Articoli di riviste: "Genome cloning"

1

Potgieter, A. C., A. D. Steele e A. A. van Dijk. "Cloning of complete genome sets of six dsRNA viruses using an improved cloning method for large dsRNA genes". Journal of General Virology 83, n. 9 (1 settembre 2002): 2215–23. http://dx.doi.org/10.1099/0022-1317-83-9-2215.

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Cloning full-length large (>3 kb) dsRNA genome segments from small amounts of dsRNA has thus far remained problematic. Here, a single-primer amplification sequence-independent dsRNA cloning procedure was perfected for large genes and tailored for routine use to clone complete genome sets or individual genes. Nine complete viral genome sets were amplified by PCR, namely those of two human rotaviruses, two African horsesickness viruses (AHSV), two equine encephalosis viruses (EEV), one bluetongue virus (BTV), one reovirus and bacteriophage Φ12. Of these amplified genomes, six complete genome sets were cloned for viruses with genes ranging in size from 0·8 to 6·8 kb. Rotavirus dsRNA was extracted directly from stool samples. Co-expressed EEV VP3 and VP7 assembled into core-like particles that have typical orbivirus capsomeres. This work presents the first EEV sequence data and establishes that EEV genes have the same conserved termini (5′ GUU and UAC 3′) and coding assignment as AHSV and BTV. To clone complete genome sets, one-tube reactions were developed for oligo-ligation, cDNA synthesis and PCR amplification. The method is simple and efficient compared to other methods. Complete genomes can be cloned from as little as 1 ng dsRNA and a considerably reduced number of PCR cycles (22–30 cycles compared to 30–35 of other methods). This progress with cloning large dsRNA genes is important for recombinant vaccine development and determination of the role of terminal sequences for replication and gene expression.

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Cochrane, Ryan R., Stephanie L. Brumwell, Arina Shrestha, Daniel J. Giguere, Samir Hamadache, Gregory B. Gloor, David R. Edgell e Bogumil J. Karas. "Cloning of Thalassiosira pseudonana’s Mitochondrial Genome in Saccharomyces cerevisiae and Escherichia coli". Biology 9, n. 11 (26 ottobre 2020): 358. http://dx.doi.org/10.3390/biology9110358.

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Algae are attractive organisms for biotechnology applications such as the production of biofuels, medicines, and other high-value compounds due to their genetic diversity, varied physical characteristics, and metabolic processes. As new species are being domesticated, rapid nuclear and organelle genome engineering methods need to be developed or optimized. To that end, we have previously demonstrated that the mitochondrial genome of microalgae Phaeodactylum tricornutum can be cloned and engineered in Saccharomyces cerevisiae and Escherichia coli. Here, we show that the same approach can be used to clone mitochondrial genomes of another microalga, Thalassiosira pseudonana. We have demonstrated that these genomes can be cloned in S. cerevisiae as easily as those of P. tricornutum, but they are less stable when propagated in E. coli. Specifically, after approximately 60 generations of propagation in E. coli, 17% of cloned T. pseudonana mitochondrial genomes contained deletions compared to 0% of previously cloned P. tricornutum mitochondrial genomes. This genome instability is potentially due to the lower G+C DNA content of T. pseudonana (30%) compared to P. tricornutum (35%). Consequently, the previously established method can be applied to clone T. pseudonana’s mitochondrial genome, however, more frequent analyses of genome integrity will be required following propagation in E. coli prior to use in downstream applications.

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Takeuchi, T., Q. V. Neri, M. Cheng, Z. Rosenwaks e G. D. Palermo. "Successful cloning of the male genome". Fertility and Sterility 88 (settembre 2007): S75. http://dx.doi.org/10.1016/j.fertnstert.2007.07.250.

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Xi, J., D. Graham, K. Wang e M. Estes. "Norwalk virus genome cloning and characterization". Science 250, n. 4987 (14 dicembre 1990): 1580–83. http://dx.doi.org/10.1126/science.2177224.

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Takahashi, Seiya. "Animal Cloning: Reprogramming the Donor Genome". Journal of Mammalian Ova Research 21, n. 3 (2004): 74–81. http://dx.doi.org/10.1274/jmor.21.74.

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Zhao, Xinping, Rod A. Wing e Andrew H. Paterson. "Cloning and characterization of the majority of repetitive DNA in cotton (Gossypium L.)". Genome 38, n. 6 (1 dicembre 1995): 1177–88. http://dx.doi.org/10.1139/g95-156.

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Repetitive DNA elements representing 60–70% of the total repetitive DNA in tetraploid cotton (Gossypium barbadense L.) and comprising 30–36% of the tetraploid cotton genome were isolated from a genomic library of DNA digested with a mixture of four blunt-end cutting restriction enzymes. A total of 313 clones putatively containing nuclear repetitive sequences were classified into 103 families, based on cross hybridization and Southern blot analysis. The 103 families were characterized in terms of genome organization, methylation pattern, abundance, and DNA variation. As in many other eukaryotic genomes, interspersed repetitive elements are the most abundant class of repetitive DNA in the cotton genome. Paucity of tandem repeat families with high copy numbers (>104) may be a unique feature of the cotton genome as compared with other higher plant genomes. Interspersed repeats tend to be methylated, while tandem repeats seem to be largely unmethylated in the cotton genome. Minimal variation in repertoire and overall copy number of repetitive DNA elements among different tetraploid cotton species is consistent with the hypothesis of a relatively recent origin of tetraploid cottons.Key words: genome analysis, genome evolution, tandemly repetitive DNA sequences, interspersed repetitive DNA sequences, polyploid.

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Aswidinnoor, H., R. J. Nelson, J. F. Dallas, C. L. McIntyre, H. Leung e J. P. Gustafson. "Cloning and characterization of repetitive DNA sequences from genomes of Oryza minuta and Oryza australiensis". Genome 34, n. 5 (1 ottobre 1991): 790–98. http://dx.doi.org/10.1139/g91-123.

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The value of genome-specific repetitive DNA sequences for use as molecular markers in studying genome differentiation was investigated. Five repetitive DNA sequences from wild species of rice were cloned. Four of the clones, pOm1, pOm4, pOmA536, and pOmPB10, were isolated from Oryza minuta accession 101141 (BBCC genomes), and one clone, pOa237, was isolated from Oryza australiensis accession 100882 (EE genome). Southern blot hybridization to different rice genomes showed strong hybridization of all five clones to O. minuta genomic DNA and no cross hybridization to genomic DNA from Oryza sativa (AA genome). The pOm1 and pOmA536 sequences showed cross hybridization only to all of the wild rice species containing the C genome. However, the pOm4, pOmPB10, and pOa237 sequences showed cross hybridization to O. australiensis genomic DNA in addition to showing hybridization to the O. minuta genomic DNA.Key words: rice, genome-specific repetitive sequences, Oryza.

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Devor, Eric J., Lingyan Huang, Abdusattor Abdukarimov e Ibrokhim Y. Abdurakhmonov. "Methodologies for In Vitro Cloning of Small RNAs and Application for Plant Genome(s)". International Journal of Plant Genomics 2009 (15 giugno 2009): 1–13. http://dx.doi.org/10.1155/2009/915061.

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The “RNA revolution” that started at the end of the 20th century with the discovery of post-transcriptional gene silencing and its mechanism via RNA interference (RNAi) placed tiny 21-24 nucleotide long noncoding RNAs (ncRNAs) in the forefront of biology as one of the most important regulatory elements in a host of physiologic processes. The discovery of new classes of ncRNAs including endogenous small interfering RNAs, microRNAs, and PIWI-interacting RNAs is a hallmark in the understanding of RNA-dependent gene regulation. New generation high-throughput sequencing technologies further accelerated the studies of this “tiny world” and provided their global characterization and validation in many biological systems with sequenced genomes. Nevertheless, for the many “yet-unsequenced” plant genomes, the discovery of small RNA world requires in vitro cloning from purified cellular RNAs. Thus, reproducible methods for in vitro small RNA cloning are of paramount importance and will remain so into the foreseeable future. In this paper, we present a description of existing small RNA cloning methods as well as next-generation sequencing methods that have accelerated this research along with a description of the application of one in vitro cloning method in an initial small RNA survey in the “still unsequenced” allotetraploid cotton genome.

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Suzuki, Tetsuya, Tomohito Tsukamoto, Eiko Sakai, Hiroyuki Mizuguchi e Hiroyuki Mizuguchi. "Sequence search for cloning and genome editing". Drug Delivery System 35, n. 3 (25 luglio 2020): 255–59. http://dx.doi.org/10.2745/dds.35.255.

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Cockburn, Andrew F., Sharon E. Mitchell e Jack A. Seawright. "Cloning of the mitochondrial genome ofAnopheles quadrimaculatus". Archives of Insect Biochemistry and Physiology 14, n. 1 (1990): 31–36. http://dx.doi.org/10.1002/arch.940140104.

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Curran, James F., e Charles R. Stewart. "Cloning and mapping of the SP01 genome". Virology 142, n. 1 (aprile 1985): 78–97. http://dx.doi.org/10.1016/0042-6822(85)90424-6.

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Heyman, John A., Jeremiah Cornthwaite, Luis Foncerrada, Jeremiah R. Gilmore, Erin Gontang, Kristen J. Hartman, Cathy L. Hernandez et al. "Genome-Scale Cloning and Expression of Individual Open Reading Frames Using Topoisomerase I-Mediated Ligation". Genome Research 9, n. 4 (1 aprile 1999): 383–92. http://dx.doi.org/10.1101/gr.9.4.383.

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The in vitro cloning of DNA molecules traditionally uses PCR amplification or site-specific restriction endonucleases to generate linear DNA inserts with defined termini and requires DNA ligase to covalently join those inserts to vectors with the corresponding ends. We have used the properties of Vaccinia DNA topoisomerase I to develop a ligase-free technology for the covalent joining of DNA fragments to suitable plasmid vectors. This system is much more efficient than cloning methods that require ligase because the rapid DNA rejoining activity of Vaccinia topoisomerase I allows ligation in only 5 min at room temperature, whereas the enzyme’s high substrate specificity ensures a low rate of vector-alone transformants. We have used this topoisomerase I-mediated cloning technology to develop a process for accelerated cloning and expression of individual ORFs. Its suitability for genome-scale molecular cloning and expression is demonstrated in this report.

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Lapitanz, Nora L. V. "Organization and evolution of higher plant nuclear genomes". Genome 35, n. 2 (1 aprile 1992): 171–81. http://dx.doi.org/10.1139/g92-028.

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The knowledge gained from studies on eukaryotic genome organization is important for understanding how genomes function and evolve, and it provides the basis for designing strategies for manipulating genomes. Hence, numerous studies on this subject have been conducted over the years, utilizing a variety of methods. In the recent decade, several techniques have been developed that allow the study of eukaryotic genome organization at different levels. Molecular techniques including molecular cloning, DNA sequencing, restriction fragment length polymorphism mapping, in situ hybridization, and pulsed field gel electrophoresis together provide a means of obtaining a comprehensive and detailed view of eukaryotic genomes. This paper summarizes recent findings on the organization and evolution of the nuclear genomes of higher plants, with emphasis on representative species with varying genome sizes, including Arabidopsis thaliana, tomato, maize, and wheat. Common, as well as unique, features in the organization of repeated DNA sequences and low copy sequences in these genomes are described and their evolutionary significance discussed.Key words: genome organization, evolution, higher plants, repeated DNA sequences, low copy number sequences.

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Moniruzzaman, M., Yun Zhong, Zhifeng Huang e Guangyan Zhong. "Having a Same Type IIS Enzyme’s Restriction Site on Guide RNA Sequence Does Not Affect Golden Gate (GG) Cloning and Subsequent CRISPR/Cas Mutagenesis". International Journal of Molecular Sciences 23, n. 9 (28 aprile 2022): 4889. http://dx.doi.org/10.3390/ijms23094889.

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Golden gate/modular cloning facilitates faster and more efficient cloning by utilizing the unique features of the type IIS restriction enzymes. However, it is known that targeted insertion of DNA fragment(s) must not include internal type IIS restriction recognition sites. In the case of cloning CRISPR constructs by using golden gate (GG) cloning, this narrows down the scope of guide RNA (gRNA) picks because the selection of a good gRNA for successful genome editing requires some obligation of fulfillment, and it is unwanted if a good gRNA candidate cannot be picked only because it has an internal type IIS restriction recognition site. In this article, we have shown that the presence of a type IIS restriction recognition site in a gRNA does not affect cloning and subsequent genome editing. After each step of GG reactions, correct insertions of gRNAs were verified by colony color and restriction digestion and were further confirmed by sequencing. Finally, the final vector containing a Cas12a nuclease and four gRNAs was used for Agrobacterium-mediated citrus cell transformation. Sequencing of PCR amplicons flanking gRNA-2 showed a substitution (C to T) mutation in transgenic plants. The knowledge derived from this study could widen the scope of GG cloning, particularly of gRNAs selection for GG-mediated cloning into CRISPR vectors.

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15

Marshall, E. "Human Genome Project: Panel Urges Cloning Ethics Boards". Science 275, n. 5296 (3 gennaio 1997): 22a—0. http://dx.doi.org/10.1126/science.275.5296.22a.

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Merriam, J., M. Ashburner, D. Hartl e F. Kafatos. "Toward cloning and mapping the genome of Drosophila". Science 254, n. 5029 (11 ottobre 1991): 221–25. http://dx.doi.org/10.1126/science.1925579.

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Brown, Kevin E., Spencer W. Green, M. Gerard O'Sullivan e Neal S. Young. "Cloning and Sequencing of the Simian Parvovirus Genome". Virology 210, n. 2 (luglio 1995): 314–22. http://dx.doi.org/10.1006/viro.1995.1348.

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Frangeul, Lionel, Karen E. Nelson, Frank Kunst, Philippe Glaser, Antoine Danchin e Carmen Buchrieser. "Cloning and assembly strategies in microbial genome projects". Microbiology 145, n. 10 (1 ottobre 1999): 2625–34. http://dx.doi.org/10.1099/00221287-145-10-2625.

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Rideout III, W. M. "Nuclear Cloning and Epigenetic Reprogramming of the Genome". Science 293, n. 5532 (10 agosto 2001): 1093–98. http://dx.doi.org/10.1126/science.1063206.

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20

Ro, Hyeon-Su, Hyung Pyo Hong, Byung Hoon Kho, Sujin Kim e Bong Hyun Chung. "Genome-wide cloning and characterization of microbial esterases". FEMS Microbiology Letters 233, n. 1 (aprile 2004): 97–105. http://dx.doi.org/10.1016/j.femsle.2004.01.046.

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Rethwilm, A., G. Darai, A. Rösen, B. Maurer e R. M. Flügel. "Molecular cloning of the genome of human spumaretrovirus". Gene 59, n. 1 (gennaio 1987): 19–28. http://dx.doi.org/10.1016/0378-1119(87)90262-9.

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Merriam, J., M. Ashburner, DL Hartl e FC Kafatos. "Toward cloning and mapping the genome of Drosophila". Science 254, n. 5029 (11 ottobre 1991): 221–25. http://dx.doi.org/10.1126/science.254.5029.221.

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An ultimate goal of Drosophila genetics is to identify and define the functions of all the genes in the organism. Traditional approaches based on the isolation of mutant genes have been extraordinary fruitful. Recent advances in the manipulation and analysis of large DNA fragments have made it possible to develop detailed molecular maps of the Drosophila genome as the initial steps in determining the complete DNA sequence.

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Gemmell, Neil J., Axel Janke, Patrick S. Western, Jaclyn M. Watson, Svante Pääbo e Jennifer A. Marshall Graves. "Cloning and characterization of the platypus mitochondrial genome". Journal of Molecular Evolution 39, n. 2 (agosto 1994): 200–205. http://dx.doi.org/10.1007/bf00163808.

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Kanda, Teru, Yuki Furuse, Hitoshi Oshitani e Tohru Kiyono. "Highly Efficient CRISPR/Cas9-Mediated Cloning and Functional Characterization of Gastric Cancer-Derived Epstein-Barr Virus Strains". Journal of Virology 90, n. 9 (17 febbraio 2016): 4383–93. http://dx.doi.org/10.1128/jvi.00060-16.

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ABSTRACTThe Epstein-Barr virus (EBV) is etiologically linked to approximately 10% of gastric cancers, in which viral genomes are maintained as multicopy episomes. EBV-positive gastric cancer cells are incompetent for progeny virus production, making viral DNA cloning extremely difficult. Here we describe a highly efficient strategy for obtaining bacterial artificial chromosome (BAC) clones of EBV episomes by utilizing a CRISPR/Cas9-mediated strand break of the viral genome and subsequent homology-directed repair. EBV strains maintained in two gastric cancer cell lines (SNU719 and YCCEL1) were cloned, and their complete viral genome sequences were determined. Infectious viruses of gastric cancer cell-derived EBVs were reconstituted, and the viruses established stable latent infections in immortalized keratinocytes. While Ras oncoprotein overexpression caused massive vacuolar degeneration and cell death in control keratinocytes, EBV-infected keratinocytes survived in the presence of Ras expression. These results implicate EBV infection in predisposing epithelial cells to malignant transformation by inducing resistance to oncogene-induced cell death.IMPORTANCERecent progress in DNA-sequencing technology has accelerated EBV whole-genome sequencing, and the repertoire of sequenced EBV genomes is increasing progressively. Accordingly, the presence of EBV variant strains that may be relevant to EBV-associated diseases has begun to attract interest. Clearly, the determination of additional disease-associated viral genome sequences will facilitate the identification of any disease-specific EBV variants. We found that CRISPR/Cas9-mediated cleavage of EBV episomal DNA enabled the cloning of disease-associated viral strains with unprecedented efficiency. As a proof of concept, two gastric cancer cell-derived EBV strains were cloned, and the infection of epithelial cells with reconstituted viruses provided important clues about the mechanism of EBV-mediated epithelial carcinogenesis. This experimental system should contribute to establishing the relationship between viral genome variation and EBV-associated diseases.

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Tomita, Satoshi, Kenji Tsuge, Yo Kikuchi e Mitsuhiro Itaya. "Targeted Isolation of a Designated Region of the Bacillus subtilis Genome by Recombinational Transfer". Applied and Environmental Microbiology 70, n. 4 (aprile 2004): 2508–13. http://dx.doi.org/10.1128/aem.70.4.2508-2513.2004.

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ABSTRACT A method for positional cloning of the Bacillus subtilis genome was developed. The method requires a set of two small DNA fragments that flank the region to be copied. A 38-kb segment that carries genes ppsABCDE encoding five enzymes for antibiotic plipastatin synthesis and another genome locus as large as 100 kb including one essential gene were examined for positional cloning. The positional cloning vector for ppsABCDE was constructed using a B. subtilis low-copy-number plasmid that faithfully copied the precise length of the 38-kb DNA in vivo via the recombinational transfer system of this bacterium. Structure of the copied DNA was confirmed by restriction enzyme analyses. Furthermore, the unaltered structure of the 38-kb DNA was demonstrated by complementation of a ppsABCDE deletion mutant.

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Budak, Hikmet, Robert C. Shearman e Ismail Dweikat. "Evolution of Buchloë dactyloides based on cloning and sequencing of matK, rbcL, and cob genes from plastid and mitochondrial genomes". Genome 48, n. 3 (1 giugno 2005): 411–16. http://dx.doi.org/10.1139/g05-002.

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Buffalograss (Buchloë dactyloides (Nutt.) Englem), a C4 turfgrass species, is native to the Great Plains region of North America. The evolutionary implications of buffalograss are unclear. Sequencing of rbcL and matK genes from plastid and the cob gene from mitochondrial genomes was examined to elucidate buffalograss evolution. This study is the first to report sequencing of these genes from organelle genomes in the genus Buchloë. Comparisons of sequence data from the mitochondrial and plastid genome revealed that all genotypes contained the same cytoplasmic origin. There were some rearrangements detected in mitochondrial genome. The buffalograss genome appears to have evolved through the rearrangements of convergent subgenomic domains. Combined analyses of plastid genes suggest that the evolutionary process in Buchloë accessions studied was monophyletic rather than polyphyletic. However, since plastid and mitochondrial genomes are generally uniparentally inherited, the evolutionary history of these genomes may not reflect the evolutionary history of the organism, especially in a species in which out-crossing is common. The sequence information obtained from this study can be used as a genome-specific marker for investigation of the buffalograss polyploidy complex and testing of the mode of plastid and mitochondrial transmission in genus Buchloë.Key words: buffalograss, evolution, organelle genomes, turfgrass.

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Banin, Andrew N., Michael Tuen, Jude S. Bimela, Marcel Tongo, Paul Zappile, Alireza Khodadadi-Jamayran, Aubin J. Nanfack et al. "Development of a Versatile, Near Full Genome Amplification and Sequencing Approach for a Broad Variety of HIV-1 Group M Variants". Viruses 11, n. 4 (1 aprile 2019): 317. http://dx.doi.org/10.3390/v11040317.

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Near full genome sequencing (NFGS) of HIV-1 is required to assess the genetic composition of HIV-1 strains comprehensively. Population-wide, it enables a determination of the heterogeneity of HIV-1 and the emergence of novel/recombinant strains, while for each individual it constitutes a diagnostic instrument to assist targeted therapeutic measures against viral components. There is still a lack of robust and adaptable techniques for efficient NFGS from miscellaneous HIV-1 subtypes. Using rational primer design, a broad primer set was developed for the amplification and sequencing of diverse HIV-1 group M variants from plasma. Using pure subtypes as well as diverse, unique recombinant forms (URF), variable amplicon approaches were developed for NFGS comprising all functional genes. Twenty-three different genomes composed of subtypes A (A1), B, F (F2), G, CRF01_AE, CRF02_AG, and CRF22_01A1 were successfully determined. The NFGS approach was robust irrespective of viral loads (≥306 copies/mL) and amplification method. Third-generation sequencing (TGS), single genome amplification (SGA), cloning, and bulk sequencing yielded similar outcomes concerning subtype composition and recombinant breakpoint patterns. The introduction of a simple and versatile near full genome amplification, sequencing, and cloning method enables broad application in phylogenetic studies of diverse HIV-1 subtypes and can contribute to personalized HIV therapy and diagnosis.

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Blake, Nancy K., Ben R. Lehfeldt, Matt Lavin e Luther E. Talbert. "Phylogenetic reconstruction based on low copy DNA sequence data in an allopolyploid: The B genome of wheat". Genome 42, n. 2 (1 aprile 1999): 351–60. http://dx.doi.org/10.1139/g98-136.

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Study of bread wheat (Triticum aestivum) may help to resolve several questions related to polyploid evolution. One such question regards the possibility that the component genomes of polyploids may themselves be polyphyletic, resulting from hybridization and introgression among different polyploid species sharing a single genome. We used the B genome of wheat as a model system to test hypotheses that bear on the monophyly or polyphyly of the individual constituent genomes. By using aneuploid wheat stocks, combined with PCR-based cloning strategies, we cloned and sequenced two single-copy-DNA sequences from each of the seven chromosomes of the wheat B genome and the homologous sequences from representatives of the five diploid species in section Sitopsis previously suggested as sister groups to the B genome. Phylogenetic comparisons of sequence data suggested that the B genome of wheat underwent a genetic bottleneck and has diverged from the diploid B genome donor. The extent of genetic diversity among the Sitopsis diploids and the failure of any of the Sitopsis species to group with the wheat B genome indicated that these species have also diverged from the ancestral B genome donor. Our results support monophyly of the wheat B genome.Key words: wheat evolution, phylogenetics, DNA sequencing.

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Kathir, Pushpa, Matthew LaVoie, William J. Brazelton, Nancy A. Haas, Paul A. Lefebvre e Carolyn D. Silflow. "Molecular Map of the Chlamydomonas reinhardtii Nuclear Genome". Eukaryotic Cell 2, n. 2 (aprile 2003): 362–79. http://dx.doi.org/10.1128/ec.2.2.362-379.2003.

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ABSTRACT We have prepared a molecular map of the Chlamydomonas reinhardtii genome anchored to the genetic map. The map consists of 264 markers, including sequence-tagged sites (STS), scored by use of PCR and agarose gel electrophoresis, and restriction fragment length polymorphism markers, scored by use of Southern blot hybridization. All molecular markers tested map to one of the 17 known linkage groups of C. reinhardtii. The map covers approximately 1,000 centimorgans (cM). Any position on the C. reinhardtii genetic map is, on average, within 2 cM of a mapped molecular marker. This molecular map, in combination with the ongoing mapping of bacterial artificial chromosome (BAC) clones and the forthcoming sequence of the C. reinhardtii nuclear genome, should greatly facilitate isolation of genes of interest by using positional cloning methods. In addition, the presence of easily assayed STS markers on each arm of each linkage group should be very useful in mapping new mutations in preparation for positional cloning.

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KELLER, BEAT, CATHERINE FEUILLET e NABILA YAHIAOUI. "Map-based isolation of disease resistance genes from bread wheat: cloning in a supersize genome". Genetical Research 85, n. 2 (aprile 2005): 93–100. http://dx.doi.org/10.1017/s0016672305007391.

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The genome of bread wheat is hexaploid and contains 1·6×1010 bp of DNA, of which more than 80% is repetitive sequences. Its size and complexity represent a challenge for the isolation of agronomically important genes, for which we frequently know only their position on the genetic map. Recently, new genomic resources and databases from genome projects have simplified the molecular analysis of the wheat genome. The first genes to be isolated from wheat by map-based cloning include three resistance genes against the fungal diseases powdery mildew and leaf rust. In this review, we will describe the approaches and resources that have contributed to this progress, and discuss genomic strategies that will simplify positional cloning in wheat in the near future.

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Mizuno, Carolina Megumi, Francisco Rodriguez-Valera, Inmaculada Garcia-Heredia, Ana-Belen Martin-Cuadrado e Rohit Ghai. "Reconstruction of Novel Cyanobacterial Siphovirus Genomes from Mediterranean Metagenomic Fosmids". Applied and Environmental Microbiology 79, n. 2 (16 novembre 2012): 688–95. http://dx.doi.org/10.1128/aem.02742-12.

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ABSTRACTCellular metagenomes are primarily used for investigating microbial community structure and function. However, cloned fosmids from such metagenomes capture phage genome fragments that can be used as a source of phage genomes. We show that fosmid cloning from cellular metagenomes and sequencing at a high coverage is a credible alternative to constructing metaviriomes and allows capturing and assembling novel, complete phage genomes. It is likely that phages recovered from cellular metagenomes are those replicating within cells during sample collection and represent “active” phages, naturally amplifying their genomic DNA and increasing chances for cloning. We describe five sets of siphoviral contigs (MEDS1, MEDS2, MEDS3, MEDS4, and MEDS5), obtained by sequencing fosmids from the cellular metagenome of the deep chlorophyll maximum in the Mediterranean. Three of these represent complete siphoviral genomes and two represent partial ones. This is the first set of phage genomes assembled directly from cellular metagenomic fosmid libraries. They exhibit low sequence similarities to one another and to known siphoviruses but are remarkably similar in overall genome architecture. We present evidence suggesting they infect picocyanobacteria, likelySynechococcus. Four of these sets also define a novel branch in the phylogenetic tree of phage large subunit terminases. Moreover, some of these siphoviral groups are globally distributed and abundant in the oceans, comparable to some known myoviruses and podoviruses. This suggests that, as more siphoviral genomes become available, we will be better able to assess the abundance and influence of this diverse and polyphyletic group in the marine habitat.

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Volpi e Silva, Nathalia, e Nicola J. Patron. "CRISPR-based tools for plant genome engineering". Emerging Topics in Life Sciences 1, n. 2 (15 settembre 2017): 135–49. http://dx.doi.org/10.1042/etls20170011.

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Molecular tools adapted from bacterial CRISPR (clustered regulatory interspaced short palindromic repeat) adaptive immune systems have been demonstrated in an increasingly wide range of plant species. They have been applied for the induction of targeted mutations in one or more genes as well as for directing the integration of new DNA to specific genomic loci. The construction of molecular tools for multiplexed CRISPR-mediated editing in plants has been facilitated by cloning techniques that allow multiple sequences to be assembled together in a single cloning reaction. Modifications of the canonical Cas9 protein from Streptococcus pyogenes and the use of nucleases from other bacteria have increased the diversity of genomic sequences that can be targeted and allow the delivery of protein cargos such as transcriptional activators and repressors. Furthermore, the direct delivery of protein–RNA complexes to plant cells and tissues has enabled the production of engineered plants without the delivery or genomic integration of foreign DNA. Here, we review toolkits derived from bacterial CRISPR systems for targeted mutagenesis, gene delivery and modulation of gene expression in plants, focusing on their composition and the strategies employed to reprogramme them for the recognition of specific genomic targets.

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Huang, Li, Steven A. Brooks, Wanlong Li, John P. Fellers, Harold N. Trick e Bikram S. Gill. "Map-Based Cloning of Leaf Rust Resistance Gene Lr21 From the Large and Polyploid Genome of Bread Wheat". Genetics 164, n. 2 (1 giugno 2003): 655–64. http://dx.doi.org/10.1093/genetics/164.2.655.

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Abstract We report the map-based cloning of the leaf rust resistance gene Lr21, previously mapped to a generich region at the distal end of chromosome arm 1DS of bread wheat (Triticum aestivum L.). Molecular cloning of Lr21 was facilitated by diploid/polyploid shuttle mapping strategy. Cloning of Lr21 was confirmed by genetic transformation and by a stably inherited resistance phenotype in transgenic plants. Lr21 spans 4318 bp and encodes a 1080-amino-acid protein containing a conserved nucleotide-binding site (NBS) domain, 13 imperfect leucine-rich repeats (LRRs), and a unique 151-amino-acid sequence missing from known NBS-LRR proteins at the N terminus. Fine-structure genetic analysis at the Lr21 locus detected a noncrossover (recombination without exchange of flanking markers) within a 1415-bp region resulting from either a gene conversion tract of at least 191 bp or a double crossover. The successful map-based cloning approach as demonstrated here now opens the door for cloning of many crop-specific agronomic traits located in the gene-rich regions of bread wheat.

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34

Kubát, Z. "Chromosome walking with BAC clones as a method of genome mapping". Plant, Soil and Environment 53, No. 10 (7 gennaio 2008): 447–50. http://dx.doi.org/10.17221/2198-pse.

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Current sequencing projects are often based on random sequencing of genomic libraries followed by contig assembly by means of bioinformatics tools. This approach is convenient for whole genome sequencing projects. Chromosome walking described here is suitable for mapping and sequencing of short genomic regions in species where whole genome sequencing is not possible or for cloning gene from its closest known marker. This method is based on searching for overlapping BAC clones specific for the genomic region of interest.

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35

Vichera, Gabriel, Ramiro Olivera e Daniel Salamone. "Oocyte genome cloning used in biparental bovine embryo reconstruction". Zygote 21, n. 1 (5 aprile 2012): 21–29. http://dx.doi.org/10.1017/s0967199412000081.

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SummaryOocyte genome cloning is a method by which haploid maternal embryos are obtained in such a way that parthenogenetic haploid blastomeres from these embryos can be considered as a clone of the original gamete. Our objective was to generate oocyte genome replicates and use them to reconstruct biparental embryos by fusion with haploid male hemizygotes. Furthermore, we generated biparental homogeneous transgene-expressing embryos using parthenogenetic haploid blastomeres that expressed a transgene (EGFP). In the first experiment, parthenogenetic haploid embryos were generated by incubation of oocytes in ionomycin and 6-dimethylaminopurine (DMAP) with a 3 h interval to permit their second polar body extrusion. The cleavage rate was 87.3%. To generate transgene-expressing blastomeres, activated oocytes were injected with pCX–EGFP–liposome complexes 3 h post ionomycin exposure, resulting in a cleavage rate of 84.4%. In the second experiment, haploid parthenogenetic blastomeres that were positive or negative for EGFP expression were used to reconstruct biparental embryos. Cleavage and blastocyst rates for the reconstructed embryos were 78.4% and 61.1% and 10.8% and 8.4%, using EGFP-positive or -negative blastomeres, respectively (P < 0.05). All of the reconstructed embryos showed EGFP expression, with 96.6% of them showing homogenic expression. Oct-4 expression in the reconstructed blastocysts displayed a similar pattern as IVF-blastocyst controls. In conclusion, our results proved that it is possible to use oocyte genome replicates to reconstruct biparental bovine embryos and that this technique is efficient to generate homogeneous transgene-expressing embryos.

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36

Bremer, C. W., H. Huismans e A. A. Van Dijk. "Characterization and cloning of the African horsesickness virus genome". Journal of General Virology 71, n. 4 (1 aprile 1990): 793–99. http://dx.doi.org/10.1099/0022-1317-71-4-793.

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37

Jander, Georg, Susan R. Norris, Steven D. Rounsley, David F. Bush, Irena M. Levin e Robert L. Last. "Arabidopsis Map-Based Cloning in the Post-Genome Era". Plant Physiology 129, n. 2 (1 giugno 2002): 440–50. http://dx.doi.org/10.1104/pp.003533.

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38

Barrera-Saldaña et al., Hugo A. "REVISITING MOLECULAR CLONING TO SOLVE GENOME SEQUENCING PROJECT CONFLICTS". Journal of Microbiology, Biotechnology and Food Sciences 6, n. 5 (1 aprile 2017): 1157–60. http://dx.doi.org/10.15414/jmbfs.2017.6.5.1157-1160.

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39

Montgomery, G. W., J. Wicks, Z. Z. Zhao, D. R. Nyholt, N. G. Martin, P. A. W. Rogers e S. A. Treloar. "026.Endometriosis — linkage, positional cloning and genome wide association". Reproduction, Fertility and Development 16, n. 9 (2004): 26. http://dx.doi.org/10.1071/srb04abs026.

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Endometriosis is a complex disease which affects up to 10% of women in their reproductive years. Common symptoms include severe dysmenorrhea and pelvic pain. The disease is associated with subfertility and some malignancies. Genetic and environmental factors both influence endometriosis. The aim of our studies is to identify genetic variation contributing to endometriosis and define pathways leading to disease. We recruited a large cohort of affected sister pair (ASP) families where two sisters have had surgically confirmed disease and conducted a 10�cM genome scan. The results of the linkage analysis identified one chromosomal region with significant linkage and one region of suggestive linkage. The regions implicated by these studies are generally of the order of 20–30�cM and include several hundred genes. Locating the gene or genes contributing to disease within the region is a challenging task. The best approach to the problem is association studies using a high density of SNP markers. The recent development of human SNP maps and high throughput SNP genotyping platforms makes this task easier. We have developed high throughput SNP typing at QIMR using the Sequenom MassARRAY platform. The method allows multiple SNP assays to be genotyped on the same sample in a single experiment. Throughput and genotyping costs depend critically on this level of multiplexing and we routinely genotype 6–8 SNPs in a single assay. We are using bioinformatics and functional approaches to develop a priority list of genes to screen early in the project. SNP markers in these genes are being genotyped using the MassARRAY platform to search for genes contributing to endometriosis. In the future, genome wide association studies with our families may locate additional genes contributing to endometriosis.

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40

Vichera, Gabriel, Ramiro Olivera, Pablo Sipowicz, Martín Radrizzani e Daniel Salamone. "Sperm genome cloning used in biparental bovine embryo reconstruction". Reproduction, Fertility and Development 23, n. 6 (2011): 769. http://dx.doi.org/10.1071/rd10252.

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The generation of androgenetic haploid embryos enables several haploid blastomeres to be obtained as identical copies of a single spermatozoon genome. In the present study, we compared the developmental ability of bovine androgenetic haploid embryos constructed by different methods, namely IVF and intracytoplasmic sperm injection (ICSI) before and after oocyte enucleation. Once obtained, the blastomeres of these androgenetic haploid embryos were used as male genome donors to reconstruct biparental embryos by fusion with matured oocytes. To verify the cytoplasmic contribution of androgenetic haploid blastomeres, we used spermatozoa incubated previously with exogenous DNA that coded for a green fluorescent protein gene (pCX-EGFP) and the enhanced green fluorescent protein (EGFP)-positive androgenetic haploid blastomeres generated were fused with mature oocytes. Of the reconstructed embryos reaching the cleavage and blastocyst stages, 85.1% and 9.0%, respectively, expressed EGFP (P > 0.05). EGFP expression was observed in 100% of reconstructed embryos, with 91.2% exhibiting homogenic expression. To confirm sperm genome incorporation, androgenetic haploid blastomeres generated by ICSI prior to enucleation and using Y chromosome sexed spermatozoa were used for biparental embryo reconstruction. Incorporation of the Y chromosome was confirmed by polymerase chain reaction and fluorescence in situ hybridisation analysis. In conclusion, the results of the present study prove that it is possible to use sperm genome replicates to reconstruct biparental bovine embryos and that it is a highly efficient technique to generate homogeneous transgene-expressing embryos.

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41

Lee, Robert W., Carole Dumas, Claude Lemieux e Monique Turmel. "Cloning and characterization of the Chlamydomonas moewusii mitochondrial genome". Molecular and General Genetics MGG 231, n. 1 (dicembre 1991): 53–58. http://dx.doi.org/10.1007/bf00293821.

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42

Brookes, Anthony J., e David J. Porteous. "Coincident sequence cloning: a new approach to genome analysis". Trends in Biotechnology 10 (1992): 40–44. http://dx.doi.org/10.1016/0167-7799(92)90166-s.

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43

Russell, Robyn J., e Steven J. Robbins. "Cloning and molecular characterization of the myxoma virus genome". Virology 170, n. 1 (maggio 1989): 147–59. http://dx.doi.org/10.1016/0042-6822(89)90362-0.

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44

Fu, Rongdian, e Gerrit Voordouw. "ISD1, an Insertion Element from the Sulfate-Reducing Bacterium Desulfovibrio vulgaris Hildenborough: Structure, Transposition, and Distribution". Applied and Environmental Microbiology 64, n. 1 (1 gennaio 1998): 53–61. http://dx.doi.org/10.1128/aem.64.1.53-61.1998.

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ABSTRACT Insertion element ISD1, discovered when its transposition caused the insertional inactivation of an introducedsacB gene, is present in two copies in the genome ofDesulfovibrio vulgaris Hildenborough. Southern blot analysis indicated at least two insertion sites in the sacBgene. Cloning and sequencing of a transposed copy of ISD1indicated a length of 1,200 bp with a pair of 44-bp imperfect inverted repeats at the ends, flanked by a direct repeat of the 4-bp target sequence. AAGG and AATT were found to function as target sequences. ISD1 encodes a transposase from two overlapping open reading frames by programmed translational frameshifting at an A6G shifty codon motif. Sequence comparison showed that ISD1 belongs to the IS3 family. Isolation and analysis of the chromosomal copies, ISD1-A and ISD1-B, by PCR and sequencing indicated that these are not flanked by direct repeats. ISD1-A is inserted in a region of the chromosome containing the gapdh-pgk genes (encoding glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase). Active transposition to other loci in the genome was demonstrated, offering the potential of a new tool for gene cloning and mutagenesis. ISD1 is the first transposable element described for the sulfate reducers, a large and environmentally important group of bacteria. The distribution of ISD1 in genomes of sulfate-reducing bacteria is limited. A single copy is present in the genome of D. desulfuricans Norway.

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45

Daud, Hassan Mat, e J. P. Gustafson. "Molecular evidence for Triticum speltoides as a B-genome progenitor of wheat (Triticum aestivum)". Genome 39, n. 3 (1 giugno 1996): 543–48. http://dx.doi.org/10.1139/g96-069.

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In polyploid wheat, the origin of the B-genome donor has remained relatively unknown in spite of a number of investigations attempting to identify the parental species. A project was designed to isolate and clone a genome-specific DNA sequence from Triticum speltoides L. to determine if that species could be the B-genome donor. A cloning scheme involving the prescreening of 1-kb fragments followed by colony, dot blot, and Southern blot hybridization screenings was used to isolate a speltoides-specific sequence (pSp89.XI). The methods used allowed for rapid isolation of a genome-specific sequence when screened against total DNA from closely related species. Subsequent analyses showed that the sequence was barely detected in any of the other genomes of the annual Sitopsis section. The results of dot blot and Southern blot analyses established that (i) the sequence pSP89.XI, specific to T. speltoides relative to the other species of the Sitopsis section, was present in the genomes of tetraploid and hexaploid wheat, (ii) the relative abundance of pSp89.XI seemed to decrease from the diploid to the polyploid wheats, and (iii) the existence of a related, but modified B genome in polyploid wheat compared with that in modern T. speltoides was probable. Key words : genome-specific, DNA.

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46

Parkin, I. AP, D. J. Lydiate e M. Trick. "Assessing the level of collinearity between Arabidopsis thaliana and Brassica napus for A. thaliana chromosome 5". Genome 45, n. 2 (1 aprile 2002): 356–66. http://dx.doi.org/10.1139/g01-160.

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This study describes a comprehensive comparison of chromosome 5 of the model crucifer Arabidopsis with the genome of its amphidiploid crop relative Brassica napus and introduces the use of in silico sequence homology to identify conserved loci between the two species. A region of chromosome 5, spanning 8 Mb, was found in six highly conserved copies in the B. napus genome. A single inversion appeared to be the predominant rearrangement that had separated the two lineages leading to the formation of Arabidopsis chromosome 5 and its homologues in B. napus. The observed results could be explained by the fusion of three ancestral genomes with strong similarities to modern-day Arabidopsis to generate the constituent diploid genomes of B. napus. This supports the hypothesis that the diploid Brassica genomes evolved from a common hexaploid ancestor. Alignment of the genetic linkage map of B. napus with the genomic sequence of Arabidopsis indicated that for specific regions a genetic distance of 1 cM in B. napus was equivalent to 285 Kb of Arabidopsis DNA sequence. This analysis strongly supports the application of Arabidopsis as a tool in marker development, map-based gene cloning, and candidate gene identification for the larger genomes of Brassica crop species.Key Words: comparative mapping, Brassica species, model crucifer, genome evolution, genome duplication.

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47

Cockram, James, Rhian M. Howells e Donal M. O’Sullivan. "Segmental chromosomal duplications harbouring group IV CONSTANS-like genes in cereals". Genome 53, n. 3 (marzo 2010): 231–40. http://dx.doi.org/10.1139/g09-101.

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Comparative mapping is an important component of map-based cloning in large-genome cereal species. We describe evidence of a segmental chromosomal duplication harbouring CONSTANS-like genes in barley that predates the divergence of the Oryzoideae (rice) and Pooideae (brachypodium, barley, wheat) clades, and discuss the implications of such events for comparative mapping and QTL cloning in temperate cereal crops.

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48

Barker, Christopher S., John W. Wills, James A. Bradac e Eric Hunter. "Molecular cloning of the Mason-Pfizer monkey virus genome: Characterization and cloning of subgenomic fragments". Virology 142, n. 2 (aprile 1985): 223–40. http://dx.doi.org/10.1016/0042-6822(85)90331-9.

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49

Smith, Cassandra L., e Charles R. Cantor. "Evolving strategies for making physical maps of mammalian chromosomes". Genome 31, n. 2 (15 gennaio 1989): 1055–58. http://dx.doi.org/10.1139/g89-181.

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Two types of physical maps are described: restriction maps made by top down approaches using enzymes that cut the genome infrequently, and complete libraries, made by bottom up approaches using fingerprinting of randomly selected cloned DNA. Construction of such maps for mammalian chromosomes is complicated by the mosaic nature of mammalian genomes, and extensive polymorphisms at the cleavage sites of most enzymes that yield large DNA fragments. However, it appears that both of these potential difficulties can be turned into advantages by new mapping strategies. When combined with yeast artificial chromosome cloning and polymerase chain reaction amplification methods, these approaches should soon yield complete maps of many human chromosomes.Key words: fingerprinting, restriction maps, DNA polymorphisms, human genome.

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50

Mittapalli, O., L. Rivera-Vega, B. Bhandary, M. A. Bautista, P. Mamidala, A. P. Michel, R. H. Shukle e M. A. R. Mian. "Cloning and characterization of mariner-like elements in the soybean aphid, Aphis glycines Matsumura". Bulletin of Entomological Research 101, n. 6 (12 maggio 2011): 697–704. http://dx.doi.org/10.1017/s0007485311000253.

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AbstractSoybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is currently the most important insect pest of soybean (Glycine max (L.) Merr.) in the United States and causes significant economic damage worldwide, but little is known about the aphid at the molecular level. Mariner-like transposable elements (MLEs) are ubiquitous within the genomes of arthropods and various other invertebrates. In this study, we report the cloning of MLEs from the soybean aphid genome using degenerate PCR primers designed to amplify conserved regions of mariner transposases. Two of the ten sequenced clones (designated as Agmar1 and Agmar2) contained partial but continuous open reading frames, which shared high levels of homology at the protein level with other mariner transposases from insects and other taxa. Phylogenetic analysis revealed Agmar1 to group within the irritans subfamily of MLEs and Agmar2 within the mellifera subfamily. Southern blot analysis and quantitative PCR analysis indicated a low copy number for Agmar1-like elements within the soybean aphid genome. These results suggest the presence of at least two different putative mariner-like transposases encoded by the soybean aphid genome. Both Agmar1 and Agmar2 could play influential roles in the architecture of the soybean aphid genome. Transposable elements are also thought to potentially mediate resistance in insects through changes in gene amplification and mutations in coding sequences. Finally, Agmar1 and Agmar2 may represent useful genetic tools and provide insights on A. glycines adaptation.

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