OVO transcription factors function antagonistically in the Drosophila female germline.

OVO controls germline and epidermis differentiation in flies and mice. In the Drosophila germline, alternative OVO-B and OVO-A isoforms have a common DNA-binding domain, but different N-termini. We show that these isoforms are transcription factors with opposite regulatory activities. Using yeast one-hybrid assays, we identified a strong activation domain within a common region and a counteracting repression domain within the OVO-A-specific region. In flies, OVO-B positively regulated the ovarian tumor promoter, while OVO-A was a negative regulator of the ovarian tumor and ovo promoters. OVO-B isoforms supplied ovo(+) function in the female germline and epidermis, while OVO-A isoforms had dominant-negative activity in both tissues. Moreover, elevated expression of OVO-A resulted in maternal-effect lethality while the absence of OVO-A resulted in maternal-effect sterility. Our data indicate that tight regulation of antagonistic OVO-B and OVO-A isoforms is critical for germline formation and differentiation.

The three dominant female-sterile mutations of the Drosophila ovo gene are point mutations that create new translation-initiator AUG codons.

The Drosophila ovo gene, which encodes a putative transcription factor (Ovo) with TFIIIA-like zinc fingers, is required for female germline survival and proper oogenesis. Three dominant female-sterile ovoD mutations cause ovarian abnormalities that define an allelic series, with ovoD1 displaying the stronger phenotype and ovoD3 the weaker. We report here that all three ovoD mutations are point mutations that create new in-frame methionine codons in the 5' part of ovo. There are two types of overlapping ovo transcription units, ovo alpha and ovo beta. By using various ovo-lacZ reporter genes, we determined that the long Ovo isoforms starting at methionine M1, present in transcripts ovo alpha, are expressed at low levels only in mature oocytes. Short Ovo isoforms are translated from methionine M373, the first in-frame start codon present in transcript ovo beta, and correspond to the activity defined by recessive loss of function ovo mutations. The new AUGs created in ovoD mutations all are located upstream of the M373 initiation site. Our results support the hypothesis that they can substitute for M373 as translation starts and initiate the synthesis of Ovo proteins that have extra amino acids at their N termini. We propose that premature expression of long Ovo protein isoforms occurs in ovoD mutants and interferes with wild-type Ovo function in controlling female germline differentiation.

ovo, a Drosophila gene required for ovarian development, is specifically expressed in the germline and shares most of its coding sequences with shavenbaby, a gene involved in embryo patterning.

Genetic analyses of Drosophila oogenesis have revealed the central role of ovo, a gene required for differentiation of the female germline. A number of recessive ovo mutations also affect the shavenbaby (svb) function required for late embryo patterning, suggesting a tight structural link between ovo and svb. By using various genomic probes for in situ hybridization to wild type and mutant embryos, we show that ovo indeed shares most of its coding sequences with svb. svb expression is detected early in the presumptive head region and later in each segment. It requires control elements located upstream of the ovo genomic region. ovo expresses abundant maternal RNAs which are uniformly distributed in early cleavage embryos. A fraction that lacks an alternative ovo-specific protein coding region (ORF 2b) is detected in pole cells. Expression of an ovo-specific lacZ reporter gene (ovoB) shows that ovo encodes a nuclear protein present in the germline of both sexes. Zygotic ovoB expression is first detected in embryos at around stage 17 and persists up to the adult stage. Our data show that the germline specific expression of ovo in females correlates with its function in oogenesis. This expression, however, is also observed in males in which ovo is not required.

Production of dominant female sterility in Drosophila melanogaster by insertion of the ovoD1 allele on autosomes: use of transformed strains to generate germline mosaics.

We have cloned a 7 kb genomic fragment containing the dominant female-sterile mutation ovoD1. This fragment confers to transgenic females a sterility phenotype, the severity of which depends both on the genetic background and the ratio of ovoD1 product to ovo+ product. Females containing two copies of the ovoD1 transgene, or those containing one recessive null allele at the ovo locus, are about as sterile as ovoD1 females. Twenty transformed strains were obtained and five of them were tested and shown to be excellent tools for identifying a germline clone of cells sustaining mitotic recombination on the autosomes. One of the tested strains carries an insert on chromosome 4, which enabled us to show that mitotic recombination on that chromosome is not a rare event: it is in fact frequent enough for the maternal effects of the zygotic lethal mutations cubitus interruptus Dominant (ciD) and l(4)29 to be studied.

The ovo gene of Drosophila encodes a zinc finger protein required for female germ line development.

As defined by dominant and recessive ovo mutations, the ovo gene is required for development of the Drosophila female germ line, and does not exert any function in males or in somatic tissues. However, reversion of dominant ovo mutations can result in new phenotypes that are not related to the female germ line: the svb and lzl mutations affect cuticle and eye development, respectively. We have identified a 7.2 kb genomic fragment that rescues ovo mutations in transgenic Drosophila and thus contains all sequences necessary for ovo+ function. This fragment has been sequenced almost in its entirety, defining the ovo locus at the molecular level. Multiple copies of the same fragment also rescue the lzl mutation. They do not rescue svb mutations, in agreement with genetic evidence that the svb function requires additional, more distal sequences. Nevertheless, a number of transposable element insertions that induce a svb phenotype interrupt the coding sequence of ovo. Taken together, the genetic and molecular data indicate the existence of a complex locus, where the ovo and svb functions depend on overlapping coding sequences but distinct regulatory elements. The data also suggest a model for the lzl phenotype. Expression of ovo at the RNA level is detectable at stage 8 of oogenesis in nurse cells and persists through the rest of oogenesis and in early embryogenesis. The ovo transcript encodes a protein of at least 1209 amino acids with four zinc fingers, suggesting that ovo might be a transcription factor required for female germ line maintenance and gametogenesis.

Mobilization of the gypsy and copia retrotransposons in Drosophila melanogaster induces reversion of the ovo dominant female-sterile mutations: molecular analysis of revertant alleles.

The ovo locus is required for the maintenance of the female germ line in Drosophila melanogaster. In the absence of an ovo gene, males are completely normal but females have no germ-line stem cells. Three dominant mutations at the ovo locus, called ovo, were observed to revert towards recessive alleles at high frequency when ovo males were crossed to females of the strain y v f mal. We have found that this strain contains an inordinately high number of gypsy transposable elements, and crossing it with the ovo strains results in the mobilization of both gypsy and copia, with high-frequency insertions into the ovo locus: of 16 revertants examined 12 have gypsy and four have copia inserted at 4E, the ovo cytological site. Using gypsy DNA as a tag we have cloned 32 kb of wild-type DNA sequences surrounding a gypsy insertion and characterized molecular rearrangements in several independent revertants: in 10 of them gypsy appears to be inserted into the same site. The orientation of gypsy is strictly correlated with whether the neighbouring lozenge-like mutation appears in the revertants. A distal limit of the ovo locus was molecularly determined from the breakpoint of a deletion affecting closely flanking regions.

A polyoma-derived plasmid vector maintained episomally in both E. coli and mouse hepatoma cells.

We describe a recombinant plasmid, pBBPY1, containing polyoma virus sequences which persists episomally in mouse hepatoma (MH) cells and can be shuttled between these cells and bacteria. This plasmid is composed of a subgenomic fragment of a polyoma virus mutant that includes two origins of replication; sequences of plasmid pML2; the xanthine-guanine phosphoribosyltransferase gene of Escherichia coli (Ecogpt) under the control of SV40 early-region promoter and RNA processing signals, providing a dominant selectable marker for mammalian transfection. MH cells from colonies growing in HAT medium (hypoxanthine, aminopterin and thymidine) were found to contain vector DNA molecules in an episomal state, the majority of them unrearranged. When HAT-selective pressure was applied for only 3 days, the resulting cells contained up to 50-100 copies of intact plasmid, i.e. 20-fold more than cells grown under standard selection conditions with continuous HAT-selective pressure. Contrary to standard conditions, transient selection does not alter the epithelial morphology nor ability of transfected hepatoma cells to produce albumin.

Immunofluorescence analysis of reexpression and activation: the origin of phenotypic diversity of rat hepatoma-mouse fibroblast hybrid colonies.

The capacity of young hybrid colonies between 2s rat hepatoma cells and mouse L-fibroblasts to reexpress rat albumin and become activated for mouse albumin production, was examined at the level of individual cells using immunofluorescent staining of intracellular albumin. Most of the colonies that happened to reexpress albumin were observed to present a stable, homogeneous, hepatoma-like morphology, visible long before the first signs of albumin reexpression. These colonies switch from an extinct to an albumin-producing state between one and four weeks after fusion. Colonies that do not express albumin, present an epithelial or fibroblastic morphology. Karyologic analysis of hybrid clones representative of the various morphologic types revealed that their phenotypic diversity is correlated with their rat over mouse chromosome ratio. The results suggest that the potential of hybrid colonies to express albumin is determined at the time of nuclear fusion in the heterokaryons, possibly by the number of parental genomes participating in the formation of the mother hybrid cell. Double immunofluorescent staining of rat and mouse albumin in the same cell has been used to determine whether reexpression and activation are correlated phenomena or appear independently in any single cell. The analysis demonstrates that activation of mouse albumin never takes place without reexpression of the previously expressed rat albumin gene, while the converse is frequent.

Immunofluorescence analysis of the time-course of extinction, reexpression, and activation of albumin production in rat hepatoma-mouse fibroblast heterokaryons and hybrids.

We have used a combination of a sensitive immunocytochemical stain for intracellular albumin, and Hoechst 33258 dye for identification of parental nuclei to investigate the time-course of extinction, reexpression, and activation of albumin production in fusion products of 1s (hyperdiploid) or 2s (hypertetradiploid) rat hepatoma cells with mouse fibroblasts (L cells or embryonic cells). In all combinations, the initial event is extinction of albumin production. Extinction occurs immediately after fusion when the mouse fibroblast is a normal embryonic (senescent?) cell. In the case of an L cell, rat albumin is synthesized and secreted during the first 12 h after fusion; no production of mouse albumin occurs. Thereafter, albumin production ceases. 8-12 d after fusion, young hybrid colonies are found to resume the synthesis of rat albumin (reexpression), and several days later the production of mouse albumin begins (activation). The patterns of reexpression and activation indicate (a) that chromosome loss is not necessary for either event to occur and (b) that the cells active in the synthesis of mouse albumin are a subpopulation of those cells already engaged in the production of rat albumin. We conclude that (a) extinction is mediated by diffusible factor(s) from the L-cell parent that act in the hepatoma nucleus to prevent the formation of new albumin messenger RNA; (b) reexpression and activation are gene dosage-dependent but extinction is not; and (c) previously active genes are more rapidly expressed than previously silent ones.

Structural studies of yeast flavocytochrome b2: cooperative roles of the alpha and beta globules in the formation of the flavin-binding sites.

The purpose of the study reported here was the localization of the heme binding sites on the two globular fragments, alpha and beta, of the 'cleaved' form of the flavocytochrome b2 chain. These fragments were partially resolved by means of molecular sieving under denaturing conditions (3 M or 6 M guanidine in the presence of 2-mercaptoethanol). They were then renatured in the presence of excesses of FMN and protoheme. The protoheme was found to be quantitatively bound to the alpha subunit, confirming previous findings. The flavin binds neither to alpha alone nor to beta alone, but only to the reassociated alphabeta protomer. the results are discussed in terms of the possible occurrence of gene fusion in the formation of the complex flavocytochrome chain of this very particular L-lactate cytochrome c reductase found specifically in yeasts.

Energy requirements for biosynthesis of DNA in Escherichia coli. Role of membrane-bound energy-transducing ATPase (coupling factor).

A mutant of Escherichia coli missing energy-transducing ATPase and known to be defective in a variety of membrane functions from earlier studies (Yamamoto, T. H., Mével-Ninio, M. and Valentine, R. C. (1973) Biochim. Biophys. Acta 314, 267-275; Thipayathasana, P. and Valentine, R. C. (1974) Biochim. Biophys. Acta 347, 464-468; Mével-Ninio, M. and Yamamoto, T. (1974) Biochim. Biophys. Acta 357, 63-66) has been found to be blocked for anaerobic DNA synthesis. The rate of anaerobic DNA synthesis in the mutant, measured as radioactive adenine incorporation into the alkali-resistant fraction of whole cells, is about 1/6 the rate of DNA synthesis in the wild type culture under similar conditions. Addition of NO-3- or O-2 restores DNA biosynthesis in the mutant. The entry of radioactive adenine is not appreciably affected in the mutant by anaerobiosis. It is concluded that coupling factor plays a role in some step(s) of DNA biosynthesis.