The characterisation of trypanosomal type 1 DnaJ-like proteins
- Authors: Ludewig, Michael Hans
- Date: 2010
- Subjects: Molecular genetics , Molecular chaperones , Protozoa , Heat shock proteins , Trypanosoma
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4126 , http://hdl.handle.net/10962/d1015205
- Description: Trypanosomes are protozoans, of which many are parasitic, and possess complex lifecycles which alternate between mammalian and arthropod hosts. As is the case with most organisms, molecular chaperones and heat shock proteins are encoded within the genomes of these protozoans. These proteins are an integral part of maintaining the structural integrity of proteins during normal and stress conditions. Heat shock protein 40 (Hsp40) is a co-chaperone of heat shock protein 70 (Hsp70) and in some cases can act as a chaperone. These proteins work together to bind non-native polypeptide structures to prevent unfolded protein aggregrate formation in times of stress, translocate proteins across organelle membranes, and transport unsalvageable proteins to proteolytic degradation by the cellular proteasome. Hsp40s are divided into four types based on their domain structure. Analysis of the nuclear genomes of eight trypanosomatid species revealed that less than 10 of the approximate 70 Hsp40 sequences per genome were Type 1 Hsp40s, many of which contained putative orthologues in the other seven trypanosomatid genomes. One of these Type 1 Hsp40s from T b. brucei, Trypanosoma brucei DnaJ 2 (Tbj2), was functionally characterised in T brucei brucei. RNA interference knockdown of expression in T brucei brucei showed that cells deficient in Tbj2 displayed a severe inhibition of the growth of the cell population. The levels of the Tbj2 protein population in T brucei brucei cells increases after exposure to 42°c and the protein was found to have a generalized cytoplasmic subcellular localization at 37°c. These findings provide evidence that Tbj2 is an orthologue of Yeast DnaJ 1 (Y dj l), an essential S. cerevisiae protein. Hsp40s interact with their partner Hsp70s through their J-domain. The amino acids of the J-domain important for a functional interaction with Hsp70 were examined in Trypanosoma cruzi DnaJ 2 (Tcj2) (the orthologue of Tbj2) and T cruzi DnaJ protein 3 (Tcj3) by testing their ability to substitute for Y dj l in Saccharomyces cerevisae and for DnaJ in Escherichia coli. In both systems, the positively charged amino acids of Helix II and III of the J-domain disrupted the functional interaction of these Hsp40s with their partner Hsp70s. Substitutions in Helix I and IV of the J-domains of Tcj2 and Tcj3 produced varied results in the two different systems, possibly suggesting that these helices serve to define with which Hsp70s a given Hsp40 can interact. The inability of an Hsp40 and an Hsp70 to interact functionally does not necessarily mean a total absence of physical interaction between these proteins. The amino acid substitution of the histidine in the HPD motif (H34Q) of the J-domain of Tcj2 and Tcj3 removed the ability of these proteins to interact functionally with S. cerevisiae Hsp70 (Ssal) in vivo. However, preliminary binding studies using the quartz crystal microbalance with dissipation monitoring (QCM-D) show that Tcj2 and Tcj2(H34Q) both physically interact with M sativa Hsp70 in vitro. This study is the first report to provide evidence that certain trypanosoma! Type 1 Hsp40s are essential proteins. Futhermore, the interaction of these Hsp40s with Hsp70 identified important features of the functional interface of this chaperone machinery.
- Full Text:
- Date Issued: 2010
- Authors: Ludewig, Michael Hans
- Date: 2010
- Subjects: Molecular genetics , Molecular chaperones , Protozoa , Heat shock proteins , Trypanosoma
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4126 , http://hdl.handle.net/10962/d1015205
- Description: Trypanosomes are protozoans, of which many are parasitic, and possess complex lifecycles which alternate between mammalian and arthropod hosts. As is the case with most organisms, molecular chaperones and heat shock proteins are encoded within the genomes of these protozoans. These proteins are an integral part of maintaining the structural integrity of proteins during normal and stress conditions. Heat shock protein 40 (Hsp40) is a co-chaperone of heat shock protein 70 (Hsp70) and in some cases can act as a chaperone. These proteins work together to bind non-native polypeptide structures to prevent unfolded protein aggregrate formation in times of stress, translocate proteins across organelle membranes, and transport unsalvageable proteins to proteolytic degradation by the cellular proteasome. Hsp40s are divided into four types based on their domain structure. Analysis of the nuclear genomes of eight trypanosomatid species revealed that less than 10 of the approximate 70 Hsp40 sequences per genome were Type 1 Hsp40s, many of which contained putative orthologues in the other seven trypanosomatid genomes. One of these Type 1 Hsp40s from T b. brucei, Trypanosoma brucei DnaJ 2 (Tbj2), was functionally characterised in T brucei brucei. RNA interference knockdown of expression in T brucei brucei showed that cells deficient in Tbj2 displayed a severe inhibition of the growth of the cell population. The levels of the Tbj2 protein population in T brucei brucei cells increases after exposure to 42°c and the protein was found to have a generalized cytoplasmic subcellular localization at 37°c. These findings provide evidence that Tbj2 is an orthologue of Yeast DnaJ 1 (Y dj l), an essential S. cerevisiae protein. Hsp40s interact with their partner Hsp70s through their J-domain. The amino acids of the J-domain important for a functional interaction with Hsp70 were examined in Trypanosoma cruzi DnaJ 2 (Tcj2) (the orthologue of Tbj2) and T cruzi DnaJ protein 3 (Tcj3) by testing their ability to substitute for Y dj l in Saccharomyces cerevisae and for DnaJ in Escherichia coli. In both systems, the positively charged amino acids of Helix II and III of the J-domain disrupted the functional interaction of these Hsp40s with their partner Hsp70s. Substitutions in Helix I and IV of the J-domains of Tcj2 and Tcj3 produced varied results in the two different systems, possibly suggesting that these helices serve to define with which Hsp70s a given Hsp40 can interact. The inability of an Hsp40 and an Hsp70 to interact functionally does not necessarily mean a total absence of physical interaction between these proteins. The amino acid substitution of the histidine in the HPD motif (H34Q) of the J-domain of Tcj2 and Tcj3 removed the ability of these proteins to interact functionally with S. cerevisiae Hsp70 (Ssal) in vivo. However, preliminary binding studies using the quartz crystal microbalance with dissipation monitoring (QCM-D) show that Tcj2 and Tcj2(H34Q) both physically interact with M sativa Hsp70 in vitro. This study is the first report to provide evidence that certain trypanosoma! Type 1 Hsp40s are essential proteins. Futhermore, the interaction of these Hsp40s with Hsp70 identified important features of the functional interface of this chaperone machinery.
- Full Text:
- Date Issued: 2010
The establishment of a virus free laboratory colony of Cryptophlebia leucotreta (False Codling Moth) and characterisation of Cryptophlebia leucotreta Granulovirus (CrleGV) genes
- Authors: Ludewig, Michael Hans
- Date: 2003
- Subjects: Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control , DNA viruses , Agricultural pests -- Biological control , Baculoviruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3957 , http://hdl.handle.net/10962/d1004016 , Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control , DNA viruses , Agricultural pests -- Biological control , Baculoviruses
- Description: Cryptophlebia leucotreta is an economically important agricultural pest throughout Sub-Saharan Africa. CrleGV has been considered as an alternative to chemical control of this pest due to its host specificity and innocuous nature towards vertebrates. A CrleGV free laboratory colony of C. leucotreta would be useful for the isolation of genotypically pure strains of the CrleGV and for virulence comparisons between isolates. It is preferable to have a full characterisation of CrleGV prior to its registration and release into the environment as a biopesticide. A laboratory colony of C. leucotreta, set up at Rhodes University, containing a low level of infection indicated that CrleGV is vertically transmitted. To establish a virus free laboratory colony of C. leucotreta, a solution of 3.5% sodium hypochlorite and 1% Tween 20 was used to surface decontaminate C. leucotreta eggs for removal of transovum CrleGV from the laboratory colony. No apparent infection by CrleGV was induced by subjecting larvae to stress. PCR of DNA extracted from larvae using CTAB failed to detect virus in the laboratory colony. This detection protocol was able to detect down to 60 fg (480 genome copies of CrleGV). The possibility of low-level virus remaining in the colony requires monitoring of genotypic purity of virus manipulated in the colony. Sequencing of Bam HI/KpnI fragments produced a preliminary sequence of the granulin region of CrleGV. This preliminary sequence supports the trend that the gene organisation of the granulin region of the granuloviruses infecting the family Tortricidae is conserved.
- Full Text:
- Date Issued: 2003
- Authors: Ludewig, Michael Hans
- Date: 2003
- Subjects: Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control , DNA viruses , Agricultural pests -- Biological control , Baculoviruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3957 , http://hdl.handle.net/10962/d1004016 , Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control , DNA viruses , Agricultural pests -- Biological control , Baculoviruses
- Description: Cryptophlebia leucotreta is an economically important agricultural pest throughout Sub-Saharan Africa. CrleGV has been considered as an alternative to chemical control of this pest due to its host specificity and innocuous nature towards vertebrates. A CrleGV free laboratory colony of C. leucotreta would be useful for the isolation of genotypically pure strains of the CrleGV and for virulence comparisons between isolates. It is preferable to have a full characterisation of CrleGV prior to its registration and release into the environment as a biopesticide. A laboratory colony of C. leucotreta, set up at Rhodes University, containing a low level of infection indicated that CrleGV is vertically transmitted. To establish a virus free laboratory colony of C. leucotreta, a solution of 3.5% sodium hypochlorite and 1% Tween 20 was used to surface decontaminate C. leucotreta eggs for removal of transovum CrleGV from the laboratory colony. No apparent infection by CrleGV was induced by subjecting larvae to stress. PCR of DNA extracted from larvae using CTAB failed to detect virus in the laboratory colony. This detection protocol was able to detect down to 60 fg (480 genome copies of CrleGV). The possibility of low-level virus remaining in the colony requires monitoring of genotypic purity of virus manipulated in the colony. Sequencing of Bam HI/KpnI fragments produced a preliminary sequence of the granulin region of CrleGV. This preliminary sequence supports the trend that the gene organisation of the granulin region of the granuloviruses infecting the family Tortricidae is conserved.
- Full Text:
- Date Issued: 2003
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