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PubMed1986

The evolution of the alpha- and beta-globin gene clusters in human populations.

DNA analysis of the alpha- and beta-globin gene clusters has revealed substantial variability between individuals and populations. As well as restriction enzyme site and length polymorphisms, variation in gene copy number and type is observed. Because of this extensive polymorphism DNA analysis offers a highly informative method of studying genetic affinities between human populations. Haplotypes, consisting of a set of restriction enzyme polymorphisms distributed along the cluster, have been developed for both loci. Analysis of the molecular basis of numerous beta-thalassaemia alleles has revealed, in general, different sets of mutations in different populations, indicating that these postdate the racial divergence. Recent microepidemiological studies on the distribution of alpha-thalassaemia support the hypothesis that this condition, like the beta s-mutation, has been selected because it confers protection against malaria. Population-specific DNA polymorphisms at these and other loci promise to be of considerable value to genetic anthropology.

Genetics, Population↗
PubMed1987

Multiple copies of the amdS gene of Aspergillus nidulans cause titration of trans-acting regulatory proteins.

It has been established that a plasmid containing the amdS gene of Aspergillus nidulans may be used to transform amdS+ strains by selecting for increased utilization of acetamide as sole nitrogen source. Analysis of transformants has shown that multiple tandem copies of the plasmid can be integrated into the chromosome, commonly at sites other than the amdS locus. While the transformed phenotype was relatively stable through mitotic and meiotic divisions evidence was found for variation in plasmid copy number presumably due to unequal recombination events. Expression of the integrated amdS genes was related to copy number, and the amdS RNA produced was similar in size to wild-type RNA. Evidence for titration of the product of the regulatory gene amdR by multiple copies of amdS was found. No titration of the product of the areA gene was observed, and amdS expression was still dependent on areA function. Multiple copies of the amdI9 mutation resulted in poor growth on acetate. This was not observed in the case of the amdS+ gene. The cis-acting amdI9 mutation causes increased facB dependent acetate induction of amdS expression. Titration of the facB gene produce by amdI9 DNA, but not by amdS+ DNA, therefore suggested that the mutation results in increased affinity for the facB gene product.

Amidohydrolases↗
PubMed1995

Multidrug resistance and P-glycoproteins in parasitic protozoa.

Drug resistance has emerged as a devasting impediment to the treatment and control of diseases of parasitic origin. The underlying mechanisms that contribute to this drug resistance in field isolates, however, are poorly understood. Members of the P-glycoprotein gene (pgp) family have been identified, cloned, and sequenced in Plasmodia, Leishmania, and Entamoeba, and variations in pgp copy number and/or expression have been implicated as a basis for drug resistance in each of these genera. The spectrum of drugs to which parasitic protozoa containing amplified pgp genes and/or transcripts are refractory range from a phenotype similar to that observed with multidrug-resistant mammalian cells to those that are completely distinct. The availability of molecular probes to pgp genes provides valuable reagents to dissect the role of pgp gene amplification and overexpression in mediating drug resistance in parasitic protozoa and to determine the physiological function of P-glycoproteins in this clinically consequential group of human pathogens.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

A new LexA-based genetic system for monitoring and analyzing protein heterodimerization in Escherichia coli.

Interactions between proteins affect a wide variety of biological processes, such as signal transduction and control of gene expression. In order to facilitate the study of protein-protein interactions we have developed a new method for specifically detecting the heterodimerization of two heterologous proteins in the bacterium Escherichia coli. The assay is based on the simultaneous use of protein fusions with an altered specificity and a wild-type LexA repressor DNA-binding domain. We have tested this system with two well known eukaryotic dimerization domains (the Fos and Jun leucine zippers). The two interacting proteins were, respectively, fused to a wild-type and a mutant LexA DNA-binding domain. Their hetero-association is specifically measured by the transcriptional repression of a reporter gene (lacZ) controlled by a hybrid operator containing a wild-type half-site (CTGT) and a mutated operator half-site (CCGT). The hybrid operator/lacZ construct was integrated into the chromosome of the reporter strain (SU202) to avoid possible artefacts due to variations in plasmid copy number. This method should be particularly useful in those cases where one or both partners are also able to form homodimers, since the assay described here is sensitive only to the formation of heterodimers. Furthermore, this assay gives rise to a screenable red/white phenotype on MacConkey-lactose indicator plates, allowing for a genetic study of the specificity of the interaction.

Bacterial Proteins↗
PubMed1987

A novel arrangement of sequence elements surrounding the rDNA promoter and its spacer duplications in tsetse species.

Variation in organization and sequence of the rDNA of six species of tsetse fly (Glossina) has been investigated. Several novel tsetse-specific features have been uncovered. Like many other species the spacer is composed of subrepeats, which in some species contain duplications of the true promoter at the spacer-ETS boundary. In tsetse, however, the first 90 base-pairs of the external transcribed spacer (ETS) (that is, +1 to +90 after transcription initiation) is the 3' end of the last subrepeat. The absence of a "unique" region between the last subrepeat and the ETS suggests that the tsetse rDNA unit may consist of multiple true promoters, that is there is no single ETS boundary. Furthermore, interspecific comparisons show that the 90 base-pair region is part of a conserved 202 base-pair region, consisting of 72 base-pairs upstream from the initiation site and a further 40 base-pairs downstream, which is shared by all promoters other than the last. In genera other than tsetse, subrepeat lengths between species are generally similar; in tsetse they differ due to (1) variation in copy-number of the subsubrepeat motif A9T6CAG, and (2) the presence of large regions flanked by direct simple repeats such as GA5 or TGGTCTC. Slippage-like mechanisms are probably responsible for (1), and recombination and subsequent excision involving the direct repeats for (2). Different structural and sequence variants are seen to be homogenized in the family and fixed in each species, reflecting continual unequal crossing-over. However, notwithstanding this process of differentiation, the available comparisons also reveal that there are two small conserved regions between Glossina and Drosophila: one is part of the promoter and the other is an ETS processing site. Such intergeneric and interspecific differences are discussed in relation to the problem of the maintenance of several essential functions within the rDNA repeating unit despite the continual differentiation of the unit into novel arrangements.

Animals↗
PubMed1995

Spinocerebellar ataxia, type 3 (SCA3) is genetically identical to Machado-Joseph disease (MJD).

Spinocerebellar ataxia, type 3 (SCA3) and Machado-Joseph disease (MJD) are two clinically distinct representatives of the heterogeneous group of autosomal dominant cerebellar ataxias. Assignment of the disease genes to the same region of the long arm of chromosome 14 in both SCA3 and MJD suggested that these two disorders are genetically identical. The recent identification of a trinucleotide (CAG) repeat expansion in a gene underlying MJD facilitates assessment of this hypothesis. We analysed the MJD gene in members of a family with characteristic features of SCA3 and no symptoms typical of MJD. We found the same trinucleotide repeat expansion within the gene that was previously described in patients with MJD. The findings demonstrate that SCA3 and MJD are genetically identical in spite of their pronounced clinical differences. Furthermore, we demonstrate a striking variation in the copy number of the CAG repeat among affected members of the same family.

Chromosome Mapping↗
PubMed1981

Distribution of IS5 in bacteria.

Four different strains of Escherichia coli and several other bacteria were examined by Southern analysis for the presence of the insertion element IS5 and IS5-like sequences. Variations in the copy number, degree of homology and restriction pattern of the IS5-like sequence were found among the different organisms. The number and distribution of IS5 sequences do not appear to correlate with the evolutionary relationship of the bacteria in which they occur.

Bacillus subtilis↗
PubMed2006

High mutation rates have driven extensive structural polymorphism among human Y chromosomes.

Although much structural polymorphism in the human genome has been catalogued, the kinetics of underlying change remain largely unexplored. Because human Y chromosomes are clonally inherited, it has been possible to capture their detailed relationships in a robust, worldwide genealogical tree. Examination of structural variation across this tree opens avenues for investigating rates of underlying mutations. We selected one Y chromosome from each of 47 branches of this tree and searched for large-scale variation. Four chromosomal regions showed extensive variation resulting from numerous large-scale mutations. Within the tree encompassed by the studied chromosomes, the distal-Yq heterochromatin changed length > or = 12 times, the TSPY gene array changed length > or = 23 times, the 3.6-Mb IR3/IR3 region changed orientation > or = 12 times and the AZFc region was rearranged > or = 20 times. After determining the total time spanned by all branches of this tree (approximately 1.3 million years or 52,000 generations), we converted these mutation counts to lower bounds on rates: > or = 2.3 x 10(-4), > or = 4.4 x 10(-4), > or = 2.3 x 10(-4) and > or = 3.8 x 10(-4) large-scale mutations per father-to-son Y transmission, respectively. Thus, high mutation rates have driven extensive structural polymorphism among human Y chromosomes. At the same time, we found limited variation in the copy number of Y-linked genes, which raises the possibility of selective constraints.

Chromosomes, Human, Y↗
PubMed2000

Monitoring phase-specific gene expression in Histoplasma capsulatum with telomeric GFP fusion plasmids.

Dimorphism is an essential feature of Histoplasma capsulatum pathogenesis, and much attention has been focused on characteristics that are unique to the saprophytic mycelial phase or the parasitic yeast phase. Recently, we identified a secreted calcium-binding protein, CBP, that is produced in large amounts by yeast cells but is undetectable in mycelial cultures. In this study, the green fluorescent protein (GFP) was established as a reporter in H. capsulatum to study regulation of CBP1 expression in cultures and in single cells grown under different conditions and inside macrophages. One GFP version that was optimized for human codon usage yielded highly fluorescent Histoplasma yeast cells. By monitoring GFP fluorescence during the transition from mycelia to yeast, we demonstrated that the CBP1 promoter is only fully active after complete morphological conversion to the yeast form, indicating for the first time that CBP1 is developmentally regulated rather than simply temperature regulated. Continuous activity of the CBP1 promoter during infection of macrophages supports the hypothesis that CBP secretion plays an important role for Histoplasma survival within the phagolysosome. Broth cultures of Histoplasma yeasts carrying a CBP-GFP protein fusion construct were able to secrete a full-length fluorescent fusion protein that remained localized within the phagolysosomes of infected macrophages. Additionally, a comparison of two Histoplasma strains carrying the CBP1 promoter fusion construct either epichromosomally or integrated into the chromosome revealed cell-to-cell variation in plasmid copy number due to uneven plasmid partitioning into daughter cells.

Animals↗
PubMed1983

Molecular basis of length polymorphism in the human zeta-globin gene complex.

The length polymorphism between the human zeta-globin gene and its pseudogene is caused by an allele-specific variation in the copy number of a tandemly repeating 36-base-pair sequence. This sequence is related to a tandemly repeated 14-base-pair sequence in the 5' flanking region of the human insulin gene, which is known to cause length polymorphism, and to a repetitive sequence in intervening sequence (IVS) 1 of the pseudo-zeta-globin gene. Evidence is presented that the latter is also of variable length, probably because of differences in the copy number of the tandem repeat. The homology between the three length polymorphisms may be an indication of the presence of a more widespread group of related sequences in the human genome, which might be useful for generalized linkage studies.

Base Composition↗
PubMed1995

From population to genome: ecogenetics of Leishmania (Viannia) braziliensis and L. (V.) peruviana.

The size polymorphism of nine chromosomes, recognized by specific probes, was analysed in populations of Leishmania (Viannia) braziliensis and L. (V.) peruviana from various Peruvian biogeographical units. Interpretation of the polymorphism, by statistical and phenetic methods, led to the identification of five consensus (alpha- and beta-tubulin) and four variable chromosomes. The dynamics of the variable chromosomes were studied. The promoter role of the environment on their polymorphism was indicated by: (1) the discrimination of L. braziliensis (forest) and L. peruviana (Andes) by the size of the chromosome containing the gp63 genes; and (2) the fact that, within L. peruviana, the polymorphism of the variable chromosomes revealed a strong eco-geographical structuring of parasite populations, accompanied by increasing chromosomal dissimilarity along a cline from north to south. The adaptative significance of the polymorphism of the variable chromosomes was suggested by: (1) a correlation between chromosomal polymorphism and phenotype variability (lesion type in patients and virulence in vitro); and (2) the association between the decrease in size of the gp63-containing chromosome from L. braziliensis to L. peruviana, and a rearrangement of the gp63 genes, probably accompanied by a decrease in their copy number. As chromosomal variation was shown to be more dependant on eco-geographical differences than isoenzymatic variation, chromosome variation and enzyme variation probably differ in adaptative significance.

Adaptation, Biological↗
PubMed2004

Stable transgene expression in tumors and metastases after transduction with lentiviral vectors based on human immunodeficiency virus type 1.

The relatively low efficiency of target cell transduction and variations in the stability of transgene expression by retroviral vectors based on the Moloney murine leukemia virus (MoMLV) are major impediments to the use of such vectors in cancer gene therapy approaches. The present study was designed to investigate the stability and efficiency of transgene expression in human lung and breast cancer cell lines transduced with vectors based on human immunodeficiency virus type 1 (HIV-1) in vitro and in vivo in nude mouse models of metastasis. H460 lung carcinoma cells and MDA-MB-231 breast carcinoma cells were transduced with lentiviral vectors encoding enhanced green fluorescent protein (EGFP) and beta-galactosidase (beta-Gal), respectively. Transduced H460 cells were administered to nude mice by either intravenous or subcutaneous injection and MDA-MB-231 cells were implanted orthotopically into the mammary fat pad of such mice to induce primary tumor and metastatic lung tumor formation. High-level EGFP expression was maintained in transduced H460 cells in metastatic lung nodules for up to 6 weeks and transgene expression in vitro persisted for at least 23 days after retrieval of EGFP-positive H460 cells from the lungs of tumor-bearing mice and subsequent cultivation in vitro. Likewise, beta-Gal expression levels in metastatic MDA-MB-231 cells in lungs remained high for up to 11 weeks. Southern blot analyses carried out with DNA from lung nodules showed that proviral DNAs in H460 cells were maintained stably over many cell generations and during subsequent reimplantation in vivo. However, molecular analyses revealed variations in transgene copy numbers and expression levels among individual lung clones. These results demonstrate the usefulness of HIV-1-based lentiviral vectors for sustained and stable transgene expression in human lung and breast cancer cell lines in vitro and in vivo.

Animals↗
PubMed2001

Gene dosage analysis in Silver-Russell syndrome: use of quantitative competitive PCR and dual-color FISH to estimate the frequency of duplications in 7p11.2-p13.

Silver-Russell syndrome (SRS) describes a heterogeneous malformation syndrome mainly characterized by intrauterine and postnatal growth retardation (IUGR/PNGR). Approximately 10% of SRS cases have been associated with maternal uniparental disomy (matUPD) 7. This suggests the involvement of at least one imprinted gene on chromosome 7 in the pathogenesis of SRS. Additionally, two familial and one single SRS patients have been published with an interstitial duplication in 7p11.2-p13, including the genes GRB10 and IGFBP1; IGFBP3 was investigated in only one case revealing duplication; conversely, double gene dosage of EGFR was excluded in all 3 patients. Two further cytogenetically abnormal cases, one with a paracentric inversion (7)(p14p12) and one with matUPD7/partial trisomy for 7p13-q11, confirmed that the proximal short arm of chromosome represents an interesting region possibly harboring (a) candidate gene(s) for SRS. Although previously published investigations on the genes GRB10, IGFBP1, IGFBP3, and EGFR report neither disease-relevant mutations nor abnormal imprinting patterns, the SRS cases with chromosomal duplications suggest that variation of gene copy number might be a further type of mutation. To obtain meaningful results on the frequency of duplications in proximal 7p, we screened 32 SRS patients using quantitative PCR assays for GRB10, IGFBP1, IGFBP3, and EGFR. The data were confirmed by dual-color fluorescence in situ hybridization (FISH) of spot check samples. Results obtained by both methods exclude duplications in all analyzed patients and indicate an overall percentage of duplication among SRS patients between 2.4% (GRB10) and 5% (IGFBP1). By testing and evaluating quantitative competitive PCR for various loci, we developed a practical approach for gene dosage analysis which can be easily established for routine purposes.

Cell Nucleus↗
PubMed1997

Characteristics and distribution of large tandem duplications in brook stickleback (Culaea inconstans) mitochondrial DNA.

Most animal mitochondrial DNAs (mtDNAs) range in size from 15 to 18 kb, but increased sizes up to approximately 40 kb are occasionally found. We investigated large size variation in mtDNA of the brook stickleback fish, Culaea inconstans, and characterized four large (2.7-5.8 kb) tandem duplications. Duplications differ in size, frequency of occurrence, and degree of associated heteroplasmy, but each includes the control region and one or more adjacent genes. Duplications are correlated with two mtDNA lineages sampled from 31 populations. L1 duplications (3.2-4.8 kb) were present in all lineage I individuals (n = 121, 19 populations); 53 fish were heteroplasmic due to variation in the copy number of a tandemly repeated 270-bp sequence within the duplicated region. In contrast, duplications, L2, L3, and L4 (2.7-5.8 kb) occurred in only 117 of 174 lineage II fish, in eight of 14 populations. Nine fish with L3 or L4 duplications were heteroplasmic, possessing some mtDNAs that lacked duplications (normal-length mtDNAs). Heteroplasmy in L2 was associated with a small variable region near the ND5 gene. Phylogenetic analysis of restriction sites in Culaea mtDNAs and haplotype-defining sequence differences present in both copies argue for multiple independent events that gave rise to three of the four duplications.

Animals↗
PubMed1989

The Caenorhabditis elegans genome contains monomorphic minisatellites and simple sequences.

Many species have been shown to contain tandemly repeated short sequence DNA known as minisatellites and simple sequence motifs. Due to allelic variation in the copy number of the repeat unit these loci are usually highly polymorphic. Here we demonstrate the presence of sequences in the genome of the nematode Caenorhabditis elegans which are homologous to two sets of short sequence DNA. However, when two independent strains were compared no polymorphism for these sequences could be detected.

Animals↗
PubMed1989

Epstein-Barr virus episome-based promoter function in human myeloid cells.

Epstein-Barr virus (EBV) episomal replicons offer an expeditious means for amplifying transfected genes in human cells. A panel of EBV episomes was constructed to assess the relative utility of five distinct eukaryotic promoter elements for high level and inducible gene expression in stably transfected human myeloid leukemia cells. The Rous sarcoma virus 3' long terminal repeat (LTR) was most highly suited for EBV episome-based gene expression, whereas the lymphopapilloma virus and the SV40 early regulatory elements exhibited substantially lower activities. Chemically responsive promoter elements, such as the SV40 early, human metallothionein IIA and rat GRP78 gene promoters, retained their inducibility when EBV episome-based. Differences in gene expression obtained with the episomes reflected differential promoter activity rather than significant variations in episome copy numbers per cell. These observations provide guidelines for the optimal design of EBV episomal expression vectors for human expression work.

Cell Differentiation↗
PubMed1991

Sequence and expression of potato U2 snRNA genes.

Plant UsnRNA multigene families show a high degree of sequence variation among individual gene members. The potato U2snRNA gene family consists of between twenty-five and forty genes. Four potato U2snRNA gene variants have been isolated. Despite the sequence variation in coding and flanking regions, all maintain the conserved U2snRNA secondary structure and all contain the plant UsnRNA promoter elements: the upstream sequence element (USE) and TATA-like box in the -70 and -30 regions respectively. In RNase A/T1 protection analyses, one of the genes, PotU2-22, protected high levels of full length U2snRNA transcripts in potato leaf, stem, root and tuber RNA. Thus, PotU2-22 or genes with identical coding regions, are highly expressed in these potato organs and therefore represent a major subset of functional U2snRNA genes. Similar expression levels of the PotU2-22 sequence variant were also found in four genetically different potato cultivars and also in tobacco, a species closely related to potato, suggesting conservation of the coding regions of expressed U2snRNA genes. A second gene, PotU2-4, protected very low levels of full length transcripts while a third gene, PotU2-11, was not expressed in the potato organs analysed. The relative expression levels of the gene variants may reflect individual gene differences in, for example, the USE and TATA regulatory elements, or variations in gene copy number.

Animals↗
PubMed2006

CAPweb: a bioinformatics CGH array Analysis Platform.

Assessing variations in DNA copy number is crucial for understanding constitutional or somatic diseases, particularly cancers. The recently developed array-CGH (comparative genomic hybridization) technology allows this to be investigated at the genomic level. We report the availability of a web tool for analysing array-CGH data. CAPweb (CGH array Analysis Platform on the Web) is intended as a user-friendly tool enabling biologists to completely analyse CGH arrays from the raw data to the visualization and biological interpretation. The user typically performs the following bioinformatics steps of a CGH array project within CAPweb: the secure upload of the results of CGH array image analysis and of the array annotation (genomic position of the probes); first level analysis of each array, including automatic normalization of the data (for correcting experimental biases), breakpoint detection and status assignment (gain, loss or normal); validation or deletion of the analysis based on a summary report and quality criteria; visualization and biological analysis of the genomic profiles and results through a user-friendly interface. CAPweb is accessible at http://bioinfo.curie.fr/CAPweb.

Chromosome Breakage↗