Meiotic behaviour of individual chromosomes in allotriploid Alstroemeria hybrids.
Kamstra, S.A. ; Jong, J.H.S.G.M. de; Jacobsen, E. ; Ramanna, M.S. ; Kuipers, A.G.J. - \ 2004
Heredity 93 (2004)1. - ISSN 0018-067X - p. 15 - 21.
in-situ hybridization - monosomic addition lines - backcross derivatives - somatic hybrids - distant hybrid - dna probes - recombination - plants - cytogenetics - progenies
Chromosome association and chiasma formation were studied in pollen mother cells at metaphase I of four allotriplod BC1 plants (2n=3x=24) obtained from the backcross of the hybrid Alstroemeria aurea x A. inodora with its parent A. inodora. We distinguished the chromosomes of both parental species by genomic in situ hybridization (GISH), whereas the individual chromosomes were identified on the basis of their multicolour FISH banding patterns obtained after a second hybridization with two species-specific satellite repeats as probes. All the four BC1 plants possessed two genomes of A. inodora and one of A. aurea. Variable numbers of recombinant chromosomes, resulting from meiotic recombination in the interspecific hybrid, were present in these plants. The homologous A. inodora chromosomes generally formed bivalents, leaving the homoeologous A. aurea chromosomes unassociated. High frequencies of trivalents were observed for the chromosome sets that contained recombinant chromosomes, even when the recombinant segments were small. Chromosome associations in the trivalents were restricted to homologous segments. The implications of the absence of homoeologous chromosome pairing on gamete constitution and prospects for introgression in Alstroemeria are discussed.
Isolation and molecular characterization of cathepsin L-like cysteine protease cDNAs from Western flower thrips (Frankliniella occidentalis)
Kuipers, A.G.J. ; Jongsma, M.A. - \ 2004
Comparative Biochemistry and Physiology. B, Biochemistry and Molecular Biology 139 (2004)1. - ISSN 1096-4959 - p. 65 - 75.
l-like enzyme - drosophila-melanogaster - cell-line - proteinase-inhibitors - functional expression - pichia-pastoris - gene family - cloning - beetle - purification
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.
Molecular basis of Colorado potato beetle adaptation to potato plant defence at the level of digestive cysteine proteinases
Gruden, K. ; Kuipers, A.G.J. ; Guncar, G. ; Slapar, N. ; Strukelj, B. ; Jongsma, M.A. - \ 2004
Insect Biochemistry and Molecular Biology 34 (2004)4. - ISSN 0965-1748 - p. 365 - 375.
crystal-structure - sequence alignment - inhibitors - larvae - proteases - papain - specificity - expression - mechanism - complex
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 K(i)s. 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 call 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. (C) 2004 Elsevier Ltd. All rights reserved.
Characterisation of distant Alstroemeria hybrids: application of highly repetitive DNA sequences from A. ligtu spp. ligtu
Shujun Zhou, ; Jeu, M.J. de; Visser, R.G.F. ; Kuipers, A.G.J. - \ 2003
Annals of Applied Biology 142 (2003)3. - ISSN 0003-4746 - p. 277 - 283.
in-situ hybridization - molecular cytogenetics - interspecific hybrids - physical organization - ovule culture - sequences - heterochromatin - localization - family - aurea
Clones from a Sau3A family of eight highly repetitive sequences previously isolated from a genomic DNA library of Alstroemeria ligtu ssp. ligtu were sequenced and found to be highly conserved. A trinucleotide microsatellite repeat [GCA](3-4) was present. A second, unrelated, Sau3A repeat was also characterised. Southern analysis proved that the isolated repeats were specific for the A. ligtu subspecies and could not be detected in other Chilean or Brazilean Alstroemeria species. As shown by in situ hybridisation, the Sau3A family and the unrelated Sau3A repeat co-localised at distinct sites along most chromosomes of Alstroemeria ligtu ssp. ligtu and Alstroemeria ligtu ssp. simsii. The present set of species-specific repetitive sequences enables the identification of A. ligtu chromosomes, and thus the tracking of chromosome transmission to interspecific hybrids and their progeny.
Molecular characterization and physical localization of highly repetitive DNA sequences from Brazilian Alstroemeria species
Kuipers, A.G.J. ; Kamstra, S.A. ; Jeu, M.J. de; Jacobsen, E. - \ 2002
Chromosome Research 10 (2002)5. - ISSN 0967-3849 - p. 389 - 398.
in-situ hybridization - distant hybrid - heterochromatin - cytogenetics - genomes - family
Highly repetitive DNA sequences were isolated from genomic DNA libraries of Alstroemeria psittacina and A. inodora. Among the repetitive sequences that were isolated, tandem repeats as well as dispersed repeats could be discerned. The tandem repeats belonged to a family of interlinked Sau3A subfragments with sizes varying from 68-127 bp, and constituted a larger HinfI repeat of approximately 400 bp. Southern hybridization showed a similar molecular organization of the tandem repeats in each of the Brazilian Alstroemeria species tested. None of the repeats hybridized with DNA from Chilean Alstroemeria species, which indicates that they are specific for the Brazilian species. In-situ localization studies revealed the tandem repeats to be localized in clusters on the chromosomes of A. inodora and A. psittacina: distal hybridization sites were found on chromosome arms 2PS, 6PL, 7PS, 7PL and 8PL, interstitial sites on chromosome arms 2PL, 3PL, 4PL and 5PL. The applicability of the tandem repeats for cytogenetic analysis of interspecific hybrids and their role in heterochromatin organization are discussed.