A rapid method for extraction of cotton (Gossypium spp.) genomic DNA suitable for RFLP or PCR analysis

Extraction of high-quality genomic DNA fromGossypium (cotton) species is difficult due to high levels of polysaccharide, oxidizable quinones, and other interfering substances. We describe a procedure that consistently permits isolation of cotton genomic DNA of satisfactory size and quality for RFLP and PCR analysis, as well as for most routine cloning applications. Several antioxidants, phenol-binding reagents, and phenol oxidase inhibitors are used throughout the procedure, and most polysaccharides are eliminated early in the procedure by isolation of nuclei.     

Influence of HIV-1 Genomic RNA on the Formation of Gag Biomolecular Condensates

Biomolecular condensates (BMCs) play an important role in the replication of a growing number of viruses, but many important mechanistic details remain to be elucidated. Previously, we demonstrated that the pan-retroviral nucleocapsid (NC) and HIV-1 pr55^Gag (Gag) proteins phase separate into condensates, and that HIV-1 protease (PR)-mediated maturation of Gag and Gag-Pol precursor proteins yields self-assembling BMCs that have HIV-1 core architecture. Using biochemical and imaging techniques, we aimed to further characterize the phase separation of HIV-1 Gag by determining which of its intrinsically disordered regions (IDRs) influence the formation of BMCs, and how the HIV-1 viral genomic RNA (gRNA) could influence BMC abundance and size. We found that mutations in the Gag matrix (MA) domain or the NC zinc finger motifs altered condensate number and size in a salt-dependent manner. Gag BMCs were also bimodally influenced by the gRNA, with a condensate-promoting regime at lower protein concentrations and a gel dissolution at higher protein concentrations. Interestingly, incubation of Gag with CD4⁺ T cell nuclear lysates led to the formation of larger BMCs compared to much smaller ones observed in the presence of cytoplasmic lysates. These findings suggest that the composition and properties of Gag-containing BMCs may be altered by differential association of host factors in nuclear and cytosolic compartments during virus assembly. This study significantly advances our understanding of HIV-1 Gag BMC formation and provides a foundation for future therapeutic targeting of virion assembly.     

Low genetic diversity and population connectivity fuel vulnerability to climate change for the Tertiary relict pine Pinus bungeana

Endemic species are important components of regional biodiversity and hold the key to understanding local adaptation and evolutionary processes that shape species distributions. This study investigated the biogeographic history of a relict conifer Pinus bungeana Zucc. ex Endl. confined to central China. We examined genetic diversity in P. bungeana using genotyping-by-sequencing and chloroplast and mitochondrial DNA markers. We performed spatial and temporal inference of recent genetic and demographic changes, and dissected the impacts of geography and environmental gradients on population differentiation. We then projected P. bungeana's risk of decline under future climates. We found extremely low nucleotide diversity (average π 0.0014), and strong population structure (global F_ST 0.234) even at regional scales, reflecting long-term isolation in small populations. The species experienced severe bottlenecks in the early Pliocene and continued to decline in the Pleistocene in the western distribution, whereas the east expanded recently. Local adaptation played a small (8%) but significant role in population diversity. Low genetic diversity in fragmented populations makes the species highly vulnerable to climate change, particularly in marginal and relict populations. We suggest that conservation efforts should focus on enhancing gene pool and population growth through assisted migration within each genetic cluster to reduce the risk of further genetic drift and extinction.     

IMPRINTING EFFECTS OF THREE AMINO ACIDS (ALANINE,LYSINE AND GLYCINE) AND THEIR OLIGOPEPTIDES INTETRAHYMENA PYRIFORMIS. DATA FROM THE HORMONE AND HORMONE RECEPTOR EVOLUTION

Hormonal imprinting takes place at the first encounter of the hormone and receptor, and results in a changed binding capacity and reaction of the cell and its progeny generations. The imprinting effect of three amino acids and their oligopeptides is studied using fluorescent-labelled peptides. Glycine and lysine could provoke positive imprinting (increased binding in the progeny generations) for their own peptides, but alanine could not. Mostly positive imprinting was provoked by glycine and lysine peptides for their own peptides of different chain length. The optimal chain length provoking self-imprinting was four for glycine, two for lysine and three for alanine. Except in this case, alanine was neutral or provoked mostly negative imprinting. After reaching the optimal chain length, there is a decline in binding. Evolutionary conclusions are discussed.     

Crystallization of alpha 1-acid glycoprotein

A possible link between cellular cyclic AMP content and Na+K+ATPase activity was investigated in homogenates of rat kidney. Enzyme kinetics of Mg2+ and Na+K+ATPase were run in the presence of cyclic AMP, dibutyryl cAMP and compounds expected to elevate cyclic AMP levels such as forskolin, a potent adenylate cyclase activator, IBMX, an inhibitor of phosphodiesterases, and the beta-agonist isoproterenol. Medullary Na+K+ATPase is strongly inhibited by cyclic AMP whereas cortical Na+K+ATPase was stimulated in the same conditions. The correlation between ATPase activity and cellular cyclic AMP content supports the concept of a possible regulation of the enzyme by cyclic AMP.     

Structure and expression of the lignin O-methyltransferase gene from Zea mays L.

The isolation and characterization of cDNA and homologous genomic clones encoding the lignin O-methyltransferase (OMT) from maize is reported. The cDNA clone has been isolated by differential screening of maize root cDNA library. Southern analysis indicates that a single gene codes for this protein. The genomic sequence contains a single 916 bp intron. The deduced protein sequence from DNA shares significant homology with the recently reported lignin-bispecific caffeic acid/5-hydroxyferulic OMTs from alfalfa and aspen. It also shares homology with OMTs from bovine pineal glands and a purple non-sulfur photosynthetic bacterium. The mRNA of this gene is present at different levels in distinct organs of the plant with the highest accumulation detected in the elongation zone of roots. Bacterial extracts from clones containing the maize OMT cDNA show an activity in methylation of caffeic acid to ferulic acid comparable to that existing in the plant extracts. These results indicate that the described gene encodes the caffeic acid 3-O-methyltransferase (COMT) involved in the lignin biosynthesis of maize.     

Isolation of a cloned DNA segment containing a ribosomal protein gene of Drosophila melanogaster

A Drosophila genomic DNA library in the vector Charon 4 was screened using cDNA derived from the small (6S-12S) poly(A)+ mRNA of 2-6-h-old Drosophila embryos. This fraction of mRNA is enriched for ribosomal protein-coding sequences. The selected recombinants were hybridized to total mRNA under conditions which allowed for isolation of homologous mRNAs. The mRNA from these RNA/DNA hybrids was eluted and translated in vitro. The translation products were analyzed by one- and two-dimensional electrophoresis with authentic ribosomal proteins as standards. One cloned DNA segment was found to contain a ribosomal protein gene, and a sequence which hybridizes strongly to at least 5 other ribosomal protein mRNAs.