Upstream and downstream cis-acting elements for cleavage at the L4 polyadenylation site of adenovirus-2

A study of the cis-acting elements involved in the 3' end formation of the RNAs from the major late L4 family of adenovirus-2 was undertaken. Series of 5' or 3' end deletion mutants and mutants harboring either internal deletions or substitutions were prepared and assayed for in vitro cleavage. This first allowed the demonstration of a sequence, located at -6 to -29, relative to AAUAAA, whose deletion or substitution reduces cleavage efficiency at the L4 polyadenylation site two to three fold. This upstream efficiency element 5' AUCUUUGUUGUC/AUCUCUGUGCUG 3' is constituted of a partially repeated 12 nucleotide long, UCG rich sequence. The activities of the 2 sequence elements in cleavage are additive. We also searched for regulatory sequences downstream of the L4 polyadenylation site. We found that the deletion or substitution of a 30 nucleotide long UCG rich sequence, between nucleotides +7 and +35 relative to the cleavage site and harboring a UCCUGU repeat reduces cleavage efficiency at least ten fold. A GUUUUU sequence, starting at +35 had no influence. Thus, the usage of the L4 polyadenylation site requires down-stream sequences different from the canonical GU or U boxes and is regulated by upstream sequence elements.     

Influenza virus NS1 protein interacts with the cellular 30 kDa subunit of CPSF and inhibits 3'end formation of cellular pre-mRNAs

Inhibition of the nuclear export of poly(A)-containing mRNAs caused by the influenza A virus NS1 protein requires its effector domain. Here, we demonstrate that the NS1 effector domain functionally interacts with the cellular 30 kDa subunit of CPSF, an essential component of the 3' end processing machinery of cellular pre-mRNAs. In influenza virus-infected cells, the NS1 protein is physically associated with CPSF 30 kDa. Binding of the NS1 protein to the 30 kDa protein in vitro prevents CPSF binding to the RNA substrate and inhibits 3' end cleavage and polyadenylation of host pre-mRNAs. The NS1 protein also inhibits 3' end processing in vivo, and the uncleaved pre-mRNA remains in the nucleus. Via this novel regulation of pre-mRNA 3' end processing, the NS1 protein selectively inhibits the nuclear export of cellular, and not viral, mRNAs.     

Auxiliary downstream elements are required for efficient polyadenylation of mammalian pre-mRNAs

We have previously identified a G-rich sequence (GRS) as an auxiliary downstream element (AUX DSE) which influences the processing efficiency of the SV40 late polyadenylation signal. We have now determined that sequences downstream of the core U-rich element (URE) form a fundamental part of mammalian polyadenylation signals. These novel AUX DSEs all influenced the efficiency of 3'-end processing in vitro by stabilizing the assembly of CstF on the core downstream URE. Three possible mechanisms by which AUX DSEs mediate efficient in vitro 3'-end processing have been explored. First, AUX DSEs can promote processing efficiency by maintaining the core elements in an unstructured domain which allows the general polyadenylation factors to efficiently assemble on the RNA substrate. Second, AUX DSEs can enhance processing by forming a stable structure which helps focus binding of CstF to the core downstream URE. Finally, the GRS element, but not the binding site for the bacteriophage R17 coat protein, can substitute for the auxiliary downstream region of the adenovirus L3 polyadenylation signal. This suggests that AUX DSE binding proteins may play an active role in stimulating 3'-end processing by stabilizing the association of CstF with the RNA substrate. AUX DSEs, therefore, serve as a integral part of the polyadenylation signal and can affect signal strength and possibly regulation.     

Affinity cleavage at the metal-binding site of phosphoenolpyruvate carboxykinase

Chicken liver phosphoenolpyruvate carboxykinase (PEPCK) was rapidly inactivated by micromolar concentrations of ferrous sulfate in the presence of ascorbate at pH 7.4. Omitting ascorbate or replacing the Fe2+ with Mn2+ or Mg2+ gives no inactivation. Mn2+, Mg2+, or Co2+ at 100-fold molar excess over Fe2+ offered complete protection from Fe2+/ascorbate-induced inactivation. The substrates PEP and GTP, but not OAA, GDP, or CO2, offered full protection from inactivation. The addition of 5 mM EDTA stopped further inactivation of the enzyme. Thermodynamic studies indicate that the inactive enzyme no longer binds Mn2+ but still had high affinity for GTP indicating that the inactivation process was specific for the metal site. A decrease in cysteine content was observed over time following PEPCK treatment with Fe2+ and ascorbate. The apparent first-order rate constant for free sulfhydryl loss (0.085 +/- 0.005 min-1) is similar to the apparent first-order rate constant for inactivation (0.067 +/- 0.005 min-1). Amino acid composition analysis revealed that cysteic acid was generated upon Fe2+/ascorbate addition to PEPCK. Native chicken liver PEPCK has an Mr of 67 kDa. SDS-PAGE of the inactivated enzyme showed the presence of two new bands at 31.7 and 35.3 kDa indicating that PEPCK was specifically cleaved at a single site. The rate of cleavage was slower than the rate of inactivation and fully inactivated enzyme was only 50% cleaved. The Fe2+/ascorbate-catalyzed inactivation was not solely due to protein cleavage. The protein fragments generated by cleavage were separated by C4 reverse phase HPLC. The cleavage exposed a new N-terminus which was identified to be the 35.3 kDa C-terminal half of PEPCK. Sequencing of the fragments indicated that the site of cleavage was between Asp296 and Ile297. These results indicate that Asp296 is involved in metal chelation. This agrees with previous studies [Hlavaty, J. J., & Nowak, T. (1997) Biochemistry 36, 3389-3403] that suggested that Asp295 and Asp296 are involved in metal binding.     

RNA structure is a critical determinant of poly(A) site recognition by cleavage and polyadenylation specificity factor

Sequence conservation among mammalian poly(A) sites is limited to the sequence AAUAAA, coupled with an amorphous downstream U- or GU-rich region. Since these sequences may also occur within the coding region of mRNAs, additional information must be required to define authentic poly(A) sites. Several poly(A) sites have been shown to contain sequences outside the core elements that enhance the efficiency of 3' processing in vivo and in vitro. The human immunodeficiency virus type 1, equine infectious anemia virus, and adenovirus L1 3' processing enhancers have been shown to promote the binding of cleavage and polyadenylation specificity factor (CPSF), the factor responsible for recognition of AAUAAA, to the pre-mRNA, thereby facilitating the assembly of a stable 3' processing complex. We have used in vitro selection to examine the mechanism by which the human immunodeficiency virus type 1 3' processing enhancer promotes the interaction of CPSF with the AAUAAA hexamer. Surprisingly, RNAs selected for efficient polyadenylation were related by structure rather than sequence. Therefore, in the absence of extensive sequence conservation, our results strongly suggest that RNA structure is a critical determinant of poly(A) site recognition by CPSF and may play a key role in poly(A) site definition.     

Structural variation induced by different nucleotides at the cleavage site of the hammerhead ribozyme

The hammerhead ribozyme is capable of cleaving RNA substrates at 5' UX 3' sequences (where the cleavage site, X, can be A, C, or U). Hammerhead complexes containing dC, dA, dI, or rG nucleotides at the cleavage site have been studied by NMR. The rG at the cleavage site forms a Watson-Crick base pair with C3 in the conserved core of the hammerhead, indicating that rG substrates inhibit the cleavage reaction by stabilizing an inactive conformation of the molecule. Isotope-edited NMR experiments on the hammerhead complexes show that there are different short proton-proton distances between neighboring residues depending upon whether there is a dC or dA at the cleavage site. These NMR data demonstrate that there are significant differences in the structure and/or dynamics of the active-site residues in these hammerhead complexes. Molecular dynamics calculations were used to model the conformations of the cleavage-site variants consistent with the NMR data. The solution conformations of the hammerhead ribozyme-substrate complexes are compared with the X-ray structure of the hammerhead ribozyme and are used to help understand the thermodynamic and kinetic differences among the cleavage-site variants.     

Identification of a stem-loop structure important for polyadenylation at the murine IgM secretory poly(A) site

We have previously shown that a distal GU-rich downstream element of the mouse IgM secretory poly(A) site is important for polyadenylation in vivo and for polyadenylation specific complex formation in vitro. This element can be predicted to form a stem-loop structure with two asymmetric internal loops. As stem-loop structures commonly define protein RNA binding sites, we have probed the biological activity of the secondary structure of this element. We show that mutations affecting the stem of the structure abolish the biological activity of this element in vivo and in vitro at the level of cleavage and polyadenylation specificity factor/cleavage stimulation factor complex formation and that both internal loops contribute to the enhancing effect of the sequence in vivo. Lead (II) cleavage patterns and RNase H probing of the sequence element in vitro are consistent with the predicted secondary structure. Furthermore, mobility on native PAGE suggests a bent structure. We propose that the secondary structure of this downstream element optimizes its interaction with components of the polyadenylation complex.     

B-lineage regulated polyadenylation occurs on weak poly(A) sites regardless of sequence composition at the cleavage and downstream regions

Early/memory and plasma B-cell lines and fibroblasts were analyzed for their ability to use a 5' proximal (variant) versus a 3' distal (constant) poly(A) site, in the absence of a competing splice, from a set of related constructs. The proximal:distal poly(A) site use (P:D ratio) of the resulting cytoplasmic poly(A)+ mRNA is a measure of poly(A) site strength. In this context the immunoglobulin gamma2b secretory-specific poly(A) site showed a P:D ratio of 1:1 in plasma cells, 0.43:1 in early/memory B-cells and an intermediate value in fibroblasts. Meanwhile, a construct with a proximal SV40 early-like poly(A) site produced mRNA with a P:D ratio of >50:1 in all cell types. Alterations in the region downstream of the proximal poly(A) addition site and at the site itself resulted in changes in the P:D ratio. However, these poly(A) sites, all with a P:D ratio of < or = 5:1, were used most efficiently in plasma cells. Constructs totally devoid of immunoglobulin sequences, but containing heterologous poly(A) sites producing mRNA with P:D ratios of < or = 5:1, were also used more efficiently in plasma cells. We therefore conclude that weak poly(A) sites, regardless of sequence composition, are used more efficiently in plasma cells than in the other cell types.     

U1 snRNP inhibits pre-mRNA polyadenylation through a direct interaction between U1 70K and poly(A) polymerase

The brain damage produced by unilateral cerebral hypoxia-ischemia in the immature rat results from major alterations in cerebral energy metabolism and glucose utilization which begin during the course of the insult and proceed into the recovery period. Consistent with a lack of pathology, the alterations in the hemisphere contralateral to the carotid artery ligation are transient and return to normal within 24 h of recovery, whereas the hemisphere ipsilateral to the ligation exhibits both early and late responses, and infarction. The facilitative glucose transporter proteins mediate glucose transport across the blood-brain barrier (55 kDa GLUT1), and into neurons and glia (GLUT3 and 45 kDa GLUT1), and demonstrate both early and late responses to perinatal hypoxia-ischemia. This study employed in situ hybridization histochemistry to investigate the temporal and regional patterns of GLUT1 and GLUT3 gene expression following a severe (2.5 h) hypoxic-ischemic insult in the 7-day old rat brain. Enhanced GLUT1 mRNA expression was apparent in cerebral microvessels of both hemispheres and remained elevated in the ipsilateral hemisphere through 24 h of recovery, consistent with our previous observation of increased microvascular 55 kDa GLUT1 protein. The expression of the neuronal isoform, GLUT3, was enhanced in penumbral regions, such as piriform cortex and amygdala, but was rapidly reduced in the affected areas of cortex, hippocampus and thalamus, reflecting necrosis. The late response, observed at 72 h of recovery, was characterized by extensive necrosis in the ipsilateral hemisphere, loss of GLUT3 expression, and a gliotic reaction including increased GLUT1 in GFAP-positive astrocytes. This study demonstrates that cerebral hypoxia-ischemia in the immature rat produces both immediate-early and long-term effects on the glucose transporter proteins at the level of gene expression.     

Synthesis and cleavage of oligodeoxynucleotides containing a 5-hydroxyuracil residue at a defined site

Oxidation and hydrolysis of a cytosine residue can lead to the formation of 5-hydroxyuracil in DNA. The biological consequences of this modification are not fully understood. To facilitate biochemical and biophysical studies aimed at elucidating the effects of this modification in DNA, we have developed a solid-phase synthetic method for the placement of 5-hydroxyuracil residues at defined sites in oligodeoxynucleotides. This method is based upon the enhanced acidity of the 5-hydroxyl proton which allows selective aqueous acetylation. Under standard aqueous ammonia deprotection conditions, however, we observed that 5-hydroxyuracil residues are lost substantially from synthetic oligonucleotides. Substitution of aqueous ammonia with methanolic potassium carbonate and the use of phosphoramidite derivatives with alternatively protected amino groups allow synthesis of oligonucleotides containing 5-hydroxyuracil and all normal bases in high yield. The composition of the oligodeoxynucleotides prepared by this method has been verified by enzymatic digestion followed by high-performance liquid chromatography (HPLC) analysis as well as acid hydrolysis followed by GC/MS analysis. The location of the 5-hydroxyuracil residue is demonstrated by selective permanganate oxidation of the 5-hydroxyuracil residue followed by beta-elimination. We have also probed a synthetic oligonucleotide containing a unique 5-hydroxyuracil residue with uracil DNA N-glycosylase, previously reported to remove this lesion from DNA.     

A H1 hemagglutinin of a human influenza A virus with a carbohydrate-modulated receptor binding site and an unusual cleavage site

Two receptor binding variants of the influenza virus A/Tübingen/12/85 (H1N1) were separated by their different plaque formation in MDCK cells. Hemagglutination of variant I was restricted to red blood cells of guinea pigs, whereas variant II also hemagglutinated chicken cells. The variants differed also in their ability to bind to alpha 2,6-linked sialic acid. Evidence is presented that this difference is determined by a complex carbohydrate side chain at asparagine131 near the receptor binding site which is absent in variant II. With both variants, the arginine found at the cleavage site of all other human isolates analyzed so far was replaced by lysine.     

Study on site specific cleavage of RNA

The precursor of an RNA molecule from bacteriophage T4 infected Escherichia coli cell (p2Sp1 RNA) has the ability to cleave itself. It has been found that the site specific RNA cleavage reaction occurred at the pyridine-adenosine sequence in the presence of a monovalent cation and a non-ionic detergent. In order to investigate the mechanism of this cleavage reaction, we designed a RNA oligonucleotide (UUUAUU) and this RNA was cleavage activity at the U-A sequence.     

HSP70-2 heat-shock protein of mouse spermatogenic cells

A novel hepta-substituted beta-cyclodextrin bearing the methoxyethylamine group linked to the upper cyclodextrin rim was successfully used as a chiral selector for enantiomeric separation of non-steroidal anti-inflammatory drugs (NSAIDs) and phenoxypropionic acid herbicides (PPAHs). Separation parameters such as pH and concentration were found to have major influences on enantiomeric resolution of the NSAIDs and PPAHs. Results indicate that heptakis(6-methoxyethylamine-6-deoxy)-beta-cyclodextrin [beta-CD-OMe (VII)] performs exceptionally well for the enantiomeric resolution of NSAIDs: indoprofen and fenoprofen (Rs = 11 and 14, respectively). In addition, baseline enantiomeric separation of a mixture of six pairs of PPAHs was achieved in under 30 min. Compared to other cationic beta-cyclodextrins reported in the literature, the beta-CD-OMe (VII) showed improved selectivity for both classes of the aforementioned anionic racemates.     

Thyrotropin receptor cleavage at site 1 involves two discontinuous segments at each end of the unique 50-amino acid insertion

Among the glycoprotein hormone receptors, only the thyrotropin receptor (TSHR) cleaves (at two sites) into disulfide-linked A and B subunits. A 50-amino acid insertion unique to the TSHR ectodomain (residues 317-366) plays no role in ligand binding or signal transduction, but its deletion abrogates cleavage at Site 1, closely upstream of the insertion. We sought to define the region within the 50-amino acid tract involved in TSHR cleavage at Site 1. Mutation of small segments within this region previously failed to prevent cleavage at Site 1. We, therefore, divided the 50-amino acid insertion into quartiles and deleted each one individually (TSHR residues 317-327, 328-338, 339-350, and 351-362). As determined by covalent cross-linking of 125I-TSH to intact cells expressing the mutant receptors, none of these deletions prevented TSHR cleavage at Site 1. Neither did larger deletions of quartiles 1 + 2, 2 + 3, and 3 + 4. However, qualitative differences in the extent of receptor cleavage suggested that quartiles 1 and 4 were playing a greater role in cleavage at Site 1 than were the middle two quartiles. In support of this hypothesis, deletion of these two discontinuous segments almost completely eliminated TSHR cleavage at Site 1. In conclusion, intramolecular cleavage at Site 1 requires the presence of the N-terminal and C-terminal quartiles of the 50-amino acid insertion unique to the TSHR. Taken together with previous observations, our data suggest that this tract may provide a discontinuous binding site for a protease that clips the TSHR at Site 1.     

Cell-cycle-dependent nuclear translocation of HSP70 in amphibian embryonic cells

The Unified Parkinson's Disease Rating Scale (UPDRS) is widely used to assess Parkinson's disease (PD) disability but its metric properties have not been extensively studied. We investigated the factor structure and internal consistency of the Motor Examination section of the UPDRS in a sample of 294 consecutive patients with idiopathic PD who were assessed while in the "on" state. There was a high degree of internal consistency. Factor analysis revealed six clinically distinct factors, three bradykinesia measures (axial/gait, right and left), one rigidity measure, and two tremor measures (rest and postural). These factors accounted for 78% of the variance. Total Motor Examination scores and selected factors correlated well with other examiner-determined global ratings of PD disability (Hoehn and Yahr stage and Schwab-England Activities of Daily Living score). These results suggest that the Motor Examination section of the UPDRS provides a useful measure of PD function as well as severity measures of six clinical disability domains.     

Detection of potato virus Y P1 protein in infected cells and analysis of its cleavage site

We report a patient with congenital homozygous factor V deficiency in whom a large pseudotumour in the right upper leg was successfully surgically excised under continuous substitution with fresh-frozen plasma.     

Mutational analysis of the encephalomyocarditis virus primary cleavage

Sixteen substitution mutations of the conserved DvExNPGP sequence, implicated in cardiovirus and aphthovirus primary polyprotein cleavage, were created in encephalomyocarditis virus cDNA, expressed, and characterized for processing activity. Nearly all the mutations severely decreased the efficiency of the primary cleavage reaction during cell-free synthesis of viral precursors, indicating a stringent requirement for the natural sequence in this processing event. When representative mutations were tested in full-length genomic contexts, they were lethal and no revertants were observed. Not only were the primary cleavage reactions deficient in these polyproteins, but subsequent cleavage of P1 by endogenous or exogenous 3C pro was also impaired. This indicates that primary cleavage has a role in the proper processing of the viral capsid precursor.     

卡介苗热休克蛋白70对肿瘤细胞裂解物免疫原性的影响

目的:观察卡介苗热休克蛋白70(BCGHSP70)对肿瘤细胞裂解物(TCL)免疫原性的影响,为TCL相关肿瘤疫苗的制备提供依据.方法:应用冻融法制备B16黑色素瘤细胞裂解物B16TCL,将BCGHSP70与B16TCL在体外混合制备BCGHSP70-B16TCL.将C57BL/6小鼠随机分为BCGHSP70-B16TCL组、PBS组、BCGHSP70组和B16TCL组,分别于第1和14天皮下免疫BCGHSP70-B16TCL、PBS、BCGHSP70和B16TCL.于末次免疫后第7天处死小鼠,MTT法观察不同免疫组小鼠脾淋巴细胞在B16TCL体外刺激后的增殖情况,计算刺激指数(SI).结果:应用冻融法能够使B16细胞完全裂解,所制备的B16TCL中含有丰富的蛋白成分.与PBS组、BCGHSP70组和B16TCL组比较,BCGHSP70-B16TCL组免疫小鼠脾脏明显增大;BCGHSP70-B16TCL免疫组SI高于PBS免疫组(P=0.00)、B16TCL免疫组(P=0.01)和BCGHSP70免疫组(P=0.00).结论:BCGHSP70 能够增强B16TCL的免疫原性,提示BCGHSP70可能作为有效的免疫佐剂用于TCL相关肿瘤疫苗的研究.