INVESTIGATIONGenome Rearrangements Caused by Depletionof Essential DNA Replication Prote insin Saccharomyces cerevisiaeEdith Cheng,* Jessica A. Vaisica, ** Jiongwen Ou,* Anastasia Baryshnikova,' Yong Lu,Frederick P. Roth,4.* and Grant W. Brown*.*Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada, 'Donnelly Centre for Cellular andBiomolecular Research, Toronto, Ontario M5S 3E1, Canada, *Banting and Best Department of Medical Research and Departmentof Molecular Genetics, University of Toronto, Toronto, Ontario M5G 1L6, Canada, SDepartment of Biological Chemistry andMolecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, and *"Samuel Lunenfeld Research Institute, Mt.Sinai Hospital, Toronto, Ontario M5G 1X5, CanadaABSTRACT Genetic screens of the collection of ~4500 deletion mutants in Saccharomyces cerevisiae have identified the cohort ofnonessential genes that promote maintenance of genome integrity. Here we probe the role of essential genes needed for genomestability. To this end, we screened 217 tetracycline-regulated promoter alleles of essential genes and identified 47 genes whosedepletion results in spontaneous DNA damage. We further showed that 92 of these 217 essential genes have a role in suppressingchromosome rearrangements. We identified a core set of 15 genes involved in DNA replication that are critical in preventing bothspontaneous DNA damage and genome rearrangements. Mapping, classification, and analysis of rearrangement breakpoints indicatedthat yeast fragile sites, Ty retrotransposons, tRNA genes, early originsfeatures at breakpoints when essential replication genes that suppress chromosome rearrangements are down regulated. We proposemechanisms by which depletion of essential replication proteins can lead to double-stranded DNA breaks near these features, whichare subsequently repaired by homologous recombination at repeated elementsreplication, and replication termination sites are commonCCURATE transmission of the genome is essential fornormal cell growth and survival. As such, cells havedeveloped elaborate mechanisms to prevent errors in replication and to respond to spontaneous DNA damage that canlead to genomic instability (Kolodner et al. 2002; Branzeiand Foiani 2007, 2009, 2010; Harper and Elledge 2007;Cimprich and Cortez 2008). The failure to repair the genomein an error-free manner can result in chromosome abnormal-ities that underlie many human diseases, including cancers(Kolodner et al. 2002; McKinnon and Caldecott 2007; Aguileraand Gomez-Gonzalez 2008). Therefore, defining the genesthat contribute to genome maintenance will be useful inunderstanding disease development and in designing newstrategies for therapeutics. However, to date, a comprehensivecuration of genes that function to suppress genome instabil-ity is incomplete.Yeast is an ideal model for genomic studies due to theconservation of gene functions and biological pathways be-tween yeast and humans. Phenotypic screens conducted withthe Saccharomyces cerevisiae nonessential gene deletion collec-tion (Giaever et al. 2002) have aided in the annotation andfunctional characterization of nonessential genes involved inthe suppression of spontaneous DNA damage (Huang et al.2003; Huang and Kolodner 2005; Shor et al. 2005; Alvaroet al. 2007) and in the suppression of spontaneous chromosomerearrangements (Smith et al. 2004; Yuen et al. 2007; Andersenet al 2008). However, since the deletion of essential genescauses lethality, similar genome-wide screening approaches toidentify the complete set of genes that suppress spontaneousDNA damage and chromosome rearrangements require collec-tions of conditional alleles of essential genes.Systematic collections of conditional alleles have beengenerated in several ways, including the replacement of nativepromoters with a tetracycline-regulated promoter (MnaimnehCopyright 2012 by the Genetics Society of Americadoi: 10.1534/genetics.112.141051Manuscript received April 9, 2012; accepted for publication May 28, 2012Supporting information is available online at http//www.genetics.org/contentsuppl/2012/06/05/genetics. 112.141051 DC1'Corresponding author: Donnelly Centre for Cellular and Biomolecular Research, 160College St, Toronto, ON MSS 3E1, Canada. E-mail: grant.brown@utoronto.caGenetics, Vol. 192, 147-160 September 2012147 et al. 2004; Yu et al. 2006), destabilization of target genemRNAs through the insertion of a selectable marker in the3'-UTR of essential genes (Schuldiner et al. 2005), systematicaddition of a heat-inducible degron to the amino terminus ofthe protein product (Labib et al. 2000), systematic generationof novel temperature-sensitive alleles (Ben-Aroya et al2008), and systematic integration of existing temperature-sensitive alleles (Li et al. 2011). Despite the availability ofseveral essential gene collections, no one collection is com-plete, suggesting that complementary approaches usinga number of screening strategies and multiple types of con-ditional alleles will be necessary to identify all of the es-sential genes that function to suppress genomic instabilityHere we describe a series of screens to identify essentialgenes that function to suppress genome instability, using thecollection of tetracycline-regulated promoter replacementalleles (Tet alleles) of essential genes (Mnaimneh et al2004). We screened 217 Tet alleles of essential genes whosedepletion caused accumulation in S or G2 phases of the cellcycle (Yu et al. 2006) and identified 47 with elevated levelsof spontaneous DNA damage. A second screen performedwith the same Tet alleles identified 92 essential genes thatsuppress the formation of chromosome rearrangements, wholechromosome deletions, and gene conversions. We quantifiedthe levels of each type of mutation in 15 strains that exhibitedboth elevated levels of spontaneous DNA damage and chro-mosome rearrangements following the depletion of an essen-tial gene. Mapping of rearrangement breakpoints in sevenrepresentative mutants from this set revealed several uniquepreviously described for Rad52-YFP (Lisby et al. 2004; Lisbyand Rothstein 2004; Chang et al. 2005). Ddc2 foci werequantified in at least 100 cells for each strain. Ddc2 foci inwild-type cells were analyzed four times and used to calcu-late a standard deviation. Tet allele strains that had Ddc2foci levels that were at least three standard deviations greaterthan wild type were scored as positive.illegitimate mating assaysTet allele strains and the R1158 wild-type strain were grownin parallel for 24 hr on YPD solid media either containing orlacking 10 Hg/ml of doxycycline. A standard mating assaywas performed with tester strains MCY13 (MATa , legiti-mate mating) and MCY14 (MATa, illegitimate mating) onthe same media conditions that the strains were grown.Diploids were isolated by replica plating on minimal media.In the quantitative form of this mating assay, Tet allelestrains and R1158 wild-type strain were grown in parallelfor 24 hr in YPD liquid media containing or lacking 10 ug/mldoxycycline. Strains were mixed with fivefold excess ofMCY13, MCY14, or 1225a (MATa his4 thr4) tester strainsand plated on YPD solid media. After 5 hr, cells were col-lected, resuspended in water, and plated on diploid selectionmedia. Independent illegitimate diploids were isolated afterthe mating of the Tet allele strains with the 1225a testerstrain. For each mating experiment, 100 diploids were iso-lated and tested for their ability to grow in the presence orabsence of histidine or threonine. This assay was repeated twotimes. Viability of each strain following growth in doxycyclinewas confirmed by plating on YPD. Only MCM7 (10 %), NUF2(30%), and UBC9 (50% ) had

Question
Asked Nov 15, 2019
17 views

what is the purpose of this experiment and the hypothesis of the paper?

INVESTIGATION
Genome Rearrangements Caused by Depletion
of Essential DNA Replication Prote ins
in Saccharomyces cerevisiae
Edith Cheng,* Jessica A. Vaisica, ** Jiongwen Ou,* Anastasia Baryshnikova,' Yong Lu,
Frederick P. Roth,4.* and Grant W. Brown*.*
Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada, 'Donnelly Centre for Cellular and
Biomolecular Research, Toronto, Ontario M5S 3E1, Canada, *Banting and Best Department of Medical Research and Department
of Molecular Genetics, University of Toronto, Toronto, Ontario M5G 1L6, Canada, SDepartment of Biological Chemistry and
Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, and *"Samuel Lunenfeld Research Institute, Mt.
Sinai Hospital, Toronto, Ontario M5G 1X5, Canada
ABSTRACT Genetic screens of the collection of ~4500 deletion mutants in Saccharomyces cerevisiae have identified the cohort of
nonessential genes that promote maintenance of genome integrity. Here we probe the role of essential genes needed for genome
stability. To this end, we screened 217 tetracycline-regulated promoter alleles of essential genes and identified 47 genes whose
depletion results in spontaneous DNA damage. We further showed that 92 of these 217 essential genes have a role in suppressing
chromosome rearrangements. We identified a core set of 15 genes involved in DNA replication that are critical in preventing both
spontaneous DNA damage and genome rearrangements. Mapping, classification, and analysis of rearrangement breakpoints indicated
that yeast fragile sites, Ty retrotransposons, tRNA genes, early origins
features at breakpoints when essential replication genes that suppress chromosome rearrangements are down regulated. We propose
mechanisms by which depletion of essential replication proteins can lead to double-stranded DNA breaks near these features, which
are subsequently repaired by homologous recombination at repeated elements
replication, and replication termination sites are common
CCURATE transmission of the genome is essential for
normal cell growth and survival. As such, cells have
developed elaborate mechanisms to prevent errors in repli
cation and to respond to spontaneous DNA damage that can
lead to genomic instability (Kolodner et al. 2002; Branzei
and Foiani 2007, 2009, 2010; Harper and Elledge 2007;
Cimprich and Cortez 2008). The failure to repair the genome
in an error-free manner can result in chromosome abnormal-
ities that underlie many human diseases, including cancers
(Kolodner et al. 2002; McKinnon and Caldecott 2007; Aguilera
and Gomez-Gonzalez 2008). Therefore, defining the genes
that contribute to genome maintenance will be useful in
understanding disease development and in designing new
strategies for therapeutics. However, to date, a comprehensive
curation of genes that function to suppress genome instabil-
ity is incomplete.
Yeast is an ideal model for genomic studies due to the
conservation of gene functions and biological pathways be-
tween yeast and humans. Phenotypic screens conducted with
the Saccharomyces cerevisiae nonessential gene deletion collec-
tion (Giaever et al. 2002) have aided in the annotation and
functional characterization of nonessential genes involved in
the suppression of spontaneous DNA damage (Huang et al.
2003; Huang and Kolodner 2005; Shor et al. 2005; Alvaro
et al. 2007) and in the suppression of spontaneous chromosome
rearrangements (Smith et al. 2004; Yuen et al. 2007; Andersen
et al 2008). However, since the deletion of essential genes
causes lethality, similar genome-wide screening approaches to
identify the complete set of genes that suppress spontaneous
DNA damage and chromosome rearrangements require collec-
tions of conditional alleles of essential genes.
Systematic collections of conditional alleles have been
generated in several ways, including the replacement of native
promoters with a tetracycline-regulated promoter (Mnaimneh
Copyright 2012 by the Genetics Society of America
doi: 10.1534/genetics.112.141051
Manuscript received April 9, 2012; accepted for publication May 28, 2012
Supporting information is available online at http//www.genetics.org/content
suppl/2012/06/05/genetics. 112.141051 DC1
'Corresponding author: Donnelly Centre for Cellular and Biomolecular Research, 160
College St, Toronto, ON MSS 3E1, Canada. E-mail: grant.brown@utoronto.ca
Genetics, Vol. 192, 147-160 September 2012
147
help_outline

Image Transcriptionclose

INVESTIGATION Genome Rearrangements Caused by Depletion of Essential DNA Replication Prote ins in Saccharomyces cerevisiae Edith Cheng,* Jessica A. Vaisica, ** Jiongwen Ou,* Anastasia Baryshnikova,' Yong Lu, Frederick P. Roth,4.* and Grant W. Brown*.* Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada, 'Donnelly Centre for Cellular and Biomolecular Research, Toronto, Ontario M5S 3E1, Canada, *Banting and Best Department of Medical Research and Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5G 1L6, Canada, SDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, and *"Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, Ontario M5G 1X5, Canada ABSTRACT Genetic screens of the collection of ~4500 deletion mutants in Saccharomyces cerevisiae have identified the cohort of nonessential genes that promote maintenance of genome integrity. Here we probe the role of essential genes needed for genome stability. To this end, we screened 217 tetracycline-regulated promoter alleles of essential genes and identified 47 genes whose depletion results in spontaneous DNA damage. We further showed that 92 of these 217 essential genes have a role in suppressing chromosome rearrangements. We identified a core set of 15 genes involved in DNA replication that are critical in preventing both spontaneous DNA damage and genome rearrangements. Mapping, classification, and analysis of rearrangement breakpoints indicated that yeast fragile sites, Ty retrotransposons, tRNA genes, early origins features at breakpoints when essential replication genes that suppress chromosome rearrangements are down regulated. We propose mechanisms by which depletion of essential replication proteins can lead to double-stranded DNA breaks near these features, which are subsequently repaired by homologous recombination at repeated elements replication, and replication termination sites are common CCURATE transmission of the genome is essential for normal cell growth and survival. As such, cells have developed elaborate mechanisms to prevent errors in repli cation and to respond to spontaneous DNA damage that can lead to genomic instability (Kolodner et al. 2002; Branzei and Foiani 2007, 2009, 2010; Harper and Elledge 2007; Cimprich and Cortez 2008). The failure to repair the genome in an error-free manner can result in chromosome abnormal- ities that underlie many human diseases, including cancers (Kolodner et al. 2002; McKinnon and Caldecott 2007; Aguilera and Gomez-Gonzalez 2008). Therefore, defining the genes that contribute to genome maintenance will be useful in understanding disease development and in designing new strategies for therapeutics. However, to date, a comprehensive curation of genes that function to suppress genome instabil- ity is incomplete. Yeast is an ideal model for genomic studies due to the conservation of gene functions and biological pathways be- tween yeast and humans. Phenotypic screens conducted with the Saccharomyces cerevisiae nonessential gene deletion collec- tion (Giaever et al. 2002) have aided in the annotation and functional characterization of nonessential genes involved in the suppression of spontaneous DNA damage (Huang et al. 2003; Huang and Kolodner 2005; Shor et al. 2005; Alvaro et al. 2007) and in the suppression of spontaneous chromosome rearrangements (Smith et al. 2004; Yuen et al. 2007; Andersen et al 2008). However, since the deletion of essential genes causes lethality, similar genome-wide screening approaches to identify the complete set of genes that suppress spontaneous DNA damage and chromosome rearrangements require collec- tions of conditional alleles of essential genes. Systematic collections of conditional alleles have been generated in several ways, including the replacement of native promoters with a tetracycline-regulated promoter (Mnaimneh Copyright 2012 by the Genetics Society of America doi: 10.1534/genetics.112.141051 Manuscript received April 9, 2012; accepted for publication May 28, 2012 Supporting information is available online at http//www.genetics.org/content suppl/2012/06/05/genetics. 112.141051 DC1 'Corresponding author: Donnelly Centre for Cellular and Biomolecular Research, 160 College St, Toronto, ON MSS 3E1, Canada. E-mail: grant.brown@utoronto.ca Genetics, Vol. 192, 147-160 September 2012 147

fullscreen
et al. 2004; Yu et al. 2006), destabilization of target gene
mRNAs through the insertion of a selectable marker in the
3'-UTR of essential genes (Schuldiner et al. 2005), systematic
addition of a heat-inducible degron to the amino terminus of
the protein product (Labib et al. 2000), systematic generation
of novel temperature-sensitive alleles (Ben-Aroya et al
2008), and systematic integration of existing temperature-
sensitive alleles (Li et al. 2011). Despite the availability of
several essential gene collections, no one collection is com-
plete, suggesting that complementary approaches using
a number of screening strategies and multiple types of con-
ditional alleles will be necessary to identify all of the es-
sential genes that function to suppress genomic instability
Here we describe a series of screens to identify essential
genes that function to suppress genome instability, using the
collection of tetracycline-regulated promoter replacement
alleles (Tet alleles) of essential genes (Mnaimneh et al
2004). We screened 217 Tet alleles of essential genes whose
depletion caused accumulation in S or G2 phases of the cell
cycle (Yu et al. 2006) and identified 47 with elevated levels
of spontaneous DNA damage. A second screen performed
with the same Tet alleles identified 92 essential genes that
suppress the formation of chromosome rearrangements, whole
chromosome deletions, and gene conversions. We quantified
the levels of each type of mutation in 15 strains that exhibited
both elevated levels of spontaneous DNA damage and chro-
mosome rearrangements following the depletion of an essen-
tial gene. Mapping of rearrangement breakpoints in seven
representative mutants from this set revealed several unique
previously described for Rad52-YFP (Lisby et al. 2004; Lisby
and Rothstein 2004; Chang et al. 2005). Ddc2 foci were
quantified in at least 100 cells for each strain. Ddc2 foci in
wild-type cells were analyzed four times and used to calcu-
late a standard deviation. Tet allele strains that had Ddc2
foci levels that were at least three standard deviations greater
than wild type were scored as positive.
illegitimate mating assays
Tet allele strains and the R1158 wild-type strain were grown
in parallel for 24 hr on YPD solid media either containing or
lacking 10 Hg/ml of doxycycline. A standard mating assay
was performed with tester strains MCY13 (MATa , legiti-
mate mating) and MCY14 (MATa, illegitimate mating) on
the same media conditions that the strains were grown.
Diploids were isolated by replica plating on minimal media.
In the quantitative form of this mating assay, Tet allele
strains and R1158 wild-type strain were grown in parallel
for 24 hr in YPD liquid media containing or lacking 10 ug/ml
doxycycline. Strains were mixed with fivefold excess of
MCY13, MCY14, or 1225a (MATa his4 thr4) tester strains
and plated on YPD solid media. After 5 hr, cells were col-
lected, resuspended in water, and plated on diploid selection
media. Independent illegitimate diploids were isolated after
the mating of the Tet allele strains with the 1225a tester
strain. For each mating experiment, 100 diploids were iso-
lated and tested for their ability to grow in the presence or
absence of histidine or threonine. This assay was repeated two
times. Viability of each strain following growth in doxycycline
was confirmed by plating on YPD. Only MCM7 (10 %), NUF2
(30%), and UBC9 (50% ) had <100% viability following
growth in doxycycline.
rearrangement structures. Sequence features, including Ty ret-
rotransposons and DNA replication origins and termination
zones, correlated with the rearrangements identified. We pro-
pose a central role for DNA replication proteins in suppressing
the formation of chromosome breaks that promote chromo-
Array comparative genome hybridization
Genomic DNA was extracted (Qiagen) from independent
illegitimate diploids and wild-type diploids isolated from the
mating assay. CGH on a microarray was performed as
previously described (Dion and Brown 2009) using S. cerevisiae
whole genome tiling microarrays (Affymetrix). Signal in
tensities of the experimental and wild-type control sam-
ples were normalized and compared using tiling analysis
software (Affymetrix). Genomic patterns were mapped and
analyzed using the integrated genome browser software
(Affymetrix).
some rearrangements.
Materials and Methods
Yeast strains and media
Tet allele strains were constructed as described previously
(Mnaimneh et al. 2004). The genotype of the wild-type
Tet allele strain, R1158, is MATa URA3::CMV-tTA his341
leu240 met1540. Using standard genetic methods, 217 MATa
Tet allele strains were engineered to contain YFP-Ddc2
marked with a nourseothricin (Nat) resistance gene. Gen
otypes for strains used in this study are listed in Table S6.
The essential genes that were studied are listed in Table S1
and Table S2. Standard yeast media and growth conditions
were used unless otherwise specified (Sherman 1991).
CHEF gel electrophoresis and Southern blot analysis
Contour-clamped homogenous electric field (CHEF) gels
were used to examine intact chromosomes of illegitimate
diploids isolated from the mating assay. CHEF gel analysis
was performed as described previously (Desany et al
1998). A 1.2 % agarose gel was run at 8 V/cm using pulse
times of 120 sec for 30 hr at 14° in 0.5x TBE buffer. PCR-
Fluorescence microscopy
Tet allele strains were grown in YPD liquid media at 30°,
Samples were divided into two cultures and grown in par
allel in the presence and absence of 10 Hg/ml doxycycline
for 4 additional hours at 23°. Intracellular localization of
Ddc2-YFP was determined by fluorescence microscopy as
purified fragments were radio labeled by random priming
(Stratagene) and used as hybridization probes for Southern
blot analysis. PCR primers designed for probe construction
are listed in Table S7.
E. Cheng et al
148
help_outline

Image Transcriptionclose

et al. 2004; Yu et al. 2006), destabilization of target gene mRNAs through the insertion of a selectable marker in the 3'-UTR of essential genes (Schuldiner et al. 2005), systematic addition of a heat-inducible degron to the amino terminus of the protein product (Labib et al. 2000), systematic generation of novel temperature-sensitive alleles (Ben-Aroya et al 2008), and systematic integration of existing temperature- sensitive alleles (Li et al. 2011). Despite the availability of several essential gene collections, no one collection is com- plete, suggesting that complementary approaches using a number of screening strategies and multiple types of con- ditional alleles will be necessary to identify all of the es- sential genes that function to suppress genomic instability Here we describe a series of screens to identify essential genes that function to suppress genome instability, using the collection of tetracycline-regulated promoter replacement alleles (Tet alleles) of essential genes (Mnaimneh et al 2004). We screened 217 Tet alleles of essential genes whose depletion caused accumulation in S or G2 phases of the cell cycle (Yu et al. 2006) and identified 47 with elevated levels of spontaneous DNA damage. A second screen performed with the same Tet alleles identified 92 essential genes that suppress the formation of chromosome rearrangements, whole chromosome deletions, and gene conversions. We quantified the levels of each type of mutation in 15 strains that exhibited both elevated levels of spontaneous DNA damage and chro- mosome rearrangements following the depletion of an essen- tial gene. Mapping of rearrangement breakpoints in seven representative mutants from this set revealed several unique previously described for Rad52-YFP (Lisby et al. 2004; Lisby and Rothstein 2004; Chang et al. 2005). Ddc2 foci were quantified in at least 100 cells for each strain. Ddc2 foci in wild-type cells were analyzed four times and used to calcu- late a standard deviation. Tet allele strains that had Ddc2 foci levels that were at least three standard deviations greater than wild type were scored as positive. illegitimate mating assays Tet allele strains and the R1158 wild-type strain were grown in parallel for 24 hr on YPD solid media either containing or lacking 10 Hg/ml of doxycycline. A standard mating assay was performed with tester strains MCY13 (MATa , legiti- mate mating) and MCY14 (MATa, illegitimate mating) on the same media conditions that the strains were grown. Diploids were isolated by replica plating on minimal media. In the quantitative form of this mating assay, Tet allele strains and R1158 wild-type strain were grown in parallel for 24 hr in YPD liquid media containing or lacking 10 ug/ml doxycycline. Strains were mixed with fivefold excess of MCY13, MCY14, or 1225a (MATa his4 thr4) tester strains and plated on YPD solid media. After 5 hr, cells were col- lected, resuspended in water, and plated on diploid selection media. Independent illegitimate diploids were isolated after the mating of the Tet allele strains with the 1225a tester strain. For each mating experiment, 100 diploids were iso- lated and tested for their ability to grow in the presence or absence of histidine or threonine. This assay was repeated two times. Viability of each strain following growth in doxycycline was confirmed by plating on YPD. Only MCM7 (10 %), NUF2 (30%), and UBC9 (50% ) had <100% viability following growth in doxycycline. rearrangement structures. Sequence features, including Ty ret- rotransposons and DNA replication origins and termination zones, correlated with the rearrangements identified. We pro- pose a central role for DNA replication proteins in suppressing the formation of chromosome breaks that promote chromo- Array comparative genome hybridization Genomic DNA was extracted (Qiagen) from independent illegitimate diploids and wild-type diploids isolated from the mating assay. CGH on a microarray was performed as previously described (Dion and Brown 2009) using S. cerevisiae whole genome tiling microarrays (Affymetrix). Signal in tensities of the experimental and wild-type control sam- ples were normalized and compared using tiling analysis software (Affymetrix). Genomic patterns were mapped and analyzed using the integrated genome browser software (Affymetrix). some rearrangements. Materials and Methods Yeast strains and media Tet allele strains were constructed as described previously (Mnaimneh et al. 2004). The genotype of the wild-type Tet allele strain, R1158, is MATa URA3::CMV-tTA his341 leu240 met1540. Using standard genetic methods, 217 MATa Tet allele strains were engineered to contain YFP-Ddc2 marked with a nourseothricin (Nat) resistance gene. Gen otypes for strains used in this study are listed in Table S6. The essential genes that were studied are listed in Table S1 and Table S2. Standard yeast media and growth conditions were used unless otherwise specified (Sherman 1991). CHEF gel electrophoresis and Southern blot analysis Contour-clamped homogenous electric field (CHEF) gels were used to examine intact chromosomes of illegitimate diploids isolated from the mating assay. CHEF gel analysis was performed as described previously (Desany et al 1998). A 1.2 % agarose gel was run at 8 V/cm using pulse times of 120 sec for 30 hr at 14° in 0.5x TBE buffer. PCR- Fluorescence microscopy Tet allele strains were grown in YPD liquid media at 30°, Samples were divided into two cultures and grown in par allel in the presence and absence of 10 Hg/ml doxycycline for 4 additional hours at 23°. Intracellular localization of Ddc2-YFP was determined by fluorescence microscopy as purified fragments were radio labeled by random priming (Stratagene) and used as hybridization probes for Southern blot analysis. PCR primers designed for probe construction are listed in Table S7. E. Cheng et al 148

fullscreen
check_circle

Expert Answer

Step 1

The given experiment was performed to rearrange the segments of the DNA to study its effect on expression of different genes.

Step 2

The purpose of the given study is to analyze the effect of different gene products on DNA stability and its maintenance. Instable DNA also lead to many lethal diseases including cancer. Many researches has been conducted in the past to study the factors that contribute in DNA maintenance and stability. Many collections ...

Want to see the full answer?

See Solution

Check out a sample Q&A here.

Want to see this answer and more?

Solutions are written by subject experts who are available 24/7. Questions are typically answered within 1 hour.*

See Solution
*Response times may vary by subject and question.
Tagged in

Science

Biology

Genetics

Related Biology Q&A

Find answers to questions asked by student like you
Show more Q&A
add
question_answer

Q: How Big Should Each Offspring Be? Can you please use examples for the reading,

A: According to Elgar and Berrigan, there is a negative correlation between size of offspring and clutc...

question_answer

Q: Construct a map of the chromosome, with the most accurate map distances, from the following recombin...

A: Genetic linkage is explained as the closeness of genes on a chromosome (closely packed DNA molecule)...

question_answer

Q: Describe meningitis and the causes

A: Meningitis can be described as the inflammation of the meninges. Meninges are the coverings of the b...

question_answer

Q: When constructing a phylogenetic tree, what problem would horizontal gene transfer pose?

A: Phylogenetic tree:It is also known as dendrogram. It is a diagram that shows the evolutionary associ...

question_answer

Q: Cell reproduction, Miosis and Mitosis.

A: Cell reproduction is the process by which cells divide to form new cells.Each time a cell divides, i...

question_answer

Q: What are the outputs from the light Reactions

A: The light-dependent reactions of photosynthesis take place on the thylakoid membranes. The inside of...

question_answer

Q: Discuss the relationship of the adrenal glands in the sympathetic nervous system

A: Adrenal glands are endocrine glands that are driven by the sympathetic nervous system. It is innerva...

question_answer

Q: If the genetic code were overlapping, how many complete codons would the followingsequence encode be...

A: The process by which mRNA is translated to the amino acid sequence is termed as translation. It occu...

question_answer

Q: In a disorder called gyrate atrophy, cells in the retina begin to degenerate in late adolescence, ca...

A: Gyrate atrophy, an autosomal recessive disorder, which is characterized by chorioretinal degeneratio...