Biochemistry and Molecular Biology  

Medical Molecular Biology Group | Chromatin and Transcription Laboratory | Cytokine Gene Transcription Laboratory | Leukocyte Signalling and Regulation Laboratory | Nuclear Signalling Laboratory | Gene Targeting | Autoimmunity / Genetic Manipulation Laboratory | Protein NMR Laboratory | Computational Molecular Biology and Drug Design Group | Membrane Physiology and Biophysics Group | Muscle Research Group | Staff

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.

Within the Cytokine Molecular Biology - Signalling Program, 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 a project of the Centre for Molecular Structure and Function in Functional Genomics 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.

Ian Young
Division Head

Essays

Medical Molecular Biology Group

Leader: Professor Ian Young

The role of IL-5 and eosinophils in reproductive performance has been investigated with S Robertson (University of Adelaide) and in host defence against Trichinella spiralis infections with S Collins (McMaster University, Ontario). A new 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 tissued damage which occurs in asthma and allergy.

The receptor system for IL-5 is shared with two related cytokines, IL-3 and GM-CSF, which are also involved in the regulation of blood cell formation and inflammation. One of the major problems in cell signalling is to understand how these cytokines regulate blood cell growth and function by binding and activating their cell-surface receptors. In a collaborative project with P.Carr and D.Ollis (Research School of Chemistry) using X-ray crystallography we have determined the structure of the complete extracellular domain of the beta common receptor which is the major signalling entity of the IL-5 receptor and is central to the signalling of all three cytokines. Its novel dimer configuration gives new insights into receptor activation. Further structural studies should provide opportunities to develop drugs capable of controlling this important receptor systemwhich could be useful in treating asthma, allergy and cancer.

The group has also participated in another project in the area of functional genomics. With H Campbell (Research School of Biological Science) the functions of two interesting genes, flightless and small optic lobes, with functions in development and behaviour in Drosophila are being investigated in mammals by gene targeting in mice.

GENE REGULATION AND CELL SIGNALLING

Chromatin and Transcription Laboratory
Dr David Tremethick

 

Chromatin and transcriptional regulation during development Introduction- The fate of the eukaryotic cell at all stages of its life cycle is dependent upon the accurate readout of genes encoded by DNA. For example, the development of a single cell into multicellular organism requires precise temporal and spatial regulation of gene transcription. Consequently, certain diseases and developmental disorders are often associated with, and likely to be caused by, aberrant gene expression.

It has become increasingly clear over the last decade that eukaryotic gene regulation at the level of transcription is strictly connected to the structural organisation of the genome. The basis for this structural organisation is the nucleosome. Our overall aim is to understand how chromatin structure contributes to the regulation of transcription during development.

Remarkably, a typical eukaryotic cell contains approximately two meters of DNA, which can be squeezed into a nucleus of about 20 nm in diameter. This packaging of DNA is achieved by a hierarchical scheme of folding and compaction into a protein-DNA ensemble called chromatin. At the first level of organisation, approximately two superhelical turns of DNA is wrapped around a protein complex consisting of eight histone molecules. This complete unit, the nucleosome, forms the basic building block of chromatin and is further reorganised into a regular array to form a chromatin fibre. How this fibre subsequently folds into higher order structures is not yet understood. This protein-induced folding of DNA into a complex three-dimensional structure has profound implications with respect to understanding how gene expression is regulated.

As a result of this compaction of the eukaryotic genome, the conformation and accessibility of DNA is dramatically altered. The compaction of a gene into chromatin clearly impedes the transcription process. The cell has therefore devised mechanisms which reversibly de-compact or remodel chromatin to allow transcription factor binding by altering the stability of protein-DNA interactions in underlying nucleosomes. The multi-subunit structure of the nucleosome (consisting of four histone-dimer subunits) is ideally suited for performing these opposing roles; nucleosomes are both stable enough to compact DNA while at the same time labile enough to allow access of DNA to transcription factors. This lability can be enhanced by targeted modification of the histone proteins or by changing the biochemical composition of the nucleosome. The hypothesis that we are examining is that the stability of protein-DNA interactions in underlying nucleosomes can be altered by the replacement of major histone types with specific histone variants.

There are two important stages with regards to the accurate transcriptional regulation of a gene. The establishment of gene activity, which for most genes occurs during early mammalian development, and the subsequent maintenance of this gene activity throughout many rounds of cell division during the life of the organism. Major global transitions in chromosome and chromatin structure occur early in development when cell lineage and tissue-specific transcriptional patterns of gene expression are established. Very little is known about these structural changes and the mechanisms by which these changes differentially regulate gene transcription. However, it is clear that chromatin plays a fundamental causal role in determining patterns of gene activity.

Background- One potential way the structure and function of nucleosomes can be altered (to facilitate or inhibit DNA-dependent processes) is by the localised incorporation of specific histone variants into chromatin. In the context of histone variants, a common theme is the greater variation of histones H1, H2A, and H2B compared to histones H3 and H4 (no variants of histone H4 exist). This reflects the essential role of histone H3 and H4 in nucleosome assembly and positioning. However, histones H2A / H2B (and histone H1) play a fundamental role in nucleosome-mediated transcriptional repression.

Actively transcribed genes appear to be deficient in H2A / H2B and can exchange (in contrast to histone H3/H4) from chromatin in vitro and in vivo. In chromatin reconstitution assays, depletion of H2A / H2B, leaving the H3 /H4 tetramer, facilitates transcription factor binding and activation of transcription. Thus, it can be expected that modulation in the structure of the nucleosome, and higher-order structures, through variation in the structure of the H2A / H2B dimer may influence nuclear functions such as transcription, replication and repair.

Our work focuses on a variant of histone H2A referred to as H2A.Z. The presence and high level of conservation from yeast to man (across species, the amino acid sequence of H2A.Z is more conserved than the amino acid sequence of major H2A) shows that H2A.Z plays an important and specific role in chromosome function. This function is essential since in Drosophila and Tetrahymena null mutants die. Recently, in collaboration with Ian Lyons from the University of Adelaide, we also found that the H2A.Z gene is essential for mouse survival with the defect occurring early in development around the time of implantation. However, despite being essential, nothing is known about the specific functional and structural consequences of having H2A.Z incorporated into chromatin.

To begin to understand why H2A.Z is essential for survival, we adopted an in vivo approach. To search for the unique feature(s) of H2A.Z required for its function, we performed amino acid swap experiments in which residues unique to Drosophila H2A.Z were replaced with equivalently positioned histone H2A residues. Mutated H2A.Z genes encoding modified versions of this histone were transformed into Drosophila and tested for their ability to rescue null mutant lethality. Most interestingly, we discovered that the unique and essential feature of H2A.Z lies outside the histone fold in the carboxy-terminal domain. This C-terminal region maps to a short ?-helix in H2A that is buried deep inside the nucleosome. A region immediately adjacent to this short ?-helix, located at the surface of the nucleosome, was also found to be important for adult Drosophila survival. Together, this region forms part of a docking domain, a domain involved in stabilising the interaction between the H2A/H2B dimer with the H3/H4 tetramer. Based on these results, our prediction is that H2A.Z would alter the stability of the nucleosome potentially weakening the interaction between the H2A.Z/H2B dimer with the H3/H4 tetramer.

Recently, we tested this prediction by solving the crystal structure of a nucleosome containing H2A.Z (in collaboration with Karolin Luger from Colorado State University). The overall structure is similar to the previously reported structure containing major H2A. However, consistent with our prediction, distinct localised changes in the docking domain result in a subtle destabilisation between the dimer and the tetramer. Potentially even more significant, the amino changes in the docking domain of H2A.Z results in an altered nucleosomal surface that includes a metal ion, a more extensive acidic patch, and a larger hole in the centre of the nucleosome. Our current favoured hypothesis is that these surface changes may create a highly specific interaction interface for other nuclear proteins like chromatin remodelling factors. Future Aims- The overall aim of our investigation is to link, for the first time, the in vivo function of H2A.Z in mammalian development with an in vitro analysis of the effect of H2A.Z on chromatin structure and transcriptional activation. We will test the hypothesis that H2A.Z is incorporated in to the chromatin of active genes, and that the structural change brought about by H2A.Z is important for its function by permitting the high-level transcription required for early stages of development.

The specific aims of this project are to investigate whether:

Cytokine Gene Transcription Laboratory
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 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 foreign and not belonging to self is known as a lymphocyte. A 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 or proteins 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 intra cellular 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 know as chromatin. The basic building block of chromatin is known as a nucleosome and is a highly ordered protein:DNA complex. 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 II. 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. The following projects are designed to address these questions.

1. A transgenic approach to studying GM-CSF gene transcription. (Jim Cakouros, Dr Renu Mital and Donna Woltring)

In order to answer the latter question above 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 these same transcription factors and implies that it may be a highly conserved and efficient switching device. It appears that the virus has made good use of the mechanisms that work for cellular genes. Further experiments have shown us that this region is 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 immune activation. We are dissecting the molecular events involved with further transgenic work in collaboration with Dr Robyn Slattery (JCSMR) and using a biochemical approach as described below.

2. Assembly of chromatin remodeling complexes on the GM-CSF promoter (Dr Adele Holloway)

The results described above indicated to us that a specific region of the GM-CSF gene is involved in reading T cell signals when the gene in encased within chromatin. We are now asking whether this region of the gene (the NF-kB/Sp-1 binding sites) is involved in recruiting protein complexes that can alter the structure of chromatin by remodeling nucleosomes on the DNA. The approaches taken here involve both in vitro and in vivo experiments. The biochemical experiments are designed to ask if this part of the GM-CSF gene can recruit remodeling factors to DNA in a multiprotein complex. Experiments have so far shown us that some known remodeling activities can be recruited to the GM-CSF gene by interacting with transcription factors that bind to the Sp-1/NF-kB region. This is an exciting finding because for the first time it gives us a handle on the molecular mechanism of chromatin remodeling on inducible cytokine genes. We also want to determine if there are novel components in these chromatin-remodeling complexes and for this purpose we are using a functional proteomics approach. This approach allows us to determine unknown protein components using mass spectrometry and database searching.

We now hope to confirm these finding by using assays that are designed to find whether these proteins actually bind to the GM-CSF gene in the cell nucleus in response to T cell activation. If these experiments are successful it will allow us to examine not only the specific proteins that are recruited but also the timeframe and the dynamics of these interactions.

3. Development of a novel method to assess chromatin remodeling across inducible gene in vivo. (Dr Sudha Rao and Song Yong)

One of the limitations of studying chromatin structure on real genes in primary cells is the need for large amounts of cellular material and the cumbersome non-quantitative nature of the assays. Dr Sudha Rao, therefore, set out to establish a novel Real Time PCR-based assay to examine chromatin structure across inducible cytokine genes in T cells. This method has been spectacularly successful and has allowed us to determine the precise regions of the interleukin -2 (IL-2) gene that undergo chromatin remodeling in response to T cell activation. This has now been achieved in T cell lines, primary CD4 T cells and T cells derived from a mouse where T cell responses can be highly controlled. This latter work is in collaboration with Dr Gitta Stockinger (NIMR, London) and will allow us to determine the possible role of chromatin remodeling in the development of T cell memory.

When T cells are activated in an immune response they develop into effector cells that are involved in the production of cytokines and the elimination of the invading organism. Following the initial response, a population of so-called memory cells develops in the animal. These cells are characterized by their ability to respond faster than their naïve counterparts to a stimulus. This is the basis of vaccination strategies. Part of this phenomenon may be based on alterations to chromatin across specific genes during the initial response that are then maintained in the memory cells. It is now possible for us to test this hypothesis with the novel assay described above. The attraction of this method is that it can be easily applied to any gene of known sequence by simply designing new PCR primers.

4. Dissecting the chromatin structure of the IL-2 gene. (Joanne Attema)

We can to some extent reconstitute chromatin and the structures that assemble on active genes 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 in collaboration with Dr Ray Reeves (Washington State University) 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 IL-2 in 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 we have previously 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.

More recent work has shown that when the IL-2 gene is assembled in vitro into chromatin (to mimic more closely how a gene would look in the nucleus), then HMGI(Y) and not other proteins tested can bind to the nucleosome-assembled DNA. This protein is, therefore, a prime candidate for a component of the displacement/remodeling machinery for chromatin and we are currently investigating these events in vitro. It is intriguing that a similar situation appears to be in place in the IL-2 receptor alpha gene, which is coregulated with IL-2 in T cells. And together affect T cell proliferation.

The approaches and projects outlined above are designed ultimately to define the molecular steps that are necessary to activate a cytokine gene in its native context in T cells.

Leukocyte Signalling and Regulation Laboratory
Dr Paul Foster

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

The worldwide incidence, morbidity, and mortality of allergic asthma and allergies are increasing at a dramatic rate. Deaths from asthma have now reached over 180,000 worldwide annually. 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.

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, directing 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:

Nuclear Signalling Laboratory
Professor David Jans

Targeting Proteins to the Nucleus: Development, Viral Disease and Therapy

Animal and plant cells differ from bacteria in that they are compartmentalised. Rather than being a simple "bag of enzymes" where chemical reactions take place rather haphazardly, animal and plant cells are partitioned into highly specialised membrane-bound structures called organelles, such as the nucleus or mitochondrion, which carry out specific functions largely in isolation from the rest of the cell. The cell requires specific "address systems" to target the specific molecules that are required in these organelles to their correct site, and this involves targeting signals and transport systems that recognise them.

We are interested in the nucleus because it is where the cellular DNA is located, and where the very important process of transcription takes place. Because protein synthesis occurs in the cytoplasm, proteins which are required in the nucleus such as those regulating transcription, need to be specifically transported from the cytoplasm into the nucleus. Generally speaking, these proteins require specific targeting signals called nuclear localisation sequences (NLSs) in order to be able to interact with the cellular nuclear transport machinery, and subsequently localise in the nucleus. Specific proteins, the importins or karyopherins (the NLS "receptors"), recognise the NLSs, and mediate "docking" at the nuclear pore followed by interaction with other cellular factors to effect energy-dependent translocation through the pore and into the nucleus. The regulation of nuclear import of proteins such as those controlling transcription (transcription factors - TFs) or growth (eg. cancer-related proteins or "oncogene" products) is central to important cellular processes such as differentiation and oncogenesis (cancer).

Among other techniques, we use high resolution digital imaging approach called confocal laser scanning microscopy to analyse transport at the level of single cells. The importance of nuclear import to eukaryotic cell function has led us to attempt to examine the mechanisms by which nuclear protein import is regulated. We have demonstrated that nuclear transport is not only dependent on targeting signals (i.e. NLSs), but also can be regulated by phosphorylation (the covalent attachment of phosphate groups to proteins, carried out by protein kinase enzymes in the cell). In the case of the Drosophila (fruit-fly) TF Dorsal, which plays a critical role in fly development, we found that a cAMP-dependent protein kinase (PK-A) site near the Dorsal NLS enhances Dorsal nuclear import, through increasing the affinity of the interaction with importins. In collaboration with David Stein at the Albert Einstein College of Medicine (New York), we demonstrated that mutations preventing PK-A phosphorylation result in lethality, indicating that the enhancement of Dorsal nuclear import by PK-A is essential for normal Drosophila development. We have also shown that phosphorylation-mediated enhancement of importin binding applies to the nuclear localising large tumour antigen (T-ag) protein from the DNA-tumour virus SV40, which play a key role in the viral replicative cycle. Specific phosphorylation thus can enhance or, as we and others have shown, inhibit NLS-dependent nuclear transport of TFs; hormonal/growth factor/cytokine signals modulate gene expression through regulating phosphorylation at such sites, thereby specifically controlling the nuclear entry of TFs or other signalling molecules such as protein kinases. Whilst we are interested in delineating other mechanisms of regulation of NLS-dependent nuclear protein important in developmental and disease systems, the real challenge is to demonstrate how this may work in the context of the whole cell, which harbours a myriad of other nuclear transport substrates and pathways competing with one another for access to the nucleus. Clearly in this context, a mechanism to effect high affinity interaction with the nuclear import machinery may be critical in the face of the imposing volume of proteins and other molecules required constitutively in large amounts in the nucleus such as ribosomal subunits, histones, and RNA binding proteins.

We have more recently been examining the nuclear import of proteins from the causative agents of auto-immune deficiency syndrome (AIDS) - the HIV-1 virus - and Dengue fever (Dengue virus), which is of significance in tropical Australia. We have found that certain viral proteins localise in the nucleus as part of the viral infectious cycle, and that they do so through importin-independent pathways that are quite distinct from those used by normal cellular proteins i.e. viruses may use additional mechanisms to access the nucleus. If our observations prove correct, and we are able to understand how these viral proteins localise in the nucleus, we should be able to devise new therapeutic strategies to block the viral nuclear import pathways, and thereby block viral infection.

Understanding of the mechanisms regulating nuclear protein import enables their application in targeting therapeutic molecules to the nucleus. 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 abnormal error of metabolism), or alternatively, toxins can be efficiently targeted to the nucleus of tumour cells in cancer therapy applications. We are currently developing strategies using modular conjugate molecules containing modified NLSs with these applications in mind.

TRANSGENIC ANIMAL RESEARCH

Gene Targeting
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 behaviour 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 totipotent 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 into 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 ES cells which are at different stages of investigation. These include mouse models of asthma, nerve re-generation, xenograft rejection, parasite-host relationships, hypertension, drug de-toxification and cancer.

Autoimmunity/Genetic Manipulation Laboratory
Dr Robyn Slattery

Type I diabetes affects 0.3% of people in Australia. The strongest inherited trait associated with this disease is the expression of certain markers on cells in the body known as HLA (or MHC in mice). These markers usually act as traffic lights to the immune system telling it when to stop and when to go. Normally these markers signal a red-light to the immune system so that it does not attack our own cells, and only when there is an infection do these markers send a green signal to the immune system that the infected cells should be destroyed. For reasons which are not well understood, the immune system in diabetes patients perceives these markers on the insulin-producing cells as sending a green signal when there is no apparent infection. This results in the immune system attacking and destroying the body's own insulin-producing cells. When this happens the body is unable to absorb sugar and so a patient becomes diabetic.

The research carried out in the AGM laboratory aims to determine where the faulty signal is initiated i.e. is the 'faulty' signal sent by the beta-cells of the pancreas to a normal immune system, or alternatively, are normal beta-cells being attacked by a 'faulty' immune system? The answers to these questions are of fundamental importance to understanding whether disease may recur in patients transplanted with healthy islets, and therefore whether transplantation needs to be combined with modification of the immune system by vaccination to prevent disease recurrence.

By using a very new genetic engineering tool called the cre-lox recombination system, it is now possible to generate mice which have lost the traffic signal molecule (HLA) from pancreatic beta-cells. Previous genetic engineering methods have concentrated on deleting HLA genes from all cells of the animal. The unique approach of the cre-lox recombination system is that it allows us to distinguish between the role of the HLA molecules on the beta-cells versus on the cells of the immune system.

PROTEIN STRUCTURE AND FUNCTION

Protein NMR Laboratory
Dr Marco G Casarotto

Nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography are the two most powerful structural techniques used in 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, mechanistic and biochemical information not accessible by other techniques. One of the primary roles of our group is to use NMR based techniques 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. A number of diverse and medically relevant projects are currently underway involving mosquito-borne diseases such as malaria and Ross River Fever as well as other relevant diseases prevalent in our society including heart disease, Aids and asthma. Our aim is to use information derived from NMR to understand the structural basis of biomolecules involved in these diseases. Since many of the projects are national and international collaborative efforts, an integrated approach involving other techniques complementary to NMR such as molecular biology, molecular modelling and a range of biophysical techniques 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 a extensive ranges of diseases such as cancer, malaria, and bacterial infections and we have used NMR structural, data, molecular biology and enzymology to determine how this enzyme functions. Work done so far has enabled us to unravel the mechanistic detail required for this enzyme to function. Such information will prove vital in allowing us to design more effective drugs which will be selective in fighting a whole host of diseases.

Many membrane proteins are essential components 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. As a result of this work we have designed a series of peptides which have the ability to regulate calcium levels in both skeletal and cardiac muscle. Such peptide therapies may hold the key to designing new drugs which be beneficial in the treatment of heart failure, malignant hypothermia and central core disease.

Computational Molecular Biology and Drug Design Group
Dr JE Gready

Our 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.

The scale and richness of data coming from the genomics revolution 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.

The future challenge of biological research will be to exploit the post-genomic information flood. 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 we 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 side chains 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 of how enzymes work, surprisingly elusive knowledge but necessary for drug design and protein engineering applications. We are currently studying three enzymes: dihydrofolate reductase, lactate dehydrogenase, and Rubisco.

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 three-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. Understanding the molecular basis of dysfunctions in these systems of protein families is important as they underlie many of the human diseases that are most difficult to treat. 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 (that is, drug) to target one site or mode of action (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 - that 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.

Membrane Physiology and Biophysics
Professor Peter W Gage

All cells are encapsulated by a thin lipid surface membrane that separates and protects the cell contents from the outside world. This surface membrane is studded with special proteins that let selected ions pass across an otherwise impenetrable barrier. These ion pathways, called ion channels, are responsible for a broad spectrum of functions such as transmission of electrical signals in nervous systems, initiation of immune responses and cell division. Cells also contain ion channels in internal membranes, around mitochondria and nuclei for example, but their functions here are less understood. Many clinically used drugs such as calcium channel blockers and anaesthetics interact specifically with ion channels but we do not yet know how. Research in our group is aimed at understanding several very different kinds of ion channnels

A target for general anaesthetics, tranquillisers and anti-epileptic drugs. The ubiquitous inhibitory neurotransmitter, gamma-aminobutyric acid (GABA), binds to GABA receptors present on most nerve cells and opens channels that are targets for clinically important drugs such as barbiturates, benzodiazepines and some general anaesthetics. We are studying the properties of individual "native" channels in nerve cells, with particular interest in how drugs affect their behaviour. We are also making our own channels using an expression system (baculovirus/insect (Sf9) cells). We are finding that changes in single amino acids can have major effects on channel function and on the response of the receptors to drugs. These results should help us understand how the normal receptor functions and to find new and better drugs.

Ion channels formed by virus proteins

It has recently been found that a small protein from Influenza A virus forms ion channels essential for viral reproduction and that a clinically useful anti-influenza drug stops the virus by blocking the channel. We are exploring the idea that small proteins from other viruses that have surface membranes may form ion channels that play an important role in the virus life cycle. The proteins are expressed in bacteria, purified and put into artificial membranes. We have also developed other ways of testing for ion channel function. We have found that proteins from several viruses (influenza and HIV-1) form channels for sodium and potassium ions. Our aim is to determine the sites essential for ion transfer using site-directed mutagenesis. It may then be possible to use drugs that interact with these sites to stop virus replication. It is also hoped that the simple nature of these proteins will give us more information about how ion channels work

Ion channels that open during oxygen-deprivation

Following a heart attack (coronary occlusion), and in some other abnormal conditions, the electrical currents that produce an orderly sequence of contractions in the heart can become erratic but we don't really know why. The heart beats irregularly and patients can die because the heart no longer pumps sufficient blood to vital organs. This is a major cause of death following a heart attack. Some antiarrhythmic drugs can help subdue the arrhythmias but none yet available is considered ideal. We have demonstrated the presence of a new kind of sodium channel in heart muscle that we believe has an important role in initiating electrical impulses. This channel is blocked by a widely used class of antiarrhythmic drugs and becomes more active during hypoxia. This may be the cause of abnormal electrical impulses and arrhythmias during hypoxia or ischaemia e.g. following coronary artery occlusion. We are now looking for the link between hypoxia and hyper-activity of this sodium channel. If we are successful, our work will provide a new kind of target for new, more selective, antiarrhythmic drugs.

Muscle Research Group
Professor Angela Dulhunty

The research of the Group is dedicated to understanding the cellular mechanisms that underlie changes in cytoplasmic calcium concentration in general, and more specifically those mechanisms which trigger contraction following an electrical signal on the surface membrane of skeletal and cardiac muscle fibres. Several different approaches are used to tackle this problem.

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

The ubiquitous ryanodine receptor calcium release channel is found in the membranes of intracellular calcium stores and is the major calcium release pathway from these stores in many cell types. Although regulation of cytoplasmic calcium concentrations is basic to the function of all cells, the mechanisms controlling ryanodine receptor activity are not well understood.

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

The ability of internal calcium stores to sequester and release calcium depends on: 1, a calcium uptake mechanism; 2, a calcium release mechanism; 3, channels to allow a counter current to flow with calcium, to prevent large changes in the potential difference across the membrane, which would impede the calcium fluxes; 4, phosphate concentrations in the store lumen. All four components are essential for the proper regulation of cytoplasmic calcium concentrations. The counterion channels, although essential, have been studied far less than the calcium uptake and release pathways. We have recently discovered that there are two types of anion channels in sarcoplasmic reticulum. One of these channels is highly regulated by a number of substances which are present in vivo, including cytoplasmic calcium concentration, inositol polyphosphates, ATP and pH. This highly regulated channel also conducts sulphate and phosphate ions. This is the first description of a divalent anion channel in sarcoplasmic reticulum and an ion channel which provides a pathway for phosphate movement in and out of the calcium store.

Excitation Contraction Coupling

The Muscle Research Group was largely responsible for much of the basic work on voltage-dependence of excitation-contraction coupling in mammalian skeletal muscle. However, the basic mechanism of excitation-contraction coupling in skeletal muscle is still not properly understood. We know that depolarization of the surface membrane activates a voltage sensor which is a part of the dihydropyridine receptor in the transverse tubule membrane. The loop between the second and third transmembrane segment of the dihydropyridine receptor is thought to be involved in transmitting the depolarisation-evoked signal to the ryanodine receptor and one serine residue in the 2/3 loop appears to be essential for activation of ryanodine receptors. We are currently looking at the ability of peptides containing different sequences to specifically activate skeletal and cardiac ryanodine receptor ion channels. Future experiments will examine the interactions between activating peptides and other co-proteins, especially the FKBPs and triadin, so that a model can be developed of the in vivo activation of the ryanodine receptor by the dihydropyridine receptor during excitation-contraction coupling.

Staff - Division of Biochemistry and Molecular Biology

Professor and Head of Division:
IG Young, MSc (Melb), PhD (ANU)

School Technical Manager: (until June)
R Ayling ECC (until June)
School Technical Support Officer: (from June)
R Taylor (from June)

Divisional Administrator:
J Lee
Assistant administrator:
S Lavender (until June)
L Hardy (from July)
Laboratory Stores Attendant:
J Burum

Autoimmunity/Genetic Manipulation Laboratory
Senior Fellow and Leader:
R Slattery, BSc (Hons), PhD (Melb)
Laboratory Technician:
S Palmer, BSc (Hons)
Laboratory Assistants
J Kofler, BSc (Hons)
E O'Neil (casual)
S Young (casual)

Biological NMR Laboratory
Fellow and Leader:
M Casarotto, Bsc (Hons) (Melb), PhD (Melb)
Laboratory Technician
T Sutherland (From October)


Chromatin and Transcriptional Regulation Laboratory
Fellow and Leader:
D Tremethick, BSc (Hons) (Syd), PhD (Macq)
Postdoctoral Fellow:
P Ridgway (BSc McMaster Uni. Canada), MSc (Queens Uni. Canada) PhD (ANU)
Jun Fan, BSc (Fundan, China), MSc (Fudan, China) PhD (Auckland NZ)
Technical Officer:
L Hyman BSc (UC)

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] (until June)
R Hornig, BSc Dipl (Basel) PhD (ETH Zur) (until August)
H Mauser, PhD, (Erlangen) [DAAD Postdoctoral Fellow]
SJ Ohms, MBChB Dipl (Obstet.) BEng MEng PhD (Auck)
RK Schmidt, BS (Nebraska) PhD (Cornell) [ARC Postdoctoral Fellow: part-time until October]
J. Zuegg, Dipl-Ing Dr (Graz) (until September)
Visiting Fellows:
PJ Harvey, BAppSc (QUT) BSc (Hons) PhD (Griff)
APL Rendell, BSc (Durham) PhD (USyd)
A Bliznyuk, BSc PhD (Novosibirsk)
K Sagarik, BSc MSc (Mahidol) PhD (Innsbruck)
Research Officer:
DL Diedrich, BS (Mich State) PhD (Penn State)
Visiting Scholar:
A Evers BSc Dipl (Koln) (until May)

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) (Until October)
S Rao BSc (Hons), PhD (Kings College London)
Technical Officer:
D Woltring, Ass Dip Biol (CIT), Ass Dip Path. (CIT), BSc (ANU)

Gene Targeting Laboratory
Fellow and Head
KI Matthaei, BSc (Hons) (UNSW), PhD (ANU)
Visiting fellow
WK Whitten BVSc (Hons), BSc, DSc.
Laboratory technicians
VW Damcevski, Ass Dip App Science (CIT).
HI Taylor
MJ Newhouse BSc. Grad Dip (ANU)
S Young (part time).
LK Langton BSc. ANU, AssDipSc (Pathology) (part time) (from October)

Leukocyte Signalling and Regulation Laboratory
Senior Fellow and Head:
P Foster, BSc(Hons) (WA), PhD (ANU)
Postdoctoral Fellows:
S Hogan, BSc(Hons), PhD (ANU) (from November 2000)
S Mahalingham, BSc (Hons), M.Phil (Uni Malayasia), PhD (ANU)
D Webb, BAppSc, PhD (UC)
Visiting Fellows
M Denborough, MD, ChB (Capetown), MD (Melb), DPhil (Oxon), DSc (Melb), FRCP
J Mattes, Physician Children's University Hospital Freiburg, Germany
Laboratory Technician:
A Koskinen, Assoc Dip Med Sci
Visiting Scholar:
AL Pereira De Siqueira, Department of Immunology, ICBIV
University of Sao Paulo (until October 00)

Medical Molecular Biology
Professor and Leader:
I Young, MSc (Melb), PhD (ANU)
Research Assistant
S Ford, BA, MSc (Qld)
Technical Officers
A Church, BSc (Hons)
S Gustin, Ass Dip Biology (CIT), Ass Dip Pathology (CIT), BSc (Hons) (until July 2000)
I Walker, BSc (Hons), (Curtin) (from July)
U Weideman (Tech.College Cert. Germany) (until Sept)
J Olsen BSc (From October 31s)

Membrane Physiology and Biophysics
Professor and Leader:
PW Gage, MB CHB (NZ), PHD (ANU) DSC (NSW), FAA
Visiting Fellows:
A Premkumar BSc (Hons), MSc (India), PhD (ANU)
G Ewart BSc (Hons), PhD (ANU)
Postdoctoral Fellow:
A Hammarström, BSc (Hons), PhD (Monash)
Visiting Scholar
S Gaul, Heidelberg, Germany (until April 2000)
Research Assistant:
J Curmi, (BOptom)(UNSW)
Technical Officer:
A Everitt, BSc (ANU)

Muscle Research Group
Professor and Group Leader:
A Dulhunty, BSc (Syd), PhD, DSc (NSW)
Postdoctoral Fellow:
J Hart, BSc (Hons), PhD (Monash) (until February)
Visiting Fellows:
D Laver, BSc (Hons), PhD (UNSW), (ARC)
Senior Technical Officer:
S Pace, BSc (UTS)
Technical Officer:
S Curtis, BSc, PLTC (half time)
Y Karunasekara
G Lenz
Laboratory Technician:
J Stivala

Nuclear Signalling Laboratory
Professor and Leader
D Jans, BSc (Hons) (Melb), PhD (ANU)
Research Fellow
Mark H.C. Lam BSc (Hons) Melb., PhD (Melb), (Peter Doherty Fellow)
D Zhang MBBS (China), MD (China), PhD Melb. (from Nov 00)
Technical Officer:
P Jans Technical Diploma (Lausanne) (part-time)
A John BSc (Hons) (UC)
A A Jadeer BApplSci. (UC)
Visiting Scholar:
N Balsgart BSc (Aarhui Uni. Denmark)



Biomolecular Resource Facility
Head:
P Milburn, BSc (HOAS), PhD (Sheffield)
Senior Technical Officers
K McAndrew,Ass.Dip.App.Sci.(UC)
C McCrae,Biol.Tech.Cert.(CIT)
Technical Officers:
M Torronen
Administrative Assistant
S Moore



Divisional Visiting Fellows
WLF Armarego, PhD, DSc (Lond), FRSC, FRACI
G Barlin, PhD, DSc (Sydney) FRANC
JF Morrison, BSc (Syd), MSc (Qld), Dphil (Oxford), DSc, (Protein Biochemistry)
G Laver, BSc, MSc, (Melb) PhD, FRS
G Cox, BSc, PhD (Melb), FAA
FEW Gibson, BSc, DSc (Melb), MA, Dphil (Oxon), FAA, FRS
D Shaw, BSc (WA), PhD (Cantab)

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