Center for Human Genome
Los Alamos National
P.O. Box 1663
Los Alamos, NM
Larry L. Deaven
*Now at University of California,
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
In 1997 Lawrence Berkeley National
Laboratory, Lawrence Livermore National Laboratory, and Los Alamos National
Laboratory began collaborating in a Joint Genome
Institute to implement high-throughput sequencing [see Human
Genome News 8(2), 12].
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.
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.
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 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.
SASE sequencing of 1.5 million bases from the p13 region of human chromosome
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
Sequencing and analysis of a novel pericentromeric duplication of a gene-rich
cluster between 16p11.1 and Xq28 (in collaboration with Baylor College
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.
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
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.
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.
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
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
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.