P-element-mediated enhancer detection applied to the study of oogenesis in Drosophila

Development ◽  
1989 ◽  
Vol 107 (2) ◽  
pp. 189-200 ◽  
Author(s):  
U. Grossniklaus ◽  
H.J. Bellen ◽  
C. Wilson ◽  
W.J. Gehring
Keyword(s):  
Germ Line ◽  
Expression Patterns ◽  
Cell Types ◽  
Regulatory Elements ◽  
P Element ◽  
Regulatory Sequences ◽  
Transcriptional Regulatory ◽  
Morphological Criteria ◽  
Enhancer Detection ◽  
New Strategies

We have stained the ovaries of nearly 600 different Drosophila strains carrying single copies of a P-element enhancer detector. This transposon detects neighbouring genomic transcriptional regulatory sequences by means of a beta-galactosidase reporter gene. Numerous strains are stained in specific cells and at specific stages of oogenesis and provide useful ovarian markers for cell types that in some cases have not previously been recognized by morphological criteria. Since recent data have suggested that a substantial number of the regulatory elements detected by enhancer detection control neighbouring genes, we discuss the implications of our results concerning ovarian gene expression patterns in Drosophila. We have also identified a small number of insertion-linked recessive mutants that are sterile or lead to ovarian defects. We observe a strong correlation with specific germ line staining patterns in these strains, suggesting that certain patterns are more likely to be associated with female sterile genes than others. On the basis of our results, we suggest new strategies, which are not primarily based on the generation of mutants, to screen for and isolated female sterile genes.

scholarly journals Probing spermatogenesis in Drosophila with P-element enhancer detectors

Development ◽  
1992 ◽  
Vol 114 (1) ◽  
pp. 89-98 ◽  
Author(s):  
P. Gonczy ◽  
S. Viswanathan ◽  
S. DiNardo
Keyword(s):  
Germ Line ◽  
Expression Patterns ◽  
Cell Types ◽  
P Element ◽  
Structural Reorganization ◽  
Germ Cell Differentiation ◽  
Distinct Cell ◽  
Morphological Criteria ◽  
Cyst Cells ◽  
A Cell

Formation of motile sperm in Drosophila melanogaster requires the coordination of processes such as stem cell division, mitotic and meiotic control and structural reorganization of a cell. Proper execution of spermatogenesis entails the differentiation of cells derived from two distinct embryonic lineages, the germ line and the somatic mesoderm. Through an analysis of homozygous viable and fertile enhancer detector lines, we have identified molecular markers for the different cell types present in testes. Some lines label germ cells or somatic cyst cells in a stage-specific manner during their differentiation program. These expression patterns reveal transient identities for the cyst cells that had not been previously recognized by morphological criteria. A marker line labels early stages of male but not female germ cell differentiation and proves useful in the analysis of germ line sex-determination. Other lines label the hub of somatic cells around which germ line stem cells are anchored. By analyzing the fate of the somatic hub in an agametic background, we show that the germ line plays some role in directing its size and its position in the testis. We also describe how marker lines enable us to identify presumptive cells in the embryonic gonadal mesoderm before they give rise to morphologically distinct cell types. Finally, this collection of marker lines will allow the characterization of genes expressed either in the germ line or in the soma during spermatogenesis.

Delimiting regulatory sequences of the Drosophila melanogaster Ddc gene

1986 ◽  
Vol 6 (12) ◽  
pp. 4548-4557
Author(s):  
J Hirsh ◽  
B A Morgan ◽  
S B Scholnick
Keyword(s):  
Drosophila Melanogaster ◽  
Germ Line ◽  
Regulatory Elements ◽  
P Element ◽  
Developmental Expression ◽  
Upstream Region ◽  
Regulatory Sequences ◽  
Flanking Sequences

We delimited sequences necessary for in vivo expression of the Drosophila melanogaster dopa decarboxylase gene Ddc. The expression of in vitro-altered genes was assayed following germ line integration via P-element vectors. Sequences between -209 and -24 were necessary for normally regulated expression, although genes lacking these sequences could be expressed at 10 to 50% of wild-type levels at specific developmental times. These genes showed components of normal developmental expression, which suggests that they retain some regulatory elements. All Ddc genes lacking the normal immediate 5'-flanking sequences were grossly deficient in larval central nervous system expression. Thus, this upstream region must contain at least one element necessary for this expression. A mutated Ddc gene without a normal TATA boxlike sequence used the normal RNA start points, indicating that this sequences is not required for start point specificity.

scholarly journals Delimiting regulatory sequences of the Drosophila melanogaster Ddc gene.

1986 ◽  
Vol 6 (12) ◽  
pp. 4548-4557 ◽  
Author(s):  
J Hirsh ◽  
B A Morgan ◽  
S B Scholnick
Keyword(s):  
Drosophila Melanogaster ◽  
Germ Line ◽  
Regulatory Elements ◽  
P Element ◽  
Developmental Expression ◽  
Upstream Region ◽  
Regulatory Sequences ◽  
Flanking Sequences

We delimited sequences necessary for in vivo expression of the Drosophila melanogaster dopa decarboxylase gene Ddc. The expression of in vitro-altered genes was assayed following germ line integration via P-element vectors. Sequences between -209 and -24 were necessary for normally regulated expression, although genes lacking these sequences could be expressed at 10 to 50% of wild-type levels at specific developmental times. These genes showed components of normal developmental expression, which suggests that they retain some regulatory elements. All Ddc genes lacking the normal immediate 5'-flanking sequences were grossly deficient in larval central nervous system expression. Thus, this upstream region must contain at least one element necessary for this expression. A mutated Ddc gene without a normal TATA boxlike sequence used the normal RNA start points, indicating that this sequences is not required for start point specificity.

Distinct expression patterns detected within individual tissues by the GAL4 enhancer trap technique

Genome ◽  
1996 ◽  
Vol 39 (1) ◽  
pp. 174-182 ◽  
Author(s):  
Kerstin Gustafson ◽  
Gabrielle L. Boulianne
Keyword(s):  
Gene Expression ◽  
Transcriptional Control ◽  
Germ Line ◽  
Expression Patterns ◽  
Cell Types ◽  
Enhancer Trap ◽  
P Element ◽  
Immunocytochemical Staining ◽  
Specific Cell ◽  
Drosophila Genome

To identify genes that are expressed in specific cell types or tissues during development, we generated enhancer-trap lines in which the yeast transcriptional activator, GAL4, was mobilized throughout the Drosophila genome. The GAL4 lines are part of a two-part system involving GAL4 and its target, the upstream activating sequence (UAS). Detection of GAL4 expression patterns was achieved by crossing individual GAL4 lines with flies carrying the reporter gene lacZ under the transcriptional control of the UAS followed by histochemical and immunocytochemical staining. Here, we present the results of this screen and the characterization of GAL4 lines that show distinct patterns of gene expression during Drosophila development, including embryogenesis, oogenesis, and imaginai disc development. However, we were unable to identify GAL4 lines that were expressed within the germ line or during early embryogenesis. Furthermore, consistent with previous results, we found that the GAL4 enhancer trap technique had a much lower frequency of transposition than has been reported for lacZ enhancer trap screens. Taken together, these results demonstrate both the strengths and weaknesses of the GAL4 enhancer trap technique for identifying unique patterns of gene expression during development. Key words : GAL4, enhancer trap, Drosophila, P element.

A variant octamer motif in a Xenopus H2B histone gene promoter is not required for transcription in frog oocytes

1991 ◽  
Vol 11 (2) ◽  
pp. 641-654
Author(s):  
C Hinkley ◽  
M Perry
Keyword(s):  
Cell Cycle ◽  
Transcription Initiation ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Histone Gene ◽  
Site Directed Mutagenesis ◽  
Regulatory Sequences ◽  
Chromosomal Dna ◽  
Transcriptional Regulatory ◽  
Octamer Motif

Xenopus oocytes, arrested in G2 before the first meiotic division, accumulate histone mRNA and protein in the absence of chromosomal DNA replication and therefore represent an attractive biological system in which to examine histone gene expression uncoupled from the cell cycle. Previous studies have shown that sequences necessary for maximal levels of transcription in oocytes are present within 200 bp at the 5' end of the transcription initiation site for genes encoding each of the five major Xenopus histone classes. We have defined by site-directed mutagenesis individual regulatory sequences and characterized DNA-binding proteins required for histone H2B gene transcription in injected oocytes. The Xenopus H2B gene has a relatively simple promoter containing several transcriptional regulatory elements, including TFIID, CBP, and ATF/CREB binding sites, required for maximal transcription. A sequence (CTTTACAT) in the H2B promoter resembling the conserved octamer motif (ATTTGCAT), the target for cell-cycle regulation of a human H2B gene, is not required for transcription in oocytes. Nonetheless, substitution of a consensus octamer motif for the variant octamer element activates H2B transcription. Oocyte factors, presumably including the ubiquitous Oct-1 factor, specifically bind to the consensus octamer motif but not to the variant sequence. Our results demonstrate that a transcriptional regulatory element involved in lymphoid-specific expression of immunoglobulin genes and in S-phase-specific activation of mammalian H2B histone genes can activate transcription in nondividing amphibian oocytes.

A Drosophila GATA family member that binds to Adh regulatory sequences is expressed in the developing fat body

Development ◽  
1993 ◽  
Vol 119 (3) ◽  
pp. 623-633 ◽  
Author(s):  
T. Abel ◽  
A.M. Michelson ◽  
T. Maniatis
Keyword(s):  
Fat Body ◽  
Regulatory Elements ◽  
Drosophila Embryo ◽  
Cdna Clones ◽  
Regulatory Sequences ◽  
Mammalian Development ◽  
Sequence Element ◽  
Gata Factors ◽  
Transcriptional Regulatory ◽  
Gata Family

We have identified a Drosophila transcription factor that binds a sequence element found in the larval promoters of all known alcohol dehydrogenase (Adh) genes. DNA sequence analysis of cDNA clones encoding this protein, box A-binding factor (ABF), reveals that it is a member of the GATA family of transcriptional regulatory factors. ABF-binding sites within the D. mulleri and D. melanogaster larval Adh promoters function as positive regulatory elements and in cotransfection experiments, ABF functions as a transcriptional activator. In further support of a role for ABF in the regulation of Adh expression, ABF mRNA is expressed in the embryonic fat body, a tissue that contains high levels of Adh mRNA. Our studies demonstrate that the fat body develops from segmentally repeated clusters of mesodermal cells, which later expand and coalesce to form the mature fat body. These observations establish ABF as the earliest known fat body precursor marker in the Drosophila embryo. Together with the established role of GATA factors during mammalian development, these results suggest that ABF may play a key role in the organogenesis of the fat body.

Developmentally distinct MYB genes encode functionally equivalent proteins in Arabidopsis

Development ◽  
2001 ◽  
Vol 128 (9) ◽  
pp. 1539-1546 ◽  
Author(s):  
M.M. Lee ◽  
J. Schiefelbein
Keyword(s):  
Functional Relationship ◽  
Expression Patterns ◽  
Cell Types ◽  
Myb Transcription Factor ◽  
Regulatory Sequences ◽  
Myb Gene ◽  
Distinct Cell ◽  
Transcription Factor Genes ◽  
Myb Genes ◽  
Multicellular Organisms

The duplication and divergence of developmental control genes is thought to have driven morphological diversification during the evolution of multicellular organisms. To examine the molecular basis of this process, we analyzed the functional relationship between two paralogous MYB transcription factor genes, WEREWOLF (WER) and GLABROUS1 (GL1), in Arabidopsis. The WER and GL1 genes specify distinct cell types and exhibit non-overlapping expression patterns during Arabidopsis development. Nevertheless, reciprocal complementation experiments with a series of gene fusions showed that WER and GL1 encode functionally equivalent proteins, and their unique roles in plant development are entirely due to differences in their cis-regulatory sequences. Similar experiments with a distantly related MYB gene (MYB2) showed that its product cannot functionally substitute for WER or GL1. Furthermore, an analysis of the WER and GL1 proteins shows that conserved sequences correspond to specific functional domains. These results provide new insights into the evolution of the MYB gene family in Arabidopsis, and, more generally, they demonstrate that novel developmental gene function may arise solely by the modification of cis-regulatory sequences.

Accurate Identification of Active Transcriptional Regulatory Elements from Global Run-On and Sequencing Data

2014 ◽  
Author(s):  
Charles G Danko ◽  
Stephanie L Hyland ◽  
Leighton J Core ◽  
Andre L Martins ◽  
Colin T Waters ◽  
...  
Keyword(s):  
Multiple Scales ◽  
Regulatory Element ◽  
Cell Types ◽  
Regulatory Elements ◽  
Support Vector ◽  
Sequencing Data ◽  
Accurate Identification ◽  
Single Experiment ◽  
Transcriptional Regulatory Elements ◽  
Transcriptional Regulatory

Identification of the genomic regions that regulate transcription remains an important open problem. We have recently shown that global run-on and sequencing (GRO-seq) with enrichment for 5′-capped RNAs reveals patterns of divergent transcription that accurately mark active transcriptional regulatory elements (TREs), including enhancers and promoters. Here, we demonstrate that active TREs can be identified with comparable accuracy by applying sensitive machine-learning methods to standard GRO-seq and PRO-seq data, allowing TREs to be assayed together with transcription levels, elongation rates, and other transcriptional features, in a single experiment. Our method, called discriminative Regulatory Element detection from GRO-seq (dREG), summarizes GRO-seq read counts at multiple scales and uses support vector regression to predict active TREs. The predicted TREs are strongly enriched for marks associated with functional elements, including H3K27ac, transcription factor binding sites, eQTLs, and GWAS-associated SNPs. Using dREG, we survey TREs in eight cell types and provide new insights into global patterns of TRE assembly and function.

scholarly journals Retroviral Transcriptional Regulation and Embryonic Stem Cells: War and Peace

2014 ◽  
Vol 35 (5) ◽  
pp. 770-777 ◽  
Author(s):  
Sharon Schlesinger ◽  
Stephen P. Goff
Keyword(s):  
Dna Sequences ◽  
Regulatory Networks ◽  
Es Cells ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Cell Types ◽  
Endogenous Retroviruses ◽  
Regulatory Sequences ◽  
Cellular Genes ◽  
Wide Range

Retroviruses have evolved complex transcriptional enhancers and promoters that allow their replication in a wide range of tissue and cell types. Embryonic stem (ES) cells, however, characteristically suppress transcription of proviruses formed after infection by exogenous retroviruses and also of most members of the vast array of endogenous retroviruses in the genome. These cells have unusual profiles of transcribed genes and are poised to make rapid changes in those profiles upon induction of differentiation. Many of the transcription factors in ES cells control both host and retroviral genes coordinately, such that retroviral expression patterns can serve as markers of ES cell pluripotency. This overlap is not coincidental; retrovirus-derived regulatory sequences are often used to control cellular genes important for pluripotency. These sequences specify the temporal control and perhaps “noisy” control of cellular genes that direct proper cell gene expression in primitive cells and their differentiating progeny. The evidence suggests that the viral elements have been domesticated for host needs, reflecting the wide-ranging exploitation of any and all available DNA sequences in assembling regulatory networks.

scholarly journals Bmp2 Transcription in Osteoblast Progenitors Is Regulated by a Distant 3′ Enhancer Located 156.3 Kilobases from the Promoter

2007 ◽  
Vol 27 (8) ◽  
pp. 2934-2951 ◽  
Author(s):  
Ronald L. Chandler ◽  
Kelly J. Chandler ◽  
Karen A. McFarland ◽  
Douglas P. Mortlock
Keyword(s):  
Risk Factor ◽  
Osteoblast Differentiation ◽  
Expression Patterns ◽  
Artificial Chromosome ◽  
Regulatory Elements ◽  
Bone Morphogenetic Protein 2 ◽  
Regulatory Function ◽  
Regulatory Sequences ◽  
Bac Clone ◽  
Osteoblast Progenitor

ABSTRACT Bone morphogenetic protein 2 (encoded by Bmp2) has been implicated as an important signaling ligand for osteoblast differentiation and bone formation and as a genetic risk factor for osteoporosis. To initially survey a large genomic region flanking the mouse Bmp2 gene for cis-regulatory function, two bacterial artificial chromosome (BAC) clones that extend far upstream and downstream of the gene were engineered to contain a lacZ reporter cassette and tested in transgenic mice. Each BAC clone directs a distinct subset of normal Bmp2 expression patterns, suggesting a modular arrangement of distant Bmp2 regulatory elements. Strikingly, regulatory sequences required for Bmp2 expression in differentiating osteoblasts, as well as tooth buds, hair placodes, kidney, and other tissues, are located more than 53 kilobases 3′ to the promoter. By testing BACs with engineered deletions across this distant 3′ region, we parsed these regulatory elements into separate locations and more closely refined the location of the osteoblast progenitor element. Finally, a conserved osteoblast progenitor enhancer was identified within a 656-bp sequence located 156.3 kilobases 3′ from the promoter. The identification of this enhancer should permit further investigation of upstream regulatory mechanisms that control Bmp2 transcription during osteoblast differentiation and are relevant to further studies of Bmp2 as a candidate risk factor gene for osteoporosis.

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