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PubMed1981

Molecular cloning of the SUF2 frameshift suppressor gene from Saccharomyces cerevisiae.

A genetic approach to the molecular cloning of frameshift suppressor genes from yeast is described. These suppressors act by suppressing +1 G:C base-pair insertion mutations in glycine or proline codons. The cloning regimen involves an indirect screen for yeast transformants which harbor a functional suppressor gene inserted into the autonomously replicating "shuttle" vector YEp13, followed by transfer of the hybrid plasmid from yeast into Escherichia coli. Using this procedure a 10.7-kb DNA fragment carrying the SUF2 frameshift suppressor gene has been isolated. This suppressor acts specifically on +1 G:C insertions in proline codons. When inserted into an integrative vehicle and reintroduced into yeast by transformation, this fragment integrates by homologous recombination in the region of the SUF2 locus on chromosome III. A large proportion of the fragment overlaps with another cloned DNA segment which carries the closely linked CDC10 gene. The SUF2 fragment carries at least two tRNA genes. The SUF2 gene and one of the tRNA genes are located on a 0.85-kb restriction fragment within the 10.7-kb segment. A method is also described for the isolation of DNA fragments carrying alternative alleles of the SUF2 locus. Using this procedure, the wild-type suf2+ allele has been cloned.

Alleles↗
PubMed1993

Ectopic expression of genes during chicken limb pattern formation using replication defective retroviral vectors.

A gene transfer method to ectopically express genes during chicken limb pattern formation using replication defective retroviral vectors has been established. Spherical non-proliferating (mitomycin C treated) aggregates of clonal retrovirus producing cells were grafted directly into developing chicken wing buds. The cell aggregates had to be placed in direct contact with the highly proliferative cells of the wing bud to promote efficient in vivo infection of embryonic cells by the released retroviral particles. The majority of grafts resulted in widespread expression of a reporter gene (encoding bacterial beta-galactosidase) during limb pattern formation and early limb bud outgrowth without affecting morphogenesis. This method provides a novel approach to study the effects of ectopic gene expression on limb pattern formation. Possible future applications to study other developmental processes are discussed.

Animals↗
PubMed1995

Intermediate filaments in disease.

Intermediate filaments are major structural proteins encoded by a large multigene family. Their tissue-specific expression makes them important in studies of development, differentiation and pathology. Most intermediate filaments are keratins; recent discoveries of keratin mutations in a range of genetic skin disorders have clarified their role as providing essential structural support for cells in different physical settings.

DNA↗
PubMed1991

Photosynthetic reaction centers: interfacing molecular genetics and optical spectroscopy.

In the elucidation of the mechanism by which certain photosynthetic bacteria convert light into chemical energy, genetics has become intertwined with biophysical techniques. While X-ray crystallography has yielded an atomic resolution structure of the photosynthetic reaction center (RC), optical spectroscopy remains the most important technique for screening mutants. Newly developed imaging devices and genetic techniques should enable biophysicists to characterize rapidly the spectra of extremely large numbers of RC and light harvesting (LH) antennae mutants. The intrinsic pigments of the RC and LH antennae act as spectroscopic reporters for assembly and function of these integral membrane proteins. To optimize this genetics/spectroscopy interface, new algorithms that relate the structure of the genetic code to the physico-chemical properties of the amino acids are being developed to design libraries of mutants.

Photosynthetic Reaction Center Complex Proteins↗
PubMed1992

Enter a new post-translational modification: D-amino acids in gene-encoded peptides.

The post-translational processing of peptides plays a key role in conferring biological activity on those peptides. Recently, ribosomally made peptides that contain D-amino acids at specific positions have been discovered in microorganisms as well as in vertebrates and invertebrates. This points to yet another strategy of circumventing stereochemical limitations imposed by the genetic code and conveying biological activity to otherwise inert molecules.

Amino Acid Sequence↗
PubMedDate not supplied

A model of motor control of the nematode C. elegans with neuronal circuits.

OBJECTIVE: Living organisms have mechanisms to adapt to various conditions of external environments. If we can realize these mechanisms on the computer, it may be possible to apply methods of biological and biomimetic adaptation to the engineering of artificial machines. This paper focuses on the nematode Caenorhabditis elegans (C. elegans), which has a relatively simple structure and is one of the most studied multicellular organisms. We aim to develop its computer model, artificial C. elegans, to analyze control mechanisms with respect to motion. Although C. elegans processes many kinds of external stimuli, we focused on gentle touch stimulation. METHODS: The proposed model consists of a neuronal circuit model for motor control that responds to gentle touch stimuli and a kinematic model of the body for movement. All parameters included in the neuronal circuit model are adjusted by using the real-coded genetic algorithm. Also, the neuronal oscillator model is employed in the body model to generate the sinusoidal movement. The motion velocity of the body model is controlled by the neuronal circuit model so as to correspond to the touch stimuli that are received in sensory neurons. CONCLUSION: The computer simulations confirmed that the proposed model is capable of realizing motor control similar to that of the actual organism qualitatively. By using the artificial organism it may be possible to clarify or predict some characteristics that cannot be measured in actual experiments. With the recent development of computer technology, such a computational analysis becomes a real possibility. The artificial C. elegans will contribute for studies in experimental biology in future, although it is still developing at present.

Algorithms↗
PubMed2005

Selenocysteine in proteins-properties and biotechnological use.

Selenocysteine (Sec), the 21st amino acid, exists naturally in all kingdoms of life as the defining entity of selenoproteins. Sec is a cysteine (Cys) residue analogue with a selenium-containing selenol group in place of the sulfur-containing thiol group in Cys. The selenium atom gives Sec quite different properties from Cys. The most obvious difference is the lower pK(a) of Sec, and Sec is also a stronger nucleophile than Cys. Proteins naturally containing Sec are often enzymes, employing the reactivity of the Sec residue during the catalytic cycle and therefore Sec is normally essential for their catalytic efficiencies. Other unique features of Sec, not shared by any of the other 20 common amino acids, derive from the atomic weight and chemical properties of selenium and the particular occurrence and properties of its stable and radioactive isotopes. Sec is, moreover, incorporated into proteins by an expansion of the genetic code as the translation of selenoproteins involves the decoding of a UGA codon, otherwise being a termination codon. In this review, we will describe the different unique properties of Sec and we will discuss the prerequisites for selenoprotein production as well as the possible use of Sec introduction into proteins for biotechnological applications. These include residue-specific radiolabeling with gamma or positron emitters, the use of Sec as a reactive handle for electophilic probes introducing fluorescence or other peptide conjugates, as the basis for affinity purification of recombinant proteins, the trapping of folding intermediates, improved phasing in X-ray crystallography, introduction of 77Se for NMR spectroscopy, or, finally, the analysis or tailoring of enzymatic reactions involving thiol or oxidoreductase (redox) selenolate chemistry.

Biotechnology↗
PubMed2006

Time left in the mouse.

Evidence suggests that the online combination of non-verbal magnitudes (durations, numerosities) is central to learning in both human and non-human animals [Gallistel, C.R., 1990. The Organization of Learning. MIT Press, Cambridge, MA]. The molecular basis of these computations, however, is an open question at this point. The current study provides the first direct test of temporal subtraction in a species in which the genetic code is available. In two experiments, mice were run in an adaptation of Gibbon and Church's [Gibbon, J., Church, R.M., 1981. Time left: linear versus logarithmic subjective time. J. Exp. Anal. Behav. 7, 87-107] time left paradigm in order to characterize typical responding in this task. Both experiments suggest that mice engaged in online subtraction of temporal values, although the generalization of a learned response rule to novel stimulus values resulted in slightly less systematic responding. Potential explanations for this pattern of results are discussed.

Animals↗
PubMed2004

Function of genetically encoded pyrrolysine in corrinoid-dependent methylamine methyltransferases.

Methanogenesis from trimethylamine, dimethylamine or monomethylamine is initiated by a series of corrinoid-dependent methyltransferases. The non-homologous genes encoding the full-length methyltransferases each possess an in-frame UAG (amber) codon that does not terminate translation. The amber codon is decoded by a dedicated tRNA, and corresponds to the novel amino acid pyrrolysine in one of the methyltransferases, indicating pyrrolysine to be the 22nd genetically encoded amino acid. Pyrrolysine has the structure of lysine with the (epsilon)N in amide linkage with a pyrroline ring. The reactivity of the electrophilic imine bond is the basis for the proposed function of pyrrolysine in activating and optimally orienting methylamine for methyl transfer to the cobalt ion of a cognate corrinoid protein. This reaction is essential for methane formation from methylamines, and may underlie the retention of pyrrolysine in the genetic code of methanogens.

Amides↗
PubMed2003

Encodamers: unnatural peptide oligomers encoded in RNA.

Conventional display libraries are generally limited to the 20 naturally occurring amino acids. Here, we demonstrate that novel unnatural amide-linked oligomers can be constructed and encoded in an attached RNA for the purpose of mRNA display library design. To do this, we translated templates of various lengths in a protein synthesis system modified to promote sense codon suppression. Unnatural residues were escorted to the ribosome as chemically acylated tRNAs added to the translation mixture. Our experiments reveal that unnatural peptide oligomers ("encodamers") consisting of an N-substituted amino acid are readily generated as mRNA-peptide fusions with excellent stepwise efficiency. The N-substituted polyamides have strikingly improved proteolytic stability relative to their naturally encoded counterparts. Overall, our work indicates that the ribosome can be used as a synthesis platform to generate encoded combinatorial chemistry outside the universal genetic code.

Base Sequence↗
PubMed2006

Replacement surgery with unnatural amino acids in the lock-and-key joint of glutathione transferase subunits.

Proteins contain amino acid residues essential to structure and function. Ribosomal protein synthesis is typically limited to the 20 amino acids of the genetic code, but posttranslational chemical modifications can greatly expand the diversity of side chain functionalities. In this investigation, a natural aromatic residue in the lock-and-key joint at the subunit interface of the dimeric glutathione transferase P1-1 was replaced by an S-alkylcysteine residue to give a functional enzyme. Introduction of Cys in the key position inactivates the enzyme, but subsequent alkylation of this residue enhances the catalytic efficiency up to 27,000-fold. Combinatorial modification of Cys by a mixture of reagents facilitated identification of an n-butyl group as the most efficient activator. Alkylation also enhanced binding affinity for active-site ligands and stabilized the enzyme against chemical denaturation and thermal inactivation.

Alkylation↗
PubMed2005

Reversed-phase liquid chromatography/mass spectrometry analysis of reduced monoclonal antibodies in pharmaceutics.

A reversed-phase LC/MS method was developed for reduced antibodies that provides efficient separation of light chain and two variants of heavy chain containing N-terminal glutamine and pyroglutamic acid. The best separation was achieved on Zorbax CN and Varian Pursuit DiPhenyl columns eluted with increasing percentage of n-propanol and acetonitrile in 0.1% trifluoroacetic acid. Although glutamine was genetically coded for the N-terminal residue of heavy chain of a monoclonal antibody used in this study, we found that most of it (70%) was converted to pyroglutamate during production. The conversion process continued in vitro and was monitored by the method. Deconvoluted electrospray ionization mass spectrum of the heavy chain revealed the glycosylation profile of a single N-linked sugar including a-, mono-, and di-galactosylated biantennary glycans and a 5-mannose sugar form.

Amino Acid Sequence↗
PubMed2005

Endocarditis caused by Propionibacterium acnes: an easily ignored pathogen.

Propionibacterium acnes, which usually considered as a contaminant, has been found to be an emerging pathogen in human diseases. We describe a case of prosthetic valve related endocarditis caused by P. acnes. Sequencing of the genetic coding of 16S ribosomal RNA was used to identify the pathogen and random amplified polymorphic DNA patterns further confirmed the persistent bacteraemia, which help to determine the diagnosis.

Anti-Bacterial Agents↗
PubMed2003

A "polyORFomic" analysis of prokaryote genomes using disabled-homology filtering reveals conserved but undiscovered short ORFs.

Prokaryote gene annotation is complicated by large numbers of short open reading frames (ORFs) that arise naturally from genetic code design. Historically, many hypothetical ORFs have been annotated as genes in microbes, usually with an arbitrary length threshold (e.g. greater than 100 codons). Given the use of such thresholds, what is the extent of genuine undiscovered short genes in the current sampling of prokaryote genomes? To assess rigorously the potential under-annotation of short ORFs with homology, we exhaustively compared the polyORFome--all possible ORFs in 64 prokaryotes (53 bacteria and 11 archaea) plus budding yeast--to itself and to all known proteins. The novelty of our analysis is that, firstly, sequence comparisons to/between both annotated and un-annotated ORFs are considered, and secondly a two-step disabled-homology filter is applied to set aside putative pseudogenes and spurious ORFs. We find that un-annotated homologous short ORFs (uhORFs) correspond to a small but non-negligible fraction of the annotated prokaryote proteomes (0.5-3.8%, depending on selection criteria). Moreover, the disabled-homology filter indicates that about a third of uhORFs correspond to putative pseudogenes or spurious ORFs. Our analysis shows that the use of annotation length thresholds is unnecessary, as there are manageable numbers of short ORF homologies conserved (without disablements) across microbial genomes. Data on uhORFs are available from http://pseudogene.org/polyo

Conserved Sequence↗
PubMed2004

Genomic determinants of protein folding thermodynamics in prokaryotic organisms.

Here we investigate how thermodynamic properties of orthologous proteins are influenced by the genomic environment in which they evolve. We performed a comparative computational study of 21 protein families in 73 prokaryotic species and obtained the following main results. (i) Protein stability with respect to the unfolded state and with respect to misfolding are anticorrelated. There appears to be a trade-off between these two properties, which cannot be optimized simultaneously. (ii) Folding thermodynamic parameters are strongly correlated with two genomic features, genome size and G+C composition. In particular, the normalized energy gap, an indicator of folding efficiency in statistical mechanical models of protein folding, is smaller in proteins of organisms with a small genome size and a compositional bias towards A+T. Such genomic features are characteristic for bacteria with an intracellular lifestyle. We interpret these correlations in light of mutation pressure and natural selection. A mutational bias toward A+T at the DNA level translates into a mutational bias toward more hydrophobic (and in general more interactive) proteins, a consequence of the structure of the genetic code. Increased hydrophobicity renders proteins more stable against unfolding but less stable against misfolding. Proteins with high hydrophobicity and low stability against misfolding occur in organisms with reduced genomes, like obligate intracellular bacteria. We argue that they are fixed because these organisms experience weaker purifying selection due to their small effective population sizes. This interpretation is supported by the observation of a high expression level of chaperones in these bacteria. Our results indicate that the mutational spectrum of a genome and the strength of selection significantly influence protein folding thermodynamics.

Archaea↗
PubMed2005

The impact of DNA damage, genetic mutation and cellular responses on cancer prevention, longevity and aging: observations in humans and mice.

Over the past 5 years, data collected from the mouse suggest that pathways important for either preventing or resolving DNA damage are longevity assurance mechanisms whose critical overall function is somatic cell maintenance, a necessary part of cancer prevention. These pathways include those that reduce DNA damage levels caused by exogenous sources, replication errors and by-products of cellular respiration. Unresolved DNA damage leads to permanent mutations in the genetic code that may be oncogenic. Therefore, pathways that resolve DNA damage are important anti-cancer mechanisms. As an important line of defense, there are a variety of pathways that repair DNA damage. In addition, there are anti-cancer pathways that respond to DNA damage by either preventing cellular replication or inducing cell death. Genes in these pathways, termed longevity assurance genes (LAG), code for proteins that reduce cancer incidence and as a result assures a sufficiently long health span needed for reproduction. Data from mouse models, many that were originally designed to study cancer, are showing that a potential consequence of DNA damage and responses to DNA damage is aging; these models support the hypothesis that at least some aspects of normal aging are the consequence of anticancer mechanisms designed to deal with damaged DNA.

Animals↗