Biochem #6 Flashcards
1
Q
where is DNA mostly found?
A
Primarily found in the nucleus in chromosomes but some is found in mitochondria or chloroplasts.
2
Q
nucleosides
A
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.
3
Q
nucleotides
A
formed when one or more phosphate groups are attached to the C-5’ of a nucleoside.
Named based on how many phosphate groups are attached.
• High energy compound because of energy of repulsion among closely associated negative charges on the phosphate groups
Building blocks of DNA
4
Q
ribose vs. deoxyribose
A
nucleic acid is DNA
• Typically double stranded
5
Q
discuss the sugar phosphate backbone
A
o The backbone of DNA is composed of alternating sugar and phosphate groups; determines directionality of DNA and is always read 5’-3’.
o Phosphates carry a negative charge; thus the backbone is negatively charged.
o DNA strands run antiparallel to each other.
o Enzymes that replicate and transcribe DNA only work in the 5’-3’ direction.
o 5’ end has an OH or phosphate group bonded to the C-5’ sugar while the 3’ end has a free OH on the C-3’ sugar.
6
Q
DNA
A
sugar + phosphate + nitrogenous base
7
Q
the DNA backbone is ______ charged
A
negatively
8
Q
what are the purines?
A
adenine and guanine, 2 rings
9
Q
what are the pyrimadines?
A
uracil, thymine cytosine, 1 ring
10
Q
is uracil and thymine found in DNA or RNA?
A
uracil is found in RNA and thymine is found in DNA
11
Q
characteristics of the bases found in DNA and RNA?
A
Are all Aromatic, have extra stability, delocalized pi electrons can travel throughout the entire compound using available molecular orbitals.
12
Q
factors determining aromaticity
A
- The compound is cyclic
- The compound is planar
- The compound is conjugated (has altering single and double bonds, or lone pairs, creating at least one unhybridized p-orbital for each atom in the ring)
- The compound has 4n + 2 (where n is any integer) pi electrons. Huckel’s rule
13
Q
describe the watson and crick model of DNA
A
o Double helix: two linear polynucleotide chains of DNA are wound together in a spiral orientation along a common axis. Key features:
1. The two strands are antiparallel
2. Sugar-phosphate backbone is on the outside, while the nitrogenous bases are on the inside
3. Complementary base pairing:
• A-T with two hydrogen bonds
• C-G with three hydrogen bonds (stronger)
• These H bonds and hydrophobic interactions among bases stabilize the structure.
4. %A=%T and %G=%C. %A + %T = %G + %C
14
Q
double helix
A
two linear polynucleotide chains of DNA are wound together in a spiral orientation along a common axis.
15
Q
what are the key features of the DNA double helix?
A
Key features:
1. The two strands are antiparallel
2. Sugar-phosphate backbone is on the outside, while the nitrogenous bases are on the inside
3. Complementary base pairing:
• A-T with two hydrogen bonds
• C-G with three hydrogen bonds (stronger)
• These H bonds and hydrophobic interactions among bases stabilize the structure.
16
Q
how many hydrogen bonds between C and G?
A
3
17
Q
how many hydrogen bonds between A and T?
A
2
18
Q
In the DNA helix, the sugar-phosphate backbone is on the ____, while the nitrogenous bases are on the ___
A
outside
inside
19
Q
what is Z-DNA?
A
o Left handed helix
o Turn every 4.6 nm and 12 bases per turn
o No biological activity
20
Q
denatured
A
melt the double helix into singular strands (needed for replication and transcription)
Covalent bonds between sugars are not broken in the backbone but hydrogen bonds are disrupted.
bases are separated
21
Q
reannealed
A
repairing of the denatured strands of DNA.
22
Q
probe DNA
A
DNA with known sequence used in laboratory processes
23
Q
chromatin
A
formed when DNA is wrapped around a group of small basic proteins called histones.
24
Q
heterochromatin
A
compact, not as accessible, appears dark under a microscope (more compact), not transcriptionally active
25
euchromatin
dispersed chromatin, appears light under a microscope, active DNA, uncondensed
26
nucleoproteins
proteins that associate with DNA.
27
telomeres
formed at the end of DNA with the simple repeating unit (TTAGGG) which allows for DNA information not to be lost with each round of replication.
28
telomerase
some of the sequence is lost in each round of replication and this can replace it.
o High GC content prevents unraveling of DNA
29
centromeres
region of DNA found in the center of chromosomes
o Look constricted due to presence of heterochromatin.
o Connect sister chromatids during cell division.
30
sister chromatids are separated during ____ or _____
mitosis or meiosis II
31
sister chromatids are held together at the _____
centromere
32
RNA translation reads codons in the ____
5 to 3 direction
33
RNA and DNA polymerase produce strands in the ___ direction by reading in the ____
5 to 3
| 3 to 5 direction
34
how does DNA replication begin?
Begins at origins of replication, proceeds in both directions creating replication forks on both sides.
35
how many origins of replication for bacterial chromosome? How many replication forks are there?
1 origin of replication with 2 replication forks that eventually meet. (double-stranded circular DNA)
36
sister chromatids
chromatids are created and held together at the centromere before being separated during mitosis/meiosis
37
helicase
unwinds the DNA, generating two single stranded template strands ahead of the polymerase
38
single -stranded DNA-binding proteins
bind to the unraveled strand, preventing both the reassociation of the DNA strands and the degradation of DNA by nucleases (the unwound DNA single strands are very "sticky")
39
DNA topoisomerases
prevent supercoiling of the DNA by acting ahead of the helicase and nicking strands to allow relaxation of the torsional pressure.
40
parental strands serve as templates for the generation of new ____ strands
daughter
41
semiconservative model of DNA
Semiconservative: one parent strand is retained in each of the two resulting identical double-stranded DNA molecules.
42
DNA polymerase
responsible for reading the DNA template, or parental strand, and synthesizing the new daughter strand
Reads in the 3’ to 5’ direction in order to synthesize is the 5’ to 3’ direction.
43
leading strand
strand that is copied in a continuous fashion, in the same direction of the advancing replication fork.
44
lagging strand
strand that is copied in a direction opposite of the replication fork. If the replication fork moves to the left then the lagging strand replication is moving to the right for that particular strand back toward the OG origin of replication.
Utilizes okazaki fragments
45
DNA cannot be synthesized ____ so an ____ must first be laid down by ___.
de-novo
RNA primer
primase
46
RNA primers are constantly being added to the ____
Constantly being added to the lagging strand, whereas the leading strand only needs one primer.
47
with the addition of each new nucleotide, what is released?
a free PPi, add an incoming 5’ deoxyribonucleotide triphosphate
48
Compare eukaryotes and prokaryotes which DNA pol add nucleotides
Pro: DNA Pol III
Euk: DNA Pol alpha, delta, epsilon (leading and lagging strand)
49
Compare eukaryotes and prokaryotes what removes RNA primer
Pro: DNA Pol I
Euk: RNase H
50
Compare eukaryotes and prokaryotes what replaces RNA primer with DNA
prok: DNA Pol I
Euk: DNA pol delta
51
what seals the DNA that is added in place of the RNA primer?
ligase
52
what sequence is repeated at the ends of chromosomes?
TTAGGG (which is located at the telomeres)
53
cancer cells
proliferate excessively because they are able to divide without stimulation (signals from other cells) from other cells and are no longer subject to the normal controls of cell proliferation.
54
metastasis
a migration to distant tissues by the bloodstream or lymphatic system
55
oncogenes
mutated genes that cause cancer. Stepping on the gas pedal. Promote the cell cycle.
• Proto-oncogenes: mutated to make oncogenes.
56
tumor suppressor genes
encode proteins that inhibit the cell cycle or participate in DNA repair processes
• Normally stop tumors but when they are mutated they can no longer do this.
57
proofreading
the two double-stranded DNA molecules pass through a part of the DNA Pol and are checked.
Notices a lack of stability, excises the incorrect base, and adds a new one.
Many more errors in the lagging strand because DNA ligase does not have the proofreading ability.
Knows the parent strand by amount of methylation
58
mismatch repair
machinery in the G2 phase that detects and removes errors from the S phase.
59
nucleotide excision repair
(NER)
• Eliminates thymine dimers (which are formed due to UV light)
• Cut and patch process.
• Excision endonuclease makes nicks in the phosphodiester bond at the location of the lesion and removes the oligonucleotide, DNA pol acts in 5' to 3' direction, DNA ligase seals.
60
base excision repair
* Repairs small, non-helix distorting mutations in bases.
* Glycosylase enzyme removes the base, leaves behind apurinic/apyrimidinic (AP) site, abasic site.
* AP endonuclease removes the damaged sequence from the DNA and then DNA pol and DNA ligase fill and seal.
61
methods to multiple DNA
PCR, gene cloning
62
recombinant DNA
DNA that has been formed artificially by combining constituents from different organisms.
DNA that can be multiplied and then used for analysis or altering genes and proteins.
63
DNA cloning
technique that can produce large amounts of a desired sequence. Produce large amount of homogenous DNA for other applications.
Make a recombinant vector (plasmid) that has the DNA sequence of interest.
Transfer to host bacterium
The bacteria are then grown, isolate colony with the recombinant vector (vector can have antibiotic resistance to aid selection)
• Vector should also have at least one origin of replication.
Grow this colony big, then either: 1 make them induce gene expression 2 lyse and reisolate the replicated recombinant vectors
• Restriction enzymes: release the cloned DNA from the vector
o Enzymes that recognize specific double-stranded DNA sequences.
o Cut at palindromic sequences: Ex: GAATTC
64
restriction enzymes
DNA cloning
release the cloned DNA from the vector
o Enzymes that recognize specific double-stranded DNA sequences.
o Cut at palindromic sequences: the 5' to 3' sequence of one strand is identical to the 5' to 3' sequence of the other strand
Ex: 5' GAATTC 3'
3' CTTAAG
65
DNA libraries
produced via DNA cloning, large collections of known DNA sequences which can equate to the genome of an organism.
66
genomic libraries
contain large fragments of DNA, and include both coding and noncoding regions of the genome.
67
complementary DNA (cDNA)
): libraries are constructed by reverse transcribing processed mRNA
Lacks introns, expression libraries
Known gene is not split so it is more reliable for things like vaccines and producing recombinant proteins.
68
hybridization
the joining of complementary base pair sequences. Can be DNA-DNA or DNA-RNA.
69
polymerase chain reaction (PCR)
an automated process that can produce millions of copies of a DNA sequence without amplifying the DNA in bacteria.
Used to identify criminal suspects, familial relationships, and disease-causing bacteria and viruses.
Knowing sequences next to the desired region allows them to be amplified
Requires primers: complementary to the DNA that flanks the region of interest. Also requires nucleotides and DNA pol.
DNA is denatured, replicated, then cooled so that it is reannealed. Repeated many times.
The polymerase is from a bacteria that thrives in warm environments.
70
sanger sequencing
Sanger sequencing, also known as the “chain termination method”, is a method for determining the nucleotide sequence of DNA.
Utilizes dideoxyribonucleotide to tell the order of DNA.
The sample will eventually contain many fragments that each terminate with a modified base. Separated by gel electrophoresis and then the last base on each one is read and put in order.
71
gel electrophoresis
all DNA will migrate toward the anode (+) of an electrochemical cell.
• Uses agarose gel.
• The longer the strand the slower it will migrate.
72
southern blot
A Southern blot is a method used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization
utilizes gel electrophoresis, used to detect the presence and quantity of various DNA strands in a sample.
• DNA is cut by restriction enzymes and then separated by gel electrophoresis. The DNA fragments are then carefully transferred to a membrane and the membrane is probed with probes labeled with radioisotopes or indicator proteins which indicate the presence of a desired sequence.
73
gene therapy
offers potential cures for individuals with inherited diseases.
Intended for diseases in which a given gene is mutated or inactive, giving rise to pathology.
First successful case of gene therapy was for SCID in 1990.
Most use viral vectors to insert the normal copy of the gene into the patient’s cells.
Must be carefully inserted by the virus into the host genome as well because if it is inserted into the wrong place then it can cause cancer.
74
transgenic mice
altered at their germ line by introducing a cloned gene into fertilized ova or into embryonic stem cells.
can design an animal model for a human disease this way.
75
transgene
cloned gene that is introduced.
• Could be a disease gene, study the disease in the mouse
• Inject the gene into a fertilized ovum and then implant into a foster mother. If successful, offspring will have the gene and also in the offspring’s germ line.
76
stem cells and mice
use embryonic stems cells which you know which genes are successfully introduced. Altered stem cells are injected into developing blastocyst and then surrogate mother.
o Chimera: has patches of cells because it contains cells from the transgene and also the original blastocyst cells.
Obvious in coat color.
77
knockout mice
a gene has been intentionally deleted (knocked out)
78
how do you go from nucleoside to nucleotide?
add one more more phosphate groups to the carbon to C5 of a nucleoside
79
adenosine + a few phosphate groups =
ATP or ADP
| adenosine triphosphate
80
DNA and RNA strands have an overall ___ charge because of ____
negative
| the negative charge on the phosphates
81
in DNA, nucleotides are added onto the ____ end during DNA synthesis
3'
82
the DNA sequence is always written in the ____ direction
5' to 3'
83
what features provide stability to the double helix?
hydrogen bonds between bases on opposite strands
| hydrophobic interactions between the bases
84
chargraff's rule
the total number of purine bases will equal the total number of pyrimadine bases.
85
what is Huckel's rule
it is one of the requirements for aromaticity: 4n+2 where n is a positive integer and the total is the number of pi electrons
86
DNA with a right handed helix forms what type of DNA?
B-DNA
| turns every 3.4 nm and contains 10 bases per turn
87
what part of DNA provides binding sites for regulatory proteins?
the major and minor groove
88
what are some common things used to denature DNA or can denature DNA?
heat, alkaline pH, and chemicals like formaldehyde and urea
89
denatured, single stranded DNA can be _____ if the denaturing condition is slowly removed
reannealed
90
the aromaticity of the bases in DNA makes them ___ and ___
stable and unreactive
91
when does Chargraff's rule NOT hold?
for RNA (because there is no complementarity)
92
what is the name of the small basic proteins that DNA is wrapped around in the nucleus?
histone
93
what are the roles of the 5 histone proteins?
DNA wraps around the 2 copies each of H2A, H2B, H3, and H4 and then H1 seals off the DNA as it enters and leaves the nucleosome.
94
nucleosome
complex of DNA wrapped histones
95
describe chromosome structure during S phase and M phase of the cell cycle
during S phase (DNA replication), the chromosome is unwound which makes the process more efficient
During M phase (mitosis), the chromosomes condense so they can be separated properly.
96
what is the repeated sequence of DNA used by telomeres?
TTAGGG
97
what are the two roles of telomeres
end hangs of DNA so that DNA is not lost during replication
| they have high GC content which acts to tie off the end of DNA
98
what is the name of specialized proteins that assist DNA polymerase?
replisome or replication complex
99
Role of DNA polymerase alpha delta and epsilon
work together to synthesize the leading and lagging strand.
100
Role of DNA polymerase delta
fills gaps where the RNA primer was removed
101
Role of DNA polymerase gamma
replicates mitochondrial DNA
102
Role of DNA polymerase beta and epsilon together
DNA repair
103
Role of DNA polymerase delta and epsilon
form the sliding clamp
104
sliding clamp
helps to strengthen the interaction between the DNA polymerases and the template strand
105
does DNA have telomeres?
no, circular DNA
106
DNA polymerase cannot finish synthesis at the ____ end
5'
107
is the leading or lagging strand more prone to errors?
lagging strand because it has to start and stop the process more times and more RNA primers must be removed and replaced.
108
compared to a car... oncogenes are like _____ while mutated tumor suppressor genes are like _____
stepping on the gas
| cutting the breaks
109
compare oncogenes and tumor suppressor genes in terms of mutations necessary to cause cancer
oncogenes only need mutation in one allele while tumor suppressor genes usually need mutation in both,
110
where is the proofreading mechanism during DNA synthesis?
it is in the DNA polymerase
111
why is DNA ligase a problem when it comes to proofreading?
it lacks a proofreading ability
112
when DNA polymerase undertakes proofreading, how does it know which strand is the parent strand and which strand is the daughter strand?
the strand with more methylation has been around longer and is thus the parent strand. Can sense this.
113
when does nucleotide and base excision repair occur?
during G1 and G2 phase
114
DNA is synthesized in the ____ to ___ direction, adding onto the ___!!!
5' to 3' direction, adding onto the 3'
115
DNA polymerase reads in the __ to ___ direction
3' to 5'
116
base excision repair occurs after ____
a base is converted to an incorrect base
117
compare the key enzymes in nucleotide excision repair and base excision repair and
Nucleotide: excision endonuclease
Base: glycosylase enzyme removes base, AP endonuclease removes damaged sequence,
Dna pol and ligase used for both
118
compare the types of lesions in nucleotide and base excision repair
nucleotide excision repair lesions distort the double helix while base excision repair lesions do not
119
what is the goal of DNA cloning
the goal is to produce a large quantity of homogenous DNA for other applications (can also produce large quantities of a protein of interest too)
120
what is the natural source of restriction enzymes?
bacteria
121
what are two types of DNA libraries and how are they different?
genomic libraries: whole genome introns and exons cDNA (expression) libraries: just exons
122
what is one problem with genomic libraries?
genes might be split up onto different vectors.
123
what are primers for PCR
DNA sequences that flank the intended target of amplification
124
what are all the materials required for PCR
primers, dNTPs, and DNA polymerase
125
what is the preferred gel for DNA electrophoresis?
agarose gel
126
what are the materials for DNA sequencing
template DNA, primers, DNA polymerase, and dNTPs
| AND ddNTPs
127
describe problem if transgene is recessive
cannot control the number of copies inserted into the genome of a transgenic mouse.
128
what conditions cause Z-DNA to form?
high GC-content or high salt concentration
129
does prokaryotic DNA have nucleosomes?
no
130
name the aromatic amino acids
FYW
131
cDNA is formed from a _____ strand by ____
processed mRNA strand by reverse transcription
