Glycoprotein biosynthesis in Saccharomyces cerevisiae. Isolation and characterization of the gene encoding a specific processing alpha-mannosidase.

We have isolated the gene from Saccharomyces cerevisiae encoding an alpha-mannosidase of unique specificity which catalyzes the removal of one mannose residue from Man9GlcNAc to produce a single isomer of Man8GlcNAc (Jelinek-Kelly, S., and Herscovics, A. (1988) J. Biol. Chem. 263, 14757-14763). Amino acid sequence information was obtained and corresponding degenerate oligonucleotide primers were synthesized for polymerase chain reactions on yeast genomic DNA. The labeled polymerase chain reaction products were used to screen a S. cerevisiae genomic library in YEp24, and positive clones of different lengths with similar restriction maps were isolated. A 4.6-kilobase fragment which hybridized with the probes was sequenced. It contained a 1650-base pair open reading frame encoding peptide sequences corresponding to the amino acid sequences of the purified alpha-mannosidase. The gene, designated MNS1, encodes a 549-amino acid polypeptide of calculated molecular size 63,017 Da produced by an mRNA species of approximately 1.7 kilobases. The protein possesses a putative noncleavable signal sequence near its N-terminal region which probably acts as a transmembrane domain. It has three potential N-glycosylation sites and a calcium-binding consensus sequence. Its amino acid sequence is homologous to the recently isolated cDNA from rabbit liver alpha-1,2 mannosidase which can transform Man9GlcNAc to Man5GlcNAc (Moremen, K. W., Schutzbach, J. S., Forsee, W. T., Neame, P., Bishoff, J., Lodish, H. F., and Robbins, P. W. (1990) Glycoconjugate J. 7, 401). Overexpression of the MNS1 gene caused an 8-10-fold increase in specific alpha-mannosidase activity. Disruption of the MNS1 gene resulted in undetectable specific alpha-mannosidase activity but no apparent effect on growth. These results demonstrate that MNS1 is the structural gene for the specific alpha-mannosidase and that its activity is not essential for viability.     

Cloning and sequence analysis of the Escherichia coli metH gene encoding cobalamin-dependent methionine synthase and isolation of a tryptic fragment containing the cobalamin-binding domain

A gene encoding cobalamin-dependent methionine synthase (EC 2.1.1.13) has been isolated from a plasmid library of Escherichia coli K-12 DNA by complementation to methionine prototrophy in an E. coli strain lacking both cobalamin-dependent and -independent methionine synthase activities (RK4536:metE, metHH). Maxicell expression of a series of plasmids containing deletions in the metH structural gene was employed to map the position and orientation of the gene on the cloned DNA fragment. A 6.3-kilobase EcoRI-SalI fragment containing the gene was cloned into the sequencing vector pGEM3B for double-stranded DNA sequencing; the MetH coding region consists of 3372 nucleotides. The enzyme was purified from an overproducing strain of E. coli harboring the recombinant plasmid, in which the level of methionine synthase was elevated 30- to 40-fold over wild-type E. coli. Recombinant enzyme is a protein of 123,640 molecular weight and has a turnover number of 1,450 min-1 in the standard assay. These values are to be compared with previously reported values of 133,000 for the molecular weight and 1,240-1,560 min-1 for the turnover number of the homogenous enzyme purified from a wild-type strain of E. coli B (Frasca, V., Banerjee, R. V., Dunham, W. R., Sands, R. H., and Matthews, R. G. (1988) Biochemistry 27, 8458-8465). Limited proteolysis of the native enzyme with trypsin resulted in loss of enzyme activity but retention of bound cobalamin on a peptide fragment of 28,000 molecular weight. This fragment has been shown to extend from residue 643 to residue 900 of the 1124-residue deduced amino acid sequence.     

Isolation and analysis of the Neurospora crassa Cyt-21 gene. A nuclear gene encoding a mitochondrial ribosomal protein

The Neurospora crassa nuclear mutant cyt-21-1 (originally 297-24; Pittenger, T.H., and West, D.J. (1979) Genetics 93, 539-555) has a defect leading to gross deficiency of mitochondrial small ribosomal subunits. Here, we have cloned the cyt-21+ gene from a N. crassa genomic library, using the sib selection procedure (Akins, R. A., and Lambowitz, A. M. (1985) Mol. Cell Biol. 5, 2272-2278). The genomic clone contains a short split gene encoding a basic protein of 107 amino acid residues. This protein shows strong homology to Escherichia coli ribosomal protein S-16. Comparison of mutant and wild-type mitochondrial ribosomal proteins (Kuiper, M. T. R., Holtrop, M., Vennema, H., Lambowitz, A. M., and de Vries, H. (1988) J. Biol. Chem. 263, 2848-2852) indicates that the cyt-21 gene encodes N. crassa mitochondrial ribosomal protein S-24. The expression of the cyt-21+ gene is regulated such that the level of the putative cyt-21+ mRNA is increased about 5-fold when mitochondrial protein synthesis is inhibited. We suggest that this reflects part of a general mechanism for coordinately activating Neurospora nuclear genes that encode mitochondrial constituents in response to impaired mitochondrial function. This is the first report of the cloning and characterization of a mitochondrial ribosomal protein gene from N. crassa.     

Isolation and sequence of a gene encoding a methionine-rich 10-kDa zein protein from maize

We have isolated the gene encoding a methionine-rich 10-kDa zein protein from a lambda EMBL3 maize genomic 'mini' library of the inbred line BSSS-53 and determined its nucleotide sequence. The sequence matches perfectly with a cDNA clone from the inbred line W22 (which has the same restriction fragment length polymorphism as many inbred lines tested) indicating that we have isolated a functional storage protein gene that is very conserved in maize. This comparison also excludes any splicing of any precursor mRNA and therefore any presence of introns. A number of potential regulatory sequences have been located in the flanking regions. The 10-kDa-zein gene represents the last size class in the zein multigene family to be characterized. Its structure allows us now to re-examine the relationship of all the zein proteins and also to compare the structure of a new class of storage proteins that are rich in methionine, an essential amino acid in livestock fodder.     

Identification of the crossing-over point of a hybrid gene encoding human glycophorin variant Sta. Similarity to the crossing-over point in haptoglobin-related genes

One of the human glycophorin variants, Stones (Sta), has been shown to be the product of a hybrid gene of which the 5'-half derived from the glycophorin B (GPB) gene whereas the 3'-half derived from the glycophorin A (GPA) gene. The present study reveals the crossing-over point of this hybrid gene from the analysis of polymerase chain reaction products. The genomic sequences encompassing the region corresponding to exon 3 to exon 4 of GPA were amplified by polymerase chain reaction with oligonucleotide primers synthesized according to GPA and GPB genomic sequences (Kudo, S., and Fukuda, M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4619-4623). After subcloning the products, the nucleotide sequences derived from GPA, GPB, and putative Sta genes were determined. Comparison of the nucleotide sequences of GPA, GPB, and Sta genes indicate that the crossing-over took place 200 base pairs upstream from the first nucleotide of exon 4. Intriguingly, the nucleotide sequence surrounding the putative crossing-over point is homologous to the crossing-over point proposed for haptoglobin genes (Maeda, N., McEvoy, S.M., Harris, H.F., Huisman, T.H.J., and Smithies, O. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 7395-7399). These results suggest strongly that homologous recombination through unequal crossing-over can be facilitated by specific genomic elements, such as those in common in these two crossing-over events. The present study also revealed that this Sta individual has a variant GPA gene; substitution of adenine for guanine at the nucleotide for codon 39 results in substitution of lysine for arginine at amino acid 39, and loss of an SstI restriction site.     

Isolation, cloning, and sequencing of the Salmonella typhimurium ddlA gene with purification and characterization of its product, D-alanine:D-alanine ligase (ADP forming)

A gene coding for D-alanine:D-alanine (D-Ala-D-Ala) ligase (ADP forming) (EC 6.3.2.4) activity has been isolated from a lambda library of Salmonella typhimurium DNA. Insertion mutations in the gene indicate that the gene is not essential for growth of the bacterium. The encoded enzyme was purified from an overproducing strain of S. typhimurium. D-Ala-D-Ala ligase is a protein of 39,271 molecular weight and has a kcat of 644 min-1 at pH 7.2. A 2.4-kilobase SalI-SphI fragment containing the gene was sequenced, and the ddlA gene consists of 1092 nucleotides. The gene sequence was compared to the sequence of the ddl gene of Escherichia coli [Robinson, A. C., Kenan, D. J., Sweeney, J., & Donachie, W. D. (1986) J. Bacteriol. 167, 809-817]. Because of differences between the S. typhimurium gene and the E. coli ddl gene, the S. typhimurium gene has been named ddlA.     

A gene encoding the proteolipid subunit of Sulfolobus acidocaldarius ATPase complex

An analysis of genes for the major two subunits of the membrane-associated ATPase from an acidothermophilic archaebacterium, Sulfolobus acidocaldarius, suggested that it belongs to a different ATPase family from the F1-ATPase (Denda, K., Konishi, J., Oshima, T., Date, T., and Yoshida, M. (1988) J. Biol. Chem. 263, 17251-17254). In the same operon of the above two genes we found a gene encoding a very hydrophobic protein of 101 amino acids (Mr = 10,362). A proteolipid was purified from the membranes of this bacteria in which partial amino acid sequences matched with the sequence deduced from the gene. Significant amino acid sequence homology and a similar hydropathy profile appeared when the sequence was compared with the 8-kDa proteolipid subunit of F0F1-ATPases. It is about 30 amino acids larger than the 8-kDa proteolipid and has a small (11-amino acid) repeat sequence. However, it is distinct from the 16-kDa proteolipid subunit of an eukaryotic vacuolar H+-ATPase (Mandel, M., Moriyama, Y., Hulmes, J.D., Pan, Y.-E., Nelson, H., and Nelson, N. (1988) Proc. Natl. Acad. Sci. U.S.A. 85,5521-5524).     

A possible receptor-binding function for the N-terminus of connective tissue activating peptide III

Connective tissue activating peptide III (CTAP-III) is an 85-residue peptide which has been purified from platelets and shown to possess mitogenic activity toward a variety of fibroblastic cell lines. beta-Thromboglobulin (beta TG) is an 81-residue peptide which is derived from CTAP-III by cleavage of the N-terminal tetrapeptide Asn-Leu-Ala-Lys which results in the loss of mitogenic activity. The near-UV CD spectra for the two proteins indicated that the conformations as well as the electronic environments of the two disulfide bonds, and also of the single aromatic tyrosine residue, were similar in CTAP-III and beta TG. However, differences in the far-UV CD spectra of these proteins indicated a substantial decrease in alpha-helical content for beta TG (29%) as compared to CTAP-III (38%). Structure prediction analysis also suggested that the longer N-terminal segment of CTAP-III may form an alpha-helix. The N-terminal region of beta TG, which lacks this tetrapeptide, was predicted to be in an unordered, or possibly a turn, conformation. This predicted structural difference appears to be due to the high helix-forming potential of the N-terminal tetrapeptide Asn-Leu-Ala-Lys in CTAP-III. These results suggest a possible structural role for the N-terminal region of CTAP-III in the expression of the biologic activities of this protein. On the basis of these studies, a reasonable hypothesis to account for the difference in mitogenic activity between beta TG and CTAP-III is that the N-terminal region must be helical for receptor binding to occur.     

Bacterial expression and characterization of proteins derived from the chicken calmodulin cDNA and a calmodulin processed gene

Both normal chicken calmodulin (CaM) and a CaM-like mutant protein have been expressed in bacteria, isolated and evaluated with respect to several physical and biological properties. The mutant CaM is derived from a CaM-like gene that lacks intervening sequences and probably evolved from a CaM-processed gene (Stein, J. P., Munjaal, R. P., Lagacé, L., Lai, E. C., O'Malley, B. W., and Means, A. R. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 6485-6489). The mutant CaM protein contains 16 of the 19 amino acids encoded by the CaM-like gene. Normal chicken CaM produced in bacteria is identical to rat CaM by all criteria tested except that it is not trimethylated. The protein product of the CaM-like gene has been termed CaML and exhibits properties which are very similar to CaM despite the presence of 16 amino acid substitutions. CaML binds Ca2+ as evidenced by Ca2+-dependent binding to phenothiazine- and phenyl-Sepharose affinity resins and a Ca2+-dependent electrophoretic mobility shift which is similar to but distinct from CaM. CaML cross-reacts with a monospecific CaM antibody and has an immunodilution curve which is identical to bacterially synthesized CaM. Finally, CaML can maximally activate rat brain phosphodiesterase but with altered kinetic parameters as compared to CaM. These data suggest that the nucleotide substitutions in the putative CaM processed gene are not random but are selected to retain CaM-like functions in the encoded protein. Such a mechanism may exist for other processed genes.     

Isolation and characterization of the nuclear gene encoding the Rieske iron-sulfur protein (RIP1) from Saccharomyces cerevisiae

The nuclear gene encoding the Rieske iron-sulfur protein of the cytochrome bc1 complex of the mitochondrial respiratory chain has been isolated and characterized from Saccharomyces cerevisiae. We used a segment of the iron-sulfur protein gene from Neurospora crassa (Harnisch, U., Weiss, H., and Sebald, W. (1985) Eur. J. Biochem. 149, 95-99) to detect the yeast gene by Southern analysis. Five different but overlapping clones were then isolated by probing a yeast genomic library carried on YEp 13 by colony lift hybridization. Several approaches confirmed that the isolated DNA contained the gene for the Rieske iron-sulfur protein. The yeast gene, which contains no introns, can be expressed in Escherichia coli. A 900-base pair HindIII-EcoRI fragment was subcloned into pUC19 and directed the synthesis of immunodetectable protein. The gene was also identified by disruption of its chromosomal copy by homologous integration. A 400 base pair PstI-EcoRI fragment cloned adjacent to a HIS3 marker in pUC18 was used as an integrating vector. HIS+ transformants were obtained which were unable to grow on the nonfermentable carbon source glycerol. Southern analysis of the respiration deficient (gly-) strains confirmed that the chromosomal copy of the gene was disrupted, and immunoblots of extracts of the transformants indicated a lack of iron-sulfur protein. A respiration-deficient integrant was transformed to GLY+ by a 2-kilobase pair HindIII-BglII fragment, including a complete copy of the gene, carried on a multicopy episomal vector. Immunoblots with monoclonal antibodies to the iron-sulfur protein indicated overproduction of the protein in the complemented strain and revealed expression of approximately equal amounts of mature iron-sulfur protein and of a protein approximately 3 kDa larger than the mature protein in the complemented strain. A 1.2-kilobase pair segment of DNA from the clone which complemented the disrupted strains was sequenced and found to contain an open reading frame of 645 nucleotides, capable of encoding a 21,946-dalton protein. The gene is flanked by consensus signal sequences for initiation and termination which are common in yeast and is preceded by a possible upstream activating sequence. Amino acid sequence analysis of the amino-terminal end of the mature iron-sulfur protein agreed exactly with that predicted by the nucleotide sequence starting at Lys-31.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cloning and nucleotide sequence of the structural gene encoding for human tryptophanyl-tRNA synthetase.

A structural gene encoding bovine (b) tryptophanyl-tRNA synthetase (WRS) has recently been cloned and sequenced [Garret et al., Biochemistry 30 (1991) 7809-7817]. Using part of this sequence as a hybridisation probe we have cloned and sequenced a structural gene encoding human polypeptide highly homologous with two mammalian proteins, bWRS [Garret et al., Biochemistry 30 (1991) 7809-7817; EMBL accession No. X52113] and rabbit peptide chain release factor [Lee et al., Proc. Natl. Acad. Sci. USA 87 (1990) 3508-3512]. Identification of the sequence encoding a human WRS is based on (i) the presence of 'HIGH' and 'KMSKS' structural motifs typical for class-I aminoacyl-tRNA synthetases [Eriani et al., Nature 347 (1990) 203-206]; (ii) coincidence of the number of SH groups per subunit estimated experimentally [Muench et al., Science 187 (1975) 1089-1091] and deduced from the cDNA sequence (six in both cases); (iii) close resemblance of two WRS polypeptides sequenced earlier [Muench et al., Science 187 (1975) 1089-1091] and the predicted structure in two different regions.     

A cellulose-binding domain-fused recombinant human T cell connective tissue-activating peptide-III manifests heparanase activity

The chemokine connective tissue-activating peptide (CTAP)-III, which belongs to the leukocyte-derived growth factor family of mediators, was previously shown to be mitogenic for fibroblasts. However, it has recently been shown that CTAP-III, released from platelets, can act like a heparanase enzyme and degrade heparan sulfate. This suggests that CTAP-III may also function as a proinflammatory mediator. We have successfully cloned CTAP-III from a lambdagt11 cDNA library of PHA-activated human CD4(+) T cells and produced recombinant CTAP-III as a fusion protein with a cellulose-binding domain moiety. This recombinant CTAP-III exhibited heparanase activity and released degradation products from metabolically labeled, naturally produced extracellular matrix. We have also developed polyclonal and monoclonal antibodies, and these antibodies against the recombinant CTAP-III detected the CTAP-III molecule in human T cells, polymorphonuclear leukocytes, and placental extracts. Thus, our study provides tools to examine further immune cell behavior in inflamed sites rich with extracellular moieties and proinflammatory mediators.     

Cloning and heterologous expression of a Methanococcus vannielii gene encoding a selenium-binding protein

The activation and incorporation of selenium into selenocysteine containing selenoproteins has been well established in an Escherichia coli model system but there is little specific information concerning the transport and intracellular trafficking of selenium in biological systems in general. A selenium transport role is a possible function of a novel 42 kDa selenium-binding protein that recently was purified from Methanococcus vannielii. The gene encoding a monomer of this protein (Sbp) has been cloned, sequenced and heterologously expressed in E. coli. The 8.8 kDa gene product contains 81 amino acids. The recombinant Sbp (rSbp) protein was shown to bind selenium from added selenite. The bound selenium appeared predominantly in dimeric and tetrameric forms of the protein. The gene encoding Sbp occurs in an operon that contains a carbonic anhydrase gene and selenocysteine-containing formate dehydrogenase genes, suggesting possible roles in selenium-dependent formate metabolism.