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Human gene mapping.

It is now possible to map the human genome completely with a set of closely linked markers. Over 500 coding genes have been cloned and localized, as have approximately 2000 anonymous DNA fragments, most of which recognize two-allele polymorphisms that are caused by single base changes which alter the recognition site for a restriction enzyme (restriction fragment length polymorphisms). Most human chromosomes have been mapped, with markers in defined order placed approximately 10 map units apart. Chromosomes X and 21 are particularly well mapped, with over 200 probes ordered on X. The strategy during the next few years will encompass moving from a linkage map to a set of overlapping cosmid or phage clones, and finally to a complete sequence of regions of chromosomes and entire chromosomes. A complete sequence of the human genome should transform our understanding of development, the control of gene expression, and the parameters of genetic disease.

Chromosome Mapping↗

Three variant introns of the same general class in the mitochondrial gene for cytochrome oxidase subunit 1 in Aspergillus nidulans.

The oxiA gene of Aspergillus nidulans, coding for cytochrome oxidase subunit 1, is shown by DNA sequencing to contain three introns. An AUG start codon is not present at the beginning of the sequence, suggesting that either another codon, possibly the four base codon AUGA, is used for initiation or there is a further short intron between the true start codon and the beginning of the recognisable coding region. The second and third introns have long open reading frames, which could code for maturase proteins. The lack of conservation of amino acid sequence in the putative region of proteolytic cleavage for maturase formation suggests that the first conserved decapeptide may act as the recognition signal for protein processing. The third intron is remarkably (70%) homologous to the second intron of the cytochrome oxidase subunit 1 gene of Schizosaccharomyces pombe and both are located in exactly the same position. The third Aspergillus intron has an in-frame insertion of a 37-bp GC-rich DNA sequence which is now flanked by a 5-bp repeat, a well-known feature of transposable elements. All three introns in the oxiA gene have a 'core' RNA secondary structure found in a class of introns fitting the RNA splicing model of Davies et al. (1982). This core RNA structure may play a catalytic as well as a structural role in intron splicing. A sequence within the intron could act as a guide to align the splice sites of two of the introns in accordance with the model of Davies et al.

Aspergillus nidulans↗

Generation of cytotoxic T lymphocytes by the H-2-encoded E molecules.

Primary CML was generated in strain combinations 4R anti-2R, R107 anti-3R, 7R anti-9R, and GD anti-R101-combinations differing only in the chromosomal interval between the I-A subregion and the Ss locus. No CML could be obtained in any of the reciprocal combinations of these strains. This unidirectionality of the CML reaction correlates with the expression or nonexpression of the E molecules encoded by this interval: the reaction occurred in combinations in which the responder strain lacked and the stimulator strain expressed the E molecules in the cell membrane. The CML reaction was positive when tested on LPS-stimulated blast cells but weak on Con A-stimulated blasts and negative on Ia-negative tumor cells. The reaction could partially be inhibited by monoclonal antibodies to the Ia.m7 determinant presumably carried gy the E alpha chain; it was not inhibited by monoclonal antibodies specific for Ia determinants carried by the A molecule. Cytotoxic lymphocytes specific for a particular combination of E beta and E alpha chains reacted with all cells expressing the particular E beta chain, no matter what the origin of the E alpha chain associated with the E beta chain was. Attempts to generate cytotoxic lymphocytes specifically reactive with allotypic determinants on E alpha chains failed. In F1 hybrids expressing one type of E alpha chain and two types of E beta chain, the single E alpha chain was found to associate with both beta chains, producing two types of E molecule. We conclude from these experiments that the CML determinants detected in the strain combinations used are encoded by the same loci as those coding for the serologically detectable Ia determinants. The CML determinants are carried by the E beta chains; the E alpha chain does not contribute in any way to the specificity of determinant recognition by the cytotoxic lymphocytes. No evidence for allotypic variation of the E alpha chain as detected by the CML assay could be found in this study.

Animals↗

The effects of intravenous diazepam and hyoscine upon recognition memory.

Diazepam and hyoscine are known to have amnesic effects when administered intravenously. Nevertheless, they achieve these effects by entirely different neurochemical mechanisms, and so the question arises whether they can be differentiated in terms of their effects upon different categories of encoding operations. Sixteen normal volunteers received intravenous administrations of diazepam, hyoscine, and saline following a double-blind procedure. Both drugs completely eliminated the subjects' ability to discriminate between successive lists of words in a test of recognition memory. Moreover, both drugs also impaired the subjects' ability to reject homophones or synonyms of presented words. Thus, both diazepam and hyoscine appeared to impair the use of list tags, phonemic coding, and semantic coding in storing individual stimulus items in long-term memory.

Adult↗

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↗

Expression and function of Toll-like receptor 2 in canine blood phagocytes.

Toll-like receptors (TLRs) are a family of highly conserved pattern recognition receptors (PRR) of mammals that participate in the activation of innate immune responses against microbial infections. Among these receptors, TLR2 is essential for the recognition of conserved structural components of bacteria, protozoa and fungi. Until now, expression of TLR2 in dogs has not been investigated. In this work we describe a partial sequence of the gene coding for canine TLR2 and show that TLR2 mRNA is constitutively expressed in canine blood PMNs. We also show that stimulation of purified PMNs with lipoteichoic acid (LTA), a ligand of TLR2, leads to the release of proinflammatory chemokine IL-8. Furthermore, TLR2 protein is easily detectable by flow cytometry on the canine peripheral blood granulocyte and monocyte cell surface, and slightly on lymphocytes. These findings suggest that, also in dogs as in humans the initial antibacterial response of PMNs could be elicited through engagement of TLR2.

Animals↗

Repeats mimic pathogen-associated patterns across a vast evolutionary landscape.

An emerging hallmark of many human diseases is transcription of typically silenced repetitive DNA containing pathogen-associated molecular patterns (PAMPs). These PAMPs engage the innate immune system via pattern recognition receptors (PRRs)-a phenomenon known as viral mimicry. We propose a statistical physics framework to quantify viral mimicry by measuring "selective forces" that enrich PAMPs compared to a genome-wide reference distribution. We validate our predictions by identifying repeats that bind different PRRs and show potential viral mimics in different repeat families across eukaryotic genomes, suggesting shared mechanisms drive emergence and retention. We propose two non-exclusive evolutionary hypotheses. The first "repeat-centric" hypothesis posits PAMPs are integral to the repeat life cycle and are therefore enriched as they mediate repeat expansion. The second "organism-centric" hypothesis proposes viral mimicry functions as a cell-intrinsic feedback mechanism for sensing and reacting to transcriptional dysregulation, which provides a selective pressure to maintain PAMPs in genomes.

Humans↗

Endogenous lectins as targets for drug delivery.

To minimize side effects of drugs it would be ideal to target them exclusively to those cell types which require treatment. As a means to this end prototypical cellular recognition systems pique our interest to devise biomimetic strategies. Since oligosaccharides of glycoconjugates outmatch other information-carrying biomolecules (proteins, nucleic acids) in theoretical storage capacity by far, work on the sugar code can spark off development of effective targeting devices. Conjugation of custom-made glycan epitopes to proteins or biocompatible non-immunogenic polymeric scaffolds produces neoglycoconjugates with purpose-adaptable properties. In the interplay with endogenous receptors such as lectins, suitable oligosaccharides such as histo-blood group trisaccharides as parts of neoglycoconjugates have already proven their practical applications in histopathology. Elucidation of the structure of cell lectins with currently five main families aids to tailor ligand characteristics rationally. They include the types of functional groups and their topological presentation to optimize the bimolecular binding as well as the optimal spatial clustering and spacer characteristics to exploit cooperativity. Indeed, the potent trivalent cluster glycosides designed for the C-type asialoglycoprotein receptors furnish an instructive example how to turn the theoretical guideline on ligand modification into nM-affinity. By placing emphasis on tissue lectins as targets of neoglycoconjugate-mediated drug delivery, the long-term perspective is opened to likewise test members of these families themselves for routing of therapeutic payloads, aiming at cell addressins. This review illustrates the conceivable potential which work on the sugar code with custom-made neoglycoconjugates and tissue lectins can have in store for drug delivery.

Animals↗

Importance of a single base pair for discrimination between intron-containing and intronless alleles by endonuclease I-BmoI.

Homing endonucleases initiate mobility of their host group I introns by binding to and cleaving lengthy recognition sequences that are typically centered on the intron insertion site (IS) of intronless alleles. Because the intron interrupts the endonucleases' recognition sequence, intron-containing alleles are immune to cleavage by their own endonuclease. I-TevI and I-BmoI are related GIY-YIG endonucleases that bind a homologous stretch of thymidylate synthase (TS)-encoding DNA but use different strategies to distinguish intronless from intron-containing substrates. I-TevI discriminates between substrates at the level of DNA binding, as its recognition sequence is centered on the intron IS. I-BmoI, in contrast, possesses a very asymmetric recognition sequence with respect to the intron IS, binds both intron-containing and intronless TS-encoding substrates, but efficiently cleaves only intronless substrate. Here, we show that I-BmoI is extremely tolerant of multiple substitutions around its cleavage sites and has a low specific activity. However, a single G-C base pair, at position -2 of a 39-base pair recognition sequence, is a major determinant for cleavage efficiency and distinguishes intronless from intron-containing alleles. Strikingly, this G-C base pair is universally conserved in phylogenetically diverse TS-coding sequences; this finding suggests that I-BmoI has evolved exquisite cleavage requirements to maximize the potential to spread to variant intronless alleles, while minimizing cleavage at its own intron-containing allele.

Alleles↗

Peptide inhibitors of West Nile NS3 protease: SAR study of tetrapeptide aldehyde inhibitors.

A series of inhibitors related to the benzoyl-norleucine-lysine-arginine-arginine (Bz-nKRR) tetrapeptide aldehyde was synthesized. When evaluated against the West Nile virus (WNV) NS3 protease, the measured IC(50) ranges from approximately 1 to 200 microM. Concurrently, a modeling study using the recently published crystal structure of the West Nile NS3/NS2B protease complex (pdb code 2FP7) was conducted. We found that the crystal structure is relevant in explaining the observed SAR for this series of tetrapeptides, with the S1 and S2 pockets being the key peptide recognition sites. In general, a residue capable of both pi-stacking and hydrogen bonding is favored in the S1 pocket, while a positively charged residue is preferred in the S2 pocket. This study not only confirms the importance of the NS2B domain in substrate-based inhibitor binding of WNV, it also suggests that the crystal structure would provide useful guidance in the drug discovery process of related Flavivirus proteases, given the high degree of homology.

Aldehydes↗

Recognition of the four Watson-Crick base pairs in the DNA minor groove by synthetic ligands.

The design of synthetic ligands that read the information stored in the DNA double helix has been a long-standing goal at the interface of chemistry and biology. Cell-permeable small molecules that target predetermined DNA sequences offer a potential approach for the regulation of gene expression. Oligodeoxynucleotides that recognize the major groove of double-helical DNA via triple-helix formation bind to a broad range of sequences with high affinity and specificity. Although oligonucleotides and their analogues have been shown to interfere with gene expression, the triple-helix approach is limited to recognition of purines and suffers from poor cellular uptake. The subsequent development of pairing rules for minor-groove binding polyamides containing pyrrole (Py) and imidazole (Im) amino acids offers a second code to control sequence specificity. An Im/Py pair distinguishes G x C from C x G and both of these from A x T/T x A base pairs. A Py/Py pair specifies A,T from G,C but does not distinguish AT from T x A. To break this degeneracy, we have added a new aromatic amino acid, 3-hydroxypyrrole (Hp), to the repertoire to test for pairings that discriminate A x T from T x A. We find that replacement of a single hydrogen atom with a hydroxy group in a Hp/Py pairing regulates affinity and specificity by an order of magnitude. By incorporation of this third amino acid, hydroxypyrrole-imidazole-pyrrole polyamides form four ring-pairings (Im/Py, Py/Im, Hp/Py and Py/Hp) which distinguish all four Watson-Crick base pairs in the minor groove of DNA.

Amino Acids↗

Detection and differentiation of causative fungi of onychomycosis using PCR amplification and restriction enzyme analysis.

BACKGROUND: Onychomycosis, a fungal nail infection, has become one of the most important dermatophytoses. Unfortunately, a predictably successful diagnostic approach to onychomycosis does not yet exist. OBJECTIVE: The purpose of this study was to develop a deoxyribonucleic acid (DNA)-based diagnostic method to improve the sensitivity and specificity of the detection and differentiation of the pathogenic fungi of onychomycosis. METHODS: We attempted to detect fungi in the nail using polymerase chain reaction (PCR) primer systems that were designed in conserved sequences of the small ribosomal subunit 18S-rRNA genes shared by most fungi, and differentiated between species by restriction enzyme analysis of the amplified product. RESULTS: Fragments of the gene coding for 18S-rRNA were amplified successfully from medically important fungi species, but not from normal nails. Restriction fragment length polymorphism patterns using HaeIII endonuclease were sufficiently different to allow the recognition of individual species. CONCLUSIONS: The PCR-restriction enzyme analysis method appears to be a more sensitive detection and identification technique for onychomycosis than conventional methods, and has considerable diagnostic value.

Arthrodermataceae↗

A mutated intron sequence codes for an antigenic peptide recognized by cytolytic T lymphocytes on a human melanoma.

We have identified an antigen recognized on a human melanoma by autologous cytolytic T lymphocytes. It is encoded by a gene that is expressed in many normal tissues. Remarkably, the sequence coding for the antigenic peptide is located across an exon-intron junction. A point mutation is present in the intron that generates an amino acid change that is essential for the recognition of the peptide by the anti-tumor cytotoxic T lymphocytes. This observation suggests that the T-cell-mediated surveillance of the integrity of the genome may extend to some intronic regions.

Alleles↗

Transport of p-nitrophenyl-alpha-maltoside by the maltose transport system of Escherichia coli and its subsequent hydrolysis by a cytoplasmic alpha-maltosidase.

In wild-type Escherichia coli the activity of the maltose transport system is dependent on a periplasmic maltose-binding protein. It has been possible, however, to isolate mutants in which transport activity is mediated by the membrane components of the system and is no longer dependent on the periplasmic binding protein. In this manuscript we show that in these binding protein-independent strains, p-nitrophenyl-alpha-maltoside is a potent inhibitor of maltose transport. In contrast, p-nitrophenyl-alpha-maltoside is only a weak inhibitor of maltose transport in wild-type bacteria. In addition, we show that p-nitrophenyl-alpha-maltoside is transported by the binding protein-independent strains but not by wild-type bacteria. We were able to detect transport of this compound because there is a cytoplasmic enzyme that cleaves p-nitrophenyl-alpha-maltoside. This enzyme has not previously been described. We show that although the synthesis of this enzyme is subject to the same regulation as the components of the maltose regulon, and is MalT dependent, it is not coded for by a known mal gene. We refer to this enzyme as alpha-maltosidase. These results strengthen our proposal that the membrane components of the maltose transport system comprise a recognition site for maltose and related substrates.

ATP-Binding Cassette Transporters↗

mRNA splicing regulates human papillomavirus type 11 E1 protein production and DNA replication.

The papillomavirus replicative helicase E1 and the origin recognition protein E2 are required for efficient viral DNA replication. We fused the green fluorescent protein (GFP) to the human papillomavirus type 11 E1 protein either in a plasmid with the E1 coding region alone (nucleotides [nt] 832 to 2781) (pGFP-11E1) or in a plasmid containing both the E1 and E2 regions (nt 2723 to 3826) and the viral origin of replication (ori) (p11Rc). The former supported transient replication of an ori plasmid, whereas the latter was a self-contained replicon. Unexpectedly, these plasmids produced predominantly a cytoplasmic variant GFP or a GFP-E1 E4 protein, respectively. The majority of the mRNAs had an intragenic or intergenic splice from nt 847 to nt 2622 or from nt 847 to nt 3325, corresponding to the E2 or E1 E4 messages. pGFP-11E1dm and p11Rc-E1dm, mutated at the splice donor site, abolished these splices and increased GFP-E1 protein expression. Three novel, alternatively spliced, putative E2 mRNAs were generated in higher abundance from the mutated replicon than from the wild type. Relative to pGFP-11E1, low levels of pGFP-11E1dm supported more efficient replication, but high levels had a negative effect. In contrast, elevated E2 levels always increased replication. Despite abundant GFP-E1 protein, p11Rc-E1dm replicated less efficiently than the wild type. Collectively, these observations show that the E1/E2 ratio is as important as the E1 and E2 concentrations in determining the replication efficiency. These findings suggest that alternative mRNA splicing could provide a mechanism to regulate E1 and E2 protein expression and DNA replication during different stages of the virus life cycle.

DNA Replication↗

Interference with olfactory memory by visual and verbal tasks.

It has been claimed that olfactory memory is distinct from memory in other modalities. This study investigated the effectiveness of visual and verbal tasks in interfering with olfactory memory and included methodological changes from other recent studies. Subjects were allocated to one of four experimental conditions involving interference tasks [no interference task; visual task; verbal task; visual-plus-verbal task] and presented 15 target odours. Either recognition of the odours or free recall of the odour names was tested on one occasion, either within 15 minutes of presentation or one week later. Recognition and recall performance both showed effects of interference of visual and verbal tasks but there was no effect for time of testing. While the results may be accommodated within a dual coding framework, further work is indicated to resolve theoretical issues relating to task complexity.

Adolescent↗

Structure of the gene for a carbohydrate-binding receptor unique to rat kupffer cells.

The gene encoding the rat Kupffer cell receptor for fucose and galactose (Lehrman, M. A., and Hill, R. L. (1986) J. Biol. Chem. 261, 7419-7425 and Lehrman, M. A., Haltiwanger, R. S., and Hill, R. L. (1986) J. Biol. Chem. 261, 7426-7432) has been isolated and characterized. The nucleotide sequence of the 9.7-kilobase gene and of 1 kilobase of 5'-flanking sequence was determined. The 5' end of the receptor coding sequence was determined by primer extension and S1 mapping of the receptor transcript. Knowledge of the transcription start site and the 5'-flanking sequence allowed potential promoter elements to be identified. The Kupffer cell receptor coding sequence is encoded by seven exons that are split by six introns. The divisions among domains in the receptor can be correlated with divisions among exons in the gene. The carbohydrate recognition domain is encoded by three exons similar to those in the rat hepatic lectin, human Fc receptor for IgE, and rat cartilage proteoglycan core protein genes. A model for the evolution of the vertebrate lectin genes that have been analyzed is presented.

Amino Acid Sequence↗