C-myc基因表达调控的研究

本文利用cDNA探针,以Southern杂交在C-myc 5′上游区域发现了一段活跃转录的序列(文中称“旁侧基因”)。对该基因克隆及进一步分析表明,能与cDNA杂交的序列存在于距C-myc 5′端约2.4kb的Sma Ⅰ片段中。Northern杂交显示,该旁侧基因在不同组织均有表达,产物为5.8kb,推断该基因为15～3.5kb。以旁侧基因部分序列为探针,在不同种属的基因组中检测出单拷贝序列,提示该基因在进化上的保守性。RNase Mapping分析发现此旁侧基因与C-myc转录方向相同,转录终止在C-myc5’上游约2.4～3.4kb区域。根据基因领域效应及本文结果,我们推测,在正常细胞中,由于旁侧基因的领域效应,使C-myc保持相对静止状态,而在肿瘤细胞中,染色体转位或原病毒插入,破坏了旁侧基因的领域效应,使C-myc表达增高。     

Lactogenic hormones increase epidermal growth factor messenger RNA content of mouse mammary glands.

Epidermal growth factor (EGF) is known to stimulate mammary epithelial proliferation, has been identified in milk and is expressed in lactating mammary epithelia. This study examined hormonal control of EGF mRNA in mammary glands of mice. Prepro-EGF mRNA (4.7 kb) was detected during lactation (and increased significantly during this period), whereas a smaller EGF-like RNA (.5 kb) was at highest levels in mammary glands of virgin and pregnant mice. The 4.7 kb RNA was polyadenylated, whereas .5 kb RNA was not. In mammary gland organ cultures from steroid-primed mice, the combinations of insulin + hydrocortisone and insulin + prolactin + hydrocortisone increased both prepro-EGF and beta-casein mRNA expression. When hydrocortisone was present there was a decrease in mammary gland content of EGF-like RNA (.5 kb band). We conclude that prepro-EGF mRNA expression in mouse mammary tissue is under the control of the lactogenic hormones prolactin and hydrocortisone.     

幽门螺旋杆菌感染与胃癌中Shh和C-myc表达的关系

为了探讨胃癌中幽门螺旋杆菌(Hp)感染和Sonic Hedgehog(Shh)、C-myc表达,它们之间的相关性以及胃癌发生的可能机制,采用免疫组化法检测89例胃癌组织及20例正常胃上皮组织中Shh及C-myc的表达。并采用快速尿素酶试验,组织病理学检测两种方法检查Hp。实验结果显示,胃癌组织Shh的表达要明显高于正常上皮组织,二者之间有显著差异(P<0.05);胃癌组织C-myc的表达水平也高于正常胃上皮组织,二者之间有显著差异(P<0.05);Hp阳性的C-myc阳性表达率明显高于Hp阴性,二者之间有显著差异(P<0.05);Shh表达阳性率在Hp阳性和阴性胃癌中无显著差异(P>0.05)。结果提示,胃癌的发生与癌基因Shh及C-myc的过度表达有关,Hp感染的致癌机制中可能有癌基因C-myc参与。     

In vivo analysis of DNase I hypersensitive sites in the human CFTR gene.

BACKGROUND: The cystic fibrosis transmembrane conductance regulator gene (CFTR) shows a complex pattern of expression. The regulatory elements conferring tissue-specific and temporal regulation are thought to lie mainly outside the promoter region. Previously, we identified DNase I hypersensitive sites (DHS) that may contain regulatory elements associated with the CFTR gene at -79.5 and at -20.5 kb with respect to the ATG and at 10 kb into the first intron. MATERIALS AND METHODS: In order to evaluate these regulatory elements in vivo we examined these DHS in a human CFTR gene that was introduced on a yeast artificial chromosome (YAC) into transgenic mice. The 310 kb human CFTR YAC was shown to restore the pheno-type of CF-null mice and so is likely to contain most of the regulatory elements required for tissue-specific expression of CFTR. RESULTS: We found that the YAC does not include the -79.5 kb region. The DHS at -20.5 kb is present in the chromatin of most tissues of the transgenic mice, supporting its non-tissue-specific nature. The DHS in the first intron is present in a more restricted set of tissues in the mice, although its presence does not show complete concordance with CFTR expression. The intron I DHS may be important for the higher levels of expression found in human pancreatic ducts and in lung submucosal glands. CONCLUSION: These data support the in vivo importance of these regulatory elements.     

Distal and proximal cis-linked sequences are needed for the total expression phenotype of the mouse alcohol dehydrogenase 1 (Adh1) gene

Mouse alcohol dehydrogenase 1 (Adh1) gene expression occurs at high levels in liver and adrenal, moderate levels in kidney and intestine, low levels in a number of other tissues, and is undetectable in thymus, spleen and brain by Northern analysis. In transgenic mice, a minigene construct containing 10 kb of upstream and 1.5 kb of downstream flanking sequence directs expression in kidney, adrenal, lung, epididymis, ovary and skin but promotes ectopic expression in thymus and spleen while failing to control expression in liver, eye, intestine and seminal vesicle. Cosmids containing either 7 kb of upstream and 21 kb of downstream or 12 kb of upstream and 23 kb of downstream sequence flanking genetically marked Adh1 additionally promotes seminal vesicle expression suggesting downstream or intragenic sequence controls expression in this tissue. However, expression in liver, adrenal, or intestine is not promoted. The Adh1(a) allele on the cosmid expresses an enzyme electrophoretically distinct from that of the endogenous Adh1(b) allele, and presence of the heterodimeric enzyme in expressing tissues confirms that transgene activity occurs in the same cell-type as the endogenous gene. Transgene expression levels promoted by cosmids were at physiologically relevant amounts and exhibited greater copy-number dependence than observed with minigenes. Transgene mRNA expression correlated with expression measured at the enzyme level. A bacterial artificial chromosome containing 110 kb of 5'- and 104 kb of 3'-flanking sequence surrounding the Adh1 gene promoted expression in tissues at levels comparable to the endogenous gene most importantly including liver, adrenal and intestinal tissue where high level Adh1 expression occurs. Transgene expression in liver was in the same cell types as promoted by the endogenous gene. Although proximal elements extending 12 kb upstream and 23 kb downstream of the Adh1 gene promote expression at physiologically relevant levels in most tissues, more distal elements are additionally required to promote normal expression levels in liver, adrenal and intestinal tissue where Adh1 is most highly expressed.     

Analysis of tissue-specific expression of human type II collagen cDNA driven by different sizes of the upstream region of the beta-casein promoter

To investigate the ability of 1.8 kb or 3.1 kb bovine beta-casein promoter sequences for the expression regulation of transgene in vivo, transgenic mice were produced with human type II collagen gene fused to 1.8 kb and 3.1 kb of bovine beta-casein promoter by DNA microinjection. Five and three transgenic founder mice were produced using transgene constructs with 1.8 kb and 3.1 kb of bovine beta-casein promoters respectively. Founder mice were outbred with the wild type to produce F1 and F2 progenies. Total RNAs were extracted from four tissues (mammary gland, liver, kidney, and muscle) of female F1 transgenic mice of each transgenic line following parturition. RT-PCR and Northern blot analysis revealed that the expression level of transgene was variable among the transgenic lines, but transgenic mice containing 1.8 kb of promoter sequences exhibited more leaky expression of transgene in other tissues compared to those with 3.1 kb promoter. Moreover, Western blot analysis of transgenic mouse milk showed that human type II collagen proteins secreted into the milk of lactating transgenic mice contained 1.8 kb and 3.1 kb of bovine beta-casein promoter. These results suggest that promoter sequences of 3.1 kb bovine beta-casein gene can be used for induction of mammary gland-specific expression of transgenes in transgenic animals.     

真核细胞起始因子4E与C-myc在甲状腺乳头状癌中的表达

目的观察真核细胞起始因子4E(eukaryotic initiation factor4E,eIF4E)及C-myc在甲状腺乳头状癌中的表达,并探讨两者间的相关性及它们与甲状腺乳头状癌的侵袭、转移等临床病理特征间的关系。方法采用免疫组化EnVin-sion法检测66例甲状腺乳头状癌、20例结节性甲状腺肿伴乳头状增生及上述病例的病变周围正常甲状腺组织中eIF4E和C-myc的表达情况。结果在正常甲状腺、结节性甲状腺肿伴乳头状增生及甲状腺乳头状癌中,eIF4E的阳性率分别为11.6%(10/86)、50.0%(10/20)和84.8%(56/66)(P<0.01),C-myc的阳性率分别为21.0%(18/86)、50%(10/20)和78.8%(52/66)(P<0.01)。eIF4E仅与乳头状癌的腺外侵袭和淋巴结转移有关(均P<0.05),C-myc仅与乳头状癌的淋巴结转移有关(P<0.01)。eIF4E及C-myc在乳头状癌中的表达呈正相关(rs=0.327,P<0.05)。结论与癌旁正常组织和良性病变相比,eIF4E和C-myc在甲状腺乳头状癌中存在过表达。eIF4E的过表达与甲状腺乳头状癌的腺外侵袭和淋巴结转移有关,C-myc的过表达与甲状腺乳头状癌的淋巴结转移有关,提示二者在甲状腺乳头状癌的进展中可能发挥重要的作用。