Scanning from an independently specified branch point defines the 3' splice site of mammalian introns

During pre-messenger RNA splicing the lariat branch point in mammalian introns is specified independently of the 3' splice site by the sequence surrounding the branch point and by an adjacent downstream polypyrimidine tract. The 3' splice site is dispensable for spliceosome assembly and cleavage at the 5' splice site, and is itself determined by a scanning process that recognizes the first AG located 3' of the branch point/polypyrimidine tract, irrespective of distance or sequence environment.     

Both subunits of U2AF recognize the 3' splice site in Caenorhabditis elegans

Introns are defined by sequences that bind components of the splicing machinery. The branchpoint consensus, polypyrimidine (poly(Y)) tract, and AG at the splice boundary comprise the mammalian 3' splice site. Although the AG is crucial for the recognition of introns with relatively short poly(Y) tracts, which are termed 'AG-dependent introns', the molecule responsible for AG recognition has never been identified. A key player in 3' splice site definition is the essential heterodimeric splicing factor U2AF, which facilitates the interaction of the U2 small nuclear ribonucleoprotein particle with the branch point. The U2AF subunit with a relative molecular mass (Mr 65K) of 65,000 (U2AF65) binds to the poly(Y) tract, whereas the role of the 35K subunit (U2AF35) has not been clearly defined. It is not required for splicing in vitro but it plays a critical role in vivo. Caenorhabditis elegans introns have a highly conserved U4CAG/ R at their 3' splice sites instead of branch-point and poly(Y) consensus sequences. Nevertheless, C. elegans has U2AF, 12). Here we show that both U2AF subunits crosslink to the 3' splice site. Our results suggest that the U2AF65-U2AF35 complex identifies the U4CAG/R, with U2AF35 being responsible for recognition of the canonical AG.     

低频电磁场频率对半连铸高强Al合金组织结构的影响

利用光学显微镜和透射电镜研究了在低频电磁场作用下,电磁场频率对半连铸直径100mm铝合金铸锭显微组织结构的影响·结果表明:与常规铸锭相比,当电流为100A,频率为15Hz时,晶粒尺寸有所减小,并且等轴晶数目增多·当频率增到25Hz时,晶粒细化最明显·随频率继续增大,晶粒尺寸又有所增大;在电流为100A,电磁场频率为25Hz时,晶内位错密度较高,沉淀相尺寸较小,析出相主要为颗粒状的η′,η相和小片状的T相,此时晶界两侧无析出带宽度为100nm左右;当电磁场频率为15Hz时,晶内沉淀相尺寸有所增大,析出相主要为片状和长板条状的η相、片状的T相和颗粒状的η′相,晶界两侧无析出带宽度增到300nm左右...     

Nucleotide sequence of the origin of replication in bacteriophage phiX174 RF DNA

The gene A protein of bacteriophage phiX174 has been used in vitro to convert phiX RFI DNA into the relaxed RFII form by nicking the viral strand. The nucleotide sequence at the 3' end of the nick has been determined as -- T G C T C C C C C A A C T T Goh. This sequence gives the exact position of the origin of phiX RF DNA replication.     

用计算机对人类TSPYl基因P53结合位点的鉴定

根据p53下游基因在其调节区域（启动子或内含子）含有与P53蛋白特异性结合的一致性序列5’-RRRCWWGYYYN（0-13）RRRCWWGYYY-3’，R—G或A，W—T或A，Y—C或T，N—A，C，T，G。用计算机对人类基因组中P53结合位点进行了研究，发现Y染色体上的TSPY1基因内含子中含有这样的一致性序列5’-GGGCTAGTTTtgGAGCTAGCCT-3’，意味着TSPY1基因有可能是一个p53下游基因。     

Preferential cis-syn thymine dimer bypass by DNA polymerase eta occurs with biased fidelity

Human DNA polymerase eta (Pol eta) modulates susceptibility to skin cancer by promoting DNA synthesis past sunlight-induced cyclobutane pyrimidine dimers that escape nucleotide excision repair (NER). Here we have determined the efficiency and fidelity of dimer bypass. We show that Pol eta copies thymine dimers and the flanking bases with higher processivity than it copies undamaged DNA, and then switches to less processive synthesis. This ability of Pol eta to sense the dimer location as synthesis proceeds may facilitate polymerase switching before and after lesion bypass. Pol eta bypasses a dimer with low fidelity and with higher error rates at the 3' thymine than at the 5' thymine. A similar bias is seen with Sulfolobus solfataricus DNA polymerase 4, which forms a Watson-Crick base pair at the 3' thymine of a dimer but a Hoogsteen base pair at the 5' thymine (ref. 3). Ultraviolet-induced mutagenesis is also higher at the 3' base of dipyrimidine sequences. Thus, in normal people and particularly in individuals with NER-defective xeroderma pigmentosum who accumulate dimers, errors made by Pol eta during dimer bypass could contribute to mutagenesis and skin cancer.     

Xyloadenosine analogue of (A2'p)2A inhibits replication of herpes simplex viruses 1 and 2

Molecules of the structure ppp(A2'p)2A containing a 2' leads to 5' phosphodiester bond, commonly abbreviated as 2-5A, are synthesized in interferon-treated virally-infected cells and have been implicated in several systems as contributing to interferon's antiviral activity. The 2-5A binds to and subsequently activates an endogenous endonuclease, ultimately resulting in degradation of RNA. We have been interested in the use of 2-5A analogues to achieve antiviral activity without the use of interferon. For this approach to be successful, analogues must be synthesized with an increased stability (native 2-5A is rapidly degraded by cellular phosphodiesterases) and with increased ability to enter intact cells. Removal of the highly-negative charged 5' terminal phosphates from ppp(A2'p)2A results in formation of the 'core' species, (A2'p)2A, which should be able to penetrate intact cells more readily. While Kimchi et al. have shown that 2-5A core has an antimitogenic effect in mouse spleen lymphocytes and 3T3 fibroblasts, Williams and Kerr have reported lack of antiviral activity against Semliki Forest virus or encephalomyocarditis virus by exogenously-administered 2-5A core. We have previously determined that (xyloA2'p)2xyloA (abbreviated as xylo 2-5A core), the xyloadenosine analogue of the 5'-terminally dephosphorylated 2-5A core, is over 100 times more stable than the parent 2-5A core species. We now report that this xylo 2-5A core inhibits replication of herpes simplex viruses 1 and 2 in vitro, with greater than 100 times the activity of the parent 2-5A core. The mechanism of antiviral action of the 2-5A core analogue appears to involve a pathway different from that activated by the parent 5' triphosphorylated 2-5A species.     

低聚噻吩衍生物的合成及液晶性能

设计合成了一系列新型低聚噻吩衍生物N,N′-双烷烃基-5,5″-二溴基-2,2′:5′2″-三噻吩4,4″-二酰胺(DNCnDBr3T,n＝5,8,16,18)．示差扫描量热法测定及偏光光学显微镜观察的结果显示,DNCl8DBr3T、DNCl6DBr3T、DNC8DBr3T具有近晶A相液晶性质．研究结果表明,烷基链的长度以及分子间氢键对于液晶的形成起着重要作用．另外,还考察了DNCnDBr3T(n＝8,16,18)的光、电性能以及与有机溶剂的凝胶化性能．     

中缅树鼩肥满度、身体组成和消化道形态的比较

为了阐明不同区域中缅树鼩(Tupaia belangeri)的体重、肥满度、身体组成和消化道形态是否存在地理差异,对中国分部的中缅树鼩进行了采样,样本来自于云南省(河口、昆明、勐腊、大理、腾冲和片马)、贵州省(兴义)、四川省(西昌)、广西壮族自治区(大新和乐业)和海南省以及中国医学科学院医学生物学研究所实验室繁殖的F1代个体,对其体重、胴体重、体长、内脏器官和消化道的长度、重量进行了测定。结果表明:海南地区中缅树鼩的体重、胴体重和肥满度较大,而昆明地区的较小;海南和大新地区中缅树鼩的褐色脂肪组织和肝脏重量较大,昆明地区的较小,身体组成的其他指标均表现出显著的地区差异;大新地区中缅树鼩的消化道总长度显著大于其他种群,片马地区的最小,片马和腾冲地区中缅树鼩的总消化道含内容物重显著大于其他种群,实验室繁殖个体较小,消化道各指标也出现了地区差异。以上所有结果说明,不同地区的中缅树鼩在体重、肥满度、身体组成和消化道形态方面出现了地理差异,而这些差异可能和其适应不同的栖息环境有关。     

折叠超立方体的广义3-连通度

设图G是一个连通图,S⊆V(G)。图G的一棵S-斯坦纳树是一棵包含S中所有顶点的树T=(V ',E '),使得S⊆V '。如果连接S的两棵斯坦纳树T和T ',满足E(T)∩E(T ')=且V(T)∩V(T ')=S,则称T和T '是内部不交的。定义κ(S)为图G中内部不相交S-斯坦纳树的最大数目。广义k-连通度(2≤k≤n)定义为κ_k(G)=min{κ(S)|S⊆V(G)且|S|=k},显然,κ₂(G)=κ(G)。证明了κ₃(FQ_n)=n,其中FQ_n是n-维折叠超立方体。     

Identification of D segments of immunoglobulin heavy-chain genes and their rearrangement in T lymphocytes

The finding that the diversity (D) and joining (JH) but not the variable (VH) DNA segments of mouse immunoglobulin heavy-chain genes are joined in the DNA of some cloned cytolytic T cells, led to identification and sequencing of three different D DNA segments. Two segments identified on the embryo DNA carry on both the 5' and 3' sides two sets of characteristic sequences separated by a 12-base pair spacer, which have been implicated as recognition signals for a recombinase. The third segment, identified in a form joined with a JHDNA segment in a T cell, carries the recognition signal on the 5' side. These results support the 12/23-base pair model for somatic generation of immunoglobulin V genes, and rule out the possibility that the cytolytic T cells use assembled VH, D and JH sequences to encode their antigen receptors.     

软件开发方法探讨

软件开发方法自20世纪70年代提出后，前后经历两个大的发展阶段：结构化软件开发方法与面向对象的软件开发方法，后者比前者的需求分析更接近问题域，而且可维护性、可重用性等软件质量指标都有了实质性的突破，然而面向对象的软件开发方法的可重用度还不够，借鉴硬件的发展，软件能否也朝着组装模式发展呢?这就是基于构件的软件开发方法的初衷．     

A role for branchpoints in splicing in vivo

The nucleotides immediately surrounding intron/exon junctions of genes transcribed by RNA polymerase B can be derived from 'consensus' sequences for donor and acceptor splice sites by only a few base changes. Studies in vivo have underlined the importance of these junction nucleotides for splicing. In higher eukaryotes, no evidence has been found for specific internal intron sequences involved in splicing. However, the recent discovery that, in vitro, introns are excised in a lariat form where the 5' end of the intron is joined via a 2'-5'-phosphodiester linkage to an A residue (branchpoint acceptor) close to the 3' end of the intron, suggests that internal intron sequences may nonetheless be important for splicing. Indeed, in yeast nuclear genes, the internal sequence 5'-TACTAAC-3' (or close homologue) is essential for splicing in vivo. A proposed consensus sequence for branchpoints in mammalian introns is 5'-CT(A/G)A(C/T)-3'. This sequence resembles the essential yeast internal sequence. Are branchpoints involved in the splicing of introns of higher eukaryotes in vivo? We show here that a branchpoint sequence from a human globin gene (5'-CTGACTCTCTCTG-3') greatly enhances the efficiency of splicing of a 'synthetic' intron in HeLa cells. A mutated branchpoint sequence, 5'-CTCCTCTCTCTG-3', in which the branchpoint acceptor nucleotide A has been deleted and the neighbouring purine G mutated to a C, does not exhibit this enhancing capability. We conclude that branchpoints have an important function in the splicing process in vivo.     

Functional recognition of the 3' splice site AG by the splicing factor U2AF35

In metazoans, spliceosome assembly is initiated through recognition of the 5' splice site by U1 snRNP and the polypyrimidine tract by the U2 small nuclear ribonucleoprotein particle (snRNP) auxiliary factor, U2AF. U2AF is a heterodimer comprising a large subunit, U2AF65, and a small subunit, U2AF35. U2AF65 directly contacts the polypyrimidine tract and is required for splicing in vitro. In comparison, the role of U2AF35 has been puzzling: U2AF35 is highly conserved and is required for viability, but can be dispensed with for splicing in vitro. Here we use site-specific crosslinking to show that very early during spliceosome assembly U2AF35 directly contacts the 3' splice site. Mutational analysis and in vitro genetic selection indicate that U2AF35 has a sequence-specific RNA-binding activity that recognizes the 3'-splice-site consensus, AG/G. We show that for introns with weak polypyrimidine tracts, the U2AF35-3'-splice-site interaction is critical for U2AF binding and splicing. Our results demonstrate a new biochemical activity of U2AF35, identify the factor that initially recognizes the 3' splice site, and explain why the AG dinucleotide is required for the first step of splicing for some but not all introns.     

ATP-induced helicase slippage reveals highly coordinated subunits

Helicases are vital enzymes that carry out strand separation of duplex nucleic acids during replication, repair and recombination. Bacteriophage T7 gene product 4 is a model hexameric helicase that has been observed to use dTTP, but not ATP, to unwind double-stranded (ds)DNA as it translocates from 5' to 3' along single-stranded (ss)DNA. Whether and how different subunits of the helicase coordinate their chemo-mechanical activities and DNA binding during translocation is still under debate. Here we address this question using a single-molecule approach to monitor helicase unwinding. We found that T7 helicase does in fact unwind dsDNA in the presence of ATP and that the unwinding rate is even faster than that with dTTP. However, unwinding traces showed a remarkable sawtooth pattern where processive unwinding was repeatedly interrupted by sudden slippage events, ultimately preventing unwinding over a substantial distance. This behaviour was not observed with dTTP alone and was greatly reduced when ATP solution was supplemented with a small amount of dTTP. These findings presented an opportunity to use nucleotide mixtures to investigate helicase subunit coordination. We found that T7 helicase binds and hydrolyses ATP and dTTP by competitive kinetics such that the unwinding rate is dictated simply by their respective maximum rates V(max), Michaelis constants K(M) and concentrations. In contrast, processivity does not follow a simple competitive behaviour and shows a cooperative dependence on nucleotide concentrations. This does not agree with an uncoordinated mechanism where each subunit functions independently, but supports a model where nearly all subunits coordinate their chemo-mechanical activities and DNA binding. Our data indicate that only one subunit at a time can accept a nucleotide while other subunits are nucleotide-ligated and thus they interact with the DNA to ensure processivity. Such subunit coordination may be general to many ring-shaped helicases and reveals a potential mechanism for regulation of DNA unwinding during replication.