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(and prevalence; total prevalence = 31.5-73 per 1000)
Diseases caused by alterations in the small cytoplasmic mitochondrial chromosome
Gene/Chromosome disorder plus other factors, like environmental factors
Prevalence of congenital malformations = 20-50 per 1000
diabetes
obesity
hypertension
heart disease
birth defects (cleft lip and palate),
(P= 6-9 per 1000)
Defective chromosome (number and/or structure)
Down syndrome (Trisomy 21)
Caused by mutant genes - either a single allele or a pair of alleles e.g. sickle cell trait/sickle cell anemia
X-Linked
P= 0.5-2 per 1000
Duchenne’s Muscular Dystrophy
Hemophilia A
Incongenentia Pigmenti
Vitamin-D resistant Rickets
Autosomal
Recessive
(P= 3-9.5 per 1000)
Cystic Fibrosis
Tay Sachs
Dominant
(P= 2-2.5 per 1000)
Neurofibromatosis
Familial Hypercholesteremia
Regulation of p53 activity
P53 induces apoptosis-
P53 role in apoptosis - intrinsic pathway
Evasion of apoptosis as a hallmark for cancer
The balance between cell proliferation and apoptosis is influenced by
Tumour Suppressor Genes
genes that encode proteins that normally suppress tumor formation
Oncogenes
genes that contribute to the development of cancer
Caspase Cascade
2 tiers of caspase activation during apoptosis:
Effector caspases
carry out apoptosis
(caspases 3, 6, and 7)
Initiator caspases
activated through the apoptosis-signaling pathways
(caspases 2, 8, 9, and 10)
destroy essential cellular proteins, leading to controlled cell death
Intrinsic (Mitochondria) pathway
activation of caspase 9
activation of the downstream effector caspases 3, 6, and/or 7
The primary function of the apoptosome seems to be multimerization and allosteric regulation of the catalytic activity of caspase 9
Cytochrome c binds the adaptor apoptotic protease activating factor-1 (Apaf-1), forming a large multiprotein structure known as the apoptosome
BCL-2 Family
Protein Classes
Anti-apoptotic proteins:
BCL-2 and BCL-XL act to prevent permeabilization of the outer mitochondrial membrane by inhibiting the action of the pro-apoptotic Bcl-2 proteins Bax and/or Bak
Pro-apoptotic proteins:
Upon membrane permeabilization, cytochrome c and the pro-apoptotic proteins (SMAC/DIABLO) are then able to translocate from the inter-membraner space of the mitochondria into the cytosol
those that only have the BH3 domain, such as Bid, Bad, Bim, Bmf, PUMA, and NOXA
– permeabilization of the mitochondrial membrane
Several BH domains (Bax and Bak)
contain signature domains of homology called BCL-2 homology (BH) domains
(termed BH1, BH2, BH3, and BH4)
cells with defects are forced to commit suicide
P53 protein senses DNA damage
-blocks Cdk2 stops progression of cell cycle in G1
ATM protein:
-maintain normal telomere length
interrupt cell cycle when damage is detected
-detects DNA damage
especially double-strand breaks
patients very high risk of cancer
(~100 fold increase)
the same disease name
ATM (Ataxia telangiectasia mutated)
DNA Replication Checkpoints
damaged template, protein complexes bound to DNA, poor supply of NTPs = barriers in replication
stall replication forks
insert stalled fork
can rearrange, break, collapse through disassembly of the replication complex
-during the replication of the millions or billions of DNA base pairs
Spindle Checkpoints
-trigger apoptosis if the damage is irreparable
-arrest the cell in metaphase until all the kinetochores are attached correctly (M checkpoint)
-detect improper alignment of the spindle itself and block cytokinesis
-detect any failure of spindle fibers to attach to kinetochores
DNA Damage Checkpoints
-sense DNA damage before the cell enters S phase (G1 checkpoint)
Inhibition of Cdk1 – prevent the cell to go from G2 to mitosis
Damage too severe – cell enters apoptosis
-sense DNA damage after S phase (G2 checkpoint)
Inhibition of Cdk2- stops cell cycle progression until the damage is repaired
M Control
Anaphase-promoting complex (APC)
Destroys B cyclins
attaching to ubiquitin-target for degradation by proteasomes
Separation of chromatids depends on the breakdown of the cohesins
Anaphase begins when the APC destroys securin
Separase is kept inactive by the securin (inhibitory chaperone)
Cohesin breakdown is caused by a protease called Separase
Allows sister chromatids at the metaphase plate to separate and move to the poles (anaphase)
Is activated by the M-phase promoting factor
M-phase promoting factor
These events take the cell to the metaphase of mitosis
mitotic B cyclins bound to M-phase Cdk (Cdk1) lead to
Cessation of all gene transcription
Breakdown of the nuclear envelope
Assembly of the mitotic spindle
CD1
Cyclin B
S Control
Mechanisms
Reactions leading to S phase and M phase
DNA replication continues
mitotic cyclins begin to rise (in G2)
- cyclin E is destroyed
Cdk activation/inactivation activity
Cyclin E binds to Cdk2
S-phase promoting factor (SPF)
prepare the cell to duplicate its DNA
enters the nucleus
cyclin A bound to Cdk2
CD2
Cyclins E and A
G1 Control
Mechanism
REgulation of the G1-S transition by p53
G1 Cyclins bind to their Cdks
cell prepares for replication
Proteins Involved
CD4, 6
Cyclin D
Meiosis Summary
Meiosis II
Segregation of the different paternal or maternal alleles of each gene
The 2 daughter cells from meiosis I divide to form 4 haploid cells with 23 chromosomes
Similar to an ordinary mitosis except the chromosome number is haploid
Meiosis I
Cytokinesis
-cell divides into 2 daughter cells and enters mitotic interphase
there is no S phase between the first and second meiotic divisions
-oogenesis
secondary oocytes receives almost all the cytoplasm and the reciprocal product becomes the first polar body
spermatogenesis
the cytoplasm is more or less equally distributed between the new cells
Telophase I
-the 2 sets of chromosomes are grouped at the opposite poles
Anaphase I
-errors can occur
meiotic arrest or cell death
missegregation of chromosomes
nondisjunction
-original paternal and maternal chromosome sets are sorted into random combinations (223)
-e.g a typical chromosome will have 3 to 5 segments alternately paternal and maternal in origin
-chromosome number is reduced in half
disjunction takes place
2 members of each bivalent move apart and the centromeres are drawn to opposite poles
Metaphase I
-paired chromosomes align themselves on the equatorial plane
-spindle forms
-nuclear membrane disappears
Prophase I
Diakinesis
Chromosomes reach maximal condensation
Diplotene
The 2 homologs are held together at chiasmata (crosses)
- ~ 50 in spermatocytes
2 components of each bivalent now begin to separate
Synaptonemal complex begins to break down
Pachytene
Meiotic crossing over takes place
Homologues appear as a bivalent (tetrad)
Chromosomes tightly coiled
Zygotene
Chromosomes held together by a synaptonemal complex
homologous chromosomes begin to align along their length
pairing/synapsis
Leptotene
Sister chromatids closely aligned
chromosomes replicated in S phase become visible and condensate
M - Mitosis
Telophase
The nuclei resume interphase appearance
Two daughters nuclei are formed
Nuclear membrane forms
Chromosomes decondensate
Anaphase
The chromatids become daughter chromosomes, moving at the poles
Chromosomes separate at the centromere
Begins suddenly
Metaphase
Facilitate the analysis of chromosomal abnormalities
Balanced by equal forces from the kinetochore
Chromosomes arranged in the equatorial plane of the cell
Chromosomes reach maximum condensation
Prometaphase
Continue condensation of the chromosomes
Chromosomes move towards the midway between the poles
= congression
Chromosomes attach by the kinetochores to microtubules of the mitotic spindle
Chromosomes disperse within the cell
Nuclear membrane breaks up
Prophase
Centrosomes gradually move to the poles of the cell
Pair of microtubule organizing center = centrosomes form foci
Beginning of the formation of the mitotic spindle
Gradual condensation of the chromosomes
Begins mitosis
G2 - Gap
preparation for mitosis
individual chromosomes begin to condense and become visible under microscope
cells enlarge
no DNA synthesis
- RNA and protein synthesis continues
at the end of S phase
S - Synthesis
synthesis of DNA and duplication of the centrosomes
DNA synthesis
- DNA content of the cell doubled
each chromosome replicates
– two sister chromatids
chromatids held together by the centromeres
associate with proteins=kinetochore
chromatids have at the end the telomeres
identical copy of the original DNA double helix
begins at hundreds-thousands of sites
origins of DNA replication
follows G1 phase
G1 - Gap
Liver cells
Cell damage?
may enter G0 but return to G1
some cells pass through this stage in hours, others in days or years
immediately after mitosis or G0
growth and preparation of the chromosomes for replication
G0 - Gap
quiescence can be either:
Permanent
Example:
Red blood cells too?
Nerve cells
Most lymphocytes in human blood are in Go
but with proper stimulation reenter the cell cycle in G1
never reenter the cell cycle, but carry their function until they die
terminally differentiated
Temporary
Cells in Go often called “quiescent
active repression of genes needed for mitosis
still attack pathogens
still have secretion
e.g. polymorphisms giving rise to blue eyes versus brown eyes, straight hair versus curly hair
observable characteristics of an individual that have developed under the combined influences of the individual’s genotype and the effects of environmental factors
HapMap
Health Benefits of the HapMap
Genetic variants contributing to longevity or resistance to disease could be identified, leading to new therapies with widespread benefits
Medical treatments could be customized, based on a patient's genetic make-up, to maximize effectiveness and minimize side effects
Cancer, stroke, heart disease, diabetes, depression, and asthma - result from the combined effects of a number of genetic variants and environmental factors
Identifying haplotypes can help in association studies (compare the haplotypes in individuals with a disease to the haplotypes of a comparable group of individuals without a disease )
Construction of the HapMap
Selected Populations
The DNA samples for the HapMap have come from a total of 270 people
Thirty U.S. trios provided samples - collected in 1980 from U.S. residents with northern and western European ancestry
In China, 45 unrelated individuals from Beijing provided samples
In Japan, 45 unrelated individuals from the Tokyo area provided samples
The Yoruba people of Ibadan, Nigeria, provided 30 sets of samples from two parents and an adult child
“Tag” SNPs within haplotypes are identified that uniquely identify those haplotypes
Adjacent SNPs that are inherited together are compiled into “haplotypes”
Single nucleotide polymorphisms (SNPs) are identified in DNA samples from multiple individuals
Hapmap Project is designed to provide information that other researchers can use to link genetic variants to the risk for specific diseases
A Hapmap is catalog of common genetic variants
how they are distributed among people within populations and among populations in different parts of the world
where they occur in our DNA
what these variants are,
Derived from Genetic Sequences
SNPs
Haploptypes
Origin of Haplotypes
all humans today are descended from ancestors who lived in Africa about 150,000 years ago
Some of the segments of the ancestral chromosomes occur as regions of DNA sequences that are shared by multiple individuals
Over the course of many generations, segments of the ancestral chromosomes in an interbreeding population are shuffled through repeated recombination events
Genetic variants that are near each other tend to be inherited together
E.g. all of the people who have an A rather than a G at a particular location in a chromosome can have identical genetic variants at other SNPs in the chromosomal region surrounding the A
These regions of linked variants are known as haplotypes
There are about 10 million common SNPs in a person's chromosomes
Changes in the DNA sequences – may increase the risk to high blood pressure, cancer, other diseases
Similar among 2 individuals but about every 1,000 nucleotides, the sequences will differ
Genotyping Analysis
High-throughput SNP genotyping
Used in association studies
process of quickly and cost-effectively identification of the SNP in many individuals in a given population
detection of the genotypes of individual SNPs
exact description of the genetic constitution of an individual, with respect to a single trait or a larger set of traits
e.g. changes associated with disease or risk of disease
e.g. changes resulted from damage by external agents like radiation or viruses
Stable Polymorphisms
Inherited variation and polymorphism translated in polymorphic proteins
Are clinically important:
Transplantation
Blood Transfusion
Hemolytic Disease of the Newborn
Clinical Relevance
Major Histocompatibility complex
3 Classes of Clusters (I, II, III)
class I and class II genes correspond to the human leucocyte antigen genes (HLA)
class III unrelated to HLA genes
class III unrelated to HLA genes
Some genes are associated with diseases
hemochromatosis
congenital adrenal hyperplasia
Genes present within the MHC complex but are functionally unrelated to HLA class I and II
Class II
Encode integral membrane cell surface proteins
Heterodimers
Beta subunit
alpha subunit
3 polymorphic Class II molecules, each consisting of an α and β chain
HLA-DR
3 α loci, 9 β loci,
each with multiple alleles - many combinations
HLA-DQ
34 α, 96 β alleles
3264 combinations
HLA-DP
27 α, 133 β alleles
3591 combinations
Class I Genes
class I and class II genes correspond to the human leucocyte antigen genes (HLA)
Encode proteins that are integral part of the plasma membrane of all nucleated cells
2 polypeptide subunits
another polypeptide, β2-microglobulin
a variable heavy chain
3 polymorphic class I α chain genes:
HLA-C
439 alleles
HLA-B
1178 alleles
HLA-A
767 alleles
MHC – composed of a large cluster of genes located on the short arm of chromosome 6
Blood groups and their polymorphisms
Rh System
The name comes from Rhesus monkeys – used for the experiments leading to the this system discovery
Hemolytic Diseases of the Newborn
Treatment:
Rh immune globulin at 28 to 32 weeks of gestation and again after pregnancy
If a Rh-negative pregnant woman – is carrying an Rh-positive fetus, hemolytic disease of the newborn can result:
The mother forms antibodies that will return to the fetus and damage the fetal red blood cells
Small amounts of fetal blood cross the placental barrier and reach the maternal blood stream
Phenotypes
Rh-negative individuals
Frequency of Rh negative varies enormously: and
0.5% among Japanese
7% in African Americans
~17% in Whites,
do not express the antigen
Rh-positive individuals
who express, on their red blood cells, the antigen Rh-D, a polypeptide encoded by a gene RHD on chromosome 1
Rh factors are glycoproteins encoded by alleles at 3 loci (C,D,E) exhibiting dominant/recessive expression
E
D
THey are encoded by the gene RHD which is found on chromosome 1
The D locus products are the most significant in terms of immune response
C
ABO Blood Groups
Blood Donation
Preferably a patient receives blood of his or her own ABO group
ABO compatibility of donor and recipient – essential to graft survival
Multiallelism:
4 phenotypes
Group O
Antigens Present: None
Antibodies Present: Anti-A, Anti-B
Group AB
Distribution: South America; North America
Antigens Present: A and B Antigens
Antibodies Present: None
Group B
Distribution: Asia
Antigens Present: B Antigen
Antibodies Present: Anti-A
Group A
Distribution: Europe, Australia, Northern Canada
Antigens Present: A antigen
Antibodies Present: Anti-B
Three alleles, two of which (A and B) are codominant and the third (O) is recessive (silent)
The O allele has a single base-pair deletion which causes a frame-shift mutation that eliminates the transferase activity in type O individuals
There is a four-nucleotide sequence difference between A and B alleles resulting in amino acid changes that alter the specificity of the galactosyl transferase encoded by the ABO gene
ABO polysaccharide antigens are exquisitely immunogenic
Determine by one gene on chromosome 9
– that encodes a galactosyl transferase
Inherited variation and polymorphism in DNA
Variable Number Tandem Repeats
Insertion-deletion polymorphisms
Short segments of 2, 3 or 4 to 6 bp repeated
Are prone to deletion, insertion, or duplication
Are used as molecular markers for kindship and in population studies
Have high mutation rate
become useful for examining relationships among individuals and breeding groups within populations
ensures high level of polymorphism
attributed to relatively high rates of error during during
recombination (unequal crossover)
DNA replication (slippage) and
are sections of DNA composed of short motifs (e.g. CA, GTG, TGCT etc) arranged in tandem
One common example - (CA)n repeat, where n is variable between alleles
Are repeating sequences of 1-5,6 base pairs of DNA
can be used as genetic markers in
DNA fingerprinting
DNA fingerprinting is widely used for individual identification
testing of paternity
the remains of victims and military personnel
suspects in criminal cases
Only identical twins show the same pattern
Probes are selected that identify VNTRs at many different loci
The most informative markers have several alleles, so that no two unrelated individuals would exhibit the same pattern upon electrophoresis
Each probe would generate a complex, unique pattern based on the VNTRs it picks up
Probes can be designed to detect many of these VNTR loci simultaneously
DNA sequences in the VNTR polymorphs, scattered throughout the genome are somewhat homologous to each other
forensic investigations,
linkage analysis of genomes
Each variant acts as an inherited allele
A short nucleotide sequence is organized as a tandem repeat
can have different length between individuals
These sequences are found on different chromosomes
Single Nucleotide polymorphisms (SNPs)
A source of variation in a genome
A subset of ~10% of the most frequent SNPs chosen to serve as the markers for a high-density map of the human genome (hHapMap)
Since the haploid genome is 3 × 109 bp, 3 million differences between any two randomly chosen individuals
1 in 1000 bp differ in any two randomly chosen humans
a single base difference in DNA
Transversion substitution
between a purine and pyrimidine
Transition substitution
between purines (A,G) or between pyrimidines (C,T)
Consists of large amounts of the chemical deoxyribonucleic acid (DNA) – contains the genetic information needed for all aspects of a functional human organism
what are dna ratios ? 1 chromosome per mitoch? and 23 pairs per nucleus?
37 genes
24 RNA-encoding genes
22 tRNAs
corresponding to each AA
2 rRNAs (16S and 23S)
13 Protein-encoding genes
All other mitochondrial proteins (enzymes of the citric acid cycle, DNA and RNA polymerases)
imported into mitochondria via chaperone proteins
(hsp60, hsp 70)
synthesized on cytoplasmic ribosomes
encoded by nuclear DNA,
The 13 protein-encoding genes are subunits of enzymes of oxidative phosphorylation (OXPHOS):
1 subunit (cytochome b) of the cytochrome c oxidoreductase complex
2 subunits of the F0 ATPase complex
3 subunits of the cytochrome oxidase complex
7 subunits of the NADH dehydrogenase complex
All mtDNA genes are maternally inherited in humans
All mtDNA genes contain only exons
The Genome includes
Repeated Sequences
Segmental duplications
can span hundreds of kilobase pairs
5% of the genome
Interspersed Repeated Sequences
LINEs (long interspersed nuclear elements) family
found in about 850,000 copies per genome
20% of the genome
-are up to 6-7kb in length
SINEs (short interspersed nuclear elements): Alu family
more than a million Alu family members in the genome
10% of human DNA
are about 300 base pairs in length
Clustered Repeated Sequences
Microsatellites
Minisatellites
long arrays of repeats
found in large inert regions on:
Chromosome 7
Chromosome 16
Chromosome 9
Chromosome 1
α-Satellite DNA
at the centromere of each human chromosome
copies of 171-base pair unit
can be several million base pairs
several percent of the DNA content of an individual chromosome
-=tandem repeats
Satellite DNAs
10-15% of the genome
Unique Sequences
Types
Non-repetitive DNA that is neither intron nor coding
Genes
Epigenetics & Epigenetic Control
Human Epigenome Project (HEP)
it constitutes the main and so far missing link between genetics, disease and the environment that is widely thought to play a decisive role in the etiology of human pathologies
Methylation is the only flexible genomic parameter that can change genome function under exogenous influence
methylation variable positions (MVPs) are common epigenetic markers
aims to identify, catalogue and interpret genome-wide DNA methylation patterns of all human genes in all major tissues
Affects the transcription of protein-encoding and RNA-encoding genes
Gene expression can be altered (to produce altered trait or disase) by chemical modification of
Histones (via acetylation, phosphorylation, ubiquitinization)
Chromatin remodeling complexes
control the local structure of chromatin
large multiprotein complexes containing helicases, which can unwind DNA double helices
works in conjunction with DNA methylation
Methods
Ubiquitination
Phosphorylation
Methylation
Acetylation
Acetylation of histone increases the likelihood of neighboring DNA segments to be transcrib
DNA (via methylation)
involved in genomic imprinting
Genomic imprinting involved in normal development and several diseases
Methylation of cytosine nucleotides
induces silencing
decreases the probability of that segment being transcribed
Gene Families
Dispersed
Exon-intron pattern is closely conserved –
more base changes have accumulated in the intron sequences than in the exons
Genes within each cluster are more similar in sequence -
evolved by duplication over last 100 million years
Two clusters code for closely related globin chains expressed at different development stages from embryo to adult
Genes encoding related functional proteins can be dispersed on a single chromosome or on 2 or more different chromosomes
Clusters
Multiple Clusters
located throughout the genome
e.g. Immunoglobulin superfamily
hundreds of genes encoding a conserved domain of ~ 70 aa which provide a distinct structural protein motif;
Compound Clusters
E.g. Major Histocompatibility Complex - HLA genes
related and unrelated genes are clustered on a single chromosome
Single Clusters
genes are controlled by a single expression control locus
e.g. Ig H chain
close cluster-
genes are organized as tandem repeated array
Gene superfamily:
a group of genes that exhibit low sequence homology, but they are similar in the encoded protein function and structure
(e.g. Ig super family, globin super family, myosins, G-protein receptor super family)
Classic gene family
a group of genes that exhibit a high degree of sequence homology over most of the gene length
Gene families thought to have arisen by duplication of primitive precursor genes as long as 500 million years ago
Many genes belong to families of closely related DNA sequences
RNA-encoding genes
3 major classes
Regulatory
Riboswitch RNA (site on mRNA)
Anti-sense RNA (block mRNA)
Micro RNA (miRNA; block mRNA)
Regulatory (usually inhibitory)
Transfer (tRNA)
Ribosomal (rRNA) and splicesomal (snRNA)
can alter gene expression, and also produce altered traits or disease
produce non-protein translated RNAs
can profoundly alter normal gene expression and hence produce an altered trait or disease
not a protein
Protein-encoding genes
-mutation or alteration in the DNA sequence results in an altered trait or disease
-gene encodes the message transcribed to pre-RNA, to mRNA, which is translated into a product protein
Components
Introns
Some genes lack introns
e.g. histones
There is a direct correlation between gene size and intron size
typically several are found in most genes
total size of which frequently exceeds that of exons
highly variable in size
Non-coding sequences
Exons
small proportion of whole genome
200bp
Protein/RNA encoding regions
Size can be variable
Examples
2400kb
Dystrophin gene
45 kb
LDL gene
1.7 kb
Insulin Gene
Promoters and regulatory elements can be sites of mutation
enhancers, silencers, locus control regions
(either at 5’ or 3’ of the gene or in its introns)
Adjacent nucleotide sequences – provide “start” and “stop” signals for the synthesis of mRNA transcribed
a sequence of DNA that is required for production of a functional product (polypeptide or RNA molecule)
Most single-copy DNA is found in short stretches, interspersed with various repetitive DNA families
Makes up about half of the DNA in the genome
Chromatin
during cell division – chromatin condenses –visible microscopically as discrete structures
Chromatin Packing
Chromosomes pass through stages of condensation and decondensation into a DNA mitotic chromosome that is 10,000x shorter than its extended length.
Decondensed
Interphase Nucleus
most decondensed stage – interphase
Distinctive chromosome puffs arise and old puffs recede as new genes are expressed and old ones are turned off
Chromatin loops decondense at loop domain when the genes are expressed
Achieved by
RNA polymerase
CHromatin remodelling complexes
Histone modifying enzymes
Each loop contains -10,000bp of DNA
Stages of Condensation
Whole Mitotic Chromosome
Condensed section of chromosome
700nm
Loops
Solenoids packed into loops attached at intervals of about 100,000 base pairs to a protein scaffold (H1)
Solenoid Fibres
Most condensed phase
-30 nm diameter
Nucleosomes compacted into a secondary helical structure
Nucleosome Fibre
beads-on-a-string
-10 to 11 nm
140 bp of DNA
Five types of histones –packing of chromatin:
H1
binds to DNA in the internucleosomal spacer region and participates in compaction
H2A, H2B, H3 and H4
Variation
Histone Code
The pattern of major and specialized histones + their modifications
Specific to cell types
Histones H3 and H4 can be modified – post-translational modifications
– can change the properties of nucleosomes that contain them
There are specialized histones – can substitute for H3 and H2A
– specific characteristics to the DNA
2 copies of H2A, H2B, H3 and H4 form an octamer around which a segment of DNA double helix winds
There is 20-60 base pair “spacer”
~140 base pairs of DNA associated with each histone core
Core dna has linker dna on both ends around the histone
nucleosomes
110 A across
55A tall
Double Helix
2nm in width
distributed throughout the nucleus, relatively homogenous under the microscope
Genomic DNA - complexed with chromosomal proteins (histones and nonhistones)
Chromosomes
found in nucleus and nucleolus
Structure
Each chromosome consists of a single, continuous DNA double helix
3 Forms
Z-DNA
B-DNA
This form is the one usually found under physiological conditions
A-DNA
1 helical turn is 3.4nm
2 nm in diameter
Minor and Major Grooves along length
– 46 DNA molecules = 6 billion nucleotides
Human chromosomes showing the centromeres and well-defined chromatids
Active or inactive centromeres surrounded by flanking heterochromatin
Karyotype
are studied in Cytogenetics for their clinical relevance with regards to
-Prenatal Diagnosis
-Cancer Cytogenetics
-Gene Mapping and Identification
-Clinical Diagnosis
Contain the genes aligned at specific position or locus
Specific number and morphology for each species
Chromosomes complement
98% non-coding DNA
2% of the nuclear genome – coding DNA
~ 3000 RNA-encoding genes (DNA to RNA; no translation to protein)
~27,000 protein-encoding genes (DNA to mRNA to protein)
46 chromosomes:
x and Y sex chromosomes
22 autosomes
Nutrigenomics
Study of molecular relationships between nutrition and the response of genes
Pharmacogenetics
Study of genetic basis for variation in drug response
Clinical genetics
Application of genetics to diagnosis & patient care
Developmental genetics
Study of the genetic control of development
Population genetics
Study of genetic variation in human population & the factors that determine allele frequencies
Genomics
Study of the genome, its organization & functions
Molecular & Biochemical genetics
Study of the structure & function of individual genes
Cytogenetics
Study of chromosomes, their structure & inheritance
Cytogenetic analysis
Subtopic
Performed in cells capable of growth and rapid division in culture (e.g. T lymphocytes)
Cell types used for chromosomal analysis
Sources include:
Solid tumors
Bone marrow
Fibroblast cultures
Chorionic villus
Amniotic fluid
Peripheral blood ( T lymphocytes)
Clinical indications for cytogenetic test
Cancer
Useful for diagnosis or prognosis
Chromosomal aberrations in almost all cancers
Family history
Chromosome abnormality in a first-degree relative
Advanced age of a pregnant woman
Should be offered as a prenatal test
Increased risk of chromosomal aberrations in women older than 35 years
Fertility problems
Women presenting amenorrhea
Couples with recurrent miscarriages
Couples with history of infertility
Stillbirth and neonatal deaths
Important for genetic counseling and prenatal diagnosis in future pregnancies
Abnormalities of early growth and development
Ambiguous genitalia
Mental retardation
Failure to thrive
Developmental delays
Multiple congenital malformations
Chromosomal abnormalities
Chromosomal disorders
Observed in
2% of pregnancies of women older than 35 years
20% of second trimester spontaneous abortions
~50% of spontaneous first-trimester abortions
Occur in ~1 of every 150 live births
Chromosomes nomenclature
Chart of Chromosome Nomenclature
Chromosomes classification
Acrocentric
Submetacentric
Metacentric
Banding
Example - Idiogram
detection of deletions, duplications
correct identification of individual chromosomes
-specific chromosome bands
Basic terminology for banded chromosomes - Paris 1971
International System for Human Cytogenetic Nomenclature (ISCN) – nomenclature reports
Chromosomes morphology
p=petit
q=queue
Telomeres
reduction in telomerase and decrease in number repeats important in aging and cell death
maintained by enzyme – telomerase
telomere consists of tandem repeats TTAGGG
seal chromosomes and retain chromosome integrity
tip of each chromosome
Centromeres
Divides the chromosome into
long (q=queue) arms
short (p=petit) arms
movement during cell division
Definitions
Karyotype
test to identify and evaluate chromosomes in different cell types for their
Shape
Size
Number
Clinical Cytogenetics
the study of the chromosomes and their abnormalities
Deals with the subset of human genetic variation that is of significance in the practice of medicine & in medical research (refers to abnormal hereditary patterns/variations that lead to pathological conditions).
Study of variation & heredity in human beings (refers to normal hereditary patterns).
Watson & Crick
1953-62
Established the molecular structure of the "gene" encoded by DNA ,
the basic chemical units of hereditary
Concerns the chemical nature of the gene itself: how genetic information is encoded, replicated and expressed
Charles Darwin
Feb 12 1809 to Apr 19 1882
Theory of Evolution
Came up with Theory of Evolution which rests on 3 principles:
Principle of Selection
Some forms are more successful at surviving and reproducing than other forms in a given environment
Principle of Heredity
Offspring resemble their parents more than they resemble unrelated individuals
Principle of Variation
Among individuals within any population, there is a variation in morphology, physiology and behavior
The voyage of the beagle led to the origin of species
Explores the genetic composition of individual members of the same species(population) and how that composition changes over time and geographic space
Involved with species variations and survival risks
G. J. Mendel
July 20 1822 to Jan 6 1884 - FATHER OF GENETICS
Experiments on Plant Hybridization – presented in 1865
Significance
Mendel’s work is still recognized as the foundation of modern genetics
Originally formulated for cultivated peas, Mendelian laws can also be applied to determine the pattern of transmission of inherited human diseases
Mendelian Laws
The Principle of Incomplete Dominance
when the heterozygote has a phenotype intermediate between the phenotypes of the two homozygotes
e.g. Fruit Colour in Eggplant
a) P generation: PP purple x pp white
b) F1: Pp violet crossed with self
c) F2: 1 PP purple: 2 Pp violet: 1: pp white
Conclusion:
genotypic ratios and phenotypic ratios remain the same.
Fruit colour in eggplant is inherited as an incompletely dominant characteristic
The principle of independent assortment
when two alleles separate, their separation is independent of the separation of alleles at other loci
Experiment: Do alleles coding different traits separate independently Methods P generation Round Yellow Seeds (RRYY) x wrinkled Green seeds (rryy) F1 – Round, Yellow Seeds (RrYy) crossed with self F2 – 9 round yellow: 3 round green: 3 wrinkled yellow: 1 wrinkled green; Conclusion: the allele encoding colour separated independently from the allele encoding seed shape, producing a 9:3:3:1 phenotypic ratio in the F2 progeny;
The principle of segregation
two alleles of a locus on homologous chromosomes separate when gametes are formed
e.g. R, round, r, wrinkled
Encompasses the basic heredity and how traits are passed from one generation to another
Demonstrated the transmission of genetic traits from one generation to the next
Mendelian Conclusions
A trait may not show up in an individual but can still be passed on to the next generation
An individual inherits one such unit from each parent for each trait
The inheritance of each trait is determined by "units" or "factors" (now called genes) that are passed on to descendents unchanged
Procedure
F1 crossed with P1 homozygous recessive to reveal ½ dominant and ½ recessive phenotypes in the F2
e.g. Yy yellow x yy green = ½ Yy yellow, ½ yy green
F1 was crossed with self to reveal ¾ dominant and ¼ recessive phenotypes in the F2.
e.g. Yy yellow x Yy yellow = ¼ YY yellow, ½ Yy yellow, and ¼ yy green
Homozygous Dominant and Recessive Parental Generation (P) crossed to make F1 progeny which was all dominant phenotype
e.g. Yellow pea (YY) x Green (yy) = 100% Yy, yellow progeny
The Human Genome Project
The project was started by the National Institutes of Health and the U.S. Department of Energy in an effort to reach six set goals:
Addressing the ethical, legal, and social issues (ELSI) that may arise from the project
Transferring technologies to private sectors
Improving the tools used for data analysis
Storing all found information into databases
Determining sequences of chemical based pairs in human DNA
Identifying all genes in human DNA
(initial estimate were approximately 100,000 genes which was reduced as research progressed)– only 25,000 genes have true known functions.
the project finished in April 2003, taking only thirteen years (50th years anniversary from DNA double helix structure by Watson & Crick)
was a molecular genetics project thatbegan in 1990 and was projected to take fifteen years to complete
Allan Maxam and Walter Gilbert (Harvard) and Frederick Sanger (U.K. Medical research Council) independently develop methods for sequencing DNA
Paul Berg - and co-workers create the first recombinant DNA molecule
the correct number of human chromosomes were specified – led to the discovery in 1959 that Down syndrome is caused by an extra chromosome 21
Hershey-Chase - identified DNA (rather than protein) as the genetic material of viruses, further evidence that DNA was the molecule responsible for inheritance
Experiment with E.Coliand bacterial chromosome plus phage and phage chromosome
1. Phage attaches to E. Coli and injects chromosme
2. Bacterial Chromosome break down and the phage chromosome replicates
3. Expression of Phage genes produces Phage structural component
4. Progeny Phage particles assemble
5. Bacterial wall lyses releasing progeny phages.
Avery, McLeod and McCarthy - demonstrated that the transforming factor in bacterial transformation of non-virulent to virulent form was DNA
Phoebus A. Levene - indicated nucleic acids contained four nitrogenous bases - thymine, cytosine, guanine and adenine (RNA contained uracil, but not thymine)
Fredrick Griffith - reported the phenomenon which came to be known as TRANSFORMATION - transformed non-virulent Streptococcus pneumonia to a virulent form by cell-free materials from virulent bacteria
Johannsen coined the term “gene” as unit of heredity
Garrod described alkaptonuria as the first “inborn error of metabolism”
Landsteiner discover ABO blood group
Edmund Wilson - suggested that inheritance might result from the transmission of chemical compounds - proposed nucleic acid or protein as the candidate
The lack of knowledge of the nucleic acids and their versatility, combined with the advances in protein chemistry, led to the belief that "protein" is the right candidate
Fredrich Miescher - extracted a substance he called "nuclein" composed of protein and nucleic acid from the cell nuclei
Mendel - published the work on inheritance but was overlooked for many years until 1900
