A clone coding for Schizophyllum commune beta-glucosidase: homology with a yeast beta-glucosidase

Three identical clones coding for a partial sequence of the Schizophyllum commune beta-glucosidase were isolated from a cDNA library in lambda gt11, using polyclonal antibody to the enzyme. The identity was confirmed by comparison of the amino-terminus of a peptide from a protease lys-C digest with the sequence inferred from the cDNA sequence. A comparison of the sequence with that reported for a beta-glucosidase from Candida pelliculosa revealed a region in the latter with 43% identity in amino acid sequence. There was also a similarity in the S. commune beta-glucosidase to an active site sequence proposed for a S. commune endoglucanase, suggesting the possibility of a common catalytic mechanism for these two glucolytic enzymes.     

Cloning, characterization, and nucleotide sequence of a gene encoding Microbispora bispora BglB, a thermostable beta-glucosidase expressed in Escherichia coli.

Genomic DNA fragments encoding beta-glucosidase activities of the thermophilic actinomycete Microbispora bispora were cloned into Escherichia coli. Transformants expressing beta-glucosidase activity were selected by their ability to hydrolyze the fluorogenic substrate 4-methylumbelliferyl-beta-D-glucoside. Two genes encoding beta-glucosidase activity were isolated and distinguished by restriction analysis, Southern hybridization, and the substrate specificities of the encoded enzymes. One gene, bglB, encoded a beta-glucosidase that was expressed intracellularly in E. coli. It exhibited a molecular mass of approximately 52,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and 51,280 Da by nondenaturing gradient PAGE, a pI of 4.6, and temperature and pH optima of 60 degrees C and 6.2, respectively. Cloned BglB showed greater activity against cellobiose than against aryl-beta-D-glucosides and was thermostable, retaining about 70% of its activity after 48 h at 60 degrees C. BglB activity is activated two- to threefold in the presence of 2 to 5% (0.1 to 0.3 M) glucose. The DNA sequence of the 2.2-kb insert carrying bglB has been determined. An open reading frame which codes for a protein of 473 amino acids with a predicted molecular mass of 52,227 Da showed significant homology (40 to 47% identity) with beta-glucosidases from glycosal hydrolase family 1.     

Cloning, characterization, and nucleotide sequence of a gene encoding Microbispora bispora BglB, a thermostable beta-glucosidase expressed in Escherichia coli.

Genomic DNA fragments encoding beta-glucosidase activities of the thermophilic actinomycete Microbispora bispora were cloned into Escherichia coli. Transformants expressing beta-glucosidase activity were selected by their ability to hydrolyze the fluorogenic substrate 4-methylumbelliferyl-beta-D-glucoside. Two genes encoding beta-glucosidase activity were isolated and distinguished by restriction analysis, Southern hybridization, and the substrate specificities of the encoded enzymes. One gene, bglB, encoded a beta-glucosidase that was expressed intracellularly in E. coli. It exhibited a molecular mass of approximately 52,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and 51,280 Da by nondenaturing gradient PAGE, a pI of 4.6, and temperature and pH optima of 60 degrees C and 6.2, respectively. Cloned BglB showed greater activity against cellobiose than against aryl-beta-D-glucosides and was thermostable, retaining about 70% of its activity after 48 h at 60 degrees C. BglB activity is activated two- to threefold in the presence of 2 to 5% (0.1 to 0.3 M) glucose. The DNA sequence of the 2.2-kb insert carrying bglB has been determined. An open reading frame which codes for a protein of 473 amino acids with a predicted molecular mass of 52,227 Da showed significant homology (40 to 47% identity) with beta-glucosidases from glycosal hydrolase family 1.     

The bglA gene of Aspergillus kawachii encodes both extracellular and cell wall-bound beta-glucosidases

We cloned the genomic DNA and cDNA of bglA, which encodes beta-glucosidase in Aspergillus kawachii, based on a partial amino acid sequence of purified cell wall-bound beta-glucosidase CB-1. The nucleotide sequence of the cloned bglA gene revealed a 2,933-bp open reading frame with six introns that encodes an 860-amino-acid protein. Based on the deduced amino acid sequence, we concluded that the bglA gene encodes cell wall-bound beta-glucosidase CB-1. The amino acid sequence exhibited high levels of homology with the amino acid sequences of fungal beta-glucosidases classified in subfamily B. We expressed the bglA cDNA in Saccharomyces cerevisiae and detected the recombinant beta-glucosidase in the periplasm fraction of the recombinant yeast. A. kawachii can produce two extracellular beta-glucosidases (EX-1 and EX-2) in addition to the cell wall-bound beta-glucosidase. A. kawachii in which the bglA gene was disrupted produced none of the three beta-glucosidases, as determined by enzyme assays and a Western blot analysis. Thus, we concluded that the bglA gene encodes both extracellular and cell wall-bound beta-glucosidases in A. kawachii.     

Comparison of cDNA-derived protein sequences of the human fibronectin and vitronectin receptor alpha-subunits and platelet glycoprotein IIb

The fibronectin receptor (FnR), the vitronectin receptor (VnR), and the platelet membrane glycoprotein (GP) IIb-IIIa complex are members of a family of cell adhesion receptors, which consist of noncovalently associated alpha- and beta-subunits. The present study was designed to compare the cDNA-derived protein sequences of the alpha-subunits of human FnR, VnR, and platelet GP IIb. cDNA clones for the alpha-subunit of the FnR (FnR alpha) were obtained from a human umbilical vein endothelial (HUVE) cell library by using an oligonucleotide probe designed from a peptide sequence of platelet GP IIb. cDNA clones for platelet GP IIb were isolated from a cDNA expression library of human erythroleukemia cells by using antibodies. cDNA clones of the VnR alpha-subunit (VnR alpha) were obtained from the HUVE cell library by using an oligonucleotide probe from the partial cDNA sequence for the VnR alpha. Translation of these sequences showed that the FNR alpha, the VnR alpha, and GP IIb are composed of disulfide-linked large (858-871 amino acids) and small (137-158 amino acids) chains that are posttranslationally processed from a single mRNA. A single hydrophobic segment located near the carboxyl terminus of each small chain appears to be a transmembrane domain. The large chains appear to be entirely extracellular, and each contains four repeated putative Ca2+-binding domains of about 30 amino acids that have sequence similarities to other Ca2+-binding proteins. The identity among the protein sequences of the three receptor alpha-subunits ranges from 36.1% to 44.5%, with the Ca2+-binding domains having the greatest homology. These proteins apparently evolved by a process of gene duplication.     

Structure of the cDNA encoding transcobalamin I, a neutrophil granule protein

Transcobalamin I (TCI) is a member of the R binder family of vitamin B12 binding proteins. It is a major protein constituent of secondary granules in neutrophils. We have isolated and characterized full length cDNA clones encoding TCI in order to determine whether its expression is coordinately regulated with the appearance of secondary granules and whether it is consequently a useful marker of granulocyte development. Partial amino acid sequences of human R protein were obtained from tryptic digestion fragments. Using the polymerase chain reaction, a partial TCI cDNA probe was isolated by selective amplification of a region of cDNA located between two oligonucleotides deduced from the available partial amino acid sequences. The amplified probe was then used to obtain full length clones from a granulocyte cDNA library. Identity of the clones was confirmed by matching DNA sequence to known peptide amino acid sequence. TCI is transcribed to a single 1.5-kilobase mRNA species. The predicted protein sequence is 433 amino acids long. We have compared the sequence of TCI to that of rat intrinsic factor. The two proteins have areas of extensive homology which implicate regions potentially important for vitamin B12 binding. TCI mRNA was present in late neutrophil precursors but absent from uninduced and induced HL60 cells.     

A protein with homology to the C-terminal repeat sequence of Octopus rhodopsin and synaptophysin is a member of a multigene family in Dictyostelium discoideum

Monoclonal antibodies were raised against a protein with a molecular mass of 24 kDa that has been described as a membrane-associated, actin binding protein from Dictyostelium discoideum [( 1985) J. Cell Biol. 100, 727-735]. Using these monoclonal antibodies we isolated from a lambda gt11 expression library cDNA clones coding for this protein. The cDNA deduced amino acid sequence revealed the presence of an unusual carboxy-terminus which has homologies to the C-termini of Octopus rhodopsin and synaptophysin. This part of the protein sequence contains 5 direct repeats with the motif GYP (P)Q(P). Southern and Northern blots showed that this sequence is present in a series of Dictyostelium genes transcribed in all stages of development.     

CRRP: a guinea pig protein, identified by sequence homology to human CR1, which contains two short consensus repeat motifs and appears not to be transmembrane or secreted.

cDNA from the C4b-binding site of the human C3b/C4b receptor (CR1) was used to find homologous sequences in the guinea pig. This cDNA identified an 18S mRNA species in guinea pig spleen, but not liver. Probing of a guinea pig spleen cDNA library identified clones with identical 1.5-kb inserts, which also hybridized to mRNA in spleen, but not liver. Sequence analysis of the insert revealed a single long open-reading frame coding for a 20,000 Mr protein consisting of two short consensus repeat motifs homologous to human CR1, and unique sequence at the amino- and carboxy-terminals of the short consensus repeats. This sequence did not encode peptides with features of transmembrane domains or signal peptides. Antibody to this complement receptor-related protein-beta galactosidase fusion protein recognized a 20,000 Mr protein in SDS lysates of guinea pig spleen, lymph node, lymphocytes, neutrophils, and peritoneal macrophages. Immunoprecipitation of human serum by this antibody revealed an 180,000 Mr protein reacting both with the anti-guinea pig protein antibody and with anti-human CR1 antibody. Immunoprecipitation of guinea pig serum revealed no protein reacting with the anti-guinea pig protein antibody. Tissue staining of cultured peritoneal macrophages with this antibody showed intracellular staining, as opposed to membrane staining obtained with anti-guinea pig Ig antibody. The lack of membrane expression was confirmed by surface protein radiolabeling experiments and by fluorescent staining of surface proteins. Thus, we have identified a guinea pig protein with homology to human CR1, which may have an unusual property for this class of proteins in that it appears to be intracellular.     

cDNA cloning and deduced amino acid sequence of microvitellogenin, a female specific hemolymph and egg protein from the tobacco hornworm, Manduca sexta

Microvitellogenin is a female-specific yolk protein from the tobacco hornworm moth Manduca sexta. A cDNA library was constructed from poly(A)+ RNA isolated from adult female fat body. cDNA clones of mRNA for microvitellogenin were isolated by using antiserum against microvitellogenin. Northern blot analysis of poly(A)+ RNA isolated from different life stages and sexes reveals that mRNA coding for microvitellogenin is only present in adult female fat body. Immunoprecipitation of the protein product translated from hybrid selected mRNA indicates that the cDNA clone is specific for microvitellogenin. The complete nucleotide sequence of the 834-base pair cDNA insert has been determined by the dideoxy chain termination method. The cDNA sequence predicts that microvitellogenin is a protein of 232 residues with a calculated molecular weight of 26,201. The cDNA also predicts an amino-terminal extension of 17 residues which are not present in the mature form. This sequence appears to be a signal peptide. A comparison of the translated amino acid sequence with the sequences in the National Biomedical Foundation protein library did not establish any sequence homology with other known proteins.     

Cloning and sequence analysis of beta-4 cDNA: an integrin subunit that contains a unique 118 kd cytoplasmic domain

The alpha 6 beta 4 complex is a member of the integrin superfamily of adhesion receptors. A human keratinocyte lambda gt11 cDNA library was screened using a monoclonal antibody directed against the beta 4 subunit. Two cDNAs were selected and subsequently used to isolate a complete set of overlapping cDNA clones. The beta 4 subunit consists of 1778 amino acids with a 683 amino acid extracellular domain, a 23 amino acid transmembrane domain and an exceptionally long cytoplasmic domain of 1072 residues. The deduced amino-terminal sequence is in good agreement with the published amino-terminal sequence of purified beta 4. The extracellular domain contains five potential N-linked glycosylation sites and four cysteine-rich homologous repeat sequences. The extracellular part of the beta 4 subunit sequence shows 35% identify with other integrin beta subunits, but is the most different among this class of molecules. The transmembrane region is poorly conserved, whereas the cytoplasmic domain shows no substantial identity in any region to the cytoplasmic tails of the known beta sequences or to other protein sequences. The exceptionally long cytoplasmic domain suggests distinct interactions of the beta 4 subunit with cytoplasmic proteins.     

Molecular cloning, characterization, and expression of a human 14-kDa lectin

Full length cDNAs coding for a 14-kDa beta-galactoside binding lectin have been isolated from HL-60 cells and human placenta. Oligonucleotide probes based on a pentapeptide present in several partial sequences of homologous human lectins were used to screen a lambda GT10 HL-60 cDNA library. The HL-60 cDNA clones that were isolated were used to design a synthetic primer representing the 3'-untranslated region of the HL-60 lectin. This primer was then used to synthesize a lambda GT10 human placenta cDNA library, and restriction fragments of the HL-60 cDNA clones were used to screen the library. The cDNA clones for both HL-60 and placenta lectin had identical sequences with short 5'- and 3'-untranslated regions and coded for a 135-amino acid protein which lacks a hydrophobic signal peptide sequence. Biochemical data show that, despite the presence of a possible N-linked glycosylation site, the protein is not glycosylated. Northern and Southern blot analyses indicate that the 14-kDa lectin is encoded for by a single gene. The lectin cDNA was expressed in Escherichia coli and biologically active protein was purified from cell lysates by affinity chromatography.     

Monocyte adherence results in selective induction of novel genes sharing homology with mediators of inflammation and tissue repair

Adherence of monocytes to endothelial cells or extracellular matrices is likely to play a critical role in triggering monocyte activation in extravascular sites of infection, chronic inflammatory disorders, tissue damage and neoplastic growth. We have constructed a cDNA library from monocytes adhered for 30 min on plastic and have screened it by differential hybridization for mRNA rapidly induced by adherence. Two of the cDNA isolated have been identified as IL-1 beta and superoxide dismutase. Sequence data from three other adherence specific clones demonstrates the presence of ATTTA mRNA instability sequences in their 3' untranslated regions signifying inflammation-associated genes. The deduced amino acid sequences indicate the presence of open reading frames with sequence homologies to a family of host defense cytokines, one of them being identified as IL-8. Of the 14 clones initially identified, 4 have been analyzed for induction of mRNA expression. Although 3 of the 4 clones were equally induced by PMA and LPS under nonadherent conditions, all 4 cDNA showed distinct patterns of induction with adherence to extracellular matrix components or endothelial cells. The deduced amino acid sequence homologies indicate that we have isolated cDNA that code for three unique gene products. These cDNA belong to a gene family of early host defense cytokines involved in inflammation and cell growth, but which are differentially regulated by adherence to different surfaces.     

Abundant mRNAs specific to the developing cotton fibre

 Five fibre-specific cDNA clones were isolated by differential screening of a cDNA library from cotton fibres, a developmentally synchronous population of non-dividing cells. The genes corresponding to these cDNAs were expressed preferentially in fibre cells and exhibited differing patterns of temporal expression during fibre development. One cDNA encoded a lipid transfer protein (LTP), and a second encoded a member of a group of well-characterised proline-rich proteins (PRP) from plants. The presence of signal peptide-encoding sequences suggests that both the LTP and the PRP are targeted to the extracellular matrix of the fibre, and a role is envisaged for each in cell elongation. Sequence analysis showed that a third clone was similar to a previously reported fibre-specific sequence of unknown function, whilst the remaining two cDNA clones showed no sequence similarity to previously reported plant nucleic acids. Received: 24 September 1996 / Accepted: 18 October 1996     

Identification of Trichinella spiralis early antigens at the pre-adult and adult stages

Three expression cDNA libraries from Trichinella spiralis worms 14 h, 20 h and 48 h post-infection (p.i.) were screened with serum from pigs experimentally infected with 20,000 T. spiralis muscle larvae. Twenty-nine positive clones were isolated from the 14 h p.i. cDNA library, corresponding to 8 different genes. A putative excretory-secretory protein similar to that of T. pseudospiralis was identified. Three clones corresponded to a T. spiralis serine proteinase inhibitor known to be involved in diverse functions such as blood coagulation and modulation of inflammation. Screening of the 20 h p.i. cDNA library selected 167 positive clones representing 12 different sequences. The clone with the highest redundancy encoded a small polypeptide having no sequence identity with any known proteins from Trichinella or other organisms. Fourteen clones displayed sequence identity with the heat shock protein (HSP) 70. HSPs are produced as an adaptive response of the parasite to the hostile environment encountered in the host intestine but their mechanism of action is not yet well defined. From the 48 h p.i. T. spiralis cDNA library, 91 positive clones were identified representing 7 distinct sequences. Most of the positive clones showed high similarity with a member of a putative T. spiralis serine protease family. This result is consistent with a possible major role for serine proteases during invasive stages of Trichinella infection and host-parasite interactions.     

Isolation of a large number of novel mammalian genes by a differential cDNA library screening strategy.

As part of the ongoing human and mouse genome projects, the aim of this study was to isolate novel, previously uncharacterized, genes from mouse testis. Two approaches were compared for their effectiveness in isolating novel genes: random, vs differential, complementary DNA (cDNA) cloning methods. In the differential approach, only the cDNA clones containing rare sequences (as determined by preliminary clone hybridization) are further analyzed; in the random approach, cDNA clones are isolated at random from the cDNA library. More than two hundred cDNA clones altogether were analyzed, using a PCR-mediated amplification and sequencing strategy. A comparison of these sequences to nucleic acid and protein sequence databases, revealed that 84% of the isolated rare cDNA clones represented new, previously uncharacterized mouse genes. In contrast, less than 63% of the cDNA clones isolated at random from cDNA libraries, contained novel genes. Thus, the probability of isolating new, previously uncharacterized, mammalian genes from cDNA libraries can be markedly improved by focusing efforts on clones containing rare sequences.     

cDNA of a Novel mRNA Expressed Predominantly in Mouse Kidney

We examined embryonic carcinoma (EC) cells for a potential prototype molecule of C3, the third component of complement. PCR primers, corresponding to the base sequence derived from the C3 cDNA of several species, were used for PCR amplification of the EC cell cDNA. All the PCR products obtained had the same sequence and showed no sequence homology to C3. Subsequently, cDNA clones were isolated from a mouse liver cDNA library using the PCR product as a probe. Unexpectedly, neither the base sequence of the cDNA clones nor the amino acid sequence deduced from the cDNA showed homology to C3, although partial homology was observed to a number of sequences from EST databases. We designated this new clone NCU-G1. Northern hybridization experiments revealed that NCU-G1 is expressed constitutively not only in the mouse fetus but also in various mouse tissues, and is most abundant in the kidney cortex.     

Type XIV collagen is a variant of undulin.

We have isolated undulin, an extracellular matrix protein associated with the surface of collagen fibrils, from chicken embryos. The protein showed a molecular mass of about 600 kDa and is composed of three 210-kDa subunits linked by reducible as well as non-reducible bonds. In contrast to human undulin which reportedly is devoid of collagenous sequences, the chicken protein contained a short triple-helical segment that was sensitive to digestion by bacterial collagenase. Screening of an expression library with affinity-purified antibodies yielded two cDNA clones specific for chicken undulin. Analysis of the amino acid sequence deduced from the nucleotide sequence of these clones showed that the human and the chicken protein shared 71% sequence identity. At the amino-terminus both polypeptides contained several similar repeats related to the type III modules found in fibronectin. Towards the carboxyl terminus, however, the two sequences diverged substantially from each other. While the human sequence terminated in a proline-rich segment, the chicken sequence continued with a domain related to von Willebrand factor, with a domain similar to the noncollagenous domain NC4 of type IX collagen and with a typical collagenous triple helix. A short segment of this sequence was found to be identical with the published sequence of a bovine peptide derived from type XIV collagen. Our protein must therefore represent chicken type XIV collagen. One way to explain these results is the possibility that undulin exists in at least two alternatively spliced variants, one lacking the collagenous domain, as described initially for human undulin, and one containing the triple-helical domain, as found in type XIV collagen.