Microbial Genetics Dr.

Ramillo-Cruz Review of past discussions: Microbes of Medical Importance Charact eristic Virus es Bacteri a Fungi Proto zoa and Helmi nths Yes 1525 DNA and RNA Eukary otic 80S Presen t Flexibl e memb rane Prokaryotes and Eukaryotes Characteristic Prokary otes DNA within a No nuclear membrane Mitotic division No DNA associated with histones Chromosome number Membrane-bound organelles Size of ribosome No One No 70S Eukaryot es Yes Yes Yes More than one Yes 80S

Cells Size ( m) Nucleic acid Nucleu s Riboso mes Mitoch ondria Nature of outer surface

No 0.02 0.2 DNA or RNA None Absent Absent Protei n capsid and lipopro tein envelo pe None Lysog eny

Yes 15 DNA and RNA Prokaryo tic 70S Absent Rigid wall containi ng peptidog lycan

Yes 310 DNA and RNA Eukar yotic 80S Prese nt Rigid wall contai ning chitin

Motility Some None Most Method Binary Buddi Sexual of fission ng life replicat cycles ion Viruses No cells. Proteins only. Bacteria since bacteria is prokaryotic, it has no nuclear membrane. The nucleus of the bacteria is called nucleiod since it has no nuclear membrane. Ribosomes are absent in Viruses but in Bacteria 70s, smaller size of Ribosomes. (This is very important itong size na to in comparison with the eukaryotes because size na yan would distinguish kung anung aatakihin ng antibiotics.) Mitochondria and other cell membrane organelles are absent in bacteria as well as sa viruse. Where does metabolism occur in the prokaryotes? In the Cytoplasm Replication is Binary fission in bacteria

Cell wall Yes No containing peptidoglycan DNA without nuclear membrane nucleiod Mitotic Division Eukaryotes Prokaryotes anung division? Binary Fission DNA not associated with histones in Prokaryotes. Division is a bit messy compared to Eukaryotes. Chromosome number in Prokaryotes is only 1. More than 1 in Eukaryotes. Since double ang chromosome sa Eukaryotes ang tawag ay Diploid. No membrane-bound organelle Haploid Ribosome is only small Eukaryotes has no Peptidoglycan unlike the Prokaryotes Question 1: While performing the Gram stain, you forgot the Iodine treatment step. Which of the ff is correct? A. All the cells will be blue B. All the cells will be decolorized C. All the cells will be purple D. All the cells will be red E. Half will be red, half will be blue D. Iodine nagseserve as Mordant. Mordant mas pinapakapit niya yung kulay which is yung una sa gram staining is cystal violet. Crystal violet is the dye. Pag namiss ang iodine which is yung Mordant, pag gagamit ka ng alcohol maeerase lang yung Crystal Violet so magkukulay ka ulit ng Safranin Red kaya ang matitirang kulay

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nalang ay safranin red. Assignments lalabas sa test. Microbial Genetics Question 2: What is a Gene? A. The total of the genetic material in a cell B. The site on a DNA molecule that carries the code for a certain cell function C. Extra-chromosomal unit of heredity D. Diploid for prokaryotes Ans. A. Genome B. Gene is a group of nucleotides, nucleotide base pairs, codons that codes or carries code for a certain cell function. For example: a Gene for Replication, Gene for transfer, gene for enzyme. It has to have a function. C. Plasmid - Extra-chromosomal, outside the chromosome. D. Diploid for prokaryotes wrong. It should be Haploid

Ruptured E. coli cell showing DNA

Viruses 4 or 5 genes, E. coli 4228 genes, Human ~ 31,000 genes Chromosome: single circular double stranded DNA. Dito naka encode ang totality of the replication, metabolism of a bacteria. All of the essential genes are carried on a single long piece of circular double stranded DNA. 2k-4k genes. Genes for cell growth and replication. Plasmids: Extra-chromosomal, walang kinalaman yan sa chromosome. It can replicate on its own. It codes for transfer of resistance and other proteins and enzymes as well. small Dna circle. Replicate independently from chromosome. Can exist as 1 or many copies. Can carry genes for toxins and proteins that promote transfer of plasmids. Contain mobile DNA sequence( transposons).

If you rupture a bacteria like the E. coli magllyse siya lalabas ang genetic material. Nucleic acids are located in the nucleoid of bacteria, and the nucleus of eukaryotes. There are 2 kinds of nucleic acids: RNA & DNA

3-D Image of DNA

E. Coli = 4.6 million nucleotide pairs (~1mm) Human = 3 billion nucleotide pairs (2nm wide by 2 meters long)

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DNA (deoxyribonucleic acid) is made of subunits called nucleotides. Nucleotides are made of 3 components. These 3 components are linked together with a covalent bond Maintains the code with high degree of fidelity. (double strand assures accurate replication) Provides a method for introducing a high degree of variety. (unlimited variety of sequences possible)

3.

2, DEOXYRIBONUCLEIC ACID Ribose - A five carbon sugar that functions as part of the DNA backbone (ie. structural). 2, Deoxy means without oxygen on the number 2 carbon atom. COMPONENT Nitrogen Bases NITROGEN CONTAINING BASES Function: express genetic information. composition : o 2 PURINES: ADENINE (A) GUANINE (G) double ring structures o 2 PYRIMIDINES: THYMINE(T) CYTOSINE(C) single ring structures

Question 3: What are the 3 Components of DNA Nucleotides? A. Hydrogen group, Deoxyibose sugar, Nitrogen bases B. Phosphate group, Ribose sugar, Nitrogen bases C. Phosphate Group, Deoxyribose sugar, Adenine-guanine-uracil-cytosine bases D. Phosphate Group, Deoxyribose sugar, Adenine-guanine-thymine-cytosine bases First component is the Phosphate Group, first part ng nucleic acid, structure ng base pairs. Second component is the Ribose sugar for the DNA is Deoxyribose. No oxygen on the second carbon. Third component would be the Nitrogen Bases. 2 Purines and 2 Pyrimidines. If you pair it, always magkapair ang guanine and thymine. For RNA guanine and Uracil. COMPONENT 1 Phosphate

DNA bond

1.

Phos phate group - Phosphate functions as a structural part of nucleic acids. 2. COMPONENT Ribose Sugar

DNA is a double helix (there are 2 strands of DNA) which are intertwined with 5 base pairs per turn. DNA has complimentarity that is A always bonds with T and G always with C DNA is always antiparallel. The 2 strands of DNA are always oriented in opposite directions. ( 5 PO3 end 3 OH end)

RNA RIBONUCLEIC ACID Similar to DNA except:

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1. RNA is single stranded 2. RNA has a ribose sugar instead of deoxyribose. (Oxygen on #2 C). 3. RNA has URACIL (u) instead of thymine 4. RNA is always shorter than DNA, ~ 1,000 nucleotides in length

2. Each cell receives 1 parental strand and 1 daughter strand. (semiconservative replication)

Question 4-6: Match function of each RNA 4. rRNA - B 5. tRNA - C 6. mRNA - A A. Template for protein synthesis B. site of protein synthesis C. carries amino acids to the ribosome during protein synthesis 4. rRNA (ribosomal) - comprises the ribosome ((60% of a ribosome is made of RNA, the rest is protein). 5. tRNA (transfer) carries amino acids to the ribosome during protein synthesis. Also known as the ANTICODON 6. mRNA (messenger) - a complimentary strand of RNA equal in size to 1 gene (normally ~1,000 nucleotides). CODON - coded info from DNA (bound for the ribosome) Central Dogma of Biology

Similar replication for bacteria and human cell. Maguunwind yung double helix ng DNA. Then there will be a replication, may leading strand and lagging strand. Parent chromosome unwinds with the use of enzyme Helicase then a daughter chromosome will be formed until makabuo ng 2 separate chromosomes. One would be the old and one would be the new. Pag nagseparate na doon lang magkakaroon ng binary fission ang bacteria.

As the two replication forks meet, the two new chromosomes separateeach containing one new and one old strand

DNA REPLICATION 1. Where 2 parental strands of DNA are copied into 2 daughter strands. Rate = 1,000 nucs per seconds without error. This leads to binary fission in bacteria. Cell Division = 2 daughter cells

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DNA unwinds using the enzyme DNA Helicase b. SSBP holds the 2 strands apart (single strand binding proteins) c. Note: 2 replication forks. DNA replication is considered bi-directional replication. What keeps the helical strand stable? Hindi siya ulit magwiwind. Single stranded binding protein. Questions 7-9: Match 7. DNA Polymerase (POL III) -C 8. DNA exonuclease (POL I) -A 9. DNA ligase -B A. removes any mistaken base pairs B. seals any gaps and joins the 2 strands together. C. enzyme that synthesizes 2 nucleotide strands d. Polymerization requires DNA Polymerase (POL III) which is an enzyme that synthesizes 2 nucleotide strands (daughter strands) from 2 parental (templates) strands. e. DNA exonuclease (POL I) removes any mistaken base pairs. f. DNA ligase seals any gaps and joins the 2 strands together.

a.

Putol-putol siya and eventually DNA Polymerase digest this Primer then magpproduce siya ng DNA which will be combined by the Ligase.

Steps in Replication

5 3 Prime Leading strand, 3 5 Prime Lagging Strand Video: The DNA polymerase that produces the lagging strand has just finished an Okazaki fragment. The clamp that keeps the lower DNA polymerase attached to the lagging strand dissociates and the DNA polymerase temporarily releases the lagging strand template DNA. As the DNA helicase continues to unwind the parental DNA, the Primase becomes activated and synthesizes a short RNA primer on a growing lagging strand. The DNA polymerase binds to the DNA again and becomes locked in the polymerase uses the RNA primer to begin a short copy of the lagging strand template DNA. The polymerase stops when it reaches the RNA primer of the preceding the Okazaki fragments and the entire cycle repeats.

DNA Replication Enzymes at Work

THE CENTRAL DOGMA OF BIOLOGY There are 3 parts to the flow of information in all cells. Transcription Translation DNA ------------- mRNA----- protein Replication Question 10: What is the enzyme responsible for making mRNA?

Leading strand derederecho ang pagreplicate Lagging strand hindi derederecho, What produces RNA Primer? Primase, RNA Polymerase. What happen is nagpproduce muna si Primase ng RNA Primer.

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Answer: RNA Polymerase B. TRANSCRIPTION

1. 2nd part of the central dogma of biology 2. 1st step in gene expression (i.e.protein synthesis). 3. The cells genetic plan contained in DNA is transcribed into a complimentary base sequence called messenger RNA (mRNA). 4. The region of DNA that produces or serves as a template for mRNA is called a gene. A gene normally consists of around 1,000 base pairs. It is the smallest segment of DNA that codes for mRNA. Transcription is the process by which the DNA is copied into RNA and the first step in gene expression. It begins with factors assembling at the start of the gene that is a linear sequence of DNA. The assembled factors include RNA polymerase. 7. Example: DNA A T G C C G DNA T A C G G C mRNA A UG C C G 8. mRNA is a blueprint of DNA or a transcript or code. 9. One code word consists of three letters.

C. TRANSLATION

1. Translation is the 3rd part of the central dogma of biology (2nd step in gene expression or protein synthesis). 2. After transcription, the coded information in mRNA is translated into an enzyme (protein). 3. This process takes place on the ribosome. Note that the ribosome is made of rRNA and protein. 4. The mechanics of translation Initiation; mRNA bumps into the small subunit and triggers the two ribosomal subunits to bind together. The first tRNA anticodon (UAC) carrying the amino acid methionine hydrogen bonds with the codon AUG on mRNA Elongation The second tRNA binds to the second code word on mRNA. A peptide bond forms between the two amino acids. The first tRNA leaves, and the enzyme translocase moves the ribosome down one code word of mRNA at a time. This repeats ~ 300X. In termination, one of three possible stop codons is reached. The last tRNA falls away and the two ribosomal subunits fall apart. Video: the ribosome has selected the correct amino acids that specify mRNA. An aminoacyl tRNA bound to EFTU (elongation factor TU) enters the free A site on the ribosome. If the anticodon of the charged tRNA does not match th codon in mRNA the tRNA is rejected. The process of trial and error repeats until the correct tRNA is identified. EFTU hydrolizes its bound GTP and dissociates. If the tRNA is correctly matched and remain bound for long enough time it is committed to the protein synthesis. The ribosome paralyzes the formation of new peptide

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bond and undergoes dramatic conformational change. Elongation factor G binds to the ribosome. Hydrolysis of GTP by elongation factor G switches the ribosome back to the state in which it can accept the next incoming tRNA.

Transfer RNA Structure Stop codons: UAA, UAG, UGA There are antibiotics that block certain processes in the genetic process of microorganism.

tRNA STRUCTURE tRNA utilizes the information in mRNA to determine the sequence of amino acids in a protein. tRNA has a cloverleaf shape. The amino acid end binds one specific amino acid in the cytoplasm. The anticodon end pairs with the codon on mRNA

Translation Blockers Streptomycin (SM) blocks assembly of the ribosome during initiation. Chloroamphenicol (CA) blocks peptide bond formation during elongation. Tetracycline TC blocks the 2nd site on the ribosome during elongation. Erythromycin EM blocks translocase during elongation. The protein synthesis by the 70s ribosome represents an important target of antimicrobial action. Pagadikit pa lang nung 2 ribosome nahihinto na ni Streptomycin. Tetracyline blocks Amino acid site. Letter A. GENE REGULATION Examples from E. coli Inducer example is lactose (lac operon) Repressor tryptophan (trp operon) How can genes be turned off and on? Why do microorganism needs to regulate gene expression? Para magkaroon ng balance sa metabolism. They need to regulate it if theres a change in the environment Ex. Inducer for lactose, the lac operon it will only turn on if may lactose present in the environment Trpytophan repressor naman siya ibig sabihin on going. If theres a tryptophan present in the environment

Question 11: How many codons encoding for the 20 Amino Acids? A. 61 codons B. 3 nonsense (or stop codons) C. 64 codons D. 60 codons 20 Amino acids 61 codons Plus 3 codons that will code sa stop codons but its not an amino acid.. Total codons : 64

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the gene expression will stop. Like negative feedback. Lac operon is specifically and well studied in the E. coli Lac operon is a group of genes coding for lactose operon. Lactose operon has 3 enzymes - Read. Read sa book microorganisms and yung metabolism nila. Ano yung mga aerobic, unaerobic at anong metabolism ginagamit ng microorganism na yun.

due to powerful mutagens; chemical or physical Question 12-14: Match 12. silent mutations -B 13. Missense mutations -C 14. Nonsense Mutations -A A. single base substitutions that yield a stop codon B. single base substitution in the 3rd base nucleotide position of a codon. NO change in amino acid. C. single base substitution in 1st or 2nd base nucleotide position. Results in a changed amino acid silent mutations - single base substitution in the 3rd base nucleotide position of a codon. This results in NO change in amino acid. Note that the first 2 letters of the genetic code are the most critical. missense mutations - single base substitution in 1st or 2nd base nucleotide position. This results in a changed amino acid. A change in one amino acid usually will have little effect depending on where in the polypeptide it occurs. nonsense mutations - single base substitutions that yield a stop codon. Note: there are 3 nonsense codons in the genetic code = NO PROTEIN

Repression - Trp operon

III. 5 Ways of Creating Genetic Diversity in Bacteria A. Mutations B. Transformation C. Conjugation D. Transposition E. Transduction Microorganisms adjust depende kung anong environment meron siya. In humans only mutation. Mutations Changes in the nucleotide sequence usually due to an error in DNA replication. These occur naturally at low levels (also known as spontaneous mutations); or by the effects of chemical agents called mutagens; or by physical agents like radiation. Results: Most mutations are neutral they have no effect on the polypeptide. Some mutations result in a less active product; Less often an inactive product; Very few mutations are beneficial. Types of Mutations Point mutations - a one base change in DNA. There are 3 types Frame Shift Mutations - the addition or deletion of 1 or more bases. These are

READ A. Chemical mutagens - (used in research to study mutagenesis). There are 3 kinds of chemical mutagens. 1. alkylating agents. Adds alkyl group, CnH(2n+1) Ex. formalin, nitrogen, mustard, and ethylene oxide (reacts with G changing it to bind with T). 2. base analogs. Mimics a nitrogen base. Ex. AZT is a modified sugar that substitutes for T. Ex. 5 - bromouracil binds with A or G. 3. intercalating agents. Inserts into DNA and pushes bases apart. Ex. AFLATOXIN - a chemical produced by peanut and grain molds. The mold is Aspergillus flavus (fungus). B. Physical mutagens: 1. nonionizing radiation - Causes the formation of T= T dimers. UV light @ 260 nm. 2. Ionizing radiation - damages DNA by causing the formation of free radicals leading to mutations. 3 Ex. X-rays. Gamma rays from radioactive fallout penetrates the body. Alpha rays from inhaled dust containing radioactive fallout. (ginagamit na panglinis sa hospital)

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B. TRANSFORMATION The passage of homologous DNA from a dead donor cell to a living recipient cell. Occurs in Streptococcus pneumoniae. When S. pneumo dies the DNA can be absorbed by a living S. pneumo and recombined into the chromosome. The gene for capsule formation is obtained in this way, as is a gene for penicillin resistance. Discovered in 1929 by Fredrick Griffith. Bacteria pag pinatay with antibiotic or naturally died, it releases its chromosome that carries the gene. Then pag narelease yung chromosome it incorporates to other bacteria or recipient bacteria which incorporates sa sarili niyang chromosome. Its like a natural recombination. Once the recipient bacteria has the old chromosome of the donor bacteria and it carries resistance to particular antibiotic, ok siya, resistant na siya ngayon.

plasmid will break, magkakaron ng replication, then magttransfer yung gene to recipient bacteria. Once the transfer is complete, magdedetach silang dalawa, magiging similar na sila. So if this bacteria carries the resistance, example, Methicillin magiging resistant na din siya. 5. Hfr (High Frequency Recombination) a. Hfr- bacterial plasmid integrates into the chromosome. b. Medical Problem: Hfr antibiotic resistance genes are passed during binary fission (every time the cell divides). Therefore, antibiotic resistance spreads very rapidly! c. When Hfr mate with F bacteria, only the bacterial genes cross NOT plasmid genes. Genetic diversity results in this case due to recombination. TRANSPOSITION 1. Transposons (jumping genes) are big chunks of DNA that randomly excise and relocate on the chromosome. 2. Transposons were discovered in 1950 by Barbara McLintock in corn. 3. Causes antibiotic resistance in Staph. aureus, the famous methicillin resistant Staphlococcus aureus (MRSA) strain! Enzyme that promotes transposon is non-specific. Any cycle of the enzyme of the chromosome pwede mangyare yan kaya jumping genes ang tawag. TRANSDUCTION the transfer of genetic material from donor bacteria to recipient bacteria via a transducing agent (virus!). Bacterial viruses are called bacteriophage. 1. Discovered in 1952 by Zinder & Lederberg. 2. Two kinds of transduction: generalized and specialized. Transfer of genetic information using virus.

QUESTION 15: C. CONJUGATION 1. A mating process between a donor F+ (bacteria with fertility factor =plasmid) and an F- recipient cell. 2. Occurs in Gram - enteric bacteria like E.coli 3. Discovered in 1946 by Joshua Lederberg and Edward Tatum. 4. Plasmids carry genes that are nonessential for the life of bacteria. Ex. gene for pili (sex pilus). Ex. plasmid replication enzymes. Ex. Medical Problem: R-Factor = antibiotic resistance! Theres a transfer of genetic material kadalasan galing sa plasmid carries antibiotic resistance. Normal - Sex plasmid transfer (usually ~20 of 100 genes). a. Requires a sex pilus a. F + bacteria transmits a copy of the plasmid to F- bacteria. This converts the Fcell into an F + cell. Medical Problem: The R factor (antibiotic resistance) on the F factor is transmitted! Donor bacteria carries resistance for antibiotic. No receptor proteins, no attraction between the two. The F pilus detects proteins, receptor proteins and this will attach this proteins. Once attached, magoopen, magjjoin sila then the

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bacteria at makukuha ng ibang virus. Yung nakalabas na virus na may dala nh chromosome nung isang bacteria magiinfect ulit ng 2nd bacteria. Pag ininfect niya ang maiibigay niya sa recipient bacteria would be the chromosome of the other bacteria. If the bacteria na nalyse kanina has gene coding for antibiotic resistance na naibigay niya sa isang bacteria, pati ito magkakaron ng antibiotic resistance. Transduction: think of virus that infects bacteria Specialized Transduction Generalized Transduction

2. Generalized transduction: Starts with the LYTIC CYCLE where a T- even phage infects E.coli killing the host cell, and synthesizing 2,000 copies of itself. The T-even phage randomly packages bacterial DNA in a few defective phages. Once a T even phage infects another E. coli, this genetic information can be recombined into the host cell without causing the lytic cycle. New genetic information is thereby transduced from one bacteria to another. Virus infects particular bacteria, pumasok yung viral genome then nagreplicate yung ibang virus sa loob, once nagreplicate siya mallyse, una masisira muna ang chromosome ng

Specialized transduction Lambda phage infects E.coli. The phage does not lyse the cell immediately. Instead it integrates into chromosome of the bacteria as a prophage and remains dormant. This is called the LYSOGENIC CYCLE. Phage genes are replicated and passed to all daughter cells until the bacteria is under environmental stress, from lack of nutrients, etc. Then phage gene will excise from the nucleoid and enter the

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LYTIC CYLE taking one adjacent gene for galactose metabolism. The gal transducing phage (lambda) makes ~ 2,000 copies of itself with the gal gene, and infects other E.coli. When gal integrates into the nucleoid of other E. coli, it may provide these bacteria with a new capacity to metabolize galactose. Difference lang niya is yung maibibigay na gene sa recipient bacteria may kasamang gene ng virus. Combination of the phage DNA ang bacterial DNA. Magllyse eventually pero late na.

6. 7.

Plants with beneficial qualities (genetically-modified foods) Novelty Blue Roses, GloFish They have been used to study and model cancer (the oncomouse), obesity, heart disease, diabetes, arthritis, substance abuse, anxiety, aging and Parkinson disease.[19] Potential cures can be tested against these mouse models.

Comparison of Bacteriophage 3. Comparison of bacteriophage transduction in E.coli. Generalized Specialized T even phage lambda phage lytic cycle lysogenic random packaging specific gal gene Lytic- mallyse agad Lysogenic- mag ggrow muna. RECOMBINATION Incorporation of extrachromosomal DNA into the chromosome(Chromosome of the another bacteria or plasmid of that particular bacteria) Types: Homologous and nonhomologous Homologous: occurs b/w closely related DNA sequences and generally substitutes one sequence dor another. Nonhomologous(illegitimate): occurs b/w dissimilar DNA sequence and produces insertions and deletions Genetic Engineering Aka Recombinant DNA Technology Different from traditional selective breeding Different from cloning Manipulation of the DNA to change its structure Manipulation of DNA by people. Purpose: to produce something useful Some Uses of Genetic Engineering 1. Insulin, HGH, vaccine production using bacteria 2. Using bacteria to clean up toxic wastes 3. Replacement of defective human genes (e.g. in SCID) 4. EPO production using hamster ovary cells 5. Production of Oncomouse, Knockout Mouse, etc., for research purposes

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