three polypeptide α chains

arranged in a triple helical structure

that results from repeat of amino acids Gly-X-Y

Gly-X-Y

Glycine is smallest amino acid with H side chain

X is usually Proline

Y is usually HydroxyLysine orHydroxyProline

(which arise from post-translational modification)

GLY is so small due it Hydrogen as its side chain

→ so it is tolerated when the helix is coming together

mutations replacing it with a larger amino acid

yield disastrous consequences

3 groups of collagen

and their Types [#s only]

1. Fibril forming–“classic” collagens that are found in supporting elements of high tensile strength [Types 1-3]

2. Network Forming – form a 3-D mesh rather than distinct fibrils

[Types 4 & 7]

3. Fibril- Associated – bind to the surface of collagen fibrils. Linking them to one another [Types 9, 12]

Type I- skin bone, tendon, blood vessels, cornea

Type II- Cartilage, intervertebral disk, vitreous body

Type III- blood vessels, fetal skin

Type IV- basement membrane

Type VII- Beneath stratified squamous epithelia

Type IX- Cartilage

Type XII- Tendon, ligaments, some other tissues

How many types of collagen is in the body?

How many genes encode for collagen?

about 30 different types of collagen

MANY collagen genes scattered throughout genome

• Usually have about 50 exons
• On interior of molecule many are 54 bp exons repeats
  • may have resulted from duplication of single parental exon to muliple exons

Genes for pro α 1 and pro α 2 chains are transcribed into mRNA

Collagen mRNA is translated on RER ribosomes in the cytosol

into prepro α polypeptide chains

that are extruded into the endoplasmic reticulum,

where the signal sequence is removed

Select proline and lysine residues are

hydroxylated by prolyl or lysyl hydroxylase

(requires vitamin C and iron as cofactors)

Select hydroxylysine residues are glycosylated

with glucose and galactose

by glucosyl transferase or galactosyl transferase.

(Precursor molecule = Procollagen (not mature) –

has residues at both C and N terminal end

that don’t form triple helix structure)

Collagen molecules begin self assembly process

(3 pro-α chains assemble)

and form intrachain and interchain disulfide bonds

at the C-terminal polypeptide extension (stablilizing molecule)

Procollagen molecule Sent to Golgi-

packaged for secretion → excreted into extracellular matrix

Step 4.

After select hydroxylysine residues are glycosylated with glucose and galactose by glucosyl transferase or galactosyl transferase

After secretion→

N terminal and C terminal end are cleaved off

(by procollagen peptidases) → mature collagen

Step 8

N terminal and C terminal end are cleaved off

(by procollagen peptidases)

(1) Mature collagen spontaneously come together

to form fibrils in an ordered, parallel, overlapping array

(2) Adjacent molecules are arranged in a staggered pattern

to give added strength

(by intramolecular cross links

between neighboring collagen molecules )

What enzymes are needed immediately aftercollagen translation

(within the ER)?

What are there co-factors?

Prolyl hydroxylase and lysyl hydroxylase

→ hydroxylase proline and lysine

respectively require Fe 2+ and ascorbic acid (vitamin C) as cofactors

Lysyl Oxidase (requires copper as a cofactor)

oxidatively deaminates some of the lysyl and hydroxylysyl residues, resulting in reactive aldehydes

The reactive aldehydes can condense with lysyl or hydroxylysyl in neighboring collagen molecules to form covalent cross-links

Dietary deficiency of copper or ability to utilize copper properly

→ defect in collagen because it can’t effectively cross-link with itself

X-linked Recessive

caused by mutations in the ATP7A gene

that is responsible for production of the ATPase enzyme

that regulates copper levels in the body

- decreased supply of copper

can reduce the activity of numerous copper-containing enzymes

that are necessary for the structure and function of bone, skin, hair, blood vessels and the nervous system

non-genetically based collagen disorder

due to dietary deficiency of vitamin C

(Primates and guinea pigs cannot synthesis vitamin C on their own)

disease prevalent in areas where malnutrition is common

easily bruised skin, bleeding gums, “corkscrew hair”,

loose teeth, poor wound healing

James Lind (1747) – on a voyage from England to Plymouth, Mass

1753 Lind published his work

Greens and fruits with vitamin C are best remedy for scurvy

*48 years later – British navy declares sailors should have daily ration of lime juice

Osteogenesis Imperfecta (OI) [Types 1-4]

Ehlers-Danlos Syndrome

Type 2,3,4 OI

• Most result from substitutions in gene for COL1A1 or COL1A2
• Gly --> another amino acid with a bulky side chain in Type 1 collagens which prevents correct folding into triple helix
  
  • Missense mutation → collagen structure is improper

Type 1 OI

• usually a result of a nonsense mutation, resulting in a premature stop codon
•  causes Nonsense mediated decay and decreased collagen
  • collagen is formed properly – there is just less of
    

Severity of OI Types: II>III>IV> I -but clinical features vary greatly (symptoms are not the same for all affected individuals)

Type 4 is one of most severe

because it results in arterial or uterine rupture

Type 4 [Vascular EDS]

Describe elastin.

Where is it found in the body?

rubbery connective tissue that

can be stretched and bent in any direction

to several times its length

and then returned to original size and shape

Found in walls of large arteries, lungs, and elastic ligaments

What is the precuror to elastin?

How many genetic types of elastic exist?

An insoluble protein polymer synthesized from precursor, tropoelastin

Only one genetic type unlike collagen

Rich in glycine, proline, and lysine

only has a little hydroxyproline, and NO hydroxylysine

How does crosslinking makes elastin rubbery?

What kind of link is formed?

Some of the lysyl side chains are oxidatively deaminated

by lysyl oxidase, forming allysine residues

Three of the allysyl side chains plus one unaltered lysyl side chain form a desmosine cross-link

This cross-linking helps make elastin

an extensively interconnected rubbery network

Autosomal Dominant

linked to FBN1 gene on chromosome 15

FBN1- encodes for fibrillin protein

essential for formation of elastic fibers found in connective tissue

(without fibriillin, many tissues are weakend, which can have severe consequences, such a resulting in rupture of arterial walls)

(often in media – athletes who had cardiac problems and died on sidelines)

very tall, long limbs, arachnodactyl (long fingers),

dilated aorta (susceptible to aortic rupture),

often pectus excavatum (concave chest)

What is Osteogenesis Imperfecta (OI) also known as?

How many people in US are affected?

Osteogenesis Imperfecta (OI) = “Brittle bone disease”

20,000-50,000 people in US are affected

presents in infancy or early childhood (least severe)

lead almost normal lives

blue or grayish sclera but any bone deformity is mild if at all

most severe- usually die in utero or shortly after birth

Severe respiratory problems due to underdeveloped lungs

Severe bone deformity and small stature

short in stature

sometimes have blue sclera

impaired hearing

loose joints (cartilage around tendons don’t form properly)

bone deformities

short in stature

white sclera

partial hearing loss

mild or moderate bone deformity

Physical symptoms of Fragile X Syndrome

long face, big ears, and hyperextensible joints

due to a connective tissue defect

Males display moderate to severe retardation

Females show learning disabilities to mild retardation.

Fragile X Syndrome is an X-linked dominant disorder

with variable penetrance.

While Fragile X Syndrome is X-linked dominant,

it does not show the classic pattern of inheritance.

It may seem as if the mutation develops randomly within a genogram.

  Because the parents of the affected individual may be pre-mutation carriers who display nonpenetrance in that generation.

Pre-mutation males never have affected daughters.

Why is there variable penetrance of Fragile X Syndrome

in different families?

The FMR-1 gene, whose absence causes Fragile X Syndrome,

has CGG repeats in the 5’ untranslated region (UTR).

A normal person has between 8-50 repeats

Women with children who have Fragile X Syndrome have

50-100 CGG repeats, a pre mutation.

When the gene is passed from a permutation mother to her child, the number of repeats increases in a process called expansion

to over 1000.

If a mother has over 90 repeats then

there is a 100% chance of the child having FX Syndrome

while if she has 70 repeats then the odds are reduced to 50%.

This large number of repeats causes

aberrant methylation of the promoter region 

prevents the mRNA from being transcribed

and the protein from being created

FX is a loss of function disorder.

The FMR1 protein has two functions that we need to know.

(1) acts as an RNA binding protein

and so modulates the translation of many mRNAs.

*important in neurons and in the CNS, --> cause of mental retardation.

(2)  presence of nuclear localization signal & nuclear export signal indicates mRNA shuttle function

Fragile X Syndrome (FX) --> FMR-1 gene

Myotonic Dystrophy (DM) --> [image]DM1 gene

Huntington ’s disease (HD) --> Huntington

Mild physical symptoms

cataracts and prefrontal baldness

More severe symptoms

myotonia, muscular dystrophy, and cardiac arrhythmia.

People with this illness display normal intelligence in contrast to the retardation seen in Fragile X Syndrome.

The cause of this disease is CTG triplet expansion

in the 3’ untranslated region of the DM1 gene.

The size of the repeats expands every generation,

Subsequent generations have worse symptoms and the age of onset is earlier.

The worsening of symptoms and earlier age of onset with each generation is called anticipation.

Under 40 CTG repeats is normal

while over 40 CTG repeats cause symptoms to appear

Smaller expansions cause milder symptoms and later onset

while larger expansions cause worse symptoms and earlier age of onset.

How does a large number of CTG repeats

in the 3' UTR of the DM-1 gene cause Myotonic Dystrophy?

The defect here is the gain of function of the

large expanded DM1 mRNA.

The CTG expansions cause a gain of function toxic mRNA.

10% of DM is caused by an expansion of the CCTG sequence

in the first intron of the DM2 gene,

also known as ZNF9 or zinc finger 9 gene.

B/c same disease is caused by expansion at two different genes,

indicates that the repeats are responsible for the disease

and not the specific protein product.

A mutation at more than one gene that causes the same disease is called locus heterogeneity.

The DM1 gene is homologous to a protein kinase gene

and so predicts protein kinase function.

What is not known is which protein it phosphorylates

but this does not matter because normal protein is made

in normal amounts in affected patients.

What is the range of repeats and associated symptoms

in Huntington’s disease?

Less than 40 CAG repeats are normal

more than 40 repeats --> symptoms of Huntington’s disease.

After 100 CAG repeats, it is lethal.

The more CAG repeats there are, the earlier the onset of the disease.

This is a gain of function toxic protein.

Autosomal Dominant

The function of the normal HD gene is unknown.

The mutant gene has CAG repeats in the open reading frame of the gene.

CAG codes for glutamine so the mutant

protein has a large poly glutamine stretch.

Unlike anticipation seen in DM,

anticipation in HD only affects age of onset

and does not worsen the symptoms.

The progression of the illness is the same in HD for all individuals.

X linked mutation

Expansion of CAG triplets in the coding region of the AR gene

Adult onset spinal bulbar muscular atrophy

Motor neuropathy

but compatible with long life in affected males

Heterozygous Advantage

Sickle cell hemoglobin (HbS)

homozygoes has a clear disadvantage, but

heterozygotes for the sickle cell trait demonstrate partial malaria resistance

Complete testicular feminization -

(1) loss or gain of function ?

(2) symptoms?

Loss of function of AR (promoter mutation)

Causes XY females.

Testosterone is produced by XY person but the receptor does not detect it.

An allele is an alternative version of a gene sequence

that occupies a specific locus

A mutation is a heritable change in a DNA sequence,

which creates an allele.

A polymorphism is a heritable change in a DNA sequence

whose frequency is too high to be sustained by mutation.

Infinite population size (or at least more than 1000)

Random mating

No selection either for or against particular genotypes

No mutation (no introduction of new alleles)

No migration into or out of the population

(or at least at a frequency less than 10^-6)

p + q = 1

p² + 2pq + q² = 1

Expected frequency of heterozygotes (carriers)

for an autosomal recessive disease? (e.g. Cystic Fibrosis)

q

disease frequency in males = mutant allele frequency

because males have only one copy

2q (roughly estimated)

both heterozygotes and homozygous mutant genotypes

express the disease phenotype

... RANDOM fluctuations in allele frequencies.

Aka Genetic Drift

... quickly reduce genetic variability.

Aka Genetic Bottleneck

caused by catastrophic event

or some other pressure that reduces the population and is NOT selective

.. new DNA alleles being fixed or lost.

not caused by selection

Stratification (religious/social barriers; geographical barriers)

Assortive mating (mate choosing for shared specific traits)

Consanguineous mating

This is confirmed via multiple distinct haplotypes.

A haplotype is the markers on a single chromosome

that are very closely linked to mutation carried over many generations.

Medically improved survival will have

little impact in mutant allele frequencies

Medically improved survival will lead an

immediate increase in mutant allele frequencies

In very serious dominant and X-linked diseases,

why do new mutations represent a significant portion of disease?

Very serious dominant and X-linked diseases ...

(1) reduce fitness to 0 (lethal before reproductive age) or

(2) significantly decreased reproductive frequency (0<1)

What is your prediction about the contribution of

new mutations to recessive diseases?

More or less frequent than dominant diseases.

Less frequent.

In order for a new mutations to give a recessive disease it must arise in a heterozygote for the same disease. In contrast a new mutation can give rise to a dominant disease In almost all members of the population.

NEW MUTATIONS are very rare. Typically less than 1 in a million individuals

What happens when new alleles are introduced in small populations?

Name an example of it.

Founder Effect

Huntington’s disease has a

worldwide incidence of approximately 4 out of 100,000.

However, a small fishing village in Venezuela

has about 40% of its population at risk for the disease.

Specific alleles or markers at different loci

(not necessarily on the same chromosome)

that are linked more (or less) often than expected by chance

Age of the markers

Distance between alleles/markers

Rate of recombination

Population structure

Genetic drift

GJB2 gene mutation (for connexin 26) is associated with ...

Describe the mutation?

Is it associated with a specific or an array of SNPS?

Is it an example of Founder Effect or Mutational Hot Spot?

50-80% of autosomal, recessive congenital hearing loss.

G deletions in a specific run of G's were almost always found in association

with a specific nearby single nucleotide polymorphism (SNP).

Thus, the condition can be attributed to a founder effect rather than a spontaneous event at a mutational hot spot.

Where are plasma proteins found?

What concentration?

How much is albumin?

Plasma proteins are found in the blood serum

circulating at about 60-80 g/L

of which 45 g/L is usually albumin.

Albumin

Alpha 1, alpha 2, beta, gamma globulin

1. The concentration of the proteins in the plasma is

HIGHER than the concentration in the extracellular fluid

2. Synthesized in the liver or the reticuloendothelial system

(the spleen, bone marrow, and lymph)

3. Function is mediated through the vascular system

4. Presence of these proteins in the plasma is NOT a result of tissue injury

Albumin is the most abundant and first

The other four were  named in order

as alpha 1 & 2, beta, and gamma globulins,

which each represent a class of proteins.

Cirrhosis or other liver damamge

Normal [albumin] is 45g/L

1. TRANSPORT

2. OSMOTIC PRESSURE

3. IRON TRANSPORT AND OXIDATION

4. HEMOGLOBIN METABOLISM

5. PROTEASE INHIBITORS

6. IMMUNE RESPONSE

7. COAGULATION

“The only important humans procreate intensely & constantly.”

580 aa

66,000 MW

1.Transport Ca++,

bilirubin, fatty acids, and medicines such as penicillin and aspirin

2. MAINTAINS OSMOTIC PRESSURE, nearly 80% of it.

As such, a drop in albumin levels leads to edema.

3. Nutrition

It can be broken down into its constituent amino acids

and reassembled into other proteins

in places like the liver, in order to complete protein synthesis.

It forms approximately 10% of the nutritional value.

occurs in liver and kidney disease

and is characterized by a LOW albumin count

in kidney dysfunction, albumin is secreted into urine

very rare

seen in cases where you have severe dehydration

As such, the amount of plasma protein hasn't actually increased,

rather the concentration in the blood plasma has increased

Describe Iron Transport and Oxidation.

What enzyme facilitates in the process?

In the liver,

iron exists in the ferrous state (Fe2+)

and needs to be oxidized to the ferric state

in order for transferrin to pick it up.

Ceruloplasmin

1. Binds Cu2+.

In the presence of Fe2+, it gets reduced -> Cu+

coupled with oxidation the Fe2+ -> Fe3+

2. Carries copper to enzymes that require it as a cofactor

3. Removes Cu2+ to prevent toxicity

Describes Wilson's Disease.

Describes its symptoms.

Wilson's Disease - congenital condition in which the patient has

low levels of ceruloplasmin.

Cu2+ to accumulate in the brain and liver

due to a lack of binding,

which causes neurological dysfunction and liver disease as a result.

Describes Atransferrinemia.

Describes its treatment.

Atransferrinemia is a congenital syndrome in which there are

low levels of transferrin.

Children who are afflicted with this condition need to be treated with

repeated blood transfusions.

Haptoglobin - combines with the globin molecule,

and then transports it to the liver

where the iron is stored and the

globulin is excreted

Hemopexin - combines with the heme molecule,

transporting it to the liver and then

storing the iron from the heme

HDL and LDL lipoproteins

thyroxine-binding protein

(2 subtypes, prealbumin binding protein and globulin binding protein)

retinol binding protein

(which functions in the transport of vitamins)

steroid binding protein

(which binds and transports steroids)

Name 2 protease inhibitors that are plasma proteins.

What are the functions of protease inhibitors?

Alpha-1 Antitrypsin

inhibits collagenase and elastase activity
from degrading extracellular matrix

low levels can also cause emphysema

Alpha-2 macroglobulin & Alpha-1 chymotrypsin

also protease inhibitors

Protease inhibitors prevent

degradation of synthesized protein products.

Gamma globulins

because they act as antibodies.

Fibrinogen

in the plasma as a fibrous protein (NOT GLOBULAR)

acts as a precursor to fibrin* in the clotting cascade

Fibrin* on the other hand is NOT considered a plasma protein.

Blood clotting cascade is a series of ...

Many active clotting factors are ...

... zymogen activations (made active by cleavage of 1 or more peptide bonds)

.. serine proteases (have serine residue on active site)

Injury activates which factor?

Is this intrinsic or extrinsic?

Injury activates factor XII (Hageman factor).

Intrinisic pathway

Trauma activates which factor?

Is this intrinsic or extrinsic?

Trauma activates Factor VII,

which releases tissue factor from blood vessels.

Extrinsic pathway

Intrinsic (last step): VIIIa + IXa convert X to Xa

Extrinsic (last step): VIIa and tissue factor convert X to Xa

Common (1st step):

Va + Xa convert prothrombin to thrombin

Vitamin K adds ... to ...

How does this affect Prothrombin?

How does this affect other factors.

Vitamin K adds a γ-carboxy group to the glutamate

Vitamin K adds γ-carboxy group to prothrombin-glutamate –

gives prothrombin negative charge

Vitamin K also activates Factors VII, IX, X

by also adding a γ-carboxy group to the glutamate of these factors

Prothrombin with negative charge

attracted to the positively charged clot (Ca2+ ions)

platelet membranes aggregate at clot site and have positive charge (Ca2+ ions)

Coumadin (warfarin) and dicumarol

inhibit action of vitamin K

so clotting factors are not attracted to the site of platelet aggregation

Describe the structural components of fibrinogen ...

and their contribution to fibringen's overall structure.

three types of chains

Aα, Bβ, and γ (gamma)

A and B chains

have negative charge

causes fibrinogen to have an extended structure

α, β, γ chains

form monomers and aggregate to form fibrin

fibrin has a globular structure

What causes the release of A and B peptides in fibrinogen?

At what sites are they cleaved?

Thrombin cleaves fibrinogen

at arginine-glycine sites

causes release of A and B peptides (fibrinopeptides)

Visualized structural components of

Tissue Type Plasminogen Activation (TPA)

In regulation of clotting, what forms an irreversible complex with thrombin?

What does it block?

Antithrombin III forms an irreversible complex with thrombin

also complexes with & blocks IXa, Xa, XIa, XIIa

What is instrumental in dissolving clots?

Describe its function.

TPA: tissue type plasminogen activator

clot binds to the Kringle domain

serine protease domain breaks plasminogen down -> plasmin

plasmin lyses the clot

Discuss treatment for a heart attack.

What is given to the patient in hospital?

What is given for home?

TPA -> lyse clot

stenting -> open up the vessels

heparin -> prevent future clotting

after sent home,

sometimes with Lovenox

usually with Plavix and aspirin

prevent platelet aggregation

Coumadin -> prevent clotting

by antagonizing Vit. K

Any exact multiple of N, the haploid number of chromosomes.

A normal somatic cell is diploid,

i.e. 2N where N-23 for humans

Any exact multiple of N greater than 2

3N = triploid

4N = tetraploid

Any non-euploid number of chromosomes

Trisomy: The presence of extra copy of a single chromosome

Monosomy: The absence of a single chromosome

Results in monosomic and trisomic cell lineages

within somatic tissues

Results in 2 disomic

and 2 nullosomic cells

Results in 2 normal,

1 disomic, and 1 nullosomic cell

What is the most common trisomy?

How common is it?

Trisomy 21 (Down Syndrome), the most common trisomy

(1/600 – 1/1000 live births).

Who discovered the cause of Down Syndrome?

When was it first identified as a disease?

In 1959, Jerome Lejeune.

First identified by John Langdon Down in 1866.

47, XXY in males.

47, XXX

45, X [Turner]

in females.

[47, XYY was discovered later)

Older women are more likely to give birth to children with Down Syndrome, but the number of births are much higher for younger women. Therefore, many children with Down Syndrome are born to young women.

Multiple integrated tests avalaible in 2nd trimester.

A 1st trimester specialized ultrasound test (nuchal translucency test) also is available.

47, XXX arises predominantly from maternal errors.

Meiosis 1 errors > Meiosis 2 errors.

47, XYY can only arise from male meiosis 2 errors

.

Where does locus-specific probes bind?

Where do alphoid or centromeric repeat probe bind?

Chromosome-specific painting probe bind?

The locus-specific probe binds in one place

  alphoid or centromeric repeat probe binds to alpha repeats

chromosome-specific painting probe can bind to many segments.

Note: A centromere-less chromosome does not count as a chromosome and will not contribute to N.

1. Simple sequence repeats (A)n, (CA)n, (CAG)n

2. Functional tandem repeats:

a. Structural: telomeres and centromeres

b. Transcribed: ribosomal RNA genes

(Multiple copies needed for when protein synthesis is increased.

3. Duplications of large chromosomal segments

4. Transposons (sines, lines, retroviral elements)

5. Pseudo-genes (transposed copies)

Repeat sequences account for more than 50% of human DNA.

The repeats are relics of evolutionary transpositions.

What transposons are abundant in introns and between genes?

What are common examples of each?

Lines and Sines are abundant in introns and between genes.

Lines = Long interspersed Sequence Element

Most common Line (transposon) is L1

Sines = Small Interspersed Sequence Element

Alu sequences are most common

~300 bp long but > 10% of the genome

L1 transposon integrase transcribes the Alu DNA

then reverse transcribes the RNA

to insert the DNA into chromosomes.

How can Transposons contribute to mutations

and chromosomal rearragements?

New insertions can inactivate a gene or alter a gene expression

Recombination between ancient relics can

rearrange DNA in chromosomes or cause deletions

The most common retrotransposon is Erv

.

What are Pseudogenes?

How are they produced?

Intronless pseudo-genes are produced

using a reverse transcribed mRNA substrate,

similar to SINE transposition.

Pseudogenes may also be the result of gene duplication in the DNA.

Before the eutherian expansion,

X and Y were identical autosomes.

(Many hundred million years ago.)

Hypothesis: inversions, deletions, and other rearrangements

reduced the similarity between X and Y.

The chromosomes became so different that recombination

between X and Y during meiosis was sharply reduced.

Describe WAGR Syndrome.

What causes it?

1. Wilms tumor – a solid renal tumor (8% of childhood cancers), Dominance inheritance, tumor suppressor.

2. Aniridia - eye defect due to deficiency in a transcription factor associated with eye development.

3. Genitourinary Anomalies - another phenotypic manifestation of the Wilms tumor gene

4. Mental Retardation Syndrome – deficiency in gene ancient gene of unknown function (also present in C. elegans)

Deletions or duplications of multiple genes may produce contiguous gene syndromes. The patient displays a group of phenotypes due to loss of multiple functions from adjacent genes.

Duplications formed by unequal crossing over

during homologous recombination.

Requires a region of homology.

Unequal crossing over at either

(1) between red and green pigment genes

in a region of homology between genes

--> severe red/green color blindness.

(2) within red and green pigment genes (opsins)

--> moderate red/green color blindness

Red and green opsins are 95% similar

These can form by recombination between repetitive sequences.

Usually there is no abnormal phenotype

unless the breakpoint interrupts a gene.

Paracentric inversions: within one arm.

Meiotic recombination within the inversion produces 2 inviable gametes –

one with two centromeres and one without any centromeres.

Both have a deletion and duplication.

Pericentric inversions: include the centromere.

Meiotic recombination within the inversion produces gametes that each have a duplication and deletion, but both have just one centromere.

What are the most common autosomal trisomies

(in decreasing order of frequency)?

Which chromosome variations are more common and less harmful?

How many miscarriages are due to chromosome variations?

Sex chromosome variations are more common in live babies

and are often less harmful than autosomal abnormalities.

Half of miscarriages are due to chromosome abnormalities.

Why can't red blood cells be analyzed for chromosome abnormalities?

What are most commonly used for study?

Only nucleated cells can be analyzed,

therefore RBCs CANNOT be used in chromosomal analysis.

Blood lymphocytes, amniocytes, fibroblasts, and bone marrow cells.

Describe the process of study lymphocytes for chromosome abnormalities.

What stain is most commonly used?

o Blood sample is added to culture and removed of all RBC

o T-Cell division is stimulated and cells are frozen at metaphase stage

o Cells are lysed and placed onto slides

where they are stained by a variety of different stains

depending on the focus of the analysis.

Giemsa stain

What is chromosome analysis called?

How are they created?

Karyotype.

Created from photographs of stained chromosomes

& banding patterns are examined

Fluorescence in situ hybridization - (FISH)

used to identify a specific genetic change such as trisomies

or microscopic deletions in metaphase or interphase cells

Spectral Karyotyping (SKY) – Whole Chromsome Paint

used when looking for lost or addition of material via color analysis

Complex rearrangements where there are many chromosomal changes

Comparative genomic hybridization - (CGH)

used to detect subtle changes that are

not normally seen on a routine chromosomal analysis

Cytogenetic Nomenclature

What does nomenclature show?

What are the symbols for variants?

How does one identify variants?

Chromosome numbers, sex chromosomes, variants

Variants include: Translocation, Deletion, Duplication, Inversion

(T, Del, Dup, Inv)

Can identify variants by analyzing the bands of the chromosome

via Karyotyping

Prenatal

intrauterine growth retardation (IUGR)

multiple congenital anomalies (MCA)

hydrops/edema

Can test using aminocentisis

Birth - Unusual facial features, MCA, mental retardation (MR), growth defects (Short Stature – SS), learning disabilities (LD), ADHD

Adult - LD, infertility, spontaneous abortion (SAB) or stillbirths

"Fools Believe Genes are Disposable"

F – Facial features

B – Birth defects and physical variations

G – Growth retardation including head growth

D – Development problems

Which trisomies have been known to demonstrate mosiacism?

Which is the only survivable trisomy in mosiacism?

Mosaicism occurs because some cells recognize the abnormality

and correct it while others do not, creating milder symptoms

Trisomy 21 Mosaicism – some cells have trisomy 21, some are normal – 1%

Trisomy 16 Mosaicism is lethal in its non-mosaic form

More often seen in miscarriages where the fetus’ chromosomes are analyzed

Wolf Hirschhorn disease has what chromosome abnormality?

DiGeorge Syndrome, aka Velocardiofacial Syndrome?

Wolf Hirschhorn (4p-)

caused by a Cytogenetic Deletion

a terminal deleton

DiGeorge Syndrome, Velocardiofacial Syndrome

caused by a mircrodeletion

Chromosome 22q11.2 Deletion

(CATCH 22)

Name an example of a Cytogenetic Duplications.

Name an example of disease caused by Unbalanced Translocations.

Duplication 22q11

Partial Trisomy 1q and Monosomy 14q

Are chromosome variations usually associated with Mental Retardation?

Is an increased risk for developmental delays?

Unlike autosomal aneuploidies,

sex chromosome variations are usually NOT associated with MR.

Yes, there is an increased risk for LD or developmental delays,

and in some cases fertility problems.

Which is most common cytogenetic abnormality at conception,

but only 1% survive to term?

What is 47, XXY?

Is there increased risk for LD? Other symptoms?

Klinefelter Syndrome.

Yes, Increased risk for LD.

Occasional breast enlargement, small testes,

and infertility seen in adolescents and adults.

What is XXX?

Is there increased risk for LD? Other symptoms?

Triple XXX Syndrome

Yes, increased risk for Learning disabilities.

No physical features at birth but increased risk for premature ovarian failure.

X: Autosome Translocations can interfere with what process?

Which diseases can manifest as a result?

X Inactivation.

Can results in expression of an X-linked recessive condition in a female

(ex: colorblindness, hemophilia, Duchenne muscular dystrophy)

Can happen in women with an X:autosome translocation leading to cellular selection or non-viability and activation of only the X that has the autosomal material attached

Can be seen in girls with Turner Syndrome

Manifesting heterozygotes may result from unfavorable or skewed X-inactivation for single gene disorders

such as Duchenne muscular dystrophy or hemophilia (blending disorder)

Instead of 50/50 inactivation skewing can occur in which there are more cells that have the wrong X inactivation therefore exhibiting X-linked recessive genes

If a normal adult comes to your office with a history of recurrent pregnancy losses, what do you want to rule out?

Who would you test?

Balanced translocation would be ruled out

Test both parents

If the cause was due to translocations,

the couple may have more kids with Down’s syndrome

Purine <--> Pyrimadine

e.g A<->C

Purine <-> purine 

A<->G

Pyrimidine <-> pyrimidine

C<->T

GAG in 6th codon is changed to GTG

GLU is substituted by VAL

What does an addition/deletion cause?

How does it affect function?

Where would it have the least detrimental affect?

Frameshift mutation.

often leads to null phenotype – loss of function -

only place where tolerated at all is if it occurs at

very end of polypeptide chain

How can you get an in-frame deletions/insertions?

Does it have a phenotypic change?

- add or delete 3 bases.

only localized change -

may or may not have a phenotypic consequence –

depending on where the change occurred

(ie if the missing amino acids had an important function)

Missense (silent)

interaction of substrate-enzyme unchanged = function of enzyme is intact -

Missense (altered)

affects interaction of substrate/enzyme = non functional enzyme → phenotypic consequence

Temperature sensitive

alteration of enzyme-substrate interaction at a certain temperatures

but normal at others

(ie normal at 23 degrees C but nonfunctional at 42 degrees C)

whether protein has function or not depends on temp

What is an exact reversion?

When is it seen?

How common is it?

- ie in sickle cell, GAG→ GTG.

An exact reversion would be if GTG reverted back to GAG

giving normal phenotype.

This is extremely rare in nature and although it can be done in lab,

has never been found in nature

What is the most common method for overcoming mutation?

Give two types of this method.

Suppression.

Intragenic suppression -

2nd mutation pushes enzyme back to somewhat original position

so that you have some function

Extragenic suppression – substrate changes to fit mutant version of enzyme

Most likely to revert are point reversions (base subsitutions)

mostly done by having compensating 2nd site mutations.

extragenic suppression > intragentic suppression > Exact reversion

What is Juctional Epidermolysis Bullosa?

How is this an example of somatic mosiacism?

Recessive disorder due to homozygous defect in LAMB3 gene

which codes for laminin-332

patches of healthy skin sometimes seen

when LAMB3 has reverted back to wild type function

by intragenic 2nd site mutations

1. Polymerase errors on normal templates that escape fidelity constraints

2. Misreplication at repetitive sequences due to strand-slippage

3. Misreplication at damaged template sites

4. Transposable Genetic Elements (TGE’s)

Polymerase and straind-slippage is a result of normal function.

Polymerase selectivity -

high selectivity increases fidelity by 3 orders of magnitude

(ie Watson-Crick base pairing alone has a fidelity of 10^-2

(99% A will pair with T but 1% it wont)

and Selectivity of dNTP ternary complex raises that fidelity to 10^-5)

other components that build on this fidelity will eventually bring it up to → 10^-10

Polymerase Proofreading:

the 3'->5' exonuclease activity to excise error.

contributes 2 magnitudes to fidelity (10^-5 → 10^-7)

Note: can only occur +1 after site of mismatch

Mismatch Repair

1. recognize newly synthesized DNA vs template stand

accomplished by simple chemical means;

mature DNA is fully methylated

(by enzyme DAM methylase *not important )

* newly synthesized DNA has not yet been methylated

→ Hemi-methylated DNA*

2. cut out “new” DNA and “refill” with correct DNA -

must have hemimethylated DNA and must be in vicinity

(anything between 10 bp – 2kb) of mismatch

Contributes another 2 orders of magnitude to fidelity (10^-7 → 10^-9)

OVERALL replication error rate 10^-10

(last order of magnitude added by other mechanisms)

(1) need hemimethylated site (anything between 10 bp – 2kb) of mismatch

(2) activates enzyme complex: MutH, MutL , MutS, ATP in bacteria

(3) acts on hemimethylated site, nicks at site which is NOT methylated

(4) Exonuclease (Exo VII) expands nick to site of mismatch

(5) DNA pol III holoenzyme refills gap with new DNA

Contributes another 2 orders of magnitude to fidelity (10^-7 → 10^-9)

Familial non-polyposis colon cancer (HNPCC = lynch syndrome)

Patients are defective in mismatch repair genes (ie hMSH2)

Autosomal dominant inheritance for cancer predisposition 

that manifests by loss of heterozygosity.

Occurs in 1 in 1000 times

incorrect reannealing so that 3rd C loops out and 4th C aligns to 3rd G => misaligned primer – but 2 more C’s will be still added for 4th and 5th G so that upper stand has 6 C’s and lower strand has 5 G’s upper strand when replicated will give rise to +1 mutation while the lower strand will give rise to WT •

Slippage only occurs with repetitive sequences

Deamination

C→ U

Oxidation: oxygen radicals react with

Guanine creating → 8-oxoG

Depurination: Guanine in DNA is hydrolyzed at base -

bond holding base to sugar phosphate backbone is broken

so that template base alone has been lost ( but backbone is intact)

=> results in an abasic site or apurinic site

It causes G-> 8-OxoG -

When 8-OxoG is formed instead of Guanine

it templates for Adenine instead of Cytosine

normal is in anti confromation

but in 8-Oxo G, the base has rotated along axis to syn conformation –

which favors H- bonding with A not C

 “Hoogsteen pairing”

What types of replication errors occur at

3 major examples of Spontaneous DNA Damage?

Deamination →translation

8-oxoG → transversion

abasic site → ¾ times wrong base will be entered

How can a transposable genetic elements damage DNA?

How common is this?

- TGE- specialized DNA sequence of elements capable of moving from one DNA location to another;

If insertion point is within a gene, that gene gets inactivated

transpositions rare

45% of human genome is TGEs- most are defective for transposition

Hemophilia A caused by

disruption of Factor VIII gene

by L1, a TGE like DNA element

- UV (sunlight); ionizing radiations (x-rays, radon’s, cosmic radiation)

chemicals

cultural artifacts: cooking, smoking

natural food carcinogens

Heat (deamination, depurination)

Oxygen radicals

Reactive metabolites

What test is used to detect for mutagens?

How does it work?

Ames Test

short term assay

can be determined overnight

start with bacterium that lacks histidine synthesis - grow His (–) bacteria in His (+) rich media - spread on two plates(with His (-) agar): control and Test sample o His (-) cells will not form colonies on agar lacking histidine - incubate both over night at 37 degrees C o in control most should die o In test sample there should be many colonies if sample has mutagen present ( * When reversion to His (+) status occurs, they can form colonies on agar without histidine

1. Excision repair – 2 types

a. Nucleotide excision repair (NER)

i.  Bulk repair

ii. Transcription-coupled repair

b. Base excision repair (BER)

2. Non-excisive (direct) repair

3. DNA double-stranded break (DSB) repair

Excision Repair Pathways

1. Nucleotide excision repair (NER):

A repair endonuclease recognizes a lesion and cuts the DNA strand at the offending site by cleaving a phosphodiester bond.

Removal of several nucleotides creates a gap that is 12-30 nucleotides long.

DNA polymerase fills in the gap, and the remaining nick is sealed by DNA ligase.

2. Base excision repair (BER):

A DNA glycosylase recognizes and removes a specific modified base (e.g. 8-oxoguanine) via cleavage of a glycosidic bond, creating an abasic site.

Other proteins cut the DNA at the abasic site, creating a gap that is 1-2 (<10) nucleotides long.

DNA polymerase fills the gap, and DNA ligase patches it.

Name a disease that results as a failure of nucleotide excision repair.

Describe the condition

Xeroderma pigmentosum (XP).

Symptoms - photosensitivity, early cancers

Pathway/function - Nucleotide excision repair

Genes - XPA thru XPG

Of all the DNA repair pathways,

why does impaired NER cause such severe consequences vs. BER?

NER is a general repair pathway that is activated whenever a

bulky distortion changes the shape of the DNA helix.

It is effective against hundreds of different types of DNA lesions,

so loss of this mechanism has widespread consequences.

By contrast, BER is a specific repair pathway. Repair of a lesion by BER requires cleavage by a glycosylate unique to that type of lesion.

Thus, the scope of any defect in BER machinery is much more limited.

What is "Transcription-Coupled" Excision Repair?

Describe the mechanism.

What proteins are important for recuitment of repair machinery.

This is when actively transcribed genes are preferentially repaired.

RNA Pol stall at site of damage,

leads to recruitment of NER/BER machinery.

CS-A & CS-B proteins are important for recruitment.

Cockayne Syndrome

Symptoms - photosensitivity, premature senility, developmental delays

Pathway/function - Transcription-coupled Excision Repair

Genes -    CS-A, CS-B,

XPB, XPD, XPG

TTD 

autosomal recessive disorder

Symptoms - photosensitivity, premature senility, "brittle hair"

Pathway/function - Repair, transcription

Genes -    XPD, XPB,

[helicase components of transcription/repair factor TFIIH]

TTD-A (p8)

Of all the XP genes, which genes is attributed to XP, TTD, and CS?

How are mutations of this gene different for each gene?

XPD.

Complete loss of function in XPD causes XP

(which has the most severe defects),

while a smaller mutation can cause CS or TTD.

What are examples of Non-Exicisive Repair of DNA?

What is done in each case?

Photoreactivation.

UV dimmers (T=T) are corrected by Photolyase.

DNA Alkyl Transferase.

Correcting damage by an alkylating mutagen

that causes G -> O6-methylguanine,

O6-methylguanine DNA methyltransferase removes the CH3- from G.

DSB repair occurs via 2 major pathways:

homologous recombination and non-homologous DNA end joining.

In non-homologous DNA end joining, 

ends must be protected by a protein cap. ( Ku70/80.)

Next, the protected DNA ends are

brought together by joining microhomologies in the end sequences.

Finally, DNA ligase seals the two ends together.

Bloom Syndrome (defective BLM coding fro RecQ-like helicase) &

Werner Syndrome (defective WRN coding fro RecQ-family helicase)

Bloom Syndrome.

Symptoms - photosensitivity, growth arrest.

Pathway/function - DNA helicase.

Gene - BLM

Werner Syndrome

autosomal recessive disorder

Symptoms - premature aging

Pathway/function - DNA helicase / nuclease

Genes -    WRM

Describe Hereditary Colon Cancer

[HNPCC, Lynch Syndrome]

Hereditary Colon Cancer

[HNPCC, Lynch Syndrome]

Symptoms - non-polyposis colon cancer

Pathway/function - mismatch correction

Genes -    hMSH2, hMLH, etc

Xerderma Pigmentosum variant

Symptoms - photosensitivity, early cancers

Pathway/function - Specialized DNA polymerase

Genes -   hRAD301 (Pol Eta)

Li-Farumeni Syndrome

Symptoms - cancer susceptibility

Pathway/function - DNA damage response

Genes -   p53

Familial Breast Cancer

Symptoms - breast and ovarian cancer

Pathway/function - dsDNA break repair pathways

Genes -   BRCA1, BRCA2, ATM

What is the Guardian of the Genome?

What is its function and what disease is caused by its misfunction?

p53

In levels of low/moderate DNA damage,

it increases p21 and induces cell cycle arrest and DNA Repair mechanism

In levels of irreversible/excessive DNA damage,

it induces apoptosis

When depurination results in a abasic site,

what DNA polymerases are used to continue polymerization?

DNA Pol iota and DNA Pol zeta are used

Iota places a random base on the new strand

Zeta places a random base on the both strands

When UV damage results in a T=T dimer,

what DNA polymerases are used to continue polymerization?

DNA Pol eta is used

Eta places AA on the new strand opposite the T=T dimer

Note: defect in this action results in XP variant

Glucose -> G-6-P

[Hexokinase]
F-6-P -> F-1,6-BiP

[Phosphofructokinase]
PEP -> Pyruvate

[Pyruvic kinase]

Glucose -> G-6-P

[Hexokinase]

F-6-P -> F-1,6-BiP

[Phosphofructokinase]

1,3 Biphosphogylcerate -> 3PGA

[phosphoglycerate kinase]

PEP -> Pyruvate

[Pyruvic kinase]

Activators: high AMP, high F-2,6-BiP

Inhibitors: high ATP, high citrate

In aerobic systems, its goes to the ETC.

In anaerobic systems,

Pyruvate -> Lactate

[lactate dehydrogenase]

DHAP -> alpha-glycero P

[alpha-glycero P

dehydrogenase]*

alpha-glycero P -> trigylcerides

*site for regeneration of NAD+

What enzymes begin the first step in glycolysis?

When is the alternative enzyme used?

Hexokinase

or occasionaly glucokinase (in liver)

glucokinase is used when having a big dinner

has lower Km (affinity) for glucose

G6P -> Glucose

[Glucose 6 Phoshatase]

F-1,6-BiP -> F-6-P

[Frucose 1,6 Bisphoshatase]

Pyruvate -> Oxaloacetate -> Malate -> Oxaloacetate -> PEP

[Pyruvate Carboxylase]                          [PEP Carboxykinase]

At what steps in Pyruvate -> PEP

does the sugars move across the mitochondrial membrance?

Pyruvate moves to mitochondria before becoming Oxaloacetate

[pyruvate carboxylase is in the mitochondria]

Oxaloacetate -> Malate -> Oxaloacetate

[Malate crosses back to cytosol]

{done by NAD oxidation then reduction}

What is the first steps to begin

pyruvate -> pep?

1. mechanism is first activated by acetyl CoA

2. this cause the carboxylation of biotin,

which releases pyruvate decarboxylase in the mitochondria

3. pyruvate then moves into the mitochondria

Insulin inhibits activity of gluconeogensis.

Cortisol activates activity of gluconeogensis.

Name regulators of the gluconeogenic enzyme,

fructose 1,6 bisphoshpatase.

F 2,6 BiP inhibits fructose 1,6 bisphoshpatase

AMP inhibits fructose 1,6 bisphoshpatase

Citrate activates fructose 1,6 bisphoshpatase

OPPOSITE to Phosphoructokinase

How is the glycolyic/gluconeogenic regulator

F 2,6 BiP generated?

F6P ----> F 2,6 BiP

[Phosphofructokinase 2]

will lead to F 1,6 BiPhosphatase inhibition

i.e, no gluconeogensis

F 2,6 BiP ----> F6P

[Fructo 2,6 Bisphosphatase]

will lead to PFK1 inhibition

i.e, no glucolysis

"Can I Keep Selling Sex for Money, Officer?"

Citrate

Isocitrate

Succinyl CoA

Succinate

Fumarate

Malate

Oxaloacetate

Cowboy Actors in Kansas Should See Foreign Movies

Citrate Synthase

Aconitatse

Isocitrate Dehydrogenase

alpha-Ketoglutarate Dehydrogenase complex

Succinyl CoA Thiokinase

Succinate Dehydrogenase

Fumarate

Malate Dehydrogenase

Isocitrate Dehydrogenase

alpha-Ketoglutarate Dehydrogenase

Which enzyme in TCA is the only site of

net reduction of FAD?

3 NADH

1 FADH2

What is the only activators of the Pyruvate Dehyrdogenase/TCA cycle

and where do they act.

ADP & Ca++

ADP -> Pyruvate Dehyrdogenase

ADP & Ca++ -> Isocitrate Dehydrogenase

Ca++ -> alpha-Ketoglutarate Dehydrogenase

Glycerol phosphate shuttle & Malate aspartate shuttle

shuttle NADH acrros the mitochondrial membrane

Describe

Malate aspartate shuttle

Malate aspartate shuttle

Malate <--> Oxaloacetate

[Malate dehydrogenase]

Oxaloacetate <--> alpha-Ketoglutarate or Aspartate

[aminotransferase]

alpha-Ketoglutarate or Aspartate <--> Glutamate

[aminotransferase]

Glutamate <--> alpha-Ketoglutarate or Aspartate

[aminotransferase]

Describe

Glycerol phosphate shuttle

Glycerol phosphate shuttle

DHAP <--> Glycerol 3 -P

[Glycercophosphate dehydrogenase]

Note: Driven by FAD oxidation towards ETC

& NAD reduction

"Cows in Kansas"

Citrate Synthase

Isocitrate DH

alpha-Ketoglutarate DH

"High Profile People Act Too Glamorous, Picture Posing Every Place":

Hexokinase
Phosphoglucose isomerase
Phosphofructokinase (PFK)
Aldase A
Triose phosphate isomerase
Glyceraldehyde-3-phosphate dehydrogenase
Phosphoglycerate mutase
Enolase
Pyruvate kinase

"Goodness Gracious, Father Franklin

Did Go By Picking Pumpkins (to) Prepare Pies":

Glucose
Glucose-6-P
Fructose-6-P
Fructose-1,6-diP
Dihydroxyacetone-P
Glyceraldehyde-P
1,3-Biphosphoglycerate
3-Phosphoglycerate
2-Phosphoglycerate (to)
Phosphoenolpyruvate [PEP]
Pyruvate

· 'Did', 'By' and 'Pies' tell you the first part of those three: di-, bi-, and py-.
· 'PrEPare' tells location of PEP in the process.

Order of ETC [Oxidative Phosphorylation]

Complex 1 [NAD/NADH, FMNH] + Complex 2 [FAD/FADH2]

--->CoQ {Ubiquinone}-->

Complex 3 [cyto b, Fe3+/2+]

-->cyto c {Fe3+/2+}-->

 Complex 4 [cyto a, Fe3+/2+] --> 1/2 O2 + H2O

energy from that fuels Complex 5 [ATP Synthase]

ATP Transport takes ATP out of mitochondria