Single-copy DNA Research Articles

Fluorescent in situ hybridization with transposable element probes to mitotic chromosomal heterochromatin of Drosophila.

The technique of in situ hybridization of DNA probes to Drosophila chromosomes has been initially applied to the salivary gland polytene chromosomes and is now routinely used for mapping single-copy and repetitive DNA sequences, such as transposable elements, to the euchromatic regions of these chromosomes. However, most of the heterochromatin normally escapes cytogenetic analyses on polytene chromosomes because it is organized in a poorly differentiated cytological structure called the chromocenter. This peculiar organization does not allow a detailed mapping of DNA clones to heterochromatin. Such a limitation can be overcome by the fluorescent in situ hybridization (FISH) technique on mitotic chromosomes of D. melanogaster, where heterochromatin has been extensively characterized by banding techniques and subdivided into several cytologically diverse regions. Digital images of FISH signals and DAPI staining can be separately recorded by CCD camera, pseudocolored, and merged using specific software for image analysis. The visualization of the signals and DAPI banding pattern on a single chromosome enables the mapping of a given sequence to specific cytological regions of mitotic heterochromatin. This method has initially proven successful in the detection and mapping of transposable element clusters in the heterochromatin of D. melanogaster and has been used to study the distribution of repeated and even single-copy sequences.

The arbuscular mycorrhizal fungus Glomus intraradices is haploid and has a small genome size in the lower limit of eukaryotes

The genome size, complexity, and ploidy of the arbuscular mycorrhizal fungus (AMF) Glomus intraradices was determined using flow cytometry, reassociation kinetics, and genomic reconstruction. Nuclei of G. intraradices from in vitro culture, were analyzed by flow cytometry. The estimated average length of DNA per nucleus was 14.07 ± 3.52 Mb. Reassociation kinetics on G. intraradices DNA indicated a haploid genome size of ∼16.54 Mb, comprising 88.36% single copy DNA, 1.59% repetitive DNA, and 10.05% fold-back DNA. To determine ploidy, the DNA content per nucleus measured by flow cytometry was compared with the genome estimate of reassociation kinetics. G. intraradices was found to have a DNA index (DNA per nucleus per haploid genome size) of approximately 0.9, indicating that it is haploid. Genomic DNA of G. intraradices was also analyzed by genomic reconstruction using four genes (Malate synthase, RecA, Rad32, and Hsp88). Because we used flow cytometry and reassociation kinetics to reveal the genome size of G. intraradices and show that it is haploid, then a similar value for genome size should be found when using genomic reconstruction as long as the genes studied are single copy. The average genome size estimate was 15.74 ± 1.69 Mb indicating that these four genes are single copy per haploid genome and per nucleus of G. intraradices. Our results show that the genome size of G. intraradices is much smaller than estimates of other AMF and that the unusually high within-spore genetic variation that is seen in this fungus cannot be due to high ploidy.

Relative rates of nucleotide substitution in frogs.

Accurate estimation of relative mutation rates of mitochondrial DNA (mtDNA) and single-copy nuclear DNA (scnDNA) within lineages contributes to a general understanding of molecular evolutionary processes and facilitates making demographic inferences from population genetic data. The rate of divergence at synonymous sites ( K(s)) may be used as a surrogate for mutation rate. Such data are available for few organisms and no amphibians. Relative to mammals and birds, amphibian mtDNA is thought to evolve slowly, and the K(s) ratio of mtDNA to scnDNA would be expected to be low as well. Relative K(s) was estimated from a mitochondrial gene, ND2, and a nuclear gene, c-myc, using both "approximate" and likelihood methods. Three lineages of congeneric frogs were studied and this ratio was found to be approximately 16, the highest of previously reported ratios. No evidence of a low K(s) in the nuclear gene was found: c-myc codon usage was not biased, the K(s) was double the intron divergence rate, and the absolute K(s) was similar to estimates obtained here for other genes from other frog species. A high K(s) in mitochondrial vs. nuclear genes was unexpected in light of previous reports of a slow rate of mtDNA evolution in amphibians. These results highlight the need for further investigation of the effects of life history on mutation rates.

BIBAC and TAC clones containing potato genomic DNA fragments larger than 100 kb are not stable in Agrobacterium.

Development of efficient methods to transfer large DNA fragments into plants will greatly facilitate the map-based cloning of genes. The recently developed BIBAC and TAC vectors have shown potential to deliver large DNA fragments into plants via Agrobacterium-mediated transformation. Here we report that BIBAC and TAC clones containing potato genomic DNA fragments larger than 100 kb are not stable in Agrobacterium. We tested the possible factors that may cause instability, including the insert sizes of the BIBAC and TAC constructs, potato DNA fragments consisting of highly repetitive or largely single-copy DNA sequences, different Agrobacterium transformation methods and different Agrobacterium strains. The insert sizes of the potato BIBAC and TAC constructs were found to be critical to their stability in Agrobacterium. All constructs containing a potato DNA fragment larger than 100 kb were not stable in any of the four tested Agrobacterium strains, including two recA deficient strains. We developed a transposon-based technique that can be used to efficiently subclone a BAC insert into two to three BIBAC/TAC constructs to circumvent the instability problem.

Nuclear Gene Sequences from a Late Pleistocene Sloth Coprolite

The determination of nuclear DNA sequences from ancient remains would open many novel opportunities such as the resolution of phylogenies, the sexing of hominid and animal remains, and the characterization of genes involved in phenotypic traits. However, to date, single-copy nuclear DNA sequences from fossils have been determined only from bones and teeth of woolly mammoths preserved in the permafrost [1]. Since the best preserved ancient nucleic acids tend to stem from cold environments [2, 3], this has led to the assumption that nuclear DNA would be retrievable only from frozen remains. We have previously shown that Pleistocene coprolites stemming from the extinct Shasta sloth (Nothrotheriops shastensis, Megatheriidae) contain mitochondrial (mt) DNA from the animal that produced them as well as chloroplast (cp) DNA from the ingested plants [4]. Recent attempts to resolve the phylogeny of two families of extinct sloths by using strictly mitochondrial DNA has been inconclusive [5]. We have prepared DNA extracts from a ground sloth coprolite from Gypsum Cave, Nevada, and quantitated the number of mtDNA copies for three different fragment lengths by using real-time PCR. We amplified one multicopy and three single-copy nuclear gene fragments and used the concatenated sequence to resolve the phylogeny. These results show that ancient single-copy nuclear DNA can be recovered from warm, arid climates. Thus, nuclear DNA preservation is not restricted to cold climates.

Direct molecular haplotyping of long-range genomic DNA with M1-PCR.

Haplotypes, combinations of several phase-determined polymorphic markers, are extremely valuable for studies of disease association and chromosome evolution. Here we describe a technique called M1-PCR (M for "multiplex" and 1 for "single-copy DNA molecules") that enables direct molecular haplotyping of several polymorphic markers separated by as many as 24 kb. A genomic DNA sample first is diluted to approximately single-copy. The haplotype is directly determined by simultaneously genotyping several polymorphic markers in the same reaction with a multiplex PCR and base extension reaction. This approach does not rely on pedigree data and does not require previous amplification of the entire genomic region containing the selected markers.

Nuclear DNA analyses in genetic studies of populations: practice, problems and prospects.

Population-genetic studies have been remarkably productive and successful in the last decade following the invention of PCR technology and the introduction of mitochondrial and microsatellite DNA markers. While mitochondrial DNA has proven powerful for genealogical and evolutionary studies of animal populations, and microsatellite sequences are the most revealing DNA markers available so far for inferring population structure and dynamics, they both have important and unavoidable limitations. To obtain a fuller picture of the history and evolutionary potential of populations, genealogical data from nuclear loci are essential, and the inclusion of other nuclear markers, i.e. single copy nuclear polymorphic (scnp) sequences, is clearly needed. Four major uncertainties for nuclear DNA analyses of populations have been facing us, i.e. the availability of scnp markers for carrying out such analysis, technical laboratory hurdles for resolving haplotypes, difficulty in data analysis because of recombination, low divergence levels and intraspecific multifurcation evolution, and the utility of scnp markers for addressing population-genetic questions. In this review, we discuss the availability of highly polymorphic single copy DNA in the nuclear genome, describe patterns and rate of evolution of nuclear sequences, summarize past empirical and theoretical efforts to recover and analyse data from scnp markers, and examine the difficulties, challenges and opportunities faced in such studies. We show that although challenges still exist, the above-mentioned obstacles are now being removed. Recent advances in technology and increases in statistical power provide the prospect of nuclear DNA analyses becoming routine practice, allowing allele-discriminating characterization of scnp loci and microsatellite loci. This certainly will increase our ability to address more complex questions, and thereby the sophistication of genetic analyses of populations.

Novel PAX6 Binding Sites in the Human Genome and the Role of Repetitive Elements in the Evolution of Gene Regulation

Pax6 is a critical transcription factor in the development of the eye, pancreas, and central nervous system. It is composed of two DNA-binding domains, the paired domain (PD), which has two helix-turn-helix (HTH) motifs, and the homeodomain (HD), made up from another HTH motif. Each HTH motif can bind to DNA separately or in combination with the others. We identified three novel binding sites that are specific for the PD and HD domains of human PAX6 from single-copy human genomic DNA libraries using cyclic amplification of protein binding sequences (CAPBS) and electrophoretic mobility shift assays (EMSAs). One of the binding sites was found within sequences of repetitive Alu elements. However, most of the Alu sequences were unable to bind to PAX6 because of a small number of mismatches (mostly in CpG dinucleotide hot spots) in the consensus Alu sequences. PAX6 binding Alu elements are found primarily in old and intermediate-aged Alu subfamilies. These data along with our previously identified B1-type Pax6 binding site showed that evolutionarily conserved Pax6 has target sites that are disparate in primates and rodents. This difference indicates that human and mouse Pax6-regulated gene networks may have evolved through these lineage-specific repeat elements.

Genetic Uniformity Among Isolates of Peronospora tabacina, the Tobacco Blue Mold Pathogen

ABSTRACT As a first step toward analysis of genetic variation and population structure in Peronospora tabacina, we used a collection of random genomic DNA fragments to survey for restriction fragment length polymorphisms (RFLPs) in DNA from a collection of isolates from Kentucky and other tobacco-growing regions of the United States. Also included in the study were isolates from the wild tobacco species, Nicotiana repanda, and from ornamental tobacco, N. alata. In a preliminary survey using DNA from 10 pathogen isolates, no polymorphisms were detected at six single-copy DNA loci using 22 probe-enzyme combinations. Moderately repetitive and highly repetitive regions of the genome were also remarkably similar between isolates, with only 6 of 15 different probes identifying genetic differences. Some of the polymorphic probes were then used to analyze a larger collection of isolates, most of which were from Kentucky. This resulted in the identification of very few additional polymorphisms, indicating that the population of P. tabacina that infects the Kentucky tobacco crop is genetically very homogeneous. The low level of polymorphism detected in this study overall, suggests that genetic variability may be lacking in P. tabacina populations throughout the United States. Two of the RFLP markers gave hybridization patterns that were consistent with P. tabacina being diploid. Frequencies of alleles at these loci and linkage disequilibrium between different marker loci indicated that genetic recombination does not occur frequently in the pathogen population. DNA polymorphisms that were identified in this study enabled us to differentiate the pathogen population into at least 10 haplotypes. One isolate was analyzed in detail and was shown to be genetically stable through several rounds of single-spore isolation and through several pathogenic cycles.

Molecular marker analysis of 24- and 25-chromosome plants obtained from Solanum tuberosum L. subsp. andigena (2n = 4x = 48) pollinated with a Solanum phureja haploid inducer.

Clones with 24 or 25 chromosomes were obtained by pollinating an Andean cultivated tetraploid potato (Solanum tuberosum subsp. andigena clone 94H94, 2n = 4x = 48) with the Solanum phureja haploid-inducer clone 1.22. Their genetic composition was analyzed in an RAPD assay using 135 decamer primers and in an RFLP assay using 45 single-copy DNA probes. In total, 22 RAPD and 20 RFLP markers were found to be specific to S. phureja. None of these markers were found in the 24- and 25-chromosome clones. RFLP genotypes for the 45 RFLP loci were further determined for each clone. Genotypes of the 24-chromosome clones were characterized using two alleles randomly selected from four alleles of the parental tetraploid clone for almost all RFLP loci. Five 25-chromosome clones had extra alleles for all of the RFLP loci of chromosomes 4, 8, 10, 11, and 12, respectively, suggesting primary trisomy for one of these chromosomes. Clones with genotypes showing double reduction were also identified. Therefore, the obtained clones likely originated from random samples of female gametes, and hence are euhaploids or aneuhaploids of S. tuberosum subsp. andigena, strongly supporting parthenogenesis to be a primary mechanism for haploid induction in potato.

The abundant class of nemis repeats provides RNA substrates for ribonuclease III in Neisseriae

About 2% of the Neisseria meningitidis genome is made up by nemis, short DNA sequences which feature long terminal inverted repeats (TIRs). Most nemis are interspersed with single-copy DNA and are found at close distance from cellular genes. In this work, we demonstrate than RNAs spanning nemis of different length and sequence compositions are specifically cleaved at hairpins formed by nemis termini by total cellular lysates derived from both Escherichia coli and Neisseria lactamica strains. The use of cellular extracts from E. coli strains impaired in the activity of known ribonucleases let to establish that cleavage at nemis TIRs is specifically mediated by the endoribonuclease RNase III. Data set the base for the identification of all of the neisserial genes that are regulated by RNase III because of their physical association with nemis DNA.

Distribution and characterization of microsatellites in the emu (Dromaius novaehollandiae) genome.

This study generates data concerning the genome of a flightless species of bird, the emu. We examined and ultimately rejected the following hypotheses: (1) Microsatellites are randomly distributed throughout the emu genome. (2) The relative order of abundance of dinucleotides will be constant across genomes. (3) Interspersion distances for a given dinucleotide will be equal across vertebrate genomes. (4) In all genomes, a dinucleotide will be more frequent than any trinucleotide. (5) The percentage of single-copy DNA will remain the same in emus as in other volant birds. A cosmid library representing 4.48% of the emu genome was probed with 23 microsatellites. Hybridizations were scored on a scale of 0-3. The average insert size, approximately 40 kb, was used to determine frequency and interspersion. The cosmid library was probed with genomic DNA to determine the percent single copy. Co-occurrence frequencies and confidence intervals were compared to expected using chi-squared. The genome is estimated to contain a microsatellite repeat every 48 kb. Of 1632 clones probed for single-copy DNA, 643 displayed maximal hybridization, 220 displayed moderate hybridization, and 202 had minimal hybridization. After 3 days, 567 showed no hybridization.

Speciation via introgressive hybridization in East African cichlids?

Speciation caused by introgressive hybridization occurs frequently in plants but its importance remains controversial in animal evolution. Here we report a case of introgressive hybridization between two ancient and genetically distinct species of Lake Tanganyika cichlids that led to the formation of a new species. Neolamprologus marunguensis contains mtDNA haplotypes from both parental species varying on average by 12.4% in the first section of the control region and by 5.2% in a segment of the cytochrome b gene. All individuals have almost identical DNA sequences in the flanking regions of the single-copy nuclear DNA locus TmoM27, and show a mosaic of alleles derived from both parental lineages in six microsatellite loci. Hence, our finding displays another mode of speciation in cichlid fishes. The increase of genetic and phenotypic diversity due to hybridization may contribute to the uniquely rapid pace of speciation in cichlids.

Single-copy nuclear DNA sequences obtained from noninvasively collected primate feces.

Noninvasively collected primate feces have been shown to provide a useful source of mitochondrial DNA for sequencing and nuclear microsatellite DNA for size analysis. In this study, single-copy nuclear DNA sequences were obtained from noninvasively collected fecal samples of two species of wild tamarins, Saguinus fuscicollis and S. mystax, in the context of a project on the functional utility of color vision. Noninvasive genotyping of the X-linked opsin gene is important for future studies of selection and adaptation at this locus in a number of primate species. The wide range of techniques that can now be applied successfully to DNA extracted from feces introduces a broad spectrum of potential genetic studies that can be undertaken on primates, without the need for intrusive or invasive methods.

Evidence of two genetically deeply divergent species of warthog, Phacochoerus africanus and P. aethiopicus (Artiodactyla: Suiformes) in East Africa

Two species of warthogs (Phacochoerus), differing by the number of functional incisors, were described in the Holocene fossil record: the common warthog (P. africanus), widespread in sub-Sahar-an Africa, and the Cape, or desert warthog (P. aethiopicus), which was considered extinct since 1896, but was recently rediscovered in East Africa by morphological analyses. Mitochondrial and single-copy nuclear DNA sequences show that common and desert warthogs belong to two deeply divergent monophyletic lineages, that might have originated in the last part of the Pliocene. The finding of two genetically divergent extant species of warthogs highlights the importance of molecular methods applied to the knowledge and conservation of biodiversity in Africa, to uncover the tempo and mode of its species evolution.

DNA Fingerprint Probe from Mycosphaerella graminicola Identifies an Active Transposable Element

ABSTRACT DNA fingerprinting has been used extensively to characterize populations of Mycosphaerella graminicola, the Septoria tritici blotch pathogen of wheat. The highly polymorphic DNA fingerprints of Mycosphaerella graminicola were assumed to reflect the action of transposable elements. However, there was no direct evidence to support that conclusion. To test the transposable element hypothesis, the DNA fingerprint probe pSTL70 was sequenced, along with three other clones from a subgenomic library that hybridized with pSTL70. Analysis of these sequences revealed that pSTL70 contains the 3' end of a reverse transcriptase sequence plus 29- and 79-bp direct repeats. These are characteristics of transposable elements identified in other organisms. Southern analyses indicated that either the direct-repeat or reverse-transcriptase sequences by themselves essentially duplicated the original DNA fingerprint pattern, but other portions of pSTL70 contained single-copy DNA. Analysis of 60 progeny from a sexual cross between two Dutch isolates of Mycosphaerella graminicola identified several new bands that were not present in the parents. Thus, the putative transposable element probably is active during meiosis. Tests of single-spore isolates revealed gains or losses of one or more DNA fingerprint bands in 4 out of 10 asexual lines derived from isolate IPO94269. Therefore, DNA fingerprint patterns produced by the putative transposable element were capable of changes during asexual reproduction of this isolate. Probe pSTL70 did not hybridize at high stringency to genomic DNAs from other fungi related to Septoria and Mycosphaerella. These results indicate that the DNA fingerprint probe pSTL70 most likely identifies a transposable element in Mycosphaerella graminicola that may have been acquired recently, and appears to be active during both sexual and asexual reproduction.

Whole-genome organization and functional properties of miniature DNA insertion sequences conserved in pathogenic Neisseriae

The chromosome of pathogenic Neisseriae is peppered by members of an abundant family of small DNA sequences known as Correia elements. These DNA repeats, that we call nemis (for neisseria miniature insertion sequences) can be sorted into two major size classes. Both unit-length (154–158 bp) and internally rearranged (104–108 bp) elements feature long terminal inverted repeats (TIRs), and can potentially fold into robust stem-loop structures. Nemis are (or have been) mobile DNA sequences which generate a specific 2-bp target site duplication upon insertion, and strictly recall RUP, a repeated DNA element found in Streptococcus pneumoniae. The subfamilies of 26L/26R, 26L/27R, 27L/27R and 27L/26R elements, found by wide-genome computer surveys in both the Neisseria meningitidis and the Neisseria gonorrhoeae genomes, originate from the combination of TIRs which vary in length (26–27 bp) as in sequence content (L and R types). In both species, the predominant subfamily is made by the 26L/26R elements. The number of nemis is comparable in the N. meningitidis Z2491 (A serogroup) and the MC58 (B serogroup) strains, but is sharply reduced in the N. gonorrhoeae strain F1090. Consequently, several genes which are conserved in the two pathogens are flanked by nemis DNA in the meningococcus genome only. More than 2/3 of nemis are interspersed with single-copy DNA, and are found at close distance from cellular genes. Both primer extension and RNase protection data lend support to the notion that nemis are cotranscribed with cellular genes and subsequently processed, at either one or both TIRs, by a specific endoribonuclease, which plausibly corresponds to RNase III.

Molecular Characterization and Gene Content of Breakpoint Boundaries in Patients with Neurofibromatosis Type 1 with 17q11.2 Microdeletions

Homologous recombination between poorly characterized regions flanking the NF1 locus causes the constitutional loss of approximately 1.5 Mb from 17q11.2 covering > or =11 genes in 5%-20% of patients with neurofibromatosis type 1 (NF1). To elucidate the extent of microheterogeneity at the deletion boundaries, we used single-copy DNA fragments from the extreme ends of the deleted segment to perform FISH on metaphase chromosomes from eight patients with NF1 who had large deletions. In six patients, these probes were deleted, suggesting that breakage and fusions occurred within the adjacent highly homologous sequences. Reexamination of the deleted region revealed two novel functional genes FLJ12735 (AK022797) and KIAA0653-related (WI-12393 and AJ314647), the latter of which is located closest to the distal boundary and is partially duplicated. We defined the complete reading frames for these genes and two expressed-sequence tag (EST) clusters that were reported elsewhere and are associated with the markers SHGC-2390 and WI-9521. Hybrid cell lines carrying only the deleted chromosome 17 were generated from two patients and used to identify the fusion sequences by junction-specific PCRs. The proximal breakpoints were found between positions 125279 and 125479 in one patient and within 4 kb of position 143000 on BAC R-271K11 (AC005562) in three patients, and the distal breakpoints were found at the precise homologous position on R-640N20 (AC023278). The interstitial 17q11.2 microdeletion arises from unequal crossover between two highly homologous WI-12393-derived 60-kb duplicons separated by approximately 1.5 Mb. Since patients with the NF1 large-deletion syndrome have a significantly increased risk of neurofibroma development and mental retardation, hemizygosity for genes from the deleted region around the neurofibromin locus (CYTOR4, FLJ12735, FLJ22729, HSA272195 (centaurin-alpha2), NF1, OMGP, EVI2A, EVI2B, WI-9521, HSA272196, HCA66, KIAA0160, and WI-12393) may contribute to the severe phenotype of these patients.

Construction of a bacterial artificial chromosome (BAC) library for citrus and identification of BAC contigs containing resistance gene candidates

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.

Heterogeneous methylation of the O(6)-methylguanine-DNA methyltransferase promoter in immortalized IMR90 cell lines.

Transcriptional silencing of the DNA repair protein, O6-methylguanine-DNA methyltransferase (MGMT), occurs only in malignant or transformed cell lines, and such MGMT-deficient cells are hypersensitive to chemotherapeutic alkylating agents such as 1, 3-bis (2-chloroethyl)-1-nitrosourea (BCNU) and temozolomide. Previously we demonstrated in a panel of established cell lines that the lack of gene expression correlated with methylation <hot spots> within the CpG island in the MGMT 5' gene flank. Now, we investigated the relationship between CpG methylation, MGMT suppression and drug-sensitivity in normal, diploid MGMT-expressing IMR90 cells and five immortalized sublines (AA, EE, J, KK and Pool), four of which have silenced MGMT. As expected, the MGMT-expressing parental cells were most drug-resistant and free of promoter methylation, whereas the MGMT-silenced immortal sublines were more drug-sensitive and promoter-methylated. Surprisingly, the sole MGMT-positive immortal subline, (AA) showed some promoter methylation although it was relatively drug-resistant; and an apparently MGMT-negative subline, (EE) showed unexpectedly low levels of methylation. We determined if these discrepancies were due to heterogeneity (cellular or allelic) and if this reflected transitional states between expressing and silenced phenotypes. Analysis of the methylation status of <hot spot> CpGs by genomic sequencing of cloned single copy DNA confirmed heterogeneity in both these sublines. With increasing cell culture passage, CpG methylation progressively increased with a concomitant trend to a completely MGMT-silenced phenotype in these sublines.