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Results for “second generation sequencing”

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PubMed1995

Methylation and expression of a metallothionein promoter ovine growth hormone fusion gene (MToGH1) in transgenic mice.

We have examined transgene methylation in the DNA from the livers of a pedigree of mice carrying three copies of an integrated MToGH1 transgene. Utilizing the methylation-sensitive isoschizomers Msp I and Hpa II, Southern blot analysis revealed that all second generation animals derived from a transgenic female had hypermethylated DNA, whereas first generation animals sired by a transgenic male displayed a range of methylation phenotypes ranging from no methylation to hypermethylation of the transgene sequences. Of the mice that exhibited hypermethylation of the transgene in CpG dinucleotides (CmCGG), a minority of these animals also exhibited apparent CpC methylation (i.e. inhibition of Msp I cutting, presumably blocked by methylation of the outer C of CCGG). Methylation was also examined in the inner C of CC(A/T)GG sequences in the MToGH1 transgene using the isoschizomer pair BstN I and EcoR II. A minority of MToGH1 animals in the F1 generation showed clear evidence of methylation in these sites as well as in the inner and outer Cs of CCGG sites. An examination of MToGH1 expression in terms of oGH levels in serum revealed that there was a high degree of variation in the levels of circulating oGH between animals of this pedigree. There was a weak inverse relationship between the serum level of oGH and the extent of methylation of the transgene. In particular, mice exhibiting CpC together with CpG methylation were found to have very low levels of circulating oGH. Our results highlight the nature and complexity of epigenetic factors associated with transgene sequences which may ultimately influence expression of introduced genes in the mammalian genome.

Animals↗

PubMed2005

Explosive lineage-specific expansion of the orphan nuclear receptor HNF4 in nematodes.

The nuclear receptor superfamily expanded in at least two episodes: one early in metazoan evolution, the second within the vertebrate lineage. An exception to this pattern is the genome of the nematode Caenorhabditis elegans, which encodes more than 270 nuclear receptors, most of them highly divergent. We generated 128 cDNA sequences for 76 C. elegans nuclear receptors, confirming that these are active genes. Among these numerous receptors are 13 orthologues of nuclear receptors found in arthropods and/or vertebrates. We show that the supplementary nuclear receptors (supnrs) originated from an explosive burst of duplications of a unique orphan receptor, HNF4. This origin has specific implications for the role of ligand binding in the function and evolution of the nematode supplementary nuclear receptors. Moreover, the supplementary nuclear receptors include a group of very rapidly evolving genes found primarily on chromosome V. We propose a model of lineage-specific duplications from a chromosome on which duplication and substitution rates are highly increased. Our results provide a framework to study nuclear receptors in nematodes, as well as to consider the functional and evolutionary consequences of lineage-specific duplications.

Animals↗

PubMed2007

Standardize and compare contrast-enhanced ultrasonographic digital images obtained with different technologies: how to overcome the subjectivity.

OBJECTIVE: This study was conducted to compare digital images obtained with cadence contrast pulse sequencing (CPS) and coherent contrast imaging (CCI) technologies for contrast-enhanced ultrasonography (CEUS). METHODS: A CEUS study on 17 focal liver lesions was performed using CPS and CCI technologies with a second-generation contrast media. The lesion/liver ratio and conspicuity index were then calculated and compared with Adobe Photoshop 6.0. RESULTS: Lesion/liver ratio and conspicuity index using CCI ranged from 1.3 to 7.1 (mean value, 3) and 19 to 127 (mean value, 58), respectively; by using CPS, we obtained results ranging from 2 to 19.1 (mean value, 8.9) and 57 to 164 (mean value, 109.2). Lesion/liver ratio and the conspicuity index for the lesions using CPS showed significantly (p < 0.0001) superior results than those obtained using CCI. CONCLUSION: The computed analysis with standardization allows an objective evaluation of digital images of CEUS. CPS technology resulted in better lesion conspicuity compared to CCI during CEUS study on focal liver lesions.

Contrast Media↗

PubMed2005

The latest phospholipase C, PLCeta, is implicated in neuronal function.

Members of the phosphoinositide-specific phospholipase C (PLC) family have key roles in cell signalling. In response to many extracellular stimuli, such as hormones, neurotransmitters, antigens and growth factors, PLCs catalyse the hydrolysis of phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P(2)], thereby generating two well-established second messengers, inositol (1,4,5)-trisphosphate and diacylglycerol. Eleven PLC isozymes encoded by different genes have been identified in mammals and, on the basis of their structure and sequence relationships, have been classified into five families designated PLCbeta (1-4), PLCgamma (1 and 2), PLCdelta (1, 3 and 4), PLCepsilon (1) and PLCzeta (1). All PLCs contain the catalytic X and Y domain, in addition to other regulatory domains including the C2 domain and the EF-hand domain. In 2005, four groups independently identified an entirely new family of PLCs--eta1 and eta2--using data mining of mammalian genomes. The properties of the PLCeta enzyme suggest that it might act as a Ca(2+) sensor, in particular, functioning during formation and maintenance of the neuronal network in the postnatal brain.

Animals↗

PubMed1997

[Development of a biological diagnosis for hepatitis C].

The agent responsible of more than 80% of the parenterally transmitted non-A-non-B hepatitis, designated hepatitis C virus (HCV), was identified in 1988 thanks to the isolation of the first cDNA reactive clone which was derived from experimentally infected chimpanzees RNA. The recombinant protein expressed from this clone located in the NS4 region of the HCV, allowed to establish the whole HCV-RNA sequence and to develop the first generation enzyme-linked immunoassays for detecting anti-HCV antibodies. Subsequently, other recombinant viral antigens derived from other HCV genome regions, were produced and used to develop second generation screening and confirmatory assays (1991). At least, thanks to a better presentation of the epitopes, since 1993 the performance of third generation reagents have been improved. Nevertheless, in spite of those modifications, the performance of both screening and confirmatory assays still needs to be improved. Moreover, in cases of difficulties of HCV infection diagnosis with serological tests, HCV-RNA detection with genomic amplification tools represents a useful alternative.

Animals↗

PubMed2001

A novel MVMp-based vector system specifically designed to reduce the risk of replication-competent virus generation by homologous recombination.

Recent work highlights the potential usefulness of MVM-based vectors as selective vehicles for cancer gene therapy (Dupont et al, Gene Therapy, 2000; 7: 790-796). To implement this strategy, however, it is necessary to develop optimized methods for producing high-titer, helper-free parvovirus stocks. Recombinants of MVMp (rMVMp) are currently generated by transiently co-transfecting permissive cell lines with a plasmid carrying the vector genome and a helper plasmid expressing the capsid genes (replaced with a foreign gene in the vector genome). The resulting stocks, however, are always heavily contaminated with replication-competent viruses (RCV), which precludes their use in vivo and particularly in gene therapy. In the present work we have developed a second-generation MVMp-based vector system specifically designed to reduce the probability of RCV generation by homologous recombination. We have constructed a new MVMp-based vector and a new helper genome with minimal sequence overlap and have used the degeneracy of the genetic code to further decrease vector-helper homology. In this system, the left homologous region was almost completely eliminated and the right sequence overlap was reduced to 74 nt with only 61% homology. We were thus able to substantially reduce ( approximately 200 x), but not completely eliminate, generation of contaminating viruses in medium-scale rMVMp preparations. Since the remaining sequence homology between the new vector and helper genomes is weak, our results suggest that contaminating viruses in this system are generated by nonhomologous recombination. It is important to note, unlike the autonomously replicating helper viruses produced from the first-generation vector/helper genomes, the contaminating viruses arising from the new packaging system cannot initiate secondary infection rounds (so they are not 'replication-competent viruses'). Our findings have important implications for the design of new MVMp-based vectors and for the construction of trans-complementing packaging cell lines.

Animals↗

PubMed1987

Coupling of photoexcited rhodopsin to inositol phospholipid hydrolysis in fly photoreceptors.

Fly photoreceptor membranes were used to test the effect on defined biochemical reactions of light and of compounds causing photoreceptor excitation. Complementary electrophysiological studies examined whether putative second messengers excite the fly photoreceptor cells. This analysis revealed the following sequence of events: photoexcited rhodopsin activates a G protein by facilitating GTP binding. The G protein then activates a phospholipase C that generates inositol trisphosphate, which in turn acts as an internal messenger to bring about depolarization of the photoreceptor cell. Binding assays of GTP analogs and measurements of GTPase activity showed that there are 1.6 million copies of G protein per photoreceptor cell. The GTP binding component is a 41-kDa protein, and the light-activated GTPase is dependent on photoconversion of rhodopsin to metarhodopsin. Analysis of phospholipase C activity revealed that this enzyme is under stringent control of the G protein, that the major product formed is inositol trisphosphate, and that this product is rapidly hydrolyzed by a specific phosphomonoesterase. Introduction of inositol trisphosphate to the intact photoreceptor cell mimics the effect of light, and bisphosphoglycerate, which inhibits inositol trisphosphate hydrolysis, enhances the effects of inositol trisphosphate and of dim light. The interaction of photoexcited rhodopsin with a G protein is thus similar in both vertebrate and invertebrate photoreceptors. These G proteins, however, activate different photoreceptor enzymes: phospholipase C in invertebrates and cGMP phosphodiesterase in vertebrates.

Animals↗

PubMed1989

Pelizaeus-Merzbacher disease: tight linkage to proteolipid protein gene exon variant.

Pelizaeus-Merzbacher disease (PMD) is a human X chromosome-linked dysmyelination disorder of the central nervous system for which the genetic defect has not yet been established. The jimpy mutation jp of the mouse is an X chromosome-linked disorder of myelin formation. The mutation is at an intron/exon splice site in the mouse gene for proteolipid protein (PLP). With the jimpy mouse mutation as a precedent, we focused our attention on the human PLP gene, which is found at Xq22. The polymerase chain reaction was used to amplify the exons of the PLP gene of an affected male from a large Indiana PMD kindred. DNA sequencing showed a C----T transition at nucleotide 40 of the second exon. An affected third cousin also showed this sequence variation, while two unaffected male relatives (sons of an obligate carrier female) had the normal cytidine nucleotide. Allele-specific oligonucleotides were used to generate data for linkage studies on the above mentioned PMD kindred. Our results show tight linkage (theta = 0) of PMD to PLP with a lod (logarithm of odds) score of 4.62. In six other unrelated PMD kindreds, only the normal-sequence oligonucleotide hybridized, which indicates genetic heterogeneity. The radical nature of the predicted amino acid change (proline to leucine), suggests that the PMD-causing defect may have been delineated in one kindred.

Base Sequence↗

PubMed1998

Effect of major deletions in the V1 and V2 loops of a macrophage-tropic HIV type 1 isolate on viral envelope structure, cell entry, and replication.

Two HIV-1 envelope mutant proteins were generated by introducing deletions in the first and second hypervariable gp120 regions (V1 and V2 loops, respectively) of a macrophage-tropic primary HIV-1 isolate, SF162, to study the effect of the deleted sequences on envelope structure, viral entry, and replication potentials. The first mutant lacked 17 amino acids of the V1 loop and the latter 30 amino acids of the V2 loop. A comparison of the immunochemical structure of the wild-type and mutant monomeric and virion-associated gp120 molecules revealed that the V1 and V2 loop deletions differentially altered the structure of the V3 loop, the CD4-binding site, and epitopes within conserved regions of gp120. Regardless of differences in structure, both mutated envelope proteins supported viral replication into peripheral blood mononuclear cells to levels comparable to those of the wild-type SF162 virus. However, they decreased the viral replication potential in macrophages, even though they did not alter the coreceptor usage of the viruses. These studies support and extend previous observations that a complex structural interaction between the V1, V2, and V3 loops and elements of the CD4-binding site of gp120 controls entry of virus into cells. The present studies, however, suggest that the effect of the V1 and V2 loops in viral entry is cell dependent.

Amino Acid Sequence↗

PubMed1987

Characterization of a highly polymorphic region 5' to JH in the human immunoglobulin heavy chain.

A cloned DNA segment 1.25 kilobases (kb) upstream from the joining segments of the human heavy chain immunoglobulin gene revealed extensive polymorphic variation at this locus, and the polymorphic pattern was stably transmitted to the next generation. Genomic restriction analysis showed that the polymorphism was caused by insertions/deletions within an MspI/BamHI fragment. Sequencing of one allele, 848 base pairs (bp) long, revealed eleven 50-base-pair tandem repeats. A second allele, 648 bp long, was cloned from a human genomic cosmid library, sequenced, and found to contain four fewer repeats than the first allele. A survey of 186 chromosomes from unrelated individuals of primarily northern European descent revealed at least six alleles.

Alleles↗

PubMed1988

Effects of turbulence on signal intensity in gradient echo images.

Although the appearance of laminar vascular flow in magnetic resonance (MR) images has been characterized, there is no general agreement about the effect of turbulent flow on MR signal intensity. This study uses a fast scan gradient echo pulse sequence to evaluate nonpulsatile turbulent flow in two different models. The first model simulated flow in normal vascular structure. It generated nonpulsatile, laminar and turbulent flow in straight, smooth-walled Plexiglas tubes. The second model simulated flow through a vascular stenosis. It generated nonpulsatile, laminar, and turbulent flow through an orifice. Velocities and flow rates ranged from low physiologic to well above the physiologic range (velocity = .3 to 280 cm/second, flow rate from .15 to 40 L/minute). Transition from laminar to turbulent flow was observed with dye streams. Turbulent flow in straight, smooth-walled vessels was not associated with a decrease in MR signal intensity even at the highest velocities and flow rates studied. The transition from laminar to turbulent flow through an orifice is not associated with a decrease in gradient echo signal intensity. As the intensity of the turbulent flow increases, however, there is a threshold above which signal intensity decreases linearly as turbulence increases (r = .97). This study suggests that flow in normal vascular structures should not be associated with decreased signal intensity in gradient echo images. Turbulent flow through areas such as valves, valvular lesions or vascular stenoses, may be associated with a decrease in gradient echo signal intensity.

Blood Flow Velocity↗

PubMed1991

Statistical analysis of collagen alignment in ligaments by scale-space analysis.

Injuries to ligaments in the knee are common in sports and other physical activities. Some clinical methods are available for qualitatively evaluating the degree of ligament injury and healing. It is, however, desirable to objectively assess the healing of ligaments and to predicate optimal treatment on quantitative measurements of their structure. Information such as areas of coverage and spatial orientations of collagen fibrils, for example, may provide important information about the internal structure of ligament tissues. Since normal ligament tissues are made up of collagen fibrils which are highly organized, they can be considered as oriented piecewise linear patterns. In this paper, we propose a computational technique for statistical analysis of collagen alignment in ligament images using the scale-space approach. In this method, a ligament image is preprocessed by a sequence of filters which are second derivatives of two-dimensional Gaussian functions with different scales. This gives a set of zero-crossing maps (the scale space) from which a stability map is generated. Significant linear patterns are captured by analyzing the stability map. The directional information in terms of orientation distributions of the collagen fibrils in the image and the area covered by the fibrils in specific directions are extracted for statistical analysis. Examples illustrating the performance of this method with scanning electron microscope images of the collagen fibrils in healing rabbit medial collateral ligaments are presented in this paper.

Animals↗

PubMed1996

Bicistronic vector for the creation of stable mammalian cell lines that predisposes all antibiotic-resistant cells to express recombinant protein.

We have developed an improved vector for the stable expression of recombinant protein in mammalian cells. In this vector, designated pCIN, both the recombinant cDNA and the neomycin phosphotransferase selection marker are transcribed from a single promoter element. To facilitate translation of the second open reading frame, the encephalomyocarditis virus internal ribosome entry site has been inserted into the expression cassette immediately before the start codon of this sequence. We report the use of this vector to generate stable cell lines expressing the human 5-HT1Da serotonin receptor and show that following transfection and clonal selection, all ten cell lines characterized express similar and high levels of receptor (1.5-11.9 pmol receptor/mg protein). Use of pCIN should permit the rapid and efficient production of stable mammalian cell lines for the characterization of recombinant protein, as this vector appears to predispose all transfected cells to express such protein.

Animals↗

PubMed2003

[Detection of novel nucleotide substitutions resulting amino acid substitutions in TEM type beta-lactamase gene isolated from a clinically isolated extended spectrum beta-lactamases (ESBLs) productive Escherichia coli].

We screened Escherichia coli (E. coli) from specimens submitted to the Showa University hospital clinical laboratory that showed an advanced resistance to third generation cephems (Cefotaxime or Ceftazidime), as candidate extended spectrum beta-lactamases(ESBLs) producing strains. Among the candidates, two strains showed the characteristics of class A ESBLs in their susceptibility to Cefmetazole, a second generation cephem, and their resistance was inhibited by the addition of clavulanic acid. Further, we detected the TEM-type gene in one of the two E. coli strains. By determining the nucleotide sequence of the whole coding region of the TEM gene, two nucleotide substitutions with amino acid substitutions 82Val-->Ile, and 182Ala-->Val were identified.

Adult↗

PubMed1999

Recently integrated human Alu repeats: finding needles in the haystack.

Alu elements undergo amplification through retroposition and integration into new locations throughout primate genomes. Over 500,000 Alu elements reside in the human genome, making the identification of newly inserted Alu repeats the genomic equivalent of finding needles in the haystack. Here, we present two complementary methods for rapid detection of newly integrated Alu elements. In the first approach we employ computational biology to mine the human genomic DNA sequence databases in order to identify recently integrated Alu elements. The second method is based on an anchor-PCR technique which we term Allele-Specific Alu PCR (ASAP). In this approach, Alu elements are selectively amplified from anchored DNA generating a display or 'fingerprint' of recently integrated Alu elements. Alu insertion polymorphisms are then detected by comparison of the DNA fingerprints generated from different samples. Here, we explore the utility of these methods by applying them to the identification of members of the smallest previously identified subfamily of Alu repeats in the human genome termed Ya8. This subfamily of Alu repeats is composed of about 50 elements within the human genome. Approximately 50% of the Ya8 Alu family members have inserted in the human genome so recently that they are polymorphic, making them useful markers for the study of human evolution.

Animals↗

PubMed2004

Covarion structure in plastid genome evolution: a new statistical test.

Covarion models of molecular evolution allow the rate of evolution of a site to vary through time. There are few simple and effective tests for covarion evolution, and consequently, little is known about the presence of covarion processes in molecular evolution. We describe two new tests for covarion evolution and demonstrate with simulations that they perform well under a wide range of conditions. A survey of covarion evolution in sequenced plastid genomes found evidence of covarion drift in at least 26 out of 57 genes. Covarion evolution is most evident in first and second codon positions of the plastid genes, and there is no evidence of covarion evolution in third codon positions. Therefore, the significant covarion tests are likely due to changes in the selective constraints of amino acids. The frequency of covarion evolution within the plastid genome suggests that covarion processes of evolution were important in generating the observed patterns of sequence variation among plastid genomes.

Codon↗

PubMedDate not supplied

Molecular analysis of potato virus X isolates in relation to the potato hypersensitivity gene Nx.

The coat protein gene of potato virus X is known to affect the outcome of interactions between different strains of the virus and potato plants carrying the Nx resistance gene. To analyze the role of the coat protein in interactions with Nx hosts, we used the potato virus X strain PVXDX, which induces a hypersensitive response on potato cultivars carrying the Nx resistance gene and the strain PVXDX4, which was originally derived from PVXDX and which overcomes Nx-mediated resistance. Sequencing of cloned coat protein genes representing the strains PVXDX and PVXDX4 showed that they differed at a single nucleotide. This change results in the substitution of glutamine-78 in the PVXDX coat protein for proline in PVXDX4. We constructed hybrid viral genomes by replacing the coat protein gene of a full-length clone of isolate PVXUK3 with the corresponding sequence from either PVXDX or PVXDX4. Progeny virus, derived from in vitro transcripts of these hybrid clones, showed that the single nucleotide difference between the coat protein genes of isolates PVXDX and PVXDX4 was sufficient to alter the outcome of the interaction between the virus and potato plants carrying the resistance gene Nx. Additional coat protein mutants generated in planta from transcript-derived inocula induced an intermediate host response on Nx potato cultivars which is influenced by the presence of a second, PVX-specific, resistance gene in the host plant genome.

Base Sequence↗

PubMed1993

Sorbitol dehydrogenase. Full-length cDNA sequencing reveals a mRNA coding for a protein containing an additional 42 amino acids at the N-terminal end.

A cDNA clone encoding rat sorbitol dehydrogenase (SDH) was isolated from a rat testis lambda ZAP II cDNA library. The full-length cDNA insert contained 2277 base pairs (bp), starting 182 bp upstream from an ATG codon where translation to the active enzyme SDH is presumed to be initiated. A second ATG codon, however, was found 126 bp upstream, aligned in the same reading frame as that of the active enzyme. Therefore, the coding sequence for SDH can be translated into an additional 42-amino-acid polypeptide linked to the N-terminal amino acid of the enzyme, generating a pre-sorbitol dehydrogenase. The sequence data indicate that the nucleotide environment around this ATG codon is more favorable towards it being the actual open reading frame (ORF) for a pre-SDH than the ATG codon preceding the nucleotide sequence for SDH. Since no known SDH starts with the additional 42 amino acids, it may be that post-translational removal of this polypeptide accompanies the release of the active enzyme. Next, the 3' untranslated region of the cDNA contained a non-coding 1021 bp downstream from the TAA stop codon. The latter sequence included three putative poly(A) signals: one at nucleotides 1362-1367, the second at nucleotides 1465-1470, and the third at nucleotides 2212-2217 [17 bp away from the poly(A) tail]. In addition to the above findings we also report a variance in one of the amino acids in the SDH cDNA sequence. This variance occurs at position 957-960, where threonine is coded for instead of aspartic acid; in the rat testis SDH cDNA, we find the sequence is ACG instead of GAC, as was reported for the rat liver SDH cDNA. Northern-blot hybridization analysis showed that SDH mRNA is a doublet, one band of 4 kb and the other of 2.3-2.4 kb, in both the rat liver and the rat lens, further confirming that the isolated SDH cDNA constituted a full-length cDNA.

Amino Acid Sequence↗