Nora J. Besansky

The white Gene of Ceratitis capitata: A Phenotypic Marker for Germline Transformation

Reliable germline transformation is required for molecular studies and ultimately for genetic control of economically important insects, such as the Mediterranean fruit fly (medfly) Ceratitis capitata. A prerequisite for the establishment and maintenance of transformant lines is selectable or phenotypically dominant markers. To this end, a complementary DNA clone derived from the medfly white gene was isolated, which showed substantial similarity to white genes in Drosophila melanogaster and other Diptera. It is correlated with a spontaneous mutation causing white eyes in the medfly and can be used to restore partial eye color in transgenic Drosophila carrying a null mutation in the endogenous white gene.

Cloning and characterization of the white gene from Anopheles gambiae

A 14 kb region of genomic DN A containing the X‐linked Anopheles gambiae eye colour gene, white, was cloned and sequenced. Genomic clones containing distinct white+ alleles were polymorphic for the insertion of a small transposable element in intron 3, and differed at 1% of nucleotide positions compared. Sequence was also determined from a rare 2914 bp cDNA. Comparison of cDNA and genomic sequences established an intron‐exon structure distinct from Drosophila white. Despite a common trend in Anopheles and Drosophila of weak codon bias given low levels of gene expression, codon usage by Anopheles gambiae white was strongly biased. Overall amino acid identity between the predicted mosquito and fruitfly proteins was 64%, but dropped to 14% at the amino terminus. To correlate phenotypically white‐eyed strains of A. gambiae with structural lesions in white, five available strains were analysed by PCR and Southern blotting. Although these strains carried allelic mutations, independently generated by gamma radiation (three strains) or spontaneous events (two strains), no white lesions were detected. Significantly, another non‐allelic X‐linked mutation, causing an identical white‐eyed phenotype, has been correlated with a structural defect in the cloned white gene (Benedict et al., 1995). Taken together, these observations suggest that the white‐eyed mutants analysed in the present study carry mutations in a second eye colour gene and are most likely white+.

Q: a new retrotransposon from the mosquito Anopheles gambiae

A new family of retrotransposons (RTPs) without long terminal repeats (LTRs), designated Q, has been isolated from the malaria vector Anopheles gambiae. The nucleotide sequence of a complete element G‐22, was determined and analysed. Approximately 4.5 kb long, 0–22 contains two long overlapping open reading frames (ORFs) that potentially encode proteins with nucleic acid binding and reverse transcriptase domains similar to those of non‐LTR RTPs previously described. The 3 end is characterized by variable numbers of the triplet repeat TAA, immediately following a polyadenylation signal. In situ hybridization of nurse cell polytene chromosomes revealed about twenty labelled sites distributed over all arms and diffuse hybridization to the chromocentre. Cross‐hybridizing sequences with the same internal structure occur in all members of the A. gambiae complex. Genomic Southerns of wild A. gambiae specimens probed with Q suggest that Q is or recently was capable of retrotransposition.

Pegasus, a small terminal inverted repeat transposable element found in the white gene of Anopheles gambiae.

Pegasus, a novel transposable element, was discovered as a length polymorphism in the white gene of Anopheles gambiae. Sequence analysis revealed that this 535 bp element was flanked by 8 bp target site duplications and 8 bp perfect terminal inverted repeats similar to those found in many members of the Tcl family. Its small size and lack of long open reading frames preclude protein coding capacity. Southern analysis and in situ hybridization to polytene chromosomes demonstrated that Pegasus occurs in approximately 30 copies in the genomes of An. gambiae and its sibling species and is homogenous in structure but polymorphic in chromosomal location. Characterization of five additional elements by sequencing revealed nucleotide identities of 95% to 99%. Of 30 Pegasus-containing phage clones examined by PCR, only one contained an element exceeding 535 bp in length, due to the insertion of another transposable element-like sequence. Thus, the majority, if not all, extant Pegasus elements may be defective copies of a complete element whose contemporary existence in An. gambiae is uncertain. No Pegasus-hybridizing sequences were detected in nine other anophelines and three culicines examined, suggesting a very limited taxonomic distribution.

The Tryptophan oxygenase gene of Anopheles gambiae

The Anopheles gambiae gene encoding tryptophan oxygenase, a homolog of the Drosophila melanogaster vermilion gene, has been molecularly cloned and characterized. Unlike Drosophila, where it is X-linked, the A. gambiae gene maps to chromosome 2R, subdivision 12E, by in situ hybridization to the polytene chromosomes. Of the six introns present, four are positioned identically to those of the Drosophila homolog, one is similarly positioned, and one is novel. A 1 955 nt cDNA potentially encodes a 392 amino acid protein of an estimated 45 kDa. Amino acid comparisons between the deduced protein and previously known tryptophan oxygenases revealed 74% identity between Anopheles and Drosophila, and 53% identity between Anopheles and nematode or mammalian proteins. Northern analysis detected a developmentally regulated transcript about 2 kb in length. Since this gene is known to control adult eye color in other flies, its cloning from A. gambiae provides the basis for a dominant phenotypic marker for germline transformation, one whose expression, unlike that of white, is not cell autonomous.

Molecular Perspectives on the Genetics of Mosquitoes

Publisher Summary This chapter presents molecular perspectives on the genetics of mosquitoes. The chapter reviews various recent molecular studies performed on mosquitoes of the genera Anopheles, Culex, and Aedes. These genera include the principal vectors of three major groups of human pathogens: malaria parasites of the genus Plasmodium, filarial worms of the genera Wuchereria and Brugia, and numerous arboviruses. Anophelines are the only mosquitoes known to transmit human malaria parasites, a group of organisms that may be responsible for more morbidity and mortality worldwide than any other human pathogen. Anophelines also transmit filarial worms as do Culex and Aedes species. The chapter also discusses how molecular techniques are used to reveal genetic differentiation, both for the identification of cryptic species and for the understanding of population structure and evolution. The chapter describes the genome organization on the basis of reassociation kinetics, molecular cloning, and genome mapping. The chapter discusses the molecular biology of specific physiological systems, including insecticide resistance, immune mechanisms, oogenesis, and salivation. The identification of endogenous mobile elements and potential transformation systems for both cell lines and embryos are also described in the chapter.

The white gene from the yellow fever mosquito, Aedes aegypti

We report the cloning and primary characterization of both cDNA and genomic fragments from the white gene of the yellow fever mosquito, Aedes aegypti. Comparisons of the conceptual translation product with white genes from four other species within the order Diptera show that the Ae. aegypti gene is most similar to the white gene of the mosquito vector of human malaria, Anopheles gambiae (86% identity and 92% similarity). The analysis of the primary sequence of genomic DNA at the 5′‐end of the coding region revealed the presence of an intron that is also present in An. gambiae, but not in the vinegar fly, Drosophila melanogaster. The isolated clones of the Ae. aegypti white gene will enable the construction of a marker gene for use in the development of a germline transformation system for this species.

The Anopheles gambiae tryptophan oxygenase gene expressed from a baculovirus promoter complements Drosophila melanogaster vermilion.

An Anopheles gambiae cDNA encoding tryptophan oxygenase was placed under the control of the constitutive baculovirus promoter, ie-1. The chimeric construct, expressed transiently in vermilion (tryptophan oxygenase) mutants of Drosophila melanogaster, partially rescued adult eye color. The successful genetic complementation by this construct demonstrated both the proper function of the tryptophan oxygenase product and the effectiveness of the ie-1 promoter in directing expression of foreign genes in live insects. The functionality of An. gambiae tryptophan oxygenase in a higher fly fulfils predictions based on its structural conservation throughout millions of years of independent evolution.

THE ANOPHELES ALBIMANUS WHITE GENE : MOLECULAR CHARACTERIZATION OF THE GENE AND A SPONTANEOUS WHITE GENE MUTATION

We have cloned and characterized the white gene of Anopheles albimanus. Comparison of the deduced amino acid sequence of this white gene with its homologs from six species of Diptera show that the An. albimanus gene is most similar to the white gene of An. gambiae (92% identity). A spontaneous white-eyed mutant An. albimanus was caused by an approximately 10 kb insertion into a CT dinucleotide repeat region of intron 2 of the white locus. The flanks of this insertion are long (at least 400 bp), nearly perfect inverted terminal repeat sequences. This cloned white gene should be useful as a marker for germ line transformation of An. albimanus.

An Anopheles gambiae cDNA predicts a protein similar to a yeast Suil translation factor

The nucleotide (nt) sequence of a cDNA cloned from the mosquito Anopheles gambiae was determined. The amino acid (aa) sequence of the deduced protein was 56% identical (60/108 aa) to the recently discovered translation initiation factor Suil of yeast, suggesting that the two proteins are homologs and have similar functions. Database searches also revealed strong similarity to other sequences, including the deduced gene products of cDNAs from organisms as diverse as nematodes, humans and plants. The functions of these putative proteins are unknown, but their homology to Suil suggests that they represent an important component of the eukaryotic translation initiation complex.