A NotI-EcoRV promoter library for studies of genetic and epigenetic alterations in mouse models of human malignancies

Aberrant promoter methylation and associated chromatin changes are primarily studied in human malignancies. Thus far, mouse models for human cancer have been rarely utilized to study the role of DNA methylation in tumor onset and progression. It would be advantageous to use mouse tumor models to a greater extent to study the role and mechanism of DNA methylation in cancer because mouse models allow manipulation of the genome, study of samples/populations with a homogeneous genetic background, the possibility of modulating gene expression in vivo, the statistical power of using large numbers of tumor samples, access to various tumor stages, and the possibility of preclinical trials. Therefore, it is likely that the mouse will emerge as an increasingly utilized model to study DNA methylation in cancer. To foster the use of mouse models, we developed an arrayed mouse NotI-EcoRV genomic library, with clones from three commonly used mouse strains (129SvIMJ, FVB/NJ, and C57BL/6J). A total of 23,040 clones representing an estimated three- to fourfold coverage of the mouse genome were arrayed in 60 x 384-well plates. We developed restriction landmark genomic scanning (RLGS) mixing gels with 32 plates to enable the cloning of methylated sequences from RLGS profiles run with NotI-EcoRV-HinfI. RLGS was used to study aberrant methylation in two mouse models that overexpressed IL-15 or c-Myc and developed either T/NK-cell leukemia or T-cell lymphomas, respectively. Careful analysis of 198 sequences showed that 188 (94.9%) identified CpG-island sequences, 132 sequences (66.7%) had homology to the 5' regions of known genes or mRNAs, and all 132 NotI-EcoRV clones were located at the same CpG islands with the predicted promoter sequences. We have also developed a modified pGL3-based luciferase vector that now contains the NotI, AscI, and EcoRV restriction sites and allows the rapid cloning of NotI-EcoRV library fragments in both orientations. Luciferase assays using NotI-EcoRV clones confirmed that the library is enriched for promoter sequences. Thus, this library will support future genetic and epigenetic studies in mouse models.     

Development of an efficient maintenance and screening system for large-insert genomic DNA libraries of hexaploid wheat in a transformation-competent artificial chromosome (TAC) vector

Three large-insert genomic DNA libraries of common wheat, Triticum aestivum cv. Chinese Spring, were constructed in a newly developed transformation-competent artificial chromosome (TAC) vector, pYLTAC17, which accepts and maintains large genomic DNA fragments stably in both Escherichia coli and Agrobacterium tumefaciens. The vector contains the cis sequence required for Agrobacterium-mediated gene transfer into grasses. The average insert sizes of the three genomic libraries were approximately 46, 65 and 120 kbp, covering three haploid genome equivalents. Genomic libraries were stored as frozen cultures in a 96-well format, each well containing approximately 300-600 colonies (12 plates for small library, four for medium-size library and four for large library). In each of the libraries, approximately 80% of the colonies harbored genomic DNA inserts of >50 kbp. TAC clones containing gene(s) of interest were identified by the pooled PCR technique. Once the target TAC clones were isolated, they could be immediately transferred into grass genomes with the Agrobacterium system. Five clones containing the thionin type I genes (single copy per genome), corresponding to each of the three genomes (A, B and D), were successfully selected by the pooled PCR method, in addition to an STS marker (aWG464; single copy per genome) and CAB (a multigene family). TAC libraries constructed as described here can be used to isolate genomic clones containing target genes, and to carry out genome walking for positional cloning.     

Construction,characterization, and screening of a transformation-competent artificial chromosome library of peach

As a genome model of fruit trees, peach (Prunus persica [L.] Batch) has advantages for studying structural and functional genomics. Okubo, a traditional peach variety used as a parent in Asian peach breeding, displays economically valuable agronomic traits. To develop an efficient platform for peach gene cloning and genomic research, a large-insert genomic DNA library of Okubo was constructed in a transformation-competent artificial chromosome (TAC) vector, pYLTAC7, which can accept and stably maintain large genomic DNA fragments in bothEscherichia coli andAgrobacterium tumefaciens. The TAC library contains 41,472 recombinant clones with an average insert size of approximately 42 kb, and it is equivalent to 6 haploid peach genomes. The TAC library was stored in 2 ways: one copy as frozen cultures in 108 pieces of 384-well plates and another copy as bulked pools in 36 pieces of 96-well plates, each well containing 12 individual clones. The lack of hybridization signal to chloroplast and mitochondrial genes indicated that the TAC library had no significant cytoplast organelle DNA contamination. TAC clones were stable inE. coli cells until generation 100 and stable in bothE. coli andA. tumefaciens. Twenty-one clones containing the polygalacturonase-inhibiting protein (PGIP) gene were detected by using pooled PCR in the TAC library. Positive clones can be used for peach PGIP gene cloning and functional analysis. The library is well suited for gene cloning and genetic engineering in peach.     

A PCR-based method for isolation of genomic DNA flanking a known DNA sequence

We describe a simple PCR-based method for the isolation of genomic DNA that lies adjacent to a known DNA sequence. The method is based on the directional cloning of digested genomic DNA into the multiple cloning site of a pUC-based plasmid to generate a limited genomic library. The library is plated onto a number of selective LA plates which are incubated overnight, and recombinant plasmid DNA is then isolated from resistant colonies pooled from each plate. PCR amplification is performed on the pooled recombinant plasmid DNAs using primers specific for the pUC vector and the known genomic sequence. The combination of efficient directional cloning and bacterial transformation gives relative enrichment for the genomic sequence of interest and generates a simple DNA template, enabling easy amplification by PCR.     

Restriction landmark genomic scanning (RLGS) in fungi

RLGS is a technique to detect DNA polymorphism using restriction sites as landmarks. It identifies the landmarks through direct end-labeling, two-dimensional electrophoresis and autoradiography, giving a profile with many spots to allow the scanning of numerous DNA loci. We successfully performed the technique on fungi using isolates ofColletotrichum acutatum andC. gloeosporioides in anamorphic Ascomycotina,Rhizopus oryzae in Zygomycotina,Phytophthora nicotianae in Mastigomycotina (or Oomycota) andRhizoctonia solani in anamorphic basidiomycotina. RLGS of total genomic DNA digested with three restriction enzymes,Not, I,EcoR V andMbo I, reproducibly gave specific profiles of ca. 400 to 1.600 spots for each isolate. A polymorphic spot appearing to reflect a genetic difference between the twoColletotrichum species was found in the profiles of the isolates. No other common spots were found in any combination of isolates of the twoColletotrichum species, and thus the other spots on the profiles were regarded as unique to each isolate. These results indicated that RLGS could be applied, as a powerful fingerprinting technique based on genetic information from the whole genomic DNA, to search for useful DNA markers for taxonomic and genomic studies on many fungal species.     

Spot Mapping on the Standard Profile of Restriction Landmark Genomic Scanning (RLGS) of Sorted Chromosome 20 Using Methylation-Insensitive Enzyme

We established the spot mapping system on a restriction landmark genomic scanning (RLGS) profile using sorted chromosome as RLGS material. In this mapping system, we can mapped RLGS spots physically, regardless of their polymorphism, using methylation-insensitive enzymes in all RLGS steps. Here, we report that we identified 28 spots derived from human chromosome 20 on an RLGS profile, and that number was in good agreement with the number predicted from the length of the chromosome 20.     

Two-dimensional screening of the Wageningen chicken BAC library

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     

Epigenetics: application of virtual image restriction landmark genomic scanning (Vi-RLGS)

Restriction landmark genomic scanning (RLGS) is a powerful method for the systematic detection of genetic mutations in DNA length and epigenetic alteration due to DNA methylation. However, the identification of polymorphic spots is difficult because the resulting RLGS spots contain very little target DNA and many non-labeled DNA fragments. To overcome this, we developed a virtual image restriction landmark genomic scanning (Vi-RLGS) system to compare actual RLGS patterns with computer-simulated RLGS patterns (virtual RLGS patterns). Here, we demonstrate in detail the contents of the simulation program (rlgssim), based on the linear relationship between the reciprocal of mobility plotted against DNA fragment length and Vi-RLGS profiling of Arabidopsis thaliana.     

Analysis of CpG islands of trophoblast giant cells by restriction landmark genomic scanning

Rat trophoblast giant cells each contain at least 100 times more genomic DNA per nucleus than diploid cells. This unusual phenomenon appears to be of interest in relation to the molecular mechanism of cell differentiation and gene expression in the placenta. In the present study, we analyzed the CpG islands of trophoblast giant cells by restriction landmark genomic scanning (RLGS) using the methylation-sensitive landmark enzymes, Not I and Bss HII. More than 1,000 and 1,900 spots were detected by RLGS using Not I and Bss HII, respectively, in the placental junctional zone, where more than 90% of genomic DNA is present in the cells with higher DNA content. Of these, 97% (1,009 spots) and 99% (1,911 spots) of the spots found in the junctional zone showed an identical pattern and identical intensity with those of diploid cell controls, for which genomic DNA was extracted from the labyrinth zone and maternal kidney. Therefore, the giant cells are basically polyploid. More importantly, 24 tissue-specific spots were detected by RLGS using Not I. Subsequent cloning and sequencing of four typical spots of the genomic DNA confirmed that these DNA fragments contained abundant CpG dinucleotides and showed characteristics of CpG islands. Of these 24 spots, there were ten spots specific for the placenta, and three of them were specific for the junctional zone, indicating that methylation status of CpG islands in the placental tissue differed between the junctional zone and labyrinth zone. These results suggest that multiple rounds of endoreduplication and modification of CpG islands by cytosine methylation occur during the differentiation process of giant cells. Dev. Genet. 22:132–140, 1998. © 1998 Wiley-Liss, Inc.     

An automatic image analysis system for RLGS films

The RLGS (Restriction Landmark Genome Scanning) method was originally developed as a powerful method for enabling viewing of thousands of restriction landmarks. It offers a tool for obtaining information about genetic loci, with a single RLGS profile displaying approximately 2000 restriction landmarks as spots. One of the most useful applications is RLGS spot mapping, which allows the efficient, low-cost construction of the genetic map of any organism. However, analyses of the profiles depend mainly on human visual observation and are tedious and laborious. Although several commercially available image analyzing systems for profile comparison have been examined, they cannot be used for the RLGS spot mapping system owing to the background characteristics of the RLGS profiles, unsatisfactory rates of correspondence, and inefficient correction of informative genetic data. We therefore developed a novel automatic image analysis system for RLGS spot mapping, using an original algorithm based on the binary image transferred from the original RLGS profile. This system was employed for identifying non-polymorphic and parental strain-specific polymorphic spots of the F₁ mouse profile and yielded efficient initial screening of RLGS profiles. Received: 5 December 1997 / Accepted: 6 April 1998     

Development of new transformation-competent artificial chromosome vectors and rice genomic libraries for efficient gene cloning

The transformation-competent artificial chromosome vector (TAC) system has been shown to be very useful for efficient gene isolation in Arabidopsis thaliana (Proc. Natl. Acad. Sci. USA 96 (1998) 6535). To adapt the vector system for gene isolation in crops, two new TAC vectors and rice genomic libraries were developed. The new vectors pYLTAC17 and pYLTAC27 use the Bar gene and Hpt gene driven by the rice Act1 promoter as the plant selectable markers, respectively, and are suitable for transformation of rice and other grasses. Two representative genomic libraries (I and II) of an Indica rice variety Minghui63, a fertility restorer line for hybrid rice, were constructed with pYLTAC17 using different size classes of partially digested DNA fragments. Library I and library II consisted of 34,560 and 1.2 x 10(5) clones, with average insert sizes of approximately 77 and 39 kb, respectively. The genome coverage of the libraries I and II was estimated to be about 5 and 11 haploid genome equivalents, respectively. Clones of the library I were stored individually in ninety 384-well plates, and those of the library II were collected as bulked pools each containing 30-50 clones and stored in eight 384-well plates. A number of probes were used to hybridize high-density colony filters of the library I prepared by an improved replicating method and each detected 2-9 positive clones. A method for rapid screening of the library II by pooled colony hybridization was developed. A TAC clone having an 80 kb rice DNA insert was successfully transferred into rice genome via Agrobacterium-mediated transformation. The new vectors and the genomic libraries should be useful for gene cloning and genetic engineering in rice and other crops.     

High-throughput fingerprinting of bacterial artificial chromosomes using the snapshot labeling kit and sizing of restriction fragments by capillary electrophoresis

We have developed an automated, high-throughput fingerprinting technique for large genomic DNA fragments suitable for the construction of physical maps of large genomes. In the technique described here, BAC DNA is isolated in a 96-well plate format and simultaneously digested with four 6-bp-recognizing restriction endonucleases that generate 3' recessed ends and one 4-bp-recognizing restriction endonuclease that generates a blunt end. Each of the four recessed 3' ends is labeled with a different fluorescent dye, and restriction fragments are sized on a capillary DNA analyzer. The resulting fingerprints are edited with a fingerprint-editing computer program and contigs are assembled with the FPC computer program. The technique was evaluated by repeated fingerprinting of several BACs included as controls in plates during routine fingerprinting of a BAC library and by reconstruction of contigs of rice BAC clones with known positions on rice chromosome 10.     

Identification and isolation of transcribed human X chromosome DNA sequences

A human X chromosome specific phage library has been used as a source of X-specific genomic DNA clones which hybridize with cellular RNA. Random cDNA clones were mapped for X chromosome sequence localization and 8 were identified as hybridizing to X chromosome Hind III fragments. All eight also hybridized with autosomal Hind III fragments. The X chromosome genomic sequences corresponding to two of these cDNA clones were isolated from a phage library constructed with the Hind III endonuclease digest products of X enriched DNA. One genomic DNA segment, localized to the short area of the X, shared sequence homology with at least one region of the human Y chromosome. The methodology developed represents a rapid means to obtain a specific genomic DNA clone from a single chromosome when multiple different genomic loci homologous to an expressed DNA sequence exist.     

Sequence-tagged microsatellite profiling (STMP): a rapid technique for developing SSR markers

We describe a technique, sequence-tagged microsatellite profiling (STMP), to rapidly generate large numbers of simple sequence repeat (SSR) markers from genomic or cDNA. This technique eliminates the need for library screening to identify SSR-containing clones and provides an ~25-fold increase in sequencing throughput compared to traditional methods. STMP generates short but characteristic nucleotide sequence tags for fragments that are present within a pool of SSR amplicons. These tags are then ligated together to form concatemers for cloning and sequencing. The analysis of thousands of tags gives rise to a representational profile of the abundance and frequency of SSRs within the DNA pool, from which low copy sequences can be identified. As each tag contains sufficient nucleotide sequence for primer design, their conversion into PCR primers allows the amplification of corresponding full-length fragments from the pool of SSR amplicons. These fragments permit the full characterisation of a SSR locus and provide flanking sequence for the development of a microsatellite marker. Alternatively, sequence tag primers can be used to directly amplify corresponding SSR loci from genomic DNA, thereby reducing the cost of developing a microsatellite marker to the synthesis of just one sequence-specific primer. We demonstrate the utility of STMP by the development of SSR markers in bread wheat.     

Sequence-tagged microsatellite profiling (STMP): a rapid technique for developing SSR markers

We describe a technique, sequence-tagged microsatellite profiling (STMP), to rapidly generate large numbers of simple sequence repeat (SSR) markers from genomic or cDNA. This technique eliminates the need for library screening to identify SSR-containing clones and provides an approximately 25-fold increase in sequencing throughput compared to traditional methods. STMP generates short but characteristic nucleotide sequence tags for fragments that are present within a pool of SSR amplicons. These tags are then ligated together to form concatemers for cloning and sequencing. The analysis of thousands of tags gives rise to a representational profile of the abundance and frequency of SSRs within the DNA pool, from which low copy sequences can be identified. As each tag contains sufficient nucleotide sequence for primer design, their conversion into PCR primers allows the amplification of corresponding full-length fragments from the pool of SSR amplicons. These fragments permit the full characterisation of a SSR locus and provide flanking sequence for the development of a microsatellite marker. Alternatively, sequence tag primers can be used to directly amplify corresponding SSR loci from genomic DNA, thereby reducing the cost of developing a microsatellite marker to the synthesis of just one sequence-specific primer. We demonstrate the utility of STMP by the development of SSR markers in bread wheat.     

Direct construction of a chromosome-specific NotI linking library from flow-sorted chromosomes.

A linking library consists of genomic DNA fragments which contain a specific rare restriction enzyme site; such clones are very useful as probes in pulsed field gel electrophoresis and in mapping and cloning large regions of DNA. However, identifying those linking clones which map to a certain chromosomal region can be laborious. Therefore, we have developed a straightforward procedure for constructing a linking library directly from flow-sorted chromosomes. As a test of the approach, a NotI linking library was constructed from the chromosome 17 fraction of a flow-sort of human chromosomes, using only 70 ng of DNA. Thirteen of sixteen linking clones were mapped to chromosome 17, suggesting that the library is highly enriched for this chromosome. This method should be generally applicable to other chromosomes and enzymes as well.     

Generation of a high-quality P1 library of Arabidopsis suitable for chromosome walking

Using improved techniques, a representative P1 library of Arabidopsis was constructed and characterized. Megabase genomic DNA was prepared from nuclei and partially digested with Sau3AI. DNA fragments of 75–100 kb were selected by size fractionation in low melting agarose, concentrated by a spot-evaporation/dialysis method, and cloned in the pAd10sacBII P1 vector. The library contains 10 080 clones individually stored in microtiter plates. With an average insert size of about 80 kb, the library represents about eight haploid genomic equivalents of this plant. This library can be screened rapidly by dot hybridization of plate and well position pools. Characterization of the library by restriction analysis, screening with RFLP probes, RFLP mapping of insert end sequences, and chromosome walking shows that the library is of high quality with respect to insert site, completeness, and absence of chimeric artifacts. With this library a contig of about 600 kb has been constructed in the cer9 locus region. This P1 library is expected to be useful for genome mapping and gene cloning in Arabidopsis research programs.