Background Information

Over a century ago, Gregor Mendel established many of the basic laws of inheritance. In the years following Mendel’s discoveries, his work was confirmed and extended by many geneticists. During this time an increased knowledge about DNA structure, and the events of mitosis and meiosis, led to a greater understanding of the chromosomal basis for inheritance which explained many of Mendel’s hypotheses. For example, Mendel demonstrated that genes on different chromosomes, or unlinked genes, are inherited by independent assortment. However, when following the inheritance of linked genes–genes on the same chromosomegametes are often produced during meiosis that contain chromosomes with different combinations of alleles compared to the parent chromosomes.

One of the key events of meiosis that has a profound effect on genetic inheritance occurs during prophase of meiosis I. In this stage, homologous chromosomes pair together, in a process called synapsis, to form a tetrad. Once paired, a reciprocal exchange or swapping of DNA occurs between segments of DNA on the nonsister chromatids of each homologue. This exchange of DNA is called crossing over or homologous recombination. Crossing over results in the mixing of alleles between the two homologues. After a single crossover event, each chromosome contains DNA obtained from its homologue. Crossing over can produce new combinations of linked alleles that were not present on the parental chromosomes thus crossing over is one of the important cellular events responsible for the tremendous genetic variation in offspring that arise from gamete formation by meiosis and subsequent sexual reproduction. Offspring that arise from recombinant gametes produced by crossing over are called recombinants because they express phenotypes that are different from the parents. Geneticists refer to recombination frequency as a measure of the number of crossover events that occur between two loci on a chromosome. On average, one to four crossover events occur between most homologous chromosomes during meiosis in humans. Unfortunately, crossover events can occasionally occur between nonhomologous chromosomes. These events are called translocations. Translocations are the underlying molecular cause of certain types of human genetic disorders. For example, one type of leukemia called chronic myelogenous leukemia arises from a translocation of DNA between chromosome 21 and chromosome 14.

The position of DNA exchange between nonsister chromatids during crossing over is called the chiasma (pl., chiasmata). Chiasmata can be visualized with an electron microscope as X-shaped configurations where DNA from the homologues overlaps and a complex of enzymes carries out the reactions that result in DNA exchange. Because chiasmata formation and crossing over is, in part, a chance event, the probability of a single crossover event between two loci on a single chromosome is directly proportional to the distance between the two loci. For example, alleles that are far apart on a chromosome are more likely to be involved in crossing over, compared to two closely space loci, because the probability that chiasmata will form between the distant loci is high. Genes that are closer together on the same chromosome will tend to be inherited together because the probability of chiasma formation and crossing over between closely spaced loci is relatively low. Hence, the distance between genes on a chromosome can be estimated by studying the results of genetic linkage studies based on recombination frequencies. Recall that recombinants that result from crossing over between two linked genes can be counted by studying the offspring of a cross between an individual who is a heterozygote for both genes with an individual that is (double) homozygote for both genes. Using recombination frequency values from such a cross, the relative location of the two genes on the same chromosome can be used to develop a genetic map of a chromosome or linkage map, which is an approximate measure of the linear arrangement of loci on a chromosome. Geneticists refer to all of the genes that are connected together on the same chromosome as a linkage group.

In a genetic map, the distance between loci is indicated in map units called centimorgans (cM). One centimorgan is equivalent to a 1% recombination frequency. One centimorgan is equal to approximately 1 megabase (Mb) or one million base pairs of DNA. For two closely spaced loci, maps units are equivalent to the percent of recombinants produced; however, for loci that are far apart, double exchanges of DNA between the two loci (double-crossovers) maintain the allele combinations of the parent effectively reducing the number of recombinants. In this case, map distance is underestimated by comparison of recombination frequencies alone. Map distance must then be measured by multiplying the total number of recombinants by 100 then dividing this value by the total number of offspring.

One of the first geneticists to study recombination, and suggest the use of recombination data as a way to map genes and chromosomes, was Sir Thomas Morgan. In the early 1900s, Morgan was studying inheritance in Drosophila melanogaster when he noticed that white-eyed male flies would occasionally appear in a line of flies with wild-type eye color. Morgan hypothesized that the molecular explanation for genetic linkage was the location of a gene on a chromosome, and that the development of recombinant offspring was the result of crossing over during meiosis. A student of Thomas Morgan’s, Alfred H. Sturtevant, extended Morgan’s observations when he discovered that recombination frequencies between linked genes are additive. For example, when studying recombination frequencies between three linked genes, the location and order of these genes on a chromosome can be arranged in a linear fashion according to the recombination frequency between each locus. The total recombination frequencies between all three loci represents the total length (in centimorgans) occupied by this linkage group.

It is obviously not possible to study the inheritance of linked traits in humans by purposely designing crosses between individuals and carrying out experiments analyzing large numbers of offspring. How then can the inheritance of human genetic disorders be studied? And how can human gene maps and chromosome maps be developed? One technique that is particularly useful for determining the mode of inheritance for a human disorder involves developing a pedigree. A pedigree represents a family tree of genetics. In a pedigree, individuals in a family are shown according to their phenotype for a given trait. When a pedigree is developed, symbols are used to signify each family member. A female is represented as a circle and a male is represented as a square. The symbol for an individual who expresses the phenotype for a certain genetic trait is shaded with a particular color. Parents are connected together by a horizontal line while vertical lines from the parents indicate the offspring produced from these parents. Geneticists refer to offspring as sibs, an abbreviation for siblings. Sibs are connected to each other by a horizontal line (sibship line). Sibs are shown from left to right in the order in which they are born. In some pedigrees the sibs may be numbered.

By carefully studying the appearance of a particular phenotype in several generations of individuals, especially in a large pedigree, it is often possible to determine the mode of inheritance of that trait. For example, it may be possible to establish whether the trait is inherited by autosomal recessive, autosomal dominant, or sex-linked inheritance. Each of the pedigrees in PedigreeLab traces the inheritance of a single trait. There are limitations to studying inheritance by pedigree analysis. Typically pedigrees tend to follow small numbers of individuals and they may often lack information about certain family members. Although a pedigree is a useful technique for evaluating the mode of transmission for a trait, it is not as reliable as carrying out crosses that produce large numbers of offspring. Also, pedigree analysis of phenotypes alone cannot be used to develop chromosome maps.

Relatively recent advances in molecular biology techniques, combined with pedigree analysis, make it possible to map genes to chromosomes with a high degree of accuracy. One important technique that is widely used to identify the location of a gene is called restriction fragment length polymorphism (RFLP) analysis. This technique is based on the fact that specific breaks or cuts in the nucleotide sequence of a piece of DNA can be made using restriction enzymes. Restriction enzymes cut DNA at specific nucleotide sequences, usually from four to six base pairs in length. For example, the restriction enzyme EcoRI recognizes and cuts between the G and A in both strands of DNA containing the sequence GAATTC. A large number of different restriction enzymes with known recognition sequences are readily available commercially. Because, with the exception of identical twins, no two individuals have the exact same nucleotide sequence of DNA in their genome, cutting the DNA from two different individuals with the same enzymes will yield different patterns of variable-length DNA fragments, or RFLPs. An individual’s RFLP pattern represents a unique "DNA fingerprint" due to allelic variations in that person’s genome. These RFLP patterns are inherited by offspring, and individuals can be assigned a genotype depending on whether they are homozygous or heterozygous for restriction enzyme sites on a particular RFLP.

In humans, RFLPs can be used to test for the presence or absence of a particular allele, especially if the nucleotide sequence of the gene in question is known. If an individual contains a mutation that affects the DNA sequence of a restriction site (either by eliminating a site or by creating a new cutting site), then cutting this DNA with a restriction enzyme will produce different restriction fragments compared with DNA containing the wild-type allele. In this technique, chromosomal DNA is isolated from a tissue sample such as skin, hair, or white blood cells. The DNA is then cut into fragments (RFLPs) with one or more restriction enzymes. These fragments are separated by gel electrophoresis. Following electrophoresis, the RFLPs are transferred to a nylon filter that binds the DNA. The filter is then mixed together with a radioactive DNA probe that hybridizes to complementary sequences in RFLPs on the filter. This procedure is called Southern blotting. Fragments bound to the probe are visualized by exposing the Southern blot to X-ray film. Exposing the film to the radioactive probe produces dark bands on the film. The exposed film is called an autoradiogram.

What if the nucleotide sequence for a gene is not known? While RFLP analysis is useful for determining genotypes by searching for the presence or absence of a known allele, RFLP analysis must be combined with other techniques when the nucleotide sequence of a gene is not known. How can geneticists find a gene that is responsible for causing a rare genetic disorder? It is possible to look for linkage between the gene causing the disorder and another gene only if other genes close to the mutant gene have already been discovered. Quite frequently, genes near the mutant gene of interest are not known. In these cases, one way to study linkage to map a mutant gene depends on sequences of chromosomal DNA called markers. A marker is typically a sequence that is known to be located at a specific location on a chromosome. Sometime markers include known genes that have been already been identified, but more commonly, markers are short non-protein-coding sequences of DNA. One very valuable group of human chromosome markers are called microsatellites. Microsatellites are usually tandem repeats of dinucleotides (e.g., CACACA) that are highly repeated from individual to individual. Because they are scattered throughout the genome, microsatellites are excellent markers for determining and mapping how close an unknown mutant gene may be to a microsatellite marker on many different human chromosomes. A marker can be detected by hybridizing short pieces of radioactive DNA, called probes, that are complementary to the nucleotide sequence of the marker.

RFLP analysis using markers has proven to be a very powerful technique for mapping chromosomes. Piecing together overlapping RFLPs and other markers has enabled biologists to develop extensive linkage maps of chromosomes in the human genome. In excess of 5000 markers representing different loci in the genome have been identified and are available for use by scientists who are searching for genes that cause inherited disorders in humans. With so many markers available, radioactive probes for many different markers can be hybridized to RFLP fragments from individuals who have a certain disease. RFLP patterns from many individuals with a disease can be compared with each other and compared against RFLP patterns for normal, healthy individuals to search for markers that are linked to the same chromosome as the defective gene.

Phenotypic information gathered from pedigrees combined with RFLP analysis provides very powerful tools that can be applied by geneticists and molecular biologists who are attempting to identify and locate mutant alleles that cause human diseases. The gene that causes cystic fibrosis was one of the first genes for a human genetic disorder that was mapped using RFLP techniques. Cystic fibrosis is an autosomal recessive disease in which individuals suffer from severe respiratory problems, among other symptoms. The cystic fibrosis gene, located on chromosome 7, codes for a protein that normally functions to pump chloride ions out of many body cells. Once the chromosome containing the cystic fibrosis gene was identified, it was possible to study recombination between the cystic fibrosis gene and linked markers to pinpoint the exact locus of the cystic fibrosis gene. Refer to Figure 1 on page 17 for an example of RFLP patterns generated for an individual who is heterozygous for an autosomal recessive condition.

Statistical analysis can be applied to the recombination data generated by RFLP studies to determine the probability that a marker and trait are linked. Based on crossing over, recombination data between a marker and a trait can be assigned an LOD (log of odds) score, which is a statistical comparison of the probability that RFLP data is due to linked loci and the probability that RFLP data is due to unlinked loci. A standard LOD score of 3.0 has been established as the threshold value for linkage. A LOD of 3.0 is equal to approximately a 20:1 ratio that favors linkage. A LOD value of 3.0 or higher is good evidence that two loci are linked, and the recombination frequency can then be used assign the trait a map unit position on a chromosome. A LOD score of less than 3.0, with a recombination frequency of approximately 50%, indicates a high probability that the trait and probe are unlinked. These techniques have been used to pinpoint the loci for a number of different single genes that cause disease in humans. In addition to cystic fibrosis, loci for Huntington’s disease, Duchenne muscular dystrophy, Alzheimer’s disease, and Parkinson’s disease, among many others, have been identified. These traits are a few of the 22 traits that you can study in PedigreeLab. And pinpointing the location for a rare trait is the ultimate goal that you will strive for using PedigreeLab!

With PedigreeLab you will choose a gene from a list of mutant genes that cause actual genetic disorders in humans. Most of these disorders are relatively rare. To determine the location of the gene for the trait that you are following in PedigreeLab, you will first study sample pedigrees to develop a hypothesis on the mode of inheritance for the trait. You will then search large family databases of RFLP data that were generated with DNA probes to different markers on human chromosomes. One of your challenges will be to find a probe for a marker that is linked to the gene for the trait that you are studying. Once you have done this, you will analyze RFLP and pedigree data from families that you will choose as ideal families for generating recombination data. You will be looking for recombination between the locus for the marker and the trait that you are studying. This will be a particularly challenging aspect of PedigreeLab because you must consider parents with the proper arrangement of the marker and trait on a set of homologous chromosomes to be able to detect recombination in the offspring.

With PedigreeLab you will count recombinants and nonrecombinants based on RFLP analysis and the genotypes that you have assigned to individuals in a pedigree. Recombination frequency and LOD values are tallied for you. If you see a LOD score of >3.0, then it is likely that the marker you chose and the trait are linked. You can then use the recombination frequency data to assign a position for the trait on the same chromosome as the marker. If, however, your LOD score is <3.0, and recombination frequency is approximately 50%, then the probability is high that the trait and marker are unlinked and you'll need to start your gene hunting again with a different probe!


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