Biomedical subjects

E Knust

Publications and source records attributed to E Knust.

49 records · Page 3Linked to original sources

PubMed1989

The molecular genetics of early neurogenesis in Drosophila melanogaster.

The extent of neurogenesis in Drosophila is under the control of the so-called neurogenic genes, named for their mutant phenotype of causing neural hyperplasia. Their wild-type products appear to be responsible for a signal chain that decides the fate of ectodermal cells in the embryo. Various kinds of data, from cell transplantation experiments as well as from genetic and molecular analyses, suggest that the proteins encoded by the genes Notch and Delta may act at the membrane of the signal-transmitting cells to provide a ligand to a still unknown receptor molecule; in contrast, the locus of Enhancer of split codes for several functions related to the transduction and further processing of the signal.

Animals↗

PubMed1989

Closely related transcripts encoded by the neurogenic gene complex enhancer of split of Drosophila melanogaster.

Genetic evidence suggests that E(spl), one of the neurogenic loci of Drosophila, is a gene complex comprising an as yet incompletely established number of transcription units. In order to correlate the various transcription units with E(spl) functions, wild-type flies were transformed with genomic DNA encoding the transcription unit m8 from the mutant E(spl)D, which was known to be altered in embryos carrying this mutant allele. Transformants show the same dominant enhancement of the spl phenotype as E(spl)D itself. Since m8 has a virtually identical pattern of expression as m4, m5 and m7, we have determined the sequence of these four transcripts. The deduced protein products of m5, m7 and m8 exhibit extensive sequence homology with each other. All three encode a sequence similar to one of the conserved domains of representatives of the vertebrate myc gene family which is also present in the deduced protein sequences of the Drosophila achaete-scute gene complex. Sequence analysis of the m8 transcription unit in the E(spl)D mutation revealed several DNA lesions. One of the lesions is a deletion in the region upstream of the transcription start site. Another lesion is a deletion in the coding region that leads to a shorter protein which, in addition, differs in its carboxy-terminal end from the wild-type protein by the presence of nine amino acids.

Amino Acid Sequence↗

PubMed1987

Molecular analysis of the neurogenic locus Enhancer of split of Drosophila melanogaster.

Enhancer of split [E(spl)], one of the neurogenic loci of Drosophila, is located in bands 96F8-13. One hundred and fifty kilobases of genomic DNA, spanning the E(spl) locus, were cloned by chromosomal walking. DNA heterogeneities associated with eleven E(spl) mutations, including three Pr alleles, were mapped to a region of 36 kb, and an additional one outside of this region. One of these mutations is a deletion of 34 kb that causes severe neural hyperplasia of homozygous embryos with complete penetrance. Mutations associated with DNA polymorphisms mapping within smaller regions do not lead to a fully penetrant neurogenic phenotype. The 36-kb region encodes 11 major transcripts, which exhibit distinct temporal and/or spatial patterns of expression. The expression of one of these transcripts is modified in two different mutants. In addition, one of the mutants [E(spl)] shows another transcript, which is not present in the wild-type and co-exists with the remaining transcripts. We suggest that more than one of the 11 transcripts are necessary for a normal function of the E(spl) locus. The spatial distribution of four of these RNAs, which exhibit almost identical patterns of expression, strongly suggests that the encoded proteins are required for the process of segregation of neural and epidermal lineages.

Journal Article↗

PubMed1987

The neurogenic gene Delta of Drosophila melanogaster is expressed in neurogenic territories and encodes a putative transmembrane protein with EGF-like repeats.

The decision of an ectodermal cell to take on a neural or an epidermal fate depends on its interactions with the neighbouring cells. In Drosophila melanogaster, the available evidence suggests that a regulatory signal necessary for epidermal commitment is built by the products of the so-called neurogenic genes. We have cloned 180 kb of genomic DNA surrounding the neurogenic gene Delta (Dl). Restriction fragment-length polymorphisms were mapped to a region of 25 kb. These 25 kb of DNA are assumed to contain essential parts, or all, of the Dl gene. Northern blots detect two developmentally regulated transcripts, of 5.4 and 4.6 kb, which are associated with the region where the mutants map. Serveral cDNA clones were recovered from embryonic cDNA libraries by homology to the 25 kb of genomic DNA. The complete sequence of a cDNA clone containing an insert of 4.73 kb was determined. The conceptual translation of the longest open reading frame yields a protein of 880 amino acids. This protein displays characteristics of a membrane protein, with intercellular, transmembrane and extracellular domains. The extracellular domain contains a tandem array of nine EGF-like repeats. In in situ hybridizations to tissue sections, transcripts homologous to Dl are detected in all territories with neurogenic abilities, e.g. the neurogenic ectoderm and the primordia of the sensory organs. Initially all cells of these neurogenic territories express Dl, but later on transcription of Dl becomes restricted to the cells that have adopted the neural fate. The topological specificity in the transcription of Dl corresponds to the one expected for a regulatory signal that mediates epidermal commitment.

Journal Article↗

PubMed1987

EGF homologous sequences encoded in the genome of Drosophila melanogaster, and their relation to neurogenic genes.

The function of the neurogenic genes of Drosophila melanogaster is required for a normal pattern of commitment of neural and epidermal progenitor cells. In the course of searching for a molecular basis for the functional interrelationships that exist between the neurogenic genes, fragments of cloned DNA from the genes master mind (mam), Delta (Dl), Enhancer of split [E(spl)] and Notch (N) were hybridized to each other. Strong cross-hybridization was observed between a fragment of the Dl gene and a fragment of the N gene encoding a peptide with homology to several proteins of mammals, including the epidermal growth factor (EGF). Sequencing of this Dl fragment revealed an open reading frame encoding four EGF-like repeats with homology to the repeats found in the N gene. Screening genomic and cDNA libraries under conditions of reduced stringency with Dl and N probes that encode EGF-like repeats uncovered several cross-hybridizing clones, suggesting that other Drosophila genes may also encode such peptides. Part of a cross-hybridizing cDNA clone, derived from a gene located at position 95F on the third chromosome, was sequenced and found to encode five repeats with homology to those encoded by N and Dl. Preliminary evidence on the spatial pattern of transcription indicates that the gene at position 95F is regulated in its expression, as it is transcribed in all ectodermal derivatives, with the exception of the central nervous system. Indirect evidence suggests that this clone may derive from the crumbs (crb) gene, which is likely to be an hitherto unknown neurogenic gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1987

The enhancer of split locus and neurogenesis in Drosophila melanogaster.

Enhancer of split (E(spl)) is one of a group of so-called neurogenic genes of Drosophila. We describe two different types of E(spl) alleles, dominant and recessive, which exert opposite effects on both central and peripheral nervous system development. The only extant dominant allele determines a reduction in the number of central neurons and peripheral sensilla; this phenotype is not reduced by a normal complement of wild-type alleles. Since animals carrying a triploidy for the wild-type locus develop similar defects, the dominant allele is probably the result of a gain-of-function mutation. Several recessive alleles, obtained as revertants of the dominant allele, are loss-of-function mutations and determine considerable neural hyperplasia. The present evidence suggests that neural defects of E(spl) mutants are due to defective segregation of neural and epidermal lineages, leading to neural commitment of less or of more cells than in the wild type, depending upon whether the animals carry the dominant or any of the recessive alleles, respectively. Therefore, E(spl) formally behaves as a gene switching between neural and epidermal pathways.

Alleles↗

PubMed1984

Predominant immediate-early transcripts of human cytomegalovirus AD 169.

Transcription of the human cytomegalovirus genome (strain AD 169) was investigated at the immediate-early (IE) time after infection, by using cycloheximide to suppress virus-specific protein synthesis. In total cell RNA, four predominant IE transcripts were found which were encoded by one contiguous region of the long unique segment between map units 0.06 and 0.16 in prototype arrangement of human cytomegalovirus AD 169 DNA. Analysis by Northern blot hybridizations demonstrated that the transcripts possessed a size of 1.9, 2.2, 2.3, and 5.0 kilobases, respectively. Coding sequences and directions of transcriptions were mapped by Northern blots and hybridizations with oligodeoxythymidylic acid-primed and randomly primed cDNA. The 1.9-, 2.2-, and 2.3-kilobase RNAs were found in the polyadenylated fraction of IE RNA exclusively; in contrast, a part of the 5.0-kilobase RNA appeared polyadenylated, although the majority of the same transcript was found in the nonpolyadenylated pool. Also, different from the other IE genes, the DNA coding for the 5.0-kilobase IE RNA was transcribed in high quantities during the late phase of virus replication, suggesting an exemption from the temporal regulation of herpesvirus transcription.

Base Sequence↗

PubMed1984

Immediate-early transcription of Herpesvirus saimiri.

Transcription of Herpesvirus saimiri was characterized during the initial phases of productive infection by Northern blot analyses and hybridizations of radioactive cDNA with cloned fragments of virion L-DNA. Under conditions of immediate-early transcription, e.g., blocking of viral protein synthesis by cycloheximide, a single cytoplasmic polyadenylated viral RNA of 2.7 kilobases was found in infected cells. The sequence coding for this RNA was between map units 0.89 and 0.93; it was transcribed from right to left in prototype arrangement of M-DNA. The immediate-early mRNA of lytically infected cells appeared to be very similar, if not identical, to the single viral RNA species found in lymphoid cells transformed by H. saimiri.

Animals↗

PubMed1983

Cloning of Herpesvirus saimiri DNA fragments representing the entire L-region of the genome.

Purified particles of Herpesvirus saimiri, a potent tumor-eliciting virus of primates, contain genomic DNA molecules (145-170 kb) consisting of a unique L-DNA region (112 kb) which is flanked by variable stretches of repetitive sequences (H-DNA). Restriction fragments representing the entire L-DNA of H. saimiri strain No. 11 were cloned in plasmid and bacteriophage vectors. The internal fragments of L-DNA generated by the enzymes EcoRI and KpnI were inserted into plasmid pACYC184, cosmid pJC81, or bacteriophage lambda derivative Charon 4A. The terminal parts of L-DNA, including the junctions between repetitive DNA and unique sequences, were cloned between the cleavage sites for KpnI and SmaI in the plasmid vector pWD7, which was constructed for this purpose. Molecular cloning allowed us to confirm and modify, in part, the existing cleavage maps of H. saimiri DNA. It provides a basis for future studies on virus replication and oncogenic transformation.

Bacteriophage lambda↗

PubMed1983

Molecular cloning and physical mapping of murine cytomegalovirus DNA.

Murine cytomegalovirus (MCMV) Smith strain DNA is cleaved by restriction endonuclease HindIII into 16 fragments, ranging in size from 0.64 to 22.25 megadaltons. Of the 16 HindIII fragments, 15 were cloned in plasmid pACYC177 in Escherichia coli HB101 (recA). The recombinant plasmid clones were characterized by cleavage with the enzymes XbaI and EcoRI. In addition, fragments generated by double digestion of cloned fragments with HindIII and XbaI were inserted into the plasmid vector pACYC184. The results obtained after hybridization of 32P-labeled cloned fragments to Southern blots of MCMV DNA cleaved with HindIII, XbaI, EcoRI, BamHI, ApaI, ClaI, EcoRV, or KpnI allowed us to construct complete physical maps of the viral DNA for the restriction endonucleases HindIII, XbaI, and EcoRI. On the basis of the cloning and mapping experiments, it was calculated that the MCMV genome spans about 235 kilobase pairs, corresponding to a molecular weight of 155,000,000. All fragments were found to be present in equimolar concentrations, and no cross-hybridization between any of the fragments was seen. We conclude that the MCMV DNA molecule consists of a long unique sequence without large terminal or internal repeat regions. Thus, the structural organization of the MCMV genome is fundamentally different from that of the human cytomegalovirus or herpes simplex virus genome.

Animals↗

PubMed1983

Virus-specific transcription in a Herpesvirus saimiri-transformed lymphoid tumor cell line.

Herpesvirus saimiri-transformed lymphoid tumor cell lines contain nonintegrated covalently closed circular viral DNA molecules in high multiplicity. One of those cell lines, 1670, carries large viral DNA circles (202 kilobase pairs) with two stretches of repetitive DNA (70.8% G + C) that are interspersed between two segments of unique DNA (36% G + C). Since it was not known if there is any viral gene expression in H. saimiri-transformed cells, we initiated a study of transcription in cell line 1670. cDNA was generated by reverse transcription of cellular RNA and hybridized with cloned virion DNA fragments. The experiments indicated that appreciable transcription is restricted to a single segment of unique DNA. This sequence is present once only in the circular viral DNA and corresponds to unique DNA between map units 0.89 and 0.93 of virion DNA. By Northern blot hybridizations with labeled cloned probes of virion unique DNA, one predominant virus-specific polyadenylated transcript of, at most, 2.7 kilo-bases could be detected in tumor cell line 1670. The direction of transcription was determined by hybridization with randomly primed cDNA and, in parallel, with oligodeoxythymidylate-primed cDNA probes. Apparently, the patterns of virus-specific RNA synthesis in the H. saimiri-transformed cells are clearly distinct from the transcription program in other herpesvirus transformation systems analyzed before.

Animals↗

PubMed1981

Analysis of nuclear proteins in primary spermatocytes of Drosophila hydei: The correlation of nuclear proteins with the function of the Y chromosomal loops.

The protein content of spermatocyte nuclei from X/Y males and mutants of D. hydei which lack different Y chromosomal loop forming sites, was compared with that of X/0 males in 14C/3H double labelling experiments. Proteins of 45,000, 52,000, 54,000, 66,000, 80,000, 84,000 and 170,000 Dalton are found to be enriched in nuclei containing two or more active Y chromosomal loop forming sites. These proteins are also present in the nuclei of X0 males. In the complete absence of the Y-chromosomal loops proteins of 35,000, 46,000, 58,000 and 110,000 Dalton become enriched in the spermatocyte nuclei. - Analysis of the nuclear RNP of spermatocytes led to the isolation of an hnRNP-containing fraction with an S-value of greater than 900S (RNP-PP), - In the RNP-PP of XY males labelled protein material associated with hnRNA is enriched by a factor of approximately 3 in respect to the X0 genotype. The nuclear RNP has a heterogenous buoyant density in CsCl of rho = 1.33 to 1.43 g/cm3. RNase T1 treatment of the crude nuclear RNP from XY males prior to sucrose gradient analysis shows that the 66,000 Dalton protein which is also strongly enriched in the nuclei in the presence of active Y chromosomal loop forming sites, is the main protein associated with protected RNA-sequences of 80-120- 300 nucleotides in length. Competitive nitrocellulose filter binding assays reveal that the 66,000 Dalton protein predominantly forms in 2 M NaCl stable RNA/protein complexes with the poly A+hnRNA of the RNP-PP. Those RNP complexes have a buoyant density of rho = 1.43 g/cm3 in CsCl. The results are discussed in relation to the nuclear structure and the function of the Y chromosomal loops during spermatogenesis in Drosophila hydei.

Animals↗

PubMedDate not supplied

Control of spindle orientation in Drosophila by the Par-3-related PDZ-domain protein Bazooka.

BACKGROUND: The orientation of the mitotic spindle influences the asymmetric distribution of cytoplasmic determinants and the positioning of the sibling cell, and therefore has important influences on cell-fate determination and patterning of the embryo. Both the establishment of an axis of polarity and the adjustment of this axis with respect to the coordinates of the embryo have to be controlled. None of the genes identified so far that are involved in these processes seems to have been conserved between flies and nematodes. RESULTS: Here, we show that the bazooka gene encodes a protein with three putative protein-interaction motifs known as PDZ domains and is the first Drosophila representative of the par gene family of Caenorhabditis elegans, members of which are required for establishment of anterior-posterior polarity of the nematode embryo. The bazooka RNA and protein were found to be restricted to the apical cortical cytoplasm of epithelial cells and neuroblasts. Embryos that were mutant for bazooka frequently failed to coordinate the axis of cell polarity with that of the embryo. This was manifested as defective spindle orientation and mispositioning of the daughter cell after division. CONCLUSIONS: The Drosophila gene bazooka is likely to be part of a regulatory mechanism required to coordinate the axis of polarity of a cell with that of the embryo. The PDZ domains of Bazooka provide several protein-protein interfaces, which possibly participate in the assembly of a multiprotein complex at the apical pole.

Amino Acid Sequence↗