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PubMed2001

The evolutionary analysis of "orphans" from the Drosophila genome identifies rapidly diverging and incorrectly annotated genes.

In genome projects of eukaryotic model organisms, a large number of novel genes of unknown function and evolutionary history ("orphans") are being identified. Since many orphans have no known homologs in distant species, it is unclear whether they are restricted to certain taxa or evolve rapidly, either because of a lack of constraints or positive Darwinian selection. Here we use three criteria for the selection of putatively rapidly evolving genes from a single sequence of Drosophila melanogaster. Thirteen candidate genes were chosen from the Adh region on the second chromosome and 1 from the tip of the X chromosome. We succeeded in obtaining sequence from 6 of these in the closely related species D. simulans and D. yakuba. Only 1 of the 6 genes showed a large number of amino acid replacements and in-frame insertions/deletions. A population survey of this gene suggests that its rapid evolution is due to the fixation of many neutral or nearly neutral mutations. Two other genes showed "normal" levels of divergence between species. Four genes had insertions/deletions that destroy the putative reading frame within exons, suggesting that these exons have been incorrectly annotated. The evolutionary analysis of orphan genes in closely related species is useful for the identification of both rapidly evolving and incorrectly annotated genes.

Animals↗
PubMed2026

Virus-induced gene editing of stomatal regulators in Nicotiana benthamiana enables rapid functional genomics.

Virus-induced gene editing (VIGE) holds promise as a rapid and scalable approach for functional genomics in plants. Here, we apply a tobacco rattle virus (TRV)-based single-guide RNA (sgRNA) delivery system to target key regulators of stomatal development in Nicotiana benthamiana using transgenic Cas9-expressing lines. sgRNAs fused to a mobile RNA element and co-delivered with TRV enabled both somatic and heritable genome editing across orthologs of STOMAGEN, EPF2, YODA, and SPEECHLESS. Somatic editing frequencies reached up to 95%, and heritable tetra-allelic mutations were recovered in multiple target genes. Mutants exhibited significant, gene-specific changes in stomatal density, with corresponding effects on leaf temperature indicative of altered evaporative cooling. Additionally, sgRNAs fused to an AmCyan reporter enabled visualization of virus-infected tissues, allowing stomatal phenotyping in edited M0 sectors. This TRV-based platform facilitates functional assessment of genes influencing stomatal patterning and offers a powerful tool for dissecting gene function in a developmentally and physiologically relevant context.

Nicotiana↗
PubMed1990

Isolation of rapidly evolving genomic sequences: construction of a differential library and identification of a human DNA fragment that does not hybridize to chimpanzee DNA.

A differential library enriched in rapidly evolving human genomic sequences was obtained by phenol-enhanced hybridization of human genomic DNA with an excess of chimpanzee DNA. A DNA fragment 110 bp in length that did not hybridize to either chimpanzee or other primate DNA was identified in this library. It was shown to be a substantially diverged member of the human beta satellite family of tandem repeats. The genomic sequences homologous to the fragment were located on the short arms of human acrocentric chromosomes by in situ hybridization. The human-specific fragment failed to hybridize with RNA from different human tissues. The human-specific fragment exhibits a remarkable level of DNA polymorphism in humans and may be used in the identification of human tissue samples, in the selection of human/rodent somatic cell hybrids containing human acrocentric chromosomes, and in the mapping of these chromosomes.

Animals↗
PubMed2004

Evidence that annexin II is not a putative membrane receptor for 1alpha,25(OH)2-vitamin D3.

The seco-steroid hormone 1alpha,25(OH)(2)-vitamin D(3) (1,25-D(3)) is known to generate biological responses via both genomic and non-genomic rapid signal transduction pathways. The calcium regulated annexin II/p11 heterotetramer (AII(2)/p11(2)] was proposed by Baran and co-authors to be the membrane receptor responsible for mediating non-genomic, rapid actions of 1,25-D(3), based on ligand affinity labeling, competition, and saturation analysis experiments. Given the cytosolic presence of both the monomeric and heterotetrameric form of AII and their functional regulation by intracellular calcium concentrations, which are known to be affected by 1,25-D(3) rapid, non-genomic activities, we investigated in vitro the affinity of [(3)H]1,25-D(3) for the AII monomer and AII(2)/p11(2) in the absence and presence of calcium using saturation analysis and gel-filtration chromatography. Using two different techniques for separating bound from free ligand (perchlorate and hydroxylapatite (HAP)) over a series of 30 experiments, no evidence for specific binding of [(3)H]1,25-D(3) was obtained with or without the presence of 700 nM exogenous calcium, using either the AII monomer or AII(2)/p11(2). However saturable binding of [(3)H]1,25-D(3) to the lipid raft/caveolae enriched rat intestinal fraction was consistently observed (K(d) = 3.0 nM; B(max) = 45 fmols/mg total protein). AII was detected in lipid raft/caveolae enriched fractions from rat and mouse intestine and ROS 17/2.8 and NB4 cells by Western blot, but incubation in the presence of exogenous calcium did not ablate 1,25-D(3) binding as reported by Baran et al. Our results suggest that AII does not bind 1,25-D(3) in a physiologically relevant manner; however, recent studies linking AII(2)/p11(2) phosphorylation to vesicle fusion and its calcium regulated localization may make AII a possible down-stream substrate for 1,25-D(3) induced rapid cellular effects.

Animals↗
PubMed2003

Tn7-mediated introduction of DNA sequences into bacmid-cloned cytomegalovirus genomes for rapid recombinant virus construction.

Our basic understanding of how viruses infect, replicate, and cause disease has been largely derived from genetic approaches in which viral sequences have been mutated and the consequences of those mutations determined. In the herpesvirus field, deletions or insertions that preclude expression of specific viral proteins or remove critical cis elements have been invaluable in identifying their overall functions. We are now ready to move to a new level of detail-mapping functional domains within viral proteins or defining cis elements to the nucleotide level. This level of detail will require mutagenesis on a new scale, with recombinant viruses containing mutations within a given locus perhaps numbering in the hundreds. Mutagenesis on this scale would be greatly facilitated by more rapid methods of recombinant virus construction. In this report, we adapted a technology employing Tn7-mediated site-specific transposition [J. Virol. 67 (1993) 4566] as a rapid and highly reliable method to introduce novel sequences into bacmid-cloned herpesvirus genomes. We show that recombinant viruses can be rapidly created and that a deletion of the human cytomegalovirus (HCMV) essential gene ie2 can be complemented in cis by reintroduction, via transposition, of an ie2 cDNA; detailed mutagenesis of the complementing ie2 gene can now follow. Tn7-mediated transposition should accelerate greatly the pace at which recombinant herpesviruses can be constructed and, thus, facilitate the use of recombinant viruses for detailed mutagenic studies of both cis- and trans-acting genetic elements.

Cells, Cultured↗
PubMed2004

Whole-genome fractionation rapidly purifies DNA from centromeric regions.

The condensed centromeric regions of higher eukaryotic chromosomes contain satellite sequences, transposons and retroelements, as well as transcribed genes that perform a variety of functions. These chromosomal domains nucleate kinetochores, mediate sister chromatid cohesion and inhibit recombination, yet their characterization has often lagged behind that of chromosome arms. Here, we describe a whole-genome fractionation technique that rapidly identifies bacterial artificial chromosome (BAC) clones derived from plant centromeric regions. This approach, which relies on hybridization of methylated genomic DNA, revealed BACs that correspond to the genetically mapped and sequenced Arabidopsis thaliana centromeric regions. Extending this method to other species in the Brassicaceae family identified centromere-linked clones and provided genome-wide estimates of methylated DNA abundance. Sequencing these clones will elucidate the changes that occur during plant centromere evolution. This genomic fractionation technique could identify centromeric DNA in genomes with similar methylation and repetitive DNA content, including those from crops and mammals.

Arabidopsis↗
PubMed1999

How valuable are model organisms for transposable element studies?

Model organisms have proved to be highly informative for many types of genetic studies involving 'conventional' genes. The results have often been successfully generalized to other closely related organisms and also, perhaps surprisingly frequently, to more distantly related organisms. Because of the wealth of previous knowledge and their availability and convenience, model organisms were often the species of choice for many of the earlier studies of transposable elements. The question arises whether the results of genetic studies of transposable elements in model organisms can be extrapolated in the same ways as those of conventional genes? A number of observations suggest that special care needs to be taken in generalizing the results from model organisms to other species. A hallmark of many transposable elements is their ability to amplify rapidly in species genomes. Rapid spread of a newly invaded element throughout a species range has also been demonstrated. The types and genomic copy numbers of transposable elements have been shown to differ greatly between some closely related species. Horizontal transfer of transposable elements appears to be more frequent than for nonmobile genes. Furthermore, the population structure of some model organisms has been subject to drastic recent changes that may have some bearing on their transposable element genomic complements. In order to initiate discussion of this question, several case studies of transposable elements in well-studied Drosophila species are presented.

Biological Evolution↗
PubMed2025

Rapid genome-wide introgression reveals fitness advantage of immigrant genotypes.

Evolutionary biology has long recognized the tendency for populations to be locally adapted to their ancestral habitat, resulting in higher resident fitness. However, immigrants can also introduce beneficial alleles. The resulting adaptive introgression is usually inferred retrospectively, rather than as a contemporary process. Here, we document exceptionally rapid ongoing adaptive introgression in a lake population of threespine stickleback (Gasterosteus aculeatus). In the first generations after a discrete immigration event, all chromosomes exhibited large increases in immigrant ancestry due to linkage disequilibrium. After a decade, the extent of introgression varied across the genome. The fastest-evolving genes included Spi1b, which enables an increased fibrosis defense against a previously common tapeworm, whose prevalence then declined dramatically. This case study highlights the capacity for immigration to supply beneficial alleles that drive rapid genome-wide evolution.

Journal Article↗
PubMed2006

Mapping tumor-suppressor genes with multipoint statistics from copy-number-variation data.

Array-based comparative genomic hybridization (arrayCGH) is a microarray-based comparative genomic hybridization technique that has been used to compare tumor genomes with normal genomes, thus providing rapid genomic assays of tumor genomes in terms of copy-number variations of those chromosomal segments that have been gained or lost. When properly interpreted, these assays are likely to shed important light on genes and mechanisms involved in the initiation and progression of cancer. Specifically, chromosomal segments, deleted in one or both copies of the diploid genomes of a group of patients with cancer, point to locations of tumor-suppressor genes (TSGs) implicated in the cancer. In this study, we focused on automatic methods for reliable detection of such genes and their locations, and we devised an efficient statistical algorithm to map TSGs, using a novel multipoint statistical score function. The proposed algorithm estimates the location of TSGs by analyzing segmental deletions (hemi- or homozygous) in the genomes of patients with cancer and the spatial relation of the deleted segments to any specific genomic interval. The algorithm assigns, to an interval of consecutive probes, a multipoint score that parsimoniously captures the underlying biology. It also computes a P value for every putative TSG by using concepts from the theory of scan statistics. Furthermore, it can identify smaller sets of predictive probes that can be used as biomarkers for diagnosis and therapeutics. We validated our method using different simulated artificial data sets and one real data set, and we report encouraging results. We discuss how, with suitable modifications to the underlying statistical model, this algorithm can be applied generally to a wider class of problems (e.g., detection of oncogenes).

Gene Deletion↗
PubMed1996

The alpha-thalassemia/mental retardation syndromes.

The chromosome-16 and the X-chromosome forms of alpha-thalassemia--ATR-16 and ATR-X--exemplify 2 important causes of syndromal mental retardation. ATR-16 is a contiguous gene syndrome which arises from loss of DNA from the tip of chromosome 16p13.3 by truncation, interstitial deletion, or unbalanced translocation. It provided the first example of a chromosome translocation that could be detected by molecular analysis but not conventional cytogenetics. It also provided the first example of a telomeric truncation giving rise to a complex genetic syndrome. In contrast ATR-X appears to be due to mutations in a trans-acting factor that regulates gene expression. Mutations in transcription factors have recently been identified in a number of genetic diseases (for example, Denys-Drash syndrome, WT1 [19]; pituitary dwarfism, PIT1 [16]; Rubinstein-Taybi syndrome, CBP [20]. Not only is this mechanism proving to be an important cause of complex syndromes but it is providing new perspectives on certain developmental pathways. XH2 may not be a classical transcription factor but it is certainly involved in the regulation of gene expression, exerting its effects on several different genes. It seems likely that other mutations in this class of regulatory proteins will be found in patients with complex disorders including mental retardation. In broader terms the 2 mechanisms described here may prove to be responsible for a significant proportion of mental retardation. However, without a feature such as alpha-thalassemia to pinpoint the area of genome or pathways involved it may prove difficult to identify other, similarly affected genes underlying other forms of mental retardation. As the human genome project and rapid genome analysis evolve this problem should become less of an obstacle. In the meantime, it is very worthwhile to continue looking for unusual clinical associations that may point to critical genes underlying human genetic disorders.

Chromosome Aberrations↗
PubMed2001

Molecular evolution of receptor-like kinase genes in hexaploid wheat. Independent evolution of orthologs after polyploidization and mechanisms of local rearrangements at paralogous loci.

Hexaploid wheat is a young polyploid species and represents a good model to study mechanisms of gene evolution after polyploidization. Recent studies at the scale of the whole genome have suggested rapid genomic changes after polyploidization but so far the rearrangements that have occurred in terms of gene content and organization have not been analyzed at the microlevel in wheat. Here, we have isolated members of a receptor kinase (Lrk) gene family in hexaploid and diploid wheat, Aegilops tauschii, and barley (Hordeum vulgare). Phylogenetic analysis has allowed us to establish evolutionary relationships (orthology versus paralogy) between the different members of this gene family in wheat as well as with Lrk genes from barley. It also demonstrated that the sequences of the homoeologous Lrk genes evolved independently after polyploidization. In addition, we found evidence for gene loss during the evolution of wheat and barley. Analysis of large genomic fragments isolated from nonorthologous Lrk loci showed a high conservation of the gene content and gene organization at these loci on the homoeologous group 1 chromosomes of wheat and barley. Finally, sequence comparison of two paralogous fragments of chromosome 1B showed a large number of local events (sequence duplications, deletions, and insertions), which reveal rearrangements and mechanisms for genome enlargement at the microlevel.

Base Sequence↗
PubMed2006

A rapid genome-scale response of the transcriptional oscillator to perturbation reveals a period-doubling path to phenotypic change.

Perturbation of the gated-synchrony system in yeast with phenelzine, an antidepressant drug used in the treatment of affective disorders in humans, leads to a rapid lengthening in the period of the genome-wide transcriptional oscillation. The effect is a concerted, genome-scale change in expression that is first seen in genes maximally expressed in the late-reductive phase of the cycle, doubling the length of the reductive phase within two cycles after treatment. Clustering of genes based on their temporal patterns of expression yielded just three super clusters whose trajectories through time could then be mapped into a simple 3D figure. In contrast to transcripts in the late-reductive phase, most transcripts do not show transients in expression relative to others in their temporal cluster but change their period in a concerted fashion. Mapping the trajectories of the transcripts into low-dimensional surfaces that can be represented by simple systems of differential equations provides a readily testable model of the dynamic architecture of phenotype. In this system, period doubling may be a preferred pathway for phenotypic change. As a practical matter, low-amplitude, genome-wide oscillations, a ubiquitous but often unrecognized attribute of phenotype, could be a source of seemingly intractable biological noise in microarray studies.

Cell Division↗
PubMed1999

Duplicated genes evolve independently after polyploid formation in cotton.

Of the many processes that generate gene duplications, polyploidy is unique in that entire genomes are duplicated. This process has been important in the evolution of many eukaryotic groups, and it occurs with high frequency in plants. Recent evidence suggests that polyploidization may be accompanied by rapid genomic changes, but the evolutionary fate of discrete loci recently doubled by polyploidy (homoeologues) has not been studied. Here we use locus-specific isolation techniques with comparative mapping to characterize the evolution of homoeologous loci in allopolyploid cotton (Gossypium hirsutum) and in species representing its diploid progenitors. We isolated and sequenced 16 loci from both genomes of the allopolyploid, from both progenitor diploid genomes and appropriate outgroups. Phylogenetic analysis of the resulting 73.5 kb of sequence data demonstrated that for all 16 loci (14.7 kb/genome), the topology expected from organismal history was recovered. In contrast to observations involving repetitive DNAs in cotton, there was no evidence of interaction among duplicated genes in the allopolyploid. Polyploidy was not accompanied by an obvious increase in mutations indicative of pseudogene formation. Additionally, differences in rates of divergence among homoeologues in polyploids and orthologues in diploids were indistinguishable across loci, with significant rate deviation restricted to two putative pseudogenes. Our results indicate that most duplicated genes in allopolyploid cotton evolve independently of each other and at the same rate as those of their diploid progenitors. These indications of genic stasis accompanying polyploidization provide a sharp contrast to recent examples of rapid genomic evolution in allopolyploids.

Biological Evolution↗
PubMed1990

Identification of dengue sequences by genomic amplification: rapid diagnosis of dengue virus serotypes in peripheral blood.

Polymerase chain reaction (PCR) was developed for the in vitro amplification of dengue virus RNA via cDNA. A fraction of the N-terminus gene of the envelope protein in the four dengue serotypes was amplified using synthetic oligonucleotide primer pairs. Amplified products were cloned and used as dengue type-specific probes in gel electrophoresis and dot-blot hybridization. We detected and characterized dengue virus serotypes in blood samples by the three-step procedure DNA-PAH consisting in cDNA priming (P), DNA amplification (A) and hybridization (H) using specific non-radiolabelled probes. Our findings showed that DNA-PAH was more rapid and sensitive in the identification of the infecting serotype than the mosquito cell cultures. Moreover, the failure of cultures to detect virus particles in sera containing few copies of viral genome or anti-dengue antibodies justified the approach of DNA-PAH to the dengue identification in clinical specimens.

Base Sequence↗
PubMed1995

A novel method for efficient amplification of whole hepatitis B virus genomes permits rapid functional analysis and reveals deletion mutants in immunosuppressed patients.

Current knowledge of hepatitis B virus (HBV) sequence heterogeneity is based mainly on sequencing of amplified subgenomic HBV fragments. Here, we describe a method which allows sensitive amplification and simplified functional analysis of full-length HBV genomes with or without prior cloning. By this method, a large number of HBV genomes were cloned from sera of six immunosuppressed kidney transplant patients. Two size classes of HBV genomes, one 3.2 kb and another about 2.0 kb in size, were found in all patients. The genome population from one serum sample was studied in detail by size analysis of subgenomic PCR fragments and sequencing. Regions with deletions and insertions were mapped in the C gene and pre-S region. Up to 100% of HBV genomes in all other immunosuppressed patients also had deletions in the C gene. Our results demonstrate the potential of the established method for the structural and functional characterization of heterogeneous populations of complete virion-encapsidated HBV DNAs and suggest that HBV genomes with C gene deletions can have a selective advantage in immunosuppressed patients.

Base Sequence↗
PubMed1994

Microsatellite genome screening: rapid non-denaturing, non-isotopic dinucleotide repeat analysis.

The study of reverse genetics has made it possible for scientists to isolate and identify the genes responsible for such human diseases as cystic fibrosis and Duchenne- and Becker-type muscular dystrophy. The expensive and time-consuming process of detecting restriction fragment length polymorphisms by Southern blotting was the only method available to localize these genes. With the discovery of polymorphic microsatellite sequences in the human genome, the speed and ease of which a genome screening can be performed has increased dramatically. This paper reports on advanced methods for detection of microsatellite-repeated sequences making the task of mapping human genes simpler, safer and more economical.

DNA, Satellite↗
PubMed2002

Efficient rescue of gutted adenovirus genomes allows rapid production of concentrated stocks without negative selection.

Gutted adenoviral (Ad) vectors have a greater cloning capacity and elicit less immune response than conventional Ad vectors. Unfortunately, clinical use of gutted vectors has been slowed by production difficulties, including low yield and a tendency for recombinant virus to emerge. These two problems are related, because expansion of dilute vector stocks requires selective pressure against helper virus. The ability to rescue gutted virus at high titer would lessen the requirement for selective pressure, thereby limiting the advantage afforded to undesirable recombinants. We tested gutted virus rescue from plasmids and from synthetic terminal protein (TP)-DNA complexes by transfection/infection or cotransfection with various forms of helper viral DNA. Optimal rescue required cotransfection of gutted and helper genomes with identical origins of replication. Transfection/infection, which introduces unequal origins, was 30 times less efficient than cotransfection of genomes that had been released from plasmid DNA and bore identical origins. Cotransfection of TP-linked genomes was several times more efficient than that of unlinked genomes, yielding average gutted viral titers above 10(7) transducing units (TU)/ml. In addition, we found that limited expression of Cre recombinase doubled the yield of gutted virus. Using these techniques, gutted viruses can be rescued at titers greater than 3 x 10(7) TU/ml, about 100 times higher than is usually achieved. Finally, we found that high-titer lysates could be serially passaged on Cre-negative cells without loss of titer, further reducing selective pressure. These methods allow large-scale production of gutted virus in three or four serial passages, while minimizing exposure to Cre recombinase.

Adenoviridae↗
PubMed1969

Evidence for multiple growing points on the genome of rapidly growing E. coli B-r.

Synchronized cultures of rapidly growing E. coli B/r cells were starved for a required amino acid at various cell ages to allow ongoing chromosome replication to be completed without initiation of new replication cycles. It has been found that when such synchronized cultures are exposed to the mutagen nitrosoguanidine, genetic markers just in the process of replication show a markedly higher mutation rate than markers elsewhere on the chromosome. The number of growing points on the chromosome at each cell age can then be determined by observing the nitrosoguanidine-induced mutation rates for specific genetic markers on the genome. These experiments indicate that there exist multiple growing points on the genome for about ten minutes during the life cycle when the cells are growing with a doubling time of 28 minutes.

Aging↗