pp. 18-32 in Human Genome Research and Society
Proceedings of the Second International Bioethics Seminar in Fukui, 20-21 March, 1992.

Editors: Norio Fujiki, M.D. & Darryl R.J. Macer, Ph.D.

Copyright 1992, Eubios Ethics Institute All commercial rights reserved. The copyrights for the employees of the US Government, are subject to other copyright arrangements. This publication may be reproduced for limited educational or academic use, however please enquire with Eubios Ethics Institute.

p. 18 in Human Genome Research and Society
Proceedings of the Second International Bioethics Seminar in Fukui, 20-21 March, 1992.

Present status of human genome research in Japan

Itaru Watanabe,
Emeritus Professor, Keio University, JAPAN

Human Genome Research was initiated by Prof. Wada of Tokyo University already over ten years ago through the Science and Technology Agency of Japan. Since that time the study of the human genome has been becoming a very hot issue. The Ministry of Education has also started the initiative in the study of the human genome by Prof. Matsubara. Now, Prof. Sakaki and Prof. Matsuda (presenting a paper instead of Prof. Honjo who unfortunately could not be present), are going to present some results of this study.

pp. 19-23 in Human Genome Research and Society
Proceedings of the Second International Bioethics Seminar in Fukui, 20-21 March, 1992.

Physical mapping of chromosome 21

Yoshiyuki Sakaki,
Professor, Institute of Medical Sciences, University of Tokyo, JAPAN

There are about 100,000 genes distributed over all the human chromosomes. These genes control different functions of our body. These chromosomes are now under study in the form of nucleotide sequences, or base-pairs. In the chromosomes there are many genes, some a single-copy genes and some are multicopy genes. Where there are no genes we can often see repetitive sequences, such as Alu and L1. In addition there are many unknown sequences. We also have centromeres and telomeres which have special characteristics and functions. The ultimate goal is to clarify the overall picture, and identify each gene and how genes work and have been organised in each chromosome.

The so-called Human Genome Project contains several different approaches. A major one is the so-called "top-down" approach (Figure 1) which starts from macro-scale mapping and ends in identifying all the genes on the genome. The top-down approach involves several steps, and currently researchers are making efforts to construct maps of the genome and also the contig of cloned DNAs (Figure 2). The other approach is the bottom up approach (Figure 1), where all the fragments are gathered in order to understand the whole. The human genome is so large that we have to first start with the top-down approach, and in the end we can combine this with the bottom-up strategy, to grasp the whole picture.

Figure 1: Approaches to human genome analysis

Figure 2: The top-down approach to human genome analysis

At the very start of the top-down approach is the mapping of markers on the genome, to set the location of points. Using these maps we further clone the corresponding segments. The mapping is the first part of this approach, and there is currently much activity on mapping in Japan. Once a map is completed we can expect many things. We can map a different variety of phenotypes, and look at genes related to them. Linkage analysis is one method (Figure 3). If we find a marker close to the disease-causing gene we can look at the location of the gene. If two points on the chromosome are close to each other there is a high probability that they will be inherited together and segregation will be rare. The frequency of segregation is used for linkage analysis. This way we can map different diseases. We can then clone the DNA and analyse it, and finally we will find the gene. One successful example of this method is cystic fibrosis, whose causative gene is located on chromosome 7. A detailed physical map of the region was made, and then several candidate genes were found and finally the true causative gene was identified.

Once the complete map is developed, we can use the same approach for other diseases. We are interested in Alzheimer's disease. One characteristic of the brains of affected people is the senile plaque. The major protein of the plaque is the -amyloid protein. We cloned the gene and sequenced the -amyloid precursor protein gene, to determine it's structure. In the case of familial Alzheimer's disease, we analysed this gene in collaboration with Dr. Miki of Osaka University. Dr Miki studies many families of Alzheimer's disease. In one of the ten families we found an abnormality in amino acid 117. We were able to find out this was the course of disease. But in other families we could not identify the cause of disease. For example the largest family of Alzheimer's we know in Japan showed no mutation in -amyloid precursor protein.

The early onset Alzheimer's disease has been studied worldwide, and the gene of this type is mapped on chromosome 21 (Figure 4). The positions are indicated in Figure. Down's syndrome has some similarity, which is trisomy 21. Therefore we started to focus on chromosome 21. The first step in making a map is to use restriction enzyme analysis, and we used the enzyme Not1 (Figure 5). One reason was that this enzyme cuts the human genome into only 3,000 fragments. This means each is about 1 million basepairs in average. This size is similar to the size used for YAC cloning. We also used several other restriction enzymes as markers. We can overlap the segments, and analyse the segments using electrophoresis and Southern blotting.

Another example of joint study is our study with Dr Ohki in Saitama Cancer Center, who has almost completed an overall physical map of chromosome 21 using Not1. This is the first Not1 restriction map for a single chromosome. Now that we have the map, we can look at other genes. One gene related to leukemia is mapped in a particular portion of chromosome 21 where a translocation occurs. The next step of mapping is cloning the corresponding region by appropriate vectors such as YACs. We use cosmid and P1 clones to make specific clones of regions of chromosome 21. We hope to have prepared all the clones which cover the chromosome in about a year.

Figure 3: Linkage analysis

Figure 4: Human chromosome 21

Figure 5: Sub-chromosomal localization of Not I linking clones

Not I linking clones isolated in this study were regionally mapped on #21 chromosome. Three clones (H1-13, H2-92 and H2-122) were mapped on contiguous Not I fragments of #21 chromosome.

Another group, Dr. Matsumoto of Nagoya University is also looking at chromosome 21, is working on the centromere area. They are analysing the unusual alphoid DNAs on the centromere region. They found some interaction with specific proteins. This area is related to the segregation of chromosomes, and it might have clinical importance. For example Down's syndrome is due to the abnormal structure of chromosome 21. We hope that when the normal segregation mechanism is clarified, we can understand why trisomies occur.

There are thus various activities on chromosome 21 in Japan. There is also research on many other chromosomes, including chromosomes 3, 5, 6, 8, 11, 21, 22, X, and Y. A major group is Dr. Yusuke Nakamura of the Cancer Institute, who are working on chromosomes 3, 5, 6, 8, and 11. They have obtained many markers on these chromosomes. Their purpose is find anti-oncogenes, tumor suppressor genes. They have looked into familial adenomatous polyposis, as will be discussed later (Prof. Nishisho). They are also trying to identify the genes related to breast and kidney cancer on chromosome 8. Chromosome 8 is interesting for Japanese, because Werner's syndrome, where advanced aging occurs, is observed in Japanese and is mapped to this chromosome. In addition there are many other genetic diseases linked to chromosome 8. Dr Tsuji's group of Niigata mapped many cosmid clones on the X chromosome. Prof. Matsuda is studying the immunological regions. By these approaches we will get overall pictures of the chromosome.

Another approach is to convert messenger RNA to complementary DNAs (cDNAs), and analyse them. In this cDNA project we get the information on the expressed genes, and Prof. Matsubara's group is doing much work on this in Japan. They have taken mRNA from a liver cell. So far more than 2,000 clones have been analysed. The frequency of expression has been analysed. Many previously unknown genes were identified. We expect the identification of more functions of the liver.

In Japan we have these two approaches, and additionally we need to develop new technology. Some groups are working on developing new technology. Furthermore, the development of bioinformatics is essential for the future of the genome project, and a five year research project organised by Prof. Kaneshisa is now in progress. There is also a group for analysis of genomes of model organisms. The mapping efforts on smaller model genomes are important in understanding approaches we can use on the human genome.

Different government agencies are supporting the Human Genome Project. Prof. Matsubara is the leader of the project supported by the Ministry of Education. The Science and Technology Agency, and Ministry of Health and Welfare have also initiated genome projects, and all projects are well coordinated. As a whole mapping is the centre of these projects. There are similar projects in various countries therefore we need international cooperation.

Another thing I would like to comment on is related to the development of technology. These projects are limited if we only have the current technology. People can work energetically for the first year of this research, but because it takes a long time to complete, people get tied of the research. We cannot complete this project by labour of human beings, automation is required to reach the final goal. Therefore the Ministry of Education has encouraged the development of new technology.


p. 24 in Human Genome Research and Society
Proceedings of the Second International Bioethics Seminar in Fukui, 20-21 March, 1992.

Watanabe: Thank you for your presentation, are there any questions?

Matsunaga: Thank you for your presentation, as far as I know, Dr. Soeda's group is working on automated analysis. They started on chromosome 21. How is this work developing?

Sakaki: I'm sorry I only concentrated on the Ministry of Education's project. As Prof. Matsunaga mentioned, Prof. Soeda's group is working on YAC cloning of chromosome 21. We are exchanging information and collaborating. They are establishing a YAC library, which we have used. The automatic sequencing machine has been made, but I am not sure whether it is operating yet.

Matsubara: I'd like to add a few comments. The ethical issue is the main topic of this seminar. At the same time since the end of last year there is a movement to insist upon the property rights of some results of the genome project, mostly from the NIH. This property right is a totally new concept, which is challenged by society. I want to ask Prof. Sakaki for your comments on this. When we analyse cDNA we will encounter new issues.

Sakaki: We are very much interested in patents. In the case of Alzheimer's disease and Down's syndrome, those genes will be related to brain. The NIH group in the USA are applying for patents on cDNAs from the brain. We have no intention of getting a profit out of these activities, but we are worried about the barriers to study.

Watanabe: Prof. Matsubara asked about cDNA patents. cDNA is not in existence in nature as it is. In some session of this seminar we should clarify what cDNA is. Perhaps we can spend ten minutes on an explanation of cDNA. Perhaps Prof. Matsubara can discuss this. Prof. Honjo was to present the next paper, but unfortunately he cannot be here with us, so Prof. Matsuda from the same laboratory will present his paper.

pp. 25-27 in Human Genome Research and Society
Proceedings of the Second International Bioethics Seminar in Fukui, 20-21 March, 1992.

Human genome research in immunology

Tasuku Honjo(1) and Fumihiko Matsuda(2),

1. Professor, Department of Medical Chemistry, Faculty of Medicine, Kyoto University, Japan

2. Professor, Center for Molecular Biology and Genetics, Kyoto University, JAPAN

Thank you for your introduction, and this opportunity. This paper will consider molecular genetics, similar to what has been discussed in the previous two papers. Research on cancer, infectious diseases, viral causes, AIDS treatment, are all areas that molecular biology has been contributing to. The analysis of hereditary diseases was discussed in Prof. Sakaki's paper. In this paper the focus will be on autoimmunological diseases such as rheumatoid arthritis, Grave's disease. The diagnosis and treatment of such diseases is important.

In vertebrates there is a very sophisticated immune system, involving B cell and T cells. We are studying the antibodies of the B cell. Our research is to clarify the diversity of antibodies, that are used to attack foreign intruders. The antibody molecule is composed of a heavy (H) chain and a light (L) chain, which are two proteins. There are two main regions of each amino acid chain, called the constant (C) and variable (V) regions. The V region functions by binding to the antigen, and it must respond to various substances. There are many different antigens and it must respond to each. The V region is very sophisticated at the gene level. Along the chromosome there is a particular arrangement of genes for both L and H chains, namely VL and JL for L chain and VH, D, JH for the H chain. The genes rearrange during the differentiation of B cells. There are many combinations of the different gene segments of immunoglobulin V regions, and there are various number of V, D, and J segments making up the diversity of the immunoglobulin. These units are randomly combined by VDJ recombination, and after this somatic mutation can frequently occur to enhance the diversity of antibody molecules and response to different antigens.

However, the number of V region segments in the human genome is not clear yet. We isolated the entire region of the VH locus so we can understand the organisation of this locus. We have been working on two aspects of molecular immunology using human genome strategies. They are the characterisation of the human immunoglobulin heavy chain (IgH) locus and isolation of the nude locus.

There are many genes in the VH locus, which encompasses about 2 Mb in the genome, and the technique to isolate them is needed. We used YACs to take large regions. First we see the total structure, then we saw the relativity between the V and the J segments. From this method we were able to see the transcription polarities of each segment. Finally, we wanted to relate autoimmune disorders to VH segments at the gene level, which might allow us to develop methods of treatment for these diseases. Biologists also have an interest in the mechanism that generates antibody diversity, so the analysis of VH organisation is useful for tracing the evolution of multigene families.

IgH genes are mapped to the distal region of the long arm of the chromosome 14 (14q32), which is estimated to encompass 2-Mb of DNA by PFGE. There are 11 constant region genes in this area. Variable region (VH) segments are classified into six different families based on nucleotide sequence homology. The total number of VH segments was estimated to be 70-100 by counting the number of restriction fragments hybridising to the six VH family probes using several restriction endonucleases. There were many VH-I and VH-III but there were few VH-II , VH-V and VH-VI family VH segments.

We set out to construct a physical map of the entire human IgH locus by isolation of cosmids and YACs, after estimating that the size would be at least 2Mb of DNA. We isolated YAC and cosmid clones which encompass in total about 2.1 Mb of DNA of this locus. Among the isolated VH segment contigs, we focused on a 10 YAC contig which covers 1 Mb downstream of the VH-D-JH region. We are jointly studying this with a group in RIKEN and a group in ICI Pharmaceuticals in the U.K.

Southern hybridization autoradiograms using VH-I and VH-III family probes were compared between an equal molar mixture of four YAC DNAs (Y24, Y6, 17H and Y20) and a control human high molecular weight DNA. Densitometry scanning analysis of hybridised fragments showed that the YAC clones contained about 80% (VH-I) and 90% (VH-III) of the total signals of control DNA. The results indicate that the total number of VH segments is probably about 100 because the four YACs contain about 70 VH segments. Therefore, we speculate that most of the VH segments are presented in the isolated 2.1 Mb of DNA which contain 101 VH segments.

We propose nomenclature of the VH segments based on the order of each segment from downstream.

From DNA of a different cell line we isolated a cosmid contig (haplotype B) containing the same DNA region as that covered by YACs, Y103 and Y20 (haplotype A). Comparison of physical maps between the two haplotypes showed that this region has extraordinary polymorphism with an additional VH segment in haplotype B. This finding was further confirmed by family study as well as RFLP analysis of different individual DNAs.

Southern hybridisation and physical mapping of the contigs show that we isolated in total 98 VH segments in the 2.1 Mb region. The number was quite different from the estimate of 69, because one band in Southern blotting may not correspond to a single VH-containing fragment. YAC clones covering the downstream 0.8 Mb region were subcloned into cosmids and mapped by restriction enzymes. At least 64 VH segments belonging to six different families were interspersed within this region. 30 out of 64 VH segments whose nucleotide sequences were determined were pseudogenes (genes which cannot be expressed).

This was interesting, because the responsiveness to antigens will be decreased if so many are nonfunctional. This is another interesting topic, from the evolutionary point of view. We compared the sequences of these genes with already published VH segments, and in the database we found 16 segments related to autoimmune diseases. These include homologies to genes expressed as autoantibodies in patients with leprosy and rheumatoid arthritis. We looked at differences in gene structure between patients with rheumatoid arthritis compared to normal. Such a gene could then be used for diagnosis of rheumatoid patients, and may be useful for developing future therapy.

We could also see a deletion of a VH region linked to generation of autoimmune anti-DNA antibodies, and we are conducting a survey of different patients and different races. We are still to get positive results, but we are attempting to gather much data to look for linkage patterns.

We would like to analysis the production of auto-antibodies associated with various diseases. For example, Grave's disease and THSF receptor, myasthenia gravis, acetylcholine receptor, auto-immune hemolytic anemia of red blood cells. We would like to see the linkage between these two factors, and to identify the auto-immune diseases connected with these anti-DNA antibodies.

In recent years it is often noted that there is gene targeting technology, so we would like to target immunoglobulin genes in transgenic mice. Then we can look at expression of different human VH genes, and make human antibodies, after destruction of the mouse immunoglobulin genes. We can produce specific human antibodies in mice that can then be used as therapeutic antibodies in humans. This is our goal.

In conclusion, we have described the human VH segments and found that most are clustered in the 0.8Mb downstream region, with the others spread over the upstream region. We also found that this region has extensive polymorphism.

Another project we have been involved with involves nude mice. The chromosomal location of the nu gene, which is responsible for hairlessness and athymus, was determined using six DNA markers (I1-3, Myhs, Acrb, Evi-2m, Mpo and Hox-2) on mouse chromosome 11. We constructed the high resolution physcial map of the six DNA markers on chromosome 11 by in situ hybridisation using fluorescence labelled cosmid probes. The results indicate the order of the centromere-(41cM)-I1-3-(3cM)-Myhs-(4cM)-Acrb-(6cM)-Evi-2-(3cM)-Mpo-(5cM)-Hox-2. We have utilised congenic nude strains and examined which of the six DNA markers were derived from the original nude mouse. We found that Evi-2 locus is linked to the nu gene in all the informative, congenic nude strains. From these data we could estimate the location of the nu gene, not only genetically but also physically within a region which spans approximately 17 Mb (9cM) between Acrb and Mpo genes.


pp. 28-32 in Human Genome Research and Society
Proceedings of the Second International Bioethics Seminar in Fukui, 20-21 March, 1992.

Watanabe: Thank for your presentations. Does anyone want to raise any questions?

Tachibana: I would like to have more explanation of anti-DNA antibodies.

Matsuda: Well, it is the antibody against DNA I will explain a little further. These cases probably occur following the release of DNA from cells, that have died. The DNA is dispersed in the blood, and antibodies are created.

Billings: Thank you for your talk, I have two questions. One is scientific and the other is more ethical, since that is why we are here. The scientific question is do you have any evidence that the pseudogenes that are found in the human VH region can act in gene conversion, as is common, for instance, in the chicken.

Matsuda: As for the possibilities of gene conversion, we are now doing computer aided research to find out whether a small part of the pseudogene can be used as the donor of gene conversion as for chicken or sheep. But, we haven't yet got evidence for this.

Billings: My second question is a little more ethical. In your presentation you mentioned leprosy, which is common as an infectious disease, as being associated with certain VH segments. This has to do in general with genetic research, what is the balance between genetic susceptibility and environmental susceptibility? Also on how our concentration on genomic research, skews our thinking about this. I wonder whether you could give an estimate of the importance of the genetic susceptibility to leprosy as related to the prevalence of the bacteria in the environment.

Matsuda: I think that autoimmune diseases can be classified into two groups. One group are the diseases of which the cause is the production of auto-antibodies, and the others such as rheumatoid arthritis or SLE or leprosy, in which the effect from the environment or infection are very important. My impression is, that about SLE or leprosy, the production of auto-antibody is not the cause of these diseases. In that case, it would be very difficult to find out the genetic linkage between disease and the existence of certain VH genes in patients.

Cohen: In one of your slides, you showed the presence of an antibody against the acetylcholine receptor, and it happened that it was a leprosy case. Do you think there was any correlation.

Matsuda: In the case of leprosy, their report, this is the antibody which has cross reactivity to acetylcholine receptor, so we don't have any data about the affinity, or whether it is something to do with leprosy. It is only a matter of binding, it may not be significant.

Takebe: Talking about the pseudo-genes, you mentioned there were 34kb in the 3.4 Mb, but did you find any in the other regions.

Matsuda: Yes, I think we can find them, but we haven't done the sequencing yet. I would like to add one more thing. The VH region of the human is on chromosome 14, but on chromosome 16, there is a region of the area of 1 Mb where there is a translocation from chromosome 14, and there is no existence of VH, so it cannot participate in the function of rearrangement. However, in that region there are three variant regions, there is no defect and it is a currently active gene. A very simple explanation to this is that there is a translocation on the gene and the VH gene still has its function. Another possibility may be there is chromosome rearrangement. In the T cell receptor there is a method for obtaining diversity which may also occur here.

Roberts: May I just pursue your answer to Dr Billings question about the effects of environmental factors and genes for antibodies. You made no mention of the other genes in the genome, I'm thinking particularly of the HLA types which were very vigorously investigated several years ago. You've mentioned in your paper rheumatoid arthritis and Graves, both of which have close associations. Can you please enlighten me as to how these other genome constituents fit into this story of the development of the immune response.

Matsuda: Many researchers reported the susceptibility to disease from HLA, and to T cell receptor genes and immunoglobulin genes. So I don't think it cannot be explained fully by immunoglobulin VH gene polymorphism, and this linkage to disease. We need other research to completing understand the link between disease and genetic susceptibility.

Watanabe: I would like to thank Prof. Sakaki and Prof. Matsuda and other people for discussion.

Tachibana: I want to ask a question on the ethical aspects. I don't know whether that is an issue for this session or not, but I would like you to make a comment because you are doing this research.

Watanabe: This is not an issue which should be discussed in this session.

Matsuda: I limited my talk to immunological study, the immunological diseases related to gene abnormality. We do not know whether it is due to the gene itself, but there are many types of abnormality. And there are many reasons, diseases caused by environment and genetics. At the present time we do not know the actual cause of these diseases, and the relative importance of genetic abnormality to some diseases.

Sakaki: In my case, I study Alzheimer's disease and other neurological diseases. As to gene abnormality, it is familial with the same symptoms occuring. There is also an environmental factor, which is stronger for Alzheimer's disease. But personally speaking, when we look at the familial type there is a very high sensitivity to the environmental factors, and the threshold in those patients is very low.

Nishisho: We have to determine the environmental susceptibility when we are discussing genetics research and this study. In familial adenomatosis, the disease that I study, there is an onset of the disease at age 30 years. Throughout the world we have found many people with the same mutation, however there are people who have very late onset such as 70 years old and others with very early onset, such as 20 years old. And they can be living in the same family, so they may have the same environmental factors. It means that there may be several genetic factors, for examples, immunoglobulin polymorphism or other factors. Analysis of the disease can not be tied to one gene abnormality, for a certain disease. We may clarify the major cause, but we still need to determine the other factors which cause the diseases. Therefore, I think that may be a more important factor than the environmental factors.

Watanabe: Well, as chairman I would like to say that we have heard that the study of DNA will be promoted, and the DNA may have some relationship with the diseases. We will be able to clarify these relationships between disease and gene abnormality as a result of this study. We do not know how much is going to be clarified. At the present time there are many more diseases to be linked to changes in the DNA. I think it is quite natural that the linkage of disease will be clarified much more in the future.

As to that point, I want to express my view. In the study of the human genome, and prior to this study there were many studies on genes and many other scientific fields. We studied the causes of diseases for example genes and environmental factors, two major reasons for the onset of the diseases. As in the case of the infectious diseases, the cause of the disease should be considered to be related to the abnormality of the gene. I think it is difficult to discount the diseases that have no relationship to genes. There is a very close relationship between genes and diseases. When the study of the human genome is further advanced, and specifically when we can relate what gene is related to specific diseases, we will clarify these relationships, and this will give a very big impact upon society.

Of course we are going to have this kind of discussion later on, but it should be stressed here that the study of the human genome is not limited to the medical field, but has very close relationship to the study of human beings themselves. When we clarify the relationship with the diseases, what will be the situation to introduce such a fact into the diagnosis or treatment? We have to determine how to deal with the new findings. In this session we wanted to have a discussion of the present situation of human genome research. These other issues will be discussed in following sections. One aspect of the study of the human genome will be the utilisation of this study to the diagnosis or for the treatment of disease. There are various methods, one method will be treatment of diseases by DNA. I think that this is a quite natural course, as Mr Tachibana mentioned, to face in the future.

Thank you very much Prof. Matsuda, and now Prof. Matsubara will talk about cDNA. There is also the issue of patents in the case of cDNA, and the question of whether the sequence of the DNA may be patented or not. This is a current issue. At the present time there are not patents on cDNA. I would like Prof. Matsubara to explain about the current knowledge of cDNA, not especially about the patents of the sequence of DNA. But, I think that this may be an important issue, not limited to the human genome, because many people may try to have everything as a patent. I think this is a very important issue in the academic field. This is not a problem for private enterprise. It is a little different from the ethical issue, but it is a very important issue. I think that the patenting of DNA sequences, not only of humans, should be discussed later. At the moment please explain about cDNA.

Matsubara: cDNA is a technical term, so I would like to present this discussion with some quick sketches. In the present discussion it was said that all living organisms share the same basic genetic information, in the case of bacteria the genome is about 5 million base pairs. The base pair is a technical term used to describe a letter of the DNA sequence, so about 5 million letters are used in the genome of the bacteria, which has about 5,000 genes. By the analysis of genomes, we can find one gene is about 1,500 - 2,000 letters. About 70-80% of the DNA are genes, what is not genes in DNA is controlling the replication of the DNA itself, and so on. Therefore there is a very high density of genes. When you go into the higher organisms, like plants, insects, fish, mammals and so on, the size of the genomes are much larger than in bacteria. About 100 million to a thousand times this, 100 billion.

In the case of humans 3 billion letters are included, which is one unit. In humans, inside the genome, about 50,000-100,000 genes are incorporated in the body. In the large gene the information sequence is broken into many regions, and by combining all these regions together you can get the information of one gene. Through the evolution, from original an organism, bacteria may have eliminated the extra regions and thus minimised the DNA that was not used in genes. In such a way the bacterial genome was constructed. However in higher organisms, genomes have some capacity for containing wasteful DNA, therefore a more complicated evolutionary process could occur.

So even in one human gene, there are much larger segments. So when you decode DNA to determine where genes are, we need to determine which parts are used to form the gene and which parts are excluded. So in the higher organisms, after transcription of DNA into RNA, a complicated splicing process is formed. After this restructuring, or splicing reactions, a mature messenger RNA is made from the combination of only the important parts and this is used for protein synthesis.

For analysis using current technology, RNA is not easy to handle compared to DNA. So we isolate the mRNA and use an enzyme reverse transcriptase to transform the mRNA to cDNA, the complementary DNA, to represent the gene. In the cDNA project we try to decode that information. In one cell there are many genes working. For example, in the case of brain, about 50,000 genes, and therefore, 50,000 different RNA are working. In liver, about 13,000 genes, and in bone marrow about 15,000 genes. Some of them are overlapping, and are called housekeeping genes. Other genes change expression during differentiation and correspond to differentiated phenotypes.

Currently we can make a cDNA library. In the case of brain, about 50,000 mRNA can be blended together in one tube, then the cDNA made. Then these cDNA can be inserted into E. coli, and E. coli can be cultured and cloned, so that it is possible to separately analyse each cDNA. It is possible to analyse all the 50,000 cDNAs. We are going to do the same process in liver and bone marrow. Dr. Craig Venter of the NIH has done some work on this, about 2,500 different mRNA were analysed. In our case in bone marrow and liver, about 2,300 mRNA have already been analysed. From this you can get the sequence of letters, and this is compared with DNA databanks to see whether it has already been registered or not, whether it is a known gene or not. Empirically, through experience about 20% of those randomly extracted and analysed correspond to known sequences, and about 80% are new gene sequences.

We have an automatic sequencing apparatus which is very efficient, but the maximum information from one sample is a sequence of about 300 letters. In total the gene may have 2000 letters, but from only 300 letters we can determine whether the gene is known or new. We are compiling the results in a catalogue, in the end all the DNA of genes working in specific organs may be determined. With the currently available technology we can identify 40,000 different types in a year. In humans about 50,000 - 100,000 different genes exist, so if you just work for a short time you can complete this analytic process. Sometimes you may encounter something very interesting, and can get a lot of interesting results. Dr Venter analysed random extracted sequences, and partitioned about 2,300 new sequences for patent approval, under instruction of the Technology Transfer Department in the NIH.

We know that they are new genes but we do not know what genes they are. In spite of this uncertainty, can you make a patent? In the science community this is an unprecedented procedure to apply for patents. It is rather against the procedure of regular science. Many people are not comfortable about this. Furthermore this analysis can be done within a short time, and if a sort of national project is initiated, all the human genes can be registered, and you can claim for patent rights within a year or two. If this is actualised, the national power will have some impact. You can create a large network for collecting everything available. This might impede the flow of information and ideas among researchers. This project might be worked not only in humans, but in birds, rice and other plants. In the case of microorganisms the structure analysed represents the gene. People are worried that all living organisms existing in the world could be exposed to patent issues. By using this unprecedented approach you can get some economic benefit and also potentially bias the framework of bio-science. Mr Alder of the NIH Technology Transfer Department had a presentation in a San Diego meeting last year, and he said the decision-maker is not scientists or ethicists, but it is an assignment for legal lawyers, and he said they could look after that assignment. Everyone is paying attention to the upcoming result. It is a very US-orientated social experimentation and development.

Watanabe: Thank you very much. In bacteria the existing gene structure is the same as cDNA, but in the case of higher developed organisms cDNA is not in the genome, in the genome there are some split fragments of the cDNA. cDNA in higher organisms seems to be a kind of artificial chemical. Therefore this is a point of the application of patents. But the point of initiation is cDNA. From cDNA the process is starting which might potentially expand greatly in the future. When it reaches that stage, what should we do? Not only do we have the issue of DNA sequencing patents, but other issues such as medical and technical issues can be developed from cDNA sequences. We are going to have more time for discussion later on.

Mr Tachibana presented one important issue. The intellectual property issue are not limited to organisations like companies or enterprises, but they can be talked about in much wider scope. Patents might work favourably for those organisations which are doing sequencing of cDNA. Anyway, cDNA raises potentially large issues. It will more directly have impact to the diagnosis and treatment of diseases. Therefore we should fully recognise that there are many potentially related important issues. We have to organise one seminar only focusing on cDNA, and develop this issue. Since time was available this morning we could expand our discussion to include cDNA. Prof. Matsubara has touched on a very important issues. Thank you.

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