Gene Expression and Biophysics Group

Pretoria, South Africa

Gene Expression and Biophysics Group

Pretoria, South Africa
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Lemgruber L.,University of Heidelberg | Lemgruber L.,Brazilian National Institute of Technology | Sant'Anna C.,Brazilian National Institute of Technology | Griffths C.,Gene Expression and Biophysics Group | And 4 more authors.
Microscopy and Microanalysis | Year: 2015

Borrelia burgdorferi sensu lato, the causative agent of Lyme disease, is transmitted to humans through the bite of infected Ixodes spp. ticks. Successful infection of vertebrate hosts necessitates sophisticated means of the pathogen to escape the vertebrates' immune system. One strategy employed by Lyme disease spirochetes to evade adaptive immunity involves a highly coordinated regulation of the expression of outer surface proteins that is vital for infection, dissemination, and persistence. Here we characterized the expression pattern of bacterial surface antigens using different microscopy techniques, from fluorescent wide field to super-resolution and immunogold-scanning electron microscopy. A fluorescent strain of B. burgdorferi spirochetes was labeled with monoclonal antibodies directed against various bacterial surface antigens. Our results indicate that OspA is more evenly distributed over the surface than OspB and OspC that were present as punctate areas. © Microscopy Society of America 2015.


Meliopoulos V.A.,University of Georgia | Andersen L.E.,University of Georgia | Birrer K.F.,University of Georgia | Birrer K.F.,Deakin University | And 17 more authors.
FASEB Journal | Year: 2012

Influenza virus encodes only 11 viral proteins but replicates in a broad range of avian and mammalian species by exploiting host cell functions. Genome-wide RNA interference (RNAi) has proven to be a powerful tool for identifying the host molecules that participate in each step of virus replication. Metaanalysis of findings from genome-wide RNAi screens has shown influenza virus to be dependent on functional nodes in host cell pathways, requiring a wide variety of molecules and cellular proteins for replication. Because rapid evolution of the influenza A viruses persistently complicates the effectiveness of vaccines and therapeutics, a further understanding of the complex host cell pathways coopted by influenza virus for replication may provide new targets and strategies for antiviral therapy. RNAi genome screening technologies together with bioinformatics can provide the ability to rapidly identify specific host factors involved in resistance and susceptibility to influenza virus, allowing for novel disease intervention strategies. © FASEB.


Henriques R.,University of Lisbon | Henriques R.,Institute Pasteur Paris | Griffiths C.,Gene Expression and Biophysics Group | Griffiths C.,University of Pretoria | And 4 more authors.
Biopolymers | Year: 2011

Live-cell fluorescence light microscopy has emerged as an important tool in the study of cellular biology. The development of fluorescent markers in parallel with super-resolution imaging systems has pushed light microscopy into the realm of molecular visualization at the nanometer scale. Resolutions previously only attained with electron microscopes are now within the grasp of light microscopes. However, until recently, live-cell imaging approaches have eluded super-resolution microscopy, hampering it from reaching its full potential for revealing the dynamic interactions in biology occurring at the single molecule level. Here we examine recent advances in the super-resolution imaging of living cells by reviewing recent breakthroughs in single molecule localization microscopy methods such as PALM and STORM to achieve this important goal. © 2011 Wiley Periodicals, Inc.


Fanucchi S.,Gene Expression and Biophysics Group | Shibayama Y.,Gene Expression and Biophysics Group | Mhlanga M.M.,Gene Expression and Biophysics Group | Mhlanga M.M.,University of Lisbon
Nucleus (United States) | Year: 2014

Chromatin loops are pervasive and permit the tight compaction of DNA within the confined space of the nucleus. Looping enables distal genes and DNA elements to engage in chromosomal contact, to form multigene complexes. Advances in biochemical and imaging techniques reveal that loop-mediated contact is strongly correlated with transcription of interacting DNA. However, these approaches only provide a snapshot of events and therefore are unable to reveal the dynamics of multigene complex assembly. This highlights the necessity to develop single cell-based assays that provide single molecule resolution, and are able to functionally interrogate the role of chromosomal contact on gene regulation. To this end, high-resolution single cell imaging regimes, combined with genome editing approaches, are proving to be pivotal to advancing our understanding of loop-mediated dynamics. © 2014 Landes Bioscience.


Fok E.T.,University of Witwatersrand | Penny C.B.,University of Witwatersrand | Mhlanga M.M.,Gene Expression and Biophysics Group | Mhlanga M.M.,University of Lisbon | And 2 more authors.
South African Journal of Science | Year: 2015

Efficient and robust genome editing tools and strategies allow for specific and exact genetic changes to be captured in model systems, thereby accelerating both forward and reverse genetics studies. The development of CRISPR/Cas9 as a facile designer nuclease toolset has allowed for defined genetic modifications to be efficiently made through homology-directed repair of targeted DNA double-stranded breaks (DSBs) using exogenous repair templates. However, traditional single DSB strategies are still relatively inefficient as the short gene conversion tracts of mammalian cell systems limit the extent of achievable gene alteration from the DSB site. In order to improve on the inefficiency, we devised a dual cut strategy, which relies on reconstituting entire deleted gene fragments to precisely modify extensive gene regions of interest. Using the CRISPR/Cas9 system, we were able to introduce targeted deletions and repair of the endogenous KRAS gene locus in cell culture. The use of two simultaneous DSBs can be employed for efficient application of homology-directed repair with a large dsDNA donor sequence, thereby improving the efficacy of deriving cells with a desired gene editing outcome. In conclusion, a multiplexed CRISPR/Cas9 editing strategy represents an efficient tool for the editing of complex, heterologous sequence tracts. © 2015. The Author(s).


Fanucchi S.,Gene Expression and Biophysics Group | Shibayama Y.,Gene Expression and Biophysics Group | Burd S.,Gene Expression and Biophysics Group | Weinberg M.S.,University of Witwatersrand | And 3 more authors.
Cell | Year: 2013

Transcription of coregulated genes occurs in the context of long-range chromosomal contacts that form multigene complexes. Such contacts and transcription are lost in knockout studies of transcription factors and structural chromatin proteins. To ask whether chromosomal contacts are required for cotranscription in multigene complexes, we devised a strategy using TALENs to cleave and disrupt gene loops in a well-characterized multigene complex. Monitoring this disruption using RNA FISH and immunofluorescence microscopy revealed that perturbing the site of contact had a direct effect on transcription of other interacting genes. Unexpectedly, this effect on cotranscription was hierarchical, with dominant and subordinate members of the multigene complex engaged in both intra- and interchromosomal contact. This observation reveals the profound influence of these chromosomal contacts on the transcription of coregulated genes in a multigene complex. © 2013 Elsevier Inc.


Barichievy S.,Gene Expression and Biophysics Group | Barichievy S.,Astrazeneca | Naidoo J.,Gene Expression and Biophysics Group | Mhlanga M.M.,Gene Expression and Biophysics Group | Mhlanga M.M.,University of Lisbon
Frontiers in Genetics | Year: 2015

On October 28th 1943 Winston Churchill said "we shape our buildings, and afterward our buildings shape us" (Humes, 1994). Churchill was pondering how and when to rebuild the British House of Commons, which had been destroyed by enemy bombs on May 10th 1941. The old House had been small and insufficient to hold all its members, but was restored to its original form in 1950 in order to recapture the "convenience and dignity" that the building had shaped into its parliamentary members. The circular loop whereby buildings or dwellings are shaped and go on to shape those that reside in them is also true of pathogens and their hosts. As obligate parasites, pathogens need to alter their cellular host environments to ensure survival. Typically pathogens modify cellular transcription profiles and in doing so, the pathogen in turn is affected, thereby closing the loop. As key orchestrators of gene expression, non-coding RNAs provide a vast and extremely precise set of tools for pathogens to target in order to shape the cellular environment. This review will focus on host non-coding RNAs that are manipulated by the infamous intracellular pathogen, the human immunodeficiency virus (HIV). We will briefly describe both short and long host non-coding RNAs and discuss how HIV gains control of these factors to ensure widespread dissemination throughout the host as well as the establishment of lifelong, chronic infection. © 2015 Barichievy, Naidoo and Mhlanga.


PubMed | Gene Expression and Biophysics Group, Astrazeneca and University of Lisbon
Type: | Journal: Frontiers in genetics | Year: 2015

On October 28th 1943 Winston Churchill said we shape our buildings, and afterward our buildings shape us (Humes, 1994). Churchill was pondering how and when to rebuild the British House of Commons, which had been destroyed by enemy bombs on May 10th 1941. The old House had been small and insufficient to hold all its members, but was restored to its original form in 1950 in order to recapture the convenience and dignity that the building had shaped into its parliamentary members. The circular loop whereby buildings or dwellings are shaped and go on to shape those that reside in them is also true of pathogens and their hosts. As obligate parasites, pathogens need to alter their cellular host environments to ensure survival. Typically pathogens modify cellular transcription profiles and in doing so, the pathogen in turn is affected, thereby closing the loop. As key orchestrators of gene expression, non-coding RNAs provide a vast and extremely precise set of tools for pathogens to target in order to shape the cellular environment. This review will focus on host non-coding RNAs that are manipulated by the infamous intracellular pathogen, the human immunodeficiency virus (HIV). We will briefly describe both short and long host non-coding RNAs and discuss how HIV gains control of these factors to ensure widespread dissemination throughout the host as well as the establishment of lifelong, chronic infection.


Fanucchi S.,Gene Expression and Biophysics Group
Nucleus (Austin, Tex.) | Year: 2014

Chromatin loops are pervasive and permit the tight compaction of DNA within the confined space of the nucleus. Looping enables distal genes and DNA elements to engage in chromosomal contact, to form multigene complexes. Advances in biochemical and imaging techniques reveal that loop-mediated contact is strongly correlated with transcription of interacting DNA. However, these approaches only provide a snapshot of events and therefore are unable to reveal the dynamics of multigene complex assembly. This highlights the necessity to develop single cell-based assays that provide single molecule resolution, and are able to functionally interrogate the role of chromosomal contact on gene regulation. To this end, high-resolution single cell imaging regimes, combined with genome editing approaches, are proving to be pivotal to advancing our understanding of loop-mediated dynamics.


PubMed | Gene Expression and Biophysics Group
Type: Journal Article | Journal: Cell | Year: 2013

Transcription of coregulated genes occurs in the context of long-range chromosomal contacts that form multigene complexes. Such contacts and transcription are lost in knockout studies of transcription factors and structural chromatin proteins. To ask whether chromosomal contacts are required for cotranscription in multigene complexes, we devised a strategy using TALENs to cleave and disrupt gene loops in a well-characterized multigene complex. Monitoring this disruption using RNA FISH and immunofluorescence microscopy revealed that perturbing the site of contact had a direct effect on transcription of other interacting genes. Unexpectedly, this effect on cotranscription was hierarchical, with dominant and subordinate members of the multigene complex engaged in both intra- and interchromosomal contact. This observation reveals the profound influence of these chromosomal contacts on the transcription of coregulated genes in a multigene complex.

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