Reverse transcriptase in the context of DNA microarray


Reverse transcriptase in the context of DNA microarray

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⭐ Core Definition: Reverse transcriptase

A reverse transcriptase (RT) is an enzyme used to convert RNA to DNA, a process termed reverse transcription. Reverse transcriptases are used by viruses such as HIV and hepatitis B to replicate their genomes, by retrotransposon mobile genetic elements to proliferate within the host genome, and by eukaryotic cells to extend the telomeres at the ends of their linear chromosomes. The process does not violate the flows of genetic information as described by the classical central dogma, but rather expands it to include transfers of information from RNA to DNA.

Retroviral RT has three sequential biochemical activities: RNA-dependent DNA polymerase activity, ribonuclease H (RNase H), and DNA-dependent DNA polymerase activity. Collectively, these activities enable the enzyme to convert single-stranded RNA into double-stranded cDNA. In retroviruses and retrotransposons, this cDNA can then integrate into the host genome, from which new RNA copies can be made via host-cell transcription. The same sequence of reactions is widely used in the laboratory to convert RNA to DNA for use in molecular cloning, RNA sequencing, polymerase chain reaction (PCR), or genome analysis.

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Reverse transcriptase in the context of Telomerase

Telomerase, also called terminal transferase, is a ribonucleoprotein that adds a species-dependent telomere repeat sequence to the 3' end of telomeres. A telomere is a region of repetitive sequences at each end of the chromosomes of most eukaryotes. Telomeres protect the end of the chromosome from DNA damage or from fusion with neighbouring chromosomes. The fruit fly Drosophila melanogaster lacks telomerase, but instead uses retrotransposons to maintain telomeres.

Telomerase is a reverse transcriptase enzyme that carries its own RNA molecule (e.g., with the sequence 3′-CCCAAUCCC-5′ in Trypanosoma brucei) which is used as a template when it elongates telomeres. Telomerase is active in gametes and most cancer cells, but is normally absent in most somatic cells.

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Reverse transcriptase in the context of Complementary DNA

In genetics, complementary DNA (cDNA) is DNA that was reverse transcribed (via reverse transcriptase) from an RNA (e.g., messenger RNA or microRNA). cDNA exists in both single-stranded and double-stranded forms and in both natural and engineered forms.

In engineered forms, it often is a copy (replicate) of the naturally occurring DNA from any particular organism's natural genome; the organism's own mRNA was naturally transcribed from its DNA, and the cDNA is reverse transcribed from the mRNA, yielding a duplicate of the original DNA. Engineered cDNA is often used to express a specific protein in a cell that does not normally express that protein (i.e., heterologous expression), or to sequence or quantify mRNA molecules using DNA based methods (qPCR, RNA-seq). cDNA that codes for a specific protein can be transferred to a recipient cell for expression as part of recombinant DNA, often bacterial or yeast expression systems. cDNA is also generated to analyze transcriptomic profiles in bulk tissue, single cells, or single nuclei in assays such as microarrays, qPCR, and RNA-seq.

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Reverse transcriptase in the context of DNA virus

A DNA virus is a virus that has a genome made of deoxyribonucleic acid (DNA) that is replicated by a DNA polymerase. They can be divided between those that have two strands of DNA in their genome, called double-stranded DNA (dsDNA) viruses, and those that have one strand of DNA in their genome, called single-stranded DNA (ssDNA) viruses. dsDNA viruses primarily belong to two realms: Duplodnaviria and Varidnaviria, and ssDNA viruses are almost exclusively assigned to the realm Monodnaviria, which also includes some dsDNA viruses. Additionally, many DNA viruses are unassigned to higher taxa. Reverse transcribing viruses, which have a DNA genome that is replicated through an RNA intermediate by a reverse transcriptase, are classified into the kingdom Pararnavirae in the realm Riboviria.

DNA viruses are ubiquitous worldwide, especially in marine environments where they form an important part of marine ecosystems, and infect both prokaryotes and eukaryotes. They appear to have multiple origins, as viruses in Monodnaviria appear to have emerged from archaeal and bacterial plasmids on multiple occasions, though the origins of Duplodnaviria and Varidnaviria are less clear.

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Reverse transcriptase in the context of Retrovirus

A retrovirus is a type of virus that inserts a DNA copy of its RNA genome into the DNA of a host cell that it invades, thus changing the genome of that cell. After invading a host cell's cytoplasm, the virus uses its own reverse transcriptase enzyme to produce DNA from its RNA genome, the reverse of the usual pattern, thus retro (backward). The new DNA is then incorporated into the host cell genome by an integrase enzyme, at which point the retroviral DNA is referred to as a provirus. The host cell then treats the viral DNA as part of its own genome, transcribing and translating the viral genes along with the cell's own genes, producing the proteins required to assemble new copies of the virus. Many retroviruses cause serious diseases in humans, other mammals, and birds.

Retroviruses have many subfamilies in three basic groups.

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Reverse transcriptase in the context of Pseudogene

Pseudogenes are nonfunctional segments of DNA that resemble functional genes. Pseudogenes can be formed from both protein-coding genes and non-coding genes. In the case of protein-coding genes, most pseudogenes arise as superfluous copies of functional genes, either directly by gene duplication or indirectly by reverse transcription of an mRNA transcript. Pseudogenes are usually identified when genome sequence analysis finds gene-like sequences that lack regulatory sequences or are incapable of producing a functional product. Pseudogenes are a type of junk DNA.

Most non-bacterial genomes contain many pseudogenes, often as many as functional genes. This is not surprising, since various biological processes are expected to accidentally create pseudogenes, and there are no specialized mechanisms to remove them from genomes. Eventually pseudogenes may be deleted from their genomes by chance of DNA replication or DNA repair errors, or they may accumulate so many mutational changes that they are no longer recognizable as former genes. Analysis of these degeneration events helps clarify the effects of non-selective processes in genomes.

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Reverse transcriptase in the context of Retrotransposon

Retrotransposons (also called Class I transposable elements) are mobile elements which move in the host genome by converting their transcribed RNA into DNA through reverse transcription. Thus, they differ from Class II transposable elements, or DNA transposons, in utilizing an RNA intermediate for the transposition and leaving the transposition donor site unchanged.

Through reverse transcription, retrotransposons amplify themselves quickly to become abundant in eukaryotic genomes such as maize (49–78%) and humans (42%). They are only present in eukaryotes but share features with retroviruses such as HIV, for example, discontinuous reverse transcriptase-mediated extrachromosomal recombination.

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Reverse transcriptase in the context of Zidovudine

Zidovudine (ZDV), also known as azidothymidine (AZT), was the first antiretroviral medication used to prevent and treat HIV/AIDS. It is generally recommended for use in combination with other antiretrovirals. It may be used to prevent mother-to-child spread during birth or after a needlestick injury or other potential exposure. It is sold both by itself and together as lamivudine/zidovudine and abacavir/lamivudine/zidovudine. It can be used by mouth or by slow injection into a vein.

Common side effects include headaches, fever, and nausea. Serious side effects include liver problems, muscle damage, and high blood lactate levels. It is commonly used in pregnancy and appears to be safe for the fetus. ZDV is of the nucleoside analog reverse-transcriptase inhibitor (NRTI) class. It works by inhibiting the enzyme reverse transcriptase that HIV uses to make DNA and therefore decreases replication of the virus.

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Reverse transcriptase in the context of Realm (virology)

In virology, realm is the highest taxonomic rank established for viruses by the International Committee on Taxonomy of Viruses (ICTV), which oversees virus taxonomy. Six virus realms are recognized and united by specific highly conserved traits:

The rank of realm corresponds to the rank of domain used for cellular life, but differs in that viruses in a realm do not necessarily share a common ancestor based on common descent nor do the realms share a common ancestor. Instead, realms group viruses together based on specific traits that are highly conserved over time, which may have been obtained on a single occasion or multiple occasions. As such, each realm represents at least one instance of viruses coming into existence. While historically it was difficult to determine deep evolutionary relations between viruses, in the 21st century methods such as metagenomics and cryogenic electron microscopy have enabled such research to occur, which led to the establishment of Riboviria in 2018, three realms in 2019, and two in 2020.

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Reverse transcriptase in the context of Pararnavirae

Revtraviricetes is a class of viruses that contains all viruses that encode a reverse transcriptase. The group includes all ssRNA-RT viruses (including the retroviruses) and dsDNA-RT viruses. It is the sole class in the phylum Artverviricota, which is the sole phylum in the kingdom Pararnavirae. The name of the group is a portmanteau of "reverse transcriptase" and -viricetes which is the suffix for a virus class.

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Reverse transcriptase in the context of Riboviria

Riboviria is a realm of viruses that includes all viruses that use a homologous RNA-dependent polymerase for replication. It includes RNA viruses that encode an RNA-dependent RNA polymerase, as well as reverse-transcribing viruses (with either RNA or DNA genomes) that encode an RNA-dependent DNA polymerase. RNA-dependent RNA polymerase (RdRp), also called RNA replicase, produces RNA (ribonucleic acid) from RNA. RNA-dependent DNA polymerase (RdDp), also called reverse transcriptase (RT), produces DNA (deoxyribonucleic acid) from RNA. These enzymes are essential for replicating the viral genome and transcribing viral genes into messenger RNA (mRNA) for translation of viral proteins.

Riboviria was established in 2018 to accommodate all RdRp-encoding RNA viruses and was expanded a year later to also include RdDp-encoding viruses. These two groups of viruses are assigned to two separate kingdoms: Orthornavirae for RdRp-encoding RNA viruses, and Pararnavirae for RdDp-encoding viruses, i.e. all reverse-transcribing viruses. Most identified eukaryotic viruses belong to the realm, including most human, animal, and plant viruses. Historically, few prokaryotic RNA viruses had been discovered to be included in the realm, but in the 2020s metagenomic and metatranscriptomic studies have discovered many prokaryotic RNA viruses.

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