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PubMed1991

Selecting high-affinity binding proteins by monovalent phage display.

Variants of human growth hormone (hGH) with increased affinity and specificity for the hGH receptor were isolated using an improved phage display system. Nearly one million random mutants of hGH were generated at 12 sites previously shown to modulate binding to the hGH receptor or human prolactin (hPRL) receptor. The mutant hormones were displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. After three to six cycles of enrichment for hGH-phage particles that bound to hGH receptor beads, we isolated hGH mutants that exhibited consensus binding sequences for the hGH receptor. Residues previously identified as important for hGH receptor binding by alanine-scanning mutagenesis were more highly conserved by this selection method. However, other residues nearby were not optimal, and by mutating them, hormone variants having greater affinity and selectivity for the hGH receptor were isolated. This approach should be useful for those who wish to modify and understand the energetics of protein-ligand interfaces.

Amino Acid Sequence↗
PubMed2000

Hypermodification of tRNA in Thermophilic archaea. Cloning, overexpression, and characterization of tRNA-guanine transglycosylase from Methanococcus jannaschii.

tRNA is structurally unique among nucleic acids in harboring an astonishing diversity of modified nucleosides. Two structural variants of the hypermodified nucleoside 7-deazaguanosine have been identified in tRNA: queuosine, which is found at the wobble position of the anticodon in bacterial and eukaryotic tRNA, and archaeosine, which is found at position 15 of the D-loop in archaeal tRNA. From homology searching of the Methanococcus jannaschii genome, a gene coding for an enzyme in the biosynthesis of archaeosine (tgt) was identified and cloned. The tgt gene was overexpressed in an Escherichia coli expression system, and the recombinant tRNA-guanine transglycosylase enzyme was purified and characterized. The enzyme catalyzes a transglycosylation reaction in which guanine is eliminated from position 15 of the tRNA and an archaeosine precursor (preQ(0)) is inserted. The enzyme is able to utilize both guanine and the 7-deazaguanine base preQ(0) as substrates, but not other 7-deazaguanine bases, and is able to modify tRNA from all three phylogenetic domains. The enzyme shows optimal activity at high temperature and acidic pH, consistent with the optimal growth conditions of M. jannaschii. The nature of the temperature dependence is consistent with a requirement for some degree of tRNA tertiary structure in order for recognition by the enzyme to occur.

Amino Acid Sequence↗
PubMed1995

Antisense oligodeoxyribonucleotide down-regulation of bcl-2 gene expression inhibits growth of the low-grade non-Hodgkin's lymphoma cell line WSU-FSCCL.

The BCL-2 gene product is involved in preventing apoptosis. The t(14,18) chromosomal translocation, which results in a fusion messenger RNA containing the entire coding region of BCL-2 and a portion of the immunoglobulin heavy chain gene, is commonly found in follicular lymphoma and appears to play a role in lymphomagenesis by inhibiting cell death. We tested the hypothesis that downregulation of BCL-2 would decrease accumulation of follicular lymphoma cells by treating the t(14,18)-carrying follicular lymphoma cell line WSU-FSCCL in vitro with antisense oligodeoxyribonucleotides (ODNs) directed against BCL-2. We found dose-dependent, sequence-specific inhibition of cell accumulation by an antisense unmodified ODN directed at codons 2 to 7, which downregulated BCL-2 protein levels. This effect was near maximal at an ODN concentration of 40 micrograms/mL (6.9 mumol/L), with minimal toxicity by control sense, reverse, and mutated antisense ODN at the same concentration. The pre-B leukemia cell line REH showed no sequence-specific growth inhibition by the antisense ODN at these concentrations, and BCL-2 protein levels were not altered. These data suggest that WSU-FSCCL may be useful in a murine model to optimize antisense ODN for potential therapeutic utility.

Base Sequence↗
PubMed1987

Site dependent time optimization of protein synthesis with special regard to accuracy.

The efficiency of protein synthesis is determined by its rate, accuracy, and energy consumption. With the energy consumption fixed, we optimize the system with respect to time and accuracy. Using an analytic model for a simple system and computer simulations for more complex systems, where also the possibility of errors is included, we demonstrate how different parts of the messenger RNA influence the protein production rate differently. The first part of the coding sequence is of major importance, since the availability of empty initiation sites is crucial, and queuing back to that region may interfere with initiation. The elongation rate at different positions depends on codon usage, on the concentrations of substrate and co-factors, and on the kinetic rate constants, including those of the proofreading branch(es). Ribosomal proofreading is a time consuming process and by allowing for more errors in the beginning of a protein, it is possible to increase the production rate of that protein. We calculate the mean translation time per functioning protein for various translation accuracies, and discuss the different strategies open to living cells.

Algorithms↗
PubMed2004

A carboxy-terminus motif of HKalpha2 is necessary for assembly and function.

BACKGROUND: The present experiments were designed to study the importance of the carboxy-terminus of HKalpha2, for both function and integrity of assembly with beta1-Na+,K+-ATPase. METHODS: For this purpose, stop codons were created, by polymerase chain reaction (PCR), at different positions in the carboxy-terminus of HKalpha2. Subsequently, chimeras between HKalpha2 and the carboxy-terminus of alpha1-Na+,K+-ATPase or with the carboxy-terminus of the gastric H+,K+-ATPase were created. Human embryonic kidney HEK-293 cells were used as expression systems for functional studies using 86Rb+ uptake and alpha/beta assembly using specific antibodies. RESULTS: The results demonstrate that the entire carboxy-terminus of HKalpha2 is required for optimal protection of the alpha/beta complex from degradation and for functionality as evidenced by 86Rb+ uptake. The results also demonstrate that there was flexibility in the sequence of the carboxy-terminus. The last two tyrosines (Y1035Y1036) of HKalpha2 could be mutated to alanines and the carboxy-terminus of HKalpha2 could be replaced by the carboxy-terminus of alpha1-Na+,K+-ATPase while preserving transport activity. CONCLUSION: The entire carboxy-terminus of HKalpha2 is required for stable assembly with beta1-Na+,K+-ATPase and functionality.

Amino Acid Sequence↗
PubMed1998

Strategies for optimizing heterologous protein expression in Escherichia coli.

The many advantages of Escherichia coli have ensured that it remains a valuable host for the efficient, cost-effective and high-level production of heterologous proteins. Here, we describe the current status of this prokaryotic expression system and focus on strategies designed to maximize the yields of recombinant proteins. Major challenges facing this expression system are also outlined.

Codon↗
PubMed1992

An in vitro system for the editing of ATP synthase subunit 9 mRNA using wheat mitochondrial extracts.

A posttranscriptional modification (C-to-U) at specific positions of plant mitochondrial mRNA leads to changes in the amino acid sequence as well as to the emergence of novel initiation or termination sites. This phenomenon, named RNA editing, has been described for several mitochondrial genes from different plant sources. We have found recently that RNA editing of the ATP synthase subunit 9 (atp9) mRNA involves eight changes including the creation of a new stop codon. In this article, we describe an in vitro system devised to follow the editing of wheat mitochondrial atp9 mRNA. Nonedited mRNA was obtained to serve as substrate for this reaction by in vitro transcription of the corresponding gene with T7 RNA polymerase. The source of conversion factor(s) was a soluble fraction obtained from purified wheat mitochondria lysed with salt and detergent. Edited RNA molecules were detected by hybridization with an end-labeled synthetic oligodeoxynucleotide probe complementary to a short region containing four editing events. Optimal conditions for the in vitro RNA editing reaction were determined. The reaction is sensitive to high temperature and protease digestion. Pretreatment with micrococcal nuclease decreased RNA editing activity in the mitochondrial extract, suggesting that a nucleic acid is necessary for the enzymatic reactions. Analysis of the edited mRNA showed that the in vitro reaction led to the same products as those observed in vivo.

Amino Acid Sequence↗
PubMed1997

Molecular cloning and nucleotide sequence of an endo-1,5-alpha-L-arabinase gene from Bacillus subtilis.

The nucleotide sequence of the gene encoding an endo-1,5-alpha-L-arabinase (protopectinase C) of Bacillus subtilis was determined by sequencing fragments amplified by the cassette-ligation-mediated PCR (CLM-PCR). The gene covering the start and stop codon was amplified by PCR with two specific primers, which were designed from the sequence data determined by CLM-PCR. An approximately 1.5-kb amplification product was cloned into the vector pUC119, forming a plasmid termed pPPC. An ORF that encodes the arabinase composed of 324 amino acids including a 33-amino-acid signal peptide was assigned. Comparison of the deduced amino acid sequence of the enzyme with that of an Aspergillus niger endoarabinase showed 37% identity in a 207-amino-acid overlap. The optimal nucleotide sequence for catabolite repression of B. subtilis was found upstream of the structural gene. In a culture of Escherichia coli DH5alpha cells harboring pPPC, no arabinase activity was detected, either intracellularly or extracellularly, suggesting that the B. subtilis promotor is not functional in this transformant. In B. subtilis IFO 3134 strain, production of protopectinase C was repressed by readily metabolizable carbohydrates. In contrast, productivity (total enzyme activity/bacterial growth) of the enzyme was increased about fourfold in the presence of 0.75 M potassium phosphate in the culture medium. The phosphate anion seemed to be involved in the stimulation of protopectinase C production in this stain.

Amino Acid Sequence↗
PubMed2000

ME-PCR for the identification of mutated K-ras in serum and bile of pancreatic cancer patients: an unsatisfactory technique for clinical applications.

Our aim was to assess the clinical reliability of mutated K-ras detection in serum or bile for the diagnosis of pancreatic cancer using ME-PCR. DNA was extracted from 1 ml serum obtained from 29 patients with pancreatic cancer and 12 control subjects. ME-PCR was optimized using a mixture of normal DNA added with different amounts of mutated DNA. The analysis of sera obtained from the 29 patients and of bile obtained from 11 pancreatic cancer patients demonstrated the presence of mutated K-ras in two (6.9%) and four cases (36%). By contrast K-ras was not amplifiable in any of the 12 serum samples obtained from healthy controls. In conclusion the DNA obtained from pancreatic cancer patients' sera is suitable for K-ras amplification and for the identification of codon 12 point mutations. However ME-PCR alone has an unsatisfactory sensitivity for the detection of pancreatic cancer using serum DNA as starting template.

Adult↗
PubMed1998

Design, total synthesis, and functional overexpression of the Candida rugosa lip1 gene coding for a major industrial lipase.

The dimorphic yeast Candida rugosa has an unusual codon usage that hampers the functional expression of genes derived from this yeast in a conventional heterologous host. Commercial samples of C. rugosa lipase (CRL) are widely used in industry, but contain several different isoforms encoded by the lip gene family, among which the isoform encoded by the gene lip1 is the most prominent. In a first laborious attempt, the lip1 gene was systematically modified by site-directed mutagenesis to gain functional expression in Saccharomyces cerevisiae. As alternative approach, the gene (1647 bp) was completely synthesized with an optimized nucleotide sequence in terms of heterologous expression in yeast and simplified genetic manipulation. The synthetic gene was functionally expressed in both hosts S. cerevisiae and Pichia pastoris, and the effect of heterologous leader sequences on expression and secretion was investigated. In particular, using P. pastoris cells, the synthetic gene was functionally overexpressed, allowing for the first time to produce recombinant Lipl of high purity at a level of 150 U/mL culture medium. The physicochemical and catalytic properties of the recombinant lipase were compared with those of a commercial, nonrecombinant C. rugosa lipase preparation containing lipase isoforms.

Amino Acid Sequence↗
PubMed1997

An Acanthamoeba polyubiquitin gene and application of its promoter to the establishment of a transient transfection system.

We have isolated and sequenced a 2388 bp polyubiquitin encoding genomic DNA from Acanthamoeba encompassing two complete and one incomplete ubiquitin units. Codon usage frequency shows extreme bias. The deduced amino acid sequences of each unit are identical to each other and the same as that deduced from a previously sequenced Acanthamoeba castellanii cDNA. The upstream region of this gene, which contained some putative regulatory modules, was recovered by PCR (polymerase chain reaction) amplification and subcloning. This upstream fragment was ligated to the CAT (chloramphenicol acetyltransferase) gene in a eukaryotic expression plasmid and successfully applied to the establishment of an Acanthamoeba transient transfection system. Transfection was performed by electroporation and the optimal voltage was 4500 volts/cm at capacitance 25 microF. DEAE-dextran (25 microg/ml) added into the electroporation buffer increased the transfection efficiency by about 45%. The CAT activity was proportional to the amount of DNA transfected and reached the peak level 48 h after transfection. CAT assays showed that the polyubiquitin gene upstream fragment contains a functional promoter which is about 2.5 times as strong as a viral RSV-LTR promoter when driving CAT expression in Acanthamoeba.

Acanthamoeba↗
PubMed2004

Multiple endocrine neoplasia type 2.

Multiple endocrine neoplasia type 2 (MEN-2) is a hereditary syndrome that is transmitted in an autosomal dominant pattern. MEN-2A, MEN-2B, and familial medullary thyroid cancer (MTC) comprise the MEN-2 syndrome. A germline mutation in the RET proto-oncogene is responsible for the MEN-2 syndrome. Recent data indicate that in 99% of MEN-2 cases, a germline RET mutation can be identified by genetic testing. The phenotypic variation of MEN-2 is diverse and partly related to the codon and specific point mutation in the RET proto-oncogene. There are increasing data on the genotype-phenotype correlations in patients with MEN-2 and this information should be used for screening at-risk patients and treatment of RET mutation carriers. All patients (especially if young) with MTC or bilateral pheochromocytoma should have a careful family history taken and genetic screening for RET germline mutations. Patients who are RET germline mutation carriers but without clinical or biochemical evidence of MTC should have a prophylactic total thyroidectomy. The optimal age of thyroidectomy should be based on the RET genotype (eg, high-risk mutations within the first year of life, intermediate-risk mutations by 5 years of age, and low-risk mutations by 10 years of age). Patients who are diagnosed with clinical or biochemical evidence of MTC should have a total or a near total thyroidectomy and at least a central neck lymph node dissection. Patients who have pheochromocytoma and a unilateral adrenal tumor on a localizing study should have a unilateral laparoscopic adrenalectomy after preoperative alpha-blockade. However, patients with bilateral adrenal tumors on localizing studies should have bilateral laparoscopic adrenalectomy. A cortical-sparing (subtotal) adrenalectomy may be considered, if technically feasible, to avoid long-term steroid dependence and to reduce the risk of Addisonian crisis. Patients with biochemical evidence of primary hyperparathyroidism should have a bilateral neck exploration and total parathyroidectomy and autotransplantation (30-60 mg of the most normal parathyroid tissue) to the nondominant forearm if asymmetric parathyroid hyperplasia is present. Rarely, patients may have only single-gland disease and excision may be performed if the other parathyroid glands are not found with biopsy to be hyperplastic. All unresected parathyroid glands should be marked with a clip because patients with MEN-2A have a high risk of persistent and recurrent primary hyperparathyroidism. Patients with familial MTC may have not manifested the other features of MEN-2A, thus these patients should have continued follow-up for pheochromocytoma and primary hyperparathyroidism.

Adrenal Gland Neoplasms↗
PubMed2005

Placing confidence limits on the molecular age of the human-chimpanzee divergence.

Molecular clocks have been used to date the divergence of humans and chimpanzees for nearly four decades. Nonetheless, this date and its confidence interval remain to be firmly established. In an effort to generate a genomic view of the human-chimpanzee divergence, we have analyzed 167 nuclear protein-coding genes and built a reliable confidence interval around the calculated time by applying a multifactor bootstrap-resampling approach. Bayesian and maximum likelihood analyses of neutral DNA substitutions show that the human-chimpanzee divergence is close to 20% of the ape-Old World monkey (OWM) divergence. Therefore, the generally accepted range of 23.8-35 millions of years ago for the ape-OWM divergence yields a range of 4.98-7.02 millions of years ago for human-chimpanzee divergence. Thus, the older time estimates for the human-chimpanzee divergence, from molecular and paleontological studies, are unlikely to be correct. For a given the ape-OWM divergence time, the 95% confidence interval of the human-chimpanzee divergence ranges from -12% to 19% of the estimated time. Computer simulations suggest that the 95% confidence intervals obtained by using a multifactor bootstrap-resampling approach contain the true value with >95% probability, whether deviations from the molecular clock are random or correlated among lineages. Analyses revealed that the use of amino acid sequence differences is not optimal for dating human-chimpanzee divergence and that the inclusion of additional genes is unlikely to narrow the confidence interval significantly. We conclude that tests of hypotheses about the timing of human-chimpanzee divergence demand more precise fossil-based calibrations.

Animals↗
PubMed1997

Escherichia coli RNase III (rnc) autoregulation occurs independently of rnc gene translation.

Control of mRNA stability is an established means of regulating gene expression. However, the detailed mechanisms by which such control is achieved are only now emerging. In particular, there remains a question about the involvement of translation. Escherichia coli ribonuclease III (RNase III) negatively autoregulates expression of its own gene (rnc) approximately 10-fold, by cleaving the untranslated leader and initiating approximately 10-fold more rapid decay of the rnc mRNA, after which RNase III plays no further role. Here, we define the mechanism of this control further. Mutations that increase rnc gene translation abolish autoregulation by increasing the stability of the RNase III-cleaved transcript RNA approximately 10-fold, with no effect on the uncleaved species. Mutations that decrease translation destabilize the rnc mRNA in the presence or absence of RNase III. In so doing, they reveal a pathway of rnc transcript decay distinct from the RNase III-dependent pathway. Stability of a 'mini-rnc' transcript containing the rnc leader and only the first two codons of the rnc gene is unaffected by decreased translation, presumably because sequences required for this pathway were removed. Importantly, this mini-rnc transcript is regulated normally by RNase III. Moreover, rnc transcripts synthesized in vitro do not decay in cell-free extracts lacking ribosomes, unless they are first cleaved by RNase III. These two results show that RNase III cleavage can initiate rnc transcript decay independently of rnc gene translation, unambiguously establishing that control of mRNA decay need not involve changes in translation. How rnc gene translation is optimized for efficient autoregulation will also be discussed.

Base Sequence↗
PubMed2005

Purification and properties of an extracellular endo-1,4-beta-xylanase from Penicillium citrinum and characterization of the encoding gene.

An extracellular endo-1,4-beta-xylanase was purified from the culture filtrate of a filamentous fungus Penicillium citrinum FERM P-15944 grown on birch-wood xylan. The purified enzyme showed a single band on SDS-PAGE with an apparent M(r) of 20,000 and had an isoelectric point below 3.5. Xylanase activity was optimal at pH 5.0 and 55 degrees C. The genomic DNA and cDNAs encoding this protein were cloned and sequenced. Southern blot analysis indicated that the xylanase gene (xynA) was present as a single copy in the genome. An open reading frame of 657 bp was interrupted by two introns of 65 and 55 bp, and encoded a presumed prepropeptide of 27 amino acids and a mature protein of 190 amino acids. Three distinct transcription start points were observed at positions -20 (A), -31 (A), and -36 (A) from the start codon. The 5'-noncoding region had a putative TATA box at nt -66 (TATAAA). The xynA cDNA was functionally expressed under the control of the alcohol oxidase I gene promoter in the methylotrophic yeast Pichia pastoris. A neighbor-joining tree showed that the P. citrinum enzyme is closely related to several other fungal xylanases belonging to the glycoside hydrolase family 11: Trichoderma reesei XYN2, Aspergillus niger xynNB, Penicillium funiculosum xynC, Penicillium sp. strain 40 xynA, Chaetomium gracile cgxB, and Aspergillus nidulans xlnA and xlnB.

Amino Acid Sequence↗
PubMed2001

Cloning of the cel9A gene and characterization of its gene product from marine bacterium Pseudomonas sp. SK38.

The yellow-pigmented bacterial strain causing green spot rot and death of layer was isolated from Porphyra dentata. This strain has been identified as Pseudomonas sp., harboring agarase, xylanase, and protease activity, as well as carboxymethyl-cellulase (CMCase). Using genomic DNA from the Pseudomonas sp. SK38 digested with Sau3AI and ligated into pBluescript II KS+, we isolated a cel gene encoding a CMCase in Pseudomonas sp. SK38. A 4.5-kb fragment was subcloned into pKR400. The structure of the cel9A gene consists of an open reading frame of 1,521 bp starting with a GTG start codon and ending with a TAG stop codon. It thus encodes 506 amino acid residues of a protein with a calculated molecular weight of 52,636 daltons plus a signal peptide of 22 amino acids. The deduced amino acid sequence of the cel9A protein is similar to the same protein of Clostridium thermocellum. It contains, in particular, the two conserved regions of the glycoside hydrolase family 9. The apparent molecular mass of the Cel9A protein is 52 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is most active at pH 6-7 and an optimal temperature of around 30 degrees C.

Amino Acid Sequence↗
PubMed1996

A generalized hidden Markov model for the recognition of human genes in DNA.

We present a statistical model of genes in DNA. A Generalized Hidden Markov Model (GHMM) provides the framework for describing the grammar of a legal parse of a DNA sequence (Stormo & Haussler 1994). Probabilities are assigned to transitions between states in the GHMM and to the generation of each nucleotide base given a particular state. Machine learning techniques are applied to optimize these probabilities using a standardized training set. Given a new candidate sequence, the best parse is deduced from the model using a dynamic programming algorithm to identify the path through the model with maximum probability. The GHMM is flexible and modular, so new sensors and additional states can be inserted easily. In addition, it provides simple solutions for integrating cardinality constraints, reading frame constraints, "indels", and homology searching. The description and results of an implementation of such a gene-finding model, called Genie, is presented. The exon sensor is a codon frequency model conditioned on windowed nucleotide frequency and the preceding codon. Two neural networks are used, as in (Brunak, Engelbrecht, & Knudsen 1991), for splice site prediction. We show that this simple model performs quite well. For a cross-validated standard test set of 304 genes [ftp:@www-hgc.lbl.gov/pub/genesets] in human DNA, our gene-finding system identified up to 85% of protein-coding bases correctly with a specificity of 80%. 58% of exons were exactly identified with a specificity of 51%. Genie is shown to perform favorably compared with several other gene-finding systems.

Chromosomes, Human↗
PubMed2003

High-level expression of a novel FMN-dependent heme-containing lyase, phenylacetaldoxime dehydratase of Bacillus sp. strain OxB-1, in heterologous hosts.

We examined the overexpression of a novel FMN-dependent heme-containing lyase, phenylacetaldoxime dehydratase (Oxd) of Bacillus sp. strain OxB-1, in Escherichia coli and Bacillus subtilis. Several plasmids were constructed to express the enzyme under the control of the lac promoter or its own promoter, together with or without nitrilase and a possible regulatory protein that is present in the wild-type genome. The enzyme was expressed using E. coli transfected with the plasmid pOxD-9OF. Expression was under the control of the lac promoter in the pUC18 vector and was much more effective when the start codon was changed from TTG to ATG. When the transfected cells were grown at 37 degrees C, the enzyme was produced mainly in inactive inclusion bodies, whereas the enzyme was largely soluble and active when the cells were grown at 30 degrees C. The production of active enzyme was markedly enhanced by increasing the volume of culture medium. This had the effect of slowing the rate of apoenzyme synthesis. A slow rate of synthesis allows for a more efficient incorporation of heme cofactor into the apoenzyme than a fast rate of synthesis. Under optimized conditions, the enzyme was produced in an active and soluble form at 15,000U/L of culture, which is about 1500-fold higher than the amount produced by the wild-type strain. Moreover, the enzyme comprised over 40% of total extractable cellular protein.

Bacillus↗