八倍体小黑麦耐盐细胞系产生的遗传机制

实验选用来源于八倍体小黑麦（×ＴｒｉｔｉｃｏｓｅｃａｌｅＷｉｔｍａｃｋ）杂种一代Ｂ、Ｃ和原种Ｆ三个品系的成对单倍体和双倍体细胞系,在１％、１．５％和２％ＮａＣｌ水平进行耐盐变异体筛选及其发生机制研究。实验和分析结果表明,未经筛选的细胞系由三类细胞组成,第一类是占大多数没有任何适盐性的细胞,第二类是具有一定适盐性的非变异体细胞,可在继代筛选中被淘汰,第三类细胞是变异体细胞;第二类细胞在杂种材料细胞系中所占比例少于原种细胞系,故杂种细胞较易筛选到耐盐变异体;比较三个级别筛选的变异体频率发现,后一级筛选的变异必须以前一级筛选的变异为基础,即耐盐变异体发生的遗传机制是基因扩增;双倍体细胞中正常同源染色体对基因扩增变异的表达有干扰作用,这种干扰因材料不同在各级筛选中呈现不稳定状态。     

Ultrastructure of freshly isolated strains of Bordetella pertussis

The study of the electronograms of B. pertussis strains isolated in the foci of pertussis revealed the existence of the morphological variants of these cells, differing in the character of the cell wall, the state of the cytoplasm, the presence of amorphous inclusions of medium electron-optical density. The morphological variants did not correlate with the character of B. pertussis colonies isolated from blood-charcoal agar. The ultrastructure of the cells belonging to the second morphological variant was similar to that of the cells from the museum strain, altered by tetracycline treatment in the course of the experiment.     

Thermodynamic models, describing formation of "bridges" between nucleic acid molecules and liquid crystals

Three variants of the model for the formation of "bridges" between the nucleic acid molecules fixed in the structure of particles of liquid-crystalline dispersions were considered. What the three variants have in common is that the bridges represent polymeric chelate cross-links consisting of alternating molecules of daunomycin and copper ions. The differences between the three variants are that in the first variant, the bridges begin and end with daunomycin molecules that form a complex by the mechanism of external binding with nucleic acids; in the second variant, the bridges begin and end with copper ions coupled with the pairs of bases of nucleic acids; and in the third variant, the bridges begin with the daunomycin molecule and end with the copper ion. For each variant, a mathematical model was constructed, which describes the formation of bridges, and equations of binding were derived. The results of calculations were compared with the experimental data. Within the framework of each variant, the values of the energy of interaction between the daunomycin molecule and the copper ion in the bridge, the energy of interaction of the daunomycin molecule with the nucleic acid, and the length of the bridge were varied. For all variants, those values of the parameters were chosen that fit best the experimental data. The theoretical curves obtained using the three variants of the model agree rather well with the family of experimental curves. The best agreement between the theoretical and experimental data was obtained when the polymeric chelate bridge includes more than two daunomycin molecules.     

Functional significance of the left adrenal vein and the gonadal veins

Owing to the investigation of changeability variants of the left adrenal, anastomosis of the inferior diaphragmatic and ovarian (testicular) veins--tributaries of the human left renal vein, three variants in inflow of the left adrenal vein in comparison to the left ovarian (testicular) vein have been revealed. In the first variant, that occurs in 70% of cases, the left adrenal vein flows into the left renal vein by 15 cm more medially from the ostium of the ovarial (testicular) vein. The second variant occurs in 11%. The left adrenal and ovarian (testicular) ostia are situated at the same level. The distance from the hilus renalis to the place, where the ostia mentioned get into the renal vein, is about 39 mm. In the third variant (19%) the left adrenal vein gets into the left renal vein nearer to the hilus renalis than the ovarian (testicular) veins. An average distance from the hilus renalis to the ostium of the left adrenal vein is 33 mm. The second variant, when the left adrenal vein gets into the left renal vein, contributes to appearance of the primary varicocele at the left. When the operation for nephrectomy is made at the left, our data make it possible to recommend a purposeful ligation of the left renal vein more laterally to the ostium of the ovarian (testicular) vein. In 80% this ligation will not disturb the function of the left adrenal vein.     

Expression and physiological role of CCN4/Wnt-induced secreted protein 1 mRNA splicing variants in chondrocytes

CCN4/Wnt-induced secreted protein 1 (WISP1) is one of the CCN (CTGF/Cyr61/Nov) family proteins. CCN members have typical structures composed of four conserved cysteine-rich modules and their variants lacking certain modules, generated by alternative splicing or gene mutations, have been described in various pathological conditions. Several previous reports described a CCN4/WISP1 variant (WISP1v) lacking the second module in a few malignancies, but no information concerning the production of WISP1 variants in normal tissue is currently available. The expression of CCN4/WISP1 mRNA and its variants were analyzed in a human chondrosarcoma-derived chondrocytic cell line, HCS-2/8, and primary rabbit growth cartilage (RGC) chondrocytes. First, we found WISP1v and a novel variant of WISP1 (WISP1vx) to be expressed in HCS-2/8, as well as full-length WISP1 mRNA. This new variant was lacking the coding regions for the second and third modules and a small part of the first module. To monitor the expression of CCN4/WISP1 mRNA along chondrocyte differentiation, RGC cells were cultured and sampled until they were mineralized. As a result, we identified a WISP1v ortholog in normal RGC cells. Interestingly, the WISP1v mRNA level increased dramatically along with terminal differentiation. Furthermore, overexpression of WISP1v provoked expression of an alkaline phosphatase gene that is a marker of terminal differentiation in HCS-2/8 cells. These findings indicate that WISP1v thus plays a critical role in chondrocyte differentiation toward endochondral ossification, whereas HCS-2/8-specific WISP1vx may be associated with the transformed phenotypes of chondrosarcomas.     

Novel intestinal splice variants of RNA-binding protein CUGBP2: isoform-specific effects on mitotic catastrophe

CUG triplet repeat-binding protein 2 (CUGBP2) is a RNA-binding protein that regulates mRNA translation and modulates apoptosis. Here, we report the identification of two splice variants (termed variants 2 and 3) in cultured human intestinal epithelial cells and in mouse gastrointestinal tract. The variants are generated from alternative upstream promoters resulting in the inclusion of additional NH(2)-terminal residues. Although variant 2 is the predominant isoform in normal intestine, its expression is reduced, whereas variant 1 is overexpressed following gamma-irradiation. All three variants bind cyclooxygenase-2 (COX-2) mRNA. However, only variant 1 inhibits the translation of the endogenous COX-2 mRNA and a chimeric luciferase mRNA containing the COX-2 3'untranslated region. Furthermore, whereas variant 1 is predominantly nuclear, variants 2 and 3 are predominantly cytoplasmic. These data imply that the additional amino acids affect CUGBP2 function. Previous studies have demonstrated that variant 1 induces intestinal epithelial cells to undergo apoptosis. However, in contrast to variant 1, the two novel variants do not affect proliferation or apoptosis of HCT116 cells. In addition, only variant 1 induced G(2)/M cell cycle arrest, which was overcome by prostaglandin E(2). Moreover, variant 1 increased cellular levels of phosphorylated p53 and Bax and decreased Bcl2. Caspase-3 and -9 were also activated, suggesting the initiation of the intrinsic apoptotic pathway. Furthermore, increased phosphorylation of checkpoint kinase (Chk)1 and Chk2 kinases and increased nuclear localization of Cdc2 and cyclin B1 suggested that cells were in mitotic transition. Taken together, these data demonstrate that cells expressing CUGBP2 variant 1 undergo apoptosis during mitosis, suggesting mitotic catastrophe.     

Relationship between morological variability of Bacillus subtilis R-623 and biosynthesis of hydrolytic enzymes

By synthetic sorbent chromatography the influence of Bacillus subtilis R-623 morphology on the qualitative and quantitative composition of alpha-amylase and proteases was studied. It was found that morphological variants of natural variability of Bac. subtilis R-623, alpha-amylase producer, differed in their cultural, morphological and physiological properties as well as in the amount of hydrolytic enzymes synthesized per unit of the cultural medium. Cells of P variant produced the highest quantity of alpha-amylase (314 U/lm) and cells of P and M variants synthesized the greatest amount of proteases (4.3 and 4.0 U/ml, respectively). Quantitative variations of alpha-amylase and proteases were measured in the cultural medium of morphological variants of Bac. subtilis R-623 during cultivation. Qualitative composition of those enzymes was determined when their content in the cultural medium was at the highest level. R variant synthesized alpha-amylase and protease, P and S variants alpha-amylase and two proteases, and P and S variants alpha-amylase and two proteases, and M variant one protease.     

Different phenotypic variants of the mouse B cell tumor A20/2J are selected by antigen- and mitogen-triggered cytotoxicity of L3T4-positive, I-A-restricted T cell clones

The L3T4+, Lyt-2-, cloned BALB/c T cell lines 5.9.24 and 5.8.6 are cytotoxic for the BALB/c B cell tumor line A20/2J. The T cell cytotoxicity against A20/2J cells could be triggered either by the specific antigen ovalbumin (OVA), which is recognized by the T cell clones in association with I-Ad determinants, or by the T cell mitogens Con A and rabbit anti-mouse brain (RaMBr) antiserum. Repeated exposure of A20/2J cells to 5.9.24 and 5.8.6 T cell cytotoxicity selected variant cell lines that had developed resistance to cytotoxicity. The variant lines could be classified into four different variant phenotypes of which three were stably maintained in vitro. The type of variant obtained appeared to be related to the nature of the ligand used to trigger T cell cytotoxicity during selection. Cytotoxicity triggered by the antigen OVA generated type 1 variants that expressed abnormally low levels of I-Ad determinants at the cell surface. Type 1 variants were resistant to OVA-triggered 5.9.24 T cell cytotoxicity, but were fully susceptible to cytotoxicity triggered by Con A or RaMBr antiserum. RaMBr-triggered cytotoxicity generated two unique types of variant cell lines: type 3 variants that were deficient in cell surface Fc receptors and resistant to 5.9.24 cytotoxicity only when triggered by RaMBr antiserum, and type 4 variants that were resistant to cytotoxicity triggered by all three ligands. One type 4 variant, the IC-1 cell line, appeared to be resistant to soluble cytotoxic factors released by 5.9.24 T cells after activation by antigen. All of these variant lines retained sensitivity to cytotoxicity by classic Lyt-2+ cytotoxic T lymphocytes (CTL), a finding that indicates that L3T4a+ T cells and Lyt-2+ CTL use different molecules to attack their target cells. The variant phenotypes were inherited by clones derived from the original cell lines. Because the variants were generated without mutagenesis, they are thought to have been derived by the immunoselection of pre-existing variant cells that arose spontaneously in the parental A20/2J cell line. It is postulated that inheritable variation of A20/2J cells may represent changes that normally occur during B cell differentiation in response to T cell signals. The variant A20/2J cell lines described here provide material for the investigation of B cell surface structures that may regulate T-B cell interactions.     

Morphological heterogeneity of Bordetella pertussis populations and clones (based on electron microscopy data)

Populations belonging to different serovars of B. pertussis museum strains and antibiotic-resistant clones obtained from them have been studied by electron microscopy. As a result, morphological heterogeneity and differences in the ultrastructure of the cells with respect to the cell-wall structure, the character of the cytoplasm, the size of the cells, cytoplasmic inclusions and intracellular links have been demonstrated and, proceeding from these data, two main morphological variants of the cells have been defined. The cells of the morphological variant characterized by the pliciform surface of the outer membrane and the pronounced periplasmic space prevail among the populations of the museum strains. The possibility of the isolation of antibiotic-resistant clones, differing in their morphological structure and functional properties from the initial population, has been shown. The morphological diversity of B. pertussis population is the necessary condition for the existence and development of microbial populations.     

Genetic Mechanism of the Occurrence of Salt-tolerant Variant of Octoploid Triticale Under Tissue and Cell Culture

Three pairs of haploid and diploid cell lines, respectively originating from three octoploid triticale lines, F1 hybrids B and C and primary strain F, were used for in vitro selection of salt-tolerant variants at 1.0%, 1.5% and 2.0% NaC1 levels, for the purpose of studying the genetic mechanism of the occurrence of salt-tolerant variant. Experimental and analytical results showed that non-selected cell lines consisted of three classes of cells. The first-class cells which accounted for the majority of a cell line had no salt adaptability, the second-class cells were non-vari-ant cells with some salt adaptability, that could be gradually eliminated through subcultures on selection medium. The third-class were variant cells. The percentage of the second-class cells in the hybrid-derived cell lines was less than that of the cell lines from homozygous primary strain, thus it was relatively easy to select the salt-tolerant variants from the hybrid-derived cell lines. In comparing the variant frequencies of the three rounds of selections it was found that the variation in second round selection was based on the variation accurred in the first round selection, and by comparing the characteristics of these variants it was proposed that a genetic mechanism played in the production of salt-tolerant variant cell could be an amplification of the salt tolerance genes with dominant effectiveness. The normal homologous chromosome in the diploid cell could interfere with the expression of the salt-tolerant variation resulted from gene amplification. Such interference varied with the different genetic background of the triticale lines and appeared inconsistent among different rounds of selections.     

Alternative splicing of mRNA of mouse interleukin-4 and interleukin-6

Interleukin-4 and interleukin-6 are multifunctional regulatory proteins, which participate both in haemopoiesis and in immunopoiesis. The alternative splicing of these interleukins in humans is known to proceed in a tissue-specific manner. Additionally, changes in splicing can also be dependent on tissue pathology. In this work, we report on the presence of alternatively spliced mRNA (IL-4delta2mRNA), lacking exon 2, in mouse bone marrow and spleen cells. We find that in unstimulated cells IL-4mRNA levels strongly dominate over IL-4delta2mRNA levels. Both increase in response to stimulation, with the concentration of the alternative variant rising earlier and faster than that of the full-length variant. In all other tissues studied dominance of IL-4delta2mRNA over the full-length variant was not observed. In addition, we find expression of three forms of IL-6 mRNA: the full-length IL-6 mRNA, IL-6Delta3 mRNA, and IL-6Delta5 mRNA in the second and third trimester placenta tissue and in the spleen of mice immunized with a high dose of sheep erythrocytes. It is anticipated that translation of these mRNA variants can generate proteins capable of binding to some subunits of the IL-6 receptor, thus possessing effector function. Alternative splicing is discussed as a source of cytokines with new regulatory properties.     

Alternative splicing of the estrogen receptor primary transcript normally occurs in estrogen receptor positive tissues and cell lines

Several laboratories have described estrogen receptor mRNA variants created by skipping internal exons. Some of the putative proteins encoded for by these variants have been functionally characterized by transfection analyses. The variant lacking exon 5 would lead, if translated, to a truncated receptor which shows dominant positive transactivation activity in the absence of hormone. It has been postulated that the variant could account for anti-estrogen resistant tumor growth and for expression of the progesterone receptor in estrogen receptor negative tumors. In order to understand the possible role this and other variants may have in the tumorigenesis of mammary tissue we have carried out a thorough analysis of variants expressed in a tumor cell line (MCF-7), in a tumor sample and in a sample of normal breast tissue derived from mammary reduction surgery. We performed rt-PCR analyses followed by hybridization with exon specific oligonucleotide probes. By these means we have detected nine different variants co-expressed in MCF-7 cells and at least the major variants were equally expressed in normal and neoplastic breast tissue. The same is true for the variant lacking exon 5 which, however, resulted to be a variant of low expression in the three samples analyzed. Variant formation appeared to be restricted to the estrogen receptor messenger since several other members of the superfamily of nuclear receptors did not show variant formation. We also have analyzed the effect of the most abundantly expressed variant, the exon 4 lacking variant, on normal estrogen receptor function, on the growth and on the response to estradiol and to tamoxifen of MCF-7 cells. Although over-expressed at high levels this variant has, if any, only marginal effects on the expression of endogenous estrogen regulated genes and on growth and response to the hormone and its antagonist. Although the lack of function of this variant cannot be extrapolated to other variants, their involvement in tumor formation appears rather unlikely since they are also expressed in normal tissue and the single variant is expressed in addition to many others, some of which might have opposing effects. Variant formation is, however, specific for the estrogen receptor and apparently regulated with tissue specificity as our expression analysis in normal mouse tissues shows. Therefore the variants probably have a physiological significance yet to be discovered.     

Characterization of Vitis vinifera L. somatic variants exhibiting abnormal flower development patterns

Mutants have proven to be a key resource for functional genomic studies in model annual plant species. In perennial plant species where mutants are difficult to generate and to screen, spontaneous somatic variants represent a unique resource to understand the genetic control of complex developmental patterns. The morphological and histological characterization of six Vitis vinifera L. somatic variants that display four different abnormal phenotypes of flower development are described here. A phenotype of reiterated reproductive meristems (RRM), with both flower and petal reiteration, was observed in a somatic variant of the cultivar Carignan. An abnormal development of reproductive organs was displayed by the unfused carpels (UFC) somatic variant of cv. Bouchalès, while a somatic variant of cv. Mourvèdre named carpel-less (CLS) developed abnormal ovules in the absence of carpels. Finally, three independent somatic variants in cvs Gamay, Morrastel, and Pinot displayed a phenotype of multiple perianth whorls (MPW). Gene expression studies showed that the expression profiles of VvMADS-box 1, 2, and 3 (putative orthologues of Arabidopsis flowering genes AG, SEP, and AGL13), were altered during grapevine flower development in the somatic variants, whereas the corresponding original cultivars displayed similar VvMADS-box gene expression profiles. Phenotypic and molecular characterization of these variants allowed the development of hypotheses on genetic functions that might be altered in most of the variants in light of the current ABCDE flower model.     

Analysis of neutralizing antigenic sites on the surface of type A12 foot-and-mouth disease virus.

A series of seven neutralizing monoclonal antibodies (nMAbs) directed against type A12 foot-and-mouth disease virus was used to generate neutralization-resistant variants. Both plaque reduction neutralization and microneutralization assays showed that the variants were no longer neutralized by the nMAbs used to generate them, although some of the variants still reacted with the nMAbs at high antibody concentrations. Results of cross-neutralization studies by both plaque reduction neutralization and microneutralization assays suggested the presence of at least one immunodominant antigenic site on the surface of type A12 foot-and-mouth disease virus, along with evidence of a second antigenic site on the viral surface. Two of the variants had reduced virulence in tissue culture as evidenced by their inability to inhibit cellular protein synthesis and a marked reduction in virus-induced cellular morphological alterations. Nucleotide sequencing of the variant genomes placed three epitopes of the major antigenic site on VP1 and the fourth epitope on VP3 and VP1. The one epitope of the minor site appears to reside only on VP1.     

An ultrastructural study of rat hepatoma cells in culture, their variants and revertants

We compared the ultrastructure of a well-differentiated rat hepatoma line (H4II) and its clonal progeny, including dedifferentiated variant cells, and revertants of the variants in which the spectrum of hepatocyte-specific functions is again expressed. The cells of the original differentiated lines and the revertants were very similar to one another. In addition, they exhibited some of the characteristics of fetal and neonatal hepatocytes. Variant cells which fail to express hepatocyte functions showed a wide range of morphological alterations accompanied by generalized disorganization. It is concluded that the loss of hepatocyte differentiation in the variants is not associated with a uniform morphological type, and that a wide range of ultrastructural phenotypes can be generated in the progeny of a single neoplastic but well-differentiated hepatocyte. Also, the expression of hepatocyte functions only occurs within a limited and organized morphological framework that includes features of young hepatocytes.     

Motility comparisons of two shape-distinctiveEntamoeba variants

Summary The motility ofEntamoeba sp. (Laredo isolate, LA) is compared to a colchicine-resistant variant (LACR) derived from it and known to exhibit physiological and morphological differences. Both variants possess contractile elements which in glycerinated cell models were activated by Mg²⁺-ATP. Short-term experiments indicated that whereas both variants are insensitive to colchicine, treatment with cytochalasin B induces a change in cell shape and loss of adhesiveness in LA cells leaving LACR relatively unaffected. Quantitative analysis of time-lapse cinematographic recordings revealed that the mean relative surface area change of LA cells was 411% per minute with a mean pseudopod extension rate of 103 m per minute. In contrast, the respective values for LACR cells were 112% per minute und 32 m per minute. Cytochalasin B treatment reduced the relative surface area change of LA cells by approximately 18-fold with a corresponding reduction in LACR cells of only 1.6-fold. Similarly, cytochalasin B inhibited pseudopod extension by 34 and 1.8-fold in LA and LACR cells respectively. Thus, we have demonstrated a clear difference in both pattern and rate of locomotion of the two variants and have shown that the colchicine resistant variant has also become relatively insensitive to treatment with CB.     

Chromosome patterns of nontransformed variants from chemically transformed Balb-3T3 cells

Nine variant cell lines isolated from cloned 7,12-dimethylbenz(a) -ahthracene transformed Balb/3T3 mouse cells by treatment with FUdR had growth parameters closely resembling nontransformed cells. Chromosome analysis of the variant lines demonstrated that six variants had a diminished number and three variants had an increased number of chromosomes compared to the parental transformed cell line. All variants had unique marker chromosomes not present in the parental transformed Balb/3T3 cells. The distribution of marker chromosomes and heterochromatin suggested that the initial event in variant formation was a reduction in chromosome number with a subsequent polyploidization of the reduced chromosome complement.