Meiotic transmission of an in vitro-assembled autonomous maize minichromosome.

Autonomous chromosomes are generated in yeast (yeast artificial chromosomes) and human fibrosarcoma cells (human artificial chromosomes) by introducing purified DNA fragments that nucleate a kinetochore, replicate, and segregate to daughter cells. These autonomous minichromosomes are convenient for manipulating and delivering DNA segments containing multiple genes. In contrast, commercial production of transgenic crops relies on methods that integrate one or a few genes into host chromosomes; extensive screening to identify insertions with the desired expression level, copy number, structure, and genomic location; and long breeding programs to produce varieties that carry multiple transgenes. As a step toward improving transgenic crop production, we report the development of autonomous maize minichromosomes (MMCs). We constructed circular MMCs by combining DsRed and nptII marker genes with 7-190 kb of genomic maize DNA fragments containing satellites, retroelements, and/or other repeats commonly found in centromeres and using particle bombardment to deliver these constructs into embryogenic maize tissue. We selected transformed cells, regenerated plants, and propagated their progeny for multiple generations in the absence of selection. Fluorescent in situ hybridization and segregation analysis demonstrated that autonomous MMCs can be mitotically and meiotically maintained. The MMC described here showed meiotic segregation ratios approaching Mendelian inheritance: 93% transmission as a disome (100% expected), 39% transmission as a monosome crossed to wild type (50% expected), and 59% transmission in self crosses (75% expected). The fluorescent DsRed reporter gene on the MMC was expressed through four generations, and Southern blot analysis indicated the encoded genes were intact. This novel approach for plant transformation can facilitate crop biotechnology by (i) combining several trait genes on a single DNA fragment, (ii) arranging genes in a defined sequence context for more consistent gene expression, and (iii) providing an independent linkage group that can be rapidly introgressed into various germplasms.

Seasonal malaria transmission and variation of anopheline density in two distinct endemic areas in Brazilian Amazonia.

Studies on seasonal anopheline fauna variation were performed in two distinct settlements in the State of Rondônia, Brazil: one at the Madeira River banks (Portuchuelo) with stable native Amazonian population; the other at an inland lumber-extracting farm (Urupá) in dry land, in which adults are mostly migrants. During a 6-yr period (1994-2000), 8,638 adult anophelines were collected: 2,684 in Urupá and 5,954 in Portuchuelo. Anopheles darlingi represented >95% of total mosquitoes caught. Dissection of 4,424 A. darlingi females yielded a very low sporozoite infection index below 0.1%. Oocysts were found in both localities in approximately 0.1% of dissected mosquitoes. Determination of the hour biting rates disclosed seasonal variations in both localities. However, in Portuchuelo, mosquito density peaked at the acme of the rainy season, whereas at Urupá it peaked in the dry season. The increase in mosquito density and incidence of malaria cases were coincident. The high mosquito densities observed in the riverine settlement of Portochuelo sector B, which permits evaluation in > 10,000 mosquitoes' bites/person/year, could explain, in spite of the low mosquito's infection index, the previously described development of natural immunity in the local population that is not observed in the dry land agroindustrial settlement of Urupá.

Bee venom phospholipase inhibits malaria parasite development in transgenic mosquitoes.

Malaria kills millions of people every year, and new control measures are urgently needed. The recent demonstration that (effector) genes can be introduced into the mosquito germ line to diminish their ability to transmit the malaria parasite offers new hope toward the fight of the disease (Ito, J., Ghosh, A., Moreira, L. A., Wimmer, E. A. & Jacobs-Lorena, M. (2002) Nature, 417, 452-455). Because of the high selection pressure that an effector gene imposes on the parasite population, development of resistant strains is likely to occur. In search of additional antiparasitic effector genes, we have generated transgenic Anopheles stephensi mosquitoes that express the bee venom phospholipase A2 (PLA2) gene from the gut-specific and blood-inducible Anopheles gambiae carboxypeptidase (AgCP) promoter. Northern blot analysis indicated that the PLA2 mRNA is specifically expressed in the guts of transgenic mosquitoes with peak expression at approximately 4 h after blood ingestion. Western blot and immunofluorescence analyses detected PLA2 protein in the midgut epithelia of transgenic mosquitoes from 8 to 24 h after a blood meal. Importantly, transgene expression reduced Plasmodium berghei oocyst formation by 87% on average and greatly impaired transmission of the parasite to naive mice. The results indicate that PLA2 may be used as an additional effector gene to block the development of the malaria parasite in mosquitoes.