Cell Biology PDF

Nucleus biology By Dr / Eman Elsaid Khalifa Lecturer of cellular and molecular biology Nile Valey University Introduction Structure of nucleus Nuclear envelope Nucleoplasm Nucleolus Chromatin (molecular structure of chromosomes) Function of nucleus ...

Nucleus biology By Dr / Eman Elsaid Khalifa Lecturer of cellular and molecular biology Nile Valey University Introduction Structure of nucleus Nuclear envelope Nucleoplasm Nucleolus Chromatin (molecular structure of chromosomes) Function of nucleus  Nucleus Nucleus is the large membrane bound organelle found in eukaryotic cells that contain genetic material in the form of multiple linear DNA molecules organized into structures called chromosomes. THE CELL NUCLEUS  History -Discovered in 1831 by Scottish botanist Robert Brown -Name (nucleus) derived from the Latin word for kernel/nut Robert Brown 1773-1858 Main Characteristics Generally found in the central region of the cell. Most of the metabolic activities which takes place in the cell is initiated by nucleus. Largest and most easily seen organelle. Majority of cells genetic material is present in nucleus. ULTRA STRUCTURE OF NUCLEUS  Classification of cells according to number of nuclei Generally a cell contains single nucleus. But sometimes 2 or more nuclei are also present. Based on Number of nucleus, cells are classified into 4 types-  Anucleated cell : in a nuclear cell, nucleus is absent. (RBCs )  Mononucleated cell : Single nucleus is present.  Binucleated cell : 2 nuclei are present. Of these, one nucleus is small (micronucleus) and other nucleus is large (macronucleus).  Multinucleated cell : contains many nuclei. Structure of nucleus Nuclear envelope Ncleoplasm nucleus Nucleolus Chromatin  Nuclear Envelope Consists of: Phospholipid bi- layer membrane Nuclear Pores Ribosomes  Nuclear envelope  Nucleus separated from the rest of the cytoplasm by a semi-permeable membrane called nuclear envelope.  It is double layered membrane made of lipoprotein.  Nuclear membrane has a fluid mosaic structure similar to that of plasma membrane.  Inner nuclear membrane is lined by a fibrous material called nuclear lamina (composed of filament protein called lamin).  Outer nuclear membrane is lined with ribosomes. Outer membrane communicates with endoplasmic reticulum at several points.  Nuclear membrane contains many pores called nuclear pores. These spores form passageways between nucleoplasm and cytoplasm.  Nuclear pore complex  The nuclear membrane contains many pores which are circular in shape.  Nuclear pores are arranged randomly in most of cells, in clusters in lymphocytes and oocytes.  The nuclear pore has a complex organization. So the entire structure of nuclear pore is called nuclear pore complex.  The nuclear pore is circular in surface view. In the center of the poor, there is a passage called central channel.  Functions of Nuclear Membrane  Protection of DNA  Nucleo-cytoplasmic material exchange  Attachment of structural elements in the cytoplasm  Attachment of nuclear component during interphase  Protein synthesis  Synthesis of chromosomal enzymes  Nucleoplasm  Nucleus is filled with homogeneous, transparent acidophilic substance known as nucleoplasm.  Chromatin threats remain suspended in nucleoplasm.  There are one or more definite structure called nucleoli in nucleoplasm.  The nucleoplasm contains organic and inorganic substances like nucleic acids, proteins, enzymes and minerals.  Chromatin reticulum or chromatin network  They are lightly stained threadlike bodies embedded in the nucleoplasm called chromonemata, which form network called chromatin network.  They represent chromosomes. Chromosome is derived from Greek language, Chrome – colour and soma – bodies. It is named so because they are coloured during staining.  During cell division chromatin network is condensed to form thick ribbon like structures called chromosomes.  Chromosomes carry long pieces of DNA which contains genetic material called genes.  Nucleolus  Nucleolus was discovered by Fontana in 1874.  They are the round spherical or oval bodies found in the nucleoplasm.  Number of nuclei differs from species to species. It depends on the number of chromosome sets in the nucleus.  Size of nucleolus is related to synthetic activity of the cell.  The important function of nucleolus is synthesis of ribosomal RNA and protein.  FUNCTIONS OF NUCLEUS  Metabolism- nucleus controls majority of the activities of cell. It is a regulatory organelle in cell metabolism.  Heredity- since the nucleus contains DNA molecules in its chromosomes, it plays a significant role in heredity.  Differentiation- it controls cell differentiation during the embryonic development.  FUNCTIONS OF NUCLEUS  RNA synthesis- the synthesis of ribosomal RNA takes place in the nucleolus.  Exchange of materials- nuclear membrane is concerned with the exchange of materials between the cytoplasm and nucleoplasm.  Nuclear membrane provides a surface for the attachment of structural elements of the cytoplasm such as microtubules and microfilaments.  Nucleus contains the initiating factor for protein synthesis.  Chemistry of nucleoproteins By Dr / Eman Elsaid Khalifa Lecturer of cellular and molecular biology Nile Valley University What are nucleoproteins? Nucleoprotein, molecule consisting of a protein linked to a nucleic acid, either DNA (deoxyribonucleic acid) or RNA (ribonucleic acid).  Histone Protein part Protamine Nucleoprotein DNA Nucleic acid RNA  Nucleotides Nucleoside Phosphate N base Pentose 2-Deoxy-D- Purines Pyrimidines D- ribose ribose Uracil Adenine Guanine Cytosine Thymine (RNA) Pentose sugar  Importance of Nucleoproteins Nucleoproteins are important for the structure and function of the cell nucleus. They are responsible for the transport of genetic material within the nucleus, and for the assembly and stability of the nuclear lamina.  Deoxyribonucleoproteins DNPs are proteins that contain DNA. DNPs are important for the replication, transcription, and translation of DNA. They are also important for the regulation of gene expression.  Functions of DNPs 1. DNP can act as a photosensitizer in photodynamic therapy (PDT) to kill cancer cells. 2. DNP can help to destroy tumor cells by causing oxidative stress. 3. DNP can increase the uptake of chemotherapy drugs by tumor cells, making them more effective at killing the cancer cells. 4. DNP can enhance the effects of radiation therapy on cancer cells. 5. DNP can help to protect normal cells from the harmful effects of radiation therapy and chemotherapy drugs.  Ribonucleoproteins are complexes of proteins and RNA molecules. The proteins can be either structural or functional. The RNA molecule can be either the messenger RNA or the ribosomal RNA. The ribonucleoprotein complex helps in the process of transcription and translation.  Functions of RNP 1. Regulating gene expression by synthesizing and degrading RNA polymerase II transcripts. 2. Regulating alternative splicing by binding to spliceosomal snRNPs. 3. Regulating translation by binding to ribosomal proteins and tRNAs. 4. Modulating the activity of other proteins by binding to regulatory RNAs (e.g. microRNAs). 5. Participating in the assembly of the spliceosome.  Functions of RNP 6. Participating in the assembly of the ribosome. 7. Regulating the activity of the small ribosomal subunit. 8. Promoting the export of mRNAs from the nucleus. 9. Regulating the stability of mRNAs. 10. Playing a role in the development of the embryo.  DNA, RNA BY Dr/ Eman Elsaid Khalifa Lecturer of cellular and molecular biology Nile valley University  Overview Chromatin is the complex of DNA and protein (Histone) that comprise eukaryotic chromosomes. The primary function of chromatin is to compress the DNA into a compact unit that will be less volume and can fit within the nucleus.  What is DNA? DNA is a nucleic acid DNA stands for Deoxyribonucleic Acid DNA – is the genetic material inside the nucleus of eukaryotic cells.  Deoxyribonucleic Acid Segment of DNA NUCLEUS GENES SEGMENTS CHROMOSOMES OF DNA What is the purpose, or function, of DNA? Stores the genetic information that instructs the cell on which proteins to make. So, DNA makes PROTEINS (both are biomolecules!) Responsible for determining all organism’s traits such as eye color, body structure, and enzyme production. Proteins are responsible for most of these traits!  The Components of DNA DNA is a long molecule made up of repeating individual units called nucleotides. Nucleotides are made up of three parts that are held together by covalent bonds: 1. Sugar 2. Phosphate Group 3. Nitrogenous Base Nitrogenous Bases DNA contains four nitrogenous bases: 1. Adenine (A) 2. Guanine (G) 3. Cytosine (C) 4. Thymine (T) In DNA, Which Bases Pair? Adenine (A) always pairs with Thymine (T) Guanine (G) always pairs with Cytosine (C) Covalent bonds  DNA is a DOUBLE HELIX or a twisted ladder. Ribonucleic Acid (RNA) RNA is SINGLE STRANDED and does not have to stay in the nucleus. RNA is not found in chromosomes because it does not carry the genetic code, however it can read the DNA code and take the information out of the  RNA Structure The building blocks of RNA are Nucleotides, just like DNA. A Nucleotide in RNA is still made of 3 important things: 1. 6-Carbon Sugar - Ribose (instead of Deoxyribose) 2. Phosphate 3. Nitrogen base there are 4 nitrogen bases in RNA, A,G,C, and U that pair together) AU CG  Types of RNA 1. Messenger RNA (mRNA) - Carries copies of instructions for the assembly of amino acids into proteins from DNA to the rest of the cell (serve as “messenger”)  Types of RNA 2.Ribosomal RNA (rRNA) – Makes up the major part of ribosomes, which is where proteins are made. Ribosomal RNA  Types of RNA 3. Transfer RNA (tRNA) - Transfers amino acids to ribosomes during protein synthesis DNA vs. RNA STRUCTURE AND FUNCTION DNA RNA  Name DeoxyriboNucleic Acid (DNA) RiboNucleic Acid (RNA) Function Long-term storage of genetic information. Used to transfer the genetic code from the nucleus to the ribosomes to make proteins. Structural Double – stranded helix. RNA is a single-strand helix. Features Composition deoxyribose sugar ribose sugar of Bases and phosphate backbone phosphate backbone Sugars adenine, guanine, cytosine, thymine bases adenine, guanine, cytosine, uracil bases Propagation DNA is self-replicating. RNA is synthesized from DNA on a needed basis. Reactivity The C-H bonds in DNA make it fairly stable, plus the The O-H bond in the ribose of RNA makes the body destroys enzymes that would attack DNA. The molecule more reactive, compared with DNA. RNA is small grooves in the helix also serve as protection, not stable under alkaline conditions, plus the large providing minimal space for enzymes to attach. grooves in the molecule make it susceptible to enzyme attack. RNA is constantly produced, used, degraded, and recycled. Ultraviolet DNA is susceptible to UV damage. RNA is relatively resistant to UV damage. Damage  By Dr / Eman Elsaid Khalifa Lecturer of cellular and molecular biology Nile Valley University DNA REPLICATION (in the nucleus) Basic Facts of DNA Replication 1. Complementary base pairing makes replication possible C-G A-T Basic Facts of DNA Replication 2. One side of DNA molecule is a template for making the other side (strand)  How does DNA replicate? 1) UNWIND: Topoisomerase unwinds the coiled strands of DNA 2) UNZIP: DNA Helicase “unzips” the strands of DNA breaking the hydrogen bonds, creating two template (parent) strands for replication 3) HOLD OPEN: Single-Strand Binding proteins (SSBs) keep strands separated 4) BASE PAIRING: DNA polymerase III bonds free nucleotides with nucleotides on each template (parent) strand using base pairing rules 5) PROOFREAD: DNA Polymerase I proofreads new strands and backtracks to correct errors 6) JOINING NUCLEOTIDES: DNA ligase bonds backbone together.  Replication Results 2 identical DNA molecules, each w/1 new strand & 1 old strand Called Semi- conservative replication  Process of DNA Replication DNA Replication Tutorial https://www.youtube.com/watch?v=Mu2bJgEZtwE Replication Movie C-G A-T DNA Template  By Dr/ Eman Elsaid Khalifa Lecturer of cellular and molecular biology Nile Valley university  Protein Synthesis The Protein-making Process https://www.youtube.com/watch?v=gG7uCskUOrA  Protein Synthesis (Gene Expression) Proteins make up all living materials  Proteins are composed of amino acids – there are 20 different amino acids Different proteins are made by combining these 20 amino acids in different combinations These amino acids come from the food we eat. Proteins we eat are broken down into individual amino acids and then simply rearranged into new proteins according to the needs and directions of our DNA.  Proteins are manufactured (made) by the ribosomes The Central Dogma Information passes from the genes (DNA) to an RNA copy of the gene, and the RNA copy directs the sequential assembly of a chain of amino acids  Steps to Make a Protein 1. Transcription DNA → RNA 2. Translation RNA → Protein (Chain of amino acids)  Step 1: Transcription A. Transcription: a complementary single strand of mRNA is copied from part of the DNA in the nucleus A. RNA Polymerase, an enzyme, unwinds DNA strand B. RNA polymerase “reads” one strand of DNA bases and makes the RNA strand c. mRNA leaves and DNA strands will coil back up RNA Polymerase I rRNA If DNA is TACCAGTTT RNA Polymerase II mRNA RNA Polymerase III tRNA mRNA will be AUGGUCAAA  mRNA editing 1. mRNA editing: cutting and splicing mRNA before it leaves the nucleus a. Introns- (intruders) “junk DNA” that doesn’t code for proteins are cut out b. Exons- “good DNA” that code for proteins stay and are expressed 2. Introns are removed and exons are spliced together. 3. Edited mRNA is sent out of nucleus to ribosome 4. The exons can be spliced together in different sequences to produce different mRNA’s = different proteins Transcription: DNA → RNA Step 2: Translation 1.How the code is read: a.Every 3 bases on mRNA represents a code for an amino acid = codon. b.Amino acids are abbreviated most times by using the first 3 letters of the amino acid’s name. Met = methonine Leu = leucine  Reading the Codon Chart Examples: AUG = Methionine CAU = Histidine UAG = Stop First Third Position Position Try these: Answers: GCU: Alanine UAC: Tyrosine CUG: Leucine UUA: Leucine This chart only works for mRNA codons. https://www.youtube.com/watch?v=LsEYgwuP6ko  Step 2: Translation Translation - Translating of a mRNA codons into a protein (amino acid chain) – Takes place on ribosomes in cytoplasm  Step 2: Translation 1. Edited mRNA attaches to a ribosome 2. As each codon of the mRNA molecule moves through the ribosome, the tRNA brings the proper amino acid to the ribosome. – Notice the anticodon on tRNA – it is complementary to the mRNA codon – The amino acids are joined together by chemical bonds called peptide bonds to build an amino acid chain called a “polypeptide”  A series of three adjacent bases in an mRNA molecule codes for a specific amino acid—called a codon. Each tRNA has 3 nucleotides Amino acid that are complementary to the codon in mRNA. Each tRNA codes for a different amino acid. Anticodon Regulation of Protein Synthesis Start codons: found at the beginning of a protein – Only one – AUG (methionine) Stop codons: found at the end of a protein (end of a polypeptide chain) Three stop codons that do not code for any amino acid therefore making the process stop: UAA, UAG,UGA  Translation Nucleus mRNA  Lysine Phenylalanine t RNA Methionine  Anticodon Ribosome mRNA Start codon  Translation Growing polypeptide chain The Polypeptide “Assembly Line” Ribosome tRNA Lysine tRNA mRNA Completing the Polypeptide mRNA Translation direction Ribosome  Roles of RNA and DNA The cell uses the vital DNA “master plan” to prepare RNA “blueprints.” The DNA molecule remains within the safety of the nucleus, while RNA molecules go to the protein- building sites in the cytoplasm—the ribosomes. Protein Synthesis Regulation of gene expression By Dr/ Eman Elsaid Khalifa Lecturer of cellular and molecular biology Nile valley university Every cell in your body contains the exact same DNA. …so why does a muscle cell have different structure and function than a nerve cell? DNA codes for… Proteins being produced can be turned off and on just like a light switch based on the needs of a cell. Chromosomes A structurally organized single piece of coiled DNA containing many different genes. Each gene will code for ONE single protein. Gene 1 Gene 2 Gene 3 Gene 4 Gene 5 Gene 6  Gene expression Gene expression is the process by which the genetic code “the nucleotide sequence “ of a gene is used to direct protein synthesis and produce the structures of the cell. Genes that code for amino acid sequences are known as 'structural genes'.  Gene expression The process of gene expression involves two main stages: Transcription: the production of messenger RNA (mRNA) by the enzyme RNA polymerase, and the processing of the resulting mRNA molecule. Translation: the use of mRNA to direct protein synthesis, and the subsequent post-translational processing of the protein molecule. Some genes are responsible for the production of other forms of RNA that play a role in translation, including transfer RNA (tRNA) and ribosomal RNA (rRNA).  A structural gene involves a number of different components: Exons. Exons code for amino acids and collectively determine the amino acid sequence of the protein product. It is these portions of the gene that are represented in final mature mRNA molecule. Introns. Introns are portions of the gene that do not code for amino acids, and are removed from the mRNA molecule before translation. Regulated Gene Expression Regulated = control the speed/amount Gene Expression = using the information from a gene to express a protein. Regulated Gene Expression = how a cell controls the speed or amount of a gene being expressed as a protein  Methods of regulation 1. Regulating transcription (DNA  mRNA) by: 1. Activator Proteins 2. Promoters 2. Splicing of mRNA Exons - Expressed 1.Regulating Transcription (DNA  mRNA) If a protein is needed by the cell, a gene coding for that protein needs to be expressed, transcription will occur. If a protein is not needed by the cell, transcription will stop.  Eukaryotic Transcription Factors Activator Protein: - a molecule that turns on transcription Transcription Factors: - molecules that allow RNA Polymerase to bind to DNA and begin transcription. Promoter: - sequence on DNA before the gene where Activator Proteins and transcription factors can bind to. Enhancers. Some transcription factors (called activators) bind to regions called 'enhancers' that increase the rate of transcription. These sites may be thousands of nucleotides from the coding sequences or within an intron. Some enhancers are conditional and only work in the presence of other factors as well as transcription factors. Silencers. Some transcription factors (called repressors) bind to regions called 'silencers' that depress the rate of transcription. Transcription will turn on When Activators and Transcription Factors are both bound to the Promoter site, RNA polymerase will bind to DNA and begin transcription. The gene will be transcribed and the protein will be translated/expressed. Splicing RNA After Transcription: Portions of the transcribed mRNA is (spliced) cut out Exons – the portions of the gene on mRNA that are cut, translated, and EXPRESS proteins Intron – the portions of the gene on mRNA that do not code for proteins and are NOT expressed The spliced Exons will then code for a Protein! Transcription Transcription is the process of RNA synthesis, controlled by the interaction of promoters and enhancers. Several different types of RNA are produced, including messenger RNA (mRNA), which specifies the sequence of amino acids in the protein product, plus transfer RNA (tRNA) and ribosomal RNA (rRNA), which play a role in the translation process.  Transcription involves four steps: 1- Initiation. The DNA molecule unwinds and separates to form a small open complex. RNA polymerase binds to the promoter of the template strand (also known as the 'sense strand' or 'coding strand'). The synthesis of RNA proceeds in a 5' to 3' direction, so the template strand must be 3' to 5'. 2- Elongation. RNA polymerase moves along the template strand, synthesising an mRNA molecule. In eukaryotes there are three RNA polymerases: I, II and III. The process includes a proofreading mechanism. 3- Termination. Termination in eukaryotes is more complicated, involving the addition of additional adenine nucleotides at the 3' of the RNA transcript (a process referred to as polyadenylation). 4- Processing. After transcription the RNA molecule is processed in a number of ways: introns are removed and the exons are spliced together to form a mature mRNA molecule consisting of a single protein-coding sequence. RNA synthesis involves the normal base pairing rules, but the base thymine is replaced with the base uracil. Translation Translation In translation the mature mRNA molecule is used as a template to assemble a series of amino acids to produce a polypeptide with a specific amino acid sequence. The complex in the cytoplasm at which this occurs is called a ribosome. Ribosomes are a mixture of ribosomal proteins and ribosomal RNA (rRNA), and consist of a large subunit and a small subunit.  translation involves four steps: Initiation. The small subunit of the ribosome binds at the 5' end of the mRNA molecule and moves in a 3' direction until it meets a start codon (AUG). It then forms a complex with the large unit of the ribosome. Elongation. Subsequent codons on the mRNA molecule determine which tRNA molecule linked to an amino acid binds to the mRNA. An enzyme peptidyl transferase links the amino acids together using peptide bonds. The process continues, producing a chain of amino acids as the ribosome moves along the mRNA molecule. Termination. Translation is terminated when the ribosomal complex reached one or more stop codons (UAA, UAG, UGA).  Post-translation processing of the protein Gene regulation is a label for the cellular processes that control the rate and manner of gene expression. A complex set of interactions between genes, RNA molecules, proteins (including transcription factors) and other components of the expression system determine when and where specific genes are activated and the amount of protein or RNA product produced.  Gene & Chromosome Mutations By Professor Sahar Ramzy In this lecture….. Mutation: Gene (point) Mutation Chromosomal Mutation Lesson Overview Mutations Types of Mutations Now and then cells make mistakes in copying their own DNA, inserting the wrong base or even skipping a base as a strand is put together. These variations are called mutations, from the Latin word mutare, meaning “to change.” Mutations are heritable changes in genetic information. Lesson Overview Mutations Types of Mutations All mutations fall into two basic categories: Those that produce changes in a single gene are known as gene mutations. Those that produce changes in whole chromosomes are known as chromosomal mutations.  Gene Mutation A spontaneous mutation is one that occurs as a result of natural processes in cells, for example DNA replication errors. These can be distinguished from induced mutations; those that occur as a result of interaction of DNA with an outside agent or mutagen that causes DNA damage. Mutagens may be of physical, chemical, or of biological origin. Mostly they act on the DNA directly, causing damage which may result in errors during replication. Although, severely damaged DNA can prevent replication and cause cell death. Lesson Overview Mutations Gene Mutations Mutations that involve changes in one or a few nucleotides are known as point mutations because they occur at a single point in the DNA sequence. They generally occur during replication. If a gene in one cell is altered, the alteration can be passed on to every cell that develops from the original one. Point mutations include substitutions, insertions, and deletions. Lesson Overview Mutations Substitutions In a substitution, one base is changed to a different base. Substitutions usually affect no more than a single amino acid, and sometimes they have no effect at all. Lesson Overview Mutations Substitutions In this example, the base cytosine is replaced by the base thymine, resulting in a change in the mRNA codon from CGU (arginine) to CAU (histidine). However, a change in the last base of the codon, from CGU to CGA for example, would still specify the amino acid arginine.  Gene (Point) Mutation  Transitions and transversions can lead to:  1- silent mutation  2- missense mutation  3- nonsense mutation  Silent mutations  Nucleotide substitutions in a protein- coding gene may or may not change the amino acid in the encoded protein.  Mutations that change the nucleotide sequence without changing the amino acid sequence are called synonymous mutations or silent mutations. Mutational changes in nucleotides that are outside of coding regions can also be silent. However, some noncoding sequences do have essential functions in gene regulation and, in this case, mutations in these sequences would have phenotypic effects. Silent mutations  Missense mutations  Nucleotide substitutions in protein-coding regions that do result in changed amino acids are called missense mutations or nonsynonymous mutations.  This type of mutation is a change in one DNA base pair that results in the substitution of one amino acid for another in the protein made by a gene.  A change in the amino acid sequence of a protein may alter the biological properties of the protein. Missense mutations  Nonsense mutations  A nucleotide substitution that creates a new stop codon is called a nonsense mutation. Because nonsense mutations cause premature chain termination during protein synthesis, the remaining polypeptide fragment is nearly always nonfunctional.  A nonsense mutation is also a change in one DNA base pair. Instead of substituting one amino acid for another, however, the altered DNA sequence prematurely signals the cell to stop building a protein. This type of mutation results in a shortened protein that may function improperly or not at all. Nonsense mutations Lesson Overview Mutations Insertions and Deletions Insertions and deletions are point mutations in which one base is inserted or removed from the DNA sequence. If a nucleotide is added or deleted, the bases are still read in groups of three, but now those groupings shift in every codon that follows the mutation. Lesson Overview Mutations Insertions and Deletions Insertions and deletions are also called frameshift mutations because they shift the “reading frame” of the genetic message. Frameshift mutations can change every amino acid that follows the point of the mutation and can alter a protein so much that it is unable to perform its normal functions.  Chromosomal mutation  Chromosomes, which carry the hereditary material, or DNA, are contained in the nucleus of each cell. Chromosomes come in pairs, with one member of each pair inherited from each parent. The two members of a pair are called homologous chromosomes. Each cell of an organism and all individuals of the same species have, as a rule, the same number of chromosomes.  Chromosomal mutation  Changes in the number, size, or organization of chromosomes within a species are termed chromosomal mutations, chromosomal abnormalities, or chromosomal aberrations. Changes in number may occur by the fusion of two chromosomes into one, by fission of one chromosome into two, or by addition or subtraction of one or more whole chromosomes or sets of chromosomes. Lesson Overview Mutations Chromosomal Mutations Chromosomal mutations involve changes in the number or structure of chromosomes. These mutations can change the location of genes on chromosomes and can even change the number of copies of some genes. There are four types of chromosomal mutations: deletion, duplication, inversion, and translocation. Lesson Overview Mutations Chromosomal Mutations Deletion involves the loss of all or part of a chromosome. Lesson Overview Mutations Chromosomal Mutations Duplication produces an extra copy of all or part of a chromosome. Lesson Overview Mutations Chromosomal Mutations Inversion reverses the direction of parts of a chromosome. Lesson Overview Mutations Chromosomal Mutations Translocation occurs when part of one chromosome breaks off and attaches to another.  NONDISJUNCTION Nondisjunction: chromosome pair fails to separate properly during meiosis Monosomy: gamete has 1 less chromosome than it should 45 chromosomes is the result Ex: Turner syndrome Missing a sex chromosome  Trisomy: Gamete has 1 more chromosome than it should Result is 47 chromosomes Ex: Down’s Syndrome Extra#21 chromosome  Down’s Syndrome (DS) Excess # 21 chromosome Prenatal testing can be done Result of chromosomal mutation 1 in 900 people born with this Likelihood of having a child with DS increases with advancing maternal age Symptoms: mental retardation, upward slant to eyes, small mouth, abnormal ear shape, decreased muscle tone No cure  Methods of Detection Chorion villi sampling: Take sample of the chorion –(membrane surrounding fetus) Chemical tests and Karyotyping performed Ultrasound: Sound waves are used to generate an image of the unborn child. Used to detect abnormalities of limbs, organs, etc. Amniocentesis: Fluid surrounding the fetus is drawn out by needle Fetal cells are collected and grown in a lab. Chromosomes can be then Karyotyped Lesson Overview Mutations Mutagens Some mutations arise from mutagens, chemical or physical agents in the environment. Chemical mutagens include certain pesticides, a few natural plant alkaloids, tobacco smoke, and environmental pollutants. Physical mutagens include some forms of electromagnetic radiation, such as X-rays and ultraviolet light. Lesson Overview Mutations Mutagens If these mutagens interact with DNA, they can produce mutations at high rates. Some compounds interfere with base-pairing, increasing the error rate of DNA replication. Others weaken the DNA strand, causing breaks and inversions that produce chromosomal mutations. Cells can sometimes repair the damage; but when they cannot, the DNA base sequence changes permanently. Lesson Overview Mutations Harmful and Helpful Mutations The effects of mutations on genes vary widely. Some have little or no effect; and some produce beneficial variations. Some negatively disrupt gene function. Whether a mutation is negative or beneficial depends on how its DNA changes relative to the organism’s situation. Mutations are often thought of as negative because they disrupt the normal function of genes. However, without mutations, organisms cannot evolve, because mutations are the source of genetic variability in a species. Lesson Overview Mutations Harmful Effects Some of the most harmful mutations are those that dramatically change protein structure or gene activity. The defective proteins produced by these mutations can disrupt normal biological activities, and result in genetic disorders. Some cancers, for example, are the product of mutations that cause the uncontrolled growth of cells. Lesson Overview Mutations Harmful Effects Sickle cell disease is a disorder associated with changes in the shape of red blood cells. Normal red blood cells are round. Sickle cells appear long and pointed. Sickle cell disease is caused by a point mutation in one of the polypeptides found in hemoglobin, the blood’s principal oxygen- carrying protein. Among the symptoms of the disease are anemia, severe pain, frequent infections, and stunted growth. Lesson Overview Mutations Beneficial Effects Some of the variation produced by mutations can be highly advantageous to an organism or species. Mutations often produce proteins with new or altered functions that can be useful to organisms in different or changing environments. For example, mutations have helped many insects resist chemical pesticides. Some mutations have enabled microorganisms to adapt to new chemicals in the environment. Lesson Overview Mutations Beneficial Effects Plant and animal breeders often make use of “good” mutations. For example, when a complete set of chromosomes fails to separate during meiosis, the gametes that result may produce triploid (3N) or tetraploid (4N) organisms. The condition in which an organism has extra sets of chromosomes is called polyploidy. Lesson Overview Mutations Beneficial Effects Polyploid plants are often larger and stronger than diploid plants. Important crop plants—including bananas and limes—have been produced this way. Polyploidy also occurs naturally in citrus plants, often through spontaneous mutations.  Thank you Any Questions  A codon wheel, which can be used to determine what amino acids are produced by a sequence of three mRNA nucleotides. This figure illustrates a substitution mutation, where a pair of bases (G-C) is changed for a different base pair (A-T).  This figure shows an insertion mutation, where a segment f DNA is added to an existing DNA molecule. A figure illustrating a deletion mutation where an existing piece of DNA is lost from DNA molecule.  This figure illustrates how altered reading frame from an insertion of a T nucleotide (in red) changes a protein product. This diagram shows the three chromosomal mutation types: chromosomal duplications, deletions, and inversions. Both mutations 1 and 3 are chromosomal deletions, as chromosome segments are missing compared to the original chromosome. However, in chromosome 2, the blue and gray segments are reversed between the parent and mutated chromosomes. This chromosome segment most likely broke off from the original chromosome and reattached to the chromosome in the opposite direction. Therefore, mutation 2 is an inversion mutation.  Example 1: Understanding Different DNA Mutation Types Which of the following is not a type of genetic mutation? A.Deletion B.Differentiation C.Insertion D.Substitution  Answer A genetic mutation is a base change in a DNA sequence. There are several genetic mutation types. If one or more bases are lost from the original DNA sequence at a specific location in the genome, this is said to be a “deletion mutation”; therefore, deletion is not the correct answer to the question. Type 1: A deletion mutation in DNA sequence OriginalDNAsequence:- ATGCCATGGACCG-Deletedbases:-ATG-MutatedDNAsequence:-ATGCCGACCG- 5′3′5′3′5′3′ Another genetic mutation type is an insertion mutation. In insertion mutations, one or more new nucleotides are inserted into the DNA sequence at a specific genomic location (type 2). Therefore, insertion is not the correct answer to the question. Type 2: An insertion mutation in a DNA sequence OriginalDNAsequence:- ATGCCATGGACCG-Insertedbases:-GC-MutatedDNAsequence:- ATGCCATGGCGACCG-5′̂̂̂̂3′5′3′5′3′ Finally, another genetic mutation is the substitution mutation. In substitution mutations, one nucleotide base is swapped for another nucleotide base in a DNA sequence (type 3), so substitution is not the correct answer to the question. Type 3: Substitution mutations in a DNA sequence OriginalDNAsequence:- ATGCCATGGACCG-Substitutedbase:-T-MutatedDNAsequence:- ATGCCATTGACCG-5′3′5′3′5′3′ Cells undergo differentiation by expressing genes that characterize specific cell types. For example, a stem cell (a nondifferentiated cell) will differentiate into a nerve or muscle cell by expressing different genes. However, a cell’s DNA sequence does not change during differentiation and therefore is not a mutation type. Thus, B is correct: differentiation is not a type of genetic mutation. Thank you Any questions Necrosis and Apoptosis  APOPTOSIS: control Intrinsic pathway (damage): Mitochondria BAX Cytochrome c release BCL-2 BAK BCL-XL BOK BCL-W BCL-Xs Pro-caspase 9 cleavage MCL1 BAD BFL1 BID DIVA B IK NR-13 BIM Pro-execution caspase (3) cleavage Several NIP3 viral BNIP3 proteins Caspase (3) cleavage of cellular proteins, nuclease activation, etc. Death  APOPTOSIS: control Receptor pathway (physiological): FAS ligand TNF Death receptors: (FAS, TNF-R, etc) Death domains Adaptor proteins Pro-caspase 8 (inactive) Caspase 8 (active) Pro-execution caspase (inactive) Execution caspase (active) MITOCHONDRIA Death Cells are balanced between life and death DAMAGE Physiological death signals DEATH SIGNAL PROAPOPTOTIC ANTIAPOPTOTIC PROTEINS PROTEINS (dozens!) (dozens!) DEATH  APOPTOSIS: important in embryogenesis Morphogenesis (eliminates excess cells): Selection (eliminates non-functional cells):  APOPTOSIS: important in embryogenesis Immunity (eliminates dangerous cells): Self antigen recognizing cell Organ size (eliminates excess cells):  APOPTOSIS: important in adults Tissue remodeling (eliminates cells no longer needed): Apoptosis Virgin mammary gland Late pregnancy, lactation Involution (non-pregnant, non-lactating) - Testosterone Apoptosis Prostate gland  APOPTOSIS: important in adults Tissue remodeling (eliminates cells no longer needed): Apoptosis Resting lymphocytes + antigen (e.g. infection) - antigen (e.g. recovery) Steroid immunosuppressants: kill lymphocytes by apoptosis Lymphocytes poised to die by apoptosis  APOPTOSIS: important in adults Maintains organ size and function: Apoptosis X + cell division Cells lost by apoptosis are replaced by cell division (remember limited replicative potential of normal cells restricts how many times this can occur before tissue renewal declines) APOPTOSIS: Role in Disease TOO MUCH: Tissue atrophy Neurodegeneration Thin skin etc TOO LITTLE: Hyperplasia Cancer Athersclerosis etc  APOPTOSIS: Role in Disease Neurodegeneration Neurons are post-mitotic (cannot replace themselves; neuronal stem cell replacement is inefficient) Neuronal death caused by loss of proper connections, loss of proper growth factors (e.g. NGF), and/or damage (especially oxidative damage) Neuronal dysfunction or damage results in loss of synapses or loss of cell bodies (synaptosis, can be reversible; apopsosis, irreversible) PARKINSON'S DISEASE ALZHEIMER'S DISEASE HUNTINGTON'S DISEASE etc.  APOPTOSIS: Role in Disease Cancer Apoptosis eliminates damaged cells (damage => mutations => cancer Tumor suppressor p53 controls senescence and apoptosis responses to damage Most cancer cells are defective in apoptotic response (damaged, mutant cells survive) High levels of anti-apoptotic proteins or Low levels of pro-apoptotic proteins ===> CANCER  APOPTOSIS: Role in Disease AGING Aging --> both too much and too little apoptosis (evidence for both) Too much (accumulated oxidative damage?) ---> tissue degeneration Too little (defective sensors, signals? ---> dysfunctional cells accumulate hyperplasia (precancerous lesions) OPTIMAL FUNCTION (HEALTH) APOPTOSIS AGING APOPTOSIS Neurodegeneration, cancer, …..  Cancer & TUMOR MARKERS 12/10/2024 1  What are the tumor markers?  Tumor markers are defined as a biochemical substance (e.g. hormone, enzymes or proteins) synthesized and released by cancer cells or produced in the host in response to cancerous substance.  They are used to monitor or identify the presence of cancerous growth.  They are different from substances produced by normal cell in quantity and quality. 12/10/2024 27  Tumor marker may be present in  Blood circulation  Body cavity fluids  Cell membranes  Cell cytoplasm  DNA 12/10/2024 28  A good tumor maker should have those properties:  1. A tumor marker should be present in or produced by tumor itself.  2. A tumor marker should not be present in healthy tissues.  3. Plasma level of a tumor marker should be at a minimum level in healthy subjects and in benign conditions. 12/10/2024 29 4. A tumor marker should be specific for a tissue, it should have different immunological properties when it is synthesized in other tissues. 5. Plasma level of the tumor marker should be in proportion to the both size of tumor and activity of tumor. 6. Half life of a tumor marker should not be very long 12/10/2024 30  7. A tumor marker should be present in plasma at a detectable level, eventhough tumor size is very small  8. The tumor marker is useful both for the prediction of the presence of the tumor and recurrence of the tumor. 12/10/2024 31  Tumor markers can be classified as respect with the type of the molecule:  1. Enzymes or isoenzymes (ALP, PAP)  2. Hormones (calcitonin)  3. Oncofetal antigens (AFP, CEA)  4. Carbonhydrate epitopes recognised by monoclonal antibodies (CA 15-3,CA 19-9, CA125)  5. Receptors (Estrogen, progesterone) 12/10/2024 32  6. Genetic changes (mutations in some oncogenes and tumor suppressor genes. Some mutations in BRCA1 and 2 have been linked to hereditary breast and overian cancer) 12/10/2024 33  Potential uses of tumor markers  Screening in general population  Differential diagnosis of symptomatic patients  Clinical staging of cancer  Estimating tumor volume  As a prognostic indicator for disease progression  Evaluating the success of treatment 12/10/2024 34   Detecting the recurrence of cancer  Monitoring reponse to therapy  Radioimmunolocalization of tumor masses 12/10/2024 35  In order to use a tumor marker for screening in the presence of cancer in asymptomatic individuals in general population, the marker should be produced by tumor cells and not be present in healthy people.  However, most tumor markers are present in normal, benign and cancer tissues and are not spesific enough to be used for screening cancer. 12/10/2024 36  Few markers are specific for a single individual tumor, most are found with different tumors of the same tissue type.  They are present in higher quantities in blood from cancer patients than in blood from both healthy subjects and patients with benign diseases. 12/10/2024 37   Some tumor markers have a plasma level in proportion to the size of tumor while some tumor markers have a plasma level in proportion to the activity of tumor.  The clinical staging of cancer is aidded by quantitiation of the marker. 12/10/2024 38  Serum level of marker reflects tumor burden.  The level of the marker at the time of diagnosis may be used as a prognostic indicator for disease progression and patient survival. After successful initial treatment, such as surgery, the marker value should decrease. The rate of the decrease can be predicted by using the half life of the marker. 12/10/2024 39  The magnitude of marker reduction may reflect the degree of success of the treatment.  In the case of recurrence of cancer, marker increases again.  Most tumor marker values correlate with the effectiveness of treatment. 12/10/2024 40   ENZYMES  Alkaline Phosphatase (ALP)  Increased alkaline phosphatase activities are seen in primary or secondary liver cancer. Its level may be helpful in evaluating metastatic cancer with bone or liver involvement. Placental ALP, regan isoenzyme, elevates in a variety of malignancies, including ovarian, lung, gastrointestinal cancers and Hodgkin’s disease. 12/10/2024 41  Prostatic acid phosphatase (PAP)  It is used for staging prostate cancer and for monitoring therapy. Increased PAP activity may be seen in osteogenic sarcoma, multiple myeloma and bone metastasis of other cancers and in some benign conditions such as osteoporosis and hyperparathyroidism. 12/10/2024 42  Prostate Specific Antigen (PSA)  The clinical use of PAP has been replaced by PSA. PSA is much more specific for screening or for detection early cancer. It is found in mainly prostatic tissue.  PSA exists in two major forms in blood circulation. The majority of PSA is complexed with some proteins. A minor component of PSA is free. 12/10/2024 43  PSA testing itself is not effective in detecting early prostate cancer. Other prostatic diseases, urinary bladder cateterization and digital rectal examination may lead an increased PSA level in serum.  The ratio between free and total PSA is an reliable marker for differentiation of prostatic cancer from benign prostatic hyperplasia. 12/10/2024 44   The use of PSA should not be together with digital rectal examination and followed by transrectal ultrasonography for an accurate diagnosis of cancer.  Serum level of PSA was found to be correlated with clinical stage, grade and metastasis 12/10/2024 45  The greatest clinical use of PSA is in the monitoring of treatment.  The PSA level should fall below the detection limit.  This may require 2-3 weeks. If it is still at a high level after 2-3 weeks, it must me assumed that residual tumor is present. 12/10/2024 46  Androgen deprivation therapy may have direct effect on the PSA level that is independent of the antitumor effect. This subject must be considered always. 12/10/2024 47  HORMONES  Calcitonin  Calcitonin is a hormone which decreases blood calcium concentration.  Its elevated level is usually associated with medullary thyroid cancer.  Calcitonin levels appear to correlate with tumor volume and metastasis.  Calsitonin is also useful for monitoring treatment and detecting the recurrence of cancer. 12/10/2024 48  However calcitonin levels are also at a high levels in some patients with cancer of lung, breast, kidney, liver and in nonmalignant conditions such as pulmonary diseases, pancreatitis, hyperparathyroidism, myeloproliferative disordes and pregnancy. 12/10/2024 49  Human Chorionic Gonadotropin (hCG)  It is a glycolprotein appears in pregnancy. Its high levels is a useful marker for tumors of placenta and some tumors of testes.  hCG is also at a high level in patients with primary testes insufficiency.  hCG does not cross the blood-brain barier. Higher levels in CSF may indicate metastase to brain. 12/10/2024 50  ONCOFETAL ANTIGENS  Most reliable markers in this group are α-fetoprotein and carcinoembryonic antigen (CEA) 12/10/2024 51  α-Fetoprotein (AFP)  α-fetoprotein is a marker for hepatocellular and germ cell carcinoma.  It is also increased in pregnancy and chronic liver diseases.  AFP is useful for screening (AFP levels greater than 1000 µg/L are indicative for cancer except pregnancy), determining prognosis and monitoring therapy of liver cancers. 12/10/2024 52  AFP is also a prognostic indicator of survival.  Serum AFP levels is less than 10 µg/L in healthy adults. Elevated AFP levels are associated with shorter survival time.  AFP and hCG combined are useful in classifying and staging germ cell tumors.One or both markers are increased in those tumors. 12/10/2024 53  Carcinoembryonic antigen (CEA)  It is a cell-surface protein and a well defined tumor marker.  CEA is a marker for colorectal, gastrointestinal, lung and breast carcinoma.  CEA levels are also elevated in smokers and some patients having benign conditions such as cirrhosis, rectal polips, ulcerative colitis and benign breast disease.  CEA testing should not be used for screening. Some tumors don’t produce CEA. It is useful for staging and monitoring therapy. 12/10/2024 54  CARBOHYDRATE MARKERS  These markers either are antigens on the tumor cell surface or are secreted by tumor cells.  They are high-molecular weight mucins or blood group antigens. Monoclonal antibodies have been developed against these antigens.  Most reliable markers in this group are CA 15-3, CA 125 and CA19-9. 12/10/2024 55  CA 15-3  CA 15-3 is a marker for breast carcinoma. Elevated CA 15-3 levels are also found in patients with pancreatic, lung, ovarian, colorectal and liver cancer and in some benign breast and liver diseases.  It is not useful for diagnosis. It is most useful for monitoring therapy. 12/10/2024 56  CA 125  Although CA 125 is a marker for ovarian and endometrial carcinomas, it is not specific. CA 125 elevates in pancreatic, lung, breast, colorectal and gastrointestinal cancer, and in benign conditions such as cirrhosis, hepatitis, endometriosis, pericarditis and early pregnancy.  It is useful in detecting residual disease in cancer patients following initial therapy. 12/10/2024 57  A preoperative CA 125 level of less than 65 kU/L is associated with a greater 5 y survival rate than is a level greater 65 kU/L.  It is also useful in differentiating benign from malignant disease in patients with ovarian masses.  In the detection of recurrence, use of CA 125 level as an indicator is about 75 % accurate. 12/10/2024 58  CA 19-9  CA 19-9 is a marker for both colorectal and pancreatic carcinoma.However elevated levels were seen in patients with hepatobiliary, gastric, hepatocellular and breast cancer and in benign conditions such as pancreatitis and benign gastrointestinal diseases.  CA 19-9 levels correlate with pancreatic cancer staging.  It is useful in monitoring pancreatic and colorectal cancer. 12/10/2024 59  Elevated levels of CA 19-9 can indicate recurrence before detected by radiography or clinical findings in pancreatic and colorectal cancer. 12/10/2024 60  PROTEIN MARKERS  Most reliable markers in this group are β2- microglobulin, ferritin, thyroglobulin and immunoglobulin.  β2-microglobulin  β2-microglobulin is a marker for multiple myeloma, Hodgkin lymphoma. It also increases in chronic inflammation and viral hepatitis. 12/10/2024 61  Ferritin  Ferritin is a marker for Hodgkin lymphoma, leukemia, liver, lung and breast cancer.  Thyroglobulin  It is a useful marker for detection of differentiated thyroid cancer. 12/10/2024 62   Immunoglobulin: Monoclonal immunoglobulin has been used as marker for multiple myeloma for more than 100 years.  Monoclonal paraproteins appear as sharp bands in the globulin area of the serum protein electrophoresis.  Bence-Jones protein is a free monoclonal immunoglobulin light chain in the urine and it is a reliable marker for multiple myeloma. 12/10/2024 63  RECEPTOR MARKERS  Estrogen and progesterone receptors are used in breast cancer as indicators for hormonal therapy.  Patients with positive estrogen and progesterone receptors tend to respond to hormonal treatment.  Those with negative receptors will be treated by other therapies. 12/10/2024 64   Hormone receptors also serve as a prognostic factors in breast cancer. Patients with positive receptor levels tend to survive longer. 12/10/2024 65  Cytoplasmic estrogen receptors are now routinely measured in samples of breast tissue after surgial removal of a tumor. Of patients with breast cancer, 60 % have tumors with estrogen receptor.  Approximately two thirds of patients with estrogen receptor (+) tumors respond to the hormonal therapy. 5% of patients with estrogen receptor (-) tumors respond to the hormonal therapy. 12/10/2024 66   Progesterone receptor testing is a useful adjunt to the estrogen receptor testing. Because progesterone receptor synthesis appears to be dependent on estrogen action.  Measurement of progesterone receptors provides a confirmation that all the steps of estrogen action are intact. Indeed breast cancer patients with both progesterone and estrogen receptor (+) tumors have a higher response rate to hormonal therapy. 12/10/2024 67  C-erbB2 (HER-2 Neu)  It is receptor for epidermal growth factor (EGF) but it doesn’t contain EGF binding domain. It serves as a co-receptor in EGF action  In the case of increased expression of C- erbB2 leads the oto-activation and increased signal transduction 12/10/2024 68  GENETIC CHANGES  Four classes of genes are implicated in development of cancer:  1) protooncogenes which are responsible for normal cell growth and differentiation  2) tumor suppressor genes Alterations on these genes may lead tumor development. 3)apoptosis-related genes are responsible for regulation of apoptosis  4)DNA repair genes  which are involved in recognition and repair of damaged DNA. 12/10/2024 69  Susceptible protooncogenes:  K-ras, N-ras mutations are found to be correlated acute myeloid leukemia, neuroblastoma 12/10/2024 70  Susceptible DNA repair genes:  BRCA1 and BRCA2 are specific genes in inherited predisposition for developing breast and over cancer, and mutations on these genes are newly measured in some laboratories.  Mismatch-repair genes are mutated in some colon cancers 12/10/2024 71  Susceptible tumor suppressor genes:  Retinoblastoma gene  P53 gene  P21 gene  Those genetic markers are very new and not routinely measured in laboratories. 12/10/2024 72  Chromosomal translocation  c-myc gene has been found to be translocated from 8.chromosom to 14. chromosom and than become activated in Burkitt’s lymphoma.  myc gene encodes a DNA-binding protein which stimulates cell dividing. 12/10/2024 73   In chronic myeloid leukemia, there is a translocation between chromosomes 9 and 22. 12/10/2024 Naglaa Alhusseini 74 12/10/2024 75 12/10/2024 76 12/10/2024 77 Recombinant DNA Technology http://www.free-powerpoint-templates-design.com  Recombinant DNA technology  Involves using enzymes and various laboratory techniques to manipulate and isolate DNA segments of interest  This method can be used to combine (or splice) DNA from different species or to create genes with new functions  The resulting copies are often referred to as recombinant DNA 2 September 2023  Molecular cloning Is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms 2 September 2023  Molecular cloning Importance of molecular cloning 1. Isolation, purification, production of large amount of specific gene, which allow characterization and manipulation of the genes and their products 2. Gene sequencing, and consequently detection of the amino acid sequence of the corresponding protein 3. Transferring valuable genes from undesirable organisms to defined and safe organisms in order to produce useful items 4. By altering a cloned gene's sequence, genetic engineers can literally create new, potentially beneficial biological products. 2 September 2023  Molecular cloning Steps of gene cloning 1. Isolation and fragmentation of the DNA source to separate specific gene. This is carried out by using specific Restriction Enzyme 2. Joining the DNA fragments to a cloning vector , treated by the same restriction enzyme, is carried out by using Ligase enzyme forming recombinant DNA 3. Incorporation into host: the recombinant DNA in a test tube can be introduced into a host organism which is then replicated. Insertion of the recombinant DNA in the organism is carried out by:  Transformation (Transfection): can be carried out by CaCl2 method or electroporation  Transduction 2 September 2023  Molecular cloning Steps of gene cloning (cont.) 4. Detection and purification of the desired clone 5. Production of large number of cells containing the desired gene for Industrial Microbiology or for isolation of the cloned DNA 2 September 2023  BamHI site Bacteria pBR322 Isolation of DNA Digestion by BamHI rest. Gene Cloning endonuclease Separation of the desired gene by System the same restrict. pBR322 endonuclease DNA Ligase Desired gene pBR322 2 September 2023 Recombinant DNA (Hybrid)  Transformation Or pBR322 transfection Host cell Recombinant DNA Recombinant bacteria Gene Cloning (Clone cell) System (cont.) Selection of clone cell and cultivation in controlled media 2 September 2023  I. Vectors Vector is a molecule of DNA into which passenger DNA can be cloned forming recombinant DNA. The vector and passenger DNA are covalently joined by ligation Types of Vectors: 1. Cloning Vector 2. Expression Vector 3. Secretion Vector 4. Shuttle Vector 2 September 2023  1. Cloning Vector A cloning vector is used to acquire multiple copies of the foreign DNA fragment (Gene of interest) Main Characters of the vector 1. Easily introduced into the host 2. Able to replicate in the host (Has high copy Number) 3. Have unique sites for the action of a variety of restriction endonucleases 4. Have marker genes such as antibiotic resistance 2 September 2023  1. Cloning Vector Types of cloning Vectors: Cloning vectors A.Plasmids D. Others B. Bacteriophages C. Cosmids 2 September 2023  A. Plasmid The main properties of plasmid as cloning vectors include: 1. Small in size 2. Circular DNA 3. Independent origin of replication 4. Multiple copy number 5. Selectable markers Examples : Plasmid pBR322 , pUC series 2 September(pUC18 2023 & pUC19) Cloning Vectors 1. Plasmid pBR322 General features: 1. Small size (4361 bp) 2. High copy number (20-30 copies/ cell) 3. An origin of replication (Ori) 4. Several unique recognition sequences (EcoRI, BamHI) 5. Two genes that confer resistance to different antibiotics (tet & amp) 6. Easily inserted in host cell by transformation 2 September 2023 Insertional inactivation Is used to detect the presence of foreign DNA within the plasmid 2 September 2023  Cloning system using pBR322 plasmid Vector + Desired gene Ligation Vector + Recombinant DNA Host cells Transformation Recombinant bacteria Host cells Recombinant bacteria With recombinant DNA with original vector (Right clone) Resist to Amp. Resist to Amp. and Sensitive to and tetr. sen2sSietipvteembteor 202T3etr. Tetr.and Amp. Diagram of Replica Technique 2 September 2023 2 September 2023 A. 2. pUC18 plasmid cloning vector It has:  One ampR gene (Marker gene)  N-terminal fragment of β-galactosidase (lacZα) gene which contain multiple cloning site various restriction sites for many restriction endonucleases are present 2 September 2023 A. 3. pUC19 plasmid cloning vector It has:  One ampR gene  N-terminal fragment of β-galactosidase (lacZ α) gene which contain multiple cloning site various restriction sites for many restriction endonucleases are present 2 September 2023  pUC18 and pUC19 plasmid *pUC18 and pUC19 are similar but different in multiple cloning sites Principle of selecting the right clone Blue white screen Chromosome Plasmid 2 September 2023 alpha complementation The vector has a short portion of the β- galactosidase gene (the alpha fragment), and the bacterial chromosome has the rest of the gene If both give rise to proteins, the subunits combine to form functional β-galactosidase If DNA is inserted into the plasmid-borne gene segment, the encoded subunit is not made and β-galactosidase is not produced When β-galactosidase is expressed, the bacteria can degrade X-gal, which turns the bacterial colony blue. If a piece of DNA is inserted into the alpha fragment gene, the bacteria cannot split X-gal and stay white 2 September 2023  Cloning system using pUC18 or 19 plasmid Vector + Desired gene Ligation Vector + Recombinant DNA Host cells Transformation Recombinant bacteria Host cells Recombinant bacteria With recombinant DNA with original vector (Right clone) Resist to Amp. Sensitive to Amp. Resist to Amp. W2hSietpetemcboerlo 2023 nies. No growth. 2. pUC18 and pUC19 plasmid Detection of the right clone by using pUC18 or pUC19 vector  pUC18 vector similar to pUC19, but the MCS region is reversed.  Each of them has one ampR gene (ampicillin resistance gene), and an N-terminal fragment of β-galactosidase (lacZα) gene of E. coli.  The multiple cloning site (MCS) region is split into the lacZα gene (codons 6–7 of lacZα are replaced by MCS), where various restriction sites for many restriction endonucleases are present.  The lacZα gene codes for β-galactosidase where its N-terminal fragment contains several recognition sites for various restriction enzymes.  The plasmid “vector” is introduced into a bacterial cell by transformation, where it can multiply and express itself.  The lac Zα fragment, whose synthesis can be induced by isopropyl thiogalactoside (IPTG), is capable of intra-allelic complementation with a defective form of β-galactosidase enzyme encoded by host chromosome (mutation lacZDM15 in E. coli JM109, DH5α and XL1-Blue strains). 2 September 2023 2. pUC18 and pUC19 plasmid Detection of the right clone by using pUC18 or pUC19 vector (cont.)  In the presence of IPTG in growth medium, bacteria synthesize both fragments of the enzyme which hydrolyze X-gal (5- bromo-4-chloro-3-indolyl- beta-D-galactopyranoside) and form blue colonies when grown on media where it is supplemented.  When a foreign piece of DNA of choice is introduced into it by inserting it into place in MCS region. The recombinant cells can be differentiated from cells which have not taken up the plasmid by growing it on media with ampicillin. Only the cells with the plasmid containing the ampicillin resistance (ampR) gene will survive.  Furthermore, the transformed cells containing the plasmid with the gene of our interest can be distinguished from cells with the plasmid but without the gene of interest, just by looking at the color of the colony they make on agar media supplemented with IPTG and X-gal. Recombinants are white, whereas non-recombinants are blue.  A foreign piece of DNA of choice can be introduced into it by inserting it into place in MCS region. 2 September 2023 2. pUC18 and pUC19 plasmid Detection of the right clone by using pUC18 or pUC19 vector (cont.)  The cells which have taken up the plasmid can be differentiated from cells which have not taken up the plasmid by growing it on media with ampicillin. Only the cells with the plasmid containing the ampicillin resistance (ampR) gene will survive.  Furthermore, the transformed cells containing the plasmid with the gene of our interest can be distinguished from cells with the plasmid but without the gene of interest, just by looking at the color of the colony they make on agar media supplemented with IPTG and X-gal. Recombinants are white, whereas non-recombinants are blue. This is the most notable feature of pUC19. 2 September 2023  Recombinant DNA technology has several promising applications in dentistry, including: Gene Therapy: Used to treat genetic disorders affecting oral tissues, such as amelogenesis imperfecta, which affects enamel formation Tissue Engineering: Helps in the regeneration of bone and periodontal tissues by introducing specific genes that promote tissue growth and repair. Disease Diagnosis: Enhances the detection of oral diseases by identifying genetic markers associated with conditions like oral cancer and periodontal disease. Antibiotic Resistance: Develops genetically engineered bacteria that can produce antimicrobial peptides to combat oral infections resistant to traditional antibiotics. Tooth Development: Studies the genetic factors involved in tooth development to better understand and potentially correct developmental anomalies. Caries Prevention: Investigates genes that influence the composition of saliva and plaque formation to develop targeted preventive treatments for dental caries.  Applications Cont. Regenerative Medicine: Uses Vaccine Development: recombinant DNA to create Develops vaccines for oral scaffolds that support the pathogens by introducing growth of new cells and tissues genes encoding antigens for dental implants and other that stimulate an immune restorative procedures. response. Personalized Dentistry: Drug Delivery Systems: Tailors dental treatments based Creates genetically engineered on an individual's genetic vectors for targeted delivery of profile, improving the therapeutic agents directly to effectiveness and outcomes of affected areas in the oral dental care. cavity. Thank You