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In this issue...
DOE '99 Oakland Highlights
In the News
Ethical, Legal, and Social Issues
Web, Other Resources, Publications
Meeting Calendars & Acronyms
Efficient interpretation of the functions of human genes and other DNA sequences requires that resources and strategies be developed to enable large-scale investigations across genomes. Goals include studies into genome expression and control, creation of mutations that cause loss or alteration of function in such nonhuman organisms as the mouse, and development of experimental and computational methods for protein analyses. Some highlights of HGP functional genomics projects follow.
Eddy Rubin's group (Lawrence Berkeley National Laboratory, LBNL) uses the laboratory mouse to examine gene function, particularly for mouse genes similar to those found in human genomic regions sequenced at the Joint Genome Institute (JGI). Rubin described use of the Cre Lox system to create mice having several large gene deletions in a 4.5-Mb stretch of mouse chromosome 11 syntenic with human chromosome 5q31. The deleted region contains nine genes of unknown function. The majority of mice homozygous for the deletion exhibited triglyceride levels about tenfold greater than control animals and died prematurely. These deletion mice may prove useful in studying triglyceride metabolism, which is important for understanding atherosclerosis in humans. Insertion of a human YAC containing about 120 kb of the deleted region successfully corrected the high-triglyceride condition and produced mice having normal lifespans. Three candidate genes are present in the YAC, including one with homology to a liver-specific, transporter-like protein previously characterized in the rat. Rubin's group also is investigating the functions of evolutionarily conserved noncoding sequences in the human 5q31 region. This region is biologically interesting because it carries a family of cytokine genes, which are important regulators of the immune response. Rubin's data suggests that a 400-bp conserved element in this region is involved in regulating the expression of the human (interleukin) IL4 and IL13 genes.
High-Throughput Mouse Mutagenesis, Phenotyping
Eugene Rinchik (Oak Ridge National Laboratory) discussed progress in creating a large mouse-mutation resource for function studies. The strategy, based on recovered recessive phenotypes, involves chemical mutagenesis of specific chromosomal regions, broad-based phenotype screening (in house and through a statewide consortium), and correlation of specific DNA sequences with phenotypes.
The group has been characterizing regions of mouse chromosome 7 while recovering recessive single-gene mutations induced by the powerful mutagen N-ethyl-N-nitrosourea (ENU) and mapped by two-generation hemizygosity screens with radiation-induced deletions. New work involves three-generation homozygosity strategies to induce mutations in proximal chromosome 7 (human 19q homology), mid-chromosome 7 (human 15q homology), and mid-to-distal chromosome 15 (human 8q, 22q, and 12q homologies). The emphasis is on developing genetic reagents that enable any mutation to be maintained and used by a wide variety of investigators without the need for molecular genotyping.
Rinchik also described the high-throughput goals of the Tennessee Mouse Genome Consortium (TMGC; http://tnmouse.org). TMGC combines ORNL's resources and experience in mouse genetics with academic and clinical expertise across the state to induce and detect mouse-gene mutations. The goal is to create human-disease models and perform gene-function studies.
Mouse-Human Comparative Sequence Analysis
Lisa Stubbs' team (JGI-LLNL) is comparing human chromosome 19 sequence (19q13.4), generated by the JGI, with a region of mouse chromosome 7 containing similar genes. The goal is to annotate the human sequence with information on gene function extrapolated from mouse genetics and biology.
The group is focusing on imprinted genes in these areas to find regulatory elements that control imprinting processes in humans and mice. Imprinted genes, which tend to be clustered and may share regulatory regions, are expressed differently depending on which parent contributed the allele. Stubbs reported identification of Zim1, a new maternally expressed gene located in both species next to the paternally expressed Peg3 gene. About 100 human genes are thought to be imprinted, and 10 or more new imprinted genes may be located in the 19q13.4 mutation region.
Phage Display Identification of Target Protein
A poster presentation by Andrew Bradbury's team (Los Alamos National Laboratory) described the use of phage display in functional genomics. Phage display offers the possibility of selecting single-chain antibodies (and the genes encoding them) from libraries of 1012 or more different polypeptides on the basis of their abilities to bind target proteins. This technology will enable derivation of ligands that recognize protein products of all human genes, and the ligands will be used to characterize proteins and protein complexes.
Ribozyme Inactivation of Target Gene Expression
Another method of determining gene function is to begin with an interesting cellular function and work back to the gene. Jack Barber's team at Immusol, Inc., has taken this approach using ribozymes (RZs), which are RNA molecules that can be engineered to cleave and inactivate other RNA molecules in a sequence-specific fashion. RZs can be designed to selectively inactivate the expression of any target gene ("gene knockdown") and therefore its corresponding protein. Immusol has developed a combinatorial library of RZ genes, delivered in viral vectors, to use as probes for finding and then cloning genes. The RZ gene library is delivered into large numbers of tissue culture cells (one RZ gene per cell for each RZ gene in the library), followed by selection for individual cells that have lost a particular function. Barber reported cloning the first gene, a tumor suppressor, with no prior sequence information. The group now is identifying genes involved in regulating cancer gene expression and hepatitis C virus replication.
One Gene But Many Proteins
Complicating the study of gene function is the fact that multiple proteins can arise from a single gene. This can be the result of alternate splicing and editing of the mRNA, post-translational protein modification, or alternative translation. Nonstandard translation involves "recoding," in which ribosomes change the coding of an mRNA by frame shifting, changing a codon's meaning, or skipping over some message regions instead of translating them all. Ray Gesteland (University of Utah) described a pilot project using electrospray liquid chromatography mass spectrometry (MS) to catalog the number of each mRNA species' protein products from the 500 genes in yeast mitochondria. The project's ultimate goal is to develop ways to monitor all proteins expressed during physiological events such as development.
Richard Smith (Pacific Northwest National Laboratory, PNNL) further described how the complexities of the proteome--all the proteins expressed by an organism-- cannot be accounted for by DNA sequences alone. He pointed out that, in contrast to an organism's virtually static and well-defined genome, the proteome continually changes in response to external and internal events.
Smith's group is exploring ways to characterize the proteome directly by combining the speed of capillary isoelectric focusing electrophoresis, which separates proteins by charge, and the mass accuracy and sensitivity obtainable with Fourier transform ion cyclotron resonance MS. He described its initial application to several completely sequenced prokaryotes including E. coli, expressing confidence in the technique's applicability to larger genomes.
The electronic form of the newsletter may be cited in the following style:
Human Genome Program, U.S. Department of Energy, Human Genome News (v10n3-4).
The Human Genome Project (HGP) was an international 13-year effort, 1990 to 2003. Primary goals were to discover the complete set of human genes and make them accessible for further biological study, and determine the complete sequence of DNA bases in the human genome. See Timeline for more HGP history.