Cofilin/ADF is required for cell motility during Drosophila ovary development and oogenesis.

The driving force behind cell motility is the actin cytoskeleton. Filopodia and lamellipodia are formed by the polymerization and extension of actin filaments towards the cell membrane. This polymerization at the barbed end of the filament is balanced by depolymerization at the pointed end, recycling the actin in a 'treadmilling' process. One protein involved in this process is cofilin/actin-depolymerizing factor (ADF), which can depolymerize actin filaments, allowing treadmilling to occur at an accelerated rate. Cofilin/ADF is an actin-binding protein that is required for actin-filament disassembly, cytokinesis and the organization of muscle actin filaments. There is also evidence that cofilin/ADF enhances cell motility, although a direct requirement in vivo has not yet been shown. Here we show that Drosophila cofilin/ADF, which is encoded by the twinstar (tsr) gene, promotes cell movements during ovary development and oogenesis. During larval development, cofilin/ADF is required for the cell rearrangement needed for formation of terminal filaments, stacks of somatic cells that are important for the initiation of ovarioles. It is also required for the migration of border cells during oogenesis. These results show that cofilin/ADF is an important regulator of actin-based cell motility during Drosophila development.

Drosophila ovary morphogenesis: analysis of terminal filament formation and identification of a gene required for this process.

The adult ovary of Drosophila is composed of approximately 20 parallel repetitive structures called ovarioles. At the anterior tip of each ovariole is a stack of 8-9 disc-shaped cells, called the terminal filament. Ovariole morphogenesis starts with the formation of the terminal filaments. Using two enhancer trap markers for terminal filament cells, we show that terminal filaments form in a progressive manner from medial to lateral across the ovary and that the number of terminal filament cells in a developing stack increases gradually. This process occurs during the second half of the third larval instar. One of these enhancer trap mutations, which is in the bric à brac gene, demonstrates that this gene is necessary for terminal filament formation and that a terminal filament cell cluster is required for ovariole morphogenesis to take place.

The BTB domain of bric à brac mediates dimerization in vitro.

The gene bric à brac (bab) is required for the proper development of the limbs and ovary in Drosophila melanogaster. bab encodes a BTB domain (also called a POZ domain), an approximately 115-amino-acid conserved motif found primarily in the N termini of zinc finger proteins. In this paper, we show that the BTB domain of bab can mediate protein dimerization in vitro. In addition, we demonstrate that the first 51 amino acids of the bab BTB domain are sufficient for dimerization, and we identify amino acids within this region that are required for binding.

Mechanisms of cell rearrangement and cell recruitment in Drosophila ovary morphogenesis and the requirement of bric à brac.

The Drosophila ovary consists of repeated units, the ovarioles, where oogenesis takes place. The repetitive structure of the ovary develops de novo from a mesenchymal cell mass, a process that is initiated by the formation of a two-dimensional array of cell stacks, called terminal filaments, during the third larval instar. We have studied the morphogenetic process leading to the formation of terminal filaments and find that this involves recruitment, intercalation and sorting of terminal filament cells. Two other types of cell stacks that participate in ovary morphogenesis, the basal stalks and interfollicular stalks, also form by cell rearrangement utilizing a convergence and extension mechanism. Terminal filament formation depends on the Bric à brac protein, which is expressed in the nuclei of terminal filament cells and is cell autonomously required. Disruption of terminal filament formation, together with defects of basal and interfollicular stalk development, leads to disruption of ovariole formation and female sterility in bric à brac mutants.

The BTB domain, found primarily in zinc finger proteins, defines an evolutionarily conserved family that includes several developmentally regulated genes in Drosophila.

The Drosophila bric à brac protein and the transcriptional regulators encoded by tramtrack and Broad-Complex contain a highly conserved domain of approximately 115 amino acids, which we have called the BTB domain. We have identified six additional Drosophila genes that encode this domain. Five of these genes are developmentally regulated, and one of them appears to be functionally related to bric à brac. The BTB domain defines a gene family with an estimated 40 members in Drosophila. This domain is found primarily at the N terminus of zinc finger proteins and is evolutionarily conserved from Drosophila to mammals.

string of pearls encodes Drosophila ribosomal protein S2, has Minute-like characteristics, and is required during oogenesis.

The first allele of string of pearls (sop) was isolated as a recessive female sterile mutant in a P element enhancer trap screen. Oogenesis in homozygous sop females arrests at approximately stage 5. In addition, homozygous flies of both sexes have Minute-like characteristics that include reduced bristles, delayed development and larval lethality. sop maps to 30D/E on chromosome 2L and encodes the Drosophila homolog of eukaryotic ribosomal protein S2. The gene is present in a single copy in the Drosophila genome and the level of mRNA present in mutant animals is reduced. The identification of a mutant allele that blocks development at a mid-stage of oogenesis may indicate that sop has a specific developmental role during oogenesis in addition to its general role in protein synthesis as a component of the small ribosomal subunit.

Pattern formation in the limbs of Drosophila: bric à brac is expressed in both a gradient and a wave-like pattern and is required for specification and proper segmentation of the tarsus.

We have identified the gene bric à brac and show that it is required for pattern formation along the proximal-distal axis of the leg and antenna of Drosophila. In bric à brac mutant legs, the bristle pattern of the three central tarsal segments is transformed towards the pattern of the most proximal tarsal segment. In addition, bric à brac mutant legs and antennae have segmentation defects. bric à brac encodes a nuclear protein that shares a highly conserved domain with two transcription factors from Drosophila. bric à brac function is dosage dependent and is required in a graded manner for the specification of tarsal segments. The graded requirement for bric à brac correlates with its graded expression pattern, suggesting that the concentration of BRIC A BRAC protein specifies segment identity in the tarsus.

Splicing of the Drosophila P element ORF2-ORF3 intron is inhibited in a human cell extract.

P element transposition in Drosophila melanogaster is regulated by germline-specific splicing of the P element ORF2-ORF3 intron. This regulation has been shown to depend on a cis-acting sequence located in the exon 12-31 bases from the 5' splice site. Mutations within this sequence disrupt the regulation and result in splicing of the ORF2-ORF3 intron in all tissues, indicating that the sequence is required to inhibit splicing of this intron in the soma. We now show that a trans-acting factor in a human (HeLa) cell extract can inhibit splicing of the intron, suggesting that this regulatory mechanism is conserved from flies to humans.

Identification of a cis-acting sequence required for germ line-specific splicing of the P element ORF2-ORF3 intron.

P element transposition in Drosophila melanogaster is limited to the germ line because the third intron (the ORF2-ORF3 intron) of the P element transcript is spliced only in germ line cells. We describe a systematic search for P element sequences that are required to regulate the splicing of the ORF2-ORF3 intron. We have identified three adjacent mutations that abolish the germ line specificity and allow splicing of this intron in all tissues. These mutations define a 20-base regulatory region located in the exon, 12 to 31 bases from the 5' splice site. Our data show that this cis-acting regulatory sequence is required to inhibit the splicing of the ORF2-ORF3 intron in somatic cells.

Construction, stable transformation, and function of an amber suppressor tRNA gene in Drosophila melanogaster.

Drosophila melanogaster strains with a stably incorporated amber suppressor tRNA gene have been generated. A tRNATyr gene was site specifically mutated to produce an anticodon sequence that recognizes the amber codon and then introduced into Drosophila by using P-element-mediated transformation. Transformants from four integration events were recovered. Two integrations resulted in both male and female sterility, whereas the other two resulted in male sterility but female fertility. Strains derived from the two female-fertile integration events were shown to have a low level of amber-suppressing activity by their ability to suppress an amber mutation in a chloramphenicol acetyltransferase gene.

Analysis of the cis-acting requirements for germ-line-specific splicing of the P-element ORF2-ORF3 intron.

P-element transposition is limited to the germ line because the element's third intron is only spliced in germ line cells. We show that a 240-bp fragment containing this 190-bp intron can confer germ line specificity when placed in the context of another gene. We find that the cis-acting regulatory sequences required for germ line regulation map near to, but not at, the 5' or 3' splice junctions.

Evidence for Drosophila P element transposase activity in mammalian cells and yeast.

Drosophila P element transposase expression is limited to the germline by tissue-specific splicing of one of its three introns. Removal of this intron by mutagenesis in vitro has allowed both P element excision and transposition to be detected in Drosophila somatic tissues. In order to determine if P element transposase can function in other organisms, we have expressed modified P elements either lacking one intron or lacking all three introns in mammalian cells and yeast, respectively. Using an assay for P element excision, we have detected apparent excision events in cultured monkey cells. Furthermore, expression of the complete P element cDNA is lethal to Saccharomyces cerevisiae cells carrying a mutation in the RAD52 gene, indicating that double-stranded DNA breaks are generated, presumably by transposase action.

Tissue specificity of Drosophila P element transposition is regulated at the level of mRNA splicing.

We show that the germline specificity of P element transposition is controlled at the level of mRNA splicing and not at the level of transcription. In the major P element RNA transcript, isolated from somatic cells, the first three open reading frames are joined by the removal of two introns. Using in vitro mutagenesis and genetic analysis we demonstrate the existence of a third intron whose removal is required for transposase production. We propose that this intron is only removed in the germline and that its removal is the sole basis for the germline restriction of P element transposition.

Identification and immunochemical analysis of biologically active Drosophila P element transposase.

We have identified proteins encoded by P transposable elements expressed in transformed Drosophila tissue culture cells. Two proteins have been identified by immunochemical techniques. One, an 87,000 dalton polypeptide, is encoded by a P element mRNA lacking the third (ORF2-ORF3) intervening sequence. The other protein, a 66,000 dalton polypeptide, is encoded by an mRNA that retains the third intron and is found in somatic tissues. Furthermore, tissue culture cell lines expressing the 87,000 dalton polypeptide are able to catalyze both the precise and imprecise excision of a nonautonomous P element. The 87,000 dalton protein is encoded by sequences from all four P element open reading frames. Taken together, these data strongly suggest that the 87,000 dalton polypeptide is the P element transposase.

Synthesis of an ochre suppressor tRNA gene and expression in mammalian cells.

We have used site-specific mutagenesis to change the anticodon of a Xenopus laevis tyrosine tRNA gene so that it would recognize ochre codons. This tRNA gene is expressed when amplified in monkey cells as part of a SV40 recombinant and efficiently suppresses termination at both the ochre codon separating the adenovirus 2 hexon gene from a 23-kd downstream gene and the ochre codon at the end of the NS1 gene of influenza virus A/Tex/1/68. Termination at an amber codon of a NS1 gene of another influenza virus strain was not suppressed by the (Su+) ochre gene suggesting that in mammalian cells amber codons are not recognized by ochre suppressor tRNAs. Finally, microinjection into mammalian cells of both (Su+) ochre tRNA genes and selectible genes containing ochre nonsense mutations gives rise to colonies under selective conditions. We conclude that it should be possible to isolate a wide assortment of mammalian cell lines with ochre suppressor activity.

Characterization of tRNA precursor splicing in mammalian extracts.

Transcription of a Xenopus laevis tRNATyr gene and splicing of the transcript have been studied in HeLa cell extracts. This tRNATyr gene has a 13-base intervening sequence and is expressed as mature tRNA when transfected into mammalian cells. The tRNATyr gene is transcribed under conditions of low concentrations of magnesium and ATP, but is processed by splicing only when both of these cofactors are added at higher concentrations. The endonucleolytic activity of the tRNA-splicing system in the HeLa extract produces exons with 3'-phosphate and 5'-hydroxyl groups. The 3'-phosphate is retained during the ligation reaction and forms the phosphodiester bond in the mature tRNA. Retention of the 3'-phosphate during tRNA splicing differs from the more extensively studied process in yeast extracts where a phosphate group from an ATP cofactor is used to form the phosphodiester bond joining the exons. Thus, eucaryotic organisms can splice tRNA precursors by at least two distinguishable mechanisms.

Functional suppression in mammalian cells of nonsense mutations in the herpes simplex virus thymidine kinase gene by suppressor tRNA genes.

A nonsense mutation (UAG) in the thymidine kinase gene of herpes simplex virus type 1 can be suppressed in vivo to produce active thymidine kinase by prior infection with a defective simian virus 40 stock which acts as a vector to introduce a functional suppressor tRNA gene into mammalian cells in culture. The suppression is specific for UAG, but not UGA or missense, mutants and restores thymidine kinase activity to 20 to 40% of the wild-type level. These results suggest that many cell lines susceptible to simian virus 40 infection may be transiently converted to a suppressor-positive phenotype for use in the genetic study of mammalian viruses.