Hammerhead ribozymes targeted to the FBN1 mRNA can discriminate a single base mismatch between ribozyme and target

Hammerhead ribozymes are catalytic RNA molecules that can act in trans, with ribozyme and substrate being two different oligoribonucleotides with regions of complementarity. Mutations in the gene for fibrillin-1 (FBN1) cause Marfan syndrome. The majority of mutations are single-base changes, many of which exert their effect via a dominant-negative mechanism. Previously we have shown that an antisense hammerhead ribozyme, targeted to the FBN1 mRNA can reduce deposition of fibrillin to the extracellular matrix of cultured fibroblasts, suggesting it may be possible to utilize ribozymes to down regulate the production of mutant protein and thus restore normal fibrillin function. This might be achieved by the mutation creating a ribozyme cleavage site that is not present in the normal allele, however this is likely to limit the number of mutations that could be targeted. Alternatively, it might be possible to target the mutant allele via the ribozyme binding arms. To determine the potential of ribozymes to preferentially target mutant FBN1 alleles via the latter approach, the effect of mismatches in helix I of a hammerhead ribozyme, on the cleavage of fibrillin (FBN1) mRNA was investigated. A single base mismatch significantly reduced ribozyme cleavage efficiency both in vitro and in vivo. The discrimination between fully-matched and mismatched ribozyme varied with the length of helix I, with the discrimination being more pronounced in ribozymes with a shorter helix. These data suggest that it should be possible to design hammerhead ribozymes that can discriminate between closely related (mutant and normal) target RNAs varying in as little as a single nucleotide, even if the mutation does not create a ribozyme cleavage site.     

Investigation of techniques for measuring lattice mismatch in a rhenium containing nickel base superalloy

Three techniques for measuring γ/γ′ lattice mismatch have been examined in a Ni-base superalloy subjected to two different aging heat treatments. The techniques used for measuring lattice mismatch were X-ray diffraction, convergent beam electron diffraction (CBED) and interface dislocation analysis. Additionally, a scanning transmission electron microscope equipped with an X-ray energy dispersive spectroscopy (EDS) system has been used to examine phase compositions. From this study, it has been determined that the X-ray diffraction and CBED yield similar results for room temperature lattice mismatch, although care must be taken in applying the CBED technique due to the complex strain fields present in high volume %γ′ alloys. The dislocation analysis technique gives larger negative values of mismatch. It is believed that these latter values represent those which exist at the aging temperature.     

The liquid film migration in a sintered Fe-Cr-C base alloy

In an Fe-5Cr-lMo-2Cu-0.5P-3C (wt pct) alloy prepared by liquid-phase sintering at 1120 °C, the intergranular liquid films migrate during cooling and isothermal heat treatment at temperatures where the solid and liquid phases coexist. The liquid film migration (LFM) occurs extensively even during rapid cooling, apparently because of a large driving force. The migrating liquid films solidify to form network carbides. In the regions swept by the migrating liquid films, the concentrations of the substitutional solute atoms are slightly different from those in the original grains. When cooled rapidly, martensite is abundant in these regions. It is possible that the driving force for LFM in this alloy stems in part from the C concentration change associated with the concentration changes of the slowly diffusing substitutional solute atoms.     

Enhanced discrimination of single nucleotide polymorphisms by artificial mismatch hybridization

A 53-year-old patient had a complex ventricular arrhythmia, which most likely was an intermittent pure (nonmodulated) parasystole, due to protection limited to the first part of the cycle coexisting with exit block. However, other interpretations of the observations were possible. Foremost among these was modulated parasystole with partial protection or with an attenuated or nondetectable early delaying phase, with exit block. Interestingly, the parasystole appeared to have fulfilled the dynamic rules regarding the number of sinus beats interposed between manifest parasystolic beats, as has been reported for pure or weakly modulated parasystole. This case corroborates unusual manifestations of an arrhythmia, which because of its newly found complexities and various possible interpretations seems to be discussed with decreasing frequency in most textbooks on general cardiology.     

Analysis of DNA mismatch repair proteins in human medulloblastoma

During replication, the primary function of the eukaryotic DNA mismatch repair (MMR) system is to recognize and correct mismatched base pairs within the DNA helix. Deficiencies in MMR have been reported previously in cases of hereditary nonpolyposis colorectal cancer and sporadic tumors occurring in a variety of tissues including gliomas. Furthermore, recent evidence indicates that the MMR system may be involved in mediating therapeutic sensitivity to alkylating agents. In this study, 22 neoplastic tissue samples from 22 patients who underwent surgical resection for medulloblastoma, a common cerebellar tumor of childhood, were assayed for the presence or absence of MMR polypeptides using Western blot and immunohistochemical techniques. Results from these experiments indicate that the MMR system is not commonly deficient in medulloblastoma.     

Recognition of a non-standard base pair by thermostable DNA polymerases

Fluoride is a potent preventive agent of caries. It acts at two levels: local and systemic. At the local level it prevents the surface demineralisation of enamel by strengthening the superficial layers, and inhibiting the local bacterial activity. At the systemic level it improves the resistance of the enamel by forming with calcium, fluorapatite which is stronger than hydroxyapatite. Main sources are naturally fluorinated water, artificially enriched water, fluorinated salt, and fluoride medication. Recommended mean daily intake is 0.05 mg/kg, the risk of dental fluorosis occurring when daily intake exceeds 1 mg/kg. The large benefits of regular fluoride supplementation on children dental health must not live down the risk of dental fluorosis resulting from over administration. Therefore when prescribing fluoride to a child, the practitioner must carefully examine his/her possible other fluoride sources.     

多语言机译系统中高质量语义单元库形成方法

讨论构建多自然语言互译机译系统所需的高质量、可扩充、完备的、无可弃、无重复、无非正常歧义的多语统一语义单元知识库.在构建过程中采用类型特征分类方法有效降低计算复杂性,使去重复的计算量降低一半,去可弃的计算量降到O(βN)(N是语义单元库规模,β是有界数,β<C,C是常数).全部算法都可以在多核处理机上以常数效率地实现.同时讨论了语义单元的再分解和自然语言种类的增多时语义单元知识库的扩充方法.该知识库不仅用于多自然语言互译系统,还可作为自然语言理解和处理的基础知识库.     

The mutK gene of Vibrio cholerae: a new gene involved in DNA mismatch repair

A new gene, mutK, of Vibrio cholerae, encoding a 19-kDa protein which is involved in repairing mismatches in DNA via a presumably methyl-independent pathway, has been identified. The product of the mutK gene cloned in either high- or low-copy-number vectors can reduce the spontaneous mutation frequency of Escherichia coli mutS, mutL, mutU, and dam mutants. The spontaneous mutation frequency of a chromosomal mutK knockout mutant was almost identical to that of wild-type V. cholerae cells, indicating that when the methyl-directed mismatch repair is blocked, the repair potential of MutK becomes apparent. The complete nucleotide sequence of the mutK gene has been determined, and the deduced amino acid sequence showed three open reading frames (ORFs), of which the ORF3 represents the mutK gene product. The mutK gene product has no significant homology with any of the proteins deposited in the EMBL data bank. ORF2, located upstream of mutK, encodes a 14-kDa protein which has more than 70% homology with a hypothetical protein found only downstream of the E. coli vsr gene. ORF1, located farther upstream of mutK, has more than 80% homology with a major cold shock protein found in several bacteria. Downstream of mutK, a partial ORF having 60% homology with an RNA methyltransferase has been identified. The mutK gene has recently been positioned in the ordered cloned DNA map of the genome of the V. cholerae strain from which the gene was isolated (10).     

Destabilization of tracts of simple repetitive DNA in yeast by mutations affecting DNA mismatch repair

The genomes of all eukaryotes contain tracts of DNA in which a single base or a small number of bases is repeated. Expansions of such tracts have been associated with several human disorders including the fragile X syndrome. In addition, simple repeats are unstable in certain forms of colorectal cancer, suggesting a defect in DNA replication or repair. We show here that mutations in any three yeast genes involved in DNA mismatch repair (PMS1, MLH1 and MSH2) lead to 100- to 700-fold increases in tract instability, whereas mutations that eliminate the proof-reading function of DNA polymerases have little effect. The meiotic stability of the tracts is similar to the mitotic stability. These results suggest that tract instability is associated with DNA polymerases slipping during replication, and that some types of colorectal cancer may reflect mutations in genes involved in DNA mismatch repair.     

Formation and migration of avian primordial germ cells

A retrospective review of 22 patients who sustained snowblower injuries to the hand was performed. There were 17 men and 5 women, ranging in age from 20 to 68 years (average age, 39.7 years). Fifty percent were manual laborers, 25% were unemployed, 15% were office workers, and 10% were not categorized. The dominant hand was involved in 86% of patients. In all patients, injuries occurred during an attempt to unclog manually the snowblower of wet snow. Patients were evaluated initially in the emergency room, where their wounds were irrigated and debrided, subungual hematomas drained, and nail bed lacerations repaired. Patients with more extensive injuries were taken to the operating room for definitive treatment including open or closed reduction of fractures, fingertip replacement as composite grafts or skin grafts, revision amputations, tenorrhaphies, and digital nerve repairs. All injuries occurred distal to the metacarpophalangeal joints. Only 1 patient sustained an injury to the proximal phalanx. Ten patients injured only 1 finger, 6 patients injured 2 fingers, and 6 patients injured 3 fingers. The middle and ring fingers were most commonly injured (39.6% and 33.3% respectively), followed by the index and little fingers (16.7% and 8.3% respectively), and the thumb (2.1%). Phalangeal fractures were the most common type of injury, occurring in 29.2% of patients, and usually involved the distal phalanx. This was followed in frequency by nail bed injuries (22.9%), amputations (22.9%), tendon lacerations (14.6%), soft-tissue avulsions (6.3%), and digital nerve injuries (4.2%). Snowblower injuries can involve bone, soft tissue, nail bed structures, nerves, and tendons, and may even result in amputation of one or several fingers. These injuries are localized to the distal portions of the fingers. The middle and ring fingers are most commonly involved, with relative sparing of the thumb. Fractures are the most frequent injury, followed by nail bed injuries and amputations. Snowblower injuries are often managed as open fractures with intravenous antibiotics; irrigation and debridement; and repair of bone, soft tissue, and nail bed structures.     

RecA binding to a single double-stranded DNA molecule: a possible role of DNA conformational fluctuations

Most genetic regulatory mechanisms involve protein-DNA interactions. In these processes, the classical Watson-Crick DNA structure sometimes is distorted severely, which in turn enables the precise recognition of the specific sites by the protein. Despite its key importance, very little is known about such deformation processes. To address this general question, we have studied a model system, namely, RecA binding to double-stranded DNA. Results from micromanipulation experiments indicate that RecA binds strongly to stretched DNA; based on this observation, we propose that spontaneous thermal stretching fluctuations may play a role in the binding of RecA to DNA. This has fundamental implications for the protein-DNA binding mechanism, which must therefore rely in part on a combination of flexibility and thermal fluctuations of the DNA structure. We also show that this mechanism is sequence sensitive. Theoretical simulations support this interpretation of our experimental results, and it is argued that this is of broad relevance to DNA-protein interactions.     

DNA modeller: an interactive program for modelling stacks of DNA base pairs on a microcomputer

DNA Modeller is a microcomputer program for interactively manipulating up to 20 bp in a DNA double helical arrangement. It calculates the van der Waals and electrostatic energies of base-base interactions using the AMBER potential, minimizes the energy with respect to the pair (buckle, propeller, opening, shear, stretch, stagger) and step (tilt, roll, twist, shift, slide, rise) parameters, calculates lengths of the canonical hydrogen bonds between the complementary bases, and calculates interatomic distances between the successive base pairs. Input/output files are simple lists of the step and pair parameters or lists of the atom specifications (N1, C2, etc.) and their Cartesian coordinates (compatible with the Desktop Molecular Modeller*.mol files). The program is supplied with a readbrk utility which transforms PDB/NDB to the *.mol format readable by DNA Modeller. The DNA crystal structures deposited in the PDB or NDB databases can thus be analyzed, and their bases visualized and interactively manipulated. In addition, DNA Modeller can calculate the base pair and step geometrical parameters and interaction energies. A plotter utility creates wire mono or stereo pictures of the bases. This program is designed for IBM-compatible computers working under DOS or can run as a DOS application under MS Windows 3.x or Merge (SCO Unix DOS emulator).     

Impaired mismatch extension by a herpes simplex DNA polymerase mutant with an editing nuclease defect

The D368A mutation within the 3'-5'-exonuclease domain of the herpes simplex type 1 DNA polymerase inactivates this nuclease and severely interferes with virus viability. Compared with the wild type enzyme, the D368A mutant exhibits substantially elevated rates of incorrect nucleotide incorporation, as measured in a LacZ reversion assay. This high rate occurs in the presence of high levels of dNTPs, a condition that forces the enzyme to extend mismatched primers. Hence, the mutant fails to correct many misincorporations that are removed in the wild type. In addition, the mutant shows a much reduced ability to replicate DNA templates primed with a 3'-mismatch as compared with wild type. This extension defect also appears more severe than observed for replicases which naturally lack editing nucleases. Based on these findings, we suggest that the inability of the D368A herpes simplex mutant polymerase to replicate beyond a mismatched base pair severely inhibits viral replication.     

Formation of detonation coatings applied on an iron base

Conclusions The results of x-ray diffraction analysis indicate a complex multiphase composition of the areas adjoining the zone of contact of the coating with the base related to occurrence of the and -structural transformations. Mutual mixing of the materials of the coating and base in the liquid phase leads to alloying and stabilization of the high temperature modification of iron (-Fe) and cobalt (-Co).The structural transformation ( -Fe) has a significant influence on the processes of formation of detonation coatings of powders of pure metals (Co-Ni) applied on iron and leads to a reduction in the level of mechanical properties of the joint.In the coating itself significant mixing of the sprayed metals occurs including different mechanisms of mass transfer such as mass transfer in the liquid and solid phases [1] with a depth of penetration of more than 10 m.On the boundary of the joint of the coating with the iron base relatively weak diffusion interaction of the contacting metals is observed. The width of this zone (5–10 m) is significantly less than that in spraying of a Co-Ni coating on an aluminum base [1].Translated from Poroshkovaya Metallurgiya, No. 10(298), pp. 60–65, October, 1987.     

Characterization of distinct human endometrial carcinoma cell lines deficient in mismatch repair that originated from a single tumor

The role of specific mismatch repair (MMR) gene products was examined by observing several phenotypic end points in two MMR-deficient human endometrial carcinoma cell lines that were originally isolated from the same tumor. The first cell line, HEC-1-A, contains a nonsense mutation in the hPMS2 gene, which results in premature termination and a truncated hPMS2 protein. In addition, HEC-1-A cells carry a splice mutation in the hMSH6 gene and lack wild-type hMSH6 protein. The second cell line, HEC-1-B, possesses the same defective hMSH6 locus. However, HEC-1-B cells are heterozygous at the hPMS2 locus; that is, along with carrying the same nonsense mutation in hPMS2 as in HEC-1-A, HEC-1-B cells also contain a wild-type hPMS2 gene. Initial recognition of mismatches in DNA requires either the hMSH2/hMSH6 or hMSH2/hMSH3 heterodimer, with hPMS2 functioning downstream of damage recognition. Therefore, cells defective in hPMS2 should completely lack MMR (HEC-1-A), whereas cells mutant in hMSH6 only (HEC-1-B) can potentially repair damage via the hMSH2/hMSH3 heterodimer. The data presented here in HEC-1-B cells illustrate (i) the reduction of instability at microsatellite sequences, (ii) a significant decrease in frameshift mutation rate at HPRT, and (iii) the in vitro repair of looped substrates, relative to HEC-1-A cells, illustrating the repair of frameshift intermediates by hMSH2/hMSH3 heterodimer. Furthermore, the role of hMSH2/hMSH3 heterodimer in the repair of base:base mismatches is supported by observing the reduction in base substitution mutation rate at HPRT in HEC-1-B cells (hMSH6-defective but possessing wild-type hPMS2), as compared with HEC-1-A (hMSH6/hPMS2-defective) cells. These data support a critical role for hPMS2 in human MMR, while further defining the role of the hMSH2/hMSH3 heterodimer in maintaining genomic stability in the absence of a wild-type hMSH2/hMSH6 heterodimer.     

一种[011]取向镍基单晶合金的蠕变断裂

研究了[011]取向的镍基单晶高温合金在750~980℃温度范围和200~680 MPa应力下的蠕变断裂特征.在扫描电镜上对各种实验状态下的蠕变断口和纵向剖面进行了详细观察.研究发现：在低温750℃和中温870℃不同初始蠕变应力条件下,枝晶间区亚晶界处不规则γ＇/γ界面是裂纹主要萌生场所,这些已萌生的裂纹在与外加应力轴垂直的（011）面上沿〈110〉和〈100〉两个方向扩展;980℃不同初始应力条件下,裂纹主要在合金中显微疏松孔洞处萌生,沿与外应力轴垂直的方向扩展.观察750℃和870℃不同应力状态蠕变试样的纵向剖面,对亚晶界区不规则γ＇相面积分数的测量和计算表明,用面积分数表征该合金[011]取向在中低温状态下的蠕变损伤程度是可行的.