Actin Genes in the Mediterranean Fruit Fly, Ceratitis Capitata

We have undertaken the study of actin gene organization and expression in the genome of the Mediterranean fruit fly (medfly), Ceratitis capitata. Actin genes have been extensively characterized previously in a wide range of eukaryotic organisms, and they have valuable properties for comparative studies. These genes are typically highly conserved in coding regions, represented in multiple copies per genome and regulated in expression during development. We have isolated a gene in the medfly using the cloned Drosophila melanogaster 5C actin gene as a probe. This medfly gene detects abundant messages present during late larval and late pupal development as well as in thoracic and leg tissue preparations from newly emerged adults. This pattern of expression is consistent with what has been seen for actin genes in other organisms. Using either the D. melanogaster 5C actin gene or the medfly gene as a probe identifies five common cross reacting EcoRI fragments in genomic DNA, but only under less than fully stringent hybridization conditions.     

The chorion genes of the medfly, Ceratitis capitata. II. Characterization of three novel cDNA clones obtained by differential screening of an ovarian library

We have isolated three new chorion cDNA clones from a Ceratitis capitata ovarian library. Their isolation was accomplished by differential screening of the library using as probes 32P-labeled poly(A)+ mRNAs obtained from hand-staged medfly choriogenic versus prechoriogenic follicles. RNA blot hybridization analysis revealed that the genes corresponding to these clones have unique temporal profiles of mRNA accumulation, restricted to specific choriogenic stages. In addition, in vitro translation products encoded by these cDNAs approximately comigrated with polypeptides synthesized de novo in culture by choriogenic follicles. All three genes are located in regions of the medfly genome that are specifically amplified in female ovaries. DNA sequence analysis has revealed that one of these clones is derived from a homolog of the Drosophila melanogaster s38 chorion gene. It appears that, although D. melanogaster and C. capitata are separated by at least 120 million years of evolution, the mechanisms by which chorion genes are expressed and regulated during development have been well maintained. We suggest that the regulatory elements controlling the expression of sex-specific (e.g., chorion) genes may be isolated and used to construct transgenic medfly strains from which females could be eliminated by negative selection; such strains could be used as part of an effort to control this agricultural pest.     

Endogenous viral elements in animal genomes

Integration into the nuclear genome of germ line cells can lead to vertical inheritance of retroviral genes as host alleles. For other viruses, germ line integration has only rarely been documented. Nonetheless, we identified endogenous viral elements (EVEs) derived from ten non-retroviral families by systematic in silico screening of animal genomes, including the first endogenous representatives of double-stranded RNA, reverse-transcribing DNA, and segmented RNA viruses, and the first endogenous DNA viruses in mammalian genomes. Phylogenetic and genomic analysis of EVEs across multiple host species revealed novel information about the origin and evolution of diverse virus groups. Furthermore, several of the elements identified here encode intact open reading frames or are expressed as mRNA. For one element in the primate lineage, we provide statistically robust evidence for exaptation. Our findings establish that genetic material derived from all known viral genome types and replication strategies can enter the animal germ line, greatly broadening the scope of paleovirological studies and indicating a more significant evolutionary role for gene flow from virus to animal genomes than has previously been recognized.     

Cchobo, a hobo-related sequence in Ceratitis capitata

A hobo-related sequence, Cchobo, with high similarity to the Drosophila melanogaster HFL1 and hobo₁₀₈ elements was isolated from the medfly. Thirteen PCR-derived clones, which share 97.9–100% DNA identity, were sequenced, seven of which do not show frame-shift or stop codon mutations in their conceptual translations. The consensus sequence has 99.7% DNA identity with the D. melanogaster hobo element HFL1. In a phylogenetic analysis with other hobo-related elements, Cchobo clusters with the HFL1 and hobo₁₀₈ elements from D. melanogaster and hobo-related elements from D. simulans, D. mauritiana and Mamestra brassicae. These elements may have undergone horizontal transfer in the recent past. The genomic distribution of Cchobo was studied by FISH to mitotic and polytene chromosomes, which revealed that Cchobo is distributed within both the heterochromatin and euchromatin. Intra- and interstrain polymorphisms were detected both at euchromatic and heterochromatic sites. These findings suggest that active copies of the element may be present in the medfly genome.     

An integrated genetic and cytogenetic map for the Mediterranean fruit fly, <Emphasis Type="Italic">Ceratitis capitata</Emphasis>, based on microsatellite and morphological markers

A genetic map based on microsatellite polymorphisms and visible mutations of the Mediterranean fruit fly (medfly), Ceratitis capitata is presented. Genotyping was performed on single flies from several backcross families. The map is composed of 67 microsatellites and 16 visible markers distributed over four linkage groups. Fluorescence in situ hybridization of selected microsatellite markers on salivary gland polytene chromosomes allowed the alignment of these groups to the second, fourth, fifth and sixth chromosome. None of the markers tested showed segregation either with the X or the third chromosome. However, this map constitutes a substantial starting point for a detailed genetic map of C. capitata. The construction of an integrated map covering the whole genome should greatly facilitate genetic studies and future genome sequence projects of the species.     

The chorion genes of the medfly, Ceratitis capitata, I: Structural and regulatory conservation of the s36 gene relative to two Drosophila species.

We have used low stringency screening with the Drosophila melanogaster s36 chorion gene to recover its homologue from genomic and cDNA libraries of the medfly, Ceratitis capitata. The same gene has also been recovered from a genomic library of D. virilis. The medfly s36 gene shows similar developmental specificity as in Drosophila (early choriogenesis). It is also specifically amplified in ovarian follicles; this is the first report of chorion gene amplification outside the genus Drosophila. Alignments of s36 sequences from three species show that, in addition to its regulatory conservation, the s36 gene is extensively conserved in sequence, in a region corresponding to a central protein domain, and in short regions of 5' flanking DNA that might correspond to cis-regulatory elements.     

Repetitive A-T rich DNA sequences from the Y chromosome of the Mediterranean fruit fly, Ceratitis capitata.

Copies of a repetitive DNA sequence distributed over 90% of the length of the long arm of the Y chromosome of the Mediterranean fruit fly, Ceratitis capitata (medfly), have been characterized. Sequencing reveals that these repeats, ranging in size from approximately 1.3 to 1.7 kb, are A-T rich overall (67%). In most cases the repeat units appear to occur in tandemly linked arrays. The repeat copies also all contain a highly similar internal region, approximately 200 bp in length, with a more extreme A-T content bias. This internal region, designated as the AT element, exhibits an A-T content of at least 83%. This exceeds what has been described for any comparable element among invertebrates. Using primers designed from the DNA sequence, PCR amplification of an internal region encompassing the AT element also reveals that these sequences are present only in the male genome in different strains of the medfly.     

Intragenomic Distribution and Stability of Transposable Elements in Euchromatin and Heterochromatin of Drosophila melanogaster: Elements with Inverted Repeats Bari 1, hobo, and pogo

The elements of the Bari 1, hobo, and pogo transposon families that are located in euchromatin, heterochromatin, and on the Y chromosome have been identified, and their stability has been assessed by Southern blot analysis. The fraction of heterochromatic elements appears to be distinctive of all transposon families tested, except for Bari 1. Evidence for instability of heterochromatic elements is described. The analysis of unstable elements in different Drosophila stocks suggests that the host genome contributes to the stability/instability of transposon families. Received: 21 August 1996 / Accepted: 24 March 1997     

Diaspora, a large family of Ty3 - gypsy retrotransposons in Glycine max, is an envelope-less member of an endogenous plant retrovirus lineage

Background  

The chromosomes of higher plants are littered with retrotransposons that, in many cases, constitute as much as 80% of plant genomes. Long terminal repeat retrotransposons have been especially successful colonizers of the chromosomes of higher plants and examinations of their function, evolution, and dispersal are essential to understanding the evolution of eukaryotic genomes. In soybean, several families of retrotransposons have been identified, including at least two that, by virtue of the presence of an envelope-like gene, may constitute endogenous retroviruses. However, most elements are highly degenerate and are often sequestered in regions of the genome that sequencing projects initially shun. In addition, finding potentially functional copies from genomic DNA is rare. This study provides a mechanism to surmount these issues to generate a consensus sequence that can then be functionally and phylogenetically evaluated.     

Genetic variation between strains of the Mediterranean fruit fly, Ceratitis capitata, detected by DNA fingerprinting.

DNA fingerprinting has been used to detect genetic variation in the Mediterranean fruit fly, Ceratitis capitata. Three different probes have been identified that can be used to detect DNA restriction fragment length polymorphisms between strains of this species. The strains used in this study differ only in terms of their geographic origin or genetic background. One of the probes used is the bacteriophage vector M13, and the other two are repetitive sequences derived from the medfly genome based on a weak homology to M13. Within a strain, each probe produces a consistent restriction fragment profile that is not affected by the method or timing of DNA extraction. Between strains, when M13 is used as a probe, an average of 10% of the observable bands are polymorphic. Use of the medfly genomic sequences as a probe increases the proportion of polymorphic bands between strains up to 30%. The fact that genetic differences between even such closely related strains can be reliably detected by this method holds great promise for studies of insect pests including the ability to monitor the movements of pest species, determining the extent of genetic variation in pest populations, and in making identifications from otherwise unidentifiable material.     

The Drosophila alcohol dehydrogenase gene may have evolved independently of the functionally homologous medfly, olive fly, and flesh fly genes

cDNAs for alcohol dehydrogenase (ADH) isozymes were cloned and sequenced from two tephritid fruit flies, the medfly Ceratitis capitata and the olive fly Bactrocera oleae. Because of the high sequence divergence compared with the Drosophila sequences, the medfly cDNAs were cloned using sequence information from the purified proteins, and the olive fly cDNAs were cloned by functional complementation in yeast. The medfly peptide sequences are about 83% identical to each other, and the corresponding mRNAs have the tissue distribution shown by the corresponding isozymes, ADH-1 and ADH-2. The olive fly peptide sequence is more closely related to medfly ADH-2. The tephritid ADHs share less than 40% sequence identity with Drosophila ADH and ADH-related genes but are >57% identical to the ADH of the flesh fly Sarcophaga peregrina, a more distantly related species. To explain this unexpected finding, it is proposed that the ADH: genes of the family Drosophilidae may not be orthologous to the ADH: genes of the other two families, Tephritidae and Sarcophagidae.     

High efficiency, site-specific excision of a marker gene by the phage P1 cre-loxP system in the yellow fever mosquito, Aedes aegypti

The excision of specific DNA sequences from integrated transgenes in insects permits the dissection in situ of structural elements that may be important in controlling gene expression. Furthermore, manipulation of potential control elements in the context of a single integration site mitigates against insertion site influences of the surrounding genome. The cre-loxP site-specific recombination system has been used successfully to remove a marker gene from transgenic yellow fever mosquitoes, Aedes aegypti. A total of 33.3% of all fertile families resulting from excision protocols showed evidence of cre-loxP-mediated site-specific excision. Excision frequencies were as high as 99.4% within individual families. The cre recombinase was shown to precisely recognize loxP sites in the mosquito genome and catalyze excision. Similar experiments with the FLP/FRT site-specific recombination system failed to demonstrate excision of the marker gene from the mosquito chromosomes.     

Transposition of elements of the 412, copia and 297 dispersed repeated gene families in Drosophila.

The stability of elements of three different dispersed repeated gene families in the genome of Drosophila tissue culture cells has been examined. Different amounts of sequences homologous to elements of 412, copia and 297 dispersed repeated gene families are found in the genomes of D. melanogaster embryonic and tissue culture cells. In general the amount of these sequences is increased in the cell lines. The additional sequences homologous to 412, copia and 297 occur as intact elements and are dispersed to new sites in the cell culture genome. It appears that these elements can insert at many alternative sites. We also describe a DNA sequence arrangement found in the D. melanogaster embryo genome which appears to result from a transposition of an element of the copia dispersed repeated gene family into a new chromosomal site. The mechanism of insertion of this copia element is precise to within 90 bp and may involve a region of weak sequence homology between the site of insertion and the direct terminal repeats of the copia element.     

Presence of dUTPase in the Various Human Endogenous Retrovirus K (HERV-K) Families

Various retroviruses have been shown to encode dUTPase. The overall phylogeny of dUTPase is unclear, though. The human genome contains a significant amount of human endogenous retroviruses (HERV) representing fossilized sequences of ancient exogenous retroviruses. A few HERV families have been reported to harbor dUTPase domains. We surveyed the various HERV families for the presence of dUTPase and found that ancestors of all HERV-K families but one encoded dUTPase. With two exceptions phylogenetic analysis shows a monophyletic origin of dUTPase for the different HERV-K dUTPases. Sequences of consensus dUTPase domains suggest that the various exogenous ancestors of HERV-K once encoded active enzymes. Our analysis provides informations on dUTPase phylogeny and further shows that endogenous retroviruses provide important informations regarding retrovirus evolution.     

Sequence composition, organization, and evolution of the core Triticeae genome

We investigated the composition and the basis of genome expansion in the core Triticeae genome using Aegilops tauschii, the D-genome donor of bread wheat. We sequenced an unfiltered genomic shotgun (trs) and a methylation-filtration (tmf) library of A. tauschii, and analyzed wheat expressed sequence tags (ESTs) to estimate the expression of genes and transposable elements (TEs). The sampled D-genome sequences consisted of 91.6% repetitive elements, 2.5% known genes, and 5.9% low-copy sequences of unknown function. TEs constituted 68.2% of the D-genome compared with 50% in maize and 14% in rice. The DNA transposons constituted 13% of the D-genome compared with 2% in maize. TEs were methylated unevenly within and among elements and families, and most were transcribed which contributed to genome expansion in the core Triticeae genome. The copy number of a majority of repeat families increased gradually following polyploidization. Certain TE families occupied discrete chromosome territories. Nested insertions and illegitimate recombination occurred extensively between the TE families, and a majority of the TEs contained internal deletions. The GC content varied significantly among the three sequence sets examined ranging from 42% in tmf to 46% in trs and 52% in the EST. Based on enrichment of genic sequences, methylation-filtration offers one option, although not as efficient as in maize, for isolating gene-rich regions from the large genome of wheat.     

Y enriched and Y specific DNA sequences from the genome of the mediterranean fruit fly,Ceratitis capitata

DNA sequences that are enriched or specific to the genome of the male medfly, Ceratitis capitata, have been isolated using a differential hybridization approach. Twelve phage clones from a genomic library have been identified that consistently display more intense hybridization with a genomic DNA probe from males as opposed to one from females. Southern DNA blot analysis reveals that these recombinant clones contain at least one EcoRI fragment that is either specific to the male genome, or more highly represented in it, as compared with the female genome. These EcoRI fragments, when used as probes, all generate a similar pattern of intense multiple bands in genomic DNA of males. This suggests the presence of repetitive sequences that are at least partially homologous in these regions of the genome that are specific to or enriched in males. In situ hybridization to mitotic chromosomes confirms a Y chromosomal origin for the male specific repetitive sequences. Data on the genomic organization, representation and evolutionary conservation of these sequences that are specific to or enriched in males are presented. Studies of the genomic organization and representation of flanking sequences that are not male specific are presented as well.by R. Appels     

On the origins of medfly invasion and expansion in Australia

As a result of their rapid expansion and large larval host range, true fruit flies are among the world's most important agricultural pest species. Among them, Ceratitis capitata has become a model organism for studies on colonization and invasion processes. The genetic aspects of the medfly invasion process have already been analysed throughout its range, with the exception of Australia. Bioinvasion into Australia is an old event: medfly were first captured in Australia in 1895, near Perth. After briefly appearing in Tasmania and the eastern states of mainland Australia, medfly had disappeared from these areas by the 1940s. Currently, they are confined to the western coastal region. South Australia seems to be protected from medfly infestations both by the presence of an inhospitable barrier separating it from the west and by the limited number of transport routes. However, numerous medfly outbreaks have occurred since 1946, mainly near Adelaide. Allele frequency data at 10 simple sequence repeat loci were used to study the genetic structure of Australian medflies, to infer the historical pattern of invasion and the origin of the recent outbreaks. The combination of phylogeographical analysis and Bayesian tests showed that colonization of Australia was a secondary colonization event from the Mediterranean basin and that Australian medflies were unlikely to be the source for the initial Hawaiian invasion. Within Australia, the Perth area acted as the core range and was the source for medfly bioinvasion in both Western and South Australia. Incipient differentiation, as a result of habitat fragmentation, was detected in some localized areas at the periphery of the core range.