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Name of dsRNA (or dsDNA) where all strands are identical

Name of dsRNA (or dsDNA) where all strands are identical


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What is the name of dsRNA (or DNA) where all component strands are identical (i.e. where the complex consists of multiple copies of the same ssRNA)?

Example: 2 identical ssRNAs forming a dsRNA

C C G C G G C G G | | | | . | | | | G G C G G C G C C

Furthermore, is there a different name for it depending on the degree of complementarity (full Watson-Crick complementarity vs. full complementarity with non-Watson-Crick basepairs vs. lower complementarity due to bulges etc)?

My thoughts

You probably wouldn't say "RNA homon-mer" (for the example above "homo dimer/2-mer") since I assume that would be referring to a 2-nucleotide long ssRNA?

Can you use the terms dimer, trimer, tetramer, 5-mer, 6-mer etc in multiple ways; (a) referring to the binding of nucleotides to each other to form a polymer/n-mer of nucleotides i.e. polyribonucleic acid, and (b) to whole ssRNA or dsRNA binding to each other to form a "dimer"?

You also probably wouldn't refer to it as a "homo duplex" since that refers to something related to chromosomal crossover.


Such a nucleic acid sequence is described as a palindrome or palindromic.

Wikipedia has an entry for palindromic sequence in which it is defined as:

A palindromic sequence is a nucleic acid sequence on double-stranded DNA or RNA wherein reading 5' (five-prime) to 3' (three prime) forward on one strand matches the sequence reading 5' to 3' on the complementary strand with which it forms a double helix. This definition of palindrome thus depends on complementary strands being palindromic of each other.

It should be noted that this is a description (rather than a name) that is usually applied to segments of larger nucleic acid molecules or to synthetic constructs. I am not aware of a generic name for short dsRNA (or dsDNA) like the one in the question as they are not, to my knowledge, found in Nature and so there has been no reason to devise a name for them. Thus, there are no names for variants either. If you wish to write an article about such things you would need to invent your own terms, but keep well clear of '-mers' (see use of e.g. tetramer).

The sequence presented in the question does not conform to the Wikipedia definition, above, as it has a central mismatch and is therefore not a perfect double-helix. However, as the use of the term palindrome for nucleic acids is a somewhat cavalier extension of the general use of the English word palindrome (an example of which is “ABLE WAS I ERE I SAW ELBA”), I would regard a further minor modification of the Wikipedia author's definition as unexceptionable.


The specific sequence you have posted is not strictly a palindrome since the reverse complement of the top strand is not the same as the bottom strand. Although there is no central body making these definitions, a better term, in my opinion, would be inverted repeat, of which palindromes are a subset (inverted repeats with no intervening sequence). Of course, the final say would be its predominant usage in the literature (of which I am not overly familiar).

Furthermore, the specific sequence you have shown forms an internal loop, though there is no requirement that internal loops be formed from inverted repeats.


RNA virus

An RNA virus is a virus which has (ribonucleic acid) RNA as its genetic material. [1] The nucleic acid is usually single-stranded RNA (ssRNA) but it may be double-stranded RNA (dsRNA). [2] Notable human diseases caused by RNA viruses include the common cold, influenza, SARS, MERS, COVID-19, Dengue Virus, hepatitis C, hepatitis E, West Nile fever, Ebola virus disease, rabies, polio, mumps, and measles.

The International Committee on Taxonomy of Viruses (ICTV) classifies RNA viruses as those that belong to Group III, Group IV or Group V of the Baltimore classification system of classifying viruses and does not consider viruses with DNA intermediates in their life cycle as RNA viruses. [3] Viruses with RNA as their genetic material which also include DNA intermediates in their replication cycle are called retroviruses, and comprise Group VI of the Baltimore classification. Notable human retroviruses include HIV-1 and HIV-2, the cause of the disease AIDS.

All RNA viruses encoding an RNA-directed RNA polymerase, known as of May 2020, form a monophyletic group now known as the realm Riboviria. [4] The majority of such RNA viruses fall into the kingdom Orthornavirae and the rest have a positioning not yet defined. [5] The realm does not contain all RNA viruses: Deltavirus, Asunviroidae, and Pospiviroidae are taxa of RNA viruses that have been mistakenly included in 2019, [a] but corrected in 2020. [6]


Contents

Bacteriophages occur abundantly in the biosphere, with different genomes, and lifestyles. Phages are classified by the International Committee on Taxonomy of Viruses (ICTV) according to morphology and nucleic acid.

ICTV classification of prokaryotic (bacterial and archaeal) viruses [1]
Order Family Morphology Nucleic acid Examples
Belfryvirales Turriviridae Enveloped, isometric Linear dsDNA
Caudovirales Ackermannviridae Nonenveloped, contractile tail Linear dsDNA
Myoviridae Nonenveloped, contractile tail Linear dsDNA T4, Mu, P1, P2
Siphoviridae Nonenveloped, noncontractile tail (long) Linear dsDNA λ, T5, HK97, N15
Podoviridae Nonenveloped, noncontractile tail (short) Linear dsDNA T7, T3, Φ29, P22
Halopanivirales Sphaerolipoviridae Enveloped, isometric Linear dsDNA
Haloruvirales Pleolipoviridae Enveloped, pleomorphic Circular ssDNA, circular dsDNA, or linear dsDNA
Kalamavirales Tectiviridae Nonenveloped, isometric Linear dsDNA
Levivirales Leviviridae Nonenveloped, isometric Linear ssRNA MS2, Qβ
Ligamenvirales Lipothrixviridae Enveloped, rod-shaped Linear dsDNA Acidianus filamentous virus 1
Rudiviridae Nonenveloped, rod-shaped Linear dsDNA Sulfolobus islandicus rod-shaped virus 1
Mindivirales Cystoviridae Enveloped, spherical Segmented dsRNA Φ6
Petitvirales Microviridae Nonenveloped, isometric Circular ssDNA ΦX174
Tubulavirales Inoviridae Nonenveloped, filamentous Circular ssDNA M13
Vinavirales Corticoviridae Nonenveloped, isometric Circular dsDNA PM2
Unassigned Ampullaviridae Enveloped, bottle-shaped Linear dsDNA
Bicaudaviridae Nonenveloped, lemon-shaped Circular dsDNA
Clavaviridae Nonenveloped, rod-shaped Circular dsDNA
Finnlakeviridae dsDNA FLiP [10]
Fuselloviridae Nonenveloped, lemon-shaped Circular dsDNA
Globuloviridae Enveloped, isometric Linear dsDNA
Guttaviridae Nonenveloped, ovoid Circular dsDNA
Plasmaviridae Enveloped, pleomorphic Circular dsDNA
Portogloboviridae Enveloped, isometric Circular dsDNA
Spiraviridae Nonnveloped, rod-shaped Circular ssDNA
Tristromaviridae Enveloped, rod-shaped Linear dsDNA

It has been suggested that members of Picobirnaviridae infect bacteria, but not mammals. [11]

Another proposed family is "Autolykiviridae" (dsDNA). [12]

In 1896, Ernest Hanbury Hankin reported that something in the waters of the Ganges and Yamuna rivers in India had a marked antibacterial action against cholera and it could pass through a very fine porcelain filter. [13] In 1915, British bacteriologist Frederick Twort, superintendent of the Brown Institution of London, discovered a small agent that infected and killed bacteria. He believed the agent must be one of the following:

  1. a stage in the life cycle of the bacteria
  2. an enzyme produced by the bacteria themselves, or
  3. a virus that grew on and destroyed the bacteria [14]

Twort's research was interrupted by the onset of World War I, as well as a shortage of funding and the discoveries of antibiotics.

Independently, French-Canadian microbiologist Félix d'Hérelle, working at the Pasteur Institute in Paris, announced on 3 September 1917, that he had discovered "an invisible, antagonistic microbe of the dysentery bacillus". For d’Hérelle, there was no question as to the nature of his discovery: "In a flash I had understood: what caused my clear spots was in fact an invisible microbe… a virus parasitic on bacteria." [15] D'Hérelle called the virus a bacteriophage, a bacteria-eater (from the Greek phagein meaning "to devour"). He also recorded a dramatic account of a man suffering from dysentery who was restored to good health by the bacteriophages. [16] It was D'Herelle who conducted much research into bacteriophages and introduced the concept of phage therapy. [17]

More than a half a century later, in 1969, Max Delbrück, Alfred Hershey, and Salvador Luria were awarded the Nobel Prize in Physiology or Medicine for their discoveries of the replication of viruses and their genetic structure. [18] [ relevant? ]

Phage therapy Edit

Phages were discovered to be antibacterial agents and were used in the former Soviet Republic of Georgia (pioneered there by Giorgi Eliava with help from the co-discoverer of bacteriophages, Félix d'Herelle) during the 1920s and 1930s for treating bacterial infections. They had widespread use, including treatment of soldiers in the Red Army. However, they were abandoned for general use in the West for several reasons:

  • Antibiotics were discovered and marketed widely. They were easier to make, store, and to prescribe.
  • Medical trials of phages were carried out, but a basic lack of understanding raised questions about the validity of these trials. [19]
  • Publication of research in the Soviet Union was mainly in the Russian or Georgian languages and for many years, was not followed internationally.

The use of phages has continued since the end of the Cold War in Russia, [20] Georgia and elsewhere in Central and Eastern Europe. The first regulated, randomized, double-blind clinical trial was reported in the Journal of Wound Care in June 2009, which evaluated the safety and efficacy of a bacteriophage cocktail to treat infected venous ulcers of the leg in human patients. [21] The FDA approved the study as a Phase I clinical trial. The study's results demonstrated the safety of therapeutic application of bacteriophages, but did not show efficacy. The authors explained that the use of certain chemicals that are part of standard wound care (e.g. lactoferrin or silver) may have interfered with bacteriophage viability. [21] Shortly after that, another controlled clinical trial in Western Europe (treatment of ear infections caused by Pseudomonas aeruginosa) was reported in the journal Clinical Otolaryngology in August 2009. [22] The study concludes that bacteriophage preparations were safe and effective for treatment of chronic ear infections in humans. Additionally, there have been numerous animal and other experimental clinical trials evaluating the efficacy of bacteriophages for various diseases, such as infected burns and wounds, and cystic fibrosis associated lung infections, among others. [22]

Meanwhile, bacteriophage researchers have been developing engineered viruses to overcome antibiotic resistance, and engineering the phage genes responsible for coding enzymes that degrade the biofilm matrix, phage structural proteins, and the enzymes responsible for lysis of the bacterial cell wall. [4] [5] [6] There have been results showing that T4 phages that are small in size and short-tailed, can be helpful in detecting E.coli in the human body. [23]

Therapeutic efficacy of a phage cocktail was evaluated in a mice model with nasal infection of multidrug-resistant (MDR) A. baumannii. Mice treated with the phage cocktail showed a 2.3-fold higher survival rate than those untreated in seven days post infection. [24] In 2017 a patient with a pancreas compromised by MDR A. baumannii was put on several antibiotics, despite this the patient's health continued to deteriorate during a four-month period. Without effective antibiotics the patient was subjected to phage therapy using a phage cocktail containing nine different phages that had been demonstrated to be effective against MDR A. baumannii. Once on this therapy the patient's downward clinical trajectory reversed, and returned to health. [25]

D'Herelle "quickly learned that bacteriophages are found wherever bacteria thrive: in sewers, in rivers that catch waste runoff from pipes, and in the stools of convalescent patients." [26] This includes rivers traditionally thought to have healing powers, including India's Ganges River. [27]

Other Edit

Food industry – Since 2006, the United States Food and Drug Administration (FDA) and United States Department of Agriculture (USDA) have approved several bacteriophage products. LMP-102 (Intralytix) was approved for treating ready-to-eat (RTE) poultry and meat products. In that same year, the FDA approved LISTEX (developed and produced by Micreos) using bacteriophages on cheese to kill Listeria monocytogenes bacteria, in order to give them generally recognized as safe (GRAS) status. [28] In July 2007, the same bacteriophage were approved for use on all food products. [29] In 2011 USDA confirmed that LISTEX is a clean label processing aid and is included in USDA. [30] Research in the field of food safety is continuing to see if lytic phages are a viable option to control other food-borne pathogens in various food products.

Dairy industry – Bacteriophages present in the environment can cause fermentation failures of cheese starter cultures. In order to avoid this, mixed-strain starter cultures and culture rotation regimes can be used. [31]

Diagnostics – In 2011, the FDA cleared the first bacteriophage-based product for in vitro diagnostic use. [32] The KeyPath MRSA/MSSA Blood Culture Test uses a cocktail of bacteriophage to detect Staphylococcus aureus in positive blood cultures and determine methicillin resistance or susceptibility. The test returns results in about five hours, compared to two to three days for standard microbial identification and susceptibility test methods. It was the first accelerated antibiotic-susceptibility test approved by the FDA. [33]

Counteracting bioweapons and toxins – Government agencies in the West have for several years been looking to Georgia and the former Soviet Union for help with exploiting phages for counteracting bioweapons and toxins, such as anthrax and botulism. [34] Developments are continuing among research groups in the U.S. Other uses include spray application in horticulture for protecting plants and vegetable produce from decay and the spread of bacterial disease. Other applications for bacteriophages are as biocides for environmental surfaces, e.g., in hospitals, and as preventative treatments for catheters and medical devices before use in clinical settings. The technology for phages to be applied to dry surfaces, e.g., uniforms, curtains, or even sutures for surgery now exists. Clinical trials reported in Clinical Otolaryngology [22] show success in veterinary treatment of pet dogs with otitis.

The SEPTIC bacterium sensing and identification method uses the ion emission and its dynamics during phage infection and offers high specificity and speed for detection. [35]

Phage display is a different use of phages involving a library of phages with a variable peptide linked to a surface protein. Each phage genome encodes the variant of the protein displayed on its surface (hence the name), providing a link between the peptide variant and its encoding gene. Variant phages from the library may be selected through their binding affinity to an immobilized molecule (e.g., botulism toxin) to neutralize it. The bound, selected phages can be multiplied by reinfecting a susceptible bacterial strain, thus allowing them to retrieve the peptides encoded in them for further study. [36]

Antimicrobial drug discovery – Phage proteins often have antimicrobial activity and may serve as leads for peptidomimetics, i.e. drugs that mimic peptides. [37] Phage-ligand technology makes use of phage proteins for various applications, such as binding of bacteria and bacterial components (e.g. endotoxin) and lysis of bacteria. [38]

Basic research – Bacteriophages are important model organisms for studying principles of evolution and ecology. [39]

Bacteriophages may have a lytic cycle or a lysogenic cycle. With lytic phages such as the T4 phage, bacterial cells are broken open (lysed) and destroyed after immediate replication of the virion. As soon as the cell is destroyed, the phage progeny can find new hosts to infect. Lytic phages are more suitable for phage therapy. Some lytic phages undergo a phenomenon known as lysis inhibition, where completed phage progeny will not immediately lyse out of the cell if extracellular phage concentrations are high. This mechanism is not identical to that of temperate phage going dormant and usually, is temporary.

In contrast, the lysogenic cycle does not result in immediate lysing of the host cell. Those phages able to undergo lysogeny are known as temperate phages. Their viral genome will integrate with host DNA and replicate along with it, relatively harmlessly, or may even become established as a plasmid. The virus remains dormant until host conditions deteriorate, perhaps due to depletion of nutrients, then, the endogenous phages (known as prophages) become active. At this point they initiate the reproductive cycle, resulting in lysis of the host cell. As the lysogenic cycle allows the host cell to continue to survive and reproduce, the virus is replicated in all offspring of the cell. An example of a bacteriophage known to follow the lysogenic cycle and the lytic cycle is the phage lambda of E. coli. [40]

Sometimes prophages may provide benefits to the host bacterium while they are dormant by adding new functions to the bacterial genome, in a phenomenon called lysogenic conversion. Examples are the conversion of harmless strains of Corynebacterium diphtheriae or Vibrio cholerae by bacteriophages, to highly virulent ones that cause diphtheria or cholera, respectively. [41] [42] Strategies to combat certain bacterial infections by targeting these toxin-encoding prophages have been proposed. [43]

Attachment and penetration Edit

Bacterial cells are protected by a cell wall of polysaccharides, which are important virulence factors protecting bacterial cells against both immune host defenses and antibiotics. [44] To enter a host cell, bacteriophages bind to specific receptors on the surface of bacteria, including lipopolysaccharides, teichoic acids, proteins, or even flagella. This specificity means a bacteriophage can infect only certain bacteria bearing receptors to which they can bind, which in turn, determines the phage's host range. Polysaccharide-degrading enzymes, like endolysins are virion-associated proteins to enzymatically degrade the capsular outer layer of their hosts, at the initial step of a tightly programmed phage infection process. Host growth conditions also influence the ability of the phage to attach and invade them. [45] As phage virions do not move independently, they must rely on random encounters with the correct receptors when in solution, such as blood, lymphatic circulation, irrigation, soil water, etc.

Myovirus bacteriophages use a hypodermic syringe-like motion to inject their genetic material into the cell. After contacting the appropriate receptor, the tail fibers flex to bring the base plate closer to the surface of the cell. This is known as reversible binding. Once attached completely, irreversible binding is initiated and the tail contracts, possibly with the help of ATP, present in the tail, [5] injecting genetic material through the bacterial membrane. [46] The injection is accomplished through a sort of bending motion in the shaft by going to the side, contracting closer to the cell and pushing back up. Podoviruses lack an elongated tail sheath like that of a myovirus, so instead, they use their small, tooth-like tail fibers enzymatically to degrade a portion of the cell membrane before inserting their genetic material.

Synthesis of proteins and nucleic acid Edit

Within minutes, bacterial ribosomes start translating viral mRNA into protein. For RNA-based phages, RNA replicase is synthesized early in the process. Proteins modify the bacterial RNA polymerase so it preferentially transcribes viral mRNA. The host's normal synthesis of proteins and nucleic acids is disrupted, and it is forced to manufacture viral products instead. These products go on to become part of new virions within the cell, helper proteins that contribute to the assemblage of new virions, or proteins involved in cell lysis. In 1972, Walter Fiers (University of Ghent, Belgium) was the first to establish the complete nucleotide sequence of a gene and in 1976, of the viral genome of bacteriophage MS2. [47] Some dsDNA bacteriophages encode ribosomal proteins, which are thought to modulate protein translation during phage infection. [48]

Virion assembly Edit

In the case of the T4 phage, the construction of new virus particles involves the assistance of helper proteins that act catalytically during phage morphogenesis. [49] The base plates are assembled first, with the tails being built upon them afterward. The head capsids, constructed separately, will spontaneously assemble with the tails. During assembly of the phage T4 virion, the morphogenetic proteins encoded by the phage genes interact with each other in a characteristic sequence. Maintaining an appropriate balance in the amounts of each of these proteins produced during viral infection appears to be critical for normal phage T4 morphogenesis. [50] The DNA is packed efficiently within the heads. The whole process takes about 15 minutes.

Release of virions Edit

Phages may be released via cell lysis, by extrusion, or, in a few cases, by budding. Lysis, by tailed phages, is achieved by an enzyme called endolysin, which attacks and breaks down the cell wall peptidoglycan. An altogether different phage type, the filamentous phage, make the host cell continually secrete new virus particles. Released virions are described as free, and, unless defective, are capable of infecting a new bacterium. Budding is associated with certain Mycoplasma phages. In contrast to virion release, phages displaying a lysogenic cycle do not kill the host but, rather, become long-term residents as prophage.

Communication Edit

Research in 2017 revealed that the bacteriophage Φ3T makes a short viral protein that signals other bacteriophages to lie dormant instead of killing the host bacterium. Arbitrium is the name given to this protein by the researchers who discovered it. [51] [52]

Given the millions of different phages in the environment, phage genomes come in a variety of forms and sizes. RNA phage such as MS2 have the smallest genomes, of only a few kilobases. However, some DNA phage such as T4 may have large genomes with hundreds of genes the size and shape of the capsid varies along with the size of the genome. [53] The largest bacteriophage genomes reach a size of 735 kb. [54]

Bacteriophage genomes can be highly mosaic, i.e. the genome of many phage species appear to be composed of numerous individual modules. These modules may be found in other phage species in different arrangements. Mycobacteriophages, bacteriophages with mycobacterial hosts, have provided excellent examples of this mosaicism. In these mycobacteriophages, genetic assortment may be the result of repeated instances of site-specific recombination and illegitimate recombination (the result of phage genome acquisition of bacterial host genetic sequences). [55] Evolutionary mechanisms shaping the genomes of bacterial viruses vary between different families and depend upon the type of the nucleic acid, characteristics of the virion structure, as well as the mode of the viral life cycle. [56]

The field of systems biology investigates the complex networks of interactions within an organism, usually using computational tools and modeling. [57] For example, a phage genome that enters into a bacterial host cell may express hundreds of phage proteins which will affect the expression of numerous host gene or the host's metabolism. All of these complex interactions can be described and simulated in computer models. [57]

For instance, infection of Pseudomonas aeruginosa by the temperate phage PaP3 changed the expression of 38% (2160/5633) of its host's genes. Many of these effects are probably indirect, hence the challenge becomes to identify the direct interactions among bacteria and phage. [58]

Several attempts have been made to map protein–protein interactions among phage and their host. For instance, bacteriophage lambda was found to interact with its host, E. coli, by dozens of interactions. Again, the significance of many of these interactions remains unclear, but these studies suggest that there most likely are several key interactions and many indirect interactions whose role remains uncharacterized. [59]

Metagenomics has allowed the in-water detection of bacteriophages that was not possible previously. [60]

Also, bacteriophages have been used in hydrological tracing and modelling in river systems, especially where surface water and groundwater interactions occur. The use of phages is preferred to the more conventional dye marker because they are significantly less absorbed when passing through ground waters and they are readily detected at very low concentrations. [61] Non-polluted water may contain approximately 2×10 8 bacteriophages per ml. [62]

Bacteriophages are thought to contribute extensively to horizontal gene transfer in natural environments, principally via transduction, but also via transformation. [63] Metagenomics-based studies also have revealed that viromes from a variety of environments harbor antibiotic-resistance genes, including those that could confer multidrug resistance. [64]

Although phage do not infect humans, there are countless phage particles in the human body, given our extensive microbiome. Our phage population has been called the human phageome, including the "healthy gut phageome" (HGP) and the "diseased human phageome" (DHP). [65] The active phageome of a healthy human (i.e., actively replicating as opposed to nonreplicating, integrated prophage) has been estimated to comprise dozens to thousands of different viruses. [66] The role of bacteriophage in human health remains poorly understood, but preliminary studies indicate that common bacteriophage are found on average in 62% of healthy individuals, while their prevalence was reduced by 42% and 54% on average in patients with ulcerative colitis (UC) and Crohn’s disease (CD). [65] This indicates that phage may be required to control the population of both normal and pathogenic bacteria in the human gut microbiome.

Among the countless phage, only a few have been studied in detail, including some historically important phage that were discovered in the early days of microbial genetics. These, especially the T-phage, helped to discover important principles of gene structure and function.


The Baltimore Classification System

The most commonly and currently used system of virus classification was first developed by Nobel Prize-winning biologist David Baltimore in the early 1970s. In addition to the differences in morphology and genetics mentioned above, the Baltimore classification scheme groups viruses according to how the mRNA is produced during the replicative cycle of the virus.

Group I viruses contain double-stranded DNA (dsDNA) as their genome. Their mRNA is produced by transcription in much the same way as with cellular DNA, using the enzymes of the host cell.

Group II viruses have single-stranded DNA (ssDNA) as their genome. They convert their single-stranded genomes into a dsDNA intermediate before transcription to mRNA can occur.

Group III viruses use dsRNA as their genome. The strands separate, and one of them is used as a template for the generation of mRNA using the RNA-dependent RNA polymerase encoded by the virus.

Group IV viruses have ssRNA as their genome with a positive polarity, which means that the genomic RNA can serve directly as mRNA. Intermediates of dsRNA, called replicative intermediates, are made in the process of copying the genomic RNA. Multiple, full-length RNA strands of negative polarity (complementary to the positive-stranded genomic RNA) are formed from these intermediates, which may then serve as templates for the production of RNA with positive polarity, including both full-length genomic RNA and shorter viral mRNAs.

Group V viruses contain ssRNA genomes with a negative polarity, meaning that their sequence is complementary to the mRNA. As with Group IV viruses, dsRNA intermediates are used to make copies of the genome and produce mRNA. In this case, the negative-stranded genome can be converted directly to mRNA. Additionally, full-length positive RNA strands are made to serve as templates for the production of the negative-stranded genome.

Group VI viruses have diploid (two copies) ssRNA genomes that must be converted, using the enzyme reverse transcriptase, to dsDNA the dsDNA is then transported to the nucleus of the host cell and inserted into the host genome. Then, mRNA can be produced by transcription of the viral DNA that was integrated into the host genome.

Group VII viruses have partial dsDNA genomes and make ssRNA intermediates that act as mRNA, but are also converted back into dsDNA genomes by reverse transcriptase, necessary for genome replication.

The characteristics of each group in the Baltimore classification are summarized in the table above with examples of each group.


Acknowledgments

We thank K. Finkelstein for experimental assistance. This research was supported by the NSF, the NIH and the NBTC at Cornell. This work is based upon research conducted at the Cornell High Energy Synchrotron Source (CHESS) which is supported by the NSF and the NIH/NIGMS. We also made use of the Cornell NanoScale Facility, a member of the National Nanotechnology Infrastructure Network, which is supported by the NSF. and CNF at Cornell. CHESS is supported by NSF and NIH/NIGMS.


FAQs siRNA/RNAi/dsRNA

Each siRNA molecule is synthesized as two ssRNA oligonucleotides between 11 and 40 nucleotide in length, which are then aligned to form a duplex. The siRNA duplex typically has 2 DNA nucleotides overhanging at each 3’-end. For more information about RNA synthesis, read our RNA FAQs.

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What is the difference between synthesis scale and final yield?

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Please note that OD260 values are a measure of total nucleotides´ optical density. Hence, neither purity nor amount of ordered substance are transparently reflected. For simplification and exemplification reasons look at the following:

1 OD of the 20mer 5´CAT CGT ATT CGA TGC TAC GT 3´

translates into approximately 5 nmol.

1 OD of the 40mer 5´CAT CGT ATT CGA TGC TAC GT CAT CGT ATT CGA TGC TAC GT 3´

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When I place an order for a larger number of oligos, sometimes some of them are delayed – why is this?

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metabion's RNA oligonucleotides are delivered deprotected and purified – ready to use. They are RNAse free, but as RNA is highly susceptible to degradation by exogenous RNAses introduced during handling, it is essential that you conduct all handling steps under sterile, RNAse free conditions. Never handle RNA without wearing gloves. RNAse free reagents, barrier pipette tips and tubes should be used!

For more information, please read FAQ: How should siRNA oligonucleotides be stored?

How should siRNA/ RNAi/ duplex RNA oligonucleotides be stored?

RNA duplexes are significantly more resistant to nucleases than a single strand RNA oligonucleotide. However, maintaining sterile, RNAse free conditions is always recommended as a precaution. Dried pellets are stable at room temperature for 2-4 weeks, but should be placed at -20°C or -80°C for long term storage. Under these conditions, dry RNA is stable for at least one year. If you want to store your RNA in solution re-suspend the delivered pellet in an RNAse-free solution buffered at pH 7.4 - 7.6 and store at -20°C or less. We recommend that RNAs are re-suspended at a convenient stock concentration and stored in small aliquots to avoid multiple freeze thaw cycles.

Are there guidelines that should be taken into account when designing siRNA/ RNAi/ duplex RNA?

You may want to consider the following:

  • Check the antisense oligo sequence for targeting specificity. The sequence should not complement any off-target mRNA. To do this, you can use metabions’ oligocalculator (link).
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  • Make sure your sequence is free of hairpins and self-complementary regions. These might affect both the synthesis and the hybridization of your oligo to the target.
  • More than six of the same consecutive bases (i.e. GGGGGGG) can be problematic and reduce final yields.

Why Mass-Check and when MALDI- or ESI-ToF?

Metabion is dedicated to reliably deliver high quality products. While every production step is performed in light of achieving best quality, the product is released only if it passes our final inspection. Mass Spectrometry has become the state-of-the-art technology for verifying the integrity of oligonucleotides, and metabion has been the first custom oligo house who introduced routine mass checks into its operations. Each and every oligo is characterized by either MALDI- or ESI-ToF and stringent release criteria are applied.

Mass Spectrometry allows for the most sensitive detection of low-level by-products/impurities such as

  • n-1/n-x oligos
  • Depurination
  • Incomplete Deprotection
  • Acrylonitrile adducts
  • High Salt Content Identification

Moreover, it is the fastest and most efficient way to identify potential product mix-ups.

We run two different types of Mass Spectrometry (MS) instruments in order to cope best with quality and quantity/throughput issues determined by the specifications of the respective oligo/analyte. While each instrument type precisely characterizes oligonucleotides in terms of composition through direct molecular weight measurement, their field of application is diligently adjusted to suitability considerations.

MALDI-ToF instruments typically have a higher throughput, while the limits of using this technique become manifest, if it comes to analyzing long oligonucleotides, or oligos carrying certain photo-labile modifications (e.g.common quenchers like BHQ®s, Dabcyl used in DLPs).

ESI-ToF is less efficient in terms of throughput but perfectly compensates for resolution issues with long oligos as well as for a potential detrimental laser impact on labile/photosensitive modifications – thus being a "natural" complement to MALDI-ToF analysis.

Comparison MALDI-ToF and ESI-ToF
Qualification Criteria MALDI-ToF ESI-ToF
< 60 nts + +
> 60 nts - ++
Photosensitive Modified Oligos - +
Wobble Oligos - +
Throughput ++ +
n-1/n-x Detection + +
Incomplete Deprotection + +
Depurination + +
Mass Accuracy ++ ++

Synthetic oligonucleotide purification is particularly challenging because of the small differences in size, charge and hydrophobicity between the full-length product and impurities, which often co-elute.

For improved analysis of complex samples like long and/or multiple labeled oligos, metabion offers liquid chromatography (LC) coupled with electrospray ionization mass spectrometry (ESI-MS). The mass spectrometer is connected to a high pressure liquid chromatography (HPLC) system, which allows premium analyte characterization via chromatographical separation, followed by respective molecular weight determination. With this system, the mass of oligonucleotides between 2 and 220 bases can be analysed with high accuracy, resolution and sensitivity. Our expert production team will take care of the method (MALDI or ESI ToF) that best applies to your sample.

What is the difference between preparative and analytical HPLC?

Preparative High Pressure Liquid Chromatography (HPLC) deals with isolating the separated components of a sample, and can be done on small-, mid- and large scale operations. In other words, the objective of a preparative HPLC is isolating and purifying a product. Practically, the sample goes from the detector into a fraction collector or it is collected manually.

Analytical HPLC refers to the processes of separating and identifying the components of a sample. It is usually a small-scale process, whose objective is the qualitative and quantitative determination of a compound. The sample goes from the detector into waste.

metabion offers analytical HPLC as an additional (optional) quality control method, complementing our Mass-Check QC, which is performed by default on all our oligos.

For product/quality documentation please see FAQ: What kind of documentation do I get with my RNA oligos?

How can I order metabion siRNA/ RNAi/ duplex RNA?

There are two ways of ordering:

  1. The preferred way is order transmission through our Web Order Portal for most convenient online shopping.
  2. You can order by sending us an e-mail with our pre-formatted excel order file as attachment. Download respective Order Form
    Duplex RNA
    download xlsx »
    Also available in our web order system (WOP)
    When you write your email, please make sure to address the following questions in the excel template:

  • Name of the siRNA?
  • Sequence of the siRNA in 5’-3’ orientation?
  • Yield range
  • Delivery form? (dry / in water/ buffer + concentration)
  • Modifications?

If you are a new customer, please additionally provide us with

  • Your shipping and billing address
  • Any other information like Purchase Order number, VAT number (VAT only for customers resident in the EU) etc

In case you opt to transmit orders via email using your own format(s), we need to alert you that above mentioned information are obligatory for processing your order. Due to extra efforts necessary for individual order format transfer into our system, order processing will take longer as compared to preferred web orders and pre-formatted emails.

What kind of documentation do I get with my siRNA/ RNAi/ duplex RNA?

The label on the siRNA tube shows basic information like oligo name, name of person who made the order, siRNA sequence including modifications, oligo ID, amount of RNA (OD260 and nmol), Tm, and molecular weight.

In addition, you will receive a technical data sheet containing more detailed information on the physical-chemical properties of the oligo, such as base composition, base count, purification grade, amount of RNA in nmol, Tm and molecular weight. Additionally, you will also receive a hard copy of the Mass-Check documentation/traces of each single strand of the duplex. The following terminology is used for differentiating between offered QC options including respective documentation coverage in our order forms and on supporting documents delivered with the products:

Mass Check
Standard quality control performed on each and every oligo. Either MALDI- or ESI-ToF, subject to the "nature of the oligo", and metabion internal procedures. This service is free of charge and the Mass Check traces will be provided.

MALDI-ToF
Explicitly ordered and performed MALDI-ToF check. Product delivered with MALDI-ToF traces. Additional charges apply.

ESI-ToF
Explicitly ordered and performed ESI-ToF check. Product delivered with ESI-ToF traces. Additional charges apply.

Do I need to anneal duplex RNA after resuspension?

While some manufacturers state that re-annealing is generally not necessary, we do recommend to re-anneal the dissolved duplex using a suitable protocol. For your convenience, we recommend to follow our guidelines prepared for download.

How are RNA oligonucleotides and RNA duplexes shipped?

All oligonucleotides, whether single-stranded or double-stranded oligos, are provided as dried pellets and shipped at ambient temperature. While being stable at room temperature for 2-4 weeks, they should be placed at -20°C or at lower temperature upon receipt.


Summary

  1. Viruses can store their genetic information in six different types of nucleic acid which are named based on how that nucleic acid eventually becomes transcribed to the viral mRNA.
  2. (+) and (-) strands of nucleic acid are complementary. Copying a (+) stand gives a (-) strand copying a (-) stand gives a (+) strand.
  3. Only (+) strands of viral RNA can be translated into viral protein.
  4. Regarding the enzymes involved in nucleic acid replication, the "dependent" part of the name refers what type of nucleic acid is being copied. The "polymerase" part of the name refers what type of nucleic acid is being synthesized.

Ustilago Maydis Viruses

Replication and Transcription

Like all dsRNA viruses, UmV has a capsid-associated transcriptase that makes the viral plus strands. However, little is known of the nature of the transcripts or the mechanism of transcription. One peculiarity of this system is the presence of dsRNAs (L, 354–355 bp) entirely homologous to the 3′ ends of the plus strands of their cognate dsRNAs (M). Presumably, like all viral dsRNAs, these are the result of replication of plus strands within nascent viral particles. It is not clear if the source of the L plus strands is internal transcription initiation or processing of M plus strands. Normal-sized L plus strands (with exactly the predicted 5′ ends) are produced either by the viral transcriptase or by RNAP II transcription of cDNA clones in Ustilago maydis or heterologous systems, implying that a cleavage event must be responsible for production of the L segments.

This cleavage must be either an inherent property of the cognate M plus strand or cleavage by a ubiquitous nuclease activity recognizing a universal feature of the M plus strands. No ribozyme activity can be demonstrated in the cognate M plus strands in vitro. However, all the cognate M plus strands have a peculiar predicted secondary structure, in which the predicted L plus strand includes a very long predicted hairpin stem (23 perfect base pairs in P4M2) immediately following the predicted cleavage site (which is highly conserved). These should make ideal substrates for the recently described cellular machinery (dicer) responsible for siRNA production, which is ubiquitous in eukaryotes.

The function of the L segments is obscure: production of toxin proceeds perfectly well from cDNA constructs lacking the L encoding region, in either homologous or heterologous systems, and the P1M2 segment (encoding the KP1 toxin) has no cognate L segment. The L segments must include a packaging signal, but it is not clear what prevents its inclusion in an M plus strand sequence lacking the 3′ L sequence, a situation that does exist with P1M2. In the closely related virus ScV, a sequence as small as 18 bases serves as the packaging signal.

Transcription in dsRNA viruses may be either semiconservative (resulting in displacement of the parental plus strand) or conservative (de novo synthesis of plus strands). Transcription in reovirus and ScV is conservative and in bacteriophage phi6 is semiconservative. An interesting difference in the structure of the viral RdRp may be responsible for this. The phi6 RdRp has an insertion of some 20 amino acids within the N-terminal F motif, an insertion that is absent in the reovirus RdRp (and absent in all the other known RdRp and reverse transcriptase structures). None of the totivirus, chrysovirus, or partitivirus RdRps has a phi6-like insertion most may therefore be conservative transcriptases. However, UmVP1M2 has been reported to be semiconservatively transcribed.


Past Systems of Classification

Viruses contain only a few elements by which they can be classified: the viral genome, the type of capsid, and the envelope structure for the enveloped viruses. All of these elements have been used in the past for viral classification (Table 1 and Figure 1). Viral genomes may vary in the type of genetic material (DNA or RNA) and its organization (single- or double-stranded, linear or circular, and segmented or non-segmented). In some viruses, additional proteins needed for replication are associated directly with the genome or contained within the viral capsid.

Table 1. Virus Classification by Genome Structure and Core
Core Classifications Examples
RNA Rabies virus, retroviruses
DNA Herpesviruses, smallpox virus
Single-stranded Rabies virus, retroviruses
Double-stranded Herpesviruses, smallpox virus
Linear Rabies virus, retroviruses, herpesviruses, smallpox virus
Circular Papillomaviruses, many bacteriophages
Non-segmented: genome consists of a single segment of genetic material Parainfluenza viruses
Segmented: genome is divided into multiple segments Influenza viruses

Figure 1. Viruses are classified based on their core genetic material and capsid design. (a) Rabies virus has a single-stranded RNA (ssRNA) core and an enveloped helical capsid, whereas (b) variola virus, the causative agent of smallpox, has a double-stranded DNA (dsDNA) core and a complex capsid. (credit “rabies diagram”: modification of work by CDC “rabies micrograph”: modification of work by Dr. Fred Murphy, CDC credit “small pox micrograph”: modification of work by Dr. Fred Murphy, Sylvia Whitfield, CDC credit “smallpox photo”: modification of work by CDC scale-bar data from Matt Russell)

Viruses can also be classified by the design of their capsids (Table 2 and Figure 2). Capsids are classified as naked icosahedral, enveloped icosahedral, enveloped helical, naked helical, and complex. The type of genetic material (DNA or RNA) and its structure (single- or double-stranded, linear or circular, and segmented or non-segmented) are used to classify the virus core structures (Table 2).

Table 2. Virus Classification by Capsid Structure
Capsid Classification Examples
Naked icosahedral Hepatitis A virus, polioviruses
Enveloped icosahedral Epstein-Barr virus, herpes simplex virus, rubella virus, yellow fever virus, HIV-1
Enveloped helical Influenza viruses, mumps virus, measles virus, rabies virus
Naked helical Tobacco mosaic virus
Complex with many proteins some have combinations of icosahedral and helical capsid structures Herpesviruses, smallpox virus, hepatitis B virus, T4 bacteriophage

Figure 2. Transmission electron micrographs of various viruses show their structures. The capsid of the (a) polio virus is naked icosahedral (b) the Epstein-Barr virus capsid is enveloped icosahedral (c) the mumps virus capsid is an enveloped helix (d) the tobacco mosaic virus capsid is naked helical and (e) the herpesvirus capsid is complex. (credit a: modification of work by Dr. Fred Murphy, Sylvia Whitfield credit b: modification of work by Liza Gross credit c: modification of work by Dr. F. A. Murphy, CDC credit d: modification of work by USDA ARS credit e: modification of work by Linda Stannard, Department of Medical Microbiology, University of Cape Town, South Africa, NASA scale-bar data from Matt Russell)


Composition and Structure of the Nucleic Acids: DNA & RNA

Read this article to learn about Composition and Structure of the Nucleic Acids: DNA & RNA !

There are two major classes of nucleic acids: DNA and RNA.

Both are composed of un-branched chains of units called nucleotides, each of which contains:

(1) A nitrogenous base (either a purine or pyrimidine),

In RNA, the pentose is ribose, whereas in DNA it is 2-deoxyribose. Both DNA and RNA contain the purine nitrogenous bases adenine (abbreviated A) and guanine (G) and the pyrimidine cytosine (C), but in DNA a second pyrimidine is thymine (T), whereas in RNA it is uracil (U).

A number of other nitrogenous bases have been identified in DNA and RNA, but these occur much less frequently. The phosphoric acid component of each nucleotide is, of course, chemically identical in both nucleic acids. These relationships are summarized in Table 7-1, and the corresponding chemical formulas are shown in Figure 7-3.

The pentose of each nucleotide unit is simulta­neously bonded through its number 1 carbon atom to the nitrogenous base (forming a nucleoside) and through its number 5 carbon atom to phosphoric acid. The structures of the four deoxyribonucleotides of DNA together with the specific numbering system used to identify each constituent atom are shown in Figure 7-4. Successive nucleotides of DNA and RNA are joined together by phosphodiester linkages involv­ing the 5′-phosphate group of one nucleotide unit and the 3′-hvdroxyl group of the neighboring unit (Figure 7-5).

The “backbone” of a nucleic acid molecule is formed by the repeating sequence of pentose and phosphate groups, and this is the same in all molecules. What dis­tinguishes one DNA (or RNA) molecule from another is the specific sequence of purine and pyrimidine bases present in the chain of nucleotides and the total num­ber of nucleotides (i.e., the size of the molecule). Each chain of nucleotides is called a polynucleotide.

Structure of DNA:

At one time, it was believed that the four purines and pyrimidine’s of DNA occurred in approximately equal amounts in the molecule. However, the studies of E, Chargaff and others in the late 1940s showed that this was not the case. Instead, they found that the relative amounts of the nitrogenous bases varied between spe­cies but were constant within a species.

The constancy noted within a species was maintained regardless of the tissue or organ from which the DNA was isolated. Furthermore, the relative amounts of the nitrogenous bases were similar in closely related species and quite different in unrelated species.

Chargaff also made the following extremely important finding. Regardless of the species used as the source of DNA, the molar ratios of adenine and thymine were always very close to unity, and the same was true for guanine and cytosine. No such constant relationship could be demonstrated for any other combination of nitrogenous base pairs. This implied that for some reason, every molecule of DNA contained equal amounts of adenine and thy­mine and also equal amounts of guanine and cytosine.

Using chemical information of this sort, together with the results of X-ray crystallographic studies of DNA, J. D. Watson, F. H. C. Crick, M. H. F. Wilkins, and R. Franklin proposed a model for the structure of DNA in the early 1950s. They suggested that a mole­cule of DNA consists of two helical polynucleotide chains wound around a common axis to form a right- handed “double helix.”

In contrast to the arrange­ment of amino acid side chains in helical polypeptides (where the side chains are directed radially away from the helix axis), the purine and pyrimidine bases of each polynucleotide chain were directed inward toward the center of the double helix so that they faced each other.

On the basis of stereo-chemical studies, Watson and Crick further suggested that the only possible ar­rangement of the nitrogenous bases within the double helix that was consistent with its predicted dimen­sions was that in which a purine always faced a pyrimidine, for the diametric distance between the two polynucleotide chains is too small to accommodate two juxtaposed purines.

Which purine was matched with which pyrimidine became clear from a consider­ation of which pairs would be able to form the hydro­gen bonds necessary to stabilize the double-helical structure. Accordingly, Watson and Crick concluded that adenine must be matched with thymine and guanine with cytosine.

This conclusion was, of course, in agreement with the chemical findings of Chargaff (see above)—in fact, Chargaff’s data may have been critical to the development of Watson and Crick’s pro­posals. The manner in which hydrogen bonds are formed between adenine and thymine and between guanine and cytosine is shown in Figure 7-6.

Al­though individually weak, the great number of these bonds contributes appreciably to the stability of the double-helical structure. In addition, the double helix is stabilized by hydrophobic bonds between neighbor­ing nitrogenous bases of each polynucleotide chain. Certain other features of the structure of DNA should be noted.

The two polynucleotide chains that make up the molecule are antiparallel. That is, begin­ning at one end of the molecule and progressing toward the other, successive nucleotides of one chain are joined together by 3’→5′ phosphodiester linkages, whereas the complementary nucleotides of the other chain are joined by 5’→3′ phosphodiester linkages. This antiparallel arrangement is depicted diagrammatically in Figure 7-7.

Right-handed, double-helical DNA can exist in either of two principal forms: these are called A-DNA and B-DNA. The two forms differ primarily in the positioning of the nitrogenous bases around the axis of the double helix and in the numbers of bases per helical turn. In B-DNA, there are 10 base pairs per turn of the helix, each turn sweeping out 3.4 nm (34 A) of linear translation (see Fig. 7-8) in A- DNA, there are 11 bases per helical turn, each turn sweeping out 2.8 nm (28 A) of linear translation.

Thus in the A form, the double helix has a greater diameter. In B-DNA the complementary bases lie in a plane that is perpendicular to the axis of the helix, whereas in A- DNA, the planes of successive base pairs are tilted rel­ative to the helical axis. It is the B form of the DNA that is believed to predominate in cells, although the A form may exist in DNA-RNA hybrids.

The two polynucleotides are twisted around one an­other in such a way as to produce two helical grooves in the surface of the molecule these are called the ma­jor and minor grooves (Fig. 7-8). The floor of the ma­jor groove is lined with oxygen and nitrogen atoms that could form hydrogen bonds with the amino acid side chains of proteins.

Indeed, it is the association of specific proteins with DNA that is believed to be in­volved in regulating gene expression. In­teraction of water molecules with the atoms lining the floor of the minor groove is believed to contribute to the stability of the B form.

Replication of DNA:

One of the intrinsic properties of the genetic material is its capacity for replication. The manner in which DNA satisfies this requirement is apparent from the nitrogenous base pairing required in the model. Be­cause the sequence of bases in one polynucleotide chain automatically determines the sequence of bases in the other, it is clear that one-half of a molecule (i.e., one of the two helices) contains all the information necessary for constructing a whole molecule.

For ex­ample, if we know that the sequence of bases along one polynucleotide chain of DNA is A T G A C, and so on, then the complementary sequence in the other chain must be T A C T G, and so on. Therefore, if the double helix were unwound, each separate polynucleo­tide chain could act as a template for the production of a new, complementary chain.

The result would be two identical double helices where there was only one be­fore. Of course, one-half of each new double helix would be represented by one of the original polynucle­otide chains. The basic features of this process are shown in Figures 7-9 and 7-10. A detailed description of the mechanism by which the replication of DNA oc­curs, together with its experimental basis.

Denaturation and Renaturation of DNA:

DNA is readily denatured by extremes of tempera­ture and pH. The denaturation takes the form of an unwinding of the double helix as hydrogen bonds be­tween complementary bases are disrupted. This form of DNA denaturation is referred to as melting and produces separate DNA strands. Solutions of DNA absorb ultraviolet light (UVL) having a wavelength of 260 nm. When the temperature of a native solution of DNA is elevated, the resulting melting is accompanied by an increase in UVL absorption.

This hyperchromic effect occurs because the purines and pyrimidines of separated strands can absorb more light energy than when they are part of a double helix. Some viruses contain a single-stranded form of DNA (see later), and because this form does not exhibit the hyperchromic effect, it is readily distinguished from double- stranded forms.

When DNA is melted thermally, denaturation be­gins in regions of the double helix that are rich in A-T base pairs and progressively shifts to regions of greater and greater G-C content. This is because the two hydrogen bonds holding each A-T pair together can be broken more easily (hence, at a lower tempera­ture) than the three hydrogen bonds holding each G-C pair together. A quantitative measure of the change in UVL absorbance that takes place as the temperature of a DNA solution is slowly elevated is called a melt­ing curve (Fig. 7-11).

The point in the melting curve at which the change from double-stranded to single- stranded DNA is half complete is called the Tm value and is characteristic of a particular source of DNA. The species specificity that is characteristic of DNA melting curves reflects differences in the G-C and A-T compositions of different kinds of DNA (Fig. 7-12).

Thermally denatured DNA can be re-natured by lowering the temperature of the solution, whereby separated strands recombine to form double helices as hydrogen bonds between complementary bases are re­formed. This re-annealing can be monitored as a de­crease in UVL absorption by the DNA solution. The capacity for denatured DNA to re-anneal can be used to assess the size of an organism’s genome and the complexity of the DNA that is present.

When reannealing studies are to be performed, the isolated DNA is first broken by shearing force into lengths of several hundred to several thousand nucleo­tide pairs. The double-helical DNA is then thermally denatured yielding single strands. A known concen­tration of the single-stranded DNA is then incubated at the reannealing temperature (usually about 25° be­low the Tm) and the reannealing rate is determined from the rate of change in UVL absorbance.

A large genome reanneals more slowly than a small genome because there is a greater number and variety of DNA fragments. Thus, each fragment takes a longer time to “seek out” and anneal with its complementary partner. The kinetics of DNA renaturation (Fig. 7-12) is represented by a curve relating the percentage of reassociated fragments to the “C0t number” (i.e., the concentration of DNA in moles of nucleotides per liter (C0) times the reaction time (t) in seconds).

As seen in Figure 7-12, viral DNA (curve c) reanneals more rap­idly than prokaryote DNA (curve d), and the latter re­anneals more quickly than eukaryote DNA (curve e). The discovery of a eukaryotic DNA fraction in mam­malian cells that reanneals unexpectedly rapidly (curve b) revealed for the first time the existence in the genomes of higher organisms of repetitive DNA sequences.

In Figure 7-12, curve shows the reannealing rate of a solution containing a mixture of synthetically produced polyuridylic acid (a nucleotide chain with only uracil bases) and poly- adenylic acid strands. Even though uracil is not usu­ally found in DNA, like thymine it can form hydrogen bonds with adenine and does so in many RNA mole­cules (see below).

For about 25 years following the original establish­ment of the Watson – Crick Model of DNA structure, it was presumed that all naturally occurring double- helical DNA was right-handed. However, in 1979 A. Rich confirmed earlier observations reported by F. M. Pohl and T. Jovin that a left-handed form of DNA also exists. As in right-handed DNA, the two helices are held together by complementary base pairing and the strands are antiparallel. Because the sugar- phosphate backbones of the two polynucleotides trace a zigzag course around the axis of the helices (Fig. 7- 13), this left-handed DNA has been called Z-DNA.

Though the structure of Z-DNA originally proposed by Rich was based on studies of DNA crystals pro­duced in the laboratory, Z-DNA has since been identi­fied in the chromosomes of a number of eukaryotic cells. A variety of indirect evidence also implies that Z-DNA is a normal constituent of animal cells, plant cells, and bacteria. Unlike B-DNA,’Z-DNA is highly immunogenic, making it possible to readily produce antibodies against Z-DNA. The reaction of these antibodies with DNA isolated from a variety of cell types implies the presence of the Z form. Natu­rally occurring Z-DNA-binding proteins have also been isolated from a number of cells.

The Z form of DNA appears to coexist with the B form in the same DNA molecules. Indeed it is be­lieved that DNA can “flip” between the B and Z forms in those regions of a double helix that are rich in se­quences having alternating purines and pyrimidines. These sequences appear to occur in selective regions of a cell’s DNA, lending credence to the idea that switches in DNA helicity between right-handed and left-handed forms may be involved in selective gene expression.

“Single-Stranded” DNA:

Although in nearly every case so far studied, DNA consists of two polynucleotide chains twisted about one another to form a double helix, it is now apparent that in a few bacterial viruses (i.e., the φ X 174 and S13 E. coli phages) DNA exists as a single polynucleotide chain. This was initially suspected when chemical analyses of the nitrogenous base contents of these vi­ral DNAs revealed that the amounts of adenine and thymine, as well as guanine and cytosine, were not equal.

During reproduction of these viruses, the single-stranded DNA (referred to as the ” + strand”) is injected into the host bacterial cell, where it acts as a template for the reproduction of a complementary polynucleotide chain (called the “- strand”) these two polynucleotides combine to form a conventional double helix, which then serves as the template for the production of additional + strands. The newly pro­duced + strands are then enclosed in the viral protein coats to form new virus particles.

Structure of RNA:

RNA and DNA differ chemically in two notable ways: in RNA, ribose is the pentose (not deoxyribose as in DNA) and the pyrimidine uracil oc­curs in place of thymine (Fig. 7-14). Early chemical analyses of the nitrogenous base contents of RNAs from various sources revealed that the A:U and G:C molar ratios were quite different from the A:T and G:C molar ratios of DNA. On this basis, it was con­cluded that RNA occurs as a single polynucleotide chain.

This contention is also supported by physico- chemical studies, but it should be noted that there are some viral RNAs that are double stranded. Although only one polynucleotide chain is usually present, RNA does possess regions of double-helical coiling where the single chain loops back upon itself.

These regions are stabilized by the formation of hydrophobic bonds between neighboring bases (as in DNA) and also by the formation of hydrogen bonds between guanine and cytosine and between adenine and uracil.

In RNA, A and U can form two hydrogen bonds similar to the two bonds formed between A and T in DNA. As in DNA, the portions of the RNA strand that are twisted around each other to form a double helix are antiparallel. In double-helical RNA, the helices and the complementary base pairs are arranged in much the same manner as in A-DNA.

Synthesis of RNA:

Except perhaps in the case of the reproduction of cer­tain RNA viruses, the synthesis of RNA appears ‘to be directed by DNA and is called tran­scription. The formation of the RNA polynucleotide takes place using the base sequence along only one of the two deoxyribonucleotide helices of DNA (produc­ing a temporary RNA-DNA hybrid) as a template and results in the release of a single, complementary poly­ribonucleotide chain in which the base uracil occurs in place of thymine (Figure 7-14).

Replication of DNA and RNA Viruses:

The viruses may be divided into two classes: viruses whose genetic complement consists of DNA and vi­ruses whose genetic complement consists of RNA. In cells infected with DNA viruses, the infecting viral DNA is replicated, forming new viral DNA that is then transcribed into RNA this RNA is then trans­lated into viral protein (Fig. 7-15a).

The newly pro­duced viral DNA and viral proteins combine in the as­sembly of new, complete virus particles that are released upon lysis of the host cell. A latent state can also be established in which the viral DNA is incorpo­rated into the host cell’s genome, being replicated and distributed along with the host cell’s native DNA, un­til it is transcribed once again into additional viral RNA and then into viral proteins.

For most RNA viruses (e.g., poliomyelitis, influ­enza, common cold, etc.), DNA involvement is essen­tially bypassed. For example, during the infection of a cell with polio virus, the single-stranded RNA (called a + strand) enters the host cell, where it acts as a tem­plate for the synthesis of complementary – strands. The latter are then employed in the proliferation of new + strands and these are translated into viral pro­teins (Fig. 7-15b).

The mechanism described above is varied in several other viruses in which the RNA is either double stranded (e.g., reoviruses, in which only one of the two RNA strands produced during replication is tran­scribed) or the infecting single RNA strand is comple­mentary (rather than identical) to the newly produced viral RNAs that are to be translated into viral pro­teins (e.g., Sendai virus, Newcastle disease virus, etc.)

Yet another mechanism exists in the case of the RNA tumor viruses (e.g., Rous sarcoma virus). These viruses do not transfer information from RNA to RNA, but rather from RNA to DNA and then to new RNA. The viral RNA is employed as a template for the synthesis of DNA by the infected cell (a phenome­non that is called “reverse transcription”). Some of the resulting “viral DNA” may be incorporated into the genome of the host cell, establishing what is called the provirus state.

The provirus state has been suggested as the basis of a number of different RNA virus-induced and DNA virus-induced cancers. According to this view, one or more of the provirus genes—which are normally re­pressed by the host cell—may become derepressed and cause the production of an oncogenic (i.e., cancer- causing) substance that alters the cell’s normal prop­erties or behavior. Such a change may be delayed for a number of generations, depending on the period of la­tency.

During the 1960s and early 1970s, the so-called “central dogma” of molecular biology was the orderly and unidirectional flow of information encoded in the base sequences of a cell’s DNA to RNA and then to protein, that is,

The discovery of reverse transcription by certain RNA viruses in which the information of RNA is passed on to DNA has necessitated a reexamination of that dogma and raised the question of whether a similar in­teraction between RNA and DNA might normally oc­cur in cells (i.e., cells not infected by viruses) under specific conditions (e.g., during cellular differentia­tion). The central dogma might more appropriately be represented as,

Types of Cellular RNA:

Cells contain three major functional types of RNA: ribosomal RNA (abbreviated rRNA), messenger RNA (mRNA), and transfer RNA (tRNA). All these are transcribed from DNA and are engaged in mediat­ing the expression of the genetic message of DNA by participating in the synthesis of the cell’s proteins. It has already been noted that RNA occasionally serves as the genetic material of viruses.

Of the cellular RNAs, rRNA is the most abundant, accounting for up to 85% of the total RNA of the cell. Only three “or four- different kinds of rRNA are present in cells, and these are confined for the most part to the cell’s ribosomes. mRNA accounts for about 5 to 10% of the cell’s RNA and is much more heteroge­neous with respect to size and nitrogenous base con­tent than the rRNAs.

This results from the relation­ship (see below) between the chain lengths and base sequences of mRNAs and the variable sizes and pri­mary structures of polypeptides synthesized in a cell. Most mRNA occurs in the cytoplasm, where it tran­siently combines with ribosomes during protein syn­thesis.

About 10 to 20% of the cell’s RNA is tRNA. All tRNA molecules are similar in size and typically con­tain 75 nucleotide units. In spite of these similarities, a single cell may contain about 60 species of tRNA dif­fering in their base sequences. Because most of the tRNA is recovered in the cytoplasmic (i.e., soluble) phase of disrupted cells following centrifugation, tRNA is also called soluble RNA (i.e., sRNA).

In view of its small size and relative ease of isolation, tRNA has been more extensively studied than the other two ribonucleic acids, and the specific primary, secondary, and tertiary structures of many tRNAs have already been determined. tRNAs contain moderate amounts of unusual nucleotides such as ribothymidine, dihydrouridine, pseudouridine, and methylguanosine. These are formed by modification of the four common RNA bases after the tRNA has been synthesized from the four unmodified ribonucleotides. The modified bases play a crucial role in establishing the unique spa­tial organization of these molecules.

Although the mechanisms of DNA replication and protein synthesis are considered in depth, it is appropriate that a brief account­ing of the functional relationships among the nucleic acids and between nucleic acids and proteins be made at this time. Inheritable information is encoded in the various nitrogenous base sequences possessed by the cell’s DNA, and by the process of transcription and translation these base sequences are employed to specify the primary structures of all proteins, pro­duced by the cell.

Most important among these pro­teins are the enzymes that catalyze and regulate the myriad of chemical reactions characterizing the cell’s metabolism. Therefore, the information of DNA con­fined essentially to the cell nucleus manifests itself primarily in the cytoplasm as the synthesis of a unique assemblage of proteins.

The replication of DNA that precedes mitotic cell division and the equal distribu­tion of the duplicated DNA among the progeny cells provides for the passage of complete sets of informa­tion from one generation of cells to another. In addi­tion to serving as templates for their own replication, the nucleotide sequences of DNA are used during transcription to produce complementary base se­quences of RNA.

The resulting RNAs then serve as in­termediaries in translating the original message into protein. Of paramount importance in this process are the mRNA molecules whose base sequences directly determine the primary structures of the polypep­tides. These mRNA molecules leave the nucleus of the cell following their synthesis and attach in the cyto­plasm to one (or several) ribosome.

The rRNA of each ribosome is believed to play a role in this attachment. tRNA molecules also produced in the nucleus enter the cytoplasm, where they combine with specially ac­tivated amino acids (distinct tRNAs exist for each spe­cies of amino acid). The resulting complexes, directed by the base sequence of mRNA attached to the ribo­somes, sequentially deposit their amino acids in the growing polypeptide chains.


Watch the video: Virus Life Cycle for Different Viral Genomes dsDNA, ssDNA, dsRNA, ssRNA, + sense, - sense MCAT (January 2023).