Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “third generation sequencing”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,045 records · Page 58Linked to original sources
PubMed1995

The Drosophila 63F early puff contains E63-1, an ecdysone-inducible gene that encodes a novel Ca(2+)-binding protein.

Pulses of ecdysone at the end of Drosophila larval development dramatically reprogram gene expression as they signal the onset of metamorphosis. Ecdysone directly induces several early puffs in the salivary gland polytene chromosomes that, in turn, activate many late puffs. Three early puffs, at 2B5, 74EF, and 75B, have been studied at the molecular level. Each contains a single ecdysone primary-response gene that encodes a family of widely expressed transcription factors. We report here a molecular characterization of the 63F early puff. Unexpectedly, we have found this locus to be significantly different from the previously characterized early puff loci. First, the 63F puff contains a pair of ecdysone-inducible genes that are transcribed in the larval salivary glands: E63-1 and E63-2. Second, E63-1 induction in late third instar larvae appears to be highly tissue-specific, restricted to the salivary gland. Third, E63-1 encodes a novel Ca(2+)-binding protein related to calmodulin. The discovery of an ecdysone-inducible Ca(2+)-binding protein provides a foundation for integrating steroid hormone and calcium second messenger signaling pathways and generates an additional level for potential regulation of the ecdysone response.

Amino Acid Sequence↗
PubMed2002

High-throughput GLGI procedure for converting a large number of serial analysis of gene expression tag sequences into 3' complementary DNAs.

Serial analysis of gene expression (SAGE) is a powerful technique for genome-wide analysis of gene expression. However, two-thirds of SAGE tags cannot be used directly for gene identification for two reasons. First, many SAGE tags match several known expressed sequences, owing to the short length of SAGE tag sequences. Second, many SAGE tags do not match any known expressed sequences, presumably because the sequences corresponding to these SAGE tags have not been identified. These two problems can be solved by extension of the SAGE tags into 3' complementary DNAs (cDNAs) by use of the GLGI technique (generation of longer cDNA fragments from SAGE tags for gene identification). We have improved the original GLGI technique into a high-throughput procedure for simultaneous conversion of a large number of SAGE tags into corresponding 3' cDNAs. The whole process is simple, rapid, low-cost, and highly efficient, as shown by our use of this procedure for analyzing hundreds of SAGE tags. In addition to identifying the correct gene for SAGE tags with multiple matches, GLGI can be used for large-scale identification of novel genes by converting novel SAGE tags into 3' cDNAs. Applying this high-throughput procedure should accelerate the rate of gene identification significantly in the human and other eukaryotic genomes.

Animals↗
PubMed1977

Germ line integration of moloney leukemia virus: effect of homozygosity at the m-mulV locus.

Mice genetically transmitting the exogenous Moloney leukemia virus (M-MuLV) have been previously derived. These animals carried one copy of M-MuLV DNA in their germ line and were heterozygous for the M-MuLV locus (Jaenisch, 1976). Experiments were performed to investigate whether homozygosity at the M-MuLV locus would be compatible with normal development. Animals heterozygous for the M-MuLV locus were mated [female (+/-) X male(+/-)] and the genotype of the offspring was analyzed. Molecular hybridization experiments revealed three classes of offspring carrying two copies (++), one copy (+/-) and no (--) M-MuLV-specific DNA sequences, respectively, in their liver DNA. Genetic experiments indicated that males of the first class transmitted the virus to 100% of their offspring, males of the second class to 50% and males of the third class not at all when mated with normal females. These results demonstrated that homozygosity at the M-MuLV locus has no detectable effect on normal development of the animals and that the M-MuLV gene is transmitted from one generation to the next strictly according to Mendelian expectations. Development of M-MuLV-induced leukemia is not influenced by the genotype of these animals--that is, animals carrying two or one copies of M-MuLV in their germ line or animals congenitally infected from the mother developed disease at similar rates.

Animals↗
PubMed2006

Modification of the trypsin-dependent cleavage activation site of the human metapneumovirus fusion protein to be trypsin independent does not increase replication or spread in rodents or nonhuman primates.

The contribution of cleavage activation of the fusion F protein of human metapneumovirus (HMPV) to replication and pathogenicity in rodents and nonhuman primates was investigated. Recombinant HMPVs were generated in which the naturally occurring trypsin-dependent cleavage sequence (R-Q-S-R downward arrow) was replaced by each of three sequences whose cleavage in vitro does not depend upon added trypsin. Two of these were multibasic sequences derived from avian metapneumovirus type A (R-R-R-R) or type C (R-K-A-R), with the former containing the consensus furin protease cleavage motif (R-X-R/K-R downward arrow). The third one (R-Q-P-R) was derived from a recently described trypsin independent HMPV isolate (J. H. Schickli, J. Kaur, N. Ulbrandt, R. R. Spaete, and R. S. Tang, J. Virol. 79:10678-10689, 2005). To preclude the possibility of conferring even greater virulence to this significant human pathogen, the modifications were done in an HMPV variant that was attenuated by the deletion of two of the three envelope glycoproteins, SH and G. Each of the introduced cleavage sequences conferred trypsin independent F cleavage and growth to HMPV in vitro. However, they differed in the efficiency of trypsin independent growth and plaque formation in vitro: R-R-R-R > R-K-A-R > R-Q-P-R > R-Q-S-R. The R-R-R-R mutant was the only one whose growth in vitro was not augmented by added trypsin, indicative of highly efficient trypsin independent cleavage. When inoculated intranasally into hamsters, there was essentially no difference in the magnitude of replication in the upper or lower respiratory tract between the mutants, and virus was not detected in organs outside of the respiratory tract. Evaluation of the most cleavage-efficient mutant, R-R-R-R, in African green monkeys showed that there was no detectable change in the magnitude of replication in the upper and lower respiratory tract or in immunogenicity and protective efficacy against HMPV challenge. These results suggest that cleavage activation is not a major determinant of HMPV virulence.

Amino Acid Sequence↗
PubMed2000

Analyzing human random time generation behavior: a methodology and a computer program.

In the present article, it is argued that in addition to the traditional random generation tasks discussed by Towse and Neil (1998), random time interval generation tasks should be considered as useful alternatives, because they allow a better empirical control of the executive task component in dual-task situations. First, a framework for discussing randomness over time is presented. Then, the article goes on to present three methods for the analysis of such tasks. A first method is based on the correlation between the intervals produced. The second method calculates the approximate entropy, and the third method converts the time sequences into binary sequences and estimates the statistical properties of the sequence on the basis of these binary data. A principal components analysis on 19 different measures based on 1,381 sequences produced in a number of single-task and dual-task experiments shows that the proposed measures form two general clusters, one related to output probability, perseveration, and alternation, and one related to sequential commonalities. The article also briefly describes a computer program that implements these methodologies.

Humans↗
PubMed2006

Pure and mixed genetic lines of Saccharomyces bayanus and Saccharomyces pastorianus and their contribution to the lager brewing strain genome.

The yeast species Saccharomyces bayanus and Saccharomyces pastorianus are of industrial importance since they are involved in the production process of common beverages such as wine and lager beer; however, they contain strains whose variability has been neither fully investigated nor exploited in genetic improvement programs. We evaluated this variability by using PCR-restriction fragment length polymorphism analysis of 48 genes and partial sequences of 16. Within these two species, we identified "pure" strains containing a single type of genome and "hybrid" strains that contained portions of the genomes from the "pure" lines, as well as alleles termed "Lager" that represent a third genome commonly associated with lager brewing strains. The two pure lines represent S. uvarum and S. bayanus, the latter a novel group of strains that may be of use in strain improvement programs. Hybrid lines identified include (i) S. cerevisiae/S. bayanus/Lager, (ii) S. bayanus/S. uvarum/Lager, and (iii) S. cerevisiae/S. bayanus/S. uvarum/Lager. The genome of the lager strains may have resulted from chromosomal loss, replacement, or rearrangement within the hybrid genetic lines. This study identifies brewing strains that could be used as novel genetic sources in strain improvement programs and provides data that can be used to generate a model of how naturally occurring and industrial hybrid strains may have evolved.

Beer↗
PubMed2006

Enhanced stability of recombinant keratinocyte growth factor by mutagenesis.

Native sequence keratinocyte growth factor (KGF) is fairly unstable, as manifested by the loss of the monomeric native protein accompanied by the accumulation of aggregated species during storage at moderate temperatures. Several different types of analogs were generated and the storage stability of the protein assessed. In the first type of analog one or more of the five cysteinyl residues in KGF were replaced; in the second class the N-terminal residues that included the first disulfide bond were deleted. Both of these types of analogs involved removal of the disulfide bond between cysteines 1 and 15. The third group involved mutating one of the basic amino acids located in a cluster of positive charges (involved in heparin binding) around Arg144 to a neutral or acidic amino acyl residue. Among the cysteine replacement analogs, the double mutation of Cys1 and 15 to Ser resulted in significantly increased stability without compromising the mitogenic activity, while Cys to Ser mutations at other positions were either destabilizing or had no effect. Deletion of the 15, 23 or 27 N-terminal amino acyl residues also increased the stability of the protein. The activity of the analogs was not affected by the deletion of 15 or 23 amino acids, but it was significantly decreased upon removal of the 27 N-terminal amino acyl residues. Much greater stability was achieved by mutation of the basic amino acids, especially Arg144, to Glu or Gln, but this increase in stability was accompanied by large decrease in activity. The analog with the 23 N-terminal amino acyl residues deleted represents one of the best compromises between increased stability and retention of activity.

Amino Acid Sequence↗
PubMed1990

Isolation and characterization of the third complement component of axolotl (Ambystoma mexicanum).

1. Using a monoclonal anti-human C3 antibody and a polyclonal anti-cobra venom factor antibody as probes, a protein homologous to the mammalian third complement component (C3) was purified from axolotl plasma and found to be axolotl C3. 2. Axolotl C3 consists of two polypeptide chains (Mr = 110,000 and 73,000) linked by disulfide bonds. An internal thiolester bond in the alpha chain was identified by the incorporation of [14C]methylamine and NH2-terminal sequence from the C3d fragment of C3. 3. Digestion of C3 by trypsin resulted in the cleavage of both the alpha and beta chains, generating fragments with a cleavage pattern similar to that of human C3. 4. The amino acid composition of axolotl C3 and the amino acid sequences of the thiolester site (and the surrounding amino acids), the cleavage site for the C3-convertase, and one of the factor I cleavage sites are similar to C3 from other vertebrates. 5. In contrast to human C3, which has concanavalin A binding carbohydrates on both the alpha and beta chains, only the beta chain of axolotl C3 contains such carbohydrates.

Ambystoma↗
PubMed2003

Tumour suppressors--a fly's perspective.

For a century, the little fruitfly Drosophila melanogaster has taught generations of geneticists about how genes control the development of a multicellular organism. More recently, Drosophila has begun to contribute more directly towards our understanding of human disease [Bernards A, Hariharan IK. Of flies and men-studying human disease in Drosophila. Curr Opin Genet Dev 2001, 11, 274-278]. It is capable of doing this because it shares many disease-related genes with us. For example, the Drosophila genome sequencing project has revealed that two thirds of the genes implicated in human cancers have a counterpart in the fly genome [Adams MD, Celniker SE, Holt RA, et al. The genome sequence of Drosophila melanogaster. Science 2000, 287, 2185-2195, Fortini ME, Skupski MP, Boguski MS, Hariharan IK. A survey of human disease gene counterparts in the Drosophila genome. J Cell Biol 2000, 150, F23-30]. In particular, the fly has homologues of the Retinoblastoma protein (pRb) and of p53, two prototypical tumour suppressors which are inactivated in the majority of human tumours. Here, we will compare the fly's tumour suppressors with their human counterparts and we will review recent advances in our understanding of how these factors function in the fly.

Animals↗
PubMed2002

Diversity in the serine recombinases.

Most site-specific recombinases fall into one of two families, based on evolutionary and mechanistic relatedness. These are the tyrosine recombinases or lambda integrase family and the serine recombinases or resolvase/invertase family. The tyrosine recombinases are structurally diverse and functionally versatile and include integrases, resolvases, invertases and transposases. Recent studies have revealed that the serine recombinase family is equally versatile and members have a variety of structural forms. The archetypal resolvase/invertases are highly regulated, only affect resolution or inversion and they have an N-terminal catalytic domain and a C-terminal DNA binding domain. Phage-encoded serine recombinases (e.g. phiC31 integrase) cause integration and excision with strictly controlled directionality, and have an N-terminal catalytic domain but much longer C-terminal domains compared with the resolvase/invertases. This high molecular weight group also contains transposases (e.g. TnpX from Tn4451). Other transposases, which belong to a third structurally different group, are similar in size to the resolvase/invertases but have the DNA binding domain N-terminal to the catalytic domain (e.g. IS607 transposase). These three structural groups represented by the resolvase/invertases, the large serine recombinases and relatives of IS607 transposase correlate with three major groupings seen in a phylogeny of the catalytic domains. These observations indicate that the serine recombinases are modular and that fusion of the catalytic domain to unrelated sequences has generated structural and functional diversity.

Bacteria↗
PubMed1973

T7 early RNAs are generated by site-specific cleavages.

Transcription of T7 DNA by purified Escherichia coli RNA polymerase without added factors produces long RNA molecules that begin near the left end of T7 DNA and terminate at the end of the early region. An endonuclease has been isolated from uninfected E. coli that cleaves these long RNAs at five specific sites to generate RNA molecules essentially the same as the early T7 RNAs observed in vivo. This sizing factor, which may be RNase III, can act during or after RNA synthesis. Synthesis of early RNA chains has been shown to start at three strong initiators, spaced about 150-200 base-pairs apart near the left end of T7 DNA. Thus, the five cleavages by sizing factor generate the five early messenger RNAs of T7 plus three overlapping RNAs from the promoter region. RNA chains that are started at two of the strong initiators begin with A; those started at the third begin with G. A few minor initiators have also been observed, from which only short chains seem to be synthesized. Their locations in T7 DNA have not been mapped. Rho factor does not appear to be needed to generate any of these early T7 RNAs.

Adenosine Triphosphate↗
PubMed1998

Expression of the Kir2.1 (inwardly rectifying potassium channel) gene in the human placenta and in cultured cytotrophoblast cells at different stages of differentiation.

The aim of this study was to investigate whether the Kir2.1 gene is expressed by the human placenta throughout pregnancy and in cytotrophoblast cells at different stages of differentiation in culture. RNA was extracted from cytotrophoblast cells isolated from term placentas and maintained in culture for 18, 66 and 114 h and from first, second and third trimester placentas. Using the reverse transcriptase-polymerase chain reaction (RT-PCR) with gene-specific primers, a cDNA product of 1.2 kb, as expected for Kir2.1 gene expression, was detected in all the RNA samples from cytotrophoblast cells and from placentas. The RT-PCR products were verified by sequencing and by detection of the expected transcript size for the Kir2.1 mRNA at 5.6-5.7 kb on Northern blots, using the 1.2 kb cDNA generated by RT-PCR. Northern blot quantification, using a control 28S rRNA probe, showed no significant difference in Kir2.1 mRNA expression between any of the three stages of cytotrophoblast cell differentiation studied (ANOVA; n = 3 RNA samples from each stage). These data demonstrate that the Kir2.1 gene is expressed by the human placenta and, specifically, by cytotrophoblast cells, at all stages of development and differentiation.

Cell Differentiation↗
PubMed2004

Flow profiling of a surface-acoustic-wave nanopump.

The flow profile in a capillary gap and the pumping efficiency of an acoustic micropump employing surface acoustic waves is investigated both experimentally and theoretically. Ultrasonic surface waves on a piezoelectric substrate strongly couple to a thin liquid layer and generate a quadrupolar streaming pattern within the fluid. We use fluorescence correlation spectroscopy and fluorescence microscopy as complementary tools to investigate the resulting flow profile. The velocity was found to depend on the applied power approximately linearly and to decrease with the inverse third power of the distance from the ultrasound generator on the chip. The found properties reveal acoustic streaming as a promising tool for the controlled agitation during microarray hybridization.

Acoustics↗
PubMed2025

Fishing for a reelGene: evaluating gene models with evolution and machine learning.

Assembled genomes and their associated annotations have transformed our study of gene function. However, each new annotated assembly generates new gene models. Inconsistencies between annotations likely arise from biological and technical causes, including pseudogene misclassification, transposon activity, and intron retention from sequencing of unspliced transcripts. To evaluate gene model predictions, we developed reelGene, a pipeline of machine learning models focused on (1) transcription boundaries, (2) mRNA integrity, and (3) protein structure. The first two models leverage sequence characteristics and evolutionary conservation across related taxa to learn the grammar of conserved transcription boundaries and mRNA sequences, while the third uses the conserved evolutionary grammar of protein sequences to predict whether a gene can produce a protein. Evaluating 1.8 million transcript models in Zea mays ssp. mays (maize), reelGene classified 28% as incorrectly annotated or non-functional. We find that reelGene classifies 92.2% of genes in the maize proteome and 99.2% of genes within the maize classical gene list as functional. reelGene also provides a way to further investigate genome biology- for instance, reelGene indicates that 10.3% of dispensable genes in B73 are functional, and within retained duplicate genes, reelGene identifies a 30% bias toward the retention of the M1 subgenome when one copy is functional and the other is non-functional. As an annotation-evaluating tool, reelGene is directly applicable to species of the Andropogoneae tribe, including other important crops like sorghum and miscanthus. As a community resource, reelGene has been integrated onto MaizeGDB both as a browser track and as an individual Shiny App, allowing researchers to evaluate gene model accuracy and further investigate genome biology.

Machine Learning↗
PubMed1989

RNA polymerase II subunit RPB3 is an essential component of the mRNA transcription apparatus.

To improve our understanding of RNA polymerase II, the gene that encodes its third-largest subunit, RPB3, was isolated from a lambda gt11 DNA library by using antibody probes. The RPB3 DNA sequence predicts a 318-amino-acid protein whose sequence was confirmed, in part, by microsequence analysis of the gel-purified RNA polymerase II subunit. RPB3 was found to be an essential single-copy gene that is tightly linked to HIS6 on chromosome IX. An RPB3 temperature-sensitive mutant that arrested growth after three to four generations at the restrictive temperature was isolated. When the mutant was shifted to the restrictive temperature, RNA polymerase II could no longer assemble, previously assembled functional enzyme was depleted, and mRNA levels were consequently reduced. These results demonstrate that RPB3 is an essential component of the mRNA transcription apparatus. Finally, the RPB3 protein is similar in sequence and length to RPC5, a subunit common to RNA polymerases I and III, suggesting that these subunits may play similar roles in RNA polymerases I, II, and III.

Amino Acid Sequence↗
PubMed1962

The operant control of vocalization in the dog.

Control over the vocal responses of three dogs was established using operant-conditioning procedures. Several points of interest were observed in the data. First, fixed-ratio schedules of reinforcement generated a vocal response topography which was similar in detail to that of a "motor" bar-nosing response. Second, vocal responding was brought under the control of external visual stimuli as a result of differential reinforcement. Third, good stimulus control was maintained on a multiple schedule containing a vocal-response component and a bar-response component. Fourth, the stimulus control on the multiple schedule transferred with minimal disruption to a chain schedule requiring a sequence of 10 bar responses followed by 10 vocal responses. Fifth, because vocal and bar responses are not mutually exclusive, concurrent responding tended to develop on the chain schedule. These results were discussed with reference to the advisability of applying the terms operant and respondent to unconditioned behavior, and, particularly, to unconditioned verbal behavior.

Animals↗
PubMed1992

STP1, a gene involved in pre-tRNA processing, encodes a nuclear protein containing zinc finger motifs.

STP1 is an unessential yeast gene involved in the removal of intervening sequences from some, but not all, families of intervening sequence-containing pre-tRNAs. Previously, we proposed that STP1 might encode a product that generates pre-tRNA conformations efficiently recognized by tRNA-splicing endonuclease. To test the predictions of this model, we have undertaken a molecular analysis of the STP1 gene and its products. The STP1 locus is located on chromosome IV close to at least two other genes involved in RNA splicing: PRP3 and SPP41. The STP1 open reading frame (ORF) could encode a peptide of 64,827 Da; however, inspection of putative transcriptional and translational regulatory signals and mapping of the 5' ends of mRNA provide evidence that translation of the STP1 ORF usually initiates at a second AUG to generate a protein of 58,081 Da. The STP1 ORF contains three putative zinc fingers. The first of these closely resembles both the DNA transcription factor consensus and the Xenopus laevis p43 RNA-binding protein consensus. The third motif more closely resembles the fingers found in spliceosomal proteins. Employing antisera to the endogenous STP1 protein and to STP1-LacZ fusion proteins, we show that the STP1 protein is localized to nuclei. The presence of zinc finger motifs and the nuclear location of the STP1 protein support the model that this gene product is involved directly in pre-tRNA splicing.

Amino Acid Sequence↗
PubMed1999

Molecular cloning and characterization of two sets of alpha-theta genes in the rat alpha-like globin gene cluster.

The highly heterogeneous rat hemoglobin system was investigated at the gene level. Two regions of the alpha-like globin gene cluster from a Wistar rat were isolated. Four lambda Dash recombinant clones carrying rat alpha-like globin genes were localized on two distinct gene regions. A region of approximately 16kb was found to contain the 5'-IIalpha1-psi theta 1-3' loci, and another of approximately 24kb the 5'-IIalpha2-psi theta2-psiI alpha3-3' loci. Both IIalpha1 and IIalpha2 are considered to be active, coding the IIalpha-globin chain. The nt sequences of IIalpha1 and IIalpha2 are identical except for six nt in the non-coding region. The psiI alpha3 locus is a truncated pseudogene. The putative promoter region of an alpha-like globin gene is joined directly to the third exon, homologous to that of Ialpha-globin cDNA. psi theta1 and psi theta2 are also pseudogenes, as evidenced by several deletions located in the protein-coding regions of these loci. The psi theta1 and psi theta2 loci exhibit extensive homology, but the restriction maps of these genes and their flanking regions differ considerably. Genomic Southern blot analyses of the total liver DNA from six rats showed the existence of three theta-globin-related genes, including psi theta1 and psi theta2. These results indicate that the two gene regions investigated are not allelic variants, but may be generated by block duplication. This is the first report of the existence of rodent theta-globin genes.

Animals↗