Facilitated DNA transfer to rat submandibular gland in vivo and GRP-Ca gene regulation

1995 ◽  
Vol 268 (6) ◽  
pp. G1074-G1078 ◽  
Author(s):  
B. C. O'Connell ◽  
K. G. Ten Hagen ◽  
K. W. Lazowski ◽  
L. A. Tabak ◽  
B. J. Baum
Keyword(s):  
Luciferase Activity ◽  
Firefly Luciferase ◽  
Upstream Region ◽  
Luciferase Expression ◽  
Upstream Sequence ◽  
Luciferase Gene ◽  
Base Pairs ◽  
Firefly Luciferase Gene ◽  
Rat Submandibular Gland

The internalization of DNA can be facilitated by adenovirus infection. Using the replication-deficient adenovirus, Ad-dl312, and a plasmid-based firefly luciferase gene as a reporter, we have optimized the uptake and expression of DNA in rat submandibular glands in vivo. Luciferase expression is transient and peaked at approximately 18 h after infection. Luciferase activity increased with plasmid concentration and was greatest at 10(9) to 10(10) plaque-forming units of Ad-dl312 per gland. We next examined the expression in vivo of plasmids containing deletions of the glutamine/glutamic acid-rich protein (GRP-Ca isoform) gene upstream region linked to a chloramphenicol acetyltransferase (CAT) reporter. Constructs with 9.4, 6.3, and 2.7 kb and 17 base pairs of upstream sequence gave relative CAT activities of 100, 30, 7.6, and 38.5, respectively. With the 9.4-kb GRP-Ca construct, CAT was preferentially expressed in acinar cells, which is characteristic of GRP. This gene transfer approach should prove useful in the further study of gene expression in salivary glands and other organs.

scholarly journals In vivo analysis of the myosin heavy chain IIB promoter region

1998 ◽  
Vol 274 (3) ◽  
pp. C681-C687 ◽  
Author(s):  
Steven J. Swoap
Keyword(s):  
Reporter Gene ◽  
Luciferase Activity ◽  
Fiber Type ◽  
Firefly Luciferase ◽  
Renilla Luciferase ◽  
Luciferase Reporter ◽  
Base Pairs ◽  
Specific Expression ◽  
Mhc Iib

The myosin heavy chain (MHC) IIB gene is preferentially expressed in fast-twitch muscles of the hindlimb, such as the tibialis anterior (TA). The molecular mechanism(s) for this preferential expression are unknown. The goals of the current study were 1) to determine whether the cloned region of the MHC IIB promoter contains the necessary cis-acting element(s) to drive fiber-type-specific expression of this gene in vivo, 2) to determine which region within the promoter is responsible for fiber-type-specific expression, and 3) to determine whether transcription off of the cloned region of the MHC IIB promoter accurately mimics endogenous gene expression in a muscle undergoing a fiber-type transition. To accomplish these goals, a 2.6-kilobase fragment of the promoter-enhancer region of the MHC IIB gene was cloned upstream of the firefly luciferase reporter gene and coinjected with pRL-cytomegalovirus (CMV) (CMV promoter driving the renilla luciferase reporter) into the TA and the slow soleus muscle. Firefly luciferase activity relative to renilla luciferase activity within the TA was 35-fold greater than within the soleus. Deletional analysis demonstrated that only the proximal 295 base pairs (pGL3IIB0.3) were required to maintain this muscle-fiber-type specificity. Reporter gene expression of pGL3IIB0.3 construct was significantly upregulated twofold in unweighted soleus muscles compared with normal soleus muscles. Thus the region within the proximal 295 base pairs of the MHC IIB gene contains at least one element that can drive fiber-type-specific expression of a reporter gene.

Transcriptional regulation of the Enterobacter cloacae UW4 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene (acdS)

2001 ◽  
Vol 47 (4) ◽  
pp. 359-367 ◽  
Author(s):  
Jiping Li ◽  
Bernard R Glick
Keyword(s):  
Transcriptional Regulation ◽  
Luciferase Activity ◽  
Growth Promotion ◽  
Regulatory Protein ◽  
Acc Deaminase ◽  
Enterobacter Cloacae ◽  
Upstream Region ◽  
Luciferase Expression ◽  
Luciferase Gene ◽  
The One

Based on DNA sequence analysis and 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, the region of DNA immediately upstream of the Enterobacter cloacae UW4 ACC deaminase gene (acdS) contains several features that appear to be involved in its transcriptional regulation. In the present study, the 5' upstream region of acdS was cloned into the promoter-probe vector, pQF70, which carries the promoterless luciferase gene (luxAB), and luciferase expression was monitored. The data obtained from studying the expression of the luciferase gene showed that (i) a leucine responsive regulatory protein (LRP)-like protein encoded within the upstream region is located on the opposite strand from acdS under the control of a promoter stronger than the one responsible for acdS transcription, (ii) luciferase gene expression required both ACC and the LRP-like protein, (iii) luciferase expression was increased three-fold under anaerobic conditions, consistent with the involvement of a fumarate-nitrate reduction (FNR)-like regulatory protein box within the upstream region, and (iv) the addition of leucine to the growth medium decreased luciferase activity in the presence of ACC and increased luciferase activity in the absence of ACC, consistent with leucine acting as a regulator of the expression of the LRP-like protein.Key words: plant growth promotion, ethylene, ACC deaminase, regulation, Enterobacter cloacae.

scholarly journals Firefly luciferase gene: structure and expression in mammalian cells.

1987 ◽  
Vol 7 (2) ◽  
pp. 725-737 ◽  
Author(s):  
J R de Wet ◽  
K V Wood ◽  
M DeLuca ◽  
D R Helinski ◽  
S Subramani
Keyword(s):  
Mammalian Cells ◽  
Firefly Luciferase ◽  
Full Length ◽  
Expression Vectors ◽  
Luciferase Expression ◽  
Luciferase Gene ◽  
S1 Nuclease ◽  
Reading Frame ◽  
Mammalian Expression ◽  
Firefly Luciferase Gene

The nucleotide sequence of the luciferase gene from the firefly Photinus pyralis was determined from the analysis of cDNA and genomic clones. The gene contains six introns, all less than 60 bases in length. The 5' end of the luciferase mRNA was determined by both S1 nuclease analysis and primer extension. Although the luciferase cDNA clone lacked the six N-terminal codons of the open reading frame, we were able to reconstruct the equivalent of a full-length cDNA using the genomic clone as a source of the missing 5' sequence. The full-length, intronless luciferase gene was inserted into mammalian expression vectors and introduced into monkey (CV-1) cells in which enzymatically active firefly luciferase was transiently expressed. In addition, cell lines stably expressing firefly luciferase were isolated. Deleting a portion of the 5'-untranslated region of the luciferase gene removed an upstream initiation (AUG) codon and resulted in a twofold increase in the level of luciferase expression. The ability of the full-length luciferase gene to activate cryptic or enhancerless promoters was also greatly reduced or eliminated by this 5' deletion. Assaying the expression of luciferase provides a rapid and inexpensive method for monitoring promoter activity. Depending on the instrumentation employed to detect luciferase activity, we estimate this assay to be from 30- to 1,000-fold more sensitive than assaying chloramphenicol acetyltransferase expression.

Firefly luciferase gene: structure and expression in mammalian cells

1987 ◽  
Vol 7 (2) ◽  
pp. 725-737 ◽  
Author(s):  
J R de Wet ◽  
K V Wood ◽  
M DeLuca ◽  
D R Helinski ◽  
S Subramani
Keyword(s):  
Mammalian Cells ◽  
Firefly Luciferase ◽  
Full Length ◽  
Expression Vectors ◽  
Luciferase Expression ◽  
Luciferase Gene ◽  
S1 Nuclease ◽  
Reading Frame ◽  
Mammalian Expression ◽  
Firefly Luciferase Gene

The nucleotide sequence of the luciferase gene from the firefly Photinus pyralis was determined from the analysis of cDNA and genomic clones. The gene contains six introns, all less than 60 bases in length. The 5' end of the luciferase mRNA was determined by both S1 nuclease analysis and primer extension. Although the luciferase cDNA clone lacked the six N-terminal codons of the open reading frame, we were able to reconstruct the equivalent of a full-length cDNA using the genomic clone as a source of the missing 5' sequence. The full-length, intronless luciferase gene was inserted into mammalian expression vectors and introduced into monkey (CV-1) cells in which enzymatically active firefly luciferase was transiently expressed. In addition, cell lines stably expressing firefly luciferase were isolated. Deleting a portion of the 5'-untranslated region of the luciferase gene removed an upstream initiation (AUG) codon and resulted in a twofold increase in the level of luciferase expression. The ability of the full-length luciferase gene to activate cryptic or enhancerless promoters was also greatly reduced or eliminated by this 5' deletion. Assaying the expression of luciferase provides a rapid and inexpensive method for monitoring promoter activity. Depending on the instrumentation employed to detect luciferase activity, we estimate this assay to be from 30- to 1,000-fold more sensitive than assaying chloramphenicol acetyltransferase expression.

Cytokinin-Enhanced Regeneration of Plants From Microprojectile Bombarded Sugarcane Meristematic Tissue

1994 ◽  
Vol 21 (5) ◽  
pp. 603 ◽  
Author(s):  
RL Gambley ◽  
JD Bryant ◽  
NP Masel ◽  
GR Smith
Keyword(s):  
Luciferase Activity ◽  
Firefly Luciferase ◽  
Target Tissue ◽  
Luciferase Gene ◽  
Meristematic Tissue ◽  
Sugarcane Cultivar ◽  
Shoot Initiation ◽  
Cytokinin Treatment ◽  
Suitable Target ◽  
Firefly Luciferase Gene

Sugarcane plants expressing the firefly luciferase gene were regenerated from microprojectile-bombarded meristematic tissue. The concentration and type of cytokinin added to the basal media, both prior to bombardment and during regeneration, significantly affected the number of plantlets produced and the percentage of plantlets expressing luciferase. Maximum shoot initiation from meristematic tissue of sugarcane cultivar Q137 was achieved on media supplemented with N6-benzylaminopurine (BAP). There were differences in the numbers of shoots initiated from different sugarcane cultivars, and some optimisation for each cultivar was required. No luciferase activity was observed in plantlets regenerated in the absence of cytokinin. Sugarcane meristematic tissue preconditioned by exposure to cytokinin appears to be a suitable target tissue for the introduction of genes of agronomic importance into sugarcane. This approach should be readily adaptable to most commercial varieties of sugarcane with minor modification to the cytokinin treatment necessary for optimal regeneration.

Synthesis of 7′-[ 123 I]iodo- d -luciferin for in vivo studies of firefly luciferase gene expression

2004 ◽  
Vol 14 (5) ◽  
pp. 1161-1163 ◽  
Author(s):  
Sang-Yoon Lee ◽  
Yearn Seong Choe ◽  
Kyung-Han Lee ◽  
Jeewoo Lee ◽  
Yong Choi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Firefly Luciferase ◽  
In Vivo Studies ◽  
Luciferase Gene ◽  
Firefly Luciferase Gene

Isolation and Characterization of Broad-Spectrum Disease-Resistant Arabidopsis Mutants

Genetics ◽  
2002 ◽  
Vol 160 (4) ◽  
pp. 1661-1671
Author(s):  
Klaus Maleck ◽  
Urs Neuenschwander ◽  
Rebecca M Cade ◽  
Robert A Dietrich ◽  
Jeffery L Dangl ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Systemic Acquired Resistance ◽  
Acquired Resistance ◽  
Constitutive Expression ◽  
Firefly Luciferase ◽  
Marker Genes ◽  
Arabidopsis Mutants ◽  
Isolation And Characterization ◽  
Firefly Luciferase Gene

Abstract To identify Arabidopsis mutants that constitutively express systemic acquired resistance (SAR), we constructed reporter lines expressing the firefly luciferase gene under the control of the SAR-inducible PR-1 promoter (PR-1/luc). After EMS mutagenesis of a well-characterized transgenic line, we screened 250,000 M2 plants for constitutive expression of the reporter gene in vivo. From a mutant collection containing several hundred putative mutants, we concentrated on 16 mutants lacking spontaneous hypersensitive response (HR) cell death. We mapped 4 of these constitutive immunity (cim) mutants to chromosome arms. Constitutive expression of disease resistance was established by analyzing responses to virulent Peronospora parasitica and Pseudomonas syringae strains, by RNA blot analysis for endogenous marker genes, and by determination of salicylic acid levels in the mutants. The variety of the cim phenotypes allowed us to define distinct steps in both the canonical SAR signaling pathway and a separate pathway for resistance to Erysiphe cichoracearum, active in only a subset of the mutants.

Estrogen-inducible binding of a nuclear factor to the vitellogenin upstream region

1988 ◽  
Vol 8 (10) ◽  
pp. 4557-4560
Author(s):  
O Bakker ◽  
J N Philipsen ◽  
B C Hennis ◽  
G Ab
Keyword(s):  
Dimethyl Sulfate ◽  
Upstream Region ◽  
Nuclear Factor ◽  
Base Pairs ◽  
Exonuclease Iii ◽  
Factor I ◽  
Nuclear Factor I ◽  
Vitellogenin Gene

The estrogen-dependent binding of a protein to the upstream region of the chicken vitellogenin gene was detected by using in vivo dimethyl sulfate, genomic DNase I, and in vitro exonuclease III footprinting. The site is located between base pairs -848 and -824, and its sequence resembles that of the nuclear factor I binding site. The results suggest that a nuclear factor binding to this site is involved in the regulation of the vitellogenin gene.

scholarly journals Efficient mobilization of E1-deleted adenovirus type 5 vectors by wild-type adenoviruses of other serotypes

2002 ◽  
Vol 83 (6) ◽  
pp. 1311-1314 ◽  
Author(s):  
Hendrik J. Rademaker ◽  
Mohamed A. Abou El Hassan ◽  
Gijs A. Versteeg ◽  
Martijn J. W. E. Rabelink ◽  
Rob C. Hoeben
Keyword(s):  
Hepg2 Cells ◽  
Adenovirus Type ◽  
Firefly Luciferase ◽  
Luciferase Gene ◽  
Wild Type ◽  
Genome Packaging ◽  
Firefly Luciferase Gene ◽  
Adenovirus Vectors ◽  
Initiation Of Dna Replication ◽  
Adenovirus Type 5

Mobilization of replication-deficient adenovirus vectors can lead to spread and shedding of the vector. Here we show that in cultured HepG2 cells wild-type (wt) adenoviruses of subgroup A (Ad12), B (Ad7, 11 and 16), C (Ad1, 2 and 5) and E (Ad4) can efficiently mobilize Ad5CMVluc, a ΔE1ΔE3-Ad5 vector carrying the firefly luciferase gene as reporter. In addition, we show that Ad5CMVluc can be propagated on Ad12E1-transformed human embryonic retinoblasts. This provides evidence that expression of the E1 region of Ad12 is sufficient for mobilizing ΔE1-Ad5-derived vectors. Thus, in therapeutic applications of replication-defective Ad vectors any active Ad infection is of potential concern, independent of the serotype involved. To prevent vector mobilization by wt Ads, new vectors should be developed in which essential functions such as the initiation of DNA replication and genome packaging are restricted.

Sign in / Sign up

Export Citation Format

Share Document