Gene Expression

the multistep process that ultimately results in the production of a functional gene product, either ribonucleic acid (RNA) or protein.

- Regulation of gene expression occurs primarily at the level of transcription in both prokaryotes and eukaryotes

• - Mediated through the binding of trans-acting proteins to
• cis-acting regulatory elements on the DNA

• - In Eukaryotes, regulation also occurs through
• 1- modifications to the DNA
• 2- Posttranscriptional and
• 3- posttranslational events

The lactose (lac) operon contains the genes that code for three proteins involved in the breakdown of the disaccharide, lactose

• - 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

The trp operon contains genes needed for the synthesis of tryptophan

• It is regulated by 
• Positive and negative control .
• Attenuation

mRNA synthesis that escaped repression by trp is terminated before completion.

When ribosomal proteins are in excess, they bind the Shine-Dalgarno sequence on their own polycistronic mRNA, preventing ribosomes from binding.

• 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

• Regulation of iron metabolism
• Gene-silencing process of RNA interference

• 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

• - 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
• 

• - 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

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

• 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

Method to confirm the DNA sequence

• 
• 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.

• - 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.

• "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

• 
• - 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

• 
• DNA template that contains the DNA region (target) to be
• amplified.
• 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

• 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

• 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.

• mRNA expression
• 1- Northern Blots
• 2- Microarrays

• Protein expression
• 1- ELISA
• 2- Western blots
• 3- Proteomics

ELISA and Western blots are commonly used to detect exposure to HIV by measuring the amount of anti-HIV antibodies in patient’s blood sample.

• • 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.

• E.coli, S. cerevisiae or in
• Animal cell lines (mainly CHO or BHK cells).

Manipulation of a protein’s amino acid sequence.

• 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

• 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