Structure analysis and performance of a microbial community from a contaminated aquifer involved in the complete reductive dechlorination of 1,1,2,2-tetrachloroethane to ethene

An anaerobic microcosm set up with aquifer material from a 1,1,2,2-tetrachloroethane (TeCA) contaminated site and amended with butyrate showed a complete TeCA dechlorination to ethene. A structure analysis of the microbial community was performed by fluorescence in situ hybridization (FISH) with already available and on purpose designed probes from sequences retrieved through 16S rDNA clone library construction. FISH was chosen as identification tool to evaluate in situ whether the retrieved sequences belong to primary bacteria responsible for the biodegradative reactions. FISH probes identified up to 80% of total bacteria and revealed the absence or the marginal presence of known TeCA degraders and the abundance of two well-known H(2)-utilizing halorespiring bacteria, Sulfurospirillum (32.4 +/- 8.6% of total bacteria) and Dehalococcoides spp. (14.8 +/- 2.8), thereby providing a strong indication of their involvement in the dechlorination processes. These results were supported by the kinetic and thermodynamic analysis which provided indications that hydrogen was the actual electron donor for TeCA dechlorination. The specific probes, developed in this study, for known dechlorinators (i.e., Geobacter, Dehalobacter, and Sulfurospirillum species) represent a valuable tool for any future in situ bioremediation study as well as a quick and specific investigation tool for tracking their distribution in the field.     

Modeling the interaction of a neutral polymer with small ions in solution. A theoretical study of ion binding to a neutral polymer

The interaction between NaCl and 1,2-dimethoxyethylene is used to model ion binding to a neutral polymer. The relative binding energies involving different ion-polymer structures were calculated using minimal basis STO-3G and split valence 3-21Gab-initio and CNDO/2 semi-empirical wavefunctions. The results obtained are consistent with an adsorption model in which the cation is primarily adsorbed on the oxygen sites. The counter ion is then bound by the charged polymer in a form consistent with an ion pair structure. These results are consistent with recent experimental studies on polyoxyethylene (POE). Additional calculations were performed to include specific interactions with the liquid environment. Electrostatic solvation effects, introduced through the self-consistent reaction field (SCRF) model, appear to be significant in the stabilization of a solvent-separated ion-pair structure. However, the global stabilization produced by both specific and electrostatic solvations predicts the preferential stabilization of an externally hydrated intimate ion pair.Dedicated to the memory of the late Professor Luis Sepulveda (1988).CMCA Contribution No 3.     

Generation of a MOR‐CreER knock‐in mouse line to study cells and neural circuits involved in mu opioid receptor signaling

Mu opioid receptor (MOR) is involved in various brain functions, such as pain modulation, reward processing, and addictive behaviors, and mediates the main pharmacologic effects of morphine and other opioid compounds. To gain genetic access to MOR‐expressing cells, and to study physiological and pathological roles of MOR signaling, we generated a MOR‐CreER knock‐in mouse line, in which the stop codon of the Oprm1 gene was replaced by a DNA fragment encoding a T2A peptide and tamoxifen (Tm)‐inducible Cre recombinase. We show that the MOR‐CreER allele undergoes Tm‐dependent recombination in a discrete subtype of neurons that express MOR in the adult nervous system, including the olfactory bulb, cerebral cortex, striosome compartments in the striatum, hippocampus, amygdala, thalamus, hypothalamus, interpeduncular nucleus, superior and inferior colliculi, periaqueductal gray, parabrachial nuclei, cochlear nucleus, raphe nuclei, pontine and medullary reticular formation, ambiguus nucleus, solitary nucleus, spinal cord, and dorsal root ganglia. The MOR‐CreER mouse line combined with a Cre‐dependent adeno‐associated virus vector enables robust gene manipulation in the MOR‐enriched striosomes. Furthermore, Tm treatment during prenatal development effectively induces Cre‐mediated recombination. Thus, the MOR‐CreER mouse is a powerful tool to study MOR‐expressing cells with conditional gene manipulation in developing and mature neural tissues.     

Regulation of c‐Ret,GFRα1, and GFRα2 in the substantia nigra Pars compacta in a rat model of Parkinson's disease

Glial cell line‐derived neurotrophic factor (GDNF) family members have been proposed as candidates for the treatment of Parkinson's disease because they protect nigral dopaminergic neurons against various types of insult. However, the efficiency of these factors depends on the availability of their receptors after damage. We evaluated the changes in the expression of c‐Ret, GFRα1, and GFRα2 in the substantia nigra pars compacta in a rat model of Parkinson's disease by in situ hybridization. Intrastriatal injection of 6‐hydroxydopamine (6‐OHDA) transiently increased c‐Ret and GFRα1 mRNA levels in the substantia nigra pars compacta at 1 day postlesion. At later time points, 3 and 6 days, the expression of c‐Ret and GFRα1 was downregulated. GFRα2 expression was differentially regulated, as it decreased only 6 days after 6‐OHDA injection. Triple‐labeling studies, using in situ hybridization for the GDNF family receptors and immunohistochemistry for neuronal or glial cell markers, showed that changes in the expression of c‐Ret, GFRα1, and GFRα2 in the substantia nigra pars compacta were localized to neurons. In conclusion, our results show that nigral neurons differentially regulate the expression of GDNF family receptors as a transient and compensatory response to 6‐OHDA lesion. © 2002 Wiley Periodicals, Inc. J Neurobiol 52: 343–351, 2002     

玉米mir1基因在玉米和薏苡中的比较物理定位

玉米基因mir1编码一种抗秋季黏虫的半胱氨酸蛋白酶。利用RFLP作图mir1基因被定位在玉米第 6号染色体短臂上 ,但它在第 6号染色体短臂上的物理位置还不知道。实验以mir1和 4 5SrDNA为探针 ,通过双色荧光原位杂交技术确定了mir1基因在玉米细胞分裂中期和粗线期第 6号染色体上的物理位置。Southern杂交结果表明 ,在薏苡基因组中存在mir1基因的同源序列 ,进一步利用荧光原位杂交的方法确定mir1基因的同源序列定位于薏苡第 7号染色体长臂的近末端 ,其信号与着丝粒的百分距离为 73 33± 0 15。     

The development of a large nucleolus during oogenesis in Acanthocyclops vernalis (Crustacea, Copepoda) and its possible relationship to chromatin diminution

The development of a single, very large (25-35 microns diameter) nucleolus during oogenesis in the crustacean Acanthocyclops vernalis is described. The nucleolus is the site of ribosomal RNA production in the egg, as shown by in situ hybridization, and apparently the only source, as accessory cells are not observed. Ribosomal DNA amplification, as manifested by the presence of multiple nucleoli, is also not observed. Silver staining and C-banding suggest that chromosomal regions other than the nucleolar organizer are involved with the elaboration of the nucleolus. These observations, along with what is known about the nature of the DNA lost during the developmental process of chromatin diminution in this organism, suggest a relationship between the large oocyte nucleolus and the DNA lost during diminution.     

Persistence of chromosomal alterations affecting the 1cen-q12 region in a human lymphoblastoid cell line exposed to diepoxybutane and mitomycin C

Multicolor fluorescence in situ hybridization (FISH) with tandem-labeling probes for the 1cen-q12 region is a potential biomarker for the detection of structural chromosomal aberrations (CAs) in human cells. To determine the suitability of this technique for biomonitoring humans exposed to 1,3-butadiene (BD) and to characterize the alterations induced as well as their stability over time, the human lymphoblastoid cell line AZH-1 was treated with 5 μM diepoxybutane (DEB) or the positive control mitomycin C (MMC; 0.1 μM) for 24 h. Following the removal of the test chemicals, cell cultures were grown for an additional 19 days in the absence of the test compound. Using the tandem FISH technique, aliquots from the main cultures were examined for the induction of CAs affecting the 1cen-q12 region at various intervals. A significant increase in chromosomal breakage/exchanges affecting the 1cen-q12 region was seen in both the DEB- and MMC-treated interphase and metaphase cells. The damage peaked at approximately 48 h following the addition of the test compound and declined with time. However, at day 20, the frequency of aberrant cells was still significantly higher than the control levels. For comparison, the frequency of micronuclei (MN) formed and their origin was determined using the cytochalasin B-modified MN assay and FISH with a pancentromeric probe. Showing a similar pattern, the frequency of centronere-negative MN peaked at 48 h, but however was not significantly elevated above control levels at 20 days. At early time points, aberrations detected using the FISH assay consisted of nearly equal proportions of unstable- and stable-type aberrations, while at the later time points, translocations were the predominant aberration type. In addition, the use of tandem-label FISH in combination with BrdU-immunfluorescence staining, showed that almost identical frequencies of structural aberrations could be seen in actively replicating and non-replicating cell populations. These studies indicate that a small but significant proportion of the alterations detected using this FISH technique persists over time and that this technique may be valuable for biomonitoring chromosomal alterations in BD-exposed populations.     

滨麦抗条锈病基因的染色体定位和分子标记

从滨麦与普通小麦杂交后代中筛选到一条抗条锈病的小滨麦品系93784。以滨麦基因组DNA为探针的荧光原位杂交结果表明,93784是小麦与滨麦的小片段易位系,易位的滨麦染色体片段位于一对小麦染色体的短臂端部,利用该易位系构建了F2分离群体,进行F2单株成株期抗条锈鉴定,抗性分析证明,小滨麦93784中的抗条锈病基因是单基因控制的,位于滨麦染色体的易位片段上,命名为YrLm。进一步采用24对TaqⅠ（T1-T4）/PstⅠd(P1-P6)引物组合对抗感亲本及F2分离群体进行AFLP分析,筛选出一个与抗条锈病基因YrLm连锁的AFLP分子标记,经克隆和测序,该标记片段长度为205bp,定名为P1T3205。     

Localized expression of a proline-rich protein gene in juvenile and mature ivy petioles in relation to rooting competence

Cell division and root initiation of excised juvenile, mature and half-expanded mature (My) leaf petioles of Hedera helix L. cultured in vitro were studied to determine whether these processes were correlated with localized expression of a proline-rich protein (PRP) gene. Petioles of all three types showed cell divisions at day 5 of culture in auxin-treated petioles but not in non-auxin-treated petioles. No cell division occurred in non-auxin-treated petioles even after day 9 of culture. Juvenile and one population of My auxin-treated petioles formed root primordia after 9 days of culture. Mature petioles and another population of My petioles formed only callus in response to auxin. The spatial and temporal expression pattern of a gene encoding a PRP was analyzed by in situ hybridization. The PRP mRNA was not detectable in petioles of any developmental phase immediately after excision. In both juvenile and mature petioles the PRP mRNA preferentially accumulated in the phloem parenchyma, the inner cortex adjacent to the phloem, and in cells surrounding ducts. Cell division was not required for PRP gene expression since both auxin-treated and non-treated juvenile and mature petioles had expression. Steady state levels of PRP mRNA were much lower in juvenile relative to mature petioles cultured in vitro. Auxin treatment reduced the steady state levels of PRP mRNA in My petioles but not in mature or juvenile petioles. These data are consistent with an inverse relationship between competence to form adventitious roots and PRP mRNA levels in the specific cell types from which root primordia form. Alternatively, the PRP mRNA level may serve as a molecular marker for developmental plasticity for root initiation.     

Genome Analysis of a Natural Hybrid with 2n= 63 Chromosomes in the Genus Elytrigia Desv. (Poaceae) Using the GISH Technique

Abstract: Genomic in situ hybridization (GISH), using genomic DNA probes from Thinopyrum elongatum (E genome, 2 n = 14), Th. bessarabicum (J genome, 2 n = 14), Pseudoroegneria stipifolia (S genome, 2 n = 14), Agropyron cristatum (P genome, 2 n = 28) and Critesion californicum (H genome, 2 n = 14), was used to identify the genome constitution of a natural hybrid population morphologically close to Elytrigia pycnantha and with somatic chromosome number of 2 n = 63. The GISH results indicated the presence of a chromosomal set more or less closely related to the E, P, S and H genomes. In particular, two sets of 14 chromosomes each showed close affinity to the E genome of Th. elongatum and to the P genome of A. cristatum. However, they included 2 and 10 mosaic chromosomes, respectively, with S genome specific sequences at their centromeric regions. Two additional sets (28 chromosomes) appeared to be very closely related to the S genome of Ps. stipifolia. The last genome involved (7 chromosomes) is related to the H genome of C. californicum but includes one chromosome with S genome-specific sequences around the centromere and two other chromosomes with a short interstitial segment also containing S genome related sequences. On a basis of GISH analysis and literature data, it is hypothesized that the natural 9-ploid hybrid belongs to the genus Elytrigia and results from fertilization of an unreduced gamete (n = 42) of E. pycnantha and a reduced gamete (n = 21) of E. repens. The genomic formula SSSSP^SP^SE^SE^SH^S is proposed to describe its particular genomic and chromosomal composition.     

玉米中抗病基因myb1和NDR1同源序列的荧光原位杂交物理定位

以烟草和拟南芥中的单拷贝抗病基因ｍｙｂ１和ＮＤＲ１作探针,利用荧光原位杂交的方法分别对这两个基因在玉米（Ｚｅａ ｍａｙｓ Ｌ．）和烟草（Ｎｉｃｏｔｉａｎａ ｔａｂａｃｕｍ Ｌ．）、玉米和拟南芥（Ａｒａｂｉｄｏｐｓｉｓ ｔｈａｌｉａｎａ（Ｌ．）Ｈｅｙｎｈ．）中的同源性做了研究。杂交结果表明ｍｙｂ１和ＮＤＲ１的同源序列分别位于玉米第８、５染色体,单个信号位置表明０这两个基因的同源序列在玉米基因组中只有     

Comparative mapping of sheep inhibin subunit βb to chromosome 2 in sheep and cattle by fluorescence in situ hybridization

A cosmid clone containing the complete sheep inhibin subunit β_B gene (INHBB) was assigned to sheep and cattle homologous chromosome bands 2q31-q33 by fluorescence in situ hybridization. The assignment of INHBB in sheep excludes another candidate gene as the site of the Fec^B mutation.     

Design of species-specific oligonucleotide probes for the detection of Bacteroides and Parabacteroides by fluorescence in situ hybridization and their application to the analysis of mouse caecal Bacteroides-Parabacteroides microbiota

Aims: To develop species‐specific monitoring techniques for rapid detection of Bacteroides and Parabacteroides inhabiting the mouse intestine by fluorescence in situ hybridization. Methods and Results: The specificity of oligonucleotide probes was evaluated by fluorescence whole‐cell hybridization. Oligonucleotide probes specific for each species hybridized only with the target bacteria. Using these probes, caecal Bacteroides–Parabacteroides microbiota of conventional mice and specific pathogen‐free (SPF) mice from three different breeders were analysed. It was shown that Bacteroides acidifaciens Group‐1, Group‐2 and Group‐3 were dominant in conventional mice and SPF mice from two out of three breeders. Bacteroides vulgatus and Parabacteroides distasonis were detected in one of these two SPF breeding colonies in addition to Bact. acidifaciens. SPF mice of the remaining breeder harboured characteristic Bacteroides–Parabacteroides microbiota, consisting of Bacteroides sp. ASF519 and Bacteroides caccae. Conclusions: Bacteroides acidifaciens is the dominant and most typical species in the mouse Bacteroides–Parabacteroides microbiota. The Group‐3 was identified as a novel group and revealed to occupy a major niche together with Bact. acidifaciens Group‐1 and Group‐2. Significance and Impact of the Study: The species‐specific probe set developed in this study was the efficient tool for rapid detection of target bacterial groups inhabiting the mouse intestine. The results of this study provide important new information on the mouse Bacteroides–Parabacteroides community.     

Characterization of the KNOX class homeobox genes Oskn2 and Oskn3 identified in a collection of cDNA libraries covering the early stages of rice embryogenesis

For identification of genes involved in embryogenesis in the model cereal rice, we have constructed a collection of cDNA libraries of well-defined stages of embryo development before, during and after organ differentiation. Here, we focus on the possible role of KNOX (maize Knotted1-like) class homeobox genes in regulation of rice embryogenesis. Three types of KNOX clones were identified in libraries of early zygotic embryos. Two of these, Oskn2 and Oskn3, encode newly described KNOX genes, whereas the third (Oskn1) corresponds to the previously described OSH1 gene. In situ hybridizations showed that during the early stages of embryo development, all three KNOX genes are expressed in the region where the shoot apical meristem (SAM) is organizing, suggesting that these genes are involved in regulating SAM formation. Whereas OSH1 was previously proposed to function also in SAM maintenance, Oskn3 may be involved in patterning organ positions, as its expression was found to mark the boundaries of different embryonic organs following SAM formation. The expression pattern of Oskn2 suggested an additional role in scutellum and epiblast development. Transgenic expression of Oskn2 and Oskn3 in tobacco further supported their involvement in cell fate determination, like previously reported for Knotted1 and OSH1 ectopic expression. Whereas Oskn3 transformants showed the most pronounced phenotypic effects during vegetative development, Oskn2 transformants showed relatively mild alterations in the vegetative phase but a more severly affected flower morphology. The observation that the KNOX genes produce similar though distinct phenotypic reponses in tobacco, indicates that their gene products act on overlapping but different sets of target genes, or that cell-type specific factors determine their precise action.     

Ecophysiology of polyphosphate-accumulating organisms and glycogen-accumulating organisms in a continuously aerated enhanced biological phosphorus removal process

Aims: To investigate the ecophysiology of populations of polyphosphate-accumulating organisms (PAO) and glycogen-accumulating organisms (GAO) in communities of a novel acetate fed process removing phosphate from wastewater. Attempts were made to see if acetate could be replaced by an alternative carbon source which did not support the growth of the GAO. Methods and Results: A continuously aerated sequencing batch reactor was operated with different acetate feed levels. Fluorescence in situ hybridization (FISH) showed that Defluviicoccus GAO numbers increased at lower acetate feed levels. With FISH/microautoradiography (MAR) both detected morphotypes of Defluviicoccus assimilated a wider range of substrates aerobically than Accumulibacter PAO. Their uptake profile differed from that reported for the same phylotype in full scale anaerobic : aerobic EBPR plants. Conclusions: This suggests that replacing acetate with another substrate is unlikely to provide Accumulibacter with a selective advantage in this process. Why Defluviicoccus appeared to out-compete Accumulibacter at lower acetate concentrations was not clear. Data suggest physiological and morphological diversity may exist within a single Defluviicoccus phylotype. Significance and Impact of the Study: This study implies that the current FISH probes for Defluviicoccus GAO may not reveal the full extent of their biodiversity, and that more information is required before strategies for their control can be devised.