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PubMed1994

An amplifiable DNA region from the Mycoplasma hyorhinis genome.

A novel amplifiable genomic region that displays variability in the number of tandem copies of a 1,368-bp DNA sequence (designated RS-2) was discovered among individual clonal derivatives within Mycoplasma hyorhinis broth-grown cell populations. Clonal isolates representing variant subpopulations from the original broth culture were of a single size variant, and although continued culture under a variety of growth conditions did not result in further amplification of RS-2, evidence for deletion events which reduced RS-2 copy number, presumably by homologous recombination, was obtained. RS-2 homologous sequences were identified in all M. hyorhinis strains tested, but only the tissue culture-derived strains GDL-1 and GDL-2 showed variability in genomic dosage. The RS-2 nucleotide sequence established that each tandem copy is flanked by direct repeats of a 20-bp sequence and suggested a possible mechanism for its original duplication as the initial phase of a genetic amplification process. The coding strand was defined by PCR amplification of a reverse transcriptase-generated cDNA, and its sequence revealed that RS-2 encodes a 456-residue internal, highly cysteine-rich domain of a larger M. hyorhinis protein whose coding sequence initiates and terminates in unique genomic sequences several hundred base pairs from RS-2 on either side of it. Changes in RS-2 copy number maintain the reading frame, and therefore the coding capacity, for this predicted size-variant protein.

Amino Acid Sequence↗

PubMed1993

Comparative analyses of heterochromatin in Microtus: sequence heterogeneity and localized expansion and contraction of satellite DNA arrays.

Southern blotting, C-banding, base-specific fluorochrome staining, and fluorescence in situ hybridization were used to analyze the constitutive heterochromatin in eight species and subspecies of arvicolid rodents (genus Microtus). Autosomal centromeric regions portrayed considerable variability between species in the amount of C-band-positive material present; e.g., M. chrotorrhinus showed relatively little, whereas M. cabrerae exhibited extensive centromeric staining. Autosomal interstitial C-bands were noted in M. guentheri and two subspecies of M. ochrogaster. All Y chromosomes examined were predominately or completely heterochromatic, as were substantial portions of the giant X chromosomes in three species (M. agrestis, M. cabrerae, and M. chrotorrhinus). Hoechst 33258 staining (with its affinity for AT binding sites) showed bright fluorescence on the heterochromatin of the sex chromosomes of M. agrestis and moderate fluorescence on those of M. cabrerae and M. chrotorrhinus; however, only the heterochromatin of M. cabrerae and M. chrotorrhinus hybridized with an AT-rich satellite DNA probe (MSAT-160) isolated from M. chrotorrhinus. Hoechst 33258-bright autosomal centromeres of M. arvalis and M. cabrerae also hybridized to the probe, whereas the Hoechst 33258-bright Y chromosomes of M. arvalis and M. guentheri did not. Two pairs of autosomes in M. guentheri are comprised of six distinct regions, based upon C-banding, Hoechst 33258 staining, chromomycin A3/distamycin A staining, and in situ hybridization. The centromeric regions of acrocentric autosomes known to retain conserved G-banding patterns may exhibit variable hybridization intensity when different species or subspecies are compared. M. ochrogaster portrays considerable intersubspecific variability in the size and location of autosomal telomeric and interstitial C-bands that are also sites of hybridization. These latter two findings illustrate that dramatic differences in copy number of the tandem satellite array can exist at homologous chromosomal positions both within and between species.

Animals↗

PubMed2006

DNA microarray and proteomic strategies for understanding alcohol action.

This article summarizes the proceedings of a symposium presented at the 2005 annual meeting of the Research Society on Alcoholism in Santa Barbara, California. The organizer was James M. Sikela, and he and Michael F. Miles were chairs. The presentations were (1) Genomewide Surveys of Gene Copy Number Variation in Human and Mouse: Implications for the Genetics of Alcohol Action, by James M. Sikela; (2) Regional Differences in the Regulation of Brain Gene Expression: Relevance to the Detection of Genes Associated with Alcohol-Related Traits, by Robert Hitzemann; (3) Identification of Ethanol Quantitative Trait Loci Candidate Genes by Expression Profiling in Inbred Long Sleep/Inbred Short Sleep Congenic Mice, by Robnet T. Kerns; and (4) Quantitative Proteomic Analysis of AC7-Modified Mice, by Kathleen J. Grant.

Alcoholism↗

PubMed2002

The evolution of the vertebrate beta-globin gene promoter.

Complexity analysis is capable of highlighting those gross evolutionary changes in gene promoter regions (loosely termed "promoter shuffling") that are undetectable by conventional DNA sequence alignment. Complexity analysis was therefore used here to identify the modular components (blocks) of the orthologous beta-globin gene promoter sequences of 22 vertebrate species, from zebrafish to humans. Considerable variation between the beta-globin gene promoters was apparent in terms of block presence/absence, copy number, and relative location. Some sequence blocks appear to be ubiquitous, whereas others are restricted to a specific taxon. Block similarities were also evident between the promoters of the paralogous human beta-like globin genes. It may be inferred that a wide variety of different mutational mechanisms have operated upon the beta-globin gene promoter over evolutionary time. Because these include gross changes such as deletion, duplication, amplification, elongation, contraction, and fusion, as well as the steady accumulation of single base-pair substitutions, it is clear that some redefinition of the term "promoter shuffling" is required. This notwithstanding, and as previously described for the vertebrate growth hormone gene promoter, the modular structure of the beta-globin promoter region and those of its paralogous counterparts have continually been rearranged into new combinations through the alteration, or shuffling, of preexisting blocks. Some of these changes may have had no influence on promoter function, but others could have altered either the level of gene expression or the responsiveness of the promoter to external stimuli. The comparative study of vertebrate beta-globin gene promoter regions described here confirms the generality of the phenomenon of sequence block shuffling and thus supports the view that it could have played an important role in the evolution of differential gene expression.

Amino Acid Sequence↗

PubMed1985

Variable numbers of pepsinogen genes are located in the centromeric region of human chromosome 11 and determine the high-frequency electrophoretic polymorphism.

A panel of 26 mouse-human somatic cell hybrids containing different human chromosome complements was analyzed with a cloned human pepsinogen cDNA probe to determine the chromosomal location and the number of genes encoding these proteins. A complex containing variable numbers of pepsinogen genes was localized to the centromeric region of human chromosome 11 (p11----q13). Examination of somatic cell hybrids containing single copies of chromosome 11 and the corresponding human parental cell lines revealed a restriction fragment length polymorphism determined by pepsinogen haplotypes that contained two or three genes, respectively. Concurrent studies of DNA from individuals exhibiting the most common pepsinogen electrophoretic phenotypes with exon-specific probes demonstrated that the absence of one gene among the different restriction fragment patterns correlated with the absence of one specific isozymogen (Pg 5). Thus, our studies demonstrate that this genetic polymorphism involving intensity variation of individual pepsinogen isozymogens results from chromosome haplotypes that contain different numbers of genes. The regional localization of this polymorphic gene complex will facilitate detailed linkage analysis of human chromosome 11.

Centromere↗

PubMed2002

Diversity and varietal classification of Hibiscus syriacus L. with the heterogeneity within retrotransposon-like elements.

Retrotransposons are present in multi-copy numbers that are integrated into plant genomes with considerable heterogeneous sequences within a single plant and between plant species, which allows the use of retrotransposons as additional sources of DNA polymorphism. A primer design for the sequence-tagged specific site and cleaved amplified polymorphic sequences (STS-CAPs) that are derived from retrotransposon-like sequences was developed for the molecular marker analysis in Hibiscus syriacus. This method was applied for the detection of sequence variations of intact retrotransposons that exist in plant genomes, which resulted in higher polymorphisms than in the amplified fragment length polymorphism (AFLP). Through STS-CAPs, specific fingerprinting data among H. syriacus varieties can be easily distinguished and generated with reproducible results. It could also be adapted to any species that possess multi-copy retrotransposons for varietal identification as well as the assessment of genetic relationships.

DNA Fingerprinting↗

PubMed2001

An insulator element from the sea urchin Hemicentrotus pulcherrimus suppresses variation in transgene expression in cultured tobacco cells.

Specialized DNA sequences known as insulators protect genes from both the positive and negative influences of nearby chromatin. Many insulators have been identified in various species; however, few function in multiple species. We have shown that an insulator from the Ars (arylsulfatase) gene of the sea urchin Hemicentrotus pulcherrimus functions in plant cells. Normally, expression of an introduced chimeric GUS gene is inactivated in approximately 30% of transformed tobacco BY2 clones. Transgenes containing the Ars insulator, however, were expressed in all transformed tobacco BY2 cells. The insulator did not affect the copy number, the chromosomal position of transgene integration or maximum expression levels. These results suggest that the insulator functions to suppress the variation normally associated with transgene expression in tobacco BY2 cells.

Animals↗

PubMed2003

Quantitative analysis of human mitochondrial DNA using a real-time PCR assay.

OBJECTIVES: Known for their ability to inhibit the human DNA polymerase-gamma, nucleoside analogues induce toxic effects on mitochondria ranging from increased serum lactate levels to fatal lactic acidosis. DNA polymerase-gamma ensures the mitochondrial DNA (mtDNA) replication and, thus, its inhibition leads to the decrease of the mtDNA. We describe a real-time PCR assay for mtDNA quantification associating DNA extraction procedures applied on peripheral blood mononuclear cells (PBMCs) and subcutaneous adipose tissues and to study the antiretroviral effect on mitochondria. METHODS: Total DNA was extracted from PBMCs and subcutaneous adipose tissues. Nuclear and mitochondrial genes were amplified to determine the number of copies of mtDNA per cell using a cyt-b recombinant plasmid as standard control. We analysed eight HIV-infected asymptomatic patients never treated, four patients who had been treated for 6 months with highly active antiretroviral therapy (HAART) and six non-infected donors. RESULTS: The mtDNA quantification gave rise to reproducible results as the mean coefficients of variation were 1.09% for replicates of samples undertaken 10 times within the same run, and 5.78% and 3.7% for replicates tested in five different runs at 1:100 and 1:1000 dilutions, respectively. Median levels of mtDNA in PBMCs of healthy donors, naive and treated HIV-infected patients were 2.94, 2.78 and 1.93 log HIV-1 RNA copies/mL, respectively. Whereas DNA from PBMCs was shown to be devoid of inhibitors, subcutaneous adipose tissues needed an extra treatment as they were found to be highly inhibited. CONCLUSIONS: The method generated consistent and reproducible results and was successfully applied to DNAs extracted from PBMCs and subcutaneous adipose tissues with adapted extraction. The mtDNA changes in PBMCs were found to be fast as they fall off after 6 months' therapy, decreasing from 2.78 to 1.93 log copies/mL.

Adipose Tissue↗

PubMed1991

A specific point mutation in the mitochondrial genome of Caucasians with MELAS.

The mitochondrial DNA (mtDNA) of Japanese patients suffering from the syndrome of mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) exhibits a specific heteroplasmic A----G transition in the tRNA(Leu) at position 3243. In this study, we investigated mtDNA from skeletal muscle, cardiac muscle, brain, liver, diaphragm, fibroblasts and blood cells of four Caucasians with MELAS, one younger healthy sister of two MELAS patients, and eleven controls. We found that 1) the mutation was present in all investigated tissues of Caucasians with MELAS but not in controls, 2) within a single patient, the tissue-specific variation of the copy number of mutated mtDNA covered the same range as in the skeletal muscle of different patients, 3) the mutation was also present in the blood cells of the healthy sister of two MELAS siblings.

Acidosis, Lactic↗

PubMed1996

Plasmodium falciparum: molecular characterization of multidrug-resistant Cambodian isolates.

Clinical resistance to many therapies for malaria is a rapidly evolving problem in most endemic areas, particularly, Southeast Asia. Recent studies have suggested linkages between the mdr-like genes of Plasmodium falciparum and resistance to quinoline containing compounds. Other studies have found an association between allelic polymorphisms in the DHFR gene and antifol resistance in these parasites. The purpose of this study was to further examine these associations in recent isolates from Cambodia. DNA sequences and gene copy number of the pfmdrl and the DHFR-TS gene in 10 Cambodian isolates were analyzed and correlated with the drug sensitivity pattern. No new intragenic alleles were detected in the pfmdrl gene by a full-length DNA sequence analysis of the L-14/Cambodia clone. The allelic variations seen in pfmdrl in these isolates did not correlate with chloroquine resistance as previously reported. The full-length sequence of the DHFR latter findings may be correlated with high-level resistance to the antifolate drugs as has been previously described. None of the Cambodian isolates presented gene amplification in either pfmdrl or DHFR-TS genes.

Alleles↗

PubMed1998

Multiple copies of the ALA-D gene are located at the Lv locus in Mus domesticus mice.

Incremental differences in delta-aminolevulinate dehydratase (ALA-D; the second enzyme of the heme biosynthetic pathway) activity among inbred mouse strains can be attributed to variation in the number of copies of the ALA-D gene. We have cloned and characterized the Lv locus from an inbred mouse strain (DBA/2J) that has three times the normal ALA-D activity levels. The entire 12-kb ALA-D gene plus 16 kb of flanking DNA are found in 28-kb tandemly repeating units. We used the derived nucleotide sequence surrounding the internal junction of the repeats to survey wild-caught mice and demonstrate that multiple copies of the ALA-D gene occur in 7 of 24 worldwide locations of Mus domesticus mice. Data are consistent with a model that high lead (Pb) in the environment may be providing a selective advantage to mice harboring multiple copies of the ALA-D gene, since the enzyme is potently inhibited by lead.

Animals↗

PubMed1991

Evolutionary lines among Salmonella enteritidis phage types are identified by insertion sequence IS200 distribution.

A survey was made of the presence, copy number and location of the Salmonella-specific DNA insertion element IS200, within the genomes of the 27 phage type strains of Salmonella enteritidis. All the phage type strains contained copies of IS200 revealed by genomic Southern blot hybridizations with a 300-bp DNA probe internal to the element. Restriction site variation around IS200 insertion sites was examined. Three fundamental patterns of hybridization corresponding to chromosomal IS200 loci were found. In terms of population genetics, these 'IS200 profiles' correspond to clonal lineages of recent evolutionary origin, and underline the phage-typing scheme for epidemiological subdivision of S. enteritidis. The molecular analysis is consistent with genetic selection pressures which are apparent in the observed epidemiological distribution of S. enteritidis, since each clonal lineage contained one of the phage types of major clinical importance in the U.K.

Biological Evolution↗

PubMed1999

Interphasic analysis of aneuploidy in cancer cell lines using primed in situ labeling.

The primed in situ (PRINS) labeling technique has been adapted to chromosomal screening of interphasic tumoral cells. A panel of ten chromosome-specific alpha-satellite DNA primers was used to evaluate numerical chromosome abnormalities in two colon cancer cell lines (Caco-2 and HT-29) and in three of their subpopulations (PF11, TC7, and HT29-MTX). In each cell line, the copy number distribution for different chromosomes showed different patterns. The observation of significant variations in the chromosome constitutions between subpopulations derived from the same original tumor suggests the common occurrence of chromosome copy number heterogeneity in tumoral cell lines. This study demonstrates that the PRINS procedure offers a simple and reliable method for in situ chromosomal screening, which could be efficiently used for karyotypic analysis of tumoral cells.

Aneuploidy↗

PubMed1993

Expression of a retroposon-like sequence upstream of the putative Trypanosoma brucei variant surface glycoprotein gene expression site promoter.

We have cloned the region spanning the putative promoter from two variant surface glycoprotein gene expression sites that are at each end of chromosome M4 of Trypanosoma brucei IsTat 7. Both expression sites contain a retroposon-like sequence (ESR) pseudogene whose 3' end is approximately 30 bp upstream of the putative expression site promoter. The ESRs from both expression sites share considerable sequence homology and are related to LINE-like elements, especially the T. brucei ingi retroposon. Other ESRs are located on large, but not intermediate or mini-, chromosomes in the IsTaR 1 serodeme, and the total copy number is 10 to 20, similar to that estimated for variant surface glycoprotein expression sites. No DNA rearrangements in the vicinity of the ESR and putative expression site promoter were detected following antigenic switches in the IsTaR 1 serodeme. ESR transcripts are present in bloodstream, but not procyclic, forms. Variation in transcript size and sequence between bloodstream variant antigenic types implies that only the ESR from the active expression site is transcribed. This pattern of expression reflects that of sequences downstream of the putative expression site promoter, suggesting that the region of coordinately controlled expression extends upstream of this promoter.

Amino Acid Sequence↗

PubMed2001

Mitochondrial pseudogenes are pervasive and often insidious in the snapping shrimp genus Alpheus.

Here we show that multiple DNA sequences, similar to the mitochondrial cytochrome oxidase I (COI) gene, occur within single individuals in at least 10 species of the snapping shrimp genus Alpheus. Cloning of amplified products revealed the presence of copies that differed in length and (more frequently) in base substitutions. Although multiple copies were amplified in individual shrimp from total genomic DNA (gDNA), only one sequence was amplified from cDNA. These results are best explained by the presence of nonfunctional duplications of a portion of the mtDNA, probably located in the nuclear genome, since transfer into the nuclear gene would render the COI gene nonfunctional due to differences in the nuclear and mitochondrial genetic codes. Analysis of codon variation suggests that there have been 21 independent transfer events in the 10 species examined. Within a single animal, differences between the sequences of these pseudogenes ranged from 0.2% to 20.6%, and those between the real mtDNA and pseudogene sequences ranged from 0.2% to 18.8% (uncorrected). The large number of integration events and the large range of divergences between pseudogenes and mtDNA sequences suggest that genetic material has been repeatedly transferred from the mtDNA to the nuclear genome of snapping shrimp. Unrecognized pseudogenes in phylogenetic or population studies may result in spurious results, although previous estimates of rates of molecular evolution based on Alpheus sister taxa separated by the Isthmus of Panama appear to remain valid. Especially worrisome for researchers are those pseudogenes that are not obviously recognizable as such. An effective solution may be to amplify transcribed copies of protein-coding mitochondrial genes from cDNA rather than using genomic DNA.

Animals↗

PubMed1997

Tissue-specific selection for different mtDNA genotypes in heteroplasmic mice.

Mammalian mitochondrial DNA (mtDNA) is a highly polymorphic, high-copy-number genome that is maternally inherited. New mutations in mtDNA segregate rapidly in the female germline due to a genetic bottleneck in early oogenesis and as a result most individuals are homoplasmic for a single species of mtDNA. Sequence variants thus accumulate along maternal lineages without genetic recombination. Most of the extant variation in mtDNA in mammalian populations has been assumed to be neutral with respect to selection; however, comparisons of the ratio of replacement to silent nucleotide substitutions between species suggest that the evolution of mammalian mtDNA is not strictly neutral. To test directly whether polymorphic mtDNAs behave as neutral variants, we examined the segregation of two different mtDNA genotypes in the tissues of heteroplasmic mice. We find evidence for random genetic drift in some tissues, but in others strong, tissue-specific and age-related, directional selection for different mtDNA genotypes in the same animal. These surprising data suggest that the coding sequence of mtDNA may represent a compromise between the competing demands of different tissues and point to the existence of unknown, tissue-specific nuclear genes important in the interaction between the nuclear and mitochondrial genomes.

Age Factors↗

PubMed2006

A BAC library for the goldfish Carassius auratus auratus (Cyprinidae, Cypriniformes).

A goldfish (Carassius auratus auratus) bacterial artificial chromosome genomic library (BAC library) was constructed from one aquarium-bred male specimen (tetraploid, 4n=100, genome size=3.52 pg/cell). The library consists of 128,352 positive clones with an average insert size of 150.4 kb, covering the genome 11-fold. All clones were spotted onto nylon filters and thus are available for screening of genomic regions of interest, such as candidate genes, gene families, or large-sized syntenic DNA regions of cyprinid species. Preliminary screens with two genes were conducted with hybridizing probes to the genes RAG1 and lgi1. RAG1 is a single-copy gene in zebrafish and is duplicated in C. a. auratus. We found a very close correlation between the number of positive BAC clones and the expected library coverage. Two copies of lgi1 were found in zebrafish. We have detected four different copies in C. a. auratus, not in the expected abundance, which indicates some variation in the coverage of the BAC library. The preliminary screens indicate that many duplicated genes that resulted from the ancient fish-specific genome duplication persist in the tetraploid goldfish genome. Hence, the BAC library will provide a useful resource for the future work on comparative genomics, polyploidy, diploidization, and evolutionary genomics in fishes.

Animals↗

PubMed1994

Structural analysis of two length variants of the rDNA intergenic spacer from Eruca sativa.

Restriction enzyme analysis of the rRNA genes of Eruca sativa indicated the presence of many length variants within a single plant and also between different cultivars which is unusual for most crucifers studied so far. Two length variants of the rDNA intergenic spacer (IGS) from a single individual E. sativa (cv. Itsa) plant were cloned and characterized. The complete nucleotide sequences of both the variants (3 kb and 4 kb) were determined. The intergenic spacer contains three families of tandemly repeated DNA sequences denoted as A, B and C. However, the long (4 kb) variant shows the presence of an additional repeat, denoted as D, which is a duplication of a 224 bp sequence just upstream of the putative transcription initiation site. Repeat units belonging to the three different families (A, B and C) were in the size range of 22 to 30 bp. Such short repeat elements are present in the IGS of most of the crucifers analysed so far. Sequence analysis of the variants (3 kb and 4 kb) revealed that the length heterogeneity of the spacer is located at three different regions and is due to the varying copy numbers of repeat units belonging to families A and B. Length variation of the spacer is also due to the presence of a large duplication (D repeats) in the 4 kb variant which is absent in the 3 kb variant. The putative transcription initiation site was identified by comparisons with the rDNA sequences from other plant species.

Base Sequence↗