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PubMed2001

The murine genome contains one functional gene and two pseudogenes coding for the 16 kDa proteolipid subunit of vacuolar H(+)-ATPase.

We cloned the genomic genes encoding the murine 16 kDa subunit (proteolipid, PL16) of vacuolar H(+)-ATPase (V-ATPase) and determined their nucleotide sequences. At least three independent genes were found in the murine genome. One gene consisted of three exons and was largely identical in sequence to that of PL16 cDNA reported previously (Hanada et al., Biochem. Biophys. Res. Commun. 176 (1991) 1062). In the 5'-flanking region of this gene, several possible transcriptional cis-elements were found. TATA and CAAT sequences were not found, which is characteristic for promoters of house-keeping genes. The other two genes identified did not contain introns. One of these genes had an open reading frame that potentially encoded PL16 but contained six amino acid substitutions and a frame-shift mutation that would result in a truncated protein unable to participate in V-ATPase activity. The other gene had the same sequence in the reading frame as that in the cDNA. However, this gene contained a polyA sequence at the same position where polyA is normally added to mRNA. The gene also had 15 bp repetitive sequences near the transcription initiation site and next to the polyA sequence. These observations suggest that this gene may have been generated by the insertion of reverse-transcribed double-stranded cDNA, as is usually observed for pseudogenes. In conclusion, there is a single functional PL16 gene and two pseudogenes in the murine genome. It is unlikely that PL16 isoforms contribute to variation in V-ATPase function.

Amino Acid Sequence↗

PubMed1996

Why sequence the genome of Mycobacterium tuberculosis?

Radical measures are required to prevent the grim predictions of the World Health Organisation for the deterioration of the global tuberculosis epidemic in the next century from becoming reality. Study of the nerve centre of the tubercle bacillus, its genome, by means of systematic deoxyribonucleic acid sequence analysis will provide a wealth of information about Mycobacterium tuberculosis that will undoubtedly fuel the next generation of research. In the coming year, this highly cost-effective approach will deliver an unprecedented amount of knowledge to catalyse the development of new, more efficient diagnostic tools and therapeutic interventions to detect, control and, ultimately, eliminate tuberculosis.

Animals↗

PubMed1978

Developmental biochemistry of cotton seed embryogenesis and germination: x. Nitrogen flow from arginine to asparagine in germination.

The enzymic basis for the flow of nitrogen from arginine to asparagine during the first 3 days of germination has been measured in extracts from cotton (Gossypium hirsutum) cotyledons. Evidence that asparagine synthetase regulates asparagine accumulation in germination (for transport to the axis) is presented. Further, evidence that the bulk of the nitrogen passed from one generation to the next in dicots is through an asparagine cycle involving the following sequence asparagine --> arginine --> storage protein --> arginine --> asparagine is discussed.

Journal Article↗

PubMed1987

Spatial distribution of twitch and tonic fibres in a snake muscle one myofibre thick.

The spatial distribution of twitch and tonic fibres in a snake muscle one myofibre thick (ventral costocutaneous) has been investigated. It was found that small groups of like fibres were favoured at the expense of larger groups when compared with sequences generated by a computer in which fibres were distributed randomly. The number of times that like fibres occurred next to one another was used as another measure of their spatial distribution. The number of adjacencies of like fibres was less than the expected number of adjacencies determined both by random sequences and by probability theory. The expected number of adjacencies determined by means of random sequences and a priori probability theory were almost identical. This dispersion of like fibres may reflect processes that occur during muscle development and may have functional implications.

Animals↗

PubMed2004

From ORFeome to biology: a functional genomics pipeline.

As several model genomes have been sequenced, the elucidation of protein function is the next challenge toward the understanding of biological processes in health and disease. We have generated a human ORFeome resource and established a functional genomics and proteomics analysis pipeline to address the major topics in the post-genome-sequencing era: the identification of human genes and splice forms, and the determination of protein localization, activity, and interaction. Combined with the understanding of when and where gene products are expressed in normal and diseased conditions, we create information that is essential for understanding the interplay of genes and proteins in the complex biological network. We have implemented bioinformatics tools and databases that are suitable to store, analyze, and integrate the different types of data from high-throughput experiments and to include further annotation that is based on external information. All information is presented in a Web database (http://www.dkfz.de/LIFEdb). It is exploited for the identification of disease-relevant genes and proteins for diagnosis and therapy.

Animals↗

PubMed1998

Sequence peculiarity of gnetalean legumin-like seed storage proteins.

The development of seeds as a specialized organ for the nutrition, protection, and dispersal of the next generation was an important step in the evolution of land plants. Seed maturation is accompanied by massive synthesis of storage compounds such as proteins, starch, and lipids. To study the processes of seed storage protein evolution we have partially sequenced storage proteins from maturing seeds of representatives from the gymnosperm genera Gnetum, Ephedra, and Welwitschia-morphologically diverse and unusual taxa that are grouped in most formal systems into the common order Gnetales. Based on partial N-terminal amino acid sequences, oligonucleotide primers were derived and used for PCR amplification and cloning of the corresponding cDNAs. We also describe the structure of the nuclear gene for legumin of Welwitschia mirabilis. This first gnetalean nuclear gene structure contains introns in only two of the four conserved positions previously characterized in other spermatophyte legumin genes. The distinct phylogenetic status of the gnetalean taxa is also reflected in a sequence peculiarity of their legumin genes. A comparative analysis of exon/intron sequences leads to the hypothesis that legumin genes from Gnetales belong to a monophyletic evolutionary branch clearly distinct from that of legumin genes of extant Ginkgoales and Coniferales as well as from all angiosperms.

Amino Acid Sequence↗

PubMed2004

Human chromosome 7 circa 2004: a model for structural and functional studies of the human genome.

Human chromosome 7 is arguably amongst the most comprehensively characterized segments of the human genome. By microscopic examination, it belongs to the medium-sized group C submetacentric class, and historical studies involving chromosome-length measurements estimated that it accounts for approximately 5.3% of the human genome (or 160 Mb). Early successes in molecular genetics led to the identification of some of its biomedically important genes, including the T-cell receptor and homeobox families as well as the erythropoietin and cystic fibrosis genes. The Human Genome Project brought chromosome 7-specific and genome-wide initiatives, generating a wealth of genomic resources that have revealed the presence of over 350 disease-associated genes. Two distinct assemblies of the chromosome 7 sequence have been generated-one based largely on mapped large-insert clones and the other based on an integrated whole-genome shotgun sequencing strategy. These two sequences are mainly identical (<1% difference), and both estimate the unit length of chromosome 7 to be just over 158 Mb, remarkably similar to the originally predicted size. Systematic annotation efforts have anchored to the sequence, amongst many features, over 900 known genes and some 1000 other gene structures, as well as over 650 chromosomal breakpoints identified in patients with characterized phenotypic differences. Chromosome 7 has also been shown to contain the highest content of intra-autosomal segmental duplications in the human genome. The orthologous regions of roughly 22 Mb of chromosome 7 are currently being sequenced in multiple other vertebrate species. Examining these comparative sequence data, in conjunction with the other accumulating genomic information about these regions and the rest of the chromosome, should provide a model for the next generation of structural and functional analyses of the human genome.

Chromosomes, Human, Pair 7↗

PubMed1998

Orphan G-protein-coupled receptors: the next generation of drug targets?

The pharmaceutical industry has readily embraced genomics to provide it with new targets for drug discovery. Large scale DNA sequencing has allowed the identification of a plethora of DNA sequences distantly related to known G protein-coupled receptors (GPCRs), a superfamily of receptors that have a proven history of being excellent therapeutic targets. In most cases the extent of sequence homology is insufficient to assign these 'orphan' receptors to a particular receptor subfamily. Consequently, reverse molecular pharmacological and functional genomic strategies are being employed to identify the activating ligands of the cloned receptors. Briefly, the reverse molecular pharmacological methodology includes cloning and expression of orphan GPCRs in mammalian cells and screening these cells for a functional response to cognate or surrogate agonists present in biological extract preparations, peptide libraries, and complex compound collections. The functional genomics approach involves the use of 'humanized yeast cells, where the yeast GPCR transduction system is engineered to permit functional expression and coupling of human GPCRs to the endogenous signalling machinery. Both systems provide an excellent platform for identifying novel receptor ligands. Once activating ligands are identified they can be used as pharmacological tools to explore receptor function and relationship to disease.

Animals↗

PubMed1998

Sequential phosphorylation of Tau by glycogen synthase kinase-3beta and protein kinase A at Thr212 and Ser214 generates the Alzheimer-specific epitope of antibody AT100 and requires a paired-helical-filament-like conformation.

AT100 is a monoclonal antibody highly specific for phosphorylated Tau in Alzheimer paired helical filaments. Here we show that the epitope is generated by a complex sequence of sequential phosphorylation, first of Ser199, Ser202 and Thr205 (around the epitope of antibody AT8), next of Thr212 by glycogen synthase kinase (GSK)-3beta (a proline-directed kinase), then of Ser214 by protein kinase A (PKA). Conversely, if Ser214 is phosphorylated first it protects Thr212 and the Ser-Pro motifs around the AT8 site against phosphorylation, and the AT100 epitope is not formed. The generation of the AT100 epitope requires a conformation of tau induced by polyanions such as heparin, RNA or poly(Glu), conditions which also favor the formation of paired helical filaments. The Alzheimer-like phosphorylation can be induced by brain extracts. In the extract, the kinases responsible for generating the AT100 epitope are GSK-3beta and PKA, which can be inhibited by their specific inhibitors LiCl and RII, respectively. A cellular model displaying the reaction with AT100 is presented by Sf9 insect cells transfected with Tau. Knowledge of the events and kinases generating the AT100 epitope in cells might allow us to study the degeneration of the cytoskeleton in Alzheimer's disease.

Alzheimer Disease↗

PubMed1984

Microinjection of simian adenovirus SA7 (C-8) DNA into the mouse zygotes: differential distribution of viral DNA in organs.

A simian adenovirus SA7 (C-8) DNA was microinjected into fertilized mouse eggs. Thirty-five mice derived from eggs injected with SA7 DNA were screened for the presence of the adenovirus genome in their liver DNA. Eighteen of these mice contained the virus-specific sequences. SA7 DNA was detected in some tissues, but in all cases, viral sequences were absent from muscle and heart DNA. Viral DNA was inherited by 50-70% of the next generation. One mouse that contained about 1 copy of SA7 DNA per haploid genome has been shown to pass it on to five generations, although the integrated viral DNA sustained a considerable structural change between F1 and F3. RNA analysis in various organs of 12 mice has shown the transcription of SA7 DNA to be very infrequent: only in the kidney of one mouse and in the spleen of another did RNA contain SA7-specific sequences.

Adenoviridae↗

PubMed2000

In vitro selection of aptamers that act with Zn2+.

An in vitro selection was carried out with Zn2+ to isolate novel RNA molecules, zinc-dependent aptamers, that bind to HIV-1 Tat protein. RNAs bound to Tat were collected by using a nitrocellulose filter from a library of random RNAs and regenerated to the next generation of the RNA library by subsequent reverse transcription, polymerase chain reaction, and transcription. Sequences of the selected RNAs were determined after 6 and 12 rounds of the selection. The control clones after normal selection procedure with Mg2+ had a consensus UUG that resembled essential sequences of TAR or Arg aptamers. On the other hand, many unique sequences were revealed from a library selected with Zn2+ and the RNA with most abundant sequence (clone 31) bound to Tat tightly only when Zn2+ existed. The secondary structure of clone 31 RNA was predicted by using a computational prediction with our thermodynamic parameters and enzymatic scission of the RNA. Several model RNAs were prepared and the binding property of these RNAs to Tat were investigated. As a result, all the model RNAs did not reproduce the binding property of clone 31. Therefore, the Tat aptamer that acts with Zn2+ should require a relatively longer region of the sequence which is able to offer tertiary cooperation of several motifs for the binding.

Base Sequence↗

PubMed2005

Metabolic engineering of the mevalonate and non-mevalonate isopentenyl diphosphate-forming pathways for the production of health-promoting isoprenoids in tomato.

The genetic manipulation of both the mevalonic acid (MVA) and methylerythritol-4-phosphate (MEP) pathways, leading to the formation of isopentenyl diphosphate (IPP), has been achieved in tomato using 3-hydroxymethylglutaryl CoA (hmgr-1) and 1-deoxy-d-xylulose-5-phosphate synthase (dxs) genes, respectively. Transgenic plants containing an additional hmgr-1 from Arabidopsis thaliana, under the control of the cauliflower mosaic virus (CaMV) 35S constitutive promoter, contained elevated phytosterols (up to 2.4-fold), but IPP-derived isoprenoids in the plastid were unaltered. Transgenic lines containing a bacterial dxs targeted to the plastid with the tomato dxs transit sequence resulted in an increased carotenoid content (1.6-fold), which was inherited in the next generation. Phytoene and beta-carotene exhibited the greatest increases (2.4- and 2.2-fold, respectively). Extra-plastidic isoprenoids were unaffected in these lines. These data are discussed with respect to the regulation, compartmentalization and manipulation of isoprenoid biosynthetic pathways and their relevance to plant biotechnology.

Journal Article↗

PubMed2004

Dynamic allocation of visual attention during the execution of sequences of saccades.

Laboratory tasks used to study vision and attention usually require steady fixation, while natural visual processing occurs during the brief pauses between successive saccades. We studied vision and attentional allocation during intersaccadic pauses as subjects made repetitive sequences of saccades. Displays contained six outline squares located along the perimeter of an imaginary circle (diam 4 degrees). Saccades were made in sequence to every other square. The visual task was to identify the orientation (2AFC) of a Gabor test stimulus that appeared briefly (91 ms) along with superimposed noise in one of the squares during a randomly selected intersaccadic pause. Gabor location was cued in advance and noise frames were presented in all squares. Contrast thresholds during intersaccadic pauses were as much as 2-3 times higher than during steady fixation with comparable cueing. Thresholds improved over time during the intersaccadic pause, and the lowest extrafoveal thresholds (statistically indistinguishable from those at the same locations during steady fixation) were found for the location that was to be the target of the next saccade in the sequence. These results show that vision during intersaccadic pauses varies over space and time due to changes in the distribution of attention, as well as to visual suppression that may be related to the execution of the saccades themselves. Generation of sequences of accurate saccades encouraged a strategy of attentional allocation in which resources were dedicated primarily to the goal of the next saccade, leaving little attention for processing objects at other locations.

Attention↗

PubMed2004

Development of rationally designed nucleic acid signatures for microbial pathogens.

The detection and identification of microbial pathogens are critical challenges in clinical medicine and public health surveillance. Advances in genome analysis technology are providing an unprecedented amount of information about bacterial and viral organisms, and hold great potential for pathogen detection and identification. In this paper, a rational approach to the development and application of nucleic acid signatures is described based on phylogenetically informative sequence features, especially single nucleotide polymorphisms. The computational tools that are available to enable the development of the next generation of microbial molecular signatures for clinical diagnostics and infectious disease surveillance are reviewed and the impact on public health and national security will be discussed.

Bacteria↗

PubMed2003

Molecular determinants of high-affinity drug binding to HERG channels.

Human ether-a-go-go-related gene (HERG) subunits mediate a K+ current that is required for normal repolarization of the cardiac action potential. The unintentional inhibition of HERG currents by numerous medications results in prolongation of the QT interval, as measured on the electrocardiogram, and is associated with increased risk of patients suffering from life-threatening cardiac arrhythmias. QT interval prolongation is considered a major safety concern by worldwide drug regulatory bodies, and early detection of new compounds with this undesirable side effect has become an important objective for pharmaceutical companies. New studies are shedding light on the structural basis of drug binding and the gating-dependent repositioning of key residues in the inner cavity of HERG, which are responsible for the unusual sensitivity of HERG to pharmacological agents. Insights from these studies may help develop novel strategies to reduce the proarrhythmic potential of the next generation of drugs.

Amino Acid Sequence↗

PubMed1997

Adeno-associated virus type 2 DNA replication in vivo: mutation analyses of the D sequence in viral inverted terminal repeats.

The adeno-associated virus type 2 (AAV) genome contains inverted terminal repeats (ITRs) of 145 nucleotides. The terminal 125 nucleotides of each ITR form palindromic hairpin (HP) structures that serve as primers for AAV DNA replication. These HP structures also play an important role in integration as well as rescue of the proviral genome from latently infected cells or from recombinant AAV plasmids. Each ITR also contains a stretch of 20 nucleotides, designated the D sequence, that is not involved in HP structure formation. We have recently shown that the D sequence plays a crucial role in high-efficiency rescue, selective replication, and encapsidation of the AAV genome and that a host cell protein, designated the D sequence-binding protein (D-BP), specifically interacts with this sequence (X.-S. Wang, S. Ponnazhagan, and A. Srivastava, J. Virol. 70:1668-1677, 1996). We have now performed mutational analyses of the D sequences to evaluate their precise role in viral DNA rescue, replication, and packaging. We report here that 10 nucleotides proximal to the HP structure in each of the D sequences are necessary and sufficient to mediate high-efficiency rescue, replication, and encapsidation of the viral genome in vivo. In in vitro studies, the same 10 nucleotides were found to be required for specific interaction with D-BP, but viral Rep protein-mediated cleavage at the functional terminal resolution site is independent of these sequences. These data suggest that AAV replication and terminal resolution functions can be uncoupled and that the lack of efficient replication of AAV DNA may not be a consequence of impaired resolution of the viral ITRs. These studies further illustrate that the D sequence-D-BP interaction plays an important role in the AAV life cycle and indicate that it may be possible to develop the next generation of AAV vectors capable of encapsidating larger pieces of DNA.

DNA Replication↗

PubMed1986

Consecutive DNA terminator sequencing by using enzymatically generated primers.

An improved strategy for the dideoxy chain termination sequencing of M13 recombinant DNA using enzymatically generated primers is presented. It involves synchronous extension of the universal primer with the Klenow fragment of DNA polymerase I at an average rate of 200 deoxynucleoside triphosphates per minute to the immediate downstream region of a preselected restriction enzyme site. Subsequent cleavage with the restriction enzyme generates the short primers with homogeneous 5' ends which can be used for further sequencing. The next restriction sites are selected in the newly sequenced regions of DNA by means of a microcomputer. By repeating this primer extension-cleavage-sequencing strategy sequences altogether about 6 kb long from several recombinant single-stranded M13 DNAs have been determined by using twenty restriction enzymes with different specificities.

Base Sequence↗

PubMed1996

Evolving cooperation: the role of individual recognition.

To evaluate the role of individual recognition in the evolution of cooperation, we formulated and analyzed genetic algorithm model (EvCo) for playing the Iterated Prisoner's Dilemma (IPD) game. Strategies compete against each other during each generation, and successful strategies contribute more of their attributes to the next generation. Each strategy is encoded on a 'chromosome' that plays the IPD, responding to the sequences of most recent responses by the interacting individuals (chromosomes). The analysis reported in this paper considered different memory capabilities (one to five previous interactions), pairing continuities (pairs of individuals remain together for about one, two, five, or 1000 consecutive interactions), and types of individual recognition (recognition capability was maximal, nil, or allowed to evolve between these limits). Analysis of the results focused on the frequency of mutual cooperation in pairwise interactions (a good indicator of overall success in the IPD) and on the extent to which previous responses by the focal individual and its partner were associated with the partner's identity (individual recognition). Results indicated that a fixed, substantial amount of individual recognition could maintain high levels of mutual cooperation even at low pairing continuities, and a significant but limited capability for individual recognition evolved under selection. Recognition generally increased mutual cooperation more when the recent responses of individuals other than the current partner were ignored. Titrating recognition memory under selection using a fitness cost suggested that memory of the partner's previous responses was more valuable than memory of the focal's previous responses. The dynamics produced to date by EvCo are a step toward understanding the evolution of social networks, for which additional benefits associated with group interactions must be incorporated.

Algorithms↗