Subterminal Sites Research Articles

Cis-acting signals modulate the efficiency of programmed DNA elimination in Paramecium tetraurelia.

In Paramecium, the regeneration of a functional somatic genome at each sexual event relies on the elimination of thousands of germline DNA sequences, known as Internal Eliminated Sequences (IESs), from the zygotic nuclear DNA. Here, we provide evidence that IESs’ length and sub-terminal bases jointly modulate IES excision by affecting DNA conformation in P. tetraurelia. Our study reveals an excess of complementary base pairing between IESs’ sub-terminal and contiguous sites, suggesting that IESs may form DNA loops prior to cleavage. The degree of complementary base pairing between IESs’ sub-terminal sites (termed Cin-score) is positively associated with IES length and is shaped by natural selection. Moreover, it escalates abruptly when IES length exceeds 45 nucleotides (nt), indicating that only sufficiently large IESs may form loops. Finally, we find that IESs smaller than 46 nt are favored targets of the cellular surveillance systems, presumably because of their relatively inefficient excision. Our findings extend the repertoire of cis-acting determinants for IES recognition/excision and provide unprecedented insights into the distinct selective pressures that operate on IESs and somatic DNA regions. This information potentially moves current models of IES evolution and of mechanisms of IES recognition/excision forward.

Genome Mutation Revealed by Artificial Hybridization between Chrysanthemum yoshinaganthum and Chrysanthemum vestitum Assessed by FISH and GISH

Present study has been done to investigate artificial interspecific crossability between Japanese Chrysanthemum yoshinaganthum (2n=36) and Chinese C. vestitum (2n=54), which were cultured in vitro and in vivo and characterization of their artificial hybrid chromosomes and type of changes assessed by FISH and GISH. GISH was applied by using biotin-labeled total genomic DNA probe of C. vestitum, which were mixed with blocking DNA of C. yoshinaganthum. Approximately 18 yellow-green colored chromosomes of C. vestitum were detected by the probe, approximately 18 yellow-red- mixed colored chromosomes could be common chromosomes of the two species, and nine chromosomes were relatively red of Ch. yoshinaganthum that were not detected by the probe. The expected average of FISH six signals of 5S rDNA sites and ten of 45S rDNA were observed on the chromosomes of three and six hybrid plants, respectively. Multicolor FISH signals showed unexpected average of seven and 14 yellow signals of 5S rDNA sites on seven and thirteen chromosomes simultaneous with ten and 11 red signals of 45S rDNA sites on ten and 11 chromosomes which were detected by the probes respectively. FISH mapping of the 5S rDNA at terminal sites was detected in hybrid chromosomes, for the first time. Yellow-color signals of the telomeres were detected by the biotin-labeled probe of the PCR-amplified telomeric probe in interphase and terminal sites in metaphase. All chromosomes that showed terminal signals except four chromosomes showed subterminal sites of telomeres indicating the presence of translocations.

Karyotypic Stability in Asparagus (Asparagus officinalis L.) Cultivars Revealed by rDNA in situ Hybridization.

Four asparagus cultivars were cytologically studied by conventional staining and in situ hybridization, in order to evaluate both the variability among cultivars and the distribution pattern of 18S-25S rRNA genes. The mitotic and meiotic analysis with conventional chromosome staining did not show heteromorphisms or differences between cultivars. In situ hybridization with 18S and 25S rDNA probes showed 6 chromosomes with 18S-25S rDNA sites in the 4 studied genotypes. The New Jersey, Waltam Washington and G4 × 14 cultivars presented 4 chromosomes with larger subterminal sites and 2 with interstitial sites of smaller size. In the Deco cultivar, heteromorphism was verified in the size of the rDNA sites, which were larger and subterminal in 3 chromosomes, and small and interstitial in 3 other chromosomes. The results suggest that asparagus cultivars may present only small differences in the number and distribution of rDNA loci.

Molecular Cytogenetics ofMusaSpecies, Cultivars and Hybrids: Location of 18S-5.8S-25S and 5S rDNA and Telomere-like Sequences

The physical sites of 18S-5.8S-25S and 5S rRNA genes and telomeric sequences in theMusaL. genome were localized by fluorescentin situhybridization on mitotic chromosomes of selected lines. A single major intercalary site of the 18S-5.8S-25S rDNA was observed on the short arm of the nucleolar organizing chromosome in each genome. AA and BB genome diploids had a single pair of sites, triploids had three sites while a tetraploid hybrid had four sites. The probe is useful for quick determination of ploidy, even using interphase nuclei from slowly growing tissue culture material. Variation in the intensity of signals was observed among heterogeneousMusalines indicating variation in the number of copies of the 18S-5.8S-25S rRNA genes. Eight subterminal sites of 5S rDNA were observed in Calcutta 4 (AA) while Butohan 2 (BB) had six sites; some were weaker in both genotypes. Triploid lines showed six to nine major sites of 5S rDNA of widely varying intensity and near the limit of detection. The diploid hybrids had five to nine sites of 5S rDNA while the tetraploid hybrid had 11 sites. The telomeric sequence was detected as pairs of dots at the ends of all the chromosomes analysed but no intercalary sequences were seen. The molecular cytogenetic studies ofMusausing repetitive and single copy DNA probes should yield insight into the genome and its evolution and provide data forMusabreeders, as well as generating genetic markers inMusa.

Binding sites for maize nuclear proteins in the subterminal regions of the transposable element Activator.

Genetic data suggest that transposition of the maize element Activator (Ac) is modulated by host factors. Using gel retardation and DNase I protection assays we identified maize proteins which bind to seven subterminal sites in both ends of Ac. Four DNase I-protected sites contain a GGTAAA sequence, the other three include either GATAAA or GTTAAA. The specificity of the maize protein binding to Ac was verified by using a synthetic fragment containing four GGTAAA motifs as probe and competitor in gel retardation assays. All seven binding sites are located within regions required in cis for transposition. A maize protein binding site with the same sequence has previously been identified in the terminal inverted repeats of the maize Mutator element. Thus, the protein, that recognizes this sequence is a good candidate for a regulatory host factor for Ac transposition.

The role of subterminal sites of transposable element Ds of Zea mays in excision.

Transposition depends on DNA sequences located at or near the termini of the transposon. In the maize transposable element Ds, these sequences were studied by site-directed mutagenesis followed by a transient excision assay in Petunia protoplasts. The transposase-binding AAACGG motifs found in large numbers in the element are important, but none of them is in itself indispensable, for excision. However, mutation of an isolated motif at the 3' end considerably reduced excisability. The inverted termini were confirmed to be indispensable. Point mutations in regions outside the inverted termini of Ds and not located in the transposase-binding motifs had, in some cases, a pronounced effect on excision frequency. The implications of these findings are discussed.

The En/Spm transposable element of Zea mays contains splice sites at the termini generating a novel intron from a dSpm element in the A2 gene.

The A2 locus of Zea mays, identified as one of the genes affecting anthocyanin biosynthesis, was cloned using the transposable elements rcy and dSpm as gene tags. The A2 gene encodes a putative protein of 395 amino acids and is devoid of introns. Two a2-m1 alleles, containing dSpm insertions of different sizes, were characterized. The dSpm element from the original state allele has perfect termini and undergoes frequent transposition. The element from the class II state allele is no longer competent to transpose. It has retained the 13 bp terminal inverted repeat but has lost all subterminal sites at the 5' end, which are recognized by tnpA protein, the most abundant product of the En/Spm transposable element system. The relatively high A2 gene expression of one a2-m1 allele is due to removal of almost all dSpm sequences by splicing. The slightly altered A2 enzyme is still functional as shown by complementation of an a2 mutant with the corresponding cDNA. The 5' and 3' splice sites are constituted by the termini of the dSpm element; it therefore represents a novel intron of the A2 gene.

A Novel Replicative Form DNA of Aleutian Disease Virus: the Covalently Closed Linear DNA of the Parvoviruses

The analysis of replicative form (RF) DNA of Aleutian disease virus (ADV) by alkaline gel electrophoresis revealed that all RF DNA species segregate into DNA single strands which represent integral multiples of a genome equivalent. This demonstrates that as with other autonomous parvoviruses, the virion and complementary DNA strands are frequently linked by hairpin structures and that also, nicks are present at subterminal sites. Approximately 50% of the 5'-terminal hairpins contain a subterminal nick whereas no nick is detectable in the 3'-terminal hairpin. This finding together with the presence of nicks in the 3' palindrome sequence of the dimer RF DNA (D RF DNA) bridge fragment is the first experimental proof for the so far hypothetical substrate specificity of a nickase. A novel DNA structure was identified in the monomer (M) RF DNA population. This molecule, designated 'monomer covalently closed linear RF DNA' (Mccl RF DNA), consists of a continuous, self-complementary, circular polynucleotide chain of twice the genome length. It was directly visualized by electron microscopy that denatured ADV M RF DNA is a single-stranded circular molecule of twice the genome length with covalently closed terminal hairpins on either end. Alkaline gradient centrifugations, enzymic assays and electrophoretic techniques confirmed the proposed structure. Moreover, evidence was obtained that the D RF DNA species contains an analogous Dccl RF DNA. It is suggested that the newly described Mccl RF DNA form is an important intermediate common to the DNA replication of all autonomously replicating parvoviruses.

Synaptonemal complex spreading in Allium cepa and A. fistulosum

The general features and fine structure of homologous chromosome alignment and pairing have been investigated in two species of Allium (A. fistulosum and A. cepa), which have similar karyotypes but very different patterns of chiasma distribution. Although there is no support for the occurrence of a general pre-meiotic alignment of homologous chromosomes, both species show some alignment of homologues as an immediate prelude to synaptonemal complex (SC) formation. In both species pairing usually commences at sub-terminal sites and is succeeded by numerous separate intercalary initiations of pairing in interstitial and distal regions and then in proximal regions. The last parts to pair, in both species, are pericentromeric and telomeric regions. There is, therefore, no evident relationship between the sequence of pairing and chiasma distribution in these species. Regularly alternating convergences and divergences of aligned axial cores (ACs), termed multiple association sites, are frequently observed. It is proposed that these represent potential pairing initiation sites and from observations on their spatial distribution it is argued that they may be evenly distributed through most of the genome. Small spherical or ellipsoid nodules are found at association sites and between closely aligned ACs which persist in the SC segments present during zygotene, but most of them disappear abruptly at the end of zygotene. These are termed zygotene nodules (ZN) and it is proposed that they are involved in matching corresponding sites on homologous chromosomes as well as possibly having a recombinational role. Their composition, structure, mode of action and relationship to pachytene recombination nodules are at present unknown.