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BIOM2202 > Gene Regulation > Flashcards

Gene Regulation Flashcards

1
Q

Promotors

A

Regulate gene expression through interaction with

proteins

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2
Q

Promotors are held to DNA via

A

Hydogen bonds

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3
Q

Sigma factors facilitate promoter activation:

A

Sigma factors bind RNA polymerase to coordinate

major gene expression programs

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4
Q

Sigma factor binds at…

A

RNA polymerase: -35 pos.

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5
Q

Strand seperation occurs at:

A

-10 pos. A=T rich

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6
Q

The lac promotor is weak under normal conditions. T/F

A

True

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7
Q

Sigma promotes…

A

Initiation of transcription

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8
Q

2a1b1b’1w –>

A

Core polymerase

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9
Q

2a1b1b’1w+sigma –>

A

Holoenzyme

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10
Q

Sigma is released at translation. T/F

A

False. Sigma is released at transcription

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11
Q

s(D):

A

“housekeeping” genes

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12
Q

s(S):

A

stationary phase/starvation

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13
Q

s(H):

A

Heat shock

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14
Q

s(E):

A

Extreme heat shock

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15
Q

s(I):

A

Iron deficiency

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16
Q

s(F):

A

Flagellar genes

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17
Q

s(N):

A

Nitrogen Deficiency

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18
Q

Under good conditions, s(D) binds to…

A

RNA polymerase

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19
Q

Under harsh conditions, ___ binds to RNA polymerase

A

s(S)

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20
Q

Housekeeping Genes:

A
  • RNA molecules
  • rRNA molecules
  • Ribosomal proteins
  • RNA polymerase subunits
  • Enzymes catalysing metabolic processes
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21
Q

Housekeeping genes are continually being expressed. T/F

A

True

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22
Q

Housekeeping genes are not constituative. T/F

A

False. Housekeeping genes are constitutive genes

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23
Q

Alarmone is coded by:

A

ppGpp

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24
Q

ppGpp:

A

-Binds to RNA polymerase
- Reduces affinity of RNApol for Housekeeping genes.
- Stationary sigma factors and other stress sigmas can now
compete for binding

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25
Q

pppGpp synthetase is composed of:

A

RelA & SpoT

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26
Q

RelA + uncharged tRNA are involved in:

A

amino acid starvation

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27
Q

SpoT - glucose are consistent with:

A

Glucose starvation

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28
Q

SpoT + glucose are consistent with:

A

Glucose abundance

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29
Q

SpoT enzyme activity reverses depending on availability

of glucose. T/F

A

True

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30
Q

Sigma factors are regulated by:

A
  • Regulation of gene transcription
  • Regulation of mRNA stability
  • Inhibition of translation
  • Inactivation by proteolysis
  • Activation by removing inhibitory N-terminal
    amino acid extensions
  • Inhibition by high affinity binding of anti sigma
    factors
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31
Q

anti-sigma factors are regulated by…

A

phosphorylation &

anti anti-sigma factors

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32
Q

During starvation (amino acid or glucose)…

A

Alarmone is produced via RelA or SpoT

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33
Q

During cellular starvation…

A
  1. s(D) released from core RNA polymerase
  2. s(S) binds to core polymerase to create starvation response
    holoenzyme
  3. RNA polymerase will bind to -35 & -10 regions of lac promoter
    » Primed for de-repression by allolactose
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34
Q

The lac operon works via…. repression

A

Catabolite repression

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35
Q

If lactose is available:

A
  1. Allolactose (modified form of lactose) signals lactose
    availability
  2. Allolactose binds to lac operon repressor

–> Prevents inhibition of lac operon transcription

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36
Q

If glucose is not available:

A
  1. Cyclic AMP (cAMP) is a specific glucose starvation
    signal
  2. cAMP binds to CRP (cyclic AMP receptor protein)

–> The lac -35 sequence does not match consensus

  1. CRP + s(S) promotes strong binding of holoenzyme
    – Facilitates transcription
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37
Q

During RNA processing, RNA is…

A

Capped at the 5’ end and polyadenalated at the 3’ end

- Loses introns

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38
Q

Prokaryotic RNA is capped at the 5’ end and polyadenalated at the 3’ end and has its introns spliced out. T/F

A

False. Eukaryotic only

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39
Q

Transcription occurs in prokaryotes when…

A
  • The negative regulatory molecules such as the lac repressor protein have been removed from the vicinity of a gene and;
  • Positive regulatory molecules such as the catabolite activator protein (CAP)/cyclic AMP complex have bound to it
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40
Q

Transcription factors:

A

Positive and negative regulator proteins bind to specific regions of the DNA and stimulate or inhibit transcription

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41
Q

The formation of the mRNA is mediated by tiny nuclear organelles called _____.

A

spliceosomes

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42
Q

An mRNA that is rapidly degraded….

A

Must be replenished by additional transcription; otherwise, the polypeptide it encodes will cease to be synthesized

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43
Q

Lactose is a(n)….. for gene transcription

A

Lactose is an inducer for gene transcription

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44
Q

Heat shock proteins are found in…

A

Prokaryotes and eukaryotes

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45
Q

The most conserved polypeptides are the…

A

Heat-shock proteins (HSP70(kd))

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46
Q

Steroid hormones are derived from:

A

Cholesterol

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47
Q

Insulin is a…

A

Peptide hormone

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48
Q

Enhancers exhibit three fairly general properties:

A

(1) they act over relatively large distances—up to several thousand base pairs from their regulated gene(s);
(2) their influence on gene expression is independent of orientation—they function equally well in either the normal or inverted orientation within the DNA; and
(3) their effects are independent of position—> they can be located upstream, downstream, or within an intron of a gene and still have profound effects on the gene’s expression

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49
Q

RNAi:

A

RNA interferase

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50
Q

In the cytoplasm, siRNAs and miRNAs become incorporated into ribonucleoprotein particles, T/F

A

True

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51
Q

Where does ppGpp come from?

A
  • It is a modified GDP nucleotide. An extra
    pyrophosphate is added to the 3’ position by the
    enzyme RelA or SpoT.
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52
Q

What does ppGpp do?

A

– It reduces the affinity of RNApol for s(D) so that
(S) or other sigma factors can bind to RNApol.

This down-regulates transcription of housekeeping genes and ­
up-regulates transcription of stress (other) response genes

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53
Q

Heat stress –>

A

Phosphorylation of HSTF
– Induction of response genes in 30 seconds
– Suppression of housekeeping genes in 300 seconds
– Regulated cell-by-cell

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54
Q

Induced proteins, e.g. hsp70…

A

facilitate restoration and reuse of proteins, stabilisation of membranes and changes to gene expression

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55
Q

Eukaryotic mRNAs may be either monogenic or multigenic. T/F

A

True

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56
Q

Prior to translation, eukaryotic DNA undergoes 3 important processes:

A
  1. 7-Methyl guanosine caps are added to the 5′ ends of the primary transcripts.
  2. Poly(A) tails are added to the 3′ ends of the transcripts
    –> generated by
    cleavage rather than by termination of chain extension.
  3. When present, intron sequences are spliced out of transcripts
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57
Q

The population of primary transcripts in a nucleus is called….

A

Heterogeneous nuclear RNA (hnRNA) because of the large variation in the sizes of the RNA molecules present

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58
Q

Most hnRNA are introns and therefore spliced out. T/F

A

True

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59
Q

RNA polymerase I is located…

A

in a distinct region of the nucleolus where rRNAs are synthesized and combined with ribosomal proteins.

RNA polymerase I catalyzes the synthesis of all ribosomal RNAs except the small 5S rRNA.

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60
Q

RNA polymerase II transcribes…

A

Nuclear genes that encode proteins and perhaps other genes specifying hnRNAs.

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61
Q

RNA polymerase III catalyzes…

A

The synthesis of the transfer RNA molecules, the 5S rRNA molecules, and small nuclear RNAs.

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62
Q

siRNAs interact with various proteins to…

A

Modify (condense or extend) chromosome structue

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63
Q

RNA polymerases can initiate transcription by themselves. T/F

A

False

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64
Q

RNA editing may occur via:

A
  1. By changing the structures of individual bases and;

2. By inserting or deleting uridine monophosphate residues.

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65
Q

Five snRNAs, called _____ are involved in nuclear pre- mRNA splicing as components of the spliceosome

A

U1, U2, U4, U5, and U6,

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66
Q

Enhancers/distal elements:

A
  • Sites of binding for special transcription factors
  • Spread across 1,000 - 10,000 bp (or more) of DNA
  • Upstream of promoter and proximal control elements
  • Sometimes in introns
  • Rarely downstream of transcribed region of gene
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67
Q

Basal transcription factor assembly process:

A
  • TFIID binds to TATA box & bends DNA sharply
  • Other basal factors assemble
  • RNA Polymerase II holoenzyme binds to TFIID
  • Bent DNA promotes
    ‘melting in’ of
    RNA polymerase II
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68
Q

If the gene has only promoter plus proximal elements,

transcription initiation will be:

A

infrequent

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69
Q

Mediator complex (~30 subunits)

A
  • Bends DNA of regulatory region
  • Brings distal elements to the promoter
  • Allows special transcription factors to facilitate RNA
    Polymerase II assembly at promoter
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70
Q

Special Transcription Factors are always positive. T/F

A

False

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71
Q

The DNA molecules in prokaryotic and viral chromosomes are organized into negatively supercoiled domains. T/F

A

True

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72
Q

Bacterial chromosomes contain circular molecules of DNA organized into…

A

about 50 domains.

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73
Q

Histones are ____ charged

A

Histones are positively charged

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74
Q

Non-chromosmal histone proteins are ______

A

Negatively charged

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75
Q

Genetics:

A
  • Heritable information encoded in
    the nucleotide sequence
    of DNA
  • Evolutionary experiences
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76
Q

Epigenetics:

A
  • semi-heritable information encoded as molecular
    tags on the DNA and associated proteins
    – Life experiences
    – Developmental programs
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77
Q

Nucleosomes consist of how many histone proteins?

A

8

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78
Q

Both nucleosome construction and

compaction are readily reversed. T/F

A

True

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79
Q

Histones can be removed in the inactive state only. T/F`

A

False. Histones can only be removed in active state

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80
Q

Nucleosomes must be

accessible –>

A
  • H1 released by phosphorylation

- Nucleosomes toggle between active and inactive states

81
Q

H3 is modified by…

A

acetylation and methylation

82
Q

K9 acetylation =

A

active

83
Q

K9 methylation –>

A

inactive

84
Q

K4 methylation enhances…

A

active state

85
Q

Reversible acetylation:

A
  • Histone acetyl transferase (HAT) (adds acetyl group)

- Histone deacetylase (HDAC) (removes acetyl group)

86
Q

Reversible methylation:

A
  • Histone methyl transferase (HMT) (adds methyl group)

- Histone demethylase (HDM) (removes methyl group)

87
Q

H3K9 acetylation:

A

gene (potentially) active

88
Q

H3K9 methylation:

A

gene inactive

89
Q

H3K9 methylation leads to DNA

methylation T/F

A

True

90
Q

Heterochromatin:

A
  • Centromeric and telomeric DNA
  • Highly methylated
  • Highly condensed (dark staining, i.e. heterochromatic)
91
Q

Dispersed repeats:

A
  • Viruses and transposons
  • Dispersed throughout the genome
  • Inactivated by methylation
  • Become reactivated when host under stress
92
Q

Methylation of transposons is maintained…

A

– In somatic cells
– In gametes
– Across generations

93
Q

4 Types of methylation:

A
  1. Dynamic gene regulation
    - Erased in early embryogenesis, re-established
    during lifetime
  2. Imprinting
    - Erased and re-established early in gamete formation
  3. Suppression of transposons (by piRNA signalling)
    - Maintained indefinitely
    - but erased under stress
  4. X chromosome inactivation
    - Cell-by-cell decision in early female embryo
    - X from male inactivated in placenta (in mice)
94
Q

DNA is reactivated via localised removal of

histone proteins. T/F

A

True

95
Q

snRNAs:

A

Small nuclear RNAs that excise introns from sequence

96
Q

Micro RNAs (miRNAs)….

A

Block the expression of complementary or partially com- plementary mRNAs by either;

  1. Causing their degradation
  2. Repressing their translation.
97
Q

All 5 types are xRNAs are produced by…

A

Transcription

98
Q

All 5 types of xRNA molecules are translated alongside the DNA. T/F

A

False

99
Q

The central dogma of molecular biology is:

A
  • Genetic information flows from DNA to DNA

- From DNA to RNA during transcription, and from RNA to protein during translation.

100
Q

Transcription involves the:

A

Synthesis of an RNA transcript complementary to one strand of DNA of a gene.

101
Q

Translation is the:

A

Conversion of information stored in the sequence of nucleotides in the RNA transcript into the sequence of amino acids in the polypeptide gene product, according to the specifications of the genetic code.

102
Q

RNAi is used extensively to…

A
  • Silence genes or to turn down or turn off their expression in C. elegans, D. melanogaster, and many plants
103
Q

Knockout mutations of genes in the mouse can be produced by…

A

Inserting foreign DNAs into chromosomal genes by homologous recombination.

104
Q

T-DNA or transposon insertions provide:

A

A source of null mutations of genes.

105
Q

RNA interference:

A
  • Blocking gene expression with double-stranded RNA

- Can be used to dissect biological processes by inhibiting the functions of specific genes

106
Q

Short interfering RNAs and microRNAs are:

A
  • Produced from larger double-stranded precursors by the action of Dicer-type endonucleases.
107
Q

In RNA-Induced Silencing Complexes (RISCs), siRNAs and miRNAs become:

A
  1. Single stranded so they can target complementary sequences in messenger RNA molecules.
  2. Messenger RNA that has been targeted by siRNA is cleaved,
  3. mRNA that has been targeted by miRNA is prevented from serving as a template for polypeptide synthesis.
108
Q

Hundreds of genes for miRNAs are present in eukaryotic genomes. T/F

A

True

109
Q

Transposons and transgenes may stimulate the synthesis of siRNAs. T/F

A

True

110
Q

RNA interference is used as a research tool to…

A

Knock out or knock down the expression of genes in cells and whole organisms

111
Q

Transgene expression is
sometimes suppressed together with
endogenous gene copies:

A
  • Suppression can spread to grafted, nontransgenic tissue
112
Q

Transgene expression is:

A

RNA mediated

113
Q

Transgene =

A

Gene integrated into the genome in a genetic
engineering experiment.

A transformed gene

114
Q

The suppressed plant transgenes were

methylated such that:

A

Transcription was inhibited
OR
Transcripts were degraded

115
Q

siRNA (also called RNAi)

A

Leads to transcript degradation

116
Q

miRNA is a natural cellular transcript that acts by:

A

Blocking translation

117
Q

RNA signals can travel between cells. T/F

A

True

118
Q

RNA signals to the germ line. T/F

A

True

119
Q

What is the remaining untranslated RNA?

A
  • mRNA
  • 5’ UTR,
  • 3’ UTR &
  • introns
120
Q

Which of the following is untrue regarding histone 3?

A

It is highly divergent across species.

121
Q

Small RNA molecules that function as adaptors between amino acids and the codons in mRNA during translation are known as:

A

tRNA

122
Q

Short ~21 nucleotide single-stranded RNAs that are cleaved from small hairpin-shaped precursors and block the expression of complementary or partially complementary mRNAs by either causing their degradation or repressing their translation are known as:

A

miRNA

123
Q

RNA polymerase binds to a specific nucleotide sequence near the start of transcription known as a/an:

A

Promotor

124
Q

What is true regarding E.coli?

A

-10 and -35 regions show the most conservation between promoters

125
Q

The sigma subunit initially recognizes and binds to which of the following sequences?

A

-35

126
Q

Sigma factors:

A

Compete to interact with RNA polymerase

127
Q

SpoT:

A
  • Is a starvation signal.
  • It is a nucleotide.
  • It is found E. coli.
128
Q

What is untrue regarding SpoT?

A

It can be degraded by RelA.

relA makes magic spot , spoT can degrade magic spot ppGpp

129
Q

Genes that are continually being expressed in most cells are referred to as:

A

Constituative genes

130
Q

Genes whose expression is turned on in response to a substance in the environment are known as:

A

Inducible genes

131
Q

Enzymes that are involved in anabolic pathways are typically encoded by:

A

Repressible genes

132
Q

In prokaryotes, genes with related functions often are present in coordinately regulated genetic units called what?

A

Operons

133
Q

What happens when a repressor is bound to the operator of a gene?

A

RNA polymerase is prevented from transcribing the structural genes in the operon

134
Q

Glucose is the preferred energy source for E. coli and represses the utilization of other sugars. T/F

A

True

135
Q

The CAP protein is involved in catabolite repression of the lac operon. T/F

A

True

136
Q

RNA polymerase is the effector molecule that enables CAP to bind to the promoter. T/F

A

False

137
Q

The CAP protein and its effector molecule exert positive control over the transcription of the lac operon. T/F

A

True

138
Q

High glucose concentrations decrease the intracellular concentration of the effector molecule of the CAP protein. T/F

A

True

139
Q

Operons:

A

Improve the efficiency of gene regulation in prokaryotes

140
Q

If gene expression regulation occurs during transcription, where in a eukaryotic cell does this regulation take place?

A

Nucleus

141
Q

Positive and negative regulator proteins that bind to specific regions of the DNA and stimulate or inhibit transcription in eukaryotes are known as:

A

Transcription factors

142
Q

Steroid hormones:

A
  • They are small, lipid-soluble molecules derived from cholesterol
  • Testosterone and estrogen are examples
  • They have little or no trouble passing through cell membranes
  • They interact with cytoplasmic or nuclear proteins called hormone receptors
143
Q

Insulin is a steroid hormone. T/F

A

False

144
Q

The more HREs present, the more vigorous transcription

A

The more HREs present, the more vigorous transcription

145
Q

Which of the following is true regarding eukaryotic special transcription factors?

A

Most transcription factors have dimerization domains

146
Q

Enhancers are:

A

Short DNA sequences to which special transcription factors bind

147
Q

Which of the following is true of enhancer sequences?

A

They can act over very large distances

148
Q

A type of post-transcriptional gene regulation in which small RNA molecules interfere with gene expression is known as:

A

RNA intererence

149
Q

Which of the following molecules participates in RNA interference?

A

siRNA

150
Q

Which type of enzyme produces siRNA molecules in eukaryotes?

A

Dicer enzymes

151
Q

The protein complex that contains a small guide RNA molecule and degrades mRNA is known as:

A

RNA Induced Silencing Complex

152
Q

RISC-associated RNAs that result in mRNA cleavage are usually termed:

A

siRNAs

153
Q

When the RNA within the RISC pairs imperfectly with its target sequence, the mRNA is usually not cleaved; instead, translation of the mRNA is inhibited. RISC- associated RNAs that most frequently have this effect are…

A

miRNA

154
Q

Puffs in polytene chromosomes are associated with:

A

Transcription

155
Q

In transcribed DNA, the nucleosomes are altered to facilitate the action of the RNA polymerase in a process known as:

A

Chromatin remodelling

156
Q

A mixture of normal and mutant characteristics in the same individual due to abnormal functioning of euchromatic genes that are artificially transposed to a heterochromatic environment, is called:

A

Position-effect variegation

157
Q

Increased gene expression may be achieved by which of the following?

A

Amplifying a gene

158
Q

All of the genes on an inactivated X-chromosome are transcriptionally silent. T/F

A

False

159
Q

X-chromosome inactivation begins at the X-inactivation center

Inactive X-chromosomes can be easily identified in mammalian cells

The Barr body represents an inactive X-chromosome.

Inactive X-chromosomes have a different pattern of distribution of acetylated histone.

All of these statements are:

A

True

160
Q

Which of the following is not a mechanism of dosage compensation?

A

Dose moderation

161
Q

Uncharged tRNA molecules:

A

Can initiate the stringent response

162
Q

Basal transcription factors…

A

Promote assembly of RNA polymerase subcomplexes

163
Q

Special transcription factors can:

A

Influence flower development

164
Q

Epigenetic modification of gene expression can:

A

Can extend the range of a genetically encoded phenotype

165
Q

The signaling role of small RNA molecules was not recognized for many years because

A

Small RNAs were to be considered degraded mRNA

166
Q

In multicellular Eukaryotes:

A

Transposon activity is epigenetically suppressed

Most DNA methylation is removed in progenitor germ cells

167
Q

In eukaryotes DNA methylation occurs on:

A

CG or CNG sequences

168
Q

Eukaryotic gene regulation does not integrate signals from the

A

DNA strand separation at the TATA box

169
Q

Eukaryotic promoters lack the equivalent of the strong lac operon repressor for all but which of the following reasons:

A

There is no mechanism for high affinity binding of proteins to DNA in eukaryotes

170
Q

RNA dependent RNA Polymerase:

A

Amplifies the siRNA signal

171
Q

Imprinted genes provide an example of:

A

Incomplete penetrance

172
Q

Most DNA methylation in somatic cells

A

Is associated with heterochromatin

173
Q

Environmentally induced DNA methylation is not associated with:

A

Frequent transmission through meiosis

174
Q

Small RNA molecules are effective regulators of gene expression for all of the following reasons except:

A

They form active dimers which destroy mRNA

175
Q

List the components of a cell-free protein synthesising system, and explain the experimental set-up when such a system was used to deduce the genetic code using poly(C) as synthetic RNA.

A

Requirements: amino acids, tRNA, ribosomes, ATP, GTP and mRNA template
Process:
Grind E.coli cells with powdered alumina to give cell lysate
Centrifuge to pellet the cell wall
Collect cell lysate (includes tRNA, ribosomes, enzymes & cofactors)
Treat with DNase to stop de novo RNA synthesis
Existing cellular RNA will degrade (RNA is relatively unstable)
Add synthetic RNA template + one 14C labelled amino acid

176
Q

Why is the genetic code said to be “standard” but not “universal”? Illustrate your answer with a comment on the genetic code in Candida albicans.

A

The genetic code is standard because in all species, DNA is made of nucleotides, which form codons to make proteins. Moreover, the same codon will encode the same amino acid in almost every organism. However, there are exceptions like in Candida Albicans Ser encodes for Leucine (CUG –> CTG)

177
Q

List the components of a cell-free protein synthesising system, and explain the experimental set-up required to use such a system to deduce the amino acid encoded by the codon CUC.

A

Components required: tRNA, Ribosomes, DNase, ATP, GTP, Amino acids, Radioactive Amino Acid which is being tested for, Various synthetic RNAs with known sequences (eg. poly(UC))

Experiment: Lyse E. coli, then add DNase to destroy DNA and unstable RNA, leaving Ribosomes and tRNA, then add ATP+GTP+AAs+Radioactive AA, with addition of synthetic RNA a polypeptide will be formed, measuring the radioactivity of the formed polypeptide will allow for the AA encoded by CUC to be deduced after performing this test with multiple different synthetic RNA templates.

178
Q

With the aid of a diagram, explain the process of nucleotide excision repair in E. coli when applied to the removal of a thymine dimer.

A
  1. Nucleotide-excision repair is catalyzed by three proteins (uvrA, B, and C)
  2. 2x UvrA and 1x UvrB recognizes damaged DNA and binds to the site of the lesion.
  3. Using ATP, this complex bends DNA, causes a conformational change in UvrB, and releases the UvrA dimer.
  4. UvrC binds. UvrB and UvrC then cleave on the 3′ and 5′ sides of the damaged site, respectively.
  5. The oligonucleotide is then released using UvrD helicase. The gap is filled by DNA Pol 1 (using the other strand as template) and is sealed by ligase.
179
Q

A base substitution in an enzyme-encoding ORF changes a codon from CTG to CAG, but has no effect on the function of the enzyme subsequently produced. Give two terms that could be used to describe this mutation, and the definition of these terms, and why they are applicable in this case.

A

Non synonomous, neutral mutation

180
Q

You culture a Saccharomyces cerevisiae frameshift mutant that is a lysine auxotroph, plate it on minimal media and identify a single prototrophic colony that has acquired a suppressor mutation. What is the most likely type of suppressor mutation that would have occurred, and why?

A

Intragenic frameshift - The first mutation resulted in an altered ORF.

This could have been due to 1 or 2 nucleotides being added to or deleted from the sequence.

If there is an addition or deletion of 1 or 2 nucleotides elsewhere on the gene, it may restore the original ORF, hence an intragenic suppressor mutation must have occurred

181
Q

What is an amber mutation?

A

When a Tyr codon mutates to recognise a STOP (TAG) codon Tyr → STOP

182
Q

How are antibody genes assembled?

A

Antibodies are made up of two ‘light’ polypeptide chains and two ‘heavy’ chains.

The light chain is encoded via somatic recombination between three different gene segments.

The LKVK segment encodes the leading polypeptide and a variable region of the light chain, which changes for each different type of antibody.

The JK segment encodes another variable region which ‘joins’ to the LKVK segment. Finally, the CK segment encodes the ‘constant’ section of the light chain which is the same in all antibodies.

When these three segments undergo recombination, an antibody is formed.

183
Q

What must happen for an antibody to form?

A

The LKVK, JK, and the CK segment must undergo recombination

184
Q

Describe how studies using the Drosophila lozenge locus revealed that recombination could occur within a gene:

A

Oliver took two independent mutants, one with glassy eye and the other with spectacle eyes and crossed them to create WT recombinant at a low rate.

  • Oliver has rejected the idea of reversions, since reversion frequency is much less than 0.2%. Also, the markers that he put around the lozenge gene were recombinant as well.

Clarence P. Oliver’s 1940 experiment:
He had two Drosophila mutants, both mutations being in the lozenge locus: a glassy eye (lzg) and a spectacle eye (lzs). When lzg/lzs females were crossed with lzg or lzs males, 0.2% were WT.

185
Q

The F factor plasmid forms Hfr’s in Salmonella typhimurium at many fewer sites and at a much lower frequency than in Escherichia coli. Based upon the way Hfr’s are formed, what would be a simple explanation for this difference between S. typhimurium and E. coli?

A

Recall that most Hfr’s arise via homologous recombination between IS sequences (IS2, IS3, and Tn1000) that are present on both the F-plasmid and on the E. coli chromosome. The S. typhimurium chromosome lacks copies of these IS elements, so any Hfr’s that arise must do so via transposition.

186
Q

The proA, proB, and proC genes are required for the biosynthesis of proline. Given a donor strain with a Hfr integrated between the proA+ proB+ and proC+ genes as shown below, and a proC- StrR recipient, how could you isolate a F’ proC+?

A

Because the proC gene would be the very last region to be transferred by the Hfr, it would only be transferred by the Hfr very rarely. Therefore, you could select for such rare events by mating into a proC auxotroph and selecting for growth on minimal medium without proline

187
Q

A new mutant was isolated that is StrR and unable to use acetate as a carbon source (ace). To determine where the mutation maps, it was mated with the four different StrS ace+ Hfr donor strains shown below. Arrowheads indicate the location and direction of transfer from each different Hfr.

a) What is the selection for exconjugants in this experiment?
b) What is the counterselection against the donor cells in this experiment?
c) Given the results in the following table, where does the ace mutation map? Indicate the map position in minutes.

A

a) Growth on acetate as a sole C-source.
b) Streptomycin resistance.
c) About 95 min, in the region transferred early between Hfr-1 and Hfr-3.

188
Q

How would you isolate an F’ Met+ from this Hfr? The Hfr strain can carry any genetic markers you choose, but be sure to indicate what they are.

A

Mate the Hfr with an F- met- recA StrR recipient, selecting Met+ StrR. The recA mutation in the recipient will prevent inheritance of Met+ directly via the Hfr, and the StrR provides a counter-selection against the donor.

189
Q

The F-plasmid is about 100 kb. Because P22 HT only packages about 45 kb of DNA, it cannot package the entire F plasmid. However, when a Tetracycline sensitive (TetS) recipient is infected with P22 HT grown on a strain with a F-plasmid marked with Tetracycline resistance (TetR), it is possible to obtain rare TetR transductants. Suggest a likely explanation for this result.

A

Recall that transduction of plasmids requires packaging of a linear concatemer of the plasmid DNA and recircularization of the plasmid DNA in the host cell. Thus, the simplest explanation is that the TetR colonies arise by transduction of rare, spontaneous deletions of the F plasmid. The deletion mutants would have to reduce the size of the F plasmid to less than 45 kb (allowing sufficient terminal redundancy for recircularization by homologous recombination in the recipient), without removing the TetR marker or the genes required for vegetative replication

190
Q

Scientists have recently isolated a phage from Hong Kong sewage that grows on E. coli. The phage acts as a generalized transducing phage on some strains of E. coli but it acts as a specialized transducing phage on other strains of E. coli. How could you distinguish generalized transduction from specialized transduction using simple genetic tests? Indicate any donor or recipient strains you would use and how you would do the experiment.

A

Generalised transduction - many different chromosomal markers will be transduced so you can try transducing several different auxotrophic recipients to prototrophy with the phage.

Specialised transduction - only markers adjacent to the integrated phage will be transduced,

191
Q

How does tryptophan depletion in Escherichia coli promote transcription of the trp operon?

A
  • The trp operon is a group of genes that encode biosynthetic enzymes for the amino acid Tryptophan (Trp). It is found in E.Coli
  • When Trp levels is low, the trp operon is expressed (turned on), and when Trp levels are high the trp operon is repressed (turned off)
  • Therefore if Trp is low or depleted, the trp operon will promote transcription
  • This is achieved by the trp repressor not attaching to the DNA or block transcription, and thus RNA polymerase is free to transcribe, thus promoting transcription when Trp levels are low.
192
Q

When plenty of glucose and all amino acids are available, would you expect a strain of E. coli with a mutated SpoT gene to grow well or poorly? Why?

A

The function of SpoT gene is to synthesise ppGpp (ppGpp is induced during stress conditions I.e. starvation response - sigma factor S binds to RNA polymerase to repress homekeeping function). Mutated SpoT gene means that the magic spot or ppGpp cannot be synthesised nor degraded.

193
Q

Which type of systemic signalling molecule will pass through the cell membrane more readily, steroid or peptide? Why?

A

Steroids will pass through the membrane more readily as it is a lipophilic molecule. Since the membrane is made up of lipids, steroid can pass through due to its hydrophobic composition

Peptides cannot pass through the molecule as it is not lipophilic - it’s composition is too big. As a result, it needs a transporter so that it can cross the membran

194
Q

Explain how DNA is made accessible locally through chromatin remodeling without affecting the remainder of the chromosome.

A

Through chromatin remodeling it is possible to access a specific section of DNA. Phosphorylation of H1 histone, the histone protein involved in tightly packing nucleosomes, expanding the nucleosomes. After this other histone proteins, particularly H3, may be released through acetylation on lysine 9 to bring the nucleosome to an active state. Active state = histones can be removed = DNA can be accessed locally without affecting the rest of the chromosome. Methylation on lysine 4 can enhance this state.

195
Q

Small RNAs participate in inhibition of translation, transcript degradation and chromatin condensation. How can such a simple molecule achieve so much?

A

Such simple molecule can achieve so much by:
Large double strand RNA dices the RNA into a shorter ds complex of 20~ kB long
The RNAs assemble into ribonucleoprotein
Small interfering RNA is unwound to produce RNA-induced silencing complex called RISC
RISC targets a sequence in mRNA that is complementary to INTERFERING RNA
RISC interfering RNA base pairs with its target in messenger rna
If there is perfect base pairing (siRNA), the mRNA is degraded and cleaved. If the base pairing is imperfect (miRNA), protein synthesis will be inhibited and mRNA is blocked
Also, piRNA (piwi-interacting RNA) directs heterochromatin formation. This is the “chromatin condensation” part of the question

196
Q

Explain the relationship between epigenetics, transposons and adaptive genetic change.

A

The primary method of controlling transposon expression is by methylation (by methyltransferases), which essentially makes the transposon silent. Transposon and viral DNA methylation is maintained through gametes, embryogenesis and across generations. However, under stress, demethylation mobilises these sections of DNA, resulting in their expression.

197
Q

Describe the role of the three most common domains in a special transcription factor and how they interact with their target factors to regulate transcription.

A
  1. DNA binding - recognises the 4-8bp of enhancers
  2. RNA polymerase binding (Activation Domain) - interacts with mediators or RNA pol II
  3. Dimerisation - most TFs are inactive as monomers so need to dimerise to be active
198
Q

The lac Operon is

transcribed only when…

A

Lactose is Present and Glucose is Absent

BIOM2202 (5 decks)

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