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21.
Eric Cundliffe 《Journal of industrial microbiology & biotechnology》1991,7(3):157-161
Originally presented as an Invited Lecture at the 1990 Society for Industrial Microbiology Annual Meeting in Orlando, Florida. 相似文献
22.
Donald E. Kizer Joseph A. Clouse Boyd A. Howell Bettye Cox David P. Ringer 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1982,697(3):353-362
We tested, in rat liver, the postulate that free polysomes were precursors of membrane-bound polysomes. Three methods were used to isolate free and membrane-bound ribosomes from either post-nuclear or post-mitochondrial supernatants of rat liver. Isolation and quantitation of 28 S and 18 S rRNA allowed determination of the 40 S and 60 S subunit composition of free and membrane-bound ribosomal populations, while pulse labeling of 28 S and 18 S rRNA with [6-14C]orotic acid and inorganic [32P]phosphate allowed assessment of relative rates of subunit renewal. Throughout the extra-nuclear compartment, 40 S and 60 S subunits were present in essentially equal numbers, but, free ribosomes contained a stoichiometric excess of 40 S subunits, while membrane-bound ribosomes contained a complementary excess of 60 S subunits. Experiments with labeled precursors showed that throughout the extra-nuclear compartment, 40 S and 60 S subunits accumulated isotopes at essentially equal rates, however, free ribosomes accumulated isotopes faster than membrane-bound ribosomes. Among free ribosomes or polysomes, 40 S subunits accumulated isotopes faster than 60 S subunits, but, this relationship was not seen among membrane-bound ribosomes. Here, 40 S subunits accumulated isotope more slowly than 60 S subunits. This distribution of labeled precursors does not support the postulate that free polysomes are precursors of membrane-bound polysomes, but, these data suggest that membrane-bound polysomes could be precursors of free polysomes. 相似文献
23.
Laura M. Haynes Zachary M. Huttinger Andrew Yee Colin A. Kretz David R. Siemieniak Daniel A. Lawrence David Ginsburg 《The Journal of biological chemistry》2022,298(12)
Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor superfamily of proteins, is unique among serine protease inhibitors for exhibiting a spontaneous conformational change to a latent or inactive state. The functional half-life for this transition at physiologic temperature and pH is ∼1 to 2 h. To better understand the molecular mechanisms underlying this transition, we now report on the analysis of a comprehensive PAI-1 variant library expressed on filamentous phage and selected for functional stability after 48 h at 37 °C. Of the 7201 possible single amino acid substitutions in PAI-1, we identified 439 that increased the functional stability of PAI-1 beyond that of the WT protein. We also found 1549 single amino acid substitutions that retained inhibitory activity toward the canonical target protease of PAI-1 (urokinase-like plasminogen activator), whereas exhibiting functional stability less than or equal to that of WT PAI-1. Missense mutations that increase PAI-1 functional stability are concentrated in highly flexible regions within the PAI-1 structure. Finally, we developed a method for simultaneously measuring the functional half-lives of hundreds of PAI-1 variants in a multiplexed, massively parallel manner, quantifying the functional half-lives for 697 single missense variants of PAI-1 by this approach. Overall, these findings provide novel insight into the mechanisms underlying the latency transition of PAI-1 and provide a database for interpreting human PAI-1 genetic variants. 相似文献
24.
25.
Zhang J Tanha J Hirama T Khieu NH To R Tong-Sevinc H Stone E Brisson JR MacKenzie CR 《Journal of molecular biology》2004,335(1):49-56
We describe a novel type of molecule in which single-domain antibodies (sdAbs) isolated from a nai;ve llama single domain antibody library are linked to an oligomerization domain to generate high-avidity, antigen-binding reagents. An sdAb is fused to the B-subunit of Escherichia coli verotoxin, or shiga-like toxin, which self-assembles to form a homopentamer and results in simultaneous sdAb pentamerization and introduction of avidity. Molecular modeling indicated that this fusion protein (PDB: 1OJF), termed pentabody, has structural flexibility for binding to surface-presented antigen. In the instance of an sdAb specific for a peptide antigen, pentamerization resulted in a dramatic increase in functional affinity for immobilized antigen. The pentabody was expressed in high yield in E.coli in a non-aggregated state, and exhibited excellent thermostability and protease resistance. This technology provides a relatively rapid means of generating novel antigen-binding molecules that bind strongly to immobilized antigen. It is expected that pentavalent sdAbs will have general applicability in proteomics, immunochemical staining, cancer diagnosis and other applications in which antigens are presented multivalently. 相似文献
26.
To investigate genes involved in cancer metastasis, mRNA differential display was used to compare the levels of gene expression of two cancer sublines derived from prostate carcinoma cell PC-3M that had different metastatic potentials. The differentially expressed genes were confirmed by Northern blot, and sequenced. The full-length cDNA of a tumor metastasis suppressor gene (TMSG-1) was obtained by using EST assembling and verified by RT-PCR and sequencing. The results showed that expression levels of TMSG-1 were lower in the highly metastatic cell line 1E8, compared with the non-metastatic cell line 2B4. The difference was significant. Full-length cDNA of TMSG-1 was about 2 kb, containing an open reading frame that encoded a protein of 230 amino acids. GenBank Blastn showed no marked homology with known genes. The functional prediction of amino acids sequence encoded by TMSG-1 gene indicated TMSG-1 protein was transmembrane protein, with 3 transmembrane domains, 3 putative protein kinase phosphorylatio 相似文献
27.
Thymidylate synthase (TS), an essential enzyme for catalyzing the biosynthesis of thymidylate, is a critical therapeutic target in cancer therapy. Recent studies have shown that TS functions as an RNA-binding protein by interacting with two different sequences on its own mRNA, thus, repressing translational efficiency. In this study, peptides binding TS RNA with high affinity were isolated using mRNA display from a large peptide library (>1013 different sequences). The randomized library was subjected up to twelve rounds of in vitro selection and amplification. Comparing the amino acid composition of the selected peptides (12th round, R12) with those from the initial random library (round zero, R0), the basic and aromatic residues in the selected peptides were enriched significantly, suggesting that these peptide regions might be important in the peptide-TS mRNA interaction. Categorizing the amino acids at each random position based on their physicochemical properties and comparing the distributions with those of the initial random pool, an obvious basic charge characteristic was found at positions 1, 12, 17 and 18, suggesting that basic side chains participate in RNA binding. Secondary structure prediction showed that the selected peptides of R12 pool represented a helical propensity compared with R0 pool, and the regions were rich in basic residues. The electrophoretic gel mobility shift and in vitro translation assays showed that the peptides selected using mRNA display could bind TS RNA specifically and inhibit the translation of TS mRNA. Our results suggested that the identified peptides could be used as new TS inhibitors and developed to a novel class of anticancer agents. 相似文献
28.
Abhilasha Singh Mathuriya 《Critical reviews in biotechnology》2016,36(5):788-802
Magnetotactic bacteria (MTB) represent a heterogeneous group of Gram-negative aquatic prokaryotes with a broad range of morphological types, including vibrioid, coccoid, rod and spirillum. MTBs possess the virtuosity to passively align and actively swim along the magnetic field. Magnetosomes are the trademark nano-ranged intracellular structures of MTB, which comprise magnetic iron-bearing inorganic crystals enveloped by an organic membrane, and are dedicated organelles for their magnetotactic lifestyle. Magnetosomes endue high and even dispersion in aqueous solutions compared with artificial magnetites, claiming them as paragon nanomaterials. MTB and magnetosomes offer high technological potential in modern science, technology and medicines. This review focuses on the applicability of MTB and magnetosomes in various areas of modern benefits. 相似文献
29.
将胰岛素受体底物-1(IRS-1)的PH结构域(pleckstrin homology domain)编码序列克隆到融合表达载体pRSETA中,阳性克隆经IPTG诱导,表达出氨基端带6个连续组氨酸残基的融合蛋白。经检测,表达的目的蛋白一部分以可溶形式存在。利用Ni-NTA金属螯合亲和层析法在非变性条件下对表达的目的蛋白进行纯化,纯度大于98%。将纯化的目的蛋白包被于聚苯乙烯平皿上作为靶蛋白,经过4轮淘筛,得到能够与IRS-1的PH结构域相结合的重组噬菌体克隆;从中随机挑选出50个克隆进行DNA序列测定,对获得的短肽序列进行了分析。并通过ELISA检测了这些克隆与IRS-1的PH结构域的结合活性。 相似文献
30.
小麦杂交种与亲本发育早期种子的基因表达差异及其与杂种优势关系的初步研究 总被引:34,自引:4,他引:30
为深入探讨小麦杂种优势形成的分子机理,选取3个冬小麦品种(系)为一组亲本,3个为另一组亲本,配制了正反交18个杂交组合,以授粉后6d的杂交和自交种子为材料,应用mRNA差异显示技术(DDRT—PCR)研究了小麦杂交当代种子与其亲本自交种子基因的表达差异,并与杂种优势进行相关分析。为降低DDRT—PCR技术假阳性的不利影响,对每个引物组合均作了两次PCR扩增,在处理数据时,仅统计能重复出现的条带。结果发现:杂交种和亲本之间的基因表达模式有8类共15种:(1)单亲沉默型(2种),(2)单亲一致型(2种),(3)正交或反交沉默型(2种),(4)正交或反交特异型(2种),(5)正交或反交单亲一致型(4种),(6)杂交种特异型(1种),(7)双亲共沉默型(1种),(8)表达一致型(1种)。分析发现,小麦杂交种和亲本间存在显著的表达差异。在差异表达类型中,杂交种特异型和双亲共沉默型比例最低。对上述15种表达模式与杂种优势进行相关分析,结果表明,表达一致型与各产量性状杂种优势之间的相关均不显著,说明杂种优势是由某些有表达差异的基因造成。9个产量性状均能检测到一种以上与其显著或极显著相关的基因表达模式,有些性状受正负相关效应的共同影响;沉默型(包括单亲沉默型、正交或反交沉默型和双亲共沉默型)和正交或反交单亲一致型在杂种优势形成中发挥重要作用。这些研究表明,在种子发育早期,基因的差异表达与杂种优势形成之间可能存在较为复杂的关系。 相似文献