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If I examine a dead cell, can I be sure that it has not a nucleus?
And what about the other organelles?
If you're looking at cause of death by standard apoptosis, to initiate the process, cytochrome c will be released from the mitochondria, bind with apaf-1, and undergo a structural change resulting in what looks like a biochemical ninja star (figure 4). This will activate caspase-9 which will activate other executor caspases, eventually creating an avalanche of protein-degrading enzymes that will leave the structure and organelles of the cell looking like paper mache. Regarding the nucleus, the complexes of enzymes meant to maintain dna integrity and structure will be degraded, the DNA's network influence on the cell will become largely inert, and the dna will be condensed and broken apart by nucleases. The membrane of the nucleus is full of proteins and ribosomes, and these will be degraded too. If you are looking to simply identify an inert nucleus by its skeleton, you may not have much success either - here are a few good images from a nice study showing the progressive destruction of a nuclear structure.
No, Really, mRNA Vaccines Are Not Going To Affect Your DNA
The short version: There is no plausible way that mRNA vaccines are going to alter your DNA. It would violate basically everything we know about cell biology.
Recently, I’ve gotten an influx of questions about how we can really be sure that mRNA vaccines will not affect our DNA. In my prior post on the subject I wrote:
Another concern raised has been the idea that mRNA can somehow alter the host’s genome. That would actually be super cool and be huge for gene therapy (and I could finally give myself the giant bat wings I’ve always wanted) but this is not so. This is ordinarily impossible except if there is also a reverse transcriptase enzyme present that produces DNA from the RNA template, which is how retroviruses work. There is no such risk with any mRNA vaccine candidate. mRNA vaccines act entirely within the cytosol of the cell- they do not go near the nucleus where all the DNA is. That’s actually a major advantage of RNA-based vaccines over DNA ones.
I gave this response in large part because I felt that the detailed discussion of reverse transcription, nuclear trafficking, the endocytic pathway, and the other 11 or so advanced cell biology topics that I would have to invoke to give this a rigorous answer was too complex to be of benefit to the average person wanting to know simply whether or not this is possible. However, I had a flurry of questions about “what ifs” relating to retroviruses or hepadnaviruses (hepatitis B), and I can grant that this response doesn’t address that, so here I will attempt to answer that as explicitly and with minimal complexity as I am capable.
To simplify the discussion so as to avoid having to explain the phases of phospholipid bilayers and the molecular composition of the lipid nanoparticle as it relates to stability (discussed in 1, 2, 3, 4), I will ask readers take for granted that mRNA vaccines are endocytosed and liberated (and this) into the cytoplasm of the cell.
Firstly, for mRNA to affect your DNA, at a minimum we need to establish that it would need to gain access to the DNA in question. There are two subcellular compartments where this can be accomplished. The first is the nucleus, so let’s start with a discussion of the trafficking of cargo in the nucleus. The nucleus of the cell is an isolated compartment with pore complexes (NPCs) that impose limits on the size of the particles that can freely enter. RNA is readily transported out as transcription occurs within the nucleus but the ribosomes required to produce proteins are in the cytosol or on the rough endoplasmic reticulum. This process is mediated by several accessory proteins which you can see on the left. Note however that there isn’t any physiological circumstance in which one might need RNA from the cytosol to be transported back to the nucleus. RNA is synthesized within the nucleus. Viruses which have a nuclear phase in their replication cycle have to have various tricks to be able to allow their RNA payload to enter. Though RNA is not readily transported into cells, proteins can be. This occurs via a network of proteins called importins (see figure 5–23C above). Proteins containing an amino acid sequence called the nuclear localization sequence (NLS there are 2 common ones) are able to bind the importins, which can then transport them across the nuclear pore complex as shown on the left. RNA viruses often have replication cycles that do not require access to the nucleus, but there are some exceptions. Influenza viruses for example are RNA viruses that have their genomes associated with ribonucleoproteins, and these ribonucleoproteins express nuclear localization signals that facilitates the entry of their genomic RNA into the nucleus. mRNA vaccines, on the other hand, are not associated with any proteins. Once inside the cytosol, the mRNA is naked and exposed to the harsh environment of ribosomes and exonucleases which destroy the mRNA in a matter of hours (at most). There is no conceivable mechanism by which mRNA can spontaneously be trafficked into the nucleus. Being made of nucleotides, it cannot contain a nuclear localization sequence.
The other relevant compartment would be the mitochondrion. Mitochondria are actually vestigial bacteria with their own genomes, and it’s thought that billions of years ago an ancient bacteria tried to consume the ancestor of the mitochondria but lacked the machinery to actually do the digesting and the two established a symbiotic relationship. Since that instance, the mitochondria have been an essential feature of our cell’s biologies. This allowed the mitochondria to develop an extremely reduced genome containing only 37 genes (most of the genes relevant to mitochondrial function are still in the nucleus). Mitochondria have their own ribosomes and even their own genetic code (sort of). There is also a specialized process for the clearance of diseased mitochondria called mitophagy, which is the subject of many excellent reviews e.g. this, this, and this.
The collective conclusion from our understanding of these biological process is that a naked mRNA in the cytosol has no potential to end up in a cellular compartment that contains our own DNA means that, irrespective of the presence or absence of other factors, there is no chance of harm to the DNA from the mRNA vaccine. But still people wanted to ask me about reverse transcriptases so let’s discuss those.
The process of going from RNA to DNA (the exact opposite of what the central dogma of molecular biology dictates) is known as reverse transcription, and it is carried out with an enzyme called a reverse transcriptase (which are a really interesting group of enzymes). In general, reverse transcription is performed by a few different genetic entities: retroviruses, hepadnaviruses, telomeres, and retrotransposons. These are worth defining.
- Retroviruses are viruses who have an RNA genome, from which they create a DNA copy through reverse transcription that then integrates into the cell of the host (by which I mean, literally inserts itself into the host cell’s genome and becomes a permanent part of it, in the form of a sequence called a provirus). The proviral sequence itself can then be transcribed in the host cell to produce viral proteins and particles that can go on to spread to the next cell. The most famous retrovirus is HIV-1.
- Hepadnaviruses are DNA viruses which have gapped genomes (there is one complete DNA strand and another partial DNA strand which is linked to a pregenomic RNA), and unlike retroviruses, do not integrate into the genome of the host cell they infect. The most famous example is Hepatitis B virus, for which multiple effective vaccines exist.
- Telomeres are structures present at the ends of human chromosomes which are maintained by a protein complex called telomerase that uses a reverse transcriptase called TERT to maintain them. The reasons this is necessary are discussed in Figure 9–12 on the left. They are about 5–15 kilobases long normally, and shortening results in arrest of cell growth and replication (senescence), or can even trigger cell death by apoptosis.
- Retrotransposons are actually the most abundant component of our genome. The human genome contains about 21,000–27,000 genes (the number you get depends on how precisely you define a gene and which source you consult), which span 40–48 million base pairs, but this accounts for only about 1.5% of the 3.2 billion total base pairs. Retrotransposons account for about 2 billion base pairs. There are several kinds of retroelements, which are worth discussing further:
- SINEs (short interspersed nuclear elements) which encode short transcripts like tRNAs, and cannot function without a LINE-encoded protein.
- LINEs (long interspersed nuclear elements) which encode a reverse transcriptase formed from the ORF1 and pol genes which can copy itself and other LINE and SINE elements into other regions of the genome.
- About 5–8% of the human genome is also composed of human endogenous retroviruses, HERVs, which also fall into the category of retrotransposons, more specifically LTR (long terminal repeats) retrotransposons (more on this shortly). HERVs contain 3 genes: gag (“group antigens,” which encodes a polyprotein that is cleaved into the structural proteins of the resultant retrovirus), pol (the reverse transcriptase needed for the virus to replicate), and env (envelope, which encodes the protein that gives the viral particles their shape).
- More broadly, the term retroelement refers to genetic sequences that have moved from one region of the genome to another via reverse transcription, and these include retrotransposons, and processed pseudogenes. Processed pseudogenes refer to the sequences of processed mRNA that lack introns that have been inserted via reverse transcription (we know they had to be inserted into the genome via reverse transcription in large part because they lack introns). They are incapable of producing any gene product.
- The only retrotransposons that can move through the genome (literally copy their DNA to new sites where it was not initially present) are the LINEs and SINEs, and of these, only a few are able to accomplish this. HERVs are stuck where they are, and processed pseudogenes are as well.
Telomerases evolved as a solution to the end replication problem. Nascent (new) DNA strands are synthesized with a leading strand and a lagging strand because the DNA polymerases have a very restricted directionality in that they must travel 3’ to 5’ with respect to the template strand. This creates a problem because the DNA is oriented antiparallel (the strands are parallel but one strand is oriented in the direction opposite to the other), so to make both strands at the same time, a single DNA polymerase would have to manage to concurrently travel what would be a Sisyphean length for it in opposite directions (imaging trying to simultaneously run east and west for 10 miles). To deal with this dilemma, one of the strands is synthesized as a leading strand with a polymerase traveling down the strand uninterrupted for many nucleotides (formally the term is “processively”), and a lagging strand in which fragments of DNA (called Okazaki fragments) are consistently generated that are complementary to the other strand that get ligated (fused) together. The dilemma is that because our chromosomes are not circular, there will always be a missing fragment once we reach the 3’ end of the chromosome, and thus each replication cycle of the DNA will cause the size of the genome to shrink, eventually with the potential to hit genes important for biological function. This is known as the end replication problem.
To your left you see a telomerase complex with its favorite telomerase RNA. The ends of the chromosome contain structures called telomeres, which are repetitive, short, palindromic sequences that get copied many times, until the gaps between the strands are filled for a length of about 5000 to 15000 nucleotides. The production of telomeric DNA occurs via a large protein complex called telomerase, which makes use of TERT (telomerase reverse transcriptase), a reverse transcriptase that takes an RNA template to make the palindromic DNA sequences. Importantly, cells eventually do lose their telomerase function, which is thought to represent a safeguard against cancer (cells that express telomerase at high levels can continue dividing- and therefore accumulating mutations, some of which might be harmful- indefinitely, and thus in most cells after about 50 divisions, the cells will cease to divide telomerase is notably expressed at high levels in stem cells). In practice, mice which have no functional telomerase will have substantial chromosomal shortening within 3 generations and by the fourth generation end up unable to reproduce. Here now, I have to shatter all your preconceived ideas about how RNA works. When speaking about DNA and RNA, we have a tendency to use the term “strand” which conjures up an image of a thread. The thread is relatively linear, it may curve, but the structure is relatively boring. This is a reasonable approximation of most DNA, as DNA can have basically one of 3 structures called A, B, and Z (there are rarer ones though such as i-motifs, and DNAzymes can do weird things). RNA on the other hand, is a much freer spirit when it comes to structure. RNA folds into complex shapes with all sorts of structural motifs in a manner not dissimilar to proteins, in that the structure of a protein relates directly to its function. What this means is: specific RNAs do specific things depending on how they fold, which depends on their sequence. To your right you can see a detailed diagram of telomerase RNA bound to the telomerase complex. That curvy thing with bars like a ladder and bubbles is the telomerase RNA. TERT, the reverse transcriptase of telomerase, binds the telomerase RNA at the core domain and a region called CR4/CR5. I won’t get into the other components of the complex but you can read in detail about how it works here and here. Immediately below the diagram to your right you can see how telomerase works to extend the 3’ cap of the chromosome through the aid of a repetitive, palindromic RNA sequence: CAAUCCCAAUC, which reproduces on the DNA a repeating “GGGTTA” to form a telomere with a length of about 5,000–15,000 nucleotides. For this to work, a bunch of things have to go right but solely for TERT to be able to recognize telomerase RNA there needs to be: the template for reverse transcription (the palindromic sequence CCCAAU), the pseudoknot domain (the core domain in the diagram), a stem–loop that interacts with TERT (CR4/CR5), and a 3′ element required for RNA stability (CR7). This is a very specific set of constraints and mRNA vaccines would have to be designed to have them (see image above for standard organization of an mRNA vaccine). Ribosomes also have intrinsic mRNA helicase activity that destroys such structures so that they can be read and processed for the synthesis of a protein. Additionally, the mature human telomerase RNA is 451 nucleotides in length. The mRNA from these vaccines is approximately 1200–1300 nucleotides long. It is too large to function as a telomerase RNA in humans (there are some animals which have telomerase RNAs of that size but we are not one of them) and given how precisely the telomerase RNA must fold, it is unlikely to assume the required structures for recognition and binding of the telomerase.
I initially considered discussing in detail the reverse transcriptases of the hepadnaviruses (i.e. hepatitis B) and retroviruses (i.e. HIV and HERVs) but the discussion quickly became inaccessible. Suffice it to say, reverse transcriptases are not capable of picking up any random RNA and generating a DNA from it. They require an RNA sequence to prime the reaction. For retroviruses, there is a tRNA that is stolen from the host cell and packaged into the virion. Furthermore, in the retroviruses, reverse transcription occurs within a nucleocapsid which allows dNTPs (the building blocks of DNA) in, but cannot permit something as large as an entirely separate RNA molecule spanning about 1200 bases. Reverse transcription by hepadnaviruses is similar in principle, requiring a pregenomic RNA segment that is chemically linked to the DNA of the hepadnavirus. Reverse transcription will not occur spontaneously with just any RNA. Even for RT-PCR reactions, the reaction requires the binding of an oligodeoxythymidine sequence to the polyA tail of the mRNA in question. Additionally, there’s a secondary requirement here to be able to “change” the DNA of the host: to actually manipulate it in some way. In the case of the hepadnaviruses this doesn’t really happen. The hepadnavirus genome gets into the nucleus and forms a covalently closed circular DNA with its own associated histones, essentially a small, separate chromosome. It doesn’t touch the host’s DNA. In the case of retroviruses, the DNA gets integrated into the host chromosome, and the effect depends on where it gets integrated. HIV for example has a strong bias for inserting itself into genes, which can be problematic if, for example, the gene produces a protein important for the maintenance of genome integrity (which could lead to cancer if left unchecked). The development of cancer from such a process however, cannot simply occur without many other things going wrong, like for instance a massive death of helper T cells that critically impairs the ability of the immune system to conduct surveillance of cells for evidence of malignancy and kill them, as happens in HIV. Now, should we choose to ignore everything thus far established about how cell biology works, including the need for a primer to initiate the reverse transcriptase reaction, and allow that a retrovirus readily permits integration of the resultant spike protein RBD or entire spike protein gene into the host, this would simply lead to the insertion of a gene that may be able to make the spike protein or just the RBD (depending on where it inserted and whether it could recruit transcriptional machinery), which would only serve to present to the immune system a foreign protein that it has been primed to respond against, and subsequently kill the cell. Also, as they are being delivered by an intramuscular injection, the cells in question would most likely be a muscle cell (which you can lose without loss of any eloquent function) or a dendritic cell (which you could also lose without any loss of significant immunological function).
To conclude, and I really hope this ends it:
Cell Nucleus Structure
A cell nucleus is surrounded by a double membrane, known as the nuclear envelope. This membrane covers and protects the DNA from physical and chemical damage. In doing so, the membrane creates a separate environment to process the DNA in. The outer membrane is in contact with the cytoplasm, and connects in some places to the endoplasmic reticulum. The inner membrane connects to the nuclear lamina. This nuclear framework inside the cell nucleus helps it maintain its shape. There is also evidence that this scaffolding of proteins helps form a matrix to transport and distribute products within and out of the nucleus. Nuclear pores create passages through the nuclear membrane, and allow products of the cell nucleus to enter the cytoplasm or endoplasmic reticulum. The pores also allow some specific macromolecules and chemicals from the cytoplasm to pass back into the cell nucleus. These macromolecules are needed to synthesize DNA and RNA, and are needed for the creation of new proteins and macromolecules within the cell nucleus. In a stained nucleus, a dark spot can be seen. This spot is the nucleolus. Within the nucleolus, the several different parts of ribosomes are produced and exported. These structures can be seen in the following image.
While the cell nuclei of plants and animals differ in subtle ways, their main purpose and general activities remain the same. The cell nucleus is responsible for producing two main products to support the efforts of each cell. The first, messenger RNA, or mRNA, is the product of transposing a gene coding for a specific protein from the DNA structure to the RNA structure. This shorter mRNA strand can exit the nucleus and enter the cytoplasm. When a ribosome picks up this mRNA, it will translate this mRNA into the language of proteins and create a long strand of amino acids. This strand will then be folded into a functional protein, which may serve one of a thousand different roles. Examples of the differences between plant and animal cell nuclei can be seen below.
Genetic Material: Properties and Evidence | Cell Biology
A living cell is composed of several inorganic and organic components. Among them, one will obviously act as genetic material responsi­ble for controlling hereditary characters. Iden­tification of this genetic material remained con­troversial for a long time.
Now if any component is to be genetic mate­rial, it must fulfil a number of basic properties:
i. Genotypic function or replication or auto-synthesis.
ii. Phenotype function or expression or hetero catalysis.
The first property states that the genetic material must be capable of storing hereditary information and replicate with high efficiency in successive cell generations forming the basis for transmission of hereditary characteristics it controls.
The second property is a fundamental one involved in gene action which through a series of chemical reactions results in the ultimate expression of the characteristics within the organism. The third property states that the genetic material does undergo occasional heritable changes called mutation.
It creates variations among the organisms besides recombination. Variations, on the other hand, are the important source of raw materials for evolution.
Besides the above-mentioned important properties of genetic material, the gene substance also shows the following additional properties:
a. To control the innumerable diversities in the characteristics of organism available in nature, the genetic material must show a very wide diversity in form.
b. Since phenotype character is the final ex­pression of a chain of reactions initiated at the gene level, obviously the genetic mate­rial must be a chemically unique entity.
Before 1900 several biologists proposed that hereditary material must be in the chromosome of the cell nucleus. In 1903, Sutton and Bovery postulated that genes were located in chro­mosome. In eukaryotic system, chromosomes are made of mainly protein and nucleic acid (DNA and RNA) and one of them obviously constitute the genetic material.
But which one would be the most suitable candidate for the position of genetic material remained contro­versial for a long time.
Early molecular biol­ogists have assigned the properties of genetic material to the chromosomal proteins because they found nucleic acid too simple to carry genetic information. Besides this, nucleic acid is made of monotonous chemical components like sugar, phosphate and base.
On the other hand, protein showed a highly complex struc­ture composed of a variety of amino acid. So a wide range of diversities is possible in protein structure to fulfill the diversity required in the genetic material for controlling the countless diversities in the characteristic of organism.
The controversy about the assignment of gene substance either to chromosomal protein or to nucleic acid, existed up to 1950 when finally it was unanimously accepted that the genetic information resides in the nucleic acids rather than in proteins.
More specifically, several elegant experiments showed that DNA is the genetic material of most microorganisms and higher organisms. Later on, RNA was found to be the genetic material of some viruses where DNA is absent.
Evidence of Genetic Material:
The concept that DNA or RNA is the genetic material of most organisms has been developed and supported by following evidence:
I. Direct Evidence:
(a) Transformation in Pneumococcus:
The first direct evidence showing that the genetic material is DNA rather than protein or RNA was published by O. T. Avery, C. M. Macleod and M. McCarthy in 1944. They discovered that the substance of the cell respon­sible for the phenomenon of transformation in the bacterium Diplococcus pneumoniae is DNA.
Transformation is the mode of exchange or transfer of genetic information (recombination) from one strain of bacterium to another strain of bacterium without involving any direct con­tact between them. The process of transforma­tion was first discovered by Frederick Griffith in 1928.
This was called as Griffith’s ef­fect. The experiment of Griffith demonstrated transformation but he could not recognise the transforming principle.
Different strains of Pneumococci shows the genetic variability that can be recognised by existence of different phenotypes. Griffith ini­tially conducted his experiment on two strains of pneumococci which were phenotypically dis­tinct.
When they are grown artificially on nutrient agar medium, they form two types of colonies:
The cells of strains forming smooth (S) colonies have a smooth glittering appearance due to presence of strain-specific polysaccharides (a polymer of glucose and glucuronic acid) capsule. Such strains are able to produce pneumonia and are called virulent.
The polysaccharide capsule is required for virulence since it protects the bacterial cell against phagocytosis by leucocytes. But the cells of stain lack this capsule and they produce dull rough (R) colonies. Such stains are termed as avirulent since they cannot produce pneumonia.
Therefore smooth (S) and rough (R) phenotypic characteristic are directly related to the presence or absence of the capsule and this trait is known to be genetically determined.
Both S and R forms occur in several subtypes and are designated as S I, S II, S III, etc. and R I, R II, R III, etc., respectively, on the basis of antigen properties of the polysaccha­rides present in their capsule. This property ultimately depends on the genotype of the cell.
The experiments of Griffith (Fig. 12.1) are briefly described below stepwise on the basis of his observation:
Griffith injected live cells of the viru­lent type III S into mice, all the mice died due to pneumonia and live type III S cells were recovered from the serum of blood of the dead bodies of mice.
When live cells of the avirulent type II R were injected into a separate group of mice, none of the mice died and live type II R were isolated from the serum of blood of all mice.
When mice were injected with heat-killed virulent type III S pneumococci alone, again none of the mice died, showing that virulence is lost after heat-killing.
When mice were injected heat-killed type III S pneumococci (virulent when alive) plus live type II R pneumococci (non-virulent), some of the mice died due to pneumonia pneumococci cells isolated from the dead mice were of the type III S.
Since it is known that non-capsulate type R cells can mutate back to virulent encapsulated type S cells, the resulting cell will be type II S, not type III S. Thus the transformation of non- virulent type II R cells to virulent type III S cells cannot be explained by mutation, rather, some component of the dead type III S cells (the “transforming principle”) must convert living type II R cells to type III S.
This leads to a change in the trait of cells and helps to bring some new characters in the transformed cell. Hence the transforming principle must contain some genetic material.
(b) Transforming Principle is DNA:
Avery, Macleod and McCarthy experimentally proved that the transforming principle was DNA. They showed that if DNA extract from type IIS pneumococci was mixed with type IIR pneumococci in vitro, some of the pneumococci were transformed to type III S.
But DNA extract from type III S may be contaminated with a few molecules of proteins, RNA and this contaminating protein and RNA may be responsible for transformation from type II R to type III S. So Avery, Macleod and McCarthy demonstrated the most definitive experiment using bacterial culture system and specific enzymes that degrade DNA, RNA and protein.
In separate experiments (Fig. 12.2) DNA extract from type III S cells was treated with:
i. DNAse which degrades DNA.
ii. RNAse which degrades RNA.
iii. trypsin, a protease which degrades protein and then tested the treated DNA extract for its ability to transform type II R pneu­mococci to type III S.
iv. They observed that the treatment with RNAse or trypsin had no effect on the abil­ity of the DNA extract to transform type II R to type III S. But DNAse treatment destroyed the transforming activity of the DNA preparation and II R cells were not transformed into III S cells. This established beyond any doubt that DNA is the transforming principle.
But these findings of Avery and co-workers was not able to explain the molecular mechanism of transformation. So some biologists were unable to appreciate the significance of these findings and they were hesitant in accepting them as an incontrovertible evidence for DNA being the genetic material.
(c) The Experiment of Hershey-Chase:
Another direct evidence indicating that DNA is the genetic material was demonstrated by A. D. Hershey and M. Chase in 1952. They first studied the life cycle of T2 bacteriophages of Escherichia coli. T2 bacteriophages are composed of hexagonal box-like head coat and tail made of protein. The DNA is packed inside the proteinaceous head coat.
Bacteriophages are acellular and do not contain cytoplasm, organelles and nucleus. The DNA is present in high pure form and is not associated with RNA and protein. Bacteriophage are obligate parasite since they can reproduce only within bacterium using as host cell.
Hershey and Chase showed that, during the reproduction of bacteriophages, the DNA of the phage entered the host cell whereas most of the protein head and tail portion remained absorbed on the outside of the cell. Hence it is strongly implied that the genetic information necessary for viral reproduction was present in DNA.
DNA con­tains phosphorus (P) but no sulphur (S), while proteins of head and tail contains sulphur (S) but no phosphorus.
Hershey and Chase were able to specifically label the phage DNA by its growth in a medium containing the radioactive isotope of phosphorus, i.e., 32 P in place of normal phosphorus. Similarly, in another group of phage, the protein coats were labelled by growth in a medium containing radioactive sulphur 35 S in place of normal sulphur.
E. coli cells were then infected with 32 P labelled T2 bacteriophage and, after being allowed 10 minutes for infection, they were agitated in a blender which sheared off the phase coats. The phase coats and the infected cells were then separated by centrifugation (Fig. 12.3).
Radioactivity was then measured of the sed­iment and in phage coat suspension. Most of the radioactivity was found in the cells. When the same experiment was done using phage with 35 S-labelled protein coat, most of the radioactivity was found in the suspension of phage coats very little entered the host cells.
Since phage reproduction (both DNA synthesis) occurs inside the infected cells, and, since only the phage DNA enters the host cell, the DNA—not the protein—must carry the genetic information. As a result of the findings of Hershey and Chase led to the universal acceptance of DNA as the genetic material.
(d) Bacterial Conjugation:
Another direct evidence for DNA as the genetic material comes from the phenomenon of conju­gation of bacteria. Conjugation was discovered by J. Lederberg and E. I.
Tatum in 1946. During conjugation DNA is transferred from a donor bacterial cell to a recipient bacterial cell through conjugation tube that forms between them. The donor cell—also called male—contains a F factor or fertility factor whereas recipient cells—or fe­male cells, lack F factor, i.e., F – cell (Fig. 12.4).
In male, the F factor can exist in two dif­ferent states:
(2) Integrated state (Fig. 12.4) where the F factor is inserted with main DNA and thus the male become Hfr male (Fig. 12.5).
The F factor is a mini-circular DNA molecule. Beadle and Tatum observed that when a F + male E. coli cell conjugated with a F – female E. coli cell, an unidirectional transfer of F + factor of male cell to F – or female cell took place, so that the latter was covered into a F + or male strain.
The F factor is actually a fragment of DNA molecule that replicates during transfer. Thus mixing a population of F + or Hfr cells with a population of F + cells results in virtually all the cells in the new population becoming F + or Hfr (Fig. 12.6).
Ii. Indirect Evidence:
The fact that DNA is the genetic material of higher organisms has also been supported by some indirect evidences:
The genetic substance should have a fixed location within the cell. If it has no fixed location, then the genes are not able to function properly. It is known that the DNA, as a gene sub­stance, is always located primarily within the chromosome in the nucleus of the eukaryotic cell.
The specific location of DNA can be stud­ied in situ by the Feulgen reaction—which is re­garded as the most specific one for DNA. Feul­gen staining stains chromosome magenta colour against the clear cytoplasmic background. This technique has shown that DNA entirely remains restricted to the chromosome and it forms the major component of chromosomes.
Various macromolecules present within the cell are continuously being anabolised and catabolized. But this is not desirable for a genetic substance containing valuable hereditary information. If it happens, the genetic function will be lost. Of all the macro- molecules in the cell, DNA is the metabolically stable.
(c) Sensitivity to Mutagens:
Mutation is an important characteristic feature of the genetic material. The agents capable of inducing mutation are called mutagens. Different types of radiation (UV-ray, X-ray, y-ray) and a variety of chemical compounds acts as mutagens. When the cells of an organism are treated with mutagens, they cause a change in the structure of gene.
Since genes are DNA segments, the gene mutation include changes in the number and arrangement of nucleotide. Sometimes muta­tion causes the breaks in the DNA molecule. The changes in the DNA structure ultimately reflect the changes of the organism’s hereditary character. Therefore sensitivity of DNA to mutagens is an indirect evidence for DNA being the genetical materials.
One of the striking features of the genetic ma­terial is the correlation between DNA content and the number of chromosome sets. Various quantitative assay methods have shown that diploid cells contain twice as much DNA as do haploid cells of the same species (Table 12.1).
Similarly, tetraploid and octaploid cells con­tains four times and eight times DNA as com­pared with DNA content of the haploid cells. Even the DNA content of sperm cells shows a correlation with the same or different tissues of different organisms (Table 12.2).
Thus the parallelism of behaviour in DNA and chromosome indirectly indicates that DNA is the genetic material.
(e) RNA as Genetic Material:
The genome of viruses may be DNA or RNA. Most of the plant viruses have RNA as their hereditary material. Fraenkel-Conrat (1957) conducted experiments on tobacco mosaic virus (TMV) to demonstrate that in some viruses RNA acts as genetic material.
TMV is a small virus composed of a single molecule of spring-like RNA encapsulated in a cylindrical protein coat. Different strains of TMV can be identified on the basis of differences in the chemical composition of their protein coats. By using the appropriate chemical treatments, proteins and RNA of RNV can be separated.
Moreover, these processes are reversible by missing the protein and RNA under appropriate conditions—reconstitution will occur yielding complete infective TMV particles. Fraenkel-Conrat and Singer took two differ­ent strains of TMV and separated the RNAs from protein coats, reconstituted hybrid viruses by mixing the proteins of one strain with the RNA of the second strain, and vice versa.
When the hybrid or reconstituted viruses were rubbed into live tobacco leaves, the progeny viruses produced were always found to be phenotypically and genotypically identical to the parental type from where the RNA had been isolated (Fig. 12.7). Thus the genetic information of TMV is stored in the RNA and not in the protein.
Cell size and numbers
An adult human body contains about 60 trillion (60 x 10 12 ) cells. Most of these cells are so small that a microscope is necessary to see them. The small size of cells fulfills a distinct purpose in the functioning of the body. If cells were larger, many of the processes that cells perform could not occur efficiently. Such a large cell has a large volume, which is much larger than surface area. Since nutrients enter the cell via the surface, only a relatively small amount of nutrients could enter the cell. Put another way, a cell would likely starve, since the nutrient supply could not keep pace with the nutrient demand of the cell. In a small cell, the correspondingly smaller volume means that the available nutrient level is usually sufficient to support cell survivial and growth.
Another reason for the small size of cells is that control of cellular processes is easier in a small cell than in a large cell. Cells are dynamic, living things. Cells transport substances from one place to another, reproduce themselves, and produce various enzymes and chemicals for export to the extracellular environment. All of these activities are accomplished under the direction of the nucleus, the control center of the cell. If the nucleus had to control a large cell, then this direction might break down. Substances transported from one place to another would have to traverse great distances to reach their destinations reproduction of a large cell would be an extremely complicated endeavor and products for export would not be as efficiently produced. Smaller cells, because of their more manageable size, are much more efficiently controlled than larger cells.
Neural Stem Cells
There has long been a consensus in the scientific community that once neurons have died, there is no way to replace them. Other cells of the body, like skin cells and blood cells are replaced when their stem cells divide to give new skin and blood cells respectively. Neurons, it was thought, did not have its special pool of stem cells.
In the 1980s, Fernando Nottebohm at Rockefeller University questioned this notion, and found stem cells in the adult brain of songbirds. These stem cells are called neural stem cells. Since then, we&rsquove found neural stem cells in rats, mice, monkeys, and even humans.
Neural stem cells aren&rsquot everywhere in the brain. They are only found in two nooks &ndash the anterior sub-ventricular zone (SVZ) in the forebrain, and the subgranular zone in the hippocampus. These stem cells cannot form long distance connection, so major regeneration after a severe injury is not possible.
This is seen as a partial explanation for the occasional recovery in patients with serious brain injuries. This process takes time, and is not a constant part of brain maintenance, so it is still important to avoid any damage to the brain and spinal cord at all costs.
These neural stem cells or NSCs are primarily active while the brain is initially developing as an infant, but many of them cease to function as we age. Some of the cells remain active throughout our lives, differentiating into different types of cells, including astrocytes, oligodendrocytes and neurons.
Neural stem cells are very intriguing for researchers, who are now isolating these cells and trying to determine their mechanism for turning their replication functions on and off. Researchers could potentially apply their findings to healing or treating the brain, especially when it comes to neurodegenerative diseases like Alzheimer&rsquos or dementia, in ways we thought were impossible.
The nervous system is the seat of consciousness and control in the human body understanding how it works, and what makes nerve cells different from other cells, provides yet another glimpse into the incredible complexity of our existence!
80 Comparing Meiosis and Mitosis
Mitosis and meiosis, which are both forms of division of the nucleus in eukaryotic cells, share some similarities, but also exhibit distinct differences that lead to their very different outcomes. Mitosis is a single nuclear division that results in two nuclei, usually partitioned into two new cells. The nuclei resulting from a mitotic division are genetically identical to the original. They have the same number of sets of chromosomes: one in the case of haploid cells, and two in the case of diploid cells. On the other hand, meiosis is two nuclear divisions that result in four nuclei, usually partitioned into four new cells. The nuclei resulting from meiosis are never genetically identical, and they contain one chromosome set only—this is half the number of the original cell, which was diploid.
The differences in the outcomes of meiosis and mitosis occur because of differences in the behavior of the chromosomes during each process. Most of these differences in the processes occur in meiosis I, which is a very different nuclear division than mitosis. In meiosis I, the homologous chromosome pairs become associated with each other, are bound together, experience crossover between homologous chromosomes, and line up in the center of the cell with spindle fibers from opposite spindle poles attached to each centromere. All of these events occur only in meiosis I, never in mitosis.
Homologous chromosomes move to opposite poles during meiosis I so the number of sets of chromosomes in each nucleus-to-be is reduced from two to one. For this reason, meiosis I is referred to as a reduction division. There is no such reduction in mitosis.
Meiosis II is much more similar to a mitotic division. In this case, duplicated chromosomes line up at the center of the cell. One sister chromatid is pulled to one pole and the other sister chromatid is pulled to the other pole. If it were not for the fact that there had been crossovers, the two products of each meiosis II division would be identical as in mitosis instead, they are different because there has always been at least one crossover per chromosome. Meiosis II is not a reduction division because, although there are fewer copies of the genome in the resulting cells, there is still one set of chromosomes, as there was at the end of meiosis I.
Cells produced by mitosis will function in different parts of the body as a part of growth or replacing dead or damaged cells. Mitosis typically occurs in somatic cells, but they may be involved in asexual reproduction in some organisms. Cells produced by meiosis will only participate in sexual reproduction.
Figure 1 Meiosis and mitosis are both preceded by one round of DNA replication however, meiosis includes two nuclear divisions. The four daughter cells resulting from meiosis are haploid and genetically distinct. The daughter cells resulting from mitosis are diploid and identical to the parent cell.
How does foreign DNA affect or utilize a cells nucleus?
Biology With the DNA based J&J vaccine coming out for COVID-19, it got me thinking how foreign DNA affects or utilizes the cells nucleus?
What goes on in a cell nucleus and how does it affect our own DNA, when there is a foreign invader?
How do cells protect itself from foreign DNA? Or can it even protect itself? In the case of the vaccine, the DNA is a good guy, but in other cases, it can be a bad guy?
So how does this whole cell nucleus work with foreign DNA?
First of all, DNA can't enter a cell or nucleus on its own. In many cases (e.g. vaccines), a virus is used that injects a strand of DNA into a cell.
As far as I know, once a strand of DNA is inside a nucleus, what happens next depends on what it codes for. If it codes for a virus, this virus can be produced within the cell. This happens when a person is infected by a virus. In the case of the J&J vaccine, the DNA only codes for one protein of Covid19. Then, this one protein is synthesised, but the person won't get sick. Then, if the strand of DNA is not integrated into the genome of the cell, the strand is eventually degraded.
Lastly, the immune system can recognise cells infected by viruses and can kill these cells.
So let me see if I understand this correctly.
Assuming DNA gets inside the nucleus, any set of DNA can hijack the nucleus of a cell and generate whatever structures it needs to create. In the case of our own DNA, stuff for our own cells, but in the case COVID-19, the spike protein.
What happens to virus DNA after it makes more viruses?
What happens to the vaccine DNA after it makes the spike protein?
Can foreign DNA live in the nucleus forever?
Okay, let me try to answer your question, but bear with me a moment, because I need to tell you about a lot of cell biology first.
First, let's talk about DNA, RNA, and Protein.
You probably already know of DNA. DNA, deoxyribonucleic acid, is a long, double helix-shaped string of A's, T's, C's and G's. On an atomic and molecular level, DNA is just a long string of bits. But from an informational perspective, when we study how the DNA gets used by a cell, it becomes obvious that the information it contains is organized into many discrete blocks, where each block is read together as a single unit to make a single product or a limited number of variants. We call these blocks of information Genes.
The product of these Genes are Proteins. Proteins are long strings of amino acids, and unlike strings of DNA which are made of 4 different letters, there are 20 different amino acid letters possible for each spot on a protein string. These strings of proteins spontaneously fold into a specific shape based on the amino acids along the chain, like a rope studded with magnets snarling up onto itself. The shape of the protein carries out a specific function, playing its part in the complex biochemical ballet of life. Proteins are made by molecular ticker-tape reader machines called Ribosomes. In order for the DNA of a gene to be made into protein, DNA gets read and transcribed into Messenger RNA (mRNA), a different format which uses A, U, C, G instead of DNA's ATCG. mRNA is a transient messenger molecule, a short-lived copy which feeds into the Ribosomes that create the amino acid chains that turn into proteins, but the mRNA itself naturally gets destroyed and recycled by the cell eventually.
DNA makes RNA, RNA makes Protein. This rule is what biologists call the Central Dogma, coined in 1958 by Francis Crick, one of the co-discoverers of the structure of DNA. It turned out to be a regrettably bad name for many reasons, one of which is that exceptions to this turned out to be plentiful, but it is broadly true almost all of the time.
So, that's enough about DNA, let's talk about how your immune system works.
When an unpleasant microbe gets into your body, intent on feasting upon your cells and reproducing inside you, your body has two lines of defense. The first line of defense is your innate immune system: it doesn't care what's attacking, it reacts to anything foreign, any signs of damage, and to alarm signals produced by cells that are under attack. Your innate immune system does a lot of things: it ramps up inflammation (fever), raising the temperature to stress the attacker, produces noxious and destructive compounds, and rallies an army of carnivorous defenders to clean up dead & dying cells, eat the attacking microbes, and harvest pieces of their proteins for analysis.
Your innate immune system buys time for your adaptive immune system. Specialized immune cells take the harvested protein pieces and get to work making antibodies: little Y-shaped proteins that circulate in your blood and specifically identify and stick to those attacker's proteins, tagging the microbes en-masse and flagging them for extermination. Antibodies often stick in a way that gums up the proteins critical for the microbe's life cycle, too. Once the active infection of microbes is wiped out, our immune system keeps those antibodies on file, so that production of those antibodies can ramp up right away if the microbe's ever encountered again and future attacks will get squished before they can do any noticeable damage at all.
Vaccination is our way of hijacking this process to grant us immunity while skipping the potentially deadly or disabling active infection necessary to get there naturally. The first vaccines, for smallpox, polio, and so on, were what we call live-attenuated vaccines. For vaccinating people against smallpox, we infected people with cowpox, a much more benign cousin of the smallpox virus which was close enough that immunity to cowpox gets smallpox squished at the front gates. For others, we used weakened or crippled but otherwise still infectious microbes: they wouldn't cause severe sickness or death, but they would do enough damage and trigger our immune system enough to establish antibodies, which protects us against the fully deadly virus in the wild.
Today, we've mostly forgotten how much vaccination changed the world and our relationship with disease. For the first time, we had the ability to give ourselves permanent protection from entire classes of terrifying diseases that have been killing and crippling people for longer than history remembers. And many of these were viral diseases for which the previous generation's miracle wonderdrug, antibiotics, could do nothing against.
Even though they revolutionized the world, these first-generation vaccines had obvious downsides. The two most glaring were that, first, taking a live, deadly microbe and weakening it is an inherently inconsistent process, and second, even a weakened virus could sporadically cause a bad case of the disease, especially in people whose immune systems were already weak.
So we developed vaccines where we deliver completely inactive and dead microbes, and then after that we made vaccines which only deliver the specific critical protein or protein fragment of the microbe that the microbe needs to attack us.
The newly approved Johnson and Johnson vaccine is part of a new generation of vaccines that include the mRNA-based Pfizer and Moderna vaccines. The new idea is this: instead of delivering the microbe's critical protein itself, we deliver the DNA or RNA blueprint for how to make one, your own cells make a bunch of it, your innate and adaptive immune systems react to it, and your adaptive immune system eventually learns to kill it on sight.
The J&J vaccine uses a DNA fragment that contains the gene for the covid spike protein, encased inside an adenovirus-like particle. Like a real live adenovirus, these virions are able to attach to cells and inject their DNA contents into a cell. Unlike a real live adenovirus, they don't contain genes for making more viral DNA molecules and virus particles, they instead contain the gene for the covid spike protein.
Once the DNA's inside your cell, it gets read just like any other piece of DNA, and mRNA messenger transcripts are created. Those mRNAs go into ribosomes, and spike proteins get made.
If the invading genes were actually a real foreign invader, like, for example, the SARS-Cov-2 virus itself, what would happen would be much the same. The covid virus's shell fuses with the receptor on the surface of our cell like a key opening a lock, and the viral RNA genome gets dumped into the cell. It gets read and the cell starts manufacturing viral spikes, capsids, and more viral RNA genomes, and these parts self-assemble into viral particles until the cell exhausts all its energy and resources and dies, blowing apart as all the new viral particles inside get scattered into the rest of your body.
- Personal protective equipment (sterile gloves, laboratory coat, safety visor)
- Waterbath set to appropriate temperature
- Microbiological safety cabinet at appropriate containment level
- CO2 incubator
- Inverted phase contrast microscope
- Pre-labelled flasks
- Bring adherent and semi-adherent cells into suspension using trypsin/EDTA as described previously and resuspend in a volume of fresh medium at least equivalent to the volume of trypsin. For cells that grow in clumps centrifuge and resuspend in a small volume and gently pipette to break up clumps.
- Under sterile conditions remove 100-200 μL of cell suspension.
- Add an equal volume of Trypan Blue (dilution factor =2) and mix by gentle pipetting.
- Clean the hemocytometer.
- Moisten the coverslip with water or exhaled breath. Slide the coverslip over the chamber back and forth using slight pressure until Newton’s refraction rings appear (Newton’s refraction rings are seen as rainbow-like rings under the coverslip).
- Fill both sides of the chamber with cell suspension (approximately 5-10 μL) and view under an inverted phase contrast microscope using x20 magnification.
- Count the number of viable (seen as bright cells) and non-viable cells (stained blue). Ideally >100 cells should be counted in order to increase the accuracy of the cell count (see notes below). Note the number of squares counted to obtain your count of >100.
- Calculate the concentration of viable and non-viable cells and the percentage of viable cells using the equations below.
Do dead cells always contain no nucleus? - Biology
|All cells, whether they are prokaryotic or eukaryotic, have some common features. These common features are:|
DNA, the genetic material contained in one or more chromosomes and located in a nonmembrane bound nucleoid region in prokaryotes and a membrane-bound nucleus in eukaryotes
Plasma membrane, a phospholipid bilayer with proteins that separates the cell from the surrounding environment and functions as a selective barrier for the import and export of materials
Cytoplasm, the rest of the material of the cell within the plasma membrane, excluding the nucleoid region or nucleus, that consists of a fluid portion called the cytosol and the organelles and other particulates suspended in it
1. The genetic material (DNA) is localized to a region called the nucleoid which has no surrounding membrane.
2. The cell contains large numbers of ribosomes that are used for protein synthesis.
3. At the periphery of the cell is the plasma membrane. In some prokaryotes the plasma membrane folds in to form structures called mesosomes, the function of which is not clearly understood.
4. Outside the plasma membrane of most prokaryotes is a fairly rigid wall which gives the organism its shape. The walls of bacteria consist of peptidoglycans. Sometimes there is also an outer capsule. Note that the cell wall of prokaryotes differs chemically from the eukaryotic cell wall of plant cells and of protists.