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21.
Frauke Behrens Uwe Claussen Leslie M. Iyer Eric D. Green Bernhard Horsthemke Robert Williamson Clare Huxley Charles Coutelle 《Chromosome research》1997,5(4):215-220
Centromeres remain the least characterized regions of human chromosomes because they have a very high content of repetitive DNA. Here, we describe a micro-dissection library from the centromeric region of human chromosome 7 and its use for generating sequence tagged sites (STSs). The library contains about 1500 clones with an average insert size of 150 bp and only about 15% of the clones harbour repetitive human DNA. Seven clones hybridizing to alphoid DNA were found to correspond to a fragment of the D7Z2 alphoid array on chromosome 7, thus confirming the origin of the library. A number of clones not containing known repetitive DNA were used to generate STSs that identified yeast artificial chromosomes (YACs) and in turn allowed the STSs to be placed on the physical map. One STS is located between the two Genethon genetic markers closest to the centromere on the q side. Another STS was located 3–4 cM away in 7q11.2, while a third identified YACs containing both low-copy and alphoid sequences that are not yet mapped but are clearly centromeric. The library therefore comprises a collection of sequences from the centromeric region of chromosome 7 that can be used to generate STSs and to map the entire centromeric region.This revised version was published online in November 2005 with corrections to the Cover Date. 相似文献
22.
cDNA arrays are a powerful tool for the identification of differentially expressed genes in malignant tumors. We used this technique to study the gene expression profiles of anaplastic large cell lymphoma (ALCL) and Hodgkin's disease (HD). Gene expression of 11 lymphoma cell lines was analyzed covering 1176 cDNA sequences. Comparing these data to the expression profiles of B- and T-lymphocytes, we identified 27 genes that were deregulated in all cell lines or in a particular entity. For the establishment of gene expression profiles the 27 genes were assigned to four groups composed of genes deregulated in (i) all lymphoma cell lines, (ii) ALCL and HD, (iii) only HD, and (iv) ALCL exclusively. Our results indicate that ALCL and HD share the differential expression of at least five genes. In addition, both entities are characterized by the differentially deregulated expression of four genes in HD and seven genes in ALCL. Because the expression profiling was performed on cell lines, further studies are needed to clarify the biological significance of the differentially expressed genes. 相似文献
23.
CLONING AND EXPRESSION OF cDNA FOR HUMAN LYMPHO-TOXIN 总被引:1,自引:0,他引:1
人淋巴毒素(hLT)系由淋巴细胞经抗原或有丝分裂原活化后产生的一类细胞因子,它具有抗瘤、抗病毒活性和许多重要的免疫调节作用,是一种非常有前途的生物制剂。近年来发现的膜相关型淋巴毒素更提示hLT可能具有尚未被揭示的免疫调节活性。因此,克隆人LTcDNA并在大肠杆菌表达重组hLT,对于hLT的开发利用和研究其功能都具有重要意义。本实验按照公布的hLTcDNA序列,经计算机分析并结合实验要求设计并合成一对PCR引物,采用RT-PCR技术从PHA/PMA活化24h的人T细胞系Jurkat细胞总RNA扩增出一541bpDNA片段;经α-互补筛选,质粒小量快速抽提,限制性内切酶酶切鉴定,将该片段定向克隆于pUC18、pUC19质粒载体。限制性内切酶图谱分析和Sanger双脱氧链终止法序列测定表明:该DNA片段与公布的人淋巴毒素cDNA序列完全一致。它包括编码人淋巴毒素成熟肽的全部cDNA序列。再进一步将该cDNA片段克隆于原核表达载体pBV220,经地高辛标记探针菌落原位杂交筛选,限制性内切酶酶切鉴定方向,筛选出一阳性重组子pBV-hLT。SDS-PAGE和Westernbloting分析表明:经温控诱导,该重组菌成功 相似文献
24.
Histidine-rich protein genes and their transcripts in Plasmodium falciparum and P. lophurae 总被引:2,自引:0,他引:2
M Wallach D F Cully L O Haas W Trager G A Cross 《Molecular and biochemical parasitology》1984,12(1):85-94
The presence of histidine-rich protein (HRP) related genes and gene products in Plasmodium falciparum was demonstrated using a synthetic pentahistidine-encoding oligonucleotide and a cloned HRP cDNA probe prepared from the avian parasite P. lophurae. In Northern blotting experiments, two knobby clones of P. falciparum were found to contain a 3500 nucleotide RNA species that hybridized with the oligonucleotide and HRP cDNA probes. As this component had the expected size for an mRNA encoding an 80-90 kDa protein and was absent from two knobless clones of P. falciparum, we concluded that it represented a 'knob protein' mRNA. Using the restriction enzyme EcoRI, three identical cross-hydribizing HRP gene fragments were found in the DNA of both knobby and knobless clones of P. falciparum. These fragments differed in size from those present in P. lophurae. These results suggest that the absence of knob protein mRNA in knobless clones is not due to loss of the corresponding gene(s). 相似文献
25.
We report three possibly disease-causing point mutations in one of the inner-ear-specific genes, KIAA1199. We identified an R187C mutation in one family, an R187H mutation in two unrelated families, and an H783Y mutation in one sporadic case of nonsyndromic hearing loss. In situ hybridization indicated that the murine homolog of KIAA1199 mRNA is expressed specifically in Deiters cells in the organ of Corti at postnatal day zero (Pn) P0 before the onset of hearing, but expression in those cells disappears by day P7. The signal of KIAA1199 was also observed in fibrocytes of the spiral ligament and the spiral limbus through to P21, when the murine cochlea matures. Thus, the gene product may be involved in uptake of potassium ions or trophic factors with a particular role in auditory development. Although the R187C and R187H mutations did not appear to affect subcellular localization of the gene product in vitro, the H783Y mutation did present an unusual cytoplasmic distribution pattern that could underlie the molecular mechanism of hearing impairment. Our data bring attention to a novel candidate for hearing loss and indicate that screening of mutations in inner-ear-specific genes is likely to be an efficient approach to finding genetic elements responsible for deafness.Nucleotide sequence data reported herein are available in the DDBJ/EMBL/GenBank databases; for details, see the electronic eatabase section of this article. 相似文献
26.
用噬菌体表面展示技术筛选与肝癌细胞系结合的抗体模拟肽 总被引:1,自引:0,他引:1
目的:从噬菌体展示肽库中,筛选可与肝癌细胞特异性结合的抗体模拟肽。方法:通过生物淘选使噬菌体富集。利用ELISA法,鉴定噬菌体单克隆原种的亲和性,并进行统计学分析。通过竞争ELISA,分析筛选所得抗体模拟肽的结合位点,并进一步分析抗体模拟肽的序列组成。结果:随着淘选次数的增加,出现噬菌体的富集。ELISA的结果显示,相对于正常肝细胞,筛选所得环状7肽对肝癌细胞系SMMC7721和BEL7402均有良好的结合活性(P<0.05),且与SMMC7721细胞的结合活性明显优于与BEL7402细胞的亲和性(P<0.05)。在α=0.01的水平上,7肽单克隆噬菌体原种可明显与scFv竞争结合SMMC7721细胞(0.005
相似文献
27.
28.
Biological properties and gene expression associated with metastatic potential of human osteosarcoma 总被引:4,自引:0,他引:4
Nakano T Tani M Ishibashi Y Kimura K Park YB Imaizumi N Tsuda H Aoyagi K Sasaki H Ohwada S Yokota J 《Clinical & experimental metastasis》2003,20(7):665-674
Lung metastasis has a great influence on the prognosis of patients with osteosarcoma. We previously established two high-metastatic
sublines, M112 and M132, from the HuO9 human osteosarcoma cell line by in vivo selection. In this study, we newly isolated a high-metastatic subline, H3, and three low-metastatic sublines, L6, L12 and
L13, from HuO9 by the dilution plating method. Three high-metastatic sublines produced more than 200 metastatic nodules in
the lung, while three low-metastatic sublines produced no or few nodules after injection of 2 × 106 cells into the tail vein of nude mice. There were significant differences in the motility and invasiveness between high-
and low-metastatic sublines, whereas the growth rates in vitro and the tumorigenicity in vivo showed no correlation with their metastatic abilities. Early adherence to culture plates was significantly lower in two of
three low-metastatic sublines, which occupied smaller surface areas on the culture plates than other sublines did. Comparison
of the expression of 637 cancer-related genes by cDNA microarray revealed that seven genes were differentially expressed between
high- and low-metastatic sublines. Among them, five genes (AXL, TGFA, COLL7A1, WNT5A, and MKK6) were associated with adherence, motility, and/or invasiveness. These results suggest that the differences in motility/invasiveness
and adhesive abilities are key determinants of lung metastasis in osteosarcoma.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
29.
Jan Schmeller Michael Wessolly Elena Mairinger Sabrina Borchert Thomas Hager Thomas Mairinger Kurt Werner Schmid Jeremias Wohlschlaeger Robert F.H. Walter Fabian D. Mairinger 《Pathology, research and practice》2019,215(2):381-386
Introduction
The usage of formalin-fixed paraffin embedded (FFPE) tissue is characterized by its long shelf-life and simple handling. Therefore it is the most commonly available tissue specimen in routine diagnostics and histological studies. Formaldehyde fixation may result in RNA degradation and cross linking with proteins, while storage conditions also affect RNA integrity. The present study was designed to investigate the influence of these factors on RNA analysis.Design
FFPE-derived RNA from sections of 23 patients with spontaneous pneumothoraxes was used. Unstained sections of FFPE tissue were stored at various temperatures (?80?°C, ?20?°C, 4?°C, 24?°C) prior to RNA extraction. The potential impact on RNA quality of semi-automatic and manual RNA isolation and three different deparaffinization agents (mineral oil, xylene and d-limonene) were compared.Results
The storage temperature of FFPE sections affects RNA concentration and fragmentation, with the optimal storage temperature below -20?°C. The RNA extracted with d-limonene shows equivalent quality to the RNA extracted using more toxic standard agents. The manual isolation provides a higher RNA yield compared to the semi-automatic isolation. However, no differences in the amount of longer RNA fragments were observed. Furthermore, the semi-automatic isolation showed an enhanced RNA quality.Conclusion
FFPE sections not directly used for RNA extraction should be stored below -20?°C to increase quality and yield of the RNA. Usage of semi-automatic isolation produces superior results and simplifies routine processes by having less hands-on-time. Replacement of toxic xylene by d-limonene may contribute to improved occupational safety while not influencing analytical results. 相似文献30.
目的差异筛选人肝癌凋亡细胞cDNA文库。方法消减杂交和点杂交相结合的噬菌斑原位杂交法筛选cDNA文库,首先采用(-)cDNA探针杂交,挑取(-)的噬菌斑克隆,再用(-)和(+)两种cDNA探针与初筛出的噬菌斑克隆杂交的差异筛选方法。结果得到4个充分孤立的噬菌斑克隆,其插入片段长度为1.5kb左右。结论该方法简便、快速,是差异筛选cDNA文库的一种较为可行的简便方法。 相似文献