Identification of Elements Essential for Transcription in Brugia malayi Promoters

Higazi, Tarig B.; Oliveira, Ana de; Katholi, Charles R.; Shu, Limin; Barchue, Joseph; Lisanby, Mark W.; Unnasch, Thomas R.

doi:10.1016/j.jmb.2005.08.014

Cited by 19 publications

(58 citation statements)

References 45 publications

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“…The parental clone of the promoter for the BmRPS12 gene (BmRPS12(−641 to −1)/luc) was prepared in the reporter vector pGL3 basic, as previously described (Higazi et al, 2005). Deletions of the 5′ domain of the parental construct and the repeat domains were prepared by restriction enzyme mediated inverse PCR, essentially as previously described (Shu et al, 2003).…”

Section: Preparation Of Deletion and Substitution Mutants Of The Bmrpmentioning

confidence: 99%

“…Furthermore, studies have demonstrated B. malayi can be transiently transfected using both biolistic and microinjection methods (Higazi et al, 2002). Biolistic transfection has been used to explore both promoter structure and trans-splicing in B. malayi (Higazi et al, 2002(Higazi et al, , 2005Shu et al, 2003;Higazi and Unnasch, 2004;Liu et al, 2007). These studies have suggested that the promoter structure of this parasite is relatively unique.…”

Section: Introductionmentioning

confidence: 99%

“…These studies have suggested that the promoter structure of this parasite is relatively unique. For example, detailed mapping studies of the promoter of the B. malayi 70 kDa heat shock protein (BmHSP70) revealed that this promoter contained four essential domains, ranging in size from 6 to 22 nucleotides (nt) (Higazi et al, 2005). The two most distal domains encoded a binding site for the heat shock transcription factor and a putative binding site for a GAGA transcription factor, motifs that are found in many other HSP70 promoters.…”

Section: Introductionmentioning

confidence: 99%

“…The two most distal domains encoded a binding site for the heat shock transcription factor and a putative binding site for a GAGA transcription factor, motifs that are found in many other HSP70 promoters. However, none of these essential domains contained sequences found in the core domain of a typical eukaryotic promoter, such as CAAT or TATAA boxes (Higazi et al, 2005). The largest essential domain was located at positions −53 to −32 relative to the start of the open reading frame (ORF) and included the splice leader (SL) addition site.…”

Section: Introductionmentioning

confidence: 99%

“…These data suggest that the regulatory domains of the BmHSP70 promoter were similar to those found in other eukaryotes, but that the core promoter domain exhibited features that appeared to be distinct from those found in most other wellcharacterised eukaryotic promoters. An analysis of two additional promoters of two B. malayi highly transcribed genes, the first encoding the B. malayi homologue of the 12 kDa peptide of the small subunit of the ribosome (the BmRPS12 gene) and the second encoding a putative RNA binding protein (the BmRBP1 gene) demonstrated that both were active in promoting transcription in the transient transfection system, whilst also lacking features commonly found in most eukaryotic core promoters (Higazi et al, 2005). Together, these studies suggested that the core domains of B. malayi promoters may lack many of the conserved elements found in most eukaryotic promoters.…”

Section: Introductionmentioning

confidence: 99%

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Characterization of the promoter of the Brugia malayi 12kDa small subunit ribosomal protein (RPS12) gene

Oliveira

Katholi

Unnasch

2008

International Journal for Parasitology

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Unravelling gene regulatory mechanisms in human filarial parasites will require an understanding of their basic promoter structure. Only a single promoter from a human filarial parasite has been characterised in detail, the 70 kDa heat shock promoter of Brugia malayi (BmHSP70). This promoter was found to lack features found in a typical eukaryotic promoter. To determine if this was unique to the BmHSP70 promoter, a detailed analysis was undertaken of the promoter for the B. malayi small subunit 12 kDa ribosomal protein (BmRPS12) gene. The BmRPS12 promoter contained a unique tandem repeat structure. Deletion of these repeats resulted in the loss of 80% of promoter activity. Block replacement mutagenesis identified five regions outside the repeat which were essential for promoter activity. No predicted binding sites for proteins that normally associate with the typical eukaryotic core promoter domains were found in the essential domains or the repeat region. However, the repeat region contained many putative binding sites for GATAA transcription factor family proteins. Of 20 upstream domains of other ribosomal protein genes, one contained a repeat structure similar to that found in the BmRPS12 promoter, and the majority encoded putative GATAA transcription factor binding sites. This study demonstrates that the BmRPS12 promoter, like the BmHSP70 promoter, is distinct from a typical eukaryotic promoter.

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Section: Preparation Of Deletion and Substitution Mutants Of The Bmrpmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Characterization of the promoter of the Brugia malayi 12kDa small subunit ribosomal protein (RPS12) gene

Oliveira

Katholi

Unnasch

2008

International Journal for Parasitology

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Biolistic Transformation of Brugia Malayi

Higazi

Unnasch

2012

Methods in Molecular Biology

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Biolistics has become a versatile tool for direct gene transfer to various cell and tissue types. Following its successful use on the parasitic nematode Ascaris suum, we developed and evaluated biolistics in the transfection of the model filarial parasite Brugia malayi. Biolistics was proven to be an efficient strategy for transfection of all life stages of the parasite and paved the way for studies on elements essential for promoter function and gene regulation of filarial parasites. Here we present a biolistics protocol for the transfection of B. malayi based on the Biolistics PDS 1000/He system and gold microcarriers.

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Functional analysis of putative operons in Brugia malayi

Liu

Oliveira

Chauhan

et al. 2010

International Journal for Parasitology

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Operons are a common mode of gene organization in Caenorhabditis elegans. Similar gene arrangements suggest that functional operons may exist in Brugia malayi. To definitively test this hypothesis, a bicistronic reporter vector consisting of an upstream firefly luciferase gene and a downstream renilla luciferase gene was constructed. The genome was then surveyed to identify 16 gene pairs that were likely to represent operons. Two of four domains upstream of the 5' gene from these clusters exhibited promoter activity. When constructs replicating the promoter and intergenic arrangement found in the native putative operon were transfected into embryos, both firefly and renilla activities were detected, while constructs with the promoter alone or intergenic region alone produced no activity from the downstream reporter. These data confirm that functional operons exist in B. malayi. Mutation of three U-rich element homologues present in one of the operons resulted in a decrease in downstream renilla reporter activity, suggesting that these were important in mRNA maturation. Hemi-nested reverse transcriptase-PCR assays demonstrated that while the mRNA encoding the native downstream open reading frame of one operon contained an SL1 spliced leader at its 5' end, the renilla gene mRNA produced from the corresponding transgenic construct did not.

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Identification of Elements Essential for Transcription in Brugia malayi Promoters

Cited by 19 publications

References 45 publications

Characterization of the promoter of the Brugia malayi 12kDa small subunit ribosomal protein (RPS12) gene

Characterization of the promoter of the Brugia malayi 12kDa small subunit ribosomal protein (RPS12) gene

Biolistic Transformation of Brugia Malayi

Functional analysis of putative operons in Brugia malayi

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