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G M Rubin

Publications and source records attributed to G M Rubin.

At least 163 records · Page 9Linked to original sources
PubMed1989

cis-acting DNA sequence requirements for P-element transposition.

The P transposable element of Drosophila melanogaster has a complex array of cis-acting DNA sequences necessary for efficient transposition. At the 3' end these sequences extend over more than 150 bp and include 11- and 31-bp sequences found repeated in inverted orientation at the 5' end. The P element's 5' end, however, cannot function as its 3' end. When two 3' P-element ends are present, the more proximal end is used preferentially. We found also that the duplication of the target site does not appear to play a role in forward transposition.

Animals↗
PubMed1988

rough, a Drosophila homeobox gene required in photoreceptors R2 and R5 for inductive interactions in the developing eye.

The rough mutation in Drosophila disrupts an early stage of ommatidial assembly in the developing eye imaginal disc. Analysis of somatic mosaics suggests that rough gene function is required only in photoreceptors R2 and R5. However, these cells apparently differentiate normally in mutant eye discs and it is the cells that are subsequently added to the ommatidium that behave aberrantly. The rough gene was isolated by P-element transposon tagging, and the mutant can be rescued by transformation with an 8.6 kb genomic fragment. The rough transcription unit is 4.3 kb and consists of three exons that are joined in a 1.3 kb mRNA. The predicted rough protein is 350 amino acids long and contains a homeobox. Taken together, our results suggest that the role of the rough gene product may be to regulate the sending of signals by photoreceptors R2 and R5 to their neighbors in the developing ommatidia.

Amino Acid Sequence↗
PubMed1988

Drosophila melanogaster as an experimental organism.

The fruit fly Drosophila melanogaster has been used as an experimental organism in studies of genetics since the early 1900s. It is now widely used not only in classical and molecular genetics but also, with many new biochemical, cell biological, and physiological techniques, to research problems requiring a multidisciplinary approach, such as those of developmental biology and neurobiology.

Animals↗
PubMed1988

Ectopic expression of a minor Drosophila opsin in the major photoreceptor cell class: distinguishing the role of primary receptor and cellular context.

We have used P-element-mediated transformation to introduce the cloned Rh1 rhodopsin gene into the germ line of Drosophila and fully rescue the visual phenotype of mutant ninaE flies. A transcriptional fusion between the ninaE promoter and the structural gene for a minor opsin (Rh2) that is not normally expressed in the R1-R6 photoreceptor cells was used to demonstrate that Rh2 rhodopsin can photoactivate the R1-R6 transduction cascade, but with different spectral sensitivity. In addition, we show that two mutants that specifically affect the R1-R6 cells, ninaA and rdgB, do not directly affect expression of the ninaE gene.

Animals↗
PubMed1988

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.

Animals↗
PubMed1988

The Drosophila ninaC locus encodes two photoreceptor cell specific proteins with domains homologous to protein kinases and the myosin heavy chain head.

The fruit fly Drosophila melanogaster has been extensively used to identify genes required for photoreceptor cell function. We show that the ninaC gene, originally isolated as a Drosophila visual mutation with an electrophysiological phenotype, encodes two novel cytoskeletal proteins. We identified the DNA sequences encoding the ninaC gene by rescuing the electrophysiological phenotype using P-element-mediated germ line transformation. The ninaC locus is expressed as two extensively overlapping mRNAs encoding proteins of 1135 and 1501 amino acids. Both proteins contain a putative protein kinase domain joined to a domain homologous to the head region of the myosin heavy chain and are spatially restricted to photoreceptor cells.

Amino Acid Sequence↗
PubMed1988

Identification and purification of a Drosophila protein that binds to the terminal 31-base-pair inverted repeats of the P transposable element.

We have used DNase I footprinting and partially fractionated nuclear extracts from Drosophila Kc tissue culture cells to identify DNA-binding proteins that interact with the terminal repeats of P transposable elements. We have identified a binding activity that interacts specifically with a region of the 31-base-pair terminal inverted repeats that is directly adjacent to the duplication of target site DNA. Binding occurs to both the 5' and 3' inverted terminal repeats irrespective of the sequence of the duplicated target DNA. UV photochemical crosslinking studies suggest that the binding activity resides in a polypeptide of 65-70 kDa. Biochemical fractionation and oligonucleotide affinity chromatography have been used to purify the binding activity to near homogeneity and identify a polypeptide of 66 kDa in the highly purified preparations. The site to which binding occurs is included in a region absolutely required for P element transposition, suggesting that this binding protein may be a cellular factor involved in P element transposition.

Animals↗
PubMed1988

Partial revertants of the transposable element-associated suppressible allele white-apricot in Drosophila melanogaster: structures and responsiveness to genetic modifiers.

The eye color phenotype of white-apricot (wa), a mutant allele of the white locus caused by the insertion of the transposable element copia into a small intron, is suppressed by the extragenic suppressor suppressor-of-white-apricot (su(wa] and enhanced by the extragenic enhancers suppressor-of-forked su(f] and Enhancer-of-white-apricot (E(wa]. Derivatives of wa have been analyzed molecularly and genetically in order to correlate the structure of these derivatives with their response to modifiers. Derivatives in which the copia element is replaced precisely by a solo long terminal repeat (sLTR) were generated in vitro and returned to the germline by P-element mediated transformation; flies carrying this allele within a P transposon show a nearly wild-type phenotype and no response to either su(f) or su(wa). In addition, eleven partial phenotypic revertants of wa were analyzed. Of these, one appears to be a duplication of a large region which includes wa, three are new alleles of su(wa), two are sLTR derivatives whose properties confirm results obtained using transformation, and five are secondary insertions into the copia element within wa. One of these, waR84h, differs from wa by the insertion of the most 3' 83 nucleotides of the I factor. The five insertion derivatives show a variety of phenotypes and modes of interaction with su[f) and su(wa). The eye pigmentation of waR84h is affected by su(f) and E(wa), but not su(wa). These results demonstrate that copia (as opposed to the interruption of white sequences) is essential for the wa phenotype and its response to genetic modifiers, and that there are multiple mechanisms for the alteration of the wa phenotype by modifiers.

Alleles↗
PubMed1988

P element insertions and rearrangements at the singed locus of Drosophila melanogaster.

DNA from the singed gene of Drosophila melanogaster was isolated using an inversion between a previously cloned P element at cytological location 17C and the hypermutable allele singed-weak. Five out of nine singed mutants examined have alterations in their DNA maps in this region. The singed locus is a hotspot for mutation during P-M hybrid dysgenesis, and we have analyzed 22 mutations induced by P-M hybrid dysgenesis. All 22 have a P element inserted within a 700-bp region. The precise positions of 10 P element insertions were determined and they define 4 sites within a 100-bp interval. During P-M hybrid dysgenesis, the singed-weak allele is destabilized, producing two classes of phenotypically altered derivatives at high frequency. In singed-weak, two defective P elements are present in a "head-to-head" or inverse tandem arrangement. Excision of one element results in a more extreme singed bristle phenotype while excision of the other leads to a wild-type bristle phenotype.

Alleles↗
PubMed1988

Analysis of the promoter of the Rh2 opsin gene in Drosophila melanogaster.

We have analyzed the cis-acting regulatory sequences of the Drosophila melanogaster Rh2 gene that encodes the protein component of a rhodopsin which is expressed in ocellar photoreceptor cells. DNA fragments containing the start point of transcription of the Rh2 gene were fused to either the Escherichia coli chloramphenicol acetyltransferase (CAT) or lacZ (beta-galactosidase) genes and introduced into the Drosophila germline by P-element-mediated transformation. Expression of the E. coli genes was then used to assay the ability of various sequences from the Rh2 gene to confer upon the indicator genes the Rh2 pattern of expression. Fragments containing between 4.3 kb and 183 bp upstream of the start of transcription plus the first 32 bp of the 5'-untranslated leader were found to result in nearly identical levels of head-specific CAT expression. Deletion of Rh2 sequences distal to position -112 bp resulted in loss of detectable CAT expression from these Rh2/CAT fusion constructs. We have, therefore, defined a region essential for head-specific expression of the Rh2 gene to a region extending from -183 to -112. We have determined the DNA sequence of the Rh2 promoter from -448 to +32 and have found an 11-bp sequence which is also present in the upstream flanking sequences of two other photoreceptor-specific genes (ninaE and ninaC). By histochemical staining of beta-galactosidase expressed under the control of the Rh2 promoter and by analyzing the effect of the ocelliless mutation on the expression of an Rh2/CAT fusion gene, we have been able to demonstrate that this promoter is active in ocelli.

Animals↗
PubMed1988

Isolation and characterization of Drosophila cAMP-dependent protein kinase genes.

We have used mammalian probes to clone genes encoding the catalytic (C) and type I regulatory (RI) components of the cAMP-dependent protein kinase in Drosophila. Both Drosophila gene products are very similar in amino acid sequence (RI, 71%; C, 82%) to their respective mammalian counterparts, implying homologous activity. A single Drosophila type I regulatory subunit gene is the source of at least three distinct transcripts originating from different promoters and spliced to a common body that would encode a full-length analog and two amino-terminally truncated variants of the mammalian RI protein. The RI locus also includes two intronic genes of unknown function. A single highly conserved catalytic subunit gene (DC0) was found that codes for a single polypeptide. It was used to isolate 11 further more distantly related apparent protein kinase genes. Two of these genes (DC1 and DC2) are sufficiently similar to DC0 in sequence (45% and 49% amino acid identity, respectively) that they could conceivably encode products of overlapping function. Two further genes are very similar in sequence to bovine cGMP-dependent protein kinase. The remaining putative gene products include amino acid sequence motifs characteristic of serine-threonine protein kinases but cannot, from the available data, be defined as homologous to specific protein kinases of other organisms.

Amino Acid Sequence↗
PubMed1988

Nucleotide sequence and structure of the sevenless gene of Drosophila melanogaster.

In the Drosophila melanogaster mutant sevenless, the R7 photoreceptor in each of the repeating units, or ommatidia, of the compound eye fails to form. We have determined the nucleotide sequence and structure of the sevenless transcription unit. Overlapping cDNA clones from an eye imaginal disc library were isolated, and these together with corresponding genomic regions were sequenced. The positions of the major and two minor transcription start sites were mapped. The gene encodes a putative cell-surface receptor, the unmodified form of which is predicted as 288 kD, bearing a cytoplasmic tyrosine kinase domain. Several structural features distinguish sevenless from other tyrosine kinase receptors, most notably the large size of its extracellular domain. Moreover, unlike the insulin or epidermal growth factor (EGF) receptors, a putative amino-terminal signal sequence does not immediately follow the initiating methionine codon. Rather, a 21-amino-acid hydrophobic sequence is found 56 amino acids after the likely initiating methionine codon, suggesting that this sequence may function as an amino-terminal anchor. If so, the sevenless protein would be expected to have its 2001-amino-acid extracellular domain anchored as a loop at either end in the membrane. Finally, we have generated a number of transformant lines that carry either a 16.3-kb genomic fragment that extends approximately 950 bp upstream of the transcription start sites or constructs in which a subset of the introns present in the genomic sevenless DNA have been removed. The degree of rescue of the sevenless mutant phenotype has been assayed in these lines using morphological and behavioral assays.

Amino Acid Sequence↗
PubMed1987

Localization of the sevenless protein, a putative receptor for positional information, in the eye imaginal disc of Drosophila.

The Drosophila gene sevenless encodes a putative trans-membrane receptor required for the formation of one particular cell, the R7 photoreceptor, in each ommatidium of the compound eye. Mutations in this gene result in the cell normally destined to form the R7 cell forming a non-neuronal cell type instead. These observations have led to the proposal that the sevenless protein receives at least part of the positional information required for the R7 developmental pathway. We have generated antibodies specific for sevenless and have examined expression of the protein by light and electron microscopy. sevenless protein is present transiently at high levels in at least 9 cells in each developing ommatidium and is detectable several hours before any overt differentiation of R7. The protein is mostly localized at the apices of the cells, in microvilli, but is also found deeper in the tissue where certain cells contact the R8 cell. This finding suggests that R8 expresses a ligand for the sevenless protein.

Animals↗
PubMed1987

Disconnected: a locus required for neuronal pathway formation in the visual system of Drosophila.

Mutations at the X-linked disconnected locus of D. melanogaster lead to the failure of adult photoreceptor axons to innervate their target cells in the developing optic lobes of the third instar larva, resulting in flies that have rudimentary optic ganglia. The cascade of epigenetic events leading to the adult disconnected phenotype is caused by the misrouting of a larval pioneer nerve, Bolwig's nerve, during embryonic development. In the disconnected mutant this nerve fails to recognize and establish stable connections with its correct synaptic partners. In addition, disconnected affects both the proper aggregation and the movement of the Bolwig neurons to their final location in the embryo. Finally, similar but more subtle defects can be found in a subset of other peripheral neurons in the thoracic and abdominal segments. The different aspects of the phenotype suggest that the disconnected gene plays a role in neuronal cell recognition.

Animals↗
PubMed1987

Sevenless, a cell-specific homeotic gene of Drosophila, encodes a putative transmembrane receptor with a tyrosine kinase domain.

The determination of cell fates during the assembly of the ommatidia in the compound eye of Drosophila appears to be controlled by cell-cell interactions. In this process, the sevenless gene is essential for the development of a single type of photoreceptor cell. In the absence of proper sevenless function the cells that would normally become the R7 photoreceptors instead become nonneuronal cells. Previous morphological and genetic analysis has indicated that the product of the sevenless gene is involved in reading or interpreting the positional information that specifies this particular developmental pathway. The sevenless gene has now been isolated and characterized. The data indicate that sevenless encodes a transmembrane protein with a tyrosine kinase domain. This structural similarity between sevenless and certain hormone receptors suggests that similar mechanisms are involved in developmental decisions based on cell-cell interaction and physiological or developmental changes induced by diffusible factors.

Amino Acid Sequence↗
PubMed1987

Concurrent intravenous administration of a labeled tracer to determine the oral bioavailability of a drug exhibiting Michaelis-Menten metabolism.

The theoretical accuracy of concurrent administration of labeled intravenous tracer and oral doses to estimate the bioavailability of drugs exhibiting Michaelis-Menten kinetics was determined by computer simulation. The simulation model consisted of sampling and hepatic compartments with elimination occurring by hepatic metabolism according to the venous equilibration model. The relationships between error in bioavailability estimation and dose, metabolic activity (Vmax), first-order absorption rate constant (ka), and volume of distribution (V) and the fraction of the dose absorbed were examined. Error was hypothesized to be relatively low when conditions result in a relatively constant value of clearance after oral dosing or when the concentration-time curves after intravenous and oral dosing are similar. The results were consistent with these hypotheses and, under most conditions, error was less than 15%. The effects, on error, of altering the intravenous tracer dose input and having a lag time in absorption of drug from the oral dose were also determined. In general, accuracy was improved by delaying administration of the iv tracer for a time equal to 50% of the oral dose peak time or by administering the tracer dose by constant-rate infusion from the time of oral dosing to the peak time. Lag time in absorption of the oral dose was shown to often result in overestimates in bioavailability of greater than 50%.

Administration, Oral↗
PubMed1987

Analysis of the promoter of the ninaE opsin gene in Drosophila melanogaster.

We have analyzed the cis-acting regulatory sequences of the ninaE gene. This gene encodes the major Drosophila melanogaster opsin, the protein component of the primary chromophore of photo-receptor cells R1-R6 of the adult eye. DNA fragments containing the start point of transcription of the ninaE gene were fused to either the Escherichia coli chloramphenicol acetyltransferase or lacZ (beta-galactosidase) gene and introduced into the Drosophila germline by P-element-mediated transformation. Expression of the E. coli genes was then used to assay the ability of various sequences from the ninaE gene to confer the ninaE pattern of expression. Fragments containing between 2.8 kb and 215 bp of the sequences upstream of the start of transcription plus the first 67 bp of the untranslated leader were able to direct nearly wild-type expression. We have identified three separable control regions in the ninaE promoter. The first, which has the properties of an enhancer element, is located between nucleotides -501 and -219. The removal of this sequence had little effect on promoter function; this sequence appears to be redundant. However, it appears to be able to substitute for the second control region which is located between nucleotides -215 and -162, and which also affects the level of output from this promoter. Removal of these two control regions resulted in a 30-fold decrease in expression; however tissue specificity was not affected. The third control region, located downstream from nucleotide -120, appears to be absolutely necessary for promoter function in the absence of the first two regulatory sequences. Examination of larvae containing fusion genes expressing beta-galactosidase suggests that the ninaE gene is also expressed in a subset of cells in the larval photoreceptor organ.

Acetyltransferases↗