Acid phosphatase-11, a tightly linked molecular marker for root-knot nematode resistance in tomato: from protein to gene,using PCR and degenerate primers containing deoxyinosine

With a view to cloning the root-knot nematode resistance gene Mi in tomato by chromosome walking, we have developed a molecular probe for the tightly linked acid phosphatase-1 (Aps-1) locus. The acid phosphatase-1 allozyme (APS-1¹), encoded by the Aps-1 ¹ allele originating from Lycopersicon peruvianum, was purified to apparent homogeneity from tomato roots and suspension cells. Microsequencing of CNBr and tryptic peptides generated from APS-1¹ provided a partial amino acid sequence, which accounted for approximately 23% of the protein and revealed two stretches of homology with soybean proteins KSH3 and VSP27, comprising 22 matches within 26 amino acid residues. The partial amino acid sequence information enabled us to isolate a 2.4 kb genomic Aps-1 ¹ sequence by means of the polymerase chain reaction (PCR), primed by degenerate pools of oligodeoxyribonucleotides, synthesized on the basis of the amino acid sequences. Synthesis of the 2.4 kb PCR product was specific for genomic templates carrying the L. peruvianum Aps-1 ¹ allele. Crucial to the priming specificity and the synthesis of the 2.4 kb genomic sequence was the use of degenerate primer pools in which the number of different primer species was limited by incorporating deoxyinosine phosphate residues at three and four base ambiguities. In using cDNA as a template, a 490 bp sequence was obtained, indicating a high proportion of intron sequences in the 2.4 kb genomic Aps-1 ¹ sequence. The Aps-1 ¹ origin of the PCR product was confirmed by RFLP (restriction fragment length polymorphism) analysis, using both a chromosome 6 substitution line and a pair of nearly isogenic lines, differing for a small chromosomal region around the Aps-1/Mi loci.     

南方菜豆花叶病毒外壳蛋白基因序列的分析

用重组DNA技术及序列分析法测定了南方菜豆花叶病毒RNA基因组3′端1,000个碱基的序列,以及由此序列推导出的整个外壳蛋白的氨基酸顺序,它与巳报导的基本上一致。介绍了用DNA的寡核苷酸水解混合物作为起始引物,以3′端不含PolyA尾巴且不能加上PolyA的病毒RNA作为模板合成互补DNA,及进一步无性繁殖此cDNA的方法。     

Spectral sensitivity of photoreceptors mediating phase-shifts of circadian rhythms in retinally degenerate CBA/J (rd/rd) and normal CBA/N (+/+) mice

Light-dark cycles are the most important time cue for the circadian system to entrain the endogenous circadian clock to the environmental 24 h cycle. Although photic entrainment of circadian rhythms is mediated by the eye in mammals, photoreceptors implicated in circadian photoreception remain unknown. In our previous study, retinally degenerate CBA/J (rd/rd) mice were found to have lower circadian photo-sensitivity for phase-shifting the locomotor activity rhythms than normal CBA/N(+/+) mice. In the present study, the spectral sensitivity for phase-shifting the rhythms was examined in order to characterize the photopigments involved in circadian photoreception of these mice. The spectral sensitivity of CBA/J-rd/rd mice clearly fitted to the Dartnall nomogram for a retinal₁-based pigment with a maximum at 480 nm, while the best fitted nomogram had a maximum at 500 nm in CBA/N- +/+ mice. These results suggest that circadian photopigments involved in CBA/J-rd/rd and CBA/N- +/+ mice may be different.     

Detection and Identification of Yersinia pestis by Polymerase Chain Reaction (PCR) Using Multiplex Primers

A PCR method for detection of Yersinia pestis-virulence determinants by the use of multiplex primers was developed. Four pairs of oligonucleotide primers were designed from each gene of three kinds of virulent plasmids and a chromosomal DNA; 60-Md plasmid-located gene (caf1) encoding Y. pestis-specific capsular antigen fraction 1, a Y. pestis-specific region of a yopM gene encoded on 42-Md virulent plasmid, a plasminogen activator gene (pla) encoded on Y. pestis-specific 7-Md plasmid and an invasin protein gene (inv) encoded on chromosomal DNA. This multiplex-primer system was specific for the detection of Y. pestis among pathogenic Yersinia species and other enterobacteriaceae having antigens common to Y. pestis. Since this method is simple and safe, it will be useful to identify and confirm Y. pestis in cases of emergency and for the surveillance of epidemics.     

Enhanced detection of polymorphic DNA by multiple arbitrary amplicon profiling of endonuclease-digested DNA: identification of markers tightly linked to the supernodulation locus in soybean

Multiple endonuclease digestion of template DNA or amplification products can increase significantly the detection of polymorphic DNA in fingerprints generated by multiple arbitrary amplicon profiling (MAAP). This coupling of endonuclease cleavage and amplification of arbitrary stretches of DNA, directed by short oligonucleotide primers, readily allowed distinction of closely related fungal and bacterial isolates and plant cultivars. MAAP analysis of cleaved template DNA enabled the identification of molecular markers linked to a developmental locus of soybean (Glycine max L. Merrill). Ethyl methane sulfonate (EMS)-induced supernodulating, near-isogenic lines altered in the nts locus, which controls nodule formation, could be distinguished from each other and from the parent cultivar by amplification of template pre-digested with 2–3 restriction enzymes. A total of 42 DNA polymorphisms were detected using only 19 octamer primers. In the absence of digestion, 25 primers failed to differentiate these soybean genotypes. Several polymorphic products co-segregated tightly with the nts locus in F₂ families from crosses between the allelic mutants nts382 and nts1007 and the ancestral G. soja Sieb. & Succ. PI468.397. Our results suggest that EMS is capable of inducing extensive DNA alterations, probably around discrete mutational hot-spots. EMS-induced DNA polymorphisms may constitute sequence-tagged markers diagnostic of specific genomic regions.     

RAPD fingerprints for identification and for taxonomic studies of elite poplar (Populus spp.) clones

RAPD (Random Amplified Polymorphic DNA) fingerprints have recently been used to estimate genetic and taxonomic relationships in plants. In this study RAPD analysis was performed on 32 clones belonging to different species of the genus Populus. Of these, 25 clones are registered in several countries for commercial use and, altogether, cover almost 50% of the worlds cultivated poplars. DNA was prepared from leaves and amplified by PCR using random oligonucleotide primers. Amplification products were separated by agarose-gel electrophoresis to reveal band polymorphisms. Four primers out of the 18 tested, were selected on the basis of the number and frequency of the polymorphisms produced. With these a total of 120 different DNA bands were reproducibly obtained, 92% of which were polymorphic. The polymorphisms were scored and used in band-sharing analyses to identify genetic relationships. With a few but interesting exceptions, these are consistent with the present taxonomy of the genus Populus and with the known predigrees of cultivated poplars. Moreover, the results show that RAPD analysis allows one to discriminate among all tested clones and can, therefore, be recommended as a convenient tool to defend plant breeders rights.     

A family of serine protease genes expressed in adult buffalo fly (Haematobia irritans exigua)

Gene fragments encoding serine proteases expressed in adult buffalo fly (Haematobia irritans exigua) were amplified from cDNA using generic oligonucleotide PCR primers, based on conserved residues surrounding the active-site His and Ser amino acids found in all serine proteases. The PCR product consisted of a broad band extending from about 450 by to 520 bp, which suggested that the PCR product actually consisted of numerous DNA fragments of slightly variable sizes. Seventeen independent clones of these fragments, each with an insert of approximately 480 bp, were digested with HaeIII. Comparison of restriction fragment patterns indicated that 13 of these clones harboured different PCR products. This was confirmed by DNA sequence analysis of 9 clones. Each of the sequenced clones contained an open reading frame which included structurally conserved regions characteristic of the serine protease superfamily. This study reveals the expression of a large and highly variable repertoire of serine proteases in adult buffalo fly. Importantly, these data also demonstrate the utility of such an approach in obtaining DNA probes for use in further investigations of gene family organization and expression, as well as providing recombinant antigens in the form of fusion proteins which may be used as candidates for vaccine production.     

Comparison of the conserved region in the dnaA gene from three mollicute species

Abstract Polymerase chain reaction was carried out to amplify the conserved region (789 bp in the case of Mycoplasma capricolum ) of the dnaA gene (1350 bp in the case of M. capricolum ) of 15 representatives of the class Mollicutes using degenerate oligonucleotide primers. The dnaA gene fragments were amplified from M. mycoides subsp. capri, Spiroplasma apis and S. citri . The amino acid sequences deduced from the nucleotide sequences of the amplified fragments showed very low similarities to those of the corresponding regions of four walled bacteria. The values of similarity between any two of the three mollicute species were lower than those between any two of the four walled bacteria.