Identification of the Tapasin gene in the chicken major histocompatibility complex

 The Tapasin molecule plays a role in the assembly of major histocompatibility complex (Mhc) class I molecules in the endoplasmic reticulum, by mediating the interaction of class I-β₂-microglobulin dimers with TAP. We report here the identification of the Tapasin gene in the chicken Mhc (B complex). This gene is located at the centromeric end of the complex, between the class II B-LBI and B-LBII genes. Like its human counterpart it comprises 8 exons, but features a significantly reduced intron size as compared to the human gene. Chicken Tapasin codes for a transmembrane protein with a probable endoplasmic reticulum retention signal. Exons IV and V, and possibly exon III, code for separate domains that are related to the immunoglobulin (Ig) superfamily (this relationship was so far unrecognized for human Tapasin domain IV which has lost its two cysteines). Two different cDNAs corresponding to the Tapasin gene were isolated, possibly related to alternative splicing events; the Ig-like domain encoded by exon IV is missing in one of the cDNAs, suggesting either that this domain is not necessary for the protein to perform its function, or that the two alternatively spliced cDNAs are translated into two functionally different forms of the protein. Received: 8 July 1998 / Revised: 5 October 1998     

Universal primers for the PCR-mediated amplification of DNA β

DNA β is an approx 1350 nucleotide, single-stranded DNA molecule which has been shown to be associated with some monopartite geminiviruses of the genus Begomovirus. This component requires the helper begomovirus for replication in the cells of host plants and for insect transmission, possibly by trans-encapsidation. Sequence comparisons of the two available DNA β sequences has identified a highly conserved region upstream of a predicted hairpin structure. Abutting primers designed to this conserved region allows PCR-mediated amplification of the full-length DNA β component from total nucleic acid extracts isolated from infected plants originating from a variety of geographically distinct sources and host plants.     

Some remarks on molecular biology

The binding energy of a very long molecular chain, composed of different classes of molecules, depends in general on the order of the molecules. It is shown that under very general conditions there exists for a givenbrutto chemical composition of a chain, a class of chains which is characterized by a total binding energy which is equal to the total binding energy of any other prescribed chain of different composition within the limits of unsharpness of the energy level. This establishes a criterion formapping of a class of configurations of long chain molecules on another class. To the extent that a mapping constitutes a generalized code those results contribute to the theory of molecular codes. Applying to our results the results of a previous paper (1959,Bull. Math. Biophysics,21, 309–326), we arrive at the conclusion that the self-replication of a living molecule may be the property not of a particular structure but of classes of structures.     

A Novel Theory on the Origin of the Genetic Code: A GNC-SNS Hypothesis

We have previously proposed an SNS hypothesis on the origin of the genetic code (Ikehara and Yoshida 1998). The hypothesis predicts that the universal genetic code originated from the SNS code composed of 16 codons and 10 amino acids (S and N mean G or C and either of four bases, respectively). But, it must have been very difficult to create the SNS code at one stroke in the beginning. Therefore, we searched for a simpler code than the SNS code, which could still encode water-soluble globular proteins with appropriate three-dimensional structures at a high probability using four conditions for globular protein formation (hydropathy, α-helix, β-sheet, and β-turn formations). Four amino acids (Gly [G], Ala [A], Asp [D], and Val [V]) encoded by the GNC code satisfied the four structural conditions well, but other codes in rows and columns in the universal genetic code table do not, except for the GNG code, a slightly modified form of the GNC code. Three three-amino acid systems ([D], Leu and Tyr; [D], Tyr and Met; Glu, Pro and Ile) also satisfied the above four conditions. But, some amino acids in the three systems are far more complex than those encoded by the GNC code. In addition, the amino acids in the three-amino acid systems are scattered in the universal genetic code table. Thus, we concluded that the universal genetic code originated not from a three-amino acid system but from a four-amino acid system, the GNC code encoding [GADV]-proteins, as the most primitive genetic code. Received: 11 June 2001 / Accepted: 11 October 2001     

Identification of new HLA class I region genes by sample sequencing

 Although many human major histocompatibility genes have been identified, relatively few have been localized to the class I region. We searched for new class I region genes by sample sequencing, a process in which short stretches of random genomic sequence are generated from cosmids and then compared with sequences deposited in nucleotide databases. Four class I region cosmids were isolated for sample sequencing by screening a chromosome 6 specific cosmid library with probes derived from specific class I region genes or with overlapping class I region yeast artificial chromosomes. Cosmids were sonnicated to produce fragments of 0.5 – 1 kilobases, subcloned, and sequenced using an automated sequencer. Sequences were then compared with nucleotide sequences deposited in the GenBank databases using the BLASTN algorithm. A number of potential new class I region genes were identified, including a cDNA with similarity to the tre oncogene, the trans-activating factor SC1 (TCF19), and a member of the interferon inducible 1 – 8 gene family. These observations suggest that sample sequencing is an efficient method for identifying new class I region genes, which can be applied to other regions of the genome and to other species, and support previous observations that the class I region contains a variety of genes other than those encoding HLA antigens. Received: 10 December 1996 / Revised: 7 January 1997     

Chaotic dynamics of information processing: The “magic number seven plus-minus two” revisited

In a well-known collection of his essays in cognitive psychology Miller (The Psychology of Communication. Penguin, 1974) describes in detail a number of experiments aiming at a determination of the limits (if any) of the human brain in processing information. He concludes that the ‘channel capacity’ of human subjects does not exceed a few bits or that the number of categories of (one-dimensional) stimuli from which unambiguous judgment can be made are of the order of ‘seven plus or minus two’. This ‘magic number’ holds also, Miller found, for the number of random digits a person can correctly recall on a row and also the number of sentences that can be inserted inside a sentence in a natural language and still be read through without confusion. In this paper we propose a dynamical model of information processing by a self-organizing system which is based on the possible use of strange attractors as cognitive devices. It comes as an amusing surprise to find that such a model can, among other things, reproduce the ‘magic number seven plus-minus two’ and also its variance in a number of cases and provide a theoretical justification for them. This justification is based on the optimum length of a code which maximizes the dynamic storing capacity for the strings of digits constituting the set of external stimuli. This provides a mechanism for the fact that the ‘human channel’, which is so narrow and so noisy (of the order of just a few bits per second or a few bits per category) possesses the ability of squeezing or ‘compressing’ practically an unlimited number of bits per symbol—thereby giving rise to a phenomenal memory.     

A Blind Empiricism Against the Coevolution Theory of the Origin of the Genetic Code

Ronneberg et al. (Proc Natl Acad Sci USA 97:13690–13695, 2000) recently suggested abandoning the coevolution theory of genetic code origin on the basis of two pieces of evidence. They (1) criticize the use of several pairs of amino acids in a precursor–product relationship to support this theory and (2) suggest a new set of codes in which to investigate the statistical bases of the coevolution theory, reaching the conclusion that this theory is not statistically validated in this set. In this paper I critically analyze the robustness of these conclusions. Observations and arguments lead to the belief that the pairs of amino acids in a precursor–product relationship originally used by the coevolution theory are such, or may at least be interpreted as such, and are therefore a manifestation of this theory. Furthermore, the new set of codes that Ronneberg et al. suggest is open to criticism and is thus substituted by the set of amino acid permutation codes, in which even the pairs of amino acids they favor end up by supporting the coevolution theory. Overall, the analysis seems to show that the paper by Ronneberg et al. is of minor scientific value while the coevolution theory seems to be one of the best theories at our disposal for explaining the evolutionary organisation of the genetic code and is, contrary to their claims, statistically well validated. Received: 21 February 2001 / Accepted: 22 May 2001     

The sheep CD1 gene family contains at least four CD1B homologues

We identified four cDNA sequences encoding sheep homologues of the CD1 molecule. The sheep sequences were selected from λgt11 thymocyte cDNA libraries by hybridization with a humanCD1C probe and a homologous sheep probe. TheSCD1B-42 andSCD1A25 sequences encode complete CD1 molecules. The third sequence,SCD1B-52, which is closely related toSCD1B-42 and may be an allele, has the sequence encoding the α3 region precisely deleted. The fourth sequence,SCD1T10, is truncated at the 5′ end. All four sequences are related to the humanCD1B and domestic rabbitCD1B-like sequences at both nucleotide and amino acid level. Comparison of the derivedCD1 amino acid sequences with the sequence of major histocompatibility complex class I molecules showed that the sheep CD1 molecules, like human CD1 molecules, lack most of the conserved class I residues known to be involved in interaction with 132-microglobulin and the CD8 molecule. They do not contain the peptide docking residues involved in anchoring peptides in the peptide binding groove of class I molecules. Southern hybridization of sheep DNA with a sheepCD1 exon 4/ga3 probe showed that the sheep genome encodes at least sevenCD1 genes. The implications of these analyses for CD1 function are discussed. The nucleotide sequence data reported in this paper have been submitted to the EMBL/GenBank nucleotide sequence databases and have been assigned the accession numbers Z36890 (SCD1A25), Z36891 (SCD1B-42), Z36892 (SCD1B-52), and X90567 (SCDIT10)     

Residue 3 of β2-microglobulin affects binding of class I MHC molecules by the W6/32 antibody

 Previous studies of class I MHC molecules have shown that the owl monkey (Aotus) possesses at least two variants of the β₂-microglobulin (β₂m) protein. These two variants have different isoelectric points, and exhibit differential reactivity with the monoclonal antibody W6/32. We report cDNA sequences of the B2m gene, from W6/32-positive and W6/32-negative Aotus cell lines. The two β₂m variants we identified exhibit a single amino acid difference at position three. An arginine residue at position 3 was correlated with W6/32 reactivity, whereas histidine was associated with non-reactivity. W6/32 reactivity was conferred to a W6/32-negative Aotus cell line when it was transfected with the B2m from the W6/32-positive cell line. Residue 3 of β₂m is located at the surface of the class I molecule. It is also close to position 121 of the MHC class I heavy chain, which has previously been shown to influence W6/32 antibody binding. We conclude that W6/32 binds a compact epitope on the class I molecule that includes both residue 3 of β₂m and residue 121 of the heavy chain. We examined the distribution of the two β₂m motifs in a sample Aotus population using an allele-specific polymerase chain reaction assay. The pattern of β₂m segregation we observed matches that which was defined previously by serology. Additionally, we identified laboratory-born hybrid animals who possess both variants of β₂m. Received: 1 April 1998 / Received: 3 July 1998     

Allelic repertoire of the humanMHC class IMICA gene

 The hallmark of the classical major histocompatibility complex (MHC) class I molecules is their astonishing level of polymorphism, a characteristic not shared by the nonclassical MHC class I genes. A distinct family of MHC class I genes has been recently identified within the human MHC class I region. The MICA (MHC class I chain-related A) gene in this family is a highly divergent member of the MHC class I family and has a unique pattern of tissue expression. We have sequenced exons encoding the extracellular α1, α2, and α3 domains of the MICA gene from twenty HLA homozygous typing cell lines and four unrelated individuals. We report the identification of eleven new alleles defined by a total of twenty-two amino acid substitutions. Thus, the total number of MICA alleles is sixteen. Interestingly, a tentative superimposition of MICA variable residues on the HLA-A2 structure reveals a unique pattern of distribution, concentrated primarily on the outer edge of the MICA putative antigen binding cleft, apparently bordering an invariant ligand binding site. Received: 13 May 1996 / Revised: 29 May 1996     

A divergent non-classical class I gene conserved in salmonids

 Complementary DNA for two class I genes of the rainbow trout, Oncorhynchus mykiss, were characterized. MhcOnmy-UBA*01 is similar to Onmy-UA-C32 and the classical major histocompatibility complex class I genes of other fish species, whereas Onmy-UAA*01 is divergent from all class I genes so far characterized. Onmy-UAA*01 is expressed at lower levels than Onmy-UBA*01. Although Onmy-UAA*01 exhibits restriction fragment length polymorphism on Southern blotting, the encoded protein is highly conserved. Two allotypes, which differ only by substitution at amino acid position 223 of the α₃ domain, have been defined. Onmy-UAA*01 has an exon-intron organization like other class I genes and contains a Tc1-like transposon element in intron III. Orthologues of Onmy-UAA*01 have been characterized in four other species of salmonid. Between four species of Oncorhynchus, UAA*01 proteins differ by only 2–6 amino acids, whereas comparison of Oncorhynchus with Salmo trutta (brown trout) reveals 14–16 amino acid differences. The Onmy-UAA*01 gene has properties indicative of a particularly divergent non-classical class I gene. Received: 22 September 1998 / Revised: 24 November 1998     

RIG‐I detects infection with live Listeria by sensing secreted bacterial nucleic acids

Immunity against infection with Listeria monocytogenes is not achieved from innate immune stimulation by contact with killed but requires viable Listeria gaining access to the cytosol of infected cells. It has remained ill‐defined how such immune sensing of live Listeria occurs. Here, we report that efficient cytosolic immune sensing requires access of nucleic acids derived from live Listeria to the cytoplasm of infected cells. We found that Listeria released nucleic acids and that such secreted bacterial RNA/DNA was recognized by the cytosolic sensors RIG‐I, MDA5 and STING thereby triggering interferon β production. Secreted Listeria nucleic acids also caused RIG‐I‐dependent IL‐1β‐production and inflammasome activation. The signalling molecule CARD9 contributed to IL‐1β production in response to secreted nucleic acids. In conclusion, cytosolic recognition of secreted bacterial nucleic acids by RIG‐I provides a mechanistic explanation for efficient induction of immunity by live bacteria.     

Selection and characterization of DNA aptamers with binding selectivity to Campylobacter jejuni using whole-cell SELEX

The need for pre-analytical sample processing prior to the application of rapid molecular-based detection of pathogens in food and environmental samples is well established. Although immunocapture has been applied in this regard, alternative ligands such as nucleic acid aptamers have advantages over antibodies such as low cost, ease of production and modification, and comparable stability. To identify DNA aptamers demonstrating binding specificity to Campylobacter jejuni cells, a whole-cell Systemic Evolution of Ligands by EXponential enrichment (SELEX) method was applied to a combinatorial library of FAM-labeled single-stranded DNA molecules. FAM-labeled aptamer sequences with high binding affinity to C. jejuni A9a as determined by flow cytometric analysis were identified. Aptamer ONS-23, which showed particularly high binding affinity in preliminary studies, was chosen for further characterization. This aptamer displayed a dissociation constant (K _d value) of 292.8 ± 53.1 nM with 47.27 ± 5.58% cells fluorescent (bound) in a 1.48-μM aptamer solution. Binding assays to assess the specificity of aptamer ONS-23 showed high binding affinity (25–36%) for all other C. jejuni strains screened (inclusivity) and low apparent binding affinity (1–5%) with non-C. jejuni strains (exclusivity). Whole-cell SELEX is a promising technique to design aptamer-based molecular probes for microbial pathogens without tedious isolation and purification of complex markers or targets.     

Cloning and Expression of the Pseudomonas Gene for Utilization of d-Leucine in Escherichia coli

Two genes of Pseudomonas putida (IFO 12996) which code for enzymes participating in amino acid metabolism, were cloned in Escherichia coli C600 using pBR322 as a vector. pST7549 is a 7.9 kb hybrid plasmid DNA which is composed of four SalI fragments (0.3, 1.4, 1.9 and 4.3 kb), and codes for β-isopropylmalate dehydrogenase (EC 1.1.1.85) in l-leucine biosynthesis. The enzyme activity in the crude extract from E. coli C600 bearing pST7549 was 80 ~ 90% lower than that of E. coli K12 or P. putida. When the foreign SalI fragments derived from P. putida were subcloned, a 1.9 kb SalI fragment was found to encode β-isopropylmalate dehydrogenase and it did not contain the promoter of P. putida DNA. Plasmid pST6961 has a 1.8 kb insert derived from the P. putida DNA in the SalI site of pBR322. E. coli cells carrying this recombinant plasmid show no leucine racemase activity and no d-leucine transaminase activity, but five-times higher d-leucine oxidation activity than the host strain, E. coli. Enzymological studies have suggested that plasmid pST6961 codes for d-amino acid dehydrogenase, a key enzyme in d-amino acid metabolism.     

Phylogenetic Affinity of Mitochondria of Euglena gracilis and Kinetoplastids Using Cytochrome Oxidase I and hsp60

The mitochondrial DNA-encoded cytochrome oxidase subunit I (COI) gene and the nuclear DNA-encoded hsp60 gene from the euglenoid protozoan Euglena gracilis were cloned and sequenced. The COI sequence represents the first example of a mitochondrial genome-encoded gene from this organism. This gene contains seven TGG tryptophan codons and no TGA tryptophan codons, suggesting the use of the universal genetic code. This differs from the situation in the mitochondrion of the related kinetoplastid protozoa, in which TGA codes for tryptophan. In addition, a complete absence of CGN triplets may imply the lack of the corresponding tRNA species. COI cDNAs from E. gracilis possess short 5′ and 3′ untranslated transcribed sequences and lack a 3′ poly[A] tail. The COI gene does not require uridine insertion/deletion RNA editing, as occurs in kinetoplastid mitochondria, to be functional, and no short guide RNA-like molecules could be visualized by labeling total mitochondrial RNA with [α-³²P]GTP and guanylyl transferase. In spite of the differences in codon usage and the 3′ end structures of mRNAs, phylogenetic analysis using the COI and hsp60 protein sequences suggests a monophyletic relationship between the mitochondrial genomes of E. gracilis and of the kinetoplastids, which is consistent with the phylogenetic relationship of these groups previously obtained using nuclear ribosomal RNA sequences. Received: 5 March 1996 / Accepted: 31 July 1996     

Universal primers for the PCR-mediated amplification of DNA 1: a satellite-like molecule associated with begomovirus-DNA beta complexes

DNA 1 is a single-stranded DNA molecule of approximately 1370 nucleotides. It is associated with monopartite geminiviruses of the genus Begomovirus, which require a DNA β component for symptomatic infection. The DNA 1 molecule requires the helper begomovirus for movement in plants, but is capable of self-replication. We designed two abutting primer pairs (DNA101/DNA102 and UN101/UN102) to conserved sequences of DNA 1. This allowed polymerase chain reaction-mediated amplification of the full-length molecule from total nucleic acid extracts produced from various host plants from geographically distinct, worldwide locations. These primers are useful both as diagnostic probes and for producing full-length infectious clones for in planta studies.     

Time versus size: which characteristic of a neural response carries more information?

 Considering a variety of quite different candidate neural codes, Cheng and Wasserman [(1996) Biol Cybern 75, 93–103, 105–115] reported data which suggested that amplitude codes transmitted information more faithfully than temporal codes. Reanalyzing their data, the present study measured how well size and time represented information resident within the same neural response feature, namely the response peak. Responses from photoreceptor cells and optic nerve fibers in the peripheral visual system of Limulus polyphemus were therefore re-examined using signal detectability values provided by receiver-operating-characteristic analyses to compare how well the timing and the amplitude of the response peak represent information within a single cell and how well they transmit information between cells. Data were available for several cells in several different light-adaptation states onto which several different test-flash intensities had been superimposed. The present reanalysis of these data replicated the results yielded earlier by the peak candidate code, and compared them with the data produced by two different measurements of the timing of the same response peak. A relative-timing code was derived from measurements of the time that elapsed between the moment when the response exceeded a criterion potential and when it reached its peak. An absolute-timing code was derived from the time that elapsed between test flash onset and the peak. The results clearly indicated that the peak code represented information within cells better than either of the two timing codes. However, both peak and absolute-timing codes clearly transmitted the available information between cells more faithfully than the relative-timing code. These data lead to two conclusions. First, that the same response feature, when measured in different ways, can produce remarkably different information processing outcomes. In this particular case, time represented information resident within a cell less well than did size. Second, that the fidelity of information transmission between nerve cells may be relatively independent of the quantity of information resident within any particular cell. Received: 5 November 2001 / Accepted in revised form: 8 July 2002 Acknowledgements. We are deeply indebted to Huiqi Yin for her technical assistance with all the calculations and the graphics. We also sincerely thank Tony Hosking, who wrote the computer programs that extracted the code data for this project, and Brendan Marr, who assisted with data analysis. Correspondence to: G.S. Wasserman (e-mail: codelab@purdue.edu, Fax: +1-765-4961264)     

The Evolution of the Genetic Code Revisited

The evolution of the genetic code in terms of the adoption of new codons has previously been related to the relative thermostability of codon–anticodon interactions such that the most stable interactions have been hypothesised to represent the most ancient coding capacity. This derivation is critically dependent on the accuracy of the experimentally determined stability parameters. A new set of parameters recently determined for B-DNA reveals that the codon–anticodon pairs for the codes in non-plant mitochondria on the one hand and prokaryotic and eukaryotic organisms on the other can be unequivocally divided into two classes – the most stable base steps define a common code specified by the first two bases in a codon while the less stable base steps correlate with divergent usage and the adoption of a 3-letter code. This pattern suggests that the fixation of codons for A, G, P, V, S, T, D/E, R may have preceded the divergence of the non-plant mitochondrial line from other organisms. Other variations in the code correlate with the least stable codon–anticodon pairs. Presented at: National Workshop on Astrobiology: Search for Life in the Solar System, Capri, Italy, 26 to 28 October, 2005.     

Searching of Code Space for an Error-Minimized Genetic Code Via Codon Capture Leads to Failure, or Requires At Least 20 Improving Codon Reassignments Via the Ambiguous Intermediate Mechanism

The standard genetic code (SGC) has a fundamental error-minimizing property which has been widely attributed to the action of selection. However, a clear mechanism for how selection can give rise to error minimization (EM) is lacking. A search through a space of alternate codes (code space) via codon reassignments would be required, to select a code optimized for EM. There are two commonly discussed mechanisms of codon reassignment; the Codon Capture mechanism, which proposes a loss of the codon during reassignment, and the Ambiguous Intermediate mechanism, which proposes that the codon underwent an ambiguous phase during reassignment. When searching of code space via the Codon Capture mechanism is simulated, an optimized genetic code can rarely be achieved (0–3.2% of the time) with most searches ending in failure. When code space is searched via the Ambiguous Intermediate mechanism, under constraints derived from empirical observations of codon reassignments from extant genomes, the searches also often end in failure. When a local minimum is avoided and optimization is achieved, 20–41 sequential improving codon reassignments are required. Furthermore, the structures of the optimized codes produced by these simulations differ from the structure of the SGC. These data are challenges for the Adaptive Code hypothesis to address, which proposes that the EM property was directly selected for, and suggests that EM is simply a byproduct of the addition of amino acids to the expanding code, as described by the alternative ‘Emergence’ hypothesis.     

The optimality of the standard genetic code assessed by an eight-objective evolutionary algorithm

Background

The standard genetic code (SGC) is a unique set of rules which assign amino acids to codons. Similar amino acids tend to have similar codons indicating that the code evolved to minimize the costs of amino acid replacements in proteins, caused by mutations or translational errors. However, if such optimization in fact occurred, many different properties of amino acids must have been taken into account during the code evolution. Therefore, this problem can be reformulated as a multi-objective optimization task, in which the selection constraints are represented by measures based on various amino acid properties.

Results

To study the optimality of the SGC we applied a multi-objective evolutionary algorithm and we used the representatives of eight clusters, which grouped over 500 indices describing various physicochemical properties of amino acids. Thanks to that we avoided an arbitrary choice of amino acid features as optimization criteria. As a consequence, we were able to conduct a more general study on the properties of the SGC than the ones presented so far in other papers on this topic. We considered two models of the genetic code, one preserving the characteristic codon blocks structure of the SGC and the other without this restriction. The results revealed that the SGC could be significantly improved in terms of error minimization, hereby it is not fully optimized. Its structure differs significantly from the structure of the codes optimized to minimize the costs of amino acid replacements. On the other hand, using newly defined quality measures that placed the SGC in the global space of theoretical genetic codes, we showed that the SGC is definitely closer to the codes that minimize the costs of amino acids replacements than those maximizing them.

Conclusions

The standard genetic code represents most likely only partially optimized systems, which emerged under the influence of many different factors. Our findings can be useful to researchers involved in modifying the genetic code of the living organisms and designing artificial ones.