scholarly journals Identification of an Embryonic Cell-Specific Region within the Pineapple SERK1 Promoter

Genes ◽  
2019 ◽  
Vol 10 (11) ◽  
pp. 883 ◽  
Author(s):  
Luan ◽  
He ◽  
Xie ◽  
Chen ◽  
Mao ◽  
...  
Keyword(s):  
Somatic Embryogenesis ◽  
Transcription Initiation ◽  
Promoter Activity ◽  
Specific Activity ◽  
Marker Gene ◽  
Immature Embryo ◽  
Embryonic Cell ◽  
Regulatory Sequence ◽  
Regulatory Sequences ◽  
Embryonic Callus

Plant tissue culture methods, such as somatic embryogenesis, are attractive alternatives to traditional breeding methods for plant propagation. However, they often suffer from limited efficiency. Somatic embryogenesis receptor kinase (SERK)1 is a marker gene of early somatic embryogenesis in several plants, including pineapple. It can be selectively induced and promotes a key step in somatic embryogenesis. We investigated the embryonic cell-specific transcriptional regulation of AcSERK1 by constructing a series of vectors carrying the GUS（Beta-glucuronidase） reporter gene under the control of different candidate cis-regulatory sequences. These vectors were transfected into both embryonic and non-embryonic callus, and three immature embryo stages and the embryonic-specific activity of the promoter fragments was analyzed. We found that the activity of the regulatory sequence of AcSERK1 lacking −983 nt ~−880 nt, which included the transcription initiation site, was significantly reduced in the embryonic callus of pineapple, accompanied by the loss of embryonic cell-specific promoter activity. Thus, this fragment is an essential functional segment with highly specific promoter activity for embryonic cells, and it is active only from the early stages of somatic embryo development to the globular embryo stage. This study lays the foundation for identifying mechanisms that enhance the efficiency of somatic embryogenesis in pineapple and other plants.

Both upstream and intron sequence elements are required for elevated expression of the rat somatic cytochrome c gene in COS-1 cells

1988 ◽  
Vol 8 (1) ◽  
pp. 35-41
Author(s):  
M J Evans ◽  
R C Scarpulla
Keyword(s):  
Cytochrome C ◽  
Cell Lines ◽  
Transcription Initiation ◽  
Promoter Activity ◽  
Physiological Role ◽  
Regulatory Elements ◽  
T Antigen ◽  
Regulatory Sequences ◽  
Region I ◽  
Region Ii

To investigate the transcriptional control of nuclear-encoded respiratory genes in mammals, we have performed a deletional analysis of cis-acting regulatory sequences in the rat somatic cytochrome c gene. Three major regions are required for maximal expression of the transfected gene in kidney cell lines CV-1 and COS-1. One of these, region III (+71 to +115 from the transcription initiation site), is an unusual intragenic controlling element found in the 5' end of the first intron, while the other two, region I (-191 to -165) and region II (-139 to -84), define the upstream promoter. Region II contains two consensus CCAAT boxes and mediates a constitutive level of expression in both cell lines. In contrast, regions I and III are both required for the increased promoter activity observed in COS-1 cells compared with promoter activity observed in CV-1 cells, and the regions function individually as competitors with the full promoter for trans-acting factors or complexes. Region III contains a perfect octanucleotide homology with region I in addition to a consensus Sp1-transcription-factor-binding site. Promoter stimulation in COS-1 cells can be duplicated in CV-1 cells by cotransfecting with a T-antigen-producing vector, but purified T antigen does not bind anywhere in the cytochrome c promoter. A control promoter from the mouse metallothionein I gene is similarly activated in T-antigen-producing cells only in the presence of zinc, which activates its upstream regulatory sites. We conclude that T antigen stimulates these cellular promoters through the activation or induction of cellular factors or complexes that mediate their effects through promoter-specific regulatory elements. Cytochrome c promoter regions activated in this system may play a physiological role in controlling gene expression.

Identification of a negative regulatory element that inhibits c-mos transcription in somatic cells

1992 ◽  
Vol 12 (5) ◽  
pp. 2029-2036
Author(s):  
S S Zinkel ◽  
S K Pal ◽  
J Szeberényi ◽  
G M Cooper
Keyword(s):  
Transcription Initiation ◽  
Regulatory Element ◽  
Somatic Cells ◽  
Regulatory Sequence ◽  
Regulatory Sequences ◽  
Flanking Sequence ◽  
3T3 Cells ◽  
Rnase Protection ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

We have used transient expression assays to identify a cis-acting region in the 5' flanking sequence of murine c-mos which, when deleted, allows expression from the c-mos promoter in NIH 3T3 cells. This negative regulatory sequence, located 400 to 500 nucleotides upstream of the c-mos ATG, also inhibited expression from a heterologous promoter. In addition to NIH 3T3 cells, the c-mos negative regulatory sequence was active in BALB/3T3 cells, PC12 rat pheochromocytoma cells, and A549 human lung carcinoma cells. Site-specific mutagenesis identified three possibly interacting regions that were involved in negative regulatory activity, located around -460, -425, and -405 with respect to the ATG. RNase protection analysis indicated that once the negative regulatory sequences were deleted, transcription in NIH 3T3 cells initiated from the same transcription initiation sites normally utilized in spermatocytes, approximately 280 nucleotides upstream of the ATG. Deletions beyond the spermatocyte promoter, however, allowed transcription initiation from progressively downstream c-mos sequences. Deletion or mutation of sequences surrounding the oocyte promoter at -53 also had little effect on expression of c-mos constructs in NIH 3T3 cells. Therefore, the major determinant of c-mos expression in NIH 3T3 cells was removal of the negative regulatory sequence rather than the utilization of a unique promoter. The c-mos negative regulatory sequences thus appear to play a significant role in tissue-specific c-mos expression by inhibiting transcription in somatic cells.

scholarly journals Both upstream and intron sequence elements are required for elevated expression of the rat somatic cytochrome c gene in COS-1 cells.

1988 ◽  
Vol 8 (1) ◽  
pp. 35-41 ◽  
Author(s):  
M J Evans ◽  
R C Scarpulla
Keyword(s):  
Cytochrome C ◽  
Cell Lines ◽  
Transcription Initiation ◽  
Promoter Activity ◽  
Physiological Role ◽  
Regulatory Elements ◽  
T Antigen ◽  
Regulatory Sequences ◽  
Region I ◽  
Region Ii

To investigate the transcriptional control of nuclear-encoded respiratory genes in mammals, we have performed a deletional analysis of cis-acting regulatory sequences in the rat somatic cytochrome c gene. Three major regions are required for maximal expression of the transfected gene in kidney cell lines CV-1 and COS-1. One of these, region III (+71 to +115 from the transcription initiation site), is an unusual intragenic controlling element found in the 5' end of the first intron, while the other two, region I (-191 to -165) and region II (-139 to -84), define the upstream promoter. Region II contains two consensus CCAAT boxes and mediates a constitutive level of expression in both cell lines. In contrast, regions I and III are both required for the increased promoter activity observed in COS-1 cells compared with promoter activity observed in CV-1 cells, and the regions function individually as competitors with the full promoter for trans-acting factors or complexes. Region III contains a perfect octanucleotide homology with region I in addition to a consensus Sp1-transcription-factor-binding site. Promoter stimulation in COS-1 cells can be duplicated in CV-1 cells by cotransfecting with a T-antigen-producing vector, but purified T antigen does not bind anywhere in the cytochrome c promoter. A control promoter from the mouse metallothionein I gene is similarly activated in T-antigen-producing cells only in the presence of zinc, which activates its upstream regulatory sites. We conclude that T antigen stimulates these cellular promoters through the activation or induction of cellular factors or complexes that mediate their effects through promoter-specific regulatory elements. Cytochrome c promoter regions activated in this system may play a physiological role in controlling gene expression.

scholarly journals Muscle-specific activity of the skeletal troponin I promoter requires interaction between upstream regulatory sequences and elements contained within the first transcribed exon.

1990 ◽  
Vol 10 (7) ◽  
pp. 3468-3482 ◽  
Author(s):  
W Nikovits ◽  
J H Mar ◽  
C P Ordahl
Keyword(s):  
Skeletal Muscle ◽  
Transcription Initiation ◽  
Troponin I ◽  
Specific Activity ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Deletion Analysis ◽  
Regulatory Sequences ◽  
Mrna Levels ◽  
Cat Gene

Expression of the skeletal troponin I (sTnI) gene is regulated transcriptionally in a muscle-specific fashion. We show here that the region of the sTnI gene between -160 and +61 (relative to the transcription initiation site) is able to direct expression of the bacterial chloramphenicol acetyltransferase (CAT) gene is muscle cultures at a level approximately 100 times higher than in fibroblast cultures. RNA analysis demonstrated that transcription of the CAT gene was initiated at the same site as transcription of the endogenous sTnI gene and that CAT activity levels were approximately proportional to CAT mRNA levels. Deletion analysis demonstrated that the region between nucleotides -160 and -40 contained sequences essential for full promoter activity. Surprisingly, 3' deletion analysis indicated that the first exon (-6 to +61) of the sTnI gene was also required for full activity of the sTnI promoter in skeletal muscle cells. Chimeric promoter experiments, in which segments of the sTnI and the herpes simplex virus thymidine kinase promoter were interchanged, indicated that reconstitution of a muscle-specific promoter required inclusion of both the upstream and exon I regions of the sTnI gene. Exon I, and the region immediately upstream, showed DNase protection over sequence motifs related to those found in other genes, including the tar region of human immunodeficiency virus type 1. These results demonstrate that expression of the sTnI promoter in embryonic skeletal muscle cells requires complex interaction between two separate promoter regions, one of which resides within the first 61 transcribed nucleotides of the gene.

scholarly journals The LIM-Homeodomain Proteins Isl-1 and Lhx3 Act with Steroidogenic Factor 1 to Enhance Gonadotrope-Specific Activity of the Gonadotropin-Releasing Hormone Receptor Gene Promoter

2006 ◽  
Vol 20 (9) ◽  
pp. 2093-2108 ◽  
Author(s):  
Anne Granger ◽  
Christian Bleux ◽  
Marie-Laure Kottler ◽  
Simon J. Rhodes ◽  
Raymond Counis ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Specific Activity ◽  
Receptor Gene ◽  
Marker Gene ◽  
Gene Promoter ◽  
Dominant Negative ◽  
R Gene ◽  
Specific Expression ◽  
Steroidogenic Factor 1 ◽  
Lim Homeodomain

Abstract The GnRH receptor (GnRH-R) plays a central role in mammalian reproductive function throughout adulthood. It also appears as an early marker gene of the presumptive gonadotrope lineage in developing pituitary. Here, using transient transfections combined with DNA/protein interaction assays, we have delineated cis-acting elements within the rat GnRH-R gene promoter that represent targets for the LIM-homeodomain (LIM-HD) proteins, Isl-1 and Lhx3. These factors, critical in early pituitary development, are thus also crucial for gonadotrope-specific expression of the GnRH-R gene. In heterologous cells, the expression of Isl-1 and Lhx3, together with steroidogenic factor 1 (SF-1), culminates in the activation of both the rat as well as human GnRH-R promoter, suggesting that this combination is evolutionarily conserved among mammals. The specificity of these LIM-HD factors is attested by the inefficiency of related proteins, including Lhx5 and Lhx9, to activate the GnRH-R gene promoter, as well as by the repressive capacity of a dominant-negative derivative of Lhx3. Accordingly, targeted deletion of the LIM response element decreases promoter activity. In addition, experiments with Gal4-SF-1 fusion proteins suggest that LIM-HD protein activity in gonadotrope cells is dependent upon SF-1 binding. Finally, using a transgenic model that allows monitoring of in vivo promoter activity, we show that the overlapping expression of Isl-1 and Lhx3 in the developing pituitary correlates with promoter activity. Collectively, these data suggest the occurrence of a specific LIM-HD pituitary code and designate the GnRH-R gene as the first identified transcriptional target of Isl-1 in the anterior pituitary.

scholarly journals Identification of a negative regulatory element that inhibits c-mos transcription in somatic cells.

1992 ◽  
Vol 12 (5) ◽  
pp. 2029-2036 ◽  
Author(s):  
S S Zinkel ◽  
S K Pal ◽  
J Szeberényi ◽  
G M Cooper
Keyword(s):  
Transcription Initiation ◽  
Regulatory Element ◽  
Somatic Cells ◽  
Regulatory Sequence ◽  
Regulatory Sequences ◽  
Flanking Sequence ◽  
3T3 Cells ◽  
Rnase Protection ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

We have used transient expression assays to identify a cis-acting region in the 5' flanking sequence of murine c-mos which, when deleted, allows expression from the c-mos promoter in NIH 3T3 cells. This negative regulatory sequence, located 400 to 500 nucleotides upstream of the c-mos ATG, also inhibited expression from a heterologous promoter. In addition to NIH 3T3 cells, the c-mos negative regulatory sequence was active in BALB/3T3 cells, PC12 rat pheochromocytoma cells, and A549 human lung carcinoma cells. Site-specific mutagenesis identified three possibly interacting regions that were involved in negative regulatory activity, located around -460, -425, and -405 with respect to the ATG. RNase protection analysis indicated that once the negative regulatory sequences were deleted, transcription in NIH 3T3 cells initiated from the same transcription initiation sites normally utilized in spermatocytes, approximately 280 nucleotides upstream of the ATG. Deletions beyond the spermatocyte promoter, however, allowed transcription initiation from progressively downstream c-mos sequences. Deletion or mutation of sequences surrounding the oocyte promoter at -53 also had little effect on expression of c-mos constructs in NIH 3T3 cells. Therefore, the major determinant of c-mos expression in NIH 3T3 cells was removal of the negative regulatory sequence rather than the utilization of a unique promoter. The c-mos negative regulatory sequences thus appear to play a significant role in tissue-specific c-mos expression by inhibiting transcription in somatic cells.

scholarly journals Methylation Analysis of CpG Islands in Pineapple SERK1 Promoter

Genes ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 425
Author(s):  
Aiping Luan ◽  
Chengjie Chen ◽  
Tao Xie ◽  
Junhu He ◽  
Yehua He
Keyword(s):  
Methylation Status ◽  
Cpg Islands ◽  
Embryonic Cell ◽  
Regulatory Mechanisms ◽  
Ananas Comosus ◽  
Regulatory Sequences ◽  
Methylation Analysis ◽  
Embryonic Callus ◽  
Methylation Inhibitor ◽  
Traditional Breeding

Somatic embryogenesis (SE) is a more rapid and controllable method for plant propagation than traditional breeding methods. However, it often suffers from limited efficiency. SERK1 promotes SE in several plants, including pineapple (Ananas comosus L.). We investigate the embryonic cell-specific transcriptional regulation of AcSERK1 by methylation analysis of CpG islands in AcSERK1 regulatory sequences. This revealed differences in the methylation status of CpG islands between embryonic callus and non-embryonic callus; the methylation inhibitor 5-azaC increased AcSERK1 expression and also accelerated SE. These findings indicate that the expression of AcSERK1 is regulated epigenetically. This study lays the foundation for further analysis of epigenetic regulatory mechanisms that may enhance the efficiency of SE in pineapple and other plants.

CHARACTERIZATION OFTHE 5’FLANKING REGION FOR THE HUMAN FIBRINOGEN β GENE

1987 ◽  
Author(s):  
P Huber ◽  
J Dalmon ◽  
M Laurent ◽  
G Courtois ◽  
D Thevenon ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Transcription Initiation ◽  
Genomic Library ◽  
Hepatoma Cell ◽  
Regulatory Sequence ◽  
Hepatoma Cell Line ◽  
Regulatory Sequences ◽  
Potential Candidate ◽  
Human Fibrinogen ◽  
Flanking Region

Fibrinogen is coded by three separate genes located in a 50kb region of chromosome 4 and organized in a α - β - γ orientation with an inversion of the gene 3- A human genomic library was constructed using the EMBL4 phage and screened with cDNA probes coding for human fibrinogen Aα, Bβ and γ chains. Clones, covering the fibrinogen locus,were identified, and their organization was analyzed by means of hybridization and restriction mapping. Among these clones one recombinant phage containing the β gene and large 5’ and 3’ -flanking sequences was isolated.To identify the regulatory sequences Dpstream from the human β gene, a 1.5 kb fragment of the immediate 5’-flanking region was sequenced. The SI mapping experiments revealed three transcription initiation sites. PotentialTATA and CAAT sequences were identified upstream the initiation start points at the positions -21 and -58 from the first initiation start point.Comparison of this sequence with that previously reported for the same region upstream from the human γ gene revealed no significant homology which suggests that the potential promoting sequences of these genes are different. In contrast, comparison of the 5’flanking regions of human and rat β genes showed more than 80% homology for 142 bp upstream from the gene. This highly conserved region is a potential candidate for a regulatory sequence of the human β gene.To verify this activity, a β fibrinogen minigene was constructed by deletion of the internal part of the normal gene and including 3.4kb of the 5’flanking region and 1.4kb of the 3’flanking region. The minigene was transfected into HepG2, a human hepatoma cell line, to show whether the 5’flanking region of the human fibrinogen gene contains DNA sequences sufficient for efficient transcription in HepG2. Constructions of several parts of the sequenced 5’flanking region of the human β gene with the gene of the chloramphenical acetyl transferase have been also transfected in the HepG2 cells to determine the specificity of the gene expression and to localize the sequences controlling the transcription of the gene.

Muscle-specific activity of the skeletal troponin I promoter requires interaction between upstream regulatory sequences and elements contained within the first transcribed exon

1990 ◽  
Vol 10 (7) ◽  
pp. 3468-3482
Author(s):  
W Nikovits ◽  
J H Mar ◽  
C P Ordahl
Keyword(s):  
Skeletal Muscle ◽  
Transcription Initiation ◽  
Troponin I ◽  
Specific Activity ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Deletion Analysis ◽  
Regulatory Sequences ◽  
Mrna Levels ◽  
Cat Gene

Expression of the skeletal troponin I (sTnI) gene is regulated transcriptionally in a muscle-specific fashion. We show here that the region of the sTnI gene between -160 and +61 (relative to the transcription initiation site) is able to direct expression of the bacterial chloramphenicol acetyltransferase (CAT) gene is muscle cultures at a level approximately 100 times higher than in fibroblast cultures. RNA analysis demonstrated that transcription of the CAT gene was initiated at the same site as transcription of the endogenous sTnI gene and that CAT activity levels were approximately proportional to CAT mRNA levels. Deletion analysis demonstrated that the region between nucleotides -160 and -40 contained sequences essential for full promoter activity. Surprisingly, 3' deletion analysis indicated that the first exon (-6 to +61) of the sTnI gene was also required for full activity of the sTnI promoter in skeletal muscle cells. Chimeric promoter experiments, in which segments of the sTnI and the herpes simplex virus thymidine kinase promoter were interchanged, indicated that reconstitution of a muscle-specific promoter required inclusion of both the upstream and exon I regions of the sTnI gene. Exon I, and the region immediately upstream, showed DNase protection over sequence motifs related to those found in other genes, including the tar region of human immunodeficiency virus type 1. These results demonstrate that expression of the sTnI promoter in embryonic skeletal muscle cells requires complex interaction between two separate promoter regions, one of which resides within the first 61 transcribed nucleotides of the gene.

scholarly journals MicroRNA Zma-miR528 Versatile Regulation on Target mRNAs during Maize Somatic Embryogenesis

2021 ◽  
Vol 22 (10) ◽  
pp. 5310
Author(s):  
Eduardo Luján-Soto ◽  
Vasti T. Juárez-González ◽  
José L. Reyes ◽  
Tzvetanka D. Dinkova
Keyword(s):  
Somatic Embryogenesis ◽  
Immature Embryo ◽  
Inverse Correlation ◽  
Total Rna ◽  
Target Mrnas ◽  
Sequence Complementarity ◽  
Low Levels ◽  
Embryogenic Callus Induction ◽  
Regenerated Plantlets ◽  
Polysome Fraction

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the accumulation and translation of their target mRNAs through sequence complementarity. miRNAs have emerged as crucial regulators during maize somatic embryogenesis (SE) and plant regeneration. A monocot-specific miRNA, mainly accumulated during maize SE, is zma-miR528. While several targets have been described for this miRNA, the regulation has not been experimentally confirmed for the SE process. Here, we explored the accumulation of zma-miR528 and several predicted targets during embryogenic callus induction, proliferation, and plantlet regeneration using the maize cultivar VS-535. We confirmed the cleavage site for all tested zma-miR528 targets; however, PLC1 showed very low levels of processing. The abundance of zma-miR528 slightly decreased in one month-induced callus compared to the immature embryo (IE) explant tissue. However, it displayed a significant increase in four-month sub-cultured callus, coincident with proliferation establishment. In callus-regenerated plantlets, zma-miR528 greatly decreased to levels below those observed in the initial explant. Three of the target transcripts (MATE, bHLH, and SOD1a) showed an inverse correlation with the miRNA abundance in total RNA samples at all stages. Using polysome fractionation, zma-miR528 was detected in the polysome fraction and exhibited an inverse distribution with the PLC1 target, which was not observed at total RNA. Accordingly, we conclude that zma-miR528 regulates multiple target mRNAs during the SE process by promoting their degradation, translation inhibition or both.

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