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1.
We describe a new approach to in vitro DNA recombination termed the Separate-Mixing method in this study. The reaction process
of this method consists of two stages: at the first stage the reaction was implemented in two parallel teams, which generated
random recombination by template-switching of growing poly-nucleotides from primers in the presence of unidirectional single-stranded
DNA fragments used as templates, and then both teams were mixed together for further extension and recombination of DNA sequences
at the second stage. Due to this particular strategy, the reaction process was also accompanied by two other processes of
DNA shuffling and StEP simultaneously. Two AdoMet synthetase genes, sam2 from Saccharomyces cerevisiae and metK from Escherichia coli, which have only 56% homology on the DNA level, were used for recombination with the Separate-Mixing method. DNA recombination
was available after a single round of reaction. When 10 randomly selected recombinants were sequenced, an unshuffled parental
clone was not found, nor was unexpected insertion, deletion, or rearrangement detected. An evolved gene, sam’, was obtained after screening and selection, which could obviously increase the accumulation of AdoMet in S. cerevisiae.
Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 3, pp. 546–553.
This article was submitted by the authors in English. 相似文献
2.
DNA改组的最新动态及应用前景 总被引:1,自引:0,他引:1
DNA改组(DNA shuffling)是目前最方便、有效的一种分子水平的体外定向进化技术,该技术同倾向错误PCR (Error-prone PCR) 相结合,通过对单基因或相关基因家族的靶序列进行多轮随机诱变、重组和高通量的筛选,可以有效富集正突变,去除负突变,提高突变文库的丰度,创造新基因和获得期望功能的蛋白质。DNA改组技术已在新药物等领域取得了广泛的应用,极大地推动了现代生物科学和生物技术的发展。该技术同计算机强大的数据分析系统相结合,将会为后基因组学的发展提供强有力的技术平台。 相似文献
3.
4.
M. Iwano K. Sakamoto G. Suzuki M. Watanabe S. Takayama K. Fukui K. Hinata A. Isogai 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1998,96(6-7):751-757
The physical localization of the S-glycoprotein (SLG) locus in the chromosome of Brassica campestris L. ‘pekinensis’ cv ‘Kukai’ was visualized by multi-color fluorescent in situ hybridization (McFISH). ‘Kukai’, which is an F1 hybrid between two parental lines, T-17 and T-18, has two SLG genes from both T-17 and T-18. In this study, a 1.3-kb DNA fragment was amplified from the genomic DNA of T-17 by PCR using
a set of primers specific to the class-I SLG. From the genomic DNA of T-18, no DNA fragment was amplified using these primers. In the genomic Southern hybridization,
a cloned PCR product hybridized with the genomic DNA of T-17 or F1 but not with that of T-18. The PCR product had a sequence homology of approximately, 85% to another class-I SLG gene, SLG-9. Therefore, the PCR product from T-17 was named SLG-17, as it is thought to be a member of the class-I SLG. Using SLG-17 as the probe, FISH was carried out to visualize the position of the SLG locus. McFISH was also carried out simultaneously using the SLG-17 and SLG-9 genes as probes. The SLG-17 gene was detected as a doublet signal at the interstitial region close to the end of a small chromosome, with the signal
site being identical to that of SLG-9. Therefore, it is concluded that the SLG-17 gene is localized at the interstitial region close to the end of the chromosome derived from T-17 in Brassica campestris L. ‘pekinensis’ cv ‘Kukai’.
Received: 18 September 1997 / Accepted: 6 October 1997 相似文献
5.
Antonín Dreiseitl 《Biologia》2011,66(5):762-767
Resistance to causal agents of diseases is an important varietal characteristic that influences the management practice of
crop plants and thus production costs of commodities. At present, almost all European barley varieties possess at least one
major gene for resistance to powdery mildew. After hybridizing selected parental varieties, resistance genes often segregate
in subsequent generations and, therefore, some varieties comprise lines that differ in the number or combinations of resistance
genes. The objective of this research was to describe the various methods available for postulating resistance genes to pathogens
in heterogeneous varieties using resistance to powdery mildew of barley as an example. Four spring barleys (‘Orbit’, ‘Malva’,
‘Tocada’ and CLE 233), and a six-row variety of winter barley, F 12872, were screened. For postulating resistance genes, several
testing procedures and many Blumeria graminis f.sp. hordei isolates were used. Minimum amounts of seed were determined and different methods of obtaining homogeneous seed samples from
heterogeneous varieties were compared. It was found that ‘Orbit’ and ‘Malva’ are composed of three and ‘Tocada’, CLE 233 and
F 12872 of two lines with different resistances to powdery mildew. Problems of postulating resistance genes in heterogeneous
varieties and the advantages of testing leaf segments instead of young plants are discussed. 相似文献
6.
Prashant G. Golegaonkar Haydar Karaoglu Robert F. Park 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2009,119(7):1281-1288
An incompletely dominant gene conferring resistance to Puccinia hordei, Rph14, identified previously in an accession of Hordeum vulgare, confers resistance to all known pathotypes of P. hordei in Australia. Knowledge of the chromosomal location of Rph14 and the identification of DNA markers closely linked to it will facilitate combining it with other important leaf rust resistance
genes to achieve long lasting resistance. The inheritance of Rph14 was confirmed using 146 and 106 F3 lines derived from the crosses ‘Baudin’/‘PI 584760’ (Rph14) and ‘Ricardo’/‘PI 584760’ (Rph14), respectively. Bulk segregant analysis on DNA from the parental genotypes and resistant and susceptible DNA bulks using
DArT markers located Rph14 to the short arm of chromosome 2H. DArT marker bPb-1664 was identified as having the closest genetic association with Rph14. PCR based marker analysis identified a single SSR marker, Bmag692, linked closely to Rph14 at a map distance of 2.1 and 3.8 cm in the ‘Baudin’/‘PI 584760’and ‘Ricardo’/‘PI 584760’ populations, respectively. 相似文献
7.
Shuffled antibody libraries created by in vivo homologous recombination and yeast surface display 总被引:1,自引:0,他引:1
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Homologous recombination in yeast can be exploited to reliably generate libraries of >107 transformants from a pool of PCR products and a linearized plasmid vector. Homology in the PCR insertion products drives shuffling of these genes in vivo by yeast homologous recombination. Two scFvs that share 89.8% homology were shuffled in vivo by homologous recombination, and chimeric genes were generated regardless of whether or not one of the scFv PCR products lacked 5′ homology to the cut vector and the second scFv PCR product lacked 3′ homology to the cut vector, or both PCR products had both 5′ and 3′ homology to the cut vector. A majority of the chimeras had single crossovers; however, double and triple crossovers were isolated. Crossover points were evenly distributed in the hybrids created and homology of as little as two nucleotides was able to produce a chimeric clone. The numbers of clones isolated with a given number of crossovers was approximated well by a Poisson distribution. Transformation efficiencies for the chimeric libraries were of the order of 104–105 transformants per microgram of insert, which is the same order of magnitude as when a single PCR product is inserted alone into the display vector by homologous recombination. This method eliminates ligation and Escherichia coli transformation steps of previous methods for generating yeast-displayed libraries, requires fewer PCR cycles than in vitro DNA shuffling and, unlike site-specific recombination methods, allows for recombination anywhere that homology exists between the genes to be recombined. This simple technique should prove useful for protein engineering in general and antibody engineering, specifically in yeast. 相似文献
8.
Zhentu Ying Thomas L. Davenport Taifang Zhang Raymond J. Schnell Cecile L. Tondo 《Plant Molecular Biology Reporter》2009,27(3):374-380
‘Hass’ is the most popular avocado (Persea americana Mill.) cultivar in the world. It has been characterized as a crop requiring cross-pollination. However, the potential extent
of self-pollination and the most effective pollen donors (best cross-pollinizing cultivars) have not been determined. In this
study, 56 markers were screened against ‘Hass’ and nine commonly used pollinizing cultivars grown in southern California:
‘Bacon,’ ‘Ettinger,’ ‘Fuerte,’ ‘Harvest,’ ‘Lamb Hass,’ ‘Marvel,’ ‘Nobel,’ ‘Sir Prize,’ and ‘Zutano.’ Seventeen microsatellite,
i.e., simple sequence repeat (SSR) markers, were found to be very promising for paternity analysis. Four highly informative
SSR markers were selected to accurately and unequivocally identify pollen parents of ‘Hass’ fruit from an orchard interplanted
with these pollinizing cultivars. From 2003 to 2006, 7,984 ‘Hass’ fruit were analyzed for their paternity. Overall, the pollen
parents of 99.55% of the analyzed fruit could be unequivocally identified with a single multiplex polymerase chain reaction
(PCR). Only 36 fruits (<0.45%) required a second PCR reaction to reach unequivocal identification of the pollen parents. 相似文献
9.
In vitro recombination of homologous genes (family shuffling) has been proposed as an effective search strategy for laboratory evolution of genes and proteins. Few data are available, however, on the composition of shuffled gene libraries, from which one could assess the efficiency of recombination and optimize protocols. Here, probe hybridization is used in a macroarray format to analyze chimeric DNA libraries created by DNA shuffling. Characterization of hundreds of shuffled genes encoding dioxygenases has elucidated important biases in the shuffling reaction. As expected, crossovers are favored in regions of high sequence identity. A sequence-based model of homologous recombination that captures this observed bias was formulated using the experimental results. The chimeric genes were found to show biases in the incorporation of sequences from certain parents, even before selection. Statistically different patterns of parental incorporation in genes expressing functional proteins can help to identify key sequence-function relationships. 相似文献
10.
Lijun Chai Xiaoxia Ge Manosh Kumar Biswas Qiang Xu Xiuxin Deng 《Plant Cell, Tissue and Organ Culture》2011,104(1):1-11
‘Shatian’ pummelo (Citrus grandis Osbeck), one of the main citrus cultivars in China, is self-incompatible, and its pollen tubes are believed to be arrested
in style after self-pollination.We have characterized one ‘Shatian’ pummelo mutant, named ‘Zigui shatian’ pummelo. The mutant
pummelo had identical DNA ploidy level, morphology (leaf shape, stoma size and density, pollen shape and size) and developmental
progress of pistil and male organs to that of the common ‘Shatian’ pummelo. However, unlike the common ‘Shatian’ pummelo,
‘Zigui shatian’ is self-compatible since its pollen tubes can self-pollinate allowing for successful fertilization. Histological
analyses of ‘Shatian’ pummelo further verified abnormal post-zygotic development which led to seed abortion. Simple sequence
repeats (SSR) analysis revealed polymorphism in 1 of the 120 primers screened showing that ‘Zigui shatian’ and ‘Shatian’ pummelo
are different at the DNA level. Taken together, these data suggested mutant ‘Zigui shatian’ pummelo might be derived from
‘Shatian’ pummelo with self-sterility by self-incongruity after self-fertilization. 相似文献
11.
Exon shuffling is an essential molecular mechanism for the formation of new genes. Many cases of exon shuffling have been reported in vertebrate genes. These discoveries revealed the importance of exon shuffling in the origin of new genes. However, only a few cases of exon shuffling were reported from plants and invertebrates, which gave rise to the assertion that the intron-mediated recombination mechanism originated very recently. We focused on the origin of new genes by exon shuffling and retroposition. We will first summarize our experimental work, which revealed four new genes in Drosophila, plants, and humans. These genes are 106 to 108 million years old. The recency of these genes allows us to directly examine the origin and evolution of genes in detail. These observations show firstly the importance of exon shuffling and retroposition in the rapid creation of new gene structures. They also show that the resultant chimerical structures appearing as mosaic proteins or as retroposed coding structures with novel regulatory systems, often confer novel functions. Furthermore, these newly created genes appear to have been governed by positive Darwinian selection throughout their history, with rapid changes of amino acid sequence and gene structure in very short periods of evolution. We further analyzed the distribution of intron phases in three non-vertebrate species, Drosophila melanogaster, Caenorhabditis elegans, and Arabidosis thaliana, as inferred from their genome sequences. As in the case of vertebrate genes, we found that intron phases in these species are unevenly distributed with an excess of phase zero introns and a significant excess of symmetric exons. Both findings are consistent with the requirements for the molecular process of exon shuffling. Thus, these non-vertebrate genomes may have also been strongly impacted by exon shuffling in general. 相似文献
12.
V. G. M. Bus D. Chagné H. C. M. Bassett D. Bowatte F. Calenge J.-M. Celton C.-E. Durel M. T. Malone A. Patocchi A. C. Ranatunga E. H. A. Rikkerink D. S. Tustin J. Zhou S. E. Gardiner 《Tree Genetics & Genomes》2008,4(2):223-236
Woolly apple aphid (WAA; Eriosoma lanigerum Hausm.) can be a major economic problem to apple growers in most parts of the world, and resistance breeding provides a sustainable
means to control this pest. We report molecular markers for three genes conferring WAA resistance and placing them on two
linkage groups (LG) on the genetic map of apple. The Er1 and Er2 genes derived from ‘Northern Spy’ and ‘Robusta 5,’ respectively, are the two major genes that breeders have used to date
to improve the resistance of apple rootstocks to this pest. The gene Er3, from ‘Aotea 1’ (an accession classified as Malus sieboldii), is a new major gene for WAA resistance. Genetic markers linked to the Er1 and Er3 genes were identified by screening random amplification of polymorphic deoxyribonucleic acid (DNA; RAPD) markers across DNA
bulks from resistant and susceptible plants from populations segregating for these genes. The closest RAPD markers were converted
into sequence-characterized amplified region markers and the genome location of these two genes was assigned to LG 08 by aligning
the maps around the genes with a reference map of ‘Discovery’ using microsatellite markers. The Er2 gene was located on LG 17 of ‘Robusta 5’ using a genetic map developed in a M.9 × ‘Robusta 5’ progeny. Markers for each of
the genes were validated for their usefulness for marker-assisted selection in separate populations. The potential use of
the genetic markers for these genes in the breeding of apple cultivars with durable resistance to WAA is discussed. 相似文献
13.
14.
An Ruisheng Bai Xiaodong Grewal Parwinder S. 《World journal of microbiology & biotechnology》2011,27(3):727-730
We describe a reliable method for the production of fusion PCR products that can be used to transform the wild-type bacteria
to replace target genes for mutagenesis studies. The relevant gene fragments and selective cassette are first amplified separately
by PCR using primers that produce overlapping ends. As economic Taq DNA polymerase is disappointed in producing overlap ends
due to adding an extra 3′-end ‘A’ base which potentially blocks the successful fusion of the amplified fragments, we use a
new primer design strategy to overcome this disadvantage by introducing an additional ‘A’ base in the overlap primers. The
amplified gene fragments were then separately cloned into a pGEM-T easy vector and re-amplified with the aid of a universal
primer T7/SP6. This procedure enables performing nested PCR with the outmost primers in the fusion reaction to reliably splice
the gene fragments into a single molecule with all sequences in the desired order. 相似文献
15.
Reducing mutational bias in random protein libraries 总被引:2,自引:0,他引:2
The success of protein optimization through directed molecular evolution depends to a large extent on the size and quality of the displayed library. Current low-fidelity DNA polymerases that are commonly used during random mutagenesis and recombination in vitro display strong mutational preferences, favoring the substitution of certain nucleotides over others. The result is a biased and reduced functional diversity in the library under selection. In an effort to reduce mutational bias, we combined two different low-fidelity DNA polymerases, Taq and Mutazyme, which have opposite mutational spectra. As a first step, random mutants of the Bacillus thuringiensis cry9Ca1 gene were generated by separate error-prone polymerase chain reactions (PCRs) with each of the two polymerases. Subsequent shuffling by staggered extension process (StEP) of the PCR products resulted in intermediate numbers of AT and GC substitutions, compared to the Taq or Mutazyme error-prone PCR libraries. This strategy should allow generating unbiased libraries or libraries with a specific degree of mutational bias by applying optimal mutagenesis frequencies during error-prone PCR and controlling the concentration of template in the shuffling reaction while taking into account the GC content of the target gene. 相似文献
16.
M. Senda Y. Onodera T. Mikami 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1998,96(6-7):964-968
The mitochondrial atpA gene sequence of the normal fertile sugarbeet (cv ‘TK81-0’) exists in one full-length version and one truncated version,
both of which are present in normal stoichiometry and have a 406-bp segment in common. The PCR approach as well as prolonged
exposure of Southern blots indicates that the products of the recombination across the 406-bp repeat are present in substoichiometric
amounts in the ‘TK81-0’ genome. Intriguingly, one of these substoichiometric sequence arrangements was revealed to be preferentially
amplified in an evolutionary lineage that led to a cytoplasmic male-sterile variant [I-12CMS(2)] in wild beets. We also found
the 406-bp repeat to be part of a 6.5-kb repeat in the mitochondrial genome of I-12CMS(2). This 6.5-kb duplication is likely
to involve recombination between two sets of repeats (the above-mentioned 406-bp repeat and a 7-bp repeat) in an ancestral
beet mitochondria.
Received: 4 October 1997 / Accepted: 31 October 1997 相似文献
17.
18.
P. Vain B. Worland M. C. Clarke G. Richard M. Beavis H. Liu A. Kohli M. Leech J. Snape P. Christou H. Atkinson 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1998,96(2):266-271
We have used a genotype-independent transformation system involving particle gun bombardment of immature embryos to genetically
engineer rice as part of a programme to develop resistance to nematodes. Efficient tissue culture, regeneration, DNA delivery
and selection methodologies have been established for elite African varieties (‘ITA212’, ‘IDSA6’, ‘LAC23’, ‘WAB56-104’). Twenty-five
transformed clones containing genes coding for an engineered cysteine proteinase inhibitor (oryzacystatin-IΔD86, OC-IΔD86), hygromycin resistance (aphIV) and β-glucuronidase (gusA) were recovered from the four varieties. Transformed plants were regenerated from all clones and analysed by PCR, Southern
and western blot. Detectable levels of OC-IΔD86 (up to 0.2% total soluble protein) in plant roots were measured in 12 out of 25 transformed rice lines. This level of
expression resulted in a significant 55% reduction in egg production by Meloidogyne incognita.
Received: 4 August 1997 / Accepted: 22 August 1997 相似文献
19.
Genome scanning for resistance-gene analogs in rice, barley, and wheat by high-resolution electrophoresis 总被引:31,自引:8,他引:23
X. M. Chen R. F. Line H. Leung 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1998,97(3):345-355
Genes cloned from diverse plants for resistance to different pathogens have sequence similarities in domains presumably involved
in pathogen recognition and signal transduction in triggering the defense response. Primers based on the conserved regions
of resistance genes often amplify multiple fragments that may not be separable in an agarose gel. We used denaturing polyacrylamide-gel
electrophoresis to detect PCR products of plant genomic DNA amplified with primers based on conserved regions of resistance
genes. Depending upon the primer pairs used, 30–130 bands were detected in wheat, rice, and barley. As high as 47%, 40%, and
27% of the polymorphic bands were detected in rice, barley, and wheat, respectively, and as high as 12.5% of the polymorphic
bands were detected by certain primers in progeny from a cross of the wheat cultivars ‘Stephens’ and ‘Michigan Amber’. Using
F6 recombinant inbred lines from the ‘Stephens’בMichigan Amber’ cross, we demonstrated that polymorphic bands amplified with
primers based on leucine-rich repeats, nucleotide-binding sites and protein kinase genes, were inherited as single loci. Linkages
between molecular markers and stripe rust resistance genes were detected. This technique provides a new way to develop molecular
markers for assessing the genetic diversity of germplasm based upon potential candidate resistance genes in diverse species.
Received : 5 September 1997 / Accepted : 6 November 1997 相似文献
20.
X. Wang R. Trigiano M. Windham B. Scheffler T. Rinehart J. Spiers 《Tree Genetics & Genomes》2008,4(3):461-468
Abundant, codominant simple sequence repeats (SSRs) markers can be used for constructing genetic linkage maps and in marker-assisted
breeding programs. Enrichment methods for SSR motifs were optimized with the ultimate aim of developing numerous loci in flowering
dogwood (C. florida L.) genome. Small insert libraries using four motifs (GT, CT, TGG, and AAC) were constructed with C. florida ‘Cherokee Brave’ deoxyribonucleic acid (DNA). Colony polymerase chain reaction (PCR) of 2,208 selected clones with three
primers we reported previously indicated that 47% or 1,034 of the clones harbored one of the four targeted SSR motifs. Sequencing
the putative positive clones confirmed that nearly 99% (1,021 of 1,034) of them contained the desired motifs. Of the 871 unique
SSR loci, 617 were dinucleotide repeats (70.8%), and 254 were trinucleotide or longer repeats (29.2%). In total, 379 SSR loci
had perfect structure, 237 had interrupted, and 255 had compound structure. Primer pairs were designed from 351 unique sequences.
The ability of the 351 SSR primer pairs to amplify specific loci was evaluated with genomic DNA of ‘Appalachian Spring’ and
‘Cherokee Brave’. Of these primers, 311 successfully amplified product(s) with ‘Cherokee Brave’ DNA, 21 produced weak or faint
products, and 19 did not amplify any products. Additionally, 218 of the 311 primers pairs revealed polymorphisms between the
two cultivars, and 20 out of 218 primers detected an average of 13.7 alleles from 38 selected Cornus species and hybrids. These SSR loci constitute a valuable resource of ideal markers for both genetic linkage mapping and
gene tagging of flowering dogwood.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献