Isolation and comparative mapping of a human chromosome 20-specific alpha-satellite DNA clone.

We have isolated and characterized a human genomic DNA clone (PZ20, locus D20Z2) that identifies, under high-stringency hybridization conditions, an alphoid DNA subset specific for chromosome 20. The specificity was determined using fluorescence in situ hybridization. Sequence analysis confirmed our previously reported data on the great similarity between the chromosome 20 and chromosome 2 alphoid subsets. Comparative mapping of pZ20 on chimpanzee and gorilla chromosomes, also performed under high-stringency conditions, indicates that the alphoid subset has ancestral sequences on chimpanzee chromosome 11 and gorilla chromosome 19. However, no hybridization was observed to chromosomes 21 in the great apes, the homolog of human chromosome 20.     

Detection of mutations and DNA polymorphisms using whole genome Southern Cross hybridization.

We report a general method for the detection of restriction fragment length alterations associated with mutations or polymorphisms using whole genomic DNA rather than specific cloned DNA probes. We utilized a modified Southern Cross hybridization to display the hybridization pattern of all size-separated restriction fragments from wild-type Caenorhabditis elegans to all the corresponding fragments in a particular mutant strain and in a distinct C. elegans variety. In this analysis, almost all homologous restriction fragments are the same size in both strains and result in an intense diagonal of hybridization, whereas homologous fragments that differ in size between the two strains generate an off-diagonal spot. To attenuate the contribution of repeated sequences in the genome to spurious off-diagonal spots, restriction fragments from each genome were partially resected with a 3' or 5' exonuclease and not denatured, so that only the DNA sequences at the ends of these fragments could hybridize. Off-diagonal hybridization spots were detected at the expected locations when genomic DNA from wild-type was compared to an unc-54 mutant strain containing a 1.5 kb deletion or to a C. elegans variety that contains dispersed transposon insertions. We suggest that this modified Southern Cross hybridization technique could be used to identify restriction fragment length alterations associated with mutations or genome rearrangements in organisms with DNA complexities as large as 10(8) base pairs and, using rare-cutting enzymes and pulse-field gel electrophoresis, perhaps as large as mammalian genomes. This information could be used to clone fragments associated with such DNA alterations.     

Y enriched and Y specific DNA sequences from the genome of the mediterranean fruit fly,Ceratitis capitata

DNA sequences that are enriched or specific to the genome of the male medfly, Ceratitis capitata, have been isolated using a differential hybridization approach. Twelve phage clones from a genomic library have been identified that consistently display more intense hybridization with a genomic DNA probe from males as opposed to one from females. Southern DNA blot analysis reveals that these recombinant clones contain at least one EcoRI fragment that is either specific to the male genome, or more highly represented in it, as compared with the female genome. These EcoRI fragments, when used as probes, all generate a similar pattern of intense multiple bands in genomic DNA of males. This suggests the presence of repetitive sequences that are at least partially homologous in these regions of the genome that are specific to or enriched in males. In situ hybridization to mitotic chromosomes confirms a Y chromosomal origin for the male specific repetitive sequences. Data on the genomic organization, representation and evolutionary conservation of these sequences that are specific to or enriched in males are presented. Studies of the genomic organization and representation of flanking sequences that are not male specific are presented as well.by R. Appels     

Characterization and chromosomal distribution of a novel satellite DNA sequence of Japanese quail (Coturnix coturnix japonica)

A novel satellite DNA sequence of Japanese quail (Coturnix coturnix japonica) was isolated from genomic DNA digested with restriction endonuclease, Bg/II. Sequence analysis of three different-size clones revealed the presence of a tandem array of a GC-rich 41 bp repeated element. This sequence was localized by fluorescence in situ hybridization (FISH) primarily to microchromosomes of Japanese quail (2n = 78); approximately 50 of the 66 microchromosomes showed positive signals, although hybridization signals were also detected on chromosomes 4 and W. This satellite DNA did not cross-hybridize with genomic DNA of chicken (Gallus gallus) and Chinese painted quail (Excalfactoria chinensis) under moderately stringent conditions, suggesting that this class of repetitive DNA sequences was species specific and fairly divergent in Galliformes species.     

Chromosome-specific organization of human alpha satellite DNA

Restriction endonuclease analysis of human genomic DNA has previously revealed several prominent repeated DNA families defined by regularly spaced enzyme recognition sites. One of these families, termed alpha satellite DNA, was originally identified as tandemly repeated 340- or 680-base pair (bp) EcoRI fragments that hybridize to the centromeric regions of human chromosomes. We have investigated the molecular organization of alpha satellite DNA on individual human chromosomes by filter hybridization and in situ hybridization analysis of human DNA and DNA from rodent/human somatic cell hybrids, each containing only a single human chromosome. We used as probes a cloned 340-bp EcoRI alpha satellite fragment and a cloned alpha satellite-containing 2.0-kilobase pair (kbp) BamHI fragment from the pericentromeric region of the human X chromosome. In each somatic cell hybrid DNA, the two probes hybridized to a distinct subset of DNA fragments detected in total human genomic DNA. Thus, alpha satellite DNA on each of the human chromosomes examined--the X and Y chromosomes and autosomes 3, 4, and 21--is organized in a specific and limited number of molecular domains. The data indicate that subsets of alpha satellite DNA on individual chromosomes differ from one another, both with respect to restriction enzyme periodicities and with respect to their degree of sequence relatedness. The results suggest that some, and perhaps many, human chromosomes are characterized by a specific organization of alpha satellite DNA at their centromeres and that, under appropriate experimental conditions, cloned representatives of alpha satellite subfamilies may serve as a new class of chromosome-specific DNA markers.     

Application of an immunoprecipitation procedure to the study of SV40 tumor antigen interaction with mouse genomic DNA sequences.

Simian Virus 40 (SV40) large T antigen is a DNA binding protein with high affinity for segments of the viral genome. To find out whether T antigen also binds to sequences of genomic cellular DNA we mixed T antigen and SAU 3 A restricted mouse DNA under stringent DNA binding conditions. Resulting protein-DNA complexes were immunoprecipitated using T antigen specific monoclonal or polyclonal antibodies. The DNA fragments in the immunoprecipitates were cloned in plasmid vectors. Four plasmid clones were selected for a detailed investigation of the inserted mouse DNA fragments. Nucleotide sequencing and DNase I footprint experiments showed that T antigen binds to sites in these fragments consisting of two tandemly oriented G(A)AGGC pentamers separated by AT rich spacers of different lengths. The cellular binding sites are very similar in their architecture to the SV40-DNA binding site I. The isolated cellular DNA fragments with T antigen binding sites occur only once or a few times in the mouse genome. Our data help to further define the structure of T antigen's DNA binding sites. The genetic functions of the isolated cellular DNA elements are not known.     

Maize DNA enrichment by representational difference analysis in a maize chromosome addition line of oat

 The recent recovery of maize (Zea mays L.) single-chromosome addition lines of oat (Avena sativa L.) from oat x maize crosses has provided novel source materials for the potential isolation of maize chromosome-specific sequences for use in genetic mapping and gene cloning. We report here the application of a technique, known as representational difference analysis (RDA), to selectively isolate maize sequences from a maize chromosome-3 addition line of oat. DNA fragments from the addition line and from the oat parent were prepared using BamHI digestion and primer ligation followed by PCR amplification. A subtractive hybridization technique using an excess of the oat parental DNA was employed to reduce the availability for amplification of DNA fragments from the addition lines that were in common with the ones from the oat parental line. After three rounds of hybridization and amplification, the resulting DNA fragments were cloned into a plasmid vector. A DNA library containing 400 clones was constructed by this method. In a test of 18 clones selected at random from this library, four (22%) detected maize-specific repetitive DNA sequences and nine (50%) showed strong hybridization to genomic DNA of maize but weak hybridization to genomic DNA of oat. Among these latter nine clones, three detected low-copy DNA sequences and two of them detected DNA sequences specific to chromosome 3 of maize, the chromosome retained in the source maize addition line of oat. The other eight out of the 13 clones that had strong hybridization to maize DNA detected repetitive DNA sequences or high-copy number sequences present on most or all maize chromosomes. We estimate that the maize DNA sequences were enriched from about 1.8% to over 72% of the total DNA by this procedure. Most of the isolated DNA fragments detected multiple or repeated DNA sequences in maize, indicating that the major part of the maize genome consists of repetitive DNA sequences that do not cross-hybridize to oat genomic sequences. Received: 18 November 1997 / Accepted: 12 March 1998     

ERIC-PCR fingerprinting-based community DNA hybridization to pinpoint genome-specific fragments as molecular markers to identify and track populations common to healthy human guts

Bacterial populations common to healthy human guts may play important roles in human health. A new strategy for discovering genomic sequences as markers for these bacteria was developed using Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR fingerprinting. Structural features within microbial communities are compared with ERIC-PCR followed by DNA hybridization to identify genomic fragments shared by samples from healthy human individuals. ERIC-PCR profiles of fecal samples from 12 diseased or healthy human and piglet subjects demonstrated stable, unique banding patterns for each individual tested. Sequence homology of DNA fragments in bands of identical size was examined between samples by hybridization under high stringency conditions with DIG-labeled ERIC-PCR products derived from the fecal sample of one healthy child. Comparative analysis of the hybridization profiles with the original agarose fingerprints identified three predominant bands as signatures for populations associated with healthy human guts with sizes of 500, 800 and 1000 bp. Clone library profiling of the three bands produced 17 genome fragments, three of which showed high similarity only with regions of the Bacteroides thetaiotaomicron genome, while the remainder were orphan sequences. Association of these sequences with healthy guts was validated by sequence-selective PCR experiments, which showed that a single fragment was present in all 32 healthy humans and 13 healthy piglets tested. Two fragments were present in the healthy human group and in 18 children with non-infectious diarrhea but not in eight children with infectious diarrhea. Genome fragments identified with this novel strategy may be used as genome-specific markers for dynamic monitoring and sequence-guided isolation of functionally important bacterial populations in complex communities such as human gut microflora.     

Interaction of DNA with nuclear scaffolds in vitro

We have previously identified a number of specific DNA fragments called SARs (scaffold-associated regions) that are associated with the nuclear scaffold and define the basis of DNA loops. We demonstrate that cloned DNA fragments containing SAR sequences bind to nuclear scaffolds in vitro with the same specificity as have genomic SAR fragments. This specific interaction is observed with the biochemically complex type I scaffolds. These scaffolds are composed of the nuclear lamina proteins and a set of other proteins that forms the internal network of these structures. So-called type II scaffolds, which are composed primarily of the lamina proteins and lack the proteins of the internal network, do not bind the SAR fragments at a detectable level. Competition experiments show that different SARs share common structural elements and can bind to the same sites on the nuclear scaffold, although with different affinities. Moreover, the SAR binding sites appear to be evolutionarily conserved, as all the Drosophila SARs also bind with identical specificity to nuclear scaffolds derived from rat liver nuclei. These Sar interaction studies were carried out with lithium 3,5-diiodosalicylate-extracted nuclei. Interestingly, scaffolds prepared by high-salt extraction also bind the genomic and exogenously added SAR fragments specifically. However, the endogenous transcribed sequences, as opposed to the same fragments added as purified DNA, associate randomly with these scaffolds.     

A related moderately repetitive DNA family in the nematodes Ascaris lumbricoides and Panagrellus silusiae.

Digestion of genomic DNA from the nematodes Panagrellus silusiae and Ascaris lumbricoides with restriction endonuclease BamH1 releases a 0.7 kilobase (kb) fragment. The 0.7 kb fragment from both nematodes was cloned onto E. coli plasmid pUC19. Using representative clones as DNA hybridization probes, it was found that (i) the BamH1 fragments cross-hybridize; (ii) a ladder-effect with multiples of 0.7 kb was evident in both species after hybridization to genomic DNA and (iii) the genomic copy number of BamH1 elements is 150 and 195 for P. silusiae and A. lumbricoides respectively. DNA sequence analysis of the inserts, AL700-1 and PS700-1, revealed nucleotide blocks with over 85% similarity. No open reading frames are present in either DNA fragment. Neither fragment hybridizes to genomic DNA from Caenorhabditis elegans. Northern blot hybridization indicated that the 0.7 kb element is transcribed into poly(A)(-)-RNA in P. silusiae; but, is not transcribed in adult Ascaris muscle. Thus, P. silusiae and A. lumbricoides share a homologous, tandemly arrayed, moderately repetitive DNA family.     

Specificity of six gene sequences for the detection of the genus Brucella by DNA amplification

M. DA COSTA, J.-P. GUILLOU, B. GARIN-BASTUJI, M. THIÉBAUD AND G. DUBRAY. 1996. DNA extracted from all Brucella species, reference and vaccine strains were amplified by PCR using primers specific for the genes encoding a 31-kDa Brucella protein, the heat shock proteins (DnaJ, DnaK, HtrA and GroEL) and 16S RNA. No difference was found between Brucella species and biovars with all primer pairs used, even after restriction enzyme analysis of the amplified fragments. The specificity of the amplified products was confirmed by hybridization with a digoxigenin 3'-labelled specific probe and by PCR using 98 non- Brucella micro-organisms' DNA. Only Ochrobactrum anthropi and Phyllobacterium spp. yielded a PCR product by using 31-kDa DnaK, DnaJ, GroEL and 16S RNA primers. After hybridization and restriction analysis, 16S RNA fragments of 3301 and 3331 O. anthropi strains showed a total similarity to those from Brucella. A similar result was shown with DnaJ fragments obtained with 3301 strain of O. anthropi after Eco RI digestion.     

Conserved polynucleotide sequences among the genomes of papillomaviruses.

The DNAs of different members of the Papillomavirus genus of papovaviruses were analyzed for nucleotide sequence homology. Under standard hybridization conditions (Tm - 28 degrees C), no homology was detectable among the genomes of human papillomavirus type 1 (HPV-1), bovine papillomavirus type 2 (BPV-2), or cottontail rabbit (Shope) papillomavirus (CRPV). However, under less stringent conditions (i.e., Tm - 43 degrees C), stable hybrids were formed between radiolabeled DNAs of CRPV, BPV-1, or BPV-2 and the HindIII-HpaI A, B, and C fragments of HPV-1. Under these same conditions, radiolabeled CRPV and HPV-1 DNAs formed stable hybrids with HincII B and C fragments of BPV-2 DNA. These results indicate that there are regions of homology with as much as 70% base match among all these papillomavirus genomes. Furthermore, unlabeled HPV-1 DNA competitively inhibited the specific hybridization of radiolabeled CRPV DNA to bpv-2 DNA fragments, indicating that the homologous DNA segments are common among these remotely related papillomavirus genomes. These conserved sequences are specific for the Papillomavirus genus of papovaviruses as evidenced by the lack of hybridization between HPV-1 DNA and either simian virus 40 or human papovavirus BK DNA under identical conditions. These results indicate a close evolutionary relationship among the papillomaviruses and further establish the papillomaviruses and polyoma viruses as distinct genera.     

Cloning of a species-specific DNA probe from Onchocerca gibsoni

A genomic library of Onchocerca gibsoni has been prepared in the vector lambda-gt10 and has been screened for specific DNA sequences by hybridization with radiolabelled total genomic DNA from a number of Onchocerca species. A clone--fOGI--has been isolated which does not interact with DNA prepared from O. gutturosa, O. lienalis, O. ochengi, O. cervicalis or O. volvulus (both Liberian and Mexican isolates). In addition, no hybridization is observed with host (cattle) DNA. fOGI can detect as little as 100-200 pg of O. gibsoni DNA. It is thus concluded that fOGI has the sensitivity to detect microfilariae of O. gibsoni found in the skin of cattle and the specificity to differentiate them from closely related species living in the same environment.     

Direct cloning by covalent attachment of probe DNA to target DNA.

A novel cloning procedure which makes use of covalent attachment of probe DNA to specific target DNA is reported. We show that specific gene fragments found in complex genomes such as the human genome can be cloned directly from a pool of genomic DNA with very high efficiency. This direct cloning method totally eliminates certain steps in current cloning procedures such as construction of DNA libraries and colony (plaque) hybridization. The resulting process has made cloning methods simpler and more time efficient, while achieving high cloning efficiency due to the stable nature of the probe-target DNA complex through covalent bonding. Most importantly, since clones are directly obtained from a pool of genomic DNA, the isolated clones are considered to be faithful copies of the original genes. This has apparently solved the problem of isolating clones with misincorporated bases or chimeric DNA, both of which are often encountered in cloning processes using PCR or other methods involving in vitro DNA synthesis.     

Study of stringency conditions for human papillomavirus DNA detection on cell lines, frozen and paraffin-embedded tissue sections by in situ hybridization with biotinylated probes

In situ hybridization was mainly used for typing human papillomavirus (HPV) in paraffin-embedded or frozen sections under stringent conditions (SC). We tested 5 different conditions of stringency with biotinylated HPV 1, 2, 16 and 18 probes on 3 cell lines (Sihà and CaSki with HPV16, HeLa with HPV18) by varying the concentration of formamide in the hybridization mixture and washings in order to determine the stringency conditions to be used to assess the presence of HPV and its typing: A-low stringency, hybridization at 35 degrees C below the melting temperature of DNA (Tm-35 degrees C) and washings without formamide; B-low stringency, hybridization and washings at Tm-35 degrees C; C-medium stringency, hybridization at Tm-35 degrees C and washings at Tm-12 degrees C; D-high stringency, hybridization at Tm-12 degrees C and washing without formamide; E-very high stringency, hybridization and washings at -12 degrees C. This study showed that HPV typing required a high stringency. On the contrary, under non stringent conditions (NSC), each cell line was positive with the heterologous probes. When 3 to 5 stringency conditions were assayed on 4 frozen samples, similar results were obtained. Typing required high stringency conditions whereas NSC allowed HPV detection. Furthermore, this study demonstrated the specificity of the reaction in lesions positive with more than one type. Stringent (Tm-12 degrees C) and non stringent (Tm-35 degrees C) conditions of hybridization were further applied to 57 biopsy sections (17 frozen and 40 paraffin-embedded specimens) from typical wart lesions and lesions suspected of HPV.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amelogenin gene similarity in vertebrates: DNA sequences encoding amelogenin seem to be conserved during evolution

Summary Mouse amelogenin cDNA was used in hybridization assays with genomic DNA, cut with the restriction enzyme Eco RI, from the edentulous chicken (Gallus domesticus), the monophyodont mouse (as control), diphyodont man, and the polyphyodont fishes Atlantic salmon (Salmo salar) and seawolf (Anarrhichas lupus). The hybridization assay was performed under stringent conditions with non-radioactive probes. Hybridization was obtained with mouse (6.4-kb band), man (9-kb and 13-kb bands), and seawolf (18-kb band) genomic DNA. This demonstrates DNA sequence similarities between these species, and supports the theory that DNA sequences encoding enamel proteins appear to be highly conserved during the evolution of vertebrates. Lack of hybridization in salmon and chicken may be due to sequence divergencies or structural differences in an amelogenin gene analog, or it may be that no amelogenin gene is present in these animals.     

Isolation and characterization of a species-specific DNA probe for Candida albicans.

As a model for the isolation of species-specific sequences of DNA, we isolated and characterized a species-specific DNA fragment from the Candida albicans genome. We used a series of differential colony hybridization experiments, in which a C. albicans genomic library was hybridized with genomic DNA probes from related organisms to minimize the number of potentially specific C. albicans DNA fragments to be tested. Six clones were tested by dot blot analysis, and one of them, a 1348 bp EcoRI DNA fragment, was confirmed as specific for C. albicans. This species-specific fragment could be utilized as a DNA probe for rapid, sensitive, and specific diagnosis of C. albicans DNA in clinical specimens.     

Magnetic bead-based solid phase for selective extraction of genomic DNA

Magnetic bead-based solid phases are widely used for the separation of nucleic acids from complex mixtures. The challenge to selectively separate specific DNA molecules (via complementary hybridization) in a single step is the selection of a linker between the capture probe and the solid support that can be exposed to high temperatures in the presence of a high salt media. This article presents a general platform for the fabrication of a magnetic bead-based selective solid phase that can be used for subtractive hybridization or sequence capture applications. Phosphorus dendrimers are used for the first time as linkers in a magnetic bead-based selective solid phase for capture of genomic DNA. Aside from providing a high loading capacity, they render a stable bond between the capture probe and the surface under the high temperature and salt conditions required for denaturation and capture to proceed in a single step. The thermal stability of the solid phase under these conditions is first demonstrated by hybridizing a Cy3-labeled target. The selective capture of DNA targets in a single step is then demonstrated by subtractive hybridization of fragmented human genomic DNA. The specificity and selectivity of the solid phase are demonstrated by the recovery of adenovirus serotype 4 DNA spiked into the human DNA target. The effect of steric and electrostatic constraints was also investigated by using dendrimers of different generations that vary in their size and the number of branches. The results demonstrate that this platform can be used for single-step subtractive hybridization applications with better performance over the conventional two-step method using streptavidin-coated magnetic beads.