Analysis of rice (Oryza sativa L.) genome using pulsed-field gel electrophoresis and rare-cutting restriction endonucleases

TheOryza sativa (rice) genome is small (600 to 900 megabase pairs) when compared to that of other monocotyledonous plants. Rice was the first of the major cereals to be successfully transformed and regenerated. An RFLP map with approximately 300 markers is readily available, and the DNA content per map unit is only two to three times that ofArabidopsis thaliana. Rice is also the main staple food for the majority of peoples in the world. We developed techniques for the preparation of intact genomic DNA from Indica and Japonica subspecies of rice, used statistical methods to determine which restriction endonucleases are rare-cutting, and used pulsed-field gel electrophoresis (PFE) to separate large fragments of rice DNA. Southern hybridization to blotted rice PFE gels was used to show that the digests were complete. The long-term goal of our work is to generate an integrated genetic/physical map for the rice genome, as well as helping to establish rice as a model for map-based gene cloning and genome analysis.     

High intensity and blue light regulated expression of chimeric chalcone synthase genes in transgenic Arabidopsis thaliana plants

Summary To establish a genetic system for dissection of light-mediated signal transduction in plants, we analyzed the light wavelengths and promoter sequences responsible for the light-induced expression of the Arabidopsis thaliana chalcone synthase (CHS) promoter fused to the -glucuronidase (GUS) marker gene. Transgenic A. thaliana lines carrying 1975, 523, 186, and 17 by of the CHS promoter fused to the GUS gene were generated, and the expression of these chimeric genes was monitored in response to high intensity light in mature plants and to different wavelengths of light in seedlings. Fusion constructs containing 1975 and 523 by of CHS promoter sequence behaved identically to the endogenous CHS gene under all conditions. Expression of these constructs was induced specifically in response to high intensity white light and blue light. The response to blue light was seen in the presence of the P_fr form of phytochrome. Fusion constructs containing 186 by of promoter sequence showed reduced basal levels of expression and only weak stimulation by blue light but were induced significantly by high intensity white light. These analyses showed that the expression of the A. thaliana CHS gene is responsive to a specific blue light receptor and that sequences between — 523 and — 186 by are required for optimal basal and blue light-induced expression of this gene. The experiments lay the foundation for a simple genetic screen for light response mutants.     

Spectrin, red cell shape and deformability. II. The antagonistic action of spectrin and sialic acid residues in determining membrane curvature in genetic spectrin deficiency in mice

In a companion paper, the shapes of spectrin deficient mouse erythrocytes were described; in contrast to previous assumptions, spherules with tethered microvesicles rather than true "spherocytes" were found. Thence, spectrin deficient mouse erythrocytes are endowed with an excess of surface area for the given volume but the membrane is assuming a highly positive curvature. Observations during and after the action of enzymes cleaving the red cell surface charge (Neuraminidase, Trypsin, Chymotrypsin) showed that the previously positive membrane curvature, as well as the tendency of the membrane to flow into fingerlike protrusions was completely abolished. The erythrocytes of the spectrin deficient, desialylated mouse erythrocytes assumed a variety of shapes, often discocytic or even stomatocytic, i.e. their membrane presented with negative curvature. However, while these desialylated membranes could be easily deformed (elongated) by shear flow they did not recoil elastically into any definitive configuration after removal of the deforming forces. It is concluded from these observations that spectrin (acting on the inner interface between membrane and cytoplasm) and sialic acid residues (acting on the outer interface between membrane and plasma) exert antagonizing effects on membrane curvature and membrane bending elasticity. Sialic acid residues, strongly charged and situated on the outer side of the cell, produce positive membrane curvature; this observation can most readily be explained by assuming that this mechanical effect is caused by repulsive coulombic forces expanding the outer half of the bilayer. To explain the effect of the spectrin-complex in counteracting positive or in producing negative membrane curvature, a similar expansive coulombic force acting between the highly charged residues has been postulated. Thence, a model for explaining the overall elastic behaviour of the normal mammalian red cell is developed which is based on the assumption of elastic interactions of proteinacous membrane components coupled to the lipid bilayer of the membrane.     

Organization of the ribosomal ribonucleic acid genes in various wild-type strains and wild-collected strains of Neurospora

Summary The organization of the ribosomal DNA (rDNA) repcat unit in the standard wild-type strain of Neurospora crassa, 74-OR23-1A, and in 30 other wild-type strains and wild-collected strains of N. crassa, N. tetrasperma, N. sitophila, N. intermedia, and N. discreta isolated from nature, was investigated by restriction enzyme digestion of genomic DNA, and probing of the Southern-blotted DNA fragments with specific cloned pieces of the rDNA unit from 74-OR23-1A. The size of the rDNA unit in 74-OR23-1A was shown to be 9.20 kilobase pairs (kb) from blotting data, and the average for all strains was 9.11+0.21 kb; standard error=0.038; coefficient of variation (C.V.)=2.34%. These data indicate that the rDNA repeat unit size has been highly conserved among the Neurospora strains investigated. However, while all strains have a conserved HindIII site near the 5 end of the 25 S rDNA coding sequence, a polymorphism in the number and/or position of HindIII sites in the nontranscribed spacer region was found between strains. The 74-OR23-1A strain has two HindIII sites in the spacer, while others have from 0 to at least 3. This restriction site polymorphism is strain-specific and not species-specific. It was confirmed for some strains by restriction analysis of clones containing most of the rDNA repeat unit. The current restriction map of the 74-OR23-1A rDNA repeat unit is presented.     

Evidence for androgen independence of male mounting behavior in white-crowned sparrows (Zonotrichia leucophrys gambelii)

Previous experiments have shown that expression of mounting behavior in sexually inexperienced, adult male white-crowned sparrows does not require elevated plasma levels of androgen; adult males maintained on nonstimulatory short days mount sexually receptive females. The experiments reported here demonstrate that (1) sexually inexperienced, prepubertal males maintained on nonstimulatory short days show very low mounting rates in response to female sexual displays; (2) these males exhibit high mounting rates when exposed to stimulatory long days but androgen treatment on short days is ineffective in stimulating high mounting rates; and (3) prepubertal castration has no effect on the expression of mounting behavior by photostimulated adult males. Thus, there is no evidence that mounting behavior of adult male white-crowned sparrows depends on androgen.     

Mutation analysis of the cystic fibrosis transmembrane regulator gene in native American populations of the southwest

We report DNA and clinical analyses of cystic fibrosis (CF) in two previously unstudied, genetically isolated populations: Pueblo and Navajo Native Americans. Direct mutation analysis of six mutations of the CFTR gene--namely, delta F508, G542X, G551D, R553X, N1303K, and W1282X--was performed on PCR-amplified genomic DNA extracted from blood samples. Haplotype analyses with marker/enzyme pairs XV2c/TaqI and KM19/PstI were performed as well. Of the 12 affected individuals studied, no delta F508 mutation was detected; only one G542X mutation was found. None of the other mutations was detected. All affected individuals have either an AA, AC, or CC haplotype, except for the one carrying the G542X mutation, who has the haplotype AB. Clinically, six of the affected individuals examined exhibit growth deficiency, and five (all from the Zuni Pueblo) have a severe CF phenotype. Four of the six Zunis with CF are also microcephalic, a finding not previously noted in CF patients. Our DNA data have serious implications for risk assessment of CF carrier status for these people.     

Dietary folate affects the response of rats to nickel deprivation

Because vitamin B₁₂ and Ni are known to interact and because of the similar metabolic roles of vitamin B₁₂ and folate, an experiment was performed to determine the effect of dietary folate on Ni deprivation in rats. A 2×2 factorially arranged experiment used groups of nine weanling Sprague-Dawley rats. Dietary variables were Ni, as NiCl₂·6H₂O, 0 or 1 μg/g; and folic acid, 0 or 2 mg/kg. The basal diet, based on skim milk, contained less than 20 ng Ni/g. After 54 d, an interaction between dietary Ni and folate affected several variables including erythrocyte folate, plasma amino acids, and femur trace elements. For example, folate deprivation decreased erythrocyte folate; folate supplementation to the Ni-supplemented rats caused a larger increase in erythrocyte folate concentration than did folate supplementation to the Ni-deprived rats. Also, dietary Ni affected several plasma amino acids important in one-carbon metabolism (e.g., Ni deprivation increased the plasma concentrations of glycine and serine). This study shows that dietary Ni, folate, and their interaction can affect variables associated with one-carbon metabolism. This study does not show a specific site of action of Ni but it indicates that Ni may be important in processes related to the vitamin B₁₂-dependent pathway in methionine metabolism, possibly one-carbon metabolism. US Department of Agriculture, Agricultural Research Service, Northern Plans Area is an equal opportunity/affirmative action employer and all agency services are available without discrimination.     

Genetic Analysis of Hispanic Individuals with Cystic Fibrosis

We have performed molecular genetic analyses of Hispanic individuals with cystic fibrosis (CF) in the southwestern United States. Of 129 CF chromosomes analyzed, only 46% (59/129) carry ΔF508. The G542X mutation was found on 5% (7/129) of CF chromosomes. The 3849+10kbC→T mutation, detected primarily in Ashkenazi Jews, was present on 2% (3/129). R1162X and R334W, mutations identified in Spain and Italy, each occurred on 1.6% (2/129) of CF chromosomes. W1282X and R553X were each detected once. G551D and N1303K were not found. Overall, screening for 22 or more mutations resulted in detection of only 58% of CF transmembrane conductance regulator gene mutations among Hispanic individuals. Analysis of KM19/XV2c haplotypes revealed an unusual distribution. Although the majority of ΔF508 mutations are on chromosomes of B haplotypes, the other CF mutations are on A and C haplotypes at higher-than-expected frequencies. These genetic analyses demonstrate significant differences between Hispanic individuals with CF and those of the general North American population. Assessment of carrier/affected risk in Hispanic CF individuals cannot, therefore, be based on the mutation frequencies found through studies of the general population but must be adjusted to better reflect the genetic makeup of this ethnic group. Further studies are necessary to identify the causative mutation(s) in this population and to better delineate genotype/phenotype correlations. These will enable counselors to provide more accurate genetic counseling.