Bi190-2013 Lecture 9Genetic Screens (cont.)

Chromosomes

C. elegans EGF-receptor signaling:a branched signaling pathway

LET-23

EGF-R

SEM-5 Grb2 LET-341 SOS

LET-60 RAS

[IP3]

Ovulation Vulval development

ITR-1 IP3 Receptor

PLCγ

[IP2]

[PIP2]

Extragenic suppressors of partially defective LET-23

activated:

LET-60 Ras

loss of:

UNC-101 = clathrin adaptor medium chain

SLI-1 = cbl proto-oncogene homolog

GAP-1 = GAP homolog

Gregg Jongeward, Junho Lee & Charles Yoon

Enhancers of sli-1 mutations

loss of:

UNC-101 = clathrin adaptor medium chain

ARK-1 = Ack-related Kinase

GAP-1 = GAP homolog

Junho Lee, Chris Lacenere & Neil Hopper

LET-23

EGF-R

SEM-5 Grb2 LET-341 SOS

LET-60 RAS

[IP3]

Ovulation Vulval development

ITR-1 IP3 Receptor

PLCγ

[IP2]

[PIP2]

LET-23

EGF-R

SEM-5 Grb2 LET-341 SOS

LET-60 RAS

[IP3]

Ovulation Vulval development

ITR-1 IP3 Receptor

PLCγ

[IP2]

[PIP2]

let-23(lf)

LET-23

EGF-R

SEM-5 Grb2 LET-341 SOS

LET-60 RAS**

[IP3]

Ovulation Vulval development

ITR-1 IP3 Receptor

PLCγ

[IP2]

[PIP2]

let-23(lf)

let-60(gf)

LET-23

EGF-R

SEM-5 Grb2 LET-341 SOS

LET-60 RAS

[IP3]

Ovulation Vulval development

ITR-1 IP3 Receptor**

PLCγ

[IP2]

[PIP2]

let-23(lf)

itr-1(gf)

LET-23

EGF-R

SEM-5 Grb2 LET-341 SOS

LET-60 RAS**

[IP3]

Ovulation Vulval development

ITR-1 IP3 Receptor**

PLCγ

[IP2]

[PIP2]

let-23(lf)

let-60(gf)

itr-1(gf)

Analyzing and Building Chromosomes

Three types of DNA sequence required for eukaryotic chromosomes

Replication origin (many per chromosome) functions in interphase to duplicate DNA.Centromere (one per chromosome) functions in mitosis for chromosome segregation.Telomeres (two per chromosome) cap the chromosome ends.

The kinetochore forms in mitosis at the centromere

eukaryotic origins of replication

In Situ Hybridization of Human Chromosomes With a Probe Against the Telomere Repeat

Selection of Functional Elements in Budding Yeastyeast functional assays for (a) ARS

Selection of Functional Elements in Budding Yeastyeast functional assays for (b) centromere

Selection of Functional Elements in Budding Yeastyeast functional assays for (c) telomere

Szostak & Blackburn (1982)

Tetrahymena rDNA: 21 kb small linear molecule

Bam H1Bam H1

+Bgl II

Tel

TelTel

Bgl IIBgl II Bam H1Bam H1

LEU2+ars1Col E1 oriAmpR

Of 15 Leu+ transformants, 14 were mitotically unstable; 1integrant. ~20 copies per cell, and LINEAR! by R-Mapping

HIS3+

CCCCAA

GGGGTT

Clone a yeast telomere:

T.TEL LEU2 yeast DNA

yeast DNA, cut with Pvu I into ~2000 pieces32 should be telomeres (1/65)

Of 60 colonies, 56 had plasmid (mitotically unstable)Pick 3 that had a small single Pvu I fragment---A yeast TEL

Pvu I

5´ ... C G A T^C G ... 3´ 3´ ... G C^T A G C ... 5´

Murray & Szostak

YLp4

LEU2+CEN3ARS1Col E1 oriAmpR

TEL

TEL

transform yeast

Plate 100 colonies/platereplica plate to -Leucount fraction Leu+

Plasmid %cells Segr. copies/cell with plasmid Freq. circle - 5 0.34 50

circle CEN 90 0.02 1

linear CEN 61 0.11 15

linear - 72 0.16 50

TEL inhibits CEN function; yeast chr are 10-100 fold longeradd more DNA, stability increases!

Pedigree analysis todetermine the segregationfrequency, the fraction of divisions at which only one daughter receivesthe plasmid

Telomere Repeats of Some Organisms

NB – 3’ overhang is G-rich

human artificial chromosomes[Harrington….Willard, 1997)

linear, mitotically stableCENTELORI

transfect in alpha-satellite, TTAGGG (TEL), selectable markerand obtain a minichromosome.After 240 generations with no selection, 29-100% had the mini99.5% segregation frequency.

Tandem repeats of alpha satellite DNA constitute a primate centromere

[Schueler, Higgins, Rudd, Gustashaw and Willard, Science (2001)]

(A) Chromosome rearrangements define the CEN as (171bpα)12

(B) Artificial chromosomes with 85 Kb alpha satellite DNA (red) bind the centromere protein CENP-E (green). Overlap of both is yellow.

Centromere Sequences Differ Between Organisms

Epigenetic model: something else besides DNA sequence may define a centromere

Analysis of CEN Mutations by DNA Sequence Replacement in Budding Yeast

(Rothstein)

ds replacement DNA

ds chromosomal DNA homology homology

ds chromosomal DNA with replacement

sectoring assays

ADE2 ADE3 White

ade2 ADE3 Red

ADE2 ade3 White

ade2 ade3 White

Hieter-Davisade2(ochre); pSUP11 (och suppressor)

copies/cell colony color0 Red1 Pink2 White

sectoring assays

ADE2 ADE3 White

ade2 ADE3 Red

ADE2 ade3 White

ade2 ade3 White

Koshland-Hartwellade2 ade3 ; pADE3(hypomorph)

copies/cell colony color0 White1 Pink2 Red

Doug Koshland

Balancer A!

m!

X!+!

mutagenize!

B non-A!

X!

F1!

X!F2!

m!F3!

m! !

Balancer A!

Balancer B!

Balancer B!Balancer A!

Balancer B!

m!Balancer B!

m!Balancer B!

Non-B non-A!

X!m/Bal!

How to turn any organism into a genetic system

Loss-of-functionReduction of function Gain of functionSite of actionTime of actionGene set; gene expressionCis-regulationComplementationScreens for functiondouble perturbations