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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↗

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↗

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↗

PubMed2001

Expression and post-transcriptional regulation of maize transposable element MuDR and its derivatives.

The transposition of Mu elements underlying Mutator activity in maize requires a transcriptionally active MuDR element. Despite variation in MuDR copy number and RNA levels in Mutator lines, transposition events are consistently late in plant development, and Mu excision frequencies are similar. Here, we report previously unsuspected and ubiquitous MuDR homologs that produce both RNA and protein. MuDR transcript levels are proportional to MuDR copy number, and homolog transcript levels increase in active Mutator lines. A subset of homologs exhibits constitutive transcription in MuDR(-) and epigenetically silenced MuDR lines, suggesting independent transcriptional regulation. Surprisingly, immunodetection demonstrated nearly invariant levels of MuDR and homolog protein products in all tested Mutator and non-Mutator stocks. These results suggest a strict control over protein production, which might explain the uniform excision frequency of Mu elements. Moreover, the nonfunctional proteins encoded by homologs may negatively regulate Mutator activity and represent part of the host defense against this transposon family.

Base Sequence↗

PubMed1988

Studies of a novel repetitive sequence family in the genome of mice.

A new middle repetitive sequence is described in the mouse genome. It has been revealed with a recombinant clone isolated from a Mus musculus BamHI gene library constructed in pBR322 and containing an insertion of 1.73 kb. When digests of genomic DNA were subjected to Southern blot hybridization, using the 1.73-kb insert as probe, we obtained a light smear and discrete bands, indicating a dispersion in the mouse genome of this sequence. This 1.73-kb sequence seems to be a part of a greater repetitive sequence at least 6 kb in length. The sizes of the bands hybridizing with the 1.73-kb insert are similar when compared between different laboratory strains but differ remarkably between the two species M. musculus and Mus caroli. We have shown also a great variation in the copy number of the sequence studied between these two species. When rat DNA is probed with the 1.73-kb insert, no hybridization is observed. Subcloning of the 1.73-kb sequence in three fragments has pointed out that the reiteration was not homogeneous along the 1.73-kb sequence. The 1.73-kb clone was sequenced and compared with other interspersed repetitive sequences, previously described in the rodent genome, and no homology was found.

Animals↗

PubMed1994

Size heterogeneity among antigenically related Giardia lamblia variant-specific surface proteins is due to differences in tandem repeat copy number.

Giardia lamblia undergoes antigenic variation by modulating the expression of the different genes that comprise the trophozoite's variant-specific surface protein (VSP) repertoire. We studied an epitope that is conserved among VSPs expressed by cloned trophozoite lines derived from the independent G. lamblia isolates WB, G3M, Be-2, and CAT. The epitope recognized by monoclonal antibody 6E7 lies entirely within the region of tandemly repeated 65-amino-acid units that is characteristic of these size-variant VSPs. Northern (RNA) hybridization, cDNA cloning, and DNA sequence analysis indicate that size heterogeneity among these VSPs is due to differences in the number of repetitive units.

Amino Acid Sequence↗

PubMed1986

Short repeats cause heterogeneity at genomic terminus of bovine herpesvirus 1.

Analysis of the genomes of different bovine herpesvirus 1 strains revealed a UL terminal HindIII fragment differing in size (from 2.4 to 2.8 kilobases). This fragment polymorphism occurred in the DNA of a wild-type isolate, in highly passaged, apathogenic tissue culture derivatives, and in plaque-purified substrains. This heterogeneity was due to variations in the copy number of a 14-base-pair tandem repeat comprising the base sequence 5'-GCTCCTCCTCCCTC-3', which also exists, with some differences, in other short reiteration sequences of herpes simplex virus type 1, Epstein-Barr virus, and related human cellular DNA. Furthermore, the tandem repeat array was located in close proximity to the left end of the viral genome and may functionally be involved in viral replication.

Animals↗

PubMed1995

Adenovirus type 12-induced fragility of the human RNU2 locus requires U2 small nuclear RNA transcriptional regulatory elements.

Infection of human cells with oncogenic adenovirus type 12 (Ad12) induces four specific chromosome fragile sites. Remarkably, three of these sites appear to colocalize with tandem arrays of genes encoding small, abundant, ubiquitously expressed structural RNAs--the RNU1 locus encoding U1 small nuclear RNA (snRNA), the RNU2 locus encoding U2 snRNA, and the RN5S locus encoding 5S rRNA. Recently, an artificial tandem array of the natural 5.8-kb U2 repeat unit has been shown to generate a new Ad12-inducible fragile site (Y.-P. Li, R. Tomanin, J. R. Smiley, and S. Bacchetti, Mol. Cell. Biol. 13:6064-6070, 1993), demonstrating that the U2 repeat unit alone is sufficient for virally induced fragility. To identify elements within the U2 repeat unit that are required for virally induced fragility, we generated cell lines containing artificial tandem arrays of the entire 5.8-kb repeat unit, an 834-bp fragment spanning the U2 gene alone, or the same 834-bp fragment from which key U2 transcriptional regulatory elements had been deleted. The U2 snRNA coding regions within each artificial array were marked by an innocuous single base change (U to C at position 87) so that the relative expression of supernumerary and endogenous U2 genes could be monitored by a primer extension assay. We find that artificial arrays of both the 5.8- and the 0.8-kb U2 repeat units are fragile but that arrays lacking either the distal sequence element or both the distal and the proximal sequence elements of the promoter are not. Surprisingly, variations in repeat copy number and/or transcriptional activity of the artificial arrays do not appear to correlate with the degree of Ad12-inducible fragility. We conclude that U2 transcriptional regulatory elements are required for virally induced fragility but not necessarily U2 snRNA transcription per se.

Adenovirus Infections, Human↗