Identification and characterization of a novel repressor site in the human tumor necrosis factor alpha gene.

In human monocytic cell lines, tumor necrosis factor alpha (TNF alpha) expression is induced by phorbol myristate acetate (PMA). We have identified positive and negative cis-acting elements in the TNF alpha promoter by deletion analysis. Here we present the initial characterization of the repressor element. The repressor element was shown to function in either orientation and at various distances upstream from the positive element of the TNF alpha promoter. The TNF alpha repressor site (TRS) has been localized to a 25 bp region between base pairs -254 and -230 in the promoter. This region contains a 10 bp sequence with homology to the binding site of the activator protein AP-2. Mutation of the 6 C's of this 10 bp AP-2-like site abolish TRS repressor function. However, this AP-2-like site is not a binding site for AP-2 protein based on gel retardation analysis. In addition, a well-characterized AP-2-binding site placed upstream of the positive element of the TNF alpha gene did not cause repression. Therefore, this repression is very likely mediated by a novel protein(s) which interacts with the AP-2 consensus site in the TRS.     

The adaptive acid tolerance response in root nodule bacteria and Escherichia coli

Root nodule bacteria and Escherichia coli show an adaptive acid tolerance response when grown under mildly acidic conditions. This is defined in terms of the rate of cell death upon exposure to acid shock at pH 3.0 and expressed in terms of a decimal reduction time, D. The D values varied with the strain and the pH of the culture medium. Early exponential phase cells of three strains of Rhizobium leguminosarum (WU95, 3001 and WSM710) had D values of 1, 6 and 5 min respectively when grown at pH 7.0; and D values of 5, 20 and 12 min respectively when grown at pH 5.0. Exponential phase cells of Rhizobium tropici UMR1899, Bradyrhizobium japonicum USDA110 and peanut Bradyrhizobium sp. NC92 were more tolerant with D values of 31, 35 and 42 min when grown at pH 7.0; and 56, 86 and 68 min when grown at pH 5.0. Cells of E. coli UB1301 in early exponential phase at pH 7.0 had a D value of 16 min, whereas at pH 5.0 it was 76 min. Stationary phase cells of R. leguminosarum and E. coli were more tolerant (D values usually 2 to 5-fold higher) than those in exponential phase. Cells of R. leguminosarum bv. trifolii 3001 or E. coli UB1301 transferred from cultures at pH. 7.0 to medium at pH 5.0 grew immediately and induced the acid tolerance response within one generation. This was prevented by the addition of chloramphenicol. Acidadapted cells of Rhizobium leguminosarum bv. trifolii WU95 and 3001; or E. coli UB1301, M3503 and M3504 were as sensitive to UV light as those grown at neutral pH.     

Time-course of Biosynthesis of Phenolics and Lignin in Roots of Wheat Genotypes Differing in Manganese Efficiency and Resistance to Take-all Fungus

Inhibition of lignin biosynthesis in Triticum aestivum L. rootsby Mn deficiency has been suggested as the mechanism of reducedresistance of Mn-deficient wheat roots to infection by the take-allfungus (Gaeumannomyces graminis var. tritici). This study evaluatedphenolics and lignin accumulation in roots of wheat genotypesdiffering in Mn efficiency (measured as growth and yield inMn-deficient soils) and take-all resistance. Seedlings of theMn-inefficient, take-all sensitive genotype Bayonet and theMn-efficient, more take-all resistant genotype C8MM were grownin nutrient solution without added Mn for 18 d and then transferredto a Mn-deficient sandy soil fertilized with Mn at 0 or 30 mgkg^-1. Both genotypes had Mn-deficient roots and shoots at thetime of transfer to the soil. Roots of both genotypes were inoculatedwith the take-all fungus 0, 1, 3 and 7 d after transfer. Twenty-fourhours after inoculation, take-all fungus penetrated the rootstele of take-all sensitive Bayonet but not of more resistantC8MM wheat. Rates of phenolics and lignin accumulation in rootsdeclined steadily during growth in soil for up to 8 d, werehigher in mature, fully differentiated parts of the root systemcompared to distal, younger root tissue, and were higher inBayonet than in C8MM. Manganese fertilization did not significantlyinfluence rates of phenolics and lignin accumulation but reduceddepth of radial penetration by hyphae in both genotypes. Therate of phenolics accumulation was positively (r = 0·91to 0·96) correlated with the rate of lignin accumulation.Mn-efficient C8MM had a higher rate of lignin accumulation perunit of phenolics than Mn-inefficient Bayonet over a wide rangeof phenolics synthesis rates. From this we suggest that C8MMhas a more efficient mechanism for conversion of phenolics tolignin, the trait which appears related to higher take-all resistanceof this genotype.Copyright 1994, 1999 Academic Press Gaeumannomyces graminis var. tritici, lignin, manganese, phenolics, resistance, roots, Triticum aestivum     

Familial Site-specific Ovarian Cancer Is Linked to BRCA1 on 17q12-21

In a study of nine families with “site-specific” ovarian cancer (criterion: three or more cases of epithelial ovarian cancer and no cases of breast cancer diagnosed at age <50 years) we have obtained evidence of linkage to the breast-ovarian cancer susceptibility gene, BRCA1 on 17q12-21. If the risk of cancer in these families is assumed to be restricted to the ovary, the best estimate of the proportion of families linked to BRCA1 is .78 (95% confidence interval .32–1.0). If predisposition to both breast and ovarian cancer is assumed, the proportion linked is 1.0 (95% confidence interval .46–1.0). The linkage of familial site-specific ovarian cancer to BRCA1 indicates the possibility of predictive testing in such families; however, this is only appropriate in families where the evidence for linkage to BRCA1 is conclusive.     

The Preparation of Subcellular Organelles from Mouse Liver in Self-Generated Gradients of Iodixanol

This paper reports the use of a new density gradient compound, Iodixanol, for the resolution of the major organelles from mouse liver. A major advantage of Iodixanol over other iodinated density gradient media is its ready ability to form self-generated gradients. Gradient-forming conditions have been modulated to provide optimal recoveries of Golgi membranes, lysosomes, mitochondria, and peroxisomes. The organelles were isolated in high yield (80-90% of gradient input) and high purity. Nycodenz and Iodixanol were compared using preformed gradients. Iodixanol provided resolution superior to that of Nycodenz, notably of peroxisomes and mitochondria and the separation of lysosomes from endoplasmic reticulum. Because Iodixanol does not interfere significantly with marker enzyme activities, gradient fractions can be analyzed without removal of the gradient medium.     

Use of Genetic and Physical Mapping to Locate the Spinal Muscular Atrophy Locus between Two New Highly Polymorphic DNA Markers

The gene for autosomal recessive forms of spinal muscular atrophy (SMA) has recently been mapped to chromosome 5ql3, within a 4-cM region between the blocks D5S465/D5S125 and MAP-1B/D5S112. We identified two new highly polymorphic microsatellite DNA markers—namely, AFM265wf5 (D5S629) and AFM281yh9 (D5S637)—which are the closest markers to the SMA locus. Multilocus analysis by the location-score method was used to establish the best estimate of the SMA gene location. Our data suggest that the most likely location for SMA is between locus D5S629 and the block D5S637/D5S351/MAP-1B/D5S112/D5S357. Genetic analysis of inbred SMA families, based on homozygosity by descent and physical mapping using mega-YACs, gave additional information for the loci order as follows: cen–D5S6–D5S125/D5S465–D5S435–D5S629–SMA–D5S637–D5S351–MAP–1B/D5S112–D5S357–D5S39–tel. These data give the direction for bidirectional walking in order to clone this interval and isolate the SMA gene.     

Bloom Syndrome and Maternal Uniparental Disomy for Chromosome 15

Bloom syndrome (BS) is an autosomal recessive disorder characterized by increases in the frequency of sister-chromatid exchange and in the incidence of malignancy. Chromosome-transfer studies have shown the BS locus to map to chromosome 15q. This report describes a subject with features of both BS and Prader-Willi syndrome (PWS). Molecular analysis showed maternal uniparental disomy for chromosome 15. Meiotic recombination between the two disomic chromosomes 15 has resulted in heterodisomy for proximal 15q and isodisomy for distal 15q. In this individual BS is probably due to homozygosity for a gene that is telomeric to D15S95 (15q25), rather than to genetic imprinting, the mechanism responsible for the development of PWS. This report represents the first application of disomy analysis to the regional localization of a disease gene. This strategy promises to be useful in the genetic mapping of other uncommon autosomal recessive conditions.     

Fine mapping of the Autosomal Dominant Split Hand/Split Foot Locus on Chromosome 7, Band q21.3-q22.1

Split hand/split foot (SHFD) is a human developmental defect characterized by missing digits, fusion of remaining digits, and a deep median cleft in the hands and feet. Cytogenetic studies of deletions and translocations associated with this disorder have indicated that an autosomal dominant split hand/split foot locus (gene SHFD1) maps to 7q21-q22. To characterize the SHFD1 locus, somatic cell hybrid lines were constructed from cytogenetically abnormal individuals with SHFD. Molecular analysis resulted in the localization of 93 DNA markers to one of 10 intervals surrounding the SHFD1 locus. The translocation breakpoints in four SHFD patients were encompassed by the smallest region of overlap among the SHFD-associated deletions. The order of DNA markers in the SHFD1 critical region has been defined as PON–D7S812–SHFD1–D7S811–ASNS. One DNA marker, D7S811, detected altered restriction enzyme fragments in three patients with translocations when examined by pulsed-field gel electro-phoresis (PFGE). These data map SHFD1, a gene that is crucial for human limb differentiation, to a small interval in the q21.3-q22.1 region of human chromosome 7.     

Polymorphic Admixture Typing in Human Ethnic Populations

A panel of 257 RFLP loci was selected on the basis of high heterozygosity in Caucasian DNA surveys and equivalent spacing throughout the human genome. Probes from each locus were used in a Southern blot survey of allele frequency distribution for four human ethnic groups: Caucasian, African American, Asian (Chinese), and American Indian (Cheyenne). Nearly all RFLP loci were polymorphic in each group, albeit with a broad range of differing allele frequencies (δ). The distribution of frequency differences (δ values) was used for three purposes: (1) to provide estimates for genetic distance (differentiation) among these ethnic groups, (2) to revisit with a large data set the proportion of human genetic variation attributable to differentiation within ethnic groups, and (3) to identify loci with high δ values between recently admixed populations of use in mapping by admixture linkage disequilibrium (MALD). Although most markers display significant allele frequency differences between ethnic groups, the overall genetic distances between ethnic groups were small (.066–.098), and <10% of the measured overall molecular genetic diversity in these human samples can be attributed to “racial” differentiation. The median δ values for pairwise comparisons between groups fell between .15 and .20, permitting identification of highly informative RFLP loci for MALD disease association studies.