Biomedical subjects

John A Dangerfield

Publications and source records attributed to John A Dangerfield.

4 recordsLinked to original sources

PubMed2006

Enhancement of the StreptoTag method for isolation of endogenously expressed proteins with complex RNA binding targets.

StreptoTag is a novel affinity chromatography-based method for the isolation of high- and low-affinity RNA binding proteins. Originally it was shown possible to isolate recombinant protein from yeast or bacterial extracts using small, specific, well-characterised RNA binding targets. Here we show that using an enhanced aptamer it is not only possible to efficiently immobilise large, highly structured RNA binding targets onto the streptomycin columns but also that the StreptoTag method can be used for the isolation and purification of endogenously expressed regulatory proteins, with relatively low abundance, from eukaryotic extracts. As an example for this we uncover the identity of a karyophilic cellular protein which specifically binds to an area within the large, highly folded structure that characterises the mRNA from the unique 3' region (U3) of the mouse mammary tumour virus (MMTV) long terminal repeat (LTR). Hence, this method is now suitable for the quick and efficient isolation and identification of novel RNA binding proteins such as regulatory factors.

Amino Acid Sequence↗

PubMed2006

Mutation of the Rev-binding loop in the human immunodeficiency virus 1 leader causes a replication defect characterized by altered RNA trafficking and packaging.

An internal RNA loop, located within the packaging signal of human immunodeficiency virus 1, that resembles the Rev-responsive element (RRE) closely was identified previously. Subsequent in vitro studies confirmed that the loop, termed loop A, could bind Rev protein specifically. Its proximity to the major splice donor has suggested a role for Rev-loop A interaction supplementary to or preceding that of the Rev-RRE interaction. To investigate this further in a replication-competent provirus, loop A was mutated to decrease its affinity for Rev. Impairing the Rev-loop A interaction led to reduced nuclear export of viral genomic RNA. RNA packaging decreased by approximately 30%. Viral protein production and export of virus particles appeared normal; however, the virus was severely replication-deficient. The loop A sequence, which is 98% conserved amongst viral isolates, is implicated in several cis-acting functions critical to virus viability.

Animals↗

PubMed2005

MMTV accessory factor Naf affects cellular gene expression.

Mouse mammary tumour virus (MMTV) encodes a viral superantigen (Sag) and a negative acting factor (Naf) which share parts of their coding sequence. Using 2-dimensional gel electrophoresis (2D-DIGE), we could show that at least 10 different cellular proteins were differentially expressed in Naf positive cells. Also, luciferase reporter expression was down-regulated in Naf expressing cells independent of the promoter used and further experiments suggested that this effect was due in part to a decrease in cellular growth rates. Although in Naf positive cells expression of the major sag containing transcript was strongly induced by the synthetic glucocorticoid dexamethasone, the hormone analogue neither influenced luciferase expression nor mRNA expression of selected cellular proteins identified by 2D-DIGE. Taken together, these data support the previous finding that Naf and Sag have separable activities and suggest that Naf may play a role in modulating host cell gene expression during MMTV infection.

Animals↗

PubMed2005

HIV-1 Rev can specifically interact with MMTV RNA and upregulate gene expression.

We present evidence that the HIV-1 Rev protein can heterologously regulate expression of the simple beta retrovirus mouse mammary tumour virus (MMTV). Up to 10-fold upregulation was seen in a functional assay system when specific MMTV sequences were substituted for the HIV-1 Rev responsive element (RRE). RNA gel shift analysis showed that purified recombinant Rev could specifically bind to MMTV unique region 3 prime (U3) RNA and that these sequences could compete for wild-type Rev-RRE binding approximately 20-fold more efficiently than a non-specific competitor RNA. Using a combination of in silico and deletion mutation analyses, it was not possible to define any single specific secondary structure responsive to Rev, suggesting that a structure or combination of structures that only form in the context of the complete U3 transcript is/are required to interact with Rev. Taken together, these results suggest that HIV-1 Rev can directly bind to MMTV RNA as well as mediate upregulation of MMTV gene expression.

Animals↗