原子力显微镜在双微体形态学研究中的应用

原子力显微术(atomic force microscopy,AFM)是一种新型的纳米显微技术,由于其拥有标本制备简单、分辨率高等优点,因此常用于细胞超微结构的观察。双微体(double minute chromosomes,DMs)是基因扩增的主要表现形式,经常出现在肿瘤细胞及耐药细胞中,可使肿瘤细胞获得生存优势或产生耐药性,因此对双微体进行研究可使人类了解肿瘤的生长特性及其抗药性的产生机理。为寻找一种研究双微体的有效方法,本实验利用原子力显微镜对小鼠耐氨甲喋呤细胞3T3R500中的双微体进行观察,在获得双微体高分辨AFM形态图的同时,还对双微体的大小进行了测量,发现细胞中双微体大小存在差异。此外,就原子力显微镜在双微体研究中的一些技术细节进行了探讨。实验结果表明原子力显微术是研究双微体的一种有效手段。     

Analysis of the replication mode of double minutes using the PCC technique combined with BrdUrd labeling

A cultured line of neuroblastoma cells (NB) was found to contain double minute chromosomes (DMs). DMs have been reported to be acentric and, therefore, to be segregated randomly into daughter cells without separating their sister elements. When NB cells were fused with Chinese hamster metaphase cells, prematurely condensed chromosomes (PCCs) were induced. DMs seen together with G₂ PCCs appeared to be closely paired, dot-like structures resembling DMs observable in metaphase cells. In contrast, DMs in G₁ cells showed a tendency to become single as the stage progressed so that the majority of DMs in late G₁ cells were actually no longer double. DMs in S-phase cells, however, again appeared double. These results clearly indicate why DMs are invariably double and never assume a quadruple configuration in metaphase cells in spite of their non-disjunctional segregation at anaphase. Such a characteristic mode of DM replication was further confirmed by a 5-bromo-2-deoxyuridine (BrdUrd) labeling experiment: when NB cells were exposed to BrdUrd for two successive rounds of DNA replication prior to PCC induction, half of the resulting single G₁ minutes as well as G₁ PCCs stained dark and the other half stained light after staining for sister chromatid differentiation.     

Structural analysis of heavy ion radiation-induced chromosome aberrations by atomic force microscopy

Heavy ion radiation (high linear energy transfer, LET, radiation) induces various types of chromosome aberration. In this report, we describe a new method employing an atomic force microscope (AFM) for nanometer-level structural analysis of chromosome damage induced by heavy ion irradiation. Metaphase mouse chromosomes with chromatid gap or chromatid breaks induced by heavy ion irradiation were marked under a light microscope. Then the detailed structure of chromosomes of Giemsa-stained or unstained samples was visualized by the AFM. The height data of chromosomes obtained by AFM provided useful information to distinguish chromatid gaps and breaks. A fibrous structure was observed on the unstained chromosome, the average width of which was about 45.8 nm in the image of AFM. These structures were considered to be 30-nm fibers on the chromosome. The structure of the break point regions induced by neon- or carbon-ion irradiation was imaged by AFM. In some cases, the fibrous structure of break points was detected by AFM imaging after carbon ion irradiation. These observations indicated that AFM is a useful tool for analysis of chromosome aberrations induced by heavy ion radiation.     

Establishment of the diploid gynogenetic hybrid clonal line of red crucian carp × common carp

This study investigated the gynogenetic cytobiological behavior of the third gynogenetic generation (G3), which was generated from the diploid eggs produced by the second gynogenetic generation (G2)of red crucian carp × common carp, and determined the chromosomal numbers of G3, G2×scatter scale carp and G2×allotetraploid hybrids of red crucian carp × common carp. The results showed that the diploid eggs of G2 with 100 chromosomes, activated by UV-irradiated sperm from scatter scale carp and without the treatment for doubling the chromosomes, could develop into G3 with 100 chromosomes.Similar to the first and second gynogenetic generations (G1 and G2), G3 was also diploid (2n=100) and presented the hybrid traits. The triploids (3n=150) and tetraploids (4n=200) were produced by crossing G2 with scatter scale carp, and crossing G2 with allotetraploids, respectively. The extrusion of the second polar body in the eggs of G2 ruled out the possibility that the retention of the second polar body led to the formation of the diploid eggs. In addition, we discussed the mechanism of the formation of the diploid eggs generated by G2. The establishment of the diploid gynogenesis clonal line (G1, G2 and G3) provided the evidence that the diploid eggs were able to develop into a new diploid hybrid clonal line by gynogenesis. By producing the diploid eggs as a unique reproductive way, the diploid gynogenetic progeny of allotetrapioid hybrids of red crucian carp × common carp had important significances in both biological evolution and production application.     

Scanning Near-field Optical/Atomic Force Microscopy detection of fluorescence in situ hybridization signals beyond the optical limit

Fluorescence in situ hybridization (FISH) is widely used in molecular biological study. However, high-resolution analysis of fluorescent signals is theoretically limited by the 300-nm resolution optical limit of light microscopy. As an alternative to detection by light microscopy, we used Scanning Near-field Optical/Atomic Force Microscopy (SNOM/AFM), which can simultaneously obtain topographic and fluorescent images with nanometer-scale resolution. In this study, we demonstrated high-resolution SNOM/AFM imaging of barley chromosome (Hordeum vulgare, cv. Minorimugi) FISH signals using telomeric DNA probes. Besides detecting the granular structures on chromosomes in a topographic image, we clearly detected fluorescent signals in telomeric regions with low-magnification imaging. The high-resolution analysis suggested that one of the telomeric signals could be observed by expanded imaging as two fluorescent regions separated by approximately 250 nm. This result indicated that the fluorescent signals beyond the optical limit were detected with higher resolution scanning by SNOM/AFM.     

Cytogenetic stability of chicken T-cell line transformed with Marek's disease virus: atomic force microscope, a new tool for investigation

The Marek's disease virus (MDV) integration may induce a novel organization of chromatin architecture with a modified genetic expression. In our opinion it is worthwhile trying to relate cytogenetic stability to functional modifications. Recently, atomic force microscopy technique was applied to study the structure of chromosomes at a nanoscale level. This high resolution allows to investigate the different structure of chromatin in order to study cytogenetic stability and chromosome aberrations due to MDV insertion. In this paper data are presented indicating a duplication [78,WZ,dup(1p)(p22-p23)] and a deletion [78,WZ cht del(3)(q2.10)] of Chromosomes 1 and 3 relatively. Relationships between GTG (G-bands by Trypsin using Giemsa) bands and the topography of chromosomes are also discussed, naming them Topographic Banding. The architecture of chromosomes observed by AFM can be related to the data obtained with classic banding techniques thus overcoming the optical resolution limits. The presence of chromatin bridges between sister chromatids at most of the heterochromatic regions is also evidenced. Besides, we present different studies of the longitudinal and transversal symmetry of the hetero and euchromatic regions to clearly demonstrate a different underlying architecture of these regions. It is indeed evident that the heterochromatic bands are more symmetrical than euchromatic bands.     

Super-stretched pachytene chromosomes for fluorescence in situ hybridization mapping and immunodetection of DNA methylation

Meiotic pachytene chromosome-based fluorescence in situ hybridization (FISH) mapping is one of the most important tools in plant molecular cytogenetic research. Here we report a simple technique that allows stretching of pachytene chromosomes of maize to up to at least 20 times their original size. A modified Carnoy's II fixative (6:1:3 ethanol:chloroform:acetic acid) was used in the procedure, and proved to be key for super-stretching of pachytene chromosomes. We demonstrate that super-stretched pachytene chromosomes provide unprecedented resolution for chromosome-based FISH mapping. DNA probes separated by as little as 50 kb can be resolved on super-stretched chromosomes. A combination of FISH with immunofluorescent detection of 5-methyl cytosine on super-stretched pachytene chromosomes provides a powerful tool to reveal DNA methylation of specific chromosomal domains, especially those associated with highly repetitive DNA sequences.     

Cell cycle-specific changes in the ultrastructural organization of prematurely condensed chromosomes

Prematurely condensed chromosomes (PCC) of HeLa cells synchronized in different phases of the cell cycle were analyzed by high-resolution scanning electron microscopy. The purpose of this study was to examine changes in the arrangement of the basic 30-nm chromatin fiber within interphase chromosomes associated with progression through the cell cycle. These studies revealed that highly condensed metaphase chromosomes and early G1-PCC consisted of tightly packed looping fibers. Early to mid G1-PCC were more extended and exhibited gyres suggestive of a despiralized chromonema. Further attenuation of PCC during progression through G1 was associated with a gradual transition from packed looping fibers to single extended longitudinal fibers. This process occurs prior to the initiation of DNA synthesis which appears to be localized within single longitudinal fibers. Following replication of a chromosome segment, extended longitudinal fibers were rapidly reorganized into packed looping fiber clusters concomitant with the formation of a multifibered chromosome axis. This results in the characteristic “pulverized” appearance of S-PCC when viewed by light microscopy. Subsequently, adjacent looping fiber domains coalesce, resulting in the uniformly packed, looping fiber arrangement observed in G2-PCC. Spiralization of the chromonema during the G2-mitotic transition results in the formation of highly compact metaphase chromosomes.     

Effects of gamma-radiation on the organisation of polytene chromosomes ofGlyptotendipes salinus Michailova (Chironomidae,Diptera)

A series of laboratory experiments onGlyptotendipes salinus were carried out in order to assess cytogenetic effects of different doses of gamma-radiation on polytene chromosomes, isolated from salivary glands. Chrinonomid larvae (III–IV larval stage) were irradiated with doses varying from 0.05 to 1.00 Gy (5–100 rad) and were bred under laboratory conditions until the fourth larval stage. Cytogenetic slides were analyzed for an estimation of occurrence of changes in the organization of the polytene chromosomes caused by gamma-radiation. A specific heterochromatin effect was found in certain chromosomes of the investigated species after 1.00 Gy irradiation. Decondensation of the centromeric heterochromatin and increased functional activity of Balbiany ring 2 were observed in the fourth (G) chromosome. Regression of the nucleolus of the first (AB) chromosome was detected.     

Rabl's model of the interphase chromosome arrangement tested in Chinise hamster cells by premature chromosome condensation and laser-UV-microbeam experiments

Summary In 1885 Carl Rabl published his theory on the internal structure of the interphase nucleus. We have tested two predictions of this theory in fibroblasts grown in vitro from a female Chinese hamster, namely (1) the Rabl-orientation of interphase chromosomes and (2) the stability of the chromosome arrangement established in telophase throughout the subsequent interphase. Tests were carried out by premature chromosome condensation (PCC) and laser-UV-microirradiation of the interphase nucleus. Rabl-orientation of chromosomes was observed in G1 PCCs and G2 PCCs. The cell nucleus was microirradiated in G1 at one or two sites and pulse-labelled with ³H-thymidine for 2h. Cells were processed for autoradiography either immediately thereafter or after an additional growth period of 10 to 60h. Autoradiographs show unscheduled DNA synthesis (UDS) in the microirradiated nuclear part(s). The distribution of labelled chromatin was evaluated in autoradiographs from 1035 cells after microirradiation of a single nuclear site and from 253 cells after microirradiation of two sites. After 30 to 60h postincubation the labelled regions still appeared coherent although the average size of the labelled nuclear area fr increased from 14.2% (0h) to 26.5% (60h). The relative distance dr, i.e. the distance between two microirradiated sites divided by the diameter of the whole nucleus, showed a slight decrease with increasing incubation time. Nine metaphase figures were evaluated for UDS-label after microirradiation of the nuclear edge in G1. An average of 4.3 chromosomes per cell were labelled. Several chromosomes showed joint labelling of both distal chromosome arms including the telomeres, while the centromeric region was free from label. This label pattern is interpreted as the result of a V-shaped orientation of these particular chromosomes in the interphase nucleus with their telomeric regions close to each other at the nuclear edge. Our data support the tested predictions of the Rabl-model. Small time-dependent changes of the nuclear space occupied by single chromosomes and of their relative positions in the interphase nucleus seem possible, while the territorial organization of interphase chromosomes and their arrangement in general is maintained during interphase. The present limitations of the methods used for this study are discussed.Part of this work is included in the doctoral thesis of H. Baumann to be submitted to the Faculty of Biology of the University of HeidelbergPart of this work is included in the doctoral thesis of V. Teuber to be submitted to the Faculty of Medicine of the University of Freiburg i. Br.     

Influence of sodium butyrate on the induction of radiation-induced chromosomal aberrations and sister chromatid exchanges in Chinese hamster ovary (CHO) cells.

CHO cells were pre-treated with sodium butyrate (SB) for 24 h and then X-irradiated in G1. Metaphases were scored for the induction of chromosomal aberrations and sister chromatid exchanges (SCEs). The data were compared with those obtained after irradiation of cells not pre-treated with SB and showed that SB has different effects on the endpoints examined. The frequencies of dicentric chromosomes were elevated and of small acentric rings (double minutes, DMs) reduced. These results are discussed to be a consequence of conformational changes in hyperacetylated chromatin which could lead to more interchromosomal and to less intrachromosomal exchanges. SB itself induces a few SCEs but suppresses the induction of SCEs by X-rays. We assume that a minor part of radiation induced SCEs are 'false' resulting from structural chromosomal aberrations, such as inversions, induced in G1. Inversions are the symmetrical counterparts of DMs. If inversions are suppressed by SB treatment to a similar extent as DMs a small reduction of SCEs by SB can be expected.     

Structure of amplified DNA, analyzed by pulsed field gradient gel electrophoresis

Pulsed field gradient electrophoresis allows the separation of large DNA molecules up to 2,000 kilobases (kb) in length and has the potential to close the resolution gap between standard electrophoresis of DNA molecules (smaller than 50 kb) and standard cytogenetics (larger than 2,000 kb). We have analysed the amplified DNA in four cell lines containing double minute chromosomes (DMs) and two lines containing homogeneously staining regions. The cells were immobilized in agarose blocks, lysed, deproteinized, and the liberated DNA was digested in situ with various restriction endonucleases. Following electrophoretic separation by pulsed field gel electrophoresis, the DNA in the gel was analysed by Southern blotting with appropriate probes for the amplified DNA. We find that the DNA in intact DMs is larger than 1,500 kb. Our results are also compatible with the notion that the DNA in DMs is circular, but this remains to be proven. The amplified segment of wild-type DNA covers more than 550 kb in all lines and possibly up to 2,500 kb in some. We confirm that the repeat unit is heterogeneous in some of the amplicons. In two cell lines, however, with low degrees of gene amplification, we find no evidence for heterogeneity of the repeats up to 750 (Y1-DM) and 800 kb (3T6-R50), respectively. We propose that amplicons start out long and homogeneous and that the heterogeneity in the repeat arises through truncation during further amplification events in which cells with shorter repeats have a selective advantage. Even if the repeats are heterogeneous, however, pulsed field gradient gels can be useful to establish linkage of genes over relatively short chromosomal distances (up to 1,000 kb). We discuss some of the promises and pitfalls of pulsed field gel electrophoresis in the analysis of amplified DNA.     

C-banding of Peromyscus constitutive heterochromatin persists following histone hyperacetylation

Approx. 35% of the DNA of cultured cells from the cactus mouse, Peromuscus eremicus, is contained in highly condensed constitutive heterochromatin which can be visualized in metaphase chromosomes stained by the C-band technique. Previous studies have shown this constitutive heterochromatin to contain a large proportion of underacetylated, arginine-rich histones, the majority of which can be hyperacetylated when cells are treated with butyrate. In order to determine whether this simulation of the acetylated state of euchromatin alters the cytological properties of constitutive heterochromatin as well, chromosomes from butyrate-treated cells have been examined. Because of the paucity of cells in butyrate-treated cultures, prematurely condensed chromosomes (PCCs) were produced from butyrate-treated cells by fusion with mitotic cells. In these PCCs, both the highly condensed nature and the ability to C-band were preserved in the hyperacetylated constitutive heterochromatin, suggesting that the subset of arginine-rich histones which is refractory to acetylation in the presence of butyrate may be responsible for the maintenance of the heterochromatic state. In addition, PCC analyses indicated that butyrate arrests Peromyscus cells in both the G1 and G2 phases of the cell cycle and confirmed the late-replicating pattern of constitutive heterochromatin.     

Visualization by atomic force microscopy and FISH of the 45S rDNA gaps in mitotic chromosomes of Lolium perenne

The mitotic chromosome structure of 45S rDNA site gaps in Lolium perenne was studied by atomic force microscope (AFM) combining with fluorescence in situ hybridization (FISH) analysis in the present study. FISH on the mitotic chromosomes showed that 45S rDNA gaps were completely broken or local despiralizations of the chromatid which had the appearance of one or a few thin DNA fiber threads. Topography imaging using AFM confirmed these observations. In addition, AFM imaging showed that the broken end of the chromosome fragment lacking the 45S rDNA was sharper, suggesting high condensation. In contrast, the broken ends containing the 45S rDNA or thin 45S rDNA fibers exhibited lower density and were uncompacted. Higher magnification visualization by AFM of the terminals of decondensed 45S rDNA chromatin indicated that both ends containing the 45S rDNA also exhibited lower density zones. The measured height of a decondensed 45S rDNA chromatin as obtained from the AFM image was about 55–65 nm, composed of just two 30-nm single fibers of chromatin. FISH in flow-sorted G2 interphase nuclei showed that 45S rDNA was highly decondensed in more than 90% of the G2/M nuclei. Our results suggested that a failure of the complex folding of the chromatin fibers occurred at 45S rDNA sites, resulting in gap formation or break.     

Cytogenetic analysis of sperm from a man heterozygous for a pericentric inversion, inv (3) (p25q21).

Human sperm chromosomes were studied in a man heterozygous for a pericentric inversion of chromosome 3(p25q21). The pronuclear chromosomes were analyzed after in vitro penetration of golden hamster eggs. A total of 144 sperm were examined: 69.2% were chromosomally balanced and 30.8% were recombinant. Of the balanced complements, the proportion with a normal chromosome 3 (37.6%) was approximately equal to the proportion with an inverted 3 (31.6%). Of the recombinant complements, the proportion of sperm with a duplication q/deletion p (17.3%) was approximately equal to the reciprocal event of duplication p/deletion q (13.5%). The recombinant chromosome 3 with a duplication q and deletion p has been observed in several abnormal children, but the duplication p/deletion q has never been reported. My results demonstrate that both recombinant chromosomes are produced as expected from an unequal number of crossovers within an inversion loop. In all likelihood the duplication p/deletion q chromosome is an early embryonic lethal because of the amount of genetic material deleted. The proportions of X-bearing (48.9%) and Y-bearing sperm (51.1%) were not significantly different from the expected 1:1 ratio. There was no evidence for an interchromosomal effect. Of the three inversions studied by human sperm chromosome analysis, recombinant chromosomes have been observed only in this case.     

Amplification of genome regions in the somatic cells of mammals resistant to colchicine. III. Localization of the amplified genes in minute chromatin bodies

Small chromatin bodies (SCB) were revealed in Djungarian hamster cells resistant to colchicine. They looked like single bodies or like clusters of small particles. SCB were localized both in nucleus and cytoplasm. Similar formations were earlier observed in oocytes of insects with amplified extrachromosomal rDNA genes. DNA in the SCB was able to replicate not only during the S phase but also during other phases of the cell cycle. The restriction analysis showed that in cells with SCB DNA amplified sequences were replicated autonomously too. These data indicate that SCB in colchicine-resistant cells contain amplified genes. Besides, SCB double-minute chromosomes (DMs) were observed in some resistant sublines. In one of them, DMs were the only karyotypic alteration. The relationship between SCB, chromosomal homogeneously staining regions (HSRs) and DMs was studied. Single SCB and DMs appeared at the early stage of the development of colchicine-resistance (the level of drug resistance is 16-22). Selection of variants 170-220-fold resistant to colchicine was usually accompanied by the decrease in the cell number with SCB and DMs and by the increase in the amount of cells containing the chromosomes with HSRs. During the further enhancement of drug resistance (700-750), some decrease in the number of cells with HSRs and the appearance of the great number of cells containing large groups of SCB were found. The loss of colchicine-resistance observed during cultivation in colchicine free medium was accompanied by the disappearance of HSRs, emergence of SCB and DMs and further elimination of SCB and DMs from cells. The quantity of autonomously replicating amplified DNA fragments after digestive by HindIII was increased with the enhancement of SCB number in cultures.     

茅舍血厉螨核型及染色体的C带、G带的研究

本文首次报道了一种革螨——茅舍血厉螨核型及染色体C带、G带的研究。用剖腹取卵法、玻璃纸压片、Giemsa染色,经分析茅舍血厉螨的核型,单倍体n=7,二倍体2n=14。 用氢氧化钡—吉姆萨技术显示茅舍血厉螨染色体C带。在第1、2、4、5染色体上出现恒定的C带部分,第3、6、7染色体上出现不恒定的C带部分。根据C带带型,茅舍血厉螨着丝点的位置可分为近中区域(sm),近端区域(St),末端区域(t)和末端点(T)四类。 G带分析用胰蛋白酶—吉姆萨技术显示。 本文对茅舍血厉螨的核型、革螨染色体研究中螨卵的收集方法和染液的改进、C带带型与着丝粒位置的确定和G带显带问题进行了讨论。     

Molecular structure and evolution of double-minute chromosomes in methotrexate-resistant cultured mouse cells.

To determine whether microscopically visible double-minute chromosomes (DMs) are derived from submicroscopic precursors, we monitored the amplification of the dihydrofolate reductase (DHFR) gene in 10 independent isolates of methotrexate (MTX)-resistant mouse cells. At every other doubling in MTX concentration, the cells were examined both microscopically, to detect the presence of microscopically visible DMs, and by pulsed-field gel electrophoresis and hybridization to a DHFR-specific probe, to detect submicroscopic DMs. One of the cloned MTX-resistant isolates was examined in detail and was shown to originally contain amplified DHFR genes on circular DMs measuring 1 and 3 Mb in size; additionally, metaphase chromosome preparations from this cloned isolate were examined and were shown to contain microscopically visible DMs too large to enter a pulsed-field gel. During stepwise selection for increasing levels of MTX, the smaller DMs (not microscopically visible) were shown to be preferentially amplified, whereas the larger (microscopically visible) ones decreased in relative numbers. Rare-cutting NotI digestion patterns of total genomic DNA that includes the DMs containing the DHFR gene suggest that the DMs increase in copy number without any further significant rearrangements. We saw no evidence from any of the 10 isolates to suggest that microscopically visible DMs are formed from smaller submicroscopic precursors.     

A Centromeric Satellite Sequence in the Pacific Oyster (Crassostrea gigas Thunberg) Identified by Fluorescence In Situ Hybridization

A highly repetitive satellite sequence was previously identified in the Pacific oyster Crassostrea gigas Thunberg. The sequence has 168 bp per unit, present in tandem repeats, and accounts for 1% to 4% of the genome. We studied the chromosomal location of this satellite sequence by fluorescence in situ hybridization (FISH). A probe was made by polymerase chain reaction and incorporation of digoxigenin-11-dUTP. Hybridization was detected with fluorescein-labeled antidigoxigenin antibodies. FISH signals were located at centromeric regions of 7 pairs of the Pacific oyster chromosomes. No interstitial site was found. Signals were strong and consistent on chromosomes 1, 2, 4, and 7, but weak or variable on chromosomes 5, 8, and 10. No signal was observed on chromosomes 3, 6, and 9. Our results showed that this sequence is clearly a centromeric satellite, disputing its previous assignment to the telomeric and submetacentric regions of 2 chromosomes. No signal was detected in the American oyster (Crassostrea virginica Gmelin). Received February 2, 2001; accepted May 24, 2001     

Quantitative Trait Loci Mapping of Leaf Morphological Traits and Chlorophyll Content in Cultivated Tetraploid Cotton

Genetic mapping provides a powerful tool for quantitative trait loci （QTL） analysis at the molecular level. A simple sequence repeat （SSR） genetic map containing 590 markers and a BCI population from two cultivated tetraploid cotton （Gossypium hirsutum L.） cultivars, namely TM-1 and Hai 7124 （G. barbadense L.）, were used to map and analyze QTL using the composite interval mapping （CIM） method. Thirty one QTLs, 10 for lobe length, 13 for lobe width, six for lobe angle, and two for leaf chlorophyll content, were detected on 15 chromosomes or linkage groups at logarithm of odds （LOD）≥2.0, of which 15 were found for leaf morphology at LOD≥3.0. The genetic effects of the QTL were estimated. These results are fundamental for marker-assisted selection （MAS） of these traits in tetraploid cotton breeding.