2. DNA MARKER
A genetic marker/ DNA marker is a DNA sequence with a specific
location on a chromosome.
Genetic markers help in various ways as its link an inherited disease
with the responsible gene.
DNA segments which are repeated again and again in DNA.
Genetic markers are used to track the inheritance of different genes
whose approximate location is known.
3.
4. β-Galactosidase
The enzyme beta-galactosidase was first mentioned in the literature
by Beijerinck exactly a hundred years ago.
β-galactosidase is also called lactase, beta-gal or β-gal belongs to the
family of glycoside hydrolase enzymes.
It catalyzes the hydrolysis of β galactosides into monosaccharides
through the breakage of glycosidic bond.
β-galactosides include carbohydrates containing galactose where the
glycosidic bond present above the galactose molecule.
6. Inhibitors of β-galactosidase
Beta-gal is inhibited by L-ribose,
Non-competitive inhibitor iodine,
Competitive inhibitors
2-phenylethyl 1-thio-beta-D-galactopyranoside (PETG),
D-galactonolactone,
isopropyl thio-beta-D-galactoside (IPTG),
galactose
7. β-Galactosidase Assay
β-Gal as a reporter gene
Protein product is stable and resistant to proteolytic degradation.
Enzyme is easily assayed.
Easy and rapid method to assay β-gal activity in transfected cells.
No expensive equipment.
8.
9. Families of β-galactosidase
Most of the work done on β-galactosidase is derived from E. coli. However
β-gal can be found in many plants especially in fruits, mammals, yeast,
bacteria, and fungi.
β-galactosidase genes may be differ in the length of their coding sequence
and the length of proteins formed by amino acids.
According to the sequence the β-galactosidase is divided into four families:
GHF-1 (Glycoside hydrolase)
GHF-2
GHF-35
GHF- 42
10. Conti ….
E. Coli belongs to GHF-2, all plants belong to GHF-35,
and Thermus thermophilus (gram negative bacteria) belongs
to GHF-42.
Fruits can express multiple β-gal genes. There are
approximately 7 β-gal genes expressed in tomato fruit
development that have amino acid similarity between 33%
and 79%.
12. Structure
The 1,023 amino acids of E. coli β-galactosidase were sequenced in
1983, and its determined structure eleven years later in 1994.
The protein is a 464-kDa homotetramer.
Each unit of β-galactosidase consists of five domains.
• Domain 1
is a jelly-roll type β-barrel
13. • Domain 2 and 4
are fibronectin type III-like barrels
• Domain 5
a novel β-sandwich
• Domain 3
is a distorted TIM-type barrel, lacking the fifth helix with a distortion in
the sixth strand. The third domain contains the active site.
15. Domain 1, blue; Domain 2, green; Domain 3, yellow; Domain 4, cyan; Domain 5, red.
Lighter and darker shades of a given color are used to distinguish the same domain in
different subunits.
16. Mechanism of reaction
It can go through reaction called transgalactosylation
to make allolactose.
Allolactose can also be break to form
monosaccharides. It can also hydrolyze lactose
into galactose and glucose which will go
into glycolysis.
17.
18. Functions
Hydrolyzes the β-glycosidic bond formed between a galactose and its
organic moiety.
In E. coli, the lacZ gene is the structural gene for β-galactosidase;
which is present as part of the inducible system lac operon.
β-galactosidase is vital for organisms as it is a key provider in the
production of energy and a source of carbons through the break
down of lactose to galactose and glucose.
It is also important for the lactose intolerant community as it is
responsible for making lactose-free milk and other dairy products.