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PubMed1995

A novel pea mitochondrial in vitro transcription system recognizes homologous and heterologous mRNA and tRNA promoters.

To elucidate the mechanism involved in the transcription initiation process in mitochondria of dicotyledonous plants, an in vitro transcription system was established for pea (Pisum sativum L.). The partially purified mitochondrial protein extract initiates transcription on homologous pea templates as well as on heterologous mitochondrial DNA from other dicot plant species. In vitro transcription begins within the nonanucleotide 5'-(-7)CRTAAGAGA(+2)-3' (transcription start site is underlined) conserved at most of the identified transcription initiation sites in dicot plant mitochondria. The in vitro initiation at promoters of protein as well as of tRNA coding genes indicates a common mode of transcription initiation for different types of RNA. The competent recognition of different heterologous templates supports a general functional role of the conserved nonanucleotide within mitochondrial promoters of dicotyledonous plants. Initial studies of the promoter structure by deletion analysis in the 5' region of the pea atp9 promoter show that in addition to the conserved nonanucleotide, which is essential for transcription initiation in vitro, sequences up to 25 nucleotides upstream of the transcription start site are necessary for an efficient initiation event.

Base Sequence↗

PubMed1988

Ribosomal gene structure, variation and inheritance in maize and its ancestors.

We have examined the structure of nuclear genes coding for ribosomal RNAs in maize and its wild relatives, the teosintes and Tripsacum. Digestion of the rDNA (genes coding for 18S, 5.8S and 26S RNAs) with 15 restriction endonucleases (with six base pair recognition sites) yields essentially a single map for the approximately 10,000 repeat units within an individual plant or species. Both length and site variation were detected among species and were concentrated in the intergenic spacer region of the rDNA repeat unit. This result is in agreement with patterns of rDNA change observed among wheat and its relatives (Triticeae), and among vertebrate species. Digestion of these nuclear DNAs with BamHI and subsequent hybridization with a 5S RNA gene-specific probe allowed determination of the size of the 5S gene repeat unit in maize, teosintes, and Tripsacum. Groupings in the genus Zea were characterized by distinct repeat unit types five Tripsacum species examined shared a 260 base pair major repeat unit type. Additionally, several other restriction endonuclease cleavage patterns differentiated among the 5S DNAs within the genus Zea. The rDNA and 5S DNA restriction site variation among the species can be interpreted phylogenetically and agrees with biochemical, karyotypic, and morphological evidence that places maize closest to the Mexican teosintes. For both gene arrays, contributions from each parental genome can be detected by restriction enzyme analysis of progeny from crosses between maize and two distantly related teosintes, Zea luxurians or Zea diploperennis, but certain teosinte arrays were underrepresented in some of the hybrids.

Blotting, Southern↗

PubMed2002

Evolution of eukaryotic translation elongation and termination factors: variations of evolutionary rate and genetic code deviations.

Translation is carried out by the ribosome and several associated protein factors through three consecutive steps: initiation, elongation, and termination. Termination remains the least understood of them, partly because of the nonuniversality of the factors involved. To get some insights on the evolution of eukaryotic translation termination, we have compared the phylogeny of the release factors eRF1 and eRF3 to that of the elongation factors EF-1alpha and EF-2, with special focus on ciliates. Our results show that these four translation proteins have experienced different modes of evolution. This is especially evident for the EF-1alpha, EF-2, and eRF1 ciliate sequences. Ciliates appear as monophyletic in the EF-2 phylogenetic tree but not in the EF-1alpha and eRF1 phylogenetic trees. This seems to be mainly because of phylogeny reconstruction artifacts (the long-branch attraction) produced by the acceleration of evolutionary rate of ciliate EF-1alpha and eRF1 sequences. Interaction with the highly divergent actin found in ciliates, or on the contrary, loss of interaction, could explain the acceleration of the evolutionary rate of the EF-1alpha sequences. In the case of ciliate eRF1 sequences, their unusually high evolutionary rate may be related to the deviations in the genetic code usage found in diverse ciliates. These deviations involve a relaxation (or even abolition) of the recognition of one or two stop codons by eRF1. To achieve this, structural changes in eRF1 are needed, and this may affect its evolutionary rate. Eukaryotic translation seems to have followed a mosaic evolution, with its different elements governed by different selective pressures. However, a correlation analysis shows that, beneath the disagreement shown by the different translation proteins, their concerted evolution can still be made apparent when they are compared with other proteins that are not involved in translation.

Animals↗

PubMed1997

How to tackle genetic loci predisposing to atherosclerosis?

To identify major genes influencing the complex disease process of atherosclerosis, the strategy for collection of study materials should be designed with care to enrich the genetic factors. The tools for an efficient gene search are provided by the Human Genome Program; current genetic maps with dense marker sets provide a basis for genome-wide scans, and close to complete physical maps and identification of coding regions of all human genes within next few years offer the scaffolding for the final recognition of genes predisposing to atherosclerosis. The statistical methods applicable in the initial gene search of complex diseases have developed during recent years including now exact modifications of association analysis, also advanced multipoint analyses applicable in both parametric (linkage analysis) and nonparametric (affected sib-pair) methods and maximizing the information extractable from individual genotypes. Genome scans in the relevant animal models will often guide to important genomic regions, and genetically modified animals will be of essential importance for final understanding of the molecular pathogenesis of atherosclerosis.

Animals↗

PubMed1984

Non-cytotoxic alloantibodies defined by the EA rosette inhibition assay.

Sera from both transfused individuals and pregnant women mediated inhibition of Fc-rosette formation. Both normal blood B lymphocytes and chronic lymphocytic leukemia cells were used as targets. Inhibition was not related to the presence or absence of lymphocytotoxic antibodies. When tested against a panel of B lymphocytes these sera displayed selective reactivity in keeping with the recognition of allospecific determinants. No association between the target antigen(s) and classically defined MHC-coded structures was evident. Heteroantibodies to both class I and class II MHC structures as well as beta 2-microglobulin also mediated FcR blockade. However, unlike the alloantisera tested, these antibodies displayed no restriction in their reactivity toward individual target cells.

Antibodies, Heterophile↗

PubMed1990

Psychophysics of reading. XI. Comparing color contrast and luminance contrast.

Text can be depicted by luminance contrast (i.e., differences in luminance between characters and background) or by color contrast (i.e., differences in chromaticity). We used a psychophysical method to measure the reading speeds of eight normal and ten low-vision subjects for text displayed on a color monitor. Reading speed was measured as a function of luminance contrast, color contrast (derived from mixtures of red and green), and combinations of the two. When color contrast is high, normal subjects can read as rapidly as with high luminance contrast (greater than 300 words/min). Curves of reading speed versus contrast have the same shape for the two forms of contrast and are superimposed when contrast is measured in multiples of a threshold value. When both color and luminance contrast are present, there is no sign of additive interaction, and performance is determined by the form of contrast yielding the highest reading rate. Our findings suggest that color contrast and luminance contrast are coded in similar ways in the visual system but that the neural signals used in letter recognition are carried by different pathways for color and luminance. We found no advantages of color contrast for low-vision reading. For text composed of 6 degrees characters, all low-vision subjects read better with luminance contrast than with color contrast.

Adult↗

PubMedDate not supplied

Gene therapy: models to study the immunological way.

Gene engineering to enhance tumour immunogenicity and elicit curative responses against established tumours and tumour recurrences has become an attractive prospect. Gene engineering enables new genes to be selectively inserted into the genome of a tumour cell, or the construction of new fusion plasmids coding tumour antigens and immunomodulatory molecules. The rationale behind current research is to enhance the immune recognition of tumour antigens through their association with the molecules on which immune recognition depends. The immunotherapy data obtained in many experimental tumour systems provide a realistic assessment of the potential and limits of this technological approach. Experimental vaccination of rodents has been shown to induce a significant immune memory, even against poorly immunogenic tumours, that can prevent tumour growth and cure initial metastases, but is poorly effective against established tumours. Its use in tumour prevention is a fresh dawning perspective.

Animals↗

PubMed1972

T4 transfer RNAs: codon recognition and translational properties.

T4 phage transfer RNAs recognize the previously assigned codons for a given amino acid; however, they tend to prefer code words that are less well recognized by their host. The phage transfer RNAs are more effective in polypeptide synthesis with T4 rather than Escherichia coli messenger RNAs.

Acylation↗

PubMed2005

Pitch ranking ability of cochlear implant recipients: a comparison of sound-processing strategies.

Pitch ranking of sung vowel stimuli, separated in fundamental frequency (F0) by half an octave, was measured with a group of eleven Nucleus 24 cochlear implant recipients using different sound coding strategies. In three consecutive studies, either two or three different sound coding strategies were compared to the Advanced Combinational Encoder (ACE) strategy. These strategies included Continuous Interleaved Sampling (CIS), Peak Derived Timing (PDT), Modulation Depth Enhancement (MDE), F0 Synchronized ACE (FOSync), and Multi-channel Envelope Modulation (MEM), the last four being experimental strategies. While pitch ranking results on average were poor compared to those expected for most normal hearing listeners, significantly higher scores were obtained using the MEM, MDE, and FOSync strategies compared to ACE. These strategies enhanced coding of temporal F0 cues by providing deeper modulation cues to F0 coincidentally in time across all activated electrodes. In the final study, speech recognition tests were also conducted using ACE, CIS, MDE, and MEM. Similar results among all strategies were obtained for word tests in quiet and between ACE and MEM for sentence tests in noise. These findings demonstrate that strategies such as MEM may aid perception of pitch and still adequately code segmental speech features as per existing coding strategies.

Acoustic Stimulation↗

PubMed1986

Visual recognition of equivalent tactile-kinesthetic and verbal information in short-term memory.

Short-term retention of equivalent tactile-kinesthetic and verbal information was compared, as well as subjects' ability to construct, and visually recognize information. Sixty undergraduate subjects participated, 30 in each of two groups. The first group received information through the fine movements of the subject's right index finger, whereas the second group was presented exactly the same information verbally coded. Both groups had to recognize visually each of 10 mentally constructed patterns, one at a time, depicted as drawings of a target and four distractors. Although both groups performed above chance level, there was a significant difference in the total number of correct recognitions, favoring the verbal one. It is concluded that: the transformation to a visual code is easier for the verbal system than for the kinesthetic one, perhaps because experience in coding visual information from verbal descriptions is greater. If we want to make meaningful comparisons between motor and verbal short-term memory, it is desirable to use tasks as similar as possible for both conditions. The present work suggests one way to achieve the above.

Adult↗

PubMed1995

Production of recombinant bovine enterokinase catalytic subunit in Escherichia coli using the novel secretory fusion partner DsbA.

Enterokinase (EK) is a heterodimeric serine protease which plays a key role in initiating the proteolytic digestion cascade in the mammalian duodenum. The enzyme acts by converting trypsinogen to trypsin via a highly specific cleavage following the pentapeptide recognition sequence (Asp)4-Lys. This stringent site specificity gives EK great potential as a fusion protein cleavage reagent. Recently, a cDNA encoding the catalytic (light) chain of bovine enterokinase (EKL) was identified, characterized, and transiently expressed in mammalian COS cells. We report here the production of EKL in Escherichia coli by a novel secretory expression system that utilizes E. coli DsbA protein as an N-terminal fusion partner. The EKL cDNA was fused in-frame to the 3'-end of the coding sequence for DsbA, with the two domains of the fusion protein separated by a linker sequence encoding an enterokinase recognition site. Active, processed recombinant EKL (rEKL) was generated from this fusion protein via an autocatalytic cleavage reaction. The enzymatic properties of the bacterially produced rEKL were indistinguishable from the previously described COS-derived enzyme. Both forms of rEKL were capable of cleaving peptides, polypeptides and trypsinogen with the same specificity exhibited by the native heterodimeric enzyme purified from bovine duodena. Interestingly, rEKL activated trypsinogen poorly relative to the native heterodimeric enzyme, but was superior in its ability to cleave artificial fusion proteins containing the (Asp)4-Lys recognition sequence.

Amino Acid Sequence↗

PubMed2001

Peptidoglycan recognition proteins: a novel family of four human innate immunity pattern recognition molecules.

The innate immune system recognizes microorganisms through a series of pattern recognition receptors that are highly conserved in evolution. Insects have a family of 12 peptidoglycan recognition proteins (PGRPs) that recognize peptidoglycan, a ubiquitous component of bacterial cell walls. We report cloning of three novel human PGRPs (PGRP-L, PGRP-Ialpha, and PGRP-Ibeta) that together with the previously cloned PGRP-S, define a new family of human pattern recognition molecules. PGRP-L, PGRP-Ialpha, and PGRP-Ibeta have 576, 341, and 373 amino acids coded by five, seven, and eight exons on chromosomes 19 and 1, and they all have two predicted transmembrane domains. All mammalian and insect PGRPs have at least three highly conserved C-terminal PGRP domains located either in the extracellular or in the cytoplasmic (or in both) portions of the molecules. PGRP-L is expressed in liver, PGRP-Ialpha and PGRP-Ibeta in esophagus (and to a lesser extent in tonsils and thymus), and PGRP-S in bone marrow (and to a lesser extent in neutrophils and fetal liver). All four human PGRPs bind peptidoglycan and Gram-positive bacteria. Thus, these PGRPs may play a role in recognition of bacteria in these organs.

Amino Acid Sequence↗

PubMed2003

Letter visibility and the viewing position effect in visual word recognition.

The ease with which printed words are recognized depends on the position at which the eyes initially fixate the word. In this study, we examined to what extent recognition performance for each fixation position depends on the average visibility of the word's constituent letters. Experiment 1 measured recognition performance to single letters embedded in strings of Xs (lengths of 5 and 7) for all combinations of letter position and initial fixation position in the string. In Experiment 2, recognition performance was measured for five-letter and seven-letter words as a function of initial fixation position in the word. Whereas average letter visibility showed a symmetric function in Experiment 1, the word recognition data of Experiment 2 showed the typical asymmetric curve. Combining the letter visibility data with measures of lexical constraint using absolute letter-in-string positions failed to capture the pattern in the word data. An alternative measure of constraint based on relative position coding of letters generated more accurate predictions.

Adult↗

PubMed1990

Molecular biology of diazepam binding inhibitor peptide.

Complementary DNA (cDNA) clones containing the entire coding sequence for Diazepam Binding Inhibitor (DBI) peptide, a 10-kDa precursor of putative natural ligands of benzodiazepine recognition sites, were isolated from rat, human and cow libraries. The sequence of all these clones is highly conserved; however, the N-terminal sequence predicted by the human DBI clone differed from that of the other two clones. DBI cDNA, utilized as hybridization probe in Southern blot analysis, revealed that DBI of both human and rat might be encoded by a multiple family of 4-6 genes. Furthermore, we have used in situ chromosomes hybridization to map human DBI genes. The results indicate that a human DBI gene is localized on chromosome 2 and that three of the four hybridization signals detected by the human DBI probe are located on three other chromosomes. These findings raise a question as whether multiple DBI genes encode for different molecular forms of DBI. In the attempt to test this hypothesis, cow cDNA and human genomic libraries were screened with DBI cDNA. In this paper I report the isolation of clones from these libraries which, although hybridizing well to DBI cDNA, possess a low percentage of homology (46.7%), randomly distributed within the coding region of DBI cDNA. Whether or not these clones encode for peptides sharing the same physiological role as DBI is under investigation.

Amino Acid Sequence↗

PubMed2005

[Comparison study on the methods for finding borders between coding and non-coding DNA regions in rice].

Entropy-based divergence measures have provided an impelling tool in evaluating sequence complexity, predicting CpG island, and detecting borders between coding and non-coding DNA regions etc. In this paper, two new divergence measures: the alpha-KL divergence and the alpha-Jensen-Shannon divergence were defined and a coarse-graining vector of amino acids- corresponding codons was proposed according to codons GC-content, in order to improve the computational approach to finding borders between coding and non-coding in rice. A comparison of the accuracies gained by different vectors (the Jensen-Shannon divergence, the Jensen-Renyi divergence, the alpha-KL divergence and the alpha-Jensen -Shannon divergence) showed that recognition efficiency based on the new information measures with the vector coarse-graining increase by 4-5 times than that of Bernaola's method in the 'stop codon' of coding regions in rice.

Algorithms↗

PubMed1995

The picture superiority effect in a cross-modality recognition task.

Words and pictures were studied and recognition tests given in which each studied object was to be recognized in both word and picture format. The main dependent variable was the latency of the recognition decision. The purpose was to investigate the effects of study modality (word or picture), of congruence between study and test modalities, and of priming resulting from repeated testing. Experiments 1 and 2 used the same basic design, but the latter also varied retention interval. Experiment 3 added a manipulation of instructions to name studied objects, and Experiment 4 deviated from the others by presenting both picture and word referring to the same object together for study. The results showed that congruence between study and test modalities consistently facilitated recognition. Furthermore, items studied as pictures were more rapidly recognized than were items studied as words. With repeated testing, the second instance was affected by its predecessor, but the facilitating effect of picture-to-word priming exceeded that of word-to-picture priming. The finds suggest a two- stage recognition process, in which the first is based on perceptual familiarity and the second uses semantic links for a retrieval search. Common-code theories that grant privileged access to the semantic code for pictures or, alternatively, dual-code theories that assume mnemonic superiority for the image code are supported by the findings. Explanations of the picture superiority effect as resulting from dual encoding of pictures are not supported by the data.

Adult↗

PubMed1996

Generation of new DNA binding specificity by truncation of the type IC EcoDXXI hsdS gene.

The hsdS subunit of a type IC restriction-modification enzyme is responsible for the enzyme's DNA binding specificity. Type I recognition sites are characterized by two defined half-sites separated by a non-specific spacer of defined length. The hsdS subunit contains two independent DNA binding domains, each targeted towards one DNA half-site. We have shown previously that the 5' half of hsdS can code for a functional substitute of the full-length hsdS. Here we demonstrate that the 3' half of the gene, when fused to the appropriate transcriptional and translational start signals, also codes for a peptide which imparts DNA binding specificity to the enzyme. About half the natural hsdS size, the mutant peptide contains a single DNA recognition domain flanked by one copy of each internal repeat found in the full-length hsdS. Deletion of either repeat sequence results in loss of activity. Like the 5' hsdS mutant, the 3' mutant recognizes an interrupted palindrome, GAAYN(5)RTTC, suggesting that two truncated subunits participate in DNA recognition. Co-expression of the 5' hsdS mutant and the 3' hsdS mutant along with hsdM regenerates the wild-type methylation specificity. Thus, there is a free assortment of subunits in the cell.

Adenine↗

PubMedDate not supplied

Obcells as proto-organisms: membrane heredity, lithophosphorylation, and the origins of the genetic code, the first cells, and photosynthesis.

I attempt to sketch a unified picture of the origin of living organisms in their genetic, bioenergetic, and structural aspects. Only selection at a higher level than for individual selfish genes could power the cooperative macromolecular coevolution required for evolving the genetic code. The protein synthesis machinery is too complex to have evolved before membranes. Therefore a symbiosis of membranes, replicators, and catalysts probably mediated the origin of the code and the transition from a nucleic acid world of independent molecular replicators to a nucleic acid/protein/lipid world of reproducing organisms. Membranes initially functioned as supramolecular structures to which different replicators attached and were selected as a higher-level reproductive unit: the proto-organism. I discuss the roles of stereochemistry, gene divergence, codon capture, and selection in the code's origin. I argue that proteins were primarily structural not enzymatic and that the first biological membranes consisted of amphipathic peptidyl-tRNAs and prebiotic mixed lipids. The peptidyl-tRNAs functioned as genetically-specified lipid analogues with hydrophobic tails (ancestral signal peptides) and hydrophilic polynucleotide heads. Protoribosomes arose from two cooperating RNAs: peptidyl transferase (large subunit) and mRNA-binder (small subunit). Early proteins had a second key role: coupling energy flow to the phosphorylation of gene and peptide precursors, probably by lithophosphorylation by membrane-anchored kinases scavenging geothermal polyphosphate stocks. These key evolutionary steps probably occurred on the outer surface of an 'inside out-cell' or obcell, which evolved an unambiguous hydrophobic code with four prebiotic amino acids and proline, and initiation by isoleucine anticodon CAU; early proteins and nucleozymes were all membrane-attached. To improve replication, translation, and lithophosphorylation, hydrophilic substrate-binding and catalytic domains were later added to signal peptides, yielding a ten-acid doublet code. A primitive proto-ecology of molecular scavenging, parasitism, and predation evolved among obcells. I propose a new theory for the origin of the first cell: fusion of two cup-shaped obcells, or hemicells, to make a protocell with double envelope, internal genome and ribosomes, protocytosol, and periplasm. Only then did water-soluble enzymes, amino acid biosynthesis, and intermediary metabolism evolve in a concentrated autocatalytic internal cytosolic soup, causing 12 new amino acid assignments, termination, and rapid freezing of the 22-acid code. Anticodons were recruited sequentially: GNN, CNN, INN, and *UNN. CO2 fixation, photoreduction, and lipid synthesis probably evolved in the protocell before photophosphorylation. Signal recognition particles, chaperones, compartmented proteases, and peptidoglycan arose prior to the last common ancestor of life, a complex autotrophic, anaerobic green bacterium.

Anticodon↗