RECOMBINANT DNA METHODOLOGIES, GENE EXPRESSION, PROTEIN FUNCTION

SPECTROPHOTOMETER:

- Measuring absorbance at 260nm

- A solution of 50 ug/ml of double stranded DNA will have an absorbance of 1.0
- Presence of phenol and proteins can affect this estimate
- proteins contamination can be verified by the absorbance ration of 260/280 =1.8
- highly purified DNA shows an absorbance ratio of 260/230 = 1.8 – 2.0
- highly purified RNA shows an absorbance ration of 260/230= 2.0 -2.2

VISUALIZING DNA BANDS IN AN AGAROSE GEL BY EtBr STAINING AND ULTRVIOLET IRRADIATION:

- agarose gel soak in 0.5 ug/ml solution of EtBr for 15 minutes

CARCINOEMBRYONIC ANTIGEN – RELATED CELL ADHESION MOLECULE 5 (CEACAM5) N DOMAIN:

- it has 7 domains ( IgV -like N domain and 6 IgC – like domains, A1, B1, A2, B2, A3 and B3)
highly glycosylated
- it has located on cell surface (lipid membrane), GPI anchored
- Its function is to meditate cell adhesion and is involved in downstream signalling. Engages in
both homophilic and heterophilic interactions
- Its significance is to overexpress proteins that is involved in the formation of malignant
tumours. Hence, used as a carcinogenic marker for carcinomas

WHAT DO THE GENES ENCODE?

- Gel electrophoresis of PCR product

- PCR of DNA sample
- Transforming using TOPO-TA into NEB
- Selection of transformants
- Growth of colonies in LBCK
- Mini prep of overnight cultures
- EcoRI digestion to check for successful transformation
- MegaBLAST - TNFR2 – Tumour Necrosis Factor Superfamily Member 1B
- BLASTn – human pancreatic mucin mRNA, complete cds

DNA REPLICATION:

- New DNA strands form prior to mitosis or meiosis during the S phase of the cell cycle
- Replication conducted by DNA polymerase and its entourage in the nucleus
- Starts with a short RNA primer that is extended by DNA polymerase in the 5’ to 3’ direction
only
- DNA helicase opens the double stranded DNA and DNAP follows
- The leading strand is made continuously and the lagging strand is made discontinuously in
pieces of 1000 bases called Okazaki fragments which are joined together by ligase

TRANSCRIPTION:

- Makes RNA copies of one DNA strand for translation and other purposes and occurs in the
nucleus
- Conducted by RNA polymerase and its entourage that help RNAPII to bind to promoter
regions on one strand of DNA
 - Promoters upstream of genes are bound by TATA binding protein or TBP that binds to TATA
boxes and recruit RNAPII and its entourage of general transcription factors and positions the
RNAPII so that it can begin downstream of the promoter
- Once the initiation complex forms, the DNA is opened up and transcription begins
downstream of the promoter
- Transcription proceeds by assembly of RNA in a 5’ to 3’ direction
- The RNA is identical to the coding strand
- Transcription proceeds until the transcription stop position Is reached when the premRNA
transcript terminates and is released

RNA PROCESSING:

- There are three steps in RNA processing that occur in the nucleus before the processed
mRNA is exported to the cytoplasm for translation by the ribosomes
A. Addition of the cap structure
B. Splicing of the introns
C. Addition of the poly A tail

TRANSLATION:

- Makes proteins and occurs in the cytoplasm

- Conducted by ribosomes, tRNA using mRNA as a template with a triplet nucleotide codon
usage
- Start when the small subunit of the ribosome binds an mRNA and tracks along
- When the ribosome finds the first AUG in any reading frame, a tRNA with an attached
methionine is inserted and the large ribosomal subunit attaches
- The mRNA is translated codon by codon to the first stop codon in the same reading frame as
the start codon
- At the stop codon, there is no corresponding tRNA so the complex falls apart and releases
the protein

LEVELS OF PROTEIN STRUCTURE

- Primary structure – sequence of amino acids forms the polypeptide chain

- Secondary structure – the primary chain forms spirals and sheets
- Tertiary structures – superimposed on secondary, alpha helices or beta sheets are folded up
to form a compact globular molecule held together by intramolecular bonds
- Quaternary structure – two or more polynucleotide chains, each with its own tertiary
structure, combine to form a functional protein

THE GENETIC CODE:

- There are bases and there is a triplet code so that there are 64 codons
- There are three stop codons and there are no tRNAs that correspond to these codons so the
ribosome falls apart and releases the new protein
- tRNA is a small molecule that contains a region called the anticodon that recognizes and
binds to its complementary codon in mRNA
- the tRNA also contains a region at the other end of the molecule that binds to the amino
acid specified by the anticodon and codon combination

GENE REGULATION:
 - all genes are present in all cells but not all genes are transcribed in all cells due to various
types of gene regulation
- housekeeping genes expressed in all cells (actin, myosin, ribosomes) are on in all cells and
have simple promoters that are on all the time
- regulated genes are usually repressed by DNA binding proteins in most cells
- in appropriate cells they are induced by sequence specific DNA binding proteins expressed
only in those cells
- regulated genes can have many promoter regions binding specific DNA binding proteins and
they tend to have long, complicated promoters

CONSTRUCTION AND EXPRESSION VECTOR:

- XbaI and Bam HI restriction enzymes were chosen to cut TNF insert out of Pcr4-TOPO for
cloning into Pet-15b expression vector
- His tag present at amino acid positions 5-10
- Pet-15b selected as expression vector due to Ampicillin resistance gene for selection and
inducibility by IPTG

CYTOKINE STORM:

- Coronavirus infects lung cells

- Immune cells including macrophages identify the virus and produce cytokines
- Cytokines attract more immune cells such as WBC which in turn produce more cytokines
creating a cycle of inflammation that damages the lung cells
- Damage can occur through the formation of fibrin
- Weakened blood vessels allow fluid to seep in and fill the lung cavities leading to respiratory
failure

ANTI TNF ALPHA THERAPIES FOR COVID 19 PATIENTS:

- Antibodies bind and neutralize TNF – alpha which is no longer capable of binding TNFR1/2
- Used to treat severe inflammatory disease