BIOC15Fall2013 Lecture 12 Notes: Chromosome mutations and Cancer and Lecture 13 Notes: Cancer and Deletion mapping: using chromosome mutations Chromosome mutations o Variations in chromosome structure or chromosome number o Variations in chromsomes number: o Aneuploidy  chromosome number is not a multiple of the haploid chromosome #, resulting from loss or gain of one or more chromosomes, ex: trisomy o Monoploidy (haploidy)  contains one copy of each homolog o Polyploidy  more than the normal diploid number of chromosome sets, ex: triploidy o Variation in chromosome structure: o Duplication  increase in the number of copies of a chromosomal region o Inversion  180 degree rotation of a chromosomal region o Translocation:  Non-reciprocal unequal exchanged between non homologous chromosomes  Reciprocal  parts of 2 non homologous chromosome trade places o Deletion  removal of a segment of DNA o Transposition  movement of short DNA segments from one position in the genome to another Cancer cells and karyotypic instability o These are changes that produce genomic and karyotypic instability o Defects in the DNA replication machinery  there are higher rates of mutation among cells with defective DNA repair machinery (mismatch repair and damage repair) because replication error persist o Increased rate of chromosomal aberrations  broken chromosomes, multiple copies of chromosomes, deletions of large chromosomal segments or a whole chromosome fidelity of chromosome reproduction is decreased in tumour cells Types of duplications o Tandem duplications  duplications arranged one after the other (same order or reversed order) o o o o o Nontandem (dispersed duplications)  same order or reverse order chromosome breakage can produce duplications according to one scenario, nontandem duplications could be produced by insertion of a fragment elsewhere on the homologous chromosome o Unequal crossing over can increase or decrease copy number o duplication of the X chromosome polytene region 16A causes Bar eyes o unequal pairins and corssing over during meiosis in females homozygous for this duplication produce chromosomes that have either one copy of region 16A (normal eyes) or 3 copies of 16A (causing the more abnormal double-bar eyes o 1 Deletions o the loss of a segment within one chromosome and the juxtaposition of the two segments on either side of the deleted segment o ex: cri du chat syndrome Inversions o chromosomal rearrangement in which the chromosome is broken 2x and flipped 180 degrees between being rejoined o creates loops in polytene chromosomes that reveal the breakpoint of the inversions o o o in an inversion loop one chromosomal region rotates to conform to similar region in the other homolog crossing over with an inversion loop produces aberrant recombinant chromatids Naming chromosomal aberrations o Duplication -> Dp(3L) = duplication in left arm of chromosome 3 o In(I) = inversion in chromosome 1 o T(3R) = translocation in the right arm of chromosome 3 o Df(3R) = deficiency in the right arm of chromosome 3 Translocation o the segment of one chromosome moved to another chromosome o translocated chromsomes are stained in red and green  reciprocal translocation o o Robertsonian translocations can reshape genomes  reciprocal translocation in acrocentric chromsomes such that the long chromosome becomes the small one and vice versa o Down syndrome and Robertsonian translocation o one way of getting 3 chromosomes 21 (trisomy 21 which is Down Syndrome) is through translocation between chromosomes 21 and 14 o so you end up with 2 ‘free’ 21 chromosomes, and the one attached to the 14, making 3 total 2 o Philadelphia chromosome (Ph) o associated with chronic myelogenous leukemia (CML) o used to be called leucocythaemia  many white blood cells o characterized by reciprocal translocation between chromosomes 9 and 22 o o o o o o this rearrangement makes an abnormal hybrid gene composed of part of the c-abl gene and part of the bcr gene the hybrid gene encodes an abnormal fused protein that disrupts control of cell division The protein encoded by c-abl is a protein tyrosine kinase, an enzyme that adds phosphate groups to tyrosine amino acids on other proteins. This enzyme is an essential part of the set of signals that dictate cell growth and division  Normal cells closely regulate the activity of the c-abl protein, blocking its function most of the time but activating it in response to stimulation by growth factors in the environment. By contrast, the fused protein encoded by bcr/c-abl in cells carrying the translocation is not amenable to regulation  It is always active, even in the absence of growth factor, and this leads to runaway cell division  cancer In a translocation homozygote, chromosomes, segregate normally during meiosis I o If the breakpoint of a reciprocal translocation do not affect the gene function, there are no genetic consequences in homozygotes o Chromosome pairing in a translocation heterozygote o in a translocation heterozygote, the two haploid sets of chromosomes carry different arrangements of DNA o chromosome pairing during prophase I of meiosis is maximized by formation of a cruciform structure  3 segreggation patterns are possible 3 o o balanced gametes are produced by alternate segregation and not by adjacent 1 or adjacent 2 segregation Reciprocal interchromosomal translocation o position effect: the expression of the gene changes because of its changed position in the genome o o When a chromosomal rearrangement such as an inversion of a segment of DNA places the gene next to highly compacted heterochromatin near the centromere, the gene's expression may cease  gene expression may be silenced in some cells and not in others Burkitt Lymphoma o Associated with reciprocal translocation between chromosomes 8 and 14 o Charachterized by change in neighbours for the MYC gene leading to cell growth and division that is not controlled (cancer) o Cancer o uncontrolled growth in a mass of cells may result in transformation such that the cell growth is no longer restrainged by contact inhibition  oncogenesis o cell cycle is controlled by the concentration of cyclin-dependent kinases at regular checkpoint  control activity of other proteins by phosphorylating them o we are going to consider: proto-onco genes, tumor suppressor genes, mutator genes o cancer producing mutations are of 2 general types 4 o o o o o o o oncogene  a gene whose action stimulated unregulated cell proliferation example 1: on the Ph chromosome, ABL gene within new location gives BCR-ABL oncogene that expresses a tyrosine kinase continuously  this causes growth of white blood cells example 2: Burkitt lymphoma MYC oncogene in the translocation is overexpressed leading to uncontrolled growth example 3: c-kit (cellular in this case canine kit) is a truncated protein tyrosine kinase involved in osteosarcoma in dogs (bone cancer) tumour suppressor genes  suppress the normal cell growth if mutated they lead to uncontrolled cell growth example retinoblastoma RBI Evidence from mouse models that cancer is caused by several mutations o transgenic mice with dominant mutations in the myc gene and in the ras gene = increased mutation rate when mutation found in both genes and earlier onset of cancer due to that o the myc oncogene produces tumours slowed than the ras oncogene but when put together, the rate is higher than either one on their own o o mice with recessive mutation in the p53 gene  heterozygous individual have a later onset in life than homozygous individuals Retinoblastoma o among patients with hereditary retinoblastoma: o patients develop tumours in both eyes during early childhood o there is often a deletion of chromosome 13q14.1 to q14.2 o you have one inherited mutation  heterozygous, Rb/rb o and thenthroughout life a second mutation happens where Rb is mutated to rb 5 o Some families have a certain genetic predisposition to certain types of cancer o retinoblastoma is caused by mutation in the RB gene o individuls who inherit one copy of the RB- allele are prone to cancer of the retina o during proliferation of retinal cells, the RB+ allele is lost or mutated o tumours develop as a clone of the RB-/RB- cells o Cancer is though to arise by successive mutations in a clone of proliferating cells o proliferating cells = cells that divide quickly/frequently o o o first mutation leads to an abnormal cell and so on until a malignant cell is formed this does not have to happen in the same gene though, one mutation in one gene combined with another mutation in another gene can still result in uncontrolled cell growth, and therefore cancer Mutator genes o render cells unable to repair mismatched DNA during replication o example: hereditary nonpolyposis colon cancer o one of the 4 Human mismatch repair genes is mutated  loss of function  disables the ability to repair a mismatch that occurs during replication and many mutations accumulate What is the evidence that cancers are genetic diseases? o Chromosome mutations are associated with some cancers o In the table below, gene EWS appears frequently in cancers, so it is considered to be associated with cancer 6 o o o o o o chromosome mutations are associated with some cancers the familial pattern of cancer  some families have more of a cancer than others cells of a tumor that is malignant are also capable of uncontrolled growth viruses an induce cancer  HPV – turns proto-oncogenes into oncogenes exposure to mutagens increases the incidence of cancer in a population Lecture 13 Notes: Cancer and Deletion mapping: using chromosome mutations Aneuploidy in the human population o incidence of abnormal phenotypes caused by aberrant chromosome organization or number is 0.004% o half of spontaneously aborted fetuses have chromosome abnormalities o incidence of abnormal phenotypes caused by single-gene mutations is 0.010% o o monosomic females survive because in humans there is X inactivation anyways, so you technically only need one X chromosome to survive Preparation of microarray and samples for comparative genomic hybridization (CGH) o o CGH detects duplications, deletions and aneuploidy o BAC clones representing the human genome are spotted in order onto a microarray. 7 o o o (b) The genomic sample to be tested is labeled with one color dye (here, red), and the control genome sample is labeled with a second color dye (yellow). (c) The two samples are mixed together, denatured, and then incubated on the microarray. (d) Automated analysis of each spot on the microarray detects the ratio of the two dyed probes that hybridize. Orange indicates a 1 : 1 ratio; other colors indicate deletion (0.5 : 1 ratio; yellow) or duplication (1.5 : 1 ratio; red) of BAC clone sequences in the test sample. Make the map o a geneticist crosses a heterozygous female with the X linked genes y, w, and f to a male that was yellow body, white eyes and forked bristles. The offsprings are given in the table. o X+++Xywf x Xywf Y o o o o o o o o this is cis coupling because parents were +++ and ywf which results in 2 changing places the order of the genes is y – w – f RF y to w o Parents were yw or ++ so recombinants = y+ or +w o RF = 23 + 22 +3 + 2 / 1000 x 100% = 5% RF w to f o Parents were ++ or wf so recombinants are = w+ or +f o RF = 49+46+3+2 / 1000 x 100% = 10% Total RF o Parents were yf or ++ so recombinants = y+ or +f o RF = 23+22+49+46+3+2 / 1000 x 100% = 14.5% Coefficient of coincidence = (observed double cross over freq./ 1000) / (expected freq. / 1000) o = 5/1000 / 0.05 (RF y to w) x 0.1 (RF w to f) = 1 interference = 1 – c.o.c = 0 Using a map o o o o o o o o o o o o The map of chromosome 7 above shows the positions (approximately) of the CFTR gene and two genetic markers used to locate it. The two genetic markers D7S15 & PON have allele variants,(let’s call them d and p respectively) associated with cystic fibrosis. Assume that a carrier female is a heterozygote for the CFTR gene and both markers in cis- conformation and she produces 4000 gametes. Given the coefficient of coincidence of 0.50 what gametes would she be expected to produce and how many of each gamete genotype would be expected? C.o.c = observed freq. of double cross overs / expected freq. of double cross overs Observed # = 0.5 (5/100 x 10/100)  these numbers are from RF on the map Observed # = 10 individuals out of the 4000 will be double cross overs Now you have 3900 individuals left to account for RF (p to cftr) = 10 So + p cftr and d++ recombinants / 4000 x 100% = 10 Recombinants = 400 400 – 10 for the double cross overs = 390 and since there are 2 classes of the recombinants then 195 each RF (d to p) = 5 so recombinants = 200 200 – 10 for thedouble crossovers = 190/2 for each type = 95 each 8 o o finally for the parentals, we just account for the missing offspring (4000 – 190 – 390 – 5)/2 = 1707.5 o Chromosome exchange o Curt Stern used a chromosome mutation as a physical marker  shortened X chromosome and an X chromosome with a translocated piece of Y chromosome o Carnation = car and bar-eye = B o o o o One X chromosome carried mutations producing carnation eyes (a dark ruby color, abbreviated car) that were kidney-shaped (Bar); in addition, this chromosome was marked physically by a visible discontinuity, which resulted when the end of the X chromosome was broken off and attached to an autosome. The other X chromosome had wild-type alleles (+) for both the car and the Bar genes, and its physical marker consisted of part of the Y chromosome that had become connected to the X-chromosome centromere. Deletion mapping o Deletion mapping of period (per) o Pseudodominance  recessive alleles appear dominant over a deletion o Example: per mutants show pseudodominance with deletion 258-11 o The three mutants in per are on the X chromosome and lie within deletion 258-11 which spans bands 3A to 3C on the polytene chromosome Transposons o Most transposons contains inverted repeats (IR) of 10-200 bp long at each end and also contains o Gene encoding transposase, which recognizes the IRs and cuts at border between the IR and genomic DNA o o P element is transposed to new locations  causes insertions and sometimes deletions (if the P element is just moved, rather than replicated and then moved to new location) Gene duplication and Hox genes o Homeobox (Hox) gene found in flies, mice and humans  Pax3 is a mouse Hox gene o They are a group of related genes that control the body plan of the embryo along the anterior-posterior axis o After segments have formed, the Hox proteins determine the type of segment structures (like antennae and wings in fruit flies) that will form a given segment  they do not form the actual segments themselves, they just conferm the identity o The organization of Hox genes on the chromosome is the same as the order of their expression along the anteriorposterior axis of the developing animal (collinear) 9