Chromosome walking and jumping
Chromosome walking and jumping are genetic mapping techniques that were historically employed to locate defective genes associated with hereditary diseases. These methods gained prominence in the late 1960s when researchers sought to identify and manipulate DNA segments within the human genome. Chromosome walking involves selecting a cloned DNA fragment near the target gene and repeatedly subcloning it to progressively screen genomic libraries for closer fragments. This process, while effective, can be slow, particularly in regions with repetitive DNA sequences. In contrast, chromosome jumping allows researchers to traverse larger segments of DNA more quickly by joining the ends of large DNA segments to form a circular structure, enabling them to bypass difficult areas and jump significant distances along the chromosome.
These techniques significantly contributed to the discovery of several disease-causing genes, including those linked to cystic fibrosis and Huntington's disease. However, the completion of the Human Genome Project in 2003 has largely rendered these methods obsolete for human gene mapping, although they continue to be useful in studying genes in other organisms, such as agricultural plants. Additionally, the ethical implications of identifying disease-causing mutations underscore the complexities involved in genetic testing and reproductive choices. Overall, chromosome walking and jumping have played crucial roles in advancing our understanding of genetics and developing potential treatments for various hereditary conditions.
Subject Terms
Chromosome walking and jumping
SIGNIFICANCE: Chromosome walking and jumping were once used as mapping methods to find defective genes that cause hereditary diseases. Although these techniques have been rendered obsolete by the completion of the Human Genome Project, they have assisted in curing diseases, seeking preventive measures, and detecting genetic carriers.
Gene Hunting
The science of molecular genetics began in the early 1950s when Alfred Hershey and Martha Chase conducted a series of experiments that proved that DNA did indeed carry life’s hereditary information. This discovery was soon followed by James Watson and Francis Crick’s determination that the structure of DNA was that of a double helix that could “unzip” and make copies of itself. By the late 1960s, researchers began to actively seek the knowledge to identify, isolate, and manipulate certain sections of DNA within the human genome.
Around this time, several geneticists autonomously recognized the possibilities of and jumping to locate genes. Hans Lehrach suggested such techniques at the European Molecular Biology Laboratory, and Sherman Weissman proposed similar methods at Yale University. Weissman’s student Francis S. Collins elaborated his mentor’s chromosome-jumping concepts. Interested in identifying disease-causing genes, Collins sought to examine sizable areas of genetic material for unknown genes believed to be responsible for triggering erratic biochemical behavior. As a result of Collins’s work, investigators began to adopt the chromosome-jumping procedure as a reliable, efficient molecular biology tool. This novel exploratory method enabled researchers to span chromosomes expeditiously and bypass repetitive or insignificant genetic information. Based on Collins’s chromosome-jumping technology, gene searching became less time-consuming and resulted in the identification of defective genes that code for abnormal proteins and cause such diseases as cystic fibrosis. Understanding the nature of such mutations makes the development of treatments and cures more likely and can lead to the ability to detect the presence of the mutated gene in carriers.
Procedure
Geneticists initiate chromosome walking and jumping by collecting genetic samples from people who have a specific disease and from their close relatives. For walking, researchers select a cloned DNA fragment from a that contains the marker closest to the gene being sought. A small part of the cloned DNA fragment that is on the end nearest the gene being sought is subcloned. The subcloned fragment is then used to screen the genomic library for a clone with a fragment closer to the gene. Then a small part of this new cloned fragment is subcloned to be used to screen for the next closer fragment. This series of steps is repeated as many times as needed, until a fragment is found that appears to contain a gene. This fragment is carefully analyzed, and if it does contain the gene of interest, the process is halted; if not, chromosome walking is continued. Chromosome walking is slow, and repetitive DNA sequences or regions that do not appear in the library can halt the process.
Another method used to maneuver to genes more quickly and to bypass troublesome regions of DNA that cannot be easily mapped by chromosome walking, such as those containing repetitive DNA, is chromosome jumping. Using chromosome jumping, researchers can travel the same distance they can using chromosome walking but they are able to advance farther along the chromosome in less time because this method uses much larger fragments. Chromosome jumping is achieved by selecting a large DNA segment from the area where geneticists believe the desired gene is located and joining the ends to form a circle. This moves DNA sequences together that naturally would occur at distances of several kilobases. Researchers cut out and clone these junctions into a phage vector, and the various junction segments are then used to form libraries. Researchers then use probes from the DNA sample to seek clones with matching start and end sequences and jump along the chromosome. After each jump, bidirectional walking is often done in the new region. A combination of and walking can be done until the gene is found.
Gene Discovery
Collaborating with Lap-Chee Tsui and researchers at Toronto’s Hospital for Sick Children, Collins examined DNA from patients suffering from cystic fibrosis. Tsui realized that the CFTR gene was located on chromosome 7. Since that chromosome consists of 150 million DNA base pairs, chromosome walking toward the CFTR gene would be a very slow process that would take approximately eighteen years to complete. After Tsui contacted Collins, his colleague at the University of Michigan, the two researchers devised a technique for jumping along the chromosome. Tsui and Collins estimated that jumping along the chromosome would be five to ten times faster than walking because it would allow researchers to cover 100,000 to 200,000 DNA bases at one time. In addition, areas on the chromosome that might otherwise be difficult to cross could simply be jumped over. Using markers Tsui made from chromosome 7 library fragments, they applied the chromosome jumping technique and scanned the genetic material to target where they should use chromosome walking to find the CFTR gene.
They discovered the CFTR gene in 1989. Analysis revealed that the mutation is a deletion of DNA base pairs. This gene codes the transmembrane conductance regulator protein. Tsui determined that the shape of the protein and how it functions are affected by the mutated gene’s coding. The abnormal cystic fibrosis transmembrane conductance regulator protein is unable to create a release channel to remove chloride and sodium from cells. Mucus builds up, adhering to lungs and organs, and bacteria proliferate. Cystic fibrosis is the most frequent fatal hereditary disease in Caucasians. Geneticists estimate that one in twenty-nine white Americans has a recessive CFTR gene, and one in three thousand white babies are born with cystic fibrosis. Internationally, researchers associated with Tsui’s Toronto-based consortium continue to study DNA fragments for additional CF gene mutations and have detected at least one thousand distinct mutations.
Impact
Chromosome walking and jumping have been utilized to find other disease-causing genes. Collins and his team identified the tumor-producing neurofibromatosis gene in 1990. Three years later, they located the gene for Huntington’s disease (Huntington’s chorea), an extreme neurological disorder. This method also detected the location on the X chromosome of the choroideremia gene, which causes gradual blindness, mostly in males, as the retina and choroid coat degenerate. Investigating Duchenne muscular dystrophy, Louis Kunkel at the Harvard Medical School used chromosome walking to detect the absence of a gene on the X chromosome that codes the dystrophin protein for muscles. Not all genes found by these methods are linked to diseases. Andrew Sinclair and his team in London applied chromosome walking to seek the gene that signals development of testes in many embryonic mammals. Although these techniques are useful, they raise ethical concerns. As genes with disease-causing mutations are identified, people can undergo testing to determine whether they carry the mutations. This information can affect reproductive choices, particularly if both partners have a recessive allele for a potentially lethal disease. Fetal material can be genetically analyzed, resulting in complex decisions to continue or terminate a pregnancy if the fetus has the mutation.
Once the mapping of the human genome was completed, however, geneticists arrived at a time when they no longer needed to depend on chromosome walking and jumping as tools to seek human genes. Investigators continue to use walking and jumping, however, to locate genes of other organisms, particularly such agricultural plants as rice and wheat. The Human Genome Project, an international, collaborative scientific research program with primary end goals of identifying, mapping, and understanding the entire human genome, was completed in April, 2003, fifty years after the discovery of the double helix structure. Due to the development of improved technologies for accelerating the elucidation of the genome, this project was completed years earlier than what was originally anticipated. Approximately 25,000 human genes have been identified and mapped through the Human Genome Project. This project has provided the entire world with a resource of information that has revolutionalized the field of medicine and biological research.
The completion of the has significantly eased the task of locating and analyzing the mechanism of actions of genes involved in complex diseases. Understanding the molecular basis of a disease can ultimately lead to new ways to diagnose and treat patients. The ability to understand the pathophysiology of a disease on a molecular level has led to the development of more specific and effective drug treatments. More recent developments in novel detection methods have led to significant advances in the ability to provide more rapid, efficient, and less expensive methods of DNA sequencing.
Key terms
- genomic librarya group of cloned DNA fragments representative of an organism’s genome
- kilobase pairs (kb)a measurement of 1,000 base pairs in DNA
- markera unique DNA sequence with a known location with respect to other markers or genes
- repetitive DNAnucleotide sequences, usually noncoding, that are present in many copies in a eukaryotic genome
Bibliography
Adams, Jill. “Sequencing Human Genome: The Contributions of Francis Collins and Craig Venter.” Nature Education 1.1 (2008). Print.
Caccabelli, Tomeo. Human Genome: Components, Structural/Functional Disorders & Ethical Issues. New York: Nova Science, 2013. Print.
Fanen, Pascale, et al. "Genetics of Cystic Fibrosis: CFTR Mutation Classifications Toward Genotype-Based CF Therapies." Intl. Journ. of Biochemistry & Cell Biology 52 (2014): 94–102. Print.
Gelehrter, Thomas D., Francis S. Collins, and David Ginsburg. Principles of Medical Genetics. 2d ed. Baltimore: Williams, 1998. Print.
Ishtiaq, Muhammad, Mahnoor Muzammil, and Mehwish Maqbool. "Chromosome Jumping." In Genetic Engineering: Volume 1: Principles, Mechanism, and Expression. Tariq Ahmad Bhat and Jameel M. Al-Khayri, eds. Apple Academic P, 2023.
Liu, Dongyou. "Miscellaneous Applications: Forward and Reverse Genetics." In Handbook of Molecular Biotechnology. Dongyou Liu, ed. CRC Press, 2024.
Metzker, M. L. “Emerging Techniques in DNA Sequencing.” Genome Research 15 (2005): 1767–76. Print.
Rommens, Johanna M., Michael C. Iannuzzi, et al. “Identification of the Cystic Fibrosis Gene: Chromosome Walking and Jumping.” Science 8 Sept. 1989: 1059–65. Print.
Tropp, Burton E. Molecular Biology: Genes to Proteins. 4th ed. Sudbury: Jones & Bartlett Learning, 2012. Print.
Tsui, Lap-Chee, et al., eds. The Identification of the CF (Cystic Fibrosis) Gene: Recent Progress and New Research Strategies. New York: Plenum, 1991. Print.