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PubMed2003

Expression of Barstar as a selectable marker in yeast mitochondria.

We describe a new and potentially universal selection system for mitochondrial transformation based on bacterial genes, and demonstrate its feasibility in Saccharomyces cerevisiae. We first found that cytoplasmically synthesized Barnase, an RNase, interferes with mitochondrial gene expression when targeted to the organelle, without causing lethality when expressed at appropriate levels. Next, we synthesized a gene that uses the yeast mitochondrial genetic code to direct the synthesis of the specific Barnase inhibitor Barstar, and demonstrated that expression of this gene, BARSTM, integrated in mtDNA protects respiratory function from imported barnase. Finally, we showed that screening for resistance to mitochondrially targeted barnase can be used to identify rare mitochondrial transformants that had incorporated BARSTM in their mitochondrial DNA. The possibility of employing this strategy in other organisms is discussed.

Bacterial Proteins↗
PubMed2002

Development of the facial midline.

"Intellectual excellence lies in having faith in the observation of apparently nontranscendental and unimportant facts. To observe an anatomic element calmly, with an open, analytical spirit, and with spiritual freedom, can lead to an explosive vortex of new knowledge."-Miguel Orticochea, M.D.(1) Traditional descriptive embryology based upon the interaction of frontonasal, lateral nasal, and medial nasal prominences is incapable of explaining the three-dimensional development of the facial midline. The internal structure of the nose and that of the oronasal midline can best be explained by the presence of paired A fields originating from the prechordal mesendoderm, associated with the nasal and optic placodes, supplied by the internal carotid artery, and sharing a common genetic coding with the prosomeres of the forebrain. Mesial drift of these fields leads to fusion of their medial walls; this in turn provides bilateral functional matrics within which form the orbits ethmoids, lacrimals, turbinates, premaxillae, vomerine bones, and the cartilages of the nose. This two-part paper reports six lines of evidence supporting the field theory model of facial development: (1) An apparent watershed exists in the midline of the base between the territories of the internal and external carotid systems. Isolation of the ICA in injected fetal specimens confirmed that the demarcation was distinct and restricted to the embryonic nasal capsule. (2) Field theory explains the developmental anatomy of the contents of the nasal capsule. (3) The neuromeric model of CNS development provides a genetic basis for the anatomy and behavior of fields. (4) Mutants for the Dlx5 gene demonstrate A field deletion patterns. These experiments relate the nasal placode to the structures of the A fields. (5) Separate regions of the original nasal placodes give rise to neurons, which are dedicated to separate sensory and endocrine systems. The A fields constitute the pathways by which these neurons reach the brain. (6) Non-cleft lip-related cleft palate, holoprosencephaly, and the Kallmann syndrome are clinical models that demonstrate the effects of anatomic disturbances within the A fields.

Animals↗
PubMed1991

Synthetic gene design to investigate the role of cis-acting DNA structural elements in regulation of gene expression in vivo.

To delineate the DNA structural elements responsible for transcriptional control in vivo, we have developed a novel approach taking advantage of the degeneracy of the genetic code. Using synthetic oligonucleotides as structural cassettes we have been able to replace, within a gene, segments of DNA coding for the same amino acid sequence but capable of adopting unusual DNA structures and monitor the effect of such structural elements on gene expression in vivo. We find that the presence of an inverted repeat sequence, with a potential to adopt cruciform structure, within the beta-galactosidase gene down regulates its expression in vivo.

Amino Acid Sequence↗
PubMed2003

Class 1 and class 2 integrons in poultry carcasses from broiler house and poultry processing environments.

Integrons have been identified as major genetic contributors to the dissemination of antimicrobial resistance in bacteria. The objective of this study was to examine the prevalence of integrons in poultry processing at the broiler house and in processing plants. Class 1 and class 2 integrons were found throughout the processing environment. Of the two classes of integrons, class 1 was the most prevalent in all processing areas. The levels of both classes of integrons decreased from the farm to the processing plant. Within the chiller tank in the processing plant, the persistence of these sequences appears to be related to the free chlorine concentration of the chiller tank water. The variable regions of the amplified integrons showed size diversity (from 680 to 2,000 bp), suggesting diversity in types of antibiotic-resistance-coding gene cassettes. The presence of the class 1 and class 2 integrons in the chlorinated chiller tank suggests that these sequences are capable of withstanding this critical step in the reduction of microbial loads on poultry carcasses. The persistence of the integron gene sequences on the farm and throughout processing highlights the stability of these transmissible antibiotic-resistance-coding nucleotide sequences and their potential role as reservoirs of antibiotic-resistance-coding genetic elements within the poultry rearing and processing environments.

Animals↗
PubMed1980

Evolution of the amino acid substitution in the mammalian myoglobin gene.

Multivariate statistical analyses were applied to 16 physical and chemical properties of amino acids. Four of these properties; volume, polarity, isoelectric point (charge), and hydrophobicity were found to explain adequately 96% of the total variance of amino acid attributes. Using these four quantitative measures of amino acid properties, a structural discriminate function in the form of a weighted difference sum of squares equation was developed. The discriminate function is weighted by the location of each particular residue within a given tertiary structure and yields a numerical discriminate or difference value for the replacement of these residues by different amino acids. This resulting discriminate value represents an expression of the perturbation in the local positional environment of a protein when an amino acid substitution occurs. With the use of this structural discriminate function, a residue by residue comparison of the known mammalian myoglobin sequences was carried out in an attempt to elucidate the positions of possible deviations from the known tertiary structure of sperm whale myoglobin. Only 11 of the 153 residue positions in myoglobin demonstrated possible structural deviations. From this analysis, indices of difference were calculated for all amino acid exchanges between the various myoglobins. All comparisons yielded indices of difference that were considerably lower than would be expected if mutations had been fixed at random, even if the organization of the genetic code is taken into consideration. On the basis of these results, it is inferred that some form of selection has acted in the evolution of mammalian myoglobins to favor amino acid substitutions that are compatible with the retention of the original conformation of the protein.

Amino Acid Sequence↗
PubMed1998

Models of amino acid substitution and applications to mitochondrial protein evolution.

Models of amino acid substitution were developed and compared using maximum likelihood. Two kinds of models are considered. "Empirical" models do not explicitly consider factors that shape protein evolution, but attempt to summarize the substitution pattern from large quantities of real data. "Mechanistic" models are formulated at the codon level and separate mutational biases at the nucleotide level from selective constraints at the amino acid level. They account for features of sequence evolution, such as transition-transversion bias and base or codon frequency biases, and make use of physicochemical distances between amino acids to specify nonsynonymous substitution rates. A general approach is presented that transforms a Markov model of codon substitution into a model of amino acid replacement. Protein sequences from the entire mitochondrial genomes of 20 mammalian species were analyzed using different models. The mechanistic models were found to fit the data better than empirical models derived from large databases. Both the mutational distance between amino acids (determined by the genetic code and mutational biases such as the transition-transversion bias) and the physicochemical distance are found to have strong effects on amino acid substitution rates. A significant proportion of amino acid substitutions appeared to have involved more than one codon position, indicating that nucleotide substitutions at neighboring sites may be correlated. Rates of amino acid substitution were found to be highly variable among sites.

Amino Acid Substitution↗
PubMed2005

Transfer RNA gene recruitment in mitochondrial DNA.

Transfer RNA (tRNA) is the adaptor molecule that mediates recognition of the codon sequence in mRNA and enables its translation into the appropriate amino acid. Accordingly, phylogenetic relationships among tRNA genes are often thought to recapitulate the evolution of the genetic code. However, it has been demonstrated experimentally that one tRNA gene can be replaced with a copy of another carrying a single mutation in its anticodon sequence. In this article, we show that such "gene recruitment" has occurred recently and repeatedly in the mitochondrial genome of the demosponge Axinella corrugata and appears to be a common phenomenon in the evolution of the tRNA multigene family.

Animals↗
PubMed2005

Dynamics of transcription and mRNA export.

Understanding the different molecular mechanisms responsible for gene expression has been a central interest of molecular biologists for several decades. Transcription, the initial step of gene expression, consists of converting the genetic code into a dynamic messenger RNA that will specify a required cellular function following translocation to the cytoplasm and translation. We now possess an in-depth understanding of the mechanism and regulations of transcription. By contrast, an understanding of the dynamics of an individual gene's expression in real time is just beginning to emerge following recent technological developments.

Active Transport, Cell Nucleus↗
PubMed1995

Phylogenetic analysis of the aminoacyl-tRNA synthetases.

Numerous aminoacyl-tRNA synthetase sequences have been aligned by computer and phylogenetic trees constructed from them for the two classes of these enzymes. Branching orders based on a consensus of these trees have been proposed for the two groups. Although the order of appearance can be rationalized to fit many different scenarios having to do with the genetic code, the invention of a system for translating nucleic acid sequences into polypeptide chains must have predated the existence of these proteins. In the past, a variety of schemes has been proposed for matching amino acids and tRNAs. Most of these have invoked direct recognition of one by the other, whether or not the anticodon was involved. Often ignored is the possibility of a nonprotein (presumably RNA) matchmaker for bringing the two into conjunction. If such had been the case, then the contemporary aminoacyl-tRNA synthetases could have entered the system gradually, each specific type replacing its matchmaking RNA counterpart in turn. A simple displacement scheme of this sort accommodates the existence of two different families of these enzymes, the second being introduced well before the first had undergone sufficient genetic duplications to specify the full gamut of amino acids. Such a scheme is also consistent with similar amino acids often, but not always, being the substrates of enzymes with the most similar amino acid sequences.

Amino Acid Sequence↗
PubMed1991

Assembly of a class I tRNA synthetase from products of an artificially split gene.

The aminoacyl-tRNA synthetases arose early in evolution and established the rules of the genetic code through their specific interactions with amino acids and RNA molecules. About half of these tRNA charging enzymes are class I synthetases, which contain similar N-terminal nucleotide-fold-like structures that are joined to variable domains implicated in specific protein-tRNA contacts. Here, we show that a bacterial synthetase gene can be split into two nonoverlapping segments. We split the gene for Escherichia coli methionyl-tRNA synthetase (a class I synthetase) at several sites near the interdomain junction, such that one segment codes for the nucleotide-fold-containing domain and the other provides determinants for tRNA recognition. When the segments are folded together, they can recognize and charge tRNA, both in vivo and in vitro. We postulate that an early step in the assembly of systems to attach amino acids to specific RNA molecules may have involved specific interactions between discrete proteins that is reflected in the interdomain contacts of modern synthetases.

Amino Acid Sequence↗
PubMed1997

Glaucoma, apoptosis, and neuroprotection.

The demise of the retinal ganglion cell represents the final common pathway of glaucomatous vision loss. Various studies demonstrate that ganglion cells die by the mechanism of apoptosis in conditions such as experimental animal models of glaucoma and optic nerve transection, and in human glaucoma. Apoptosis is a basic cell death mechanism noted in a number of neurodegenerative conditions. It constitutes a genetically coded "suicide" program activated when cells are no longer needed or have been seriously damaged, and is typified by rapid phagocytosis without inflammation. These cells demonstrate characteristic morphological changes on electron microscopy: nuclear chromatin condensation, compaction of cytoplasmic organelles, and membrane blebbing. Neurotrophin withdrawal and excitotoxic neurotransmitters have been implicated in apoptosis in ganglion cells damaged by glaucoma. Understanding the cellular and molecular biological events involved in ganglion cell death may lead to novel approaches to the treatment of glaucoma.

Animals↗
PubMed1996

And the next 50 years? The future of recombinant DNA technology in oral medicine.

As we celebrate this spectacular 50th anniversary, fluoridation continues to be the most effective public health strategy to reduce the disease burden of dental caries. Curiously, while H. Trendley Dean and his colleagues at the National Institutes of Health were investigating the effects of fluoride on tooth enamel in the mid-1930s, two young boys, one in London and the other in Chicago, were growing up to become the catalysts for another "biological revolution." These two very talented individuals, James Watson and Francis Crick, would later meet by accident at Cambridge and produce their seminal discovery published in April 1953 as a letter in Nature, a one-page article provoking an international scientific adventure to understand living organisms in terms of the structure and function of deoxyribonucleic acid (DNA), a universal genetic code and a rationale for the applications of recombinant DNA technology (rDNA) in fields as diverse as agriculture, energy, industry, and health. As we now reflect upon the triumphs from fluoridation and ponder the next 50 years and the complexities of craniofacial, oral, and dental diseases, it becomes increasingly evident that recombinant DNA technology coupled with health promotion, disease prevention, and public education offers the promise for remarkable advances in prevention, diagnosis, and therapeutics in oral medicine.

Animals↗
PubMed1989

[Spinal muscular atrophy: disappearance of RNA fluorescence of degenerating motor neurons. An acridine orange study].

The histochemical distribution of nucleic acids has been studied in degenerating motor neurons of 9 children who died with spinal muscular atrophy, using the fluorochrome acridine orange. Ribonucleic acid (RNA) fluorescence disappeared abruptly from involved motor neurons without chromatolysis, attenuation of intensity, or other intermediate transitions that follow axotomy or hypoxic insults. We found a nearly identical pattern in 3 adults with amyotrophic lateral sclerosis. The findings in older subjects are complicated, however, by the presence of cytoplasmic lipofuscin. The autofluorescence of this pigment is inhibited by acridine orange. Our results support the hypothesis that spinal muscular atrophy is a disturbance of the genetically coded mechanism that arrests the programmed physiological death of surplus motor neuroblasts after a certain time in embryonic life, so that the normal lethal developmental process becomes pathological by persisting postnatally. A failure of RNA transcription seems to be primary and results in a failure of synthesis of neurotransmitters, of enzymes, and of cytoplasmic proteins. Consequent inanition leads to cell death.

Acridine Orange↗
PubMed2000

[From ready-made to tailor-made--future possibilities for individualized drug therapy].

Knowledge of the genetic code offers great promise for individualised drug therapy. Recent advances in molecular biology and biotechnology have led to new possibilities in the prediction of therapeutic effects and risks of adverse reactions based upon the patient's individual genotype. In addition, these techniques are the basis of gene therapy and are a prerequisite for the development of many other new drugs. This article presents the current status in these fields and discusses to which extent the present promises can be expected to be realised in the future.

Drug Therapy, Computer-Assisted↗
PubMed1993

Molecular cloning and genomic organization of a gene for luciferin-binding protein from the dinoflagellate Gonyaulax polyedra.

The circadian expressed luciferin-binding protein (LBP) gene from the marine bioluminescent alga Gonyaulax polyedra represents the first dinoflagellate gene that has been cloned and sequenced at both cDNA and genomic levels. Starting with a fragment from the 3'-end of the LBP cDNA that was found by immunoscreening of a cDNA library, genomic clones were obtained by the inverse polymerase chain reaction technique. Full-length cDNA clones were selected by screening a cDNA library by plaque hybridizations and by polymerase chain reaction amplifications. The LBP sequence has a 2004-nucleotide open reading frame coding for a protein of 668 amino acids (approximately 75 kDa). The reading frame and identity of the clone were confirmed by the sequence of an octapeptide obtained from a purified fragment of CNBr-treated LBP. A variant LBP cDNA was found to differ in sequence by approximately 11% at the DNA level. The untranslated regions of the mRNA are 111 nucleotides (5'-untranslated region) and 158 nucleotides (3'-untranslated region) long, respectively. The LBP gene contains no introns and exhibits certain features not typical for a eukaryotic gene. Its promoter does not include the typical TATA box within approximately 50 nucleotides upstream of the transcription start site, and the usual poly(A+) signal (AAUAAA) is not present on the end of the LBP mRNA. The copy number of the gene is very high (approximately 1000 copies/cell). However, the universal genetic code and conserved positions relevant for the translational apparatus are maintained.

Amino Acid Sequence↗
PubMed1976

Purification and sequence analysis of the mRNA coding for an immunoglobulin heavy chain.

A mutant cell line (IF2) derived from the mouse myeloma MOPC 21 has been used for the isolation and sequence analysis of H-chain mRNA. The IF2 cells synthesise an H-chain of reduced size in which the CH1 homology region is missing. Sizing of the IF2 H-chain mRNA and wild-type H-chain mRNA revealed that the deletion is expressed at the mRNA level. The mutant H-chain mRNA sedimented at 16-S, enabling effective resolution from 18-S ribosomal RNA. In experiments using IF2 cells labelled with [32P]phosphate, the 16-S mRNA was purified by oligo(T)-cellulose chromatography. Polyacrylamide gel analysis of the poly(A)-containing fraction showed the presence of a single radioactive band. Comparison of the mobility of this band relative to markers of known molecular weight revealed that the molecule contained about 1600 nucleotides. Digestion of the 32-P-labelled mRNA with T1 ribonuclease and two-dimensional fractionation of the resulting oligonucleotides yielded a 'finger-print' suitable for a preliminary sequence analysis. By using the established amino acid sequence of the IF2 H-chain and a knowledge of the genetic code, 14 oligonucleotides were assigned within the constant region and four within the variable region of the IF2 H-chain. This sequence data accounts for 19.5% of the coding region. Several other oligonucleotides, which could not be assigned within the coding region but which occurred in approximately molar yield, have also been partially characterised. These oligonucleotides are presumably derived from the untranslated regions of mRNA.

Amino Acid Sequence↗
PubMed2004

The ambush hypothesis: hidden stop codons prevent off-frame gene reading.

Coding sequences lack stop codons, but many stops appear off-frame. Off-frame stops (stops in -1 and +1 shifted reading frames, termed hidden stops) terminate frame-shifted translation, potentially decreasing energy, and resource waste on nonfunctional proteins. Benefits may include reduced waste elimination costs and avoidance of potentially cytotoxic frame-shifted products. Our "ambush" hypothesis suggests that hidden stops are sometimes selected for. Codons of many amino acids can contribute to hidden stops, depending on the synonymous position state and adjacent codons. In vertebrate mitochondria, 31.75% of all amino acid combinations can form hidden stops. Codons with more potential to form hidden stops have greater usage frequency and bias in their favor among synonymous codons. Among primates, predicted mitochondrial rRNA secondary structure stability correlates negatively with the number of hidden stops in the mitochondrial genome. The taxonomic distribution of genetic codes suggests that +1 frameshifts might be more frequent than -1 frameshifts. This is confirmed by analyses of primate mitochondrial genomes: species with unstable rRNAs have more +1 stops, but the correlation is weak for -1 stops. High hidden stop density seems to be an adaptation in species with slippage prone ribosomes (unstable rRNAs). Hidden stops may thus compensate for reduced efficiency of some parts of the biosynthetic machinery. Some experimental data confirm our hypothesis: gene expression increases with the experimentally manipulated number of stops in the promoter region of a gene, suggesting biotechnological applications.

Animals↗
PubMed2002

Bacterial genomics: potential for antimicrobial drug discovery.

The sequencing of entire bacterial genomes is becoming increasingly routine, promising to revolutionise approaches to identifying putative antimicrobial drug targets. In silico methods can be used to identify putative gene products by comparing sequences of biochemically characterised enzymes and proteins with data produced by sequencing projects. Comparative genomics between a pathogenic bacterium versus nonpathogen as well as pathogen versus host can identify molecular targets that would be ideal for future investigation. The aim of these comparisons would be to identify genes that code for pathogenicity factors in the bacterium or genes essential for bacterial survival. The latter set of genes includes those that are nonfunctional or redundant in the host as well as genes absent from the host but essential in the pathogen. The products of these genes would be ideal targets for antimicrobial compounds. If compounds could be generated that disrupt the pathogen's ability to thrive but not affect the host, since there is a lack of the targeted protein, they could prove to be powerful therapeutics. An elegant example illustrating the power of comparative genomics involves comparison of the pathways of bacterial and eukaryotic aminoacyl-tRNA synthesis. Comparison of pathogenic bacterial genomes shows that many bacteria lack the genes encoding either one or two specific aminoacyl-tRNA synthetases, enzymes involved in ensuring correct aminoacylation of tRNA for subsequent translation of the genetic code. Bacteria have an alternative pathway by which amide aminoacyl-tRNAs are formed. Comparative genomics has demonstrated that this pathway is uniquely prokaryotic/archaeal and also relatively widely found in pathogenic bacteria, indicating the potential of the catalytic enzymes of the pathway as targets for novel antimicrobial drugs.

Animals↗