lab practical 3 Flashcards
what is an operon
it is a group of genes regulated by a single gene, the expression of these genes is regulated together
what are the components of an operon?
more than one gene, a single promoter, regulatory binding sites
what is the function of the genes in the Lac operon?
they are involved in the breakdown of lactose as a energy source in the absence of glucose
what enzyme is encoded by the LacZ gene?
Beta-galactosidase: it breaks down lactose to yield glucose and galactose which can be broken down to further yield glucose
what is the function of beta galactosidase
it cleaves the beta bond in lactose to yield glucose and galactose
what does the LacZ gene do
it codes for beta galactosidase which break down lactose
what does the CAP activator do
it binds to the activator site when in the absence of glucose
what does the repressor do
it binds to the operator to suppression transcription when lactose is not present
what is the operator
it is the regulatory element where the repressor binds in the absence of lactose
what is the promoter
it is the site where RNA polymerase binds to begin transcription
under what conditions is the Lac operon active?
it is active in the absence of glucose snd presence of lactose
does E.coli prefer to use glucose or lactose as an energy source? why
glucose, it is the preferred source of energy as it can directly enter glycoylsis
why does the lac operon only want to activate in the presence of lactose and absence of glucose
it is a waste of energy to synthesize the genes/proteins needed for lactose metabolism
under what conditions does the CAP activator bind to the CAP?
it binds when there is no glucose (high levels of cAMP)
under what conditions does the CAP activator come off the CAP
when there is glucose present and there are low levels of cyclic AMP
under what conditions does the repressor come off the operator
when in the presence of lactose (allolactose inducer)
under what conditions does the repressor bind to the operator
in the absence of lactose
under what conditions will the lac operon be active and transcribe downstream genes
in the absence of glucose (CAP on) and presence of lactose (repressor off)
if you have no glucose or lactose, what is bound
CAP and repressor
if you have glucose and no lactose, what is bound
repressor on
if you have glucose and lactose, what is bound
no CAP, no repressor
can the strain of E.coli utilize sucrose as an energy source
no, it does not contain the enzyme needed to break down sucrose
why did we add toluene to the cultured cells?
it pokes holes in the membrane without denaturing the proteins and allows beta-galactosidase to leak out
How was the ONPG substrate used to measure beta-galactosidase activity?
ONPG has a similar bond to lactose and will be cleaved into ONP which produces a yellow color that can be used to measure enzyme activity
how does a spectrophotometer work
a specific wavelength of light is used to shine through a sample and the amount of light absorbed is detected
what is the induction ratio?
enzyme activity/cell for lactose/ enzyme activity/cell for water
what is a silent mutation
it is a point mutation where there is no amino acid change and no subsequent affect on protein function
what is a missense mutation
it is a mutation where a single base change leads to a different amino acid, which may or may not affect protein function
what is a nonsense mutation
it is a mutation where a single base change leads to a early stop codon which terminates translation of the protein early
what is an insertion/deletion?
it is when a single/ few nucleotides are inserted or deleted from the sequence
what is a frame shift mutation
when more/less than three bases are added/deleted from the sequence, it results in a frameshift where all codons downstream of the mutation are read differently and this results in a change of all the amino acids
if three nucleotides are inserted, what is the consequence on the amino acid sequence
there will be no change in the reading frame, but will result in the gain or loss of one amino acid
how can a mutation occurring outside of the coding region for a gene affect gene expression
a mutation outside the coding region may affect gene expression by affecting the promoter, regulatory elements, untranslated regions, and splice sites
what does SNP stand for
they are “small nucleotide polymorphisms”
what are SNPs
they are base substitutions that serve as molecular markers and are used to index the amount of variation that exists between individuals
why are SNPs important to the study of human?
1.) SNPS are linked to Mendelian mutations which are inherited diseases that come from a single nucleotide changes
2) may be linked to disease causing genes and can be redictive of a certain phenotype
How are SNPS related to diseases?
some can be found directly in a gene that is involved in a disease or they may be indirectly related and exist nearby disease-causing genes and can be predictive of certain phenotype
are most SNPs directly involved in causing a phenotype or condition
no, most SNPs are found nearby a disease causing gene and can be predictive of having a disease
what is the goal of GWAS
to discover genetic risk factors for many diseases and to identify SNPs that may be predictors of disease
SNPS are
common variants with small effects
what is a haplotype
it is a set of closely linked genetic markers or DNA variations on a chromosome that tend to be inherited together
what are haplotypes used for
they are used to correlate particular SNPs that are tightly linked and tend to be inherited together
clusters of SNPS can be used as predictors for a disease/phenotype
what is the purpose of genetic testing
to diagnose or rule out potential genetic disorders for patients or their offspring
what is newborn screening
it is genetic testing performed immediately after the birth of the child
what is fetal testing
it is genetic testing that is performed prior to the birth of the child
what is predictive testing? is it 100% accurate
it is performed on individuals who are pre-symptomatic but may be at risk for developing a disease
it is not 100% accurate due to external risk factors like environment
describe the method of preimplantation genetic diagnosis
a cell from the early cleavage stage of an embryo can be removed and assayed for various genetic disorders
embryos without the disorder can be selected for implantation
how does CGH (comparative genomic hybridization) be used to detect chromosomal; duplications or deletions
reference DNA will be partitioned and immobilized onto a solid glass support where patient DNA and control DNA will be differentially fluorescently labeled and hybridized with the reference DNA on the slide—differences in the fluorescence patterns between the control DNA and the patient DNA can be used to detect abnormalities
why might a person use DTC genetic testing services?
DTC companies offer affordable genetic tests that allow patients to get insight into their risk for disease without going through a physician who generally requires clinical indications of disease or family histories of disease
list several reasons why one might be cautious when using DTC companies for genetic testing
1) concerns for privacy being accessed by third parties
2) DTC companies do not test for all known variants involved in disease so an individual who appears to not be at risk might actually be
3) misinterpretation of results by the consumer
4) lack of clinical support
5) less regulation involved
what is the purpose of FISH
it confirms the presence of a duplication or deletion mutation by using a fluorescent probe that binds to specific regions of chromosomes
what will FISH show if a deletion is resent
the fluorescent probe will bind to only one copy of the chromosome instead of two and this will result in only one signal rather than 2
in the example that shows a patient with a deletion on chromosome 22, what do the green dots represent? what do the red dots represent?
the green dots are the control dots which are region in the DNA where the patient is known to NOT have a deletion. The red dot is representative of the known deletion region and since there is only one red dot, this confirms that a deletion occurred on one of the chromosomes and not both
what is the study of cytogenetics?
it is the study of the chromosomal organization in an organism. It is used to determine whether there are large-scale chromosomal mutations
what is a karyotype
it is a visual representation of an organism’s complete set of chromosomes and is arranged by size, shape, and binding pattern
what are the steps in generating a karyotype
cells are placed into a tissue culture medium and are induced to enter mitosis with an inducing agent
the cells are arrested near metaphase by stopping the formation of the spindle apparatus
the cells are lysed with a hypotonic solution so the chromosomes can leak out
the cells are fixed onto slides and are stained for analysis
why are cells induced to enter mitosis
when cells enter mitosis they begin to condense which will make them easier to visualize
why are cells arrested at metaphase
at metaphase, the cells are maximally condensed and will be easier to visualize
how are the cells arrested at metaphase
a chemical is added which binds to the micro-tubule forming proteins which prevents the formation of a spindle apparatus
why is karyotyping of fetal cells when a mother is older particularly important?
when the mother is older, the risk for nondisjunction events during meiosis is greater
how are G banding patterns on fixed chromosomes produced?
Giemsa stains differentially stain AT and GC regions of DNA to create dark and light bands on chromosomes
why are the chromosomes treated with trypsin prior to the Giemsa staining?
trypsin digests proteins (including histones) which helps expose DNA for staining
what is a metacentric positioning
the centromere is at the middle
what is submetacentric positioning
the centromere is 1/3 down
what is arocentric positoning
the centromere is located near the end of the chromosome
what is telomeric positioning
the centromere is located at the very end, this is not found in humans
what is a chromosomal duplication
it is where a chromosomal segment is present in many copies
what is a chromosomal deletion
it is where a chromosomal segment is missing
what is the effect of chromosomal duplications or deletions
since abnormal amounts of the genes will be produced, it will affect multiple traits
what are chromosomal inversions
chromosomal segments are flipped around in orientation IN THE SAME chromosome
what are chromosomal translocations
a chromosomal segment moves to another chromosome or a different location in the same chromosome, this may be a reciprocal relationship and segments may swap with eachother
what is the effect of inversions and translocations
If the breaking points at the ends of the segments are in the MIDDLE of genes, it will interrupt the coding sequence and may lead to the loss of the coding sequences
for individuals with one normal and one rearranged chromosome, what could happen?
during meiosis one, there may be abnormal synapsis of homologous regions
what is a polyploid
it is a genome mutation where there is an additional or loss of one or more of every chromosome in the set
give two examples of polyploid
triploid, 3n
tetraploid 4n
what is an aneuploid
it is when there are 1-2 extra or missing chromosomes, but the rest are still 2n
give three examples of aneuploid
trisomy 21: down syndrome
Kleinfelter syndrome: 2X chromosomes (XXY)
Turner syndrome (monosomy) where females are missing an X chromosome
what type of mutation is down syndrome
it is a trisomy aneuploid, there are three chromosome 21s
what type of mutation is kleinfelter
it is a trisomy in males where they have two X chromosomes (XXY)
what type of mutation is Turner Syndrome
it is a monosomy in females where they are missing one X chromosome
what is nondisjunction
it is when chromosomes fail to separate in meiosis or mitosis
how does nondisjunction occur in meiosis 1
the homologs fail to segregate
how does nondisjunction occur in meiosis 2
the sister chromatids fail to separate
if nondisjunction occurs in meiosis 1, where do the two chromosomes in the gamete come from?
different homologs
if nondisjunction occurs in meiosis 2, where do the two chromosomes in the gamete come from
the same homolog
in GWAS, what do the statistical tests do?
they determine whether a particular SNP is statistically found more often than expected in individuals with the disease
