DNA AND GENETICS
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Two chromatids joined together by centromere -- form one chromosome. The chromosomes are composed of nucleoprotein called chromatin. Consists of --DNA, RNA, basic proteins called histones and non-histone proteins. Under electron microscope, the chromatin fibre appears to have a structure like a ‘String of beads’. According to the nucleosome solenoid model proposed by Kornberg and Thomas (1974), the beaded string is made of repeating units called nucleosomes.
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•Each nucleosome is a bead-like particle in the chromatin structure. •It is made up of two components, namely,a)Central core b) DNA segment of certain length. The central core is called an octamer because it is made of 8 molecules of histone proteins The double stranded DNA is wound around the octamer core. The segment of DNA molecule in between the nucleosome (beads) are called the linker DNA in the chromatin. This gives ‘beads on a string’ appearance to chromatin fibre. www.indiandentalacademy.com .
•The string of beads condenses further to produce a 100 Å thick nucleosome fibre. •The nucleosome fibre then supercoils to produce a 300350 Å thick solenoid fibre. Solenoid represents structure of chromatin fibre in the chromosome Homologus chromosomes: Sexually reproducing diploid organism develops from a diploid zygote (2n). The zygote is formed when a haploid (n) male and a haploid (n) female gametes fuse at the time of fertilization. Thus, the diploid individual receives two sets of chromosomes: one through the male gamete (paternal set) and the other trough the female gamete (maternal set). For every chromosome in the paternal set, there is a similar looking chromosome present in maternal set. Such similar chromosomes from paternal and maternal sets have identical gene loci and are called homologous chromosomes www.indiandentalacademy.com
•The genes for opposing or alternative factors are called as alleles. They occupy the same locus on homologous chromosomes e.g. Tall and dwarf represent allelic forms of the same gene. •The chromosome constitution of a species is called karyotype. This karyotype varies with respect to chromosome number and morphology for every species. •The Genotype of an individual is his genetic constitution. The phenotype is the expression of the genotype as a morphological, biochemical or physiological trait. The term genome refers to the full DNA content of the chromosome set.
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The human chromosomes: -The 46 chromosomes of normal human somatic cells constitute 23 homologous pairs -One member of each chromosome pair is inherited from the father; the other from the mother and one of each pair is transmitted to each child. Twenty-two pairs are alike in males and females and hence are called autosomes. The two sex chromosomes, the remaining pair, differ in male and female and are of major importance in sex determination
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KARYOTYPING It is a procedure in which chromosomes are stained and analyzed by microscopic examination and photographed. Further the chromosomes are cut out from these photomicrographs and arranged in pairs according to the standard classification. The complete picture is called ‘karyotype’
www.indiandentalacademy.com Fig. --: The Paris classification.
I) The original classification - Denver Classification Devised in 1960 at a meeting of cytogeneticists in Denver, Colorado. Distinguished seven chromosome groups identified by the letters ‘A’ through ‘G’ on the basis of their overall length and centromere position. The location of the centromere or primary constriction is a constant feature of each chromosome. Human chromosomes can be classified by centromere position into three types; 1. Metacentric if the centromere is central, 2.Submetacentric if it is somewhat off-center 3.Acrocentric if it is near one end.
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Types of metaphase chromosomes. Telocentric chromosomes are not found in human beings.
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II) The Paris classification: •Devised in 1971 at the Paris conference, •Identification of chromosomes based on various banding patterns. •Today this nomenclature is accepted all over the world. •According to this, both P and q arms consist of regions which are numbered 1, 2 and 3, starting from centromere. The regions are further subdivided into bands so as to give precise location e.g. RBI (Retinoblastoma) locus is situated on chromosome 13. Its precise location is 13q 14 i.e. fourth band on the first region of long arm of chromosome 13.
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Application of karyotyping: 1) Clinical diagnosis: especially indicated, in patients with congenital malformations involving multiple systems, mental retardation or in cases of ambiguous genitalia. 2) Gene mapping: proper localization of human genes to their specific positions on chromosomes. 3) Role in cancer: The detection of Philadelphia chromosome in patients of chronic myelogenous leukemia (CML) 4) Repeated fetal loss: On chromosome analysis, the couple may reveal a chromosomal defect in any one of the partner. Chromosomal aberrations account for a sizeable number of spontaneous abortions in the first trimester of pregnancy. www.indiandentalacademy.com
5) Prenatal diagnosis: Chromosome analysis of chorion villous samples and amniotic cells may reveal a chromosome abnormality in the fetus warranting medical termination of pregnancy. 6) Polymorphisms: Minor heritable differences in the banding pattern are seen, especially for chromosome 1, 9, 16 and Y chromosomes These polymorphisms may be used to trace individual chromosome through families. Thus they can serve as a markers in family studies or for determination of the source of the abnormal gamete in chromosome abnormalities
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Structure of nucleic acid Nucleic acid consists of a long chain of molecules called nucleotides. Each nucleotide molecule in turn consists of a nitrogenous base, a five carbon sugar and phosphorus molecule. Nucleic acids--two types. Deoxyribonucleic acid or DNA and ribonucleic acid or RNA DNA contains sugar called deoxyribose and RNA contains sugar called ribose DNA -- mainly found in chromosomes (exception mitochondrial DNA) and RNA--chiefly found in nucleolus and in cytoplasm The nitrogenous bases are of two types – purine and pyrimidine. The purine bases are adenine (A) and guanine (G); and the pyrimidine bases are thymine (T), cytosine (C) and uracil (U). www.indiandentalacademy.com
RNA differs from DNA basically in three respects: 1) It has sugar ribose in place of deoxyribose of DNA. 2) Of the four bases, three are common in DNA and RNA. They are adenine, cytosine and guanine. The fourth base in RNA is uracil instead of thymine in DNA. 3) RNA molecule is usually single stranded while DNA has two strands. Types of RNAs: They are of three types: • m RNA – messanger RNA • t RNA – transfer RNA • r RNA – ribosomal RNA www.indiandentalacademy.com
Process of information transmission from DNA to RNA (transcription and translation): Transcription: It is a process whereby information is transmitted from DNA to the messenger RNA
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Translation: It is a process of translating information from mRNA into protein synthesis.
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GENE – A UNIT OF HEREDITY Mendel during his experiments on garden Peas used the term ‘Factors’ which an individual receives from his parents In 1909 a Danish Botanist, Johansen, introduced the term ‘gene’ for these factors. Later in 1910 Morgan from his experiments on Drosophila showed that ‘genes are parts of chromosome’. -Until 1977 gene was thought to be a segment of DNA molecule possessing a code for amino acids sequence of a polypeptide chain. This model, however appears to be inadequate to elucidate the precise mechanism of gene operation.
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Normal gene structure: In genes, coding sequences are split by intervening sequences. • The exon-intron patterns of split genes appear to be strikingly conserved during evolution • Within exons, alterations in sequence occur slowly, approximately at the rate of 10-9 substitutions per codon per generation through natural selection. • A typical gene contains a ‘promoter’ region, which extends for about 200 bp upstream of the transcription start site at nucleotide +1. -The TATA box is responsible for correctly locating the site at which transcription starts. Three other commonly found elements within the promoter affect the efficiency of transcription. - GC box (GGGCGG), which binds the transcription factor SP1. www.indiandentalacademy.com
-CAAT box (GGCCAATCT), which binds the transcription factor CTF and NF-1. Normally found around position – 80 relative to the transcription start site. -Oct box (ATTTGCAT), which binds the transcription factors Oct 1 and Oct – 2. •Enhancers are the regions of DNA that operate to stimulate the basal level of transcription from its promoter •Response elements induce genes in response to particular signals. •Silencers produce negative control where genes are turned off by the binding of proteins to elements •Some genes, termed ‘housekeeping genes’, are expressed in most tissues mot of the time and are responsible for functions likely to be necessary in any cell. •The overall level of transcription of a gene is the outcome of the different influences exerted by the promoter and www.indiandentalacademy.com enhancers
Code: In 1971, Nirenberg and Matthaei proved in vitro that three consecutive bases of mRNA code for a specific amino acid. This is known as triplet code or codon. Triplet code: The basic function of gene --direct synthesis of proteins. There are 20 different amino acids in proteins. Stored within DNA molecule is genetic information in the form of a triplet code, which is a sequence of three bases that codes for one amino acid. As there are four bases A, T, G and C there can be 43 = 64 such combinations. The sequence of these three bases is called genetic code or codon. For some amino acids there is more than one triplet code. In such cases codes are sometimes called degenerate.. Three of the 64 codons are ‘nonsense’ designating termination of message and no amino acid will be coded. www.indiandentalacademy.com
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MUTATION: It is defined as any change in sequence of genomic DNA. In the normal course DNA replication is highly precise, however, any error that involves this process is copied in subsequent replications. Mechanism of mutations:- Mutation occurs by any of the following three mechanisms: Substitution, deletion, insertion:
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Causes of mutation: 1) Radiation: Ionizing radiation is by far the most potent mutagen known. The effect of X-ray in increasing the rate of mutation was first observed by Muller (1927). 2) Chemical mutagenesis: Many chemicals are known to be carcinogenic; most of these are now known to be mutagenic as well. The first example, found by Auerbach in 1941, was mustard gas, an alkylating agent that produces single base substitution in DNA. 3) Spontaneous mutations: Some mutations are spontaneous errors in replication that evade the proof reading function of the DNA polymerases that synthesize new polynucleotides at the replication fork. www.indiandentalacademy.com
Effect of mutations on gene product: They are as follows: 1) Chain termination mutations: DNA transcription normally ceases when a termination codon is reached. A mutation, which creates a termination codon, can cause premature cessation of transcription. In another situation, if the mutation destroys a termination codon then it allows transcription to continue till the next termination of codon is reached. 2) Splice mutations: In this, mutation involves a normal mechanism by which introns are excised and exons are spliced together during the formation of a messenger RNA. It leads to complete failure of synthesis of the gene product. 3) Mutations involving regulatory sequences: Mutations that involve CAT box and TATA box regions upstream of the structural gene can cause reduced transcription of sequence. www.indiandentalacademy.com
GENE MAPPING: METHODS TO LOCALIZE GENES ON CHROMOSOMES Gene mapping can be broadly divided into two. 1) Chromosome mapping i.e. assigning a particular gene or a DNA sequence to a specific chromosome. 2) The second is finer piece of information. It includes the detailed structure of the gene i.e. ‘DNA mapping’. Chromosome mapping can be achieved by either a) Somatic cell hybridization or b) Fluorescent in situ hybridization
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a) Somatic cell hybridization: This has proved rewarding in genetic linkage. In this technique somatic cells from two different species are fused together under favourable conditions and cultured.
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when human and mouse cells are cultured together, some of the cells form heterokaryons (fused cells with separate nuclei). The nuclei may then fuse to form true hybrid cells. The hybrid cells continue to proliferate in culture but for some unknown reasons, the number of human chromosomes they contain is progressively reduced The fact that it is the human chromosome, not the mouse ones, that are preferentially lost is the basis of the usefulness of somatic cell hybrids for human linkage studies For example:Clones of somatic cell hybrids are selected in which there are certain combinations of human chromosomes, as shown. The three clones are examined for expression of given phenotype. www.indiandentalacademy.com
Chromosome 1
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Clone A
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Clone B
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Clone C
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If the phenotype is expressed in all the three clones, its locus is on chromosome 1; if it is expressed in clones A and B but not C, its locus is on chromosome 2; and so on Panel of hybrid clones covering all 24 human chromosomes (22 autosomes, X and Y) are available, so it is usually unnecessary to make new hybrids to look for linkage www.indiandentalacademy.com
2) Fluorescent in situ hybridization (FISH) This has revolutionized the concept of chromosome analysis. It is based upon the unique ability of a portion of single stranded DNA, the probe to anneal or hybridize with its complementary target DNA sequence located in the genome. This probe is conjugated with a fluorescent label and hence can be visualized under uv light. Various types of chromosome specific probes can be used. Some of them are specific for the centromere of chromosome or for a particular portion of a chromosome. On application to a metaphase spread this probe hybridizes to that chromosome.
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