Exam 3 Flashcards
(330 cards)
1
Q
Genome
A
All the DNA present in a cell or virus
2
Q
Genotype
A
specific set of genes an organisms possesses
3
Q
Major targets for antibiotics
A
- cell wall
- protein synthesis
- DNA/RNA replication
- tetrahydrofolic acid biosynthesis
4
Q
Phenotype
A
collection of observable characteristics
5
Q
Fred Griffith was able to observe what phenomenon?
A
transformation
6
Q
Nucleoside
A
nitrogenous base & 5 C sugar
7
Q
Nucleotide
A
nucleoside-phosphate
8
Q
360 turn of the helix = ____ bases
A
10
9
Q
Most RNA molecules are ____ stranded
A
single stranded
10
Q
Double stranded RNA can be found in
A
viral genomes
11
Q
Small regulatory RNA (sRNA)
A
- any RNA that is not making proteins
- not translated
- functional as RNA molecules
12
Q
DNA replication is
A
semi-conservative
13
Q
DNA polymerase
A
synthesizes complementary strand of DNA
14
Q
T / F : DNA synthesis occurs only in one direction
A
T
15
Q
New bases are only added to the ___ end of a DNA molecule
A
3’
16
Q
What does DNA polymerase require?
A
- template
-primer - dNTPs
17
Q
Template
A
directs synthesis of complementary strand
18
Q
Primer
A
DNA or RNA strand
19
Q
dNTPS
A
dATP, dTTP, dCTP, dGTP
20
Q
Helicases
A
unwind DNA strands
21
Q
Single stranded binding proteins (SSB)
A
keeps strands apart for replication to occur
22
Q
DNA gyrase / topoisomerases
A
alter DNA supercoiling to relieve tension from rapid unwinding of double helix
23
Q
Ligase
A
enzyme that attaches two pieces of single stranded DNA together
24
Q
Leading strand is synthesized
A
continuously
25
Primase
synthesizes short complementary strands of RNA (~10 nucleotides) to serve as primers needed by DNA polymerase
26
Okazaki fragments
the short fragments of the lagging strand
27
DNA polymerase I
removes and replaces RNA primer with DNA
28
DNA ligase
forms a bond between 3'-hydroxyl of the growing strand and the 5'-phosphate of an Okazaki fragment
29
Exonuclease activity goes in what direction?
3' to 5'
30
Catenates
form when the two circular daughter chromosomes do not separate
31
Okazaki fragments hinder
replication of ends of chromosomes
32
Coding strand
top strand
33
Template strand
- bottom strand
- read in 3' to 5' direction
34
Promoter is located ____ of the gene
upstream
35
Leader sequence is also known as
5' untranslated region (5' UTR)
36
Shine-Dalgarno
- ribosome binding site
-located within 5' UTR
- typically 6-10 nucleotides upstream of start codon
37
N-formylmehtionine
a modified amino acid used to initiate protein synthesis in bacteria
38
Stop codons
TAG, TAA, TGA, where coding region stops/translation stop site
39
T / F : no introns/exons in bacterial genes
T
40
Monocistronic
- encodes for one gene
- one gene = one protein
- eukaryotes
41
Polycistronic
- bacteria
- directions for >1 polypeptide contained within one mRNA molecule
42
2 subunits of RNA polymerase
core enzyme and sigma factor
43
Holoenzyme
core enzyme + sigma factor
44
Transcription is carried out by
RNA polymerase
45
Promoter
site where RNA polymerase binds to initiate transcription
- site that sigma factor binds to
46
T / F : transcription termination is at stop codons
F
47
Describe how Rho-independent transcription termination works
- no proteins involved
- DNA sequence alone results in termination
- RNA forms stem loop structure
- knocks RNA polymerase off the DNA
48
Describe how Rho-dependent transcription termination works
- Rho binds to mRNA at the rut site
- moves along mRNA in direction of RNA polymerase
- RNA polymerase pauses at Rho terminator
- Rho catches up and knocks RNA polymerase off
49
Final step in expression of protein encoding genes is
translation
50
T / F : double strands are not always antiparallel
F
51
Start codon
- start site for translation
- typically AUG
52
Sense codons
61 codons that specify amino acids
53
Code degeneracy
up to six different codons can code for the same amino acid
54
Codons UAA and UAG can code for
- stop
- amino acids
55
How many naturally occurring amino acids are there?
20
56
Rare amino acids encoded by some microbes
Selenocysteine (Sec, U, UGA), Pyrrolysine (Pyl, O, UAG)
57
If GUG and UUG are first
they are start codons and encode f-Met
58
N terminus
like 5' end
59
C terminus
like 3' end
60
Polyribosome
complex of mRNA with several ribosomes attached to it
61
T / F : transcription and translation cannot be coupled in bacteria and archaea
F
62
tRNA has terminal ____ sequence
CCA
63
Charged tRNA
tRNA with amino acid attached
64
tRNA without amino acid
uncharged
65
Aminoacyl-tRNA synthetases
catalyzes attachment of amino acid to tRNA
66
rRNA
- contributes to structure of ribosome
- 16S rRNA
- 23S rRNA
67
16S rRNA
- binds to Shine Dalgarno site on mRNA for protein synthesis initiation
- binds initiation factor 3 (protein needed for translation initiation) and 3' end of charged tRNA
68
23S rRNA
ribozyme catalyzes peptide bond formation
69
What is usually removed after protein synthesis?
N-formylmethionine
70
Core enzyme
composed of 5 proteins and catalyzes RNA synthesis
71
Sigma Factor
has no catalytic activity but helps the core enzyme recognize the DNA at start of genes (promoter)
72
Transcription termination
occurs when core RNA polymerase dissociates from template DNA
- site where RNA synthesis ends
73
Translation
synthesis of polypeptide directed by sequence of nucleotides in mRNA (5' to 3')
74
Polyribosome
complex of mRNA with several ribosomes
75
Site of translation
ribosome
76
Two approaches to regulation
- regulation of gene expression
- alter activity of enzymes and proteins
77
Constitutive genes
housekeeping genes that are continuously expressed by the cell
78
Inducible genes
- genes that are typically off but can be switched on
- function in catabolic pathways
79
Repressible genes
- genes that are typically on but can be shut off
- repressible enzymes function in anabolic pathways
80
T / F: inducible enzymes are present only when their substrate is available
T
81
Inducer
effector molecule
82
Beta galactosidase hydrolyses ____ into ___ and ____
lactose, galactose and glucose
83
Beta galactosidase gene is only "on" when ____ is available
lactose
84
Typically, enzymes that function in biosynthetic pathways are products of
repressible genes
85
Induction and repression occur because of the activity of ____ containing ____
regulatory proteins, DNA binding domains
86
Regulatory proteins bind to DNA and can do one of what two things?
inhibit transcription or promote transcription
87
Repressors
inhibit transcription
88
Activators
promote transcription
89
Negative transcriptional control (overview and result)
- binding of regulatory protein (repressor) at DNA regulatory site (operator) inhibits initiation of transcription
- mRNA expression is reduced
90
_____ or _____ alter activity of repressor
inducers, corepressors
91
Positive control (overview and result)
- binding of a regulatory protein (activator) at a regulatory region on DNA (activator binding sites) promotes transcription initiation
- mRNA synthesis is increased
92
Inactive protein is activated by
inducer
93
Active protein is inactivated by
inhibitor
94
T / F: enzymes of a catabolic pathway are not only needed when the preferred substrate is available
F
95
3 structural genes coding for lactose uptake and metabolism
lacZ, lacY, lacA
96
Control of Lac operon is (+) or (-) ?
has negative and positive control
97
Expression of Lac operon is repressed by
Lacl repressor
98
____ of Lacl form and bind to three operator sites
tetramers
99
Allolactose binds ____
Lacl repressor
100
Catabolite activator protein
- regulates lac operon in response to presence or absence of glucose
101
When ___ is present, lac operon will not be activated
glucose
102
When ____ is absent, lac operon will be activated
glucose
103
Tryptophan operon consists of how many structural genes?
5
104
Trp operon only functions in the absence of ____
tryptophan
105
Attenuation
termination of transcription within the leader region
106
Trp experiences what type of regulation of transcription elongation?
attenuation
107
T/ F: transcription (attenuation) terminates after the gene is transcribed
F
108
Attenuation of the trp operon
- the trp leader sequence contains a short peptide called TrpL
- during transcription, the trp mRNA leader sequence forms secondary structure
- 2 hairpin loops form (regions 1 and 2, regions 3 and 4)
- regions 3 and 4 form transcription terminator
109
T/F: in attenuation of the trp operon, transcription and translation are coupled
T
110
TrpL has ___ Trp codons
2
111
What happens if amount of charged Trp tRNA is low?
ribosome stalls at region 1
112
Where does the anti-terminator form when there is a low amount of charged Trp tRNA?
2 and 3
113
What happens if amount of charged Trp tRNA is high?
ribosome does not stall at region 1
114
Where does the anti-terminator form when there is a high amount of charged Trp tRNA?
3 and 4
115
Riboswitches
a specialized form of transcription attenuation
116
Folding of ___ also known as _____ determines if transcription will continue/terminate
leader sequence, the riboswitch
117
Folding pattern altered in response to mRNA binding of a(n)
effector molecule
118
T / F: cis-acting regulatory elements are part of the same RNA transcript they regulate
T
119
Small RNAs (sRNAs)
- typically are complementary (antisense) to mRNA and function by base pairing
- may inhibit or enhance translation
- trans-acting RNAs
120
T/ F: sRNAs function as mRNA, tRNA, or rRNA
F
121
Why are global regulators important for bacteria?
because they must respond rapidly to wide variety of changing conditions
122
Regulon
set of genes or operons controlled by a common regulatory protein
123
Types of regulation global regulatory systems use
- regulatory proteins
- alternative sigma factors
- phosphorelay systems
- two component signal transduction systems
124
Many genes and operons are turned on or off in response to ____
environmental conditions
125
Common system used by bacteria to regulate gene expression in response to environmental conditions are _____
two-component signal transduction systems (TCSTS)
126
T / F: two component regulatory system is found in all three domains of life
T
127
Two proteins of the two component regulatory system
sensor kinase and response-regulatory protein
128
Sensor kinase (histidine kinase)
- extracellular receptor for sensing
- intracellular communication domain
129
Response-regulatory protein
- intracellular protein
- activated by sensor kinase
- DNA binding protein
^ activator, repressor
130
Activation of sensor kinase results in
phosphorylation of histidine
131
In RRP, phosphate group is transferred from ___ to ___
His (H), Asp (D)
132
EnvZ
phosphorylated in high osmolarity
133
OmpR
phosphorylated and regulates transcription
134
The OmpR/EnvZ two component signal transduction system
regulates expression of outer membrane proteins (OmpC and OmpF) depending on osmolarity
135
OmpC
makes smaller pore in membrane, lower levels of diffusion into the cell
136
OmpF
makes larger pore in membrane, allows more diffusion of solutes
137
High osmolarity leads to
autophosphorylation of EnvZ ---> --> --> OmpR activates OmpC
138
Low osmolarity leads to
no phosphorylation of EnvZ, no activation of OmpC
139
T / F: machinery involved in replicating and expressing archaeal genomes is more like that of eukaryotes than that of bacteria
T
140
DNA polymerase catalyzes synthesis of
complementary strand of DNA
141
Template
directs synthesis of complementary strand
142
Primer
DNA or RNA strand
143
dNTPs
dATP, dTTP, dCTP, dGTP
144
Eukaryotic chromosomes are ____ while bacterial are ___
linear, circular
145
Polymerase enzymes require
- template
- primer
- dNTPs
146
Bacterial chromosome is ___ Mb
2.5
147
Eukaryotic chromosomes are wound around
histone proteins
148
3 DNA polymerases responsible for eukaryotic DNA replication
- α-primase
- Pol ε
- Pol δ
149
Pol δ
appears to synthesize the lagging strand
150
Pol ε
appears to synthesize the leading strand
151
α-primase
lays down an RNA/DNA hybrid starting point
152
Chromosome ends may be digested by
DNAses
153
What are some challenges presented by linear DNA molecules?
- chromosome ends may be digested
- DNA ends can fuse with other DNA molecules
- the "end replication problem"
154
Telomerase
ribonucleoprotein complex enzyme that forms the telomeres and maintains them
155
Telomerase has an internal _______ that partially matches the _______
RNA template, G-tail sequence
156
Telomerase uses the _____ of the G tail as a DNA synthesis starting point
3' OH
157
The internal RNA sequence is used as the template for
DNA synthesis
158
The _______ activity of telomerase increases the length of the G-tail
reverse transcriptase
159
Lengthening the G-tail creates enough room to make a(n) ____ on the _____
RNA primer, lagging strand
160
T / F: archaeal chromosomes are more similar to eukaryotes than bacteria
F
161
T / F: some archaea have histones associated with their chromosomes
T
162
Archaeal chromosomes, like bacterial, are ___
circular
163
Archaeal and bacterial chromosomes are similar in ____ and ___
size, structure (circular)
164
Most archaea have how many origin of replication?
one
165
_________ in archaea are similar to those of eukaryotes
replisome proteins
166
___ family use the same family of DNS polymerases as eukaryotes
B
167
T / F: on a molecular level, transcription is very similar in all organisms
T
168
What direction is RNA produced in?
5' to 3'
169
Promoter is located at the ____
start of the gene
170
Two characteristics of a promoter
- recognition/binding site for RNA polymerase
- orients polymerase
171
The bacterial RNA polymerase is known as
holoenzyme
172
Holoenzyme is composed of
sigma factor, core enzyme
173
Sigma factor
has no catalytic activity but helps the core enzyme recognize the start of genes
174
How many polymerase do bacteria have for transcription?
1, have multiple sigma factors
175
Where does eukaryotic transcription occur?
nucleus
176
Exons
regions coding for protein that end up in the mRNA
177
Introns
code for RNA that is never translated into protein
178
T / F: most eukaryotes have singlet protein-coding genes (monocistronic transcripts)
T
179
3 major types of RNA polymerase
RNA pol II
RNA pol I
RNA pol III
180
RNA polymerase II catalyzes
mRNA synthesis
181
RNA polymerase I catalyzes
rRNA synthesis
182
RNA polymerase III catalyzes
tRNA synthesis
183
T / F: promoters do not differ between eukaryotes and bacteria
F
184
Elements that define the core promoter
- TATA box
- BRE (TFIB recognition element)
- Inr (initiator element)
- DPE (downstream promoter element)
185
T / F: eukaryotic and archaeal promoters share sequences
T
186
T / F: no sigma factor in eukaryotes or archaea
T
187
Eukaryotes and archaea use _____ instead of sigma factors
transcription factors
188
Transcription factors
bind to DNA and line the polymerase up to it correctly
189
How must initial transcripts be modified before they are ready to be translated?
- 5' cap of 7-methylguanosine added
- removal of introns/splicing together exons
- addition of 3' poly a tail
190
Spliceosome
large complex of proteins and RNA molecules unique to eukaryotes, removes introns
191
Alternative splicing
different ways of joining exons together
192
T / F: alternative splicing can be found in any domain
F, only in eukaryotes
193
What mechanism allows for a smaller number of genes to code for a greater number of products in cells?
alternative splicing
194
Similarities between archaeal transcription and eukaryotic
- RNA polymerase resembles polymerase II
- archaeal promoters and binding of the RNA polymerase
- no sigma factors
195
Similarities between archaeal transcription and bacterial
- transcription occurs in the cytoplasm
- coupling of transcription/translation
- mRNA is polycistronic
- introns are rare
196
Introns in archaea and bacteria are thought to be excised by
different methods, maybe something like tRNA splicing in eukaryotes
197
Eukaryotic translation requires more or less initiation factors for proper positioning on the mRNA?
more
198
Eukaryotic ribosome size:
Bacterial ribosome size:
80S
70S
199
Eukaryotic initiation factors bind to
the 5' cap
200
PABPs bind to
3' poly-A tail
201
The cap/tail protein complexes form a _____, activating the _____
bridge, mRNA molecule
202
Initiator tRNA (Met) interacts with ____ to form ____
40S ribosomal subunit, 43S subunit
203
43S binds to the activated ______
(bridged) mRNA
204
____ is recruited after _____ to begin elongation steps
60S, 43S unit scans the mRNA for start codon
205
T / F: elongation and termination are similar in eukaryotes and bacteria
T
206
Polysomes
observed in eukarya, more than one ribosome translating the same mRNA molecule
207
T / F: no N-formylmethionine in archaeal translation
T
208
Transcription and translation are ____ in archaea
coupled
209
Polycistronic transcripts
each coding region translated into separate protein
210
Regulation of cellular processes can take place at what 3 levels
transcriptional, translational, posttranslational
211
Activator binding sites include
upstream activating sequences, and enhancers
212
Upstream activating sequences (UASs)
near the promoter of the gene regulated
213
Enhancers
- either upstream or downstream of the promoter
- exert effects from a distance
214
Repressor binding sites
silencers
215
Histones act as a _____ in transcription
road block
216
Acetylation of histones _____ transcription
promotes
217
Methylation of histones _____ transcription
represses
218
T / F: histone acetylation is a gene regulation/control mechanism unique to eukaryotes
T
219
Examples of regulatory mechanisms for gene expression
antisense RNAs that function at the translation level, riboswitches, sRNA molecules
220
Riboswitches
- in bacteria: transcription elongation/translation
- control whether or not RNA splicing occurs
221
sRNA molecules
regulate gene expression via alternative splicing modulation
222
Other RNA based regulatory mechanisms in eukaryotes
- micro RNAs (miRNAs)
- small interfering RNAs (siRNAs)
223
siRNAs
- form complexes with proteins including argonaute (AGO)
- function at translational level
- prevent translation initiation and elongation
- can also degrade mRNAs
224
Levels of gene regulation
- transcription initiation
- splicing efficiency
- mRNA trafficking
- mRNA stability/degradation
- translational rate
- post-translational modification
225
T / F: regulation of transcription initiation in archaea has similar machinery to eukaryotes
T
226
Mutations
stable, heritable changes in sequence of bases in DNA
227
Most common kind of mutation:
point
228
Point mutations
- single nucleotide polymorphism, single nucleotide is changed (A to G)
- insertion or deletion (AAAA to AAA or AAA to AAAA)
229
consequence of mutation depends on ___ and ___
type and location
230
If a mutation occurs within a gene, it is called a(n)
coding DNA sequence, CDS, mutation
231
If a mutation is between two genes, it is called a(n)
intergenic (IG) mutation
232
An SNP CDS mutation would result in what?
a silent mutation, no amino acid change OR missense, amino acid change
233
An SNP IG mutation would result in what?
- no effect
- alter gene expression
- sRNAs
234
An Indel CDS mutation would result in what?
a frameshift, multiple amino acid changes/stop codon
235
An Indel IG mutation would result in what?
- no effect
- alter gene expression
- sRNAs
236
Consequences of mutations manifest as
phenotypic changes
237
What are consequences of a change in protein sequence?
- loss of function
- gain of function
238
Error rate of DNA polymerase
10E-9 errors per base
239
Errors per genome replication (bacteria)
5Mbp
240
Mismatch repair
- type of excision repair
- mismatch correction enzyme (MutS) scans newly synthesized DNA for mismatched pairs
- mismatched pairs removed and replaced by DNA pol and DNA ligase
- repair system uses methylation to "know" which strand is correct
241
DNA methylation
used by E. coli mismatch repair system to distinguish old DNA strands from new DNA strands
242
The E. coli mismatch repair system cuts out the mismatch from the ____ strand
unmethylated
243
Vertical gene transfer
transfer of genes from parents to progeny
244
Genetic variation in VGT is primarily due to
- fusion of male and female gametes
- crossing over between sister chromatids during meiosis
245
Source of genetic variability for eukaryotes
sexual reproduction
246
Genetic variability (in prokaryotes) primarily due to
horizontal gene transfer
247
HGT differs from VGT in what ways?
- transfer of genes from one independent, mature organism to another
- genes can be transferred to the same or different species
- if transfer is stable, recipient acquires function of transferred gene
248
What does it mean for DNA to be stable?
self replicating, able to be integrated onto chromosome
249
Integrating into the host genome occurs by
recombination
250
2 major types of recombination
homologous recombination, site specific recombination
251
What is the most common type of recombination?
homologous
252
Homologous recombination
- occurs between two long sequences of DNA with same/similar sequence
- double-strand break occurs
- adjacent molecules are rejoined to each other
- involves the RecA protein
253
Site-specific recombination is important in
insertion of viral genome into host chromosomes and transposons
254
3 important differences between site-specific recombination and homologous
- does not require long regions of DNA homology
- recombination occurs at specific target sites in DNA molecules
- mediated by specific enzymes called recombinases
255
2 major types of transmissible DNA
- transposable elements
- plasmids
256
3 mechanisms for HGT
- conjugation
- transformation
- transduction
257
Transposition
process by which small segments of DNA move about the genome
258
Insertion sequences
simplest transposable elements
259
Composite transposons (Tn)
transposable elements which contain "extra" genes, flanked by two IS
260
Unit transposons are between ___ and ___
IS, composite Tn
261
Unit transposons
like expanded IS but can contain "extra" genes
262
Inverted repeats
- short stretch of identical nucleotides in opposite orientation
- marks the ends of the IS/Tn
263
Transposase
enzyme responsible for site specific transposition
264
Direct repeats
- host DNA that is the site for insertion
- duplicated during insertion
265
2 mechanisms of action for transposition
1. simple transposition (cut and paste)
2. replicative transposition (copy and paste)
266
Simple transposition
- cut and paste
- transposase catalyzes excision of TE
- TE is bound by transposase and migrates to new insertion site
- new target site is cleaved and TE ligated into site
- target site is replicated in the process (DR)
267
Replicative transposition
- original transposon remains at parental site in DNA
- copy is inserted in target DNA
- retrotransposons
268
Plasmids
- small, extrachromosomal circular DNA molecules
- exist and replicate independently of chromosome
- contain non essential genes
269
Episomes
can integrate reversibly into the host chromosome
270
Conjugative plasmids
can transfer copies of themselves to other bacteria during conjugation
271
Retrotransposons
- RNA copy of TE is made
- reverse transcribed into DNA
- integrates at new site
272
Plasmid is replicated by
rolling circle method
273
F factor codes for
sex pilus and all machinery needed for plasmid transfer
274
F plasmid is an
episome
275
Integration of F plasmid occurs at
insertion sequences
276
When F factor is not integrated into chromosome, cell is called ____
F+
277
When F factor is integrated into chromosome, cell is called ____
HFr
278
T / F: conjugation cannot occur between HFr and F- strains
F
279
F' plasmid forms when
the F factor incorrectly excises from the host chromosome
280
T / F: in F' conjugation, some of the F factor is left behind in the host chromosome and/or some host genes have been removed along with some of the F factor
T
281
Transformation
uptake of extracellular DNA by a bacterial cell
282
Competent cells
bacterial cells that have the ability to take up DNA
283
Competent cells are ____ when they take up DNA
transformed
284
DNA being transformed may be ___ or ___
circular (plasmid) or linear (fragment of genomic DNA)
285
For linear DNA to be stably transformed, it must
integrate into the chromosome
286
Transduction
the transfer of bacterial genes by viruses
287
Lytic
host cell is destroyed
288
Lysogenic
viral DNA integrates into the host genome (becoming prophage)
289
Two potential cycles of transduction
lytic and lysogenic
290
Phage mediated transduction
- host genes transferred by bacteriophage
- typically an "accidental" process
- results from errors in phage life cycle
291
2 types of phage mediated transduction
- generalized transduction
- specialized transduction
292
Generalized transduction
- occurs because of errors in lytic cycle
- any part of bacterial genome can be transferred
- during viral assembly, fragments of host DNA mistakenly packaged into phage head
293
Specialized transduction
- occurs because of errors in lysogenic cycle
- occurs when prophage is incorrectly excised
- new virions contain part of the viral genome plus host DNA surrounding viral integration site
- host DNA is introduced into recipient cell
- can recombine into genome or integrate with partial phage genome
- host DNA transferred is that surrounding viral integration site
294
Generalized transducing particles
- bacterial DNA wrapped in viral proteins
- no viral DNA
- can be easily transferred to another bacterial cell
295
Phage lambda specialized transduction
recipient becomes gal+
296
Mechanisms of drug resistance
- modification of target enzyme or organelle
- inactivation of drug
- efflux pumps
- use of alternative pathways or proteins
297
How does antibiotic resistance occur?
- mutations (arise spontaneously and are then selected for)
- horizontal gene transfer of resistance genes
298
Resistance genes can be found on
- bacterial chromosomes
- plasmids
- transposons
299
Genetic engineering
deliberate modification of organism's genetic information by directly changing the sequence of nucleic acids in its genome
300
Recombinant DNA
artificially created DNA sequences, results from combining 2 strands of DNA together
301
Cloning
generating a large number of genetically identical DNA molecules
302
Biotechnology
use of biological organisms to form useful products
303
Industrial microbiology
use of microbes to manufacture important compounds
304
What are the 2 main reasons you might want to express a foreign gene in a host cell?
- to determine its function
- to purify the protein
305
Recombinant DNA technologies
- restriction enzymes
- genetic cloning
- PCR
-DNA sequencing
306
Restriction enzymes (RE)
- recognize and bind specific sequences in DNA called recognition sites
307
Type II RE cleave
DNA at/around the site
308
Type I and III cleave
a defined distance from this site
309
Cleavage with restriction enzymes may produce
blunt or sticky ends
310
Palindromes
same going forward as it is going backwards
311
Vectors
carriers of foreign DNA
312
4 types of cloning vectors
- phages and viruses
- cosmids
- artificial chromosomes
- plasmids (most commonly used)
313
Cloning a gene
- foreign DNA combined with/inserted into a cloning vector
- newly created plasmid is a recombinant DNA molecule
- inside a new host cell plasmid vector replicates
- maintains foreign DNA fragment (gene)
314
Characteristics of good cloning vectors
- replicate autonomously
- easy to purify
315
Requirements for vectors
- origin of replication
- selectable marker (ex: antibiotic resistance gene)
316
Multiple cloning site (MCS)
- site where cloned gene to be inserted into the plasmid vector
- contains many unique RE sites
- may contain selection gene
317
PCR
technique that enables DNA amplification
318
Specificity of PCR arises from
DNA primers
319
Oligonucleotides
short DNA molecules that flank the DNA sequence being amplified, serve as primers for DNA polymerase
320
PCR cycle
- DNA is denatured
- primers anneal to target DNA
- target DNA is synthesized (amplified)
- repeat 34x
321
PCR reaction mix contains:
- primers
- template DNA (target)
- thermostable DNA polymerase (taq)
- dNTPs (dATP, dCTP, dTTP, dGTP)
322
Number of copies of DNA produced in PCR reaction =
2^n, where n = how many cycles were done
323
Uses of PCR
- simplifies gene cloning
- generates DNA fragments
- may amplify environmental genes without culturing the microbes
- diagnostic purposes
324
Limitations of PCR
- mutations due to Taq polymerase
- cannot have a completely unknown sample
325
Most common method for determining DNA sequences
Sanger DNA sequencing
326
How does Sanger sequencing work?
referred to as the chain-termination DNA sequencing method, uses dideoxynucleoside triphosphates (ddNTP), strand synthesis terminates when ddNTP is incorporated
327
Sanger sequencing original method
newly synthesized DNA strands were labeled with radioactive atom, electrophoresed and detected with X ray film
328
Sanger sequencing, modern method
automated sequencing, uses four different fluorescent color dyes instead of radio-labeled ddNTP, electrophoresis and laser beam determines order
329
NGS (next generation DNA sequencing)
sequences millions of DNA strands simultaneously
330
Sanger vs NGS (bp comparison)
Sanger: 1,000 bp/reaction
NGS: 10-100 billion bp/reaction
