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Recombinant DNA phage

Posted by star on 2018-09-20 23:30:07
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Homologous recombination is not restricted to eukaryotic organisms. Viruses and bacteria also recombine their genomes. Studies of HR in these biological systems have provided important information about the physical nature and enzymology of HR. Recall from Chapter 8 that λ phages produce plaques on Escbericbia coli lawns. Plaque morphology is determined by several λ phage genes, which can be used for recombination studies.

Infection of E. coli with a pair of λ phages that differ in plaque morphology gives parental and recombinant plaque types. Matthew Meselson and Jean Weigle coinfected E. coli with a pair of λ phages that differed in genetic markers and in DNA density. One λ phage was used to infect bacterial cells cultured in a "light" medium that contained nutrients with normal isotopes 12C and 14N, while the other λ phage was used to infect bacterial cells cultured in a "heavy" medium that contained nutrients with the heavy isotopes 13C and 15N.

The λ phages released when the cells in each culture lysed were used to coinfect E. coli cultured in "light" medium. The cell lysate containing the progeny λ phages was centrifuged in a cesium chloride gradient, which separated the λ phages based on density. The λ phages were collected and then tested for parental or recombinant genotype. The major conclusion of this experiment was that recombinant DNA can be formed by breakage and rejoining as indicated by the appearance of genetic recombinants that had both heavy and light DNA.



Homologous recombination repairing DNA

Posted by star on 2018-09-20 18:48:45
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    Homologous recombination not only forms the basis for much of the genetic diversity among progeny of common parentage, but it is also essential for correct segregation of homologous chromosome pairs at the first meiotic division. When HR does not occur, homologous chromosomes are not held together and therefore segregate randomly at meiosis I, giving rise to meiotic products that are missing chromosomes or have extra copies of chromosomes. Such grossly aneuploid gametes are not functional. In somatic cells, HR is needed to repair double-strand breaks that arise from exogenous DNA damage sources such as x-rays and from endogenous sources such as defective DNA topoisomerases. Homologous recombination can also be used to repair single-stranded DNA gaps at replication forks. Homologous recombination can restart replication forks that have stalled at a lesion on the template DNA strand or can reinitiate a replication fork that has collapsed at a nick or other single strand interruption on the template DNA strand. Last, HR is needed to maintain telomeres, the specialized ends of chromosomes, when the enzyme telomerase that normally takes care of replicating telomeres is missing.

    In the early days of genetics after the work of Gregor Mendel was rediscovered, two observations led to the concept of linkage the idea that genes can be genetically and physically linked and hence will not segregate randomly from each other during the first division of meiosis.

    Reginald Punnett indicating that two sweet pea traits tended to segregate with each other more often than expected by random segregation. This phenomenon was called coupling. However linkage was not complete, and sometimes nonparental meiotic products or gametes were recovered, These were called nonparental or recombinant gametes and were the product of meiotic crossing over or recombination. The term crossing over was coined by Thomas Hunt Morgan.......

The steps of the paleoelongation differ from those of eukaryotes

Posted by star on 2018-09-19 01:58:40
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    The machinery required for the elongation stage of DNA synthesis in the arcnaea is very similar to that used by the bacteria and eukaryotes. Lagging and leading strand synthesis are coordinated. Processive DNA synthesis requires tethering DNA polymerase to a sliding clamp. The archaeal sliding clamp is similar in sequence and structure to PCNA in eukaryotes. The clamp loader appears to also be similar to its eukaryotic counterpart. Two different kinds of archaeal DNA polymerases, PolB and FolD, participate in the elongation process; however, not all archaeal species have PolB.

    It is not yet clearwhat specific contributions each of these polymerases make to DNA replication. Perhaps one participates in leading strand synthesis and the other in lagging strand synthesis. The archaeal counterparts of eukaryotic Fen-1 and RNase H remove RNA from the 5'-ends of the Okazaki fragments and DNA ligase joins the fragments. Archaeal ligase, like its eukaryotic counterpart, requires ATP for ligation. It therefore differs from the NAD+-dependent bacterial ligase.

    The archaeal replication system has one major surprise. Thermophiles require an ATP-dependent reverse gyrase to introduce positive supercoils. The reverse gyrase works by introducing transient nicks into a single strand and so functions by a different mechanism from the bacterial gyrase. Thermophiles may require reverse gyrase because they grow at high temperatures that tend to unwind DNA. Undoubtedly the archaeal replication machinery will have other surprises in store as we learn more about its components and how they work together. Perhaps the most remarkable surprise of all is how similar the replication process is in all three domains of life.



Cas9 Targeted DNA Cutting

Posted by star on 2018-09-19 01:56:52
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    The working principle of this system is that crRNA(CRISPR-pried RNA) binds to tracrRNA/crRNA by base pairing to form a tracrRNA/crRNA complex, which directs the nuclease Cas9 protein to be paired with crRNA. The sequence target point cuts double-stranded DNA. Through the artificial design of these two RNAs, sgRNA(sigle-Guide RNA) can be modified to form a guiding effect, which is sufficient to guide Cas9's fixed-point DNA cutting.

    As an RNA-guided dsDNA binding protein, Cas9 effect nuclease is the first known unifying factor that can collectively locate RNA, DNA, and proteins, thus having great potential for transformation. The protein is fused with Cas9 nuclease-free Cas9 and expresses an appropriate sgRNA that can target any dsDNA sequence, and the end of the sgRNA can be attached to the target DNA without affecting the binding of Cas9. Therefore, Cas9 can bring any fusion protein and RNA to any dsDNA sequence, which has great potential for research and transformation of organisms.



The role of Orc1/Cdc6 in MCM

Posted by star on 2018-09-18 01:53:23
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    Some archaeal chromosomes appear to have a single origin of replication whereas others have two or more origins. Homologs of some of the proteins involved in the initiation of eukaryotic DNA synthesis have been identified in archaeal genomes. However, different archaeal species appear to have ditftrent variants of the replication machinery components. These differences may reflect the great variation in environmental conditions under which the archaea live.

    Tne archaea produce a single protein, designated Ore1/Cdc6 which binds to the archaeal origin of replication. This protein share some sequences in common with the eukaryotic ORC. Once bound to the origin, Ore1/Cdc6 recruits MCM to the origin. The MCM helicase appears to work in the same way as the eukaryotic helicase. The helicase opens and unwinds the double-stranded DNA.

    RPA (single-stranded DNA binding protein) binds to the exposed single-stranded DNA.

    Primase associates with the RPA· DNA complex and synthesizes the short RNA primers required to initiate DNA synthesis. Then DNA polymerase associates with the replication bubble, initiating rapid and processive bidirectional DNA synthesis.



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