Region-specific Libraries Research Articles

Transposable elements in the nurse cell chromocenter DNA of Drosophila orena (Diptera, Drosophilidae)

A region-specific library is constructed by microdissection of chromocenter DNA polytene chromosomes from Drosophila orena nurse cells. Fragments of the library are cloned in a plasmid vector to yield 133 clones. In total, 76 clones are sequenced. A search for resemblance between the fragments cloned and transposable elements known in the genomes of different Drosophila species is performed with the Repeat-Masker program in RepBase. It is found that 26 out of 33 clones homologous to regions of various transposable elements share a resemblance with regions of LTR retrotransposons (Gypsy, Idefix, Zam, Roo, Quasimodo, Stalker, HMS Beagle, Copia, Tv1, Circe, and DM297) and four with LINE regions (DMCR1A, G3, Helena, and I). Other clones show a significant degree of resemblance to regions of DNA transposons, polintons, and helitrons: DNAREP1_DM, Polinton-1_DY, and Helitron-1_DYak. One clone is similar to the MINIME-DN region belonging to mini-me non-LTR retrotransposons.

Analysis of the chromocenter DNA composition in polytene chromosomes of Drosophila orena ovarian nurse cells

Chromocenter DNA fragments of polytene chromosomes of Drosophila orena ovarian nurse cells were cloned from a region-specific library (Dore 1) in a plasmid vector to yield 133 clones. A total of 76 clones were selected and sequenced. The total length of the sequenced fragments was 23940 bp. Analysis with several software packages revealed various repetitive sequences among the fragments of the Dore 1 library, including mobile genetic elements (25 fragments homologous to various LTR retrotransposons, five fragments homologous to LINEs, three fragments homologous to Helitrons, one fragment homologous to Polinton, and one fragment homologous to the mini-me non-LTR retrotransposon), four minisatellites, a satellite (SAR_DM), the (TATATG)n simple sequence repeat, and a low-complexity T-rich repeat. Sequences homologous to protein-coding genes were also found in the Dore 1 library. Various repetitive DNA sequences and gene homologs were identified as conserved sequences of pericentric heterochromatin of polytene chromosomes of ovarian nurse cells in nine species of the melanogaster species subgroup.

Molecular and cytogenic analysis of pericentromeric heterochromatin in ovarian nurse cells of Drosophila melanogaster subgroup species

Evolutionary rearrangements of pericentromeric heterochromatin among Drosophila melanogaster subgroup species have been investigated. Region-specific DNA library from Drosophila orena ovarian nurse cell chromocenter has been obtained by microdissection of polytene chromosomes. The probe was localized on ovarian nurse cells chromosomes of D. melanogaster subgroup species using fluorescent in situ hybridization. Sequences homologous to the sequences of the DNA-probe were found in chromocenter and pericentromeric regions of D. orena polytene chromosomes; and in all pericentromeric regions of other species with several exceptions. So, there was no labeling on one of the arms of D. simulans chromosome 2 but such sequences were present on telomere of D. erecta chromosome 3 and in regions adjacent to the brightly DAPI-stained heterochromatic blocks of D. yakuba, D. santomea and D. teissieri chromosomes 2 and 3. At S6 stage (secondary reticulate nucleus), the labeled chromatin in D. orena could be found mostly within a restricted area and no such chromatin could be detected throughout the rest of the nucleus. On the contrary, the labeled DNA was spread diffusely in the nuclei of other species at this stage.

A 7 Mb duplication at 22q13 in a girl with bipolar disorder and hippocampal malformation

We identified a duplication of 22q13.1-q13.2 in a 10-year-old girl and demonstrated that this duplication was the recombinant product of a maternal intrachromosomal insertion. Phenotypic characteristics included prominent forehead, small low-set ears, hypertelorism, epicanthal folds, small palpebral fissures, short philtrum, and syndactyly. MRI of the brain revealed high signal abnormalities in the periventricular white matter, a hypoplastic corpus callosum, under-rotated hippocampus on the left and atrophic hippocampus on the right. Since age 5, the child's behavior has shown cyclic maniacal episodes with severely disorganized mood and behavior. Psychiatric and cognitive assessment led to a diagnosis of bipolar disorder not otherwise specified, manic episodes, attention deficit hyperactivity disorder and moderate mental retardation. Array-CGH revealed an interstitial duplication of 6.9 Mb at chromosome 22q: dup(22)(q13.1q13.2). FISH using BAC clones confirmed the array-CGH results and demonstrated that the duplication was inverted. G-banding analysis in the proposita's mother revealed a banding pattern suggestive of an intrachromosomal insertion, as demonstrated by dual-color FISH with BACs that were duplicated in the proposita and multicolor-banding (MCB) based on microdissection derived region-specific libraries for chromosome 22. Our findings suggest that in both seemingly de novo deletions and duplications, the parent transmitting the imbalance should be investigated for possible balanced rearrangements. This report reinforces previous evidence that chromosome imbalances, and thus gene dosage effects, may be at the basis of some psychiatric disorders. Stringent correlations between submicroscopic imbalances, specific behavioral phenotypes and brain imaging will possibly help in dissecting complex behavioral traits.

Biological Dosimetry in cases of accidental and occupational exposure to ionising radiation: State of the art

f.darroudi@lumc.nl 1. Abstract: The importance of detecting risks at the low doses (<5cGy) and low dose rates in radiation protection is well recognised. Similar issues surround the acquisition of knowledge on common genetic factors that might determine inter-individual differences in low dose cancer risk. These aspects are of continuing importance in respect of social/economic policy relating to the industrial and medical uses of ionising radiation, and for risk assessment among people occupationally are being exposed to low and/or high LET radiation, such as astronauts, pilots, stewardess and nuclear power plant workers, as well as victims of radiation accidents. In order to emphasis on the acquisition of fundamental knowledge and the development of low dose as well as high dose risk models for low LET radiation (i.e. gamma-rays and X-rays) and high LET radiation (i.e. heavy ions, alpha-particles, neutrons), several biological assays were developed and attempts were made to investigate formation of radiation induced chromosome aberrations and induction of genomic instability in human following acute and/or chronic exposure. Newly obtained data indicate that (a) Premature chromosome condensation assay is a unique method to be used for immediate dose assessment at low does (5≥Gy) as well as high doses (≥3 Gy), and can accurately discriminate between wholeand partial-body exposure. Therefore, this is the method of choice for biodosimetry in cases of mass casualties and accidental over-exposure to high doses of ionising radiation (b) Fluorescence in situ hybridization (FISH) technique using chromosome, chromosome-arm, chromosome region, centromere and telomere specific DNA libraries has improved the resolution of detecting all classes of radiation induced chromosomal interand intra-changes. Consequently, has increased significantly the accuracy and detection limit of biological dosimetry. FISH-based translocation assay has the potential to assess biodosimetry in cases of accidental as well as occupational exposures to ionising radiation, either immediately following exposure, and in particular, retrospectively by defining accumulative effects to red bone marrows. (c) Further modification of FISH assay by either combining chromosome, centromeres and telomeres specific probes or the M-FISH assay revealed distinct finger-prints, such as insertions and complex translocations for high LET radiation in comparison to low LET radiation. KeywordsIonising radiation, Biological dosimetry, Chromosomal aberrations, PCC, FISH-based translocation, I. The importance of dose assessment in human: Considering the notion that chromosomal instability is the hallmark of cancer, it is essential to develop and validate different biological assays for dose-assessment in cases of occupational and accidental over-exposure to ionising radiation of different qualities. High doses of ionizing radiation clearly produce deleterious consequences in human, including, but not exclusively, cancer induction. Therefore, it is essential to estimate chromosomal

Further delineation of complex chromosomal rearrangements in fertile male using multicolor banding

BackgroundComplex chromosomal rearrangements (CCRs) are defined as structural chromosomal rearrangements with at least three breakpoints and exchange of genetic material between two or more chromosomes. Complex chromosomal translocations are rarely seen in the general population but the frequency of occurrence is anticipated to be much higher due balanced states with no phenotypic presentation. Here, we report a severely mentally retarded fertile male patient in whom further delineation of CCR involving chromosomes 1, 4 and 2 was carried out by using high resolution multicolor banding (MCB) technique. As a FISH based novel chromosome banding approach, high resolution MCB allows for the differentiation of chromosome region specific areas at band and subband levels.ResultsCytogenetic studies using high resolution banding of the proband necessitated further delineation of the breakpoints because of their uncertainty: 46,XY,t(1;4;2)(p21~31;q31.3;q31). After using high resolution MCB based on microdissection derived region-specific libraries, the exact nature of chromosomal rearrangements for chromosomes 1, 2 and 4 were revealed and these breakpoints were located on 1p31.1, 1q24.3 and 4q31.3 giving rise to a balanced situation.ConclusionFurther delineations are certainly required to provide detailed information about the relationship between balanced CCRs and their phenotypes in order to offer proper counseling to the families concerned. Carriers must be investigated with high resolution banding and molecular cytogenetic techniques to determine the exact locations of the breakpoints. High resolution MCB is an alternative and an efficient method to other FISH based chromosome banding techniques and can serve in clarifying the nature of CCR.

Characterization of a PttRPS18 promoter active in the vascular cambium region of hybrid aspen.

By screening 273 hybrid aspen plants transformed with a luciferase-based promoter trap T-DNA vector, one plant was found in which the reporter gene (luxF2) was activated only in cells of the cambial region, i.e., vascular cambium, phloem and differentiating xylem. Southern blot analysis revealed that this plant denoted #24 had a single T-DNA insert. The chromosomal regions flanking the T-DNA were cloned by plasmid rescue. A 757 bp DNA fragment, originating from the rescued plasmid and covering the genomic region immediately upstream from the right-border sequence of the T-DNA, was used as a probe to isolate the corresponding chromosomal region from a wild-type hybrid aspen genomic library. A hybrid aspen small ribosomal protein gene, PttRPS18, was then isolated. By screening a wt cambial region-specific cDNA library, two cDNA clones encoding a putative 152 amino acid PttRPS18 protein were isolated. Comparison of the DNA sequence immediately flanking the T-DNA insert in #24 with the corresponding wild-type sequence showed that only a minor deletion occurred during the T-DNA integration. Northern analysis revealed that the PttRPS18 gene was expressed mainly in the cambial region. By RT-PCR and DNA sequencing analysis, the exact structures of the PttRPS18 and luxF2 transcripts were determined. Finally, the hybrid aspen PttRPS18 promoter was fused to the uidA reporter gene and transformed into hybrid aspen plants. Histochemical analysis of GUS activity showed that the PttRPS18 promoter was expressed in the cambial region in the same manner as the luciferase reporter gene in the initial screening.

Microdissection based high resolution multicolor banding for all 24 human chromosomes

The multicolor-banding (MCB) approach allows the differentiation of chromosome region specific areas at the band and sub-band level and is based on region-specific microdissection libraries producing changing fluorescence intensity ratios along the chromosomes. The latter are used to assign different pseudocolors to specific chromosomal regions. We present the complete set of 138 region-specific microdissection libraries for the entire human genome and the resulting MCB patterns for all human chromosomes at the 450-550 band level. In the present work, the creation and handling of the microdissection libraries is detailed for the first time. Additionally, the unique possibilities of the MCB technique to adjust the pseudocolor bands according to the necessities of the studied case is presented in exemplarity. In conclusion, the MCB-technique is a high resolution alternative to other FISH based chromosome banding approaches and suited to clarify, which changes appeared in complex chromosomal rearrangements.

Reorganization of the X chromosome in voles of the genus Microtus

Comparative chromosomal analysis is a powerful tool in the investigation of the mechanisms of chromosomal evolution. The accuracy of the analysis depends on the availability of region-specific markers to follow the fate of the particular chromosomal region through the evolution of species. We have assigned 12 unique sequences to the euchromatic part of the vole X chromosome, which serve as reliable markers of chromosomal segments. Together with region-specific libraries and GTG banding, these markers allow us to delineate the homologous regions of the X chromosomes in five species of the genus Microtus. We found that X chromosomes of these species differ by numerous rearrangements and all rearrangements are clustered at specific breakpoints. Moreover, these breakpoints were found to colocalise with repetitive and/or duplicated DNA sequences. We suggest that clusters of repeated and/or duplicated DNA sequences have played a crucial role in the formation of rearrangement hot spots during evolution of the X chromosome in the subgenus Microtus.

Isolation of lambda and YAC clones from defined regions of the rye genome.

A dispersed, rye-specific element has been used to isolate clones of rye origin from wheat plants containing only a single rye chromosome arm or segment. In this way a set of 23 YAC clones has been isolated from the short arm of rye chromosome 1 (1RS). This technique was extended to isolate clones from a small region of 1RS that contains a large number of agronomically important genes. The targeted cloning method allowed the isolation of 26 classes of lambda clones representing about 5% of the region. Ten of the lambda clones could be mapped to segments within this region. A third example of the application of this technique involved the isolation of clones from a very small but fully functional rye chromosome, the midget chromosome. These clones have allowed the confirmation of the origin of the midget from 1RL, and may provide a tool for the isolation of structural elements of cereal chromosomes. This technique allows the identification of clone libraries for any rye chromosome or chromosome arm, since substitution, addition and translocation lines are available for all rye chromosomes. Furthermore, the technique allows isolation of clones derived from segments of the rye genome recombined into wheat. The method is technically simple and both lambda and YAC libraries can be constructed. Synteny between the genomes of the cereals allows region-specific libraries from rye to be used to target regions of the wheat and barley genomes.

Detailed analysis of a 17q21 microdissection library by sequence bioinformatics and isolation of region-specific clones.

A region-specific microdissection library originating from human chromosome 17q21, was constructed using the MboI linker-adaptor microcloning technique. DNA sequencing of 241 microclones resulted in the identification of 74 novel coding sequences, paralogs of known genes, and known, but previously unmapped, genes or expressed sequence tags that were "virtually" mapped to chromosome 17q21. By pooling the microclones as multiplexed hybridization probes, and by virtue of their origin on 17q21, we were able to identify approximately 150 P1 clones from the human Reference Library Data Base P1 Library that potentially map to chromosome 17q21. Verification of the 17q21 location of 16 P1 clones was accomplished by PCR analysis with STS primer pairs to known 17q21 genes or by FISH. Our results demonstrate the substantial advantage of combining the sequence analysis of microclones with multiplex hybridization strategies for gene discovery and mapping specific gene rich regions of the genome.

Construction and characterization of three region-specific microdissection libraries for human chromosome 18.

Three region-specific libraries for the entire human chromosome 18 were constructed using microdissection and Mbol linker-adaptor microcloning techniques. The libraries included 18pter-p11.1 (designated 18P library), 18q11.1-q12.3 (18Q1 library), and 18q21.1-qter (18Q2 library). Samples of the microclones from each library were analyzed in detail. The insert sizes ranged between 50-600 bp, with a mean of 180-220 bp for the three libraries. The libraries contained approximately 40-60% microclones with unique sequence inserts. More than 30 unique sequence microclones from each library were analyzed by Southern blot hybridization to demonstrate that they are human specific and were derived from chromosome 18. The human genomic HindIII fragments hybridized to each microclone were determined and microclones cross-hybridized to rodent species were identified. These region-specific libraries and the unique sequence microclones from the libraries are useful reagents for (1) isolating highly polymorphic microsatellite markers for refined linkage analysis, (2) identifying corresponding YAC, BAC or other clones with large inserts for contig assembly and high resolution physical mapping, (3) isolating cDNA clones from the dissected region, and (4) convenient sequencing of the microclones to prepare high density markers and sequence-tagged sites (STSs). Such applications have been demonstrated in a series of similarly constructed microdissection libraries from other regions of the human genome.

Complete set of eleven region-specific microdissection libraries for human chromosome 2.

The construction and characterization of 11 region-specific libraries for the entire human chromosome 2 have been completed, including four libraries for the short arm and six libraries for the long arm, plus a library for the centromere region. These libraries were constructed using the chromosome microdissection and microcloning technology. Eight libraries have been described previously. This paper presents the final three libraries: 2q21-q22 (designated 2Q5 library), 2q11-q14 (2Q6). and 2p11.1-q11.1 (2CEN). The sizes of the dissected regions ranged between 20 and 30 Mb, with the centromere region of about 4 Mb. All these libraries are large, potentially comprising hundreds of thousands of recombinant microclones. Between 77% and 97% of the microclones were shown to derive from respective dissected regions. From 26 to 66 unique sequence microlones were isolated and characterized in detail for each library. The microclones have short inserts, ranging between 50 and 600 bp, with a mean of about 200 bp. The short inserts can be conveniently sequenced as STSs to provide high density probes for the dissected region. A plasmid sub-library containing at least 20,000 microclones, and usually more, has been prepared from each library and deposited to ATCC for general distribution. The libraries have been used effectively in constructing high resolution physical maps and for contig assembly, as well as in positional cloning of disease genes assigned to the dissected region. Comparing to other chromosomes with detailed mapping information and densely populated probes, chromosome 2 remains largely under-exploited. The availability of a complete set of region-specific libraries and unique sequence microclones from the libraries should provide valuable resources for genome analysis, high resolution physical mapping, region-specific cDNA isolation, and positional cloning for chromosome 2.

Three region-specific microdissection libraries for the long arm of human chromosome 2, regions q33-q35, q31-q32, and q23-q24

Three region-specific libraries have been constructed from the long arm of human chromosome 2, including regions 2q33-35 (2Q2 library), 2q31-32 (2Q3) and 2q23-24 (2Q4). Chromosome microdissection and the MboI linker-adaptor microcloning techniques were used in constructing these libraries. The libraries comprised hundreds of thousands of microclones in each library. Approximately half of the microclones in the library contained unique or low-copy number sequence inserts. The insert sizes ranged between 50 and 800 bp, with a mean of 130-190 bp. Southern blot analysis of individual unique sequence microclones showed that 70-94% of the microclones were derived from the dissected region. 31 unique sequence microclones from the 2Q2 library, 31 from 2Q3, and 30 from 2Q4, were analyzed for insert sizes, the hybridizing genomic HindIII fragment sizes, and cross-hybridization to rodent species. These libraries and the short insert microclones derived from the libraries should be useful for high resolution physical mapping, sequence-ready reagents for large scale genomic sequencing, and positional cloning of disease-related genes assigned to these regions, e.g. the recessive familial amyotrophic lateral sclerosis assigned to 2q33-q35, and a type I diabetes susceptibility gene to 2q31-q33.

A region-specific microdissection library for human chromosome 2p23→p21 and the analysis of an interstitial deletion of 2p21

A region-specific library of human chromosome 2p23-->p21 was constructed using microdissection and microcloning techniques. Analysis of 94 single-copy microclones from the library showed that 64% were derived from the dissected region. Ten microclones were further mapped to the 2p21 region using a patient with an interstitial deletion of 2p21 and displaying holoprosencephaly, an abnormal embryonic development in midbrain and midface.

A microdissection library of the rat renal carcinoma gene region.

Predisposition to hereditary renal carcinoma in the Eker rat involves a mutation of a putative tumor suppressor gene within chromosome band 10q12. We describe the identification of three unique polymorphic sequences in the vicinity of this locus following the microdissection, construction and characterization of a region-specific DNA library for rat chromosome band 10q12.

Analysis of region-specific library constructed by sequence-independent amplification of microdissected fragments surrounding weaver (wv) gene on mouse chromosome 16.

The C3-C4 region of mouse chromosome 16 was microdissected and amplified directly by sequence-independent amplification (SIA). The SIA product was proved to originate from the microdissected region by fluorescence in situ hybridization (FISH) and was cloned into the PCR II vector (mean insert size 506 bp). Colony hybridization showed that about 59% of the clones contained either unique or low copy number sequences. Southern blot analysis of 100 unique clones demonstrated that 50 clones hybridized with single (33 clones) or multiple (17 clones) bands on blots of DNA from a hamster-mouse hybrid cell line that contains mouse chromosome 16, 13 clones hybridized with mouse but not with the hamster-mouse hybrid DNA, 19 clones contained repetitive sequences, and the remaining 18 clones failed to yield bands. One third of the 100 unique clones hybridized to human genomic DNA. Thirty-three clones were sequenced. None of them was found in GenBank. Our results demonstrate that this relatively simple method of microdissection and cloning can produce a library of good quality.

Construction and characterization of region-specific microdissection libraries and single-copy microclones for short arm of human chromosome 2

The short arm of human chromosome 2, comprising approximately 93 million bp, has been divided into four regions to construct region-specific microdissection libraries to facilitate physical mapping and gene cloning. These four regions include 2p23-p25 (designated 2P1), 2p21-p23 (2P2), 2p14-p16 (2P3), and 2p11-p13 (2P4). Together with three previously constructed microdissection libraries of 2P1, 2P2 and 2P4, a fourth library for the region 2p14-p16 (2P3) has been constructed and characterized to complete all four region-specific libraries for the entire 2p. The 2P3 library is very large, potentially comprising 1,000,000 recombinant microclones with insert sizes ranging between 50 and 800 bp and a mean of 250 bp. Approximately 40% of the microclones contain unique sequences. Of the 77 single-copy microclones analyzed, 66 clones (86%) hybridized to both human and chromosome 2 DNAs, indicating that they were derived from human and are chromosome 2 specific. The hybridizing HindIII genomic fragments for the 66 microclones have also been determined.

A region-specific microdissection library for human chromosome 2p23-p25 and the analysis of an interstitial deletion of 2p23.3-p25.1.

A region-specific library for human chromosome 2p23-p25 was constructed using microdissection and polymerase chain reaction (PCR)-mediated microcloning techniques. This library is large, comprising 300,000 recombinant microclones. The insert sizes range between 50-600 base pairs (bp) with a mean of 200 bp. About 50%-60% of the clones contain unique or very low copy number sequence inserts as determined by their weak or no hybridization to total human DNA. A subset of 48 microclones that did not hybridize to total human DNA after colony hybridization was analyzed, and 26 (54%) clones were shown to contain single-copy inserts and hybridize to human chromosome 2 DNAs, indicating that they are human chromosome 2 specific. The human genomic fragments identified by these clones after cleavage with HindIII have also been characterized. The single-copy microclones were used to analyze an interstitial deletion in the 2p23.3-p25.1 region--46,XY, del(2) (pter-->p25.1::p23.3-->qter)--previously reported in a patient with severe growth and mental retardation and multiple anomalies. Of the 26 microclones analyzed, 14 clones were mapped to the deletion region. The availability of the 2p23-p25 region-specific library and the probes derived from the library should be valuable for fine structure physical mapping analysis and the cloning of disease-related genes localized to the region. These studies also demonstrate the efficiency with which useful probes can be quickly generated for genome studies and for positional cloning.

Preparative in Situ Hybridization: Selection of Chromosome Region-Specific Libraries on Mitotic Chromosomes

We have developed preparative in situ hybridization (Prep-ISH) of complex DNA populations to mitotic chromosomes as a means of generating chromosome region-specific DNA subpopulations. Prep-ISH is a combination of two cytogenetic techniques: in situ hybridization of DNA molecules to mitotic chromosomes and chromosome microdissection. Here, we present test cases demonstrating the feasibility of this approach on mouse and human genomes, using single nuclei, single chromosomes, or single chromosomal subregions to assess sensitivity, specificity, and representation of the Prep-ISH technique. Prep-ISH has a number of applications in studies of gene expression and genome organization, including efficient cytogenetic sorting of tissue-specific cDNAs and genomic DNA libraries. In addition, Prep-ISH is likely to dramatically reduce the number of candidate genes to aid in gene discovery efforts and to improve efficiency of developing transcription maps and YAC and cosmid contigs through defined cytogenetic regions.