Center for Human Genome Studies 
Los Alamos National Laboratory 
P.O. Box 1663 
Los Alamos, NM 87545 

Larry L. Deaven 
Acting Director 
505/667-3912, Fax: -2891 
ldeaven@telomere.lanl.gov 

Lynn Clark 
Technical Coordinator 
505/667-9376, Fax: -2891 
clark@telomere.lanl.gov  

Robert K. Moyzis 
Director, 1989­97* 
 

*Now at University of California, Irvine 
  
 
 
 

In lieu of individual abstracts, research projects and investigators at the LANL Center for Human Genome Studies are represented in this narrative. More information can be found on the center's Web site. 
 
 

 

Chromosome 16  Physical Map  Part 1 (80k JPG)

Chromosome 16  Physical Map  Part 2 (79k JPG)

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  

Flow cytometer   (18kb JPG)

 

Biological research was initiated at Los Alamos National Laboratory (LANL) in the 1940s, when the laboratory began to investigate the physiological and genetic consequences of radiation exposure. Eventual establishment of the national genetic sequence databank called GenBank, the National Flow Cytometry Resource, numerous related individual research projects, and fulfillment of a key role in the National Laboratory Gene Library Project all contributed to LANL's selection as the site for the Center for Human Genome Studies in 1988. 

Center Organization and Activities 

The LANL genome center is organized into four broad areas of research and support: Physical Mapping, DNA Sequencing, Technology Development, and Biological Interfaces. Each area consists of a variety of projects, and work is distributed among five LANL Divisions (Life Sciences; Theoretical; Computing, Information, and Communications; Chemical Science and Technology; and Engineering Sciences and Applications). Extensive interdisciplinary interactions are encouraged. 

Physical Mapping  
The construction of chromosome- and region-specific cosmid, bacterial artificial chromosome (BAC), and yeast artificial chromosome (YAC) recombinant DNA libraries is a primary focus of physical mapping activities at LANL. Specific work includes the construction of high-resolution maps of human chromosomes 5 and 16 and associated informatics and gene discovery tasks. 

Accomplishments 

• Completion of an integrated physical map (see below, left) of human chromosome16 consisting of both a low-resolution YAC contig map and a high-resolution cosmid contig map. With sequence tagged site (STS) markers provided on average every 125,000 bases, the YAC-STS map provides almost-complete coverage of the chromosome's euchromatic arms. All available loci continue to be incorporated into the map. 
• Construction of a low-resolution STS map of human chromosome 5 consisting of 517 STS markers regionally assigned by somatic-cell hybrid approaches. Around 95% mega-YAC­STS coverage (50million bases) of 5p has been achieved. Additionally, about 40million bases of 5q mega-YAC­STS coverage have been obtained collaboratively. 
• Refinement of BAC cloning procedures for future production of chromosome-specific libraries. Successful partial digestion and cloning of microgram quantities of chromosomal DNA embedded in agarose plugs. Efforts continue to increase the average insert size to about 100,000 bases. 

DNA Sequencing 
DNA sequencing at the LANL center focuses on low-pass sample sequencing (SASE) of large genomic regions. SASE data is deposited in publicly available databases to allow for wide distribution. Finished sequencing is prioritized from initial SASE analysis and pursued by parallel primer walking. Informatics development includes data tracking, gene- discovery integration with the Sequence Comparison ANalysis (SCAN) program, and functional genomics interaction. 

Accomplishments

• SASE sequencing of 1.5 million bases from the p13 region of human chromosome 16.
• Discovery of more than 100 genes in SASE sequences. 
• Generation of finished sequence for a 240,000-base telomeric region of human chromosome7q. From initial sequences generated by SASE, oligonucleotides were synthesized and used for primer walking directly from cosmids comprising the contig map. Complete sequencing was performed to determine what genes, if any, are near the 7q terminus. This intriguing region lacks significant blocks of subtelomeric repeat DNA typically present near eukaryotic telomeres. 
• Complete single-pass sequencing of 2018 exon clones generated from LANL's flow-sorted human chromosome 16 cosmid library. About 950 discrete sequences were identified by sequence analysis. Nearly 800 appear to represent expressed sequences from chromosome 16. 
• Development of Sequence Viewer to display ABI sequences with trace data on any computer having an Internet connection and a Netscape World Wide Web browser. 
• Sequencing and analysis of a novel pericentromeric duplication of a gene-rich cluster between 16p11.1 and Xq28 (in collaboration with Baylor College of Medicine). 

Technology Development  
Technology development encompasses a variety of activities, both short and long term, including novel vectors for library construction and physical mapping; automation and robotics tools for physical mapping and sequencing; novel approaches to DNA sequencing involving single-molecule detection; and novel approaches to informatics tools for gene identification. 

Accomplishments 

• Development of SCAN program for large-scale sequence analysis and annotation, including a translator converting SCAN data to GIO format for submission to Genome Sequence DataBase. 
• Application of flow-cytometric approach to DNA sizing of P1 artificial chromosome (PAC) clones. Less than one picogram of linear or supercoiled DNA is analyzed in under 3 minutes. Sizing range has been extended down to 287 base pairs. Efforts continue to extend the upper limit beyond 167,000 bases. 
• Characterization of the detection of single, fluorescently tagged nucleotides cleaved from multiple DNA fragments suspended in the flow stream of a flow cytometer (see picture at left). The cleavage rate for ExoIII at 37°C was measured to be about 5 base pairs per second per M13 DNA fragment. To achieve a single-color sequencing demonstration, either the background burst rate (currently about 5 bursts per second) must be reduced or the exonuclease cleavage rate must be increased significantly. Techniques to achieve both are being explored. 
• Construction of a simple and compact apparatus, based on a diode-pumped Nd:YAG laser, for routine DNA fragment sizing. 
• Development of a new approach to detect coding sequences in DNA. This complete spectral analysis of coding and noncoding sequences is as sensitive in its first implementations as the best existing techniques. 
• Use of phylogenetic relationships to generate new profiles of amino acid usage in conserved domains. The profiles are particularly useful for classification of distantly related sequences. 

Biological Interfaces  
The Biological Interfaces effort targets genes and chromosome regions associated with DNA damage and repair, mitotic stability, and chromosome structure and function as primary subjects for physical mapping and sequencing. Specific disease-associated genes on human chromosome 5 (e.g., Cri-du-Chat syndrome) and on 16 (e.g., Batten's disease and Fanconi anemia) are the subjects of collaborative biological projects. 

Accomplishments 

• Identification of two human 7q exons having 99% homology to the cDNA of a known human gene, vasoactive intestinal peptide receptor 2A. Preliminary data suggests that the VIPR2A gene is expressed. 
• Identification of numerous expressed sequence tags (ESTs) localized to the 7q region. Since three of the ESTs contain at least two regions with high confidence of homology (~90%), genes in addition to VIPR2A may exist in the terminal region of 7q. 
• Generation of high-resolution cosmid coverage on human chromosome 5p for the larynx and critical regions identified with Cri-du-Chat syndrome, the most common human terminal-deletion syndrome (in collaboration with Thomas Jefferson University). 
• Refinement of the Wolf-Hirschhorn syndrome (WHS) critical region on human chromosome 4p. Using the SCAN program to identify genes likely to contribute to WHS, the project serves as a model for defining the interaction between genomic sequencing and clinical research. 
• Collaborative construction of contigs for human chromosome16, including 1.05 million bases in cosmids through the familial Mediterranean fever (FMF) gene region (with members of the FMF Consortium) and 700,000 bases in P1 clones encompassing the polycystic kidney disease gene (with Integrated Genetics, Inc.). 
• Collaborative identification and determination of the complete genomic structure of the Batten's disease gene (with members of the BDG Consortium), the gamma subunit of the human amiloride-sensitive epithelial channel (Liddle's syndrome, with University of Iowa), and the polycystic kidney disease gene (with Integrated Genetics). 
• Participation in an international collaborative research consortium that successfully identified the gene responsible for Fanconi anemia type A.

Patents, Licenses, and CRADAs 
• Rhett L. Affleck, James N. Demas, Peter M. Goodwin, Jay A. Schecker, Ming Wu, and Richard A. Keller, "Reduction of Diffusional Defocusing in Hydrodynamically Focused Flows by Complexing with a High Molecular Weight Adduct," United States Patent, filed December 1996. 
• R.L. Affleck, W.P. Ambrose, J.D. Demas, P.M. Goodwin, M.E. Johnson, R.A. Keller, J.T. Petty, J.A. Schecker, and M.Wu, "Photobleaching to Reduce or Eliminate Luminescent Impurities for Ultrasensitive Luminescence Analysis," United States Patent, S-87, 208, accepted September 1997. 
• J.H. Jett, M.L. Hammond, R.A. Keller, B.L. Marrone, and J.C. Martin, "DNA Fragment Sizing and Sorting by Laser-Induced Fluorescence," United States Patent, S.N.75,001, allowed  May 1996. 
• James H. Jett, "Method for Rapid Base Sequencing in DNA and RNA with Three Base Labeling," in preparation. 
• Development license and exclusive license to LANL's DNA sizing patent obtained by Molecular Technology, Inc., for commercialization of single-molecule detection capability to DNA sizing. 

Future Plans 

LANL has joined a collaboration with California Institute of Technology and The Institute for Genomic Research to construct a BAC map of the p arm of human chromosome 16 and to complete the sequence of a 20-million­base region of this map.  In its evolving role as part of the new DOE Joint Genome Institute, LANL will continue scaleup activities focused on high-throughput DNA sequencing. Initial targets include genes and DNA regions associated with chromosome structure and function, syntenic break-points, and relevant disease-gene loci. 

A joint DNA sequencing center was established recently by LANL at the University of New Mexico. This facility is responsible for determining the DNA sequence of clones constructed at LANL, then returning the data to LANL for analysis and archiving.