An analysis of the relative activities of a number of promoter constructs from genes which are expressed during late pollen development as determined by particle bombardment

Summary The promoters of a tobacco actin gene, a tobacco pectate lyase, a tobacco and maize polygalacturonase and aBrassica S-locus related gene have been fused to the-glucuronidase reporter gene and their activities determined by biolistic transient assay in tobacco pollen. In stably transformed tobacco all the transgenes with the exception of Cauliflower Mosaic Virus-35S--glucuronidase appear to express efficiently in maturing pollen. Transient assay analysis showed that the tobacco pectate lyase and the polygalacturonase constructs were 8x more active than the tobacco actin construct, and that the tobacco polygalacturonase construct was some 33x more active than the maize polygalacturonase construct. Constructional manipulations that altered the lengths of the 5-untranslated leaders including one which resulted in the removal of a 490 bp leader intron had little effect on the observed level of expression. However, the alteration of the context of the ATG from A/TnnATGG to CnnATGT resulting in a 70% reduction in the observed levels of activity, was obtained with the pectate lyase and polygalacturonase promoters. An identical reductional was also observed in transgenic plant populations transformed with the polygalacturonase transgenes.Abbreviations GUS -glucuronidase - LUC luciferase - NosTer nopaline synthase terminator - CaMV Cauliflower Mosaic Virus - UTL untranslated leader - PCR polymerase chain reaction - PG polygalacturonase - Npg tobacco polygalacturonase - Pl pectate lyase - Ac actin     

Homology between the ribosomal DNA of Escherichia coli and mitochondrial DNA preparations of maize is principally to sequences other than mitochondrial rRNA genes

E. coli ribosomal DNA has been used to probe maize mitochondrial DNA. It hybridizes primarily with chloroplast ribosomal DNA sequences and with fungal and bacterial sequences which may contaminate the mtDNA preparations. It also hybridizes to the chloroplast 16S ribosomal RNA gene sequence present in the mitochondrial genome (1) as well as to the mitochondrial 18S ribosomal RNA gene sequence. Weak sequence homology was detected between E. coli rDNA and the mitochondrial 26S ribosomal RNA gene.     

Identification of the C2-1H histidine NMR resonances in chloramphenicol acetyltransferase by a 13C-1H heteronuclear multiple quantum coherence method

Chloramphenicol acetyltransferase (CAT) was used to assess the feasibility of study of specific proton resonances in an enzyme of overall molecular mass 75,000, [ring 2-13C]Histidine was selectively incorporated into the type III chloramphenicol acetyltransferase (CATIII) using a histidine auxotroph of E. coli. Heteronuclear multiple and single quantum experiments were used to select the C2 protons in the histidyl imidazole ring. One- and two-dimensional spectra revealed six signals out of a total of seven histidine residues in CATIII. pH titration, chemical modification and ligand binding were used to demonstrate that the signal from H195, the histidine at the active site, is not among those observed. Nevertheless, this work demonstrates that selective isotopic enrichment and multiple quantum coherence techniques can be used to distinguish proton resonances in a protein of high molecular mass.     

Determination of the solution structures of domains II and III of protein G from Streptococcus by 1H nuclear magnetic resonance.

We have used 1H nuclear magnetic resonance spectroscopy to determine the solution structures of two small (61 and 64 residue) immunoglobulin G (IgG)-binding domains from protein G, a cell-surface protein from Streptococcus strain G148. The two domains differ in sequence by four amino acid substitutions, and differ in their affinity for some subclasses of IgG. The structure of domain II was determined using a total of 478 distance restraints, 31 phi and 9 chi 1 dihedral angle restraints; that of domain III was determined using a total of 445 distance restraints, 31 phi and 9 chi 1 dihedral angle restraints. A protocol which involved distance geometry, simulated annealing and restrained molecular dynamics was used to determine ensembles of 40 structures consistent with these restraints. The structures are found to consist of an alpha-helix packed against a four-stranded antiparallel-parallel-antiparallel beta-sheet. The structures of the two domains are compared to each other and to the reported structure of a similar domain from a protein G from a different strain of Streptococcus. We conclude that the difference in affinity of domains II and III for IgG is due to local changes in amino acid side-chains, rather than a more extensive change in conformation, suggesting that one or more of the residues which differ between them are directly involved in interaction with IgG.     

Sequential 1H NMR assignments and secondary structure of an IgG-binding domain from protein G

Protein G is a member of a class of cell surface bacterial proteins from Streptococcus that bind IgG with high affinity. A fragment of molecular mass 6988, which retains IgG-binding activity, has been generated by proteolytic digestion and analyzed by 1H NMR. Two-dimensional DQF-COSY, TOCSY, and NOESY spectra have been employed to assign the 1H NMR spectrum of the peptide. Elements of regular secondary structure have been identified by using nuclear Overhauser enhancement, coupling constant, and amide proton exchange data. The secondary structure consists of a central alpha-helix (Ala28-Val44), flanked by two portions of beta-sheet (Val5-Val26 and Asp45-Lys62). This is a fundamentally different arrangement of secondary structure from that of protein A, which is made up of three consecutive alpha-helices in free solution (Torigoe et al., 1990). We conclude that the molecular mechanisms underlying the association of protein A and protein G with IgG are different.     

Pollen specific cDNA clones from Zea mays.

We have cloned and sequenced four pollen-specific cDNAs. None of the clones are complete at their 5' ends. One of the clones shows significant homology to the tomato fruit-ripening polygalacturonase and to a pollen-specific polygalacturonase from Oenothera. The other three clones have no significant homologies to any reported sequence.     

The serine acetyltransferase from Escherichia coli. Over-expression, purification and preliminary crystallographic analysis

An expression vector has been constructed which increases the expression of serine acetyltransferase (SAT) from E. coli to 17% of the soluble cell protein. A novel purification procedure, using dye-affinity chromatography, allows purification of SAT to homogeneity. The enzyme has been crystallised from polyethylene glycol, in the presence of L-cysteine (an inhibitor of SAT). The crystals which diffract to beyond 3.0 A resolution are of the tetragonal spacegroup P4(1)2(1)2 (or P4(3)2(1)2) with cell dimensions a = b = 123 A, c = 79 A. Since ultracentrifugation and gel-filtration experiment indicate that purified SAT is a tetramer, there appears to be one-half tetramer in the asymmetric unit (Vm = 2.55 A3/Da).     

Operation Crayweed: Ecological and sociocultural aspects of restoring Sydney’s underwater forests

Operation Crayweed focuses on the restoration of underwater forests that disappeared from the coastline of Sydney, Australia’s largest city, 40 years previously. We show how a combination of science, hands‐on restoration, community engagement and art has helped the project to reach its goals as well as raise awareness about the importance of underwater kelp forests that are experiencing global decline.