Genetices 2024 PDF

Definitions Genetics: branch of biology that study Genes, Genetic variation and Heredity in living organisms Classical genetics: old branch of genetics based only on visible results of reproductive acts based on experiments of Mendel Molecu...

Definitions Genetics: branch of biology that study Genes, Genetic variation and Heredity in living organisms Classical genetics: old branch of genetics based only on visible results of reproductive acts based on experiments of Mendel Molecular genetics New field of genetics that studies structure & function of Nucleic acids at molecular level Chromosomes structures, replication, gene expression  Molecular The nucleic acids GENETICS DNA and RNA are the carrier of genetic material in all living organisms. Nucleic acids have two important functions Ability to express genetic traits through Ability to duplicate itself transfer to progeny by chromosomal replication transcription to mRNA translation into proteins.  The Structure of DNA DNA consists of nucleotides 3 parts backbone of alternating phosphate & sugar (2-deoxyribose) Nitrogenous base Purines : adenine (A), guanine (G) Pyrimidines: cytosine (C), thymine (T) A paired with T (2 hydrogen bonds) G paired with C (3 hydrogen bonds) nucleotides are joined by phospho- diester bond  DNA is double helix 2 chains of nucleotides coiled around each other the 2 standards are complementary The 2 chains are antiparallel (i.e., their sugar-phosphate backbones are oriented in opposite directions) 5' to 3' direction 3' to 5' direction In prokaryotes DNA is circular, super-coiled molecule associated with basic proteins (histone-like) In eukaryotes DNA is more organized contain histone proteins & coiled into repeating units known as nucleosomes.  RNA RNA is single strand coil back on itself RNA composed of the sugar ribose instead of 2- deoxyribose Uracil (U) instead of thymine (T) There are 3 main types of RNA Ribosomal (rRNA) Transfer (tRNA) Messenger (mRNA)  Types of RNA 1. Messenger RNA (mRNA) Carry message direct protein synthesis in ribosomes mRNA formed by transcription of DNA Transcription depend on RNA polymerase initiates RNA synthesis at promoter site on gene stop when termination codon 2. Ribosomal reached. RNA (rRNA) rRNAs are components of ribosomes They are made from large precursors → Enzymatically cleaved to → 16s, 23s and 5s rRNA  3. Transfer RNA (tRNA) Carry amino acids during protein synthesis tRNAs can distinguish different amino acids Each type of tRNA is covalently bound to one of the 20 aa. Each tRNA has a triplet of nucleotides called 'anticodon‘ binds to triplet nucleotides on mRNA, called codon during protein synthesis. When aa is attached to tRNA tRNA is said to be charged.  DNA REPLICATION Each strand of DNA serves as template for production of another complementary strand This pattern of replication is responsible for maintaining (conserving) proper sequence of bases on DNA molecule The pattern of DNA replication is described as 'semiconservative‘ produce 2 copies of DNA molecule each copy contained one original strand and one new strand  Replication starts at a point called 'origin of replication' by separation of the two strands The replication origin specific segment of DNA molecule consisting of about 245 bp. Replication fork area of DNA molecule where strand separation occurs and the synthesis of new DNA takes place.  A replicon consists of origin of replication and DNA that is replicated from that origin Bacterial chromosome has single replicon (one bubble). Eukaryotes have multiple replicons (several bubbles exist) to efficiently replicate the relatively large molecules within a reasonable time Polymerization of nucleotides occur in 5' to 3' direction for original strand Leading strand (3' 5‘) A challenge in DNA replication is how to achieve 5‘ to 3' polymerization in the opposite direction from the template strand which is itself is from 5'-3' direction (lagging strand) This problem is solved by having different modes of polymerization for the two growing strands  Leading strand Lagging strand Formed continuously Formed from 5‘ 3' end in small from the 5' to 3'end fragments Okazaki fragments linked together by ligase enzyme  DNA replication DNA replication carried out using DNA polymerases cells in all organisms contain multiple highly specialized DNA polymerases Bacteria 5 DNA polymerases I, II, III, IV, V Yeast 8 DNA polymerases humans at least 15 DNA polymerases The rate of DNA synthesis 750-1000 bp/second in Prokaryotes 50-100 bp/second in Eukaryotes  Polymerase III main polymerase in DNA replication process To initiate replication DNA polymerase require presence of primer short strand of RNA to which growing polynucleotide chain is covalently attached Polymerase I has exonuclease activity to remove mismatched nucleotide & adds correct nucleotide repair process Proofreading removal of incorrect nucleotides immediately after they are added to growing DNA during replication process. The proofreading function of DNA polymerase I improves fidelity of replication to one error in every 109 -1010 bp  Steps of DNA replication in E. coli (1) DnaA protein binds to origin of replication (Ori C) DNA replication starts by separation of the two strands. (2) Helicase(dnaB) bind replication fork to unwind the 2 DNA strands at same time topoisomerases (DNA gyrase) relieve the tension caused by unwinding process (3) Single-stranded DNA binding proteins (SSBs) keep the single stranded region of the template DNA apart (4) Primase (dnaG) synthesize small RNA molecule (~10 nucleotides) act as primer for DNA synthesis (5) DNA polymerase III synthesizes complementary strand of DNA according to base-pairing rules The direction of DNA synthesis is from 5' to 3' end of the newly formed strand On one strand (leading strand), synthesis is continuous on the other (lagging strand) discontinuous synthesis (Okazaki fragments) are generated DNA synthesis is bi-directional 2 replication forks in opposite directions from origin of replication (6) DNA polymerase I removes primer and fills gaps that result from RNA deletion. (7) DNA ligases join DNA fragments to form a complete DNA strand DNA replication is extraordinary complex; at least 30 proteins are required to replicate the E. coli chromosome.  Lecture 2 Gene expression  THE GENE EXPRESSION DNA sequence define amino acid sequence in protein The 4 DNA bases (A, T, C, G) code for 20 amino acids Each Codon represented by 3 bases Provide (43) 64 different codons. 61 of codons specify amino acids called sense codons. 3 codons UGA, UAG and UAA do not specify amino acids called non-sense (stop) codons. Some amino acids are encoded by more than one codon called degeneracy  Gene EXPRESSION DNA Transcription mRNA Translation RNA Synthesis protein synthesis  GENE AND ITS STRUCTURE The gene is unit of heredity of living organism It is linear sequence of nucleotides with fixed start and end points encodes protein, tRNA, or rRNA Genes are not overlapping (With few exceptions). In prokaryotes coding sequence is continuous (few bacterial genes are interrupted). In eukaryotes most genes have coding sequences (exons) that are interrupted by non-coding sequences (introns).  Regions of Genes coding protein Template strand contain coding information direct RNA synthesis (1) Pomoter sequence found upstream of coding region (5` UTR) act as recognition/binding site for RNA polymerase Recognition site Binding site Site of initial association with RNA Sequence that favors DNA unwinding polymerase (35 bases). before transcription (10 bases). (2) Leader transcribed sequence that is not translated recognition site for ribosome (Shine-Dalgarno (SD) sequence) AGGAGG in E. coli  (3) Coding region begins immediately downstream of leader sequence Starts with 3'TAC5‘ transcribed into mRNA codon 5'AUG3‘ translated into N-formyl-methionine in bacteria, or methionine in eukaryotes. (4) Trailer region: transcribed but non-translated region located immediately downstream of translation terminator (stop codon) and before the transcription terminator. (5) Operator: sites where DNA- regulatory proteins (repressor) bind to inhibit gene expression. Regions of Genes Genes coding tRNA and rRNA The tRNA genes Parts Promoter, leader, coding region, and trailer region. more than one tRNA may be made from a single transcript in such case the tRNAs are separated by a non-coding spacer region which is removed after transcription. The rRNA genes Have the same parts like tRNA genes, all rRNA molecules are transcribed as a single large transcript cut up after transcription giving final rRNA products  Transcription in Eukaryotes 3 major RNA polymerases RNA polymerase II catalyze mRNA RNA polymerase I synthesis catalyze rRNA synthesis RNA polymerase III catalyze tRNA synthesis  Transcription in prokaryotes Prokaryotic mRNA can code for one polypeptide (monogenic) many polypeptides (polygenic). Many prokaryotic genes are organized in Operons group of genes that have related functions & transcribed as one unit beside coding regions, mRNA molecules have untranslated regions Leader sequences & Trailer regions Prokaryotes has One RNA polymerase Multi-subunit enzyme synthize all RNA types RNA polymerase consists of core enzyme (α2,β,β' subunits) catalytic subunit The sigma subunit (σ-factor) is not catalytic helps core enzyme bind DNA at promoter sites.  Transcription in E. coli In transcription only gene(s) is copied (not entire genome) The transcription needs 3 major steps Initiation, elongation & termination Initiation Binding of core polymerase to promotor (on template strand) facilitates by σ-factor Core polymerase + σ-factor called RNA Holoenzyme In E. coli promotor consists of 2 conserved sequences TTGACG at -35 element (Recognition) TATAAT at -10 element (Binding) Binding of holoenzyme to the 2 sequences form complex RNA polymerase initiate transcription by itself not require primase the enzyme synthesize short RNA molecule 10 bp  Transcription in E. coli Elongation After synthesis 10 bp of RNA σ-factor is ejected and the enzyme move in 5’-3’ direction continuously synthesizing RNA. The synthesized RNA exit from RNA exit channel. Termination Transcription is terminated due to specific sequence in DNA The terminator DNA contains repeat which cause pairing transcript RNA form hairpin loop structure Invert repeat followed by larger number of TTTTTTTT(~8 bp) on template DNA uracil appear in RNA. The load of hair pin structure is not tolerated RNA get separated from RNA-DNA hetero-duplex.  Modifications of mRNA 2 modifications for mRNA to protect from exonucleases. 1. Adenylic acid added to 3' end produce poly-A sequence ~200 nucleotides long (poly-A tail) 2. 7-methylguanosine is added to 5' end (5' cap). Eukaryotic gene contain coding sequences (exons) separated by non-coding sequences (introns) both transcribed Introns should be removed by process called RNA splicing. Splicing is catalyzed by RNA molecules called ribozymes.  Translation (protein synthesis) Translation synthesis of polypeptide chain directed by mRNA sequence The sequence “read” in groups of 3 nucleotides (codon) by tRNA that carry complementary anti-codon. Ribosome (site of translation) perform Codon-anticodon hydrogen bonding Formation of covalent bond between neighboring amino acids. Protein synthesis Starts at 5’ end of mRNA with start codon (AUG) Ends with non-sense (termination codon) UAG, UAA, or UGA  Translation in prokaryotes Performed by 70S ribosomes Polyribosome complex of mRNA + several ribosomes during protein synthesis Synthesis of protein divided into 3 stages Initiation of protein Elongation of Termination of synthesis polypeptide chain protein synthesis require peptidyl transferase Require termination codon Require initiation codon and elongation factor and IF  Initiation of protein synthesis Initiation of translation in bacteria begins when 30S subunit binds to SD sequence (on mRNA) complementary to 5-6 b of 16S rRNA in 30S subunit. GTP bound to 30S subunit and initiation factor (IF) promote attachment of the first aminoacyl tRNA N-formylmethionyl-tRNA in bacteria Methionyl-tRNA in eukaryotes. anticodon on tRNA binds to AUG start codon on mRNA. 50S subunit bind to both 30S subunit & N- formylmethionyl tRNA on (P-site).  Elongation of polypeptide chain Elongation involves addition of extra amino acids to growing polypeptide chain. Subsequent tRNA(s) associate with mRNA in ribosome codon following start codon determines which of aminoacyl-tRNA comes next. aminoacyl tRNA, elongation factor, & GTP molecule form complex that binds to ribosome. hydrolysis of GTP causes aminoacyl tRNA to bind strongly to (A-site) on 50S subunit Peptidyltransferase catalyzes formation of peptide bond between N- formylmethionine and the new amino acid  The formation of peptide bond cleaves the N-formylmethionine from its tRNA the 2 amino acids are joined and attached to the tRNA in A-site. uncharged tRNA in P-site hydrolyze causing the move of peptidyl tRNA from the A-site to P-site. Amino acids are added to form polypeptide chain by repeating elongation steps  Termination of protein synthesis Translation ends when ribosome comes to a non-sense (termination) codon (UAA, UAG, and UGA). release factors bind to ribosome when it comes in front of non-sense codon stimulates peptidyl transferase to cleave newly formed peptide from tRNA A protein called dissociation factor binds to 30S subunit causes the ribosome to dissociate into its 2 subunits.  Post-translation modifications Protein splicing part of polypeptide chain is removed before folding (modification in some proteins). Proteins self-folded to give special regions helix & sheets 2ry structure Molecular chaperones are special helper proteins that aid polypeptide folding to its proper shape 3ry structure  Regulation of protein synthesis Induction & repression of gene expression Synthesis of enzymes involved in catabolic pathways is inducible initial substrate of pathway is the inducer induction increases amount of mRNA encoding the enzymes Synthesis of enzymes involved in anabolic pathways is repressible end product of pathway acts as repressor Repression decreases amount of mRNA encoding the enzymes. Also rate of mRNA synthesis is controlled by repressor proteins bind to specific sites on DNA called operator In bacteria, a set of genes controlled by one operator or promoter is called an operon Regulon is group of genes or operons controlled by one regulatory protein  Lecture 3 Microbial variation  Microbial variations The genetic information of a bacterial cell is contained in a single circular chromosome carry thousands of genes. Total number of genes in the cell is known as cell’s “genome” The genome of cell determines its genotype heritable characteristics Characters expressed in given environment called phenotype. Replication of DNA is usually very accurate process progeny have the same characters of parent cell Sometimes Variations from parent cell appear  MICROBIAL VARIATION If permanent change happen genotypic variation Irreversible heritable not associated with specific environmental conditions If change is not permanent Phenotypic variation reversible related to environmental condition change in expression not involves changes in genetic material. Examples of phenotypic variation lack of pigmentation of Serratia marscesens colonies grown at 37oC the pigment re-appear when grown at 22oC. Loss of Salmonella spp flagella, when it is grown in presence of phenol appear again in absence of phenol  Causes of Genotypic variation Permanent heritable changes in genetic material Result from mutation acquisition (transfer) Mutation Permanent change in genetic information often result from errors in replication (spontaneous) or induced by exposure to mutagen (inducible) Mutations can lead to base pair substitutions (point mutation) or Frameshift Deletion or insertion of nucleotides alter codon reading frame different protein  Types of mutations Tautomeric shift spontaneous isomerization of bases alternative hydrogen-bonding form Transition substitution of one purine (A, G) for another, or one pyrimidine (T, C) for another Transversion substitution of purine for pyrimidine or vice versa Recombination exchanging & rearranging regions of DNA Mutation can be induced by substances called mutagens (mutagenic agents)  Mutagenic agents (1) incorporated into DNA during replication Base analogs 5-bromouracil (5-BU) replace T pairs with G 2-aminopurine replace A pairs with C (2) Alkylating agents Most potent mutagens e.g. nitrosamine and ethylene oxide cause Cross linking stop replication DNA break specific mis-pairing (3) Intercalating agents e.g. acridine dyes inserted between bases of helix distort DNA structure induce single nucleotide pair insertion or deletion  (4) Chemicals that react with bases giving product with altered H-bonding. *Nitrous acid bases deamination alters the way in which bases pair introduces mutations into DNA molecules during DNA replication. *Nitrous acid has effect on 3 DNA bases cytosine changed to uracil (U) pair A adenine changed to hypoxanthine (HX) pair G guanine changed to xanthine (X) pair C  (5) Radiation severely damage DNA UV powerful mutagen absorbed by T & C activates pyrimidines to form dimers formation of dimers distorts DNA and interferes with DNA replication and transcription. If not corrected leads to cell death cells have number of repair systems to correct the distortion. The repair mechanisms are error prone Mistakes are made at rate of about 1 in 107 -108 repairs Ionizing radiations X-rays, gamma rays & high energy particles are very potent mutagens.  DNA Repair Microorganisms have different mechanisms to repair DNA damage including (1) Excision repair damage in one strand damaged area is excised, producing single-stranded gap then the gap is filled by DNA polymerase I and DNA ligase. (2) Recombination repair restores DNA that has damage in both strands by recombination with an undamaged molecule (this frequently occurs in rapidly dividing cells where there is another copy of the chromosome not damaged). (3) Removal of damage without removing or replacing bases limited to repair of certain kinds of damage (e.g. photo-reactivation remove thymine dimers).  Genetic transfer among bacteria In prokaryotes, fragment of the donor’s genome (exogenote) can be transferred and the corresponding segment of the recipient is called the endogenote. The recipient’s chromosome acquires new genes and looses corresponding genes. 3 mechanisms of inter-species (horizontal) gene transfer Transformation Conjugation Transduction  1.Transformation In transformation small piece of DNA is taken up from medium and sometimes integrated in bacterial chromosome. Exogenote naked DNA (plasmid or DNA fragment) taken from the environment. Competent cells special type of cells that are able to take exogenous DNA (prepared by different methods)  2. Conjugation Process by which large number of genes can be transferred to the recipient cell through cell-to-cell contact without cell fusion. Conjugation also called bacterial mating DNA transfer is mediated by sex pili produced by donor cell attach to specific receptor on recipient cell  2. Conjugation Most conjugation involve plasmid DNA called conjugative plasmids (F factor) contain genes known as tra (transfer) genes. Cells carry F plasmid called F+, those which do not are F- Conjugative plasmids mostly seen in gram negative bacteria, but also in certain gram positive bacteria. A single stranded copy of the plasmid is transferred to recipient cell both cells made complementary strand. F plasmid can integrate in bacterial chromosome by recombination Recombination of the plasmid into the chromosome results in production of Hfr cells (high frequency recombination). Conjugation is promoted by tra genes of integrated F plasmid.  Recombinants occur 1000 times more often in Hfr mating than in F+ mating. The order of transfer of chromosomal genes will depend upon site of attachment of the F plasmid in the chromosome. The probability of transfer of given gene decreases exponentially with its distance from the transfer origin.  3. Transduction Transduction is the process by which large number of genes (around10) can be transferred by bacteriophage that contains chromosomal or plasmid DNA taken from the host cell (donor) during previous replication cycle There are three types of transduction Generalized Restricted (specialized) Abortive  Generalized transduction Sensitive bacterium exposed to virulent phage infection occurs and lytic cycle will take place. defective phage formed (at rate of 1x10-6) these particles have been accidentally filled with similar bacterial DNA. This piece of DNA equal in size to normal phage DNA and contain 30-150 genes. These genes may then be incorporated in the chromosome of the new host cell Phages randomly pick up any portion of host chromosome they can transfer any gene at approximately the same frequency.  Restricted “specialized” transduction Temperate phages undergo lysogenic cycle the phage genome integrated into chromosome “prophage” and replicate only with the cell chromosome at cell division. The bacterial cells carrying prophages are called “lysogenized cells”. Sometimes spontaneously (low frequency) or after a stimulus (heat, radiation) the inserted prophage is detached from cell chromosome and lytic cycle occurs. After replication, the whole progeny phages (defective phage) will carry these bacterial genes and transfer it to new host cells. As prophage has specific site on bacterial chromosome gene transfer restricted to area near to this site restricted transduction  Generalized transduction Restricted transduction Done by Lytic phage Lysogenic phage Transferred Equal to phage genome in size Smaller pieces of DNA DNA size Integration in randomly pick up any portion of has specific site on bacterial host genome host chromosome chromosome gene transfer is restricted to those near this site Abortive transduction In some cases of restricted transduction, the exogenate is not integrated in bacterial chromosome but instead stay in the cytoplasm. Exogenate is circularized and transmitted to the next generation of cells with or without replication. This phenomenon is called “abortive transduction”.  Transposition Transposition movement of pieces of DNA in the genome. Transposons (Tn) are segments of DNA that can jump from place to place in the genome. Transposon size 2000-20000 bp able to direct synthesis of copies of themselves Transposons often carry antibiotic resistance genes Conjugative transposons can move between bacteria through the process of conjugation Effects of transposable elements (1) Mutagenesis cause deletion of genetic material, or arrest of translation due to termination sequences. (2) Insertion of F plasmids into chromosome. (3) Generation of plasmids with resistance genes.  Recombination A process by which nucleic acid molecules are rearranged or combined to produce new nucleotide sequence. In eukaryotes recombination occurs during meiosis In Bacteria There are a number of mechanisms by which DNA molecules recombine with each other (1) General (homologues) recombination Occurs between homologous regions of DNA (2) Sit-specific recombination non-homologous insertion of DNA into a chromosome. Occurs during viral genome integration into host catalyzed by enzymes specific for the virus and its host.