Ian G. Macreadie

Biogenesis of mitochondria: the mitochondrial gene (aap1) coding for mitochondrial ATPase subunit 8 in Saccharomyces cerevisiae

A mitochondrial gene (denoted aap1) in Saccharomyces cerevisiae has been characterized by nucleotide sequence analysis of a region of mtDNA between the oxi3 and oli2 genes. The reading frame of the aap1 gene specifies a hydrophobic polypeptide containing 48 amino acids. The functional nature of this reading frame was established by sequence analysis of a series of mit- mutants and revertants. Evidence is presented that the aap1 gene codes for a mitochondrially synthesized polypeptide associated with the mitochondrial ATPase complex. This polypeptide (denoted subunit 8) is a proteolipid whose size has been previously assumed to be 10 kilodaltons based on its mobility on SDS-polyacrylamide gels, but the sequence of the aap1 gene predicts a molecular weight of 5,815 for this protein.

Sequence of the small double-stranded RNA genomic segment of infectious bursal disease virus and its deduced 90-kDa product

The smaller dsRNA segment of the genome of infectious bursal disease virus (IBDV) encodes a single polypeptide of approximately 90 kDa (VP1). The consensus nucleotide sequence, derived from independent and overlapping cDNA clones, contains a single open reading frame which begins with an exact Kozak sequence and could encode a polypeptide of 878 amino acid residues. It has been suggested that VP1 could be the viral RNA-dependent RNA polymerase. A comparison of the predicted amino acid sequence of this protein with those of other DNA-dependent and ssRNA-dependent RNA polymerases has failed to reveal any homology between VP1 and the conserved regions in these enzymes. It is possible that the polypeptide encoded by the IBDV virus may represent a new class of polymerases which are involved in the replication of double-stranded RNA genomes.

Aβ aggregation and possible implications in Alzheimer's disease pathogenesis

•  Introduction •  Amyloid Structure •  Mechanism of Amyloid aggregation •  Aβ: a natively unfolded protein? •  Ambiguities in synthetic Ab studies •  Formation of Amyloid plaques •  Role of Ab in AD Pathogenesis •  Conclusion

Passive protection against infectious bursal disease virus by viral VP2 expressed in yeast

Infectious bursal disease virus (IBDV), a pathogen of major economic importance to the worlds poultry industries, causes a severe immunodepressive disease in young chickens. Maternal antibodies are able to protect the progeny passively from IBDV infection. The gene encoding the IBDV host-protective antigen (VP2) has been cloned and expressed in yeast resulting in the production of an antigen that very closely resembles native VP2. When injected into specific pathogen free chickens a single dose of microgram quantities of the yeast derived antigen induces high titres of virus neutralizing antibodies that are capable of passively protecting young chickens from infection with IBDV.

High-frequency binding of IgE to the Der p allergen expressed in yeast

The production of allergens from cDNA clones will provide a clonally pure source of material for experimental and perhaps clinical studies. Attempts to produce the major mite allergen, Der p I, in a highly antigenic form in bacteria have, to date, had limited success. In this study, a high level of production of Der p I from a Cup1 gene cassette from pYELC5-13T in Saccharomyces cerevisiae is described. Although the protein was insoluble, it could be readily solubilized in a urea solution and remained in solution when it was returned to more physiologic buffers. An amount equivalent to about 1 mg/L of yeast culture could then be isolated by affinity chromatography with an immobilized monoclonal antibody. This product reacted strongly with IgE in 9/11 sera from mite-allergic patients compared to the 50% reactivity achieved for Der p I previously produced as a fusion by bacteria. Similarly, the intensity of binding and ability to absorb out Der p I specificities were much greater for the yeast, pYELC5-13T, product. Studies with monoclonal antibodies also demonstrated the yeast, Der p I, had a high degree of antigenicity, although clear differences with the native allergen were demonstrated. The high frequency of reactivity with IgE of the pYELC5-13T formally demonstrates that a single gene product of Der p I is a major allergen and demonstrates that even for Der p I, which is synthesized from a proenzyme, considerable antigenicity can be obtained by expressing the mature protein.

Antimalarial Drug Development and New Targets

The Molecular Approaches to Malaria (MAM2000) conference, Lorne, Australia, 2-5 February 2000, brought together world-class malaria research scientists. The development of new tools and technologies - transfection, DNA microarrays and proteomic analysis - and the availability of DNA sequences generated by the Malaria Genome Project, along with more classic approaches, have facilitated the identification of novel drug targets, the development of new antimalarials and the generation of a deeper understanding of the molecular mechanism(s) of drug resistance in malaria. It is hoped that combinations of these technologies could lead to strategies that enable the development of effective, efficient and affordable new drugs to overcome drug-resistant malaria, as discussed at MAM2000 and outlined here by Ian Macreadie and colleagues.

A C-terminal domain of HIV-1 accessory protein Vpr is involved in penetration, mitochondrial dysfunction and apoptosis of human CD4+ lymphocytes

We have previously shown that expression of HIV-1 vpr in yeast results in cell growth arrest and structural defects, and identified a C-terminal domain of Vpr as being responsible for these effects in yeast.1 In this report we show that recombinant Vpr and C-terminal peptides of Vpr containing the conserved sequence HFRIGCRHSRIG caused permeabilization of CD4+ T lymphocytes, a dramatic reduction of mitochondrial membrane potential and finally cell death. Vpr and Vpr peptides containing the conserved sequence rapidly penetrated cells, co-localized with the DNA, and caused increased granularity and formation of dense apoptotic bodies. The above results suggest that Vpr treated cells undergo apoptosis and this was confirmed by demonstration of DNA fragmentation by the highly sensitive TUNEL assay. Our results, together with the demonstration of extracellular Vpr in HIV infected individuals,2,3 suggest the possibility that extracellular Vpr could contribute to the apoptotic death and depletion of bystander cells in lymphoid tissues4,5 during HIV infection.

Copper transport and Alzheimer’s disease

This brief review discusses copper transport in humans, with an emphasis on knowledge learned from one of the simplest model organisms, yeast. There is a further focus on copper transport in Alzheimer’s Disease (AD). Copper homeostasis is essential for the well-being of all organisms, from bacteria to yeast to humans: survival depends on maintaining the required supply of copper for the many enzymes, dependent on copper for activity, while ensuring that there is no excess free copper, which would cause toxicity. A virtual orchestra of proteins are required to achieve copper homeostasis. For copper uptake, Cu(II) is first reduced to Cu(I) via a membrane-bound reductase. The reduced copper can then be internalised by a copper transporter where it is transferred to copper chaperones for transport and specific delivery to various organelles. Of significance are internal copper transporters, ATP7A and ATP7B, notable for their role in disorders of copper deficiency and toxicity, Menkes and Wilson’s disease, respectively. Metallothioneins and Cu/Zn superoxide dismutase can protect against excess copper in cells. It is clear too, increasing age, environmental and lifestyle factors impact on brain copper. Studies on AD suggest an important role for copper in the brain, with some AD therapies focusing on mobilising copper in AD brains. The transport of copper into the brain is complex and involves numerous players, including amyloid precursor protein, Aβ peptide and cholesterol.

Improved shuttle vectors for cloning and high-level Cu2+ -mediated expression of foreign genes in yeast

New yeast episomal vectors having a high degree of utility for cloning and expression in Saccharomyces cerevisiae are described. One vector, pYEULlacZ, is based on pUC19 and employs the pUC19 multiple cloning site for the selection of recombinants in Escherichia coli by lacZ inactivation. In addition, the vector contains two genes, URA3 and leu2-d, for selection of the plasmid in ura3 or leu2 yeast strains. The presence of the leu2-d gene appears to promote replication at high copy numbers. The introduction of CUP1 cassettes allows these plasmids to direct Cu(2+)-regulated production of foreign proteins in yeast. We show the production of a helminth antigen as an example of the vector application.

Expression and analysis of the NS2 protein of influenza A virus

SummaryInfluenza NS2 protein was expressed inSaccharomyces cerevisiae using a copper-inducible promoter. The protein produced had a molecular weight of 13 kDa, was reactive with anti-NS2 antiserum and was localised to the yeast cell nucleus. Two-hybrid analysis identified a direct protein-protein interaction between NS2 and the M2 protein of the virus, involving the C-terminal 163 residues of M1. A filter-binding assay localised the M1 binding region to the C-terminal 70 amino acids of NS2.