I. General Genetics A. General concepts 1. Genetic information is contained in the nucleotide sequence of DNA (sometimes RNA).

Transcription

1 I. General Genetics A. General concepts 1. Genetic information is contained in the nucleotide sequence of DNA (sometimes RNA). Amino acids are specified by a triplet codon. a. Griffith expt.: killed virulent + living non-virulent Strep. pneumoniae killed mice (transformation) b. Avery used cell components from virulent strains to determine that DNA was genetic material c. Hershey and Chase used radiolabelled T2 bacteriophage ( 32 P DNA and 35 S protein) to show that during viral infection, DNA entered the cell and protein remained outside. 2. A gene is a nucleotide sequence that codes for a polypeptide, trna, or rrna. Promoters and operators are controlling regions of the gene. 3. Most bacterial genes have 4 parts: promoters, leaders, coding regions, and trailers 4. Mutations are stable, heritable alterations in the gene sequence that usually, but not always, produce a phenotypic change. Mutations can be spontaneous or induced by specific mutagens. 5. Organisms have mechanisms to maintain and repair genetic information. Some changes will still occur, providing the potential for evolutionary change. 6. genetics = study of inheritance or heredity a. biological properties are passed from parents to offspring b. these traits determined by expression and variation of genes (1) gene = fundamental unit of heredity responsible for a given trait (2) segment of DNA that contains information to make a protein or RNA molecule (3) genome = sum total of all genetic material of a cell c. chromosomes = cellular structures that contain the genes 7. genomes vary in size a. smallest viruses = 4-5 genes b. E. coli = 3,000 genes c. human cell = 100,000 genes (46 chromosomes) 8. Central dogma: DNA mrna protein 9. genotype = genetic makeup 10. phenotype = actual, expressed properties a. genotype represents potential properties b. phenotype represents manifestation of the genotype B. Mutations 1. mutation = permanent, inheritable change in the genetic information a. loss, addition or rearrangement in base sequence b. wild type = strain with non-mutated characteristic c. mutant strain = strain with a mutated characteristic 2. mutant strains can show difference in morphology, nutritional characteristics, genetic control mechanisms, resistance to chemicals, temperature preference, nearly any type of enzymatic function a. useful for tracking genetic events, determining genetic organization, mapping

2 genes b. detected by replica plating or biochemical indicators c. many mutations are neutral (no phenotypic change) (1) usually single nucleotide substitution, "corrected" by degeneracy of the genetic code (2) mutation could also effect nonvital portion of protein 3. different types of mutations a. conditional mutations = expressed only under specific environmental conditions b. auxotroph = mutant missing an enzyme in a key anabolic pathway (prototroph = strain able to grow on minimal medium) c. point mutation = involves single base pair (affects only a single gene) (1) transition = purine substituted for purine, pyrimidine for pyrimidine (2) transversion = purine for pyrimidine or vice versa (3) frameshift mutations = insertion or deletion so that the natural order of message is shifted (4) silent = codon changes, but not amino acid (degeneracy) (5) missense mutations = amino acid substitution in a protein (6) nonsense mutation = stop codon in middle of sequence (early termination) d. loss or change in entire or large portion of chromosome (deletion or insertion) 4. spontaneous mutation = random change in DNA arising from mistakes in replication or detrimental effects of natural background radiation on DNA a. rate varies in different species (1) low = (2) high = 10-5 b. high rate of reproduction allow ready observation 5. induced mutations = arise from exposure to mutagens a. mutagen = physical or chemical agent that interact with DNA is a disruptive manner b. valuable laboratory tool for studying metabolism, physiology, and molecular biology 6. Detection and isolation of mutants a. direct selection b. replica plating (1) auxotrophs (2) temperature sensitive 7. Carcinogen testing (Ames test) a. mutational reversion assay with specific strains of Salmonella typhimurium (1) different mutations in histidine biosynthesis operon (2) cell wall mutations to make cells more permeable to test chemicals (3) altered repair mechanisms to enhance errors b. bacteria and test compound mixed in molten top agar with trace histidine (1) auxotrophs grow slightly (allow DNA replication) (2) revertants (due to mutagenesis) will continue to grow and produce visible colonies after a couple of days (3) colony counts compared to controls

3 c. the more revertants, the greater the relative mutagenicity of the compound d. sometimes liver extract added to molten agar to promote transformations that occur in mammals II. Recombination and plasmids A. recombination = process of combining genetic material from 2 organisms to produce a genotype different from either parent (exchange of DNA between different genes) 1. recombination is important in bacterial populations as a means of increasing genetic diversity 2. recombination is also an important tool for creation of new strains useful in biotechnology B. Plasmids = small, circular DNA molecules that can exist independently of host chromosomes (extrachromosomal) 1. have their own replication origins 2. contain few genes (usually < 30) 3. not essential to host 4. curing = elimination of plasmid from host cell C. conjugation = transfer of DNA between bacteria in direct contact 1. depends on plasmids (free or integrated) 2. Lederberg and Tatum mixed multiple auxotrophs and plated on minimal medium to demonstrate genetic transfer 3. Davis demonstrated the need for contact by separating strains with a glass filter 4. F factor = fertility plasmid; plays a major role in conjugation a. donors have F (F + ), produce sex pilus, transfer the plasmid to recipients (F - ), b. Hfr (high frequency of recombination) cell has the F factor integrated into the chromosome c. Hfr strains transfer part of their chromosome during conjugation (1) origin of transfer lies within the F gene, so recipient does not contain complete F unless entire chromosome is transferred (2) through recombination, transferred DNA incorporated into chromosome at homologous site (3) linear transfer, useful for mapping genes by using interrupted mating (location related to time before transfer) d. F plasmid is F plus chromosomal genes (deintegration of F plasmid) (1) functions similar to F+ (2) provides diploid, important to determining dominant gene and mapping 5. R factor = plasmids that contain genes that code for enzymes that can destroy or modify antibiotics a. can have single or multiple resistance factors b. conjugative, but not as rapidly as F factors 6. col plasmids code for bacteriocins = proteins that destroy other bacteria a. some are conjugative b. some can carry resistance genes 7. plasmids can also carry genes that increase an organisms virulence or increase metabolic versatility D. transformation = transfer of genes as "naked" DNA in solution

4 1. DNA in environment (released from lysed cells) can be taken up by related bacteria and incorporated into the chromosome by recombination 2. occurs naturally in a few genera of bacteria and can be induced in others a. works best when donor DNA from closely related species b. when recipient can take up DNA, it is said to be competent 3. can be very useful for genetic engineering 4. classic case involve Streptococcus pneumoniae a. capsule formation is required for virulence b. dead virulent + live avirulent = live virulent + live avirulent E. transduction = transfer of DNA between bacteria via a bacteriophage 1. in the lytic cycle, a phage infects a host, takes over machinery, produces new phages, and lyses the host a. 4 stages: (1) attachment (adsorption and penetration) (2) synthesis (3) assemby (4) lysis b. usually called virulent bacteriophages 2. temperate phages are lysogenic a. genome integrates with chromosome and is replicated as part of cell b. under appropriate conditions, phages are reproduced c. new phages are released when the cell lysis d. synthesis of lysogenic phage particles = induction 3. generalized transduction a. phage attaches to bacterium and injects its DNA b. DNA directs synthesis of new phages c. during infection, bacterial chromosome breaks apart and some fragments can be packaged inside the phage d. later infections transfer bacterial genes to new cells e. normal replication results in lysis but if the phage carries bacterial instead of viral DNA, lysis does not occur (defective phage) 4. specialized (restricted) transduction = specific regions of bacterial DNA are transferred a. requires temperate phage (lysogenic cycle) b. phage DNA includes bacterial DNA when viral replication occurs F. transposons = transposable genetic elements = jumping genes = genes that move (transposition) around the chromosome 1. small segments of DNA that can move (be transposed) from one region of DNA molecule to another a ,000 bp b. Barbara McClintock studied them in maize, but they occur in all organisms 2. all transposons carry the information for their own transfer a. simplest are insertion sequences (IS elements) (1) contain the gene for transposase (catalyzes the cutting and ligating of DNA) and recognition sites

5 b. recognition sites are short regions of DNA in inverted repeats that the enzyme recognizes as recombination sites between the transposon and the chromosome 3. composite transposons carry other genes, like toxins or antibiotic resistance 4. usually the original transposon remains at the parental site, while the replicate inserts elsewhere 5. target sites are cut, transposon (ds) inserted, and ss gaps filled in III. Genetic engineering or Recombinant DNA technology A. recombinant DNA technology = intentional recombination of genes from different sources by artificial means 1. genetic engineering = creation of new genetic varieties of organisms 2. restriction endonucleases = cut double-stranded DNA at specific locations (ligases rejoin) a. usually used by cell to destroy foreign (phage) DNA b. EcoR1: 5' GAATTC 3' c. HindIII: (Haemophilus influenzae d) 5' AAGCTT 3' 3. cloning vector = carrier of gene to be recombined 4. markers = selectable phenotypic traits 5. southern blot = DNA-DNA hybridization on nitrocellulose filter a. DNA fragments separated by AGE, denatured, transferred to NC filter b. filter soaked in solution with radioactive probe (labelled gene fragment) c. autoradiography reveals band containing the gene of interest B. Synthetic DNA 1. oligonucleotides ( 30 nucleotides) are RNA or DNA sequences synthesized in the lab 2. site-directed mutagenesis = insertion of constructed sequence into ss region of gene 3. pcr = method for high volume synthesis of DNA fragment a. denatured fragment is replicated b. replicates are denatured and replicated c. cycle is repeated as necessary d. high temperature for denaturation requires thermostable DNA polymerase (Taq from Thermus aquaticus or pol from Thermococcus litoralis) IV. History of Bacterial Systematics A. Systematics = comparative study of the diversity of organisms in order to establish a logical system to describe and classify them 1. taxonomy = science of classification, nomenclature, and identification of organisms 2. nomenclature = assigning names to units described in a classification system a. rules assigned by international committees (1) bacteria covered in bacteriological code (2) fungi and algae covered in botanical code (3) protozoa covered in zoological code (4) viruses covered in virological code b. name may reflect physiological feature or name of an individual 3. identification = applying classification system and nomenclature to an unknown organism in order to assign a proper name and position within a classification system 4. classification = ordering or placing organisms into groups based on their

6 relationships a. artificial schemes = based on observable (phenotypic) features (1) may not accurately reflect genetic similarities (2) may not correspond to genetic flow of events (3) homologously dissimilar (genetic) organisms may be analogously similar (phenotype) (a) classification on a phenotypic trait, like pigmentation, could produce a taxonomic group of genetically unrelated bacteria (b) many groups established on morphological and physiological characteristics are now considered to be of uncertain taxonomic affinity b. natural schemes = based on evolutionary (genetic) relatedness (1) evolutionary relatedness difficult to discern due to lack of a fossil record (2) many bacteria are unculturable in the laboratory (3) limitations slowly being overcome by use of RNA and DNA analyses (a) many decisions still subjective, i.e., degrees of relatedness (b) results in lumpers and splitters c. Organisms are in essence historical documents; their structure at all levels reflects their evolutionary history. Carl Woese, The Prokaryotes B. Bacterial phylogeny the only truly scientific foundation of classification is to be found in appreciation of the available facts from a phylogenetic point of view. Only in this way can the natural interrelationships of the various bacteria be properly understood... it cannot be denied that the studies in comparative morphology made by botanists and zoologists have made phylogeny a reality. Under these circumstances it seems appropriate to accept the phylogenetic principle also in bacteriological classification. Kluyver and van Niel, 1936 a. attempts to develop a natural system of classification was doomed to failure (1) bacteria are morphologically simple (2) bacteria do not have meaningful developmental stages (3) bacterial fossils (not discovered at that time) are phylogenetically uninformative b. phenotypic characteristics chosen for classification (1) emphasis on morphology (2) physiological traits suspect due to ease of bacterial adaptation 2. classification schemes were workable, but had obvious shortcomings, especially as the majority of information used represented relatively superficial properties of bacteria 3. bacterial evolution became a predominantly philosophical subject, greatly upsetting many of the key workers in bacterial physiology and ecology C. The molecular revolution 1. molecular sequences are tremendously informative in that they are easy to interpret (essentially linear), well defined, and generally comprise hundreds or thousands of reasonably independent characters a. number of possible sequences is enormous (1) extensive homology = evolutionary relatedness

7 (2) convergence is a moot point (consider reversion of frameshift mutations) b. by extrapolation, molecular genealogies can be constructed based on the extent of differences among sequences that correspond in different organisms to the same function 2. DNA base ratios were used extensively, generally with more weight than appropriate 3. conserved proteins (cytochromes, ferredoxins) were sequenced 4. oligonucleotide cataloging (late 1970's) gave first real indication of bacterial phylogeny D. Molecular Chronometers 1. in different organisms, there exist different (but related) molecular sequences that correspond to what appears to be the same molecular function 2. this implies that most of the net changes that become fixed over time are selectively neutral (i.e., no phenotypic or selective consequence) 3. such changes occur randomly in time (more or less), and so can be used to measure time in a relative sense 4. this evolutionary clock can be used to infer evolutionary histories and relationships 5. most useful molecular chronometers are molecules like ribosomal RNA a. universal, constant, highly conserved functions established at early stages of evolution b. functions are not affected by changes in the organism s environment (1) except for physical parameters like intracellular ph and temperature (2) near constancy of secondary structure, even between kingdoms c. large molecules, so they hold considerable information and are less erratic than smaller molecules d. rrna is easy to isolate, do not seem subject to lateral gene transfer, and they can be sequenced directly (cloning not required) E. Construction of phylogenetic trees 1. align molecular sequences a. ideally, all homologous sequences correspond b. actually, equate statistically significant sequence similarity with homology 2. numerical taxonomy = construction of classification trees based on measures of similarity or differences a. distances between groups shown on dendograms indicate degrees of relatedness b. evolutionary distance = based on number of positions that differ between two pairs of sequences c. parsimony = based on minimum number of differences and where they occur (doesn t account for multiple changes at a site) d. distance is simpler, parsimony also takes the route into account F. Endosymbiote theory 1. hypothesis that some organelles evolved from bacteria a. mitochondria and chloroplasts are bacterial in size b. both contain closed, circular DNA and reproduce semiautonomously c. ribosomes similar to bacterial ribosomes; similar sequences in rrna and trna 2. Protozoan flagellate Cyanophora paradoxa has photosynthetic organelle (cyanellae) with structures similar to cyanobacteria

8 a. some peptidoglycan in cell walls b. DNA < cyanobacteria, about same as chloroplasts c. lacks OM that cyanobacteria have