Retinoids down-regulate telomerase and telomere length in a pathway distinct from leukemia cell differentiation

Human telomerase, a cellular reverse transcriptase (hTERT), is a nuclear ribonucleoprotein enzyme complex that catalyzes the synthesis and extension of telomeric DNA. This enzyme is specifically activated in most malignant tumors but is usually inactive in normal somatic cells, suggesting that telomerase plays an important role in cellular immortalization and tumorigenesis. Terminal maturation of tumor cells has been associated with the repression of telomerase activity. Using maturation-sensitive and -resistant NB4 cell lines, we analyzed the pattern of telomerase expression during the therapeutic treatment of acute promyelocytic leukemia (APL) by retinoids. Two pathways leading to the down-regulation of hTERT and telomerase activity were identified. The first pathway results in a rapid down-regulation of telomerase that is associated with retinoic acid receptor (RAR)-dependent maturation of NB4 cells. Furthermore, during NB4 cell maturation, obtained independently of RAR by retinoic X receptor (RXR)-specific agonists (rexinoids), no change in telomerase activity was observed, suggesting that hTERT regulation requires a specific signaling and occurs autonomously. A second pathway of hTERT regulation, identified in the RAR-responsive, maturation-resistant NB4-R1 cell line, results in a down-regulation of telomerase that develops slowly during two weeks of all-trans retinoic acid (ATRA) treatment. This pathway leads to telomere shortening, growth arrest, and cell death, all events that are overcome by ectopic expression of hTERT. These findings demonstrate a clear and full dissociation between the process of tumor cell maturation and the regulation of hTERT mRNA expression and telomerase activity by retinoids. We propose telomerase expression as an efficient and selective target of retinoids in the therapy of tumors.

Extreme morphological and ecological homoplasy in tropical salamanders

Fossorial salamanders typically have elongate and attenuated heads and bodies, diminutive limbs, hands and feet, and extremely elongate tails. Batrachoseps from California, Lineatriton from eastern México, and Oedipina from southern México to Ecuador, all members of the family Plethodontidae, tribe Bolitoglossini, resemble one another in external morphology, which has evolved independently. Whereas Oedipina and Batrachoseps are elongate because there are more trunk vertebrae, a widespread homoplasy (parallelism) in salamanders, the genus Lineatriton is unique in having evolved convergently by an alternate “giraffe-neck” developmental program. Lineatriton has the same number of trunk vertebrae as related, nonelongated taxa, but individual trunk vertebrae are elongated. A robust phylogenetic hypothesis, based on sequences of three mtDNA genes, finds Lineatriton to be deeply nested within a clade characterized by generalized ecology and morphology. Lineatriton lineolus, the only currently recognized taxon in the genus, shows unanticipated genetic diversity. Surprisingly, geographically separated populations of L. lineolus are not monophyletic, but are sister taxa of different species of the morphologically generalized genus Pseudoeurycea. Lineatriton, long thought to be a unique monospecific lineage, is polyphyletic. Accordingly, the specialized morphology of Lineatriton displays homoplasy at two hierarchical levels: (i) with respect to other elongate lineages in the family (convergence), and (ii) within what is currently recognized as a single taxon (parallelism). These evolutionary events are of adaptive significance because to invade the lowland tropics salamanders must be either arboreal or fossorial; the repeated evolution of elongation and attenuation has led to multiple lowland invasions.

Microsatellite spreading in the human genome: evolutionary mechanisms and structural implications.

Microsatellites are tandem repeat sequences abundant in the genomes of higher eukaryotes and hitherto considered as "junk DNA." Analysis of a human genome representative data base (2.84 Mb) reveals a distinct juxtaposition of A-rich microsatellites and retroposons and suggests their coevolution. The analysis implies that most microsatellites were generated by a 3'-extension of retrotranscripts, similar to mRNA polyadenylylation, and that they serve in turn as "retroposition navigators," directing the retroposons via homology-driven integration into defined sites. Thus, they became instrumental in the preservation and extension of primordial genomic patterns. A role is assigned to these reiterating A-rich loci in the higher-order organization of the chromatin. The disease-associated triplet repeats are mostly found in coding regions and do not show an association with retroposons, constituting a unique set within the family of microsatellite sequences.

Purification, cloning, and functional expression of sucrose:fructan 6-fructosyltransferase, a key enzyme of fructan synthesis in barley.

Fructans play an important role in assimilate partitioning and possibly in stress tolerance in many plant families. Sucrose:fructan 6-fructosyltransferase (6-SFT), an enzyme catalyzing the formation and extension of beta-2,6-linked fructans typical of grasses, was purified from barley (Hordeum vulgare L.). It occurred in two closely similar isoforms with indistinguishable catalytic properties, both consisting of two subunits with apparent masses of 49 and 23 kDa. Oligonucleotides, designed according to the sequences of tryptic peptides from the large subunit, were used to amplify corresponding sequences from barley cDNA. The main fragment generated was cloned and used to screen a barley cDNA expression library. The longest cDNA obtained was transiently expressed in Nicotiana plumbaginifolia protoplasts and shown to encode a functional 6-SFT. The deduced amino acid sequence of the cDNA comprises both subunits of 6-SFT. It has high similarity to plant invertases and other beta-fructosyl hydrolases but only little to bacterial fructosyltransferases catalyzing the same type of reaction as 6-SFT.

Detection of HIV-1 RNA by nucleic acid sequence-based amplification combined with fluorescence correlation spectroscopy

Nucleic acid sequence-based amplification (NASBA) has proved to be an ultrasensitive method for HIV-1 diagnosis in plasma even in the primary HIV infection stage. This technique was combined with fluorescence correlation spectroscopy (FCS) which enables online detection of the HIV-1 RNA molecules amplified by NASBA. A fluorescently labeled DNA probe at nanomolar concentration was introduced into the NASBA reaction mixture and hybridizing to a distinct sequence of the amplified RNA molecule. The specific hybridization and extension of this probe during amplification reaction, resulting in an increase of its diffusion time, was monitored online by FCS. As a consequence, after having reached a critical concentration of 0.1–1 nM (threshold for unaided FCS detection), the number of amplified RNA molecules in the further course of reaction could be determined. Evaluation of the hybridization/extension kinetics allowed an estimation of the initial HIV-1 RNA concentration that was present at the beginning of amplification. The value of initial HIV-1 RNA number enables discrimination between positive and false-positive samples (caused for instance by carryover contamination)—this possibility of discrimination is an essential necessity for all diagnostic methods using amplification systems (PCR as well as NASBA). Quantitation of HIV-1 RNA in plasma by combination of NASBA with FCS may also be useful in assessing the efficacy of anti-HIV agents, especially in the early infection stage when standard ELISA antibody tests often display negative results.

How the owl resolves auditory coding ambiguity

The barn owl (Tyto alba) uses interaural time difference (ITD) cues to localize sounds in the horizontal plane. Low-order binaural auditory neurons with sharp frequency tuning act as narrow-band coincidence detectors; such neurons respond equally well to sounds with a particular ITD and its phase equivalents and are said to be phase ambiguous. Higher-order neurons with broad frequency tuning are unambiguously selective for single ITDs in response to broad-band sounds and show little or no response to phase equivalents. Selectivity for single ITDs is thought to arise from the convergence of parallel, narrow-band frequency channels that originate in the cochlea. ITD tuning to variable bandwidth stimuli was measured in higher-order neurons of the owl’s inferior colliculus to examine the rules that govern the relationship between frequency channel convergence and the resolution of phase ambiguity. Ambiguity decreased as stimulus bandwidth increased, reaching a minimum at 2–3 kHz. Two independent mechanisms appear to contribute to the elimination of ambiguity: one suppressive and one facilitative. The integration of information carried by parallel, distributed processing channels is a common theme of sensory processing that spans both modality and species boundaries. The principles underlying the resolution of phase ambiguity and frequency channel convergence in the owl may have implications for other sensory systems, such as electrolocation in electric fish and the computation of binocular disparity in the avian and mammalian visual systems.

The nucleocapsid protein specifically anneals tRNALys-3 onto a noncomplementary primer binding site within the HIV-1 RNA genome in vitro

HIV type 1 (HIV-1) specifically uses host cell tRNALys-3 as a primer for reverse transcription. The 3′ 18 nucleotides of this tRNA are complementary to a region on the HIV RNA genome known as the primer binding site (PBS). HIV-1 has a strong preference for maintaining a lysine-specific PBS in vivo, and viral genomes with mutated PBS sequences quickly revert to be complementary to tRNALys-3. To investigate the mechanism for the observed PBS reversion events in vitro, we examined the capability of the nucleocapsid protein (NC) to anneal various tRNA primer sequences onto either complementary or noncomplementary PBSs. We show that NC can anneal different full-length tRNAs onto viral RNA transcripts derived from the HIV-1 MAL or HXB2 isolates, provided that the PBS is complementary to the tRNA used. In contrast, NC promotes specific annealing of only tRNALys-3 onto an RNA template (HXB2) whose PBS sequence has been mutated to be complementary to the 3′ 18 nt of human tRNAPro. Moreover, HIV-1 reverse transcriptase extends this binary complex from the proline-specific PBS. The formation of the noncomplementary binary complex does not occur when a chimeric tRNALys/Pro containing proline-specific D and anticodon domains is used as the primer. Thus, elements outside the acceptor-TΨC domains of tRNALys-3 play an important role in preferential primer use in vitro. Our results support the hypothesis that mutant PBS reversion is a result of tRNALys-3 annealing onto and extension from a PBS that specifies an alternate host cell tRNA.

Functional organization of the hippocampal memory system

In humans declarative or explicit memory is supported by the hippocampus and related structures of the medial temporal lobe working in concert with the cerebral cortex. This paper reviews our progress in developing an animal model for studies of cortical–hippocampal interactions in memory processing. Our findings support the view that the cortex maintains various forms of memory representation and that hippocampal structures extend the persistence and mediate the organization of these codings. Specifically, the parahippocampal region, through direct and reciprocal interconnections with the cortex, is sufficient to support the convergence and extended persistence of cortical codings. The hippocampus itself is critical to the organization cortical representations in terms of relationships among items in memory and in the flexible memory expression that is the hallmark of declarative memory.

A high-resolution physical map of human chromosome 11.

The development of a highly reliable physical map with landmark sites spaced an average of 100 kbp apart has been a central goal of the Human Genome Project. We have approached the physical mapping of human chromosome 11 with this goal as a primary target. We have focused on strategies that would utilize yeast artificial chromosome (YAC) technology, thus permitting long-range coverage of hundreds of kilobases of genomic DNA, yet we sought to minimize the ambiguities inherent in the use of this technology, particularly the occurrence of chimeric genomic DNA clones. This was achieved through the development of a chromosome 11-specific YAC library from a human somatic cell hybrid line that has retained chromosome 11 as its sole human component.To maximize the efficiency of YAC contig assembly and extension, we have employed an Alu-PCR-based hybridization screening system. This system eliminates many of the more costly and time-consuming steps associated with sequence tagged site content mapping such as sequencing, primer production, and hierarchical screening, resulting in greater efficiency with increased throughput and reduced cost. Using these approaches, we have achieved YAC coverage for >90% of human chromosome 11, with an average intermarker distance of <100 kbp. Cytogenetic localization has been determined for each contig by fluorescent in situ hybridization and/or sequence tagged site content. The YAC contigs that we have generated should provide a robust framework to move forward to sequence-ready templates for the sequencing efforts of the Human Genome Project as well as more focused positional cloning on chromosome 11.