共查询到20条相似文献,搜索用时 31 毫秒
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Z. Deng Q. Tao Y.-L. Chang S. Huang P. Ling C. Yu C. Chen F. G. Gmitter Jr. H.-B. Zhang 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2001,102(8):1177-1184
A BAC library was constructed from the genomic DNA of an intergeneric Citrus and Poncirus hybrid. The library consists of 24,576 clones with an average insert size of 115 kb, representing approximately seven haploid
genome equivalents and is able to give a greater than 99% probability of isolating single-copy citrus DNA sequences from this
library. High-density colony hybridization-based library screening was performed using DNA markers linked to the citrus tristeza
virus (CTV) resistance gene and citrus disease resistance gene candidate (RGC) sequences. Between four and eight clones were
isolated with each of the CTV resistance gene-linked markers, which agrees with the library’s predicted genome coverage. Three
hundred and twenty-two clones were identified using 13 previously cloned citrus RGC sequences as probes in library screening.
One to four fragments in each BAC were shown to hybridize with RGC sequences. One hundred and nine of the RGC BAC clones were
fingerprinted using a sequencing gel-based procedure. From the fingerprints, 25 contigs were assembled, each having a size
of 120–250 kb and consisting of 2–11 clones. These results indicate that the library is a useful resource for BAC contig construction
and molecular isolation of disease resistance genes.
Received: 22 May 2000 / Accepted: 25 September 2000 相似文献
3.
A. C. J. Frijters Z. Zhang M. van Damme G.-L. Wang P. C. Ronald R. W. Michelmore 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1997,94(3-4):390-399
Existing bacterial artificial chromosome (BAC) vectors were modified to have unique EcoRI cloning sites. This provided an additional site for generating representative libraries from genomic DNA digested with
a variety of enzymes. A BAC library of lettuce was constructed following the partial digestion of genomic DNA with HindIII or EcoRI. Several experimental parameters were investigated and optimized. The BAC library of over 50,000 clones, representing one
to two genome equivalents, was constructed from six ligations; average insert sizes for each ligation varied between 92.5
and 142 kb with a combined average insert size of 111 kb. The library was screened with markers linked to disease resistance
genes; this identified 134 BAC clones from four regions containing resistance genes. Hybridization with low-copy genomic sequences
linked to resistance genes detected fewer clones than expected from previous estimates of genome size. The lack of hybridization
to chloroplast and mitochondrial sequences demonstrated that the library was predominantly composed of nuclear DNA. The unique
EcoRI site in the BAC vector should allow the integration of BAC cloning with other technologies that utilize EcoRI digestion, such as AFLPTM markers and RecA-assisted restriction endonuclease (RARE) cleavage, to clone specific large EcoRI fragments from genomic DNA.
Received: 5 August 1996 / Accepted: 23 August 1996 相似文献
4.
A cDNA of Japanese flounder (Paralichthys olivaceus) CC chemokine designated as Paol-SCYA104 was cloned and sequenced. The cDNA contains an opening reading frame of 315 nucleotides encoding 104 amino acid residues. The full gene was cloned and sequenced from a BAC library. It has a length of approximately 750 bp from the start codon to the stop codon and is composed of four exons and three introns. Four cysteine residues are conserved in the same positions as those of mammalian and fish CC chemokines. Paol-SCYA104 gene was expressed in several organs, including peripheral blood leukocytes (PBLs), head kidney, trunk kidney, and spleen. The recombinant Paol-SCYA104 was expressed in Escherichia coli and the expressed protein was partially purified. The recombinant Paol-SCYA104 was able to attract Japanese flounder PBLs in a microchemotaxis chamber. On the other hand, a negative control, the fraction of the control cells carrying an expression vector lacking the Paol-SCYA104 cDNA, did not show chemotactic activity. These results indicate that Paol-SCYA104 probably acts as a CC chemokine. 相似文献
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Takeshi Omasa Yihua Cao Joon Young Park Yasuhiro Takagi Shuichi Kimura Hidenori Yano Kohsuke Honda Shuichi Asakawa Nobuyoshi Shimizu Hisao Ohtake 《Biotechnology and bioengineering》2009,104(5):986-994
Chinese hamster ovary (CHO) cell lines are widely used for scientific research and biotechnology. A CHO genomic bacterial artificial chromosome (BAC) library was constructed from a mouse dihydrofolate reductase (DHFR) gene‐amplified CHO DR1000L‐4N cell line for genome‐wide analysis of CHO cell lines. The CHO BAC library consisted of 122,281 clones and was expected to cover the entire CHO genome five times. A CHO chromosomal map was constructed by fluorescence in situ hybridization (FISH) imaging using BAC clones as hybridization probes (BAC‐FISH). Thirteen BAC‐FISH marker clones were necessary to identify all the 20 individual chromosomes in a DHFR‐deficient CHO DG44 cell line because of the aneuploidy of the cell line. To determine the genomic structure of the exogenous Dhfr amplicon, a 165‐kb DNA region containing exogenous Dhfr was cloned from the BAC library using high‐density replica (HDR) filters and Southern blot analysis. The nucleotide sequence analysis revealed a novel genomic structure in which the vector sequence containing Dhfr was sandwiched by long inverted sequences of the CHO genome. Biotechnol. Bioeng. 2009; 104: 986–994. © 2009 Wiley Periodicals, Inc. 相似文献
6.
S. Allouis X. Qi S. Lindup M. D. Gale K. M. Devos 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2001,102(8):1200-1205
A bacterial artificial chromosome (BAC) library was constructed using nuclear DNA from pearl millet (Pennisetum glaucum), and used as a resource for the isolation of microsatellite sequences. The library contains a total of 159,100 clones with
an average insert size of 90 kb, and corresponds to 5.8 haploid genome equivalents. The BAC library was pooled for screening
by the polymerase chain reaction (PCR) as well as robotically gridded on high-density filters. PCR-based screening of a subset
of the library (4.7 haploid genome equivalents) using five sequence-tagged site (STS) and six microsatellite markers identified
between 2 and 11 positives superpools (5.4 on average). The frequency of BAC clones carrying inserts of chloroplast DNA was
estimated to be less than 1% by hybridisation with a rice chloroplast probe.
Received: 30 January 2000 / Accepted: 16 October 2000 相似文献
7.
Identification and differential expression of microRNAs during metamorphosis of the Japanese flounder (Paralichthys olivaceus) 总被引:2,自引:0,他引:2
Background
MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs of 20–25 nucleotides that play a key role in diverse biological processes. Japanese flounder undergo dramatic metamorphosis in their early development. The metamorphosis is characterized by morphological transformation from a bilaterally symmetrical to an asymmetrical body shape concomitant with extensive morphological and physiological remodeling of organs. So far, only a few miRNAs have been identified in fish and there are very few reports about the Japanese flounder miRNA.Methodology/Principal Findings
Solexa sequencing technology was used to perform high throughput sequencing of the small RNA library from the metamorphic period of Japanese flounder. Subsequently, aligning these sequencing data with metazoan known miRNAs, we characterized 140 conserved miRNAs and 57 miRNA: miRNA* pairs from the small RNA library. Among these 57 miRNA: miRNA* pairs, twenty flounder miRNA precursors were amplified from genomic DNA. We also demonstrated evolutionary conservation of Japanese flounder miRNAs and miRNA* in the animal evolution process. Using miRNA microarrays, we identified 66 differentially expressed miRNAs at two metamorphic stages (17 and 29 days post hatching) of Japanese flounder. The results show that miRNAs might play a key role in regulating gene expression during Japanese flounder metamorphosis.Conclusions/Significance
We identified a large number of miRNAs during flounder metamorphosis, some of which are differentially expressed at two different metamorphic stages. The study provides an opportunity for further understanding of miRNA function in the regulation of flounder metamorphosis and gives us clues for further studies of the mechanisms of metamorphosis in Japanese flounder. 相似文献8.
Gutman W Pawełkowicz M Woycicki R Piszczek E Przybecki Z 《Cellular & molecular biology letters》2008,13(1):74-91
Cloning using bacterial artificial chromosomes (BACs) can yield high quality genomic libraries, which are used for the physical
mapping, identification and isolation of genes, and for gene sequencing. A BAC genomic library was constructed from high molecular
weight DNA (HMW DNA) obtained from nuclei of the cucumber (Cucumis sativus L. cv. Borszczagowski; B10 line). The DNA was digested with the HindIII restriction enzyme and ligated into the pCC1BAC vector. The library consists of 34,560 BAC clones with an average insert
size of 135 kb, and 12.7x genome coverage. Screening the library for chloroplast and mitochondrial DNA content indicated an
exceptionally low 0.26% contamination with chloroplast DNA and 0.3% with mitochondrial DNA. 相似文献
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A maize bacterial artificial chromosome (BAC) library from the European flint inbred line F2 总被引:2,自引:0,他引:2
D. M. O’Sullivan P. J. Ripoll M. Rodgers K. J. Edwards 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2001,103(2-3):425-432
We report here the construction and characterisation of a BAC library from the maize flint inbred line F2, widely used in
European maize breeding programs. The library contains 86,858 clones with an average insert size of approximately 90 kb, giving
approximately 3.2-times genome coverage. High-efficiency BAC cloning was achieved through the use of a single size selection
for the high-molecular-weight genomic DNA, and co-transformation of the ligation with yeast tRNA to optimise transformation
efficiency. Characterisation of the library showed that less than 0.5% of the clones contained no inserts, while 5.52% of
clones consisted of chloroplast DNA. The library was gridded onto 29 nylon filters in a double-spotted 8 × 8 array, and screened
by hybridisation with a number of single-copy and gene-family probes. A 3-dimensional DNA pooling scheme was used to allow
rapid PCR screening of the library based on primer pairs from simple sequence repeat (SSR) and expressed sequence tag (EST)
markers. Positive clones were obtained in all hybridisation and PCR screens carried out so far. Six BAC clones, which hybridised
to a portion of the cloned Rp1-D rust resistance gene, were further characterised and found to form contigs covering most of this complex resistance locus.
Received: 30 August 2000 / Accepted: 6 December 2000 相似文献
10.
S. Zhu C. A. Saski H. R. Boerma J. P. Tomkins J. N. All W. A. Parrott 《Plant Molecular Biology Reporter》2009,27(2):229-235
Positional cloning of an insect-resistance quantitative trait locus (QTL) requires the construction of a large-insert genomic
DNA library from insect-resistant genotypes. To facilitate cloning of a major defoliating insect-resistance QTL on linkage
group M of the soybean genetic map, a bacterial artificial chromosome (BAC) library for PI 229358 was constructed and characterized.
The HindIII BAC library contains 55,296 clones with an average insert size 131 kb. This library represents a 6-fold soybean haploid
genome equivalents, allowing a 99.8% probability of recovering any specific sequence of interest in soybean. BAC filters were
screened with a genomic DNA probe Sat_258sc2 obtained through genome walking from flanking sequences of a simple sequence
repeat (SSR) marker, Sat_258, which links to the insect-resistance QTL. Thirteen BAC clones were identified positive for Sat_258sc2,
and two of them were confirmed to carry Sat_258. The results suggest that this library is useful in positional cloning of
the major insect-resistance QTL, and the approach presented here can be used to screen a BAC library for a SSR marker without
requiring the creation of BAC pools. 相似文献
11.
A bacterial artificial chromosome (BAC) library of the genomic DNA of Coprinus cinereus strain MP#2 was constructed using the BAC vector pBACTZ, which carries the C. cinereus trp1 gene. The library consists of 1536 clones. Analysis of inserts in some of the clones suggested that the library covers five times the C. cinereus genome. Screening of the BAC clones using ten markers mapped on nine different chromosomes also indicated that the library is likely to cover the whole length of the genomic DNA. We show an example of transformation of C. cinereus with BACs containing inserts of longer than 170kb. 相似文献
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Haiying Liang Eric G. Fang Jeffrey P. Tomkins Meizhong Luo David Kudrna Hye Ran Kim K. Arumuganathan Shaying Zhao James Leebens-Mack Scott E. Schlarbaum Jo Ann Banks Claude W. dePamphilis Dina F. Mandoli Rod A. Wing John E. Carlson 《Tree Genetics & Genomes》2007,3(3):215-225
Liriodendron tulipifera L., a member of the Magnoliaceae, occupies an important phylogenetic position as a basal angiosperm that has retained numerous
putatively ancestral morphological characters, and thus has often been used in studies of the evolution of flowering plants
and of specific gene families. However, genomic resources for these early branching angiosperm lineages are very limited.
In this study, we describe the construction of a large-insert bacterial artificial chromosome (BAC) library from L. tulipifera. Flow cytometry estimates that this nuclear genome is approximately 1,802 Mbp per haploid genome (±16 SD). The BAC library
contains 73,728 clones, a 4.8-fold genome coverage, with an average insert size of 117 kb, a chloroplast DNA content of 0.2%,
and little to no bacterial sequences nor empty vector content clones. As a test of the utility of this BAC library, we screened
the library with six single/low-copy genic probes. We obtained at least two positive clones for each gene and confirmed the
clones by DNA sequencing. A total of 182 paired end sequences were obtained from 96 of the BAC clones. Using BLAST searches,
we found that 25% of the BAC end sequences were similar to DNA sequences in GenBank. Of these, 68% shared sequence with transposable
elements and 25% with genes from other taxa. This result closely reflected the content of random sequences obtained from a
small insert genomic library for L. tulipifera, indicating that the BAC library construction process was not biased. The first genomic DNA sequences for Liriodendron genes are also reported. All the Liriodendron genomic sequences described in this paper have been deposited in the GenBank data library. The end sequences from shotgun
genomic clones and BAC clones are under accession DU169330–DU169684. Partial sequences of Gigantea, Frigida, LEAFY, cinnamyl alcohol dehydrogenase, 4-coumarate:CoA ligase, and phenylalanine ammonia-lyase genes are under accession DQ223429–DQ223434.
Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. 相似文献
13.
Songhun Jang Hang Liu Jianguo Su Feng Dong Feng Xiong Lanjie Liao Yaping Wang Zuoyan Zhu 《Marine biotechnology (New York, N.Y.)》2010,12(3):261-266
Bacterial artificial chromosome (BAC) library is an important tool in genomic research. We constructed two libraries from
the genomic DNA of grass carp (Ctenopharyngodon idellus) as a crucial part of the grass carp genome project. The libraries were constructed in the EcoRI and HindIII sites of the vector CopyControl pCC1BAC. The EcoRI library comprised 53,000 positive clones, and approximately 99.94% of the clones contained grass carp nuclear DNA inserts
(average size, 139.7 kb) covering 7.4× haploid genome equivalents and 2% empty clones. Similarly, the HindIII library comprised 52,216 clones with approximately 99.82% probability of finding any genomic fragments containing single-copy
genes; the average insert size was 121.5 kb with 2.8% insert-empty clones, thus providing genome coverage of 6.3× haploid
genome equivalents of grass carp. We selected gene-specific probes for screening the target gene clones in the HindIII library. In all, we obtained 31 positive clones, which were identified for every gene, with an average of 6.2 BAC clones
per gene probe. Thus, we succeeded in constructing the desired BAC libraries, which should provide an important foundation
for future physical mapping and whole-genome sequencing in grass carp. 相似文献
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Y. Yu J. P. Tomkins R. Waugh D. A. Frisch D. Kudrna A. Kleinhofs R. S. Brueggeman G. J. Muehlbauer R. P. Wise R. A. Wing 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2000,101(7):1093-1099
Modern cultivated barley is an important cereal crop with an estimated genome size of 5000 Mb. To develop the resources for
positional cloning and structural genomic analyses in barley, we constructed a bacterial artificial chromosome (BAC) library
for the cultivar Morex using the cloning enzyme HindIII. The library contains 313344 clones (816 384-well plates). A random sampling of 504 clones indicated an average insert
size of 106 kbp (range=30–195 kbp) and 3.4% empty vectors. Screening the colony filters for chloroplast DNA content indicated
an exceptionally low 1.5% contamination with chloroplast DNA. Thus, the library provides 6.3 haploid genome equivalents allowing
a >99% probability of recovering any specific sequence of interest. High-density filters were gridded robotically using a
Genetix Q-BOT in a 4×4 double-spotted array on 22.5-cm2 filters. Each set of 17 filters allows the entire library to be screened with 18432 clones represented per filter. Screening
the library with 40 single copy probes identified an average 6.4 clones per probe, with a range of 1–13 clones per probe.
A set of resistance-gene analog (RGA) sequences identified 121 RGA-containing BAC clones representing 20 different regions
of the genome with an average of 6.1 clones per locus. Additional screening of the library with a P-loop disease resistance
primer probe identified 459 positive BAC clones. These data indicate that this library is a valuable resource for structural
genomic applications in barley.
Received: 20 September 1999 / Accepted: 25 March 2000 相似文献
17.
Azusa Okumura Masanori Kobayashi Hiroshi Kitajima Yuji Yasukochi Go Suzuki Ken Sahara 《Entomological Science》2019,22(2):167-172
Karyotype evolution in one of the most diverse and species‐rich group of insects, moths and butterflies (Lepidoptera), has interesting features that remain to be resolved. Recent studies showed that fluorescence in situ hybridization using bacterial artificial chromosome clones (BAC‐FISH) is an efficient cytogenetic method for identification and gene mapping of lepidopteran chromosomes. Using comparative mapping by BAC‐FISH, extensive synteny of genes was revealed between chromosomes of different lepidopteran species based on Bombyx mori genomic information. However, this comparative mapping has been done only in representatives of advanced groups of Lepidoptera. Here we constructed a BAC library of Endoclita excrescens, which belongs to the primitive lepidopteran family Hepialidae. High molecular weight DNA for the library construction was prepared from the pupae by using a rapid nuclear isolation method known in plants. The BAC clones of E. excrescens contain 66.6 kb inserts on average. The successful application of BAC‐FISH showed that the BAC library of E. excrescens is a useful tool for comparative gene mapping on chromosomes of this species. 相似文献
18.
Heng Zhu Sangdun Choi Andrea K. Johnston Rod A. Wing Ralph A. Dean 《Fungal genetics and biology : FG & B》1997,21(3):337-347
Magnaporthe grisea(Hebert) Barr causes rice blast, one of the most devastating diseases of rice (Oryza sativa) worldwide. This fungus is an ideal organism for studying a number of aspects of plant–pathogen interactions, including infection-related morphogenesis, avirulence, and pathogen evolution. To facilitateM. griseagenome analysis, physical mapping, and positional cloning, we have constructed a bacterial artificial chromosome (BAC) library from the rice infecting strain 70-15. A new method was developed for separation of partially digested large-molecular-weight DNA fragments that facilitated library construction with large inserts. The library contains 9216 clones, with an average insert size of 130 kbp (>25 genome equivalents) stored in 384-well microtiter plates that can be double spotted robotically on to a single nylon membrane. Several unlinked single-copy DNA probes were used to screen 4608 clones in the library and an average of 13 (minimum of 6) overlapping BAC clones was found in each case. Hybridization of total genomic DNA to the library and analysis of individual clones indicated that ≈26% of the clones contain single-copy DNA. Approximately 35% of BAC clones contained the retrotransposon MAGGY. The library was used to identify BAC clones containing a adenylate cyclase gene (mac1). In addition, a 550-kbp contig composed of 6 BAC clones was constructed that encompassed two adjacent RFLP markers on chromosome 2. These data show that the BAC library is suitable for genome analysis ofM. grisea.Copies of colony hybridization membranes are available upon request. 相似文献
19.
Two-dimensional screening of the Wageningen chicken BAC library 总被引:10,自引:0,他引:10
Richard P.M.A. Crooijmans Julia Vrebalov Rosilde J.M. Dijkhof Jan J. van der Poel Martien A.M. Groenen 《Mammalian genome》2000,11(5):360-363
We have constructed a Bacterial Artificial Chromosome (BAC) library that provides 5.5-fold redundant coverage of the chicken
genome. The library was made by cloning partial HindIII-digested high-molecular-weight (HMW) DNA of a female White Leghorn chicken into the HindIII site of the vector pECBAC1. Several modifications of standard protocols were necessary to clone efficiently large partial
HindIII DNA fragments. The library consists of 49,920 clones arranged in 130 384-well plates. An average insert size of 134 kb
was estimated from the analysis of 152 randomly selected BAC clones. The average number of NotI restriction sites per clone was 0.77. After individual growth, DNA was isolated of the pooled clones of each 384-well plate,
and subsequently DNA of each plate was isolated from the individual row and column pools. Screening of the Wageningen chicken
BAC library was performed by two-dimensional PCR with 125 microsatellite markers. For 124 markers at least one BAC clone was
obtained. FISH experiments of 108 BAC clones revealed chimerism in less than 1%. The number of different BAC clones per marker
present in the BAC library was examined for 35 markers which resulted in a total of 167 different BAC clones. Per marker the
number of BAC clones varied from 1 to 11, with an average of 4.77. The chicken BAC library constitutes an invaluable tool
for positional cloning and for comparative mapping studies.
Received: 26 October 1999 / Accepted: 6 January 2000 相似文献
20.
L. Wilfert M. Muñoz Torres C. Reber-Funk R. Schmid-Hempel J. Tomkins J. Gadau P. Schmid-Hempel 《Insectes Sociaux》2009,56(1):44-48
The primitively social bumblebee Bombus terrestris is an ecological model species as well as an important agricultural pollinator. As part of the ongoing development of genomic
resources for this model organism, we have constructed a publicly available bacterial artificial chromosome (BAC) library
from males of a field-derived colony. We have shown that this library has a high coverage, which allows any particular sequence
to be retrieved from at least one clone with a probability of 99.7%. We have further demonstrated the library’s usefulness
by successfully screening it with probes derived both from previously described B. terrestris genes and candidate genes from another bumblebee species and the honeybee. This library will facilitate genomic studies in
B. terrestris and will allow for novel comparative studies in the social Hymenoptera.
Received 12 March 2008; revised 13 September 2008; accepted 17 September 2008.
R. Schmid-Hempel: contact for data repository of this BAC library 相似文献