the multistep process that ultimately results in the production of a functional gene product, either ribonucleic acid (RNA) or protein.
Where regulation of gene ene expression happen and how it is mediated?
- Regulation of gene expression occurs primarily at the level of transcription in both prokaryotes
- Mediated through the binding of trans-acting proteins
- cis-acting regulatory elements on the DNA
- - In Eukaryotes, regulation also occurs through
- 1- modifications to the DNA
- 2- Posttranscriptional and
- 3- posttranslational events
Function of lactose (lac) operon in prokaryotic
The lactose (lac) operon contains the genes that code for three proteins involved in the breakdown of the disaccharide, lactose
What are the three genes that code for 3 proteins in lactose
(lac) operon in prokaryotic?
- - lac Z gene codes for B-galactosidase,
- which hydrolyzes lactose to galactose and glucose
- - lac Y gene, which codes for a permease
- facilitates the movement of lactose into the cell
- - lac A gene that codes for thiogalactoside transacetylase
- function is unknown
Function of trp operon in prokaryotic
The trp operon contains genes needed for the synthesis of tryptophan
How trp operon is regulated in prokaryotic ?
- It is regulated by
- Positive and negative control .
mRNA synthesis that escaped repression by trp is terminated before completion.
Translation is also a site of prokaryotic gene regulation in prokaryotic
When ribosomal proteins are in excess, they bind the Shine-Dalgarno sequence on their own polycistronic mRNA, preventing ribosomes from binding.
Differences between gene regulation of Eukaryotic Gene Expression and Prokaryotic Gene Expression
- Gene regulation is more complex in eukaryotes.
- Operons are not present
- Coordinate regulation of the transcription of genes located on different chromosomes can be achieved through the Binding of trans-acting proteins to cis-acting elements
- How long an mRNA remains in the cytosol before it
- is degraded influences
- how much protein product can be produced from it
mRNA stability is important for
- Regulation of iron metabolism
- Gene-silencing process of RNA interference
Gene expression in eukaryotes is also influenced by
- Availability of DNA to the transcriptional apparatus
- Amount of DNA
- Arrangement of the DNA
Small DNA molecules capable of self-replication
Engineered DNA molecules generated
Restriction Endonucleases Definition and the function
- - Recognize short stretches of DNA (4-8 bp) that contain specific nucleotide sequences
- - These sequences are palindromes
- Biological function is to protect cells from foreign DNA.
- Specific nucleotide sequences when read in the 5-3 direction, the sequence on the top strand is identical to the bottom strand.
- Restriction endonnuclease recognize plaindrome
Sticky or cohesive ends
- - TaqI form staggered cuts that produce sticky or cohesive ends
- - DNA ligase, join the sticky ends of a DNA fragment
- HaeIII: produce fragments that have "blunt"ends that are double stranded
DNA sequence cut by restriction enzyme/endonulcease is called a restriction site
Difference between enzyme that recognizes a specific four-base-pair and enzyme that recognizes six base pairs
Enzyme that recognizes a specific
- Four-base-pair sequence produces many cuts in the DNA molecule, one every 44 bp.
- Six-base-pairs produces fewer cuts, one every46 bp, longer pieces
Cloning into a Plasmid
- Occur in bacteria
- Much smaller than chromosomal DNA
- Carry their own origin of replication
- Easy to manipulate and identify
- Many copies per cell
- Carry antibiotic resistant genes
Sanger dideoxy method
Method to confirm the DNA sequence
Why do we use dideoxynucleotides in Sanger dideoxy method?
- We use Dideoxynucleotides (ddNTP) in DNA chain Termination
- Dideoxynucleotides are essentially the same as nucleotides except they contain a H group on the 3’ carbon instead of (OH).
- When integrated into a sequence, it prevents the addition of further nucleotides.
- This occurs because a phosphodiester bond cannot form between the dideoxynucleotide and the next incoming nucleotide, and thus the DNA chain is terminated.
What are the Methods to confirm the DNA sequence? Similarities ?
- - The Maxam–Gilbert chemical sequencing method
- - The Sanger–Coulson enzymatic sequencing method.
- - The Automated Sequncers non-enzymatic sequncing, sequencing by hybridization.
- Both involve the ultimate generation of a full set of
- fragments of the DNA strand to be sequenced.
What do you add in the 4 tubes in Sanger's method?
- "G" tube: all four dNTP's, ddGTP and DNA polymerase
- "A" tube: all four dNTP's, ddATP and DNA polymerase
- "T" tube: all four dNTP's, ddTTP and DNA polymerase
- "C" tube: all four dNTP's, ddCTP and DNA polymerase
What is Polymerase chain reaction (PCR) used for?
- - PCR is used to amplify a specific region of a DNA strand (the DNA target)
- - Amplify DNA fragments of up to ~10 kp (kilo base pairs)
- - Some techniques - fragments up to 40 kb in size
Component of PCR
- DNA template that contains the DNA region (target) to be
- Two primers that are complementary to the 3' (three prime) ends of the DNA target.
- Taq polymerase or another DNA polymerase with a temperature optimum at around 70 °C.
- Deoxynucleoside triphosphates (4dNTP)(dATP- dGTP- dCTP- dTTP), the building-blocks from which the DNA polymerase synthesizes a new DNA strand.
- Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.
- Divalent cations, magnesium or manganese ions; Mg2+ is used
Advantage of PCR over Cloning
- Speed- amplify millions of copies in few hours- irrespective of sample size
- Sensitive – DNA sequence present in an individual cell can be amplified and studied.
- PCR process is less difficult than cloning
Applications of PCR
- Comparison of normal cloned gene with an uncloned mutant form of the gene.
- Detection of low abundance nucleic acid sequences (HIV)
- Forensic analysis of DNA samples (crimes)
- Prenatal diagnosis and carrier detection of cystic fibrosis.
Techniques to Analysis of Gene Expression
- mRNA expression
- 1- Northern Blots
- 2- Microarrays
ELISA and Western blots
- Protein expression
- 1- ELISA
- 2- Western blots
- 3- Proteomics
are commonly used to detect exposure to HIV
by measuring the amount of anti-HIV antibodies
in patient’s blood sample.
Geneomic DNA will contain..................... if the targe therapeutic protein is eukaryotic
- • If the target therapeutic protein is eukaryotic, the genomic DNA will contain both coding (exon) and non-coding (intron) sequences,
- whereas the cDNA will be a reflection of the exons only.
Where does the Recombinant protein produced?
- E.coli, S. cerevisiae or in
- Animal cell lines (mainly CHO or BHK cells).
Manipulation of a protein’s amino acid sequence.
Advantages of Protein engineering?
- Understanding of the link between a polypeptide’s amino acid sequence and its structure.
- Studying the relationship between structure and its function.
- Tailor structural or functional attributes of therapeutic and other commercially important proteins
Reasons for Primary Non-function of Islet Grafts
- 1- Transplantation In Liver Sinusoids
- Transplantation Under Kidney
- 2- Hypoxia to the inner core of islets due to inadequate revascularization
- 3- Inflammation and immune mediated cell death and loss of islet function