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PubMed1986

Evolutionarily selected replication origins: functional aspects and structural organization.

A selective replicative pressure occurs during the evolution of simian virus 40 variants. When the replication origin is duplicated as an inverted repeat, there is a dramatic enhancement of replication. Having regulatory sequences located between the inverted repeat of ori magnifies their enhancing effect on replication. A passage 20 variant and a passage 45 variant containing three pairs of an inverted repeat of ori replicated more efficiently than a passage 13 variant containing nine copies of ori arranged in tandem. A 69-base-pair cellular sequence inserted between inverted repeats of ori of both passage 40 and 45 variants enhanced simian virus 40 DNA replication. Differences in replication efficiencies became greater as the total number of replicating species was increased in the transfection mixture, under conditions where T antigen is limiting. In a competitive environment, sequences flanking the replication origin may be inhibitory to replication.

Animals↗

PubMed1991

Genetic variation in mouse apolipoprotein A-IV due to insertion and deletion in a region of tandem repeats.

We have detected three unique apolipoprotein A-IV (apoA-IV) charge isoforms in strains of commensal mice. The cDNA sequences for one representative of each isoform (Mus domestesticus strains C57BL/6J and 129/J and Mus castaneus) revealed a polymorphism within a series of four imperfect repeats encoding the sequence Glu-Gln-Ala/Val-Gln. Insertions or deletions of 12 nucleotides within this repetitive region have given rise to three genotypes characterized by three (129), four (C57BL/6), or five (M. castaneus) copies of the repeat unit. To ascertain the extent of this variation among other species of the Mus genus, we sequenced this region of apoA-IV cDNAs from eight additional M. domesticus inbred strains and from five wild-derived Mus species. All eight additional M. domesticus strains examined had four repeat units, as found in C57BL/6. Among wild-derived mice, however, one species (Mus spretus) had three repeats, two species (Mus cookii and Mus cervicolor) had four repeats, and two species (Mus hortulanus and Mus minutoides) had five repeats. A lack of correlation between the number of repeat units and the phylogeny of Mus species indicates that independent mutations may have occurred throughout the evolution of specific mouse lineages. We suggest that the repetitive nature of the polymorphic sequence may predispose this region to slippage errors during DNA replication, resulting in frequent deletion/insertion mutations.

Amino Acid Sequence↗

PubMed1999

The electrophoretic karyotype of Entamoeba histolytica.

Although knowledge about gene organization and transcription control in the protozoan parasite Entamoeba histolytica has increased substantially during the last few years, questions remain open about ploidy, organization and number of chromosomes in this human pathogen. To get insight into these questions conditions were elaborated to consistently separate E. histolytica chromosomes using pulsed-field gel electrophoresis. Southern blot analyses indicated variations in number and size of homologous chromosomes between various E. histolytica isolates, but only minor differences were observed between clones of a given isolate. Depending on the isolate used 31-35 chromosomes were identified ranging in size from 0.3 to 2.2 megabases. The assignment of 68 independent cDNA probes to the chromosomes of three axenically cultured E. histolytica isolates identified 14 linkage groups, which suggested a haploid genome-size of < or =20 megabases. As single copy probes bound to as many as four chromosome-sized bands, it is most likely that E. histolytica has a functional ploidity of at least 4.

Animals↗

PubMed1992

Genetic divergence between the Wistar-Kyoto rat and the spontaneously hypertensive rat.

A method of restriction fragment length polymorphism (RFLP) analysis was used to estimate the amount of genetic divergence between the spontaneously hypertensive rat (SHR) strain and the Wistar-Kyoto (WKY) strain. DNA from each strain was digested with eight restriction endonucleases and hybridized with six single copy gene sequences. The number of hybridization bands in each digestion was used to estimate the total number of bases analyzed and RFLPs were scored as single mutations. Divergence was then estimated by dividing the number of mutations by the number of bases analyzed. In a total of 808 bases analyzed in WKY rats, a minimum of 13 mutations were scored in SHR, which yields a nucleotide divergence of 1 change per 62 bp. This is an extremely high amount of divergence given the known origin of these two strains and is comparable to the maximum divergence possible between unrelated humans.

Animals↗

PubMed2003

Variability of HXT2 at the protein and gene level among the Saccharomyces sensu stricto group.

Variability of HXT2 at the protein and gene level was investigated among Saccharomyces sensu stricto and other yeast species. Results showed that the HXT2 gene is probably present in yeast genera other than Saccharomyces, suggesting that this gene is widely distributed in the yeast world. Chromosomal analyses indicated the stable location of HXT2 on the same chromosome and with the same copy number throughout the entire sensu stricto group. Results of the immunoblotting assay demonstrated that all strains tested (with the exception of S. cerevisiae DBVPG 6042) exhibited a lower level of Hxt2p expression than that shown by laboratory wild-type. Moreover, Hxt2p expression seems to reinforce the taxonomical differences between the two pairs of species (S. cerevisiae and S. paradoxus vs. S. pastorianus and S. bayanus) within the sensu stricto group of the genus of Saccharomyces that also reflect their different ecological niche.

Chromosomes, Fungal↗

PubMed2000

The potential of soil microorganisms to mineralize atrazine as predicted by MCH-PCR followed by nested PCR.

The potential of soil microorganisms to mineralize atrazine was studied in soil samples collected from fields with various histories of atrazine application. In contrast to many previous studies, which showed no atrazine mineralization activity, all the tested soils mineralized atrazine regardless of their atrazine application history. However, the delay before mineralization and the variation in the subsequent mineralization rate were in agreement with the initial copy number of the atrazine dechlorinaze gene, and the proliferation rate of the degraders. Soils from corn fields, which had up to 100 copies of the atzA gene per gram of soil, had a lag period of 4-5 days before atrazine mineralization started, and final mineralization percentages ranged from 40% to 54%. However, soils from fields that were never amended with atrazine had much longer lag periods (more than 17 days), which decreased after enrichment of the degrader population with high concentrations of atrazine for 15 days. Generally the mineralization rate and the atzA gene copy number increased after the enrichment period. The atrazine mineralization potential was measured by PCR of genes from the atrazine mineralization pathway. Magnetic capture hybridization was the most efficient of the two tested methods for purifying target DNA of PCR inhibitors, without reducing the copy number of the required fragment. Nested PCR proved to be the most effective method for predicting the exact potential of the soil to mineralize the pollutant even without enrichment of a small population with the target genes. This method can complement microcosm studies and eliminate futile efforts when the potential to mineralize the pollutant does not exist in the soil.

Atrazine↗

PubMed2001

Diversity and dynamics of the minichromosomal karyotype in Trypanosoma brucei.

The genome of African trypanosomes contains a large number of minichromosomes. Their only proposed role is in the expansion of the parasites' repertoire of telomeric variant surface glycoprotein (VSG) genes as minichromosomes carry silent VSG gene copies in telomeric locations. Despite their importance as VSG gene donors, little is known about the actual composition of the minichromosomal karyotype and the stability of its inheritance. In this study we show, by using high-resolution pulsed-field electrophoresis, that a non-clonal trypanosome population contains an extremely diverse pattern of minichromosomes, which can be resolved into less complex clone-specific karyotypes by non-selective cloning. We show that the minichromosome patterns of such clones are stable over at least 360 generations. Furthermore, using DNA markers for specific minichromosomes, we demonstrate the mitotic stability of these minichromosomes within the population over a period of more than 5 years. Length variation is observed for an individual minichromosome and is most likely caused by a continuous telomeric growth of approximately 6 bp per telomere per cell division. This steady telomeric growth, counteracted by stochastic large losses of telomeric sequences is the most likely cause of minichromosome karyotype heterogeneity within a population.

Animals↗

PubMed1993

Variability and evolution of highly repeated DNA sequences in the genus Beta.

Satellite DNA from wild beet species was separated from restriction endonuclease digested genomic DNA by polyacrylamide gel electrophoresis. Two nonhomologous HaeIII satellite DNA repeats were cloned from the wild beet Beta trigyna. The type I repeat is 140-149 bp long and AT rich, while the type II is 162 bp in size and GC rich. A third repetitive HaeIII element cloned from the related wild beet B. corolliflora was shown to be organized as a HinfI satellite DNA family in the cultivated beet B. vulgaris ssp. vulgaris and the wild beet B. vulgaris ssp. maritima. This type III satellite monomer is 149 bp long and contains a high number of short direct subrepeats. The monomer was found in different genomic organizations and copy numbers in all sections of the genus Beta indicating an amplification early in the phylogeny. The HaeIII repeats from B. trigyna are characterized by a lower variability and form long tandem arrays in the genomes of Corollinae species. The investigation of the distribution of all three sequence families provided data that may contribute to the solution of taxonomic problems of the genus Beta and be useful in the characterization of hybrids and derived lines with alien wild beet chromosomes.

Base Sequence↗

PubMed1999

Use of pulsed-field gel electrophoresis for molecular epidemiologic and population genetic studies of Mycobacterium tuberculosis.

Pulsed-field gel electrophoresis (PFGE) is a powerful molecular biology technique which has provided important insights into the epidemiology and population biology of many pathogens. However, few studies have used PFGE for the molecular epidemiology of Mycobacterium tuberculosis. A laboratory protocol was developed to determine the typeability, stability, and reproducibility of PFGE typing of M. tuberculosis. Formal data-analytical techniques were used to assess the genetic diversity elucidated by PFGE analyses using four separate restriction enzymes and by IS6110 RFLP analyses, as well as to assess the concordance among these typing methods. One hundred epidemiologically characterized clinical isolates of M. tuberculosis were genotyped with four different PFGE enzymes (AseI, DraI, SpeI, and XbaI), as well as by RFLP analysis with IS6110. Identical patterns were found among 34 isolates known to be genetically related, suggesting that the PFGE protocol is robust and reproducible. Among 66 isolates representing population-sampled cases, heterozygosity and information content dependency estimates indicate that all five genotyping systems capture quantitatively similar levels of genetic diversity. Nevertheless, comparisons between PFGE analyses and IS6110 typing reveals that PFGE provided more discrimination among isolates with fewer than five copies of IS6110 and less clustering in isolates with five or more copies. The comparisons confirm the hypothesis that the resolution of IS6110 RFLP genotyping is dependent upon the number of IS6110 elements in the genome of isolates. The general concordance among the results obtained with four independent enzymes suggests that M. tuberculosis is a clonal organism. The availability of a robust genotyping technique largely independent of repetitive elements has implications for the molecular epidemiology of M. tuberculosis.

DNA Transposable Elements↗

PubMed1995

Quantitative analysis of comparative genomic hybridization.

Comparative genomic hybridization (CGH) is a new molecular cytogenetic method for the detection of chromosomal imbalances. Following cohybridization of DNA prepared from a sample to be studied and control DNA to normal metaphase spreads, probes are detected via different fluorochromes. The ratio of the test and control fluorescence intensities along a chromosome reflects the relative copy number of segments of a chromosome in the test genome. Quantitative evaluation of CGH experiments is required for the determination of low copy changes, e.g., monosomy or trisomy, and for the definition of the breakpoints involved in unbalanced rearrangements. In this study, a program for quantitation of CGH preparations is presented. This program is based on the extraction of the fluorescence ratio profile along each chromosome, followed by averaging of individual profiles from several meta phase spreads. Objective parameters critical for quantitative evaluations were tested, and the criteria for selection of suitable CGH preparations are described. The granularity of the chromosome painting and the regional inhomogeneity of fluorescence intensities in metaphase spreads proved to be crucial parameters. The coefficient of variation of the ratio value for chromosomes in balanced state (CVBS) provides a general quality criterion for CGH experiments. Different cutoff levels (thresholds) of average fluorescence ratio values were compared for their specificity and sensitivity with regard to the detection of chromosomal imbalances.

Chromosome Banding↗

PubMed1994

Amplification of the ancient murine Lx family of long interspersed repeated DNA occurred during the murine radiation.

We identified and characterized the relics of an ancient rodent L1 family, referred to as Lx, which was extensively amplified at the time of the murine radiation about 12 million years ago, and which we showed was ancestral to the modern L1 families in rat and mouse. Here we have extended our analysis of the Lx amplification by examining more murine and nonmurine species for Lx sequences using both blot hybridization and the polymerase chain reaction for a total of 36 species. In addition we have determined the relative copy number and sequence divergence, or age, of Lx elements in representative murine genera. Our results show that while Lx sequences are confined to murine genera, the extent of the amplification was different in the different murine lineages, indicating that the amplification of Lx did not precede, but was coincident with, the murine radiation. The implications of our findings for the evolutionary dynamics of L1 families and the utility of ancestral amplification events for systematics are discussed.

Animals↗

PubMed2001

[Localization and analysis of 1A6 gene by dual-color fluorescence in situ hybridization on gastric carcinoma tissue and tumor cell lines].

OBJECTIVE: To locate 1A6 gene on the chromosome and study its copy number by dual-color fluorescence in situ hybridization (FISH) on three tumor cell lines and 22 gastric tumors patients. METHODS: The single-color FISH of small size biotin-labeled cDNA cloned in plasmid was used to locate the 1A6 gene on one of the chromosomes in C group. Genomic DNA of 1A6 gene was screened by polymerase chain reaction (PCR) in PAC library according to the sequence. The florenscence of green and orange was incorporated into the target gene-1A6 cloned in PAC and chromosome-12-specific alpha-satellite repeat DNA by nick translation, followed by dual-color FISH. 1A6 gene was located in the metaphase chromosome of the normal peripheral lymphocytes. 1A6 gene and chromosome 12 copy numbers were analyzed on touch slide of gastric cancer tissue and cell lines. RESULTS: 1A6 gene was located in 12q23.2-23.3. The chromosome 12/1A6 signal ratio was 0.96-1.01 in breast, ovarian and gastric cancer cell lines. The ratio was 0.93-1.11 in gastric cancer tissue touch slides. The number of chromosome 12 is disomic (87.7%), triploid (7.4%) in BMI cell line; multisomic(100%), including pentasomic (67.6%) in SKOV3; the multisomic (83.1%), including trisomic (71%) in SGC823. There are highly disomic rate in 86. 4% (19/22) of patients. CONCLUSION: 1A6 gene is in 12q23.2-23.3. Neither 1A6 amplification nor deletion in gastric cancer tissue and three cell lines was found. Further study is needed for the understanding of chromosome 12 copy number variation in different cancer cell lines.

Chromosome Mapping↗

PubMed1989

Ag-NOR staining and in situ hybridization of rDNA in the chromosomes of the South American camelids.

The location and frequency of Ag-stained NORs and sites of rDNA hybridization were studied in the chromosomes of the South American camelids. In the four camelids these regions occur distally on chromosomes 18, 21, and 27 and the smallest biarmed elements. Quantitative analysis of NOR distribution showed variations between both cells and species. In llama, guanaco and alpaca the NORs number averaged 6 per cell, this being higher than in vicuña where the average was 3. Relative frequencies of NOR-bearing chromosomes in the four camelids were similar. Yet, in vicuña virtual absence of NOR sites on one of the smallest biarmed pairs was observed. The rDNA sites assessed in llama and vicuña by in situ hybridization with cloned 18S DNA were coincident with the NOR locations and with the frequencies characteristics for each species. Moreover, varying the exposure time of the autoradiographs, labeling patterns specific for each camelid were observed. Grain counts on individual chromosomes indicated that under our conditions one month exposure is enough to demonstrate all the rDNA sites available in the complement of llama. Conversely, at least two months are necessary to show the total sites existing in vicuña. Most probably this finding reflects the presence of variations in the amount of copies of the ribosomal genes per chromosome.

Animals↗

PubMed2004

Somatic variation plays a key role in the evolution of the Vf gene family residing in the Vf locus that confers resistance to apple scab disease.

A cluster of four receptor-like genes has been previously identified in the Vf locus of the crabapple Malus floribunda clone 821 that confers resistance to five races of the fungal pathogen Venturia inaequalis, the casual agent of apple scab disease. Pairwise comparisons of the four Vf paralogs in both promoter and coding regions reveal their timeline evolutionary history. The four Vf paralogs have evolved from four ancient Vf members resulting from two sequential duplication events of a single Vf progenitor initially present in the Malus genome. The coding sequences of the four Vf paralogs are characterized with high numbers of unique polymorphic nucleotides, a number of short duplications/deletions, various deletions of complete LRR copy units, and a casual insert of a transposon-like element. Significant high ratios of nonsynonymous to synonymous substitutions, Ka/Ks, are observed in the putative ligand binding residues in the LRR domains. No sequence exchange between the four Vf paralogs is observed. Compared with promoter regions, only nucleotide substitutions are dramatically elevated in the coding regions. The results presented in this study strongly indicate that the Vf locus is under strong and steady horizontal selective pressures imposed by the fungal pathogen V. inaequalis, and divergent selection on somatic variations plays a key role in shaping the resistance specificity.

Ascomycota↗

PubMed1998

Appropriate mammalian expression systems for biopharmaceuticals.

Process development for biopharmaceuticals is dictated by product quality, drug safety and economy of the manufacturing process. Not surprisingly, these factors also play a key role in the evaluation of mammalian cell expression systems to be used in the production of pharmacologically active glycoproteins. To date, the most prominent candidates for efficient expression of glycoproteins are mammalian cell lines such as mouse fibroblast cells (C 127-BPV), Chinese hamster ovary cells (CHO-DHFR, CHO-NEOSPLA, CHO-GS), mouse myeloma cells (NSO-GS) as well as transgenic animals carrying c-DNA or genomic DNA which codes for the protein of interest. The expression titer in the case of glycoproteins is mainly determined by the promoter construct, the site of integration into the chromosome, the copy number and the type of protein in question. Based on expression titer, CHO-NEOSPLA and NSO-GS expression systems are most effective in the production of monoclonal antibodies and, to a lesser extent, of recombinant DNA derived proteins. However, based on overall product yield, expression of recombinant DNA derived proteins in transgenic animals is by far the most promising system. Therefore, for proteins required in large quantities, transgenic expression systems offer an attractive choice. However, cost of goods for products for which the dosage or the overall annual quantities are low, is dominated by downstream processing, filling, lyophilization and packaging and not by the fermentation process. Such proteins are preferentially produced by classical mammalian cell culture systems. Concerns which have to be addressed with respect to drug safety in the transgenic animal approach are the size of the herd, genetic stability from animal to animal, variation in productivity and in impurity profiles during lactation periods, microbial, viral, mycoplasma and prion contaminants, the dependence on health status and the life span of the animal. In a number of cases glycosylation of the protein is relevant for the prevention of immunogenicity of the protein, the pharmacological activity, the pharmacokinetic profile, solubility and stability against proteolysis. The glycosylation pattern, depending on protein structure, is influenced by the enzymatic system of the host cell as well as by fermentation conditions. Therefore, selection of host cells and culture conditions must take into account the requirement for a specific and stable glycosylation pattern. For the assessment of glycovariants, a number of protein analytical methods such as peptide mapping, isoelectric focusing, oligosaccharide mapping, MALDI-TOF (matrix assisted laser desorption mass spectrometry-time of flight), capillary electrophoresis and specific potency assays are available. In our experiments, glycosylation of proteins expressed in CHO cells was demonstrated to be very stable. Only extreme process times, cultivation methods and ammonium ion concentrations had an influence on the glycosylation profile. Among the three products investigated--tissue plasminogen activator (t-PA), interferon omega and soluble intercellular adhesion molecule (s-ICAM)--t-PA expressed the most stable glycosylation pattern. Only at extreme ammonium concentrations an increase of mannose-5 structures was observed, whereas biantennary complex structures were reduced. On the other hand, interferon omega and s-ICAM showed greater susceptibility to increased ammonium concentrations and to adherent cultivation. Such conditions induced quantitative changes to the glycosylation pattern favoring the appearance of higher branched structures. Short cultivation times resulted in more heterogenous oligosaccharide structures. Since the glycosylation of the three proteins is different in the same host cell, the amino acid sequence of the protein apparently influences the glycosylation pattern and its sensitivity to culture conditions. In NSO-mouse myeloma cells, production of s-ICAM is two times as high as in CHO cells

Animals↗

PubMed2004

Genome-wide comparative analysis of the transposable elements in the related species Arabidopsis thaliana and Brassica oleracea.

Transposable elements (TEs) are the major component of plant genomes where they contribute significantly to the >1,000-fold genome size variation. To understand the dynamics of TE-mediated genome expansion, we have undertaken a comparative analysis of the TEs in two related organisms: the weed Arabidopsis thaliana (125 megabases) and Brassica oleracea ( approximately 600 megabases), a species with many crop plants. Comparison of the whole genome sequence of A. thaliana with a partial draft of B. oleracea has permitted an estimation of the patterns of TE amplification, diversification, and loss that has occurred in related species since their divergence from a common ancestor. Although we find that nearly all TE lineages are shared, the number of elements in each lineage is almost always greater in B. oleracea. Class 1 (retro) elements are the most abundant TE class in both species with LTR and non-LTR elements comprising the largest fraction of each genome. However, several families of class 2 (DNA) elements have amplified to very high copy number in B. oleracea where they have contributed significantly to genome expansion. Taken together, the results of this analysis indicate that amplification of both class 1 and class 2 TEs is responsible, in part, for B. oleracea genome expansion since divergence from a common ancestor with A. thaliana. In addition, the observation that B. oleracea and A. thaliana share virtually all TE lineages makes it unlikely that wholesale removal of TEs is responsible for the compact genome of A. thaliana.

Arabidopsis↗

PubMed1998

Ribosomal RNA gene copy number and nucleolar-size polymorphisms within and among chicken lines selected for enhanced growth.

Ribosomal (r) DNA genotypes (rRNA gene copy number) and nucleolar phenotypes (nucleoli number and size) were studied in dam and sire commercial broiler pure lines from three primary breeder sources. Thirteen lines were studied to determine whether directionally selected broiler pure lines contain higher numbers of rRNA genes than a control line unselected for performance traits. Eight of the 13 lines exhibited rRNA gene copy averages between 261 and 331 copies, three lines had averages between 365 and 380, and two lines had average copy numbers equal to or greater than 450 rRNA genes. The overall source copy number average from one breeder company exhibited a value (402 rRNA genes) significantly different from the control value (300 rRNA genes). Nucleoli number and relative-size were examined in 9 of the 13 lines to establish ploidy and determine the population incidence of nucleolar size polymorphisms. All of the individuals examined for nucleolar phenotype expressed two nucleoli, indicating that gene copy number variation in those lines was generally unrelated to haploidy, aneuploidy, or polyploidy. A high frequency of individuals exhibited nucleolar size polymorphisms (line values of 57 to 87%). The results suggest that multiple nucleolus organizer region (NOR) types are segregating within and among broiler pure lines and that these NOR types contain variable numbers of rRNA genes that differ in nucleogenesis capacity.

Animals↗

PubMed1999

Potential phylogenetic utility of the low-copy nuclear gene pistillata in dicotyledonous plants: comparison to nrDNA ITS and trnL intron in Sphaerocardamum and other Brassicaceae.

We report the potential phylogenetic utility of DNA sequence data from the last 700 bp of a ca. 1-kb intron of the MADS-box gene pistillata from a sampling of Sphaerocardamum species and other Brassicaceae. These results are compared with nrDNA ITS and the chloroplast trnL intron for the same taxa to demonstrate the potential phylogenetic utility of this pistillata intron and to identify potential historically independent sequences for an ongoing study of relationships within Sphaerocardamum. Analyses of the DNA sequence data for Brassicaceae indicated that pairwise divergences and potentially informative characters were higher in the pistillata intron (0.6-30.8%, 284 characters) and ITS (0-24%, 94 characters) than in the chloroplast trnL intron (0-4.2%, 17 characters). A comparison of Sphaerocardamum sequences identified low divergences and numbers of informative characters for trnL intron (0-2.4%, 1 character) and nrDNA ITS (0-2.5%, 2 characters) and substantially more variation among the pistillata sequences (0.15-3.7%, 19 characters). Phylogenetic analyses of these pistillata sequences fully resolve ingroup relationships without character conflict. Results of pistillata PCR amplifications from a broader dicot sample showed that some primers may be useful in amplifying orthologous pistillata sequences. Ultimately this pistillata intron may be a valuable source of phylogenetic characters at lower taxonomic levels.

Arabidopsis Proteins↗