Nuclear DNA of myocardial cells in the periphery of infarctions and scars.

In 20 hearts the enlargement of muscle fibers and the increase in nuclear DNA is studied in the periphery of fresh and healed infarctions. The data of attack is verified by ECG records in most cases, five had passed undiagnosed. Selective cytophotometry is performed by measuring the largest nucleus in each of 50 fields of comparable density. Since significant differences in polyploidization exist between the control regions of hearts of similar weight primarily regions of the same heart are compared. No differences are observed in five hearts with infarctions about one week old. After five to six months the enlargement of the fibers is significaant (P less than 0.0005) while the increase in polyploidy is still slight (P less than 0.15). In all cases with scars one to nine years old the enlargement of fibers is significant (P less than 0.0005) while the increase in DNA is significant in most of them (5 X P less than 0.0005, 3 X P less than 0.005, 2 X P less than 0.01). In a case with two lesions, one posterior five (P less than 0.0005) and one anterior seven years old (P less than 0.0005), there is still slight increase in polyploidy with age (P less than 0.1). In the five undiagnosed cases only one has no significant increase in DNA. There is no correlation found between the enlargement of fibers and the increase in polyploidy in this limited number of cases.     

The development of polyploidy in two classes of rat liver nuclei

Two classes of nuclei from livers of Sprague-Dawley rats were isolated, one pelleting in 2.3 M sucrose (H nuclei) and the second class sedimenting through 1.6 and 1.8 M sucrose and banding at the 1.8/2.3 M sucrose interface (L nuclei) of a three-step discontinuous gradient. In younger animals, the L nuclear fraction was the major fraction, but the percentage of nuclei found in the L fraction decreased as the animals grew. Nuclear ploidy was determined by flow microfluorometry using propidium iodide as a DNA stain. Both the H and L nuclear fractions contained diploid, tetraploid and octaploid nuclei; but the degree of polyploidy was greater in the H fraction. Concomitant with the change in distribution of nuclei between the H and L fractions with increasing age was a progressive increase in the degree of polyploidy in the H fraction. Polyploidy did not increase linearly with age in the H nuclear fraction but increased in cycles marked by large changes in the numbers of nuclei found in H and L nuclear fractions. By 12 weeks of age, 4n-H nuclei were the largest single population of nuclei in rat liver. These observations suggested that the shift of liver nuclei from the L fraction to the H fraction was associated with the development of polyploidy and with the differentiation of hepatocytes.     

The relationship of DNA synthesis to protein accumulation in the cell nucleus

The relationship between DNA synthesis and protein accumulation in cell nucleus and cytoplasm has been investigated by the use of a combination of ultramicrointerferometric and ultramicrospectrophotometric methods. 5-Fluoro-2'-deoxyuridine (FUdR) inhibited DNA synthesis, resulting in inhibition of cell proliferation in G-1 and early S-phase. However, synthesis and accumulation of protein continued in the presence of FUdR, as indicated by a 54% increase in the average dry mass value per individual cell during 18-hour exposure to FUdR; due primarily to protein accumulation in the cytoplasm, the average cytoplasmic dry mass increased by as much as 85%, while the dry mass of the nucleus increased by only 21%. The dry mass values of individual nuclei were well-correlated to the nuclear DNA content throughout the period of exposure to FUdR. In contrast to the continued accumulation of protein in the cytoplasm during inhibition of DNA synthesis, protein accumulation in the nucleus was inhibited. When cells were released from inhibition of DNA synthesis by the addition of 2'-deoxythymidine, the nuclear DNA content and nuclear dry mass increased in near-synchrony, there being some evidence that DNA synthesis was initiated somewhat prior to initiation of increase in nuclear dry mass. Thus, it appears that DNA synthesis (or an increase in nuclear DNA content) is intimately related to the regulation of protein accumulation in the nucleus.     

Polyploidy: significance for cardiomyocyte function and heart aerobic capacity

Somatic polyploidy, defined as genome multiplication, was found in all differentiated mammalian tissues. The highest level of such a polyploidy was found in the myocardium. This phenomenon was shown to be associated with changes in the pattern of gene expression. Hence, polyploidization may create cells with new physiology. The effect of polyploidy on the heart function has never been studied. The aim of the present study was to investigate the effect of polyploidy on cardiomyocyte functioning and heart aerobic capacity. DNA and the total protein content, nucleolar activity reflecting the rate of rRNA synthesis and, consequently, ribosome biogenesis, were measured in ventricular myocytes isolated from the human and from 21 mammalian species by image cytometry and microscopic morphometry. The total protein content was estimated after staining slides with naphtol-yellow dye. For measurement of DNA and nucleolar area, staining with Hoechst and AgNO3 was applied. Cardiac aerobic capacity was evaluated by the heart mass to body mass ratio. A negative correlation between the heart index and the average cell ploidy was revealed (r = -0.79; P < 0.0001). The average genome number per myocyte was registered to be higher by approximately 35% in the sedentary mammals, with the heart index about 0.4% from body mass, than in the athletes with heart index about 0.6% of body mass. Polyploidization was shown to be associated with a sharp decrease in the protein/DNA ratio in cardiomyocytes. As a result, cardiomyocytes in the athletic mammals with poorly polyploid hearts have much higher protein content per genome than do cells in the sedentary species with highly polyploid hearts. Surprisingly, despite decreased protein/DNA ratio, the nucleolar area per genome significantly increased with polyploidization, indicating the imbalance between the cellular protein content and the rate of ribosome biogenesis. Such an imbalance should obviously impair cardiac function, because the additional genomes take some valuable space and biological resources from the cell, which could have been otherwise directed to the maintenance of cardiomyocyte contractile machinery. It is generally accepted that somatic polyploidy is associated with oxidative stress and energetic starvation. Thus, we suppose that additional genomes may serve for cardiomyocyte protection from oxidative damage in the hearts.     

Cytologic observations on the effects of metallocene dichlorides on human fibroblasts cultivated in vitro

The effects of the organometallic cytostatic agents titanocene dichloride (TDC) and vanadocene dichloride (VDC) and of the inorganic cytostatic drug cis-diamminedichloroplatinum(II) (DDP) on the morphologic appearance of human embryonal fibroblasts cultivated as monolayers in vitro were analyzed by light and electron microscopy. All three substances induced similar structural changes which consisted of nuclear as well as cytoplasmic alterations. Many fibroblasts enlarged but the nuclear volume increased more extensively than that of the cytoplasm. The nuclear envelopes underwent invagination so that segmented nuclei were formed and the nucleoli increased in size and density. Within the cytoplasm there was evidence of conspicuous protein synthesis, characterized morphologically by an increase in the size and number of mitochondria, Golgi apparatuses and of cisternae of the rER. The phenomena observed are interpreted as indicating unbalanced cell growth characterized by a selective inhibition of DNA synthesis, coupled with progressive RNA and protein syntheses.     

Characterization of the DNA from the dinoflagellate Crypthecodinium cohnii and implications for nuclear organization.

Although dinoflagellates are eucaryotes, they possess many bacterial nuclear traits. For this reason they are thought by some to be evolutionary intermediates. Dinoflagellates also possess some unusual nuclear traits not seen in either bacteria or higher eucaryotes, such as a very large number of identical appearing, permanently condensed chromosomes suggesting polyteny or polyploidy. We have studied the DNA of the dinoflagellate Crypthecodinium cohnii with respect to DNA per cell, chromosome counts, and renaturation kinetics. The renaturation kinetic results tend to refute extreme polyteny and polyploidy as the mode of nuclear organization. This organism contains 55-60% repeated, interspersed DNA typical of higher eucaryotes. These results, along with the fact that dinoflagellate chromatin contains practically no basic protein, indicate that dinoflagellates may be organisms with a combination of both bacterial and eucaryotic traits.     

Age-associated change in mitochondrial DNA damage

There is an age-associated decline in the mitochondrial function of the Wistar rat heart. Previous reports from this lab have shown a decrease in mitochondrial cytochrome c oxidase (COX) activity associated with a reduction in COX gene and protein expression and a similar decrease in the rate of mitochondrial protein synthesis. Damage to mitochondrial DNA may contribute to this decline.

Using the HPLC-Coularray system (ESA, USA), we measured levels of nuclear and mitochondrial 8-oxo-2'-deoxyguanosine (8-oxodG) from 6-month (young) and 23-month-old (senescent) rat liver DNA. We measured the sensitivity of the technique by damaging calf thymus DNA with photoactivated methylene blue for 30s up to 2h. The levels of damage were linear over the entire time course including the shorter times which showed levels comparable to those expected in liver. For the liver data, 8-oxodG was reported as a fraction of 2-deoxyguanosine (2-dG). There was no change in the levels of 8-oxodG levels in the nuclear DNA from 6 to 23-months of age. However, the levels of 8-oxodG increased 2.5-fold in the mitochondrial DNA with age. At 6 months, the level of 8-oxodG in mtDNA was 5-fold higher than nuclear and increased to approximately 12-fold higher by 23 months of age. These findings agree with other reports showing an age-associated increase in levels of mtDNA damage; however, the degree to which it increases is smaller. Such damage to the mitochondrial DNA may contribute to the age-associated decline in mitochondrial function.     

DNA repair and aging in mouse liver: 8-oxodG glycosylase activity increase in mitochondrial but not in nuclear extracts

8-oxo-deoxyguanosine (8-oxodG) is one of the major DNA lesions formed upon oxidative attack of DNA. It is a mutagenic adduct that has been associated with pathological states such as cancer and aging. Base excision repair (BER) is the main pathway for the repair of 8-oxodG. There is a great deal of interest in the question about age-associated accumulation of this DNA lesion and its intracellular distribution, particularly with respect to mitochondrial or nuclear localization. We have previously shown that 8-oxodG-incision activity increases with age in rat mitochondria obtained from both liver and heart. In this study, we have investigated the age-associated changes in DNA repair activities in both mitochondrial and nuclear extracts obtained from mouse liver. We observed that 8-oxodG incision activity of mitochondrial extracts increases significantly with age, from 13.4 + or - 2.2 fmoles of oligomer/100 microg of protein/16 h at 6 to 18.6 + or - 4.9 at 14 and 23.7 + or - 3.8 at 23 months of age. In contrast, the nuclear 8-oxodG incision activity showed no significant change with age, and in fact slightly decreased from 11.8 + or - 3 fmoles/50 microg of protein/2 h at 6 months to 9.7 + or - 0.8 at 14 months. Uracil DNA glycosylase and endonuclease G activities did not change with age in nucleus or mitochondria. Our results show that the repair of 8-oxodG is regulated differently in nucleus and mitochondria during the aging process. The specific increase in 8-oxodG-incision activity in mitochondria, rather than a general up-regulation of DNA metabolizing enzymes in those organelles, suggests that this pathway may be up regulated during aging in mice.     

The Growth of the Nucleus in Dividing and Non-dividing Cells of the Pea Root

The growth of the nucleus and the cell in the pea root was followedthrough the mitotic cycle and subsequently in post-mitotic developmentby comparing cells and nuclei from the meristem, at differentstages of interphase, and cells and nuclei from two regionsof the enlarging zone of the root. Measurements of cell andnuclear volumes were made in sections of fixed roots. Measurementsof nuclear volume, DNA content, and dry mass were made on isolatednuclei. Growth in the mitotic cycle was characterized by a doublingof DNA and nuclear dry mass and a five-fold increase of nuclearvolume. Since cell volume doubled, a differential hydrationof cytoplasm and nucleus is inferred. Post-mitotic growth wascharacterized by a four-fold or greater increase in cell volume,with vacuolation and a continued increase of cytoplasmic constituents,but a cessation of nuclear growth except by uptake of water;the only increase in nuclear dry matter appeared to be in cellsbecoming endopolyploid. The concentration of dry matter in thenucleus fell as the nuclei enlarged in the mitotic cycle andin post-mitotic growth. The relationships between the measuredparameters are examined to see whether they might be indicativeof causal relationships.     

基于核酸定量的核质分离质控方案

目的:拟建立一种方便快捷、经济有效的细胞核质分离鉴定方法。方法:本研究从DNA水平进行核质分离鉴定,选择GAPDH、ND1分别作为细胞核和细胞质标志基因,并根据GAPDH及ND1序列保守区设计引物,基于荧光定量PCR方法定性检测核质分离的效果。随后将本鉴定方法应用于其他种类细胞(BEAS-2B细胞、GT1-7细胞)及组织(小鼠心脏、肝脏、大脑)的核质分离鉴定。结果:在293T细胞应用该鉴定方法鉴定核质分离效果,结果显示:GAPDH、ND1在核组、质组中的含量存在明显差异,其中核标志基因GAPDH在细胞核中的比例达到了95%以上,质标志基因ND1在细胞质中的比例也达到了90%左右,这与从RNA水平及蛋白水平鉴定核质分离的结果一致。在其他种类的细胞(BEAS-2B细胞、GT1-7细胞)及组织(小鼠心脏、肝脏、大脑)应用该方法结果显示:细胞核组分中质标志基因ND1含量比293T细胞的有所增加,但仍可以实现核质分离鉴定。结论:本研究所建立的核质分离质控方法可以实现从DNA水平进行核质分离的鉴定,该方法更加经济、快捷。     

Hybridization and genome size evolution: timing and magnitude of nuclear DNA content increases in Helianthus homoploid hybrid species

Hybridization and polyploidy can induce rapid genomic changes, including the gain or loss of DNA, but the magnitude and timing of such changes are not well understood. The homoploid hybrid system in Helianthus (three hybrid-derived species and their two parents) provides an opportunity to examine the link between hybridization and genome size changes in a replicated fashion. Flow cytometry was used to estimate the nuclear DNA content in multiple populations of three homoploid hybrid Helianthus species (Helianthus anomalus, Helianthus deserticola, and Helianthus paradoxus), the parental species (Helianthus annuus and Helianthus petiolaris), synthetic hybrids, and natural hybrid-zone populations. Results confirm that hybrid-derived species have 50% more nuclear DNA than the parental species. Despite multiple origins, hybrid species were largely consistent in their DNA content across populations, although H. deserticola showed significant interpopulation differences. First- and sixth-generation synthetic hybrids and hybrid-zone plants did not show an increase from parental DNA content. First-generation hybrids differed in DNA content according to the maternal parent. In summary, hybridization by itself does not lead to increased nuclear DNA content in Helianthus, and the evolutionary forces responsible for the repeated increases in DNA content seen in the hybrid-derived species remain mysterious.     

Determination of the functional domains involved in nucleolar targeting of nucleolin.

Nucleolin (713 aa), a major nucleolar protein, presents two structural domains: a N-terminus implicated in interaction with chromatin and a C-terminus containing four RNA-binding domains (RRMs) and a glycine/arginine-rich domain mainly involved in pre-rRNA packaging. Furthermore, nucleolin was shown to shuttle between cytoplasm and nucleolus. To get an insight on the nature of nuclear and nucleolar localization signals, a set of nucleolin deletion mutants in fusion with the prokaryotic chloramphenicol acetyltransferase (CAT) were constructed, and the resulting chimeric proteins were recognized by anti-CAT antibodies. First, a nuclear location signal bipartite and composed of two short basic stretches separated by eleven residues was characterized. Deletion of either motifs renders the protein cytoplasmic. Second, by deleting one or more domains implicated in nucleolin association either with DNA, RNA, or proteins, we demonstrated that nucleolar accumulation requires, in addition to the nuclear localization sequence, at least two of the five RRMs in presence or absence of N-terminus. However, in presence of only one RRM the N-terminus allowed a partial targeting of the chimeric protein to the nucleolus.     

Distribution of chromosome material during division of giant nuclei by fragmentation in rodent trophoblast. Morphologic and cytophotometric study]

A study was made of a population of secondary giant cells (in the placenta of white rats and mice), of which a rather high polyploidy (128c--1024c) is characteristic, and which remains viable up to the end of pregnancy. At a certain stage of cell differentiation, some giant nuclei, looking as interphase nuclei, are divided into numerous smaller nuclear fragments bound with nuclear membranes. Two ways of division have been described: by a progressive budding of small nuclei into the cytoplasm, and the total division of the original nucleus into numerous tightly contracting nuclear fragments. Multinuclear cells originating from the nuclear fragmentation rather soon degenerate. The cytophotometrical measurement of the DNA amount in newly formed fragments has shown their ploidy extending from 1 to 32c, di-, three-, tetra-, and octoploid nuclei predominating. The distribution of chromosomal markers of the interphase nuclei (nucleoli, heterochromatinous blocks of nucleolus-forming chromosomes) confirms the photometrical evidence on the trends of chromosome fragmentation into genes. The fragmentation of the giant nucleus is preceded by a complex rearrangement of genetical material in the original nucleus, resulting in becoming polygenomal from polytene, with individual genomes separating to be segregated again, during division.     

Nuclear DNA Metabolism in the Tip of the Cortical Strands of Viscum album L

The tips of the cortical strands of Viscum album were investigatedby autoradiographic and cytophotometric methods. It is shown that DNA synthesis and mitotic activity of the sub-apicalmeristematic cells are constant during the whole year. Theirnuclear DNA content varies from 2C to 4C values. The elongated cells which cover the meristematic zone are characterizedby no DNA synthesis, no mitotic activity and a 2C nuclear DNAcontent. They are ‘blocked’ in the presyntheticphasc G₁ of their cellular cycle, without polyploidy. The relationship between polyploidy and secretion is discussed. Viscum album, mistletoe, nucleus, DNA, polyploidy     

叶绿体基因infA-rpl36区域在小麦族物种中的序列变异分析

利用小麦叶绿体基因组中infA-rpl36区域的序列设计引物, 对小麦族(Triticeae)的12个二倍体和多倍体的物种进行了PCR扩增和序列测定, 获得了长度为584～603 bp的12条DNA序列。序列分析表明, 供试物种在infA-rpl36基因间隔区的核苷酸变异明显高于基因编码区。基因编码区核苷酸序列同源性高达97%, 表明了目标片段具有高度的保守性。但在5个物种的infA编码区出现了较大的插入、缺失突变, 导致推导的氨基酸序列也发生了很大的变化, 证实了infA基因是叶绿体基因组中最活跃的基因之一, 而rpl36基因的变异较小, 说明不同叶绿体基因的进化速度是不同的。基于测定序列建立的种系树分析发现, 多倍体物种中间偃麦草(Thinopyrum intermedium)具有多种不同的细胞质起源, 与核基因组一样在进化上较为复杂。     

The Relative Concentration of Solids in the Nucleolus, Nucleus, and Cytoplasm of the Developing Nerve Cell of the Chick

Growing and differentiating nerve cells of the fifth cranial ganglion of the chick embryo were studied by several means. During the period of 70 hours to 11 days of incubation (Hamburger-Hamilton stages 19 to 37) average cell mass increased more than 4.5 times while cells changed from relatively undifferentiated neuroblasts to morphologically characteristic nerve cells with long processes. By making simplifying assumptions about thickness of nucleus and nucleolus, relative to cytoplasmic thickness, it was possible to calculate solute concentration of nucleus and nucleolus relative to that of the cytoplasm from measurements of optical retardations through living cells. Differences in relative solute concentration were observed in nucleolus, cytoplasm, and nucleoplasm in the approximate ratio 1.2:1.0:0.8, respectively. The ratio remained essentially constant during the growth period examined despite the fact that the cell components grow at markedly different rates. This suggests that solid concentrations are physical characteristics of nucleus, nucleolus, and cytoplasm which are maintained even during rapid growth and differentiation. By cytochemical means it was demonstrated that mass increase in the nucleus is not associated with increase in deoxyribonucleic acid. Both ribonucleic acid and protein are in greater concentration in nucleolus and cytoplasm than in the nucleoplasm. Electron microscopy shows interruptions in the nuclear envelope as well as an approximately even distribution of electron density in nucleus and cytoplasm. It is pointed out that consistent differences in solid concentration can exist on either side of the nuclear envelope even though it contains "pores." Implications of these data are discussed.     

Nuclear DNA variation, chromosome numbers and polyploidy in the endemic and indigenous grass flora of New Zealand

BACKGROUND AND AIMS: Little information is available on DNA C-values for the New Zealand flora. Nearly 85 % of the named species of the native vascular flora are endemic, including 157 species of Poaceae, the second most species-rich plant family in New Zealand. Few C-values have been published for New Zealand native grasses, and chromosome numbers have previously been reported for fewer than half of the species. The aim of this research was to determine C-values and chromosome numbers for most of the endemic and indigenous Poaceae from New Zealand. SCOPE: To analyse DNA C-values from 155 species and chromosome numbers from 55 species of the endemic and indigenous grass flora of New Zealand. KEY RESULTS: The new C-values increase significantly the number of such measurements for Poaceae worldwide. New chromosome numbers were determined from 55 species. Variation in C-value and percentage polyploidy were analysed in relation to plant distribution. No clear relationship could be demonstrated between these variables. CONCLUSIONS: A wide range of C-values was found in the New Zealand endemic and indigenous grasses. This variation can be related to the phylogenetic position of the genera, plants in the BOP (Bambusoideae, Oryzoideae, Pooideae) clade in general having higher C-values than those in the PACC (Panicoideae, Arundinoideae, Chloridoideae + Centothecoideae) clade. Within genera, polyploids typically have smaller genome sizes (C-value divided by ploidy level) than diploids and there is commonly a progressive decrease with increasing ploidy level. The high frequency of polyploidy in the New Zealand grasses was confirmed by our additional counts, with only approximately 10 % being diploid. No clear relationship between C-value, polyploidy and rarity was evident.