Ch 11 gene regulations how is cloning possible
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Ch. 11: Gene regulations How is cloning possible?. Every cell has the same chromosomes Then….. Why does a heart muscle cell look different from a skin cell? Organisms respond to their environment by altering gene expression Central question: what regulates gene expression?. Differentiation.

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Ch. 11: Gene regulations How is cloning possible?

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Ch 11 gene regulations how is cloning possible

Ch. 11: Gene regulationsHow is cloning possible?

  • Every cell has the same chromosomes

  • Then….. Why does a heart muscle cell look different from a skin cell?

    • Organisms respond to their environment by altering gene expression

  • Central question: what regulates gene expression?


Differentiation

Differentiation

0

  • Differentiation is controlled by turning specific sets of genes on or off


Dna packing

DNA Packing

0

  • eukaryotic chromosomes condense during prophase of Mitosis

  • helps regulate gene expression by preventing transcription

    • Nucleosomes

    • Tight helical fiber =

    • Supercoil = coiling of the tight helical fiber


Ch 11 gene regulations how is cloning possible

0

Metaphase

chromosome

Tight helical fiber

(30-nm diameter)

DNA double helix

(2-nm diameter)

Linker

“Beads on

a string”

Nucleosome

(10-nm

diameter)

Histones

Supercoil

(300-nm diameter)

700 nm

Animation: DNA Packing


X chromosome inactivation

X-chromosome inactivation

0

  • female mammals

  • one of the two X chromosomes is highly compacted and transcriptionally inactive (Barr body)

  • Occurs early in embryonic development, thus all cellular descendants have the same inactivated chromosome

  • Tortoiseshell fur coloration is due to inactivation of X chromosomes in heterozygous female cats


Ch 11 gene regulations how is cloning possible

0

Early embryo

Two cell populations

in adult

Cell division

and random

X chromosome

inactivation

Orange

fur

Active X

X chromosomes

Inactive X

Inactive X

Allele for

orange fur

Black fur

Active X

Allele for

black fur


Eukaryotic gene expression

Eukaryotic gene expression

0

  • Each gene has its own promoter and terminator

  • Are controlled by interactions between numerous regulatory proteins and control sequences


Ch 11 gene regulations how is cloning possible

0

  • Regulatory proteins

    • Transcription factors - help RNA polymerase bind to the promoter

    • Activators –

    • Silencers -

  • Control sequences

    • Promoter

    • Enhancer

      • Related genes located on different chromosomes can be controlled by similar enhancer sequences

Animation: Initiation of Transcription


Ch 11 gene regulations how is cloning possible

Enhancers

Promoter

0

Gene

DNA

Activator

proteins

Transcription

factors

Other

proteins

RNA polymerase

Bending

of DNA

Transcription


Alternative rna splicing

Alternative RNA splicing

0

  • Can involve removal of an exon with the introns on either side

Animation: RNA Processing


Ch 11 gene regulations how is cloning possible

0

Exons

4

1

3

2

5

DNA

4

1

3

2

RNA

transcript

5

RNA splicing

or

4

1

2

1

5

3

2

mRNA

5


Small rnas control gene expression

Small RNAs control gene expression

0

  • RNA interference (RNAi)

    • small, complementary RNAs bind to mRNA transcripts, blocking translation

  • MicroRNA (miRNA)

    • MicroRNA + protein complex binds to complementary mRNA transcripts, blocking translation

Animation: Blocking Translation

Animation: mRNA Degradation


Ch 11 gene regulations how is cloning possible

0

Protein

miRNA

1

miRNA-

protein

complex

2

Target mRNA

4

3

Translation blocked

OR

mRNA degraded


Ch 11 gene regulations how is cloning possible

0

  • Control of gene expression also occurs with

    • Breakdown of mRNA

    • Initiation of translation

    • Protein activation

    • Protein breakdown


Ch 11 gene regulations how is cloning possible

0

Ex. Insulin formation

Folding of

polypeptide and

formation of

S—S linkages

Cleavage

Active form

of insulin

Initial polypeptide

(inactive)

Folded polypeptide

(inactive)


Epigenetic inheritance

Epigenetic Inheritance

This can be accomplished by acetylation or methylation of histones


Regulation of chromatin structure

Regulation of Chromatin Structure

Chemical modification of histone tails can affect the configuration of chromatin and thus gene expression

Histone

tails

DNA

double helix

(a) Histone tails protrude outward from a nucleosome


Ch 11 gene regulations how is cloning possible

Addition of methyl groups to certain bases in DNA is associated with reduced transcription in some species

Acetylated histones

Unacetylated histones

(b) Acetylation of histone tails promotes loose chromatin structure that permits transcription


Ch 11 gene regulations how is cloning possible

NUCLEUS

0

Chromosome

DNA unpacking

Other changes to DNA

Gene

Gene

Transcription

Exon

RNA transcript

Intron

Addition of cap and tail

Splicing

Tail

mRNA in nucleus

Cap

Flow through

nuclear envelope

mRNA in cytoplasm

CYTOPLASM

Breakdown of mRNA

Broken-

down

mRNA

Translation

Polypeptide

Cleavage / modification /

activation

Active protein

Breakdown

of protein

Broken-

down

protein


Why so much control over gene expression

Why so much control over gene expression?

0

  • It allows cells to respond appropriately to their environment

  • Signal transduction pathways convert messages received at the cell surface to responses within the cell via gene expression

  • Three steps:

    • Reception –

    • Amplification/transduction –

    • Response - transcription factor is activated, enters nucleus, transcribes specific genes


Ch 11 gene regulations how is cloning possible

Signaling cell

0

Signaling

molecule

Plasma

membrane

1

Receptor

protein

2

3

Target cell

Relay

proteins

Transcription

factor

(activated)

4

Nucleus

DNA

5

Transcription

mRNA

New

protein

6

Translation


Ch 11 gene regulations how is cloning possible

0

  • Cloning: How? Nuclear transplantation

    • Replacing the nucleus of an egg cell with a nucleus from an adult somatic cell. Allow embryo to form. Embryo can be used in:

      • Reproductive cloning

      • Therapeutic cloning

        • Grow embryonic stem cells in culture

        • Induce stem cells to differentiate and grow into organs, tissues, etc.


Ch 11 gene regulations how is cloning possible

0

Donor

cell

Reproductive

cloning

Nucleus from

donor cell

Implant blastocyst in

surrogate mother

Clone of

donor is born

Remove

nucleus

from egg

cell

Add somatic cell

from adult donor

Grow in culture

to produce an

early embryo

(blastocyst)

Therapeutic

cloning

Remove embryonic

stem cells from

blastocyst and

grow in culture

Induce stem

cells to form

specialized cells


To clone or not to clone

To clone or not to clone….

0

  • Benefits of reproductive cloning?

  • Disadvantages of cloning?


Human stem cell research

Human stem cell research

0

  • Ethical concerns with reproductive cloning

  • Ethical concerns with therapeutic cloning?

    • Benefits:

    • Human embryos – have the greatest potential to give rise to all cell types

    • Adult stem cells (bone marrow) or cord blood cells

      • can give rise to many but not all types of cells


Ch 12 dna technology

Ch 12: DNA Technology

  • DNA profiling

  • Genetically modified organisms/recombinant DNA technology

  • Gene therapy

  • Genomics


Ch 11 gene regulations how is cloning possible

0

1. DNA profiling = analysis of DNA fragments to determine whether they come from a particular individual

  • 3 steps:

    • .

    • Amplify (copy) markers for analysis –

    • Compare sizes of amplified fragments by gel electrophoresis


1 select genetic marker to analyze

1. Select genetic marker to analyze

0

  • Short tandem repeats (STRs) are genetic markers used in DNA profiling

    • STRs =

    • STR analysis compares the lengths of STR sequences at specific regions of the genome

    • Current standard for DNA profiling is to analyze 13 different STR sites


Ch 11 gene regulations how is cloning possible

0

STR site 2

STR site 1

Crime scene DNA

Number of short tandem

repeats match

Number of short tandem

repeats do not match

Suspect’s DNA


Ch 11 gene regulations how is cloning possible

0

2. Amplify the DNA sample

  • Polymerase chain reaction (PCR) = method of amplifying a specific segment of a DNA molecule

  • Relies upon a pair of primers =

  • Repeated cycle of steps for PCR:

    • Sample is heated to separate DNA strands

    • Sample is cooled and primer binds to specific target sequence

    • Target sequence is copied with DNA polymerase


Ch 11 gene regulations how is cloning possible

0

Cycle 1

yields 2 molecules

Cycle 2

yields 4 molecules

Cycle 3

yields 8 molecules

Genomic

DNA

3

3

3

5

5

5

5

DNA

polymerase adds

nucleotides

to the 3 end

of each primer

Cool to allow

primers to form

hydrogen bonds

with ends of

target sequences

2

3

Heat to

separate

DNA strands

1

3

5

3

5

Target

sequence

5

5

3

5

3

5

3

Primer

New DNA


3 gel electrophoresis

3. Gel electrophoresis

0

  • separates DNA molecules based on size

    • DNA samples placed at one end of a porous gel

    • Current is applied and DNA molecules move from the negative electrode toward the positive electrode

    • DNA fragments appear as bands, visualized through staining or radioactivity or fluorescence

Video: Biotechnology Lab


Ch 11 gene regulations how is cloning possible

0

Mixture of DNA

fragments of

different sizes

Longer

(slower)

molecules

Power

source

Gel

Shorter

(faster)

molecules

Completed gel


Ch 11 gene regulations how is cloning possible

0

Crime scene

Suspect 1

Suspect 2

1

DNA isolated

DNA of selected

markers amplified

2

Amplified DNA

compared

3


Ch 11 gene regulations how is cloning possible

0

Mixture of DNA

fragments

Longer

fragments

move slower

A “band” is a

collection of DNA

fragments of one

particular length

Power

source

Shorter

fragments

move faster

DNA attracted to +

pole due to PO4– groups


Applications of dna profiling

Applications of DNA profiling

0

  • Forensics - to show guilt or innocence

  • Establishing paternity

  • Identification of human remains

  • Species identification

    • Evidence for sale of products from endangered species


2 recombinant dna technology genetically modified organisms

2. Recombinant DNA technology/ Genetically Modified organisms

0

  • Recombinant DNA is formed by joining DNA sequences from two different sources:

    • .

    • .

      • Bacterial Plasmids (small, circular DNA molecules independent of the bacterial chromosome) are often used as vectors


Recombinant cells and organisms can mass produce gene products

Recombinant cells and organisms can mass-produce gene products

0

  • Common prokaryotic host: E. coli bacterium

    • Has many advantages in gene transfer, cell growth, and quantity of protein production

  • Common eukaryotic hosts:

    • Yeast: S. cerevisiae

    • “Pharm” animals

      • Will secrete gene product in milk


Ch 11 gene regulations how is cloning possible

0


Ch 11 gene regulations how is cloning possible

0

  • Advantages of recombinant DNA products


Ch 11 gene regulations how is cloning possible

0

  • Genetically modified (GM)

  • Transgenic organisms contain at least one gene from another species


Ch 11 gene regulations how is cloning possible

Agrobacterium tumefaciens

Plant cell

DNA containing

gene for desired trait

1

3

2

Ti

plasmid

Recombinant

Ti plasmid

Introduction

into plant

cells

Insertion of gene

into plasmid

Regeneration

of plant

DNA carrying new gene

Plant with new trait

Restriction site


Ch 11 gene regulations how is cloning possible

Pros?

  • GM plants

  • GM animals


Ch 11 gene regulations how is cloning possible

Cons?

0


3 gene therapy

3. Gene therapy

0

  • One possible procedure:

    • insert functional gene into a virus

    • virus delivers the gene to an affected cell

    • Viral DNA & gene insert into the patient’s chromosome

    • Return the cells to the patient for growth and division


Ch 11 gene regulations how is cloning possible

Cloned gene

(normal allele)

0

Insert normal gene

into virus

1

Viral nucleic acid

Retrovirus

Infect bone marrow

cell with virus

2

Viral DNA inserts

into chromosome

3

Bone marrow

cell from patient

Bone

marrow

Inject cells

into patient

4


4 genomics

4. Genomics

0

  • Genomics =

  • Applications:

    • Evolutionary relationships: Genomic studies showed a 96% similarity in DNA sequences between chimpanzees and humans

    • Medical advancement: Functions of human disease-causing genes have been determined by comparisons to similar genes in yeast


Ch 11 gene regulations how is cloning possible

0


Human genome project

Human Genome Project

0

  • Goals:

    • To determine the nucleotide sequence all DNA in the human genome

    • To identify the location and sequence of every human gene


Ch 11 gene regulations how is cloning possible

0

  • Results of the Human Genome Project

    • 21,000 genes in 3.2 billion nucleotide pairs

    • Only 1.5% of the DNA codes for proteins

    • The remaining 88.5% of the DNA contains

      • Control regions (promoters, enhancers)

      • Unique noncoding DNA

      • Repetitive DNA


Ch 11 gene regulations how is cloning possible

Exons (regions of genes coding for protein

or giving rise to rRNA or tRNA) (1.5%)

0

Introns and

regulatory

sequences

(24%)

Repetitive

DNA that

includes

transposable

elements

and related

sequences

(44%)

Unique

noncoding

DNA (15%)

Repetitive

DNA

unrelated to

transposable

elements

(15%)


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