Genome Mapping




Keywords



The major interest of our mapping group is the construction of integrated physical and transcriptional maps of the human genome with a special focus on chromosome 22 . We are using a variety of different cloning systems (plasmids, cosmids, P1s, YACs) for the detailed molecular analysis of chromosomal regions of medical or biological interest. Recently, we started using the Bacterial Artificial Chromosomes (BACs) to assemble extended clone contigs in several chromosomal regions (21q22.3, 22q12-qter, 22cen, various telomeres) and for the detailed cytogenetic analysis of chromosome aberrations by fluorescence in situ hybridization (FISH) .


Chromosome 22


Several clinically relevant disorders and neoplasias are associated with anomalies involving chromosome 22. There are a number of important regions and/or genes involved in a variety of tumors (ALL, CML, Burkitt's lymphoma, Ewing's sarcoma, soft tissue clear cell sarcoma, dermatofibrosarcoma, meningiomas, NF2, acoustic neuromas) and several developmental disorders (DiGeorge syndrome, CATCH22, VCF syndrome, cat eye syndrome).

The majority of the known chromosome 22-specific markers are assigned to the more proximal part of the long arm, enabling the establishment of high-coverage physical maps. However, due to low marker density, the distal portion remains less characterized, leaving the largest gap in 22q13. Terminal deletions in several clinical cases were reported, a critical region for the del(22)(q13.3) syndrome has been defined and cases of mental retardation were linked to subtelomeric regions on chromosome 22. Therefore, our major focus is to identify and clone new markers from chromosome 22 (Muellenbach et al. 1994 ) and to characterize and map clones from chromosome 22-specific libraries (ICRF, Lawrence Livermore) (Blin et al. 1993, Cancer Genet Cytogenet 70: 108-111). Isolating cosmids containing CA-repeats for STS mapping and DNA sequencing of an individual clone resulted in the precise mapping of new transcription units (e.g. RNA-polymerase II subunit) to chromosome 22q13.1 (Pusch et al. 1996a and 1996b ).


An integrated physical BAC-map of chromosomal region 22q13.1-qter


The construction of integrated physical and genetic linkage maps is of basic interest in the ongoing effort to map and sequence the entire human genome. Contiguously overlapping sets of genomic clones spanning long regions of the genome that are defined by anchor points on a genetic linkage map will substatially facilitate the localization and identification of new disease genes and thus allow focused studies of gene expression on a molecular level. High-density physical maps relate the information from genetic linkage analysis to detailed molecular characterization of genes, transcripts and chromosome structure and provide genomic DNA templates for sequencing individual chromosomes.

Long-range physical mapping can most efficiently be established using cloning systems capable of carrying very large fragments of exogenous DNA in a single clone. For our studies we decided to use the Bacterial Artificial Chromosome (BAC) system developed by Shizuya et al. (1992, PNAS USA 89: 8794-8797), which provides several advantages over established cloning vectors like cosmids or Yeast Artificial Chromsomes (YACs). High stability, minimal chimerism and ease of purification of large inserts (> 300 kb) characterize the BAC vector system as a suitable source of intact DNA fragments for constructing large-scaled detailed physical maps of genomic regions. Recently, in collaboration with M. Simon and H. Shizuya, CalTech, Pasadena, CA, U.S.A. we constructed an integrated physical map of the distal half of the long arm of chromosome 22, which provides the backbone for complete physical coverage and the genomic DNA material for reliable sequencing of this particular genomic area (Schmitt et al. 1996b ). Presently, we are filling the remaining gaps to achieve contiguous physical coverage of the distal area of chromosome 22 all the way down to the 22q telomere.

In another project the usefulness of the BAC-system for structural and functional genome analysis was further demonstrated by mapping the third trefoil peptide gene (hITF/TFF3) to 21q22.3 (Schmitt et al. 1996a ) and the detailed structural analysis of this gene cluster (Beck et al. 1996 , Goett et al. 1996 ).


Heterochromatin-euchromatin-borderlines of the human genome


We are also interested in the organization, evolution and function of repetitive DNA sequences within the human genome. The highly condensed heterochromatic areas defining the pericentric regions of eukaryotic chromosomes consist of tandem arrays of several types of repetitive DNA families including satellite DNAs. Some or all of them may be involved in the function of the centromere as a major element of chromosome segregation. We identified a new repetitive element (48 bp repeat, D22Z3/phom48) from the pericentric region of chromosome 22 with further members of this repeat family on other acrocentric chromosomes. To gain more information on sequence composition of chromosome 22 heterochromatin we analysed the spacing of several of its repetitive DNA units (Muellenbach et al. 1996 ).

For further structural analysis of heterochromatic DNA sequences we constructed BAC contigs in centromeric areas, which are characterized by the presence of various types of repetitive elements. With sets of overlapping BAC clones spanning pericentric and short arm regions of chromosome 22 (CenBACs) we are examining the cluster distribution of various repeat elements and are attempting to define the borderlines between heterochromatic and euchromatic chromosomal areas. DNA sequencing of individual clones gave us more information on the nature of repetitive clusters and revealed the presence of single copy sequences within stretches of repetitive DNA.

Screening the total human BAC library with telomeric and subtelomeric repeat sequences resulted in the isolation of clones representing the very ends of various chromosomes (TeloBACs) . FISH experiments showed a chromosome- (or chromosome-subgroup-)specifity for most of the TeloBACs. Our goal is to establish telomere-specific markers, which will provide excellent DNA material for the analysis of chromosomal rearrangements in telomeric areas by FISH and also for the sequencing of individual chromosome ends.


Molecular and cytogenetic analysis of chromosomal aberrations


Our set of well mapped cosmid and BAC clones from different genomic locations in combination with chromosome-specific centromeric repeats and painting probes has proven to be very helpful for cytogenetic studies of various types of chromosomal aberrations by fluorescence in situ hybridization (FISH). Several clinical cases displaying ring formations, translocated or deleted chromosomal fragments have been analysed on a molecular level supporting clinical diagnosis (Schmitt et al. 1994 , Schmitt et al. 1997 , Schroeder et al. 1997 ), and in addition, PCR analysis of deletions and point mutations was performed (Wundrack et al. 1994 and 1996 ).


Transcription mapping


Presently, we are screening our genetically localized BAC contigs from chromosomes 21 and 22 and several telomeres for the presence of new, transcribed sequences by exon trapping. Preliminary results indicate new transcriptional units in the most proximal and distal BAC clones. In addition, clones from a chromosome 22 hncDNA library (collaboration with E. Meese) are being mapped and sequenced. These data shall contribute to the public cDNA databases and transcription maps.



People involved


For contact link to the staff page Move to staff page


Scientific Cooperations






Back to our Homepage Homepage

Last updated: 23 January 1997

This page is maintained by Holger Schmitt and Jose Carlos Machado
For comments or suggestions please e-mail Mail Holger(holger.schmitt@uni-tuebingen.de)