Cerevisiae and Other Fungi: Principles of Gene Manipulation by Liuzengran Hebei University of Economics and Business

CHAPTER 9
Cloning in Saccharomyces cerevisiae and other fungi
Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business
Demands Need to master the specialist vectors, including speciality
be familiar with plasmids used in fungi gene manipulations and S. cerevisiaes features Need to know the transformation methods of fungi, the dual-bait (two-hybrid system how to identify genes encoding particular cellular activities
Class hour: 4h
Homeworks
One : Explain S. cerevisiae Special position in gene engineering Two : Compare the eukaryotic expression and prokaryotic expression
Main contents 9.1 Introduction 9.2 Plasmid vectors for use in fungi 9.3 Expression of cloned genes 9.4 Overexpression of proteins in fungi
9.5 Specialist vectors

9.6 Identifying genes encoding particular cellular activities
9.7 Determining functions associated with particular genes

9.1 Introduction
9.1.1 Property of yeast expression system(6) 1 The analysis of eukaryotic DNA sequences The analysis of the rearranged sequences must be As eukaryotic DNA sequences has been taken out of the facilitated by cloning but recipient bacteria eukaryotes DNA in and reintroduced prokaryotes using into a eukaryotic vectors donor.
2 there are many functions common to eukaryotic cells which are absent from prokaryotes
Localization() of ATP-generating systems to Mitochondria(), association of DNA with histones, mitosis() and meiosis(), and obligate differentiation() of cells. The genetic control of such functions must be assessed in a eukaryotic environment.
3 The cellular biochemistry and regulation of yeast are like those higher eukaryotes.
Signal transduction and transcription regulation by mammalian steroid ( receptors can be mimicked in strains of S. cerevisiae expressing receptor sequences
4 many homologues genes of humans in yeast

e.g. those involved in cell division
Yeast be a very good substitute host for studying the structure and function of eukaryotic gene products
5 Yeast cells easier to grow and easier to manipulate than plant and animal cells. 6 No special virus, no metabolic toxin
Used in food industry hundreds ,safety host
7 Large-scale fermentation process simple, cost low
FDA Generally Recognized As Safe GRAS

9.1.2 reasons for cloning genes in yeast (2) the potential use of yeast as a cloning host for the overproduction of proteins of commercial value. Yeast offers a number of advantages, such as the ability to glycosylate proteins during secretion and the absence of pyrogenic toxins(. The ability to clone in yeast without introducing bacterial sequences (by using proper vectors) is particularly beneficial.
The ability to clone large pieces of DNA. YACs offer a convenient way to clone large DNA fragments
9.1.3 Transformation methods

Like E. coli
Fungi are not naturally transformable and artificial means have to be used for introducing foreign DNA. Spheroplast method
Lithium Acetate Electroporation method
Spheroplast method The cell wall is removed enzymically and the spheroplasts are fused with ethylene glycol( in the presence of DNA and CaCl2. The spheroplasts are allowed to generate new cell walls in a stabilizing medium containing 3% agar.
Whole-cell method by alkali metal ion Li+PEGshock DNA DNA DNA80
Electroporation method
be a simpler and more convenient method Cells transformed by electroporation can be selected on the surface of solid media, thus facilitating subsequent manipulation.
All methods been applied to a wide range of yeasts and filamentous fungi.
9.3 Plasmid vectors for use in fungi Saccharomyces plasmids were developed from Escherichia coli plasmid vectors. E. coli plasmids are the foundation for the construction of the Saccharomyces yeast cloning vectors. Saccharomyces sequences were added to the E. coli vectors to create E. coli/yeast shuttle vectors
Yeast vector
yeast episomal plasmids (YEps), yeast replicating plasmids (YRps), Yeast centromere () plasmids (YCps) All three autonomous plasmid vectors are maintained in yeast as circular DNA molecules.
yeast artificial chromosomes(YACs)

yeast integrating plasmids
9.3.1 common features of the four kinds of vectors contain unique target sites replicate in E. coli, often at high copy number. it is necessary to amplify the vector important DNA in E. coli before transformation of the ultimate yeast recipient. employ markers that can be selected readily in yeast. selectable marker genes
selectable marker genes

antifungal agents like kanamycin (also called G418 or neomycin) and hygromycin Nutritional genes The four most widely used markers: His3, Leu2, Trp1 , Ura3. complement the corresponding mutations in E. coli as well
an appropriate host : contain recessive mutant alleles of the nutritional genes

Concept specific chromosomal DNA fragments

1) Telomeres structures A unique structures at the ends of chromosomes Telomere structure has evolved as a device to preserves the integrity of the ends of DNA
2) centromere sequences have the same distinctive chromatin( )structure that occurs in the centromere region of yeast chromosomes
CENDNA
3) ars
0.8--1.5kb
autonomously replicating sequence.
Acts as an origin of replication

ARSYRp 2YEp
Summary
Plasmid in E coli
Yeast gene fragment YIp
Yeast 2 YEp Recognised as genomic DNA
Yeast ARS YRp
Yeast CEN
YCp Two copies TEL
Clone large fragments
YAC
9.3.2 YIp plasmid A YIP plasmid consists of the basic E. coli vector plus a yeast selectable marker gene, but does not contain a Saccharomyces origin of replication.
YIP plasmids must integrate into a chromosome in order to be replicated at each cell division.
9.3.3 Yeast episomal plasmids YEp A YEp plasmid contains an origin of replication derived from 2 circle in the basic YIP vector. Constructing strategy an E. coli cloning vector the naturally occurring yeast 2 m plasmid.
6.3 kb
9.3.4 Yeast replicating YRp plasmids constructed by adding Saccharomyces origin of replication derived ARS to YIP vector
transform yeast very efficiently, the transformants are very unstable

often left in the mother nucleus. Principles of Gene Manipulation by liuzengran
Hebei University of Economics and Business
9.3.5 Yeast centromere plasmids
A YCp plasmid contains a centromere sequence, CEN, added to a YRp vector.
The plasmids are treated like mini chromosomes by the dividing yeast cell. attach to spindle fibers are transmitted to both mother and daughter cells in mitosis and meiosis, are mitotically stable in the absence
of selective pressure./
segregate during meiosis in a Mendelian manner./ are found at low copy number in the host cell./
9.3.6 Yeast artificial chromosomes YACs / to carry large chromosomal DNA fragments / useful in cloning fragments for various genome sequencing projects and for positional cloning studies Principles of Gene Manipulation by liuzengran
Key regions
TEL, yeast telomeres; ARS 1, autonomously replicating sequence; URA3 and TRP1, yeast marker genes CEN 4, centromere from chromosome 4; Amp, ampicillin-resistance determinant of pBR322; ori, origin of replication of pBR322.
advantage
their stability increases as the size of the insert unlike the other increases. plasmid vectors,
there is no practical limitation to the size of a YAC . they are essential tools in any genome-sequencing project.
major difference between YAC and YCp vectors
inserting two copies of a sequence derived from Saccharomyces telomere DNA

Construction of a yeast artificial chromosome containing large pieces of cloned DNA.
9.3.7 Retro-virus-like vectors The genome of S. cerevisiae contains 3040 copies of a 5.9 kb mobile genetic element called Ty This transposable element shares many structural and functional features with retroviruses
Structure of a typical Ty element. two identical terminal 334 bp repeats sequence. delta a central region element long open reading frames contains a promoter sequences recognized by the transposing enzyme.
The complementary DNA can transpose to many sites in the host DNA.
in vitro the delta promoter sequence of Ty element Promoters derived from phosphoglycerate kinase or galactose-utilization genes
introduced into yeast on high-copy-number vectors

Ty element is overexpressed the formation of large numbers of virus-like particles accumulate in the cytoplasm
pGAL and P are yeast promoters, (delta sequences) the red region represents the cloned gene.
Structure of the multicopy plasmid used for inserting a modified Ty element, carrying a cloned gene, into the yeast chromosome.
Properties of the different yeast vectors.

YIp
Cloned genes can be used in conventional genetic analysis by means of recombination using YIp vectors or linearized YRp vectors
Complementation can be carried out using YEp,YRp, YCp or YAC vectors, but there are a number of factors which make YCps the vectors of choice.
YEp
YRp
YCp
YAC
Ty
9.4 Expression of cloned genes
promoter
Four structural elements in the average yeast promoter several sequences at the upstream transcription-initiation activating site. sequences upstream repressing sequences
Structure of typical yeast promoters.

9.5 Overexpression of proteins in fungi 9.5.1 in Saccharomyces cerevisiae
promoters
induced by glucose ADH1: alcohol dehydrogenase PGK(GAP): glyceraldehyde-3- phosphate dehydrogenase Now many native and engineered promoters available Differ in strength, regulation and induction ratio.
The ideal promoter is tightly regulated have a high induction ratio minimizes the selection of nonexpressing cells Fit to express proteins toxic to the host cell
the growth phase separated

the induction phase
Promoter of GAL1 gene ideal promoter most widely used in yeast
Galactose regulation well studied a model system for eukaryotic transcriptional regulation
galactose addition induce
1000-fold reach 1% of total mRNA

GAL1 mRNA
GAL1 gene
repressed
addition
so glucose
in glucose-grown cultures, induction occurs when glucose depleted.

9.5.2 in Pichia pastoris methylotrophic() extremely popular hosts to over express heterologous proteins. reasons the alcohol oxidase (AOX1) promoter: one of the strongest and most regulatable promoters. Plasmids: may stably integrate expression at specific sites in the genome ( single or multiple copies). the strains: can be cultivated to very high density.
9.6 Specialist vectors Many different specialist yeast vectors have been developed
features
incorporate the useful features found in the corresponding E. coli vectors e.g. an f1 origin : to permit sequencing of the cloned gene product as a purification fusion
V5, X press and 6XHis encode epitopes which can be readily detected and purified by affinity chromatography.
to facilitate DNA sequencing
for high copy for auxotrophic selection on minimal medium
blasticidin resistance for dominant selection in any strain
for low copy in yeast
Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business (YES Saccharomyces
specialized vectors for use in vectors)
c-myc encode epitopes which can be readily detected and purified by affinity chromatography.
Pichia
E. coli
the zeocin-resistance gene
specialized vectors used in Pichia

discussion Two kinds of vectors : secretion surface display.
destined for the cell surface or 9.6.1.1 Secretion process for export from the cell
proteins on endoplasmic reticulum synthesized
9.6.1 Secreted vectors
translocated
transported to
into endoplasmic reticulum
the Golgi body
processing
Packaging into secretory vesicles
constitutively or in response to an external signal
Fusion with the plasma membrane
9.6.1.2 destination of the Secreted protein a few end up in the growth medium,
invertase and acid phosphatase,
mating pheromone factor and the killer toxin.
The most cross the plasma membrane
remain within the periplasmic space or become associated with the cell wall.
9.6.1.3 Secretion peptide Polypeptides destined for secretion have a hydrophobic amino-terminal extension is cleaved from the mature protein within the endoplasmic reticulum. is responsible for translocation to the endoplasmic reticulum
9.6.2 Yeast surface display vectors makes use of the cell surface receptor -agglutinin (Aga)
epitope
two-subunit glycoprotein anchors to the cell wall by a covalent linkage.
The 725-residue subunit Aga1p
is linked to Aga1p by two disulphide bonds. The 69-residue binding subunit Aga2p
Usually
the cloned gene product is sandwiched between two simple epitopes
haemagglutinin epitope
c-myc epitope.
Yeast surface display of heterologous proteins.

Process to achieve surface display the gene inserted at the C terminus of a Aga2p gene under the control of the GAL1 promoter at vector.
carrying a chromosomal copy of the Aga1p gene under the control of the GAL1 promoter.
transformed into a yeast strain If the cloned gene been inserted in the correct translational reading frame a fusion protein with the Aga2p subunit synthesized
associate with the Aga1p subunit in the secretory pathway

exported to the cell surface
9.7 Detecting proteinprotein interactions The two-hybrid system has become a major tool in the study of proteinprotein or proteinligand interactions the two-hybrid system. concept
Interaction between the test protein (bait) and a protein encoded by one of the library plasmids (prey) led to transcriptional activation of a reporter gene
The GAL4 protein has separate domains for the binding to UAS DNA and for transcriptional activation the DNA-binding domain DBD of GAL4 protein fused to a test protein the transcription- activation domain TAD of GAL4 protein, fused to protein encoded by a library of yeast genomic DNA fragments.
Plasmid 1 Plasmid 2
Both fusion gene constructions are expressed from high-level constitutive promoters so that the proteins are abundantly made under commonly used growth conditions. Principles of Gene Manipulation by liuzengran
Strategy to detect interacting proteins by two-hybrid system
is the upstream activating sequence for the yeast GAL genes, which binds the GAL4 protein
DNAbinding domain
transcription-activation domain
Interaction
Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business detected by expression of a GAL1 lacZ gene
fusion
Application of the two-hybrid method To demonstrate whether two proteins interact
and under what conditions they interact.

to test whether mutations in either protein affect the interaction and thereby identify the specific residues involved in the interaction. to screen a library for novel proteins that
interact with the bait fusion protein.
Example To make the prey library 1 random genomic DNA fragments (or cDNA fragments) are fused to the Gal4 activation domain using the prey vector. 2 The library is transformed into a yeast host cell containing the reporter gene and the bait fusion gene. 3 Transformants are selected and then all are screened for expression of the reporter gene.
If the reporter gene is IucZ, one can screen for blue colonies on X-gal plates.
9.8 Identifying genes encoding particular activities How to connect specific biochemical activities with particular genes?
a yeast vector
carrying the GST gene under the control of the CUP1 promoter.
GST: glutathione- S-transferase
number of different DNA fragments bearing yeast ORFs were cloned in this vector to generate GSTORF fusions. 6144 constructs were made and transformed back into S. cerevisiae.
correlate genes with activity
Method for selecting GST open reading-frame fusions.
the GSTORF fusions are readily purified by affinity chromatography using immobilized glutathione.
1 different transformants were dispensed into 6496 well microtitre plates (64 96 = 6144).
64 pools each with 96 GSTORF fusions 2 Initially, all the clones from each plate were pooled and the 64 pools analysed for a specific biochemical activity.
3 a plate was identified as having the desired activity 4 pools of clones were made from each of the 8 rows and each of the 12 columns of wells the 20 pools reassayed.
If only one clone expresses the desired activity

5 one row and one column be identified
6 the point of intersection identifies the wanted clone.

7 Sequencing of the ORF then allows identification of the gene corresponding to the activity.
9.9 Determining functions associated with particular genes Function determining involves to determine
the expression profile of the gene, the subcellular location of the protein, the phenotype of a null strain lacking the protein. Conditional alleles of the gene are created as an additional tool.
a multifunctional, transposon-based system The transposons carries a reporter gene (galactosidase or green fluorescent protein) lacking a promoter. In-frame fusions between a yeast coding region and the transposon can be detected by -galactosidase activity or fluorescence. The transposon insertion creates a truncation of the gene, generating a null phenotype.
Schematic representation of the transposons, and the derived HAT tag elements, for use in yeast. Each transposon carries the tetracyclineresistance determinant, a functional yeast URA3 gene, and the res element from transposon Tn3. The function of the res element is to resolve transposition cointegrates.
yeast genes are cloned in an E. coli strain that overexpresses the Tn3 transposase. The transposon, carried on a derivative of the sex factor F, is introduced by mating and transposition ensues.
Yeast DNA containing the transposon is excised from the E. coli vector and transformed back into yeast, with selection made for URA3 activity.
Two types of transformants are obtained. recombination occurs at the URA3 locus, no reporter activity is seen. If integration occurs at the locus of the gene carrying the transposon, reporter activity is retained but gene function is lost.
This reporter activity can be used to study the expression of the cloned gene under different growth conditions.
An alternative approach to the large-scale mutational analysis of the yeast genome
individually delete each gene using a polymerase chain reaction (PCR)based methodology
Knockout strain collection.
(a)The bar-coded deletion cassette is generated in two rounds of PCR. bar-codes 20 bp long common sequence elements that allow for the PCR amplification 45 bp sequences that are homologous to sequences flanking the gene of interest.
(b) The deletion cassette is transformed into diploid yeast strains. The 45 bp of yeast sequences allows one copy of the gene of interest to be replaced with the deletion cassette
(c) can be sporulated and dissected to generate a collection of haploid mutant strains,
Homozygous mutant strains
(e) are made by the selective matings of haploid segregants.


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CHAPTER 9

Cloning in Saccharomyces cerevisiae and other fungi

Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

Demands Need to master the specialist vectors, including speciality

Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

9.5 Specialist vectors

9.7 Determining functions associated with particular genes

Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

4 many homologues genes of humans in yeast

7 Large-scale fermentation process simple, cost low

Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

FDA Generally Recognized As Safe GRAS

Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

9.1.3 Transformation methods

Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

Whole-cell method by alkali metal ion Li+PEGshock DNA DNA DNA80

Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

yeast artificial chromosomes(YACs)

Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

selectable marker genes

an appropriate host : contain recessive mutant alleles of the nutritional genes

Concept specific chromosomal DNA fragments

Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

autonomously replicating sequence.

Acts as an origin of replication

Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

Yeast 2 YEp Recognised as genomic DNA

Yeast ARS YRp

Clone large fragments

Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

transform yeast very efficiently, the transformants are very unstable

9.3.5 Yeast centromere plasmids

A YCp plasmid contains a centromere sequence, CEN, added to a YRp vector.

Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

major difference between YAC and YCp vectors

inserting two copies of a sequence derived from Saccharomyces telomere DNA

Construction of a yeast artificial chromosome containing large pieces of cloned DNA.

Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

introduced into yeast on high-copy-number vectors

Properties of the different yeast vectors.

Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

9.4 Expression of cloned genes

Structure of typical yeast promoters.

9.5 Overexpression of proteins in fungi 9.5.1 in Saccharomyces cerevisiae

the growth phase separated

Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

Promoter of GAL1 gene ideal promoter most widely used in yeast

Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

galactose addition induce

1000-fold reach 1% of total mRNA