Schizosaccharomyces pombe or fission yeast has been called micromammal due to the potential application of the knowledge derived from the yeast in the physiology of higher eukaryotes. Fission yeast has been consolidated as an excellent model for the study of highly conserved cellular processes. The possibility of using haploid or diploid strains facilitates the analysis of the dominant or recessive phenotype of an allele as well as its function, making it a model of first choice for the development of any investigation in eukaryotes cells. With a growing community that employs fission yeast as a model system for the study of numerous cellular processes, it has motivated the simultaneous development of molecular tools that facilitate the study of genes and proteins in the yeast. In this review, we present the most used molecular techniques in fission yeast for the analysis of genes, its characterization, as well as the determination of its function.
- fission yeast
- gene replacement
- gene expression
- yeast two-hybrid
In its haploid state and in favorable conditions,
Under conditions of nutrient restriction especially nitrogen, cells become arrested in G1, and if the two sexual types (
2. Gene replacement
In fission yeast, gene deletion or one-step gene deletion by gene replacement via homologous recombination is probably the most used molecular tool in the functional characterization of the function of the gene and the protein. Gene disruption is a genetic analysis strategy to achieve gene modifications, generation of tagged protein fusions, genetic expression placed under the control of a regulated promoter, specific mutations, insertions, and deletion [28, 29]. Gene replacement by homologous recombination in
Gene replacement requires a switch construction that contains 5′ and 3′ homologous regions of the target locus that flank a selection marker gene, and its efficiency in homologous recombination depends largely on the size of these regions . The genetic construct is incorporated into the cells by transformation, then the reporter gene that will be used for gene replacement is inserted into the target gene due to the presence of terminal homologous regions in the construct, thus eliminating a large fragment of the target gene and incorporating instead, the reporter gene. At the beginning of the use of this technique, the protocol was based on obtaining the homologous regions to the target gene by digestion to flank a selection marker gene that was obtained from a plasmid containing the desired selection gene as well as the restriction sites resulting from the digestion of the homologous regions.
For this method, it was essential that DNA fragments share the restriction site for subsequent linkage. With the advance in molecular biology, methods based on PCR were developed . The PCR strategy was improved by Wang et al. , and the protocol described the generation of construction switch gene called two-step PCR. Four oligonucleotides are required for the amplification of the homologous regions of the target gene to eliminate or modify. These PCR fragments can be called AB located in the 5′ region and CD located in the 3′ region of the target gene. A novel strategy was a little modification in the 3′ antisense oligonucleotide from AB region and 5′ sense oligonucleotide from CD region, which contains a short complementary sequence and a single restriction site to facilitate the link of the two products generated in the first PCR and then forming a product that serves as template for the second PCR. The final PCR product can be called ABCD and is cloned into a plasmid. At the same time, the gene marker selection used in the gene replacement like leu1 or ura4+ is amplified by PCR with oligonucleotides including the same restriction site in both ends, used in the 3′ antisense oligonucleotide from AB region and 5′ sense oligonucleotide from CD region of target gene to replacement. The PCR product of marker gene is incorporated into a cloning vector, and then it’s digested with the unique restriction enzyme selected. Finally, the plasmid containing ABCD fragment is digested with the same restriction enzyme used to prepare the marker gene and linked to produce the AB-selection marker gene-CD gene deletion cassette.
In order to achieve the gene modification, a one-step gene deletion technique by pop-in homologous must be performed . The gene deletion cassette is transformed into a yeast strain with a deletion in the endogenous gene selected like ura+ (ura4-D18) to the gene replacement  by the lithium acetate protocol . Then, it is efficiently targeted to its homologous location in the chromosome DNA. Moreover, it is widely known that the efficiency of homologous recombination is greatly stimulated if the incoming DNA sequence has free ends. The DNA flanking to the marker gene, on each side, recombines with the genome, inserting the marker gene into the target gene, therefore disrupting or completely replacing it.
It has been reported that the optimal length of homologous sequences to achieve an efficient elimination of the gene is 80–100 pb. Nevertheless, high efficiency in mutagenesis directed for
In addition, to make the functional analyses of various genes as well as minimize incidental recombination events between DNA sequences within the marker gene and a chromosomal sequence, gene deletion cassettes consisting entirely of heterologous DNA sequences have been designated. Those gene deletion cassettes even allow multiple gene deletions to be performed. Because the incorporation of
3. Heterologous protein expression
Fission yeast is a very popular system for protein expression with potential biotechnological applications. The choice of yeasts for the purification of proteins, their structural analysis, and the generation of mutants aimed at knowing the function of proteins is based on the shared conserved biological processes as cell cycle progression, protein turnover, vesicular trafficking, and signal transduction with cells of higher eukaryotes [39, 40]. In yeasts, the appropriate expression of proteins with the posttranslational modifications required allows to obtain the correct protein structure and function. So, the use of yeast in the industrial production of enzymes employed in food, medicine and health, environment, and other applications has been proposed [41, 42]. To fulfill this purpose, “humanized yeast model systems” have been created as tools to study the molecular mechanisms involved in chronic degenerative diseases such as neurological disorders [43, 44]. Due to the accessibility of the yeast to simple genetic and environmental manipulations, it reduced complexity compared to the mammalian models.
Fission yeast is an excellent system to study the complex intracellular mechanisms underlying neurodegenerative diseases such as Alzheimer’s disease (AD). Heterologous expression of Tau and Aβ can provide new insights into the pathobiology of these proteins in vivo as well as the screening of compounds that may be useful in treatment and/or prevention of AD . Recently, it was reported that ginger (dietary condiment) fermented with
Numerous expression vectors have been used in molecular studies on
The use of antibiotics to induce genes to antibiotic resistance genes as selection markers into the yeast plasmid is very frequent. The kanamycin/G418, hygromycin B, phleomycin/bleomycin, and nourseothricin/clonNat are excellent markers in fission yeast . Relative to auxotrophy, new markers such as ade7, his1, his2, his3, his5, arg3, arg12, lys1, lys2, and tyr1 are being developed [51, 52, 53, 54, 55].
However, ade6, his3+, LEU2, and ura4+ remain the most widely used markers for the selection of multi-copy vectors in common use. The pDUAL series and pJK148 vectors have been used to achieve the conversion of the leucine auxotrophy of leu1.32 to leucine prototrophy to select integration at the leu1 locus by recombination as well as pJK210 has been used to rescue ura4.294 to target integration at the ura4 locus .
In regard to the promoters used in the cloning vectors to protein expression, there are many promoters between the most used such as
To solve the problem, other promoters were generated to avoid the inactivation of the promoter nmt1 in the YES culture medium. The promoters of the 276-bp eno and 273-bp gpd were modified from eno101 and gpd3 genes in
As it was mentioned, there are other constitutive promoters widely used in
Therefore, it is necessary to find more efficient promoters for high-expression proteins in
The pREP series vectors are general-purpose episomal vectors widely used in fission yeast research that contains a replication origin ARS1, ura4+, or LEU2 as the selective marker and kan, nat, hph, and bsd genes as a second type of marker of resistance to the specific antibiotics G418, clonNAT, hygromycin B, and blasticidin S, respectively. The latter are used routinely during chromosomal integration. The pREP vectors have been modified to produce novel and versatile plasmids pREP1 and pREP41. pREP1 contains a promoter derived from the gene nmt1. pREP41 contains a moderate-activity promoter (Pnmt41), whereas pREP81 contains a weaker promoter (Pnmt81). pREP vectors that contain ura4+ along with Pnmt1, Pnmt41, and Pnmt81 are named pREP2, pREP42, and pREP82, respectively . The dominant selection marker genes kan, nat, hph, and bsd, which confer resistance against the specific antibiotics G418, clonNAT, hygromycin B, and blasticidin S, respectively, are used routinely during chromosomal integration [66, 67, 68, 69].
Other important kinds of vectors of
The pYZ vectors are derivatives from the pREP series, which were designated for general purposes of cloning and large scale random gene cloning, as well as for allowing positive identification of cloning gene insertion and fusion to the GFP gene for analysis of gene expression. The pYZ vectors were constructed by inserting an
The pREP1, pREP41, pREP81, and pSGA plasmids were generated from the pREP series called pYZ1N, pYZ41N, pYZ81N (N represents an additional
The HIV-1 protease (PR) is a viral enzyme encoded by vpr gene that was initially expressed in
In the molecular analysis of the Zika virus (ZIKV) infection, a large-scale molecular cloning and functional characterization of the viral proteins were performed. The Zika virus (ZIKV) is the causal agent of the microcephaly and the Guillain-Barré syndrome after the viral infection. However, there is insufficient knowledge about how ZIKV viral proteins are involved in cell damage. So,
Other interesting series of vectors are those that were produced as the pREP-X vectors that lack an ATG start codon . Between them, pREP3X (promoter strength high), pREP41X (promoter strength medium), and pREP81X (promoter strength low), the three vectors lack tags and used Leu2 as marker. The pSLF vectors contain N-terminal or C-terminal triple hemagglutinin (3× HA) epitope tag. Between them, pSLF173 (promoter strength high), pSLF273 (medium), and pSLF373 (low), all of them contain 3xHA as tag and use ura4+ as the selective marker and the inducible promoter nmt1. From the pREP-X series were constructed several vectors with the purpose of being utilized for high-throughput functional analysis of heterologous genes in
There are many expression vectors constructed containing a destination cassette suitable for high-throughput cloning of target genes via the gateway system. There are vectors with N-terminal tagging such as the pDES173N, 273 N, and 373 N series, which add a 3XHA tag with the ura4+ gene as marker, and the vectors were constructed from the pSLF173, 273, and 373 vectors. The pDES175N, 275 N, and 375 N series add a GFP tag with the LEU2 marker, and those plasmids were built from the pSLF175, 275, and 375 vectors. The pDES177N, 277 N, and 377 N vectors add a GFP tag using ura4+ as marker selection. The pDES5XN, 45XN, and 85XN series add a RFP tag, with the LEU2 marker, which were derived from the pSLF5X, 45X, and 85X vectors. The pDES179, 279, and 379 series add a RFP tag, with the ura4+ marker, which were derived from the pSLF179, 279, and 379 vectors .
There are vectors with C-terminal tagging; those in the pDes173C, 273C, and 373C series add a 3XHA tag with ura4+ as marker, and the plasmids were constructed from the pSLF173, 273, and 373 vectors. The pDEs175C, 275C, and 375C series add a GFP tag with the LEU2 as marker, and those were constructed from the pSLF175, 275, and 375 vectors. The pDEs179C, 279C, and 379C series that add an RFP tag with the ura4+ marker were constructed from the pSLF179, 279, and 379 plasmids [77, 78]. These vectors exposed above lead the protein expression with N-terminal or C-terminal tagged, useful for the affinity purification or the functional analysis of target genes .
In 2013, an interesting series of vectors was described to PCR-based epitope tagging and gene disruption. The vectors developed were pFA6a-LEU2MX6, pFA6a-his3MX6, and pFA6a-ura4MX6. All of them were designed from the pFA6a-MX6-based plasmid (which contains antibiotic-resistance markers as kan) for amplification of gene-targeting DNA cassettes and integration into specific genetic loci, allowing expression of proteins fused to 12 tandem copies of the Pk (V5) (epitope from the P and V proteins of the paramyxovirus SV5), or 5 tandem copies of the FLAG epitope with a glycine linker. All vectors can use the LEU2, his3+, and ura4 + genes as selection markers. Also, some vectors as pFA6a-G9–5FLAG-kanMX6 and pFA6a-G11–5FLAG-kanMX6 were created, which were generated for studies of proteins when the direct epitope tagging compromises protein conformation and/or function. Other vectors were constructed to add a green fluorescent protein (GFP(S65 T)) or a monomeric red fluorescent protein (mRFP) genomic tagging as FA6A-GFP-bleMX6 .
Between the PK-tagging vectors are the pFA6a-6 × GLY-V5-(marker) and C-terminal FLAG-tagging vectors using KanMX6 and hphMX4 as markers. The FLAG-tagging vectors with N-terminal and C-terminal tags included the pFA6a-6 × GLY-FLAG-(maker), with kanMX6, hphMX6, natMX6, bleMX6, and his3MX6 as possible markers. Between the GFP-tagging vectors are pFA6a-GFP(S65 T)-(maker) and N-terminal and C-terminal GFP(S65 T)-tagging, which include kanMX6, hphMX6, natMX6, bleMX6, and ura4MX6. Also, some disruption plasmids as pFA6a-(maker), which has been used for gene deletions using kanMX6, hphMX6, natMX6, bleMX6, ura4MX6, his3MX6, and LEU2MX6, were constructed .
A novel system to cloning several DNA fragments, into a plasmid, is the Golden Gate shuffling method. Golden Gate cloning [80, 81, 82] is a modular cloning system and was set up for simultaneous overexpression of multiple genes. Some of the applications of the Golden Gate that have been tested in
The use of this methodology for the construction of pREP1-type plasmids that expressed GOI-FPtag was reported
In first place, modules were prepared using the pREP1 vector . A segment from pREP1, which includes ars1 and Amp, was amplified by PCR with a pair of oligonucleotides containing BsaI and NotI sites. A typical expression plasmid for
Recently, pheromone-inducible expression vectors for were developed
In some cases of the heterologous protein expression, the better way to obtain the right protein production host is through its ability to secrete high titers of properly folded post-translationally processed and active recombinant proteins into the culture media. Proteins secreted in their native hosts will also be secreted in the culture medium. Some signal sequences used to secrete the protein into the extracellular space include α-MF and SUC2 invertase. Both are derived from
One of the major problems to the correct production and purification of heterologous proteins from fission yeast is the proteolytic degradation of the recombinant gene product by host-specific proteases. To avoid that problem, a protease-deficient disruptant was constructed set by disruption of 52
In the first place, the protease-deficient disruptant was obtained, which was amplified from genomic DNA of the
To analysed the levels of the secretory production of human growth hormone (hGH), that its known to be a proteolytically sensitive model protein. The results indicated that some of the resultant disruptants were effective in reducing hGH degradation. Although in some cases, added inhibitors of proteasas like Antipain, bestatin, Chymostatin, E-64, Leupeptin, pepstatin, Phosphoramidon, EDTA, aprotininto avoid protein degradation were necessary. Eight protease coding genes useful for reducing degradation of recombinant proteins [isp6 (subtylase type 9 proteinase), pgp1 (endopeptidase), psp3 (subtylase type peptidase), sxa2 (serine carboxypeptidase), ppp51 (aminopeptidase), ppp53 were identified (zinc metallopeptidase), ppp60 (metalloprotease) and ppp80 (peptidase)], the use of a strain lacking the aforementioned enzymes allowed a high level of recombinant hGH production. This publication raised the need to evaluate different proteases to identify those that are the best candidates for the production of recombinant proteins, as well as for functional screening, specification, and modification of proteases in
In relation to the methods for the transformation of
4. Mutants to analyze the function of genes
The use of mutants to analyze the function of genes has been a tool widely used in
In the case of the essential genes, a lethal phenotype is frequently observed. To achieve the study of essential genes, there are two strategies. First, the mutations or gene deletions are created in the diploid state and then the synthetic lethality is studied in the haploid state. Sometimes, it’s possible to observe a slow-growth phenotype, in which haploid cells can partially survive without function of the inactivated gene. Second, the creation of the conditional lethal mutations allows to study a relatively normal gene function under permissive conditions, and then the loss of function is observed under nonpermissive conditions. The most used conditional mutants are the temperature sensitivity, sensitivity to DNA-damaging agents, sensitivity to drugs and inhibitors, and dependence on amino acids or certain carbon sources for viability. Three methods highly used to produce mutants are gene knockouts, random mutagenesis, and site-directed mutagenesis .
The CRISPR/Cas system is a bacterial defense mechanism, and its main function is to identify and degrade exogenous nucleic acid sequences . CRISPR-CAs is organized in an operon, which codes the CAS proteins, and a series of identical repeated sequences separated by other sequences known as spacers, which are recognized by intruding DNA molecules . A part of the nucleic acid stranger is incorporated into the spacer’s zone of the operon using the Cas proteins, which degrade the strange DNA. Next, the transcription of CRISPR-Cas generates a precursor CRISPR-RNA or pre-crRNA, which is then processed to generate crRNAs of small size, which are complementary to the sequence of the foreign DNA. In the last known phase of interference, Cas proteins, using as a guide to crRNAs, detect intruding sequences and degrade them .
The CRISPR/Cas technology allows to identify a specific segment of DNA, remove, or replace it using always the same tools: a duplex RNA with the copy of the DNA to be identified (sgARN) and a short sequence adjacent to the proto-spacer (PAM) that will bind to DNA and stabilize the protein Cas9, protein with endonuclease activity, and helicase guided by the sgARN that separates and cuts the two strands of DNA. A Cas9-gRNA plasmid expressing the active Cas9 enzyme and sgRNA, as well as another plasmid with donor DNA for each deletion are required. The CRISPR-Cas technology allows targeting of multiple genetic manipulations to the same strain, it avoids indirect physiological effects, and it limits the perturbation of the local chromatin and transcriptional environment to the gene manipulation of interest. In fission yeast, this technique has allowed to produce genetic modifications as point mutation knock-in, endogenous N-terminal tagging, and genomic sequence deletion .
Recently, a web-tool called CRISPR4P CRISPR for
A gap-repair-based CRISPR/Cas9 procedure allows to efficiently knockin a point mutation in fission yeast. The rpl42-P56Q mutation confers cycloheximide resistance (CYHR) . Employing this technique, a CCC codon for proline was changed, and with the use of a pair of 90-nt complementary oligos as donor DNA, the gap repair procedure achieved a high editing efficiency (84%).
Using the CRISPR-Cas9, yeast strains, functional and successfully complemented with the markers ura4-D18, leu1-Δ0, his3- Δ0, and lys9-Δ0, were created. To achieve the goal, all the components were assembled with the “BsaI-pad,” a single 42 bp region containing two BsaI cutting sites to produce the plasmids pYZ182, pYZ183, and pYZ184 with nmt1, nmt41, and nmt81 cassettes, respectively. Using that design, the marker genes ura4, leu1, his3, and lys9 were integrated separately. Later, the plasmids were transformed into yeast .
Recently, the type VI CRISPR system, Cas13a from Leptotrichia shahii (LshCas13a), was employed to introduce genetic changes on the DNA, disrupting or editing to target and knockdown endogenous gene transcripts with different efficiencies in
RNA interference (RNAi) is a highly conserved eukaryotic gene regulatory mechanism, which uses small noncoding RNAs to mediate posttranscriptional gene silencing as a host defense mechanism. It was described that
siRNA is generated by the Dicer family endoribonuclease Dcr1, from double-stranded noncoding RNA that is complementary to heterochromatin. The siRNA duplex is loaded onto a non–chromatin-associated complex called Argonaute, small interfering RNA chaperone (ARC), which contains the Ago1 endoribonuclease. The loading of the siRNA duplex onto the Ago1 subunit requires the two ARC-specific subunits, Arb1 and Arb2, which also inhibit the release of the passenger strand . Thus, this complex changes its subunits’ composition to form a chromatin-associated effector complex called RNA-induced transcriptional silencing (RITS) . The RITS complex is composed of Ago1, now binding single-stranded siRNA as a guide for target recognition, and the two RITS-specific subunits: Chp1 and Tas3. Chp1 uses a chromodomain to recognize H3K9me, whereas Tas3 bridges Ago1 and Chp1 .
To analyze the role of the RNAi in fission yeast, the lacZ fission yeast system was employed. With this system, it was possible to know that the gene inhibition is dependent on the dose of the antisense RNA, the size of the antisense transcript, as well as the targeted region. Any of them can affect the efficacy of target gene inhibition. The generation of dsRNA through either intermolecular or intramolecular hybridization is central to make the antisense RNA-mediated gene silencing in
An interesting proposal to analyze the role of the siRNAs in
7. Yeast two-hybrid system
The yeast two-hybrid system (Y2H) is a method widely employed to study the physical interaction of proteins by the downstream activation of a reporter gene. Considering that many eukaryotic transcription factors are organized in a modular way with at least two domains, it is possible to separate them into their domains .
In this assay, two plasmids are created; the first is named the bait plasmid including the DNA-binding domain of a transcription factor joined to one of the proteins to analyze and it is named Bait. In this vector, a selection marker is included such as HIS3, ADE2 (Gal4 system), or LEU2 (LexA system with binding sites for the DNA-binding domain). The second vector is named prey including the activation domain of the transcription factor joined to the second protein to study in the interaction, named Prey. As in the other vector, a different selection marker is included. When the Bait and Prey proteins are put together by protein interaction, they restored the organization of the transcription factor, and then they can activate the transcription of the reporter gene as the
Gal4 is a transcriptional activator in yeast that binds to UAS (upstream activation domain), a specific DNA sequence, and activates transcription in the presence of galactose. The separation of Gal4 in two fragments produces N-terminal DNA-binding domain (DBD) and C-terminal transcriptional activation domain (AD), but did not activate transcription in the presence of galactose until both domains are associated to reconstitute a fully functional Gal4. Some disadvantages of the assay consider that in some cases, it’s necessary to modify the bait proteins because a protein with both DNA-binding and transcriptional activating properties is possible to be found. Some fused proteins may not be able to enter or be expressed in the yeast nucleus. The GAL4 BD has its own nuclear localization signal (NLS). If the GAL4-based Y2H system fails, the interaction could be analyzed and detected successfully using a LexA-based Y2H system [110, 111].
The Y2H system has been widely used. In
To the assay, the entire
8. DNA microarray
DNA microarray is an orderly set of segments of genes that are immobilized on a surface called chip. The DNA arrangements allow the massive study of the gene expression of an organism, and it allows to know the differences of gene expression between two samples of RNA in a given cellular condition. In cells that present some mutation or elimination in some genes or cells derived from individuals with some infectious disease or not, the microarrays allow the identification of sets of genes related to the gene or genes under study or the condition of disease. Comparing RNA prepared from diseased cells and normal cells can lead to the identification of sets of genes that play key roles in diseases. Genes that are overexpressed or underexpressed in the diseased cells often present excellent targets for therapeutic drugs.
The application of DNA microarray technology requires a genomic library conformed by a set of DNA segment derived from each of the genes of the model of interest, which is generated from PCR products or synthetic oligonucleotides, as well as the design and construction of the arrangement, to determine the physical location and accurate identification for the analysis and interpretation of gene expression data. Microarray analysis requires total RNA extraction from control and the problem obtained by any strategy optimized for certain cell type . Total RNA control and the problem should be submitted to retrotranscription incorporating uracil marked with a fluorescent molecule as dUTP-Cy3, dUTP-Cy5, dUTP-Alexa 555, dUTP-Alexa 647, and biotin, among others. The labeling of the cDNA must be differentiable between the two tissues to be analyzed . The hybridization of the microarray containing probe sets that represent a finite number of transcripts is carried out. Fluorescence reading is obtained with a microarray reader. The quantification of the signal produced by the fluorescence of the spots allows to calculate for each point the mean density value of the nucleotides marked cDNA (g. e. of Alexa555, Alexa647) and the average value of the background. To identify the genes expressed differentially in the experiment, it is necessary to perform a statistical analysis, from the normalization of the data. The goal is to analyze those genes that move away from normalization through the value of Z . The genes with the value of Z > 2 present a statistically significant change between the experimental condition and the control (genes with greater or lesser expression). . Easy and useful software for data analysis of microarrays is GenArise (computer unit of the Institute of Cellular Physiology of UNAM (http://www.ifc.unam.mx/genarise/).
From the data that record a significant change, it is necessary to determine its association to some biological processes by clustering analysis for gene expression .
With this molecular tool, it was possible to analyze in fission yeast the effect of Spc1, a mitogen-activated protein kinase in the stress responses. Spc1 is an activator of transcription factors that control gene expression in response to extracellular stimuli and is also known to interact with the translation machinery. Using microarrays of Affymetrix GeneChip Yeast Genome 2.0 Array, it was possible to know the set of genes that is regulated by SPC1, and this analysis was carried out without and with a stress condition to evaluate the effect of the wild-type SPC1 kinase and Spc1K49R, a mutant of this enzyme. Spc1 and Spc1K49R were separately overexpressed in
In 2016, the role of the putative NO dioxygenase SPAC869.02c (Yhb1) and the S-nitrosoglutathione reductase Fmd2 was analyzed. Both proteins are NO-detoxification enzymes. In the study, it was found that exogenous NO protects
The fission yeast
9. Next-generation sequencing
Next-generation sequencing (NGS) involves the parallel mass sequencing of thousands of DNA fragments. Sample processing for NGS can be summarized as follows: First, nucleic acid extraction (DNA or RNA). Second, selection of the type of NGS sequencing (targeted sequencing, whole exome sequencing, and whole genome sequencing). Third, library generation by DNA fragmentation, ligation of adaptors, and amplification and sample enrichment. Fourth, template generation or cluster generation according to the platform of sequencing. Fifth, sequencing (using a specific platform as Illumina, PacBio). Sixth, data analysis. Data analysis includes the quality evaluation of the sequence, alignment to reference sequence to identify some possible variations such as single nucleotide polymorphism (SNP) or insertion-deletion (indel) identification, phylogenetic or metagenomic analysis, as well as the identification, interpretation, and classification of pathogenic variants [122, 123].
Splicing is an essential step in eukaryotic gene expression. Introns are excised by the spliceosome, composed of five uridine-rich small nuclear RNAs (U1, U2, U4, U5, and U6 snRNAs) and several polypeptides. To characterize the U2·U5·U6 complex of
There are some other techniques to study several aspects of the physiology of
Finally, it is necessary to mention that Rallis & Bähler offered to the world pombe community an excellent review showing the relevance of