The use of DNA from phage M13 for the analysis of interindividual polymorphism of human DNA as demonstrated by a population study in Krasnodar city

Hypervariable "minisatellite" regions detected in human genome by wild-type M13 DNA were found to have high polymorphism and somatic stability. Analysis of individual specific patterns of 34 human DNAs from Krasnodar population is presented. The observed length of fragments ranged from 2 to 6 kb. The mean frequency of a fragment in the population under study is p = 0.247 +/- 0.171, the mean number of fragments per individual being x = 9.35 +/- 1.95. The mean probability of individual identification is calculated to be 1-2.10(-6) = 0.999998.     

Analysis of genetic distances between populations using human DNA "fingerprints" detected by a phage M13 DNA probe]

The frequencies of different electrophoretic bands in DNA fingerprints detected by phage M13 DNA probe in two populations of the Kirov district were determined. The DNA polymorphisms observed in these two populations were compared with each other and with those of the Krasnodar populations, and pseudogenetic distances were calculated. The mean genetic distance between two Kirov populations was 0.072, this between every Kirov and Krasnodar population being 0.21 and 0.22.     

Analysis of DNA polymorphism in populations of the Volga-Ural region using genome fingerprinting with phage M13 DNA]

The hyperpolymorphism of minisatellite DNA hybridizing with DNA of bacteriophage M13 was analyzed in seven Turkic and Finno-Ugric populations from the Volga-Urals region. In total, hybridization revealed 80 BspRI genomic DNA fragments ranging in size from 1.7 to 10 kb; the average frequency of an individual fragment was 0.299 +/- 0.020. The average number of hybridization fragments per pattern (varying from 14 to 20 in different populations) and frequencies of individual fragments showed significant interpopulation differences. Parameters of this polymorphic system were assumed to reflect phenotypic diversity of populations. Genome fingerprinting with the use of phage M13 can be employed in the studies of population genetic structure and differentiation and in forensic medicine, for more accurate personal identification.     

DNA "fingerprints" and segregation analysis of multiple markers in human pedigrees

Tandem-repetitive DNA hybridization probes based on a putative human recombination signal detect multiple polymorphic minisatellite fragments in human DNA. The genetic complexity of the resulting individual-specific DNA "fingerprints" was investigated by studying a large sibship affected by neurofibromatosis and a more extensive pedigree segregating for two different hemoglobinopathies. The segregation of up to 41 different heterozygous DNA fragments from each parent could be analyzed in a single sibship, using two different repeat probes. Most of these variable DNA fragments could not be paired as alleles, to an extent which suggests that the DNA fingerprints are together derived from approximately 60 heterozygous loci (approximately 120 variable fragments), only a proportion of which can be scored in a given individual. Two or three of the DNA fragments detected by one probe showed tight linkage and may be derived from long minisatellite(s) that are cleaved to produce more than one polymorphic DNA fragment. Excluding allelic and linked DNA fragments, almost all remaining scorable fragments segregated independently, allowing up to 34 unlinked loci to be examined simultaneously. These loci are scattered over most or all of the human autosomes. Minisatellite probes are therefore suitable for rapid marker generation and can be applied to linkage analysis in human pedigrees.     

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 DNA fingerprints for cell-line individualization.

DNA fingerprints of 46 human cell lines were derived using minisatellite probes for hypervariable genetic loci. The incidence of 121 HaeIII DNA fragments among 33 cell lines derived from unrelated individuals was used to estimate allelic and genotypic frequencies for each fragment and for composite individual DNA fingerprints. We present a quantitative estimate of the extent of genetic difference between individuals, an estimate based on the percentage of restriction fragments at which they differ. The average percent difference (APD) among pairwise combinations from the population of 33 unrelated cell lines was 76.9%, compared with the APD in band sharing among cell lines derived from the same individual (less than or equal to 1.2%). Included in this survey were nine additional cell lines previously implicated as HeLa cell derivatives, and these lines were clearly confirmed as such by DNA fingerprints (APD less than or equal to 0.6%). On the basis of fragment frequencies in the tested cell line population, a simple genetic model was developed to estimate the frequencies of each DNA fingerprint in the population. The median incidence was 2.9 X 10(-17), and the range was 2.4 X 10(-21) to 6.6 X 10(-15). This value approximates the probability that a second cell line selected at random from unrelated individuals will match a given DNA fingerprint. Related calculations address the chance that any two DNA fingerprints would be identical among a large group of cell lines. This estimate is still very slight; for example, the chance of two or more common DNA fingerprints among 1 million distinct individuals is less than .001. The procedure provides a straightforward, easily interpreted, and statistically robust method for identification and individualization of human cells.     

Herpesvirus saimiri DNA in tumor cells--deleted sequences and sequence rearrangements.

Herpesvirus saimiri DNA in continuous lymphoblastoid cell lines obtained from viral induced tumors in marmosets has been analyzed by gel electrophoresis of restricted DNA. Southern transfer to nitrocellulose filters, and hybridization to 32P-labeled viral DNA or DNA fragments. The viral DNA fragments EcoRI-G, -H, -D, and -I, KpnI-A, and BamHI-D and -E were not detected in Southern transfers of DNA from the nonproducing 1670 cell line. For each restriction endonuclease, a new fragment appeared, consistent with a 13.0-megadalton deletion of viral DNA sequences. This deletion encompassed 35 to 48 +/- 0.6 megadaltons from the left end of the unique DNA region. A sequence arrangement map is presented for the major population of H. saimiri DNA sequences in the 1670 cell line. Although H. saimiri DNA in the nonproducing 70N2 cell line can be distinguished from viral DNA in the 1670 cell line by several criteria, the same sequences were found to be deleted in the major population of viral DNA molecules. Unlike 1670 and 70N2 cells, restricted DNA from the virus-producing cell lines 77/5 and 1926 contained all of the DNA fragments present in the parental virion DNA. DNA from 1670, 70N2, and 77/5 cells contained additional viral DNA fragments that did not comigrate with any virion DNA fragments. Most of these unexplained fragments were confined to or highly enriched in partially purified circular or linear DNA fractions. DNA from tumor cells taken directly from a tumor-bearing animal contained viral DNA indistinguishable from the parental virion DNA by the assay conditions used. These results indicate that viral DNA sequence rearrangements can occur upon cultivation of tumor cells in vitro and that excision of DNA sequences from the viral genome may play a role in establishing the nonproducing state of some tumor cell lines.     

Interindividual hyperpolymorphism of autosomal satellites III of human DNA

Hypervariability of DNA restriction fragments from pericentromeric heterochromatin detected by autosomal "classic" DNA of satellite III has been demonstrated in this work. Using hybridization probe of satellite III DNA localized predominantly on the chromosome 9 strong interindividual differences in the sets of polymorphic DNA restriction fragments inherited from both paternal and maternal sides as well as intraspecies amplifications of some variants of the satellite III were observed. The number and intensity of restriction bands are identical in both sexes. It is suggested that strong interindividual DNA variability may be largely specified by high level of DNA spot mutability in satellites III. Pericentromeric "classic" satellites III may serve as efficient markers for identification of individuals and for molecular-genetic mapping of human genome pericentromeric regions.     

Homologies between nuclear and plastid DNA in spinach

Summary Homologies between spinach nuclear (n) DNA and Chloroplast (pt) DNA, have been detected with a clone bank of spinach ptDNA as hybridization probes to restriction fragments of nDNA prepared from purified root nuclei. Every cloned fragment of ptDNA showed homologies to discrete restriction fragments of nDNA, different from those of ptDNA, indicating integration of these homologies into nDNA. While most ptDNA clones were relatively large and probably contained several genes, sequence homologies were also found to the cloned plastid gene for RuBP carboxylase and the subunit of ptATPase. Many of the homologies in nDNA occur in regions of the genome that are highly methylated and are not digested by the methylation sensitive restriction endonucleases HpaII and MspI. In contrast these enzymes cleave ptDNA into small fragments which allows the nDNA homologies to be distinguished in total root DNA. The sequence homologies observed were not due to contaminating non nuclear sequences as shown by hybridization to mitochondrial (mt) and bacterial DNAs. The total amount of homology to ptDNA in nDNA is equivalent to about five copies of the plastome per haploid nuclear genome. The homologies generally appear to be in individual segments of less than 2 kbp in length, integrated into several different places in the genome.On sabbatical leave from Department of Botany, University College, Dublin, Ireland     

DNA associated with nucleosomes in plants.

50 to 55% of tobacco and barley nuclear DNA is accessible to micrococcal endonuclease digestion. The DNA fragments resulting from a mild endonuclease treatment are multiples of a basic unit of 194 +/- 6 base pairs in tobacco and 195 +/- 6 base pairs in barley. After extensive digestion, a DNA fragment of approximately 140 base pairs is predominant. Hence the "extra-core" or "linker"-DNA is 55 base pairs long. Other fragments having 158 and less than 140 base pairs are present as well. Treatment with DNase I results in multiples of 10 bases when analysed under denaturating conditions. These results show that the general organization of the DNA within the nucleosomes is about the same in higher plants as in other higher eukaryotes.     

DNA Fingerprinting and Analysis of Population Structure in the Chestnut Blight Fungus, Cryphonectria Parasitica

We analyzed DNA fingerprints in the chestnut blight fungus, Cryphonectria parasitica, for stability, inheritance, linkage and variability in a natural population. DNA fingerprints resulting from hybridization with a dispersed moderately repetitive DNA sequence of C. parasitica in plasmid pMS5.1 hybridized to 6-17 restriction fragments per individual isolate. In a laboratory cross and from progeny from a single perithecium collected from a field population, the presence/absence of 11 fragments in the laboratory cross and 12 fragments in the field progeny set segregated in 1:1 ratios. Two fragments in each progeny set cosegregated; no other linkage was detected among the segregating fragments. Mutations, identified by missing bands, were detected for only one fragment in which 4 of 43 progeny lacked a band present in both parents; no novel fragments were detected in any progeny. All other fragments appeared to be stably inherited. Hybridization patterns did not change during vegetative growth or sporulation. However, fingerprint patterns of single conidial isolates of strains EP155 and EP67 were found to be heterogenous due to mutations that occurred during culturing in the laboratory since these strains were first isolated in 1976-1977. In a population sample of 39 C. parasitica isolates, we found 33 different fingerprint patterns with pMS5.1. Most isolates differed from all other isolates by the presence or absence of several fragments. Six fingerprint patterns each occurred twice. Isolates with identical fingerprints occurred in cankers on the same chestnut stems three times; isolates within the other three pairs were isolated from cankers more than 5 m apart. The null hypothesis of random mating in this population could not be rejected if the six putative clones were removed from the analysis. Thus, a rough estimate of the clonal fraction of this population is 6 in 39 isolates (15.4%).     

Distribution of DNA replication origins between matrix-attached and loop DNA in mammalian cells

Using a previously developed procedure (Gencheva et al. [1996] J Biol Chem 271:2608-2614), we isolated a DNA fraction consisting of short fragments originating from the regions of initiation of DNA synthesis from exponentially growing Chinese hamster ovary cells. This fraction arbitrarily designated as "collective origin fraction" was labeled in vitro and used to probe the abundance of origin containing sequences in preparations of matrix-attached and loop DNA isolated by two different procedures from Chinese hamster ovary cells. Alternatively, an individual DNA replication origin sequence - a 478-bp long DNA fragment located at about 17-kb downstream of the dihydrofolate reductase gene - was used to probe the same matrix-attached and loop DNA fractions. The results with both the collective and individual DNA replication origins showed that there was random distribution of the origin sequences between DNA attached to the matrix and DNA from the loops.     

Isolation and analysis of DNA markers specific to human chromosome 15.

Chromosome-specific DNA markers provide a powerful approach for studying complex problems in human genetics and offer an opportunity to begin understanding the human genome at the molecular level. The approach described here for isolating and characterizing DNA markers specific to human chromosome 15 involved construction of a partial chromosome-15 phage library from a human/Chinese hamster cell hybrid with a single human chromosome 15. Restriction fragments that identified unique- and low-copy loci on chromosome 15 were isolated from the phage inserts. These fragments were regionally mapped to the chromosome by three methods, including Southern analysis with a mapping panel of cell hybrids, in situ hybridization to metaphase chromosomes, and quantitative hybridization or dosage analysis. A total of 42 restriction fragments of unique- and low-copy sequences were identified in 14 phage. The majority of the fragments that have been characterized so far exhibited the hybridization pattern of a unique locus on chromosome 15. Regional mapping assigned these markers to specific locations on chromosome 15, including q24-25, q21-23, q13-14, q11-12, and q11. RFLP analysis revealed that several markers displayed polymorphisms at frequencies useful for genetic linkage analysis. The markers mapped to the proximal long arm of chromosome 15 are particularly valuable for the molecular analysis of Prader-Willi syndrome, which maps to this region. Polymorphic markers in this region may also be useful for definitively establishing linkage with one form of dyslexia. DNA probes in this chromosomal region should facilitate molecular structural analysis for elucidation of the nature of instability in this region, which is frequently associated with chromosomal aberrations.     

DNA profiling by detection of repetitive nucleotide sequences on human chromosome 6

Genetic/genomic polymorphism, i.e. variations in DNA sequences are ideally assayed by direct nucleotide sequencing of a gene region or other homologous segment of the genome. An easier and cheaper approach, however, if the variants are analyzed by hybridization technology using restriction fragment length polymorphisms (RFLPs) or by detection of the number of tandem repeats (VNTR) of small DNA segments, the "minisatellites". In this study we describe results of the DNA analysis of repetitive sequences of human 6th chromosome by the application of a chemiluminescent labeled probes. The allele frequency distribution of polymorphic DNA sequences has been determined in unrelated individuals. The isolated genomic DNA was cut with Pst I restriction enzyme, size fractionated on agarose gel and hybridized with a chemiluminescent labeled D6 S132 probe. At this locus the Pst I cleaved DNA fragments are ranging from 1841 to 6098 base pairs (bp). Specific genetic pattern was characterized by more frequent fragments (3313 and 3884 bp), and the rarely occurring ones (clustered between 1841-2595 and 5227-6098 bp). Our study provides a further possibility for characterization of individual genomic patterns.     

Genetic determinants of antibiotic resistance in enterobacteria

Antibiotic resistance of enterobacterial strains from population isolated in Krasnodar region is rather often controlled by the "plasmid" genes. The conclusion is based on using the colony hybridization with [32P]-DNA fragments of plasmids, carrying the genetic determinants of antibiotic resistance, as a method for antibiotic resistance, genes screening. Kanamycin resistance in the majority of strains is coded by APH (3') II gene, streptomycin resistance by APH (3") gene, chloramphenicol resistance by CATI, sulphonilamide resistance by DHPS type II gene. Tetracycline resistance of the studied enterobacterial strains is not connected with the widespread genetic determinants of a new class tetracycline resistance.     

Sequence organization and expression of a yeast plasmid DNA.

Saccharomyces cerevisiae strain A364A D5 contains circular double-stranded DNA molecules of 6230 +/- 30 base pairs (2mu DNA) which are present in 68 copies per cell and make up 2.4% of the haploid genome. About 0.4% of non-poly A containing yeast RNA hybridizes to the yeast DNA circles. When denatured and then self-annealed, the DNA molecules assume a characteristic "dumbbell" shape in the electron microscope indicating that each circle possesses a non-tandem inverted repeat sequence of 630 +/- 10 base pairs. Eco-RI digestion of purified 2mu DNA yields 4 fragments on an agarose gel whose combined molecular mass is twice that of the monomer circle, suggesting that there are 2 populations of circles, each of the same molecular weight. Representatives of each population have been separated by cloning in Escherichia coli via the bacterial plasmid pSC101. Heteroduplex analysis of the cloned circles show that the 2 different populations arise because of intramolecular recombination between the inverted repeat sequences. Acrylamide gel patterns of polypeptides synthesized in bacterial mini-cells containing the hybrid plasmids between 2mu DNA and pSC101 are significantly different than the pattern obtained from mini-cells containing pSC101 alone.     

Studies on DNA bound to histones in chromatin]

Regions of DNA protected by histones against the action of DNAse 1 in the chromatin were isolated. Such DNA fragments ("subhistones" DNA) have 80% double helix structure, their nucleotide composition is close to that of total DNA, and their sedimentation constant is within the range of 2-2.7S for completely denatured molecules. Kinetics of renaturation of "subhistone" DNA was studied: within a wide range of Cot values, renaturation curves of total and "subhistone" DNA are almost identical. According to the data on hybridization with nuclear d-RNA, "subhistone" DNA is transcribed in the cell. The data obtained witness for uniform character of distribution of histones along the DNA chain in the chromatin. DNA sites which are active in RNA synthesis seem to be bound to histones as well as the non-active ones. No significant difference was found in the hybridization of "subhistone" DNA from rat liver and thymus with ibver nuclear RNA.     

Divergence, hybridization, and recombination in the mitochondrial genome of the human pathogenic yeast Cryptococcus gattii

The inheritance of mitochondrial genes and genomes are uniparental in most sexual eukaryotes. This pattern of inheritance makes mitochondrial genomes in natural populations effectively clonal. Here, we examined the mitochondrial population genetics of the emerging human pathogenic fungus Cryptococcus gattii . The DNA sequences for five mitochondrial DNA fragments were obtained from each of 50 isolates belonging to two evolutionary divergent lineages, VGI and VGII. Our analyses revealed a greater sequence diversity within VGI than that within VGII, consistent with observations of the nuclear genes. The combined analyses of all five gene fragments indicated significant divergence between VGI and VGII. However, the five individual genealogies showed different relationships among the isolates, consistent with recent hybridization and mitochondrial gene transfer between the two lineages. Population genetic analyses of the multilocus data identified evidence for predominantly clonal mitochondrial population structures within both lineages. Interestingly, there were clear signatures of recombination among mitochondrial genes within the VGII lineage. Our analyses suggest historical mitochondrial genome divergence within C. gattii , but there is evidence for recent hybridization and recombination in the mitochondrial genome of this important human yeast pathogen.     

A Schizosaccharomyces pombe artificial chromosome large DNA cloning system.

The feasibility of using the fission yeast, Schizosaccharomyces pombe , as a host for the propagation of cloned large fragments of human DNA has been investigated. Two acentric vector arms were utilized; these carry autonomously replicating sequences ( ars elements), selectable markers ( ura4(+) or LEU2 ) and 250 bp of S. pombe terminal telomeric repeats. All cloning was performed between the unique sites in both vector arms for the restriction endonuclease Not I. Initially the system was tested by converting six previously characterized cosmids from human chromosome 11p13 into a form that could be propagated in S.pombe as linear episomal elements of 50-60 kb in length. In all transformants analysed these cosmids were maintained intact. To test if larger fragments of human DNA could also be propagated total human DNA was digested with Not I and size fractionated by pulsed field gel electrophoresis (PFGE). Fractions of 100-1000 kb were ligated to Not I-digested vector arms and transformed into S.pombe protoplasts in the presence of lipofectin. Prototrophic ura+leu+transformants were obtained which upon examination by PFGE were found to contain additional linear chromosomes migrating at between 100 and 500 kb with a copy number of 5-10 copies/cell. Hybridization analyses revealed that these additional bands contained human DNA. Fluorescent in situ hybridization (FISH) analyses of several independent clones indicated that the inserts were derived from single loci within the human genome. These analyses clearly demonstrate that it is possible to clone large fragments of heterologous DNA in fission yeast using this S.p ombe artificial chromosome system which we have called SPARC. This vector-host system will complement the various other systems for cloning large DNA fragments.