Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

Molecular Genetics > Exam 1 > Flashcards

Exam 1 Flashcards

You may prefer our related Brainscape-certified flashcards:
Biology 101 flashcards
1
Q

genome

A
  • the complete set of genetic material in the organism
  • hereditary material of the organism
  • composed of DNA
  • includes DNA of chromosomes and any DNA in organelles (eukaryotes) or plasmids (prokaryotes)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

chromosome

A
  • a discrete unit of the genome carrying many genes
  • each chromosome consist of a very long molecule of duplex DNA
  • plus approximately equal mass of proteins
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

Number of chromosomes in different organisms

A
  • humans - 46
  • drosophila - 8
  • corn - 20
  • bacteria - 1 (circular)
  • male jack jumper ant - 1
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

gene

A

a section of DNA on a chromosome that encodes for genetic information

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

structural gene

A

a gene that encodes any RNA or polypeptide product other than a regulator

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

allele

A

one of the several alternative forms of a gene

  • slightly different DNA sequence
  • hair color; height
  • may have different alleles from mother and father
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

locus

A
  • the position on a chromosome at which the gene for a particular trait resides
  • it may be occupied by any one of the alleles for a gene
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

genetic recombination

A
  • the rearrangement of DNA sequences by the breakage and rejoining of chromosomes
  • due to such processes as crossing over in meiosis or transposition
  • the consequences of such rearrangements is a novel combinations of alleles in the offspring that carry recombinant chromosomes
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

nucleotide

A

makes up DNA and RNA

  • 5-carbon sugar
  • phosphate attached to 5’ carbon of sugar
  • nitrogenous base attached to 1’ carbon
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

DNA structure

A

deoxyribose sugar (2’-H)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

RNA structure

A

ribose sugar (2’-OH)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

nucleoside

A

contains

  • a nitrogenous base linked to the 1’ carbon of a pentose sugar
  • no phosphate attached
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

purines

A
  • nine atoms - guanine and adenine
  • larger than pyrimidines
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

pyrimidines

A
  • cytosine and thymine in DNA
  • uracil and thymine in RNA
  • smaller than purines
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

DNA is a double helix

A
  • a double helix consisting of two polynucleotide chains
  • chains run antiparallel
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

nitrogenous base pairing

A
  • the nitrogenous bases of each chain are flat purine or pyrimidine rings
  • they face inward with the sugar-phosphate forming the external background
  • the bases pair with one another by hydrogen bonding to form only A-T or G-C pairs
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

how many hydrogen bonds form between A and T?

A

2

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

how many hydrogen bonds form between G and C?

A

3

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

the phosphates provide a strong ______ charge

A

negative (in solution)
In Vitro, charge is neutralized by:

  • sodium ions
  • positively charged proteins
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

physical structure of DNA

A
  • diameter of the helix = 20A
  • one complete turn = 34A
  • 10 bp per turn (about 10.4 in solution)
  • 1A (Angstrom) = 0.1nm
  • major and minor groove
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

forms of DNA

A

A-form

  • dehydrated DNA
  • shorter and thicker

B-form

  • average structure
  • right-handed helix turns clockwise along the axis
  • found in aqueous conditions

Z-form

  • left-handed helix
  • long and narrow
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

RNA

A
  • single stranded
  • has ribose as the sugar (2’ OH)
  • purines (A and G)
  • pyrimidines (C and uracil)
  • not as stable as DNA
  • no base pair hydrogen bonds
  • ribose -OH is more reactive
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

prion

A
  • a proteinaceous infectious agent
  • behaves as an inheritable trait even though it contains no nucleic acid
  • one example is PrP^Sc, the agent of scrapie in sheep and bovine spongiform encephalopathy (mad cow disease)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

central dogma

A
  • information cannot be transferred from protein to protein, or protein to nucleic acid
  • translation is unidirectional
  • RNA may be converted into DNA by reverse transcription
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Q

DNA polymerase

A

an enzyme that synthesizes DNA from a DNA template

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
26
Q

RNA polymerase

A

an enzyme that synthesizes RNA using a DNA template

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
27
Q

reverse transcriptase

A

an enzyme that synthesizes DNA using an RNA template
- used by some viruses

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
28
Q

intermolecular base pairing

A
  • complementary base pairing between two different strands of nucleic acids
  • DNA to DNA
  • DNA to RNA
  • RNA to RNA
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
29
Q

intramolecular base pairing

A

complementary base pairing between different sections of the same nucleic acid

  • RNAs
  • tRNA
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
30
Q

nucleic acids anneal by complementary base pairing

A
  • heating causes the two strands of a DNA duplex to separate or denature
  • melting temperature (Tm)
    • the midpoint of the temperature range for denaturation
  • reduce temperature
    • complementary single strands can renature or anneal (hybridize)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
31
Q

filter hybridization

A
  • denature a known DNA and attach to a solid filter
  • denature unknown DNA in solution
  • mix - if DNAs have similar sequences, they will anneal
  • the ability of two single stranded nucleic acids to hybridize is a measure of their complementarity
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
32
Q

hybridization can occur with

A
  • DNA-DNA
  • DNA-RNA
  • RNA-RNA

it can be intermolecular or intramolecular

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
33
Q

mutations

A

changes in the sequence of DNA

may occur

  • spontaneously
  • or induced by mutagens
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
34
Q

mutagens

A
  • chemicals that can cause mutations
  • radiation (UV, gamma)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
35
Q

point mutation

A

changes a single base pair
may be due to

  • chemical conversion of one base into another
  • or errors that occur during replication
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
36
Q

transition

A

a type of point mutation

  • replaces a G-C base pair with an A-T base pair or vice versa
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
37
Q

transversion

A

type of point mutation

  • replaces a purine with a pyrimidine, such as changing A-T to T-A
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
38
Q

insertions/deletions of larger DNA segments results from:

A

the movement of transposable elements (DNA segments that can be inserted in chromosomes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
39
Q

forward mutations

A

alter the function of a gene

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
40
Q

back mutations (revertants)

A

reverse their effects

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
41
Q

insertions can revert by ____

A

deletion of the inserted material

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
42
Q

can deletions revert?

A

no

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
43
Q

one gene one enzyme hypothesis

A
  • suggested by Beadle and Tatum in 1940s
  • a gene is a stretch of DNA encoding one or more isoforms of a single polypeptide chain
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
44
Q

heteromultimer

A

a molecular complex (such as a protein) composed of different subunits

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
45
Q

homomultimer

A

a molecular complex (such as a protein) composed of identical subunits

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
46
Q

one gene one polypeptide hypothesis

A

a modified version

  • a gene is responsible for the production of a single polypeptide
  • then polypeptides are put together to form the enzyme

but: most genes do not encode polypeptides, but encode structural or regulatory RNAs

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
47
Q

a locus can have many different mutant alleles

A
  • multiple alleles
  • wild type = w+ (red eye)
  • various mutants: W^h (honey eye)
  • since have 2 homologous chromosomes
  • allows for heterozygotes with any pairs of combination of alleles
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
48
Q

a locus can have more than one wild-type allele

A
  • a locus may be polymorphic in alleles
  • no individual allele is considered to be the only wild type
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
49
Q

the genetic code is triplet

A
  • it’s the code on mRNA (DNA>mRNA>protein)
  • the genetic code is read in triplet nucleotides called codons
  • the triplets are non-overlapping and are read from a fixed starting point

each codon triplet codes for:
- a specific amino acid
- or a stop codon

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
50
Q

can codons code for the same amino acid?

A

yes

ex: UUU and UUC code for Phenylalanine

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
51
Q

effects of mutations

A
  • insertion or deletion of bases
  • cause a shift in the triplet sets after the site of mutation
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
52
Q

frameshift mutations

A
  • insertion or deletion of three bases (or multiples of three)
  • inserts or deletes amino acids
  • but reading (or AA sequence) remains the same after the third insertion/deletion
  • they happen after the deletion of 4 bases
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
53
Q

open reading frame (ORF)

A
  • a sequence of DNA consisting of triplet codons that can be translated into a string of amino acids
  • starts with an initiation codon and end with a termination (stop) codon
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
54
Q

every coding sequence has ___ possible reading frames

A

three

  • usually only one of the three possible reading frames is translated
  • the other two are closed by frequent termination signals (stop codons)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
55
Q

requirements for protein synthesis

A
  • functional mRNA
  • ribosome - a large complex of ribosomal RNA and proteins that synthesize polypeptides using an mRNA template
  • tRNAs
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
56
Q

tRNA

A
  • a tRNA has an anticodon sequence that is complementary to the codon representing an amino acid
  • each tRNA molecule is linked to that amino acid
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
57
Q

what really matters in molecular genetics?

A
  • protein coding genes
  • regulatory sequences
  • epigenetics
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
58
Q

differences between humans and chimpanzees are most caused by ___

A

gene regulation

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
59
Q

genes have DNA control sites

A
  • proteins that regulate gene transcription bind to control sites next to the coding regions
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
60
Q

cis

A

sites located on the same DNA strand

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
61
Q

trans

A

sites located on different DNA strands

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
62
Q

proteins are trans-acting but sites on DNA are cis-acting

A
  • all gene products (RNA or polypeptides) are trans-acting
  • they can act on any copy of a gene in the cell
  • regulatory proteins are trans-acting
    • they act on any gene regulatory region
    • copies of the same protein can act on both homologous alleles
  • a cis-acting DNA site controls expression of the adjacent DNA
    • but does not influence the homologous allele on the other chromosome
  • a mutation in the control site of a gene is cis-acting
    • affects the adjacent gene
    • does not affect the homologous allele
  • a trans-acting mutation in a gene for a regulatory protein affects both alleles of a gene that it controls
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
63
Q

original meaning of genetic engineering

A
  • cloning genes by placing a gene DNA from one organism into another DNA or organism to allow it to be replicated
    -ex: placing a mouse
    enzyme gene into a
    bacterial plasmid
  • creates recombinant DNA
    • a DNA molecule
      composed of sequences
      from two (or more)
      different sources
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
64
Q

genetic engineering now

A
  • direct manipulation of an organism’s genome through the use of biotechnology to insert or delete genes
  • often involves the production and use of recombinant DNA to transfer genes between organisms
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
65
Q

restriction endonuclease

A
  • enzyme that recognizes short specific sequences of DNA and cleaves the duplex
  • it cleaves sometimes at the target site, sometimes elsewhere, depending on type of enzyme
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
66
Q

nucleases

A
  • hydrolyze phosphodiester bonds
  • separates the nucleotides
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
67
Q

endonuclease

A
  • nuclease that cleaves phosphoester bonds within a nucleic acid chain
  • breaks the chain
  • it may be specific for RNA or for single-stranded or double-stranded DNA
  • cleave within the strand
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
68
Q

exonuclease

A
  • nuclease that cleaves phosphoester bonds one at a time from the end of a polynucleotide chain
  • chews off nucleotides from the end
  • it may be specific for either the 5’ or 3’ end of DNA or RNA
  • cleave at the terminal nucleotide
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
69
Q

nucleases can be:

A
  • broad specificity
    e.g. exonuclease that cleaves any nucleotide from the end of DNA
    e.g. pancreatic RNase = cleaves RNA after any pyrimidine
  • sequence specific - restriction endonucleases
    -Type I, II, and III
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
70
Q

Type II restriction endonucleases

A
  • most common
  • many derived from bacteria
    • EcoRI from E.coli
  • recognition sites are 4-8 bp
  • sites are typically inversely palindromic
    • reads the same forward and backward
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
71
Q

restriction enzymes

A

cut DNA in two different ways:

  • staggered cut
    -leaves “sticky ends” of complementary bases
  • blunt ds cut
    -no sticky ends
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
72
Q

restriction mapping

A
  • a map can be generated by using the overlaps between the fragments generated by different restriction enzymes
  • used to find sites of restriction enzymes in your DNA
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
73
Q

cloning DNA

A

cloning
- to make an identical copy of something
- DNA, Dolly the sheep etc.

  • cloning DNA uses recombinant DNA

what you need to clone DNA
- an insert : the gene or DNA fragment you want to clone
- a cloning vector

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
74
Q

bacterial plasmids

A
  • bacteria have two different types of DNA
    1) circular chromosome: genes for the bacteria to function
    2) plasmids
  • small circular dsDNA
  • often contain survival genes
  • antibiotic resistance - ARGs
  • self replicating
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
75
Q

cloning vector

A
  • a genetically engineered modified plasmid
  • DNA that can be used to propagate an incorporated DNA sequence in a host cell
  • often derived from a plasmid or a bacteriophage (virus that attacks a bacteria)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
76
Q

engineered plasmid vectors contain:

A
  • replication origins
  • selectable markers
  • known restriction enzyme sites
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
77
Q

replication origins

engineered plasmid vectors

A
  • ORI (origin of replication initiation)
  • where DNA replication can start
  • allows replication of the plasmid
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
78
Q

selectable markers

engineered plasmid vectors

A
  • allows you to identify cells that contain the plasmid
  • Ampicillin Antibiotic resistance gene
  • lacZ gene
79
Q

known restriction enzyme sites

A
  • multiple cloning site (MCS)
    e.g EcoRI
80
Q

multiple cloning site (MCS)

A
  • a plasmid section containing a series of tandem restriction endonuclease sites used in cloning vectors for creating recombinant molecules
81
Q

the DNA fragment insert

A
  • fragments of genomic DNA that has been treated with restriction enzymes
    • size selected from an agarose gel
  • a PCR fragment
    • specific DNA selected and amplified by PCR
  • DNA fragments synthesized in the lab
82
Q

cloning steps

A
  1. cut the insert DNA and the plasmid DNA with the SAME restriction enzyme
    - easiest if use an enzyme that makes sticky ends
  2. mix the insert and plasmid
    - allows sticky ends to hybridize
    - insert sticky ends anneals to the sticky ends of the plasmid
  3. add DNA ligase enzyme
    - seals the phosphodiester bonds
  4. the plasmid circle is now
    - sealed
    - may contain the insert
    - may be larger than before

Note: the insert has disrupted the lacZ gene

83
Q

transformation of bacteria

A
  • use the bacteria to replicate the plasmid
    transformation
  • the process of introducing the DNA into a cell
83
Q

competence

A

method for transformation

competent cell = capable of taking in DNA

  1. high salt wash of calcium chloride (CaCl2)
    - creates small holes or pores in the bacteria cell wall
    - allows the DNA to bind to the bacteria to be taken into the cell
  2. electroporation
    - add the bacteria and the plasmid to a small chamber
    - add an electrical current to the liquid
    - creates small holes or pores in the cell wall
    - allows the DNA to be taken into the cell

note: both methods will transform only a fraction of the cells
grow the bacteria on agar with Ampicillin (only bacteria with plasmid will grow)

84
Q

blue-white cloning vector

A
  • plasmid has lacZ gene
    -codes for bacterial gene for beta-galactosidase enzyme
    -cleaves lactose sugar
    -also will cleave X-gal
  • multiple cloning site is in the middle of the lacZ gene
    -cloning a DNA insert into the MCS will disrupt the lacZ gene
85
Q

result of blue-white cloning vector

A
  • bacteria without the plasmid will not grow
    why? - agar has ampicillin and only bacteria with plasmid will survive
  • bacteria with the empty plasmid
    -colonies will be blue
  • bacteria with the insert, has disrupted lacZ
    -colonies will be white
  • so white colonies are the ones we want!
86
Q

possible problems with transformation

A
  1. plasmid inserted into the plasmid
    - disrupts the lacZ = white colonies
    - but no insert
  2. insertion of random, non-target DNA
    - disrupts the lacZ = white colonies

solution:

  • collect the plasmids
  • restriction enzymes to remove the insert
  • agarose gel to confirm size of the insert
  • sequence the insert
87
Q

other cloning vectors exist

A

if need to clone larger DNA segments

  • bacteriophage vector
    -infects the bacteria and inserts the DNA into the bacterial chromosome
    -package larger DNA segment
    -can get the bacteria to express or produce the gene product
  • cosmid
    -plasmid with insertion sequences of a bacteriophage
    -combines best of both
  • YAC (yeast artificial chromosome)
    -use yeast as the host
88
Q

expression vectors

A
  • control regions of genes contain promoters
    -the region of DNA where the RNA polymerase binds to start transcription
  • for genes in plasmids/vectors to be:
    -to be replicated = need ORI
    -to be expressed (the protein produced) = need a promoter
  • expression vectors contain promoters that allow transcription of any cloned gene
89
Q

two types of expression vectors

A
  1. continuously active (constitutive) promoters
  2. inducible promoters
90
Q

continuously active (constitutive) promoters

A
  • if it is in the vector, the inserted DNA will be transcribed and the protein produced ALL THE TIME
  • use to have the bacteria/yeast/eukaryotic cell produce your protein
  • recombinant protein: protein produced from recombinant DNA
  • e.g. recombinant insulin produced by biotech company
91
Q

inducible promoters

A
  • the inserted DNA will only be transcribed when the promoter is turned on
  • so, transform the bacteria/yeast/eukaryotic cell
  • then you determine when to turn on the gene expression
  • provide a chemical/hormone/etc. that turns on the promoter
92
Q

reporter genes

A
  • a gene attached to a promoter and/or your gene
  • its product is an easily identified protein
  • can show when and where the gene or promoter is activated
  • used to measure promoter activity or tissue-specific expression
93
Q

lacZ reporter

A
  • insert your gene between the plasmid expression promoter and the lacZ gene
    -when your protein is produced, it will be linked to beta-galactosidase enzyme
  • transform your bacteria/yeast/mammalian cell
  • add X-gal
  • wherever your protein is found, blue stain will be seen

your protein attached to B-galactosidase

94
Q

localized expression of a protein

A
  • insert your promoter and gene in front of the lacZ gene
    -your gene and the lacZ gene are under control of your promoter
    -when your promoter is activated, your protein will be linked to beta-galactosidase enzyme
  • transform - add X-gal
  • wherever your protein is produced, blue stain will be seen
95
Q

other reporter proteins

A
  • green fluorescent protein (GFP)
    -protein from the Aequorea victoria jellyfish
    -is a green fluorescing protein
  • genetically mutated variations
    -YFP - yellow
    -CFP - cyan
96
Q

fluorescent microscopy

A
  • fluorescent molecules absorb light at excitation wavelength
  • give off light at emission wavelength
    -lower energy
    -longer wavelength
97
Q

common fluorochromes

A
  • DAPI - stains DNA
  • CFP
  • GFP
  • FITC - replaced by Cy2/Alexa488
98
Q

detecting nucleic acids

A

fluorescent DNA and RNA stains
- ethidium bromide
- DAPI
- propidium iodide

  • bind to the nucleic acid and fluoresce
  • stain ALL nucleic acid
  • cannot distinguish your gene from all other DNA
99
Q

detecting specific DNAs

A
  • based on the specific DNA sequence
  • use nucleic acids hybridization and complementary base pairing
  • probe
    -short DNA or RNA segment of known sequence of the gene you are looking for
    -radioactively label the short DNA used to identify a complementary binding DNA or RNA
100
Q

specific hybridization with a probe

A
  • synthesizes a short DNA sequence that matches your gene
  • label the probe
101
Q

labeling probes

A
  1. radioactive phosphorous
    - can use radioactive tritium
  • end labeling
    -use phosphorous and a kinase to add the phosphate to the end
  • labeling by incorporating a 32P labeled nucleotide
    -DNA synthesis using DNA polymerase
    -polymerase chain reaction (PCR)
  1. fluorescent labeled nucleotides
102
Q

denaturing DNA

A

separating the DNA duplex strands

  • must overcome the stable hydrogen bonds of base pairing
  • more G-C content = more stable
  • high temperature “melts” the DNA
  • low salt concentrations
    -high salt = Na+ stabilizes the phosphate backbone
    -low salt = negative charged phosphates in backbone repel each other
103
Q

nucleic acid detection

A

autoradiography
a method of capturing an image of radioactive materials on a photographic film

  • ethidium bromide stains all DNA
  • autoradiograph shows only the radioactive probe labeled DNA binding to the complementary DNA
104
Q

DNA separation techniques

A

agarose gel electrophoresis

  • uses agarose as a matrix gel
    -can use polyacrylamide
  • uses an electric current to cause the DNA to migrate toward a positive charge
  • remember - DNA has a negative charge due to phosphate backbone
  • separates DNA fragments by size (smaller travel faster = end up further)
  • compare to migration of known DNA size standards
105
Q

difficult to probe agarose gels:

A
  • transfer the separated DNAs to:
    -a stronger matrix
    -an easier to manipulate matrix
  • nitrocellulose membrane
    -good but easily cracks
  • nylon membrane
    -strong
106
Q

southern blotting

A
  • invented by Dr. Edwin Southern
  • transfer of DNA from a gel to a membrane (in alkaline solution to denature the DNA)
  • followed by detection of specific sequences by hybridization with a labeled probe
107
Q

northern blotting

A
  • involves the transfer of separated RNA from a gel to a membrane
108
Q

western blotting

A
  • separation of proteins on a sodium dodecyl sulfate (SDS) gel
  • transfer to a nitrocellulose membrane
  • detection of proteins of interest using antibodies
  • proteins are separated by size
109
Q

isolating mRNA

A
  • many types of RNA
    1. primary RNA transcripts
    2. mRNA
    3. tRNA
    4. rRNA
    5. small heterogeneous RNAs
  • mRNA always have a poly-adenylated tail
  • string of deoxyAdenines added to the 3’ end
  • can isolate mRNA from everything else by using Oligo (dT) bound to beads
  • short strands of deoxy Thymidine
110
Q

oligo (dT) column

A
  • oligo (dT) bound to Sepharose beads
  • packed into a small column
  • add RNA mixture to the column
  • mRNA binds to the Oligo(dT)
  • wash out the other RNAs to pass through
  • then pass through denaturing solution to wash out mRNA
111
Q

polymerase chain reaction (PCR)

A
  • developed by Kary Mullis - Nobel prize in 1993
  • continuous cycling of
  • denature DNA
  • hybridize primers
  • allow polymerase to copy the DNA
  • repeat 20-40 times
  • exponential amplification of a desired sequence
112
Q

PCR requires:

A
  • DNA primers that flank the gene you want
  • single stranded primers complementary to 3’–>5’ end of gene on each strand of DNA
  • primers run 5’–>3’
  • 18 to 25 bases long
  • easily synthesized (purchased)
113
Q

polymerases require:

A
  • a DNA or RNA template
  • a free 3’ end to add nucleotides to
    -satisfy this using primers
  • must know sequence to make the primers
  • target DNA
    -genomic DNA containing a gene you want
    -pieces of DN of a gene you want
  • Taq polymerase
    -from Thermus aquaticus bacterium
    -lives in very hot springs
    -very temp resistant
    -works best at 72 degrees
  • excess of the four deoxynucleotides (dNTPs) to make DNA
114
Q

PCR steps

A

A. mix template, primers, Taq, and dNTPs
B. heat to 95-100 degrees

  • melts (denatures) the DNA template
  • 15 seconds

C. rapidly cool to temperature optimal for primers to anneal to template

  • too low = primers anneal anywhere
  • too high - primers won’t anneal
  • is different for each set of primers
  • 30 seconds

D. heat to optimal temperature for Taq polymerase to function

  • 72 degrees
  • 30 seconds

Result:
- one new copies of the target gene
- plus the original copy

repeat 20-40 cycles

  • 2nd cycle - 4 copies of the gene
  • 3rd cycle - 8 copies of the gene, now see copies that are only the gene plus the primers
  • by 20 cycles - almost all are the gene+primers only, up to a million copies
115
Q

PCR primers

A
  • must be long enough to be specific for your DNA only
  • usually ~20 nucleotide sequence is specific
  • for any 20 nucleotide sequence
    -occurs ONCE in 4^20 nucleotides
    -human genome is 3.2x10^9 base pairs
    -so will occur only once in the genome
    -many use 23 to 25 nucleotides for primers
116
Q

uses for PCR

A
  • MUST know nucleotide sequences at the ends of the DNA to be amplified
  • amplify short DNAs without having to use plasmids and cloning
    -works great for single genes or gene sequences
    -does not work for large DNAs or genomic DNA
    -generally best with 100 to 500 bp of DNA
  • isolate DNA segments from mutants
  • excellent for producing DNAs for cloning in E.coli
  • use to label DNA for probes
    -add fluorescent dNTP
  • identify bacteria/virus/infection
117
Q

reverse transcription

A
  • transcribes single-strand RNA into single-strand complementary DNA (cDNA)
  • from retroviruses
  • transcribe viral RNA into cDNA
  • integrate viral DNA into the host genome
  • requires a DNA primer
    -short strand of DNA complementary to the RNA to be copied
118
Q

reverse transcriptase primers

A
  1. oligo(dT) primer for mRNA
  2. random primers for any RNA
    - mixture of short random sequences (usually hexomers)
  3. gene-specific primers for specific RNAs
    - determined from the gene sequence
119
Q

reverse transcription-PCR (rtPCR)

A
  • isolate RNA
  • reverse transcribe to cDNA
    -use oligo(dT) for mRNA
    -makes cDNA from ALL mRNA only
  • add specific PCR primers, dNTPs, and Taq polymerase
    -first round copies ONLY the specific cDNA to be dsDNA
    -subsequent rounds amplify the specific cDNA
120
Q

real-time PCR, or quantitative (qPCR)

A
  • detects the PCR products during PCR amplification
  • fluorescent dye to label dsDNA
  • allows monitoring the increase in fluorescent labeled PCR products
  • is more sensitive and quantitative than conventional PCR
  • can now make copies for jsut about any gene by cloning and expanding E.coli or PCR
121
Q

can we study more than one gene at a time?

A

Yes! DNA microarrays

  • backwards of the southern/northern blots
  • immobilize the known DNAs (different genes) on a membrane
  • then add the labeled unknown nucleic acid sample
  • see where the labeled nucleic acids bind (hybridize)
122
Q

DNA arrays

A
  • initially, 30-100 different DNAs “spotted” on membranes and dried
  • 1000 DNAs spotted onto glass slides
  • thousands to millions of DNAs spotted onto silicon chips
  • use fluorescent labels instead of autoradiography
    -allows better resolution
    -automated optical microscopy for detection
  • allows comparisons
    -experimental to control
    -disease versus normal
123
Q

DNA microarrays

A
  • label control mRNA/cDNA with green during reverse transcription
  • label experimental mRNA/cDNA with red
  • mix together and add to the microarray
  • ssDNAs bind to specific gene DNAs on the DNA chip
  • wash and scan
124
Q

Bacterial genes

A
  • usually continuous sequences of nucleotides
  • encodes the amino acids to create a polypeptide
    NOT TRUE FOR EUKARYOTES
125
Q

eukaryotic genes are interrupted

A
  • the coding sequences are not continuous
  • coding regions of eukaryotic genes are interrupted by segments of non coding regions

note: not necessarily true for yeasts

126
Q

exons

A

the DNA sequences of the gene that code for the gene product (polypeptide or RNA)i

127
Q

intron

A
  • a segment of DNA that does not code for the product
  • lie between exons
128
Q

primary (RNA) transcript

A
  • the original unmodified RNA product that is transcribed
  • contains the exons and the introns
  • is only a precursor RNA
  • it is NOT the final mRNA
129
Q

making the mature transcript

A
  • a mature mRNA is made by RNA splicing of the primary RNA transcript
  • the process of
    -excising introns from RNA
    -then connecting the exons into a continuous mRNA
130
Q

primary RNA transcript is equal to the ____ ____ minus ____

A

primary RNA transcript is equal to the entire gene minus the control DNA sequences

131
Q

mRNA is just the

A

exons

132
Q

removal of introns by RNA splicing occurs in __ in individual RNA molecules

A

cis

  • affects only within the same RNA
  • does not affect other RNAs
  • exons remain in the same order in mRNAs as in DNA, but distances along the gene are not the same as those of mRNA or polypeptide products
133
Q

effect of mutations

A
  • mutations in exons can affect polypeptide sequence
  • mutations in introns do not directly affect the polypeptide sequence
  • but may affect RNA processing
  • mutations at the exon-intron junctions may affect the splicing event
  • point mutations may yield stop codons affecting the final protein
134
Q

how are introns detected in genes?

A
  • compare the gene to the mRNA
  • usually compare DNA gene to cDNA of the mRNA

restriction mapping
- treat gene DNA and cDNA with the same restriction enzyme(s)
- restriction sites in the gene and mRNA/cDNA are the same in exons
- restriction sites in introns are missing in the mRNA/cDNA

  • introns can also be detected by sequencing the gene and the cDNA, then matching the two together
135
Q

does an intron have an open reading frame?

A

no
an intact open reading frame is created in the mRNA sequence by removing the introns

136
Q

gene structure of other DNAs

A
  • genes coding for polypeptides, rRNA, and tRNA can all have introns
  • introns have been found in every class of eukaryote
  • introns are rare in prokaryotic genes
  • animal mitochondria do not have introns
  • but some plants, fungi, and protists do
137
Q

members of gene families have a common gene organization

A

e.g. mammalian genes for dihydrofolate reductase

  • the number of exons and introns is maintained
  • the relative position of the introns to the exons is maintained
  • but the length of the exons and especially the introns can vary
138
Q

exon versus intron sequences

A
  • normally, exon sequences do not vary much from species to species for the same gene
  • sequences of introns show differences from species to species for the same genes

why?

  • introns lack of selective pressure to produce a polypeptide with a useful sequence
  • while exons must produce a useful product
    (not always true for exons producing beneficial mutations)
139
Q

some DNA sequences encode more than one polypeptide

A

1. alternative start codons in the same reading frame

  • alternative initiation or termination codons allows multiple variants of a polypeptide chain
  • produces a short form and full-length form of the polypeptide

2. overlapping gene - a gene in which part of the sequence is found within part of the sequence of another gene

  • different polypeptides can be produced from the same sequence of DNA
  • the mRNA is read in different reading frames (as two overlapping genes) so sequence is different
  • found in some viral and mitochondrial genes

3. alternative splicing of the primary transcript

  • one gene may be alternative spliced to exclude exons or choose between alternative exons
  • yields otherwise identical polypeptides, differing by the presence or absence of certain regions
140
Q

some exons correspond to protein functional domains

A
  • a discrete part of an amino acid sequence that has a particular function (e.g. the immunoglobulin domain)
  • exons may be functional building blocks of genes
  • genes that share related exons may code for proteins with similar functions
  • possibly suggests a common exon ancestry
  • low-density lipoprotein (LDL) - gene has 18 exons
  • gene has similar exons as
  • complement 9
  • EGF
141
Q

gene family

A
  • a set of genes within a genome that encodes identical or related proteins of RNAs
  • the members were derived by duplication of an ancestral gene
  • followed by accumulation of changes in sequence between the copies
  • most often the members are related but not identical
142
Q

superfamily

A
  • a set of genes all related by presumed descent from a common ancestor, but now showing considerable variation
  • myoglobin = oxygen binding protein in animals
  • similar amino acid sequence to alpha globin and beta globin
  • leghemoglobins = oxygen binding protein in legume plants

together make up the Globin Superfamily

143
Q

members of a gene family have a common gene organization

A
  • similar exons
  • Leghemoglobin gene has an extra intron
  • suggests they are descended from a single ancestral gene
144
Q

orthologous genes (orthologs)

A
  • related genes in different species
  • should share common features that preceded their evolutionary separation
  • rat has two different genes for insulin
  • one similar to chicken insulin gene
  • one missing an intron
  • suggests the 1-intron gene evolved from the other
145
Q

genome

A
  • the complete set of DNA sequences in the genetic material of an organism
  • it includes the sequence of each chromosome plus any DNA in organelles
146
Q

transcriptome

A
  • the complete set of RNAs present in a cell, tissue, or organism
  • its complexity is due mostly to mRNAs, but it also includes noncoding RNAs
  • mRNAs, tRNAS, rRNAS, microRNAs, etc
147
Q

proteome

A
  • the complete set of proteins expressed by the entire genome
  • also could be the proteins expressed by a cell at any one time
148
Q

interactome

A
  • the complete set of protein complexes and protein-protein interactions present in a cell, tissue, or organism
  • multiproteins or complexes
  • DNA and RNA polymerase haloenzymes
  • enzymes clustered into metabolic pathways
149
Q

linkage maps

A
  • based on the frequency of recombination between genetic markers
  • monitoring genetic cross-over occurrence and the resulting phenotype
150
Q

restriction maps

A
  • based on the physical distances between markers
  • using restriction enzymes
151
Q

sequencing genomes

A

DNA is sequenced to identify the position of functional genes, introns, exons, etc.

152
Q

polymorphism

A

variation in sequence between individuals can be seen:

  • at the phenotypic level when a sequence affects gene function or a characteristic (variations in eye color)
  • at the restriction fragment level when it affects a restriction enzyme target site
  • at the sequence level by direct analysis of DNA
  • however, a different in gene sequence may not result in a difference in phenotype
153
Q

changes in sequence at a single locus…

A

may change the DNA sequence but:

  • NOT change the polypeptide sequence
    -redundant codons
    -changes in introns
  • change the polypeptide sequence but NOT change the polypeptide function
    -amino acids with similar characteristics
  • may change the polypeptide function
  • result in altered polypeptides that are not functional
154
Q

single nucleotide polymorphism (SNP)

A
  • a polymorphism caused by a change in a single nucleotide
  • responsible for most of the genetic variation between individuals
155
Q

haplotype

A

the particular combination of alleles in a defined region of some chromosome; in effect, the genotype is miniature

156
Q

nonrepetitive DNA

A

generally encodes for polypeptides

157
Q

repetitive DNA

A
  • sequences present at more than one copy in the haploid genome
  • larger genomes within a taxonomic group do not contain more genes but have large amounts of repetitive DNA
  • most bacteria have all nonrepetitive DNA
  • larger animals and plants have large amounts of repetitive DNA
158
Q

why have repetitive (junk) DNA?

A
  • sequences without any apparent function
  • may have functions we do not understand??
    -gene control regions (promoters)
    -code for microRNA
  • a large part of moderately repetitive DNA can be made up of transposons
    -short sequences of DNA (up to ~5000 bp)
    -have the ability to move to new locations in the genome
    -can make more copies of themselves
159
Q

which eukaryotic organelles have DNA?

A

mitochondria and chloroplasts

160
Q

extranuclear genes

A
  • genes that reside outside the nucleus, in organelles such as mitochondria and chloroplasts
  • organelle genomes are usually (but not always) circular molecules of DNA
  • mitochondrial DNA
  • chloroplast DNA (cpDNA or ctDNA)
161
Q

animal cell mtDNA

A
  • typically encodes 13 proteins, 2 rRNAs, and 22 tRNAs
  • proteins are ones involved in respiration/electron transport Complexes I to IV
162
Q

mitochondria and chloroplasts evolved by endosymbiosis

A
  • both mitochondria and chloroplasts are descended from bacteria ancestors
  • most of the mitochondrial and chloroplast genes have been transferred to the nucleus during the organelles evolution
  • mitochondria originated by an endosymbiotic event when a bacterium was captured by a eukaryotic cell
163
Q

where did introns come from?

A
  • not found in bacterial genomes
  • hypothesis = the earliest genes did not contain introns
    -introns were subsequently added to some genes
  • how? possibly they have always been an important part of the gene
    -splice sites, providing the correct reading frame, etc.
163
Q

the human genome has fewer genes than originally expected

A
  • originally estimated that the human genome contained 30-40,000 genes
  • sequencing revealed the actual number is ~20,000
  • only 1% of the human genome is exons
  • 24% are introns
  • so genes are only about 25% of the genome
  • most is repetitive DNA
164
Q

exon shuffling

A
  • the hypothesis that genes have evolved by the recombination of various exons encoding functional protein domains
    -creating proteins with greater function and value
  • most successful shufflings were once where the exons was flanked by intron sequences
    -containing 5’ and 3’ splicing sites on either side of the exon
  • exons were inserted into large introns
  • however, only 1 in 3 chance of a correct reading frame
165
Q

repeated sequences account for more than ___% of the human genome

A

50%

  • the majority of repeated sequences are copies of nonfunctional transposons
  • Pseudogenes
  • tandem repeats at the centromere and telomeres
166
Q

mouse genome has ~22,000 genes

A
  • 12% (~3000) code for RNAs
    -rRNAs, tRNAs, regulatory RNAs, etc.
  • 4.8% (1200) are pseudogenes
167
Q

pseudogene

A
  • stable but inactive genes derived by mutation of an ancestral active gene
  • often inactive due to mutations that stop transcription or translation (or both)
168
Q

what percent of proteins are essential for ALL life and what are they called

A

21%
housekeeping genes

169
Q

housekeeping genes

A
  • transcription and translation
  • metabolism
  • transport
  • DNA replication and modification
  • protein folding and degradation
170
Q

morphological complexity evolves by adding new gene functions

A
  • as morphological complexity increases, additional genes are needed with increased complexity
  • most of the genes that are unique to vertebrates/animals are concerned with the immune or nervous systems
171
Q

striking feature of the human genome

A
  • there are many more unique proteins compared to other eukaryotes
  • but relatively few unique protein domains
  • most protein domains are common to animals
  • the greatest proportion of unique proteins are the transmembrane and extracellular proteins
  • cell-cell communication secreted proteins and receptors
172
Q

gene duplication contributes to genome evolution

A
  • exons can be modules for building new genes
  • exon could be copies and used in another gene
  • provides new function
    • enzyme function
    • structural function
  • entire gene (exons + introns) is duplicated
  • duplicated genes can then allow mutations to collect and evolve
  • becomes a new gene/function
  • or becomes a pseudogene
  • as long as the original gene stays functional
173
Q

pseudogenes have lost their original functions

A
  • copies of functional genes with altered or missing regions
  • produce polypeptides that are nonfunctional or have altered functions
174
Q

gene clusters

A
  • a group of adjacent genes that are identical or related
  • may be simply two adjacent identical genes to hundreds of identical genes in a tandem array
  • e.g. Immunoglobulin genes have:
  • ~300 variable region gene segments
  • 20 D region segments
  • 6 J region segments
  • 9 heavy chain region segments
175
Q

why do some genes cluster?

A
  • tandem repeats due to a need for large amount of the product
  • rRNA for protein synthesis
  • histone proteins for replicated DNA
176
Q

heterochromatin

A
  • regions of chromosomes that are permanently tightly coiled
  • DNA is not active
177
Q

euchromatin

A
  • parts of chromosomes that are less tightly coiled
  • contain most of the active or potentially active genes
178
Q

nucleolus

A

the region in the nucleus where:

  • the rRNAs are produced
  • the rRNA and proteins are put together to form ribosomes
179
Q

parts of the nucleolus

A

Fibrillar core
- where the rRNA is transcribed from the DNA template (rDNA)

Granular cortex
- area around the fibrillar core
- where the rRNAs and proteins are actually put together to form the ribosome

180
Q

nucleolus is NOT an organelle but rather ____

A

a region where chromosomes “stick” their DNA to pre-make ribosomes

each nucleus can have many nucleoli, but usually only have one or two

181
Q

ribosome

A
  • composed of **rRNA(( and protein (ribonucleoprotein)
  • two subunits:
    1. large subunit (60S)
  • 5S rRNA
  • 28S rRNA
    2. small subunit (40S)
  • 18S rRNA
182
Q

genes for rRNA form ____ ____ of the same transcription unit

A

tandem repeats

  • rRNA is encoded by a large number of identical genes that are tandemly repeated to form one or more clusters
183
Q

ribosomal DNA (rDNA)

A
  • DNA where the ribosomal genes are located
  • the genes in an rDNA cluster all have an identical sequence
  • each rDNA cluster organization
  • transcription units for a precursor containing both the major rRNAs alternating with nontranscribed spacers
184
Q

nontranscribed spacers

A
  • shorter repeating units whose number varies so that the lengths of individual spacers are different
  • allow several RNA polymerases to attach and transcribe rRNAs at the same time
185
Q

the genome contains highly repetitive DNA

A

very short sequences of DN repeated many times in tandem and in large clusters

  • also called satellite DNA
  • no coding function
  • usually less than 10% of the genome
186
Q

satellite DNAs

A
  • DNA that consists of many tandem repeats (identical or related) of a short basic repeating unit
    • repeating unit is ~100bp or more
  • usually located in heterochromatin
  • commonly found at the centromeres of chromosomes
  • suggests it has a structural role in chromosome segregation in mitosis and meiosis
187
Q

mini and microsatellite DNA

A
  • DNAs of tandemly repeated copies of a short repeating sequence
  • minisatellite DNA - length of repeating unit is ~10 to 100 bp
  • microsatellite DNA - length of repeating unit is usually less than 10 bp
  • the number of repeats varies between individual genomes
188
Q

the length of the mini- and microsatellites:

A
  • varies quite a bit between individuals
  • very consistent for a single individual
  • therefore mini- and microsatellites lengths are unique for an individual
  • one set of sizes inherited from the mother
  • a different set of sizes inherited from father
  • we can use these unique mini- and microsatellites to identify individuals
189
Q

DNA profiling

A
  • PCR of a person’s DNA with a mixture of primers for unique sequences flanking the microsatellite DNAs
  • PCR amplify these microsatellite DNAs
  • creates a DNA fingerprint of the microsatellites for that person
  • these DNA fingerprints are unique for that person
190
Q

forensic DNA profiling

A
  • collect DNA from crime scene
  • collect DNA from suspects
  • analyze microsatellite DNA fingerprints
  • compare for matches
191
Q

DNA profiling used to establish paternity

A
  • collect DNA from mother, child, and potential fathers
  • child’s DNA fingerprint is inherited from mother and father
  • child’s DNA fingerprint will match with both

Molecular Genetics (2 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions