EUKARYOTIC GENE EXPRESSION

DNA PACKING•Histones•Nucleosomes (1st)•30nm fibers (2nd)•Looped domains (3rd)•Heterochromatin –

not transcribed,metaphase chromatid

•Euchromatin – open,

actively transcribed

•Chromosome location specific

Repetitive DNA (tandem repeats)◦ 10-15% in mammals◦ short sequences (10) repeated in series, 100,000’s

times◦ Fragile X: 100’s instead of 30 triplet repeats◦ Huntington’s: CAG repeats

# of repeats correlates with severity and age of onset.

◦ Regular, mini & micro satellites Interspersed Repetitive DNA

◦ Scattered, 25-40%, similar but not identical◦Alu elements: family of sequences 5% primate

genome, about 300 bp, many transcribed; most transposons

Telomeres & centromeres

Useful in fingerprinting

MULTIGENE FAMILY:Collection of similar or identical genes

Salamander RNA3 kinds of rRNAafter processing,S-sedimentationRates due to differences in density

Chromosome puff

NON-IDENTICAL MULTIGENE FAMILY2 related families that code for globins

4 subunits,2α & 2β

Nonfunctional, very similar to

functional genes

• Gene Amplification: the temporary increase in the number of copies of a gene– rRNA in amphibians– rRNA in developing ovum, large # of

ribosomes burst of protein synthesis after fertilization

– Extra copies cannot replicate & are broken down

• Selective Gene Loss: occurs in certain insects, whole chromosomes or parts of chromosomes may be lost early in development.

Transposons: can prevent normal functioning, may ↑ or ↓production, or be activated, 10% of human genome

Retrotransposons: use an RNA intermediate Immunoglobulin Genes: code for antibodies,

genes become rearranged as immune cells differentiate

Retrotransposon Movement: like retrovirus reproduction, can populate the genome in huge numbers

DNA Rearrangementmaturation of an immunoglobulin gene

The joining of V, J & C regions of DNA in random combinations enormous variety of antibody-producing lymphocytes

•Hundreds of V regions•Several Junction regions•1-2 Constant regions

CONTROL OF GENE EXPRESSION

•Cellular differentiation – divergence in form & function as cells become specialized during development•3-5% expressed at any given time (liver vs skin)•DNA Methylation – genes not expressed, methyl group (CH3) attached

•Barr body – heavy methylation, inactive X, heterochromatin•Genomic Imprinting – turning off alleles

•Histone Acetylation – acetyl groups on histone a.a. causing shape change, grip DNA less tightly, easier access to genes for transcription

OPPORTUNITIES FOR CONTROLOF GENE EXPRESSION

1.DNA packing, methylation, acetylation

2.Transcription (most important)3.RNA Processing4.Transport to cytoplasm5.Degradation of mRNA6.Translation7.Cleavage, Chemical

modification, Transport to cellular destination

8.Degradation of protein

Transcription factors Equivalent to repressors & activators Bind to specific sites (TATA box) May be near promoter Optimum binding – 50 ish factors “read” DNA without unzipping to find appropriate

gene (zinc fingers & leucine zippers)Promoter, Enhancer, Suppressor sequences

Transcription initiation – controlled by transcription factors (proteins) that interact w/ DNA & each other.Typical Eukaryotic Gene – promoter, terminator, distal & proximal control elements (key to high levels of transcription)

Promoter regions bind to RNA polymerases I,II,III (tRNA’s)

Model for enhancer action

Activator proteins bind to enhancer sequencesDNA bends, activators closer to promoter

Protein-binding domains attach to transcription factors, form active transcription initiation complex on promoter

DNA Binding Domain: 3-D part of a transcription factor that binds to DNA

Found in many regulatory proteins

α helix β sheetheld by zinc atom

2 α helices w/ spaced leucines coil

mRNA processing – mG cap? poly A tail? Alternative splicing – same primary

transcript, different introns & exons mRNA degradation – prokaryotic mRNA’s

last a few minutes; eukaryotic- hours, can be days even weeks (hemoglobin)

Translation inhibited by masked mRNA prior to fertilization (activated in embryo)

Protein processing & degradation

ALTERNATIVE mRNA SPLICING

PROTEIN DEGRADATION

EUKARYOTIC vs PROKARYOTIC

Genes spread out1 promoter = 1 geneMany intronsProcessing2 copies of DNA (2n)Paired, rod shaped

chrom.3 polymerasesNucleus, separation of

transcription/translation

Operons1 promoter=multiple

genesLack intronsNo processing1 copy of DNA (n)Single circular chrom.1 polymeraseNo nucleus,

simultaneous transcription/translation

Mutations in genes that regulate growth & division, chemical carcinogens, physical mutagens (X-rays, viruses)

Oncogenes – cancer causing Proto-oncogenes – normal gene oncogene Tumor-suppressor genes – prevent uncontrolled

division ras gene – proto-oncogene p53 gene – tumor suppressor gene

Proto-oncogenes Oncogenes

ras gene•G protein•Relays growth signal•Result – stimulation of cell cycle

p53 – transcription factor, activates p21, product blocks CDK’s

MODEL: DEVELOPMENT OF COLORECTAL CANCER

DNA TECHNOLOGY

TERMINOLOGY

Recombinant DNA – DNA in which genes from two different sources are combined in vitro into the same molecule.

Genetic engineering – direct manipulation of genes for practical purposes.

Biotechnology – manipulation of organisms or their components to make useful products.

Gene cloning – method for preparing well defined, gene sized pieces of DNA in multiple identical copies.

BACTERIAL PLASMIDS FOR CLONING

RESTRICTION ENZYMES-Cut up foreign DNA. -Endonucleases

RESTRICTION SITE-Recognition sequence-Usually symmetrical

RESTRICTION FRAGMENT-Piece of DNA cut by specific enzyme

STICKY END-Single strand end of restriction fragment

Cloning a human gene in a bacterial

plasmid

USING A NUCLEIC ACID PROBE TO IDENTIFY A

CLONED GENE

Cloned gene of interest on a plasmid, probe is short length of radioactive single stranded DNA complimentary to part of gene.2) Result – single stranded DNA stuck to filter paper3) Probe DNA hybridizes with complimentary DNA on filter4) Filter laid on photographic film, radioactive areas expose film (autoradiography)

MAKING COMPLEMENTARY DNA (cDNA) FOR A EUKARYOTIC GENE

Expression vector - Cloning vector w/ prokaryotic promoter upstream of restriction site where eukaryotic gene can be inserted.

cDNA – made in vitro using mRNA template & reverse transcriptase (DNA w/o introns)

Yeast artificial chromosomes (YACs) – vectors that combine the essentials of a eukaryotic chromosome ( origin for replication, centromere, 2 telomeres) w/ foreign DNA.Electroporation – application of electric pulse to solution containing cells creating temporary hole in membrane allowing DNA to enter.Genomic library – complete set of thousands of recombinant plasmid clones, each carrying copies of a particular segment from the initial genome.cDNA library – library containing a collection of genes (represents only part of a cell’s genome – only the genes that were transcribed in the starting cells)

TERMINOLOGY

Shown - 3 of the thousands of “books” in the library. Each is a bacterial clone containing one particular variety of foreign genome fragment in its recombinant plasmid

The same 3 foreign genome fragments in a phage library

PCR

Polymerase chain reaction

•Technique to quickly amplify (copy many times) a piece of DNA w/o using cells

•Start w/ double stranded DNA (“target”), add to polymerase, nucleotide supply & primers

•5 minutes per cycle

DNA ANALYSIS & GENOMICS

Genomics – the study of whole sets of genes and their interactions

Gel electrophoresis – separates macromolecules (nucleic acids or proteins) based on size, electrical charge and other physical properties.

Southern blotting – hybridization technique that enables researchers to determine the presence of certain nucleotide Sequences in a sample of DNA.

Restriction fragment length polymorphisms RFLPs - differencesin DNA sequence on homologous chromosomes that can result in different restriction fragment patterns. Scattered abundantlythroughout genomes

GEL ELECTROPHORESIS

3 DNA samples placed in wells.

Electrodes attached & voltage applied

Negatively chargedDNA migrates toward positive electrode.

Longer fragments travel more slowly

DNA samples arranged in bands along a “lane” according to size.

Shorter fragments travel farthest

Using restriction fragment patterns to distinguish DNA from different alleles

a) 2 homologous segments w/ different alleles

b) Electrophoresis separates the fragments; allele 1has 3 fragments, allele 2 has 2

c) Addition of binding dye, fragments fluoresce pink; shown: 6 samples cut w/ a restriction enzyme

RESTRICTION FRAGMENT ANALYSIS BY SOUTHERN BLOTTING

DNA denatured & transferred to paper

Probe complimentary to gene of interest

Radioactivity exposes film, image forms – bands w/ DNA base-pairs w/ probe

MAPPING GENOMES AT THE DNA LEVEL

• Human Genome Project – effort to map the entire human nucleotide sequence for each chromosome.

•Genetic (Linkage) Mapping – construction of a linkage map using various genetic markers.

•Physical Mapping: Ordering DNA Fragments

−chromosome walking: make fragments that overlap, then use probes of the ends to find the overlaps

−Bacterial artificial chromosome (BAC): artificial version of a bacterial chromosome that can carry inserts of 100,000 – 500,000 base pairs

•DNA Sequencing – determining the nucleotide sequence of a DNA segment or an entire genome. 3 sequencing methods

•Alternative Approaches to Whole-Genome Sequencing

1) Prepare probe to match 3’ end2) Cut starting DNA w/ 2 restrictionenzymes & clone fragments3) Use probe 1 to screen library II for DNA fragments that overlap the known gene4) Isolate DNA from tagged clone,prepare probe 2 to match 3’ end ofthat segment.5) Use probe 2 to screen library I for an overlapping fragment farther along6) Repeat 4&5 with new probes &Alternating libraries to “walk” down DNA7) Result – DNA map w/ series of known markers (sequences) in a Known order & separated by knowndistances

Chromosomewalking

4 portions each incubated w/ • primer•DNA polymerase•4 deoxyribonucleoside triphosphates: dATP, dGTP, dCTP, dTTP•Different one of the 4 nucleotides in modified dideoxy (dd) form

SANGER METHOD

Eventually, a set of labeled strandsof various lengthsis generated.

4 portions each incubated w/ •primer

•DNA polymerase•4 deoxyribonucleoside triphosphates: dATP, dGTP, dCTP, dTTP•Different one of the 4 nucleotides in modified dideoxy (dd) form

Synthesis of new strands beginsat primer & continues until a dideoxyribonucleotide is incorporated, which prevents further synthesis.

SANGER METHODSANGER METHOD

New strands separated by electrophoresis

Sequence can be read from bands on autoradiograph and original template sequence deduced.Longest fragment ends with a ddG, so G must be the last base in the sequence

SANGER METHOD

ALTNERATIVE STRATEGIES FOR SEQUENCING AN ENTIRE GENOME- The arrangement of DNA fragments in order depends on their having overlapping regions

Cut DNA of chromosome into small fragments

Clone fragments in plasmid or phage vectors

Sequence fragments

Assemble overall sequence

DNA microarray assay for gene expression

Researcher simultaneously test all the genes expressed in particular tissue for hybridization with an array of short DNA sequences representing thousands of genes.

Fluorescence intensity indicates relative amount of mRNA in tissue.

Practical Applications of DNA Technology

Diagnosis of Diseases – PCR & labeled probes Human Gene Therapy – replacing defective genes Pharmaceutical Products – vectors, vaccines Forensics - DNA fingerprinting, simple tandem repeats Environmental Uses – mining, sewage treatment,

detoxifying microbes (oil spills etc.) Agricultural Uses – transgenic organisms, “pharm”

animals, Ti plasmid, herbicide resistant crops Genetically Modified Organisms – safety & ethical

questions

Using RFLP markers to detect presence of disease causing alleles

Gene Therapy

DNA Fingerprinting

Using the Ti plasmid as a vector for genetic engineering