应用荧光原位杂交技术检测康柏西普基因在CHO细胞染色体上的整合状态

CHO细胞是常用的哺乳动物表达工程细胞。外源基因整合至CHO细胞染色体后,在大规模蛋白质生产过程中,由于相关压力撤除,外源基因存在丢失的可能,因此有必要对其整合稳定性进行检测。康柏西普(conbercept)是一个能够特异性结合VEGF-A的各种异构体、VEGF-B以及PlGF,从而发挥抗血管生成活性的融合蛋白。康柏西普目前已在美国进入Ⅲ期临床试验。本文运用荧光原位杂交对康柏西普基因在CHO细胞的整合状态进行了检测,发现经过4和19次传代后,康柏西普基因依然能稳定整合在基因组上,并且呈现出3个特点：(1)分布在一条染色体上,而不是多条染色体上;(2)分布在较长的染色体上;(3)在同一染色体上有较多拷贝数。同时,荧光定量PCR结果证明基因拷贝数无明显改变,ELISA检测证明蛋白表达水平亦无明显改变。上述实验证明在经过19次传代以后,康柏西普基因仍然稳定整合在基因组中,并可活跃表达,为康柏西普大规模生产及产品质控提供了有力依据。     

莲C_0t-1 DNA的荧光原位杂交分析

为分析中国莲C_0t-1 DNA在其中期染色体上的分布,从中国莲基因组DNA中分离出C_0t-1 DNA,将基因组和所分离的C_0t-1 DNA用生物素标记后作探针,对中国莲染色体进行原位杂交。杂交结果用耦联有荧光素Cy3的生物素抗体检测,发现在每对染色体上均显示出特定的荧光原位杂交带。同时分析了FISH和GISH信号分布的异同。基于C_0t-1 DNA荧光原位杂交带型及染色体型,构建了中国莲核型。     

莲C0t-1 DNA的荧光原位杂交分析

为分析中国莲Cot-1DNA在其中期染色体上的分布,从中国莲基因组DNA中分离出Cot-1DNA,将基因组和所分离的Cot-1DNA用生物素标记后作探针,对中国莲染色体进行原位杂交。杂交结果用耦联有荧光素Cy3的生物素抗体检测,发现在每对染色体上均显示出特定的荧光原位杂交带。同时分析了FISH和GISH信号分布的异同。基于Cot-1DNA荧光原位杂交带型及染色体型,构建了中国莲核型。     

苜蓿核糖体基因物理定位及染色体荧光分带

利用核糖体基因为探针对,二倍体和四倍体苜蓿(Medicago sativa)进行原位杂交,结果表明,45s在四倍体、二倍体种中总是以单位点位于核仁组织区,5s则有2～3个位点;以二倍体种的基因组DNA为探针的原位杂交表明,蓝花苜蓿(M.coerulea)和黄花苜蓿(M.falcata)均能与四倍体染色体进行杂交,仅杂交信号强弱的染色体数目有差别;荧光染料DAPI使苜蓿的染色体显示带纹,蓝花苜蓿的DAPI带与C-带基本一致.文章对四倍体苜蓿的可能来源进行了讨论.     

Chromosomal domains of chimpanzee are diverged from human as revealed by in situ hybridization using human genomic probe

Utilization of repetitive DNA probes to assess the taxonomic affinity between related species has become the most powerful tool in evolutionary biology today. Consequently, tremendous strides have recently been made towards establishing the phylogenetic relationship of humans with chimpanzee. We employed human genomic proe (P5080 B.5) to identify the degree of divergence of chimpanzee genome from humans. A small protion of structurally distinct genomic areas in chimpanzee could be identified by fluorescencein situ hybridization (FISH) technique when compared to human DNA. The genomic divergence is confined mainly to the chromosomal ends in chimpanzee and may be an important phylogenetic characteristic in human evolution.     

In situ hybridization to metaphase chromosomes in six species ofPhaseolus andVigna using ribosomal DNA as the probe

In situ hybridization with a biotin-labeled rice ribosomal DNA (rDNA) probe to the somatic metaphase chromosomes of six species ofPhaseolus andVigna (P. angularis, P. calcaratus, P. coccineus, P. vulgaris, V. sesquipedalis andV. sinensis) was done to determine the sites of rDNA. Hybridization signals were present in the terminal and subterminal chromosome regions of each of the six species. The number of rDNA sites was two inP. angularis andP. calcaratus, four inP. coccineus andP. vulgaris, and six inV. sesquipedalis andV. sinensis.     

The pachytene chromosomes of maize as revealed by fluorescence in situ hybridization with repetitive DNA sequences

A repetitive DNA sequence, ZmCR2.6c, was isolated from maize based on centromeric sequence CCS1 of the wild grass Brachypodium sylvaticum. ZmCR2.6c is 309 bp in length and shares 65% homology to bases 421–721 of the sorghum centromeric sequence pSau3A9. Fluorescence in situ hybridization (FISH) localized ZmCR2.6c to the primary constrictions of pachytene bivalents and to the stretched regions of MI/AI chromosomes, indicating that ZmCR2.6c is an important part of the centromere. Based on measurements of chromosome lengths and the positions of FISH signals of several cells, a pachytene karyotype was constructed for maize inbred line KYS. The karyotype agrees well with those derived from traditional analyses. Four classes of tandemly repeated sequences were mapped to the karyotype by FISH. Repeats 180 bp long are present in cytologically detectable knobs on 5L, 6S, 6L, 7L, and 9S, as well as at the termini and in the interstitial regions of many chromosomes not reported previously. A most interesting finding is the presence of 180-bp repeats in the NOR-secondary constriction. TR-1 elements co-exist with 180-bp repeats in the knob on 6S and form alone a small cluster in 4L. 26S and 5S rRNA genes are located in the NOR and at 2L.88, respectively. The combination of chromosome length, centromere position, and distribution of the tandem repeats allows all chromosomes to be identified unambiguously. The results presented form an important basis for using FISH for physical mapping and for investigating genome organization in maize. Received: 29 June 1999 / Accepted: 10 November 1999     

Localization of the T-DNA on marker chromosomes in transformed tobacco cells by in situ hybridization

Summary Chromosome and molecular analyses were conducted on tobacco cells which had been transformed by the T-DNA of the Ti-plasmid. These analyses showed that there were specific chromosome rearrangements in the transformed cells (marker chromosomes). There was a positive correlation between the number of marker chromosomes per cell and the oncogenic potential of the transformed cells. However, we show, using the Southern hybridization method, that the T_L fragment of T-DNA, but not the T_R, clearly hybridizes with nuclear DNA. In situ hybridization was used to locate the insertion site of T-DNA: the hybridization signal was found on a small metacentric chromosome. This chromosome may occur single or translocated onto other chromosomes, to make marker chromosomes. Thus, by locating the T-DNA, we have confirmed the correlation between the marker chromosomes and the oncogenic potential.     

Physical mapping of ribosomal RNA genes in peonies (Paeonia,Paeoniaceae) by fluorescent in situ hybridization: implications for phylogeny and concerted evolution

Physical maps of the 18S–5.8S–26S ribosomal RNA genes (rDNA) were generated by fluorescent in situ hybridization for five diploid Paeonia species, P. delavayi and P. rockii of section Moutan, and P. emodi, P. tenuifolia, and P. veitchii of section Paeonia. Of five pairs of mitotic chromosomes, rDNA loci were mapped near the telomeres of chromosomes 3, 4, and 5 of P. rockii and P. tenuifolia, chromosomes 2, 3, 4, and 5 of P. delavayi, and all five pairs of chromosomes of P. emodi and P. veitchii. Combining this information with the previously obtained rDNA maps of P. brownii and P. californica of section Oneapia, we hypothesized that the most recent common ancestor of extant peony species had three rDNA loci located on chromosomes 3, 4, and 5. Increase in number of rDNA loci occurred later in each of the three sections, and the increase from three to four loci represents a parallel gain of an rDNA locus on chromosome 2 in P. delavayi of section Moutan and P. brownii of section Oneapia. The increase in number of rDNA loci likely resulted from the translocation of rDNA repeats from chromosomes bearing rDNA loci to chromosomes without them; such translocation is probably facilitated by the telomeric location of rDNA loci. For allotetraploid peony species lacking polymorphism in sequences of the internal transcribed spacers (ITS) of rDNA, the rDNAs derived from divergent diploid parents may have been homogenized through concerted evolution among at least six rDNA loci in the allotetraploids. Chromosomal location of rDNA loci has a more substantial impact on the tempo of concerted evolution than the number of loci.     

Nucleologenesis: use of non-isotopic in situ hybridization and immunocytochemistry to compare the localization of rDNA and nucleolar proteins during mitosis

Using in situ hybridization and immunocytochemistry during interphase and mitosis, we have compared the distribution of ribosomal DNA (rDNA) to that of the nucleolar proteins fibrillarin and RNA polymerase I. During interphase, nucleolar proteins were localized at sites throughout the nucleolus while the bulk of rDNA was localized in a single restricted nucleolar area. During metaphase and anaphase, all six NORs were detected by in situ hybridization, Ag-staining, or by the immunolocalization of RNA polymerase I. During telophase, rDNA and RNA polymerase I were found in a distinct subset of the prenucleolar bodies (PNBs) which obviously must contain the nucleolar organizers. Other numerous PNBs are smaller in size and do not contain detectable amounts of rDNA or RNA polymerase I. Therefore, reconstruction of the nucleolus originates in telophase-specific domains which contain both rDNA and RNA polymerase I.     

Recombinant chromosomes of advanced backcross plants between Allium cepa L. and A. fistulosum L. revealed by in situ hybridization

Cytological analysis of (Allium cepa L.×Allium fistulosum L.)×A. cepa L. F₁BC₃ plants revealed most plants were diploid with 16 chromosomes. Karyotypes of these plants showed recombinant chromosomes. Fluorescence and genomic in situ hybridization patterns of interspecific F₁ hybrid and F₁BC₃ plants revealed A. fistulosum chromosomes or chromosomal segments. A highly repetitive 376-bp DNA sequence and genomic DNA of A. fistulosum revealed similar telomeric hybridization sites when hybridized onto A. fistulosum chromosomes. Cytogenetic evidence showed that A. fistulosum DNA has recombined into the A. cepa genome. Received: 20 October 1999 / Accepted: 11 November 1999     

Fluorescence in situ hybridization using an old world monkey Y chromosome specific probe combined with immunofluorescence staining on rhesus monkey tissues.

To date, there is no commercially available Y chromosome probe that can be used for fluorescence in situ hybridization (FISH) for the male rhesus monkey. We have recently generated a probe for FISH with high specificity to the short arm of the rhesus monkey Y chromosome. In this study, we further describe a method that keeps the integrity of tissue-specific antigenic structures for immunofluorescence staining subsequent to FISH on paraffin-embedded rhesus monkey tissues. We have examined this technique in combination with an epithelial cell-specific marker, cytokeratin 8/18 (CK8/18), on various tissues, including jejunum, liver, kidney, and pancreas. CK8/18 and Y chromosome signals were distinctly seen simultaneously on epithelial cells from the same tissue section from male but not female monkeys. These studies indicate that our FISH immunofluorescence technique can be reliably used to identify and phenotype male cells in paraffin-embedded rhesus monkey tissues.     

A new multicolor fluorescence in situ hybridization probe set directed against human heterochromatin: HCM-FISH

A new multicolor fluorescence in situ hybridization (mFISH) probe set is presented, and its possible applications are highlighted in 25 clinical cases. The so-called heterochromatin-M-FISH (HCM-FISH) probe set enables a one-step characterization of the large heterochromatic regions within the human genome. HCM-FISH closes a gap in the now available mFISH probe sets, as those do not normally cover the acrocentric short arms; the large pericentric regions of chromosomes 1, 9, and 16; as well as the band Yq12. Still, these regions can be involved in different kinds of chromosomal rearrangements such as translocations, insertions, inversions, amplifications, and marker chromosome formations. Here, examples are given for all these kinds of chromosomal aberrations, detected as constitutional rearrangements in clinical cases. Application perspectives of the probe set in tumors as well as in evolutionary cytogenetic studies are given.     

Visualization of ribosomal DNA loci in spore interphasic nuclei of glomalean fungi by fluorescence in situ hybridization

 Fluorescence in situ hybridization (FISH) was applied to interphasic nuclei isolated from spores of four species of AM fungi : Scutellospora castanea, Glomus mosseae, Glomus intraradices and Gigaspora rosea. Ribosomal DNA loci were visualized using digoxigenin-labeled 25 S rDNA probes obtained by nested PCR. Several hybridization sites were detected per nucleus and an internuclear variability was observed in the number of loci. This is the first report of successful application of FISH to analyse the genomes of glomalean fungi. Accepted: 16 September 1998     

Interphase fluorescence in situ hybridization mapping: a physical mapping strategy for plant species with large complex genomes

The chromatin in interphase nuclei is much less condensed than are metaphase chromosomes, making the resolving power of fluorescence in situ hybridization (FISH) two orders of magnitude higher in interphase nuclei than on metaphase chromosomes. In mammalian species it has been demonstrated that within a certain range the interphase distance between two FISH sites can be used to estimate the linear DNA distance between the two probes. The intephase mapping strategy has never been applied in plant species, mainly because of the low sensitivity of the FISH technique on plant chromosomes. Using a CCD (charge-coupled device) camera system, we demonstrate that DNA probes in the 4 to 8 kb range can be detected on both metaphase and interphase chromosomes in maize. DNA probes pA1-Lc and pSh2.5·SstISalI, which contain the maize locia1 andsh2, respectively, and are separated by 140 kb, completely overlapped on metaphase chromosomes. However, when the two probes were mapped in interphase nuclei, the FISH signals were well separated from each other in 86% of the FISH sites analyzed. The average interphase distance between the two probes was 0.50 µm. This result suggests that the resolving power of interphase FISH mapping in plant species can be as little as 100 kb. We also mapped the interphase locations of another pair of probes, ksu3/4 and ksu16, which span theRp1 complex controlling rust resistance of maize. Probes ksu3/4 and ksu16 were mapped genetically approximately 4 cM apart and their FISH signals were also overlapped on metaphase chromosomes. These two probes were separated by an average of 2.32 µm in interphase nuclei. The possibility of estimating the linear DNA distance between ksu3/4 and ksu16 is discussed.     

Effect of zinc on cadmium influx and toxicity in cultured CHO cells

Addition of zinc lowers the toxicity level of cadmium in cultured CHO cells. Cell survival and protein synthesis were used to measure the cellular toxicity of cadmium.¹⁰⁹Cd was used to measure cadmium uptake by the cells. The results suggest that these class IIB transition metals, zinc and cadmium, share a common transport mechanism. Thus, the antagonism appears to involve a reduction in the influx of cadmium due to the presence of zinc.     

Assessment of the state of activity of individual bacterial cells by hybridization with a ribosomal RNA targeted fluorescently labelled oligonucleotidic probe

Abstract A fluorescently labelled oligonucleotide probe complementary to an evolutionarily conserved domain of the small subunit ribosomal RNA sequence has been used with an image analysis equipment to quantify cellular rRNA contents during the successive phase of a bacterial culture ( Escherichia coli ). The amount of hybridization increased steadily upon inoculation of stationary phase bacteria into a new medium, while cell divisions were delayed. This hybridization signal was abruptly reduced by 50% at the onset of the exponential growth phase during which it then remained stable. A further slow decrease took place during stationary phase. The amount of rRNA per cell is thus maximal immediately before the beginning of active cell division. The implications of these results are discussed in terms of bacterial ecology.