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PubMedDate not supplied

[Rate limitations in the elongation working cycle and the action mechanisms of GTP-complexed elongation protein factors].

Kinetic aspects of the peptide chain elongation process, proper role and the working mechanisms of the GTP-complexed protein elongation factors are discussed. High rates of the codon-dependent binding of aminoacyl-tRNA and translocation are shown to need the mutually exclusive properties of the ribosomal A centre which in the absence of some additional events seems to be unable to possess simultaneously these properties. A centre of translating ribosome is postulated to have a character of dynamic structure providing unsimultaneous consecutive optimization of the aminoacyl-tRNA binding and translocation conditions in accordance with the principle "either binding or translocation". According to this suggestion the rate of elongation is limited by the rate of reversible changes of the A centre structure fitting into the scheme A in equilibrium with B. Each step of this scheme is specifically promoted by corresponding GTP-complexed protein factor. Thus, elongation factors are suggested to be specific modulators of the A centre affinity for the codon-appropriate tRNA and to play a role of complex ligands carrying out an allosteric regulation of the ribosomal functional activity.

Codon↗

PubMed1997

Further characterization of HLA homozygous typing cell lines at the LMP2 polymorphic codon 60 by an ARMS typing method.

LMP2 is a subunit of the 20S proteasome within the cellular cytosolic compartment that is thought to cleave proteins into approximately 9 amino acid long oligopeptides. It is hypothesized that changes in the low molecular mass protease (LMP) gene sequence may alter the activity or specificity in which the LMP genes cleave peptides. Currently, the typing method for LMP2 involves polymerase chain reaction (PCR), restriction enzyme digestion, and gel electrophoresis. To help reduce the cost and cumbersomeness of this method, a new typing method was adapted for the LMP2 gene. To establish this new amplification refractory mutation system (ARMS) typing method, primers have been defined, amplification conditions optimized, and control cell lines sequenced to validate testing parameters. Results are listed for selected 10th and 11th International Histocompatibility Workshop homozygous cell lines.

Cell Line↗

PubMed1997

Molecular genetic characterization of the L-lactate dehydrogenase gene (ldhL) of Lactobacillus helveticus and biochemical characterization of the enzyme.

The Lactobacillus helveticus L-(+)-lactate dehydrogenase (L-LDH) gene (ldhL) was isolated from a lambda library. The nucleotide sequence of the ldhL gene was determined and shown to have the capacity to encode a protein of 323 amino acids (35.3 kDa). The deduced sequence of the 35-kDa protein revealed a relatively high degree of identity with other lactobacillar L-LDHs. The highest identity (80.2%) was observed with the Lactobacillus casei L-LDH. The sizes and 5' end analyses of ldhL transcripts showed that the ldhL gene is a monocistronic transcriptional unit. The expression of ldhL, studied as a function of growth, revealed a high expression level at the logarithmic phase of growth. The ldhL gene is preceded by two putative -10 regions, but no corresponding -35 regions could be identified. By primer extension analysis, the ldhL transcripts were confirmed to be derived from the -10 region closest to the initiation codon. However, upstream of these regions additional putative -10/-35 regions could be found. The L-LDH was overexpressed in Escherichia coli and purified to homogeneity by two chromatographic steps. The purified L-LDH was shown to be a nonaliosteric enzyme, and amino acid residues involved in allosteric regulation were not conserved in L. helveticus L-LDH. However, a slight enhancement of enzyme activity was observed in the presence of fructose 1,6-diphosphate, particularly at neutral pH. A detailed enzymatic characterization of L-LDH was performed. The optimal reaction velocity was at pH 5.0, where the kinetic parameters K(m), and Kcat for pyruvate were 0.25 mM and 643 S-1, respectively.

Amino Acid Sequence↗

PubMed1996

Nucleotide substitution patterns can predict the requirements for drug-resistance of HIV-1 proteins.

The enzyme reverse transcriptase (RT) plays a fundamental role in the replication of the human immunodeficiency virus type 1 (HIV-1) and several antiviral agents that target this key enzyme have been developed. Unfortunately, treatment of patients with RT inhibitors results in the appearance of drug-resistant variants with specific mutations in the RT protein. We hypothesized that if "difficult' resistance mutations (e.g. transversions/double-hits) are consistently observed at certain positions, it is likely that "easier' nucleotide substitutions (transitions/single-hits) at that codon do not result in a drug-resistant and/or active RT enzyme. In this study, we examined codon changes involved in RT drug resistance against nucleoside and non-nucleoside inhibitors and listed all easier substitutions, which apparently were not selected, either due to reduced enzyme RT activity or lack of drug resistance. These predictions on the requirements for resistance were confirmed by published mutational data on RT variants. We also propose that differences in mutation type can explain the order of appearance of substitutions in case multiple amino acid changes are required for optimal fitness. Differences in mutation pattern have been reported for drug-resistant HIV-1 variants selected in tissue culture compared with variants found in treated patients. In contrast to the in vivo situation, a relatively small population size is handled in in vitro tissue culture systems and this may limit the chances of creating a resistance mutation. Indeed, inspection of the codon changes indicates that the in vitro culture system is more strongly biased towards the relatively easy nucleotide substitutions. These results suggest that the nucleotide substitution pattern can provide important information on RT drug resistance.

Anti-HIV Agents↗

PubMed1989

The extension reached by the minimization of the polarity distances during the evolution of the genetic code.

The level reached by the optimization of the polarity distances during the evolution of the genetic code was investigated. The results, although not conclusive, indicate that this optimization level is higher than the data reported in the literature. The results seem compatible with the reaching of an evolutionary minimum, with respect to the optimization of the polarity distances, by the genetic code during its formation.

Amino Acids↗

PubMed1994

Polymerase chain reaction-single strand conformation polymorphism analysis of the p53 gene in paraffin-embedded surgical material from human renal cell carcinomas.

p53 tumour suppressor gene mutations were studied in 118 renal cell carcinomas using paraffin-embedded surgical material. Optimal results were obtained with analysis of exon lengths between 150 and 200 base pairs for polymerase chain reaction. Single strand conformation polymorphism and sequencing analysis revealed only two point mutations (2/118, 2%): one involving codon 135; TGC-->TTC (cysteine-->phenylalanine) and the other codon 175; CGC-->CAC (arginine-->histidine). Both of these cases were classified as granular cell subtype on microscopic observation. The data suggest that the p53 tumour suppressor gene is not related to tumour initiation, promotion, or progression of renal cell carcinomas. However, there is the possibility that granular cell type carcinomas may have a different genetic background from clear cell type renal neoplasms.

Base Sequence↗

PubMed1989

Effect of spermine on the efficiency and fidelity of the codon-specific binding of tRNA to the ribosomes.

Binding of the yeast Tyr-tRNA and Phe-tRNA to the A site, and the binding of their acetyl derivatives to the P site of poly(U11,A)-programmed Escherichia coli ribosomes was studied. Spermine stimulated the rate of binding of both tRNAs at least threefold, enabling more than 90% final saturation of both ribosomal binding sites. The effect is observed when the tRNAs, but not ribosomes or poly(U11,A), are preincubated with polyamine. Regardless of the binding site, optimal saturation was reached at spermine/tRNA molar ratios of 3 for tRNA(Phe) and 5 for tRNA(Tyr). The same low spermine/tRNA ratios were previously reported to stabilize the conformation of these tRNAs in solution. On the other hand, the messenger-free, EF-Tu- and EF-G-dependent polymerization of lysine from E. coli Lys-tRNA is drastically reduced, while the poly(A)-directed polymerization is stimulated by spermine through a wide range of Mg2+ concentrations. Misreading of UUU codons as isoleucine, assayed by the A-site binding of E. coli Ile-tRNA, is also inhibited by spermine. All these results demonstrate that spermine increases the efficiency and accuracy of a series of macromolecular interactions leading to the correct incorporation of an amino acid into protein, at the same time preventing some unspecific or erroneous interactions. From the analogy with its known structural effects, it can be inferred that spermine does so by conferring on the tRNA a specific biologically functional conformation.

Binding Sites↗

PubMed1988

Physical map and polyhedrin gene sequence of Lymantria dispar nuclear polyhedrosis virus.

Restriction maps of the 166.6-kb genome of Lymantria dispar multiply-enveloped nuclear polyhedrosis virus (LdMNPV clone g) were constructed for BamHI, BglII, EcoRI, EcoRV, HindIII and KpnI, using cosmid pVK102 and pBluescript vectors. Southern hybridizations indicated that the LdMNPV genome contains five dispersed regions of intragenomic sequence homology. The polyhedrin gene of LdMNPV was located within BglII-E and the sequence of the 735-nucleotide (nt) coding region and 678 nt of flanking DNA was determined. A conserved 14-nt sequence, associated with transcriptional start points in other polyhedrins, was identified at 44 to 57 nt upstream from the start codon. The deduced polyhedrin amino acid (aa) sequence showed a high degree of homology with a previously determined protein sequence for LdMNPV polyhedrin (89%) and with deduced amino acid sequences for three other MNPV polyhedrins (74%). Optimal alignment of the four sequences indicated that LdMNPV polyhedrin possesses a single aa insertion at residue 4 and a single aa deletion at residue 164.

Amino Acid Sequence↗

PubMed1994

Optimal infectivity in vitro of human immunodeficiency virus type 1 requires an intact nef gene.

The replication competence of human immunodeficiency virus type 1 genomes containing mutations in the nef open reading frame was evaluated in continuous cell lines. Mutants that contained a deletion in the nef open reading frame, premature termination codons, or missense mutations in the N-terminal myristoylation signal were constructed. The replication of these mutants was tested in three ways. First, plasmid genomes were used to transfect T-lymphoblastoid cells. Second, low-passage posttransfection supernatants were used to infect cells with a relatively low virus input. Third, high-titer virus stocks were used to infect cells with a relatively high virus input. These experiments demonstrated a 100- to 10,000-fold decrement in p24 production by the nef mutants compared with that by the wild-type virus. The greatest difference was obtained after infection with the lowest virus input. The myristoylation signal was critical for this positive effect of nef. To investigate the mechanism of the positive influence of nef, nef-positive and nef-minus viruses were compared during a single cycle of replication. These single-cycle experiments were initiated both by infection with high-titer virus stocks and by transfection with viral DNA. Single-cycle infection yielded a three- to fivefold decrement in p24 production by nef-minus virus. Single-cycle transfection yielded equal amounts of p24 production. These results implied that nef does not affect replication after the provirus is established. In support of these results, viral production from cells chronically infected with nef-positive or nef-minus viruses was similar over time. To determine whether the effect of nef was due to infectivity, end point titrations of nef-positive and nef-minus viruses were performed. nef-positive virus had a greater infectivity per picogram of HIV p24 antigen than nef-minus virus. These data indicated that the positive influence of nef on viral growth rate is due to an infectivity advantage of virus produced with an intact nef gene.

Amino Acid Sequence↗

PubMed1995

High expression of synthetic human interferon-gamma cDNA in E. coli.

Human interferon-gamma (IFN-gamma) cDNA was synthesized, and it makes the usage of favorable codons in E. coli. The authors got 9 different expression plasmids which contain the synthetic IFN-gamma-cDNA and have different spaces between SD sequence and ATG. The free energies G0f298 in the formation of stable secondary structure in the translation initiation region (TIR) are different in various expression plasmids. One of them, pLY4-gamma 5, can highly yield INF-gamma which will be about 60%-80% of the total bacterial proteins, such a high expression was hardly noted in literature. The reasons of high expression in this work are optimal spaces between SD and ATC, favorable delta G0f298, favourable condons usage for E. coli.

Amino Acid Sequence↗

PubMed1999

Overexpression of salt-tolerant glutaminase from Micrococcus luteus K-3 in Escherichia coli and its purification.

A high-expression plasmid, pKSGHE3-1, containing the salt-tolerant glutaminase (EC 3.5.1.2) from marine bacterium Micrococcus luteus K-3 was constructed. pKSGHE3-1 was made by inserting the DNA fragment (1.43 kb) containing the structural gene synthesized by polymerase chain reaction into the downstream region of the tac promoter of expression vector pKK223-3. The translational start codon was located 10 bases downstream of the Shine-Dalgarno sequence (AGGA) of pKK223-3. Escherichia coli JM109 transformed with pKSGHE3-1 exhibited more than 190-fold higher glutaminase activity than M. luteus K-3 under optimal culture conditions. The enzyme was purified to homogeneity through three column chromatography steps with a final yield of 17.1%. The recombinant enzyme showed the same enzymatic properties, including salt tolerance, as those of M. luteus K-3. This glutaminase expression system allows the production of sufficient quantities of glutaminase for basic structure-function studies including chemical modification and future X-ray crystallization analysis.

Bacterial Proteins↗

PubMed1997

Green fluorescent protein expressed by recombinant pseudorabies virus as an in vivo marker for viral replication.

We isolated and characterized a pseudorabies virus (PrV) mutant expressing an engineered green fluorescent protein (GFP) optimized for expression in human cells. The GFP DNA was inserted in the non-essential glycoprotein G (gG) gene of the attenuated PrV strain Bartha. The coding sequence was cloned in frame behind the first seven codons of the gG gene under control of the strong gG promotor. On excitation with blue light, live cells infected with the recombinant PrV B80eGFP exhibited bright fluorescence when examined microscopically using filters for FITC fluorescence. In fixed samples detection sensitivity was increased by immunofluorescence using an anti-GFP antibody. Specifically labelled PrV mutants have been used successfully as transsynaptic circuit tracers for definition of central command neurons in the brain (Jansen et al., 1995. Central command neurons of the sympathetic nervous system: basis of the fight-or-flight response. Science 270, 644-646). Availability of this recombinant allows the study of even more complex interactions using differentially labelled PrV mutants, and provides a means to monitor viral replication and spread without destruction of the cell.

Animals↗

PubMed2025

Optimized genomic editing of a common Duchenne muscular dystrophy mutation in patient-derived muscle cells and a new humanized mouse model.

Duchenne muscular dystrophy (DMD) is a fatal X-linked, recessive disease caused by mutations in the DMD gene encoding dystrophin, a membrane-associated protein necessary for maintaining muscle structure and function. One of the common DMD mutations is the deletion of exon 52 (Δ52), which introduces a premature stop codon in exon 53, preventing the expression of functional dystrophin protein. Patients with this mutation could benefit from skipping or reframing exon 53 to restore the dystrophin open reading frame. In this study, we investigated the efficacy of single-cut CRISPR gene editing with Staphylococcus pyogenes Cas9 (SpCas9)-LRVQR to restore dystrophin expression in patient-derived induced pluripotent stem cells (iPSCs) and a newly generated humanized DMD mouse model. We compared two injection routes for adeno-associated virus (AAV) serotype 9 to deliver gene-editing components to neonatal mice: intraperitoneal (IP) and facial vein (FV) injection. We observed efficient restoration of dystrophin protein expression across multiple skeletal muscle groups and the heart. The AAV9-mediated CRISPR single-cut approach ameliorated key DMD hallmarks, including histopathological phenotypes, impaired grip strength, and elevated serum creatine kinase levels. Our optimized strategies for dystrophin restoration in humanized DMD mice with exon 52 deletion represent a promising treatment for DMD.

AAV↗

PubMed1985

Optimizing the expression in E. coli of a synthetic gene encoding somatomedin-C (IGF-I).

Double-stranded DNA encoding the human hormone somatomedin-C (SMC) has been synthesized. This synthetic gene has been inserted into a plasmid bearing the strong leftward promoter (PL) of bacteriophage lambda and expressed in E. coli. Codons for the N-terminal region of SMC which maximized the hormone's synthesis were chosen in an SMC-lac z fusion assay. The amounts of SMC accumulated in E. coli were influenced by mutations at two chromosomal loci, lon and htpR.

ATP-Dependent Proteases↗

PubMed1996

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↗

PubMed1996

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↗

PubMed1994

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↗

PubMedDate not supplied

[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↗