Showing papers on "Nuclear DNA published in 1988"

Normal oxidative damage to mitochondrial and nuclear DNA is extensive.

Abstract: Oxidative damage to DNA can be caused by excited oxygen species, which are produced by radiation or are by-products of aerobic metabolism The oxidized base, 8-hydroxydeoxyguanosine (oh8dG), 1 of approximately 20 known radiation damage products, has been assayed in the DNA of rat liver oh8dG is present at a level of 1 per 130,000 bases in nuclear DNA and 1 per 8000 bases in mtDNA Mitochondria treated with various prooxidants have an increased level of oh8dG The high level of oh8dG in mtDNA may be caused by the immense oxygen metabolism, relatively inefficient DNA repair, and the absence of histones in mitochondria It may be responsible for the observed high mutation rate of mtDNA

Activation of programmed cell death in the rat ventral prostate after castration.

Abstract: 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.

Replicative intermediates of hepatitis B virus in HepG2 cells that produce infectious virions.

Abstract: Clonal cells derived from HepG2 cells transfected with a plasmid containing hepatitis B virus (HBV) DNA secrete hepatitis B surface antigen particles, nucleocapsids, and virions (M. A. Sells, M.-L. Chen, and G. Acs, Proc. Natl. Acad. Sci. USA 84:1005-1009, 1987) which elicit acute hepatitis in chimpanzees (G. Acs, M. A. Sells, R. H. Purcell, P. Price, R. Engle, M. Shapiro, and H. Popper, Proc. Natl. Acad. Sci. USA 84:4641-4644, 1987). We report here the initial characterization of the viral nucleic acids produced in this culture system. Kinetic analyses of nuclear, cytoplasmic, and extracellular HBV DNAs were performed by Southern blotting with radiolabeled HBV strand-specific probes. The results from these analyses indicate that at the stationary cellular growth phase, there is a dramatic increase in the rate at which HBV DNA accumulates. Incomplete double- and single-stranded forms of the HBV genome were detected in the nuclear and cytoplasmic fractions as well as in the extracellular medium. In addition, the nuclear DNA apparently includes multiple complete copies of the HBV genome chromosomally integrated and full-length covalently closed circular HBV DNA. Multiple HBV-specific polyadenylated RNAs with lengths of 3.5, 2.5, and 2.1 kilobases were identified by Northern (RNA) blot analysis. S1 nuclease mapping and primer extension identified a single 3' end and multiple unique initiation sites corresponding to nucleotides just 5' to the pre-S1 region, as well as upstream and within the pre-S2 and precore regions. The nucleic acid profile obtained from these analyses is essentially a facsimile of that obtained by studying liver tissue from HBV-infected individuals.

Studies of human mitochondria. Steady-state levels and metabolic properties of the mitochondrial tRNAs. Injection of mitochondria into human cells leads to a rapid replacement of the endogenous mitochondrial DNA

Abstract: The steady-state levels and metabolic properties of mitochondrial tRNAs have been analyzed in Hela cells and correlated with the function of the tRNAs for organelle-specific protein synthesis. DNA excess hybridization experiments utilizing separated strands of mitochondrial DNA (mtDNA) and purified tRNA samples from exponential cells long-term labeled with [^(32)P] orthophosphate have revealed a steady-state level of 6 x 10^5 tRNA molecules per cell, with threefourths being encoded in the heavy (H)-strand and one-fourth in the light (L)-strand. Hybridization of the tRNAs with a panel of M13 clones of human mtDNA containing, in most cases, single tRNA genes and a quantitation of two dimensional electrophoretic fractionations of the tRNAs have shown that the steady-state levels of tRNA^F and tRNA^V are two to three times higher than the average level of the other H-strand-encoded tRNAs and three to four times higher than the average level of the L-strand-encoded tRNAs. Similar experiments carried out with tRNAs from cells labeled with very short pulses of [5-^3H] uridine have indicated that the rates of formation of the individual tRNA species are proportional to their steady state amounts. Therefore, the 15-fold to 60-fold higher rate of transcription of the tRNA^V and tRNA^F genes (transcribed with the rDNA transcription unit) relative to the other H-strand tRNA genes (transcribed with the whole H-strand transcription unit) and the 13-fold to 20-fold higher rate of transcription of the L-strand tRNA genes relative to the H-strand tRNA genes (other than tRNA^V and tRNA^F genes) are not reflected in the rates of formation of the corresponding tRNAs. The available data indicate that the majority of tRNA^V and tRNA^F transcribed from the rDNA transcription unit are degraded as they are excised from the primary transcripts. It also seems likely that the majority of the L-strand-encoded tRNAs are degraded before they are excised from the short-lived polycistronic transcripts. Furthermore, a role of the aminoacyl tRNA synthetases in stabilizing the different tRNA species at relatively uniform levels is suggested. A comparison of the steady-state levels of the individual tRNAs with the corresponding codon usage for protein synthesis, as determined from the DNA sequence and the rates of synthesis of the various polypeptides, has not revealed any significant correlation between the two parameters. In other experiments, isolated human mitochondria containing a mitochondrial DNA (mtDNA)-coded chloramphenicol resistance marker were injected at an average dose of less than one into sensitive human cells partially depleted of their mtDNA by ethidium bromide treatment. Under selective conditions, the mitochondria became established in the recipient cells with a frequency greater than 2 to 3 x 10^(-3). A rapid and, in some cases, complete replacement of the resident mtDNA by the exogenous mtDNA took place in the transformants, as shown by multiple mtDNA and nuclear DNA polymorphisms. Intracellular mtDNA selection played a crucial role in this replacement, with significant implications for mitochondrial genetics.

Activation of a Ca2+-Mg2+-dependent endonuclease as an early event in castration-induced prostatic cell death.

Abstract: Previous studies have demonstrated that castration-induced androgen withdrawal results in the fragmentation of prostatic DNA into nucleosomal oligomers, and this process comprises an early event in the activation of programed cell death in the rat ventral prostate. This DNA fragmentation could be due to changes in the chromatin conformation increasing its sensitivity to preexisting nucleases and/or to increases in the activity of the nucleases themselves. However, comparative kinetic analysis of in vitro DNA fragmentation induced by exogenous nucleases did not reveal any differences in the sensitivity of prostatic chromatin between intact and castrated rats. In contrast to these negative findings, using [3H] DNA as an exogenous substrate, it was shown that within the first day following castration there was a twofold increase in a Ca2+-Mg2+-dependent nuclease activity without a concomitant increase in other nuclear nucleases. This Ca2+-Mg2+-dependent nuclease activation occurred coincidental with the initial increase in nuclear DNA fragmentation following castration and preceded the enhanced appearance of morphological changes characteristic of dying cells (i.e., apoptosis), as well as the major increase in prostatic DNA loss. These results suggest that castration-induced androgen deprivation leads to a sequential activation of a Ca2+-Mg2+-dependent nuclease leading to the fragmentation of the genome into discrete nucleosomal-sized fragments of DNA, subsequently followed by the fragmentation of the nucleus itself (i.e., apoptosis) and eventually with the complete digestion of the nucleosomal oligomers into component nucleotides (i.e., DNA loss). Since the castration-induced nuclease is dependent upon calcium ions for maximal activity, a potential role of intracellular calcium in the early events activating prostatic cell death was investigated. Acute disturbances in intracellular calcium homeostasis within the ventral prostate by means of a potent calcium influx blocker, nifedipine, simultaneous with castration, resulted in a significant delay in the biochemical and morphological changes associated with prostatic cell death (i.e., prostatic weight loss, prostatic DNA loss, and DNA fragmentation). These results point to a potential role of intracellular calcium levels in the mechanism of activation of castration-induced death of the androgen-dependent epithelial cells in the ventral prostate.

Prognostic significance of DNA measurements in 409 consecutive breast cancer patients.

Abstract: Four hundred nine consecutive breast cancer patients were studied retrospectively. Microspectrophotometric DNA measurements were performed using archival, fine-needle slide preparations upon which the primary diagnoses had been based 8 to 13 years earlier. The DNA distribution patterns of the tumor cell populations were analyzed according to various criteria and the cytochemical data were correlated to the clinical course, defined as distant recurrence-free survival. The results demonstrated a strong relationship between nuclear DNA content of the breast cancer cells and prognosis. Tumors exhibiting DNA values within the limits of normal tissues (DNA euploidy) were found to be correlated with a favorable prognosis. In contrast, tumors with increased and scattered DNA values (DNA aneuploidy) were found indicative of poor prognosis. This was found to be the case regardless whether the percentage of cells above 2.5c or 5c, DNA index/modal value, or the histogram typing according to Auer et al were utilized to discriminate low-grade from high-grade malignant cases. All of these DNA variables were also shown to be significantly correlated. With the aid of the Cox regression method, the additional prognostic value of any given variable was tested against the others. The statistical analyses showed that the histogram typing gives significant prognostic information in addition to that provided by any other variable. In conclusion, the current study demonstrates that tumor nuclear DNA content is a strong indicator of prognosis in patients suffering from invasive breast adenocarcinoma. However, the results also show that simple determination of the stemline position is not the optimal DNA measure of intrinsic tumor malignancy potential. The fraction of cells scattered outside the modal peaks of the histograms are of utmost importance for adequate cytochemical malignancy grading in breast carcinomas.

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

Abstract: 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.

Predictive value of nuclear DNA content in breast cancer in relation to clinical and morphologic factors. A retrospective study of 227 consecutive cases

Abstract: The predictive value of nuclear DNA content in breast cancer in relation to clinical and morphologic factors was studied in 227 consecutive cases of invasive breast adenocarcinomas with follow-up periods of 8 to 13 years. The results show that, with the use of Cox multivariate analysis nuclear DNA content provided significant prognostic information additional to that given by all other clinical and histomorphologic variables taken together. This fact indicates that the DNA content of breast cancer cells reflects biological properties, associated with the malignant behavior of the tumor, other than those determining the stage of the disease. Nuclear DNA content was strongly correlated to histopathologic grading of the ductal carcinomas, with poorly differentiated tumors more likely to be aneuploid. On the other hand, no clear correlation was found to exist between nuclear DNA content and axillary node status, indicating that these two factors are independent prognostic parameters. It is noteworthy that DNA content provided additional prognostic information within both the node-negative and node-positive patient groups. In summary, the results shown here indicate that nuclear DNA content, as an objective biological marker of tumor aggressiveness, can significantly improve our prognostic capabilities within the currently designated stages.

Flow cytometric DNA analysis of neuroblastoma and ganglioneuroma. A 10-year retrospective study.

Abstract: Retrospective quantitative DNA analysis was done on 147 samples from 89 patients with neuroblastoma and ganglioneuroma using flow cytometry. In the neuroblastoma patients, nuclear DNA content was found to be a stable tumor marker irrespective of site (primary versus metastatic) and despite changes with time in tumor progression, maturation, or therapy. The occurrence of DNA aneuploidy, which was detected in 60% of the neuroblastoma patients, paralleled other favorable indicators and was highly associated with survival (P less than 0.001). Of clinical stage, age, primary site, sex, and DNA content, only stage and DNA content correlated with survival. Those patients with favorable stage and DNA aneuploidy had higher survival rates. Further, favorable stage and the presence of DNA aneuploidy were independent prognostic indicators. Abnormal DNA content was also detected in samples from ganglioneuromas in which significant numbers of ganglion cell nuclei were recovered. These results indicate a striking difference between neuroblastoma and adult tumors in which DNA aneuploidy is generally a poor prognostic sign and provide a molecular link between ganglioneuromas and their malignant counterparts.

7-Methylguanine adducts in DNA are normally present at high levels and increase on aging: analysis by HPLC with electrochemical detection.

Abstract: The 7-methylguanine adduct in the DNA of rat liver is determined as an indicator of exposure to exogenous and endogenous methylating agents. A method for the analysis of 7-methylguanine adducts has been developed by combining the selectivity of separation of reversed-phase HPLC with the specificity and high sensitivity of electrochemical detection. The sensitivity of the method is about 10,000-fold that of optical methods and is sufficient to determine the endogenous background of DNA methylation. DNA from the liver of normal young rats (6 months old) contains 7-methylguanine at a level of 1 residue per 31,000 bases in mitochondrial DNA and 1 residue per 105,000 bases in nuclear DNA. These levels increase about 2.5-fold in old rats (24 months old). We attribute this strikingly high level of adducts to endogenous methylation, which could contribute to aging and cancer.

Repair of alkylated purines in the hepatic DNA of mitochondria and nuclei in the rat

Abstract: Further evidence for the preferential interaction of carcinogens with mitochondrial DNA (mtDNA) has been obtained. In rats treated with high doses of N-nitrosodimethylamine (NDMA) or N-nitroso-N-butylurea (NBU), hepatic mtDNA contains 1.4 times more O6-methyl-2'-deoxyguanosine (O6-MedG) or 2.3 times more O6-butyl-2'-deoxyguanosine (O6-BudG) than does nuclear DNA (nDNA). The kinetics of removal of O6-MeG from mtDNA and nDNA are similar at both high (20 mg/kg) and low (2 mg/kg) doses of NDMA, and the removal of O6-MeG can be increased by pretreating the animals with 2-acetylaminofluorene (AAF), indicating that O6-MeG is repaired in the mitochondrion by a mechanism similar to that which functions in the nucleus. In contrast, O6-BudG is removed very slowly from mtDNA and rapidly from nDNA, an observation which is consistent with the absence of a nucleotide excision mechanism in the mitochondrion and the repair of O6-BudG, predominantly by an excision mechanism, in the nucleus of mammalian cells. A 23-kd methyltransferase (MT) protein, similar to the one found in the nucleus, has been isolated from hepatic mitochondria and is present in mitochondria from which the outer membrane has been removed. It is suggested that O6-MeG, but not O6-BuG can be repaired from mtDNA by a MT protein that is nuclear encoded and transported across the mitochondrial membrane.

A head-to-tail tandem organization of hsp70 genes in Trypanosoma cruzi

Abstract: We describe the isolation and characterization of the T. cruzi hsp 70 DNA coding region which was found to be formed by multigene copies organized in a tandem array in a head-to-tail manner. The restriction pattern of one of the repetition units within the largest clone obtained from the genomic library, clone Tc70.6, shows that the hsp70 coding region should be formed by at least seven identical copies of 2.5 kb. We have found, however, the presence of restriction polymorphisms (Pvu II) within these repeats. Subsequent analysis of the time course of nuclear DNA digestion has revealed that the copy number per haploid genome could be as greater as 10. The analysis of the DNA and amino acid sequence of a fragment (70%) of one of the repetition units has shown the existence of a high homology with all the hsp70 genes of other organisms. The protein sequence homology of the fragment analyzed is as high as 88% when compared with that of the T. brucei hsp70. On the other hand, there are significant restriction site variations between both. The T. cruzi hsp70 contains at the C-terminal end a tetrapeptide repeat of the structure (GMPG)9.

Preferential synthesis of plastid DNA and increased replication of plastids in cultured tobacco cells following medium renewal.

Abstract: During the culture of tobacco BY 2 cells derived from Nicotiana tabacum L. cv. Bright Yellow 2, morphological changes of plastid (pt) nucleoids and their replication were examined by fluorescence microscopy after staining with 4′6-diamidino-2-phenylindole. Upon transfer to fresh medium, the fluorescence intensity originating from pt nucleoids increased markedly. Copy numbers of ptDNA per cell calculated from the quantitative data by super-sensitive microspectroscopy increased 11-fold within 1 d of culture to reach 11 000, then decreased gradually to 1 000 after one week of culture. Autoradiography by labelling with [3H]thymidine showed that DNA synthesis in plastids occurred exclusively during the first day of culture, whereas nuclear DNA synthesis was observed from the first to the sixth day of culture. Replication of plastids was most frequently observed on the second day. Thereafter the formation of starch granules predominated in plastids up to the fifth day of culture, but the starch granules disappeared in the stationary-phase cells. The meaning of such preferential synthesis of ptDNA upon transfer to fresh medium is discussed in relation to the interaction between plastids and nuclei.

Correlation between flow cytometric determination of nuclear DNA content and chromosome number in somatic hybrids within Brassicaceae.

Abstract: Fourty-one somatic hybrids from two species combinations, Brassica oleracea + B campestris and B napus + Eruca sativa, were analysed for chromosome number and nuclear DNA content The DNA content was measured in a flow cytometer using two internal standards as references and when related to the chromosome number a correlation of 091 was found The chromosome number of the hybrids could be determined with an accuracy of ±10% by using flow cytometry, and the smallest statistically significant difference in DNA content between two individuals was 023 pg DNA/cell

Single-copy DNA-DNA hybridizations among five species of Laminaria (Phaeophyceae): Phylogenetic and biogeographic implications

Abstract: DNA-DNA hybridizations between single-copy nuclear DNA fromLaminaria digitata and total DNA fromL. saccharina, L. Hyperborea, L. rodriguezii, L. ochroleuca andChorda filum, respectively, show that these species ofLaminaria are genotypically closely related.Chorda filum is only distantly related withL. digitata. Based on the thermal elution patterns of the DNA hybrids, as quantified by ΔTm(e) values, it is hypothesized that all five species ofLaminaria evolved at about the same time from their most recent common ancestor some 15–19 Ma ago. This phylogenetic hypothesis is discussed in relation to the history of modern laminarialean distribution patterns.

Transmission of gametic nuclei through a fertilization tube during mating in a primitive dinoflagellate, Prorocentrum micans Ehr

Abstract: In the dinoflagellate Prorocentrum micans Ehr., changing the culture medium (switching from Erdschreiber9s to Provasoli9s medium) provokes the appearance of individuals that are morphologically different from the normal vegetative forms. Observations and microspectrofluorimetric measurements made in vivo of the relative amounts of nuclear DNA show that these forms are sexual forms; unlike the situation in dino-flagellate species that are known to be sexual, the male and female gametes of P. micans do not fuse. Cells playing the role of isogametes and containing q DNA pair and form a fertilization tube by means of which a donor cell (♂) injects its nucleus into a recipient cell (♀). After conjugation, the zygote containing 2 q DNA replicates and thereafter contains 4 q DNA. Two successive meiotic divisions lead to the formation of a tetrad in which each nucleus contains q DNA. Cells released from the tetrad seem to be adapted to Provasoli9s medium and vegetative divisions occur again. The characteristics of sexual reproduction in those dinoflagellates in which this phenomenon has been described are reviewed and discussed.

Selective loss of mitochondrial DNA after treatment of cells with ditercalinium (NSC 335153), an antitumor bis-intercalating agent.

Abstract: Ditercalinium (NSC 335153), a bifunctional intercalating molecule with antitumor activity, is found to express its toxicity through a mechanism of action completely different from that of other monointercalating agents. Electron microscopic observation of ditercalinium-treated cells shows a drastic alteration of mitochondrial structure. Cells deficient in mitochondrial respiration (GSK3 cells) isolated by A. Franchi et al. (Int. J. Cancer, 27: 819–827, 1981) are about 25-fold more resistant than cells deficient in glycolysis (DS7 cells) isolated by J. Pouyssegur et al. (Proc. Natl. Acad. Sci. USA, 77: 2698–2701, 1980). Revertants have been isolated from GSK3 cells. In these cells, the sensitivity to ditercalinium has been recovered with mitochondrial respiration. Ditercalinium treatment of L1210 leukemic mouse cells leads to a specific elimination of mitochondrial DNA detected by DNA-DNA hybridization. No measurable alteration of nuclear DNA is observed. In contrast, the monomeric analogue of ditercalinium only alters nuclear DNA and does not change the mitochondrial DNA content. The activity of cytochrome c oxidase, an enzyme which contains a subunit coded by the mitochondrial DNA, decreases exponentially in treated cells with a half-life of 24 h, corresponding to the turnover of the enzyme. These results suggest that ditercalinium exerts a specific cytotoxic effect at the level of mitochondrial DNA. This action could account for the delayed cytotoxicity induced by this compound.

DNA amounts and chromatin compactness in Vicia

Abstract: 2C DNA amounts and areas of chromatin were determined with a M 86 Vickers microdensitometer in 56 species of Vicia (x=5, 6, 7), exhibiting large differences in chromosome size. There were significant differences between the species both in DNA content and chromatin area. The nuclear DNA amounts range from 3.85 to 27.07 pg. DNA distribution appears discontinuous; species cluster into distinct groups and the average nuclear DNA amount separating each successive pair is approximately the same (2.23 pg). The compaction of DNA in interphase nuclei increases with increasing DNA amount, which is, at least partly, due to a disproportionate increase in the heterochromatin relative to the euchromatin component of DNA. Comparisons of DNA readings at various stages of the cell cycle show that the DNA amounts are underestimated by microdensitometry in nuclei with high DNA density. Estimation of relative DNA content and area of individual chromosomes were made in twelve species. The results show that changes in DNA content within chromosomes affect the degree of metaphase coiling in an orderly fashion.

Estimation of genetic divergence in meloidogyne mitochondrial DNA.

Abstract: Restriction fragments from purified mitochondrial DNA can be readily detected following rapid end-labeling with [alpha-(3)(2)]nucleoside triphosphates and separation by gel electrophoresis. Mitochondrial DNA from 12 populations of Meloidogyne species was digested with 12 restriction enzymes producing more than 60 restriction fragments for each species. The mitochondrial genome of M. arenaria is the most genetically distinct of the four species compared. M. arenaria shows approximately 2.1-3.1% nucleotide sequence divergence from the mitochondrial genomes of M. javanica, M. incognita, and M. hapla. Among the latter three species, interspecific estimates of sequence divergence range from 0.7 to 2.3%. Relatively high intraspecific variation in mitochondrial restriction fragment patterns was observed in M. hapla. Intraspecific variation in M. incognita resulted in sequence divergence estimates of 0.5-1.0%. Such polymorphisms can serve as genetic markers for discerning mitochondrial DNA genotypes in nematode populations in the same way that allozymes have been used to discern nuclear DNA genotypes.

Functional role of a highly repetitive DNA sequence in anchorage of the mouse genome

Abstract: The major portion of the eukaryotic genome consists of various categories of repetitive DNA sequences which have been studied with respect to their base compositions, organizations, copy numbers, transcription and species specificities; their biological roles, however, are still unclear A novel quality of a highly repetitive mouse DNA sequence is described which points to a functional role: All copies (approximately 50,000 per haploid genome) of this DNA sequence reside on genomic Alu I DNA fragments each associated with nuclear polypeptides that are not released from DNA by proteinase K, SDS and phenol extraction By this quality the repetitive DNA sequence is classified as a member of the sub-set of DNA sequences involved in tight DNA-polypeptide complexes which have been previously shown to be components of the subnuclear structure termed 'nuclear matrix' From these results it has to be concluded that the repetitive DNA sequence characterized in this report represents or comprises a signal for a large number of site specific attachment points of the mouse genome in the nuclear matrix

Constraints upon the organisation and evolution of chromosomes in Allium.

Abstract: In terms of chromosome morphology, karyotype organisation, taxonomy and genetic relationship as judged from chromosome pairing in the Fl hybrid, A. cepa and A.fistulosum are two closely related species. But large variation in nuclear DNA amounts has occurred during the evolution of the two species. A comparison of the molecular composition of DNA in the two species has confirmed that the excess DNA acquired during evolution was predominantly repetitive sequences (sequences which do not encode genetic information). However, its distribution within the chromosome complements was equal in all chromosomes irrespective of the differences in chromosome size. The even distribution of the excess DNA within complements suggests strong constraints underlying evolutionary changes in genome organisation. The nature of the constraints is discussed, and it is shown that such constraints can influence the direction of karyotype evolution during speciation.

Effects of Antileukemia Agents on Nuclear Matrix-bound DNA Replication in CCRF-CEM Leukemia Cells

Abstract: The effects of various antileukemic agents on DNA replication associated with the nuclear matrix were investigated in CCRF-CEM leukemia cells. Residual nuclear matrices were prepared by sequential treatment of nuclei with 1.5 m NaCl, DNase I, and Triton X-100 and contained 1–5, 10, and 37% of the total nuclear DNA, protein, and phospholipid, respectively. In control cells pulse-labeled for 45 s with [3H]thymidine, the specific activity of nascent DNA was four-fold greater in the nuclear matrix fraction relative to the specific activity of the high salt-soluble (nonmatrix) DNA fraction. Pulse-labeling and reconstitution experiments indicated that this enrichment of newly replicated DNA on the nuclear matrix did not result from aggregation of nascent DNA with the matrix. A 2-h incubation of tumor cells with either 0.1 µm teniposide (VM-26), 0.2 µm VM-26, or 0.5 µm amsacrine (m-AMSA) reduced the relative specific activity of nascent DNA on the nuclear matrix by 59, 61, and 54%, respectively, compared to control cells. In contrast hydroxyurea and cytosine arabinoside, at concentrations that markedly inhibited total nuclear DNA synthesis, did not decrease the relative specific activity of newly replicated DNA on the matrix. The results provide evidence that the antiproliferative effects of the DNA topoisomerase II inhibitors, VM-26 and m-AMSA, are localized on the nuclear matrix of CCRF-CEM leukemia cells.

Chromosome numbers, genome sizes, cell volumes and evolution of snake-head fish (family Channidae)

Abstract: Chromosome number, C-value and cell volume studies were carried out on three species of the genus Channa, viz., C. punctatus, C. striatus and C. gachua. The chromosome number, karyotypic structure and DNA content per cell along with cell volume are reported and described. A series of chromosomal rearrangements are established in three different karyotypes along with polyploidy. Both pericentric inversion and Robertsonian fusion played a major role in chromosome rearrangements. The nuclear DNA content of these three species is within 19-29% of the present-day placental mammals, and is thus lower than the median amount for fishes in general and teleosts in particular. Their lower DNA content suggests that the three species of the family Channidae are highly specialized, and this is supported by their known morphologic, reproductive, behavioural and ecological characteristics. The evolutionary significance of these chromosomal rearrangements, their origin and their mode of establishment are discussed. A probable phylogenetic model based on karyotype, C-value and chromosomal rearrangements of the genus is presented.

On the mechanism of cytoplasmic male sterility in the 447 line of Vicia faba.

Abstract: The trait of cytoplasmic male sterility, expressed in plants bearing the 447 cytoplasm of Vicia faba, is uniquely and positively correlated with the presence of a linear double-stranded RNA molecule (dsRNA) 16.7 kb in size. Restriction enzyme digestion profiles of mitochondrial DNA isolated from fertile and cytoplasmic malesterile (CMS) lines do show a limited number of specific differences in fragment intensities and mobilities. However, mitochondria isolated from the progeny of the cross CMS × Restorer line contain DNA with an identical restriction profile as the male-sterile parent: moreover, subsequent generations are completely and permanently fertile, even upon segregation of the nuclear restoration gene. Southern hybridizations, using cDNA clones as probes, reveal homology between the CMS-associated dsRNA and the nuclear genome of both sterile and fertile lines. The regions cloned, representing approximately 22% of the total dsRNA sequence, show no homology to organelle DNA. We have not been able to stably transmit the dsRNA to fertile lines of V. faba or any other plant species, using a variety of standard virological techniques.