Cloning and expression of a glucoamylase gene from Lactobacillus amylovorus ATCC 33621 in Escherichia coli

Summary The glucoamylase gene from Lactobacillus amylovorus was cloned and expressed in Escherichia coli. A genomic DNA library from Lactobacillus amylovorus was prepared by partially digesting genomic DNA with EcoRI and ligating random fragments to the EcoRI digested cloning vector, pZErO-1.1. Three E. coli transformants expressing glucoamylase were identified using a probe prepared from the STA2 glucoamylase gene from Saccharomyces cerevisiae var. diastaticus. The physical maps of the recombinant plasmids were constructed. These plasmids contained inserts of about 5.2 Kb, 5.9 Kb and 6.4 Kb respectively. Temperature and pH optima of 45°C and 6.0, respectively, were obtained for both recombinant and purified wild type glucoamylases. Also, the enzymes were found to be thermolabile at temperatures above 50°C.     

Synthesis of biodegradable emulsifier using lipase in anhydrous pyridine

Summary Synthesis of a bioemulsifier using a lipase from Pseudomonas sp. with fructose and vinyl laurate was carried out in anhydrous pyridine. The synthetic product was identified as laurylfructose with an emulsifying activity on various hydrocarbons, edible oils and petroleum oils. The compound reduced the surface tension of distilled water from 72 mN/m to 29 mN/m and the interfacial tension of water/n-hexadecane from 50 mN/m to 6 mN/m.     

Association of specific leaf weight, an estimate of chlorophyll, and chlorophyll concentration with apparent photosynthesis in soybean

Increasing specific leaf weight (SLW) may improve leaf apparent photosynthesis (AP) in soybean [Glycine max (L.) Merr.] but screening for SLW and AP is laborious. The Objectives of this study were (i) to determine the time course of SLW and chlorophyll concentration in experimental lines selected for differences in SLW and (ii) to evaluate the potential use of the Minolta 502 SPAD meter as a rapid estimator of SLW, AP and chlorophyll concentration in leaves of soybean. In 1991 and 1992, sixteen experimental lines representing extremes in SLW were grown at Urbana, IL, and West Lafayette, IN, with three replications at each location. SPAD values, SLW and AP were measured at the R2 (full flower), R4 (full pod) and R5 (beginning seed) growth stages. In 1992 SLW, SPAD values and chlorophyll concentration were measured weekly. Seasonal patterns of SPAD values, SLW, and chlorophyll concentration were very similar through R5. After R5, SLW continued to increase but SPAD values and chlorophyll concentration declined. SPAD values and SLW were highly correlated at the R2, R4 and R5 stages at both locations and in both years. Environmental conditions during this research were not suitable for maximum AP expression, which is likely why AP and SPAD values were correlated only at the R4 growth stage at Urbana in 1992. SPAD measurements were consistent across diverse environments and effectively separated the high SLW lines from the low SLW lines. Measuring with the Minolta 502 SPAD meter is rapid, simple and non-destructive and could be an alternative method for direct selection for SLW.Abbreviations AP- leaf apparent photosynthesis - CV- coefficient of variation - Rug- leaf rugosity - SLW- specific leaf weight - SPAD-L- SPAD value of the most recently expanded terminal leaflet - SPAD-P- SPAD value of leaflet used to measure AP - SPAD-S- SPAD value of leaflets used to measure SLW - SPAD-U- SPAD value of the terminal leaflet one node above the most recently expanded terminal leaflet The US Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged     

Isolation of a root-specific cDNA encoding a ns-LTP-like protein from the roots of bean (Phaseolus vulgaris L.) seedlings

A root-specific cDNA clone, PVR3, was isolated from a bean (Phaseolus vulgaris L.) root cDNA library by a differential screening procedure. The nucleotide sequence of PVR3 contains an open reading frame coding for an 11.14 kDa polypeptide of 102 amino acid residues; the first 25 amino acids correspond to the sequence characteristic of a signal peptide. Comparison of the deduced PVR3 polypeptide sequence with the polypeptide sequences of previously cloned genes indicates that PVR3 may encode a ns-LTP-like protein. Molecular modelling of the PVR3 protein predicts that it has a three-dimensional structure that is similar to the three-dimensional model determined from the maize ns-LTP. The PVR3 mRNA accumulated mainly in the roots of young seedlings. It can be detected at low levels in flowers, but it is not detected in other organs. Genomic Southern blot analysis indicates that the genomic DNA corresponding to PVR3 cDNA is encoded by a single gene or small gene family in the bean genome.     

Ca2+ and Mg2+-binding and a putative calmodulin type Ca2+-binding site in Synechococcus Photosystem II

Thylakoids and Photosystem II particles prepared from the cyanobacterium Synechococcus PCC 7942 washed with a HEPES/glycerol buffer exhibited low rates of light-induced oxygen evolution. Addition of either Ca²⁺ or Mg²⁺ to both thylakoids and Photosystem II particles increased oxygen evolution independently, maximal rates being obtained by addition of both ions. If either preparation was washed with NaCl, light induced O₂ evolution was completely inhibited, but re-activated in the same manner by Ca²⁺ and Mg²⁺ but to a lower level. In the presence of Mg²⁺, the reactivation of O₂ evolution by Ca²⁺ allowed sigmoid kinetics, implying co-operative binding. The results are interpreted as indicating that not only Ca²⁺, but also Mg²⁺, is essential for light-induced oxygen evolution in thylakoids and Photosystem II particles from Synechococcus PC 7942. The significance of the reactivation kinetics is discussed. Reactivation by Ca²⁺ was inhibited by antibodies to mammalian calmodulin, indicating that the binding site in Photosystem II may be analogous to that of this protein.Abbreviation HEPES n-2-Hydroxyethylpiperazine--2-ethane sulphonic acid     

Characterization of a cDNA encoding cysteine proteinase inhibitor from Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower buds

A cDNA encoding a new phytocystatin isotype named BCPI-1 was isolated from a cDNA library of Chinese cabbage flower buds. The BCPI-1 clone encodes 199 amino acids resulting in a protein much larger than other known phytocystatins. BCPI-1 has an unusually long C-terminus. A BCPI-1 fusion protein expressed in Escherichia coli strongly inhibits the enzymatic activity of papain, a cysteine proteinase. Genomic Southern blot analysis revealed that the BCPI gene is a member of a small multi-gene family in Chinese cabbage. Northern blot analysis showed that it is differentially expressed in the flower bud, leaf and root.     

Co-translational trimerization of the reovirus cell attachment protein.

The reovirus cell attachment protein, sigma1, is a trimer with a 'lollipop' structure. Recent findings indicate that the N-terminal fibrous tail and the C-terminal globular head each possess a distinct trimerization domain. The region responsible for N-terminal trimerization (formation of a triple alpha-helical coiled-coil) is located at the N-terminal one-third of sigma1. In this study, we investigated the temporality and ATP requirement of this trimerization event in the context of sigma1 biogenesis. In vitro co-synthesis of the full-length (FL) and a C-terminally truncated (d44) sigma1 protein revealed a preference for homotrimer over heterotrimer formation, suggesting that assembly at the N-terminus occurs co-translationally. This was corroborated by the observation that polysome-associated sigma1 chains were trimeric as well as monomeric. Truncated proteins (d234 and d294) with C-terminal deletions exceeding half the length of sigma1 were found to trimerize post-translationally. This trimerization did not require ATP since it proceeded normally in the presence of apyrase. In contrast, formation of stable FL sigma1 trimers was inhibited by apyrase treatment. Collectively, our data suggest that assembly of nascent sigma1 chains at the N-terminus is intrinsically ATP independent, and occurs co-translationally when the ribosomes have traversed past the midpoint of the mRNA.     

DNA polymerase epsilon may be dispensable for SV40- but not cellular-DNA replication.

The contributions of DNA polymerases alpha, delta, and epsilon to SV40 and nuclear DNA syntheses were evaluated. Proteins were UV-crosslinked to nascent DNA within replicating chromosomes and the photolabelled polymerases were immunopurified. Only DNA polymerases alpha and delta were detectably photolabelled by nascent SV40 DNA, whether synthesized in soluble viral chromatin or within nuclei isolated from SV40-infected cells. In contrast, all three enzymes were photolabelled by the nascent cellular DNA. Mitogenic stimulation enhanced the photolabelling of the polymerases in the alpha>delta>epsilon order of preference. The data agree with the notion that DNA polymerases alpha and delta catalyse the principal DNA polymerisation reactions at the replication fork of SV40 and, perhaps, also of nuclear chromosomes. DNA polymerase epsilon, implicated by others as a cell-cycle checkpoint regulator sensing DNA replication lesions, may be dispensable for replication of the small, fast propagating virus that subverts cell cycle controls.