The role of transcriptional activation in the function of the Drosophila myb gene.

Vertebrate myb genes encode DNA-binding proteins that regulate transcription and have been implicated in regulation of cell proliferation, differentiation, and apoptosis. We have demonstrated that the single myb gene in Drosophila melanogaster, Dm myb, is required for the G(2)/M transition of the cell cycle and for suppression of endoreduplication. Recently, it has become apparent that the family of proteins containing Myb-related DNA-binding domains is much larger than originally believed and that the biochemical properties and functions of these proteins are diverse. We undertook studies to characterize the biochemical properties of the Drosophila Myb protein (DMyb). We now provide evidence that in addition to having homology with the vertebrate Myb proteins, the Drosophila Myb protein (DMyb) shares its biochemical properties. DMyb binds to a similar consensus sequence and activates transcription from a reporter construct regulated by vertebrate Myb proteins. We also show that DMyb proteins carrying mutations corresponding to previously isolated mutant alleles of Dm myb are less active as transcriptional activators than wild-type DMyb, indicating that a decrease in transcriptional activation ability is likely to cause the mutant phenotypes.

Probable mechanisms underlying interallelic complementation and temperature-sensitivity of mutations at the shibire locus of Drosophila melanogaster.

The shibire locus of Drosophila melanogaster encodes dynamin, a GTPase required for the fission of endocytic vesicles from plasma membrane. Biochemical studies indicate that mammalian dynamin is part of a complex containing multiple dynamin subunits and other polypeptides. To gain insight into sequences of dynamin critical for its function, we have characterized in detail a collection of conditional and lethal shi alleles. We describe a probable null allele of shi and show that its properties are distinct from those of two classes of lethal alleles (termed I and II) that show intergroup, interallelic complementation. Sequenced class I alleles, which display dominant properties, carry missense mutations in conserved residues in the GTPase domain of dynamin. In contrast, the sequenced class II alleles, which appear completely recessive, carry missense mutations in conserved residues of a previously uncharacterized "middle domain" that lies adjacent to the GTPase region. These data suggest that critical interactions mediated by this middle domain are severely affected by the class II lethal mutations; thus, the mutant sequences should be very useful for confirming the in vivo relevance of interactions observed in vitro. Viable heteroallelic combinations of shi lethals show rapid and reversible temperature-sensitive paralytic phenotypes hitherto only described for the ts alleles of shi. When taken together with the molecular analysis of shi mutations, these observations suggest that the GTPase domain of dynamin carries an intrinsically temperature-sensitive activity: hypomorphic mutations that reduce this activity at low temperatures result in conditional temperature-sensitive phenotype. These observations explain why screens for conditional paralytic mutants in Drosophila inevitably recover ts alleles of shi at high frequencies.

Drosophila myb is required for the G2/M transition and maintenance of diploidy.

The myb proto-oncogenes are thought to have a role in the cell division cycle. We have examined this possibility by genetic analysis in Drosophila melanogaster, which possesses a single myb gene. We have described previously two temperature-sensitive, recessive lethal mutants in Drosophila myb (Dm myb). The phenotypes of these mutants revealed a requirement for myb in diverse cellular lineages throughout the course of Drosophila development. We now report a cellular explanation for these findings by showing that Dm myb is required for both mitosis and prevention of endoreduplication in wing cells. Myb apparently acts at or near the time of the G2/M transition. The two mutant alleles of Dm myb produce the same cellular phenotype, although the responsible mutations are located in different functional domains of the gene product. The mutant phenotype can be partially suppressed by ectopic expression of either cdc2 or string, two genes that are known to promote the transition from G2 to M. We conclude that Dm myb is required for completion of cell division and may serve two independent functions: promotion of mitosis, on the one hand, and prevention of endoreduplication when cells are arrested in G2, on the other.

myb provides an essential function during Drosophila development.

Vertebrate c-myb encodes a transcription factor thought to play an important role in the cell cycle. To gain further insight into myb function, we have been studying a related gene in Drosophila. We found that Drosophila myb is abundantly expressed throughout development in all mitotically active tissues, at lower levels in some postmitotic tissues, but not at detectable levels in polyploid larval tissues. We performed genetic screens to isolate recessive lethal mutations in the chromosomal region that includes the myb gene. We obtained two temperature-sensitive alleles of myb, demonstrating that the gene provides an essential function. Examination of the mutant phenotype revealed that Drosophila myb is important for both embryonic and imaginal development and that myb serves a role in the development of many tissues and during oogenesis.

The Drosophila clathrin heavy chain gene: clathrin function is essential in a multicellular organism.

The clathrin heavy chain (HC) is the major structural polypeptide of the cytoplasmic surface lattice of clathrin-coated pits and vesicles. As a genetic approach to understanding the role of clathrin in cellular morphogenesis and developmental signal transduction, a clathrin heavy chain (Chc) gene of Drosophila melanogaster has been identified by a combination of molecular and classical genetic approaches. Using degenerate primers based on mammalian and yeast clathrin HC sequences, a small fragment of the HC gene was amplified from genomic Drosophila DNA by the polymerase chain reaction. Genomic and cDNA clones from phage libraries were isolated and analyzed using this fragment as a probe. The amino acid sequence of the Drosophila clathrin HC deduced from cDNA sequences is 80%, 57% and 49% identical, respectively, with the mammalian, Dictyostelium and yeast HCs. Hybridization in situ to larval polytene chromosomes revealed a single Chc locus at position 13F2 on the X chromosome. A 13-kb genomic Drosophila fragment including the Chc transcription unit was reintroduced into the Drosophila genome via P element-mediated germline transformation. This DNA complemented a group of EMS-induced lethal mutations mapping to the same region of the X chromosome, thus identifying the Chc complementation group. Mutant individuals homozygous or hemizygous for the Chc1, Chc2 or Chc3 alleles developed to a late stage of embryogenesis, but failed to hatch to the first larval stage. A fourth allele, Chc4, exhibited polyphasic lethality, with a significant number of homozygous and hemizygous offspring surviving to adulthood. Germline clonal analysis of Chc mutant alleles indicated that the three tight lethal alleles were autonomous cell-lethal mutations in the female germline. In contrast, Chc4 germline clones were viable at a rate comparable to wild type, giving rise to viable adult progeny. However, hemizygous Chc4 males were invariably sterile. The sterility was efficiently rescued by an autosomal copy of the wild-type Chc gene reintroduced on a P element. These findings suggest a specialized role for clathrin in spermatogenesis.

Expression during Drosophila development of DER, a gene related to erbB-1 and neu: correlations with mutant phenotypes.

We used in situ hybridization to study expression of the DER gene during Drosophila development. DER encodes a transmembrane cell-surface receptor with a cytoplasmic protein-tyrosine kinase domain, and resembles the vertebrate genes that encode the EGF receptor and the neu protein. We examined most stages of development in the Drosophila life cycle and found a substantial correlation between DER expression and the phenotypes associated with a variety of mutant alleles. Of particular note were high levels of expression in the primordia of the mouth parts, which are the embryonic tissues most sensitive to reductions in DER activity; discrete expression in a subset of neural cells essential for construction of the axonal scaffold, a structure that is deformed in mutant embryos; uneven expression in the eye disc, the development of which is abnormal in both mild hypomorphs and hypermorphs; and expression in the follicular epithelial cells of the ovary, which are responsible for producing the eggshell of developing oocytes and do so aberrantly in the mildest hypomorphs. However, DER transcripts were also detected in a subset of tissues that have not been reported to be abnormal in mutant organisms. Our findings agree with and extend recently reported results for the DER protein, indicating that DER is regulated primarily at the level of transcription, in contrast to previous suggestions. We conclude that the phenotypes displayed by recessive mutants can be attributed to loss of DER function in the affected tissues.

Cloning and characterization of the scalloped region of Drosophila melanogaster.

Viable mutants of the scalloped gene (sd) of Drosophila melanogaster exhibit defects that can include gapping of the wing margin and ectopic bristle formation on the wing. Lethal sd alleles characterized in the present study now implicate this gene in a genetic function essential for normal development. In order to further characterize the developmental role of this gene, we have undertaken to clone and characterize the region where sd maps. A P[ry+] transposon insertion at 13F associated with sd[ry+2216] served as the starting point for a 42-kb chromosomal walk. Molecular lesions associated with viable and lethal sd alleles were characterized by genomic hybridization analysis as a means of defining the extent of the gene. DNA rearrangements associated with 11 viable sd alleles map to a 2-kb interval which appears to be a "hot spot" for P element activity. Four of five recessive lethal sd mutations were mapped by denaturing gradient gel electrophoresis to a region 12-14 kb away from the region of viable lesions. In a sd+ genotype, at least two structurally related and developmentally regulated transcripts hybridize to the genomic region where several sd lethal alleles have been localized. A viable mutation, sd58, used for comparison in the transcript analysis, makes at least two slightly smaller transcripts that also hybridize to this region. Preliminary analysis of cDNA clones has identified three structurally related transcripts that hybridize to this genomic region. The 5' end of these transcripts extends into the 2-kb genomic region wherein DNA rearrangements were seen in the P element rearrangements. We favor the view that the transcripts represented by these cDNA clones are products of the sd gene. If this is true, the sd gene would include genomic sequences extending over at least 14 kb of the described chromosomal walk, and would appear to be subject to alternative splicing.

A gene related to the proto-oncogene fps/fes is expressed at diverse times during the life cycle of Drosophila melanogaster.

The proto-oncogene fps/fes encodes a distinctive type of protein-tyrosine kinase. We identified a Drosophila gene (dfps85D) whose product resembles the proteins encoded by vertebrate fps/fes and the closely related gene fer. dfps85D is located at chromosomal position 85D10-13 and is unlikely to correspond to any previously defined genetic locus in Drosophila melanogaster. Expression of the gene is entirely zygotic in origin and occurs throughout the life cycle. But hybridization in situ revealed that the pattern of expression is specialized and evolves in a provocative manner. The most notable feature of expression is the diversity of developmental periods, tissues, and cells in which it occurs. In some tissues, expression is transient; in others, it is continuous. Expression occurs in both mitotic and terminally differentiated tissue and, at various times in development, is prominent in imaginal disks, gut, muscle, testes, ovaries, retina, and other neural tissues. It appears that the use of dfps85D is more diversified than that of other Drosophila protein-tyrosine kinases reported to date and contrasts sharply with the restricted expression of fps itself in vertebrates. The detailed description of expression provided here will help guide the search for mutants in dfps85D.

Diverse expression of dsrc29A, a gene related to src, during the life cycle of Drosophila melanogaster.

We used in situ hybridization to study the RNA expression of the dsrc29A gene during Drosophila development. This gene encodes two proteins differing at their amino termini. Both gene products contain a protein-tyrosine kinase domain and resemble the protein encoded by vertebrate src. We examined most stages of development in the Drosophila life cycle: embryos, third instar larvae, pupae and adults. Our results revealed that dsrc29A expression is specialized throughout development, being prominent at various times in neural tissue, phagocytic cells, dorsal vessel, ovaries, gut, developing salivary glands, imaginal discs and disc derivatives. These findings confirm and extend previous results for the distribution of dsrc29A protein, indicating that the regulation of this gene is primarily at the level of transcription. In some tissues expression is transient, whereas in others, it is continuous, and expression occurs in proliferative, differentiating and differentiated tissue. These patterns of expression demonstrate how a single protein-tyrosine kinase might play diverse roles at different times during development. Comparison of the expression of dsrc29A and other members of the protein-tyrosine kinase gene superfamily reveals that the genes are expressed in distinctive but sometimes overlapping patterns.

Isolation of the proto-oncogene c-myb from D. melanogaster.

We have isolated the proto-oncogene c-myb from Drosophila melanogaster. This gene is represented by a single locus at position 13E-F on the X chromosome, and is expressed in early embryos by transcription into two polyadenylated RNAs with lengths of approximately 3.0 and 3.8 kb. The gene may encode a protein with a molecular weight of at least 55,000 that shares a domain with c-myb (chicken) in which 91 of 125 (or 73%) of the amino acids are identical in the Drosophila and chicken genes. These findings represent the first rigorous identification of a Drosophila proto-oncogene that can encode what may be a nuclear protein, and they set the stage for a genetic analysis of how c-myb serves the normal organism.

A drug-resistant mutation in the ribosomal DNA of Tetrahymena.

A mutation that confers resistance to the drug paromomycin is shown to be in the structural gene that codes for the ribosomal RNA in Tetrahymena. This observation was made by exploiting a variant of the ribosomal DNA that distorts amplification of this locus when a new somatic nucleus develops during conjugation. Because the allelic forms of this locus have a restriction endonuclease site polymorphism, it was possible to correlate drug resistance with presence of a specific allele. The genetic results have been confirmed by sequence analysis (presented elsewhere). Thus, the crosses presented here provide a unique opportunity to identify mutations in the ribosomal DNA.

Pituitary-adrenal function in rats chronically exposed to cold.

Exposure to cold for 2 weeks was used to assess the effects of a sustained stimulus on pituitary-adrenal function in male rats. The diurnal peak in plasma and adrenal corticosterone was advanced by 4 h during the first 24 h of exposure to cold but returned to its usual time (2000 h) by the next day. Plasma ACTH and corticosterone levels were generally greater at all times during the 24-h cycle in animals exposed to cold for up to 2 weeks, with the greatest increase occurring consistently at the time of peak. When rats exposed to cold for 1 week were returned to a normal 24 C environment, plasma corticosterone tended to increase. Plasma ACTH and plasma and adrenal corticosterone responses to a superimposed acute provocative stimulus (ip saline injection) were faster, greater, and more sustained in rats exposed to cold for 3 or 7 days. Similarly, the compensatory adrenal hypertrophy response to unilateral adrenalectomy was greater in cold-exposed rats. Such animals were also more resistant to pituitary-adrenal suppression by prednisolone. In contrast, there was no change in the sensitivity of the adrenal to exogenous ACTH. The results suggest that chronic exposure to cold causes a sustained activation of central mechanisms that regulate pituitary ACTH secretion as well as extra-pituitary mechanisms that regulate adrenal size; it reduces the effectiveness of negative feedback mechanisms, but does not alter those involved in the regulation of adrenal rhythmicity or adrenal sensitivity to ACTH.

The genome of Plasmodium falciparum. I: DNA base composition.

Some structural properties of the DNA of Plasmodium falciparum were studied thoroughly using several techniques. Its G+C content was found to be extremely low (17-19%), the lowest reported for a living organism. The DNA seems to be composed only of the four major bases as no methylated bases were detected. This DNA had a Tm value of 62.5 degrees C and its denaturation profile showed no marked intramolecular heterogeneity.

Sequence-specific fragmentation of macronuclear DNA in a holotrichous ciliate.

Macronuclear DNA from the protozoan G. chattoni, a holotrichous ciliate, was analyzed. Most, if not all, of the macronuclear DNA is subchromosomal, ranging in size from above 100 kb down to 2.1 kb, with molecules in the lower molecular weight range being resolvable by gel electrophoresis into reproducible, specific, discrete size classes. A prominent class of linear 9.3 kb molecules consists of single free rRNA genes. Upon denaturation of total macronuclear DNA was found as single-stranded circles. Sequence analysis of showed that a minimum of 38 tandem repeats of the sequence CCCCAA is present in inverted orientation at each end of most or all Glaucoma macronuclear DNA molecules, including the rDNA. This sequence must therefore be recognized during site-specific fragmentation of chromosomes in macronuclear development.