1. Chromosomes: Structure and Function
MOLECULAR GENETICS (856)

2. Changes in Chromatin Structure
Polytene chromosomes are giant chromosomes found
in certain tissues of Drosophila and some other organisms.
These large, unusual chromosomes arise when repeated
rounds of DNA replication take place without accompanying
cell divisions, producing thousands of copies of DNA that lie
side by side.
Chromosomal puffs—localized swellings of the chromosome. Each puff is a region of the chromatin that has relaxed its structure, assuming a more open state. If radioactively labeled uridine is added to a Drosophila larva, radioactivity accumulates in chromosomal puffs, indicating that they are regions of active transcription.
Appearance of puffs at particular locations on the chromosome can be stimulated by exposure to hormones and other compounds that are known to induce the transcription of genes at those locations. This correlation between the occurrence of transcription and the relaxation of chromatin at a puff site indicates that chromatin structure undergoes dynamic change associated with gene activity.

3. Chromosomal puffs in condensed Drosophila chromosome show states of de-condensing in expressed regions
DNA packaging is not simple. If most of the DNA exists as “packaged”, how does the cell know to “unpackage” the right part of the genome for gene expression? (e.g. shown here is the “unpackaged” portion of the chromosome that is currently being expressed (i.e. making RNA)

4. A several experiments indicating that chromatin structure changes with gene activity is sensitivity to DNase I, an enzyme that digests DNA.
The ability of this enzyme to digest DNA depends on chromatin structure: when DNA is tightly bound to histone proteins, it is less sensitive to DNase I, whereas unbound DNA is more sensitive to digestion by DNase I.
The results of experiments that examine the effect of DNase I on specific genes show that DNase sensitivity is correlated with gene activity.
For example, globin genes code for hemoglobin in the erythroblasts (precursors of red blood cells) of chickens. The forms of hemoglobin produced in chick embryos and chickens are different and are encoded by different genes (Fig.a).
No hemoglobin is synthesized in chick embryos in the first 24 hours after fertilization. If DNase I is applied to chromatin from chick erythroblasts in this first 24-hour period, all the globin genes are insensitive to digestion (Fig.b).

5. Changes in Chromatin Structure
From day 2 to day 6 after fertilization, after hemoglobin synthesis has begun, the globin genes become sensitive to DNase I, and the genes that code for embryonic hemoglobin are the most sensitive (Fig c).
After 14 days of development, embryonic hemoglobin is replaced by the adult forms of hemoglobin. The most sensitive regions now lie near the genes that produce the adult hemoglobins (Fig.d).
DNA from brain cells, which produce no hemoglobin, remains insensitive to DNase digestion throughout development (Fig.e).

6. Method: Sensitivity to DNase I was tested on different tissues and at different times in development
DNase I sensitivity is correlated with the transcription of globin genes in erythroblasts of chick embryos.
The U gene codes for embryonic hemoglobin;
the D and A genes code for adult hemoglobin.

7. Changes in Chromatin Structure
In summary, when genes become transcriptionally active, they also become sensitive to DNase I, indicating that the chromatin structure is more exposed during transcription.
What is the nature of the change in chromatin structure that produces chromosome puffs and DNase I sensitivity?
In both cases, the chromatin relaxes; histones loosen their grip on the DNA. One process that appears to be implicated in changing chromatin structure is acetylation, a reaction that adds chemical groups called acetyls to the histone proteins.
Enzymes called acetyltransferases attach acetyl groups to lysine amino acids at one end (called a tail) of the histone protein.
This modification reduces the positive charges that normally exist on lysine and destabilizes the nucleosome structure, and so the histones hold the DNA less tightly. Proteins taking part in transcription can then bind more easily to the DNA and carry out transcription.

8. Histones are subject to modification which affect gene regulation
Exposed regions of the histone are subject to modification
These modification influence the structure of chromatin and it regulator properties
Some modification
Acetylation
Methylation
Phosphorylation

9. How are chromosome organized in a nucleus
Staining pattern results in different color specific for each chromosome
Chromosomes occupy discrete territories in the cell nucleus
Chicken nuclei

10. Features of human chromosome territories
specific location of p and q arms and specific territories
Transparent view
Three dimensional view of chromsome territories
Same specific territories for the active and inactive X chromosome

11. Where are gene-rich and gene-poor regions of chromosome located
Gene-poor chromosome located at nuclear periphery
So generally silent regions of chromosomes are located at the nuclear periphery
Gene-rich chromosome located in nuclear interior

12. Essential Functional elements of Chromosomes
A centromere
A pair of telomeres
Origins of replication

13. Chromosomes as functioning organelles Centromere
Chromosome fragments
that lack a
centromere are lost in mitosis.
Chromosomes as functioning organelles Centromere
A constricted region of the chromosome where spindle fibers attach and is essential for proper movement of the chromosome in mitosis and meiosis .
The first centromeres to be isolated from Yeast (small, linear chromosomes).

14. Chromosomes as functioning organelles Centromere
Centromeric sequences are the binding sites for proteins that function as the kinetochore, a complex that assembles on the centromere and to which the spindle fibers attach.
Kinetochores play a central role in this process, by controlling assembly and disassembly of the attached microtubules and, through the presence of motor molecules, by ultimately driving chromosome movement.

15. Chromosomes as functioning organelles Centromere
In simple eukaryotes, the sequences that specify centromere function are very short.
Yeast Saccharomyces cerevisiae the centromere element (CEN) is about 110 bp long, comprising two highly conserved flanking elements of 9 bp and 11 bp and a central AT-rich segment of about 80–90 bp.
The centromeres of such cells are interchangeable - a CEN fragment derived from one yeast chromosome can replace the centromere of another with no apparent consequence.
In mammals, centromeres comprise hundreds of kilobases of repetitive DNA, some nonspecific and some chromosome-specific.
Centromeres consist of particular sequences repeated many times. This nucleotide sequence is found in the point centromere of Saccharomyces cerevisiae.

16. Chromosomes as functioning organelles Centromere
Variations in centromeric sequences
Diffuse Centromeres, spindle fibers attach along the entire length of the chromosome.
Localized centromeres; spindle fibers attach at a specific place on the chromosome. Appear constricted, but there also can be secondary constrictions at places that do not have centromeric functions.
Classes of localized centromeres
Point centromeres
Smaller and more compact. DNA sequences are both necessary and sufficient to specify centromere identity and function in organisms with point centromeres)
Budding yeast (Saccharomyces cerevisiae) encompasses 125 bp of DNA.
Regional centromeres (large amounts of DNA and are often packaged into heterochromatin).
Most of the centromere is made up of short sequences of DNA that are repeated thousands of times in tandem. Within these repeats are “islands” of more complex sequence, primarily transposable element sequences.

17. Chromosomes as functioning organelles Centromere
Centromeric DNA shows remarkable sequences heterogeneity.
Universally marked by the presence of a centromere-specific variant of histone H3, generically known as CenH3 (the human form of CenH3 is named CENP-A).
At centromeres, CenH3/CENP-A replaces the normal histone H3 and is essential for attachment to spindle microtubules.
Depending on centromere organization different numbers of spindle microtubules can be attached.

20. Chromosomes as functioning organelles: origins of replication
Origins of replication are the sites where DNA synthesis begins;
Eukaryotic chromosomes have multiple origin of replication.
Eukaryotic origins of replication; yeast, where the presence of a putative replication origin can be tested by a genetic assay.
Bacterial replication origin in the plasmid does not function in yeast, therefore the few plasmids that transform at high efficiency must possess a sequence within the inserted yeast fragment that confers the ability to replicate extrachromosomally at high efficiency - that is an autonomously replicating sequence (ARS) element.

21. Chromosomes as functioning organelles: origins of replication
ARS elements are thought to derive from authentic origins of replication and, in some cases, this has been confirmed by mapping a specific ARS element to a specific chromosomal location and demonstrating that DNA replication is indeed initiated at this location.
ARS elements extend for only about 50 bp and consist of an AT-rich region which contains a conserved core consensus and some imperfect copies of this sequence.
ARS elements contain a binding site for a transcription factor and a multiprotein complex is known to bind to the origin.

22. Chromosomes as functioning organelles: origins of replication
Mammalian replication origins are less well defined because of the absence of a genetic assay.
There are speculations that replication can be initiated at multiple sites over regions tens of Kb long.
Computer analysis of regions encompassing several eukaryotic origins of replication, including some human and other mammalian examples, identified a consensus DNA sequence WAWTTDDWWWDHWGWHMAWTT where W = A or T; D = A or G or T; H = A or C or T; and M = A or C

23. Chromosomes as functioning organelles Telomeres
Telomeres are specialized structures, comprising DNA and protein, which cap the ends of eukaryotic chromosomes.
Functions
Maintaining the structural integrity of a chromosome.
Ensuring complete replication of the extreme ends of chromosomes.
Helping establish the three-dimensional architecture of the nucleus and/or chromosome pairing.
The ability of telomerase to replicate a chromosome end depends on the unique molecular structure of the telomere.

24. Chromosomes as functioning organelles Telomeres
Eukaryotic telomeres consist of a long array of tandem repeats. One DNA strand contains TG-rich sequences and terminates in the 3′ end; the complementary strand is CA-rich.
Highly conserved in evolution - there is considerable similarity in the simple sequence repeat,
Example
TTGGGG (Paramecium), TAGGG (Trypanosoma) TTTAGGG (Arabidopsis) and TTAGGG (Homo sapiens)

25. Chromosomes as functioning organelles Telomeres
First isolated from the protozoan Tetrahymena thermophila and possess multiple copies of the sequence:

26. Human Telomeres
The (TTAGGG)" array of a human telomere spans about kb.
A very large protein complex shelterin, or the telosome contains several components that recognize and bind to telomeric DNA.
Two telomere repeat binding factors (TRFl and TRF2) bind to double-stranded TTAGGG sequences.
G-rich strand has a Single-stranded overhang at its 3' end that is typically nucleotides long.
This can fold back and form base pairs with the other, C-rich, strand to form a telomeric loop known as theT-loop.
Protect the telomere DNA from natural cellular mechanisms that repair double-stranded DNA breaks.

28. Chromosomes as functioning organelles Telomeres
Telomeres have now been isolated from protozoans, plants, humans, and other organisms; most are similar in structure.

29. Chromosomes as functioning organelles Telomere Structure
The G-rich strand often protrudes beyond the complementary C-rich strand at the end of the chromosome.
The length of the telomeric sequence varies from chromosome to chromosome and from cell to cell, suggesting that each telomere is a dynamic structure that actively grows and shrinks.
The telomeres of Drosophila chromosomes are different in structure.
They consist of multiple copies of the two different retrotransposons , Het-A and Tart, arranged in tandem repeats.
Apparently, in Drosophila, loss of telomere sequences during replication is balanced by transposition of additional copies of the Het-A and Tart elements.

30. Cytogenetics Structure and properties of chromosomes,
Chromosomal behaviour during mitosis and meiosis,
Chromosomal influence on the phenotype and the factors
that cause chromosomal changes.
Related to disease status caused by abnormal chromosome
number and/or structure.

31. Methods for chromosomal analysis: Karyotyping and Banding
The collection of all the chromosomes is referred to as a Karyotype.
The method used to analyze the chromosome constitution of an individual, known as chromosome banding.
Chromosomes are displayed as a karyogram.

32. Obtaining and preparing cells for
chromosome analysis
Cell source:
Blood cells
Skin fibroblasts
Amniotic cells / chorionic villi
Increasing the mitotic index
- proportion of cells in mitosis using colcemid
Synchronizing cells to analyze prometaphase chromosomes

33. Key Procedure In the case of peripheral (venous) blood
A sample is added to a small volume of nutrient medium containing phytoheamagglutinin, which stimulates T lymphocytes to divide.
The cells are cultured under sterile conditions at 37C for about 3 days, during which they divide, and colchicine is then added to each culture.
This drug has the extremely useful property of preventing formation of the spindle, thereby arresting cell division during metaphase, the time when the chromosomes are maximally condensed and therefore most visible.
Hypotonic saline is then added, which causes the red blood cells to lyze and results in spreading of the chromosomes, which are then fixed , mounted on a slide and stained ready for analysis

34. PREPARATION OF CHROMOSOMES

35. Karyotype Analysis Following Steps are involved;
Counting the number of cells, sometimes referred as metaphase spread
Analysis of the banding pattern of each individual chromosome in selected cells.
Total chr. Count is determined in cells, but if mosaicism is suspected then 30 or more cell count will be undertaken.
Detailed analysis of the banding pattern of the individual chromosomes is carried out in approx. 3-5 metaphase spread, which shows high quality banding.
The banding pattern of each chromosome is specific and shown in the form of Idiogram.

36. MITOTIC CHROMOSOMAL SPREAD

38. Chromosome Banding
Chromosome banding is developed based on the presence of heterochromatin and euchromatin.
Heterochromatin is darkly stained whereas euchromatin is lightly stained during chromosome staining.

39. Types of chromosome banding
G-banding
C-banding
Q-banding
R-banding
T-banding

40. G-Banding
- G-banding is obtained with Giemsa stain following digestion of
chromosomes with enzyme trypsin.
-- It is a mixture of methylene blue and eosin.
- It is specific for the phosphate groups of DNA and attaches
itself to regions of DNA where there are high amounts of
adenine-thymine bonding.
- Yields a series of lightly and darkly stained bands – the dark
regions tend to be heterochromatic, late-replicating and AT
rich.
- The light regions tend to be euchromatic, early-replicating
and GC rich .

41. G-Banding
G-banding of human female metaphase chromosomes

42. Q-Banding
- Q-banding is a fluorescent pattern obtained using quinacrine for
staining. The pattern of bands is very similar to that seen in G-
banding.
- Chromosomes are stained with a fluorescent dye which binds
preferentially to AT-rich DNA, such as Quinacrine.
- Quinacrine banding (Q-banding) was the first staining method
used to produce specific banding patterns for mammalian
chromosomes.
- It is especially useful for distinguishing the Y chromosome.

43. Q-Banding
Q-banding of human male metaphase chromosomes

44. R-Banding
R-banding is the reverse of G-banding (the R stands for "reverse").
The chromosomes are heat-denatured in saline before being stained with Giemsa. The heat treatment denatures AT-rich DNA.
Dark regions are euchromatic (guanine-cytosine rich regions) and the bright regions are heterochromatic (thymine-adenine rich regions).
Telomeres are stained well by this procedure.

45. R-Banding
R-banding of human female metaphase chromosomes

46. T-Banding
Identifies a subset of the R bands which are especially concentrated at the telomeres.
The T bands are the most intensely staining of the R bands and are visualized by employing either a particularly severe heat treatment of the chromosomes prior to staining with Giemsa, or a combination of dyes and fluorochromes.

47. C-Banding Chromosomes of mouse Chromosomes of human female
C-banding stains the constitutive heterochromatin, which usually lies near the centromere. The chromosomes are typically exposed to denaturation with a saturated solution of barium hydroxide, prior to Giemsa staining.
Chromosomes of mouse
Chromosomes of human female

49. Molecular Cytogenetics

50. Molecular cytogenetics locates specific DNA sequences on chromosomes
Analysis of the gross structural organization of chromosomes
Higher resolution analyses
Target DNA Sequence
Probe
probes are often nucleotides long and are chemically synthesized.

51. High Resolution Karyotype
Advantages
“Whole genome scan”
Relative low cost
Disadvantages
Labor intensive
Detection above 5 Mb

52. Molecular Methods for chromosomal analysis Molecular Cytogenetics
Fluorescent in situ Hybridization (FISH)
Chromosome painting
Comparative Genomic Hybridization (CGH)
Molecular karyotyping and Multiplex FISH(M-FISH)
Spectral Karyotyping
Array CGH

53. In situ hybridization
In situ hybridization, DNA probes can be used to determine the chromosomal location of a gene or the cellular location of an mRNA in a process called in situ hybridization.
The name is derived from the fact that DNA or RNA is visualized while it is in the cell (in situ).
The maximum resolution of conventional FISH on metaphase chromosomes is several megabases.
Prometaphase chromosomes can permit 1 Mb resolution.

54. Fluorescent IN SITU Hybridization (FISH)
A technology in which labeled nucleic acid sequence/ probes are used for the visualization of specific DNA or RNA sequences on mitotic chromosome preparations or in interphase cells.
Fluorescently labeled DNA probes to detect or confirm gene or chromosome abnormalities that are generally beyond the resolution of routine Cytogenetics.
The sample DNA (metaphase chromosomes or interphase nuclei) is first denatured, a process that separates the complimentary strands within the DNA double helix structure.
The fluorescently labeled probe of interest is then added to the denatured sample mixture and hybridizes with the sample DNA at the target site as it reanneals (or reforms itself) back into a double helix.
The probe signal can then be seen through a fluorescent microscope and the sample DNA scored for the presence or absence of the signal.
Concept: A simple procedure for mapping genes and other DNA sequences is to hybridize a suitable labeled DNA probe against chromosomal DNA that has been denatured in situ.

55. Fluorescent in situ Hybridization
Originally, probes were radioactively labeled and detected with autoradiography, but now many probes carry attached fluorescent dyes that can be seen directly with the microscope (Fig.a).
Fluorescent probes are used to mark the locations of specific gene sequences on chromosomes.

56. Different types of FISH Probes
Centromeric Probes; consist of repetitive DNA sequences found in and arround the centromere of a specific chromosomes.
Used for rapid diagnosis of trisomies 13, 18, 21.
Chomosomes specific unique sequence probes; specific for a particular single locus. Locus specific probes for chromosomes 13q14 and the critical region for down syndrome on chr.21(21q q22.2), X and Y chromosomal abnormalities.
Telomeric probes; Complete set of telomeric probes for all 24 chromosomes, used for subtelomeric abnormalities (deletions, translocations).
Whole chromosome paint probes; consist of a cocktail of probes obtained from different parts of a particular chromosome, used for ring chromosomes and translocations.

57. Different types of Fish Probes
Probes derived from flow- sorted chromosomes; Because of their size and DNA composition, chromosomes bind different amount of fluorescent dyes, some of which bind specifically to GC sequences and others to AT sequences.
This property of differential binding allows chromosomes to be separated by the process of flow cytometry or fluorescent activated cell sorting (FACS). This will stained the metaphase chromosomes with a florescent DNA- binding dye and then projecting them across a laser beam which excites the chromosome to fluoresce. This fluorescence intensity is measured and analyzed by a computer that draws up a distribution histogram of chromosomes size called as a flow karyotype.

58. Green signals indicate positive hybridization of a YAC from human 3q26
Green signals indicate positive hybridization of a YAC from human 3q26.3 to metaphase chromosomes from a patient with Cornelia syndrome with a balanced translocation (breakpoints at 3q26.3 and 17q23.1). Red signals indicate simultaneous hybridization with a chromosome 17 centromere probe. The YAC spans the 3q26.3 breakpoint (green signals on one normal chromosome 3 + the two translocation chromosomes). One translocation chromosome is small and carries a chromosome 17 centromere (red signal); the other has a chromosome 3 centromere and is about the same size as the normal chromosome 3.