Estimating female malaria mosquito age by quantifying Y-linked genes in stored male spermatozoa.

Vector control strategies are among the most effective measures to combat mosquito-borne diseases, such as malaria. These strategies work by altering the mosquito age structure through increased mortality of the older female mosquitoes that transmit pathogens. However, methods to monitor changes to mosquito age structure are currently inadequate for programmatic implementation. Female mosquitoes generally mate a single time soon after emergence and draw down spermatozoa reserves with each oviposition cycle. Here, we demonstrate that measuring spermatozoa quantity in female Anopheles mosquitoes is an effective approach to assess mosquito age. Using multiplexed qPCR targeted at male spermatozoa, we show that Y-linked genes in female mosquitoes are exclusively found in the spermatheca, the organ that houses spermatozoa, and the quantity of these gene sequences significantly declines with age. The method can accurately identify mosquitoes more than 10 days old and thus old enough to potentially transmit pathogens harbored in the salivary glands during blood feeding. Furthermore, mosquito populations that differ by 10% in daily survivorship have a high likelihood of being distinguished using modest sample sizes, making this approach scalable for assessing the efficacy of vector intervention control programs.

Extensive copy-number variation of the human olfactory receptor gene family.

As much as a quarter of the human genome has been reported to vary in copy number between individuals, including regions containing about half of the members of the olfactory receptor (OR) gene family. We have undertaken a detailed study of copy-number variation of ORs to elucidate the selective and mechanistic forces acting on this gene family and the true impact of copy-number variation on human OR repertoires. We argue that the properties of copy-number variants (CNVs) and other sets of large genomic regions violate the assumptions of statistical methods that are commonly used in the assessment of gene enrichment. Using more appropriate methods, we provide evidence that OR enrichment in CNVs is not due to positive selection but is because of OR preponderance in segmentally duplicated regions, which are known to be frequently copy-number variable, and because purifying selection against CNVs is lower in OR-containing regions than in regions containing essential genes. We also combine multiplex ligation-dependent probe amplification (MLPA) and PCR to assay the copy numbers of 37 candidate CNV ORs in a panel of approximately 50 human individuals. We confirm copy-number variation of 18 ORs but find no variation in this human-diversity panel for 16 other ORs, highlighting the caveat that reported intervals often overrepresent true CNVs. The copy-number variation we describe is likely to underpin significant variation in olfactory abilities among human individuals. Finally, we show that both homology-based and homology-independent processes have played a recent role in remodeling the OR family.

Human subtelomeric WASH genes encode a new subclass of the WASP family.

Subtelomeres are duplication-rich, structurally variable regions of the human genome situated just proximal of telomeres. We report here that the most terminally located human subtelomeric genes encode a previously unrecognized third subclass of the Wiskott-Aldrich Syndrome Protein family, whose known members reorganize the actin cytoskeleton in response to extracellular stimuli. This new subclass, which we call WASH, is evolutionarily conserved in species as diverged as Entamoeba. We demonstrate that WASH is essential in Drosophila. WASH is widely expressed in human tissues, and human WASH protein colocalizes with actin in filopodia and lamellipodia. The VCA domain of human WASH promotes actin polymerization by the Arp2/3 complex in vitro. WASH duplicated to multiple chromosomal ends during primate evolution, with highest copy number reached in humans, whose WASH repertoires vary. Thus, human subtelomeres are not genetic junkyards, and WASH's location in these dynamic regions could have advantageous as well as pathologic consequences.

Elevated rates of sister chromatid exchange at chromosome ends.

Chromosome ends are known hotspots of meiotic recombination and double-strand breaks. We monitored mitotic sister chromatid exchange (SCE) in telomeres and subtelomeres and found that 17% of all SCE occurs in the terminal 0.1% of the chromosome. Telomeres and subtelomeres are significantly enriched for SCEs, exhibiting rates of SCE per basepair that are at least 1,600 and 160 times greater, respectively, than elsewhere in the genome.

Myc regulates VEGF production in B cells by stimulating initiation of VEGF mRNA translation.

Deregulated c-myc gene expression is associated with many human and animal cancers. Myc overexpression promotes the growth of blood and lymphatic vessels, which is due in part to induction of growth factors including vascular endothelial growth factor (VEGF). We determined that the P493-6 human B-cell line increases VEGF production 10-fold upon Myc overexpression. Myc overexpression in avian B cells similarly resulted in high level VEGF production. Real-time RT-PCR analyses showed that Myc did not alter the VEGF mRNA content of these cell lines, indicating that a post-transcriptional mechanism regulates VEGF production. VEGF mRNA translation was examined by RT-PCR analysis of monosome and polysome sucrose gradient fractions from Myc-on and Myc-off P493-6 cells. Myc increased VEGF mRNA translation initiation, as VEGF mRNA loading onto polysomes increased 14-fold in Myc-on cells, and the number of ribosomes loaded per VEGF mRNA increased threefold. This translational regulation is specific to VEGF mRNA, as total polysomes show the same sucrose gradient profile in Myc-on and Myc-off cells, with no change in the percent ribosomes in polysomes, or in the number of ribosomes per polysomal mRNA. Myc stimulates VEGF production by a rapamycin- and LY294002-sensitive pathway, which does not involve alteration of eIF4E activity.

Reduced Myc overexpression and normal B-cell differentiation mediate resistance to avian leukosis virus lymphomagenesis.

Avian leukosis virus (ALV) induces bursal lymphoma in tumor-susceptible chicken strains after proviral integration within the c-myc gene, and subsequent expansion of Myc-overexpressing lymphocytes within transformed follicles. Line 6(3) strain chickens are resistant to ALV tumorigenesis, largely failing to develop Myc-transformed follicles, although they show similar levels of ALV infection and integration as lymphoma-susceptible strains. Immunohistochemical analysis determined that the transformed follicles that do arise in lymphoma-resistant birds show much lower and more variable Myc overexpression than those of susceptible birds. This reduced Myc overexpression fails to block B-cell differentiation in resistant birds, while high Myc consistently blocks development at a late embryo stage in susceptible birds. This failure of Myc to block differentiation results in a normal pattern of posthatching bursal emigration in resistant transformed follicles, while transformed follicles of susceptible birds grow rapidly due to blocked emigration. Forced Myc overexpression produces transformed follicles in resistant birds, indicating that resistant lymphocytes can tolerate high Myc expression. The coding sequence and expression of the endogenous c-myc gene is the same in resistant and susceptible birds, suggesting that genetic resistance is instead mediated by reduced ALV LTR enhancer-driven transcription in the target lymphocytes of resistant birds.