Genetic and molecular characterization of a variegating hsp70-lacZ fusion gene in the euchromatic 31B region of Drosophila melanogaster

Genome ◽  
2001 ◽  
Vol 44 (4) ◽  
pp. 698-707
Author(s):  
Patrick Morcillo ◽  
Ross J MacIntyre
Keyword(s):  
Drosophila Melanogaster ◽  
Heat Shock ◽  
Fusion Gene ◽  
Position Effect ◽  
P Element ◽  
Lacz Fusion ◽  
Position Effect Variegation ◽  
Salivary Gland Cells ◽  
P Element Transformation

A hsp70–lacZ fusion gene introduced into Drosophila melanogaster at the euchromatic 31B region by P-element transformation displayed a variegated expression with respect to the lacZ fusion protein in the salivary gland cells under heat-shock conditions. The variegation is also reflected by the chromosome puffing pattern. Subsequent transposition of the 31B P element to other euchromatic positions restored wild-type activity, that is, a nonvariegated phenotype. A lower developmental temperature reduced the amount of expression under heat-shock conditions, similar to genes undergoing position-effect variegation (PEV). However, other modifiers of PEV did not affect the expression pattern of the gene. These results show a novel euchromatic tissue-specific variegation that is not associated with classical heterochromatic PEV.Key words: Drosophila, euchromatic position effect, heat shock construct.

scholarly journals A heat-shock-activated cDNA encoding GAGA factor rescues some lethal mutations in the Drosophila melanogaster Trithorax-like gene

2001 ◽  
Vol 78 (1) ◽  
pp. 13-21 ◽  
Author(s):  
H. GRANOK ◽  
B. A. LEIBOVITCH ◽  
S. C. R. ELGIN
Keyword(s):  
Drosophila Melanogaster ◽  
Heat Shock ◽  
Position Effect ◽  
Efficient Production ◽  
Position Effect Variegation ◽  
Chromosomal Protein ◽  
Gaga Factor ◽  
Genetic Rescue ◽  
Regulation Of Synthesis

GAGA factor is an important chromosomal protein involved in establishing specific nucleosome arrays and in regulating gene transcription in Drosophila melanogaster. We developed a transgenic system for controlled heat-shock-dependent overexpression of the GAGA factor 519 amino acid isoform (GAGA-519) in vivo. Efficient production of stable protein from these transgenes provided genetic rescue of a hypomorphic Trithorax-like (Trl) lethal allele to adulthood. Nevertheless, supplemental GAGA-519 did not suppress position effect variegation (PEV), a phenomenon commonly used to measure dosage effects of chromosomal proteins, nor did it rescue other lethal alleles of Trl. The results suggest requirements for the additional isoforms of GAGA factor, or for more precise regulation of synthesis, to carry out the diverse functions of this protein.

Mutations in ash1 and trx enhance P-element-dependent silencing in Drosophila melanogaster

Genome ◽  
2016 ◽  
Vol 59 (8) ◽  
pp. 527-540
Author(s):  
Allen McCracken ◽  
John Locke
Keyword(s):  
Drosophila Melanogaster ◽  
Spontaneous Mutation ◽  
Position Effect ◽  
P Element ◽  
Position Effect Variegation ◽  
Genetic Modifiers ◽  
Gypsy Element ◽  
P Elements ◽  
Opposite Action ◽  
Dose Dependent

In Drosophila melanogaster, the mini-w+ transgene in Pci is normally expressed throughout the adult eye; however, when other P or KP elements are present, a variegated-eye phenotype results, indicating random w+ silencing during development called P-element-dependent silencing (PDS). Mutant Su(var)205 and Su(var)3-7 alleles act as haplo-suppressors/triplo-enhancers of this variegated phenotype, indicating that these heterochromatic modifiers act dose dependently in PDS. Previously, we recovered a spontaneous mutation of P{lacW}ciDplac called P{lacW}ciDplacE1 (E1) that variegated in the absence of P elements, presumably due to the insertion of an adjacent gypsy element. From a screen for genetic modifiers of E1 variegation, we describe here the isolation of five mutations in ash1 and three in trx that enhance the E1 variegated phenotype in a dose-dependent and cumulative manner. These mutant alleles enhance PDS at E1, and in E1/P{lacW}ciDplac, but suppress position effect variegation (PEV) at In(1)wm4. This opposite action is consistent with a model where ASH1 and TRX mark transcriptionally active chromatin domains. If ASH1 or TRX function is lost or reduced, heterochromatin can spread into these domains creating a sink that diverts heterochromatic proteins from other variegating locations, which then may express a suppressed phenotype.

scholarly journals The dose of a putative ubiquitin-specific protease affects position-effect variegation in Drosophila melanogaster.

1996 ◽  
Vol 16 (10) ◽  
pp. 5717-5725 ◽  
Author(s):  
S Henchoz ◽  
F De Rubertis ◽  
D Pauli ◽  
P Spierer
Keyword(s):  
Drosophila Melanogaster ◽  
Position Effect ◽  
Transcript Level ◽  
Open Reading Frame ◽  
P Element ◽  
Position Effect Variegation ◽  
Reading Frame ◽  
Effect Variegation ◽  
Specific Protease ◽  
Ubiquitin Specific Protease

A dominant insertional P-element mutation enhances position-effect variegation in Drosophila melanogaster. The mutation is homozygous, viable, and fertile and maps at 64E on the third chromosome. The corresponding gene was cloned by transposon tagging. Insertion of the transposon upstream of the open reading frame correlates with a strong reduction of transcript level. A transgene was constructed with the cDNA and found to have the effect opposite from that of the mutation, namely, to suppress variegation. Sequencing of the cDNA reveals a large open reading frame encoding a putative ubiquitin-specific protease (Ubp). Ubiquitin marks various proteins, frequently for proteasome-dependent degradation. Ubps can cleave the ubiquitin part from these proteins. We discuss the link established here between a deubiquitinating enzyme and epigenetic silencing processes.

scholarly journals The AT-Hook Protein D1 Is Essential for Drosophila melanogaster Development and Is Implicated in Position-Effect Variegation

2002 ◽  
Vol 22 (4) ◽  
pp. 1218-1232 ◽  
Author(s):  
Nathalie Aulner ◽  
Caroline Monod ◽  
Guillaume Mandicourt ◽  
Denis Jullien ◽  
Olivier Cuvier ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Position Effect ◽  
D1 Protein ◽  
P Element ◽  
Centromeric Heterochromatin ◽  
Position Effect Variegation ◽  
Chromosomal Protein ◽  
Mrna Levels ◽  
Satellite Repeats ◽  
Effect Variegation

ABSTRACT We have analyzed the expression pattern of the D1 gene and the localization of its product, the AT hook-bearing nonhistone chromosomal protein D1, during Drosophila melanogaster development. D1 mRNAs and protein are maternally contributed, and the protein localizes to discrete foci on the chromosomes of early embryos. These foci correspond to 1.672- and 1.688-g/cm3 AT-rich satellite repeats found in the centromeric heterochromatin of the X and Y chromosomes and on chromosomes 3 and 4. D1 mRNA levels subsequently decrease throughout later development, followed by the accumulation of the D1 protein in adult gonads, where two distributions of D1 can be correlated to different states of gene activity. We show that the EP473 mutation, a P-element insertion upstream of D1 coding sequences, affects the expression of the D1 gene and results in an embryonic homozygous lethal phenotype correlated with the depletion of D1 protein during embryogenesis. Remarkably, decreased levels of D1 mRNA and protein in heterozygous flies lead to the suppression of position-effect variegation (PEV) of the white gene in the white-mottled (wm4h ) X-chromosome inversion. Our results identify D1 as a DNA-binding protein of known sequence specificity implicated in PEV. D1 is the primary factor that binds the centromeric 1.688-g/cm3 satellite repeats which are likely involved in white-mottled variegation. We propose that the AT-hook D1 protein nucleates heterochromatin assembly by recruiting specialized transcriptional repressors and/or proteins involved in chromosome condensation.

scholarly journals Genetics of P-Element Transposition Into Drosophila melanogaster Centric Heterochromatin

Genetics ◽  
2003 ◽  
Vol 165 (4) ◽  
pp. 2039-2053 ◽  
Author(s):  
Alexander Y Konev ◽  
Christopher M Yan ◽  
David Acevedo ◽  
Cameron Kennedy ◽  
Elaina Ward ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Position Effect ◽  
Molecular Genetic ◽  
Phenotypic Selection ◽  
Single Copy ◽  
P Element ◽  
Position Effect Variegation ◽  
Entry Points ◽  
Drosophila Heterochromatin ◽  
Genetic Entry

Abstract Heterochromatin is a major component of higher eukaryotic genomes, but progress in understanding the molecular structure and composition of heterochromatin has lagged behind the production of relatively complete euchromatic genome sequences. The introduction of single-copy molecular-genetic entry points can greatly facilitate structure and sequence analysis of heterochromatic regions that are rich in repeated DNA. In this study, we report the isolation of 502 new P-element insertions into Drosophila melanogaster centric heterochromatin, generated in nine different genetic screens that relied on mosaic silencing (position-effect variegation, or PEV) of the yellow gene present in the transposon. The highest frequencies of recovery of variegating insertions were observed when centric insertions were used as the source for mobilization. We propose that the increased recovery of variegating insertions from heterochromatic starting sites may result from the physical proximity of different heterochromatic regions in germline nuclei or from the association of mobilizing elements with heterochromatin proteins. High frequencies of variegating insertions were also recovered when a potent suppressor of PEV (an extra Y chromosome) was present in both the mobilization and selection generations, presumably due to the effects of chromatin structure on P-element mobilization, insertion, and phenotypic selection. Finally, fewer variegating insertions were recovered after mobilization in females, in comparison to males, which may reflect differences in heterochromatin structure in the female and male germlines. FISH localization of a subset of the insertions confirmed that 98% of the variegating lines contain heterochromatic insertions and that these schemes produce a broader distribution of insertion sites. The results of these schemes have identified the most efficient methods for generating centric heterochromatin P insertions. In addition, the large collection of insertions produced by these screens provides molecular-genetic entry points for mapping, sequencing, and functional analysis of Drosophila heterochromatin.

scholarly journals Analysis of subtelomeric heterochromatin in the Drosophila minichromosome Dp1187 by single P element insertional mutagenesis.

Genetics ◽  
1992 ◽  
Vol 132 (3) ◽  
pp. 737-753 ◽  
Author(s):  
G H Karpen ◽  
A C Spradling
Keyword(s):  
Insertional Mutagenesis ◽  
Position Effect ◽  
P Element ◽  
Centromeric Heterochromatin ◽  
Small Subset ◽  
Position Effect Variegation ◽  
Subtelomeric Heterochromatin ◽  
P Elements ◽  
Salivary Gland Cells ◽  
And Function

Abstract We investigated whether single P element insertional mutagenesis could be used to analyze heterochromatin within the Drosophila minichromosome Dp1187. Forty-five insertions of the P[lacZ,rosy+] element onto Dp1187 (recovered among 7,825 transpositions) were highly clustered. None was recovered in centromeric heterochromatin, but 39 occurred about 40 kb from the distal telomere within a 4.7-kb hotspot containing tandem copies of a novel 1.8-kb repetitive DNA sequence. The DNA within and distal to this region lacked essential genes and displayed several other properties characteristic of heterochromatin. The rosy+ genes within the inserted transposons were inhibited by position-effect variegation, and the subtelomeric region was underrepresented in polytene salivary gland cells. These experiments demonstrated that P elements preferentially transpose into a small subset of heterochromatic sites, providing a versatile method for studying the structure and function of these chromosome regions. This approach revealed that a Drosophila chromosome contains a large region of subtelomeric heterochromatin with specific structural and genetic properties.

The Drosophila melanogaster sir2+ Gene Is Nonessential and Has Only Minor Effects on Position-Effect Variegation

Genetics ◽  
2003 ◽  
Vol 163 (3) ◽  
pp. 931-937 ◽  
Author(s):  
Stefan U Åström ◽  
Thomas W Cline ◽  
Jasper Rine
Keyword(s):  
Drosophila Melanogaster ◽  
Life Span ◽  
Transcriptional Start Site ◽  
Position Effect ◽  
Developmental Rate ◽  
P Element ◽  
Position Effect Variegation ◽  
Lethal Mutations ◽  
Effect Variegation ◽  
Dependent Protein

Abstract Five Drosophila melanogaster genes belong to the highly conserved sir2 family, which encodes NAD+-dependent protein deacetylases. Of these five, dsir2+ (CG5216) is most similar to the Saccharomyces cerevisiae SIR2 gene, which has profound effects on chromatin structure and life span. Four independent Drosophila strains were found with P-element insertions near the dsir2 transcriptional start site as well as extraneous linked recessive lethal mutations. Imprecise excision of one of these P elements (PlacW 07223) from a chromosome freed of extraneous lethal mutations produced dsir217, a null intragenic deletion allele that generates no DSIR2 protein. Contrary to expectations from the report by Rosenberg and Parkhurst on their P-mobilization allele dSir2ex10, homozygosity for dsir217 had no apparent deleterious effects on viability, developmental rate, or sex ratio, and it fully complemented sir2ex10. Moreover, through a genetic test, we ruled out the reported effect of dSir2ex10 on Sex-lethal expression. We did observe a modest, strictly recessive suppression of whitem4 position-effect variegation and a shortening of life span in dsir2 homozygous mutants, suggesting that dsir2 has some functions in common with yeast SIR2.

Varied expression of a Y-linked P[w+] insert due to imprinting in Drosophila melanogaster

Genome ◽  
2000 ◽  
Vol 43 (2) ◽  
pp. 285-292 ◽  
Author(s):  
Bethany S Haller ◽  
R C Woodruff
Keyword(s):  
Drosophila Melanogaster ◽  
Y Chromosome ◽  
Genetic Background ◽  
Position Effect ◽  
P Element ◽  
Parental Origin ◽  
Position Effect Variegation ◽  
Reporter System ◽  
Heterochromatin Protein 1 ◽  
Parental Female

During gametogenesis, a gene can become imprinted affecting its expression in progeny. We have used the expression of a Y-linked P[w+]YAL transposable DNA element as a reporter system to investigate the effect of parental origination on the expression of the w+ insert. Expression of w+ was greater in male progeny when the Y chromosome, harboring the insert, was inherited from the parental male rather than from the parental female. Imprinting was not due to a genetic background influence in the males, since the only difference among the males was the parental origin of the Y chromosome. It was also observed that the genetic background can affect imprinting, since w+ expression was also higher in males when the Y was derived from C(1)DX attached-X parental females rather than from C(1)RM attached-X parental females. Though the heterochromatic imprinting mechanism is unknown, a mutated Heterochromatin Protein 1 (HP1) gene, which is associated with suppression of position-effect variegation, increases expression of the w+ locus in the P[w+]YAL insert, indicating that HP1 may play a role in Y chromosome packaging. Key words: Drosophila melanogaster, heterochromatin, HP1, imprinting, P-element, Y chromosome.

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