Identification of c-myc promoter-binding protein and X-box binding protein 1 as interleukin-6 target genes in human multiple myeloma cells.

Interleukin-6 (IL-6) is implicated in the in vivo proliferation of malignant plasma cells in multiple myeloma. To define the molecular basis of the IL-6-induced mitogenic response in myeloma cells, we applied STAR (subtractive transcriptional amplification of mRNA), a new differential expression analysis technology, to isolate mRNAs preferentially expressed in IL-6-treated versus untreated cultures of the factor-responsive myeloma cell line U266. From the resulting collection of STAR clones, sequence information was obtained for a total of 72 distinct transcripts. Of these, 29 were found to correspond to known genes, 22 matched expressed sequence tags in public databases and 21 showed no sequence similarity to any existing entries. Among the known genes uncovered in the screen were those encoding proteins that function in cell division, cell signalling and gene/protein expression. Northern blot analysis documented that two transcription factor genes chosen for further study, c-myc promoter-binding protein (MBP-1) and X-box binding protein 1 (XBP-1), were up-regulated in U266 cells about 3-fold relative to the cell cycle-dependent beta-actin gene 12 h after IL-6 treatment. Both genes were also similarly up-regulated by IL-6 in factor-dependent ANBL-6 myeloma cells. These results indicate that MBP-1 and XBP-1 are IL-6 genes in myeloma cells; as such, they may play a role in IL-6-mediated growth control in multiple myeloma.

A nucleic acid sequence-based amplification system for detection of Listeria monocytogenes hlyA sequences.

A nucleic acid sequence-based amplification system primarily targeting mRNA from the Listeria monocytogenes hlyA gene was developed. This system enabled the detection of low numbers (< 10 CFU/g) of L. monocytogenes cells inoculated into a variety of dairy and egg products after 48 h of enrichment in modified listeria enrichment broth.

Expression and characterization of soluble human erythropoietin receptor made in Streptomyces lividans 66.

A gene encoding the extracellular domain of the human erythropoietin receptor (EPO-R) was constructed using oligonucleotides, with a view to maintaining preferred codon usage for the Streptomycetes. The gene was subcloned into a multicopy Streptomyces-Escherichia coli shuttle vector, pCAN46 (derived from pIJ680), containing a strong constitutive promoter from the S. fradiae aph gene, a signal peptide coding region derived from the protease B gene of S. griseus, and a transcription terminator sequence also derived from the S. fradiae aph gene. Extracellular expression of authentic EPO-R by S. lividans was demonstrated using SDS-PAGE and Western blot analysis, followed by direct amino terminal sequencing of the purified product. Specific binding of S. lividans-expressed EPO-R to recombinant human glycosylated EPO was demonstrated using BIAcore (surface plasmon resonance) analysis and native gel shift assays.

Isolation and characterization of a gene encoding a chymotrypsin-like serine protease from Streptomyces lividans 66.

A gene encoding a Streptomyces lividans homologue of the chymotrypsin-like serine protease (SAM-P20) of Streptomyces albogriseolus was isolated using the Streptomyces griseus prtB gene as a hybridization probe. Nucleotide sequence analysis of a representative clone uncovered the possible presence of a sequence of 900 nucleotides encoding 300 amino acids, including a putative "prepro" region of 115 amino acids. Alignment of the predicted amino acid sequence of this putative gene with other members of the family of Streptomyces extracellular chymotrypsin-like proteases indicated a high degree of homology in all cases, especially with the SAM-P20 protease. This gene product has been identified as the second member of a potentially larger family of SAL (SAM-P20-like) proteases in S. lividans 66.

Cloning and analysis of a gene from Streptomyces lividans 66 encoding a novel secreted protease exhibiting homology to subtilisin BPN'.

Amino-terminal degradation has been observed for many of the secreted heterologous proteins produced by S. lividans 66. We, therefore, set out to characterize the relevant proteinases and their genes. A tripeptide chromogenic substrate was used to identify a gene that was shown to encode a secreted protein which removed tripeptides from the amino terminus of extracellular proteins (tripeptidyl aminopeptidase, Tap; Butler et al. 1995). This activity was removed by a homologous gene deletion replacement and the ability of the S. lividans strain to remove N-terminal tripeptides was greatly reduced, but still significant. When the tap-deleted strain was used as a host for the rescreening of a S. lividans 66 genomic DNA library, a number of other genes encoding proteases with aminopeptidase activities were discovered. One clone (P5-4) produced a 45-kDa secreted protein (Ssp), which showed activity against Ala-Pro-Ala-beta-naphthylamide (APA-beta NH-Nap) substrate. Further analysis of the cloned DNA showed an open-reading frame encoding a protein larger than 45 kDa. Direct Edman degradation of the secreted protein confirmed that it was encoded within the cloned DNA and probably processed from a larger precursor. Protein sequence analysis revealed a striking homology to subtilisin BPN' in three regions around the active-site residues suggesting that the protein is a serine protease. As expected, the protease activity was inhibited by phenylmethylsulphonyl fluoride. Mutant strains with most of the ssp gene deleted exhibited reduced activity against APA-beta NH-Nap substrate compared to their non-deleted parental strains.

Isolation and characterization of two genes encoding proteases associated with the mycelium of Streptomyces lividans 66.

A strain of Streptomyces lividans 66 deleted for a major tripeptidyl aminopeptidase (Tap) was used as a host to screen an S. lividans genomic library for clones overexpressing activity against the chromogenic substrate Ala-Pro-Ala-beta-naphthylamide. In addition to reisolation of the tap gene, clones representing another locus, slpD, were uncovered. slpD was analyzed by deletion subcloning to localize its functional sequence. Nucleotide sequence determination revealed an open reading frame encoding a 55-kDa protein exhibiting significant amino acid sequence homology to Tap, particularly around the putative active-site serine residue. No secreted protein was observed for strains harboring the slpD clone, but inspection of the predicted protein sequence revealed a putative lipoprotein signal peptide (signal peptidase II type), suggesting a mycelial location for the SlpD proteinase. In an attempt to isolate an endoprotease known to be active against some heterologous proteins, a second clone was isolated by using a longer substrate (t-butyloxycarbonyl [Boc]-APARSPA-beta-naphthylamide) containing a chemical blocking group at the amino terminus to prevent aminopeptidase cleavage. This locus, slpE, appeared to also encode a 55-kDa mycelium-associated (lipoprotein) proteinase, whose predicted protein sequences showed significant amino acid homology to Tap and SlpD, particularly around the putative active-site serine residues. Chromosomal integration and deletion analysis in both the wild-type and Tap-deficient backgrounds appeared to indicate that SlpD was essential for viability and SlpE was required for growth on minimal media.

Cloning and characterization of a gene encoding a secreted tripeptidyl aminopeptidase from Streptomyces lividans 66.

The gene encoding a tripeptidyl aminopeptidase (Tap) from Streptomyces lividans was cloned by using a simple agar plate activity assay. Overexpression of the cloned gene results in the production of a secreted protein which has an apparent subunit molecular weight of 55,000 and is responsible for the major amino-terminal degradative activity in culture broths of S. lividans strains. A DNA sequence analysis revealed a potential protein-encoding region of the size expected to encode the observed protein, which contained a sequence that exhibited significant homology around a putative active site serine residue observed for lipases, esterases, and acyl transferases. Preceding the amino terminus of the secreted protein was a predicted signal peptide of 36 amino acids followed by a tripeptide, which could be autocatalytically removed from a secreted Tap precursor. The transcriptional start site for the gene was mapped by primer extension. Mutant strains of S. lividans lacking detectable Tap activity were able to grow and sporulate normally. Cross-species hybridization experiments showed that DNA homologs of the tap gene are present in most of the Streptomyces strains tested.

Fidelity of nucleic acid amplification with avian myeloblastosis virus reverse transcriptase and T7 RNA polymerase.

The Nucleic Acid Sequence-Based Amplification (NASBA) process involves alternate steps of DNA synthesis from an RNA template and RNA synthesis from a DNA template, using avian myeloblastosis virus (AMV) reverse transcriptase and T7 RNA polymerase, respectively. The overall fidelity of the amplification process was determined by sequence analysis of cloned DNA products of NASBA reactions. An error frequency of less than 0.3% was observed in cloned DNA products from two different segments of the HIV-1 gag gene. Partial substitution of GTP with ITP in the NASBA reaction did not significantly change the fidelity of the process. An error rate of 2 x 10(-4) was calculated for the combined effects of both polymerases.

The aminopeptidase N-encoding pepN gene of Streptomyces lividans 66.

The gene (pepN) encoding an aminopeptidase N (PepN) has been cloned from Streptomyces lividans. This was done using either leucine-beta-naphthylamide or arginine-beta-naphthylamide in a liquid overlayer on colonies growing on agar medium to screen for overproduction of the ability to hydrolyse the substrates. The nucleotide sequence of pepN was determined and shown to encode a 95-kDa protein, which displayed significant homology to PepN proteins from other organisms. Analysis of the overproduced proteinase confirmed that this protein was located intracellularly as a monomeric active species. PepN is a metallo-exopeptidase cleaving next to Leu, Arg and Lys in peptide-bond-containing substrates.

Intracellular aminopeptidases in Streptomyces lividans 66.

We have investigated the aminopeptidase activities present in Streptomyces lividans strains. The majority of these activities proved to be intracellular with multiple active species. Two aminopeptidase P genes were identified to be responsible for the ability to hydrolyze amino terminal peptide bonds adjacent to proline residues. Two other broad spectrum aminopeptidases were found to display homology at both the DNA and protein levels. One showed significant homology to PepN proteins, particularly around the putative zinc-binding residues which are important for catalysis. The second broad spectrum activity was not analyzed in detail but showed a different spectrum of substrate specificity to that of PepN.

Quantification of HIV-1 RNA in plasma using NASBA during HIV-1 primary infection.

Quantification of HIV-1 viral RNA in plasma was achieved by competitive co-amplification of a dilution series of in vitro generated RNA using the nucleic acid sequence based amplification (NASBA) technology. This 1.5 kilobase in vitro RNA, comprising the gag and part of the pol region, differs only by sequence-randomization of a 20 nt fragment from the wild-type RNA, ensuring equal efficiency of amplification. In model systems the accuracy of this method is within one log. Application of the Q-NASBA to plasma samples of a patient with a primary HIV-1 infection shows good concordance of the HIV-1 RNA profile with the p24 antigen profile. However, the HIV-1 RNA determination is more sensitive than the p24 antigen determination. Peak values of HIV-1 RNA are around 10(8) RNA molecules per ml plasma at the moment of seroconversion. Quantitative nucleic acid detection methods, like Q-NASBA, allow the monitoring of HIV-1 RNA during the course of infection which might have predictive value for disease development.

Cloning and characterisation of an aminopeptidase P-encoding gene from Streptomyces lividans.

An aminopeptidase P (PepP)-encoding gene has been cloned from Streptomyces lividans 66 by screening for overexpression of activity using the chromogenic substrate Gly-Pro-beta-naphthylamide as a liquid overlayer on colonies growing on agar medium. The pepP gene was localised by deletion mapping, and the nucleotide sequence was determined. The deduced amino acid sequence was found to display significant similarity to Escherichia coli PepP. The partially purified S. lividans enzyme had a 50-kDa subunit and was present as a homodimer. Direct Edman degradation of the purified protein confirmed that pepP encoded the observed intracellular PepP.

Cloning of genetic loci involved in endoprotease activity in Streptomyces lividans 66: a novel neutral protease gene with an adjacent divergent putative regulatory gene.

A skimmed-milk clearing assay was used to identify, in a multicopy Streptomyces lividans 66 genomic library, DNA fragments that lead to increased expression of protease activity in S. lividans 66. Three independent loci were identified. The majority class (slpA, which represented 68 of 71 clones) produced large zones of clearing. Two other classes (designated slpB and slpC) showed smaller zones than slpA. Subcloning and deletion analysis of the slpA locus delineated the relevant DNA to within a 2.5 kilobase pair fragment. DNA sequence analysis revealed a structural gene associated with the appearance of an extracellular protein in the culture medium. The derived amino acid sequence indicated the presence of a zinc-binding motif, which was previously noted to be characteristic of metalloprotease enzymes. However, the relatively small size of the protein (apparent molecular weight 20,000-24,000) suggests that it represents a novel class of neutral proteases distinct from the thermolysin-type enzymes. An adjacent divergent open reading frame was identified and shown to cause a significant increase in protease activity when present together with the protease structural gene on a multicopy plasmid in S. lividans 66. The derived amino acid sequence of this open reading frame showed homology with previously characterized regulatory proteins of the LysR family of transcriptional regulator proteins.

Characterization and structure of genes for proteases A and B from Streptomyces griseus.

Protease A and protease B are extracellular proteins which are secreted by Streptomyces griseus. The genes encoding protease A (sprA) and protease B (sprB) were isolated from an S. griseus genomic library by using a synthetic oligonucleotide probe. Fragments containing sprA and sprB were characterized by hybridization and demonstration of proteolytic activity in Streptomyces lividans. Each DNA sequence contains a large open reading frame with the coding region of the mature protease situated at its carboxy terminus. The amino terminus of each reading frame appears to encode a 38-amino-acid signal peptide followed by a 76- or 78-amino-acid polypeptide, a propeptide, which is joined to the mature protease. Strong homology between the coding regions of the protease genes suggests that sprA and sprB originated by gene duplication.

Heterogeneity of the 5' terminus of hen ovalbumin messenger ribonucleic acid.

The 5'-terminal sequence of hen ovalbumin mRNA was investigated using a novel labeling method. Ovalbumin mRNA was purified by hybridization to complementary DNA coupled to cellulose. The mRNA thus purified was shown to be 97.9% pure by hybridization with plasmid DNA containing sequences to the messengers coding for conalbumin and ovomucoid, the next two most abundant messengers of oviduct. After digestion with RNase T1 and alkaline phosphatase, 5'-terminal capped oligonucleotides were selected by binding to anti-m7G-Sepharose. These were then labeled using RNA ligase and [5'-32P]pCp, separated by two-dimensional gel electrophoresis, and sequenced by partial digestion with base-specific ribonucleases. A nested set of three capped oligonucleotides was identified. Their structures and relative abundances were m7GpppAUACAG, 3% m7GpppACAUACAG, 61+; and m7GpppGUACAUACAG, 36%.