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I am sorry to bother with this question (i study genetics for about few hours, because I need to understand my data) and I am really confused about these two terms, because I dont know if the books uses word "reverse" interchengeably or what is going on here.
The main problem I am totally confused about are words reverse complementarity and complementarity of two DNA strands. Since I know that strands runs in antiparallel directions and two DNA strands are complementary to each other: i.e. sequence ACTCTG is complementary to TGAGAC and vice versa.
But my question is: are two DNA strands reverse complementary to each other? Because at least in two books I have read that two strands of DNA are. But if I would apply it to my previous example and use reversion, then no way I can get to my other strand. Is the word reverse in this context use as antiparalel (5' - 3' and 3' - 5') or am I missing something and it's true that if I take the whole DNA strand, I can use complement and reverse it and it will be my other strand?
Again I am sorry to ask this really basic question, but I am so confused about this terms. Nice day to all and thanks if someone will have time to answer my question.
I agree that the definitions can be somewhat confusing when first encountered. Each nucleotide has a complement A-T, C-G. But the DNA strand are reverse-complementary because when aligned from 5'->3' they are not (necessarily) complementary.
DNA strand 5'-ATCCGG-3'
Since we want to write all sequences in the same direction we must call the strand reverse-complementary
DNA complementarity against reverse complementarity - Biology
Reverse and/or complement DNA sequences. Separate sequences with line returns. Complementarity will follow the IUPAC convention.
|Base||Name||Bases Represented||Complementary Base|
|B||not A||C G T||V|
|D||not C||A G T||H|
|H||not G||A C T||D|
|V||not T/U||A C G||B|
|N||Unknown||A C G T||N|
hjm/projects/tacg/ (no longer online)
* Thanks to Joost Kolkman at Maxygen who pointed out that revcomp(S)=S and revcomp(W)=W in the source above (no longer online), revcomp(S) was W and vice-versa, which is is incorrect. I knew that but hadn't verified.
Understanding BLAST and Reverse Complementary RNA, and Discussing miRcore’s Future Leadership
During the March 24th meeting, members were introduced to BLAST, the Basic Local Alignment Search Tool ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ). The focus was on using Nucleotide BLAST to match RNA sequences from a file type called FASTA to RNA sequences in the NCBI database. FASTA files contain a name for each sequence and the order of the nucleotides in the sequence. Lines from a FASTA file can be copy-pasted into BLAST, which will provide a visual summary of the matches that it has found for the input sequence. It also lists out information for each individual sequence match. During the meeting, the concept of reverse complementary DNA sequences was reviewed, in order to explain why BLAST will label some sequence matches as “Plus/Plus” and others as “Plus/Minus.” DNA has two strands (it is a double helix) that go in opposite directions. One strand is designated the “positive” strand, and one is “negative.” DNA has a 5’ end and a 3’ end. When RNA is transcribed from the positive strand, it will be transcribed from the 3’ end towards the 5’ end of the DNA. The 5’ end of the RNA will match up with the 3’ end of the DNA, and vice versa. The RNA goes in the reverse direction compared to the DNA, but its base pairs still match (e.g. G to C). The reverse complementary RNA for a positive strand DNA sequence will be identical to the corresponding negative strand DNA sequence. For the example below, the reverse complementary RNA for the positive strand, read from the 5’ end, would be CAUCCU . . . the same as the negative strand, only with T’s replaced by U’s.
BLAST finds all the matches it knows, from both positive and negative strand DNA and RNA this explains why some matches are Plus/Plus and others are Plus/Minus. However, another element of complexity is that RNA is processed by having parts removed, i.e. introns. So not all the RNA that is initially transcribed from the DNA makes it into the final RNA product. Depending on which introns are removed from the RNA, different “isoforms” (versions) can be created. The process of creating isoforms by processing the RNA differently is called “alternative splicing.” BLAST finds many different matches for the RNA sequence input that may be slightly different, or have gaps, due to alternative splicing.
Image: In number 2, “RNA processing,” the blue loops that are removed from the red pre-mRNA are the introns.
Image from miRcore summer camp slideshows.
The meeting concluded with suggestions from members on how miRcore leadership could be determined in the future, and what qualities a good miRcore leader should have. Members proposed a mix of the application and election processes in order to choose leaders, and qualifications such as experience, attendance, and, of course, compassion. Some commented that the list of good leadership qualities generated at the meeting included traits that were at odds with each other (being assertive versus being open-minded), but agreed that character was an important determining factor for good leadership. The discussion is still open, as miRcorers determine the direction of their organization in the years to come.
DNA complementarity against reverse complementarity - Biology
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Almost every cell in the body has the same DNA, but different cell types, such as neurons and muscle cells, express different genes because only certain genes are transcribed into messenger RNA, or mRNA, in each cell. In the laboratory, mRNAs can be used as a template to synthesize complementary DNA, cDNA, to study gene expression. A common method is to extract RNA from cells, then isolate the mRNA from other types of RNA, like ribosomal RNA or transfer RNA, by running the sample over a column of beads with stretches of thymine nucleotides attached.
These bind to the poly-A tail, a chain of adenine nucleotides specifically present on the 3-prime ends of eukaryotic mRNA. The other types of RNA do not bind and are washed away.
After the mRNA is isolated, a poly-T primer is bound to the poly-A tail, providing a starting point for reverse transcriptase enzymes to transcribe a single-stranded cDNA from the mRNA. Chemicals, such as RNase enzymes, are then added to degrade the RNA.
DNA polymerase enzymes are then used to synthesize a strand complementary to the cDNA, resulting in double-stranded cDNA, which can be inserted into a bacterial or viral vector and used in molecular biology research.
15.13: Complementary DNA
Only genes that are transcribed into messenger RNA (mRNA) are active, or expressed. Scientists can, therefore, extract the mRNA from cells to study gene expression in different cells and tissues. The scientist converts mRNA into complementary DNA (cDNA) via reverse transcription. Because mRNA does not contain introns (non-coding regions) and other regulatory sequences, cDNA&mdashunlike genomic DNA&mdashalso allows researchers to directly determine the amino acid sequence of the peptide encoded by the gene.
cDNA can be generated by several methods, but a common way is to first extract total RNA from cells, and then isolate the mRNA from the more predominant types&mdashtransfer RNA (tRNA) and ribosomal (rRNA). Mature eukaryotic mRNA has a poly(A) tail&mdasha string of adenine nucleotides&mdashadded to its 3&rsquo end, while other types of RNA do not. Therefore, a string of thymine nucleotides (oligo-dTs) can be attached to a substrate such as a column or magnetic beads, to specifically base-pair with the poly(A) tails of mRNA. While mRNA with a poly(A) tail is captured, the other types of RNA are washed away.
Next, reverse transcriptase&mdasha DNA polymerase enzyme from retroviruses&mdashis used to generate cDNA from the mRNA. Since, like most DNA polymerases, reverse transcriptase can add nucleotides only to the 3&rsquo end of a chain, a poly(T) primer is added to bind to the poly(A) tail to provide a starting point for cDNA synthesis. The cDNA strand ends in a hairpin loop. The RNA is then degraded&mdashcommonly with alkali treatment or RNase enzymes&mdashleaving the single-stranded cDNA intact.
A second DNA strand complementary to the cDNA is then synthesized by DNA polymerase&mdashoften using the hairpin loop of the first cDNA strand or a nicked piece of the mRNA as a primer.
The resulting double-stranded cDNA can be inserted into bacterial or viral vectors and cloned using standard molecular biology techniques. A cDNA library&mdashrepresenting all the mRNAs in the cells or tissue of interest&mdashcan also be constructed for additional research.
Pray, Leslie A. &ldquoThe Biotechnology Revolution: PCR and the Use of Reverse Transcriptase to Clone Expressed Genes.&rdquo Nature Education 1, no. 1 (2008): 94. [Source]
Thank you for reading this highlight of the new pre-print “Improved architectures for flexible DNA production using retrons across kingdoms of life” from the Shipman Lab at the Gladstone Institutes in San Francisco. If the thought of becoming a molecular tool builder sounds more exciting than optimizing ad revenue, you should consider applying to work with the Shipman Lab! The young group is looking for new members, and this is a chance to get in on the ground floor with a lab doing some of the most exciting and innovative work in synthetic biology.
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This is clearly a simplistic classification, but can be an interesting question to consider. One really exciting protein-centric research program is that of Mohammed AlQuraishi, who just started a new lab at Columbia with the bold vision of simulating a cell with structural resolution by 2050. Such systems-level “structural cell biology” will be incredible to see.
The paper is "Refactoring bacteriophage T7" from the Endy Lab. This is an arbitrary example, and I’m not going to detail the history of synthetic biology, DNA-based programming languages, or the entire field of DNA computing.
Template switching by a group II intron reverse transcriptase: biochemical analysis and implications for RNA-seq
The reverse transcriptases (RTs) encoded by mobile group II intron and other non-LTR-retro-elements differ from retroviral RTs in being able to template switch from the 5’ end of one template to the 3’ end of another without pre-existing complementarity between the donor and acceptor nucleic acids. Here, we used the ability of a thermostable group II intron RT (TGIRT GsI-IIC RT) to template switch directly from synthetic RNA template/DNA primer duplexes having either a blunt end or a 3’-DNA overhang end to establish a complete kinetic framework for the reaction and identify conditions that more efficiently capture acceptor RNAs or DNAs. The rate and amplitude of template switching are optimal from starter duplexes with a single nucleotide 3’-DNA overhang complementary to the 3’ nucleotide of the acceptor RNA, suggesting a role for non-templated nucleotide addition of a complementary nucleotide to the 3’ end of cDNAs synthesized from natural templates. Longer 3’-DNA overhangs progressively decrease the rate of template switching, even when complementary to the 3’ end of the acceptor template. The reliance on a single base pair with the 3’ nucleotide of the acceptor together with discrimination against mismatches and the high processivity of the enzyme enable synthesis of full-length DNA copies of nucleic acids beginning directly at their 3’ end. We discuss possible biological functions of the template-switching activity of group II intron and other non-LTR-retroelements RTs, as well as the optimization of this activity for adapter addition in RNA-and DNA-seq.
- Relatively few antiviral chemotherapeutic agents are currently available and they are only somewhat effective against just a few limited viruses.
- Many antiviral agents resemble normal DNA nucleosides molecules and work by inhibiting viral DNA synthesis.
- Some antiviral agents are protease inhibitors that bind to a viral protease and prevent it from cleaving the long polyprotein from polycistronic genes into proteins essential to viral structure and function.
- Some antiviral agents are entry inhibitors that prevent the virus from either binding to or entering the host cell.
- Antiviral agents are available for only a few viruses, including certain influenza viruses, herpes viruses, cytomegaloviruses, hepatitis C viruses, and HIV.
- Certain interferon cytokines have been produced by recombinant DNA technology and several are used for certain severe viral infections.
250+ TOP MCQs on Reverse Transcriptase PCR and Answers
Molecular Biology Multiple Choice Questions on “Reverse Transcriptase PCR”.
1. Reverse transcriptase produces DNA from RNA.
Explanation: Production of DNA from RNA is the reverse process of transcription. Such DNA produce will not be exactly same as its parent gene as it will lack the introns as it is converted from the RNA. This reaction is facilitated by the enzyme reverse transcriptase and the DNA thus formed is known as the complementary DNA or the cDNA.
2. Which of the following serves as the first primer in RT – PCR for eukaryotic RNA?
A. Oligo A
B. Oligo T
C. Oligo G
D. Oligo C
Explanation: All RNAs have a poly – A tail added to its 3’ end as the post transcriptional modification. Thus, during reverse transcription this poly – A tail is used as the template strand and a primer of oligo T that is used for the synthesis of the cDNA strand.
3. In RT – PCR the enzyme deoxynucleotydil transferase adds poly – G residues in the __________
A. 5’ end of RNA
B. 3’ end of RNA
C. 3’ end of cDNA
D. 5’ end of cDNA
Explanation: During the synthesis of the complementary strand for the cDNA molecule, its 3’ end is unknown. Thus no primer can be formed which can synthesize its complementary strand. Here the enzyme deoxynucleotydil transferase adds poly – G residues in the 3’ end of cDNA so that oligo C primer can be used for the synthesis of its complementary strand.
4. The digestion of mRNA during RT – PCR is carried out by the enzyme ____________
B. RNase H
C. Bal 31
Explanation: Exonuclease and endonuclease both restrict DNA fragments and Bal 31 is a type of exonuclease. RNase H brings about the mRNA digestion during RT – PCR.
5. How many primers are used in the process of reverse transcriptase amplification?
Explanation: The first primer of oligo – T is used during the reverse transcription of the mRNA to cDNA. The cDNA produced is a single stranded molecule thus synthesis of its complementary strand is necessary. A poly G/C tail is added to its 3’ end and thus the second primer is used against this poly G/C tail for the synthesis of the cDNA duplex.
6. From which organism is the enzyme reverse transcriptase isolated?
Explanation: Certain viruses have RNA as their genetic material. These viruses carry an extra protein for the synthesis of cDNA so that it can be incorporated efficiently into the host chromosome. This enzyme used by the viruses is a reverse transcriptase. Examples of such viruses are retrovirus, human papiloma virus, etc.
7. The cDNA library provides several advantages over RT – PCR.
Explanation: The use of 5’ specific primer eliminates the risk of amplification of partial cDNAs. Thus the procedure is so sensitive that the total cellular RNA can be used for RT – PCR, and mRNA separation step is unnecessary. Thus, RT – PCR offers several advantages over cDNA library for the isolation of a specific gene provided enough information is available.
8. ________________ is a modification of RT – PCR.
A. Overlap extension PCR
B. Inverse PCR
C. Thermal cycle sequencing PCR
Reverse Transcription (cDNA Synthesis)
The synthesis of DNA from an RNA template, via reverse transcription, produces complementary DNA (cDNA). Reverse transcriptases (RTs) use an RNA template and a short primer complementary to the 3' end of the RNA to direct the synthesis of the first strand cDNA, which can be used directly as a template for the Polymerase Chain Reaction (PCR). This combination of reverse transcription and PCR (RT-PCR) allows the detection of low abundance RNAs in a sample, and production of the corresponding cDNA, thereby facilitating the cloning of low copy genes. Alternatively, the first-strand cDNA can be made double-stranded using DNA Polymerase I and DNA Ligase. These reaction products can be used for direct cloning without amplification. In this case, RNase H activity, from either the RT or supplied exogenously, is required.
Many RTs are available from commercial suppliers. Avian Myeloblastosis Virus (AMV) Reverse Transcriptase and Moloney Murine Leukemia Virus (M-MuLV, MMLV) Reverse Transcriptase are RTs that are commonly used in molecular biology workflows. M-MuLV Reverse Transcriptase lacks 3´ &rarr 5´ exonuclease activity. ProtoScript ® II Reverse Transcriptase is a recombinant M-MuLV reverse transcriptase with reduced RNase H activity and increased thermostability. It can be used to synthesize first strand cDNA at higher temperatures than the wild-type M-MuLV. The enzyme is active up to 50°C, providing higher specificity, higher yield of cDNA and more full-length cDNA product, up to 12 kb in length.
The use of engineered RTs improves the efficiency of full-length product formation, ensuring the copying of the 5' end of the mRNA transcript is complete, and enabling the propagation and characterization of a faithful DNA copy of an RNA sequence. The use of the more thermostable RTs, where reactions are performed at higher temperatures, can be very helpful when dealing with RNA that contains high amounts of secondary structure.
For help comparing the RTs and cDNA Synthesis reagents available, view our RT/cDNA Synthesis selection chart.
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Part 2: Extended Edition: Discovering Reverse Transcriptase
00:00:1520 I am David Baltimore.
00:00:1620 I am a professor at the California Institute of Technology -- Caltech.
00:00:2302 And I'm here to talk about viruses.
00:00:2516 I've worked on viruses, now, for 50+ years.
00:00:3014 And I find them fascinating.
00:00:3420 And I'll concentrate on HIV because it's the virus most people know.
00:00:4114 And of course it's the scourge of the 20th and 21st century.
00:00:4706 So, I call viruses a separate kingdom of the living world.
00:00:5112 They're not really a kingdom, but they are so different from any other organism that
00:00:5713 they should be considered very separate.
00:00:5927 They're extremely tiny.
00:01:0200 They can only duplicate themselves by penetrating into living cells and diverting the
00:01:0924 cells' activities towards the ends that are dictated by the genetics of the virus.
00:01:1617 And thus they are obligate cellular parasites.
00:01:2017 Viruses can only exist once cells. could only exist once cells have evolved.
00:01:2818 They can have either RNA or DNA as their genetic material the rest of the biological world
00:01:3409 uses DNA.
00:01:3803 And they can grow in all kinds of cells: in animal cells, in bacteria, in plants.
00:01:4527 And the fact that so many of them are RNA viruses suggests that they're left over from
00:01:5220 an RNA world, which was an intermediary in the evolution of the world we know today,
00:02:0016 which is based on both DNA and RNA.
00:02:0418 The central dogma of molecular biology hasn't changed in. since the 1950s, when it was
00:02:1208 first enunciated by Francis Crick.
00:02:1803 And that is that DNA can duplicate itself, it can be transcribed into RNA, and that RNA
00:02:2421 can in turn be a template for making protein.
00:02:2812 And protein is what makes life work.
00:02:3204 All of our cells are built out of proteins.
00:02:3415 All of the events of our lives are determined by proteins.
00:02:4113 In 1970, I was working on a virus, and had the good fortune to make a discovery that
00:02:4724 changed the central dogma slightly.
00:02:5126 We discovered reverse transcription: RNA giving rise to DNA.
00:02:5918 And that reverse transcription actually gives rise to a very large amount of the DNA
00:03:0904 of us and of most higher organisms.
00:03:1215 The discovery of reverse transcription filled out our understanding of the logic of
00:03:1827 how viruses multiply.
00:03:2210 And let me describe that logic by focusing on the most important product that's
00:03:2905 made during a virus infection, which is messenger RNA.
00:03:3405 Messenger RNA encodes protein.
00:03:3714 And those proteins, now viral proteins, can take over a cell, can make more virus particles.
00:03:4701 They are the essence of the virus infection process.
00:03:5205 And so, the. the nucleic acid, the DNA or RNA of the virus, is holding the instructions
00:04:0006 for making those proteins.
00:04:0212 And the question is, how do those instructions get to making protein?
00:04:0804 And there are really six ways that that can happen, depending on exactly how the instructions
00:04:1511 are transmitted.
00:04:1800 And those six ways distinguish one class of viruses from another class of viruses.
00:04:2502 So, the most simple, straightforward ones are the double-stranded DNA viruses,
00:04:3112 class I viruses.
00:04:3419 Those simply make messenger RNA just like we make messenger RNA in our normal cells,
00:04:4203 because we carry double-stranded DNA as our hereditary material.
00:04:4721 Then there are single-stranded DNA viruses.
00:04:5011 They have to become double strands -- those are the class II viruses -- and then
00:04:5524 they can make messenger RNA.
00:04:5812 And then they have to make, again, those single strands to be the genetic material of
00:05:0228 those viruses.
00:05:0408 Then there are RNA viruses.
00:05:0711 There are double-stranded RNA viruses, very similar to the double-stranded DNA viruses
00:05:1226 except they're RNA.
00:05:1515 And they make messenger RNA from their double-stranded RNA.
00:05:2010 There are two kinds of single-stranded RNA viruses.
00:05:2402 They can either carry the sense strand, which we call the plus strand, of genetic material,
00:05:3314 in which case they can encode protein by just putting that into the cell.
00:05:4021 Or they can, in the class V viruses, carry the minus strand, the complementary strand.
00:05:4706 That's sense-less until it's copied into a plus strand.
00:05:5204 And so it needs to be copied before it can make protein.
00:05:5720 And the virus particle has to carry a polymerase to do that.
00:06:0328 And then there's the reverse transcription process, in which an RNA is carried by the virus,
00:06:0821 but it. it's copied into, first single-stranded DNA, then double-stranded DNA, and that then
00:06:1419 is the template to make messenger RNA.
00:06:1728 So, that gives us six different kinds of viruses.
00:06:2122 I call them. it's called the Baltimore classification because we first enunciated it back in 1970,
00:06:3213 and many textbooks use it as a way of organizing. thinking about viruses.
00:06:3726 And. but it really isn't a classification.
00:06:4206 It's a statement of how viruses fit their style of handling genetic information
00:06:4922 into the needs of the central dogma.
00:06:5204 Now, how many viruses are there?
00:06:5501 There are an uncountable number of viruses.
00:06:5810 Every animal in the animal kingdom has its own set of viruses every bacterium has
00:07:0326 its own set of viruses.
00:07:0604 There are certainly a million viruses that are easily distinguishable, one from another,
00:07:1213 and many estimates have been made that are much higher than that.
00:07:1809 Because viruses can only grow inside cells, they have to get into a cell.
00:07:2322 But once they get in there, they can very quickly take over the cell.
00:07:2822 Some viruses, particularly of bacteria, have a life cycle of only 20 or 30 minutes.
00:07:3701 And even mammalian viruses take only a few hours to make enormous numbers of copies of themselves.
00:07:4618 They can continue to live only if they are passed on from one organism to another because,
00:07:5210 being obligate parasites, they can't live out in the world on their own.
00:07:5717 And so every virus is a story of transmission.
00:08:0205 We know that transmission in humans can involve sneezing or touching a surface that
00:08:0814 a sick person has touched.
00:08:1109 But for cats, the transmission is different, for insects the transmission is different,
00:08:1902 and that's part of the evolution of viruses.
00:08:2106 They evolve to become part of the living style of their hosts.
00:08:2826 If viruses are very particular to a species, like the human species, then we can
00:08:3513 actually get rid of them because they have no reservoir outside of us.
00:08:4013 And so if we can vaccinate the whole population of the world, then we can get rid of that virus,
00:08:4720 because we make ourselves immune to it.
00:08:5121 And we've done that with smallpox.
00:08:5317 And we've almost done that with polio, and they believe that within a couple of years
00:09:0023 polio will be eliminated from the Earth.
00:09:0514 Now, viruses growing inside cells, for them to get out of cells, they've got to do something.
00:09:1413 And they do one of two things, generally.
00:09:1712 Either they bet. bud out from the surface of cells, and that's what this picture shows,
00:09:2422 or they grow up to large numbers inside the cell and then they burst the cell open.
00:09:3215 And that liberates virus particles from the cell interior into wherever it is, into the
00:09:3806 blood system. bloodstream in the humans, for instance.
00:09:4416 Viruses only made sense when molecular biology was born as a science, because they're
00:09:5007 so small that the only really important thing that they have is their genetic instructions,
00:09:5528 their DNA or their RNA.
00:09:5822 They generally protect that in a coat, but the coat is. is largely inert, except for
00:10:0422 helping them get into the next infected cell, or next cell that they're going to infect.
00:10:1317 Some of them have a few other proteins that help initiate the infection.
00:10:1903 And then, we're now learning about viruses that are very big and complicated -- surprising.
00:10:2526 They're actually of the size of bacteria.
00:10:2827 But what distinguishes them is that they require to be inside a cell to multiply, whereas bacteria
00:10:3823 can multiply as free-living organisms.
00:10:4403 The way we detect viruses is by what's called a plaque assay.
00:10:4926 We have a lawn of susceptible cells.
00:10:5321 They could be mammalian cells they could be bacterial cells.
00:10:5808 We put a few virus particles down.
00:11:0028 Those virus particles chew up the lawn from a center where they land, and they form
00:11:0715 a plaque.
00:11:0915 And if you count the number of plaques, you know how many viruses were there.
00:11:1426 And if you make. if you put down twice as many viruses, you get twice as many plaques,
00:11:2028 so it's a highly quantitative test, and bacterial genetics was done using those tasks.
00:11:2917 those techniques.
00:11:3023 Here, we can see tiny plaques made by a kind of bacterial virus, or huge plaques made
00:11:4100 by poliovirus plated on human cells.
00:11:4528 A distinction which I find very important among viruses is the distinction between
00:11:5301 equilibrium viruses and non-equilibrium viruses.
00:11:5816 Equilibrium viruses have. are viruses that have been, for a very long time, parasites
00:12:0617 of a particular species.
00:12:0820 And so they're highly adapted to that species, to the lifestyle of that species, to the cells
00:12:1405 of that species.
00:12:1623 And what happens is that they evolve to actually be less lethal, because they don't want to
00:12:2209 kill off their hosts.
00:12:2313 If they killed off every member of the species, that virus would have. would die itself.
00:12:2926 And so, they cause enough disease to help their spread, but they don't.
00:12:3706 like causing sneezing or coughing. but they don't kill most people.
00:12:4222 The common cold virus is a very good example of a. an equilibrium virus in humans.
00:12:5007 Then, there are non-equilibrium viruses.
00:12:5324 Non-equilibrium viruses are viruses that have jumped from one species to another.
00:13:0024 They're not yet adapted to that new host.
00:13:0316 They've only been with that new host for a while, even decades.
00:13:1021 And they can be extremely lethal, because this is not a host they care about.
00:13:1521 And so they haven't evolved to be non-lethal in that host.
00:13:2203 They may in fact spread poorly, or they could spread very well.
00:13:2625 They may be very efficient, very inefficient.
00:13:2919 There's enormous variation.
00:13:3218 But those are the viruses that cause us the biggest problems, because those viruses
00:13:3902 can be lethal.
00:13:4002 So, if we talk about equilibrium viruses, we talk about polio.
00:13:4503 Polio, in its day, caused. was. was lethal to a very small fraction of the people
00:13:5312 who were infected by it.
00:13:5509 Most people didn't know they were infected -- 90%+.
00:13:5900 A few people actually were paralyzed by it.
00:14:0226 And a few people were actually killed by it.
00:14:0620 And that's in the nature of an equilibrium virus, largely non-lethal, although occasionally lethal.
00:14:1422 Smallpox actually was. is. is a virus that's lethal to a fair number of people,
00:14:2226 and you really want to get it when you're young and get over it.
00:14:2625 And that was the nature of vaccination against smallpox in the very early days.
00:14:3424 The common cold virus we mentioned.
00:14:3822 Measles virus, another one, actually causes quite severe infections to a few people.
00:14:4514 Most people get over it after being in bed for a couple of days.
00:14:5013 Herpes viruses are all around us and are. keep infecting people, but are generally non-lethal.
00:15:0001 Now, non-equilibrium viruses.
00:15:0122 The most famous one of those is influenza virus.
00:15:0613 It's a natural virus of birds.
00:15:1006 In birds, it's generally not very lethal.
00:15:1328 It comes to humans and it's lethal to older people and very young people.
00:15:2114 And it's really a serious disease for adults to get.
00:15:2722 And that's why we try to vaccinate against it, although our vaccines are nowhere near
00:15:3219 as good as we'd like them to be.
00:15:3609 HIV is a virus that recently came to us from chimpanzees, but in fact it's not even
00:15:4403 a natural virus of chimpanzees.
00:15:4507 It seems to be a natural virus of monkeys.
00:15:5013 And they gave it to chimpanzees, which in turn gave it to us.
00:15:5527 SARS is a virus, probably, from bats.
00:16:0101 Ebola is a virus almost certainly from bats.
00:16:0422 Hantaan was from rodents.
00:16:0811 Those have been very lethal to humans, but generally in contained areas, because they.
00:16:1511 they haven't evolved to be able to spread very effectively.
00:16:1915 Now, let me turn to HIV, because it's a virus that we've heard so much about and that
00:16:2901 has been such a devastating virus to human beings.
00:16:3325 In. in the last few decades, the spread of the human immunodeficiency virus
00:16:4102 throughout the world has been a constant source of fear and news.
00:16:4909 It came into our midst, really, in the early 1980s.
00:16:5401 Before that, it was in human beings, but it was in very limited populations, mainly in Africa.
00:17:0213 Somewhere around the 1980s, it started spreading, coincident with travel.
00:17:0824 It was taken on airplanes in people from its. its source in Africa into Europe,
00:17:1609 into the United States.
00:17:2006 Making sense of. of HIV depended on having discovered the reverse transcriptase.
00:17:2715 So, our work in 1970 actually set the base for finding HIV, because HIV. it uses
00:17:3528 a reverse transcriptase, and it was that enzyme that revealed the nature of that virus.
00:17:4217 So, let me talk about the discovery of reverse transcription for a minute.
00:17:4917 In 1960, I began working on viruses.
00:17:5406 And I realized that in order to understand this lifecycle of viruses the thing to do
00:18:0117 was to examine the enzymes that made viral DNA or RNA.
00:18:0909 And so we actually discovered a number of polymerases in viruses all through the 1960s.
00:18:2104 And then in 1969, we found the polymerases that. that allow negative-strand viruses
00:18:3226 to be made.
00:18:3413 And they, as I mentioned earlier, are in the virus particle.
00:18:3717 And that suggested that if we were going to understand cancer inducing viruses,
00:18:4208 maybe we should look in the virus particle.
00:18:4600 Howard Temin, in the 1960s, had speculated that RNA. that the RNA tumor viruses --
00:18:5127 the viruses that have RNA as their genetic material but cause cancer -- might actually copy their
00:19:0124 RNA into DNA.
00:19:0318 But nobody could find clear evidence that that was true.
00:19:0800 And so in 1970, when we said, let's look in the virus particle, we found the enzyme
00:19:1520 that copies RNA into DNA.
00:19:1801 It was actually, for me, because I was a. had a background in that kind of experimentation,
00:19:2315 a very simple experiment.
00:19:2425 It took a matter of days to discover it.
00:19:2917 And in 1975, Howard Temin and I, along with Renato Dulbecco, who worked on DNA viruses,
00:19:3712 were honored with a Nobel Prize, because the work that the three of us had done
00:19:4309 really set the stage for understanding the genetic nature of cancer.
00:19:4713 And that was a big deal, scientifically, and actually has turned out to be a big deal in
00:19:5513 our treatment of cancer.
00:19:5822 It's nostalgic for me to remember back to the circumstances of doing those experiments
00:20:0607 because I was thinking about this diagram and realized that the.
00:20:1306 the tumor-inducing viruses didn't fit into it.
00:20:1613 And so got ahold of a stock of those viruses to test whether they might have a polymerase.
00:20:2506 And as I said, in a few days, found what we were looking for.
00:20:3200 The reverse transcriptase is an enzyme, now, that's understood in great detail.
00:20:3815 You can see how the DNA or RNA are threaded through the. the polymerase from its structure.
00:20:5122 So, let me say a few words about what the discovery of reverse transcription allowed.
00:21:0009 It allowed various kinds of understanding, and it allowed various kinds of technology.
00:21:0621 The understanding was the understanding that RNA tumor viruses are retroviruses, that they
00:21:1324 reverse transcribe their genome and integrate it into the host cell.
00:21:1911 And that really said that cancer was a consequence of genetic alteration of cells.
00:21:3026 And the other thing is that reverse transcription allowed biotechnologists to capture the
00:21:4104 genetic information from individual genes as messenger RNA that was reverse transcribed in the laboratory.
00:21:5103 And that became a central methodology of biotechnology.
00:21:5704 We also learned from work on the structure of the human genome that as much as 45% of
00:22:0621 the human genome arose by reverse transcription over historic time, mainly by the
00:22:1427 copying of mobile genetic elements, which are like viruses except they don't have an extracellular existence.
00:22:2301 They simply are part of cellular life.
00:22:2719 And finally, the discovery of reverse transcription set the base for, ten years later, the discovery
00:22:3503 of. that. that the AIDS epidemic was being caused by the spread of a virus, HIV,
00:22:4520 and that that virus was a retrovirus, like the tumor viruses that up to then were
00:22:5217 the only ones known.
00:22:5506 So, it's been particularly gratifying to me that this discovery has reverberated down
00:23:0326 through time in so many different ways.
00:23:0626 And it really speaks to the importance of basic research.
00:23:1217 So, if we look at the life cycle of a retrovirus -- any retrovirus -- what it does is to
00:23:2028 make its way into a cell by fusing to the surface of the cell and popping the core of the virus
00:23:3009 into the cell itself.
00:23:3222 There, that core acts as an enzyme to make a DNA copy of the RNA in the virus.
00:23:4120 That in turn integrates itself, forming what's called the provirus.
00:23:4624 And the provirus then acts as a template to make proteins. to make RNA to make proteins.
00:23:5512 And those proteins come together to form new virus particles that bud out from the
00:24:0002 surface of the cell and become a free-living virus looking for a new potential host.
00:24:0705 It's a very interesting life cycle because the virus is actually finding its way
00:24:1324 into the chromosomes of the cell, turning itself from a free-living virus into genes of the cell.
00:24:2112 And that is a unique and remarkable way of the virus hiding out, allowing its transmission
00:24:3124 from one cell to the next cell to the next cell without the virus having to do anything,
00:24:3724 because every time the cell duplicates it duplicates the proviral DNA.
00:24:4426 Here it is budding off the surface of a cell.
00:24:4910 And so, let me just talk for a minute about how awful the AIDS epidemic has been,
00:24:5804 how powerful HIV has been.
00:25:0107 This. these are statistics from 2005.
00:25:0510 And I put them in here because that was sort of the peak of the epidemic.
00:25:1009 By that time, 65 million people had been infected all around the world, the bulk in Africa.
00:25:1701 There were 25 million who had been killed by the virus.
00:25:2222 40 million people were living with HIV, many in India, many in China, but the bulk in.
00:25:3005 in Africa.
00:25:3322 There were, at that time, 13,000 infections per day, 5 million people infected every year.
00:25:4427 It was a really devastating epidemic for the world.
00:25:5111 Now, if we look at it now, or at 2015, when the last statistics were available,
00:26:0108 what we see is an epidemic that is waning some but still really serious.
00:26:0801 So by now, 75 million people have been infected, 35 million have been killed, and there are
00:26:1528 still on the order of 40 million people living with AIDS.
00:26:2100 I've shown the 2005 numbers in parentheses.
00:26:2700 But there are now, as opposed to 2005, 18 million people on antiretrovirals,
00:26:3325 chemicals that prevent the growth of retroviral. of retroviruses.
00:26:4100 And those people are largely very healthy, and are also not transmitting virus,
00:26:4718 because the virus has been knocked down so effectively.
00:26:5207 And that's part of the reason why transmissions are down.
00:26:5524 So, the number of infections is now about 50% of what it was.
00:27:0210 The number dying is only 30% of what it was.
00:27:0819 And this. these retrovirals. antiretrovirals are making all the difference.
00:27:1414 But they're extremely expensive, even expensive in Africa, although much cheaper than here.
00:27:2406 And it's increasing the lifespan of people who are infected, it's lowering the infection rates,
00:27:2926 but it's not clear that it is the final answer.
00:27:3508 The final answer would be to make a vaccine that could prevent the transmission of HIV.
00:27:4306 And we. many, many other people in the scientific community are working on finding a way
00:27:5013 to vaccinate people.
00:27:5113 But this virus, as opposed to any other virus, has been a recalcitrant to our attempts to
00:27:5809 make a vaccine.
00:28:0126 So, HIV is the classic non-equilibrium virus.
00:28:0610 It's, as I say, endemic in African monkeys.
00:28:1015 It got a foothold in the human populations maybe 90 years ago.
00:28:1612 For a long time, it was found only in villages in Africa.
00:28:2107 Then, as. as transportation became more and more commonly used to take people
00:28:3006 from one continent to another, it seeded other continents and became a worldwide epidemic.
00:28:3800 It grows in one of the key cell types of our immune system, and so it actually knocks out
00:28:4419 our immune systems.
00:28:4609 And that's what. why it's a lethal virus.
00:28:4910 We die of some infection, not of HIV infection.
00:28:5807 And with that, I've finished with this discussion of viruses and of HIV.
- Part 1: Introduction to Viruses