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PubMed1997

Relational mapping of MYCN and DDXI in band 2p24 and analysis of amplicon arrays in double minute chromosomes and homogeneously staining regions by use of free chromatin FISH.

MYCN amplification has been observed in diverse neuronal tumors including neuroblastoma, retinoblastoma, and small cell carcinoma of the lung, and has been correlated with a poor prognosis in advanced-stage neuroblastomas. Recent studies have shown a co-amplification of DDXI, a DEAD box gene, and MYCN in retinoblastoma and neuroblastoma. DDXI has been mapped to within a megabase of the MYCN gene in band 2p24. In the present study, the relational map of DDXI and MYCN by fluorescence in situ hybridization (FISH) mapping to metaphase cells and extended free chromatin fibers indicated that DDXI is telomeric to MYCN. Dual-color FISH analysis of amplicons within arrays of extended chromatin fibers was performed to examine the physical relationship of MYCN and DDXI within double minute chromosomes (dmins) and homogeneously staining regions (hsrs). No regular reiterated amplicon repeat unit was present in the hsrs, but detailed analysis of the configurations of DDXI and MYCN within each array indicated that multiple rearrangements generated a complex hsr amplicon structure. Similarly, analysis of a cell line bearing dmins showed that a composite amplicon structure involving deletions and/or duplications of MYCN and DDXI is a feature of dmin formation. These data are consistent with a molecular mechanism involving many rearrangements during the evolution of gene amplification, resulting in complex amplicon structures with distinct changes in relative gene copy number and considerable variation in intragenic distances between coamplified genes.

Blotting, Southern↗

PubMed2004

Gene expression microarray analysis and genome databases facilitate the characterization of a chromosome 22 derived homogeneously staining region.

Karyotype and fluorescence in situ hybridization (FISH) analyses previously identified a homogeneously staining region (HSR) derived from chromosome 22 in OV90, an epithelial ovarian cancer (EOC) cell line. Affymetrix expression microarrays in combination with the UniGene and Human Genome Browser databases were used to identify the candidate genes comprising the amplicon of the HSR, based on comparison of expression profiles of OV90, EOC cell lines lacking HSRs and primary cultures of normal ovarian surface epithelial (NOSE) cells. A group of probe sets displaying a minimum 3-fold overexpression with a high reliability score (P-call) in OV90 were identified which represented genes that mapped within a 1-2 Mb interval on chromosome 22. A large number of probe sets, some of which represent the same genes, displayed no evidence of overexpression and/or low reliability scores (A-call). An investigation of the probe set sequences with the Affymetrix and Sanger Institute Chromosome 22 Group databases revealed that some of the probe sets displaying discordant results for the same gene were complementary to intronic sequences and/or the antisense strand. Microarray results were validated by RT-PCR. Genomic analysis suggests that the HSR was derived from the amplification of a 1.1 Mb interval defined by the chromosomal map positions of ZNF74 and Hs.372662, at 22q11.21. The deduced amplicon is derived from a complex region of chromosome 22 that harbors low-copy repeats (LCRs). The amplicon contains 18 genes as likely targets for gene amplification. This study illustrates that large-scale expression microarray analysis in combination with genome databases is sufficient for deducing target genes associated with amplicons and stresses the importance of investigating probe set design before engaging in validation studies.

Cell Line, Tumor↗

PubMed2026

Genetic interactions between PIWI subfamily genes and hobo transposons modulate Drosophila melanogaster lifespan under chronic low-intensity irradiation.

In recent decades, there has been active research into how ionizing radiation at low doses, an inevitable factor in human activity, affects aging processes and which molecular genetic mechanisms underlie this influence. This study investigates the effects of mutations in PIWI subfamily genes (piwi and aub), which regulate transposable elements, on the lifespan of Drosophila melanogaster under conditions of genome instability induced by hobo transposons and chronic low-intensity irradiation (20 cGy). It is shown that dysfunction of piwi and aub modulates the activity of hobo transposons, increasing the frequency of their excisions/transpositions and recombinogenic activity, as confirmed by phenotypic and PCR analyses. The presence of hobo transposons in the genome elevates the spontaneous level of DNA fragmentation in ovarian cells, and chronic irradiation enhances this effect, leading to increased DNA damage in somatic and germline cells of most studied strains. Despite increased genetic instability and reduced fertility in some genotypes, the combined presence of mutations and hobo transposons paradoxically increases lifespan both under control conditions and after irradiation. Analysis of the interaction between genetic factors reveals a predominantly antagonistic, and in one case synergistic, effect on lifespan, depending on the type of mutation, the structure of the hobo transposons (full-size or defective copies), sex, and irradiation conditions. These results demonstrate the complex interplay between systems controlling transpositional activity and stress-induced processes that affect key viability parameters.

Animals↗

PubMed2005

Two retrotransposons maintain telomeres in Drosophila.

Telomeres across the genus Drosophila are maintained, not by telomerase, but by two non-LTR retrotransposons, HeT-A and TART, that transpose specifically to chromosome ends. Successive transpositions result in long head-to-tail arrays of these elements. Thus Drosophila telomeres, like those produced by telomerase, consist of repeated sequences reverse transcribed from RNA templates. The Drosophila repeats, complete and 5'-truncated copies of HeT-A and TART, are more complex than telomerase repeats; nevertheless, these evolutionary variants have functional similarities to the more common telomeres. Like other telomeres, the Drosophila arrays are dynamic, fluctuating around an average length that can be changed by changes in the genetic background. Several proteins that interact with telomeres in other species have been found to have homologues that interact with Drosophila telomeres. Although they have hallmarks of non-LTR retrotransposons, HeT-A and TART appear to have a special relationship to Drosophila. Their Gag proteins are efficiently transported into diploid nuclei where HeT-A Gag recruits TART Gag to chromosome ends. Gags of other non-LTR elements remain predominantly in the cytoplasm. These studies provide intriguing evolutionary links between telomeres and retrotransposable elements.

Animals↗

PubMed1986

Binding of purified wild-type and mutant pi initiation proteins to a replication origin region of plasmid R6K.

The three replication origins of the antibiotic resistance plasmid R6K require for their activity in Escherichia coli a DNA segment containing seven 22 base-pair direct repeats and a plasmid-encoded initiation protein (pi). The pi protein functions in the negative control of R6K replication, in addition to its requirement for the initiation of replication. Construction of a plasmid containing the pi structural gene (pir) downstream from the inducible pR promoter of bacteriophage lambda provided high levels of production of pi protein in E. coli. The pi protein was purified and shown to possess general DNA binding properties with a preference for DNA fragments containing the gamma origin of replication, the operator region of the pir gene and the R6K beta-origin region. Velocity sedimentation analysis indicates that the pi protein exists as a dimer in its native form. Agarose gel electrophoresis analysis of pi-gamma-origin complexes suggests that one pi dimer binds to each copy of the 22 base-pair direct repeats in the gamma origin region. Purified mutant pi protein obtained from a temperature-sensitive initiation mutant (pir 105-ts) exhibited temperature-sensitive binding activity to the gamma-origin region, whereas two mutant proteins exhibiting a high copy number phenotype were unaltered (pir104-cop) or slightly reduced (pir1-cop) in binding activity. The patterns of DNase I protection and enhancement were similar for the wild-type and mutant proteins examined.

Binding Sites↗

PubMed1983

Prenatal lethalities in mice homozygous for human growth hormone gene sequences integrated in the germ line.

The mutagenic potential of recombinant DNA in the germ line was investigated in the descendants of mice obtained from eggs injected with the human growth-hormone gene in a pBR322 vector. Six positive animals (including one mosaic) were produced and had 1-20 copies of the donor sequences in unique and often complex integration patterns indicative of a single or an interrupted insertion. All were heterozygotes and transmitted the foreign insert to their progeny, forming six new HUGH strains. Matings between heterozygotes yielded viable healthy homozygotes in four of the strains. However, in the HUGH/3 and HUGH/4 strains, no postnatal homozygotes were found and litter sizes at birth were small. These two independent cases of mutation, both homozygous recessive prenatal lethals, are attributable to disruption of native sequences by alien ones. They constitute the first instances of insertional mutagenesis due to integration of recombinant DNA in the germ line of the mouse. The mutants provide new possibilities for molecular identification of gene functions necessary for normal development.

Animals↗

PubMed2005

[Primary hepatic localization of the PPNET (primitive peripheral neuroectodermal tumors). Case report].

Our purpose is to study, through the case of a patient operated for right hepatic tumour, the clinical, radiological, anatomopathologic and therapeutic aspects of the peripheral primitive neuroectodermal tumours (PPNET). This tumour (PPNET) is a neoplasm belonging to the Ewing's family tumours, whose histology is similar. Its diagnosis requires the contribution of histopathology, immunohistochimy and cytogenetic studies. The primary hepatic localization of this rare tumour (our case), has never been reported. The treatment, in theory copied on that of the Ewing's sarcoma, is complex and not yet codified, which makes it another disappointing aspect of this disease whose prognosis remains dark.

Diagnosis, Differential↗

PubMed2005

Functional NifD-K fusion protein in Azotobacter vinelandii is a homodimeric complex equivalent to the native heterotetrameric MoFe protein.

The MoFe protein of the complex metalloenzyme nitrogenase folds as a heterotetramer containing two copies each of the homologous alpha and beta subunits, encoded by the nifD and the nifK genes respectively. Recently, the functional expression of a fusion NifD-K protein of nitrogenase was demonstrated in Azotobacter vinelandii, strongly implying that the MoFe protein is flexible as it could accommodate major structural changes, yet remain functional [M.H. Suh, L. Pulakat, N. Gavini, J. Biol. Chem. 278 (2003) 5353-5360]. This finding led us to further explore the type of interaction between the fused MoFe protein units. We aimed to determine whether an interaction exists between the two fusion MoFe proteins to form a homodimer that is equivalent to native heterotetrameric MoFe protein. Using the Bacteriomatch Two-Hybrid System, translationally fused constructs of NifD-K (fusion) with the full-length lambdaCI of the pBT bait vector and also NifD-K (fusion) with the N-terminal alpha-RNAP of the pTRG target vector were made. To compare the extent of interaction between the fused NifD-K proteins to that of the beta-beta interactions in the native MoFe protein, we proceeded to generate translationally fused constructs of NifK with the alpha-RNAP of the pTRG vector and lambdaCI protein of the pBT vector. The strength of the interaction between the proteins in study was determined by measuring the beta-galactosidase activity and extent of ampicillin resistance of the colonies expressing these proteins. This analysis demonstrated that direct protein-protein interaction exists between NifD-K fusion proteins, suggesting that they exist as homodimers. As the interaction takes place at the beta-interfaces of the NifD-K fusion proteins, we propose that these homodimers of NifD-K fusion protein may function in a similar manner as that of the heterotetrameric native MoFe protein. The observation that the extent of protein-protein interaction between the beta-subunits of the native MoFe protein in BacterioMatch Two-Hybrid System is comparable to the extent of protein-protein interaction observed between the NifD-K fusion proteins in the same system further supports this idea.

Azotobacter vinelandii↗

PubMed1983

Transfer of band 3, the erythrocyte anion transporter, between phospholipid vesicles and cells.

Band 3, the anion transport protein of human erythrocyte membranes, can be transferred from cells to liposomes and from liposomes back to cell membranes, retaining function and native orientation. After incubation with cells, sonicated phosphatidylcholine vesicles bind a transmembrane protein that comigrates with band 3 on sodium dodecyl sulfate-polyacrylamide gels. Like native red cell band 3, the vesicle-bound protein is cleaved by chymotrypsin into 65- and 30-kdalton fragments and is not cleaved by trypsin. The protein can be cross-linked by copper-phenanthroline oxidation either before or after transfer to vesicles; in either case, the vesicle fractions contain high molecular weight material that is dissociated into 95-kdalton species by mercaptoethanol. Band 3-vesicle complexes contain no detectable cell lipid and are specifically permeable to anions. Greater than 99% of their anion uptake can be blocked by the band 3 inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS). Red cells whose band 3 function has been blocked irreversibly by DIDS or eosin maleimide regain part of their anion permeability upon incubation with band 3-vesicle complexes. Under the conditions employed, an average of one copy of functional band 3 is delivered to half of the cells, increasing by 2.3-fold the number of cells containing functional anion transporters. Incubation of pure lipid vesicles or red cell membrane buds with either normal red cells or eosin maleimide inhibited cells has no detectable effect on the cells' anion permeability.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2003

Structural roles of subunit cysteines in the folding and assembly of the DNA packaging machine (portal) of bacteriophage P22.

The DNA packaging machine (portal assembly) of bacteriophage P22 is constructed from 12 copies of a multidomain 725-residue subunit comprising a complex alpha/beta fold. The portal subunit contains four cysteines (Cys 153, Cys 173, Cys 283, and Cys 516), which produce distinctive Raman markers in the spectral interval 2500-2600 cm(-1) originating from S-H bond-stretching vibrations diagnostic of S-H...X hydrogen-bonding interactions. The Raman spectrum is unique in the capability to characterize cysteine sulfhydryl interactions in proteins and shows that portal cysteine environments are significantly altered by assembly (Rodriguez-Casado et al. (2001) Biochemistry 40, 13583-13591). We have employed site-directed mutagenesis, size-exclusion chromatography, and Raman difference spectroscopy to characterize the roles of portal cysteines in subunit folding and dodecamer assembly. The stability of the portal monomer is severely reduced by a Cys --> Ser point mutation introduced at either residue 173 or 516. In the case of C516S, the destabilized monomer still forms portal rings, as visualized by negative-stain electron microscopy, whereas portal ring formation cannot be detected for C173S, which forms aberrant aggregates. The C283S mutant is a hyperstable monomer that is defective in portal ring formation. Interestingly, Cys 283 is suggested by secondary structure homology with the phi29 portal to be within a domain involved in DNA translocation. Conversely, the phenotype of the C153S mutant is close to that of the wild-type protein, implying that the sulfhydryl moiety of Cys 153 is not essential to formation of the native subunit fold and productive assembly dynamics. The present results demonstrate that cysteines of the P22 portal protein span a wide range of sulfhydryl hydrogen-bonding strengths in the wild-type assembly, that three of the four sulfhydryls play key roles in portal protein stability and assembly kinetics, and that substitution of a mutant seryl interaction (O-H...X) for a wild-type cysteinyl interaction (S-H...X) can either stabilize or destabilize the native fold depending upon sequence context.

Amino Acid Substitution↗

PubMedDate not supplied

Stable expression of two variable surface glycoproteins by cloned Trypanosoma equiperdum.

African trypanosomes are thought to evade the host immune system by periodically changing their variable surface glycoprotein (VSG). VSG genes are activated by a complex process involving the duplicative transposition of silent basic copy genes to one of several expression sites. These expression-linked copies (ELCs) of the VSG genes are also subject to regulation within expression sites by as yet unknown mechanisms. It is generally assumed that trypanosomes can express only one VSG gene at a time. Nevertheless, the finding that they contain multiple VSG gene expression sites suggests that multiple expression is possible. We show here that Trypanosoma equiperdum can stably express two VSG genes in a simple axenic culture system and that both antigens are present on the cell surface. The two antigens do not co-cap or form heterodimers. Their corresponding genes show no cross-hybridization and are situated in different telomere-linked expression sites. Northern blot analysis reveals that both genes are active in the double expressors.

Animals↗

PubMed2006

Microdeletion encompassing MAPT at chromosome 17q21.3 is associated with developmental delay and learning disability.

Recently, the application of array-based comparative genomic hybridization (array CGH) has improved rates of detection of chromosomal imbalances in individuals with mental retardation and dysmorphic features. Here, we describe three individuals with learning disability and a heterozygous deletion at chromosome 17q21.3, detected in each case by array CGH. FISH analysis demonstrated that the deletions occurred as de novo events in each individual and were between 500 kb and 650 kb in size. A recently described 900-kb inversion that suppresses recombination between ancestral H1 and H2 haplotypes encompasses the deletion. We show that, in each trio, the parent of origin of the deleted chromosome 17 carries at least one H2 chromosome. This region of 17q21.3 shows complex genomic architecture with well-described low-copy repeats (LCRs). The orientation of LCRs flanking the deleted segment in inversion heterozygotes is likely to facilitate the generation of this microdeletion by means of non-allelic homologous recombination.

Adolescent↗

PubMed2003

Genetic variation in organisms with sexual and asexual reproduction.

The genetic variation in a partially asexual organism is investigated by two models suited for different time scales. Only selectively neutral variation is considered. Model 1 shows, by the use of a coalescence argument, that three sexually derived individuals per generation are sufficient to give a population the same pattern of allelic variation as found in fully sexually reproducing organisms. With less than one sexual event every third generation, the characteristic pattern expected for asexual organisms appear, with strong allelic divergence between the gene copies in individuals. At intermediary levels of sexuality, a complex situation reigns. The pair-wise allelic divergence under partial sexuality exceeds, however, always the corresponding value under full sexuality. These results apply to large populations with stable reproductive systems. In a more general framework, Model 2 shows that a small number of sexual individuals per generation is sufficient to make an apparently asexual population highly genotypically variable. The time scale in terms of generations needed to produce this effect is given by the population size and the inverse of the rate of sexuality.

Gene Frequency↗

PubMed1997

The ovine model of neuronal ceroid lipofuscinosis (NCL): its contribution to understanding the pathogenesis of Batten disease.

Development of the ovine model of NCL has been pivotal to our present understanding of the ceroid lipofuscinoses. Analyses of isolated storage product have shown it to be composed of identifiable chemical species of which subunit c of mitochondrial ATP synthase is dominant (ca 50%). It is an extremely hydrophobic protein and failure to catabolise it may be associated with a propensity to form paracrystalline structures with lipids that cannot be degraded by the normal complement of lysosomal enzymes. However, the putative biochemical defect must be related to it and may reside within the mitochondria. Multiple copies of subunit c help form the transmembrane Fo complex which, with partially immobilised lipids, forms an Fo complex domain. This may need to be disassembled in an orderly fashion before proteolysis of subunit c can occur. It is postulated that the primary defect may involve disassembly of the Fo complex domain which may involve more than one step.

ATP Synthetase Complexes↗

PubMed1986

Assessing children's memory productions of the Rey-Osterrieth Complex Figure.

This report describes developmental changes in children's memory productions of the Rey-Osterrieth Complex Figure and in the relation of memory to copy productions, and a method for evaluating these productions that is sensitive to parameters relevant to neuropsychological diagnosis. Based on protocols from a standardization sample of 454 children between the ages of 5 and 14 (Waber & Homes, 1985), a system was devised for objectively evaluating organization, production style, and accuracy. Normative findings are described and implications for the use of this instrument in the neuropsychological assessment of children discussed.

Adolescent↗

PubMed1999

Abnormal folate metabolism and mutation in the methylenetetrahydrofolate reductase gene may be maternal risk factors for Down syndrome.

BACKGROUND: Down syndrome, or trisomy 21, is a complex genetic disease resulting from the presence of 3 copies of chromosome 21. The origin of the extra chromosome is maternal in 95% of cases and is due to the failure of normal chromosomal segregation during meiosis. Although advanced maternal age is a major risk factor for trisomy 21, most children with Down syndrome are born to mothers <30 y of age. OBJECTIVE: On the basis of evidence that abnormal folate and methyl metabolism can lead to DNA hypomethylation and abnormal chromosomal segregation, we hypothesized that the C-to-T substitution at nucleotide 677 (677C-->T) mutation of the methylenetetrahydrofolate reductase (MTHFR) gene may be a risk factor for maternal meiotic nondisjunction and Down syndrome in young mothers. DESIGN: The frequency of the MTHFR 677C-->T mutation was evaluated in 57 mothers of children with Down syndrome and in 50 age-matched control mothers. Ratios of plasma homocysteine to methionine and lymphocyte methotrexate cytotoxicity were measured as indicators of functional folate status. RESULTS: A significant increase in plasma homocysteine concentrations and lymphocyte methotrexate cytotoxicity was observed in the mothers of children with Down syndrome, consistent with abnormal folate and methyl metabolism. Mothers with the 677C-->T mutation had a 2.6-fold higher risk of having a child with Down syndrome than did mothers without the T substitution (odds ratio: 2.6; 95% CI: 1.2, 5.8; P < 0.03). CONCLUSION: The results of this initial study indicate that folate metabolism is abnormal in mothers of children with Down syndrome and that this may be explained, in part, by a mutation in the MTHFR gene.

Adult↗

PubMed2006

Structural variants: changing the landscape of chromosomes and design of disease studies.

The near completeness of human chromosome sequences is facilitating accurate characterization and assessment of all classes of genomic variation. Particularly, using the DNA reference sequence as a guide, genome scanning technologies, such as microarray-based comparative genomic hybridization (array CGH) and genome-wide single nucleotide polymorphism (SNP) platforms, have now enabled the detection of a previously unrecognized degree of larger-sized (non-SNP) variability in all genomes. This heterogeneity can include copy number variations (CNVs), inversions, insertions, deletions and other complex rearrangements, most of which are not detected by standard cytogenetics or DNA sequencing. Although these genomic alterations (collectively termed structural variants or polymorphisms) have been described previously, mainly through locus-specific studies, they are now known to be more global in occurrence. Moreover, as just one example, CNVs can contain entire genes and their number can correlate with the level of gene expression. It is also plausible that structural variants may commonly influence nearby genes through chromosomal positional or domain effects. Here, we discuss what is known of the prevalence of structural variants in the human genome and how they might influence phenotype, including the continuum of etiologic events underlying monogenic to complex diseases. Particularly, we highlight the newest studies and some classic examples of how structural variants might have adverse genetic consequences. We also discuss why analysis of structural variants should become a vital step in any genetic study going forward. All these progresses have set the stage for a golden era of combined microscopic and sub-microscopic (cytogenomic)-based research of chromosomes leading to a more complete understanding of the human genome.

Chromosomes↗

PubMed1995

Determination of NMDA NR1 subunit copy number in recombinant NMDA receptors.

Co-expression of wild-type and mutated NMDA NR1 (N598R) subunits in Xenopus oocytes has been used to determine the stoichiometry of the NMDA receptor-channel. When expressed together, wild-type NR2A and mutant NR1 (N598R) subunits produced channels with a main conductance of 2.6 pS and a sublevel of 1.2 pS. These conductances were clearly different from those obtained from wild-type NR1 and wild-type NR2A channels which gave characteristic 50 pS events with a 40 pS sublevel. When wild-type and mutant NR1 subunits were co-expressed together with NR2A subunits a different channel type with a main conductance of 15.2 pS and a sublevel of 11.4 pS was obtained, as well as the 'all wild-type' and 'all mutant' channels described above. These results indicate that there are likely to be two copies of the NR1 subunit in each NMDA receptor complex.

Animals↗