Gene mapping on maize pachytene chromosomes by in situ hybridization

A sensitive in situ hybridization technique which was effective for mapping genes of low copy number on human metaphase chromosomes was used for gene mapping on maize pachytene chromosomes. A cloned genomic EcoR1 fragment of 10.8 kb, containing most or all of the sequence encoding the Waxy locus mRNA, was used as the probe. Southern DNA blotting analyses performed by Shure et al. (1983) indicated that the Waxy locus was a single copy sequence. In our in situ hybridization experiment, the probe hybridized to a specific site on chromosome 9. Labeling at this site was detected in 48.6% of 154 randomly selected copies of chromosome 9. To test the sensitivity of the method, subclones of the fragment with insert sizes of 6.6, 4.7, 3.5, 2.3, 1.9 and 0.8 kb were used for in situ hybridizations. Labeling efficiency for each probe was determined. The data showed that a single copy probe of 1.9 kb could be detected at the correct position in 18% of 183 randomly selected number 9 chromosomes.     

Adsorption of ionized and neutral pentachlorophenol to phosphatidylcholine membranes

We have studied adsorption of pentachlorophenol (PCP) to phosphatidylcholine (PC) membranes by measuring the electrophoretic mobility of multilayered lipid vesicles in PCP solutions. PC vesicles become negatively charged due to the adsorption of ionized PCP, and we have found that their zeta potential depends upon the ionic strength and pH of the aqueous suspension. We have shown that the experimental results can be adequately accounted for in terms of a two-component Langmuir-Stern-Grahame adsorption model assuming that the 'PCP adsorption sites' are occupied either by the neutral (HA) or the ionized (A-) species. The characteristics of adsorption isotherms of the PCP - PC membrane are as follows: the association constants are KA = 55,000 dm3/mol, KHA = 279,000 dm3/mol; 4.3 PC molecules make up each PCP adsorption site at saturation; the linear partition coefficients are beta HA = (15.5 +/- 0.7) x 10(-5) m and beta A = (3.0 +/- 0.3) x 10(-5) m. The properties of PCP adsorption isotherms for PC membranes predict an increased pKa value of membrane-bound PCP, which has been observed in related studies.     

Cloning and characterization of LOS1, a Saccharomyces cerevisiae gene that affects tRNA splicing.

Saccharomyces cerevisiae strains carrying los1-1 mutations are defective in tRNA processing; at 37 degrees C, such strains accumulate tRNA precursors which have mature 5' and 3' ends but contain intervening sequences. Strains bearing los1-1 and an intron-containing ochre-suppressing tRNA gene, SUP4(0), also fail to suppress the ochre mutations ade2-1(0) and can1-100(0) at 34 degrees C. To understand the role of the LOS1 product in tRNA splicing, we initiated a molecular study of the LOS1 gene. Two plasmids, YEpLOS1 and YCpLOS1, that complement the los1-1 phenotype were isolated from the YEp24 and YCp50 libraries, respectively. YEpLOS1 and YCpLOS1 had overlapping restriction maps, indicating that the DNA in the overlapping segment could complement los1-1 when present in multiple or single copy. Integration of plasmid DNA at the LOS1 locus confirmed that these clones contained authentic LOS1 sequences. Southern analyses showed that LOS1 is a single copy gene. The locations of the LOS1 gene within YEpLOS1 and YCpLOS1 were determined by deletion and gamma-delta mapping. Two genomic disruptions of the LOS1 gene were constructed, i.e., an insertion of a 1.2-kilobase fragment carrying the yeast URA3 gene, los1::URA3, and a 2.4-kilobase deletion from the LOS1 gene, los1-delta V. Disruption or deletion of most of the LOS1 gene was not lethal; cells carrying the disrupted los1 alleles were viable and had phenotypes similar to those of cells carrying the los1-1 allele. Thus, it appears that the los1 gene product expedites tRNA splicing at elevated temperatures but is not essential for this process.     

Optimization by simulating molecular evolution

Based on the analogy between mathematical optimization and molecular evolution and on Eigen's quasi-species model of molecular evolution, an evolutionary algorithm for combinatorial optimization has been developed. This algorithm consists of a versatile variation scheme and an innovative decision rule, the essence of which lies in a radical revision of the conventional philosophy of optimization: A number of configurations of variables with better values, instead of only a single best configuration, are selected as starting points for the next iteration. As a result the search proceeds in parallel along a number of routes and is unlikely to get trapped in local optima. An important innovation of the algorithm is introduction of a constraint to let the starting points always keep a certain distance from each other so that the search is able to cover a larger region of space effectively. The main advantage of the algorithm is that it has more chances to find the global optimum and as many local optima as possible in a single run. This has been demonstrated in preliminary computational experiments.     

Purification and partial characterisation of two abscisic-acid-responsive proteins induced in cultured embryos ofPisum sativum L.

When pea (Pisum sativum L.) embryos were cultured on low osmotica, with or without added abscisic acid (ABA), there was very little change in the total mRNA translation products resolved by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The only marked alteration was an increase in production of two low-molecular-weight proteins. The purification and partial characterisation of these two ABA-responsive seed proteins (ABR17 and ABR18) is described. Both proteins were purified to homoeneity, as judged by SDS-PAGE, from embryos cultured in the presence of ABA. Antisera were raised against both proteins. Each serum cross-reacted with the other protein, indicating that the proteins are closely related. Their apparent molecular masses (M_rs) were estimated to be 17200 (ABR17) and 18100 (ABR18) by SDS-PAGE, and 26000 by gel filtration. Both proteins were heterogeneous on isoelectric focusing. Neither protein was detected (by immunoblotting or immunoprecipitation of cell-free translation products) in embryos grown in vivo at early to mid-development stages but both were present in embryos late in development. These proteins appear to be produced late in seed development but are capable of being induced early in development by culturing embryos in vitro and are markedly enhanced by ABA.     

Microtubules in maize leaf protoplasts in relation to donor tissue and in vitro culture

Summary Maize (Zea mays) leaf protoplasts were isolated from various leaves of two-week (4-leaf) seedlings and from sections of the third leaf blades. Microtubules (MTs) were visualized using immunofluorescence microscopy. Only freshly isolated protoplasts from the third and fourth leaf blades contained MTs, with protoplasts from the fourth leaf containing the most i.e. 13% of fourth-leaf protoplasts contained MTs. In general, protoplasts with fewer and smaller chloroplasts had more MTs. Initially 90–95% of protoplasts from basal portions of leaves had MTs but the percentage decreased slightly during culture particularly after 10 days. The antioxidant n-propyl gallate was beneficial in maintaining MT content. Few protoplasts from older sections intitially contained MTs but in all sections at least some protoplasts regained a significant MT content during culture (e.g., 10% of protoplast from the tip section possessed microtubules after 7 days of culture). Far fewer MTs were observed in individual leaf protoplasts than those isolated from suspension culture.Abbreviations BMS Black Mexican Sweet - MT microtubule - MtSB microtubule stabilizing buffer - PBS phosphate buffered saline     

Some physicochemical properties of rice mitochondrial DNA

Summary Certain physicochemical properties of rice mitochondrial DNA (mtDNA) were determined. Certain low-molecular-weight mtDNA bands were found in addition to the major mtDNA band. Rice mtDNA appeared in the electron microscope as a collection of linear molecules with heterogeneous length in the range of 1–156 kb. The major distribution area was 60–105 kb. A small fraction (less than 5%) of rice mtDNA was found in the form of a circular molecule. Some molecules had the appearance of being supercoiled. Replication fork structures were found in both circular and linear mtDNA molecules. In one rice species, Jin Nante, 15 different circular molecules were found. Rice mtDNA was digested with different restriction enzymes. The total molecular weight of rice mtDNA was calculated to be about 300 kb according to the data of restriction enzyme digestion and electron microscopy.