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OLD "MBS 601" > Exam 3 > Flashcards

Exam 3 Flashcards

1
Q

What are assays that determine RNA secondary structures?

A

RNAse Digestion
DMS-seq
SHAPE-seq

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2
Q

What are the assays that determine RNA abundance?

A

(q) RT-PCR
PT-PCR
RNA-seq

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3
Q

What is the assay that determines promoter/consensus sequences?

A

CHIP-seq

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4
Q

What is the assay that determines rate of transcription?

A

GRO-seq

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5
Q

What are the 3 enzymes used in RNAse digestion assay and what do they do?

A

RNAse V1: cleaves dsRNA
RNAse 1: cleaves ssRNA
RNAse T: cleaves ssRNA

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6
Q

If you were using RNAse V1 to determine the secondary structure what would you see on the gel if the temperature was high?

A

nothing: RNAse V1cleaves dsRNA so it would all be denatured

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7
Q

If you were using RNAse 1 to determine the secondary structure what would you see on the gel if the temperature was high?

A

everything: RNAse 1 cleaves ssRNA with would be all of the RNA at high temps

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8
Q

What is the modification of DMS-seq?

A

modify A and C in ssRNA

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9
Q

Is DMS-seq genome wide or sensitive?

A

genome wide

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10
Q

What is special about DMS-seq?

A

it can be done in vivo and in vitro

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11
Q

In DMS-seq where does the RT polymerase stop?

A

at all the modifications of the A and Cs

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12
Q

Are there DMS signals present at dsRNA in DMS-seq charts?

A

no; it only deals with ssRNA

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13
Q

DMS signals in DMS-seq represent what?

A

ssRNA

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14
Q

What is the most accurate assay at predicting RNA secondary structures?

A

SHAPE-seq

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15
Q

What is the modification of SHAPE-seq?

A

2’ OH on ssRNA

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16
Q

What is special about SHAPE-seq that makes it so specific?

A

single nucleotide resolution (observes each base)

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17
Q

SHAPE-seq and DMS-seq have the same mechanism but what 2 things are different about them?

A
  1. SHAPE-seq has single nucleotide resolution (more specific)
  2. different modifications
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18
Q

What RNA abundance assays are relatively quantitative?

A

(q) RT-PCR
RNA-seq

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19
Q

(q) RT-PCR and RT-PCR are the same except for what 2 things…

A

(q) RT-PCR is quantitative and measures every cycle while RT-PCR is not

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20
Q

(q) RT-PCR and RT-PCR is genome wide or sensitive?

A

sensitive

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21
Q

What RNA abundance assay uses fluorescence?

A

(q) RT-PCR

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22
Q

What is the equation in (q)RT-PCR to determine the fold of a sample?

A

2 ^ (cycle # - cycle #)

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23
Q

What RNA abundance assay is the most commonly used?

A

RNA-seq

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24
Q

Is RNA-seq genome wide or sensitive?

A

genome-wide

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25
Q

What assay can detect intron splicing?

A

RNA-seq

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26
Q

Why do you need the whole genome sequenced for RNA-seq?

A

you must be able to match the sequenced fragments to their location in the genome (aids in identification of splice sites)

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27
Q

What are the dips in RNA-seq charts represent?

A

introns/splicing

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28
Q

Is CHIP-seq genome wide or sensitive?

A

genome-wide

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29
Q

What does CHIP-seq use to tag RNA?

A

antibodies

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30
Q

what does CHIP-seq idenitfy?

A

location of proteins that bind to DNA (like sigma factor)

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31
Q

Why do you need the whole genome sequenced for CHIP-seq?

A

you need to be able to match the sequenced fragments that had the protein bound to it to its location in the genome

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32
Q

CHIP-seq chart shows letters at positions in the genome; what does this help identify?

A

consensus sequence

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33
Q

What is the modification in GRO-seq?

A

changing UTP to BrUTP

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34
Q

What does GRO-seq use to tag BrUTP?

A

antibodies

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35
Q

What assays use antibody tagging?

A

GRO-seq and CHIP-seq

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36
Q

Is GRO-seq genome wide or sensitive?

A

sensitive

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37
Q

What assay looks at strands being actively transcribed by RNA polymerase?

A

GRO-seq

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38
Q

What assay needs different adaptors added to each end of fragments and why?

A

GRO-seq
needed to identify the direction of transcription

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39
Q

What does the first positive peak in GRO-seq graphs show?

A

proximal pausing

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40
Q

What 3 things did GRO-seq discover?

A
  1. proximal pausing
  2. bi-direction transcription
  3. transcription (bi-directional) at enhancers even though they don’t code for anything
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41
Q

What 3 things are special to RNA making it less stable?

A
  1. 2’ OH
  2. single stranded
  3. uracil (incorrect base pairing)
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42
Q

How many hydrogen bonds does G-C, A-U and G-U have?

A

G-C: 3
A-U: 2
G-U: 2

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43
Q

Out of G-C, G-U and A-U what is their strength ranking?

A

G-C (strongest)
A-U
G-U (weakest)

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44
Q

What is the most common unusual base in RNA?

A

pseudouridine

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45
Q

unusual bases are most common in what RNAs?

A

tRNAs

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46
Q

What is the purpose of unusual bases in RNA?

A

increase stability

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47
Q

What RNA assays are sensitive and not genome wide

A

(q)RT-PCR
GRO-seq

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48
Q

What does Non-Watson-Crick base pairing triple helixes do to RNA?

A

increase stability

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49
Q

What is the difference between an internal loop or multi-loop in RNA secondary structure?

A

multi-loop has more than 2 branches coming off it

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50
Q

What is different about A form RNA?

A

shorter and fatter

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51
Q

What is an example of a G-quadruplex?

A

GGGG (telomeres)

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52
Q

Are secondary or tertiary RNA structures more stable?

A

secondary (lower free E)

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53
Q

What proves structure = function in RNA?

A

ncRNA secondary structure are more conserved than their sequences

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54
Q

______________ are ribonucleoproteins which guide RNA molecules to their appropriate destinations

A

signal recognition particles (SRP)

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55
Q

eukaryotic SRPs are made of _____ RNA

A

7SL

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56
Q

What is pRNA?

A

packaging RNA

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57
Q

What are ribozymes?

A

catalytic RNA

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58
Q

What do natural ribozymes do?

A

formation of peptide bonds
cleave phosphodiester bonds

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59
Q

What do artificial ribozymes do?

A

glycosidic bond formation
RNA phosphorylation

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60
Q

What happens if RNA is a ribozyme AND a riboswitch?

A

1 sequence with 2 structures with different functions

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61
Q

What are riboswitches?

A

sensitive to environmental changes

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62
Q

What are aptamers?

A

RNA that binds to specific ligands

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63
Q

What is special about RNA in the RNA v. Lipid world that show they were first?

A

store genetic information and catalytic reactions

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64
Q

What were the first enzymes?

A

ribozymes

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65
Q

what RNA was the first that lead to life?

A

self-replicating RNA

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66
Q

What is transcription?

A

RNA synthesized from DNA template

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67
Q

what performs transcription polymerization?

A

RNA polymerase

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68
Q

What are 3 similarities between RNA and DNA polymerase?

A

require a template
goes in 5’–3’
adds single nucleotides at a time

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69
Q

Does RNA polymerase require a primer?

A

No (DNA polymerase does)

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70
Q

Can transcription happen outside of S phase in prokaryotes?

A

yes

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71
Q

Is RNA or DNA polymerase more error prone?

A

RNA polymerase

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72
Q

Are transcription and translation coupled in eukaryotes?

A

No (it is in prokaryotes)

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73
Q

How many RNA polymerases does bacteria have?

A

1

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74
Q

How many subunits does bacterial RNA polymerase have?

A

5

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75
Q

What is the prokaryotic holoenzyme of RNA polymerase?

A

the active form of RNA polymerase (sigma factor bound)

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76
Q

What is not always attached to prokaryotic RNA polymerase?

A

sigma factor

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77
Q

What does the prokaryotic RNA polymerase sigma factor do?

A

directs polymerase to promoter

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78
Q

Do all prokaryotes have the same number of sigma factors?

A

No all different

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79
Q

Does bacterial RNA polymerase have a helicase?

A

No, uses Mg2+ isomerase to unwind DNA

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80
Q

What are the 4 steps of bacterial transcription initation?

A
  1. RNA pol sigma factor recognizes and binds to promoter (closed complex)
  2. DNA winds by Mg2+ isomerazation (open complex) transcription bubble
  3. first phosphodiester bond formed (unstable ternary complex)
  4. release of sigma factor (stable ternary complex)
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81
Q

0

A

0

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82
Q

Do sigma factors have to bind to exact consensus sequences?

A

No, they can vary slightly

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83
Q

Why is it beneficial for bacteria to have different variations of consensus sequences?

A

allows different levels of that genes expression and different times

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84
Q

The more the consensus sequence matches the original…

A

the stronger the binding of the sigma factor = more transcription

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85
Q

0

A

0

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86
Q

What specifically on the promoter does the sigma factor recognize?

A

consensus sequence

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87
Q

The _____ domain on bacterial RNA polymerase binds the UP element, thereby strengthening RNA Pol’s binding affinity to the promoter

A

alpha - CTD

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88
Q

Upstream promoters are present on ________ expressed genes

A

highly expressed genes

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89
Q

What does DNA footprinting identify?

A

RNA polymerase binding site (sigma factor)

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90
Q

What is abortive initiation in bacteria?

A

RNA polymerizes a few times but then it is stuck because sigma factor is still attaching it to promoter

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91
Q

What is promoter escape?

A

in abortive initiation polymerase is able to escape and polymerize because sigma factor released

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92
Q

in bacteria, _________ forms ahead and behind transcription bubble during elongation

A

positive supercoils

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93
Q

In bacteria, is transcription a smooth process?

A

No (proximal pausing)

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94
Q

What stalls RNA polymerase in transcription in bacteria?

A

strong GC stem loop

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95
Q

In bacterial Intrinsic termination, what causes the RNA polymerase to fall off and release transcript?

A

UUUUUUU

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96
Q

How does Rho-dependent termination in bacteria happen?

A

GC stem loop stalls polymerase, Rho factor catches up to polymerase, Rho melts off the transcript and polymerase

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97
Q

Why is post-transcriptional modification in bacteria rare?

A

transcription and translation coupled

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98
Q

What is a post-transcriptional modification in bacteria?

A

polyadenylation

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99
Q

Polyadenylation in bacteria _______ the mRNA while in eukaryotes it _______ it

A

bacteria - destabilizes
eukaryotes stabilizes it

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100
Q

All rRNA is derived from a single precursor in bacteria. Why is this possible?

A

rRNA is not translated into a protein so it has time to be modified into different types of rRNA

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101
Q

What degrades bacterial RNA?

A

endosomes and exosomes

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102
Q

Why is the polyA tail added to bacterial mRNA?

A

bacteria have hairpins at the end of mRNA so polyA allows exonucleases to degrade mRNA

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103
Q

What is constitutive gene expression?

A

expressed all the time

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104
Q

What is repressible gene expression?

A

normally off

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105
Q

What is inducible gene expression?

A

normally on

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106
Q

What regulation is most important in bacteria?

A

transcription regulation

107
Q

What kind of system is Lac operon?

A

inducible – negative and positive regulation

108
Q

What Lac gene makes the repressor?

A

Lac I

109
Q

What happens to cAMP levels when lactose is present?

A

high

110
Q

What happens to CAP when lactose is present?

A

bound

111
Q

What happens to RNA polymerase when lactose is present?

A

binds to promoter

112
Q

What happens to Lac genes when lactose is present?

A

transcribed

113
Q

What happens to Lac repressor when lactose is present?

A

allolactose blocks binding site so it doesn’t bind to operator

114
Q

What does Lac Z transcribe?

A

B-gala

115
Q

What does Lac Y transcribe?

A

perm

116
Q

What does Lac A transcribe?

A

transA

117
Q

What happens to cAMP levels lactose is absent?

A

low

118
Q

What happens to CAP when lactose is absent?

A

not bound

119
Q

What happens to RNA polymerase when lactose is absent?

A

not bound

120
Q

What happens to Lac genes when lactose is absent?

A

not transcribed

121
Q

What happens to Lac repressor when lactose is absent?

A

binds to operator

122
Q

Do does cAMP bind to CAP when lactose is present?

A

yes

123
Q

Does cAMP bind to CAP when lactose is absent?

A

no

124
Q

In the presence of glucose and no lactose look like what?

A

the same cycle as no lactose present

125
Q

In the presence of glucose AND lactose, what happens to CAP?

A

not bound

126
Q

In the presence of glucose AND lactose, what happens to the level of cAMP?

A

low

127
Q

In the presence of glucose AND lactose, what happens to the repressor?

A

not bound

128
Q

In the presence of glucose AND lactose, what happens to RNA polymerase?

A

weakly binds

129
Q

In the presence of glucose AND lactose, what happens to the Lac genes?

A

little transcription

130
Q

What type of system is Trp?

A

repressible system with 2 negative regulations

131
Q

When Trp is high in the cell, what happens to the repressor?

A

Trp binds to repressor and they bind to operator

132
Q

When Trp is high in cell, what happens to RNA polymerase?

A

not bound

133
Q

What happens to Trp genes when Trp is high in cell?

A

no transcribed

134
Q

When Trp is low in the cell, what happens to the repressor?

A

there is no Trp to bind to repressor, so repressor doesn’t bind

135
Q

When Trp is low in cell, what happens to RNA polymerase?

A

binds

136
Q

What happens to Trp genes when Trp is low in cell?

A

transcribed

137
Q

What is the second line of defense for Trp operon?

A

attenuation control

138
Q

When Trp levels are high, what happens to the leader sequence?

A

hairpin loop is formed = no transcription

139
Q

When Trp levels are high in a cell, what happens to the level of Trp-tRNA?

A

increase

140
Q

When Trp levels are low in a cell does the polymerase stall at the leader sequence or pass through?

A

stall

141
Q

What are riboswitches?

A

RNA that changes its secondary structure

142
Q

Riboswitches are common in ___________

A

bacteria

143
Q

sRNA bind ____________ to mRNA

A

complementary

144
Q

one __RNA can regulate multiple target genes

A

sRNA (small RNA)

145
Q

What sequence allows attenuation control of Trp operon?

A

leader sequence (can adopt 2 structures)

146
Q

What does RNA polymerase I in eukaryotes transcribe?

A

rRNA

147
Q

What does RNA polymerase II in eukaryotes transcribe?

A

mRNA
snoRNA
miRNA/siRNA
sRNA
lncRNA

148
Q

What does RNA polymerase III in eukaryotes transcribe?

A

tRNA
sRNA

149
Q

How many subunits does eukaryotic RNA polymerase II have?

A

12

150
Q

How are eukaryotic II and prokaryotic RNA polymerases similar

A

structure

151
Q

What assays use antibody tagging?

A

CHIP-seq
GRO-seq

152
Q

What does transcription factor TFIID do in eukaryotic RNA polymerase II?

A

recognize TATA box (promoter)

153
Q

What does transcription factor TFIIH do in eukaryotic RNA polymerase II?

A

unwinds DNA and releases polymerase from promoter (phosphorylation of CTD tail)

154
Q

Is there a universal promoter sequence for eukaryotes?

A

no

155
Q

Where are core promoters?

A

close to start site

156
Q

Where are proximal promoters?

A

distant from start site

157
Q

Where are distal promoters?

A

far from start site

158
Q

How do eukaryotes fine tune levels of expression using promoters?

A

combinations of proximal and core promoters

159
Q

How are proximal promoters used even though they far not near start site?

A

DNA folds and uses a mediator (loops form; neighborhood)

160
Q

TFII___ binds to TATA box via the _____

A

TFIID
TBP

161
Q

TFIIH has ______ activity and ________ CTD causing the release of mediator allowing polymerase to move

A

helicase
phosphorylation

162
Q

What transcription factor in eukaryotes is important for promoter selection?

A

TFIID

163
Q

TBP binding to promoter causes __________ in DNA making unwinding easier

A

bend

164
Q

What are the 4 steps of eukaryotic transcription initation?

A
  1. TFIID bind to TATA box
  2. TFIIB is recruited to TATA box
  3. RNA polymerase recruited with TFIIF
  4. TFIIE and TFIIH join complex = complete pre-initiation complex
165
Q

Eukaryotic transcription elongation requires…

A

elongation factors

166
Q

eukaryotic RNA polymerase always does proximal ________

A

pausing

167
Q

When does 7mGTP capping first happen in eukaryotes?

A

elongation

168
Q

When does splicing start in eukaryotes?

A

elongation

169
Q

What are the 3 ways nucleosomes are dealt with in eukaryotes?

A
  1. chaperons displace nucleosomes
  2. polymerase can move around them
  3. co-transcriptional modification of histones can displace them
170
Q

What are the co-transcriptional modifications in eukaryotes?

A

5’ capping
splicing
polyadenylation

171
Q

Explain the CTD cycle?

A

CTD contain a repeat with Serines that get phosphorylated and will recruit certain factors during transcription

172
Q

What does Ser5-P do in transcription of eukaryotes?

A

recruits capping and splicing factors

173
Q

What does Ser2-P do in transcription of eukaryotes?

A

recruits polyadenylation and termination factors

174
Q

When is Ser5-P most abundant in eukaryotic transcription?

A

initiation/elongation because it recruits capping and splicing factors

175
Q

When is Ser2-P most abundant in eukaryotic transcription?

A

elongation/termination because it recruits polyadenylation and termination factors

176
Q

What facilitates co-transcriptional modifications in eukaryotic transcription?

A

CTD

177
Q

What is the first transcription modification to be done in eukaryotes?

A

7-methylG capping

178
Q

Are 5’ caps added to all RNA polymerase II transcripts in eukaryotes?

A

yes

179
Q

What kind of bond is made in 5’ capping?

A

5’-5’ phosphodiester

180
Q

Why are 5’-5’ phosphodiester bonds used in 5’ capping?

A

resistant to exonucleases

181
Q

________ group is added to the 2nd and 3rd nucleotides after the 5’ cap

A

methyl group

182
Q

5’ capping aids in the first ________

A

intron splicing

183
Q

Are polyA tails added to all RNA polymerase II transcripts?

A

no

184
Q

Does intron splicing in eukaryotes have to be precise?

A

yes; it could cause a frameshift

185
Q

are there more intron or exons?

A

introns

186
Q

What mainly does the splicing on introns in eukaryotes?

A

spliceosomes

187
Q

In major intron splicing in eukaryotes, what is recognized at the 5’ end and 3’ end?

A

5’ – GU (T)
3’ – AG

188
Q

In minor intron splicing in eukaryotes, what is recognized at the 5’ end and 3’ end?

A

5’ – AU (T)
3’ – AC

189
Q

Exon and intron boundaries are marked by ______

A

CTD

190
Q

What are the steps of the spliceosome process?

A
  1. A (branch point) is pushed out of sequence by snRNA
  2. A pairs with G farther up the sequence (beginning of intron) forming a loop
  3. loop is removed (intron)
  4. splice site is marked by exon junction complex (EJC)
191
Q

What is different about group II self-splicing compared to normal splicing?

A

same as regular but there is no spliceosome complex

192
Q

What is different about group I self-splicing compared to normal splicing?

A

uses free G instead of A = no loop
no complex

193
Q

Why do mammals have alternative splicing forms?

A

allows for diversity of the function of 1 mRNA (can be tissue specific)

194
Q

What are the 3 options of transcription termination in eukaryotes?

A
  1. allosteric model
  2. torpedo model
  3. combination
195
Q

How does the transcription termination allosteric model work in eukaryotes?

A

RNA poly II is destabilized by the conformational change of sequence and falls off

196
Q

How does the transcription termination torpedo model work in eukaryotes?

A

exonuclease degrades RNA from the 5’ end and displaces RNA polymerase II

197
Q

What does the post-transcriptional base-editing cause?

A

change in protein sequence

198
Q

Base editing in eukaryotes in most common in what type of RNAs?

A

tRNAs

199
Q

What is A to I base editing done by?

A

ADARs

200
Q

A to I base editing is involved in ______ processing

A

miRNA

201
Q

What is A to I base editing important for?

A

nervous system and development

202
Q

What is C to U base editing done by?

A

APOBECs

203
Q

mRNA methylation is the only modification that is ____________

A

transient (reversible)

204
Q

in mRNA methylation, what are readers?

A

recognize and bind to modified bases

205
Q

in mRNA methylation, what are writers?

A

modify bases

206
Q

in mRNA methylation, what are erasers?

A

reverse modification

207
Q

What are the 3 examples of mRNA quality control in eukaryotes?

A
  1. non-stop decay
  2. no-go decay
  3. nonsense-mediated decay
208
Q

When is the eukaryotic mRNA quality control non-stop decay recruited?

A

recruited when mRNA doesn’t have a stop codon

209
Q

When is the eukaryotic mRNA quality control no-go decay recruited?

A

when ribosome starts translation then stalls because an intron is not removed

210
Q

When is the eukaryotic mRNA quality control nonsense-mediated decay recruited?

A

when stop codon is added before the real stop codon

211
Q

Decapping and deadenylation of mRNAs in eukaryotes exposes them to what?

A

exonucleasees

212
Q

95% of RNA is ______

A

noncoding RNA

213
Q

miRNA/siRNA does what to translation

A

suppresses it

214
Q

How does miRNA/siRNA suppress translation?

A

attaches to mRNA complementary

215
Q

What happens if miRNA/siRNA attaches itself 100% complementary to mRNA?

A

degrades mRNA

216
Q

What happens if miRNA/siRNA attaches itself less than 100% complementary to mRNA?

A

suppresses translation

217
Q

________ strand of miRNA attaches itself to mRNA

A

leader strand

218
Q

lncRNA is ___________ expressed

A

lowly

219
Q

lncRNA forms a _____________ with DNA to recruit factors

A

triple helix

220
Q

Does lncRNA usually affect transcription (nucleus) or translation?

A

transcription

221
Q

circRNA sequesters __________

A

miRNA

222
Q

cirRNA are resistant to _________

A

exonucleases

223
Q

circRNA are produced by ________

A

back splicing

224
Q

what are tRFs?

A

fragments of tRNAs

225
Q

What do tRF regulate?

A

translation in stress conditions

226
Q

What is the use for siRNA in research?

A

can downregulate genes

227
Q

What is the use for miRNA in research?

A

mimics other miRNAs (tissue specific)

228
Q

How does the SELEX process work?

A

finds RNA that will bind to a specific molecule

229
Q

What is the advantage of the SELEX process?

A

can be used on live cells

230
Q

What are the 4 advantages of RNA therapeutics?

A

safer
faster
personalized
tackle difficult targets

231
Q

what are the 2 challenges of RNA therapeutics?

A

degrades quickly
delivery (charged and large)

232
Q

Do prokaryotes have introns?

A

no

233
Q

Regulation in multicellular organisms is important for cell _____________

A

specialization

234
Q

What do master transcriptional regulators control?

A

cell identity

235
Q

miRNA is transcribed by which RNA polymerase?

A

II

236
Q

What is the most abundant RNA in a cell?

A

rRNA

237
Q

What is RNAse P?

A

ribozyme

238
Q

_____RNA is a signal recognition particle (SRP) in eukaryotes

A

7SL

239
Q

What catalyzes the spliceosome?

A

U6 snRNA

240
Q

What does U1 snRNA do?

A

binds to sequence complementary and aids spliceosome

241
Q

In prokaryotes, what is the first thing to unwind?

A

-10 (pribnow) box

242
Q

The -10 (pribnow) box is the first thing to unwind in prokaryotes. What is its sequence rich in to aid in winding?

A

AT

243
Q

_________ act to turn off expression in both inducible and repressible operon

A

repressors

244
Q

RPB-1 contain the ___________

A

CTD

245
Q

______ phosphorylates Ser5-P for release of the mediator

A

TFIIH

246
Q

What polymerase makes lncRNA?

A

II

247
Q

________ phosphorylates CTD

A

TFIIH

248
Q

Bacteria only need _____ transcription factor. What is it?

A

1
sigma factor

249
Q

Lac repressor is a protein or lipid?

A

protein

250
Q

Lactose controls _________
Glucose controls _______

A

Lac repressor
CAP

251
Q

What charge do histone N terminal tails?

A

positive

252
Q

Are histone N-terminal tails structured of unstructured?

A

unstructured

253
Q

Where are histone modification most present on the histone?

A

N terminal tail

254
Q

Can the charge of N-terminal tails of histones be changed?

A

yes

255
Q

Do all lncRNA have 5’ caps?

A

yes

256
Q

What is the function of snoRNAs?

A

base modification in rRNA and tRNAs

257
Q

What RNAs are double stranded?

A

siRNA and miRNAs

258
Q

tRNA transcripts are processed by…..

A

RNAse P, endonuclease, ligase

259
Q

can methylated mRNAs be translated anymore?

A

yes

260
Q

What is the catalytic part of the minor spliceosome?

A

U6atac

261
Q

What is U11?

A

the non catalytic part of the minor spliceosome

262
Q

What is 4.5SL RNA?

A

bacterial SRP

263
Q

What is U2 snRNA?

A

non catalytic part of major spliceosome

OLD "MBS 601" (8 decks)

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