scholarly journals Structure and tissue-specific expression of the Drosophila melanogaster organellar-type Ca2+-ATPase gene

1995 ◽  
Vol 310 (3) ◽  
pp. 757-763 ◽  
Author(s):  
A Magyar ◽  
E Bakos ◽  
A Váradi
Keyword(s):  
Drosophila Melanogaster ◽  
Transcription Initiation ◽  
Initiation Site ◽  
Drosophila Genome ◽  
Coding Region ◽  
Specific Expression ◽  
S1 Nuclease ◽  
Protein Coding ◽  
Atpase Gene ◽  
High Level

A 14 kb genomic clone covering the organellar-type Ca(2+)-ATPase gene of Drosophila melanogaster has been isolated and characterized. The sequence of a 7132 bp region extending from 1.1 kb 5′ upstream of the initiation ATG codon over the polyadenylation signal at the 3′ end has been determined. The gene consists of nine exons including one with an exceptional size of 2172 bp representing 72% of the protein coding region. Introns are relatively small (< 100 bp) except for the 3′ intron which has a size of 2239 bp, an exceptionally large size among Drosophila introns. Five of the introns are in the same positions in Drosophila, Artemia and rabbit SERCA1 Ca(2+)-ATPase genes. There is only one organellar-type Ca(2+)-ATPase gene in the Drosophila genome, as was shown by Southern-blot analysis [Váradi, Gilmore-Hebert and Benz (1989) FEBS Lett. 258, 203-207] and by chromosomal localization [Magyar and Váradi (1990) Biochem. Biophys. Res. Commun. 173, 872-877]. Primer extension and S1-nuclease assays revealed a potential transcription initiation site 876 bp upstream of the translation initiation ATG with a TATA-box 23 bp upstream of this site. Analysis of the 5′ region of the Drosophila organellar-type Ca(2+)-ATPase gene suggests the presence of potential recognition sequences of various muscle-specific transcription factors and shows a region with remarkable similarity to that in the rabbit SERCA2 gene. The tissue distribution of expression of the organellar-type Ca(2+)-ATPase gene has been studied by in situ RNA-RNA hybridization on microscopic sections. A low mRNA abundance can be detected in each tissue of adult flies, suggesting a housekeeping function for the gene. On the other hand a pronounced tissue specificity of expression has also been found as the organellar-type Ca(2+)-ATPase is expressed at a very high level in cell bodies of the central nervous system and in various muscles.

scholarly journals Structure and tissue-specific expression of the human metallothionein IB gene.

1986 ◽  
Vol 6 (6) ◽  
pp. 2149-2157 ◽  
Author(s):  
A Heguy ◽  
A West ◽  
R I Richards ◽  
M Karin
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Transcription Initiation ◽  
Cell Types ◽  
Initiation Site ◽  
Predicted Amino Acid Sequence ◽  
Functional Protein ◽  
Specific Expression ◽  
Cis Acting ◽  
High Level

The human metallothionein (MT) IB gene (hMT-IB) is located in a region of human DNA containing at least four tandemly arranged MT genes. As deduced from its sequence, hMT-IB is likely to encode a functional protein. However, the predicted amino acid sequence differed from the hMT-I amino acid sequence in four positions. Most remarkable was the presence of an additional cysteine. Like other MT genes, hMT-IB has at least two copies of the metal-responsive element upstream from the transcription initiation site. These elements probably are responsible for the metal responsiveness of the hMT-IB promoter, leading to inducible expression of fused heterologous genes. Unlike the hMT-IIA and hMT-IA genes described previously, which are expressed in many different cell types, a high level of expression of the endogenous hMT-IB gene could be detected only in human hepatoma and renal carcinoma cell lines. Therefore, this is the first MT gene described which exhibits tissue specificity of expression. This specificity is controlled by a cis-acting mechanism involving methylation, since incubation of nonexpressing cells with an inhibitor of DNA methylation led to activation of the hMT-IB gene. In support of this notion, we found that the 5' flanking region of the hMT-IB gene was highly methylated in HeLa cells, a nonexpressing cell type, but it was not methylated in a hepatoma (expressing) cell line.

Structure and tissue-specific expression of the human metallothionein IB gene

1986 ◽  
Vol 6 (6) ◽  
pp. 2149-2157
Author(s):  
A Heguy ◽  
A West ◽  
R I Richards ◽  
M Karin
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Transcription Initiation ◽  
Cell Types ◽  
Initiation Site ◽  
Predicted Amino Acid Sequence ◽  
Functional Protein ◽  
Specific Expression ◽  
Cis Acting ◽  
High Level

The human metallothionein (MT) IB gene (hMT-IB) is located in a region of human DNA containing at least four tandemly arranged MT genes. As deduced from its sequence, hMT-IB is likely to encode a functional protein. However, the predicted amino acid sequence differed from the hMT-I amino acid sequence in four positions. Most remarkable was the presence of an additional cysteine. Like other MT genes, hMT-IB has at least two copies of the metal-responsive element upstream from the transcription initiation site. These elements probably are responsible for the metal responsiveness of the hMT-IB promoter, leading to inducible expression of fused heterologous genes. Unlike the hMT-IIA and hMT-IA genes described previously, which are expressed in many different cell types, a high level of expression of the endogenous hMT-IB gene could be detected only in human hepatoma and renal carcinoma cell lines. Therefore, this is the first MT gene described which exhibits tissue specificity of expression. This specificity is controlled by a cis-acting mechanism involving methylation, since incubation of nonexpressing cells with an inhibitor of DNA methylation led to activation of the hMT-IB gene. In support of this notion, we found that the 5' flanking region of the hMT-IB gene was highly methylated in HeLa cells, a nonexpressing cell type, but it was not methylated in a hepatoma (expressing) cell line.

Transcriptional and posttranscriptional regulation of maize mitochondrial gene expression

1991 ◽  
Vol 11 (1) ◽  
pp. 533-543
Author(s):  
R M Mulligan ◽  
P Leon ◽  
V Walbot
Keyword(s):  
Dna Sequences ◽  
Transcription Initiation ◽  
Posttranscriptional Regulation ◽  
Rank Order ◽  
Mitochondrial Gene ◽  
Initiation Site ◽  
Gene Copy ◽  
Promoter Strength ◽  
Protein Coding

Lysed maize mitochondria synthesize RNA in the presence of radioactive nucleoside triphosphates, and this assay was utilized to compare the rates of transcription of seven genes. The rates of incorporation varied over a 14-fold range, with the following rank order: 18S rRNA greater than 26S rRNA greater than atp1 greater than atp6 greater than atp9 greater than cob greater than cox3. The products of run-on transcription hybridized specifically to known transcribed regions and selectively to the antisense DNA strand; thus, the isolated run-on transcription system appears to be an accurate representation of endogenous transcription. Although there were small differences in gene copy abundance, these differences cannot account for the differences in apparent transcription rates; we conclude that promoter strength is the main determinant. Among the protein coding genes, incorporation was greatest for atp1. The most active transcription initiation site of this gene was characterized by hybridization with in vitro-capped RNA and by primer extension analyses. The DNA sequences at this and other transcription initiation sites that we have previously mapped were analyzed with respect to the apparent promoter strengths. We propose that two short sequence elements just upstream of initiation sites form at least a portion of the sequence requirements for a maize mitochondrial promoter. In addition to modulation at the level of transcription, steady-state abundance of protein-coding mRNAs varied over a 20-fold range and did not correlate with transcriptional activity. These observations suggest that posttranscriptional processes are important in the modulation of mRNA abundance.

Signals for transcription initiation and termination in the Saccharomyces cerevisiae plasmid 2 micron circle

1985 ◽  
Vol 5 (10) ◽  
pp. 2770-2780
Author(s):  
A Sutton ◽  
J R Broach
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Unknown Function ◽  
Transcription Initiation ◽  
Coding Region ◽  
S1 Nuclease ◽  
Plasmid Maintenance ◽  
Transcriptional Regulatory ◽  
Polyadenylation Sites ◽  
Plasmid Genes ◽  
Extension Analysis

By S1 nuclease protection experiments and primer extension analysis, we determined precisely the cap and polyadenylation sites of transcripts from the four genes of the yeast 2 micron circle plasmid, as well as those of other plasmid transcripts of unknown function. In addition, we used deletion analysis to identify sequences necessary for polyadenylation in plasmid transcripts. Our results indicate that plasmid genes constitute independent transcription units and that plasmid mRNAs are not derived by extensive processing of precursor transcripts. In addition, we found that the D coding region of 2 micron circle is precisely encompassed by a polyadenylated transcript, suggesting that this coding region constitutes a functional plasmid gene. Our identification of the position of plasmid polyadenylation sites and of sequences necessary for polyadenylation provides support for a tripartite signal for polyadenylation as proposed by Zaret and Sherman (K.S. Zaret and F. Sherman, Cell 28:563-573, 1982). Finally, these data highlight salient features of the transcriptional regulatory circuitry that underlies the control of plasmid maintenance in the cell.

Identification of upstream and intragenic regulatory elements that confer cell-type-restricted and differentiation-specific expression on the muscle creatine kinase gene

1988 ◽  
Vol 8 (7) ◽  
pp. 2896-2909 ◽  
Author(s):  
E A Sternberg ◽  
G Spizz ◽  
W M Perry ◽  
D Vizard ◽  
T Weil ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Regulatory Element ◽  
Simian Virus 40 ◽  
Muscle Cells ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Simian Virus ◽  
Cell Type ◽  
Specific Expression ◽  
Developing Muscle

Terminal differentiation of skeletal myoblasts is accompanied by induction of a series of tissue-specific gene products, which includes the muscle isoenzyme of creatine kinase (MCK). To begin to define the sequences and signals involved in MCK regulation in developing muscle cells, the mouse MCK gene has been isolated. Sequence analysis of 4,147 bases of DNA surrounding the transcription initiation site revealed several interesting structural features, some of which are common to other muscle-specific genes and to cellular and viral enhancers. To test for sequences required for regulated expression, a region upstream of the MCK gene from -4800 to +1 base pairs, relative to the transcription initiation site, was linked to the coding sequences of the bacterial chloramphenicol acetyltransferase (CAT) gene. Introduction of this MCK-CAT fusion gene into C2 muscle cells resulted in high-level expression of CAT activity in differentiated myotubes and no detectable expression in proliferating undifferentiated myoblasts or in nonmyogenic cell lines. Deletion mutagenesis of sequences between -4800 and the transcription start site showed that the region between -1351 and -1050 was sufficient to confer cell type-specific and developmentally regulated expression on the MCK promoter. This upstream regulatory element functioned independently of position, orientation, or distance from the promoter and therefore exhibited the properties of a classical enhancer. This upstream enhancer also was able to confer muscle-specific regulation on the simian virus 40 promoter, although it exhibited a 3- to 5-fold preference for its own promoter. In contrast to the cell type- and differentiation-specific expression of the upstream enhancer, the MCK promoter was able to function in myoblasts and myotubes and in nonmyogenic cell lines when combined with the simian virus 40 enhancer. An additional positive regulatory element was identified within the first intron of the MCK gene. Like the upstream enhancer, this intragenic element functioned independently of position, orientation, and distance with respect to the MCK promoter and was active in differentiated myotubes but not in myoblasts. These results demonstrate that expression of the MCK gene in developing muscle cells is controlled by complex interactions among multiple upstream and intragenic regulatory elements that are functional only in the appropriate cellular context.

Identification of the principal promoter sequence of the c-H-ras transforming oncogene: deletion analysis of the 5'-flanking region by focus formation assay

1987 ◽  
Vol 7 (8) ◽  
pp. 2933-2940
Author(s):  
H Honkawa ◽  
W Masahashi ◽  
S Hashimoto ◽  
T Hashimoto-Gotoh
Keyword(s):  
Dna Sequences ◽  
Promoter Sequence ◽  
Ras Oncogene ◽  
Coding Region ◽  
Focus Formation ◽  
S1 Nuclease ◽  
Protein Coding ◽  
Consensus Sequences ◽  
Flanking Region ◽  
Virus C

A number of deletion mutants were isolated, including 5', 3', and internal deletions in the 5'-flanking region of the human cellular oncogene related to the Harvey sarcoma virus (c-H-ras), and their transforming activities were examined in NIH 3T3 cells. DNA sequences which could not be detected without losing transforming activity were localized to a relatively short stretch upstream of the region which showed homology to the 5'-flanking region of v-H-ras oncogene. S1 nuclease analysis indicated that there were two clusters of mRNA start sites at positions that were about 1,371 and 1,298 base pairs upstream of the first coding ATG. The minimum region required for promoter function was estimated to be a 51-base-pair-long (or less) DNA segment. The promoter was GC rich (78%) and did not contain the consensus sequences that are usually observed in PolII-directed promoters but contained a GC box within which one of the mRNA start sites was included. In addition, two sets of positive and negative elements seemed to be located between the promoter and the protein-coding region, which appeared to influence positively and negatively, respectively, the efficiency of transformation with the c-H-ras oncogene.

scholarly journals Signals for transcription initiation and termination in the Saccharomyces cerevisiae plasmid 2 micron circle.

1985 ◽  
Vol 5 (10) ◽  
pp. 2770-2780 ◽  
Author(s):  
A Sutton ◽  
J R Broach
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Unknown Function ◽  
Transcription Initiation ◽  
Coding Region ◽  
S1 Nuclease ◽  
Plasmid Maintenance ◽  
Transcriptional Regulatory ◽  
Polyadenylation Sites ◽  
Plasmid Genes ◽  
Extension Analysis

By S1 nuclease protection experiments and primer extension analysis, we determined precisely the cap and polyadenylation sites of transcripts from the four genes of the yeast 2 micron circle plasmid, as well as those of other plasmid transcripts of unknown function. In addition, we used deletion analysis to identify sequences necessary for polyadenylation in plasmid transcripts. Our results indicate that plasmid genes constitute independent transcription units and that plasmid mRNAs are not derived by extensive processing of precursor transcripts. In addition, we found that the D coding region of 2 micron circle is precisely encompassed by a polyadenylated transcript, suggesting that this coding region constitutes a functional plasmid gene. Our identification of the position of plasmid polyadenylation sites and of sequences necessary for polyadenylation provides support for a tripartite signal for polyadenylation as proposed by Zaret and Sherman (K.S. Zaret and F. Sherman, Cell 28:563-573, 1982). Finally, these data highlight salient features of the transcriptional regulatory circuitry that underlies the control of plasmid maintenance in the cell.

scholarly journals Hormonally mediated negative regulation of human pro-opiomelanocortin gene expression after transfection into mouse L cells.

1985 ◽  
Vol 5 (9) ◽  
pp. 2443-2453 ◽  
Author(s):  
A Israel ◽  
S N Cohen
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
Negative Regulation ◽  
Coding Region ◽  
Transient Expression Assay ◽  
S1 Nuclease ◽  
Protein Coding ◽  
Pomc Gene ◽  
Simplex Virus ◽  
Nuclease Mapping

We report results indicating that expression and hormonally controlled negative regulation of the human pro-opiomelanocortin (POMC) gene in mouse fibroblasts can be accomplished by the placement nearby of a simian virus 40 enhancer sequence. Expression resulting from correctly initiated transcription required the enhancer in cis both in cells stably transfected with the POMC gene and in a transient expression assay with constructs that fused that POMC promoter region to the protein-coding region of the herpes simplex virus thymidine kinase (TK) gene. Negative regulation of POMC transcription by glucocorticoids was demonstrated in transiently infected cells by assaying for TK activity encoded by the POMC-TK fusion constructs and by quantitative S1 nuclease mapping. The sequences responsible for such regulation were shown to be contained within a DNA segment that extends 670 base pairs upstream from the cap site for POMC mRNA.

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 In vitro synthesis and processing of a maize chloroplast transcript encoded by the ribulose 1,5-bisphosphate carboxylase large subunit gene.

1985 ◽  
Vol 5 (10) ◽  
pp. 2733-2745 ◽  
Author(s):  
L Hanley-Bowdoin ◽  
E M Orozco ◽  
N H Chua
Keyword(s):  
Transcription Initiation ◽  
Large Subunit ◽  
Plastid Dna ◽  
Subunit Gene ◽  
Coding Region ◽  
Protein Coding ◽  
Bisphosphate Carboxylase ◽  
Transcription In Vitro ◽  
Maize Chloroplast

The large subunit gene (rbcL) of ribulose 1,5-bisphosphate carboxylase was transcribed in vitro by using maize and pea chloroplast extracts and a cloned plastid DNA template containing 172 base pairs (bp) of the maize rbcL protein-coding region and 791 bp of upstream sequences. Three major in vitro RNA species were synthesized which correspond to in vivo maize rbcL RNAs with 5' termini positioned 300, 100 to 105, and 63 nucleotides upstream of the protein-coding region. A deletion of 109 bp, including the "-300" 5' end (the 5' end at position -300), depressed all rbcL transcription in vitro. A plasmid DNA containing this 109-bp fragment was sufficient to direct correct transcription initiation in vitro. A cloned template, containing 191 bp of plastid DNA which includes the -105 and -63 rbcL termini, did not support transcription in vitro. Exogenously added -300 RNA could be converted to the -63 transcript by maize chloroplast extract. These results established that the -300 RNA is the primary maize rbcL transcript, the -63 RNA is a processed form of the -300 transcript, and synthesis of the -105 RNA is dependent on the -300 region. The promoter for the maize rbcL gene is located within the 109 bp flanking the -300 site. Mutagenesis of the 109-bp chloroplast sequence 11 bp upstream of the -300 transcription initiation site reduced rbcL promoter activity in vitro.

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