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At least 739 records · Page 41Linked to original sources
PubMed1989

An efficient strategy for cloning 5' extremities of rare transcripts permits isolation of multiple 5'-untranslated regions of rat tryptophan hydroxylase mRNA.

The 5' end mapping of rat tryptophan hydroxylase (TPH) mRNA indicated a diversity in 5'-untranslated regions. Corresponding sequences were isolated by a variant of the Polymerase Chain Reaction, recently designated as 'anchor PCR', and a 'cRNA enrichment' procedure. The latter circumvents the limitations of 'anchor PCR', which failed to yield minor TPH sequences: this novel strategy allows purification of specific DNA fragments by elimination of the unspecific products, generated by the PCR, which prevent further amplification. Analysis of TPH sequences strongly suggests that TPH mRNAs are synthesized from at least two promoters, the proximal one exhibiting two 'CCAAT homologies'.

Animals↗
PubMed1989

Mutations in the RNA polymerase recognition sequence of the Klebsiella pneumoniae nifH promoter permitting transcriptional activation in the absence of NifA binding to upstream activator sequences.

Positive control of the wild-type Klebsiella pneumoniae nifH promoter by the NifA protein requires that NifA is bound at the upstream activator sequence (UAS). By introducing base substitutions at -15 to -17 in the RNA polymerase recognition sequence of the nifH promoter, positive control by a form of NifA unable to bind to the UAS was greatly increased when compared to the wild-type promoter. Transcriptional activation still required the rpoN encoded sigma factor and was initiated at the same nucleotide as in the wild-type promoter. Mutations at -15 to -17 suppressed the requirement that the UAS should be located on the correct face of the DNA helix with respect to the RNA polymerase recognition sequence in order that titration of NifA and efficient activation occur. This result supports the suggestion that upstream bound NifA interacts with the RNA polymerase-RpoN complex. To examine the minimal carboxy terminal sequences required for the positive control function of NifA a series of carboxy terminal deletions were constructed. Efficient positive control at a UAS-independent promoter was only observed in deletions which did not extend beyond the proposed boundary separating the carboxy terminal NifA DNA-binding domain from its central domain.

Amino Acid Sequence↗
PubMed1994

Pokeweed antiviral protein (PAP) mutations which permit E.coli growth do not eliminate catalytic activity towards prokaryotic ribosomes.

Pokeweed antiviral protein (PAP) has N-glycosidase activity towards both eukaryotic and prokaryotic ribosomes. This is in marked contrast with the A chains of type 2 ribosome inactivating proteins (RIPs) such as ricin and abrin, which inactivate only eukaryotic ribosomes. A recent report described spontaneous mutations in PAP that implicated specific amino acids to be involved in determining the activity of PAP towards prokaryotic ribosomes. As part of an ongoing study into RIP--ribosome interactions these mutations were specifically recreated in a PAP clone encoding the mature 262 amino acid PAP sequence. Mutants were tested for their N-glycosidase activity by analysing the integrity of eukaryotic and prokaryotic ribosomes after mutant protein expression. Mutations of F196Y and K211R, either individually or within the same clone, were active toward both classes of ribosome, indicating that these amino acid positions are not involved in differentiating ribosomal substrates. Mutation R68G led to a protein that appeared to be inactive towards prokaryotic ribosomes, but also very poorly active towards eukaryotic ribosomes. This mutation is currently under further investigation.

Base Sequence↗
PubMed1996

Pbx-1 Hox heterodimers bind DNA on inseparable half-sites that permit intrinsic DNA binding specificity of the Hox partner at nucleotides 3' to a TAAT motif.

Heterodimers between the Pbx/Exd and Hox/HOM-C classes of homeodomain proteins bind regulatory elements in tissue-specific and developmentally regulated genes. In this work, we characterize the half-site bound by both Pbx1 and Hox proteins on a prototypic element (TGATTAAT) and determine how the orientation of the Hox protein contributes to the DNA binding specificity of Pbx-Hox heterodimers. We demonstrate that the Hox protein binds the 3' TAAT sequence as its recognition core and exhibits sequence-specific binding at positions 3' to the TAAT core. Unfavored sequences at this position, such as two cytosines, abrogate binding to the element. The upstream Pbx1 core sequence, TGAT, must immediately juxtapose the Hox core. This geometry maintains the preference of Hox/HOM-C proteins for a T base at position -1, as T represents the fourth position of the Pbx1 core, and suggests that this T base is bound by both Pbx1 and Hox proteins, Pbx1 binding in the major grove and the Hox protein binding in the minor grove. Pbx1 also exhibits base selectivity 5' to its TGAT recognition sequence.

Base Sequence↗
PubMed1996

Multiplex Cre/lox recombination permits selective site-specific DNA targeting to both a natural and an engineered site in the yeast genome.

Variant lox sites having an altered spacer region (heterospecific lox sites) are not proficient for Cre-mediated recombination with the canonical 34 bp loxP site, but can recombine with each other. By placing different heterospecific lox sites at different genomic locations, Cre can catalyze independent DNA recombination events at multiple loci in the same cell without concern that unwanted inter-locus recombination events will be generated. Such heterospecific lox sites also allow Cre to specifically target efficient integration of exogenous DNA to endogenous lox-like sequences that naturally occur in the genome. Specific targeting occurs only with a DNA vector carrying a heterospecific lox site in which the spacer region has been redesigned to match the 'spacer' region of the targeted chromosomal element. Moreover, in cells expressing a catalytically active Cre recombinase, naturally occurring lox-like sequences can exhibit almost 20% mitotic recombination. Thus, in the same cell, heterospecific lox sites can be used independently at multiple loci for integration, for deletion and for enhanced mitotic recombination, thereby increasing the repertoire of genomic manipulations catalyzed by the Cre recombinase.

Base Sequence↗
PubMed2003

Yeast Ume6p repressor permits activator binding but restricts TBP binding at the HOP1 promoter.

Ume6p plays essential roles in the regulation of early meiotic genes in Saccharomyces cerevisiae. Ume6p exerts repression via recruitment of the Sin3p-Rpd3p histone deacetylase and Isw2p chromatin remodeling complexes. The transcriptional step that is ultimately inhibited by Ume6p is unknown. Here, in vivo footprinting shows that transcriptional activators Hap1p and Abf1p occupy upstream sites in repressed and derepressed promoters. In contrast, chromatin immunoprecipitation shows that TATA box-binding protein (TBP)- promoter binding is reduced upon repression of HOP1. Fusion of TBP to a zinc cluster DNA binding domain relieves repression at a HOP1 promoter modified to include the zinc cluster target site. We suggest that TBP binding is inhibited through chromatin modification by the Sin3p-Rpd3p and Isw2p complexes recruited by Ume6p.

Binding Sites↗
PubMed2006

Expression of Cre recombinase during transient phage infection permits efficient marker removal in Streptomyces.

We report a system for the efficient removal of a marker flanked by two loxP sites in Streptomyces coelicolor, using a derivative of the temperate phage phiC31 that expresses Cre recombinase during a transient infection. As the test case for this recombinant phage (called Cre-phage), we present the construction of an in-frame deletion of a gene, pglW, required for phage growth limitation or Pgl in S.coelicolor. Cre-phage was also used for marker deletion in other strains of S.coelicolor.

Bacteriophages↗
PubMed1994

Failure of GM-CSF to permit dose-escalation in an every other week dose-intensive regimen for advanced breast cancer.

BACKGROUND: In an attempt to improve dose intensity and therapeutic effectiveness in breast cancer, GM-CSF was incorporated into a multi-drug every other week chemotherapy regimen that had been previously reported to have promising activity, but whose dose-limiting toxicity was neutropenia. PATIENTS AND METHODS: A Phase I-II study in patients with locally advanced or metastatic breast cancer was initiated using GM-CSF and a 5-drug chemotherapy regimen employing oral cyclophosphamide daily for 7 days and doxorubicin, vincristine, methotrexate, 5-fluorouracil, and leucovorin IV every 2 weeks for 10 courses. In the first 8 patients, GM-CSF in escalating doses (1-20 micrograms/kg s.c. per day) was given on days 8-13 of each 2 week cycle. In the last 12 patients, GM-CSF was given on days 3-14 of each cycle in an attempt to improve its effectiveness by prolonging treatment duration. RESULTS: The regimen was poorly tolerated. Only 10 patients completed all 10 courses of treatment, and most of those required dose delays and/or reductions. GM-CSF failed to reduce neutropenia when given by either schedule. Furthermore, thrombocytopenia was severe and progressive, especially with the more prolonged GM-CSF schedule in which the mean lowest nadir platelet count was 15,000/microliters. Anemia, fatigue, mucositis, and neutropenic fevers were also common, and dose escalations were not possible in any patient. Central venous catheter complications were also common. Complete or partial remissions were observed in 15 of 20 patients, but response durations were brief. CONCLUSIONS: GM-CSF in two different schedules failed to ameliorate myelosuppression when used in combination with this multiple drug, every other week regimen. Neutropenia and, especially, thrombocytopenia remained dose limiting.

Adult↗
PubMed1979

Phosphorylation of uterine smooth muscle myosin permits actin-activation.

Myosin was purified from ovine uterine smooth muscle. The 20,000 dalton myosin light chain was phosphorylated to varying degrees by an endogenous Ca2+ dependent kinase. The kinase and endogenous phosphatases were then removed via column chromatography. In the absence of actin neither the size of the initial phosphate burst nor the steady state Mg2+-dependent ATPase activity were affected by phosphorylation. However, phosphorylation was required for actin to increase the Mg2+-dependent ATPase activity and for the myosin to superprecipitate with actin. Ca2+ did not affect the Mg2+-dependent ATPase activity in the presence or absence of action or the rate or extent of superprecipitation with actin once phosphorylation was obtained. These data indicate that: 1) phosphorylation of the 20,000 dalton myosin light chain controls the uterine smooth muscle actomyosin interaction, 2) in the absence of actin, phosphorylation does not affect either the ATPase of myosin or the size of the initial burst of phosphate and, 3) Ca2+ is important in controlling the light chain kinase but not the actomyosin interaction.

Actins↗
PubMed1983

Construction of a lambda packageable ColE1 vector which permits cloning of large DNA fragments: cloning of thyA gene of Escherichia coli.

A small packageable plasmid depending on the ColE1 replicon was constructed from ColE1-gal-cos lambda:: Tn3, pKY2113 (T. Nakamura et al. (1981) J. Biochem. 90, 1013), and named pKY2662. This plasmid carries ampicillin resistance and colicin E1 immunity genes as selective markers, and has neither mobilization function nor movable transposon. The molecular size of pKY2662 is 8.7 kb, and it has a single cleavage site each for BamHI, Bg/II, ClaI, EcoRI, HpaI, PstI, and Tth111I. By using pKY2662 as a vector, a 32 kb Escherichia coli DNA fragment covering thyA, recC, recB, and argA genes was cloned. This new small cosmid is among the most efficient vectors hitherto found for in vitro cloning of large DNA fragments.

Bacteriophage lambda↗
PubMed2002

An engineered IN-1 F(ab) fragment with improved affinity for the Nogo-A axonal growth inhibitor permits immunochemical detection and shows enhanced neutralizing activity.

The myelin axonal growth inhibitor NI-220/250 (Nogo-A) has attracted considerable attention in elucidating the mechanisms that account for the lack of plasticity in the adult central nervous system. The cognate monoclonal antibody IN-1, which was obtained prior to the molecular characterization of its Nogo-A antigen, has played a crucial role in this respect. However, this murine IgM/kappa antibody does not only provide an inappropriate format for in vivo studies, its low antigen affinity has also hampered the thorough structure-function analysis of its neutralizing effect toward the Nogo-A inhibitor on a molecular basis. We describe here the affinity maturation of a bacterially produced functional IN-1 F(ab) fragment via protein engineering. A soluble fragment of Nogo-A derived from the central exon 3 of its gene, which was prepared by secretion into the periplasm of Escherichia coli, served as a target in these experiments. After repeated cycles of site-directed random mutagenesis and screening, the mutant II.1.8 of the IN-1 F(ab) fragment was obtained, carrying five side chain substitutions within CDR-L3. Its dissociation constant for the complex with the recombinant Nogo-A fragment was determined in surface plasmon resonance measurements as approximately 1 microM. The affinity of the unmutated IN-1 F(ab) fragment was 8-fold lower. The engineered F(ab) fragment appeared to be well suited for the specific detection of Nogo-A in immunochemical assays and for the histochemical staining of myelin-rich tissue sections. Most importantly, its concentration-dependent neutralizing effect on the Nogo-A inhibitory activity was significantly enhanced in cell culture. This study confirms Nogo-A to be the antigen of the IN-1 antibody and it demonstrates increased potential of the engineered F(ab) fragment as a reagent for promoting axonal regeneration in vivo.

Amino Acid Substitution↗
PubMed2005

A new yeast display vector permitting free scFv amino termini can augment ligand binding affinities.

Yeast surface display and sorting by flow cytometry are now widely used to direct the evolution of protein binding such as single-chain antibodies or scFvs. The available commercial yeast display vector pYD1 (Invitrogen) displays the protein of interest flanked on the N-terminus by Aga2, the disulfide of which binds the myristylated surface membrane protein Aga1. We have noted that two anti-CD3epsilon scFvs expressed as fusion proteins suffer a 30- to 100-fold loss of affinity when placed NH(2) terminal to either truncated toxins or human serum albumin. In the course of affinity maturing one of these scFv (FN18) using pYD1 we noted that the affinity towards the ectodomain of monkey CD3epsilongamma was too low to measure. Consequently we rebuilt pYD1 tethering the scFv off the NH(2) terminus of Aga2. This display vector, pYD5, now gave a positive signal displaying FN18 scFv with its ligand, monkey CD3epsilongamma. The apparent equilibrium association constant of the higher affinity scFv directed at human CD3epsilongamma increased approximately 3-fold when displayed on pYD5 compared with pYD1. These data show that for certain yeast-displayed scFvs a carboxy-tethered scFv can result in increased ligand-scFv equilibrium association constants and thereby extend the low range of affinity maturation measurements.

Amino Acid Sequence↗