1. Significance of microbial
    1. microoganisms includes prokaryotes and (mainly unicellular) eukaryotes
      1. unique features of prokaryotes with respect to genetics
        1. nucleus vs. nucleoid
        2. haploid vs. diploid and the significance of it
          1. diploid organisms have genes that come in sets of two-one paternal and one maternal copy. The two copies are similar, but not identical. We call each member of the set an allele.
          2. prokaryotes are haploid-each gene is present in only a single copy (for the most part). Not only that, but bacteria multiply (divide) by binary fission (asexual reproduction), so in principle, each progeny cell is a clone of the parent. It would be logical to think that the term allele would not make senses when applied to bacteria. But in reality, mutations happen. Progeny cells may not be clones. This is just a way of saying that progeny bacterial cells might have variant genes (alleles).
          3. Examples- alleles of lac and trp genes
            1. wild-type cell
            2. binary fission produces clones, but mutations can arise
          4. mutations in trp gene creates a new trp allele. If the mutation inactivates the gene, then the mutant is a trp auxotroph. (the wild-type cell is a trp prototroph). Likewise for the lac gene.

  1. Nomenclature of bacterial genetics
    1. Wild-type
    2. Mutants and Mutations
      1. Analogy to car motor and removing engine parts to see their function.
      2. Types of mutations
        1. null mutants completely knock out a function, completely eliminate the activity of a gene product. (e.g., a dead battery) Leaky mutants are not complete in their elimination of function (a weak battery)
        2. some mutants express wild-type phenotype under certain conditions.
          1. These are conditional mutants, the most common of which is temperature-sensitive (Ts) mutant. Example would be an engine that has a weak hose. The car will run as long as the temperature is low. Likewise, a Ts mutant will have a protein with normal activity as long as the temp is low; high temp will cause the protein to unfold and not work.
          2. suppressors we will leave for later
      3. phenotype and genotype
    3. Notations related to genotype and phenotype
      1. Phenotype designation is three letters. First is capitalized, and it is not italicized.
        1. example, Trp+ is a cell that can make the amino acid tryptophan. We also use this notation for proteins. A cell is Trp+ because it has many proteins, designated TrpA, TrpB, TrpC, etc.
        2. a cell incapable of making tryptophan would be Trp-
      2. The thing is, the Trp- cell would be so because of a mutation, a change in its genotype
        1. that mutant would be denoted trp or trp-.
        2. Now, we should note that not all wild type genes are written down as part of the genotype of a organism. This would involve thousands of genes. We only denote the ones we are interested in.
        3. Be aware that the phenotype is just the genotype converted to three letters, first capitalized, no italics.
    4. Mobile DNA and horizontal gene transfers
      1. vertical vs. horizontal transfer
      2. plasmids and transposons
      3. potential for partial diploids in "haploid" organisms
        1. haploid and diploid notations
          1. we typically say that a bacterial cell contains only a single set of genes. So it is considered haploid. In some cases, bacteria can contain more than one copy of a particular gene, and are said to be partial or merodiploids
          2. these are denoted by the two genes with a slash between them (trp+/trp-)
          3. mutations can be dominant or recessive, just like in eukaryotes.
    5. antibiotic resistance
      1. superscript r for resistant (e.g., ampr)
      2. superscript s for sensitive (amps)
    6. to repeat and summarize - mutations can give rise to different alleles
        1. meaning of allele in eukaryotes is pairs of genes, each gene being found on one of each of the homologous chromosomes (autosomes)
        2. in bacteria, allele means a variant of a gene that can arise by mutation. A single gene in bacteria may have several or many different mutations. Each is said to be an allele of that gene, and often is denoted by a number based on the order that the mutations were described. E.g., trp-58 and trp-79. If we know what gene the allele is in, we add the gene letter to the designation (atpE3). Also, we now can easily figure out which amino acid is changed by the mutation, so that we may designate an allele as atpE(lys258->pro).
    7. Ways to study mutations
      1. selections allow only the mutants to grow. Very powerful.
      2. screens allow both wild-type and mutants to grow. But a screen will allow you to tell the mutant from the wild-type somehow.
        1. MacConkey plates
        2. the advantages of screens over selections
          1. because selections kill off the unwanted cells, it is possible to analyze billions of colonies for a single mutant
          2. screens are limited to the number of colonies that can be resolved on a plate (500 at most), so to screen a billion colonies would take at least 2 million plates.  You'd be tired.
    8. Complementation analysis
      1. rationale
        1. remember, a negative phenotype (like Trp-) can arise from mutations in any of the genes that encode enzymes associated with that phenotype (like enzymes of the tryptophan biosynthetic pathway)
        2. suppose you have three Trp- mutants (trp1, trp2, trp3).  How do you find out whether they are in the same gene or in different genes? You do complementation analysis.
      2. method
        1. prepare a set of merodiploids containing all of the possible genotype pairs, and then check the phenotypes of the merodiploids
        2. eg.  merodiploid        Phenotype
          1. trp1/trp2            Trp+
          2. trp1/trp3            Trp-
          3. trp2/trp3            Trp+
        3. if a positive phenotype is restored by a merodiploid, then we say that the two mutations "complement" each other
        4. finally, if mutations complement each other, then we can safely assume that they are in different genes.  I'll tell you why in class. If mutations do not complement each other (i.e., the negative phenotype persists in the merodiploid), then we assume that they are in the same gene.
        5. so, how many genes do we have represented by the three mutants?  Which mutants are in the same gene? Email me the answer here for all kinds of extra credit