[Detection of maize centromeric repeats in the relatives of maize using fluorescence in situ hybridization].

In order to analyze the conservation of maize centromeric satellite DNA (CentC) and centromeric retrotransposon (CRM) in the subspecies and relatives of Zea mays, dual fluorescence in situ hybridization (FISH) was used to detect the existence and distribution of the above two repetitive sequences in Zea mays ssp. mexicana, Z. diploperennis, Z. perennis, Tripsacum dactyloides, Coix lacryma-jobi, and Sorghum bicolor. In Z. mays ssp. mexicana, Z. diploperennis, and Z. perennis, both CentC and CRM probes produced strong or relatively strong signals in the centromeric regions of all chromosomes. There was an obvious variation in the intensity of hybridization signals on different chromosomes, indicating that different centromeres have different amounts of CentC and CRM sequences. In some centromeres, the intensity of CentC signals differed from that of CRM signals and was free from overlapping. In T. dactyloides, only weak CentC and CRM signals were detected in the centromeric regions of most chromosomes, while in C. lacryma-jobi and S. bicolor only relatively strong or strong CRM signals primarily located in the centromeric regions were detected. This result indicates that CentC is highly conserved among the subspecies of Z. mays and the species of Zea, and has high conservation in Tripsacum, a genus that is most closely related to Zea, and CRM is conserved among the species of grass family either closely or distantly related to Zea.

An optimal method of DNA silver staining in polyacrylamide gels.

DNA silver staining has widely been used to detect DNA fragments in polyacrylamide gels with high sensitivity. We developed an optimal method for DNA silver staining on polyacrylamide gels. The novel procedure can be completed within 10 min instead of over 20 min with the conventional methods. The sensitivity is significantly improved by the silver-ion sensitizer (Eriochrome black T (EBT)) and the minimum of 0.11 and 1.75 ng of DNA amount can be detected in denaturing and nondenaturing polyacrylamide gel, respectively. Compared with the conventional silver staining methods, the improved optimal method can save time and display high sensitivity, color uniformity, and long storage time of the staining gels.

[Advances in research of the structure and function of plant centromeres].

Centromeres are the chromosomal domains necessary for faithful chromosome segregation and transmission during mitosis and meiosis in eukaryotes. In the last decade, centromeres in some plant species including Arabidopsis, rice and maize have been deeply studied at molecular level. Centromeric DNAs evolve rapidly and are little conserved among various plants, but the types of centromeric DNA sequences and their organization patterns within centromeres are basically similar in plants. Plant centromeres are usually composed of clusters of tandemly arrayed satellite repeats that are interspersed with centromere-specific retrotransposons. In contrast to centromeric DNA, structural and transient centromeric/kinetochoric proteins are conserved among eukaryotes including plants. As the cases in other eukaryotes, the presence of CENH3 (centromeric histone H3)-containing nucleosomes is the fundamental feature of plant functional centromeres, and CENH3 plays critical roles in the identity and maintenance of plant centromeric chromatin.

Visualization of the ribosomal DNA (45S rDNA) of Indica rice with FISH on some phases of cell cycle and extended DNA fibers.

The ribosomal DNA (45S rDNA) behaviors during the cell cycle were analyzed on interphase nuclei, prophases, metaphases, pachytene chromosomes and extended DNA fibers in rice (Oryza, sativa ssp. indica cv. Guangluai No.4) by using high-resolution fluorescent in situ hybridization (FISH). The results show that 45S rDNA is located at the ends of short arms of chromosomes 9 and 10. But the signals are much more intense on chromosome 9 than on chromosome 10 in metaphase. Pachytene chromosome has rDNA signal arrays on chromosome 9. Different phases are described and discussed. These results indicate that the activity ofrDNA at individual loci may also vary through the cell cycle in rice. On extended DNA fibers, 45S rDNA signals appear as strings of numerous red spots, but some signals are missed in some regions, probably result from weak signals or intergenic spacers.

Visualization of the ribosomal DNA (45S rDNA) of Indica rice with FISH on some phases of cell cycle and extended DNA fibers

The ribosomal DNA (45S rDNA) behaviors during the cell cycle were analyzed on interphase nuclei, prophases, metaphases, pachytene chromosomes and extended DNA fibers in rice (Oryza,sativa ssp.indica cv.Guangluai No.4) by using high-resolution fluorescent in situ hybridization (FISH). The results show that 45S rDNA is located at the ends of short arms of chromosomes 9 and 10. But the signals are much more intense on chromosome 9 than on chromosome 10 in metaphase. Pachytene chromosome has rDNA signal arrays on chromosome 9. Different phases are described and discussed. These results indicate that the activity of rDNA at individual loci may also vary through the cell cycle in rice. on extended DNA fibers, 45S rDNA signals appear as strings of numerous red spots, but some signals are missed in some regions, probably result from weak signals or intergenic spacers

Cytogenetic comparisons between A and G genomes in Oryza using genomic in situ hybridization.

The genomic structures of Oryza sativa (A genome) and O. meyeriana (G genome) were comparatively studied using bicolor genomic in situ hybridization (GISH). GISH was clearly able to discriminate between the chromosomes of O. sativa and O. meyeriana in the interspecific F1 hybrids without blocking DNA, and co-hybridization was hardly detected. The average mitotic chromosome length of O. meyeriana was found to be 1.69 times that of O. sativa. A comparison of 4,6-diamidino-2-phenylindole staining showed that the chromosomes of O. meyeriana were more extensively labelled, suggesting that the G genome is amplified with more repetitive sequences than the A genome. In interphase nuclei, 9-12 chromocenters were normally detected and nearly all the chromocenters constituted the G genome-specific DNA. More and larger chromocenters formed by chromatin compaction corresponding to the G genome were detected in the hybrid compared with its parents. During pachytene of the F1 hybrid, most chromosomes of A and G did not synapse each other except for 1-2 chromosomes paired at the end of their arms. At meiotic metaphase I, three types of chromosomal associations, i.e. O. sativa-O. sativa (A-A), O. sativa-O. meyeriana (A-G) and O. meyeriana-O. meyeriana (G-G), were observed in the F1 hybrid. The A-G chromosome pairing configurations included bivalents and trivalents. The results provided a foundation toward studying genome organization and evolution of O. meyeriana.

[The relationship between abnormity of synaptonemal complex and male fertility impairment in human].

It was said that 11% of all men showed their infertility. The genetic origins of male infertility may be classified into three main groups: chromosome abnormalities, microdeletions and gene mutations. Growing literature has shown that spermatogenesis failure or reproductive failure (pregnancy wastages) occurred in male carriers of chromosomal distortion/aberration. But the mechanisms remain largely unsolved. Synaptonemal complex (SC) of human spermatocytes from such carriers offers new information. This review aims to summarize recent development of SC analysis for male infertile diagnosis and sum up our results obtained recently. The relationship between male infertility/sterility and SC abnormity was discussed and reviewed as following five aspects. (1) The association of XY-bivalent and the rearranged autosomes interfere with or affect, by their contact, X chromosome normal functions. (2) Male infertility is related to the incomplete pairing in break regions of rearranged autosomes. (3) SC fragmentation, lateral elements (LEs) swelling and pairing disorder result in spermatogenic failure. (4) This heterosynapsis at early stage of meiosis in rearranged autosomes, results in unbalanced germs and pregnancy wastages. (5) Gene mutations of SC proteins result in male infertility.

CPD banding patterns and identification of 45S rDNA sites in tomato.

In this study, we performed sequentially combined PI and DAPI (CPD) staining and FISH with two different 45S rDNA clones on meiotic pachytene and mitotic metaphase chromosomes in tomato. Ten red CPD bands were shown on eight pachytene bivalents, and 12 bands were shown on six pairs of mitotic metaphase chromosomes. The CPD bands exhibited on mitotic metaphase chromosomes corresponded to the prominent bands exhibited on the pachytene chromosomes. The distinctive CPD bands, which could be constantly and clearly detected using the CPD staining procedure we improved, provided new landmarks for chromosome identification in tomato. FISH with the tomato 45S rDNA clone revealed very strong signal(s) in the satellite(s) on the short arm of chromosome 2 as well as weak signals in five CPD banded regions at pachytene or four pairs of CPD banded regions at metaphase chromosomes. However, FISH with pTa71 plasmid only revealed signals in the satellite. Considering the difference in sequence between the two rDNA clones, we inferred that only the satellite contains the coding regions of 45S rDNA unit in tomato. The property of CPD bands as well as the DNA sequences probably involved in the five CPD banded regions was discussed.

FISH analysis of pachytene chromosome and DNA fiber of telomere sequence in rice (Oryza sativa L. indica).

Telomere sequences were analyzed by using pachytene chromosome and extended DNA fiber FISH in rice (Oryza, sativa ssp. indica cv. -Guangluai No.4). Pachytene FISH results showed that most of chromosome ends possess the telomere tandem repeats, but the signals on different chromosomes were not the same in intensity. Fiber FISH results indicated that the longest string of beads was 6.55 microm, while the shortest one was 1.82 microm long,which were equal to 16.44 and 4.56 kb correspondingly based on a stretching factor of 2.51 kb/microm. The average value of signal length was 3.62 +/- 1.32 microm, i.e. 9.09 +/- 3.31 kb. It could be estimated that the longest and the shortest as well as the average value were equal to 2,349,651 and 1298 +/- 473 of copy number respectively.

[Flow cytometric analysis and sorting of plant chromosomes.].

Flow cytometry refers to the technique where the measurement of the physical and chemical characteristics of particles including chromosomes, nuclei and cells is made as the particles pass in a fluid stream through a measuring point surrounded by an array of detectors. FCM (flow cytometry) analysis has played an important role in human genome projects. This technique is now increasingly used to establish flow karyotypes, sort chromosomes, map genes and construct DNA libraries in plants. This paper reviewed progress in flow cytometric analysis of plant genomes.

[Analysis of synaptonemal complex from a carrier with 46,XY,t(11;18) balanced translocation].

Over ten years ago, two male patients of pregnancy wastage, who were the carriers of 4;6 and 4;13 reciprocal translocations, respectively, were analyzed with synaptonemal complexes (SCs) by de Perdigo et al. They suggested that the pregnancy wastage should be caused by heterosynapsis, which reciprocal translocations resulted in. The heterosynapsis might avoid spermatogenesis failure, but easily induced non-disjunction of some chromosomes and led to occurrence of unbalanced gametes. A new male patient of the pregnancy wastage was detected in China three years ago. Karyotyping of the patient showed 11;18 reciprocal translocation. Analysis of synaptonemal complexes (SCs) of the patient was performed using the EM technology of SC surface spreading, SDS treatment, AgNO3 staining. The SCs of 30 spermatocytes at pachytenes were analyzed. The SCs in electron micrographs were measured by the method published by de Perdigo et al. (1991). The results showed that each of the observed spermatocytes displayed 20 autosomal bivalents, a quadrivalent 11;18 and a sexual bivalent. Of the 30 spermatocytes, 21 each exhibited a quadrivalent of adequate quality for measurement and interpretation. The 21 quadrivalents were classified into three types: type I, quadrivalents with fully paired arms with the expected cross-configuration, two quadrivalents were of this type. Type II, quadrivalents with an evident heterosynapsis, forming five SC segments (four fully pairing arms and one middle pairing region), six quadrivalents were of this type. Type III, quadrivalents with four paired arms, but with asynapsed regions around the breakpoints, thirteen quadrivalents were of this type. Based on configuration, quadrivalents were classified into cis-configuration with above three types and trans-configuration with above two types except type I. Each of the analyzed quadrivalents showed four synaptic arms, which respectively paired between chromosome 11 and t18 (the translocated 18;11), 18 and t18, 18 and t11 (the translocated 11;18), and 11 and t11, and the arms were designated as A, B, C, and D correspondingly. The measurements of A and D as well as B and C observed in different cells were taken by calculating the percentages of their lengths in comparison to the length of chromosome 11 and 18, respectively. So were the middle synaptic regions. Measurements of SCs revealed that the location of the putative breakpoints estimated from the fully paired quadrivalents were different from those determined by G-band analysis for mitotic chromosomes. The length of the paired and unpaired segments varied from one quadrivalent to another. For instance, in the case of fully paired A and D arms, the breakpoint was located near to 45.59% and 38.53% of the length of the normal chromosome 11, the breakpoint from the mitotic chromosome was estimated to be near to 45%. The location of the putative breakpoints estimated from the No. 1 quadrivalent was consistent with the breakpoint from the mitotic chromosome. This suggested that only this quadrivalent showed complete homologous pairing. Obviously, the lengths of middle pairing region in type II quadrivalent were different from one quadrivalent to another. The middle pairing region/the length of the normal chromosome 11 is 4.11% - 18%, that/the length of the normal chromosome 18 is 8% - 41.1%. The middle pairing regions should be the heterologous pairing regions in quadrivalents. The quadrivalents with unsynaptic regions also showed partially heterologous synapsis. In 14 of 21 quadrivalents, the paired regions overlapped the mitotic breakpoint position in chromosome 11. In 20 of 21 quadrivalents, the paired regions overlapped the chromosome 18 of mitotic breakpoint position in one or two arms. As synapsis can proceed homologously only up to the breakpoints, a heterosynapsis apparently occurred in 20 quadrivalents. The length of the paired and unpaired segments varied from one quadrivalent to another, and the measurements of the arms of the quadrivalents could not be used to determine breakpoints. The unpaired segment showed a thick and sometimes a split aspect similar to that of the sexual bivalent. Of the 20 quadrivalents with heterosynapsis, four were at early pachytene stages, fourteen at the middle and two at the late, respectively. The heterosynapsis, which occurred on the early pachytene, was without previous homosynapsis. This was different from the classical "synaptic adjustment" during the late pachrtene of spermatocytes. Many researches on reciprocal translocation associated with pregnancy wastage have been reported. It is most possible that the wastage detected in this study was related to the t(11;18). Normal synapsis finishes at early pachytene, while in the observed proband, most of the quadrivalents still left unsynaptic parts during middle and late pachytene stages. Obviously, the synapsis was delayed or incomplete. The unsynaptic regions distributed mainly in the central regions. The central regions perhaps completed the synapsis more late, therefore they had more chances to leave the unsynaptic parts. In addition, the results of SC analysis showed that heterosynapsis occurred wildly and was exhibited from early to late pachytene stages. It has been reported that crossing over could be inhibited by heterosynapsis in mice, man and boar, and normal segregation needs existence of crossing over. Therefore, heterosynapsis may interfere in normal segregation. Both unsynapsis and heterosynapsis could induce incidence of unbalanced gametes. In general, the unbalanced gametes are lethal and could not take part in fertilization, once they fertilize with normal eggs, the risk of pregnancy wastage or other genetic disorders will exist certainly. Our results showed that the heterosynapsis occurred widely, so a poor risk of pregnancy wastages caused by reciprocal translocation probably is not unusual.

[The production and multi-color genomic in situ hybridization identification of maize-Z. perennis substituted material].

Genus Zea. L was composed of two sections: sect. Luxuriantes Doebley & Iltis including Z. dipperennis, Z. luxurians and Z. perennis, and sect. Zea. mays consisting only one species, annual Z. mays. To improve the biodiversity of germ-plasm in maize breeding, the study of transferring maize relatives gene into common maize were performed. Firstly, interspecific hybrids between maize (Zea. L) (2n = 20) and Z. perennis (2n = 40) were produced with the aim of transferring desirable horticultural traits from Z. perennis to maize. The F1 of maize x Z. perennis (2n = 30) plant had the most frequent configuration of 5 III + 5 II + 5 I, which were sterile and difficult to produce progeny because of genomic affinities. However,few F2 individuals of maize x Z. perennis could be obtained by some specially treatments, and one maize-like F2 plant were obtained, which were used as a female parent in backcrossing with maize parent. Twelve F2 x P1 ( BC1 F2 ) plants were obtained and then self-crossed to produce self-crossing generation of F2 x P1 (2n = 20) ( BC1 F3). The phenotypic characters of parents, F1 (2n = 30) hybrids, F2 and F2 x P1 were investigated, such as plant height, flowering, leaf shape and tillers. To further verify the genomic organization of maize-Z. perennis material, maize (inbred line 48-2) and BC1 F3 chromosomes, the root tip cells were analyzed by Multi-color GISH. We probed maize and BC1 F3 chromosomes with the probe mixture containing biotin-labeled Z. perennis genomic DNA and digoxigenin-labeled maize genomic DNA, the maize spread exhibited 10 chromosomes with yellow signals and the other 10 chromosomes carried green fluorescing bands. However, although the BC1 F3 was 2n = 20 in all cases. Multi-color GISH images revealed that 17 chromosomes had uniform signals similar to maize chromosomes, but dispersed red signals over the remaining three chromosomes were observed, which indicated that the 3 chromosomes originated from Z. perennis, and they were smaller than maize chromosomes. Data obtained from multi-color GISH images indicated that BC1 F3 was probably a substituted material from maize-Z. perennis.

A new chromosome fluorescence banding technique combining DAPI staining with image analysis in plants.

In this study, a new chromosome fluorescence banding technique was developed in plants. The technique combined 4',6-diamidino-2-phenylindole (DAPI) staining with software analysis including three-dimensional imaging after deconvolution. Clear multiple and adjacent DAPI bands like G-bands were obtained by this technique in the tested species including Hordeum vulgare L., Oryza officinalis, Wall & Watt, Triticum aestivum L., Lilium brownii, Brown, and Vicia faba L. During mitotic metaphase, the numbers of bands for the haploid genomes of these species were about 185, 141, 309, 456 and 194, respectively. Reproducibility analysis demonstrated that banding patterns within a species were stable at the same mitotic stage and they could be used for identifying specific chromosomes and chromosome regions. The band number fluctuated: the earlier the mitotic stage, the greater the number of bands. The technique enables genes to be mapped onto specific band regions of the chromosomes by only one fluorescence in situ hybridisation (FISH) step with no chemical banding treatments. In this study, the 45S and 5S rDNAs of some tested species were located on specific band regions of specific chromosomes and they were all positioned at the interbands with the new technique. Because no chemical banding treatment was used, the banding patterns displayed by the technique should reflect the natural conformational features of chromatin. Thus it could be expected that this technique should be suitable for all eukaryotes and would have widespread utility in chromosomal structure analysis and physical mapping of genes.

[Synaptonemal complexes analysis in male fertility impairment in two men].

Synaptonemal complexes (SCs) of male fertility impairment were analysed in two patients. In case one, his testicular histology was normal and there were 20% abnormal pachytene spermatoeytes, showing unsynaptic and broken SCs. It resulted in spermatogensis with unbalanced chromosomes and pregnancy wastages. In case two, he had no sperms basically. G-banded chromosome analysis of lymphocytes showed normal chromosomal karyotype, but triple fragment, lateral element swelling and fragment breakages of SCs were observed in his spermatocytes. The mechanisms of infertility,impairment of fertility for the observed men were discussed in this paper.

Expressions of human p53 and c-myc gene homologues during caryopsis development in maize.

Tumor suppressor gene p53 and proto-oncogene c-myc have been proved to be highly conserved and participate in many PCD processes in animals. In maize, proteins and RNAs related to p53 and c-myc have already been reported and the sequences homologous to these two genes have also been localized onto maize chromosomes by FISH. In this study, using immunohistochemistry we investigated the expression patterns of maize genes homologous to human p53 and c-myc during caryopsis development stages in maize. In a giving stage after pollination, p53 homologue showed high levels in the antipodal cells, integument, immature endosperm, ovary wall, tracheary elements, and aleurone layer, while c-myc homologue showed low levels in these tissues, only before pollination showed high expression in polar nucleus. The results of TUNEL assay demonstrated that TUNEL positive signals were detected where p53 homologue showed high expression levels. In animal cells, p53 shows reverse function with that of c-myc, so does it in maize basically. These results demonstrated that p53 and c-myc homologues might play some important roles in PCD during caryopsis development in maize. There may be conserved mechanisms for PCD in animals and in high plants.

Genomic in situ hybridization analysis for identification of introgressed segments in alloplasmic lines from Zea mays x Zea diploperennis.

In this study, the tested four alloplasmic inbred lines, a2-4, a2-5, b1-1 and b2-1 were propagated from the same disease resistant individual in the parthenogenetic progenies of Zea mays L. cv. Lu 9 x Zea diploperennis (DP). All the lines except a2-5 were resistant to Helminthosporium turcium Pass and H. maydis Nisik. Introgressed DP segments in these lines were detected by both Southern hybridization and genomic in situ hybridization (GISH). The results of Southern hybridization showed that DP species-specific DNA sequences had been introgressed into the genomes of alloplasmic lines. The Southern hybridization band patterns in all of the tested lines were consistent with those of DP. Genomic in situ hybridization (GISH) signals were detected on 7 different chromosome pairs in lines a2-4 and a2-5, on 5 chromosome pairs in b1-1 and on 4 chromosome pairs in b2-1. The features of introgression, and disease resistant genes in the introgressed segments, as well as the gene silence or elimination in some alloplasmic lines are discussed.

Quantitative chromosome map of Coix lacryma-jobi L. by imaging methods.

Prometaphase chromosomes of Coix lacryma-jobi L. were quantitatively analyzed based on their distribution patterns of DAPI signals. The DAPI signals showed prominent uneven distribution along the chromosomes. Based on the DAPI signal patterns, a quantitative chromosome map was constructed for the first time in C. lacryma-jobi. The quantitative chromosome map will offer the foundation for genome analysis of C. lacryma-jobi.

Characterization of the early stages of programmed cell death in maize root cells by using comet assay and the combination of cell electrophoresis with annexin binding.

An emerging topic in plant biology is whether plant cells display similar elements of programmed cell death (PCD) as animal cells do. We have studied cell death in maize roots exposed to cold stress by using fluorescence microscopy, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL), DNA gel electrophoresis, single cell gel electrophoresis (SCGE), cell electrophoresis, and annexin binding techniques. The results showed that cell death in maize root cells triggered by cold stress was accompanied by a subset of features characteristic of animal PCD such as nuclear condensation and fragmentation, and oligonucleosomal DNA fragmentation. In addition to DNA laddering and TUNEL positivity, a "comet" pattern indicative of DNA breakage appeared as short as after one day of treatment. The maize root cell PCD process was also accompanied by an increase in negative surface charge of the dying cells due to exposure of phosphatiolylserine (PS) from inner to outer membrane. After annexin binding, however, the enhanced electrophoretic mobility (EPM) of the dying cells decreased nearly to normal values. This result suggests that the combination between cell electrophoresis and annexin binding provides a quantitative method for monitoring PS exposure during plant PCD.

Fluorescence in situ hybridization analysis of alien genes in Agrobacterium-mediated Cry1A(b)-transformed rice.

The transgene in Agrobacterium-mediated Cry1A(b)-transgenic rice plants has been detected and its chromosomal location determined by fluorescence in situ hybridization (FISH). Eight of the nine transgenic lines tested showed hybridization signals. Signals were located on regions of the chromosome in which fraction length (FL) values varied from 26.2 (near the centromere) to 95.2 (distal regions). No signal was found on regions where the fraction length was less than 26.2, while six of the nine signals detected were located on regions with FL values of 75.3 or over. This demonstrates that Agrobacterium-mediated genes can integrate into multiple sites distributed in different parts of the chromosome, but that distal regions are the preferred sites and regions near the centromeres are colder for T-DNA integration. The donor DNA of the transformation was divided into two parts, labelled separately as probes for two-colour FISH. Results show that the transformed DNA sequences remained linked in the recipient genome. The relationship between integration position and transgene silencing, known as the 'position effect', is discussed.