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PubMed2000

Variation and phylogenetic utility of the Arabidopsis thaliana Rps2 homolog in various species of the tribe Brassiceae.

Our objective was to analyze the evolutionary paths of cultivated diploid Brassica species and a few related wild species (tribe Brassiceae) in relation to Arabidopsis thaliana (tribe Arabidae), using the Rps2 sequence. Rps2 confers resistance to Pseudomonas syringae in A. thaliana. We found that similar to Arabidopsis, the Rps2 homolog in Brassica species is present in a single copy. Primers based in the Rps2 sequence amplified Rps2 homologs from the other species. Maximum-parsimony analysis based on number of nucleotide substitutions yielded a single tree, grouping the species as expected from other evolutionary inferences. Age of divergence between the two tribes was within the range of previous estimates. Indels in the different sequences were also useful for distinguishing some of the species. The Rps2 gene is a useful phylogenetic tool for more comprehensive studies of the species of Brasicaceae.

Amino Acid Sequence↗

PubMed1996

Telomere exchange can be an important mechanism of variant surface glycoprotein gene switching in Trypanosoma brucei.

Trypanosoma brucei undergoes antigenic variation by changing its Variant Surface Glycoprotein (VSG) coat. Although there are up to a thousand VSG genes, only one is transcribed at a time from a telomeric VSG expression site. Switching can involve DNA rearrangements exchanging the active VSG gene, or transcriptional activation of a new expression site and transcriptional silencing of the old one. Determining the mechanism mediating a switch has not always been easy, as the many virtually identical copies of VSG gene expression sites complicate transcriptional analysis. To overcome this problem, we have used bloodstream form T. brucei with a single copy VSG gene in an active expression site marked with a hygromycin resistance gene. We allowed these transformants to undergo switching of the active VSG gene, via three different experimental methods. We were able to select large numbers of switched trypanosomes from a single infected mouse using a new microtitre-dish based procedure developed for this purpose. The drug sensitivity of the switched trypanosomes allowed us to determine the transcriptional state of the marked expression site, and polymerase chain reaction (PCR) amplification was used to determine whether the single copy drug resistance gene and VSG gene present in the marked expression site had been retained. These studies showed that telomere exchange, which has been considered rare, can in some cases be an important mechanism of VSG gene switching. We describe 4 telomere exchange events between the active VSG 221 expression site and 4 different chromosomes.

Animals↗

PubMed2005

Genotype-phenotype correlation of SMN locus genes in spinal muscular atrophy patients from India.

Spinal muscular atrophy has been classified into four groups based on the age of onset and clinical severity of the disease. Homozygous deletion in SMN1 gene causes the disease but the clinical severity may be modified by copy number of homologous gene SMN2 as well as the extent of deletion at SMN locus. In the view of scarcity of genotype and phenotype correlation data from India, this study has been undertaken to determine that correlation in SMA patients by using the SMN and NAIP genes and two polymorphic markers C212 and C272 located in this region. Two to four alleles of the markers C212 and C272 were observed in normal individuals. However, majority of Type I patients showed only one allele from both markers whereas in Type II and III patients, 2-3 alleles were observed. The SMN2 copy number in our type III patients showed that patients carry 3-5 copies of SMN2 gene. Our results suggest that extent of deletions encompassing H4F5, SMN1, NAIP and copy number of SMN2 gene can modify the SMA phenotype, thus accounting for the different clinical subtypes of the disease.

Adolescent↗

PubMed2006

Structural variation in the human genome.

The first wave of information from the analysis of the human genome revealed SNPs to be the main source of genetic and phenotypic human variation. However, the advent of genome-scanning technologies has now uncovered an unexpectedly large extent of what we term 'structural variation' in the human genome. This comprises microscopic and, more commonly, submicroscopic variants, which include deletions, duplications and large-scale copy-number variants - collectively termed copy-number variants or copy-number polymorphisms - as well as insertions, inversions and translocations. Rapidly accumulating evidence indicates that structural variants can comprise millions of nucleotides of heterogeneity within every genome, and are likely to make an important contribution to human diversity and disease susceptibility.

Genetic Variation↗

PubMed1997

Repetitive sequence families in Alces alces americana.

High-resolution derivative melting was used to obtain detailed distributions of local (G + C) contents in a number of ruminant DNAs. Profiles over low (G + C) regions [20-36% (G + C)] are congruent for all ruminants. This region represents 45-50% of the nuclear DNA content and primarily contains intergenic and intron sequences. The high (G + C) region, where most coding sequences are found [38-68% (G + C)], is marked by satellite bands denoting the presence of transcriptionally inert, tandemly repetitive sequence families. These bands can be analyzed for the abundance, base composition, and sequence divergence of satellite families with relatively high precision. Band patterns are unique to each species; even closely related species can be readily distinguished by their base distribution profiles. Variations in nuclear DNA contents in ruminants, determined by flow cytometry, are primarily due to variations in abundances of these repetitive sequence families. Thus, A. alces (moose) is found to have 8.85 +/- 0.2 pg DNA/cell, 25% more than the average in ruminants, while the base distribution curve indicates the presence of an unusually abundant satellite of 52.6% (G + C). The size (1 kb) and sequence of this satellite corresponds to satellite-I of other cervids, and in consequence it is designated Alces-I. The sequence of a cloned repeat of Alces-I has a length of 968 bp, a (G + C) content of 52.6%, and contributes 35%, or almost 3 million copies to the nuclear DNA, exceeding by approximately 300% the average array size of this repeat family in related cervids. In situ hybridization indicates the repeat is distributed throughout centromeric regions of all 62 acrocentric autosomes. Alces-I has much greater-than-expected numbers of GG, GA, and AG and far fewer numbers of TA and CG duplets, characteristics of all tandem repeats. The sequence is judged to be orthologous with satellite-I sequences from Rangifer tarandus (caribou), Capreolus capreolus (roe deer), Muntiacus muntjac (Chinese muntjac) and Muntiacus reevesi (Indian muntjac), as well as Antilocapra americana (pronghorn), and the bovids Bos taurus and Ovis aries. A tentative tree for the five cervids is in excellent agreement with one proposed on the basis of morphological characteristics. Differences from a consensus sequence indicate transversions exceed transitions by almost twofold, suggesting that substitutions occur randomly, or nearly so.

Animals↗

PubMed1981

The role of chromosome 15 in murine leukemogenesis. I. Contrasting behavior of the tumor vs. normal parent-derived chromosomes No. 15 in somatic hybrids of varying tumorigenicity.

G-banding analysis was carried out on a series of hybrids derived from the fusion of a chromosome 15-trisomic murine T-cell leukemia of AKR origin and normal diploid fibroblasts or lymphocytes of the CBT6T6 strain. Due to the 14;15 translocation involved in the generation of the T6 marker, the chromosomes No. 15 and 14 derived from the normal and the tumor parent can be distinguished cytogenetically. Highly tumorigenic, in vitro maintained hybrids, and high-tumorigenic segregants of originally low-tumorigenic in vitro hybrids, selected by in vivo passage, showed a similar cytogenetic pattern. It was characterized by the amplification of the tumor-derived chromosomes No. 15 from the expected 3 to 5.5 +/- 0.2 copies and a concomitant decrease of the normal derived T(14;15)6 from 2 copies to 0.9 +/- 0.2. All other autosomes except No. 14 showed only minor random variations, around the expected number of 4 copies. The tumor-derived chromosome 14 was amplified from the expected 2 to 3 copies. The low-tumorigenic hybrids showed the opposite pattern with a decrease in the number of the tumor-derived 15 chromosome from 3 to 2.6 +/- 0.1 and the maintenance of the two normal parent derived T(14;15)6 chromosomes. These findings suggest the existence of a qualitative difference between the 15 chromosomes derived from the tumor vs. the normal parent, due to mutation or proviral DNA insertion in the tumor-derived homologue. Amplification of the change locus and a decrease in the dosage of its normal counterpart appear to favor tumorigenicity.

Animals↗

PubMed1985

An evolutionary comparison of homologous mitochondrial plasmid DNAs from three Neurospora species.

We have discovered a mitochondrial DNA plasmid in N. crassa 516 (Roanoke, LA) which is homologous to those previously described from N. intermedia 435 (Fiji) and N. tetrasperma 2510 (Hanalei, HA). Subsequent analysis by DNA-DNA hybridization showed that 6 of 14 other Louisiana N. crassa isolates possessed plasmids homologous to these three plasmids, but at lower copy number. Plasmids from the three named strains were studied to examine possible plasmid diversity within each isolate, the extent of the homology between the plasmids, and the possibility that these plasmids could be inherited separately from their host mitochondria. Comparison of cloned plasmids and covalently closed circular mitochondrial DNA showed that only one plasmid line was present in each of the three intensively studied isolates. DNA-DNA hybridization and restriction endonuclease site mapping showed that the mitochondrial plasmids from the three species were very similar; most of the variation was due to presumed nucleotide substitutions. Plasmids judged identical by our analysis were found in different species. The distribution of the homologous plasmids in nature and the presence of these identical plasmids in different species, suggested that these plasmids could be transmitted between isolates independently of their host mitochondria.

Biological Evolution↗

PubMed1996

The clp (CS31A) operon is negatively controlled by Lrp, ClpB, and L-alanine at the transcriptional level.

Biosynthesis of the Escherichia coli CS31A surface antigen was subject to phase-variation control and repressed by L-alanine. Nucleotide sequence analysis of the clp operon, encoding the biosynthesis of CS31A, revealed the presence of a regulatory gene, clpB. The amino acid sequence of the regulatory protein ClpB showed similarity to the primary structure of PapB, FaeB and AfaA, involved in the regulation of expression of Pap, K88, and Afa-3 fimbriae, respectively. The clp regulatory region contained two deoxyadenosine methylase sites (GATC-I and GATC-II). The leucine-responsive regulatory protein (Lrp) was required for specific methylation inhibition of the GATC-II site. The activity of the clp promoter was monitored in a clp-lacZYA single-copy fusion. The cloned DNA used in this study did not contain a related papl gene. In these conditions, we showed, as expected, that phase variation did not occur. However, transcription of the clp operon was negatively controlled by ClpB and Lrp, and was maximal in the absence of Dam methylase. In the presence of AfaF, a Papl equivalent, the phase-variation control was restored. We concluded that two regulatory mechanisms were superimposed to control the clp expression. Phase variation, mediated by Lrp and a Papl equivalent, controlled the number of cells producing CS31A in a single colony. The second mechanism, described in this report, was mediated by ClpB and Lrp and controls the level of CS31A produced by a single cell. Furthermore, we showed that L-alanine reduced, by about twofold, the clp promoter activity independently of a Papl equivalent, ClpB, Lrp or Dam methylase. In addition, the presence of L-alanine prevented the phase-variation control mediated by AfaF.

Alanine↗

PubMed2003

Divergence of reiterated sequences in a series of genital isolates of herpes simplex virus type 1 from individual patients.

Both serotypes of herpes simplex virus (HSV), HSV-1 and HSV-2, are aetiological agents of genital herpes, although genital herpes caused by HSV-1 recurs less frequently. The HSV-1 genome contains a number of short, tandemly repeated sequences, and some reiterated sequences can serve as sensitive markers for the differentiation of HSV-1 strains. In the present study, variation in reiterations (assumed to be due to different copy numbers of tandemly repeated sequences) was examined in HSV-1 isolates from genital lesions from the same individual. Six sets (three primary-recurrence sets and three multiple-recurrence sets) of HSV-1 isolates were analysed: the primary-recurrence set consisted of two isolates (one isolated at a primary episode and the other at a recurrent episode) from the same individual; the multiple-recurrence set consisted of plural isolates from different episodes of recurrence in the same individual. Variations in length of the major DNA fragment, containing reiteration I (within the a sequence) and/or reiteration IV (within introns of genes US1 and US12), were detected between isolates of each multiple-recurrence set, but not of the primary-recurrence set. Thus, HSV-1 isolates of multiple-recurrence sets are assumed to have diverged more widely within each set than those of primary-recurrence sets, probably because of more rounds of virus DNA replication. This divergence of reiterations seems to indicate a forward step in the division of HSV-1 from a common ancestor into different lineages.

Cohort Studies↗

PubMed1998

Quantification of G protein mRNA using reverse transcription and competitive PCR with a colorimetric microplate assay.

This paper reports an assay for the quantification of levels of specific mRNA for the alpha subunits of the inhibitory G proteins G alpha i-1, G alpha i-2, and G alpha i-3. The assay employs reverse transcription and competitive polymerase chain reaction (PCR) coupled to enzyme-linked oligonucleotide sorbent assay for differential detection and quantification of PCR products. The assay was conducted with conventional thermal block PCR cyclers as well as rapid air microcapillary cyclers. The detection stage consists of three steps using synthetic oligonucleotides, commercially available reagents and a conventional 96-well plate absorbance reader at settings of 450 and 630 nm. The assay is: (1) rapid, requiring about 3 h for quantification of PCR products; (2) safe, being non-radiometric; (3) relatively simple; (4) highly sensitive, being capable of detecting less than 10 initial copies of target cDNA; (5) precise, resolving two-fold differences in initial copy numbers of specific sequences as low as 10(-20) mol; (6) linear over a 3 log range, with two-fold differences in the quantity of cDNA producing consistent reductions in quantity of specific cDNA detected; and (7) reproducible, intra-assay and inter-assay coefficients of variation being 11.9 and 14.7%, respectively.

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↗

PubMed2006

Use of 16S rRNA and rpoB genes as molecular markers for microbial ecology studies.

Several characteristics of the 16S rRNA gene, such as its essential function, ubiquity, and evolutionary properties, have allowed it to become the most commonly used molecular marker in microbial ecology. However, one fact that has been overlooked is that multiple copies of this gene are often present in a given bacterium. These intragenomic copies can differ in sequence, leading to identification of multiple ribotypes for a single organism. To evaluate the impact of such intragenomic heterogeneity on the performance of the 16S rRNA gene as a molecular marker, we compared its phylogenetic and evolutionary characteristics to those of the single-copy gene rpoB. Full-length gene sequences and gene fragments commonly used for denaturing gradient gel electrophoresis were compared at various taxonomic levels. Heterogeneity found between intragenomic 16S rRNA gene copies was concentrated in specific regions of rRNA secondary structure. Such "heterogeneity hot spots" occurred within all gene fragments commonly used in molecular microbial ecology. This intragenomic heterogeneity influenced 16S rRNA gene tree topology, phylogenetic resolution, and operational taxonomic unit estimates at the species level or below. rpoB provided comparable phylogenetic resolution to that of the 16S rRNA gene at all taxonomic levels, except between closely related organisms (species and subspecies levels), for which it provided better resolution. This is particularly relevant in the context of a growing number of studies focusing on subspecies diversity, in which single-copy protein-encoding genes such as rpoB could complement the information provided by the 16S rRNA gene.

Bacteria↗

PubMed1984

Copy number and the stability of 2-micron circle-based artificial plasmids of Saccharomyces cerevisiae.

The copy number and stability of artificial 2-micron circle-based plasmids have been accurately measured in [Cir+] and [Cir0] strains of Saccharomyces cerevisiae. We conclude that (i) instability and copy number vary greatly from plasmid to plasmid; (ii) instability and copy number are negatively correlated--that is, high copy number is associated with low instability; (iii) it is difficult to reconcile this variability with a strict and direct system of copy number control; (iv) instabilities are much higher than expected from random partition and the observed copy numbers: this may imply partition which is less efficient than random. Even so, (v) the partitioning of 2-micron circle-like plasmids is more efficient than that of ARS-based plasmids, which hints at the existence of a system for the (inefficient) distribution of 2-micron circles.

Chimera↗

PubMed2004

Classification and strength measurement of stationary-phase promoters by use of a newly developed promoter cloning vector.

When an Escherichia coli culture changes from exponential growth to the stationary phase, expression of growth-related genes levels off, while a number of stationary-phase-specific genes are turned on. To gain insight into the growth phase-dependent global regulation of genome transcription, we analyzed the strength and specificity of promoters associated with the stationary-phase genes. For the in vivo assay of promoter activity, 300- to 500-bp DNA fragments upstream from the translation initiation codon were isolated and inserted into a newly constructed doubly fluorescent protein (DFP) vector. The activity of test promoters was determined by measuring the green fluorescent protein (GFP). To avoid the possible influence of plasmid copy number, the level of transcription of reference promoter lacUV5 on the same plasmid was determined by measuring the red fluorescent protein (RFP). Thus, the activities of test promoters could be easily and accurately determined by determining the GFP/RFP ratio. Analysis of the culture time-dependent variation of 100 test promoters indicated that (i) a major group of the stationary-phase promoters are up-regulated only in the presence of RpoS sigma; (ii) the phase-coupled increase in the activity of some promoters takes place even in the absence of RpoS; and (iii) the activity of some promoters increases in the absence of RpoS. This classification was confirmed by testing in vitro transcription by using reconstituted RpoD and RpoS holoenzymes.

Bacterial Proteins↗

PubMed1998

Variation in genome organization of the plant pathogenic fungus Colletotrichum lindemuthianum.

The genome structure of Colletotrichum lindemuthianum in a set of diverse isolates was investigated using a combination of physical and molecular approaches. Flow cytometric measurement of genome size revealed significant variation between strains, with the smallest genome representing 59% of the largest. Southern-blot profiles of a cloned fungal telomere revealed a total chromosome number varying from 9 to 12. Chromosome separations using pulsed-field gel electrophoresis (PFGE) showed that these chromosomes belong to two distinct size classes: a variable number of small (< 2.5 Mb) polymorphic chromosomes and a set of unresolved chromosomes larger than 7 Mb. Two dispersed repeat elements were shown to cluster on distinct polymorphic minichromosomes. Single-copy flanking sequences from these repeat-containing clones specifically marked distinct small chromosomes. These markers were absent in some strains, indicating that part of the observed variability in genome organization may be explained by the presence or absence, in a given strain, of dispensable genomic regions and/or chromosomes.

Ascomycota↗

PubMed1991

Mapping of DNA instability at the fragile X to a trinucleotide repeat sequence p(CCG)n.

The sequence of a Pst I restriction fragment was determined that demonstrate instability in fragile X syndrome pedigrees. The region of instability was localized to a trinucleotide repeat p(CCG)n. The sequence flanking this repeat were identical in normal and affected individuals. The breakpoints in two somatic cell hybrids constructed to break at the fragile site also mapped to this repeat sequence. The repeat exhibits instability both when cloned in a nonhomologous host and after amplification by the polymerase chain reaction. These results suggest variation in the trinucleotide repeat copy number as the molecular basis for the instability and possibly the fragile site. This would account for the observed properties of this region in vivo and in vitro.

Base Sequence↗

PubMed1994

Molecular genetic analyses of the Tyrolean Ice Man.

An approximately 5000-year-old mummified human body was recently found in the Tyrolean Alps. The DNA from tissue samples of this Late Neolithic individual, the so-called "Ice Man," has been extracted and analyzed. The number of DNA molecules surviving in the tissue was on the order of 10 genome equivalents per gram of tissue, which meant the only multi-copy sequences could be analyzed. The degradation of the DNA made the enzymatic amplification of mitochondrial DNA fragments of more than 100 to 200 base pairs difficult. One DNA sequence of a hypervariable segment of the mitochondrial control region was determined independently in two different laboratories from internal samples of the body. This sequence showed that the mitochondrial type of the Ice Man fits into the genetic variation of contemporary Europeans and that it was most closely related to mitochondrial types determined from central and northern European populations.

Animals↗

PubMed2002

Phylogeny of Gaertnera Lam. (Rubiaceae) based on multiple DNA markers: evidence of a rapid radiation in a widespread, morphologically diverse genus.

Phylogenetic relationships among 28 of the 68 species of the paleotropical genus Gaertnera (Rubiaceae) and two related genera were inferred from nucleotide sequence variation in four nuclear DNA (nDNA) markers: the internal transcribed spacers of nuclear rDNA (ITS), the large and small copies of phosphoenolpyruvate carboxylase (PepC-large and PepC-small), and triose phosphate isomerase (Tpi). Phylogenetic analysis of the combined nDNA dataset suggested that Gaertnera is monophyletic, but genetic variation among species was insufficient to reconstruct well-supported relationships within the genus. This was counter to expectations based on the very distinct morphologies and widespread distribution of the genus (West Africa to Sulawesi). Molecular clock analyses suggested variable dates of origin for Gaertnera depending upon the calibration method used. The most plausible calibration implies that Gaertnera migrated to Africa during the early Tertiary, possibly via a boreotropical land bridge and suggests that Gaertnera started to radiate 5.21 +/- 0.14 million years ago. This implies that range expansion in the group has occurred via a number of long-distance dispersal events rather than vicariance. The molecular clock estimate in turn estimated an unusually rapid lineage diversification rate within the radiation of 0.717-0.832 species/million years, comparable to those estimated for radiations on oceanic islands. Although low interspecific competition levels may have contributed to the diversification of Gaertnera on Mauritius, the mechanisms driving the rapid radiation of the group in other parts of its range remain elusive.

Base Sequence↗