Human Genome And Karyotype

Question 1

What is another term for genome size?

Answer 1

C-value.

Humans: 3.2X10^9 BP of DNA in each somatic cell

Question 2

How many genes do we have?

Answer 2

22,000

Question 3

When is DNA packed into chromosomes? What is it referred to during other times?

Answer 3

During mitosis.

Chromatin (DNA+histones)

Question 4

What is DNA wrapped twice around 8 core histone proteins?

Answer 4

Nucleosome

Question 5

When does chromatin condense into chromosomes?

Answer 5

During prophase of mitotic cell division (after replication)

Question 6

After DNA is replicated, what do they form into?

Answer 6

Sister chromatids (attached at centromere)

Question 7

How does increased genome complexity arise?

Answer 7

1. Duplication of existing sequences followed by divergence and selection
2. Incorporation of DNA from other species (lateral transfer)

Question 8

What was/is the ENCODE project responsible for?

Answer 8

Encyclopedia of DNA elements

Mapped genome in 80 diff cell types:

• > transcripes ans protein encoding exons
• > chromatin (histone) modification and DNA methylation
• > DNAse hypersensitivity (binding of regulatory factors exposes DNA to cleavage while DNA in nucleosomes is protected)

->Binding of 100 known TF, RNA Pol II/III and other proteins

Question 9

What are some conclusions from the ENCODE project?

Answer 9

Chromatin has 7 functional states

60-75% is transcribed into RNA

Non-coding transcripts (regulatory role) are nearly as abundant as protein-encoding genes.

At least 80% of genome is likely to be functional

Question 10

How many SNPs are in the genome?
Insertions/Deletions?
Block substitutions?
Inversions?
Copy Number variations?

Answer 10

3,000,000

800,000 indels

50,000 block

100 inversion

50-100 small and large (alpha amylase varies from 3-14 tandem copies)

Question 11

What do repetitive sequences consist of?

Answer 11

1. Tandem repeats (genes or blocks of genes)
2. Short repeats (each present in many copies)
3. Retrotransposons - repeats which are products of reverse transcriptions

**Repeats are difficult to sequence

Question 12

Why are repeats substrates/’hot spots’ for recombination?

Answer 12

Similiar/identical in nucleotide sequence.

Recombination between them may cause inversion, duplication, or deletions.

Question 13

What are examples of recombination?

Answer 13

Red-green color blindness
Rh-factor
Velocardiofacial syndrome
Hemophilia A (x-linked)

Question 14

What is red-green color blindness caused by?

Answer 14

Recombination between duplicated genes with almost identical sequences on x-chromosomes.

Misalignment in chromosomes leads to only one gene being expressed in color blind pts.

Misalignment followed by meiosis (trait gets missed)

X with only single receptor gene -> inability to distinguish red/green

X with 1 red + 2 green receptors (still be phenotypically normal)

Red (long receptor)
Green (medium)

Question 15

Where does recombination occur in a microdeletion or segemental aneuploidy syndrome? What are some examples?

Answer 15

Occurs between large repeats resulting in deletion of a block of DNA that contains multiple genes

DiGeorge(Velocardiofacial Syndrome)
->del 22q11; failure of pharyngeal pouches to develop (parathyroid, thymus, cardiac defects)

Prader-Willi and Angelman syndrome
->del 15q11-13

Dx with FISH probe for deleted region

Question 16

Why are DNA satellites called that?

Answer 16

Old technique when DNA was fractionated by density , reoeats would form a small satellite seen next to DNA peak b/c they differ from it in base composition

Question 17

Where do satellite sequences occur most frequently?

Answer 17

Mostly at centromeres and telomeres

Question 18

What are micro-satellites used most frequently for?

Answer 18

Identigy specific chromosomes in genetic counseling

Question 19

What are retroransposons?

Answer 19

I.e. Transcription by RNA Pol II,

MRNA gets converted to dscDNA via reverse transcription & that gets integrated into a new genomic site

Estimated for up to 25% of increased complexity of human genome.

The inserted rtRNA into DNA can disrupt a gene at the integration site.

Question 20

What kind of retrotransposons exist?

Answer 20

LINE - long interspersed nuclear elements; mRNAs encoding reverse transcriptase

SINE - short interspersed nuclear elements; copies of short cellular RNA

–>Alu sequences (1/kb,, contain a restriction site more Alu1, unique to human DNA

Pseudogenes - copies of cellular mRNAs, not transcribed because they lack promoter sequences.

Question 21

When should we consider a diagnosis of chromosome abnormality?

Answer 21

1. Problems with physical or mental development
2. Infertility, spontaneous abortion, still birth
3. Pregnancy in woman 35 and older
4. Cancer

Question 22

What are some karyotyping techniques used?

Answer 22

1. G banding: Giemsa staining creates pattern of dark and light bands unique to each chromosome
2. FISH: detects changes too small to see in g banding
3. Comparitive genomic hybridization (CGH)L detects deletions or duplications even if their location is not known

Only can see changes that a technique is able to pick up on

Question 23

How do we do G-banding technique? Why is colchicine important for this technique?

Answer 23

Incubate cells in colchicine (binds tubulin and prevent spindle function arresting cell in metaphase)

Chromosomes condense steadily during prolonged metaphase. Over time number of cells in mitosis increases, but number of visual bands decrease

Stain with Giemsa dye

Std Karyotype: 500-800 bands per haploid set of chromosomes

Question 24

How do we identify chromosomes based on their G bands?

Answer 24

Size (1largest-22smallest)
Centromere position
Dividing pattern

Question 25

what is p arm? Q arm?

Answer 25

``` P= petite Q= queue (long arm) ```

Question 26

What are metacentric chromosomes? Sub-metacentric? Acrocentric?

Answer 26

``` Meta = centromere occur in middle (Chr3) Sub-meta = Off center centromere (Chr 18) Acrocentric = centromere located very close to telomere, still some DNA on the other side ``` Telocentric - not present in humans, always some DNA btwn centromere and telomere

Question 27

What is g-banding resolution good for?

Answer 27

Detect structural change regardless of nature or location, but: - Detects only relatively large changes on chromosome - Lower resolution limit is one band - 500-800 bands for metaphase G banding - 1band is about 4-7MB; 45 or so genes Smaller changes -> need FISH or CGH

Question 28

What is a requirement of FISH?

Answer 28

Need to know what sequence you are looking for! Chromatin/chromosomes are fixed to slode and probe binds to DNA of complenetary site

Question 29

What is difference between interphase FISH and metaphase FISH?

Answer 29

Interphase: happens on chromatin, faster b/c we can do it on clinical samples. LOWER RESOLUTION (DNA isnt condensed) Metaphase: chromosomes, requires a culture to amplify cell number and then incubate in colchicine

Question 30

What is general sequence of FISH?

Answer 30

Usually have at least 2 probes. One is used for locating chromosome (internal control) other is used for detecting the critical region. FISH wont rule out minor changes like a SNP

Question 31

What is a disadvantage of FISH detection?

Answer 31

Only detects presence/absence/position of DNA to which the probe binds Cannot detect single nucleotide changes

Question 32

What is comparative genome hybridization?

Answer 32

Array of oligonucleotides immobilized at different positions on a glass slide (microarray) complementary to sequences spaced across genome.

Question 33

What are some strengths of CGH? Limitations?

Answer 33

Can detect very small changes anywhere in genome, we do not need to know where to look Detects only increase/decrease in copy number -->deletions/duplications CANNOT DETECT REARRANGEMENTS W/O GAIN or LOSS (inversions or translocations)

Question 34

Are diploid conceptions with two maternal or two paternal chromosome sets viable?

Answer 34

NO

Question 35

What is euploidy? | Aneupolidy?

Answer 35

Euploidy: normal number of chromosomesl 22 pairs of autosomes and one pair of sex chromosomes. (46, XX or 46,XY) Aneuploidy: extra or missing chromosomes Missing-> monosomy (2N-1) Trisomy-> extra (2N+1) Most are lethal

Question 36

What are the aneuploidys that aren't lethal?

Answer 36

Exception: Monosomy X Trisomy 13,18,21 Triploidy (3N) = 2 sperm fertilizing 1 egg = 3 complete sets of chromosomes = lethal)

Question 37

Give karyotypes for Male w/ DS F w/ trisomy 13 Kleinfeelter syndrome (male) Female with Turner syndrome

Answer 37

47, XY, +21 47, XX, +13 47, XXY 45, X

Question 38

What are the abnormalities of chromosome structure?

Answer 38

Translocation: genetic material moved from one chromosome to another ->Reciprocal = two chromosomes exchange segments ->Non-reciprocal = movement of DNA from one chromosome to another Inversion: segment is inverted in respect to rest of chromosome

Question 39

How do we identify and name abnormal chromosome identity?

Answer 39

Chromosome centromere stays constant.

Question 40

Where do the most common translocations happen?

Answer 40

Acrocentric chromosomes Short (p) arms of these chromosomes contain only genes for ribosomal RNA (rDNA gene copy number is very large)

Question 41

What are robertsonian translocations?

Answer 41

45, XX, -14, -21, +rob(14q;21q) Carrier usually has a normal phenotype. Don't lose DNA b/c q arms maintained. P arms have the mRNA copy numbers. Maintain 1 normal copy of 14/21

Question 42

What are the lethal gametes produced by Robertsonian translocation?

Answer 42

Trisomy 14 [rob(14;21)+14] Trisomy 21 [rob(14;21)+21]

Question 43

What is the karyotype of isochromosome 21?

Answer 43

45, XX, t(21;21)(q10,q10) This is viable since it is diploid for 21q. ->gametes recieve either i21q or no chromosome 21) Fertilization leads to -Trisomy 21 (only viable in Down Syndrome) -Monosomy 21 (lethal)

Question 44

How would we label a pericentric conversion? How can we tell a pericentric inversion from a paracentric?

Answer 44

46, XY, inv(6)(p23;q21) May be phenotypically normal Pericentric INVOLVES THE CENTROMERE Paracentric inversion -> breakpoints occur in the same arm

Question 45

How would we karotype a duplication? | Deletion?

Answer 45

46, XX, dup8(q13->qter) ; ter=terminus 46, XY, del19(q13.1,q13.3)