Ancient DNA research goes nuclear. A new technique to extract sequence data from nuclear DNA may reveal exciting new insights into evolution and phylogeny.
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Animals↗
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Flow cytometric DNA analysis of parathyroid glands. Relationship between nuclear DNA and pathologic classifications.
Nuclear DNA contents of 95 paraffin-embedded parathyroid glands (2 carcinomas, 56 adenomas, 10 primary and 17 secondary chief cell hyperplasias, and 10 normal glands) were determined by flow cytometric analysis. All normal parathyroid glands and secondary hyperplasias, 80% of the primary hyperplasias, and 73% of the adenomas had diploid DNA patterns, with 15% or less tetraploid cells. Twenty-one percent of the adenomas, 29% of the primary hyperplasias, and all carcinomas had diploid and tetraploid DNA distribution patterns, with greater than 15% of the cells in the tetraploid region. One of the carcinomas (Figure 4B, Case 2) had an additional near-triploid aneuploid peak. Three of the adenomas (5.4%) had near-triploid aneuploid patterns. One of the patients with carcinoma (Figure 4A, Case 1) died, 32 months after initial diagnosis, of disease-related causes. The remaining patient with carcinoma (Case 2) had a 47-month disease-free interval. All of the patients with hyperplastic and adenomatous glands are free of disease, after a mean follow-up interval of 25 months. This study indicated that flow cytometric analysis of nuclear DNA content does not complement conventional pathologic methods in distinguishing between parathyroid gland chief cell hyperplasia, adenoma, or carcinoma; however, it did suggest the possibility that parathyroid adenomas and primary chief cell hyperplasias may contain a subset of tumors that could manifest biologic malignancy if allowed to progress untreated.
Adolescent↗
Sequence organization of animal nuclear DNA.
Animal nuclear genomes contain DNA sequences of various degrees of repetition. These sequences are organized in highly ordered fashions; repetitive and nonrepetitive sequences either alternate in short periods, i.e., short [0.2-0.4 kilobases (kb) long] repeats are flanked by nonrepetitive sequences less than 2 kb long, or in longer periods, with repetitive and/or nonrepetitive sequences extending for several kilobases. There are two main categories of genome organization, namely those exhibiting short-period interspersion and those that do not. There are arguments for and against a regulatory role of short interspersed repetitive sequences. Besides the merely 'statistical' kinetic approach by conventional reassociation kinetics, sequence organization has been studied by restriction endonuclease mapping and nucleotide sequencing. Such studies have revealed some general features of the organization of the eukaryotic gene and its transcripts, namely possible 'promoters', 'leaders', 'introns', 'exons', 'flanking sequences', 'caps', ribosome-binding sites, and poly(A) sequences. This paper discusses how these elements of a gene might serve regulatory roles in its expression.
Animals↗
A comparative study on hepatocellular carcinoma between South Africans and Japanese from the viewpoint of nuclear DNA content.
Nuclear deoxyribonucleic acid (DNA) content of hepatocellular carcinoma (HCC) in 41 South African and 47 Japanese patients at autopsy was analysed by dual-wavelength microspectrophotometry. The DNA distribution patterns were classified as type I, II, III or IV and as low ploidy (types I, II) or high ploidy (types III, IV), according to the degree of dispersion. We found a significantly higher incidence of high ploidy in South African HCC than in Japanese HCC. Moreover, type IV was significantly more frequent among South Africans than among the Japanese. These findings demonstrate that large differences in biological characteristics and clinical behaviour of HCC between South Africa and Japan may reflect differences in DNA distribution patterns which we observed between these two races.
Adolescent↗
In situ microfluorometry of whole kinetoplast and nuclear DNA in a single cell of Trypanosoma gambiense and Trypanosoma cruzi.
In situ microfluorometry of whole kinetoplast DNA (K-DNA) and nuclear DNA (N-DNA) from a single trypomastigote cell of Trypanosoma gambiense (strain Wellcome) and Trypanosoma cruzi (strain Tulahuen) was attempted by using the microfluorometer combined with a photon counter. The results obtained were summarized as follows; 1. The K-DNA value of 2K 1N (2 kinetoplasts and 1 nucleus) T. gambiense was about twofold as much as that of 1K 1N on. 2. The N-DNA value of 2K 1N T. gambiense was a little less than two times as much as that of 1K 1N T. gambiense, showing that the N-DNA had already bee increased nearly twofold before the nuclear division was initiated. 3. The K-DNA value of 1K 1N T. cruzi was about fivefold as much as that of 1K 1N T. gambiense. The N-DNA value was contrarily lower than that of T. gambiense, being approximately 70% of the latter. 4. Percent K-DNA/N-DNA value was, in average, about 7 in either 1K 1N or 2K 1N T. gambiense and about 50 in T. cruzi. 5. Percent K-DNA/N-DNA value in individual trypomastigote cell was variable, showing that the increase of K-DNA and N-DNA was not synchronized at least in the two species of trypanosomes used here. 6. The fact that K-DNA and N-DNA values varied from cell to cell although they do not divide, suggested the possibility of DNA increase in the trypomastigote of T. cruzi.
Animals↗
The localization of nuclear DNA helicase II in different nuclear compartments is linked to transcription.
Nuclear DNA helicase II (NDH II) is a member of the DEAH superfamily of helicases and functions as a pre-mRNA- and mRNA-binding protein in human cells. Here we report for the first time that human NDH II is associated with the nucleolus of transformed and nontransformed cells as shown by immunofluorescence and by ultrastructural studies. When RNA polymerase II (POL II) transcription is inhibited, NDH II highly accumulates in the nucleolus and shows predominant association with subdomains in DFC and in a portion of GC attached to DFC. Furthermore, these subdomains completely co-localize with mRNA-binding protein TLS. In addition, we show that nucleolar accumulation of NDH II is closely related to G(0)-phase growth arrest in human fibroblasts. Thus, the nucleolar localization of NDH II depends upon the metabolic state of the cell. Based on the data we propose that NDH II operates in both nucleoplasmic and nucleolar mode, and that its redistribution reflects accumulations indicating a possible cycling of NDH II between nucleoplasm and the nucleolus. The nucleolus can serve as a temporary storage or recycling center for NDH II. Possible functions of NDH II in pre-rRNA biogenesis, or in nucleolar mRNA metabolism, are also discussed.
Adenosine Triphosphatases↗
[Malignancy grading in breast cancer as assessed by nuclear DNA content].
The relationship between nuclear DNA content measured by cytofluorometry and the prognostic factors, especially the histological lymph node metastasis was investigated in patients with breast cancer. Nuclear DNA content was measured in 82 cases to evaluate its clinical significance concerning the malignancy grading. Histograms of DNA content were classified into two basic ploidy patterns based on presence or absence of a prominent peak at the 2c (diploid) region. D type (diploid type) which had a prominent peak at the 2c region resembled the histogram pattern of normal cells more closely than N type (non-diploid type). The rate of lymph node metastasis of N type (57.4%) was significantly higher than that of D type (28.6%) (p less than 0.05). The rate of lymph node metastasis of N type was higher than D type regardless of the tumor size or histological types. The five year cumulative survival rate of D type (91.7%) was significantly higher than that of N type (68.2%) (p less than 0.05). Despite the lymph node metastasis, the survival rate of D type was higher than that of N type. From a view point of the relationship between nuclear DNA content and lymph node metastasis, breast cancers of D type might be much safer indications for modified radical mastectomy. From this study, it was suggested that the measurement of nuclear DNA content of the breast cancer would bring the important informations about the malignancy grading and decision of the operative procedure.
Adult↗
Nuclear DNA density of parathyroid lesions.
Nuclear DNA density analysis was carried out on six normal control human parathyroid glands and forty-six pathological lesions of human parathyroid tissue. The pathological glands had originally been classified as twenty hyperplasias, twenty-one adenomas, and five carcinomas in accordance with clinical-pathological information. Specimens had been obtained through the Surgical Pathology Department of the University Hospital, London, in the form of either imprints from the cut surface of a lesion, or as cell separation samples from a paraffin block. Nuclear DNA ploidy analysis of the normal glands showed, as expected, a diploid cell population. Sixty-one percent of nuclei of the carcinomas were above 2C, extending to 7C. There was a significant difference in nuclear DNA content between normal and benign tissue versus tissue classified as carcinoma. No significant difference in nuclear DNA ploidy could be established between the benign adenoma and hyperplasia tissue; however, marked significant DNA ploidy differences were found between tissue from the latter two and carcinoma. DNA ploidy from cell image analysis using Feulgen's reaction can be useful in establishing a differential diagnosis between carcinoma and benign lesions.
Adenocarcinoma↗
Evidence that protein disulfide isomerase (PDI) is involved in DNA-nuclear matrix anchoring.
DNA-nuclear matrix (NM) anchoring plays a critical role in the organization of DNA within the nucleus and in functional access to DNA for transcription, replication, and DNA repair. The cellular response to oxidative stress involves both gene expression and DNA repair. We, therefore, determined if changes in the oxidative-reductive environment can affect DNA-NM anchoring. The present study used two approaches to study the effect of the reducing agent DTT on DNA-NM anchoring. First, the relative stringency of the DNA-NM attachment was determined by measuring the ability of NM attached DNA loops to undergo supercoiling changes. Second, the effects of DTT on the association of nuclear proteins with DNA were determined by cisplatin crosslinking. When nucleoids (nuclear matrices with attached DNA loops) were prepared from HeLa cells with 1 mM dithiothreitol (DTT), supercoiled DNA loops unwound more efficiently compared with control in the presence of increasing propidium iodide (PI) concentrations. In addition, the rewinding of DNA supercoils in nucleoids treated with DTT was inhibited. Both effects on DNA supercoiling ability were reversed by diamide suggesting that they are dependent on the oxidation state of the protein thiols. When DTT treated nucleoids were isolated from gamma-irradiated cells, the inhibition of DNA supercoil rewinding was equal to the sum of the inhibition due to DTT and gamma-rays alone. Nucleoids isolated from heat-shocked cells with DTT, showed no inhibition of DNA rewinding, except a small inhibition at high PI concentrations. Nuclear DNA in DTT-treated nuclei was digested faster by DNase I than in untreated nuclei. These results suggest that DTT is altering DNA-NM anchoring by affecting the protein component(s) of the anchoring complex. Extracting NM with increasing concentrations of DTT did not solubilize any protein to a significant extent until measurable NM disintegration occurred. Therefore, we determined if 1 mM DTT affected the ability of 1 mM cisplatin to crosslink proteins to DNA. Isolated nuclei were treated with 1 mM DTT for 30 min or left untreated prior to crosslinking with 1 mM cisplatin for 2 h at 4 degrees C. The ability of capsulation to crosslink DNA to proteins per se, did not appear to be affected by 1 mM DTT because relative amounts of at least four proteins, 69, 60, 40, and 35 kDa, were crosslinked to DNA to the same extent in DTT-treated and untreated nuclei. However, protein disulfide isomerase (PDI) crosslinked to DNA in untreated nuclei, but did not crosslink DNA in nuclei that were treated with 1 mM DTT; 1 mM DTT did not affect the intranuclear localization of PDI. Thus, DTT appears to alter the conformation of PDI, as suggested by the DTT-induced change in DNA association, but not its NM association. These results also imply that DNA-NM anchoring involves the redox state of protein sulfhydryl groups.
Cisplatin↗
Stage C prostatic adenocarcinoma: flow cytometric nuclear DNA ploidy analysis.
Flow cytometric nuclear DNA ploidy analysis was used to study pathologic stage C prostatic adenocarcinoma (pT3, N0, M0) in 146 patients who underwent radical retropubic prostatectomy and bilateral pelvic lymphadenectomy between 1967 and 1981. Of these tumors, 46% had a DNA diploid pattern, 47% had a DNA tetraploid pattern, and 7% had a DNA aneuploid pattern. Abnormal ploidy patterns were associated more frequently with histologic high-grade tumors than with low-grade tumors. Considered alone, DNA ploidy pattern showed a strong association with subsequent prognosis. The median interval to progression for tumors with DNA tetraploid and DNA aneuploid patterns was 7.8 and 3.5 years, respectively. For the DNA diploid tumors, only 23% progressed within 18 years, the longest follow-up. At 10 years, only 10% of patients with DNA diploid tumors had died of prostatic cancer, in comparison with 28% of the DNA tetraploid and 36% of the DNA aneuploid groups (P less than 0.01). By analysis of a combination of histologic tumor grade and nuclear DNA ploidy pattern, an even stronger association with prognosis was demonstrated. For the 38 patients with histologic low-grade and DNA diploid tumors, progression-free survival was 92% at 10 years, in comparison with 57% for 23 patients with low-grade DNA nondiploid tumors. Patients with high-grade tumor had a poorer prognosis whether the DNA ploidy pattern was diploid or nondiploid. Nuclear DNA ploidy pattern is an important and independent prognostic variable for patients with pathologic stage C prostatic cancer treated by radical prostatectomy.
Adenocarcinoma↗
Flow cytometric analysis of the nuclear DNA content of hepatoblastoma.
The nuclear DNA content of 15 hepatoblastoma cases was determined in paraffin-embedded tissues by flow cytometry. The DNA index (DI) was calculated, and the ploidy pattern of nuclear DNA was estimated. The correlation between the ploidy pattern and clinicopathologic findings was studied, and the prognostic significance of the ploidy pattern was investigated. An aneuploid pattern was seen in 50% of the lesions with histologic embryonal and anaplastic types. It was not seen in the fetal type. In the tumors with combined epithelial components, the fetal-type component had a diploid pattern in all five cases. The embryonal-type component was associated with aneuploidy in two of five cases. In aneuploid tumors, vascular invasion (tumor emboli in the vessels) was observed more frequently. The prognosis of the patients with an aneuploid tumor was significantly poorer. These results indicate that nuclear DNA ploidy pattern analysis might be useful in investigating the prognosis of hepatoblastoma.
Carcinoma, Hepatocellular↗
Organisation of inverted repeat sequences in hamster cell nuclear DNA.
Hamster cell nuclear DNA is shown to contain inverted repeat (foldback) sequences, in some respects similar to the foldback fraction in DNA from other animal cell types. Using electron microscopy the majority of foldback duplexes are shown to be located in simple hairpin-like DNA structures, formed from individual pairs of complementary inverted repeated sequences 50--1000 nucleotides in length, in some cases arranged in tandem, and in other cases separated by intervening sequences, up to 16000 nucleotide residues long. In addition, a novel class of foldback structure, referred to as 'bubbled hairpins' is reported, which appear to be formed from clusters of inverted repeat sequences that are separated from adjacent clusters of complementary inverted repeats by large intervening sequences which vary in length from 5000 to over 20000 nucleotide residues. Due to the special pattern of distribution of these latter inverted repeat sequences, 'bubbled hairpins' are observed only in long foldback DNA. Evidence is presented that the distribution of foldback sequences in hamster cell DNA is highly ordered. The lengths of the intervening single chains in foldback structures appear to vary non-randomly. This gives rise to a localised periodic pattern of organisation that is believed to be a consequence of regular alternating arrangements of foldback and non-foldback sequences in the segments of DNA from which foldback structures are derived.
Animals↗
Nuclear DNA helicase II unwinds both DNA and RNA.
Nuclear DNA helicase II (NDH II) has been purified to near-homogeneity by exploiting its high affinity to poly[(rI).(rC)]-agarose. The purified enzyme was obtained as two catalytically active forms of 130- and 100-kDa molecular mass, respectively. After treatment with cyanogen bromide, the separated polypeptides displayed very similar digestion patterns. Thus, the 100-kDa form most likely is a proteolytic product of the 130-kDa polypeptide. For DNA unwinding, NDH II could use any of the four rNTPs or dNTPs with Km values between 20 and 100 microM. DNA unwinding was stimulated up to 20-fold by substrates that contained single-stranded 3'-tails. NDH II-catalyzed DNA unwinding was strongly inhibited by RNA, but was little affected by DNA. The strongest RNA inhibitor, poly[(rI).(rC)], was also the strongest effector of the NTPase activity of NDH II. The binding constant for poly[(rI).(rC)] binding was about 2 x 10(7) M-1; the minimal binding site size was determined as 16 nucleotides. In agreement with its high affinity to RNA, NDH II unwound double-stranded RNA. RNA unwinding required the presence of a nucleoside triphosphate and a divalent cation (Mg2+). Thus, like the prototypic replicative helicase large T antigen of simian virus 40, NDH II may function in both DNA and RNA unwinding.
Adenosine Triphosphatases↗
Heterogeneity of centronuclear myopathy shown by flow cytometry of nuclear DNA.
Determination of nuclear DNA by flow cytometry of skeletal muscle biopsies of three unrelated male patients with centronuclear (myotubular) myopathy demonstrated, in two patients, a normal pattern and, in one case, a near haploid DNA content in c. 60% of all nuclei, indicating muscle tissue mosaicism.
Adult↗
Mitochondrial DNA synthesis in mouse L cells temperature sensitive in nuclear DNA replication.
Temperature-sensitive (ts) A 1S9 mouse L cells continue to synthesize double-stranded covalently closed mitochondrial (mt) DNA at a temperature (38.5 degrees C) which is nonpermissive for chromosomal DNA replication. The amount of mt DNA made appears to be quantitatively linked to nuclear DNA synthesis. Nuclear DNA replication proceeds normally for 6-8 h after the cells are shifted to 38.5 degrees C, and then declines to reach a minimum at 20-24 h. The level of mt DNA synthesis remains high during this period and decreases once the ts lesion has been established.
Animals↗
Mapping sequences in loops of nuclear DNA by their progressive detachment from the nuclear cage.
Nuclear DNA is organised into loops, probably by attachment to a supramolecular structure. We describe a method which enables us to map the position of sequences within a loop relative to the point of attachment. Nuclear DNA is isolated unbroken by lysing HeLa cells in 2M NaCl to release structures which retain many of the morphological features of nuclei. Their DNA is supercoiled and so must remain unbroken and looped during lysis. Nucleoids are digested to various degrees with a restriction endonuclease and the cages - and any associated DNA - sedimented free from unattached DNA. The cage-associated DNA is purified and completely fragmented using the same restriction endonuclease. Equal weights of fragmented DNA are separated by gel electrophoresis, transferred to a filter and the relative amounts of the alpha, beta and gamma globin genes on the filter determined by hybridisation to the appropriate probes. The alpha genes, unlike the beta and gamma genes, resist detachment from the cage and so must lie close to the point of attachment to the cage. Our ability to map these genes implies that sequences cannot be attached at random to the cage; rather, specific sequences must be attached, so looping the DNA.
Base Sequence↗
The prognostic significance of nuclear DNA content in chondrosarcoma.
The nuclear DNA content in 48 chondrosarcomas and 4 chondromas was determined. Grading according to O'Neal-Ackerman was also performed. All chondromas were found to be diploid (normal nuclear DNA content). Twenty-two of the chondrosarcomas showed the same DNA distribution curve as the chondromas and were characterized as diploid, while 26 were found to be hyperploid (increased nuclear DNA content). A comparative study of grade and ploidy in relation to ten-year survival and metastasis rates showed that ploidy determinations in chondrosarcomas give significant prognostic information, even better than that obtained by conventional histopathologic grading. In this study, diploid chondrosarcomas were found to be associated with a significantly more favorable clinical course than were hyperploid chondrosarcomas. The prognostic relevance of these ploidy determination was further confirmed by analyzing the ploidy level of selected cases characterized bydifferent clinical courses. Thus, the tumors from 13 (93%) of 14 patients with a highly malignant clinical course (death within two years) and the tumors at the primary site in 17 (81%) of 21 metastasizing chondrosarcomas were found to be hyperploid. Direct measurements on four metastases revealed that all were hyperploid.
Adolescent↗
Fluorescence cytophotometric measurements of nuclear DNA in adenocarcinoma of the lung; relation of proliferative activity and DNA ploidy to prognosis.
Nuclear DNA measurements on specimens of adenocarcinomas of the lung obtained at surgery from 22 patients showed that in 8 tumors the cells were near diploid and slowly proliferating. These 8 tumors displayed a higher degree of histopathologic differentiation than did the remaining 14 adenocarcinomas. In 3 of the 14 more poorly differentiated tumors the cells were also near diploid but had more S-phase DNA values, indicating increased proliferative activity. In 11 tumors aneuploid DNA stem line or lines were noted. Among patients with slowly growing near-diploid tumors the frequency of tumor recurrence and/or metastases (1 of 8) was lower than among those with more rapidly proliferating near-diploid tumors and aneuploid tumors (9 of 14). Nuclear DNA histogram patterns yielded information about the tumor proliferation activity, modal DNA value and occurrence of aneuploid DNA values. Furthermore, prognostic information about the likelihood of tumor recurrence after surgical treatment was obtained. This information can be utilized in the choice of therapy and in prognostic evaluation.
Adenocarcinoma↗