Structure of the human alpha 1-acid glycoprotein gene: sequence homology with other human acute phase protein genes.

We have determined the sequence coding for human alpha 1-acid glycoprotein from two independently isolated cDNA clones and a genomic clone. The aminoacid sequences deduced from the three clones, deriving from three different individuals, are identical. Southern blot analysis on human DNA indicates that there are at least two genes coding for alpha 1-AGP. We propose that alpha 1-AGP found in plasma is a mixture of the products of these two different genes. This is the simpler explanation for the heterogeneity in the aminoacid composition in purified alpha 1-AGP observed by Schmid et al. (1). DNA sequence comparison with cDNA clones coding for human alpha 1-antitrypsin and haptoglobin shows a conserved sequence within the 5' untranslated region which may play a role in the acute phase response.     

Molecular structure of the immunity gene and immunity protein of the bacteriocinogenic plasmid Clo DF13.

The nucleotide sequence of the Clo DF13 DNA region comprising the immunity gene has been determined. We also elucidated the aminoacid sequence of the 40 N-terminal and 7 C-terminal aminoacids of the purified immunity protein. From analysis of the data obtained we were able to locate the immunity gene between 11.7 and 14.5% on the Clo DF13 map, and to determine the complete aminoacid sequence of the immunity protein. It was observed that the Clo DF13 immunity gene encodes an 85 aminoacid protein and is transcribed in the same direction as the cloacin gene. These experimental data support our model, presented elsewhere, which implicates that the cloacin and immunity genes of Clo DF13 are coordinately transcribed from the cloacin promoter. We also present DNA sequence data indicating that an extra ribosome binding site precedes the immunity gene on the polycistronic mRNA. This ribosome binding site might explain the fact that in cloacinogenic cells more immunity protein than cloacin is synthesized. The comparison of the complete aminoacid sequence of the Clo DF13 immunity protein, with the aminoacid sequence data of the purified, comparable Col E3 immunity protein revealed that both proteins have extensive homologies in primary and secondary structure, although they are exchangeable only to a low extent in vivo and in vitro. It was also observed that a lysine residue was modified in immunity protein isolated from excreted bacteriocin complexes.     

Molecular cloning and characterization of human kinectin.

We have identified a human cDNA that is homologous to the chicken kinectin, a putative receptor for the organelle motor kinesin. The human cDNA clone hybridized to a single 4.6-kb mRNA species that codes for a protein of 156 kDa molecular mass. The predicted primary translation product contains an N-terminal transmembrane helix followed by a bipartite nuclear localization sequence and two further C-terminal leucine zipper motifs. In addition, the aminoacid sequence revealed a large region (327-1362) of predicted alpha-helical coiled coils. A monoclonal antibody CT-1 raised against a GST-kinectin fusion protein produced a perinuclear, endoplasmic reticulum-like staining pattern in diverse cell types from different species, indicating evolutionary conservation. Monoclonal antibody CT-1 and anti-chicken kinectin antibodies cross-reacted both in Western blotting and immunoprecipitation with a 160-kDa protein, confirming the antigenic identity of this 160-kDa protein with chicken kinectin. Epitope tagging studies revealed that the nuclear localization sequence motif of kinectin is not functional. Furthermore, a truncated kinesin cDNA lacking the N-terminal hydrophobic domain revealed a nonspecific cytoplasmic staining pattern. Together the data suggest that kinectin is an integral membrane protein anchored in the endoplasmic reticulum via a transmembrane domain.     

Nucleotide sequence of soybean chloroplast DNA regions which contain the psb A and trn H genes and cover the ends of the large single copy region and one end of the inverted repeats.

The soybean chloroplast psb A gene (photosystem II thylakoid membrane protein of Mr 32 000, lysine-free) and the trn H gene (tRNAHisGUG), which both map in the large single copy region adjacent to one of the inverted repeat structures (IR1), have been sequenced including flanking regions. The psb A gene shows in its structural part 92% sequence homology with the corresponding genes of spinach and N. debneyi and contains also an open reading frame for 353 aminoacids. The aminoacid sequence of a potential primary translation product (calculated Mr, 38 904, no lysine) diverges from that of spinach and N. debneyi in only two positions in the C-terminal part. The trn H gene has the same polarity as the psb A gene and the coding region is located at the very end of the large single copy region. The deduced sequence of the soybean chloroplast tRNAHisGUG is identical with that of Zea mays chloroplasts. Both ends of the large single copy region were sequenced including a small segment of the adjacent IR1 and IR2.     

Structural analysis of the human HOX4A homeobox gene.

The HOX4A gene, one of a cluster of homeobox-containing genes on human chromosome 2, has been isolated by screening a genomic cosmid library with the HOX4B cDNA probe. The amino acid sequence was predicted according to the conceptual translation of 13 homology groups of human HOX genes (1). The HOX4A gene consists of at least two exons separated by a long intron of 1860 bp. The HOX4A protein predicted from the nucleotide sequence of the HOX4A gene is comprised of 416 amino acid residues. Comparison of the predicted HOX4A protein with the HOX2G protein revealed three regions of sequence similarity: an N-terminal octapeptide, a hexapeptide (pre-box) upstream of the homeodomain, and the homeodomain at the C-terminus.     

Cloning and sequencing of the human homeobox gene HOX4A.

The HOX4A gene, one of the homeobox-containing genes on human chromosome 2, has been isolated by screening a genomic cosmid library with a HOX4B cDNA probe. The HOX4A gene consists of at least two exons separated by a long intron of 1860 bp. According to conceptual translation, the HOX4A protein is predicted to be composed of 416 amino acid residues. Interestingly, the HOX4A protein has a sequence, Pro-Ala-Ser-Gln-Ser-Pro-Glu-Arg-Ser, eight amino acids downstream from the homeodomain, which is similar to that containing a phosphorylation site in pp60c-src, Pro-Ala-Ser-Gln-Thr-Pro-Asn-Lys-Thr. However, the HOX2G protein, which exhibits a paralogous relationship with the HOX4A protein, does not possess the sequence which is similar to that in pp60c-src. A comparison of the predicted HOX4A protein with the HOX2G protein revealed four regions of amino acid sequence similarities: an N-terminal tetrapeptide, a pentapeptide (pre-box) upstream of the homeodomain, the homeodomain and a C-terminal octapeptide.     

Nucleotide sequence of the EcoRI E fragment of adenovirus 2 genome.

The entire nucleotide sequence of the Ad.2 EcoRI E fragment has been determined using the Maxam and Gilbert method. This sequence of 2222 bp, which maps between coordinate 83.4 and 89.7 contains information relative to the early 3 region and to the fiber gene. Altogether with fragment EcoRI D which has been recently sequenced, they cover the entire Early 3 region in which several mRNA were mapped. The aminoacid sequence of the 16K and 14K protein is deduced. The localization of the 14.5K mRNA directing the synthesis of the third E3 known protein is discussed, as well as the hypothetical existence of three other early 3 proteins, which would have a molecular weight of 11K. The initiator ATG triplet of the fiber protein has been found at coordinate 86.1, it is followed up to the end of the fragment by an open reading frame allowing deduction of 80% of the aminoacid sequence of this protein. Sequences known to be frequently present at the border of exon sequence were used to tentatively localize the additional "Z" late leader.     

Microheterogeneity of the mammalian high mobility group (HMG) proteins 1 and 2 investigated by reverse-phase high performance liquid chromatography

Microheterogeneity within the high mobility group (HMG)-1 and HMG-2 groups of nonhistone chromatin proteins has been investigated using reverse-phase high-performance liquid chromatography (RP-HPLC) under conditions (acetonitrile elution with 0.1% trifluoroacetic acid (TFA) as the counter ion) which separate proteins primarily on the basis of differences in their overall hydrophobicity. RP-HPLC proved to be a fast and efficient means for separating multiple subspecies of both the HMG-1 and HMG-2 proteins from both crude nuclear extracts and from ion-exchange column "purified" protein samples obtained from different types of mammalian cell nuclei. In crude nuclear extracts at least eight different HMG-2 protein species (two major and six minor), but only one major HMG-1 species, could be resolved by RP-HPLC. Three of the minor HMG-2 protein species could be isolated in "pure" form from crude extracts in one RP-HPLC step whereas under the same conditions the two major HMG-2 peaks (as well as the other minor species) were contaminated with either HMG-1 or HMG-3 (a degradation product of HMG-1). In crude extracts the major HMG-1 fraction always seems to be contaminated with one of the HMG-2 subfractions. RP-HPLC analysis of apparently "pure" protein preparations isolated by ion-exchange chromatography techniques revealed that "pure" HMG-1 can be resolved into at least three different protein species and "pure" HMG-2 into at least four different species. Amino acid analyses of different resolvable forms of the HMG proteins were not inconsistent with the suggestion that at least some of these may be primary sequence variants of the individual proteins, but other possibilities also exist.     

Molecular structure and function of the bacteriocin gene and bacteriocin protein of plasmid Clo DF13.

In this paper we present the complete nucleotide sequence of the bacteriocin gene of plasmid Clo DF13. According to the predicted aminoacid sequence the bacteriocin, cloacin DF13, consists of 561 aminoacids and has a molecular weight of 59,293 D. To obtain insight into the structure and function of specific parts of the cloacin molecule, we constructed a hydration profile and we predicted the secondary structure of the protein. According to our predictions, the N-terminus of cloacin DF13 (corresponding to the first 150-180 aminoacids) is relatively hydrophobic and is rich in glycine residues. The data obtained support previous findings that the N-terminal part of cloacin DF13 is involved in translocation of this protein across the cell membrane. The C-terminal part of the cloacin protein is rich in positively charged aminoacids; this might reflect the RNase activity located within this domain. A comparison of the bacteriocin genes and corresponding proteins of Clo DF13 and Col E1 did not reveal any homology at the level of either the nucleotide or the aminoacid sequence. The codon usage of both genes, however, exhibits striking similarities. The sequence data obtained during this study enabled us to present the nucleotide sequence of the entire cloacin operon. The structure of this operon and the regulation of expression of the genes, located within this operon, is discussed.     

Various physico-chemical properties of the Yersinia pestis capsular protein

Some properties of the structure of Y. pestis capsular antigen macromolecules have been studied. The aminoacid composition of F1 protein, the aminoacid sequence of the N-terminal fragment of antigen polipeptide chain were determined. Some peculiarities in the dissociation of capsular antigen macromolecules have been studied. The formation of the product resulting from unterminated thermodissociation of F1 protein oligomeric form, consisting of four subunits, has been registered. The aspects of F1 protein association are discussed.     

Cloning of cDNA encoding rat TCP-1.

We have isolated and sequenced a cDNA encoding a rat homolog of the mouse t-complex polypeptide 1 (TCP-1). Its deduced gene product is a polypeptide of 556 amino acids, with a predicted Mr of 60,341. The similarity between mouse Tcp-1 and the rat homolog is about 94.0% at the nucleotide level and 97.1% at the amino acid level showing the evolutionary conservation of this protein. The similarity of the amino acid sequence of the rat TCP-1 is not significantly biased to any of those from wild (TCP-1B) or from t-haplotype mice (TCP-1A). From a comparison of deduced amino acid sequences of eukaryotic TCP-1 proteins, we found highly conserved domains. Southern blot analysis revealed that there are at least two similar sequences to Tcp-1 in the rat, one is a structural gene and the other seems to be a processed pseudogene.     

Ni(II) and Cu(II) binding with a 14-aminoacid sequence of Cap43 protein, TRSRSHTSEGTRSR

The tetradecapeptide containing the 10 aminoacid repeated sequence on the C-terminus of the Ni(II)-induced Cap43 protein, was analyzed for Ni(II) and Cu(II) binding. A combined pH-metric and spectroscopic UV-VIS, EPR, CD and NMR study of Ni(II) and Cu(II) binding to the blocked CH3CO-Thr-Arg-Ser-Arg-Ser-His-Thr-Ser-Glu-Gly-Thr-Arg-Ser-Arg-NH2 (Ac-TRSRSHTSEGTRSR-Am) peptide, modeling a part of the C-terminal sequence of the Cap43 protein, revealed the formation of octahedral complexes involving imidazole nitrogen of histidine, at pH 5.5 and pH 7 for Cu(II) and Ni(II), respectively; a major square planar 4N-Ni(II) complex (about 100% at pH 9, log K* = -28.16) involving imidazole nitrogen of histidine and three deprotonated amide nitrogens of the backbone of the peptide was revealed; a 3N-Cu(II) complex (maximum about 70% at pH 7, log K*=-13.91) and a series of 4N-Cu(II) complexes starting at pH 5.5 (maximum about 90% at pH 8.7, log K* = -21.39 for CuH(-3)L), were revealed. This work supports the existence of a metal binding site at the COOH-terminal part of the Cap43 peptide.     

Sequence homologies between p36, the substrate of pp60src tyrosine kinase and a 67 kDa protein isolated from bovine aorta

A 67 kDa actin-binding protein was isolated from bovine aorta. Partial amino acid sequence determination of two large thermolysin peptides were used to compare 67 kDa bovine aorta protein and p36 the substrate of pp60src tyrosine kinase. Sequence analysis shows that 67 kDa bovine aorta protein shares common domains with p36 and possesses the consensus aminoacid sequences of mammalian Ca2+-dependent membrane-binding protein and p36/gelsolin.     

Gene expression in alloploids: genetic control of lipopurothionins in wheat

Lipopurothionins are complexes of basic polypeptides and polar lipids found in petroleum ether extracts of wheat endosperm. Location of the structural genes for the protein moiety and of genes probably controlling the lipid moiety has been achieved by analysis of compensated nulli-tetrasomic and ditelosomic lines of Triticum aestivum L. cv. Chinese Spring, as well as of other genetic stocks. There are two electrophoretic variants of the apoprotein designated α and β purothionins. Structural genes for α purothionins are located in the long arm of chromosomes 1B and 1D, and for the β variant in the long arm of 1A. These genes have been tentatively designated Pur-A1, Pur-B1, and Pur-D1. The aminoacid composition of purified α and β purothionins from Triticum aestivum (genomes AABBDD) and T. durum (AABB), and of β purothionin from T. monococcum (AA) is also consistent with this conclusion and suggests that the α purothionin encoded by gene Pur-B1 probably differs from that encoded by gene Pur-D1 in at least three positions of the aminoacid sequence. A gene (or genes) located in the short arm of chromosome 5D markedly affects the level of lipopurothionin but does not affect apoprotein synthesis. It is concluded that they control the lipid moiety which is required for solubility in petroleum ether.     

Localization of sites modified during inactivation of the bovine heart mitochondrial F1-ATPase by quinacrine mustard using [3H]aniline as a probe

The aziridinium of purified quinacrine mustard at 50 microM inactivates the bovine heart mitochondrial F1-ATPase with a pseudo-first order rate constant of 0.07 min-1 at pH 7.0 and 23 degrees C. An apparent Kd of 27 microM for the enzyme-reagent complex was estimated from the dependence of the rate of inactivation on the concentration of quinacrine mustard. The pH inactivation profile revealed that deprotonation of a group with a pKa of about 6.7 is necessary for inactivation. The amount of reagent incorporated into the protein increased linearly with the extent of inactivation. Complete inactivation was estimated to occur when 3 mol of reagent were incorporated/mol of F1. Enzyme, in which steady state ATPase was inactivated by 98% by quinacrine mustard, hydrolyzed substoichiometric ATP with zero order kinetics suggesting that residual activity is catalyzed by F1 in which at least one beta subunit is modified. By exploiting the reactivity of the aziridinium of covalently attached reagent with [3H] aniline, sites modified by quinacrine mustard were labeled with 3H. Isolation of radioactive cyanogen bromide peptides derived from F1 inactivated with the reagent in the presence of [3H]aniline which were identified by sequence analysis and sequence analyses of radioactive tryptic fragments arising from them have revealed the following. About two thirds of the radioactivity incorporated into the enzyme during inactivation is apparently esterified to one or more of the carboxylic acid side chains in a CNBr-tryptic fragment of the beta subunit with the sequence: 394DELSEEDK401. The remainder of the radioactivity is associated with at least two sites within the cyanogen bromide peptide containing residues 293-358 of the beta subunit. From these results it is concluded that inactivation of F1 by the aziridinium of quinacrine mustard is due, at least in part, to modification of one or more of the carboxylic acid side chains in the DELSEED segment of the beta subunit and possibly also to modification of unspecified amino acid side chains between residues 302-356 of the beta subunit.