Division of Molecular Medicine

The Division of Molecular Medicine is comprised of several groups and laboratories that pursue fundamental research into cellular, molecular and genetic processes of relevance to medicine. Common medical problems investigated by members of the Division include cancer, diabetes, autoimmune disease and mental illness. Several of the groups with overlapping interests have formed collaborative research programs within and between the separate Divisions.

These programs focus on specific areas and generate synergies that could not be achieved independently. For example, the Integrative Genetics Program is pooling the expertise of several groups in order to address fundamental questions regarding the biological and clinical significance of genetic diversity. These studies have implications for understanding of the genotype/environmental interactions that appear to have a role in many disorders such as common forms of mental illness that are characterized by anxiety and depression, Parkinson's disease and different forms of cancer.

Another major program in the Division is focused on the development and regulation of the immune system and the molecular and physiological analysis of autoimmunity and its contribution to the pathogenesis of diseases such as diabetes and multiple sclerosis.

The mutagenesis program within the Medical Genome Centre has generated a range of mutant mice with novel phenotypes. The characacterization of these mice is revealing pathways involved in the development of the immune system and cancer. The success of this program has attracted major funding from the Wellcome Trust, Juvenile Diabetes Foundation and the Australian Government's Major National Research Facilities program for the establishment of a National Phenomics Centre.

The groups within the Division continuously review their research findings to identify those aspects with potential clinical applications. Many of the groups have well established collaborations with hospital based researchers to facilitate the transfer, to the clinic, of advances made in fundamental research.

Professor Philip Board, Head of Division

Developmental Physiology Group

Two projects using fetal lambs were concerned with applying the novel system for in vitro development of gut associated lymphoid tissue described in earlier reports and with investigation of a recent observation of putative relocation of thymic function in extra-thymic lymphoid tissue. Following the disestablishment of the Group in April, these projects have been continued in other laboratories.

The development of miniature gut associated lymphoid organelles in tissue culture by mixing single cell suspensions of B lymphocytes, intestinal epithelium and fibroblasts replicates many of the processes essential for the formation of primary lymphoid tissue (in which lymphocyte proliferation occurs without any requirement for exposure to antigen). Recent research has been directed both to further exploration of primary lymphoid organ function and to exploiting the miniature organ system to examine functions of secondary lymphoid tissues (lymphocyte proliferation occurs only in response to exposure to antigen). Investigation of primary lymphoid function currently includes examination of organelle B cells for the occurrence of hypermutation. The system has now been successfully adapted from the fetal lamb to the chick embryo to achieve the formation, in tissue culture, of the Bursa of Fabricius, a primary lymphoid organ responsible for generation of the B cell repertoire in the chick. The significance of the Bursa extends considerably beyond the confines of avian immunology as it is the archetypal system on which the current understanding of mammalian B cell immunology is based. It is now feasible to probe avian and mammalian B cell ontogeny in parallel using a common technique.

The gut lymphoid tissue culture system is currently in use to examine secondary lymphoid tissue function in two situations. Firstly, colonization of fetal lamb organelles by Mycobacterium avium is of interest because the entry of this bacterium through the gut lymphoid tissue initiates Johnes disease, currently the sheep disease of greatest economic impact in Australia. The entry of the same organism via this route also represents one of the commonest and most intractable opportunistic infections of human patients with AIDS. In a second set of experiments, chick organelles have been utilized to grow pathogenic viruses for which no culture system had previously been available.

The second project, which was initiated before disestablishment of the Group, is based on the observation that, following microsurgical removal of the thymus gland (exclusively responsible for generation of the T cell repertoire) from fetal lambs early in gestation, thymic functions appear to be taken over by a specific cervical lymph node. With the benefit of hindsight, this relocation suffices to explain the longstanding observation that thymectomy has less impact on immunological development in fetal lambs than in mice. The current project has as its objective delineation of the processes responsible for establishment of thymic functions in a lymph node. Successful accomplishment of this objective affords the possibility of applying the new knowledge to re-establishing thymic function in the peripheral lymphoid tissues of paediatric AIDS patients in whom the thymus has been destroyed.

Peter McCullagh

Transplantation Immunology Laboratory

Insulin-dependent or Type 1 diabetes in humans results from an autoimmune disease which selectively destroys insulin-producing beta cells of the islets of Langerhans. Islets are normally found in large numbers, scattered throughout the pancreas. The transplantation of pancreatic islet tissue represents a treatment which would restore physiological control of blood sugar levels thereby preventing the secondary microvascular complications associated with current insulin therapy. Islet tissue transplants, however, are faced with destruction via two possible mechanisms: rejection and autoimmune disease which returns to attack the transplanted tissue. From a practical viewpoint, there is a vast shortage of human pancreases available for transplantation. Use of a non-primate animal species (e.g. pig) for pancreas donation would offer the bonus of an abundant supply of islets for replacement therapy. However, ethical concerns have been raised about the possible transmission of pig viruses (e.g. endogenous retrovirus) from the transplant tissue to diabetic human recipients. This cross-species transmission is known as 'xenozoonoses'.

Although immunosuppressive drugs are currently used to prevent the immunological rejection of life-saving organ transplants e.g. kidney and heart, such therapy can severely compromise the bodys immune defenses and render patients susceptible to fatal infections. This potential risk cannot be justified in patients with Type 1 diabetes because the immunosuppressive treatment could lead to far worse health problems. For this reason, the laboratory's research focuses on identifying the immune mechanisms responsible for the destruction of pancreatic islet tissue following transplantation to both different recipient animal species ('xeno'- grafts) and to genetically different members of the same species ('allo'- grafts). This information is being used to develop safe, non-toxic strategies for preventing transplant destruction i.e. thereby eliminating the need for systemic or toxic immunosuppression. Approaches currently under investigation include: gene therapy using viral vectors expressing genes for protective proteins (cytokines), cellular co-transplants engineered to produce protective proteins (e.g. cytokines) in the local vicinity of the islet transplant and blockade of the signals which attract cells of the immune system to sites of injury or invasion by foreign material.

Ultimately the safety of pig islet transplantation needs to be established for this approach to be recognised as an improved treatment for Type 1 diabetes. Computerised databases of viral sequences (Bioinformatics) are being used to help assess whether transmission of pathogens, e.g. viruses, from transplanted pig islet tissue to recipient tissues (xenozoonoses) occurs and to design strategies for investigating the potential role of such viruses in immune recognition of xenogenic transplants.

Studies in the laboratory have focused on the immunobiology of pancreatic islet xenotransplantation and allotransplantation in mice.

Charmaine Simeonovic

Lack of stable transmission of porcine C-type endogenous retrovirus (PERV) to host tissues after pig islet tissue xenotransplantation in fetal lambs
The xenotransplantation of animal organs and tissues offers a possible solution to the shortage of human organs and tissues available for clinical transplantation. Pigs remain the preferred donor species because of their near-human size and their already established capacity to be bred in large numbers for meat consumption by the human population. Nevertheless, there exist both immunological and ethical barriers to this otherwise attractive concept. Achievements in establishing xenograft survival have been offset by growing ethical concerns over the potential for transmission of porcine pathogens, particularly endogenous retroviruses, from the donor tissue to recipient tissues i.e. xenozoonoses. Endogenous retroviruses have been ear-marked for concern because of their known capacity to remain non-pathogenic in their native host but to have the potential to become pathogenic in atypical hosts, often inducing leukemias and other malignancies.

Three classes of porcine endogenous retrovirus (PERV), a member of the mammalian C-type retrovirus genus, have been identified: PERV A, PERV B and PERV C (9-11). Pig islet tissue (fetal proislets) carry and express PERV A, PERV B and PERV C nucleotide sequences. To determine whether PERV transmission to host tissue occurs after pig proislet xenotransplantation, two recipient models were studied: (i) the fetal lamb in utero (at 54-56 days of gestation) which has an immature immune system and represented an environment of rapidly dividing host cells, and (ii) the NODscid mouse which is an immunoincompetent host and thus ensured long-term xenograft survival. Nested PCR was used to analyse PERV sequences in fetal lamb liver and spleen at 5-84 days post-transplant and in xenografts, host liver and spleen harvested from NODscid recipient mice at 96 days post-transplant. During the early 5 to 23 day post-transplant period in fetal lambs, PERV A, B or C sequences were detected in 4/12 fetal lamb liver samples in the absence of pig COII sequences, indicating PERV transmission and the integration of PERV sequences into the genome of host liver. Transmission of PERV sequences to fetal lamb liver was not demonstrated at 84 days post-transplant and no transmission was detected in fetal lamb spleen at any time. Detection of pig COII DNA sequences in 2/12 fetal lamb liver samples and 3/12 fetal lamb spleen at 5 days post-transplant suggested that pig cells had migrated to host lamb tissues, thereby establishing pig cell microchimerism. PERV envelope (env) RNA sequences were detected in only 1/12 fetal lamb liver samples and in no fetal lamb spleen samples, suggesting that xenogeneic host cells represented a sub-optimal environment for PERV replication. Pig cell microchimerism but not transmission of PERV DNA sequences or PERV env RNA (PERV replication) was demonstrated in NODscid recipient mice. In contrast to long-term xenograft survival in NODscid recipient mice, pig proislet xenografts were completely rejected by 23 days in fetal lamb recipients. The differences observed in PERV transmission between the two models may be due to the increased susceptibility of rapidly dividing fetal lamb cells to retrovirus infection. Based on the observed limited potential for PERV replication in recipient cells, pig cell migration to host tissues i.e. cell-cell contact, represents the likely mechanism responsible for PERV transmission to host tissues. In fetal lambs, the lack of long-term PERV transmission and the rejection of pig proislet xenografts correlated with the known kinetics for the establishment of host immunocompetence. We therefore suggest that absence of stable PERV transmission in fetal lamb recipients is due to the immunological destruction of PERV-infected host cells. Prevention of PERV transmission to xenogeneic host tissues is likely to require inhibition of PERV receptor function and/or the integration of PERV nucleotide sequences into the host genome rather than inhibition of PERV replication.

David Mann, Adrian Gibbs, Peter McCullagh, J Dennis Wilson and Charmaine Simeonovic

The role of chemokines and their receptors in the rejection of pig islet tissue xenografts.
The limited availability of human organs and tissues for allotransplantion has made it necessary to search for alternative sources of donor tissue. The use of animals as organ donors for xenotransplantation may represent a possible solution to this problem. However, xenograft rejection remains the main obstacle preventing this therapy from becoming a viable option for patients. The mechanism of xenograft rejection, and the process of inflammatory cell recruitment to the graft site must be clearly understood for an appropriate anti-rejection therapy to be developed. The mechanism by which inflammatory cells are recruited to pig islet tissue (proislet) xenografts was investigated by examining the intragraft mRNA expression of murine a- and b- chemokines in CBA/H mice from days 3-10 post-transplant. Xenograft rejection was associated with early intragraft transcript expression for MCP-1 (3-5 days), IP-10 (3-4 days) and MIP-1a (3-5 days) and subsequent expression of eotaxin (days 4-10) , MIP-1 b (days 4,5) and RANTES (days 4-6) mRNA. This pattern was consistent with the early recruitment of macrophages (MCP-1, MIP-1 a), the influx of CD4 T cells (MCP-1, MIP1 a, MIP-1 b, IP-10 and RANTES) and the characteristic infiltrate of eosinophils (eotaxin and RANTES) associated with islet xenograft rejection. Inhibition of b -chemokine signaling in CCR2-/- mice (which lack the major co-receptor for MCP-1) resulted in delayed rejection, retarded macrophage and CD4 T cell recruitment and enhanced eosinophil influx, compared to wildtype mice; delayed rejection and inhibition of leukocyte infiltration was more transient in xenografts harvested from CCR5-/- mice. The obstruction to leukocyte migration into xenografts in CCR2-/- hosts was associated with delayed intragraft expression of MCP-1 and RANTES mRNA; absence of MCP-1/CCR2-mediated signaling led to enhanced intragraft expression of MCP-1, MIP-1a and MIP-1b mRNA. These findings suggest that MCP-1 plays an important role in regulating macrophage and CD4 T cell infiltration to xenograft sites via the CCR2 signaling pathway. Additional treatment of xenografted CCR2-/- transplant recipients with anti-IL-4 and anti-IL-5 mAbs further delayed xenograft rejection demonstrating the potential for combined anti-rejection strategies in facilitating pig islet xenotransplantation.

Michelle Solomon, William Kuziel and Charmaine Simeonovic

Molecular Genetics Group

Because human beings are constantly exposed to a variety of environmentally derived chemicals that effect the normal function of our cells, tissues and organs, we have evolved a complex group of enzymes that detoxify these compounds and provide an important layer of protection against their deleterious effects. It is now very clear that an individual’s genetically determined complement of detoxication enzymes has a significant influence on their response to a variety of therapeutic drugs and environmentally derived toxins. The work of the Molecular Genetics Group is aimed at gaining a fundamental understanding of the molecular and biochemical mechanisms that underlie individual responses to such compounds. One of the major research interests of the Molecular Genetics Group is the role played by the glutathione-linked enzymes such as the glutathione transferases (GSTs) in the metabolism and detoxification of therapeutic drugs and environmentally derived carcinogens and toxins.

The GSTs are a large family of enzymes and previous studies have shown that they can be subdivided into a number of different classes that have characteristic structural variations, substrate preferences and sites of expression. The GSTs function by conjugating glutathione to the target chemical thereby making it more water soluble and making it recognisable by an export pump that expels glutathione conjugates from cells.

Genetically determined deficiency in the expression of some GSTs can be a risk factor for lung, stomach and skin cancer. In contrast, over expression of GSTs has been associated with resistance to cancer chemotherapy. Genetic variations that cause subtle changes in GST function can be clinically important. For example, we found that a variant form of glutathione transferase GSTP1 that works with different substrates was associated with the occurrence of Parkinson's disease in patients who had been exposed to pesticides.

To gain a comprehensive understanding of the genetic diversity in response to environmental toxins and therapeutic drugs it will be necessary to identify all the enzymes involved in detoxication processes and to identify the common genetic variants of these enzymes that contribute to functional differences. The recent expansion of the Expressed Sequence Tag database (EST) to include more than a million DNA sequences encoding copies of most active human genes has provided a remarkable resource for the identification of new genes and polymorphisms. We have developed novel screening strategies that have successfully identified several new glutathione transferase gene families and a number of novel polymorphisms.

The new enzymes discovered by this data mining approach have been shown to catalyse unique detoxification reactions and to participate in metabolic pathways not previously attributed to the action of glutathione transferases. For example we recently discovered that a GST we have termed Omega can inhibit ryanodine receptor calcium release channels in the heart. This enzyme also plays a role in the metabolism of arsenic. Another GST we have discovered and called Zeta is involved in the metabolism of compounds such as dichloracetic acid (DCA) which is known to cause cancer in mice. Significantly, DCA is a contaminant of chlorinated drinking water.

The strategies we have developed for screening the EST database can be readily applied to other genes and gene families and will be of great value in the identification of new genes and polymorphic variants of pharmacogenetic interest.

Philip Board

Ubiquitin Laboratory

The Ubiquitin Laboratory investigates the small protein ubiquitin; its role in the destruction of other proteins (proteolysis) in the cell; and in the consequences of aberrant proteolysis due to defects in the ubiquitin system.

Ann-Maree Catanzariti with supervisor, Dr Rohan Baker
Ann-Maree Catanzariti, Dr Rohan Baker, pb

Complex processes such as cell growth, development, and gene expression, are controlled by proteins that are only required transiently and must be rapidly destroyed to control their activity. This is accomplished by attaching multiple ubiquitin proteins to the target, which "marks" it for destruction by a large proteolytic complex called the proteasome. Most of our research focuses on a family of enzymes called ubiquitin-specific proteases (USPs), that have the ability to cleave ubiquitin from such marked proteins, and thus slow or prevent their destruction. In this context, some USPs can be considered "proof-reading" components of the system, safeguarding a target from inappropriate ubiquitination (and thus destruction), and imposing a further level of regulation on the pathway.

We study USP enzymes using yeast as a model system, both because it is very amenable to genetic and biochemical studies, and also because the ubiquitin system is so highly conserved between yeast and humans, that insights we gain in yeast can be applied to our studies on mouse and human enzymes. Current work centres on Ubp15, one of a family of 16 USPs in yeast. Ubp15 binds directly to yeast multi-drug resistance (mdr) proteins, and Ubp15 can regulate drug sensitivity of cells, presumably by affecting function and/or degradation of mdr proteins. We are extending this work to the study of drug resistance in mouse and human cells.

We also use mice as a model organism to study human disease. We have previously identified a USP that functions as an oncogene in mice; that is, it causes cancer when overproduced. Others have linked the human version of this USP with certain types of lung cancer. We and our collaborators have shown that this USP interacts with the Retinoblastoma tumour-suppressor protein and other related tumour suppressors, which function to prevent cell growth by keeping transcription factors (proteins that control gene expression) in check. The human genome project has revealed at least 31 USPs in humans; one of these is a very close relative of this oncogenic USP in both mouse and human cells, and we have shown it also interacts with the Retinoblastoma protein. Our current efforts are aimed at determining the functional significance of these interactions; how these USPs traffic to the nucleus; and if the closely-related USP is also an oncogene. We are also studying novel USPs identified by the genome project.

Our results show that USPs can play important roles in regulating the ubiquitin pathway, and through this, processes such as cell growth. Aberrations in the function of these USPs may lead to cancer and disease.

Rohan Baker

Human Genetics Group

My research is aimed at understanding human genetic variation as it relates to disease. I take a broad view, recognising the importance of the evolutionary forces that have shaped the structure of the human genome and the nature of human genetic diversity. Our investigations fall into two areas: Mechanisms of gene and genome evolution, and the nature of human genetic and genomic variation.

Gene and genome evolution
In mammals genetic novelty arises through genetic and evolutionary processes such as mutation, gene duplication, gene conversion, genetic drift and natural selection, occurring in large and structurally complex genomes. Information relating to genome and protein function, the processes by which functional novelty has arisen and patterns of species evolution are contained within DNA and protein sequences. We use a comparative approach to the interpretation of this information, investigating differences in genomes, genes and the proteins they encode, among related species, particularly humans and other primates.

Human genetic and genomic variation
Medical genetics has largely been focused on understanding single gene defects. Less attention has been given to genetic disorders that have a polygenic basis, such as mental illness, cardiovascular disease and diabetes. Although these are more important from a public health point of view, they are technically more difficult to investigate. This situation is changing largely through the technological advances of the human genome project. Associated with this change is an increased recognition of the importance of the normal range of human genetic variation, and its importance in relation to disease prevention. Understanding the relationship between molecular genotypes and disease phenotypes is becoming more dependent on population-based rather than family-based investigations. Our research is directed at understanding the basis of a number of genetic disorders, and of the patterns of normal genetic variation in human populations. One particular focus is the genetic basis of personality variation, which is associated with a predisposition to common forms of mental illness involving depression and anxiety. This work is done in collaboration with the NHMRC Centre for Mental Health Research.

Simon Easteal

Medical Genome Centre

Many of the health problems we face today - cancer, autoimmune diseases, allergy, cardiovascular disease, osteoporosis, - stem from a discordance between genetics and environment. Our genetic code was selected to survive for a shorter period of time in a very different world, and it is inevitably out of step with the lifestyles we lead today. In some cases we know what to do to fix this imbalance, like putting on a hat and sunscreen to offset genetic susceptibility to the sun. For many cancers and numerous other diseases such as diabetes, rheumatoid arthritis, osteoporosis or obesity, the solutions are not so straightforward.

Rapid advances in gene technology are bringing us to the point of visualizing the imbalance between our genetic code and lifestyle/environment. A worldwide effort has already produced a map and determined the spelling of the forty-thousand genes in our genetic 'dictionary'- collectively referred to as our genome. The epic challenge for the next decade is to decipher what these gene words mean. How are they combined into the molecular language that guides the cells in our body and determines how well we cope with different environmental stresses? Which gene products are good targets for new drugs to prevent or cure common diseases? Differences in the spelling of those gene words underlie differences in susceptibility to modern diseases and divergent responses to particular therapies, and gene-fingerprinting technology now allows a list of spelling differences to be compiled from each person's unique genetic dictionary. This great opportunity to advance health care hinges on establishing the function of genes and how they interact.

To decipher the functional meaning of genes and their contextual interactions, laboratory mice represent a crucial Rosetta stone. The mouse genome has also now been sequenced, and almost all of the genes in mice match a human counterpart with only small changes in their spelling. While surprising given the large differences in external appearance, the mouse and human genetic languages are in fact no more different than Chaucer's English and modern English. This underlying similarity makes it possible to define the function of human health genes in the mouse in a way that is impossible in humans.

The strategy being followed is genome-wide mutagenesis in the mouse at an unparalleled scale. In the last year, the Medical Genome Centre has successfully analysed a large panel of new mutant strains to reveal functions of many key genes. These strains were derived from libraries of laboratory mice in which single letters in the spelling of many of the genes in the mouse genetic dictionary have been changed by random chemical mutagenesis. The ENU1 library comprised 185 pedigrees of ENU mutagenised C57BL/6 mice, bred over 3 generations to yield at least 25 potentially recessive homozyogous offspring in each pedigree. Collectively, we estimate that this library scanned 15,000 loss of function mutations. By visual inspection and high-throughput screening of blood, this library yielded many new mutant mouse models to discover and understand genes controlling obesity and diabetes, cardiovascular disease, kidney disease, limb and spine abnormalities, eye function, melanocyte function, coordination, seizures, dermatitis, colitis, and disorders of lymphocyte development such as autoimmunity, immunodeficiency, and leukemia/lymphoma.

State-of-the-art facilities for transgenic mouse production, sperm freezing, and database tracking of genetic and phenotypic data have been developed to support this resource and provide a service to researchers. The chromosomal location of the defective gene in each of the mutant strains has been determined, employing an efficient strategy for gene mapping many mutant strains in parallel. In many of the strains, the defective gene has been identified through resequencing of mRNA, revealing critical functional domains in the proteins these genes encode. In parallel, we have defined the precise cellular abnomalities brought about by each mutation by cell and biochemical analysis. From the immunological screen on blood cells, we have shown that the genome-wide mutagenesis approach yields clusters of ten or more mutant strains from a library of 185 families, to reveal key functional elements at many successive steps in the molecular pathway of T cell activation. To use the dictionary analogy, this approach is ideally suited to the problem of how individual genes are linked together to form a molecular language, because it identifies many of the key genes in the molecular sentences that cells use to communicate and respond to stress, rather than individual gene words in isolation.

To build on our genome-wide mutagenesis technology, new projects and screens have been started in the last year. With Prof John Bell and Dr Richard Cornall at Oxford University, a collaborative Programme Grant from The Wellcome Trust began in April 2001, employing sophisticated immune cell challenges and sensitization with a T cell receptor transgene to identify a broad range of genes controlling the immune response to antigen. In September 2001, a Special Programme funded by the Juvenile Diabetes Research Foundation and the NH&MRC is using a pair of transgenes in the mutagenized mouse stock to sensitize the discovery of genes that control diabetes. In August 2001, a consortium of the ANU, Monash University, the Garvan Institute, and the University of Queensland was awarded funds from the Commonwealth Government Major National Research Facilities program to establish a much larger National Facility, the Australian Phenomics Facility. Design and construction of the facility on the ANU campus will begin early in 2002.

Chris Goodnow

Mining the genome for disease genes
Earlier this year the sequence of our DNA, the human genome, was completed. Now the genome sequences from other organisms including the laboratory mouse are nearing completion. These accomplishments are unquestionably major milestones in the history of biomedical research. Nevertheless, completion of the human genome sequence represents only the first step in unlocking the information encoded in our DNA. The challenge we now face is to identify all genes, the unique pieces of DNA in the genome that encode proteins, within the vast genome sequence and to decipher how these genes enable our bodies to grow, develop and fight disease.

This is a tremendous challenge. It is estimated that the genomes of humans and mice contain at least 40,000 genes and currently we only understand the importance of a fraction of these genes. At the Medical Genome Centre of the John Curtin School of Medical Research we have developed a process that represents a powerful, new approach for discovering genes important in disease. This process in effect pinpoints genes in the genome based on their importance in specific physiological, developmental and disease processes. It is difficult to accurately predict the importance of genes to specific physiological and disease processes from computer analyses or in vitro methods such as tissue culture. Because of these limitations, our process utilises the gold-standard laboratory model for human physiology and disease - the mouse.

The process we have developed, called genome-wide chemical mutagenesis, is initiated by subtly altering the DNA code of hundreds of genes simultaneously in a laboratory mouse by treating it with the chemical ethyl-nitroso-urea (ENU). The ENU has its effect on genes by inducing random point changes or mutations in the chemical sequence of DNA across the genome. These point mutations have the potential to affect the function of proteins encoded by the chemically altered genes and ultimately cause significant changes in physiology or disease susceptibility. These gene altering point mutations are not apparent in the initial ENU-treated mouse, but they can be detected in his offspring by screening them for specific characteristics or 'phenotypes' that indicate that a gene critical to a physiological or disease process has been mutated. Many of the screens we use are very similar to screens done in a hospital on human patients. Once a mouse is identified with a disease phenotype, we are then able to identify the gene that was mutated in that mouse by using a combination of genetic mapping and DNA sequencing techniques. Most importantly, by starting with a mouse that exhibits a disease phenotype we know that the gene mutated in that mouse is critical to the development of that disease. Thus, this process allows us to unequivocally identify genes involved in different disease processes.

Using this approach, we have identified over 50 new strains of mice that exhibit phenotypes directly related to human disease. These phenotypes include immunodeficiency, late and early onset obesity, diabetes, neurological disorders and cancer. We have now mapped and characterised many of the mutated genes that result in the observed disease phenotypes. From these studies it is clear that many of the genes identified using these mice have never been characterised. In addition, this approach has led to the discovery of important new functions of previously identified genes in critical processes such as red blood cell development. Genome-wide chemical mutagenesis is allowing us to mine deeply into the genome for genes that are critical to many different disease processes. The information derived from this genetic excavation will be critical in the development of new therapeutic strategies for the treatment of a number of human diseases.

Keats Nelms

Australian Cancer Research Foundation Genetics Laboratory

A one million dollar grant from the Australian Cancer Research Foundation in 1997 made possible the establishment of the ACRF Genetics Laboratory in the Medical Genome Centre. The laboratory's focus is on developing genome-wide mutagenesis resources for studying candidate human cancer genes and novel laboratory mouse models to accelerate cancer research at both the basic and clinical ends of the spectrum.

Four new mouse strains to illuminate cancer genes have been obtained from the ENU1 forward genetics library. One of these strains, Plastic, carries a single dominant gene defect that results in acute T cell lymphoblastic leukemia, providing a valuable model for understanding this important form of childhood leukemia. Peter Papathanasiou has mapped and identified the mutation in this strain, which creates a single change in a protein that appears to be frequently defective in human T cell lymphoblastic leukemia. The cancer protein normally regulates expression of many other genes in blood cells. He has shown that the protein is critical for the production of red blood cells, because it is necessary to maintain expression of receptors for stem cell growth factors as the cells differentiate.

Adele Loy has mapped the mutation in another cancer-prone strain, Bblast, which carries a semi-dominant gene defect that results in leukemia, lymphoma, osteosarcoma, chondrosarcoma and teratocarcinoma. These cancers arise because the mice carry a single misspelling of a protein called Tumor suppressor p53. Tsp53 is the most frequent defect in a wide range of human cancers, and controls a range of tumour and ageing processes. The Bblast strain therefore provides a valuable model for investigating cancers of many types. A third strain, carrying a purely recessive susceptibility to lymphoma and primitive myeloid leukemias, is currently in the gene mapping and identification phase.

Progress continued on a separate discovery project aimed at illuminating genes regulating solid tumours of the skin and cervix. This project involves a consortium with Dr Douglas Hanahan at the University of California in San Francisco, who has developed a sensitized mouse strain for skin and cervical cancer research, and Dr Simon Foote at the Walter and Eliza Hall Institute. The project is mapping cancer inhibitory genes between two inbred mouse strains, C57BL/6 and FVB, and conducting genome-wide mutagenesis to illuminate skin cancer inhibitory genes.

Genes and functions discovered by these efforts will be available to the research community to help in three key areas:

-primary prevention, where illuminating patterns of inherited susceptibility will help to resolve environmental risk factors and target preventive measures

-early diagnosis, where alterations in gene spelling and expression pattern will help to distinguish cancer cells early and predict the best course for treatment

-therapy and prevention of secondary tumours, where the genes identified will provide new targets for drugs and new avenues for chemotherapeutic or immunological intervention

Goodnow Laboratory

Our research aims to understand how immune cells make a fundamental decision: either to fight or to disarm. The process of deciding which immune cells should fight and which should disarm is key to our ability to resist infection and parasitism. Mistakes in this process result in autoimmune diseases, allergy, lymphoma, and leukemia. Moreover, drugs and other ways to alter fight or disarm decisions are sorely needed to improve the success of organ transplantation and treatment of autoimmune diseases and metastatic cancer.

The immune system is made up of billions of immune cells called lymphocytes. By a remarkable gene shuffling process akin to a poker machine, each lymphocyte carries a unique receptor, enabling every lymphocyte to detect a different set of molecules termed antigens. Some lymphocytes have receptors for foreign antigens that are unique parts of the molecular makeup of different infectious organisms. When these rare lymphocytes bind a foreign antigen during an infection, they receive a signal to fight. The lymphocyte first multiplies to make many clones of itself, and then the cells elaborate destructive compounds that neutralize the infectious antigen.

By chance, other lymphocytes carry receptors for self antigens, ie. parts of our own normal tissues and body fluids. When a lymphocyte binds a self antigen it normally receives a signal to disarm. Instead of multiplying and producing destructive compounds, the lymphocyte either commits cell suicide by apoptosis or the cell disarms itself by becoming functionally tolerant, ie. less responsive to antigens and less able to multiply or produce destructive compounds.

For a long time it was not possible to see how self-reactive lymphocytes disarm themselves. Our laboratory has developed ways to visualize this process in genetically modified laboratory mice called transgenic mice. By studying cells in the transgenic mice, we have discovered that each immune cell must run through a complex series of fight or disarm checkpoints before it can be fully launched into an immune response. In some ways, the process resembles the sequence of fight/disarm decisions in a military missile launch, which serve a similar purpose of preventing friendly fire.

Members of the laboratory are deciphering different fight/disarm checkpoint processes, using a combination of biochemistry, cellular immunology, genetic analysis, and transgenesis. At each of these checkpoints, we are focussing much of our work on elucidating how it is that antigen receptors on lymphocytes can trigger several different cell fates ranging from cell proliferation to cell death. Two examples of our work are summarized below.

T cell tolerance and destruction of pancreatic islets
The cellular and molecular processes regulating CD4 T cell responses to self antigens that are restricted to specific organs (organ-specific tolerance and autoimmunity) are being investigated through an NIH and JDRF-funded project involving a unique transgenic mouse model. The core of this model is a “TCR-transgenic” mouse where most CD4 T cells react with a model antigen, and where the model antigen is made by pancreatic islet beta cells. The islet-reactive T cells are normally regulated so that they can cause inflammation of the pancreatic islets, but this inflammation does not progress to diabetes or autoantibody production. This regulation is broken by one or more genes from the NOD strain, an excellent animal model for spontaneous Type 1 diabetes in humans, but it has been difficult to identify what regulatory processes are primarily affected. In the last year, Dr Sylvie Lesage has discovered that one of the main actions of the diabetes susceptibility genes is to dramatically reduce the efficiency of islet-reactive T cell deletion in the thymus. Some of the diabetes genes act within the islet reactive T cells, interfering with the normal processes that censor these forbidden clones of cells. By revealing the primary cell regulatory processes that are disturbed by diabetes genes, this experimental model provides a unique opportunity to define the molecular pathways regulating diabetes and other autoimmune diseases.

Memory receptor tail
Our ability to resist infection stems from a cardinal property of the immune system, namely an ability to mobilise a much higher and faster antibody response when the antigens that make up a virus or bacteria are seen a second time. This phenomenon of immunological memory lasts for years after infection or immunisation, but its cellular and molecular basis is still poorly understood. Mr Stephen Martin has defined a key role for the 'BCR Tail sequence' in promoting memory-level antibody responses. It has been known for many years that B cells switch portions of the antibodies they produce from IgM to IgG as part of the memory process. Among the many changes that result from switching to IgG, one intriguing change is in the sequence and length of the antibody segment that serves as a receptor for antigen- the transmembrane and cytoplasmic tail. Through a sophisticated combination of transgenic mice and in vivo immunological analyses, Steve has shown that the memory-type IgG tail dramatically increases the number of progeny cells that are formed by proliferation of initially rare B cells that react to antigen. This finding explains the memory phenomenon by a localizing it to a precise sequence of amino acids and a specific process- augmenting the clonal burst. This finding has wide implications for vaccination, allergy and autoimmunity, where B cells bearing the memory tail are formed.

Chris Goodnow