Chapter 6: DNA and Biotechnology Flashcards
What is a nucleoside? A nucleotide?
A nucleotide is a building block of nucleic acids (DNA and RNA) consisting of a sugar molecule, a nitrogenous base, and one or more phosphate groups.
While a nucleoside is a molecule containing only a sugar molecule and a nitrogenous base, without the phosphate group.
Book description:
Nucleosides are composed of a five carbon sugar (pentose) bonded to a nitrogenous base and are formed by covalently linking the base to C-1’ of the sugar.
Nucleotides are formed when one or more phosphate groups are attached to C-5’ of a nucleoside.
Is ATP a nucleoside or nucleotide?
ATP is a nucleotide as it is adenosine bonded to three phosphate groups.
By definition, a nucleotide is formed when one or more phosphate groups are attached to the C-5’ of a nucleoside (which by definition are composed of a 5 carbon sugar, pentose, bonded to a nitrogenous base formed by covalently bonding the base to C-1’ of the sugar).
Why is ATP a high energy compound?
Why is this special?
ATP is a high energy compound because of the energy associated with the repulsion between closely associated negative charges on the phosphate groups.
This is special because breaking is usually endothermic and bond making is usually exothermic. ATP is a biologically relevant exception to this. Due to all the negative charges in close proximity, removing the term terminal phosphate from ATP, actually releases energy, which powers our cells.
Bond breaking is usually endothermic, bond formation is usually exothermic. ATP is a biologically relevant exception to this rule. Talk about it.
Due to all the negative charges in close proximity on ATP, removing the terminal phosphate from ATP actually release his energy, powering our cells.
How are nucleic acids classified?
Nucleic acids are classified according to the pentose they contain.
If the pentose is ribose, the nucleic acid is RNA.
If the pentose is deoxyribose, the nucleic acid is DNA.
What are the two chemically distinct forms of nucleic acids within eukaryotic cells?
Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)
List the common bases, nucleosides, and nucleotides?
Which direction is DNA read from? Why?
What is the overall charge of DNA?
The backbone of DNA is composed of alternating sugar and phosphate groups. It determines the directionality of DNA and is always read from 5’ to 3’.
DNA is formed as nucleotides are joined by 3’-5’ phosphodiester bonds ( a phosphate group linked to three carbon of one sugar to the five phosphate of the next incoming sugar in the chain.
Phosphates carry a negative charge, thus DNA and RNA strands have an overall negative charge.
How will this molecule be written?
Write them in 5’ to 3’ direction.
This molecule will be:
Forward: 5’-ATG-3’
Backward: 3’-GTA-5’
Position of phosphates may be shown: pApTpG
“d” may be used for deoxyribose: dAdTdG
There are two families of nitrogen containing bases found in nucleotides. What are they called?
Purines and pyrimidines
There are two mnemonics in the book for remembering the types and structures for nitrogenous bases. What are they?
CUT the PYe (C U and T are PYrimidines). Pie has one ring of crust, pyrimidines have one ring.
PURe As Gold (A and G are PURines). Gold wedding rings, it takes two at a wedding, PURines have two rings in their structure.
What defines an aromatic compound?
The compound is:
Cyclic
Planar
Conjugated (has alternating single and multiple bonds, creating at least one unhybridized p orbital for each atom in the ring)
Obeys Huckels rule (the compound has has 4n + 2 pi electrons, where n is any positive integer)
What is Huckels rule? Do it for benzene, cyclobutadiene.
4n+2 pi electrons. If n is a positive integer, the compound is aromatic.
What is a heterocyle?
Heterocyles are ring structures that contain at least two different elements in the ring.
Is DNA parallel or anti parallel?
Is the sugar phosphate backbone on the outside or inside?
What always pairs with adenine? Guanine? Which pairing is the strongest?
DNA is anti parallel. The strand are oriented in the opposite direction. When one strand has polarity 5’-3’ down the page, the other strand has 5’-3’ up the page.
The sugar phosphate backbone is on the outside of the helix with nitrogenous bases on the inside.
Adenine always pairs either thymine, guanine always pairs with cytosine.
G-C has three hydrogen bonds and is the strongest base pair interaction.
What is Chargaff rule of base pairing?
If a sample of DNA has 10% G, what is the percent of T?
In double stranded DNA, purines equal pyrimidines:
%A=%T
%G=%C
If a sample of DNA has 10% G, what is the percent of T?
10% G equals 10% C, leaving 80% A and T, therefor 40% T.
Image of base paring of DNA
Write the complimentary strand for 5’-ATCG-3’
5’-CGAT-3’
Why are the major and minor grooves on DNA important?
The major and minor grooves provide the binding sites for regulatory proteins.
The helix in beta DNA makes a turn every 3.4 nm. How many base pairs are in that span?
About 10
What is DNA denaturation?
What substances and methods are commonly used to denature DNA?
Do these methods disrupt the covalent bonds of the backbone structure?
Disrupting the hydrogen binding between the strands is known as denaturation.
Heat, alkaline pH, formaldehyde, and urea are commonly used to denature DNA.
Denaturing DNA does not disrupt the covalently bonded backbone structure of DNA.
MCAT concept check DNA structure page 204 question 1
What is the difference between nucleoside and nucleotide?
Nucleosides contain a five carbon sugar, pentose, and nitrogenous base. Nucleotides are composed of a nucleoside one to three phosphate groups.
MCAT concept check DNA structure page 204 question 2
What are the bases pairing rules according to the Watson Crick model?
A pairs with T (in DNA) and U (in RNA), using two hydrogen bonds.
C pairs with G, using three hydrogen bonds.
MCAT concept check DNA structure page 204 question 3
What are the three major structural differences between DNA and RNA?
DNA contains deoxyribose, while RNA contains ribose.
DNA contains thymine, RNA contains uracil.
DNA is double stranded, while RNA is single stranded.
MCAT concept check DNA structure page 204 question 4
How does the aromaticity of purines and pyrimidines underscore their genetic function?
The aromaticity of nucleic acids make these compounds very stable and unreactive.
Stability is important for storing genetic information and avoiding spontaneous mutations.
MCAT concept check DNA structure page 204 question 5
If a strand of RNA contained 15% cytosine, 15% adenine, 35% guanine, and 35% uracil, would this violate Chargaff’s rule? Why?
This does not violate Chargaff’s rule. RNA is single-stranded, and thus the complementarity seen in DNA does not hold true.
For single-stranded RNA, percent C does not necessarily equal percent G; a does not necessarily equal percent U.
How many bases of DNA are in each human cell?
Over 6 billion
How many chromosomes is DNA divided up in humans?
23 pairs of chromosomes for a total of 46 chromosomes. 23 from each parent.
The DNA that makes up a chromosome is wound around a group of small, basic proteins called _________, forming ________.
Histones, chromatin.
What are nucleosomes composed of?
Nucleosomes are composed of DNA wrapped around histone proteins.
What are nucleoproteins? What is one example of a nucleoprotein?
Histones are one example of nucleoproteins (proteins that associate with DNA).
Most other nucleoproteins are acid soluble in tend to stimulate processes, such as transcription.
What is heterochromatin?
A small percentage of the chromatin remains compacted during interphase and is referred to as heterochromatin.
Heterochromatin often consists of DNA with highly repetitive sequences.
What is euchromatin?
The dispersed chromatin during interphase is called euchromatin, which appears light under light microscopy.
Chromatin contains genetically active DNA.
How do we tell the difference between heterochromatin and euchromatin?
Heterochromatin is dark, dense, and silent.
Euchromatin is light, uncondensed, and expressed.
What is a telomere?
What is the sequence of a telomere?
What is the second function of a telomere?
DNA replication cannot extend all the way to the end of a chromosome. This will result in losing sequences and information with each round of replication.
The solution to this is telomeres. A sequence of TTAGGG at the end of the DNA.
The second function of telomeres: they “knot off” the end of chromosomes as they have a high G-C content. Recalling that G-C has three hydrogen bonds and A-T (or U) has only two.
What is telomerase?
Where is higher activity of telomerase found?
Some of the sequence of telomeres is lost in each round of replication and can be replaced by the enzyme telomerase.
Telomerase is more highly expressed in rapidly dividing cells.
How do telomeres “knot off” the end of a chromosome?
Telomeres have a high content of guanine (TTAGGG). This makes a high content of guanine-cytosine pairing, which has three hydrogen bonds (adenine-tyrosine has two).
What is a centromere?
What are centromeres composed of?
Centromeres are regions of DNA Found in the center of chromosomes.
Centromeres are composed of heterochromatin, containing high content of guanine and cytosine.
MCAT concept check eukaryotic chromosome organization 6.2 page 210 question 1
What are the five histone proteins found in eukaryotic cells?
Which one is not part of the histone core around which DNA wraps to form chromatin?
The five histone proteins are:
H1, H2A, H2B, H3 and H4.
H1 is the only one not in the histone core. It is known as the “linker histone” and is the histone component that binds to the nucleosome core particle and plays a critical role in chromatin structure and DNA accessibilty.
MCAT concept check eukaryotic chromosome organization 6.2 page 210 question 2
MCAT concept check eukaryotic chromosome organization 6.2 page 210 question 3
What property of telomeres and centromeres allow them to stay tightly traveled, even when the rest of DNA is uncondensed?
High guanine and cytosine increases hydrogen bonding, making the association between DNA strands very strong at telomeres and centromeres.
How many base pairs does the human genome have?
About 3 billion base pairs (6 billion bases total)
What is the replisome, or replication complex?
The replisome or replication complex is a set of specialized proteins that assist the DNA polymerases.
What is the origin of replication? What are replication forks? Daughters strands?
Regarding replication of DNA, compare and contrast the sites of origin and forking that occurs for prokaryotes and eukaryotes.
The bacterial chromosome is a closed, double stranded, circular DNA molecule with the single origin of replication. Thus, there are two replication forks that move away from each other in opposite direction around the circle. The two replication forks eventually meet, resulting in the production of two identical, circular molecules of DNA.
Eukaryotes replication must copy many more bases compared to prokaryoticic replication and is a slower process. Each eukaryotic chromosome contains one linear molecule of double stranded DNA having multiple origins of replication.
What enzyme is responsible for unwinding DNA?
Helicase is the enzyme responsible for unwinding the DNA, generating two single-stranded template strands ahead of the polymerase.
Any antibiotic ending with -floxacin (ciprofloxaxin, moxifloxacin, ofloxacin, etc) is called a fluoroquinolone (flora-quinn-alone).
Fluoroquinolone targets prokaryotic topoisomerase DNA gyrase.
How do fluoroquinolones stop bacterial growth? What type of infection are fluoroquinolones typically used for?
Fluoroquinolones stops the bacteria from replicating its DNA and slows the infection.
Fluoroquinolones are used for pneumonia and certain infections in the genitourinary system.
What is the genitourinary system?
The genitourinary system (urogenital system) encompasses both urinary and reproductive systems.
Free DNA strands are sticky, in a molecular sense, so there are proteins that hold the strands apart. What are those proteins called?
Single stranded DNA binding proteins bind to the unraveled strand, preventing both the reassociated of the DNA strands and the degradation of DNA by nucleases.
What are nucleases?
A nuclease is an enzyme that cleaves the phosphodiester bonds between nucleotides in nucleic acids (DNA or RNA), acting like “molecular scissors” to break down these molecules.
What is supercoiling?
What enzyme alleviates supercoiling?
Supercoiling occurs when coiling strain is applied to a strand of DNA as it unwinds during the replication process (by helicase).
DNA topoisomerases introduce negative supercoils that counteract the supercoiling effect. They do so by working ahead of helicase, nicking one or both strands, relieving torsional pressure.
Why is the replication process considered semi conservative?
The parental strand serves as a template for the generation of new daughter strands.
This is a semi conservative process because one parental strand is retained in each of the two resulting identical double stranded DNA.
Talk about DNA polymerase, leading strand, and lagging strand, in general.
DNA polymerases read the DNA template and synthesize the new daughter strands reading in the 3’ to 5’ direction. DNA polymerases therefor construct the strand in the 5’ to 3’ direction.
The leading strand can be read immediately (3’ to 5’) and a continuous copy can be produced (5’ to 3’).
The lagging strand introduces a complication. To overcome this, Okazaki fragments are made, which are short segments of DNA that are ligased together to eventually form a copy that is read in the 3’ to 5’ direction and built in the 5’ to 3’ direction.
Regarding DNA replication, why do the single DNA parent strands need primase?
The first step in DNA replication is to lay down an RNA primer. This allows a site for DNA polymerase to anchor onto to begin copying.
Why do lagging strands have more RNA primers on them than leading strands?
Primase synthesize a short primer, roughly 10 nucleotides long, in the 5’ to 3’ direction to start replication on each strand.
These short RNA sequences are constantly being added to the lagging strand because each Okazaki fragment must start with new primer.
The leading strand requires only one primer, in theory.
Steps and Proteins involved with DNA replication
Which direction to the following processes read along DNA?
DNA polymerase
DNA synthesis
DNA repair
DNA transcription
DNA translation
DNA polymerase READING direction is 3’-5’.
Synthesis, repair, transcription, and translation occur 5’-3’.
Why do chromosomes shorten over time?
DNA polymerase cannot complete synthesis of the 5’ end of the strand. Every time the DNA synthesis is carried out, chromosomes become a little shorter. This is why telomeres (high GC content TTAGGG)
MCAT concept check DNA replication 6.3 page 216 question 1
Do this card until you know it well.
MCAT concept check DNA replication 6.3 page 216 question 2
Between the leading strand and lagging strand, which is more prone to mutations? Why?
The lagging strand is more prone to mutations because it must constantly start and stop the process of DNA replication. Additionally, it contains many more RNA primers, all of which must be removed and filled in with DNA.
MCAT concept check DNA replication 6.3 page 216 question 3
What is the function of a telomere?
Telomeres are the ends of eukaryotic chromosomes and contain repetitive sequences of non-coding DNA.
Telomeres protect the chromosome from losing important genes from the incomplete replication of the 5’ end of the DNA strand.
Do prokaryotes have telomeres?
There is no need for telomeres in the prokaryotic genome as there are no ends of DNA to protect (their DNA is circular).
The outcome of oncogenes and mutated tumor suppressor genes is the same (cancer), but the actual cause is different. Speak.
Oncogenes promote the cell cycle while mutated tumor suppressors can no longer slow the cell cycle.
Oncogenes are like an accelerator.
Mutated tumor suppressor is cutting the brakes.
What are oncogenes?
What product do oncogenes typically encode?
What was the first gene discovered in this category?
Are they considered dominant?
Oncogenes are mutated genes that cause cancer. Proto-oncogenes are oncogenes before they are mutated.
Oncogenes primarily encode cell cycle related proteins. The abnormal alleles encode proteins that are more active than normal proteins, promoting rapid cell cycle advancement. A mutation and only one copy is sufficient to promote tumor growth and is therefore considered dominant.
The first discovered was src (named after sarcoma, a category of connective tissue cancers).
What are tumor suppressor genes?
Name a couple.
Why are they sometimes referred to as antioncogenes?
Are they considered dominant or recessive?
Tumor suppressor genes, like p53 or Rb (retinoblastoma), encode proteins that inhibit the cell cycle or participate in DNA repair processes.
They function to stop tumor progression, so they are sometimes referred to as antioncogenes.
The loss of tumor suppression activity promotes cancer.
In activation of both alleles is necessary for the loss of function even one copy of the normal protein can function to inhibit tumor formation. Multiple mutations are required.
They are considered recessive.
What is DNA proofreading?
Incorrectly paired bases produce unstable hydrogen bonds which can be detected by DNA polymerase.
DNA polymerase can also detect the level of methylation to determine which is the correct strand: the parent strand has more methylation.
Closing of the gaps with DNA ligase lacks proofreading, creating a higher likelihood for mutation on the lagging strand.
What is mismatch repair?
Genes in eukaryotes, prokaryotes?
In G2 phase of the cell cycle, there are genes that encode for enzymes which detect and remove errors introduced in replication (during S phase). This is called mismatch repair.
MSH2, MLH1 in euk.
MutS and MutL in prok.
What is nucleotide excision repair (NER)?
What phase of the cell cycle do NER do?
Thymine residues can form dimers when exposed to ultraviolet light, called thymine dimers, which distort the shape of the DNA.
NER repairs this by cutting and patching.
The bulge is recognized. Excision endonuclease removes the oligonucleotide, DNA polymerase fills the gap in 5’-3’ direction (reading in the 3’-5’ direction).
G1 and G2 phase.
What is base excision repair (BER)?
Can cytosine become uracil? How?
BER are detection systems for small, non helical deforming mutations. ONLY REMOVES THE BASE.
Example. Cytosine can reanimate to uracil via thermal energy. Uracil does not belong in DNA.
Detected then removed by glycosylase enzyme which creates apurinic/apyrimidinic (AP) site because glycosylase only removed the base. (Also called an abasic site).
AP endonuclease replaced with proper base.
BER do not hydrolyze the sugar phosphate backbone, they replace the bases attached to them to create AP sites (abasic sites).
MCAT concept check DNA repair 6.4 page 221 question 1
What is the difference between an oncogene and a tumor suppressor gene?
Oncogene accelerates cell growth and division, tumor suppressor gene mutations is like cutting the brakes.
MCAT concept check DNA repair 6.4 page 221 question 2
How does DNA polymerase recognize which strand is the template strand once the daughter strand is synthesized?
The parent strand is more heavily methylated.
MCAT concept check DNA repair 6.4 page 221 question 3
Page 218
MCAT concept check DNA repair 6.4 page 221 question 4
What is the structural difference in the types of lesions corrected by nucleotide excision repair versus those corrected by base excision repair?
What is recombinant DNA technology?
Allows a DNA fragment from any source to be multiplied by either gene cloning or polymerase chain reaction (PCR).
Describe DNA cloning and restriction enzymes.
DNA cloning produces a large amount of a desired sequence.
Mutate DNA of interest forming a recombinant vector (usually bacterial or viral plasma that can be transferred to a host bacterium after insertion of the DNA of interest)
Isolate a colony containing the recombinant vector.
Dabble in a gene for antibiotic resistance, so that the antibiotics can then kill off all of the colonies that do not contain the recombinant vector.
Grow the colony in large quantities.
Restriction enzymes (restriction endonucleases) recognize specific double strand DNA sequences (which are palindromic and can therefore be excised as a double stranded unit)
What are exon and intron dna sequences?
Exon are coding
Intron are non coding
What are genomic libraries?
Genomic libraries contain large fragments of DNA and include both coding (exon) and non-coding regions (intron) of the genome.
What are expression libraries?
What is cDNA?
Expression libraries are cDNA libraries that only contain exon (or coding) genes.
cDNA is complimentary DNA of an mRNA sequence.
Table comparing and contrasting genomic and expression (cDNA) libraries
Regarding recombination, what is hybridization?
Hybridization is the joining of complementary base pair sequences.
What is PCR?
What is a primer?
PCR is an automated process that can produce millions of copies of DNA sequences without amplifying the DNA in bacteria.
During the process of polymerase chain reaction, the DNA of interest is denatured, replicated, and then cool to allow reannealing of the daughter strands with the parent strands. Every time this process is repeated it doubles the amount of DNA until enough copies of the DNA sequence are available for further testing.
PCR requires primers that are complementary to the DNA that flank the region of interest. Primers have a high GC content for stability.
The process requires heat, so DNA polymerase from Thermus aquaticus is used.
Describe DNA gel electrophoresis.
DNA is negatively charged, so will be attracted to the anode.
Agarose gel is used (rather than polyacrylamide gel)
The longer the strand, the slower it will migrate.
What is southern blotting?
Performed with gel electrophoresis, southern blotting is used to detect the presence and quantity of various DNA strands in a sample.
Southern blotting is a molecular biology technique to detect specific DNA sequences by transferring DNA fragments from a gel onto a membrane and then probing with a labeled DNA sequence.
Why are dideoxyribonucleotides used in DNA sequencing?
Dideoxyribonucleotides contain a H at C-3 rather than a hydroxyl group, and when added to a sequence, DNA polymerase can no longer add to the chain.
What is gene therapy?
Gene therapy is intended for disease diseases in which a given gene is mutated or inactive, giving rise to pathology.
By transferring a normal copy of the gene into the effective tissues, the pathology should be fixed, essentially curing the individual.
Image shows a model therapeutic human gene therapy using retrovirus as a vector (retroviral gene therapy).
What are transgenic and knockout mice?
Once DNA has been isolated, it can be introduced into eukaryotic cells.
Transgenic mice are altered at their germline by introducing a clone gene into fertilized over or into embryonic stem cells.
The cloned gene that is introduced is referred to as a transgene.
If the transgene is a disease producing allele, the transgenic mice can be used to study the disease process.
Knock out mice are where a gene has been intentionally deleted.
Why are transgenic studies better suited for dominant gene effects and not as well suited for recessive disease?
Transgenic and knockout mice approach is useful for studying dominant gene effects because the number of copies of the gene that insert into the genome cannot be controlled; the transgenic mice may each contain a different number of copies of the transgene.
What is a chimera? How is this useful for transgenic study and application?
Using embryonic stem cells for developing transgenic mice can create chimeras.
Cloned genes are introduced into cultures, and one can select for cells with the transgene successfully inserted.
These altered stem cells are injected into developing blastocyst and implanted into surrogates.
The blastocyst itself is thus composed of two types of stem cells: the ones containing the transgene and the original blastocyst that lack the transgene: this is a chimera.
This property can be readily apparent (spotting fur for example) allowing for easy identification of the chimera. Breed these to produce mice that are heterozygote and homozygous for a transgene.
Safety and ethics of DNA recombination and biotechnology.
MCAT concept check DNA recombination and biotechnology 6.5 page 230 question 1
MCAT concept check DNA recombination and biotechnology 6.5 page 230 question 2
MCAT concept check DNA recombination and biotechnology 6.5 page 230 question 3
MCAT concept check DNA recombination and biotechnology 6.5 page 230 question 4
MCAT mastery DNA and Biotechnology page 192 question 1
MCAT concept check DNA recombination and biotechnology 6.5 page 230 question 2
Which side of DNA is responsible for the negative charge?
The negative charge in DNA is primarily due to the phosphate groups in the sugar-phosphate backbone, and this backbone has a directionality, with the 5’ end having a free phosphate group and the 3’ end having a free hydroxyl group
MCAT concept check DNA recombination and biotechnology 6.5 page 230 question 3
MCAT concept check DNA recombination and biotechnology 6.5 page 230 question 4
MCAT concept check DNA recombination and biotechnology 6.5 page 230 question 5
MCAT concept check DNA recombination and biotechnology 6.5 page 230 question 6
MCAT concept check DNA recombination and biotechnology 6.5 page 230 question 7
MCAT concept check DNA recombination and biotechnology 6.5 page 230 question 8
MCAT concept check DNA recombination and biotechnology 6.5 page 230 question 9
MCAT concept check DNA recombination and biotechnology 6.5 page 230 question 10
MCAT concept check DNA recombination and biotechnology 6.5 page 230 question 11
MCAT concept check DNA recombination and biotechnology 6.5 page 230 question 12
MCAT concept check DNA recombination and biotechnology 6.5 page 230 question 13
MCAT concept check DNA recombination and biotechnology 6.5 page 230 question 14
MCAT concept check DNA recombination and biotechnology 6.5 page 230 question 15
Do prokaryotes have nucleosomes?
No, prokaryotes, such as bacteria, do not have nucleosomes, which are the basic units of DNA packaging in eukaryotes, because they lack the histone proteins that form nucleosomes.
