Functional RNA polymerase II promoters in solitary retroviral long terminal repeats (LTR-IS elements).

LTR-IS elements are middle repetitive sequences in the mouse genome with structural features of solitary retroviral LTRs. In order to get some insight in the possible functional role of these sequences the promotor activity of two LTR-IS representatives differing by 105 bp in their U3 region was investigated. Gene fusions between LTR-IS sequences and the bacterial gene coding for chloramphenicol acetyl transferase (CAT) were transfected into mouse 3T6 cells and the expression of CAT was measured. It is shown that the LTR-IS sequences represent weak RNA polymerase II promoters which require enhancement by cis-or trans-activating factors.     

Promiscuous trans activation of gene expression by an Epstein-Barr virus-encoded early nuclear protein.

We identified an Epstein-Barr virus (EBV) gene product which functions in transient-expression assays as a nonspecific trans activator. In Vero cells, cotransfection of the BglII J DNA fragment of EBV together with recombinant constructs containing the bacterial chloramphenicol acetyltransferase (CAT) gene gave up to a 100-fold increased expression of CAT activity over that in cells transfected with the recombinant CAT constructs alone. The BglII J fragment acted promiscuously, in that increased CAT synthesis was observed regardless of whether the promoter sequences driving the CAT gene were of EBV, simian virus 40, adenovirus, or herpes simplex virus origin. Cleavage of cloned BglII-J plasmid DNA before transfection revealed that activation was dependent upon the presence of an intact BMLF1 open reading frame. This was confirmed with subclones of BglII-J and with hybrid promoter-open reading frame constructs. This region of the genome is also present in the rearranged P3HR-1-defective DNA species, and defective DNA clones containing these sequences produced a similar activation of CAT expression in cotransfection experiments. The heterogeneous 45-60-kilodalton polypeptide product of BMLF1 may play an important regulatory role in expression of lytic-cycle proteins in EBV-infected lymphocytes.     

Transient gene expression control: effects of transfected DNA stability and trans-activation by viral early proteins.

The effects of trans-acting factors and transfected DNA stability on promoter activity were examined with chloramphenicol acetyl transferase (CAT) transient expression analysis. With cotransfection into CV-1P and HeLa cells, simian virus 40 T antigen, adenovirus E1a, and herpes-virus IE proteins were compared for their ability to trans-activate a variety of eucaryotic promoters constructed into CAT plasmids. T antigen and the IE protein were promiscuous activators of all the promoters tested [the simian virus 40 late promoter, the adenovirus E3 promoter, the alpha 2(I) collagen promoter, and the promoter of the Rous sarcoma virus long terminal repeat]. Conversely the E1a protein was specific, activating only the adenovirus E3 promoter and suppressing the basal activity of the other promoters. This specificity of activation by E1a contrasted with the high activity generated by all of the promoter-CAT plasmids when transfected into 293 cells, which endogenously produce E1a protein. Examination of transfected 293 cells determined that they stabilized much greater amounts of plasmid DNA than any other cells tested (CV-1P, COS, NIH-3T3, KB). Thus the high activity of nonadenovirus promoter-CAT plasmids in 293 cells results from the cumulative effect of basal promoter activity from a very large number of gene copies, not from E1a activation. This conclusion was supported by similar transfection analysis of KB cell lines which endogenously produce E1a protein. These cells stabilize plasmid DNA at a level comparable to that of CV-1P cells and, in agreement with the CV-1P cotransfection results, did not activate a nonadenovirus promoter-CAT plasmid. These results indicate that the stability of plasmid DNA must be considered when transient gene expression is being compared between cell lines. The use of relative plasmid copy numbers for the standardization of transient expression results is discussed.     

Multi-gene gateway clone design for expression of multiple heterologous genes in living cells: conditional gene expression at near physiological levels

Using Multisite Gateway five-DNA-fragment constructs vectors that enable expression of two tandemly situated cDNAs on a single plasmid were developed. Heterologous protein production in cells was achieved by modulating respective cDNA expression to pre-determined and different levels. Optimization of cDNA expression at near physiological protein levels was achieved using promoters from four cell cycle-dependent genes. In comparison with conventionally available promoters, EF-1alpha or CMV, the promoters used in this study were able to modulate cDNA expression levels over a magnitude of approximately 10 or 100-fold, respectively. In transiently transfected cells, two different proteins (CPalpha1 and CPbeta2), which form a heterodimer, each labeled with a different-colored fluorescent protein, were successfully synthesized at pre-determined levels from their respective cDNAs. The above vectors were designed to contain an FRT/Flp recombination site for integration onto chromosomes and for establishment of stable clones in HeLa cells by site-specific recombination. In the stable transformant cells produced only about 4% of the protein production levels measured in the transiently transformed cells. The biological significance of these observations is discussed.     

cDNA cloning and structure of mouse putative Ah receptor.

Mouse cDNA clones for a putative Ah receptor have been isolated from a cDNA library of mRNA from Hepa-1 cells by an oligonucleotide probe produced by PCR with a pair of primers which was synthesized according to the reported N-terminal sequence of 26 amino acids. The cDNA clones encode a polypeptide of 805 amino acids with a helix-loop-helix motif and with some similarity to a certain region designated PAS of Drosophila Per and Sim, and human Arnt protein. Cotransfection of an expression vector of the Ah receptor with a reporter plasmid pMC6.3k consisting of CYP1A1 promoter and CAT structural gene into CV-1 cells enhanced the CAT expression in response to added 3-methylcholanthrene.     

Isolation and analysis of a rat genomic clone containing a long terminal repeat with high similarity to the oncomodulin mRNA leader sequence

The structure of a novel long terminal repeat (LTR) from an intracisternal A particle (IAP) DNA element in the rat (Sprague-Dawley) genome was determined. This LTR has a total length of 313 base pairs (bp). Several structural features typical for retroviral LTR promoters were identified, including a "CCAAT" box, a "TATA" box, a polyadenylation signal, and a polyadenylation site. The LTR is flanked by 3-bp inverted repeats, and it consists of the three typical LTR regions, U3, R, and U5. U3 contains 213 bp, R 46 bp, and U5 54 bp, which is within the usual size range of IAP LTRs. A sequence of 60 bp in the U3 region reveals considerable similarity to a murine IAP LTR U3 element, which is known to interact with nuclear proteins. A sequence of 69 bp in the U5 and R regions has 83 and 93% similarities to an endogenous retroviral LTR from Syrian hamster and to the cDNA leader sequence of (Buffalo) rat oncomodulin, respectively. Oncomodulin is an "EF-hand" Ca2+-binding protein and appears in many human and rodent tumors and in cells with tumor-like properties but not in normal tissues. We postulate that in the rat the tumor-specific expression of oncomodulin is controlled by a retroviral LTR promoter.     

食管癌细胞抗失巢凋亡基因UBCH7的发现与鉴定

失巢凋亡(Anoikis)是细胞失去与细胞外基质(Extra-cellular matrix,ECM)粘附时发生的特殊形式的凋亡,是机体维持组织稳态的关键机制之一。抗失巢凋亡能力的获得是肿瘤细胞发生远处转移的前提条件之一。为了鉴定与食管癌细胞抗失巢凋亡相关的基因,文章首先构建食管癌细胞系的逆转录病毒文库,感染对失巢凋亡敏感的NIH3T3细胞,利用感染病毒cDNA文库的混合细胞系进行软琼脂集落形成实验,挑取在悬浮条件下仍可生长成为较大集落的细胞单克隆(潜在具有抗失巢凋亡能力的细胞),通过逆转录病毒载体特异的引物PCR扩增失巢凋亡抗性克隆基因组中的插入cDNA片段,以此获得食管癌细胞系cDNA文库中潜在的具有失巢凋亡抗性的基因。经测序发现其中一个失巢凋亡克隆中整合的cDNA片段包括人UBCH7/UBE2L3基因全长的编码序列(开放阅读框)。利用携带pMSCV-UBCH7的逆转录病毒感染NIH3T3细胞进行验证,结果显示细胞失巢凋亡抗性增强,并且在具有高转移潜能的食管癌细胞系MLuC1中降调UBCH7表达可减弱其失巢凋亡抗性。这些结果表明,UBCH7/UBE2L3是一个与食管癌失巢凋亡抗性相关的基因。     

食管癌细胞抗失巢凋亡基因UBCH7的发现与鉴定

失巢凋亡(Anoikis)是细胞失去与细胞外基质(Extra-cellular matrix, ECM)粘附时发生的特殊形式的凋亡, 是机体维持组织稳态的关键机制之一。抗失巢凋亡能力的获得是肿瘤细胞发生远处转移的前提条件之一。为了鉴定与食管癌细胞抗失巢凋亡相关的基因, 文章首先构建食管癌细胞系的逆转录病毒文库, 感染对失巢凋亡敏感的NIH3T3细胞, 利用感染病毒cDNA文库的混合细胞系进行软琼脂集落形成实验, 挑取在悬浮条件下仍可生长成为较大集落的细胞单克隆(潜在具有抗失巢凋亡能力的细胞), 通过逆转录病毒载体特异的引物PCR扩增失巢凋亡抗性克隆基因组中的插入cDNA片段, 以此获得食管癌细胞系cDNA文库中潜在的具有失巢凋亡抗性的基因。经测序发现其中一个失巢凋亡克隆中整合的cDNA片段包括人UBCH7/UBE2L3基因全长的编码序列(开放阅读框)。利用携带pMSCV-UBCH7的逆转录病毒感染NIH3T3细胞进行验证, 结果显示细胞失巢凋亡抗性增强, 并且在具有高转移潜能的食管癌细胞系MLuC1中降调UBCH7表达可减弱其失巢凋亡抗性。这些结果表明, UBCH7/UBE2L3是一个与食管癌失巢凋亡抗性相关的基因。