1. Mutations

2. Methods used to study mutations
Gross chromosomal changes-
deletions, insertions, inversions, translocations
Cytology- microscopy- karyotype
Small mutations
Small deletions, insertions and point mutations
Recombinant DNA technologies

3. Chromosomes and chromosome rearrangements
Cytogenetics is the study of genetics by visualizing chromosomes. This area of research is germane to several areas of biological research.
Cytogenetics has been fundamental to understanding the evolutionary history of a species (for example, although the Chimp and the human are morphologically very different, at the level of the chromosome (and DNA sequence) they are extremely similar.
H = human
C= chimp
G = Gorilla
O = Orang utang

4. Karyotype
Chromosomes are classified by size, centromere position and banding pattern:
Shown below is the human karyotype (description of the chromosome content of a given species)
Karyotype is the chromosome description of length, number, morphology.
Karyotype analysis is extremely important in medicine. Alternations in karyotypes are linked to birth defects and many human cancers.
Metacentric- centromere in the middle
Acrocentric- centromere off center
telocentric centromere at one end

5. Banding patterns
Specialized stains produce unique banding patterns along each chromosome. Banding patterns are extremely useful for detecting abnormalities in chromosome structure.
For many of the chromosome stains- the molecular basis of the banding patterns is unclear. Nonetheless these techniques remain fundamental in many areas of genetic research

6. MU to bp
Genetic maps are based on recombination frequencies and describe the relative order and relative distance between linked genes.
Remember genes reside on chromosomes.
So what we would like to know is where are the genes located on the chromosomes
What does this mean in terms of chromosomes and DNA?

7. Chromosome loss/gain
Chromosome instability-
Elevated gain or loss of complete chromosomes
Very Frequent in tumors
Frequent in in vitro fertilized embryos
Increase with age of parent
Males have 2% rate of aneuploidy during gamete formation
Females have 15% rate of aneuploidy (that increases to 60% by age 50) (25% of all conceptions result in miscarriage due to aneuploidy usually at implantation stage)
Gross chromosomal rearrangements during in vitro fertilization. 40% of embryos carried entire chromosome imbalance
Entire chromosome aneuploidy in human adults:
Only chromosome 13, 18, 21 and X/Y

8. Gross chromosomal changes
The Cri du chat syndrome in humans is a result of a deletion in the short arm of chromosome 5. This was determined by comparing banding patterns with normal and Cri du Chat individuals
Types of chromosome rearrangements that can be studied by karyotype analysis:
GROSS CHROMOSOMAL CHANGES
Deletions, Duplications, Inversions, Translocations

9. Copy Number Variation of chromosomes
CNV- copy number variation of entire chromosomes or of chromosome segments
5% of an individuals genome displays CNV
Synuclein gene (involved in membrane stability of neurons). CNV is involved in Parkinsons
Many cancers- malignant cells most often gain additional copies of chromosome segments- genes in these segments are mis-expressed and this leads to mis-expression of other genes.
Tissues of identical twins show copy number differences at several loci (Bruder et al 2008)
Different tissues of single individual show copy number differences (Piotrowski et al., 2008)
Gross chromosomal rearrangements during in vitro fertilization
55% of embryos carried terminal imbalance (sub-telomere loss) (Vanneste et al., microarray based screen of IVF 35 embryo)

10. A____B____C________D____E____F
DDIT
Normal Chromosome
A____B____C________D____E____F
Deletions (deficiency)
A____B____C________D____F
Duplications
A____B____C________D____E____E____F
Inversions
A____B____C________E____D____F
Translocation
A____B____C________D____E____F
H____I____J________K____L
A____B____C________D____L
H____I____J________K____E____F

11. Deletions
Deletions are often detected cytologically by comparing banding patterns between the normal and the partially deleted chromosomes
Deleted
segment
Chromosome no
female
deletion
chromosome1
Band
46,XX, del(1)(q24q31)
Female with a deletion of chromosome 1 on the long arm (q) between bands q24 to q31.

12. In many instances deletions are too small to be detected cytologically
In many instances deletions are too small to be detected cytologically. In these instances genetic/molecular techniques are used.
Since cytological deletions remove a contiguous set of genes, there is a high probability that an essential gene will be deleted. Therefore deletions will survive as heterozygotes and not homozygotes.
A____B________C____D
Normal
A____B________C____D
A____________C____D
Homologous deletion
(Lethal?)
A____________C____D
A____________C____D
Heterologous deletion
(NOT Lethal)
A____B________C____D

13. Consequences of deletions
A+_____B+_____C+___________D+
Normal
A+_____B+_____C+___________D+
In individuals heterozygous for the deletion, pairing is disrupted in the regions surrounding the deletion. Therefore recombination is also significantly reduced in these regions. B+
A+____/ \_____C+___________D+
A+___________C+___________D+
Genotype
A+_____b______c____________D+
Normal
A+_____B+_____C+___________D+
A deletion on one homologue unmasks recessive alleles on the other homologue. The effect is called pseudo- dominance.
A+____b______c____________D+
A+___ _____C+___________D+

14. Deletions in X
Females in Drosophila XX
Males in Drosophila XY or XO
Deletion series phenotype
sick
dead
sick

15. Changes in chromosome structure
Deletions:
Hemizygosity from large deletions results in lethality- even the smallest cytologically defined deletions take out tens of 1,000's of bps and are likely to remove essential genes.
2. Organisms can tolerate hemizygosity from small but not large deletions. The reason for this is not entirely clear and is placed under the rubric of disrupting the overall ratio of gene products produced by the organism

16. xxxxxxxx

17. Duplications
A____B____C________D____E____F
normal
A____B____C________D____E____E____F
Duplication
Individuals bearing a duplication possess three copies of the genes present in the duplicated region.
In general, for a given chromosomal region, organisms tolerate duplications much better than deletions.
46,XY, dup(7)(q11.2q22)
Male with a duplication of chromosome 7 on the long arm (q) between bands 11.2 to 22

18. Tandem Duplications
Tandem duplications- Important class of duplications!!!
This is a case in which the duplicated segment lies adjacent to the original chromosomal segment
A B C D E A B C B C B C B C D E
Once a tandem duplication arises in a population, even more copies may arise because of asymmetrical pairing at meiosis.
Remember when the homologs pair during prophase of meiosis I, they line up base-pair for base pair. Duplications lead to mistakes in this pairing mechanism

19. Pairing of duplicated segments
Proper pairing:
A____B____C____B____C____D____E
A____B____C____B____C____D____E
A____B____C____B____C____D____E
A____B____C____B____C____D____E
Inappropriate pairing:
A____B____C____B____C____D____E
A____B____C____B____C____D____E
A____B____C____B____C__ __D____E
A____B____C____B____C__ __D____E

20. Pairing of duplicated segments
Tandem duplications expand by mistakes in meiosisI during pairing a b c d e a A b B A B C D E c C d D e E
Sister chromatids
Sister chromatids

21. Pairing of duplicated segments
Normal pairing of chromosomes
pairing of chromosomes in repetitive regions
Pairing of duplicated segments

22. Pairing of duplicated segments
Tandem duplications expand by mistakes in meiosis during pairing
Paired non-sister chromatids A B C D E a b c d e 22

23. Crossover in mispaired duplicated segments
What happens if you get a crossover after mis-pairing in meiosisI? A B C D A B C D a b c d a b c d A B C D E a b c d e A B C D A B C D A B C D A B C D

24. The four meiotic products of a crossover between regions B and C:
A-B-C-B-C-D-E
A-B-C-D-E
A-B-C-B-C-B-C-D-E
This process may repeat itself many times, such that a small fragment of the genome is repeated 10,000 times.

25. An example of this is near the centromeres of the Drosophila genome:
If you look at the DNA sequence in this region it consists of small 5-10 bp sequences (AATAC)n repeated 1,000s of times. It is believed to have arisen from unequal crossing over.
Repetitive DNA- cell does not like it- They try to reduce recombination of repetitive DNA by packaging the DNA with proteins to form heterochromatin- cold spots of recombination along the chromosome

26. Duplications provide additional genetic material capable of evolving new function. For example in the above situation if the duplication for the B and C genes becomes fixed in the population- the additional copies of B and C are free to evolve new or modified functions.
This is one explanation for the origin of the tandemly repeated globin genes in humans. Each of these has a unique developmental expression pattern and provides a specialized function.
The hemoglobin in fetus has a higher affinity for oxygen since it acquires its oxygen from maternal hemoglobin via competition

27. xxxxxx

28. Inversion
Chromosomes in which two breaks occur and the resulting fragment is rotated 180 degrees and reinserted into the chromosome.
Inversions involve no change in the amount of genetic material when they occur and therefore they are often genetically viable and show no abnormalities at the phenotypic level.
Gene fusions may occur
Inversions are defined as to whether they span the centromere
Paracentric inversions do not span the centromere:
A B C D E
A B D C E
Pericentric inversions span the centromere:
A C B D E
In a pericentric inversion one break is in the short arm and one in the long arm. Therefore an example might read 46,XY,inv(3)(p23q27).
A paracenteric inversion does not include the centromere and an example might be 46,XY,inv(1)(p12p31).

29. Homologs which are heterozygous for an inversion have difficulties pairing in meiosis.
During pairing homologous regions associate with one another. Consequently individuals heterozygous for an inversion will form a structure known as an inversion loop.
Crossover within inverted region?
A---B---C----D---E----F----G
A’--B’---C’---D’--E’---F’---G’
A---B---C----D----E---F---G
A’--B’---C’---E’---D’--F’---G’
D E
D’ E’
A B C
F G
A’ B’ C’
‘F G’

30. The consequence of crossover within a paracentric inversion
a-b-c
d-e
f-g
d-e
a-b-c
f-g
a-b-c
e-d
f-g
During meiosis, pairing leads to formation of an inversion loop
This is a problem if crossing over occurs within the inversion
D E
D’ E’
A B C
F G
A’ B’ C’
‘F G’
A-B-0-C-D-E’-C’--0--B’-A’ dicentric-fragmentation
G-F-E-D’-F’-G’ acentric- no segregation

31. The consequence of crossover within a pericentric inversion (one that spans the centromere).
a-b-c
d-e
f-g
d-e
a-b-c
f-g
a-b-c
e-d
f-g
During meiosis, pairing leads to formation of an inversion loop
This is a problem if crossing over occurs within the inversion
D E
D’ E’
A B C
F G
A’ B’ C’
‘F G’
A-B-C-D-0-E’-C’-B’-A’ fragment
G-F-E-0-D’-F’-G’ fragment

32. Inversions
Paracentric inversion crosses over with a normal chromosome, the resulting chromosomes are an acentric, with no centromeres, and a dicentric, with 2 centromeres.
The acentric chromosome isn't attached to the spindle, so it gets lost during cell division, and the dicentric is usually pulled apart (broken) by the spindle pulling the two centromeres in opposite directions. These conditions are lethal.
Pericentric inversion crosses over with a normal chromosome, the resulting chromosomes are duplicated for some genes and deleted for other genes. (They do have 1 centromere apiece though).
The gametes resulting from these do not produce viable progeny.
Thus, either kind of inversion has lethal results when it crosses over with a normal chromosome.
The only offspring that survive are those that didn't have a crossover or crossed over in regions outside the inversion.
Thus when you count the offspring you only see the non-crossovers, so it appears that crossing over has been suppressed.

33. What are the consequences of crossing-over in an individual homozygous for an inversion?
Genotype for normal individual
A______B______0______C______D______E______F______G
Genotype of an individual heterozygous for an inversion:
A______B______0______C______F______E______D______G
Genotype of an individual homozygous for an inversion:

34. Translocations
A segment from one chromosome is exchanged with a segment from another chromosome.
Chromosome 1
A B C D E F
Chromosome 2
O P Q R S T
Reciprocal translocation
A B C D S T
O P Q R E F
This is more specifically called a reciprocal translocation and like inversions (and unlike duplications and deficiencies) no genetic material is gained or lost in a reciprocal translocation.
Non-reciprocal translocations may also occur

35. Philadelphia chromosome: t(9;22)(q34;q11).
long arms of chromosome 7 and 21 have broken off and switched places. So you can see a normal 7 and 21, and a translocated 7 and 21.
This individual has all the material needed, just switched around (translocated), so they should have no health problems. However there can be a problem when this person has children.
Remember that when the gametes are made, each parent gives one of each chromosome pair. What would happen if this person gave the normal seven and the 21p with 7q attached?
There are three copies of 7q instead of two. And there is only one copy of 21q
t(11;18)(q21;q21) translocation between chromosomes 11 and 18 at bands q21 and q21
Philadelphia chromosome: t(9;22)(q34;q11).

36. Pairing after translocations
As with inversions, individuals heterozygous for a reciprocal translocation will exhibit abnormalities in chromosome pairing
A B C D E F
A B C D S T
O P Q R S T
O P Q R E F
Notice this individual has the normal amount of genetic material
(two copies of each gene).
However it is rearranged.
If the translocated fragment contains a centromere, you could get dicentri and acentric chromosomes
How will translocated chromosomes pair in meiosis?

37. Homologous regions associate with one another.B C D E F S T R Q P O N1 T1 T2 N2
Homologous regions associate with one another.
These chromosomes will follow Mendel's rule of independent of assortment. In this instance one must focus on the centromere
There are three possible patterns of segregation.
Normal Pairing of 10 chromosomes in maize
Chr8-9 translocation

38. Segregation after translocations
Alternate segregation:
ｷ N1 and N2 segregate to one pole
ｷ T1 and T2 segregate the other pole
These gametes have the normal haploid gene content: one copy of each gene and are normal
Adjacent segregation:
ｷ N1 and T1 segregate to one pole
ｷ T2 and N2 segregate to the other pole
These gametes are anueploid: they are missing some genes and duplicated for other genes.
Adjacent segregation
ｷ N1 and T2 segregate to one pole
ｷ N2 and T1 segregate to one pole
Therefore, in a translocation heterozygote, some of the gametes are viable and some are inviable. A B C D E F S T R Q P O N1 T1 T2 N2

39. Reciprocal translocations result in genes that are known to map to different chromosomes but behave as linked genes.
Under normal circumstances genes E and R assort independently because they are on different chromosomes. However in a translocation they will behave as closely linked genes and segregate together. A B C D E F S T R Q P O N1 T1 T2 N2

40. Translocations (and inversion) breakpoints sometimes disrupt an essential gene. That is the break occurs in the middle of a gene.
In fact because of this, a number of specific translocations are causally associated with specific human cancers.
The inherited disease Duchenne muscular dystrophy was mapped through a translocation that specifically disrupted this gene.

41. abl/bcr. Fusion protein
abl/bcr Fusion protein Chronic myelogenous and acute lymphotic leukemia
ALK/NPM Fusion Large cell lymphomas
HER2/neu Fusion Breast and cervical carcinomas
MYH11/CBFB Fusion Acute myeloid leukemia
ML/RAR Fusion Acute premyelocytic leukemia
ERG/TMPRSS2 Fusion prostate cancer
Gene fusion -prostate cancer -ERG merges with a prostate-specific gene called TMPRSS2. ERG is a transcription factors

42. 3D chromosome/nucleus affects gross chromosomal alterations
Deletions/inversions
Translocations

43. Xxxx