Genetic Networks - Systems Biology Concepts and Insights - page 110

example, genetic suppression represents an asymmetric
positive interaction whereby the double mutant exhibits
increased fitness relative to the sickest single mutant
( Figure 6.1A ). Genetic suppression between two interacting
loss-of-function (LOF) deletion alleles often indicates that
the suppressor gene functions as a negative regulator of
a pathway associated with the interacting gene [17] . Our
recent global analysis of the yeast genetic network revealed
a large number of LOF suppression interactions, indicating
that positive interactions between genes within different
complexes or pathways are surprisingly prevalent [17] .
On the other hand, genes encoding members of the
same non-essential protein complex are frequently con-
nected by symmetric genetic interactions, presumably
because once the function of the complex is disrupted by
the removal of the first component, the phenotype cannot be
made worse by the removal of additional components
( Figure 6.1A ) [19

fundamental properties of eukaryotic cells at a molecular
level and has served as a primary test bed for the development
of most functional genomic methodologies. Large-scale
genetic and phenotypic analyses have been made possible
through the development and availability of a comprehensive
collection of deletion mutants where each of the ~6000 yeast
open reading frames was replaced with a dominant, drug-
resistance marker flanked by unique synthetic 'barcode'
sequences [5] . This systematic endeavour defined the set of
~1000 essential genes and generated a set of ~5000 viable
deletion mutants [5,6] . The systematic mapping of genetic
interactions was first made possible through the development
of methodologies that take advantage of the yeast non-
essential gene deletion collection.

Genetic Interaction Mapping Technologies
Synthetic genetic array (SGA) is an automated method that
combines arrays of either non-essential gene deletion
mutants, or conditional alleles of essential genes, with
robotic manipulations for high-throughput construction of
haploid yeast double mutants and identification of genetic
interactions ( Box 6.1 ) [3,4] . In its first large-scale appli-
cation, SGA methodology was used to cross 132 query
genes to the complete array of ~5000 viable haploid dele-
tion mutants, resulting in a genetic interaction network
consisting of ~1000 genes and ~4000 synthetic lethal/sick
interactions [3] . This initial analysis provided important
insight into fundamental genetic network properties and
topology. For example, although generally rare, synthetic

21] . The relationship between genetic
and physical interactions is discussed in greater detail
below (see Exploring Genetic Interaction Networks).

e

EXPERIMENTAL APPROACHES TO MAP
GENETIC INTERACTION NETWORKS
IN YEAST
The Yeast Non-Essential Gene Deletion
Collection
With its elegant and straightforward genetics, Saccharo-
myces cerevisiae is a powerful model system to dissect the

BOX 6.1 Synthetic Genetic Array (SGA)
In a typical SGA screen a MATa mutant strain carries

a 'query' mutation, marked with the dominant drug-
resistance marker natMX4 (closed black circle), is crossed to
an array of ~5000 viable MATa deletion mutants or condi-
tional alleles of essential genes, with each mutation marked
with a kanMX4 resistance cassette (closed red circle). The
CAN1 gene, which encodes an arginine permease, is
replaced in the SGA query strain with a MATa haploid-
specific reporter,

query deletion mutant is pinned onto the
MATa non-essential gene deletion collection on rich media and
incubated at 30 C.
Diploid selection: Resulting MATa/

Mating: A MATa

zygotes are pinned
onto rich media containing kanamycin and nourseothricin and
incubated at 30 C to select heterozygous diploid double
deletion mutants.
Sporulation: Diploids are transferred to sporulation medium
and incubated for 5 days at room temperature.
MAT

a

:: STE2pr-
Sp_his5. The query strain also carries a deletion of the lysine
permease gene LYP1. Following mating, diploid selection
and sporulation, meiotic progeny are grown on media con-
taining the G418 and nourseothricin and lacking histidine to
select MATa haploid double mutants. In addition to positive
selection, the media is also supplemented with canavanine
and thialysine (toxic analogs of arginine and lysine, respec-
tively) in order to counter-select against unsporulated diploid
mutants that are heterozygous for CAN1 and LYP1 deletion
mutations. The SGA procedure is briefly described below.
For a more detailed protocol and media composition, refer
to Baryshnikova et al. [119] .

STE2pr-Sp_his5 such as can1D

a haploid selection: Spores are grown on synthetic
medium lacking histidine and containing canavanine and thia-
lysine (SD-his

thialysine), which selects for MATa
haploid cells expressing the MATa-specific reporter, can1D

þ canavanine þ

::

STE2pr-Sp_his5 and selects against unsporulated diploids.
Single mutant selection: Colonies are transferred to SD-

his thialysine growth medium supplemented

with G418 to select for MATa haploids harboring the deletion
array mutation.
Double mutant selection: Haploid double mutants are
selected following growth on (SD-his

þ canavanine þ

þ canavanine þ thialysine)

medium containing both G418 and nourseothricin.