Somatic breakpoints,distribution of repetitive DNA and non-LTR retrotransposon insertion sites in the chromosomes of <Emphasis Type="Italic">Chironomus piger</Emphasis> Strenzke (Diptera,Chironomidae)

Structural aberrations, their frequency and distribution as well as distribution of the tandem repetitive minisatellite DNA clusters of Alu and Hinf elements and two retroelements, the LINE NLRCth1 and the SINE CTRT1, were analyzed in the genome of the chironomid C. piger Strenzke larvae from a Bulgarian population. A consistent somatic variability in the structure of the polytene chromosomes was detected, showing that the C. piger genome is more actively rearranging than supposed before. Breakpoints were concentrated in proximal parts of chromosomes significantly more often than in distal parts. By FISH analysis we could detect only one locus containing Alu elements and 38 Hinf cluster loci which appear to be dispersed equally all over the chromosomes. The retrotransposons NLRCth1 and CTRT1 are present only in a few loci, but highly variant among different individuals. The mean number of NLRCth1 sites per individual was 18.4 ± 2.09 and of CTRT1 was 54.8 ± 8.42. A third of breakpoint locations were close to or coincide with a locus occupied by a retroelement (either NLRCth1 or CTRT1). Nineteen percent of breakpoints coincided with Hinf repetitive DNA elements. Some breakpoints were identical in the two sibling species C. piger and C. riparius Meigen (syn.: C. thummi thummi) and are considered as conserved hot spots of chromosome breakage.     

Effect of Environmental Pollution on the Chromosomal Variability of Chironomus Riparius

Different frequencies of chromosomal alterations in salivary gland polytene chromosomes AB, CD and EF were described in larvae of Chironomus riparius (syn. Chironomus thummi) from the trace metal-polluted station of Santena on the river Banna, near Turin, and from the unpolluted station of Corio (40 Km from Turin) which was taken as a reference area. In a sample of 56 larvae from Santena, no specimen with the standard karyotype in all cells of the salivary glands was found. Different types of aberrations were found: 33 paracentric and five pericentric inversions, three deficiencies, four amplified sections and one chromatid break. Fifteen out of the 38 inversions and two amplified sections appeared to be inherited, while all the other aberrations were somatic. Most of the aberrations' breakpoints were located on both sides of the centromere regions, where constitutive heterochromatin is present. Also functional alterations were observed (mainly telomere and centromere decondensations and nine novel puffs). In a sample of 49 larvae of a population from the well-preserved area of Corio only six somatic and one inherited paracentric inversions were found. These results suggest that the strong destabilization of the genomes of C. riparius larvae from Santena could be a reaction to the activity of the toxic substances present in the polluted sediments of the river Banna. This revised version was published online in August 2006 with corrections to the Cover Date.     

Agrobacterium-mediated transformation of Citrus stem segments and regeneration of transgenic plants

Summary A method for Agrobacterium-mediated transformation of Citrus and organogenic regeneration of transgenic plants is reported. Internodal stem segments were co-cultured with Agrobacterium harboring binary vectors that contained the genes for the scorable marker ß-glucuronidase (GUS) and the selectable marker NPT-II. A low but significant percentage ( 5%) of the shoots regenerated in the presence of 100 g/ml kanamycin were GUS⁺. Polymerase chain reaction (PCR) analysis confirmed that GUS⁺ shoots contained T-DNA. Two plants established in soil were shown to be transgenic by Southern analysis.     

Effects of tidewater on the feeding ecology of hooded crane (<Emphasis Type="Italic">Grus monacha</Emphasis>) and conservation of their wintering habitats at Chongming Dongtan,China

Dongtan (or East Tideland) on Chongming Island in China is an important wintering site of Grus monacha (hooded crane). Field observations were conducted in Dongtan in the wintering season of 2000–2001 to determine how tidal fluctuation affects the habitat use and behavior of G.monacha. Scirpus mariqueter (sea-bulrush) was the dominant species in the tidal vegetation; and its corms were the major food source for G.monacha. The corms in the outer zone (far from the dyke) were buried underground shallower than those in the inner zone (near the dyke) because of erosion by tidewater. For easy access to food, G.monacha tended to select the outer zone and the edge of tidal creeks as its foraging sites for the majority of time spent on the tideland. Regression analysis revealed that distance of the foraging sites to the dyke was correlated with tidal height. The cranes did not appear to show territorial behavior, and tended to form large-sized flocks, especially at high tide, because of the effect of the tidewater and the non-uniform distribution of food. Thus, the height of the tide is an important factor responsible for the habitat selection of G.monacha, which was also the major cause of the cranes selecting only the natural tideland at Dongtan on Chongming Island. Unfortunately, the habitats of G.monacha are limited, and even threatened, at Dongtan because the tideland is frequently reclaimed. Appropriate measures should be taken to prevent the tideland being further reclaimed, otherwise the natural wintering grounds of G.monacha will soon disappear.     

Novel methylation at GpC dinucleotide in the fish Sparus aurata genome

To date, vertebrate DNA has been found methylated at the 5 position of cytosine exclusively in dinucleotide CpG or CpNpG stretches. On the the other hand, we determined that cytosine was methylated unusually in dinucleotide GpC at 5-GGCC-3 sequences in the teleost Sparus aurata EcoRI satellite DNA family. This finding is the first example of methylated GpC sequences in the eukaryotic genomes. At this regard, we have examined the relative methylation levels at this site of the highly repetitive EcoRI satellite DNA family from Sparus aurata different tissues. The EcoRI repeat was remarkably more methylated in male germ cells but hypomethylated in female germ cells at the Hae III restriction site ( GpC). The novel modification and the differential methylation pattern suggest that EcoRI satellite could have a structural and/or functional role at the centromeres of Sparus aurata.     

Nucleotide sequence of the lig gene and primary structure of DNA ligase of Escherichia coli

Summary The DNA ligase of Escherichia coli catalyses the NAD-dependent formation of phosphodiester linkages between 5-phosphoryl and 3-hydroxyl groups in DNA. It is essential for DNA replication and repair of damaged DNA strands. We determined the nucleotide sequence of the lig gene of Escherichia coli coding for DNA ligase and flanking regions. The coding frame of the gene was confirmed by the amino acid composition and the amino- and carboxyl-terminal amino acid sequences of the purified ligase. The ligase consists of 671 amino acid residues with a molecular weight of 73,690.On leave from Takara Shuzo Co., Ltd., Kyoto, Japan     

A comprehensive characterization of single-copy T-DNA insertions in the Arabidopsis thaliana genome

T-DNA flanking sequences were isolated from 112 Arabidopsis thaliana single-copy T-DNA lines and sequence mapped to the chromosomes. Even though two T-DNA insertions mapped to a heterochromatic domain located in the pericentromeric region of chromosome I, expression of reporter genes was detected in these transgenic lines. T-DNA insertion did not seem to be biased toward any of Arabidopsis' five chromosomes. The observed distribution of T-DNA copies in intergenic sequence versus gene sequence (i.e. 5-upstream regions, coding sequences and 3-downstream regions) appeared randomly. An evaluation of T-DNA insertion frequencies within gene sequence revealed that integration into 5-upstream regions occurred more frequently than expected, whereas insertions in coding sequences (exons and introns) were found less frequently than expected based on random distribution predictions. In the majority of cases, single-copy T-DNA insertions were associated with small or large rearrangements such as deletions and/or duplications of target site sequences, deletions and/or duplications of T-DNA sequences, and gross chromosomal rearrangements such as translocations. The accuracy of integration was similarly high for both left- and right-border sequences. These results may be called upon when making detailed molecular analyses of transgenic plants or T-DNA induced mutants.     

Isolation of repetitive clones from human muscle cDNA library

Human fetal muscle cDNA library was screened with a-myosin heavy chain gene fragment containing Alu sequences. Two cDNA clones AI and BII with 1.8 and 3 kb inserts respectively were chosen for further characterization by means of RNA and DNA hybridization procedures and sequencing. The clones appeared to contain repetitive sequences as well as single copy regions. They are actively transcribed in different stages of myogenic development but not in the liver. DNA sequence analysis of short stretches from both clones revealed no sequence homology to any other published DNA sequences.     

Aggressive and Foraging Behavioral Interactions Among Ruffe

The ruffe, Gymnocephalus cernuus, is a nonindigenous percid in the Great Lakes. Ruffe are aggressive benthivores and forage over soft substrates. Laboratory studies in pools (100cm diameter, 15cm water depth) were conducted to determine whether fish density (low=2, medium=4, high=6 ruffe per pool) changed foraging and aggressive behaviors with a limited food supply of chironomid larvae. All fish densities demonstrated a hierarchy based on aggressive interactions, but ruffe were most aggressive at low and high fish densities. Time spent in foraging was lowest at the low fish density. The best forager at the low fish density was the most aggressive individual, but the second most aggressive fish at the medium and high fish density was the best forager and also the one chased most frequently. A medium fish density offered the best energetic benefits to ruffe by providing the lowest ratio of time spent in aggression to that spent foraging. Based on our results, ruffe should grow best at an intermediate density. With high ruffe densities, we would also expect disparity in size as the more aggressive fish are able to garner a disproportionate amount of the resources. Alternatively, as the Great Lakes are a fairly open system, ruffe could migrate out of one area to colonize another as populations exceed optimal densities.     

Comparative study ofTriticum aestivum andT. timopheevi genomes using C-banding technique

The somatic chromosomes ofTriticum timopheevi and those of two varieties ofT. aestivum, Chinese Spring and Bezostaya-1, have been identified by a Giemsa staining technique. The data suggest thatT. timopheevi and tetraploid wheats had a common ancestor from which their genomes differentiated due to chromosomal aberrations and the increase of heterochromatin in the chromosomes of theT. timopheevi G-genome. The differences between the chromosomes of the AB and AG genomes result in substitutions and large translocations between these chromosomes in interspecific hybrids.     

The heat shock cognate 80 gene of tomato is flanked by matrix attachment regions

Matrix attachment regions (MARs) are thought to participate in the organization and segregation of independent chromosomal loop domains. Although there are several reports on the action of MARs in the context of heterologous genes, information is more limited on the role of MARs associated with plant genes. Transgenic studies suggest that the upstream, intron and downstream regions of the developmentally regulated heat shock cognate 80 gene (HSC80) of tomato participate in chromatin organization. In this study, we tested the in vitro affinity of the HSC80 gene to chromosomal scaffolds prepared from shoot apices of tomato. We found that a 1.5 kb upstream region and a 1.4 kb downstream region, but not the intron region, are MARs. These MARs interact with tomato and pea scaffolds and bind regardless of the expression status of HSC80 in the tissue from which the nuclei were isolated. Comparison to two known yeast MARs ARS1 and CENIII, showed that the HSC80 5MAR binds more avidly to tomato scaffolds than ARS1, while no binding of CENIII was observed. Competition binding between the two HSC80 MARs indicated that the 5 MAR can outcompete the 3 MAR and not vice versa. Last, we observed that the interaction of the 3 MAR with the scaffold could result in an electrophoretic mobility shift resistant to SDS, protease, and phenol treatment. In conclusion, MARs whose binding properties can be clearly differentiated are closely flanking the HSC80 gene. The discovery of MARs in regions which have a distinct function in HSC80 transgenes but not in transient expression assays, is consistent with a chromosomal scaffold role in HSC80 gene regulation.     

AluElements: Repetitive DNA as Facilitators of Chromosomal Rearrangement

Alu repeats are the most common type of repetitive DNA sequences dispersed throughout the human genome. Technical advances in the field of cytogenetics and molecular biology have facilitated the analysis of epithelial tumors and hematologic malignancies which has led to the observation of Alu elements in and near sites often involved in chromosomal rearrangements. Repair mechanisms of double strand breaks (DSB) such as homol-ogous recombination (HR) may rely on the sequence homology of Alu repeats, potentially leading to chromosomal rearrange-ments. Databases have confirmed the strong association between Alu repeats, specifically the 26 bp consensus sequence and chro-mosomal regions involved in deletions and translocations. Although the Alu repetitive sequence is a potential "hotspot" during homologous recombination, there are other cellular mech-anisms that may play a more prominent role in the initiation of chromosomal rearrangements.     

Satellited chromosomes,systematics and phylogeny inTaraxacum (Asteraceae)

A survey is made of the occurrence, nature and frequency of satellited chromosomes in the agamospermous genusTaraxacum. Species belonging to the 10 sections thought to be most primitive in the genus lack satellited chromosomes. In most other sections, a characteristic satellited chromosome is seen with a large euchromatic region distal to the presumed nucleolar oraniser region (NOR). In sections of a precursor type, there is always one chromosome of this Taraxacum type per haploid genome. In sections thought to be of an advanced type the number of such satellited chromosomes is very unstable, sometimes even within the same tissue. In sectionHamata, two such satellited chromosomes are invariably found in triploids. This finding strongly supports the integrity of this section, suggests that the species of the section are monophyletic, and have evolved from a single ancestor subsequent to the occurrence of obligate agamospermy. In three sections of the genus, satellited chromosomes of the conventional type with a very small distal euchromatic region distal to the NOR are reported for the first time in the genus.     

Highly repetitive DNA sequence in parthenogeneticArtemia

Summary The study of the structural organization of the eukaryotic genome is one of the most important tools for disclosing the evolutionary relationships between species.Artemia (Crustacea, Phyllopoda) offers a very interesting model for speciation studies. The genus, distributed all over the world, comprises both bisexual sibling species and parthenogenetic populations, exhibiting different chromosome numbers (diploidy, polyploidy, and heteroploidy).Digestion of genomic DNA of the parthenogeneticArtemia sp. from Tsing-Tao (China) with the restriction enzymes Eco RI and Alu I reveals that a highly repetitive sequence of 133 bp is present. The Eco RI fragment has been cloned and characterized by genomic organization. The distribution of the Eco RI family of repeats was also studied in several bisexual and parthenogeneticArtemia populations and compared with an Alu I repetitive fragment previously identified inArtemia franciscana.     

Site specific differentiation in metal tolerance in the midge Chironomus riparius (Diptera,Chironomidae)

Metal tolerance in Chironomus riparius (Diptera) populations from contaminated streams was studied by comparing the effects of cadmium, zinc and iron on first generation laboratory reared midges. First instar larvae were exposed for four days, after which surviving larvae were counted and their length measured. Larvae from two highly polluted sites, kept under control conditions, grew substantially slower than those from other populations. All populations showed the same growth responses to increased zinc concentrations, but differences were found in the responses to both cadmium and iron. Since population differentiation was demonstrated in first generation laboratory animals, it is suggested that the differences between populations of C. riparius have a genetic basis.     

Interstitial deletions of repetitive DNA blocks in dicentric human Y chromosomes

Cytogenetic analysis of aberrant human Y chromosomes was done by fluorescence in situ hydbridization (FISH) with Y specific repetitive DNA probes. It revealed an interstitial deletion of different DNA blocks in two dicentric chromosome structures. One deletion includes the total alphoid DNA structure of one centromeric region. The second deletion includes the total repetitive DYZ5 DNA structure in the pericentromeric region of one short Y arm. Both dicentric Y chromosomes were iso(Yp) chromosomes with break and fusion point located in Yq11, the euchromatic part of the long Y arm. Their phenotypic appearance was abnormal, resembling small monocentric Yq-chromosomes in metaphase plates. Mosaic cell lines, usually included in karyotypes with dicentric Y chromosomes, were not observed. It is assumed that both deletion events suppress the kinetochore activity in one Y centromeric region and thus stabilize its dicentric structure. Local interstitial deletion events had not been described in dicentric human Y chromosomes, but are common in dicentric yeast chromosomes. This raises the question of whether deletion events in dicentric human chromosomes are rare or restricted to the Y chromosome or also represent a general possibility for stabilization of a dicentric chromosome structure in human.     

Relationship between cyclic AMP level and accumulation of carotenoid pigments in Neurospora crassa

Dark grown mycelial cells of Neurospora crassa bearing mutant genes crisp-I or frost and having a decreased level of cyclic adenosine 3,5-monophosphate contained more carotenoid pigments than the cells with wild alleles of these genes. A transient decrease of the cyclic AMP occurred following photoinduction of carotenoid synthesis during its lag-period. Its intensity correlated with the increase of carotenoid pigment level due to photoinduction. No correlation in the content of cyclic guanosine 5-phosphate with both constitutive level of carotenoids and its photoinduced increase was observed.     

Comparative cytogenetic variability of polytene chromosomes from salivary glands of Chironomus riparius Mg., 1804 (Diptera, Chironomidae) from two polluted biotopes in Bulgaria and Russia

The structural-functional variation of Chironomus riparius salivary gland polytene chromosomes was studied in two geographically isolated Palearctic regions, Bulgaria (village Pancharevo) and Russia (St. Petersburg). The two biotopes, where larvae were collected, were polluted with various heavy metals from anthropogenic sources. Hereditary paracentric heterozygous inversions were characteristic of the Russian population, whereas somatic paracentric or pericentric heterozygous inversions were more common in the Bulgarian one. All inversions occurred at low frequencies. Other aberrations found in the two populations included somatic deletions resulting in a pompon structure of chromosome IVG, heterozygous translocation between chromosomes IVG and IIIF, enlargement of individual disks, and the appearance of a heterozygous block close to the centromere of chromosome IVG. In addition, changes in functional activity of the nucleolus organizer and Balbiani rings (BRc/BRb) were observed. Several aberration breakpoints proved to coincide with satellites of the Alu and Hinf families.     

Analysis of regions essential for the function of chromosomal replicator sequences from Yarrowia lipolytica

Summary Two DNA segments exhibiting ARS (autonomously replicating sequence) activity in the dimorphic yeast Yarrowia lipolytica were cloned from its chromosome on an integrative LEU2 plasmid. These ARS segments, designated YlARS1 and YlARS2, conferred on the hybrid plasmids high transformation efficiency and enabled extrachromosomal transmission of the plasmids in 1 or 2 copies per yeast cell under selective conditions. Deletion analysis showed that at least 728–1003 by for YlARS1 and 1377–1629 by for YlARS2 were required for full function. Both of these regions contained two 10/11 matches to an ARS core consensus in Saccharomyces cerevisiae, whereas neither was similar to the S. cerevisiae centromere sequence. Significantly, both YlARS elements contained at, or close to, their boundaries a 13 bp sequence, 5-TATATTCAAGCAA-3, which resembles the cleavage site for topoisomerase II in Drosophila. A central 524 by ClaI fragment of YlARS2 contained four stretches of a 17 bp direct repeat sequence, 5-GAAAAACAAAAACAGGC-3, and exhibited the electrophoretic behavior typical of bent DNA.