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PubMed1998

Relocation of urf a from the mitochondrion to the nucleus cures the mitochondrial mutator phenotype in the fission yeast Schizosaccharomyces pombe.

In previous papers we have reported the characterisation of mitochondrial mutator mutants of Schizosaccharomzyces pombe. In contrast to nuclear mutator mutants known from other eucaryotes, this mutator phenotype correlates with mutations in an unassigned open reading frame (urf a) in the mitochondrial genome. Since an efficient biolistic transformation system for fission yeast mitochondria is not yet available, we relocated the mitochondrial urf a gene to the nucleus. As host strain for the ectopic expression, we used the nonsense mutant ana(r)-6, which carries a premature stop codon in the urf a gene. The phenotype of this mutant is characterised by continuous segregation of progeny giving rise to fully respiration competent colonies, colonies that show moderate growth on glycerol and a fraction of colonies that are unable to grow on glycerol. The phenotype of this mutant provides an excellent tool with which to study the effects on the mutator phenotype of ectopic expression of the urf a gene. Since a UGA codon encoding tryptophan is present in the original mitochondrial gene, we constructed two types of expression cassettes containing either the mitochondrial version of the urf a gene (mt-urf a) or a standard genetic code version (nc-urf a; UGA replaced by UGG) fused to the N-terminal import leader sequence of the cox4 gene of Saccharomyces cerevisiae. We show that the expression of the mt-urf a gene in its new location is able to cure, at least in part, the phenotype of mutant ana(r)-6, whereas the expression of the nc-urf a gene completely restores the wild-type (non-mutator) phenotype. The significant similarity of the urf a gene to the mitochondrial var1 gene of S. cerevisiae and homologous genes in other yeasts suggests that the urf a gene product might be a ribosomal protein with a dual function in protein synthesis and maintenance of mitochondrial DNA integrity.

Amino Acid Sequence↗
PubMed1999

The mitochondrial genome: structure, transcription, translation and replication.

Mitochondria play a central role in cellular energy provision. The organelles contain their own genome with a modified genetic code. The mammalian mitochondrial genome is transmitted exclusively through the female germ line. The human mitochondrial DNA (mtDNA) is a double-stranded, circular molecule of 16569 bp and contains 37 genes coding for two rRNAs, 22 tRNAs and 13 polypeptides. The mtDNA-encoded polypeptides are all subunits of enzyme complexes of the oxidative phosphorylation system. Mitochondria are not self-supporting entities but rely heavily for their functions on imported nuclear gene products. The basic mechanisms of mitochondrial gene expression have been solved. Cis-acting mtDNA sequences have been characterised by sequence comparisons, mapping studies and mutation analysis both in vitro and in patients harbouring mtDNA mutations. Characterisation of trans-acting factors has proven more difficult but several key enzymes involved in mtDNA replication, transcription and protein synthesis have now been biochemically identified and some have been cloned. These studies revealed that, although some factors may have an additional function elsewhere in the cell, most are unique to mitochondria. It is expected that cell cultures of patients with mitochondrial diseases will increasingly be used to address fundamental questions about mtDNA expression.

Cells, Cultured↗
PubMed2002

An unnatural base pair for incorporating amino acid analogs into proteins.

An unnatural base pair of 2-amino-6-(2-thienyl)purine (denoted by s) and pyridin-2-one (denoted by y) was developed to expand the genetic code. The ribonucleoside triphosphate of y was site-specifically incorporated into RNA, opposite s in a template, by T7 RNA polymerase. This transcription was coupled with translation in an Escherichia coli cell-free system. The yAG codon in the transcribed ras mRNA was recognized by the CUs anticodon of a yeast tyrosine transfer RNA (tRNA) variant, which had been enzymatically aminoacylated with an unnatural amino acid, 3-chlorotyrosine. Site-specific incorporation of 3-chlorotyrosine into the Ras protein was demonstrated by liquid chromatography-mass spectrometry (LC-MS) analysis of the products. This coupled transcription-translation system will permit the efficient synthesis of proteins with a tyrosine analog at the desired position.

Amino Acid Sequence↗
PubMed2005

Intracellular protein degradation: from a vague idea thru the lysosome and the ubiquitin-proteasome system and onto human diseases and drug targeting.

Between the 1950s and 1980s, scientists were focusing mostly on how the genetic code is transcribed to RNA and translated to proteins, but how proteins are degraded has remained a neglected research area. With the discovery of the lysosome by Christian de Duve, it was assumed that cellular proteins are degraded within this organelle. Yet, several independent lines of experimental evidence strongly suggested that intracellular proteolysis is largely nonlysosomal, but the mechanisms involved remained obscure. The discovery of the ubiquitin-proteasome system resolved the enigma. We now recognize that degradation of intracellular proteins is involved in regulation of a broad array of cellular processes, such as cell cycle and division, regulation of transcription factors, and assurance of the cellular quality control. Not surprisingly, aberrations in the system have been implicated in the pathogenesis of human disease, such as malignancies and neurodegenerative disorders, which led subsequently to an increasing effort to develop mechanism-based drugs.

Adenosine Triphosphate↗
PubMed2001

A dependent-rates model and an MCMC-based methodology for the maximum-likelihood analysis of sequences with overlapping reading frames.

We present a model and methodology for the maximum-likelihood analysis of pairwise alignments of DNA sequences in which two genes are encoded in overlapping reading frames. In the model for the substitution process, the instantaneous rates of substitution are allowed to depend on the nucleotides occupying the sites in a neighborhood of the site subject to substitution at the instant of the substitution. By defining the neighborhood of a site to extend over all sites in the codons in both reading frames to which a site belongs, constraints imposed by the genetic code in both reading frames can be taken into account. Due to the dependency of the instantaneous rates of substitution on the states at neighboring sites, the transition probability between sequences does not factorize and therefore cannot be obtained directly. We present a Markov chain Monte Carlo procedure for obtaining the ratio of two transition probabilities between two sequences under the model considered, and we describe how maximum-likelihood parameter estimation and likelihood ratio tests can be performed using the procedure. We describe how the expected numbers of different types of substitutions in the shared history of two sequences can be calculated, and we use the described model and methodology in an analysis of a pairwise alignment of two hepatitis B sequences in which two genes are encoded in overlapping frames. Finally, we present an extended model, together with a simpler approximate estimation procedure, and use this to test the adequacy of the former model.

Base Sequence↗
PubMed1989

Human insulin: DNA technology's first drug.

The history, biologic activity, and immunogenicity of human insulin are described. Recombinant human insulin first entered clinical trials in humans in 1980. At that time, the A and B chains of the insulin molecule were produced separately and then combined by chemical techniques. Since 1986, a different recombinant process has been used. The human genetic coding for proinsulin is inserted into Escherichia coli cells, which are then grown by fermentation to produce proinsulin. The connecting peptide is cleaved enzymatically from proinsulin to produce human insulin. Studies indicate that there are no important differences between pork insulin and human insulin in terms of therapeutic efficacy and disposition after intravenous administration. Recombinant human insulin has a faster onset of action and lower immunogenicity than pork or beef insulin. Diabetic patients may have an improvement in glucose concentrations when their therapy is switched from animal-source insulin to human insulin. Such a change usually requires a dosage adjustment, which must be determined by a physician. Pharmacists are responsible for educating patients concerning all insulin products and for preventing patients from interchanging insulin products. The availability of human insulin as the first pharmaceutical product manufactured through recombinant DNA technology, however, has had little effect on the pharmacist's role in the care of such patients. The production of human insulin through recombinant DNA technology represents an important advance in the treatment of patients with diabetes.

Genetic Engineering↗
PubMed2002

Rescue of a mitochondrial deficiency causing Leber Hereditary Optic Neuropathy.

A G to A transition at nucleotide 11778 in the ND4 subunit gene of complex I was the first point mutation in the mitochondrial genome linked to a human disease. It causes Leber Hereditary Optic Neuropathy, a disorder with oxidative phosphorylation deficiency. To overcome this defect, we made a synthetic ND4 subunit compatible with the "universal" genetic code and imported it into mitochondria by adding a mitochondrial targeting sequence. For detection we added a FLAG tag. This gene was inserted in an adeno-associated viral vector. The ND4FLAG protein was imported into the mitochondria of cybrids harboring the G11778A mutation, where it increased their survival rate threefold, under restrictive conditions that forced the cells to rely predominantly on oxidative phosphorylation to produce ATP. Since assays of complex I activity were normal in G11778A cybrids we focused on changes in ATP synthesis using complex I substrates. The G11778A cybrids showed a 60% reduction in the rate of ATP synthesis. Relative to mock-transfected G11778A cybrids, complemented G11778A cybrids showed a threefold increase in ATP synthesis, to a level indistinguishable from that in cybrids containing normal mitochondrial DNA. Restoration of respiration by allotopic expression opens the door for gene therapy of Leber Hereditary Optic Neuropathy.

Adenosine Triphosphate↗
PubMed2004

Epigenetic reprogramming during early development in mammals.

Epigenetic modifications serve as an extension of the information content by which the underlying genetic code may be interpreted. These modifications mark genomic regions and act as heritable and stable instructions for the specification of chromatin organisation and structure that dictate transcriptional states. In mammals, DNA methylation and the modification of histones account for the major epigenetic alterations. Two cycles of DNA methylation reprogramming have been characterised. During germ cell development, epigenetic reprogramming of DNA methylation resets parent-of-origin based genomic imprints and restores totipotency to gametes. On fertilisation, the second cycle is triggered resulting in an asymmetric difference between parental genomes. Further epigenetic asymmetry is evident in the establishment of the first two lineages at the blastocyst stage. This differentiative event sets the epigenetic characteristics of the lineages as derivatives of the inner cell mass (somatic) and trophectoderm (extra-embryonic). It is the erasure and subsequent re-tracing of the epigenetic checkpoints that pose the most serious obstacles to somatic nuclear transfer. Elaboration of the mechanisms of these interactions will be invaluable in our fundamental understanding of biological processes and in achieving substantial therapeutic advances.

Animals↗
PubMed1991

Catalytic function of DNA topoisomerase II.

Although the genetic code is defined by a linear array of nucleotides, it is the three-dimensional structure of the double helix that regulates most of its cellular functions. Over the past two decades, it has become increasingly clear that aspects of this three-dimensionality which reflect topological relationships within the double helix (i.e., superhelical twisting, knotting, or tangling) influence virtually every facet of nucleic acid physiology. In vivo, DNA topology is modulated by ubiquitous enzymes known as topoisomerases. The type II enzyme is essential to the eukaryotic cell and is required for unlinking daughter chromosomes and maintaining chromosome structure. Moreover, topoisomerase II also has been identified as the primary cellular target for several widely used antineoplastic drugs. Before the physiological functions of topoisomerase II can be effectively dissected or its drug interactions fully exploited, it is imperative to understand the mechanism by which this important enzyme carries out its catalytic cycle.

Adenosine Triphosphate↗
PubMed2003

X-ray crystal structures of the WT and a hyper-accurate ribosome from Escherichia coli.

Protein biosynthesis on the ribosome requires accurate reading of the genetic code in mRNA. Two conformational rearrangements in the small ribosomal subunit, a closing of the head and body around the incoming tRNA and an RNA helical switch near the mRNA decoding site, have been proposed to select for complementary base-pairing between mRNA codons and tRNA anticodons. We determined x-ray crystal structures of the WT and a hyper-accurate variant of the Escherichia coli ribosome at resolutions of 10 and 9 A, respectively, revealing that formation of the intact 70S ribosome from its two subunits closes the conformation of the head of the small subunit independent of mRNA decoding. Moreover, no change in the conformation of the switch helix is observed in two steps of tRNA discrimination. These 70S ribosome structures indicate that mRNA decoding is coupled primarily to movement of the small subunit body, consistent with previous proposals, whereas closing of the head and the helical switch may function in other steps of protein synthesis.

Base Sequence↗
PubMed2007

Are transversion mutations better? A Mutagenesis Assistant Program analysis on P450 BM-3 heme domain.

Directed evolution represents a versatile tool to tailor enzyme properties to needs in industrial applications and to understand structure-function relationships. Genetic diversity is commonly generated using error-prone PCR. Exploration of sequence space by random mutagenesis strongly favors transitions when enzyme-based mutagenesis methods are employed (Wong, T. S., Zhurina, D., Schwaneberg, U., Comb. Chem. High Throughput Screen. 2006, 9, 271-288). The genetic code has been organized in a manner that limits chemical diversity when a single transition mutation occurs in a codon (Wong, T. S., Roccatano, D., Schwaneberg, U., Biocatal. Biotransformation 2006, in press). Are transitions more beneficial than transversions for adapting biocatalysts to non-natural process conditions? In a statistical analysis performed with the Mutagenesis Assistant Program (MAP), we compared the consequences of transition and transversion bias on amino acid substitution patterns of the P450 BM-3 heme domain. For the analysis, we used a recently introduced benchmarking system consisting of a protein structure indicator, an amino acid diversity indicator with a codon diversity coefficient, and a chemical diversity indicator. A detailed analysis for the P450 BM-3 heme domain showed that an ideal transversion bias generates more diverse amino acid substitution patterns with a significantly different chemical composition than an ideal transition bias. Emphasis is given on the theoretical analysis with a brief discussion on potential implication of transition and transversion bias in directed evolution experiments.

Algorithms↗
PubMed2001

Bubble propagation in a helicoidal molecular chain.

We study the propagation of very large amplitude localized excitations in a model of DNA that takes explicitly into account the helicoidal structure. These excitations represent the "transcription bubble," where the hydrogen bonds between complementary bases are disrupted, allowing access to the genetic code. We propose these kinds of excitations in alternative to kinks and breathers. The model has been introduced by Barbi et al. [Phys. Lett. A 253, 358 (1999)], and up to now it has been used to study on the one hand low amplitude breather solutions, and on the other hand the DNA melting transition. We extend the model to include the case of heterogeneous chains, in order to get closer to a description of real DNA; in fact, the Morse potential representing the interaction between complementary bases has two possible depths, one for A-T and one for G-C base pairs. We first compute the equilibrium configurations of a chain with a degree of uncoiling, and we find that a static bubble is among them; then we show, by molecular dynamics simulations, that these bubbles, once generated, can move along the chain. We find that also in the most unfavorable case, that of a heterogeneous DNA in the presence of thermal noise, the excitation can travel for well more than 1000 base pairs.

Base Composition↗
PubMed2005

SiteFind: a software tool for introducing a restriction site as a marker for successful site-directed mutagenesis.

BACKGROUND: Site-directed mutagenesis is a widely-used technique for introducing mutations into a particular DNA sequence, often with the goal of creating a point mutation in the corresponding amino acid sequence but otherwise leaving the overall sequence undisturbed. However, this method provides no means for verifying its success other than sequencing the putative mutant construct: This can quickly become an expensive method for screening for successful mutations. An alternative to sequencing is to simultaneously introduce a restriction site near the point mutation in manner such that the restriction site has no effect on the translated amino acid sequence. Thus, the novel restriction site can be used as a marker for successful mutation which can be quickly and easily assessed. However, finding a restriction site that does not disturb the corresponding amino acid sequence is a time-consuming task even for experienced researchers. A fast and easy to use computer program is needed for this task. RESULTS: We wrote a computer program, called SiteFind, to help us design a restriction site within the mutation primers without changing the peptide sequence. Because of the redundancy of genetic code, a given peptide can be encoded by many different DNA sequences. Since the list of possible restriction sites for a given DNA sequence is not always obvious, SiteFind automates this task. The number of possible sequences a computer program must search through increases exponentially as the sequence length increases. SiteFind uses a novel "moving window" algorithm to reduce the number of possible sequences to be searched to a manageable level. The user enters a nucleotide sequence, specifies what amino acid residues should be changed in the mutation, and SiteFind generates a list of possible restriction sites and what nucleotides must be changed to introduce that site. As a demonstration of its use, we successfully generated a single point mutation and a double point mutation in the wild-type sequence for Krüppel-like factor 4, an epithelium-specific transcription factor. CONCLUSION: SiteFind is an intuitive, web-based program that enables the user to introduce a novel restriction site into the mutated nucleotide sequence for use as a marker of successful mutation. It is freely available from http://www.utmb.edu/scccb/software/sitefind.html.

Algorithms↗
PubMed2005

Loss of editing activity during the evolution of mitochondrial phenylalanyl-tRNA synthetase.

Accurate selection of amino acids is essential for faithful translation of the genetic code. Errors during amino acid selection are usually corrected by the editing activity of aminoacyl-tRNA synthetases such as phenylalanyl-tRNA synthetases (PheRS), which edit misactivated tyrosine. Comparison of cytosolic and mitochondrial PheRS from the yeast Saccharomyces cerevisiae suggested that the organellar protein might lack the editing activity. Yeast cytosolic PheRS was found to contain an editing site, which upon disruption abolished both cis and trans editing of Tyr-tRNA(Phe). Wild-type mitochondrial PheRS lacked cis and trans editing and could synthesize Tyr-tRNA(Phe), an activity enhanced in active site variants with improved tyrosine recognition. Possible trans editing was investigated in isolated mitochondrial extracts, but no such activity was detected. These data indicate that the mitochondrial protein synthesis machinery lacks the tyrosine proofreading activity characteristic of cytosolic translation. This difference between the mitochondria and the cytosol suggests that either organellar protein synthesis quality control is focused on another step or that translation in this compartment is inherently less accurate than in the cytosol.

Adenosine Triphosphate↗
PubMed2005

Untangling the effects of codon mutation and amino acid exchangeability.

Determining the relative contributions of mutation and selection to evolutionary change is a matter of great practical and theoretical significance. In this paper, we examine relative contributions of codon mutation rates and amino acid exchangeability on the frequencies of each type of amino acid difference in alignments of distantly related proteins, alignments of closely related proteins, and among human SNPs, using a model that incorporates prior estimates of mutation and exchangeability parameters. For the operational exchangeability of amino acids in proteins, we use EX, a measure of protein-level effects from a recent statistical meta-analysis of nearly 10,000 experimental amino acid exchanges. EX is both free of mutational effects and more powerful than commonly used "biochemical distance" measures (1). For distant protein relationships, mutational effects (genetic code, transition/transversion bias) and operational exchangeability (EX) account for roughly equal portions of variance in off-diagonal values, the complete model accounting for R2 = 0.35 of the variance. For human/chimpanzee alignments representing closely related proteins relationships, mutational effects (including CpG bias) account for 0.52 of the variance; adding EX to the model increases this to 0.67. For natural variation in human proteins, the variance explained by mutational effects alone, and by mutational effects and operational exchangeability are, respectively, 0.66 and 0.70 for SNPs in HGVBase, and 0.56 and 0.60 for disease-causing missense variants in HGMD. Thus, exchangeability has a stronger relative effect for distant protein evolution than for the cases of closely related proteins or of population variation. A more detailed model for the hominid data suggests that 1) there is a threshold in EX below which substitutions are highly unlikely to be accepted, corresponding to roughly 30 % relative protein activity; 2) selection against missense mutants is a slightly convex function of protein activity, not changing much as long as protein activity is low; and 3) the probability of disease-causing effects decreases nearly linearly with EX.

Amino Acid Substitution↗
PubMed2003

Nucleic acid-based techniques for post-transcriptional regulation of molecular targets.

PURPOSE OF REVIEW: Messenger RNA, transfer RNA and ribosomal RNA were defined long ago as essential components for transmission of genetic code from DNA. However, there are many other, less commonly recognized RNAs, such as ribozymes and small interfering RNAs, which are distinguished by their ability to inhibit RNA function. This review describes the basic molecular concepts and potential therapeutic applications of RNA inhibition by a variety of molecules, including ribozymes, antisense oligonucleotides, aptamers and small interfering RNAs. RECENT FINDINGS: A tremendous amount of data has recently emerged about double-stranded small interfering RNAs, which bind and degrade corresponding messenger RNAs by a process called RNA interference. Though native small interfering RNAs have been shown to be biologically relevant in animals and plants, synthetic types have rapidly become powerful tools for post-transcriptional inhibition of specific gene products to determine functional consequences in simple organisms and in-vitro model systems. More established means of RNA inhibition, such as with ribozyme and antisense strategies, continue to be viable options for in-vitro experiments, and form the basis for many ongoing clinical trials. SUMMARY: Ribozymes, antisense oligonucleotides, aptamers and small interfering RNAs are potentially useful reagents for in-vitro investigation and for treatment of kidney and hypertension diseases.

Animals↗
PubMed2006

Bursts of nonsynonymous substitutions in HIV-1 evolution reveal instances of positive selection at conservative protein sites.

The fixation of a new allele can be driven by Darwinian positive selection or can be due to random genetic drift. Identifying instances of positive selection is a difficult task, because its impact is routinely obscured by the action of negative selection. The nature of the genetic code dictates that positive selection in favor of an amino acid replacement should often cause a burst of two or three nucleotide substitutions at a single codon site, because a large fraction of amino acid replacements cannot be achieved after just one nucleotide substitution. Here, we study pairs of successive nonsynonymous substitutions at one codon in the course of evolution of HIV-1 genes within HIV-1 populations inhabiting infected individuals. Such pairs are more numerous and more clumped than expected if different substitutions were independent and than what is observed for pairs of successive synonymous substitutions. Bursts of nonsynonymous substitutions in HIV-1 evolution cannot be explained by mutational biases and must, therefore, be due to positive selection. Both reversals, exact or imprecise, of fixed deleterious mutations and acquisitions of amino acids with new properties are responsible for the bursts. Temporal clumping is strongest at codon sites with a low overall rate of nonsynonymous evolution, implying that a substantial fraction of replacements of conservative amino acids are driven by positive selection. We identified many conservative sites of HIV-1 proteins that occasionally experience positive selection.

Amino Acid Substitution↗
PubMed1997

A sequence-specific polymerase chain reaction assay for mitochondrial DNA polymorphisms in human platelets and white cells.

BACKGROUND: Because mitochondria are abundant in white cells and are also present in platelets, polymorphic sequences in mitochondrial DNA (mtDNA) represent a unique target for polymerase chain reaction (PCR)-based detection of donor material. STUDY DESIGN AND METHODS: A PCR assay was developed that uses sequence-specific primers (SSP) focused on two continent-specific mtDNA polymorphisms. Results were validated by the use of informative restriction endonucleases. Three commercially available methods to extract mtDNA from white cell-reduced human platelets was compared. In preparation for in vivo studies, in vitro mixing studies designed to mimic transfusion were conducted to investigate the performance of the SSP-PCR assay. RESULTS: The gene sequences of two representative examples of amplicons obtained with the new SSP-PCR matched the sequence expected from the published genetic code. Fifteen individuals were classified as either positive (n = 6) or negative (n = 9) for the Asian polymorphism by the use of published primers known to flank the polymorphic site followed by digestion with appropriate restriction enzymes. Results with SSP-PCR were nearly perfectly concordant with those of restriction enzyme analysis. Although the use of three DNA extraction methods allowed the preparation of mtDNA that was suitable for PCR, large and consistent differences (ranging from 10- to 1000-fold) in endpoint sensitivity were found. In vitro mixing studies reproducibly documented that the SSP-PCR assay could detect as little as 1 percent of donor platelets mixed with recipient blood. CONCLUSION: PCR-SSP can be reliably used to identify human mtDNA polymorphisms. By optimization of the method of mtDNA extraction, the sensitivity of PCR-SSP assay was greatly increased. This assay should prove useful in investigations of allogeneic platelet transfusions without cell labeling. It may also be applied to studies of the donor cell microchimerism that follows transfusion or transplantation.

Asian People↗