1) The document describes genetic and recombinant DNA techniques for isolating and characterizing genes, including the study of mutations, cloning DNA into vectors, constructing cDNA libraries, and screening libraries.
2) Key techniques discussed are the use of dominant and recessive mutations to identify gene function, restriction enzymes for cleaving DNA, ligation for joining DNA fragments, transformation of E. coli for cloning DNA, and hybridization for screening cDNA libraries.
3) The techniques allow researchers to generate mutants, isolate genes, characterize protein function, and determine interactions between gene products.
Genes, Genomics, and Chromosomes computational biology introduction .pptMohamedHasan816582
The 5 ß-globin genes are derived from an ancestral ß-globin gene via gene duplication. Over time, these genes accumulated adaptive mutations via sequence drift resulting in the specialized species of ß-globin proteins. Genomic DNA also contains nonfunctional DNA sequences called pseudogenes that are derived from gene duplication or reverse transcription and integration of cDNA sequences made from mRNA (covered below). ß-globin pseudogenes contain introns and thus were derived by gene duplication. Over time these genes became nonfunctional also due to sequence drift. Because they are not harmful, pseudogenes remain in the genome, marking a gene duplication event in an earlier ancestor.
The ß-globin gene cluster on chromosome 11 is shown in Fig. 6.4a. The ß-globin genes are expressed in different stages of life. , Ag, and Gg are expressed during different trimesters of fetal development (next slide). ß expression begins around birth & continues throughout adult life. Fetal hemoglobin molecules made with the d and G or A polypeptides have a higher affinity for O2 than maternal hemoglobin, facilitating O2 transfer to the fetus.
Higher eukaryotes contain far more noncoding DNA between genes than bacteria and simple eukaryotes (Fig. 6.4). The region of human genomic DNA containing the ß-globin gene cluster shown in the figure actually is a relatively "gene-rich" region of human DNA. Some regions known as gene-poor "deserts" also occur. Higher eukaryotes also contain a larger amount of intron DNA. Although one-third of human DNA is transcribed into pre-mRNA, 95% ends up being degraded after RNA splicing reactions. On average, the typical exon is 50-200 bp in length, while the median length of introns is 3.3 kb in human genes.
DNA fingerprinting is a method for identifying individuals based on their minisatellite DNA (Fig. 6.7). It was developed in the mid-80s and is widely used in forensics, paternity analysis, and for research purposes. In the method, minisatellite DNA from a genomic DNA specimen is amplified by PCR using primers that bind to unique sequences flanking minisatellite repeat units. Bands corresponding to each minisatellite locus then are separated on gels. Although satellite DNA is highly conserved in sequence, the number of tandem copies at each loci is highly variable between individuals. This results from unequal crossing over during formation of gametes in meiosis. Due to the variation in the number of repeats at each locus, different individuals can be readily distinguished based on banding patterns.
Interspersed repeat DNA comprises the largest fraction of repetitious DNA in eukaryotic genomes. This DNA, which is also called moderately repeated DNA makes up ~45% of human genomic DNA. Interspersed repeat DNA is composed of partial and complete transposon sequences or "mobile DNA". Mobile DNAs were discovered by Barbara McClintock in the 1940s. These sequences move by "transposition". Transpositions in germ line cells are inhe
Immerse yourself in a captivating world epigenetics with our comprehensive PDF guide, this document serves as an insightful resource for both beginners and seasoned enthusiasts seeking a deeper understanding of the molecular mechanisms that influence gene expression and cellular function.
Unlock the secrets behind the heritable changes in gene activity that go beyond the DNA sequence, as we explore the dynamic interplay between genetics and environmental factors. This PDF delves into the fascinating realm of epigenetic modifications, including DNA methylation, histone modification, and non-coding RNA, shedding light on their pivotal roles in regulating gene expression and cellular identity.
Download "what is Epigenetics" document now and embark on a journey that transcends the traditional boundaries of genetics, exploring the intricate tapestry of epigenetic regulation that influences life at its very core.
this is all of the information that I have please help Lab 5 In.pdfambikacomputer4301
this is all of the information that I have please help Lab 5 Introduction - Genetic mapping See
figure 5.1 for a schematic of the fly the cross you initially started with, you'll either crosses you
have been working on. Two labs ago, map the distance between the w gene and the m you set up
a pair of reciprocal parental crosses, gene, or between the w gene and the y gene. between mutant
and wild type flies (fig. 5.1a). You had one of two different mutant strains, each with two mutant
phenotypes - either white eyes all F2 individuals will receive only recessive (w) and miniature
wings (m), or white eyes (w) alleles from this parent (fig. 5.1d, orange and and yellow body (y).
The phenotypes of the F1 yellow chromosomes). Because of this, the flies should have indicated
to you that all mutant phenotype of each F2 fly will tell you which phenotypes in question are x-
linked recessive alleles (mutant or WT) were inherited from the (fig. 5.1b). heterozygous female
F1 parent (fig. 5.1d, dark and light blue chromosomes). The first F2 fly Last lab, you used the F1
flies from one of shown in figure 5.1d inherited 'a B' from the your parental crosses to set up an
F1 cross (fig. heterozygous parent and will end up with the consequence. After crossing two pure
breeding this F2, you observe a ABphenotype and parents, F1 offspring will be heterozygous for
therefore know that this fly received 'A B' from nearly all genes in question - the exception is X -
the heterozygous parent. linked genes in the male offspring. Since the Y chromosome is
equivalent to recessive alleles for The goal of genetic mapping is to X-linked genes, these F1
males are recessive for determine the likelihood of cross over between all X-linked genes and act
as a test cross. The two loci/genes. If we score the phenotypes of a heterozygous F1 females and
recessive F1 males large F2 population from our crosses, we can (test cross) can be used to map
the distance determine the recombination frequency of your between the genes causing the two
phenotypes two genes. of your parental mutant female. Depending on
e) F2 phenotype scoring: f) Recombination frequency: Eigure 5.1: Schematic of your Drosophila
crosses, See text of lab 5 intro for description.
For a given F1 gamete for the F2 individual it number of flies, it is easy to calculate the creates),
if no cross over occurs between the two recombinant frequency between your two genes genes in
question, the F2 phenotype will be the (fig. 5.1ef ). same as one of the original parents - either
fully WT or double mutant in this case. In figure 5.1d Recall from last time that a lower
recombinant these have blue chromosomes of a single colour. frequency is observed when
genetic map If a crossover does occur between the two genes, distances are small. When genes
are close to the F2 fly will have a phenotype unlike either of each other, there's a narrow range
on the the parents - a recombinant phenotype (shown chromosome for a random crossover to
land.
Genes, Genomics, and Chromosomes computational biology introduction .pptMohamedHasan816582
The 5 ß-globin genes are derived from an ancestral ß-globin gene via gene duplication. Over time, these genes accumulated adaptive mutations via sequence drift resulting in the specialized species of ß-globin proteins. Genomic DNA also contains nonfunctional DNA sequences called pseudogenes that are derived from gene duplication or reverse transcription and integration of cDNA sequences made from mRNA (covered below). ß-globin pseudogenes contain introns and thus were derived by gene duplication. Over time these genes became nonfunctional also due to sequence drift. Because they are not harmful, pseudogenes remain in the genome, marking a gene duplication event in an earlier ancestor.
The ß-globin gene cluster on chromosome 11 is shown in Fig. 6.4a. The ß-globin genes are expressed in different stages of life. , Ag, and Gg are expressed during different trimesters of fetal development (next slide). ß expression begins around birth & continues throughout adult life. Fetal hemoglobin molecules made with the d and G or A polypeptides have a higher affinity for O2 than maternal hemoglobin, facilitating O2 transfer to the fetus.
Higher eukaryotes contain far more noncoding DNA between genes than bacteria and simple eukaryotes (Fig. 6.4). The region of human genomic DNA containing the ß-globin gene cluster shown in the figure actually is a relatively "gene-rich" region of human DNA. Some regions known as gene-poor "deserts" also occur. Higher eukaryotes also contain a larger amount of intron DNA. Although one-third of human DNA is transcribed into pre-mRNA, 95% ends up being degraded after RNA splicing reactions. On average, the typical exon is 50-200 bp in length, while the median length of introns is 3.3 kb in human genes.
DNA fingerprinting is a method for identifying individuals based on their minisatellite DNA (Fig. 6.7). It was developed in the mid-80s and is widely used in forensics, paternity analysis, and for research purposes. In the method, minisatellite DNA from a genomic DNA specimen is amplified by PCR using primers that bind to unique sequences flanking minisatellite repeat units. Bands corresponding to each minisatellite locus then are separated on gels. Although satellite DNA is highly conserved in sequence, the number of tandem copies at each loci is highly variable between individuals. This results from unequal crossing over during formation of gametes in meiosis. Due to the variation in the number of repeats at each locus, different individuals can be readily distinguished based on banding patterns.
Interspersed repeat DNA comprises the largest fraction of repetitious DNA in eukaryotic genomes. This DNA, which is also called moderately repeated DNA makes up ~45% of human genomic DNA. Interspersed repeat DNA is composed of partial and complete transposon sequences or "mobile DNA". Mobile DNAs were discovered by Barbara McClintock in the 1940s. These sequences move by "transposition". Transpositions in germ line cells are inhe
Immerse yourself in a captivating world epigenetics with our comprehensive PDF guide, this document serves as an insightful resource for both beginners and seasoned enthusiasts seeking a deeper understanding of the molecular mechanisms that influence gene expression and cellular function.
Unlock the secrets behind the heritable changes in gene activity that go beyond the DNA sequence, as we explore the dynamic interplay between genetics and environmental factors. This PDF delves into the fascinating realm of epigenetic modifications, including DNA methylation, histone modification, and non-coding RNA, shedding light on their pivotal roles in regulating gene expression and cellular identity.
Download "what is Epigenetics" document now and embark on a journey that transcends the traditional boundaries of genetics, exploring the intricate tapestry of epigenetic regulation that influences life at its very core.
this is all of the information that I have please help Lab 5 In.pdfambikacomputer4301
this is all of the information that I have please help Lab 5 Introduction - Genetic mapping See
figure 5.1 for a schematic of the fly the cross you initially started with, you'll either crosses you
have been working on. Two labs ago, map the distance between the w gene and the m you set up
a pair of reciprocal parental crosses, gene, or between the w gene and the y gene. between mutant
and wild type flies (fig. 5.1a). You had one of two different mutant strains, each with two mutant
phenotypes - either white eyes all F2 individuals will receive only recessive (w) and miniature
wings (m), or white eyes (w) alleles from this parent (fig. 5.1d, orange and and yellow body (y).
The phenotypes of the F1 yellow chromosomes). Because of this, the flies should have indicated
to you that all mutant phenotype of each F2 fly will tell you which phenotypes in question are x-
linked recessive alleles (mutant or WT) were inherited from the (fig. 5.1b). heterozygous female
F1 parent (fig. 5.1d, dark and light blue chromosomes). The first F2 fly Last lab, you used the F1
flies from one of shown in figure 5.1d inherited 'a B' from the your parental crosses to set up an
F1 cross (fig. heterozygous parent and will end up with the consequence. After crossing two pure
breeding this F2, you observe a ABphenotype and parents, F1 offspring will be heterozygous for
therefore know that this fly received 'A B' from nearly all genes in question - the exception is X -
the heterozygous parent. linked genes in the male offspring. Since the Y chromosome is
equivalent to recessive alleles for The goal of genetic mapping is to X-linked genes, these F1
males are recessive for determine the likelihood of cross over between all X-linked genes and act
as a test cross. The two loci/genes. If we score the phenotypes of a heterozygous F1 females and
recessive F1 males large F2 population from our crosses, we can (test cross) can be used to map
the distance determine the recombination frequency of your between the genes causing the two
phenotypes two genes. of your parental mutant female. Depending on
e) F2 phenotype scoring: f) Recombination frequency: Eigure 5.1: Schematic of your Drosophila
crosses, See text of lab 5 intro for description.
For a given F1 gamete for the F2 individual it number of flies, it is easy to calculate the creates),
if no cross over occurs between the two recombinant frequency between your two genes genes in
question, the F2 phenotype will be the (fig. 5.1ef ). same as one of the original parents - either
fully WT or double mutant in this case. In figure 5.1d Recall from last time that a lower
recombinant these have blue chromosomes of a single colour. frequency is observed when
genetic map If a crossover does occur between the two genes, distances are small. When genes
are close to the F2 fly will have a phenotype unlike either of each other, there's a narrow range
on the the parents - a recombinant phenotype (shown chromosome for a random crossover to
land.
The number of sequenced genes having unknown function continues to climb with the continuing decrease in the cost of genome sequencing. In Reverse Genetics (RG), functions of known genes are investigated with targeted modulation of gene activity, and hypothesis regarding gene function directly tested in vivo. Several RG approaches like insertional mutagenesis, fast neutron mutagenesis, TILLING and RNA interference have led to the identification of mutations in candidate genes and subsequent phenotypic analysis of these mutants.
Okabe et al. (2011) employed TILLING technique to screen six ethylene receptor genes in tomato (SlETR1–SlETR6) and two allelic mutants of SlETR1 (Sletr1-1 and Sletr1-2) with reduced ethylene response were identified. Using fast neutron mutagenesis, Li et al. (2001) obtained arabidopsis deletion mutants for bZIP transcription factor viz. AHBP 1b and OBF 5, a key regulator for systemic acquired resistance but their role were compensated by other regulatory factors in mutants. Terada et al. (2007) successfully blocked the expression of the Adh 2 gene through homologous recombination followed by transgenesis in rice however phenotype could not be determined since no differences were observed between wild and transgenic plants. RNA interference (RNAi) works as sequence-specific gene regulation and has been used in determination of function of many genes. Saurabh et al. (2014) reviewed the impact of RNAi in crop improvement and found its application in improvement of nutritional aspects, biotic and abiotic stresses, morphol¬ogy, crafting male sterility, enhanced secondary metabolite synthesis.
In addition, new advances in technology and reduction in sequencing cost may soon make it practical to use whole genome sequencing or gene targeting like ZFN technology and TAL effectors technology on a routine basis to identify or generate mutations in specific genes. Scholze and Boch (2011) mentioned that TAL effectors technology is more specific and predictable than ZFN. RG techniques have their own advantages and disadvantages depending on the species being targeted and the questions being addressed. Finally, with the continuous development of new technologies, the most efficient RG technique in the future may involve high throughput direct sequencing of part or complete genomes of individual plants followed by efficient novel tools to determine the function for utilization in crop improvement.
Direct Lineage Reprogramming: Novel Factors involved in Lineage ReprogrammingAhmed Madni
Direct linage reprogramming has got a major focus in biomedical field. The production of specific functional cell type from totally different cell lineage is called lineage reprogramming. In other words, it is induction of functional cell type from another linage without passing through intermediate stage of pluripotent.
describe the tumor suppressor genes and examples for downloading the presentation, more presentations , infographics and blogs visit :
studyscienceblog.wordpress.com
This presentation provide knowledge about Gene Expression & its regulation in brief.
i hope it gives some information about gene expression in your academic time.
In this presentation mentioned - Lac Operon and its expressor.
A powerful non-transgenic reverse genetics method that combines chemical mutagenesis with PCR based screening to identify point mutations in regions of interest.
EcoTILLING is a molecular technique that is similar to TILLING, except that its objective is to uncover natural genetic variation as opposed to induced mutations.
Palestine last event orientationfvgnh .pptxRaedMohamed3
An EFL lesson about the current events in Palestine. It is intended to be for intermediate students who wish to increase their listening skills through a short lesson in power point.
The number of sequenced genes having unknown function continues to climb with the continuing decrease in the cost of genome sequencing. In Reverse Genetics (RG), functions of known genes are investigated with targeted modulation of gene activity, and hypothesis regarding gene function directly tested in vivo. Several RG approaches like insertional mutagenesis, fast neutron mutagenesis, TILLING and RNA interference have led to the identification of mutations in candidate genes and subsequent phenotypic analysis of these mutants.
Okabe et al. (2011) employed TILLING technique to screen six ethylene receptor genes in tomato (SlETR1–SlETR6) and two allelic mutants of SlETR1 (Sletr1-1 and Sletr1-2) with reduced ethylene response were identified. Using fast neutron mutagenesis, Li et al. (2001) obtained arabidopsis deletion mutants for bZIP transcription factor viz. AHBP 1b and OBF 5, a key regulator for systemic acquired resistance but their role were compensated by other regulatory factors in mutants. Terada et al. (2007) successfully blocked the expression of the Adh 2 gene through homologous recombination followed by transgenesis in rice however phenotype could not be determined since no differences were observed between wild and transgenic plants. RNA interference (RNAi) works as sequence-specific gene regulation and has been used in determination of function of many genes. Saurabh et al. (2014) reviewed the impact of RNAi in crop improvement and found its application in improvement of nutritional aspects, biotic and abiotic stresses, morphol¬ogy, crafting male sterility, enhanced secondary metabolite synthesis.
In addition, new advances in technology and reduction in sequencing cost may soon make it practical to use whole genome sequencing or gene targeting like ZFN technology and TAL effectors technology on a routine basis to identify or generate mutations in specific genes. Scholze and Boch (2011) mentioned that TAL effectors technology is more specific and predictable than ZFN. RG techniques have their own advantages and disadvantages depending on the species being targeted and the questions being addressed. Finally, with the continuous development of new technologies, the most efficient RG technique in the future may involve high throughput direct sequencing of part or complete genomes of individual plants followed by efficient novel tools to determine the function for utilization in crop improvement.
Direct Lineage Reprogramming: Novel Factors involved in Lineage ReprogrammingAhmed Madni
Direct linage reprogramming has got a major focus in biomedical field. The production of specific functional cell type from totally different cell lineage is called lineage reprogramming. In other words, it is induction of functional cell type from another linage without passing through intermediate stage of pluripotent.
describe the tumor suppressor genes and examples for downloading the presentation, more presentations , infographics and blogs visit :
studyscienceblog.wordpress.com
This presentation provide knowledge about Gene Expression & its regulation in brief.
i hope it gives some information about gene expression in your academic time.
In this presentation mentioned - Lac Operon and its expressor.
A powerful non-transgenic reverse genetics method that combines chemical mutagenesis with PCR based screening to identify point mutations in regions of interest.
EcoTILLING is a molecular technique that is similar to TILLING, except that its objective is to uncover natural genetic variation as opposed to induced mutations.
Palestine last event orientationfvgnh .pptxRaedMohamed3
An EFL lesson about the current events in Palestine. It is intended to be for intermediate students who wish to increase their listening skills through a short lesson in power point.
Model Attribute Check Company Auto PropertyCeline George
In Odoo, the multi-company feature allows you to manage multiple companies within a single Odoo database instance. Each company can have its own configurations while still sharing common resources such as products, customers, and suppliers.
Honest Reviews of Tim Han LMA Course Program.pptxtimhan337
Personal development courses are widely available today, with each one promising life-changing outcomes. Tim Han’s Life Mastery Achievers (LMA) Course has drawn a lot of interest. In addition to offering my frank assessment of Success Insider’s LMA Course, this piece examines the course’s effects via a variety of Tim Han LMA course reviews and Success Insider comments.
Biological screening of herbal drugs: Introduction and Need for
Phyto-Pharmacological Screening, New Strategies for evaluating
Natural Products, In vitro evaluation techniques for Antioxidants, Antimicrobial and Anticancer drugs. In vivo evaluation techniques
for Anti-inflammatory, Antiulcer, Anticancer, Wound healing, Antidiabetic, Hepatoprotective, Cardio protective, Diuretics and
Antifertility, Toxicity studies as per OECD guidelines
How to Make a Field invisible in Odoo 17Celine George
It is possible to hide or invisible some fields in odoo. Commonly using “invisible” attribute in the field definition to invisible the fields. This slide will show how to make a field invisible in odoo 17.
The French Revolution, which began in 1789, was a period of radical social and political upheaval in France. It marked the decline of absolute monarchies, the rise of secular and democratic republics, and the eventual rise of Napoleon Bonaparte. This revolutionary period is crucial in understanding the transition from feudalism to modernity in Europe.
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Home assignment II on Spectroscopy 2024 Answers.pdf
13-miller-chap-5a-lecture.ppt
1. Chap. 5 Molecular Genetic Techniques
(Part A)
Topics
• Genetic Analysis of Mutations to Identify and Study Genes
• DNA Cloning and Characterization
Goals
Learn about genetic
and recombinant
DNA methods for
isolating genes and
characterizing the
functions of the
proteins they
encode.
Use of RNA interference (RNAi) in analysis
of planarian regeneration
3. Importance of Mutations in Gene Analysis
One of the most important ways in which the function of a gene can be
learned is by the study of a mutant in which the gene has been
inactivated. Currently, mutants can be generated by classical “forward”
genetic methods, and by more modern “reverse” genetic approaches (Fig.
5.1). In forward genetic analyses one generates a mutant organism and
then uses molecular biological techniques to isolate the mutant gene and
characterize the protein responsible for the phenotype of the mutant. In
reverse genetic approaches, a gene is inactivated and the function of the
gene is learned by study of the properties of the mutant organism.
4. Alleles-Different versions (sequences) of a gene.
Mutant-Newly created allele made by mutagenesis.
Genotype-The complete set of alleles for all genes carried by
an individual.
Wild type-Standard reference genotype. Most common allele
for a certain trait.
Phenotype-Observable trait specified by the genotype.
Point mutation-A change in a single base pair (e.g., a G.C to
A.T transition).
Silent mutation-A point mutation in a codon that does not
change the specified amino acid.
Missense mutation-A point mutation that changes the encoded
amino acid.
Nonsense mutation-A point mutation that introduces a
premature stop codon into the coding sequence of a gene.
Recessive & dominant mutant alleles-(next slide)
Genetics Terms
5. Recessive and Dominant Mutant Alleles
Diploid organisms have two copies of each gene; haploid organisms
(e.g., some unicellular organisms) contain only one. A recessive
mutant allele must be present in two copies (be homozygous) to
cause a phenotype in a diploid organism (Fig. 5.2). Only one copy
of a recessive allele must be present for the phenotype to be
observable in a haploid organism. In contrast, a dominant mutant
allele needs to be present in only one copy (heterozygous) in a
diploid organism for the phenotype to be observable. Most
recessive alleles cause gene inactivation and phenotypic loss of
function. Some dominant alleles change or increase activity causing
a gain of function. However, a dominant affect can be caused by
gene inactivation if two copies of the gene are needed for proper
function (haplo-insufficiency). Lastly, a dominant negative mutation
refers to a situation where the product of the mutant gene
inactivates the product of the wild-type gene. This can occur if a
gene encodes one subunit of an oligomeric protein.
6. Review of Mitosis
Mating experiments provide
important information about gene
function. These experiments
demand a thorough knowledge of
meiosis and production of
gametes (sperm & egg cells in
higher eukaryotes). In Fig. 5.3,
mitosis is described to contrast
it with meiosis. In mitosis, one
round of DNA replication in a
diploid somatic cell is followed by
one cell division. The paternal
and maternal homologous
chromosomes (homologs) first are
duplicated. The sister
chromatids then are separated
by a cell division. The daughter
cells end up with one copy of
each paternal and maternal
chromosome and are diploid (2n).
7. Review of Meiosis
In meiosis, one round of DNA
replication in a diploid germ cell is
followed by two cell divisions,
resulting in four haploid gametes
(Fig. 5.3). Paternal and maternal
homologous chromosomes first are
copied as in mitosis. However, after
alignment (synapsis) and crossing
over (recombination) of homologous
chromosomes, paternal and maternal
chromosomes are randomly
segregated between the daughter
cells formed in the first cell
division. Subsequently, the sister
chromatids of each chromosome are
separated in a second cell division,
which produces the gametes (1n).
The two sets of gametes each
contain a random assortment of the
paternal and maternal chromosomes.
8. Identification of Dominant Mutations
Dominant mutations can be
identified by mating strains
that each are homozygous
for two alleles of a given
gene. Because all gametes
from each parent are of
one type (Fig. 5.4a), all
members of the first filial
generation from the cross
(F1) necessarily will be
heterozygotes. If the
mutation is dominant, all F1
offspring will display the
mutant phenotype. On self
crossing of F1 cells, 3/4 of
the second filial generation
(F2) will display the mutant
phenotype, if it is dominant.
9. Identification of Recessive Mutations
Recessive mutations also can
be identified by mating
strains that are homozygous
for two alleles of a given
gene. Again, because all
gametes from each parent are
of one type (Fig. 5.4b), all
members of the F1 generation
from the cross necessarily
will be heterozygotes. None
of the F1 offspring will
display the mutant phenotype
if it is recessive. On self
crossing of F1 cells, only 1/4
of the F2 generation will
display the phenotype, if it is
recessive.
10. Analysis of Mutant Alleles in Yeast
The yeast Saccharomyces
cerevisiae is an ideal
experimental organism for
analysis of dominant and
recessive alleles. First, cells
can exist in either a haploid
or diploid state. Second,
haploid cells occur in two
mating types (a and a) that
are useful for performing
crosses. The diploid cells
resulting from matings can
be examined to determine if
a mutation is dominant or
recessive (Fig. 5.5). Finally,
haploid cells can be
regenerated by meiotic
sporulation of diploid cells
grown under starvation
conditions.
11. Use of Conditional Mutations to Study
Essential Genes
The study of essential genes
(needed for life) requires special
genetic screening techniques. In
diploid organisms, such as the
fruit fly Drosophila, lethal
mutations in essential genes can
be maintained in the diploid state
and identified by inbreeding
experiments. In haploid
organisms, such as haploid yeast
(Fig. 5.6), defects in essential
genes can be isolated and
maintained through the use of
conditional mutations. Very often, conditional
mutations that display temperature-sensitive
(ts) phenotypes are used. ts mutations often
result from substitution mutations that cause
an essential protein to be unstable and
inactive at high (nonpermissive), but not low
(permissive) temperatures. A number of yeast
cell-division cycle (cdc) mutants have been
isolated via this technique (Fig. 5.6).
12. Complementation Analysis of Recessive
Mutations
Many processes, including
cell division involve the
combined actions of
multiple genes. Thus, a
genetic screen for
mutations affecting such
processes will turn up a
collection of genes.
Through mating haploid
yeast containing the
defective genes, one can
establish in the diploid
cells whether the
mutations fall in the same
or separate genes. As
shown in Fig. 5.7, diploid
cells will grow under
nonpermissive conditions if
the mutations reside in
different genes (the wild
type genes complement
the defective ones).
However, diploids with two
defective copies of the
same gene will not survive.
13. Double Mutant Analysis of Biosynthetic
Pathways
Genetic experiments can be used to determine the order in
which gene products act in carrying out a process. Double
mutant analysis can be applied to order the enzyme-catalyzed
steps in a metabolic pathway. As shown in Fig. 5.8a, the
accumulation of intermediate 1 in the double mutant strain
indicates enzyme A operates prior to enzyme B.
14. Suppressor Mutations
Suppressor mutation analysis is a powerful tool for identifying
proteins that interact with one another in the performance of a
certain cellular process. In genetic suppression, a loss of function
mutation in Protein A is corrected by a compensating mutation in
Protein B. The resulting gain of function phenotype of the double
mutant results from the recreation of interaction sites between
two proteins that are disrupted by each individual amino acid
substitution mutation (Fig. 5.9a).
15. Synthetic Lethal Mutations
The analysis of synthetic lethal mutations also is an important
tool for identifying proteins that must interact to carry out a
cellular process (Fig. 5.9b). It also is a powerful method to
identify proteins that function in redundant pathways needed for
the production of an essential cell component (Fig. 5.9c). Unlike
suppressor mutations, synthetic lethal double mutants display a
loss of function phenotype.
16. Intro to DNA Cloning by Recombinant
DNA Methods
To study a gene, one must first prepare and purify its DNA in
relatively large amounts. This is accomplished via the
recombinant DNA (rDNA) technology method known as DNA
cloning. In cloning, a DNA molecule of interest is spliced into a
vector such as a bacterial plasmid or virus forming a rDNA
molecule which can be propagated in bacterial cells such as E.
coli. After replication and amplification of the rDNA in the
bacterium, it is purified for sequencing and other manipulations
used in gene characterization.
17. DNA Cleavage by Restriction Enzymes
Restriction enzymes are nucleases that are very important in
rDNA technology. These enzymes make double-stranded cuts in
DNA molecules at specific 4-8 bp palindromic (two-fold
symmetrical) sequences called restriction sites. Many restriction
enzymes make staggered cuts in DNA molecules resulting in
single-stranded complementary sticky ends (Fig. 5.11). Sticky-
ended fragments can be readily joined together to synthesize
rDNA molecules (Fig. 5.12). In many cases, cleavage at the
restriction site is blocked by methylation of bases in the site.
18.
19. Joining of DNA Molecules by Ligation
Plasmid vectors containing a
DNA of interest (e.g.,
genomic DNA) can be
readily constructed by
ligating restriction
fragments to vector DNA
that has been digested with
the same restriction enzyme
(Fig. 5.12). Base-pairing
between the complementary
sequences of the sticky
ends aligns the fragments
for covalent linkage by a
DNA ligase, typically T4
DNA ligase. This enzyme
uses 2 ATP to provide
energy for joining the 3'-
hydroxyl and 5'-phosphate
groups of the base-paired
fragments together in 2 new
3'-5' phosphodiester bonds.
Note, all restriction
enzymes produce a 5'-
phosphate and 3'-hydroxyl
group at the cut site.
20. E. coli Plasmid Cloning Vectors
Plasmids are autonomously replicating circular DNAs found in
bacterial cells. Naturally occurring plasmids contain an origin of
replication (ori) for propagation in the host cell and one or more
genes that specify a trait that may be useful to the host. Cloning
vectors are plasmids that have been genetically engineered to
reduce unneeded DNA and to introduce selectable markers such
as antibiotic resistance genes (e.g., ampr) that are used to force
cells to maintain the plasmid. Polylinker sequences that encode
several unique restriction sites for cloning purposes also are
engineered into these vectors (Fig. 5.13).
21. Cloning of DNA in Plasmid Vectors
An overview of the steps required
for DNA cloning in a plasmid
vector is presented in Fig. 5.14.
In Step 1, the DNA of interest
is ligated into a plasmid cloning
vector. In Step 2, the
recombinant plasmid is introduced
into E. coli host cells by
transformation. In Step 3, cells
that have taken up the plasmid
are selected on antibiotic
(ampicillin) agar. In Step 4, the
transformed cells replicate their
chromosomal and plasmid DNA
and multiply to form a colony.
Cells in the colony contain the
cloned DNA and are themselves
clones. The rDNA plasmid then is
harvested by growing a larger
culture of the cells.
1
2
3
4
4
22. Construction of cDNA Libraries (Part 1)
A genomic DNA library is a collection of cloned DNA fragments
representing all of the DNA of an organism. A cDNA library
(complementary DNA), is a collection of cloned DNA fragments
corresponding to all mRNAs transcribed in a certain tissue or
organism. Libraries can be constructed using plasmid cloning
vectors. To construct a cDNA library, one begins by isolating
mRNA from the cell or tissue of interest (Fig. 5.15). Because many
genes are transcribed at a low frequency, it is best to start with a
cell/tissue that expresses the
gene of interest at a relatively
high level. cDNAs are
transcribed from a mRNA
template by a retroviral enzyme
known as reverse transcriptase
(RT). In Step 1, mRNA isolated
by oligo-dT affinity
chromatography is hybridized via
its 3' poly(A) tail to an oligo-dT
primer. In Step 2, RT
synthesizes the first cDNA
strand. In Step 3, RNA is
destroyed and a poly(dG) tail is
added by terminal transferase.
In Step 4, the cDNA is
hybridized to an oligo-dC
primer. (Go to next slide).
23. Construction of cDNA Libraries (Part 2)
In Step 5, a DNA
polymerase is used to
synthesize the second
strand of the cDNA. In
Step 6, EcoRI sites that
might be present within
the mRNA coding region
are protected by
methylation using EcoRI
methylase. In Step 7,
unmethylated EcoRI
linkers, that encode
EcoRI restriction sites,
are ligated to the ends
of the fragment. In Step
8a, the cDNA is cleaved
with EcoRI restriction
enzyme, generating
sticky-ended cDNA
fragments. (See next
slide).
24. Construction of cDNA Libraries (Part 3)
In the last steps of cDNA library construction, the plasmid vector
is cut with EcoRI restriction enzyme (Step 8b), and then the
EcoRI-cut cDNA and plasmid are ligated together (Step 9).
Finally, the E. coli host strain is transformed and cells are plated
(Step 10) on selective medium. To be complete, both genomic and
cDNA libraries for higher eukaryotes must contain on the order of
a million individual clones.
25. Screening cDNA Libraries
To screen a plasmid library (Fig.
5.16), colonies representing each
cloned DNA first are plated on a
number of petri plates. Library
DNA then is lifted onto
nitrocellulose membranes which
serve as replicas of the plates.
Bound DNA is denatured and
hybridized with a radioactively-
labeled single-strand DNA probe
(next slide). After washing, spots
corresponding to colonies containing
the DNA of interest are detected
by autoradiography. Because not all
DNA gets lifted onto the
membranes, DNA for the clone can
be purified from the residual colony
on the original plate. Note, that
oligonucleotide probes must only be
~ 20 nucleotides long to recognize
unique sequences even in genomic
DNA. The probe sequence can be
derived from genome sequencing
databases, or designed based on
the known sequence of a protein.
26. DNA Detection by
Membrane Hybridization
The general method for
screening a membrane-bound
DNA sample for a gene of
interest is illustrated in Fig.
5.16. This involves fixation of
single-stranded DNA to the
membrane, hybridizing the
fixed DNA to a labeled DNA
probe complementary to the
gene of interest, removal of
un-hybridized probe by
washing, and detection of the
specifically hybridized probe
by autoradiography, etc.
27. Construction of a Yeast
Genomic Library in a
Shuttle Vector
Plasmids known as E. coli-yeast
shuttle vectors (Fig. 5.17a) can
replicate in both organisms.
Shuttle vectors contain 1) origins
of replication for both species
(ori, E. coli; ARS, yeast), 2)
markers for selection in E. coli
(ampr) and yeast (URA3), and 3) a
CEN sequence that ensures stable
replication and segregation in
yeast. The method for
construction of a yeast genomic
library in a E. coli-yeast shuttle
vector is illustrated in Fig. 5.17b.
A total of ~105 clones is needed
to include all genes, if the genomic
DNA is cut into fragments of
about 10 kb in length.
28. Screening by Functional Complementation
A yeast genomic library can be screened by the technique of
functional complementation to isolate the cloned version of a gene
of interest (Fig. 5.18). First, all recombinant plasmids from the
library are isolated from E. coli, pooled, and used to transform
haploid ura3- yeast that carry a conditional lethal ts copy of the
gene of interest. Transformants are selected by plating on
uracil-deficient agar at the permissive temperature. Second,
transformants are replica plated onto agar and incubated at the
nonpermissive temperature to identify colonies carrying a wild
type version of the gene of interest. Only cells containing the
library copy of the wild type gene can survive at high
temperature.