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At least 1,063 records · Page 59Linked to original sources
PubMed1999

Human minisatellites, repeat DNA instability and meiotic recombination.

Minisatellites include some of the most variable loci in the human genome and are superb for dissecting processes of tandem repeat DNA instability. Single DNA molecule analysis has revealed different mutation processes operating in the soma and germline. Low-level somatic instability results in simple intra-allelic rearrangements. In contrast, high frequency germline instability involves complex gene conversions and is therefore recombinational in nature, almost certainly occurring at meiosis. To determine whether true meiotic crossovers occur at human minisatellites, we have used polymorphisms near the repeat array to recover recombinant DNA molecules directly from sperm DNA. Analysis of minisatellite MS32 has revealed an intense and highly localised meiotic crossover hotspot centred upstream of the array, the first example of a human hotspot defined at the molecular level. This hotspot extends into the beginning of the repeat array, resulting in unequal and equal crossovers. Array crossovers occur much less frequently than array conversions but appear to arise by a common process, most likely by alternative processing of a recombination initiation complex. The location of MS32 at the boundary of a recombination hotspot suggests that this locus has evolved as a by-product of localised meiotic recombination activity, and that minisatellites might in general mark recombinationally proficient hotspots or hot domains in the genome. Finally, sperm crossover analysis makes it possible to explore the molecular rules that govern human meiotic recombination, and to detect phenomena such as meiotic drive that could provide a possible connection between recombination and DNA sequence diversity itself.

DNA↗
PubMed2001

Strain-specific TCR repertoire selection of IL-4-producing Thy-1 dull gamma delta thymocytes.

Thy-1 dull gamma delta thymocytes constitute an unusual subset of mature TCR gamma delta cells which share with NK T cells the expression of cell surface markers usually associated with activated or memory cells and the simultaneous production of high levels of IL-4 and IFN-gamma upon activation. In DBA / 2 mice, Thy-1 dull gamma delta thymocytes express a restricted repertoire of TCR that are composed of the V1 gene product mainly associated with V6.4 chains exhibiting very limited junctional sequence diversity. In this study we have characterized this gamma delta T cell population in different strains of mice and show that Thy-1 dull gamma delta thymocytes are present in every strain tested, albeit at different frequencies. Moreover IL-4 production by gamma delta thymocytes is mainly confined to the Thy-1 dull population in every strain tested. Finally, the repertoire of TCR expressed by Thy-1 dull gamma delta thymocytes varies in different strain of mice, although a biased expression of Vgamma1 and Vdelta6 chains was observed in all strains studied. However, the extent of junctional diversity of the V1 and V6 chains expressed by Thy-1 dull gamma delta thymocytes varied from oligoclonal in DBA/2 mice to polyclonal in FVB/N mice. Thy-1 dull gamma delta thymocytes from mouse strains such as C3H/HeJ and BALB/c contain cells with diverse Vdelta6(D)Jdelta junctions together with cells with relatively homogeneous Vdelta6(D)Jdelta junctions, similar to those found in DBA/2. Thus, the Thy-1 dull gamma delta population appears to contain two subsets of cells which differ in the diversity of their TCR.

Animals↗
PubMed2001

Receptor revision plays no major role in shaping the receptor repertoire of human memory B cells after the onset of somatic hypermutation.

In order to determine whether V gene replacement accompanies somatic hypermutation in the germinal center (GC) reaction in the human, we analyzed V(kappa)J(kappa) and V(lambda)J(lambda) joints and the kappa-deleting element in single lambda(+) naive and post GC B cells for rearrangements at the kappa and lambda loci. Among 265 lambda(+) post GC B cells, not a single unequivocal and only two potential examples of a cell that switched to lambda light chain expression after accumulation of (unfavorable) mutations in its productive V(kappa) rearrangement were observed. Taking the PCR efficiency into account, the frequency of such cells is likely below 3 %. In addition, heavy and light chain gene rearrangements were amplified and sequenced from the oligoclonal population of IgD-only peripheral blood post GC B cells which display extensive intraclonal sequence diversity. Among 61 IgD-only B cells belonging to 15 clones with intraclonal diversity, no combination of V gene rearrangements indicating receptor revision during clonal expansion was observed. Moreover, among 124 and 49 V(H) genes amplified from IgD-only and class-switched B cells, respectively, not a single example of V(H) revision through V(H) hybrid generation was detected. These results suggest that in the human GC reaction V gene replacement either does not usually accompany somatic hypermutation or is mostly counterselected.

B-Lymphocytes↗
PubMed2004

Mitochondrial phylogeny and systematics of baboons (Papio).

Baboons (Papio, s.s.) comprise a series of parapatric allotaxa (subspecies or closely related species) widely distributed in sub-Saharan Africa. Despite extensive studies of their ecology, morphology, and behavior, disagreement about their phylogenetic relationships continues, as expressed in the current coexistence of at least three major, competing taxonomic treatments. To help resolve this situation, we sequenced approximately 900 bases of mitochondrial DNA of 40 individuals from five of the widely recognized "major" allotaxa. Total sequence diversity (>5%) is high compared to most primate species. Major mitochondrial clades correspond to recognized allotaxa, with the important exception that haplotypes from yellow and olive baboons form a single, monophyletic clade within which the two allotaxa do not comprise mutually exclusive clusters. The major clades fall unambiguously into the pattern: (chacma (Guinea (hamadryas (yellow + olive)))). This phylogeny does not support taxonomies that oppose hamadryas to all other baboons ("desert" vs. "savanna"), but is compatible with the view that all definable allotaxa should be recognized as coordinates, either as "phylogenetic" species or "biological" subspecies. The close relationship and unsegregated distribution of haplotypes from Kenyan and Tanzanian yellow and olive baboons are unexplained, but may reflect introgression across the documented hybrid zone. The overall phylogeny, when combined with paleontological data, suggests a southern African origin for extant Papio baboons, with all extant lineages sharing a common mitochondrial ancestor at approximately 1.8 Ma.

Africa↗
PubMed1990

Differential effects of gelsolins on tissue culture cells.

Gelsolins, prepared from a number of different sources, showed similar severing activity on F-actin in vitro or on stress fibers of detergent-extracted cells but differed in their effects on actin in stress fibers of microinjected cells. When human gelsolin isolated from plasma was injected into cells in a Ca(++)-containing buffer, stress fibers were degraded, the cellular morphology was changed, and numerous actin patches appeared. These effects were particularly striking when the Ca(++)-insensitive N-terminal proteolytic fragment of this gelsolin was injected. By contrast, Ca(++)-sensitive gelsolins isolated from human platelets, pig stomach smooth muscle and pig plasma showed no comparable activity. Furthermore, the Ca(++)-independent N-terminal proteolytic fragments prepared from these gelsolins also had no effect despite their in vitro actin severing activity. Most striking was the finding that human plasma gelsolin expressed in E. coli did not degrade stress fibers, in contrast to the same protein isolated from plasma; nor was there any stress fiber disruption observed with the N-terminal half of human gelsolin expressed in Escherichia coli. The different behavior of these gelsolins in cells cannot be explained by sequence diversity between plasma and cytoplasmic forms, nor by variability in the Ca++ sensitivity of the preparations. It suggests the presence of factors, as yet unidentified, that may regulate gelsolin activity in the cytoplasm of living cells and discriminate between gelsolins of different origin. Such discrimination could be achieved as a result of post-translational modification of the gelsolin; only in this way can differences between apparently identical proteins isolated from human plasma and expressed in E. coli be reconciled.

Actin Cytoskeleton↗
PubMed1995

Cross-recognition by T cells of an epitope shared by two unrelated mycobacterial antigens.

The molecular mimicry represented by cross-recognition of determinants shared by unrelated antigens by antibodies or T cells is of broad immunological interest. In this study, we analyzed the cross-recognition by CD4+ T cells of a peptide epitope shared by two mycobacterial proteins of diverse sequence, represented by the 19-kDa antigen of Mycobacterium tuberculosis and the 28-kDa antigen of Mycobacterium leprae. This epitope was immunodominant with respect to the 19-kDa antigen, but cryptic in relation to the 28-kDa antigen. The cross-reactive epitope cores were identified by Pepscan window analysis and found to be eight residues long in both antigens (residues 69-76 and 127-134). Alignment of these octameric sequences revealed two identical and five conservatively related amino acids. Within the epitope core, two residues (73Asn and 76Ile) were identified as critical for recognition on the basis of inhibition of the cross-reactive T cell proliferative response using singly substituted analog peptides. These results suggest that T cell cross-reactive epitopes can exist in proteins with apparently not more than random levels of sequence homology. Their potential for unsuspected cross-sensitization may play a role in the maintenance of T cell memory, in the pathogenesis of autoimmune diseases and possibly in a wide range of host immune responses to infectious pathogens.

Amino Acid Sequence↗
PubMed1986

Lymphoblastoid cell lines and Burkitt-lymphoma-derived cell lines differ in the expression of a second Epstein-Barr virus encoded nuclear antigen.

Twenty-seven Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines and 27 EBV-carrying Burkitt-lymphoma-derived lines were analyzed for expression of the second EBV-encoded nuclear antigen (EBNA-2) by immunoblotting and anticomplement immunofluorescence with EBNA-2-specific sera. While all lymphoblastoid cell lines expressed EBNA-2, only 10 of the 27 BL lines were EBNA-2-positive. Comparison of the EBNA-2 coding BamHI W-, Y- and H-fragments of EBV-DNA in the different cell lines by restriction enzyme analysis suggests that EBNA-2 negativity is due either to sequence diversity or to a deletion in the BamHI WYH region.

Antigens, Viral↗
PubMed1985

Transposition in plants: a molecular model.

A molecular model for transposition of plant transposable elements is described. This process may occur via excision and re-integration of the element. Excision generates DNA sequence diversity which suggests the participation of DNA repair enzymes in the healing of the donor molecule.

DNA Transposable Elements↗
PubMed2006

The guanidino-group modifying enzymes: structural basis for their diversity and commonality.

The guanidino-group modifying enzyme (GME) superfamily contains many drug targets, including metabolic enzymes from pathogenic microorganisms as well as key regulatory proteins from higher eukaryotes. These enzymes, despite their diverse sequences, adopt the common alpha/beta propeller fold and catalyze the modification of (methylated) guanidino groups. Our structural superposition and structure-based alignment for the GMEs have identified key residues that are involved in the catalysis and substrate binding. We have shown that conserved guanidino-carboxyl interactions are utilized in two different ways; the acidic residues in the catalytic site form hydrogen bonds to the substrate guanidino group, and the enzyme Arg residues at several key positions recognize the carboxyl group of the substrate and fix its orientation. Based on this observation, we have proposed rules for classifying the GME sequences and predicting their molecular function from the conservation of the key acidic and Arg residues. Other novel motifs have been identified, which involve residues that are not in direct contact with the substrate but are likely to stabilize the active-site conformation through hydrogen-bonding networks. In addition, we have examined the domain architecture of the GMEs. Although most members consist of a single catalytic domain, fold recognition analysis has identified a likely bifunctional enzyme from a cyanobacterium. It has also revealed common immunoglobulin-like beta-sandwich domains found in the enzymes that recognize protein substrates. These findings will be useful for predicting the precise mechanism of action for potential novel targets and designing therapeutic compounds against them.

Amidinotransferases↗
PubMed2007

Strategies for high-throughput comparative modeling: applications to leverage analysis in structural genomics and protein family organization.

The technological breakthroughs in structural genomics were designed to facilitate the solution of a sufficient number of structures, so that as many protein sequences as possible can be structurally characterized with the aid of comparative modeling. The leverage of a solved structure is the number and quality of the models that can be produced using the structure as a template for modeling and may be viewed as the "currency" with which the success of a structural genomics endeavor can be measured. Moreover, the models obtained in this way should be valuable to all biologists. To this end, at the Northeast Structural Genomics Consortium (NESG), a modular computational pipeline for automated high-throughput leverage analysis was devised and used to assess the leverage of the 186 unique NESG structures solved during the first phase of the Protein Structure Initiative (January 2000 to July 2005). Here, the results of this analysis are presented. The number of sequences in the nonredundant protein sequence database covered by quality models produced by the pipeline is approximately 39,000, so that the average leverage is approximately 210 models per structure. Interestingly, only 7900 of these models fulfill the stringent modeling criterion of being at least 30% sequence-identical to the corresponding NESG structures. This study shows how high-throughput modeling increases the efficiency of structure determination efforts by providing enhanced coverage of protein structure space. In addition, the approach is useful in refining the boundaries of structural domains within larger protein sequences, subclassifying sequence diverse protein families, and defining structure-based strategies specific to a particular family.

Computational Biology↗
PubMed1999

Genomic sites of topoisomerase II activity determined by comparing DNA breakage enhanced by three distinct poisons.

To define the sites of topoisomerase II activity in two genomic regions of Drosophila melanogaster Kc cells, we have investigated in vivo DNA cleavage sites stimulated by three poisons with diverse sequence specificity, clerocidin, VM-26 and dh-EPI (an anthracycline analog). DNA cleavage was studied by PFGE (pulse-field gel electrophoresis), standard gel electrophoresis, and by genomic primer extension. Poisons stimulated specific intensity patterns of cleavage in the two genomic regions studied. At the centromeric satellite III DNA, fragments of about 270-310 and 385-430 kb could be detected specifically after treatment with clerocidin, suggesting a complex DNA loop organisation, which may correspond with a centromere-specific higher-order chromatin structure. Clerocidin-dependent DNA fragmentation was detectable by PFGE but not by standard agarose gel electrophoresis; while VM-26-dependent cleavage was detected with either method, dh-EPI was ineffective at this locus. Thus, clerocidin DNA cleavage sites were rarer than those of VM-26 at the satellite locus. In the histone H2A-H2B intergenic region, clerocidin and dh-EPI stimulated cleavage whereas VM-26 was only weakly effective. Some clerocidin cleavage sites did not undergo spontaneous reversion, indicating that this agent can stimulate irreversible cleavage in vivo. Direct genomic sequencing showed that many clerocidin and dh-EPI sites, although distinct, mapped to the transcription start and to the proximal promoter of the H2A gene, suggesting that the region is highly accessible to topoisomerase II. Thus, the enzyme may play a role in maintaining a highly accessible chromatin structure under normal cell growth conditions, possibly mediated by specialised protein complexes. This study demonstrates that the use of distinct poisons greatly improves the definition of genomic sites of topoisomerase II activity.

Animals↗
PubMed1989

Intraspecific genetic variability in mitochondrial DNA of the screwworm fly (Cochliomyia hominivorax).

Mitochondrial DNA variability has been analyzed in the primary screwworm fly (Cochliomyia hominivorax) using restriction endonuclease fragment patterns and restriction site mapping. A total of 30 different screwworm lines originating from Texas to Costa Rica and the Island of Jamaica was examined using 15 restriction endonucleases. Eleven of the restriction enzymes revealed polymorphism and yielded 16 mitochondrial genotypes or haplotypes. Two of the haplotypes were widely distributed, haplotype 1 being found scattered across southern Mexico and haplotype 2 along the west coast of Mexico. Haplotype 1 also appeared paired with several other haplotypes in mixed lines that were most likely the result of collecting an egg mass to which more than one female had contributed or to some form of contamination by haplotype 1 after introduction into the laboratory. These lines became fixed before single insects were examined and thus it is impossible to rule out heteroplasmy. The other 14 haplotypes were found in only a single locale and 12 of these were found in only one line. The average sequence diversity among 27 mainland lines was about 0.5%. The two Jamaican lines and one east coast mainland line differed from the others by greater than 2%. The pattern of geographical distribution, a small number of apparently recurring haplotypes and a substantial number (75%) of the haplotypes unique, bears similarities to patterns observed in other insects such as Drosophila. The high frequency of unique genotypes in southern Mexico suggests a population with a very reduced gene flow, which may have had a positive effect on the sterile male release control program.

Animals↗
PubMedDate not supplied

Magnetic resonance angiography of the abdominal aorta.

Magnetic resonance angiography (MRA) of the abdominal aorta was performed in 36 patients using the "bright-blood" technique on the basis of a series of flow-compensated breath-hold 2 D-GE pulse sequences. Diverse diseases included complete occlusion, stenosis, atherosclerotic or dissecting aneurysm and renal artery stenosis. Coronal and axial projection angiograms [maximum intensity projection (MIP) algorithm] and individual GE images were compared with DSA and contrast-enhanced computed tomography (CT). Our data showed a good correlation of MR and digital subtraction arteriography (DSA) or CT resp. in all cases, when both rotating MIP angiograms and individual GE images were analyzed. Thus, MR is suggested to be a useful noninvasive diagnostic method for the abdominal aorta, particularly in preoperative staging of aneurysms, aortic occlusive disease, and stenosis of the proximal main renal artery. Drawbacks of the method are a signal loss in slow or turbulent flow conditions, and unsatisfactory spatial resolution in small vessels.

Adult↗
PubMed2000

Evolutionary dynamics of HTLV-I.

Using mathematical models to describe the in vivo dynamics of HTLV-I infection, an explanation is offered for the slow rate of evolution of HTLV-I relative to HIV-1. In agreement with experimental findings, it is assumed that cell activation is required for successful replication in T helper cells and that HTLV-I induces a significant degree of bystander activation. It is found that the rate of evolution of HTLV-I is limited by the restricted availability of activated uninfected T cells, both at high and low proviral loads. This limits the within-host sequence diversity of HTLV-I and may therefore account for the slow rate of evolution of the virus in the population. Specific differences in the in vivo dynamics of HTLV-I and HIV-1 are identified which may account for the discrepancy in the rate of evolution of these two retroviruses.

Evolution, Molecular↗
PubMed2006

Combination DNA plus protein HIV vaccines.

A major challenge in developing an HIV vaccine is to identify immunogens and delivery methods that will elicit balanced humoral and cell mediate immunities against primary isolates of HIV with diverse sequence variations. Since the discovery of using protein coding nucleic acids (mainly DNA but also possible RNA) as a means of immunization in the early 1990s, there has been rapid progress in the creative use of this novel approach for the development of HIV vaccines. Although the initial impetus of using DNA immunization was for the induction of strong cell-mediated immunity, recent studies have greatly expanded our understanding on the potential role of DNA immunization to elicit improved quality of antibody responses. This function is particularly important to the development of HIV vaccines due to the inability of almost every previous attempt to develop broadly reactive neutralizing antibodies against primary HIV-1 isolates. Similar to the efforts of developing cell mediated immunity by using a DNA prime plus viral vector boost approach, the best antibody responses with DNA immunization were achieved when a protein boost component was included as part of the immunization schedule. Current experience has suggested that a combination DNA plus protein vaccination strategy is able to utilize the benefits of DNA and protein vaccines to effectively induce both cell-mediated immunity and antibody responses against invading organisms.

Animals↗
PubMed1997

A de novo recombination in the ABO blood group gene and evidence for the occurence of recombination products.

We have encountered a paternity case where exclusion of the putative father was only observed in the ABO blood group (mother, B; child, A1; putative father, O), among the many polymorphic markers tested, including DNA fingerprints and microsatellite markers. Cloning a part of the ABO gene, PCR-amplified from the trio's genomes, followed by sequencing the cloned fragments, showed that one allele of the child had a hybrid nature, comprising exon 6 of the B allele and exon 7 of the O1 allele. Based on the evidence that exon 7 is crucial for the sugar-nucleotide specificity of A1 and B transferases and that the O1 allele is only specified by the 261G deletion in exon 6 of the consensus sequence of the A1 allele, we concluded that the hybrid allele encodes a transferase with A1 specificity, resulting, presumably, from de novo recombination between the B and O1 alleles of the mother during meiosis. Screening of random populations demonstrated the occurrence of four other hybrid alleles. Sequencing of intron VI from the five hybrid alleles showed that the junctions of the hybrid alleles were located within intron VI, the intron VI-exon 7 boundaries, or exon 7. Recombinational events seem to be partly involved in the genesis of sequence diversities of the ABO gene.

ABO Blood-Group System↗
PubMed1999

Contribution of gene conversion in the evolution of the human beta-like globin gene family.

Gene conversion is referred to as one of two types of mechanisms known to act on gene families, mainly to maintain their sequence homogeneity or, in certain cases, to produce sequence diversity. The concept of gene conversion was established 20 years ago by researchers working with fungi. A few years later, gene conversion was also observed in the human genome, i.e. the gamma-globin locus. The aim of this article is to emphasize the role of genetic recombination, particularly of gene conversion, in the evolution of the human beta-like globin genes and further to summarize its contribution to the convergent evolution of the fetal globin genes. Finally, this article attempts to re-examine the origin and spread of specific mutations of the beta-globin cluster, such as the sickle cell or beta-thalassemia mutations, on the basis of repeated gene conversion events.

Animals↗
PubMed2000

Hypertension and single nucleotide polymorphisms.

Hypertension is a common, complex disease phenotype that has been intensively studied to identify susceptibility loci in humans. Candidate genes continue to be uncovered via genetic analysis in model organisms through linkage analysis with families and/or sib-pairs and through association studies using sequence variants in genes that play a role in key pathways regulating blood pressure in humans, such as the renin-angiotensin system (RAS). Recent studies exploring the sequence diversity in human candidate genes suggest that the distribution and organization of single nucleotide polymorphisms (SNPs) within and among human populations is complex. Issues related to the use of SNPs in analyzing the genetic determinants of hypertension are illustrated using recent studies on the angiotensin-converting enzyme (ACE).

Angiotensin-Converting Enzyme Inhibitors↗