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Category: Microbial Genetics and Molecular Biology
DNA Polymorphisms in Gatekeeper and Guardian Genes, Page 1 of 2
< Previous page | Next page > /docserver/preview/fulltext/10.1128/9781555816704/9781555813192_Chap30-1.gif /docserver/preview/fulltext/10.1128/9781555816704/9781555813192_Chap30-2.gifAbstract:
This chapter focuses solely on the human as an experimental organism. The data discussed are derived from unique experimental approaches. There is substantial evidence that cancer segregates in many additional families, albeit at a reduced frequency compared with that for the more severe syndromes. There is also epidemiological evidence for significant variation in DNA repair capacity among individuals in the population and evidence that those with mildly reduced capacity may be more likely to exhibit a cancer predisposition. A great many publications have proposed associations between specific genetic variants (polymorphisms) in DNA repair and/or damage response genes and a cancer predisposition. Evidence documenting the impact of a polymorphism on protein function is generally lacking. Thus, the appreciation of a specific role for the variant proteins in disease, while logical in theory, remains an important aspect of DNA repair and mutagenesis that is still under development.
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The NCBI discovery space. The figure illustrates how records in the dbSNP database are cross-annotated within other internal information resources. More information is available at http://www.ncbi.nlm.nih.gov/About/primer/snps.html (Adapted from NCBI and from reference 265 .)
Relationship between SNPs and the HapMap. (A) SNPs are identified in DNA samples from various populations. (B) Adjacent SNPs that are inherited as blocks called haplotypes. (C) Tag SNPs within haplotypes are those markers that uniquely distinguish the different haplotypes for a larger set of markers. For example, by genotyping the three tag SNPs shown in the figure, each of the four haplotypes shown can be identified. (Adapted from NCBI at http://www.hapmap.org/whatishapmap.html.en.)
Repair-related genes mapping in close proximity to the XPC gene on human chromosome 3p25.1-25.3.
Genotyping by differential array hybridization. (A) A representative fluorescence image of an entire oligonucleotide probe array following hybridization is shown. (B) Scheme for genotyping of an Arabidopsis polymorphism in the Columbia and Landsberg erecta ecotypes on a variant-detector high-density oligonucleotide probe array. The array is designed to interrogate not only the polymorphic site (marked with an asterisk) by using four 25-mer probes that have an A, C, G, or T at the center position (N) but also five flanking bases on either side, adding to the specificity of the assay. The target DNA hybridizes most strongly to a sequence representing a perfect match. Therefore, the probe with the correct base at each center position will produce the strongest hybridization signal. (C) Actual and schematic hybridization patterns are shown for homozygous Columbia (T/T genotype) (top), homozygous Landsberg erecta (C/C genotype) (bottom), and a heterozygous recombinant (T/C genotype) (center). Hybridization of the T allele to the C allele variant-detector array (or the C allele on the T variant-detector array) yields a strong hybridization signal only when the base is varied at the polymorphic site, restoring the correct sequence match. Variation at a second site left or right of the polymorphic site completely disrupts hybridization of an allele on the opposite variant-detector array, since the allele now has a twobase mismatch, but only partially affects hybridization on the correct variant-detector array for some sites, since these are the only mismatches. (Adapted from reference 197 .)
Schematic of the mismatch repair detection (MRD) procedure. MRD utilizes two vectors that are identical except for a 5-bp deletion in the gene coding for Cre recombinase on one of the two vectors. DNA fragments, such as exons, are cloned in the vector containing the active Cre and grown in a bacterial strain that lacks the Dam methylase. These clones, referred to as the standard panel, are made only once and serve as sequence comparison templates for exons from each person to be tested. (A) Heteroduplex formation. DNA fragments being tested are PCR amplified from each individual and pooled. In the example presented, samples are tested for a total of 35 different sequences (illustrated by shades of gold). Linearized methylated vector DNA with a 5-bp deletion within the Cre gene is shown as a grey line, and the 5-bp deletion is shown as a black square. Unmethylated standards are shown in black circles and inserts, with colors and numbers corresponding to the 35 different fragments amplified from the test sample. The components are placed in a single tube, denatured, and reannealed to form heteroduplexes between the unmethylated single-stranded standard, complementary PCR product, and vector with the deleted Cre gene. Hemimethylated heteroduplex circles are ligated with Taq ligase, unligated molecules are removed after treatment with exonuclease III, and the circles are transformed into an Escherichia coli strain (MS) carrying an F’ factor with Tetr and Strs genes flanked by two lox sites. This strain carries a streptomycin resistance gene on its chromosome. (B) Identification of mismatches. Heteroduplex molecules without mismatch (i.e., no variation between the standard and the DNA fragment that is being tested) will replicate normally, and both plasmids carrying the active and inactive Cre will be present. The active Cre protein (grey circles) recombines the two lox sites, leading to the loss of the Tetr and Strs genes and leaving the cells tetracycline sensitive and streptomycin resistant. In the presence of a mismatch in the heteroduplex, MMR occurs and the unmethylated strand carrying the deleted Cre gene is degraded. These cells cannot recombine the two lox sites, retain the Tetr and Strs genes on the F’ factor, and survive in the presence of tetracycline but not streptomycin (the streptomycin-sensitive allele is dominant over the resistant allele). (C) Identification of the fragment content of variant and nonvariant pools. Growing the same transformation mixture on two plates containing either tetracycline or streptomycin facilitates the production of pools representing clones that contain variation and those that do not. If the assorted inserts designated 1 to 35 vary in size, then analysis and comparison of the pools by restriction digestion and/or gel electrophoresis provide a means to identify those inserts that are represented by variation. (Adapted from reference 72 .)
Double D-loop formation assay. (A) A DNA “incoming” oligonucleotide (oligo) is converted to a recombinant filament with RecA in the presence of ATPγ-S. (B) RecA catalyzes the pairing and strand invasion of a duplex, creating a D-loop intermediate. (C) The D-loop intermediate is stabilized, and a double D-loop is produced by an “annealing” oligonucleotide complementary to the displaced strand. (D) The resulting double D-loop is sufficiently stable to remain intact following removal of all RecA protein. (Adapted from reference 230 .)
Comparisons of the DNA repair capacity measured for women with (Breast) and without (Controls) breast carcinoma. The capacity was measured in lymphocytes, using the host cell reactivation assay. Values are expressed as the mean, and 1 standard error of the mean is shown by the error bars. (Adapted from reference 223 .)
Relationship between donor age and DNA repair capacity, using a UV-irradiated pCMVcat plasmid reactivation assay. Activity was determined using fibroblast cell lines from 20 normal donors. The mean standard error of the mean of triplicate determinations is shown for each donor. The dashed line is the least-squares linear-regression line, and the colored zone illustrates the 95% confidence interval. CAT, chloramphenicol acetyltransferase. (Adapted from reference 180 .)
Comparing the two-hit and the haploinsufficiency models of tumorigenesis. (Top) In the two-hit model, tumors result when both alleles of a tumor suppressor gene (TSG) are consecutively inactivated. (Bottom) In the haploinsufficiency model, reduced expression from only one functional allele leads to an altered cellular environment that might confer a selective growth advantage, increased genetic instability, or a new specific cellular phenotype. Such phenotypes might be realized following additional tumor-promoting events such as the acquisition of mutations in different tumor suppressor genes or oncogenes. (Adapted from reference 75 .)
Model for the dependence of biological functions on protein concentration. Roles in embryonic development (dashed line) and tumor suppression (solid line) are illustrated for several tumor suppressor genes. Arrows depict theoretical protein concentrations for haploid and diploid gene copy numbers. (A) p53 and p27 are examples of genes not required for development; however, haploidy lends to tumor predisposition. (B) RB1 is required for both embryonic development and tumor suppression, but haploidy accommodates the former and nearly the latter. (C) Haploinsufficiency for a gene like CBFA2/RUNX1/AML1 may hence be barely sufficient to fully support development and tumor suppression. For the last two panels, loss of the normal allele would affect development. However, for all three panels, loss of the normal allele would contribute to a tumorigenic state. (Adapted from reference 52 .)
The duration of smoking increases the risk of lung cancer. This effect is more dramatic in heterozygotes and homozygotes with the CYP1A1 MsPI polymorphism than for homozygote “normal” individuals. (Adapted from reference 319 .)
The most extensively characterized XPD gene polymorphisms, Asp312Asn and Lys751Gln, are at sites that are not conserved. Shades of gold illustrate regions of greatest conservation, while grey identifies nonconservative changes.
Kaplan-Meier curves of the XPD Asp312Asn polymorphism (A), the XRCC1 Arg399Gln polymorphism (B), and the number of variant alleles from both polymorphisms (C). An increase in the number of variant alleles is associated with higher mortality. (Adapted from reference 94 .)
Luciferase (LUC) activities by Lys751Gln XPD genotypes. Subjects homozygous for the Gln751 allele show a lower mean DNA repair capacity (as indicated by the horizontal line) than do wild-type Lys751 or heterozygous individuals. Significant scatter among data points is seen. (Adapted from reference 222 .)
Figure 30-15 Assessment of the DNA repair activity of an XPC cDNA, deleted for exon 12 using a host cell reactivation assay. (A) Increased expression of luciferase activity in XPC cells was seen following cotransfection of full-length XPC cDNA expression vector but was not seen on cotransfection with the XPC cDNA expression vector with a deletion of exon 12 (p-XPC-deletion Ex12) or with the empty vector (pcDNA3). (B) Reduction in the expression of luciferase in normal cells was seen by cotransfection with an XPC cDNA expression vector with a deletion of exon 12. (Adapted from reference 137 .)