Division of Biochemistry and Molecular Biology

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 viral diseases, in understanding asthma and allergy, in anaesthetic complications and in diabetes.

The Membrane Proteins in Health and Disease Program studies the function of membrane ion channels involved in nerve conduction, the control of muscle contraction and in the replication of viruses.

This work includes the examination of how the bioactivity of these channels is governed by molecular shape and recognition. The program is also investigating new approaches for controlling HIV. The Division also has a strong group working on the prediction of protein structure, the Computational Proteomics and Therapy Design Group.

In the Cytokines and Inflammation Program, the Cytokine Molecular Biology and Signalling Group has continued its collaborative studies on the structure of the cytokine receptor system involved in IL-3, IL-5 and GM-CSF signalling with the X-ray Crystallography Group of the Research School of Chemistry. The work of this group also interfaces with that of the Allergy and Inflammation Research Group and of the Gene Targeting Laboratory which use transgenic animal models to study the key cytokines and cells involved in asthma and allergy.

The Division also has a strong focus on how genes are regulated, with high quality research on nuclear translocation, chromatin function and cytokine gene transcription. This work, which is carried out by the Gene Regulation in Development and Immunity Program, includes studies on the signals which regulate gene expression during the early development of an organism and during immune responses to foreign antigens. Another major focus of the program is the mechanisms regulating the import of proteins from the cytoplasm to the nucleus. This research may allow the development of new approaches for the treatment of cancer and viral diseases.

A novel protein regulating the cytoskeleton of cells, which is essential for early embryonic development, is being studied in a collaborative project between members of the Division and the Research School of Biological Sciences. This project in functional genomics exploits the high conservation of the fundamental life processes between simpler organisms like the fruit fly and more complex mammalian species to give a better understanding of early mammalian development.

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 its services with the introduction of a microarray facility. A new head, Ms Karen Edwards has been appointed to manage the Facility.

Expertise in gene-targeting and transgenic animal research is provided by the Gene Targeting Laboratory and the Autoimmunity and Genetics Laboratory. Such approaches are necessary in generating the much-needed link between molecular studies and integrative whole animal research. The Gene Targeting Laboratory is involved in collaborations with other scientists in the School and elsewhere involving research into asthma, nerve growth, hypertension and embryonic development. The Autoimmunity and Genetics Laboratory has pioneered the use of a new genetic engineering tool called cre-lox recombination and is using it to investigate the causation of diabetes.

Professor David Jans left the Division in December to take up a position at Monash University. His creative research energy will be missed by many in the School. We wish him and Patricia Jans well in their new research location.

Professor Ian Young, Head of Division

Membrane Proteins in Health and Disease

Membrane Physiology and Biophysics

Our research is focused on the structure and function of ion channels. What are ion channels? All cells are encapsulated by a thin lipid surface membrane that separates and protects the cells 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 wide variety 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 they change channel function. Many major pharmaceutical companies are showing increasing interest in ion channels as a new drug target. Research in our group is aimed at understanding several very different kinds of ion channels.

Ion channels in the brain
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 such as general anaesthetics, tranquillisers and anti-epileptics affect their behaviour. We are also synthesizing these channels by introducing the cDNA into foreign cells that are tricked into making the GABA receptor protein and functioning channels. In this way we can introduce changes in single amino acids and study their effect. We are finding that small changes can have major effects on channel function and on the response of the receptors to drugs. Our results are helping us to understand how the normal GABA receptor functions and to find new and better drugs to produce anaesthesia or relief from anxiety.

Ion channels formed by virus proteins
It has been known for about 10 years 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 these ion channels. We have been exploring the idea that small proteins from other viruses may form ion channels that play an important role in the virus life cycle. The proteins are expressed in bacteria, purified and inserted 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 that allow movement of sodium and potassium ions through them. Our aim is to determine the sites essential for these ion movements using site-directed mutagenesis. It may then be possible to find drugs that interact with these sites and stop virus replication. It is also hoped that the simple nature of these proteins will give us more information about how ion channels work in general.

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: they have unwanted side effects.

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 many currently used 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.

Peter W Gage

Muscle Research

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 concentration is basic to the function of all cells, the mechanisms controlling ryanodine receptor activity are not well understood.

We are examining the regulation of 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 and 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.

Angela Dulhunty

Biomolecular Structure Laboratory

Dr Casarotto. JCSMR Photography One of the fascinating properties of biological molecules is their remarkable ability to trigger a range of biological responses by adopting distinct three-dimensional structures. The range of structural diversity forms the basis of a wide range of scientific disciplines ranging from molecular recognition and drug design through to protein folding and design. The Biomolecular Structure Laboratory at the JCSMR seeks to carry out research that explores how the structural properties of biological molecules can impact on biological process involved in health and disease. Through our close ties with biomedical researchers and clinicians at the JCSMR our laboratory is perfectly placed to examine how bioactivity is governed by molecular shape and recognition.

Although the main focus of our research is from a structural perspective, an integrated approach involving complementary techniques such as molecular biology, kinetics and molecular modeling are routinely employed in the laboratory. Research projects currently under investigation relate to a wide range of diseases and applications including cancer, malaria, heart disease, muscular dystrophy and virus related illnesses such as AIDS and Ross River fever. A number of projects are currently the focus of our research efforts; these include (1) the structure, specificity and mechanism of enzyme systems and includes dihydrofolate reductase, glutathione-S-transferases and chitinases (2) structural and functional studies of muscle related proteins (3) the role of ion channels in virus associated proteins.

The mechanism by which enzyme systems function is central to the development of effective therapies associated with these systems. The enzyme dihydrofolate reductase is the target for an extensive range 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. Enzymes such as glutathione S-transferase are involved in the metabolism of chemical toxins and mutagens as well as of therapeutic agents. A detailed understanding of their specificity and mechanism is crucial if one is to be able to predict the metabolism of foreign compounds. Chitinases are sugar degrading enzymes that specifically target chitin. Both chitin and chitinase are widespread in nature, occurring in a range of organisms and are, consequently, of major biotechnological interest. We are actively involved in the structural study of a chitin binding domain and chitinase with the view of investigating its binding and inhibitory properties.

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 and peptide analogues 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 may be beneficial in the treatment of a range of muscle related diseases such as heart failure, malignant hypothermia and muscular dystrophy.

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.

Facilities
The laboratory is well equipped with access to state-of-the-art facilities including two high field Nuclear Magnetic Resonance spectrometers (Varian, Inova 500 & 600) and a network of Silicon Graphics workstations running the latest structure related software. Other facilities include stopped-flow instrumentation capable of performing CD, fluorescence and UV kinetic analysis which is housed in a well appointed molecular biology laboratory.

Marco G Casarotto

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Computational Proteomics

Proteins--life's machines in health and disease

Dr Jill Gready, JCSMR Photography Of the several kinds of matter in the living cell, proteins are the main doers. They act as little machines to carry out the amazing variety of functions in the cell, such as enzymes catalyzing the chemical reactions, receptors accepting and transmitting signals for communication between cells, and transcription and repressor factors turning genes on and off. Many diseases arise from mutations or deletions of genes, either transmitted genetically or arising during the life of the animal, which abolish or cripple the cellular functions of the proteins they encode/specify.

However, very few such mutations, or even deletions, impair cellular functions sufficiently to cause disease, and most have no effect at all! Protein machines, and the networks in which they work together, are apparently quite robust.

In order to understand this apparent paradox, it is necessary to understand how proteins work. This is the study of protein structure, function and evolution. Focussing on individual proteins, we need to know which parts are critical to maintain their 'working parts' and enable them to fulfil their functions, i.e. the link between structure and function. This requires a detailed knowledge of protein structure and mechanism. Like inanimate machines, proteins are assembled from components to create a specific shape. In the case of proteins, the assembly is disarmingly simple - just a linear chain, of variable length, where each position contains one of only 20 building blocks, the amino acids. This sequence adopts a 3-dimensional structure, i.e. the shape, specific for that protein. Although proteins need to be in their correct 3-D structure, often at exquisite levels of precision, in order for them to 'work', very few amino acids in the sequence are absolutely necessary to maintain structure and function. Most can be substituted by similar amino acids (although not all at once!), or many even replaced by any of the other 20 amino acids or entirely deleted. Hence, the robustness to mutation.

My Group's work is concerned with understanding how particular proteins work, and how they are related by evolution to proteins of similar shape which work similarly (homologous proteins) or differently (analogous proteins), both within an animal or across species. Our work aims to capture the essence of a protein, to define its essential features and how it works both structurally and energetically, using a combination of computer-based and experimental approaches. The very large protein molecule provides a scaffold which positions the functional groups of the essential amino acids in a specific way with respect to each other. This arrangement involves only a relatively small region of the protein, and has a specially adapted chemical environment. The environment has evolved to minimize the energy required to promote binding of small-molecule substrates, agonists or antagonists, or other proteins, or lower barriers for enzyme reactions. Often, cunningly, this protein design improves cellular efficiency by driving reactions in a particular direction or creating pathways for enzyme-reaction products or other 'signals' to move to their next 'station'. These details, and hence the essence of the protein, vary greatly from protein to protein, and, although Nature adapts and re-uses some 'designs', each protein problem has unique features. Such detailed understanding provides us with the means to suggest innovative and often simpler solutions to therapy design and protein engineering, than the usual trial-and-error extrapolations and mimicking based on straightforward structure-based principles.

A computer-generated model of how a prion protein might bind to a cell membrane. This shows two single protein molecules, or monomers, bound to each other as a double molecule, or dimer, and anchored to the membrane surface in a vertical position.

Among our current applications we are investigating how prion protein (PrP), which is associated with Creutzfeldt-Jakob disease (CJD) in man and mad-cow disease (BSE), has evolved its ambiguous structure. Understanding both its normal and abnormal functions, provides a secure basis for designing agents to minimize the latter. Another project in collaboration with the Australian Red Cross Blood Service on the blood-plasma protein, mannose-binding lectin (MBL), aims to refine therapeutic use of MBL in patients with inherited or acquired (e.g. AIDS) immune deficiency. MBL is the body’s first line of defence against bacteria in the blood. Understanding the specificity of MBL for binding complex sugars on the surfaces of different types and strains of bacterial pathogens, would allow MBL therapy to be targeted in cases where it would be most efficacious against pathogens. Of our two enzyme projects, one on the mechanism of dihydrofolate reductase involves the design of inhibitors as cytotoxic drugs against cancer and microbial infections. The second project in collaboration with Prof John Andrews in the Research School of Biological Sciences aims to understand why the photosynthetic CO₂-fixing enzyme, Rubisco, is so inefficient, by characterizing the mechanisms of its complex multistep catalytic chemistry. Our goal is to undertake a fundamental redesign of Rubisco, in a way very different from other work being done in the field of artificial photosynthesis.

Jill Gready

Cytokines and Inflammation

Allergy Research and Inflammation Group

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. Allergic disease of the gastrointestinal tract in response to food allergens is also of growing concern in developed countries. One of the major precipitating factors of asthma, particularly in children, is viral infections of the respiratory tract.

A predominant clinical feature of allergy disease is a persistent inflammation at the site of disease. In asthma, inflammation is localized to the airway wall. Currently, it is thought that inflammatory cells (white blood cells) induce asthma by releasing substances that damage the lining of the airways and induce constriction (narrowing of the airways), mucus production and remodeling of tissue. The inflammatory response in the asthmatic lung and in other allergic diseases is a very complex mixture of cells and molecules and it is not clear which factors play the major role in inducing disease.

Allergic asthma is recognized as a chronic inflammatory disease of the airways that is characterized by reversible airways obstruction in association with aberrant CD4+ T helper 2 (Th2 cell) lymphocyte responses to common environmental stimuli. Indeed, the hallmark features of allergic asthma, elevated serum immunoglobulin E (IgE), mucus hypersecretion, eosinophilia and enhanced bronchial reactivity (airways hyperreactivity [AHR]) to non-specific spasmogenic stimuli have all been linked to the effector functions of Th2 cytokines (e.g. interleukin- (IL)-4, 5, 9, 10 and 13). Collectively, it is these pathogenic processes that are thought to promote airways obstruction in asthma, which predisposes to wheezing, shortness of breath and life-threatening limitations in airflow. Th2 mediated immune processes have also linked to the pathogenesis of allergic diseases of the gastrointestinal tract. Viral infections of the lung play a key role in exacerbating asthma and eosinophils and T -lymphocytes are also thought to underpin viral induced asthmatic episodes.

Research in our Group focuses on two major areas

Identifying the key cells and molecules, which underpin the pathogenesis of allergic disease of the lung, skin and gastrointestinal tracts and those that underpin the pathogenesis of respiratory viral infections.

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 and allergy and viral-induced disease of the lung.

Our experimental approach is integrative, employing state-of-the-art molecular genetic techniques in association with model systems to identify the role of inflammatory cells and molecules in the events that underpin disease. Projects within the group focus on the role of individual Th2 cytokines in disease pathogenesis and characterization of the downstream signaling pathways employed by theses molecules to induce disease.

Paul Foster

Cytokine Molecular Biology and Signalling Group

Interleukin-5 (IL-5) is a hormone-like protein which plays a key role in the regulation of immune responses to helminth parasites and to allergens. This cytokine regulates inducible white blood cell formation in response to infection by helminth parasites or to allergen exposure and is principally responsible for controlling the eosinophilic inflammation characteristic of asthma and allergy. This inflammatory response is primarily orchestrated by the inducible production of IL-5 by T lymphocytes which results in enhanced production of eosinophils in the bone marrow. IL-5 then cooperates with other cytokines like eotaxin which promote the migration of the eosinophils to the affected site. Our group has had a long term interest in defining the biological role, regulation and signalling mechanism for IL-5. It is expected that these fundamental studies will provide opportunities for the development of novel therapeutic approaches for the treatment of allergic disease and also information relevant to understanding some leukemias.

Collaborative studies with the Allergy and Inflammation Research Group and the Gene Targeting Laboratory on the role of IL-5 in allergic lung disease have continued. The present emphasis is to define the roles played by IL-5 and eotaxin in regulating eosinophilic inflammation and airways hyperresponsiveness using mice deficient in one or both cytokines. The results indicate that eosinophils play a key role in the orchestration of allergic inflammation and airways dysfunction.

Studies have also continued on the mechanisms regulating IL-5 gene 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. Jun Wang has established transient expression systems in both mouse and human T cells and carried out comprehensive transactivation studies to identify the transcription factors and the binding sites involved in the regulation of the mouse and human IL-5 genes. The results suggest the formation of an enhanceosome type complex in the proximal promoter region involving the transcription factors GATA-3, AP-1 and Ets and possibly also HMGI(Y) which regulates inducible and tissue-specific gene expression. The involvement of MAP kinase pathways in IL-5 induction has also been studied.

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 Paul Carr and David 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. The receptor was expressed in insect cells and its crystallization and derivatization involved extensive use of site-directed mutagenesis to improve crystal quality and to solve the phase problem. The novel dimer configuration of the receptor gives new insights into receptor activation. James Murphy is using site-directed mutagenesis to define the residues of the beta common receptor which are involved in forming the activated receptor complex. Ian Walker has shown that the membrane form of the beta common receptor is also a dimer. Peter Fineran, Alice Church, Sally Ford and Jane Olsen have prepared the activated complex of the closely related beta-IL-3 receptor. Further structural studies should give a better understanding of the process of receptor activation and provide opportunities to develop drugs capable of controlling this important receptor system. Such drugs could be useful in treating asthma, allergy or cancer.

The group has also participated in another project in the area of functional genomics. with Hugh Campbell (Research School of Biological Sciences) and the Gene Targeting Laboratory. In this work, the functions of the mammalian homologues of two interesting genes, flightless and small optic lobes, with functions in development and behaviour in the fruit fly Drosophila are being investigated by gene targeting in mice. Investigations to date have shown a function for mammalian flightless in early embryonic development, remarkably analogous to its role in Drosophila. This cross-organism approach uses information gained from studies with the simpler fruit fly to better understand development and brain function in mammals.

Ian Young

Gene Targeting Laboratory

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 particularit is important to study the function of genes and their derangements 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 chimeric offspring that 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 chimera with a normal mouse gives rise to an animal carrying 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 the ability to study the function of a cloned gene in the context of the whole mammal by creating mutant mice defective in specific genes. 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. Moreover, it is possible to also add genes using similar processes resulting in 'transgenic' mice. Our laboratory has generated a number of different 'knockout' and 'transgenic' mouse mutants using C57BL/6 or BALB/c ES cells that 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.

Klaus Matthaei

Gene Regulation in Development and Immunity

Nuclear Signalling Laboratory

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 copying DNA into mRNA or transcription takes place (see schematic of illustration). Because protein synthesis occurs out of the nucleus 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 a 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 (cells with nuclei) 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 chemical bonding of phosphate groups to proteins, carried out by protein kinase enzymes in the cell).

We and others have shown that specific phosphorylation sites can enhance or 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. 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 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 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.

David Jans

Cytokine Gene Expression Laboratory

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 intracellular 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. The following projects are designed to address these questions.

Assembly of chromatin remodeling complexes on the GM-CSF promoter (Dr Adele Holloway)
One of the cytokine genes that we study in the lab is that encoding granulocyte-macrophage colony stimulating factor (GM-CSF). This cytokine is important in immune and inflammatory responses because it is required for activation of macrophages and dendritic cells. We have previously found that a specific region of the GM-CSF gene is involved in reading T cell signals when the gene is 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 involved 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. We have determined that the remodeling complexes are recruited by the NF-kB protein, RelA, binding to its cognate sites on this region of the promoter. 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. The components of the remodeling complexes that are recruited to the promoter were also determined and we found that members of the ATP-dependent remodeling complexes known as Swi/Snf proteins are recruited by NF-kB. 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. To determine if these in vitro results are relevant in cells, we have examined the role of NF-kB proteins in remodeling of chromatin across the GM-CSF promoter and found that the remodeling activity is dependent on the presence of NF-kB proteins in the nucleus. We now hope to confirm these finding by using assays that are designed to determine whether the NF-kB and Swi/Snf 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.

Frances Shannon

Development of a novel method to assess chromatin remodeling across inducible gene in vivo.
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. The attraction of this method is that it can be easily applied to any gene of known sequence by simply designing new PCR primers.This method has been very 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. Combining this method with inhibitors of specific signaling pathways and with mice lacking specific transcription factors, we have determined some of the requirements for chromatin remodeling across the IL-2 gene in primary T cells. This has led us to the important finding that c-Rel, again a member of the NF-kB family of transcription factors, is critical for chromatin remodeling at the IL-2 gene promoter.

Sudha Rao and Donna Woltring

Dissecting the chromatin structure of the IL-2 gene.
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. Another interesting finding to arise from these studies is that the IL-2 promoter assembles a precisely positioned nucleosome across several of the important transcription factor binding sites and as stated above blocks the binding of many transcription factors. We have found evidence for a similar positioned nucleosome in T cells. When the T cells are activated, then this positioned nucleosome appears to be remodeled, allowing transcription factors access, assembly of functional trancription activation complexes and eventually RNA polymerase activity to generate mRNA.

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.

Joanne Attema

Chromatin and Transcriptional Regulation Laboratory

Chromatin and transcriptional regulation during development

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 mm 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 are 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 regard 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.

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 alpha-helix in H2A that is buried deep inside the nucleosome. A region immediately adjacent to this short alpha-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. In part, consistent with our prediction, distinct localised changes in the docking domain result in a subtle destabilisation between the dimer and the tetramer however, there is also a stablisation between the two H2A.Z molecules at the back of the nucleosome. Consequently, at this stage it is not clear whether H2A.Z increases or decreases the overall stability of the nucleosome. 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 and/or modulate nucleosome-nucleosome interactions.

Most recently we tested the prediction that incorporation of H2A.Z into chromatin alters intra- and/or inter- nucleosomal interactions. This was determined by examining whether this histone variant alters the folding pathway from an extended nucleosomal array to highly compacted heterochromatin. In summary, we carried out the first biochemical analysis of a homogenous preparation of H2A.Z-containing nucleosomal arrays. H2A.Z uniquely effects chromatin condensation; intra-nucleosomal interactions are accentuated while the formation of condensed heterochromatin is inhibited. These data suggest that a major function of H2A.Z is to regulate the conformational equilibria of the chromatin fibre to promote specialised functional chromosomal domains including a domain that is poised for transcriptional activation.

Based on our structural studies, the overall aim of our investigation is to link the in vivo function of H2A.Z in mammalian development with an in vitro analysis of the effect of H2A.Z on transcriptional activation. We will test whether the inhibition of heterochromatin formation by H2A.Z can activate transcription. We will also investigate whether H2A.Z can specifically recruit chromatin modifying enzymes to promoter regions. These results will determine whether H2A.Z is incorporated into the chromatin of active genes, and if these structural changes brought about by H2A.Z are 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

in vitro assembled H2A.Z containing chromatin facilitates transcriptional activation.
H2A.Z containing chromatin recruits specific nuclear proteins.
the docking domain of H2A.Z, and specific amino acid residues within the docking domain, are essential for early mouse development.
H2A.Z is associated with, and responsible for, gene activation in vivo.
mechanisms exist that target H2A.Z to specific regions in chromatin.

David Tremethick

How are genes turned on and off?
All cells in the human body contain a blueprint for producing specialized or 'differentiated' cell types such as those found in the liver, heart or brain. This blueprint, termed the genome, is composed of DNA that codes for unique proteins to perform highly specialized functions. The Human Genome Project has provided scientists with a copy of this blueprint leading to a wealth of information on the composition of all human genes.

The question that continues to intrigue scientists, however, is how a cell decides to follow a pathway leading to a particular cell type such as those found in heart rather than in brain. The answer lies in understanding how the heart genes are turned on while brain genes are turned off. Failure to correctly establish and maintain these gene expression patterns can lead to developmental defects or aberrant cell growth resulting in cancer. Moreover, if we knew the answer to this question we could encourage cells along a desired pathway to establish new heart muscle cells to repair damaged tissue or nerve cells to treat patients with neuro-degenerative diseases. The goal of our research is to define the signals that regulate patterns of gene expression during differentiation and development of a complex organism from a single cell. We believe that a series of small proteins called histones that wrap up the genes may hold the key to answer this perplexing question.

Genes (DNA) and histone proteins are assembled in a step-wise fashion to form chromatin: a highly ordered and dynamic structure. Chromatin efficiently packages DNA into the tiny cell nucleus and is a powerful regulator of gene expression by a switching mechanism. Chromatin switches genes on and off by physically blocking or exposing different parts of the genetic blueprint. Since histone proteins are abundant and intimately interact with DNA they have the potential to regulate intricate patterns of gene expression in unison as “master controllers” of the genome. One way to alter the activity of chromatin is to change its makeup by replacing the major histone types with naturally occurring variants. The focus of our research is one such variant, H2A.Z, which we have shown, in collaboration with Ian Lyons at the University of Adelaide, to be essential for early mammalian development. Evidence to date suggests that H2A.Z is a critical factor associated with both active regions of the genome and chromosome stability. To investigate the role of H2A.Z during differentiation and development we employ two model systems: mouse embryonic stem cells and the Xenopus toad embryo.

Embryonic stem cells are isolated from early embryos and have the potential to develop into any cell type. The ability to grow large numbers of these cells in culture and readily manipulate their genetic makeup means that stem cells are a powerful tool to investigate the signals leading to differentiation. In these cells we have established a system to regulate the expression of H2A.Z during differentiation. The switching of H2A.Z on and off and its location in the cell is efficiently monitored by the Green Fluorescent Protein from jellyfish (see photographs). With this approach we are identifying gene expression profiles during differentiation along specific cell lineages using a new micro-array technique. This technology allows us to examine the whole blueprint of the genome at one time by computer analysis under conditions when H2A.Z is depleted.

One obstacle in the investigation of the function of essential genes is the inability to use the standard method of creating knockout organisms, with a specific gene deleted or knocked out, since the essential gene is needed for survival. An alternative approach is to disrupt the gene of interest so that it produces much more protein than usual or produces protein at the incorrect time or place in the cell. We are using this strategy in frog embryos. They are easy to handle, and the developmental stages are easily monitored in a culture dish. In collaboration with Genevieve Almouzni at the Curie Institute in Paris we are identifying developmental defects arising from disruption of H2A.Z. This information will improve our understanding of the initiation and maintenance of developmental gene expression profiles. With this knowledge we are learning how the same cell blueprint can result in the multitude of highly unique cells functioning together in a complex organism.

Patricia Ridgway

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Autoimmunity and Genetics Laboratory

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.

Robyn Slattery