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PubMed1990

Similar myocardial effects of aging and hypertension.

Aging has sometimes been referred to as 'blunted hypertension', and hypertension as 'accelerated aging'. Systolic arterial pressure increases with aging; the increase encompasses both the clinically normal and hypertensive ranges of pressure and is due in part to arterial stiffening. Cardiac changes that occur with aging in otherwise healthy individuals can occur at a younger age in clinically hypertensive individuals and can be produced in young animals by experimental hypertension. Such cardiac adaptations in hypertension and aging include left ventricular hypertrophy and prolonged Ca2+ activation of contractile proteins, leading to a prolonged force-bearing capacity. This is mediated by a prolongation of the cytosolic Ca2+ transient, due to a reduced rate of Ca2+ sequestration by the sarcoplasmic reticulum. The action potential is also prolonged. The resultant delayed contractile relaxation, in part, leads to a slower velocity of early diastolic left ventricular filling; however, this is offset by enhanced atrial filling. Cardiac muscle of the aging and hypertensive heart are also less sensitive to beta-adrenergic stimulation. These myocardial adaptations with aging and hypertension, in part, are controlled from within the genome and permit a relatively normal heart volume and ejection fraction in the presence of chronically elevated afterload.

Action Potentials↗
PubMed1993

Functions for DNA methylation in vertebrates.

DNA methylation is ancestrally a mechanism for neutralizing potentially damaging DNA elements in the genome. The genomes of most multicellular organisms contain a small fraction of methylated DNA that contains the methylated elements, whereas the organism's own genes remain free of methylation. Vertebrates are exceptional among animals in that their genomes, including genes, are predominantly methylated. They retain the ability to inactivate viral DNA but have recruited the DNA methylation system for new functions. Widespread low-density methylation can contribute to lowering of the level of transcriptional "noise" from cryptic or inappropriate promoters. This may be the major advantage of DNA methylation in these organisms and may be sufficiently beneficial to offset the disadvantage of m5C mutability. The other novel feature of DNA methylation in vertebrates is the capacity to de novo methylate certain CpG islands, causing long-term strong repression. These evolutionary innovations may explain the high complexity of vertebrate organs and cell types.

5-Methylcytosine↗
PubMedDate not supplied

An embryonal carcinoma multiple phenotype locus maps to the proximal position of the mouse X chromosome.

A mutant embryonal carcinoma cell line, NR1-6, was established subsequent to retroviral insertion. The insertion was shown to be causative for a number of aberrant properties associated with the mutant cells. Analysis of >17 kb of the insertion site flanking region failed to reveal any homology between this locus and any reported sequence with the exception of one EST of unknown function and a few repetitive elements including B1 element and a CA dinucleotide repeat. CA repeats occur commonly in the mouse genome and usually show size variation. In this study, we mapped this multiphenotype locus using CA repeat polymorphism and Jackson Laboratory's interspecific backcross panels. The locus maps to the proximal end of the X chromosome between MGI offsets 1.5 and 4.5 and has been designated DXUalb1. There are several interesting candidate genes within this region. Analyses of their expression pattern may lead us to a better understanding of the molecular regulation of the variant mutant phenotypes.

Animals↗
PubMed2026

Cohesin cofactor dosage sets the rate of loop extrusion, rendering genome folding tunable yet vulnerable to genetic disruption.

Genome folding is not static but emerges from dynamic processes that control transcription, replication, recombination, and repair. DNA loop extrusion by cohesin is central to genome organization, yet it remains unclear how cells can tune extrusion kinetics to achieve precise and functional chromosome folding patterns. Here, we show that extrusion rate acts as a tunable biophysical parameter in cells, quantitatively dialed by the respective dosage of the cohesin cofactors NIPBL and PDS5. Modulation of extrusion rate can offset changes in cohesin lifetime to buffer steady-state chromosome structure and transcriptional states, even in the face of abnormal extrusion dynamics. These findings provide a long-sought mechanistic basis for the genetic interactions between cohesin cofactors and for the molecular origin of haploinsufficiency in cohesinopathies, such as Cornelia de Lange syndrome.

Cell Cycle Proteins↗
PubMed1999

DNA technology in the clinical laboratory.

OBJECTIVES: To review the advances in clinically useful molecular biological techniques and to identify their applications in clinical practice, as presented at the Eighth Annual William Beaumont Hospital Symposium. DATA SOURCES: The 10 manuscripts submitted were reviewed, and their major findings were compared with literature on the same topic. STUDY SELECTION: Two manuscripts addressed specimen (nucleic acid) stability, 2 described novel analytic approaches, 3 discussed detection of B- or T-cell clonality in lymphoproliferative disorders, and 3 reported the frequency of a variety of genetic polymorphisms found in cardiac disorders. DATA SYNTHESIS: DNA from dried blood spots is stable and may be purified rapidly for amplification and mutation analysis. RNA is much less stable, and a variety of methods may be used to reduce ribonuclease degradation of enteroviral RNA. False-negative reactions may be reduced by genomic amplification of ligated padlock probes by cascade rolling circle or polymerase chain reaction. A multiplex polymerase chain method using fluorescence-labeled products that separate both the wild-type and mutant hemochromatosis gene alleles by capillary gel electrophoresis represents another approach for detecting the 2 major missense mutations (C282Y and H63D) in hemochromatosis. Southern blotting and polymerase chain reaction have been used to detect B- and T-cell clonality in lymphoproliferative diseases, including mantle cell lymphoma and lymphoma of the breast. Genetic polymorphisms in a variety of coagulation factors and platelet glycoprotein IIIa are associated with ischemic heart disease. CONCLUSIONS: As the Human Genome Project continues to define disease-associated mutations, the number of clinically useful molecular pathologic techniques and assays will expand. Clinical outcome analysis is still required to document a decrease in the patient's length of stay to offset the cost of introducing molecular biological assays in the routine clinical pathology laboratory.

Clinical Laboratory Techniques↗
PubMed1999

Exploring the impact of extended phenotype in stratified samples.

We have performed initial nonparametric sib-pair genome scans in the early (N = 52) and late (N = 53) onset subgroups of the COGA pedigrees, stratified near the median value of pedigree mean age of onset for ALDX1 diagnosis of alcoholism. Because the early group contained a higher proportion of smokers, traits of alcoholism, smoking, and addiction (defined as either alcoholism or smoking) were examined. Subgroups and phenotypic definitions influenced initial linkage results, corrected for the number of analyzed traits. Evidence for linkage to the ALDX1 alcoholism phenotype at the ADH3 functional candidate gene was increased in the late onset subgroup (Bonferroni corrected significance level < 0.002), as compared with the unstratified sample that replicated COGA linkage obtained in the same analysis; there was no evidence for linkage at this locus in the early onset subgroup. The theoretical implication of this result is that the loss of power due to contracting sample size through stratification may in some cases be more than offset by extraction of a more homogeneous subgroup from the etiologically complex trait.

Adolescent↗
PubMed1992

Sequencing by hybridization: towards an automated sequencing of one million M13 clones arrayed on membranes.

An immediately applicable variant of the sequencing by hybridization (SBH) method is under development with the capacity to determine up to 100 million base pairs per year. The proposed method comprises six steps: (i) arraying genomic or cDNA M13 clones in 864-well plates (wells of 2 mm); (ii) preparation of DNA samples for spotting by growth of the M13 clones or by polymerase chain reaction (PCR) of the inserts using standard 96-well plates, or plates having as many as 864 correspondingly smaller wells; (iii) robotic spotting of 13,824 samples on an 8 x 12 cm nylon membrane, or correspondingly more, on up to 6 times larger filters, by offset printing with a 96 or 864 0.4 mm pin device; (iv) hybridization of dotted samples with 200-2000 32P-labeled probes comprising 16-256 10-mers having a common 8-mer, 7-mer, or 6-mer in the middle (20 probes per day, each hybridized with 250,000 dots); (v) scoring hybridization signals of 5 million sample-probe pairs per day using storage phosphor plates; and (vi) computing clone order and partial-to-complete DNA sequences using various heuristic algorithms. Genome sequencing based on a combination of this method and gel sequencing techniques may be significantly more economical than gel methods alone.

Bacteriophage M13↗
PubMed2004

The analysis of stress-induced duplex destabilization in long genomic DNA sequences.

We present a method for calculating predicted locations and extents of stress-induced DNA duplex destabilization (SIDD) as functions of base sequence and stress level in long DNA molecules. The base pair denaturation energies are assigned individually, so the influences of near neighbors, methylated bases, adducts, or lesions can be included. Sample calculations indicate that copolymeric energetics give results that are close to those derived when full near-neighbor energetics are used; small but potentially informative differences occur only in the calculated SIDD properties of moderately destabilized regions. The method presented here for analyzing long sequences calculates the destabilization properties within windows of fixed length N, with successive windows displaced by an offset distance d(o). The final values of the relevant destabilization parameters for each base pair are calculated as weighted averages of the values computed for each window in which that base pair appears. This approach implicitly assumes that the strength of the direct coupling between remote base pairs that is induced by the imposed stress attenuates with their separation distance. This strategy enables calculations of the destabilization properties of DNA sequences of any length, up to and including complete chromosomes. We illustrate its utility by calculating the destabilization properties of the entire E. coli genomic DNA sequence. A preliminary analysis of the results shows that promoters are associated with SIDD regions in a highly statistically significant manner, suggesting that SIDD attributes may prove useful in the computational prediction of promoter locations in prokaryotes.

Base Pairing↗
PubMed2001

Structure, chromosomal localization, and expression of the gene for mouse ecto-mono(ADP-ribosyl)transferase ART5.

Mono(ADP-ribosyl)transferases regulate the function of target proteins by attaching ADP-ribose to specific amino acid residues in their target proteins. The purpose of this study was to determine the structure, chromosomal localization, and expression profile of the gene for mouse ecto-ADP-ribosyltransferase ART5. Southern blot analyses indicate that Art5 is a single copy gene which maps to mouse chromosome 7 at offset 49.6 cM in close proximity to the Art1, Art2a and Art2b genes. Northern blot and RT-PCR analyses demonstrate prominent expression of Art5 in testis, and lower levels in cardiac and skeletal muscle. Sequence analyses reveal that the Art5 gene encompasses six exons spanning 8 kb of genomic DNA. The 5' end of the Art5 gene overlaps with that of the Art1 gene. A single long exon encodes the predicted ART5 catalytic domain. Separate exons encode the N-terminal leader peptide and a hydrophilic C-terminal extension. Sequencing of RT-PCR products and ESTs identified six splice variants. The deduced amino acid sequence of ART5 shows 87% sequence identity to its orthologue from the human, and 37 and 32% identity to its murine paralogues ART1 and ART2. Unlike ART1 and ART2, ART5 lacks a glycosylphosphatidylinositol-anchor signal sequence and is predicted to be a secretory enzyme. This prediction was confirmed by transfecting an Art5 cDNA expression construct into Sf9 insect cells. The secreted epitope-tagged ART5 protein resembled rat ART2 in exhibiting potent NAD-glycohydrolase activity. This study provides important experimental tools to further elucidate the function of ART5.

ADP Ribose Transferases↗
PubMed1998

A third-generation lentivirus vector with a conditional packaging system.

Vectors derived from human immunodeficiency virus (HIV) are highly efficient vehicles for in vivo gene delivery. However, their biosafety is of major concern. Here we exploit the complexity of the HIV genome to provide lentivirus vectors with novel biosafety features. In addition to the structural genes, HIV contains two regulatory genes, tat and rev, that are essential for HIV replication, and four accessory genes that encode critical virulence factors. We previously reported that the HIV type 1 accessory open reading frames are dispensable for efficient gene transduction by a lentivirus vector. We now demonstrate that the requirement for the tat gene can be offset by placing constitutive promoters upstream of the vector transcript. Vectors generated from constructs containing such a chimeric long terminal repeat (LTR) transduced neurons in vivo at very high efficiency, whether or not they were produced in the presence of Tat. When the rev gene was also deleted from the packaging construct, expression of gag and pol was strictly dependent on Rev complementation in trans. By the combined use of a separate nonoverlapping Rev expression plasmid and a 5' LTR chimeric transfer construct, we achieved optimal yields of vector of high transducing efficiency (up to 10(7) transducing units [TU]/ml and 10(4) TU/ng of p24). This third-generation lentivirus vector uses only a fractional set of HIV genes: gag, pol, and rev. Moreover, the HIV-derived constructs, and any recombinant between them, are contingent on upstream elements and trans complementation for expression and thus are nonfunctional outside of the vector producer cells. This split-genome, conditional packaging system is based on existing viral sequences and acts as a built-in device against the generation of productive recombinants. While the actual biosafety of the vector will ultimately be proven in vivo, the improved design presented here should facilitate testing of lentivirus vectors.

Animals↗
PubMed2002

SST: an algorithm for finding near-exact sequence matches in time proportional to the logarithm of the database size.

MOTIVATION: Searches for near exact sequence matches are performed frequently in large-scale sequencing projects and in comparative genomics. The time and cost of performing these large-scale sequence-similarity searches is prohibitive using even the fastest of the extant algorithms. Faster algorithms are desired. RESULTS: We have developed an algorithm, called SST (Sequence Search Tree), that searches a database of DNA sequences for near-exact matches, in time proportional to the logarithm of the database size n. In SST, we partition each sequence into fragments of fixed length called 'windows' using multiple offsets. Each window is mapped into a vector of dimension 4(k) which contains the frequency of occurrence of its component k-tuples, with k a parameter typically in the range 4-6. Then we create a tree-structured index of the windows in vector space, with tree-structured vector quantization (TSVQ). We identify the nearest neighbors of a query sequence by partitioning the query into windows and searching the tree-structured index for nearest-neighbor windows in the database. When the tree is balanced this yields an O(logn) complexity for the search. This complexity was observed in our computations. SST is most effective for applications in which the target sequences show a high degree of similarity to the query sequence, such as assembling shotgun sequences or matching ESTs to genomic sequence. The algorithm is also an effective filtration method. Specifically, it can be used as a preprocessing step for other search methods to reduce the complexity of searching one large database against another. For the problem of identifying overlapping fragments in the assembly of 120 000 fragments from a 1.5 megabase genomic sequence, SST is 15 times faster than BLAST when we consider both building and searching the tree. For searching alone (i.e. after building the tree index), SST 27 times faster than BLAST. AVAILABILITY: Request from the authors.

Algorithms↗
PubMed2004

The hyperthermophile protein Sso10a is a dimer of winged helix DNA-binding domains linked by an antiparallel coiled coil rod.

Sso10a is a member of a group of DNA-binding proteins thought to be important in chromatin structure and regulation in the hyperthermophilic archaeon Sulfolobus solfataricus. We have determined the structure of Sso10a to 1.47A resolution directly with unlabelled native crystals by a novel approach using sulfur single-wavelength anomalous scattering (SAS) from a chromium X-ray source. The 95 amino acid residue protein contains a winged helix DNA-binding domain with an extended C-terminal alpha-helix that leads to dimerization by forming a two-stranded, antiparallel coiled-coil rod. The winged helix domains are at opposite ends of the extended coiled coil with two putative DNA-recognition helices separated by 55A and rotated by 83 degrees. Formation of stable dimers in solution is demonstrated by both analytical ultracentrifugation and differential scanning calorimetry. With a T0 of 109 degrees C, Sso10a is one of the most stable two-stranded coiled coils known. The coiled coil contains a rare aspartate residue (D69) in the normally hydrophobic d position of the heptad repeat, with two aspartate-lysine (d-g') interhelical ion pairs in the symmetrical dimer. Mutation of D69 to alanine resulted in an increase in thermal stability, indicating that destabilization resulting from the partially buried aspartate residue cannot be offset by ion pair formation. Possible DNA-binding interactions are discussed on the basis of comparisons to other winged helix proteins. The structure of Sso10a provides insight into the structures of the conserved domain represented by COG3432, a group of more than 20 hypothetical transcriptional regulators coded in the genomic sequences of both crenarchaeota and euryarchaeota.

Amino Acid Sequence↗
PubMed2025

Dosage sensitivity of the loop extrusion rate confers tunability to genome folding while creating vulnerability to genetic disruption.

Genome folding is not static, but emerges from dynamic processes that control transcription, replication, recombination, and repair. DNA loop extrusion by cohesin is central to genome organization, yet it remains unclear how cells can tune extrusion kinetics to achieve precise and functional chromosome folding patterns. Here we discover extrusion rate acts as a tunable biophysical parameter in cells, quantitatively dialed by the respective dosage of the cohesin cofactors NIPBL and PDS5. Modulation of extrusion rate can offset changes in cohesin lifetime to buffer steady-state chromosome structure and transcriptional states, even in the face of abnormal extrusion dynamics. These findings provide a long-sought mechanistic basis for the genetic interactions between cohesin cofactors and the molecular origin of haploinsufficiency in cohesinopathies, such as Cornelia de Lange syndrome.

Journal Article↗
PubMed2004

An exonic splicing enhancer offsets the atypical GU-rich 3' splice site of human apolipoprotein A-II exon 3.

Human apolipoprotein A-II (apoA-II) intron 2/exon 3 junction shows a peculiar tract of alternating pyrimidines and purines (GU tract) that makes the acceptor site deviate significantly from the consensus. However, apoA-II exon 3 is constitutively included in mRNA. We have studied this unusual exon definition by creating a construct with the genomic fragment encompassing the whole gene from apoA-II and its regulatory regions. Transient transfections in Hep3B cells have shown that deletion or replacement of the GU repeats at the 3' splice site resulted in a decrease of apoA-II exon 3 inclusion, indicating a possible role of the GU tract in splicing. However, a 3' splice site composed of the GU tract in heterologous context, such as the extra domain A of human fibronectin or cystic fibrosis transmembrane conductance regulator exon 9, resulted in total skipping of the exons. Next, we identified the exonic cis-acting elements that may affect the splicing efficiency of apoA-II exon 3 and found that the region spanning from nucleotide 87 to 113 of human apoA-II exon 3 is essential for its inclusion in the mRNA. Overlapping deletions and point mutations (between nucleotides 91 and 102) precisely defined an exonic splicing enhancer (ESEwt). UV cross-linking assays followed by immunoprecipitation with anti-SR protein monoclonal antibodies showed that ESEwt, but not mutated ESE RNA, was able to bind both alternative splicing factor/splicing factor 2 and SC35. Furthermore, overexpression of both splicing factors enhanced exon 3 inclusion. These results show that this protein-ESE interaction is able to promote the incorporation of exon 3 in mRNA and suggest that they can rescue the splicing despite the noncanonical 3' splice site.

Apolipoprotein A-II↗
PubMed1998

SnoRNA-guided ribose methylation of rRNA: structural features of the guide RNA duplex influencing the extent of the reaction.

Eukaryotic rRNAs contain a large number of ribose-methylated nucleotides of elusive function which are confined to the universally conserved rRNA domains. Ribose methylation of these nucleotides is directed by a large family of small trans -acting guide RNAs, called box C/D antisense snoRNAs. Each snoRNA targets precisely one of the nucleotides to be methylated within the pre-rRNA sequence, through transient formation of a 10-21 bp regular RNA duplex around the modification site. In this study we have analyzed how different features of the double-stranded RNA guide structure affect the extent of site-specific ribose methylation, by co-expressing an appropriate RNA substrate and its cognate tailored snoRNA guide in transfected mouse cells. We show that an increased GC content of the duplex can make up for the inhibitory effects of a helix truncation or for the presence of helix irregularities such as a mismatched pair or a bulge nucleotide. However, some helix irregularities dramatically inhibit the reaction and are not offset by further stabilization of the duplex. Overall, the RNA duplex tolerates a much larger degree of irregularity than anticipated, even in the immediate vicinity of the methylation site, which offers new prospects in the search for additional snoRNA guides. Accordingly, a few snoRNA-like sequences of uncertain status detected in the yeast Saccharomyces cerevisiae genome now appear as likely bona fide ribose methylation guides.

Animals↗
PubMed2001

Cellular osmoregulation: beyond ion transport and cell volume.

All cells are characterized by the expression of osmoregulatory mechanisms, although the degree of this expression is highly variable in different cell types even within a single organism. Cellular osmoregulatory mechanisms constitute a conserved set of adaptations that offset antagonistic effects of altered extracellular osmolality/environmental salinity on cell integrity and function. Cellular osmoregulation includes the regulation of cell volume and ion transport but it does not stop there. We know that organic osmolyte concentration, protein structure, cell turnover, and other cellular parameters are osmoregulated as well. In this brief review two important aspects of cellular osmoregulation are emphasized: 1) maintenance of genomic integrity, and 2) the central role of protein phosphorylation. Novel insight into these two aspects of cellular osmoregulation is illustrated based on two cell models, mammalian kidney inner medullary cells and teleost gill epithelial cells. Both cell types are highly hypertonicity stress-resistant and, therefore, well suited for the investigation of osmoregulatory mechanisms. Damage to the genome is discussed as a newly discovered aspect of hypertonic threat to cells and recent insights on how mammalian kidney cells deal with such threat are presented. Furthermore, the importance of protein phosphorylation as a core mechanism of osmosensory signal transduction is emphasized. In this regard, the potential roles of the 14-3-3 family of phospho-protein adaptor molecules for cellular osmoregulation are highlighted primarily based on work with fish gill epithelial cells. These examples were chosen for the reader to appreciate the numerous and highly specific interactions between stressor-specific and non-specific pathways that form an extensive cellular signaling network giving rise to adaptive compensation of hypertonicity. Furthermore, the example of 14-3-3 proteins illustrates that a single protein may participate in several pathways that are non-specific with regard to the type of stress and, at the same time, in stress-specific pathways to promote cell integrity and function during hypertonicity.

Journal Article↗
PubMed2002

On Richerson and Boyd's model of cultural evolution by sexual selection.

Richerson and Boyd proposed a model of sexual selection in which both the male trait and the female preference are culturally transmitted. We generalize their model by introducing sex-dependent oblique transmission rates and a fairly comprehensive female preference rule. The model differs markedly from the standard genetic models in that the male trait and the female preference are uncorrelated. Hence, there can be no "sexy son" effect to offset the assumed fertility cost to choosy females. Nevertheless, the cultural processes can support a stable polymorphic equilibrium at which the choosy females are present. Also of interest are the cyclical dynamics observed in the neighborhood of the internal equilibrium.

Cultural Evolution↗
PubMed2024

Interspecific transfer of genetic information through polyploid bridges.

Hybridization blurs species boundaries and leads to intertwined lineages resulting in reticulate evolution. Polyploidy, the outcome of whole genome duplication (WGD), has more recently been implicated in promoting and facilitating hybridization between polyploid species, potentially leading to adaptive introgression. However, because polyploid lineages are usually ephemeral states in the evolutionary history of life it is unclear whether WGD-potentiated hybridization has any appreciable effect on their diploid counterparts. Here, we develop a model of cytotype dynamics within mixed-ploidy populations to demonstrate that polyploidy can in fact serve as a bridge for gene flow between diploid lineages, where introgression is fully or partially hampered by the species barrier. Polyploid bridges emerge in the presence of triploid organisms, which despite critically low levels of fitness, can still allow the transfer of alleles between diploid states of independently evolving mixed-ploidy species. Notably, while marked genetic divergence prevents polyploid-mediated interspecific gene flow, we show that increased recombination rates can offset these evolutionary constraints, allowing a more efficient sorting of alleles at higher-ploidy levels before introgression into diploid gene pools. Additionally, we derive an analytical approximation for the rate of gene flow at the tetraploid level necessary to supersede introgression between diploids with nonzero introgression rates, which is especially relevant for plant species complexes, where interspecific gene flow is ubiquitous. Altogether, our results illustrate the potential impact of polyploid bridges on the (re)distribution of genetic material across ecological communities during evolution, representing a potential force behind reticulation.

Polyploidy↗