Bioinformatic analysis and expression of Psc3 intronic sequences

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Bioinformatic analysis and expression of psc3 intronic sequences

Prediction of structural features within psc3 introns

The overall strategy of predicting structures within the introns of the psc3 gene was to first identify orthologues of the encoded protein in diverse fungal species, retrieve the cDNA for multiple alignments in order to identify and confirm introns within Psc3, and then conduct structure prediction analyses on intron consensus sequences.

Identification of orthologous proteins in fungi

The first step in the identification and construction of the S. pombe psc3 introns was to create a subset of all homologous proteins within fungi through searches of publicly accessible protein sequence databases. Searches were conducted using BLASTP as part of the BLAST (Lopez, 2003) toolkit available from the European Bioinformatics Institute (www.ebi.ac.uk). BLAST searches employed default settings (matrix BLOSUM62, topcombo 1, normal sensitivity, 1xE10) and the Psc3 protein as query template. The corresponding cDNA and genomic DNA sequence for each Psc3 orthologue was also retrieved for sequence alignment purposes. In cases where an organism contained multiple Psc3 paralogues, only the paralogue with the highest score was used.

Identification of orthologous introns in fungal cohesins

In this study, an orthologous intron was defined as a DNA sequence that was found between two corresponding exons of the orthologous psc3 gene, as long as this sequence was present in the majority of fungal species retrieved in the BLASTP search. Orthologous introns in the psc3 genes from the various fungal species were identified by BLASTN using the intronic sequences of S. pombe psc3. Orthologous intronic sequences were confirmed by mapping them to the corresponding positions within the primary sequences. Alignment of putative intron sequences with the reverse-translated cDNA sequences was conducted using the multiple alignment tool of MEGA (Tamura et al., 2011). In cases where the psc3 orthologous gene did not contain any introns or did not possess the corresponding intron, these sequences were excluded from subsequent analyses. All of non-excluded genes were divided into five groups based on the phylogenetic distance.

Prediction of structure within introns of psc3

The realignment of intron sequences for the purposes of structure prediction was done for each of the five groups individually using ClustalOmega (Sievers et al., 2011). To predict the most stable RNA structure for each intron, sequence alignments were uploaded to RNAalifold program via the web server http://rna.tbi.univie.ac.at/cgi-bin/RNAalifold.cgi. The predictions were performed using RIBOSUM scoring and minimum free energy with partition function (Bernhart et al., 2008).

Detection of ncRNA transcripts from psc3 intronic sequences by PCR

Strains and culture and storage conditions of S. pombe

The list of strains used in this study is given in Table 2.1. Long-term storage of all S. pombe strains was by freezing in YES media containing 25% v/v glycerol. The wild-type strain used in this work was genotype h-ade6-M26 ura4-D18 leu1-32 (BP90, McFarlane Collection). Routine growth of S. pombe strains, unless specified otherwise, was done using a rich yeast extract-based medium (YES), which contained 5 g/l of yeast extract, 28 g/l of Glucose and 250 mg/l of each supplement Adenine, Uracil, Leucine, Histidine and lysine. Solid YES for cell growth on petri dishes was made with the addition of agar at 14 g/l. and incubated it at 30oC for 3 days.

Isolation of total RNA

Wild-type cells were grown in 25 ml of YES at 30oC with shaking at 200 r.p.m until they had reached on O.D.600 = 0.2. Cells were harvested by centrifugation for 2 minutes at 2,000 r.p.m in a bench-top centrifuge capable of holding 50 ml conical centrifuge tubes. The supernatant was decanted and the cells frozen in liquid N2. Total RNA was extracted by the hot phenol/chloroform method according to (Lyne et al., 2003) and described in more detail here. Frozen cells (~50 mg) were thawed on ice for 5 minutes, resuspended in 1 ml of pre-chilled DEPC-treated water (0.1% w/v DEPC) and transferred to 2 ml microfuge tubes. The cells were pelleted by centrifugation for 10 second at 5,000 r.p.m and washed once in DEPC water and collected by centrifugation. A volume of 750 µl of TES (10 mM Tris pH 7.5; 10 mM EDTA pH 8; 0.5% SDS) was added to the tube and the cells were gently resuspended with a pipette, and then 750 µl of pre-chilled acidic phenol-chloroform (Sigma P-1944) was added and the cells mixed by vortex, and incubated at 65°C for 1 hour in a water bath or heating block.
The sample was placed on ice for 1 minute, vortexed for 20 seconds, and then centrifuged at 14,000 r.p.m at 4°C for 15 minutes. A volume of 700 µl of the supernatant was transferred to a 2 ml, yellow phase-lock tube, which held 700 µl of acidic phenol-chloroform. The solution was mixed by inverting the tube and then centrifuged at 14,000 r.p.m at 4°C for 5 minutes. An aliquot of 700 µl of the top phase of the tube was transferred to a 2 ml, yellow phase-lock tube containing 700 µl of chloroform:isoamyl alcohol (24:1)
(Sigma C-0549). The solution was mixed by inverting the tube and the phases separated by centrifugation at 14,000 r.p.m at 4°C for 5 minutes. A volume of 500 µl of the supernatant was transferred to a 2 ml Eppendorf tubes containing 1.5 ml of 100% EtOH (-20°C) and 50 µl of 3 M NaAc, pH 5.2, vortexed for 10 seconds, and the RNA precipitated by incubating the tube at -20°C overnight. The following morning the RNA was collected by centrifugation at 14,000 r.p.m at room temperature for 10 minutes. The supernatant was removed, the RNA pellet washed with 500 µl of pre-chilled 70% EtOH (made with DEPC water) and then centrifuged at 14,000 r.p.m at room temperature for 1 minute to collect the RNA. The RNA sample was dried for 10-15 minutes by inverting the tube on a clean tissue. A volume of 100 µl of DEPC-treated water was added to the tube and it was kept at room temperature for 10 minutes and then resuspended by pipette.

Quality and quantity of RNA

The quality and quantity of RNA was determined by the absorbance measurements at 260 nm and 280 nm. The absorbance at 260 nm was used to quantify the amount of RNA according to 1 O.D. corresponding to a concentration of 40 µg/ml. The ratio of O.D.260/O.D.280 provided the measure of the purity of RNA with a good quality preparation having a ratio between 1.6 and 2.0 (Sambrook et al., 1989). The quality of RNA was also visualized by agarose formaldehyde gel electrophoresis using standard protocols.

Reverse Transcription

Potentially contaminating DNA was removed from total RNA samples by adding 1 μl DNAse (1 U/μl) to 1 μg of total RNA in a total volume of 10 μl. Samples were incubated at 37°C for 30 minutes followed by the addition of 1µl of Stop solution and a further incubation of 10 minutes incubation at 65°C to inactivate the DNase. First-strand cDNA was produced by random-priming from 1 μg of total RNA using the QuantiTectTM Reverse Transcription kit according to manufacturer’s instructions (QIAGEN Ltd.). Each reaction mix consisted of 2 µl of 1x gDNA Wipeout Buffer, 1 μg of DNAse-treated total RNA, which was brought to a final volume of 14 μl using RNase-free water. The sample was incubated for 2 minutes at 42°C and then it was transferred immediately to ice. A volume of 1 μl of QuantiscriptTM (Qiagen Ltd.) reverse transcriptase, 4 μl of 5x QuantiscriptTM RT Buffer and 1 μl of RT Primer Mix were combined and added to the 14 μl of RNA template, mixed and incubated at 42°C for 15 minutes. The sample was incubated at 95°C for 3 minutes to inactivate the reverse transcriptase and the sample was diluted to an estimated concentration of 10 ng/µl for use in PCR reactions. The remainder of the total RNA sample was stored at -20°C until needed.

Design of primers for PCR

Three different types of primer pairs were designed to determine the expression of intronic sequences of the psc3 gene, an exon pair with the forward and reverse primers spanning the intron, a junction pair covering the exon/intron boundaries of each intron, and only intron primers. Primers for ncRNA genes were made as a control to test for expression of unrelated ncRNAs. Primers were designed using the OligoPerfect™ primer design tool from Invitrogen (tools.lifetechnologies.com) using genomic DNA sequences from Pombase (www. Pombase.org) with the constraint that all would have an annealing temperature of approximately 57oC. All PCR primers were obtained from eurofins Genomics (http://www.eurofinsgenomics.eu). The primers used in this study are given in Tables 2.2 and 2.3.

Polymerase Chain Reaction

All primers were tested by PCR on appropriate DNA or cDNA templates to ensure that the primers generated the expected size of fragments. The PCR reactions were prepared in a volume of 10 μl comprised of 1x GoTaq Flexi Buffer, 3 mM magnesium chloride, 0.2 mM of each dNTP, 0.05 µM of forward and reverse primers, 0.25 U of GoTaq DNA Polymerase, 10 ng of cDNA or DNA, and brought to the final volume with DEPC-treated water. PCR was conducted on a MJ Research thermocycler with the following cycling parameters: an initial denaturation step of one cycle of 1 minutes at 94°C, 34 cycles of 15 seconds at 94oC, 15 seconds at 55oC, and 15 seconds at 72°C, followed by an extension period of 72ºC for 5 minutes.

Visualisation of PCR products by UREA-PAGE

Polyacrylamide gels were used in preference to agarose gels for determining the amplification success of fragments less than 100 bp. amplicons. All PAGE was run using the MiniproteanTM gel electrophoresis system from BioRad. Gels were poured at a thickness of 1.5 mm with a final composition of 1x TBE buffer (1 M Tris, pH 7.5, 0.9 M boric acid, 0.01 M EDTA ), 15% bis-acrylamide and 8 M urea, 0.06 % v/v ammonium persulfate, and 0.05% TEMED. The gel was pre-run for 15-30 minutes at 200 V using 1x TBE. PCR samples were prepared in 1x Nucleic Acid Sample Loading Buffer (BioRad, #161-0767), loaded into the wells and the gel run at 200 V until just before the blue exited the gel.

Generation of mutant strains of S. pombe

Biological Material

Three different strains of E. coli were used in this project. These strains had the pFA6a-KanMX6, pFA6a-NatMX6 and pAW1 plasmids used to amplify the marker genes used for gene replacement mutagenesis of S. pombe. For long-term storage, bacterial cultures containing 25% glycerol were kept at -80oC.
Bacteria were growth on LB-agar plates consisting of 10 g/l (w/v) of tryptone, 5 g/l (w/v) of NaCl and 15 g/l (w/v) of agar or in liquid LB media without agar for isolation of nucleic acids. Agar plates and liquid cultures were incubated at 37oC with liquid cultures being agitated at 160 r.p.m in a rotary shaker. All powdered media was dissolve in MilliQTM H2O and sterilized by autoclaving. For screening cells by antibiotic resistance, media were allowed to cool to 55°C before addition of the appropriate antibiotic (100 µg/ml of Ampicillin, 50 µg/ml kanamycin or 100 µg/ml nourseothricin). The background genotype of S. pombe for mutagenesis and how it was grown is provided in Section 2.1.2.1.

Isolation of plasmid DNA from E. coli

Strains of E. coli were grown in LB broth containing the appropriate antibiotic at 37oC with rotation at 150 r.p.m until an O.D.600 = 0.5 was reached. Plasmid DNA was extract from a total of 3 ml of the culture using the alkaline lysis method according to (Sambrook et al., 1989). The bacterial pellet was resuspended in 100 µl of ice-cold Solution I (25 mM Tris-HCl, pH8.0, 50 mM glucose, 10 mM of EDTA) and allowed to sit for 5 minutes at room temperature. To the tube was added 200 µl of fresh Solution II (0.2 N NaOH, 1% w/v SDS) and the sample was mixed by gentle agitation of the tube for 10 seconds. The tube was incubated on ice for 5 minutes and then 150 µl of ice-cold Solution III (3 M KAc, pH 4.8), was added to the tube and the sample gently mixed by vortex for 10 seconds with the tube inverted. The sample was centrifuged in an Eppendorf centrifuge at 4°C for 5 minutes at 14,000 r.p.m. The supernatant was transfer to 2.0 ml PhaseLockTM microfuge tube (5 Prime GmbH, Hamburg, Germany) and an equal volume of phenol / chloroform was added to the tube. The sample was mixed by vortex and then centrifuged for 2 minutes at 14,000 r.p.m in a microcentrifuge. The supernatant was transfer to fresh tube and two volumes of 100% ethanol (-20°C) was added. The sample was mixed by vortex and allowed to incubate at room temperature for 2 minutes. The tube was centrifuged for 10 minutes at room temperature to pellet the DNA and the supernatant was removed by pipette and discarded. A volume of 500 µl of 70% (v/v) ethanol in H2O (kept at -20°C) was added to the pellet, the sample gently mixed by inverting the tube several times, and the DNA pelleted by centrifugation for 5 minutes at room temperature. The supernatant was decanted and the sample was dried by standing the tube in an inverted position on a paper tissue. The DNA was resuspended in 50 µl of TE buffer, pH 8.0. To each sample was added 1 µl of 10 mg/ml RNase A (Novagen., UK), and the tube was incubated at 37oC for 15 minutes. The concentration and quality of DNA was estimated by absorbance measurements at 260 nm and 280 nm using a spectrophotometer, and an aliquot of the DNA was routinely visualised for quality by agarose gel electrophoresis and stained with SafeViewTM (NBS Biologicals, Cambridgeshire, UK).

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Design of primers for amplification of transformation templates

The primer sequences used to generate the fragments for gene replacement by homologous recombination of hid1+, hid2+ and hid3+ were obtained using the primer design strategy specified in (1998) and which is publically available as a design tool at http://128.40.79.33/cgi-bin/PPPP/pppp_deletion.pl. Each primer was 120 bases in length and contained a 20 base segment corresponding to the cassette sequence flanking the marker genes linked to 100 bases of a gene specific sequence (Table 2.4). The marker genes used in this project were the auxotrophic gene ura4+, the aminoglycoside phosphotranspherase (KanR), and the nourseothricin acetyltransferase (NatR) gene. The plasmid templates from which the marker genes were amplified were pAW1 containing the ura4 gene (Watson et al., 2008) , the pFA6A-KanMX and pFA6A-NatMX (Hentges et al., 2005).

Amplification and preparation of templates for gene replacement by homologous recombination.

Isolated plasmid containing the appropriate marker gene was linearized with an appropriate restriction enzyme to improve the PCR efficiency. pAW1, pFA6a-kanMX6 and pFA6a-natMX6 were cut with Hind III, NdeI and EcoRI, respectively. Each restriction digest consisted of 5 µg of DNA, 1x restriction buffer, and 3 Units/µg of DNA restriction enzyme in a total volume of 40 µl. Digests were conducted at 37°C for 1-2 hours and the efficiency of cutting was determined by agarose gel electrophoresis.
The PCR reactions to amplify the transformation sequences were prepared in following manner for a reaction mix of 400 μl: 50 μl of each primer (Stock 5 μM), 200 μl of MyTaqTM HS Red Mix (BIOLINE), and 50 μl of plasmid DNA (10 ng/µl) mixed with 100 μl MilliQTM purified water. This was mixed was divided into 8 PCR tubes and the reactions performed using an MJ Research thermocycler with the following cycling conditions: an initial heating of one cycle at 95°C for 1 minutes, 34 cycles of 15 seconds at 95ºC, 15 seconds at 52.2°C the appropriate annealing temperature and 45 seconds at 72°C. The PCR reaction was terminated with elongation at 72ºC for 5 minutes. The PCR products were purified according to the method of (1998). An equal volume of phenol/chloroform was added to a phase lock tube containing the pooled PCR reaction mix. The sample was mix gently by inverting the tube repeatedly and the sample centrifuged at 14,000 r.p.m at room temperature for 5 minutes. The aqueous layer was transferred to clean microfuge tube and 0.1 volumes of 0.1 M NaCl and then three volumes of 100% ethanol (-20°C) were added to the tube. The tubes were placed at -80°C for two hours in order to facilitate the precipitation of DNA. The sample was placed at room temperature for 5 minutes and the DNA collected by centrifugation at 14,000 r.p.m at 2°C for 15 minutes. The supernatant was removed and the DNA pellet wash with 500 µl of 70% EtOH (kept at -20°C), mixed gently to minimize perturbation of the pellet and then centrifuged at 14,000 r.p.m at 2°C for 5 minutes. The supernatant was removed and the sample was allowed to dry for 5 minutes by inversion on a clean tissue. A volume of 13 µl of 1 x TE buffer was added to the sample and the DNA was used directly for transformation of S. pombe cells.

Transformation of S. pombe cells

S. pombe cells were transformed with the PCR fragments using the standard LiAc/TE transformation protocol according to (Bähler et al., 1998; Keeney and Boeke, 1994). Cells transformed with fragments carrying the NatR marker were plated onto two YES plates containing 100 μg/ml clonNATTM (Werner BioAgents, Jena Germany) and the plates were incubate at 30°C for 3 days. The colonies were re-streaked onto fresh YES plates containing clonNAT, allowed to grow for 3 days. Cells transformed with fragments carrying the KanR marker were plated onto two YES plates and incubated at 30°C for 18 hours. When the lawn of cells was visible the plates were replicated onto fresh YES plates containing 100 μg/ml G418 Sigma-Aldritch Ltd) and incubated at 30°C for 2-3 day. The largest colonies were re-streaked onto fresh YES plates containing G418. Cells transformed with fragments carrying the ura4+ marker were plated onto two EMM plates without uracil and incubated at 30°C for 3 days. The colonies were re-streaked onto fresh EMM plates without uracil.

Verification of gene replacements

The success of mutant strain creation by gene replacement was verified both by genotyping the mutant strains and demonstrating the lack of corresponding transcript.

Isolation of genomic DNA from S. pombe

All strains were grown in liquid cultures of 5 ml of YES to late log-phase, and 3 ml of cells were harvested by consecutive centrifugation in a microcentrifuge spun at 6,000 r.p.m for 5 minutes. The supernatant was removed and the pellet was washed in 1 ml of sterile distilled H2O containing 0.1% (w/v) Sodium azide. DNA was isolated from cell using the WizardTM Genomic DNA isolation kit according to manufacturer’s instructions (Promega). In brief, the solution was centrifuged at 6,000 r.p.m for 2 minutes to pellet the cells and the supernatant was removed. The cells were resuspended thoroughly in 300 μl of a buffer containing 50 mM Na2HPO4, 11.5 g/l citric acid, 40 mM EDTA, 1 M sorbitol, and 0.1 mg/ml Zymolyase® 20T (Amsbio) and the tubes incubated at 37°C for 30 minutes to digest the cell wall. After cooling the tubes to room temperature (~ 2 minutes), the cells were again pelleted by centrifugation in microfuge at 13,000 r.p.m for 2 minutes, and the supernatant discarded. Next, 300 μl of Nuclei Lysis Solution was added to the tubes, the pellets were resuspended by gentle mixing and 100 μl of Protein
Precipitation Solution was added. The tubes were vortexed vigorously for 20 seconds and the tubes incubated on ice for 5 minutes. After centrifugation for 2 minutes and the supernatant containing the DNA was transferred to a clean 1.5 ml microcentrifuge tube containing 300 μl of isopropanol at ambient temperature and gently mixed by inverting the tubes until the DNA precipitate became visible. The DNA was collected by centrifugation, the supernatant was removed, and the pellet was washed with 300 μl of room temperature 70% ethanol by inverting the tubes several times. The DNA pellet was recollected by centrifugation for 2 minutes, the ethanol was discarded, and the pellet was allowed to dry by leaving the tube inverted on clean absorbent paper for at least 10 minutes. The DNA was dissolved in 50 μl of DNA Rehydration Solution to which was added 1.5 μl of a 1 mg/ml RNase Solution and the tubes incubated at 37°C for 15 minutes. Finally, the DNA was left to rehydrate overnight at 4°C.

 Mutant genotyping and expression analysis by PCR

Four sets of primers were created to verify replacement of the endogenous hid gene by the selectable marker gene: a primer pair flanking the complete insertion site, two pairs of primers consisting of genome-specific and marker specific oligonucleotide to amplify the junction regions at both ends, internal cDNA primers to demonstrate removal of the hid gene, and a primer pair for marker gene insertion. All primers were designed online using the Primer3 program (http://biotools.umassmed.edu/bioapps/primer3_www.cgi) and the S. pombe gene sequences from Pombase as templates. The list of primers is given in Table 2.5. PCR was conducted using 10 ng of genomic DNA as described in Section 2.1.2.6. Total mRNA isolation and RT-PCR were conducted as described in Sections 2.1.2.2 to 2.1.2.4. The success of PCR reactions was determined visually by agarose gel electrophoresis.

Table of contents :

I. Chapter 1: General Introduction 
1.1. Nucleic acids and general transcription
1.2 Gene expression and its regulation
1.2.1 The transcriptional machinery
1.2.2 Chromatin structure
1.3. The basic biology of cancer
1.3.1 Genes involved in carcinogenesis: Oncogenes and tumour-suppressor genes
1.4 Gene expression in cancer
1.4.1 Regulation of transcriptional by Ubiquitin
1.4.2 Ubiquitination and Transcription Factor activity
1.5 Introduction to the Golgi apparatus
1.5.1 Golgi apparatus in yeast
1.5.2 Signaling through the Sterol receptor element binding protein (SREBP) pathway
1.5.3 GOLPH3
1.5.4 GOLPH3 function and cancer
1.6. Human HID1 and its role in cancer development
1.7 Introduction to non-coding RNAs
1.7.1 Classes of ncRNA
1.7.1.1 Ribosomal and transfer RNAs
1.7.1.2 Regulatory ncRNAs
1.7.1.2.1 .LncRNAs
1.7.1.2.2 sncRNAs
1.7.1.2.2.1 miRNAs, siRNAs and piRNAs
1.7.1.2.3 snoRNAs
1.7.2 General features of ncRNA function
1.7.3 ncRNAs and cancer
1.7.4 Modeling small RNA structures and determining expression
1.8 RNAseq as a gene expression tool
1.9 S. pombe as a model organism
1.9.1 The cell cycle in S. pombe
1.10 Objectives of the work and strategy of the thesis
II. Chapter 2: Materials and Methods 
2.1 Bioinformatic analysis and expression of Psc3 intronic sequences
2.1.1 Prediction of structural features within Psc3 introns
2.1.1.1 Identification of orthologous proteins in fungi
2.1.1.2 Identification of orthologous introns in fungal cohesins
2.1.1.3 Prediction of structure within introns of Psc3
2.1.2 Detection of ncRNA transcripts from Psc3 intronic sequences by PCR
2.1.2.1 Growth of S. pombe
2.1.2.2. Isolation of total RNA
2.1.2.3 Quality and quantity of RNA
2.1.2.4 Reverse Transcription
2.1.2.5 Design of primers for PCR
2.1.2.6 Polymerase Chain Reaction
2.1.2.7 Visualisation of PCR products by UREA-PAGE
2.2 Generation of mutant strains of S. pombe
2.2.1 Biological Material
2.2.2 Isolation of plasmid DNA from E. coli
2.2.3 Design of primers for amplification of transformation templates
2.2.4 Amplification and preparation of templates for gene replacement by homologous recombination.
2.2.5 Transformation of S. pombe cells
2.2.6 Verification of gene replacements
2.2.6.1 Isolation of genomic DNA from S. pombe
2.2.6.2 Mutant genotyping and expression analysis by PCR
2.2.6.3 Transmission electron microscopy
2.3 Characterisation of gene expression
2.3.1 Preparation of material and isolation of mRNA
2.3.2 Calculation of gene correlations
2.3.3 Primer design for quantitative PCR of genes correlated to the Hids
2.3.4 Gene expression by RT-qPCR
2.3.4.1 Analysis of RT-qPCR data
2.3.5 RNAseq and differential expression analysis
2.3.6 Small size selected RNAseq and novel ncRNA transcript detection
2.3.7 snoRNA prediction from novel ncRNA transcripts
2.4 General Data Analysis.
III. Chapter 3: Production of hidΔ mutants of S. pombe 
3.1 Introduction
3.2 Results
3.2.1 Creation of mutants by gene replacement
3.2.2 Verification of gene deletion and replacement by PCR-genotyping and RT-PCR:
3.2.3 Construction of single mutants
3.2.3.1 Confirmation of strains lacking Hid1
3.2.3.2 Confirmation of strains lacking Hid2
3.2.3.3. Confirmation of strains lacking Hid3
3.2.4 Identification of Vector Control strains
3.2.5 Construction of double mutants
3.2.6 Initial physiological studies of gene-replacement mutants
3.2.6.1 Morphology of single mutants
3.2.6.2 Characterisation of growth properties.
3.2.4.3 Preliminary ultrastructural characterisation of the mutants
3.3 Discussion
IV. Chapter 4: Transcriptional properties of the hid1Δ and hid3Δ mutants of S. pombe 
4.1. Introduction
4.2 Results
4.2.1 Evaluation of HsHID1 as a Tumour Suppressor gene
4.2.2 Comparative expression of hid genes using RT-qPCR
4.2.3 Evaluating the potential regulation of co-expressed genes by RT-PCR
4.2.4 Gobal analysis of gene expression changes in hid1Δ and hid3Δ by RNAseq
4.2.4.1 Strategy of sample selection and evaluation of RNA quality
4.2.4.2 RNAseq reveals greatest differences in gene expression for hid3Δ
4.2.4.3. Validation of RNAseq data by qRT-PCR
4.2.4.4. Analysis of correspondence with genetic interaction data provided in PomBase.
4.2.4.5. Finding biological function through gene ontololgies
4.2.4.6. Changes in expression of specific genes
4.3 Discussion
V. Chapter 5: Analysis of structural features of introns and their expression in fission yeast based on the pcs3 gene family 
5.1. Introduction
5.2. Results
5.2.1. Identifying orthologous introns in fungal cohesins
5.2.2. Alignment of orthologous introns and secondary structure prediction
5.2.3. Investigation of intron expression by RT-PCR
5.2.4. Investigating the potential for intron expression by RNAseq
5.3 Discussion
VI. Chapter 6: General Discussion and Outlook 
6.1 Outlook and Future Work

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