Unit 2 Flashcards
imperfect base pairing with single stranded DNA probe occurs under what conditions
lower temperatures
a collection of methods used to isolate, manipulate, amplify, re organize, and analyze genes at the molecular level
Recombinant DNA technology or molecular genetics
perfect base pairing with a single stranded DNA probe occurs under what conditions
higher temperature
cut dsDNA in palindromic sequences
restriction enzymes
most useful function of restriction enzymes
leave staggered ends, with 5’ or 3’ overhangs
why are staggered ends created by restricted enzymes useful
they can be efficiently ligated to complementary ends with T4 DNA ligase
how do restriction enzymes serve as landmarks on DNA sequences
used for linkage analysis
RFLPs
cut at specific pattern points
why do you use gel electrophoresis
to separate nucleic acids based on size
voltage moves negatively charged molecule toward the positive anode
Vehicles used to move, manipulate, and amplify cloned genes/fragments of genes. Altered versions of naturally occurring DNA elements used to transfer genetic information
vectors
which vector/host carries the smallest insert size range in molecular cloning (go from smallest to largest, there are five)
plasmids in bacteria/yeast then bacteriophage lambda in bacteria then cosmids in bacteria then BACs in bacteria and YACs in yeast are the biggest
YAC
yeast artificial chromosome, can be tricked into carrying the largest amount of recombinant DNA
how does bacteriophage lambda spread into the growth medium
lysis
a large collection of random inserts for a genome of interest in a cloning vector
library
cDNA library doesn’t contain ___
introns
where does DNA come from in a cDNA library
directly from a tissue via mRNA/reverse transcriptase, so you only get mRNA being expressed in that specific tissue
allows you to amplify known sequence of RNA or DNA from complex mixtures of nucleic acids even if the copies of that sequence are extremely inabundant
PCR
how does PCR work
reiterative rounds of primer directed DNA replication employing a thermophilic DNA polymerase
cycles of heating and cooling (heat to separate strands, cool to hybridize primers and synthesize DNA)
binomial expansion using PCR
amount of DNA made= # starting templates x 2^# cycles
product then analyzed by gel electrophoresis
method to determine size and quantity of purified DNA
DNA gel electrophoresis
method used to detect presence and size of a specific DNA sequence in a complex mixture/sample
Southern blot/assay
method used to amplify a specific DNA sequence from a complex mixture/sample
PCR
method used to amplify a specific RNA sequence from a complex mixture
rtPCR (reverse transcriptase)
method used to amplify multiple specific sequences from a complex mixture in a single PCR reaction
multi plex PCR
method used to detect the presence and size of a specific RNA sequence in a complex mixture/sample
Northern blot/assay
method used to separate proteins by size/molecular weight
denaturing gel electrophoresis of proteins
method used to separate proteins based on size/shape and charge at a particular pH
non-denaturing/native protein gel electrophoresis
method used to separate proteins based on pI (pH at which protein is not charged)
isoelectric focusing
method used to detect presence, size/shape, and abundance of specific protein in a complex mixture/sample
Western blot/assay
method used to detect the presence and localization of a protein in a fixed tissue/cell sample
Immuno Fluorescence Microscopy
method used to determine the location and dynamics of a protein of interest fused to GFP
GFP Fluorescence
method used to determine the presence and relative abundance of all mRNA species in different samples/cells/tissues etc
micro array based expression analysis
method used to down regulate the expression of a specific gene in a cell/tissue
RNAi/morpholinos
specific use for non-denaturing/native protein gel electrophoresis
can be used to compare proteins with mutations in charge amino acids such as certain mutations in hemoglobin that cause sickle cell disease
at high pH, a protein is (+/-) charged
negatively
at low pH, the protein is (+/-) charged
positively charged
two dimensional protein gel electrophoresis-protemics
is a combo of:
gel electrophoresis with mass spec
2D protein gel electrophoresis shows what specifically
all the proteins expressed in a cell or tissue and how they are post translationally modified
antibodies raised in animals by injecting them with the antigen of interest
primary antibody
antibodies raised against the constant regions of antibodies from a different animal species
secondary antibodies
labeled antibodies can be used as
molecular tags
immuno fluorescence microscopy analysis can give information about cells involved in which disease (example)
muscular dystrophy
what do micro array expression experiments tell us
relative expression levels of all genes in the genome under specific conditions
how this expression pattern changes when the system is perturbed
identifies groups of coordinately regulated genes that may be functionally related
provides a diagnostic tool to type a tissue/tumor/disease state
how we handle all the data from micro array expression experiments
cluster analysis
-compare all expression patterns to each other, join patterns that are most similar (the painting example), compare joined patterns to all other unjoined patterns and repeat repeat repeat
cluster analysis and display of genome wide expression patterns
hierarchical clustering
next thing in DNA methods after micro arrays phase out
next generation sequencing–whole genome sequencing
introduction of dsRNA corresponding to a cellular mRNA will induce degradation of the mRNA and downregulation
RNA interference aka RNAi, discovered in C elegans
what is the purpose of RNAi
can suppress dominant alleles or diseases due to inappropriate over expression
short single stranded oligos complementary to a transcript you want to knock down
inhibit expression by blocking progress of the ribosome/translation or by blocking a splice site
morpholinos
can be fused to any gene of interest and used to study the expression and/or localization of the gene product in living cells
Green Fluorescent Protein GFP (from jellyfish)
method that provides the greatest sensitivity for detecting specific nucleic acid sequences
PCR
method that can be used to clone genes
PCR
recognition site for a restriction enzyme
palindromic sequence
method used to detect specific nucleotide sequences in both Northern and Southern blots
nucleic acid hybridization
method used to create a large set of plasmids that contain random inserts of the expressed genes from a tissue of interest
construction of cDNA library
method used to determine if a patient with muscular dystrophy is making normal dystrophin protein in their muscle cells
western blot
method used to amplify and clone a specific exon from the dystrophin gene of a patient with muscular dystrophy
PCR
method used to examine the gene expression profile in the skeletal muscle cells of a patient with muscular dystrophy
DNA micro arrays
why use a control probe when trying to detect a deletion in FISH
because you need to rule out technical error, such as the probe just didn’t bind, although the sequence was present. the control probe proves that it is working, but there is something missing
FISH is best used for:
detailed information about a specific locus (not the whole genome)
three types of FISH probes
repeat sequences, single copy DNA (subtelomere FISH), chromosome painting (multicolor)
type of probes isolated from centromere or telomere regions
repeat sequences
what do centromere probes detect
gain or loss of specific chromosomes
syndromes identified using repeat sequence FISH probe
Down Syndrome, Turner Syndrome
FISH probe isolated from cloned DNA of a disease-causing gene or a fragment of DNA of known location associated with a particular gene
unique sequence aka single copy
FISH probes that are DNA sequences from the distal ends of the chromosomes in the regions proximal to the actual telomere regions
subtelomere probes
why use subtelomere probes for FISH
these are very gene rich regions
probes that are actually a cocktail of many unique DNA fragments from along the entire length of a chromosome such that following hybridization, the entire chromosome fluoresces
chromosome painting probes aka whole chromosome paints WCP
when is chromosome painting in FISH most useful
when identifying complex rearrangements or marker chromosomes
accuracy limitations when using FISH to detect deletions
most deletions are very large and the probes are much smaller, a deletion may be present that cannot be detected by the FISH probe designated for the disease
how do you use FISH when you want more info about a developmental delay
check for a subtelomeric microdeletion
what are caused by deletion or duplication in regions of the genome with clusters of closely associated genes whose normal functions are generally unrelated
continguous gene syndromes
contiguous gene syndrome caused by deletion of elastin gene
Williams syndrome
characteristics of _____ contiguous gene syndrome: aortic stenosis, thickening of skin, renal anomalies, low IQ, excellent music skills but terrible with math, outgoing and friendly, blue sclera, stellate iris
Williams
contiguous gene syndrome with presentations of learning disabilities, cleft lip/palate, facial anomalies, cardiac anomalies, weak immune system, difficulty feeding at birth
Velocardiofacial syndrome VCFS
how can a parent carry the VCFS deletion?
15% of the time, parent carries the deletion but may not be clinically abnormal–huge phenotypic variability
how is VCFS deletion caused
unequal crossing over
compensation on homologous chromosome
gene arrays tell you:
specific DNA sequences of interest
expression arrays tell you:
gene products to understand which genes are being expressed in a particular cell type at a particular time
clinical application of expression arrays
can create tumor profiles to show genes that are upregulated for each type of tumor to aid with diagnosis
what method is used to view the copy number variants in association with the chromosomes
chromosome microarray
gold standard for test used to identify problems that are not suited to just one disorder (seemingly unrelated presentations)
chromosome microarray
problem with next generation sequencing
more data than we know how to interpret, sequencing is complicated and so is reading the results
goal of panels in clinical sequencing
to get manageable amounts of data back
things to keep in mind when ordering genome sequencing (4)
- is the lab accredited/the test validated/
- it takes a long time (about 2.5 weeks)
- cost is 3K-5K
- do the patients have insurance?
what type of abnormalities will karyotype analysis detect
numerical and structural (large) abnormalities
what are the pros and cons of molecular diagnostics
pros: well defined, specific, can detect very small mutations
cons: need to know what you’re looking for already-targeted testing
what size mutations does FISH recognize
medium sized
targeted testing
pros and cons of microarray
pro: genome wide screen for small to large mutations
con: will not detect balanced rearragements
pros and cons of DNA sequencing
pro: high resolution, detect mutations on a single base level
con: trying to interpret all the data
which technology would be best to diagnose autism?
since autism is related to a wide variety of genes and none of the mutations have been associated with numerical or structural abnormalities (so no FISH or karyotype), you need to use microarray analysis
the study of development between fertilization and birth
embryology
study of embryonic and other developmental processes
developmental biology
what percent of all embryos are spontaneously aborted
50%
weeks 1-8 of human pregnancy when organ primordia are established
embryogenesis
the embryonic period is followed by the ___ period of continued differentiation and growth
fetal
system used by embryologists to describe the apparent maturity of embryos based on external features
Carnegie stages
system used by clinicians to describe the maturity of an embryo, refers to the length of time since the last ovulation before pregnancy
postovulatory age
by the end of the first week of embryogenesis, the blastocyst begins:
implantation into the uterine wall
zygote > cleavage > _____ > blastocyst
morula
how is the first cleavage division initiated
fertilization
what is the result of cleavage divisions
increase in number of cells but no change in size of the zygote
process during cleavage that segregates inner cells from outer cells
compaction
how is the compact ball after the third cleavage division stabilized
tight junctions that form between the outside cells of the ball
inner cells of the morula
inner cell mass ICM
the inner cell mass gives rise to:
tissues in the embryo proper
outer cells of the morula give rise to the
trophoblast that later contributes to the placenta
during a process called ______, the trophoblast cells secrete fluid into the morula to create a blastocoel, an internal cavity
cavitation
what is the function of the zona pellucida around the morula
protection
cells that can give rise to all of the cell types that make up the body
pluripotent
from which cells are embryonic stem cells derived?
inner cell mass
trophoblast cells contribute to ____ tissues
extraembryonic
______ are pluripotent cells that give rise to the embryo
inner cell mass
cells that can differentiate into any cell type including extraembryonic tissues
totipotent
cells at the morula stage of development are considered (pluripotent/totipotent)
totipotent
why does the blastocyst hatch from the zona pellucida
in order to attach to the endometrium/uterine wall
what does the zona pellucida prevent before the embryo reaches the uterus
implantation in oviduct walls
what is the mechanism of hatching from the zona pellucida
a trypsin like protease lyses a hole in the fibrillar matrix of the zona and the embryo squeezes out through that hole
uterine epithelium
endometrium
what type of embryonic cells attach to the uterine wall epithelium
trophoblast
molecules called ____ (carbohydrate binding proteins) on trophoblast cells interact with carbohydrate receptors on the endometrium to mediate attachment of the blastocyst to the uterus
L-selectin
after initial binding of the embryo to the endometrium, trophoblast cells express _____ proteins that bind uterine collagen, fibroconectin, and laminin to keep the blastocyst bound to the uterine wall
integrin
once in contact with the endometrium, the trophoblast secretes _____ enzymes to digest the ECM of the uterine tissue, enabling the blastocyst to bury itself within the uterine wall
protease
tethering of the blastocyst to the uterine wall
implantation
abnormal blastocyst implantation is called:
ectopic pregnancy
the trophoblast differentiates into two layers: the ___ and ___
cytotrophoblast and the syncyiotrophoblast
an inner layer of mononucleated cells that differentiates from the trophoblast
cytotrophoblast
an outer mutlinucleated layer that lacks distinct cell boundaries that differentiates from the trophoblast
syncytiotrophoblast
how does the cytotrophoblast adhere to the endometrium
adhesion molecules
cytotrophoblast cells contain ___ enzymes that enable them to enter the uterine wall and remodel the uterine blood vessels so that the maternal blood bathes the fetal blood vessels
proteolytic
on day __, the cells of the ICM differentiate into two layers: the ____ and the ____, which form a flat bilaminar disc
day 9
hypoblast and epiblast
on day __, the cells of the trophoblast differentiate into the cytotrophoblast and the syncytiotrophoblast
8
the amniotic cavity forms within which layer that has differentiated from the ICM
epiblast
what type of role does the hypoblast play after it has differentiated from the ICM
supportive role
what is the purpose of amniotic fluid
surrounds the embryo to protect it from abrupt movement and keeps it from drying out
the maternal portion of the placenta
the uterine endometrium
the fetal portion of the placenta
the chorion
how do soluble substances diffuse from the mother to the fetus
through villi
fertilization results in the formation of a (diploid/haploid) zygote and activates:
diploid
cleavage division
the process of ____ forms a tight ball of cells that separate into inner and outer layers in the embryo
compaction
the process of ____ transforms the bilaminar disc into three germ layers
gastrulation
three goals of gastrulation
1- bring inside the embryo areas destined to form endodermal organs
2- surround the embryo with cells capable of forming ectoderm
3- place mesodermal cells in proper positions in between
gastrulation begins with the formation of the ________ (2 words) on the surface of the epiblast
primitive streak
the primitive streak becomes a narrow groove with a structure called the primitive ___ surrounding a primitive ___ at the cephalic end
node
pit
what is the role of the primitive node during gastrulation
it is an organizing center
____ cells (which layer of cells) migrate into the primitive streak
epiblast
inward movement of epiblast cells toward the primitive streak
invagination
invaginating epiblast cells give rise to which two germ layers
endoderm and mesoderm
what is Hansen’s node (primitive node)
organizer tissue-the node tissue expresses genes that induce formation of the embryo
the ____ layer gives rise to the epidermal surface of skin, the CNS, and the neural crest (ex pigment cells)
ectoderm
the ___ layer gives rise to the notocord, bone tissue, RBCs, facial muscle, and the tubule cells of the kidney
mesoderm
the ___ layer gives rise to the digestive tube, pharynx, respiratory tube
endoderm
the anterior-posterior axis is signaled by cells at the cranial margin of the embryonic disc, an area called the:
anterior visceral endoderm (AVE)
AVE expresses genes that direct formation of what body part
head
when are body axes established
before or during gastrulation
what gene establishes left/right body axes
BMP4
____ is the process by which the neural plate forms the neural tube
neurulation
neurulation divides the ectoderm into three distinct domains:
surface ectoderm (epidermis), neural crest (nervous system), and neural tube (brain and spinal cord)
how many sites of neural tube closure are there
three
what does neural tube closure depend on
genes and dietary factors such as cholesterol and folate
neural tube closure defect results in
spina bifida
specific location of neural tube closure defect that causes spina bifida
posterior neurospore
condition in which the forebrain remains in contact with amniotic fluid and degenerates, always fatal
anacephaly
complete failure of closure along the entire neural tube results in
craniorachischisis
which dietary supplement can prevent neural tube defects
folic acid
free cells that can migrate away from their epithelial layer linkage
mesenchymal
what type of cells undergo an epithelial-to-mesenchymal transition
neural crest cells
___ neural crest cells produce craniofacial cartilage, bone, neurons, glia, and connective tissue
cranial (cephalic)
____ neural crest is a subregion of cranial neural crest that develops into melanocytes, neurons, cartilage, and connective tissue. Also gives rise to large arteries
cardiac
____ neural crest migrates to form dorsal root ganglia containing sensory neurons or sympathetic ganglia and the adrenal medulla
trunk
____ neural crest form the parasympathetic ganglia of the gut
enteric (vagal and sacral)
how do neural crest cells migrate
on specific paths
why is the second week of embryogenesis regarded as the week of 2’s
the trophoblast differentiates into cytotrophoblast and synctiotrophoblast
the inner cell mass forms the hypoblast and the epiblast
_____: when the epiblast cells give rise to endoderm, ectoderm, mesoderm
gastrulation
origin of embryonic stem cells
inner cell mass
when does implantation of the blastocyst occur
one week post fert
cardiac neural crest cell migrate into the heart to contribute to which structures
septum of the outflow tract that separate the aorta and pulmonary artery
there is a (high/low) level of genetic similarity among different animals
high. 40% of human genes are present in flies and worms!
what percentage of human genes are present in mice
92%
genes that can function interchangeably during development of different species
homologous–functions the same no matter the organism
how do genes drive development?
the genetic material is identical in every cell, but different cells express different sets of genes
_____ _____: all cells contain the same set of genes
genetic equivalence
_______ provides evidence that all cells contain the same genes (experiment)
somatic nuclear transfer (cloning)
____: only a small percentage of the genome is expressed in each cell type (turn off wrong genes and turn on right genes)
differential gene expression
four level of gene expression level regulation
differential gene transcription
selective nuclear RNA processing
selective mRNA translation
differential protein modification
what does RNA FISH show you?
detects mRNA expression in cells or tissues (only cells expressing heart genes, for example, will be colored)
producing many cells from one
cell proliferation
creating cells with different characteristics at different positions
cell specialization
coordinating the behavior of one cell with that of its neighbors
cell interactions
rearranging the cells to form structured tissues and organs
cell movement
when one group of cells changes the behavior of an adjacent set of cells
induction
the two components to every inductive interaction
inducer and responder (responder must have the ability to respond to the signal=competence)
responder’s ability to respond to signal from inducer
competence
transcription factor in the ectoderm that makes ectoderm competent to respond to inductive signals from the optic vesicle (allows it to form eye lens)
Pax6
autosomal dominant mutations in Pax6 cause:
aniridia (affects iris, intraocular pressure, lens, cornea, optic nerve)
homozygous loss of Pax6 results in:
fatal condition, complete failure of entire eye formation
cell-cell signaling using proteins that are secreted into the EC space to deliver signals to neighboring cells
paracrine signaling
cell-cell signaling achieved by direct contact between inducing and responding cells
juxtracrine
paracrine signaling molecules that cause concentration-dependent effects
morphogens
a morphogen can specify more than one cell type by forming:
a concentration gradient
what organism is used to test whether a molecule can function as a morphogen
zebrafish
when a cell has competence to respond to an inducer, how are signals then received and interpreted in the cell?
signaling cascades/transduction pathways
___ signaling pathway is an example of a signal transduction cascade: secreted molecules bind receptors at cell surface of responding cells, proteins are then phosphorylated and enter the nucleus where they modulate gene transcription
transforming growth factor B (TGFB)
the two gradients that tell neural tube cells what to do
Bmp/wnt
Shh
high concentration of Bmp/Wnt along the neural tube indicates which side (dorsal/ventral)
dorsal
high concentration of Shh along the neural tube signals which side (dorsal/ventral)
ventral
normal left/right organ symmetry
situs solitus
completely reversed left/right organ symmetry
situs inversus
type of abnormal left/right organ symmetry in which the organs are abnormally abnormal (not a complete reversal)
heterotaxy
asymmetric gene expression of the ____ gene is greater on the left side
Nodal
in normal embryos, Nodal is expressed on the left _______
lateral plate mesoderm
what is the role of cilia in establishing right/left axes
cells in the node on its internal face have cilia beat in a helical fashion to drive fluid toward the left side
syndrome with bronchitis, infertility, and situs inversus
Kartagener’s syndrome (cilia defect)
asymmetric fluid flow caused by cilia creates:
a morphogen gradient that orients the left/right sides of the body
how is Lefty activated
activated by Nodal signaling cascade
what does Lefty do
restricts/inhibits asymmetric domain of Nodal signaling
the transcription factor ___ is expressed on the left side of the developing heart, gut, and brain, and is thought to regulate expression of genes that mediate asymmetric morphogenesis of these organs
Pitx2
method used when looking for specific mutations within genes (but don’t use for something like CF, which has 1000’s of possible mutations)
gene array
many mutations, gain or loss of DNA at base level, related to a disease, and may be able to use it to identify disease causing mutations when compared to normal
copy number variation
mitchondrial diseases occur mainly in organs like (three), where there is a high level of _____
nerve, brain, muscle
ox/phos
MOI of mitochondrial diseases
may show autosomal or X linked or matrilineal inheritance
male affected with mitochondrial disease (will/will not) transmit to children
will not
refers to a population of mitochondria that all have the same genetic composition–all mitochondria are the same within a given cell
homoplasmy
when there are two or more different populations of mitochondria present in a cell
heteroplasmy
how can there be different levels of expression of the same mitochondrial disease within a family
heteroplasmy–mother has a low frequency of mutant cells but passes higher frequencies on to her children
what is a mechanism that explains why mitochondrial disorders are often progressive with late onset
replicative segregation
why is there a high mutation rate in mit DNA
oxidative environment creates damage to DNA and causes a high frequency of mutations
acquired mutations in mit DNA (are/are not) passed on to children
are not–occur in somatic cells
what should you look for if there are unexplained neurological defects
mitochondrial disease
three reasons why diagnosis of mit disease is complicated
heteroplasmy/variable expression
maternal inheritence
progressive/late onset
use of DNA tech (PCR, RFLP, sequencing, etc) to obtain info on the genetic identity of an individual and how that relates to a criminal, medical, or scientific investigation
forensic DNA analysis
what does mitochondrial DNA analysis tell you in forensics
maternal inheritance-family identity
NOT individual identity
cons of using nuclear DNA for forensics
linear, degrades quickly
what do you need when doing a DNA paternity test
minimum of two probes
mother’s DNA
child’s DNA
STR
short tandem repeat DNA
benefit of nuclear (genomic) DNA forensics
identify an individual
how many individuals are necessary to show mitochondrial DNA relationships
at least two additional (mom and sibling preferably)
most common parasitic diseases in the US
trichomoniasis, giardiasis, cryptosporidiosis, toxoplasmosis
immature stages of certain parasites must partially develop inside of another species, such as an insect, snail, mammal. these are called:
intermediate hosts
host in which sexual reproduction occurs for the parasites
definitive
host in which asexual phase of lifecycle occurs for the parasite
intermediate host
why are parasites so difficult to get rid of
complicated life cycles
another name for definitive host for a parasite
principal
two major categories of parasites
parasitic protozoa-unicellular
parasitic helminths-multicellular
five ways in which host mammals can be infected by parasites
1-direct ingestion of infective larvae, eggs, cyst
2-eating the intermediate host (problem with raw fish)
3-parasite actively penetrates the principal host (hookworms)
4-parasite may be maternally transmitted (toxoplasma-kitty litter)
5-vector borne transmission (insect bite)
IgE
humoral response element as a result of parasite, allergy, autoimmune
helminth therapy
for autoimmune disease and allergies
what immune response do parasites elicit
cell mediated
what immune response can achieve complete immunity from a parasite
adaptive
which immune response results in the destruction and removal of the parasite, thus preventing the establishment of an infection
innate/nonspecific (cytokines, phagocytosis, activate complement system)
most important survival strategy that parasites have
complicated lifecycle–each stage has a different antigen
parasite strategies to evade adaptive immune responses
antigenic concealment
antigenic variation
antigenic shedding
antigenic mimicry (mimic host molecules)
in the intermediate host, you can observe the parasite in which forms
development from asexual stage to sexual stage before being transmitted to definitive host
most frequent of human prion diseases
Creutzfeldt-Jacob Disease
causes of CJD
sporadic (85-95% of cases)
familial (7-10%)
iatrogenic (1%)
symptoms of sCJD
spongiform encephalopathy
RAPID onset of Alzheimer’s type loss of brain function
death within a year of onset
irregular involuntary contraction of a muscle seen in sCJD
myoclonus
what causes prion diseases
infectious proteins
difference between PrPc and PrPsc
alpha helix => protein misfolding => beta sheet
sc=scrapie form, leads to prion disease
what does PrPc normally do
involved in maintaining the brain’s white matter, regulating innate immune cells, responses to oxidative stress, neuron formation
how does PrPsc form arise
from mutation or exogenous source (meat, blood)
PrP
prion protein
what type of protein is the PrP
membrane protein
what results from PrPc misfolding (clinical presentation)
encephalopathy
__ (#) different types of prions
four
how do you distinguish the different types of prions
Western blot ratio of banding patterns
what is the purpose of prion fragmentation
liberates new ends to allow for amplification
allows the dissemination of infectious material
gold standard for sCJD diagnosis
brain biopsy–look for spongiform vacuoles
what population is most at risk for sCJD
elderly
vast majority of sCJD cases occur in which race
white
characteristics of Variant CJD (vCJD)
type 4 prion
bovine-to-human transmission of BSE (muscle and milk DO NOT contain infectious BSE)
difference between vCJD and sCJD symptoms
vCJD progresses slower
vCJD peripheral pathogenesis (involvement of lymphoreticular tissues)
staining difference between vCJD and sCJD
vCJD: plaques
sCJD: diffuse
mean age at onset for vCJD
29
vCJD has a (long/short) incubation period
long 10-20 years
only way to sterilize against prions
auto claving
do prions contain genetic material
no
how were viruses discovered to be distinct from bacteria (experiment)
bacteria could be filtered but viruses passed through into the filtrate
why can viruses not be cultured
they are obligate intracellular parasites–can only replicate in cells
definition of a virus
capsid encoding organism
protein shell that encapsulates the nucleic acid genome
capsid
the particle encoded by a virus genome
virion
three gene modules of virus genome
1-capsid protein
2-replicon
3-host cell interacting factors
structural proteins encoded by the virus genome
capsid, some replication proteins
non structural proteins encoded by the virus genome
host interacting factors (they are not packaged into the virion)
virion structure
genome + capsid +/- envelope
what type of virions lack an envelope
naked virions
what is a virion envelope made of
lipid bilayer from the host cell
three ways of classifying viruses
host cell (eukaryotic or prokaryotic, plant/insect/animal) genome type (RNA or DNA, single or double stranded) virion structure (enveloped or naked, helical or icosahedral or complex)
virion component found in the center
nucleocapsid
virion component: gel in between the capsid and the envelope
tegument
virion component right under the envelope
matrix
virion component name for spikes embedded in the membrane
glycoprotein
virion component not embedded in the membrane but sticking out from it
spike or fiber
adenovirus appearance
icosahedral, contains fibers, DNA
poxvirus appearance
helical, DNA genome
flu virus structures
helical, glycoproteins, lipid bilayer, segmented RNA genome
HIV structure
icosahedral, glycoproteins, mRNA, morphs into a trapezoid capsid
limiting factor of virus lifestyle
Ro infectivity of virus
virus strategy for survival (three things)
1-capsid
2-genome
3-transmission to a new host
____ consist of a few genes encased in a protein shell that may be enveloped by a membrane
virions
what do viruses need in order to grow? (six things)
1-right host (tropism) 2-cells with the right receptors (susceptible) 3-appropriate environment (permissive) 4-biosynthesis machinery 5-abundant building blocks 6-time to finish replication
step of virus replication with interactions between virions and tissues
recognition (1)
step in virus replication with binding of a virion surface molecule to its specific cellular receptor
attachment (2)
step of virus replication with internalization of the virion into the cell
entry (3)
what happens to the virus when replication is incomplete
abortive infection
cells that a virus can enter
susceptible
cells that support virus replication and virion synthesis
permissive
preferred cell type for virus infection
tissue tropism
preferred species for virus infection
host range
virus entry method: engulfment of entire virion into cell via receptor-mediated endocytosis/pinocytosis/phagocytosis
penetration
viral entry into a cell when the virion envelope fuses with plasma membrane, leaving parts of the virion behind
fusion
step of viral replication when there is a release of the genome into the cell
uncoating
uncoating marks the beginning of the _____ phase, past which you cannot culture the virus anymore
eclipse
what factors regulate mRNA synthesis in the virus
viral and host transcription factors
viral mRNA’s are translated into what three types of proteins by the host machinery
ribosomes, tRNAs, amino acids
what do RNA viruses use to make mRNA and genomes
RNA dependent RNA Polymerase
what stage of viral replication marks the end of the eclipse phase
assembly of the virion
how the virion is assembled
first the capsid forms an empty shell
then viral DNA is inserted into the capsid
how to enveloped viruses acquire their membranes
from a cellular source–ER, Golgi, plasma membrane
how does a virus kill a cell
enters cell, duplicates itself
cell explodes to release the viruses
the number of infectious viral progeny from a single round of replication
burst size
name for virus growth curve
single step
phase of virus growth curve when virus particles are made and can infect other cells
maturation and release
cell lysis produces viral _____
plaques
how are infections virions measured
PFUs-plaque forming units
the entry of enveloped viruses into cells involves expression of ______ on the cell surface
specific receptors
how do enveloped viruses obtain their membranes
from a cellular compartment
virion envelopes are contructed from (viral/cell) glycoproteins and (viral/cell) membranes
viral glycoproteins
cell membranes
how do virus membrane proteins reach the site of envelopment
secretory pathways
MOI fragile X
X linked dominant with reduced penetrance (80% in males and 30% in females)
cause of FRAXA
expansion of a CGG triplet repeat
disease that affects pre mutation male in family with FRAXA
Fragile X related Tremor/ataxia
disease that affects pre mutation female in family with FRAXA
premature ovarian failure
when a larger PCR product which does not amplify as well as a shorter product is not detected in 35 cycles
allelic drop out
diseases that involve mutations in the alpha or beta globin chain genes which complex together to carry oxygen in the blood
hemoglobinopathies
_____ result when mutations at alpha or beta globin chain genes cause an imbalance in the quantity (rather than quality) of the two chains
Thalassemias
heterozygous sickle cell advantage
sickle cell trait, confers resistance to malaria
results from genes involving the beta globin genes that lead to a deficiency of beta globin
beta thalassemia
how are the alpha and beta globin gene clusters coordinated
locus control regions
what results in individuals with one normal alpha gene and one copy of Hb Constant Spring and two deleted alpha globin genes
deficiency leads to beta globin tetramer formation
number of CF disease causing mutations
1900
delta F508
most common CF mutation
HD is due to a (gain/loss) in function
gain
FRAXA is due to a (gain/loss) in function
loss due to expansion of CGG repeat
triplet repeats in FRAXA account for phenomenon of
anticipation
a normal person usually has _ beta globin genes
2-one each on chromosome 11
a Fragile X Normal Transmitting male has a risk of
developing FXTAS (greater than 1/3 risk) his daughters will be carriers who are at risk of having affected children
molecular mechanism of the triplet repeat expansion responsible for FRAXA
mutation leading to a deficiency via methylation and gene silencing
a slight difference on a southern blot for a male child with possible FRAXA shows
normal transmitting male–modest increase in copy number but no methylation
most common cause of inherited mental retardation
Fragile X
Fragile X arises from what type of mutations
dynamic
CF has (allelic/locus) heterogeneity
allelic
Fragile X presentations
MR, large ears and head, long face, large testicles in males
how do you detect the triplet expansion involved in Fragile X
PCR and/or southern blot
three classes of Fragile X mutations
normal, pre mutation, full mutation
FXTAS and POF occur through (gain/loss) of function
gain
how many bands does a Fragile X pre mutation carrying female have on a southern blot
four (as opposed to normal two)
what characterizes Fragile X full mutation on a southern blot
smear
on what chromosome are the alpha globin genes clustered
11
site of erythropoiesis in a fetus
spleen and liver
types of Hb in fetus
alpha and gamma
types of Hb in adult
alpha and beta
presentation of sickle cell disease
anemia, failure to thrive, splenomegaly, repeated infections, painful swelling of hands and feet
compensation used in beta thalassemia
fetal hemoglobin remains turned on (gamma)
most people have _ functional alpha globin genes
four
homozygous deletion of alpha globin gene leads to:
hydrops fetalis
mutation in a normal termination codon so that longer unstable alpha globin chain is produced
Hb Constant spring–instead of folding into globin, remains a chain and results in tetramers of beta
problem with beta tetramers (Hb)
do not let go of oxygen
a baby that tastes salty when kissed is a sign of
CF
4 ways for things to go wrong in CF
protein is absent, not processed properly, not regulated properly, not delivered
the variation in copy number is in some way related to stability such that the higher copy number the more likely to expand, thus the mutability of a product of mutation is different from that of its precursor
dynamic mutation
disease becomes more severe with every generation
dynamic mutation
single gene mutation birth defect with ocular defects, facial abnormalities
Axenfeld-Rieger Syndrome
2 specific gene mutations in Axenfeld-Rieger Syndrome that code for proteins that affect eye developent
PITX2, FOXC1
holoprosencephaly is caused by (a single gene mutation/multiple causes)
multiple factors (chromosomal abnormalities, environmental conditions such as maternal diabetes and alcohol intake, gene mutations)
genes that have been implicated in holoprosencephaly are part of the ___ pathway
SHH–important for development of forebrain (SHH, GLI2, PTCH)
where is the SHH expressed in the developing embryo
at Hensen node, the floor plate of the neural tube, the early gut endoderm, limb buds, throughout notocord
what is the first example of when we understood that an infectious agent in the mother would cause birth defects
congenital rubella (German measles) syndrome-cataracts in baby
how does hyperthermia (fever) during pregnancy affect baby
can interfere with neurulation and cause neural tube defects
first known case of a pharmacological agent that caused birth defects
thalidomide (a sedative used to treat nausea in pregnant women)
what did thalidomide cause in babies
limb malformation
the study of birth defects
teratology
agents that cause birth defects
teratogens
susceptibility depends on three factors:
1-development stage at time of exposure
2-dose and duration of exposure
3-genotype of embryo; gene-environment interactions
most sensitive period for inducing birth defects
weeks 3-8 during embryogenesis
leading cause of mental retardation
alcohol
most serious type of Fetal Alcohol Spectrum Disorders
Fetal Alcohol Syndrome FAS
presentation of FAS
structural defects, growth deficiency, intellectual disability
small head, narrow upper lip, low nose bridge, flat midface
most debilitating feature of FAS
defective brain development
what happens when pregnant moms take SSRIs
slight increase in congenital heart defects (but what would be the alternative)
most common birth defect (1% of all live births)
congenital heart defect
____ defect: a hole in the heart
septal
an opening in the wall (septum) that separate the left and right atria
atrial septal defect
a hole in the part of the septum that separates the ventricles
ventrictular septal defect
a condition where the heart is positioned on the right side of the thorax instead of the left
dextrocardia
how does dextrocardia occur
problem with left/right patterning (cilia> L/R signal > nodal > cascade > gene expression)
result of reversed nodal signaling cascade
situs inversus
result of correct nodal signaling cascade
situs solitus
result of bilateral nodal signaling cascade
heterotaxy
septal defects, double outlet right ventricle, and transposition of the great arteries all can result from:
heterotaxy
when the aorta rises from the right ventricle instead of the left
double outlet right ventricle
when the two main arteries carrying blood out of the heart (pulm and aorta) are switched
transposition of great arteries
detection of congenital heart defects
sometimes ultrasound
cyanosis, rapid breathing, fatigue, poor blood circulation
the ____ tract of the embryonic heart gives rise to the great arteries
outflow
a defect in which a single common blood vessel comes out of the heart instead of pulm and aorta
truncus arteriosus (in 22q11.2 deletion syndrome)
four heart defects of tetralogy of Fallot in 22q11.2 deletion syndrome
1-ventricular septal defect
2-pulmonary stenosis
3-overriding aorta (enlarged)
4-ventricular hypertrophy (heart trying to compensate)
___ cells migrating from the dorsal neural tube into the arterial pole participate in separation of the outflow tract
cardiac neural crest cells
how does the 22q11.2 deletion affect cardiac neural crest cell development
genes located in that deletion region code for proteins that regulate cardiac neural crest cell development
when does the neural tube close (by which week of pregnancy)
by the 6th week (so not at risk of not closing if you get a fever after the 6th week)
functions of proteins are dependent on what two factors
polymer length and aa composition, which specify 3D structure
the content of proteins within the cell at any given time
proteome
critical residues responsible for nucleophilic attack on the substrate in HIV protease
Asp26
similar in structure to a natural peptide substrate of the HIV protease, but non hydrolyzable
tipranivir anti HIV drug
disease characterized by extensive deposits of misfolded protein (amyloid fibers) in the brain
AD
beta sheet phi and psi values
close to 180 degrees
alpha helix phi and psi values
close to 0 degrees
dark areas on a Ramachandran map mean
that these phi and psi values are allowed, all structures must satisfy this plot
lots of black area on a Ramachandran map means that this protein is
flexible
most flexible aa residue
glycine
pKa of Asp
4.0
pKa of Glu
4.0
pKa of His
6.5
pKa of Cys
8.5
pKa of Lys
10.0
pKa of Arg
12.0
pKa of carboxy terminus
4.0
pKa of amino terminus
8.0
(high/low) pKa binds H+ tightly
high
(high/low) pKa binds H+ weakly
low
pH at which half the ionizing groups are protonated, half are deprotonated
pKa
when the pHs want to (retain/give up) protons
retain, lots of H+ in solution forces H+ onto ionizing group
when the pH>pKa, aa’s want to (retain/give up) protons
give up, low bulk of H+ in solution draws off the H+
why is His used as a catalytic residue by enzymes engaging in acid-base catalysis
pKa is 6.5 so it can accept or donate protons at physiological pH
how does Cys introduce crosslinks
remove a hydride from SH group, make disulfide links
what type of bonding forces determine primary protein structure
covalent
what type of bonding forces determine secondary, tertiary, and quaternary structure
non covalent
function of Glu in ATP synthase Fo ring
proton binds the Glu and rides it like a merry go round
function of Pro in ATP synthase Fo ring
kink
what limits phi and psi angles
steric clashes
how do proteins fold so as to maximize vdw’s forces?
almost no empty space between atoms
what force contributes to protein structure more than any other force
hydrophobic effect
origin of hydrophobic effect
formation of clathrates by water is energetically unfavorable
what secondary structure of proteins expresses a peptide group H bonding pattern with the CO of residue i bonded to NH of residue i+4
alpha helix
three ways that the alpha helix is stabilized
backbone-backbone interactions
backbone- side chain interactions
side chain-side chain interactions (i, i+4)
aa that prefers to form an alpha helix
Ala
aa’s that prefer to break alpha helices
Pro (lacks NH to hydrogen bond), Gly (flexible)
characteristics and examples of aa’s that are medium level helix breakers
bulky or Beta branched
Val, Thr, Trp, Phe
lose much rotational freedom when in a helix
helix indifferent aa’s
long, straight chains
Arg, Lys, Glu
amphipathic alpha helix side chain arrangement
every fourth is hydrophilic, on the outside
every fourth is hydrophobic, on the inside
aa used to relieve steric clashes at beta turns
gly
aa used to make a kink at beta turns
pro
what is random about loops and random coils
not periodic
where are random coils found
usually at protein surface, hydrophilic residues
short stretches of secondary structure that usually require additional structures for stability
motifs
continuous stretches of polypeptide that are linked together by flexible loops, stable in isolation after the rest of the protein has been removed
domain
what holds coiled-coil domains together
hydrophobic effect, knobs into holes packing, electrostatic interactions
what forms the leucine zipper
C terminal half of GCN4 transcription factor is a classical heptad Leu/Val repeat every 4th position
a protein domain that only folds when it binds its target ligand
intrinsically disordered protein domain
