Genetics
INDIAN DENTAL ACADEMY Leader in continuing dental education www.indiandentalacademy.com
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• Recombinant DNA • Patterns of inheritance • Biochemical genetics • Population studies • Human genome project • Cloning
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The Beginning • Gregor Mendel – Pisum sativum – pairs of contrasting characteristics in the garden pea. – Tall or dwarf, yellow or green seeds, violet or white flowers etc.
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Early Genetics • Carvings in stone – 6000 years old • Haemophillia - 1500 years ago • Regnier de Graaf – male and the female parent transmitted genetic characteristics to the off spring.
• Pierre Louis Moreau de Maupertuis -1700s – hereditary particles – one from each parent www.indiandentalacademy.com
Mendel’s Experiments TT
tt
F1
Tt
F2
TT
F3
All Dominant
Tt Same as F2
Tt
tt All Recessive
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Mendel’s Laws • Mendel’s 1st law, or the Law of Segregation • 2 factors for a specific character • parent transmits only one to offspring • matter of chance as to which unite
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Mendel’s Laws
Female Gametes
• Punnet’s Square
Male Gametes T
t
T
TT
Tt
t
Tt
tt
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Mendel’s Laws • The law of Unit Inheritance – Before Mendel’s time it was believed that the characteristics of parents blended into the offspring. (Darwin) • Blending did not occur. • Characteristics of one parent may not appear in one generation (F1) but may reappear in the next generation (F2). www.indiandentalacademy.com
Mendel’s Laws • Law of Independent assortment – Members of different gene pairs assort to the gametes (sex cells) independently of one another. Tt T t • Random recombination
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Mendel’s Laws • Work of Mendel was not widely noticed – • Darwin’s theory – based on inheritance But mechanism not known • Rediscovered – – Vries - Holland, – Correns - Germany and – Tschermak - Austria www.indiandentalacademy.com
Terminology • Homozygous – an individual who has the same factors for a particular characteristic. (eg-TT or yy) • Heterozygous – individual with different factors (Tt or Yy) – character that is manifested - dominant, – and the other - recessive .
• The term gene - Danish botanist Johannsen, represents the hereditary factors. • The genes responsible for contrasting characters are called alleles.
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Human Genetics • Family Studies – pedigree • Albinism, Polydactyly (early 1700s) and Hemophilia (early 1800s). Consanguineous marriages. • Effects of Nature and Nurture 1800s by Galton – Hereditary improvement of men and animals by selective breeding – eugenics. www.indiandentalacademy.com
Human Genetics • 1900s Sir Garrod • Alkaptonuria - dark urine • Children were usually normal, but the disorder could reappear later in the descendents. • Mendelean recessive type of inheritance • Excretion of homogentisic acid which is usually metabolized in normal individuals. www.indiandentalacademy.com
Human Genetics • Connection between gene and enzyme • This was the first time that the idea that genes control the synthesis of enzymes arose. • Landsteinter ABO blood groups Blood group genetics
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Nucleic Acid • First isolated as early as 1869 by a Swiss doctor named Meicher. • Rich in phosphorous • Nuclein • 1892 – postulated that it is the hereditary material.
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Nucleic Acid • Protein was the basic substance of life • Importance in protein formation was not appreciated until the work of Griffith and later Avery, Macleod and McCarthy (on pneumococci) and Hershey and Chase (using bacteriophages). www.indiandentalacademy.com
Nucleic Acid - Structure • long chains of molecules called nucleotides • Each nucleotide is composed of :– A nitrogenous base – A sugar molecule, and – A phosphate molecule
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Nucleic Acid - Structure • The nitrogenous bases are of 2 types – purines & pyrimidines. • The purines include – adenine and guanine • The pyrimidines include – cytosine, thymine and uracil.
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Nucleic Acid - Structure • Nucleic acids – 2 types (according to sugar) • Ribose Ribonucleic acid or RNA - nucleolus and cytoplasm • Deoxyribose Deoxyribonucleic acid or DNA – nucleus
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Nucleic Acid - Structure Structure of DNA Requirements – 1. Versatile. 2. Produce identical replica is formed at each cell division.
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Structure of DNA
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Structure of DNA • Purine ↔ Pyrimidine • Guanine ↔ cytosine • Adenine ↔thymine
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Replication • Semi-conservative method. • Complementary chain is formed • Daughter cell – one parent strand – one new strand www.indiandentalacademy.com
Genetic Code • Genes code the sequence of the amino acids • 4 bases 20 amino acids • 3 bases are essential for coding the amino acids. • Triplet code = codon www.indiandentalacademy.com
Genetic Code
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Transcription • Information from DNA messenger – RNA • Complementary bases are found in the RNA. – Cytosine with guanine, – thymine with adenine, and – adenine with uracil www.indiandentalacademy.com
Translation • mRNA associates with ribosomes • Proteins formed around mRNA template • Amino acids collected from cytoplasm (AA + ATP) ↔ tRNA Ribosome www.indiandentalacademy.com
“Central dogma”
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A Closer Look at Genes • But about 80% of human DNA does not code for proteins. • The coding part of the DNA is known as exons, and the intervening noncoding sequences are called introns.
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A Closer Look at Genes • These genes have a regulatory effect. • In prokaryots (without nucleus) – Operon = operator gene and structural gene – Remote regulator gene • Produces repressor. – Repressor can be inactivated by inducer.
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A Closer Look at Genes • In humans – certain flanking regions referred to as enhancers and promoters. • Structure of Globin gene – CAT box = CCAAT – TATA box = TATA – End flanking region = AATAAA
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A Closer Look at Genes • Exon-intron pattern of a particular gene appears to be conserved during evolution • Two introns at precisely the same locations since their appearance 500 million years ago • Alterations in exons are slow, and mutations are rarely retained • Changes in the introns occur much more rapidly www.indiandentalacademy.com
Structure of Chromosomes • There are several meters of DNA in a human body, and the total length of the chromosomes is less than a millimeter. • Finch and Klug suggested the “Solenoid model”
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Structure of Chromosomes • Each DNA duplex is coiled around itself – primary coiling • This is couled around histone ‘beads’ – secondary coiling – called nucleosomes • Nucleosomes are coiled to form chromatin fibres, around a protein matrix or scaffold – tertiary coiling • Chromatin fibres are coiled to form loops – quaternary coiling • The loops are further wound in a tight helix to form the chromosome – that can be seen under a microscope. www.indiandentalacademy.com
Structure of Chromosomes
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Human Chromosomes • 46 chromosomes in the normal human – 23 pairs. – 22 pairs are alike in males and females – known as autosomes – 1 pair differs – the sex chromosomes.
• Pair of chromosomes are microscopically indisdtinguishable, except the x and y chromosomes. • Y is smaller than x, but the 2 are thought to have a homologous short segment. www.indiandentalacademy.com
Studying Chromosomes • cells to be studied must be able to grow and divide rapidly. • WBC cultures - usually short lived • skin cultures – for biochemical and histochemical studies
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Studying Chromosomes Cells Culture
phytohemagglutinin (mitogenic agent)
Cells rapidly dividing colchicine Metaphase hypotonic solution Staining
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Studying Chromosomes
Chromosome Spread
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Studying Chromosomes •1960 - Denver classification •7 chromosome groups (A through G) based on length and centromere position.
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Studying Chromosomes
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Studying Chromosomes
Paris Classification www.indiandentalacademy.com
Studying Chromosomes • The location of the centromere can be used to classify the chromosomes – – Metacentric – central centromere – Submetacentric - off –centre – Acrocentric – towards one end – Telocentric – terminal centromere (does not occur in man)
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Studying Chromosomes • Acrocentric - small masses of chromatin known as satellites attached to their short arms by narrow stalks (secondary constrictions) • Stalks contain the genes for 18S and 28S ribosomal RNA (rRNA). • The rRNA transcribed from these areas, along with 5S rRNA (from another location), is utilized in the nucleolus to synthesize ribosomes. www.indiandentalacademy.com
Studying Chromosomes • • • •
Autoradiography radioactive thymidine cell divisions are stopped not all chromosomes replicate at the same time. But this process is laborious and time consuming, and is rarely used.
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Studying ChromosomesStaining • After 1970 • Q banding - quinacrine mustard or related compounds – fluroscence microscopy.
• G banding – widely used – tripsin to denature the protein – Giemsa stain – dark bands correspond to the bright Q bands www.indiandentalacademy.com
Studying ChromosomesStaining • R banding – – – –
less widely used heat treated then stained with Giemsa results are the REVERSE of G banding
• C banding – centromere – regions of the chromosome containing constrictive heterochromatin – secondary constrictions of chromosomes 1, 9, 16
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Studying ChromosomesStaining • NOR staining – – ammoniacal silver to stain the nucleolus
• High resolution banding -– used for staining cells in prophase – shows much more bands than the metaphase staining.
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Studying Chromosomes- Medical applications •
Clinical diagnosis – congenital malformations, mental retardation disorders of sexual development etc.
•
Linkage and Mapping – Assignment of specific human genes to their chromosomal positions.
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Studying Chromosomes- Medical applications •
Polymorphisms – Minor heritable differences in chromosomes are common, especially in chromosomes 1, 9, and 16 and the Y chromosome.
•
Highly specific genetic marker
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Studying Chromosomes- Medical applications • Chromosomes and Neoplasia – Philadelphia chromosome • Reproductive problems • Prenatal Diagnosis – Amniocentesis – Useful in older pregnant women, and families with a history of chromosomal abnormalities. www.indiandentalacademy.com
Mitosis • • •
Nuclear material is conserved in the daughter cell Cytoplasm seem to split Nuclear division – 4 stages – – – –
Prophase Metaphase Anaphase Telophase www.indiandentalacademy.com
Mitosis • Interphase chromosome divides longitudinally into 2 daughter chromosomes, or chromatids, which remain attached to each other at the centromere.
Interphase
G2 M S
G1
G1 S G2 M
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Mitosis • Prophase – chromosomes can be seen and easily discerned – chromatids can be seen – centriole, – each one migrates to the opposite pole of the cell – nuclear membrane disappears and the nucleus begins to loose its identity.
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Mitosis • Metaphase – chromosomes are maximally contracted and deeply staining – 2 dimensional metaphase plate – spindle is now formed – (microtubles of protein) – spindle fibres centrioles to kinetochores (sites of attachment at the centromere)
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Mitosis • Prophase – chromosomes can be seen and easily discerned – chromatids can be seen – centriole, – each one migrates to the opposite pole of the cell – nuclear membrane disappears and the nucleus begins to loose its identity.
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Mitosis • Anaphase – centromeres divide – spindles contract – actin-myosin interactions?
• Telophase – daughter chromosomes arrive at the poles – Cytokinesis – chromosomes unwind – nuclear membrane www.indiandentalacademy.com
Meiosis • 2 phases – – Meiosis I – the reduction division – Meiosis II – an ordinary mitosis, without DNA replication
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Meiosis I - Prophase • Leptotene – chromosomes begin to condense – consist of alternating thick and thin regions – (chromomeres), characteristic for each chromosome. www.indiandentalacademy.com
Meiosis I - Prophase • Zygotene – pairing (synapsis) – bivalents
• Pachytene – chromomeres become more prominent – bivalent – actually a tetrad – crossing over occurs
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Meiosis I - Prophase • Diplotene – – Two components of the bivalent begin to separate – Centromere of each chromosome remains intact – Chromatids seem to be contact at several places, called chiasmata
• Diakinesis – more condensation www.indiandentalacademy.com
Meiosis I • Metaphase I – – the nuclear membrane disappears, and the chromosomes move to the equatorial plane.
• Anaphase I – 2 members of the bivalent disjoin, and one member goes to each pole
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Meiosis II • Resembles mitosis, but without DNA replication • Without an interphase • Centromeres divide, and the sister chromatids disjoin, passing to opposite poles and produce 2 daughter cells.
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Crossing Over • Reorganization of genes among the chromosomes hence increases genetic variability. • Chiasmata sites of cross over • 2 chromatids take part in any crossover. But all 4 chromatids of the bivalent may be simultaneously involved in crossovers at different sites. www.indiandentalacademy.com
Crossing Over • Crossing over in mitosis is much less common • Important effect in case of heterozygous cells
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Crossing Over • Sometimes crossing over can also occur between the sister chromatids. • Bloom Syndrome – growth retardation, prenatally and postnatally, and a butterfly rash is seen on the face
• But the correlation of these findings to the sister chromatid exchange is unknown.
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Recombinant DNA Technology • Genetic Engineering • Portion of DNA cut introduced into another host cultured • Discovery on restriction endonucleases (Smith in 1970). • A restriction endonulease is an enzyme that cuts DNA at specific sequences, called restriction sites. Over 200 such enzymes are known. www.indiandentalacademy.com
Recombinant DNA Technology • Principles of the technique – Use of the restriction enzyme to cut away a DNA fragment which includes certain gene/genes. – Incorporation of these fragments into a carrier or vector. – Transformation of a host organism eg-E. coli by the vector. – Culturing of the host organism in a suitable medium. – Selection of the bacteria containing the relavent DNA fragment.
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Recombinant DNA Technology • Cleavage of the DNA is done with the restriction endonucleases.
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Recombinant DNA Technology • DNA fragment vector. – Phage, or a plasmid (circular mass of DNA in bacteria, which replicate independent of the main bacterial chromosomes).
• The vector DNA is usually cleaved with the same enzyme as the DNA fragment, and the complementary base pairs that result are combined together.
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Recombinant DNA Technology • Bactriophages larger fragments of DNA, • cosmids – which are a combination of plasmids and phages Even larger fragments
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Recombinant DNA Technology • Vector is incorporated into a host bacteria (usually E. coli).
• Bacteria is cultured
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Recombinant DNA Technology • Southern Blot Technique
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Recombinant DNA Technology Applications • Analysis of gene structure • Restriction fragment length polymorphisms (RFLPs) – small changes in the nucleotide sequences without phenotypic effects (small deletions or insertions)
• length of the fragments generated by a particular restriction enzyme will be different, • inherited as Mendelian characters – As genetic markers – which can be used to study genetic structure – similar to blood groups and serum proteins. – In the prenatal diagnosis of any genetic disorder which is linked to an RFLP, or to identify carriers of the disorder. www.indiandentalacademy.com
Recombinant DNA Technology Applications •
In detecting the products of various genes
•
In producing probes to diagnose viral diseases like Hepatitis B or HIV
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Recombinant DNA Technology Therapeutic Applications • Biosynthesis of gene products – no antigenic properties, and free from contamination by HIV etc.
• Gene therapy – The missing gene is created and introduced into the host – single gene disorders • most genetic problems are multifactorial, this seems to have only limited application. www.indiandentalacademy.com
Genetics – Part II Dr. Punit Thawani
Mutations • Muller – Mutation of fruit flies by X-rays • 3 types – Substitution – Deletion – Insertion
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Mutations • Single Base Substitutions – alter the triplet codon – one amino acid to be replaced
• Deletions and insersions – if a single base pair is deleted or inserted, the entire frame of the DNA strand gets shifted
• Single base substitutions – proteins produced • Frame shift mutations – no proteins www.indiandentalacademy.com
Mutations • Chain termination mutations – – Termination codons can be added prematurely or be deleted
• Splice Mutations – – These interfere with the way introns are removed from the messenger RNA.
• Mutations in regulatory sequences – These affect the TATA box and the CAT box regions of the gene. www.indiandentalacademy.com
Inheritance • Diseases in families – The family history • Environmental factors play a role – Multifactorial disorders
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Inheritance • Single Gene disorders [1 in 2000 or less] • Chromosome disorders [7 in 1000] • Multifactorial disorders
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Inheritance •
Indications that a condition has a genetic etiology (Neel and Schull 1954) 1. Occurrence of a disease in definite proportions in families when environmental factors can be ruled out. 2. Absence of disease in unrelated lines 3. Characteristic age of onset, absence of precipitating factors
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Inheritance 4. More in monozygotic than dizygotic twins. 5. Demonstration of characteristic phenotype and chromosomal abnormality, with or without family history.
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Inheritance – Single gene disorders Dominant Autosomal Recessive Dominant Sex-linked (Gonosomal)
Recessive www.indiandentalacademy.com
Inheritance – Single gene disorders • Autosomal dominant – Rare – Patient usually heterozygous – Parent affected – New mutation – Achondroplasia, Osteogenesis imperfecta – Porphyria variegata – one couple (1688) – ½ the children affected – irrespective of sex www.indiandentalacademy.com
Inheritance – Single gene disorders • Experssivity – polydactyly • Non – penetrance • Sex influence – Gout – Presenile baldness – “Eunuchs neither get gout nor grow bald” Hippocrates
• Viral etiology? – Alzheimer’s disease. www.indiandentalacademy.com
Inheritance – Single gene disorders • Pattern of autosomal dominant inheritance
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Inheritance – Single gene disorders • Autosomal Recessive inheritance – Both sexes – homozygous – Extremely rare – heterozygous are normal A
a
A
AA
Aa
a
Aa
aa
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Inheritance – Single gene disorders • Consanguineous marriages – Chance that cousins will carry the same genes is 1 in 8 – The rarer the disease more probability of consanguineous marriage of parents. – Eg – Alkaptonuria
• Most common autosomal recessive disorder – Cystic fibrosis – 1 in 22 is a carrier. www.indiandentalacademy.com
Inheritance – Single gene disorders • Pattern of autosomal recessive inheritance
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Inheritance – Single gene disorders • Intermediate inheritance – Sickle cell trait – recessive/dominant?
• Codominance – Blood group AB
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Inheritance – Single gene disorders • Sex-Linked inheritance – X Linked – Y Linked
• Female must be homozygous • Male is hemizygous (since only 1 X chr.) • Eg – Haemophillia.
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Inheritance – Single gene disorders • Females with hemophillia – XO (Turner’s syndrome) – Homozygous – Mutation during gametogenesis – Manifesting heterozygote.
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Inheritance – Single gene disorders • Pattern of X linked recessive inheritance
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Inheritance – Single gene disorders • X linked dominant – Similar to autosomal dominant – Male transmits disease to all daughters but none of his sons – Vit. D resistant Rickets Xh
X
X
Y
XXh
XY
XXh
XY
X
X
X
Y
XX
XY
XX
XY
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Inheritance – Single gene disorders • Y linked inheritance – Hairy pinna – Transmitted from father to all his sons – Females not affected
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Inheritance – Single gene disorders • • • •
Establishing modes on inheritance Autosomal dominant – Verical pattern Autosomal recessive – Horizontal Sex linked – Oblique if male does not produce offspring
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Inheritance – Single gene disorders • Multiple alleles – Some characters may have more than one allele – Blood group genes • A1 A2 B O – Any one may be transmitted to the offspring.
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Inheritance – Chromosomal Abnormalities Chromosomal Abnormalities
Autosomes
Numerical
Sex Chromosomes
Structural
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Inheritance – Chromosomal Abnormalities • Numerical abnormalities – Aneuploidy – Polyploidy
• Monosomy – Lethal – one infant with monosomy 21
• Trisomy – Non-disjunction – Lejeune (1959) – Down’s syndrome – Patau’s syndrome – trisomy 13 – Edward’s syndrome – trisomy 18 www.indiandentalacademy.com
Inheritance – Chromosomal Abnormalities • Structural abnormalities – Translocations – exchange of segments – Deletion – loss of a segment.
• Translocation www.indiandentalacademy.com
Inheritance – Chromosomal Abnormalities • Gametes – (14/21) 14 14/21
14 14/21
21
21 21
Carrier
14/21 14 14
Down’s syndrome
21
Normal Monosomy - death www.indiandentalacademy.com
Inheritance – Chromosomal Abnormalities • Reciprocal translocation
• Person is normal • Gametes produced are abnormal
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Inheritance – Chromosomal Abnormalities • Deletions – Results in partial monosomy
• Lejeune – ‘cri du chat’ • Deletion of short arm of chromosome 5
• Formation of ring chromosomes www.indiandentalacademy.com
Inheritance – Chromosomal Abnormalities • Sex chromosome abnormalities – Kleinfelter’s syndrome – XXY – Turner’s syndrome – XO – Multiple X – XYY males
• Structural abnormalities – Isochromosome – long X
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Inheritance – Multifactorial • More than one gene involved + environment • Familial tendency – Incidence in family more than in general population – Less common than unifactorial disorders
• Normal traits – intelligence, skin colour, blood pressure, etc. • Abnormal traits – schizophrenia, diabetes, peptic ulcer, ischemic heart disease, ankylosing spondylitis etc. www.indiandentalacademy.com
Inheritance – Multifactorial • Relatives generally have higher incidence than general population
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Inheritance – Multifactorial • The incidence is greater among relatives of individuals with more severe form of the disease – Pt. with bilateral cleft lip – 6% – Pt. with unilateral cleft lip – 2.5%
• Similarly, subsequent children have more chance of being affected.
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Inheritance – Multifactorial Disorder • Heritability – proportion of the total variation of a Asthma character which can be attributed to CL/CP genetic factors. – Greater the heritability, greater the genetic component.
Heritability (%) 80 76
Hypertension
62
Peptic Ulcer
37 www.indiandentalacademy.com
Homeobox Genes • Development of a head-tail axis. • According to their location – cells differentiate – under the regulatory effect of homeotic or homeobox genes.
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Homeobox Genes • These genes contain a specific 180 base pair region called the homeobox. • Produces proteins – transcription factors – bind to DNA and regulate its expression. • First experiments on Drosophilla melanogaster later found in vertebrates.
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Homeobox Genes • Hox genes form “Hox codes” which specify the position of cells. • Anterior to posterior arrangement
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Homeobox Genes
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Homeobox Genes • In mice – disruption of Hox a-2 – Lack of derivatives of 2nd branchial arch – Some 2nd arch structures changed to 1st arch structures • Stapes missing – 2 malleus’
• Ectopic expression of Hox d-4 – Occipital bones to vertebrae
• Deletion of Hox a-3 and Hox d-3 – Atlas is deleted www.indiandentalacademy.com
Homeobox Genes • Each branchial arch exhibits a specific combination of Hox gene expression • No Hox genes been detected in the brain
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Homeobox Genes • Msx -1 development of secondary palate and tooth. – familial tooth agenesis – missing 2nd premolar and 3rd molar • Studies in Finnish families (Nieminen et al 1995).
• Msx- 2 Craniosynostosis • SHH Patterning of Neural crest and neural tube – Affects midline structures • Hytertelorism www.indiandentalacademy.com
Cloning • Cloning is the process of making a genetically identical organism through nonsexual means. • First animal cloned in 1997, at the Roslin Institute in Scotland
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Cloning
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Cloning • Proposed benefits of cloning – Use of clones as donors – cloning only organs – Saving endangered species –Noah the gaur – Cloning of stem cells
• Claims of first cloned baby born on 26 Dec 2002.
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Human Genome Project • Begun formally in 1990, the U.S. Human Genome Project is a 13-year effort coordinated by the U.S. Department of Energy and the National Institutes of Health • Expected completion date –sometime in 2003
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Human Genome Project • Identify all human genes • Determine the entire base pair sequence • Store the information in databases
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Human Genome Project • Study of various non human organisms – E.Coli, fruit fly, mice
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Human Genome Project • Molecular Medicine – Knowledge of which genes cause which disorders – Based on the causes, specific treatments can be devised.
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Human Genome Project • Microbial Genomics – Use of bacteria useful in energy production (like photosynthesis), toxic waste reduction, and industrial processing. – Better understanding of micro-organisms and hence development of new drugs
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Human Genome Project • Risk Assessment – Genetic basis to variable response to toxins, x-rays, cigarette smoke, and susceptibility to cancer.
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Human Genome Project • Anthropology and Evolution – By studying mutations and variations in genes
• DNA forensics • Agriculture, animal breeding etc.
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