乙型肝炎病毒的核心启动子各区段功能的研究

将一系列核心启动子区的缺失突变引入乙肝病毒（ＨＢＶ）线性转录单元,从病毒的抗原,ＲＮＡ以及子代ＤＮＡ等各个水平,分析了各缺失突变对前基因组ＲＮＡ和前核心ＲＮＡ转录的影响,对核心启动子各片段的功能进行了深入的研究。Ｃ片段缺失后检测不到ｅ抗原和前核心ＲＮＡ,却仍有核心抗原和前基因组ＲＮＡ的合成以及病毒子代ＤＮＡ的复制;而Ｂ３片段缺失后ｅ抗原和核心抗原均有显著下降,但仍能检测到两种ｍＲＮＡ的合成和病毒子     

Turn-on of Inactive Genes by Promoter Recruitment in ESCHERICHIA COLI: Inverted Repeats Resulting in Artificial Divergent Operons

We have characterized two rearrangements consisting of inverted repeats of the argE gene. The promoters (p) of argE and of argCBH face each other over an internal operator. The rearrangements were obtained as reactivations of argE in a strain harboring an argEp deletion on a lambda darg prophage. In both cases the repeat included argE and argCBHp on either side of a unique sequence; the result is a divergent operon in which each copy of argCBHp reads into the adjacent argE repeat. In one case, the pair of repeats adjoins the silent parental gene, forming a triplication (comes from leads to comes from). The other rearrangement consists of a single argE palindrome, but the whole prophage is rearranged into an inverted repeat, analogous to certain lambda dv's. Both structures could be explained by breakage of a replication fork passing argE and by inaccurate rejoining of strands. The lambda dv-like rearrangement would result from breakage at both replication forks of a phage or prophage replicating during transient release of immunity. The triplication would imply breaking of a chromosomal replication fork, formation of a cyclic intermediate by recombination between the daughter duplex molecules and reinsertion into the parental argE gene. Formation of a triplication by replication errors involving appropriate strand switchings and branch migrations can not be excluded however.     

A Leader Intron of a Soybean Elongation Factor 1A (eEF1A) Gene Interacts with Proximal Promoter Elements to Regulate Gene Expression in Synthetic Promoters

Introns, especially the first intron in the 5’ untranslated region (5’UTR), can significantly impact gene expression via intron-mediated enhancement (IME). In this study, we demonstrate the leader intron of a soybean elongation factor 1A (eEF1A) gene (GmScreamM8) was essential for the high activity of the native promoter. Furthermore, the interaction of the GmScreamM8 leader intron with regulatory element sequences from several soybean eEF1A promoters was studied using synthetic promoters, which consisted of element tetramers upstream of a core promoter used to regulate a green fluorescent protein (gfp) reporter gene. Element tetramers, placed upstream of a GmScreamM8 core promoter, showed very high activity using both transient expression in lima bean cotyledons and stable expression in soybean hairy roots, only if the native leader intron was included, suggesting an interaction between intronic sequences and promoter elements. Partial deletions of the leader intron showed that a 222 bp intronic sequence significantly contributed to very high levels of GFP expression. Generation of synthetic intron variants with a monomeric or trimeric repeat of the 222 bp intronic sequence, yielded almost two-fold higher expression compared to the original intron, while partial deletion of the 222 bp intronic repeated sequence significantly decreased gene expression, indicating that this intronic sequence was essential for the intron-element interaction enhancement.     

牛泡沫病毒反式激活因子在内部启动子上应答元件的研究

泡沫病毒的基因转录依赖至少两个不同的启动子：ＬＴＲ调节病毒结构蛋白的表达,而内部启动子（ＩＰ）则起始调节蛋白ｍＲＮＡ的转录。牛泡沫病毒（ＢＦＶ）在ｅｎｖ与３’ＬＴＲ之间有两个重叠的开放阅读框架ｏｒｆ－１和ｏｒｆ－２,分别编码ＢＦＶ ＯＲＦ－１、ＯＲＦ－２等多种调节蛋白。这此蛋白中ＢＦＶ ＯＲＦ－１为转录激活因子,称为Ｔａｓｏ Ｔａｓ对ＬＴＲ及ＩＰ均有反式激活作用。ＢＦＶ中第二类启动子ＩＰ的存在反映     

诱导方式对P_L启动子控制下人工合成心钠素28肽基因转录动态变化的影响

本文用含有人工合成28肽心钠素基因质粒的大肠杆菌(TAP106-pYX40),分别进行温度诱导和丝裂霉素C诱导,提取菌体中的总RNA,与γ-~(32)P标记的心钠素基因片段进行RNA打点杂交,并通过测定杂交阳性信号的总灰度值(Totalgray value),建立了人心钠素基因特异mRNA相对定量方法,从而观察到温度诱导对P_L启动子控制下的心钠素基团转录发生得慢,特异的mRAN产量较低,但转录所持续的时间长,而丝裂霉素C诱导对P_L启动子控制下的心钠素基因转录发生得快,特异mRNA产量高,但持续的时间短。     

Nitro-Tyrosine as Promoter of Free Radical Damage in a DNA Model System

Nitro-tyrosine considerably promotes the degradation of DNA, when incubated with Cu²⁺ and ascorbate in oxygenated aqueous solution. This deleterious process requires oxygen and can be inhibited with catalase, indicating that H₂O₂ is involved, via the reduction of oxygen. Menadione and 2,4,6-trinitro-benzenesulfonate, known to catalyze particularly fast such reduction of oxygen, were only slightly more active than nitro-tyrosine. Degradation of DNA can be explained by a site-specific Fenton type reaction of H₂O₂ with the DNA-Cu⁺ complex.

DNA-Cu⁺ + H₂O₂ → DNA' ' 'OH + Cu²⁺ + OH^?

Copper-chelating agents (EDTA and penicillamine) prevent DNA degradation, whereas OH-scavengers (t-butanol) are ineffective. The deleterious activity of nitro-tyrosine (and of other nitroaromatics) in the DNA model system may indicate important toxicological implications, since aromatic nitration is a significant mode of action of nitrogen dioxide.