Genetics Exam 1

what are the 4 fundamental properties of genetic material?

Genetic material must…

1. contain complex information

2. encode a phenotype

3. replicate faithfully

4. have the capacity to vary

What is the main structural difference between ribose and deoxyribose sugars?

deoxyribose has a hydrogen bonded on the 2’ carbon, but ribose has a 2’ hydroxyl group that makes it less stable

identify the purines and pyrimidines

• purines (6 & 5 rings structure): Adenine, Guanine

• pyrimidines (6 ring structure): Cytosine, Thyamine, Uracil

What is polarity?

polarity is the direction that synthesis follows from 5’ to 3’ (5’ phosphate to 3’ hydroxyl)

what is a polynucleotide?

a single strand of DNA or RNA

Chargaff’s Rule

• A bonds with T (2 hydrogen bonds)

• C bonds with T (3 hydrogen bonds)

• this makes strands complimentary

• C+T = A+G

Identify the 2 supercoiling enzymes in prokaryotes

1. topoisomerase 1 - one strand of DNA is nicked and induces coiling

2. topoisomerase 2 - both strands nicked and induces coiling

Explain DNA packaging between prokaryotes and eukaryotes

Prokaryotes (bacteria)

• first use topoisomerase enzymes to nick and supercoil the DNA

• then looped with looping proteins

Eukaryotes

• first DNA wraps around histone proteins and is locked into place by linger protein

• histones bunch and form nucleosomes

• nucleosomes pack together to form chromatin (DNA+proteins) that then forms chromosome

identify and explain the 2 levels of chromatin organization

1. euchromatin - less condensed, lots of genes (arms of chromosome)

2. heterochromatin - compact, few genes (telo/centromeres of chromosome)

what core histones make up nucleosomes?

8 total (2 of each): H2A, H2B, H3, H4

What are the 3 models of DNA replication?

1. conservative

2. semiconservative

3. dispersive

What type of replication model does DNA go through?

semiconservative; means that each daughter cell contains an old/original strand and a new strand

What are the 3 requirements for DNA Replication?

1. template of single stranded DNA (unwound double stranded DNA)

2. substrates (dNTPs) to be assembled into a new nucleotide strand

3. enzymes that assemble substrates into double stranded DNA molecule

What proteins expose single stranded DNA at the origin for prokaryotic replication? (AKA what proteins start the process of DNA replication?)

• initiator proteins start process at origin by binding to oriC

• helicase unzips DNA into single strands by breaking hydrogen bonds between bases at replication forks

• single stranded binding proteins (SSBPs) stabilizes single stranded DNA during replication

• gyrase (topoisomerase II) relieves tension ahead of replication fork by breaking and rejoining DNA strands

Explain the process of adding a nucleotide to a DNA strand during replication

1. The 3’-OH group on the pentose sugar attacks the 5’ (alpha) phosphate on the phosphate group of the incoming dNTP

2. The remaining 2 phosphates are cleaved off

3. A phosphodiester bond forms between the nucleotides on the new DNA strand

What proteins actually synthesize DNA during replication?

• primase (RNA polymerase) provides primers for leading and lagging strands

• DNA polymerase III adds the nucleotides to the strands (exonuclease removes any wrong nucleotides)

• DNA polymerase I replaces primers with nucleotides once synthesis is complete (also has exonuclease)

what are the functions of polymerase and exonuclease?

• polymerase - adds nucleotides in 5’ to 3’ direction

• exonuclease - removes nucleotides in both 5’ to 3’ and 3’ to 5’

what is the function of ligase?

ligase connects the okazaki fragments of the lagging strands during DNA replication so that there is a continuous molecule

how many origins are present for eukaryotic DNA replication?

multiple origins “fire” at different times but all must eventually synthesize the entire genome

What are the steps in eukaryotic DNA replication?

1. Initiation

2. Unwinding

3. Elongation

4. Termination

What occurs during initiation of eukaryotic DNA replication?

• Origins are licensed (approved) for replication by the origin recognition complex (ORC)

• Helicases initiate replication

What occurs during unwinding of eukaryotic DNA replication?

• DNA helicase unwinds strands

• SSBPs stabilize single stranded DNA during replication

• Gyrase (Topoisomerases) removes supercoiling ahead of replication forks

what occurs during elongation in eukaryotic DNA replication?

• DNA polymerase alpha initiates synthesis by generating primers

• DNA polymerase delta synthesizes lagging strands

• DNA polymerase epsilon synthesizes leading strand

• DNA polymerase gamma synthesizes mitochondrial DNA

what is the function of DNA polymerase alpha?

generates primers

what is the function of DNA polymerase delta?

synthesizes lagging strands

what is the function of DNA polymerase epsilon?

synthesizes leading strand

what is the function of DNA polymerase gamma?

synthesizes mitochondrial DNA

True or False: all DNA polymerases have proofreading activity

True

What occurs during termination of eukaryotic DNA replication?

• There is an end-replication problem—primers cannot be replaced with nucleotides because there is no 3’-OH group for nucleotides to attach

• When primers are removed, there is a 3’ overhang which shortens the chromosome at telomeres

• However, some but not all cells have a specialized process to replicate telomere ends

For some cells, how do telomere ends replicate after termination?

• Telomerase contains the complementary telomere sequence and uses reverse transcriptase to synthesize DNA from RNA

• The complimentary RNA template pairs under the 3’ overhang (telomere)

• The RNA template moves along the DNA, adding nucleotides to extend the strand

• Telomerase (RNA) is removed

• Synthesis replaces the telomerase’s absence on the complimentary strand

What is the purpose of telomerase?

Telomerase extends the DNA due to the removal of primers during DNA replication in eukaryotes. However, this mechanism doesn’t occur in all cells.

What is the purpose of PCR?

replicates DNA exponentially by amplifying a small amount of DNA

What are the steps of PCR?

1. Melting (Denaturing)

2. Annealing

3. Extension

What occurs during the melting (denaturing) stage of PCR?

DNA is heated until the two strands are separated

What occurs during the annealing stage of PCR?

DNA is cooled to allow primers to stick/anneal to their complimentary sequences

What occurs during the extension stage of PCR?

The DNA and primers are heated so that DNA polymerase synthesizes new DNA strands. This creates two new double stranded DNA molecules.

What are the requirements for PCR?

1. Template DNA—DNA of interest that is amplified

2. Substrates—dNTPs

3. Enzyme—DNA polymerase

4. Primers—single stranded polynucleotides designed to target the change

5. Salts/Buffer, H2O, Mg++

Which are the sense and anti-sense strands in PCR?

The sense strand is the top strand while the anti-sense strand is the bottom one (anti-parallel). However, during annealing the primers go on the separate strands than they are originally associated/labeled.

What are some guidelines for primer design in PCR?

Length:

• 20-24 bases in length

• 50% GC

• avoid repeated stretches of bases

Melting Temp:

• between 55-65 degrees C

• TmC = 4(#GC) + 2(#AT)

• Primers should be close im Tm of each other

Polarity

• 5’ to 3’

What is the purpose of dideoxy/sanger sequencing?

‘reads’ the nucleotide sequence of target DNA base by base, but terminates synthesis because the dideoxynucleotide lacks a 3’-OH

define polycistronic and monocistronic

• polycistronic - multiple genes can be transcribed at once (in prokaryotes)

• monocistronic - generally one gene can be transcribed at once (in eukaryotes)

List and explain the 3 main classes of RNA in both bacterial and eukaryotic cells

1. Ribosomal RNA (rRNA) - structural and functional components of the ribosome

2. Messenger RNA (mRNA) - carries genetic code for proteins

3. Transfer RNA (tRNA) - incorporates amino acids into polypeptide chain for protein formation

What is transcription?

synthesizing RNA from a DNA template

list and explain the strands of RNA during transcription

• template (non-coding) strand - strand that is transcribed from DNA into RNA (AKA antisense)

• non-template (coding) strand - not transcribed (AKA sense)

What are the 3 requirements for transcription?

1. template of single stranded DNA

2. substrates (rNTPs) that are assembled into a new RNA polynucleotide

3. “transcription complex” or RNA polymerase and factor(s) - recognize and synthesize substrates into an RNA strand by ‘reading’ DNA template

What are the 3 steps of transcription?

1. Initiation

2. Elongation

3. Termination

Explain initiation of transcription in prokaryotes

• the sigma factor and the core enzyme of RNA polymerase join to form a holoenzyme

• The holoenzyme (AKA transcription complex) binds the promoter and unwinds DNA

• RNA polymerase base pairs at the +1 and begins synthesis of RNA strand

Explain elongation of transcription in prokaryotes

• the promoter escapes once the transcription bubble is formed and synthesis starts

• the template strand is used for RNA synthesis

• transcription occurs until it reaches a terminator

Explain termination of transcription in prokaryotes

• RNA polymerase stops synthesizing RNA and dissociates from the DNA template

• The newly synthesized RNA strand is released through either rho-dependent or independent mechanisms