I. Gene transfer and genetic engineering (Chapter 8) 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. 3. 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. 4. Organisms have mechanisms to maintain and repair genetic information. Some changes will still occur, providing the potential for evolutionary change. 5. 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 6. genomes vary in size a. smallest viruses = 4-5 genes b. E. coli = 3,000 genes c. human cell = 100,000 genes (46 chromosomes) 7. Central dogma: DNA! mrna! protein 8. genotype = genetic makeup 9. phenotype = actual, expressed properties a. genotype represents potential properties b. phenotype represents manifestation of the genotype B. 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 C. 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 D. transformation = transfer of genes as "naked" DNA in solution 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. electroporation = electrically shocking the cells to open pores in the membrane and let in naked DNA 5. 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. geno me 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. 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 G. 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. 700-40,000 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 b. recognition sites are short regions of DNA in inverted repeats t hat the enzyme recognizes as recombination sites between the transposon and t he 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 H. Genetic engineering or Recombinant DNA technology 1. genetic engineering = creation of new genetic varieties of organisms 2. genetic fusion = coupling two genes to the same promoter 3. protoplast fusion = removing the cell walls and combining bacteria with different traits in one culture 4. gene amplification = increasing the number of a particular gene to overproduce
the product of that gene 5. recombinant DNA technology = intentional recombination of genes from different sources by artificial means a. transgenic = combining genes from different species b. involves three processes: (1) in vitro manipulation of genes (2) recombination of another organism s DNA with bacterial DNA in a phage or plasmid (3) cloning = production of identical progeny c. restriction endonucleases = cut double-stranded DNA at specific locations (ligases rejoin) (1) usually used by cell to destroy foreign (phage) DNA (2) EcoR1: 5' GAATTC 3' (3) HindIII: (Haemophilus influenzae d) 5' AAGCTT 3' d. cloning vector = carrier of gene to be recombined e. markers = selectable phenotypic traits 6. 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 I. Synthetic DNA 1. oligonucleotides ("d30 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) J. Applications of recombinant DNA 1. medical use focuses on production of human hormones by microorganisms a. insulin b. human growth hormone c. blood clotting or anticlotting factors d. vaccines e. forensic applications f. gene therapy 2. industrial applications focus on strain improvement or creation of strains with added metabolic capabilities a. adding enzymes to yeast (1) malolactic fermentation (2) amylase b. bioremediation
c. production of chemical stocks 3. agricultural applications focus on increasing yields a. bioinsecticides b. higher yields from plants c. resistance of plants to adverse conditions and herbicides d. self-fertilizing (nitrogen fixation) 4. hybridoma a. fusion of a myeloma (bone cancer cell) with an antibody-producing white blood cell (1) long-lived antibody producing hybrid cell (2) make monoclonal antibodies (one particular type) b. other such combinations of cells from higher organisms has tremendous potential in medical, industrial, and agricultural areas K. The risk from genetically modified organisms seems low 1. no recombinant bacterium has infected workers 2. recombinant organisms do not survive in nature (can t compete) 3. intentional inoculation of volunteers did not produce illness 4. But what possibilities exist with intentional creation of bioweapons?