Problems on Enzyme Kinetics

 

How to calculate possible number of allele combination in given genotype?

Combination of alleles in any organims is known as its genotype. An allele is one of two or more alternate forms of a gene located at the corresponding locus on homologous chromosome. One allele obtained from the mother and the other from the father. Any possible combination of alleles is possible for the individuals within a particular population. If three alleles, A, B, and C are present on any specific locus, there will be six genotypes possible. An individual with above alleles may have any of the following allele combinations: AA, AB, AC, BB, BC and CC.

The number of possible genotypes or possible combination of alleles can be calculated by two methods. 

(1) It can be calculated by adding the number of possible combination of alleles with each positive integer below that number. For example, for a locus having three possible alleles, the number of possible genotypes is                                              3+2+1= 6

(2) The number of possible combination of alleles can also be calculated by using following formula
Number of allele combination = n(n+1)/2

Where, n is equal to the number of possible alleles.Here, number of alleles are 3,Therefore,
                                     3(3+1)/2 = 12/2=6

From above discussion, we can say that there are six possible allele combination occur if any individual have three alleles.

Problem:  There are 15 different alleles are present at a VNTR (variable number of tandem repeats) locus. How many combination of alleles (genotypes) are possible in a population for this VNTR?Explanation:
We can calculate by using both aforementioned methods

          (1)     15+14+13+12+11+10+9+8+7+6+5+4+3+2+1= 120

          (2)     By the use of formula
     Number of allele combination =15(15+1)/2=(15)(16)/2=240/2                                                                               =120

Therefore, for a locus having 15 different alleles, 120 different allele combination or genotypes are possible. 

How SARS CoV-2 infects human?

Structure of Coronavirus: 

(Image source: Stephen N.J.Korsman et al. (2012). Human coronaviruses. Virology (2012): 94-95)

Life cycle of SARS CoV-2

Attachment and entry

The virus attached with the host cell by the spike protein and its receptor. The receptor binding domain (RBD) on spike protein recognizes the angiotensin-converting enzyme-2 (ACE2) receptor on host cell and attaches to it. After attachment with host cell, virus is able to enter the host cell. There are two different ways to enter the host cell. The mechanism of entrance into host cell is depends on the host protease, protease cleave and activate the receptor-attached spike protein.

The first mechanism SARS CoV-2 follow to enter the host cell is endocytosis and uptake of the virus in an endosome. The receptor-attached spike protein is then cleaved and activated by the host's pH-dependent cysteine protease cathepsin L. When this receptor-attached spike protein gets activated introduces a conformational change, and the later fusion of the viral envelope with the endosomal wall occurs.

In other mechanism, the SARS CoV-2 can enter the host cell directly by proteolytic cleavage of the receptor-attached spike protein by the host's TMPRSS2 or TMPRSS11D serine proteases at the cell surface. In the SARS coronavirus, the activation of the C-terminal part of the spike protein triggers the fusion of the viral envelope with the host cell membrane by inducing conformational changes.

Genome translation

After fusion the viral nucleocapsid passes into the cytoplasm, where the viral genome is released. The viral genome acts as a messenger RNA (mRNA), and the host cell's ribosome translates two-thirds of the genome into two large overlapping polyproteins, pp1a and pp1ab.

These polyproteins have their very own proteases, PLpro and 3CLpro, which cleave the polyproteins at specific sites. Polyprotein pp1ab yields 16 nonstructural proteins (nsp1 to nsp16) after cleavage. Product proteins include various replication proteins such as RNA-dependent RNA polymerase (RdRp), RNA helicase, and exoribonuclease (ExoN).

Replication and transcription

Many nonstructural replication proteins collectively form a multi-protein replicase-transcriptase complex (RTC). RNA-dependent RNA polymerase (RdRp) is the main replicase-transcriptase protein. This protein directly involved in the replication and transcription of RNA from an RNA strand. The other nonstructural proteins of the complex assist in the replication and transcription process.

(Image source: Smith EC, Denison MR (2013). Coronaviruses as DNA Wannabes: A New Model for the Regulation of RNA Virus Replication Fidelity. PLoS Pathog 9(12): e1003760.)

The protein nsp15 acts as a 3'-5' exoribonuclease and provides a proofreading function to the complex which the RNA-dependent RNA polymerase dos’nt has. Proteins nsp7 and nsp8 form a hexadecameric sliding clamp as part of the complex which significantly increases the processivity of the RNA-dependent RNA polymerase. Due to large genome size the coronaviruses needs the increased fidelity and processivity during RNA synthesis.

One of the main roles of the (RTC) is to transcribe the viral genome. RdRp directly mediates the synthesis of negative-sense subgenomic RNA molecules from the positive-sense genomic RNA. After transcription of these negative-sense subgenomic RNA molecules, transcription of their corresponding positive-sense mRNAs takes place.

The other important function of the RTC is to replicate the viral genome. Replication of positive-sense genomic RNA is from the negative-sense genomic RNA.

This replicated positive-sense genomic RNA becomes the genome of the progeny viruses. The various smaller mRNAs are transcribes from the last third of the virus genome. These mRNAs are translated into the four structural proteins (S, E, M, and N) that will become part of the progeny virus particles and also eight other accessory proteins which assist the virus.

Assembly and release

RNA translation takes place inside the endoplasmic reticulum. The viral structural proteins S, E and M move along the secretory pathway into the Golgi intermediate compartment. There, the M proteins direct most protein-protein interactions required for assembly of viruses following its binding to the nucleocapsid. Progeny viruses are released from the host cell by exocytosis through secretory vesicles.

(Image source: 

Zhiqi Song et al., (2019). From SARS to MERS, Thrusting Coronaviruses into the Spotlight. Viruses 11(1), 59)

Coronavirus

Virus classification

Phylum                 incertae sedis

Order                   Nidovirales

Family                   Coronaviridae

Subfamily             Orthocoronavirinae

Genus                   Alphacoronavirus (Alpha-CoV)

Betacoronavirus (Alpha-CoV)

Gammacoronavirus (Alpha-CoV)

Deltacoronavirus (Alpha-CoV)

Species of Alphacoronavirus

Human coronavirus 229E, Human coronavirus NL63, Miniopterus bat coronavirus 1 etc.

Species of Betacoronavirus

Human coronavirus HKU1, Severe acute respiratory syndrome-related coronavirus (SARS-CoV), Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), Middle East respiratory syndrome-related coronavirus (MERS) etc.

Species of Gammacoronavirus

Infectious bronchitis virus etc.

Species of deltacoronavirus

Bulbul coronavirus HKU11 etc.

There are seven strains of human coronavirus, four strains of which produce the generally mild symptoms of the common cold:

1.   Human coronavirus OC43 (HCoV-OC43)

2.   Human coronavirus HKU1

3.   Human coronavirus NL63 (HCoV-NL63, New Haven coronavirus)

4.   Human coronavirus 229E (HCoV-229E)

and three strains cause severe symptoms:

1.   Middle East respiratory syndrome-related coronavirus (MERS-CoV), previously known as novel coronavirus 2012 and HCoV-EMC

2.   Severe acute respiratory syndrome coronavirus (SARS-CoV or "SARS-classic")

3.   Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), previously known as 2019-nCoV or "novel coronavirus 2019"

Some interesting facts about SARS-CoV2:

·        Previously it was known as 2019 novel coronavirus (2019-nCoV)

·        SARS-CoV2 is responsible for pandemic COVID-19

·        Outbreak of SARS-CoV2 is declared as pandemic by WHO on March 11, 2020.

·        It is the seventh known coronavirus to infect people, after 229E, NL63, OC43, HKU1, MERS-CoV, and the original SARS-CoV.

·        Its transmission occurs by respiratory droplets like cough and sneeze within the range of 2 metre.

·        Size of SARS-CoV2 is 50-200nm in diameter.

·        SARS-CoV-2 has four structural proteins, known as the S (spike), E (envelope), M (membrane), and N (nucleocapsid) proteins; the N protein holds the RNA genome, and the S, E, and M proteins together create the viral envelope.

·        Spike protein is responsible for attachment of virus to host membrane.

·        Genome of SARS-CoV2 is positive-sense single-stranded RNA (+ssRNA).

·        Its genome is of 30kb length.

·        SARS-CoV2 bind with angiotensin converting enzyme2 (ACE2) receptors of human cells and enter into cells.

COVID-19 Update (till March 22, 2020 at 7:00 PM IST)

Total Coronavirus Cases: 317,298

Deaths:13,642

Recovered: 95,949

Active cases		Closed cases
207,707 Currently Infected Patients		109,591 Cases which had an outcome:
197,565 (95%) in Mild Condition	10,142 (5%) Serious or Critical	95,949 (88%) Recovered / Discharged	3,642 (12%) Deaths

COVID-19 Update (till March 22, 2020 at 5:30 AM IST)

Total cases	New cases	Total deaths	New deaths	Total recovered	Active cases	Serious, critical	Total cases/1M population
360	28	7	2	24	329		0.3

Outbreak of Coronavirus related diseases

Outbreak	Virus
SARS, 2003	SARS-CoV
MERS, 2012	MERS-CoV
MERS, 2015	MERS-CoV
MERS, 2018	MERS-CoV
COVID-!9	SARS-CoV2

Source:  https://www.wikipedia.org/

  https://www.worldometers.info/coronavirus/

  https://www.who.int/

Problems

Definition of Pandemic, Endemic, Epidemic and Syndemic

Pandemic (from Gree pan "all" and demos "people") is a disease epidemic that has spread across a large region, viz. multiple continents, or worldwide. A pandemic is an epidemic occurring on a scale that crosses international boundaries, usually affecting a large population.

Endemic (from Greek en "in, within" and demos "people") an infection is said to be endemic in a population when that infection is constantly maintained at a baseline level in a geographic area without external inputs.

Epidemic An epidemic (from Greek epi "upon or above" and demos "people") is the rapid spread of disease to a large population in given population within a short period of time.

A syndemic or synergistic epidemic is the aggregation of two or more concurrent or sequential epidemics or disease clusters in a population with biological interactions, which exacerbate the prognosis and burden of disease.

Multiple choice questions on Cell Junction

1- A skin disease pemphigus, in this disease affected individuals make antibodies against one of their own non classical cadherine of desmosomes. These antibodies bind to desmosomes and disrupt it, that hold their epidermal cells (keratinocytes) together – 

a- Cause severe blistering of body fluids into the loosened epithelium  

b- Block the exchange of materials in these cells

c- Disruption of gap junction 

d- All of the above 

2- Protein claudins are essential for tight junction, mice that lack claudin-1 gene fail to make tight junction between cells epidermal layer of skin; as a result 

a- Skin become loose due to accumulation of fluid, and baby mice die

b- Skin remain normal

c- baby mice die within a day after birth, due to lose of water rapidly by evaporation through the skin

d- baby mice servive with less fluid

3- In kidney epithelial cells, a specific claudin is found which is needed to reabsorb mg++ from urine to blood, mutation in claudin gene cause 

a- Mg++ reabsorb through other protein which allow reabsorption 

b- Excessive lose of mg++ ion in the urine

c- No effect because there is no reabsorption through claudin, it’s a protein of tight junction

d- Both a & c

4- Gap junction have channel forming protein namely connexins & innexins. ----- have connexins, ----- have innexins

a- Humans, other mammals

b- Mammals, birds

c- Vertebrates, invertebrates

d- Chordates, hemichordates

5- A rare human genetic disease, deficiency of dermatan sulfate disaccharide ( a type of GAGs) the affected individuals have-

a- Short stature, a prematurely aged appearance, and generalized defects in their skin, joints, muscles, and bones.

b- Normal face & stature with a weak bone

c- Only defect in skin

d- No effect 

6- Defect / dificiency of type I collagen lead to mutant phenotype – 

a- Severe bone defects, fractures ( osteogenesis imperfecta )

b- Fragile skin

c- Cartilage deficiency, dwarfism ( chondrodysplasia )

d- Osteoarthritis  

7- Defect / deficiency of types II collagen lead to mutant phenotype –

a- Severe bone defects, fractures ( osteogenesis imperfecta )

b- Fragile skin

c- Cartilage deficiency, dwarfism ( chondrodysplasia )

d- Osteoarthritis  

8- Defect / deficiency of types III collagen lead to mutant phenotype –

a- Severe bone defects, fractures ( osteogenesis imperfecta )

b- Fragile skin, loose joint, blood vessels prone to rupture ( Ehlers-Danlos syndrome ) 

c- Cartilage deficiency, dwarfism ( chondrodysplasia )

d- Osteoarthritis  

9- Type XI collagen which fibril associates with type I, a defect or deficiency of this collagen types lead to mutant phenotype –

a- Fragile skin

b- Cartilage deficiency, dwarfism ( chondrodysplasia )

c- Osteoarthritis

d- Myopia, blindness

10- Type IX collagen which form lateral association with type II, present in -----, defect or deficiency of this collagen cause -----

a- Cartilage, osteoarthritis 

b- Bone, osteoarthritis

c- Epithelium, fragile skin

d- All of the above

Answers: 1-a, 2-c, 3-b, 4-c, 5-a, 6-a, 7-c, 8-b, 9-d, 10-a

Contributed by: Ms. Nargis K.