Thermophilic fungi can thrive in temperatures extending up to 62°C (143°F). As the only representatives of eukaryotic organisms that can grow at such extreme temperatures, the thermophilic fungi are valuable experimental systems for investigators. Although widespread in terrestrial habitats, they have remained underexplored compared to thermophilic species of bacteria. You are most interested in the molecular details of DNA replication in a newly isolated thermophilic fungus you have maintained in your laboratory. You are anxious to identify its DNA replicator sequence. You intend to use the same method utilized by researchers studying both prokaryotic and eukaryotic DNA replication to identify sequences that can serve as replicator. Describe in detail the screen you would use to identify potential DNA replicator sequences. Use pictures to illustrate if necessary, but include text to explain. Solution DNA replicator sequences are the sequences where DNA replication starts. These are also called Autonomous Replicative sequences (ARS). For a genetic screen to work, there are two methods: i) Auxotrophic strains: strains that can not grow on a particular media ii) Reporter assays: strains containg a reporter gene like beta-galactosidase or GFP In this particular assay we need to use auxotrophic strains because replication is associated with survival and not with protein expression. So, firstly, we will do random mutagenesis and select for the strains that do not grow in absence of a crucial nutrient (For eg: Uracil). One can obtain this mutant strain by replica plating the newly obtained irradiated fungus (having random mutations) on nutrient rich media and media lacking uracil. The colonies that do not grow on media lacking uracil can be taken for further analysis. Complementary assay will help to identify the gene responsible for this mutation. Let us assume it is Ura. Now we will generate overlapping restriction fragments of the genome of this fungus by using different restriction enzymes (atleast two) for a brief period of time. These fragments can be cloned in a shuttle vector (plasmid) alongwith the wild type gene Ura that was identified in the complementary assay but having no replicative sequence of its own. So when the cell divides, the plasmid can not replicate with the cell unless it has an external ARS. However, if the restriction fragment cloned with Ura has ARS in it, then this plasmid can replicate along with the cell and will get distributed equally among the daughter cells. As the original cells do not have gene Ura to make Uracil, the cells containing the plasmid having ARS only can replicate and form colonies. Now these plasmids can be sequenced to find out the sequences and the most common sequences in various plasmids of the surviving colonies can be termed as ARS in that particular species. The accuracy of the sequence obtained will depend upon the median lengths of DNA sequences obtained while restriction digestion and fals.

1. Thermophilic fungi can thrive in temperatures extending up to 62°C (143°F). As the only
representatives of eukaryotic organisms that can grow at such extreme temperatures, the
thermophilic fungi are valuable experimental systems for investigators. Although widespread in
terrestrial habitats, they have remained underexplored compared to thermophilic species of
bacteria.
You are most interested in the molecular details of DNA replication in a newly isolated
thermophilic fungus you have maintained in your laboratory. You are anxious to identify its
DNA replicator sequence.
You intend to use the same method utilized by researchers studying both prokaryotic and
eukaryotic DNA replication to identify sequences that can serve as replicator. Describe in detail
the screen you would use to identify potential DNA replicator sequences. Use pictures to
illustrate if necessary, but include text to explain.
Solution
DNA replicator sequences are the sequences where DNA replication starts. These are also called
Autonomous Replicative sequences (ARS). For a genetic screen to work, there are two methods:
i) Auxotrophic strains: strains that can not grow on a particular media
ii) Reporter assays: strains containg a reporter gene like beta-galactosidase or GFP
In this particular assay we need to use auxotrophic strains because replication is associated with
survival and not with protein expression. So, firstly, we will do random mutagenesis and select
for the strains that do not grow in absence of a crucial nutrient (For eg: Uracil). One can obtain
this mutant strain by replica plating the newly obtained irradiated fungus (having random
mutations) on nutrient rich media and media lacking uracil. The colonies that do not grow on
media lacking uracil can be taken for further analysis. Complementary assay will help to identify
the gene responsible for this mutation. Let us assume it is Ura.
Now we will generate overlapping restriction fragments of the genome of this fungus by using
different restriction enzymes (atleast two) for a brief period of time. These fragments can be
cloned in a shuttle vector (plasmid) alongwith the wild type gene Ura that was identified in the
complementary assay but having no replicative sequence of its own. So when the cell divides,
the plasmid can not replicate with the cell unless it has an external ARS. However, if the
restriction fragment cloned with Ura has ARS in it, then this plasmid can replicate along with the
cell and will get distributed equally among the daughter cells. As the original cells do not have
gene Ura to make Uracil, the cells containing the plasmid having ARS only can replicate and
form colonies. Now these plasmids can be sequenced to find out the sequences and the most

2. common sequences in various plasmids of the surviving colonies can be termed as ARS in that
particular species. The accuracy of the sequence obtained will depend upon the median lengths
of DNA sequences obtained while restriction digestion and false positive rates and false negative
rates due to sequencing depth and availability of multiple ARS.