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.     

Differences between the catabolism and tumour distribution of intact monoclonal antibody (791T/36) and its Fab/c fragment in mice with tumour xenografts revealed by the use of a residualizing radiolabel (dilactitol-125I-tyramine) and autoradiography

Summary Radioiodine-labelled 791T/36 monoclonal antibody (mAb) and its Fab/c fragment, consisting of one Fab arm and the Fc portion, have identical whole-body survival curves in BALB/c mice (t1/2 = 3.75 days). Therefore, these two forms of this antibody provide a suitable model for studying the role of valency in the targeting efficiency of antibodies to tumours in vivo. 791/T36 antibody and its Fab/c fragment were labelled either by direct iodination using the iodogen method (¹²⁵I) or by dilactitol-¹²⁵I-tyramine (¹²⁵I-DLT), a residualizing label, which accumulates in the cells involved in degradation of the carrier protein. In tumour-bearing nude mice, the percentage of injected dose of mAb or Fab/c fragment reaching the specific 791T tumour was similar, and these proteins appeared to be catabolized at a similar rate in this tissue. mAb, but not the Fab/c fragment, was found to be very actively catabolized by the liver and spleen of tumour-bearing mice compared to control nude mice, this probably resulting from clearance of immune complexes. This effect was most pronounced when the mAb was labelled with¹²⁵I-DLT, the percentage of injected dose of mAb reaching the spleen and liver being higher than the percentage of injected dose reaching the tumour. This effect was not seen with the Fab/c fragment. Autoradiographic studies on tumour sections, which exhibit antigenic sites throughout the tumour mass, showed that the Fab/c fragment was already homogeneously distributed in the tumour 12 h after injection whereas the whole antibody was mainly localized at the periphery of the tumour. Those results suggest a binding site barrier effect. Overall, these results indicate that the highest valency and affinity may not be the optimal choice for mAb to be used for therapeutic purposes.     

Hydrogen evolution by subchloroplast preparations of photosystem II from pea and spinach,

Covalent coupling of bovine rhodopsin to CPG-thiol glass was used for separation of CNBr peptides. It is shown that cysteine residues 322 and 323 in the C-terminal cytoplasmic fragment of rhodopsin are modified with palmitic acid.     

利用单克隆抗体鉴定人甲胎蛋白(HAFP)抗原结构可分性

本文利用两株针对HAFP分子不同抗原决定簇的单克隆抗体,鉴定HAFP酶解片断的抗原抗体反应性质,并同完整HAFP分子进行比较。结果表明,酶解片断上失去了一株单克隆抗体所对应的分子部份,完整保留着另一株单克隆抗体所识别的抗原决定簇,从而证实HAFP分子某些抗原结构之间具有可分割性。     

Measurement of Substance P Metabolites in Rat CNS

A procedure based on ion-exchange chromatography for chemical separation and radioimmunoassays for quantitation of substance P (SP), the SP(1-7), and C-terminal fragments, respectively, has been developed. The procedure allows the determination of these fragments in the presence of large (i.e., 50- to 100-fold) excess of parent compound. The chemical identity of isolated SP and fragments was studied with preparative electrophoresis on dilute agarose gel and with HPLC. The activity identified as SP(1-7) comigrated with the authentic standard whereas practically all activity isolated as C-terminal fragments comigrated with SP(5-11). The levels of C-terminal fragments in rat brain areas rich in SP and in spinal cord were 1-2% of those of parent compound. The levels of SP(1-7) were always higher, in the spinal cord markedly higher (three to five times). Postmortem storage of samples from brain and spinal cord indicated that SP(1-7) levels fell more rapidly than those of SP or C-terminal fragments.     

Restriction fragment polymorphisms as probes for plant diversity and their development as tools for applied plant breeding

Summary Maize and tomato cDNA clones have been hybridized in Southern blotting experiments to plant genomic DNA prepared from different lines to detect restriction fragment polymorphisms (RFPs). In maize we have found that a high degree of genetic variability is present, even among domestic inbred lines. Most randomly chosen maize cDNA clones can be used to detect elements of this variability. Similar levels of polymorphism are observed when genomic DNA is digested with any of a number of different restriction enzymes and probed with individual clones. When a clone is hybridized to genomic DNAs prepared from several different maize lines, a number of different alleles are often detected at a single locus. At the same time one clone can often detect more than one independently segregating locus by cross hybridization to related sequences at other loci. As expected these markers are inherited as simple codominant Mendelian alleles from one generation to the next and colinkage of these markers can be demonstrated in the progeny from a heterozygous parent. In similar studies with tomato, remarkably different results were found. Few RFPs were demonstrable among domestic Lycopersicon esculentum lines although a higher level of variability could be detected when comparing esculentum with its wild Lycopersicon relatives. These results are discussed in relation to the applied uses of RFPs in plant breeding as well as the inherent variability of different plant genomes.This work was supported in part by funds from Sandoz Ltd. (Basel, Switzerland) and its subsidiary company, Northrup King Co. (Minneapolis, Minn., U.S.A.) as well as by NSF SBIR grant #BSR-8360870.     

Streptozotocin diabetes results in increased responsiveness of adipocyte lipolysis to glucagon

Adipocytes from streptozotocin-diabetic rats are approximately 50-times more sensitive to the lipolytic action of glucagon. This change is only perceived in the presence of a small quantity of adenosine deaminase which itself has little effect on basal lipolysis. Insulin treatment restores glucagon sensitivity to normal.     

第Ⅷ凝血因子基因内限制性片段长度多态性(RFLP)的研究及其在甲型血友病基因连锁分析中的应用

本文系统地研究了广东地区汉族人群中FⅧ:C基因内BclⅠ,XbaⅠ和BgⅡ位点RFLP的基因频率。多态性位点BclⅠ,XbaⅠ及BglⅠ的切点阳性率分别为63.5%、43.5%和100%。对Bcll和Xbal多态性切点连锁情况研究显示,19.5%的Bcll切点阳性纯合子为Xbal切点杂合子,证明联合应用此两位点RFLP可以把甲型血友病基因连锁分析的有效率提高到65.9%。用RFLP连锁分析对两例甲型血友病家系中的女性进行了致病基因携带者检测,对另一例家系进行了基因产前诊断。     

Primate evolution of a human chromosome 1 hypervariable repetitive element

Summary The clone designated hMF #1 represents a clustered DNA family, located on chromosome 1, consisting of tandem arrays displaying a monomeric length of 40 bp and a repetition frequency of approximately 7×10³ copies per haploid genome. The sequence hMF #1 reveals multiple restriction fragment length polymorphisms (RFLPs) when human genomic DNA is digested with a variety of 4–6-bp recognition sequence restriction enzymes (i.e., Taq I, Eco RI, Pst I, etc.). When hamster and mouse genomic DNA was digested and analyzed, no cross-species homology could be observed. Further investigation revealed considerable hybridization in the higher primates (chimpanzee, gorilla, and orangutan) as well as some monkey species.The evolutionary relationship of this repetitive DNA sequence, found in humans, to that of other primates was explored using two hybridization methods: DNA dot blot to establish copy number and Southern DNA analysis to examine the complexity of the RFLPs. Homology to the hMF #1 sequence was found throughout the suborder Anthropoidea in 14 ape and New and Old World monkey species. However the sequence was absent in one species of the suborder Prosimii. Several discrepancies between established evolutionary relationships and those predicted by hMF #1 exist, which suggests that repetitive elements of this type are not reliable indicators of phylogenetic branching patterns. The phenomenon of marked diversity between sequence homologies and copy numbers of dispersed repetitive DNA of closely related species has been observed inDrosophila mice,Galago, and higher primates. We report here a similar phenomenon for a clustered repeat that may have originated at an early stage of primate evolution.     

Identification of Brassica oleracea monosomic alien chromosome addition lines with molecular markers reveals extensive gene duplication

Summary Chromosomes of Brassica oleracea (2n=18) were dissected from the resynthesized amphidiploid B. napus Hakuran by repeated backcrosses to B. campestris (2n=20), creating a series of monosomic alien chromosome addition line plants (2n=21). Using morphological, isozyme and restriction fragment length polymorphism markers (RFLPs), 81 putative loci were identified. Of nine possible synteny groups, seven were represented in the 25 monosomic addition plants tested. Sequences homologous to 26% of the 61 DNA clones utilized (80% were cDNA clones) were found on more than one synteny group, indicating a high level of gene duplication. Anomalous synteny associations were detected in four 2n=21 plants. One of these plants showed two markers from one B. oleracea chromosome associated with a second complete B. oleracea synteny group, suggesting translocation or recombination between non-homologous chromosomes in Hakuran or the backcross derivatives. The other three 2n=21 plants each contained two or more B. oleracea synteny groups, suggesting chromosome substitution.