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134-460: An important application of the Cre-lox system is excision of selectable markers in gene replacement. Commonly used gene replacement strategies introduce selectable markers into the genome to facilitate selection of genetic mutations that may cause growth retardation. However, marker expression can have polar effects on the expression of upstream and downstream genes. Removal of selectable markers from

268-554: A double helix . The polymer carries genetic instructions for the development, functioning, growth and reproduction of all known organisms and many viruses . DNA and ribonucleic acid (RNA) are nucleic acids . Alongside proteins , lipids and complex carbohydrates ( polysaccharides ), nucleic acids are one of the four major types of macromolecules that are essential for all known forms of life . The two DNA strands are known as polynucleotides as they are composed of simpler monomeric units called nucleotides . Each nucleotide

402-439: A in cis cassette exchange event. The loxP mutants are created to avoid these problems. During genetic recombination, a Holliday junction is formed between the two strands of DNA and a double-stranded break in a DNA molecule leaves a 3’OH end exposed. This reaction is aided with the endonuclease activity of an enzyme. 5’ Phosphate ends are usually the substrates for this reaction, thus extended 3’ regions remain. This 3’ OH group

536-495: A 5’ hydroxyl group. This process occurs in the active site of two out of the four recombinase subunits present at the synapse tetramer. If the 5’ hydroxyl groups attack the 3’-phosphotyrosine linkage one pair of the DNA strands will exchange to form a Holliday junction intermediate. Cre recombinase plays important roles in the life cycle of the P1 bacteriophage . Upon infection of a cell

670-445: A buffer to recruit or titrate ions or antibiotics. Extracellular DNA acts as a functional extracellular matrix component in the biofilms of several bacterial species. It may act as a recognition factor to regulate the attachment and dispersal of specific cell types in the biofilm; it may contribute to biofilm formation; and it may contribute to the biofilm's physical strength and resistance to biological stress. Cell-free fetal DNA

804-413: A cell makes up its genome ; the human genome has approximately 3 billion base pairs of DNA arranged into 46 chromosomes. The information carried by DNA is held in the sequence of pieces of DNA called genes . Transmission of genetic information in genes is achieved via complementary base pairing. For example, in transcription, when a cell uses the information in a gene, the DNA sequence is copied into

938-490: A chemical signal or a heat shock. These targeted DNA changes are useful in cell lineage tracing and when mutants are lethal if expressed globally. The Cre-Lox system is very similar in action and in usage to the FLP-FRT recombination system. Cre-Lox recombination is a special type of site-specific recombination developed by Dr. Brian Sauer and patented by DuPont that operated in both mitotic and non-mitotic cells, and

1072-482: A circular loop of DNA whilst intervening DNA between two loxP sites that are opposingly orientated will be inverted. The enzyme requires no additional cofactors (such as ATP ) or accessory proteins for its function. The enzyme plays important roles in the life cycle of the P1 bacteriophage, such as cyclization of the linear genome and resolution of dimeric chromosomes that form after DNA replication . Cre recombinase

1206-445: A complementary RNA sequence through the attraction between the DNA and the correct RNA nucleotides. Usually, this RNA copy is then used to make a matching protein sequence in a process called translation , which depends on the same interaction between RNA nucleotides. In an alternative fashion, a cell may copy its genetic information in a process called DNA replication . The details of these functions are covered in other articles; here

1340-497: A deletion event. If loxP sites are on different chromosomes it is possible for translocation events to be catalysed by Cre induced recombination. Two plasmids can be joined using the variant lox sites 71 and 66. Cre recombinase can be synthesized by the corresponding gene under the direction of cell-specific promoters, including promoters under the control of doxycycline. An additional level of control can be achieved by using his Cre recombinase, engineered to reversibly activate in

1474-409: A directional core sequence where recombination can occur. When cells that have loxP sites in their genome express Cre, a recombination event can occur between the loxP sites. Cre recombinase proteins bind to the first and last 13 bp regions of a lox site forming a dimer . This dimer then binds to a dimer on another lox site to form a tetramer . Lox sites are directional and the two sites joined by

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1608-492: A double helix can thus be pulled apart like a zipper, either by a mechanical force or high temperature . As a result of this base pair complementarity, all the information in the double-stranded sequence of a DNA helix is duplicated on each strand, which is vital in DNA replication. This reversible and specific interaction between complementary base pairs is critical for all the functions of DNA in organisms. Most DNA molecules are actually two polymer strands, bound together in

1742-428: A full set of the mitochondrial genes. Each human mitochondrion contains, on average, approximately 5 such mtDNA molecules. Each human cell contains approximately 100 mitochondria, giving a total number of mtDNA molecules per human cell of approximately 500. However, the amount of mitochondria per cell also varies by cell type, and an egg cell can contain 100,000 mitochondria, corresponding to up to 1,500,000 copies of

1876-500: A genetic tool to control site specific recombination events in genomic DNA. This system has allowed researchers to manipulate a variety of genetically modified organisms to control gene expression, delete undesired DNA sequences and modify chromosome architecture. The Cre protein is a site-specific DNA recombinase that can catalyse the recombination of DNA between specific sites in a DNA molecule. These sites, known as loxP sequences, contain specific binding sites for Cre that surround

2010-439: A helical fashion by noncovalent bonds; this double-stranded (dsDNA) structure is maintained largely by the intrastrand base stacking interactions, which are strongest for G,C stacks. The two strands can come apart—a process known as melting—to form two single-stranded DNA (ssDNA) molecules. Melting occurs at high temperatures, low salt and high pH (low pH also melts DNA, but since DNA is unstable due to acid depurination, low pH

2144-571: A higher number is also possible but this would be against the natural principle of least effort . The phosphate groups of DNA give it similar acidic properties to phosphoric acid and it can be considered as a strong acid . It will be fully ionized at a normal cellular pH, releasing protons which leave behind negative charges on the phosphate groups. These negative charges protect DNA from breakdown by hydrolysis by repelling nucleophiles which could hydrolyze it. Pure DNA extracted from cells forms white, stringy clumps. The expression of genes

2278-667: A long-standing puzzle known as the " C-value enigma ". However, some DNA sequences that do not code protein may still encode functional non-coding RNA molecules, which are involved in the regulation of gene expression . Some noncoding DNA sequences play structural roles in chromosomes. Telomeres and centromeres typically contain few genes but are important for the function and stability of chromosomes. An abundant form of noncoding DNA in humans are pseudogenes , which are copies of genes that have been disabled by mutation. These sequences are usually just molecular fossils , although they can occasionally serve as raw genetic material for

2412-409: A narrower, deeper major groove. The A form occurs under non-physiological conditions in partly dehydrated samples of DNA, while in the cell it may be produced in hybrid pairings of DNA and RNA strands, and in enzyme-DNA complexes. Segments of DNA where the bases have been chemically modified by methylation may undergo a larger change in conformation and adopt the Z form . Here, the strands turn about

2546-431: A process called translation . Within eukaryotic cells, DNA is organized into long structures called chromosomes . Before typical cell division , these chromosomes are duplicated in the process of DNA replication, providing a complete set of chromosomes for each daughter cell. Eukaryotic organisms ( animals , plants , fungi and protists ) store most of their DNA inside the cell nucleus as nuclear DNA , and some in

2680-442: A radius of 10 Å (1.0 nm). According to another study, when measured in a different solution, the DNA chain measured 22–26 Å (2.2–2.6 nm) wide, and one nucleotide unit measured 3.3 Å (0.33 nm) long. The buoyant density of most DNA is 1.7g/cm . DNA does not usually exist as a single strand, but instead as a pair of strands that are held tightly together. These two long strands coil around each other, in

2814-416: A second protein when read in the opposite direction along the other strand. In bacteria , this overlap may be involved in the regulation of gene transcription, while in viruses, overlapping genes increase the amount of information that can be encoded within the small viral genome. DNA can be twisted like a rope in a process called DNA supercoiling . With DNA in its "relaxed" state, a strand usually circles

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2948-412: A series of short loops. Helices A & E are involved in the formation of the recombinase tetramer with the C terminus region of helix E known to form contacts with the C terminal domain of adjacent subunits. Helices B & D form direct contacts with the major groove of the loxP DNA. These two helices are thought to make three direct contacts to DNA bases at the loxP site. The carboxy terminal domain of

3082-445: A simple TTAGGG sequence. These guanine-rich sequences may stabilize chromosome ends by forming structures of stacked sets of four-base units, rather than the usual base pairs found in other DNA molecules. Here, four guanine bases, known as a guanine tetrad , form a flat plate. These flat four-base units then stack on top of each other to form a stable G-quadruplex structure. These structures are stabilized by hydrogen bonding between

3216-421: A single loxP site two active sites are present. Cre mediated recombination requires the formation of a synapse in which two Cre-LoxP complexes associate to form what is known as the synapse tetramer in which 4 distinct active sites are present. Tyr 324 acts as a nucleophile to form a covalent 3’-phosphotyrosine linkage to the DNA substrate. The scissile phosphate (phosphate targeted for nucleophilic attack at

3350-472: A vast number of organisms, making it a particularly useful tool in scientific research. Studies carried out in 1981 by Sternberg and Hamilton demonstrated that the bacteriophage ' P1 ' had a unique site specific recombination system. EcoRI fragments of the P1 bacteriophage genome were generated and cloned into lambda vectors . A 6.5kb EcoRI fragment (Fragment 7) was found to permit efficient recombination events. The mechanism of these recombination events

3484-480: Is a temperate phage that causes either a lysogenic or lytic cycle when it infects a bacterium. In its lytic state, once its viral genome is injected into the host cell, viral proteins are produced, virions are assembled, and the host cell is lysed to release the phages, continuing the cycle. In the lysogenic cycle the phage genome replicates with the rest of the bacterial genome and is transmitted to daughter cells at each subsequent cell division. It can transition to

3618-482: Is a widely used tool in the field of molecular biology . The enzyme's unique and specific recombination system is exploited to manipulate genes and chromosomes in a huge range of research, such as gene knock out or knock in studies. The enzyme's ability to operate efficiently in a wide range of cellular environments (including mammals, plants, bacteria, and yeast) enables the Cre-Lox recombination system to be used in

3752-465: Is also an important process that viruses, such as bacteriophages, adopt to integrate their genetic material into the infected host. The virus, called a prophage in such a state, accomplishes this via integration and excision. The points where the integration and excision reactions occur are called the attachment (att) sites. An attP site on the phage exchanges segments with an attB site on the bacterial DNA. Thus, these are site-specific, occurring only at

3886-480: Is also important as low expression of Cre recombinase tends to result in non-parallel recombination. Non-parallel recombination is especially problematic in a fate mapping scenario where one recombination event is designed to manipulate the gene under study and the other recombination event is necessary for activating a reporter gene (usually encoding a fluorescent protein) for cell lineage tracing. Failure to activate both recombination events simultaneously confounds

4020-514: Is based on double cross-integration of non-replicating vectors into the genome. Furthermore, recombination systems such as Cre-lox are widely used, mostly in eukaryotes. The versatile properties of Cre recombinase make it ideal for use in many genetic engineering strategies. As such, the Cre lox system has been used in a wide variety of eukaryotes, including plants. Multiple variants of loxP, in particular lox2272 and loxN, have been used by researchers with

4154-498: Is broken. This reaction synthesizes the Holliday junction discussed earlier. In this fashion, opposite DNA strands are joined. Subsequent cleavage and rejoining cause DNA strands to exchange their segments. Protein-protein interactions drive and direct strand exchange. Energy is not compromised, since the protein-DNA linkage makes up for the loss of the phosphodiester bond, which occurred during cleavage. Site-specific recombination

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4288-447: Is called intercalation . Most intercalators are aromatic and planar molecules; examples include ethidium bromide , acridines , daunomycin , and doxorubicin . For an intercalator to fit between base pairs, the bases must separate, distorting the DNA strands by unwinding of the double helix. This inhibits both transcription and DNA replication, causing toxicity and mutations. As a result, DNA intercalators may be carcinogens , and in

4422-435: Is called a polynucleotide . The backbone of the DNA strand is made from alternating phosphate and sugar groups. The sugar in DNA is 2-deoxyribose , which is a pentose (five- carbon ) sugar. The sugars are joined by phosphate groups that form phosphodiester bonds between the third and fifth carbon atoms of adjacent sugar rings. These are known as the 3′-end (three prime end), and 5′-end (five prime end) carbons,

4556-445: Is composed of one of four nitrogen-containing nucleobases ( cytosine [C], guanine [G], adenine [A] or thymine [T]), a sugar called deoxyribose , and a phosphate group . The nucleotides are joined to one another in a chain by covalent bonds (known as the phosphodiester linkage ) between the sugar of one nucleotide and the phosphate of the next, resulting in an alternating sugar-phosphate backbone . The nitrogenous bases of

4690-434: Is dependent on ionic strength and the concentration of DNA. As a result, it is both the percentage of GC base pairs and the overall length of a DNA double helix that determines the strength of the association between the two strands of DNA. Long DNA helices with a high GC -content have more strongly interacting strands, while short helices with high AT content have more weakly interacting strands. In biology, parts of

4824-480: Is done by a special group of enzymes called Resolvases. RuvC is just one of these Resolvases that have been isolated in bacteria and yeast. For many years, it was thought that when the Holliday junction intermediate was formed, the branch point of the junction (where the strands cross over) would be located at the first cleavage site. Migration of the branch point to the second cleavage site would then somehow trigger

4958-470: Is elongated (that is, bases are added) with the help of DNA Polymerase. The pairing of opposite strands is what constitutes the crossing-over or Recombination event, which is common to all living organisms, since the genetic material on one strand of one duplex has paired with one strand of another duplex, and has been elongated by DNA polymerase. Further cleavage of Holliday Intermediates results in formation of Hybrid DNA. This further cleavage or ‘resolvation’

5092-530: Is excised; however if the two loxP sites are in the opposite orientation, the floxed sequence is inverted. If there exists a floxed donor sequence, the donor sequence can be swapped with the original sequence. This technique is called recombinase-mediated cassette exchange and is a very convenient and time-saving way for genetic manipulation. The caveat, however, is that the recombination reaction can happen backwards, rendering cassette exchange inefficient. In addition, sequence excision can happen in trans instead of

5226-413: Is given below; 'N' indicates bases which may vary, and lowercase letters indicate bases that have been mutated from the wild-type. The 13 bp sequences are palindromic but the 8 bp spacer is not, thus giving the loxP sequence a certain direction. Usually loxP sites come in pairs for genetic manipulation. If the two loxP sites are in the same orientation, the floxed sequence (sequence flanked by two loxP sites)

5360-400: Is highly unstable, and the strand on which it is present must find its complement. Since homologous recombination occurs after DNA replication, two strands of DNA are available, and thus, the 3’ OH group must pair with its complement, and it does so, with an intact strand on the other duplex. Now, one point of crossover has occurred, which is what is called a Holliday Intermediate. The 3’OH end

5494-411: Is influenced by how the DNA is packaged in chromosomes, in a structure called chromatin . Base modifications can be involved in packaging, with regions that have low or no gene expression usually containing high levels of methylation of cytosine bases. DNA packaging and its influence on gene expression can also occur by covalent modifications of the histone protein core around which DNA is wrapped in

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5628-432: Is introduced by enzymes called topoisomerases . These enzymes are also needed to relieve the twisting stresses introduced into DNA strands during processes such as transcription and DNA replication . DNA exists in many possible conformations that include A-DNA , B-DNA , and Z-DNA forms, although only B-DNA and Z-DNA have been directly observed in functional organisms. The conformation that DNA adopts depends on

5762-422: Is nothing special about the four natural nucleobases that evolved on Earth. On the other hand, DNA is tightly related to RNA which does not only act as a transcript of DNA but also performs as molecular machines many tasks in cells. For this purpose it has to fold into a structure. It has been shown that to allow to create all possible structures at least four bases are required for the corresponding RNA , while

5896-517: Is rarely used). The stability of the dsDNA form depends not only on the GC -content (% G,C basepairs) but also on sequence (since stacking is sequence specific) and also length (longer molecules are more stable). The stability can be measured in various ways; a common way is the melting temperature (also called T m value), which is the temperature at which 50% of the double-strand molecules are converted to single-strand molecules; melting temperature

6030-428: Is recreated by an enzyme called DNA polymerase . This enzyme makes the complementary strand by finding the correct base through complementary base pairing and bonding it onto the original strand. As DNA polymerases can only extend a DNA strand in a 5′ to 3′ direction, different mechanisms are used to copy the antiparallel strands of the double helix. In this way, the base on the old strand dictates which base appears on

6164-455: Is similar in structure to the domain in the Integrase family of enzymes isolated from lambda phage . This is also the catalytic site of the enzyme. loxP (locus of X-over P1) is a site on the bacteriophage P1 consisting of 34 bp . The site includes an asymmetric 8 bp sequence, variable except for the middle two bases, in between two sets of symmetric, 13 bp sequences. The exact sequence

6298-428: Is the sequence of these four nucleobases along the backbone that encodes genetic information. RNA strands are created using DNA strands as a template in a process called transcription , where DNA bases are exchanged for their corresponding bases except in the case of thymine (T), for which RNA substitutes uracil (U). Under the genetic code , these RNA strands specify the sequence of amino acids within proteins in

6432-516: Is the largest human chromosome with approximately 220 million base pairs , and would be 85 mm long if straightened. In eukaryotes , in addition to nuclear DNA , there is also mitochondrial DNA (mtDNA) which encodes certain proteins used by the mitochondria. The mtDNA is usually relatively small in comparison to the nuclear DNA. For example, the human mitochondrial DNA forms closed circular molecules, each of which contains 16,569 DNA base pairs, with each such molecule normally containing

6566-489: Is to allow the cell to replicate chromosome ends using the enzyme telomerase , as the enzymes that normally replicate DNA cannot copy the extreme 3′ ends of chromosomes. These specialized chromosome caps also help protect the DNA ends, and stop the DNA repair systems in the cell from treating them as damage to be corrected. In human cells , telomeres are usually lengths of single-stranded DNA containing several thousand repeats of

6700-657: The DNA sequence . Mutagens include oxidizing agents , alkylating agents and also high-energy electromagnetic radiation such as ultraviolet light and X-rays . The type of DNA damage produced depends on the type of mutagen. For example, UV light can damage DNA by producing thymine dimers , which are cross-links between pyrimidine bases. On the other hand, oxidants such as free radicals or hydrogen peroxide produce multiple forms of damage, including base modifications, particularly of guanosine, and double-strand breaks. A typical human cell contains about 150,000 bases that have suffered oxidative damage. Of these oxidative lesions,

6834-533: The P1 bacteriophage . The enzyme uses a topoisomerase I -like mechanism to carry out site specific recombination events. The enzyme (38 kDa) is a member of the integrase family of site specific recombinase and it is known to catalyse the site specific recombination event between two DNA recognition sites ( LoxP sites ). This 34 base pair (bp) loxP recognition site consists of two 13 bp palindromic sequences which flank an 8bp spacer region. The products of Cre-mediated recombination at loxP sites are dependent upon

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6968-406: The amino-acid sequences of proteins is determined by the rules of translation , known collectively as the genetic code . The genetic code consists of three-letter 'words' called codons formed from a sequence of three nucleotides (e.g. ACT, CAG, TTT). In transcription, the codons of a gene are copied into messenger RNA by RNA polymerase . This RNA copy is then decoded by a ribosome that reads

7102-420: The mitochondria as mitochondrial DNA or in chloroplasts as chloroplast DNA . In contrast, prokaryotes ( bacteria and archaea ) store their DNA only in the cytoplasm , in circular chromosomes . Within eukaryotic chromosomes, chromatin proteins, such as histones , compact and organize DNA. These compacting structures guide the interactions between DNA and other proteins, helping control which parts of

7236-419: The 3′ and 5′ carbons along the sugar-phosphate backbone confers directionality (sometimes called polarity) to each DNA strand. In a nucleic acid double helix , the direction of the nucleotides in one strand is opposite to their direction in the other strand: the strands are antiparallel . The asymmetric ends of DNA strands are said to have a directionality of five prime end (5′ ), and three prime end (3′), with

7370-591: The 5′ end having a terminal phosphate group and the 3′ end a terminal hydroxyl group. One major difference between DNA and RNA is the sugar, with the 2-deoxyribose in DNA being replaced by the related pentose sugar ribose in RNA. The DNA double helix is stabilized primarily by two forces: hydrogen bonds between nucleotides and base-stacking interactions among aromatic nucleobases. The four bases found in DNA are adenine ( A ), cytosine ( C ), guanine ( G ) and thymine ( T ). These four bases are attached to

7504-447: The Cre recombinase, resulting in Cre becoming specifically activated by tamoxifen. In the absence of tamoxifen, CreER will result in the shuttling of the mutated recombinase into the cytoplasm. The protein will stay in this location in its inactivated state until tamoxifen is given. Once tamoxifen is introduced, it is metabolized into 4-hydroxytamoxifen, which then binds to the ER and results in

7638-531: The Cre-loxP system is used to cause circularization of the P1 DNA. In addition to this Cre is also used to resolve dimeric lysogenic P1 DNA that forms during the cell division of the phage. Inducible Cre activation is achieved using CreER (estrogen receptor) variant, which is only activated after delivery of tamoxifen . This is done through the fusion of a mutated ligand binding domain of the estrogen receptor to

7772-435: The DNA are transcribed. DNA is a long polymer made from repeating units called nucleotides . The structure of DNA is dynamic along its length, being capable of coiling into tight loops and other shapes. In all species it is composed of two helical chains, bound to each other by hydrogen bonds . Both chains are coiled around the same axis, and have the same pitch of 34 ångströms (3.4  nm ). The pair of chains have

7906-460: The DNA double helix that need to separate easily, such as the TATAAT Pribnow box in some promoters , tend to have a high AT content, making the strands easier to pull apart. In the laboratory, the strength of this interaction can be measured by finding the melting temperature T m necessary to break half of the hydrogen bonds. When all the base pairs in a DNA double helix melt,

8040-497: The DNA polymerase beta gene was deleted in T cells based on DNA blotting. It was unclear whether only one allele in each T-cell or 50% of T cells had 100% deletion in both alleles. Researchers have since reported more efficient Cre-Lox conditional gene mutagenesis in the developing T cells by the Marth laboratory in 1995. Incomplete deletion by Cre recombinase is not uncommon in cells when two copies of floxed sequences exist, and allows

8174-661: The Holliday intermediate), coupled to an isomerisation event that is responsible for switching the strand cleavage specificity. Site-specific recombination (SSR) involves specific sites for the catalyzing action of special enzymes called recombinases. Cre, or cyclic recombinase, is one such enzyme. Site-specific recombination is, thus, the enzyme-mediated cleavage and ligation of two defined deoxynucleotide sequences. A number of conserved site-specific recombination systems have been described in both prokaryotic and eukaryotic organisms. In general, these systems use one or more proteins and act on unique asymmetric DNA sequences. The products of

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8308-401: The RNA sequence by base-pairing the messenger RNA to transfer RNA , which carries amino acids. Since there are 4 bases in 3-letter combinations, there are 64 possible codons (4  combinations). These encode the twenty standard amino acids , giving most amino acids more than one possible codon. There are also three 'stop' or 'nonsense' codons signifying the end of the coding region; these are

8442-491: The TAG, TAA, and TGA codons, (UAG, UAA, and UGA on the mRNA). Cell division is essential for an organism to grow, but, when a cell divides, it must replicate the DNA in its genome so that the two daughter cells have the same genetic information as their parent. The double-stranded structure of DNA provides a simple mechanism for DNA replication . Here, the two strands are separated and then each strand's complementary DNA sequence

8576-475: The absence of tamoxifen, due to leakage of a few Cre recombinase molecules into the nucleus which, in combination with very sensitive reporters, results in unintended cell labelling. CreER(T2) was developed to minimize tamoxifen-independent recombination and maximize tamoxifen-sensitivity. Cells alter their phenotype in response to numerous environmental stimuli and can lose the expression of genes typically used to mark their identity, making it difficult to research

8710-442: The axis of the double helix once every 10.4 base pairs, but if the DNA is twisted the strands become more tightly or more loosely wound. If the DNA is twisted in the direction of the helix, this is positive supercoiling, and the bases are held more tightly together. If they are twisted in the opposite direction, this is negative supercoiling, and the bases come apart more easily. In nature, most DNA has slight negative supercoiling that

8844-407: The canonical bases plus uracil. Twin helical strands form the DNA backbone. Another double helix may be found tracing the spaces, or grooves, between the strands. These voids are adjacent to the base pairs and may provide a binding site . As the strands are not symmetrically located with respect to each other, the grooves are unequally sized. The major groove is 22 ångströms (2.2 nm) wide, while

8978-409: The carboxy domain and this helix is reputed to play a role in mediating interactions with other subunits. Crystal structures demonstrate that this terminal N helix buries its hydrophobic surface into an acceptor pocket of an adjacent Cre subunit. The effect of the two-domain structure is to form a C-shaped clamp that grasps the DNA from opposite sides. The active site of the Cre enzyme consists of

9112-467: The case of thalidomide, a teratogen . Others such as benzo[ a ]pyrene diol epoxide and aflatoxin form DNA adducts that induce errors in replication. Nevertheless, due to their ability to inhibit DNA transcription and replication, other similar toxins are also used in chemotherapy to inhibit rapidly growing cancer cells. DNA usually occurs as linear chromosomes in eukaryotes , and circular chromosomes in prokaryotes . The set of chromosomes in

9246-581: The cell (see below) , but the major and minor grooves are always named to reflect the differences in width that would be seen if the DNA was twisted back into the ordinary B form . In a DNA double helix, each type of nucleobase on one strand bonds with just one type of nucleobase on the other strand. This is called complementary base pairing . Purines form hydrogen bonds to pyrimidines, with adenine bonding only to thymine in two hydrogen bonds, and cytosine bonding only to guanine in three hydrogen bonds. This arrangement of two nucleotides binding together across

9380-425: The cell type of choice. Models thus obtained are more likely to mimic the physiological situation. The Cre protein (encoded by the locus originally named as "Causes recombination", with "Cyclization recombinase" being found in some references) consists of 4 subunits and two domains: The larger carboxyl ( C-terminal ) domain, and smaller amino ( N-terminal ) domain. The total protein has 343 amino acids . The C domain

9514-619: The chromatin structure or else by remodeling carried out by chromatin remodeling complexes (see Chromatin remodeling ). There is, further, crosstalk between DNA methylation and histone modification, so they can coordinately affect chromatin and gene expression. For one example, cytosine methylation produces 5-methylcytosine , which is important for X-inactivation of chromosomes. The average level of methylation varies between organisms—the worm Caenorhabditis elegans lacks cytosine methylation, while vertebrates have higher levels, with up to 1% of their DNA containing 5-methylcytosine. Despite

9648-405: The cleavage site) is coordinated by the side chains of the 3 amino acid residues of the catalytic triad ( Arg 173, His 289 & Trp 315). The indole nitrogen of tryptophan 315 also forms a hydrogen bond to this scissile phosphate. (n.b A Histidine occupies this site in other tyrosine recombinase family members and performs the same function). This reaction cleaves the DNA and frees

9782-442: The combination of different Cre actions (transient or constitutive) to create a " Brainbow " system that allows multi-colouring of mice's brain with four fluorescent proteins. Another report using two lox variants pair but through regulating the length of DNA in one pair results in stochastic gene activation with regulated level of sparseness. Cre recombinase Cre recombinase is a tyrosine recombinase enzyme derived from

9916-538: The complex relationship between specific cells/circuits and behaviors for brain research, has promoted the NIH to initiate the NIH Blueprint for Neuroscience Research Cre-driver mouse projects in early 2000. To date, NIH Blueprint for Neuroscience Research Cre projects have created several hundreds of Cre driver mouse lines which are currently used by the worldwide neuroscience community. Cre-Lox recombination involves

10050-480: The conditions found in cells, it is not a well-defined conformation but a family of related DNA conformations that occur at the high hydration levels present in cells. Their corresponding X-ray diffraction and scattering patterns are characteristic of molecular paracrystals with a significant degree of disorder. Compared to B-DNA, the A-DNA form is a wider right-handed spiral, with a shallow, wide minor groove and

10184-400: The conserved catalytic triad residues Arg 173, His 289, Arg 292 as well as the conserved nucleophilic residues Tyr 324 and Trp 315. Unlike some recombinase enzymes such as Flp recombinase, Cre does not form a shared active site between separate subunits and all the residues that contribute to the active site are found on a single subunit. Consequently, when two Cre molecules bind at

10318-503: The contribution of certain cell types to disease. Therefore, researchers often use transgenic mice expressing CreER recombinase induced by tamoxifen administration, under the control of a promoter of a gene that marks the specific cell type of interest, with a Cre-dependent fluorescent protein reporter. The Cre recombinase is fused to a mutant form of the oestrogen receptor, which binds the synthetic oestrogen 4-hydroxytamoxifen instead of its natural ligand 17β-estradiol. CreER(T2) resides within

10452-405: The creation of new genes through the process of gene duplication and divergence . A gene is a sequence of DNA that contains genetic information and can influence the phenotype of an organism. Within a gene, the sequence of bases along a DNA strand defines a messenger RNA sequence, which then defines one or more protein sequences. The relationship between the nucleotide sequences of genes and

10586-446: The cytoplasm and can only translocate to the nucleus following tamoxifen administration, allowing tight temporal control of recombination. The fluorescent reporter cassette will contain a promoter to permit high expression of the fluorescent transgene reporter (e.g. a CAG promoter) and a loxP flanked stop cassette, ensuring the expression of the transgene is Cre-recombinase dependent and the reporter sequence. Upon Cre driven recombination,

10720-449: The cytoplasm called the nucleoid . The genetic information in a genome is held within genes, and the complete set of this information in an organism is called its genotype . A gene is a unit of heredity and is a region of DNA that influences a particular characteristic in an organism. Genes contain an open reading frame that can be transcribed, and regulatory sequences such as promoters and enhancers , which control transcription of

10854-483: The daughter cells of the host will lose the plasmid. The Cre-lox recombination system prevents these situations by unlinking the rings of DNA by carrying out two recombination events (linked rings -> single fused ring -> two unlinked rings). It is also proposed that rolling circle replication followed by recombination will allow the plasmid to increase its copy number when certain regulators (repA) are limiting. A classical strategy for generating gene deletion variants

10988-405: The double helix (from six-carbon ring to six-carbon ring) is called a Watson-Crick base pair. DNA with high GC-content is more stable than DNA with low GC -content. A Hoogsteen base pair (hydrogen bonding the 6-carbon ring to the 5-carbon ring) is a rare variation of base-pairing. As hydrogen bonds are not covalent , they can be broken and rejoined relatively easily. The two strands of DNA in

11122-442: The edges of the bases and chelation of a metal ion in the centre of each four-base unit. Other structures can also be formed, with the central set of four bases coming from either a single strand folded around the bases, or several different parallel strands, each contributing one base to the central structure. In addition to these stacked structures, telomeres also form large loop structures called telomere loops, or T-loops. Here,

11256-481: The end of an otherwise complementary double-strand of DNA. However, branched DNA can occur if a third strand of DNA is introduced and contains adjoining regions able to hybridize with the frayed regions of the pre-existing double-strand. Although the simplest example of branched DNA involves only three strands of DNA, complexes involving additional strands and multiple branches are also possible. Branched DNA can be used in nanotechnology to construct geometric shapes, see

11390-411: The enzyme consists of amino acids 132–341 and it harbours the active site of the enzyme. The overall structure of this domain shares a great deal of structural resemblance to the catalytic domain of other enzymes of the same family such as λ Integrase and HP1 Integrase. This domain is predominantly helical in structure with 9 distinct helices (F−N). The terminal helix (N) protrudes from the main body of

11524-440: The enzyme. At both of the DNA duplexes, the bonding of the phosphate group to tyrosine residues leave a 3’ OH group free in the DNA backbone. In fact, the enzyme-DNA complex is an intermediate stage, which is followed by the ligation of the 3’ OH group of one DNA strand to the 5’ phosphate group of the other DNA strand, which is covalently bonded to the tyrosine residue; that is, the covalent linkage between 5’ end and tyrosine residue

11658-418: The focus is on the interactions between DNA and other molecules that mediate the function of the genome. Genomic DNA is tightly and orderly packed in the process called DNA condensation , to fit the small available volumes of the cell. In eukaryotes, DNA is located in the cell nucleus , with small amounts in mitochondria and chloroplasts . In prokaryotes, the DNA is held within an irregularly shaped body in

11792-437: The formation and resolution of recombination intermediate. Another factor is the length of DNA between the lox pair. Increasing the length of DNA leads to decreased efficiency of Cre/lox recombination possibly through regulating the dynamics of the reaction. Genetic location of the floxed sequence affects recombination efficiency as well probably by influencing the availability of DNA by Cre recombinase . The choice of Cre driver

11926-477: The formation and study of chimeric tissues. All cell types tested in mice have been shown to undergo transgenic Cre recombination. Independently, Joe Z. Tsien has pioneered the use of Cre-loxP system for cell type- and region-specific gene manipulation in the adult brain where hundreds of distinct neuron types may exist and nearly all neurons in the adult brain are known to be post-mitotic. Tsien and his colleagues demonstrated Cre-mediated recombination can occur in

12060-448: The functions of these RNAs are not entirely clear. One proposal is that antisense RNAs are involved in regulating gene expression through RNA-RNA base pairing. A few DNA sequences in prokaryotes and eukaryotes, and more in plasmids and viruses , blur the distinction between sense and antisense strands by having overlapping genes . In these cases, some DNA sequences do double duty, encoding one protein when read along one strand, and

12194-464: The genome by Cre-lox recombination is an elegant and efficient way to circumvent this problem and is therefore widely used in plants, mouse cell lines, yeast, etc. The system consists of a single enzyme, Cre recombinase , that recombines a pair of short target sequences called the Lox sequences. This system can be implemented without inserting any extra supporting proteins or sequences. The Cre enzyme and

12328-448: The helical axis in a left-handed spiral, the opposite of the more common B form. These unusual structures can be recognized by specific Z-DNA binding proteins and may be involved in the regulation of transcription. For many years, exobiologists have proposed the existence of a shadow biosphere , a postulated microbial biosphere of Earth that uses radically different biochemical and molecular processes than currently known life. One of

12462-442: The host from the virion is in the form of a linear double stranded DNA molecule. The Cre enzyme targets loxP sites at the ends of this molecule and cyclises the genome. This can also take place in the absence of the Cre lox system with the help of other bacterial and viral proteins. The P1 plasmid is relatively large (≈90Kbp) and hence exists in a low copy number - usually one per cell. If the two daughter plasmids get interlinked one of

12596-448: The hydration level, DNA sequence, the amount and direction of supercoiling, chemical modifications of the bases, the type and concentration of metal ions , and the presence of polyamines in solution. The first published reports of A-DNA X-ray diffraction patterns —and also B-DNA—used analyses based on Patterson functions that provided only a limited amount of structural information for oriented fibers of DNA. An alternative analysis

12730-427: The hydrolytic activities of cellular water, etc., also occur frequently. Although most of these damages are repaired, in any cell some DNA damage may remain despite the action of repair processes. These remaining DNA damages accumulate with age in mammalian postmitotic tissues. This accumulation appears to be an important underlying cause of aging. Many mutagens fit into the space between two adjacent base pairs, this

12864-412: The importance of 5-methylcytosine, it can deaminate to leave a thymine base, so methylated cytosines are particularly prone to mutations . Other base modifications include adenine methylation in bacteria, the presence of 5-hydroxymethylcytosine in the brain , and the glycosylation of uracil to produce the "J-base" in kinetoplastids . DNA can be damaged by many sorts of mutagens , which change

12998-414: The interpretation of cell fate mapping results. Inducible Cre activation is achieved using CreER (estrogen receptor) variant, which is only activated after delivery of tamoxifen . This is done through the fusion of a mutated ligand binding domain of the estrogen receptor to the Cre recombinase, resulting in Cre becoming specifically activated by tamoxifen. In the absence of tamoxifen, CreER will result in

13132-429: The location and relative orientation of the loxP sites. Two separate DNA species both containing loxP sites can undergo fusion as the result of Cre mediated recombination. DNA sequences found between two loxP sites are said to be " floxed ". In this case the products of Cre mediated recombination depends upon the orientation of the loxP sites. DNA found between two loxP sites oriented in the same direction will be excised as

13266-577: The lytic cycle by a later event such as UV radiation or starvation. Phages like the lambda phage use their site specific recombinases to integrate their DNA into the host genome during lysogeny. P1 phage DNA on the other hand, exists as a plasmid in the host. The Cre-lox system serves several functions in the phage: it circularizes the phage DNA into a plasmid, separates interlinked plasmid rings so they are passed to both daughter bacteria equally and may help maintain copy numbers through an alternative means of replication. The P1 phage DNA when released into

13400-441: The minor groove is 12 Å (1.2 nm) in width. Due to the larger width of the major groove, the edges of the bases are more accessible in the major groove than in the minor groove. As a result, proteins such as transcription factors that can bind to specific sequences in double-stranded DNA usually make contact with the sides of the bases exposed in the major groove. This situation varies in unusual conformations of DNA within

13534-516: The mitochondrial genome (constituting up to 90% of the DNA of the cell). A DNA sequence is called a "sense" sequence if it is the same as that of a messenger RNA copy that is translated into protein. The sequence on the opposite strand is called the "antisense" sequence. Both sense and antisense sequences can exist on different parts of the same strand of DNA (i.e. both strands can contain both sense and antisense sequences). In both prokaryotes and eukaryotes, antisense RNA sequences are produced, but

13668-477: The most dangerous are double-strand breaks, as these are difficult to repair and can produce point mutations , insertions , deletions from the DNA sequence, and chromosomal translocations . These mutations can cause cancer . Because of inherent limits in the DNA repair mechanisms, if humans lived long enough, they would all eventually develop cancer. DNA damages that are naturally occurring , due to normal cellular processes that produce reactive oxygen species,

13802-464: The new strand, and the cell ends up with a perfect copy of its DNA. Naked extracellular DNA (eDNA), most of it released by cell death, is nearly ubiquitous in the environment. Its concentration in soil may be as high as 2 μg/L, and its concentration in natural aquatic environments may be as high at 88 μg/L. Various possible functions have been proposed for eDNA: it may be involved in horizontal gene transfer ; it may provide nutrients; and it may act as

13936-454: The open reading frame. In many species , only a small fraction of the total sequence of the genome encodes protein. For example, only about 1.5% of the human genome consists of protein-coding exons , with over 50% of human DNA consisting of non-coding repetitive sequences . The reasons for the presence of so much noncoding DNA in eukaryotic genomes and the extraordinary differences in genome size , or C-value , among species, represent

14070-452: The original Lox site called the LoxP sequence are derived from bacteriophage P1 . Placing Lox sequences appropriately allows genes to be activated, repressed, or exchanged for other genes. At a DNA level many types of manipulations can be carried out. The activity of the Cre enzyme can be controlled so that it is expressed in a particular cell type or triggered by an external stimulus like

14204-428: The place of thymine in RNA and differs from thymine by lacking a methyl group on its ring. In addition to RNA and DNA, many artificial nucleic acid analogues have been created to study the properties of nucleic acids, or for use in biotechnology. Modified bases occur in DNA. The first of these recognized was 5-methylcytosine , which was found in the genome of Mycobacterium tuberculosis in 1925. The reason for

14338-403: The post-mitotic pyramidal neurons in the adult mouse forebrain. These developments have led to a widespread use of conditional mutagenesis in biomedical research, spanning many disciplines in which it becomes a powerful platform for determining gene function in specific cell types and at specific developmental times. In particular, the clear demonstration of its usefulness in precisely defining

14472-470: The presence of the estrogen analogue 4-hydroxy tamoxifen. This provides the advantage that the Cre recombinase is active for a short time. This prevents non-specific actions of Cre recombinase. The Cre recombinase can recognize cryptic sites in the host genome and induce unauthorized recombination, damaging host DNA. This tool is suitable for deleting antibiotic resistance genes, but above all it allows conditional knockouts that can be induced at specific times in

14606-530: The presence of these noncanonical bases in bacterial viruses ( bacteriophages ) is to avoid the restriction enzymes present in bacteria. This enzyme system acts at least in part as a molecular immune system protecting bacteria from infection by viruses. Modifications of the bases cytosine and adenine, the more common and modified DNA bases, play vital roles in the epigenetic control of gene expression in plants and animals. A number of noncanonical bases are known to occur in DNA. Most of these are modifications of

14740-412: The prime symbol being used to distinguish these carbon atoms from those of the base to which the deoxyribose forms a glycosidic bond . Therefore, any DNA strand normally has one end at which there is a phosphate group attached to the 5′ carbon of a ribose (the 5′ phosphoryl) and another end at which there is a free hydroxyl group attached to the 3′ carbon of a ribose (the 3′ hydroxyl). The orientation of

14874-466: The proposals was the existence of lifeforms that use arsenic instead of phosphorus in DNA . A report in 2010 of the possibility in the bacterium GFAJ-1 was announced, though the research was disputed, and evidence suggests the bacterium actively prevents the incorporation of arsenic into the DNA backbone and other biomolecules. At the ends of the linear chromosomes are specialized regions of DNA called telomeres . The main function of these regions

15008-404: The reciprocal exchange of DNA strands between these sites. Initiation of site-specific recombination begins with the binding of recombination proteins to their respective DNA targets. A separate recombinase recognizes and binds to each of two recombination sites on two different DNA molecules or within the same DNA strand. At the given specific site on the DNA, the hydroxyl group of the tyrosine in

15142-421: The recombinase attacks a phosphate group in the DNA backbone using a direct transesterification mechanism. This reaction links the recombinase protein to the DNA via a phospho-tyrosine linkage. This conserves the energy of the phosphodiester bond, allowing the reaction to be reversed without the involvement of a high-energy cofactor. Cleavage on the other strand also causes a phospho-tyrosine bond between DNA and

15276-411: The recombination event depend on the relative orientation of these asymmetric sequences. Many other proteins apart from the recombinase are involved in regulating the reaction. During site-specific DNA recombination, which brings about genetic rearrangement in processes such as viral integration and excision and chromosomal segregation, these recombinase enzymes recognize specific DNA sequences and catalyse

15410-732: The recombination site was named loxP ( lo cus of crossing ( x ) over, P 1). The Cre protein was purified in 1983 and was found to be a 35,000 Da protein. No high energy cofactors such as ATP or accessory proteins are required for the recombinase activity of the purified protein. Early studies also demonstrated that Cre binds to non specific DNA sequences whilst having a 20 fold higher affinity for loxP sequences and results of early DNA footprinting studies also suggested that Cre molecules bind loxP sites as dimers . Cre recombinase consists of 343 amino acids that form two distinct domains. The amino terminal domain encompasses residues 20–129 and this domain contains 5 alpha helical segments linked by

15544-482: The removal of reported cryptic splice sites , an altered stop codon , and reduced CpG content to reduce the risk of epigenetic silencing in mammals . A number of mutants with enhanced accuracy have also been identified. DNA Deoxyribonucleic acid ( / d iː ˈ ɒ k s ɪ ˌ r aɪ b oʊ nj uː ˌ k l iː ɪ k , - ˌ k l eɪ -/ ; DNA ) is a polymer composed of two polynucleotide chains that coil around each other to form

15678-437: The respective att sites. The integrase class of enzymes catalyse this particular reaction. Two factors have been shown to affect the efficiency of Cre's excision on the lox pair. First, the nucleotide sequence identity in the spacer region of lox site. Engineered lox variants which differ on the spacer region tend to have varied but generally lower recombination efficiency compared to wildtype loxP, presumably through affecting

15812-472: The roles of early genetic lesions in disease (and phenotype) maintenance. Shortly thereafter, researchers in the laboratory of Prof. Klaus Rajewsky reported the production of pluripotent embryonic stem cells bearing a targeted loxP-flanked (floxed) DNA polymerase gene. Combining these advances in collaboration, the laboratories of Drs. Marth and Rajewsky reported in 1994 that Cre-lox recombination could be used for conditional gene targeting. They observed ≈50% of

15946-409: The same biological information . This information is replicated when the two strands separate. A large part of DNA (more than 98% for humans) is non-coding , meaning that these sections do not serve as patterns for protein sequences . The two strands of DNA run in opposite directions to each other and are thus antiparallel . Attached to each sugar is one of four types of nucleobases (or bases ). It

16080-433: The second half of the pathway. This model provided convenient explanation for the strict requirement for homology between recombining sites, since branch migration would stall at a mismatch and would not allow the second strand exchange to occur. In more recent years, however, this view has been challenged, and most of the current models for Int, Xer, and Flp recombination involve only limited branch migration (1–3 base pairs of

16214-432: The section on uses in technology below. Several artificial nucleobases have been synthesized, and successfully incorporated in the eight-base DNA analogue named Hachimoji DNA . Dubbed S, B, P, and Z, these artificial bases are capable of bonding with each other in a predictable way (S–B and P–Z), maintain the double helix structure of DNA, and be transcribed to RNA. Their existence could be seen as an indication that there

16348-476: The shape of a double helix . The nucleotide contains both a segment of the backbone of the molecule (which holds the chain together) and a nucleobase (which interacts with the other DNA strand in the helix). A nucleobase linked to a sugar is called a nucleoside , and a base linked to a sugar and to one or more phosphate groups is called a nucleotide . A biopolymer comprising multiple linked nucleotides (as in DNA)

16482-478: The shuttling of the mutated recombinase into the cytoplasm. The protein will stay in this location in its inactivated state until tamoxifen is given. Once tamoxifen is introduced, it is metabolized into 4-hydroxytamoxifen, which then binds to the ER and results in the translocation of the CreER into the nucleus, where it is then able to cleave the lox sites. Importantly, sometimes fluorescent reporters can be activated in

16616-502: The single-stranded DNA curls around in a long circle stabilized by telomere-binding proteins. At the very end of the T-loop, the single-stranded telomere DNA is held onto a region of double-stranded DNA by the telomere strand disrupting the double-helical DNA and base pairing to one of the two strands. This triple-stranded structure is called a displacement loop or D-loop . In DNA, fraying occurs when non-complementary regions exist at

16750-593: The stop cassette is excised, allowing reporter genes to express specifically in cells in which the Cre expression is being driven by the cell-specific marker promoter. Since removal of the stop cassette is permanent, the reporter genes are expressed in all the progeny produced by the initial cells where the Cre was once activated. Such conditional lineage tracing has proved to be extremely useful to efficiently and specifically identify vascular smooth muscle cells (VSMCs) and VSMC-derived cells and has been used to test effects on VSMC and VSMC-derived cells in vivo. The P1 phage

16884-518: The strands separate and exist in solution as two entirely independent molecules. These single-stranded DNA molecules have no single common shape, but some conformations are more stable than others. In humans, the total female diploid nuclear genome per cell extends for 6.37 Gigabase pairs (Gbp), is 208.23 cm long and weighs 6.51 picograms (pg). Male values are 6.27 Gbp, 205.00 cm, 6.41 pg. Each DNA polymer can contain hundreds of millions of nucleotides, such as in chromosome 1 . Chromosome 1

17018-469: The sugar-phosphate to form the complete nucleotide, as shown for adenosine monophosphate . Adenine pairs with thymine and guanine pairs with cytosine, forming A-T and G-C base pairs . The nucleobases are classified into two types: the purines , A and G , which are fused five- and six-membered heterocyclic compounds , and the pyrimidines , the six-membered rings C and T . A fifth pyrimidine nucleobase, uracil ( U ), usually takes

17152-404: The targeting of a specific sequence of DNA and splicing it with the help of an enzyme called Cre recombinase . Cre-Lox recombination is commonly used to circumvent embryonic lethality caused by systemic inactivation of many genes. As of February 2019, Cre–Lox recombination is a powerful tool and is used in transgenic animal modeling to link genotypes to phenotypes. The Cre-lox system is used as

17286-437: The tetramer are parallel in orientation. The double stranded DNA is cut at both loxP sites by the Cre protein. The strands are then rejoined with DNA ligase in a quick and efficient process. The result of recombination depends on the orientation of the loxP sites. For two lox sites on the same chromosome arm, inverted loxP sites will cause an inversion of the intervening DNA, while a direct repeat of loxP sites will cause

17420-556: The translocation of the CreER into the nucleus, where it is then able to cleave the lox sites. Importantly, sometimes fluorescent reporters can be activated in the absence of tamoxifen, due to leakage of a few Cre recombinase molecules into the nucleus which, in combination with very sensitive reporters, results in unintended cell labelling. CreER(T2) was developed to minimize tamoxifen-independent recombination and maximize tamoxifen-sensitivity. In recent years, Cre recombinase has been improved with conversion to preferred mammalian codons ,

17554-430: The two separate polynucleotide strands are bound together, according to base pairing rules (A with T and C with G), with hydrogen bonds to make double-stranded DNA. The complementary nitrogenous bases are divided into two groups, the single-ringed pyrimidines and the double-ringed purines . In DNA, the pyrimidines are thymine and cytosine; the purines are adenine and guanine. Both strands of double-stranded DNA store

17688-591: Was initially used in activating gene expression in mammalian cell lines. Subsequently, researchers in the laboratory of Dr. Jamey Marth demonstrated that Cre-Lox recombination could be used to delete loxP-flanked chromosomal DNA sequences at high efficiency in specific developing T-cells of transgenic animals, with the authors proposing that this approach could be used to define endogenous gene function in specific cell types, indelibly mark progenitors in cell fate determination studies, induce specific chromosomal rearrangements for biological and disease modeling, and determine

17822-404: Was known to be unique as they occurred in the absence of bacterial RecA and RecBCD proteins. The components of this recombination system were elucidated using deletion mutagenesis studies. These studies showed that a P1 gene product and a recombination site were both required for efficient recombination events to occur. The P1 gene product was named Cre ( c yclization re combination) and

17956-526: Was proposed by Wilkins et al. in 1953 for the in vivo B-DNA X-ray diffraction-scattering patterns of highly hydrated DNA fibers in terms of squares of Bessel functions . In the same journal, James Watson and Francis Crick presented their molecular modeling analysis of the DNA X-ray diffraction patterns to suggest that the structure was a double helix. Although the B-DNA form is most common under

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