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 An Upstream Sequence Element Required for NucC-Dependent Expression of the Serratia marcescensExtracellular Nuclease

1998 ◽  
Vol 180 (22) ◽  
pp. 6064-6067 ◽  
Author(s):  
Robert H. Winslow ◽  
Bryan Julien ◽  
Richard Calendar ◽  
Gail E. Christie
Keyword(s):  
Serratia Marcescens ◽  
Binding Site ◽  
Upstream Region ◽  
Upstream Sequence ◽  
Gene Encoding ◽  
Sequence Element ◽  
Activator Proteins ◽  
Footprint Analysis ◽  
Extracellular Nuclease ◽  
Dyad Symmetry

ABSTRACT The Serratia marcescens extracellular nuclease gene,nucA, is positively regulated by the product of thenucC gene. In this study, the upstream region required for NucC-dependent nuclease expression was defined by using fusions to the gene encoding chloramphenicol acetyltransferase (cat). This sequence includes an element of hyphenated dyad symmetry identified previously as the binding site for the P2 Ogr family of activators. Footprint analysis confirmed that members of this family of activator proteins bind to this site, protecting a region between −76 and −59 relative to the start of transcription. The activator binding site in the nucA promoter lies one turn of the helix upstream from the corresponding sites in the P2 and P4 late promoters. The effects of deletions between the downstream end of the activator binding site and the putative −35 region are consistent with a strict helical phasing requirement for activation.

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 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.

Overproduction of yeast viruslike particles by strains deficient in a mitochondrial nuclease

1989 ◽  
Vol 9 (8) ◽  
pp. 3323-3331
Author(s):  
Y X Liu ◽  
C L Dieckmann
Keyword(s):  
Coat Protein ◽  
Killer Toxin ◽  
Nuclear Gene ◽  
Fold Increase ◽  
Mitochondrial Outer Membrane ◽  
Gene Encoding ◽  
Recessive Mutations ◽  
A Genome ◽  
Nonfermentable Carbon Source ◽  
Viruslike Particles

Saccharomyces cerevisiae strains are often host to several types of cytoplasmic double-stranded RNA (dsRNA) genomes, some of which are encapsidated by the L-A dsRNA product, an 86,000-dalton coat protein. Here we present the finding that nuclear recessive mutations in the NUC1 gene, which encodes the major nonspecific nuclease of yeast mitochondria, resulted in at least a 10-fold increase in amounts of the L-A dsRNA and its encoded coat protein. The effect of nuc1 mutations on L-A abundance was completely suppressed in strains that also hosted the killer-toxin-encoding M dsRNA. Both NUC1 and nuc1 strains containing the L-A genome exhibited an increase in coat protein abundance and a concomitant increase in L-A dsRNA when the cells were grown on a nonfermentable carbon source rather than on glucose, an effect independent of the increase in coat protein due to nuc1 mutations or to the absence of M. The increase in L-A expression in nuc1 strains was similar to that observed in strains with mutations in the nuclear gene encoding the most abundant outer mitochondrial membrane protein, porin. nuc1 mutations did not affect the level of porin in the mitochondrial outer membrane. Since the effect of mutations in nuc1 was to alter the copy number of the L-A coat protein genome rather than to change the level of the M toxin genome (as do mak and ski mutations), these mutations define a new class of nuclear genes affecting yeast dsRNA abundance.

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 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.

scholarly journals A Novel Gene Encoding Xanthan Lyase of Paenibacillus alginolyticus Strain XL-1

2000 ◽  
Vol 66 (9) ◽  
pp. 3945-3950 ◽  
Author(s):  
Harald J. Ruijssenaars ◽  
Sybe Hartmans ◽  
Jan C. Verdoes
Keyword(s):  
Signal Sequence ◽  
Fold Increase ◽  
Side Chain ◽  
Cloning And Sequencing ◽  
Gene Encoding ◽  
E Coli ◽  
Mature Enzyme ◽  
Locust Bean ◽  
Encoding Gene ◽  
Amino Acid Signal

ABSTRACT Xanthan-modifying enzymes are powerful tools in studying structure-function relationships of this polysaccharide. One of these modifying enzymes is xanthan lyase, which removes the terminal side chain residue of xanthan. In this paper, the cloning and sequencing of the first xanthan lyase-encoding gene is described, i.e., thexalA gene, encoding pyruvated mannose-specific xanthan lyase of Paenibacillus alginolyticus XL-1. ThexalA gene encoded a 100,823-Da protein, including a 36-amino-acid signal sequence. The 96,887-Da mature enzyme could be expressed functionally in Escherichia coli. Like the native enzyme, the recombinant enzyme showed no activity on depyruvated xanthan. Compared to production by P. alginolyticus, a 30-fold increase in volumetric productivity of soluble xanthan lyase was achieved by heterologous production in E. coli. The recombinant xanthan lyase was used to produce modified xanthan, which showed a dramatic loss of the capacity to form gels with locust bean gum.

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)

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.

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