Coalescent estimates of HIV-1 generation time in vivo

The generation time of HIV Type 1 (HIV-1) in vivo has previously been estimated using a mathematical model of viral dynamics and was found to be on the order of one to two days per generation. Here, we describe a new method based on coalescence theory that allows the estimate of generation times to be derived by using nucleotide sequence data and a reconstructed genealogy of sequences obtained over time. The method is applied to sequences obtained from a long-term nonprogressing individual at five sampling occasions. The estimate of viral generation time using the coalescent method is 1.2 days per generation and is close to that obtained by mathematical modeling (1.8 days per generation), thus strengthening confidence in estimates of a short viral generation time. Apart from the estimation of relevant parameters relating to viral dynamics, coalescent modeling also allows us to simulate the evolutionary behavior of samples of sequences obtained over time.

Chemical egg defense in a green lacewing (Ceraeochrysa smithi)

The green lacewing Ceraeochrysa smithi (Neuroptera, Chrysopidae), like other members of its family, lays its eggs on stalks, but it is unusual in that it coats these stalks with droplets of an oily fluid. The liquid consists of a mixture of fatty acids, an ester, and a series of straight-chain aldehydes. Relative to the eggs of a congeneric chrysopid that lacks stalk fluid, the eggs of C. smithi proved well protected against ants. Components of the fluid, in an assay with a cockroach, proved potently irritant. Following emergence from the egg, C. smithi larvae imbibe the stalk fluid, thereby possibly deriving nutritive benefit, defensive advantage, or both.

cut11+: A Gene Required for Cell Cycle-dependent Spindle Pole Body Anchoring in the Nuclear Envelope and Bipolar Spindle Formation in Schizosaccharomyces pombe

The “cut” mutants of Schizosaccharomyces pombe are defective in spindle formation and/or chromosome segregation, but they proceed through the cell cycle, resulting in lethality. Analysis of temperature-sensitive alleles of cut11+ suggests that this gene is required for the formation of a functional bipolar spindle. Defective spindle structure was revealed with fluorescent probes for tubulin and DNA. Three-dimensional reconstruction of mutant spindles by serial sectioning and electron microscopy showed that the spindle pole bodies (SPBs) either failed to complete normal duplication or were free floating in the nucleoplasm. Localization of Cut11p tagged with the green fluorescent protein showed punctate nuclear envelope staining throughout the cell cycle and SPBs staining from early prophase to mid anaphase. This SPB localization correlates with the time in the cell cycle when SPBs are inserted into the nuclear envelope. Immunoelectron microscopy confirmed the localization of Cut11p to mitotic SPBs and nuclear pore complexes. Cloning and sequencing showed that cut11+ encodes a novel protein with seven putative membrane-spanning domains and homology to the Saccharomyces cerevisiae gene NDC1. These data suggest that Cut11p associates with nuclear pore complexes and mitotic SPBs as an anchor in the nuclear envelope; this role is essential for mitosis.

Preselection of retrovirally transduced bone marrow avoids subsequent stem cell gene silencing and age-dependent extinction of expression of human β-globin in engrafted mice

Transcriptional silencing of genes transferred into hematopoietic stem cells poses one of the most significant challenges to the success of gene therapy. If the transferred gene is not completely silenced, a progressive decline in gene expression as the mice age often is encountered. These phenomena were observed to various degrees in mouse transplant experiments using retroviral vectors containing a human β-globin gene, even when cis-linked to locus control region derivatives. Here, we have investigated whether ex vivo preselection of retrovirally transduced stem cells on the basis of expression of the green fluorescent protein driven by the CpG island phosphoglycerate kinase promoter can ensure subsequent long-term expression of a cis-linked β-globin gene in the erythroid lineage of transplanted mice. We observed that 100% of mice (n = 7) engrafted with preselected cells concurrently expressed human β-globin and the green fluorescent protein in 20–95% of their RBC for up to 9.5 mo posttransplantation, the longest time point assessed. This expression pattern was successfully transferred to secondary transplant recipients. In the presence of β-locus control region hypersensitive site 2 alone, human β-globin mRNA expression levels ranged from 0.15% to 20% with human β-globin chains detected by HPLC. Neither the proportion of positive blood cells nor the average expression levels declined with time in transplanted recipients. Although suboptimal expression levels and heterocellular position effects persisted, in vivo stem cell gene silencing and age-dependent extinction of expression were avoided. These findings support the further investigation of this type of vector for the gene therapy of human hemoglobinopathies.

Conserved circadian elements in phylogenetically diverse algae

Circadian expression of the luciferin-binding protein (LBP) from the dinoflagellate Gonyaulax polyedra is regulated at the translational level. A small interval in the lbp 3′-untranslated region, which contains seven UG-repeats, serves as a cis-acting element to which a trans-acting factor (CCTR) binds in a circadian manner. Its binding activity correlates negatively with the circadian expression of LBP. Here I report the identification of a protein in the green alga Chlamydomonas reinhardtii that represents a CCTR analog. It binds both specifically and under control of the circadian clock to the UG-repeat region. The data show for the first time that circadian cis-elements implicated in translational regulation have been conserved during evolution.

Whole-body and intravital optical imaging of angiogenesis in orthotopically implanted tumors

The development of drugs for the control of tumor angiogenesis requires a simple, accurate, and economical assay for tumor-induced vascularization. We have adapted the orthotopic implantation model to angiogenesis measurement by using human tumors labeled with Aequorea victoria green fluorescent protein for grafting into nude mice. The nonluminous induced capillaries are clearly visible against the very bright tumor fluorescence examined either intravitally or by whole-body luminance in real time. The orthotopic implantation model of human cancer has been well characterized, and fluorescence shadowing replaces the laborious histological techniques for determining blood vessel density. Intravital images of orthotopically implanted human pancreatic tumors clearly show angiogenic capillaries at both primary and metastatic sites. A quantitative time course of angiogenesis was determined for an orthotopically growing human prostate tumor periodically imaged intravitally in a single nude mouse over a 19-day period. Whole-body optical imaging of tumor angiogenesis was demonstrated by injecting fluorescent Lewis lung carcinoma cells into the s.c. site of the footpad of nude mice. The footpad is relatively transparent, with comparatively few resident blood vessels, allowing quantitative imaging of tumor angiogenesis in the intact animal. Capillary density increased linearly over a 10-day period as determined by whole-body imaging. Similarly, the green fluorescent protein-expressing human breast tumor MDA-MB-435 was orthotopically transplanted to the mouse fat pad, where whole-body optical imaging showed that blood vessel density increased linearly over a 20-week period. These powerful and clinically relevant angiogenesis mouse models can be used for real-time in vivo evaluation of agents inhibiting or promoting tumor angiogenesis in physiological microenvironments.

A Fruit-Specific Putative Dihydroflavonol 4-Reductase Gene Is Differentially Expressed in Strawberry during the Ripening Process1

A cDNA clone encoding a putative dihydroflavonol 4-reductase gene has been isolated from a strawberry (Fragaria × ananassa cv Chandler) DNA subtractive library. Northern analysis showed that the corresponding gene is predominantly expressed in fruit, where it is first detected during elongation (green stages) and then declines and sharply increases when the initial fruit ripening events occur, at the time of initiation of anthocyanin accumulation. The transcript can be induced in unripe green fruit by removing the achenes, and this induction can be partially inhibited by treatment of de-achened fruit with naphthylacetic acid, indicating that the expression of this gene is under hormonal control. We propose that the putative dihydroflavonol 4-reductase gene in strawberry plays a main role in the biosynthesis of anthocyanin during color development at the late stages of fruit ripening; during the first stages the expression of this gene could be related to the accumulation of condensed tannins.