gene regulation and pathological development studies using mouse models
Download
Skip this Video
Download Presentation
Gene Regulation and Pathological Development Studies Using Mouse models

Loading in 2 Seconds...

play fullscreen
1 / 74

Gene Regulation and Pathological Development Studies Using Mouse models - PowerPoint PPT Presentation


  • 104 Views
  • Uploaded on

Gene Regulation and Pathological Development Studies Using Mouse models. Yaowu Zheng Transgenic Research Center [email protected] (0431)85098583. Materials and Lecturers. Oct. 9: Introduction to Gene regulation, Zheng Oct. 16: Gene expression and development, Zheng

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about ' Gene Regulation and Pathological Development Studies Using Mouse models' - hans


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
gene regulation and pathological development studies using mouse models

Gene Regulation and Pathological DevelopmentStudies Using Mouse models

Yaowu Zheng

Transgenic Research Center

[email protected]

(0431)85098583

materials and lecturers
Materials and Lecturers
  • Oct. 9: Introduction to Gene regulation, Zheng
  • Oct. 16: Gene expression and development, Zheng
  • Oct. 23: Gene expression and coagulation system, Zheng
  • Oct. 30: Gene expression and immune system, Professor Zhang
  • Nov. 6: Gene expression and cancer, Zheng
  • Nov. 13: Gene expression and vascular system, Professor Zhang
  • Nov. 20: Gene expression and obesity, Dr. Xiaodan Lu
  • Nov. 27: Gene expression and cardiovascular disease, Zheng
  • Dec. 4: Gene expression and reproduction, Yan Ji
  • Dec. 11: Transgenic technology, Zheng

Lecturers:

  • Yan Ji: Ph.D student in Zheng lab.
  • Xiaodan Lu: Postdoctoral fellow in Jilin University, former Ph.D graduate student from Zheng’s lab
  • Professor Zhang: Assistant professor, Ph.D graduate from Nanking University
  • Yaowu Zheng: Professor in Transgenic Research Center, NENU
contents
Contents
  • Basic gene regulation
  • Higher level regulation
  • Prokaryotic regulation
  • Eukaryotic regulation
  • Regulation and development
  • Gene regulation and disease
  • Application of gene regulation
  • Gene regulation studies using transgenic mice
central dogma
CENTRAL DOGMA
  • Genetic information always goes from DNA to RNA to protein

Updated Central Dogma

biological sequence information
Biological sequence information

Primary structure in living organisms

  • 3 linear biopolymers:
    • Polydeoxyribonucleotide= DNA
    • Polyribonucleotide= RNA
    • Polypeptide=Proteins
  • 3 classes of information transfer suggested by the dogma:
    • General case
      • Replication: DNA → DNA,
      • Transcription: DNA → RNA,
      • Translation: RNA → protein (general)
    • Special case
      • Reverse transcription: RNA → DNA,
      • Viral: RNA → RNA,
      • Non-natural: DNA → protein
    • Unknown case
      • protein → DNA,
      • protein → RNA,
      • protein → protein? prions
slide8

Regulation at DNA levels

Double helix Structure

prokaryotic gene regulation
Prokaryotic Gene Regulation
  • Combination of a promoter and a gene or genes is called an OPERON
  • Operon is a cluster of genes encoding related enzymes that are regulated together and performed one related processes
  • Operon consists of
    • A promoter site where RNA polyerase binds and begins transcribing the message.
      • Genes coding protein from the message (mRNA) is called structural genes.
      • One mRNA codes for more than one protein is called polycistronic.
    • An upstream region (a gene) that makes a protein called repressor. The gene codes for repressor is called regulatory gene. The repressor is like a transcription factor in eukaryotes.
  • Active repressor sits a site between promoter and structural genes and blocks transcription, This site is called the operator which works like an enhancer or silencer in eukaryotes.
types of operons
Types of Operons
  • Inducible Operon
    • e.g lac operon
  • Repressible Operon
    • e.g trp operon
trp operon
Trp Operon
  • E. coli uses several proteins encoded by a cluster of 5 genes to manufacture the amino acid tryptophan
  • All 5 genes are transcribed together as a unit called an operon, which produces a single long piece of mRNA for all the proteins
  • RNA polymerase binds to a promoter located at the beginning of the first gene and transcribe the genes in sequence
  • A trp repressor protein is made constantly (constitutively).
  • When tryptophan is present in the cell, it binds to repressor and activates it, so it can binds operator and tell operon to stop making tryptophan.
  • When tryptophan is absent, the trp repressor is inactive and can not binds to operator, so trp operon is making all the necessary proteins for tryptophan synthesis.
lac operon
Lac Operon
  • The lac Operon
    • Regulates lactose metabolism
    • It turns on when lactose is present & glucose is absent.
    • Lactose is a disaccharide, a combination of Galactose & Glucose
  • To Ferment Lactose E. coli Must:
    • Transport lactose across cell membrane
    • Separate the two sugars. To do that it needs three enzymes, it makes them all at once!
    • 3 Genes Turned On & Off Together:
      • Lac Z code for b-galactosidase
      • LacY codes for permease that allows lactose to enter cell
      • LacA code for enzyme that acetylates lactose
lac operon1
Lac Operon
  • Near the lac operon is another gene, called lacI that codes for the lac repressor protein
  • The lac repressor gene is expressed “constitutively”, meaning that it is always on.
  • Just upstream from the transcription start point in the lac operon are two regions called the operator (o) and the promoter (p).
  • Operator is the DNA sequence that repressor binds.
  • The promoter is the site where RNA polymerase binds and starts transcription.
  • Operator and promoter are “cis” or associated to lac operon.
  • Lac repressor protein is “trans” to lac operon, since the repressor is diffusible and can bind to other lac operator sequences.
  • In the presence of lactose, the repressor binds to lactose, changes conformation and floats away from the operator. RNA polymerase can bind to the promoter and transcribed z, y and a. Permease allow lactose to enter the cell, b-galactosidase digests the lactose and transacetylase modify galactose.
  • When level of lactose drops, the released repressor binds to the operator DNA again and blocks lac operon
prokaryotic translational control
Prokaryotic Translational Control
  • Shine-Dalgarno sequence is generally located 8 basepairs upstream of the start codon AUG
  • The six-base consensus sequenceAGGAGG helps to recruit the ribosomes to the mRNA to initiate protein synthesis
  • The complementary sequence (CCUCCU), called the anti-Shine-Dalgarno sequence, is located at the 3\' end of the 16SrRNA in the 30S ribosome subunit.
eukaryotic gene regulation
Eukaryotic Gene Regulation
  • A multi-cell organism may contain millions of cells.
  • Virtually every cell in your body contains 1 or 2 complete set of genes
  • But they are not all turned on at all the time or in every tissues
  • Each cell or tissue in your body expresses only a small subset of genes at any time
  • During development, different cells express different sets of genes in a precisely regulated fashion
  • Some genes are expressed only at certain conditions
multilevel gene regulation in eukaryotes
Multilevel Gene Regulation in Eukaryotes
  • Features of Eukaryotic Genomes
  • Gene structure of eukaryotes
  • The types of regulation in eukaryotes
  • Multi-level of gene expression and regulation
slide21

Chromatin

Chromosome

slide22

Nucleosome:

Two of each H2A, H2B, H3 and H4 subunits in the core

Average 200 bp

140bp in core area

genome level regulation
Genome Level Regulation
  • Epigenetic Modification
  • Developmental methylation and Acetylation
  • DNA folding and unfolding
  • X-chromosomal inactivation
  • Chromosomal remodeling
  • Replication
unpredictable gene regulation at dna levels
Unpredictable Gene Regulation at DNA Levels
  • Gene Deletion
  • Gene Duplication
  • DNA Rearrangement
  • Gene Amplification
  • Chemical Modification
  • Environmental DNA damage, like UV
dna replication
DNA Replication
  • In the Central Dogma, DNA replication occurs in order to faithfully transmit genetic material to the progeny.
  • Replication is carried out by a complex group of proteins called the replisome
  • Replisome consists of a helicase that unwinds the superhelix as well as the double-stranded DNA helix
  • DNA polymerase and its associated proteins insert new nucleotides in a sequence specific manner, like copy machine.
  • This process typically takes place during S phase of the cell cycle.
epigenetic modification
Epigenetic Modification
  • Epigenetics is the study of heritable changes in gene expression or cellular phenotype caused by changes other than underlying DNA sequence
  • Epigenetic changes are preserved when cells divide.
  • DNA methylation is important in the control of gene transcription and chromatin structure.
  • The epigenetic changes in eukaryotic biology is active in the process of cellular differentiation
  • Methylation of mRNA was demonstrated having a critical role in human energy homeostasis in 2011
dna methylation
DNA Methylation
  • Variation in methylation states of DNA can alter gene expression levels significantly.
  • Methylation variation usually occurs through the action of DNA methylases.
  • When the change is heritable, it is considered epigenetic.
  • When the change in information status is not heritable, it would be a somatic epitype.
  • The effective information content has been changed by means of the actions of a protein or proteins on DNA, but the primary DNA sequence is not altered.

CpG islands

  • Genomic regions that contain a high frequency of CG dinucleotides.
  • CpG islands particularly occur at or near the transcription start site of housekeeping genes.
  • Housekeeping gene:-
    • A gene is required for basic functions the sustenance of the cell.
    • Housekeeping genes are constitutively expressed
  • Luxury gene:
    • A gene has specialized functions expressed in large amounts in particular cell types or “inducible”.
slide28

TF

RNA pol

Unmethylated CpG island

TF

RNA pol

CH3

CH3

CH3

Methylated CpG island

Gene regulation by Methylation

Active transcription

Repressed transcription

slide29

TF

Histone modification

 Methylation

Acetylation

slide30

Mechanisms of gene regulation

  • Histone acetylation directly attracts transcription factors
  • Histone acetylation further attracts co-activator proteins
eukaryotic gene regulation1
Eukaryotic Gene Regulation
  • Key Concept:
    • Eukaryotic genes are controlled in a group or Individually at different levels
    • Have many regulatory sequences
    • Are much more complex than prokaryotic genes
comparisons
Prokaryotes

Genes controlled in group by operons

27% of E. coli genes are controlled by operons

Housekeeping genes not controlled by operons

Simultaneous transcription and translation

Eukaryotes

No operons, but they still need to coordinate regulation

Genes are fragmented into exons and introns

More kinds of control elements, many levels of control

Separated transcription and translation location and control

Epigenetic regulation and Chromatin remodeling

Tissue-specific, developmental and inducible regulation

Comparisons
slide33

Basics of eukaryotic gene regulation

  • Specific transcription factors bind to regulatory elements (enhancers and silencers) and regulate transcription
  • Regulatory elements may be tissue specific and will activate their gene only in one kind of tissue
  • Regulatory elements may be developmental and only expressed at certain stages
  • Regulatory elements may be inducible and expressed only at certain condition
  • Sometimes the expression of a gene requires the function of two or more different regulatory elements
eukaryotic gene regulation2
Eukaryotic Gene Regulation
  • Monocistronic: one mRNA, one protein
  • Basic promoter: TATA box, CAT box, GC-rich
  • Enhancers: Distance and orientation independent
  • Silencers: Distance and orientation independent
  • Transcription factors: Contain at least 2 domains:_Enhancer specific DNA binding and activation (silencing) of RNA polymerase
  • Intron and exons: Number and size variable
  • 3 RNA polymerases in eukaryotes
    • RNA pol I for ribosomal RNAs
    • RNA pol II for mRNAs
    • RNA pol III for small RNAs
mode of gene regulation
Mode of Gene Regulation
  • Developmental: When
  • Tissue-specific: Where
  • Event-induced: What
  • Intensity level: How much

Question:

How does this happen?

What decide tissue-specificity?

What decide the developmental stages?

What decide the expression level?

eukaryotic transcription
Eukaryotic Transcription
  • Promoter strength
  • Tissue-specific enhancers and silencers
  • Transcription Factors
  • Post-transcriptional regulation
  • RNA stability, RNA degradation and half life
eukaryotic promoters
Eukaryotic Promoters

Core promoter

  • Determine transcriptional start site
  • In prokaryote: -10 region
  • In eukaryote: TATA-box

Proximal elements of promoter

  • in prokaryote: -35 region
  • In eukaryote: CAAT-box, GC-box

Enhancer

  • A regulatory DNA sequence that greatly enhances the transcription of a gene. They are mostly tissue specific

Silencer

  • A DNA sequence that helps to reduce or shut off the expression of a nearby gene. They are mostly tissue-specific

Terminator

  • A DNA sequence downstream of a gene and recognized by RNA polymerase as a signal to stop transcription.
  • PolyA signals (AATAAA) tell where to cut and add poly A sequences
transcription factors
Transcription factors
  • Trans-acting factors
  • Contain at least 2 domains
    • DNA binding domain
    • Transcription activation domain

􀂄  Acidic domains,

 Glutamine-rich domains

􀂄  Proline-rich domains

  • They are proteins that bind to the cis-acting elements to control gene expression
  • Control gene expression in several ways:
    • may be expressed in a specific tissue, “tissue-specific”
    • may be expressed at specific time in development, “Developmental”
    • They may be induced by certain conditions, “inducible”
    • may be required for protein modification
    • may be activated by ligand binding, e.g. hormone receptors
slide39

HTH (helix-turn-helix)

α-helix (N-terminus)----DNA-specific

α-helix (C-terminus)----non-specific

introns and exons
INTRONS AND EXONS
  • Eukaryotic DNA differs from prokaryotic DNA in that the coding sequences are interspersed with noncoding sequences called introns
  • Sequences retain in mature mRNA are called EXONS. A mature mRNA contains 5’-UT, coding region and 3’UT.
  • Introns are spliced out, cap and polyA are added before it matures and transported to cytoplasm, where it is ready for translation
  • Introns can be very large and numerous, so some genes are much bigger than the final processed mRNA
  • Yeast has 4% genes with introns, Mammals have most genes with introns.
transcriptional regulation
Transcriptional Regulation
  • Three RNA polymerases responsible for all the transcription
  • Specific transcription factors bind to these regulatory elements and regulate transcription
  • Regulatory elements may be tissue specific and will activate their gene only in one kind of tissue
  • Regulatory elements may be developmental and only expressed at certain stages
  • Regulatory elements may be inducible and expressed only at certain condition
  • Sometimes the expression of a gene requires the function of two or more different regulatory elements
  • In eukaryotic cells the primary transcript (pre-mRNA) must be processed further in order to ensure translation.
  • This normally includes a 5\' cap, a poly-A tail and splicing.
  • Alternative splicing can contributes to the diversity of proteins any single mRNA can produce.
post transcriptional regulation
Post-Transcriptional Regulation
  • Gene Regulation through mRNA Processing
  • Exon shuffling
  • Alternative splicing
  • RNA editing
  • RNA stability
  • RNA degradation
  • RNA half life
  • mRNA Transport Control
  • RNA Interference (RNAi)
    • miRNA
    • siRNA
post transcriptional regulation1
Post transcriptional Regulation
  • RNA Splicing
    • Introns are removed during this process, GT-AG junction
    • Exons can be spliced together in different ways to generate variants and perform different functions
    • Alternate splicing is common in insects and vertebrates
  • RNA editing
    • Nucleoside modifications such as cytidine (C) to uridine (U) and adenosine (A) to inosine (I) deaminations, as well as non-templated nucleotide additions, deletions and insertions.
    • RNA editing in mRNAs effectively alters the amino acid sequence of the encoded protein so that it differs from that predicted by the genomicDNA sequence.
    • Such changes have been observed in tRNA, rRNA, mRNA and microRNA molecules of eukaryotes but not prokaryotes
  • Capping: Adding two G in 3’-5’ direction, important for ribosomal binding.
  • Polyadenylation: Adding long stretches of As, related to mRNA stability.
  • Nuclear exporting, mRNA transport control
  • RNA stability
  • RNA degradation, RNA half life
  • RNA Interference (RNAi)
    • miRNA
    • siRNA
reverse transcription
Reverse transcription
  • Reverse transcription is the transfer of information from RNA to DNA.
  • Important for RNA virus propagation
  • Occur in the case of retroviruses, such as HIV, Involves chromosomal integration
  • Occurs in eukaryotes in the case of retrotransposons and telomere synthesis.
transposons
Transposons
  • A transposable element (TE) is a DNA sequence that can change its relative position (self-transpose) within the genome of a single cell
  • The mechanism of transposition can be either "copy and paste" or "cut and paste"
  • Transposition can create phenotypically significant mutations and alter the cell\'s genome size.
  • Barbara McClintock\'s discovery of these jumping genes early in her career earned her a Nobel prize in 1983
  • Class I (retrotransposons):
    • They copy themselves from DNA to RNA by transcription, from RNA back to DNA by reverse transcription.
    • The DNA copy is then inserted into the genome in a new position.
    • Reverse transcription is catalyzed by a reverse transcriptase often coded by itself, behave very similarly to retroviruses, such as HIV.
  • Class II (DNA transposons):
    • Cut-and-paste transposition mechanism, No RNA intermediate.
    • Catalyzed by various types of transposase enzymes.
    • Transposases bind non-specifically to any target site or to specific sequence targets.
    • The transposase makes a staggered cut at the target site producing sticky ends, cuts out the DNA transposon and ligates it into the target site.
    • A DNA polymerase fills in gaps from the sticky ends and DNA ligase closes backbone resulting in target site duplication.
    • The duplications at the target site can result in gene duplication, which plays an important role in evolution
function of transposable elements
Function of transposable elements
  • From the immediate point of view, transposons have no necessary function in the cell - called, junk DNA"; or "selfish DNA", as transposons propagate on behalf of the cellular resources.
  • On a wider scale, the motility of the retrotransposable elements can be important for genome plasticity.
  • Occasional insertion into genes can disrupt the gene function and cause an inherited disease (Fig. 3C).
  • LTR and LINE elements can also change gene expression, if inserted near a gene, as LTRs and LINE 5´UTR have strong promoter activity in both directions (Fig. 3F).
eukaryotic translation
Eukaryotic Translation
  • Ribosome bind to cap site and scan down. When meet the first good ATG (with Kozak sequences), translation starts.
  • mRNA is read by the ribosome as triplet codons, usually beginning with an AUG, or initiator methionine codon downstream of the ribosome binding site.
  • Complexes of initiation factors and elongation factors bring aminoacylatedtransfer RNAs (tRNAs) into the ribosome-mRNA complex, matching the codon in the mRNA to the anti-codon on the tRNA, thereby adding the correct amino acid in the sequence encoding the gene.
  • As the amino acids are linked into the growing peptide chain, they begin folding into the correct conformation.
  • Translation ends with a UAA, UGA, or UAG stop codon.
  • The nascent polypeptide chain is then released from the ribosome as a mature protein.
  • One mRNA can only be translated once, one mRNA, one protein. Occationally (viral) ribosome can start translation from the middle of a mRNA when it contains a IRES (internal ribosomal entry sequence) sequence.
  • In some cases the new polypeptide chain requires additional processing to make a mature protein (post-translational modification).
  • The correct folding process is quite complex and may require other proteins, called chaperone proteins.
  • Occasionally, proteins themselves can be further spliced; when this happens, the inside "discarded" section is known as an intein.
translational regulation
Translational Regulation
  • Translation efficiency:
    • Ribosomal binding, Kozak sequence
    • Blocking mRNA Attachment to Ribosomes
    • Elongation
  • Coordinated translation, folding, processing, modification, translocation and secretion
  • Protein stability, N-terminal rule
  • Protein degradation, ubiquitylation
translational and post translational regulation
Translational and Post-translational Regulation
  • Translation Control
  • Blocking mRNA Attachment to Ribosomes
  • Regulation of Protein Processing
  • Protein Modification
  • Phosphorylation
    • Ser/Thr type
    • Tyr type
    • Reversible
    • Integrated signals from different pathways effectively
    • The same kind kinase or phosphatase is multiple-substrates.
    • Modified different amino acids, have different influences
slide59

Chaperone functions: Hsp70

The Fate of Proteins after translation

post translational modification
Post-translational Modification
  • Addition of functional groups
    • addition of cofactors for enhanced enzymatic activity
    • modifications of translation factors
    • addition of smaller chemical groups
  • Addition of other proteins or peptides, e.g.,dimerization
  • Changing the chemical nature of amino acids
  • Structural changes, amino acid aditing
  • Protease processing
    • Activation of proteases, e.g., coagulation factors
    • Structure maturation, e.g., Insulin
    • Signal peptide removal during translocation and secretion
  • Glycosylation: Epitope recognition
  • Acetylation: Histone modification
  • Methylation: Histone modification
  • Ubiquitylation: Direct protein to degradation
  • phosphorylation
    • Ser/Thr type, Tyr type
    • Reversible, Integrated signals from different pathways
  • Sumoylation: Small Ubiquitin-like Modifier (SUMO) proteins
    • A family of small proteins that are covalently attached to and detached from other proteins in cells to modify their function.
    • Involved in various cellular processes, such as nuclear-cytosolic transport, transcriptional regulation, apoptosis, protein stability, response to stress, and progression through the cell cycle
  • Lipidation: Addition of hydrophobic groups for membrane localization
    • myristoylation
    • palmitoylation
    • isoprenylation or prenylation
protein degradation
Protein Degradation
  • The half-lives of proteins within cells vary widely, from minutes to several days, and differential rates of protein degradation are an important aspect of cell regulation.
  • Many rapidly degraded proteins function as regulatory molecules, such as transcription factors.
  • The rapid turnover of these proteins is necessary to allow their levels to change quickly in response to external stimuli.
  • Other proteins are rapidly degraded in response to specific signals, providing another mechanism for the regulation of intracellular enzyme activity.
  • In addition, faulty or damaged proteins are recognized and rapidly degraded within cells, thereby eliminating the consequences of mistakes made during protein synthesis.
  • In eukaryotic cells, two major pathways—the ubiquitin-proteasome pathway and lysosomal proteolysis—mediate protein degradation.
the ubiquitin proteasome pathway
The Ubiquitin-Proteasome Pathway
  • The major pathway of selective protein degradation in eukaryotic cells uses ubiquitin as a marker that targets cytosolic and nuclear proteins for rapid proteolysis.
  • Ubiquitin is a 76-amino-acid polypeptide that is highly conserved in all eukaryotes (yeasts, animals, and plants).
  • Proteins are marked for degradation by the attachment of ubiquitin to the amino group of the side chain of a lysine residue.
  • Additional ubiquitins are then added to form a multiubiquitin chain and recognized and degraded by a large, multisubunit protease complex, called the proteasome.
  • Ubiquitin is released in the process, so it can be reused in another cycle.
  • It is noteworthy that both the attachment of ubiquitin and the degradation of marked proteins require energy in the form of ATP.
n end rule
N-end rule
  • The N-end rule is a rule related to ubiquitination, discovered by Alexander Varshavsky in 1986.
  • The rule states that the N-terminal amino acid of a protein determines its half-life.
  • The rule applies to both eukaryotic and prokaryotic organisms, but with different strength.
  • However, only rough estimations of protein half-life can be deduced from this \'rule\', as N-terminal amino acid modification can lead to variability and anomalies, whilst amino acid impact can also change from organism to organism.
  • Other degradation signals, known as degrons, can also be found in sequence.

Val -> 100h

Met, Gly -> 30h

Pro - > 20h

Ile -> 20h

Thr -> 7.2h

Leu -> 5.5h

Ala -> 4.4h

His -> 3.5h

Trp -> 2.8h

Tyr -> 2.8h

Ser -> 1.9h

Asn -> 1.4h

Lys -> 1.3h

Cys -> 1.2h

Asp -> 1.1h

Phe -> 1.1h

Glu -> 1.0h

Arg -> 1.0h

Gln -> 0.8h

N-terminal residues in Yeast

Met, Gly, Ala, Ser, Thr, Val, Pro - > 20 hrs (stabilising)

Ile, Glu - approx. 30 min (stabilising)

Tyr, Gln - approx. 10 min (destabilisiing)

Leu, Phe, Asp, Lys - approx. 3 min (destabilising)

Arg - approx. 2 min (destabilising)

"N"-terminal residues - approximate half-life of proteins in mammalian systems

lysosomal proteolysis
Lysosomal Proteolysis
  • The other major pathway of protein degradation in eukaryotic cells involves the uptake of proteins by lysosomes.
  • Lysosomes are membrane-enclosed organelles that contain an array of digestive enzymes, including several proteases.
  • They have several roles in cell metabolism, including the digestion of extracellular proteins taken up by endocytosis as well as the gradual turnover of cytoplasmic organelles and cytosolic proteins.
  • The containment of proteases and other digestive enzymes within lysosomes prevents uncontrolled degradation of the contents of the cell.
  • Therefore, in order to be degraded by lysosomal proteolysis, cellular proteins must first be taken up by lysosomes.
  • One pathway for this uptake of cellular proteins, autophagy, involves the formation of vesicles (autophagosomes) in which small areas of cytoplasm or cytoplasmic organelles are enclosed in membranes derived from the endoplasmic reticulum.
  • These vesicles then fuse with lysosomes, and the degradative lysosomal enzymes digest their contents.
  • The uptake of proteins into autophagosomes appears to be nonselective, so it results in the eventual slow degradation of long-lived cytoplasmic proteins.
inteins
Inteins
  • An intein is a segment of a protein able to excise itself from the chain of amino acids as they emerge from the ribosome and rejoin the remaining portions with a peptide bond.
  • This is a case of a protein affecting its own primary sequence encoded originally by the DNA of a gene.
  • Additionally, most inteins contain a Homing endonuclease or HEG domain which is capable of finding a copy of the parent gene not containing the intein nucleotide sequence.
  • On contact with the intein free copy the HEG domain initiates the DNA double-stranded break repair mechanism.
  • This process causes the intein sequence to be copied from the original source gene to the intein free gene.
  • This is an example of protein directly editing DNA sequence, as well as increasing the sequences heritable propagation.
prions
Prions
  • Reported in 1982 by Stanley B. Prusiner
  • Prions are infectious agent composed of protein in a misfolded form
  • Proteins that propagate themselves by making conformational changes in other molecules of the same type of protein.
  • This change affects behavior of the protein.
  • In fungi this change happens from one generation to the next, i.e. Protein → Protein.
  • A transfer of information through protein, prion interactions leave the sequence of the protein unchanged.
  • Causing many diseases in mammals
    • ability to catalytically convert native protein to an infectious state
    • capable of inducing a phenotypic change without a modification of the genome

Microscopic "holes" are characteristic in prion-affected tissue sections, causing the tissue to develop a "spongy" architecture.

how to study gene functions
How to study gene functions
  • High throughput sequencing has uncovered thousands of genome and predicted huge numbers of genes.
  • Biologic functions of many are still unknown.
  • Systematic study of gene functions are needed.
  • Gene level: Gene inactivation (Gene knockout)
    • Global
    • Tissue-specific
  • Transgenic Studies: over expression
  • Anti-sense RNA knock down (siRNA)
  • Protein level: Mutant protein, dominant-negative
    • Site-directed mutagenesis: amino acid change and function
    • Biochemical studies
  • In vitro (cellular level) and In vivo (animal) studies
application of gene regulation
Application of Gene Regulation
  • Gene Therapy
    • To correct mutations
    • To provide proper amount of functional proteins
    • To curb the bad gene expression
  • Transgenics
    • To facilitate research, animal models of diseases
    • To improve live stocks for better nutrition, better health, better economic values.
identification of genetic association of disease by human genome sequencing
Identification of Genetic Association of Disease by Human Genome Sequencing
  • Chromosomal studies
  • SNPs
  • Non-coding regions
  • Non-translated regions
  • Non-coding RNAs
  • Small RNAs
  • SiRNAs
  • Auto-immune diseases
aspect of gene regulation
Aspect of Gene regulation
  • Systematic: Conserved and Species-specific programming
  • Genetic: Each individual predetermined
  • Epigenetic: Predetermined and developmentally determined
    • There are several layers of regulation of gene expression.
    • One way that genes are regulated is through the remodeling of chromatin
  • Chromatin Remodeling
    • DNase I hypersensitive site
    • DNA methylation
    • Histone Methylation
    • Histone acetylation
  • Environmental: Unpredictable
homeostasis
Homeostasis
  • Genetic determinants try to maintain the balanced development internally
  • At the same time try to adapt environment changes externally
  • When the balances is lost, a pathologic development comes
    • Genetic disease
    • Acquired disease
slide72

Environment Causes of Cancer:

Viruses, bacteria infection

Smoking

 Unhealthy Food

Radiation

Chemicals, pollution

Depression

slide73

Summary

  • Gene regulation happens every where
    • Genomic level_developmental
    • Gene level
      • Transcriptional, post-transcriptional
      • Translational, post-translational
      • Protein activation and deactivation
      • Degradation and Half life
  • Gene out of control causes disease
    • One gene, one disease (protein disease, Hemoglobin)
    • One gene, many diseases (regulatory factor disease, VEGF)
    • Many genes, one disease (pathway disease)
    • Direct cause, easy to identify
    • In direct effect, hard to cure
ad