Sponsored by the U.S. Department of Energy Human Genome Program
Human Genome News Archive Edition
Human Genome News, May 1990; 2(1)
Larry L. Deaven, Life Sciences Division, Los Alamos National Laboratory
Marvin A. Van Dilla, Biomedical Sciences Division, Lawrence Livermore National Laboratory
The National Laboratory Gene Library Project (NLGLP), a coordinated research effort between Lawrence Livermore National Laboratory (LLNL) and Los Alamos National Laboratory (LANL), was formed in 1983 as a result of the development of flow cytogenetic analysis and chromosome sorting at the two laboratories. The purpose of NLGLP is to construct libraries to be used in mapping projects. Libraries are unordered DNA fragments for each human chromosome; they are created by digesting the chromosome with a given restriction enzyme.
NLGLP announced in the December issues of Science and Nature that its first group of partial digest libraries is available for testing. Laboratories to receive the libraries for chromosomes 11, 21, 22, and Y (LLNL) and 4, 5, 8, and 17 (LANL) will be selected on the basis of short proposals (proposal forms available from the authors). Proposals will be accepted in late 1990 for initial distribution of the eight additional chromosomes that make up the second group of partial digest libraries; initial distribution of the third group will take place in 1991. The availability of these libraries will be announced in future issues of this newsletter.
At first, libraries were constructed from Chinese hamster chromosomes, but as flow-sorting technology became more refined, highly purified human chromosomes could be obtained in quantities sufficient for library construction. Scientists at LANL and LLNL, believing that a set of libraries would provide useful tools for the restriction fragment length polymorphism (RFLP) approach to gene mapping and for locating human disease loci, requested that a joint project be initiated between the two national laboratories. DOE staff concurred, and NLGLP was formed. The aim of NLGLP is to construct libraries (covering each human chromosome) useful to a broad segment of the scientific community in the study of the molecular biology of genes, gene mapping, and the diagnosis of genetic diseases.
Options for constructing restriction fragment digest libraries include partial and complete digest. Partial digest libraries are made using restriction enzymes at low concentrations on purified chromosomes, so that not all enzyme recognition sites are cleaved. Complete chromosomal digest libraries result when the restriction enzyme is present at sufficiently high concentrations to cleave virtually all recognition sites.
The most urgent need for chromosome-specific libraries in 1983 was for isolating probes to locate genes responsible for human genetic diseases. Because complete digest libraries maximize the probability of finding unique sequence probes, the NLGLP Advisory Committee agreed that the first priority for the project should be the construction of a set of small-insert libraries. The construction of larger-insert, partial digest libraries was to be reconsidered when the small insert libraries were completed.
To maximize chromosomal coverage, two libraries were made for each chromosome, one cut with Eco RI (LANL) and one cut with Hind III (LLNL). Both types of libraries were cloned into Charon 21A (size acceptance range from 0 to 9 kb). The complete digest libraries for the human karyotype were constructed between 1984 and 1986. Initially, these libraries were distributed directly from LANL and LLNL; however, in February 1986 a library repository was established by NIH at the American Type Culture Collection (ATCC) in Rockville, Maryland.
Before the repository was established, approximately 1200 libraries were distributed from the two national laboratories. Since 1986, the number of yearly requests for the libraries has grown from 167 to 816, and more than 2250 complete digest libraries have been distributed.
The establishment of the Human Genome Project made the construction of partial digest libraries even more valuable than originally envisioned, since the larger inserts (up to 40 kb of human DNA in phage or cosmid vectors) provided by these libraries are more useful in physical mapping studies. NLGLP scientists began developing the necessary techniques in 1987, and the first partial digest phage library was completed that year. In 1988 the first cosmid libraries were constructed and put to use in the physical mapping projects at LANL (chromosome 16) and LLNL (chromosome 19). These libraries are useful sources of fragments for assembly of ordered clone maps by DNA fragment fingerprinting methods (Anthony Carrano, Human Genome Quarterly, Vol. 1, No. 2).
Compared to the work on complete digest libraries, the NLGLP strategy for the construction of partial digest libraries has changed in several ways. The karyotype has been divided between the two laboratories: LANL is constructing phage and cosmid libraries for chromosomes 4, 5, 6, 8, 10, 13, 14, 15, 16, 17, 20, and X; LLNL is doing the same for chromosomes 1, 2, 3, 7, 9, 11, 12, 18, 19, 21, 22, and Y.
To construct libraries from single homologs, all chromosomes are being sorted from rodent-human hybrid cells. Both laboratories are currently using the same phage vector (Charon 40); the cosmid libraries from LANL will be cloned into sCos1, and those from LLNL will be cloned into Lawrist 5. If new vectors or host strains with substantially improved properties are developed during the project, attempts will be made to use them for the remaining libraries.
Each library will be examined for purity by plaque or by colony hybridization to total rodent and total human DNA. This information will be compared with purity data derived during chromosome sorting and library construction. If results are consistent and the library appears to be of high quality, it will be distributed to several (six to ten) selected laboratories for further characterization. Phage libraries will be available as aliquots of a single amplification and cosmid libraries as aliquots of a plate amplification or as fivefold coverage arrays in microtiter plates. When data are available from the test laboratories, a decision will be made to determine whether a library should be reconstructed or go into general distribution. In parallel with this work, the laboratories are examining the feasibility of using flow-sorted chromosomes to construct yeast artificial chromosomes (YAC) libraries. Preliminary results suggest that construction of the first YAC libraries may be completed in late 1990.
Scientists in the NLGLP are attempting to construct libraries of the highest possible purity. To a large extent, purity depends on how well a human chromosome is resolved from the rodent background on a flow karyotype. This level of resolution varies considerably from one hybrid cell line to another. There has been excellent cooperation from a number of laboratories in sharing hybrid lines for library construction, and there is now a collection of cell lines that includes all the human chromosomes. However, the search is continuing for more suitable lines for chromosomes 1, 14, 15, 20, and X.
Any reader who has a hybrid containing one or more of these chromosomes and is willing to share it for library construction should contact the authors Larry Deaven at 505/665-3024 or Marvin Van Dilla at 415/422-5662.
(Project strategy planners)
The electronic form of the newsletter may be cited in the following style:
Human Genome Program, U.S. Department of Energy, Human Genome News (v2n1).
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.
Published from 1989 until 2002, this newsletter facilitated HGP communication, helped prevent duplication of research effort, and informed persons interested in genome research.