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Second strand synthesis of cDNA

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can be used to prime second strand synthesis, ..

Triplet repeats behave similarly, but are particularly suited to evolution of proteins with differing characteristics. In the clock-like gene of the fruit fly, triplet repeats 'fine tune' an insect's biological clock in response to changes in environmental temperatures. Triplet repeats are widely distributed in genomes, and their high frequency of mutation is responsible for several genetically determined disorders in humans.

cDNA templates generated by first- and second-strand synthesis from an RNA precursor ..

As the ribosome travels down the mRNA one codon at a time, another tRNA is attached to the mRNA at one of the ribosome sites. The first tRNA is released, but the that is attached to the first tRNA is now moved to the second tRNA, and binds to its amino acid. This translocation continues on, and a long chain of amino acid (protein), is formed.

Taq Polymerase builds second strand

2.2.3 Pre-Preparation for Second Strand cDNA Synthesis; ..

Tandem repeats are made of succesive identical or nearly identical (degenerate) repeat units. They vary in length of repeat unit as well as lenght of the whole repeat much, so every classification is not satisfying and must be taken "cum grano salis". The largest repeats, which tend to be composed from large repeat units are called satellites. The name satellites comes from centrifugation of DNA in density gradients. First, during DNA isolation conventional methods, DNA is subject to shear stress, with resulting DNA fragmentation (note that in vivo one G1 phase chromosome contains 1 DNA molecule). These fragments can be then centrifuged in density gradients so the DNA molecules occupy places in the gradient with the same density as the DNA molecule. Bulk of DNA will form one band. But DNA fragments with significantly different CG/AT content, caused e. g. by large monotonous repeats will form minor "satellite" bands. The denomination of satellite DNA was later broadened to incorporate similarly repetitive sequences that are not forming these satellite bands. Satellite primary repeat units are various, from GGAAT found in satellites 2 and 3 to 171 bp in alpha satellite. But these primary units are often degenerated, conaining certain irregularities. These irregularities can be periodical, forming thus secondary repeat units. Satellite DNA is abundand at centromeres and constitutive heterochromatin. Although human genome is considered completely assembled, the centromere regions and heterochromatin containing satellite sequences are not included, since the sequencing of such regions is from various reasons challenging (absence of restriction sites, difficult sequencing, almost impossible contig assembly). From the various satellites found at or near the centromere, a family of alpha-satellite repeat (with primary unit 171 bp) probably form functional core of centromeres, as they are important for kinetochore assembly during cell division (some kinetochore proteins bind to the alpha-satellite at centromere, and thus nucleate kinetochore assembly). The function of other satellites is unknown, regarded mostly as junk DNA.

Retrotransposons are most important transposable elements in the human genome. First, they are much more abundant, directly forming at least 45% of the human genome (the estimations vary, but most researchers believe, that it must be even more, since ancient retrotransposons that have been inactivated, have diverged by mutation to the point where they are unidentifiable).Second, retrotransposons are still active in the human genome.

As second strand synthesis is based on random priming, ..

The bases on one strand of DNA form base pairs with a second strand of DNA ..


Figure 2. Target primed reverse transcription (TPRT)
ORF2 protein cleaves first one DNA strand at the target (target sequence is rich in A+T and the sequence is usually similar to consensus TTAAAA, cleavage occurs between T and A on the complementary strand). The cleaved strand dissociates and binds to polyA tail of L1 mRNA (dashed orange line). Free 3´ OH group of the DNA strand primes cDNA first strand synthesis. Cleavage of the second DNA strand occurs 7-20 nt downstream of the first cut and the free 3´ OH group generated by this event is used to prime the second strand synthesis of L1 cDNA. The mechanism of second strand synthesis is not completely elucidated. The whole process ends by formation of a new DNA copy of L1, flanked by duplication of the target site.


Figure 4 Alu sequences are hyperparasites
A: Structure of the 7SL RNA gene and Alu element (left) and secondary structure of the respective RNA molecules (right). Transcription of 7SL RNA gene is directed by internal RNA polymerase III promoter (A) and enhancer (EN). Alu gene has composite internal promoter (A+B). Natural terminator of RNA polymerase III is tetranucleotide TTTT. Transcription is interrupted after first three T. 7SL RNA is composed from Alu domain (blue) and S-domain (yellow). SRP proteins 9 and 14 bind to the Alu domain, which serves for the anchorage to ribosome. Other proteins bind to the S-domain, includin protein 54, which collaborates on the signal peptide (red line) recognition. Alu RNA is formed basically by two Alu domains of 7SL RNA, with an addition of a polyA sequence.
B: Alu RNA binds to ribosome. If the ribosome is just translating ORF2 of LINE-1 mRNA (green line), the polyA tail of Alu element competes with the polyA tail of L1 for binding of nascent ORF2. PolyA binding proteins mediate the interaction. If ORF2 binds to Alu, ORF2 will reversely translate and transpose Alu instead of L1 and thus parasite on L1. If we consider L1 as a genomic parasite, Alu is a hyperparasite - i. e. parasite´s parasite. Other cellular mRNAs (blue line) can compete with the L1 mRNA for ORF2 binding too, albeit with much lower efficiency (it is estimated, that from 3000 L1 retrotranspositions, 300 would be hijacked by Alu elements and only cca 1 by another mRNA.

bind to the DNA until a second strand is ..
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  • A second round of DNA synthesis is carried ..

    - Second strand synthesis Add together 40 µl first strand reaction mixture 32 µl 5x Second Strand buffer

  • DNA helix and then pass a second strand of ..

    Second-strand DNA is ..

  • The second step in transcription initiation ..

    Second, penicillin-binding ..

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The replication fork moves at the rate of 1000 nucleotides per second


Figure 6. Satellites
A: primary units and higher order (secondary) units of tandem repeat. Probable "evolutionary history" of repeats as exemplified by GGAAT sequence. This sequence multiplicates and form thus a perfect monotone repeat. Some positions later undergo mutation (red) creating imperfect (degenerate) repeat. Then the sequence multiplicates again, but now several degenerated units multiplicate together as one unit, creating thus a perfect repetition of this larger, secondary unit (arrow). The sequence GGAAT is base of the human satellites 2 and 3. These satellites differ by the secondary unit.
B: Structure of human mitotic chromosome with respect to satellite sequences. Alpha-satellite forms heterochromatin at the core of the centromere. Besides the proteins associated with heterochromatin, alpha-satellite binding proteins assemble on the alpha satellite sequences to form inner plate of the kinetochore. Some of these proteins are associated with the centromere throughout the cell cycle. On the inner kinetochore plate assembles an outer kinetochore plate that interacts with microtubules of the mitotic spindle. Centromere is usually flanked by pericentric heterochromatin formed by other types of satellite sequences. Tips of the chromosome (telomeres) are formed by telomeric repeat TTAGGG, the adjacent subtelomeric regions are also higly repetitive.

DNA replication occurs during ..

These enzymes are needed to relieve the twisting stresses introduced into DNA strands during processes such as and .When a section of the double helix is being unwound, the remaining sections can be tangled. Type 1 toppoisomerases work by cutting the DNA helix and allowing one section to rotate; the enzyme then seals the DNA break. Type 11 cuts one DNA helix and then pass a second strand of DNA through this break, before rejoining the helix.

leading strand synthesis is thought ..

Recombination is when two DNA helices break, swap a section and then rejoin. In eukaryotes, this usually occurs during , when two are paired together in the center of the cell. This allows chromosomes to exchange genetic information and produces new combinations of genes. Genetic recombination can also be involved in DNA repair.

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