Total Nuclear DNA Research Articles

The variability of DNA fingerprints in three species of swan

The genetic variation in three species of swans was examined by DNA fingerprinting. Human minisatellite probes detected a number of highly variable fragments in the restriction digests of total nuclear DNA. Assuming bands to be independent, the average allele frequency for minisatellite loci was 0·154, 0·154 and 0·109 for Mute, Whooper and Bewick's Swans, respectively. The pattern of band-distribution showed a certain degree of species-specificity, so that the fingerprints may reflect taxonomic affinities of the species. Pedigree analysis of a single family of Mute Swans showed that most of the variable fragments were inherited in a simple Mendelian fashion, allowing the relatedness of the individuals to be determined. DNA fingerprinting potentially provides a large number of genetic markers for studying the behavioural ecology, population genetics and evolution of swans.

A comparative study of the heterochromatin of Apodemus sylvaticus and Apodemus flavicollis

The two closely related species Apodemus sylvaticus and Apodemus flavicollis (Muridae) differ in the distribution of their heterochromatin. Two major repetitive sequences known to occur in both species were isolated from A. flavicollis after digestion of total nuclear DNA with the restriction enzymes HindIII and EcoRI respectively and characterized in both species by filter hybridisation and in situ hybridisation to metaphase chromosomes. The EcoRI clone detects a dispersed repetitive sequence family in the genome of both species. Southern blot hybridisation with the HindIII satellite DNA probe reveals major similarities and minor differences in the two species. In situ hybridisation with the HindIII probe labels all chromosomes of A. flavicollis exclusively in the centromeric heterochromatin, whereas in A. sylvaticus several autosomes are also labelled distally. The labelling patterns correspond to the distribution of heterochromatin in the two species. It is concluded that the additional distal heterochromatin of A. sylvaticus contains similar sequences to those of the centromeric heterochromatin of both species. The distal heterochromatin in A. sylvaticus most likely evolved by transposition and amplification of centromeric satellite DNA elements, after the separation of the two species.

Three-dimensional analysis of the organization of human chromosome domains in human and human-hamster hybrid interphase nuclei.

This report describes the intranuclear organization of chromosomes in human-hamster hybrid nuclei and in human cell nuclei. The target chromosomes were stained using in situ hybridization with biotinylated, chromosome-specific DNA probes. Bound probe was detected with fluorescein-avidin. Hybridizations were performed to fixed nuclei in aqueous suspension in order to preserve their three-dimensional morphology. Total nuclear DNA was stained with DAPI. Three-dimensional information about the organization of DNA and probe within the nucleus was obtained by optical sectioning. The human chromosomes in human-hamster hybrid nuclei were found to be confined to 'domains' that were maintained during the cell cycle. Different spatial localization patterns of the human chromosomes were seen in interphase nuclei of two different hybrid cell lines. The positions of chromosome-specific repetitive sequences in human fibroblast interphase nuclei were also studied using probes for the telomeric region of chromosome 1p (1p36), the centromeric region of chromosome 9 (9q12) and the long arm of the Y chromosome (Yq12). These studies showed that the two 1p telomeric loci are located near the nuclear surface. The chromosome 9 centromeric loci are similarly located. Simultaneous hybridization of the chromosome 1 telomeric probe (target size approximately 200 kb; b, base) and the Y-specific probe (target size greater than 2Mb), demonstrate that the binding sites of the two probes can be distinguished in the same nucleus on the basis of domain size.

Affinity Isolation of Transcriptionally Active Murine Erythroleukemia Cell DNA Using a Cleavable Biotinylated Nucleotide Analog

We have developed an affinity technique to obtain active gene domains from murine erythroleukemia cell nuclei, based on the differential sensitivity of potentially active and inactive chromatin to DNase I. Nuclei isolated from potentially active noninduced cells and transcriptionally active induced MEL cells were treated with DNase I at concentrations which did not digest the beta-globin gene, followed by repair using a typical nick translation reaction during which a cleavable biotinylated nucleotide analog, 5-[N-biotinamido)hexanoamido-ethyl-1,3-dithiopropionyl -3-aminoallyl]-2'- deoxyuridine 5'-triphosphate (Bio-19-SS-dUTP), was inserted into DNA sequences. Following purification and digestion with EcoRI restriction endonuclease, biotinylated sequences were affinity isolated by sequential binding to streptavidin and biotincellulose. The streptavidin/biotin-cellulose complex bound up to 80% of the nick-translated DNA, which comprised a small percentage of the total nuclear DNA. Cleavage of the disulfide bond in the linker arm of the biotinylated nucleotide resulted in elution of virtually all of the affinity isolated sequences. Hybridization analysis of this fraction of DNA revealed up to a 16-fold enrichment for the active beta-globin gene, as compared with DNA which did not bind to the biotincellulose. Conversely, the inactive alpha-fetoprotein gene was barely detectable in affinity isolated DNA from noninduced cells and was 2-fold depleted in samples from induced cells.

DNA Amplification among Anther‐Derived Doubled Haploid Lines of Tobacco and Its Relationship to Agronomic Performance

Anther culture in tobacco (Nicotiana tabacum L.) has been associated with yield losses and DNA amplification without chromosome number changes among doubled haploid lines. The objectives of this study were to confirm the amplification phenomenon and to determine if increases in DNA are correlated with yield losses. Total nuclear DNA was determined for two cultivars, ‘NC95’ and ‘Coker 139’ (C139), and six doubled haploid (DH) lines derived from cultivar. The DHs were selected so that approximately equal increments of yield losses would be present among the array. The DNA analysis was performed via flow cytometry on isolated nuclei stained with the DNA‐specific fiuorochrome propidium iodide. Agronomic data also were collected on the DH lines. Results indicated that genotypic effects for nuclear DNA were highly significant. While the DNA increases observed among the NC95 genotypes were not statistically significant, doubled haploids derived from C139 had significantly more DNA per nucleus than C139. Coker 139 DHs also sustained greater yield reductions from the parental cultivar than did the NC95 DH lines. Regression analyses of meanuclear DNA values of C139 genotypes on mean leaf yields resulted in a correlation coefficient of ‐0.69; this was significant only at the 0.1 probability level. While DNA amplification among anther‐derived doubled haploids of tobacco was confirmed in one cultivar, DNA amplification and yield losses do not appear to be strongly correlated. The lack of a linear relationship between these two variables may be caused by different chromosomal amplification sites among the doubled haploid lines.

Correlation between automated karyometric measurements of squamous cell carcinoma of the esophagus and histopathologic and clinical features.

The clinical staging of esophageal carcinoma is unreliable currently, making it difficult to select patients for aggressive therapy. To further refine staging criteria, the nuclear characteristics of a series of 31 patients with squamous cell carcinoma of the esophagus were studied using a computerized image analysis system (MicroTICAS). Karyometric measurements, including total nuclear DNA content, nuclear area, and nuclear roundness were compared with various clinical and histologic variables. Nearly all tumors (30 of 31) were aneuploid. Tumors with nuclear areas greater than 70 microns2 were associated with transmural esophageal penetration (P less than 0.05) and to a lesser extent with poor survival (less than 6 months; P = 0.06). Surprisingly, nuclear ploidy did not correlate with either variable. These data support a role for nuclear analysis on preoperative biopsy specimens as an adjunct in clinical staging.

Chromosomal DNA variation, genomic constraints and recombination in Lathyrus

There is approximately a doubling of the total nuclear DNA between the 8 Lathyrus species and there are significant differences in the amounts of DNA in euchromatin and heterochromatin. Between the 8 species chiasma frequency and total nuclear DNA are not correlated but within complements it is positively correlated with the amount of DNA in the chromosomes. There is no significant correlation between chiasma frequency and euchromatin DNA nor between chiasma frequency and heterochromatin DNA among species, but among chromosomes, as with total DNA, it is positively correlated with euchromatin DNA and heterochromatin DNA. Results show that despite the large differences in DNA amounts between species there are genomic constraints underlying the frequency and distribution of chiasmata in the chromosome complements.

Different allele frequencies in Trypanosoma brucei brucei and Trypanosoma brucei gambiense populations

Restriction fragment length polymorphism (RFLP) has been analysed in Trypanosoma brucei DNA following hybridization with different DNA probes. This polymorphism seems to be due to allelic variation, and not to variation between sequence duplicates, since the genomic environment of the probed polymorphic fragments is conserved over considerable distances. In an analysis of 35 non- gambiense stocks, we found different combinations of homozygotes and heterozygotes for the four RFLP probes used, in keeping with previous observations that genetic reassortment occurs in T. b. brucei. Moreover, the non- gambiense populations from West and East Africa can be differentiated according to their characteristic allele frequencies. In sharp contrast, we found that the 49 T. b. gambiense stocks, analysed with the same probes, share the same single allelic combination and are all homozygous for each one of the four markers. This characteristic gambiense allele combination is very common among Western non- gambiense isolates, but rare or absent among Eastern ones. Two stocks isolated from man in West Africa turned out to be non- gambiense by all molecular criteria examined, including total nuclear DNA content. Taken together, these observations suggest that human serum-resistant variants may appear among the West African T. b. brucei population, and that T. b. gambiense evolved from one of these resistant variants as a man-adapted subspecies that became genetically isolated from the rest of the West African trypanosome population.

Mapping replicational sites in the eucaryotic cell nucleus.

We have used fluorescent microscopy to map DNA replication sites in the interphase cell nucleus after incorporation of biotinylated dUTP into permeabilized PtK-1 kangaroo kidney or 3T3 mouse fibroblast cells. Discrete replication granules were found distributed throughout the nuclear interior and along the periphery. Three distinct patterns of replication sites in relationship to chromatin domains in the cell nucleus and the period of S phase were detected and termed type I (early to mid S), type II (mid to late S) and type III (late S). Similar patterns were seen with in vivo replicated DNA using antibodies to 5-bromodeoxyuridine. Extraction of the permeabilized cells with DNase I and 0.2 M ammonium sulfate revealed a striking maintenance of these replication granules and their distinct intranuclear arrangements with the remaining nuclear matrix structures despite the removal of greater than 90% of the total nuclear DNA. The in situ prepared nuclear matrix structures also incorporated biotinylated dUTP into replication granules that were indistinguishable from those detected within the intact nucleus.

A molecular and cytogenetic survey of major repeated DNA sequences in tomato (Lycopersicon esculentum)

The major families of repeated DNA sequences in the genome of tomato (Lycopersicon esculentum) were isolated from a sheared DNA library. One thousand clones, representing one million base pairs, or 0.15% of the genome, were surveyed for repeated DNA sequences by hybridization to total nuclear DNA. Four major repeat classes were identified and characterized with respect to copy number, chromosomal localization by in situ hybridization, and evolution in the family Solanaceae. The most highly repeated sequence, with approximately 77000 copies, consists of a 162 bp tandemly repeated satellite DNA. This repeat is clustered at or near the telomeres of most chromosomes and also at the centromeres and interstitial sites of a few chromosomes. Another family of tandemly repeated sequences consists of the genes coding for the 45 S ribosomal RNA. The 9.1 kb repeating unit in L. esculentum was estimated to be present in approximately 2300 copies. The single locus, previously mapped using restriction fragment length polymorphisms, was shown by in situ hybridization as a very intense signal at the end of chromosome 2. The third family of repeated sequences was interspersed throughout nearly all chromosomes with an average of 133 kb between elements. The total copy number in the genome is approximately 4200. The fourth class consists of another interspersed repeat showing clustering at or near the centromeres in several chromosomes. This repeat had a copy number of approximately 2100. Sequences homologous to the 45 S ribosomal DNA showed cross-hybridization to DNA from all solanaceous species examined including potato, Datura, Petunia, tobacco and pepper. In contrast, with the exception of one class of interspersed repeats which is present in potato, all other repetitive sequences appear to be limited to the crossing-range of tomato. These results, along with those from a companion paper (Zamir and Tanksley 1988), indicate that tomato possesses few highly repetitive DNA sequences and those that do exist are evolving at a rate higher than most other genomic sequences.

Activation of Programmed Cell Death in the Rat Ventral Prostate after Castration*

The rapid involution of the rat ventral prostate after castration is an active process initiated by removal of the inhibitory effects of androgen on prostatic cell death. The present studies demonstrate that after castration-induced androgen deprivation a series of temporally discrete biochemical events are activated which result in the rapid programmed death of the subset of androgen-dependent cells within the rat ventral prostate. These biochemical steps involve 1) rapid loss of nuclear androgen receptor retention; by 12 h after castration, androgen receptors are no longer detectable in ventral prostatic nuclei; 2) an initial fragmentation of nuclear DNA into low mol wt (less than 1000 basepairs) nucleosomal oligomers which lack intranucleosomal break points; and 3) eventual complete digestion of these nucleosomal oligomers into component nucleotides. Additional studies demonstrate that activation of a Ca2+-Mg2+-dependent endonuclease is associated with this DNA fragmentation. By 4 days after castration, maximal DNA fragmentation is obtained, with 15% of the total nuclear DNA extractable as low mol wt fragments. Proteolytic enzymes are apparently not involved initially in this process, suggesting that DNA fragmentation is a discrete event in, rather than a result of, cell death. Flow cytometric analysis of nuclear DNA content demonstrated that each day after castration, a subpopulation of androgen-dependent cells in rat ventral prostate fragmented all of their genomic DNA, as opposed to the whole population of cells fragmenting an increasing portion of their DNA daily.

Endogenous polymers of ADP-ribose are associated with the nuclear matrix.

The metabolism of nuclear polymers of ADP-ribose has been implicated in several chromatin-associated processes. However, the distribution of endogenous ADP-ribose polymers in the nucleus or within different fractions of chromatin has not been studied. Using a procedure which allowed the radiolabeling and detection of endogenous polymers of ADP-ribose, we have analyzed the nuclear distribution of these polymers in untreated cells and in cells subjected to hyperthermia, N-methyl-N'-nitro-N-nitrosoguanidine, or both. When isolated nuclei from cells subjected to any of these conditions were digested with micrococcal nuclease such that 80% of the DNA was released, 90% of the total poly(ADP-ribose) remained with the micrococcal nuclease resistant chromatin fraction. When nuclear matrix fractions were prepared by exhaustive DNase I digestion in combination with three different salt extraction procedures (2 M NaCl, 300 mM (NH4)2SO4 or 25 mM lithium diiodosalicylate), the matrices contained less than 1% of the total nuclear DNA but 50 to 70% of the total poly(ADP-ribose). These data suggest that the nuclear matrix may be a major site of poly(ADP-ribose) metabolism.

Covalent DNA modification and the regulation of Mutator element transposition in maize

Analyses of the multiple genomic Mu transposable elements in active Mutator lines with several C-methylation sensitive restriction enzymes indicate that Mu elements are undermodified compared with total maize nuclear DNA. Intercrossing of diverse Mutator lines leads to a discrete hypermodification of the Mu elements in a particular plant concurrent with a loss of mutagenic and transpositional potential. The modification events observed appear to be methylation of cytosine at the 5 position in the sequences 5′-CG-3′ and 5′-CNG-3′. Some potential C-methylation sites in Mu elements show a higher degree of methylation than others. Once established, the modified Mu state, like the loss of Mutator activity, is stable on outcrossing. Crosses between active Mutator lines with unmodified Mu elements and Mutator-loss lines with modified Mu elements show partial maternal dominance for the modification event. Mutator activity may also be lost thorugh outcrossing in a mechanism not associated with any detected modification events.

Nuclear matrix-bound DNA primase. Elucidation of an RNA priming system in nuclear matrix isolated from regenerating rat liver.

Recent findings in purified systems demonstrate the universality of DNA polymerase-primase complexes which may function in the priming and continuation of eucaryotic DNA replication. In this report we characterize an in vitro, nuclear matrix-associated, priming and continuation system that can utilize either endogenous matrix-bound DNA or exogenous single-stranded DNA as template. 30-40% of total nuclear DNA primase activity was recovered in association with the isolated nuclear matrix fraction from regenerating rat liver. Matrix-bound primase catalyzed the alpha-amanitin, actinomycin D-resistant synthesis of oligonucleotide chains of 8-50 nucleotides on the endogenous template. At least a portion of the RNA primers were continued by DNA polymerase alpha with deoxynucleoside triphosphate incorporation up to 300-600 nucleotides. Nearest neighbor analysis revealed ribodeoxynucleotide covalent linkages in these RNA-DNA chains. The matrix-bound primase preferred single-stranded fd DNA as exogenous template over synthetic homopolymers and was strictly dependent on the presence of ribonucleoside triphosphates. Appropriate subfractionation revealed that the matrix-bound primase activity is exclusively localized in the nuclear matrix interior. The ability of primase and DNA polymerase to synthesize covalently linked RNA-DNA products demonstrates the potentially useful role of the nuclear matrix in vitro system for elucidating the organizational and functional properties of the eucaryotic replication apparatus in the cell nucleus.

The solution to the cytological paradox of isomorphy.

Cells with polyploid nuclei are generally larger than cells of the same organism or species with nonpolyploid nuclei. However, no such change of cell size with ploidy level is observed in those red algae which alternate isomorphic haploid with diploid generations. The results of this investigation reveal the explanation. Nuclear DNA content and other parameters were measured in cells of the filamentous red alga Griffithsia pacifica. Nuclei of the diploid generation contain twice the DNA content of those of the haploid generation. However, all cells except newly formed reproductive cells are multinucleate. The nuclei are arranged in a nearly perfect hexagonal array just beneath the cell surface. When homologous cells of the two generations are compared, although the cell size is nearly identical, each nucleus of the diploid cell is surrounded by a region of cytoplasm (a "domain") nearly twice that surrounding a haploid nucleus. Cytoplasmic domains associated with a diploid nucleus contain twice the number of plastids, and consequently twice the amount of plastid DNA, than is associated with the domain of a haploid nucleus. Thus, doubling of ploidy is reflected in doubling of the size and organelle content of the domain associated with each nucleus. However, cell size does not differ between homologous cells of the two generations, because total nuclear DNA (sum of the DNA in all nuclei in a cell) per cell does not differ. This is the solution to the cytological paradox of isomorphy.

Chromosome weights and measures in Petunia

The 2C nuclear DNA amounts in diploid Petunia species are low, ranging from 1·73 to 3·00 picograms. The nuclear dry mass and the total chromosome volume are closely correlated with the nuclear DNA amount. So also is cell size measured in pollen grains. Increase in the total nuclear DNA within the genus is achieved by the addition of equal increments of DNA to all members of the chromosome complement irrespective of their size. It is suggested that the correlations and distribution patterns of DNA changes displayed by the natural species may serve as standards for monitoring and interpreting changes in genetic material which are associated with regeneration from protoplasts and callus.

Mapping of sequence-specific chromatin proteins by a novel method: Topoisomerase I on Tetrahymena ribosomal chromatin

DNA derived from the 5′ spacers of the rRNA genes from Tetrahymena has unusual electrophoretic properties. These properties made it possible to devise a simple electrophoretic procedure for isolating specific rDNA spacer fragments from preparations of total nuclear DNA, enabling us to study DNA modifications at the level of unfractionated nuclei. We have employed the method to study the distribution of topoisomerase I binding sites on the r-chromatin (ribosomal chromatin) of Tetrahymena at the DNA sequence level. The presence of topoisomerase I in situ was detected by its ability to introduce single-strand cleavages into DNA. The positions of the cleavages were determined on DNA sequencing gels after isolation of the fragments. Topoisomerase I binding in r-chromatin is sequence specific and cleavage is confined to a 16 base-pair conserved sequence element previously determined to be a high-affinity binding site for topoisomerase I in vitro. The high degree of sequence specificity may be of important functional significance, as we find a similar sequence specificity with enzymes isolated from five evolutionarily distant species, indicating that preference for the 16 base-pair element is an intrinsic property of eukaryotic type I topoisomerases.

Specificity of non-histone protein antigens in chicken liver nuclei

1. 1. Chicken liver nuclei were fractionated by disruption with ultrasound and subsequent precipitation with divatent cations. A small, protein-rich fraction (CS), representing less than 5% of the total nuclear DNA reacted strongly with antisera to dehistonized chicken liver chromatin. 2. 2. Further fractionation of the CS by DNA-affinity chromatography yielded two DNA-binding immunoreactive proteins (approximate M r 35 and 56 kD). 3. 3. We have shown previously (Kiliańska et al., 1981, Kiliańska, 1984) that this DNA-binding component strongly inhibited the in vitro transcription of RNA.

The taxonomic structure of Phytophthora megasperma: Evidence for emerging biological species groups

Nomenclatural uncertainty surrounds P. megasperma as various authors, working with limited groups of isolates, offer their interpretations of this species based on pathology, morphology, or cytology. We compared 93 isolates, including many described by others, for classical morphological features, growth behaviour and appearance, electrophoretic pattern of total proteins, chromosome number and nuclear DNA content. Nine distinct sub-groups were distinguished. While most groups could be distinguished by each of the criteria, protein electrophoresis was the most sensitive. The groups included: ALF, pathogenic to alfalfa, n = 12–15; SOY, pathogenic to soybean, n = 12–15; CLO, pathogenic to clover, n = 11–15; DF, pathogenic to Douglas fir, n = 17–24; AC, isolated from rosaceous fruit trees; and BHR, a major group obtained from a broad range of hosts. The last two groups, distinguished primarily by protein pattern, comprised at least four karyotypes: KI, n = 12–17; KII, n = 15–23; KIII, n = 22–28; and KIV, n = 26–34. All four karyotypes occur within the BHR protein group, suggesting a polyploid series within a closely related genotype. Two broad lines of evolution are hypothesized, a legume line comprising ALF, SOY, CLO, and perhaps DF isolates, and a Broad Host Range line of AC and BHR isolates. Sub-groups within each line may represent emerging biological species, isolated by host specificity or karyotype. Taxonomic designation for the various groups must await confirmation of the hypothesis by demonstration of the extent of barriers to gene flow between the groups.

Characterization of DNA sequences associated with residual nuclei of Saccharomyces cerevisiae

We have used two different approaches to determine whether particular DNA sequences are specifically associated with high-salt-treated residual nuclei of Saccharomyces cerevisiae. First, libraries of yeast DNA in phage λ were probed with nick-translated total nuclear or residual nuclear DNA from unsynchronized yeast cells. None of the plaques gave a significantly stronger or weaker signal with the residual nuclear probe than with the total nuclear probe. Second, DNA was purified from whole nuclei or residual nuclei which had been isolated from cells in G1, G1/S, early S, or nuclear division. This DNA was “dot-blotted” and then probed with specific yeast DNA sequences. Ribosomal DNA was 2- to 3-fold enriched in residual nuclei in late G1, G1/S, and early S, and 2 μm plasmid DNA sequences were 3- to 5-fold depleted during nuclear division and early G1. However, ARS1, TRP1, CEN6, and a telomere sequence were neither enriched nor depleted at any time during the cell cycle.