A genomic BAC library and a new BAC-GFP vector to study the holocentric pest Spodoptera frugiperda.
Two genomic tools for the study of Lepidoptera and the holocentric structure of their chromosomes are presented in this paper. A bacterial artificial chromosome (BAC) library was constructed using nuclear DNA partially digested with HindIII from eggs of Spodoptera frugiperda. The library contains a total of 36,864 clones with an average insert size of 125 kb, which corresponds to approximately 11.5 genome equivalents. Hybridization screening of the library was performed with eight single-copy genes, giving an average hit of 10 clones per marker gene. Colinearity between the genome and BACs was demonstrated at the triose phosphate isomerase (tpi) locus. Probing of the library with a PCR fragment internal to the 18S ribosomal gene allowed an estimation of the rDNA locus size close to 115 repeats per haploid genome. A new vector (pBAC3.6eGFP) for transient transfection into S. frugiperda cell lines has been constructed. It is based on the BAC vector, pBAC3.6e, in which a gene encoding GFP was inserted under the control of the densovirus P9 promoter. This vector has the advantage to accommodate large genomic inserts and to be transfected in a large lepidopteran host range. It was used to construct a second BAC library from Sf9 cell nuclear DNA in order to allow a comparison between somatic and cell line genome organization.
d'Alençon
Emmanuelle
E
Laboratoire de Pathologie Comparée, Institut National de la Recherche Agronomique (INRA) UMR 1231, Centre National de la recherche Scientifique (CNRS) FRE 2689, Univ. Montpellier II, 30380 Saint Christol-les-Alès, France. alencon@ensam.inra.fr
Piffanelli
Pietro
P
Volkoff
Anne-Nathalie
AN
Sabau
Xavier
X
Gimenez
Sylvie
S
Rocher
Janick
J
Cérutti
Pierre
P
Fournier
Philippe
P
D000818:Animals; D002460:Cell Line; D002875:Chromosomes / Q000235:genetics; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004275:DNA, Ribosomal / Q000032:analysis / Q000235:genetics; D017990:Densovirus / Q000235:genetics; D004330:Drosophila / Q000235:genetics; D017344:Genes, Insect / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D015698:Genomic Library; D023281:Genomics / Q000379:methods*; D049452:Green Fluorescent Proteins; D017403:In Situ Hybridization; D007915:Lepidoptera / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics*; D010063:Ovum; D016133:Polymerase Chain Reaction; D018411:Spodoptera / Q000166:cytology / Q000235:genetics*; D014162:Transfection
D004275:DNA, Ribosomal; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D004247:DNA