Karyotyping

What is a karyotype?

Karyotype is the complete set of an individual’s chromosomes i.e.; it describes the chromosome count of an organism and what it looks like under a light microscope.

The description is based on

  • Length of the chromosome
  • Position of centromere
  • Banding pattern
  • Differences between sex chromosomes

Karyotyping is the process by which photographs of chromosomes are taken in order to determine the chromosome complement of an individual, including the number of chromosomes and any abnormalities.

Karyotyping is a part of cytogenetics.

What are chromosomes?

They are the structures that hold the genes that control and regulate our body’s functions. There are 46 chromosomes in each of our cells that occur as 23 pairs. The first 22 pairs are labelled depending on their length in a descending order. The last pair is called gonosomes or sex chromosomes labelled as X or Y.

Female: 46, XX

Male: 46, XY

Each chromosome has 2 arms, separated by a centromere, named as p (short arm) and q (long arm). The chromosomes are visualized under a light microscope on staining. When stained, the chromosomes look like a string with dark and light bands. Each chromosome is distinguished based on the banding pattern, position of centromere and the size.

Human Male – Classic Karyotype

Why is karyotyping important?

Karyotyping is a means of detecting polyploidy, aneuploidy, translocations, inversions, rings, copy number changes in the size ranging from 4 – 6 Mb and other chromosomal abnormalities.

Karyotyping or chromosome analysis is done for children, adults and fetus if a genetic abnormality is suspected. It is also performed on cancer cells to study the extent of genomic degradation.

Abnormal Karyotype – Cancer

Karyotypes are used to study cellular functions, taxonomic relationships and to gather information regarding past evolutionary events.

During the lifespan of an organism, does the karyotype change?

Yes. Karyotype is the chromosome count of an organism. Unlike replication and transcription of DNA which are highly standardized in eukaryotes, karyotypes are highly variable. Karyotype may vary within a species in different stages of life.

Some organisms have been observed to go for large scale elimination of heterochromatin over usual gene repression. Chromosome elimination in sciarid flies, chromosome diminution in Ascaris suum and X chromosome inactivation in mammals are a few examples of visible adjustments chromosome undergoes during a life span of an organism.

What are the different depictions of karyotypes?

Cytogenetics uses several techniques to visualize various aspects of the chromosome

  1. G – banding
  2. R – banding
  3. C – banding
  4. Q – banding
  5. T – banding

What is spectral karyotype?

Spectral karyotype (SKY) is a karyotype in which the homologous pairs of chromosomes are manipulated to have different colors.

Spectral Karyotype (SKY)

SKY is an advanced molecular cytogenetic technique for chromosomal analysis based on the principle of FISH. All the chromosomes are visualized simultaneously by labeling them with a combination of different colors that are spectrally distinguishable fluorochromes, but different methods are used for detecting and discriminating the different combinations of fluorescence after in situ hybridization. In SKY the images are captured by charge-coupled device (CCD) imaging and analyzed by using an interferometer attached to a epifluorescence microscope. Image processing software then assigns a pseudo color to each spectrally different combination, allowing the visualization of the individually colored chromosomes.

Thus, this technique makes it easier for the scientists to detect chromosomal abnormalities, as compared to the conventional karyotypes.

A Brief account on Sickle Cell Diseases (SCD)

SCD was first described in 1910 in a patient presenting pulmonary symptoms. Given the symptoms presented by the patient, it was unclear as to whether the blood condition was a disease sui generis or a manifestation of another disease.

Herrick coined the term “sickle-shaped” to describe the peculiar appearance of the RBC.

Linus Pauling was the first to hypothesize in 1945 that the disease might originate from an abnormality in the hemoglobin molecule. This hypothesis was validated in 1949 by the demonstration of the differential migration of sickle versus normal hemoglobin as assessed by gel electrophoresis.

Phenotype-Gene Relationships

Location

Phenotype

Phenotype

MIM number

Inheritance

Phenotype

mapping key

Gene/Locus

Gene/Locus

MIM number

11p15.4

Sickle cell anemia

603903

AR

3

HBB

141900

Genetics of SCD

In 1945, while studies by Pauling et al. established that SCD results from a defect in the hemoglobin molecule, the mode of inheritance was shown to be Autosomal Recessive.

The sickle mutation was characterized several years later by Ingram et al. as a glutamine-to-valine substitution at the sixth residue of the β-globin polypeptide.

Once the human globin genes were cloned and sequenced, a great deal of insight was available regarding their expression.

Human hemoglobin is a tetrameric molecule that consists of two pairs of identical polypeptide subunits, each encoded by a different family of genes. The human α-like globin genes (ζ, α1, and α2) are located on chromosome 16, and the β-like globin genes (ε, Gγ, Aγ, δ, and β) are located on chromosome 11.

During fetal life, the predominant type of hemoglobin is Hb F (α2γ2). During the postnatal period, Hb F is gradually replaced by Hb A (α2β2). Hb A2 (α2δ2) is minor adult-type hemoglobin that accounts for less than 2.5% of the circulating hemoglobin in normal individuals in adult life. Upon completion of the switch from Hb F to Hb A, patients with disorders of the β-globin genes start manifesting the clinical features of their diseases.

The prospect of therapeutic reactivation of Hb F production in adult life has been in large part responsible for the tremendous interest in the elucidation of the molecular mechanisms of the switch from fetal to adult hemoglobin production.

 Chromosomal organization of the α- and β-globin gene clusters.

Homozygosity for the sickle mutation (i.e., HbSS disease) is responsible for the most common and most severe variant of SCD. Several other genetic variants of SCD result from the interaction of different mutations of the human β-globin genes.

Why is the Glu to Val substitution such a big deal?

To understand why the substitution of one amino acid causes such a wide range of diseases, we have to understand the nature of the amino acids involved.

There are 20 standard amino acids found in mammalian tissues. They are classified depending on their side chain ( R group) as :

  1. Amino Acids with Polar side chain
  2. Amino acids with Non-polar side chain
  3. Amino acids with Acidic side chain
  4. Amino acids with Basic side chain

Glutamate is an amino acid that has a Polar side chain. This side chain is capable of participating in Hydrogen bonds. Thus, it is hydrophilic.

Valine is an amino acid that has a Nonpolar side chain. They neither gain nor lose protons or participate in Hydrogen bonding. Hence they are lipid-like or “oily” that promote hydrophobic interactions. In a Polar environment, they cluster together in the interior of the protein much like droplets of oil that coalesce in an aqueous environment. Thus, they fill up the interior of the folded protein and help give it its three-dimensional shape.

Therefore, when the polar glutamate is replaced by nonpolar valine at the 6th position in the 𝛃-subunit of Hemoglobin, the three dimensional folding of the protein is hampered. The 6th position that was initially supposed to interact with the environment and form H-bonds, turns inwards into the three-dimensional structure and exhibits hydrophobicity.

Pathophysiology of SCD

In addition to the obvious shape changes that result from the formation of intracellular hemoglobin polymers, the polymers can have a direct impact on the RBC plasma membrane, leading to the extracellular exposure of protein epitopes and glycolipids that are normally found inside the cell. These changes and the increased expression of adhesion molecules on the surface coils explain the increased adherence of sickle RBC to vascular endothelium which leads to Vascular Occlusion.

This image has an empty alt attribute; its file name is image-4.png
Alteration of the RBC membrane by polymers of sickle hemoglobin.

Diagnosis of SCD

The diagnosis of SCD is usually simple and rarely poses a major challenge. The major challenge in the diagnosis of sickling disorders is to identify the disease during the prenatal period, at a time when such information would be critically important in enabling a couple at risk to make an informed decision about potential termination of pregnancy. Before the advent of molecular diagnostics, the only way to make a diagnosis prenatally was to obtain a fetal blood sample for analysis, which could only be performed after the 20th week of pregnancy. By that time, the pregnancy is already too advanced to make it possible to terminate safely. With the advent of DNA diagnostics, it has become possible to make definitive diagnoses of the different sickling disorders during the first trimester by analyzing fetal DNA obtained by chorionic villus biopsy. The molecular diagnostic technology is being pushed further to allow the diagnosis to be made from a small number of fetal cells that can be harvested from the maternal circulation.

The major challenge in the diagnosis of sickling disorders is to identify the disease during the prenatal period.

An Introduction To Chinese Hamster

Chinese Hamster

Scientific Name: Cricetus griseus

Country of Origin: Mongolia and Northern China

Habitat: Steppes and rocky plains

Length: Females 8-12 cm; Males 8-14 cm

Weight: Females 25-40 g; Males 30-60 g

Life span: 18-36 months

GENERAL DESCRIPTION

  1. Chinese hamsters are small rodents with a grayish-black coat and a black dorsal stripe.
  2. Adult animals weigh approximately 39–46 g and measure approximately 9 cm in length.
  3. They live two to three years on average.
  4. Males have a relatively large scrotum, therefore females are generally kept as pets, and males were used solely for breeding and research purposes until scientists started using other rodents, albino mice, and rats.
  5. The wild color is brown with a black stripe down the spine, black and grey ticks, and a whitish belly. This coloration, combined with their lithe build and longer tail, makes them look “mousy” to some eyes and, in fact, they are members of the group called ratlike hamsters. Besides the wild color, a well-known variation is the white-spotted Chinese hamster, which often is grayish-white all over, with only a dark stripe on its back.
  6. The average gestation length is 20.5 days, with a litter size of approximately 4–5 offspring.
  7. The Chinese hamster, like the Syrian hamster, has a cheek pouch that can be used as an immunologically privileged site.

HISTORY

  • The Chinese hamster was first used as a laboratory animal to type pneumococci obtained from human patients (Hsieh, 1919). 
  • The Chinese hamster became a valuable animal model in other infectious disease and epidemiology research studies. This hamster is a known carrier of the protozoan parasite Leishmania that causes the often deadly human disease leishmaniasis, also known as Kala-azar or black fever.
  • Efforts at domestication, in the mid-20th century, resulted in the serendipitous finding that some inbred lines of Chinese hamsters develop spontaneous hereditary diabetes mellitus. This finding fostered an interest in the Chinese hamster as an animal model for diabetes and helped spur research interest in hamster genetics (Yerganian, 1959, 1985).
  • Tijo and Puck (1958) isolated Chinese hamster ovary (CHO) cells which have since been commonly used as an in vitro research tool for mutagenesis and carcinogenesis studies.
  • Subsequent laboratory investigations have led to a series of elegant research studies directed at utilizing CHO cells for the production of recombinant therapeutic proteins (Oka and Rupp, 1990).
  • During the past 20 years, CHO cells have been used to synthesize a wide array of recombinant proteins that have found clinical application in the treatment of a variety of human diseases (Chin, 2007, Jayapal et al., 2007).

    Chinese hamster ovary cell culture, phase contrast (positive) microscopy

AS LABORATORY ANIMALS

In the past, Chinese hamsters were commonly used laboratory animals, until they were replaced by the common mouse and rat, which are easier to keep and breed; however, several biotech drugs are still being produced by putting the gene for the protein into Chinese hamster ovary cells, which then produce the protein.

This species has been shown to be susceptible to a number of experimentally induced viral, bacterial, and parasitic infections. In recent years, the Chinese hamster’s contributions as a laboratory animal have been largely overshadowed by the focus on its cell lines and the role it plays in scientific research and biotechnology.

FEATURES THAT PROMOTED ITS USE IN BIOMEDICAL RESEARCH

  • Small size,
  • Polyestrous cycle,
  • Short gestation period,
  • Low chromosome number.
  • Low incidence of spontaneous and endogenous viral infections

 

CYTOGENETICS AND FETAL DEVELOPMENT

The Chinese hamster has been frequently selected for studies of chromosomal abnormalities because of a low spontaneous mutation rate and a low diploid chromosome number (2N = 22). The ten large pairs of autosomes and the two sex chromosomes can be readily differentiated.

Fertilization begins approximately 2–3 hours after ovulation and is completed within the next 4–5 hours. At 20–26 hours following ovulation, the first three cleavages occur. Four days post-ovulation, the fertilized egg reaches the blastocyst stage and enters the uterus. Implantation occurs at approximately day 5 or 6 post-ovulation (Pickworth et al., 1968). Embryonic and fetal development of the Chinese hamster has been extensively characterized. Prenatal developmental stages are similar to the developmental stages in the mouse (ten-Donkelaar et al., 1979).

Cell Lines Derived from Chinese Hamster Tissues and Organs

Puck et al. (1958) obtained a female Chinese hamster from Yerganian’s laboratory and used it to derive the original Chinese hamster ovary (CHO) cell line. Puck and his junior colleague Fa-Ten Kao, sub-cloned the hamster cells and generated the CHO-K1 cell line, which would become a standard of mammalian cell culture in the decades to come. Puck himself used to call the cells “the mammalian E. coli”.

The Chinese hamster ovary (CHO) and V-79 lung fibroblast are two cell lines derived from the Chinese hamster that has been used extensively in biomedical research (Bradley et al., 1981).

The family tree of prominent CHO cell lines. Sequenced lines are highlighted in blue. Where known, the name of those who isolated the strain and the year it was done is given in parentheses. From N.E. Lewis et al. (2013)

ADVANTAGES OF CHO CELLS

Easy to Culture

Grow well in suspension and as adherent culture, rendering the cells ideal for GMP procedures. Their tolerance to variations in pH, oxygen levels, temperature, or pressure makes them the ideal cell for large-scale culture.

High Productivity

High recombinant protein yields and specific productivity. Thanks to genetic optimization, protein yields 3-10 grams per liter of cell culture.

Defined Culture Conditions

Can be adapted for defined, serum-free culture conditions, as well as allowing for animal-free and protein-free production and better safety and stability profiles.

Various Selection Systems

Antibiotic and metabolic selection by DHFR- or glutamine synthase (GS)-deficiency to obtain stable clones of high productivity with ease.

Post-Translational Modifications

A variety of post-translational modifications of the produced biologic, often allowing allow for a biosimilar, if not human-identical products with excellent pharmaceutical activity and biocompatibility.

Genetic Cell Engineering

Well-proven genetic tools are available to optimize CHO cells, from gene introduction to knock-out, knock-down, and post-translational silencing.

FDA-Approval

Used for nearly 50 biotherapeutics already approved in the USA and EU.

Low Virus Susceptibility

Due to the hamster origin, the risk of propagation of human viruses is decreased, reducing production loss and increasing biosafety.

Filamentous Bacteriophage

First reported in 1963. It is the simplest known organism with less than 10,000 nucleotides in its DNA. The structure was studied using X Ray filter diffraction, solid state NMR and cryo-electron microscopy. Their hosts are gram negative bacteria and the virions are long, flexible filaments. They enter the bacteria by adsorbing onto the sex pili and unlike other bacteriophages, they are released from growing and dividing cells without markedly harming the cells. It is a valuable tool in the study of fundamental aspects of molecular biology and an object of interest in immunology and nanotechnology.

Life cycle of filamentous bacteriophage

Their taxonomy was described by Andre Lwoff and Paul Tournier.

Order: Tubulavirales

Family: Inophagoviridae

Genus: inophagovirus

Species: Inophagovirus bacterii

What makes FV a valuable model in molecular biology?

  1. These viruses are among the smallest viruses known, so that it is feasible to define all of the virus functions at the molecular level and to consider the interaction of the various functions: the “systems” aspect of molecular biology. 
  2. The small genome size makes it relatively easy to study the mechanics, enzymology, and regulation of DNA replication and recombination of FV. 
  3. The purified virions themselves are a plentiful source of homogeneous stable deoxyribonucleoprotein for physical studies. This type of bacterial virus is the only bacterial virus which has been crystallized in a form suitable for structure analysis with X-ray diffraction techniques. Since bacterial viruses are particularly convenient for genetic studies, this is a good system in which to analyze the effect of mutation on virus structure and morphogenesis. Analysis of the structure of the virion will provide information which may increase our understanding of deoxyribonucleoprotein in general and nucleo-histone in particular. 
  4. Study of the unusual method of virus penetration and release may lead to useful knowledge about the nature of the bacterial cell envelope. In fact, the cell membrane itself may be a morphogenetic factor in the assembly of the virion. 
  5. Infection by FV alters the metabolism of the bacterial cell without killing the cell. One may hope to learn more about the bacterial cell by studying the nature of these perturbations. Since useful information has been obtained by measuring parameters of bacterial growth after shift of temperature or medium, so may useful information be obtained by measuring such parameters after FV infection. 
  6. Certain aspects of the interaction of virus and host suggest that this system may have some relevance as a model for infection of eukaryotic cells by animal viruses, in particular oncogenic viruses.

Influence of filamentous bacteriophage on bacterial pathogens

When other phages are pathogens on their bacterial host, filamentous phages infecting bacteria are episomal replicating phages that impose only a modest burden on the host. This phage, though it requires very little from its host, it contributes significantly to its virulence and evolutionary fitness.

For example: The major means by which cholera is caused by Vibrio cholerae is secretion of the cholera toxin: an oligomeric protein encoded by two genes that are carried by the temperate bacteriophage CTX. CTX phage can transmit cholera toxin genes from one V. cholerae strain to another, one form of horizontal gene transfer. CTX phage particles are secreted from the bacteria without lysis. The CTXφ genome is 6.9 kb long. When it infects V. cholerae cells, it integrates into specific sites on either of the two chromosomes. These sites often contain tandem arrays of integrated CTXφ prophage. Cholera toxin provides an advantage to the bacterial host, as it promotes profuse diarrhea in humans which results in the dissemination of the pathogen. 

Reference

Mai-Prochnow A, Hui JG, Kjelleberg S, Rakonjac J, McDougald D, Rice SA. ‘Big things in small packages: the genetics of filamentous phage and effects on fitness of their host’. FEMS Microbiol Rev. 2015;39(4):465-487. doi:10.1093/femsre/fuu007

Origin of life

Origin of life begins with the origin of the universe, which has been estimated to be about 20 billion years ago. The Big Bang Theory is the most accepted theory regarding the origin of the planet Earth and the existence of different life forms on it. According to this theory, the universe is a result of a massive explosion that occurred 20 billion years ago. Whether it is a hypothesis or a fact, a new universe was formed. The atmospheric condition after the explosion became more stable. The temperature reduced and gases like hydrogen and helium formed which led to the formation of galaxies of today. Later, after 10 billion years, the earth was formed which was covered by water vapor, methane, carbon dioxide, and ammonia. There was no atmosphere but only gases and moisture. The powerful rays of the sun stimulated and hastened evolution. By making and breaking bonds between gas molecules, the Earth came out with a new face. After millions of years i.e., once the Earth atmosphere was stabled, the first life on earth came into existence (around 4 million years ago). There began the story of the origin of life on earth.

 

Abiogenesis

It is the generation of life from non-living matter and not precisely known as ‘ Spontaneous generation’. This theory states that complex living organisms are generated from decaying organic matter. Example: Maggots spontaneously appeared in meat.

Francesco Redi, an Italian Physicist, Naturalist, Biologist, and Poet, was the first person to disprove the theory of spontaneous generation by demonstrating that maggots came from the eggs of the flies. Unfortunately, several scientists were still unconvinced by his experiment.

Redi_experiment

Louis Pasteur, a French Biologist, Microbiologist, and Chemist, performed a similar experiment and successfully convinced the scientific population that Abiogenesis was indeed impossible.

louis

Biogenesis

Omne vivum ex vivo’ similar to ‘Omnis cellula e cellula’  which means all cells come from cells. The theory states that living things come from other living things. The term biogenesis was coined by Henry Charlton Bastian.

Chemical Evolution

In the 1920s, Russian scientist Aleksander Oparin and English scientist J. B. S. Haldane both separately proposed what’s now called the Oparin-Haldane hypothesis: That life on Earth could have arisen step-by-step from non-living matter through a process of “gradual chemical evolution.” According to their theory, life evolved in the oceans during a period when the atmosphere was reducing – containing H2, H2O, NH3, CH4, and CO2, but no free O2. Organic compounds were synthesized non-biologically by ultraviolet light energy, which in the absence of an ozone shield would penetrate the upper layers of the ocean. Without free O2 to oxidize them, these organic molecules would be stable and would accumulate in a warm, dilute broth that has been nicknamed “Haldane soup” – as proposed by J.B.S Haldane in 1929.

image_thumb111

The first living organism would be little more than a few chemical reactions wrapped up in a film or membrane to keep them from being diluted and destroyed. These organelles would absorb chemicals, grow, divide, and obtain energy by fermenting the available organic molecules around them. Photosynthesis would arise eventually as an alternative energy source when natural foods ran short. The oxygen released by photosynthesis would have the side effect of screening out the ultraviolet radiation with an ozone layer in the upper atmosphere and eventually would turn the atmosphere from reducing to oxidizing. Free oxygen would lead to the evolution of respiration and to modern eukaryotic metabolism.

Miller-Urey Experiment

In 1953, Stanley Miller and Harold Urey did an experiment to test Oparin and Haldane’s ideas. The Miller–Urey experiment was a chemical experiment that simulated the conditions thought at the time (1952) to be present on the early Earth and tested the chemical origin of life under those conditions. It established that the conditions of the early earth were sufficient to produce organic molecules like amino acids as confirmed by chromatographic analysis. In the initial experiment, Miller identified five amino acids present in the solution: Glycine, α-alanine, and β-alanine were positively identified, while aspartic acid and α-aminobutyric acid (AABA) were less certain, due to the spots being faint. miller

In a 1996 interview, Stanley Miller recollected his lifelong experiments following his original work and stated: “Just turning on the spark in a basic prebiotic experiment will yield 11 out of 20 amino acids.”

Is the experiment still meaningful?

Scientists now believe that the atmosphere of early earth was quite different from that as proposed by A.I Oparin and J.B.S Haldane i.e. not reducing, and not rich in ammonia and methane. However, a variety of experiments done in the years since have shown that organic building blocks (especially amino acids) can form from inorganic precursors under a fairly wide range of conditions. From these experiments, it seems reasonable to imagine that at least some of life’s building blocks could have formed abiotically on early Earth. However, exactly how (and under what conditions) remains an open question.

 

Complex Biological molecules

The experiment explained the formation of simple bio-molecules but further evolution depends on the polymerization or condensation of these monomers to polymers.

In the 1950s, Sidney Fox and co-workers at the University of Miami produced synthetic peptides like products of molecular mass between 4000 and 10,000 Da by heating a dry mixture of amino acids at 150℃ and 180℃. He called these ‘Proteinoids’ or Thermal proteins. The sequences of these “thermal proteinoids” are not completely random, but show some internal order. These polymers display a limited catalytic activity, probably resulting from their charged side chains of acidic and basic amino acids.  These thermal proteinoids have another interesting property. If a hot proteinoid mixture is washed with water or salt solution, microspheres of a fairly uniform 20,000-Å diameter are formed, as in the photograph to the right.

Microspheres – These are small globules of the proteinoid polymer solution, enclosed by a semipermeable proteinoid film with some of the physical properties of simple cell membranes. Microspheres shrink and swell in salt solutions of different concentrations. They will grow at the expense of dissolved proteinoid material and have been observed to bud like yeast cells to produce “daughter” microspheres. They can be induced to fission by MgCl2 or by a pH change. The enclosing film is a double layer resembling those found in soap films and artificial and natural membranes.

Fox hypothesized that proteinoid material first polymerized on hot, dry volcanic cinder cones, and then was leached into the oceans by rain to form microspheres, which then could have become the early segregated chemical systems that eventually led to protocells. But the weakness of this theory stems from the need for the high concentration of dry amino acids that didn’t fit into the concept of Haldane’s soup.

In Russia, Oparin proposed that Coacervates may be the intermediates between loose molecules and a living system.

Coacervate – is the aggregation of colloidal particles in the liquid phase that persists in the form of tiny droplets. They are capable of exchanging substances with the environment, increasing in size, and selectively concentrating compounds within them.

The spontaneous self-assembly of macromolecules into coacervates and microspheres indicates that the occurrence of similar structures under primitive conditions would give rise to a more organized membrane-bound structure containing molecules, protocells.

 

 

Membranes defined the first cell

Oparin has suggested an evolutionary scheme for protocells or “protobionts” along the lines suggested by his coacervate experiments. He proposes that in lakes or ponds with appreciable concentrations of polymerized material, coacervate droplets would be formed naturally by wave action. In general, the composition of these droplets would differ from that of the bulk solution. These “microenvironments” in time could develop into enclosed systems of chemical reactions that absorb high-energy compounds from their surroundings to perform protective reactions or other necessary syntheses.

Cell_origin_model Continue reading “Origin of life”

Marshall Warren Nirenberg

marshall
Marshall W Nirenberg

Marshall W Nirenberg is an American Biochemist and Geneticist. He shared a Nobel Prize in Physiology and Medicine (1968) with Har Gobind Khorana and Robert W Holly for “Breaking the genetic code” and describing how it operates in protein synthesis. Working independently, Khorana had mastered the synthesis of nucleic acids, and Holley had discovered the exact chemical structure of transfer-RNA.

 

After the discovery of DNA, the biologists all over the world were still wondering how the protein is created from the linear DNA and the precise role played by RNA in the cell. Marshall Nirenberg earned a Ph.D. in biological chemistry from the University of Michigan with a dissertation on the mechanism of sugar uptake in tumor cells. His interest in genetics was piqued by the interdisciplinary classes he took as a part of his preparations to develop an independent study. In 1959, He teamed up with Heinrich J. Matthaei at the National Institutes of Health and began to study the steps that relate DNA, RNA, and Proteins; much to his biochemist colleagues’ dismay who called it professional suicide as he was up against the best scientist making a breakthrough in the field of genetics with no formal training in molecular genetics.

 

EXPERIMENT WITH SYNTHETIC RNA

The experiment was initiated with a study of linear DNA (A, T, G, C) and RNA (A, U, G, C).

  1. Marshall created a synthetic RNA sequence composed only of Uracil (found only in RNA, not DNA).
  2. They created a stable cell-free extract using E.coli which is found widely in the human gut by closely following the work of Alfred Tissieres. A cell-free extract has DNA, RNA, Ribosomes, and other necessary machinery for Protein synthesis. The cell-free extract was also a controlled environment where only the desired RNA added to it would be able to synthesis the amino acid.
  3. They created 20 samples to test their work.
  4. Then added 1 type of radioactively labeled amino acid and 19 un-labelled Amino acids to the sample; varying the labeled Amino Acid in each sample.
  5. He observed that the sample with the radioactive protein was the sample that used the synthetic RNA. At that time the number of bases per codon had not been determined. So they continued to repeat the steps with synthetic RNA containing only Adenine and cysteine. Later, Using the poly U Experiment as a model, it was determined that the RNA was read as codons containing only three bases and this helped them crack the genetic code.
  6. Synthetic codon UUU – Phenylalanine and CCC – Proline; AAA – Lysine (failed because polylysine was soluble in trichloroacetic acid unlike other proteins).

This was the first step to decipher Genetic code and the first demonstration of the role of RNA as a messenger. In August 1961, at the International Congress of Biochemistry in Moscow, Nirenberg presented the poly-U experiments – first to a small group, but then at Francis Crick‘s urging, again to about a thousand attendees. The work was very enthusiastically received, and Nirenberg became famous overnight. The experiment ushered in a furious race to fully crack the genetic code.

In 1964 Nirenberg and Philip Leder, a postdoctoral fellow at NIH, discovered a way to determine the sequence of the letters in each triplet word for amino acids. By 1966 Nirenberg had deciphered the 64 RNA three-letter code words (codons) for all 20 amino acids. The language of DNA was now understood and the code could be expressed in a chart. Nirenberg’s main competition was the esteemed biochemist and Nobel laureate Severo Ochoa, who had many more people. But many at the NIH pitched in to help Nirenberg, aware that it might lead to the first Nobel prize by an intramural NIH scientist. DeWitt Stetten Jr., the NIH director who first hired Nirenberg, called this period of collaboration “NIH’s finest hour.”

 

ETHICAL CONCERNS

Nirenberg received worldwide appreciation for his work in molecular genetics.  When Deciphering the genetic code raised ethical concerns about the potential for genetic engineering. Nirenberg addressed these concerns in a famous editorial in Science entitled “Will Society Be Prepared?” in August 1967, noting “When a man becomes capable of instructing his own cells, he must refrain from doing so until he has sufficient wisdom to use this knowledge for the benefit of mankind…. [D]ecisions concerning the application of this knowledge must ultimately be made by society, and only an informed society can make such decisions wisely.” Nirenberg contended that the impulse to exploit molecular genetics could only be kept in check by sobriety and caution. When asked several decades later if society has acted “wisely” regarding genetic engineering, Nirenberg answered, “Absolutely!”

 

TRANSITION INTO NEUROBIOLOGY

As the race to decipher the genetic code came to a close in 1965, Nirenberg sought out new scientific puzzles. Many minds were still trying to unravel the mysteries of protein synthesis, but Nirenberg’s mind was on another mystery–that of the mind itself. From 1965 to 1969 Nirenberg turned his attention and his laboratory over to the field of neurobiology.  His transition into Neurobiology may seem peculiar at first but makes sense when information processing is considered. Nirenberg noticed the similarities between the two biological systems that stored information; a system that processes information by receiving it, storing it, and then relaying it: the DNA- RNA-protein system, which processes heritable, genetic information; and the brain, which processes sensory, emotional, and cognitive information.

Nirenberg studied the neural code by employing the conceptual and experimental approaches to science that proved so successful for him in his work on the genetic code. Nirenberg considered the various facets of the neural code for more than a year, but these reflections never made it into a published form. Nirenberg presented his ideas at Howard University in a 1969 lecture titled “Genetic Versus Neural Information Processing Systems”, but he was only willing to endorse the thoughts as “speculations”. While Nirenberg’s interest in the similarities between the genetic and the neural code never evolved past the conceptual stage, these ideas helped to spark his curiosity in how the brain and the nervous system develop.

 

FROM NEUROBIOLOGY TO HOMEOBOX GENES

After reading a paper by Michael Levine of Oxford University in 1987, Nirenberg found the perfect opportunity to bring his experience in genetics and neurobiology together. Since homeobox genes influence the process by which hereditary information is converted into physical characteristics during development, Nirenberg recognized that understanding their function could provide new avenues for research. He recalled that “Levine found a homeobox protein that was distributed quite remarkably in some neurons in the developing embryo and not in other neurons… [Homeobox genes are] an important class of genes and to find them quite specifically distributed in specific sets of neurons was quite a remarkable observation. At that time, there were seventeen homeobox genes that were known, that had been found in Drosophila, and it was a burgeoning field of study.” The relationship between homeobox genes and neural development presented an ideal opportunity for new discoveries.

Until his death in 2010, Dr. Nirenberg continued his research by using advanced digital scanning technology to study the genetic development of neural networks in the brains of Drosophila embryos.

References

https://profiles.nlm.nih.gov/spotlight/jj

https://www.acs.org/content/acs/en/education/whatischemistry/landmarks/geneticcode.html#poly-u-experiment

“RNA CODEWORDS AND PROTEIN SYNTHESIS, III. ON THE NUCLEOTIDE SEQUENCE OF A CYSTEINE AND A LEUCINE RNA CODEWORD ” -BY PHILIP LEDER AND MARSHALL W. NIRENBERG, NATIONAL HEART INSTITUTE, NATIONAL INSTITUTES OF HEALTH Communicated by Richard B. Roberts, October 1, 1964

Generation of code for my AIIMS 2020 Application.

Hello!

AIIMSAll India Institute Of Medical Sciences has courses not just on MBBS and Nursing but also on MSc Anatomy, Biochemistry, Biophysics, Physiology, Pharmacology, Reproductive Biology and Biotechnology. It came as a surprise to me that AIIMS offered these courses let alone know that there was a highly competitive exam for the same. The course I’m interested in is Clinical Embryology, and AIIMS happens to be one of the best colleges in India offering it. So naturally I started collecting information on the application deadlines, procedures, etc. All the websites made the application filling process fairly easy and it was easy until I reached the part where I was to generate the code to move further on my application status.

Now, This step would have been easy if there were any instructions on the website on how to generate it. But… Nope. The universe was bent on stressing me on this cause one fine night when I open to login to check the status of my application, I was shown this page

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Instead of the usual this

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The part where I needed an RUC to login nearly gave me a heart attack. I struggled for an hour trying to figure out how and where I was supposed to find the RUC. Every help website ever only said, “Go here, Click that and There you go!”. Now if only it were that simple.

After an hour as I was losing hope with the site and contemplating emailing the Administrative office, I decided to mess around on the site one last time and THAT was my saving grace. Any of you looking to know HOW the RUC is generated, Please pay attention.

Step 1 – Go to the login Page

Here, Instead of freaking out, Click on “Proceed to Basic Registration (PG Courses)”

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Step 2

You have reached the familiar log in page where you login with your ID, Password and captcha. Log in in your details.

Screenshot (19).png

Step 3 – Generation of code

Click on that link to choose your course for which you want to generate the code. The option is common for MSc Nursing, Biotechnology and other MSc courses.

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Once you have generated the code, print the page for further reference.

You will need the code to log in and complete your application, payment and place of exam selection.

Good luck with the Exam!

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