Primary Structure Data Research Articles

Hydrolytic specificity of the barley grain aspartic proteinase

We recently published the primary structure and inhibition data of the barley grain aspartic proteinase (HvAP, Hordeum vulgare aspartic proteinase)

ATP synthase of yeast mitochondria. Isolation and disruption of the ATP epsilon gene.

The nuclear gene encoding the subunit epsilon of the catalytic sector F1 of the yeast Saccharomyces cerevisiae ATP synthase was cloned and sequenced. Degenerated oligonucleotide primers were constructed from primary structure data. A part of the ATP epsilon gene was amplified by polymerase chain reaction from yeast genomic DNA. From the amplified DNA sequence a nondegenerated oligonucleotide probe was constructed and used for isolating a 2040-base pair EcoRI fragment bearing the whole gene. A 186-base pair open reading frame encoding a 62-amino acid polypeptide is described. The deduced amino acid sequence was one amino acid longer than the mature protein. A null mutant was constructed. The mutant strain was unable to grow on glycerol medium. The mutant mitochondria had no detectable oligomycin-sensitive ATPase activity. The catalytic sector appeared unstable during purification but F0-subunits were still bound to F1. The mutation promoted a highly oligomycin-sensitive uncoupling of the mitochondrial respiration rate.

Exon/intron structure of the human alpha 3(IV) gene encompassing the Goodpasture antigen (alpha 3(IV)NC1). Identification of a potentially antigenic region at the triple helix/NC1 domain junction.

The Goodpasture antigen has been identified as the non-collagenous (NC1) domain of alpha 3(IV), a novel collagen IV chain (Saus, J., Wieslander, J., Langeveld, J., Quinones, S., and Hudson, B.G. (1988) J. Biol. Chem. 263, 13374-13380). In the present study, the exon/intron structure and sequence for 285 amino acids of human alpha 3(IV), comprising 53 amino acids of the triple-helical domain and the complete NC1 domain (232 amino acids), were determined. Based on the comparison of the amino acid sequences of the alpha 1(IV), alpha 2(IV), alpha 3(IV), and alpha 5(IV) NC1 domains, a phylogenetic tree was constructed which indicates that alpha 2(IV) was the first chain to evolve, followed by alpha 3(IV), and then by alpha 1(IV) and alpha 5(IV). The exon/intron structure of these domains is consistent with this evolution model. In addition, it appears that alpha 3(IV) changed most after diverging from the parental gene. Analysis of its primary structure reveals that, at the junction between the triple-helical and NC1 domains, there exists a previously unrecognized, highly hydrophilic region (GLKGKRGDSGSPATWTTR) which is unique to the human alpha 3(IV) chain, containing a cell adhesion motif (RGD) as an integral part of a sequence (KRGDSGSP) conforming to a number of protein kinase recognition sites. Based on primary structure data, we outline new aspects to be explored concerning the molecular basis of collagen IV function and Goodpasture syndrome.

Amplification and direct sequencing of a cDNA encoding human cytosolic 3-hydroxy-3-methylglutaryl-coenzyme A synthase

Cytosolic 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) synthase (E.C. 4.1.3.5) is a highly regulated enzyme involved in isoprenoid biosynthesis and therefore a potential target for cholesterol-lowering drugs. Up to now, primary structure data have only been available for chicken, rat and hamster HMG-CoA synthase. Using in vitro amplification and direct sequencing, we have determined the nucleotide sequence of the coding region of the human cytosolic 3-hydroxy-3-methylglutaryl CoA synthase cDNA.

The complete sequence of perlecan, a basement membrane heparan sulfate proteoglycan, reveals extensive similarity with laminin A chain, low density lipoprotein-receptor, and the neural cell adhesion molecule.

A heparan sulfate proteoglycan is a component of all basement membranes. This molecule consists of three heparan sulfate side chains linked to a large core protein of approximately 400 kDa. We have isolated seven overlapping murine cDNA clones that encode the entire mRNA sequence of 12.685 kilobases of this molecule. This sequence has a single open reading frame of 3,707 amino acids that encodes for a protein of 396 kDa. Identical or near identical matchups with nine peptide sequences derived from the core protein of the molecule isolated from the Engelbreth-Holm-Swarm tumor were found with the deduced sequence. Sequence analysis and data base comparison of the deduced sequence show the protein to consist of five different domains, most of which contain internal repeats. Domain I contains a start methionine followed by a typical signal transfer sequence and a unique segment of 172 amino acids that contains the three probable sites of heparan sulfate attachment, SGD. Domain II contains four cysteine- and acidic amino acid-rich repeats that are very similar to those found in the LDL receptor and proteins such as GP330. Domain III consists of cysteine-rich and globular regions, both of which show similarity to those in the short arm of the laminin A chain. Domain IV contains 14 repeats of the immunoglobulin superfamily that are most highly similar to the immunoglobulin-like repeats in the neural cell adhesion molecule. Domain V contains three repeats with similarity to the laminin A chain G domain that are separated by epidermal growth factor-like regions not found in the laminin A chain. As the primary structural data agree with the appearance of the molecule in the electron microscope as a series of globules separated by rods, or "beads on a string," we have adopted the name perlecan for this molecule. The variety of domains in perlecan suggest multiple interactions with other molecules.

Stereochemistry of collagen*

This review article, based on a lecture delivered in Madras in 1985, is an account of the author's experience in the working out of the molecular structure and conformation of the collagen triple-helix over the years 1952-78. It starts with the first proposal of the correct triple-helix in 1954, but with three residues per turn, which was later refined in 1955 into a coiled-coil structure with approximately 3.3 residues per turn. The structure readily fitted proline and hydroxyproline residues and required glycine as every third residue in each of the three chains. The controversy regarding the number of hydrogen bonds per tripeptide could not be resolved by X-ray diffraction or energy minimization, but physicochemical data, obtained in other laboratories during 1961-65, strongly pointed to two hydrogen bonds, as suggested by the author. However, it was felt that the structure with one straight NH...O bond was better. A reconciliation of the two was obtained in Chicago in 1968, by showing that the second hydrogen bond is via a water molecule, which makes it weaker, as found in the physicochemical studies mentioned above. This water molecule was also shown, in 1973, to take part in further cross-linking hydrogen bonds with the OH group of hydroxyproline, which occurred always in the location previous to glycine, and is at the right distance from the water. Thus, almost all features of the primary structure, X-ray pattern, optical and hydrodynamic data, and the role of hydroxyproline in stabilising the triple helical structure, have been satisfactorily accounted for. These also lead to a confirmation of Pauling's theory that vitamin C improves immunity to diseases, as explained in the last section.

The primary structure of the mitochondrial genome of Saccharomyces cerevisiae — a review

We have collated and compiled all the available primary structure data on the mitochondrial genome of Saccharomyces cerevisiae. Data concern 78 500 bp, namely 92% of the “long” genomes; they are derived from several laboratory strains. Interstrain differences belong to three classes: (i) a small number of large deletions/additions, mainly concerning introns; (ii) a large number of small (10–150 bp) deletions/additions located in the intergenic sequences; (iii) 1–3 bp deletions/additions and point mutations; the interstrain sequence divergence due to the latter, is of the order of 2% for the strains compared; this low value is, however, an overestimate because of sequence mistakes.

In situ hybridization histochemistry with synthetic oligonucleotides: Strategies and methods

In situ hybridization histochemistry is a useful method for localizing specific mRNA and studying the regulation of gene expression in an anatomical context. Previously, classical recombinant DNA and microbiological techniques have been required to identify and nick-translate the cloned DNAs necessary for in situ hybridization experiments. These requirements can be circumvented by the use of synthetic oligonucleotides complementary to the mRNA of interest. Compared to cloned cDNA probes, oligonucleotides are easy to manufacture, penetrate tissue much more easily, can be made to correspond to a sequence at any point in a known cDNA structure, and allow for the design of more precise controls for in situ studies. We describe a number of considerations in oligonucleotide probe design, including unique probe design from cDNA sequences and mixed probe design from protein primary structure data. The issues of species specificity, G-C content, probe length, tissue-specific mRNA expression, repeated sequences, non-coding region specific probes, and gene family homologies are discussed in an in situ hybridization context. Alternative strategies for mixed probe design are also considered. Information on the synthesis, purification, and sequence confirmation of oligonucleotides is then presented, followed by methods for labeling and using these probes for in situ hybridization histochemistry. The special considerations of specificity controls are addressed, including combined in situ hybridization histochemistry and immunocytochemistry, competition studies, the use of multiple hybridization probes. Tm studies, and Northern analysis of extracted RNA. The current and future directions of research with this technique are considered, with emphasis on the need to improve quantitation in order to facilitate the study of gene expression and regulation at the single cell level.

Sequence organization of the mitochondrial genome of yeast — a review

We have compiled the available primary structural data for the mitochondrial genome of Saccharomyces cerevisiae and have estimated the size of the remaining gaps, which represent 12–13% of the genome. The lengths of sequenced regions and of gaps lead to a new assessment of genome sizes; these range (in round figures) from 85 000 bp for the long genomes, to 78 000 bp for the short genomes, to 74 000 bp for the supershort genome of Saccharomyces carlsbergensis. These values are 8–11 % higher than those previously estimated from restriction fragments. Interstrain differences concern not only facultative intervening sequences (introns) and mini-inserts, but also insertions/deletions in intergenic sequences. The primary structure appears to be extremely conserved in genes and orion sequences, and highly conserved in intergenic sequences. Since coding sequences represent at most 33–35% of the genome, at least two thirds of the genome are formed by noncoding and yet highly conserved sequences. The G + C level of genes or exons is 25%, and that of intronic open reading frames (ORFs) 22%; increasingly lower values are shown by intronic closed reading frames (CRFs), 20%, ori sequences, 19%, intergenic ORFs, 17.5% and intergenic sequences, 15%.

The light-harvesting polypeptides of Rhodospirillum rubrum. I. The amino-acid sequence of the second light-harvestng polypeptide B 880-beta (B 870-beta) of Rhodospirillum rubrum S 1 and the carotenoidless mutant G-9+. carotenoidless mutant G-9+.

The light-harvesting complex B 880 from Rhodospirillum rubrum S 1 (wild type) and B 870 from the carotenoidless mutant G-9+ was shown to consist mainly of an organic solvent-(chloroform/methanol-) soluble and an organic solvent-insoluble polypeptide. The isolation and separation of these two low-molecular-mass polypeptides (Mr 6101 and Mr 6079) were achieved by a two-step extraction procedure of chromatophores using in the first step chloroform/methanol containing 0.1M ammonium acetate. Following Sephadex LH-60 chromatography of this first extract a light-harvesting polypeptide (B 870-alpha) was isolated and its complete amino acid sequence was determined (R. Brunisholz et al. (1981) FEBS Lett. 129/1, 150-154, B 880-alpha: G. Gogel et al. (1983) Biochim. Biophys. Acta 746, 32-39). Upon reextraction of the chromatophore pellet with chloroform/methanol/ammonium acetate containing in addition acetic acid a second low-molecular-mass polypeptide (B 880-beta of B 870-beta) was generated. The complete amino acid sequences of the chloroform/methanol-insoluble light-harvesting polypeptide of Rs. rubrum S 1 (B 880-beta) and of Rs. rubrum G-9+ (B 870-beta) were determined. They are identical and consist of 54 amino acid residues. The conserved histidine residue within the hydrophobic stretch raises more evidence for ligand complexation of bacteriochlorophyll to this specific histidine residue which therefore possibly plays the key role in pigment-protein interactions. Both polypeptides (B 880-alpha and B 880-beta) are part of the light-harvesting complex B 880 in an apparent ratio of 1:1. Based on the primary structure data a possible arrangement of both light-harvesting polypeptides within the membrane will be discussed.

Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria. III) The primary structure of thermophilic lactate dehydrogenase from Bacillus stearothermophilus. Hydroxylamine-, o-iodosobenzoic acid- and tryptic-fragments. The complete amino-acid sequence.

Based on the partial sequence of the cyanogen bromide fragments [Tratschin, J.D., Wirz, B., Frank, G. and Zuber, H. (1983) Hoppe-Seyler's Z. Physiol. Chem. 364, 879-892], the amino-acid sequence of thermophilic lactate dehydrogenase from B. stearothermophilus was completed by the preparation and sequencing (sequenator, carboxypeptidase A and Y) of further overlapping fragments. Suitable peptide fragments were obtained by lactate dehydrogenase cleavage with hydroxylamine, o-iodosobenzoic acid and trypsin. The polypeptide chain of thermophilic lactate dehydrogenase from B. stearothermophilus consists of 317 amino-acid residues. While sequence homology with mesophilic lactate dehydrogenase of higher organisms reaches 35%, it is substantially higher with this mesophilic enzyme of bacillae (greater than 60%, B. megaterium, B. subtilis). The secondary structure elements and amino-acid residues of the active site of thermophilic lactate dehydrogenase deducted from primary structure data were compared with those from the mesophilic enzyme, the same was done for the internal sequence homology at the nucleotide-binding units. A comparative structure analysis (matrix system) based on the primary structure data of thermophilic enzyme should provide insight into the characteristic structure differences between thermophilic and mesophilic lactate dehydrogenase.

Secondary structure prediction of protamines

Protamines are rery basic, small proteins with about 70% arginine content. Molecular weight ranges from 4000 in fish to ∼6000–7000 in mammals and birds. During spermatogenesis protamines replace histones to build an extremely compact DNA protamine complex in which transcription is inhibited. Circular dichroism. X-ray diffraction and electron microscopy studies have yielded different and non compatible models for the mechanism of interaction between DNA and protamine. Chou and Fasman's method for prediction of secondary structure based on primary structure data allows us to postulate a unique structural pattern for protamines obtained from fish, mammals and birds. The main feature of the structure is the presence of β-turns which give rise to parallel chains with or without β-sheet configuration. The β-turns give a strong globular character to the molecular, the presence of α-helix being excluded from the structure. This structural model permits us to reconcile some of the existing models with the experimental data available.

Ca 2+-induced spectral changes in human prothrombin

Ca 2+ binding to human prothrombin as well as to the N-terminal region of the molecule, prothrombin fragment 1, is a highly cooperative process. This suggests that the filling of these Ca 2+ binding sites is related to a conformational change in the protein. We have attempted to characterize such a change by difference spectroscopy in the ultraviolet region and by solvent perturbation studies. Ca 2+ binding to human prothrobin is associated with a red-shift tryptophan difference spectrum, the contribution of tyrosine residues being minimal. An identical spectrum is observed upon the binding of Ca 2+ to prothrombin fragment 1. Conversely, no spectral change can be obtained with the fragment 2 region. The fact that such spectral change is not decreased in an apolar solvent (20% glycerol) supports the idea that it is due mainly to a local charge effect of Ca 2+ on chromophore. Primary structure data and comparison with similar results already performed on Factor Xa suggest that the tryptophan residue located in the vicinity of Ca 2+ binding sites and, therefore, involved in the spectral changes might be tryptophan 41. In the absence of Ca 2+, solvent perturbations of prothrombin with 20% glycerol result in a large difference spectrum involving tryptophan and tyrosine residues. This spectral change is markedly reduced in the presence of 2 mM Ca 2+, suggesting that, besides a local vicinity charge effect, the binding of Ca 2+ to prothrombin leads to an overall structural change in the protein corresponding to the burial of an average of 1.7 tryptophan and 6 tyrosine.

Physicochemical and immunological properties of a μ chain disease protein

The physicochemical and immunological properties of a μ heavy chain disease protein (BO) have been investigated. The native protein has a mol. wt of 520,000 daltons and it is constituted of 10 polypeptide chains interlinked by 30 interchain disulfide bonds. It was shown that each polypeptide chain contains the Cμ4, Cμ3 and Cμ2 domains and the 160–170 missing residues are probably located at the level of the V H−Cμ1 regions. Protein BO was shown to contain one residue of galactosamine per polypeptide chain. Primary structure data indicate that significant amino acids changes occur in the Fcμ region of this protein as compared with the Fc structure of a Waldenström IgM molecule.

A New Histone from Trout Testis

Abstract A new histone has been isolated from testis cells of the rainbow trout (Salmo gairdnerii) and the initial characterization of this protein has been performed. This histone (forming about 0.5% of the total histones), was easily dissociated from purified chromatin either by chemical modification with maleic anhydride or by extraction at low salt or trichloroacetic acid concentrations. The histone was purified either by gel filtration on Bio-Gel P-10 eluted with 0.01 n HCl or by ion exchange chromatography on carboxymethylcellulose eluted with a linear gradient of lithium chloride. The molecular weight was determined to be 14,500 by the mobility of the formylated derivative during sodium dodecyl sulfateacrylamide gel electrophoresis. Dansylation revealed proline to be the amino terminus and amino acid analyses showed the complete absence of 7 amino acids including the aromatic and sulfur-containing residues. Three amino acids, lysine, alanine, and proline, accounted for 60% of the residues in the protein. Peptide maps of the tryptic peptides of the histone showed 13 unique peptides and thus ruled out an origin by degradation of one of the major histones. The new histone resembles histone I in the high content of lysine and alanine but is more similar to histone IIb2 in its molecular weight of 14,500, a prolyl N terminus, and a lysine to arginine ratio of 3:1. Pending primary structural data which should reveal whether this histone should be classified as more similar to histone I or histone IIb2, we propose to call it histone T (for trout).

Evolutionary and phylogenetic relationships of rubredoxin-containing microbes

The primary structure of the Pseudomonas oleovorans rubredoxin, except for the placement of several residues, is presented. There is evidence for gene duplication in the Pseudomonas oleovorans rubredoxin. When the primary structures of this rubredoxin and the rubredoxins from anaerobic bacteria were compared, homologous sequences were observed. The best homology was obtained by creating an ancestral sequence and then comparing each of the rubredoxins with the ancestral sequence. This had led to the theory that proposed that all of the three rubredoxins have arisen from a common ancestor. A phylogenetic tree based on the primary structure data is presented.