Identification of Transcribed Sequences: Functional and Expression Analysis
Meeting Abstracts (November 1997)

A major goal in human genetics is to create good animal models for human inherited pathologies to: 1 - Understand the physiopathology of the disorder. 2 - Test new therapeutical approaches. Standard transgenic approaches have been mainly proposed, but they result in random integration of multiple copies of transgenes. The position effects related to the integration site often lead to low levels of gene expression, and aberrant patterns of expression. Moreover, the large copies number of tandemly repeated sequences is frequently unstable. We have thus developed, in collaboartion with C.Huxley (London), an alternative approach, using large genomic fragments (as YACs) as vectors. The large size of YACs (several hundreds of Kb) usually transfer all elements required for faithful regulation of gene expression, in quantitative as well as in qualitative terms. We have applied this technology to create an animal model of the Charcot-Marie-Tooth disease type 1A which is the most frequent inherited peripheral neuropathy in human. This disorder is caused by a 1.5 Mb duplication, which includes the gene coding the synthesis of the myelin protein PMP22. We have injected a human YAC of 560 Kb, containing PMP22 and flanking elements, in the murine oocytes. We have obtained 5 lines, which have integrated from 1 to 7 copies of the YACs. The human PMP22 gene expression has been determined, and we have demonstrated that gene expression is proportional to the copy number of the YAC integrated in the murine genome. Mice with 1/2 copies have no phenotypes. Mouse with 4 copies have a demyelinating neuropathy comparable to CMT1A. Mouse with 7 copies have a very severe peripheral neuropathy. This technique has thus proven to be extremely powerful to create a pathological phenotype. Moreover, we think that if the technique is able to create a phenotype, it can also be used to correct a phenotype. In that way, we will present data of YACs expression in human cells, and particularly the expression of CFTR gene in normal and CF cells.

Michel Fontés
INSERM U406- Fac de Médecine
27 Bd.J.Moulin
13358 MARSEILLE CEDEX5 FRANCE
telephone: 33 4 91 25 71 59
fax: 33 4 91 80 43 19
email: MICHEL.FONTES@MEDECINE.UNIV-MRS.FR

Presentation format: Platform

Chromosome 1p36 region consistently displays allelic deletions in a variety of cancer, e.g. melanoma, neuroblastoma, breast cancer, or hepatoma. We previously defined a chromosomal segment, 1p36.31 - p36.32, whose integrity is essential for tumor suppression in neuroblastoma. Applying a positional cloning strategy we have identified and cloned a novel human gene located in the neuroblastoma critical region, in the immediate vicinity of a balanced translocation breakpoint found in a primitive neuroectodermal tumor (PNET). Structural analysis of this novel gene by long range sequencing revealed 28 exons and a genomic coverage of the transcribed region of more than 200 kb. Northern blot analysis displayed a fairly wide expression pattern with predominant transcript length of 6.5 kb and a putative alternative splice product of 6.2 kb in most tissues. There is an additional 2.5 kb transcript present in skeletal muscle. The cDNA contains an open reading frame of 3522 nucleotides, encoding a putative 100 kD protein. The predicted protein sequence contained no obvious functional sequence motifs; however, database comparisons revealed a very high homology to the yeast UFD2 gene (Ubiquitin fusion degradation protein). UFD2 represents a factor involved in ubiquitin-associated protein degradation pathways. We designate the chromosome 1p36.3 located gene hsUFD2 owing to the homologies found. Initial screening attempts for mutations have revealed a gross rearrangement of this gene in at least one primary hepatoma tumor. Independent of the structural analyses we have recently started to analyze the function of hsUFD2 in order to gain insight into its possible role in malignant transformation. Functional assays employing transfection analyses in neuroblastoma tumor cell lines indicate that expression of this gene is incompatible with soft agar growth of tumor cells. Immunocytochemical and phenotypic analyses of transfected neuroblastoma clones are currently in progress.

Barbora Lubyova
Institute of Molecular Pathology (I.M.P.)
Dr. Bohr-gasse 7
Vienna, Austria A-1030
telephone: +431-79730-423
fax: +431-7987153
email: Lubyova@aimp.una.ac.at

Presentation format: Poster

The chicken genome comprises eight pairs of large autosomal ‘macrochromosomes’, Z and W sex chromosomes and thirty pairs of small ‘microchromosomes’. Work has been done which indicates that there is a higher concentration of CpG islands on the microchromosomes than is present on the macrochromosomes. Knowing that 60-70% of chicken genes are associated with CpG islands, this may imply that the microchromosomes (accounting for only 25% of the genome) are more gene dense than the macrochromosomes .

In order to test this theory, a ‘sequence sampling’ approach has been undertaken. To date, ten cosmids which have been physically assigned to chicken chromosomes by FISH, have been subcloned and a representative portion of each cosmid sequenced (70%). Five cosmids are known to map to macrochromosomes and five to microchromosomes. Each sequence is searched against blastn, blastp, dbest and dbsts databases as well as the Fugu rubribes database. CpG analysis is also carried out on each sequence. This is carried out automatically using a system based on the Pregap part of the Staden processing programme. for each cosmid, the presence of genes, gene homologies, CR1 repeats and CpG islands is noted. So far there is no indication that microchromosomes are more gene dense than their macrochromosomal counterparts. As a means of randomly finding genes within a particular region of DNA, sequence sampling is proving itself to be a powerful tool.

This work was supported by the Biotechnology and Biological research Council, UK and EC grant no. BIO4-CT95-0287, as part of the Chickmap project.

Dr. Jacqueline Smith
Roslin Institute
Roslin, Midlothian EH25 9PS
Scotland
telephone: 44 (0) 131 527 4200
fax: 44 (0) 131 440 0434
email: Jacqueline.Smith@bbsrc.ac.uk

Presentation format: Poster

We are involved in two large-scale S. cerevisiae genome projects. The first applies a random transposon insertion/tagging approach; the second will systematically create deletions of each yeast ORF.

The Transposon Insertion Project
(http://ycmi.med.yale.edu/YGAC/home.html): We intend to construct a reporter gene fusion and an epitope-tagged version of the protein for every gene in the yeast genome. This will allow us to analyze gene expression throughout the life cycle, the subcellular location of the encoded protein, and the phenotypic effect of disrupting the gene. A novel transposon system (PNAS 94: 190, 1997) has been used to create a bank of yeast strains, each with a transposon inserted at a random genomic location. This insertion allows us to analyze expression of yeast ORFs via in-frame fusions to lacZ. Using the Cre/lox system, we can modify the transposon in vivo to derive an in-frame element that leaves a 93 amino-acid epitope tag in the product of the mutagenized gene. We have found that in strains carrying insertions in essential genes, the smaller tag often does not cause lethality. Such strains should provide a rich source of conditional or hypermorphic alleles, a valuable genetic tool.

The full-length, tagged proteins are also excellent substrates for immunolocalization. Thus far, we have identified 6500 strains with vegetatively-expressed fusions, and examined the subcellular localization of epitope-tagged proteins in 2112 strains. Sixty strains have tagged proteins that localize to the nucleus; we are extending this work by immunolocalization on spread chromosomes. We have also identified tagged proteins localizing to more unusual sites, such as the bud neck, vacuolar rim and spindle apparatus.

Sequence analysis shows that in addition to finding fusions in recognized ORFs, some highly-expressed fusions occur in large ORFs that were not annotated by the systematic sequencing project, while numerous insertions correspond to fusions to smaller unn amed ORFs. Of the nuclear-localizing tagged proteins, about 60% correspond to insertions in uncharacterized ORFs; many of the remainder are in known chromatin-associated proteins or transcription factors.

The Genome Deletion Project
(http://sequence-www.stanford.edu/group/yeast_deletion_project/deletion.html): In a collaborative effort involving the USA, Canada and Europe, whole-gene deletions are being constructed for each annotated ORF in the yeast genom e. Over the next two years, our laboratory will construct 600 deletion strains. Each deletion construct bears a unique tag; hence parallel analysis of all c. 6000 strains generated will be possible, using hybridization to an Affymetrix chip carring the tag sequences. This will allow both specific target screens and studies on the enviromental interactions of all genes.

Petra Ross-Macdonald
Dept. of Biology, KBT 912
Yale University
PO Box 208103
New Haven, CT 06520-8103
telephone: (203) 432 9949
fax: (203) 432 6161
email: petra@pantheon.yale.edu
url: http://ycmi.med.yale.edu/YGAC/home.html

Presentation format: Platform

1 Dipartimento di Biologia D.B.A.F. Universita' della Basilicata, via Anzio 10, 85100 Potenza Italy 2 Liuni S - Centro di Studio sui Mitocondri e Metabolismo Energetico, C.N.R., via Orabona, 4, 70126 Bari, Italy 3 Grillo G. - Dipartimento di Biochimica e Biologia Molecolare, Universita' di Bari, via Orabona, 70126 Bari, Italy 4 Sidney Kimmel Cancer Center, 3099 Science Park Road, San Diego, CA 92121.

A computer program is described that selects a small set of short primer pairs for PCR to sample all the sequences in a list of mRNAs of interest. Such primer pairs have previously been shown to increase the probability of sampling the mRNAs of interest using RNA fingerprinting. The program selects pairs of primers that have the following properties: [1] each primer pair samples more than one sequence in the list, [2] a small set of primer pairs samples all, or nearly all, of the sequences in the list, [3] the primers have a fixed range of G+C content, [4] primer pairs are excluded that generate simulated PCR products of the same size from a number of sequences in the list, and [5] primers can be excluded that occur in other lists of sequences. In the examples presented, the primers are confined to 50-90% G+C content and primers are excluded if they occur in the hyper-abundant ribosomal RNAs, mitochondrial RNA, or dispersed transcribed repeats. Pairs of primers of eight or nine bases in length that fit such criteria are generated using four lists; 65 human cDNAs associated with DNA repair; 60 mRNAs associated with apoptosis; 44 members of the human nuclear receptor gene family; 113 members of the G-protein coupled receptor gene family. Applications to much longer lists of mRNAs will be discussed.

Michael McClelland
Sidney Kimmel Cancer Center
3099 Science Park Road
San Diego, CA 92121
telephone: 619 450 5990 ext 280
fax: 619 550 3998
email: mmcclelland@skcc.org
url: www.skcc.org
http://www.skcc.org/skcc_staff/mc_clelland/mc_clelland.html

Presentation format: Platform

Due to the enormous amount of new genomic sequences it is mandatory to preselect candidate sequences by computerized analysis prior to experimental functional analysis. This includes prediction of exons and introns as well as the identification of potential regulatory regions which usually encompass multiple regulatory elements that exert their regulatory function only within the correct context. Last year, we reported our approach to this problem and presented sucessful identification of a new LTR as an example. We have now extended our work aiming at the prediction of inherent tissue and/or cell specificity of such regions. Actins comprise one of the most commonly expressed gene families in mammalian tissues. Yet there are specialized actin genes which are either preferentially or exclusively expressed in all or only subsets of muscle cells. These expression patterns are mostly controlled at the level of transcription as is known from Jim Fickett´s work (and his excellent web-site) about muscle-specific gene expression. Therefore, the muscle-specificity of particular actin genes is most likely encoded in their promoter sequences although the most prominent muscle-specific transcription factors MEF2 and MyoD are apparently not crucial in this case although present in some of these promoters. Here, we present a pilot study focusing on the specific recognition of muscle-specific actin promoters. We developed a muscle-actin promoter model starting from a general analysis of the correlation of transcription factor binding sites (TF-sites) with these promoters and identified candidates for crucial TF-sites. Our model consists of 6 different elements and was developed on a training set of 11 sequences. This training set was already to heterogeneous in sequence to allow identification by FASTA analysis. It is muscle-actin specific and does not recognize most of the other muscle-specific promoters indicating that there are several independent ways to achieve muscle-specificity of a promoter. We analyzed more than 150 million bp from GenBank with the actin model and retrieved a total of 63 matches, 34 of which were true muscle-actin matches (54% true positives, there were only 10 false negatives). This demonstrates that specific promoter recognition against a vast background of anonymous sequences is pricipally possible.

Thomas Werner
GSF-National Research Center for Environment and Health
AG BIODV / Institute of Mammalian Genetics
Ingolstaedter Landstrasse 1
Neuherberg, Bavaria D-85758
Germany
telephone: +89-3187-4050
fax: +89-3187-4400
email: werner@gsf.de
url: http://www.gsf.de/biodvThomas WERNER

Presentation format: Platform

Many pairs of duplicated genes in yeast (Saccharomyces cerevisiae) are located within pairs of larger duplicated chromosomal regions, where a set of unrelated genes on one chromosome has a set of paralogs on another chromosome, in conserved map order and transcriptional orientation. About 50% of the yeast genome can be mapped into non-overlapping pairs of duplicated regions like this. These regions appear to be relics of a whole-genome duplication (i.e., a tetraploid stage) during yeast's evolution. Ab out 750 yeast genes (13% of its genome) are members of paralogous pairs identifiably resulting from this duplication, which we estimate occurred about 10^8 years ago. Some of the paralog pairs now have slightly divergent functions or are regulated differ ently. The genome duplication has the consequence that there is a two-to-one correspondence between many yeast genes and their orthologs in other species.

Susumu Ohno suggested in 1970 that tetraploidy played a role in increasing the gene number in vertebrates following their divergence from tunicates. Some large duplicated regions consistent with Ohno's model have recently been mapped in mammals so the st ructure of the yeast genome may provide a model relevant to mammals, although Ohno' s model remains far from proven. The genome of Caenorhabditis elegans, on the other hand, shows no sign of large duplicated chromosomal regions but instead has many tandemly repeated duplicate genes (which are virtually absent from yeast).

Gene order evolution in yeast seems to have occurred almost entirely by deletion and reciprocal translocation, with very little transposition of genes. We used computer simulations and analytical methods to estimate that the fraction of genes retained in duplicate after tetraploidy was about 8%, and that the number of "illegitimate" reciprocal translocations which broke up the original duplicated chromosomes into smaller duplicated genomic blocks was about 75. If vertebrate genomes have undergone two rounds of tetraploidy (as often proposed) and similarly small fractions of the genome were retained in duplicate after each round, it may be not be possible either to prove or disprove Ohno's hypothesis for mammals without a near-complete set of mapped and sequenced genes.

Dr. Ken Wolfe
University of Dublin, Trinity College
Department of Genetics
University of Dublin, Trinity College
Dublin 2 Ireland
telephone: +353-1-608-1253
fax: +353-1-679-8558
email: khwolfe@tcd.ie
url: http://acer.gen.tcd.ie/~khwolfe

Presentation format: Platform

Harley McAdams
Infernus
724 Esplanada Way
Stanford CA 94305
telephone: (650) 858-1864
fax: (650) 858-1886
email: mcadams@cmgm.stanford.edu

Presentation format: Platform

For the purposes of this discussion I will discuss the following attributes of genes

• where is a gene expressed ? the spatial problem, described in terms of an organism's anatomy.
• what is the (sub)-cellular localisation of a gene product ?, described in in terms of subcellular anatomy.
• when is a gene expressed ? the temporal problem, described in terms of an organism's ontogeny.
• of what larger structure is a gene product a component ?, described in terms of part-lists of the larger structures.
• what is the function of a gene product ?, described in terms of functional class, metabolic reactions and/or biochemical, cellular, physiological process.

I will illustrate one potential solution to the description of gene function designed for implementation in FlyBase, a comprehensive database of genetic and molecular data concerning Drosophila.

Michael Ashburner
European Bioinformatics Institute
Wellcome Trust Genome Campus
Hinxton, Cambridge CB10 1SD
England
telephone: +44-1223-494648
fax: +44-1223-494468
email: ashburner@ebi.ac.uk

Presentation format: Platform

In order to derive the intended benefits of this data several key issues need to be addressed. These include computational normalization, assembly of consensus sequences, identification, and correspondence to genomic sequence. We are using a combination of techniques to identify genes. BLASTN and TBLASTX (Altshul et al. 1990) searches are run on both the EST and genomic sequence data against a number of different datasets. The results are interpreted using a program called BOP written here at the center. Genie (Kulp, Reese, and Haussler) is used for gene prediction. The correlation of these analyses and a summary of the results will be presented.

Suzanna Lewis
University of California Berkeley
539 Life Sciences Addition
Berkeley, CA 94720
telephone: 510 643-0269
fax: 510 643-9947
email: suzi@fruitfly.berkeley.edu
url: fruitfly.berkeley.edu

Presentation format: Platform

presenter: George L. Gabor Miklos

The flightless region of D. melanogaster has been characterized by genomic and cDNA sequencing, reverse transcription-PCR, deletion analysis, transgenic rescues, and phenotypic dissections. It contains 12 genes, 5 of which have close human relatives, with the remaining 7 having human ESTs. Some are associated with mutant phenotypes; the human flightless homolog (in the Smith-Magenis deletion), the human SOX9 gene (campomelic dysplasia and sex determination), the human dodo homolog (cell cycle abnormalities), the human proline oxidase gene (type 1 hyperprolinaemia), and the mouse diff6 family (defects in cytokinesis). Most of the 12 multidomain proteins are absent from bacteria and half are absent from Saccharomyces cerevisiae. These multilevel data have significant implications for the transferability of functional genomics from model organisms to human beings.

George L Gabor Miklos
The Neurosciences Institute
10640 John Jay Hopkins Drive
SAN DIEGO, CALIFORNIA 92121
telephone: 619 626 2000
fax: 619 626 2079
email: miklos@nsi.edu

Presentation format: Platform

(1)Nikinmaa M., Cech J.J., Ryhanen E-L. and Salama A. (1987) Red cell function of carp (Cyptinus carpio) in acute hypoxia. J.Exptl.Biol 47 53-58.

(2)Romero M.G., Guizoran H., Pellisier B., Garcia-Romeu F. and Motais R (1996) The erythrocyte exchangers of eel (Anguilla anguilla) and Rainbow Trout (Onchorhynchus mykiss): a comparative study. J.Exptl.Biol: 199 415-426.

(3)Borgese F., Sardet C., Cappadoro M., Pouyssegur J. and Motais R (1992) Cloning and expression of a cAMP-activatable Na/H exchanger: evidence that the cytoplasmic domain mediates hormonal regulation. Proc.Natl.Acad.Sci.USA: 89 6765-6769.

(4)Borgese F., Malapert M., Fievet B., Pouyssegur J. and Motais R (1994) The cytoplasmic domain of the Na/H exchangers (NHE’s) dictates the nature of the hormonal response: Behaviour of a chimeric human NHE1/trout bNHE antiporter. Proc.Natl.Acad.Sci: 91 5 431-5435.

Dr Cheryl Wright
Department of Environmental and Evolutionary Biology
Derby Building
Brownlow st
Liverpool University
PO box 147
Merseyside L69 3BX
UK
telephone: 0151 794 4985
fax: 0151 794 5094
email: cwright@liv.ac.uk

Presentation format: Poster

We have developed and automated a method, TOGA (Total Gene expression Analysis), that utilizes a combination of nucleotide sequence and a precise fragment length near the 3’ ends of mRNA molecules to give each mRNA in an organism a unique identity, regardless of whether the mRNA has been discovered previously. The identity feature is used in PCR-based assays performed on tissue extracts of interest to determine the presence and relative concentration of nearly every mRNA in the extracts. Using automated DNA sequencing machines, the data are automatically compiled in a digital form that makes tissue comparisons facile and enables data merging and mining with other information accumulating in a wide range of genome databases and other research resources. A Netscape browser-based graphical user interface exploits these features of TOGATM to allow researchers to quickly identify mRNA expression patterns of interest in experimentally treated, diseased, and control samples, to select candidate mRNAs implicated in disease and drug action paradigms, and to instantly determine whether the particular mRNAs correspond to previously characterized species or are novel.

Karl W. Hasel
Digital Gene Technologies, Inc.
11149 North Torrey Pines Road
La Jolla, CA 92037
telephone: 619-552-1400
fax: 619-552-8625
email: chuck@dgt.com

Presentation format: Platform

Dr Tom Freeman
The Sanger Centre, Wellcome Trust Genome Campus
Hinxton, Cambs., CB10 1SA
UK.
telephone: 0044 1223 494907
fax: 0044 1223 494919
email: tcf@sanger.ac.uk

Presentation format: Platform

Genes coding for structurally and fuctionally similar proteins are often found in close physical proximity in the genome, forming gene clusters. Examples of cytokine gene clusters include the IL-1 cluster on human chromosome (HSA) 2, the TNF cluster on HSA6, the LIF-Oncostatin M cluster on HSA22, and the interleukin cluster on HSA5.

In order to clone genes of secreted and membrane-bound proteins from selected genomic regions, we combined the principles of signal and exon trapping and developed a new method, SEX trapping. Translation initiated from trapped exons coding for functional signal peptides results in the secretion of a secretory pathway-specific reporter enzyme from COS-7 cells into the cell culture medium. Using test constructs we showed that SEX trapping can identify signal sequence-containing exons from cytokine and non-cytokine genes, and from genomic inserts of widely different lengths. We applied the SEX trap method in the screening of a segment of the interleukin gene cluster on 5q31, which has been partially sequenced at LBNL. Signal sequence-containing exons of the IL-5 and IL-13 genes and a number of potential novel genes have been trapped. Thus, SEX trapping can be a useful tool in the discovery of genes from cytokine and cytokine receptor clusters, as well as in other positional cloning efforts.

Miklos Peterfy
Amgen Center, M/S 8-1-D
Thousand Oaks, CA 91320
telephone: (805)-447-6596
fax: (805)-498-8674
email: mpeterfy@amgen.com

presentation type: Poster

AgResearch Molecular Biology Unit, Department of Biochemistry and Centre for Gene Research, University of Otago, PO Box 56, Dunedin, New Zealand

presenter: Eric. A. Lord

The ovine Booroola mutation (FecB) increases ovulation rate and litter size. FecB was mapped to ovine chromosome 6 (OOV6) within a 28 cM region between epidermal growth factor (EGF) and secreted phosphoprotein-1 (SPP1). Since there were no ot her genes mapped to this region of OOV6, we have relied on genetic maps from other species as sources for positional candidates and additional loci to better define the position of FecB on OOV6. This has included genes and ESTs from human chromosome 4, to which OOV6 shares synteny, and candidate genes from the oogenesis pathway in Drosophila. Genes and ESTs assigned to HSA4 linkage and physical maps within the critical region were amplified from ovine genomic DNA and a hamster somatic cell hybrid containing OOV6. Primers for six genes were designed from the comparison of mammalian cDNA sequences. Primers pairs from 31 human ESTs were tested and four amplified similar sequences mapping to OOV6. The PCR products were used as RFLP or SSCP probes for linkage mapping in sheep gene mapping flocks and a deer interspecies hybrid mapping panel to confirm their relative positions on OOV6. To improve the efficiency of isolating sequences on OOV6 similar to human ESTs, a further 45 human brain and placental ESTs were screened with a human YAC contig that covers the critical region. The full cDNA clon es of 10 ESTs that mapped to the human YAC contig are being tested as RFLP probes.

Protein sequences of 55 Drosophila genes were screened for matches in the EST database and high similarity [P(N) The use of genetic maps from other species has benefited the Booroola mapping programme, firstly, by eliminating known genes and ESTs as potential candidates, and secondly, by adding new loci to the genetic map of OOV6. These additional loci have strengthened the synteny between OOV6 and HSA4. The genetic map in the vicinity of FecB has been better defined to allow us to proceed with physical mapping and transcriptional mapping strategies.

Eric Lord
AgResearch Molecular Biology Unit
Department of Biochemistry and Centre for Gene Research
University of Otago
PO Box 56
Dunedin, New Zealand
telephone: +64-3-4797662
fax: +64-3-4775413
email: lorde@agresearch.cri.nz
url: http://www.agresearch.cri.nz
http://biochem.otago.ac.nz:800/panzora/agresrch.html

Presentation format: Platform

Ref.: Ruttledge, M. H., Xie, Y.-G., Han, F.-Y., Peyrard, M., Collins, P., Nordenskjöld, M. and Dumanski, J. P. (1994). Deletion on chromosome 22 in sporadic meningioma. Genes, Chrom. and Cancer 10: 122-130.

Myriam Peyrard
Department of Molecular Medicine
CMM building L8:00
Karolinska Hospital
Stockholm
S-171 76 Sweden
telephone: +46-8-517 73922
fax: +46-8-517 73909
email: Myriam.Peyrard@cmm.ki.se

Presentation format: Platform

The CD3-z chain of TCR-DC3 complex plays a pivotal role in the activation of T cell responses and in the selection of the T cell repertoire. In z-knockout mice, the T cells have a profound reduction in the surface levels of TCR-CD3 complexes and these animals have poorly developed thymuses. In thymus, the cortex and the medulla are made up of different types of stromal cells that belong to epithelial or hematopoietic lineages. Thymocytes are found in tight interaction with these cells at various stages of differentiation. This complex pattern of migration is precisely regulated in time and space; it likely that part of this process is controlled by specialised cells interaction molecules expressed by stromal cells.

In order to find new genes differentially expressed between normal and z-knock out thymus, we set up two hybridizations on a same set of 3,072 genes with complex probe made from total RNA of z-knock out or wild type thymus. After quantitative measurement of the amount of hybridized probe on each colony, the intensity ratio z-knock out /wild type are calculated. 171 cDNA clones were selected showing either an increased or reduced representation in z-knock out. Additional hybridizations made with complex probes made from RNA of different cells types (such as macrophage, thymocyte, epithelial cell line under different stimulation) or tissues (lymph node, spleen...) allow to refine the selection.

Among clones presenting the most significant profiles, 10 are new or related to mouse EST. Analysed of tissue expression patterns by in situ hybridization shows a selective transcription in epithelial cells types. The sequence analysis reveals that some of these cDNA encode molecules likely involved in the dynamic organisation of thymus.

Catherine Nguyen
"TAGC" CIML INSERM-CNRS
CIML case 906
parc scientifique de luminy
13288
marseille cedex9
France
telephone: 33 4 91 26 94 82
fax: 33 4 91 26 94 30
email: nguyen@ciml;univ-mrs.fr

Presentation format: Platform

A major locus involved in linear growth has been implicated within the pseudoautosomal region (PAR1) of the human sex chromosomes. Cytogenetic studies have provided further evidence that terminal deletions of the short arms of either the X or the Y chromosome (0-700 kb) consistently lead to short stature (SS). We have constructed a cosmid contig across this region. The resulting map was used to position four breakpoints thereby reducing the critical interval for short stature to a 170 kb DNA segment. Using cDNA selection, exon amplification, and CpG island cloning, three novel genes were identified. To search for transcription units within the smallest 170 kb critical region, cDNA selection and exon amplification on six cosmids was carried out. cDNA selection on 25 different cDNA libraries proved to be unsuccessful, suggesting that genes in this interval are expressed at very low abundancy, below the sensitivity level imposed by the method. Exon amplification using the pSPL3-vector allowed the isolation of a homeobox containig exon. The low efficiency of trapped exons was due to the high number of redundant clones generated by cryptic splice sides within the vector. A new approach that we called EASE (exon amplification and selective enrichment) increased the efficiency of positive clones 20fold. Using a pSPL3 derivative vector (pSPL3b) yealded in a further enrichment of positive clones (177fold) and resulted in the isolation of three exons of a novel homeobox-containing gene, SHOX (short stature homeobox-containing gene), within the SS interval. This gene is alternatively spliced encoding proteins with different expression pattern. Mutation analysis and DNA sequencing were used to demonstrate that short stature can be caused by mutations in SHOX.

Ercole Rao
Institut für Humangenetik, INF 328, Heidelberg
Heidelberg, Germany 69120
telephone: 0049 6221 565067
fax: 0049 6221 565332
email: Ercole_Rao@krzmail.krz.uni-heidelberg.de

Presentation format: Platform

Patrick Onyango
Research Institute of Molecular Pathology (IMP),
Dr. Bohr-gasse 7, A-1030 Vienna, Austria
telephone: 0043 797 30 423
fax: 0043 1 798 71 53
email: Onyango@nt.imp.univie.ac.at

Presentation format: Platform

Intragenic Single Nucleotide Polymorphisms (SNPs) are being gathered by database screening plus automated PCR + sequencing protocols. The practical target is 10,000 SNPs within 18 months. This fundamental aspect of the research is completely dependent upon, and its structure determined by, the wealth of current database information. Details about this aspect of the research shall be presented, and examples given from its application to nuclear genes of oxidative phosphorylation. A one-step, microtitre plate formatted, fluorescence based assay for SNP screening is under development. This will provide automated genotype read-outs for 96 (and perhaps 384) samples at a time. Finally, various clinical collaborations, particularly exploiting a vast and superbly documented Swedish twin registry, will provide appropriate patient and control materials for testing.

Once this system is fully in place, association studies will be possible on a meaningful scale. Furthermore, linkage studies may be performed at far higher speeds and lower costs than with conventional microsatellite analysis. The entertaining problem then will be how to most effectively examine and interpret the large volumes of genotype data produced!

Anthony J. Brookes
Uppsala University
Department of Medical Genetics
Biomedical Centre
Box 589
S-751 23 Uppsala
Sweden
telephone: +46 (18) 471 4151
fax: +46 (18) 526 849
email: tony@medgen.uu.se
url: http://www.medgen.uu.se/

Presentation format: Platform

The ability of this RNA architecture analysis to predict the effect of antisense oligonucleotide on inhibition of mRNA in cell cultures were examined. The jellyfish green fluorescence protein (GFP) mRNA was used as a target mRNA as it provides an in vivo, real time marker for mRNA degradation as reflected by changes in fluorescence intensity. The architectural regions of GFP mRNA were analyzed and confirmed by RT-PCR assay. Antisense oligonucleotides were designed towards different regions of GFP mRNA and the changes in fluorescence were monitored by flow cytometry. Antisense inhibitors directed towards regions of low energy or open regions were shown to exhibit higher degree of inhibition than those directed towards regions of high energy values. This observation suggests that the efficacy of antisense inhibition can be improved by targeting regions of target mRNA of low energy values.

An application of this observation is to examine the intracellular action of regulatory genes by antisense inhibition and/or by over-expression to define the responsive genes. The responsive genes were then characterized changes in hybridiztion patterns on cDNA arrays and by changes in band intenisty of a RT-PCR process. Some of the responsive genes could futher control the expression of other genes as antisense inhibitors against several responsive genes were shown to alter the expression of other genes. Therefore, the responsive genes can be classified into group I and group II linked together in a cascade pattern of gene expression.

Wai-Choi Leung, Ph.D.
Tulane University School of Medicine
Dept of Pathology, 1430 Tulane Ave,
New Orleans, LA 70112
telephone: 504-588-5237
fax: 504-587-7389
email: wcleung@tmc.tulane.edu

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Departments of Biochemistry1 and Genetics2, and Howard Hughes Medical Institute1, Stanford University, Stanford CA, Synteni Inc. Fremont CA3, NCBI, Bethesda, MD4, Research Genetics, Huntsville, AL 5.

DNA microarray (chip) technology and two color fluorescent hybridization allows the efficient quantitative determination of relative gene expression on a large scale. We have generated microarrays that contain 10,000 human cDNAs robotically spotted onto treated microscope slides. For comparative hybridization, probe is generated by reverse transcription of mRNA derived from two samples each in the presence of distinguishable fluorescently tagged nucleotides. The probes are combined and hybridized in a small volume (10ul) underneath a coverslip using conventional hybridization chemistry. The arrays are read through use of a custom built confocal laser scanner that generates both a digital reconstructed image, and a quantitative measurement of gene expression in units of relative fluorescent intensity. Examples of the use of the arrays for identification of novel targets of oncogenic proteins, for determination of patterns of gene expression change occurring during induced differentiation,

Douglas T. Ross
Stanford University
Beckman Center B-435
Dept. of Biochemistry
Stanford, CA 94305-5307
telephone: 650-723-6719
fax: 650-725-6044
email: dross@cmgm.stanford.edu

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Mark Hannink
Biochemistry Department, University of Missouri
M121 Medical Science Building
One Hospital Drive
Columbia, Missouri 65212
telephone: (573)-882-7971
fax: (573)-884-4597
email: bcmarkh@muccmail.missouri.edu

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We have been pursuing the use of so-called message display technology in high-throughput transcript scanning, and established Fluorescent Differential Display system (FDD), that can survey tens of thousands of cDNAs a day. To simultaneously address the three issues described above, we are currently applied FDD to the analysis of yeast gene disruptants/overexpressers for the elucidation of functional relationships between the disrupted/overexpressed gene and those whose transcripts are modulated. To facilitate the generation of expression mutants, we developed a PCR-based promoter replacement strategy to make the expression of any genes under an artificial control. Also, the introduction of an ideal primer set and the multicapillary gel electrophoresis system are planned to accelerate the FDD analysis further.

Another novel intriguing application of FDD is the Allelic Message Display (AMD) for multiplexed imaging of allelic expression status. With AMD among two mouse strains and reciprocal F1 hybrids as well as backcrossed progenies, we have developed a novel screening method for monoallelically expressed transcripts to hunt genes subjected to genomic imprinting, a unique interpretation mode of mammalian genomes. Identification of a novel paternally expressed gene will be presented as a successful example of AMD approach.

Takashi Ito
Human Genome Center, Institute of Medical Science, University of Tokyo
4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan
Tokyo 108
Japan
telephone: 81-3-5449-5623
fax: 81-3-5449-5445
e-mail: tito@ims.u-tokyo.ac.jp

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Ataxia telangiectasia (AT) is an autosomal recessive disease characterized by progressive cerebellar ataxia, immunodeficiency, predisposition to cancer, chromosomal instability and radiosensitivity. Using positional cloning, the gene was localized to a less than 500kb interval on 11q22-23 and subsequently cloned. The gene has PI 3 kinase, rad3 and SH3 domain in addition to a leucine zipper. Recent studies suggest that the ATM protein is involved in signal transduction and forms part of the synaptonemal complex during meiosis. The gene shows strong homologies to TEL1, ESR1/MEC1 genes from yeast. The human ATM gene is contained within ~180kb of genomic DNA. It has 66 exons and the 13kb transcript encodes a protein that is 3056 a.a. and 350kDa. The mouse homologue (Atm) has 84% amino acid identity and 91% similarity to the human counterpart.

We have isolated and sequenced the pufferfish homologue from a lambda and PAC genomic library. Sequence comparison shows a strong conservation in the kinase domain and the 3’end but weaker at the 5’end. It is contained within a 16kb genomic region. Most of the exons and the splice junctions are well conserved.

Our mutation analysis of >280 homozygotes using PTT, SSCP, CSGE, HA and direct sequencing, have identified >150 mutations. PTT detects 70% of ATM mutations. Almost all common mutations were due to founder effect. One public mutation was found in two American families, one of Ashkenazi Jewish background and the other not. Some of the interesting mutations we came across were : 1) Mutations within an intron that create a new splice site, 2) Mutations within an exon that create a new splice site and delete the subsequent exon, 3) Leaky mutations that give more than one mutant RNA, 4) Both in frame and frame shift mutations. We now plan to align the sites of these human mutations in the pufferfish homologue and try to determine new functional domains.

Dr. Nitin S. Udar
Jules Stein Eye Institute
3-544 DSERC
100 Stein Plaza
UCLA School of Medicine
Los Angeles, CA 90095
telephone: 310 794 7420
fax: 310 794 7904
email: NUDAR@Pathology.Medsch.UCLA.Edu

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We isolated and analysed human, fugu and amphioxus homologues of several genes located in the G6PD region of human Xq28 and compared the degree of homology of their nucleotide and amino acid sequences, exon/intron structures and overall gene sizes. Two human, two fugu and one amphioxus rab GDI genes were studied in greatest detail. Their analysis indicated that the occurrence of two forms of rab GDIs preceded the fish-tetrapod divergence, while the amphioxus rab GDI may have evolved from the ancestor of both forms. The gene structures of all rab GDI genes studied were highly conserved. While all were generally composed of 11 exons, the amphioxus gene had one additional intron and one of the fugu genes was missing one intron. The Xq28-located human rab GDI alpha gene was 6.3 kb long and its size was similar to both fugu rab GDI genes. The autosomal human rab GDI b occupied 50 kb of genomic DNA and contained many retroelements in its introns. This size difference reflected the location of the two genes in different isochores of the human genome. The size of the amphioxus rab GDI gene was intermediate to the above genes.

The rab GDIs and several other anchor genes from our model region of Xq28 served us as starting points in the isolation and analysis of large genomic segments in the search for possible traces of ancestral chordate genomic segments or paralogous regions in vertebrate genomes arisen by genome duplications. The data indicate only partial conservation of gene colinearity within the segments studied.

Annemarie Poustka, Barnard Korn, Stephen Wiemann
German Cancer Research Center
Division of Molecular Genome Analysis
Heidelberg 69120
Germany
telephone: +49 6221 42 4702
fax: +49 6221 42 4704
email: s.wiemann@dkfz-heidelberg.de
url: http://www.dkfz-heidelberg.de/abt0840/

1 Resource Center of the German Genome Project 2 Division of Molecular Genome Analysis, German Cancer Research Center, Heidelberg 3 Max-Planck-Institute for Molecular Genetics, Berlin-Dahlem

The technology of cDNA selection has been applied to whole chromosomes through the use of chromosome specific cosmid libraries. We made use of mRNAs from 25 different human tissues as primary, non-cloned cDNA (adrenal gland, adult brain, adult skeletal muscle, bone marrow, fetal brain, fetal kidney, fetal liver, heart muscle, kidney, liver, lung, lymph node, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, small intestine, spinal cord, spleen, stomach, testis, thymus and uterus), in order to have the most complex starting material. We subjected that cDNA to fragmentation and 3’ end specific amplification to have access to all genes, not biased by their transcript sizes. This cDNA source was hybridised to the LLNLXU cosmid library, covering the X chromosome (3.7x coverage), in liquid. 23.000 positively selected cDNA clones were picked into microtiter plates and all mapped genes and EST from the human chromosome X were added to this clone collection. High density filters were generated and used for further analysis. Filters are made available through the Ressource Center of the German Genome Project. Randomly chosen clones were sequenced and mapped, either in silico or onto high density filters containing arrayed X chromosome specific YACs . In silico mapping was supported by the fact that we designed the cDNA selection to specifically clone the 3’ end of transcripts and, therefore, have immediate access to the EST databases by BlastN alignment. Results of the sequence analysis of 94 randomly chosen clones are given below.

Table 1

no database hits	39,36%
ESTs of unknown location	30,85%
Genes/ESTs mapped to human X	18,09%
repetitive sequences	5,32%
ESTs/genes located on non-X chromosomes	4,26%
background (e. g. E. coli,...)	2,13%

By this approach we have been able to quality control the established X chromosome cDNA library and to identify and map new genes at the X chromosome.

Annemarie Poustka, Barnard Korn, Stephen Wiemann
German Cancer Research Center
Division of Molecular Genome Analysis
Heidelberg 69120
Germany
telephone: +49 6221 42 4702
fax: +49 6221 42 4704
email: s.wiemann@dkfz-heidelberg.de
url: http://www.dkfz-heidelberg.de/abt0840/

In the frame of the German Genome Project, we generate cDNA libraries enriched for ‘full length’ cDNAs from human tissues with the aim to obtain complete cDNAs, from the 5’ cap structure to the poly A tail of as many as possible human genes. Today libraries have been made from fetal brain, fetal kidney, testis, skeletal muscle and spinal cord. Starting from oligo-dT primed cDNA, the first strand cDNA is amplified under long range conditions in few cycles. Cloning is done directionally into plasmid vectors. Libraries (30,000 to 120,000 clones) are picked in 384 well plates and spotted on high density filters. We initially characterize new libraries by sequencing randomly picked clones and hybridization of genes of varying size and abundance. The libraries have already been successfully used to screen for full length representations of the human MTM1 gene (3.4 kb), the human homologue of flightless (4.2 kb,) and a number of other transcripts. Currently, the libraries are used to isolate full length representations of the genes located in the chromosomal region Xq27.3 - qter.

Clones with inserts longer than 1.5 kb are pre-selected by agarose gel electrophoresis, for subsequent sequence analysis. Highly abundant genes are hybridized to the filters in order to minimize redundancy in initial EST sequencing. EST sequences are analyzed for the likelihood of the clones to be full length, e.g. by the presence of CpG clusters, in order to obtain a minimal set of full length clones for efficient complete sequence analysis. Clones identified to be full length are sequenced and further analyzed by a national consortium in the frame of the German Genome Project. We will determine 8 Mb of finished sequence comprising 3,000 - 4,000 full length cDNA sequences in the next three years. The sequences are analyzed for possible function in silico to facilitate subsequent functional analysis. All clones and data generated during the project are made publicly available via the Resource Centre of the German Genome Project (RZPD).

Annemarie Poustka, Barnard Korn, Stephen Wiemann
German Cancer Research Center
Division of Molecular Genome Analysis
Heidelberg 69120
Germany
telephone: +49 6221 42 4702
fax: +49 6221 42 4704
email: s.wiemann@dkfz-heidelberg.de
url: http://www.dkfz-heidelberg.de/abt0840/

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This work was partially financed by MURST (Italy) and EC grant BIO4-CT95-0130.

Graziano Pesole
Dept. of Biology D.B.A.F.
University of Basilicata, Italy
via Anzio 10
Potenza 70126
Italy
telephone: +39-971-474431
fax: +39-971-474439
email: graziano@area.ba.cnr.it

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Thus far, some 40,000 clones have been subjected to tag-sequencing from both ends, which were classified into about 7,300 unique cDNA species (almost half of the total genes) by comparing the 3'-tags. Most of them were mapped on the genome either by in silico mapping or hybridization to the YAC filters. We are systematically analyzing the expression patterns of the classified cDNA species using in situ hybridization on whole mount specimen of embryos, larvae and adults. Rough estimation is that 39% of the cDNA species show specific pattern of expression during embryogenesis, out of which 1/3 shows zygotic expression and 1/3 maternal expression. mRNA of 1/4 of the maternally expressed genes (about 4% of all) disappears in very early stages before gastrulation. Classifying of the expression patterns leads to identification of sets of genes which show the very similar expression patterns. Determination of the regulatory regions of these genes is also in progress. We are constructing the database named NEXTDB (the Nematode EXpression paTtern DataBase) to make the information available on the internet.

Yuji Kohara
Gene Network Lab
National Institute of Genetics
1111, Yata
Mishima 411
Japan
telephone: +81-559-81-6854
fax: +81-559-81-6855
email: ykohara@lab.nig.ac.jp

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The Jackson Laboratory, Bar Harbor; *MRC Human Genetics Unit, Edinburgh; #Edinburgh University, Edinburgh.

The process of differential gene expression generates extraordinarily complex spatio-temporal networks of gene and protein interactions. With its new focus on gene function and expression analysis, genome research is beginning to elucidate these networks to understand the molecular basis of human health and disease. The laboratory mouse will serve as a pivotal animal model in these studies. High throughput expression methods will make it possible to analyze in parallel the expression of thousands of genes in different tissues that can be derived from many different mouse strains and mutants. These experiments will provide global insights into expression profiles and molecular pathways, and lead the way to more focused expression studies using Northern and Western blot, RT-PCR, RNA in situ hybridization, and immunohistochemistry assays to determine what transcripts and proteins are produced by specific genes, and where and when these products are expressed at the cellular level.

We are developing a database of gene expression information for the laboratory mouse that can store and integrate these data and make the data freely and widely available in formats appropriate for thorough analysis. Expression patterns are described using a standardized anatomical dictionary. For in situ studies, the textual annotations are complemented with digitized images of original expression data that are indexed via the terms from the dictionary. This database system will be combined with a 3D atlas of mouse development to enable 3D graphical display and analysis of expression patterns. Integration with the Mouse Genome Database and comprehensive interconnections with other relevant databases will place the gene expression data into the larger biological and analytical context.

Expression data are and will be acquired from the literature by database editors, but primarily data will come via electronic submissions directly from research laboratories. The Gene Expression Annotator, an electronic submission system for expression data that we have developed, is currently being tested by a number of laboratories in North America and Europe with the aim of developing a user friendly system for the community at large. The Gene Expression Index, a searchable index into the expression literature for mouse development, being updated daily, is already accessible to the general public at http://www.informatics.jax.org/gxd.html. Additional data sets will be made available in the near future. The current status of the database and its future applications will be discussed.

Martin Ringwald
The Jackson Laboratory
600 Main Street
Bar Harbor, Maine 04609
telephone: (207) 288-6436
fax: (207) 288-6132
email: ringwald@informatics.jax.org

Joakim Lundeberg
Royal Institute of Technology
Department of Biochemistry
KTH-Royal Institute of Technology
Stockholm S-100 44
SWEDEN
telephone: 46 8 790 87 58
fax: 46 8 24 54 52
email: joakim.lundeberg@biochem.kth.se

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Attaching functional annotation to predicted genes in genomic sequences

Roderic Guigo
Informatica Medica
Institut Municipal d'Investigacio Medica (IMIM)
C/ Dr. Aiguader 80
Barcelona 08003
Spain
telephone: +34 3 221 1009
fax: + 34 3 221 3237
email: rguigo@imim.es
url: http://www1.imim.es/~rguigo/Welcome.html

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John H. Postlethwait
Institute of Neuroscience
University of Oregon
Eugene, OR 97403
telephone: 541-346-4538
fax: 541-346-4538
email: jpostle@oregon.uoregon.edu

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This work was partially financed by MURST (Italy) and EC grant BIO4-CT95-0130.

Graziano Pesole
Dipartimento di Biologia D.B.A.F.
via Anzio 10
POTENZA, 85100
ITALY
telephone: +39-971-474431
fax: +39-971-474439
email: graziano@area.ba.cnr.it

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Joseph H. Nadeau
Genetics Dept., Case Western Reserve University School of Medicine
10900 Euclid Ave
Cleveland, Ohio 44106
telephone: 216-368-0581
fax: 216-368-3432
e-mail: jhn4@po.cwru.edu

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The way in which candidate disease genes are identified in positional cloning projects is rapidly changing. A few years ago, one had to use methods like cDNA-selection, cDNA- library screening , identification of evolutionary conservation sequences, localization of HTF- islands or exon trapping. Currently, an increasing number of "electronic" possibilities are emerging, including the analysis of the mapped ESTs and searching databases for functional candidates. The most recent addition is the analysis of the large stretches of sequenced DNA that are emerging from the Human Genome Project. We are involved in two positional cloning projects, Wolf-Hirschhorn Syndrome (WHS) and Retinoschisis (RS), for which the entire disease gene candidate region has been, or is being, sequenced; 165 kb on 4p13 and 1.2 Mb on Xp22 respectively.

Computer analysis of the sequenced regions revealed several problems. On one hand, the diversity of databases and gene prediction programs available created very helpful resources, but on the other hand, the lack of specifically designed software made the analysis very time consuming. For example, since repeat masking was not perfect, repeated database searches had to be performed. Furthermore, detailed analyses of the results would be simplified if e.g. retrieval of a 5' EST sequence would simultaneously yield the 3' sequence or, even better, the entire batch from a "UniGene" set. dbEST contained the most valuable data resource. For many genes both human and murine transcripts were present. Some ESTs seem to be derived from priming at intronic A-rich regions, others probably derive from hnRNA (or genomic DNA), with A-rich regions at both ends. ESTs from both DNA strands were also detected; in one case this probably derived from a duplicated genomic sequence. Database searches using translations of the constructed putative gene sequences against the six frame translations of dbEST frequently identified transcripts from diverse organisms with high local similarities, probably representing new protein domains.

The electronic results were verified using RT-PCR, cDNA-library screening, exon trapping and Northern blot analysis. Both Northern analysis and RT-PCR were facilitated by the expression profile deduced from dbEST. RT-PCR was most powerful to link computer- predicted exons and to verify intron/exons borders. Identification of the gene's 5' end turned out to be the most difficult, especially since RT-PCR analysis seemed to link transcripts from directly flanking genes. For one region, containing a large open reading frame which was clearly evolutionary conserved, transcripts could never be identified, neither using RT- PCR, nor using cDNA-library screening or Northern analysis. Furthermore, database searches revealed no homologies.

Dr. Johan T. den Dunnen
Department of Human Genetics
Leiden University
Wassenaarseweg 72
2333 AL LEIDEN
the Netherlands
telephone: +31-71-5276105
fax: +31-71-5276075
email: ddunnen@ruly46.medfac.leidenuniv.nl
url: http://ruly70.medfac.leidenuniv.nl/

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Among other applications, the availability of libraries enriched for full-length cDNAs will facilitate all ongoing efforts aimed at the identification of disease-causing genes. A few methods have been described for construction of full-length cDNA libraries, which take advantage of the CAP structure present at the 5' end of intact mRNAs, to select for "full-length" molecules (1-4). However, as a general rule, these libraries have not been characterized to the extent that it would be required to determine whether most clones are truly full-length. It is noteworthy, however, that at the very least these procedures will be most valuable to generate libraries enriched for 5' ends of mRNAs. There are two potential problems. First of all, since there is no selection for bonafide 3' ends, many of the resulting clones may be truncated at the 3' end. This is so because mRNAs may be primed internally during synthesis of first-strand cDNA. Because of their smaller size, these clones may outcompete their full-length counterparts during ligation and amplification. Second, and most importantly, the differential clonability and growth properties of smaller (full-length) cDNAs versus longer (full-length) cDNAs make it very difficult to isolate long full-length cDNAs. In an effort to address these concerns, we developed an alternative strategy for construction of libraries enriched for full-length cDNAs which is based on the rationale that if cDNA is synthesized from size fractionated mRNA, it can be strictly size selected prior to cloning accordingly, thus yielding sub-libraries greatly enriched for full-length cDNAs. We have documented the feasibility of this approach by generating a number of such sub-libraries from a mixture of size-fractionated mRNA from human brain and placenta. The results indicated that the sub-libraries produced were significantly enriched for full-length cDNAs. However, detailed characterization of these libraries also pointed to some problems which we are currently attempting to solve. A critical review of the advantages and disadvantages of this procedure will be presented.

1. Carninci, P., Kvam, C., Kitamura, A., Ohsumi, T., Okazaki, Y., Itoh, M., Kamiya, M., Shibata, K., Sasaki, N., Izawa, M., Muramatsu, M., Hayashizaki, Y. and Schneider, C. (1996). High-efficiency full-length cDNA cloning by biotinylated CAP trapper. Geno mics 37: 327-336.

2. Carninci, P., Westover, A., Nishiyama, Y., Ohsumi, T., Itoh, M., Nagaoka, S., Sasaki, N., Okazaki, Y., Muramatsu, M., Schneider, C and Hayashizaki, Y. (1997). High-efficiency selection of full-length cDNA by improved biotinylated Cap trapper. DNA Resea rch 4: 61-66.

3. Kato, S., Sekine, S., Oh, S-W., Kim, N-S., Umezawa, Y., Abe, N., Yokoyama-Kobayashi, M. and Aoki, T. (1994). Construction of a human full-length cDNA bank. Gene 150: 243-250.

4. Edery, I., Chu, L.L., Sonenberg, N. and Pelletier, J. (1995). An efficient strategy to isolate full-length cDNAs based on a mRNA Cap retention procedure (CAPture). Mol. Cell. Biol. 15: 3363-3371.

Marcelo Bento Soares
The University of Iowa
451 Eckstein Medical Research Building
Iowa City, IA 52242
telephone: (319) 335-8250
fax: (319) 335-9565
email: bento-soares@uiowa.edu

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Li Zhu
CLONTECH Laboratories, Inc.
1020 E. Meadow Circle
Palo Alto, CA 94303
telephone: (650)-424-8222x1462
fax: (650)-354-0776
url: liz@clontech.com

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The nematode C. elegans will be the first animal for which the complete genome sequence will become available. This opens the possibility of new large scale functional studies of e.g. whole gene families, using loss of function and gain of function mutants and expression patterns. Thus far the method of choice for gene inactivation was a two-step approach using the transposon Tc1 (Zwaal et al., 1993, Proc. Natl. Acad. Sci. USA 90, 7431-7435). We have developed a one step method for target-selected gene inactivation in C. elegans using chemical mutagenesis (Jansen et al., 1997, Nature Genet. 17, 119-121). A permanent frozen mutant collection has been established, consisting of over 7,000 cultures, each representing approximately 150 genomes. We use PCR to selectively visualize deletions in genes of interest: primers are selected more than 3 kbp apart, so that the deletion fragment will have a selective advantage in the amplification reaction. The method is sufficiently sensitive to permit detection of a single mutant among more than 15,000 wild types. The approach has successfully been applied in our study of the function of all heterotrimeric G-protein genes in C. elegans. We will discuss our plans to scale up the method for systematic inactivation of all 17,000 C. elegans genes.

Gert Jansen
Division of Molecular Biology (H8)
The Netherlands Cancer Institute
Plesmanlaan 121
1066 CX Amsterdam
The Netherlands
telephone: #31-20-5122090
fax: #31-20-5122086
email: GJANS@NKI.NL
url: http://www.nki.nl/nkidep/h8/people/gert.html

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The mouse testis determining gene, Sry, on the Y chromosome encodes a protein with a DNA-binding (HMG box) domain at its amino end and a glutamine-rich domain at its carboxyl end. The HMG box is conserved in the Sry of other mammals. The glutamine-rich domain is encoded mostly by CAG repeats and its function is unknown. We hypothesize that the glutamine- rich domain was generated by an in-frame insertion of a repetitive sequence in the mouse ancestral Sry. It had gained a protein-protein binding function, similar to situations of the CAG expansion in the mutated genes of several neurodegenerative diseases. However, in the case of the mouse, an evolutionary adaptation results in a stable retention of the CAG repeats in Sry. Using the glutamine- rich domain as probes in farwestern blotting studies, we detected 3 specific bands at 94, 32 and 28 kDa only in testis extract and a 90 kDa in the brain extract from adult tissues. The 94, 32 and 28 kDa testicular proteins have been designated as Sry interactive protein 1 (Sip-1), 2 (Sip-2) and 3 (Sip-3) respectively. The Sips were detected in somatic cells of testicular origin and their expression was associated with spermatogenic activities. Additional studies using subcellular fractionation techniques demonstrated that both Sip-2 and -3 were predominantly present in the nuclei while Sip-1 was present in both cytoplasmic and nuclear fractions. In situ blotting and farwestern blotting of adult testis section demonstrated that indeed the Sips were preferentially localized in the interstitial and peripheral regions of the seminiferous tubules. Sips were expressed in tissues of embryos as early as 8.5 days post coitus (dpc) and in fetal gonads of both sexes at 11.5 dpc, during the time of sex determination. However, their expression patterns varied both quantitatively and qualitatively at different developmental stages. Although the exact nature of these Sry interactive proteins has yet to be defined, their detection supports our hypothesis that the mouse Sry glutamine-rich domain contributes to the biological function(s) of Sry through a protein-protein interactive role(s).

Chris Lau
Division of Cell and Developmental Genetics
Department of Medicine, VAMC-111C5
University of California, San Francisco
4150 Clement Street
San Francisco, California 94121
telephone: 415-476-8839
fax: 415-502-1613
email: clau@itsa.ucsf.edu

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i) Microdissection of 21q21 has provided a number of novel unique sequences; 70 of these have been used to screen the LLNL chromosome 21 specific cosmid library. A nonredundant subset of positive cosmids was then used in exon trapping experiments. Results indicate an approximately 8-fold lower density of putative exons and shows that these are significantly smaller and much less GC-rich than those within 21q22. Sequencing in the parent cosmids indicates that the exon trapped products are likely bona fide exons and novel.

ii) The APP gene from Fugu rubripes has been isolated and completely sequenced. Several exon prediction programs applied to the Fugu sequence show a slightly higher true positive rate and a significantly lower false positive rate than is obtained with the same programs applied to the genomic sequence of the human APP gene. Were the human APP protein now known, use of the Fugu sequence would reduce time and effort required to verify gene predictions. Conserved synteny also aided in gene discovery: <3 kb downstream of APP in the same Fugu cosmid, the GABPA transcription factor was found, a gene in humans known only to lie within 800 kb of APP.

2. Determination of the genomic structure of the Fugu APP gene revealed unusually high compaction: the Fugu introns are on average 50-fold smaller than the human homologues. APP is also the most AT-rich human gene so far analyzed in Fugu. Characterization of additional genes whose isochore location is known in human indicates that base composition can predict the relative extent of compaction of the homologue. This supports the value of Fugu analysis, particularly in AT-rich regions.

Katheleen Gardiner
Eleanor Roosevelt Institute
11899 Gaylord Street
Denver, Colorado 80206
303-333-4515
303-333-8423
gardiner@eri.uchsc.edu

Marie-Laure Yaspo
Max Planck Institut fur Molekulare Genetick
Ihnestrasse 73
Berlin D-14195
GERMANY
49-30-8413-1356
49-30-8413-1380
yaspo@mpimg-berlin-dahlem.mpg.de

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A cDNA for human RED1 was used to screen a Fugu rubripes cosmid library (constructed by Greg Elgar; archived at the German Human Genome Resource Center), identifying 4 non- overlapping cosmids. Sequence analysis revealed a family of editases. One cosmid contains the homologue of DRADS, identified by the similarity of number and organization of RNA binding doamins, and intron-exon structure. A second cosmid contains the apparent different genes (named RED1a and RED1b), each showing high homology to human RED1. The exon-intron boundaries of RED1a and RED1b are conserved with human, but intron sizes vary, both from human and between each other. Overall the protein similarity between RED1a and RED1b is 83%; in the two RNA binding domains the similarity rises to >95%.

Together, these data imply that Fugu contains at least 4 distinct A-to-I RNA editase genes, suggesting that additional editases remain to be identified in mammals. These Fugu genes also provide the material for further characterization of some unusual features observed in the human RED1 3' UTR.

Dobrimir Slavov
Eleanor Roosevelt Institute
1899 Gaylord Street
Denver, Colorado 80206
telephone: 303-333-4515
fax: 303-333-8423
email: gardiner@eri.uchsc.edu

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There are, however, similarities at the expression level between the 3q21 and 3q26 breakpoint regions. These include: i) restricted expression patterns: e.g. in 3q21, expression of the GR6 gene has been observed only in early fetal development; EVI1 exhibits fetal and tissue specificity; ii) complex alternative splicing: e.g. both GR6 and EVI1 genes display several aternative transcripts including those produced by use of splice sites within exons and read through into introns; and iii) intergenic splicing: in some normal tissues, intergenic splicing between the MDS1 and the EVI1 genes in 3q26 is observed. In AML with t(3;3), intergenic splicing between the GR6 and RPBHI genes in 3q21 and EVI1 in 3q26 is observed. The 3q21 breakpoints are up to 30 kb downstream of the 3q21 genes. These unusual features and their implications in normal expression patterns and leukemia will be discussed.

A. Rynditch, Y. Pekarsky, K. Gardiner
Eleanor Roosevelt Institute
1899 Gaylord Street
Denver, Colorado 80206
telephone: 303-333-4515
fax: 303-333-8423
email: gardiner@eri.uchsc.edu

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Shuo Lin
Institute of Molecular Medicine and Genetics
Medical College of Georgia
Augusta, Georgia 30912
telephone: 706-721-8762
fax: 706-721-8752
email: slin@mail.mcg.edu

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Ischemic stroke is a common, complex disorder caused by a combination of genetic and environmental factors. A significant genetic component to stroke predisposition has been demonstrated by both rare Mendelian inheritance, and by increased concordance in monozygotic compared with dizygotic twens. In addition, two recent studies have identified a major stroke-influencing quantitative trait locus. STR3, on rat chromosome 5, in the spontaneously hypertensive stroke-prone rat (SHRSP).

We report the use of Quantitative Expression Analysis (QEA) to compare the gene expression profiles in hearts from SHR and SHRSP rats fed a normal diet. QEA is a novel method that comprehensively and rapidly compares levels of gene expression between samples, with a limit of mRNA detection below 1 part in 125,000. QEA relies on uniform labeling and non-biased amplification of cDNA fragments, and uses information about specific sequences at the ends of the amplified products in conjunction with product lengths to assign electronically the potential genes that a given band represents (GeneCalling).

Using QEA, 12,000 fragments, derived from ~6000 genes, were compared in triplicate between heart mRNA samples of SHR and SHRSP rats. 29 differences (0.2%) of magnitude >1.5-fold were found. One gene shown by QEA to be expressed differently between SHR and SHRSP heart was atrial natriuretic factor (ANF), which maps within the segment of rat chromosome 5 containing STR3. Two fragments derived from ANF were expressed at 2-fold higher levels in SHRSP than SHR; abolition of these peaks with ANF-specific oligonucleotides confirmed the identities of these fragments (oligo poisoning). Sequence analysis of ANF from SHRSP and SHR rats revealed a substitution (G99S) that changes a highly-conserved glycine to serine residue, and that may influence peptide cleavage by the inactive prohormone. The finding that ANF is altered in expression and sequence between SHR and SHRSP rats, together with co-localization of ANF and STR3, suggests that an ANF mutation underlies STR3. Furthermore, the SHRSP allele is protective against stroke development, while the mutation observed in ANF in SHRSP rats is consistent with impaired function. Also in accord with this hypothesis is the increase in ANF expression in SHRSP heart, that may represent a consequence of a functional ANF impairment. The known role of ANF in control of vascular tone and intravascular volume, as well as the high density of ANF binding sites in the brain, are consistent with causality for STR3. Moreoever, increased ANF levels have been found in humans with acute stroke. Finally, these findings suggest the potential of the mutant ANF peptide as a preventative agent for stroke.

QEA, in combination with candidate gene mapping (positional expression cloning), offers a novel, rapid alternative to positional cloning, by identifying differentially expressed genes that map adjacent to disease loci. In common with conventional positional cloning, the comprehensive nature of QEA permits identification of disease loci without prior knowledge of pathophysiology. Positional expression cloning is anticipated to have broad applicability to animal models of inherited disease and, in particular, to multigenic disorders that defy conventional positional cloning strategies.

Richard A. Shimkets, Suresh G. Shenoy
CuraGen Corporation
555 Long Wharf Drive
New Haven, Connecticut 06511
email: skingsmo@curagen.com

Presentation format: Platform

The next phase of the Human Genome Project entails genome scale, high-throughput generation of data leading to a deeper understanding of function. The management, analysis and visualization of data generated in this phase will undoubtedly be substantially more difficult than the sequence-oriented data that forms the foundation for the first phase of the Genome Project. EpoDB is a prototype system designed to explore the issues surrounding functional analysis of differentiation using vertebrate erythropoiesis as a model system. We will describe the current capabilities and tools developed in EpoDB for information capture, representation and visualization, and their use in the analysis of gene expression during erythropoiesis. EpoDB readily extends to other pathways in hematopoiesis and other differentiating systems.

C. Overton1, J. Haas1, F. Salas1,
1312 Blockley Hall (6021)
418 Guardian Drive
Philadelphia, Pennsylvania 19104-6145
telephone: 215-573-3105
fax: 215-573-3111
email: coverton@Wcbil.humgen.upenn.edu

This work was supported by the United States Department of Energy, Office of Health and Environmental Research, under Contract No. W-31-109-ENG-38. This publication was also made possible by grant number ES07141 from the National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health. Its contents are solely the responsibility of the authors and do not necessarily represent the official view of the NIEHS, National Institutes of Health.

Gayle E. Woloschak
Argonne National Laboratory
Center for Mechanistic Biology and Biotechnology
9700 South Cass Avenue
Argonne, Illinois 60439-4833
telephone: 630-252-3312
fax: 630-252-3387
email: woloschak@anl.gov

Presentation format: Platform

Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA.

Automatic annotation of large amounts of genomic DNA sequence is, and will continue to be, a formidable challenge. Only by developing very efficient computational tools for the initial annotation of the sequence and then by treating these annotations as hypotheses and testing and verifying them in the laboratory will this problem be properly addressed. We are developing an engine which will provide a framework for the analysis and annotation of genomic DNA sequences. This system includes methods for data retrieval, visualization, data warehousing and data mining. The interface to this system is called The Genome Channel.

Results of the various analyses performed by the system are presented in this interface. A number of features including simple and complex repetitive DNA sequences, tRNAs, CpG islands, as well as the results of several gene finding programs, including a new version of GRAIL, which incorporates EST similarity in gene prediction and modeling, are included in the analysis. Links from the analysis to other data resources are also included. Currently the results from all the major human sequencing centers can be viewed in the Genome Channel.

This research was supported by the Office of Health and Environmental Research, United States Department of Energy, under contract DE-AC05-84OR21400 with Lockheed Martin Energy Systems, Inc.

Richard Mural
Life Sciences Division, Oak Ridge Natioanl Laboratory
1060 Commerce Park
Oak ridge, TN 37831
telephone: 423-576-2938
email: muralrj@ornl.gov

Presentation format: Platform

Marie-Laure Yaspo
MPIMG
Ihnestrasse 73
Berlin
D-14195 GERMANY
telephone: 49-30-8413-1356
fax: 49-30-8413-1380
email: yaspo@mpimg-berlin-dahlem-mpg.de
url: http://chr21.rz-berlin.mpg.de

Presentation format: Platform