Dr. Ray was the only one who showed up. Here is my personal study sheet. Pray hard. You'll do fine.
Review sheet for Final in CMB
5 Senogles,1 Skapek, 2 Scott, 2 Zhang, 2 Ray questions on test
Senogles-Review points and sample questions
Senogles Review included
Know pedigrees. Recognize basic forms inheritance. Know about mapping methods and be able to distinguish. Know differences kinds of cloning. Know what’s on this review, and in her objectives. Proband is first individual Identified as having the disorder. Gene is not dominant or recessive. Phenotype is what you can see. Phenotype might be more than one trait. Affected person indicates a disease phenotype or something clinically observable.
I. Human genetics
a. Autosomal and pseudoautosomal
b. X linked
c. Y linked
Sample question:
What kind of inheritance is the following? Justify your answer.
For pedigrees, explain your answers. Use the questions from her lecture 1 for each type of inheritance. EMAIL HER YOUR QUESTIONS.
II. Mapping of the genome by cytogenetic, genetic and physical methods
Sample question:
Explain how one generates a physical vs. a genetic map of a human chromosome?.
III. Positional vs traditional cloning techniques
a. Chromosome walking and jumping
b. exon identification
Sample question: Explain the difference between functional and positional cloning.
IV. Shermans paradox and resolution
V. Genetic anticipation, somatic mosaicism
Skapek
Concepts to cover:
Developmental processes
Determination
Migration
Differentiation
Cell biology of myogenic differentiation
Molecular biology of myogenic differentiation
Key myogenic transcription factors
How myogenic differentiation is controlled
Ray
2 lectures on apoptosis. He put 8 or 9 questions on Blackboard.
Look at how questions can be answered differently under different contexts.
Be able to tell the difference between necrosis and apoptosis. How is apoptosis relevant to development and disease (remember examples to justify- What happens when apoptosis increases and decreases?)
Remember BCl2 protein domains and how they work. Remember caspases and how they work.
Remember CIAPs that inhibit caspase 3 activity. Do not remember the number of amino acids, remember general mechanisms of activation and inhibition of caspases.
Look at the sample questions. Remember that Each question on the test has one compulsory part, one choice part. You will have to use creativity and your understanding to answer questions. They will not be direct.
For example, in a system you induce apoptosis with TNF-CHX (inhibits anti-apoptotic proteins, which have short half lives), one system with caspase inhibitors. Look at beginning and after certain time point. What would happen? Some cell systems, do not need CHX. TNFa induces both pathways (apoptotic and anti-apoptotic) at same time. One overrrides the other. In the papers, proteins may have different names. Gamma irradiation and chemical induction of apoptosis do not go through receptor
Remember slide on p53 and irradiated mice. Classical example of p53 and BCl2 and apoptosis. Remember the slides. Put them in your memory. He will try to find out what we know, not what we do not know.
Q.1 How would you use the understanding of the mechanisms of cell death for the treatment strategies for the diseases involving apoptosis
Q.2 How would you differentiate a cell undergoing apoptosis from necrosis ?
Q.3 Giving suitable examples discuss in brief the significance of apoptosis during development and pathogenesis
Q.4 Discuss in brief the mechanism by which TNF-α and FAS induces apoptosis.
Q.5 Describe the role of Bcl-2 family proteins in the regulation of
Apoptosis.
Q.6 Discuss in brief the mechanisms by which γ-irradiation and chemical agents induce apoptosis.
Q.7 Giving suitable example discuss the role of apoptosis in the development of cancer.
Q.8 Discuss the role of mitochondria in the regulation of apoptosis
Zhang
Review Points
1. For cell-cell adhesion lecture
a. How is cadherin connected to cytoskeleton?
b. What are the structural and functional differences between gap junction and gap junction.
c. Compare the compositions of adherent junction and desmosome
d. List the calcium-independent cell-cell adhesion molecules.
e. What are cadherin-mediated functions?
f. Illustrate the roles of E-cadherin and β-catenin in tumorigenesis and tumor progression.
2. For cell-extracellular matrix adhesion lecture
a. What are the cellular receptors of fibronectin, laminin, and collagen?
b. What are the major components of the stromal environment in connective tissue?
c. Give an example that integrin functions as a cell-cell adhesion molecule.
d. Is the RGD sequence the binding site in collagen for integrin?
e. What are the components of basement membrane?
f. Why is integrin called bi-directional signaling machinery?
Scott
Know the 6-part definition of differentiation.
Know fundamental definitions of other terms: dedifferentiation, transdifferentiation, metaplasia, terms from his cancer lectures. Be able to describe the controversy surrounding transdifferentiation and dedifferentiation. Do stem cells grow in adult tissues into which they are implanted? Do they merely fuse with the cells in the tissue? (There is an article in Science about this from several years back that marked chromosomes in the injected cells with fluorescent dye. They showed cell fusion in heart tissue). Does it depend on the tissue? Be able to state evidence to support arguments either way.
This is what I have, folks. If you need more detail, contact the professors. Have fun studying.
Saturday, February 17, 2007
Friday, February 16, 2007
Senogles 6
Senogles 6 Huntington’s Disease
1
Huntington’s is a trinucleotide repeat expansion disease, but has unique characteristics. Dementia is due to severe effects on basal ganglia of brain. 2 forms: adult onset and juvenile form. Juvenile form gave insights into inheritance
2
Emphasis on juvenile is because of insights.
3
RFLP marker assigned to chromosome 4 in 1983. One of 1st testable markers for a disease.
Marker led to cloning of disease gene. Linkage disequilibrium- indicates ancestor gene close to marker.
4
Huge pedigrees back to 1800s from families around Lake Maracaibo in Venezuela.
5
Assigned to end of chromosome 4- 5 million base pair fragment.
6
Segregation with HD: Markers land close to disease gene. Diamonds indicate blind study- did not want to give identifying information about individuals.
7
Restriction map and theoretical locus: Knew that disease mapped to this region.Wanted to clone gene.
8
Problems: big region. Instead of chromosome walking and jumping and cros-hybridization, they used exon amplification.
Make family of fragments, generate probes, screen library.
9
Take vector with known sequences on either side of multiple cloning site (commonly use a beta globulin). Clone in DNA. Get family of fragments. Transfect cell. Isolate RNA, amplify by PCR using primers for known sequence. PCR product is exon flanked by known sequence. If it has an exon, will be transcribed and can be identified. Idea of trapping- if vector has exonic sequence- RNA will result. Can sequence PCR product. Now have probe to screen CDNA library and Northern blots.
10
Using strategy, cloned a number of CDNAs. Composite is 340 KB protein. Found that in giant clone had large ORF with 2 repeats in middle of coding sequence. Poly Q, and poly P. Poly P is not related to disease. Poly Q is.
12
Repeat size is larger, but not horrendously larger. This is more subtle. Disease state centers around 45 repeats.
13
Juvenile Huntington’s: imprinting phenomenon. Repeat size and age of onset correlate.
14
Huntington’s is autosomal dominant, so affected male has an allele in the normal range, and an expanded one. For autosomal dominant disease, one parent must be affected, shows up in every generation.
Some individuals have larger repeats than parent . 8 and 10 do. Age of onset correlates to size of expanded allele.
15
In Venezuela you can get huntington’s disease homozygous. About 25% of kids are homozygous dominant. Some are heterozygous. Age onset homozygous is 6-7 yrs.
16
Sporadic mutation occurs. PCR shown. Huntington’s can be caused by sporadic increase in repeats.
17
No evidence of Huntington’s until mutation. Expansions in fragile X were in UTR. These are in code for a protein. Changes in poly Q yield changes in protein function.
18
Genetic anticipation- bigger expanded allele, earlier child diagnosed with disease. Not Mendelian phenomenon. Through mother- repeat sizes do not change all that much. Changes dramatically in gene passed down from father.
19
Scattergram of juvenile age onset vs number of repeats.
20
Age of onset is a function of repeat length- no correlation with maternal descent- is correlated with paternal in juvenile Huntington’s.
21
Large repeats have paternal origin, in contrast to Fragile X.
22
Expansion may occur during male gametogenesis. Somatic mosaicism- different numbers of repeats in different somatic cells from same patient. Different tissues are different.
23
Protein product- you get a functional protein and can observe multiple species. Proteins are made with larger repeat. Heterogeneity detectable at protein level.
24
Lots of expression in basal ganglia.
25
From a given individual, samples from cortex and cerebellum are different. Individuals cannot efficiently replicate repeats. Different size proteins are reflected.
26
Result:
Basis of Huntington’s is undefined. Cellular inclusions form in cells and cause cell death. This may be a gain-of-function mutation- as protein expands, interacts with new things and causes bad effects.
Transgenic mice- can get Huntington’s phenotype.
1
Huntington’s is a trinucleotide repeat expansion disease, but has unique characteristics. Dementia is due to severe effects on basal ganglia of brain. 2 forms: adult onset and juvenile form. Juvenile form gave insights into inheritance
2
Emphasis on juvenile is because of insights.
3
RFLP marker assigned to chromosome 4 in 1983. One of 1st testable markers for a disease.
Marker led to cloning of disease gene. Linkage disequilibrium- indicates ancestor gene close to marker.
4
Huge pedigrees back to 1800s from families around Lake Maracaibo in Venezuela.
5
Assigned to end of chromosome 4- 5 million base pair fragment.
6
Segregation with HD: Markers land close to disease gene. Diamonds indicate blind study- did not want to give identifying information about individuals.
7
Restriction map and theoretical locus: Knew that disease mapped to this region.Wanted to clone gene.
8
Problems: big region. Instead of chromosome walking and jumping and cros-hybridization, they used exon amplification.
Make family of fragments, generate probes, screen library.
9
Take vector with known sequences on either side of multiple cloning site (commonly use a beta globulin). Clone in DNA. Get family of fragments. Transfect cell. Isolate RNA, amplify by PCR using primers for known sequence. PCR product is exon flanked by known sequence. If it has an exon, will be transcribed and can be identified. Idea of trapping- if vector has exonic sequence- RNA will result. Can sequence PCR product. Now have probe to screen CDNA library and Northern blots.
10
Using strategy, cloned a number of CDNAs. Composite is 340 KB protein. Found that in giant clone had large ORF with 2 repeats in middle of coding sequence. Poly Q, and poly P. Poly P is not related to disease. Poly Q is.
12
Repeat size is larger, but not horrendously larger. This is more subtle. Disease state centers around 45 repeats.
13
Juvenile Huntington’s: imprinting phenomenon. Repeat size and age of onset correlate.
14
Huntington’s is autosomal dominant, so affected male has an allele in the normal range, and an expanded one. For autosomal dominant disease, one parent must be affected, shows up in every generation.
Some individuals have larger repeats than parent . 8 and 10 do. Age of onset correlates to size of expanded allele.
15
In Venezuela you can get huntington’s disease homozygous. About 25% of kids are homozygous dominant. Some are heterozygous. Age onset homozygous is 6-7 yrs.
16
Sporadic mutation occurs. PCR shown. Huntington’s can be caused by sporadic increase in repeats.
17
No evidence of Huntington’s until mutation. Expansions in fragile X were in UTR. These are in code for a protein. Changes in poly Q yield changes in protein function.
18
Genetic anticipation- bigger expanded allele, earlier child diagnosed with disease. Not Mendelian phenomenon. Through mother- repeat sizes do not change all that much. Changes dramatically in gene passed down from father.
19
Scattergram of juvenile age onset vs number of repeats.
20
Age of onset is a function of repeat length- no correlation with maternal descent- is correlated with paternal in juvenile Huntington’s.
21
Large repeats have paternal origin, in contrast to Fragile X.
22
Expansion may occur during male gametogenesis. Somatic mosaicism- different numbers of repeats in different somatic cells from same patient. Different tissues are different.
23
Protein product- you get a functional protein and can observe multiple species. Proteins are made with larger repeat. Heterogeneity detectable at protein level.
24
Lots of expression in basal ganglia.
25
From a given individual, samples from cortex and cerebellum are different. Individuals cannot efficiently replicate repeats. Different size proteins are reflected.
26
Result:
Basis of Huntington’s is undefined. Cellular inclusions form in cells and cause cell death. This may be a gain-of-function mutation- as protein expands, interacts with new things and causes bad effects.
Transgenic mice- can get Huntington’s phenotype.
Wednesday, February 14, 2007
Senogles 5
Senogles 5
Fragile X syndrome
Review questions are posted on Blackboard under Assignments with bullets for important points. We will review after lecture tomorrow-short. You can also email her.
Tomorrow –Huntingdon’s disease.
Today-fragile X. These are trinucleotide expansion diseases (the only new development in genetics in a long time- dseases in which a repeat of three nucleotides is generated too many times, separating a gene from its promoter, and a disease results). This mechanism explains some diseases with weird inheritance.
Decreased penetrance can occur with an X-linked dominant disease. The phenotype is not manifested the same in each individual. More severe in males. Oddball is the normal transmitting male. Flag: carrier is not affected, but subsequent generations are severely affected.
2
Sherman’s paradox will be on the exam. Understand basic evidence for #2.
3
Sherman was a clinical psychologist who discovered aberrations in pedigrees. For X-linked dominant disorder, risk should not change in pedigree for succeeding generations. Something was wrong here. This is unusual genetics.
4
X-linked dominant disorders- all female children of affected males are carriers.
5
Review slide of normal X-linked transmission.
6
Sherman’s paradox pedigree
Risk gets higher with succeeding generations. Grandchildren at higher risk than siblings of the normal transmitting male (T).
7
Look at karyotype of patient- deprive cells of folate in vitro- X chromosome fragments at fragile sites. Name of the disease comes from karyotype.
8
Used perturbation to clone by somatic cell hybrids. Breakage is always at same place on chromosome. Take panel of rodent cells and fuse human cells to them. Used fragile X patient cells. X breaks under thymidine stress and reforms with rodent cells.
9
Heterokaryon is fused cell.
Mouse tumor cell is immortal and clonal. Form heterokaryons, select, then grow fused cells on permissive medium that selects only for hybrid cells. Wind up with hybrid cells that are clonal- each group grows from single cell. Can use RT-PCR to see what human chromosomes are carried in the heterokaryons.
10
Get a rodent chromosome with a bit of a human chromosome at the end.
11
When mapped out, get this diagram. The fragile X site is near telomeric end. B is a higher resolution map. CpG islands are normally unmethylated regions 5’ to a mammalian gene. Probes used are shown in D. Hypothesis was that something odd was going on near CpG island.
12
Southern analysis of 2 fragile X pedigrees. Filled in squares are affected individuals. Females indicate various degrees of clinical manifestation. Took blood, looked at DNA. Normal individual- Eag1 should cut, indicating a non-methylated CpG island.
13
Different pedigree. Affected males and carrier females have higher fragment size from CpG islands being methylated. Region is larger in some affected males and carrier females. Two things are happening (methylation of the CpG islands and increased numbers of repeats).
14
Track through pedigree analysis and look at size of insertions- size on Southern blot gets larger (higher in gel). Gets substantially larger in terms of base pairs. The insertion is at the fragile X locus.
15
Cloned region. Circled area is CpG island. Repeat of CCG is shown by arrows.
Repeat could be basis of increase of fragment size, or a nearby gene could be changing.
17
Flanking regions did not change size. Repeat region changes, not flanking regions.
19
Examine repeat sequence in more detail. Is it polymorphic in normal individuals, or just in fragile X families?
20
Is polymorphic in normal population. Run sequencing gel to determine size of repeats. PCR through- get normal repeat size (29 repeats on average).
21
Look at fragile X pedigrees- centers on several hundred repeats.
22
Studied number of pedigrees. Found risk of expansion to full mutation was correlated through premutation of mother . Expansion occurs in carrier female. Graph shows repeat size in mother. Does not correlate with paternal allele size. Imprinting- differential expression depending on parent allele comes from.
23
This is consistent with Sherman’s observations. Expansions go through females, but only when allele is transmitted through female does risk increase. NTM (normal transmitting male) has expanded allele, but not fragile X. NTM is key. He has premutation repeat size. Size of allele correlates well with retardation in males, but X inactivation in females complicates things, producing diverse phenotypes.
24
3 is at 53/29 copies (females have 2 X chromosomes, so for a female, there can be two sets of repeat values). Daughter 4 is at 29/83 copies. 29 size does not change. Other one changes. Sons relevant DNA will not enter gel- off scale in terms of number of repeats.
Pedigree 2:
1 has 83 and 29 repeats. 3 has a reduction in size, which is possible. Repeat sizes are dynamic. Nobody knows how the expansion and reduction occur. Curent thinking is that repeats form odd structures so they can’t copy accurately.
25
premutation size is also dynamic. Normal family shown. Premutation size shown. There is flux even in premutation alleles.
26
Affected gene was cloned. Original start site in the literature was wrong. Originally reported repeat in coding region. It is actually in the 5’UTR. Called Fragile X Mental Retardation Gene (FMR).
27
Methylated CpG island and repeat size change in fragile X patients. How are they linked? Look at methylation status. By Southern blot analysis we see complete cleavage (unmethylated DNA) in normal individuals. Carrier females- methylated and unmethylated. Affected males- not digested by BssHII. They have methylated CpG island close to gene. Carrier females- half methylated. They have 2 copies. Fragile X males- CpG is completely methylated.
28
Expression or transcription of gene: there is a correlation between methylation and gene expression- inactivated by methylation.Silences DNA. Transcription of FMR1 was measured as follows: Isolated RNA, reverse transcriptased it,Used RT-PCR. No message- no band on PCR. All had HPRT housekeeping gene. N are normal individuals. Asterisks are fragile X patients- no transcript.Carriers also lack the transcription of the gene.
29
Scan gel with densitometer-2 blips for normal individual. In patient 411- no FMR1 product. Patient 412- reduced over normal population. 410 carrier female also has reduced quantities.
30
Correlates expression and methylation status.
31
Normal repeat size puts AUG close to CpG island unmethylated, allowing transcription. As repeat size gets larger, still get transcription.
Full mutation size, cell methylates CpG island and shuts down gene.
32
Resolved Sherman’s paradox with a mechanistic explanation to some degree. Repeat size gets large through mitotic expansion in the females. There is a threshold size of repeat.
Fragile X syndrome
Review questions are posted on Blackboard under Assignments with bullets for important points. We will review after lecture tomorrow-short. You can also email her.
Tomorrow –Huntingdon’s disease.
Today-fragile X. These are trinucleotide expansion diseases (the only new development in genetics in a long time- dseases in which a repeat of three nucleotides is generated too many times, separating a gene from its promoter, and a disease results). This mechanism explains some diseases with weird inheritance.
Decreased penetrance can occur with an X-linked dominant disease. The phenotype is not manifested the same in each individual. More severe in males. Oddball is the normal transmitting male. Flag: carrier is not affected, but subsequent generations are severely affected.
2
Sherman’s paradox will be on the exam. Understand basic evidence for #2.
3
Sherman was a clinical psychologist who discovered aberrations in pedigrees. For X-linked dominant disorder, risk should not change in pedigree for succeeding generations. Something was wrong here. This is unusual genetics.
4
X-linked dominant disorders- all female children of affected males are carriers.
5
Review slide of normal X-linked transmission.
6
Sherman’s paradox pedigree
Risk gets higher with succeeding generations. Grandchildren at higher risk than siblings of the normal transmitting male (T).
7
Look at karyotype of patient- deprive cells of folate in vitro- X chromosome fragments at fragile sites. Name of the disease comes from karyotype.
8
Used perturbation to clone by somatic cell hybrids. Breakage is always at same place on chromosome. Take panel of rodent cells and fuse human cells to them. Used fragile X patient cells. X breaks under thymidine stress and reforms with rodent cells.
9
Heterokaryon is fused cell.
Mouse tumor cell is immortal and clonal. Form heterokaryons, select, then grow fused cells on permissive medium that selects only for hybrid cells. Wind up with hybrid cells that are clonal- each group grows from single cell. Can use RT-PCR to see what human chromosomes are carried in the heterokaryons.
10
Get a rodent chromosome with a bit of a human chromosome at the end.
11
When mapped out, get this diagram. The fragile X site is near telomeric end. B is a higher resolution map. CpG islands are normally unmethylated regions 5’ to a mammalian gene. Probes used are shown in D. Hypothesis was that something odd was going on near CpG island.
12
Southern analysis of 2 fragile X pedigrees. Filled in squares are affected individuals. Females indicate various degrees of clinical manifestation. Took blood, looked at DNA. Normal individual- Eag1 should cut, indicating a non-methylated CpG island.
13
Different pedigree. Affected males and carrier females have higher fragment size from CpG islands being methylated. Region is larger in some affected males and carrier females. Two things are happening (methylation of the CpG islands and increased numbers of repeats).
14
Track through pedigree analysis and look at size of insertions- size on Southern blot gets larger (higher in gel). Gets substantially larger in terms of base pairs. The insertion is at the fragile X locus.
15
Cloned region. Circled area is CpG island. Repeat of CCG is shown by arrows.
Repeat could be basis of increase of fragment size, or a nearby gene could be changing.
17
Flanking regions did not change size. Repeat region changes, not flanking regions.
19
Examine repeat sequence in more detail. Is it polymorphic in normal individuals, or just in fragile X families?
20
Is polymorphic in normal population. Run sequencing gel to determine size of repeats. PCR through- get normal repeat size (29 repeats on average).
21
Look at fragile X pedigrees- centers on several hundred repeats.
22
Studied number of pedigrees. Found risk of expansion to full mutation was correlated through premutation of mother . Expansion occurs in carrier female. Graph shows repeat size in mother. Does not correlate with paternal allele size. Imprinting- differential expression depending on parent allele comes from.
23
This is consistent with Sherman’s observations. Expansions go through females, but only when allele is transmitted through female does risk increase. NTM (normal transmitting male) has expanded allele, but not fragile X. NTM is key. He has premutation repeat size. Size of allele correlates well with retardation in males, but X inactivation in females complicates things, producing diverse phenotypes.
24
3 is at 53/29 copies (females have 2 X chromosomes, so for a female, there can be two sets of repeat values). Daughter 4 is at 29/83 copies. 29 size does not change. Other one changes. Sons relevant DNA will not enter gel- off scale in terms of number of repeats.
Pedigree 2:
1 has 83 and 29 repeats. 3 has a reduction in size, which is possible. Repeat sizes are dynamic. Nobody knows how the expansion and reduction occur. Curent thinking is that repeats form odd structures so they can’t copy accurately.
25
premutation size is also dynamic. Normal family shown. Premutation size shown. There is flux even in premutation alleles.
26
Affected gene was cloned. Original start site in the literature was wrong. Originally reported repeat in coding region. It is actually in the 5’UTR. Called Fragile X Mental Retardation Gene (FMR).
27
Methylated CpG island and repeat size change in fragile X patients. How are they linked? Look at methylation status. By Southern blot analysis we see complete cleavage (unmethylated DNA) in normal individuals. Carrier females- methylated and unmethylated. Affected males- not digested by BssHII. They have methylated CpG island close to gene. Carrier females- half methylated. They have 2 copies. Fragile X males- CpG is completely methylated.
28
Expression or transcription of gene: there is a correlation between methylation and gene expression- inactivated by methylation.Silences DNA. Transcription of FMR1 was measured as follows: Isolated RNA, reverse transcriptased it,Used RT-PCR. No message- no band on PCR. All had HPRT housekeeping gene. N are normal individuals. Asterisks are fragile X patients- no transcript.Carriers also lack the transcription of the gene.
29
Scan gel with densitometer-2 blips for normal individual. In patient 411- no FMR1 product. Patient 412- reduced over normal population. 410 carrier female also has reduced quantities.
30
Correlates expression and methylation status.
31
Normal repeat size puts AUG close to CpG island unmethylated, allowing transcription. As repeat size gets larger, still get transcription.
Full mutation size, cell methylates CpG island and shuts down gene.
32
Resolved Sherman’s paradox with a mechanistic explanation to some degree. Repeat size gets large through mitotic expansion in the females. There is a threshold size of repeat.
Tuesday, February 13, 2007
Senogles 4
Senogles 4: Color Blindness
She expects us to know her learning objectives, and her tests are very straightforward. She shows references in her lectures, but we DO NOT have to read them.
1
Next 3 lectures are about the molecular basis of 3 different diseases. Humans are trichromats, and our brains mix the colors to produce what we see.
2
Opsins function as a tandem array.
Ancestor opsin duplicated and changed to produce color opsins.
6
Green deficiency-muddy brown. Cats and dogs are dichromatic.
7
Number cannot be perceived by colorblind person.
8
Rhodopsin allows dim-light vision. Cones are for color. Photoreceptors are named for color opsins they carry.
10
Bottom graph- different opsins are sensitive to different wavelengths.
11
Eye perceives light by rods and cones. Rods mediate light intensity. Cones carry distinct opsins for color receptors.
12
Rhodopsin is a G-protein-coupled receptor with 7 transmembrane spanning domains. Light is agonist causing isomerization of retinal. Cis-trans conformational change is the activation signal for the receptor.
13
Nathan Used rhodopsin as probe in eye library. Little squares indicate 6 exons for each protein he found.
14
He cloned them out and looked at protein similarity. Did stepwise comparison of proteins sequences for amino acid similarity. Amino acid identity shown in white. Differences are shown in black. Highly related proteins.
15
Probably result of gene duplication event.
16
3 reasons for tandem arrays in the human genome:
1. Histone tandem array (lots of copies of the genes for histone subunits arranged end to end) on genome because you need a lot of histones at certain points in growth. Get a lot made in a short period from master promoter.
2. BetaGlobin locus gene family: this protein has different fetus, embryo, adult forms. Region can be under coordinate regulation- region under master switch.
3. diversity in organisms. Evolutionary pressure to increase color vision for viability.
Unequal crossing over event creates 2 genes on one chromosome, none on other. The one with two may have a survival advantage.
17
Selection pressure leads to gene being maintained in original form if it is necessary for viability. If selection pressure is not so great, mutations may result in a gene with a related function or a pseudogene. There are lots of human pseudogenes. Slow and rapid are still millions of years. What was thought to happen in opsins was that rhodopsin maintained its original function and color opsins came later (18). Know this is how proteins with closely related functions are thought to be generated.
19
Photopigments underly both normal and color-deficient vision.
20
XLR=x-linked recessive.
AD=autosomal dominant
AR= autosomal recessive
Blue opsin is on chromosome 6, red and green on X.
21
Remember that affected males are related through mother- and which X male gets determines whether his vision is normal or not.
23
Nathan says take color normal individual and look a red and green opsins. He made probes for green and red opsins (at bottom). Used shorthand nomenclature as way to express data. Arrow is entire locus. Probes are A,B,C,D. Ag is the A probe for green.
He normalized DNA on the Southern blot. Intensity of bands is different. That means more copies of G on blot than R (For example, look at blot A. The bands for Ag are much darker than those for Ar. Another clue is that some color normal individuals are missing Dr. This is a key to the arrangement of the tandem array. They have to be in order on the chromosome. Dr was lost during recombination events that duplicated the green opsin gene. R gene is 5’. If it were at 3’ end you would sometimes lose parts of green.
24
Nathan speculated . Did densitometry scan on Southern blot and found it was common to have multiple copies of green. Had one red, 1 to 3 copies green.
A is color normal males. B shows mechanism for changing number of copies of green.
25
Looking at dichromats, genotypes shown across top of blot. Genomic probes shown upper left. This is a more complex analysis. Some individuals have complete loss of parts of opsins probed. You do ratio of green to red.
26
G-R+ had 2 phenotypes: missing entire Green opsin, or missing Dg. Just missing 3’ end probe.
G+R- more complex- variety phenotypes listed. All have Dg. 14 is color normal. Compare the others in this graph to him. These are “blown out” slides, so you cannot tell intensity easily.
Conclusions are based on Southern-blot data.
27
remember arrow represents entire gene region. Colored in to represent what is present.
A- color normal individual.
B- Recombination resulting in G+R-
C- Examples of how recombination results in unequal crossing over events with unusual arrays.
The data is complicated- go back and compare the slides and data. Slide 27 is a model to explain what is sitting in the genome to produce the Southern blot data. He hypothesized these structures to account for dichromatic vision. Look at analysis and blots to get it to make sense. She will not ask details of this. Understnd how got idea of 1 red to multiple copies of green and how to look at pathology of cases. How do you get the model from the data?
28
Achromatopsia is rare. No red or green.
30
X-linked trait.
31
genomic map red-green opsins. Added Z probe upstream of red locus.
32
Southern analysis again. 9 and 10 are color normal. Z missing or shortened in people with BCM. They have intact fragments for the opsins mostly, but missing 5’Z region (See data in G). Z region appears to be essential. Major control region just upstream is necessary.
Go back and look. It will make sense.
She expects us to know her learning objectives, and her tests are very straightforward. She shows references in her lectures, but we DO NOT have to read them.
1
Next 3 lectures are about the molecular basis of 3 different diseases. Humans are trichromats, and our brains mix the colors to produce what we see.
2
Opsins function as a tandem array.
Ancestor opsin duplicated and changed to produce color opsins.
6
Green deficiency-muddy brown. Cats and dogs are dichromatic.
7
Number cannot be perceived by colorblind person.
8
Rhodopsin allows dim-light vision. Cones are for color. Photoreceptors are named for color opsins they carry.
10
Bottom graph- different opsins are sensitive to different wavelengths.
11
Eye perceives light by rods and cones. Rods mediate light intensity. Cones carry distinct opsins for color receptors.
12
Rhodopsin is a G-protein-coupled receptor with 7 transmembrane spanning domains. Light is agonist causing isomerization of retinal. Cis-trans conformational change is the activation signal for the receptor.
13
Nathan Used rhodopsin as probe in eye library. Little squares indicate 6 exons for each protein he found.
14
He cloned them out and looked at protein similarity. Did stepwise comparison of proteins sequences for amino acid similarity. Amino acid identity shown in white. Differences are shown in black. Highly related proteins.
15
Probably result of gene duplication event.
16
3 reasons for tandem arrays in the human genome:
1. Histone tandem array (lots of copies of the genes for histone subunits arranged end to end) on genome because you need a lot of histones at certain points in growth. Get a lot made in a short period from master promoter.
2. BetaGlobin locus gene family: this protein has different fetus, embryo, adult forms. Region can be under coordinate regulation- region under master switch.
3. diversity in organisms. Evolutionary pressure to increase color vision for viability.
Unequal crossing over event creates 2 genes on one chromosome, none on other. The one with two may have a survival advantage.
17
Selection pressure leads to gene being maintained in original form if it is necessary for viability. If selection pressure is not so great, mutations may result in a gene with a related function or a pseudogene. There are lots of human pseudogenes. Slow and rapid are still millions of years. What was thought to happen in opsins was that rhodopsin maintained its original function and color opsins came later (18). Know this is how proteins with closely related functions are thought to be generated.
19
Photopigments underly both normal and color-deficient vision.
20
XLR=x-linked recessive.
AD=autosomal dominant
AR= autosomal recessive
Blue opsin is on chromosome 6, red and green on X.
21
Remember that affected males are related through mother- and which X male gets determines whether his vision is normal or not.
23
Nathan says take color normal individual and look a red and green opsins. He made probes for green and red opsins (at bottom). Used shorthand nomenclature as way to express data. Arrow is entire locus. Probes are A,B,C,D. Ag is the A probe for green.
He normalized DNA on the Southern blot. Intensity of bands is different. That means more copies of G on blot than R (For example, look at blot A. The bands for Ag are much darker than those for Ar. Another clue is that some color normal individuals are missing Dr. This is a key to the arrangement of the tandem array. They have to be in order on the chromosome. Dr was lost during recombination events that duplicated the green opsin gene. R gene is 5’. If it were at 3’ end you would sometimes lose parts of green.
24
Nathan speculated . Did densitometry scan on Southern blot and found it was common to have multiple copies of green. Had one red, 1 to 3 copies green.
A is color normal males. B shows mechanism for changing number of copies of green.
25
Looking at dichromats, genotypes shown across top of blot. Genomic probes shown upper left. This is a more complex analysis. Some individuals have complete loss of parts of opsins probed. You do ratio of green to red.
26
G-R+ had 2 phenotypes: missing entire Green opsin, or missing Dg. Just missing 3’ end probe.
G+R- more complex- variety phenotypes listed. All have Dg. 14 is color normal. Compare the others in this graph to him. These are “blown out” slides, so you cannot tell intensity easily.
Conclusions are based on Southern-blot data.
27
remember arrow represents entire gene region. Colored in to represent what is present.
A- color normal individual.
B- Recombination resulting in G+R-
C- Examples of how recombination results in unequal crossing over events with unusual arrays.
The data is complicated- go back and compare the slides and data. Slide 27 is a model to explain what is sitting in the genome to produce the Southern blot data. He hypothesized these structures to account for dichromatic vision. Look at analysis and blots to get it to make sense. She will not ask details of this. Understnd how got idea of 1 red to multiple copies of green and how to look at pathology of cases. How do you get the model from the data?
28
Achromatopsia is rare. No red or green.
30
X-linked trait.
31
genomic map red-green opsins. Added Z probe upstream of red locus.
32
Southern analysis again. 9 and 10 are color normal. Z missing or shortened in people with BCM. They have intact fragments for the opsins mostly, but missing 5’Z region (See data in G). Z region appears to be essential. Major control region just upstream is necessary.
Go back and look. It will make sense.
Sunday, February 11, 2007
Senogles 2 and 3
Senogles 2
Correction for Yesterday:
Pseudoautosomal Inheritance
Erratum for yesterday’s lecture: pseudoautsomal domains include only the ends
Y linked is also holandric inheritance. No daughter carriers. Pseudoautosomal has carrier daughters.
Ways to map genome:
Complementary and tell you different things. Cytogenetic mapping today, then genetic mapping and physical mapping. Genetic is mapping by inheritance. Physical map is the sequence itself. Cytogenetic map is chromosome.
2
Get this. Be able to understand what mapping CF by RFLP means.
3
Chromosomes as spread here have 2 arms. Nomenclature is standard and based on Giemsa staining.
4
R banding is reversed.
5
position is defined unequivocally in international nomenclature.
Ideograms are based on G banding of actual chromosomes.
7
Pattern FYI
8
female pattern. These gross analyses are used for a 1st test for a lot of chromosomal abnormalities.
9
Know that abnormalities exist. She does not care if you know details. Know they can be seen using cytogenetic methods.
10
Aneuploidy
Nondisjunction can occur – chromosomes do not separate in first round- one cell gets none, one gets both chromosomes. Gametes still function and can fertilize or be fertilized. If nondisjunction at second round, normal complement for 2 cells,a diploid, and a zero complement. Know that aneuploidy happens because separation does not occur properly.
Outcome- trisomy 13,18,21 are viable, but bad. Many other trisomies are lethal. XXX not as devastating. Monosomy X is Turner’s syndrome- one of few monosomies to survive.
11
Rearrangements occur with DS breaks. Repair system usually correct these breaks. Balanced- swapping pieces of DNA- no removal or deletion.
Unbalanced- duplicate or loss- serious potential effects.
12
Possible changes. Squashes or FISH can detect these changes in order. Most of the time these are silent.
13
Reciprocal translocations- no net loss or gain, just change in location. This is not recombination. This is translocation between different chromosomes.
May disrupt gene function if break is between promoter and gene. May be silent.
14
Nonreciprocal translocation- adds chromosomal material to another chromosome.
16
Tandem duplication results in repitition of segment.
17
Have knowledge of FISH analysis. #1 for cytogenetic analysis. Can “paint” whole chromosome or other things listed. Human chromosome has lots of repeat sequence. SSRs are inherited markers.
18
FISH probe is complementary only to a region of the chromosome. Family of probes can be made to entire chromosome.
22
Whole chromosome painting- probe each chromosome with a different dye emitting at different wavelength for different color. Can see deletions, increase in chromosome size.
24
Chromosome 19 has unbalanced rearrangement to attach to another chromosome.
She likes FISH analysis.
29
Genetic mapping can cause problems with understanding.
Linkage analysis- tendency for alleles close together on chromosome to be transmitted together. Can map through pedigree analysis. Close together- stay together in recombination. RFLP not used much anymore. Markers are not necessarily genes- they can be followed in pedigrees. Markers allow us to map genes to chromosomes.
30
1. relative to each other.
2. Polymorphic means difference among individuals.
RFLP and SSR are markers.
31
cM is a genetic unit. Crossing over happens a lot, so if genes are together 1% of the time, they are close together. cM and base pairs are not equivalent. To map a disease to a chromosome, must map several markers on each chromosome for inheritance.
33
Allele view. D and M could be separated by recombination. Randomly distributed- not linked.
34
Look at progeny or gametes that result. Some linkage- intermediate.
35
Marker A is not linked to disease phenotype, but marker B is.
Marker is not a predictor of disease per se, but happens to be linked. Not predictor. To be a predictor of disease, if you find marker very very close, can be used as diagnostic event. It may be linked to the disease gene, not associated with the disease itself.
36
RFLP- anything can be used as a marker. Restriction site can be used. 1bp change creates an ECOR1 cut site. RFLP site is inherited. See it a lot in literature, but not used much anymore. Get tons of fragments. Lots of work.
37
How is it done now? SSR or SRS (simple repeat sequence). Microsatellites are the repeat sequence. Repeats det by PCR and electrophoresis. Each chromosome has a different repeat number.Each individual 2 copies with different repeat sequence. Useful way to map. Marker and easier to follow than RFLPs.
38
SSRs in chromosome 1 Markers are under linkage map.
39 Physical mapping
We know the sequence, but we need to be able to navigate around.
40
Take part of human chromosome and fuse into rodent cell line- panel of hybrids with different parts of chromosome.PCR for gene of interest. Clonal lines .
STS are physical sites that are not simple repeat sequence. These are not polymorphic. These do not change and are the same in every human being. Cannot follow in genetic map because everyone has the same ones. One copy.
Fri- clone CF gene. Tues- inherited color vision, fragile X, Huntingdon’s disease.
Senogles 3
Assignment is posted.
Today we talk about positional cloning. Text reference is to cystic fibrosis (CF). Specific references are not required reading. Presented because it is good science and illustrative of how you do the technique.
3
Functional common until 1990s. Purified protein, followed activity as you purified, got to homogeneity, sequenced, got protein sequence, made oligonucleotide, screened library, got gene. You knew what you were looking for and what the activity was.
Now we look on the basis of where it maps on the chromosome with very little protein information. Positional cloning- map by genetics, then clone.
4
Knowing the DNA sequence does not help because you do not know what you are looking for.
5
2 types of mapping: see slide
6
10% codes for exons. Small part of genome.
7
Localize by linkage analysis position of disease gene you are interested in. Want to localize to greatest extent we can. Yacs and BACs can handle large pieces of DNA by cloning.
If you go to human genome project, is good resource. Can click on a region and see all the known genes in the region.
The two markers were well known.
Linkage disequilibrium- had a mutation in a chromosome. As goes through successive generations, some markers closely segregate with it. B1 stays with CF. It is in linkage disequilibrium. Suggests a founder chromosome with that marker.
11
High resolution map. Things with D are markers, polymorphic, inherited. Closer you can narrow it, less DNA you have to sequence.
12
Trying to minimize brute-force sequencing.
CpG islands tend to be islands of methylated CG that are close to promoters in coding regions. If it cross hybridizes, it is conserved and may be the candidate gene.
13
Want to isolate large fragments first. Large fragment- Mb.
14 Question: Disease- how to clone it out?
Chromosome walking- series overlapping clones. Cut up DNA. Make series of restriction fragments. Pull out and sequence along. Laborious.
15
Chromosome jumping- maximize potential hits by jumping to places from which you can sequence. Covers maximum amount of distance and bypasses unsequenceable stretches of DNA.
Circularized- A and B become close. Can sequence short segment between A and B to find the sequence of site B. Can jump, do not plan to go anywhere. You don’t really care what was between A and B to begin. Can make jumping library, and make a library of sites to walk from. Jumping randomly, then sequence from positions in both directions. Increases chances you will hit something interesting.
16
Series of jumps and where sequence from are shown.
18
Arcs are jumps, horizontal lines are sequence. Roman numerals indicate exons.
20
How do we know we have sequenced something interesting? Look for an ORF.
2 types of candidate genes.
21
Used 2 criteria for candidate genes:
Conserved in phylogenetic tree if important for viability. Used strategy for trying to clone further. Looked for CpG islands at 5’end of genes.
22
CpGs tend to fall close to beginning of genes. Housekeeping genes do not often have unmethylated CpG islands, but class 2 proteins do.
23 Found cross-hybridized exons. They probed a zooblot.
24
Found exon1 with ORF. Screened library and got 24 exons. He had sequence of something.
26
Found 12 TM domains.
30
Evidence-Does mutation correlate with disease? Is function disrupted?
31
Sequencing ladder shown. CTT was deleted. Was an in-frame deletion. Segregated with disease.
Did Southern blot. Most of homozygotes had mutation.
33
Ahead 10 years- CFTR protein can have lots of mutations. II most common. Protein never gets to cell surface.
35
Point of exercise is to understand positional cloning (need a method to identify candidate exons- looking for kinase-can look for homology with other kinase sequence, or can express in culture and look for activity). Once you have CDNA, you have to give evidence that your putative candidate gene does in fact cause the disease. Do not have to know names of markers.
Correction for Yesterday:
Pseudoautosomal Inheritance
Erratum for yesterday’s lecture: pseudoautsomal domains include only the ends
Y linked is also holandric inheritance. No daughter carriers. Pseudoautosomal has carrier daughters.
Ways to map genome:
Complementary and tell you different things. Cytogenetic mapping today, then genetic mapping and physical mapping. Genetic is mapping by inheritance. Physical map is the sequence itself. Cytogenetic map is chromosome.
2
Get this. Be able to understand what mapping CF by RFLP means.
3
Chromosomes as spread here have 2 arms. Nomenclature is standard and based on Giemsa staining.
4
R banding is reversed.
5
position is defined unequivocally in international nomenclature.
Ideograms are based on G banding of actual chromosomes.
7
Pattern FYI
8
female pattern. These gross analyses are used for a 1st test for a lot of chromosomal abnormalities.
9
Know that abnormalities exist. She does not care if you know details. Know they can be seen using cytogenetic methods.
10
Aneuploidy
Nondisjunction can occur – chromosomes do not separate in first round- one cell gets none, one gets both chromosomes. Gametes still function and can fertilize or be fertilized. If nondisjunction at second round, normal complement for 2 cells,a diploid, and a zero complement. Know that aneuploidy happens because separation does not occur properly.
Outcome- trisomy 13,18,21 are viable, but bad. Many other trisomies are lethal. XXX not as devastating. Monosomy X is Turner’s syndrome- one of few monosomies to survive.
11
Rearrangements occur with DS breaks. Repair system usually correct these breaks. Balanced- swapping pieces of DNA- no removal or deletion.
Unbalanced- duplicate or loss- serious potential effects.
12
Possible changes. Squashes or FISH can detect these changes in order. Most of the time these are silent.
13
Reciprocal translocations- no net loss or gain, just change in location. This is not recombination. This is translocation between different chromosomes.
May disrupt gene function if break is between promoter and gene. May be silent.
14
Nonreciprocal translocation- adds chromosomal material to another chromosome.
16
Tandem duplication results in repitition of segment.
17
Have knowledge of FISH analysis. #1 for cytogenetic analysis. Can “paint” whole chromosome or other things listed. Human chromosome has lots of repeat sequence. SSRs are inherited markers.
18
FISH probe is complementary only to a region of the chromosome. Family of probes can be made to entire chromosome.
22
Whole chromosome painting- probe each chromosome with a different dye emitting at different wavelength for different color. Can see deletions, increase in chromosome size.
24
Chromosome 19 has unbalanced rearrangement to attach to another chromosome.
She likes FISH analysis.
29
Genetic mapping can cause problems with understanding.
Linkage analysis- tendency for alleles close together on chromosome to be transmitted together. Can map through pedigree analysis. Close together- stay together in recombination. RFLP not used much anymore. Markers are not necessarily genes- they can be followed in pedigrees. Markers allow us to map genes to chromosomes.
30
1. relative to each other.
2. Polymorphic means difference among individuals.
RFLP and SSR are markers.
31
cM is a genetic unit. Crossing over happens a lot, so if genes are together 1% of the time, they are close together. cM and base pairs are not equivalent. To map a disease to a chromosome, must map several markers on each chromosome for inheritance.
33
Allele view. D and M could be separated by recombination. Randomly distributed- not linked.
34
Look at progeny or gametes that result. Some linkage- intermediate.
35
Marker A is not linked to disease phenotype, but marker B is.
Marker is not a predictor of disease per se, but happens to be linked. Not predictor. To be a predictor of disease, if you find marker very very close, can be used as diagnostic event. It may be linked to the disease gene, not associated with the disease itself.
36
RFLP- anything can be used as a marker. Restriction site can be used. 1bp change creates an ECOR1 cut site. RFLP site is inherited. See it a lot in literature, but not used much anymore. Get tons of fragments. Lots of work.
37
How is it done now? SSR or SRS (simple repeat sequence). Microsatellites are the repeat sequence. Repeats det by PCR and electrophoresis. Each chromosome has a different repeat number.Each individual 2 copies with different repeat sequence. Useful way to map. Marker and easier to follow than RFLPs.
38
SSRs in chromosome 1 Markers are under linkage map.
39 Physical mapping
We know the sequence, but we need to be able to navigate around.
40
Take part of human chromosome and fuse into rodent cell line- panel of hybrids with different parts of chromosome.PCR for gene of interest. Clonal lines .
STS are physical sites that are not simple repeat sequence. These are not polymorphic. These do not change and are the same in every human being. Cannot follow in genetic map because everyone has the same ones. One copy.
Fri- clone CF gene. Tues- inherited color vision, fragile X, Huntingdon’s disease.
Senogles 3
Assignment is posted.
Today we talk about positional cloning. Text reference is to cystic fibrosis (CF). Specific references are not required reading. Presented because it is good science and illustrative of how you do the technique.
3
Functional common until 1990s. Purified protein, followed activity as you purified, got to homogeneity, sequenced, got protein sequence, made oligonucleotide, screened library, got gene. You knew what you were looking for and what the activity was.
Now we look on the basis of where it maps on the chromosome with very little protein information. Positional cloning- map by genetics, then clone.
4
Knowing the DNA sequence does not help because you do not know what you are looking for.
5
2 types of mapping: see slide
6
10% codes for exons. Small part of genome.
7
Localize by linkage analysis position of disease gene you are interested in. Want to localize to greatest extent we can. Yacs and BACs can handle large pieces of DNA by cloning.
If you go to human genome project, is good resource. Can click on a region and see all the known genes in the region.
The two markers were well known.
Linkage disequilibrium- had a mutation in a chromosome. As goes through successive generations, some markers closely segregate with it. B1 stays with CF. It is in linkage disequilibrium. Suggests a founder chromosome with that marker.
11
High resolution map. Things with D are markers, polymorphic, inherited. Closer you can narrow it, less DNA you have to sequence.
12
Trying to minimize brute-force sequencing.
CpG islands tend to be islands of methylated CG that are close to promoters in coding regions. If it cross hybridizes, it is conserved and may be the candidate gene.
13
Want to isolate large fragments first. Large fragment- Mb.
14 Question: Disease- how to clone it out?
Chromosome walking- series overlapping clones. Cut up DNA. Make series of restriction fragments. Pull out and sequence along. Laborious.
15
Chromosome jumping- maximize potential hits by jumping to places from which you can sequence. Covers maximum amount of distance and bypasses unsequenceable stretches of DNA.
Circularized- A and B become close. Can sequence short segment between A and B to find the sequence of site B. Can jump, do not plan to go anywhere. You don’t really care what was between A and B to begin. Can make jumping library, and make a library of sites to walk from. Jumping randomly, then sequence from positions in both directions. Increases chances you will hit something interesting.
16
Series of jumps and where sequence from are shown.
18
Arcs are jumps, horizontal lines are sequence. Roman numerals indicate exons.
20
How do we know we have sequenced something interesting? Look for an ORF.
2 types of candidate genes.
21
Used 2 criteria for candidate genes:
Conserved in phylogenetic tree if important for viability. Used strategy for trying to clone further. Looked for CpG islands at 5’end of genes.
22
CpGs tend to fall close to beginning of genes. Housekeeping genes do not often have unmethylated CpG islands, but class 2 proteins do.
23 Found cross-hybridized exons. They probed a zooblot.
24
Found exon1 with ORF. Screened library and got 24 exons. He had sequence of something.
26
Found 12 TM domains.
30
Evidence-Does mutation correlate with disease? Is function disrupted?
31
Sequencing ladder shown. CTT was deleted. Was an in-frame deletion. Segregated with disease.
Did Southern blot. Most of homozygotes had mutation.
33
Ahead 10 years- CFTR protein can have lots of mutations. II most common. Protein never gets to cell surface.
35
Point of exercise is to understand positional cloning (need a method to identify candidate exons- looking for kinase-can look for homology with other kinase sequence, or can express in culture and look for activity). Once you have CDNA, you have to give evidence that your putative candidate gene does in fact cause the disease. Do not have to know names of markers.
Wednesday, February 7, 2007
Senogles 1
Senogles 1
Why is genetics in curriculum?
Design of course- human and Mendelian genetics and basis of disease. We need to be able to read a pedigree for following lectures. Friday- looking at positional cloning of genetic disease genes. She means her learning objectives. Know what she says.
Also know pseudoautosomal or Y-linked.
3.
Definitions she will use. Email her if you have questions.
Marker is known DNA sequence- not a gene enecessarily. Locus usually refers to a gene.
Dominant and recessive refers to traits, Not genes.
5
44 of our chromosomes are autosomes. 2 sex chromosomes.
Somatic cell is anything nongonadal. Germ cells are haploid.
6.
G bands are reproducible.
Upper arm is P, lower Q. Centromere in middle. Number corresponds to nomenclature shown here when you look up a genetic mutation or deletion for a disease.
8
Mitosis- how somatic cells reproduce. Same complement of chromosomes results in the end.
9
Meiosis- haploid complement.
10
Recombination between nonsister chromaids occurs at metaphase 1.
2 points: reduces to haploid state, and recombination in metaphase 1 scrambles information between maternal and paternal alleles.
13
Standard
Open symbol- person not affected by disease. Fillled in affected.
Proband- where pedigree begins.
16
Principle of independent segregation- each offspring independently has probability of inheriting a gene.
Each offspring independent.
17
Autosomal dominant- can be caused by new mutation, but otherwise appears every generation. No gender bias.
18 Sometimes normal mates are not shown. Proband is first person identified.
Pedigree may not show exact 50/50 segregation.
19
Phenotype may not show up until later.
22
Autosomal recessive option often left to end. Often sproadic
23
X-linked is trickier. Females compensate for gene dosage by inactivating one X chromosome in somatic tissue. Females are X mosaics.
26
X-linked dominant- affected male- affected daughters, not sons. No male to male transmission. Follow pattern- connected through female. No male to male.
29X-linked recessive- transmitted from affected male through daughters. No male to male transmission. Carrier females show in pedigree.
30
Half males affected, 25% of females carrier.
32
Obligate heterozygote is carrier. (synonym)
33
Y linked traits do occur. X and Y line up- only recombine on end portions. Not a gene dense region. Rest of chromosome cannot recombine X-Y.
34 Only males affected. See it in every generation, but only males affected. Rare pedigrees.
35
Atypical- genomic imprinting is difference in gene expression between allele from mom and one from Dad. Only a few of these. Mice have more than we do. Not completely understood- thought to be differential methylation of DNA. Imprinting- which parent the chromosome is from determines outcome.
Why is genetics in curriculum?
Design of course- human and Mendelian genetics and basis of disease. We need to be able to read a pedigree for following lectures. Friday- looking at positional cloning of genetic disease genes. She means her learning objectives. Know what she says.
Also know pseudoautosomal or Y-linked.
3.
Definitions she will use. Email her if you have questions.
Marker is known DNA sequence- not a gene enecessarily. Locus usually refers to a gene.
Dominant and recessive refers to traits, Not genes.
5
44 of our chromosomes are autosomes. 2 sex chromosomes.
Somatic cell is anything nongonadal. Germ cells are haploid.
6.
G bands are reproducible.
Upper arm is P, lower Q. Centromere in middle. Number corresponds to nomenclature shown here when you look up a genetic mutation or deletion for a disease.
8
Mitosis- how somatic cells reproduce. Same complement of chromosomes results in the end.
9
Meiosis- haploid complement.
10
Recombination between nonsister chromaids occurs at metaphase 1.
2 points: reduces to haploid state, and recombination in metaphase 1 scrambles information between maternal and paternal alleles.
13
Standard
Open symbol- person not affected by disease. Fillled in affected.
Proband- where pedigree begins.
16
Principle of independent segregation- each offspring independently has probability of inheriting a gene.
Each offspring independent.
17
Autosomal dominant- can be caused by new mutation, but otherwise appears every generation. No gender bias.
18 Sometimes normal mates are not shown. Proband is first person identified.
Pedigree may not show exact 50/50 segregation.
19
Phenotype may not show up until later.
22
Autosomal recessive option often left to end. Often sproadic
23
X-linked is trickier. Females compensate for gene dosage by inactivating one X chromosome in somatic tissue. Females are X mosaics.
26
X-linked dominant- affected male- affected daughters, not sons. No male to male transmission. Follow pattern- connected through female. No male to male.
29X-linked recessive- transmitted from affected male through daughters. No male to male transmission. Carrier females show in pedigree.
30
Half males affected, 25% of females carrier.
32
Obligate heterozygote is carrier. (synonym)
33
Y linked traits do occur. X and Y line up- only recombine on end portions. Not a gene dense region. Rest of chromosome cannot recombine X-Y.
34 Only males affected. See it in every generation, but only males affected. Rare pedigrees.
35
Atypical- genomic imprinting is difference in gene expression between allele from mom and one from Dad. Only a few of these. Mice have more than we do. Not completely understood- thought to be differential methylation of DNA. Imprinting- which parent the chromosome is from determines outcome.
Zhang notes, both lectures
Recommended reading is from Lodish. He recommends Albert’s molecular biology of the cell. Everything we will discuss is in handout. It should be good enough for the exam.
Cells are assembled into functionally coordinated assemblies called tissue in multicellular organisms. They are connected by cell-cell adhesions. Beside contacting with other cells, cells can also interact with the extracellular matrix.
Interaction categories:
1. cell-cell adhesion
2. cell-matrix adhesion
19-1:
Epithelium is full of cell-cell contact. Also interact with underlying connective tissue. Large molecules are secreted by fibroblasts. They synth and org the ECM.
Today- mechanism to make cells stick together.Epithelium has 3 sections: apical surface, baso-lateral surface, basal. Basal- interact through cell junction and non-cell junction. Interact with basal membrane through ECM adhesion . Junction functions listed in handout. Junction also active in signalling.
Skip a few pages- classification cell adhesion
Junctional mechanism means adhesion based on adhesive structures (cell-cell junction or cell-matrix junction) . Many adhesion mechanisms- nothing visible under EM (Ex: T cell interaction with APC) . When neuron extends neurites, extension based on cell-cell adhesion. Junction mechanisms listed in picture; you can tell functions from name. Tight junction also called occluding junction. Desmosome sticks cells together. Gap junction makes pores between cells for communication.
Cell-matrix adhesion: desmosome and hemidesmosomes are in skin. Desmosome connected to intermediate filaments, thicker than cytoskeleton to resist mechanical stress. Cadherin junction found in many tissues. Non junction adhesion mechanisms- no structural base to visualize under EM.
Tight junction also called occluding junction. Present in most epithelium as well as endothelium. Present at apical end of the basolateral membrane. Network sealing strands account for cell-cell adhesion. EM shows them in the intestinal lumen. The thin strands form the tight junction to seal apical end. Functions- next page.
If you are starving- tight junction is regulated to allow glucose into circulation under extreme conditions. Otherwise it does not allow glucose in.
Tight junction regulated by pore.
Regultion by assembly or disassembly- tight junction comes and goes in previous slide. For individual cells, junction subdivides plasma membrane into different compartments. Basolateral proteins cannot be freely translocated to apical surface or vice versa. Apical membrane has different functions from basolateral. Cancer cells lose tight junction and behave aberrantly due to loss of distinction.
Compsition: Every cell adhesion mechanism can be divided into 3 parts:
1. TM linker proteins called CAMs
2. cytoplasmic adaptor proteins
3. cytoskeleton.
Engage environmental elements and internal elements to create adhesion. All 3 are essential. Adaptor proteins used for regulation and bridging.
Occludin and claudin are 4 transmembrane proteins. Desmosome interacts with intermediate filaments, tight junction with actin filaments.
Extracellular: claudins and occludins. Called type III proteins (N and C terminus intracellular). Matches up with corresponding portions of protein in adjacent cells. Intracellular interact with adaptor proteins. There are many adaptor proteins. Through ZO1 and ZO2 interact with cytoskeleton. Essential components are still controversial.
GAP junction- communicating junctions. Found even in fibroblasts, epithelium, endothelium. Is channel for small molecules between 2 intracellular environments. Is nonspecific. See handout. 10 Amino acids=1 kD. Can label ubiquitin, but it will not pass, which is <1 kD. Can try injecting dye molecules of different sizes into cells and see if they pass.
Difference between gap junction and synapse- chemical in synapse has delay passing from upstream to downstream cell. No delay in gap junction. Fish escape fast not based on synapse, but on diffusion of messages through gap junction. Heart beat transduced through gap junction mechanism.
EM junction is next.
Molecular base: Gap junction formd by connexins with 4 TM domains. 6 of them form oligomer from one cell. One connexon engages another in extracellular domain.
Back to p.5
Families of CAMs here.
Cadherin is important CAM. Engages homophilic interaction. Counterreceptor is itself. Cadherin engages cadherin from another cell. Depends on Ca or Mg- divalent cation. Only cell adhesion molecule not dependent on Ca or Mg is Ig family.
Cmadh- integrins. Integrin can also engage cell-cell adhesion.
Cadherin is a type 1 TM protein. Functions dependent on Ca. Se handout.Classical form adherens junction.
How do cadherins engage homophilic interaction?Cadherin has affinity for Ca. Conformational change in absence- can be chipped off by protease. Trypsin and EDTA (chelates Ca)used to make cells confluent. Cadherin floppy without Ca. Some cells can be detached only with EDTA iif cell-cell adhesion not well developed (as in cancer cells) Prtottype is E cadherin. Extracellullarly has 5 domains. Each domain contains Ca binding seq. Membrane-proximal has higher Ca affinity than distal. Low [Ca]- binds to proximal dom first. Triggers conformational change resulting in stiffness of subdomain. Higher Ca- distal stiff. Cis interaction must occur first. Dimerization on same cell is intermediated by seq between C1 and C2. Once homodimerization accomplished. Conformation is ready for trans interaction with another pair from adjacent cell to accomplish homophilic interaction.
Functions: see handout.Cadherins serve as sorting mechanism for cells in embryogenesis. Only functions ID cells come together. Prevents intrusion from other tissues. How would you demonstrate interaction homophilically? Simple experiment. Have to find system that does not express E cadherin, express it, and observe. Fibroblasts do not express E-cadherin or other cadherins. Transfected fibroblasts and observed. Fibroblasts with E cadherin came together. Were important for cell-cell adhesion and only cells with E cadherin could be sorted to specific place.
Neural tube development from epithelial cells. E-cadherin is major one experessed in epithelial cells. Neural tube cells stopped expressing E and started expressing N cadherin. Formed 2 organs based on sorting.
Neural crest cells migrate and form lot of organs. When they migrate lose N cadherin expression. Aggregate wih expression of N cadherin again at the end.
Cadherins also play role in pathologic conditions.
When cancer loses E cadherin, can undergo epithelial-mesenchymal transition. Transfection in cancer cells to increase cadherin expression can stop cancer migration.
How cadherin connected to intracellular cytoskeleton- Slide shows classical cadherins interaction with actin. Catenins are adaptor proteins. Beta and gamma engage cytoplasmic domains of cadherins. Alpha interacts with cytoskeleton as a bridge. In some pathological conditions, level of one or more catenins is altered.
Well known signalling pathway-wnt pathway. Ligand engages frizzled receptor. Activates disheveled (names from Drosophila mutation) Dsh inactivates GSk3beta (glycogen synthase kinase) Once GSK3B activated, associates with B-catenin, binds APC (adenomatous polyposis coli protein)GSK phosphorylates N terminal B catenin and targets by APC through ubiquitination to be degraded.
If APC is mutated, B catenin accumulates in cytoplasm. Wnt signalling activation results in inactivated GSK. No B-catenin gotten rid of- accumulates in cytoplasm. Consequent accumulation of B-catenin- saturates cadherin junction. Extra will translocate interaction to nucleus. Can bind a variety of TFs to activate transcription. This is necessary during embryogenesis, but once development is finished, wnt pathway should be deactivated.
Desmosome- homophilic, Ca dependent interaction. Difference between desmosome and cadherin junction is cytoskeletal part. Linked to intermediate filaments. Plakoglobins are gamma catenins. Mutation in desmosome results in disease.Mutation in desmoplakins- blister easily.
Hemidesmosome is not cell-cell adhesion molecule. It is a is cell matrix adhesion molecule. Connects to keratins in skin.
Some molecules have no cytoskeletal structural base. Cell-cell adhesion mediated by Ig family of molecules.Ca independent interaction. Structural Ig subdomains is similar to antibody subdomains. VCAM is Ig superfamily proteins.
Zhanglecture 2
This talk about extracellular matrix and adhesion molecules necessary for ECM adhesion. Complex matrix of large molecules is the extracellular matrix. Produced by fibroblasts. Fibroblasts are migratory and use the fibers they produce to migrate around. Basal lamina is produced by epithelial layer above it.
What are the functions of the ECM?
Fill and provide order to extracellular space.
ECM has ligands for cell adhesion proteins and can communicate signals to cell about survival and differentiation and motility through these molecules. Also reservoirs for growth factors, chemokines, cytokines. Prevent growth factor effects from being too transient or abrupt- buffer them and prolong effects through persistent release. ECM is important for educating stem cells to commit for different lineages.
Provides way for cells to move within tissues. Cells migrate on collagen molecules like a railroad track.
Components ECM?
Complicated. Only going through major components. 2 parts: proteins and polysaccharides or proteoglycans.
Large space in ECM filled by proteoglycans- combination of peptides with polysaccharides. Difference between proteoglycan and glycoprotein- glycoprotein mostly from AAs. Proteoglycan- opposite. Cell culture used to be on the flask, but this is not physiological. In vivo, growth factors come from below, not above. More experiments are done these days on 3-D collagen gel to closely mimic in vivo conditions. Lots of proteoglycans included.
Structure is simple for proteoglycan- GAGs are building blocks of them.
Major component connective tissue- collagen.Basic biochem feature is Gly-X-Y triplet repetitive sequence. Y is typically proline or hydroxyproline. Enzyme prolyl hydroxylase is important for maturation of collagen. Hydroxylation required for lateral association. Each collagen- 3 chains. Form a triplex. Multiple helices for collagen fibers. Hydroxyproline negatively charged- can form H bonds between chains to provide strong bonds in helix. Enzyme to hydroxylate proline require vitamin C.
Fibrillar collagen- connective tissues.
Fibril-associated collagens have more flexibility.
Network form networks instead of fibers.
Fibril assoc collagen attach to fibrillar collagens. Triplets are interrupted by non-triplet sequence- increases flexibility.
Type IV is different- network in basement membrane.Laminin, entactin, proteoglycans, other proteins are also there. C terminal interaction is covalent. Lateral interactions are mediated by H bonds.
Goodpasture’s syndrome- affects glomerulus and lung because endothelium and epithelium interact at basement membrane with blood vessel on one side and lumen on other. Patient develops autoantibody to Alpha 3 chain of collagen IV. Antibody precipitates in lung and kidney.
Different forms basement membrane occur. The muscle form is thin and less well developed than that in other locations, but it is still important for health. It surrounds individual muscle cells. Muscular dystrophy can be caused by many factors- one is when the muscle cell cannot attach securely to basement membrane. Mutation in an integrin keeps muscle from attaching to membrane and cannot retract firmly.
Stereotype is that of epithelium and endothelium. The basement membrane in kidney is fusion of basement membrane of epithelium and endothelium.
Matrigel- constructed from basement membrane to use as 3-D culture system- from mouse tumor thans secretes lots of basement membrane components.
Called recombinant basement membrane- used to culture cancer cells, stem cells, epithelium.
Laminin- isolated from lamina. Large protein formed by 3 beta chains.
Fibronectin is a major non-collagen ECM protein. Typically basic unit is single chain. Dimerizes at C terminal through disulfide bond.Type III fibronectin subdomain is one of the most observed ones in the body. Found in lots of proteins. 3AAs- RGD form a loop projecting from Type III domain- is bonding site for integrin 5 beta1, which is fibronectin receptor.Cell binding region is Type III subdomain where it binds to integrins listed. Leukocytes use Cell Binding Site #2 to migrate into tissues.
Integrins- affinity ECM and proteins is lower than intracellular interactions. Could use affinity chromatography to isolate. Ligand to column and pass cell lysate through. Put fibronectin on column and found the cellular receptor called integrin.
Integrins function as cell-cell adhesion molecules as well.Function as dimer of alpha and beta chains. Relatively large EC domain and short cytoplasmic chains..
Collagen receptors are alpha1 and alpha2 (major ones)See sheet.
RGD binding integrins bind to fibronectins and other RGD-containing proteins. Slide is a headache- have rough concept. Do memorize the ones for collagen and laminin.
Integrins can also be used as receptor to invade cells by bacteria and viruses.
Cytoplasmic view of integrins- has transmembrane linker proteins and intracellular skeletal protein and adapter proteins. Most integrins bind to actin cytoskeleton. Actinin and talin are well-established bridging proteins.
Special case in spleen- intermediate filaments interact with alpha 6 beta 4 integrin in spleen.
Integrins are often physiologically inactive. Dangerous to be active all the time. Only want to activate in neutrophils or platelets under special circumstances. Only under wound condition do you want platelets to aggregate.
Most cells do not survive unless attached because of integrin signalling. Cancer cells have aberrant integrin signalling to avoid apoptosis.
Cells are assembled into functionally coordinated assemblies called tissue in multicellular organisms. They are connected by cell-cell adhesions. Beside contacting with other cells, cells can also interact with the extracellular matrix.
Interaction categories:
1. cell-cell adhesion
2. cell-matrix adhesion
19-1:
Epithelium is full of cell-cell contact. Also interact with underlying connective tissue. Large molecules are secreted by fibroblasts. They synth and org the ECM.
Today- mechanism to make cells stick together.Epithelium has 3 sections: apical surface, baso-lateral surface, basal. Basal- interact through cell junction and non-cell junction. Interact with basal membrane through ECM adhesion . Junction functions listed in handout. Junction also active in signalling.
Skip a few pages- classification cell adhesion
Junctional mechanism means adhesion based on adhesive structures (cell-cell junction or cell-matrix junction) . Many adhesion mechanisms- nothing visible under EM (Ex: T cell interaction with APC) . When neuron extends neurites, extension based on cell-cell adhesion. Junction mechanisms listed in picture; you can tell functions from name. Tight junction also called occluding junction. Desmosome sticks cells together. Gap junction makes pores between cells for communication.
Cell-matrix adhesion: desmosome and hemidesmosomes are in skin. Desmosome connected to intermediate filaments, thicker than cytoskeleton to resist mechanical stress. Cadherin junction found in many tissues. Non junction adhesion mechanisms- no structural base to visualize under EM.
Tight junction also called occluding junction. Present in most epithelium as well as endothelium. Present at apical end of the basolateral membrane. Network sealing strands account for cell-cell adhesion. EM shows them in the intestinal lumen. The thin strands form the tight junction to seal apical end. Functions- next page.
If you are starving- tight junction is regulated to allow glucose into circulation under extreme conditions. Otherwise it does not allow glucose in.
Tight junction regulated by pore.
Regultion by assembly or disassembly- tight junction comes and goes in previous slide. For individual cells, junction subdivides plasma membrane into different compartments. Basolateral proteins cannot be freely translocated to apical surface or vice versa. Apical membrane has different functions from basolateral. Cancer cells lose tight junction and behave aberrantly due to loss of distinction.
Compsition: Every cell adhesion mechanism can be divided into 3 parts:
1. TM linker proteins called CAMs
2. cytoplasmic adaptor proteins
3. cytoskeleton.
Engage environmental elements and internal elements to create adhesion. All 3 are essential. Adaptor proteins used for regulation and bridging.
Occludin and claudin are 4 transmembrane proteins. Desmosome interacts with intermediate filaments, tight junction with actin filaments.
Extracellular: claudins and occludins. Called type III proteins (N and C terminus intracellular). Matches up with corresponding portions of protein in adjacent cells. Intracellular interact with adaptor proteins. There are many adaptor proteins. Through ZO1 and ZO2 interact with cytoskeleton. Essential components are still controversial.
GAP junction- communicating junctions. Found even in fibroblasts, epithelium, endothelium. Is channel for small molecules between 2 intracellular environments. Is nonspecific. See handout. 10 Amino acids=1 kD. Can label ubiquitin, but it will not pass, which is <1 kD. Can try injecting dye molecules of different sizes into cells and see if they pass.
Difference between gap junction and synapse- chemical in synapse has delay passing from upstream to downstream cell. No delay in gap junction. Fish escape fast not based on synapse, but on diffusion of messages through gap junction. Heart beat transduced through gap junction mechanism.
EM junction is next.
Molecular base: Gap junction formd by connexins with 4 TM domains. 6 of them form oligomer from one cell. One connexon engages another in extracellular domain.
Back to p.5
Families of CAMs here.
Cadherin is important CAM. Engages homophilic interaction. Counterreceptor is itself. Cadherin engages cadherin from another cell. Depends on Ca or Mg- divalent cation. Only cell adhesion molecule not dependent on Ca or Mg is Ig family.
Cmadh- integrins. Integrin can also engage cell-cell adhesion.
Cadherin is a type 1 TM protein. Functions dependent on Ca. Se handout.Classical form adherens junction.
How do cadherins engage homophilic interaction?Cadherin has affinity for Ca. Conformational change in absence- can be chipped off by protease. Trypsin and EDTA (chelates Ca)used to make cells confluent. Cadherin floppy without Ca. Some cells can be detached only with EDTA iif cell-cell adhesion not well developed (as in cancer cells) Prtottype is E cadherin. Extracellullarly has 5 domains. Each domain contains Ca binding seq. Membrane-proximal has higher Ca affinity than distal. Low [Ca]- binds to proximal dom first. Triggers conformational change resulting in stiffness of subdomain. Higher Ca- distal stiff. Cis interaction must occur first. Dimerization on same cell is intermediated by seq between C1 and C2. Once homodimerization accomplished. Conformation is ready for trans interaction with another pair from adjacent cell to accomplish homophilic interaction.
Functions: see handout.Cadherins serve as sorting mechanism for cells in embryogenesis. Only functions ID cells come together. Prevents intrusion from other tissues. How would you demonstrate interaction homophilically? Simple experiment. Have to find system that does not express E cadherin, express it, and observe. Fibroblasts do not express E-cadherin or other cadherins. Transfected fibroblasts and observed. Fibroblasts with E cadherin came together. Were important for cell-cell adhesion and only cells with E cadherin could be sorted to specific place.
Neural tube development from epithelial cells. E-cadherin is major one experessed in epithelial cells. Neural tube cells stopped expressing E and started expressing N cadherin. Formed 2 organs based on sorting.
Neural crest cells migrate and form lot of organs. When they migrate lose N cadherin expression. Aggregate wih expression of N cadherin again at the end.
Cadherins also play role in pathologic conditions.
When cancer loses E cadherin, can undergo epithelial-mesenchymal transition. Transfection in cancer cells to increase cadherin expression can stop cancer migration.
How cadherin connected to intracellular cytoskeleton- Slide shows classical cadherins interaction with actin. Catenins are adaptor proteins. Beta and gamma engage cytoplasmic domains of cadherins. Alpha interacts with cytoskeleton as a bridge. In some pathological conditions, level of one or more catenins is altered.
Well known signalling pathway-wnt pathway. Ligand engages frizzled receptor. Activates disheveled (names from Drosophila mutation) Dsh inactivates GSk3beta (glycogen synthase kinase) Once GSK3B activated, associates with B-catenin, binds APC (adenomatous polyposis coli protein)GSK phosphorylates N terminal B catenin and targets by APC through ubiquitination to be degraded.
If APC is mutated, B catenin accumulates in cytoplasm. Wnt signalling activation results in inactivated GSK. No B-catenin gotten rid of- accumulates in cytoplasm. Consequent accumulation of B-catenin- saturates cadherin junction. Extra will translocate interaction to nucleus. Can bind a variety of TFs to activate transcription. This is necessary during embryogenesis, but once development is finished, wnt pathway should be deactivated.
Desmosome- homophilic, Ca dependent interaction. Difference between desmosome and cadherin junction is cytoskeletal part. Linked to intermediate filaments. Plakoglobins are gamma catenins. Mutation in desmosome results in disease.Mutation in desmoplakins- blister easily.
Hemidesmosome is not cell-cell adhesion molecule. It is a is cell matrix adhesion molecule. Connects to keratins in skin.
Some molecules have no cytoskeletal structural base. Cell-cell adhesion mediated by Ig family of molecules.Ca independent interaction. Structural Ig subdomains is similar to antibody subdomains. VCAM is Ig superfamily proteins.
Zhanglecture 2
This talk about extracellular matrix and adhesion molecules necessary for ECM adhesion. Complex matrix of large molecules is the extracellular matrix. Produced by fibroblasts. Fibroblasts are migratory and use the fibers they produce to migrate around. Basal lamina is produced by epithelial layer above it.
What are the functions of the ECM?
Fill and provide order to extracellular space.
ECM has ligands for cell adhesion proteins and can communicate signals to cell about survival and differentiation and motility through these molecules. Also reservoirs for growth factors, chemokines, cytokines. Prevent growth factor effects from being too transient or abrupt- buffer them and prolong effects through persistent release. ECM is important for educating stem cells to commit for different lineages.
Provides way for cells to move within tissues. Cells migrate on collagen molecules like a railroad track.
Components ECM?
Complicated. Only going through major components. 2 parts: proteins and polysaccharides or proteoglycans.
Large space in ECM filled by proteoglycans- combination of peptides with polysaccharides. Difference between proteoglycan and glycoprotein- glycoprotein mostly from AAs. Proteoglycan- opposite. Cell culture used to be on the flask, but this is not physiological. In vivo, growth factors come from below, not above. More experiments are done these days on 3-D collagen gel to closely mimic in vivo conditions. Lots of proteoglycans included.
Structure is simple for proteoglycan- GAGs are building blocks of them.
Major component connective tissue- collagen.Basic biochem feature is Gly-X-Y triplet repetitive sequence. Y is typically proline or hydroxyproline. Enzyme prolyl hydroxylase is important for maturation of collagen. Hydroxylation required for lateral association. Each collagen- 3 chains. Form a triplex. Multiple helices for collagen fibers. Hydroxyproline negatively charged- can form H bonds between chains to provide strong bonds in helix. Enzyme to hydroxylate proline require vitamin C.
Fibrillar collagen- connective tissues.
Fibril-associated collagens have more flexibility.
Network form networks instead of fibers.
Fibril assoc collagen attach to fibrillar collagens. Triplets are interrupted by non-triplet sequence- increases flexibility.
Type IV is different- network in basement membrane.Laminin, entactin, proteoglycans, other proteins are also there. C terminal interaction is covalent. Lateral interactions are mediated by H bonds.
Goodpasture’s syndrome- affects glomerulus and lung because endothelium and epithelium interact at basement membrane with blood vessel on one side and lumen on other. Patient develops autoantibody to Alpha 3 chain of collagen IV. Antibody precipitates in lung and kidney.
Different forms basement membrane occur. The muscle form is thin and less well developed than that in other locations, but it is still important for health. It surrounds individual muscle cells. Muscular dystrophy can be caused by many factors- one is when the muscle cell cannot attach securely to basement membrane. Mutation in an integrin keeps muscle from attaching to membrane and cannot retract firmly.
Stereotype is that of epithelium and endothelium. The basement membrane in kidney is fusion of basement membrane of epithelium and endothelium.
Matrigel- constructed from basement membrane to use as 3-D culture system- from mouse tumor thans secretes lots of basement membrane components.
Called recombinant basement membrane- used to culture cancer cells, stem cells, epithelium.
Laminin- isolated from lamina. Large protein formed by 3 beta chains.
Fibronectin is a major non-collagen ECM protein. Typically basic unit is single chain. Dimerizes at C terminal through disulfide bond.Type III fibronectin subdomain is one of the most observed ones in the body. Found in lots of proteins. 3AAs- RGD form a loop projecting from Type III domain- is bonding site for integrin 5 beta1, which is fibronectin receptor.Cell binding region is Type III subdomain where it binds to integrins listed. Leukocytes use Cell Binding Site #2 to migrate into tissues.
Integrins- affinity ECM and proteins is lower than intracellular interactions. Could use affinity chromatography to isolate. Ligand to column and pass cell lysate through. Put fibronectin on column and found the cellular receptor called integrin.
Integrins function as cell-cell adhesion molecules as well.Function as dimer of alpha and beta chains. Relatively large EC domain and short cytoplasmic chains..
Collagen receptors are alpha1 and alpha2 (major ones)See sheet.
RGD binding integrins bind to fibronectins and other RGD-containing proteins. Slide is a headache- have rough concept. Do memorize the ones for collagen and laminin.
Integrins can also be used as receptor to invade cells by bacteria and viruses.
Cytoplasmic view of integrins- has transmembrane linker proteins and intracellular skeletal protein and adapter proteins. Most integrins bind to actin cytoskeleton. Actinin and talin are well-established bridging proteins.
Special case in spleen- intermediate filaments interact with alpha 6 beta 4 integrin in spleen.
Integrins are often physiologically inactive. Dangerous to be active all the time. Only want to activate in neutrophils or platelets under special circumstances. Only under wound condition do you want platelets to aggregate.
Most cells do not survive unless attached because of integrin signalling. Cancer cells have aberrant integrin signalling to avoid apoptosis.