scholarly journals Identification of Trans-dominant Modifiers of Prat Expression in Drosophila melanogaster

Genetics ◽  
2003 ◽  
Vol 164 (4) ◽  
pp. 1419-1433
Author(s):  
Nicolas Malmanche ◽  
Denise V Clark
Keyword(s):  
Drosophila Melanogaster ◽  
De Novo ◽  
Essential Gene ◽  
Single Copy ◽  
Induced Mutations ◽  
Coding Region ◽  
Rt Pcr ◽  
Transcription Start ◽  
Eye Color ◽  
Transcription Start Sites

Abstract The first committed step in the purine de novo synthesis pathway is performed by amidophosphoribosyl-transferase (EC 2.4.2.14) or Prat. Drosophila melanogaster Prat is an essential gene with a promoter that lacks a TATA-box and initiator element and has multiple transcription start sites with a predominant start site. To study the regulation of Prat expression in the adult eye, we used the Prat:bw reporter gene, in which the Prat coding region was replaced with the brown (bw) coding region. The pale-orange eye color of a single copy of Prat:bw prompted us to use a multicopy array of Prat:bw that was derived using P transposase mutagenesis and produces a darker-orange eye color in a bwD; st genetic background. We used a 13-copy array of Prat:bw as a tool to recover dominant EMS-induced mutations that affect the expression of the transgene. After screening 21,000 F1s for deviation from the orange eye color, we isolated 23 dominant modifiers: 21 suppressors (1 Y-linked, 5 X-linked, 4 2-linked, and 11 3-linked) and 2 enhancers (1 2-linked and 1 3-linked). Quantification of their effect on endogenous Prat gene expression, using RT-PCR in young adult fly heads, identifies a subset of modifiers that are candidates for genes involved in regulating Prat expression.

Whole-Genome Effects of Ethyl Methanesulfonate-Induced Mutation on Nine Quantitative Traits in Outbred Drosophila melanogaster

Genetics ◽  
2001 ◽  
Vol 157 (3) ◽  
pp. 1257-1265 ◽  
Author(s):  
Hsiao-Pei Yang ◽  
Ana Y Tanikawa ◽  
Wayne A Van Voorhies ◽  
Joana C Silva ◽  
Alexey S Kondrashov
Keyword(s):  
Drosophila Melanogaster ◽  
Quantitative Traits ◽  
Spontaneous Mutation ◽  
Developmental Time ◽  
Ethyl Methanesulfonate ◽  
Induced Mutations ◽  
Mean Values ◽  
Eye Color ◽  
Recessive Mutations ◽  
The Mean

Abstract We induced mutations in Drosophila melanogaster males by treating them with 21.2 mm ethyl methanesulfonate (EMS). Nine quantitative traits (developmental time, viability, fecundity, longevity, metabolic rate, motility, body weight, and abdominal and sternopleural bristle numbers) were measured in outbred heterozygous F3 (viability) or F2 (all other traits) offspring from the treated males. The mean values of the first four traits, which are all directly related to the life history, were substantially affected by EMS mutagenesis: the developmental time increased while viability, fecundity, and longevity declined. In contrast, the mean values of the other five traits were not significantly affected. Rates of recessive X-linked lethals and of recessive mutations at several loci affecting eye color imply that our EMS treatment was equivalent to ∼100 generations of spontaneous mutation. If so, our data imply that one generation of spontaneous mutation increases the developmental time by 0.09% at 20° and by 0.04% at 25°, and reduces viability under harsh conditions, fecundity, and longevity by 1.35, 0.21, and 0.08%, respectively. Comparison of flies with none, one, and two grandfathers (or greatgrandfathers, in the case of viability) treated with EMS did not reveal any significant epistasis among the induced mutations.

scholarly journals Characterization of RPR1, an essential gene encoding the RNA component of Saccharomyces cerevisiae nuclear RNase P.

1991 ◽  
Vol 11 (2) ◽  
pp. 721-730 ◽  
Author(s):  
J Y Lee ◽  
C E Rohlman ◽  
L A Molony ◽  
D R Engelke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Essential Gene ◽  
Rnase P ◽  
Single Copy ◽  
Temperature Sensitive ◽  
Coding Region ◽  
Gene Encoding ◽  
Processing Steps ◽  
Potential Precursor

RNA components have been identified in preparations of RNase P from a number of eucaryotic sources, but final proof that these RNAs are true RNase P subunits has been elusive because the eucaryotic RNAs, unlike the procaryotic RNase P ribozymes, have not been shown to have catalytic activity in the absence of protein. We previously identified such an RNA component in Saccharomyces cerevisiae nuclear RNase P preparations and have now characterized the corresponding, chromosomal gene, called RPR1 (RNase P ribonucleoprotein 1). Gene disruption experiments showed RPR1 to be single copy and essential. Characterization of the gene region located RPR1 600 bp downstream of the URA3 coding region on chromosome V. We have sequenced 400 bp upstream and 550 bp downstream of the region encoding the major 369-nucleotide RPR1 RNA. The presence of less abundant, potential precursor RNAs with an extra 84 nucleotides of 5' leader and up to 30 nucleotides of 3' trailing sequences suggests that the primary RPR1 transcript is subjected to multiple processing steps to obtain the 369-nucleotide form. Complementation of RPR1-disrupted haploids with one variant of RPR1 gave a slow-growth and temperature-sensitive phenotype. This strain accumulates tRNA precursors that lack the 5' end maturation performed by RNase P, providing direct evidence that RPR1 RNA is an essential component of this enzyme.

Generation, characterization, and molecular cloning of the Noerg-1 mutation of rhodopsin in the mouse

2005 ◽  
Vol 22 (5) ◽  
pp. 619-629 ◽  
Author(s):  
LAWRENCE H. PINTO ◽  
MARTHA H. VITATERNA ◽  
KAZUHIRO SHIMOMURA ◽  
SANDRA M. SIEPKA ◽  
ERIN L. MCDEARMON ◽  
...  
Keyword(s):  
Autosomal Dominant ◽  
De Novo ◽  
Genomic Sequence ◽  
Outer Nuclear Layer ◽  
Dna Polymorphisms ◽  
Fundus Photography ◽  
Induced Mutations ◽  
Coding Region ◽  
Optomotor Response

We performed genome-wide mutagenesis of C57BL/6J mice using the mutagen N-ethyl-N-nitrosourea (ENU) and screened the third generation (G3) offspring for visual system alterations using electroretinography and fundus photography. Several mice in one pedigree showed characteristics of retinal degeneration when tested at 12–14 weeks of age: no recordable electroretinogram (ERG), attenuation of retinal vessels, and speckled pigmentation of the fundus. Histological studies showed that the retinas undergo a photoreceptor degeneration with apoptotic loss of outer nuclear layer nuclei but visual acuity measured using the optomotor response under photopic conditions persists in spite of considerable photoreceptor loss. The Noerg-1 mutation showed an autosomal dominant pattern of inheritance in progeny. Studies in early postnatal mice showed degeneration to occur after formation of partially functional rods. The Noerg-1 mutation was mapped genetically to chromosome 6 by crossing C57BL/6J mutants with DBA/2J or BALB/cJ mice to produce an N2 generation and then determining the ERG phenotypes and the genotypes of the N2 offspring at multiple loci using SSLP and SNP markers. Fine mapping was accomplished with a set of closely spaced markers. A nonrecombinant region from 112.8 Mb to 115.1 Mb was identified, encompassing the rhodopsin (Rho) coding region. A single nucleotide transition from G to A was found in the Rho gene that is predicted to result in a substitution of Tyr for Cys at position 110, in an intradiscal loop. This mutation has been found in patients with autosomal dominant retinitis pigmentosa (RP) and results in misfolding of rhodopsin expressed in vitro. Thus, ENU mutagenesis is capable of replicating mutations that occur in human patients and is useful for generating de novo models of human inherited eye disease. Furthermore, the availability of the mouse genomic sequence and extensive DNA polymorphisms made the rapid identification of this gene possible, demonstrating that the use of ENU-induced mutations for functional gene identification is now practical for individual laboratories.

scholarly journals The organization and expression of the light gene, a heterochromatic gene of Drosophila melanogaster.

Genetics ◽  
1990 ◽  
Vol 125 (1) ◽  
pp. 129-140 ◽  
Author(s):  
R H Devlin ◽  
B Bingham ◽  
B T Wakimoto
Keyword(s):  
Drosophila Melanogaster ◽  
Dna Sequences ◽  
Molecular Mapping ◽  
Single Copy ◽  
Transcription Unit ◽  
Malpighian Tubules ◽  
Centromeric Heterochromatin ◽  
Chromosome 2 ◽  
Induced Mutations ◽  
Flanking Regions

Abstract The light (lt) gene is located in the centromeric heterochromatin of chromosome 2 of Drosophila melanogaster. This gene is necessary for normal levels of pigmentation in a number of adult and larval tissues and is required for viability. Hybrid dysgenic and X-ray induced mutations have been used to identify the gene and compare its organization to that of euchromatic genes. Molecular mapping of lt mutations and its major transcripts has shown that the lt gene is at least 17 kb. By injecting cosmid clones that include this region into lt mutant embryos, we have defined a 30-kb region that can transiently rescue the pigmentation defect in the Malpighian tubules. The major transcription unit of this gene is comprised of exons that are single copy. It is unusual in its organization in having a heterogeneous array of middle repetitive DNA sequences within its intronic and flanking regions.

Paraquat selection identifies X-linked oxygen defense genes in Drosophila melanogaster

Genome ◽  
1993 ◽  
Vol 36 (1) ◽  
pp. 162-165 ◽  
Author(s):  
James M. Humphreys ◽  
Arthur J. Hilliker ◽  
John P. Phillips
Keyword(s):  
Drosophila Melanogaster ◽  
Xanthine Dehydrogenase ◽  
Oxygen Radical ◽  
Drosophila Genome ◽  
Induced Mutations ◽  
Defense Genes ◽  
Proximate Cause ◽  
Eye Color ◽  
Cuzn Superoxide Dismutase ◽  
New Alleles

We have previously shown that homozygous mutants of Drosophila melanogaster deficient in the oxygen radical scavengers, CuZn superoxide dismutase or urate, are adult viable and yet hypersensitive to the oxygen radical-generating agent, paraquat. Thus, paraquat could be used as a selective agent to identify adult-viable mutants potentially defective in other, perhaps unknown, oxygen defense functions. Here we report the successful use of paraquat hypersensitivity in the isolation of X-linked, ethylmethanesulfonate-induced mutations affecting oxygen defense in Drosophila melanogaster. Two paraquat hypersensitive mutants were identified that, by complementation analysis, were shown to be new alleles of the maroon-like gene. In addition to paraquat hypersensitivity, both alleles confer a maternally affected dark brown eye color and a complete lack of enzymatically active xanthine dehydrogenase, both of which are characteristic phenotypes of known maroon-like alleles. We conclude that the lack of xanthine dehydrogenase in these mutants leads to the absence of urate, which is the proximate cause of paraquat sensitivity. Because our search for such mutants on the X chromosome revealed two alleles of only a single selectable gene, we anticipate that the total number of major oxygen defense genes in the complete Drosophila genome may not be large.Key words: paraquat, maroon-like, xanthine dehydrogenase, oxygen defense.

scholarly journals Optimal and regulated transcription facilitates formation of damage-induced cohesion

2020 ◽  
Author(s):  
Pei-Shang Wu ◽  
Donald P. Cameron ◽  
Jan Grosser ◽  
Laura Baranello ◽  
Lena Ström
Keyword(s):  
De Novo ◽  
Transcriptional Response ◽  
Chromatin Accessibility ◽  
Regulation Of Transcription ◽  
Transcription Start ◽  
Transcription Start Sites ◽  
Expression Of Genes ◽  
Chromatid Cohesion ◽  
Active Transcription ◽  
Histone Exchange

AbstractThe SMC complex cohesin mediates sister chromatid cohesion established during replication, and damage-induced cohesion formed in response to DSBs post replication. The translesion synthesis polymerase Polη is required for damage-induced cohesion through a hitherto unknown mechanism. Since Polη is functionally associated with transcription, and transcription triggers de novo cohesion in S. pombe, we hypothesized that active transcription facilitates damage-induced cohesion in S. cerevisiae. Here, we found that expression of genes involved in chromatin assembly and positive transcription regulation were relatively enriched in WT compared to Polη-deficient cells (rad30Δ). The rad30Δ mutant showed a dysregulated transcriptional response and increased cohesin binding around transcription start sites. Perturbing histone exchange at promoters adversely affected damage-induced cohesion, similarly to deletion of RAD30. Conversely, altering chromatin accessibility or regulation of transcription elongation, suppressed the lack of damage-induced cohesion in rad30Δ cells. These results indicate that Polη promotes damage-induced cohesion through its role in transcription, and support the model that regulated transcription facilitates formation of damage-induced cohesion.

scholarly journals Epigenetic Drug Treatment Globally Induces Cryptic Transcription Start Sites Encoded in Long Terminal Repeats

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3931-3931
Author(s):  
Michael Daskalakis ◽  
David Brocks ◽  
Christopher Schmidt ◽  
Daofeng Li ◽  
Jing Li ◽  
...  
Keyword(s):  
Drug Treatment ◽  
De Novo ◽  
Protein Isoforms ◽  
Epigenetic Therapy ◽  
Transcription Start ◽  
Altered Expression ◽  
Transcription Start Sites ◽  
Fusion Transcripts ◽  
Dnmt Inhibitors ◽  
Epigenetic Drug

Abstract Epigenetic drugs are currently used for the treatment of several hematologic malignancies, but their mechanism of action remains poorly understood. By using a previously described reporter cell line for epigenetic reactivation of the DAPK1 locus, we have shown that epigenetic treatment causes transcription from uncharacterized intronic transcription start sites (TSSs), thereby generating DAPK1 mRNA with novel first exons. Based on these findings, we analyzed whether inhibition of DNA-Methyltransferases (DNMTs), Histone deacetylases (HDACs), or both resulted in the genome-wide induction of non-canonical TSSs. While epigenetic treatment altered expression of known promoter sites, we observed that both HDAC- and DNMT-inhibitors predominantly induced de novo transcription from cryptic promoters encoded in long-terminal repeat (LTR) retrotransposons. These LTR-associated 'treatment induced, not-annotated TSS' (TINATs) are currently not annotated and normally silenced in almost all cell types with the exception of testicular und thymic tissue. In the majority of cases, these TINATs arose most commonly from LTR12 elements, particularly LTR12C (which apparently provides 50% of all TINATs). TINAT activation after DNMT-inhibitors (DNMTi) coincided with DNA hypomethylation and gain in H3K4me3, H3K9ac, and H3K27ac histone marks. In contrast, HDAC-inhibitors (HDACi) induced only canonical TSSs in association with histone acetylation, but TINATs via a yet unknown mechanism. Nevertheless, both inhibitors convergently induced unidirectional transcription from identical TINAT sites. Moreover, we found a consensus GATA2 binding motif which strongly distinguished LTR12Cs with TINATs from LTR12Cs without TINATs, supporting that GATA2 is likely the upstream transcription factor responsible for TINAT activation. TINATs originating from non-canonical TSSs located within introns of protein-coding genes frequently spliced into downstream exons thereby creating LTR/non-LTR fusion transcripts that harbor novel in place of canonical exon sequence at their 5' end. The resulting transcripts encode truncated or chimeric open reading frames which translated into currently uncharacterized protein isoforms with predicted abnormal functions or immunogenic potential, the last one based on their foreign sequence and capability of being presented on MHC-class I molecules. In summary, we could show that DNMTi and/or HDACi do not predominantly alter the expression of canonical genes, but induce de novo transcription of LTRs especially of the LTR12 family, resulting in numerous fusion transcripts that encode novel protein isoforms which might have the potential to influence cell proliferation or might be an elegant explanation for the priming effect of epigenetic therapy. Ongoing experiments are investigating the functional mechanisms of TINAT reactivation upon epigenetic drug treatment and future proteomic approaches combined with T-cell cytotoxicity assays will further shed light on the interaction between epigenetic and immune therapy and the role of ERV-derived antigen presentation. Disclosures Lübbert: Janssen-Cilag: Other: Travel Funding, Research Funding; Ratiopharm: Other: Study drug valproic acid; Celgene: Other: Travel Funding.

Cloning and characterization of ERG8, an essential gene of Saccharomyces cerevisiae that encodes phosphomevalonate kinase

1991 ◽  
Vol 11 (2) ◽  
pp. 620-631
Author(s):  
Y H Tsay ◽  
G W Robinson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genomic Library ◽  
Essential Gene ◽  
Single Copy ◽  
Yeast Cells ◽  
Upstream Region ◽  
Ergosterol Biosynthesis ◽  
Temperature Sensitive ◽  
Coding Region ◽  
Phosphomevalonate Kinase

Saccharomyces cerevisiae strains that contain the ery8-1 mutation are temperature sensitive for growth due to a defect in phosphomevalonate kinase, an enzyme of isoprene and ergosterol biosynthesis. A plasmid bearing the yeast ERG8 gene was isolated from a YCp50 genomic library by functional complementation of the erg8-1 mutant strain. Genetic analysis demonstrated that integrated copies of an ERG8 plasmid mapped to the erg8 locus, confirming the identity of this clone. Southern analysis showed that ERG8 was a single-copy gene. Subcloning and DNA sequencing defined the functional ERG8 regulon as an 850-bp upstream region and an adjacent 1,272-bp open reading frame. The deduced 424-amino-acid ERG8 protein showed no homology to known proteins except within a putative ATP-binding domain present in many kinases. Disruption of the chromosomal ERG8 coding region by integration of URA3 or HIS3 marker fragments was lethal in haploid cells, indicating that this gene is essential. Expression of the ERG8 gene in S. cerevisiae from the galactose-inducible galactokinase (GAL1) promoter resulted in 1,000-fold-elevated levels of phosphomevalonate kinase enzyme activity. Overproduction of a soluble protein with the predicted 48-kDa size for phosphomevalonate kinase was also observed in the yeast cells.

Characterization of RPR1, an essential gene encoding the RNA component of Saccharomyces cerevisiae nuclear RNase P

1991 ◽  
Vol 11 (2) ◽  
pp. 721-730 ◽  
Author(s):  
J Y Lee ◽  
C E Rohlman ◽  
L A Molony ◽  
D R Engelke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Essential Gene ◽  
Rnase P ◽  
Single Copy ◽  
Temperature Sensitive ◽  
Coding Region ◽  
Gene Encoding ◽  
Processing Steps ◽  
Potential Precursor

RNA components have been identified in preparations of RNase P from a number of eucaryotic sources, but final proof that these RNAs are true RNase P subunits has been elusive because the eucaryotic RNAs, unlike the procaryotic RNase P ribozymes, have not been shown to have catalytic activity in the absence of protein. We previously identified such an RNA component in Saccharomyces cerevisiae nuclear RNase P preparations and have now characterized the corresponding, chromosomal gene, called RPR1 (RNase P ribonucleoprotein 1). Gene disruption experiments showed RPR1 to be single copy and essential. Characterization of the gene region located RPR1 600 bp downstream of the URA3 coding region on chromosome V. We have sequenced 400 bp upstream and 550 bp downstream of the region encoding the major 369-nucleotide RPR1 RNA. The presence of less abundant, potential precursor RNAs with an extra 84 nucleotides of 5' leader and up to 30 nucleotides of 3' trailing sequences suggests that the primary RPR1 transcript is subjected to multiple processing steps to obtain the 369-nucleotide form. Complementation of RPR1-disrupted haploids with one variant of RPR1 gave a slow-growth and temperature-sensitive phenotype. This strain accumulates tRNA precursors that lack the 5' end maturation performed by RNase P, providing direct evidence that RPR1 RNA is an essential component of this enzyme.

scholarly journals Identification of Human Herpesvirus 6 Latency-Associated Transcripts

2002 ◽  
Vol 76 (8) ◽  
pp. 4145-4151 ◽  
Author(s):  
Kazuhiro Kondo ◽  
Kazuya Shimada ◽  
Junji Sashihara ◽  
Keiko Tanaka-Taya ◽  
Koichi Yamanishi
Keyword(s):  
Human Herpesvirus ◽  
Immediate Early ◽  
Human Herpesvirus 6 ◽  
Coding Region ◽  
Transcription Start ◽  
Protein Coding ◽  
Transcription Start Sites ◽  
Latently Infected ◽  
Infected Cells ◽  
Herpesvirus 6

ABSTRACT Four kinds of latency-associated transcripts of human herpesvirus 6 were identified which were detected only in latently infected cells. Although they were oriented in the same direction as the immediate-early 1 and 2 (IE1/IE2) genes and shared their protein-coding region with IE1/IE2, their transcription start sites and exon(s) were latency associated.

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