RESUMEN
This paper presents a bird's-eye view of the major repeats and chromatin types of tomato. Using fluorescence in-situ hybridization (FISH) with Cot-1, Cot-10 and Cot-100 DNA as probes we mapped repetitive sequences of different complexity on pachytene complements. Cot-100 was found to cover all heterochromatin regions, and could be used to identify repeat-rich clones in BAC filter hybridization. Next we established the chromosomal locations of the tandem and dispersed repeats with respect to euchromatin, nucleolar organizer regions (NORs), heterochromatin, and centromeres. The tomato genomic repeats TGRII and TGRIII appeared to be major components of the pericentromeres, whereas the newly discovered TGRIV repeat was found mainly in the structural centromeres. The highly methylated NOR of chromosome 2 is rich in [GACA](4), a microsatellite that also forms part of the pericentromeres, together with [GA](8), [GATA](4) and Ty1-copia. Based on the morphology of pachytene chromosomes and the distribution of repeats studied so far, we now propose six different chromatin classes for tomato: (1) euchromatin, (2) chromomeres, (3) distal heterochromatin and interstitial heterochromatic knobs, (4) pericentromere heterochromatin, (5) functional centromere heterochromatin and (6) nucleolar organizer region.
Asunto(s)
Mapeo Cromosómico/métodos , ADN de Plantas/genética , Hibridación Fluorescente in Situ/métodos , Secuencias Repetitivas de Ácidos Nucleicos/genética , Solanum lycopersicum/genética , Genoma de Planta , HeterocromatinaRESUMEN
Cysteine proteases are predominant in thrips guts (TGs) and, therefore, a suitable target for selecting effective protease inhibitors against western flower thrips (Frankliniella occidentalis). We report the isolation of four full-length cysteine protease cDNA clones from thrips in a two-step PCR approach with degenerate oligonucleotides designed on conserved cathepsin L domains. At the deduced amino acid level, the clones possessed all functional and structural characteristics of cathepsin L, and showed high mutual identity and strong similarity with cathepsin L-like cysteine proteases from other insects and arthropods. Southern analysis indicated that a family of four closely related and 10-12 less-related genes encode the cathepsin L-like cysteine proteases in the thrips genome. Partial sequencing of genomic DNA demonstrated the presence of three introns in the coding DNA.
Asunto(s)
Catepsinas/genética , Cisteína Endopeptidasas/genética , Insectos/enzimología , Insectos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Catepsina L , Catepsinas/química , Biología Computacional , Cisteína Endopeptidasas/química , ADN Complementario/genética , Biblioteca de Genes , Genómica , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Alineación de SecuenciaRESUMEN
Potato synthesises high levels of proteinase inhibitors in response to insect attack. This can adversely affect protein digestion in the insects, leading to reduced growth, delayed development and lowered fecundity. Colorado potato beetle overcomes this defence mechanism by changing the composition of its digestive proteinases. The induced cysteine proteinases in the adapted gut sustain a normal rate of protein hydrolysis either by inactivating the inhibitors by cleavage or by insensitivity to the inhibitors as a result of high Kis. In this study cDNA clones of cysteine proteinases in adapted guts were isolated by nested PCR on the basis of N-terminal sequences previously determined for purified enzymes (Gruden et al., 2003). The cysteine proteinase cDNAs can be classified into three groups: intestains A, B and C. The amino acid identity is more than 91% within and 35-62% between the groups. They share 43-50% identity to mammalian cathepsins S, L, K, H, J and cathepsin-like enzymes from different arthropods. Homology modelling predicts that intestains A, B and C follow the general fold of papain-like proteinases. Intestains from each group, however, differ in some specific structural characteristics in the S1 and S2 binding sites that could influence enzyme-inhibitor interaction and thus, provide different mechanisms of resistance to inhibitors for the different enzymes. Gene expression analysis revealed that the intestains A and C, but not B, are induced twofold by potato plants with high levels of proteinase inhibitors.