Regeneration of amphidiploid plants from tissue cultures of Allium wakegi

Callus was induced from the bulb of Allium wakegi Araki on MS semisolid medium supplemented with several growth regulating substances. The calli were subcultured every 40 days. At the time of every subculture the callus was subdivided to be used for chromosome studies, plant regeneration, or continuous callus multiplication. The chromosome constitution of cells in callus and regenerated plants varied over the culture period, and at the 3rd subculture amphidiploid plants were obtained. They appeared even more frequently than amphihaploid plants in the 4th subculture. Hypoamphihaploid regenerants appeared as stumpy shoots but none of these shoots proceeded further to form a normal plant. By Giemsa C-banded karyotype, the chromosome constitution of amphidiploid plants was found to result from exact doubling of the chromosome sets of amphihaploid common species. Amphidiploid plants show better viability and growth than common plants. The possibility and the expectation of new crop plants to be developed from amphidiploid plants will be discussed.     

Overall DNA methylation and chromatin structure of normal and abnormal X chromosomes

DNA methylation patterns were studied at the chromosome level in normal and abnormal X chromosomes using an anti-5-methylcytosine antibody. In man, except for the late-replicating X of female cells, the labeled chromosome structures correspond to R- and T-bands and heterochromatin. Depending on the cell type, the species, and cell culture conditions, the late-replicating X in female cells appears to be more or less undermethylated. Under normal conditions, the only structures that remain methylated on the X chromosomes correspond to pseudoautosomal regions, which harbor active genes. Thus, active genes are usually hypomethylated but are located in methylated chromatin. Structural rearrangements of the X chromosome, such as t(X;X)(pter;pter), induce a Turner syndrome-like phenotype that is inconsistent with the resulting triple-X constitution. This suggests a position effect controlling gene inactivation. The derivative chromosomes are always late replicating, and their duplicated short arms, which harbor pseudoautosomal regions, replicate later than the normal late-replicating X chromosomes. The compaction or condensation of this segment is unusual, with a halo of chromatin surrounding a hypocondensed chromosome core. The chromosome core is hypomethylated, but the surrounding chromatin is slightly labeled. Thus, unusual DNA methylation and chromatin condensation are associated with the observed position effect. This strengthens the hypothesis that DNA methylation at the chromosome level is associated with both chromatin structure and gene expression.     

Flow microfluorometric DNA content measurements of tissue culture cells and peripheral lymphocytes

Summary The difference in DNA content of peripheral lymphocytes from normal males, normal females, and an individual with a 48 (xxxy) chromosome constitution was determined by rapid flow microfluorometric techniques. A similar comparison was performed using tissue culture fibroblasts derived from an individual with a 49 (xxxxy) chromosome constitution and WI-38 cells as a normal control. Less than 60 min were required to isolate the lymphocytes, to stain the cells fluorescently, and to measure the increased DNA content. The measured increase in DNA content is consistent with chromosome DNA analyses and chromosome length measurements.     

Cross-species chromosome painting

Comparative genomics is an important and expanding field of research, and the genome-wide comparison of the chromosome constitution of different species makes a major contribution to this field. Cross-species chromosome painting is a powerful technique for establishing chromosome homology maps, defining the sites of chromosome fusions and fissions, investigating chromosome rearrangements during evolution and constructing ancestral karyotypes. Here the protocol for cross-species chromosome painting is presented. It includes sections on cell culture and metaphase preparation, labeling of chromosome-specific DNA, fluorescent in situ hybridization (chromosome painting) and image analysis. Cell culture and metaphase preparation can take between 1 and 2 wk depending on the cell culture. Labeling of chromosome-specific DNA is completed in 1 d. Fluorescent in situ hybridization can be completed in a maximum of 4 d.     

The 47,XYY male, Y chromosome, and tooth size.

Permanent teeth of 12 individuals with a 47,XYY chromosome constitution have been examined. The tooth sizes of 47,XYY males were found to be larger than those of control males and females. In many instances the differences were statistically significant. Using these results, it was possible to conclude that a factor or factors which influence excess growth of 47,XYY males probably are in effect during prenatal life, but without doubt must be in effect very early in postnatal life. The time period needed for the achievement of final excess growth is relatively short, in the case of first permanent molars probably only from 2 1/2 to 3 1/2 years. On the basis of the finding that the Y chromosome apparently carries genes affecting tooth sizes in normal males [1], it was suggested that gene products of the extra Y chromosome could cause the observed size difference between normal and 47,XYY males. The nature of the influence of one versus two Y chromosomes on growth was discussed in terms of the possible influence of the Y chromosome on the cell divisions within the developing tooth germ.     

Genomic imprinting: male mice with uniparentally derived sex chromosomes

Although it has been known that there is an X-chromosome imprinting effect during early embryogenesis in female mammals, it remains unknown if parental origin of the X chromosome has an effect in males. Furthermore, it has not been possible to produce animals with normal sex chromosomes of uniparental origin to further evaluate such imprinting effects. We have devised a breeding scheme to produce male mice, designated XPYP males, in which both the X and Y chromosomes are paternally inherited. To our knowledge, these are the first mammals produced that have a normal sex chromosome constitution but with both sex chromosomes derived from one parent. Development and reproduction in these XPYP males and the sex ratio and chromosome constitution of their offspring appeared normal; thus there is no apparent effect in males of having both sex chromosomes derive from one parent or of having the X chromosome derived from an inappropriate parent. Although we have detected no X-chromosome imprinting effect in these males, evidence from other sources suggest that the X chromosome is parentally imprinted. Thus detection and definition of an imprint can depend on the assay used.     

Torus mandibularis in 45,X females (Turner syndrome)

Ninety-three Finnish females with a 45,X chromosome constitution, 78 first-degree female, and 37 first-degree male relatives were examined to determine the frequency and expression of torus mandibularis. The results indicate that among adults the frequency of the trait was significantly lower and the expression weaker in the 45,X females than in male control relatives. A similar trend was observed in comparison to normal females. In juveniles the trend was reversed. Our findings suggest that the sex chromosomes may have an influence on the occurrence, expression, and timing of development of the mandibular torus. Sexual dimorphism in the manifestation of torus mandibularis may result particularly from the effect of the Y chromosome on growth. © 1996 Wiley-Liss, Inc.     

Karyotype analysis of the euploid cell population of a mouse embryonic stem cell line revealed a high incidence of chromosome abnormalities that varied during culture

It is common knowledge that mouse embryonic stem cell (mESC) lines accumulate chromosomal changes during culture. Despite the wide use of mESCs as a model of early mammalian development and cell differentiation, there is a lack of systematic studies aimed at characterizing their karyological changes during culture. We cultured an mESC line, derived in our laboratory, for a period of 3 months investigating its chromosome complement at different times. About 60% of the metaphases analysed were euploid throughout the culture period but, from passage 13, only 50% of the euploid metaphases had a proper chromosome complement. The remaining 50% showed chromosome abnormalities, mainly gain or loss of entire chromosomes, both within the same passage and among different passages analysed. The very heterogeneous spectrum of abnormalities indicates a high frequency of chromosome mutations that arise continuously during culture. The heterogeneity of the aberrant chromosome constitution of 2n = 40 metaphases, observed at different passages of culture, might be due either to their elimination or to a shift towards the hypoeu- or hypereuploid population of those metaphases that accumulate further chromosome abnormalities. The stability of the frequency of eu-, hypoeu- and hypereuploid populations during culture might, however, be due to the elimination of those cells that carry a high mutational burden. Based on our results, we suggest that karyotype analysis of the euploid cell population of mESC lines is necessary when such lines are used in the production of chimeric mice, for their contribution to the germ line, or when they are differentiated into specific cell types.     

青海高原披碱草属种间天然杂种的细胞学鉴定

利用重复序列探针染色体荧光原位杂交（FISH）和基因组原位杂交（GISH）技术,对采自青海高原披碱草属种间天然杂种进行细胞学鉴定,同时结合物种分布及形态学特征,共揭示6种不同天然杂种的类型。第一类为垂穗披碱草（Elymus nutans Griseb.）和鹅观草属（Roegneria C.Koch）物种间的天然杂种,染色体数为35,染色体组成为StStYYH;第二类为垂穗披碱草和达乌力披碱草种（Elymus dahuricus Turcz.ex Griseb.）间杂种,染色体数为42,染色体组成为StStHHYY;第三类为达乌力披碱草和老芒麦（Elymus sibiricus L.）种间杂种,染色体数为35,染色体组成为StStHHY;第四类为垂穗披碱草和糙毛以礼草（Kengilia hirsuta Keng）种间杂种,染色体数为42,染色体组成为StStYYHP;第五类为垂穗披碱草和大颖草（Kengilia grandiglumis Keng）种间杂种,染色体数为42,染色体组成为StStYYHP;第六类为糙毛以礼草和赖草（Leymus secalinus（Georgi） Tzvel.）种间杂种,染色体数为35,染色体组成为StYPNsXm。研究结果为进一步研究披碱草属种间杂交渐渗提供了重要参考资料;同时鉴定出的天然杂种可以作为潜在的种质资源在牧草或生态草育种中加以利用。     

Karyotype stability in long-term callus derived plants ofCrepis tectorum L.

The study of in vitro growth of Crepis tectorum revealed 100 % callusing and 40 % plantlet regeneration. The root and leaf used as explants showed the normal diploid (2n=8) chromosome constitution. In one month old culture 95 % callus cells were diploid. The callus maintained in 2,4-D 1 mg 1-1 for two years showed 62 % diploid, 5 % tetraploid and 33 % hyperdiploid cells. The differentiation of shoot occurred in two year old calli after subeulturing in 2 mg I-1 BAP and the potentiality of regeneration was retained for more than one year. The leaf-tips of regenerated plants were homogeneous and identical to the donor plant both in number and morphology of chromosomes.     

X-X translocation in a patient with gonadal dysgenesis and the problem of phenotype-karyotype correlations

Summary A sex-chromatin-positive woman without stunted growth, but with primary amenorrhea, and some stigmas of pure gonadal dysgenesis had the chromosome constitution 45,X/46,Xt(X;X)(q27;q27). The abnormal chromosome formed a large Barr body and was late-labeling. The chromosome consisted of two X chromosomes attached by their long arms (end-to-end), both apparently having the partial distal deletion. Both centromeric regions showed C-staining but only one constriction. The chromosome is interpreted as an isodicentric with only one centromere functioning. Some problems of phenotype-karyotype correlations are discussed.     

Genetic confirmation of facioscapulohumeral muscular dystrophy in a case with complex D4Z4 rearrangments

Facioscapulohumeral muscular dystrophy (FSHD) is caused by contraction of the D4Z4 repeat on chromosome 4q. Genetic confirmation of the clinical diagnosis of FSHD is complicated by the presence of a homologous repeat on chromosome 10q and the frequent repeat exchanges between both chromosomes. Here, we describe the genetic evaluation of an FSHD patient with a complex D4Z4 allele constitution in which the potentially pathogenic allele seemingly resides on chromosome 10, despite FSHD being exclusively linked to chromosome 4. Complementary allele typing and segregation analysis confirmed the clinical diagnosis of FSHD by revealing the chromosome 4 origin of the pathogenic allele in the presence of two exchanged repeat arrays, one on chromosome 4 and one on chromosome 10, an allele constitution that cannot be identified by conventional DNA diagnosis.     

Cytogenetic effect of plant tissue culture medium with certain growth substances onAllium sativum L. meristem root tip cells

The effect of plant tissue culture medium with different concentrations and combinations of growth regulators (kinetin, indol-3-ylacetic acid, 2,4-dichlorophenoxyacetic acid) was evaluated on mitosis ofAllium sativum meristem root tip cells. Different combinations of growth regulators at low concentrations had no effect on induction of mitotic aberrations or inhibition of mitotic activity. Inhibition of mitotic activity, a tendency to chromosome stickiness and clumping and a slight increase in the frequency of mitotic aberrations were observed at higher concentrations. It may be proposed that plant tissue culture media have no direct effect on induction of mitotic aberrations in plant tissue culturesin vitro.     

Long-term storage of tissue samples for cell culture

The establishment of cultured cell lines from skin biopsies stored at -196 degrees C for periods up to 1 year has been investigated. Attempts to initiate cell cultures from the frozen tissue samples were uniformly successful. There was no alteration in chromosome constitution, morphological appearance, or specific activities of lysosomal enzymes in cells cultured from the stored samples. This process can safeguard against failure of the initial tissue culture and provide an alternate means of storing viable cells when it is impossible or impractical to initiate a cell culture immediately.     

不同细胞质的普通小麦"中国春"(Triticum aestivum var.Chinese Spring)与小偃麦杂交F1代的育性与减数分裂行为的研究

15个不同细胞质“中国春”小麦与八倍体小偃麦杂交 ,杂种F1减数分裂的染色体行为表明 :普通小麦与天蓝偃麦草的F或E组染色体之间存在着部分同源关系 ;D2 型细胞质促进部分同源染色体配对、但却抑制同源染色体配对 ;Sv 型细胞质对同源染色体或部分同源染色体的配对均有抑制作用 ;G型细胞质促进同源染色体配对。1 5个不同细胞质“中国春”小麦与六倍体小偃麦杂交 ,F1结实率很低 ,减数分裂中期的染色体行为混乱 ,单价体过多 ,或许意味着在天蓝偃麦草 (Elytrigiain termedium)与长穗偃麦草 (E .elongatum)的E组染色体之间存在着很大差别。随着回交代数的增加 ,选出G型、D2 型、Mt 型、Mu 型等细胞质雄性不育的八倍体小偃麦品系 ,其中D2 型细胞质八倍体小偃麦具有光周期敏感性雄性不育的特征 ;G型细胞质“远中 3”育性正常 ,表明八倍体小偃麦“远中 3”的E组染色体中存在G型胞质的育性恢复基因。     

Effect of donor cell cycle stage on chromatin and spindle morphology in nuclear transplant rabbit embryos.

We investigated the influence of the cell cycle stage of the nuclear donor on prematurely condensed chromatin (PCC) and spindle morphology and on chromosome constitution in rabbit nuclear transplant embryos. The configuration of PCC following nuclear transplantation with G1, early S, and late S phase donor nuclei (G1, early S, and late S transplants, respectively) was characterized in whole mounts and chromosome spreads. In addition, the influence of the donor cell cycle stage on chromosome constitution in cleavage stage-manipulated embryos was determined. Within 2 h after fusion of the donor blastomere, the recipient oocyte cytoplasm was able to induce formation de novo of a metaphase plate associated with a spindle in G1, early S, and late S transplants. Metaphase chromosomes and spindle were intact in most cases of PCC in G1 transplants. However, these structures displayed minor abnormalities in early S transplants and gross abnormalities in late S transplants, such as incomplete or absent spindle formation and incomplete chromatin condensation. Normal chromosomes were present in G1 and early S transplants, whereas chromosome abnormalities were detected in late S transplants. The results indicate that morphology of prematurely condensed G1 and early S chromatin has a minor influence on chromosome constitution of manipulated embryos. That of late S chromatin, however, affects chromosome constitution in embryos and may account for reduced development of nuclear transplant embryos when late S phase donor nuclei are used.     

Effect of charged particles of relativistic energy on the number of chromosome aberrations in human blood lymphocytes. Dose-response relationship and the RBE of protons, deuterons and helium ions

In experiments with human blood lymphocyte culture exposed to fast protons, deuterons and helium ions the frequency and types of chromosome aberrations have been studied. Fast charged particles of a relativistic energy are shown to have a pronounced harmful effect resulting in a sharply increased formation of exchange chromosome aberrations as compared to that produced by gamma-radiation. The RBE coefficients of the particles under study have been determined: their values vary depending on the type of radiation and the tests used.     

Cytological identification of two X-chromosome types in the wood lemming (Myopus schisticolor)

In the wood lemming (Myopus schisticolor) three genetic types of sex chromosome constitution in females are postulated: XX, X^*X and X^*Y (X^*=X with a mutation inactivating the male determining effect of the Y chromosome). Males are all XY. It is shown in the present paper that the two types of X chromosomes, X and X^*, exhibit differences in the G-band patterns of their short arms. In addition, it was demonstrated in unbanded chromosomes that the short arm in X^* is shorter than in X. The origin of these differences is still obscure; but they allow to identify and to distinguish the individual types of sex chromosome constitution, as of XX versus X^*X females and of X^*Y females versus XY males, on the basis of G-banded chromosome preparations from somatic cells.     

Somatic and germ-cell sex in mammals

The phenotypic sex of an individual mammal is determined by the sex of its gonads, i.e. testes or ovaries. This in turn is determined by the presence or absence of a small region of the Y chromosome, located near the X-Y pairing region in man and on the short arm of the Y chromosome in the mouse. The testis-determining region of the Y appears to exert its primary effect by directing the supporting-cell lineage of the gonad to differentiate as Sertoli cells, acting at least in part cell-autonomously. The phenotypic sex of a germ cell, i.e. whether it undergoes spermatogenesis or oogenesis, is determined at least in the mouse by whether or not it enters meiotic prophase before birth. This depends not on its own sex chromosome constitution, but on its cellular environment. A germ cell in or near normal testis cords (made up mainly of Sertoli cells) is inhibited from entering meiosis until after birth; one that escapes this inhibition will develop into an oocyte even if it is in a male animal and is itself XY in chromosome constitution.     

The effect of different cellulosic growth substrates and pH on the production of cellulolytic enzymes by Trichoderma reesei

The effect of different cellulosic growth substrates on the production of cellulolytic enzymes by Trichoderma reesei was investigated. It was observed that growth on Avicel, Solka Floc and wheat straw produced different pH/time profiles in cultures. Over a range of controlled pH it was demonstrated that the production of cellulolytic and xylanolytic activity by T. reesei is dependent on culture pH and the type of growth substrate. The effect of pH on enzyme production varies with the nature of the growth substrate. Furthermore, it was shown that the optimum culture pH and growth substrate for the production of enzyme preparations for the extensive saccharification of cellulosic materials depends on the type of material to be saccharified.