Microbial Genetics As we have looked at, information for all cellular function is from DNA, mRNA carries that info to ribosomes, rRNA codes for proteins constructed at ribosomes So – in an environmental/geological context, what is that info used for??

Genomic info Gives us:

Changing the genetic code: Mutation: Change in at least one nucleotide –Point mutations: 1 nucleotide change –Framesift mutations: base-pair change Think on how sequence is ‘read’ –ATTGGCCATAGG –ATTGGCCATAGG  Codon grouping with a start and stop coding –Frameshift  changes that mess up codon groups, mess up start/stop info – completely change the protein… –Result  Silent (codons are degenerate – amino acids coded by several possible sequences), Missence (amino acid substitution), Nonsense (shorter or longer protein)

Mutation agents: ‘Natural’ error in replication Chemical agent: –acids – esp. nitrous acid by modifying structure of adenine –Base analog – organic base similar enough to substitute in –Mutagens – cause base-pair problems or disruption of chain Physical agent: –UV light – disrupts base pairing – thymine dimerization (T- T bonds form) –Radiation damage – high energy particles disrupt electronic structure and bonding Viruses and gene transfer – adding snippets of ‘foreign’ code

Genetic repair Organisms have the machinery to repair damaged DNA Some organisms have adapted to handle doing this often, as is the case for a particular bacteria that can survive in abnormally high radiation: Deinoccocus radiodurans (can survive 1.5 million rads – 3000x what would kill us)

Microbial Identification All organisms have unique genetic codes describing their composition and function The diffference between these codes is a measure of how similar these things are to each other IF all organisms evolved from a common ancestor, then all codes of current life came FROM that common ancestor – this idea is the study of phylogentics

Construction of phylogenies What piece of the genetic code is most appropriate for determining these relationships – what should that part of DNA be responsible for??? –Homologous function (common to all organisms) –Larger is better (more info) –Rate of mutation accumulation relevant to evolution –Does it all make sense – i.e., when you look at the phylogeny does it make sense? – function and evolution??

Comparative sequence analysis Looking to compare similarity (in base 4) of sequences 1.ATTGGCCACG 2.ATTCGCCTCG 3.TGGCGCCTTT 4.ATTGGGCACG Determine the ‘degree of similitude’ between these and represent that graphically Literally hundreds of ways to do this…

What piece of DNA do we use 16S rRNA coding – info that is responsible for the 16S subunit in ribosomes –All bacteria and archea have 70S ribosomes with a 16S subunit –All bacteria and archaea need this to make proteins –Some parts accumulate mutation slow, other parts fast (evolution resolved over long time but also with good resolution) After original work of Carl Woese and colleagues

Clone Library

DNA Sequencing DNA sequencing reactions are just like the PCR reactions for replicating DNA. The reaction mix includes the template DNA, free nucleotides, an enzyme (usually a variant of Taq polymerase) and a 'primer' - a small piece of single-stranded DNA about nt long that can hybridize to one strand of the template DNA. The reaction is initiated by heating until the two strands of DNA separate, then the primer sticks to its intended location and DNA polymerase starts elongating the primer. If allowed to go to completion, a new strand of DNA would be the result. If we start with a billion identical pieces of template DNA, we'll get a billion new copies of one of its strands.

Dideoxynucleotides: We run the reactions, however, in the presence of a dideoxyribonucleotide. This is just like regular DNA, except it has no 3' hydroxyl group - once it's added to the end of a DNA strand, there's no way to continue elongating it. Now the key to this is that MOST of the nucleotides are regular ones, and just a fraction of them are dideoxy nucleotides....

Replicating a DNA strand in the presence of dideoxy-T MOST of the time when a 'T' is required to make the new strand, the enzyme will get a good one and there's no problem. MOST of the time after adding a T, the enzyme will go ahead and add more nucleotides. However, 5% of the time, the enzyme will get a dideoxy-T, and that strand can never again be elongated. It eventually breaks away from the enzyme, a dead end product. Sooner or later ALL of the copies will get terminated by a T, but each time the enzyme makes a new strand, the place it gets stopped will be random. In millions of starts, there will be strands stopping at every possible T along the way. End up with billions of strands, with representatives of every length…

Gel electrophoresis can be used to separate the fragments by size and measure them.

Tag the terminal bases with a fluorescent tag specific to each base Now can separate based on size AND see which base that size is terminated with… Start (GCGA) is based on a primer!

Gel electrophoresis separates the products with respect to size, a fluorescence detector ID’s the terminal base Yields the sequence… Works great starting from a specific place (16S rDNA for example…)

How to get more of the gene? Several different methods developed to get more of the genome (ideally the complete genome) Pyrosequencing (454) Randominzed BAC libraries Shotgun sequencing Get many fragments of the genome, trick is to find overlap and reassemble!!