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.     

Hybrid Lethal Systems in the Drosophila Melanogaster Species Complex. II. the Zygotic Hybrid Rescue (Zhr) Gene of D. Melanogaster

Hybrid females from Drosophila simulans females X Drosophila melanogaster males die as embryos while hybrid males from the reciprocal cross die as larvae. We have recovered a mutation in melanogaster that rescues the former hybrid females. It was located on the X chromosome at a position close to the centromere, and it was a zygotically acting gene, in contrast with mhr (maternal hybrid rescue) in simulans that rescues the same hybrids maternally. We named it Zhr (Zygotic hybrid rescue). The gene also rescues hybrid females from embryonic lethals in crosses of Drosophila mauritiana females X D. melanogaster males and of Drosophila sechellia females X D. melanogaster males. Independence of the hybrid embryonic lethality and the hybrid larval lethality suggested in a companion study was confirmed by employing two rescue genes, Zhr and Hmr (Hybrid male rescue), in doubly lethal hybrids. A model is proposed to explain the genetic mechanisms of hybrid lethalities as well as the evolutionary pathways.     

Maternal and Zygotic Interactions between the Abnormal Oocyte Mutation and the Scute Inversion in DROSOPHILA MELANOGASTER

The authors have studied the interaction between the abnormal oocyte mutation and an inversion of the X chromosome, In( 1)sc⁴, which has a proximal breakpoint in or near the heterochromatic region (ABO) that maternally interacts with the abo product. It has been demonstrated that the presence of X chromosomes carrying this inversion, besides a marked increase in the severity of the maternal effect of the abo mutation, produces a zygotic effect resulting in the lethality of the progeny of stocks homozygous for abo and sc⁴. These results indicate that the sc⁴ inversion carries an abnormal region indispensable for the development of abo zygotes from sc⁴;abo mothers.     

Excessive variation in Y chromosomal DNA in Rumex acetosa (Polygonaceae)

Rumex acetosa is one of the few angiosperms that possesses sex chromosomes. The same types of abundant repetitive sequences cover both heterochromatic Y chromosomes present in males. The aim of this study was to investigate genetic variation in paternally inherited Y chromosomal DNA and in maternally inherited cpDNA, and to find out whether the examined genomic regions are suited to a phylogeographic study in R. acetosa. DNA sequence polymorphisms present in the 850-bp heterochromatic segment on the Y chromosomes were compared to variation in the 409-bp long chloroplast section (trnL- trnF spacer) in R. acetosa originating from several European locations and from the Altai mountains in Russia. A great amount of genetic variation was detected within the Y chromosomal region while only four chloroplast genotypes were detected. Although the chloroplast haplotypes possessed some geographic pattern, no clear phylogeographic pattern was detected based on the variable Y chromosomes. The mean Y chromosomal nucleotide diversity among all samples equaled 6.6 %, and the mean proportion of polymorphic sites per individual equaled 8.2 % among SNP sites and 1.7 % among all sites investigated. The high number of substitutions detected in the Y chromosomal DNA shows that this heterochromatic sequence has a high mutation rate. The diversity pattern indicates that gene flow via pollen is extensive and it blurs any geographical pattern in the Y chromosomal variation. The high number of repeats and uncertainty concerning the extent of recombination between the two Y chromosomes impair the usability of the Y chromosomal segment for phylogeographic or population genetic studies.     

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.     

A Note on the Maternal Effect Mutants Daughterless and Abnormal Oocyte in DROSOPHILA MELANOGASTER

Two deficiencies for, and a dominant enhancer of, the second chromosome maternal effect mutant, "daughterless" (da), were induced with X-irradiation. Their properties were studied with respect to both da and the linked maternal effect mutant, "abnormal oocyte" (abo), with the following conclusions. (1) The most probable map positions of da and abo are: J-(1/2)-da-2(1/2)-abo, where J is a dominant marker located at 41 on the standard map. (2) The da locus is in bands 31CD-F on the polytene chromosome map; abo is to the right of 32A. (3) Because homozygous da individuals survive while individuals carrying da and a deficiency for da are lethal, it is concluded that da is hypomorphic. (4) From a weak da-like maternal effect in heterozygous da females induced by an "Enhancer of da," we have confirmed a previous report that (a) the amount of sex chromosome heterochromatin contributed by the father can influence the severity of the da maternal effect, and (b) the sex chromosome heterochromatin which influences the da effect is different from that which influences the abo effect. (5) The possibility that da and abo are in a special region of chromosome 2 concerned with the regulation of sex chromosome heterochromatin is strengthened by the observation that the Enhancer of da appears to rescue abnormal eggs produced by homozygous abo mothers. (6) The Enhancer of da is a translocation between chromosomes 2 and 3 with the second chromosome breakpoint in the basal heterochromatin; because the enhancing effect maps in this region of chromosome 2, it is possible that autosomal, as well as sex chromosomal, heterochromatin interacts with da and abo.     

Changes in abo phenotypic expression without increase in rDNA in Drosophila melanogaster

Summary Females of Drosophila melanogaster, homozygous for the abnormal oocyte mutation (abo 2; 44) produce eggs with a greatly reduced probability of developing into adults compared with those of control females. After several generations in abo homozygous stocks, the abo maternal effect is no longer observed. The progressive amelioration of the abo maternal effect in the Canton S background, into which the abo mutation was introduced, was concomitant with an increase in rDNA and variation in the rDNA restriction pattern. To clarify the relationship between the loss of the abo phenotype and the change in rDNA redundancy, we performed genetic and molecular analyses using abo stocks carrying X chromosomes of different origin and carrying different amounts of rDNA. The results we present confirm, in different genetic backgrounds, the previous observations on the behaviour of the abo mutation. However, both the amount and the restriction pattern of rDNA of the different X chromosomes studied remain unchanged after the loss of the abo phenotype. From these observations, it appears that changes in heterochromatic regions other than rDNA are responsible for the loss of the abo maternal effect.     

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.     

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.     

Genetic rescue of lethal genotypes in the mouse

Deletions of gene sequences in chromosome 7 of the mouse are known to interfere with biochemical and cellular development differentiation with lethal effects in homozygotes. The presence of the corresponding wild-type alleles in Cattanach's translocation (chromosomes 7 to X) is able to “rescue” potentially lethal females if they are made heterozygous for the translocation-carrying X chromosome. This holds true for those chromosome 7 deletions with perinatally lethal effects, whereas “rescue” is not readily accomplished with the deletions that cause early embryonic lethality. Females homozygous for the relevant deletion sequences and heterozygous for the translocation-carrying X chromosome are mosaics of two cell types: those in which the wild-type alleles included in the translocated piece complement the depleted sequences, resulting in a normal cellular phenotype, and those with the ordinary X chromosome expressing the lethal phenotype. The developmental interactions between the two cell types and their role in the mechanisms responsible for survival of females homozygous for lethal deletions are discussed. The failure of “rescue” of embryonic lethals reflects as yet unknown temporal and functional aspects of X-inactivation early embryogenesis.     

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.     

Temperature-Sensitive Mutations in DROSOPHILA MELANOGASTER Xii. the Genetic and Developmental Effects of Dominant Lethals on Chromosome 3

Out of 25,000 EMS-treated third chromosomes examined, ten dominant temperature-sensitive (DTS) lethal mutations which are lethal when heterozygous at 29 degrees C but survive at 22 degrees C were recovered. Seven of the eight mutations mapped were tested for complementation; these mutants probably define eight loci. Only DTS-2 survived in homozygous condition at 22 degrees C; homozygous DTS-2 females expressed a maternal effect on embryonic viability. Two of the mutant-bearing chromosomes, DTS-1 and DTS-6, exhibited dominant phenotypes similar to those associated with Minutes. Each of the seven mutants examined exhibited a characteristic phenotype with respect to the time of death at 29 degrees C and the temperature-sensitive period during development. Only DTS-4 exhibited dominant lethality in triploid females.     

Embryonic and imaginal requirements for wingless, a segment-polarity gene in Drosophila

In Drosophila melanogaster mutant alleles of the segmentation gene wingless fall into two classes: winglessLethal mutations are embryonic lethals with a segment-polarity phenotype; the wingless1 mutation is viable when homozygous and produces a homeotic transformation in adults. This paper further describes the embryonic lethal phenotype, and also pole-cell transplants, experiments with a temperature-sensitive mutation, and clonal analysis with a winglessLethal mutation. It is argued that the wg gene is zygotically required after gastrulation for the normal patterning of each embryonic segment. The gene is still required in the larval stages, and the cell nonautonomy of this function supports the view that the wg gene product may be involved in intercellular signaling during development.     

Detection and location of three simple sequence DNAs in polytene chromosomes from virilis group species of Drosophila

In vitro synthesized RNAs complementary to the three satellite DNAs of Drosophila virilis have been used in a series of in situ hybridization experiments with polytene chromosomes from virilis group species. Gall and Atherton (1974) demonstrated that each of the satellites of D. virilis is comprised of many repeats of a distinct, seven base pair long, simple sequence. With few exceptions, copies of each of these simple sequences are detected in the chromocenters of all virilis group species. This is true even in species which do not possess satellite DNAs at buoyant densities corresponding to those of the satellite DNAs of D. virilis. Small quantities of the three simple sequences are also detected in euchromatic arms of several different species. The same euchromatic location may contain detectable copies of one, two, or all three simple sequence DNAs. The amounts of simple sequences at each location in the euchromatin may vary between species, between different stocks of the same species, and even between individuals of the same stock. The simple sequences located in the euchromatin appear to undergo DNA replication during formation of polytene chromosomes unlike those in heterochromatin. The locations of the euchromatic sequences are not the results of single chromosomal inversion events involving heterochromatic and euchromatic breakpoints.     

Difference between two hybrid stocks of mice in the incidence of congenital abnormalities following X-ray exposure of stem-cell spermatogonia

Unbalanced (duplication/deficiency) sperm from balanced reciprocal translocations induced in spermatogonial stem cells of mice generally lead to embryonic lethality around the time of implantation. In a recent study (Generoso et al., 1985), it was found that the incidence of X-ray-induced embryonic lethality differed markedly between two hybrid stocks of irradiated male mice. A parallel difference in the frequencies of reciprocal translocations was observed cytologically in the meiocytes of irradiated males. In the present report, which is an adjunct to the study by Generoso et al. (1985), it was determined whether or not similar differences between the two stocks exist for congenital defects resulting from genetic damage to stem-cell spermatogonia. The results indicate not only an association between the frequencies of induced reciprocal translocations and congenital abnormalities, but also a parallel greater frequency of induced malformations in the (C3H × 101)F1 stock versus the (SEC × C57BL)F1 stock of males.     

Specificity of PS integrin function during embryogenesis resides in the alpha subunit extracellular domain.

We tested the ability of different integrin alpha subunits to substitute for each other during embryonic development. Two alpha subunits, which form heterodimers with the same betaPS subunit, are expressed in complementary tissues in the Drosophila embryo, with alphaPS1 expressed in the epidermis and endoderm, and alphaPS2 expressed in the mesoderm. As a result the two integrin heterodimers are present on opposite surfaces at sites of interaction between the mesoderm and the other cell layers where they are required for normal development. Using the GAL4 system, we are able to rescue fully the embryonic lethality of an alphaPS2 null mutation with a UAS-alphaPS2 transgene, but only partially with a UAS-alphaPS1 gene, due to partial rescue of both muscle and midgut phenotypes. Similarly we are able to rescue the embryonic/first instar larval lethality of an alphaPS1 null mutation gene with UAS-alphaPS1, but only partially with UAS-alphaPS2. Each UAS-alpha gene, when it contains the cytoplasmic domain from the other alpha subunit, maintains an equivalent ability to rescue its own mutation and cannot fully rescue a mutation in the other alpha. We conclude that the two alpha subunits are not equivalent and have distinct functions which reside in the extracellular domains.     

pR plasmid replication provides evidence that single-stranded DNA induces the SOS system in vivo

HP1 is a small nonhistone chromosomal protein of Drosophila melanogaster predominantly localized to the pericentric heterochromatin. We have shown previously that mutations in the HP1 coding sequences are associated with dominant suppression of heterochromatic position-effect variegation, and with recessive lethality. When fused to an Hsp70 heat shock gene promoter, the cDNA encoding HP1 supports the heat shock-inducible accumulation of HPI protein in transgenic flies; this cDNA construct complements the dominant suppression of position-effect variegation associated with mutations in the HP1 gene. Here, we report experiments demonstrating that the heat shock-driven HP1 cDNA is capable of fully rescuing the recessive lethality associated with HP1 mutations in a heat shock-dependent fashion. If heat shock-induced HP1 expression is delayed for as long as 5 days, more than half of the mutant flies still survive until adulthood, consistent with a substantial maternal contribution to embryonic and larval viability. Elevating HP1 levels as late as 7–8 days of development is sufficient to enhance variegation three-fold, suggesting that the extent of heterochromatic position effect can be modified subsequent to the initial appearance of HP1 in the nuclei of syncytial blastoderm embryos.     

pR plasmid replication provides evidence that single-stranded DNA induces the SOS system in vivo

HP1 is a small nonhistone chromosomal protein of Drosophila melanogaster predominantly localized to the pericentric heterochromatin. We have shown previously that mutations in the HP1 coding sequences are associated with dominant suppression of heterochromatic position-effect variegation, and with recessive lethality. When fused to an Hsp70 heat shock gene promoter, the cDNA encoding HP1 supports the heat shock-inducible accumulation of HPI protein in transgenic flies; this cDNA construct complements the dominant suppression of position-effect variegation associated with mutations in the HP1 gene. Here, we report experiments demonstrating that the heat shock-driven HP1 cDNA is capable of fully rescuing the recessive lethality associated with HP1 mutations in a heat shock-dependent fashion. If heat shock-induced HP1 expression is delayed for as long as 5 days, more than half of the mutant flies still survive until adulthood, consistent with a substantial maternal contribution to embryonic and larval viability. Elevating HP1 levels as late as 7–8 days of development is sufficient to enhance variegation three-fold, suggesting that the extent of heterochromatic position effect can be modified subsequent to the initial appearance of HP1 in the nuclei of syncytial blastoderm embryos.