Sponsored by the U.S. Department of Energy Human Genome Program
Human Genome News Archive Edition
Human Genome News, November 1992; 4(4)
Data Published on Chromosomes Y and 21, Leukemia Gene, Genetic Linkage Map
International progress in physical and genetic mapping has recently been reported as the U.S. Human Genome Project moves into its third year:
In two articles appearing in the October 2 issue of Science, David Page and his colleagues at the Whitehead Institute and Massachusetts Institute of Technology presented their physical map of the entire functional (euchromatic) portion of the human Y or "male" chromosome, which at 60 Mb is one of the smallest. The new map, consisting of 196 overlapping YAC clones, is expected to facilitate positional cloning of genes for a given phenotype, speed gene identification, and provide material for large-scale sequencing. The work was supported by the NIH National Center for Human Genome Research (NCHGR) to further the interim (5-year) Human Genome Project goal of constructing physical maps of all human chromosomes with markers spaced at 100-kb intervals (see Physical and Genetic Maps).
The euchromatic region includes the short arm, centromere, and proximal long arm. It contains all known genes on the Y chromosome and appears relatively constant in size. By contrast, the heterochromatic region has no known function; consists of short, repetitive sequences; and varies tremendously among males (undetectable in some and twice the size of the euchromatic region in others).
Because most of the Y chromosome does not undergo meiotic recombination, a genetic linkage map is precluded, and Y-linked gene identification has been based on physical mapping of naturally occurring deletions. Deletion mapping, in which DNA loci are ordered along the chromosome, is a practical approach because Y chromosome deletions occur fairly frequently in the population.
In the first of two Science articles,(1) Page and colleagues described deletion mapping based on sequence tagged site (STS) detection; an STS is a short DNA sequence that can be detected by the polymerase chain reaction (PCR)-a fast, sensitive assay. An STS can be mapped to a specific point that becomes a chromosomal landmark easily disseminated through publication in journals and computer databases.
The researchers assembled a set of STS probes to screen DNA from 96 patients who had either visible Y chromosome abnormalities or sex chromosome constitutions (XX males and XY females) that did not correlate with their outward sexual appearance. To establish the relative STS order, investigators compared sets of positive points (indicating the presence of an STS) and negative points for each patient.
The deletion map is expected to be useful in identifying Y chromosomal genes, studying the origin of chromosomal disorders, and tracing the evolution of the Y chromosome.
In their second Science article,(2) the researchers describe how ordered STS probes, generated during deletion mapping, provided the framework for rapid construction of the chromosomal physical map. Investigators began by preparing a library of YAC clones from the genomic DNA of a human XYYYY male to obtain a Y chromosome representation fourfold greater than could have been achieved with a comparably sized library for an XY male.
Use of a total human YAC library eliminated the labor-intensive step of constructing chromosome-specific YAC libraries. The YAC library, containing 10,368 clones with an average insert size of 650 kb, was then analyzed by "STS-content mapping" and screened for landmarks using 160 deletion-mapped STSs. YAC clones were ordered by determining overlap (common STSs). Based on their presence or absence in the YACs, 207 Y-DNA probes were assigned to 127 ordered intervals with an average spacing of about 220 kb. The authors estimate that the physical map spans at least 28 Mb; previous estimates based on cytological observations were in the 30- to 40-Mb range.
This mapping strategy enabled investigators to isolate specific YACs from a complex library, order and arrange them in larger units to form "contigs," and place the STSs along the chromosome. These ordered points on the physical map will provide landmarks for all future investigations of the Y chromosome, the authors stated. They also noted that this method can be applied to other human chromosomes if large numbers of STSs can be generated for them.
In the October 1 issue of Nature,(3) French researcher Daniel Cohen of Genethon and 35 others from 12 institutions [including American researchers Stylianos Antonarakis (Johns Hopkins School of Medicine); David Cox (University of California, San Francisco); Harold Riethman (Wistar Institute); and Katheleen Gardiner and David Patterson (Eleanor Roosevelt Institute)] report a complete physical map of the long arm of human chromosome 21. The smallest and one of the best studied of the human chromosomes, chromosome 21 has been associated with several genetic diseases, including Down's syndrome, some forms of Alzheimer's disease, amyotrophic lateral sclerosis (Lou Gehrig's disease), and progressive myoclonus epilepsy. The new physical clone map will speed identification of the chromosomal regions involved in these and other diseases after the regions are further localized by genetic linkage or cytogenetic analysis.
The map, containing 191 STSs spaced at about 220 kb, was constructed from human YAC libraries having insert sizes of 400 kb to 2 Mb. The new map contains an average of five clones in each interval between two STSs, an indication of the "robustness" of the contig.
The work was conducted at laboratories in France, the United States, Spain, and Japan. It was supported by the French Ministry of Research and Technology and the Association Francaise Contre les Myopathies through the Genethon program and by grants from NCHGR and other NIH institutes.
Researchers constructing a human chromosome 11 physical map consisting of ordered DNA clones have determined the base-pair sequence of fragments spanning the chromosomal region 11q23. The area is frequently broken in patients with some forms of childhood leukemia; this breakage may affect a gene that normally plays a role in regulating cell division in white blood cells. Leukemias arise when immature white blood cells become abnormal and grow and divide rapidly.
This work, supported by NCHGR and DOE, was reported in the October issue of Nature Genetics (4) by Glen Evans and his coworkers at the Salk Institute in La Jolla, California, and Mark Bower and Bryan Young at the Imperial Cancer Research Fund in the United Kingdom. Evans and his collaborators studied chromosomes of patients with acute leukemias, which account for almost all leukemia cases among children and young adults.
Investigators compared the sequence of the 11q23 fragment to others stored in computer databases and found it to be similar to that of the fruit fly gene trithorax (trx), which instructs cells to produce a protein having a "zinc finger" region (so called because it loops around a molecule of zinc). The trx protein regulates the transcription of developmental genes in the fruit fly.
The findings suggest that chromosome 11 breakage during translocation disrupts the function of the human trx-like gene and makes it unable to produce a normal protein.
The genetic linkage map of the entire human genome, which appears in the October 2 issue of Science,(5) results from the efforts of researchers at more than 70 laboratories worldwide; the work was sponsored largely by NCHGR and the Centre d'Etude du Polymorphisme Humain. Genetic mapping data were assembled into a single publication with chromosomes represented in a common format. Collated by Washington University (St. Louis) geneticist Helen Donis-Keller and her staff, the linkage map contains restriction fragment length polymorphism (RFLP) and microsatellite DNA markers.
According to the Science article, the major difference between a previously published genome map and the new one lies in the number and type of markers used. This map incorporates over 4 times more markers, some 300 of which are variable repeats of short (2 to 4 bp) DNA units called microsatellites.
The first human genetic linkage maps were based on protein polymorphisms (assayed by a variety of methods including serologic testing, gel electrophoresis, and enzymatic assay). However, because such markers were scarce, genetic mapping lagged before the discovery that restriction enzymes could be used to reveal DNA sequence variation among individuals (polymorphisms) and that the amount of variation was sufficient for construction of a rudimentary linkage map spanning much of the genome.
Although RFLPs have contributed greatly to advances in genetic mapping, most are not usefully informative in the small-pedigree resources typically available for disease-gene mapping. Furthermore, the best RFLPs, variable number tandem repeats (VNTRs), have limited value in disease-gene hunts because they cluster near the ends of chromosomes. RFLPs also suffer the disadvantage of assay by Southern hybridization, now considered a tedious technology that could not be scaled efficiently to high-production levels.
More recently, researchers discovered that microsatellites are abundant, with an estimated 500,000 well distributed across the human genome. Microsatellites are particularly useful for developing markers at gene sequences because a 1-Mb gene-containing segment may have several of these repeat elements along its length. In contrast, testing of candidate genes in linkage studies is severely limited because of the rarity of RFLPs near or within gene sequences.
In addition, microsatellites can be assayed by PCR, making them easily accessible from sequences published or posted in databases and quickly available to the scientific community. Collection and distribution of probes from public repositories often led to long delays, sometimes up to years. Because of their advantages, microsatellites have already become the dominant marker type. The next objective is a genome map of markers that are informative in at least 70% of the population and spaced at intervals of about 10 cM; this map is expected to consist entirely of microsatellites. The original goal of constructing a genetic linkage map with markers spaced an average of 2 to 5 cM apart now appears easily surpassable.
Because microsatellite markers are STSs, they can be used as anchors to connect the genetic linkage maps with physical maps of DNA clones. Parent clones from which the microsatellites derive will serve as probes for cytogenetic mapping. With the addition of more microsatellite markers to the genetic map, information from linkage and physical maps will converge, the Science article states. "It is anticipated that full integration of genetic, cytogenetic, and physical mapping information will be possible, thereby providing a new view of the [human] genome upon which to base future biological studies," the authors conclude.
For additional background information, see also "A Genetic Linkage Map of the Human Genome," Cell 51, 319-37 (October 23, 1987).
A physical map represents actual locations (as compared with relative locations on a genetic map) of identifiable landmarks such as STSs, restriction enzyme cutting sites, DNA repeats, and genes. Distance is measured in base pairs. Different types of physical maps vary in their resolution (level of detail), the coarsest being a map of the banding patterns on stained human chromosomes visible through a light microscope (a cytogenetic map). The contig map provides more detail and depicts the order of overlapping DNA fragments. The new maps for chromosomes Y and 21 described in the accompanying article are contig maps that contain overlapping clones spanning entire chromosomal regions.
Complete physical maps spanning the entire genome will facilitate the correlation of genetic and cytogenetic maps with the underlying DNA, offer access to clones from any region of interest, and provide material for large-scale sequencing. Physical maps for the genomes of several model organisms (such as the bacterium Escherichia coli, the yeast Saccharomyces cerevisiae, and the nematode Caenorhabditis elegans) have been completed or are nearly complete.
By correlating the inheritance of DNA markers with the appearance of biological traits in large numbers of related people, scientists can identify the chromosome on which a gene resides. Markers - DNA properties that differ among individuals and are easily identified in the laboratory - serve as signposts along a chromosome and can be genes or segments that have no known coding function but whose inheritance pattern can be followed. Such markers include the DNA microsatellite repeats described in the accompanying article.
Genetic linkage maps, which represent the relative chromosomal order and spacing of DNA markers according to their inheritance patterns, are made by determining how frequently two markers are passed together from parent to child. Closely linked markers are less likely to be separated during normal processes in gamete formation. Distances are measured in centimorgans (cM); two markers are said to be 1 cM apart if they are separated by recombination 1% of the time in humans. A genetic distance of 1 cM is roughly equal to a physical distance of 1 million base pairs (1 Mb).
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Human Genome Program, U.S. Department of Energy, Human Genome News (v4n4).
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.