The identification of nuclear proteins that bind the homopyrimidine strand of d(GA.TC)n DNA sequences, but not the homopurine strand.

Alternating d(GA.TC)(n)DNA sequences, which are abundant in eukaryotic genomes, can form altered DNA structures. Depending on the environmental conditions, the formation of (GA.GA) hairpins or [C+T(GA.TC)] and [GA(GA.TC)] intramolecular triplexes was observed in vitro. In vivo, the formation of these non-B-DNA structures would likely require the contribution of specific stabilizing factors. Here, we show that Friend's nuclear extracts are rich in proteins which bind the pyrimidine d(TC)(n)strand but not the purine d(GA)n strand (NOGA proteins). Upon chromatographic fractionation, four major proteins were detected (NOGA1-4) that have been purified and characterized. Purified NOGAs bind single-stranded d(TC)n with high affinity and specificity, showing no significant affinity for either d(GA)n or d(GA.TC)nDNA sequences. We also show that NOGA1, -2 and -3, which constitute the three most abundant and specific NOGA proteins, correspond to the single-stranded nucleic acid binding proteins hnRNP-L, -K and -I, respectively. These results are discussed in the context of the possible contribution of the NOGA proteins to the stabilization of the (GA.GA) and [GA(GA.TC)] conformers of the d(GA.TC)n DNA sequences.     

Characterization and developmental expression of single-stranded telomeric DNA-binding proteins from mung bean (Vigna radiata)

We have identified and characterized protein factors from mung bean (Vigna radiata) nuclear extracts that specifically bind the single-stranded G-rich telomeric DNA repeats. Nuclear extracts were prepared from three different types of plant tissue, radicle, hypocotyl, and root, in order to examine changes in the expression patterns of telomere-binding proteins during the development of mung bean. At least three types of specific complexes (A, B, and C) were detected by gel retardation assays with synthetic telomere and nuclear extract from radicle tissue, whereas the two major faster-migrating complexes (A and B) were formed with nuclear extracts from hypocotyl and root tissues. Gel retardation assays also revealed differences in relative amount of each complex forming activity in radicle, hypocotyl, and root nuclear extracts. These data suggest that the expression of telomere-binding proteins is developmentally regulated in plants, and that the factor involved in the formation of complex C may be required during the early stages of development. The binding factors have properties of proteins and are hence designated as mung bean G-rich telomere-binding proteins (MGBP). MGBPs bind DNA substrates with three or more single-stranded TTTAGGG repeats, while none of them show binding affinity to either double-stranded or single-stranded C-rich telomeric DNA. These proteins have a lower affinity to human telomeric sequences than to plant telomeric sequences and do not exhibit a significant binding activity to Tetrahymena telomeric sequence or mutated plant telomeric sequences, indicating that their binding activities are specific to plant telomere. Furthermore, RNase treatment of the nuclear extracts did not affect the complex formation activities. This result indicates that the single-stranded telomere-binding activities may be attributed to a simple protein but not a ribonucleoprotein. The ability of MGBPs to bind specifically the single-stranded TTTAGGG repeats may suggest their in vivo functions in the chromosome ends of plants.     

Differential ASF/SF2 activity in extracts from normal WI38 and transformed WI38VA13 cells.

The normal human fibroblast cell line WI38 and a transformed derivative, WI38VA13, differentially splice fibronectin pre-mRNA in vivo. As a first step to understand the molecular basis for this regulation of splicing, we examined the ability of WI38 and WI38VA13 nuclear extracts to splice model adenovirus and globin pre-mRNAs. Adenovirus RNA splicing was detected in WI38VA13 but not in WI38 extracts. Likewise, when supplemented with a HeLa post-nuclear supernatant (S100), human beta-globin RNA splicing was detected in WI38VA13 but not in WI38 extracts. The splicing defect in WI38 extracts was associated with a reduced ability to form splicing complexes and with a corresponding decrease in the interaction of U2 small nuclear ribonucleoprotein (snRNP) with the branchsite. These defects did not correlate with a decrease in 65 kD U2AF binding since equivalent U2AF level and activity were detected in WI38 and WI38VA13 extracts. Rather, WI38 extracts displayed reduced ASF/SF2 activity and contained a low level of 30 and 40 kD SR phosphoproteins. Moreover, addition of purified ASF/SF2 dramatically increased splicing complex formation in WI38 extracts. These results raise the possibility that variations in the level and activity of ASF/SF2 and other SR proteins play a role in the regulation of fibronectin splicing.     

Rice proteins that bind single-stranded G-rich telomere DNA

In this work, we have identified and characterized proteins in rice nuclear extracts that specifically bind the single-stranded G-rich telomere sequence. Three types of specific DNA-protein complexes (I, II, and III) were identified by gel retardation assays using synthetic telomere substrates consisting of two or more single-stranded TTTAGGG repeats and rice nuclear extracts. Since each complex has a unique biochemical property and differs in electrophoretic mobility, at least three different proteins interact with the G-rich telomere sequences. These proteins are called rice G-rich telomere binding protein (RGBP) and none of them show binding affinity to double-stranded telomere repeats or single-stranded C-rich sequence. Changing one or two G's to C's in the TTTAGGG repeats abolishes binding activity. RGBPs have a greatly reduced affinity for human and Tetrahymena telomeric sequence and do not efficiently bind the cognate G-rich telomere RNA sequence UUUAGGG. Like other telomere binding proteins, RGBPs are resistant to high salt concentrations. RNase sensitivity of the DNA-protein interactions was tested to investigate whether an RNA component mediates the telomeric DNA-protein interaction. In this assay, we observed a novel complex (complex III) in gel retardation assays which did not alter the mobilities or the band intensities of the two pre-existing complexes (I and II). The complex III, in addition to binding to telomeric sequences, has a binding affinity to rice nuclear RNA, whereas two other complexes have a binding affinity to only single-stranded G-rich telomere DNA. Taken together, these studies suggest that RGBPs are new types of telomere-binding proteins that bind in vitro to single-stranded G-rich telomere DNA in the angiosperms.     

Cytosine-block telomeric type DNA-binding activity of hnRNP proteins from human cell lines

Following the observation of the presence in mammalian nuclear extracts of a DNA binding activity quite specific for the single-stranded C-rich telomeric motif, we have isolated from the K562 human cell line by affinity chromatography and identified by mass spectrometry a number of proteins able to bind to this sequence. All of them belong to different heterogeneous nuclear ribonucleoprotein subgroups (hnRNP). Whereas many of them, namely hnRNP K, two isoforms of hnRNP I, and the factor JKTBP, appear to bind to this sequence with limited specificity after isolation, an isoform of hnRNP D (alias AUF1) and particularly hnRNP E1 (alias PCBP-1) show a remarkable specificity for the (CCCTAA)n repeated motif. Both have been obtained also as recombinant proteins expressed in Escherichia coli and have been shown to retain their binding specificity toward the C-block repeated sequence. In the light of the current knowledge about these proteins, their possible involvement in telomere functioning is discussed.     

Monoclonal antibodies showing sequence specificity in their interaction with single-stranded DNAs.

Six hybridoma cell lines which secrete monoclonal antibodies binding to nucleic acids were produced from autoimmune NZB/NZW mice. Four of the antibodies were IgG's and the other two were IgM's. Using a solid phase radioimmunoassay (SPRIA) the binding of the antibodies to over thirty different nucleic acids was estimated. All the antibodies were extremely specific. There was no detectable interaction with various RNAs, and single-stranded DNAs bound more antibodies than duplex or multi-stranded DNAs. In every case the antibodies also showed considerable sequence preferences. For example one monoclonal antibody bound to d(TTC)n but not to d(TCC)n while another interacted strongly with D(TG)n and d(CA)n but not with d(TC)n, d(GA)n or homopolymers. In other cases the patterns of sequence specificity were extremely difficult to interpret although it seems clear that monoclonal antibodies have the potential to distinguish between any two nucleic acids however similar.     

Evidence for a HeLa nuclear protein that binds specifically to the single-stranded d(CCCTAA)n telomeric motif.

In recent years several telomere binding proteins from eukaryotic organisms have been identified that are able to recognise specifically the duplex telomeric DNA repeat or the G-rich 3'-ending single strand. In this paper we present experimental evidence that HeLa nuclear extracts contain a protein that binds with high specificity to the single-stranded complementary d(CCCTAA)n repeat. Electrophoretic mobility shift assays show that the oligonucleotide d(CCCTAACCCTAACCCTAACCCT) forms a stable complex with this protein in the presence of up to 1000-fold excesses of single-stranded DNA and RNA competitors, but is prevented from doing so in the presence of its complementary strand. SDS-PAGE experiments after UV cross-linking of the complex provide an estimate of 50 kDa for the molecular weight of this protein.     

The lef-3 gene of Autographa californica nuclear polyhedrosis virus encodes a single-stranded DNA-binding protein.

The Autographa californica nuclear polyhedrosis virus (AcNPV) replicates in the nuclei of infected cells and encodes several proteins required for viral DNA replication. As a first step in the functional characterization of viral replication proteins, we purified a single-stranded DNA-binding protein (SSB) from AcNPV-infected insect cells. Nuclear extracts were chromatographed on single-stranded DNA agarose columns. An abundant protein with an apparent molecular weight of 43,000 was eluted from the columns at 0.9 to 1.0 M NaCl. This protein was not evident in extracts prepared from control cells, suggesting that the SSB was encoded by the virus. SSB bound to single-stranded DNA in solution, and binding was nonspecific with respect to base sequence, as single-stranded vector DNA competed as efficiently as single-stranded DNA containing the AcNPV origin of DNA replication. Competition binding experiments indicated that SSB showed a preference for single-stranded DNA over double-stranded DNA. To determine whether SSB was encoded by the lef-3 gene of AcNPV, the lef-3 open reading frame was cloned under the control of the bacteriophage T7 promoter. Immunochemical analyses indicated that LEF-3 produced in bacteria or in rabbit reticulocyte lysates specifically reacted with antiserum produced by immunization with purified SSB. Immunoblot analyses of infected cell extracts revealed that SSB/LEF-3 was detected by 4 h postinfection and accumulated through 48 h postinfection.     

Single-stranded DNA binding proteins isolated from mouse brain recognize specific trinucleotide repeat sequences in vitro.

Expansion of trinucleotide repeats (CAG)n and (CGG)n is found in genes responsible for certain human hereditary neurodegenerative diseases. By gel-mobility shift assay, we detected a single-stranded (AGC)n repeat-binding activity primarily in mouse brain extracts and very low or undetectable activity in other tissue extracts. Two (AGC)n-repeat binding proteins, with apparent molecular weights of 44 and 40 kDa, have been purified from mouse adult brain by a DNA affinity column and fast protein liquid chromatography. UV-cross linking of radiolabeled (AGC)n repeats with crude brain extracts and with purified two proteins of 44 and 40 kDa produced identical doublet bands, indicating that these proteins are in fact responsible for the (AGC)n-binding activity in brain extracts. We designated these two proteins TRIP-1 for the 44 kDa protein and TRIP-2 for the 40 kDa protein, where TRIP represents trinucleotide repeat-binding protein. TRIP-1 and TRIP-2 bind to a specific subset of trinucleotide repeat sequences including (AGC)n, (AGT)n, (GGC)n, and (GGT)n repeats but not to various other trinucleotide repeats. A minimum of eight (AGC) trinucleotide repeating units is required for TRIP-1 and -2 recognition and binding. The (AGC)n repeat-binding activity increases in the brain after birth and reaches a plateau within 3 weeks. In the brain, TRIP-1 and TRIP-2 may alter the function of the genes containing the expanded-trinucleotide repeats.     

Characterization of a multisubunit human protein which selectively binds single stranded d(GA)n and d(GT)n sequence repeats in DNA.

A protein which selectively binds d(GA)n and d(GT)n sequence repeats in single stranded DNA has been identified in human fibroblasts. This protein, designated PGB, has been purified at least 500-fold by ammonium sulfate precipitation followed by DEAE-Sepharose column chromatography and affinity chromatography in a column of d(GA)-Sepharose. Electrophoretic mobility shift assays revealed that the PGB protein bound most avidly d(GA)n and d(GT)n tracts of n > 5. It also bound other G-rich DNA sequence repeats, including dGn tracts, with lower affinities. It did not manifest significant binding affinities to single stranded M13 DNA, or to the homopolynucleotides poly dA, poly dC and poly dT, or to various DNA sequence repeats which do not contain G residues, such as d(A-C)n and d(TC)n. It did not bind double stranded d(T-C)n.d(GA)n tracts or other double stranded DNA sequences. In glycerol gradient centrifugation assays the d(GA)n- and the d(GT)n-binding activities cosedimented as a homogeneous protein species having an S20,w = 9.4 +/- 0.7 and an estimated native molecular weight of 190,000 +/- 7,000. UV crosslinking assays revealed that the protein contains 33.6 +/- 2.1 kd subunits which bind d(GA)n and d(GT)n sequences. However, SDS-polyacrylamide gel electrophoresis of the purified protein followed by silver staining indicated that it may also contain other subunits that do not contact the DNA. It is proposed that binding of the PGB protein to single stranded d(GA)n or d(GT)n tracts in double stranded topologically restricted DNA may stimulate strand separation and formation of triple helices or other unusual DNA structures.     

鸡胚骨骼肌组织M-CAT结合因子的初步鉴定

采用偶联ＣＡＴＴＧＣＴ寡核苷酸的ＤＮＡ亲和层析柱从发育１３ｄ鸡胚骨骼肌核抽提物中分离到两种核蛋白．ＳＤＳ－ＰＡＧＥ结果表明,被ＤＮＡ亲和柱滞留的两种核蛋白分子量分别为３０ｋＤ和３２ｋＤ．凝胶阻滞结合竞争分析显示,纯化的核蛋白可与Ｍ－ＣＡＴ共有序列ＣＡＴＴＣＣＴ特异结合．Ｓｏｕｔｈｗｅｓｔｅｒｎ印迹技术确定仅３０ｋＤ分子可直接识别、结合ＣＡＴＴＣＣＴ元件,但３２ｋＤ分子却不能．结果提示,３０ｋＤ分子为依赖ＤＮＡ的Ｍ－ＣＡＴ结合因子,３２ｋＤ分子属性有待进一步研究证实     

降血脂药物（DEHP）对酰基CoA氧化酶基因反式作用因子的影响

从对照和用ＤＥＨＰ处理的大鼠肝脏提取核蛋白,以含酰基ＣｏＡ氧化酶（ＡＯＸ）基因表达调控部位的ＤＮＡ片段和该基因的不同蛋白结合位点的ＤＮＡ片段作为核蛋白结合反应的探针,通过凝胶电泳迁移率改变实验和Ｓｏｕｔｈｗｅｓｔｅｒｎ印迹分析检查了ＤＥＨＰ对ＡＯＸ基因反式作用因子的影响。结果表明,降血脂药物ＤＥＨＰ可显著增加ＡＯＸ基因反式作用因子的含量和（或）与基因的结合活性,在转录水平上促进基因的表达。     

Proteolysis of splicing factors during rat and monkey cell fractionation.

We have investigated the ability of various rat and monkey cell lines to yield nuclear extracts that would allow splicing of a model adenovirus pre-mRNA substrate. Extracts from normal FR3T3, rat-1 and CV-1 fibroblasts were unable to assemble splicing complexes and displayed a dramatic reduction in the binding activity of the splicing factor 65 kD U2AF. These results correlated with reduced levels of 65 kD U2AF and the snRNP-associated B protein. When a battery of protease inhibitors was used during cell fractionation, increased levels of 65 kD U2AF and B proteins were detected. Most importantly, U2AF binding and complex formation were dramatically improved in FR3T3, rat-1 and CV-1 extracts. Interestingly, transformation of rat and monkey cells with the SV40 large T antigen yielded extracts active in complex formation. Similar extracts were generated following transformation of rat-1 cells with the Py middle T antigen but not with the v-fos oncogene. Only SV40-transformed FR3T3 extracts displayed splicing activity. Our results indicate that proteolysis is a major obstacle encountered during the preparation of active extracts from normal rat and monkey cells and suggest that cells transformed with T antigens manifest reduced proteolysis during fractionation.     

Structure of hnRNP D complexed with single-stranded telomere DNA and unfolding of the quadruplex by heterogeneous nuclear ribonucleoprotein D

Heterogeneous nuclear ribonucleoprotein D, also known as AUF1, has two DNA/RNA-binding domains, each of which can specifically bind to single-stranded d(TTAGGG)n, the human telomeric repeat. Here, the structure of the C-terminal-binding domain (BD2) complexed with single-stranded d(TTAGGG) determined by NMR is presented. The structure has revealed that each residue of the d(TAG) segment is recognized by BD2 in a base-specific manner. The interactions deduced from the structure have been confirmed by gel retardation experiments with mutant BD2 and DNA. It is known that single-stranded DNA with the telomeric repeat tends to form a quadruplex and that the quadruplex has an inhibitory effect on telomere elongation by telomerase. This time it is revealed that BD2 unfolds the quadruplex of such DNA upon binding. Moreover, the effect of BD2 on the elongation by telomerase was examined in vitro. These results suggest the possible involvement of heterogeneous nuclear ribonucleoprotein D in maintenance of the telomere 3'-overhang either through protection of a single-stranded DNA or destabilization of the potentially deleterious quadruplex structure for the elongation by telomerase.