Genetics Lecture 2

What type of chromosomes do eukaryotes have?

Linear chromosomes.

What type of chromosome do most prokaryotes have?

One large circular chromosome.

What are plasmids?

Small circular DNA molecules separate from the main bacterial chromosome, can replicate on their own and contain antibiotic resistance

What kind of genes are often found on plasmids?

Antibiotic resistance genes.

What is the correct form of replication

semiconvservative

What does semiconservative replication mean?

Each new DNA molecule contains one old strand and one newly synthesized strand.

What is dispersive replication

Old and New DNA would be mixed together in little section throughout the strand, patches throughout

What does conservative replication mean

Og DNA stays together and a new double helix is made

What did Meselson and Stahl prove?

DNA replication is semiconservative.

What isotopes did Meselson and Stahl use?

Heavy nitrogen 15N and light nitrogen 14N.

What occurred in Meselson and Stahl experiment?

o   Grew E. Coli in heavy Nitrogen first (contains all of the DNA bases, which is why it is heavy)

o   They moved the bacteria into normal light nitrogen and let it replicate

o   After one round of replication, there was one intermediate band

One old heavy N and one light N (more replications, the lighter it becomes)

After one round of semiconservative replication, what type of DNA is produced?

Hybrid/intermediate DNA: one heavy strand and one light strand.

After two rounds of semiconservative replication, what bands are present?

One intermediate band and one light band

In conservative replication after two rounds, what bands would appear?

One heavy band and one light band.

What is theta replication?

Circular DNA replication that forms a replication bubble and usually proceeds bidirectionally. (2 circular DNA)

Where is theta replication commonly used?

Bacterial chromosomes such as E. coli.

What is rolling-circle replication?

Replication where one DNA strand is nicked and synthesis proceeds around the circle.

o   Basically gets cut and creates free 3’ OH end and the DNA polymerases adds nucleotides at 3’ end, as new DNA is made the old strand gets pushed/displaced outward, goes in one direction, displaced strand gets copied and produces circular DNA molecules ( A LOT)

Where is rolling-circle replication commonly found?

Some plasmids and viruses.

Is rolling-circle replication usually unidirectional or bidirectional?

Unidirectional.

Why do eukaryotic chromosomes have many origins of replication?

Their chromosomes are very long, so many origins allow replication to happen faster.

What forms at each eukaryotic origin of replication?

A replication bubble with two replication forks.

What is a replication fork?

The Y-shaped region where DNA is unwound and new DNA is synthesized.

What does replication licensing do?

Ensures each origin is used only once per cell cycle.

In what direction does DNA polymerase synthesize DNA?

5′ to 3′.

In what direction is the DNA template read?

3′ to 5′

Why is one DNA strand replicated discontinuously?

Because DNA strands are antiparallel and DNA polymerase can only synthesize 5′ to 3′.

What is the leading strand?

The strand synthesized continuously toward the replication fork.

What is the lagging strand?

The strand synthesized discontinuously away from the fork

What are Okazaki fragments?

Short DNA fragments made on the lagging strand.

What does helicase do?

Unwinds DNA by breaking hydrogen bonds between the strands.

What do single-strand binding proteins do?

Keep separated DNA strands from reannealing.

What does DNA gyrase/topoisomerase do?

Relieves twisting and supercoiling ahead of the replication fork.

What does primase do?

Makes short RNA primers.

Why is a primer needed in DNA replication?

DNA polymerase needs a free 3′-OH to begin adding nucleotides.

What is the main bacterial DNA synthesis enzyme?

DNA polymerase III.

What does DNA polymerase I do in bacteria?

Removes RNA primers and replaces them with DNA.

What does DNA ligase do?

Seals nicks in the sugar-phosphate backbone.

What does proofreading do?

Removes incorrectly paired nucleotides during replication.

What direction is proofreading exonuclease activity?

3′ to 5′.

What is mismatch repair?

Repair of replication errors after DNA synthesis is completed.

Why is eukaryotic DNA replication more complicated than bacterial replication?

Eukaryotic DNA is packaged with histones into chromatin.

What happens to nucleosomes during DNA replication?

They are temporarily disrupted and then reassembled on the new DNA

What are histones used for?

Packaging DNA into chromatin.

What is the end-replication problem?

The final RNA primer on a linear chromosome cannot be completely replaced, causing chromosome shortening.

Why does the end-replication problem mainly affect eukaryotes?

Eukaryotic chromosomes are linear and have ends

What are telomeres?

Repetitive DNA sequences at the ends of chromosomes.

What does telomerase do?

Extends the 3′ end of telomeres by adding repetitive DNA

What happens to telomeres in most normal somatic cells?

They progressively shorten with each division.

In what cells is telomerase more active?

Germ cells, early embryonic cells, some stem cells, and some rapidly dividing cells

How is telomerase related to cancer?

Many cancer cells reactivate telomerase, allowing telomeres to be maintained and division to continue.

Does telomerase directly cause cell division?

No. It helps remove the telomere-shortening limit on repeated division.

What is Werner syndrome associated with?

Defective WRN helicase, telomere problems, and premature aging.

What is dyskeratosis congenita associated with?

Defective telomere maintenance, often involving telomerase-related genes. (bone marrow failure)

What is PCR used for?

Amplifying a specific DNA sequence.

What are the three PCR steps?

Denaturation, annealing, extension.

What happens during PCR denaturation?

DNA strands separate using heat. 95°C

What happens during PCR annealing?

DNA primers bind to complementary target sequences. o   50-65

What happens during PCR extension?

Taq polymerase synthesizes new DNA o   72

Why is Taq polymerase useful in PCR?

It is heat stable.

Are PCR primers RNA or DNA?

DNA.

What does high annealing temperature do to PCR specificity?

Increases specificity.

What does low annealing temperature do to PCR specificity?

Decreases specificity and can produce multiple product

What does a longer primer generally do?

Increases specificity.

What does more G/C content do to melting temperature?

Raises the melting temperature.

Why does more G/C raise melting temperature?

G-C pairs have three hydrogen bonds versus two for A-T.

What is transcription?

Making RNA from a DNA template

In what direction is RNA synthesized?

5′ to 3′.

In what direction does RNA polymerase read the template?

3′ to 5′.

What are the three main parts of a transcription unit?

Promoter, RNA-coding region, terminator.

What is the promoter?

The region where transcription machinery binds and transcription begins.

What is the RNA-coding region?

The DNA region copied into RNA.

What is the terminator?

A signal that causes transcription to stop.

What is the template strand?

The DNA strand RNA polymerase reads.

What is the nontemplate/coding strand?

The DNA strand whose sequence matches the RNA except T is replaced by U.

What does upstream mean?

Before the transcription start site.

What does downstream mean?

In the direction transcription proceeds.

What does sigma factor do in bacteria?

Helps RNA polymerase recognize the promoter

What is the bacterial RNA polymerase holoenzyme?

Core RNA polymerase plus sigma factor.

What are the main bacterial promoter consensus regions?

The -35 and -10 regions.

What is the bacterial -10 consensus sequence?

TATAAT, the Pribnow box.

What is the bacterial -35 consensus sequence?

TTGACA

What does +1 mean in transcription?

The first nucleotide that is transcribed into RNA.

Does RNA polymerase require a primer?

No.

What are the three stages of transcription?

Initiation, elongation, termination.

What is rho-independent termination?

Termination caused by an RNA hairpin followed by a stretch of uracils

What is rho-dependent termination?

Termination using the Rho protein.

What does Rho do?

Binds RNA, catches RNA polymerase, and helps release the transcript

Which RNA polymerase makes most rRNA in eukaryotes?

RNA polymerase I.

Which RNA polymerase makes pre-mRNA/mRNA in eukaryotes?

RNA polymerase II.

Which RNA polymerase makes tRNA in eukaryotes?

RNA polymerase III.

Do eukaryotes use sigma factor?

No.

What helps eukaryotic RNA polymerase II bind promoters?

Transcription factors.

What binds the TATA box?

TATA-binding protein, TBP

What do enhancers do?

Increase transcription when activator proteins bind them.

What must often happen to chromatin before eukaryotic transcription?

Chromatin must be opened or modified so the DNA is accessible.

What does mRNA do?

Carries genetic information from DNA for protein synthesis.

What does rRNA do?

Forms part of the ribosome and helps catalyze protein synthesis.

What does tRNA do?

Carries amino acids to the ribosome.