Ch 6 - DNA and Biotechnology Flashcards
1
Q
What is DNA?
A
a macromolecule that stores genetic information in all living organisms
2
Q
What is the difference between nucleoside and nucleotide?
A
- side: contain a 5-carbon sugar bonded to a nitrogenous base
- tide: nucleoside + 1-3 phosphate groups added
3
Q
What is the difference between nucleotides in DNA v RNA?
A
- DNA: contain deoxyribose and thymine
- RNA: contain ribose and uracil
4
Q
What model does DNA organization follow?
A
Watson and Crick
5
Q
What is the backbone of DNA composed of?
A
alternating sugar and phosphate groups, and is always read 5; to 3’
6
Q
How do the 2 strands of DNA relate?
A
2 strands with antiparallel polarity would into a double helix
7
Q
What is the difference between purines nad pyrimidines?
A
- purines (A and G) always pair with pyrimidines (CUT)
- in DNA, A pairs with T (via 2 H bonds) and C pairs with G (via 3 H bonds)
- RNA does not contain thymine, but contains uracil instead; thus in RNA, A pairs with U (via 2 H bonds)
8
Q
Why are purines and pyrimidines considered biological aromatic heterocycles?
A
- aromatic compounds are cyclic, planar, and conjugated, and contain 4n + 2 pi electrons (where n is any integer; Huckel’s rule)
- heterocycles are ring structures that contain at least 2 difference elements in the ring
9
Q
What are Chargaff’s rules?
A
- states that purines and pyrimidines are equal in number in DNA molecules, and that because of base-pairing, A=T and C=g
10
Q
What form are most DNA? When might Z DNA be seen?
A
- most DNA is B-DNA, forming a right hand helix
- low concentrations of Z-DNA with a zigzag shape may be seen with high GC content or high salt concentration
11
Q
What can cause denaturation of DNA
A
- heat, alkaline pH, and chemicals like formaldehyde and urea
- removal of these conditions may result in reannealing of the strands
12
Q
How many chromosomes are DNA organized into in human cells?
A
46
13
Q
What is the relationship between histone proteins, nucleosomes, and chromatin in DNA in eukaryotes?
A
- DNA is wound around histone proteins (H2A, H2B, H3, H4) to form nucleosomes, which may be stabilized by another histone protein (H1)
- as whole, DNA and its associated histones make up chromatic in the nucleus
14
Q
What is the difference between heterochromatin and euchromatin?
A
- heter: dense, transcriptionally silent DNA that appears dark under light microscopy
- eu: less dense, transcriptionally active DNA that appears light under light microscopy
15
Q
What are telomeres? What do they contain? What happens to them during replication?
A
- the ends of eukaryotic chromosomes that protect from losing important genes from incomplete replication of the 5’ end of DNA strand
- contain high GC content to prevent unraveling of the DNA
- during replication, they are slightly shortened, although this can be (partially) reversed by the enzyme telomerase
16
Q
Where are centromeres located and what do they do? Why do they have a high GC content?
A
- located in the middle of chromosomes and hold sister chromatids together until they are separated during anaphase in mitosis
- contain high GC content to maintain a strong bond between chromatids
17
Q
What is a replisome?
A
- replication complex
- a set of specialized proteins that assist the DNA polymerse
18
Q
What is the first step of replicated DNA?
A
it is first unwound at an origin of replication by helicases producing 2 replication forks on either side of the origin
19
Q
How do prokaryotes and eukaryotes differ in their origin of replication?
A
- pro have circular chromosomes that contain only one OriC
- eu have linear chromosomes that contain many OriC
20
Q
What to single-stranded DNA binding proteins do?
A
in both eukaryotes and prokaryotes; keep unwound strands of DNA from reannealing or being degraded once unwound by helicases
21
Q
What do DNA topoisomerase do?
A
- supercoiling causes torsional strain on the DNA molecule, which can be released by DNA topoisomerase, which create nicks in the DNA molecules
- both eu/prokaryotes
22
Q
What does DNA being semiconservative mean?
A
one old parent strand and one new daughter strand is incorporated into each of the 2 new DNA molecules
23
Q
What is primase?
A
DNA cannot be synthesized without an adjacent nucleotide to hook onto, so a small RNA primer is put down by primase
- both pro/eukaryotes
24
Q
What is the DNA polymerase 3? What enzyme does the same function for eukaryotes?
A
- they synthesize a new strand of DNA; they read the template DNA 3’ to 5’ and synthesize the new strand 5’ to 3’
- in eukaryotes, DNA polymerase alpha. gamma, or epsilon
25
What is the difference between the leading strand and lagging strand?
- leading: requires only one primer and can then be synthesized continuously in its entirety
- lagging: requires many primers and is synthesized in discrete sections called Okazaki fragments
26
What do DNA polymerase 1 do? What enzyme has the same function in eukaryotes?
remove RNA primers and filled gaps with DNA
| - in eukaryotes, RNase H
27
What do DNA ligase do?
fuse the DNA strands together once primer is removed and create one complete molecule
28
Where do oncogenes develop from and what do they do?
- develop from mutations of proto-oncogenes and promote cell cycling
- may lead to cancer
29
What is cancer?
defined by unchecked cell proliferation with the ability to spread by local invasion or metastasize (migrate to distant sites via the bloodstream or lymphatic system)
30
What do tumor suppressor genes code for?
proteins that reduce cell cycling or promote DNA repair, mutations of these genes can lead to cancer
31
What performs proofreading during DNA replicaiton?
DNA polymerase proofreads its work and excises incorrectly matched bases in the S phase
- the daughter strand is identified by its lack of methylation and corrected accordingly
32
Where do mismatch excision repair occur and what genes are used?
during G2 phase of the cell cycle, using genes MSH2 and MLH1
33
What does nucleotide excision repair do and where does it occur?
fixed helix-deforming lesions of DNA via a cut-and-patch process that requires an dexcisions endonuclease
- occurs in G1 and G2 phase
34
What do base excision repair do and where does it occur?
- fixes nondeforming lesions of the DNA helix by removing the base, leaving an apurinic/apyrimidinic (AP) site
- an AP endonuclease then removes the damages sequence, which can be filled in with the correct bases
- occurs in G1 and G2 phase
35
What is recombinant DNA composed of?
nucleotides from 2 different sources
36
What does DNA cloning introduce?
a fragment of DNA into a vector plasmid
37
What does a restriction enzyme do?
- used to cleave DNA before electrophoresis and Southern blotting and to introduce a gene of interest into a viral vector for gene therapy
38
What do vectors contain?
an ORiC, the fragment of interest, and at least 1 gene for Abx resistance (to permit for selection of that colony after replication)
39
What can the bacterial cell be used for once replicated?
to create a protein of interest or can be lysed to allow for isolation of the fragment of interest from the vector
40
What are DNA libraries?
large collections of known DNA sequences
41
What is a genomic library?
- contain large fragments of DNA, including both coding and noncoding regions of the genome
- cannot be used to make recombinant proteins or for gene therapy
42
What are cDNA libraries?
- contain smaller fragments of DNA and only include the exons of genes expressed by the sample tissue
- can be used to make recombinant proteins or for gene therapy
43
How are DNA molecules separated by size?
agarose gel electrophoresis
44
What is hybridization?
the joining of complementary base pair sequences
45
What is PCR?
an automated process by which millions of copies of DNA sequence can be created from a very small sample of hybridization
46
What is southern blotting?
- used to detect the presence and quantity of various DNA strands in a sample
- after electrophoresis, the sample is transferred to a membrane that can be probed with a ss-DNA molecules o look for a sequence of interest
47
What do DNA sequences use dideoxyribonucleotides for?
- to terminate the DNA chain because they lack 3'-OH group
| - the resulting fragments can be separated by gel electrophoresis and the sequence can be read directly from the gel
48
What is gene therapy?
a method of curing genetic deficiencies by introducing a functional gene with a viral vector
49
How are transgenic mice created?
by integrating a gene of interest into a germ line or embryonic stem cells of a developing mouse
- can be mated to select for the transgene
50
What are chimeras?
organisms that contain cells from 2 different lineages
51
How are knockout mice created?
by deleted a gene of interest
52
How does the aromaticity of purines and pyrimidines underscore their genetic function?
- aromaticity of nucleic acids make these compounds very stable and unreactive
- stability is important for storing genetic information and avoiding spontaneous mutations
53
What properties of telomeres and centromeres allow them to stay tightly raveled, even when the rest of DNA is uncondensed?
high GC content increases H bonding, making the association between DNA strands very strong at telomeres and centromeres
54
Which processes occur in the 5' to 3' direction?
DNA synthesis
DNA repair
RNA transcription
RNA translation (reading codons)
55
What is the difference between oncogenes and tumor suppressor genes?
they both cause cancer, but:
- oncogenes promote cell cycle while mutated (stepping on gas pedal)
- tumor suppressor genes no longer slow the cell cycle (cutting the brakes)
56
How does DNA polymerase recognize which strand is the template strand once the daughter strand is synthesized?
- the parent strand is more heavily methylated, whereas the daughter strand is barely methylated
- this allows DNA polymerase to distinguish between 2 strands during proofreading
57
What is the key structural difference in the types of lesions corrected by nucleotide excision repair v those corrected by base excision repair?
- nucleotide excision repair corrects lesions that are large enough to distort the double helix
- base excision repair corrects lesions that are small enough not to distort the double helix
58
What is the difference between the advantages of using genomic v cDNA library?
- genomic libraries include all the DNA in an organism's genome including noncoding regions; may be useful studying DNA in introns, centromeres, or telomeres
- cDNA libraries only include expressed genes from a given tissue, but can be used to express recombinant proteins or to perform gene therapy
59
During DNA sequencing, why does the DNA polymer stop growing once a dideoxyribonucleotide is added?
- dideoxyribonucleotides lack the 3-OH group that is required for DNA strand elongation
- so once a dideoxyribonucleotide is added to a growing DNA molecule, no more nucleotides can be added because dideoxyribonucleotides have no 3'-OH group with which to form a bond
60
In a single strand of nucleic acid, what bonds link nucleotides?
phosphodiester bonds bind adjacent nucleotides
61
How does GC content affect melting point of DNA sequences?
- the melting temperature of DNA is the temperature at which a DNA double helix separates into 2 single strands (denatures)
- to do this, H bonds linking the base pairs must be broken
- C-G have 3 H bonds and A-T have 2 H bonds so breaking GC would require more heat, thus higher mp
62
How is cDNA formed?
complementary DNA formed from a processed mRNA strand by reverse transcription
63
Why does prokaryotic DNA lack nucleosomes?
because it is circular and does not have histone proteins
64
Why is uracil excluded from DNA but not RNA?
Cytosine degradation results in uracil
- one common DNA mutation is the transition from cytosine to uracil in the presence of heat
- DNA repair enzymes recognize uracil and correct this error by excising the base and inserting cytosine
- RNA exists only transiently in the cell, such that cytosine degradation is insignificant
- were uracil to be used in DNA under normal circumstances, it would be impossible to tell if a base should be uracil or if it is damaged cytosine
