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Structural requirements for initiation of translation by internal ribosome entry within genome-length hepatitis C virus RNA.

Cap-independent translation of hepatitis C virus (HCV) RNA is mediated by an internal ribosomal entry segment (IRES) located within the 5' nontranslated RNA (5'NTR), but previous studies provide conflicting views of the viral sequences which are required for translation initiation. These discrepancies could have resulted from the inclusion of less than full-length 5'NTR in constructs studied for translation or destabilization of RNA secondary structure due to fusion of the 5'NTR to heterologous reporter sequences. In an effort to resolve this confusion, we constructed a series of mutations within the 5'NTR of a nearly full-length 9.5-kb HCV cDNA clone and examined the impact of these mutations on HCV translation in vitro in rabbit reticulocyte lysates and in transfected Huh-T7 cells. The inclusion of the entire open reading frame in HCV transcripts did not lead to an increase in IRES-directed translation of the capsid and E1 proteins, suggesting that the nonstructural proteins of HCV do not include a translational transactivator. However, in reticulocyte lysates programmed with full-length transcripts, there were multiple aberrent translation initiation sites resembling those identified in some picornaviruses. The deletion of nucleotides (nt) 28-69 of the 5'NTR (stem-loop IIa) sharply reduced capsid translation both in vitro and in vivo. A small deletion mutation involving nt 328-334, immediately upstream of the initiator AUG at nt 342, also resulted in a nearly complete inhibition of translation, as did the deletion of multiple intervening structural elements. An in-frame 12-nt insertion placed within the capsid-coding region 9 nt downstream of the initiator AUG strongly inhibited translation both in vitro and in vivo, while multiple silent mutations within the first 42 nt of the open reading frame also reduced translation in reticulocyte lysates. Thus, domains II and III of the 5'NTR are both essential to activity of the IRES, while conservation of sequence downstream of the initiator AUG is required for optimal IRES-directed translation.

Amino Acid Sequence

A segment-based dynamic programming algorithm for predicting gene structure.

An algorithm called segment-based dynamic programming is described for predicting gene structure from a sequence of genomic DNA. The algorithm explores the space of gene structures that satisfy junctional and frame constraints and finds the gene structure that optimizes the sum of junctional and segmental scoring functions. Junctional constraints specify acceptable sites of initiation, termination, and splicing, whereas frame constraints ensure that the total exon length is a multiple of three and that no in-frame stop codons occur within exons or at exon-exon junctions. By computing over segments, segment-based dynamic programming maintains reading frame and phase information for each segment, it can assemble exons in-frame as well as score them in-frame. The algorithm is used to quantify the computational power of constraints. Experimental results show that frame constraints reduce the size of the search space by several orders of magnitude and that cardinality constraints place an asymptotic limit on the size of the search space. The algorithm is also used to compare the accuracy of different methods for assembly and scoring. A scoring scheme based on fifth-order Markov hexamer frequencies is presented and used in three objective functions, corresponding to in-frame, frame-independent, and frame-maximal scoring strategies. Experimental results show that in-frame assembly improves specificity only slightly over frame-independent assembly, whereas in-frame scoring improves specificity substantially over frame-independent and frame-maximal scoring.

Algorithms

Nitrile hydratase gene from Rhodococcus sp. N-774 requirement for its downstream region for efficient expression.

For improvement of the production of nitrile hydratase (NHase) from Rhodococcus sp. N-774 by recombinant DNA techniques, several plasmids, each of which had a deletion of the upstream or downstream region of the genes encoding the alpha and beta subunits of NHase, were constructed. Enzyme assays of recombinant R. rhodochrous and Escherichia coli cells showed that a downstream region of the NHase genes was indispensable for the production of active NHase in both cells, but for the production of the active amidase, no genes other than the amidase structural gene were required. The nucleotide sequence of the downstream region contained a single open reading frame (Orf1188) with 396 amino acids. Orf1188 showed similarity in amino acid sequence to P47K, an open reading frame found downstream of the NHase genes from Pseudomonas chlororaphis B23, and also to the cobW gene product, which may be involved in cobalamin biosynthesis in Pseudomonas denitrificans. Because the distance between the TGA stop codon for the NHase beta-subunit and the ATG codon for Orf1188 is only 98 bp, and because production of both Orf1188 and NHase is dependent on a promoter upstream of the amidase gene, these genes appear to be co-transcribed in a polycistronic manner, forming an operon. By optimization of the culture conditions of R. rhodochrous carrying pKRNH2, which contained the amidase, NHase, and Orf1188 genes, the transformant showed the NHase activity 6-fold higher than that of the original strain, Rhodococcus sp. N-774.

Amino Acid Sequence

[Computer programs for the analysis of nucleotide sequences (MALK)].

A system for the computer analysis of nucleic acid and protein sequences ("Helix") is described. Format of the DNA sequences is EMBL--compatible and may be easily commented with the help of convenient menus. "Helix" has also following possibilities: an effective alignment of gele reading data and formation of the final sequence; simple making of recombined molecules "in calcular"; calculations of nucleotide and dinucleotide distribution along the sequence; looking for coding frames; calculations percentage of codons and amino acids in coding frames; searching for direct and inverted repeats; sequences alignment; protein secondary structure prediction; restriction mapping; DNA--protein translation. "Helix" also contain programs for RNA-structure prediction, looking for homologies throughover the EMAL bank, choosing optimal sequence for probes and searching promoters. All the programs are written at FORTRAN-77 and automatically translated into FORTRAN-4. "Helix" require only 64 kbite.

Base Sequence

Recognition of genes in human DNA sequences.

A new approach to computer-assisted gene recognition in higher eukaryote DNA is suggested. It allows one to use not only linear functions for scoring structures, but all functions satisfying natural monotonicity conditions. The algorithm constructs the set of structures guaranteed to contain an optimal structure for every function. So, it uncouples the time-consuming step of generation of this set from the fast step of structure scoring, thus making it simple to experiment with different functions. One particular scoring function, taking into account only codon usage and positional nucleotide frequencies of the splicing sites, has been implemented in the Genome Recognition and Exon Assembly Tool program, and has been tested on an independent sample of human genes, yielding 88% sensitivity and 79% specificity.

Algorithms

Transcriptional regulation of the carbon monoxide dehydrogenase gene (cdhA) in Methanosarcina thermophila.

The mechanisms of gene regulation in the phylogenetic domain Archaea are not yet understood. To examine the expression of a gene encoding a highly regulated catabolic enzyme from the methanogenic archaea, a Methanosarcina thermophila lambda gt11 chromosomal library was probed with antiserum prepared against the 89-kDa subunit of carbon monoxide dehydrogenase, an enzyme which is required for growth and methanogenesis from acetate. A 2.3-kilobase DNA fragment was isolated that encoded 300 bases of the 5'-end of cdhA, the gene which encodes the 89-kDa subunit, and 2 kilobases upstream of cdhA that included an upstream open reading frame (ORF1). Primer extension analyses determined that cdhA and ORF1 each had a single transcriptional initiation site located 370 and 9 nucleotides, respectively, 5' of the putative translation initiation codons for cdhA and ORF1. Each promoter element had sequence similarity to a consensus archaeal promoter sequence. Three discrete mRNA cdhA transcripts of 9.5, 5.6, and 4.8 kilobases and one mRNA ORF1 transcript of < 2 kilobases were identified. All four transcripts were optimally expressed in cells grown with acetate, while growth with the more energetically favorable substrates methanol and trimethylamine caused a significant reduction in levels of the cdhA and ORF1 mRNA's. The half-lives of the 5' ends of the three cdhA transcripts and entire ORF1 mRNA transcript were approximately 2 min upon addition of methanol to cells growing exponentially in medium that contained acetate. Results of this study demonstrate that transcription of both cdhA and ORF1 is highly regulated in response to substrate by this methanogenic archaeon.

Aldehyde Oxidoreductases

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

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

A comprehensive set of DnaA-box mutations in the replication origin, oriC, of Escherichia coli.

We probed the complex between the replication origin, oriC, and the initiator protein DnaA using different types of mutations in the five binding sites for DnaA, DnaA boxes R1-R4 and M: (i) point mutations in individual DnaA boxes and combinations of them; (ii) replacement of the DnaA boxes by a scrambled 9 bp non-box motif; (iii) positional exchange; and (iv) inversion of the DnaA boxes. For each of the five DnaA boxes we found at least one type of mutation that resulted in a phenotype. This demonstrates that all DnaA boxes in oriC have a function in the initiation process. Most mutants with point mutations retained some origin activity, and the in vitro DnaA-binding capacity of these origins correlated well with their replication proficiency. Inversion or scrambling of DnaA boxes R1 or M inactivated oriC-dependent replication of joint replicons or minichromosomes under all conditions, demonstrating the importance of these sites. In contrast, mutants with inverted or scrambled DnaA boxes R2 or R4 could not replicate in wild-type hosts but gave transformants in host strains with deleted or compromised chromosomal oriC at elevated DnaA concentrations. We conclude that these origins require more DnaA per origin for initiation than does wild-type oriC. Mutants in DnaA box R3 behaved essentially like wild-type oriC, except for those in which the low-affinity box R3 was replaced by the high-affinity box R1. Apparently, initiation is possible without DnaA binding to box R3, but high-affinity DnaA binding to DnaA box R3 upsets the regulation. Taken together, these results demonstrate that there are finely tuned DnaA binding requirements for each of the individual DnaA boxes for optimal build-up of the initiation complex and replication initiation in vivo.

Bacterial Proteins

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

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

Detection of K-ras mutations in stools of patients with colorectal cancer by mutant-enriched PCR.

Mutant-enriched PCR was applied to the detection of mutations at codons 12 and 13 of K-ras genes in the stools of patients with colorectal cancer. Mutations were analyzed in stool samples obtained prior to surgery. Resected tumor specimens were screened for K-ras mutations by PCR-mediated RFLP analysis. Using normal stool samples, assay conditions were adjusted to optimal sensitivity and specificity. The following specimens were included in the study: 16 stool samples corresponding to carcinomas in which K-ras mutations had been identified; 7 randomly selected stool samples corresponding to carcinomas which were negative for K-ras mutations; 1 stool sample from a patient with non-Hodgkin's lymphoma. In 13 of the 16 stool samples (81%) corresponding to tumors in which K-ras mutations had been identified previously, K-ras mutations were detected. In 2 of the 7 stool samples corresponding to tumors in which K-ras mutations had not been detected by previous PCR-mediated RFLP analysis, K-ras mutations were also present. Reanalyses of the tumors corresponding to these 2 positive stool samples by mutant-enriched PCR revealed a K-ras mutation in one of the tumors. The stool and tumor of the patient with non-Hodgkins lymphoma were negative for K-ras mutations. DNA sequence analysis revealed that, for each of the K-ras mutations identified in stool samples, identical base substitutions were present in the corresponding tumor tissue. The results indicate that tumor cells harboring K-ras mutations can be detected in the stools of patients with colorectal cancer by mutant-enriched PCR with high sensitivity and specificity. Because of the simplicity of the technique, it may be suitable for screening of stool samples for mutations of the K-ras gene.

Adult

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

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

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

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

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

[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