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.     

Reversible tyrosine phosphorylation of cdc2: dephosphorylation accompanies activation during entry into mitosis

Tyrosine phosphorylation of cdc2 is regulated in the cell cycle of mouse 3T3 fibroblasts. Phosphotyrosine in cdc2 is detectable at the onset of DNA synthesis and becomes maximal in the G2 phase of the cell cycle. Quantitative tyrosine dephosphorylation of cdc2 occurs during entry into mitosis and no phosphotyrosine is detected during the G1 phase of the cell cycle. While increasing tyrosine phosphorylation of cdc2 correlates with the formation of a cdc2/p62 complex, the tyrosine phosphorylated cdc2 is inactive as a histone H1 kinase. cdc2 is fully dephosphorylated in its most active mitotic form, yet specific tyrosine dephosphorylation of interphase cdc2 in vitro is insufficient to activate the kinase. In vivo inhibition of tyrosine dephosphorylation by exposure of cells to a phosphatase inhibitor is associated with G2 arrest, which is reversible upon the removal of the phosphatase inhibitor. Tyrosine dephosphorylation of cdc2 may be one of a number of obligatory steps in the mitotic activation of the kinase.     

The murine p53 protein blocks replication of SV40 DNA in vitro by inhibiting the initiation functions of SV40 large T antigen

We have characterized the effect of murine p53 on SV40 DNA replication in vitro. Purified wild-type murine p53 dramatically inhibited the ability of SV40 T antigen to mediate the replication of a plasmid bearing the viral origin (ori-DNA) in vitro. In contrast, polyoma ori-DNA replication in vitro was unaffected by p53. Surprisingly, both unbound p53 and SV40 T antigen-bound p53 were equally detrimental to SV40 ori-DNA replication. Thus, p53 interferes with interactions between T antigen molecules that are required for DNA synthesis. p53 inhibited the binding to and subsequent unwinding of the SV40 origin by T antigen and thus selectively blocked the initial stages of ori-DNA replication. In contrast to the nononcogenic wild-type murine p53, high concentrations of a mutant transforming p53 failed to block SV40 ori-DNA replication in vitro. These observations may provide insight into a possible role for p53 in the cell.     

recA-dependent DNA repair processes

UV-radiation-induced lesions in DNA result in the formation of: (1) excision gaps (i.e. a lesion is excised, leaving a gap), (2) daughter-strand gaps (i.e. a lesion can be skipped during replication, leaving a gap), and (3) double-strand breaks (i.e. the DNA strand opposite a gap can be cut). In Escherichia coli, the recA gene product is involved in repairs of all three types of lesions--repair of daughter-strand gaps (2) and double-strand breaks (3) constitutes post-replication repair. The evidence suggests, furthermore, that recA-dependent repair of excision gaps (1) produced in DNA replicated prior to UV irradiation (pre-replication repair) appears to occur by similar mechanisms.     

Joint report of the Third International Bovine Lymphocyte Antigen (BoLA) Workshop, Helsinki, Finland, 27 July 1986

Two hundred and eighty-two alloantisera were submitted by 20 participating laboratories from 13 countries and tested against lymphocytes of 1298 cattle. The cell panel consisted of samples from 38 Bos taurus breeds, 11 Bos taurus crossbreeds, 4 Bos indicus breeds, 6 Bos taurus x Bos indicus, and a variety of other crossbred populations. Using a standardized lymphocytotoxicity test, all 17 previously identified BoLA specificities were confirmed. The workshop produced agreement on 16 new lymphocyte alloantigenic specificities. Three of the new specificities behaved as splits of previously identified BoLA specificities. Four of the new specificities behaved as alleles at the agreed BoLA-A locus. Seven new specificities are tentatively assigned to the BoLA-A locus but require further definition. Two new specificities may represent products of a second closely-linked BoLA locus.     

Cloning and Expression of a Schwanniomyces occidentalis alpha-Amylase Gene in Saccharomyces cerevisiae

An alpha-amylase gene (AMY) was cloned from Schwanniomyces occidentalis CCRC 21164 into Saccharomyces cerevisiae AH22 by inserting Sau3AI-generated DNA fragments into the BamHI site of YEp16. The 5-kilobase insert was shown to direct the synthesis of alpha-amylase. After subclones containing various lengths of restricted fragments were screened, a 3.4-kilobase fragment of the donor strain DNA was found to be sufficient for alpha-amylase synthesis. The concentration of alpha-amylase in culture broth produced by the S. cerevisiae transformants was about 1.5 times higher than that of the gene donor strain. The secreted alpha-amylase was shown to be indistinguishable from that of Schwanniomyces occidentalis on the basis of molecular weight and enzyme properties.