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PubMed2004

The somatic generation of immune recognition. 1971.

Antibody specificity is determined by structural v-genes that code for the amino acid sequences of the variable regions of antibody polypeptide chains. The present hypothesis proposes that the germ-cells of an animal carry a set of v-genes determining the combining sites of antibodies directed against a complete set of certain class of histocompatibility antigens of the species to which this animal belongs. The evolutionary development of this set of v-genes in phylogeny is traced back to the requirements for cell to cell recognition in all metazoa. The hypothesis leads to a distinction between two populations of antigen-sensitive cells. One population consists of cells forming antibodies against foreign antigens; these lymphocytes have arisen as mutants in clones descending from lymphocytic stem cells which expressed v-genes belonging to the subset (subset S) coding for antibody against histocompatibility antigens that the individual happens to possess. The other population consists of allograft rejecting lymphocytes that express v-genes of the remaining subset (subset A) coding for antibody against histocompatibility antigens of the species that the individual does not possess. The primary lymphoid organs are viewed as mutant-breeding organs. In these organs (e.g. in the thymus), the proliferation of lymphocytes expressing the v-genes of subset S and the subsequent suppression of the cells of these "forbidden" clones, leads to the selection of mutants cells expressing v-genes that have been modified by spontaneous random somatic mutation. This process generates self-tolerance as well as a diverse population of antigen-sensitive cells that reflects antibody diversity. The proliferation in the primary lymphoid organs of lymphocytes expressing v-genes of subset A generates the antigen-sensitive cell population that is responsible for allo-aggression. The theory explains how a functional immune system can develop through a selection pressure exerted by self-antigens, starting during a period in early ontogeny that precedes clonal selection by foreign antigens. The hypothesis provides explanations for the variability of the N-terminal regions of antibody polypeptide chains, for the dominant genetic control of specific immune responsiveness by histocompatibility alleles, for the relative preponderance of antigen-sensitive cells directed against allogeneic histocompatibility antigens, for antibody-idiotypes, for allelic exclusion, for the precommitment of any given antigen-sensitive lymphocyte to form antibodies of only one molecular species and for the cellular dynamics in the primary lymphoid tissues.

Animals↗

PubMed2005

Preparation of recombinant thioredoxin fused N-terminal proCNP: Analysis of enterokinase cleavage products reveals new enterokinase cleavage sites.

C-type natriuretic peptide (CNP) acts as a paracrine hormone to dilate blood vessels and is also required for the growth of long bones. In vivo, CNP is produced by cleavage from the C-terminal end of a larger proCNP peptide. The remaining N-terminal proCNP fragment (NT-proCNP) escapes into the circulation where its concentration is much higher than that of CNP due presumably to a lower clearance rate. Our strategy to obtain large quantities of pure NT-proCNP for further physiological investigations was to express it as a fusion protein with His(6)-tagged thioredoxin followed by cleavage using enterokinase to yield NT-proCNP alone. We have successfully designed and artificially synthesized the coding sequence specifying both mouse and human NT-proCNP with built-in codon bias towards Escherichia coli codon preference. An enterokinase recognition sequence was incorporated immediately upstream of the NT-proCNP coding sequence to allow the fusion protein to be cleaved without leaving any extra residues on the NT-proCNP peptide. High levels of fusion proteins were obtained, constituting 50-58% of total bacterial proteins. Greater than 90% of recombinant thioredoxin/NT-proCNP was expressed in the soluble form and purified to near homogeneity in a single chromatographic step using nickel as the metal ion in IMAC. A time course analysis of the products released from enterokinase cleavage of the recombinant proteins by ESI-MS revealed three sensitive secondary cleavage sites: two were located on vector-associated sequences linking the thioredoxin moiety and NT-proCNP, and one at the C-terminal end of NT-proCNP. Clearly, substrate specificity of both the native and recombinant forms of enterokinase for the recognition sequence DDDDK was by no means exclusive. Hydrolysis at the unexpected LKGDR site located towards the carboxyl end on NT-proCNP was significantly more efficient than at the internally sited DDDDK target sequence. However, when this same sequence was sited internally replacing the DDDDK in another construct of thioredoxin/mouse NT-proCNP, it was found to be poorly processed by enterokinase. Our results showed that non-target sequences can be preferentially recognized over the canonical DDDDK sequence when located accessibly at the ends of proteins.

Amino Acid Sequence↗

PubMed1993

Analysis of the nucleotide and derived amino acid sequences of the SsoII restriction endonuclease and methyltransferase.

A 2648-bp fragment from the P4 plasmid of Shigella sonnei strain 47 coding for the SsoII restriction endonuclease (ENase) and methyltransferase (MTase) (recognition sequence 5'-CCNGG) was sequenced. Two divergently arranged open reading frames of 905 bp for the SsoII ENase (R.SsoII) and 1137 bp for the MTase (M.SsoII) were identified. The coding regions are separated by 110 bp. The calculated M(r) of R.SsoII (35937) and M.SsoII (42887) are in good agreement with values previously obtained by in vitro transcription-translation experiments, i.e., 35 and 43 kDa for the ENase and MTase, respectively. The M.SsoII amino acid (aa) sequence revealed a considerable similarity to m5C-MTases recognizing the related sequences--M.EcoRII, M.dcm, M.MspI, M.BsuFI, M.HpaII, and M.HhaI. Surprisingly, the greatest degree of homology has been observed between the aa sequences of M.SsoII and M.NlaX, with an unidentified recognition sequence. The multiple alignment of aa sequences helps to identify the blocks of conserved aa in variable regions of MTases. These conserved aa can play a key role in target recognition. Some aspects of evolution of m5C-MTases are discussed.

Amino Acid Sequence↗

PubMed2002

Phoneme recognition and confusions with multichannel cochlear implants: vowels.

The aim of this study was to investigate how postlingually severely or profoundly hearing-impaired adults relearn to recognize vowels after receiving multichannel cochlear implants. Vowel recognition of 19 Finnish-speaking subjects was studied for a minimum of 6 months and a maximum of 24 months using an open-set nonsense-syllable test in a prospective repeated-measure design. The responses were coded for phoneme errors, and 95% confidence intervals for recognition and confusions were calculated. The average vowel recognition was 68% (95% confidence interval = 66-70%) 6 months after switch-on and 80% (95% confidence interval = 78-82%) 24 months after switch-on. The vowels [ae], [u], [i], [o], and [a] were the easiest to recognize, and the vowels [y], [e], and [ø] were the most difficult. In conclusion, adaptation to electrical hearing using a multichannel cochlear implant was achieved well; but for at least 2 years, given two vowels with either F1 or F2 at roughly the some frequencies, confusions were drawn more towards the closest vowel with the next highest F1 or F2.

Acoustic Stimulation↗

PubMed1993

V(D)J recombination coding junction formation without DNA homology: processing of coding termini.

Coding junction formation in V(D)J recombination generates diversity in the antigen recognition structures of immunoglobulin and T-cell receptor molecules by combining processes of deletion of terminal coding sequences and addition of nucleotides prior to joining. We have examined the role of coding end DNA composition in junction formation with plasmid substrates containing defined homopolymers flanking the recombination signal sequence elements. We found that coding junctions formed efficiently with or without terminal DNA homology. The extent of junctional deletion was conserved independent of coding ends with increased, partial, or no DNA homology. Interestingly, G/C homopolymer coding ends showed reduced deletion regardless of DNA homology. Therefore, DNA homology cannot be the primary determinant that stabilizes coding end structures for processing and joining.

Animals↗

PubMed1999

Genomic sequence comparison of the human and mouse adenosine deaminase gene regions.

A challenge for mammalian genetics is the recognition of critical regulatory regions in primary gene sequence. One approach to this problem is to compare sequences from genes exhibiting highly conserved expression patterns in disparate organisms. Previous transgenic and transfection analyses defined conserved regulatory domains in the mouse and human adenosine deaminase (ADA) genes. We have thus attempted to identify regions with comparable similarity levels potentially indicative of critical ADA regulatory regions. On the basis of aligned regions of the mouse and human ADA gene, using a 24-bp window, we find that similarity overall (67.7%) and throughout the noncoding sequences (67.1%) is markedly lower than that of the coding regions (81%). This low overall similarity facilitated recognition of more highly conserved regions. In addition to the highly conserved exons, ten noncoding regions >100 bp in length displayed >70% sequence similarity. Most of these contained numerous 24-bp windows with much higher levels of similarity. A number of these regions, including the promoter and the thymic enhancer, were more similar than several exons. A third block, located near the thymic enhancer but just outside of a minimally defined locus control region, exhibited stronger similarity than the promoter or thymic enhancer. In contrast, only fragmentary similarity was exhibited in a region that harbors a strong duodenal enhancer in the human gene. These studies show that comparative sequence analysis can be a powerful tool for identifying conserved regulatory domains, but that some conserved sequences may not be detected by certain functional analyses as transgenic mice.

Adenosine Deaminase↗

PubMed2004

Changing the recognition specificity of a DNA-methyltransferase by in vitro evolution.

The gene coding for the SinI DNA-methyltransferase, a modification enzyme able to recognize and methylate the internal cytosine of the GG(A)/(T)CC sequence, was subjected to in vitro mutagenesis, DNA-shuffling and a strong selection for relaxed GGNCC recognition specificity. As a result of this in vitro evolution experiment, a mutant gene with the required phenotype was selected. The mutant SinI methyltransferase carried five amino acid substitutions. None of these was found in the 'variable region' that were thought to be responsible for sequence specificity. Three were located near the N-terminal end, preceding the first conserved structural motif of the enzyme; two were found between conserved motifs VI and VII. A clone engineered to carry out only the latter two replacements (L214S and Y229H) displays relaxed recognition specificity similar to that of the parental mutant, whereas the clone carrying only the N-terminal replacements showed a much weaker change in recognition specificity. The enzyme with two internal mutations was purified and characterized. Its catalytic activity (kcat/Km) was approximately 5-fold lower towards GG(A)/(T)CC and 20-fold higher towards GG(G)/(C)CC than that of the wild-type enzyme.

Amino Acid Substitution↗

PubMed1989

Characterization of mutations of the bacteriophage P1 mod gene encoding the recognition subunit of the EcoP1 restriction and modification system.

This study characterized several mutations of the bacteriophage P1 mod gene. This gene codes for the subunit of the EcoP1 restriction enzyme that is responsible for DNA sequence recognition and for modification methylation. We cloned the mutant mod genes into expression vectors and purified the mutant proteins to near homogeneity. Two of the mutant mod genes studied were the c2 clear-plaque mutants described by Scott (Virology 41:66-71, 1970). These mutant proteins can recognize EcoP1 sites in DNA and direct restriction but are unable to modify DNA. Methylation assays as well as S-adenosylmethionine (SAM) binding studies showed that the c2 mutants are methylation deficient because they do not bind SAM, and we conclude that the mutations destroy the SAM-binding site. Both of the c2 mutations lie within a region of the EcoP1 mod gene that is not conserved when compared with the mod gene of the related EcoP15 system. EcoP15 and EcoP1 recognize different DNA sequences, and we believe that this region of the protein may code for the DNA-binding site of the enzyme. The other mutants characterized were made by site-directed mutagenesis at codon 240. Evidence is presented that one of them, Ser-240----Pro, simultaneously lost the capacity to bind SAM and may also have changed its DNA sequence specificity.

Amino Acid Sequence↗

PubMed1994

Testing and assessment ethics for all persons: beginning and agenda.

The previous American Psychological Association (APA) assessment ethics code (APA, 1990) and the new code (APA, 1992) differ in content that is relevant to culturally sensitive practices (i.e., culture-specific assessment technologies, culture-specific service delivery styles, use of clients' first language, and recognition of non-Anglo-American belief systems). The new code falls short in areas that are in the process of development or remain controversial. Suggestions for implementation and supplementation are included.

Codes of Ethics↗

PubMed1983

Can response preparation begin before stimulus recognition finishes?

The present experiments were designed to determine whether information obtained early in the process of recognizing a stimulus can be used to begin preparing keypress responses before recognition of the stimulus has completely finished. This question is relevant to the recent debate between discrete (e.g., Sternberg, 1969a) and continuous (e.g., McClelland, 1979) models of human information processing. Stimulus sets were chosen so that recognition processes could extract incomplete preliminary information about a stimulus much faster than they could extract secondary information needed for unique stimulus identification. Discriminability of the secondary information was manipulated to vary the opportunity for response preparation based on preliminary information, with difficult secondary discriminations providing more time for response preparation than easy ones. Precues were given on some trials to allow response preparation to occur before the stimulus was presented, thereby reducing any difference in response preparation as a function of discriminability. Continuous models predict that precues should facilitate response preparation less when the secondary discrimination is difficult than when it is easy, and discrete models predict equal facilitation regardless of secondary discrimination difficulty. Evidence of response preparation was obtained with some but not all stimulus sets. The results were interpreted as support for the asynchronous discrete coding model (Miller, 1982a), in which response preparation can begin only after recognition processes have completely activated a code used in categorizing the stimulus.

Cues↗

PubMed1996

Speech recognition and direct data entry in clinical microbiology.

The aim of this study was to design a system (Vigilante) for the direct entry of urine microscopy, culture and sensitivity test results at bench level using natural continuous speech, removing the need for time-consuming report coding and keyboard entry. Graphical and audio feedback facilitated error recognition and correction, and removed the need for time-consuming report checking and validation stages. The system was evaluated over a three-month period by direct comparison of 2360 urine specimens processed by multiple operators using both Vigilante and traditional methods. The results showed that the Vigilante system reduced the time taken to enter data, improved the accuracy of reporting and reduced the time taken to issue reports. The results suggest that direct data entry using speech offers a practical, reliable and innovative alternative to more conventional means of data entry. The system is now used routinely in this laboratory to report urine microscopy, culture and sensitivity test results, and may be adapted to individual urine methodologies or other data entry tasks.

Humans↗

PubMed1988

Highly efficient positive selection of recombinant plasmids using a novel rglB-based Escherichia coli K-12 vector system.

We have developed pBR328-derived vectors which allow highly efficient positive selection of recombinant plasmids. The system is based on the rglB-coded restriction activity of Escherichia coli K-12 directed against 5-methylcytosine (5mC)-containing DNA. The vectors code for cytosine-specific, temperature-sensitive DNA methyltransferases (ts-Mtases), whose specificity elicits RglB restriction. 5mC-free vector DNA - a prerequisite to allow establishment of such plasmids in cells expressing the RglB nuclease activity - can be prepared from cultures grown at 42 degrees C. At 30 degrees C the vector plasmids are vulnerable to RglB restriction due to the expression of suicidal Mtase activity. Cloning a DNA fragment into the ts-Mtase-coding gene disrupts the lethal methylation and thus permits selection of such recombinant plasmids at 30 degrees C. The standard vector used, pBN73, contains unique recognition sites for nine restriction enzymes within the ts-Mtase-coding gene, which can be used independently or in combination for the construction of recombinant plasmids selectable by the rglB-coded activity. Plasmid pBN74, which carries the determinants for both the ts-Mtase and the RglB nuclease, contains seven unique sites within the ts-Mtase-coding gene. While selection of recombinant plasmids derived from pBN73 obligatorily requires the employment of rglB+ strains, selection of pBN74 derivatives can be performed independent of the E. coli-host genotype. It remains to be elucidated whether positive selection of pBN74-derived recombinant plasmids can also be achieved in hosts other than E. coli. Plasmids pBN73, pBN74 and the recombinants are structurally stable. Generally applicable procedures, as developed during the establishment of this vector system, are described; they allow the isolation of ts-Mtases and facilitate the cloning of genes coding for nucleases directed against 5mC-containing DNA.

Base Sequence↗

PubMed2001

Coding of concurrent vocal signals by the auditory midbrain: effects of duration.

Neural selectivity to signal duration within the auditory midbrain has been observed in several species and is thought to play a role in signal recognition. Here we examine the effects of signal duration on the coding of individual and concurrent vocal signals in a teleost fish with exceptionally long duration vocalizations, the plainfin midshipman, Porichthys notatus. Nesting males produce long-duration, multi-harmonic signals known as hums to attract females to their nests; overlapping hums produce acoustic beats at the difference frequency of their spectral components. Our data show that all midbrain neurons have sustained responses to long-duration hum-like tones and beats. Overall spike counts increase linearly with signal duration, although spike rates decrease dramatically. Neurons show varying degrees of spike rate decline and hence, differential changes in spike rate across the neuron population may code signal duration. Spike synchronization to beat difference frequency progressively increases throughout long-duration beats such that significant difference frequency coding is maintained in most neurons. The significance level of difference frequency synchronization coding increases by an order of magnitude when integrated over the entirety of long-duration signals. Thus, spike synchronization remains a reliable difference frequency code and improves with integration over longer time spans.

Animals↗

PubMed2003

RNA recognition by designed peptide fusion creates "artificial" tRNA synthetase.

The genetic code was established through aminoacylations of RNA substrates that emerged as tRNAs. The 20 aminoacyl-tRNA synthetases (one for each amino acid) are ancient proteins, the active-site domain of which catalyzes formation of an aminoacyl adenylate that subsequently reacts with the 3' end of bound tRNA. Binding of tRNA depends on idiosyncratic (to the particular synthetase) domains and motifs that are fused to or inserted into the conserved active-site domain. Here we take the domain for synthesis of alanyl adenylate and fuse it to "artificial" peptide sequences (28 aa) that were shown previously to bind to the acceptor arm of tRNAAla. Certain fusions confer aminoacylation activity on tRNAAla and on hairpin microhelices modeled after its acceptor stem. Aminoacylation was sensitive to the presence of a specific G:U base pair known to be a major determinant of tRNAAla identity. Aminoacylation efficiency and specificity also depended on the specific peptide sequence. The results demonstrate that barriers to RNA-specific aminoacylations are low and can be achieved by relatively simple peptide fusions. They also suggest a paradigm for rationally designed specific aminoacylations based on peptide fusions.

Amino Acid Motifs↗

PubMed1980

Codon recognition rules in yeast mitochondria.

The mitochondrial genome of Saccharomyces cerevisiae codes for 24 tRNAs. The nucleotide sequences of the tRNA genes suggest a unique set of rules that govern the decoding of the mitochondrial genetic code. The four codons of unmixed fmilies are recognized by single tRNAs that always have a U in the wobble position of the anticodon. The codons of the mixed families are read by two different tRNAs. Codons terminating in a C or U are recognized by tRNAs with a G and codons terminating in a G or A are recognized by tRNAs with a U in the corresponding positions of the anticodons. There are two exceptions to these rules. In the AUN family for isoleucine and methionine, the isoleucine tRNA has a G and the methionine tRNA has a C in the wobble position. The tRNA for the arginine CGN family also has an A in the wobble position of the anticodon. It is of interest that the CGN codons have not been found in the mitochondrial genes sequenced to date. The simplified decoding system of yeast mitochondria allows all the codons to be recognized by only 24 tRNAs.

Anticodon↗

PubMed1993

Mouse neurofibromatosis type 1 cDNA sequence reveals high degree of conservation of both coding and non-coding mRNA segments.

To identify evolutionary conserved domains and facilitate the recognition of potentially significant mutations in NF1 patients or tumors, we have determined the complete approximately 12 kb sequence of mouse neurofibromatosis type 1 mRNA. The sequence predicts a 2841 amino acid protein that is more than 98% identical to human neurofibromin. All but 9 of the 45 amino acid differences between mouse and human neurofibromin occur in the N-terminal half of the protein, with 16 changes clustered just upstream of the IRA-related segment. Given the high degree of sequence identity, virtually any sequence alteration in NF1 patients or tumors is potentially significant. We have also found that the 3' untranslated segment of NF1 mRNA is highly conserved, suggesting that this region may also be a target for mutations in NF1 patients.

Amino Acid Sequence↗

PubMed1997

Characterization of Mycobacterium tuberculosis LexA: recognition of a Cheo (Bacillus-type SOS) box.

The gene coding for the Mycobacterium tuberculosis homologue of LexA has been cloned and sequenced. Amino acids required for autocatalytic cleavage are conserved, whereas those important for specific DNA binding are not, when compared with Escherichia coli LexA. The transcriptional start site was mapped and a DNA sequence motif was identified which resembled the consensus Cheo box sequence involved in the regulation of DNA-damage-inducible genes in Bacillus subtilis. The M. tuberculosis-LexA protein was overexpressed in E. coli and purified by means of a His tag. The purified LexA was shown to bind to the Cheo box sequence found upstream of its own gene.

Amino Acid Sequence↗

PubMed2000

Immunization with DNA coding for gp100 results in CD4 T-cell independent antitumor immunity.

BACKGROUND: Xenogeneic DNA immunization can exploit small differences in expressed protein sequence resulting in immune recognition of self-molecules. We hypothesized that immunizing mice with xenogeneic DNA coding for the human melanosomal membrane glycoprotein gp100 would overcome immune ignorance or tolerance and result in tumor immunity. We also investigated the immunologic mechanisms of the antitumor immunity. METHODS: C57BL/6 mice were immunized with DNA coding for human gp100, mouse gp100, or control vector by gene gun. After immunization, mice were challenged with a syngeneic melanoma expressing gp100, and tumor growth was analyzed. Mice deficient in major histocompatibility complex class I or class II molecules were similarly studied to assess the immunologic mechanism of the tumor protection. RESULTS: There was significant tumor protection after vaccination with xenogeneic human gp100 DNA. Class I, but not class II, major histocompatibility complex molecules were required for tumor immunity. In addition, mice immunized with human gp100 demonstrated autoimmunity manifested as coat color depigmentation. CONCLUSIONS: Immunization with xenogeneic DNA coding for the melanosomal glycoprotein gp100 results in tumor protection and autoimmune depigmentation. These results show that xenogeneic DNA vaccines can lead to cancer immunity without CD4(+) T-cell help with potential implications for rational vaccine design.

Amino Acid Sequence↗