scholarly journals Structure and Regulation of the Salivary Gland Secretion Protein Gene Sgs-1 of Drosophila melanogaster

Genetics ◽  
1999 ◽  
Vol 153 (2) ◽  
pp. 753-762
Author(s):  
Günther E Roth ◽  
Sigrid Wattler ◽  
Hartmut Bornschein ◽  
Michael Lehmann ◽  
Günter Korge
Keyword(s):  
Drosophila Melanogaster ◽  
Tandem Repeats ◽  
Transcriptional Start Site ◽  
Regulatory Elements ◽  
Coding Region ◽  
Band Shift ◽  
Salivary Gland Secretion ◽  
Enhancer Binding Protein ◽  
Factor Secretion ◽  
Third Instar Larvae

Abstract The Drosophila melanogaster gene Sgs-1 belongs to the secretion protein genes, which are coordinately expressed in salivary glands of third instar larvae. Earlier analysis had implied that Sgs-1 is located at the 25B2-3 puff. We cloned Sgs-1 from a YAC covering 25B2-3. Despite using a variety of vectors and Escherichia coli strains, subcloning from the YAC led to deletions within the Sgs-1 coding region. Analysis of clonable and unclonable sequences revealed that Sgs-1 mainly consists of 48-bp tandem repeats encoding a threonine-rich protein. The Sgs-1 inserts from single λ clones are heterogeneous in length, indicating that repeats are eliminated. By analyzing the expression of Sgs-1/lacZ fusions in transgenic flies, cis-regulatory elements of Sgs-1 were mapped to lie within 1 kb upstream of the transcriptional start site. Band shift assays revealed binding sites for the transcription factor fork head (FKH) and the factor secretion enhancer binding protein 3 (SEBP3) at positions that are functionally relevant. FKH and SEBP3 have been shown previously to be involved in the regulation of Sgs-3 and Sgs-4. Comparison of the levels of steady state RNA and of the transcription rates for Sgs-1 and Sgs-1/lacZ reporter genes indicates that Sgs-1 RNA is 100-fold more stable than Sgs-1/lacZ RNA. This has implications for the model of how Sgs transcripts accumulate in late third instar larvae.

Transformation mapping of the regulatory elements of the ecdysone-inducible P1 gene of Drosophila melanogaster

1991 ◽  
Vol 11 (5) ◽  
pp. 2913-2917
Author(s):  
F Maschat ◽  
M L Dubertret ◽  
J A Lepesant
Keyword(s):  
Drosophila Melanogaster ◽  
Hormonal Regulation ◽  
Germ Line ◽  
Regulatory Elements ◽  
Fat Bodies ◽  
Germ Line Transformation ◽  
Third Instar Larvae

The transcription of the P1 gene is induced by 20-hydroxyecdysone in fat bodies of third-instar larvae. Germ line transformation showed that sequences between -138 to +276 contain elements required for a qualitatively correct developmental and hormonal regulation of P1 transcription. Sequences from -138 to -68 are essential for this expression.

A genetic and molecular analysis of an invectedDominant mutation in Drosophila melanogaster

Genome ◽  
1998 ◽  
Vol 41 (3) ◽  
pp. 381-390 ◽  
Author(s):  
A J Simmonds ◽  
J B Bell
Keyword(s):  
Drosophila Melanogaster ◽  
Imaginal Disc ◽  
Transcriptional Start Site ◽  
Ectopic Expression ◽  
Careful Analysis ◽  
Wing Imaginal Disc ◽  
Coding Region ◽  
Specific Expression ◽  
Additional Lesion ◽  
Normal Expression

The invected gene of Drosophila melanogaster is a homeobox-containing gene that is closely related to engrailed. A dominant gain of function allele, invectedDominant, was derived from mutagenesis of a dominant allele of vestigial, In(2R)vgW. A careful analysis of the phenotype of invectedDominant shows that it is associated with a transformation of the anterior compartment of the wing to a posterior fate. This transformation is normally limited to the wing blade itself and does not involve the remaining tissues derived from the wing imaginal disc, including the wing hinge and dorsal thorax of the fly. The ectopic expression of invected protein associated with invectedDominant correlates spatially with the normal expression pattern of vestigial in the wing imaginal disc, suggesting that control elements of vestigial are driving ectopic invected expression. This was confirmed by sequence analysis that shows that the dominant vestigial activity was eliminated by a deletion that removes the 3' portion of the vestigial coding region. This leaves a gene fusion wherein the vestigial enhancer elements are still juxtaposed immediately 5' to the invected transcriptional start site, but with the vg sequences harboring an additional lesion. Unlike recessive invected alleles, the invectedDominant allele produces an observable phenotype, and as such, should prove useful in determining the role of invected in patterning the wing imaginal disc. Genetic analysis has shown that mutations of polyhomeotic, a gene involved in regulating engrailed expression, cause a reproducible alteration in the invectedDominant phenotype. Finally, the invectedDominant allele should prove valuable for identifying and characterizing genes that are activated within the posterior compartment. A screen using various lacZ lines that are asymmetrically expressed in an anterior-posterior manner in the wing imaginal disc isolated one line that shows posterior-specific expression within the transformed anterior compartment.Key words: Drosophila, development, dominant mutation, ectopic, wings.

scholarly journals Transformation mapping of the regulatory elements of the ecdysone-inducible P1 gene of Drosophila melanogaster.

1991 ◽  
Vol 11 (5) ◽  
pp. 2913-2917 ◽  
Author(s):  
F Maschat ◽  
M L Dubertret ◽  
J A Lepesant
Keyword(s):  
Drosophila Melanogaster ◽  
Hormonal Regulation ◽  
Germ Line ◽  
Regulatory Elements ◽  
Fat Bodies ◽  
Germ Line Transformation ◽  
Third Instar Larvae

The transcription of the P1 gene is induced by 20-hydroxyecdysone in fat bodies of third-instar larvae. Germ line transformation showed that sequences between -138 to +276 contain elements required for a qualitatively correct developmental and hormonal regulation of P1 transcription. Sequences from -138 to -68 are essential for this expression.

scholarly journals Paired CRISPR/Cas9 guide-RNAs enable high-throughput deletion scanning (ScanDel) of a Mendelian disease locus for functionally critical non-coding elements

2016 ◽  
Author(s):  
Molly Gasperini ◽  
Gregory M. Findlay ◽  
Aaron McKenna ◽  
Jennifer H. Milbank ◽  
Choli Lee ◽  
...  
Keyword(s):  
Large Scale ◽  
Transcriptional Start Site ◽  
Regulatory Elements ◽  
Mendelian Disease ◽  
Regulatory Sequences ◽  
Coding Region ◽  
Endogenous Gene ◽  
Distal Regulatory Elements

AbstractThe extent to which distal non-coding mutations contribute to Mendelian disease remains a major unknown in human genetics. Given that a gene’s in vivo function can be appropriately modeled in vitro, CRISPR/Cas9 genome editing enables the large-scale perturbation of distal non-coding regions to identify functional elements in their native context. However, early attempts at such screens have relied on one individual guide RNA (gRNA) per cell, resulting in sparse mutagenesis with minimal redundancy across regions of interest. To address this, we developed a system that uses pairs of gRNAs to program thousands of kilobase-scale deletions that scan across a targeted region in a tiling fashion (“ScanDel”). As a proof-of-concept, we applied ScanDel to program 4,342 overlapping 1- and 2- kilobase (Kb) deletions that tile a 206 Kb region centered on HPRT1, the gene underlying Lesch-Nyhan syndrome, with median 27-fold redundancy per base. Programmed deletions were functionally assayed by selecting for loss of HPRT1 function with 6-thioguanine. HPRT1 exons served as positive controls, and all were successfully identified as functionally critical by the screen. Remarkably, HPRT1 function appeared robust to deletion of any intergenic or deeply intronic non-coding region across the 206 Kb locus, indicating that proximal regulatory sequences are sufficient for its expression. A sparser mutagenesis screen of the same 206 Kb with individual gRNAs also failed to identify critical distal regulatory elements. Although our screen did find programmed deletions and individual gRNAs with putative functional consequences that targeted exon-proximal non-coding sequences (e.g. the promoter), long-read sequencing revealed that this signal was driven almost entirely by rare, unexpected deletions that extended into exonic sequence. These targeted validation experiments defined a small region surrounding the transcriptional start site as the only non-coding sequence essential to HPRT1 function. Overall, our results suggest that distal regulatory elements are not critical for HPRT1 expression, and underscore the necessity of comprehensive edited-locus genotyping for validating the results of CRISPR screens. The application of ScanDel to additional loci will enable more insight into the extent to which the disruption of distal non-coding elements contributes to Mendelian diseases. In addition, dense, redundant, large-scale deletion scanning with gRNA pairs will facilitate a deeper understanding of endogenous gene regulation in the human genome.

scholarly journals The congested-like tracheae Gene of Drosophila melanogaster Encodes a Member of the Mitochondrial Carrier Family Required for Gas-Filling of the Tracheal System and Expansion of the Wings After Eclosion

Genetics ◽  
1997 ◽  
Vol 147 (4) ◽  
pp. 1755-1768 ◽  
Author(s):  
Kirsten Hartenstein ◽  
Pradip Sinha ◽  
Arati Mishra ◽  
Heide Schenkel ◽  
Istvan Török ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Tandem Repeats ◽  
Polytene Chromosomes ◽  
P Element ◽  
Coding Region ◽  
Tracheal System ◽  
Mitochondrial Carrier ◽  
Transmembrane Segments ◽  
Hatching Time ◽  
Mitochondrial Carrier Family

Abstract A recessive semi-lethal mutation resulting from the insertion of a P-lacW transposon at the cytological position 23A on the polytene chromosomes of Drosophila melanogaster was found to affect the unfolding and expansion of the wings resulting in a loss of venation and a marked decrease in their size. Lethality was polyphasic with numerous animals dying during early larval development and displaying apparently collapsed tracheal trees. The gene was therefore designated as congested-like tracheae, or colt. The colt mutation resulted from the insertion of a P-lacW transposon within the coding region of a 1.4kb transcript. Wild-type function was restored by inducing a precise excision of the P-lacWtransposon, while a deletion of the colt locus, produced by imprecise excision of the P element, showed a phenotype similar to that of the original P insert. The colt gene consists of a single exon and encodes a protein of 306 amino acids made of three tandem repeats, each characterized by two predicted transmembrane segments and a loop domain. The COLT protein shares extensive homology with proteins in the mitochondrial carrier family and particularly with the DIF-1 protein of Caenwhabditis ekgans, which has been shown to be maternally required for embryonic tissue differentiation. Our analysis revealed that zygotic colt function is dispensable for normal embryonic morphogenesis but is required for gas-filling of the tracheal system at hatching time of the embryo and for normal epithelial morphogenesis of the wings.

Transvection and Silencing of the Scr Homeotic Gene of Drosophila melanogaster

Genetics ◽  
2002 ◽  
Vol 161 (2) ◽  
pp. 733-746
Author(s):  
Jeffrey W Southworth ◽  
James A Kennison
Keyword(s):  
Drosophila Melanogaster ◽  
Chromosomal Aberrations ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Anomalous Behavior ◽  
Polycomb Group ◽  
Homeotic Gene ◽  
Polycomb Group Proteins ◽  
Sex Combs Reduced ◽  
In Cis

Abstract The Sex combs reduced (Scr) gene specifies the identities of the labial and first thoracic segments in Drosophila melanogaster. In imaginal cells, some Scr mutations allow cis-regulatory elements on one chromosome to stimulate expression of the promoter on the homolog, a phenomenon that was named transvection by Ed Lewis in 1954. Transvection at the Scr gene is blocked by rearrangements that disrupt pairing, but is zeste independent. Silencing of the Scr gene in the second and third thoracic segments, which requires the Polycomb group proteins, is disrupted by most chromosomal aberrations within the Scr gene. Some chromosomal aberrations completely derepress Scr even in the presence of normal levels of all Polycomb group proteins. On the basis of the pattern of chromosomal aberrations that disrupt Scr gene silencing, we propose a model in which two cis-regulatory elements interact to stabilize silencing of any promoter or cis-regulatory element physically between them. This model also explains the anomalous behavior of the Scx allele of the flanking homeotic gene, Antennapedia. This allele, which is associated with an insertion near the Antennapedia P1 promoter, inactivates the Antennapedia P1 and P2 promoters in cis and derepresses the Scr promoters both in cis and on the homologous chromosome.

scholarly journals PRESENCE OF A BASE LINE LEVEL OF SISTER CHROMATID EXCHANGES IN SOMATIC CELLS OF DROSOPHILA MELANOGASTER IN VIVO AND IN VITRO

Genetics ◽  
1982 ◽  
Vol 100 (2) ◽  
pp. 259-278
Author(s):  
Hideo Tsuji
Keyword(s):  
Drosophila Melanogaster ◽  
Sister Chromatid ◽  
Ganglion Cells ◽  
Sister Chromatid Exchanges ◽  
Base Line ◽  
Cell Cycles ◽  
Chromatid Exchanges ◽  
Third Instar Larvae

ABSTRACT Sister chromatid exchanges (SCEs) under in vivo and in vitro conditions were examined in ganglion cells of third-instar larvae of Drosophila melanogaster (Oregon-R). In the in vivo experiment, third-instar larvae were fed on synthetic media containing 5-bromo-2′-deoxyuridine (BrdUrd). After two cell cycles, ganglia were dissected and treated with colchicine. In the in vitro experiment, the ganglia were also incubated in media containing BrdUrd for two cell cycles, and treated with colchicine. SCEs were scored in metaphase stained with Hoechst 33258 plus Giemsa. The frequencies of SCEs stayed constant in the range of 25-150 vg/ml and 0.25-2.5 vg/ml of BrdUrd in vivo and in vitro, respectively. SCEs gradually increased at higher concentrations, strongly suggesting that at least a fraction of the detected SCEs are spontaneous. The constant levels of SCE frequency were estimated, on the average, at 0.103 per cell per two cell cycles for females and 0.101 for males in vivo and at 0.096 for females and 0.091 for males in vitro. No difference was found in the SCE frequency between sexes at any of the BrdUrd concentrations. The analysis for the distribution of SCEs within chromosomes revealed an extraordinarily high proportion of the SCEs at the junctions between euchromatin and heterochromatin; the remaining SCEs were preferentially localized in the euchromatic regions of the chromosomes and in the heterochromatic Y chromosome. These results were largely inconsistent with those of Gatti et al. (1979).

scholarly journals Genome-wide characterization of MATE gene family and expression profiles in response to abiotic stresses in rice (Oryza sativa)

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Zhixuan Du ◽  
Qitao Su ◽  
Zheng Wu ◽  
Zhou Huang ◽  
Jianzhong Bao ◽  
...  
Keyword(s):  
Oryza Sativa ◽  
Gene Family ◽  
Tandem Repeats ◽  
Expression Profiles ◽  
Functional Divergence ◽  
Expression Patterns ◽  
Regulatory Elements ◽  
Genome Wide ◽  
Comprehensive Information ◽  
Family Gene

AbstractMultidrug and toxic compound extrusion (MATE) proteins are involved in many physiological functions of plant growth and development. Although an increasing number of MATE proteins have been identified, the understanding of MATE proteins is still very limited in rice. In this study, 46 MATE proteins were identified from the rice (Oryza sativa) genome by homology searches and domain prediction. The rice MATE family was divided into four subfamilies based on the phylogenetic tree. Tandem repeats and fragment replication contribute to the expansion of the rice MATE gene family. Gene structure and cis-regulatory elements reveal the potential functions of MATE genes. Analysis of gene expression showed that most of MATE genes were constitutively expressed and the expression patterns of genes in different tissues were analyzed using RNA-seq. Furthermore, qRT-PCR-based analysis showed differential expression patterns in response to salt and drought stress. The analysis results of this study provide comprehensive information on the MATE gene family in rice and will aid in understanding the functional divergence of MATE genes.

The 5'-flanking region of the mouse thymidylate synthase gene is necessary but not sufficient for normal regulation in growth-stimulated cells

1991 ◽  
Vol 11 (2) ◽  
pp. 1023-1029
Author(s):  
Y Li ◽  
D Li ◽  
K Osborn ◽  
L F Johnson
Keyword(s):  
Thymidylate Synthase ◽  
Cultured Cells ◽  
Transcriptional Start Site ◽  
Simian Virus 40 ◽  
Normal Growth ◽  
Housekeeping Gene ◽  
Simian Virus ◽  
Coding Region ◽  
Proliferating Cells ◽  
Early Promoter

The thymidylate synthase (TS) gene is a housekeeping gene that is expressed at much higher levels in proliferating cells than in quiescent cells. We have studied the role of the TS 5'-flanking sequences in regulating the level of expression of the mouse TS gene. A variety of chimeric TS minigenes that contain different promoters linked either to the TS coding region (with or without introns) or to the chloramphenicol acetyltransferase (CAT) coding region were constructed. The activities of the minigenes were determined by transfecting them into cultured cells and measuring the levels of mRNA or enzyme derived from the chimeric genes. We found that the mouse TS promoter had about the same strength as the simian virus 40 early promoter but was significantly stronger than the herpes simplex virus thymidine kinase promoter. Stable transfection studies revealed that minigenes consisting of the normal TS promoter (extending to -1 kb), coding region, and polyadenylation signal were regulated normally in response to growth stimulation. When the TS promoter was replaced by the simian virus 40 early promoter or by a TS promoter that retained only 60 nucleotides upstream of the first transcriptional start site, the minigene was expressed constitutively. A minigene consisting of the TS promoter (extending to -1 kb) linked to the CAT coding region was also expressed constitutively. These observations indicate that sequences upstream of the transcriptional start sites of the TS gene are necessary, although not sufficient, for normal growth-regulated expression of the mouse TS gene.

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