The root-knot nematode resistance gene (Mi) in tomato: construction of a molecular linkage map and identification of dominant cDNA markers in resistant genotypes

A dominant allele at the Mi locus on chromosome 6 of tomato (Lycopersicon esculentum Mill) confers resistance to three species of root-knot nematodes (Meloidogyne). The resistance, which is associated with a localized necrotic response, was originally introduced into tomato from the wild species Lycopersicon peruvianum. As a step towards the molecular cloning of Mi, we have identified closely linked DNA markers from both cDNA and genomic DNA libraries as restriction fragment length polymorphisms (RFLPs). DNA from tomato populations segregating for nematode resistance was analyzed to generate a high-resolution genetic map of this region. Additional information on gene order was obtained by comparing the size of the introgressed L. peruvianum chromosomal segment within a collection of nematode-resistant tomato lines. Among the four cDNA markers that are tightly linked to Mi, three are dominant, i.e. L. peruvianum-specific. One cDNA marker corresponds to a gene family comprising 20-30 members, one of which is diagnostic for all nematode-resistant genotypes tested. The presence of non-homologous sequences around the Mi gene may contribute to the suppression of recombination in this region of the genome in crosses heterozygous for Mi. The potential of 'walking' from closely linked markers to Mi is discussed.     

Bleomycin resistance: a new dominant selectable marker for plant cell transformation

Summary Plant cells are sensitive to the antibiotic bleomycin, a DNA damaging glycopeptide. A bleomycin resistance determinant, located on transposon Tn5 and functional in bacteria, has been cloned in a plant expression vector and introduced into Nicotiana plumbaginifolia using Agrobacterium tumefaciens. The expression of this determinant in plant cells confers resistance to bleomycin and allows selection of transformed plant cells.     

Exclusion of epidermal growth factor and high-resolution physical mapping across the Rieger syndrome locus.

We have evaluated the 4q25-4q26 region where the autosomal dominant disorder Rieger syndrome has been previously mapped by linkage. We first excluded epidermal growth factor as a candidate gene by carrying out SSCP analysis of each of its 24 exons using a panel of seven unrelated individuals with Rieger syndrome. No evidence for etiologic mutations was detected in these individuals, although four polymorphic variants were identified, including three that resulted in amino acid changes. We next made use of two apparently balanced translocations, one familial and one sporadic, to identify a narrow physical localization likely to contain the gene or to be involved in regulation of gene function. Somatic cell hybrids were established from individuals with these balanced translocations, and these hybrids were used as a physical mapping resource for, first, preliminary mapping of the translocation breakpoints using known sequence tagged sites from chromosome 4 and then, after creating YAC and cosmids contigs encompassing the region, for fine mapping of those breakpoints. A cosmid contig spanning these breakpoints was identified and localized the gene to within approximately 150 kb of D4S193 on chromosome 4. The interval between the two independent translocations is approximately 50 kb in length and provides a powerful resource for gene identification.     

Integration of the Classical and Molecular Linkage Maps of Tomato Chromosome 6

In the past, a classical map of the tomato genome has been established that is based on linkage data from intraspecific Lycopersicon esculentum crosses. In addition, a high density molecular linkage map has recently been constructed using a L. esculentum X L. pennellii cross. As the respective maps only partially match, they provide limited information about the relative positions of classical and molecular markers. In this paper we describe the construction of an integrated linkage map of tomato chromosome 6 that shows the position of cDNA-, genomic DNA- and RAPD markers relative to 10 classical markers. Integration was achieved by using a L. esculentum line containing an introgressed chromosome 6 from L. pennellii in crosses to a variety of L. esculentum marker lines. In addition, an improved version of the classical linkage map is presented that is based on a combined analysis of new linkage data for 16 morphological markers and literature data. Unlike the classical map currently in use, the revised map reveals clustering of markers into three major groups around the yv, m-2 and c loci, respectively. Although crossing-over rates are clearly different when comparing intraspecific L. esculentum crosses with L. esculentum X L. pennellii crosses, the clusters of morphological markers on the classical map coincide with clusters of genomic- and cDNA-markers on the molecular map constructed by Tanksley and coworkers.     

Thyroglobulin structure-function: the effect of iodination on the structure of human thyroglobulin

Thyroglobulin of very low iodine content has been prepared from a single non-toxic human goitre. The initial iodine content of the protein (0.038%) has been increased to levels of 0.16% and 0.85% by in vitro treatment with thyroid peroxidase and the resulting proteins studied with respect to their intrinsic fluorescence, circular dichroism spectra and binding of the hydrophobic probe 1,8-anilinonaphthalene sulfonic acid (ANS). While significant differences were observed between levels of iodination in both the ANS binding and intrinsic fluorescence of the thyroglobulin, no significant differences in the near and far UV circular dichroism spectra of the protein as a function of iodine content were observed. These data suggest that, the iodination of thyroglobulin effects specific areas of the protein without significant disruption of its overall secondary structure.     

Familial pericentric inversion of the X chromosome [inv(X)(p11q28)]

A familial pericentric inversion of the X chromosome [46,X,inv(X)(p11q28)] and [46,inv(X)(p11q28), Y] is reported. The carriers of the inv(X) presented no clinical symptoms. Either the inverted or the normal X chromosome may be late replicating.     

Terminal uridylyl transferase of Vigna unguiculata: purification and characterization of an enzyme catalyzing the addition of a single UMP residue to the 3''-end of an RNA primer.

An enzyme which catalyzes the addition of a single UMP residue from UTP to the 3'-end of an RNA primer and which is referred to as terminal uridylyl transferase (TUT) has been extensively purified from the membrane fraction of vigna unguiculata leaves. The purification procedure involved (i) solubilization by cation depletion (ii) DEAE-Sepharose CL-6B column chromatography (iii) affinity chromatography of poly(U)-Sepharose 4B and (iv) glycerol gradient centrifugation. The molecular weight of the native enzyme was approximately 50,000 as determined by velocity sedimentation. Under conditions that were optimal for UMP-incorporation (5 mM Mg2+, low salt, 30 degrees C) TUT displayed a marked specificity for UTP as substrate, was unable to incorporate deoxyribonucleoside triphosphates and required a single-stranded oligo- or polyribonucleotide as primer. When oligoA20, tRNAasp of E. coli or alfalfa mosaic virus RNA 4 were used as primers at various substrate to primer ratio's, the vast majority of the product appeared to consist of primer molecules elongated with a single UMP residue as shown by polyacrylamide gelelectrophoresis and nearest neighbour analysis. We believe TUT to be a novel enzyme which has not been reported before and which may be a feasible tool in RNA sequencing as it enables the specific 3'-terminal labeling of RNA molecules.     

Salmonella typhimurium mutants with reduced levels of transfer ribonucleic acid-inhibitable endodeoxyribonucleolytic activity.

Two methods of mutagenesis, chemical alkylation and insertion of the tetracycline resistance transposon Tn10, were used to generate mutants of Salmonella typhimurium which had reduced levels of endodeoxyribonucleolytic activity. The chemically induced mutations defined a locus, endA, which was cotransduced with serA at a low frequency and with metK at a high frequency. Three-factor crosses revealed that metK was between serA and endA. The major endodeoxyribonucleolytic activity in crude extracts of s. typhimurium was similar to the activity of Escherichia coli endonuclease I. A Tn10 insertion mutation of endA resulted in the most severe loss of endodeoxyribonucleolytic activity among the endA alleles studied. Two of the chemically induced mutations resulted in activities which were more thermolabile than the wild-type activity.