MOLECULAR CHARACTERIZATION AND TRANSIENT EXPRESSION OF MAIZE SUS1 PROMOTER

Sucrose synthase (SS) is one of the key enzymes in plant carbohydrate metabolism. In maize, this enzyme is encoded by two genes, Sh1 and Sus1. We have isolated and determined the 5'-upstream sequence of maize Sus1 gene and compared it with the corresponding sequence in Sh1 gene. Sequence analysis revealed that there was a weak homology between the two promoters and no common sequence elements were found. To understand the differential regulation of the expression of the two genes, we constructed chimeric GUS fusions using the two promoters of SS genes. By using the biolistic system, we delivered these constructs into various plant tissues, and their transient expression was studied. Our results showed that the two promoters of SS genes directed tissue-specific expression in the same way that the two genes are expressed in vivo. The effectiveness of the expression of the constructs was recorded by counting the total blue expression units (blue spots) per shot and by fluorometric assays. High levels of GUS activity were detected in the immature embryos, young coleoptiles, and heterotrophic young leaves bombarded with the Sus–GUS construct. More than 100 expression units were observed in these tissues. Compared with the transient expression of the 35S promoter in the same tissue, Sus promoter activity was twice as high. Strong Sus–GUS expression was also detected in the aleurone cells of developing kernels. In contrast, the Sh-GUS construct was expressed only in the endosperm with an activity twice as high as that of Sus–GUS and 35S–GUS in the same tissue. The results will be discussed in terms of the physiological roles of the two SS isozymes in plant tissues.

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Identification of the Core Pollen-Specific Regulation in the Rice OsSUT3 Promoter

International Journal of Molecular Sciences ◽

10.3390/ijms21061909 ◽

2020 ◽

Vol 21 (6) ◽

pp. 1909

Author(s):

Dandan Li ◽

Rucong Xu ◽

Dong Lv ◽

Chunlong Zhang ◽

Hong Yang ◽

...

Keyword(s):

Pollen Development ◽

Ectopic Expression ◽

Gus Expression ◽

35S Promoter ◽

Crop Breeding ◽

Specific Expression ◽

Regulatory Motifs ◽

Gus Reporter ◽

Specific Regulation ◽

Promoter Deletions

The regulatory mechanisms of pollen development have potential value for applications in agriculture, such as better understanding plant reproductive regularity. Pollen-specific promoters are of vital importance for the ectopic expression of functional genes associated with pollen development in plants. However, there is a limited number of successful applications using pollen-specific promoters in genetic engineering for crop breeding and hybrid generation. Our previous work led to the identification and isolation of the OsSUT3 promoter from rice. In this study, to analyze the effects of different putative regulatory motifs in the OsSUT3 promoter, a series of promoter deletions were fused to a GUS reporter gene and then stably introduced into rice and Arabidopsis. Histochemical GUS analysis of transgenic plants revealed that p385 (from −385 to −1) specifically mediated maximal GUS expression in pollen tissues. The S region (from −385 to −203) was the key region for controlling the pollen-specific expression of a downstream gene. The E1 (−967 to −606), E2 (−202 to −120), and E3 (−119 to −1) regions enhanced ectopic promoter activity to different degrees. Moreover, the p385 promoter could alter the expression pattern of the 35S promoter and improve its activity when they were fused together. In summary, the p385 promoter, a short and high-activity promoter, can function to drive pollen-specific expression of transgenes in monocotyledon and dicotyledon transformation experiments.

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A 40-kilodalton protein binds specifically to an upstream sequence element essential for muscle-specific transcription of the human myoglobin promoter

Molecular and Cellular Biology ◽

10.1128/mcb.12.11.5024-5032.1992 ◽

1992 ◽

Vol 12 (11) ◽

pp. 5024-5032

Author(s):

R Bassel-Duby ◽

M D Hernandez ◽

M A Gonzalez ◽

J K Krueger ◽

R S Williams

Keyword(s):

Mammalian Cells ◽

Mutational Analysis ◽

Transcriptional Start Site ◽

Upstream Sequence ◽

Specific Expression ◽

Sequence Element ◽

Promoter Function ◽

Human Myoglobin

To define transcriptional control elements responsible for muscle-specific expression of the human myoglobin gene, we performed mutational analysis of upstream sequences (nucleotide positions -373 to +7 relative to the transcriptional start site) linked to a firefly luciferase gene. Transient expression assays in avian and mammalian cells indicated that a CCCACCCCC (CCAC box) sequence (-223 to -204) is necessary for muscle-specific transcription directed either by the native myoglobin promoter or by a heterologous minimal promoter linked to the myoglobin upstream enhancer region. A putative MEF2-like site (-160 to -169) was likewise necessary for full transcriptional activity in myotubes. Mutations within either of two CANNTG (E-box) motifs (-176 to -148) had only minimal effects on promoter function. We identified and partially purified from nuclear extracts a 40-kDa protein (CBF40) that binds specifically to oligonucleotides containing the CCAC box sequence. A mutation of the CCAC box that disrupted promoter function in vivo also impaired binding of CBF40 in vitro. These data suggest that cooperative interactions between CBF40 and other factors including MEF-2 are required for expression of the human myoglobin gene in skeletal muscle.

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A 40-kilodalton protein binds specifically to an upstream sequence element essential for muscle-specific transcription of the human myoglobin promoter.

Molecular and Cellular Biology ◽

10.1128/mcb.12.11.5024 ◽

1992 ◽

Vol 12 (11) ◽

pp. 5024-5032 ◽

Cited By ~ 43

Author(s):

R Bassel-Duby ◽

M D Hernandez ◽

M A Gonzalez ◽

J K Krueger ◽

R S Williams

Keyword(s):

Mammalian Cells ◽

Mutational Analysis ◽

Transcriptional Start Site ◽

Upstream Sequence ◽

Specific Expression ◽

Sequence Element ◽

Promoter Function ◽

Human Myoglobin

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AT-rich upstream sequence elements regulate spore germination-specific expression of theDictyostelium discoideum celAgene

Nucleic Acids Research ◽

10.1093/nar/23.15.3018 ◽

1995 ◽

Vol 23 (15) ◽

pp. 3018-3025 ◽

Cited By ~ 7

Author(s):

Ramachandran Ramalingam ◽

John E. Blume ◽

Kalyan Ganguly ◽

Herbert L. Ennis

Keyword(s):

Spore Germination ◽

Upstream Sequence ◽

Specific Expression ◽

Sequence Elements

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Factors Affecting Transient Gene Expression in Apple Cotyledons following Particle Bombardment

HortScience ◽

10.21273/hortsci.31.4.617a ◽

1996 ◽

Vol 31 (4) ◽

pp. 617a-617

Author(s):

Hong Y. Yang ◽

Schuyler S. Korban

Keyword(s):

Gene Transfer ◽

Transient Expression ◽

Particle Bombardment ◽

Transient Gene Expression ◽

Gus Expression ◽

Cauliflower Mosaic Virus ◽

35S Promoter ◽

Tungsten Particle ◽

Factors Affecting ◽

Efficient Gene

Developing an efficient gene transfer system for apple (Malus ×domestica L.) remains a major objective in genetic engineering efforts of this fruit crop. Transient expression of the uidA gene coding for β-glucuronidase (GUS) and driven by the cauliflower mosaic virus 35S promoter (CaMV35S) has been induced in apple cotyledonary explants of mature seeds by tungsten particle bombardment using the Particle Inflow Gun (PIG). Several factors that affect transient expression of the GUS gene in apple cotyledons were investigated. The gene transfer efficiency was monitored by recording the number of blue spots observed on explants two days following bombardment. Precultivation of cotyledons for 18 hours before bombardment significantly increased the number of blue foci. Of the three different precipitation methods tested including water, 25% PEG, and 60% glycerol, the latter was the most effective for coating DNA onto tungsten particles. Washing DNA-coated tungsten particles with 70% ethanol and resuspending in 100% ethanol significantly enhanced gene delivery to cotyledons. The amount of particles used for each bombardment also influenced GUS expression. About 0.5 mg of particles per shot resulted in the highest number of blue foci. Using larger quantity of particles (i.e., 2 mg) drastically decreased GUS expression probably due to the toxicity of tungsten particles.

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The Bradyrhizobium japonicum hsfA Gene Exhibits a Unique Developmental Expression Pattern in Cowpea Nodules

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2001.14.11.1286 ◽

2001 ◽

Vol 14 (11) ◽

pp. 1286-1292 ◽

Cited By ~ 7

Author(s):

Hye-Sook Oh ◽

Ora Son ◽

Jong-Yoon Chun ◽

Gary Stacey ◽

Myung-Sok Lee ◽

...

Keyword(s):

Nitrogen Fixation ◽

Bradyrhizobium Japonicum ◽

Regulatory Protein ◽

Gus Expression ◽

Proximal Region ◽

Developmental Expression ◽

Nodule Development ◽

Upstream Sequence ◽

Specific Expression ◽

Fusion Construct

The Bradyrhizobium japonicum host-specific fixation gene hsfA was identified as essential for nitrogen fixation on cowpea, but not required for nitrogen fixation on soybean or siratro. The DNA sequence of the hsfA promoter contains a consensus RpoN, -24/-12 binding site, suggesting the involvement of a regulatory protein that binds to an upstream activating sequence (UAS). To further explore the regulation of this interesting gene, serial deletions of the hsfA promoter were made and fused with the β-glucuronidase (GUS) gene. The HsfA3 deletion, containing 60 bp 5′ of the -24/-12 sequence, showed a similar level of GUS expression to that shown by the longest fusion construct (HsfA1), containing 464 bp of upstream sequence. In contrast, the HsfA4-GUS fusion, containing only 20 bp 5′ of the -24/-12 region, showed no GUS activity, delimiting the location of a putative UAS to a 40-bp region. During nodule development, GUS expression first appeared in nodules 12 days postinoculation (dpi) and reached a maximum level of expression in approximately 17-day-old nodules. By 28 dpi, HsfA-GUS expression had returned to a low, basal level. These data were consistent with the detection of hsfA mRNA by in situ hybridization in 17-day-old nodules, but not in 28-day-old nodules. In contrast to the stage-specific expression in cowpea, HsfA-GUS expression increased with nodule development in HsfA3-inoculated soybean. These data indicate that HsfA expression is regulated in cowpea in a unique developmental manner and that the DNA regulatory regions that control this expression are confined to a short, promoter-proximal region.

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Expression of a Rice Chlorophyll a/b Binding Protein Promoter in Sweetpotato

Journal of the American Society for Horticultural Science ◽

10.21273/jashs.132.4.551 ◽

2007 ◽

Vol 132 (4) ◽

pp. 551-556 ◽

Cited By ~ 2

Author(s):

Guo-qing Song ◽

Hideo Honda ◽

Ken-ichi Yamaguchi

Keyword(s):

Chlorophyll A ◽

Ipomoea Batatas ◽

Binding Protein ◽

Gus Expression ◽

Cauliflower Mosaic Virus ◽

35S Promoter ◽

Target Tissue ◽

Storage Roots ◽

Specific Expression ◽

Pests And Diseases

Leaves are usually the target tissue for expressing transgenes conferring resistances to herbicides, pests, and diseases. To achieve leaf-specific expression, a light-harvest chlorophyll a/b binding protein (CAB) of photosystem-II (CAB2) promoter (CAB2-p) from rice (Oryza sativa L.) and the cauliflower mosaic virus 35S promoter were fused to the β-glucuronidase (GUS) reporter and subsequently evaluated in transgenic sweetpotato [Ipomoea batatas L. (Lam.)]. The 35S promoter-directed GUS activities varied from 46.0 to 61.2 nmol 4-methyl-umbelliferyl-β-D-glucuronide (4-MU) per minute per milligram of protein in leaf, stem, primary, and storage roots. In contrast, the CAB2-p directed an uneven distribution of GUS activities (4-MU at 1.1 to 12.6 nmol·min−1·mg−1 protein); GUS activity in mature leaves was ≈12-fold as high as that in storage roots. In addition, GUS assay in leaf tissues revealed that CAB2-p enabled a developmentally controlled and light-regulated GUS expression. These results indicate that the rice CAB2-p could be used to drive leaf-specific expression of linked genes in sweetpotato.

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Probing the 14-3-3 Isoform-Specificity Profile of Interactions Stabilized by Fusicoccin A

10.26434/chemrxiv.12052869.v1 ◽

2020 ◽

Author(s):

James Frederich ◽

Ananya Sengupta ◽

Josue Liriano ◽

Ewa A. Bienkiewicz ◽

Brian G. Miller

Keyword(s):

Protein Interactions ◽

Neurological Diseases ◽

Adapter Proteins ◽

Protein Protein Interactions ◽

Specific Expression ◽

Individual Client ◽

Isoform Specificity ◽

Fusicoccin A

Fusicoccin A (FC) is a fungal phytotoxin that stabilizes protein–protein interactions (PPIs) between 14-3-3 adapter proteins and their phosphoprotein interaction partners. In recent years, FC has emerged as an important chemical probe of human 14-3-3 PPIs implicated in cancer and neurological diseases. These previous studies have established the structural requirements for FC-induced stabilization of 14-3-3·client phosphoprotein complexes; however, the effect of different 14-3-3 isoforms on FC activity has not been systematically explored. This is a relevant question for the continued development of FC variants because there are seven distinct isoforms of 14-3-3 in humans. Despite their remarkable sequence and structural similarities, a growing body of experimental evidence supports both tissue-specific expression of 14-3-3 isoforms and isoform-specific functions <i>in vivo</i>. Herein, we report the isoform-specificity profile of FC <i>in vitro</i>using recombinant human 14-3-3 isoforms and a focused library of fluorescein-labeled hexaphosphopeptides mimicking the C-terminal 14-3-3 recognition domains of client phosphoproteins targeted by FC in cell culture. Our results reveal modest isoform preferences for individual client phospholigands and demonstrate that FC differentially stabilizes PPIs involving 14-3-3s. Together, these data provide strong motivation for the development of non-natural FC variants with enhanced selectivity for individual 14-3-3 isoforms.

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Non-Viral Vectors for Gene Delivery

Nanoscience &Nanotechnology-Asia ◽

10.2174/2210681208666180110154233 ◽

2018 ◽

Vol 9 (1) ◽

pp. 4-11 ◽

Cited By ~ 5

Author(s):

Aparna Bansal ◽

Himanshu

Keyword(s):

Gene Therapy ◽

Viral Vectors ◽

Transfection Efficiency ◽

Gene Transfection ◽

Expression System ◽

Target Cells ◽

Specific Expression ◽

Naked Dna

Introduction: Gene therapy has emerged out as a promising therapeutic pave for the treatment of genetic and acquired diseases. Gene transfection into target cells using naked DNA is a simple and safe approach which has been further improved by combining vectors or gene carriers. Both viral and non-viral approaches have achieved a milestone to establish this technique, but non-viral approaches have attained a significant attention because of their favourable properties like less immunotoxicity and biosafety, easy to produce with versatile surface modifications, etc. Literature is rich in evidences which revealed that undoubtedly, non–viral vectors have acquired a unique place in gene therapy but still there are number of challenges which are to be overcome to increase their effectiveness and prove them ideal gene vectors. Conclusion: To date, tissue specific expression, long lasting gene expression system, enhanced gene transfection efficiency has been achieved with improvement in delivery methods using non-viral vectors. This review mainly summarizes the various physical and chemical methods for gene transfer in vitro and in vivo.

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Overexpression of the Oral Mucosa-Specific microRNA-31 Promotes Skin Wound Closure

International Journal of Molecular Sciences ◽

10.3390/ijms20153679 ◽

2019 ◽

Vol 20 (15) ◽

pp. 3679 ◽

Cited By ~ 3

Author(s):

Lin Chen ◽

Alyne Simões ◽

Zujian Chen ◽

Yan Zhao ◽

Xinming Wu ◽

...

Keyword(s):

Wound Healing ◽

Oral Mucosa ◽

Wound Closure ◽

Expression Patterns ◽

Skin Wound ◽

Skin Wound Healing ◽

Specific Expression ◽

Keratinocyte Proliferation

Wounds within the oral mucosa are known to heal more rapidly than skin wounds. Recent studies suggest that differences in the microRNAome profiles may underlie the exceptional healing that occurs in oral mucosa. Here, we test whether skin wound-healing can be accelerating by increasing the levels of oral mucosa-specific microRNAs. A panel of 57 differentially expressed high expresser microRNAs were identified based on our previously published miR-seq dataset of paired skin and oral mucosal wound-healing [Sci. Rep. (2019) 9:7160]. These microRNAs were further grouped into 5 clusters based on their expression patterns, and their differential expression was confirmed by TaqMan-based quantification of LCM-captured epithelial cells from the wound edges. Of these 5 clusters, Cluster IV (consisting of 8 microRNAs, including miR-31) is most intriguing due to its tissue-specific expression pattern and temporal changes during wound-healing. The in vitro functional assays show that ectopic transfection of miR-31 consistently enhanced keratinocyte proliferation and migration. In vivo, miR-31 mimic treatment led to a statistically significant acceleration of wound closure. Our results demonstrate that wound-healing can be enhanced in skin through the overexpression of microRNAs that are highly expressed in the privileged healing response of the oral mucosa.

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