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At least 217 records · Page 12Linked to original sources
PubMed2006

Telomere-mediated chromosomal truncation in maize.

Direct repeats of Arabidopsis telomeric sequence were constructed to test telomere-mediated chromosomal truncation in maize. Two constructs with 2.6 kb of telomeric sequence were used to transform maize immature embryos by Agrobacterium-mediated transformation. One hundred seventy-six transgenic lines were recovered in which 231 transgene loci were revealed by a FISH analysis. To analyze chromosomal truncations that result in transgenes located near chromosomal termini, Southern hybridization analyses were performed. A pattern of smear in truncated lines was seen as compared with discrete bands for internal integrations, because telomeres in different cells are elongated differently by telomerase. When multiple restriction enzymes were used to map the transgene positions, the size of the smears shifted in accordance with the locations of restriction sites on the construct. This result demonstrated that the transgene was present at the end of the chromosome immediately before the integrated telomere sequence. Direct evidence for chromosomal truncation came from the results of FISH karyotyping, which revealed broken chromosomes with transgene signals at the ends. These results demonstrate that telomere-mediated chromosomal truncation operates in plant species. This technology will be useful for chromosomal engineering in maize as well as other plant species.

Chromosomes, Plant↗
PubMed1992

A sarcomeric alpha-actinin truncated at the carboxyl end induces the breakdown of stress fibers in PtK2 cells and the formation of nemaline-like bodies and breakdown of myofibrils in myotubes.

In many nonmuscle cells, nonsarcomeric alpha-actinin is distributed in the dense bodies of stress fibers, adhesion plaques, and adherens junctions. In striated muscle, a sarcomeric isoform of alpha-actinin (s-alpha-actinin) is found in the Z-bands of myofibrils and subsarcolemmal adhesion plaques. To understand the role(s) of the alpha-actinin isoforms in the assembly and maintenance of such cytoskeletal structures, full-length or truncated s-alpha-actinin cDNAs were expressed in PtK2 cells and in primary skeletal myogenic cells. We found the following. (i) In transfected PtK2 cells the truncated s-alpha-actinin was rapidly incorporated into preexisting dense bodies, adhesion plaques, and adherens junctions. With time these structures collapsed, and the affected cells detached from the substrate. (ii) In myotubes the truncated s-alpha-actinin was incorporated into nascent Z-bands. Many of these progressively hypertrophied, forming nemaline-like bodies. With time the affected myofibrils fragmented, and the myotubes detached from the substrate. (iii) In both cell types the truncated s-alpha-actinin was significantly more disruptive of the cytoskeletal structures than the full-length molecule. (iv) Pools of "over-expressed" full-length or truncated protein did not self-aggregate into homogeneous, amorphous complexes; rather the exogenous proteins selectively colocalized with the same cohort of cytoskeletal proteins with which the endogenous alpha-actinin normally associates. The similarity among the hypertrophied Z-bands in transfected myotubes, the nemaline bodies in patients with nemaline myopathies, and the streaming Z-bands seen in various muscle pathologies raises the possibility that the genetically determined nemaline bodies and the pathologically induced Z-band alterations may reflect primary and/or post-translational modifications of s-alpha-actinin.

Actinin↗
PubMed1996

Truncation of the class II beta-chain cytoplasmic domain influences the level of class II/invariant chain-derived peptide complexes.

Previous studies have established that antigen presenting cells (APC) expressing major histocompatibility complex class II beta chains with truncated cytoplasmic domains are impaired in their capacity to activate T cells. While it had been widely accepted that this impairment is due to a defect in class II cytoplasmic domain-dependent signal transduction, we recently generated transgenic mice expressing only truncated class II beta chains, and functional analyses of APC from these mice revealed signaling-independent defects in antigen presentation. Here, we demonstrate that T cells primed on such transgenic APC respond better to stimulation by APC expressing truncated beta chains than by wild-type APC. This finding suggests that APC expressing truncated class II beta chains are not inherently defective in their antigen presenting capacity but, rather, may differ from wild-type APC in the peptide antigens that they present. Indeed, analysis of the peptides bound to class II molecules isolated from normal and transgenic spleen cells revealed clear differences. Most notably, the level of class II-associated invariant chain-derived peptides (CLIP) is significantly reduced in cells expressing only truncated beta chains. Prior studies have established that CLIP and antigenic peptides compete for binding to class II molecules. Thus, our results suggest that the cytoplasmic domain of the class II beta chain affects antigen presentation by influencing the level of CLIP/class II complexes.

Amino Acid Sequence↗
PubMed1996

Brush border myosin-I truncated in the motor domain impairs the distribution and the function of endocytic compartments in an hepatoma cell line.

Myosins I, a ubiquitous monomeric class of myosins that exhibits actin-based motor properties, are associated with plasma and/or vesicular membranes and have been suggested as players for trafficking events between cell surface and intracellular membranous structures. To investigate the function of myosins 1, we have transfected a mouse hepatoma cell line (BWTG3) with cDNAs encoding the chicken brush border myosin-I (BBMI) and two variants truncated in the motor domain. One variant is deleted of the first 446 amino acids and thereby lacks the ATP binding site, whereas the other is deleted of the entire motor domain and lacks the ATP and actin binding sites. We have observed (i) that significant amounts of the truncated variants are recovered with membrane fractions after cell fractionation, (ii) that they codistribute with a compartment containing alpha2-macroglobulin internalized for 30 min as determined by fluorescent microscopy, (iii) that the production of BBMI-truncated variants impairs the distribution of the acidic compartment and ligands internalized for 30 min, and (iv) that the production of the truncated variant containing the actin binding site decreases the rate of alpha2-macroglobulin degradation whereas the production of the variant lacking the ATP binding site and the actin binding site increases the rate of a2-macroglobulin degradation. These observations indicate that the two truncated variants have a dominant negative effect on the distribution and the function of the endocytic compartments. We propose that an unidentified myosin-I might contribute to the distribution of endocytic compartments in a juxtanuclear position and/or to the regulation of the delivery of ligands to the degradative compartment in BWTG3 cells.

Adenosine Triphosphate↗
PubMed1995

Stable expression of a truncated AT1A receptor in CHO-K1 cells. The carboxyl-terminal region directs agonist-induced internalization but not receptor signaling or desensitization.

Phosphorylation of serine and threonine residues in the carboxyl-terminal region of many G-protein-coupled receptors directs the rapid uncoupling from signal transduction pathways. In Chinese hamster ovary cells, we have stably expressed a truncated mutant of the angiotensin II (AT1A) receptor devoid of the carboxyl-terminal 45 amino acids, encompassing 13 serine/threonine residues. One clone, designated TL314 to indicate truncation after leucine 314, expressed a single class of angiotensin II receptors with a dissociation constant of 1.08 nM and a receptor density of 560 fmol/mg of protein (approximately 75,000 receptors/cell). A nonhydrolyzable analog of GTP accelerated the angiotensin II-induced dissociation of [125I]angiotensin II from TL314 plasma membranes 3.6-fold, indicating G-protein coupling. In TL314 cells, angiotensin II stimulated the release of intracellular calcium and the induction of mitogen-activated protein kinase activity, the level of which were comparable with the full-length AT1A receptor. The AII-stimulated calcium response was rapidly desensitized in both full-length and truncated AT1A receptors. Interestingly, angiotensin II-induced endocytosis of the truncated receptor was almost completely inhibited, suggesting that a recognition motif within the carboxyl-terminal 45 amino acids of the AT1A receptor promotes sequestration. Thus, truncation of the AT1A receptor after leucine 314 inhibits agonist-induced internalization without affecting the capacity of the expressed protein to adopt the correct conformation necessary for high affinity binding of angiotensin II, coupling to G-proteins, and activation of signal transduction pathways. The rapid desensitization and refractoriness of the angiotensin II-induced calcium transient in the TL314 cell line, in which putative carboxyl-terminal phosphorylation sites are absent, suggests that the mechanism of AT1A receptor desensitization differs from that of other prototypical G-protein-coupled receptors.

Amino Acid Sequence↗
PubMed1995

Partial truncation of the yeast RNA polymerase II carboxyl-terminal domain preferentially reduces expression of glycolytic genes.

The largest subunit of RNA polymerase II contains an essential carboxyl-terminal domain (CTD) that consists of highly conserved heptapeptide repeats with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. Yeast cells with a partially truncated CTD grow slowly, are temperature- and cold-sensitive, and are unable to fully activate transcription of some genes. Screening a yeast wild-type cDNA library by means of comparative hybridization we find that CTD truncation preferentially reduces transcription of genes encoding glycolytic enzymes. Using a newly developed dual reporter assay we demonstrate that sensitivity to CTD truncation is conferred by the glycolytic gene promoters. Expression driven by glycolytic gene promoters is reduced, on average, about 3-fold in strains with the shortest CTD growing on either fermentable or nonfermentable carbon sources. Sensitivity to CTD truncation is particularly acute for the constitutively expressed ENO1 gene, which is reduced 10-fold in a strain with only eight CTD repeats. The sensitivity of constitutive ENO1 expression argues that CTD truncation can cause defects in uninduced as well as induced transcription.

Amino Acid Sequence↗
PubMed2000

Src-mediated tyrosine phosphorylation of NR2 subunits of N-methyl-D-aspartate receptors protects from calpain-mediated truncation of their C-terminal domains.

Src-mediated tyrosine phosphorylation of N-methyl-d-aspartate receptor subunits has been shown to modify the functional properties of N-methyl-d-aspartate receptors. Moreover, calpain-mediated truncation of N-methyl-d-aspartate receptor subunits has been found to alter the structure of the receptors. In the present study, we first used immunoprecipitation with a variety of antibodies against N-methyl-d-aspartate receptor subunits and anti-phosphotyrosine antibodies to show that tyrosine-phosphorylated subunits of N-methyl-d-aspartate receptor are protected against calpain-mediated truncation of their C-terminal domains. A GST fusion protein containing the C-terminal domain of NR2A was used to identify the calpain cutting sites in the C-terminal domain. One site was identified at residues 1278-1279, corresponding to one of the preferred calpain truncation sites. This site is adjacent to a consensus sequence for Src-mediated tyrosine phosphorylation, and Src-mediated tyrosine phosphorylation of the GST-NR2A C-terminal fusion protein also inhibited calpain-mediated truncation of the fusion protein. We propose that phosphorylation of NR2 subunits and the resulting inhibition of calpain-mediated truncation of their C-terminal domains provide for the stabilization of the N-methyl-d-aspartate receptors in postsynaptic structures.

Amino Acid Sequence↗
PubMed2002

Nuclear localization of a non-caspase truncation product of atrophin-1, with an expanded polyglutamine repeat, increases cellular toxicity.

Dentatorubral and pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder similar to Huntington's disease, with clinical manifestations including chorea, incoordination, ataxia, and dementia. It is caused by an expansion of a CAG trinucleotide repeat encoding polyglutamine in the atrophin-1 gene. Both patients and DRPLA transgenic mice have nuclear accumulation of atrophin-1, especially an approximately 120-kDa fragment, which appears to represent a cleavage product. We now show that this is an N-terminal fragment that does not correspond to the previously described caspase-3 fragment, or any other known caspase cleavage product. The atrophin-1 sequence contains a putative nuclear localization signal in the N terminus of the protein and a putative nuclear export signal in the C terminus. We have tested the hypothesis that endogenous localization signals are functional in atrophin-1, and that nuclear localization and proteolytic cleavage contribute to atrophin-1 cell toxicity. In transient cell transfection experiments using a neuroblastoma cell line, full-length atrophin-1 with 26 (normal) or 65 (expanded) glutamines localized to both nucleus and cytoplasm, with no significant difference in toxicity between the normal and mutant proteins. A construct with 65 glutamine repeats encoding an N-terminal fragment (which removes an NES) of atrophin-1 similar in size to the truncation product in DRPLA patient tissue, showed increased nuclear labeling, and an increase in cellular toxicity, compared with a similar fragment with 26 glutamines. Full-length atrophin-1 with 65 polyglutamine repeats and mutations inactivating the NES also yielded increased nuclear localization and increased toxicity. These data suggest that truncation enhances cellular toxicity of the mutant protein, and that the NES is a relevant region deleted during truncation. Furthermore, mutating the NLS in the truncated protein shifted atrophin-1 more to the cytoplasm and eliminated the increased toxicity, consistent with the idea that nuclear localization enhances toxicity. In none of the experiments were inclusions visible in the nucleus or cytoplasm suggesting that inclusion formation is unrelated to cell death. These data indicate that truncation of atrophin-1 may alter its ability to shuttle between the nucleus and cytoplasm, leading to abnormal nuclear interactions and cell toxicity.

Animals↗
PubMed2002

Mass measurements of C-terminally truncated alpha-crystallins from two-dimensional gels identify Lp82 as a major endopeptidase in rat lens.

Molecular chaperone activity of lens alpha-crystallins is reduced by loss of the C terminus. The purpose of this experiment was to 1) determine the cleavage sites produced in vitro by ubiquitous m-calpain and lens-specific Lp82 on alpha-crystallins, 2) identify alpha-crystallin cleavage sites produced in vivo during maturation and cataract formation in rat lens, and 3) estimate the relative activities of Lp82 and m-calpain by appearance of protease-specific cleavage products in vivo. Total soluble protein from young rat lens was incubated with recombinant m-calpain or Lp82 and 2 mM Ca2+. Resulting fragmented alpha-crystallins were separated by two-dimensional gel electrophoresis. Eluted alpha-crystallin spots were analyzed by mass spectrometry. Cleavage sites on insoluble alpha-crystallins were determined similarly in mature rat lens nucleus and in cataractous rat lens nucleus induced by selenite. In vitro proteolysis of alphaA-crystallin by Lp82 and m-calpain produced unique cleavage sites by removing 5 and 11 residues, respectively, from the C terminus. In vivo, the protease-specific truncations removing 5 and 11 residues from alphaA were both found in maturing lens, whereas only the truncation removing 5 residues was found in cataractous lens. Other truncation sites, common to both calpain isoforms, resulted from the removal of 8, 10, 16, 17, and 22 residues from the C terminus of alphaA. Using uniquely truncated alphaA-crystallins as in vivo markers, Lp82 and m-calpain were both found to be active during normal maturation of rat lens, whereas Lp82 seemed especially active during selenite cataract formation. These C-terminal truncations decrease chaperone activity of alpha-crystallins, possibly leading to the observed increases in insoluble proteins during aging and cataract. The methodology that allowed accurate mass measurements of proteins eluted from 2D gels should be useful to examine rapidly other post-translational modifications.

Animals↗
PubMed1994

Differential perturbations in the morphogenesis of anterior structures induced by overexpression of truncated XB- and N-cadherins in Xenopus embryos.

Cadherins, a family of Ca-dependent adhesion molecules, have been proposed to act as regulators of morphogenetic processes and to be major effectors in the maintenance of tissue integrity. In this study, we have compared the effects of the expression of two truncated cadherins during early neurogenesis in Xenopus laevis. mRNA encoding deleted forms of XB- and N-cadherin lacking most of the extracellular domain were injected into the four animal dorsal blastomeres of 32-cell stage Xenopus embryos. These truncated cadherins altered the cohesion of cells derived from the injected blastomeres and induced morphogenetic defects in the anterior neural tissue to which they chiefly contributed. Truncated XB-cadherin was more efficient than N-cadherin in inducing these perturbations. Moreover, the coexpression of both truncated cadherins had additive perturbation effects on neural development. The two truncated cadherins can interact with the three known catenins, but with distinct affinities. These results suggest that the adhesive signal mediated by cadherins can be perturbed by overexpressing their cytoplasmic domains by competing with different affinity with catenins and/or a common anchor structure. Therefore, the correct regulation of cadherin function through the cytoplasmic domain appears to be a crucial step in the formation of the neural tissue.

Amino Acid Sequence↗
PubMed2005

Exact fan-beam image reconstruction algorithm for truncated projection data acquired from an asymmetric half-size detector.

In this paper, we present a new algorithm designed for a specific data truncation problem in fan-beam CT. We consider a scanning configuration in which the fan-beam projection data are acquired from an asymmetrically positioned half-sized detector. Namely, the asymmetric detector only covers one half of the scanning field of view. Thus, the acquired fan-beam projection data are truncated at every view angle. If an explicit data rebinning process is not invoked, this data acquisition configuration will reek havoc on many known fan-beam image reconstruction schemes including the standard filtered backprojection (FBP) algorithm and the super-short-scan FBP reconstruction algorithms. However, we demonstrate that a recently developed fan-beam image reconstruction algorithm which reconstructs an image via filtering a backprojection image of differentiated projection data (FBPD) survives the above fan-beam data truncation problem. Namely, we may exactly reconstruct the whole image object using the truncated data acquired in a full scan mode (2pi angular range). We may also exactly reconstruct a small region of interest (ROI) using the truncated projection data acquired in a short-scan mode (less than 2pi angular range). The most important characteristic of the proposed reconstruction scheme is that an explicit data rebinning process is not introduced. Numerical simulations were conducted to validate the new reconstruction algorithm.

Algorithms↗
PubMed1999

Recombinant chicken IFN-gamma expressed in Escherichia coli: analysis of C-terminal truncation and effect on biologic activity.

Interferon-gamma (IFN-gamma) possesses potent immunostimulatory properties, and it has recently been shown to have potential therapeutic properties. Recombinant protein technology is frequently used for commercial production of therapeutics, such as IFN. Biologically active recombinant chicken IFN-gamma (rChIFN-gamma) constructs bearing an N-terminal poly-His tag were expressed in Escherichia coli. Preparations of rChIFN-gamma contained varying ratios of a full-length and a truncated protein species (18 and 16 kDa, respectively). Amino acid sequence analysis of the full-length protein corroborated the sequence previously predicted from the cDNA sequence. Full-length rChIFN-gamma contains two cysteine residues at the C-terminus, and these were labeled by reduction and subsequent specific alkylation with fluorescent tag (5-I-AEDANS) to distinguish between full-length and C-terminally truncated forms of rChIFN-gamma. Comparative peptide mapping, amino acid sequencing, and mass spectrometry revealed that the 16 kDa protein was truncated at Lys133. It was also observed that the 18 kDa rChIFN-gamma protein was infrequently contaminated with small quantities of protein truncated at Arg141. A truncated recombinant construct (His1-Lys133) was also expressed in E. coli and had biologic activity comparable with that of the full-length construct. The 3-D structure of rChIFN-gamma was deduced by comparative modeling with bovine and human IFN-gamma crystallographic structures. Analysis of sequences and comparison of structures have revealed that the 3-D structure of rChIFN-gamma is similar to those of bovine and human molecules despite an overall amino acid identity of only 32%.

Amino Acid Sequence↗
PubMed2006

Conditions for bias from differential left truncation.

Spontaneous abortion studies that recruit pregnant women are left truncated because an unknown proportion of the source population experiences losses prior to enrollment. Unconditional logistic regression, commonly used in such studies, ignores left truncation, whereas survival analysis can accommodate left truncation and is therefore more appropriate. This study assessed the magnitude of bias introduced by fitting logistic versus Cox models using left-truncated data from a 1998 US pregnancy cohort study (n = 5,104) of trihalomethanes and spontaneous abortion. In addition, the conditions producing bias were explored by using simulated exposure data. The odds ratios and hazard ratios from the actual study differed by 10% or less. However, when the exposed women entered observation earlier on average than those unexposed, the hazard ratio was closer to the null than the odds ratio, whereas the reverse was true when the exposed entered later. The simulation suggests that bias in the odds ratio will exceed 20% when average gestational age at entry for the exposed versus the unexposed differs by 10 days or more, as has been observed regarding some socioeconomic factors, such as education and ethnicity. Cox regression can correct for left truncation and is no more difficult to perform than logistic regression.

Abortion, Spontaneous↗
PubMed1994

Defining the minimal size of catalytically active primate alpha 1,3 galactosyltransferase: structure-function studies on the recombinant truncated enzyme.

The glycosylation enzyme alpha 1,3 galactosyltransferase, which synthesizes the carbohydrate Gal alpha 1-3Gal beta 1-4GlcNAc-R, is active in non-primate mammals, prosimians and New World monkeys, but not in Old World monkeys, apes and humans. In this study, we have cloned and sequenced the enzyme expressed in a New World monkey, determined the exact size of the stem region and assessed the minimal size of catalytically active alpha 1,3 galactosyltransferase (alpha 1,3GT). Various primer sets were used in the polymerase chain reaction to generate cDNAs which coded for forms of alpha 1,3GT with deletions at the N- or C-terminal domains. The cDNA was inserted into the expression vector pPROTA which contains the coding sequence for protein A, and subsequently transfected into COS cells. The soluble chimeric products (truncated enzyme and protein A) were harvested from the cell culture medium using IgG-Sepharose beads and assayed for enzymatic activity. As many as 67 amino acids could be truncated at the amino terminal region of the luminal portion of the enzyme without affecting its catalytic activity. Truncation of 68, 69 and 74 amino acids resulted in a 50, 75 and > 95% loss in the in vitro catalytic actively, respectively. Introduction of a frameshift mutation which is characteristic of apes and human alpha 1,3GT gene resulted in the complete loss of enzyme activity. Moreover, truncation of as few as three amino acids at the carboxyl end of alpha 1,3GT resulted in complete loss of the catalytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2000

Rotational and lateral dynamics of I-A(k) molecules expressing cytoplasmic truncations.

Rotational and lateral diffusion of I-A(k) molecules with various alpha and beta chain cytoplasmic truncations known to affect class II function were measured to assess the role of cytoplasmic domains in regulating I-A(k) molecular motions. Deletion of all 12 alpha chain C-terminal residues and all 18 corresponding beta chain residues (alpha-12/beta-18) is known to abrogate translocation of protein kinase C to the nucleus upon class II cross-linking. Similarly, truncation of the entire cytoplasmic alpha chain domain and the 10 C-terminal residues of the beta chain impairs presentation of antigenic peptides to T cells. The rotational correlation time of the wild-type molecule, 11.9 +/- 2.6 micros as measured by time-resolved phosphorescence anisotropy, decreased to 7. 2 +/- 3.7 micros in the fully truncated alpha-12/beta-18 protein. Other truncated class II molecules exhibited only small changes in molecular rotation rates relative to the wild-type. The rate of lateral diffusion of the fully truncated molecule, measured with two independent methods, 2.3 x 10(-10) cm(2)/s, was comparable with that of the wild-type molecule. Thus, it appears that the alpha and beta chain cytoplasmic domains regulate the molecular motions of unperturbed I-A(k) molecules only modestly, despite the known involvement of these regions in class II signaling. Various explanations for this behavior are discussed, e.g. the possibility that class II membrane complexes are sufficiently large that association and dissociation of specific signaling proteins during antigen presentation do not significantly perturb the apparent molecular motions of the complex.

Anisotropy↗
PubMed2005

Selective inhibition of Fcepsilon RI-mediated mast cell activation by a truncated variant of Cbl-b related to the rat model of type 1 diabetes mellitus.

Ubiquitin-protein ligase Cbl-b negatively regulates high affinity IgE receptor (FcepsilonRI)-mediated degranulation and cytokine gene transcription in mast cells. In this study, we have examined the role of a truncated variant of Cbl-b related to the rat model of type 1 diabetes mellitus using the mast cell signaling model. Overexpression of the truncated Cbl-b that lacks the C-terminal region did not suppress the activation of proximal and distal signaling molecules leading to degranulation. FcepsilonRI-mediated tyrosine phosphorylation of Syk, Gab2, and phospholipase C-gamma1, and activation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAP kinase), and inhibitor of nuclear factor kappaB kinase (IKK), and generation of Rac1 are unaffected in cells overexpressing the truncated Cbl-b in the lipid raft. On the other hand, FcepsilonRI-mediated transcriptional activation of nuclear factor of activated T cells (NFAT), and transcription of interleukin-3 (IL-3) and IL-4 mRNA are inhibited by overexpression of the truncated variant of Cbl-b. This suppression parallels the re-compartmentalization of specific effector molecules in the lipid raft. These structural and functional analyses reveal the mechanism underlying the selective inhibition of cellular signaling by the truncated variant of Cbl-b related to insulin-dependent diabetes mellitus.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Rapid generation of incremental truncation libraries for protein engineering using alpha-phosphothioate nucleotides.

Incremental truncation for the creation of hybrid enzymes (ITCHY) is a novel tool for the generation of combinatorial libraries of hybrid proteins independent of DNA sequence homology. We herein report a fundamentally different methodology for creating incremental truncation libraries using nucleotide triphosphate analogs. Central to the method is the polymerase catalyzed, low frequency, random incorporation of alpha-phosphothioate dNTPs into the region of DNA targeted for truncation. The resulting phosphothioate internucleotide linkages are resistant to 3'-->5' exonuclease hydrolysis, rendering the target DNA resistant to degradation in a subsequent exonuclease III treatment. From an experimental perspective the protocol reported here to create incremental truncation libraries is simpler and less time consuming than previous approaches by combining the two gene fragments in a single vector and eliminating additional purification steps. As proof of principle, an incremental truncation library of fusions between the N-terminal fragment of Escherichia coli glycinamide ribonucleotide formyltransferase (PurN) and the C-terminal fragment of human glycinamide ribonucleotide formyltransferase (hGART) was prepared and successfully tested for functional hybrids in an auxotrophic E.coli host strain. Multiple active hybrid enzymes were identified, including ones fused in regions of low sequence homology.

Amino Acid Sequence↗
PubMed1991

Renaturation, purification, and characterization of human truncated macrophage colony-stimulating factor expressed in Escherichia coli.

A human truncated macrophage colony-stimulating factor (M-CSF) encoding the amino acid residues from 3 to 153 of the native M-CSF was expressed by using a two-cistron expression system in Escherichia coli. The truncated M-CSF found in inclusion bodies was renatured and had CSF activity. Purification, which included a QAE-ZeTa preparative cartridge concentration step followed sequentially by HPLC on TSK-gel Phenyl-5PW and TSK-gel DEAE-5PW columns, gave an overall yield of 63.8%. The purified truncated M-CSF had a specific activity of 4 x 10(7) units/mg of protein. Peptide mapping of a lysylendopeptidase digest by reversed-phase HPLC confirmed the amino acid sequence predicted from the cDNA sequence. SDS-PAGE of the purified truncated M-CSF gave a single band at 17 kDa under reducing conditions and at 32 kDa under non-reducing conditions. Activated Thiol-Sepharose 6B column chromatography and other experiments failed to detect any free cysteine residue in spite of the existence of 7 cysteine residues in the truncated M-CSF subunit. These results indicate that it is a dimeric structure linked by one or more intermolecular disulfide bonds.

Amino Acid Sequence↗