W2 Flashcards
(136 cards)
1
Q
- What are the 4 stages of mitosis, describe each.
2. conversion of 2n content of DNA into?
A
- Prophase (DNA has been replicated and the duplicated chromosomes are arranged as sister chromatids, attached at the centromere)
- Metaphase (the sister chromatids line up in preparation for cell division)
- Anaphase (sister chromatids separate)
- Telophase (cell division)
2n content of DNA –>4n preparation for cell division–>2 daughter cells (2n)
2
Q
Meiosis generate what type of cells?
How many divisions?
Describe the chromosome/sister chromatids in the process.
A
- generate haploid cells from diploid cells –
- two cell divisions are required.
- After the first division, the sister chromatids are still attached, as opposed to separated as in mitosis.
- The second cell division then separates the identical chromosomes.
3
Q
Describe Independent Assortment of Chromosomes
A
As chromosomes migrate to daughter cells during meiotic division, they do so independently of other chromosomes (otherwise, Mendelian genetics would not work)
independent assortment of nonhomologous chromosome during meiotic division I
4
Q
Crossing over in meiosis happens when? What type of chromosome is involved? The ares of crossing over is called? How many cross overs during division and per chromosome? What does this link to?
A
- prophase I of meiosis (synapsis)
- pairs of sister chromatids crossover of genetic material can occur btw chromatids
- chiasma
- 30-40 (1-2)
- genetic linkage
5
Q
Definition of:
- Metacentric
- submetacentric
- acrocentric
- telomere
- chromosome arms
A
- centromere center
- centromere non-centere
- stalk
- end of chromosome
p=short; q= long
6
Q
banding patterns of chromosomes
- two types of treatments, describe
- What allows further subdivision into what?
- Other banding techniques? (5)
A
- enzymatic tx with trypsin & stain chromosomes
- G-banding (Giemsa)–>light band=euchromatin, transcriptionally active; dark abnd= heterochromatin - Giemsa binds better to AT rich region–>subdivsion of regions & sub-region (14q21)
- Q-banding (fluorescent)
R-banding (reverse banding from Giemsa, need to heat DNA)
C-banding (highlights centromeres; remove proteins)
Spectral karyotypes (either chromosome specific, or analyze different areas of the same chromosome)
FISH – fluorescent in situ hybridization
7
Q
Chromosome & karyotype nomenclature Meaning of & example relating to chromosome 1. 1-22 2. X, Y 3. p 4. q 5. del 7. der 8. dup 9. ins 10. inv 11. / 12. t 13. ter 14. r 15. +/-
A
- autosome number
- sex chromosomes
- short arm of chrome, petit
- long arm
- deletion of chromosome material
- derivative, structurally rearranged chrom
- duplication of parts of chrom
- insertion of DNA into
- inversion of DNA within
- indicate mosaicism (2 different cell types within one individual)
- translocation (region moved are described after t symbol)
- terminal (or pter, qter)
- ring chrom
- placed before chromo #, addiitons/deletion of whole chromo
8
Q
Explain Karyotype
- 46, XY
- 47, XX, +21
- 47, XY, +21/46, XY
- 46, XY, del(4)(p14)
- 46, XX, dup(5p)
- 46, XX, inv(3)(p21;q13)
- 46, X, r(X)
A
- normal male chromosome constitution
- female with trisomy 21
- male c mosaic of trisomy 21 cells and normal cells
- male with deletion at chromosome 4 at short arm designated band 14
- woman with duplication of short arm at chromosome 5
- female with inversion on chromosome 3 between band 21 on short arm and band 13 on long arm
- female with one normal X chromosome and one ring X chromosome
9
Q
Definition of:
- euploid
- diploid
- triploidy
- aneuploid
- monosomies
- trisomies - consequences of these abnormality?
A
- human with multiple of 23 chromosomes
- 46 chromosomes
- 69 chromosomes
- does not have a multiple of 23 chromosomes
- 1 copy of a chromosome always lethal if autosomal
- a limited number allow survival - spontaneous abortion due to too many expressed genes in polyploidy, on autosomes, is detrimental to development
10
Q
What causes polyploidy?
Result from what during maternal meisos II?
Common forms? (3)
what are dignynic & diandric
A
- fertilization of same ovum by 2 or more sperm
- errors in maternal meiosis II –>egg with diploid # of chromosomes–>fertilized by a sperm with haploid # chrome.
- 69, XXX+69, XXY
- digynic=46 chrom from mom
- diandric= 46 chrom from dad (65%-75%)
11
Q
What causes aneuploidy?
- prevalance
- common causes
- related to
A
1 in 300 newborn infants are aneuploid - non-disjunction -More common in eggs > sperm -trisomies due to errors in maternal meiosis I egg suspended in meiosis II -maternal age
12
Q
Describe non-disjunction in meiotic & mitotic division
A
nondysjunction in Meosis I -chromosome are different in the gametes nondisjunction Meiosis II -chromosome are identical gametes -both lead to trisomic & monosomic zygote
mitotic –>mosaicism
- one of the cells has no disjunction event during mitosis
- will have mixture of normal and trisomic cells, monosomic usually die off
- all daughter cells with trisomy
- single cells could live with trisomy
- common in tumors
13
Q
Major Chromosomal Aneuploidy Syndromes Compatible with Live Births
- what is the chromosomal abnormality?
- clinical features?
- why does chrome 13, 18, 21 trisomies occur?
1. patau’s syndrome
2. edward’s syndrome
3. down syndrome
4. turner syndrome
5. klinefeltter’s syndrome
6. Triple X
7. XYY
A
- trisomy 13
- cleft lip & palate, several CNS anomaly, polydactyly - trisomy 18
- low birth wt, CNS anomalies, heart defects - trisomy 21
- hypotonia, characteristic facial features, developmental delays - monosomy X
- short stature, amenorrhea, lack secondary sexual development - XXY
- small testes, infertility, tall stature, learning problems - XXX
- learning disabilities, no major physical anomalies - XYY
- learning & behavior problems
13, 18, 21 are small chrom. with large sections of heterochromatin (inactive)
- 21 smallest mildest symptoms
- 13 & 18 lead to early death
14
Q
Structural Abnormalities in Chromosomes describe: 1. pericentric 2. paracentric 3. duplication 4. interstitial & terminal deletions 5. which are inversion vs.insertion vs. duplication? 6. what has genetic lose and which does not?
A
- 2.
3.
4.interstitial= middle; terminal= end - pericentric & paracentric= inversion
insertion of one part into another chrom. or duplicated from original and insert into original - deletion, isochromosome, microdeletion= genetic loss
- no genetic loss= inversion, reciprocal translocation
15
Q
What is isochromosomes?
What does it lead to?
A
- abnormal centromere division
- result in either duplication of p arm or deletion of q arm (iso-p)
- or duplication of q arm & deletion of p arm (iso-q)
- loss of genetic material
16
Q
Compare & Contrast :
Reciprocal vs. Robertsonian translocation
A
Reciprocal
- No loss of genetic material
- Carrier usually phenotypically normal; problems with gametes
- The resulting chromosomes are called derivative chromosomes
Robertsonian
- occur between acrocentric (centromere close to the end) chromosomes (13, 14, 15, 21, 22).
- short arms lost, one long arm fused generated.
- genes lost from short arms are rRNA genes, which are duplicated elsewhere in the chromosome
- carriers are phenotypically normal.
- individuals will have a karyotype of 45 chromosomes.
- problems with gamete formation.
17
Q
Gametes in reciprocal vs. robertsonian translocation
A
- both have 1/3 for normal function
- the unbalanced monosomy & trisomy gametes not viable for live
18
Q
What is microdeletion syndromes?
what is lost?
how is it detected?
example?
A
- Disorders due to a small (< 5 megabases) chromosomal deletion; very complex phenotypes observed
- Multiple genes lost via the deletion–>monosomy for a number of genes
- deletions can only be detected using specific probes to the missing region (FISH)
- Angelman and Prader Willi syndromes are example (15q11)
19
Q
Indication for prenatal diagnosis include:
Methods to obtain fetal cells for cytogenetic study include
A
- advanced maternal age (>35)
- Previous child with a chromosomal abnormality
- Family history of chromosomal abnormalities
- Abnormal prenatal screening study
Amniocentesis
Chorionic Villus sampling
Cordocentesis
20
Q
amniocentesis
when is it perfumed?
risk of what?
how is it performed?
what is being studied?
A
- Gold Standard
- 0.5% risk of procedure-induced pregnancy loss
- Perform at 15-16 weeks gestation
- Amniotic fluid is obtained using ultrasound to guide the placement of the needle in the uterus
- Remove amniotic cells, culture in lab, do cytogenetics study
21
Q
chorionic villus sampling
when is it performed?
risk?
how is it done?
A
- Performed at 10-12 weeks gestation
- Has a 0.5% risk of procedure induced pregnancy loss
- Removed cells are from the placenta (chorion), although they are contaminated with maternal cells. 4. Ultrasound is used to guide needle placement. A separation procedure is required to remove maternal cells from the sample
- Cells are grown in the lab and analyzed
22
Q
Cordocentesis what is it? when is it indicative to use? how is it done? at what gestation? risk? advantage?
A
- Umbilical blood sampling; used if inconclusive results are obtained by other procedures
- Ultrasound guides needle into the umbilical artery (obtain blood instead of amniotic fluid)
- Performed after 18 weeks of gestation
- Has a high rate of fetal loss: 1-2%
- Advantage is less time is required to obtain results (blood cells grow more rapidly)
23
Q
trancriptional initiation & elongation steps (5)
A
- binding of DNA pol
- separate DNA
- binding of 1 st nuc by base paring
- binding of 2nd nuc & 1st: PPP remains at 5’ end and PPi splits from 2nd nuc
24
Q
give a general view of a gene from 5’–>3’
-4 important structures
A
5’–>3’
- regulatory sequence
- promoter
- start point for transcription
- coding region of gene
25
Promoters--the business end of a gene
1. activator & repressors proteins are know as? and what role does it play?
2. what are the two promoter consensus sequence?
1. transcriptional factors, bind at particular sequence with high affinity that affects transcriptional efficiency
2. TTGACA upstream near -30, TATAAT downstream near -10
26
coding strand vs. template strand
1. discuss it's characteristics and complementary
2. which strand is read by RNA polymerase?
1. coding strand has the identical sequence informarion to the mRNA
- template strand is the one actually read by RNA polymerase which synthesize a complementary mRNA identical in sequence to the coding strand
27
Prok promoter recognition involve what protein?
what is this protein bound to?
what transcribe all prok genes?
how many ways can pok terminate transcription?
1. alpha protein which only recognizes promoter sequence when it is bound within the holoenzyme
2. RNA pol is a multi-subunit enzyme that transcribes all pork genes
3. two ways for termination: Rho-dependent or Rho-independent
28
Compare & contrast
| Rho dependent vs. Independent termination of transcription
Rho-dependent
1. helicase protein called rho inds RNA-DNA duplex downstream of translation STOP signal (other signal obscure by ribosomes)
2. denatures the RNA=DNA duplex using its ATP-dependent unwinding activity
Rho-independent
1. termination does not involve other secondary proteins
2. specigic G_C rich sequence signal downstream of translation STOP signal causes RNA pol to stall out over an immediately following A/T rich sequence
- DNA duplex is re-established via annealing, expel RNA from transcription bubble
29
```
Properties of EUK RNa polymerase
I, II, III
1. localization
2. cellular transcripts
3. effect of alpha amanitin
```
function of:
1. rRNA
2. miRNA
3. tRNA
4. How many RNA pol are in prok. ?
Euk:
pol I:
- nucleolus
- 18S, 5.8S, 28S, rRNA
- insensitive
pol II
- nucleoplasm
- mRNA precursors, non-coding RNAs (miRNA) precursors
- strongly inhibited
pol III
- nucleoplasm
- tRNA, 5S rRNA
- inhibted by high concentration only
1. important in translation
2. ...in gene regulation
3. --- in translation
4. PROK.: all RNA species are transcibed by 1 single RNA pol.
30
compare & contrast Prok vs. Euk cells
1. nucleus
2. chromosome
3. membrane bound organelles
4. cell wall
5. plasma membrane
6. ribosome
7. mitochondria for ox phos? translation/transcription? endocytosis?
Prok:
1. no nuclear envelope
2. single (DNA supercoiled), circular, plasmid
3. none
4. peptidoglycan cell wall, usually have cell wall
5. no carbohydrate, most lack sterols
6. 70S
7. no mitochondria for ox phis--but in cytoplasmic membrane
- translation & transcription coupled
- no receptor mediated endocytosis
EUK:
1. double membrane nuclear envelope
2. multiple
3. yes
4. cell wall in fungi & plants, no petidoglycan
5. sterols & carbohydrates
6. 80S
7. ox phos in mitochondria, transcription in nucleus, protein synthesis in cytoplasm
31
4 ways bacteria adapt
1. streamlined gene expression
2. diverse metabolism
3. rapid growth
4. high numbers
(3&4)= spontaneous mutation rate 10^-6
selection for resistance
32
1. three types of vectors
| 2. 4 mechanisms of genetic exchange in bacteria
1. prok genomes, bacteriophages, plasmids, transposons
| 2. transformation, transduction, conjugation, transposition
33
```
what is a bacteriophage?
Describe the bacteriophage:
-morphology,
-genomes, and l
-ife cycles
```
1. is a virus that infects bacteria
2. icosahedral, filamentous,
3. DNA or RNA, double or single stranded
4. virus is genetic material package into virions require host cell for replication
34
describe the lytic & lysogenic cycle
lytic
1. phage DNA attached to bacteria
2. inject phage DNA into cytoplasm
3. phage DNA direct synthesis of many new phages
4. cell lyse and release new phages
5. new phage can bind o bacterial cells
lysogenic
1. phage DNA attached to bacteria
2. inject phage DNA into cytoplasm
3. phage DNA integrate into host chromosome
4. prophage DNA is copied when cell divides
5. exposure to stress such as UV light triggers excision from host chromosome
35
what is a prophage?
where do you see it, cycle?
what's episome?
1. is a phage DNA integration into host chromosome
2. found in lysogenic cycle
3. plasmid integrate into chromosome (F+ factor)
- replication of integrated episome is synchronous with host chromosome
36
bacterial chromosome vs. plasmid
bact chromo.
- circule double stranded DNA
- 3000 gene
- one copy per cell
- highly folded
plasmid
- ciruclar double stranded DNA
- 5-100 genes
- 1-20 copies per cell
37
what is a transposon found under what condition?
why?
what is the consequences of transposition?
1. transposon are never free, they are either found on plasmid or chromosome
2. they do not have origin of replication
3.
a. inactivate genes at insertion site
b. move around gnes necessary for function
c. introduce drug resistance gene
38
describe transformation
1. DNA fragment insert into bacteria
2. uptake of DNA into bact. chromosome
3. integration by nonreciprocal recombination
or degradation unsuccessful transformation
ie. transformation of plasmid used in rDNA technology
39
what does transduction requires?
phage
40
Describe conjugation
-what is HFR (high frequency of recombination?
types of plasmids
1. plasmid in once prok transfer over via secretion bridge to another prok cell (filli transfer DNA material)
2. the receiver cell recirculate plasmid and recombined fragment
plasmids: F+ factor, RTF (resistance transfer facor),-->episome
HRF- transfer alot of host chromosome.
41
3 major things in cytosol of prok.
1. generate ATP, RNA/DNA, cell wall synthesis
2. ribosomes throughout cytoplasms 70S
3. DNA supercoiled
42
4 possible things related to the envelope layers of prok.
| -what is the difference between the envelope of gram - and + bacteria?
1. cytoplasmic membrane-->phospholipid bilayer + proteins
2. cell wall
3. capsule
4. outer membrane found in gram - bacteria
43
cytoplasmic membrane of pork. composed of?
| -7 fucntions of it
- phospholipids & proteins
1. transport nutrients
2. regulation of gene expression
3. energy generation
4. peptidoglycan synthesis & turnover
5. secretion
6. cell divison
7. sensor mechanisms
44
functions (5) of cell wall peptidoglycan
1. unique to eubacteria
2. protect cytoplasmic membrane from osmotic stress environment
3. define shape
4. cell division
5. can cause symptoms in animals (induce inflammation, cytokine release, sepsis)
45
what are cell wall made of?
how does this affect staining?
possible morphology
compare & contrast peptidoglycan btw gram - & +
1. peptidoglycan cell wall=murein= highly crosslinked mesh of peptides & polysaccharides
-NAM & NAG
2. gram + retain crystal violet color stain due to outer membrane layer
gram - has a counter stain red color
3. they can be gram -/+ rods, gram -/+ coccus
4.
Gram + cell envelope
-lipoteichoic acid & teichoic acid
-thicker peptidoglycan layer
-no outer membrane
-no LPS endotoxin activity
-has a lysine-5 gly-NH2+ bridge
-D-ala & L-glu amino acid
-stain crystal violet
-NAM & NAG
```
Gram - cell envelope
-no lipoteichoic & teichoic acid
-thinner peptidoglycan layer
-outer membrane present
-has LPS endotoxin
-no bridge
-has DAP (diaminopemelic acid)
-D-alu, L-glu amino acids
-counter stain red
-NAM & NAG
-
```
46
action of beta-lactam antibiotic
| what is a penicillin binding protein?
1. precursors crosslinked by penicellin binding protein (PBP)-->precurosrs to cell wall
2. b-lactam enters cell wall bind PBP
3. binding lead to autolysine-->break down preformed cell wall
4. b-lactam PBP cannot make cell wall
5. cell wall lose integrity-->osmotic stress will effect it
6. PBP=transpeptidase
47
pharmacologica activity of LPS from Gram -
| describe the pathway that lead to multi-organ system failure
-endotoxin is not directly responsible for effect of LPS pharmacologic activity
MOSF
1. release of endotoxin-->release acute phase cytokines (IL1, 6, TFN-a, platelet activating factor)
2. low conc, can be protective (fever, vasodilation, immune & inflammatory response
3. high level-->shock & death
4. can activate alternative complement pathway, promote high fever, hypotension, shock (vasodilation & capillary leakage), DIC due to activation of blood coagulation pathways
5. MOSF
48
compare & contrast gram +/- by each:
1. cell wall (peptidoglycan)
2. outer membrane
3. LPS
4. endotoxin lipid A
5. teichoic acid
6. lysozyme
7. antibacterial activity of penicillin
8. capsule
9. exotoxin production
Gram+ vs. Gram
1. thicker/thinner
2. none/yes
3. n/y
4. n/y
5. often/absent
6. sensitive/resitant
7. more susceptible/more resistant
8. sometimes/sometimes
9. some strain/some strains
49
phenotypic variation arises from?
which is more frequent? why?
what change occurs?
1. genotypic variation called polymorphism
2. Variation in DNA sequence in greater magnitude in frequency than variation in protein sequence, which polymorphism are responsible for many phenotypes. More DNA polymorphism outside the protein coding sequences
3. IN both DNA & protein polymorphism, it occurs when there is a change in the base pair sequence
49
phenotypic variation arises from?
which is more frequent? why?
what change occurs?
1. genotypic variation called polymorphism
2. Variation in DNA sequence in greater magnitude in frequency than variation in protein sequence, which polymorphism are responsible for many phenotypes. More DNA polymorphism outside the protein coding sequences
3. IN both DNA & protein polymorphism, it occurs when there is a change in the base pair sequence
50
5 types of DNA polymorphisms summary
1. RFLP- restriction fragment length polymorphism
2. SNP- single nucleotide polymorphism. most common
3. VNTR- variable number of tandem repeats, mini satellite
4. STR- short tandem repeats (Microsatellites)
5. CNV- copy number variation
basic principle for detection of DNA variability is differences in size or sequences of fragments
50
genetic drift
substantial departure of observed allele frequency from expected frequency from generation to generation due to small population size
50
gene flow
change of allele frquencies due to mixture and migration of populations
50
what is the amount of variation of polymorphism btw two individuals?
most variation in human traits is due to DNA polymorphism. 1 million polymorphic differences btw 2 people
1/3000 base pairs
50
Mendelian inheritance:
| Autosomal recessive
- traits usually appears equally in males & females
- and tend to skip generations
- autosomal recessive traits are more likely to appear among progeny of related parents (?)
50
```
Thalassemia is what type of mendelian?
what causes it?
what are the common outcomes?
How is it different than sickle cells?
what does it result, physiologically?
```
1. it is an example of gene expression-linked disease
2. alpha & beta thalassemias are classic recessive genetic diseases but their variety of origins -->mutations in many completely different mechanistic aspects of gene expression can lead to similar outcomes-->thalassemias are complex set of syndromes variety causatives
3. a reduction in normal amount or even complete elimination, of the relevant glob in protein in RBCs
4. sickle cell--> mutant form of beta glob in protein leads to alteration in its biochem activity
5. loss of overall oxygen carrying capacity is an obvious consequence of reduced levels or absences of proteins. Less obvious is that remaining glob in protein can aggregate abnormally-->cell destruction
50
list the four ways to detect DNA polymorphism
1. differences in RFLP
2. in number of small repetitive sequences
3. number of large repetitive sequences
4. single nucleotides
50
How is tandem repeat polymorphism created?
| how is it detected?
1. bands of differing lengths are created by different numbers of tandem repeats in DNA on two copies of chromosome. After amplification (PCR), labeling of the region that contain the polymorphism, different fragment lengths separated by electrophoresis-->autoradiogram
2. detected by differences in molecular weight
50
What can PCR amplifies?
what does PCR allow use to visualize and automate?
With automated sequencing, what can we do with it leading to the foundation of what?
1. PCR is used to amplify alleles at specific loci
2. DNA sequencing of PCR amplified alleles allows us to visualize and automate SNP, CNV, VNTR
3. automated sequencing allows us to build whole genome databases for individuals-->core of personalized medicine
50
describe the process of detecting SNP using what technique?
Microaaray
1. oligonucleotides placed or synthesized on chip
2. exposed to labeled DNA from subject
3. hybridization occurs only if oligonucleotide contains a DNA sequence that is complementary to that of the subject's DNA
4. fluorescent label marks the location of the complementary oligonucleotides sequence on chip
50
founder effect
subset of genetic drift (small founding population) and some sort of subsequent natural selection (heterozygote advantage)
50
Humans have how many genes?
how many encodes genes?
how many bp differences btw 2 individual?
how many base pair in 1 polymorphism?
1. 3 billions
2. 30,0000
3. 1 million
4. 3000 base pairs
51
Mendelian inheritance:
| X-linked dominant
- x-linked dominant traits do not skip generations
- affected males pass the trait on to all their daughters and none of their sons
- affected females (if heterozygous) pass the trait on to about half their sons and about half of their daughter
- affects both males and females
- an affected male must have an affected mother
51
Mendelian inheritance:
| X-linked recessive
-an affected males does not pass trait to his sons
-allele to a daughter who is unaffected
-and passes it to sons who are
x-linked recessive traits appear more frquently in males
-more common in males than females
-are not passed from father to son
51
Mendelian inheritance:
| X-linked dominant
- x-linked dominant traits do not skip generations
- affected males pass the trait on to all their daughters and none of their sons
- affected females (if heterozygous) pass the trait on to about half their sons and about half of their daughter
- affects both males and females
- an affected male must have an affected mother
52
what are 3 non-traditioanl modes of inheritance?
1. mitochondrial inheritance
2. anticipation and repeat expansion
3. genomic imprinting
52
Mendelian inheritance:
| Y-linked
- traits appears only in males
| - all male offspring of an affected male are affected
52
what are 3 non-traditioanl modes of inheritance?
1. mitochondrial inheritance
2. anticipation and repeat expansion
3. genomic imprinting
53
2 additional factors that affect phenotypes in Mendelian traits?
define each
1. penetrance- % of individuals having particular genotype that express the expected phenotype
2. expressivity- the degree to which a character is expressed
53
2 additional factors that affect phenotypes in Mendelian traits?
define each
1. penetrance- % of individuals having particular genotype that express the expected phenotype
2. expressivity- the degree to which a character is expressed
54
Mitochondrial maternal effect
- cytoplasmically inherited characteristics frequently exhibit extensive phenotypic variation because cells and individual offspring contain various proportions of cytoplasmic genes
- the random segregation of mitochondria and cell division and replication results in progeny cells that differ in their number of mitochondria with wild type and mutated genes
55
what is genetic anticipation? when is it expressed? what is caused by?
genetic anticipation-- is a genetic trait that becomes more strongly expressed or is expressed at an earlier stage as it is passed from generation to generation
-caused by expansion of triplet nucleotides in specific genes
56
describe amiocentesis procedure.
when is it performed in pregnancy?
what is it used for?
1procedure:
1. using ultrasound, sterile needle inserted through abdominal wall into amniotic sac
2. small amount of amniotic fluid is withdrawn through needle
3. fluid contain fetal cells which are separated from amniotic fluid
4. cultured the cell
5. chemical analysis, DNA analysis, chromosomal analysis
2. performed at 15-18 weeks
3. genetic testing
57
compare & contrast B^0 and B+ symptoms
B^0= have RBC which are small, pale (hypochronmic), frequently misshapen
B-0 thalaseemia major=Cooley's anemia
1. hemolytic anemia within 1 yr of birth
2. bone deformities in the face/enlarged spleen
3. fatigue
4. growth failure
5. shortness of breath
6. yellow skin (jaundice)
B+ thalaseemia minor symptoms:
1. no obvious symptoms unless living at high altitude or engaging in extreme exertion
58
describe chorinic villus (CVS) procedure
when is it performed?
what is it used for?
procedure
1. CVS can be done early pregnancy
2. ultrasound, catheter is inserted through vagina and cervix and into uterus
3. contact with chorion, the outer layer of placenta
4. suction removes small piece of chorion
5. cells of chorion used directly for many genetic tests, and culturing is not required
6. chemical, DNA, chromosomal analysis
2. 10-12 weeks
3. genetic testing
59
lists 11 genetic diseases and disorders that can be detected prenatally
1. chromosome abnormalities---
2. cleft lip & palate--
3. cystic fibrosis-
4. dwarfism---
5. hemophilia---
6. Lesch-Nyhan syndrome---
7. neural tube defects-
8. osteogenesis imperfecta (brittle bone)
9. phenylketonuria
10. sickle cell anemia
11. Tay-Sachs disease
60
Thalassemia is what type of mendelian?
what causes it?
what are the common outcomes?
what does it result, physiologically?
1. it is an example of gene expression-linked disease
2. alpha & beta thalassemias are classic recessive genetic diseases but their variety of origins -->mutations in many completely different mechanistic aspects of gene expression can lead to similar outcomes-->thalassemias are complex set of syndromes variety causatives
3. a reduction in normal amount or even compelte elimination, of the relevant glob in protein in RBCs
61
Humans have how many genes?
how many encodes genes?
how many bp differences btw 2 individual?
1. 3 billions
2. 30,0000
3. 1 million
62
compare & contrast B^0 and B+ symptoms
Both have RBC which are small, pale (hypochronmic), frequently misshapen
B-0 thalaseemia major=Cooley's anemia
1. hemolytic anemia within 1 yr of birth
2. bone deformities in the face/enlarged spleen
3. fatigue
4. growth failure
5. shortness of breath
6. yellow skin (jaundice)
B+ symptoms:
1. no obvious symptoms unless living at high altitude or engaging in extreme exertion
63
describe the 5' cap structure and process in Euk mRNA
2 reasons why capping is important
1. specific capping enzyme adds GTP to 5' end of mRNA in unique 5'==>5' triphosphate linkage
2. methylases modify the nucleotides post-transcriptionally at the 2-OH and N-7
3. capping occurs very soon after RNA pol II has initiated transcription
4. capping stabalizes mRNA agianst nuclease degradation from the 5' end
- essential for EUK mRNA translation initiation
64
EUK mRNA terminates via 3' polyadenlyation. Describe
2 importances of polyadenylation
1. pre-mRNA cleavage site situated just a few nucleotides 3' of AAUAAA polyadenylation recognition signal
2. non-templated chain of 100-200 A nucleotides is added to 3'OH end by poly A polymerase & ATP
3. polyadenylation stabalizes mRNA against nucleases degradation from 3'end an dplay a role in translation initiation
65
what are exons and introns?
-exons contain protein coding information that must be spliced away from introns -->maure mRNA
-introns are non coding, interupts genetic information they are spliced out from the mRNA
introns contain both variant & consensus sequences that are required for their removeal from primary transcript (40 nuc-thousandsth nts)
83
four characteristics of introns
1. all 5' splice junctions contain an invariant GU at 5' end intron
2. all 3' splice junctions contain an invariant AG 3'end of intron
3. each invariant splice junction is surrounded by short consensus sequences that contribute to the efficiency of splicing
4. an adenine (A) base 30 nt upstream splice site in essential role
84
The slicing process is catalyzed by SNURPS
1. what are they?
2. iinteraction with mRNA?
3. function?
1. snRNPs (U1, U2) are small nuclear ribonucleoproteins that consist of specific proteins bound to particular short RNA species ( snRNAs)
2. snRNAPs are attracted to sites of interaction on precursor mRNA by complementary bp btw their snRNA component and short stretches of local mRNA sequence
3. Snurps help forld the precursor mRNA into appropriate structure and catalyze the breaking and reformation of 5'3' phosphodiester linkages
- the essential A base forms an intron lariat via 2'3' nad 5' phosphodiester linkages
85
Suffixes indicate what class of drug it belongs in:
1. -afil
2. -ane
3. -aptan
4. azide
5. -caine
6. -mab
7. -mycin or -micin
8. -nib
9. -olol
10. -osin or -ocin
11. -pril
12. -sartan
13. -statin
14. -ase
15. -kin
16. -stim
17. -nib
18. -mab
19. -omab
20. -ximab
21. -zumab
22. -umab
1. cGMP phosphodiesterase 5 inhibitor
2. inhaled halogenated anesthetic
3. antagonist to ADH
4. thiazide diurectic
5. local anesthetic
6. monoclonal antibody
7. antibiotic
8. a protein tyrosine kinase inhibitor
9. beta adrenergic receptor antagonist
10. alpha 1 adrenergic receptor antagonist
11. ACE inhibitor (ACEI)
12. AT1 receptor antagonist (ARB or ARA)
13. HMG-COA reductase inhibitor
14. an enzyme
15. an interleukin
16. a colony stimulating factor
17. a protein tyrosine kinase inhibitor
18. a monoclonal antibody (AMB)
19. a mouse MAB (not commonly used in humans therapeutically)
20. a chimeric (mouse/human) MAB human constant region + mouse variable immunoglobulin regions
21. a humanized MAB (has mouse CDR regions)
22. a fully human antibody
86
what are the four key pathways to cancers(which requires multiple pathways)
1. cell growth
2. differentiation
3. DNA repair
4. genome integrity
87
Which is more common: germline mutation or soamtic mutation?
germ line is 5%
| somatic mutaiton 95%
88
Key cancer gene paradigms include the following, what are they used for?
1. NF1
2. TP53
4. APC
5. HNPCC
6. BRCA 1 & 2
7. CDKN2A
8. RET proto-oncogene
1. cell signaling, neurofibromatosis
2. genomic integrity, lung and other cancers
3. cell signaling, colon cancer
4. DNA repair (mismatched repair), colon CA
5. DNA repair, breast cancer
6. cell cycle control, familial melanoma
7. receptor tyrosine kinase, multiple endocrine neoplasia
89
oncogenes are? example?
| how are they activated and the result of it?
1. oncogenes are dominant cancer genes that causes cancer.
- when activated by mutation results in overwhelming the normal controls on cell growth/differentiation. ONly ONE copy needs to be activated (dominant mechanism)
- growth factor receptor tyrosne kinase receptors
2. proto-oncogenes-->
a. deletion or point mutation in coding sequence-->hyperactive pro made in normal amount
b. gene amplification-->normal protein greatly overproduced
c. chromosome rearrangement-->
nearby regulatory DNA sequence causes normal protein to be overproduced or fusion to actively transcribed gene greatly overproduces fusion protein; or fusion protein is hyperactive
90
what is tumor suppressor gene?
-one whose gene product is required for normalcy (recessive mechanism). loss of the gene product by mutation of BOTH alleles (Knudson two hit model) is required for tumor formation.
first mutation may be inherited or somatic
second mutation will often be gross event leading to loss of heterozygosity in the surrounding area
91
what is an operator?
| describe the operon components in Prok
- is a DNA sequence that binds the repressor
- coupled transcription & translation
- transcriptional control sequences (promoter) + genes
- single promoter for all protein products= coordinated expression
92
How is transcription initiation control in Prok?
| what is a repressor?
- repressor when bound by inducer-->loses affinity for operator sequence via conforamtional change
- repressor has it own promoter and encoded by distinct genes
93
describe the lac operon:
| The genes nad its products and function?
-transcriptional regualtion
-is an inducible gene and in its uninduced state it is actively repressed (must be induce to be active)
-has binding site for repressors
lacZ (b-galactosidase-->lactose--> glucose + galactose-->Co2+H2)+ATP)
lacY (permease-->transport of lactose into cell)
lacA (acetylation of b-galactosides)
-
94
how does lactose regulate lac expression in its absence, presence, and with glucose?
absence of lactose
- RNA pol holoenzyme cant move
- lac repressor binds 2 distinct operator sites as tetrameric complex
presence of lactose
- allolactose (isomeric form of lactose produced when loactose encountered by bacterial cells)
- allolactose=inducer bind etramers-->change in conformation--->lose ability to bind DNA-->RNA pol can move and produced mRNA
lactose + glucose
-glucose present= no transcription regardless of lactose= catabolite repression=CRP (cyclic AMP repression protein) decrease-->allolactose inactive
lactose no glucose
- cAMP-CRP binds promoter upstream recruit RNA pol (pro-pro interaction with)
- relief catabolite repression and promote active transcription
95
two requirements for transcription at lac
1. absence of repressor (derepression)
| 2. presence of activator (true activation)
96
what is CRP
cyclicAMP receptor protein
- binds to promoter activates lac operon
- DNA recognition sequences for both lac repressor and CRP promote binding of protein dimers
- bind as dimers looking for symmetry
- both lac repressor and CRP are transcription factors regulate amount of RNA pol at promoter
- consensus for CRP because it regulate other genes too
- less symmtry weaker binding
97
name the multiple sites of regulation in making genetic infor in Euk to functioning proteins at the right time in the right place
1. transcriptional control DNA-->1' RNA transcript
2. processing control 1'RNA-->mRNA in nucleus
3. transport control mRNA nuc->mRNA cytoplasm
4. mRNA degradtion control, translation control by ribosome mRNA-->pro
5. inactive or active protein degration, modification, transport control, acytylation
98
Prok vs. Euk transcription rate
Prok
-rate of pro synthesis parallels rate of mRNA transcription
EUk
-no inherent correlation btw rates of transcription and conc of mRNA in cell
99
most critical points of gene regualtion in EUk cells?
post-transcriptional contorl
- mRNA processing/alternative splicing
- mRNA stability/degradtion (concentration)
- translational control
transcription initiation control
- activation/repression by trans factors
- developmental regulation
- epigenetic control
100
```
Pharm:
what is it?
IND
NDA
BLA
```
Investigational New Drug permission to start human trials
New drug application permission to market
Biological license application permission to market
101
an estimate of how many NCE/NBE drugs enter market each year?
25 new biological/chemical entity
102
```
drug category and description
A
B
C
D
X
```
A- controlled human studies show no risk
B- animal studies sow no drug risk OR animal studies did show risk but not confirmed in human
C-animal studies adverse effects but no human studies OR no animal or human studies available
D- positive evidence of human fetal risk, BUT may be acceptable in some clinical situations
X- fetal abnormalities or risk of mother evident AND risks clearly outweigh possible benefits-- Contraindicated in pregnancy
103
Decsriibe the 4 stages of drugs from discovery to approval and marketing
Preclinical stage
-in vitro studies + animal testing
-lead compound-->efficacy selectivity mechanism
drug mechanism and toxic effects
IND (investigational new drug) permission to start clnical trial on human
Phase clnical testing
-PI: 20-100 subject safety, pharmacokinetics,
healthy population
bioavailability
biotransformation and metabolism, half life, biodistribution, elimination
```
-PII: 100-200 efficacy
disease population
evaluatie effect of drug doses
dose-dependent efficacy
modes of action
side effects
safe & effective
```
-PIII: 1000-6000 double blind study, does it work? most expensive phase
more diverse group
confirm clinical value compare to existing drugs
tracking effects of drug overtime
observed adverse side effects
ALL three deals with drug metabolism and safety assessment
NDA (new drug application)
Phase 4
- postmarketing surveillance
- patent expires 20 years after filing application
104
audioradiography
-useful in studying timing of cell activity
give animal radioactive isotope
-Allow isotope to be incorporated into tissues.
Process tissue with TEM freeze fracture.
Overlay sample with photographic emulsion; radioactive “hot spots” will precipitate silver
105
Reporter genes & reporter proteins
reporter gene is a construct made to find where about the product was produced
- engineered lab tool made of DNA containing the transcriptional promoter from a gene of study
- also has sequence of gene whose product is easily detected
- fused coding sequence to promoter
ie. sequence encoding-GFP-->protein produced has fluorescent tag
- bright visualize
- developed by mutational modification, allowing nultiple structures to be visualized simultaneously
106
Flow cytometry & FACS
-collect quantitaive information on many cells quickly
-collected cellsare flowed through the machine
and measurements are taken and recorded,
traditionally as graphs
-rapid blood sample diagnostic
oe. T helper cells in HIV, RH+, cells in anemia and leukemia pts
Fluorescence escence activated cell sorting to separate different cell type
107
IMMUNOFLUORESCENCE microscopy,
Immunofluorescence direct & indirect
Confocal microscopy
-
108
IMMUNOHISTOCHEMISTR
-antibodies probes
109
CISH (chromogenic in situ hybridization
```
companies have made
polynucleotide probes that
recognize hundreds of
DNA and RNA sequences
of various types (DNA or
RNA, human or viral,
```
110
HISTOCHEMICAL stain vs. Enzyme histochemical stain morphology
chistochemical stain” is chemical stain that leads
to pigment deposition due to molecular structure
in a tissue section (example, hematoxylin staining
chromatin in the nucleus)
• the term “enzyme histochemistry” is deposition of pigment resulting from the activity of an endogenous enzyme present in a tissue, as when a special chromogenic version of the enzyme’s substrate is applied to the slide (example, detection of ATPase in muscle).
- limited to enzyme with artificial substrates that change color when convert to products
- enzyme activity (alkaline phosphatase)
111
what is the predominate means of regulation in prok.?
control of gene transcription
112
describe three ways that produces protein isoforms in different cell types post transcriptional
1. alternative splicing-- euk. gene spliced in multiple ways allow greater variety of pro to be priduced from smaller number of genes
2. altenative polyadenylation sites
3. non-coding exons important in post-transcriptional regulation
113
how does b-thalassemia result from alternative splicing
- mutation in intron 1 provides splicing machinary with 2 splice acceptor choices
- mutant pathway ratio 9:1
- frameshift and STOP codon truncate pro-->nonfunctional
114
what is the importance of mRNA half life
-short lived mRNA likely to produce small quantity of proteins a good strategy if protein is only needed briefly for signaling
115
```
transferrin receptor synthesis
-example of what type of regulationq
-at low iron
-at high iron
what does transferrin do?
```
- transferrin helps import iron into cells
- regulate degradation of mRNA, control conc mRNA= conc translated protein
low iron-->
untranslated 3' of transferrin mRNA -->hairpin loop bind IRE-BP-->no degradation= inc transfferin to bring in iron from outside
high iron
IRE-BP loses affinity for mRNA and binds to iron, mRNA degraded-->dec transferrin=dec iron
116
ferritin synthesis
- function of ferritin?
- low iron
- high iron
-ferritin stores iron
'low iron
IRE-BO bind hairpin loop in untranslated 5' region of ferrtin mRNA prevent ribosome from accessing mRNA-->dec ferritin level
high iron
IRE-BP loses affinity for mRNA binds iron; ribosomes bind mRNA and translate it
117
what can regulate mRNA stability & mRNA translation?
| -what is the process?
-RNA interference (RNAi) via microRNA (miRNA) regulate mRNA stability & translation
- miRNA transcribed as 1' transcripts within introns of other genes
- processing occurs within nucleus & cytoplasm-->single strand species incprporated into RISC complex
- inhibition of translation or mRNA degradation depend on complementarity of miRNA to its mRNA target
- dsRNA can enter cytoplasmic pathway and lead to specific mRNA degradation-->possible therapeutic to down regulate aberrantly expressed genes (CA cells).
118
Euk. gene contain 3 important differences (vs. prok) in regulating protein synthesis
1. regulatory sequence elements tned to be compound bindmore than one TF
2. combinatorial control interaction btw TFs allow finer control of transcription
3. cell type, tissue, developmental stage specific expression of gene depend on TFs combination, regulatory elements like enhancers
119
transcriptional EUk. initiation complex
- multiple pro-pro and pro-DNA interactions
- DNa loops & bends
- in contrast to activation, genes are repress by co-repressors complex or activator by co-activator=mediators (non-DNA binding transcription factor complex
- gene regulatory sequences: HRE, Enhancer, promoter proximal segments, TATA box, initiator
- regulatory DNA binding pro specific TF: hormone receptors, transactivator
- basal transcription complex:TATA binding protein, RNA pol,
120
beta-globin regulatory elements
GATA-1: hematopoetic trans factor
EKLF is erythroid specific trans factor
-2 identical a-globin gene on chrom 16
-5 distinct b-globin on chrom 11
-a-globin expresssed throughout life
-different b-globin like genes in different stages of life
-locus control region (LCR) upstream enahncer contorls overall expresion and developmental gene switching in b-globin locus,
-has at least 4 diffent regions tha tbind to differnt proteins
-mutation in LCR results in gamma, deltabeta thalassemia due to complete regulatory failure at b-globin locus
121
hereditary peristance of fetal hb (HPFH)
- condition where loss of gamma-globin down regulation can compensate for reduced or basent b-globin expression
- point mutation in gamma globin proximal promoter region
- several deletions that eliminate delta beta genes
- hypothesis mutation lead to loss of binding of developmental-specific repressors; repressor no longer bind therefore gamma does not turn off
122
beta turn often contain what 2 amino acid?
glycine nad proline
123
4 factors that favor folding?
1. intra-molecular H- bond-->2' structure
2. hydrophobic interactions= micelles interior
3. electrostatic ionic interactions= opposite charged group attraction
4. van der waals= electron charge around atom asymmetric inducing asymmetry in another atoms electron charge such that electrostatic attraction occurs btw 2 atoms
124
2 factors favor unfolding
1. conformational entropy---only 1 folded state, but infinite # of incorrect unfolded states
2. H-bonding to solvent/h2O interferes with H bonding necessary for 2' structure
125
thermodynamic of folding
- overall free energy change of folding is negative, favorable delta G= delta H-TdeltaS
- conformational entropy largest factor contributing to destabilization of protein structure
- enthalpy favorable energy from intramolecular side chain interactions
- favorable entropy from hydrophobic interactions
126
non polar aliphatic aa
- Gly (G),
- Ala (A)
- Pro, P
127
nonpolar alipathic branched aa
-Val, V
Leu, L
Ile, I
128
nonpolar aromatic aa
Phe, F
129
slightly polar aromatic aa
Tyr, Y
| Try, W
130
polar uncharged aa
Asn, N
Gln, Q
Ser, S
Thr, T
131
sulfur containing aa
Met, M
| Cys, C
132
Acidic aa
Asp, D
| Glu, E
133
basic aa
Lys, K
Arg, R
His, H
134
list 7 ionizable side chains, their pka side chain, and at physiological pH
```
asp (aspartate)= 4 (-/-)
Glu (glutamate)=4.1 (-/-)
his=6 (variable)
cys=8.5 (disulfide)
tyr-10.5(H-bond)
lys= 10.5 (+)
arg=12.5 (+)
```
135
peptide bond why is it N-C-C-N
1. trans steric hinderance, R groups on opposite sides of bond
2. planar bond hybridization
NCCNCCNCCNCCN...
136
three syndrome resulting from micro deletion where small region on chromosome is deleted leads to monosomy for a number of genes, uses FISH to detect banding pattern changes
1. angleman's syndrome
2. prader-willi syndrome
3. retinoblastoma
