scholarly journals Exploration of an Actin Promoter-Based Transient Expression Vector to Trace the Cellular Localization of Nucleorhabdovirus Proteins in Leafhopper Cultured Cells

2018 ◽  
Vol 9 ◽  
Author(s):  
Xiao-Feng Zhang ◽  
Yunjie Xie ◽  
Haitao Wang ◽  
Juan Wang ◽  
Hongyan Chen ◽  
...  
Keyword(s):  
Transient Expression ◽  
Cellular Localization ◽  
Expression Vector ◽  
Cultured Cells ◽  
Actin Promoter

scholarly journals Monoclonal antibody specific for human nuclear proteins IEF 8Z30 and 8Z31 accumulates in the nucleus a few hours after cytoplasmic microinjection of cells expressing these proteins.

1986 ◽  
Vol 103 (6) ◽  
pp. 2083-2089 ◽  
Author(s):  
P Madsen ◽  
S Nielsen ◽  
J E Celis
Keyword(s):  
Monoclonal Antibody ◽  
Proliferating Cell Nuclear Antigen ◽  
Cellular Localization ◽  
Nuclear Translocation ◽  
Cultured Cells ◽  
Nuclear Proteins ◽  
Nuclear Antigen ◽  
Molecular Weights ◽  
Nuclear Location ◽  
Proliferating Cell

A monoclonal antibody (mAB 1C4C10) that reacts specifically with human nuclear proteins IEF 8Z30 and 8Z31 (charge variants; HeLa protein catalogue number; Bravo, R., and J. E. Celis, 1982, Clin. Chem., 28:766-781) has been microinjected into the cytoplasm of cultured cells that either express (primates) or lack these proteins (at least having similar molecular weights and pIs; other species), and its cellular localization has been determined by indirect immunofluorescence. Nuclear localization (nucleolar and nucleoplasmic) of the antibody was observed only in cells expressing these antigens, suggesting that a determinant present in IEF 8Z30 and 8Z31 is required for cytoplasm-nuclear translocation. Nuclear migration was not inhibited by cycloheximide, implying that these proteins may shuttle between nucleus and cytoplasm. The results assumed to support the signal rather than the free diffusion model are further supported by microinjection experiments using antibodies (proliferating cell nuclear antigen/cyclin, DNA) that react with nuclear components but do not recognize cytoplasmic antigens. Furthermore, they raise the possibility that some nonnuclear proteins may be transported to the nucleus by interacting with proteins harboring nuclear location signals.

Localization of Z-RNA in Normal Lens Epithelium: Middle Fibers

2000 ◽  
Vol 6 (S2) ◽  
pp. 900-901
Author(s):  
C.E. Gagna ◽  
H.R. Kuo ◽  
R. Schulz ◽  
R. Cordova ◽  
G. Crippen ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Cellular Localization ◽  
Cultured Cells ◽  
Polyclonal Antibodies ◽  
Image Analyzer ◽  
Left Handed ◽  
Z Dna ◽  
Normal Lens

The goal of this project was to analyze the cellular localization of Z-RNA, within middle fibers (MF) of the adult dog ocular lens (1.5 yr) (Fig. 1), using anti-Z-RNA IgG polyclonal antibodies. B-DNA can adopt the left-handed Z-DNA conformation in vitro (1). Right-handed A-RNA can be transformed into left-handed Z-RNA (2). Z-RNA has been studied in cultured cells (3). Evidence supports the presence of Z-DNA in vivo (1). Removal of DNA binding proteins by fixatives can initiate supercoiling which stabilizes Z-DNA sequences (1).Anti-Z-RNA polyclonal antibody probes were developed in rabbits immunized with multiple injections of Z-RNA: Br-poly[ribosomal(G-C)]. Regarding immunohistochemistry, lens tissues were fixed in Carnoy's, embedded in paraffin and sectioned (2.5 μm) (Fig. 2). Computerized image analysis was performed using a Leitz DM-RB microscope and Leica Quantiment 500 + image analyzer.

Positive and negative regulatory elements mediating transcription from the Drosophila melanogaster actin 5C distal promoter

1990 ◽  
Vol 10 (12) ◽  
pp. 6172-6180
Author(s):  
Y T Chung ◽  
E B Keller
Keyword(s):  
Drosophila Melanogaster ◽  
Transient Expression ◽  
Cultured Cells ◽  
Regulatory Elements ◽  
Positive Element ◽  
Negative Effect ◽  
Expression Activity ◽  
Replacement Experiment ◽  
Chloramphenicol Acetyltransferase Gene ◽  
Distal Promoter

The major cytoskeletal actin gene of Drosophila melanogaster, the actin 5C gene, has two promoters, the distal one of which controls synthesis of actin in a tissue- and developmental stage-specific manner. This very strong promoter has widely been used for expression of heterologous genes in cultured cells. To locate functional regulatory elements in this distal promoter, mutants of the promoter were fused to the bacterial chloramphenicol acetyltransferase gene and assayed for transient expression activity in cultured Drosophila embryonic Schneider line 2 cells. The results showed that the upstream end of the promoter extends to 522 bp from the transcription start site. In addition, there are two remote activating regions about 2 kb upstream. Between -522 and -379 are two regions that exert a strong negative effect. Downstream from these negative regions are at least six positive regions and a TATA element. The strongest positive determinant of the promoter was identified at -320 as AAAATGTG by footprinting and by a replacement experiment. When the relevant region was replaced by a synthetic sequence containing this element in a random context, the transient expression activity was restored. The sequence TGTATG located at -355 was also identified as a positive element by a similar replacement approach. Apparently the very high activity of this promoter is the result of the combined activities of multiple factors.

Construction of an expression vector containing a ?-actin promoter region for gene transfer by microinjection in red sea bream Pagrus major

2007 ◽  
Vol 73 (2) ◽  
pp. 440-445 ◽  
Author(s):  
Keitaro KATO ◽  
Masayoshi TAKAGI ◽  
Yutaka TAMARU ◽  
Shin-ichi AKIYAMA ◽  
Takafumi KONISHI ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Red Sea ◽  
Promoter Region ◽  
Expression Vector ◽  
Sea Bream ◽  
Red Sea Bream ◽  
Pagrus Major ◽  
Actin Promoter

scholarly journals Agrobacterium-mediated gene delivery and transient expression in the red macroalga Chondrus crispus

2018 ◽  
Vol 61 (5) ◽  
pp. 499-510 ◽  
Author(s):  
Anusha Devi Ramessur ◽  
John H. Bothwell ◽  
Christine A. Maggs ◽  
Sook Yee Gan ◽  
Siew Moi Phang
Keyword(s):  
Transient Expression ◽  
Red Algae ◽  
Transformation Efficiency ◽  
Bacterial Cells ◽  
Chondrus Crispus ◽  
Cultivation Medium ◽  
Intimate Association ◽  
Actin Promoter ◽  
Transfer Method ◽  
Gene Transfer Method

Abstract Molecular resources and transgenic studies in red algae are lagging behind those for green algae. The Agrobacterium-mediated gene-transfer method routinely used in plant transformation has not been fully utilised in the red algae, which, as an important source of phycocolloids, warrant more studies. In this regard, a stepwise methodology was developed for Agrobacterium-mediated transformation of the carrageenophyte Chondrus crispus using pCAMBIA 1301 and a construct featuring a codon-optimized beta-glucuronidase (GUS) reporter gene driven by the endogenous Chondrus actin promoter. The effects of several factors on transformation efficiency were investigated. An intimate association of Chondrus and bacterial cells was observed using scanning electron microscopy. GUS transient expression within Chondrus cortical and medullary cells with both expression cassettes testified to the amenability of Chondrus to Agrobacterium-mediated transformation. Darker staining, indicative of higher GUS activity, was observed with the Chondrus-specific construct, suggesting its superiority over the pCAMBIA 1301. Presence of acetosyringone, the wounding method and the type of co-cultivation medium significantly affected the transformation outcome and efficiency. The Agrobacterium-mediated transient expression presented here constitutes a first step towards tailoring a transformation strategy for Chondrus, which can serve to facilitate further transgenic studies in this important red alga.

scholarly journals Positive and negative regulatory elements mediating transcription from the Drosophila melanogaster actin 5C distal promoter.

1990 ◽  
Vol 10 (12) ◽  
pp. 6172-6180 ◽  
Author(s):  
Y T Chung ◽  
E B Keller
Keyword(s):  
Drosophila Melanogaster ◽  
Transient Expression ◽  
Cultured Cells ◽  
Regulatory Elements ◽  
Positive Element ◽  
Negative Effect ◽  
Expression Activity ◽  
Replacement Experiment ◽  
Chloramphenicol Acetyltransferase Gene ◽  
Distal Promoter

The major cytoskeletal actin gene of Drosophila melanogaster, the actin 5C gene, has two promoters, the distal one of which controls synthesis of actin in a tissue- and developmental stage-specific manner. This very strong promoter has widely been used for expression of heterologous genes in cultured cells. To locate functional regulatory elements in this distal promoter, mutants of the promoter were fused to the bacterial chloramphenicol acetyltransferase gene and assayed for transient expression activity in cultured Drosophila embryonic Schneider line 2 cells. The results showed that the upstream end of the promoter extends to 522 bp from the transcription start site. In addition, there are two remote activating regions about 2 kb upstream. Between -522 and -379 are two regions that exert a strong negative effect. Downstream from these negative regions are at least six positive regions and a TATA element. The strongest positive determinant of the promoter was identified at -320 as AAAATGTG by footprinting and by a replacement experiment. When the relevant region was replaced by a synthetic sequence containing this element in a random context, the transient expression activity was restored. The sequence TGTATG located at -355 was also identified as a positive element by a similar replacement approach. Apparently the very high activity of this promoter is the result of the combined activities of multiple factors.

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