Genetics Exam 3

chromosome condensation

• 2 meters (6.5 ft) of DNA in every cell in our body

• Large DNA molecules must be tightly condensed to fit within the nucleus of a cell

• condensation occurs in stages:

  • nucleosomes

  • solenoids

  • chromatin loops

  • condensed chromatin

  • chromatin folded around protein scaffold

• intimately associated with the cell cycle and gene expression

chromatin

• a complex of DNA and protein found in eukaryotic cells

• primary function: to package long DNA molecules into more compact, denser structures

euchromatin

• lightly packed chromatin rich in gene concentration and is most often under active transcription

heterochromatin

• tightly packed chromatin consisting mainly of genetically inactive sequences

  • faculative

  • constitutive

faculative heterochromatin

type of heterochromatin that, under the right conditions, can change to euchromatin

• gene silencing, barr bodies

constitutive heterochromatin

• type of heterochromatin

  • very gene poor, centromeres and telomeres

  • ALWAYS closed, can never be accessed

folded fiber model

• 1965 E.J. dupraw

  • whole mounts of human white blood cells

  • found few or no free fiber ends and concluded each chromatid must consist of a single fiber

  • .2nm double helix of DNA

    • Type A fiber- 1-10nm (ratio of ~6:1)

    • Type B fiber- 20-25 nm (ratio of ~10:1)

  • extensive folding of Type B forms a chromatid

  • replication form each end toward centromere

  • Condensation occurs through extensive, irregular looping and folding both longitudinally and transversely

  • Describes each chromatid as a single continuous, unbranched 20 nm20nm to 30 nm30nm nucleoprotein fiber

nucleosome model

• 1974, R.D. Kornberg; 1975 P. Oudet

  • most commonly accepted model for DNA packaging

• nucleosome

• Fundamental repeating structural unit of eukaryotic chromatin, producing the "beads-on-a-string" appearance (10 nm fiber)

nucleosome

• simplest packaging structure of all eukaryotic chromatin

• localized areas of transcription; better fit for protein biosynthesis

core histones

• H2A, H2B, H3, H4

  • consist of approximately 120 amino acids each

  • highly conserved during evolution

  • in combination, form the core particle (ocatmer)

  • VERY BASIC in charge (approx 25% lysine and arginine)

linker histones

• H1

  • consist of approximately 200 amino acids

  • tissue specific expression and NOT highly conserved

  • loosely associated with core particle

    • fairly basic

    • not considered a part of the core

10 nm fiber

• nucleosome

  • 200 bp DNA

    • 146 bp wrap core

    • 54 bp link to next nucleosome- LINKER DNA

  • reduce length by 7 times

  • linear arrangement

  • primary packaging of the chromatin

  • “beads on a string”

30 nm fiber

• type of condensation of chromatin

  • H1 histone responsible for packaging

  • solenoid

  • supercoiling- reduces length of DNA by ~7 times

solenoid

• type of condensation of chromatin

  • helical coiling of 10nm fibers consisting of 6 nucleosomes

H1 histone

• works by binding 2 distinct regions of DNA

  • linker DNA

  • portion of the 146 bp core

• indues tighter DNA wrapping around histones= ~40x

  • involved with creation of solenoid

zig-zag model

• alternative model

  • DNA backbone not flexible enough to bend between nucleosomes

  • straight linker DNA connect opposite nucleosomes

• this model allows for more compaction

• middle of “tube” has DNA

• experimental results show that both model topologies may both simultaneously be present in chromatin fiber

300nm fiber

• type of higher order coiling of chromatin

  • “chromatin loops”

  • built around a scaffold of topoisomerase 2

  • compaction level of euchromatin

700nm fiber

• final condensation of chromatin

  • DNA diameter is ~700nm

  • spiral scaffold composed of topoisomerase 2 and about 15 non-histone proteins

  • compaction level of heterochromatin

    • metaphase chomosome

chromatin formation

• cell cycle

  • I: interphase: DNA is replicated in S phase

  • P: Prophase:

    • early: chromosomes condense

    • late: nuclear envelope breaks down, mitotic spindle assembles

  • M; metaphase: mitotic spindles arrange chromosomes at cell equator

  • A: Anaphase: sister chromatids separate

  • T: Telophase: cell plate forms

biological complexity

the result of the hierarchical organization of nested levels of cells, tissues, and higher order part types interacting together

C value paradox

• excess DNA is present in the genome that does not seem to be essential for the development or evolutionary divergence of an organism

  • genome size does not correlate with organismal complexity

the onion test

• T. Ryan Gregory

• Onion

  • diploid (2n= 16)

  • haploid= 17pg

  • 17×10^6bp (has 5x more DNA)

• Human

  • diploid (2n= 46)

  • haploid= 3.5 pg

  • 3.5×10^6bp

G-value paradox

• the number of genes does not correlate with organismal complexity

  • ex: watercress and humans ~ 20,000 genes

  • coding sequences: 2-5% of the human genome

classes of nucleotide sequence

1. highly repetitive DNA sequence (HR)

2. moderately repetitive DNA sequence (MR)

3. single copy DNA sequence (unique)

highly repetitive DNA sequence

• comprises about 10% of the human genome

• most is located in heterochromatic regions around the centromere/telomere

  • “non-coding DNA regions”

• occurs as variable length modifs (5-100 bp), in long tracts of up to 100 Mb

• present at >10^6 copies per genome

• postulated functions include structural and organization role (centromeres and telomeres) to nothing more than junk

• e.g. alpha satellite DNA- from 2 to more than 30 repeats of 171 bp tandem repeats

• heterochromatin (tightly coiled)

moderately repetitive DNA sequence

• comprises about 30% of the human genome

• found mostly throughout euchromatin (faculative heterochromatin)

• average 300bp in size

• present at 10-10^5 copies per genome

• function in length mutability, modulation of transcription factor binding, spacing between promoter elements, cytosine methylation, alternative splicing, mRNA stability, transcription start and termination sites, etc.

• also includes “redundant” genes for histones, and ribosomal RNA and proteins (gene-products present in cell in large numbers)

• e.g. microsatellite DNA (repeating sequences of 2-30 bp of DNA); interspersed repetitive DNA (transposable elements)

• scattered throughout euchromatin

microsatellites

variable number of tandem repeats typically occurring in non-coding regions of the genome

• occurs through a mutation process known as “slippage recognition”

• type of moderately repetitive DNA sequence

• useful genetic markers as they tend to be highly polymorphic

  • used to sequence the human genome

  • markers for certain disease conditions

  • primary markers for DNA testing in forensic cases

single copy DNA sequence

• unique

• comprises about 1-5% of the human genome

• found throughout the euchromatin

  • “coding DNA regions = GENES”

  • ~20,000 protein coding genes

  • present at single or low copy number per genome

• euchromatin- GENES

gene

• sequence of unique nucleotides (genotype) that carry the genetic information which is to be expressed (phenotype)

  • “instruction manuals for our bodies”

  • every person has 1 copy from mother and 1 from father

• >99.5% of the DNA, number of genes, types of genes, protein products, are the same in ALL people

  • <1% of the total number of genes contribute to each person’s unique physical features

• the basic physical and functional unit of heredity

transcriptional unit

• molecular level “gene” - DNA sequence that gives rise to an RNA molecule

  • contains:

    • promoter

    • transcribed region

    • regulatory sequence

    • terminator

transcribed region

• contains exons and introns

• flanking regions

  • 5’ untranslated region

  • 3’ untranslated region

exon

“coding sequences” = phenotype

make it into mature mRNA

• part of the transcribed region of a gene

intron

• “intervening sequences” = areas of genes that generally dont code for phenotype

  • taken out of mature mRNA

  • part of the transcribed region of a gene

5’ untranslated region

• part of the transcribed region of a gene

  • mRNA that is directly upstream from the initiation codon

    • differs in prokaryotes and eukaryotes

    • mostly involved in regulation of translation

    • recruitment of ribosomal subunits

3’ untranslated region

• part of the transcribed region of a gene

  • section of mRNA that immediately follows the translation termination codon

    • influences the localization, stability, export, and translation efficiency of an mRNA

    • post-transcriptional influences on gene expression

promoter

• part of the structural view of a gene

  • DNA sequence onto which the transcription machinery binds and initiates transcription

  • ex: TATA box

TATA box

• type of core promoter

  • highly conserved sequence in DNA serving as the binding site for transcription factor binding

    • core DNA sequence= TATAAA

    • ON/OFF for transcription

  • Basal level of transcription

regulatory sequences

• enhancer/silencer regions

  • CAAT box

  • GC Box

  • RE1

• “ramped up level of transcription or silenced transcription”

CAAT box

• type of regulatory sequence

  • GGCCAATCT- consensus sequence that occurs upstream by 60-100 bases to the initial transcription site

    • typically required for inducible genes to be produced in sufficient amounts

GC box

• type of regulatory sequence (proximal region)

  • region of DNA that can be bound with proteins (activators) to activate transcription of a gene or genes

    • ramps up or silences

RE1

• type of regulatory sequence (proximal region)

  • region of DNA that binds repressor proteins preventing neuronal genes from begin expressed in non-neuronal tissues

terminator

section of nucleic acid sequence that marks the end of a gene or operon in genomic DNA during transcription

types of genes

1. solitary genes

2. duplicated genes

3. multigene families

4. psuedogenes

5. repeated genes

solitary genes

• (unique) type of gene

• a single copy of a gene (haploid situation) or two copies in diploid

• comprises the bulk of euchromatin

duplicated genes

• type of gene

  • process by which a portion of a chromosome is duplicated resulting in an additional copy of a gene

  • results in a copy of the original gene called a paralog gene

  • either of the 2 genes may mutate and change the original function of the gene

  • usually occurs due to an error during meiosis

mulitgene families

• type of gene

  • set of several similar genes, formed by duplication of a single original gene, and generally with similar biochemical functions

  • most often located in similar region of the chromosome

  • most often used or synthesized at different times

pseudogenes

• type of gene

  • dysfunctional relatives of genes that have lost their protein-coding ability

  • often the result of multiple mutations within a gene

    • most likely used to be functional (through evolution lost function)

repeated genes

• type of gene

  • multiple copies of small genes clustered throughout the genome at specific sites

  • present in high copy numbers

  • many times present in a “heat to tail” configuration

  • ex; genes for tRNA and rRNA

cell division

• process where a parental cell divides into 2 daughter cells

  • cell size is limited due to the ability of the cell to transport food and oxygen from the cell membrane to the interior of the cell

    • maximizes SA:V ratio

  • outside of the cell unable to keep up with the inside because the inside grows at a faster rate

DNA replication

• process by which genetic information is duplicated by the use of an existing strand of DNA as a template for the synthesis of a new, identical strand

• ensures that each cell in an organism has a complete and correct copy of the organism’s genome or genetic data

  • G0: resting phase

  • G1: growth phase; cell increases size for DNA synthesis

  • S- DNA REPLICATION

  • G2: growth phase; cell increases size for mitosis

  • M- cell growth sops and cells divide

Fredrick Griffith

• 1928- “Transforming principle”

• suggested that bacteria could transfer information from one to another

  • first suggestion of DNA at heritable material

Avery-Macleod and McCarty

• 1994: experimental demonstration that DNA is the substance causing bacterial transformation

  • basically confirmed Griffith’s work and isolated DNA as the heritable material

Hershey-Chase

1952- confirmed that DNA was the genetic material

• responsible for inheritance using radioactively labeled bacteriophage to infect bacteria

Franklin and Wilkins

1952: used X ray crystallography to study the structure of DNA

• image B51: X ray diffraction image of DNA

Watson and Crick

1952: used data from image B51 to propose the double helical structure of DNA

• proposed a possible copying mechanism (due to the specific pairing of bases)

• Predictions

  • the DNA helix is “unwound” during replication

  • the sequence of the “parental strand” can serve as a template for synthesis of “new strand” of DNA

  • each nucleotide of the DNA strand is copied precisely due to the complimentary base-pairing.

  • process occurs semi-conservatively

semi-conservative model

• the double stranded DNA contains one parental and one daughter strand following replication

conservative model

• both parental strands stay together after DNA replication and the daughter molecules contain all new nucleotides

dispersive model

• parental and daughter DNA are interspersed in both strands; consist of old and new strands

Meselson-Stahl Experiment

• Cultured E. coli in medium containing heavy nitrogen (15N), then switched cells to medium containing light nitrogen (14N)

• Separated DNA by density using CsCl (cesium chloride) equilibrium density gradient centrifugation:

  • Generation 0: All heavy DNA (15N/15N15N/15N)

  • Generation 1: 100%100% intermediate/hybrid density DNA (15N/14N) — ruled out the conservative model

  • Generation 2: 50%50% hybrid DNA (15N/14N) and 50%50% light DNA (14N/14N) — ruled out the dispersive model

• Supported Watson and Crick's proposal that each replicated double helix contains one conserved parental strand and one newly synthesized daughter strand

replication stages

1. initiation

2. unwinding

3. primer synthesis

4. elongation

replication initiation

• origin of replication (OriC in bacteria)

  • ~250 base pairs AT-rich region (DnaA boxes)

  • GATC methylation sites (precise timing of replication)

• initiator proteins (origin binding proteins) DNaA protein

• replication bubbles

• replication occurs bi-directionally

• replication forks

unwinding of dsDNA strand

• step 2 of replication

  • topoisomerase

  • helicase

  • single strand binding proteins

topoisomerase

• involved in step 2 of unwinding dsDNA

  • aka: gyrase

  • reduce torsional strain causing unwinding of double helix

helicase

• involved in step 2 of unwinding dsDNA

  • breaks hydrogen bonds between complimentary nucleotides

single strand binding proteins

• involved in step 2 of unwinding dsDNA

  • stabilize ssDNA until elongation beings

    • prevent ssDNA from reannealing

priming of ssDNA strand

• step 3 of replication

  • primase

  • primers are typically 10-12 bases in length

  • primers are removed following elongation and replaced with DNA nucleotides

primase

• involved in the 3rd step of transcription initiation

  • RNA polymerase that adds a ribonucleotide primer to ssDNA

transcription elongation

• 4th step of replication (DNA synthesis)

  • DNA polymerase (prok) 1,2,3,4,5

  • DNA polymerase (euk) a,d,e

• limitations:

  • DNA polymerase cannot initiate DNA synthesis by itself; requires addition of RNA primer

  • DNA polymerase can only synthesize DNA in 5’-3’ direction

• replication is semi-conservative

• replication is bi-directional

DNA polymerase

enzymes that catalyze attachment of nucleotides to make new DNA during replication

DNA polymerase 1

• prokaryotic DNA polymerase

  • removes RNA primer and starts synthesis

DNA polymerase 3

• prokaryotic DNA polymerase

  • responsible for most of replication process

DNA polymerase 2,4,5

• prokaryotic DNA polymerase

  • synthesize lagging strand

DNA polymerase alpha

• eukaryotic DNA polymerase

  • synthesizes the primer

DNA polymerase delta

• eukaryotic DNA polymerase

  • synthesize leading strand

DNA polymerase eplison

• eukaryotic DNA polymerase

  • synthesize lagging strand

Watson and Crick DNA model

• two strands of DNA

• run anti-parallel to each other

• sugar phosphate backbone

  • hydrophilic sugar and phosphate backbone on the outside with nitrogenous base inside

• nucleotides joined together by phosphodiester bonds (5’C-3’C)

• Base pairing A=T C- - - G

DNA elongation steps

1. new DNA is synthesized from deoxyribonucleotide triphosphates (dNTP’s)

2. In replication, the 3’OH group of the last nucleotide on teh strand interacts with the 5’PO4 group of the incoming dNTP

3. two phosphates are cleaved off the incoming dNTP

4. a phosphodiester bond forms between the two nucleotides

5. and phosphate ions are released

leading strand synthesis

• continuous synthesis

• goes INTO replication fork

lagging strand synthesis

• discontinuous synthesis → Okazaki fragments

• goes away from the replication fork

• DNA ligase

DNA ligase

• joins together newly synthesized DNA strands by creating a covalent phosphodiester bond

high fidelity

• exonuclease activity

  • mistakes are very rare (1 × 10^6 bases)

    • instability of mismatched bas e pairs

    • configuration of DNA polymerase 3 active site

    • proofreading/exonuclease activity of DNA polymerase 3 (backs up in the 3’-5’ direction to fix mistakes

transcription

• DNA to mRNA (specifically hnRNA- heteronuclear RNA)

  • Where: cell nucleus (eukaryotes) and sometimes mitochondria

  • When: either G1/S or G2/M (waves of transcription that coincides with different transition points during the cell cycle

• immature mRNa (hnRNA) consists of both introns and exons

• mature mRNA, introns have been removed (splicing)

• RNA polymerase makes the new mRNA strand

rules of transcription

1. only one of the two DNA strands serve as a template

   1. template strand (DNA strand that mRNA is built from)

   2. coding strand (matches mRNA except U’s are T’s in coding strand)

2. selective process- each gene has its own transcription protocol

3. always in 5’ to 3’ direction (nucleotides added at 3’ growing tip)

4. results in an RNA compliment (anti-parallel and unidirectional)

steps of transcription

0. Pre-recognition: accessibility to DNA

1. recognition: pre-initiation complex formation

2. initiation: binding of RNA polymerase complex

3. elongation: movement of RNA polymerase 2 and formation of mRNA

4. termination: cleavage of new transcript

pre-recognition

• DNA packaged in the nucleus as heterochromatin

  • facultative

  • constitutive

• Lys (K) acetylation

  • histone acetyl transferase (HAT)→ allows DNA to be read

  • histone deacetylase (HDAC) → clamps tail back down

    • DNA must be converted to euchromatin prior to transcription

    • part of gene expression regulation

• decreases positive charge of the histone core and loosens the DNA strand

transcription recognition

• pre-initiation complex formation at the promoter

  • formation of large complex of ~100 proteins (PIC) required for RNA polymerase 2 to bind

    • TATA binding protein (TBP) binds to TATA box

    • general transcription factors (e.g. TF IID) recruited by TBP

      • TBP binds to TATA → recruits other proteins and TF → formation of pre-initiation complex

trasnscription initiation

• binding of RNA polymerase complex (i.e. mediator)

  • other transcription factors (b,f,h) and RNA polymerase 2 recruited to complex

  • Mediator complex (~20 proteins)

    • ATPase and Helicase activity unwinds DNA

    • ~1 turn of DNA unwinds and forms transcription bubble

  • RNA polymerase 2 binds to the template strand via the polymerase active site

  • RNA polymerase 2 is unphosphorylated at its carboxyl end (CTD)

elongation

• movement of RNA polymerase 2 and formation of mRNA

  • RNA polymerase 2 is phosphorylated at CTD by mediator complex

  • RNA polymerase 2 traverses the template strand and creates an RNA copy

    • RNA polymerase reads template (3’-5’) and writes (5’-3)

  • exact copy of coding strand (except thymines are replaced with uracils, and the nucleotides are composed of a ribose (5C sugar)

termination

• cleavage of new transcript

  • two protein complexes carried by CTD recognize poly A signal (AAUAA)

    • CPSF (cleavage and polyadenylation specificity factor)

    • CSTF (clevage stimulation factor

  • post transcriptional processing can now occur

5’ capping

• co-transcriptional regulation

  • when a growing RNA chain is ~30 nucleotides long, a guanine cap (7-methyl guanine) is added to the 5’ end by capping enzymes

  • functions:

    • regulation of nuclear export- CBC recognized at nuclear pore

    • promotes translation- helps ribosome recognize the message

    • prevention of mRNA degradation in the cytoplasm

    • involved in intron splicing

3’ polyadenylation

• post transcriptional regulation

  • nascent RNA is cleaved by ribonuclease downstream of conserved AAUAAA site

  • Poly (A) polymerase adds adenine ribonucleotides to the 3’ end of the RNA molecule—up to 200 bases

  • not dependent on template

• functions;

1. enhances the stability of the RNA molecule

2. regulates transport to the cytoplasm

RNA processing

• co-transcriptionally and post-transcriptionally

  • splicing

  • introns

  • exons

  • spliceosome

splicing

• the mechanism by which introns are removed

introns

intervening sequences of RNA not expressed in proteins

• GU nucleotide sequence at the 5’ splice site (donor site)

• AG nucleotide sequence at the 3’ splice site (acceptor site)

• polypyrimidine tract (PPT) just upstream of the 3’ splice site

  • promotes the assembly of the spliceosome

• branch point sequence (UACUAAC)- binding site for snRNp-U2

exons

retained in mature mRNA and are the expressing sequences

spliceosome

• large complex of RNA and proteins responsible for removal of introsn from transcribed mRNA (splicing)

  • responsible for both cleavage of the intron from the RNA and ligation of the remaining exons

  • 1977: sharp and Roberts lab revealed that genes of highe rorgansimsa re “split” or present in several distnct segmetns along the DNA molecule

  • hnRNA

  • snRNP

  • mRNA

hnRNA

• pre-mRNA

• immature single stranded mRNA present in the nucleus (contains introns)

snRNP

• snurps

• small nuclear RNA molecules associated with specific ribonuclear proteins playing essential role in splicing (U1,U2,U4,U5,U6)

mRNA

• mature mRNA

• RNA that is translated to become a polypeptide chain

• hnRNA + snRNP

• written in linear form

• comma-less (no pauses)

• coded in triplets (codon)

• unambiguous (each codon only codes for 1 amino acid)

• degenerate (1 amino acid can have more than 1 codon—ex: val has 4 diffferent codons)

• specific start and stop codons

• non overlapping

• universal

spliceosome assembly

1. U1 binds to 5’ splice site

2. U2 binds to BPS

3. trimer of U4, U5, and U6 from hnRNA leaving U5 and U6 bound

4. U1 and U4 dissociate from hnRNA leaving U5 and U6 bound

5. U2 and U6 associate

6. “lariat” of hnRNA is formed from the intron

7. 5’ splice site is cleaved by U5 and U5 binds to 3’ splice site

8. 3’ splice site is celaved and U5 ligates exons togeher via ATP hydrolysis

9. snRNP’s are released along with spliced intron