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1.
以地高辛标记c-myc、c-H-ras、c-sis基因作探针,利用组织切片原位杂交的方法,检测了昆明种小鼠5周龄、10周龄和孕期卵巢,以及1天龄、5周龄和10周龄睾丸,初步研究了这3种原癌基因在生殖腺中的转录表达及其可能的功能作用。结果显示:(1)c-myc基因在孕鼠卵巢的个别卵泡的卵泡细胞中有强转录信号,但在3组卵巢的卵母细胞中及3组睾丸切片中均未见杂交信号。(2)c-H-ras基因在孕鼠卵巢黄体中有较强转录表达,但在3组卵巢的卵母细胞中及3组睾丸切片中均无杂交信号,(3)c-sis基因在10周龄睾丸曲细精管的精原细胞层和精母细胞有强转录表达,而在1天龄、5周龄睾丸及3组卵巢切片中均无杂交信号。上述结果表明:c-myc基因可能与精子发生和卵子的发育关系不大,但可能与卵泡的退化有关;c-H-ras基因可能与精子发生和卵子的发育关系也不大,但可能与妊娠黄体的形成有关;c-sis基因可能与卵子的发育无关,但可能与精子的发生密切相关。 Abstract:Using in Site Hybridization on tissue section of ovary from 5 weeks old,10 weeks old and pregnant Kunming mice,and test is from one-day old,5 weeks old,and 10 weeks old Kunming mice,we prelimmaily studied the expression and function of the proto-oncogenes c-myc,c-H-ras and c-sis.The results are as follows:(1)The strong expression of c-myc gene was found in follicular epithelium of some follicles of ovary from pregnant mice,and was not found in the oocytes of three groups of female mice and testes of three groups of male mice.(2)The strong expression of c-H-ras was observed in corpus luteum of ovary of pregnant mice,but it was not observed in any germ cells of all three groups of female mice and male mice.(3)The strong expression of c-sis gene was found in spermatogomia and spermatocytes of mature male mice,but it was not found in any germ cells of one day old and immature male mice and three groups of femala mice.These results showed that there might be no relationships between the expression of c-myc gene and spermatogenesis or oocyteatogenesis,but the expression of c-myc gene probably related to the cells degeneration of follicle;the expression of c-H-ras gene hadn’t a bearning on oogenesis and spermatogenesis,but probably related to the genesis of corpus luteum of corpus luteum of pregnant mice;the expression of c-sis gene there might be no related to the oogenesis and sperm maturation,but probably promoted the division of spermatogonim.  相似文献   

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Previous studies have revealed that Epstein-Barr virus (EBV) was closely associated with nasopharyngeal carcinoma (NPC). This study aimed to characterize the global pathways affected in the EBV-associated NPC. Combined with microdissection, gene expression profries in 22 NPCs and 10 non-tumor nasopharyngeal epithelial (NPE) tissue samples were analyzed. All NPC specimens served in the microarray analysis were positive for EBV, as judged by identification of the expression of EBV nuclear antigen 1 (EBNA1). Through gene set enrichment analysis (GSEA), we found that cell cycle pathway was the most disregulated pathway in NPC (P = 0.000, false discovery rate q-value = 0.007), which included some aberrant expressed components. We first found that overexpression of CDK4, cyclin D1, and Rb proteins, and loss of expression of proteins p16, p27, and p19 were statistically significant in NPC tissues compared with non-cancerous NPE (P〈 0.05) by real-time RT- PCR and tissue microarray. EBV-encoded small RNA-1 (EBER-1) hybridization signals in the NPC showed significant associations with the overexpression of Rb (P = 0.000), cyclin D1 (P = 0.000), CDK4 (P = 0.000), and the loss of expression of p16 proteins (P = 0.039). In the final logistic regression analysis model, EBER-1 and abnormal expression of p16, Rb, cyclin D1, and E2F6 were inde- pendent contributions to nasopharyngeal carcinogenesis. Through survival analysis, only cyclin D1 could predict the prognosis of NPC patients. These results suggested that cell cycle pathway was the most disregulated pathway in the EBV-associated NPC, and EBER-1 was closely associated with p16, CDK4, cyclin D1, and Rb. cyclin D1 could be the prognosis biomarker for NPC.  相似文献   

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参照已报道小鼠受精抗原1(FA1)基因序列设计引物,运用PCR和PCR产物克隆测序等方法,对人受精抗原1(FA1)基因进行了克隆,并对两者进行了比较,结果显示:(1)已报道的小鼠FA1基因序列在其可读框内可能存在两处错误。(2)人OTK27基因可能与小鼠FA1基因同源,即OTK27基因就是人hFA1基因,或FA1基因就是小鼠otk27基因。 Abstract:The cloning of human fertilization antigen 1 gene(FA1) ,the supposed counterpart gene of mouse fertilization antigen 1 gene (FA1),was performed using the PCR and PCR products cloned sequencing methods.The result shows that there might be two mistakes in the mouse FA1 gene open reading frame (ORF),and human OTK27 gene and mouse FA1 gene might be homogeneous genes in the two species.  相似文献   

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妊娠小鼠子宫内膜LIF基因表达的研究   总被引:9,自引:2,他引:7  
本文对妊娠第4天(Ⅰ组)、第7天(Ⅱ组)、第10天(Ⅲ组)的小鼠(各20只)子宫内膜LIF基因表达进行了研究。Ⅰ组20只小鼠子宫内膜全部存在LIF基因的表达、Ⅱ组有5只小鼠表达、Ⅲ组仅有1只小鼠表达。文中对不同孕期LIF基因的表达程度与胚胎着床的关系进行了讨论。 Abstract:Leukemia inhibitory factor(LIF)is a glycoprotein with multiple activities and is essential for blastocyst implantation in mouse.We have examined LIF gene expression in mice endometrium on day 4(group Ⅰ),day 7(group Ⅱ),day 10(group Ⅲ)of pregnancy.In group Ⅰ all had LIF gene expression,5 mice had LIF gene expression in group Ⅱ,only one mouse had LIF gene expression in group Ⅲ.We discussed the relation between level of LIF gene expression and embryonic implantation.  相似文献   

7.
宫颈癌p16基因甲基化及表达的研究   总被引:2,自引:0,他引:2  
原继荣  李钰  胡双玖  李璞 《遗传》2005,27(1):39-43
为了探讨p16基因甲基化及异常表达在宫颈癌中的意义,分别采用甲基化特异性PCR(MSP)方法检测不同病理类型和临床分期的60例宫颈癌组织中p16基因启动子区域5´CpG岛甲基化状态;采用PCR方法检测p16基因外显子1(E1)和外显子2(E2)纯合缺失情况;采用免疫组化的方法分析p16蛋白的表达缺失和减弱情况。结果显示正常对照组织及癌旁p16基因无甲基化,且无E1和E2缺失和p16蛋白表达异常。60例宫颈癌标本的甲基化率为21.67%(13/60);p16 基因缺失率为15.00%(9/60);p16蛋白表达下降或无表达为51.67%(31/60)。可见p16基因蛋白的阳性表达率随着临床分期升高呈明显下降趋势。结果提示p16基因失活在宫颈癌中多见且与病理分级密切相关。p16 基因甲基化在宫颈癌发生中起着一定作用。Abstract: To detect hypermethylation and aberrant expression of the p16 gene in cervical carcinoma (CC), methylation-specific PCR (MSP) was used to determine the methylation status of 5´CpG islands of the p16 gene, loss or decrease of p16 expression was analyzed by immunohistochemistry (IHC), and homozygous deletion of exon 1 (E1) and/or exon 2 (E2) was determined by PCR. in 60 cases of CC at different pathological grades and clinical stages. The results showed absence of methylation and presence of normal expression of the p16 gene in the control and adjacent tissues of CC. Hypermethylation, loss or decrease of expression and deletion of the p16 gene were detected in 21.67%(13/60), 51.67%(31/60) and 15.00%(9/60) of the tumor tissues, respectively. The rate of p16 expression markedly reduced with the increase of clinical stages. Our data suggested that inactivation of the p16 gene was a frequent event and positively correlated with pathological grades in CC, and that methylation of the p16 gene was an important event in carcinogenesis of CC.  相似文献   

8.
The aim of the present study is to investigate gene expression involved in the signal pathway of MAPK and death signal receptor pathway of FAS in lead- induced apoptosis of testicular germ cells. First, cell viabilities were determined by MTT assay. Second, using single cell gel-electrophoresis test (comet assay) and TUNEL staining technique, apoptotic rate and cell apoptosis localization of testicular germ cells were measured in mice treated with 0.15%, 0.3%, and 0.6% lead, respectively. Third, the immunolocalization of K-ras, c-fos, Fas, and active caspase-3 proteins was determined by immunohistochemistry. Finally, changes in the translational levels of K-ras, c-fos, Fas, and active caspase-3 were further detected by western blot analysis. Our results showed that lead could significantly induce testicular germ cell apoptosis in a dose-dependent manner (P 〈 0.01). The mechanisms were closely related to the increased expressions of K-ras, c-fos, Fas, and active caspase-3 in apoptotic germ cells. In conclusion, K-ras/c-fos and Fas/caspase-3 death signafing receptor pathways were involved in the lead-induced apoptosis of the testicular germ cells in mice.  相似文献   

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Scientific research has yet of cage size on mouse breeding to conclusively determine the optimal cage size for mice. The authors examined the effect performance and on offspring behavior, which can serve as indications of overall wellbeing. They housed breeding trios of C57BL/6Tac mice in standard or large individually ventilated cages and measured four reproductive parameters: litter size; litter survival to weaning age; average pup weight at 7, 14 and 21 days; and the number of days between litter births. They investigated the behavior of a subset of male and female pups from parents housed in cages of each size in the elevated plus maze test, the open field assay and the acoustic startle test. Cage size had no significant effect on any of the reproductive parameters measured and few or inconsistent effects on behavior in weaned pups. Lab Animal 2007,36(10)  相似文献   

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Although cell polyploidization is not an infrequent event in mammalian cells and is common in tumours, the mechanisms involved are not well understood. Using the murine B16 cell line as a model, we evaluated the role of some key proteins involved in cell cycle progression: p34cdc2, cyclin B1 and PCNA. By means of flow cytometry, we showed that both in modal- and in high-ploidy subpopulations, almost all cells were p34cdc2-positive. In the modal-ploidy subpopulation only 17.1% cells were cyclin B1-positive and 85.6% PCNA-positive; in contrast, in the high-ploidy subpopulation up to 91.8% cells were cyclin B1-positive and 97.3% cells were PCNA-positive (P < 0.001). Immunofluorescence microscopy showed that PCNA was located in the nucleus; p34cdc2, both in the nucleus and cytoplasm; and cyclin B1 yielded a cytoplasmic spotted pattern with a perinuclear reinforcement. After a 24-h incubation with 3[H]-thymidine followed by withdrawal of the isotope, high-ploidy cells remained labelled 8 days after thymidine withdrawal, in contrast to modalploidy cells. Taken together, our results suggest that polyploid cells are not quiescent, their cell cycle is longer than that of the modal-ploidy population, and they maintain cyclin B1 throughout the cycle, which may contribute to their genesis by impeding the exit from mitosis.  相似文献   

12.
The mouse FT210 cell line is a temperature-sensitive cdc2 mutant. FT210 cells are found to arrest specifically in G2 phase and unlike many alleles of cdc2 and cdc28 mutants of yeasts, loss of p34cdc2 at the nonpermissive temperature has no apparent effect on cell cycle progression through the G1 and S phases of the division cycle. FT210 cells and the parent wild-type FM3A cell line each possess at least three distinct histone H1 kinases. H1 kinase activities in chromatography fractions were identified using a synthetic peptide substrate containing the consensus phosphorylation site of histone H1 and the kinase subunit compositions were determined immunochemically with antisera prepared against the "PSTAIR" peptide, the COOH-terminus of mammalian p34cdc2 and the human cyclins A and B1. The results show that p34cdc2 forms two separate complexes with cyclin A and with cyclin B1, both of which exhibit thermal lability at the non-permissive temperature in vitro and in vivo. A third H1 kinase with stable activity at the nonpermissive temperature is comprised of cyclin A and a cdc2-like 34-kD subunit, which is immunoreactive with anti-"PSTAIR" antiserum but is not recognized with antiserum specific for the COOH-terminus of p34cdc2. The cyclin A-associated kinases are active during S and G2 phases and earlier in the division cycle than the p34cdc2-cyclin B1 kinase. We show that mouse cells possess at least two cdc2-related gene products which form cell cycle regulated histone H1 kinases and we propose that the murine homolog of yeast p34cdc/CDC28 is essential only during the G2-to-M transition in FT210 cells.  相似文献   

13.
Fission yeast cells expressing the human gene encoding the cyclin-dependent kinase inhibitor protein p21Cip1 were severely compromised for cell cycle progress. The degree of cell cycle inhibition was related to the level of p21Cip1 expression. Inhibited cells had a 2C DNA content and were judged by cytology and pulsed field gel electrophoresis to be in the G2 phase of the cell cycle. p21Cip1 accumulated in the nucleus and was associated with p34cdc2 and PCNA. Thus, p21Cip1 interacts with the same targets in fission yeast as in mammalian cells. Elimination of p34cdc2 binding by mutation within the cyclin-dependent kinase binding domain of p21Cip1 exaggerated the cell cycle delay phenotype. By contrast, elimination of PCNA binding by mutation within the PCNA-binding domain completely abolished the cell cycle inhibitory effects. Yeast cells expressing wild-type p21Cip1 and the mutant form that is unable to bind p34cdc2 showed enhanced sensitivity to UV. Cell cycle inhibition by p21Cip1 was largely abolished by deletion of the chk1+ gene that monitors radiation damage and was considerably enhanced in cells deleted for the rad3+ gene that monitors both DNA damage and the completion of DNA synthesis. Overexpression of PCNA also resulted in cell cycle arrest in G2 and this phenotype was also abolished by deletion of chk1+ and enhanced in cells deleted for rad3+. These results formally establish a link between PCNA and the products of the rad3+ and chk1+ checkpoint genes.  相似文献   

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When treated with DNA-damaging chemotherapy agents, many cancer cells, in vivo and in vitro, undergo a terminal growth arrest and acquire a senescence-like phenotype. We investigated the molecular basis for this in breast cancer cells following a 2-hour treatment with 1 muM doxorubicin. Treated cells arrested in G1 and G2 phases of the cell cycle, with concomitant reductions in S-phase and G2-M regulatory genes. p53 and p21 protein levels increased within hours after treatment and were maintained for 5 to 6 days but were reduced 8 days posttreatment, though the cells remained growth arrested. Levels of p130 rose after drug treatment, and it was the primary RB family member recruited to the S-phase promoters cyclin A and PCNA and G2-M promoters cyclin B and cdc2, remaining present for the entire 8-day time period. In contrast, p107 protein and promoter occupancy levels declined sharply after drug treatment. RB was recruited to only the PCNA promoter. In MCF-7 cells with p130 knockdown, p107 compensated for p130 loss at all cell cycle gene promoters examined, allowing cells to retain the growth arrest phenotype. Cells with p130 and p107 knockdown similarly arrested, while cells with knockdown of all three family members failed to downregulate cyclin A and cyclin B. These results demonstrate a mechanistic role for p130 and compensatory roles for p107 and RB in the long-term senescence-like growth arrest response of breast cancer cells to DNA damage.  相似文献   

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Murine cDNA clones for three cyclin D genes that are normally expressed during the G1 phase of the cell cycle were used to clone the cognate human genes. Bacteriophage and cosmid clones encompassing five independent genomic loci were partially sequenced and chromosomally assigned by an analysis of somatic cell hybrids containing different human chromosomes and by fluorescence in situ hybridization to metaphase spreads from normal peripheral blood lymphocytes. The human cyclin D1 gene (approved gene symbol, CCND1) was assigned to chromosome band 11q13, cyclin D2 (CCND2) to chromosome band 12p13, and cyclin D3 (CCND3) to chromosome band 6p21. Pseudogenes containing sequences related to cyclin D2 and cyclin D3 mapped to chromosome bands 11q13 and 6p21, respectively. Partial nucleotide sequence analysis of exons within each gene revealed that the authentic human cyclin D genes are more related to their mouse counterparts than to each other. These genes are ubiquitously transcribed in human tumor cell lines derived from different cell lineages, but are independently and, in many cases, redundantly expressed. The complex patterns of expression of individual cyclin D genes and their evolutionary conservation across species suggest that each family member may play a distinct role in cell cycle progression.  相似文献   

18.
The abundance of B-type cyclin-CDK complexes is determined by regulated synthesis and degradation of cyclin subunits. Cyclin proteolysis is required for the final exit from mitosis and for the initiation of a new cell cycle. In extracts from frog or clam eggs, degradation is accompanied by ubiquitination of cyclin. Three genes, CDC16, CDC23, and CSE1 have recently been shown to be required specifically for cyclin B proteolysis in yeast. To test whether these genes are required for cyclin ubiquitination, we prepared extracts from G1-arrested yeast cells capable of conjugating ubiquitin to the B-type cyclin Clb2. The ubiquitination activity was cell cycle regulated, required Clb2's destruction box, and was low if not absent in cdc16, cdc23, cdc27, and cse1 mutants. Furthermore all these mutants were also defective in ubiquitination of another mitotic B-type cyclin, Clb3. The Cdc16, Cdc23, and Cdc27 proteins all contain several copies of the tetratricopeptide repeat and are subunits of a complex that is required for the onset of anaphase. The finding that gene products that are required for ubiquitination of Clb2 and Clb3 are also required for cyclin proteolysis in vivo provides the best evidence so far that cyclin B is degraded via the ubiquitin pathway in living cells. Xenopus homologues of Cdc16 and Cdc27 have meanwhile been shown to be associated with a 20S particle that appears to function as a cell cycle-regulated ubiquitin-protein ligase.  相似文献   

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We have recently shown that two proteins, proliferating cell nuclear antigen (PCNA) and p21, are associated with cyclin D. Here we show that PCNA and p21 are common components of a wide variety of cyclin/cyclin-dependent kinase complexes in nontransformed cells. These include kinase complexes containing cyclin A, cyclin B, and cyclin D, associated either with CDC2, CDK2, CDK4, or CDK5. We show that PCNA and p21 form separate quaternary complex with each cyclin/CDK and that these quaternary complexes contain a substantial, if not major, fraction of the cell cycle kinases in asynchronously growing cells. These results suggest that PCNA and p21 may perform a common function for all these kinases.  相似文献   

20.
The aim of the present study was to investigate bromodeoxyuridine (BrdU) uptake and coordinated distribution of proliferating cell nuclear antigen (PCNA) and p34-cdc2-kinase, two important proteins involved in cell cycle regulation and progression. Flow cytometric analysis of marker proteins in freshly plated mouse T-lymphoma cells (Yac-1 cells), using fluorescein isothiocyanate (FITC)-labeled specific antibodies, showed PCNA distributed throughout the cell cycle with increased intensity in S-phase. PCNA is essential for cells to cycle through S-phase and its synthesis is initiated during late G1-phase before incorporation of BrdU and remains high during active DNA replication. The intensity of PCNA fluorescence increases with the duration of incubation after plating. The cdc2-kinase was detectable in all phases of the cell cycle and the G2-M-phase appears to have the maximum concentrations. The cell cycle analysis of high dose colcemid (2 μg/ml) treated Yac-1 cells showed an aneuploid or hypodiploid population. Although the G2-M-phase seems to be the dominating population in aneuploid cells, the concentrations of cdc2-kinase were variable in this phase of cell cycle. The colcemid treatment at 25 ng/ml arrested 96% of cells in S-phase and G2-M-phase, but PCNA expression was evident in a portion of the cell population in G2-M-phase. Although cells blocked in M-phase seem to have high levels of cdc2-kinase, colcemid renders them inactive. From these data, it appears that the down regulation and/or inactivation of cdc2-kinase could be responsible for the colcemid arrest of cells in M-phase.  相似文献   

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