussed in the light of this finding, may be ascribed to the nature of the particular allele studied.     

Maternal and Zygotic Sex-Specific Gene Interactions in DROSOPHILA MELANOGASTER

Sex-lethal (Sxl) is a vital, X-chromosome gene involved in Drosophila sex determination. The most striking aspect of the phenotype of daughterless (da), an autosomal maternal-effect mutation, may be explained by effects on the functioning of the Sxl gene in the zygote. In this paper, new aspects of interactions between various combinations of Sxl and da alleles are explored in order to understand better the complex da phenotype. The study focuses on the relationship between maternal and zygotic da+ gene functions, and on the relationship between aspects of the da phenotype that are sex-specific and aspects that are not. The SxlM#1 allele, which counteracts the female-specific maternal effect of da, is shown to have no effect on two other aspects of the da phenotype (one maternal, one primarily zygotic) that are not sex-specific. The female-lethal da maternal effect is shown to kill daughters even when the progeny are entirely wild-type with respect to da. Recessive mutant alleles of the two genes can interact synergistically when both are heterozygous with their wild-type alleles, disrupting the development of most of the daughters. Surprisingly, even a deficiency of the da+ locus can produce a dominant, temperature-sensitive, female-lethal maternal effect. A new class of subliminal Sxlf alleles is described. These spontaneous mutations can confuse analysis of both da and Sxl if their presence is not appreciated. Finally, conditions are described that facilitate the study of the Enhancer of daughterless mutation.     

The Genetic Factors Altered in Homozygous abo Stocks of DROSOPHILA MELANOGASTER

Females homozygous for the maternal-effect mutation abo (2-44.0) produce a large fraction of eggs which arrest during embryogenesis. Increasing doses of defined heterochromatic regions inherited by offspring of abo mothers from their fathers function zygotically to bring about a partial rescue of the abo-induced embryonic lethality. Another property of the abo mutation is that the severity of the maternal effect decreases when an abo stock is maintained in homozygous condition for a number of generations. Here, we show that the factors which change in homozygous abo stocks to result in the decrease in maternally induced embryonic lethality, act zygotically, dominantly and additively. More importantly, we show that the X and second chromosomes, but not the Y and third chromosomes, derived from homozygous abo stocks are, when inherited from males, more effective in promoting zygotic rescue of the abo-induced lethality than are the equivalent chromosomes derived from an abo stock maintained in heterozygous condition. The chromosomal locations of the factors maintained in the homozygous condition. The chromosomal locations of the factors altered in homozygous stock, as well as their behavior, strongly suggest that the same heterochromatic elements that are responsible for rescuing embryos from the abo-induced maternal effect are altered in homozygous abo flies in such a way that the maternal effect itself is less severe.     

Two Closely Linked Mutations in DROSOPHILA MELANOGASTER That Are Lethal to opposite Sexes and Interact with Daughterless

A new spontaneous mutation named Sex-lethal, Male-specific No. 1 (SxlM1) is described that is lethal to males, even in the presence of an Sxl+ duplication. Females homozygous for SxlM1 are fully viable. This dominant, male-specific lethal mutation is on the X chromosome approximately 0.007 map units to the right of a previously isolated female-specific mutation, Female-lethal, here renamed Sex-lethal, Female-specific No. 1 (SxlF1). SxlM1 and SxlF1 are opposite in nearly every repect, particularly with regard to their interaction with maternal effect of the autosomal mutation, daughterless (da). Females that are homozygous for da produce defective eggs that cannot support female (XX) development. A single dose of SxlM1 enables daughters to survive this da female-specific lethal maternal effect. A duplication of the Sxl locus weakly mimics this action of SxlM1. In contrast, SxlF1 and a deficiency for Sxl, strongly enhance the female-lethal effects of da. The actions of SxlM1 and SxlF1 are explained by a model in which expression of the Sxl locus is essential for females, lethal for males, and under the control of a product of the da locus. It is suggested that SxlM1 is a constitutive mutation at the Sxl locus.     

A New Mutant Controlling Mitotic Chromosome Disjunction in DROSOPHILA MELANOGASTER

A new mutant, mit (mitotic loss inducer), is described. The mutant is recessive and maternal in action, producing gynandromorphs and haplo-4 mosaics among the progeny of homozygous mit females. Mosaic loss of maternal or paternal chromosomes can occur. The probabilities of either maternal or paternal X chromosome loss are equal. mit has been mapped to approximately 57 on the standard X chromosome map.-Using gyandromorphs generated by mit, a morphogenetic fate map, placing the origins of 40 cuticular structures on the blastoderm surface, has been constructed. This fate map is consistent with embryological data and with the two other fate maps generated in different ways.     

E(var)3-9 of Drosophila melanogaster encodes a zinc finger protein

The importance of a gene's natural chromatin environment for its normal expression is poignantly illustrated when a change in chromosome position results in variable gene repression, such as is observed in position effect variegation (PEV) when the Drosophila melanogaster white (omega) gene is juxtaposed with heterochromatin. The Enhancer of variegation 3-9 [E(var)3-9] gene was one of over a hundred loci identified in screens for mutations that dominantly modify PEV. Haploinsufficiency for E(var)3-9 enhances omegam4 variegation, as would be expected from increased heterochromatin formation. To clarify the role of E(var)3-9 in chromosome structure, the gene has been cloned and its mutant alleles characterized. The involvement of E(var)3-9 in structure determination was supported by its reciprocal effects on euchromatic and heterochromatic PEV; E(var)3-9 mutations increased expression of a variegating heterochromatic gene in two tissue types. E(var)3-9 mutations also had a recessive phenotype, maternal effect lethality, which implicated E(var)3-9 function in an essential process during embryogenesis. Both phenotypes of E(var)3-9 mutations were consistent with its proposed function in promoting normal chromosome structure. The cloning of E(var)3-9 by classical genetic methods revealed that it encodes a protein with multiple zinc fingers, but otherwise novel sequence.     

Evidence for a Set of Closely Linked Autosomal Genes That Interact with Sex-Chromosome Heterochromatin in DROSOPHILA MELANOGASTER

It is proposed that there exists a special region in the euchromatin of the left arm of chromosome 2 (contained within sections 31-32 of the standard salivary gland chromosome map) that is defined by a set of genes, each one of which interacts with a specific sex-chromosome heterochromatic segment. The evidence for the existence of this region is, first, the exhibition, mapping, and analysis of five different maternal-effect, embryonic semi-lethals located in region 31-32. Secondly, in each case the consequence of the maternal effect is markedly influenced by the amount of X- or Y-chromosome heterochromatin carried by the progeny of mutant mothers. The nature of this interaction and possible reasons for the existence of the cluster of autosomal genes are discussed.     

The Effect of ABO Phenotypic Expression on Ribosomal DNA Instabilities in DROSOPHILA MELANOGASTER

The recessive maternal-effect mutation, abnormal oocyte (abo:2--38), reduces viability in the offspring of homozygous mutant females. Zygotes lacking specific heterochromatic segments of the X or Y chromosomes are most severely affected. We have shown that abo/abo lines can lose the capacity to express the mutant phenotype, and that elevated rDNA redundancies can be observed in such stocks (Krider and Levine 1975). In this study, we describe a microhybridization procedure that facilitates the measurement of rDNA redundancy, using a small number of adult Drosophila. We show that instability of the rDNA content persists in an abo/abo line after loss of the capacity to express the phenotype, and that changes in rDNA amounts occur between successive generations of the stock. Further, we show that the rDNA content of XO progeny from abo/abo females is elevated. The effect is directly correlated with the expression of the abo phenotype, and it is not observed in the XO progeny of abo heterozygous females or abo homozygotes from lines that do not show abo expression.     

The relationship of relative gene dose to the complex phenotype of the daughterless locus in Drosophila

The daughterless (da) gene provides an essential maternally supplied component for Drosophila sex determination and dosage compensation. In this connection, it is required as a positive regulator of a female-specific master regulatory gene, Sex-lethal (Sxl). In addition, zygotic da gene function is required for male and female viability. Thus, the phenotype da is complex; it includes both maternal and zygotic aspects, as well as both sex-specific and nonsex-specific aspects. Assessment of wild-type da function has relied on the characterization of only a single leaky mutant da allele. In order to better understand the nature of this allele and the relationships between the various aspects of its complex phenotype, tandem duplications of both the mutant and wild-type da alleles were isolated and used in a dose study of this gene's function. Three conclusions were reached: 1) by the most stringent genetic criteria, the mutant da allele is a simple hypomorph, an allele with reduced but non-zero levels of wild-type functions; 2) since increased dose of da+ had no effect on viability or progeny sex ratio, this gene seems not to be a dose-sensitive element of the X/A ratio sex determination signal; and 3) expression of the maternal da+ allele does make a contribution to the nonsex-specific developmental processes that require zygotic da+ function; however, that contribution is clearly minor. In contrast, the zygotic genotype with respect to da appears to have no effect on the expression of Sxl+ in the zygote, the sex-specific process that requires maternal da+ function.     

Interaction between maternal effect and zygotic effect mutations during maize seed development

Double fertilization of the embryo sac by the two sperm cells of a pollen grain initiates seed development. Proper development of the seed depends not only on the action of genes from the resulting embryo and endosperm, but also on maternal genes acting at two stages. Mutations with both sporophytic maternal effects and gametophytic maternal effects have been identified. A new maternal effect mutation in maize, maternal effect lethal1 (mel1), causes the production of defective seed from mutant female gametophytes. It shows reduced pollen transmission, suggesting a requirement in the male gametophyte, but has no paternal effect on seed development. Interestingly, the defective kernel phenotype of mel1 is conditioned only in seeds that inherit mel1 maternally and are homozygous for the recessive allele (endogenous to the W22 inbred line) of either of two genes, sporophyte enhancer of mel1 (snm1) or snm2, suggesting redundancy between maternally and zygotically required genes. Both mel1 and snm1 map to the short arm of chromosome 2, and snm2 maps to the long arm of chromosome 10. The mode of action of mel1 and the relationship between mel1 and snm1 and snm2 are discussed.     

Genetic variability of the tumorous-head maternal effect in Drosophila melanogaster

Summary The tumorous-head maternal effect in Drosophila melanogaster is produced by a recessive gene (tuh-1) in chromosome 1. Polymorphism exists at this locus. This maternal effect, which is part of the normal variation found in this species, is detected with the aid of a mutant gene. In the presence of the maternal effect, a semi-dominant mutant gene (tuh-3) causes homoeotic changes in the eye-antennal imaginal discs. The phenotype in the adult is known as the tumorous-head abnormality. The mutant gene, which is located in the right arm of chromosome 3, is characterized by reduced penetrance. Using the penetrance of the mutant gene as the criterion, the results of these experiments show that the level of the maternal effect activity is influenced remarkably by modifiers present in wild type strains. The assay is to mate females homozygous for tuh-1 with males homozygous for tuh-3 and to determine the percent of the offspring showing the tumorous head abnormality. Using this procedure, it was observed that parental females with various combinations of chromosomes 1 and 3 from Lausanne and Stephenville wild type strains show great variability in the level of maternal effect activity. Modifiers in chromosome 1 and 3 from the Stephenville strain increase the level of the maternal effect activity. The level is reduced if these chromosomes are replaced by those from the Lausanne strain. A major locus in chromosome 3 is in the same region occupied by clusters of functionally related genes with regulating action. These results demonstrate that the penetrance of a mutant gene, which acts during embryogenesis, is influenced by modifiers which act during oogenesis.This investigation was supported by Public Health Service Research Grant GM 18664 to Arizona State University from the National Institute of General Medical Sciences     

Polytene chromosome sof monogenic and amphogenic Chrysomya species (Calliphoridae,diptera): analysis of banding patterns and in situ hybridization with Drosophila sex determining gene sequences

Standard maps for the five banded polytene chromosomes found in trichogen cell nuclei of the monogenic blowfly Chrysomya rufifacies and the amphogenic Chrysomya pinguis are presented. The chromosomes are highly homologous in the two species; differences in banding patterns are predominantly caused by one pericentric and ten paracentric inversions. In chromosome 5 of the amphogenic Chrysomya phaonis, also analysed in this paper, an additional paracentric inversion was observed. The distribution of species specific inversions indicates that the monogenic C. rufifacies is phylogenetically older than the amphogenic species. The maternal sex realizer locus F'/f on polytene chromosome 5 of C. rufifacies is not associated with a structural heterozygosity. Chromosome pair 6 of C. rufifacies and the sex chromosome pair of C. pinguis are under-replicated in polytene nuclei; they consist of irregular chromatin granules, frequently associated with nucleolus material. Evolution of heteromorphic sex chromosomes in Chrysomya is probably correlated with heterochromatin accumulation. A search for sex determining genes in Chrysomya was initiated using sex determining sequences from Drosophila melanogaster for in situ hybridization. The polytene band 41A1 on chromosome 5 of monogenic and amphogenic Chrysomya species contains sequences homologous to the maternal sex determining gene daughterless (da). Homology to the zygotic gene Sex-lethal (Sxl) of Drosophila is detected in band 39A1 on chromosome 5 of C. rufifacies. The findings reported here are the first evidence for a possible homology between the da gene of Drosophila and the maternal sex realizer F of C. rufifacies. An hypothesis for the evolution of the maternal effect sex determination of C. rufifacies is proposed.Dedicated to Professor Dr. Fritz-Helmut Ullerich on the occasion of his 60th birthday     

Rare etiology of autosomal recessive disease in a child with noncarrier parents

A child with maple syrup urine disease type 2 (MSUD2) was found to be homozygous for a 10-bp MSUD2-gene deletion on chromosome 1. Both purported parents were tested, and neither carries the gene deletion. Polymorphic simple-sequence repeat analyses at 15 loci on chromosome 1 and at 16 loci on other chromosomes confirmed parentage and revealed that a de novo mutation prior to maternal meiosis I, followed by nondisjunction in maternal meiosis II, resulted in an oocyte with two copies of the de novo mutant allele. Fertilization by a sperm that did not carry a paternal chromosome 1 or subsequent mitotic loss of the paternal chromosome 1 resulted in the propositus inheriting two mutant MSUD2 alleles on two maternal number 1 chromosomes.     

Identification of the female-determining region of the W chromosome in Bombyx mori

The W chromosome of the silkworm Bombyx mori is devoid of functional genes, except for the putative female-determining gene (Fem). To localize Fem, we investigated the presence of W-specific DNA markers on strains in which an autosomal fragment containing dominant marker genes was attached to the W chromosome. We produced new W-chromosomal fragments from the existing Zebra-W strain (T(W;3)Ze chromosome) by X-irradiation, and then carried out deletion mapping of these and sex-limited yellow cocoon strains (T(W;2)Y-Chu, -Abe and -Ban types) from different Japanese stock centers. Of 12 RAPD markers identified in the normal W chromosomes of most silkworm strains in Japan, the newly irradiated W(B-YL-YS)Ze chromosome contained three, the T(W;2)Y-Chu chromosome contained six, and the T(W;2)Y-Abe and -Ban chromosomes contained only one (W-Rikishi). To investigate the ability of the reduced W-chromosome translocation fragments to form heterochromatin bodies, which are found in nuclei of normal adult female sucking stomachs, we examined cells of the normal type p50 strain and the T(W;2)Y-Chu and -Abe strains. A single sex heterochromatin body was found in nuclei of p50 females, whereas we detected only small sex heterochromatin bodies in the T(W;2)Y-Chu strain and no sex heterochromatin body in the T(W;2)Y-Abe strain. Since adult females of all strains were normal and fertile, we conclude that only extremely limited region, containing the W-Rikishi RAPD sequence of the W chromosome, is required to determine femaleness. Based on a comparison of the normal W-chromosome and 7 translocation and W-deletion strains we present a map of Fem relative to the 12 W-specific RAPD markers.