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DNA …
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Ahmed Tarek

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Amjad Ebrahim

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3
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5
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CAUSES :1
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Heat " UV light absorption at 260 nm "
Features :1
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Phosphodiester bonds aren't be broken

3
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Single stander higher absorbed to UV than double stander

5
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Small basic protein with high content of lysine and arginine
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Consist of 5 classes H1 , H2A, H2B, H3, H4
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The most tissue specific and species specific
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Help the packing DNA to more compact stracture
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Nucleosomes joinen by linker to form polynucleosome or
nucleofilament fiber " 30 nm "
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d- Non histones:1
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2
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b- Ori-c contain 245 base pair characterized by
1
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2
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2- In eukaryotes :It begin at multiple sites along the helix which are rich in
A T Bases
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Mechanizm :1
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2
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3- remove the mismatched base
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Gap is filled by polymerase III
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4
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3- moves in the 5`--3` direction to the termination site in
presence of ATP
4- Protein at termination site displaces the DNA template
strand
5- facilitating the dissociation of RNA molecule
6- RNA polymerase is released

1
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4 - make complementary RNA of DNA template and
recognize terminal region
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IN Eukaryotes :There are 3 large type with multiple subunits :1
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RNA Polymerase II : form mRNA
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It is an enzyme that use RNA as a template to form DNA in
the direction of " 5' --- 3' "
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It move along the template in the direction " 3'—5' "
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Retrovirus such as immunodeficiency virus " AIDZ "

Definition
It is a dictionary which corresponds the nucleotide sequence of mRNA and
the amino acid sequence on the poly peptide chain of protein
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Defination :the attachment of an amino acid to its (tRNA)
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1-(AA) + ATP ------(E)------- E-AMP-AA + pp
2-E-AMP-AA + t-RNA ------AA-tRNA + AMP + E

I-Diagnosis
A-prenatal diagnosis
1-obtaining samples from amniotic fluid by amniocentesis
2- separation of fetal cells
3-extraction of DNA from fetal cells
4-amplification of required gene
5-diagnosis of the genetic defect like dawn syndrome
B- diagnosis of genetic disorders :
Such as thalassemia and sickle cell anemia
II- treatment
Gene therapy and cell therapy
III-production of proteins
1-production of coagulation factors interleukin
2-production of hormones : insulin, GH, antidiuretic H
3-production of vaccines for hepatitis B
4- forensic medicine application
5 – under standing the pathology of diseases as molecule
level that help prediction and prevention of disease

Definition
is an experimental technique that uses genes to treat or
prevent disease
by replacing abnormal gene by normal gene
types
1-germ cell gene therapy
Is injecting of foreign genes into fertilized eggs so that the
inserted genes would be distributed among germ cells and
somatic cells
2- somatic cell gene therapy
Changing individuals genetic constitution during their life
time and this would not affect their children
Application of gene therpy
1-treatment of genetic disease
2-treatment of some malignant and infections diseases

Methods of gene therapy
1-physical : direct injection of normal DNA into diseased
muscle
2-using retrovirus and adenovirus as vectors for transfer of
normal gene into target cell eg fibroblast , hepatocyte , stem
cell ,lymphocyte

Steps of gene therapy :1-take a sample from the patient’s blood
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2-infect the lymphocytes with a retrovirus modified to carry the
normal adenosine deaminase gene
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to form synthesize adenosine deaminase
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 Methods of detection of PCR amplified
products
1-electrophorsis
2-radioimmunoassay
3-enzymatic immunoassay

 Applications of PCR :
1- diagnosis and early detection of malignant
diseases
2- diagnosis and early detection infectious
diseases like hepatitis C

3- diagnosis and early detection genetic diseases
and mutation
4- - forensic medicine application" Finger print"

A) RNA Processing :
1- The newly synthesized RNA molecule is called Primary Transcript
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2- Prokaryotic mRNA is identical its primary transcript whereas eukaryotic mRNA is
extensively modified post transcriptionally
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2- Preribosomal RNAs are cleaved to yield intermediate sized pieces of rRNA which are
trimmed to produce the rRNA species
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7-methylguanosine is linked to the 5’ terminal residue of eukaryotic mRNA
through unusual 5-5 triphosphates linkages
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The 5’ cap permits to initiation translation and helps stabilize the mRNA
Fig 10 pg :96

2-Addition of a poly – tail :
1
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2
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facilitate their exit from the nucleus and protects them from enzymatic
destruction
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The remaining exons are joined together to form mature mRNA

Mechanism of RNA splicing :
The First mechanism :
1- The specific adenine nucleotide in the intron sequence attacks the 5’ splice site
2- cuts the sugar-phosphate backbone of the RNA at this point

3- The cut intron becomes covalently linked to the adenine nucleotide
4- creating a loop in the RNA molecule

5- The released free 3’ OH end of the exon (1) sequence reacts with the start of the
exon (2) joining the two exons together
6- >> releasing the intron sequence in the shape of lariat and then degraded

The Second Mechanism :
This mechanism requires 5 small nuclear RNAs (snRNAs) combine with protein , form
small nuclear ribonucleoprotein particles (snRNps)
1- snRNps forms a splicosome with a region on primary transcript
2- The (2’-OH) group of an adenosine (A) residue in the intron attacks and forms a
phosphodiester bond with the phosphate at the 5’-end of intron 1

3- The newly – freed (3’OH of the exon ) then forms a phosphodiester bond with
the 5”end of exon 2
4- The excised intron is released as a lariat structure which is degraded
After removal of all the introns the mature mRNA molecules leave the nucleus by passing
into the cytosol through pores in the nuclear membrane

16-Effect of missense mutation
*The codon containing the changed base may code for different
amino acid
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g
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2-partially acceptable missense mutation:
The best example is HBS in which the AA glutamic in position 6
replaced by valine
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g
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17-Replication in eukaryotes
Differ from replication in prokaryotes in:
1-Multiple origin of replication versus (vs) single origin in
prokaryotes
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*Beta-polymerase DNA repair
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*Delta-polymerase  synthesize leading & okazaki fragments &
proof reading activity
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3-synthesis of new histones occurs simultaneously with Dna
replication for formation of nucleosomes
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B :The classification according to :
1 ) molecular weight

3) sensitivity to inhibitor

2 ) cellular location

4) substrate act on

18-Regulation of Regulation Of Operon
Model in presence of glucose
A-lactose operon model :
1-in prokaryotes , the genes involved in metabolic pathway are often
present in a linear array called an operon e
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,the lac operon is a
best model for regulation of lactose consumption by bacteria
(E
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The
repressor has a strong affinity for The operator
4-the z gene codes for B galactosidase (B gal)
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5-the y gene codes for permease , which increase permeability of
the cell to B-galactosides

8-The a gene codes a trans-acetylase
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(lac i gene product)
2-the repressor protein binds to the operator ,

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this interferes with progress of RNA polymerase
4
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(B) derepression state

repressor protein is synthesized (lac | gene product &
bound to operator region of lac operon )
1-

2-inducer bind to the repressor protein forming complex, so
that it can do no longer bind to the operator

3- This allows RNA polymerase to initiate transcription of
mRNA that are translated to the 3 enzymes for catabolism of
lactose

Another mechanism
Glucose CAMP activation protein called (CAP) catabolic activating
protein that facilitate the binding of RNA polymerase to promotor &
RNA transcription of this enzymes

 DNA Polymerase III :has the following activity:
1-

5" -> 3" polymerization activity that synthesis DNA

2-

5' -> 3' Repair endonuclease activity
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It check correctly of addition of nucleotide and
match to it's complementary base on template
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Hydrolytically remove the mismatched nucleotide

c
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4-

Formation of leading stander " continuously " &
lagging stander " discontinuously " in short fragment
called " okazaki "

 RNA polymerase III :It is responsible for formation of small rRNA and tRNA

1-It is a single strand product of transcription of DNA
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3- Mature mRNA is characterized by the following :-

a- Attachment of the 7-methyl guanosine triphosphates cap
to the hydroxyl group of the ribose at 5" terminal end of
mRNA, forming unusual 5" -> 5" triphosphates
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b-A Poly –A tail 3" hydroxyl terminal of mRNA
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5- a
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convert genetic information to synthsis protien
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THEN ,proteins under go
modification to be active
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2-Parts of the protein chain removed by endoproteases,>> release of an
active molecule
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G the trypisinogen, becomes activated to trypsin in the
small intestine,

B)Covalent alterations

Proteins,both enzymatic and structural, may be activated or inactivated by
covalent attachment of a variety of chemical groups
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- it increase or decrease the functional activity of the protein

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3 - Hydroxylation :1
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Methylation & acetylation of some proteins such as histones
&protein in lipid bilayer of membrane respectively

4
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occure between cysteine residues
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protect the protein molecule from denaturation in an extra
cellular environment

22-Restriction Endonucleases
Enzymes:
1-They are enzymes of bacterial origin

2-They cut DNA at a specific palindromic sequence creating
either sticky to blunt ends

Importance :1
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Used in genomic DNA library

DONE BY :



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