scholarly journals DNA-mediated transformation in Dictyostelium discoideum: regulated expression of an actin gene fusion.

1984 ◽  
Vol 4 (12) ◽  
pp. 2890-2898 ◽  
Author(s):  
W Nellen ◽  
C Silan ◽  
R A Firtel
Keyword(s):  
Dictyostelium Discoideum ◽  
Gene Fusion ◽  
Regulatory Sequences ◽  
Control Of Gene Expression ◽  
Cis Acting ◽  
Regulated Expression ◽  
Promoter Gene ◽  
Flanking Region ◽  
Average Copy ◽  
Flanking Regions

We have constructed a new vector for transformation that carries a fusion of the Dictyostelium discoideum actin 6 promoter gene and 5' flanking region with the bacterial Tn5 NeoR (KanR) gene which can confer resistance to the aminoglycoside G418. This vector can be used to transform D. discoideum cells. Approximately 200 to 2,000 transformants were obtained per 10(7) cells. Transformed cell populations carried vector DNA at an average copy number of ca. 5 per cell, and the DNA was stable for more than 40 generations in the absence of selection. We have shown that transformed cells synthesize functional kanamycin phosphotransferase and that initiation of transcription of the actin 6-NeoR gene fusion occurs at the actin 6 cap site. Moreover, analysis of RNA isolated from transformed and untransformed cells during vegetative growth and during development indicated that the actin 6-NeoR gene fusion was regulated in parallel with the endogenous actin 6 gene, suggesting that the upstream flanking regions of actin 6 contain the cis-acting regulatory sequences sufficient for differential regulation of this gene during D. discoideum development. These results indicate that this system can be used to examine control of gene expression during D. discoideum development.

DNA-mediated transformation in Dictyostelium discoideum: regulated expression of an actin gene fusion

1984 ◽  
Vol 4 (12) ◽  
pp. 2890-2898
Author(s):  
W Nellen ◽  
C Silan ◽  
R A Firtel
Keyword(s):  
Dictyostelium Discoideum ◽  
Gene Fusion ◽  
Regulatory Sequences ◽  
Control Of Gene Expression ◽  
Cis Acting ◽  
Regulated Expression ◽  
Promoter Gene ◽  
Flanking Region ◽  
Average Copy ◽  
Flanking Regions

We have constructed a new vector for transformation that carries a fusion of the Dictyostelium discoideum actin 6 promoter gene and 5' flanking region with the bacterial Tn5 NeoR (KanR) gene which can confer resistance to the aminoglycoside G418. This vector can be used to transform D. discoideum cells. Approximately 200 to 2,000 transformants were obtained per 10(7) cells. Transformed cell populations carried vector DNA at an average copy number of ca. 5 per cell, and the DNA was stable for more than 40 generations in the absence of selection. We have shown that transformed cells synthesize functional kanamycin phosphotransferase and that initiation of transcription of the actin 6-NeoR gene fusion occurs at the actin 6 cap site. Moreover, analysis of RNA isolated from transformed and untransformed cells during vegetative growth and during development indicated that the actin 6-NeoR gene fusion was regulated in parallel with the endogenous actin 6 gene, suggesting that the upstream flanking regions of actin 6 contain the cis-acting regulatory sequences sufficient for differential regulation of this gene during D. discoideum development. These results indicate that this system can be used to examine control of gene expression during D. discoideum development.

Regulated expression of a chimeric histone gene introduced into mouse fibroblasts

1985 ◽  
Vol 5 (9) ◽  
pp. 2316-2324
Author(s):  
R B Alterman ◽  
C Sprecher ◽  
R Graves ◽  
W F Marzluff ◽  
A I Skoultchi
Keyword(s):  
Histone Gene ◽  
Chimeric Gene ◽  
Regulatory Sequences ◽  
Mrna Levels ◽  
Histone Genes ◽  
Mouse Fibroblasts ◽  
Cis Acting ◽  
Regulated Expression ◽  
Chimeric Rna ◽  
The Stability

The regulated expression of a mouse histone gene was studied by DNA-mediated gene transfer. A chimeric H3 histone gene was constructed by fusing the 5' and 3' portions of two different mouse H3 histone genes. Transfection of the chimeric gene into mouse fibroblasts resulted in the production of chimeric mRNA at levels nearly equal to that of the total endogenous H3 histone mRNAs. Most chimeric RNA transcripts had correct 5' and 3' termini, and the chimeric mRNA was translated into an H3.1 protein that accumulated in the nucleus of the transfected cells. Expression of the chimeric gene was studied under several conditions in which the rate of transcription and the stability of endogenous H3 transcripts change. Chimeric mRNA levels were regulated in parallel with endogenous H3 mRNAs, suggesting that cis-acting regulatory sequences lie within or near individual histone genes. In addition to correctly initiated and terminated chimeric mRNA, we also detected a novel H3 transcript containing an additional 250 bases at the 3' end. Surprisingly, the longer transcript is polyadenylated and accumulates in the cytoplasm.

scholarly journals Evolution of lineage-specific functions in ancient cis -regulatory modules

Open Biology ◽  
2015 ◽  
Vol 5 (11) ◽  
pp. 150079 ◽  
Author(s):  
Stefan Pauls ◽  
Debbie K. Goode ◽  
Libero Petrone ◽  
Paola Oliveri ◽  
Greg Elgar
Keyword(s):  
Gene Expression ◽  
Morphological Evolution ◽  
Regulatory Sequences ◽  
Enhancer Activity ◽  
Regulatory Modules ◽  
History Of ◽  
Flanking Region ◽  
Regulatory Functions ◽  
Conserved Core ◽  
Flanking Regions

Morphological evolution is driven both by coding sequence variation and by changes in regulatory sequences. However, how cis -regulatory modules (CRMs) evolve to generate entirely novel expression domains is largely unknown. Here, we reconstruct the evolutionary history of a lens enhancer located within a CRM that not only predates the lens, a vertebrate innovation, but bilaterian animals in general. Alignments of orthologous sequences from different deuterostomes sub-divide the CRM into a deeply conserved core and a more divergent flanking region. We demonstrate that all deuterostome flanking regions, including invertebrate sequences, activate gene expression in the zebrafish lens through the same ancient cluster of activator sites. However, levels of gene expression vary between species due to the presence of repressor motifs in flanking region and core. These repressor motifs are responsible for the relatively weak enhancer activity of tetrapod flanking regions. Ray-finned fish, however, have gained two additional lineage-specific activator motifs which in combination with the ancient cluster of activators and the core constitute a potent lens enhancer. The exploitation and modification of existing regulatory potential in flanking regions but not in the highly conserved core might represent a more general model for the emergence of novel regulatory functions in complex CRMs.

Extrachromosomal replication of shuttle vectors in Dictyostelium discoideum

1985 ◽  
Vol 5 (11) ◽  
pp. 3241-3250
Author(s):  
R A Firtel ◽  
C Silan ◽  
T E Ward ◽  
P Howard ◽  
B A Metz ◽  
...  
Keyword(s):  
Dictyostelium Discoideum ◽  
Copy Number ◽  
Gene Fusion ◽  
Nuclear Dna ◽  
Wild Type ◽  
Shuttle Vectors ◽  
Transformation Vector ◽  
G418 Resistance ◽  
Average Copy ◽  
Derivatives Of

We cloned a 12.3-kilobase (kb) endogenous plasmid, Ddp1, found in several wild-type and laboratory strains of Dictyostelium discoideum into pBR322. The cloned plasmids have been used to cotransform D. discoideum cells with B10S, a transformation vector carrying a gene fusion conferring resistance to G418. Whereas B10S DNA alone appears to integrate in a tandem array, the cloned Ddp1 plasmids replicate extrachromosomally and are stably maintained in the absence of selection with an average copy number of 50 to 100 copies per cell. The Ddp1-derived plasmids can be directly recovered by transforming Escherichia coli with bulk nuclear DNA from these cells. Preliminary deletion analysis indicates that not all regions of Ddp1 are necessary for stable replication in D. discoideum. Several recombinant vectors which replicate extrachromosomally in D. discoideum were also isolated. One contains the Act6-neor gene fusion from B10S recombined into one of the cloned derivatives of Ddp1 and can be used to directly transform D. discoideum amoebae, selecting for G418 resistance. Another recombinant is only 5.6 kb and resulted from a deletion of a 16.6-kb cloned Ddp1 hybrid plasmid. An analysis of the vector DNAs present in clones derived from single D. discoideum transformants is also described.

scholarly journals Flanking Regulatory Sequences of theTetrahymena R Deletion Element Determine the Boundaries of DNA Rearrangement

1999 ◽  
Vol 19 (8) ◽  
pp. 5631-5641 ◽  
Author(s):  
Douglas L. Chalker ◽  
Antonietta La Terza ◽  
Allison Wilson ◽  
Christopher D. Kroenke ◽  
Meng-Chao Yao
Keyword(s):  
Sequence Similarity ◽  
Common Mechanism ◽  
Complex Nature ◽  
Regulatory Sequence ◽  
Regulatory Sequences ◽  
Flanking Region ◽  
The Many ◽  
The Right ◽  
Flanking Regions

ABSTRACT In the ciliate Tetrahymena thermophila, thousands of DNA segments of variable size are eliminated from the developing somatic macronucleus by specific DNA rearrangements. It is unclear whether rearrangement of the many different DNA elements occurs via a single mechanism or via multiple rearrangement systems. In this study, we characterized in vivo cis-acting sequences required for the rearrangement of the 1.1-kbp R deletion element. We found that rearrangement requires specific sequences flanking each side of the deletion element. The required sequences on the left side appear to span roughly a 70-bp region that is located at least 30 bp from the rearrangement boundary. When we moved the location of the leftcis-acting sequences closer to the eliminated region, we observed a rightward shift of the rearrangement boundary such that the newly formed deletion junction retained its original distance from this flanking region. Likewise, when we moved the flanking region as much as 500 bp away from the deletion element, the rearrangement boundary shifted to remain in relative juxtaposition. Clusters of base substitutions made throughout this critical flanking region did not affect rearrangement efficiency or accuracy, which suggests a complex nature for this regulatory sequence. We also found that the right flanking region effectively replaced the essential sequences identified on the left side, and thus, the two flanking regions contain sequences of analogous function despite the lack of obvious sequence identity. These data taken together indicate that the R-element flanking regions contain sequences that position the rearrangement boundaries from a short distance away. Previously, a 10-bp polypurine tract flanking the M-deletion element was demonstrated to act from a distance to determine its rearrangement boundaries. No apparent sequence similarity exists between the M and R elements. The functional similarity between these different cis-acting sequences of the two elements is firm support for a common mechanism controlling Tetrahymenarearrangement.

scholarly journals Regulated expression of a chimeric histone gene introduced into mouse fibroblasts.

1985 ◽  
Vol 5 (9) ◽  
pp. 2316-2324 ◽  
Author(s):  
R B Alterman ◽  
C Sprecher ◽  
R Graves ◽  
W F Marzluff ◽  
A I Skoultchi
Keyword(s):  
Histone Gene ◽  
Chimeric Gene ◽  
Regulatory Sequences ◽  
Mrna Levels ◽  
Histone Genes ◽  
Mouse Fibroblasts ◽  
Cis Acting ◽  
Regulated Expression ◽  
Chimeric Rna ◽  
The Stability

The regulated expression of a mouse histone gene was studied by DNA-mediated gene transfer. A chimeric H3 histone gene was constructed by fusing the 5' and 3' portions of two different mouse H3 histone genes. Transfection of the chimeric gene into mouse fibroblasts resulted in the production of chimeric mRNA at levels nearly equal to that of the total endogenous H3 histone mRNAs. Most chimeric RNA transcripts had correct 5' and 3' termini, and the chimeric mRNA was translated into an H3.1 protein that accumulated in the nucleus of the transfected cells. Expression of the chimeric gene was studied under several conditions in which the rate of transcription and the stability of endogenous H3 transcripts change. Chimeric mRNA levels were regulated in parallel with endogenous H3 mRNAs, suggesting that cis-acting regulatory sequences lie within or near individual histone genes. In addition to correctly initiated and terminated chimeric mRNA, we also detected a novel H3 transcript containing an additional 250 bases at the 3' end. Surprisingly, the longer transcript is polyadenylated and accumulates in the cytoplasm.

scholarly journals A GBF-binding site and a novel AT element define the minimal sequences sufficient to direct prespore-specific expression in Dictyostelium discoideum.

1994 ◽  
Vol 14 (9) ◽  
pp. 5840-5849 ◽  
Author(s):  
J A Powell-Coffman ◽  
G R Schnitzler ◽  
R A Firtel
Keyword(s):  
Dictyostelium Discoideum ◽  
Molecular Mechanisms ◽  
Gene Activation ◽  
Point Mutations ◽  
Specific Gene ◽  
Regulatory Sequences ◽  
Specific Expression ◽  
Cis Acting ◽  
Minimal Sequences

In order to better understand the molecular mechanisms of cellular differentiation in Dictyostelium discoideum, we have identified the minimum regulatory sequences of the prespore-specific gene SP60/cotC that are sufficient to confer cell-type-specific expression on a heterologous promoter. This region includes at least two essential cis-acting elements: a novel AT-rich element (or elements) and CAE3. The essential function of the AT element is confirmed through point mutations that decrease expression below the level of detection. CAE3 is one of three CA-rich elements (CAEs) required for the induction of SP60/cotC during development or in response to extracellular cyclic AMP. The CAEs have differential affinities for a specific developmentally induced nuclear activity (CAE1 > CAE2 >> CAE3). Here, we identify this activity as G-box-binding factor (GBF) and show that in vitro-transcribed and -translated GBF binds all three SP60/cotC CAEs in a sequence-specific manner. Previous studies have suggested that GBF mediates the induction of some prestalk genes, and these results demonstrate that it also has a specific role in prespore gene activation.

scholarly journals Spatially regulated expression of retrovirus-like transposons during Drosophila melanogaster embryogenesis

1994 ◽  
Vol 64 (3) ◽  
pp. 167-181 ◽  
Author(s):  
Dali Ding ◽  
Howard D. Lipshitz
Keyword(s):  
Drosophila Melanogaster ◽  
Expression Patterns ◽  
Regulatory Elements ◽  
Host Gene ◽  
Regulatory Sequences ◽  
Wild Type ◽  
Control Of Gene Expression ◽  
Spatial Expression ◽  
Regulated Expression ◽  
Type Strains

SummaryOver twenty distinct families of long terminal direct repeat (LTR)-containing retrotransposons have been identified in Drosophila melanogaster. While there have been extensive analyses of retrotransposon transcription in cultured cells, there have been few studies of the spatial expression of retrotransposons during normal development. Here we report a detailed analysis of the spatial expression patterns of fifteen families of retrotransposons during Drosophila melanogaster embryogenesis (17.6, 297, 412, 1731, 3S18, blood, copia, gypsy, HMS Beagle, Kermit/flea, mdg1, mdg3, opus, roo/B104 and springer). In each case, analyses were carried out in from two to four wild-type strains. Since the chromosomal insertion sites of any particular family of retrotransposons vary widely among wild-type strains, a spatial expression pattern that is conserved among strains is likely to have been generated through interaction of host transcription factors with cis-regulatory elements resident in the retrotransposons themselves. All fifteen families of retrotransposons showed conserved patterns of spatially and temporally regulated expression during embryogenesis. These results suggest that all families of retrotransposons carry cis-acting elements that control their spatial and temporal expression patterns. Thus, transposition of a retrotransposon into or near a particular host gene-possibly followed by an excision event leaving behind the retrotransposon's cis-regulatory sequences-might impose novel developmental control on such a host gene. Such a mechanism would serve to confer evolutionarily significant alterations in the spatio-temporal control of gene expression.

scholarly journals A GBF-binding site and a novel AT element define the minimal sequences sufficient to direct prespore-specific expression in Dictyostelium discoideum

1994 ◽  
Vol 14 (9) ◽  
pp. 5840-5849
Author(s):  
J A Powell-Coffman ◽  
G R Schnitzler ◽  
R A Firtel
Keyword(s):  
Dictyostelium Discoideum ◽  
Molecular Mechanisms ◽  
Gene Activation ◽  
Point Mutations ◽  
Specific Gene ◽  
Regulatory Sequences ◽  
Specific Expression ◽  
Cis Acting ◽  
Minimal Sequences

In order to better understand the molecular mechanisms of cellular differentiation in Dictyostelium discoideum, we have identified the minimum regulatory sequences of the prespore-specific gene SP60/cotC that are sufficient to confer cell-type-specific expression on a heterologous promoter. This region includes at least two essential cis-acting elements: a novel AT-rich element (or elements) and CAE3. The essential function of the AT element is confirmed through point mutations that decrease expression below the level of detection. CAE3 is one of three CA-rich elements (CAEs) required for the induction of SP60/cotC during development or in response to extracellular cyclic AMP. The CAEs have differential affinities for a specific developmentally induced nuclear activity (CAE1 > CAE2 >> CAE3). Here, we identify this activity as G-box-binding factor (GBF) and show that in vitro-transcribed and -translated GBF binds all three SP60/cotC CAEs in a sequence-specific manner. Previous studies have suggested that GBF mediates the induction of some prestalk genes, and these results demonstrate that it also has a specific role in prespore gene activation.

scholarly journals Extrachromosomal replication of shuttle vectors in Dictyostelium discoideum.

1985 ◽  
Vol 5 (11) ◽  
pp. 3241-3250 ◽  
Author(s):  
R A Firtel ◽  
C Silan ◽  
T E Ward ◽  
P Howard ◽  
B A Metz ◽  
...  
Keyword(s):  
Dictyostelium Discoideum ◽  
Copy Number ◽  
Gene Fusion ◽  
Nuclear Dna ◽  
Wild Type ◽  
Shuttle Vectors ◽  
Transformation Vector ◽  
G418 Resistance ◽  
Average Copy ◽  
Derivatives Of

We cloned a 12.3-kilobase (kb) endogenous plasmid, Ddp1, found in several wild-type and laboratory strains of Dictyostelium discoideum into pBR322. The cloned plasmids have been used to cotransform D. discoideum cells with B10S, a transformation vector carrying a gene fusion conferring resistance to G418. Whereas B10S DNA alone appears to integrate in a tandem array, the cloned Ddp1 plasmids replicate extrachromosomally and are stably maintained in the absence of selection with an average copy number of 50 to 100 copies per cell. The Ddp1-derived plasmids can be directly recovered by transforming Escherichia coli with bulk nuclear DNA from these cells. Preliminary deletion analysis indicates that not all regions of Ddp1 are necessary for stable replication in D. discoideum. Several recombinant vectors which replicate extrachromosomally in D. discoideum were also isolated. One contains the Act6-neor gene fusion from B10S recombined into one of the cloned derivatives of Ddp1 and can be used to directly transform D. discoideum amoebae, selecting for G418 resistance. Another recombinant is only 5.6 kb and resulted from a deletion of a 16.6-kb cloned Ddp1 hybrid plasmid. An analysis of the vector DNAs present in clones derived from single D. discoideum transformants is also described.

Sign in / Sign up

Export Citation Format

Share Document