Chapter 21- Structure and Properties of DNA Flashcards
What consists of the primary structure of DNA and RNA?
A sugar, base, and phosphate group
Sugar is deoxyribose for DNA and Ribose for RNA
nitrogenous heterocyclic Base can be pyrimidines or purines
Phosphate group signifies starting 5’ end
Phosphodiester bonds join the 3’ carbon of one sugar to 5’ carbon of next sugar
What is a nucleoside?
Consists of sugar and base
What describes the secondary structure of DNA?
Double Helix, antiparallel chains (5’to 3’ and 3’ to 5’), bases are toward the center with hydrogens bonding A-T and G-C stacked like a staircase
Phosphate groups are on the outside and gives DNA its negative charge
B form of double helix is characterized by what?
Watson-crick DNA helix, naturally occurring right handed, predominant form under physiological conditions, 10.5 bases per turn
A form of double helix is characterized by what?
similar to B-form, right handed and more compact, dehydrated form, bases are tilted 20° relative to the helical axes, 11 bases per turn
Z form of double helix is characterized by what?
left handed and has zigzag structure, forms under high salt concentration and presence of divalent cations, 12 bases per turn, CGCGCGCGCG polynucleotide in solution, may play a role in regulation of gene expression
What describes the tertiary structure of DNA?
super helical (positive supercoil from replication fork)
Denaturation of DNA is achieved by what?
Alkali or heat cause the strands to separate but does not break the phosphodiester bonds
Melting temperature (Tm) of DNA is dependent upon what?
GC content of DNA, determined when half of dsDNA converted to ssDNA, lowering ionic strength can lower Tm
How would you renature DNA after denaturation via heat?
temperature is slowly decreased and base pairs reform and complementary strands of DNA come back together
What is the central dogma of molecular biology?
DNA makes RNA which makes protein
How does 3’-deoxy-3’azidothymidine (AZT) affect DNA synthesis?
AZT is a base analog of thymidine that substitutes for thymidine and interfering with DNA replication. Used in HSV and HIV treatment
What are the most frequently methylated bases?
Guanine and cytosine
In mammalian cells how many % GC base pairs are methylated?
70%
What enzyme adds a methyl group to adenine contained in any GATC sequence?
deoxyadenosine methylase (DAM)
Dem methylase methylates what?
cytosine in the sequence CCAGG
What is the function of methylation?
Inactivation of DNA expression, protection against restriction endonucleases
What is genomic imprinting?
different methylation pattern in maternal and paternal chromosomes at CpG nucleotides
What are examples of faulty genomic imprinting?
Prader-Willi/Angelmann’s Syndrome and Beckwith-Wiedemann Syndrome
What is an example of good methylation?
Women have 2 X-chromosomes and methylate to turn off one X chromosome therefore only 1 X-chromosome is working
What is an example of bad methylation?
In fragile X-syndrome the CpG island is methylated when normally it should not. This results in the cell’s inability to copy information in the FMR1 gene and FRM protein is not synthesized
Female ability to avoid fragile X majority of the time
If large proportion of cells turn off X with the fragile X mutation then active X chromosome can produce FMR protein. Otherwise the opposite may happen and the mutated FMR1 is expressed resulting in fragile X.
Severity of fragile X dependent upon female inactivation of either good FMR1 or bad FMR1
Female ability to avoid fragile X majority of the time (mosaic female).
If large proportion of cells turn off X with the fragile X mutation then active X chromosome can produce FMR protein. Otherwise the opposite may happen and the mutated FMR1 is expressed resulting in fragile X.
Severity of fragile X dependent upon female inactivation of either good FMR1 or bad FMR1
Acycloguanosine (acyclovir) has what mechanism of action
a base analog of guanine that substitutes for guanine and interfering with DNA replication. Used in antiviral treatment
6-Mercaptopurine
base analog of hypoxanthine and guanine; used in acute leukemia treatment
5-Fluorouracil
analog of thymine, an “antimetabolite” that irreversibly inhibits thymidylate synthetase -> inhibits thymine synthesis, used in cancer treatments
2-aminopurine
analog of adenine -> can pair with T or C during DNA replication, non-mutagenic or weakly mutagenic for eukaryotes while it is mutagenic for bacteria
What are the 3 methods of DNA sequencing?
Edman degradation, Sanger Method, and Maxam-Gilbert method
The complementary base sequences Chargraff’s rules follow what?
A pairs with T, G pairs with C
Physical structure of DNA consists of what characteristics?
double stranded helical structure, antiparallel, major and minor grooves, variation of DNA base composition, can be in A, B, or Z forms
How many base pairs per helical turn in DNA?
10bp
What is diameter and gap between bases in DNA?
2.0nm diameter, 0.34nm gap between bases
Name three intercalating agents
ethidium bromide, acridine orange, actinomycin D
What do intercalating agents do?
ring molecules that resemble ring structure of base pairs -> Insert self in between DNA stacked pairs -> distorts DNA double helix -> interferes with DNA replication, transcription, and repair
Describe plasmid DNA
Circular DNA molecules, widespread among bacteria, consisting of just a few genes to more than a hundred, antibiotic resistance genes
Plasmids are transferred between bacteria via
conjugation
Negatively supercoiled plasmid DNA help alleviate…
underwound DNA
Positively supercoiled plasmid DNA helps alleviate…
overwound DNA
Topoisomerase receives supercoil stress by what?
DNA gyrase (Topo II) catalyzes the ATP dependent formation of negatively supercoiled DNA ahead of replication fork
Denaturation of DNA can be spectrophotometrically measured by
denatured OD(optical density) increased 40% at 260nm (Hyperchromic shift)
impure DNA represented by what?
impure DNA 280nm
at pH >11.5 does what to DNA?
deprotonation of bases, destroying H-bonding, denaturation
at pH <2.3 does what to DNA?
protonation of bases, disrupts base-pairing, denaturation
rate of DNA renaturation is dependent upon what?
length of DNA, short highly repetitive sequences renature faster than long non-repetitive DNA molecules
Characteristics of histones
very basic proteins containing positively charged lysines and arginines
chromatin consists of
DNA and histones
Five major classes of Histones
H1, H2A, H2B, H3, and H4
Nucleosomes are
the beadlike structure from resting nuclei, 10nm diameter
describe chromatin fibers/filament
30nm nucleoprotein fiber, a spring-shaped solenoid with about six nucleosomes per turn, stabilized by head to tail associations of the H1 histone
What are two classes of chromatin and describe differences
euchromatin ( less densely packed, active genes) and heterochromatin (tightly packed, limited gene expression)
Satellites:
very highly repetitive with repeat lengths of one to several thousand base pairs, organized as large (up to 100 million bp !) clusters in the heterochromatic regions of chromosomes, near centrosomes and telomeres; found abundantly on the Y chromosome.
Minisatellites:
are moderately repetitive, tandemly repeated arrays of moderately-sized (9 to 100 bp, but usually about 15 bp) repeats, generally involving mean array lengths of 0.5 to 30 kb, found in euchromatic regions of the genome of vertebrates, fungi and plants and are highly variable in array size.
Microsatellites:
moderately repetitive, and composed of arrays of short (2-6 bp) repeats found in vertebrate, insect and plant genomes. The human genome contains at least 30,000 microsatellite loci located in euchromatin. Copy numbers are characteristically variable within a population, typically with mean array sizes on the order of 10 to 100.
Alu family:
another class of highly repetitive sequences, 500,000 in human genome
fragile X gene (FMR1) was characterized as
containing a tandemly repeated trinucleotide sequence (CGG) near its 5’ end
The normal population varies in how many CGG repeats
6-50
How is fragile X carriers and affected individuals determined?
polymerase chain reaction (PCR) and Southern blot analysis
consequences of >200 repeats of CGG (full mutation) on the FMR1 gene
heavily CpG methylated -> transcriptional silencing of FMR1 gene -> no FMR protein
examples of recombinant DNA techniques
Purification of Nucleic Acids Enzymatic Manipulation of DNA DNA Cloning Analysis of DNA Expression of Recombinant Protein Site-directed Mutagenesis
what is a genomic library?
contains DNA fragments representing the entire genome of an organism
what is a cDNA library?
contains only complementary DNA molecules synthesized from mRNA molecules in a cell
What are DNA vaccines?
Usually consist of single or multiple genes of immunogenic proteins inserted into a commercially available DNA expression plasmid
