Cloning, quantification and characterization of a minisatellite DNA sequence from common bean Phaseolus vulgaris L.

 We describe the cloning and the characterization of a 130-bp DNA fragment, called OPG9-130, amplified from bean (Phaseolus vulgaris L.) genomic DNA. This fragment corresponds to a minisatellite DNA sequence containing seven repeats of 15 bp which differ slightly from each other in their sequence. Southern analysis showed that the core sequence of 15 bp is repeated in clusters dispersed throughout the genome. The use of this fragment as a probe allowed us to identify common bean lines by their DNA fingerprints. We suggest that OPG9-130 will be useful for line identification as well as for the analysis of genetic relatedness between bean species and lines. Received: 14 February 1998 / Accepted: 10 February 1998     

An unusual wheat insertion sequence (WIS1) lies upstream of an alpha-amylase gene in hexaploid wheat, and carries a "minisatellite" array

Summary Comparison of the 5′ flanking regions of three α-amylase genes from chromosome 6B of hexaploid wheat by heteroduplex and sequence analysis revealed the presence of a 1.6 kb stem-loop insertion sequence (WIS1) in one of them. Polymorphism among hexaploid wheat varieties suggests the relatively recent insertion/excision of this sequence from its present position. The complete sequence of the stem-loop insertion shows that it has many of the features found in transposable elements, including target site duplication and terminal inverted repeats. One unusual feature is a tandem array of direct repeats comprising a wheat “minisatellite” sequence. Both the insertion sequence and the minisatellite are found at multiple locations in the wheat genome, but the functional significance of their association in WIS1 is unknown. The minisatellite arrays share a common core structure, and long arrays are polymorphic between different hexaploid varieties.     

The transposable element Tan1 of Aspergillus niger var. awamori , a new member of the Fot1 family

 Aspergillus niger var. awamori has transposable elements that we refer to as Vader and Tan1 (transposon A. niger). Vader was identified by screening unstable nitrate reductase (niaD) mutants for insertions. Four of the isolated niaD mutants were shown to contain a small insertion element. This 437 bp insertion element, Vader, is flanked by 44 bp inverted repeats (IR) and is present in approximately 15 copies in the genomes of two A. niger strains examined. A synthetic 44 bp oligomer of the inverted repeat of Vader has now been used to clone, via the polymerase chain reaction, a 2.3 kb Tan1 element. The Tan1 element has also been isolated from a partial genomic library. Tan1 is present as a single copy in A. niger var. awamori. The Tan1 element has a unique organization: IR-ORF-IR-IR-Vader-IR. The single open reading frame (ORF) (1668 bp) encodes a putative transposase homologous to Fusarium oxysporum Fot1 and Magnaporthe grisea Pot2. Immediately 3′ to the second inverted repeat, which bounds the transposase, is a copy of the AT-rich Vader element. We hypothesize that at some stage the independent Vader element, although inactive by itself, arose from Tan1, resulting in current strains with only one copy of Tan1 providing transposase activity and numerous mobile copies of Vader dispersed in the genome. Received: 14 September 1995/Accepted: 4 June 1996     

Highly repetitive DNA sequences that are restricted to the germ line in the hagfish Eptatretus cirrhatus: a mosaic of eliminated elements

The New Zealand hagfish, Eptatretus cirrhatus, is known to eliminate parts of its chromosomes during embryogenesis from presumptive somatic cells. Electrophoresis of germ line and somatic DNAs of this species, after treatment with the restriction endonucleases DraI and EcoRI, revealed three fragments of DNA that were restricted to the germ line. DNA filter hybridization experiments demonstrated that these fragments were present almost exclusively in the germ line DNA of E. cirrhatus and that they were highly and tandemly repeated. Thus, these DNA fragments appeared to be eliminated during embryogenesis. Moreover, one fragment (a DraI fragment) cross-hybridized with the germ line DNA from other species of hagfish, namely, Eptatretus okinoseanus and Paramyxine atami. Molecular cloning and sequence analysis revealed that the DraI fragment was composed mainly of closely related sequences of 85 bp in length and that this sequence was about 75% homologous to the sequence of EEEo2 (eliminated element of E. okinoseanus 2) which is a germ line-restricted and highly repetitive sequence that was isolated previously from E. okinoseanus. The other two fragments were composed of three families of closely related sequences that were 172 bp long (designated EEEc1), 61 bp long (EEEc2) and 54 bp long (EEEc3). Fluorescence in situ hybridization experiments revealed that each eliminated element was distributed on several chromosomes that are limited to germ cells. EEEo2 was dispersed on 12 C-band-positive chromosomes. EEEc1 and EEEc3 were dispersed on all C-band-positive and several C-band-negative chromosomes. By contrast, EEEc2 was located to terminal regions of several C-band-negative chromosomes. These results suggest that the eliminated chromosomes in hagfish are mosaics of highly repeated, germ line-restricted families of DNA sequences. Received: ██; in revised form: 25 October 1997 / Accepted: ██     

Sequencing of the internal transcribed spacer region ITS1 as a molecular tool detecting variation in the Stylosanthes guianensis species complex

 The internal transcribed spacer (ITS) regions 1 and 2 of the ribosomal DNA from Stylosanthes guianensis CIAT 1283 and cv ‘Schofield’ were amplified by polymerase chain reaction using conserved ITS primers from the 18S, 5.8S and 26S ribosomal genes flanking those regions. The entire region of 683 bp long was cloned, and seven clones were sequenced. Comparison of the ITS spacer regions with published DNA sequences of other plant species revealed limited homology only; this was in contrast to their comparison with the 5.8S rDNA sequences. The ITS1 region of 45 S. guianensis accessions was amplified by PCR and sequenced on both strands using the conserved primers ITS2-ITS5. These sequences, ranging from 201 to 204 bp, were aligned to each other to assess intra-specific polymorphism. Within the S. guianensis (Aubl.) Sw. species complex, 11 DNA sequence types could be distinguished based on an insertion/deletion (indel) event and 15 single base-pair substitutions. In 1 of the S. guianensis types, two kinds of ITS1 sequence were observed in each individual, reminiscent of an incomplete homogenization of the repeat structure in this type. Polymorphisms in the sequence of the ITS1 region were used to define molecular markers for S. guianensis on the basis of PCR-restriction fragment length polymorphism and selective PCR. Received: 24 June 1997 / Accepted: 31 October 1997     

Conservation in wheat high-molecular-weight glutenin gene promoter sequences: comparisons among loci and among alleles of the GLU-B1-1 locus

 The high-molecular-weight glutenin (HMW) genes and encoded subunits are known to be critical for wheat quality characteristics and are among the best-studied cereal research subjects. Two lines of experiments were undertaken to further understand the structure and high expression levels of the HMW-glutenin gene promoters. Cross hybridizations of clones of the paralogous x-type and y-type HMW-glutenin genes to a complete set of six genes from a single cultivar showed that each type hybridizes best within that type. The extent of hybridization was relatively restricted to the coding and immediate flanking DNA sequences. Additional DNA sequences were determined for four published members of the HMW-glutenin gene family (encoding subunits Ax2^*, Bx7, Dx5, and Dy10) and showed that the flanking DNA of the examined genes diverge at approximately −1200 bp 5′ to the start codon and 200–400 bp 3′ to the stop codon. These divergence sites may indicate the boundaries of sequences important in gene expression. In addition, promoter sequences were determined for alleles of the Bx gene (Glu-B1-1), a gene reported to show higher levels of expression than other HMW-glutenin genes and with variation among cultivars. The sequences of Bx promoters from three cultivars and one wild tetraploid wheat indicated that all Bx alleles had few differences and contained a duplicated portion of the promoter sequence “cereal-box” previously suspected as a factor in higher levels of expression. Thus, the “cereal-box” duplication preceeded the origin of hexaploid wheat, and provides no evidence to explain the variations in Bx subunit synthesis levels. One active Bx allele contained a 185-bp insertion that evidently resulted from a transposition event. Received: 5 August 1997 / Accepted: 6 November 1997     

Cell-free extract(s) of Pseudomonas putida catalyzes the conversion of cyanides,cyanates, thiocyanates,formamide, and cyanide-containing mine waters into ammonia

 Our isolate, Pseudomonas putida, is known to be capable of utilizing cyanides as the sole source of carbon (C) and nitrogen (N) both in the form of free cells and cells immobilized in calcium alginate. In the present study, the cell-free extract(s) were prepared from the cells of P. putida grown in the presence of sodium cyanide. The ability of enzyme(s) to convert cyanides, cyanates, thiocyanates, formamide and cyanide-containing mine waters into ammonia (NH₃) was studied at pH 7.5 and pH 9.5. The kinetic analysis of cyanide and formamide conversion into NH₃ at pH 7.5 and pH 9.5 by the cell-free extract(s) of P. putida was also studied. The K _m and V _max values for cyanide/formamide were found to be 4.3/8 mM and 142/227 μmol NH₃ released mg protein^-1 min^-1 respectively at pH 7.5 and 5/16.67 mM and 181/434 μmol NH₃ released mg protein^-1 h^-1 respectively at pH 9.5. The study thus concludes that the cell-free extract(s) of P. putida is able to metabolize not only cyanides, cyanates, thiocyanates, and formamide but also cyanide-containing mine waters to NH₃. Received: 10 April 1995/Received revision: 24 July 1995/Accepted: 22 August 1995     

Picophytoplankton biomass, community structure and productivity in the Great Astrolabe Lagoon, Fiji

 Phytoplankton biomass, community structure and productivity of the Great Astrolabe lagoon and surrounding ocean were studied using measurements of chlorophyll concentration and carbon uptake. The contribution of picophytoplankton to biomass, productivity and community structure was estimated by size fractionation, ¹⁴C-incubation and flow cytometry analysis. Picoplankton red fluorescence was demonstrated to be a proxy for chlorophyll <3 μm. Consequently, the percentage contribution to chl a<3 μm from each picoplankton group could be calculated using regression estimated values of ψ _i (fg chl a per unit of red fluorescence). In the lagoon, average chlorophyll concentration was 0.8 mg m^-3 with 45% of phytoplankton <3 μm. Primary production reached 1.3 g C m^-2 day^-1 with 53% due to phytoplankton <3 μm. Synechococcus was the most abundant group at all stations, followed by Prochlorococcus and picoeukaryotes. At all stations, Prochlorococcus represented less than 4% of the chl a <3 μm, Synechococcus between 85 and 95%, and Picoeukaryotes between 5 and 10%. In the upper 40 m of surrounding oceanic waters, phytoplankton biomass was dominated by the >3 μm size fraction. In deeper water, the <1 μm size fraction dominated. Prochlorococcus was the most abundant picoplankton group and their contributions to the chlorophyll a<3 μm were close to that of the picoeukaryotes (50% each). Accepted: 27 May 1999     

skippy, a retrotransposon from the fungal plant pathogen Fusarium oxysporum

A retrotransposon from the fungal plant pathogen Fusarium oxysporum f. sp. lycopersici has been isolated and characterized. The element, designated skippy (skp) is 7846 by in length, flanked by identical long terminal repeats (LTR) of 429 by showing structural features characteristic of retroviral and retrotransposon LTRs. Target-site duplications of 5 bp were found. Two long overlapping open reading frames (ORF) were identified. The first ORF, 2562 by in length, shows homology to retroviral gag genes. The second ORF, 3888 bp in length, has homology to the protease, reverse transciptase. RNase H and integrase domains of retroelement pol genes in that order. Sequence comparisons and the order of the predicted proteins from skippy indicate that the element is closely related to the gypsy family of LTR-retrotransposons. The element is present in similar copy numbers in the two races investigated, although RFLP analysis showed differences in banding patterns. The number of LTR sequences present in the genome is higher than the number of copies of complete elements, indicating excision by homologous recombination between LTR sequences.     

Resistance gene analogues from rice: cloning, sequencing and mapping

 Degenerate oligonucleotide primers were designed on the basis of nucleotide-binding-site (NBS) motifs conserved between resistance genes of Arabidopsis, flax and tobacco and subsequently used as PCR primers to amplify resistance gene analogues (RGA) in rice. Primers amplified a major band of approximately 500 bp. Restriction analysis of the amplified product revealed that the band was made up of several different fragments. Many of these fragments were cloned. Sixty different cloned fragments were analysed and assigned to 14 categories based on Southern blot analysis. Fourteen clones, each representing one of the 14 categories of RGAs were mapped onto the rice genetic map using a Nipponbare ( japonica)בKasalath’ (indica) mapping population consisting of 186 F₂ lines. Of the 14 clones representing each class 12 could be mapped onto five different chromosomes of rice with a major cluster of 8 RGAs on chromosome 11. Our results indicate that it is possible to use sequence homology from conserved motifs of known resistance genes to amplify candidate resistance genes from diverse plant taxa. Received: 23 September 1998 / Accepted: 28 November 1998     

A rapeseed FAE1 gene is linked to the E1 locus associated with variation in the content of erucic acid

 The synthesis of very long chain fatty acids occurs in the cytoplasm via an elongase complex. A key component of this complex is the β-ketoacyl-CoA synthase, a condensing enzyme which in Arabidopsis is encoded by the FAE1 gene. Two sequences homologous to the FAE1 gene were isolated from a Brassica napus immature embryo cDNA library. The two clones, CE7 and CE8, contain inserts of 1647 bp and 1654 bp, respectively. The CE7 gene encodes a protein of 506 amino acids and the CE8 clone, a protein of 505 amino acids, each having an approximate molecular mass of 56 kDa. The sequences of the two cDNA clones are highly homologous yet distinct, sharing 97% nucleotide identity and 98% identity at the amino acid level. Southern hybridisation showed the rapeseed β-ketoacyl-CoA synthase to be encoded by a small multigene family. Northern hybridisation showed the expression of the rapeseed FAE1 gene(s) to be restricted to the immature embryo. One of the FAE1 genes is tightly linked to the E1 locus, one of two loci controlling erucic acid content in rapeseed. The identity of the second locus, E2, is discussed. Received: 4 April 1997 / Accepted: 30 July 1997     

A genetic linkage map of Quercus robur L. (pedunculate oak) based on RAPD, SCAR, microsatellite, minisatellite, isozyme and 5S rDNA markers

 A genetic map of Pedunculate oak (Quercus robur) was constructed based on one 5S rDNA, 271 RAPD, ten SCAR, 18 microsatellite, one minisatellite, and six isozyme markers. A total of 94 individuals from a full-sib family was genotyped. Two maps, including 307 markers, were constructed according to the “two-way pseudo-testcross” mapping strategy. Testcross markers segregating in the 1 : 1 ratio were first used to establish separate maternal (893.2 cM, 12 linkage groups) and paternal (921.7 cM, 12 linkage groups) maps. Both maps provided 85–90% genome coverage. Homologies between the male and female linkage groups were then identified based on 74 intercross markers segregating in the 3 : 1, 1 : 2 : 1 and 1 : 1 : 1 : 1 ratios (RAPDs, SCARs, SSRs, 5S rDNA and isozymes) in the hybrid progeny. In each map, approximately 18% of the studied markers showed segregation distortion. More than 60% of the skewed markers were due to an excess of heterozygote genotypes. This map will be used for: (1) studying the molecular organisation of genomic regions involved in inter- and intraspecific differentiation in oaks and (2) identification of QTLs for adaptive traits. Received: 30 January 1998 / Accepted: 12 May 1998     

Somatic mutations caused by excision of the transposable element, Tpn1, from the DFR gene for pigmentation in sub-epidermal layer of periclinally chimeric flowers of Japanese morning glory and their germinal transmission to their progeny

Pigmentation in flowers of Japanese morning glory is intense in the epidermal layer, lighter in the subepidermis, and much lighter in the internal tissues; by contrast coloration in stems occurs only in the sub-epidermal layer. The a-3 ^f mutant of Japanese morning glory bears white flowers with normal-colored flecks and sectors, and its variegation also occurs in leaves and stems. The mutable line can produce chimeric flowers pigmented uniformly in the sub-epidermal tissue and variegated in the epidermal layer, and stems of these flowers are also pigmented. Since they give selfed progeny that segregate to give a ratio of three germinal revertants bearing fully colored flowers to one flecked mutant, it has been [OR Imai (1934) has] postulated that somatic mutations in the sub-epidermal layer can be transmitted to the next generation and that the germ cells in the reproductive organs must form from the cells of the sub-epidermal layer. Recently, we found that the 6.4-kb En/Spm-related transposable element, Tpn1, resides within the DFR-B gene for anthocyanin biosynthesis in the mutable a-3 ^f line. To test whether somatic mutations caused by Tpn1 excision from the DFR-B gene in the subepidermis of periclinally chimeric flowers are transmissible to their progeny, we have examined the structure of the DFR-B region in the germinal revertants derived from the chimeric flowers and compared the sequences generated by the somatic excision of Tpn1 in periclinally chimeric flowers with those in their germinal revertants. Our results confirm that somatic mutations caused by Tpn1 excision from the DFR-B gene in the sub-epidermal tissue of chimeric flowers can be transmitted to their progeny, which results in the generation of germinal revertants.     

FISH to meiotic pachytene chromosomes of tomato locates the root-knot nematode resistance gene Mi-1 and the acid phosphatase gene Aps-1 near the junction of euchromatin and pericentromeric heterochromatin of chromosome arms 6S and 6L, respectively

 The root-knot nematode resistance gene Mi-1 in tomato has long been thought to be located in the pericentromeric heterochromatin region of the long arm of chromosome 6 because of its very tight genetic linkage (approx. 1 cM) to the markers Aps-1 (Acid phosphatase 1) and yv (yellow virescent). Using Mi-BAC clones and an Aps-1 YAC clone in fluorescence in situ hybridisation (FISH) to pachytene chromosomes we now provide direct physical evidence showing that Mi-1 is located at the border of the euchromatin and heterochromatin regions in the short arm (6S) and Aps-1 in the pericentromeric heterochromatin of the long arm (6L) close to the euchromatin. Taking into account both the estimated DNA content of hetero- and euchromatin regions and the compactness of the tomato chromosomes at pachytene (2 Mb/μm), our data suggest that Mi-1 and Aps-1 are at least 40 Mb apart, a base pair-to-centiMorgan relationship that is more than 50-fold higher than the average value of 750 kb/cM of the tomato genome. An integrated cytogenetic-molecular map of chromosome 6 is presented that provides a framework for physical mapping. Received: 24 July 1998 / Accepted: 14 August 1998     

Heterogeneity of the internal structure of PDR1, a family of Ty1/copia-like retrotransposons in pea

We characterised the extent of heterogeneity among PDR1 elements, a Ty1/copia-like retrotransposon family in pea, by restriction mapping and PCR with primers designed to amplify four functional domains. The data suggest that two main subfamilies of PDR1 differ in the size of their 5′-region. There are also sequence variants and rearranged copies which include a wide range of deletions of different sizes and deletions combined with insertions of host DNA, or inversions of various regions of the retrotransposon. A deletion hot-spot has been found at nucleotide position 394, where buffer sequences of 26 bp and 38 bp containing microsatellite motifs have been generated. There is more heterogeneity in the gag domain of PDR1 than in other functional domains, and the extent and pattern of this diversity was assessed among 56 Pisum accessions. We found a higher rate of rearrangement and sequence variation within the gag domain of PDR1 in P. fulvum and P. abyssinicum accessions than would be expected from the degree of insertion site polymorphism. A neighbour-joining phylogenetic tree constructed for gag sequences has a similar branching pattern to the equivalent insertion site tree, implying that the PDR1 family and its gag domain have coevolved with the pea genome. Combining both trees revealed clear and distinct subgroups among the Pisum ssp. Received: 17 March 1999 / Accepted: 20 July 1999     

Brittle star fauna (Echinodermata: Ophiuroidea) of the arctic northwestern Barents sea: composition,abundance, biomass and spatial distribution

 Epibenthic brittle star assemblages were investigated on the northwestern Barents Sea shelf between 81° and 77°N in July 1991. At 9 drift stations in water depths between 80 and 360 m, series of 35–71 photographs, each depicting about 1 m² of the seabed, were taken along transects of about 150- to 300-m length to assess abundances and spatial distribution patterns of adult brittle stars (disc diameter ≥1 mm). Biomass values were derived by combining abundances with size-weight relationships and size frequencies established using specimens from trawl catches. Six brittle star species were identified on the seabed images. Ophiocten sericeum was the most abundant species on shallow shelf banks (≤100 m). Up to 2,800 individuals were counted on a single photograph; median abundances per station ranged from 32 to 524 ind.m^-2 and biomass from 0.3 to 5.0 g ash-free dry weight (AFDW) m^-2. The spatial distribution along the transects (i.e. on the 100-m scale) was, however, extremely patchy. Disc diameters of O. sericeum ranged between 1.6 mm and 15.4 mm. In deeper shelf habitats (>150 m), O. sericeum was rare or absent, and Ophiacantha bidentata dominated the brittle star fauna with median densities and biomasses of 2–49 ind.m^-2 and 0.07–1.9 g AFDW m^-2, respectively. Its disc diameters ranged from 2.9 to 14.4 mm. The other species (Ophiura sarsi, Ophiopholis aculeata, Ophioscolex glacialis, Ophiopleura borealis) occurred in distinctly lower numbers. Our findings provide further evidence that brittle stars dominate epibenthic communities on Arctic shelves and locally reach very high abundances. Dense beds of Ophiocten sericeum seem to be a general phenomenon on high-Arctic shallow shelf banks. Received: 30 March 1995/Accepted: 30 June 1995     

Characterization of <Emphasis Type="Italic">TPMT</Emphasis> minisatellite locus in five ethnic groups of India

A total of 206 random, healthy individuals belonging to five distinct ethnic groups (Ezhavas, Arayas, Nairs, Vishwakarmas and Muslims) were analyzed for 18 bp VNTR repeat polymorphism present in the 5í flanking region of the Thiopurine Methyl Transferase gene (TPMT). In the present study, the population data of TPMT minisatellite was compared with the population data of other loci and the utility of minisatellite was evaluated in population studies. Human tandem repeat alleles of the TPMT minisatellite locus were characterized for the length polymorphism. The expected and observed heterozygosity did not show any significant difference. All five populations were in Hardy-Weinberg equilibrium. High polymorphism information iontent (PIC) (≥0.658) and power of discrimination (PD) (ranging from 0.775–0.860) value of this VNTR showed that this marker is informative. The combined power of discrimination of TPMT minisatellite along with other two loci studied earlier in our lab was 0.9964. The paternity exclusion power (PE) of TPMT VNTR ranged from 0.203 to 0.533 and the combined power of paternity exclusion (with two other loci D8S315 and D2S1328) was ≥0.8285. All these parameters (heterozygosity, PD, PIC, PE) of TPMT minisatellite locus showed that this marker is informative and can be used for DNA typing and population studies besides being used in clinical investigation in checking thiopurine drug sensitivity of individuals. The text was submitted by the autor in English.     

Molecular markers residing close to the Rhg4 locus conferring resistance to soybean cyst nematode race 3 on linkage group A of soybean

 The restriction fragment length polymorphism (RFLP) clone pBLT65 is a 450-nt soybean cDNA encoding a portion of the bifunctional enzyme aspartokinase-homoserine dehydrogenase (AK-HSDH). pBLT65 maps within 3.5 cM of the i locus, conferring a pigmented seed coat, on linkage group A; hence, it is closely linked to the Rhg ₄ locus conferring resistance to race 3 of the soybean cyst nematode. From this useful RFLP we developed a PCR reaction yielding polymorphic bands for use in marker-assisted breeding programs to select progeny containing the Rhg ₄ allele. The polymorphic bands were sequenced to determine the cause of the polymorphisms. Using primers 548 and 563, PCR amplification of DNA from the soybean cultivar Peking (Rhg ₄ ) yielded three DNA fragments, 1a (1160 bp), 1b (1146 bp) and 3 (996 bp). Amplification of DNA from the cultivar Kent (rhg ₄) yielded DNA fragments 2 (1020 bp), 3 (996 bp) and 4 (960 bp). Fragments 1a, 1b, 2 and 4 were also polymorphic between the soybean lines PI 290136 and BARC-2(Rj ₄ ). A segregating population of 80 F₂ and F₃ plants derived from the cross PI 290136×BARC-2 (Rj ₄ ) was used to confirm the map position of the PCR polymorphisms near the i locus, and hence the Rhg ₄ locus on linkage group A. The nucleotide sequences of fragments 1b, 3 and 4 were determined. Large and small deletions in the intronic region were responsible for the size differences of the different fragments, whereas the exon was well conserved. Received: 8 January 1998 / Accepted: 15 July 1998