Microbial Genetics A. Definitions 1. gene = discrete unit, codes for functional product eg.

protein, RNA, regulatory molecule 2 genome = bacterial chromosome -- structure which carries genes 3. genotype = actual genetic makeup 4. phenotype = expression of genes 5. DNA = deoxyribonucleic acid a. composed of 4 types of bases hooked to sugarphosphate backbone, double-stranded, helical A = adenine pairs with T = thymine G = guanine pairs with C = cytosine b. structural genes -- code for proteins c. regulatory genes -- code for molecules which regulate other genes d. genes for rRNA, tRNA -- product is a molecule of rRNA or tRNA (no translation) e. untranscribed -- spacer, punctuation f. plasmids -- small circular, extrachromosomal element same rules apply re replication or expression 6. RNA = ribonucleic acid a. composed of 4 types of bases hooked to sugar-phosphate backbone; single-stranded, linear A = adenine U = uracil G = guanine C = cytosine b. 3 types, different functions rRNA = molecule which becomes structural part of ribosome tRNA = molecule necessary for protein synthesis carries amino acids and adds them to growing polypeptide chain during translation mRNA = informational intermediate carries info from DNA to direct proper synthesis of protein

B. Processes 1. gene replication a. semiconservative DNA replication each strand of DNA serves as template for production of complementary strand of DNA b. many enzymes involved in process including: DNA polymerases, ligases, "unwinding" enzymes, proofreading and modification enzymes 2. gene expression -- using stored information a. transcription --- production of RNA's (mRNA, tRNA, rRNA) b. translation --- production of protein using informational mediary mRNA 3. transcription 3 a. using sense strand of ds DNA --- transcribe area of interest (one or more genes) produce ss RNA transcript b. requires enzyme RNA polymerase c. product may be mRNA, tRNA or rRNA 4. translation a. from info in mRNA molecule a polypeptide (protein) is produced b. mRNA is read 3 bases at a time = codon 64 possible codons for 20 amino acids provides redundancy (>1 code for an amino acid) start codon = AUG (methionine) stop codon = 3 possibilities c. tRNA molecule carries amino acid = charged tRNA recognizes codon on mRNA strand by having opposite complementary sequence known as anticodon on tRNA d. ribosomes contain 2 binding sites where 2 charged tRNA's line up opposite complementary sequence on mRNA, amino acids are attached to growing polypeptide chain uncharged tRNA molecule is released and free to pick up another amino acid in cytoplasm

C. Gene expression 1. phenotype = visible expression of genes 2. operon = organizational unit grouped arrangement of controls and structural genes (as in a biosynthetic pathway) a. promoter = site where RNA polymerase binds b. operator = on/off switch -- binds inducer or repressor molecules binding here can block passage of RNA polymerase c. structural genes = sequence of genes, codes for product; ex. enzyme 3. Induction a. turn on sequence -- usually in response to extracellular condition ex. lac operon = sequence of genes for utilization of lactose b. inducer = substance which initiates enzyme induction ex. lactose normally lactose not present, transcription of genes is blocked by a repressor protein add inducer (lactose) which combines with repressor protein --- inactivates repressor RNA polymerase now can bind to DNA and carry out transcription when inducer low or absent, repressor is free, can then bind to operator and block transcription 4. Repression a. usually final product in biosynthetic pathway can act to turn off or repress a pathway b. end-product = co-repressor binds with repressor molecule c. together there is a change in configuration, complex can now bind to DNA at operator region - this stops transcription by preventing the binding of RNA polymerase d. When end product becomes low in cell, there is less around to bind to repressor protein, therefore no blockage of RNA polymerase --- transcription resumes

D. Genetic Change 1. Mutation = chemically altered DNA sequence physical, chemical causes a. results may vary (1) no change different sequence of DNA (codon) but still get same amino acid different codon resulting in different amino acid with no effect on protein function (2) slight change different amino acid but only minor functional difference in protein function (3) drastic change different amino acid results in non-functional protein b. can be of two types (1) spontaneous -- no apparent reason ex. proofreading error, low level environmental influences natural, low-level programmed rate (2) induced -- produced by known agent ex. chemicals, radiation c. specific types of mutations (1) point mutation = 1 base change silent mutation -- no effect on protein mis-sense -- may or may not affect protein nonsense -- incomplete protein (2) deletions or insertions ---- Frame shift portion of DNA missing or portion added to original DNA sequence results in alteration of mRNA sequence produced and a different 3 base reading frame for protein synthesis

2. Mutagens = chemical or physical condition which changes DNA 4 a. Base analogs = chemicals which resemble the A, T, G, or C normally incorporated in replicating DNA during next round of DNA replication, get faulty pairing eventually the original base pair has been changed ex. A=T --- G C takes a few generation to be seen b. Chemicals which react with DNA (1) Alkylating agents --- crosslink DNA strands faulty region excised potential for mistake (faulty repair) (2) Acridine dyes -- insert between bases (intercalate) bases pushed apart during DNA replication may get extra base incorporated frame shift c. Radiation (1) Ionizing = short wavelength (X ray, cosmic rays, gamma rays) produces reactive species of chemicals which cause breaks in DNA mistakes can occur during repair (2) Non-ionizing = UV produce thymine dimers = adjacent T's covalently linked, impedes replication (3) more "hits" more repair needed greater possibility for error 3. Testing for mutagenicity = Ames test a. using bacteria -- cheap, easy, short term (2 days) b. operating under assumption that many mutagens are also carcinogens c. disadvantages: not all carcinogens are positive in Ames test not all mutagens are necessarily carcinogenic bacterial DNA is not the same as human DNA

d. procedure: (1) Salmonella typhimurium strain designed to: lack DNA repair mechanisms mutated to require histidine added for growth (2) Add mutagen + S. typhimurium + rat liver extract (3) Put on agar plates containing minimal nutrients and NO histidine (4) After incubation, count number of colonies any cells which grew have experienced a 2nd mutation in order to no longer require histidine (5) compare number of colonies grown with suspected mutagen to number of colonies in presence of known mutagen to number of colonies with no mutagen (background) (6) the higher the number of colonies, the greater the mutagenicity of test compound e. mutagen has caused 2nd mutation known as back mutation which restores cell's original capability f. cells which grew without added histidine known as revertants E. Recombination 1. Transformation -- recipient cell takes up extracellular fragment of DNA a. 1928, Fred Griffith working with Strep pneumoniae encapsulated strains appear smooth (S), are virulent non-encapsulated strains appear rough (R), avirulent R + mouse healthy S + mouse dead heat-killed S + healthy mouse heat-killed S + live R dead + mouse isolate R colonies isolate S colonies isolate no colonies

isolate R and S

5 b. mechanism = fragment of DNA from lysed cells taken up by some of the live cells (known as competent cells) (1) DNA binds to receptor site on surface of competent cell (2) segments cut into smaller pieces by enzymes (3) some piece enters cell may or may not recombine with host DNA (about 50% possibility)

2. Transduction -- requires virus carrier to move DNA from cell to cell a. lytic cycle -- results in rupture of host to free viruses (1) occasionally host DNA fragment may be packaged in virus (2) next cell infected will receive this piece of bacterial DNA (generalized transduction) b. lysogenic phage -- insert DNA which incorporates into host chromosome (1) each replication/division of host cell produces more cells carrying prophage (2) may result in new properties for host ex. carry gene for drug resistance ex. produce disease -- Corynebacterium diphtheriae, diphtheria toxin gene carried by prophage 3. Conjugation (bacterial sex) -- requires cell-to-cell contact a. plasmid-mediated (1) plasmid = (in general) circular piece of extrachromosomal DNA double-stranded, self-replicating (2) F plasmid (specifically) = F factor contains genes necessary to direct sex pilus formation and transfer of plasmid through conjugation tube (3) cell types cell + plasmid = F+ (donor)

cell - plasmid = F - (recipient) cell + integrated plasmid = Hfr

(4) transfer of genetic material F+ may transfer plasmid to F - cell and it then becomes F+ Hfr may transfer part of host genes to F - cell as the F factor does not pass through pilus first, recipient remains F-

Study Guide: Microbial Genetics 1. Know definitions of gene, genome, genotype, phenotype, structural gene, regulatory gene. 2. Know in general process of information flow : transcription and translation. 3. Know definitions of operon, promoter, operator, terminator site, induction, repression, constitutive enzymes. 4. Know process of gene expression in bacteria - operon model - and regulation by induction or repression. 5. Know definition of mutation, mutagen, recombination. 6. Know types of mutations (spontaneous and induced) and examples of classes of mutations given in class (point mutation, deletions and insertions) and consequences of each (mis-sense, non-sense, silent mutations, frame shifts). 7. Know, in general, types of mutagens and how these mutagens (alkylating agents, base analogs, acridine dyes, radiation) can act. 8. Know how mutants can be identified. 9. Understand the significance of Ames test and the rationale for its steps. 10.Know 3 ways for genetic exchange to occur in bacteria (transduction, transformation, conjugation). Be able to describe similarities and differences for these 3 methods. 11. Know natural and engineered aspects of recombinant DNA (plasmids, transposons, foreign DNA).