Telomere-specific non-LTR retrotransposons and telomere maintenance in the silkworm, <Emphasis Type="BoldItalic">Bombyx mori</Emphasis>

Most insects have telomeres that consist of pentanucleotide (TTAGG) telomeric repeats, which are synthesized by telomerase. However, all species in Diptera so far examined and several species in other orders of insect have lost the (TTAGG)_n repeats, suggesting that some of them recruit telomerase-independent telomere maintenance. The silkworm, Bombyx mori, retains the TTAGG motifs in the chromosomal ends but expresses quite a low level of telomerase activity in all stages of various tissues. Just proximal to a 6–8-kb stretch of the TTAGG repeats in B. mori, more than 1000 copies of non-LTR retrotransposons, designated TRAS and SART families, occur among the telomeric repeats and accumulate. TRAS and SART are abundantly transcribed and actively retrotransposed into TTAGG telomeric repeats in a highly sequence-specific manner. They have three possible mechanisms to ensure specific integration into the telomeric repeats. This article focuses on the telomere structure and telomere-specific non-LTR retrotransposons in B. mori and discusses the mechanisms for telomere maintenance in this insect.     

Chromosome pairing in allotetraploid hybrids of <Emphasis Type="Italic">Festuca pratensis</Emphasis> × <Emphasis Type="Italic">Lolium perenne</Emphasis> revealed by genomic <Emphasis Type="Italic">in situ</Emphasis> hybridization (GISH)

Genomic in situ hybridization (GISH) was used to make a detailed study of chromosome pairing at metaphase I (MI) of meiosis in six F(1) hybrid plants of the allotetraploid Festuca pratensis x Lolium perenne (2n = 4x = 28; genomic constitution FpFpLpLp). The mean chromosome configurations for all hybrids analysed were 1.13 univalents + 11.51 bivalents + 0.32 trivalents + 0.72 quadrivalents, and the mean chiasma frequency was 21.96 per cell. GISH showed that pairing was predominantly intragenomic, with mean numbers of L. perenne (Lp/Lp) and F. pratensis (Fp/Fp) bivalents being virtually equal at 5.41 and 5.48 per cell, respectively. Intergenomic pairing between Lolium and Festuca chromosomes was observed in 33.3% of Lp/Fp bivalents (0.62 per cell), in 79.7% of trivalents - Lp/Lp/Fp and Lp/Fp/Fp (0.25 per cell), and in 98.4% of quadrivalents - Lp/Lp/Fp/Fp and Lp/Lp/Lp/Fp (0.71 per cell). About 4.0% of the total chromosome complement analysed remained as univalents, an average 0.68 Lp and 0.45 Fp univalents per cell. It is evident that in these hybrids there is opportunity for recombination to take place between the two component genomes, albeit at a low level, and this is discussed in the context of compromising the stability of Festulolium hybrid cultivars and accounting for the drift in the balance of the genomes over generations. We speculate that genotypic differences between hybrids could permit selection for pairing control, and that preferences for homologous versus homoeologous centromeres in their spindle attachments and movement to the poles at anaphase I could form the basis of a mechanism underlying genome drift.     

Use of cross-species <Emphasis Type="Italic">in-situ</Emphasis> hybridization (ZOO-FISH) to assess chromosome abnormalities in day-6 <Emphasis Type="Italic">in-vivo</Emphasis>- or <Emphasis Type="Italic">in-vitro</Emphasis>-produced sheep embryos

Causes of chromosomal differences such as mosaicism between embryos developed in vivo and in vitro may be resolved using animal models to compare embryos generated in vivo with those generated by different production systems. The aims of this study were: (1) to test a ZOO-FISH approach (using bovine painting probes) to detect abnormal chromosome make-up in the sheep embryo model, and (2) to examine the extent of chromosome deviation in sheep embryos derived in vivo and in vitro. Cytogenetic analysis was performed on day 6 in-vivo and in-vitro derived sheep embryos using commercially available bovine chromosome painting probes for sex chromosomes X–Y and autosomes 1–29. A total of 8631 interphase and metaphase nuclei were analyzed from 49 in-vitro-derived and 51 in-vivo-derived embryos. The extent of deviation from normal ovine chromosome make-up was higher (p < 0.05) in in-vitro-produced embryos relative to in-vivo-derived embryos (65.3% vs. 19.6% respectively) mainly due to diploid–polyploid mosaicism. Polyploid cells ranged from 3n to 8n with tetraploids most predominant among non-diploid cells. The proportions of polyploid cells per mixoploid embryo in in-vitro-produced embryos ranged from 1.4% to 30.3%, in contrast to less than 10% among the in-vivo-derived embryos. It was concluded that in-vitro-derived embryos are vulnerable to ploidy change compared to their in-vivo counterparts. The application of ZOO-FISH to domestic animal embryos is an effective approach to study the chromosome complement of species for which DNA probes are unavailable.     

Chromosome elimination in the interspecific hybrid medaka between <Emphasis Type="Italic">Oryzias latipes</Emphasis> and <Emphasis Type="Italic">O. hubbsi</Emphasis>

An interspecific hybrid medaka (rice fish) between Oryzias latipes and O. hubbsi is embryonically lethal. To gain an insight into the cellular and molecular mechanisms that cause the abnormalities occurring in the hybrid medaka, we investigated the behavior of chromosomes and the expression patterns of proteins responsible for the chromosome behavior. The number of chromosomes in the hybrid embryos gradually decreased to nearly half, since abnormal cell division with lagging chromosomes at anaphase eliminated the chromosomes from the cells. The chromosome lagging occurred at the first cleavage and continued throughout embryogenesis even after the midblastula transition. Fluorescent in-situ hybridization analyses revealed that the chromosomes derived from O. hubbsi are preferentially eliminated in both O. latipes–hubbsi and O. hubbsi–latipes embryos. Whole-mount immunocytochemical analyses using antibodies against α-tubulin, γ-tubulin, inner centromere protein, Cdc20, Mad2, phospho-histone H3 and cohesin subunits (SMC1α, SMC3 and Rad21) showed that the expression patterns of these proteins in the hybrid embryos are similar to those in the wild-type embryos, except for phospho-histone H3. Phospho-histone H3 present on chromosomes at metaphase was lost from normally separated chromosomes at anaphase, whereas it still existed on lagging chromosomes at anaphase, indicating that the lagging chromosomes remain in the metaphase state even when the cell has proceeded to the anaphase state. On the basis of these findings, we discuss the cellular and molecular mechanisms of chromosome elimination in the hybrid medaka.     

Cytogenetics and genome analysis in <Emphasis Type="Italic">Brassica</Emphasis> crops

The genus Brassica contains a wide range of diploid and amphipolyploid species including some of the most important vegetable, condiment and oilseed crops worldwide. As members of the Brassicaceae family the brassicas are the closest crop relatives to the model plant Arabidopsis thaliana, and hence are major beneficiaries from the vast array of Arabidopsis molecular genetic and genomic tools and the increasingly good annotation to major Brassica crop genomes. In this review examples are shown from recent studies that demonstrate the potential for intergenome navigation from model to crop plant and for comparisons among genetic and cytogenetic maps between the model and crop species and among different crop brassicas. The use of interspecific and intergeneric hybridization for introgression of novel traits into Brassica genomes from the secondary and tertiary crucifer genepools is described. In this context the use of the Brassica triangle of three diploid species and their corresponding amphiploids as an excellent model system for studying the mechanisms and control of homeologous recombination and polyploidization is discussed from a crop breeding perspective.     

A frameshift mutation of the chloroplast <Emphasis Type="Italic">mat</Emphasis>K coding region is associated with chlorophyll deficiency in the <Emphasis Type="Italic">Cryptomeria japonica</Emphasis> virescent mutant <Emphasis Type="Italic">Wogon</Emphasis>-Sugi

Wogon-Sugi has been reported as a cytoplasmically inherited virescent mutant selected from a horticultural variety of Cryptomeria japonica. Although previous studies of plastid structure and inheritance indicated that at least some mutations are encoded by the chloroplast genome, the causative gene responsible for the primary chlorophyll deficiency in Wogon-Sugi, has not been identified. In this study, we identified this gene by genomic sequencing of chloroplast DNA and genetic analysis. Chloroplast DNA sequencing of 16 wild-type and 16 Wogon-Sugi plants showed a 19-bp insertional sequence in the matK coding region in the Wogon-Sugi. This insertion disrupted the matK reading frame. Although an indel mutation in the ycf1 and ycf2 coding region was detected in Wogon-Sugi, sequence variations similar to that of Wogon-Sugi were also detected in several wild-type lines, and they maintained the reading frame. Genetic analysis of the 19 bp insertional mutation in the matK coding region showed that it was found only in the chlorophyll-deficient sector of 125 full-sibling seedlings. Therefore, the 19-bp insertion in the matK coding region is the most likely candidate at present for a mutation underlying the Wogon-Sugi phenotype. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">Trichomonas vaginalis</Emphasis> harboring <Emphasis Type="Italic">Mycoplasma hominis</Emphasis> increases cytopathogenicity in vitro

The parasite Trichomonas vaginalis causes one of the most common non-viral sexually transmitted infections in humans. Mycoplasmas are frequently found with trichomonads but the consequences of this association are not yet known. In the present study, the effects of T. vaginalis harboring M. hominis on human vaginal epithelial cells and on MDCK cells are described. The results were analyzed by light, scanning and transmission electron microscopy, as well as using cell viability assays. There was an increase in the cytopathic effects on the epithelial cells infected with T. vaginalis associated with M. hominis compared to T. vaginalis alone. The epithelial cells exhibited an increase in the intercellular spaces, a lesser viability, and increased destruction provoked by the infected T. vaginalis. In addition, the trichomonads presented a higher amoeboid transformation rate and an intense phagocytic activity, characteristics of higher virulence behavior.     

Evaluation of hematological values in indigenous chickens infected with <Emphasis Type="Italic">Plasmodium gallinaceum</Emphasis> and <Emphasis Type="Italic">Aegyptianella pullorum</Emphasis>

Plasmodium, Haemoproteus, and leukocytozoon are the most important hematozoa in birds, which have been reported in different areas of the world. The present study was undertaken to find which blood protozoans exist in indigenous chickens in Shiraz, southern Iran and to evaluate hematological parameters in birds infected with hematozoas. Plasmodium and Aegyptianella were the two parasites found in 740 blood samples examined from indigenous chickens of which 29 (3.91%) were positive for Aegyptianella pullorum, 106 (14.32%) for Plasmodium gallinaceum, and 12 (1.62%) for A. pullorum and Plasmodium gallinaceum together. There was no significant difference between hematological parameters of non-infected and naturally infected chickens with Plasmodium gallinaceum, A. pullorum, and both (P > 0.05). Low infection of indigenous chickens with A. pullorum, Plasmodium gallinaceum, and both had no significant effects on hematological parameters (P > 0.05), which is probably due to low parasitemia rate and immunity against these two parasites.     

C-<Emphasis Type="Italic">erb</Emphasis>B-2 oncogene and <Emphasis Type="Italic">P21</Emphasis><Superscript><Emphasis Type="Italic">WAF/CIP1</Emphasis></Superscript> tumor suppressor gene expression as prognostic factors in canine mammary adenocarcinomas

To evaluate the use of c-erbB-2 oncogene and p21 ^WAF1/CIP1 suppressor gene products as the prognosis markers for canine mammary tumors, expression of these gene products were examined immunohistochemically using tumor tissues and clinical data from 96 dogs with malignant mammary tumors. Semiquantitative data was compared with histopathological grades, proliferating cell nuclear antigen (PCNA)-positive index, and clinicopathological matters. The expression c-erbB-2 protein was found in the cellular membrane and cytoplasm of neoplastic epithelial cells, and the positive index had no significant relation to the histopathological features and PCNA-positive index, except for the individual age of affected dogs (P < 0.05). The product of p21 ^WAF1/CIP1 was mostly found in cytoplasm and occasionally in the nucleus of neoplastic cells. The quantitative data had significant association to the malignancy grade and size of tumors (P < 0.05). However, that had no significant relationship to the PCNA-positive index. The present study concluded that both gene products could not apply as the direct markers to evaluate the prognosis of canine mammary tumors. The detection of c-erbB-2 product may be partly beneficial to the differential diagnosis of epithelial type of mammary cancer. The use of p21 ^WAF1/CIP1 product in prognosis of canine mammary cancer needs further investigation.     

Infestation of sand lizards (<Emphasis Type="Italic">Lacerta agilis</Emphasis>) resident in the Northeastern Poland by <Emphasis Type="Italic">Ixodes ricinus</Emphasis> (L.) ticks and their infection with <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> sensu lato

Sand lizards (Lacerta agilis) were trapped and examined for ticks from May to September in 2002 and 2003 in Northeastern Poland. A total of 233 Ixodes ricinus (L.) ticks (76 larvae and 157 nymphs) was found on 31 of 235 captured lizards (13.2%). The tick infestation is relatively low compared to that of mammals and passerine birds from the same area (Siński et al. 2006, Gryczyńska et al. 2002). Tick infestation depended on the month of capture, being the highest in spring. In autumn no ticks were recorded on any of the captured lizards. The oldest lizards carried the highest number of ticks but no differences related to sex of the host were found. All the collected ticks were analysed by PCR for the presence of Borrelia burgdorferi sensu lato, the etiological agents of Lyme disease. Spirochetes were detected in 11 out of 233 (4.7%) ticks tested. Genetic analysis confirmed that the spirochetes are members of the Borrelia afzelii, B. garinii and B. burgdorferi sensu stricto genospecies. Mixed infection were not detected. The prevalence of infection was analysed in relation to months of the capture, age and sex of the lizards, but differences were not statistically significant. The obtained results suggest that lizards are probably not B. burgdorferi reservoirs, but further studies are required to confirm this.     

Sex determination in platypus and echidna: autosomal location of <Emphasis Type="Italic">SOX3</Emphasis> confirms the absence of <Emphasis Type="Italic">SRY</Emphasis> from monotremes

In eutherian ('placental') mammals, sex is determined by the presence or absence of the Y chromosome-borne gene SRY, which triggers testis determination. Marsupials also have a Y-borne SRY gene, implying that this mechanism is ancestral to therians, the SRY gene having diverged from its X-borne homologue SOX3 at least 180 million years ago. The rare exceptions have clearly lost and replaced the SRY mechanism recently. Other vertebrate classes have a variety of sex-determining mechanisms, but none shares the therian SRY-driven XX female:XY male system. In monotreme mammals (platypus and echidna), which branched from the therian lineage 210 million years ago, no orthologue of SRY has been found. In this study we show that its partner SOX3 is autosomal in platypus and echidna, mapping among human X chromosome orthologues to platypus chromosome 6, and to the homologous chromosome 16 in echidna. The autosomal localization of SOX3 in monotreme mammals, as well as non-mammal vertebrates, implies that SRY is absent in Prototheria and evolved later in the therian lineage 210-180 million years ago. Sex determination in platypus and echidna must therefore depend on another male-determining gene(s) on the Y chromosomes, or on the different dosage of a gene(s) on the X chromosomes.     

Histopathologic and electron microscopic characterization of cutaneous leishmaniasis in <Emphasis Type="Italic">Tatera indica</Emphasis> and <Emphasis Type="Italic">Gerbillus</Emphasis> spp. infected with <Emphasis Type="Italic">Leishmania major</Emphasis>

Cutaneous leishmaniasis (CL) is a zoonotic disease transmitted between rodents and canines, mainly by Phlebotomus sand flies and man. In southern Iran, the incidence of this protozoan disease has doubled over the last decade. The present study deals with histopathological features of CL in Tatera indica and Gerbillus spp. that participate in the epidemiology of CL in southern Iran. Thirty-two trapped rodents were evaluated for any Leishmania infection using enzyme electrophoresis and polymerase chain reaction and were concomitantly studied for any histopathological changes. Histopathological studies showed that bone marrow was the tissue of choice for light and electron microscopic study of Leishmania, demonstrating the macrophages infected with the amastigote form of the parasite. This is the first report of the histopathological detection of L. major in naturally infected T. indica and Gerbillus spp in the Larestan region.     

Characterisation of <Emphasis Type="Italic">Zygosaccharomyces rouxii</Emphasis> centromeres and construction of first <Emphasis Type="Italic">Z. rouxii</Emphasis> centromeric vectors

Zygosaccharomyces rouxii is a hemiascomycetous yeast known for its high osmotolerance, the basis of which still remains unknown. By exploring the Génolevures I database, four Z. rouxii fragments homologous to Saccharomyces cerevisiae centromeres were identified. Two of them were subjected to further analysis. Their function as centromeres in Z. rouxii was proved, and they were localized to Z. rouxii chromosomes II and VII, respectively. The species-specificity of centromeres was observed; plasmids with a Z. rouxii centromere were not recognized as centromeric in S. cerevisiae, and a S. cerevisiae centromere did not function as a centromere in Z. rouxii. Constructed plasmids bearing Z. rouxii centromeres serve as the first specific centromeric plasmids, and thus contribute to the so-far limited set of genetic tools needed to study the Z. rouxii specific features.     

Stimulation of biofilm formation by insertion of <Emphasis Type="Italic">Tetrahymena pyriformis</Emphasis> wells within <Emphasis Type="Italic">Burkholderia cenocepacia</Emphasis> biofilms

Biofilm formation is an important part of the bacterial life cycle. Biofilms provide bacterial resistance to external stresses and protozoan grazing. Biofilm formation by the wild type of B. cenocepacia strain 370 in the presence of the free-living ciliate Tetrahymena pyriformis was studied. T. pyriformis grazed on planktonic bacteria and reduced the planktonic bacterial subpopulation while it noticeably stimulated biofilm formation. When cultivated alone, T. pyriformis did not form visible biofilms. Confocal laser scanning microscopy was used to demonstrate the inclusion and further destruction of protozoan cells within the biofilms formed by the bacteria. The destruction of protozoan cells was accompanied by the exit of bacteria from vacuoles and intracytoplasmic multiplication; changes in the form of protozoan cells; the demolition of internal structures; and the visual exit of the cytoplasmic content from destructing cells. Microcolonies of a characteristic round shape were revealed in the biofilms formed by B. cenocepacia in the presence of T. pyriformis. These structures were absent in the biofilms formed by B. cenocepacia alone. Insertion of protozoan cells within biofilms seems to be a driving force that promotes biofilm proliferation and influences their structure. The mortality of protozoan cells in the biofilms caused a decrease in the T. pyriformis population under conditions advantageous to B. cenocepacia biofilm formation. The mutant B. cenocepacia strain Bcb-1, which is unable to form biofilms, was isolated by plasposon mutagenesis. In contrast to the parental strain, the cocultivation with Bcb-1 bacteria improved the growth of T. pyriformis. A mutation was mapped in the ompR gene. The text was submitted by the authors in English.     

Integrated cytogenetic BAC map of the genome of the gray,short-tailed opossum, <Emphasis Type="Italic">Monodelphis domestica</Emphasis>

The generation of high-quality genome assemblies for numerous species is advancing at a rapid pace. As the number of genome assemblies increases, so does our ability to investigate genome relationships and their contributions to unraveling complex biological, evolutionary, and biomedical processes. A key process in the generation of a genome assembly is to determine and verify the precise physical location and order of the large sequence blocks (scaffolds) that result from the assembly. For organisms of relatively recent common ancestry this process may be achieved largely through comparative sequence alignment. However, as the evolutionary distance between species lengthens, the use of comparative sequence alignment becomes increasingly less reliable. Simultaneous cytogenetic mapping, using multicolor fluorescence in-situ hybridization (FISH) analysis, offers an alternative means to define the cytogenetic location and relative order of DNA sequences, thereby anchoring the genome sequence to the karyotype. In this article we report the molecular cytogenetic locations of 415 bacterial artificial chromosome (BAC) clones that served to anchor sequence scaffolds of the gray, short-tailed opossum (Monodelphis domestica) to its karyotype, which enabled accurate integration of these regions into the genome assembly.     

Description of a new species of <Emphasis Type="Italic">Tylodelphys</Emphasis> (Digenea,Diplostomidae) in the wood stork, <Emphasis Type="Italic">Mycteria americana</Emphasis> (Aves,Ciconiidae) from Argentina

During the course of a study on the endohelminth parasites of birds, specimens of an undescribed species of Tylodelphys Diesing, 1850 (Diplostomidae) were collected from the wood stork, Mycteria americana L., from Formosa Province, Argentina. Tylodelphys brevis sp. nov. can be distinguished from the other Neotropical species of this genus, T. elongata, T. americana and T. adulta, principally by the smaller size of the body (570–851 μm), by the fewer eggs in the uterus (1–2) and by the smaller ratio of body to egg length (6–8). This is the second report of an adult of the genus Tylodelphys from Argentina and the first record of a digenean species parasitizing Mycteria americana in this country.     

Redescription of <Emphasis Type="Italic">Decorataria decorata</Emphasis> (Spirurida,Acuariidae) based on nematodes from <Emphasis Type="Italic">Podiceps cristatus</Emphasis> and <Emphasis Type="Italic">P. grisegena</Emphasis> (Aves,Podicipediformes) from Bulgaria

Decorataria decorata (Cram, 1927) is redescribed on the basis of light-microscopy and SEM observations on specimens collected from the stomach of Podiceps cristatus and P. grisegena from Bulgaria. The SEM study revealed the presence of a porebearing field on each pseudolabium and a pair of spines (one dorsal and one ventral) situated between bases of the cordons. The deirids are spine-like and minute. The light-microscopy examination showed the presence of ornamentation situated under the dorsal surface of caudal alae. The occurrence of D. decorata in Bulgaria is a new geographical record.     

Molecular and cytogenetic characterization of a durum wheat–<Emphasis Type="Italic">Aegilops speltoides</Emphasis> chromosome translocation conferring resistance to stem rust

Stem rust is a serious disease of wheat that has caused historical epidemics, but it has not been a threat in recent decades in North America owing to the eradication of the alternative host and deployment of resistant cultivars. However, the recent emergence of Ug99 (or race TTKS) poses a threat to global wheat production because most currently grown wheat varieties are susceptible. In this study, we evaluated a durum wheat–Aegilops speltoides chromosome translocation line (DAS15) for reaction to Ug99 and six other races of stem rust, and used molecular and cytogenetic tools to characterize the translocation. DAS15 was resistant to all seven races of stem rust. Two durum–Ae. speltoides translocated chromosomes were detected in DAS15. One translocation involved the short arm, centromere, and a major portion of the long arm of Ae. speltoides chromosome 2S and a small terminal segment from durum chromosome arm 2BL. Thus, this translocated chromosome is designated T2BL-2SL•2SS. Cytogenetic mapping assigned the resistance gene(s) in DAS15 to the Ae. speltoides segment in T2BL-2SL•2SS. The Ae. speltoides segment in the other translocated chromosome did not harbour stem rust resistance. A comparison of DAS15 and the wheat stocks carrying the Ae. speltoides-derived resistance genes Sr32 and Sr39 indicated that stem rust resistance gene present in DAS15 is likely novel and will be useful for developing germplasm with resistance to Ug99. Efforts to reduce Ae. speltoides chromatin in T2BL-2SL•2SS are currently in progress. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.