Pharmaceutical Biotechnology Lecture 9 PDF

# Level 5, Semester 1 ## Pharmaceutical Biotechnology ### Lecture 9 #### Prof. Mona Ibrahem Shaaban Prof. of Microbiology and Immunology, Faculty of Pharmacy, Mansoura University # Microbial Genetics ## Lecture Summary - Transcription - Translation # Protein Synthesis - DNA --> mRNA --> Protein...

# Level 5, Semester 1 ## Pharmaceutical Biotechnology ### Lecture 9 #### Prof. Mona Ibrahem Shaaban Prof. of Microbiology and Immunology, Faculty of Pharmacy, Mansoura University # Microbial Genetics ## Lecture Summary - Transcription - Translation # Protein Synthesis - DNA --> mRNA --> Protein - Transcription - Translation - Central Dogma of Molecular Genetics # Triplet Code - DNA serves as a template for RNA synthesis - A codon is a sequence of three nucleotides - Codon's are translated into chains of amino-acids (polypeptides) - Image of DNA and RNA. DNA molecule is a double helix with the bases Adenine, Cytosine, Guanine and Thymine. The DNA strand is transcribed into mRNA, which is a single strand with the bases Uracil, Cytosine, Guanine and Adenine. The mRNA codon is translated into different amino acids. # mRNA Structure - mRNAs consist of a sequence of nucleotide codons that code for amino acids - The Genetic Code: 64 CODONES - 61 sense codons for 20 amino acids - Amino acid is coded for by more than one codon - 3 stop codons = nonsense codons - Note AUG, the start codon, codes for Methionine - Image of genetic code table, depicting the 64 codons and the amino acids that they code for. # Process of Transcription and Translation - Image of a DNA molecule being transcribed into mRNA. The image depicts the sense strand, template strand and the codons and anticodons. - Image depicts the translation of mRNA into polypeptide. The image shows the tRNa with the tRNA anticodon aligning with the mRNA codons. - The image also shows the translation of each codon into an amino acid. ## Transcription - One strand of DNA is used as a template strand (3-5) to form a complementary strand of mRNA (5-3). - The other DNA strand is called the sense strand. - The sense stand is similar in sequence to the mRNA strand EXCEPT T is replaced with U. - Image of a section of a gene with regions labelled: promoter, leader, coding, and termination. - Image of the transcription of a gene. The image shows the Shine-Dalgarno sequence and the start codon. ## mRNA - mRNA --> messenger RNA - RNA synthesis requires: - Promoter - RNA Polymerase - Termination Site - 5' to 3' of the DNA to form mRNA new 3 to 5 ## Transcription Process - RNA polymerase attaches to promoter DNA and begins to synthesize RNA. - This promoter site (TATAAA, called the TATA box) is not transcribed but just bind the RNA polymerase enzyme - Transcription begins at 3-TAC-5 to 5-AUG-3 ## Elongation - As the RNA grows, it separates from its DNA template, and the 2 DNA strands bind back together. ## Termination - RNA polymerase reaches a terminator sequence in the DNA that signals end of the gene. - RNA polymerase detaches from RNA and from gene. ## RNA synthesis - Image of RNA synthesis. 1. RNA polymerase binds to the promoter. 2. RNA is synthesized. 3. The site of synthesis moves along DNA. 4. Transcription reaches the terminator. 5. RNA and RNA polymerase is released and the DNA helix reforms. ## Transcription in Eukaryotic - Inside the nucleus - RNA splicing: - Introns (noncoding sequences) are cut out - Exons (coding sequences) are joined together. - Cap and tail are added to facilitate release of mRNA from nucleus, stabilize mRNA, and help ribosomes bind to it. - Cap: a single G nucleotide - Tail: 50-250 A nucleotides - Image of a eukaryotic gene, depicting introns and exons. # Translation - mRNA translation begins at start codon (AUG) and terminates at stop codon (UAA, UAG, UGA). - Includes three main steps: - Initiation - Elongation - Termination ## Initiation - mRNA binds to the small ribosomal subunit and initiator tRNA binds to start codon (AUG). - Initiator carries the amino acid methionine (Met). - Large ribosomal subunit binds to the small one. - Initiator tRNA fits into the P site of the ribosome. - The other tRNA-binding site on the ribosome (the A site) is empty, awaiting the next amino-acid-bearing tRNA. - Image of the initiation of translation. ## Elongation - New tRNA carrying an amino acid binds to mRNA codon in the A site of the ribosome. - Peptide bond formation between amino acid at P site and A site and Polypeptide separates from tRNA at P site. - The P site tRNA leaves the ribosome and the ribosome translocates the tRNA in the A site to the P site. - Image depicting the elongation process. ## Termination - A stop codon reaches the ribosome’s A site. - Stop codons (UAA, UAG, UGA) don’t code for amino acids but instead act as signals to stop translation. - The completed polypeptide is released from the last tRNA, and the ribosome subunits separate. ## Transfer of Genetic Elements - Three different natural processes by which bacteria can gain genetic material (DNA). - Transformation in which DNA is taken up from the environment. - Conjugation in which DNA (plasmid) is transferred from one bacteria to another. - Transduction in which the transfer of DNA from one bacteria to another is mediated by a bacteriophage. - Image depicting the three types of genetic transfer: transformation, transduction and conjugation. ## Transformation - Genes are transferred from one bacterium as "naked" DNA to another - Frederick Griffith (1928) - Image of Griffith's experiment with four stages. - **Transformation process:** - A fragment of DNA from the donor bacterium or degraded DNA binds to DNA binding proteins on the cell wall of a competent, living recipient bacterium - Competence: A cell that is able to take up DNA and be transformed. Competent cells bind much more DNA than do noncompetent cells. - Natural competence: special proteins play a role in the uptake and processing of DNA called recA. - Examples are, Streptococcus pneumonia, Bacillus subtilis, Hemophilus influensa, Neisseria gonorrhoeae. - **Artificial competence:** - Bacteria treated with high concentrations of calcium ions. - Small pores are produced in the membranes of cells - Image depicting the transformation process. ## Conjugation - Bacterial Conjugation there is a transfer of DNA (in a plasmid) from a donor bacterium to a recipient bacterium. - Donor cells should have a sex pilus F+. - Image of bacterial conjugation showing a sex pilus connecting two bacteria. - Image depicting conjugation: - 1. Donor and recipient bacteria - 2. DNA polymerase is transferring DNA from donor to recipient. - 3. The plasmid is integrated into the bacterial genome. - 4. The old donor and the new donor. ## Importance of Conjugation - A bacteria cell can get many new genes and new genetic properties. - In some cases these can be incorporated into the genome and become part of the genome - It play an important roll in the transfer of antibiotic resistance between bacteria. ## Transduction - DNA is transferred from one bacterium to another by a virus known as bacteriophage - Image of a bacteriophage. ## Generalized transduction - During a lytic infection, the enzymes responsible for packaging viral DNA into the bacteriophage sometimes accidentally package host DNA - Undergo genetic recombination with the DNA of the new host. - Image of generalized transduction. ## Thank You - Image of a thank you card # Level 2, Semester 3 ## General Microbiology and Immunology ### Lecture 7 A #### Prof. Mona Ibrahem Shaaban Prof. of Microbiology and Immunology, Faculty of Pharmacy, Mansoura University ## Microbial Genetics ## Lecture Summary - Structure of DNA - Characters of DNA - Replication - Transcription - Translation - Mutation # Structure and Function of Genetic Material - DNA and RNA - DNA: deoxyribonucleic acid - **Function of DNA:** - Storage of genetic information - Expression of the genetic information to proteins # DNA ## Structure of DNA - DNA is a polymer called ‘polynucleotide”. - The monomer units of DNA are nucleotides. - **Nucleotide consists of:** - 5-carbon sugar (deoxyribose). - Nitrogen containing base attached to the sugar. - A phosphate group. - Image of a nucleotide. - Image of a nucleoside. ## Types of Nucleotides - **Adenine** (A): - Purine nucleotide - Image of adenine molecular structure - **Guanine** (G): - Purine nucleotide - Image of guanine molecular structure - **Thymine** (T): - Pyrimidine nucleotide - Image of thymine molecular structure. - **Cytosine** (C): - Pyrimidine nucleotide - Image of cytosine molecular structure ## DNA Backbone - The deoxyribose sugars are joined at 3'-hydroxyl and 5'-hydroxyl groups to phosphate groups in ester links, which known as "phosphodiester" bonds. - Image of the DNA backbone. ## Characters of DNA Molecule - **Base pairing complementary sequences:** - DNA is Composed of two strands that are held together by hydrogen bond - **Complementary bases means:** - A and T forming two hydrogen bonds - G and C forming three hydrogen bonds (more stable) - Image of a DNA molecule showing complementary base pairs. ## Double Helix and Complementary Sequences of DNA - Composed of parallel strand and anti-parallel strand (one oriented 5'-3' and the other 3' – 5') - If sequence 5'-ATGTC-3' is present on one chain, the opposite chain must have the complementary sequence 3'-TACAG-5'. - Image of a double helix of DNA showing the structure of the two strands. ## Double-Stranded DNA Features - 10 base pairs per turn - One complete turn 3.4 nm - Width is 2nm ## Supercoiling - DNA can be twisted like a rope in a process called DNA super coiling. - Topoisomerases enzymes are needed to relieve the twisting. - **DNA in prokaryote:** Supercoiled called **chromosome.** - **In Eukaryote:** the DNA binds with **histones** proteins to form **nucleosome** and super coiled to form **Chromatin** - Image of a supercoiled molecule of DNA. ## Histones - Basic proteins with a positive charge that bind to DNA. ## Functions of Chromatin - Package DNA into a smaller volume to fit in the cell, - Prevent DNA damage - Control gene expression - Control gene replication - Image of a chromosome depicting chromatin. ## Chromatin, Chromosomes, Genes - **Genes:** - It is polynucleotide sequence that code for one or more functional products (polypeptide, tRNA, rRNA) - **Chromosome:** - It is a DNA molecule - In bacteria there is a single chromosome, while in Human there are 46 chromosomes (23 x 2) - **Chromatin:** - The DNA and associated proteins an supercoiled in the nucleus of eukaryotes. ## Eukaryotic Chromosome Structure - Eukaryotic genes are characterized by presence of: - Introns (noncoding sequences) - Exons (coding sequences) - Diagram of a eukaryote gene. # RNA ## Structure - RNA is a single-stranded polynucleotide - contains ribose as a sugar moiety - Uracil instead of thymine. - Image of RNA structure. - Image of Uracil and Thymine, showing the difference between the two. ## Difference Between DNA and RNA - **DNA:** - Double Helix - Deoxyribose sugar - Adenine pairs with Thymine (A-T) - Stays in nucleus - **RNA:** - Single strand - Ribose sugar - Uracil replaces Thymine! - Leaves nucleus to Cytoplasm - Image of a DNA and RNA molecule to illustrate the difference between the two. ## Types of RNA - **Messenger RNA (mRNA):** - Nucleus, migrates to ribosomes in cytoplasm - Encodes genetic information from DNA that will be translated into protein - **Transfer RNA (tRNA):** - Cytoplasm - Has an anticodon end that recognizes codons on mRNA and delivers the appropriate amino acid to a built up polypeptide chain. - **Ribosomal RNA (rRNA):** - Cytoplasm - Structural component of ribosomes ## Transfer RNA (tRNA) - Image of tRNA, showing the anticodon site and the amino acid attachment site. ## Ribosomal RNA (rRNA) - 3 grooves on the ribosome (A, P, E) - A: tRNA binding site - P: polypeptide bonding site - E: exit site - Ribosomes have 2 subunits - Image of a ribosome, showing the A, P and E sites. # Central Dogma of Life - Never happen (till now) - Reverse transcription - Transcription - Translation - DNA --> mRNA --> Protein; Proteins have regulatory and structural functions - Duplication: Genes are passed on to next generation of cells (Replication) - Information flow - Genotype --> Phenotype ## Central Dogma of Life - DNA → DNA = **Replication** - DNA → RNA = **Transcription** - RNA → Protein = **Translation** - RNA → cDNA = **Reverse Transcription** - Protein → RNA or DNA: **DOES NOT HAPPEN** - Information flows from Genotype to Phenotype # DNA Replication: Semi-Conservative Model - (Watson & Crick) - Means half of DNA molecules is conserved from the original DNA molecule - New DNA consists of one PARENTAL (original) and one NEW strand of DNA. - Image of the semi-conservative model. ## Where Does DNA Replication Start? - It begins at origins of replication (bubbles). - Prokaryotes (bacteria) have a single bubble, - Eukaryotes have many bubbles. - Image of a DNA strand undergoing replication, showing the origin of replication and the bubbles. ## DNA Replication - **DNA Gyrase** (a type of topoisomerase) relaxes the supercoiled DNA. - **DNA helicase:** separates the 2 DNA strands by break the weak hydrogen bonds. - **Single-Strand Binding Proteins (SSB):** attach and keep the 2 DNA strands separated and untwisted with formation of replication fork. - **Primase:** synthesizes a short RNA primer of 10-12 nucleotides - (Why primer is made from RNA **NOT** DNA) ## DNA Polymerase III - DNA polymerase III can only add nucleotides to the 3' end of the DNA at the RNA primer. - This causes the NEW strand to be built in a 5' to 3' direction. - It is proofreading "remove mismatched base pairs" ## Leading Strand - The Leading Strand is synthesized as a single strand in the direction 5-3 of DNA templet ## Lagging Strand - The Lagging Strand is synthesized discontinuously. - It is replicated from the replication fork toward the origin. - This strand is made in MANY short segments called Okazaki Fragments which joined together by ligase enzyme. ## DNA Replication (Step-by-step): - 1. Primase (RNA polymerase) synthesizes a short RNA primer of 10-12 nucleotides. - 2. DNA polymerase III add nucleotides in the direction of 5'-3' to form Okazaki fragments. - 3. Polymerase I: removed and replaced RNA primer is with DNA - 4. DNA ligase seals the gaps between Okazaki fragments with a phosphodiester bond. - Image of DNA replication process. ## Thank You - Image of a thank you card # Level 2, Semester 3 ## General Microbiology and Immunology ### Lecture 6 A #### Prof. Mona Ibrahem Shaaban Prof. of Microbiology and Immunology, Faculty of Pharmacy, Mansoura University ## Fungi ## Lecture Summary - Introduction to fungi - Structure - Classification - Replication # Fungi - Definition: is a group of eukaryotic nonphotosynthetic microorganisms - **Structure of fungi:** - **Fungal cell wall composition:** -Consist of polysaccharides, cellulose and/or chitin, mannan or glucan. - Structural components: - chitin microfibers - B-linked glucans - Mannoproteins (form matrix throughout wall) - Image of the fungal cell wall. ## The Plasma membrane - Structure: sterol ergosterol - Function: - Antigenic glycoproteins, agglutinins - Assist in adhesions ## Fungal nuclei - It has chromosomes - Surrounded by Nuclear membrane - microtubule-Spindle pole bodies- Centrioles ## Organelles - Other types: - Ribosomes 80 S(40S, 60S) - Endoplasmic reticulum, vacuoles, lipid bodies, microtubules, vesicles - Glycogen, lipids and trehalose # Nutrition in Fungi - Heterotrophs: that acquire their nutrients by absorption of small organic molecules from the surroundings. - Saprobes (saprophors): get their, carbon, and nitrogen directly from dead organic matter. - Biotrophic: can grow only on specific living host - NOT Autotrophic: make their food by photosynthesis - The most suitable pH of fungal growth is 4.5-6.5 # Fungal Morphology ## Yeast cells - Characters of yeast: - Shape: Unicellular, oval or spherical cells. - Morphology: - Individual yeast cells called Blastoconidia - Elongated bud called Pseudohyphae - Some yeasts produce thick-walled, spore like structures called chlamydospores - Reproduction by budding - Image of yeast cells. ## Molds - Most fungi grow as group of filaments called mycelium. - Each filament is known as "hypha" - Hyphae may "septa”. or not septated. - Image of mold hyphae. ## Dimorphic Fungi - Dimorphism; Under certain environmental conditions some fungi exhibit two different forms appearing as either 20-30 °C as molds or 37 °C as yeasts. - Image of dimorphic fungi. # Fungal Reproduction - Fungi reproduce by asexual and sexual spores. ## Asexual reproduction - **Conidiospore:** Spores arranged in chains or as single spores at the end of an aerial hypha (conidiophore) - Aspergillus spp. OR Penicillium spp. - **Sporangiospores:** Sporangiospores are present inside the sporangium (spore sac) - Rhizopus spp, Mucor spp, Absidia spp - **Arthrospore:** Formed by the fragmentation of a septate hyphae into single, thickened cells - e.g. Coccidiodes immitis - **Chlamydospores:** A thick-walled enlarged mycelium. - e.g.Candida albicans - **Blastospores:** They are formed from buds of the parent cell. - Such spores found in yeasts - Image depicting different types of asexual spores. ## Sexual spores - **Zygospores:** Fusion of two similar Cells. - Ex. Zygomycota - **Basidiospores:** A spore formed externally on a base of basidium. - There are 4 basidiospores per basidium - e.g.Basidiomycota. - **Ascospores:** - Produced in a sac like structure called an ascus. - There are 2-8 ascospores in an ascus - E.g. Ascomycota. - Penicillium and Aspergillus. - **Fungi imperfecti (Deuteromycetes):** - They have no sexual spores - Reproduce by asexual reproduction, including budding. - They grow as mycelium or as yeast-like cells. - The majority of pathogenic fungi for man and animal belong to this order. - Image depicting examples of sexual spores. ## Importance of Studying Fungi - Pathogenicity: few species have been identified as human pathogens. - Toxin production: such as mycotoxins that cause liver cancer - The metabolic activity of fungi is a damaging factor. Fungi can destroy foods and wooden structures. - Fungi can cause numerous plant diseases, in particular diseases of crops - The metabolic activity of fungi such as: - Food industry (production of bread, wine, beer, cheese and etc) - Pharmaceutical industry (e.g., production of antibiotics, enzymes, citric acid, etc) ## Comparison of Fungi and Bacteria - **Property:** - **Fungi:** - Cell membrane: Steroid present - Cell wall: Glucans; mannans; chitin - Spores: Sexual and asexual reproductive spores - **Bacteria:** - Cell membrane: Sterols absent except in Mycoplasma - Cell wall: NAM/NAG; teichoic acid - Spores: Endospores - Image of a table outlining the comparison of fungi to bacteria. ## Thank You - Image of a thank you card.