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PubMed1991

Sequence and linkage analysis of the Coxiella burnetii citrate synthase-encoding gene.

The nucleotide (nt) sequence of the Coxiella burnetii citrate synthase-encoding gene (gltA), previously cloned in Escherichia coli, was determined. The nt sequence analysis revealed an open reading frame (ORF) of 1290 bp capable of coding for a protein of 430 amino acids (aa) with a deduced Mr of 48,633. Preceding an ATG start codon, a possible transcription start point (tsp) with homology to the E. coli promoter consensus was detected. A poly-purine-rich region occurred immediately upstream from the gltA reading frame and potentially serves as a ribosome-binding site. Additionally, a G + C-rich region of dyad symmetry 3' to the translational stop codon was found that could possibly function as a Rho-independent transcriptional termination signal. A large, nearly perfect, inverted repeat was identified upstream from the gltA tsp and was shown by Southern analysis to be present in multiple copies in the C. burnetii genome. The deduced aa sequence of C. burnetii GltA was optimally aligned with enzymes from various prokaryotic sources and one eukaryotic source (pig heart). Using perfect aa identity, the C. burnetii enzyme demonstrated the greatest homology with GltA from Acinetobacter anitratum (65%). Although only 26% aa identity was seen with the pig heart enzyme, many of the residues identified in ligand binding appear to be conserved. Sequencing studies of a region centered approx. 5.6 kb upstream from gltA revealed an ORF read with opposite polarity that encodes a peptide highly homologous to the C terminus of the flavoprotein subunit of E. coli succinate dehydrogenase. This report represents the first nt sequence analysis of a gene of known function from the obligate intracellular parasite, C. burnetii.

Amino Acid Sequence↗

PubMed1991

Optimizing the promoter and ribosome binding sequence for expression of human single chain urokinase-like plasminogen activator in Escherichia coli and stabilization of the product by avoiding heat shock response.

The expression of recombinant single-chain urokinase-like plasminogen activator (rscuPA) in Escherichia coli was optimized by fusing the puk gene to different promoters and ribosome binding sequences. Comparison of the tac, trp and lambda PL promoters showed that expression was maximal under tac control. Variation in the ribosome binding sequence and its distance to the AUG start codon yielded a further slight improvement of expression. The largest increase in rscuPA expression was achieved by variations in the host strain and growth conditions. In E. coli DG75 grown at 37 degrees C maximal expression was achieved 30 min after induction and decreased gradually until 240 min after induction. Growth at 30 degrees C yielded maximal expression 60 min after induction and resulted in reduced activity at longer times. Western blot analysis of the products showed that degradation of rscuPA was much larger at 37 degrees C than at 30 degrees C. Using E. coli CAG630 carrying the htpR mutation, which avoids heat shock response, for expression of rscuPA eliminated the instability of the product at both temperatures. Expression in this strain was even more efficient than in E. coli JM101 carrying the lon mutation. It is concluded that induction of the general heat-shock response in E. coli must be avoided to obtain stabilization of rscuPA. This drastically improves the overall yield of rscuPA from recombinant E. coli strains.

Base Sequence↗

PubMed1980

Early assignments of the genetic code dependent upon protein structure.

Orgel (1972) has suggested that polynucleotides with sequences of alternating purine/pyrimidine are likely to have predominated in prebiotic conditions. Therefore, in any early template-directed protein synthesis, the number of available codons would have been limited. However, for any self-organizing system to survive and propagate, some feedback must occur from the products of the synthesis to the control of the synthetic procedure itself; i.e. the protein synthesized should have catalysed some step in the initation of template-directed synthesis. A given protein structure with a characteristic conformation and function would be optimal for the product of such a synthesis, and this in turn would limit the number of nucleotide sequences of those available able to give rise to a functioning synthetic assembly. A possible candidate for such an early polypeptide is the ferredoxin group of proteins and it is shown that with the present-day code the corresponding nucleotides do have a high percentage of alternating purine/pyrimidine sequences. Hence these combined restraints on the primitive synthetic machinery would direct the possible assignments of the genetic code helping to explain its regularity and universality.

Amino Acid Sequence↗

PubMed1991

Structural and functional effects of mutations altering the subunit interface of mitochondrial malate dehydrogenase.

Among highly conserved residues in eucaryotic mitochondrial malate dehydrogenases are those with roles in maintaining the interactions between identical monomeric subunits that form the dimeric enzymes. The contributions of two of these residues, Asp-43 and His-46, to structural stability and catalytic function were investigated by construction of mutant enzymes containing Asn-43 and Leu-46 substitutions using in vitro mutagenesis of the Saccharomyces cerevisiae gene (MDH1) encoding mitochondrial malate dehydrogenase. The mutant enzymes were expressed in and purified from a yeast strain containing a disruption of the chromosomal MDH1 locus. The enzyme containing the H46L substitution, as compared to the wild type enzyme, exhibits a dramatic shift in the pH profile for catalysis toward an optimum at low pH values. This shift corresponds with an increased stability of the dimeric form of the mutant enzyme, suggesting that His-46 may be the residue responsible for the previously described pH-dependent dissociation of mitochondrial malate dehydrogenase. The D43N substitution results in a mutant enzyme that is essentially inactive in in vitro assays and that tends to aggregate at pH 7.5, the optimal pH for catalysis for the dimeric wild type enzyme.

Acetates↗

PubMed1995

Progress in genetic screening of multiple endocrine neoplasia type 2A: is calcitonin testing obsolete?

BACKGROUND: Recent identification of RET mutations in multiple endocrine neoplasia type 2A (MEN 2A) allows a DNA-based approach to diagnosis in lieu of calcitonin sampling. To prospectively evaluate the efficacy of mutational analysis, genetic screening was performed in 124 patients (53 male, 71 female; age, 1 month to 80 years) at risk for MEN 2A referred over 3 months. METHODS: Analysis used genomic DNA and a polymerase chain reaction-based denaturing gradient gel electrophoresis strategy for mutation detection at RET codons 609, 611, 618, 620, and 634. Ninety-three of 124 patients were from established MEN 2A kindreds (group A), and screening replaced calcitonin testing. Twenty-one of 124 patients (group B) represented index cases of medullary thyroid carcinoma (MTC), and DNA analysis was performed to distinguish sporadic from hereditary disease. Ten patients (group C) had modest calcitonin elevations or had undergone thyroidectomy without confirming pathologic results, and testing was undertaken to clarify status. RESULTS: Group A: RET mutations occurred in 29 (median age, 10 years) of 93 patients, 14 of whom underwent thyroidectomy. No false-positive results were observed. Group B: five (24%) of 21 patients with seemingly sporadic MTC had RET mutations at codons 618 (one), 620 (one), or 634 (three). Group C: Nine of 10 patients with alleged MEN 2A had genetically negative results. CONCLUSIONS: Denaturing gradient gel electrophoresis reliably detects MEN 2A. Modest calcitonin elevations may lead to a false-positive diagnosis of MTC. DNA testing is the optimal approach to evaluating MEN 2A. Index cases of sporadic MTC should also undergo DNA analysis.

Adolescent↗

PubMed1982

A convenient and adaptable package of DNA sequence analysis programs for microcomputers.

We describe a package of DNA data handling and analysis programs designed for microcomputers. The package is convenient for immediate use by persons with little or no computer experience, and has been optimized by trial in our group for a year. By typing a single command, the user enters a system which asks questions or gives instructions in English. The system will enter, alter, and manage sequence files or a restriction enzyme library. It generates the reverse complement, translates, calculates codon usage, finds restriction sites, finds homologies with various degrees of mismatch, and graphs amino acid composition or base frequencies. A number of options for data handling and printing can be used to produce figures for publication. The package will be available in ANSI Standard FORTRAN for use with virtually any FORTRAN compiler.

Amino Acid Sequence↗

PubMed1988

Disruption of the CHO1 gene encoding phosphatidylserine synthase in Saccharomyces cerevisiae.

A Saccharomyces cerevisiae mutant that lacked phosphatidylserine synthase [EC 2.7.8.8] (CDP-1,2-diacyl-sn-glycerol: L-serine O-phosphatidyltransferase) completely was constructed by disrupting its structural gene, CHO1. Over two-thirds of its coding region, from the starting to the 200th codon, was replaced with a LEU2 DNA fragment. This new cho1 mutant showed no detectable synthesis of phosphatidylserine but grew slowly in a medium that contained either ethanolamine or choline. These results indicate that phosphatidylserine synthase and most probably phosphatidylserine are dispensable in S. cerevisiae but necessary for its optimal growth. Additional supplementation with myo-inositol raised the cellular content of phosphatidylinositol and improved the growth of the mutant, suggesting the importance of the negative charges of the membrane surface. The CHO1-disrupted mutant, when grown on choline, accumulated phosphatidylethanolamine to a significant level even after extensive dilution of the initial culture. It segregated prototrophic revertants that could synthesize phosphatidylethanolamine without recovery of phosphatidylserine synthesis. These results imply the presence of a route(s) for the formation of ethanolamine or its phosphorylated derivative in S. cerevisiae.

Blotting, Southern↗

PubMed1989

Control of reovirus messenger RNA translation efficiency by the regions upstream of initiation codons.

The 10 species of reovirus messenger RNA are translated in vivo with efficiencies/frequencies that differ by as much as 100-fold. The s1 mRNA, which is translated 10 times less efficiently than the s4 mRNA but 10 times more efficiently than the/1 and m1 mRNAs, has a unique BamH1 cleavage site located immediately downstream of its initiation codon. Because the reovirus mRNAs have been cloned, this provides the opportunity for placing modified and altered sequences upstream of its coding sequence. The translation efficiencies of the variant mRNAs, transcribed via the SP6 in vitro transcription system, can then be measured in the rabbit reticulocyte lysate in vitro translation system. Using this system it was found that replacing the 5'-upstream sequence of the s1 mRNA with that of the s4 mRNA increases its in vitro translation efficiency by 4-fold; that the trinucleotide immediately upstream of the s1 initiation codon renders it very weak, and that it is only slightly superior to the weakest Kozak consensus sequence; that the nature of the nucleotides further upstream than position -3 can profoundly affect translation efficiency; that the nature of this effect is in turn markedly modified by the nature of nucleotides in positions -1 to -3; and that there is a minimum optimal 5'-upstream sequence length of about 14 nucleotides. We also investigated the effect of secondary structure involvement on the ability of 5'-upstream sequences to promote translation. Two effects were noted. First, being part of moderately stable stem loops (delta G, -18 kcal/mol) decreased translation efficiency about 3-fold; second, mRNA in which only three 5'-terminal nucleotides were unpaired were translated five times less efficiently than mRNA in which six nucleotides were unpaired. Accessibility of the 5'-cap as well as secondary structure of the 5'-upstream sequences are therefore factors that affect translation efficiency. Finally, we showed that the m1 mRNA, which is transcribed very poorly in vivo, is translated very efficiently in vitro; and that its 5'-upstream sequence is as effective in increasing protein sigma 1 formation as that of s4 mRNA. Since both m1 mRNA and protein mu 2 are stable in infected cells, the reason why m1 mRNA is translated so inefficiently in vivo therefore remains unexplained.

Codon↗

PubMed1995

In vitro antibody maturation. Improvement of a high affinity, neutralizing antibody against IL-1 beta.

Abs as therapeutic antagonists should be of relatively high affinity to effectively neutralize their target Ag. Typically, Abs with nanomolar affinities to protein Ags can be obtained in vivo, however, this may not be the upper limit of affinity because the biologic process attempts to optimize Ab function, of which affinity is only one component. SK48, a high affinity neutralizing murine Fab against human IL-1 beta was used to explore the nature of Ab-Ag interactions and the potential for further affinity improvement in vitro using mutagenesis and selection via phage display. The codons of six amino acids in the third complementarity-determining region of the heavy chain (CDR3-H) were both individually and combinatorialy randomized and the resultant libraries were screened for IL-1 binding phenotype. Mutations that reduced affinity suggested that both the backbone conformation of the CDR3 loop and certain side chains are essential for binding, yet alterations to the canonical salt bridge residues at the base of the loop had minimal effect on affinity. Four rounds of selection of the phage Ab libraries on immobilized IL-1 beta gave predominantly the wild-type sequence, indicating efficient affinity maturation of this CDR in vivo. However, a twofold improvement in affinity was observed for both single and double amino acid changes at two positions in the middle of the CDR. Moreover, a 10-fold increase in affinity for IL-1 beta was achieved by combining two of the phage-selected single amino acid substitutions in CDR3-H, thereby demonstrating that a significant improvement in affinity can be achieved through CDR mutagenesis, even in a matured Ab. The increased affinity of this Fab did not, however, enhance its neutralizing activity in vitro.

Amino Acid Sequence↗

PubMedDate not supplied

[Use of the hygromycin phosphotransferase gene as the dominant selective marker for Chlamydomonas reinhardtii transformation].

The hygromycin phosphotransferase gene (hpt) from E. coli under the control of the SV40 early promoter was used as a dominant selectable marker for transformation of Chlamydomonas reinhardtii. Cells were transformed by electroporation (pulse length, 2 ms, field strength, 1 kV/cm). The culture growth phase was a crucial parameter for transformation (optimal density approximately 10(6) cells/ml). It was possible to obtain approximately 10(3) Hyg-resistant colonies under these conditions. Foreign DNA integrated into the Chlamydomonas genome was maintained for at least 8 months but the Hyg-resistant phenotype of the transformed clones was unstable. The frequency of codon usage in the hpt gene was compared with the one in Chlamydomonas nuclear genes. It is supposed that highly biased codon usage in Chlamydomonas does not preclude expression. Advantages of this selection system for studying Chlamydomonas transformation by heterologous genes are discussed.

Animals↗

PubMed1992

The glgB gene from the thermophile Bacillus caldolyticus encodes a thermolabile branching enzyme.

We have cloned the structural gene for the Bacillus caldolyticus glycogen branching enzyme (glgB) in Escherichia coli. The glgB gene consisted of a 1998 bp open reading frame (ORF) encoding a 78,087 Da protein, which was highly similar to the Bacillus stearothermophilus branching enzyme. The 5' end of a second gene that encoded a protein with extensive similarity to E. coli ADP-glucose pyrophosphorylase (ADPGP) partly overlapped the 3' end of the glgB gene. A putative promoter recognized by Bacillus subtilis RNA polymerase containing the sigma factor H (E-sigma H) preceded the genes. These data suggest that in contrast to the situation observed in B. stearothermophilus, the genes involved in glycogen synthesis in B. caldolyticus are clustered on the chromosome, and are presumably coordinately expressed during the early stages of sporulation. An incomplete third gene started upstream of B. caldolyticus glgB. This gene was highly similar to a gene found directly upstream of B. stearothermophilus glgB, which encodes a putative membrane protein with unknown function. The B. caldolyticus glgB gene was expressed in E. coli and B. subtilis. Surprisingly, the branching enzyme appeared to be thermolabile, the temperature of optimal activity being only 39 degrees C.

1,4-alpha-Glucan Branching Enzyme↗

PubMed1987

A computer program for the design of optimal synthetic oligonucleotide probes for protein coding genes.

A computer program has been written in FORTRAN 77 to locate on a protein sequence a region with optimum length and limited degeneracy in order to design artificial oligonucleotide probes for use in molecular cloning. In addition the program checks for regions of homology between this probe and any other base sequence found in nucleotide sequence data banks. There are options in the program to eliminate rare codons or to make preferential choices of bases in order to minimize the degeneracy of probes.

Algorithms↗

PubMed1983

Nucleotide sequence of a cDNA clone encoding the entire glycoprotein from the New Jersey serotype of vesicular stomatitis virus.

The nucleotide sequence of the mRNA encoding the glycoprotein from the New Jersey serotype of vesicular stomatitis virus (VSV) was determined from a cDNA clone containing the entire coding region. The sequence of 12 5'-terminal noncoding nucleotides present in the mRNA but not in the cDNA clone was determined from a primer extended to the 5' terminus of the mRNA. The mRNA is 1,573 nucleotides long (excluding polyadenylic acid) and encodes a protein of 517 amino acids. Only six nucleotides occur between the translation termination codon and the polyadenylic acid. Short homologies between the untranslated termini of this mRNA and the mRNAs of the Indiana serotype were found. The predicted protein sequence was compared with that of the glycoprotein of the Indiana serotype of VSV and with the glycoprotein of rabies virus, using a computer program which determines optimal alignment. An amino acid identity of 50.9% was found for the two VSV serotypes. Approximately 20% identity was found between the rabies virus and VSV New Jersey glycoproteins. The positions and sizes of the transmembrane domains, the signal sequences, and the glycosylation sites are identical in both VSV serotypes. Two of five serine residues which were possible esterification sites for palmitate in the glycoprotein from the Indiana serotype are changed to glycine residues in the glycoprotein from the New Jersey serotype. Because the glycoprotein of the New Jersey serotype does not contain esterified palmitate, we suggest that one or both of these residues are the probable esterification sites in the glycoprotein from the Indiana serotype.

Base Sequence↗

PubMed1989

Mature apolipoprotein AI and its precursor proApoAI: influence of the sequence at the 5' end of the gene on the efficiency of expression in Escherichia coli.

Apolipoprotein AI (ApoAI) plays a central role in the regulation of lipid metabolism. Initial attempts to express human apoAI cDNA in Escherichia coli did not yield detectable levels of the mature protein. By analyzing the efficiency of expression of apoAI-lacZ gene fusions, we have been able to show that the sequence at the 5' end of the ApoAI-coding region is a critical parameter. Indeed, silent changes in the codons for the first 8 residues of ApoAI, which did not alter the amino acid sequence, affected expression dramatically. Analysis of the corresponding mRNA steady-state levels suggested a role for differential mRNA stability in the control of apoAI expression in this system. Among all the possible alternative sequences, we have identified an optimal sequence which, when reinserted in the original expression plasmid, yields high level production of mature ApoAI. This procedure has been extended to the production of the natural variant ApoAI-Milano and the precursor proApoAI. Availability of these recombinant molecules would allow the investigation of their structural and biological features. In addition, the methodology used to optimize ApoAI expression is of general interest in assuring high expression of heterologous proteins in E. coli.

Amino Acid Sequence↗

PubMed1995

Kinetics of translation of gamma B crystallin and its circularly permutated variant in an in vitro cell-free system: possible relations to codon distribution and protein folding.

Analysis of nascent gamma B-crystallin peptides accumulating during in vitro translation in a rabbit reticulocyte lysate cell-free system was carried out. As a consequence of the irregular distribution of rare codons along the polypeptide chain of gamma B-crystallin, translation of the two-domain protein is a non-uniform process characterized by specific pauses. One of the major delays occurs during the translation of the connecting peptide between the domains. Comparing the kinetics of translation of natural gamma B-crystallin and its circularly permutated variant (with the order of the N- and C-terminal domains exchanged) reveals that the natural N-terminal domain is translated faster than the C-terminal one. Since the N-terminal domain in natural gamma B-crystallin is known to be more stable and to fold faster than the C-terminal one [E.-M. Mayr et al. (1994) J. Mol. Biol. 235, 84-88], the present data suggest that the translation rates are optimized to tune the synthesis and folding of the nascent polypeptide chain. In this connection, the pause in the linker region between the domains provides a delay allowing the correct folding of the N-terminal domain and its subsequent assistance in the stabilization of the C-terminal one.

Animals↗

PubMed1990

A generalized information function applied to the genetic code.

The problem of the partitioning of the degeneracy of the codons in the genetic code is considered in the framework of a generalized information function IG = c sigma kpk(ln pk + G(Ek] where k represents the number of codons in a specific degeneracy class and G(Ek) is an arbitrary real valued function. For G(Ek) = 0 the Shannon information function is recovered. For a particular choice of G(Ek) that takes the dominance of even degeneracies into account, it is found by direct numerical calculations that the correct degeneracy partitioning appears as optimal values of the Ig function. This results is also supported by optimization calculations in which the generalized information function is regarded as a continuous function in the degeneracy variables.

Amino Acids↗

PubMed1992

Detection of hepatitis B pre-core mutant by allele specific polymerase chain reaction.

AIM: Development of a specific polymerase chain reaction (PCR) assay for detection of the pre-core, stop codon, mutant of hepatitis B virus (HBV). METHODS: PCR primers, specific at the 3'-end for nucleotide 1896 of either the pre-core, stop codon, mutant or wild type HBV, were synthesised using published sequence data. Positive control templates for both types of virus were synthesised by the PCR, incorporating sequences specific for each virus type at the appropriate position. These templates were used to optimise the specificity of the procedure. Formalin fixed, paraffin wax embedded human tissue from acute or fulminant HBV hepatitis from Hong Kong or Oxford was then investigated for presence of mutant or wild type virus. The HBV DNA was amplified from this tissue using a two step procedure, with an initial amplification phase followed by a second diagnostic phase on optimally diluted target DNA. RESULTS: Specific detection of mutant or wild type HBV was achieved. An important factor in determining specificity was the temperature of annealing, 70 degrees C proving to be highly specific. To overcome the inherent variation of target copy number in clinical samples and to provide an intrinsic positive control, it was important to generate and standardise the amount of target HBV used for the specific PCR. Two cases of fulminant hepatitis and four cases of acute hepatitis from Hong Kong, and one case of fulminant hepatitis from Oxford, contained only wild type HBV, with no evidence of a mutant virus. CONCLUSION: This method can be applied to FFPE tissues. It is rapid, non-radioactive, and specific for the stop codon mutation at nucleotide 1896 of HBV. Preliminary investigation of a small number of cases of fulminant hepatitis from Oxford and Hong Kong showed only wild type virus. The result differs from results published from Japan and Israel.

Alleles↗

PubMed1990

Nucleotide sequence of the Synechococcus sp. PCC7942 branching enzyme gene (glgB): expression in Bacillus subtilis.

The nucleotide sequence of the Synechococcus sp. PCC7942 glgB gene has been determined. The gene contains a single open reading frame (ORF) of 2322 bp encoding a polypeptide of 774 amino acids (aa) with an Mr of 89,206. Extensive sequence similarity exists between the deduced aa sequence of the Synechococcus sp. glgB gene product and that of the Escherichia coli branching enzyme in the middle portions of the proteins (62% identical aa). In contrast, the N-terminal portions shared little homology. The sequenced region which follows glgB contains an ORF encoding 79 aa of the N terminus of a polypeptide that shares extensive sequence similarity (41% identical aa) with human and rat uroporphyrinogen decarboxylase. This suggests that the region downstream from glgB contains the hemE gene and, therefore, that the organization of genes involved in glycogen biosynthesis in Synechococcus sp. is different from that described for E. coli. A fusion gene was constructed between the 5' end of the Bacillus licheniformis penP gene and the Synechococcus sp. glgB gene. The fusion gene was efficiently expressed in the Gram+ micro-organism Bacillus subtilis and specified a branching enzyme with an optimal temperature for activity similar to the wild-type enzyme.

1,4-alpha-Glucan Branching Enzyme↗