Problem of quantitative genetics

Problem: Fruit colour of wild Solanum nigrum is controlled by two alleles of a gene (A and a). The frequency of A, p=0.8 and a, q=0.2. In a neighbouring field a tetraploid genotype of S. nigrum was found. After critical examination five distinct genotypes were found; which are AAAA, AAAa, AAaa, Aaaa and aaaa. Following Hardy Weinberg principle and assuming the same allele frequency as that of diploid population, the numbers of phenotypes calculated within a population of 1000 plants are close to one of the following: AAAA : AAAa : AAaa : Aaaa : aaaa

1. 409 : 409 : 154 : 26 : 2

2. 420 : 420 : 140 : 18 : 2

3. 409 : 409 : 144 : 36 : 2

4. 409 : 420 : 144 : 25 : 2                                                      (CSIR June 2016)

Solution:

Frequency of allele A = 0.8 and a = 0.2

	AA	Aa	Aa	aa
AA	AAAA	AAAa	AAAa	AAaa
Aa	AAAa	AAaa	AAaa	Aaaa
Aa	AAAa	AAaa	AAaa	Aaaa
aa	AAaa	Aaaa	Aaaa	aaaa

From above chart phenotypic ratio is,

AAAA : AAAa : AAaa : Aaaa : aaaa = 1 : 4 : 6 : 4 : 1

From Hardy-Weinberg principle:

p⁴ : 4p³q : 6p²q² : 4pq³ :q⁴

for AAAA= p⁴ x 1000 = (0.8)⁴ x 1000

= 409.6 » 409

for AAAa = 4p³q x 1000 = 4x(0.8)³x0.2x1000

= 409.6  » 409

for AAaa = 6p²q² x 1000 = 6x(0.8)²x(0.2)² x1000

= 153.6  » 154

for Aaaa = 4pq³ x 1000 = 4x0.8x(0.2)³ x1000

= 25.6  » 26

for aaaa = q⁴ x 1000 = (0.2)⁴ x1000

= 1.6 » 2

Number of phenotypes are 

AAAA : AAAa : AAaa : Aaaa : aaaa = 409 : 409 : 154 : 26 : 2

Option 1 is correct

Function of Vir genes

The genes responsible for the transfer of the T-DNA region into the host plant are also situated on the Ti plasmid in a region of approximately 40 kb outside the T-DNA, known as the vir (virulence) region. The genes of vir region are not transferred themselves; they only induce the transfer of T-DNA.

These genes have following functions:

Vir genes	Functions
VirA	Encodes acetosyringone (phenolic sensor) receptor protein, functions as autokinase; also activates VirG gene by phosphorylation leading to constitutive expression of all genes
VirB1-B11	Encodes membrane protein, involved in conjugal tube formation through which T-DNA is transport, VirB11 has ATPase activity
VirC	Encodes helicase enzymes, binds to the overdrive region, unwinding of T-DNA
VirD1	Topoisomerase activity- required for T-DNA processing, modulates VirD2 activity
VirD2	VirD2 is an endonuclease- nicks the right border of T-DNA, directs T-DNA through the VirB/VirD4 transfer apparatus, contain nuclear localization sequences (NLS) that promote nuclear uptake of the T-complex
VirD4	Components of transfer apparatus
VirE1	Required for VirE2 export from Agrobacterium
VirE2	Single strand binding protein (SSBP), binds to T-DNA during transfer, forms a membrane channel that transfers the T-strand through the plant plasma membrane, involved in nuclear targeting and passage through nuclear pore complex, contain nuclear localization sequences (NLS), assist nuclear uptake of the T-complex by keeping the T-strand in an unfolded state
VirF	Directs protein coating of T-DNA complex removal by proteasomal machinery
VirG	Master controller DNA binding protein, vir A activates vir G by phosphorylation, vir G dimerises and activates constitutive expression of all vir operons
VirJ	T-DNA export

Some compounds that use as herbicides

S.No.	Chemical compound	How it interferes	Mechanism
1.	DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea)	Inhibition of electron transfer	Competes with plasoquinone (QB) for binding site in PSII
2.	FCCP (cyanide-p-trifluoromethoxyphenylhydrazone;)	Uncoupling of phosphorylation from electron transfer	Hydrophobic proton carriers
3.	DNP(2,4-dinitrophenol)		Hydrophobic proton carriers
4.	Valinomycin		K+ ionophore
5.	Thermogenin		In brown adipose tissue, forms proton-conducting pores in inner mitochondrial membrane
6.	Atractyloside	Inhibition of ATP-ADP exchange	Inhibits adenine nucleotide translocase
7.	DCCD(dicyclohexylcarbodiimide)	Inhibition of ATP synthase	Blocks proton flow through Fo and CFo
8.	Aurovertin		Inhibits F1
9.	Oligomycin		inhibits Fo and CFo
10.	Venturicidin
11.	Cyanide	Inhibition of electron transfer	Inhibit cytochrome oxidase
12.	Carbon monoxide
13.	Antimycin A		Blocks electron transfer from cytochrome b to cytochrome c1
14.	Myxothiazol		Prevent electron transfer from Fe-S center to ubiquinone
15.	Rotenone
16.	Amytal
17.	Piericidin A

Source: Lehninger Principles of Biochemistry (Nelson and Cox, 6^th edition)

Mechanism of action of antibiotics – Part II

Puromycin:

Puromycin is a structural analogue of the 3′ end of aminoacyl- tRNA, but differs from tRNA as the aminoacyl residue is linked via an amide bond rather than an ester bond. Puromycin, like aminoacyl-tRNA, binds to the A site of the ribosome peptidyl-transferase center. When the A site is occupied by puromycin, peptidyl transferase links the peptide residues of the peptidyl-tRNA in the ribosomal P site covalently to puromycin. Since the amide bond cannot be hydrolyze by the ribosome, no further peptidyl transfer takes place, and the peptidyl-puromycin complex falls off the ribosome. Puromycin concentrations should be high to inhibit translation completely, because (a) the of puromycin bind with ribosome by weak bonds (b) single ribosome is able to transfer several puromycin molecules to peptidyl-puromycin, and (c) once peptidyl-puromycin has fallen off the ribosome, it does not bind again thatswhy no further antibacterial activity.

Actinomycin D:

Actinomycin D is a molecule that consists of a chromophore (fenoxazone ring) attached to two identical cyclic pentapeptides, it favors guanine-cytosine pairs and is therefore inserted between the G-C steps. Hydrogen bonds are established between the guanine 2-amino group and the carbonyl oxygen of threonine, and also between the guanine N-3 atom and the NH group of the same threonine residue, helping to stabilize the actinomycin-DNA complex. The proline, sarcosine and methylvaline residues of the pentapeptide side chain are involved in further hydrophobic interactions with the DNA minor groove. The formation of this stable actinomycin-DNA complex prevents the unwinding of the double helix which leads to inhibition of the DNA-dependent (this prevents DNA from acting as a template for RNA synthesis) RNA polymerase activity and hence transcription. Actinomycin D does not bind to single stranded DNA/RNA and at low concentration it does not affect DNA replication. As this antibiotic does not directly affect the translation rocess, protein synthesis can continue from the preexisting mRNA. Actinmycin D works with both in prokaryotes and eukaryotes.

Vancomycin:

Vancomycin is a branched tricyclic glycosylated nonribosomal peptide. Vancomycin acts by inhibiting the second stage of cell wall synthesis in susceptible bacteria. Peptidoglycan layer of the cell wall is rigid due to its highly cross-linked structure. During the synthesis of the peptidoglycan layer of bacteria vancomycin prevents incorporation of new building blocks of peptidoglycan i.e., N-acetylmuramic acid (NAM)- and N-acetylglucosamine (NAG)-peptide subunits from being incorporated into the peptidoglycan matrix; which forms the major structural component of Gram-positive cell walls. The large hydrophilic molecule is able to form hydrogen bond interactions with the terminal D-alanyl-D-alanine moieties of the NAM/NAG-peptides. This binding of vancomycin to the D-Ala-D Ala prevents the incorporation of the NAM/NAG-peptide subunits into the peptidoglycan matrix.Reformation of the peptide cross links occurs by the enzyme transpeptidase. Vancomycin after binding to the building blocks (i.e. NAG and NAM) of the peptidoglycan prevents the transpeptidase from acting on these new formed blocks and thus prevents cross-linking of the peptidoglycan layer. By doing so, vancomycin makes the peptidoglycan layer less rigid and more permeable. This causes cellular contents of the bacteria to leak out and eventually death of the bacteria.

Mechanism of action of antibiotics – Part I

Mechanism of action of antibiotics – Part I

Penicillin:

Penicillin kills susceptible bacteria by specifically inhibiting the transpeptidase that catalyzes the final step in cell wall biosynthesis, the cross-linking of peptidoglycan. Penicillin is a structural analog of the acyl-D-alanyl-D-alanine terminus of the pentapeptide side chains of nascent peptidoglycan. The membranes of many species of bacteria contain one major and several minor proteins (penicillin binding protein-PBP) which bind penicillin covalently. These proteins specifically catalyze the penicillin-sensitive hydrolysis of COOH-terminal D-alanine from the peptide chain of cell wall-related substrates. Hence, these enzymes have been given the name D-alanine carboxypeptidase (CPase). Penicillin covalently binds to CPase via an ester linkage to serine 36 which is relatively rapidly hydrolyzed. Penicillin acylates the active site of enzymes involved in cell wall biosynthesis. Thus formation of a complete cell wall is blocked, leading to osmotic lysis.

Chloramphenicol:

Chloramphenicol is a bacteriostatic antibiotic inhibits protein synthesis in bacteria. Chloramphenicol enters the bacteria by an energy-dependent process. It binds to 23S rRNA on the 50S ribosomal subunit to inhibit (competitive inhibition) the peptidyl transferase reaction. Binding of Chloramphenicol induces conformational change in the ribosome, which slows or even inhibits the incorporation of the aminoacyl tRNA and in turn the transpeptidation reaction and block protein chain elongation. It specifically binds to A2451 and A2452 residues in the 23S rRNA of the 50S ribosomal subunit, preventing peptide bond formation.

Tetracycline:

Tetracyclines are bacteriostatic and time dependent antibiotics. They enter gram negative bacteria by passive diffusion through the porin channels and gram positive bacteria and other organisms by active transport. After entering the cell, tetracyclines bind reversibly to the 30S subunit of the bacterial ribosome, blocking the binding of aminoacyl-tRNA to the acceptor site on the mRNA-ribosome complex. This inhibits addition of amino acids to the growing peptide. tetracyclines are responsible for the selective toxicity to the microbes because the carrier involved in the active transport of Tetracycline is absent in the mammalian cells and also tetracyclines do not bind to mammalian 60S or 40S ribosomes.

How to Calculate Isoelectric point

The specific pH at which the net electric charge of an amino acid is zero, known as isoelectric point or isoelectric pH, denoted as pI. Every amino acid has its own specific pI value. Every amino acid has inonizable groups i.e., COOH and NH₂. If amino acid is acidic viz. Aspartic acid and Glutamic acid it has extra COOH in R chain whereas, in case of basic amino acid viz. Arginine and Lysine, one extra NH₂ group is found in its R chain.

⁺H₃N-CH(R)-COOH           →      ⁺H₃N-CH(R)-COO^–          →         H₂N-CH(R)-COO^–

^{Net charge        +1                                               0                                                   –1}

When these ionizable groups ionize give characteristic pKa values. When amino acids having nonionizable R groups give only two pKa values i.e., pKa₁ & pKa₂ and amino acids with nonionizable R groups give three pKa values i.e., pKa₁(–COOH), pKa₂(–NH₂) and pKa_R(–COOH or –NH₂ of R chain)

These pKa values are very important in calucaltion of isoelectric point (pI).

Calculation of pI:

One can calculate pI, easily by following formula

(1)   Amino acid with nonionizable R groups

pI = (pKa₁ + pKa₂)/2

Example: pI of Alanine (pKa₁= 2.34, pKa₂= 9.69)

pI = (2.34 + 9.69)/2 = 6.01

(2)   Negatively charged (acidic) amino acid

pI = (pKa₁ + pKa_R)/2

(pKa_R = pKa value of extra –COOH in R chain)

Example: pI of Aspartic acid (pKa₁= 1.88, pKa₂= 9.60, pKa_R= 3.65 )

pI = (1.88 + 3.65)/2= 2.77

(3)   Positively charged (basic) amino acid

pI = (pKa₂ + pKa_R)/2

            (pKa_R = pKa value of extra –NH₂ in R chain)

Example: pI of Arginine (pKa₁= 2.17, pKa₂= 9.04, pKa_R= 12.48)

pI = (9.04 + 12.48)/2= 10.76

Isoelctric point (pI) of 20 standard amino acids:

Amino acid	pK1	pK2	pKR	pI
Glycine	2.34	9.60		5.97
Alanine	2.34	9.69		6.01
Proline	1.99	10.96		6.48
Valine	2.32	9.62		5.97
Leucine	2.36	9.60		5.98
Isoleucine	2.36	9.68		6.02
Methionine	2.28	9.21		5.74
Phenylalanine	1.83	9.13		5.48
Tyrosine	2.20	9.11	10.07	5.66
Tryptophan	2.38	9.39		5.89
Serine	2.21	9.15		5.68
Threonine	2.11	9. 62		5.87
Cysteine	1.96	10.28	8.18	5.07
Asparagine	2.02	8.80		5.41
Glutamine	2.17	9.13		5.65
Lysine	2.18	8.95	10.53	9.74
Arginine	1.82	9. 17	12.48	10.76
Histidine	2.17	9.04	6.00	7.59
Aspartatic acid	1.88	9.60	3.65	2.77
Glutamic acid	2.19	9. 67	4.25	3.22