Mechanisms of chromosome banding. II. Evidence that histones are not involved

C-and G-banding in mouse metaphase chromosomes is unaffected by exposure of the chromosomes to 0.2 N HCl for 4 h. Electrophoretic studies indicate that this is adequate to completely remove the histones from fixed and dried chromatin thus indicating that histones are not involved in C- or G-banding.     

Evidence that the ribosomal DNA genes of yeast are not on chromosome I

Summary Several workers have reported that most of the ribosomal DNA genes (rDNA) of the yeast Saccharomyces cerevisiae are located on chromosome I. More recently, data indicating that the yeast rDNA genes are located on chromosome XII has been presented. In this report, we present additional evidence indicating that most of the yeast rDNA genes are not on chromosome I. Starting from a diploid yeast strain, we isolated ten strains which were monosomic (2n-1) for chromosome I. We found that each of these ten strains contained two copies of the rDNA-containing chromosome. In addition, we show that the earlier evidence indicating that the yeast rDNA genes were on chromosome I cannot be explained by a difference in the yeast strains which were used in the different experiments.     

Evidence that free polysomes are not the precursors of membrane-bound polysomes in rat liver

We tested, in rat liver, the postulate that free polysomes were precursors of membrane-bound polysomes. Three methods were used to isolate free and membrane-bound ribosomes from either post-nuclear or post-mitochondrial supernatants of rat liver. Isolation and quantitation of 28 S and 18 S rRNA allowed determination of the 40 S and 60 S subunit composition of free and membrane-bound ribosomal populations, while pulse labeling of 28 S and 18 S rRNA with [6-¹⁴C]orotic acid and inorganic [³²P]phosphate allowed assessment of relative rates of subunit renewal. Throughout the extra-nuclear compartment, 40 S and 60 S subunits were present in essentially equal numbers, but, free ribosomes contained a stoichiometric excess of 40 S subunits, while membrane-bound ribosomes contained a complementary excess of 60 S subunits. Experiments with labeled precursors showed that throughout the extra-nuclear compartment, 40 S and 60 S subunits accumulated isotopes at essentially equal rates, however, free ribosomes accumulated isotopes faster than membrane-bound ribosomes. Among free ribosomes or polysomes, 40 S subunits accumulated isotopes faster than 60 S subunits, but, this relationship was not seen among membrane-bound ribosomes. Here, 40 S subunits accumulated isotope more slowly than 60 S subunits. This distribution of labeled precursors does not support the postulate that free polysomes are precursors of membrane-bound polysomes, but, these data suggest that membrane-bound polysomes could be precursors of free polysomes.     

Evidence that unrejoined DNA double-strand breaks are not predominantly responsible for chromosomal radiosensitivity of AT fibroblasts

To examine more fully the nature of chromosomal radiosensitivity in ataxia telangiectasia (AT) cells, we employed 24-color combinatorial painting to visualize 137Cs gamma-ray-induced chromosome-type aberrations in cells of two AT and one normal primary human fibroblast strains irradiated in log-phase growth. As a measure of misrejoined radiation-induced DSBs, we quantified exchange breakpoints associated with both simple and complex exchanges. As a measure of unrejoined DSBs, we quantified breakpoints from terminal deletions as well as deletions associated with incomplete exchange. For each of these end points, the frequency of damage per unit dose was markedly higher in AT cells compared to normal cells, although the proportion of total breaks that remained unrejoined was rather similar. The majority of breakpoints in both cell types were involved in exchanges. AT cells had a much higher frequency of complex exchanges compared to normal cells given the same dose, but for doses that resulted in approximately the same level of total breakpoints, the relative contribution from complex damage was also similar. We conclude that although terminal deletions and incomplete exchanges contribute to AT cell radiosensitivity, their relative abundance does not-in apparent contrast to the situation in lymphoblastoid cells-overwhelmingly account for the increased damage we observed in cycling AT fibroblasts. Thus, from a cytogenetic perspective, a higher level of unrepaired DSBs does not provide a universal explanation for the radiation-sensitive AT phenotype.     

Evidence that functional erythrocyte-type glucose transporters are oligomers

In this study we tested the hypothesis that functional erythrocyte-type glucose transporters (GLUT1) exist as oligomeric complexes by expressing chimeric transporter proteins in Chinese hamster ovary cells harboring endogenous GLUT1 transporters. The chimeric transporters were GLUT1-4c, in which the 29 C-terminal residues of human GLUT1 were replaced by the 30 C-terminal residues of rat skeletal muscle glucose transporter (GLUT4), and GLUT1n-4, containing the N-terminal 199 residues of GLUT1 and the 294 C-terminal residues of GLUT4. Endogenous GLUT1 was quantitatively co-immunoprecipitated by using an anti-GLUT4 C-terminal peptide antibody from detergent extracts of Chinese hamster ovary cells expressing either of the chimeric proteins, as detected by immunoblotting the precipitates with an anti-GLUT1 C-terminal peptide antiserum. No co-immunoprecipitation of native GLUT1 with native GLUT4 from extracts of 3T3-L1 adipocytes, which contain both these transporters, was observed with the same antibody. These data are consistent with the hypothesis that GLUT1 transporters exist as homodimers or higher order oligomers and that a major determinant of oligomerization is located within the first 199 residues of GLUT1.     

Mitochondrial DNA barcoding detects some species that are real, and some that are not

Mimicry and extensive geographical subspecies polymorphism combine to make species in the ithomiine butterfly genus Mechanitis (Lepidoptera; Nymphalidae) difficult to determine. We use mitochondrial DNA (mtDNA) barcoding, nuclear sequences and amplified fragment length polymorphism (AFLP) genotyping to investigate species limits in this genus. Although earlier biosystematic studies based on morphology described only four species, mtDNA barcoding revealed eight well-differentiated haplogroups, suggesting the presence of four new putative 'cryptic species'. However, AFLP markers supported only one of these four new 'cryptic species' as biologically meaningful. We demonstrate that in this genus, deep genetic divisions expected on the basis of mtDNA barcoding are not always reflected in the nuclear genome, and advocate the use of AFLP markers as a check when mtDNA barcoding gives unexpected results.     

Paraspeckles are subpopulation-specific nuclear bodies that are not essential in mice

Nuclei of higher organisms are well structured and have multiple, distinct nuclear compartments or nuclear bodies. Paraspeckles are recently identified mammal-specific nuclear bodies ubiquitously found in most cells cultured in vitro. To investigate the physiological role of paraspeckles, we examined the in vivo expression patterns of two long noncoding RNAs, NEAT1_1 and NEAT1_2, which are essential for the architectural integrity of nuclear bodies. Unexpectedly, these genes were only strongly expressed in a particular subpopulation of cells in adult mouse tissues, and prominent paraspeckle formation was observed only in the cells highly expressing NEAT1_2. To further investigate the cellular functions of paraspeckles, we created an animal model lacking NEAT1 by gene targeting. These knockout mice were viable and fertile under laboratory growth conditions, showing no apparent phenotypes except for the disappearance of paraspeckles. We propose that paraspeckles are nonessential, subpopulation-specific nuclear bodies formed secondary to particular environmental triggers.     

Evidence that the lizard helospectin peptides are O-glycosylated.

Six forms of helospectin (a vasoactive intestinal peptide analogue) were purified from the venom of the Heloderma horridum lizard. Their identification was performed by combining sequencing by automated Edman degradation and electrospray mass spectrometry analysis on the complete peptides and their tryptic fragments. The products resulting from the action of an O-glycosidase were also analysed. Two forms were identified as the previously named Hs1 and Hs2 of 38 and 37 amino-acid residues, respectively. Two forms corresponded to Hs1 and Hs2 O-glycosylated by a N-acetylhexosamine-hexose motif attached to the Ser32 residue. Two other forms were not completely characterized but might correspond to the O-glycosylated forms bearing a phosphate or a sulfate group. The glycosylation did not affect the capacity of the helospectins to recognize and to activate the human and the rat VPAC1 and VPAC2 receptors.     

Evidence that rice and other cereals are ancient aneuploids

Detailed analyses of the genomes of several model organisms revealed that large-scale gene or even entire-genome duplications have played prominent roles in the evolutionary history of many eukaryotes. Recently, strong evidence has been presented that the genomic structure of the dicotyledonous model plant species Arabidopsis is the result of multiple rounds of entire-genome duplications. Here, we analyze the genome of the monocotyledonous model plant species rice, for which a draft of the genomic sequence was published recently. We show that a substantial fraction of all rice genes ( approximately 15%) are found in duplicated segments. Dating of these block duplications, their nonuniform distribution over the different rice chromosomes, and comparison with the duplication history of Arabidopsis suggest that rice is not an ancient polyploid, as suggested previously, but an ancient aneuploid that has experienced the duplication of one-or a large part of one-chromosome in its evolutionary past, approximately 70 million years ago. This date predates the divergence of most of the cereals, and relative dating by phylogenetic analysis shows that this duplication event is shared by most if not all of them.     

Structure predictions of membrane proteins are not that bad

A simple generalization of the well-known hydrophobicity analysis is sufficient to predict membrane-spanning amphiphilic alpha-helices and beta-strands. The use of this method is illustrated for bacteriorhodopsin, OmpA protein and the photosynthetic reaction centre.     

Experimental Evidence that Sexual Displays are Costly for Nest Survival

Nest predation is one of the most significant limitations for successful breeding of tropical passerines. Thus, parental strategies may include choosing appropriate nest sites and behaving in ways that minimize predation. Habitat characteristics that may influence nest success include degree of nest concealment, proximity to habitat edge, plant architecture as well as several others cited in the literature. However, few studies have examined display behavior as a factor that could also influence nest survival. We experimentally tested whether sexual motor displays served as a cue for visually oriented predators to locate artificial nests in a population of blue‐black grassquits Volatinia jacarina, a Neotropical passerine that exhibits a complex sexual display and is subjected to elevated rates of nest predation. We also evaluated the effect of nest substrate on survival. Predation rate was higher for nests within territories of displaying males relative to areas without displaying males and for nests placed in shrubs relative to grasses. Predation increased sharply in the third experimental replicate, at the end of the breeding season, which suggests that predators may develop a search image for nests or may become more abundant during specific periods of the season. Avian predators appear to be the most important nest predators. Results suggest that there may be a trade‐off between the increase in fitness derived from sexual displays of males to attract potential mates and the decrease owing to predation of active nests within their territories.     

Evidence that S-formylglutathione hydrolase and esterase D polymorphisms are identical

Summary From the analyses of families, populations, and somatic cell hybrids it could be concluded that the S-formylglutathione hydrolase (FGH) and esterase D (ESD) polymorphisms are identical.