Effect of limited homology on gene conversion in a Saccharomyces cerevisiae plasmid recombination system.

Plasmids containing heteroallelic copies of the Saccharomyces cerevisiae HIS3 gene undergo intramolecular gene conversion in mitotically dividing S. cerevisiae cells. We have used this plasmid system to determine the minimum amount of homology required for gene conversion, to examine how conversion tract lengths are affected by limited homology, and to analyze the role of flanking DNA sequences on the pattern of exchange. Plasmids with homologous sequences greater than 2 kilobases have mitotic exchange rates as high as 2 x 10(-3) events per cell per generation. As the homology is reduced, the exchange rate decreases dramatically. A plasmid with 26 base pairs (bp) of homology undergoes gene conversion at a rate of approximately 1 x 10(-10) events per cell per generation. These studies have also shown that an 8-bp insertion mutation 13 bp from a border between homologous and nonhomologous sequences undergoes conversion, but that a similar 8-bp insertion 5 bp from a border does not. Examination of independent conversion events which occurred in plasmids with heteroallelic copies of the HIS3 gene shows that markers within 280 bp of a border between homologous and nonhomologous sequences undergo conversion less frequently than the same markers within a more extensive homologous sequence. Thus, proximity to a border between homologous and nonhomologous sequences shortens the conversion tract length.     

Synthesis and secretion of mucous glycoprotein by the gill of Mytilus edulis. I. Histochemical and chromatographic analysis of [14C]glucosamine bioincorporation

The ability of the isolated gill epithelium of Mytilus edulis to incorporate [14C]glucosamine as a precursor in the biosynthesis and secretion of mucous glycoproteins was investigated. Localization of mucous cells in the gill filament was achieved using histochemical staining techniques. Mucus cells containing neutral and acidic mucins were found in the lateral region, whereas mucus cells containing primarily neutral or sulfated mucins were found in the abfrontal region. Autoradiographic results showed that in both regions, the mucous cells were rich in content of the incorporated radiolabel. The secreted glycoproteins containing the incorporated radiolabel were analyzed by column chromatography using Bio-Gel P-2 and P-6. Two populations of the glycoproteins differing in molecular size were isolated. Upon alkaline reductive borohydride cleavage of the O-glycosidic linkages of the high molecular weight protein, about 70% of the radiolabel and 85% of the carbohydrate content were removed from the protein. The alkaline borohydride cleavage resulted in the formation of at least six oligosaccharide chains of various lengths of sugar units. Gas chromatographic analysis of the carbohydrate composition shows that the glycoproteins contain N-acetylglucosamine, N-acetylgalactosamine, and galactose, fucose, and mannose as the neutral monosaccharides. The above results indicate that the isolated gill epithelium of M. edulis is capable of incorporating [14C]glucosamine in the synthesis of secretable mucin-type glycoproteins.     

Recycling of membrane proteins during endo- and exocytosis in amoebae

The fate of a membrane protein of the amoeba plasmalemma was studied by means of 125I iodination by lactoperoxidase, gel electrophoresis, radioautography and gamma counting. There was only one iodinatable polypeptide group with a molecular weight (MW) of 175 000 on the external surface of the plasmalemma. Two hours or more after induced phagocytosis, isolated phagolysosomal membranes contained two other smaller polypeptides with MWs of 70 000 and 35 000, respectively, suggesting that the 175 000 polypeptide was broken down to these smaller components during endocytosis. After 22 h of induced phagocytosis, isolated plasmalemma contained a 35 000 polypeptide group in addition to the 175 000 polypeptide species. The results suggested that some of the iodinatable membrane proteins were altered and recycled during endo- and exocytosis in amoebae, while others were recycled intact.     

Detection of IgG and IgM antibodies with ELISA technique in human trichomoniasis

The direct wet mount examination of vaginal secretion, widely applied for the diagnosis of Trichomonas vaginalis infection in woman patients, is rapid and economical, however, the sensitivity of this technique is not so high. In this study enzyme-linked immunosorbent assay (ELISA) was employed for the detection of serum anti-T. vaginalis IgG and IgM antibodies from 30 vaginal trichomoniasis patients and 30 non-infected healthy persons. The results were as follows: 1. Serum ELISA-IgG value was 0.37 +/- 0.134 (Mean +/- S.D.) in vaginal trichomoniasis patients and 0.21 +/- 0.054 in healthy controls (p less than 0.005), and the sensitivity and specificity of ELISA for serum IgG antibody were 70.0% and 96.7%, respectively. 2. Serum ELISA-IgM value was 0.33 +/- 0.177 (Mean +/- S.D.) in vaginal trichomoniasis patients and 0.11 +/- 0.051 in healthy controls (p less than 0.005), and the sensitivity and specificity of ELISA for serum IgM antibody were 70.0% and 96.7%, respectively. 3. The ELISA-IgG values showed a significant correlation with ELISA-IgM values (r = 0.77, p less than 0.005). With above results, it is assumed that ELISA is a reliable method for the diagnosis of T. vaginalis infection and simultaneous measurement of serum IgG and IgM with this technique is recommended.     

Maturation-associated loss and incomplete de novo synthesis of the transferrin receptor in peripheral sheep reticulocytes: response to heme and iron

Hemin, but not iron, in the culture medium stimulates the maturation-associated loss of the transferrin receptor from sheep reticulocytes (t1/2 for loss approximately 6 hr) and its appearance in a population of externalized vesicles. A similar pattern is seen with nucleoside binding (a measure of the nucleoside transporter), where hemin increases the loss of binding activity from the cells during culture, concomitant with an increase in nucleoside binding in the externalized vesicles. Sheep reticulocytes retain the ability to synthesize the transferrin receptor, but the 35S-labeled receptors are not detected in released vesicles. Whereas hemin stimulates the loss of 35S-labeled transferrin receptors from the cell (t1/2 for loss approximately 20 hr), nonheme iron is more effective than heme. This difference in response of native and 35S-labeled receptor to hemin and iron supplements appears to be related to the differences in the two classes of receptors. Although the 35S-labeled receptor binds transferrin and both native and 35S-labeled peptides comigrate after chemical deglycosylation, the 35S-receptor is approximately 2 kD smaller than the native receptor and fails to acquire its complete size even when chased for up to 24 hr. Moreover, the 35S-labeled receptor is not expressed at the cell surface, but is retained in a nonrecycling compartment, where it is insensitive to digestion by trypsin at both 0 degrees C and 37 degrees C.     

Sites of lymph follicle formation in the draining popliteal lymph nodes of mice locally injected with antigenic and mitogenic substances

Our previous studies showed that some antigenic and mitogenic substances, when locally injected into mice, efficiently produced new lymph follicles outside pre-existing follicles in draining lymph nodes, whereas others had virtually no effect. In the present experiments, young adult male mice were injected with several antigens and mitogens in the rear footpad, and the number and development sites of newly produced lymph follicles in the draining popliteal nodes were studied using serial sections of the nodes obtained between 5 and 21 days after injection. In the unstimulated state, each popliteal node contained a limited number of lymph follicles which mostly lay in a portion of the peripheral cortex overlaying the deep cortex (this portion is referred to as the PCOU), whereas a portion of the peripheral cortex extending beyond the deep cortex (referred to as the PCBU) was underdeveloped with only occasional follicles. Mice treated with soluble PHA or fluid tetanus toxoid developed germinal centers in association with existing follicles but failed to produce new follicles. The PCBU of the draining nodes remained underdeveloped, and the number and distribution pattern of lymph follicles within a draining node were comparable to those in the control node. Animals treated with LPS (50 micrograms), Con A, alum-precipitated PHA or alum-precipitated tetanus toxoid produced significantly large numbers of new follicles outside pre-existing follicles in the draining nodes, the new follicles produced in the PCBU being generally more numerous than those in the PCOU. In these draining nodes, the peripheral cortex, comprising a number of follicles, was found to overlie the deep cortex and extend beyond the deep cortex towards the hilar region. In animals given a less effective stimulant, such as ferritin or a smaller dose of LPS (10 micrograms), the draining nodes produced a relatively small number of new follicles, most of which were formed in the PCBU. The present results indicate that in the mouse popliteal node, the PCBU is morphologically underdeveloped under normal conditions, but develops lymph follicles in response to exogenous stimuli more readily than the PCOU, and that substances efficient in inducing follicle formation can be regarded as capable of stimulating the development of the peripheral cortex.     

Saturated Molecular Map of the Rice Genome Based on an Interspecific Backcross Population

A molecular map has been constructed for the rice genome comprised of 726 markers (mainly restriction fragment length polymorphisms; RFLPs). The mapping population was derived from a backcross between cultivated rice, Oryza sativa, and its wild African relative, Oryza longistaminata. The very high level of polymorphism between these species, combined with the use of polymerase chain reaction-amplified cDNA libraries, contributed to mapping efficiency. A subset of the probes used in this study was previously used to construct an RFLP map derived from an inter subspecific cross, providing a basis for comparison of the two maps and of the relative mapping efficiencies in the two crosses. In addition to the previously described PstI genomic rice library, three cDNA libraries from rice (Oryza), oat (Avena) and barley (Hordeum) were used in this mapping project. Levels of polymorphism detected by each and the frequency of identifying heterologous sequences for use in rice mapping are discussed. Though strong reproductive barriers isolate O. sativa from O. longistaminata, the percentage of markers showing distorted segregation in this backcross population was not significantly different than that observed in an intraspecific F(2) population previously used for mapping. The map contains 1491 cM with an average interval size of 4.0 cM on the framework map, and 2.0 cM overall. A total of 238 markers from the previously described PstI genomic rice library, 250 markers from a cDNA library of rice (Oryza), 112 cDNA markers from oat (Avena), and 20 cDNA markers from a barley (Hordeum) library, two genomic clones from maize (Zea), 11 microsatellite markers, three telomere markers, eleven isozymes, 26 cloned genes, six RAPD, and 47 mutant phenotypes were used in this mapping project. Applications of a molecular map for plant improvement are discussed.     

Homoeologous relationships of rice, wheat and maize chromosomes

A set of cDNA clones, which had previously been mapped onto wheat chromosomes, was genetically mapped onto the chromosomes of rice. The resulting comparative maps make it possible to estimate the degree of linkage conservation between these two species. A number of chromosomal rearrangements, some of which must have involved interchromosomal translocations, differentiate the rice and wheat genomes. However, synteny of a large proportion of the loci appears to be conserved between the two species. The results of this study, combined with those from a recently published comparative map of the rice and maize genomes, suggest that rice, wheat and maize share extensive homoeologies in a number of regions in their genomes. Some chromosomes (e.g. chromosome 4 in rice, chromosomes 2 and 2S in wheat and maize, respectively) may have escaped major rearrangement since the divergence of these species from their last common ancestor. Comparative maps for rice, wheat and maize should make it possible to begin uniting the genetics of these species and allow for transfer of mapping information (including centromere positions) and molecular marker resources (e.g. RFLP probes) between species. In addition, such maps should shed light on the nature of chromosome evolution that accompanied the radiation of grasses in the early stages of plant diversification.     

Effect of Root Zone Temperature on the Mineral Composition of Xylem Sap and Plasma Membrane K+-Mg++-ATPase Activity of Grafted-Cucumber and -Figleaf Gourd Root Systems

Cucumber (Cucumis sativus L.) seedlings were grafted onto cucumber-(CG) or figleaf gourd- (FG, Cucurbita ficifolia Bouché)seedlings in order to determine the effect of solution temperature(12, 22, and 32°C) on the mineral composition of xylem sapand the plasma membrane K⁺-Mg⁺⁺-ATPase activities of the roots.Low solution temperature (12°C) lowered the concentrationof NO^–₃ and H₂PO^–₄ in xylem sap of CG plants butnot of FG plants. Concentrations of K⁺, Ca⁺⁺ and Mg⁺⁺ in xylemsap were less affected than anions by solution temperature.The plasma membrane of FG plants grown in 12°C solutiontemperature showed the highest K⁺- Mg⁺⁺-ATPase activity at allATP concentrations up to 3 mM and at low reaction temperatureup to 12°C, indicating resistance of figleaf gourd to lowroot temperature. (Received December 27, 1994; Accepted March 10, 1995)