The clustering of four subfamilies of satellite DNA at individual chromosome ends in Silene latifolia

The satellite DNA (satDNA) on the ends of chromosomes has been isolated and characterized in the dioecious plant Silene latifolia. BAC clones containing large numbers of repeat units of satDNA in a tandem array were isolated to examine the clustering of the repeat units. satDNA repeat units were purified from each isolated BAC clone and sequenced. To investigate pairwise similarities among the repeat units, a phylogenetic tree was constructed using the neighbor-joining algorithm. The repeat units derived from 7 BAC clones were grouped into SacI, KpnI, #11F02, and #16E07 subfamilies. The SacI and KpnI subfamilies have been reported previously. Multicolored fluorescence in situ hybridization (FISH) using SacI or KpnI subfamily probes resulted in different signal intensities and locations at the chromosomal ends, indicating that each chromosomal end has a unique composition of subfamilies of satDNA. For example, the p arm of the X chromosome exhibited signal composition similar to that on the pseudo autosomal region (PAR) of the Y chromosome, but not to that on the q arm of the X chromosome. The satDNA has not been completely homogenized in the S. latifolia genome. Each subfamily is available for a probe of FISH karyotyping.Key words: BAC library, concerted evolution, multicolored FISH, karyotyping, satellite DNA, Silene latifolia.

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Analysis of BAC clones containing homologous sequences on the end of the Xq arm and on chromosome 7 in the dioecious plant Silene latifolia

Genome ◽

10.1139/g10-008 ◽

2010 ◽

Vol 53 (4) ◽

pp. 311-320 ◽

Cited By ~ 4

Author(s):

Kotaro Ishii ◽

Yasuhito Amanai ◽

Yusuke Kazama ◽

Miho Ikeda ◽

Hiroshi Kamada ◽

...

Keyword(s):

Satellite Dna ◽

Sex Chromosome ◽

Artificial Chromosome ◽

Chromosome 7 ◽

Silene Latifolia ◽

Dioecious Plant ◽

Homologous Sequences ◽

Autosomal Region ◽

Single Primer

Silene latifolia is a model dioecious plant with morphologically distinguishable XY sex chromosomes. The end of the Xq arm is quite different from that of the Yp arm, although both are located at opposite ends of their respective chromosomes relative to a pseudo-autosomal region. The Xq arm does not seem to originate from the same autosome as the Yp arm. Bacterial artificial chromosome clone #15B12 has an insert containing a 130-kb stretch in which a 313-bp satellite DNA is repeated 420 times. PCR with a single primer revealed that this 130-kb stretch consists of three reversals of the orientation of the satellite DNA. A non-long terminal repeat retroelement and two sequences that share homology with an Oryza sativa RING zinc finger and a putative Arabidopsis thaliana protein, respectively, were found in the sequences that flank the satellite DNA. Fluorescence in situ hybridization carried out using this low-copy region of #15B12 as a probe confirmed that these sequences originated from the X chromosome and that homologous sequences exist at the end of chromosome 7. The region distal to DD44X on the Xq arm is postulated to have recombined with a region containing satellite DNA on chromosome 7 during the process of sex chromosome evolution.

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A Census of rRNA Genes and Linked Genomic Sequences within a Soil Metagenomic Library

Applied and Environmental Microbiology ◽

10.1128/aem.69.5.2684-2691.2003 ◽

2003 ◽

Vol 69 (5) ◽

pp. 2684-2691 ◽

Cited By ~ 142

Author(s):

Mark R. Liles ◽

Brian F. Manske ◽

Scott B. Bintrim ◽

Jo Handelsman ◽

Robert M. Goodman

Keyword(s):

16S Rrna ◽

Bac Library ◽

Metagenomic Library ◽

Pcr Amplification ◽

16S Rrna Genes ◽

Rrna Genes ◽

Acid Uptake ◽

Direct Pcr ◽

Bac Clone ◽

Bac Clones

ABSTRACT We have analyzed the diversity of microbial genomes represented in a library of metagenomic DNA from soil. A total of 24,400 bacterial artificial chromosome (BAC) clones were screened for 16S rRNA genes. The sequences obtained from BAC clones were compared with a collection generated by direct PCR amplification and cloning of 16S rRNA genes from the same soil. The results indicated that the BAC library had substantially lower representation of bacteria among the Bacillus, α-Proteobacteria, and CFB groups; greater representation among the β- and γ-Proteobacteria, and OP10 divisions; and no rRNA genes from the domains Eukaryota and Archaea. In addition to rRNA genes recovered from the bacterial divisions Proteobacteria, Verrucomicrobia, Firmicutes, Cytophagales, and OP11, we identified many rRNA genes from the BAC library affiliated with the bacterial division Acidobacterium; all of these sequences were affiliated with subdivisions that lack cultured representatives. The complete sequence of one BAC clone derived from a member of the Acidobacterium division revealed a complete rRNA operon and 20 other open reading frames, including predicted gene products involved in cell division, cell cycling, folic acid biosynthesis, substrate metabolism, amino acid uptake, DNA repair, and transcriptional regulation. This study is the first step in using genomics to reveal the physiology of as-yet-uncultured members of the Acidobacterium division.

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Utilization of Super BAC Pools and Fluidigm Access Array Platform for High-Throughput BAC Clone Identification: Proof of Concept

Journal of Biomedicine and Biotechnology ◽

10.1155/2012/405940 ◽

2012 ◽

Vol 2012 ◽

pp. 1-7 ◽

Cited By ~ 3

Author(s):

Peter J. Maughan ◽

Scott M. Smith ◽

Joshua A. Raney

Keyword(s):

Bac Library ◽

Pcr Amplification ◽

Artificial Chromosome ◽

Nucleotide Polymorphisms ◽

Bac Clone ◽

Pcr Screening ◽

Clone Identification ◽

Bac Clones ◽

Array Platform ◽

Fluidigm Access Array

Bacterial artificial chromosome (BAC) libraries are critical for identifying full-length genomic sequences, correlating genetic and physical maps, and comparative genomics. Here we describe the utilization of the Fluidigm access array genotyping system in conjunction with KASPar genotyping technology to identify individual BAC clones corresponding to specific single-nucleotide polymorphisms (SNPs) from an Amplicon Express seven-plate super pooledAmaranthus hypochondriacusBAC library. Ninety-six SNP loci, spanning the length ofA. hypochondriacuslinkage groups 1, 2, and 15, were simultaneously tested for clone identification from four BAC super pools, corresponding to 28 384-well plates, using a single Fluidigm integrated fluidic chip (IFC). Forty-six percent of the SNPs were associated with a single unambiguous identified BAC clone. PCR amplification and next-generation sequencing of individual BAC clones confirmed the IFC clone identification. Utilization of the Fluidigm Dynamic array platform allowed for the simultaneous PCR screening of 10,752 BAC pools for 96 SNP tag sites in less than three hours at a cost of~$0.05per reaction.

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Construction of a garlic BAC library and chromosomal assignment of BAC clones using the FISH technique

Genome ◽

10.1139/g03-012 ◽

2003 ◽

Vol 46 (3) ◽

pp. 514-520 ◽

Cited By ~ 8

Author(s):

Hye-Ran Lee ◽

Eun-Mi Eom ◽

Yong-Pyo Lim ◽

Jae-Wook Bang ◽

Dong-Hee Lee

Keyword(s):

Allium Sativum ◽

Bac Library ◽

Chromosomal Assignment ◽

Fish Analysis ◽

Average Insert Size ◽

Insert Size ◽

Bac Clone ◽

Fish Technique ◽

Bac Clones ◽

Cytogenetic Studies

For molecular and cytogenetic studies, two partial bacterial artificial chromosome (BAC) libraries of the garlic cultivar Allium sativum L. 'Danyang' were constructed using high molecular weight (HMW) garlic DNA, the pBAC1SACB1 vector, and the pIndigoBAC536 vector. The average insert size of the BAC library was about 90 kb. The sequence compositions of the BAC clones were characterized by Southern hybridization with garlic genomic DNA and a repetitive sequence clone of garlic. Two BAC clones with weak signals (thus implying mostly unique sequences), GBC2-5e and GBC2-4d, were selected for FISH analysis. FISH analysis localized the GBC2-5e (~100 kb) BAC clone on the long arm of garlic chromosome 7. The other BAC clone, GBC2-4d (~110 kb), gave rise to discrete FISH signals on a mid-size early metaphase chromosome. The FISH screening with BAC clones proved to be a useful resource for molecular cytogenetic studies of garlic, and will be useful for further mapping and sequencing studies of important genes of this plant.Key words: garlic, chromosomal assignment, Allium sativum L., FISH technique, BAC library.

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Construction and characterization of bacterial artificial chromosome library of black-handed spider monkey (Ateles geoffroyi)

Genome ◽

10.1139/g03-122 ◽

2004 ◽

Vol 47 (2) ◽

pp. 239-245 ◽

Cited By ~ 4

Author(s):

Yaping Qian ◽

Li Jin ◽

Bing Su

Keyword(s):

Bacterial Artificial Chromosome ◽

Large Scale ◽

Bac Library ◽

Artificial Chromosome ◽

Spider Monkey ◽

Ateles Geoffroyi ◽

Comparative Genomic ◽

Average Insert Size ◽

Bac Clones ◽

Genomic Study

The large-insert genomic DNA library is a critical resource for genome-wide genetic dissection of target species. We constructed a high-redundancy bacterial artificial chromosome (BAC) library of a New World monkey species, the black-handed spider monkey (Ateles geoffroyi). A total of 193 152 BAC clones were generated in this library. The average insert size of the BAC clones was estimated to be 184.6 kb with the small inserts (50-100 kb) accounting for less than 3% and the non-recombinant clones only 1.2%. Assuming a similar genome size with humans, the spider monkey BAC library has about 11× genome coverage. In addition, by end sequencing of randomly selected BAC clones, we generated 367 sequence tags for the library. When blasted against human genome, they showed a good correlation between the number of hit clones and the size of the chromosomes, an indication of unbiased chromosomal distribution of the library. This black-handed spider monkey BAC library would serve as a valuable resource in comparative genomic study and large-scale genome sequencing of nonhuman primates.Key words: black-handed spider monkeys, Ateles geoffroyi, BAC library.

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Analysis of the histone cluster in Senegalese sole (Solea senegalensis): evidence for a divergent evolution of two canonical histone clusters

Genome ◽

10.1139/gen-2016-0143 ◽

2017 ◽

Vol 60 (5) ◽

pp. 441-453 ◽

Cited By ~ 7

Author(s):

Manuel Alejandro Merlo ◽

Roger Iziga ◽

Silvia Portela-Bens ◽

Ismael Cross ◽

Nadezda Kosyakova ◽

...

Keyword(s):

Product Diversification ◽

Divergent Evolution ◽

Solea Senegalensis ◽

Senegalese Sole ◽

Bac Clone ◽

Bac Clones ◽

Different Types ◽

Priority Species ◽

First Time ◽

Canonical Histone

The Senegalese sole (Solea senegalensis) is commercially very important and a priority species for aquaculture product diversification. The main histone cluster was identified within two BAC clones. However, two replacement histones (H1.0 and H3.3) were found in another BAC clone. Different types of canonical histones H2A and H2B were found within the same species for the first time. Phylogenetic analysis demonstrated that the different types of H1, H2A, and H2B histones were all more similar to each other than to canonical histones from other species. The canonical histone H3 of S. senegalensis differs from subtypes H3.1 and H3.2 in humans at the site of residue 96, where a serine is found instead of an alanine. This same polymorphism has been found only in Danio rerio. The karyotype of S. senegalensis comprises 21 pairs of chromosomes, distributed in 3 metacentric pairs, 2 submetacentric pairs, 4 subtelocentric pairs, and 12 acrocentric pairs. The two BAC clones that contain the clusters of canonical histones were both mapped on the largest metacentric pair, and mFISH analysis confirmed the co-location with the dmrt1 gene in that pair. Three chromosome markers have been identified which, in addition to those previously described, account for 18 chromosome pairs in S. senegalensis.

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Use of a Mycobacterium tuberculosisH37Rv Bacterial Artificial Chromosome Library for Genome Mapping, Sequencing, and Comparative Genomics

Infection and Immunity ◽

10.1128/iai.66.5.2221-2229.1998 ◽

1998 ◽

Vol 66 (5) ◽

pp. 2221-2229 ◽

Cited By ~ 144

Author(s):

Roland Brosch ◽

Stephen V. Gordon ◽

Alain Billault ◽

Thierry Garnier ◽

Karin Eiglmeier ◽

...

Keyword(s):

Comparative Genomics ◽

Bacterial Artificial Chromosome ◽

Genome Mapping ◽

Sequence Data ◽

Bac Library ◽

Artificial Chromosome ◽

Excellent Correlation ◽

Sequencing Project ◽

Polysaccharide Biosynthesis ◽

Bac Clones

ABSTRACT The bacterial artificial chromosome (BAC) cloning system is capable of stably propagating large, complex DNA inserts in Escherichia coli. As part of the Mycobacterium tuberculosis H37Rv genome sequencing project, a BAC library was constructed in the pBeloBAC11 vector and used for genome mapping, confirmation of sequence assembly, and sequencing. The library contains about 5,000 BAC clones, with inserts ranging in size from 25 to 104 kb, representing theoretically a 70-fold coverage of the M. tuberculosisgenome (4.4 Mb). A total of 840 sequences from the T7 and SP6 termini of 420 BACs were determined and compared to those of a partial genomic database. These sequences showed excellent correlation between the estimated sizes and positions of the BAC clones and the sizes and positions of previously sequenced cosmids and the resulting contigs. Many BAC clones represent linking clones between sequenced cosmids, allowing full coverage of the H37Rv chromosome, and they are now being shotgun sequenced in the framework of the H37Rv sequencing project. Also, no chimeric, deleted, or rearranged BAC clones were detected, which was of major importance for the correct mapping and assembly of the H37Rv sequence. The minimal overlapping set contains 68 unique BAC clones and spans the whole H37Rv chromosome with the exception of a single gap of ∼150 kb. As a postgenomic application, the canonical BAC set was used in a comparative study to reveal chromosomal polymorphisms between M. tuberculosis, M. bovis, and M. bovis BCG Pasteur, and a novel 12.7-kb segment present in M. tuberculosis but absent from M. bovis and M. bovis BCG was characterized. This region contains a set of genes whose products show low similarity to proteins involved in polysaccharide biosynthesis. The H37Rv BAC library therefore provides us with a powerful tool both for the generation and confirmation of sequence data as well as for comparative genomics and other postgenomic applications. It represents a major resource for present and future M. tuberculosis research projects.

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Isolation of a Pericentromeric Satellite DNA Family in Chnootriba argus (Henosepilachna argus) with an Unusual Short Repeat Unit (TTAAAA) for Beetles

Insects ◽

10.3390/insects10090306 ◽

2019 ◽

Vol 10 (9) ◽

pp. 306 ◽

Cited By ~ 1

Author(s):

Pablo Mora ◽

Jesús Vela ◽

Areli Ruiz-Mena ◽

Teresa Palomeque ◽

Pedro Lorite

Keyword(s):

Repetitive Dna ◽

Satellite Dna ◽

Repeat Unit ◽

Pericentromeric Region ◽

Fish Analysis ◽

Agricultural Pests ◽

Base Pairs ◽

Satellite Dnas ◽

Repeat Units

Ladybird beetles (Coccinellidae) are one of the largest groups of beetles. Among them, some species are of economic interest since they can act as a biological control for some agricultural pests whereas other species are phytophagous and can damage crops. Chnootriba argus (Coccinellidae, Epilachnini) has large heterochromatic pericentromeric blocks on all chromosomes, including both sexual chromosomes. Classical digestion of total genomic DNA using restriction endonucleases failed to find the satellite DNA located on these heterochromatic regions. Cloning of C0t-1 DNA resulted in the isolation of a repetitive DNA with a repeat unit of six base pairs, TTAAAA. The amount of TTAAAA repeat in the C. argus genome was about 20%. Fluorescence in situ hybridization (FISH) analysis and digestion of chromosomes with the endonuclease Tru9I revealed that this repetitive DNA could be considered as the putative pericentromeric satellite DNA (satDNA) in this species. The presence of this satellite DNA was tested in other species of the tribe Epilachnini and it is also present in Epilachna paenulata. In both species, the TTAAAA repeat seems to be the main satellite DNA and it is located on the pericentromeric region on all chromosomes. The size of this satDNA, which has only six base pairs is unusual in Coleoptera satellite DNAs, where satDNAs usually have repeat units of a much larger size. Southern hybridization and FISH proved that this satDNA is conserved in some Epilachnini species but not in others. This result is in concordance with the controversial phylogenetic relationships among the genera of the tribe Epilachnini, where the limits between genera are unclear.

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The Y chromosome-specific STS marker MS2 and its peripheral regions on the Y chromosome of the dioecious plant Silene latifolia

Genome ◽

10.1139/g08-005 ◽

2008 ◽

Vol 51 (4) ◽

pp. 251-260 ◽

Cited By ~ 8

Author(s):

Kotaro Ishii ◽

Ryuji Sugiyama ◽

Megumi Onuki ◽

Yusuke Kazama ◽

Sachihiro Matsunaga ◽

...

Keyword(s):

Y Chromosome ◽

Chromosome Evolution ◽

Sex Chromosome ◽

Early Stage ◽

Silene Latifolia ◽

Segmental Duplications ◽

Average Insert Size ◽

Sts Marker ◽

Bac Clone ◽

Peripheral Regions

Sex determination in Silene latifolia uses the XX/XY system. The recent evolution of dioecy in S. latifolia provides a unique opportunity to study the early stages of Y chromosome evolution. However, the current Y chromosome map still contains many large gaps with no available markers. In this study, a sequence tagged site (STS) marker, MS2, was isolated and mapped to the same locus as L8 on the Y chromosome. To investigate the peripheral regions of MS2, a bacterial artificial chromosome (BAC) library was constructed from a male plant, and the BAC clone containing MS2 (MS2-9d12F) was isolated from 32 640 clones with an average insert size of 115 kb. A 109-kb insert of the BAC clone was analyzed. BLASTX analysis showed 11 sequences similar to some known proteins, most of which are retrotransposon-like elements. The ORF Finder predicted 9 ORFs within MS2-9d12F. RT-PCR analyses revealed that only 4 of the 9 predicted ORFs are expressed in both male and female plants. These 4 ORFs are candidates for genes having counterparts on both the X and Y chromosomes. Dot-matrix plot analysis and a BLASTN search revealed LTR-like sequences close to the retrotransposon-like elements and high similarity to 3 known genomic sequences of S. latifolia. These results suggest an accumulation of retrotransposons and segmental duplications in peripheral regions of MS2 during the early stage of sex chromosome evolution.

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Instability of bacterial artificial chromosome (BAC) clones containing tandemly repeated DNA sequences

Genome ◽

10.1139/g01-029 ◽

2001 ◽

Vol 44 (3) ◽

pp. 463-469 ◽

Cited By ~ 22

Author(s):

Junqi Song ◽

Fenggao Dong ◽

Jason W Lilly ◽

Robert M Stupar ◽

Jiming Jiang

Keyword(s):

Dna Sequences ◽

Tandem Repeats ◽

Early Stage ◽

Repeated Dna ◽

Genome Research ◽

Bac Clone ◽

Artificial Chromosomes ◽

Bac Clones ◽

The Impact ◽

Tandemly Repeated Dna

The cloning and propagation of large DNA fragments as bacterial artificial chromosomes (BACs) has become a valuable technique in genome research. BAC clones are highly stable in the host, Escherichia coli, a major advantage over yeast artificial chromosomes (YACs) in which recombination-induced instability is a major drawback. Here we report that BAC clones containing tandemly repeated DNA elements are not stable and can undergo drastic deletions during routine library maintenance and DNA preparation. Instability was observed in three BAC clones from sorghum, rice, and potato, each containing distinct tandem repeats. As many as 46% and 74% of the single colonies derived from a rice BAC clone containing 5S ribosomal RNA genes had insert deletions after 24 and 120 h of growth, respectively. We also demonstrated that BAC insert rearrangement can occur in the early stage of library construction and duplication. Thus, a minimum growth approach may not avoid the instability problem of such clones. The impact of BAC instability on genome research is discussed.Key words: repetitive DNA, large insert DNA library, genome research.

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