Biotechnology: Genes and Genomes Reviewing cells, protein ... · •Transformation: DNA is taken up...

Biotechnology: Genes and Genomes Reviewing cells, protein synthesis, etc.

© 2013 Pearson Education, Inc.

Chapter Contents

•2.1 A Review of Cell Structure

•2.2 The Molecule of Life

•2.3 Chromosome Structure, DNA

Replication, and Genomes

•2.4 RNA and Protein Synthesis

•2.5 Mutations: Causes and Consequences

•2.6 Revealing the Epigenome


2.1 A Review of Cell Structure

•All cells have these structures:

•Plasma Membrane – double-layer structure

of lipids and proteins that surrounds its outer

surface

•Cytoplasm – inner contents of a cell

between the nucleus and plasma membrane

•Ribosomes – structures in the cytoplasm

which facilitate protein synthesis

•DNA – molecules which carry genetic code


Insert table 2.1


•Comparison of Prokaryotic and

Eukaryotic Cells



•Prokaryotic Cells


Insert figure 2.2


•Eukaryotic cells (include plant and animal

cells)



•Plasma membrane: fluid, dynamic, complex

double-layered barrier made of what

macromolecules?

–Roles include cell adhesion, cell-cell communication, cell

shape, transport molecules in and out of cell, is selective

barrier

•Cytosol: nutrient-rich, gel like fluid that makes up

cytoplasm

•Organelle: compartment where chemical rxns and

cell processes take place

–Each organelle has its own biochem rxns

–Allows coordination of chem rxns within a cell


2.1 A Review of Cell Structure Work in groups to complete the table






2.2 The Molecule of Life

•Evidence that DNA is the Inherited Genetic

Material

–1869 Friedrich Miescher: "nuclein"

•Could not be broken down by proteases

•Based on this observation, would you consider it a

protein?

•Discovered nuclein had acidic properties: renamed "nucleic

acids"

•What are the 2 types of nucleic acids?



–More Evidence that DNA is the Inherited Genetic

Material

–1928 Frederick Griffith

•Two strains of bacteria (Streptococcus pneumoniae)

–Virulent disease causing smooth strain (S cells) and harmless rough

strain (R cells)

•S cells are surrounded by capsule (smooth coat)

•R cells lack the capsule

•Did experiment in which mice were injected with either a) S

strain; b) R strain; c) heat killed S strain or d) mix of heat

killed S strain and live R strain to see if mice would survive


•What are the macromolecules that make up the capsule?

•Why did they heat the S strain and not the R?

•Discuss the process of transformation in fig. d.




•Transformation: DNA is taken up by another

bacterium

•Heat treatment broke open S cells and their

capsule so DNA was released

•Living R cells took up S cell DNA which

transformed the property of R cells so they

became virulent/pathogenic

•Molecular biologists use transformation

routinely to introduce genes into bacteria for

cloning



•Definitive evidence that DNA Is the Inherited

Genetic Material

–1944 Oswald Avery, Colin MacLeod, and Maclyn McCarty

•Homogenized mixtures of bacterial cells from large batches of

Streptococcus pneumoniae

–Treated extracts with proteases; RNases; DNases

–Did transformation expts with the extracts

•Found that extracts from killed S cells treated with DNase mixed with living R

cells did not transform because DNA in S cells was degraded

•Experiment provided evidence that DNA is genetic material and proved

that it was the transforming factor in the Griffith experiments



•Follow-up questions related to Avery, MacLeod, McCarty experiment

•Why did extracts treated with proteases still maintain ability to be transformed?

•Why did extracts treated with RNases still maintain ability to be transformed?

•Why did extracts treated with DNases lose the ability to be transformed?



•DNA Structure

–Building block of DNA is the nucleotide

–Each nucleotide is composed of

•Pentose (5-carbon) sugar called deoxyribose

•Phosphate molecule

•A nitrogenous base

–The nitrogenous bases are the interchangeable

component of a nucleotide

•Each nucleotide contains one base

–Adenine (A), thymine (T), guanine (G) or cytosine (C)


•Building block of DNA = nucleotide

•What is the difference between deoxyribose and ribose sugar?




•DNA Structure

–Rosalind Franklin and Maurice Wilkens provided x-

ray crystallography data to show helical structure

–James Watson and Francis Crick used data from

Franklin and Wilkens; Chargaff base pairing rules to

develop wire model that revealed the definitive

structure of DNA

–"The Molecular Structure of Nucleic Acids: A

Structure for Deoxyribose Nucleic Acid" published in

Nature on April 25, 1953



•DNA Structure

–Nucleotides are joined together to form long strands of DNA

and each DNA molecule consists of two strands that join

together and wrap around each other to form a double helix

–Nucleotides in a strand are held together by

phosphodiester bonds

–What part of a nucleotide and adjacent nucleotide does

the phosphodiester bond connect?

–Each strand has a polarity – a 5' end and a 3' end

–Polarity refers to the carbons on what part of a

nucleotide?



•DNA Structure

–The two strands of a DNA molecule are held

together by hydrogen bonds

•Formed between complementary base pairs

•Adenine (A) pairs with thymine (T)

•Guanine (G) pairs with cytosine (C)

–The two strands are antiparallel because their

polarity is reversed relative to each other

–DNA resembles a twisted ladder


•What are the rungs of the ladder?

•What is the backbone or sides of the ladder?




•What is a gene?

•Sequence of nucleotides that provides cells with

instructions to synthesize a protein or type of RNA

•On average genes are 1000–4000 nucleotides long

•Genes influence how cells, tissues and organs

appear

–Define the term trait:

–*not all genes are used to produce a protein

•(example) State the function of genes involved in making tRNA.


2.3 Chromosome Structure, DNA


•Chromosome Structure

–Chromosomes – where cells package DNA

–Chromatin – strings of DNA and DNA-binding

proteins called histones

State of DNA inside the nucleus when the cell

is NOT dividing - long, thin, chromatin. •During cell division chromatin is coiled into fibers that wrap

around each other so chromosomes are highly coiled

•Why would chromatin be condensed during cell division

based on what you know about the number of bases in

every cell?




•Most human cells have two sets (pairs) of 23

chromosomes (paternal and maternal), or 46

chromosomes total

These are called homologous pairs

–Autosomes – chromosomes 1-22

–Sex chromosomes – chromosome pair # 23

•X and Y chromosomes

•Gametes (sex cells) contain a single set of 23

chromosomes (haploid number, n)

•All other cell in body are somatic cells

•What type of cell is a kidney cell?




•Characteristics common to eukaryotic chromosomes

•Chromosome consists of two thin, rodlike structures of DNA

called sister chromatids

–Exact replicas of each other copied during DNA replication

–During cell division, each sister chromatid is separated

–Each chromosome has a single centromere – region consisting of

intertwined DNA and protein that join sister chromatids together and also

contain proteins that attach chromosomes to microtubules

•Centromere delineates each sister chromatid into 2 arms – p and q

•Each arm of chromosome ends with a telomere- highly conserved

repetitive nucleotide sequence that attach chromosomes to nuclear

envelope

–telomeres allow cells to divide without losing genes) but during aging

and cancer progression telomeres become shortened




•Chromosome Organization

•What is a potential consequence of shortening of telomeres during

the aging process?


•Karyotype- way to study chromosome number and basic aspects of chromosome structure –Spread cells on microscope slide and treat with chemicals to release and stain chromosomes

–Chromosomes can be aligned and paired based on staining pattern and size

•Based on the karyotype, which chromosome is the largest? Explain how you came up with your answer (work in groups).

•What would a karyotype for Trisomy 21 (Down Syndrome) look like? Explain.






•DNA Replication

–Somatic cells divide by a process called mitosis

–Mitosis

•One cell divides to form two daughter cells, each with an

identical copy of the parent cell DNA

•A single skin cell would produce how many cells and

how many chromosomes/cell?

•In order to accomplish this, the DNA of the parent cell must

be copied prior to mitosis




•DNA replication

•Sex cells divide by a process called meiosis – parent cell

divides to create 4 daughter cells which can be sperm or egg

cells

•The DNA in each daughter cell is not an identical copy of the

parent cell

•Chromosome number is cut in half to the haploid number

•How many sets of chromosomes does each daughter

cell have?

•Through sexual reproduction a fertilized egg called zygote is

formed and zygote divides by mitosis to form an

embryo = complete set of 46 chromosomes




•Semiconservative Replication

–DNA replication occurs in such a manner that, after

replication, each helix contains one original (parental) DNA

strand and one newly synthesized DNA strand




•Steps in DNA Replication

1.Unwinding the DNA –Helicase enzyme breaks the hydrogen bonds between complimentary base pairs that hold the two DNA strands together; "unzips" DNA

–Single strand DNA binding proteins bind to each strand and prevent them from base pairing and reforming a double helix

–Separation of strands occurs in regions called origins of replication

2.Adding short segments of DNA 10–15 nucleotides long called DNA primers –Primase enzyme synthesizes DNA primers

–DNA primers start the replication process because they serve as binding sites for DNA Polymerase – enzyme that synthesizes new strands of DNA




•Steps in DNA Replication

3.Copying the DNA

–DNA polymerase enzyme binds to the

DNA primers

–Uses nucleotides to synthesize complementary

strands of DNA

–Always works in one direction – 5' to 3' direction

–What kind of bond is formed between the

phosphate in one nucleotide and sugar in the

previous nucleotide?




•Steps in DNA replication

•Since DNA Pol only goes in 5'–3' direction, replication along leading strand is continuous and is discontinuous along lagging strand

•Why is it discontinuous on lagging strand?

•Short Okazaki fragments are synthesized as DNA Pol works on lagging strand

•DNA primers are replaced with DNA nucleotides using DNA Pol

•Covalent bonds are formed between Okazaki fragments with DNA ligase







•Genome – all of DNA in organism's cell

•DNA contains instructions for life in form of genes

•Human genome has 20,000 genes scattered among 3 billion base pairs of DNA!

•What is the study of genomics?

•What was the purpose of the human genome project that was completed in 2003?

CENTRAL DOGMA:

http://www.wiley.com//legacy/college/boyer/04700037

90/animations/central_dogma/central_dogma.htm

http://www.wiley.com/legacy/college/boyer/0470003790/animations/central_dogma/central_dogma.htm

http://www.wiley.com/legacy/college/boyer/0470003790/animations/central_dogma/central_dogma.htm


•Transcription – genes are copied (transcribed) from DNA code into RNA code

•Translation – RNA code (exact copies of genes) is read into a protein sequence of amino acids

•Through production of RNA and protein synthesis DNA controls properties of the cell and its traits

•Give an example of a trait using the words in the above figure.

2.4 RNA and Protein Synthesis



•Transcription

–Occurs only in segments of chromosomes that contain

genes

–RNA polymerase unwinds DNA helix and copies one

strand of DNA into RNA

•Binds to a promoter region – consensus nucleotide sequence

•Transcription factor (TF) proteins are DNA binding proteins that bind to

specific regions on DNA – purpose of transcription factors is to help RNA

Pol find the promoter

–TF can act to speed up transcription (like Red Bull drink) or stop

transcription (like Tylenol PM)

•Enhancers are DNA nucleotides that also play role in transcription



•Transcription •After RNA Pol binds to promoter, it unwinds a region of DNA to separate the 2 strands

•The template strand is copied by RNA Pol

•Copies template DNA in a 5' to 3' direction into RNA

•Uses nucleotides

–Adenine, uracil, guanine, and cytosine

–A-U, C-G

–What kind of covalent bond is formed between ribonucleotides?

–At end of gene, RNA polymerase encounters the termination sequence to create loops at the end of RNA so RNA polymerase and newly formed strand of RNA are released from DNA molecule

–RNA strand is called a messenger RNA (mRNA)

–Multiple copies of mRNA are transcribed from each gene during transcription





•mRNA is not the only type of RNA that gets transcribed.

•Name 2 other types of RNA that are produced by transcription.

•Do these 2 types of RNA carry information that directly codes for synthesis of protein? Work in groups and explain.

•New class of non-protein coding RNA: microRNA



•mRNA Processing

–Initial mRNA produced is the primary transcript •Immature and not fully functional

–Three modifications before primary transcripts are ready for protein synthesis (takes place in nucleus) •RNA splicing – splice out the DNA not coding for proteins (introns) and retain the protein coding sequence of the gene (exons)

–Alternative splicing – multiple proteins produced from single gene

•3' PolyA tail – 100–300 adenine nucleotides added to protect mRNA from RNA degrading enzymes; increase its stability and availability for translation

•Addition of a 5' cap – guanine base containing methyl group allows ribosome recognition





TRANSLATION

•Components of translation – in cytoplasm

•mRNA – messenger RNA: copy of gene (acts as

messenger by carrying genetic code from nucleus to

cytoplasm where info is read into protein)

•tRNA – transfer RNA: molecules that transport

amino acids to ribosomes during protein synthesis

•rRNA – ribosomal RNA: short single stranded RNA

molecules and are components of ribosomes

•Ribosomes – site of protein synthesis



•How is mRNA read?

–Genetic code – universal language of genetics used by

virtually all living organisms

•Works in three nucleotide units of mRNA called codons

•Each codon codes for a single amino acid

•One amino acid may be coded for by more than one codon

–There are 64 codons and only 20 amino acids – degeneracy of the

genetic code

•Start codon (AUG) codes for Met and signals starting point for translation

•Stop codons – UGA, UAA, UAG – do not code for amino acid but signal

end of translation



•Read the genetic code and state which amino acid has the most codons. Explain.

• Read the genetic code and state which amino acid has the least amount of redundancy within the genetic code.



•Ribosomes and tRNA molecules

•Ribosomes – aggregates containing rRNA and protein that

make up subunits

–Each ribosome contains 2 subunits: large and small and associate to

form 2 grooves A (aminoacyl) and P (peptidyl) site into which tRNA

molecules bind and also E (exit) site which tRNA molecules leave the

ribosome

–tRNA – small molecules that fold into cloverleaf structure- has site for

amino acid attachment by aminoacyl tRNA synthetase enzyme

•Aminoacyl tRNA bind to A site of ribosome

•Opposite end of tRNA is 3 nucleotide sequence = anticodon

•Each amino acid binds to a different anticodon

• Anticodons form complimentary base pairs with what part of the

mRNA sequence?

•What are the 3 players of translation?


2.4 RNA and Protein Synthesis Stages of translation

1.Initiation – small ribosome subunit binds to 5' end of mRNA

•What part of mRNA processing allows this 5' end to be recognized by ribosome?

–Initiation protein factors help guide small ribosome subunit to mRNA

–Small ribosome subunit moves along the mRNA until the start codon is found

–Small subunit waits for correct tRNA (initator tRNA)

• Met amino acid is attached to what anticodon?

–Now large subunit binds to complex containing small subunit; initiation factors; mRNA; initiator tRNA

2. Elongation – tRNAs, carrying the correct amino acid, enter the ribosome, one at a time, as the mRNA code is read after 2 tRNAs are attached to ribosome peptidyl transferase catalyzes formation of peptide bond between 2 amino acids translocation phase: ribosome shifts so tRNA and protein move into P site

3. Termination – ribosome encounters the stop codon

–Releasing factor proteins interact stop codon to terminate translation and ribosomal subunits come apart and release the mRN with newly formed protein released into the cell





•Basics of Gene Expression Control

–Gene expression refers to the production of mRNA by a cell

•All cells of an organism contain the same genome.

•If they have the same genome, then why are pancreas

cells different from lung cells?

•Not all genes will be turned on at the same time – some will

be upregulated while others will be silenced

•Will a gene coding for a protein that is involved in

concentrating be turned on while you are in this lecture

and while you are sunbathing at the beach? Work in

groups to discuss the answer.




–Gene regulation is how genes can be turned on

and off in response to different signals

–There are several levels of gene regulation

–The fastest gene regulation is energetically the

most costly. Work in groups to explain this idea.



•Levels of gene expression

regulation




–Transcriptional regulation – controlling the amount of

mRNA transcribed from a particular gene as a way to turn

genes on or off

–Promoter is DNA sequence located upstream from the gene

•Certain sequences found in the promoter region

–TATA box and CAAT box

•RNA polymerase cannot bind to promoter region without presence of

transcription factors – DNA binding proteins

•Enhancer DNA sequences which are located very far upstream or

downstream of a gene can bind to regulatory proteins called activators


•How can transcription be inhibited? Work in groups to answer this question.





–Bacteria use operons to regulate gene expression

•Organization of bacterial genes

•Clusters of several related genes located together and

controlled by a single promoter

•Operator – region within promoter

–Can use operons to regulate gene expression in

response to their nutrient requirements

•lac operon




–Bacteria use operons to regulate gene expression

•Organization of bacterial genes

•Clusters of several related genes located together and

controlled by a single promoter

–Can use operons to regulate gene expression in

response to their nutrient requirement



•Lac operon and its players:

•Laci codes for repressor protein

•3 genes coded by lac operon: lac z codes for B Gal that breaks down

lactose into glucose and galactose; lacy codes for galacto permease

which is a membrane protein that transports lactose into the cell; lacA

codes for acteyltransferase which has a protective function

•When there is lactose, it acts as an inducer by binding to the repressor

and changing its shape so it falls off the operator and allows RNA Pol to

transcribe the 3 genes

•When there is glucose the repressor stays bound and prevents

transcription

•Why is it energetically favorable to not transcribe the 3 genes

coded by the lac operon when the bacteria is exposed to glucose

and no lactose?

•Why is it necessary to turn transcribe the 3 genes coded by the lac

operon in the presence of lactose and no glucose?



lac operon




–RNA interference – RNA mechanism of gene silencing

–Short interfering RNA (siRNA) – 22 nucleotide double

stranded non-coding RNA bind to mRNA and regulate gene

expression by "silencing" gene expression through blocking

or interfering with translation of bound mRNA

–Micro RNA – another category of small RNA molecules that

do not code for proteins

•miRNA silence gene expression by blocking translation of mRNA or

causing degradation of mRNA

•Work in groups and describe an example of how RNA interference

can be used as a gene therapy for a particular disease.


2.4 RNA and Protein Synthesis RNA interference


2.5 Mutations: Causes and Consequences

•Mutation – change in the nucleotide sequence of DNA –Major cause of genetic diversity

–Can also be detrimental

–Can be spontaneous (due to error during DNA replication) or induced due to environmental factors including X rays and UV

–They exert their effects on a cell by changing properties of the protein which affects the trait

•Types of Mutations

•Work in groups to discuss examples of each type of point mutation and how it can affect the protein structure and function –Point mutations

•Silent mutations

•Missense mutations

•Nonsense mutations

•Frameshift mutations





•Gene mutations can be inherited or acquired

–Inherited mutations are those passed on to

offspring through gametes

•Can cause birth defects or inherited diseases because the

mutation is present in the genome of all of the offspring's

cells

–Acquired mutations occur in the genome of

somatic cells

•Are not passed along to offspring

•These mutations can lead abnormalities in cell growth and

ultimately become cancerous



•Mutations are a major cause of genetic diversity

–Human genomes are approximately 99.9% identical

•0.1% differences in DNA between individuals, or one base out of every thousand –Roughly 3 million differences between different individuals

•Most have no obvious effects; other mutations strongly influence cell functions, behavior, and susceptibility to genetic diseases

•Most genetic variations are created by SNPs (single nucleotide polymorphism) – are bad when they occur in exons and change protein structure so ultimately can change protein function


2.6 Revealing the epigenome

•Epigenome – modifications in chromatin structure which DO NOT involve mutations in DNA sequence

•Changes in epigenome affect both DNA and histones

–Some epigenetic modifications are inherited and others vary from generation to generation

–Some are reversible; some are long lasting

–These changes vary between cell types in the body and in normal and diseased tissues

–Diet and environment can influence epigenome

–Involved in patterns of gene expression during development


2.6 Revealing the epigenome

•Please watch the interactive video:

http://learn.genetics.utah.edu/content/epigenetics/

twins/epigenetics.

•Then work in groups and discuss why identical twins

are different as they get older.

http://www.slideshare.net/Pammy98/teaching-biotechnology-in-the-local-high-schools-ppt

















BIOTECHNOLOGY

UNIT TWO: Raw Materials and Basic Skills

BIOTECHNOLOGY

UNIT TWO: Raw Materials and Basic Skills

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Biotechnology: Genes and Genomes Reviewing cells, protein ... · •Transformation: DNA is taken up...

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