Introduction

The research projects of the Division of Biochemistry and Molecular Biology have the common aim of gaining an understanding at the molecular level of the life processes occurring in living cells and of the derangements in these processes which result in disease. The projects relate to clinical medicine in areas such as the control of influenza and other viral diseases, in understanding asthma and allergy, in anaesthetic complications and in diabetes.

The Membrane Biology Program studies the function of membrane ion channels involved in energy transduction, nerve conduction and in viruses. The latter work is creating new opportunities for controlling the replication of viruses such as HIV. The Division also has a strong group working on structural predictions, the Computational Molecular Biology and Drug Design Group.

The Medical Molecular Biology Group has continued its collaborative study on the structure of the interleukin-5 receptor with the X-ray Crystallography Group of the Research School of Chemistry as part of the Centre for Molecular Structure and Function. Interleukin-5 is a new drug target for asthma. This work also interfaces with the research of the Leukocyte Signalling and Regulation Laboratory and the Gene Targeting Laboratory which collaborate in using transgenic animals to study allergic lung disease in mice. The Division also has a major program on Transcriptional Regulation with high quality research on nuclear translocation, chromatin function and cytokine gene transcription.

The Division has continued to participate in projects of the Centre for Molecular Structure and Function such as the Functional Genomics and Molecules to Memory initiatives with the Research School of Biological Sciences. The University's Biomolecular Resource Facility, which provides a wide range of services to molecular biologists, is also housed in the Division. It has continued to develop this year and has expanded the services available.

Expertise in gene-targeting and transgenic animal research is provided by the Gene Targeting Facility and is also present in the Autoimmunity/Gene Manipulation Laboratory. Such approaches are necessary in generating the much-needed link between molecular studies and integrative whole animal research.

Two long standing members of the Division, Professor Graeme Cox and Dr Denis Shaw retired this year. They both made many significant contributions to the Division, the School and to medical research in general. Their expertise and good humour will be greatly missed.

Leader: Dr David Tremethick

Aims: The role of chromatin and histones in controlling transcription.

The overall aim of this project is to determine the precise molecular steps involved in the transcriptional activation of a eukaryotic gene within a native chromatin environment. Elucidation of these mechanisms, at the molecular level, is crucial to our understanding of gene expression and regulation during cellular differentiation and to understand disease states such as cancer where normal gene expression patterns become altered. In addition, understanding how chromatin contributes to the regulation of gene expression is essential for understanding the pathogenesis of retrovirus, such as HIV-1, infection. HIV-1 must integrate a DNA copy of their genome into chromosomal DNA of newly infected cells. Moreover, independent of the site of integration, the virus assembles into a highly sterospecific chromatin structure indicating that this assembly process is important for its life cycle. Using HIV-1 as a model system, the specific aims of this work are to understand (1) the mechanism by which DNA is assembled into chromatin, (2) how chromatin architecture contributes to the regulation of transcription and (3) how chromatin is remodelled to allow transcription factor access. This will involve looking at nucleosome displacement, positioning, exclusion and modification.

Background:

A multicellular organism is made up of many different cell types; each cell type performing a unique function. The information that determines the function or behavior of a cell is stored in its genes (DNA). An important unanswered question in biology is how can different cells develop, carrying out different functions, when every cell within an organism contains the same genes?

Genes are copied into RNA in a process called transcription and then, this RNA is translated into protein (it is the type of protein made that determines the function of a cell). The transcription of gene is a complex process requiring many 'DNA binding' proteins. These proteins are called transcription factors. The binding of transcription factors to one gene but not to another is one mechanism by which a subset of genes can be expressed enabling a cell type to carry out its specific function.

In the nucleus of a cell DNA is not in a 'free form' but it is complexed with about an equivalent mass of protein to form a structure known as chromatin. Chromatin is a periodic structure made up of repeating, regularly spaced subunits, the subunit being the nucleosome. The DNA joining each nucleosome is known as linker DNA. It has been proposed that this packaging of DNA into chromatin may control the accessibility of genes to the binding of transcription factors. To investigate the role of chromatin structure in controlling the transcription of gene, an in vitro chromatin assembly system has been developed utilizing extracts prepared from Xenopus laevis oocytes. It was found that the assembly of a gene into chromatin does indeed prevent the binding of transcription factors to DNA. This suggests that chromatin may play a role in determining which subset genes are transcribed in a cell. This finding also suggests that for a specific gene to be transcribed, nucleosomes must be excluded, displaced or its position on the DNA altered.

Research:

In order to understand how the chromatin structure of a gene is altered to allow transcription, it is first necessary to understand how DNA is assembled into chromatin. Fractionation of the oocyte extract, and the eventual purification of all chromatin assembly components, will reveal the mechanism by which chromatin is assembled. Current fractionation work has shown that the assembly of DNA into chromatin is a sequential, time dependent process occurring in at least four steps. Two steps are required for the formation of the nucleosome, and a further two steps are needed to space these nucleosomes in order to generate a regular array.

These experiments have led to the identification of a novel nucleosome spacing factor that requires ATP to function. This protein has recently been purified and is a new type of chromosomal protein. A similar functional protein has been purified from mammalian cells. Mechanistic studies have shown that this mammalian protein spaces nucleosomes by interacting with both the nucleosome and the linker DNA. Other work has characterised the protein domains required for this interaction. Most interestingly, the binding of this nucleosome spacing protein facilitates the binding of transcription factors to DNA by modifying the structure of chromatin. Based on this and other fractionation work, it appears that during the chromatin assembly process, a time window exists which enables transcription factors to bind DNA before the formation of mature inactive chromatin. In addition, during this time window, the mammalian spacing protein can bind DNA to modify the structure of chromatin which further facilitates the transcriptional activation process. There are also other mechanisms involved in chromatin disruption. For example, transcription factors themselves may be able to modify or alter chromatin to allow transcription. We have also shown that a transcription factor known as AP-1 can completely disrupt a nucleosome to allow the subsequent binding of other transcription factors. Most interestingly, this disruption process is reversible since removal of AP-1 allows the reformation of the nucleosome. The ability of histones to rapidly refold into a nucleosome upon removal of AP-1 from the nucleoprotein complex provides an attractive mechanism by which an inducible promoter can be rapidly inactivated upon cessation of the activation signal. Importantly, more recent findings have demonstrated that this nucleosome disruption is dependent on a histone modification (acetylation). This suggests that the nucleosome itself may be an important target for signal pathways. Whether the incorporation of specific histone variants promotes chromatin modelling is also being investigated.

This work is particularly relevant to HIV-1 transcription. The replication rate of integrated HIV-1 is mainly controlled at the level transcription. The long terminal repeats, located at both ends of the integrated virus, contains all of the transcription binding factor sites needed for transcription initiation. Notable, the promoter region contains three binding sites for the transcription factor AP-1. Most interestingly, during the activation of the HIV-1 promoter in vivo, a nucleosome that covers these three AP-1 binding sites is specifically disrupted. Therefore, our finding that AP-1 can directly disrupt the structure of nucleosome in vitro may provide a molecular explanation for this in vivo observation. This hypothesis is currently being tested.

Leader: Dr M Frances Shannon

When the body is invaded by a foreign pathogen such as a virus, bacterium or parasite, the immune system is immediately triggered to attempt to eliminate the invader. The immune system consists of a network of cells each with a specific role in the destruction of the invading organism. One cell type that is involved in the specific recognition of the pathogen as an invader is known as a lymphocyte. One type of lymphocyte, called a T cell can recognize a foreign antigen when it is "presented" to it on the surface of another cell type known as the antigen presenting cell (APC). When T cells come into contact with APCs carrying foreign antigens, they become activated and respond by producing a host of signals that are important for their own growth and the activity of other cells of the immune network.

The signals produced by the T cells consist mainly of proteins, known as cytokines, and are made in response to a complex array of signaling events inside the cell. These signals trigger an event known as gene transcription, which is the starting point inside the cell nucleus for the production of new proteins. Each gene in the cell nucleus is made up of a code for the production of a particular protein. An enzyme known as RNA polymerase II transcribes this code and it is this process that is referred to as gene transcription. Each gene also contains a complex molecular switching mechanism that allows it to switch its transcription on and off in response to signaling events in the cell. The overall aim of this laboratory is define the mechanism of action of these molecular switches for cytokine genes in T cells and determine how they respond to the cellular environment. The activity of these switches can sometimes be triggered without the presence of a foreign invader. This happens in autoimmune diseases such as diabetes, rheumatoid arthritis or in cancers such as leukemia. If we can understand how these molecular switches work then we can potentially design drugs to block the signals in disease states or enhance the signals when we need to fight infection.

In the nucleus of the cell, the genes or DNA stretches that encode the cytokines needed for an immune response are normally silent, ie no protein is being made. These inactive genes are generally encased in a higher order structure known as chromatin. When the gene receives an appropriate signal, it becomes unraveled from chromatin and uses its switching mechanism to assemble a large multiprotein complex on the gene that ultimately activates the RNA polymerase. While we know a lot about the components of the switching mechanism, there are still many questions that we need to answer in order to determine how these molecular switches work in the complex milieu of the cell nucleus. How are the specific genes, required for a response, triggered to unravel themselves from chromatin? What is the structure and dynamics of the multiprotein switching complex on the gene? Another important question is whether the same controls operate in an animal as those that we have defined in the laboratory, using cells that we can easily grow in culture.

In order to answer the latter question we are using mice that have been genetically altered to express a human gene in response to the signals received by the mouse T cells. By making mutations in the control or switching regions of this gene we hope to identify the important parts of the gene that operate in a real T cell in an animal. Jim Cakouros, a PhD student, has been taking this approach to study the control of one gene that encodes a cytokine known as GM-CSF. He has recently identified a crucial region in the switching mechanism of the gene without which the gene cannot be "switched on". This was a surprise finding since all the previous work in cells in culture had not indicated an important function for this region. This region consists of binding sites for two components of the switching mechanism, known as NF-kB and Sp-1. It is interesting that the switching mechanism of the HIV virus (a virus that infects T cells) also depends on NF-kB and Sp-1. It appears that the virus has made good use of the mechanisms that work for cellular genes. Therefore, we believe that this region may be important in signaling the unraveling event from chromatin described above and has identified for us a part of the gene that is crucial in reading the signals that the cell receives in a real immune activation.

We can to some extent reconstitute this system in the laboratory and ask what are the molecular requirements for the displacement of the chromatin or the assembly of the switching complexes. Recent work by Joanne Attema and Dr Ilya Levichkin (and Dr Roy Himes, a former lab member) has shown that a small protein found in all cells is essential for the assembly of the activation complexes on several genes that encode cytokines such as GM-CSF and interleukin-2 in the T cells. This protein is known as HMGI(Y) and appears to alter the way other proteins can bind to the DNA therefore affecting complex assembly. In normal human T cells they have shown that if the amount of HMGI(Y) is altered either positively or negatively it affects the way the cells produce specific cytokines and also the speed at which the cells grow and divide. This protein is also a prime candidate for a component of the displacement machinery for chromatin and we are currently investigating these events in vitro.

Viruses that infect the immune system such as HIV rely on the transcription machinery of the cell to make the viral proteins. Proteins made by the virus can also disturb the cellular switching machinery. The HIV virus makes a protein known as Tat that is essential for high-level virus production within the cell. It does this by interacting with the cellular transcription machinery and increasing its activity specifically for the virus. Tat, however, can have adverse effects on cells, such as altering protein production from cellular genes or leading to cell death. By examining the role of Tat in T cell transcription, Dr Adele Holloway has discovered a novel interaction between Tat and a cellular protein known as PC4. PC4 is a protein that is part of the multiprotein switching machinery that assembles on a gene. By dissecting this novel protein:protein interaction Adele Holloway and Donna Woltring hope to learn more about the structure of the molecular switch that controls HIV protein production.

HIV infection leads by many mechanisms, to a massive destruction of T cells in the body and thus to the inability of the immune system to fight other infections. These secondary infections are often the cause of death in AIDS patients. At least some of this T cell destruction is brought about by a process known as programmed cell death. Some of the signals that are required for cytokine production in an immune response also lead the cell to produce a set of proteins that can prevent cell death. One family of proteins that has been implicated in preventing cell death and activating cytokine genes is known as NF-kB. By genetic manipulation, Dr Renu Mital and May Hadzantonis have established cells in culture in which NF-kB can no longer to activated. They can now use these cells to test whether NF-kB is important for preventing HIV mediated cell death.

Leader: Professor Ian Young

The group has continued its studies on the molecular and cellular biology of the cytokine interleukin-5(IL-5). The production of this hormone-like protein is linked to the immune system and to host defence against parasites. It appears to play an important role in asthma and allergy. IL-5 production occurs following the activation of T lymphocytes as part of an immune response and this cytokine stimulates the production and activity of eosinophils, a white blood cell involved in host response to parasitic infections and in allergic diseases such as asthma.

We have been trying to get a more complete understanding of the role of IL-5 in vivo through studies with a mouse engineered by gene targeting so that it is unable to produce IL-5. This work involves a continuing collaboration with K. Matthaei (Gene Targeting Laboratory, JCSMR ). The results of experiments with P. Foster and A. Ramsay (JCSMR) have previously shown a central role for IL-5 in lung damage and airways hyper-responsiveness in a mouse asthma model. More recently, different strains of inbred mice have been prepared carrying the IL-5 deficiency as well as an eotaxin /IL-5 double knockout mouse. These animals are enabling more detailed studies to be carried on the role of IL-5 and the eosinophil-specific chemokine eotaxin in allergic airways disease. The IL-5 deficient mouse has also been engineered to express the human IL-5 alpha receptor so that it will respond to human IL-5 for studies on the human IL-5 receptor.

Evidence for a role of IL-5 and eosinophils in host defence against parasites has been obtained in collaboration with C. Behm, K. Ovington and E. Milbourne (The Faculties, ANU) and S. Collins (Mc Master University, Ontario) and a role for IL-5 in an anti-tumour response involving cytotoxic T lymphocytes has been shown with V. Apostolopoulos and I McKenzie (Austin Research Institute, Melbourne).

Studies have continued on the mechanisms regulating IL-5 expression in T lymphocytes. This expression is both tissue-specific and inducible and is very relevant to the eosinophil-mediated tissue damage which occurs in asthma and allergy. Structural studies using X-ray crystallography on the IL-5 beta receptor in collaboration with P. Carr and D. Ollis (Research School of Chemistry) have now reached an exciting stage and we hope to gain new insights into the mechanism of receptor activation using this approach in the near future. The role of nuclear localisation in IL-5 signalling is being examined in collaboration with D. Jans.

The Group has participated in two other projects in the area of functional genomics. With H. Campbell (RSBS) the functions of two interesting genes concerned with development and behaviour in Drosophila are being investigated in mammals via gene targeting in mice. We are also seeking to identify genes involved in the formation of long-term memory using chickens as a model system (with S. Redman). This project is interfaced with a parallel project on memory formation in honey bees involving R. Maleska and M. Srinivasan (RSBS).

Leader: Dr Paul Foster

The aim of this research is to understand the cellular and molecular processes that underlie the development of disease processes associated with asthma.

The worldwide incidence, morbidity, and mortality of allergic asthma are increasing at a dramatic rate. In the USA alone 15 million people are thought to suffer from asthma and this disorder is now the most common cause of childhood absence from school. Deaths from asthma have now reached over 180,000 worldwide annually.

A predominant feature of the asthmatic lung is a persistent inflammation of the airway wall (infiltration of the airways with various white blood cells). Currently, it is thought that inflammatory cells induce asthma by releasing substances that damage the lining of the airways and induce constriction (narrowing of the airways). The inflammatory response in the asthmatic lung is a very complex mixture of cells and molecules and it is not clear which factors play the major role in inducing disease. Clinical investigations suggest that the inappropriate inflammatory response in the lung is driven by a lymphocyte known as a TH-2 cell. This cell is thought to direct the inflammatory response by releasing factors called cytokines that recruit other white blood cells to the airways. In particular, the recruitment of the eosinophil to the lung is thought to play a major role in the development and initiation of asthma. The eosinophil is thought to release toxic mediators onto the lining of the airways which results in tissue damage and constriction of the airways.

Research in our laboratory focuses on two major areas:

1) identifying the key cells and molecules which induce disease

2) developing strategies that will direct the immune response away from the harmful TH2 inflammatory response to that which is protective or non-responsive. The long-term goal of this research is to identify novel therapeutic approaches for the treatment of asthma.

Current research focuses on characterising the molecular mechanisms that regulate the accumlation and activation of eosinophils in the allergic lung. This work includes characterising the role TH-2 cytokines (molecules released from the TH-2 cells) such as interleukin-4 (IL-4), IL-5 and IL-13 and their receptor systems in the regulation of airways inflammation and eosinophil function. To facilitate this work genetically engineered mice which are deficient in, or over-express a specific cytokine are being employed in conjunction with mouse asthma models. This transgenic approach is also being employed to understand allergic disorders of the skin and gastrointestinal tract, and in host defense against parasitic infections.

Investigations on deviating the immune system from the harmful TH2 response to allergens focuses on the use of specific molecules that have the potential to act as vaccines. These strategies employ specific structures of allergens in association with molecules that promote non-responsiveness of the immune system.

This work is conducted in collaboration with research groups at the JCSMR, the ANU, nationally and internationally.

The second focus of our research is on malignant hyperpyrexia (MH). MH occurs in individuals with a specific inherited disorder of muscle, and presents clinically as a syndrome of life-threatening complications during general anaesthesia. The MH myopathy may also present clinically as heat stroke, death after taking neuroleptic drugs for psychiatric disorders (the neuroleptic malignant syndrome) and the sudden infant death syndrome (SIDS). The association between MH and SIDS occurs through overheating, which is an important clinical feature of MH and is also an important predisposing factor for SIDS. In collaboration (Drs Taske and Cavanaugh, The Canberra Hospital and Drs Denborough and Milburn, JCSMR) the genes which predispose to MH are being indentified and characterized, in Australian families. The aim of this research is to define the molecular basis of MH and develop a simple non-invasive genetic test to screen infants at birth for susceptibility to MH and SIDS, and individuals before general anaesthesia. This should eliminate deaths from MH, and reduce sudden infant deaths from this cause.

We continue to act as a national reference centre for the diagnosis of MH susceptibility.

Leader: Dr David A. Jans

Eukaryotic cells possess a nucleus in which the genetic information, the DNA, is stored separated from the rest of the cell. Protein synthesis occurs in the cytoplasm so that proteins which are required in the nucleus need to be specifically transported from the cytoplasm into the nucleus. Our work is focussed on the mechanisms regulating protein transport to the nucleus, which relates to many important cellular processes such as differentiation, transformation, signal transduction and the regulation of transcription and cell metabolism. The nuclear import of proteins such as those controlling transcription (transcription factors - TFs) or cancer-related viral proteins (oncogene products) is a key event in the control of gene expression.

We are using the techniques of microinjection and quantitative fluorescence microscopy to examine nuclear protein import in living cells, and thereby identify the mechanisms of regulation of this important process. We have also developed reconstituted in vitro systems to analyse nuclear protein transport at the single cell level. Our work has shown that whilst nuclear localisation is dependent on targeting sequences called nuclear localisation signals (NLSs), phosphorylation (the covalent attachment of phosphate groups to proteins) at sites near the NLSs can act as additional signals determining the rate and maximal level of nuclear transport of a particular protein. Hormonal signals can thus modulate gene expression through phosphorylation at such sites, thereby controlling the nuclear entry of particular TFs.

We are currently attempting to identify and isolate the cellular proteins interacting with phosphorylation-regulated NLSs. We have also used our quantitative approaches to characterise several novel signal-mediated nuclear inport pathways. These include DNA-binding proteins, proteins of RNA viruses such as HIV-1, and granzymes, the serine proteases involved in eliciting apoptosis (programmed suicide) of virus infected cells. In the case of viral proteins, understanding of these novel pathways may enable the development of new anti-viral (eg anti-HIV) therapies.

Understanding of the mechanisms regulating nuclear protein import may ultimately enable their application in targeting molecules of interest to the nucleus in a research or clinical setting. In the latter case, efficient and tightly regulated nuclear uptake of DNA will be very useful in gene therapy applications (eg., the introduction of normal gene copies into appropriate cells harbouring a genetically conferred error of metabolism), or alternatively, toxic molecules can be efficiently targeted to the nucleus of tumour cells in cancer therapy applications. We are currently developing a strategy of modular conjugate molecules containing modified NLSs which has thus far proved successful in the case of both of these applications.

Leader: Dr Klaus Matthaei

Creating mice with a pre-determined genetic makeup.

A major aim of modern biology is to understand how normal gene activities give rise to the structure and behavior of complex organisms. In particular it is important to study the function of genes and their derangement's involved in human diseases. In most cases however, it is impossible to achieve these studies directly in the human. It is easier therefore, to carry out such studies in a more manipulable system such as the mouse. However, natural mutations occur in a serendipitous manner, i.e. by chance. To find a mutation that mimics a particular human disease is therefore difficult. However, given a knowledge of the nucleotide sequence of a gene, it is now possible to make changes to the corresponding endogenous gene of an embryonal stem cell and to produce a mouse that is homozygous for the desired mutation. This procedure is called gene targeting.

Gene targeting involves firstly the use of recombinant DNA technology to modify a cloned gene (usually to stop the function of the gene). At the same time a cultured cell line of embryonic stem (ES) cells is generated by culturing cells from an early mouse embryo (a blastocyst). The ES cells are to tipotent and can be used to regenerate live normal animals (i.e. it is possible to select a single ES cell and produce a whole mouse from that cell, see below). Whilst in tissue culture the modified gene is introduced into the ES cells and the normal gene is replaced by the mutated (functionally inactive) gene. The modified ES cells are then micro-injected into another mouse embryo and the ES cells become integrated. These blastocysts are re-implanted in to pseudo-pregnant mice and give rise to live chimæric offspring which consist of the modified injected cells as well as the normal cells. Since the injected cells can also contribute to the testis of these mice, the breeding of a chimæra with a normal mouse gives rise to an animal carrying half of the genes of the modified stem cell including the mutated gene. Interbreeding of the heterozygous (F1) siblings finally yields transgenic animals homozygous for the desired mutation (usually a deletion or a "gene knockout" mouse). In this way co-isogenic animals can be generated, i.e. animals which are identical to the original mouse strain except that the function of a single gene has been deleted thereby allowing the study of the loss of this gene in vivo.

Gene targeting therefore allows for the first time in a mammal the ability to study the function of a cloned gene in the context of the whole organism by creating mutants defective in that gene. This is particularly important since, with gene targeting, mouse models can be created for studying human genetic diseases and also provides a powerful approach to the development of somatic gene therapy.

Our laboratory has generated a number of different "knockout" mouse mutants using C57BL/6 or BALB/c cells which are at different stages of investigation. These include mouse models of asthma, nerve re-generation, xenograft rejection, parasite-hostrelationships, drug de-toxification and cancer.

Auto-Immunity/Genetic Manipulation Laboratory

Leader: Dr Robyn Slattery

IDDM is an organ specific autoimmune disease. Although it is known that predisposition to IDDM involves the MHC, and that the disease is mediated by T cells, details of the T cell interaction with MHC remain poorly defined. Since both CD8 and CD4 T cells are necessary for disease progression it is presumed that both class I and class II MHC antigens are involved. The direct involvement of class II MHC has been demonstrated and its diabetogenic effect is thought to be mediated through the antigen presenting cell (APC) rather than the beta cell itself. However, the role of class I is less well defined. We do not know whether MHC class I expression on the beta cells themselves is important for disease, or whether its role in diabetes is on the APC like the role of class II. The role of class I expression on beta cells may be important for initiation, progression or as a final target for CD8-mediated destruction. However, until class I expression on beta cells can be separated from class I expression elsewhere it will be difficult to answer these questions. Through an understanding of the importance of the organ specific expression of class I we can understand the nature of the autoantigen/s which the class I restricted CD8 T cell sees.

Through the potential of the cre lox system to target genes in vivo it is possible to delete class I expression specifically from the beta cells while leaving class I expression in other tissues unchanged. This involves making NOD ES cells for targeting the b-2M gene in such a way that it carries recombination sequences (lox sites) flanking the gene. NOD mice resulting from ES cells targeted in this way will be crossed to transgenic NOD mice which express the cre enzyme in the beta-cells of the pancreas. Expression of the cre enzyme in the beta cells of NOD mice which carry lox sites flanking b-2M will result in the tissue specific recombination and deletion of beta-2M. These mice will lack class I expression in the b cells, but express class I normally elsewhere. It should then be possible to separate the roles of class I expression on beta cells vs on antigen presenting cells and to gain an insight into the class I-restricted CD8 mediated autoimmune destruction of the islets in IDDM.

The in vivo gene targeting system will not only be used for the study of the role of class I in IDDM, but also the role of beta cell antigens. Clearly this technology has many applications for the study of other organ specific diseases as well as in non-disease states for the understanding of the normal functioning of genes. This extends beyond the tissue specific control of gene expression to the temporal control with the use of inducible promotors. NOD mice have been created which have b 2M flanked by lox sites. These mice have been crossed to a panel of NOD mice which express cre under the control of different promotors. The resultant offspring lack expression of b 2M (and class I) from the tissue in which cre was expressed. We are currently studying these mice for evidence of diabetes.

Leader: Dr Marco G Casarotto

Nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography remain the two most powerful techniques for the structural study of biological molecules. Of these, NMR is not only capable of yielding high resolution structural information of biomolecules in solution but can also provide dynamic and biochemical information not accessible by other techniques. Over the past few years great advances have taken place in NMR and molecular biology enabling the study of large biomolecules.

One of the primary roles of the biological NMR laboratory is to foster and develop collaborative ties with members of the medical and scientific community. The laboratory is well equipped having access to two high-field NMR spectrometers (500 & 600 MHz) and a network of Silicon Graphics computer workstations. The projects currently in progress involve drug binding, mechanistic studies of enzyme systems and structural investigations of membrane related proteins. Since many of the projects are collaborative efforts within and outside the School, an integrated approach involving other techniques complementary to NMR such as molecular biology, enzymology and molecular modelling are employed.

Many of the projects are linked under the common theme of drug design and development. Listed below are a few of the projects being investigated in this laboratory.

The enzyme dihydrofolate reductase is the target for anticancer, antibacterial and antimalarial drugs and we have used NMR structural, data, molecular biology and enzymology to better understand how this enzyme works. Work done in the future will allow us to design more effective drugs which will be selective in fighting a whole host of diseases.

Many membrane proteins are essential for the survival of viruses and we are targeting several proteins which form ion channels. The aim is to design "blockers" of the ion channels based on a structural knowledge of these ion channels. This approach will give rise to a new generation of drugs to treat diseases such as HIV AIDS, hepatitis C and Ross River Fever.

For skeletal and heart muscle to function properly careful regulation of calcium levels must occur. In skeletal muscle two proteins, the dihydropyridine and ryanodine receptors interact, triggering the release of calcium. We are using high resolution NMR spectroscopy to determine how these proteins function by firstly determining the structure of various regions of these proteins and then using this structural information to determine how they interact. It is hoped that through this work we will be able to design drugs which will control calcium release and be beneficial in the treatment of heart failure.

Leader: Dr J.E. Gready

The Group's research aims to understand how the structure and function of proteins have evolved together, and how the molecular details of their structure, interactions, energetics and dynamics enable proteins to perform their functions. Such understanding can be developed only by undertaking detailed studies on particular protein problems, and then developing more general conclusions by comparative studies. While such studies have always used information from the literature, the scale and richness of data coming from the discovery-driven "Xomics" revolution – genomics, structural genomics, functional genomics and proteomics – in biological science is transforming the way in which protein structure and function problems are framed and undertaken. A particular goal of our research is to couple computer-based methods and experiment in study of particular topics within our two main themes, enzyme mechanisms and extracellular receptors. As widely recognized internationally (e.g. NIH report of June 1999 Biomedical Information Science and Technology Initiative; http://www.nih.gov/welcome/director/060399.htm), the future challenge of biological research will be to exploit the post-genomic information flood. For protein structure and function problems, this will enable us to understand how proteins and protein families operate in cellular and organ systems in normal, disease or developmental states. In our computer-based work, we are coupling computational and simulation methods with bioinformatic methods, so as to use database information, especially on the evolutionary history of proteins, to complement the molecular description of how they work.

Mechanisms of enzyme reactions: In this area, our aim is to predict the energetics and mechanisms of enzymic reactions. Of particular interest is how the enzyme environment binds substrates, products and transition states, and facilitates the reaction. This requires definition of factors such as the roles of protein sidechains in the active site, electrostatics of the whole enzyme, and enzyme-bound solvent and protons. Also of interest, is how this environment is modified and conserved by protein evolution. Much of this information, particularly energetic contributions, cannot be obtained easily or unambiguously from experiment. Information gained also enables critical assessment of current competing literature hypotheses [electrostatic pre-organization, low-energy hydrogen bonds, desolvation] of how enzymes work, surprisingly elusive knowledge but necessary for drug design and protein engineering applications. We are currently studying three enzymes. Dihydrofolate reductase, a key enzyme in DNA biosynthesis, is a major target for the antifolate class of cytotoxic drugs, which includes anti-cancer and antimicrobial agents. Our mechanistic work forms the basis of a more sophisticated approach to design of enzyme inhibitors which we have successfully tested experimentally. Lactate dehydrogenase, which is involved in energy production in the cell, is also a potential target for drugs, especially in parasites. For Rubisco [D-Ribulose-1,5-bisphosphate carboxylase/oxygenase], the enzyme catalysing the fixation of carbon dioxide in photosynthesis in plants, the key issue is to understand why it also catalyses a wasteful oxygenation reaction. If Rubisco could be re-engineered to increase its selectivity to use carbon dioxide rather than oxygen, enormous improvements in food production and extension of agriculture and forestry to more arid land would be possible.

Mechanisms of action of modular extracellular (EC) proteins: In this second area, our long-term aim is to understand how the peculiar characteristics of EC proteins, in particular membrane-bound receptors and proteins of the extracellular matrix, are related to their specialized functions in multicellular organisms. Their 3-dimensional structures are characteristically composed of discrete modules, with interlinking sequence regions of undefined structure such as repeats of a subset of amino acids. This structure derives from their recent rapid evolutionary history involving extensive duplication of genes and recombination of modules through shuffling of DNA segments (exons). This has produced large families and superfamilies of proteins divergent in both structure and function. EC receptor functions are required for intercellular communication, and defence and repair systems, in multicellular organisms, and the proteins are usually developmentally regulated and characteristic of differentiated cells in tissues and organs. Understanding the molecular basis of dysfunctions in these systems of protein families is important as they underlie many of the human diseases which are most difficult to treat. This is because the complex interactions among these systems, especially overlapping specificities for ligands producing redundancy in their actions, complicates the design of selective therapeutic agents. By contrast for enzyme inhibitors, the principle of one molecule (i.e. drug) to target one site or mode of action (i.e. the enzyme) usually works well. We are addressing these issues both by systematic studies on some EC families, and by specific studies on prion protein (PrP). PrP is the unusual protein, implicated in such brain diseases as Creutzfeldt-Jacob and "mad-cow" diseases, which apparently can exist in two conformational forms, one associated with disease. Our PrP studies have implications for understanding PrP and other "folding diseases", such as Alzheimer's, as a first step to designing therapies.

Leader: Dr Peter Jeffrey

Sedimentation analysis was the first and is still the best method for defining the size of proteins in the conditions where they are actually active , that is aqueous solutions and at the physiologically appropriate concentrations, which in the case of hormones and enzymes may be very low, or in the case of oxygen binding proteins , very high. It is nicely complementary to x-ray crystallography, nuclear magnetic resonance, and mass spectometry which yield more precise structural and size information but under much more restrictive conditions. The technique also allows interactions between specific proteins and the effects of the presence of components like other proteins and small molecules and ions on proteins in solution to be detected and defined quantitatively.

In the protein interactions laboratory we have a state of the art instrument for studying proteins in this way, the Beckman Optima-XLA analytical ultracentrifuge. The XLA with its superb optics and electronics can collect and process data from three cells run simultaneously at speeds up to 60000rpm. Centrifugal forces generated at these speeds are up to 250000g's and force molecules to sediment at rates dependent on their sizes and shapes in velocity experiments, upon their sizes only in equilibrium experiments. Measurements of sedimentation rates can give gross information about shapes of macromolecules while measurements of concentration distributions at sedimentation equilibrium give absolute molecular weights under defined solution conditions independent of molecular shape. Only 100 microlitres of solution containing 100 micrograms of material are needed and the technique is non-destructive.

Leader: Dr Denis Shaw

Patients with multiple myeloma produce large amounts of a single immunoglobulin and excrete excess light chains of that immunoglobulin as light chain dimers in their urine. Different patients produce different immunoglobulins and it is not known what antigens they would bind. The physicochemical properties of the immunoglobulin Yvo are unusual and that patient experienced related clinical complications. Hence, Professor Allen Edmundson of the Oklahoma Medical Research Foundation wanted to determine the three-dimensional structure of the immunoglobulin to try to explain the properties of the unique protein. Interpretation of X-ray crystallographic data requires a detailed knowledge of the primary amino acid sequence of the whole protein being studied. The sequence of the light chain had been determined in the USA but attempts to sequence the heavy chain were singularly unsuccessful due to the propensity of fragments of the chain to aggregate. Similar difficulties had been experienced previously with an immunoglobulin isolated from a patient coded Mcg. In that case I separated the peptide fragments from chemical and enzymic cleavage on SDS-polyacrylamide gels, modified the technique for in-gel digestion, used preferential elution of more soluble components and passively transferred the target peptides to obtain a complete overlapped sequence of the variable region of its heavy chain. The same techniques have now been applied to the present sample. The N-terminus was blocked and unlike Mcg did not respond to specific cleavage but there was a point of entry six residues into the chain and a fully overlapped sequence of the variable region was determined, including the three hyper variable complementarity-determining regions which define the specific interactions of the immunoglobulin. Also significant features of the constant region were obtained to relate that part to the observed physical properties and improve our general understanding of the structure and functional relationships of immunoglobulins.

Acting Leader: Dr Gary Ewart

A membrane, or phospholipid bilayer, separates all living cells from their environment. This fatty layer is essentially impermeable to any substance normally found in the aqueous solution surrounding cells. Compartments within cells are also surrounded by phospholipid bilayer membranes. Specialised proteins or protein complexes have evolved that prefer to locate in membranes and such proteins mediate, in a controlled fashion, the passage of ions and other essential molecules across the membrane. These processes are involved in such diverse functions as, for example, the generation of biologically useful energy from ingested food and the communication between nerve cells and between nerve and muscle cells. The understanding, at the molecular level, of these membrane processes is proving to be one of the most intractable problems in biochemistry. If any membrane system is to be manipulated rationally, either genetically or by drugs, then knowledge of the molecular mechanisms is essential.

The Membrane Biochemistry Group has joined the Membrane Physiology and Biophysics Group and the Muscle Research Group (both from Division of Neuroscience) to form the 'Membrane Biology' program. We are using a combination of advanced biophysical, biochemical and molecular biological techniques to probe the molecular mechanisms of two such membrane systems:

1) Viroporins - small virus encoded membrane proteins that form ion channels - such as the M2 and NB proteins of influenza virus, and the VPU and VPR proteins of HIV-1.

The viral proteins M2, NB, VPR, VPU have all been expressed as fusion proteins in bacteria. The purified proteins have been incorporated into planar lipid bilayers and their ion channel properties analysed and probed with site-directed mutations. These four proteins have also been expressed as unfused proteins in Escherichia coli and their effects on membrane-associated functions correlated with the ion-channel properties in the bilayer system. One of the aims of our research is to find inhibitors of these viral ion channels that may allow alternative anti-viral strategies to be developed. In that vein, we have identified a chemical (ANU 9) that inhibits the Vpu ion channel activity and have shown that ANU 9 also inhibits replication of HIV-1 in cultured human blood monocytes and macrophages.

We are also investigating viroporin-like proteins from various alphaviruses and flaviviruses to see whether they too form ion channels that have important roles for the replication of these viruses. Members of these virus groups are responsible for a wide range of diseases in humans (eg Dengue Fever and Murray Valley Encephalitis) that are currently very difficult to treat. It is our hope that drugs targeting the ion channels in these viruses will prove useful for combating the associated diseases.

2) The human GABAA receptor.

Gamma-amino-butyric acid (GABAA) is the major neuroinhibitory transmitter in the brain and is the target for many drugs. We are using an insect cell culture system and a genetically engineered virus to express the human GABAA receptor in a functional form on the cell surface. Substitution of amino acid residues in the hydrophobic domains of the subunits has had significant effects on ion conduction and on the process of desensitization. The effects of drugs have also been modified by these mutations.

Membrane Physiology and Biophsics

Group leader: Professor Peter W Gage

A target for general anaesthetics, tranquillisers and anti-epileptic drugs.

Ion channels formed by virus proteins

Ion channels that open during oxygen-deprivation

Muscle Research Group

Group Leader: Professor Angela Dulhunty

These include:
electrophysiological studies of currents through single ion channel proteins and of contraction in isolated bundles of intact muscle fibres and in skinned segments of single fibres; biochemical isolation and modification of ion channel proteins; molecular biology of ion channel proteins and proteins that regulate the calcium release channels; NMR studies of protein structure and immunoelectron microscopic studies of the distribution of proteins in membrane systems

The Ryanodine Receptor Calcium Release Channel

We are examining the regulation calcium ion flow through the ryanodine receptor by studying the currents through single channels incorporated into artificial lipid bilayers. Our specific interests are the modulation of channel activity by calcium and magnesium ions, following sulfhydryl reduction and oxidation (by oxidants such as NO), by FK-506 binding proteins (FKBPs), by co-proteins like triadin and calsequestrin and by protein-protein interactions with the skeletal muscle L-type calcium channel (an essential step in excitation-contraction coupling), which is also known as a dihydropyridine receptor (DHPR). We have identified basic mechanisms in (a) calcium magnesium regulation sites, (b) redox state and (c) FKBP in controlling the "gating" of the ion channel. Our studies have shown for the first time that small peptides, corresponding to a sequence in the DHPR, both activate and inhibit single ryanodine receptor channels, and that the activation is modified by FKBP12. These studies are continuing. Future studies will investigate the sequences in the ryanodine receptor and co-proteins, and the structural constraints, that allow regulatory interactions to proceed. We are also examining the effects of the ryanodine receptor mutation in malignant hyperthermia on single channel activity.

The distribution of the ryanodine receptor protein in the sarcoplasmic reticulum of skeletal and cardiac muscle fibres is being examined using immunoelectron microscopic techniques. We have shown that there are an unexpectedly large number of ryanodine receptors in the longitudinal sarcoplasmic reticulum of skeletal muscle fibres. These studies are continuing in both skeletal and cardiac muscle and we are investigating the important functional implications of extrajunctional ryanodine receptors in calcium regulation.

Counterion Channels in the Sarcoplasmic Reticulum Membrane

Excitation Contraction Coupling

STAFF - DIVISION OF BIOCHEMISTRY AND MOLECULAR BIOLOGY

Autoimmunity/Genetic Manipulation Laboratory
Leader:
R Slattery, PhD
Laboratory Technician:
S Palmer, BSc (Hons)
Laboratory Assistant:
J Kofler, DipApp Animal Sci

Biomolecular Resource Faculty
Head: P Milburn BSc (HOAS), PhD (Sheffield)
Senior Technical Officers: K McAndrew LC McCrae
Technical Officers: J McGovern BSc (QLD) Grad. Dip. Sci.
M Torronen
Administrative Assistant:
S Moore

Biological NMR Laboratory
Research Fellow and Leader:
MG Casarotto, Bsc (Hons) (Melb), PhD (Melb)
Visiting Fellows:
WLF Armarego, PhD, DSc (Lond), FRSC, FRACI
JF Morrison, BSc (Syd), MSc (Qld), Dphil (Oxford), DSc, (Protein Biochemistry)

Chromatin and Transcriptional Regulation Laboratory
Fellow and Leader: D Tremethick, BSc (Hons) (Syd), PhD (Macq)
Technical Officers:
L Hyman BSc (UC)
M Clarkson BSc (Hons) (Uni. Adelaide)

Computational Molecular Biology & Drug Design Group
Senior Fellow and Leader:
JE Gready, BSc (Hons) PhD (USyd) FRACI
Research Fellow:
PL Cummins, BSc (Hons) PhD (USyd)
Postdoctoral Fellows:
SP Greatbanks, BSc (UMIST) PhD (Manc) [funded by ANUSF/Fujitsu]
PJ Harvey, BAppSc (QUT) BSc (Hons) PhD (Griff) [funded by Canberra Hospital Private Practice Fund] (until July)
R Hornig, BSc Dipl (Basel) PhD (ETH Zur)
WA King, BSc PhD (UNSW) [Inter-School collaborative PDF with RSBS] (until September)
SJ Ohms, MBChB Dipl (Obstet.) BEng MEng PhD (Auck)
RK Schmidt, BS (Nebraska) PhD (Cornell) (ARC Postdoctoral Fellow)(part-time from October)
J Zuegg, Dipl-Ing Dr (Graz)
Visiting Fellows:
APL Rendell, BSc (Durham) PhD (USyd)
PJ Harvey, BAppSc (QUT) BSc (Hons)PhD (Griff) (from July)
Research Officers:
DL Diedrich, BS (Mich State) PhD (Penn State)
J Fan, BSc MSc (Fudan) PhD (Auck)
Research Assistant:
C McKinlay, Chem Cert. (Woll) BSc (Woll)

Cytokine Gene Transcription Laboratory
Senior Fellow and Leader:
M Frances Shannon, BSc (Hons), PhD (National University of Ireland)
Postdoctoral Fellows:
A Holloway, BSc (Hons), PhD (Tasmania)
R Mital, BSc, MSc, (Bombay University), PhD (Indian Institute of Technology)
I Levichkin, MS (Moscow Institute of Physics and Technology), PhD (Centre for Bioengineering, Russian Academy of Science)
S Rao, BSc (Hons), PhD (Kings College London) (from September)
Technical Officer:
D Woltring, Ass Dip Biology (CIT), Ass Dip Pathology (CIT)

Gene Targeting Laboratory
Fellow and Leader::
KI Matthaei, BSc (Hons) (UNSW), PhD (ANU)
Laboratory Technicians:
VW Damcevski, Ass Dip App Science (CIT)
HI Taylor
MJ Newhouse BSc, Grad Dip (ANU)
S Young (part time)

Leukocyte Signalling and Regulation Laboratory
Senior Fellow and Leader:
Dr P Foster, BSc(Hons) (WA), PhD (ANU)
Postdoctoral Fellows:
Dr D Webb, PhD (UC)
Visiting Fellows:
Dr M Denborough, MD, ChB (Capetown), MD (Melb), DPhil (Oxon), DSc (Melb), FRCP
Dr N Taske, BSc (Hons) (Qld), PhD (ANU) (until May)
Dr Ming Yang, West China Uni. of Medical Science, (Bachelor of Medicine)
EPI Section Henan Provincial Instit. of HYgiene and Anti Epidemic, PhD
Dr J Mattes, University Hospital Frieburg, Germany
Ms A Lucia Pereira De Siqueira, Department of Immunology, ICBIV, University of Sao Paulo
Laboratory Technician:
A Koskinen, Assoc. Dip. Med. Sci.

Medical Molecular Biology
Professor and Leader:
IG Young, MSc (Melb), PhD
Postdoctoral Fellow:
VL Ross, BA (LittB), PhD (until July)
Research Assistant: S Ford, BA, MSc (Qld)
Senior Technical Officer:
D Mann BSc (Hons) (until October)
Technical Officers: A Church (Lord) BSc (Hons)
S Gustin, Ass Dip Biology (CIT), Ass Dip Pathology (CIT), BSc (Hons)

Membrane Biochemistry Group
Group Leader (Acting) & Research Fellow:
G Ewart, BSc (Hons), PhD (ANU)
Professor:
GB Cox, BSc, PhD (Melb), FAA
Laboratory Technicians:
B Matheson
Visiting Fellow:
FWE Gibson, BSc, DSc (Melb), MA, DPhil (Oxon), FAA, FRS
Visiting Fellows:
B Cromer
L Tierney

Membrane Physiology And Biophysics
Professor and Group Leader
P. W. Gage, MB ChB (NZ), PhD (ANU) DSc (NSW), FAA
Research Fellow
B. Birnir, BS (Wash), PhD (UCLA),(until December)
Postdoctoral Fellow
A. K. M. Hamarström, BSc (Hons),
PhD (Monash) (funded by NHF)
Research Assistant
J. Curmi, (BOptom)(UNSW)
Laboratory Technician
A. Everitt BSc (ANU)
Administrative Assistant
L. Hardy (part time)

Muscle Research Group
Professor and Group Leader
A. F. Dulhunty, BSc (Syd), PhD, DSc (NSW)
Postdoctoral Fellow
K. Eager BSc (NZ), PhD (ANU)(until March)
J. Hart, BSc (Hons), PhD (Monash)
Visiting Fellows
E. Gallant, PhD (USA) D.Laver, BSc (Hons), PhD (UNSW), (ARC)(until January)
Senior Technical Officer
S. Pace, BSc (UTS)
Technical Officer
S. M. Curtis, BSc, PLTC (half time)
Laboratory Technician
J. Stivala
Administrative Assistant
L. Hardy (part time)

Nuclear Signalling Laboratory
Senior Fellow and Leader:
DA Jans, BSc (Hons) (Melb), PhD (ANU)
Technical Officer:
P Jans, Technical Diploma (Lausanne) (part-time)
Laboratory Technician:
C Barton, Associate Diploma of Applied Science in Biology
Visiting Fellow: R Lixin, MSc (China), B.M. (M.D, Wuhu Anhui), MSc (Taiyuan, Shanxi)

Protein Interactions Laboratory
Fellow and Head:
PD Jeffrey, BSc (Hons), PhD (Adel)

Protein Chemistry Laboratory
Fellow and Head:
DC Shaw, BSc (WA), PhD (Cantab)
Technical Officer
H Gajardo, BRTC

Divisional Visiting Fellow
G Laver, BSc, MSc (Melb), PhD FRS

School Visitor
E Spinner, MSc Tech, PhD, DSc (Manc), FRACI