Gabriel Macaya

An analysis of eukaryotic genomes by density gradient centrifugation

DNA preparations from 25 eukaryotes, ranging from yeast to man, were analyzed in their base composition, sedimentation coefficient, modal and mean buoyant density in CsCl. Four mammalian and two amphibian DNAs were fractionated by preparative Cs2SO4−Ag+ density gradient centrifugation. The CsCl band profile of each fraction was analyzed in terms of Gaussian curves; this allowed the buoyant densities and the relative amounts of the components present in each DNA to be assessed. Direct analysis of the CsCl band profiles of unfractionated DNA showed a satisfactory agreement with the results obtained in the combined Cs2SO4−Ag+, CsCl investigations. This simpler procedure was therefore applied to all 25 eukaryotic DNAs. The main finding of this work is the recognition of phylogenetic differences at the macromolecular level in the organization of eukaryotic genomes. The differences concerning the main band DNA are: (a) the three major components (1·697, 1·704 and 1·709 g/cm3) first observed in the main band of calf DNA by Filipski et al. (1973) were also found in the other ten mammalian DNAs investigated here; similar components appear to be present in avian genomes; in both cases, the 1·704 and 1·709 g/cm3 components are responsible for the skewness to the heavy side of the CsCl main band of these DNAs; (b) the DNAs of reptiles, amphibians and fish showed a much lower and decreasing skewness of their CsCl bands compared to both mammalian and avian DNAs; the Cs2SO4−Ag+, CsCl combined analysis of two amphibian DNAs revealed discrete components, different from those of mammals and birds; (c) essentially symmetrical bands in CsCl were exhibited by three invertebrate DNAs; (d) the DNAs from three unicellular eukaryotes exhibited perfectly symmetrical bands in CsCl and could not be resolved into discrete comoponents. A number of observations on minor and satellite DNA components collected in this work are reported.

An approach to the organization of eukaryotic genomes at a macromolecular level.

DNAs from three mammals, two amphibians, two invertebrates and a unicellular eukaryote, enzymatically degraded to molecular weights in the 1×106 to 2×106 range, were analyzed in their sedimentation coefficients, and modal and mean buoyant density of their CsCl bands. These were further analyzed in terms of Gaussian curves; a more detailed analysis of these genomes was done using the combined Cs2SO4−Ag+, CsCl approach of Thiery et al. (1976). The major components of degraded mammalian DNAs were identical, in both buoyant densities and relative amounts, to those of the undergraded preparations, with only slight density shifts for some components. In contrast, the major components of degraded amphibian DNAs showed a very different pattern compared to the undergraded DNAs; changes in buoyant densities and amounts of some components, the disappearance of some components and the appearance of the components were observed. Finally, the degraded Drosophila and Saccharomyces cerevisiae DNAs were similar to the undergraded preparations in that no components could be resolved within the main bands. Mouse DNA preparations ranging in molecular weight from 1·8×106 to over 200×106 were examined. Only very slight changes in the relative amounts of some components seemed to take place in the 1·8×106 to 66×106 molecular weight range: the buoyant densities of the components did not appear to vary in the size range, 1·8×106 to over 200×106.

THE REMARKABLE VARIETY OF PLANT RNA VIRUS GENOMES

Introduction. Most plant viruses contain an RNA genome and are traditionally classified into genera (or families) of which there are over 40. This large number reflects the formidable variety that exists among plant RNA viruses. The genomes are mostly single-stranded (ss) and of positive polarity; in a few genera, they are negative sense, ambisense, or double-stranded (ds). The different strategies used by viruses for their amplification have been described at length. However, it is often difficult to find in a summarized form the main data on genome features of all groups of plant RNA viruses. The last review article along these lines (Davies & Hull, 1982) dates from before any full-length RNA sequence of a plant RNA virus was published; never-theless, a few books have more recently included this subject (Francki et al., 1991; Matthews, 1991).

The isochore patterns of mammalian genomes and their phylogenetic implications

The compositional distributions of high molecular weight DNA fragments from 20 species belonging to 9 out of the 17 eutherian orders were investigated by analytical CsCl density gradient centrifugation and by preparative fractionation in Cs2SO4/BAMD density gradients followed by analysis of the fractions in CsCl. These compositional distributions reflect those of the isochores making up the corresponding genomes.A “general distribution” was found in species belonging to eight mammalian orders. A “myomorph distribution” was found in Myomorpha, but not in the other rodent infraorders Sciuromorpha and Histricomorpha, which share the general distribution. Two other distributions were found in a megachiropteran (but not in microchiropteran, which, again, shares the general distribution) and in pangolin (a species from the only genus of the order Pholidota), respectively.The main difference between the general distribution and all other distributions is that the former contains sizable amounts (6–10%) of GC-rich isochores (detected as DNA fragments equal to, or higher than, 1.710 g/cm3 in modal buoyant density), which are scarce, or absent, in the other distributions. This difference is remarkable because gene concentrations in mammalian genomes are paralleled by GC levels, the highest gene concentrations being present in the GC-richest isochores.The compositional distributions of mammalian genomes reported here shed light on mammalian phylogeny. Indeed, all orders investigated, with the exception of Pholidota, seem to share a common ancestor. The compositional patterns of the megachiropteran and of Myomorpha may be derived from the general pattern or have independent origins.

Multisystem Component Phenotypes of Bipolar Disorder for Genetic Investigations of Extended Pedigrees

IMPORTANCE Genetic factors contribute to risk for bipolar disorder (BP), but its pathogenesis remains poorly understood. A focus on measuring multisystem quantitative traits that may be components of BP psychopathology may enable genetic dissection of this complex disorder, and investigation of extended pedigrees from genetically isolated populations may facilitate the detection of specific genetic variants that affect BP as well as its component phenotypes. OBJECTIVE To identify quantitative neurocognitive, temperament-related, and neuroanatomical phenotypes that appear heritable and associated with severe BP (bipolar I disorder [BP-I]) and therefore suitable for genetic linkage and association studies aimed at identifying variants contributing to BP-I risk. DESIGN, SETTING, AND PARTICIPANTS Multigenerational pedigree study in 2 closely related, genetically isolated populations: the Central Valley of Costa Rica and Antioquia, Colombia. A total of 738 individuals, all from Central Valley of Costa Rica and Antioquia pedigrees, participated; among them, 181 have BP-I. MAIN OUTCOMES AND MEASURES Familial aggregation (heritability) and association with BP-I of 169 quantitative neurocognitive, temperament, magnetic resonance imaging, and diffusion tensor imaging phenotypes. RESULTS Of 169 phenotypes investigated, 126 (75%) were significantly heritable and 53 (31%) were associated with BP-I. About one-quarter of the phenotypes, including measures from each phenotype domain, were both heritable and associated with BP-I. Neuroimaging phenotypes, particularly cortical thickness in prefrontal and temporal regions as well as volume and microstructural integrity of the corpus callosum, represented the most promising candidate traits for genetic mapping related to BP based on strong heritability and association with disease. Analyses of phenotypic and genetic covariation identified substantial correlations among the traits, at least some of which share a common underlying genetic architecture. CONCLUSIONS AND RELEVANCE To our knowledge, this is the most extensive investigation of BP-relevant component phenotypes to date. Our results identify brain and behavioral quantitative traits that appear to be genetically influenced and show a pattern of BP-I association within families that is consistent with expectations from case-control studies. Together, these phenotypes provide a basis for identifying loci contributing to BP-I risk and for genetic dissection of the disorder.

Rice hoja blanca virus genome characterization and expression in vitro

No information exists on the organization and mechanisms of expression of the genome of rice hoja blanca virus (RHBV), a member of the tenuivirus group, but here we describe the first steps in its characterization. RHBV contains four ssRNA and three dsRNA species, the sizes of which were estimated by native and denaturing gel electrophoresis. Hybridization analyses using 32P-labelled riboprobes of viral and viral complementary polarities showed that unequal amounts of the two polarities of at least the smallest RNA are present in the virion, and indicated that the dsRNA species contain the same information as the ssRNA species of corresponding size. Total RHBV RNA directs the synthesis of two major proteins of 23K and 21K in vitro. RNA3 directs the synthesis of a 23K protein designated NS3, and RNA4 of a 21K protein designated NS4. The NS4 protein corresponds to the non-structural protein that accumulates in RHBV-infected rice tissue. The nuclecocapsid protein is not translated from either total RHBV RNA or any individual RHBV RNA in vitro.

Large-scale methylation patterns in the nuclear genomes of plants

Methylation was investigated in compositional fractions of nuclear DNA preparations (50-100 kb in size) from five plants (onion, maize, rye, pea and tobacco), and was found to increase from GC-poor to GC-rich fractions. This methylation gradient showed different patterns in different plants and appears, therefore, to represent a novel, characteristic genome feature which concerns the noncoding, intergenic sequences that make up the bulk of the plant genomes investigated and mainly consist of repetitive sequences. The structural and functional implications of these results are discussed.

Genetic contributions to circadian activity rhythm and sleep pattern phenotypes in pedigrees segregating for severe bipolar disorder.

Significance Characterizing the abnormalities in sleep and activity that are associated with bipolar disorder (BP) and identifying their causation are key milestones in unraveling the biological underpinnings of this severe and highly prevalent disorder. We have conducted the first systematic evaluation of sleep and activity phenotypes in pedigrees that include multiple BP-affected members. By delineating specific sleep and activity measures that are significantly heritable in these families, and those whose variation correlated with the BP status of their members, and by determining the chromosomal position of loci contributing to many of these traits, we have taken the first step toward discovery of causative genetic variants. These variants, in turn, could provide clues to new approaches for both preventing and treating BP. Abnormalities in sleep and circadian rhythms are central features of bipolar disorder (BP), often persisting between episodes. We report here, to our knowledge, the first systematic analysis of circadian rhythm activity in pedigrees segregating severe BP (BP-I). By analyzing actigraphy data obtained from members of 26 Costa Rican and Colombian pedigrees [136 euthymic (i.e., interepisode) BP-I individuals and 422 non–BP-I relatives], we delineated 73 phenotypes, of which 49 demonstrated significant heritability and 13 showed significant trait-like association with BP-I. All BP-I–associated traits related to activity level, with BP-I individuals consistently demonstrating lower activity levels than their non–BP-I relatives. We analyzed all 49 heritable phenotypes using genetic linkage analysis, with special emphasis on phenotypes judged to have the strongest impact on the biology underlying BP. We identified a locus for interdaily stability of activity, at a threshold exceeding genome-wide significance, on chromosome 12pter, a region that also showed pleiotropic linkage to two additional activity phenotypes.

Properties of DNA rosettes and their relevance to chromosome structure

We have studied the spreading conditions that lead to the formation of rosettes in DNA and chromatin preparations from the amphibians Bufo marinus and Bolitoglossa subpalmata and the bacterium Shigella. Both nuclear preparations and extensively deproteinized DNA produced rosettes. The longest fibers and the most symmetric rosettes were observed in amphibian nuclear spreadings. In this procedure purified nuclei were submitted immediately to Kleinschmidt spreading over various types of hypophase. Distilled-water hypophases were most conducive for rosette production or stability. Rosettes were observed with cytochrome C as the basic protein, but not with ribonuclease A and bovine serum albumin. We cannot prove that all rosettes are artifacts of the spreading procedure, but we believe that at least some result from the expansion of compact DNA doughnuts and other structures that are apparently formed in the presence of basic proteins in salt concentrations over 40 mM (Olins and Olins 1971; Manning 1979). The dilute hypophase requirement is explainable by the assumption that dilution and spreading effects unfold a compact precursor. Occasionally we have detected structures that appear to be intermediates in the process of doughnut unfolding and that illustrate a procedure that may give rise to rosettes.

Results of a SNP genome screen in a large Costa Rican pedigree segregating for severe bipolar disorder

We have ascertained in the Central Valley of Costa Rica a new kindred (CR201) segregating for severe bipolar disorder (BP‐I). The family was identified by tracing genealogical connections among eight persons initially independently ascertained for a genome wide association study of BP‐I. For the genome screen in CR201, we trimmed the family down to 168 persons (82 of whom are genotyped), containing 25 individuals with a best‐estimate diagnosis of BP‐I. A total of 4,690 SNP markers were genotyped. Analysis of the data was hampered by the size and complexity of the pedigree, which prohibited using exact multipoint methods on the entire kindred. Two‐point parametric linkage analysis, using a conservative model of transmission, produced a maximum LOD score of 2.78 on chromosome 6, and a total of 39 loci with LOD scores >1.0. Multipoint parametric and non‐parametric linkage analysis was performed separately on four sections of CR201, and interesting (nominal P‐value from either analysis <0.01), although not statistically significant, regions were highlighted on chromosomes 1, 2, 3, 12, 16, 19, and 22, in at least one section of the pedigree, or when considering all sections together. The difficulties of analyzing genome wide SNP data for complex disorders in large, potentially informative, kindreds are discussed.