Sequence and spatial requirements for the tissue- and species-independent 3'-end processing mechanism of plant mRNA

1994 ◽  
Vol 14 (10) ◽  
pp. 6829-6838
Author(s):  
L Wu ◽  
T Ueda ◽  
J Messing
Keyword(s):  
Transcription Unit ◽  
Cauliflower Mosaic Virus ◽  
Upstream Region ◽  
General Mechanism ◽  
Upstream Sequence ◽  
Preference Order ◽  
Spatial Positioning ◽  
Species Specific ◽  
Zein Mrna ◽  
Processing Mechanism

Two cis-regulatory regions are required for efficient mRNA 3'-end processing of the maize 27-kDa zein mRNA: a region containing a duplicated AAUGAA poly(A) signal and a region that is present upstream from it. Strict spatial positioning of these two regions is required for efficient mRNA 3'-end processing. Insertion of a stuffer sequence as short as 17 or 18 bp either between the upstream region and the two AAUGAA motifs or between the two AAUGAA motifs drastically reduced the efficiency of 3'-end processing. Mutational analyses of the nucleotide preference at the fourth position of the AAUGAA motif revealed the preference order G > A >> C or U, suggesting that AAUAAA is neither a defective nor an optimal poly(A) signal for the 27-kDa zein mRNA. As for the 3' control region of the cauliflower mosaic virus (CaMV) transcription unit, the mRNA 3'-end processing mechanism mediated by the 27-kDa zein 3' control sequence is neither tissue nor species specific. The 3' upstream sequence of the 27-kDa zein gene can functionally replace that of the CaMV transcription unit. Conversely, the CaMV upstream sequence can mediate mRNA polyadenylation in the presence of a duplicated 27-kDa zein poly(A) signal. However, instead of the proximal poly(A) signal normally used in the 27-kDa zein mRNA, the distal signal is utilized. These results suggest that a general mechanism controls the 3'-end processing of plant mRNAs and that the cis-regulatory functions mediated by their upstream regions are interchangeable.

scholarly journals Sequence and spatial requirements for the tissue- and species-independent 3'-end processing mechanism of plant mRNA.

1994 ◽  
Vol 14 (10) ◽  
pp. 6829-6838 ◽  
Author(s):  
L Wu ◽  
T Ueda ◽  
J Messing
Keyword(s):  
Transcription Unit ◽  
Cauliflower Mosaic Virus ◽  
Upstream Region ◽  
General Mechanism ◽  
Upstream Sequence ◽  
Preference Order ◽  
Spatial Positioning ◽  
Species Specific ◽  
Zein Mrna ◽  
Processing Mechanism

Two cis-regulatory regions are required for efficient mRNA 3'-end processing of the maize 27-kDa zein mRNA: a region containing a duplicated AAUGAA poly(A) signal and a region that is present upstream from it. Strict spatial positioning of these two regions is required for efficient mRNA 3'-end processing. Insertion of a stuffer sequence as short as 17 or 18 bp either between the upstream region and the two AAUGAA motifs or between the two AAUGAA motifs drastically reduced the efficiency of 3'-end processing. Mutational analyses of the nucleotide preference at the fourth position of the AAUGAA motif revealed the preference order G > A >> C or U, suggesting that AAUAAA is neither a defective nor an optimal poly(A) signal for the 27-kDa zein mRNA. As for the 3' control region of the cauliflower mosaic virus (CaMV) transcription unit, the mRNA 3'-end processing mechanism mediated by the 27-kDa zein 3' control sequence is neither tissue nor species specific. The 3' upstream sequence of the 27-kDa zein gene can functionally replace that of the CaMV transcription unit. Conversely, the CaMV upstream sequence can mediate mRNA polyadenylation in the presence of a duplicated 27-kDa zein poly(A) signal. However, instead of the proximal poly(A) signal normally used in the 27-kDa zein mRNA, the distal signal is utilized. These results suggest that a general mechanism controls the 3'-end processing of plant mRNAs and that the cis-regulatory functions mediated by their upstream regions are interchangeable.

scholarly journals Ontogenic expression of a CyI actin fusion gene injected into sea urchin eggs

Development ◽  
1987 ◽  
Vol 101 (3) ◽  
pp. 437-447
Author(s):  
K.S. Katula ◽  
B.R. Hough-Evans ◽  
R.J. Britten ◽  
E.H. Davidson
Keyword(s):  
Sea Urchin ◽  
Fusion Gene ◽  
Transcription Unit ◽  
Upstream Region ◽  
Upstream Sequence ◽  
Flanking Sequence ◽  
Bacterial Gene ◽  
Fusion Construct ◽  
Gene Coding ◽  
Sea Urchin Egg

The 5′ terminus of the CyI actin gene transcription unit of Strongylocentrotus purpuratus was located by primer extension and other procedures, and the flanking upstream region was partially sequenced and mapped. A fusion gene was constructed containing about 2.5 kb of 5′ flanking sequence, the transcribed leader sequence, and the first few codons of the CyI gene ligated to the bacterial gene coding for chloramphenicol acetyl transferase (CAT). This was micro-injected into the cytoplasm of S. purpuratus eggs, and CAT enzyme activity was measured at various stages of embryonic development. CAT synthesis was activated between 10 and 14 h postfertilization, the same time at which newly synthesized transcripts of the endogenous CyI gene first appear. The exogenous CyI.CAT fusion DNA replicated actively during cleavage, as observed previously for other DNAs injected into sea urchin egg cytoplasm. Thus the absence of CAT activity prior to 10 h postfertilization could not be due to insufficient CyI.CAT genes. The amounts of CAT enzyme produced by embryos bearing CyI.CAT deletions that lack various regions of the CyI sequence were measured. As little as 254 nucleotides of upstream CyI sequence suffice for correct temporal activation of the fusion construct, although the level of CAT enzyme produced in embryos bearing any deletion retaining less than 850 nucleotides of upstream sequence was significantly lowered compared to controls bearing the complete CyI.CAT fusion construct.

scholarly journals Transvection at the End of the Truncated Chromosome in Drosophila melanogaster

Genetics ◽  
2003 ◽  
Vol 163 (4) ◽  
pp. 1375-1387
Author(s):  
Mikhail Savitsky ◽  
Tatyana Kahn ◽  
Ekaterina Pomerantseva ◽  
Pavel Georgiev
Keyword(s):  
Drosophila Melanogaster ◽  
Homologous Chromosome ◽  
Regulatory Region ◽  
Proximal Region ◽  
The Other ◽  
Upstream Region ◽  
Upstream Sequence ◽  
Transcription Start ◽  
Promoter Proximal Region

Abstract The phenomenon of transvection is well known for the Drosophila yellow locus. Thus enhancers of a promoterless yellow locus in one homologous chromosome can activate the yellow promoter in the other chromosome where the enhancers are inactive or deleted. In this report, we examined the requirements for trans-activation of the yellow promoter at the end of the deficient chromosome. A number of truncated chromosomes ending in different areas of the yellow regulatory region were examined in combination with the promoterless y alleles. We found that trans-activation of the yellow promoter at the end of a deficient chromosome required ∼6 kb of an additional upstream sequence. The nature of upstream sequences affected the strength of transvection: addition of gypsy sequences induced stronger trans-activation than addition of HeT-A or yellow sequences. Only the promoter proximal region (within -158 bp of the yellow transcription start) was essential for trans-activation; i.e., transvection did not require extensive homology in the yellow upstream region. Finally, the yellow enhancers located on the two pairing chromosomes could cooperatively activate one yellow promoter.

Adenovirus 5 E2 transcription unit: an E1A-inducible promoter with an essential element that functions independently of position or orientation

1984 ◽  
Vol 4 (5) ◽  
pp. 875-882
Author(s):  
M J Imperiale ◽  
J R Nevins
Keyword(s):  
Transcription Initiation ◽  
Essential Element ◽  
Inducible Promoter ◽  
Initiation Site ◽  
Transcription Unit ◽  
Upstream Sequence ◽  
Wild Type ◽  
Promoter Sequences ◽  
Adenovirus 5 ◽  
Wild Type Promoter

Utilizing deletion mutants of a plasmid containing the adenovirus E2 gene, an E1A-inducible transcription unit, we determined the promoter sequences required for full expression in transient transfection assays. Wild-type expression was obtained from plasmids containing only 79 nucleotides of upstream sequence relative to the transcription initiation site. Removal of an additional nine nucleotides lowered expression 10-fold, and deletion to -59 resulted in near total loss of transcription. Wild-type levels of expression were restored to a -28 deletion mutant by insertion of the sequence from -21 to -262 from the wild-type promoter at the -28 position, in either orientation, even though when inserted in the opposite orientation the relevant sequences were ca. 270 nucleotides upstream from their normal position. Finally, this sequence could be placed at a distance of 4,000 nucleotides from the E2 cap site and still retain near total function. Thus, the E2 promoter element can function independent of orientation and position, properties characteristic of enhancer elements.

scholarly journals Identification of an enhancer-like element in the polyhedrin gene upstream region of Bombyx mori nucleopolyhedrovirus

2001 ◽  
Vol 82 (11) ◽  
pp. 2811-2819 ◽  
Author(s):  
Asha Acharya ◽  
Karumathil P. Gopinathan
Keyword(s):  
Bombyx Mori ◽  
Reporter Gene ◽  
Fold Increase ◽  
Firefly Luciferase ◽  
Upstream Region ◽  
Upstream Sequence ◽  
Gene Encoding ◽  
Viral Promoters ◽  
Enhancer Sequences ◽  
In Cis

A series of deletions in the upstream region of the gene encoding polyhedrin (polh) of Bombyx mori nucleopolyhedrovirus (BmNPV) were generated in plasmid constructs and tested for transcription. In transient transfection assays in Bombyx mori-derived BmN cells with firefly luciferase as the reporter gene, a 293 bp fragment located 1·0 kb upstream with respect to the +1 ATG of polh showed 10-fold enhancement in expression from the minimal promoter. This increase in reporter activity was observed only when the fragment was positioned in cis with respect to the promoter and not in trans. The stimulation of reporter gene expression was independent of the orientation of the fragment and was due to increased transcription from the promoter. When placed upstream of another promoter, the viral very late gene p10 promoter, the enhancer brought about a 2-fold increase in expression. The region encompassing the enhancer was itself transcriptionally active, and transcripts corresponding to both of the encoded ORFs (N-terminal regions of ORF453 and ORF327, located in opposite orientations) were detected. Two AP1 sites (TGACTCG) in the 293 bp fragment did not appear to contribute to the enhancer function. Since repeat motifs, the hallmark of conventional enhancer sequences, were absent from this fragment, it is designated as an enhancer-like element. The influence of this region of the polh upstream sequence on expression from strong, very late viral promoters has not been reported previously.

scholarly journals Regulatory elements in the upstream region of metallothionein gene in tilapia species

2021 ◽  
Vol 30 (1) ◽  
pp. 95-103
Author(s):  
Mohammad Shamimul Alam ◽  
Israt Jahan ◽  
Sadniman Rahman ◽  
Hawa Jahan ◽  
Kaniz Fatema
Keyword(s):  
Tissue Specificity ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Metal Resistance ◽  
Genomic Region ◽  
Upstream Region ◽  
Upstream Sequence ◽  
Oreochromis Aureus ◽  
Metallothionein Gene ◽  
Genomic Regions

Tilapia is a hardy fish which can survive in water bodies polluted with heavy metals. Metal resistance is conferred by higher expression of metallothionein gene (mt) in many organisms. Level, time and tissue-specificity of gene expression is regulated through transcription factor binding sites (TFBS) which may be present in the upstream, downstream, or even in the introns of a gene. So, as a candidate regulatory region, the 5’upstream sequence of mt gene in three tilapia species, Oreochromis aureus, O. niloticus and O. mossambicus was studied. The targeted region was PCR-amplified and then sequenced using a pair of custom-designed primer. A total of only 2.7% variation was found in the sequenced genomic region among the three species. Metal-related TFBS were predicted from these sequences. A total of twenty eight TFBS were found in O. aureus and twenty nine in O. mossambicus and O. niloticus. The number of metalrelated TFBS predicted in the targeted sequence was significantly higher compared to that found in randomly selected other genomic regions of same size from O. niloticus genome. Thus, the results suggest the presence of putative regulatory elements in the targeted upstream region which might have important role in the regulation of mt gene function. Dhaka Univ. J. Biol. Sci. 30(1): 95-103, 2021 (January)

Porcine OCT4 Reporter System can Monitor Species-Specific Pluripotency During Somatic Cell Reprogramming

2020 ◽  
Author(s):  
Seung-Hun Kim ◽  
Kwang-Hwan Choi ◽  
Mingyun Lee ◽  
Dong-Kyung Lee ◽  
Chang-Kyu Lee
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Fluorescent Protein ◽  
Upstream Region ◽  
Reporter System ◽  
System L ◽  
Reporter Systems ◽  
Induced Pluripotent ◽  
Species Specific ◽  
Fluorescent Protein Reporter

Abstract l Background: The present study examined the activity and function of pig OCT4 enhancer in porcine reprogramming cells. Dual fluorescent protein reporter systems controlled by the upstream regulatory region of OCT4, which is one of the master regulators for pluripotency, are widely used in studies of the mechanism of pluripotency. We analyzed how this reporter system functions in FGF- or LIF-dependent reprogrammed porcine pluripotent stem cells using the previously established porcine-specific reporter system. l Results: Porcine embryonic fibroblasts were coinfected with the pOCT4-∆PE-eGFP (DE-GFP) and pOCT4-∆DE-DsRed2 (PE-RFP) vectors, and GFP and RFP expression was verified during a DOX-dependent reprogramming process. We demonstrated that the porcine OCT4 distal enhancer and proximal enhancer were activated in different expression patterns simultaneously as the changes in the expression of pluripotent marker genes during the establishment of porcine-induced pluripotent stem cells (iPSCs). l Conclusions: Porcine OCT4 upstream region-derived dual fluorescent protein reporter systems serve as live naïve/primed pluripotency indicators for porcine induced pluripotent cell establishment. This work demonstrates the applicability of the porcine OCT4 upstream region-derived dual fluorescence reporter system, which may be applied to investigations of species-specific pluripotency in porcine-origin cells. These reporter systems may be useful tools for studies of porcine-specific pluripotency, early embryo development and embryonic stem cells.

scholarly journals Investigation of Polymorphisms in the Upstream Sequence of LIF and LIFR Genes in the Infertile Women

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Nahid Tajeddin ◽  
Ali Mohammad Ahadi ◽  
Gholamreza Javadi ◽  
Hoda Ayat
Keyword(s):  
Embryo Implantation ◽  
Single Strand Conformation Polymorphism ◽  
Sexual Cycle ◽  
Upstream Region ◽  
Control Group ◽  
Upstream Sequence ◽  
Sequencing Analysis ◽  
Genetic Changes ◽  
Infertile Women ◽  
Pcr Products

: During pregnancy, the embryo implantation stage is a highly dynamic and molecularly controlled phenomenon. Several genes are involved in the implantation process, among which the leukemia inhibitory factor (LIF) is a marker of implantation. LIF is a multi-functional cytokine located on chromosome 22. The expression of this gene is increased in the middle of the secretion phase from the sexual cycle, and any defect in its expression will cause the implantation failure. LIF receptor or LIFR gene, as the LIF receptor, consists of two membrane proteins called LIFR and GP130. LIFR acts as a signal receptor for LIF in a low-affinity level. In this study, we focused on the screening of polymorphisms in the promoter region of the LIF and LIFR genes in the infertile women using the polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) method. In this study, blood samples were collected from 100 women with primary and secondary infertility and 50 healthy women as a control group. Extraction of DNA was done by the phenol-chloroform method, and in the next step, using specific primers for upstream regions of the LIF and LIFR genes, target sequences were amplified and analyzed by the SSCP method. Finally, PCR products with different configurations were selected for sequencing analysis. The results showed two polymorphisms in the upstream region of LIF and LIFR genes of two women, but there were no genetic changes in the control group. The present study was the first in this field, and the results indicated the importance of examining such genes in infertility with an unknown cause.

scholarly journals Molecular characterization of the giant freshwater prawn (Macrobrachium rosenbergii) beta-actin gene promoter

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5701
Author(s):  
Junjun Yan ◽  
Qiang Gao ◽  
Zongbin Cui ◽  
Guoliang Yang ◽  
Yong Long
Keyword(s):  
Gene Function ◽  
Transcription Initiation ◽  
Macrobrachium Rosenbergii ◽  
Gene Promoter ◽  
Freshwater Prawn ◽  
Upstream Region ◽  
Upstream Sequence ◽  
Giant Freshwater Prawn ◽  
Beta Actin ◽  
Positive Elements

Constitutive promoters are important tools for gene function studies and transgenesis. The Beta-actin (actb1) gene promoter has been isolated from many species but remains to be cloned from the giant freshwater prawn (Macrobrachium rosenbergii). In this study, we cloned and characterized the Mractb1 gene promoter. Two alternative promoters were identified for the Mractb1 gene, which direct the generation of two transcripts with different 5′ untranslated regions. Three CpG islands were predicted in the upstream sequence, which are intimately related to transcription initiation and promoter activity. In addition to the CCAAT-box and the CArG-box, molecular dissection of the flanking sequence revealed the existence of one negative and two positive elements in the upstream region and the first intron. Finally, the Mractb1 promoter demonstrated comparative activity to the carp (Cyprinus carpio) actb1 promoter. Our investigations provide a valuable genetic tool for gene function studies and shed light on the regulation of the Mractb1 gene.

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