Identification and phylogenetic analysis of novel human endogenous retroviral sequences belonging to the HERV-H family on human X and Y chromosomes

HERV-H, a family of endogenous retroviral elements that has undergone successive expansions in the human genome, includes sequences that are expressed in placenta and T cells. With a PCR approach to the HERV-H using human monochromosomal somatic cell hybrid DNA, we identified 8 new HERV-H sequences on the X chromosome, and one novel HERV-H element, HY-1, the first reported such element on the Y chromosome, and compared these with sequences in the nucleotide sequence database. Phylogenetic analysis indicated that clone HX-1 and BAC clone 523A23 on the X chromosome were found to be in close relationship to the sequences of DJ088A21 on the human chromosome 7q31. This finding allows us to speculate that HERV-H elements may have evolved by intra-chromosomal spread. Our data may relevant to an understanding of human genomic plasticity.     

Repetitive DNA, Genome and Species Relationships in Avena and Arrhenatherum(Poaceae)

Repetitive sequences have been widely used for examining genomeand species relationships by in situ and Southern hybridization.In the present study, double-stranded DNA sequences, from denaturedDNA reannealed to Cot = 1, from Avena strigosa(2 n = 2x = 14;A genome; referred to as CotA) and Avena sativa(2n = 6 x =42; ACD genome; referred to as CotACD) were isolated with ahydroxyapatite column, and were used for in situ hybridizationon hexaploid A. sativa chromosomes. Probe CotACD labelled allchromosomes evenly throughout their length at the same intensity.Probe CotA labelled the 28 A and D genome chromosomes stronglyand the 14 C genome chromosomes weakly. Three cloned repetitivesequences, pAvKB9 (126 bp), pAvKB26 (223 bp) and pAvKB32 (721bp) were characterized in the A, B, C and D Avena genomes andthe genus Arrhenatherum using molecular and cytological methods.Clones pAvKB9 and pAvKB26 were absent from the Avena C genome,while both could identify the presence of the D genome by Southernhybridization. In situ hybridization to diploid and tetraploidAvena species revealed that the probes showed a dispersed genomicorganization and that they are present on both arms of all chromosomes.These sequences were excluded from areas where tandem repeats,such as rRNA genes and telomeres, are present. These resultsindicate the close relationship between A and D genomes andthe presence of common DNA sequences between A and C Avena genomes.All three clones hybridized to Southern blots containingArrhenatherumdigested genomic DNA, indicating Arrhenatherum’s closeaffinity to A, B and D Avena genomes. Copyright 2000 Annalsof Botany Company Cereals, DNA, hydroxyapatite, in situ hybridization, oats, reassociation kinetics, repetitive DNA     

Identification of the Genomic Constitution ofMusaL. Lines (Bananas, Plantains and Hybrids) Using Molecular Cytogenetics

The genomic constitutions of someMusaL. lines (bananas, plantainsand artificial hybrids) were identified using molecular cytogenetictechniques. Double targetin situDNA:DNA hybridization to chromosomespreads using as probes, total genomic DNA isolated from diploidMusalinesof known AA (labelled with biotin-11-dUTP) and BB (labelledwith digoxigenin-11-dUTP) genome constitution was carried out.The use of 60% acetic acid combined with heating over a flamegave high quality chromosome spreads free of cytoplasm forinsituhybridization. Total genomic A DNA labelled broad centromericregions of all 22 chromosomes of the diploid line, Calcutta4 (M. acuminataColla. ssp.burmanniccoides; A genome) with somechromosomes showing stronger hybridization. Labelled DNA fromthe B genome hybridized strongly to the centromeric regionsof all 22 chromosomes of Butohan 2 (M. balbisianaColla; B genome).The two satellited chromosomes of genome B labelled stronglywith genomic A DNA.In situhybridization of labelled A and Bgenomic DNA to metaphase chromosomes of triploid AAB and ABBcultivars discriminated between A and B genome chromosomes.The plantains Agbagba, Obino l'Ewai and Mbi Egome showed 22genome A and 11 genome B chromosomes while the cooking bananasBluggoe and Fougamou showed 11 genome A and 22 genome B chromosomes.Hybridization of labelled A and B genomic DNA to chromosomesof the hybrids showed that TMP2x 2829-62 has all 22 genome Achromosomes while TMPx 4698-1 has 33 genome A and 11 genomeB chromosomes.In situhybridization of labelled total genomicDNA to chromosomes has immense potential for identificationof chromosome origin and can be used to characterize cultivarsand hybrids produced inMusabreeding.Copyright 1997 Annals ofBotany Company Genomicin situhybridization; banana; plantain; hybrids; plant breeding; genome organization; biodiversity     

Endogenous retrovirus sequences as a novel class of tumor-specific antigens: an example of HERV-H env encoding strong CTL epitopes

Our genome consists to about 8% of human endogenous retroviral (HERV) sequences. These HERVs have been discussed to be linked to human diseases for decades. Recently, a detailed analysis of a HERV-H sequence located on chromosome Xp22.3 revealed a strong expression in a subset of gastrointestinal cancers whereas expression in normal tissues and in other cancer entities was low. In the present study, we used the reverse immunology approach to test the immunological potential of this HERV-H ORF on Xp22.3. A total of ten peptides displaying HLA-A2.1-binding motifs were selected from the predicted env protein sequence. Stimulation of peripheral T cells with retroviral peptides (RVPs) presented by autologous antigen-presenting cells clearly resulted in sustained proliferation of predominantly CD8(+) T cells. High numbers of IFN-γ-secreting T cells were detectable after several weekly stimulations with RVP mixes. Reactivity observed in RVP-Mix-stimulated cultures was attributable to RVP03, RVP09 and to a lower extend to RVP08, suggesting those to be highly immunogenic epitopes. Besides killing of RVP-loaded target cells, up to 40% specific lysis of colorectal carcinoma cell lines endogenously expressing this HERV-H Xp22.3 ORF was achieved. These data demonstrate that human T cells can be sensitized toward HERV peptides and moreover posses a high lytic potential toward HERV-H expressing CRC cells. Additionally, these data hint toward endogenous ENV protein expression followed by proteasomal degradation and presentation in the context of HLA molecules. Finally, our data strengthen the view that HERV-encoded sequences should be considered as a new class of tumor-specific antigens.     

The Close Relationship Between the A and B Genomes in Avena L. (Poaceae) Determined by Molecular Cytogenetic Analysis of Total Genomic, Tandemly and Dispersed Repetitive DNA Sequences

The genusAvena L. (Poaceae) consists of diploid, tetraploid,and hexaploid species, with the B genome known only in tetraploidspecies and the D genome in the hexaploid species. DNA:DNAinsitu hybridization, using total genomic DNA from diploidAvenastrigosa Schreb. (A_sgenome) as a probe, labelled all 28 chromosomesof the AB tetraploidAvena vaviloviana (Malz.) Mordv. stronglyand uniformly, revealing the close relationship between thesetwo genomes. Comparison of patterns of size-separated DNA restrictionfragments between the diploidA. strigosa and the tetraploidA.vaviloviana , using 32 different restriction enzymes, revealedno differences. Southern hybridization using total AB genomicDNA as a probe also gave no differences in banding patternsbetween the two genomes, even when a large excess of A genomicDNA was used as a block. From anA. vaviloviana genomic library,1800 colonies were blotted and probed sequentially with A andAB genomic DNA, but no colony was identified to be B genomespecific. DNA digests of AB genome tetraploids with restrictionenzymeHae III gave a strong band at 4.2 kb. Clone pAbKB3, derivedfrom the 4.2 kb band, was found to be part of aTy1-copia -likeretrotransposon present in A and B genome chromosomes. ClonedrRNA genes were used forin situ hybridization and showed thatdiploidA. strigosa has four major sites for 18S-25S rDNA andtwo pairs of sites for 5S rDNA (pairs on the same satellitedchromosome, on different chromosome arms), while 4xA. vavilovianahas eight major sites for 18S-25S rDNA and four pairs of sitesfor 5S rDNA (pairs on the same satellited chromosome, on differentchromosome arms). A repetitive sequence from rye pSc119.2, showeddispersed hybridization, while the telomeric sequence in clonepLT11 hybridized to telomeres. Again no discrimination was possiblebetween A and B genome chromosomes. The molecular similaritiesbetween the diploidA. strigosa and thebarbata group tetraploidsclearly indicate that thebarbata group of tetraploids arosefrom A_sdiploids through autotetraploidization. Avena ; evolution; repetitive sequences; in situ hybridization; retrotransposons; genome organization     

Hybrid Identification in Clivia(Amaryllidaceae) using Chromosome Banding and Genomic In Situ Hybridization

Giemsa C-banding and genomic in situ hybridization (GISH) wereused to identify parental genomes in hybrids of Clivia(Amaryllidaceae).Of the three groups reputed to be hybrids, onlyC. cyrtanthiflorawas shown to be of hybrid origin. The ‘German hybrids’and ‘Belgian hybrids’ were both shown to be karyotypicallyand genomically similar to C. miniata, and are either selectionsor intraspecific hybrids of that species. Successful genomedifferentiation in F₁hybrids by GISH required high stringencyand high ratios of blocking DNA to probe. The spatial dispositionof different genomes with C-band or GISH markers in the hybridswas investigated in two dimensions on the spread. In five artificiallyproduced hybrids, either C-banding or GISH was used to locatethe position of parental genomes in mitotic metaphase cells.In all cases there was a significant tendency for centromeresof the different parental genomes to occupy two distinct concentricdomains on the metaphase plate. The presence or absence of centromericheterochromatin was not correlated with genome disposition.Results show that chromosome analyses can be a useful way ofidentifying Clivia hybrids in their vegetative phase. Copyright2001 Annals of Botany Company Clivia, genomic in situ hybridization, cultivar origin, parental genome separation     

Genomic Origin and Organization of the Hybrid Poa jemtlandica(Poaceae) Verified by Genomic In Situ Hybridization and Chloroplast DNA Sequences

Chloroplast DNA sequencing and genomic in situ hybridization(GISH) were used to investigate the genomic origin and organizationof the alpine grass Poa jemtlandica. Using genomic probes ofP. alpina and P. flexuosa, GISH clearly distinguished betweenthese two putative parental genomes and thus confirmed the hybridnature of P. jemtlandica. The chloroplast trn L intron and trnL–trn F intergenic spacer (IGS) sequence genotypes ofP. flexuosa and P. jemtlandica were 100% identical but differedfrom those of P. alpina by a total of ten or 11 nucleotide substitutionsand six indels over 866 aligned positions, identifying P. flexuosaas the maternal parent of the P. jemtlandica population studiedhere and supporting a relatively recent origin of the hybrid.GISH revealed the presence of intergenomic translocations inthe hybrid genome, indicating that the two parental genomeshave undergone some rearrangements following hybridization.It is likely that some of these chromosome changes took placesoon after hybridization in order to overcome the adverse interactionsbetween the nuclear and the cytoplasmic genomes and to facilitatethe successful establishment of the newly formed hybrid. Thepresence of intergenomic chromosome changes may play an importantrole in the evolution of natural hybrids and the establishmentof new evolutionary lineages. Copyright 2000 Annals of BotanyCompany Natural hybridization, genome origin, intergenomic translocations, GISH, chloroplast DNA sequences, Poa jemtlandica     

Molecular Cytogenetics of an Amphiploid Trigeneric Hybrid between Triticum durum, Thinopyrum distichum and Lophopyrum elongatum

In situ hybridization of total genomic DNA was used to analyselines derived from an amphiploid between tetraploid wheat,Triticumdurum Desf. (2n =4x =28), and the wheatgrassesThinopyrum distichum(Thunb.) A. Löve (2n =4x =28) andLophopyrum elongatum (Host)A. Löve (2n =2x =14). A range of chromosome numbers wasdetected, arising from loss or gain of chromosomes. Total genomicDNA probes fromThinopyrum species,L. elongatum andTriticum monococcumL. were able to discriminate chromosomes from the A and B genomesof tetraploid wheat and those of wheatgrass-origin. The methoddid not discriminate the two wheatgrass genomes, J and E, indicatingtheir close similarity. Chromosomal aberrations—includingtelocentric and ring chromosomes—were frequent. Distalinter-genomic translocations of parts of A and B genome chromosomearms, unusual in wheat itself, were more frequent than translocationsbetweenT. durum and wheatgrass.In situ hybridization of an rDNAprobe most frequently revealed four sites associated with secondaryconstrictions onT. durum chromosomes and four onTh. distichumorL. elongatum chromosomes, although there was variation inthe number of loci between and within plants. Within interphaseand prophase nuclei, the three genomes were not intermixed andoften lay in distinct sectors. Wheat; hybrids; Triticum ; Triticeae; evolution; introgression; nuclear architecture; rDNA; in situ hybridization     

Chromosomal Variation in Crocus vernus Hill (Iridaceae) Investigated by in situ Hybridization of rDNA and a Tandemly Repeated Sequence

The physical localization of three tandemly-organized repetitiveDNA sequences was investigated byin situ hybridization to metaphasechromosomes of 11 Crocus vernus accessions. The sequences includedwere the 18S–25S rDNA, the 5S rDNA and a tandemly-repeatedsequence cloned from C. vernus(clone pCvKB8). Ten 2n = 8 karyotypesfrom accessions ranging across the Alps and the Pyrenees couldbe interpreted as variations of a standard karyotype. Polymorphismswere found involving size of the satellite chromosomes, extra5S rDNA sites, and extensive differences in size and numberof pCvKB8 loci. The 2 n = 16 type did not correspond to anypossible tetraploid derived from the 2 n = 8 types. Copyright2000 Annals of Botany Company Evolution, phylogeny, Crocus vernus Hill (Iridaceae), in situ hybridization, chromosomal polymorphism, karyotype evolution, repetitive DNA     

Investigations of Genome Relationships Between Leymus, Psathyrostachys and Hordeum Inferred by Genomic DNA:DNA in situ Hybridization

Genome relationships between the genera Leymus Hochst., PsathyrostachysNevski and Hordeum L. (Poaceae, Triticeae) were investigatedby fluorescent in situ hybridization using both total genomicDNA and cloned DNA sequences as probes. In hybrids between speciesof Hordeum and Leymus there was a clear differentiation betweenthe H genomes of Hordeum species and the genomes of Leymus speciesafter probing with genomic Hordeum or Leymus DNA. Chromosomesof species of Leymus and Psathyrostachys were also differentiatedby subtelomeric heterochromatic segments or by negative bandsalong their length. The number and location of 18S-5·8S-26SrRNA genes varied between the investigated genera. Unusually,L. angustus and P. stoloniformis rDNA sites were localized onboth ends of some chromosomes. Interphase nuclei of the Hordeumx Leymus hybrids had groups of chromosomes from both parentalgenomes in discrete, non-intermixed domains.Copyright 1994,1999 Academic Press Taxonomy, evolution, molecular evolution, repetitive DNA, rDNA sites, in situ hybridization, Triticeae, Leymus, Hordeum, Psathyrostachys     

Cloning and Characterization of New Repetitive Sequences in Field Bean (Vicia fabaL.)

Five new repetitive sequences have been isolated from theViciafabagenome, by cloning bands visible on agarose gel electrophoresisafter digestion of genomic DNA with various restriction enzymes.The sequences were 109 to 584 bp long, their abundance rangingfrom 5x10⁴to 5x10⁵copies per haploid genome. Southern blot andinsituhybridization revealed that four of five newly isolatedrepeats were dispersed in theV. fabagenome. One of the repeats(TIII15) showed tandem organization with several major hybridizationspots on mitotic chromosomesin situ.These sites were distributedin euchromatic as well as in heterochromatic chromosomal regions,and in several loci they were simultaneously localized withpreviously describedFokI repeated elements. The sequence ofTIII15 comprises four 26–27 bp subrepeats, but sharesno homology toFokI elements which have similar sequence organization.All newly described sequences were highly specific forV. faba,withlittle or no hybridization to DNA of otherViciaspecies, andno hybridization to DNA of other legumes tested.Copyright 1999Annals of Botany Company Vicia faba, field bean, repeated DNA sequences, FISH, PRINS, genome organization, copy number.     

In Situ Localization of Parental Genomes in a Wide Hybrid

In situ hybridization enabled DNA originating from the two parentalgenomes to be distinguished in plant hybrids. A probe of biotinylatedtotal genomic DNA from Secale africanum labelled the chromosomesof S. africanum origin but not those from Hordeum chilense inroot-tip chromosome spreads of the sexual hybrid between thetwo species. Hybridization of total genomic DNA from S. africanumto DNA on filters (dot blots) confirmed the distinction betweenDNA from Hordeum and Secale. The total genomic probe hybridizedto the whole length of the chromosomes from S. africanum remarkablyuniformly, labelling both euchromatin and heterochromatin, exceptat the centromeric region. The probe binding was visualizedas a yellow colour by the fluorescein-coupled detection systemwhich contrasted with the red fluorescing counterstain of theunlabelled chromatin. The chromosomes originating from bothparents could be seen and distinguished as red and yellow fluorescenceat all stages of the cell cycle. At interphase and prophase,the chromatin originating from the two parental genomes didnot mix. Chromosomes or groups of chromosomes occupied distinctdomains and also tended to be arranged in a Rabl configurationwith the centromeres clustered at one end of the nucleus. Wepropose calling the technique using total genomic DNA as a probe‘genomic in situ hybridization.’ Hordeum chilense, Secale africanum, hybrids, genomic in situ hybridization, DNA, repetitive sequences, chromosomes, chromosome disposition, nuclear order     

Comparison of Plant Telomere Locations using a PCR-generated Synthetic Probe

We have generated a telomere-specific probe by the polymerasechain reaction and used it to localize chromosome telomeresof ten unrelated angiosperm species in in situ. Concatenationof the simple monomers, 5'-(TTTAGGG)-3', derived from the sequenceof Arabidopsis thaliana telomeres, yielded a stable, versatileand reliable probe that gave a signal of high intensity followingfluorescence in situ hybridization. Most species, includingthose with known karyotype rearrangements, showed telomere labelonly at chromosome termini. These findings are discussed inthe context of the chromosomal events responsible for generatingand stabilizing karyotype change in plants.Copyright 1993, 1999Academic Press PCR, telomere repeat sequence (TRS), in situ hybridization, comparative physical mapping, karyotype evolution, genome organization, Lathyrus sativus L., Vicia sativa L. subsp. nigra (L.) Ehrh., Vigna radiata (L.) R. Wilczek cv. Berken, Nicotiana sylvestris Speg et Comes, Haplopappus gracilis (Nutt.) A. Gray, Gibasis pulchella (Kunth) Rafin., Tradescantia commelinoides Schultes. fil., Hordeum vulgare L. cv. Sultan., Hordeum vulgare L. cv. Tuleen 346, Milium vernale Bieb., Paphiopedilum insigne (Wall.) Pfitz     

Molecular Cytogenetics of the Genus Arabidopsis: In situ Localization of rDNA Sites, Chromosome Numbers and Diversity in Centromeric Heterochromatin

We have used in situ hybridization to determine the number ofsites of rDNA in species in the genus Arabidopsis. A. wallichii(2n = 16) has one major pair of sites and one minor pair ofsites, while A. pumila and A. griffithiana (both 2n = 32) havesix major and two minor rDNA sites. A. thaliana (2n = 10) isknown to have two pairs of rDNA sites. a highly repeated para-centromericsequence from A. thaliana, pAL1, is absent in the other threespecies. Hence the A.thaliana genome is not present (or thecentromeric DNA has evolved substantially) in the polyploidspecies A. pumila and A. griffithiana. Analysis of Arabidopsisspecies is a valuable complement to the large programmes forgenetic analysis of A. thaliana.Copyright 1993, 1999 AcademicPress Arabidopsis, centromeric DNA, maps (genetic), nuclear architecture, repetitive DNA, ribosomal DNA, rDNA, evolution, Brassicaceae, Crucifereae, in situ hybridization     

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 telomericsequences in theMusaL. genome were localized by fluorescentinsituhybridization on mitotic chromosomes of selected lines.A single major intercalary site of the 18S-5.8S-25S rDNA wasobserved on the short arm of the nucleolar organizing chromosomein each genome. AA and BB genome diploids had a single pairof sites, triploids had three sites while a tetraploid hybridhad four sites. The probe is useful for quick determinationof ploidy, even using interphase nuclei from slowly growingtissue culture material. Variation in the intensity of signalswas observed among heterogeneousMusalines indicating variationin the number of copies of the 18S-5.8S-25S rRNA genes. Eightsubterminal sites of 5S rDNA were observed in Calcutta 4 (AA)while Butohan 2 (BB) had six sites; some were weaker in bothgenotypes. Triploid lines showed six to nine major sites of5S rDNA of widely varying intensity and near the limit of detection.The diploid hybrids had five to nine sites of 5S rDNA whilethe tetraploid hybrid had 11 sites. The telomeric sequence wasdetected as pairs of dots at the ends of all the chromosomesanalysed but no intercalary sequences were seen. The molecularcytogenetic studies ofMusausing repetitive and single copy DNAprobes should yield insight into the genome and its evolutionand provide data forMusabreeders, as well as generating geneticmarkers inMusa.Copyright 1998 Annals of Botany Company Genome evolution, nucleolar organizing regions, telomeres,in situhybridization, genetic markers, banana, plantain.