Regulation of scute function by extramacrochaete in vitro and in vivo.

The pattern of adult sensilla in Drosophila is established by the dosage-sensitive interaction of two antagonistic groups of genes. Sensilla development is promoted by members of the achaete-scute complex and the daughterless gene whereas it is suppressed by whereas extramacrochaete (emc) and hairy. All these genes encode helix-loop-helix proteins. The products of the achaete-scute complex and daughterless interact to form heterodimers able to activate transcription. In this report, we show that (1) extra-macrochaete forms heterodimers with the achaete, scute, lethal of scute and daughterless products; (2) extramacrochaete inhibits DNA-binding of Achaete, Scute and Lethal of Scute/Daughterless heterodimers and Daughterless homodimers and (3) extramacrochaete inhibits transcription activation by heterodimers in a yeast assay system. In addition, we have studied the expression patterns of scute in wild-type and extramacrochaete mutant imaginal discs. Expression of scute RNA during imaginal development occurs in groups of cells, but high levels of protein accumulate in the nuclei of only a subset of the RNA-expressing cells. The pattern is dynamic and results in a small number of protein-containing cells that correspond to sensillum precursors. extramacrochaete loss-of-function alleles develop extra sensilla and correspondingly display a larger number of cells with scute protein. These cells appear to arise from those that in the wild type already express scute RNA; hence, extramacrochaete is a repressor of scute function whose action may take place post-transcriptionally.

Transcriptional activation by heterodimers of the achaete-scute and daughterless gene products of Drosophila.

The achaete-scute complex (AS-C) and the daughterless (da) genes encode helix-loop-helix proteins which have been shown to interact in vivo and to be required for neurogenesis. We show in vitro that heterodimers of three AS-C products with DA bind DNA strongly, whereas DA homodimers bind weakly and homo or heterocombinations of AS-C products not at all. Proteins unable to dimerize did not bind DNA. Target sequences for the heterodimers were found in the promoters of the hunchback and the achaete genes. Using sequences of the former we show that the DNA binding results obtained in vitro fully correlate with the ability of different combinations to activate the expression of a reporter gene in yeast. Embryos deficient for the lethal of scute gene fail to activate hunchback in some neural lineages in a pattern consistent with the lack of a member of a multigene family.

Lateral inhibition and cell fate during neurogenesis in Drosophila: the interactions between scute, Notch and Delta.

A comparison of the patterns of expression of AS-C (T3) RNA and protein suggests that an important level of regulation occurs post-transcriptionally. First, when the RNA is abundant in the early embryo the protein is barely detectable. Later, the protein starts to accumulate in only a subset of the nuclei of those cells expressing the RNA. Only the cells in the subsets become the neuroblasts. This post-transcriptional regulation is suppressed in embryos mutant for the genes Notch and Delta; where all cells expressing RNA accumulate protein. These findings suggest that deployment of T3 protein expression is one of the causal factors that assigns specific fates to the neuroblasts and, in consequence, a basis for the mechanism of lateral inhibition is proposed.

Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence.

A DNA binding and dimerization motif, with apparent amphipathic helices (the HLH motif), has recently been identified in various proteins, including two that bind to immunoglobulin enhancers (E12 and E47). We show here that various HLH proteins can bind as apparent heterodimers to a single DNA motif and also, albeit usually more weakly, as apparent homodimers. The HLH domain can mediate heterodimer formation between either daughterless, E12, or E47 (Class A) and achaete-scute T3 or MyoD (Class B) to form proteins with high affinity for the kappa E2 site in the immunoglobulin kappa chain enhancer. The achaete-scute T3 and MyoD proteins do not form kappa E2-binding heterodimers together, and no active complex with N-myc was evident. The formation of a heterodimer between the daughterless and achaete-scute T3 products may explain the similar phenotypes of mutants at these two loci and the genetic interactions between them. A role of E12 and E47 in mammalian development, analogous to that of daughterless in Drosophila, is likely.

The achaete-scute gene complex of Drosophila melanogaster comprises four homologous genes.

We have determined the nucleotide sequence of two genes of the achaete - scute complex (AS-C) and show that they are homologous to two previously sequenced members of the same locus. These four genes are interspersed with other transcription units of unknown function. We also study the expression of one of these genes by in situ hybridization and compare it with the other three genes. We suggest that the complete function ascribed to the AS-C by genetic experiments is carried out by the four homologous genes. We discuss the possible biochemical function of the AS-C in neurogenesis in the light of the homologies of the four genes with the mammalian myc family.

Phenocopies induced with antisense RNA identify the wingless gene.

Earlier work suggested that the wingless gene of Drosophila is required for cooperation within discrete groups of cells during development. We show that antisense RNA made from a 3 kb transcript produces wingless mutant phenocopies when injected into wild-type eggs, proving that this transcript executes the wingless function. In the accompanying paper, Rijsewijk et al. report cloning of a Drosophila homolog (Dint-1) of the mouse int-1 gene and show that this gene is identical to wingless+. The partial sequence of the wingless cDNA that we have isolated is identical to part of the complete Dint-1 sequence described by Rijsewijk et al.

The expression of three members of the achaete-scute gene complex correlates with neuroblast segregation in Drosophila.

The expression of a subset of homologous genes of the AS-C is required during embryogenesis and metamorphosis for proper neural development. Here we study the expression of three of these genes (T3, T4, and T5) and show that their transcripts accumulate at the blastoderm stage in periodic patterns coincident with the dorsoventral extent of the neuroectoderm. Subsequent expression is in partially overlapping patterns that correlate with the segregation of neuroblasts. These genes are not transcribed in neurons. We also show that a deficiency for the AS-C prevents the appearance of at least one identified class of neuroblasts. We propose that the AS-C is part of a network of genes responsible for the segregation of neuroblasts.

The achaete-scute gene complex of D. melanogaster: conserved domains in a subset of genes required for neurogenesis and their homology to myc.

We have determined the nucleotide sequence of two transcription units of the AS-C and shown that their potential protein coding regions share two principal domains of homology. Both domains are conserved within the myc protein family, and one of them is highly homologous with the consensus for protein tyrosine kinase substrates. We show in addition that at least one of these domains is shared with other transcription units within the AS-C, some of which were not previously known. We suggest that the AS-C encodes several homologous polypeptides, which represent a subset of a larger gene family. We propose that each member of the family functions at an equivalent stage of a unique morphogenetic operation involving the segregation of individual neural lineages from the epidermal anlage.

Biochemical and electron microscopic studies of the transcription of vaccinia DNA by RNA polymerase from Escherichia coli: localization and characterization of transcriptional complexes.

We used the prokaryotic Escherichia coli RNA polymerase to determine if vaccinia DNA might provide recognition sites for the bacterial binding and initiation. Electron microscopic studies of the interaction of E. coli RNA polymerase with vaccinia DNA and molecular hybridization analysis of the transcription products formed after 3 or 5 min of in vitro incubation showed that: there were 30-40 sites on the template where the polymerase could bind and initiate cRNA synthesis; the entire coding capacity of the genome was utilized for cRNA synthesis; transcription was asymmetric; cRNA molecules were similar in size to the transcripts synthesized by the vaccinia virus RNA polymerase in vitro and in vivo; cRNA contains sequences in common with 'pre-early', 'early', and 'late' in vivo RNA; 'self-annealing' of cRNA in the presence or absence of RNA synthesized in vitro by the virion associated RNA polymerase showed that less than 1% dsRNA product could be detected suggesting that initially the same strand(s) was copied by the viral and bacterial enzymes; no differences in the frequency with which sequences represented in the Hind III fragments of vaccinia DNA were transcripted with time of in vitro incubation could be detected. These findings strongly suggest that the bacterial enzyme might recognize truly viral promotors. With extended in vitro incubations of the E. coli RNA polymerase with vaccinia DNA the control of transcription was found to diminish. This was correlated with an increase in the size of the transcripts and the synthesis of significant amounts of self-complementary RNA, indicating that symmetrical transcription was occurring. The dsRNA species recovered after self-annealing the cRNA from a 30 min in vitro reaction mixture were found to contain sequences which hybridized to some portion of all the Hind III restriction fragments of vaccinia DNA. The methods described here might be useful for the localization and characterization of promotor sequences in the genome of vaccinia virus, as well as for studies on sequence conservation between members of the Poxvirus genus.

Molecular genetics of the achaete-scute gene complex of D. melanogaster.

The achaete-scute gene complex (AS-C), involved in differentiation of the sensory chaetes of D. melanogaster, and the yellow locus have been cloned. The yellow locus is the most distal and is followed, proximally, by the achaete and the scute loci. In the scute locus (75 kb), three transcription units separated by long stretches of DNA give rise to poly(A)+ RNAs of 1.6, 1.2, and 1.6 kb. Most DNA lesions associated with scute mutations map within the presumably untranscribed DNA. Their mutant phenotypes are stronger the closer the lesions are to the structural gene of one transcript (T4 RNA). Genetic and developmental data suggest that only this RNA is fundamental for the scute function. Its transcription might be perturbed by far removed DNA lesions. A second transcript is probably implicated in the lethal of scute embryonic function, while the third transcript is unnecessary for the differentiation of most macrochaetes. Two additional polyadenylated RNAs are transcribed from the achaete (1.1 kb) and yellow (1.9 kb) loci.

Regulation of cytoplasmic mRNA prevalence in sea urchin embryos. Rates of appearance and turnover for specific sequences.

Complementary DNA clones representing cytoplasmic poly(A) RNAs of sea urchin embryos were hybridized with metabolically labeled cytoplasmic RNA preparations and the rates of appearance and of decay for each transcript species were determined at the blastula-gastrula stage of development. The prevalence of the transcripts chosen for this study ranged, on average, from about one molecule per cell to a few hundred molecules per cell. The embryos were labeled continuously for 18 hours with [3H]guanosine, beginning at 24 hours post-fertilization. The amount of cytoplasmic [3H]poly(A) RNA that hybridized to each cloned sequence was determined and the specific activity of the [3H]GTP pool was measured in the same embryos. Rate constants for the entry of each transcript species into the cytoplasm, and for its decay were extracted from these data. The embryo transcript species identified by the cloned probes displayed a range of stabilities. Half-lives of only a few hours were measured both for a very rare sequence and for a moderately prevalent sequence. Other newly synthesized transcripts, including sequences that first appear during embryonic development, as well as sequences also represented in maternal RNA, are far more stable. We conclude that cytoplasmic RNA turnover rate is a major variable in the determination of the cytoplasmic level of expression of embryo genes. The entry rates of the transcripts into the cytoplasm also varied, from a few molecules per embryo per minute to several hundred, depending on the sequence. By comparing the mass of transcripts of a given sequence in the embryo to the mass of transcripts of that sequence accumulating as a result of new synthesis, the point at which embryo transcription accounts for the major fraction of the cytoplasmic molecules could be estimated. This calculation showed that for some sequences maternal transcripts persist well beyond gastrulation, while other embryo poly(A) RNA species are largely the product of transcription in the embryo nuclei from the blastula stage onwards. There is no single stage at which all maternal transcripts are suddenly replaced by newly synthesized embryo transcripts. Primary transcription rates were measured for two sequences by determining accumulation of label in these RNA species soon after addition of [3H]guanosine to the cultures. Comparing these rates to the cytoplasmic entry rates, we did not detect a significantly greater nuclear transcription of the sequence homologous to the cloned probe.

Electron microscopic studies of transcriptional complexes released from vaccinia cores during RNA-synthesis in vitro: methods for fractionation of transcriptional complexes.

Electron microscopic (EM) and biochemical methods were employed to study the transcriptional complexes present in detergent lysates of vaccinia virus cores actively synthesizing RNA in vitro. When processed and examined in the EM, 14 'transcriptional sites' could be observed on full-length DNA templates. Fractionation of lysates by equilibrium density centrifugation in CsSO4, chromatography on hydroxyapatite columns or by sedimentation in sucrose gradients, allowed isolation of DNA templates associated with transcripts but these manipulations often resulted in fragmentation of the DNA template or promoted the release of transcripts from the template. It is suggested that RNA transcripts remain associated with the template in regions of supercoiling. These regions, in turn, may be maintained by DNA-protein interactions which are compromised as the transcriptional complexes are fractionated and purified.

Transcripts of three mitochondrial genes in the RNA of sea urchin eggs and embryos.

cDNA clones representing mitochondrial 16 S rRNA, and mRNAs for cytochrome oxidase I and an unidentified reading frame were used to measure the prevalence and stability of these transcripts in gastrula stage embryos. The 16 S rRNA is the most prevalent embryo poly(A) RNA, and is synthesized about four times more rapidly than is the mRNA for cytochrome oxidase. The relative prevalence of the two mRNAs is largely determined by their turnover rates.

Herpes simplex virus DNA sequences in the CNS of latently infected mice.

It has been amply documented that herpes simplex virus (HSV) persists in sensory ganglia of the peripheral nervous system (PNS). In contrast, HSV latency in the central nervous system (CNS) has not been well characterized. Corneal inoculation of virus results in a productive viral infection in the CNS during the first week after inoculation, indicating that the virus can progress from the PNS to the CNS. During latency, HSV has been found by co-cultivation of CNS tissue in only a very small fraction of inoculated mice. We have used here molecular hybridization techniques to analyse the fate of viruses that reach the CNS by anatomical pathways. We show that 6 days after corneal inoculation of HSV-1 a productive viral infection was present in brain tissue as well as in peripheral ganglia in at least 90%F of the inoculated mice. The mortality during this acute phase was only 2%. In the survivors, latent HSV could be recovered by explantation from 95% of the trigeminal ganglia, but only 5% of the brain tissue explants of the same mice yielded infectious virus. However, HSV DNA sequences were detected in the brains of 30% of mice which harboured latent HSV in their trigeminal ganglia. These results suggest that viruses that progress from the PNS into the CNS are not eliminated, but are capable of establishing a latent infection in the CNS that cannot be reactivated by explantation techniques.

Procedure for purification of intact DNA from vaccinia virus.

A procedure for the isolation of intact vaccinia DNA molecules by chromatography on hydroxyapatite in the presence of 6 M urea is described. When lysates of virions containing 0.5 to 10 microgram of DNA were employed, over 95% of the viral DNA could be recovered free of poteins. Vaccinia DNA molecules isolated in this manner sedimented at 68S in neutral sucrose gradients and had an average contour length of 62.3 micrometer when examined in an electron microscope, and the DNA could be cleaved with the restriction endonuclease EcoRI and BamHI. The results of these analyses showed that intact vaccinia DNA molecules of 120 X 10(6) to 130 X 10(6) molecular weight could be obtained by the procedures described.