The marine picoplankter Synechococcus sp. WH 7803 exhibits an adaptive response to restricted iron availability

Abstract: Laboratory cultures of marine Synechococcus sp. WH 7803 were grown under conditions of restricted iron availability. The culture medium was adjusted to restrict iron availability: (i) by adding the iron chelator EDDA; (ii) by omitting iron; and (iii) by omitting both iron and EDTA. An adaptive response was observed to these iron-restricted conditions, including a decrease in cellular phycoerythrin and synthesis of a 36 kDa polypeptide in [³⁵S]methionine radiolabelled whole cell lysates separated by SDS-PAGE. The polypeptide was synthesized within 48 h of transferring exponential phase cells to the iron-restricted medium. The protein was localized to the cell membranes and not the cytoplasmic fraction.     

The rad16 gene of Schizosaccharomyces pombe: a homolog of the RAD1 gene of Saccharomyces cerevisiae.

The rad10, rad16, rad20, and swi9 mutants of the fission yeast Schizosaccharomyces pombe, isolated by their radiation sensitivity or abnormal mating-type switching, have been shown previously to be allelic. We have cloned DNA correcting the UV sensitivity or mating-type switching phenotype of these mutants and shown that the correcting DNA is encompassed in a single open reading frame. The gene, which we will refer to as rad16, is approximately 3 kb in length, contains seven introns, and encodes a protein of 892 amino acids. It is not essential for viability of S. pombe. The predicted protein is the homolog of the Saccharomyces cerevisiae RAD1 protein, which is involved in an early step in excision-repair of UV damage from DNA. The approximately 30% sequence identity between the predicted proteins from the two yeasts is distributed throughout the protein. Two-hybrid experiments indicate a strong protein-protein interaction between the products of the rad16 and swi10 genes of S. pombe, which mirrors that reported for RAD1 and RAD10 in S. cerevisiae. We have identified the mutations in the four alleles of rad16. They mapped to the N-terminal (rad10), central (rad20), and C-terminal (rad16 and swi9) regions. The rad10 and rad20 mutations are in the splice donor sequences of introns 2 and 4, respectively. The plasmid correcting the UV sensitivity of the rad20 mutation was missing the sequence corresponding to the 335 N-terminal amino acids of the predicted protein. Neither smaller nor larger truncations were, however, able to correct its UV sensitivity.     

Effect of dietary lipid supplementation on progesterone concentration and reproductive performance in suckled beef cows

Using whole cottonseed as a lipid source, silage-based diets that were isocaloric and isonitrogenous yet varied in lipid level were fed to multiparous cows. In Experiment 1, 48 cows (n = 12 per treatment) were allotted to 1 of 4 treatments where diets were formulated to supply 3.9, 4.3, 5.3 and 6.3% of total lipid. In Experiment 2, 66 cows (n = 22 per treatment) were allotted to 1 of 3 treatments where diets were formulated to supply 3.1, 5.5 and 8.3% of total lipid. Length of the first ovarian cycle, length of the first normal estrous cycle, postpartum intervals to onset of ovarian luteal activity and to first estrus were not affected by diet (P>0.10) in either experiment. Mean progesterone (P(4)) concentrations for first normal estrous cycles were not different (P>0.10) in either experiment. Anestrous periods were divided into 3 phases for analyses: Phase I) parturition to onset of ovarian luteal activity, Phase II) first ovarian luteal activity and Phase III) first normal estrous cycle. No differences were observed in P(4) concentrations during any phase of the postpartum period. In conclusion, isocaloric and isonitrogenous diets with increasing levels of lipid had no effect on reproductive performance in suckled beef in these experiments.     

Characterization of a truncated form of arrestin isolated from bovine rod outer segments.

The inactivation of photolyzed rhodopsin requires phosphorylation of the receptor and binding of a 48-kDa regulatory protein, arrestin. By binding to phosphorylated photolyzed rhodopsin, arrestin inhibits G protein (Gt) activation and blocks premature dephosphorylation, thereby preventing the reentry of photolyzed rhodopsin into the phototransduction pathway. In this study, we isolated a 44-kDa form of arrestin, called p44, from fresh bovine rod outer segments and characterized its structure and function. A partial primary structure of p44 was established by a combination of mass spectrometry and automated Edman degradation of proteolytic peptides. The amino acid sequence was found to be identical with arrestin, except that the C-terminal 35 residues (positions 370-404) are replaced by a single alanine. p44 appeared to be generated by alternative mRNA splicing, because intron 15 interrupts within the nucleotide codon for 369Ser in the arrestin gene. Functionally, p44 binds avidly to photolyzed or phosphorylated and photolyzed rhodopsin. As a consequence of its relatively high affinity for bleached rhodopsin, p44 blocks Gt activation. The binding characteristics of p44 set it apart from tryptic forms of arrestin (truncated at the N- and C-termini), which require phosphorylation of rhodopsin for tight binding. We propose that p44 is a novel splice variant of arrestin that could be involved in the regulation of Gt activation.     

Isolation and Molecular Characterization of Five Marine Cyanophages Propagated on Synechococcus sp. Strain WH7803

Five marine cyanophages propagated on Synechococcus sp. strain WH7803 were isolated from three different oceanographic provinces during the months of August and September 1992: coastal water from the Sargasso Sea, Bermuda; Woods Hole harbor, Woods Hole, Mass.; and coastal water from the English Channel, off Plymouth Sound, United Kingdom. The five cyanophage isolates were found to belong to two families, Myoviridae and Styloviridae, on the basis of their morphology observed in the transmission electron microscope. DNA purified from each of the cyanophage isolates was restricted with a selection of restriction endonucleases, and three distinguishably different patterns were observed. DNA isolated from Myoviridae isolates from Bermuda and the English Channel had highly related restriction patterns, as did DNA isolated from Styloviridae isolates from Bermuda and the English Channel. DNA isolated from the Myoviridae isolate from Woods Hole had a unique restriction pattern. The genome size for each of the Myoviridae isolates was ca. 80 to 85 kb, and it was ca. 90 to 100 kb for each of the Styloviridae isolates. Southern blotting analysis revealed that there was a limited degree of homology among all cyanophage DNAs probed, but clear differences were observed between cyanophage DNA from the Myoviridae and that from the Styloviridae isolates. Polypeptide analysis revealed a clear difference between Myoviridae and Styloviridae polypeptide profiles, although the major, presumably structural, protein in each case was ca. 53 to 54 kDa.     

Carbon Sink-to-Source Transition Is Coordinated with Establishment of Cell-Specific Gene Expression in a C4 Plant

Plants that use the highly efficient C4 photosynthetic pathway possess two types of specialized leaf cells, the mesophyll and bundle sheath. In mature C4 leaves, the CO2 fixation enzyme ribulose-1,5-bisphosphate carboxylase (RuBPCase) is specifically compartmentalized to the bundle sheath cells. However, in very young leaves of amaranth, a dicotyledonous C4 plant, genes encoding the large subunit and small subunit of RuBPCase are initially expressed in both photosynthetic cell types. We show here that the RuBPCase mRNAs and proteins become specifically localized to leaf bundle sheath cells during the developmental transition of the leaf from carbon sink to carbon source. Bundle sheath cell-specific expression of RuBPCase genes and the sink-to-source transition began initially at the leaf apex and progressed rapidly and coordinately toward the leaf base. These findings demonstrated that two developmental transitions, the change in photoassimilate transport status and the establishment of bundle sheath cell-specific RuBPCase gene expression, are tightly coordinated during C4 leaf development. This correlation suggests that processes associated with the accumulation and transport of photosynthetic compounds may influence patterns of photosynthetic gene expression in C4 plants.     

Identification of tyrosines 154 and 307 in the extracellular domain and 653 and 766 in the intracellular domain as phosphorylation sites in the heparin-binding fibroblast growth factor receptor tyrosine kinase (flg).

Four tyrosine residues have been identified as phosphorylation sites in the tyrosine kinase isoform of the heparin-binding fibroblast growth factor receptor flg (FGF-R1). Baculoviral-insect cell-derived recombinant FGF-R1 was phosphorylated and fragmented with trypsin while immobilized on heparin-agarose beads. Phosphotyrosine peptides were purified by chromatography on immobilized anti-phosphotyrosine antibody and analyzed by Edman degradation and electrospray tandem mass spectrometry. Tyrosine residue 653, which is in a homologous spatial position to major autophosphorylation sites in the catalytic domain of the src and insulin receptor kinases, is the major intracellular FGF-R1 phosphorylation site. Residue 766 in the COOH-terminus outside the kinase domain is a secondary site. Tyrosine residues 154 and 307, which are in the extracellular domain of transmembrane receptor isoforms and are in an unusual sequence context for tyrosine phosphorylation, were also phosphorylated.     

3D 13C/1H NMR-based assignments for side-chain resonances of Lactobacillus casei dihydrofolate reductase. Evidence for similarities between the solution and crystal structures of the enzyme

Summary ¹³C-based three-dimensional ¹H–¹H correlation experiments have been used to determine essentially complete ¹³C and ¹H resonance assignments for the amino acid side chains of uniformly ¹³C/¹⁵N labelled L. casei dihydrofolate reductase in a complex with the drug methotrexate. Excellent agreement is observed between these assignments and an earlier set of partial assignments made on the basis of correlating nuclear Overhauser effect and crystal structure data, indicating that the tertiary structure of the enzyme is similar in solution and in the crystal state.To whom correspondence should be addressed.     

Characterization of disulfide bond position in proteins and sequence analysis of cystine-bridged peptides by tandem mass spectrometry.

Tandem mass spectrometry employing high-energy, collisionally activated dissociation (CAD) is shown to be a useful method for sequencing through the cystine bridge of intermolecularly disulfide-bonded peptides. A characteristic triplet of intense fragment ions is observed corresponding to cleavage through and to either side of the disulfide bridge. These fragments define the masses of the linked peptides. Fragments due to peptide chain cleavage are also observed at lower abundance in the product-ion spectra and can be sufficient to sequence both of the disulfide-linked peptides without any prior knowledge of the peptide or protein sequence. Even in cases where the peptide sequence-related product-ion yields are poor, the intensities of the disulfide cleavage ions are usually sufficient to determine the molecular weights of the component cystine-bridged peptides. In this paper we demonstrate that the high-energy CAD tandem MS approach may be used to characterize disulfide-bonded peptides directly in complex enzymatic or chemical digests of native proteins. This obviates the need for individual purification of intermolecularly disulfide-linked peptides prior to analysis. The techniques are illustrated here for synthetic, inter- and intramolecularly disulfide-linked peptides and for human transforming growth factor-alpha (des-Val-Val-TGF-alpha), a compact protein containing 48 residues and three disulfides.