Biochemical evidence of a translocation between 6 RL/7 RL chromosome arms in rye (Secale cereale L.). A genetic map of 6R chromosome

Summary The segregation of different isozymic loci was investigated in backcrosses and F₂s in rye. The leucin aminopeptidase-1 (Lap-1), Aconitase-1 (Aco-1), Esterase-6 (Est-6), Esterase-8 (Est-8), and Endopeptidase-1 (Ep-1) loci were linked. The Aco-1, Est-6, and Est-8 loci have been previously located on the 6RL chromosome arm. The Lap-1 locus has been located on the 6RS chromosome arm. The results favor the gene order: Lap-1... (centromere)... Aco-1... Est-8... Est-6... Ep-1. The isoelectric focusing separations of aqueous extracts from mature embryo tissue of wheat-rye addition and substitution lines involving the chromosomes of cereal rye Secale cereale L. confirmed the gene location of locus Ep-1 on the 6RL chromosome arm. Screening of wheat-rye addition lines involving the chromosomes of Secale montanum revealed that Ep-1 locus is not located on chromosome 6R of S. montanum. These results are the first biochemical evidence of the translocation between chromosome arms 6RL/7RL in the evolution of S. cereale from S. montanum.     

High-resolution mapping of YACs and the single-copy gene Hs1pro−1 on Beta vulgaris chromosomes by multi-colour fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) is a powerful approach for physical mapping of DNA sequences along plant chromosomes. Nematode-resistant sugar beets (Beta vulgaris) carrying aBeta procumbens translocation were investigated by FISH with two differentially labelled YACs originating from the translocation. At mitotic metaphases, the translocation was identified with both YACs in the terminal region on a pair of chromosomes. Meiotic chromosomes, representing a far more extended hybridization target, were used to determine the orientation of YACs with respect to chromosomal domains in combination with chromosomal landmark probes for telomeres and centromeres. The in situ detection of plant single-copy sequences is technically difficult, and the wild beet translocation was used to explore the potential resolution of the FISH approach and to introduce the chromosomal mapping of single-copy genes into genome analysis of Beta species. An internal fragment of the nematode resistance gene Hs1 ^pro–1, 684 bp long, was detected on both chromatids of different Beta chromosomes and represents one of the shortest unique DNA sequences localized on mitotic plant chromosomes so far. Comparative chromosomal mapping of the 684 bp Hs1 ^pro–1 probe in the translocation line, a monosomic addition line and in B. procumbens revealed the origin of the wild beet translocation leading to nematode-resistant sugar beets.     

Esr,a second locus in the house mouse controlling esterase-5

Electrophoretic variation characterized by the presence (ES-5B⁺) or absence (ES-5B^–) of esterase-5B in the plasma of the house mouse has been observed. It is suggested that the expression of esterase-5B is controlled by an autosomal locus, Esr, linked to Ldr-1 on chromosome 6, in addition to the presumptive structural locus Es-5, which is located on chromosome 8. A gene order of Lyt-3-Esr-Ldr-1 was determined by two crosses.Supported by the Deutsche Forschungsgemeinschaft (SFB 46).This is communication No. 33 of a research program devoted to the investigation of cellular distribution and genetics of nonspecific esterases.     

Transgenic plants of rutabaga (Brassica napobrassica) tolerant to pest insects

Cotyledons cut from axenic seedlings were immersed inAgrobacterium tumefaciens suspension which was treated with acetosyringone and nopaline at low pH overnight. The infected cotyledon explants were cultured on MSB medium (MS salts + B₅ Vitamins) containing 6-BA 3mg/1 for 2–3 days, and transferred onto selective medium (MSB with kanamycin 50–100 mg/l). Kanamycin-resistant shoots were selected. More than 60 regenerated plants were obtained. About 60% of the plants showed high NPT II activity. Southern blot hybridization showed that some of the plants gave a positive signal with the insecticidal crystal protein gene (cry IA gene) probe, and exhibited tolerant to insects such asPieris rapae (cabbage caterpillar) in leaf feeding experiments. Kanamycin-resistance and insect-resistance were maintained in the progeny.Abbreviations 6-BA 6-benzylaminopurine - IBA indole-3-butyric acid - CryIA gene bacillus thuringiensis insecticidal crystal protein genecryIA - NPT II neomycin phosphotransferase II     

Genetic mapping with dispersed repeated sequences in the rice blast fungus: mapping the SMO locus

Summary The SMO genetic locus in strains of the fungus Magnaporthe grisea that infect weeping lovegrass, directs the formation of correct cell shapes in asexual spores, infection structures, and asci. We have identified and characterized a Smo^– strain of M. grisea that infects rice. The smo mutation in this strain segregates as a single gene mutation and is allelic to previously identified smo alleles. A marked reduction in pathogenicity co-segregates with the Smo^– phenotype, suggesting that the SMO locus plays a role in rice pathogenicity. A family of dispersed repeated DNA sequences, called MGR, have been discovered in the nuclear DNA of M. grisea rice pathogens. Genetic crosses between Smo^– rice pathogens and Smo⁺ non-rice pathogens were used to follow the segregation of the SMO locus and individual MGR sequences. Using DNA blot analysis with cloned MGR hybridization probes, we mapped the SMO locus to a chromosomal region flanked by two closely linked MGR sequences. We demonstrated that the copy number of MGR sequences could be reduced in subsequent crosses to non-rice pathogens of M. grisea, and that new MGR sequences did not occur following meiosis indicating that these sequences are stable in the genome. We conclude that restriction fragment polymorphism mapping with cloned MGR sequences as hybridization probes is an effective way to map genes in the rice blast fungus.     

Identification and physical characterization of yeast glucoamylase structural genes

Summary Each one of at least three unlinked STA loci (STA1, STA2 and STA3), in the genome of Saccharomyces diastaticus controls starch hydrolysis by coding for an extracellular glucoamylase. Cloned STA2 sequences were used as hybridization probes to investigate the physical structure of the family of STA genes in the genomes of different Saccharomyces strains. Sta⁺ strains, each carrying a single genetically defined STA locus, were crossed with a Sta^– strain and the segregation behavior of the functional locus (i.e. Sta⁺) and sequences homologous to a cloned STA2 glucoamylase structural gene at that locus were analyzed. The results indicate that in all strains examined there is a multiplicity of sequences that are homologous to STA2 DNA but that only the functional STA loci contain extensive 5 and 3 homology to each other and can be identified as residing on unique fragments of DNA; that all laboratory yeast strains examined contain extensive regions of the glucoamylase gene sequences at or closely linked to the STA1 chromosomal position; that the STA1 locus contains two distinct glucoamylase gene sequences that are closely linked to each other; and that all laboratory strains examined also contain another ubiquitous sequence that is not allelic to STA1 and is nonfunctional (Sta^–), but has retained extensive sequence homology to the 5 end of the cloned STA2 gene. It was also determined that the DEX genes (which control dextrin hydrolysis in S. diastaticus), MAL5 (a gene once thought to control maltose metabolism in yeast) and the STA genes are allelic to each other in the following manner: STA1 and DEX2, STA1 and MAL5, and STA2 and DEX1 and STA3 and DEX3.     

GnRHR gene polymorphisms and their effects on reproductive performance in Chinese goats

In this study, polymorphisms in the goat GnRHR gene exon 1 were detected by PCR-SSCP and DNA sequencing methods in 786 individuals from two different goat breeds. Two haplotypes (A and B), two observed genotypes (AA and AB), and two single nucleotide polymorphisms (SNPs) were detected, which resulted in five amino acid substitutions. The frequencies of haplotypes A and B in the two goat breeds were 0.78–0.83 and 0.17–0.22, respectively. The SNP locus was in Hardy–Weinberg disequilibrium in the two goat breeds (P < 0.05). Polymorphisms of the GnRHR gene were shown to be associated with litter size in the two goat breeds. The SNPs in the goat GnRHR gene had significant effects on litter size (P < 0.05). Therefore, these results suggest that the GnRHR gene is a strong candidate gene that affects litter size in goat.     

The use of genetic complementation in the study of eukaryotic macromolecular evolution: Rate of spontaneous gene duplication at two loci ofDrosophila melanogaster

Summary Gene duplications must play an important role in the evolutionary development of living organisms. Presented here is a general scheme that uses complementary alleles to isolate gene duplications in diploid organisms. The technique was used inDrosophila melanogaster to assess the rate of spontaneous gene duplication at two loci, maroon-like and rosy. The results indicate (1) that the rate of duplication of the maroon-like locus is on the order of 2.7×10^–6; (2) that the rate of duplication of the rosy locus is approximately 1.7×10^–4; and (3) that duplication occurs in males, suggesting that there may actually be two modes of gene duplication inDrosophila melanogaster.     

Quantitative traits associated with adaptation of three barley (Hordeum vulgare L) cultivars to suboptimal iron supply

A laboratory method was developed that allows determination of in situ net nitrification with high sensitivity and at high temporal resolution. Nitrate in soils is quantitatively converted into nitrous oxide under strictly anaerobic conditions in the presence of 10 kPa acetylene by the soil endogenous denitrifier population, with the N₂O detected by a gas chromatograph equipped with a ⁶³Ni electron capture detector. Thus, even low net nitrification rates, i.e. small net increases in soil nitrate concentrations can easily be detected. Comparison of results using this method with results obtained using the classical in situ incubation method (buried bag soil incubation) revealed excellent agreement. Application of the new method allowed both determination of the seasonal pattern of net nitrification as well as correlation analysis between in situ NO and N₂O flux rates and in situ net nitrification rates of the forest soils studied. Regardless of the forest site studied (spruce, spruce limed, beech), and during each year of a 3 years period (1995–1997), net nitrification varied strongly with season and was least during winter and greatest during summer. The long-term annual, mean rate of net nitrification for the untreated spruce site, the limed spruce site and the beech site were 1.54 ± 0.27 mg N kg^–1 sdw d^–1, 1.92 ± 0.23 mg N kg^–1 sdw d^–1 and 1.31 ± 0.23 mg N kg^–1 sdw d^–1, respectively. In situ rates of nitrification and NO and N₂O emission were strongly correlated for all sites suggesting that nitrification was the dominate source of NO as well as N₂O.     

A new family of polymorphic genes in Saccharomyces cerevisiae: α-galactosidase genes MEL1-MEL7

Summary Using genetic hybridization analysis we identified seven polymorphic genes for the fermentation of melibiose in different Mel⁺ strains of Saccharomyces cerevisiae. Four laboratory strains (1453-3A, 303-49, N2, C.B.11) contained only the MEL1 gene and a wild strain (VKM Y-1830) had only the MEL2 gene. Another wild strain (CBS 4411) contained five genes: MEL3, MEL4, MEL5, MEL6 and MEL7. MEL3-MEL7 were isolated and identified by backcrosses with Mel^– parents (X2180-1A, S288C). A cloned MEL1 gene was used as a probe to investigate the physical structure and chromosomal location of the MEL gene family and to check the segregation of MEL genes from CBS 4411 in six complete tetrads. Restriction and Southern hybridization analyses showed that all seven genes are physically very similar. By electrokaryotyping we found that all seven genes are located on different chromosomes MEL1 on chromosome II as shown previously by Vollrath et al. (1988), MEL2 on VII, MEL3 on XVI, MEL4 on XI, MEL5 on IV, MEL6 on XIII, and MEL7 on VI. Molecular analysis of the segregation of MEL genes from strain CBS 4411 gave results identical to those from the genetic analyses. The homology in the physical structure of this MEL gene family suggests that the MEL loci have evolved by transposition of an ancestral gene to specific locations within the genome.     

Genetic Alterations at Chromosome 6 Associated with Cervical Cancer Progression

Loss of heterozygosity (LOH) analysis on chromosome 6 was performed to define the genetic changes that occur in the development of squamous cell cervical cancer (SCC). Detailed analysis with 28 microsatellite markers revealed several loci with high frequency of deletions at the short (6p25, 6p22, 6p21.3) and long (6q14, 6q16–q21, 6q23–q24, 6q25, 6q27) arms of chromosome 6. Examination of microdissected 37 SCC and 22 cervical intraepithelial neoplasias (CIN) revealed allelic deletions in the HLA class I–III region (6p22–p21.3) and at subtelomeric locus 6p25-ter in more than 40% of CIN. By a combination of LOH and microdissection of multiple samples from the same tumor sections, we studied the intratumoral genetic heterogeneity of SCC, and identified clonal and subclonal allelic deletions. Half of SCC had clonal allelic deletion at D6S273, which is localized in intron of Ly6G6D (MEGT1) gene mapped in the HLA class III region. The LOH frequency at 6q in CIN cases did not exceed 20%. Allelic deletions at two loci, 6q14 and 6q16–q21, were for the first time associated with invasion and metastasis in SCC.     

Genetic and physical mapping of an hydrogenase gene cluster from Rhodobacter capsulatus

Summary An hydrogenase-deficient (Hup^–) mutant of Rhodobacter capsulatus was obtained by adventitious insertion of IS21 DNA into an hydrogenase structural gene (hup) of the wild-type strain 1310. The resulting Hup^– mutant, strain JP91, selected by its inability to grow autotrophically (Aut^– phenotype) together with other Hup^– mutant strains obtained by classical ethyl methane sulphonate mutagenesis were used in R plasmid-mediated conjugation experiments to map the hup/aut loci on the chromosome of R. capsulatus. The hup genes tested in this study were found to cluster on the chromosome in the proximity of the his-1 marker. A cluster of hup genes comprising the structural genes was isolated from a gene bank constructed in the cosmid vector pHC79 with 40 kb insert DNA. The clustered hup genes, characterized by hybridization studies and complementation analyses of the R. capsulatus Hup^– mutants, span 15–20 kb of DNA.     

A recombination hotspot in the maize A1 intragenic region

Summary We find that recombination between two alleles of the maize A1 locus that contain transposon insertions at known molecular positions can occur at 0.04–0.08 cM per kbp (centimorgan per kilobase pair), which is two orders of magnitude higher than the recombination rate for the whole maize genome. It is however, close to the rates found within the bronze locus, another maize structural gene for which both genetic and molecular data are available. This observation supports the idea that the genome consists of regions that are highly recombinogenic — in some cases, at least, structural genes — interspersed with regions that are less recombinogenic.     

Analysis of a repetitive DNA family from Arabidopsis arenosa and relationships between Arabidopsis species

We have analysed a family of highly repetitive DNA from Arabidopsis arenosa (L.) Lawalrée [syn. Cardaminopsis arenosa (L.) Hayck] composed of AT-rich tandem repeats of 166–179 bp in head to tail organization. Sequence comparison between several repeat units revealed a high level of divergence of 4.5% to 25%. The sequence family shows more than 58% homology to satellite sequences described in Arabidopsis thallana (L.) Heynh. but no homology to other satellite repeats in the Cruciferae. Within the genus Arabidopsis the satellite sequence was found to be present in A. thaliana and Arabidopsis suecica (Fries) Norrlin, but not in Arabidopsis griffithiana (Boiss.) N. Busch and Arabidopsis pumila (Stephan) N. Busch. In situ hybridization to metaphase chromosomes of A. arenosa (2n=4x=32) showed the sequence to be localized at the centromeres of all 32 chromosomes with substantial hybridization along the chromosome arms. Using Southern hybridization and in situ hybridization, we give evidence that A. suecica is a hybrid of A. thaliana and A. arenosa. A considerable reorganization of the A. thaliana satellite sequence pAL1 occurred in the hybrid genome while no molecular change of the A. arenosa repeat was observed in the hybrid. Analysis of related repeats enabled differentiation between closely related genomes and are useful for the investigation of hybrid genomes.     

X-linked mutations that give rise to overproduction of glucose-6-phosphate dehydrogenase in Drosophila melanogaster

Two X-linked mutations that give rise to overproduction of glucose-6-phosphate dehydrogenase (G6PD) were found among the progenies of isogenic strains which had been subjected to selection for high G6PD activity. Mapping of the high-activity factor in these mutants was carried out using car Zw ^B sw males of low G6PD activity. As a result, the factor mapped 0.02–0.04 unit to the left of the Zw locus. The amount of the G6PD gene was also quantitated utilizing a cloned G6PD gene as a probe, but no significant difference was found between the mutants and low-G6PD activity flies which shared the same X, second, and third chromosomes with the mutants. These findings are consistent with our notion that the mutations might be regulatory mutations, possibly resulting from the insertion of a novel class of transposable genetic elements.This research was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.     

Automated brightfield break-apart in situ hybridization (ba-ISH) application: ALK and MALT1 genes as models

Cancer diagnosis can be a complex process, which takes consideration of histopathological, clinical, immunophenotypic, and genetic features. Since non-random chromosomal translocations are specifically involved in the development of various cancers, the detection of these gene aberrations becomes increasingly important. In recent years, break-apart (or split-signal) fluorescence in situ hybridization (FISH) has emerged as an advantageous technique to detect gene translocations on tissue sections. However, FISH assays are technically challenging and require specialized fluorescence microscopes. Furthermore, the FISH signal is not stable for long term archiving due to photo bleaching. Our objective was to demonstrate the feasibility of brightfield break-apart in situ hybridization (ba-ISH) for anaplastic lymphoma kinase (ALK) and mucosa-associated lymphoid tissue translocation protein 1 (MALT1) genes as models. ALK or MALT1 break-apart probes were labeled with digoxigenin (DIG) or 2,4-dinitrophenyl (DNP) on both sides of a known gene breakpoint region and the hybridization sites were visualized with the combination of alkaline phosphatase (AP)-based blue and red detection. Therefore, normal genes are detected as purple dots by mixing blue and red colors while translocated genes are detected as isolated blue or red dots. Formalin-fixed, paraffin-embedded tonsil was used as control for the co-localized 5′ and 3′ probes. Gene translocations of ALK or MALT1 were detected as separate blue and red dots on ALCL and MALT lymphoma cases. Thus, ISH analyses of gene translocations can be conducted with a regular light microscope and the long term archiving of break-apart ISH slides can be achieved.     

The knee osteoarthritis susceptibility locus DVWA on chromosome 3p24.3 is the 5′ part of the split COL6A4 gene

In a recent study the DVWA gene located on human chromosome 3p24.3 was identified as a susceptibility locus for knee osteoarthritis in Japanese and Chinese patients (Miyamoto, Y., Shi, D., Nakajima, M., Ozaki, K., Sudo, A., Kotani, A., Uchida, A., Tanaka, T., Fukui, N., Tsunoda, T., Takahashi, A., Nakamura, Y., Jiang, Q., Ikegawa, S., 2008. Common variants in DVWA on chromosome 3p24.3 are associated with susceptibility to knee osteoarthritis. Nat. Genet. 40, 994–998). The authors concluded that DVWA codes for a novel protein containing two von Willebrand factor A (VWA) domains without a signal peptide sequence. The experimental data provided in this interesting study led to the suggestion of a mechanism for the etiology of the disease, based on an interaction between DVWA protein and β-tubulin. More recently, no significant association between DVWA and osteoarthritis was found in UK patient samples (Valdes, A.M., Spector, T.D., Doherty, S., Wheeler, M., Hart, D.J., Doherty, M., 2008. Association of the DVWA and GDF5 polymorphisms with osteoarthritis in UK populations. Ann. Rheum. Dis. Dec 3. [Epub ahead of print]), but a meta-analyses with data from individuals of white European descent from the Netherlands, the UK, Spain and Greece and the original Japanese and Chinese cohort provided evidence for a global association of one of the polymorphisms, a cysteine to tyrosine exchange (rs7639618) (Meulenbelt, I., Chapman, K., Dieguez-Gonzalez, R., Shi, D., Tsezou, A., Dai, J., Malizos, K.N., Kloppenburg, M., Carr, A., Nakajima, M., van der Breggen, R., Lakenberg, N., Gomez-Reino, J.J., Jiang, Q., Ikegawa, S., Gonzalez, A., Loughlin, J., Slagboom, E.P., 2009. Large replication study and meta-analyses of DVWA as an osteoarthritis susceptibility locus in European and Asian populations. Hum. Mol. Genet. 8, 1518–1523). However, there was no independent association with knee osteoarthritis in Europeans. Here we present information that the newly identified DVWA represents the human gene coding for the collagen VI α4 chain, which could point to a more complex disease mechanism.     

Ammonia-oxidizing bacteria on root biofilms and their possible contribution to N use efficiency of different rice cultivars

Ammonia-oxidizing bacteria (AOB) populations were studied on the root surface of different rice cultivars by PCR coupled with denaturing gradient gel electrophoresis (DGGE) and fluorescence in situ hybridization (FISH). PCR-DGGE of the ammonium monooxygenase gene (amoA) showed a generally greater diversity on root samples compared to rhizosphere and unplanted soil. Sequences affiliated with Nitrosomonas spp. tended to be associated with modern rice hybrid lines. Root-associated AOB observed by FISH were found within a discrete biofilm coating the root surface. Although the total abundance of AOB on root biofilms of different rice cultivars did not differ significantly, there were marked contrasts in their population structure, indicating selection of Nitrosomonas spp. on roots of a hybrid cultivar. Observations by FISH on the total bacterial community also suggested that different rice cultivars support different bacterial populations even under identical environmental conditions. The presence of active AOB in the root environment predicts that a significant proportion of the N taken up by certain rice cultivars is in the form of NO₃ ^–-N produced by the AOB. Measurement of plant growth of hydroponically grown plants showed a stronger response of hybrid cultivars to the co-provision of NH₄ ⁺ and NO₃ ^–. In soil-grown plants, N use efficiency in the hybrid was improved during ammonium fertilization compared to nitrate fertilization. Since ammonium-fertilized plants actually receive a mixture of NH₄ ⁺ and NO₃ ^– with ratios depending on root-associated nitrification activity, these results support the advantage of co-provision of ammonium and nitrate for the hybrid cultivar.     

High-resolution mapping of repetitive DNA by in situ hybridization: molecular and chromosomal features of prominent dispersed and discretely localized DNA families from the wild beet species Beta procumbens

Members of three prominent DNA families of Beta procumbens have been isolated as Sau3A repeats. Two families consisting of repeats of about 158 bp and 312 bp are organized as satellite DNAs (Sau3A satellites I and II), whereas the third family with a repeat length of 202 bp is interspersed throughout the genome. Multi-colour fluorescence in situ hybridization was used for physical mapping of the DNA families, and has shown that these tandemly organized families occur in large heterochromatic and DAPI positive blocks. The Sau3A satellite I hybridized exclusively around or near the centromeres of 10, 11 or 12 chromosomes. The Sau3A satellite family I showed high intraspecific variability and high-resolution physical mapping was performed on pachytene chromosomes using differentially labelled repeats. The physical order of satellite subfamily arrays along a chromosome was visualized and provided evidence that large arrays of plant satellite repeats are not contiguous and consist of distinct subfamily domains. Re-hybridization of a heterologous rRNA probe to mitotic metaphase chromosomes revealed that the 18S-5.8S-25S rRNA genes are located at subterminal position on one chromosome pair missing repeat clusters of the Sau3A satellite family I. It is known that arrays of Sau3A satellite I repeats are tightly linked to a nematode (Heterodera schachtii) resistance gene and our results show that the gene might be located close to the centromere. Large arrays of the Sau3A satellite II were found in centromeric regions of 16 chromosomes and, in addition, a considerable interspersion of repeats over all chromosomes was observed. The family of interspersed 202 bp repeats is uniformly distributed over all chromosomes and largely excluded from the rRNA gene cluster but shows local amplification in some regions. Southern hybridization has shown that all three families are specific for genomes of the section Procumbentes of the genus Beta.     

A simple method for mass isolation of protoplasts from species of Monostroma, Enteromorpha and Ulva (Chlorophyta, Ulvales)

A simple enzyme mixture containing 2% Cellulase Onozuka R–10 and1% Macerozyme R–10 prepared in deionised water supplemented with 3% NaCland 1 mM CaCl₂ was developed for isolating rapidlyprotoplasts from different species of Monostroma,Enteromorpha and Ulva. The yield fordifferent species of Monostroma ranged from 9.6 ×10⁶ to 10.2 × 10⁶ cells g^–1f. wt thallus, and forEnteromorpha from 3.48 × 10⁶ to 11.7× 10⁶ cells g^–1 f. wt and forUlva from 4.58 × 10⁶ to 26.8 ×10⁶ cells g^–1 f. wt. The overallregeneration rate of the protoplasts isolated was usually > 90% and showednormal morphogenesis. The method yields rapid mass production of viableprotoplasts with high regeneration rates.