Last Modified: 2013-02-25
Missense mutation- this point mutation causes a change in the DNA, if the base substitution results in an amino acid substitution in the synthesized protein, i.e. sickle cell anemia
- a single nucleotide is changed, resulting in a different codon that codes for a new amino acid
- the new base leads to the insertion of a different amino acid than had been coded for, possibly altering the resulting protein’s structure.
Nonsense Mutation- a base substitution resulting in a nonsense codon.
- some base substitutions prevent the synthesis of a complete functional protein
- mutations that change an amino acid to a stop codon
- ending protein synthesis and resulting in a shorter and usually nonfunctional protein.
- A change in a letter of a codon
- has no affect on amino acid sequence.
- the new base does not change the amino acid sequence coded for by the original DNA strand.
- a mutation that shifts the "reading" frame of the genetic message by inserting or deleting a nucleotide
- basically: a base is added or deleted from the DNA sequence
- (also called nucleotide substitutions)
- Involve changes to individual nucleotides in the DNA sequence; changes in one base pair
- frameshift mutations and base substitution mutations
- i.e. substitution, insertion or deletion
- a frameshift deletion at the beginning of the gene
- During protein synthesis, incorrect amino acids would be inserted from the point where the frameshift mutation occurred on; the resulting protein would most probably be nonfunctional. For this reason, a frameshift mutation at the beginning of a gene is generally the most severe type of mutation.
- insertions in certain regions of the gene can alter the splicing of introns from the precursor mRNA
- this results in mRNA that contains introns, resulting in significantly altered protein products
- ex: A splice site mutation in the Beta-globin
gene is responsible for certain cases
- if the DNA polymerase inserts the wrong nucleotide base as it synthesizes a new strand of DNA
- Some are in the promoter region, just 5’ to the coding sequences.
- interfere with the binding of the RNA polymerase to the promoter region (no transcription)
- The Ames test evaluates mutagenicity, which is the ability to induce permanent, heritable changes in the DNA. Carcinogenic compounds cause cancer.
- The Ames test is used as a preliminary screening tool. Not all compounds that give a positive Ames test are carcinogenic.
- Liver enzymes may activate some innocuous compounds, making them mutagenic
- Some compounds are innocuous until they are activated metabolically by liver enzymes.
- Some compounds are not mutagenic until they are metabolized.
- The strains used in the Ames test are not able to produce an amino acid.
- The bacteria used in the Ames test to evaluate mutagenicity are his− auxotrophs. If the Ames test is positive, these bacteria have reverted back to wild type and are his+prototrophs
- A plasmid is a small, circular DNA molecule found in bacteria in addition to the bacterial chromosome.
- To clone DNA using plasmids, molecular biologists insert DNA fragments into plasmids and then introduce the plasmids into bacteria.
- Bacteria reproduce super rapidly
- shear DNA into fragments
- insert fragments in plasmids and produce library
- sequence DNA fragments
- assemble overlapping fragments*
- sequence DNA by PCR amplification
- D genes and functions
- method of sequencing a genome
- Start with large number of clones and determine order by sequencing shotgun clones individually
- look for overlaps
- Short sequenced fragments are assembled into a whole-genome sequence by...
- creating detailed genetic & physical maps of the genome that provide known locations of genetic markers at regularly spaced intervals along each chromosome.
- These markers are later used to help align the short sequenced fragments into the correct order.
- is a better approach for repetitive sequences
- starts w/ libraries of large, overlapping DNA fragments
- requires sub cloning of large fragments into smaller clones for sequencing
- uses genetic recombination data to help arrange sequences correctly
- starts with closing and sequencing of short, random DNA fragments
- require chromosome fragments to overlap for contig assembly
- require sequences to be annotated after contig assembly
- found on different chromosomes. can recombine to porduce many combinations (Ab, AB, aB, ab)
- inheritance follows Mendel's law of independent assortment
- for 3+ do all probabilities separately and then multiply them together to find the probability of the events occurring simultaneously
- located on same chromosome, inherited together
- the closer two genes are on the same chromosome, the less likely they are to separated by a crossover event in meiosis 1
- produces gametes with recombinant chromosomes, individual chromosomes that carry genes from two different parents
- b/c all genes are linked, it is more likely that the parental allele combinations would stay together, rather than be recombined through crossover
- All possible phenotypes would occur, but a greater proportion of the offspring would have the parental phenotypes.
- some crossing over would occur; small proportion of offspring would have recombinant phenotypes
The tendency of close -together genes to segregate together
* Genes that are located quite close to each other almost always segregate together, and so are * inherited together.
( a cross over event is unlikely the closer two genes are to each other on a chromosome)
- account for DCO's that occur b/w the 2 genes that are farthest apart on the chromosome
- (1) look @ # of progeny w/ ea. of the 8 phenotypes. The 2 lowest are DCO's b/c it's less likely to occur than a SCO.
- (# of DCO's)x(2) b/c ea. represents 2 crossovers
- Can also add the 2 smaller map distances b/w the 2 gene pairs located closer together- to determine the distance b/w the 2 genes farthest apart
- the probability of a double crossover is the product of the probabilities of the single crossovers
- 0.25 x 0.05= 0.0125 or 1.25%
- recombination frequencies between linked genes along a chromosome are additive
- the recombination frequency between genes X and Z is: 25 + 5= 30 or 30%
- the frequency of recombinant gametes is half the frequency of crossing over b/c each crossover takes place between only 2 of the 4 chromatids of a homologous pair
- SCO's between two non-sister chromatids leaves two non-recombinant chromosomes in gametogenesis
- used to determine the distance between 2 genes
- can be used to determine the order of three genes on the chromosome
- to study double exchanges, three pairs of genes must be investigated, each heterozygous for two alleles
- expected frequency of D.C. gametes is much lower than that of either S.C gamete class
- incapable of growth on nutritionally deficient medium.
- don't survive under non-lab conditions.
- i.e. the strain is autotrophic for leucine and methionine means that is cannot synthesize those nutrients
- thus, it requires those 2 nutrients to be added to minimal media for growth
- ability to uptake naked DNA; natural phenomenon
- can uptake fragments of linear DNAfragmented by nucleases
- Not all cells are competent to be transformed 1/1000
- a marozygote is a partially diploid bacterial cell
- formed when the F factor and several adjacent bacterial genes are excised from the bacterial chromosome of an F' cell and transferred to an F- cell.
- to ensure that only recombinant genotypes are recovered
- selective media allows elimination of parental genotypes and recovery of only those whose genotypes result from transfer of donor genes
- selective medium are chosen for their ability to remove unwanted cells from a mixed cell pop.
- genes are transferred from donor to recipient in a linear fashion
- remember: parameter being measured in these experiments is time of transfer
- dependent on which side of the origin the gene resides
- see homework for more on this
- a bacterium that can grow on minimal medium
- contains a carbon source and a few inorganic salts
- wild-type bacteria are prototrophs
- bacterium that has a mutation in a biosynthetic pathway that prevents growth on a minimal medium
- unless the product of that pathway is aded as a supplement
- F+ carry the fertility factor (the f plasmid)
- F- lack the F plasmid
- and F+ bacterium can form a pilus to conjugate with an F- bacterium
- if it integrates into the bacterial chromosome, it will transfer a portion of the it downstream from the integration site to an F-cell
- b/c this integration allow for more frequent recombination involving the transferred chromosomal genes, bacterial strains with integrated F plasmids are called HIGH FREQUENCY RECOMBINATION strains (Hfr)
- (A) require a supplement to grow, (P) grow in the presence or absence of the supplement
- i.e. the medium that lacks phenylalanine selects for (P)'s for phenylalanine, which have the genotype phe+
- the genotypes for the other two loci may be either (P) or (A) b/c both genotypes will enable colonies to grow when the supplement is provided
- an integrated plasmid and a copy of the Hfr chromosome attached to it enter a recipient cell from an Hfr donor cell
- in an interrupted conjugation experiment on the other hand, this process is halted at various times
- they can be used to make a rough map of the loci
- method is not precise enough to distinguish between closely linked loci, which have similar entry times
- Bacterial cells that survived treatment w: 1st antibiotic (ampicillin) have taken up plasmid
- These cells are cultured- Spreading them v. thin on nutrient agar plates
- Each separate cell on the plate will grow to a genetically identical colony.
- Tiny sample of each colony is transferred onto 2nd (replica) plate in same position as colonies
- replica plating is a technique that allows the same bacterial colonies to be plates onto different media
- a sterile velvet pad is pressed onto a "master" plate to pick up bacteria from each colony
- the pad is then pressed onto new plates to reproduce the same colonies on other media
- formed by the most crossover events
- heat-killed bacteria harbor the constituents necessary to convey genetic properties to living bacteria
- Because some of the nonvirulent bacteria acquired properties of the virulent bacteria, instructions for this transformation must be carried by the virulent bacteria.
- Incubated nonvirulent cells with the complete extract
- The complete extract possessed the same ability to induce transformation in IIR bacteria as whole heat-killed IIS bacteria.
- polysaccharides are the genetic material
- Failure to transform suggests that the chemical degraded in that preparation is the one responsible for transformation.
- It was necessary that each of the two phage components, DNA and protein, be identifiable upon recovery at the end of the experiment.
- Because it was concluded that the component associated with bacteria at the end of the experiment must be the genetic material, it was critical that the component be identifiable as either DNA or protein.
- DNA is the identity of the hereditary material in phage T2.
- Because phage DNA and not protein was associated with bacteria at the end of the experiment, it could be concluded that DNA - not protein - must be the genetic material.
- Both preparations of infected bacteria would exhibit radioactivity
- Instead of being removed from the preparation, the "ghosts" would be retained. Because both bacterial preparations would include ghosts as well as viral DNA, both would be radioactive, one with P32, one with S35.g
- Genes that are constantly expressed
- i.e. I-O+Z+, I-OcZ+, I-OcZ+/F'O+, I+OcZ+/F'O+
- Enzymes that are produced continuously, regardless of the chemical makeup of the environment.
- expressed only as needed, can be turned off by certain conditions (negative regulation)
- repressed when trp present
- i.e. IsO+Z+, IsO+Z+/ F'I+
- Genes that are expressed only when their proteins are needed by the cell.
- turned on in the presence of an inducer
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