Adam S. Inglis

The effects of gibberellic acid and abscisic acid on α-amylase mRNA levels in barley aleurone layers studies using an α-amylase cDNA clone

SummaryTwo cDNA clones were characterized which correspond to different RNA species whose level is increased by gibberellic acid (GA3) in barley (Hordeum vulgare L.) aleurone layers. On the criteria of amino terminal sequencing, amino acid composition and DNA sequencing it is likely that one of these clones (pHV19) corresponds to the mRNA for α-amylase (1,4-α-D-glucan glucanohydrolase, EC 3.2.1.1.), in particular for the B family of α-amylase isozymes (Jacobsen JV, Higgins TJV: Plant Physiol 70:1647–1653, 1982). Sequence analysis of PHV19 revealed a probable 23 amino acid signal peptide. Southern hybridization of this clone to barley DNA digested with restriction endonucleases indicated approximately eight gene-equivalents per haploid genome.The identity of the other clone (pHV14) is unknown, but from hybridization studies and sequence analysis it is apparently unrelated to the α-amylase clone.Both clones hybridize to RNAs that are similar in size (∼1500b), but which accumulate to different extents following GA3 treatment: α-amylase mRNA increases approximately 50-fold in abundance over control levels, whereas the RNA hybridizing to pHV14 increases approximately 10-fold. In the presence of abscisic acid (ABA) the response to GA3 is largely, but not entirely, abolished. These results suggest that GA3 and ABA regulate synthesis of α-amylase in barley aleurone layers primarily through the accumulation of α-amylase mRNA.

Studies on the size, chemical composition, and partial sequence of the neuraminidase (NA) from type A influenza viruses show that the N-terminal region of the NA is not processed and serves to anchor the NA in the viral membrane

Abstract Intact neuraminidase (NA) molecules were isolated by detergent treatment of influenza virus particles from NA subtypes, N1, N2, N5, and N8. Incomplete NA molecules (“heads”) were isolated by Pronase digestion of virus particles from the N2, N5, and N8 strains. Sedimentation equilibrium analysis of NA heads from A/Tokyo/67 (N2) influenza virus gave a molecular weight for the tetramer of 200,000. Under nonreducing denaturing conditions these tetramers were found to dissociate into two disulphide-linked dimers of molecular weight 100,000. Using a monomer molecular weight of 50,000, amino acid and carbohydrate analyses show that the Pronase-released monomer contains approximately 397 amino acid residues and 31 carbohydrate residues. Only N -acetylglucosamine, mannose, galactose, and fucose were found suggesting that all sugar units are in N-glycosidic linkage to asparagine residues. Comparative peptide mapping and direct N-and C-terminal sequence analysis of intact NA and Pronase-released heads showed that the membrane-associated stalk region of the enzyme comes from the N-terminal region of the molecule. Maps of tryptic peptides from intact NA molecules yielded a peptide which had the same composition as the N-terminal hexapeptide (predicted from the nucleotide sequence of the NA genes), which is common to all these strains. This peptide was not present in the Pronase-released NA heads of the N2, N5, and N8 strains. Amino acid sequence analysis showed that the N-terminal sequence of intact NA was very different from that of Pronase-released heads, some 73–76 amino acids being removed by Pronase digestion. The C-terminal regions of both forms of NA were the same. The sequence data also show that protein synthesis does start at the AUG codon that is equivalent to the first ATG in the cDNA sequence of the NA gene and that the N-terminus of the NA polypeptide is not modified following its synthesis. No initiating methionine or signal peptide is cleaved off the neuraminidase molecule.

Coat protein of potyviruses. 2. Amino acid sequence of the coat protein of potato virus Y.

The amino acid sequence of the coat protein of potato virus Y (PVY), the type member of the potyvirus group, has been determined by protein sequencing techniques. The protein contains 267 amino acid residues with a calculated mol wt of 29,945. A comparison of the PVY coat protein sequence with those of tobacco etch virus (TEV) and pepper mottle virus (PeMV) predicted from nucleotide sequence data (R. F. Allison, J. G. Sorenson, M. E. Kelly, F. B. Armstrong, and W. G. Dougherty, Proc. Natl. Acad. Sci. USA82, 3969-3972, 1985; W. G. Dougherty, R. F. Allison, T. D. Parks, R. E. Johnston, M. J. Feild, and F. B. Armstrong, Virology 146,282-291, 1985) shows that sequence homology between the coat proteins from PVY and PeMV is 92% and that between PVY and TEV is 62%. These data suggest that PVY and PeMV are much more closely related than previously believed from serological studies.

Major acute phase α1-protein of the rat is homologous to bovine kininogen and contains the sequence for bradykinin: its synthesis is regulated at the mRNA level

Major acute phase protein Bradykinin Kininogen Protein‐biosynthesis regulation Acute phase reaction Inflammation

Amino acid sequence of pilin from Bacteroides nodosus (strain 198), the causative organism of ovine footrot

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Sequence interrelationships of the subunits of vicilin from pea seeds.

Serological studies and comparison of N-terminal amino acid sequences with the amino acid sequence deduced from a cDNA clone have been used to establish the sequence relationships between the subunits of the pea seed storage protein, vicilin. Subunits smaller than Mr∼50 000 (i.e., Mr 34 000, 30 000, 25 000, 18 000, 14 000, 13 000 and 12 000) show extensive homology with molecules within Mr∼50 000 group. Both the sequencing and serological data confirm earlier evidence from studies on vicilin synthesisin vivo andin vitro which indicated that the vicilin subunits smaller than Mr∼50 000 arose by endoproteolytic cleavage of parent molecules within the Mr∼50 000 group. Cleavage in different Mr 50 000 parent molecules containing either one or both of two susceptible processing sites accounts for the formation of all the vicilin subunits smaller than Mr∼50 000, with the possible exception of the Mr34 000 polypeptide. The position of these sites in the putative parents were defined by reference to a complete amino acid sequence deduced from the sequence of DNA complementary to mRNA for one member of the Mr∼50 000 group.

Sequence of a Glycine-Rich Protein from Lizard Claw: Unusual Dilute Acid and Heptafluorobutyric Acid Cleavages

The hard keratins of mammals and birds have been extensively studied over recent years and a considerable body of knowledge is now available on the structure and composition of the constituent proteins and of their arrangement within the keratin structure1. Mammalian keratins have a very characteristic structure in which filaments of about 70A diameter are embedded in a non-filamentous matrix composed of sulfur-rich and glycine-tryosine-rich proteins. The filaments are responsible for the α-type X-ray pattern. In contrast avian keratins contain only one family of proteins arranged as small filaments of about 30A diameter which are responsible for a characteristic X-ray pattern, often referred to as the feather type. No sequence homology has been found between avian and mammalian keratin proteins and it is generally considered that they represent separate evolutionary developments.

Amino acid sequence of conglutin δ, a sulfur-rich seed protein of Lupinus angustifolius L.: Sequence homology with the C-III α-amylase inhibitor from wheat

Complete amino acid sequences and disulfide cross‐link arrangements have been determined for the two subunit polypeptides (M r 9401 and 4597) ofconglutin δ, a helix‐rich seed protein from Lupinus angustifolius cv. Uniwhite. There are two intrachain disulfide bonds and a free sulfhydryl group within the large chain and two interchain disulfide bonds to the small chain. The sequences show regions enriched in glutamineglutamic acid and serine residues which were correlated by a predictive method to the high measured level of α‐helix (~ 38%). Homology was found between a cystine‐rich region ofconglutin δ and the C‐III α‐amylase inhibitor from wheat suggesting that these proteins originated from a common ancestral gene.

Single analysis for cysteine, cystine, and tryptophan in proteins

Abstract Quantitative analysis for cysteinyl, cystinyl and tryptophyl residues, as well as those of the more stable amino acids, may be made on a single column of an amino acid analyzer after acid hydrolysis of either soluble or insoluble proteins. The procedure involves determination of cysteinyl residues as S -β-[4-pyridylethyl]cysteine, and cystinyl residues as S -sulfocysteine, after hydrolysis of the pyridylethyl-protein by 4 m methanesulfonic acid. The presence of tryptamine during hydrolysis was mandatory for quantitative tryptophan recovery and did not affect the subsequent determination of cystine as S -sulfocysteine.

Amino acids analysis using phenylisothiocyanate prederivatization: Elimination of the drying steps

Results of experiments on the procedure for amino acid analysis via analysis of the phenylthiocarbamyl amino acids are reported. It was found that yields of some amino acids varied in the presence of salt and with changes in the vacuum drying steps. An improved procedure is described which includes a standard addition of salt to the hydrolysate before drying it; the redrying step is omitted and the post derivatization drying is replaced by a simple addition of heptane to the reaction mixture.