BIOL1020 Lecture Notes - Lecture 4: Bacterial Growth, Nitrogenous Base, Dna Replication

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blueclam798

20 May 2018

School

Department

Biology

Course

BIOL1020

Professor

Dr Paul Ebert

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L4 - DNA !

DNA

•Occurs in all living cells

•Stores information for the synthesis of speciﬁc proteins

•Directs RNA synthesis

•Directs protein synthesis through RNA

•Made up of nucleic acids. There are two types of nucleic acids

-RNA (Ribonucleic acid)

-DNA (Deoxyribonucleic acid)

Nucleotides !

•monomers of nucleic acids

•Made up of a nitrogenous base, a sugar and a phosphate group

(Know this diagram really well including the carbon positions)

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DNA replication

There are three possible models of replication that can happen and the following

experiment tries to ﬁnd which way DNA replicates.

The Meselson-Stahl experiment

The idea!

If DNA could be labeled so that newly

synthesized DNA could be differentiated

from DNA present at the start of the

experiment, it would be possible to identify

the mechanism of DNA replication based on

the distribution of the label in the DNA.

Materials

•centrifuge to sediment the parts of the DNA

•Bacterial growth media with and without

isotopic label

•Equipment for cell lysis & DNA isolation

•Cesium chloride (for DNA separation)

Ethidium bromide (to visualize the DNA)

The isotope used for labelling the DNA was

an isotope of nitrogen because they needed

an element that already existed in DNA and

wouldn’t be rejected.

The method !

Cell samples were lysed and

separated by centrifugation on a

cesium chloride gradient.

In the gradient, heavier molecules

move more quickly towards the

bottom of the tube than lighter

molecules

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Document Summary

Dna: occurs in all living cells, stores information for the synthesis of speci c proteins, directs rna synthesis, directs protein synthesis through rna, made up of nucleic acids. Nucleotides: monomers of nucleic acids, made up of a nitrogenous base, a sugar and a phosphate group (know this diagram really well including the carbon positions) There are three possible models of replication that can happen and the following experiment tries to nd which way dna replicates. Materials: centrifuge to sediment the parts of the dna, bacterial growth media with and without isotopic label, equipment for cell lysis & dna isolation, cesium chloride (for dna separation) The isotope used for labelling the dna was an isotope of nitrogen because they needed an element that already existed in dna and wouldn"t be rejected. Cell samples were lysed and separated by centrifugation on a cesium chloride gradient. In the gradient, heavier molecules move more quickly towards the bottom of the tube than lighter molecules.

Related Questions

Multiple answers to each question might be possible!

You decide to identify the CFTR mutation by analyzing the genomic DNA of your patients compared to healthy individuals. You specifically are looking to see whether a specific 3' gene truncation has occurred in the patients. You will determine this using hybridization techniques with samples from healthy and CF patients. Which of the following will allow you to accomplish this?

	Using an RNA probe complementary to the region not removed by the truncation.
	Using an RNA probe complementary to the region removed by the truncation.
	Using an DNA probe complementary to the region not removed by the truncation.
	Using an DNA probe complementary to the region removed by the truncation.

To conduct the hybridization experiment, you are trying to decide between using a DNA or RNA probe. Which would be ideal to use and why?

	As both are composed of nucleic acids, using either would result in identical results.
	An RNA probe because RNA has uracil bases.
	An RNA probe because it could also be used in a translation experiment.
	A DNA probe because it is more stable than RNA.
	A DNA probe because RNA cannot bind to DNA.

One step of the Hershey/Chase experiment involved blending the virus/cell mixture before centrifugation and probing the pellet for radioactivity. Why was the blending step necessary?

	To collect the bacteria at the bottom of the tube.
	To break open the bacteria to release the genome.
	To separate the bacteria from the bacteriophages.
	To be able to detect the radioactivity.

Imagine Hershey/Chase had used an RNA virus (genome composed of RNA) instead of a DNA virus in their experiment. Would radioactivity still have been found in the pellet?

	No, because only DNA can be labeled with radioactivity.
	No, because the RNA genome would not enter the bacteria upon infection.
	No, because while DNA and RNA nucleotides are similar, they are not identical.
	Yes, because DNA and RNA nucleotides are similar.
	Yes, because genome in any form (DNA, RNA, protein) would be labeled similarly.

The human genome consists mostly of non-coding DNA. Which of the following are benefits of this?

	Random DNA mutations generally won't affect RNA and protein function.
	It is faster to duplicate the genome when these are present.
	The existence of introns can lead to multiple variations of proteins encoded by a single gene.
	It is unlikely transposons would exist in the genome if there was too much protein coding DNA.

Explain the 5â to 3â directionality of a DNA stand.

	It is due to the fact that the free 5â carbon is on one end and the free 3â carbon is on the other
	It is due to the fact that new nucleotide are added to the 5â carbon of the previous nucleotide
	It is due to the fact that there are 3 phosphate groups attached to the 5â carbon
	It is due to the fact the complementary strand is 3â to 5â
	More than one of the above explain the 5â to 3â directionality

You accidentally add a mutant dNTP (which has an H instead of an OH connected to the 3â carbon) to a reaction where DNA is being replicated. Which of the following is true of this mutant dNTP?

	It can be incorporated into DNA strand but cannot form a phosphodiester bond with an incoming wild type dNTP
	It can be incorporated into a DNA strand but cannot base pair with a complementary nucleotide
	It can be incorporated into a DNA strand and can form a phosphodiester bond with an incoming dNTP, but only if it is another mutant dNTP
	It cannot be incorporated into a DNA strand.

Why does DNA polymerase utilize an RNA primer?

	DNA polymerase is unable to initiate strand synthesis but RNA polymerase can
	DNA polymerase can only add a dNTP to an rNTP
	DNA synthesis proceeds in the 3â to 5â when initiating strand synthesis
	Chromosomal DNA contains interspersed RNA fragments
	The RNA primer increases stability of the newly synthesized strand

rosehippopotamus950

	Using an RNA probe complementary to the region not removed by the truncation.
	Using an RNA probe complementary to the region removed by the truncation.
	Using an DNA probe complementary to the region not removed by the truncation.
	Using an DNA probe complementary to the region removed by the truncation.

To conduct the hybridization experiment, you are trying to decide between using a DNA or RNA probe. Which would be ideal to use and why?

	As both are composed of nucleic acids, using either would result in identical results.
	An RNA probe because RNA has uracil bases.
	An RNA probe because it could also be used in a translation experiment.
	A DNA probe because it is more stable than RNA.
	A DNA probe because RNA cannot bind to DNA.

Imagine Hershey/Chase had used an RNA virus (genome composed of RNA) instead of a DNA virus in their experiment. Would radioactivity still have been found in the pellet?

	No, because only DNA can be labeled with radioactivity.
	No, because the RNA genome would not enter the bacteria upon infection.
	No, because while DNA and RNA nucleotides are similar, they are not identical.
	Yes, because DNA and RNA nucleotides are similar.
	Yes, because genome in any form (DNA, RNA, protein) would be labeled similarly.

The human genome consists mostly of non-coding DNA. Which of the following are benefits of this?

	Random DNA mutations generally won't affect RNA and protein function.
	It is faster to duplicate the genome when these are present.
	The existence of introns can lead to multiple variations of proteins encoded by a single gene.
	It is unlikely transposons would exist in the genome if there was too much protein coding DNA.

You accidentally add a mutant dNTP (which has an H instead of an OH connected to the 3â carbon) to a reaction where DNA is being replicated. Which of the following is true of this mutant dNTP?

	It can be incorporated into DNA strand but cannot form a phosphodiester bond with an incoming wild type dNTP
	It can be incorporated into a DNA strand but cannot base pair with a complementary nucleotide
	It can be incorporated into a DNA strand and can form a phosphodiester bond with an incoming dNTP, but only if it is another mutant dNTP
	It cannot be incorporated into a DNA strand.

Andrew Murray's sister, Andrea, is adding to her brother's work on chromosomes. She is using cells that are unable to synthesize adenine (âade) and histidine (âhis). The plasmid she is currently working with consists of an origin of replication and the Ade gene.

Following her transformation of the plasmid into her yeast, what media will the cells be plated on to select for cells that have picked up the plasmid?

	Media containing histidine
	Media containing adenine
	Media lacking adenine
	Media lacking histidine

She starts by attempting to add the centromere DNA into a plasmid containing the origin of replication. Unfortunately, when adding the centromere sequence, the origin of replication is removed, thus leaving a plasmid with only a centromere and selection marker. Following plasmid transformation, what growth result will she see on her plates?

	No colonies
	Little colonies
	Big colonies

She fixes the mistakes from the previous experiment and now has a complete plasmid (selection marker, origin of replication, centromere). She then inserts telomere sequences into the plasmid. How will this impact her transformation?

	It will not impact her transformation
	Her transformation will no longer work because plasmids donât require telomeres
	She will now see much larger colonies
	She will now see fewer but larger colonies
	She will now see smaller colonies

In the Meselson/Stahl experiment, E. coli were first grown in media containing heavy nitrogen, 15N, and then transferred to light nitrogen, 14N, at the beginning of the experiment.

Imagine that their data showed that replication occurs in a conservative manner instead of semi-conservative. What fraction of the DNA helices will consist of mixed DNA after 4 rounds of replication in this case?

	None
	More than 75%
	25-50%
	51-75%
	Less than 25%

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BIOL1020 Lecture Notes - Lecture 4: Bacterial Growth, Nitrogenous Base, Dna Replication

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