This application describes studies on regulation of ribosome biosynthesis during mosquito reproduction, in the context of an overall effort to develop molecular tools to genetically interfere with disease transmission. Our research integrates in vitro studies with mosquito cell lines and in vivo studies with the intact organism to 1) understand at the molecular level, key physiological processes that control mosquito reproduction, 2) clone and characterize the genes involved, and 3) use the cloned genes to develop effective gene transfer procedures and genetic control strategies. A particular focus of this phase of research is identification of promoter elements that mediate coordinate control of gene expression, with an immediate goal of establishing basic information for construction of synthetic modular promoters to control gene expression in transfected cells and transformed mosquitoes. Mosquito reproductive physiology is central to acquisition and transmission of human pathogens by mosquito vectors. Ribosome biosynthesis provides the protein synthetic machinery essential to egg production, and the coordinate control of RNA and ribosomal protein genes with respect to one another and in response to the metabolic needs of the organism provides a model for manipulation of genes transcribed by RNA polymerases I and II. Because ribosomal genes are expressed in all cells, elucidation of molecular aspects of their regulation will facilitate extension of gene transfer technologies to the mosquito embryo. Finally, in transformed Drosophila, it has already been shown that genetic disruption of ribosome biosynthesis interferes with normal egg production.
The specific aims i nclude physiological studies on ribosomal protein and rRNA gene expression in the adult female mosquito during the acquisition of """"""""competence"""""""" following emergency from the pupa, and during the vitellogenic cycle initiated by the blood meal. This study will include psoralen crosslinking to distinguish between active and inactive genes, and analysis of the recruitment of mRNA from ribonucleoprotein particles into actively translating polysomes. Cis- and trans-acting regulatory elements from the genes we have cloned will be identified using transfected cells, gel retardation, and nuclear run-on techniques. Cloning efforts will focus on rp S6 and its regulatory elements. Phosphorylation of this highly conserved ribosomal protein is associated with control of protein synthesis, and reduced expression of rp S6 in Drosophila leads to disruption of immunity functions. These studies will contribute new information and analytical approaches to investigate gene expression and provide molecular infrastructure essential to disruption of disease transmission in genetically transformed mosquitoes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI020385-15
Application #
2376311
Study Section
Special Emphasis Panel (ZRG5-TMP (01))
Project Start
1987-01-01
Project End
2000-02-29
Budget Start
1997-03-01
Budget End
1998-02-28
Support Year
15
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Zoology
Type
Schools of Earth Sciences/Natur
DUNS #
168559177
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Fallon, Ann M; Gerenday, Anna (2010) Ecdysone and the cell cycle: investigations in a mosquito cell line. J Insect Physiol 56:1396-401
Fallon, Ann M; Li, Lei (2007) The C-terminal extension that characterizes mosquito (Diptera: Culicidae) ribosomal protein S6 is widespread among the Culicomorpha. J Med Entomol 44:608-16
Hernandez, Vida P; Fallon, A M (2007) Histone H1-like, lysine-rich low complexity amino acid extensions in mosquito ribosomal proteins RpL23a and RpS6 have evolved independently. Arch Insect Biochem Physiol 64:100-10
Zhai, Yongjiao; Fallon, Ann M (2005) PCR cloning of a histone H1 gene from Anopheles stephensi mosquito cells: comparison of the protein sequence with histone H1-like, C-terminal extensions on mosquito ribosomal protein S6. BMC Genomics 6:8
Li, Lei; Fallon, A M (2005) Recovery of cDNAs encoding ribosomal proteins S9 and L26 from Aedes albopictus mosquito cells and identification of their homologs in the malaria vector, Anopheles gambiae. Arch Insect Biochem Physiol 60:44-53
Hernandez, Vida P; Higgins, LeeAnn; Schwientek, Melinda Sue et al. (2003) The histone-like C-terminal extension in ribosomal protein S6 in Aedes and Anopheles mosquitoes is encoded within the distal portion of exon 3. Insect Biochem Mol Biol 33:901-10
Jayachandran, Gitanjali; Fallon, Ann M (2003) Ribosomal protein P0 from Aedes albopictus mosquito cells: cDNA cloning and analysis of expression. Genetica 119:1-10
Schwientek, Melinda Sue; Higgins, LeeAnn; Fallon, Ann Marie (2002) Cultured Aedes albopictus mosquito cells accumulate elongation factor-1 alpha (EF-1 alpha) during serum starvation. Insect Biochem Mol Biol 32:1055-63
Gerenday, A; Shih, K M; Herman, C C et al. (2001) Increased ribonucleotide reductase activity in hydroxyurea-resistant mosquito cells. Arch Insect Biochem Physiol 46:19-25
Niu, L L; Fallon, A M (2000) Differential regulation of ribosomal protein gene expression in Aedes aegypti mosquitoes before and after the blood meal. Insect Mol Biol 9:613-23

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