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At least 1,351 records · Page 75Linked to original sources
PubMed1995

A high-resolution genetic linkage map of the pericentromeric region of the human X chromosome.

We generated a high-resolution genetic linkage map of the pericentromeric region of the human X chromosome from approximately Xp11.4 to Xq22. This map contains 41 loci defined by 50 marker systems genotyped in the CEPH families. For this study we have generated 3 new markers (DXS1689, DXS1690, and DXS159) and 2 new primer sequences for previously described markers (PGK1P1 and AR). Using two different mapping algorithms based on genotype data alone, we developed two well-supported framework maps containing 15 and 18 markers with average interval sizes of 2.7 and 1.7 cM. The 18 marker map is [DXS426, PFC]-2.0-DXS255-1.2-DXS991-1.6-AR-1.3-DXS153-2.0-DX S106-1.5-DXS132-0.6- DXS1690-0.8-DXS453-1.4-DXS559-1.5-[PGK1, DXS56]-0.6-DXS1002-1.8-DXYS1X-2.0-DXS3-7.5-DX S458-2.8-DXS454, where the distance between adjacent loci is in centimorgans. As a third approach, we used physical mapping data to define bins for markers; this approach permitted us to place 26 markers on our framework map. Finally, we constructed a map based on the physical order of 35 markers from the fifth international workshop on human X chromosome mapping. A comparison of the physical and genetic maps indicates a relationship of 2 cM per megabase in this region, with two regions of reduced recombination. The first is around the centromere (DXZ1), and the second is in the region around PGK1 (DXS441 to DXS995). Our maps should aid in the fine-mapping of the many disease loci that localize to this region of the X chromosome.

Algorithms↗
PubMed2002

Computer aided shot reconstructions by means of individualized animated three-dimensional victim models.

In cases of lethal firearm injuries with indefinite indications concerning self-versus third-party infliction a computer enhanced reconstruction with the aim of an anatomical feasibility study can provide significant clues concerning the course of the traumatic event. To this end an exact three-dimensional geometrical model of the victim including all relevant anatomical data as well as the careful documentation of the injuries and a three-dimensional model of the characteristic outlines of the weapon true to scale is generated with the help of an animation program (POSER Version 4, Meta Creation, Egisys AG). With this animated digital three-dimensional model of the victim and the weapon a series of simulation sequences is created by variation of the body positions and the grasp of the weapon. Anatomically impossible positions in view of the physical characteristics of the victim and the site and direction of the bullet path are automatically excluded from the reconstruction. An exact match of the simulation sequence and the real injuries is a statement for a possible self-infliction of the gunshot wound.

Computer Simulation↗
PubMed2003

Analysis of trans-silencing interactions using transcriptional silencers of varying strength and targets with and without flanking nuclear matrix attachment regions.

We investigated the effect of the Rb7 matrix attachment region (MAR) on trans-silencing in tobacco plants, comparing the effects of three transgene silencer loci on ten target loci. Two of the silencer loci, C40 and C190, contain complex and rearranged transgene arrays consisting of 35S:GUS or NOS:NPTII containing plasmids. The third silencer locus, V271, was previously characterized as a complex locus containing rearranged 35S:RiN sequences. Each of these silencers can reduce 35S promoter-driven expression at other loci, albeit with varying efficiencies. The presence of MARs at a target locus does not prevent trans-silencing by the V271 silencer. However, four of seven MAR-containing loci were at least partially resistant to silencing by the C40 and C190 loci. One MAR locus was unaffected by C40, our weakest silencer, and three were silenced only when the silencer locus was maternally inherited. Silencing is progressive in the F1 and F2 generations; two days after germination there is little or no difference between seedlings derived from crosses to silencing or control lines, but seedlings containing silencer loci slowly lose expression during subsequent development. These observations are compatible with the hypothesis that a product of the silencer locus must accumulate before unlinked loci can be affected. However, our silencer loci are themselves silenced for GUS transcription, and coding region homology is not required for their effects on target loci. Our results are consistent with a model in which transcriptional silencing is triggered by transcription of sequences during the early stages of embryo or seedling development.

Gene Silencing↗
PubMed2001

One INK4 gene and no ARF at the Fugu equivalent of the human INK4A/ARF/INK4B tumour suppressor locus.

The INK4A/ARF/INK4B locus, conserved in mammals, encodes three polypeptides that regulate cell proliferation via the pRb and p53 tumour suppressor pathways. The locus is mutated in many cancers. The related, tandemly-linked INK4A and INK4B genes encode the p16(INK4A) and p15(INK4B) members of the INK4 family of cyclin-dependent kinase inhibitors which block phosphorylation of pRb, whereas the third product, ARF, derived from an alternative reading frame of INK4A, regulates p53 activity. We assessed the status of this unusual locus in the puffer fish, Fugu rubripes, and identified two INK4 genes using degenerate PCR and hybridization analyses. Sequence conservation and conservation of synteny between human and Fugu predict one gene to be an INK4A or INK4B homologue and the other an INK4D homologue. Analysis of the Fugu INK4A/B gene and the surrounding 40-kb of genomic DNA did not reveal the presence of any ARF-encoding potential or another related INK4 gene. We conclude that the gene duplication event that generated adjacent INK4A and INK4B genes and the association of ARF with the ancestral INK4A gene occurred after the divergence of the lineage leading to mammals from fish. Thus, unlike mammals, the fish p53 and pRb tumour suppressor pathways are not regulated by a single locus.

Amino Acid Sequence↗
PubMed1993

Biological roles of oligosaccharides: all of the theories are correct.

Many different theories have been advanced concerning the biological roles of the oligosaccharide units of individual classes of glycoconjugates. Analysis of the evidence indicates that while all of these theories are correct, exceptions to each can also be found. The biological roles of oligosaccharides appear to span the spectrum from those that are trivial, to those that are crucial for the development, growth, function or survival of an organism. Some general principles emerge. First, it is difficult to predict a priori the functions a given oligosaccharide on a given glycoconjugate might be mediating, or their relative importance to the organism. Second, the same oligosaccharide sequence may mediate different functions at different locations within the same organism, or at different times in its ontogeny or life cycle. Third, the more specific and crucial biological roles of oligosaccharides are often mediated by unusual oligosaccharide sequences, unusual presentations of common terminal sequences, or by further modifications of the sugars themselves. However, such oligosaccharide sequences are also more likely to be targets for recognition by pathogenic toxins and microorganisms. As such, they are subject to more intra- and inter-species variation because of ongoing host-pathogen interactions during evolution. In the final analysis, the only common features of the varied functions of oligosaccharides are that they either mediate 'specific recognition' events or that they provide 'modulation' of biological processes. In so doing, they generate much of the functional diversity required for the development and differentiation of complex organisms, and for their interactions with other organisms in the environment.

Glycoconjugates↗
PubMed1991

Malignant cell detection in Burkitt's lymphoma using third-complementarity-determining region (CDRIII), clone-specific probe developed by sequencing DNA from stored slides.

The DNA sequence of the third-complementarity-determining region (CDRIII) of the immunoglobulin heavy chain (IgH) gene in a case of Burkitt's lymphoma was determined by polymerase chain reaction (PCR) using template DNA extracted from a smear stored at room temperature for more than one year. The DNA sequence obtained from the stored slide was compared with that of DNA from a frozen lymph node biopsied at the initial presentation. The sequences were shown to be identical, implying that DNA from a smear on a stored slide can be used as a source of DNA for PCR amplification, sequencing, and development of a clone-specific probe. Using oligonucleotides generated from one of the CDRIII sequences of the IgH gene as molecular probes, a retrospective study for the malignant clone on the smears was carried out. Malignant cells were detectable in the peripheral blood at an early stage of bone marrow relapse but not in the peripheral blood or bone marrow at the initial presentation. No malignant clone was detected in the bone marrow when testicular infiltration was diagnosed by examination of a pathological specimen. Thus, the technique permits molecular analysis of hematologic malignancies of B-cell lineage in cases where fresh or frozen specimens are not available.

Base Sequence↗
PubMed1996

Involvement of topoisomerase IV and DNA gyrase as ciprofloxacin targets in Streptococcus pneumoniae.

Ciprofloxacin-resistant mutants of Streptococcus pneumoniae 7785 were generated by stepwise selection at increasing drug concentrations. Sequence analysis of PCR products from the strains was used to examine the quinolone resistance-determining regions of the GyrA and GyrB proteins of DNA gyrase and the analogous regions of the ParC and ParE subunits of DNA topoisomerase IV. First-step mutants exhibiting low-level resistance had no detectable changes in their topoisomerase quinolone resistance-determining regions, suggesting altered permeation or another novel resistance mechanism. Nine of 10 second-step mutants exhibited an alteration in ParC at Ser-79 to Tyr or Phe or at Ala-84 to Thr. Third- and fourth-step mutants displaying high-level ciprofloxacin resistance were found to have, in addition to the ParC alteration, a change in GyrA at residues equivalent to Escherichia coli GyrA resistance hot spots Ser-83 and Asp-87 or in GyrB at Asp-435 to Asn, equivalent to E. coli Asp-426, part of a highly conserved EGDSA motif in GyrB. No ParE changes were observed. Complementary analysis of two S. pneumoniae clinical isolates displaying low-level resistance to ciprofloxacin revealed a ParC change at Ser-79 to Phe or Arg-95 to Cys but no changes in GyrA, GyrB, or ParE. A highly resistant isolate, in addition to a ParC mutation, had a GyrA alteration at the residue equivalent to E. coli Asp-87. Thus, in both laboratory strains and clinical isolates, ParC mutations preceded those in GyrA, suggesting that topoisomerase IV is a primary topoisomerase target and gyrase is a secondary target for ciprofloxacin in S. pneumoniae.

Amino Acid Sequence↗
PubMed2000

[Mutation in microsatellite repeats of DNA and embryonal death in humans].

In the analysis of tetranucleotide DNA repeats inheritance carried out in 55 families with a history of spontaneous miscarriages and normal karyotypes in respect to 21 loci located on seven autosomes, 8 embryos (14.5%) demonstrating 12 cases of the presence of alleles absent in both parents were described. The study of chromosome segregation using other DNA markers permitted highly probable exclusion of false paternity as well as uniparental disomy as the reasons for parent/child allele mismatches. The high probability of paternity together with the presence of a "new" allele at any offspring locus points to the mutation having occurred during game-togenesis in one of the parents. Examination of mutation in spontaneous abortuses revealed an increased number of tandem repeat units at microsatellite loci in three cases and an decreased number of these repeats in six cases. In two abortuses, a third allele absent in both parents, which resulted from a somatic mutation that occurred during embryonic development, was observed. The prevalence of the male germline mutations, revealed during investigation of the mutation origin, was probably associated with an increased number of DNA replication cycles in sperm compared to the oocytes. In spontaneous abortuses, the mean mutation rate of the tetranucleotide repeat complexes analyzed was 9.8 x 10(-3) per locus per gamete per generation. This was about five times higher than the spontaneous mutation rate of these STR loci. It can be suggested that genome instability detected at the level of repeated DNA sequences can involve not only genetically neutral loci but also active genomic regions crucial for embryonic viability. This results in cell death and termination of embryonic development. Our findings indicate that the death of embryos with normal karyotypes in most cases is associated with an increased frequency of germline and somatic microsatellite mutations. The data of the present study also provide a practical tool for the quantitative evaluation of this phenomenon and for the analysis of the reasons for miscarriages and embryonic death in certain families.

DNA, Satellite↗
PubMed1992

The anti-DNA-associated idiotype 8.12 is encoded by the V lambda II gene family and maps to the vicinity of L chain CDR1.

The 8.12 idiotype is an anti-DNA-associated Id present on lambda L chains that are expressed at high titers in 50% of patients with systemic lupus erythematosus. Since this Id can be present on as much as a third of a patient's anti-DNA antibodies and is found in renal glomeruli, 8.12 is thought to be a marker for a subset of pathogenic anti-DNA auto-antibodies. A molecular analysis of the 8.12 positive antibodies was designed to explore the genetic basis of this Id. Monoclonal human B cell lines were generated by transformation with EBV and lambda L chain-secreting lines were analyzed for Id expression and V region gene usage. In this panel of Ig lambda cell lines, the 8.12 idiotype is encoded exclusively by members of the V lambda II gene family. The sequences of several 8.12+ and 8.12- V lambda II genes are reported here and are used to map the 8.12 Id to the vicinity of CDR1, as well as to further characterize the large and polymorphic V lambda II gene family.

Amino Acid Sequence↗
PubMed1988

Localization of two epitopes of apolipoprotein A-I that are exposed on human high density lipoproteins using monoclonal antibodies and synthetic peptides.

To understand the structure of apolipoprotein A-I, we have used an immunochemical approach and identified specific regions of apoA-I that may be exposed on the apoprotein as it exists on high density lipoprotein (HDL). Twelve mouse monoclonal antibodies specific for human apoA-I were generated from six fusions. Thirteen synthetic peptides of between 5 and 16 amino acid residues in length, which span the amino-terminal two-thirds of apoA-I, were tested for their ability to react with each of the 12 antibodies. In a competitive solid-phase radioimmunoassay, a synthetic peptide, which represented residues 1-15 of mature apoA-I, inhibited the binding of antibody AI-16 to immobilized HDL. Similarly, a synthetic peptide, which represented residues 90-105 of apoA-I, inhibited the binding of antibody AI-18 to immobilized HDL. Using systematic changes in the size and sequence of the oligopeptides, the limits and essential amino acid residues of these epitopes were defined. Comparisons of the slopes of the competition curves obtained with immunoreactive peptides, isolated apoA-I, and HDL verified that these two regions of apoA-I are exposed on the surface of apoA-I as it exists on native HDL.

Animals↗
PubMed1989

Detection of Epstein-Barr virus by in situ hybridization. Progress toward development of a nonisotopic diagnostic test.

This work presents some initial quantitation of an in situ hybridization method for detection of Epstein-Barr (EB) virus nucleic acids. The purpose is to develop evaluative criteria for diagnosis of viral presence in clinical tissue specimens. In this work simultaneous denaturation of probe and target DNA and an alkaline phosphatase conjugate to detect biotinated probe were used as described by Unger et al. For evaluation of the hybridization, a variety of cell lines, both productively and latently infected, that were hybridized in situ using nick translated 32P-labeled viral probe sequences and counted by scintillation after the method of Lawrence and Singer were used. Producer cells (B95-8) showed intense foci of staining in approximately 5% of cells, with most of the other cells showing varying staining intensity. Raji cells showed varying amounts of signal from cell to cell. Namalwa cells exhibited one spot in most cells that was decreased after cells were treated with Actinomycin D (dactinomycin, Merck Sharp & Dohme, West Point, PA). Signal was identified in only a third of these same cells after sectioning. EB virus-negative Ramos cells showed no signal. The nuclear punctate nature of the signal generated is diagnostic of infected cells, and may be a useful test for cultured cells or pathologic specimens.

Autoradiography↗
PubMed1996

Identification of antigenic regions in the GB hepatitis viruses GBV-A, GBV-B, and GBV-C.

The genomes of two novel members of the Flaviviridae associated with GB agent hepatitis (GB viruses A and B) were cloned and sequenced recently. The genome of a third novel virus (GB virus C), related to but distinct from GB viruses A and B, has also been identified and characterized. Overlapping clones encompassing the large open reading frames of these three viruses have been expressed in E. coli as CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase (CKS) fusion proteins. Bacterial lysates were subjected to Western blot analyses using sera from GB agent-infected tamarins and human sera from various individuals with or "at risk" for non-A, non-B, non-C, non-D, non-E hepatitis. Antigenic regions were identified in the putative NS3, NS4, and NS5 proteins from all three viruses. An antigenic region was also identified in the putative core protein of GB virus B. Many of the clones identified originally as encoding antigenic proteins were quite large. To map these regions more narrowly, smaller overlapping clones were generated by polymerase chain reaction (PCR), expressed as recombinant CKS fusion proteins and tested by Western blot. Additionally, a lambda gt11 expression library was generated from infectious tamarin sera and immunoscreened. These studies have identified at least three epitopes in GB virus A, five epitopes in GB virus B and four epitopes in GB virus C.

Animals↗
PubMed1990

Env and Vpu proteins of human immunodeficiency virus type 1 are produced from multiple bicistronic mRNAs.

Three size classes of human immunodeficiency virus type 1 (HIV-1) mRNAs are produced in infected cells: full-length, intermediate, and small. Here we report that the intermediate-size class of viral mRNAs is heterogeneous, consisting of at least 12 differentially spliced species. This group contains nine bicistronic mRNAs producing Env and Vpu and three mRNAs expressing only the first exon of tat. In the latter mRNAs, Env and Vpu expression is blocked by the presence of the upstream tat open reading frame. We conclude that internal initiation of translation is not the mechanism for generation of the bicistronic env mRNAs. Translation of HIV-1 mRNAs is consistent with the scanning mechanism in which Env is produced by leaky scanning from mRNAs that contain env as the second or third reading frame. Env and Vpu proteins are expressed from the same mRNAs and are coordinately regulated by Rev. This arrangement may reflect a requirement for coordinate expression of Vpu and Env.

Base Sequence↗
PubMed1993

Characterization of the left 4 kb of conjugative transposon Tn916: determinants involved in excision.

The rate-limiting step in movement of the conjugative transposon Tn916, originally identified in Enterococcus faecalis, is believed to be an excision event that generates a non-replicative circular intermediate. When present on a plasmid vector in Escherichia coli, Tn916 generally excises at a high frequency. It was reported previously that insertion of Tn5 in a region near the left end of Tn916 eliminated the ability to excise; and the mutation could be complemented in trans. In this communication the nucleotide sequence of 4 kb of Tn916 DNA connecting the recently sequenced tet(M) determinant (Su et al., 1992; Burdett, 1990) with the left end of the transposon. Ten open reading frames (ORFs) were deduced, two of which (ORF3 and ORF4) were encoded in-frame within a third (ORF2). Mutants with Tn5 insertions in the ORF1 or ORF2 (ORF3 and ORF4) were defective in excision, but could be complemented in vivo by a co-resident plasmid containing the ORF1 or ORF2 determinant, respectively. The data support the view that both ORF1 and ORF2 are essential for excision. ORF1 and ORF2 are essentially identical to determinants designated xis-Tn and int-Tn, respectively, in the closely related Tn1545. A Tn5 insertion in ORF5 eliminated conjugative transfer between E. faecalis strains. Functions for the remaining ORFs (ORF6 through ORF10) remain unknown; however, nucleotide sequences within ORF6 and ORF9 had significant homology with sequence downstream of other tet(M) determinants.

Amino Acid Sequence↗
PubMed1992

Nuclear magnetic resonance and circular dichroism studies of a triple-helical peptide with a glycine substitution.

The triple-helical conformation has the stringent amino acid sequence constraint that every third residue must be a glycine, (X-Y-Gly)n. We use nuclear magnetic resonance and circular dichroism to quantify the consequences of a substitution in the glycine position of a triple-helical peptide, and to enhance our understanding of interactions in this basic structural motif. A 30-residue peptide with a Gly----Ala change forms a stable trimer at a folding rate somewhat less than that of the unsubstituted peptide, and the substitution results in a marked decrease in thermal stability and a conformational perturbation of about 30% of the triple-helical structure. Two models were generated for this peptide, one with the alanine residues packed inside the triple helix and one with a looping out of the chain at the substitution site. Studies on the Gly----Ala peptide are useful in understanding connective tissue diseases which result from the substitution of one glycine residue in the triple-helix of fibrillar collagens.

Circular Dichroism↗
PubMed1994

Domain B protruding at the third beta strand of the alpha/beta barrel in barley alpha-amylase confers distinct isozyme-specific properties.

alpha-Amylases belong to the alpha/beta-barrel protein family in which the active site is created by residues located at the C-terminus of the beta strands and in the helix-connecting loops extending from these ends. In the alpha-amylase family, a small separate domain B protrudes at the C-terminus of the third beta strand of the (beta/alpha)8-barrel framework. The 80% identical barley alpha-amylase isozymes 1 and 2 (AMY1 and AMY2, respectively) differ in substrate affinity and turnover rate, CaCl2 stimulation of activity, sensitivity to the endogenous 21-kDa alpha-amylase/subtilisin inhibitor, and stability at low pH. To identify regions that confer these isozyme-specific variations, AMY1-AMY2 hybrid cDNAs were generated by in vivo homologous recombination in yeast. The hybrids AMY1-(1-90)-AMY2-(90-403) and AMY1-(1-161)-AMY2-(161-403) characterized in this study contain the 90-residue and 161-residue N-terminal sequences, respectively, of AMY1 and complementary C-terminal regions of AMY2. AMY1-(1-90)-AMY2-(90-403) comprises the 60-amino-acid domain B of AMY2 and resembles this isozyme in sensitivity to alpha-amylase/subtilisin inhibitor and its low affinity for the substrates p-nitrophenyl alpha-D-maltoheptaoside, amylose and the inhibitor acarbose. Only AMY1-(1-161)-AMY2-(161-403) and AMY1, which both share domain B, are stable at low pH. However, AMY2 and both hybrid AMY species, but not AMY1, show maximum enzyme activity on insoluble blue starch at approximately 10 mM CaCl2. Domain B thus determines several functional and stability properties that distinguish the barley alpha-amylase isozymes.

Acarbose↗
PubMed1998

Role of variable regions A and B in receptor binding domain of amphotropic murine leukemia virus envelope protein.

For the amphotropic murine leukemia virus (MuLV), a 208-amino-acid amino-terminal fragment of the surface unit (SU) of the envelope glycoprotein is sufficient to bind to its receptor, Pit2. Within this binding domain, two hypervariable regions, VRA and VRB, have been proposed to be important for receptor recognition. In order to specifically locate residues that are important for the interaction with Pit2, we generated a number of site-specific mutations in both VRA and VRB and analyzed the resulting envelope proteins when expressed on retroviral vectors. Concurrently, we substituted portions of the amphotropic SU with homologous regions from the polytropic MuLV envelope protein. The amphotropic SU was unaffected by most of the point mutations we introduced. In addition, the deletion of eight residues in a region of VRA that was previously suggested to be essential for Pit2 utilization only decreased titer on NIH 3T3 cells by 1 order of magnitude. Although the replacement of the amino-terminal two-thirds of VRA with the polytropic sequence abolished receptor binding, smaller nonoverlapping substitutions did not affect the function of the protein. We were not able to identify a single critical receptor contact point within VRA, and we suggest that the amphotropic receptor binding domain probably makes multiple contacts with the receptor and that the loss of some of these contacts can be tolerated.

3T3 Cells↗
PubMed2003

Clinical, genetic, and biophysical characterization of a homozygous HERG mutation causing severe neonatal long QT syndrome.

Previous studies have identified mutations in five ion channel genes as a cause of long QT syndrome, a heterogeneous disorder characterized by prolongation of the QT interval, multiform ventricular tachycardia (torsades de pointes), seizures, syncope, and sudden death. However, in these studies, the average age of initial symptoms is in the third decade of life or later, and few reports have described the genetic causes of long QT syndrome presenting in the prenatal or neonatal period. We used a candidate gene approach to identify the genetic cause of long QT syndrome in an infant whose initial manifestations were detected in utero. Direct bidirectional sequencing of long QT syndrome genes identified a previously unreported HERG missense mutation (R752Q). Three asymptomatic family members were heterozygous for R752Q, and the proband, who manifested ventricular tachycardia in utero, was homozygous. R752Q was not found in 100 normal unrelated chromosomes. Paternal DNA was unavailable for testing. Transient transfection of HERG generated robust IKr, but no current was observed for the mutant HERG. The HERG mutant, R752Q, is associated with a mild phenotype, inasmuch as family members with a heterozygous mutation appear unaffected. The homozygous mutation results in absence of functional IKr, causing a profound loss of HERG channel function, creating the equivalent of a "HERG knockout" and leading to a severe phenotype.

Base Sequence↗