Analysis Of Human Genome Research Articles

A Novel NanoPipetting Solution for the Development of High Quality BioChip Arrays for Diagnostic Applications

T he dramatic progress in the analysis of human, animal and plant genomes as well as parallel developments such as the human cancer gene anatomy project have created an enormous demand for low-cost high throughput technologies for DNA and RNA analysis. Chip-based molecular techniques if available in satisfactory quality for diagnostic applications will enable major analytical issues in health care such as predisposition, cancer, infectious diseases and others to be addressed. DNA micro-arrays have been predicted to become a key technology in molecular analysis for nearly one decade now. Although their proportion in the research market (e.g. expression profiling) has increased significantly they still play a minor role in clinical and medical diagnostic fields. Main drawbacks for the implementation of BioChips in clinical routine analysis are insufficient reproducibility, lack of standardization as well as expensive chips and detectors. With respect to BioChip production, industrial standards and uniform protocols have not yet been defined. Therefore different BioChip layouts and analytical performance cannot be compared. This article describes the development and production of DNA/RNA BioChip arrays for diagnostic applications emphasizing in particular quality controlled production processes with high reproducibility of each step. In order to achieve the required analytical performance, many of the currently used components and techniques such as substrate material, coupling chemistry, arraying, probe design, hybridization and high resolution scanning were reevaluated by IMNT and replaced by appropriate new approaches. In addition, TECAN has developed a novel nanopipetting solution based on GENESIS pipetting robot to dispense volumes as small as 0.5 nanoliters (nl).

Identification of a 5-cM region of common allelic loss on 8p12-p21 in human breast cancer and genomic analysis of the hEXT1L/EXTR1/EXTL3 gene in this locus.

The short arm of chromosome 8 is frequently lost in many human carcinomas including breast cancer, suggesting the presence of a tumor suppressor gene(s) in this region. We identified a gene termed hEXT1L/EXTR1/EXTL3 (hEXT1L hereinafter) that was mapped to chromosome bands 8p12-p21 where frequent LOHs of this region was reported in breast cancer. The existence of the third breast cancer susceptibility gene was also suggested in this region by linkage analysis. We further performed LOH analysis in 8p12-p21 in 34 breast cancers and identified a 5-cM region of common allelic loss that overlapped with the locus for positive lod score in familial breast cancer. We further analyzed genomic alterations of hEXT1L in tumors in which frequent LOHs of 8p were reported. A total of 327 cancers (313 primary tumors and 14 cancer cell lines) including 22 primary breast cancers were analyzed, but none of the tumors had somatic mutations: only one thyroid cancer patient without any family history of cancer had a 9-bp insertion in the constitutional DNA. These results suggest that mutations of hEXT1L do not play a major role in the development of sporadic cancers including breast cancer, and that other tumor suppressor gene(s) exists in the 5-cM region identified in this study.

Direct isolation of specific chromosomal regions and entire genes by TAR cloning.

Human genome analysis has been advanced considerably by the development of Yeast Artificial Chromosome (YAC) cloning systems (1). YAC techniques have made possible the isolation of megabase DNA fragments, thereby greatly simplifying the physical mapping of chromosomes and the cloning of entire genes. Overlapping YACs can be conveniently modified by homologous recombination in vivo to construct a single YAC that contains a large genomic locus (2), or to create a new YAC with alterations in specific regions (3). This approach can be used to generate YACs which, upon transfer into mammalian cells, will contribute to the functional analysis of the isolated region.

薬理学の新世紀を拓く ヒトゲノム解析計画と２１世紀の医療

The human genome project is considered to be the most important project in biology and medicine. The discovery of an entire human genes through this project should revolutionize biological medicine including molecular diagnosis of various diseases and development of novel treatment. The entire genome DNA sequence is expected to be completed by 2003, and 90% of the genes will be identified by 2001. The information will accelerate discovery of genes susceptible to or causing various diseases and contribute to screening of novel drugs that target these disease-gene products. In addition, the field of "phamacogenetics" will become more important. Phamacogenetic studies focusing on inherited variations in drug metabolism and polymorphisms in drug metabolisms of the genes encoding drug-metabolizing enzymes are also very important to determine an appropriate dose of certain drugs to obtain the maximum effect and avoid serious toxicity. In this regard, the recent world-wide effort of the SNP (single nucleotide polymorphism) project in which scientists attempt to discover 100,000 genetic variations in our genome will generate very variable resources. In this review, I will be describing the recent progress and future direction of human genome analysis and its impact on medicine and pharmacology.

PL–1 - Impact of human genome analysis on the future medicine

The human genome project is considered to be the most important project in biology and medicine. The discovery of an entire human genes through this project should revolutionize biological medicine including molecular diagnosis of various diseases and development of novel treatment. The entire genome DNA sequence is expected to be completed by 2003, and 90% of the genes will be identified by 2001. The information will accelerate discovery of genes susceptible to or causing various diseases and contribute to screening of novel drugs that target these disease-gene products. In addition, the field of phamacogenetics will become more important. Phamacogenetic studies focusing on inherited variations in drug metabolism and polymorphisms in drug metabolisms of the genes encoding drug-metabolizing enzymes are also very important to determine an appropriate dose of certain drugs to obtain the maximum effect and avoid serious toxicity. In this regard, the recent world-wide effort of the SNP (single nucleotide polymorphism) project in which scientists attempt to discover 100,000 genetic variations in our genome will generate very variable resources. In this review, I will be describing the recent progress and future direction of human genome analysis and its impact on medicine and pharmacology.

The Complete Chromosomal Organization of the Reference Strain of the Leishmania Genome Project, L. major `Friedlin'

In recent years, analysis of the genomes of many organisms has received increasing international attention. The bulk of the effort to date has centred on the Human Genome Project and analysis of model organisms such as yeast, Drosophila and Caenorhabditis elegans. More recently, the revolution in genome sequencing and gene identification has begun to impact on infectious disease organisms. Initially, much of the effort was concentrated on prokaryotes, but small eukaryotic genomes, including the protozoan parasites Plasmodium, Toxoplasma and trypanosomatids (Leishmania, Trypanosoma brucei and T. cruzi), as well as some multicellular organisms, such as Brugia and Schistosoma, are benefiting from the technological advances of the genome era. These advances promise a radical new approach to the development of novel diagnostic tools, chemotherapeutic targets and vaccines for infectious disease organisms, as well as to the more detailed analysis of cell biology and function.

Large scale ENU screens in the mouse: genetics meets genomics

The worldwide effort to completely sequence the human and mouse genome will be accomplished within the next years. The focus of current activities within the framework of human genome research is mainly on the assembly of high resolution genetic and physical maps and genomic sequencing. Cloning of new genes is getting more easy using those maps. Nevertheless, it is necessary to work on a systematic analysis of gene function. Results obtained from these efforts will be of enormous value for future biological and biomedical research. However, even the complete sequence will not in all cases reveal the molecular and cellular role of the different genes. Therefore, the next phase of the Human Genome Project will have at its core the functional analysis of genes. Those genes relevant for the diagnosis, prevention and therapy of human diseases are of particular interest. Looking at the history of life sciences, mutants have been the most important tool to obtain insight into the biological function of genes. The mouse is the model of choice for the study of inherited diseases in man. In order to meet the requirements for functional human genome analysis, we need a large number of mouse mutants similar to the collection of mutants available for other model organisms such as flys and worms. To fully apply the power of genetics, multiple alleles of the same gene such as hypomorphs or hypermorphs are required. Efficient production of mouse mutants showing specific phenotypes can be achieved by the use of ethylnitrosourea (ENU). ENU is the most powerful mutagen known and we currently see a renaissance of ENU mutagenesis. The application of ENU mutagenesis is reviewed and discussed in the context of a new era of functional genomics.

Functional expression and germline transmission of a human chromosome fragment in chimaeric mice.

Human chromosomes or chromosome fragments derived from normal fibroblasts were introduced into mouse embryonic stem (ES) cells via microcell-mediated chromosome transfer (MMCT) and viable chimaeric mice were produced from them. Transferred chromosomes were stably retained, and human genes, including immunoglobulin (Ig) kappa, heavy, lambda genes, were expressed in proper tissue-specific manner in adult chimaeric tissues. In the case of a human chromosome (hChr.) 2-derived fragment, it was found to be transmitted to the offspring through the germline. Our study demonstrates that MMCT allows for introduction of very large amounts of foreign genetic material into mice. This novel procedure will facilitate the functional analyses of human genomes in vivo.

DNA copy number changes associated with characteristic LOH in islet cell carcinomas of transgenic mice

Comparative genomic hybridization (CGH) provides a method of surveying the entire tumor genome for regional variations in DNA sequence copy number. Such variations, if found recurrently, may indicate the locations of genes that contribute to tumor development through upregulation of oncogenes (copy number increase), inactivation of tumor-suppressor genes (copy number decrease), or changes in the level of expression through gene dosage effects. Thus, CGH is a powerful tool for screening for new cancer genes. Although CGH is widely applied to human genome analysis, application to the mouse is only beginning. The present study is designed to compare results obtained by CGH with those obtained by other techniques used for analysis of the murine genome. We report CGH analysis of several control cell lines with cytogenetically established regional copy number changes, as well as analysis of 16 primary insulinomas, four tumor-derived cell lines, and three hyperplasia-derived cell lines from transgenic mice expressing the SV40 large T antigen under control of the rat insulin promoter. Loss of heterozygosity (LOH) on chromosomes 9 and 16 had previously been found to be frequent in primary insulinomas, and specimens were selected for the present study based on the LOH status of these chromosomes. We found complete concordance of the CGH results with the cytogenetically described copy number changes in the control cell lines and with the LOH on chromosomes 9 and 16 in the tumors. Thus, CGH can provide accurate data in murine systems, and it reveals that the LOH in these islet cell tumors most frequently results from deletion of one of the alleles.

Accessing genetic information with high-density DNA arrays.

Rapid access to genetic information is central to the revolution taking place in molecular genetics. The simultaneous analysis of the entire human mitochondrial genome is described here. DNA arrays containing up to 135,000 probes complementary to the 16.6-kilobase human mitochondrial genome were generated by light-directed chemical synthesis. A two-color labeling scheme was developed that allows simultaneous comparison of a polymorphic target to a reference DNA or RNA. Complete hybridization patterns were revealed in a matter of minutes. Sequence polymorphisms were detected with single-base resolution and unprecedented efficiency. The methods described are generic and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability.

Single-well genotyping of diallelic sequence variations by a two-color ELISA-based oligonucleotide ligation assay.

Single nucleotide substitutions and unique insertions/deletions are the most common form of DNA sequence variation and disease-causing mutation in the human genome. Because of the biological and medical importance of these variations, a wide array of methods have been developed for their typing. We have applied an approach that combines the amplification of polymorphic regions by the polymerase chain reaction (PCR) with a system for typing diallelic variants using an oligonucleotide ligation assay (OLA). In this report, we describe a significant advance in this technology that permits the typing of two alleles in a single microtiter well. By marking each of the allele-specific primers with a unique hapten, i.e. digoxigenin and fluorescein, each OLA reaction can be detected by using hapten specific antibodies that are labeled with different enzyme reporters, alkaline phosphatase or horseradish peroxidase. This system permits the detection of the two alleles using a high throughput format that leads to the production of two different colors. We demonstrate the specificity, sensitivity and ease of data interpretation with this system. Furthermore, we show that multiplex PCR/OLA not only increases the throughput of DNA typing but also increases its accuracy in typing diallelic sequence variations using an approach that can be broadly applied for human genome analysis (in evaluating genotype/phenotype links), in typing infectious agents and in forensic analysis.

Book Reviews

Books reviwed in this article: Human Genome Analysis Programme, Biomedical and Health Research, Vol. 8. Manuel Hallen and Andreas Klepsch Clinical Epilepsy. J. S. Duncan, S. D. Shorvon, and D. R. Fish.

A short CIC-2 mRNA transcript is produced by exon skipping.

CIC-2 is a voltage- and volume-regulated chloride channel expressed in many tissues. We have shown that CIC-2 in rat lung airways is significantly down-regulated after birth [Murray,C.B. et al. (1995) Am. J. Respir. Cell Mol. Biol., 12, 597-604]. During PCR amplification from rat lung cDNA, a second transcript was identified which is 60 bp shorter than the full length sequence. The peptide translated from this 60 bp sequence contains many positively charged amino acid residues. Rat genomic DNA sequencing showed that the 60 bp sequence is an intact exon. A 71% pyrimidine content and an AAG 3'-end splice site in the intron immediately upstream from the 60 bp sequence were identified which may account for the alternative splicing of the following exon. Human genomic sequence analyses demonstrated similar intron-exon arrangement. A high CT content and an AAG 3' acceptor site were conserved in the intron corresponding to the rat upstream intron. The presence of the full length short form transcript was confirmed in rat kidney by RT-PCR, and the ratio of the long and the short form transcripts varied significantly according to the tissues examined, with the lowest long/short form ratio found in the lung among the tissues studied. Our data demonstrated that the alternatively spliced short form (CIC-2S) is transcribed in many rat tissues, the ratio of the long/short form transcripts is lower in the lung compared with the brain, and the genomic organization in this area is conserved in rat and human.

Characterization and Expression of the Human A2a Adenosine Receptor Gene

The actions of the neurotransmitter adenosine are mediated by a family of high-affinity, G protein-coupled receptors. We have characterized the gene for the human A2a subtype of adenosine receptor (hA2aR) and determined levels of A2aR mRNA in human brain regions and nonneural tissues. Human genomic Southern blot analysis demonstrates the presence of a single gene encoding the hA2aR located on chromosome 22. Two overlapping cosmids containing the hA2aR gene were isolated from a chromosome 22 library and characterized. Southern blot and sequence analyses demonstrate that the hA2aR gene spans approximately 9-10 kb with a single intron interrupting the coding sequence between the regions encoding transmembrane domains III and IV. The sequence of the hA2aR gene diverged from the reported cDNA structure in the 5' untranslated region. This divergence appears to result from an artifact in the construction of the original cDNA library. By northern blot analysis, high expression of the hA2aR gene was identified in the caudate nucleus with low levels of expression in other brain regions. High expression was also seen in immune tissues; lesser A2aR expression was detected in heart and lung. The gene for the A2a subtype of receptor for the neurotransmitter adenosine falls in the class of intron containing G protein-coupled receptor genes. Expression in the basal ganglia is consistent with a role for the hA2aR in motor control. Activation of the A2aR may also regulate immune responses and cardiopulmonary function.

Cotranscription of two RNA coding for the cell adhesion regulator and its variant in Reh leukemia cells

Human genomic DNA analysis reveals the existence of polymorphisms at the cell molecular adhesion regulator (CMAR) locus. In order to choose between the two possible open frames deduced from the variant sequence, we have sequenced both the human 5′ non-coding region and the mouse CMAR variant DNA. We found that both mRNA species coexist in human cells.

Evaluation of an automated DNA sequencing system developed in RIKEN

For the advancement of Human Genome Project, we have developed an automated DNA sequencing system ‘HUGA-I.’ It is composed of several automated instruments and transfer robots connecting them. In this paper we describe the results of the performance evaluation test of HUGA-I. Although some of the system units showed good performances, the total performance of the HUGA-I was about 1/6 of the designed value. By revealing principal reasons of this poor performance, we would like to contribute to the automation in genome analysis, particularly in human genome analysis.

Retrovirus-like particles released from the human breast cancer cell line T47-D display type B- and C-related endogenous retroviral sequences.

The human mammary carcinoma cell line T47-D releases retrovirus-like particles of type B morphology in a steroid-dependent manner (I. Keydar, T. Ohno, R. Nayak, R. Sweet, F. Simoni, F. Weiss, S. Karby, R. Mesa-Tejada, and S. Spiegelman, Proc. Natl. Acad. Sci. USA 81:4188-4192, 1984). Furthermore, reverse transcriptase (RT) activity is found to be associated with particle preparations. Using a set of degenerate primers derived from a conserved region of retroviral pol genes, we repeatedly amplified three different retroviral sequences (MLN, FRD, and FTD) from purified T47-D particles in several RT-PCR experiments. Screening of a human genomic library and Southern blot analysis revealed that these sequences are of endogenous origin. ERV-MLN represents a multicopy family of human endogenous retroviral elements (HERVs) with two closely related copies and up to 20 more distantly related members. In contrast, ERV-FRD and ERV-FTD comprise only one copy and five to seven related elements per haploid human genome. DNA sequence analysis of the proviral pol region of ERV-MLN revealed an uninterrupted stretch of 241 amino acids that shows 65% identity with the RT of the type B-related HERV designated HERV-K10. ERV-FRD and ERV-FTD are defective type C-related HERVs. The pol gene of ERV-FRD displays a nucleotide homology of 54% to the gibbon ape leukemia virus, and the pol gene of ERV-FTD is about 67% homologous to members of the RTVL-I family of HERVs. Our results thus indicate that the retroviral particles released by the breast cancer cell line T47-D are probably generated by complementation of several endogenous proviruses and can package retroviral transcripts of different origins.

High performance computing for the human genome project

Human Genome Analysis and Image Processing are part of the ‘Grand Challenges’ in High Performance Computing. The traditional mainframe has become insufficient for these applications in Biocomputing. New scalable parallel processor systems enter the marketplace with superior price/performance. The evaluation process of such a system by an application-oriented benchmark test suite is described. The system is integrated in the client/server structure of the Deutsches Krebsforschungszentrum where ‘rightsizing’ will eliminate the mainframe completely in the near future.

Expression of human platelet-activating factor receptor gene in EoL-1 cells following butyrate-induced differentiation.

Platelet-activating factor (PAF) is a potent lipid mediator of allergic inflammation through its interaction with eosinophils. Expression of the PAF receptor is modulated by many agents, including those responsible for cell differentiation. We report here that differentiation of a human eosinophilic leukaemia cell line, EoL-1, by sodium n-butyrate is associated with induction of PAF receptor gene expression, as indicated by: PAF receptor mRNA accumulation; increases in the binding of [3H]WEB 2086, a PAF antagonist; analysis of cell-surface expression of PAF receptor protein using a monoclonal anti-(PAF receptor) antibody; and augmentation of PAF-induced increase in the intracellular concentration of calcium. Using cDNA cloning, the receptor expressed in EoL-1 cells was identified as 'Transcript 1', one of two transcripts which was previously reported from human genomic analysis (Mutoh, Bito, Minami, Nakamura, Honda, Izumi, Nakata, Kurachi, Terano and Shimizu (1993) FEBS Lett. 322, 129-134). The PAF-induced calcium response and phosphoinositide turnover were decreased by pertussis toxin (PTX) treatment, suggesting that these signals are coupled largely with PTX-sensitive G-protein(s) in EoL-1 cells. These systems may provide a useful experimental model with which to investigate the relationship between eosinophilic differentiation and PAF receptor induction, and the role of eosinophils in allergic responses.

Comparative Analysis of Mouse NotI Linking Clones with Mouse and Human Genomic Sequences and Transcripts

NotI cleavage sites are frequently associated with CpG islands that identify the 5' regulatory sites of functional genes in the genome. Therefore we analyzed a sample of 22 NotI linking clones prepared from mouse brain DNA, to determine whether these mouse NotI site associated clones could be used for comparative analysis of mouse and human genomes by cross-reaction with both mouse and human genomic DNA and RNA in Southern and Northern hybridization. We further examined whether we could establish the identity of these clones with known genes by comparing the nucleotide sequences surrounding the NotI site with the GenBank database. We observed that 70% of the clones cross-hybridized with human DNA and that 4 of 11 tested clones (36%) detected a transcript in human HeLa cells RNA whereas 73% clones (8/11) detected transcripts in mouse RNAs from one or more organs. Single pass sequence analysis was successful on 16 of 19 clones. The GC content in these sequence was very high (48.8% to 73.8%) suggesting that 12 of 16 sequenced clones contained a CpG island. Three out of 19 clones showed significant similarity with previously analyzed mouse gene sequences in GenBank, including the mouse rRNA gene family, cathepsin and the scip POU-domain genes. In addition, two sequences showed significant similarity to the human and rabbit protein phosphatase 2A-beta subunit and the human transforming growth factor-beta. Thus, 5 of 16 clones showed homology with identified genes. These results and the recent work of using RLGS methods for genetic mapping indicate that NotI linking clones can be used to efficiently cross reference a comparative analysis of the mouse and human genomic maps.