Molecular Biology uwindsor lecture 1 Flashcards
What organism serves as a model eukaryote?
yeast
What does a DNA molecule consist of?
Two complementary chains of nucleotides (sugar-phosphate backbone with nitrogenous bases)
What does the structure of DNA provide?
A mechanism for heredity
What is eukaryotic DNA packaged into?
A set of chromosomes
What do chromosomes contain?
Long strings of genes
What must each DNA molecule that forms a linear chromosome contain?
A centromere, two telomeres and replication origins.
What is a gene?
A functional unit of inheritance, usually corresponding to the segment of DNA coding for a single RNA.
What is a genome?
All of an organism’s DNA sequences.
What is a locus?
The site of the gene in the genome
What are alleles?
Alternative forms of a gene
What is a genotype?
The specific set of alleles forming the genome of an individual
What is a phenotype?
The visible character of the individual
What is a wild type allele?
The normal, naturally occurring type
What is a mutant allele?
The allele that differs from the wild type because of a genetic change (a mutation)
What is a point mutation?
Maps to a singe site in the genome, corresponding to a single nucleotide pair or a very small part of a single gene.
What is an inversion?
Inverts a segment of a chromosome.
What is a deletion?
Deletes a segment of a chromosome
What is a translocation?
Breaks off a segment from one chromosome and attaches it to another.
What is a loss of function mutation?
A mutation that causes the loss of function in the resulting protein.
What is a conditional loss-of-function mutation?
A mutation that causes the protein to lose function given a specific subset of conditions.
Why are there model organisms in molecular biology?
They are much easier to study and have great relatedness to other species at the genetic level.
What makes the yeast a model eukaryote?
The budding yeast is the simplest model system. It is good for genetics and biochemistry. It is very good from studying cell division but lacking in cell to cell signalling research opportunities.
What is commonplace when studying C. elegans?
RNAi for gene knockouts. It is a very basic eukaryote and will give inference on fate of each cell.
What makes the drosophila a model organism?
Great system for genetics and vertebrate development.
What make the xenopus (frog) a model organism?
Makes giant eggs, studying meiosis and mitotic division of embyo is easily viewed. Possible to inject substances into these. Great biochemistry system. Bad genetic system however due to it being tetrapod.
What makes the zebra fish a good model system?
Good system for genetics. Accessible model from vertebrate development.
Why is the mouse used as a model organism?
Very similar to humans at the molecular level and the mouse is the predominant mammalian model organism.
Describe the traits of DNA.
Formed of nucleotide bases hydrogen bonded to each other,these are composed of nitrogenous bases with a sugar phosphate backbone. Each strand is paired anti-parallel with a complimentary strand and forms a double helix.
What is the model of replication of DNA?
Semi-conservative replication, in which one old strand is attached to a new daughter strand.
When is karyotyping done?
During metaphase of meiosis.
What is the difference between yeast and human chromosomes?
Yeast chromosomes contain mostly genes while human chromosomes are littered with genome-wide repeats.
What is the central dogma of genetic replication?
DNA to RNA to proteins.
What is the basic structure of a eukaryotic gene?
Enhancer upstream, promoter upstream, genes with introns and exons, then stop codon, then poly-A tail.
What are exons?
Coding region of gene.
What are introns?
Non-coding region of genes that are spliced out.
What binds to the promoter?
RNA polymerase.
What binds to the enhancer?
Transcription factors.
What do transcription factors do?
Enhance promoter specificity.
What is complementation?
The ability for a mutation in two different genes, do not have to be gene families, to counteract the seperate mutations and form a wild type progeny.
What are the general steps in a genetic screen (forward genetics)?
1) Mutagenize
2) Cross to obtain individuals with mutations
3) Crosses to obtain homozygous lines
4) identify lines of phenotypic interest
5) Complementation, genetic mapping
6) Clone and sequence the gene
7) Positive or negative selection
8) Set up screen for conditional mutants
In a genetic screen, why are the bacteria mutagenized?
Large population of bacteria mutagenized so every gene in genome is hit.
Why do we have to find complements in a genetic screen?
To determine if the mutations are in the same gene or not. (If the progeny are wild type, this is indicative of mutations on different genes)
Describe the basic steps of reverse genetics.
1) Start with gene you think is of interest
2) Mutate gene
3) Use either homologous recombination, gene editing by the Cas9/CRISP method or use RNAi to knockdown gene
What are transgenics?
Introducing genes into cell or whole animals. Technique to study gene function. Can either be transfection or transformation.
What is transfection?
Genes are introduced into cells using vector DNA present in cell for given amount of time until lost from cell.
What is transformation?
DNA (competent) is brought into cell and stabily integrated, permanently there. When the cell divides, DNA is in daughter cell.
In germline cell, transgene is passed onto progeny.
What is a double mutant?
Make individual homozygous mutant for two different genes.
How does one make a double mutant?
Start with heterozygous for one gene and another heterozygous for another gene. Cross to get double mutant.
What are transgenes used for?
Used to study gene function; through overexpression, gene reporters, epitope or GFP tag and structure/function studies where you delete a specific domain of protein and test for function.
What does cloning involve?
1) Using plasmids or PCR
2) take DNA of interest and using RE cut add to cut plasmid
3) Use ligase to put in plasmid
4) Transform into bacteria
5) Positive selection
6) purify plasmid isolated from bacteria
How does purified DNA get tagged?
1) Denature DNA
2) add primers (random hexamers
3) add DNA polymerase and labelled nucleotides
4) get radioactive complimentary DNA, use as probe
5) Can use to find genes on chromosomes
What are the steps of PCR amplification?
1) Denature DNA
2) Add primers, nucleotides and taq. polymerase
3) annealing temperature
4) hybridization temperature
5) Cycle
How is a DNA library made?
Start with dsDNA
1) cleave with RE’s
2) insert DNA fragments into plasmids
3) introduce into bacteria through cloning
4) obtain library
How is a cDNA library made?
Start with RNA transcripts from genes
1) RNA splicing to form mRNAs
2) use reverse transcriptase and DNA polymerase to produce cDNA copies of mRNA’s
3) DNA cloning
4) cDNA library
What are the steps for hybridization?
Start with dsDNA
1) denature
2) slowly cool to hybridize
3) can use different temperatures to allow non-perfect binding
