D. T. Champlin, M. Frasch, H. Saumweber and J. T. Lis (1991). Characterization of a Drosophila protein associated with boundaries oftranscriptionally active chromatin. Genes Dev 5, 1611-21.
We have used indirect immunofluorescence of polytene chromosomes to examine the chromatin distribution of a 52-kD Drosophila protein designated B52. B52 is localized to transcriptionally active loci and, at the highly decondensed heat shock loci, can be seen to bracket the RNA polymerase II fluorescence signals symmetrically. We have also examined the distribution of B52 on nonpolytene chromosomes in Drosophila cell cultures with an invivo UV cross-linking method and find that, here too, B52 is associated with boundaries of transcriptionally active chromatin. The predicted primary amino acid sequence of B52 reveals two regions with similarities to a number of other proteins known to interact with nucleic acids.P. Culp, C. Nüsslein-Volhard and N. Hopkins (1991). High-frequency germ-line transmission of plasmid DNA sequences injected into fertilized zebrafish eggs. ProcNatl Acad Sci U S A 88, 7953-7.
With the goal of developing techniques for DNA insertional mutagenesis in zebrafish, we established procedures for rapidly obtaining and injecting large numbers of fertilized eggs. Using either of two plasmid constructs, we injected uncut DNA into fertilized eggs at the one- or two-cell stage. Fish hatched from injected eggs were raised to sexual maturity, and the frequency of transgenic founder fish was determined by pair-mating the fish andtesting DNA extracted from pools of their 16-hr-old offspring by the polymerase chain reaction (PCR) and then Southern analysis. Eggs injected with one of two different plasmids yielded no transgenic fish, but 7-25% (19 of 115 overall) of the eggs injected with the other plasmid transmitted the injected sequences to their offspring (F1). Of seven lines studied further, all were able to pass the foreign DNA sequences to the next (F2) generation. Inheritance in the F2 generation was Mendelian in the fivelines tested. PCR and Southern analysis indicated that the plasmid sequences were present in multiple copies, probably tandemly arranged. Two founder fish carried more than one independent integration of the plasmid sequences. The line studied in more detail was a mosaic carrying two independently segregating copies of the transgene in one germ cell and a third copy in another germ-line precursor cell. The ability to obtain and inject large numbers of zebrafish eggs combined with a high frequency ofgerm-line integration may be steps toward the goal of being able to perform insertional mutagenesis with this organism.M. Frasch (1991). The maternally expressed Drosophila gene encoding the chromatin-binding protein BJ1 is a homolog of the vertebrate gene Regulator of Chromatin Condensation, RCC1. Embo J 10, 1225-36.
Using monoclonal antibodies I have identified a nuclear protein of Drosophila, BJ1 (Mr approximately68 kd), and isolated its gene. Biochemical analysis demonstrates that the BJ1 protein is associated with nucleosomes and is released from chromatin by agents which intercalate into DNA, as previously shown for the high mobility group proteins (HMGs). On polytene chromosomes the protein is localized in all bands, with no preference for particular loci. Both the BJ1 protein and in particular the BJ1 mRNA are strongly expressed maternally. In early embryos all nuclei contain equal amounts of BJ1. Duringneuroblast formation, BJ1 mRNA becomes restricted to cells of the central nervous system, and higher protein levels are found in the nuclei of this tissue. In late embryonic stages, the mRNA almost completely disappears, but significant amounts of BJ1 protein persist until morphogenesis. The BJ1 gene encodes a 547 amino acid polypeptide featuring two different types of internal repeats. The sequence from amino acids 46 to 417 containing seven repeats of the first type has been highly conserved inevolution. 45% of the amino acids in this region are conserved in seven similar tandem repeats of the human gene Regulator of Chromatin Condensation, RCC1. The phenotype of a cell line carrying a mutation of RCC1 suggested a main function for this gene in cell cycle control. A yeast gene, SRM1/PRP20, also contains these repeats and shows 30% amino acid identity to BJ1 in this region. Mutations in this gene perturb mRNA metabolism, disrupt nuclear structure and alter the signal transduction pathway forthe mating pheromone. Complementation experiments argue for a common function of these genes in the different species. I propose that their gene products bind to the chromatin to establish or maintain a proper higher order structure as a prerequisite for a regulated gene expression. Disruption of this structure could cause both mis-expression and default repression of genes, which might explain the pleiotropic phenotypes of the mutants.R. Lehmann and C. Nüsslein-Volhard (1991). The maternal gene nanos has a central role in posterior pattern formation of the Drosophila embryo. Development 112, 679-91.
A group of maternal genes, the posterior group, is required for the development of the abdominal region in the Drosophila embryo. We have used genetic as well as cytoplasmic transfer experiments to order seven of the posterior group genes (nanos, pumilio, oskar, valois, vasa, staufen and tudor) into a functionalpathway. An activity present in the posterior pole plasm of wild-type embryos can restore normal abdominal development in posterior group mutants. This activity is synthesized during oogenesis and the gene nanos most likely encodes this activity. The other posterior group genes have distinct accessory functions: pumilio acts downstream of nanos and is required for the distribution or stability of the nanos-dependent activity in the embryo. Staufen, oskar, vasa, valois and tudor act upstream ofnanos. Embryos from females mutant for these genes lack the specialized posterior pole plasm and consequently fail to form germ-cell precursors. We suggest that the products of these genes provide the physical structure necessary for the localization of nanos-dependent activity and of germ line determinants.C. Nüsslein-Volhard (1991). The 1991 Albert Lasker Public Service Award. From egg to organism. Studies on embryonic pattern formation. Jama 266, 1848-9.C. Nüsslein-Volhard (1991a). Determination of the embryonic axes of Drosophila. Dev Suppl 1, 1-10.
The principles of embryonic pattern formation have been studied extensively in many systems using classical experimental approaches. In Drosophila, a powerful combination of genetics and transplantation experiments, as well as molecular biology, have helped to elucidate the mechanisms that operate duringoogenesis and early embryogenesis to establish a set of positional cues required for axis determination in the early embryo. In systematic searches for maternal effect mutations a small number of about 30 genes have been identified that specifically affect the process of determination of the embryonic axes. These 'coordinate' genes define four systems that determine the anteroposterior (AP) axis (three systems) and the dorsoventral (DV) axis (one system) independently. In the anteroposterior axis, theanterior system determines the segmented region of head and thorax, the posterior system determines the segmented abdominal region, and the terminal system is responsible for the formation of the nonsegmented termini at the anterior and posterior egg tips, the acron and telson. In contrast, pattern along the dorsoventral axis is determined by one system only. Although all four systems use different biochemical mechanisms, they share several properties. (1) The product of one gene in each system islocalized in a specific region of the freshly laid egg and functions as a spatial signal. (2) In each system, this spatial information finally results in the asymmetrical distribution of one gene product that functions as a transcription factor. (3) This transcription factor is distributed in a concentration gradient that defines the spatial limits of expression of one or more zygotic target genes. The combined action of these three anteroposterior systems as well as the dorsoventral system defines theexpression of zygotic target genes in at least seven distinct regions along the anteroposterior and at least three in the dorsoventral axis. These longitudinal and transverse domains provide a coarse spatial prepattern which is then further refined by the action and interaction of zygotic pattern genes.M. Ohtsubo, T. Yoshida, H. Seino, H. Nishitani, K. L. Clark, G. F. Sprague, Jr., M. Frasch and T. Nishimoto (1991). Mutation of thehamster cell cycle gene RCC1 is complemented by the homologous genes of Drosophila and S.cerevisiae. Embo J 10, 1265-73.
The RCC1 gene has been isolated from several vertebrates, including human, hamster and Xenopus. Genes similar to RCC1, namely BJ1 and SRM1/PRP20, have been isolated from the insect Drosophila and from the budding yeast Saccharomyces cerevisiae. A mutation of the RCC1 gene in the hamster BHK21 cell line, tsBN2, confers pleiotropic phenotypes, including G1 arrest andpremature induction of mitosis in cells synchronized at the G1/S boundary. Similarly, mutations of the SRM1/PRP20 gene are pleiotropic; the srm1 mutant shows G1 arrest and suppression of the mating defect of mutants lacking pheromone receptors, and the prp20 mutant shows an alteration in mRNA metabolism. Here we show that both BJ1 and SRM1/PRP20 complement the temperature sensitive phenotype of the tsBN2 cells. Like RCC1 proteins of vertebrates, the protein products of the Drosophila and yeast RCC1homologues were located in the nuclei of the mammalian cells. These results suggest that the BJ1 and SRM1/PRP20 genes are functionally equivalent to the vertebrate RCC1 genes, and that the RCC1 gene plays an important role in the regulation of gene expression in the eukaryotic cell cycle.B. M. Paterson, U. Walldorf, J. Eldridge, A. Dubendorfer, M. Frasch and W. J. Gehring (1991). The Drosophila homologue of vertebratemyogenic-determination genes encodes a transiently expressed nuclear protein marking primary myogenic cells. Proc Natl Acad Sci U S A 88, 3782-6.
We have isolated a cDNA clone, called Dmyd for Drosophila myogenic-determination gene, that encodes a protein with structural and functional characteristics similar to the members of the vertebrate MyoD family. Dmyd clone encodes a polypeptide of 332 amino acids with 82% identity to MyoD in the 41 amino acids of the putative helix-loop-helix regionand 100% identity in the 13 amino acids of the basic domain proposed to contain the essential recognition code for muscle-specific gene activation. Low-stringency hybridizations indicate that Dmyd is not a member of a multigene family similar to MyoD in vertebrates. Dmyd is a nuclear protein in Drosophila, consistent with its role as a nuclear-gene regulatory factor, and is proposed to be a transiently expressed marker for muscle founder cells. We have used an 8-kilobase promoter fragment from thegene, which contains the first 55 amino acids of the Dmyd protein, joined to lacZ, to follow myogenic precursor cells into muscle fibers with antibodies to beta-galactosidase and to Dmyd. Unlike the myogenic factors in vertebrate muscle cells, Dmyd appears to be expressed at a much lower level in differentiated Drosophila muscles, so Dmyd cannot be followed continuously as a muscle marker. This fact is reflected in the loss of Dmyd RNA expression in 12- to 24-hr embryos, a major period of earlymyogenesis, as well as in the undetectable level of the nuclear antigen in primary cultures of embryonic and adult Drosophila muscle.
R. P. Ray, K. Arora, C. Nüsslein-Volhard and W. M. Gelbart (1991). The control of cell fate along the dorsal-ventral axis of the Drosophila embryo. Development 113, 35-54.
We have analyzed the contributions made by maternal and zygotic genes to the establishment of the expression patterns of fourzygotic patterning genes: decapentaplegic (dpp), zerknüllt (zen), twist (twi), and snail (sna). All of these genes are initially expressed either dorsally or ventrally in the segmented region of the embryo, and at the poles. In the segmented region of the embryo, correct expression of these genes depends on cues from the maternal morphogen dorsal (dl). The dl gradient appears to be interpreted on three levels: dorsal cells express dpp and zen, but not twi and sna; lateral cells lack expression ofall four genes; ventral cells express twi and sna, but not dpp and zen. dl appears to activate the expression of twi and sna and repress the expression of dpp and zen. Polar expression of dpp and zen requires the terminal system to override the repression by dl, while that of twi and sna requires the terminal system to augment activation by dl. The zygotic expression patterns established by the maternal genes appear to specify autonomous domains that carry out independent developmental programs,insofar as mutations in the genes that are expressed ventrally do not affect the initiation or ontogeny of the expression patterns of the genes that are expressed dorsally, and vice versa. However, interactions between the zygotic genes specific to a particular morphological domain appear to be important for further elaboration of the three levels specified by dl. Two of the genes, dpp and twi, are unaffected by mutations in any of the tested zygotic dorsal-ventral genes, suggesting that dpp and twi arethe primary patterning genes for dorsal ectoderm and mesoderm, respectively.S. Roth, Y. Hiromi, D. Godt and C. Nüsslein-Volhard (1991). cactus, a maternal gene required for proper formation of the dorsoventral morphogen gradient in Drosophila embryos. Development 112, 371-88.
The dorsoventral pattern of the Drosophila embryo is mediated by a gradient of nuclear localization of the dorsal protein which acts as amorphogen. Establishment of the nuclear concentration gradient of dorsal protein requires the activities of the 10 maternal 'dorsal group' genes whose function results in the positive regulation of the nuclear uptake of the dorsal protein. Here we show that in contrast to the dorsal group genes, the maternal gene cactus acts as a negative regulator of the nuclear localization of the dorsal protein. While loss of function mutations of any of the dorsal group genes lead to dorsalized embryos, loss of cactusfunction results in a ventralization of the body pattern. Progressive loss of maternal cactus activity causes progressive loss of dorsal pattern elements accompanied by the expansion of ventrolateral and ventral anlagen. However, embryos still retain dorsoventral polarity, even if derived from germline clones using the strongest available, zygotic lethal cactus alleles. In contrast to the loss-of-function alleles, gain-of-function alleles of cactus cause a dorsalization of the embryonic pattern.Genetic studies indicate that they are not overproducers of normal activity, but rather synthesize products with altered function. Epistatic relationships of cactus with dorsal group genes were investigated by double mutant analysis. The dorsalized phenotype of the dorsal mutation is unchanged upon loss of cactus activity. This result implies that cactus acts via dorsal and has no independent morphogen function. In all other dorsal group mutant backgrounds, reduction of cactus function leads to embryosthat express ventrolateral pattern elements and have increased nuclear uptake of the dorsal protein at all positions along the dorsoventral axis. Thus, the cactus gene product can prevent nuclear transport of dorsal protein in the absence of function of the dorsal group genes. Genetic and cytoplasmic transplantation studies suggest that the cactus product is evenly distributed along the dorsoventral axis. Thus the inhibitory function that cactus product exerts on the nuclear transport of the dorsalprotein appears to be antagonized on the ventral side. We discuss models of how the action of the dorsal group genes might counteract the cactus function ventrally.D. St Johnston, D. Beuchle and C. Nüsslein-Volhard (1991). Staufen, a gene required to localize maternal RNAs in the Drosophila egg. Cell 66, 51-63.
The posterior group gene staufen is required both for the localization of maternal determinants tothe posterior pole of the Drosophila egg and for bicoid RNA to localize correctly to the anterior pole. We report the cloning and sequencing of staufen and show that staufen protein is one of the first molecules to localize to the posterior pole of the oocyte, perhaps in association with oskar RNA. Once localized, staufen is found in the polar granules and is required to hold other polar granule components at the posterior pole. By the time the egg is laid, staufen protein is also concentrated at theanterior pole, in the same region as bicoid RNA. GenBank accession no.: M69111D. Stein, S. Roth, E. Vogelsang and C. Nüsslein-Volhard (1991). The polarity of the dorsoventral axis in the Drosophila embryo is defined by an extracellular signal. Cell 65, 725-35.
Twelve maternal effect loci arerequired for the production of Drosophila embryos with a correct dorsoventral axis. Analysis of mosaic females indicates that the expression of the genes nudel, pipe, and windbeutel is required in the somatic tissue, presumably in the follicle cells that surround the oocyte. Thus, information coming from outside the egg cell influences dorsoventral pattern formation during embryogenesis. In transplantation experiments, the perivitelline fluid from the compartment surrounding the embryo can restoredorsoventral pattern to embryos from females mutant for nudel, pipe, or windbeutel. The positioning of the transplanted pervitelline fluid also determines the polarity of the restored dorsoventral axis. We propose that the polarizing activity, normally present at the ventral side of the egg, is a ligand for the Toll receptor. Presumably, local activation of the Toll protein by the ligand initiates the formation of the nuclear concentration gradient of the dorsal protein, thereby determining dorsoventralpattern.
M. van Lohuizen, M. Frasch, E. Wientjens and A. Berns (1991). Sequence similarity between the mammalian bmi-1 proto-oncogene and the Drosophila regulatory genes Psc and Su(z)2. Nature 353, 353-5.
The bmi-1 proto-oncogene can be activated by Moloney murine leukaemia proviral insertions in E mu-myc transgenic mice. It encodes a highly conserved nuclear protein of 324 amino acids which belongs to a familyof proteins containing a putative new zinc-finger. Another closely related member of this family is the mouse protein Mel-18. Here we report on the cloning and characterization of a homologous gene (D-bmi) from Drosophila melanogaster. Our analysis indicates that distinct domains of the mouse Bmi-1 protein, including the putative zinc-finger motif, are highly conserved within the much larger D-Bmi protein. Chromosomal localization and sequence comparison reveal that D-bmi is identical to PosteriorSex Combs (Psc) and indicate that the conserved domains between mouse bmi and Psc are also conserved within Suppressor-2 of Zeste (Su(z)2).