Human Genetics-Exam 2

Frederik Griffith

discovered bacterial transformation using different stains and types of Pneumococcus bacteria (1928)

virulent

killed the mice (s-strain)

non-virulent

did not kill the mice (r-strand)

Avery, MacLeod, McCarty

determined DNA was transforming factor (not protein, sugar, or carbs)

Erwin Chargaff

discovered the rules of base pairing

Rosaline Franklin and Maurice Wilkins

deduced overall structure of DNA using x-ray diffraction

James Watson and Francis Crick

determined the molecular structure of DNA

created 3D models, no experiments 

Nucleotides

DNA molecule with Nitrogenous base, Sugar (deoxyribose), and phosphate group

has 1-3 phosphate attached

nucleoside

nitrogenous base and sugar but no phosphate group

Purines

double-ring structure 

Guanine and Adenine

Pyrimidine

single-ring structure

Thymine and Cytosine

Phosphodiester bond

bond that holds nucleotides together. creates the sugar-phosphate backbone

DNA double helix

2 polymers of nucleotides that are antiparallel

right-handed helix

major and minor groves

Hydrogen bond

Holds bases together

A:T has 2 hydrogen bonds

C:G has 3 hydrogen bonds

Meselson and Stahl Experiment

DNA replication is semiconservative

DNA Polymerase

Catalyzes the formation of the phosphodiester bond

bidirectional: occurs in both directions

Origins of Replications

replications begins

earlier ORI has bigger circle

Consensus Sequence

similar or identical nucleotides occurring at the same position in DNA sequence in many lineages of a species or across species

replisome 

machinery that catalyzes replication 

leading strand

continuous replication 3’ - 5’

lagging strand

discontinuous replication 5’ - 3’

Okazaki Fragments

fragments from lagging strand

RNA Primer

use a short amount to get started

gets replaced with DNA later

DNA topoisomerase 

relaxes supercoiling 

Helicase

unwinds the double helix - unzipping

single strand binding proteins

prevents reannealing of separated strands

Primase

Synthesizes RNA primers

DNA polymerase III

synthesizes DNA

DNA polymerase I

removes and replaces RNA primer with DNA 

DNA ligase

joins DNA segment with phosphodiester bond

molecular glue

Telomeres

Structure at ends of DNA strand

code repeated thousands of times and folds upon itself bounded by Shelterin Complex

length decreases as we age

Ends Replication Problem

Telomeres shorten with DNA replication

Telomerase

enzyme that catalyzes telomere lengthening

-germ cells

-some somatic cells

-90% of cancer cells

Gene expression

info is encoded by gene and used to direct synthesis of functional gene product

RNA

less stable than DNA

can catalyze reactions

mostly single-stranded

has uracil

Ribozyme

RNA able to catalyze reactions

Messenger RNA (mRNA)

used to encode the sequence of amino acids in a polypeptide

Ribosomal RNA (rRNA)

RNAs that are part of the ribosome, responsible for translation

Transfer RNA (tRNA)

caries amino acids to the ribosome 

small nuclear RNA (snRNA)

RNAs that are part of the spliceosome, responsible for mRNA splicing

MicroRNA (miRNA)

regulate gene expression by affecting mRNA stability and translation, arises from single strand RNA

small interfering RNA (siRNA)

regulate gene expressing by affecting mRNA stability and translation, arises from long double strand RNA

Cis Regulatory Elements

short sequences of DNA in the promoter that are the binding sites for proteins

Transcription factors 

proteins that bind to DNA at Cis regulatory elements

general transcription factors

transcription factors located in every gene (TFIIA)

Preinitiation complex

transcription factor binds with RNA polymerase creates the

+1 nucleotide

transcription begins at this location

coding region

transcribed portion

termination region

transcription is ended

core promotor

minimum amount of DNA sequence to be transcribed by RNA polymerase

consensus sequence

group of cis regulatory elements, group of genes we know TATA

Transcription Initiation

1) preinitiation complete is built but is inactive/closed (general TFs + RNA pol)

2) TFIIH performs helicase activity (unwinding) and kinase activity (adds phosphate group catalyzes phosphorylating) to make it active/open

3) promotor escape: transcribes enough to no longer be in promotor stage 

4) General TFs dissociate form RNA pol II

Transcription Elongation

template strand: RNA pol scans 3’ → 5’ and makes complementary 

coding strand: same sequence as synthesized RNA

Transcription Termination

1) signals for RNAase

2) Torpedo RNAase cuts DNA at AAUAAA in mRNA, then chases after RNA by degrading leftover mRNA

3) when they meet, RNA pol stops transcribing

5’ Cap

1) Guanylyl transferase enzyme: 5’-5’ triphosphate linkage, G-G

2) Methyl transferase enzyme: methylation added to cap

3) Functions:

Protect from degradation

facilitate transportation

facilitate splicing

enhance translation efficiency

UTR

untranslated regions → 5’ or 3’

exons

expresses sequences, retained and transcribed by ribosome

introns

removed during splicing

Polyadenylation

polyadenylate polymerase: adds As to mRNA 3’ end (20-200)

Functions:

protect from degradation

facilitate transportation

enhance translation activity 

Splicesome

catalyzes mRNA splicing

Small nuclear ribonucleic proteins (snRNP): come together to form spliceosome, U1, U2 etc, made from small nuclear RNA (snRNA) and proteins

snRNPs bind splicing regulatory elements (SRE)

5’ splice cite: beginning of intron

branch site: middle of intron

3’ splice site: end of intron

Amino Acids

building blocks of proteins

20 common R groups

Peptide bond

links amino acids

condensation reaction

2 amino acids coming together

creases residue

N-terminus

beginning of polypeptide (H3N+)

C-terminus

end of polypeptide bond (COO-)

Primary

sequence of amino acids in polypeptide

secondary 

formation of alpha-helixes (coils) and beta-pleated sheets

Hydrogen bonds form between the backbone C=O of one strand and N-H of another

tertiary

3D shape of polypeptide, bonds between R-group and backbone 

quaternary 

combination of multiple 3D polypeptides

Translation

mRNA sequence dictates assembly of polypeptides

reads in 3 nucleotides

Ribosome

Ribosomal RNA associated with certain proteins form…

A site

aminoacyl site: the incoming tRNA binds here

P site

peptide site: tRNA with growing polymer

E site

exit site: empty tRNA

functions of rRNA

structural support and catalysis

tRNA

bring amino acids to the ribosome to match with corresponding codon

anticodon

part of tRNA that does base pairing with mRNA. matches with codon

charged tRNA

with amino acids

uncharged tRNA

without amino acids

Translation Initiation

preinitiation complex made

initiator tRNA: met

Kozak sequence 

AUG start sequence 

Translation Elongation

peptide bond formed, ribosome moved towards 3’ end

GTP hydrolysis: Ef-Tu and GDP released

Translation Termination

encounters stop codon

release factor binds A-site

polypeptide released form P-site tRNA

ribosome subunits separate from mRNA

Genetic Code

64 unique codes

synonymous: different mRNA, same amino acids

nonsynonymous: different mRNA, different amino acids

third base wobble

multiple different third nucleotide can create the same amino acid, it is relaxed

Polymerase Chain Reaction

amplifies specific DNA sequence in vitro

Required parts: template, primers, nucleotides, thermostable DNA polymerase

after 20 cycles there is over a million identical DNA molecules

2^n - 2n

Using PCR and DNA gel electrophoresis

analyze differences between individuals

variable number tandem repeats (VNTR)

PCR amplify region around VNTR, size of band corresponds to repeated number

Dideoxy DNA Sequencing (sanger sequencing)

3’ end does not have hydroxyl group

the chain stops synthesizing when dideoxynucleoside is added → polymer stops growing 

runs four different reactions each with different nucleotides

fragments separated by gel electrophoresis

Next Generation DNA Sequencing

simultaneous sequence of many small DNA fragments during synthesis

PCR used to create many rounds 

fluorescent marker highlights which base is likely to be in that position 

vivo

studying item in place

vitro

taking out item then studying it

Southern blotting

detects specific DNA segments in cell extracts separated by gel electrophoresis

probe

known nucleic acid sequence created in lab

target sequence

sequence of interest in a heterogeneous population of sequences

Chromosome Fluorescence in Situ Hybridization

probes for specific DNA sequences on chromosome

all chromosomes stained blue and the probes are fluorescently labeled

northern blotting

detects specific RNA in cell extracts separated by gel electrophoresis

RNA fluorescence in situ hybridization

detects specific sequence RNA in fixed cells and tissues

Reverse transcriptase - quantitative polymerase chain reaction (RT-qPCR) 

measures RNA abundance in “real time”

monitors abundance of one RNA segment at a time

makes complementary DNA

more RNA expression → more starting material → reaches threshold faster

RNA next generation sequencing (RNAseq)

measures all RNA in a cell at once

levels of expression

Microarray and RNAsep

represented by heat maps

red or green lines represent differentially expressed genes between two samples

brightness → high or low expression

differences in gene expression occurs across cancer cells