Gene Technologies 3.8: The Control of Gene Expression

Genome size: $3\times 10^9$ base pairs; ≈ $2\times 10^4$ genes.
Proteome: all proteins in a cell; varies over time and between cells.
In simple organisms, genome sequence can derive the proteome from the genetic code; in humans, non-coding DNA and regulatory genes mean the proteome is not directly inferred from the genome.
Sequencing methods are automated and enable screening DNA for medical problems.
Genome, proteome, and sequencing relate to DNA/RNA structure and to features of the genetic code.
Recombinant DNA technologies enable combining DNA from different organisms; based on the universal genetic code and shared transcription/translation.

Three methods to create DNA fragments:
- 1) Reverse transcription
- 2) Restriction endonucleases
- 3) Gene machine
Reverse transcription:
- A cell that naturally produces the protein of interest is used; mRNA is abundant for the protein.
- Reverse transcriptase makes DNA nucleotides complementary to the mRNA sequence, creating single-stranded cDNA.
- DNA polymerase converts the cDNA into double-stranded DNA (dsDNA).
- The cDNA is intron-free because it is based on the mRNA template.
Gene machine:
- Protein of interest determines amino acid sequence, then mRNA and DNA sequences are inferred.
- Computer checks biosafety/biosecurity; designs small overlapping oligonucleotides (short DNA fragments) that assemble into the full gene.
- DNA is assembled from oligonucleotides and then amplified (e.g., by PCR) to dsDNA; intron-free for prokaryotic transcription.
PCR (amplification) basics:
- Amplifies DNA quantity quickly and automatically; can produce up to $10^{11}$ copies in hours.
- Components: thermocycler, DNA fragment, DNA polymerase (e.g., Taq), primers, DNA nucleotides.
- PCR cycle elements: denaturing at $95^ ext{oC}$ , annealing at $55^ ext{oC}$ , synthesis at $72^ ext{oC}$ .
Modified DNA fragments for transcription:
- Promoter region: binding site for RNA polymerase to enable transcription.
- Terminator region: signals RNA polymerase to stop transcription; ensures one gene is copied per mRNA.

Enzymes cut DNA at specific recognition sequences; originated in bacteria as a defense mechanism.
Endings:
- Blunt ends: straight cuts.
- Sticky ends: staggered cuts producing exposed bases; palindromic sequences; can rejoin with complementary strands.
Enzyme activity defines where fragments are cut and how DNA pieces can be joined.

DNA fragment insertion into a vector (usually a plasmid):
- Restriction enzymes cut plasmid to create compatible sticky ends; DNA ligase seals the fragment into the plasmid.
- Resulting DNA is a recombinant plasmid.
Transformation:
- Plasmids and bacterial cells are mixed in Ca^{2+} ions; heat shock or rapid temperature changes increase membrane permeability, enabling plasmid uptake.
- Not all cells take up plasmids or inserts; selection is required to identify successful transformants.
Vectors and plasmids:
- Plasmids act as vectors to transport DNA into host cells.
- Recombinant plasmids result from ligation of the DNA fragment into a cut plasmid.

Marker genes on plasmids help identify cells that took up plasmids and, later, recombinant plasmids.
Three main marker gene categories:
- Antibiotic resistance genes
- Fluorescent protein genes (e.g., GFP)
- Enzyme-encoding genes (e.g., lacZ)
Antibiotic selection:
- Grow cells on agar with antibiotic (e.g., ampicillin); survivors indicate plasmid uptake.
- To identify recombinants, the DNA fragment is inserted into the middle of a marker gene; recombinants lose that marker function.
- Further screening: non-fluorescent colonies (for GFP) or colonies unable to turn a color change indicate recombinant plasmids.
Enzyme markers example:
- LacZ gene disrupted by insertion cannot metabolize substrate to color change; recombinant plasmids yield colorless colonies on certain indicators.
Growth and production:
- Once recombinant cells are identified, they are grown in fermenters to amplify the DNA fragment and produce the encoded product (e.g., insulin).
GFP as marker:
- GFP gene as a marker can indicate plasmid status via fluorescence.

DNA hybridisation:
- DNA is heated to separate strands, then cooled with complementary single-stranded sequences to form duplexes.
- Used to detect presence of specific alleles in medical diagnostics.
Genetic fingerprinting:
- ~95% of human DNA is introns; VNTRs (variable number tandem repeats) vary between individuals.
- Pattern of VNTRs is highly individual; used to assess genetic relationships and population variation.
- Applications: forensic science (crime scene analysis), medical diagnosis, paternity testing, and breeding programs.
DNA probes and screening:
- Short single-stranded probes labeled with radioactivity or fluorescence bind to complementary VNTRs.
- Probes reveal presence of alleles or disease-associated genes via detectable signals (X-ray or UV).
Personalised medicine and genetic counselling:
- Screening for alleles guides drug choice and health advice.
- Genetic counselling provides information after disease screening based on genotype.

Collection and extraction:
- Smallest feasible DNA sample (blood, cells, hair) is collected; DNA is extracted (cell fractionation and ultracentrifugation); PCR if needed to amplify.
Digestion:
- Restriction endonucleases cut DNA near VNTRs.
Separation:
- Gel electrophoresis separates DNA fragments by size; alkaline treatment denatures DNA strands.
Hybridisation:
- DNA probes hybridise with VNTRs; probes are labeled (radioactive or fluorescent).
Development and analysis:
- Gel dries and transfers to a nylon sheet; visualisation via X-ray or UV exposes the probe positions.
Uses:
- Determine genetic relationships, detect disease-predictive genes, and compare unknown samples (e.g., crime Scene).

Gene technology links to DNA and RNA structure and to the genetic code.
It connects to enzyme function and gene expression control (promoters, terminators, transcription).
Genetic fingerprinting links to cell fractionation and VNTR analysis for identity and relationships.
DNA probes and hybridisation enable targeted genetic screening for personalised medicine and counseling.