Quantification of hepatitis B virus DNA over a wide range from serum for studying viral replicative activity in response to treatment and in recurrent infection.

A new standardized test for hepatitis B virus (HBV) DNA with increased sensitivity and range over previous assays (30 to 10(6) HBV genomes/test) was evaluated in this study. The quantitative results from the test have been validated using international reference specimens of known titer and a reference solution hybridization test. The test has small variability considering the wide dynamic range. The CV was 14% within one experiment and 32% to 39% between independent experiments. Hepatitis B surface antigen (HBsAg)-negative, anti-HBc-positive blood donor sera (n = 25) were all negative for HBV DNA in the new test, whereas 63% (n = 19) of HBsAg-positive healthy carriers had measurable quantities of HBV DNA. In five example cases of chronic hepatitis B patients responding to alfa-interferon treatment but remaining virus positive, HBV DNA was consistently present in posttreatment sera in a titer range 4 x 10(3) to 10(6)/mL not detectable by the conventional hybridization test. In two complete responders, the HBV DNA titer decreased over six orders of magnitude to below cutoff of the test. In four liver transplant recipients with chronic hepatitis B, viral recurrence was detected by the new test at an early stage much before the clinical relapse. Unlike serology, the test was suitable also in patients under anti-HBs immunoprophylaxis. In conclusion, the new colorimetric polymerase chain reaction (PCR) test allowed thousandfold increased sensitivity in quantification of HBV DNA in patient sera. The test may have future applications in improving assessment of efficacy of antiviral treatment and guiding therapeutic interventions.

Quantitative PCR for hepatitis B virus with colorimetric detection.

A novel, sensitive colorimetric test is described for quantification of the initial number of hepatitis B virus (HBV) genomes amplified in PCR. The viral genomes are amplified together with a synthetic internal standard (IS) to correct for the variability of the efficiency factor. One of the two primers is biotinylated, and the amplified mixtures of HBV and IS DNAs are bound to streptavidin-coated microtiter plates for quantitative detection. The ratio of HBV to IS DNA is determined for each sample by hybridization with DNP-containing probes and immunoenzymatic detection. The colorimetric detection is quantitative, rapid, and accurate with a dynamic range from approximately 10(8) to > 10(11) DNA molecules. The initial number of HBV genomes in a clinical sample is interpreted from the signal ratio HBV/IS by using a standard curve, obtained from coamplification of known quantities of synthetic HBV templates with IS. The assay quantified 15 viral genomes from 10 microliters of serum, and its dynamic range was up to five orders of magnitude. After the amplification step, the assay takes > 2 hr, and the method is applicable to automation.

Quantification of hepatitis B virus DNA by competitive amplification and hybridization on microplates.

Present methods for quantification of hepatitis B virus (HBV) particles from serum samples are not sensitive enough for some recent clinical applications. We describe a test that allows quantification of HBV DNA in a broad dynamic range from less than 40 to 10(6) molecules based on competitive PCR. The specimen DNA and a known amount of an internal standard (IS) are co-amplified in the same tube with the same primers, one of which is biotinylated. The two biotinylated products can be quantified by hybridization on microplates coated with streptavidin, because their internal sequences are nonhomologous. An adequate standard curve is obtained by amplifying HBV DNA from a plasmid clone together with an IS. The ratio of amplified HBV DNA to IS DNA enables quantification of the original amount of HBV without tedious titrations of each sample with competitor. The lower limit for quantitative analysis with radioactive probes was between 4 and 40 virus particles in a 10-microliters serum samples.

Characterization of the genes of the 2,3-butanediol operons from Klebsiella terrigena and Enterobacter aerogenes.

The genes involved in the 2,3-butanediol pathway coding for alpha-acetolactate decarboxylase, alpha-acetolactate synthase (alpha-ALS), and acetoin (diacetyl) reductase were isolated from Klebsiella terrigena and shown to be located in one operon. This operon was also shown to exist in Enterobacter aerogenes. The budA gene, coding for alpha-acetolactate decarboxylase, gives in both organisms a protein of 259 amino acids. The amino acid similarity between these proteins is 87%. The K. terrigena genes budB and budC, coding for alpha-ALS and acetoin reductase, respectively, were sequenced. The 559-amino-acid-long alpha-ALS enzyme shows similarities to the large subunits of the Escherichia coli anabolic alpha-ALS enzymes encoded by the genes ilvB, ilvG, and ilvI. The K. terrigena alpha-ALS is also shown to complement an anabolic alpha-ALS-deficient E. coli strain for valine synthesis. The 243-amino-acid-long acetoin reductase has the consensus amino acid sequence for the insect-type alcohol dehydrogenase/ribitol dehydrogenase family and has extensive similarities with the N-terminal and internal regions of three known dehydrogenases and one oxidoreductase.

Random mutagenesis used to probe the structure and function of Bacillus stearothermophilus alpha-amylase.

Mutations that cover the sequence of Bacillus stearothermophilus alpha-amylase were produced by an efficient in vitro enzymatic random mutagenesis method and the mutant alpha-amylases were expressed in Escherichia coli, which also secreted the product. Ninety-eight mutants were identified by sequencing and their enzyme activities were classified into three classes: wild-type, reduced or null. A molecular model of the enzyme was constructed using the coordinates of Takaamylase A and a consensus alignment of mammalian, plant, and bacterial alpha-amylases. The location of mutant amino acids on the model indicate that mutations which destroy or decrease the catalytic activity are particularly clustered: (i) around the active site and along the substrate-binding groove and (ii) in the interface between the central alpha/beta barrel and the C-terminal domain. Exposed loops are typically tolerant towards mutations.

Crystallization of the core protein of cellobiohydrolase II from Trichoderma reesei.

Single crystals of the core protein of the cellulase cellobiohydrolase II have been grown in polyethylene glycol 6000 with the hanging drop method. Successful crystallization occurred only when 82 amino acids were removed from the N terminus by papain cleavage. Crystals belong to the space group P2(1) and have cell constants a = 49.1 A, b = 75.8 A, c = 92.9 A, beta = 103.2. The diffraction pattern extends to better than 2.0 A.

Isolation and characterization of the 3-phosphoglycerate kinase gene (pgk) from the filamentous fungus Trichoderma reesei.

The 3-phosphoglycerate kinase gene (pgk) from Trichoderma reesei was isolated by hybridization with the corresponding Saccharomyces cerevisiae PGK gene. The 1,545 nt long nucleotide sequence of the cloned gene codes for a 416 amino acid protein. The coding sequence contains two introns of 219 and 75 nt, respectively, at positions identical to those corresponding genes from the other filamentous fungi Aspergillus nidulans and Penicillum chrysogenum. This gene codes for two mRNAs of about 1.65 kb and 1.85 kb. The PGK protein of Trichoderma shows extensive homology to the PGKs of other fungi A. nidulans (77%), P. chrysogenum (73%) and Saccharomyces cerevisiae (69%). However, the PGKs of the two other filamentous fungi, A. nidulans and P. chrysogenum, seem to be more closely related to each other than to the T. reesei enzyme.

A new method for random mutagenesis of complete genes: enzymatic generation of mutant libraries in vitro.

A new efficient in vitro mutagenesis method for the generation of complete random mutant libraries, containing all possible single base substitution mutations in a cloned gene is described. The method is based on controlled use of polymerases. Four populations of DNA molecules are first generated by primer elongation so that they terminate randomly, but always just before a known type of base (before A, C, G or T respectively). Each of the four populations is then mutagenized in a separate misincorporation reaction, where the correct base can now be omitted. The regeneration of wild-type sequences can thus be efficiently avoided. Also, the misincorporating nucleotide concentrations can be optimized to give the three possible single mutations in close to equal ratio. The mutagenesis can be precisely localized within a predetermined target region of any size, and vector sequences remain intact. We have mutagenized the DNA coding for the alpha-fragment of Escherichia coli beta-galactosidase, and identified 176 different base substitution mutations by sequencing. The present method gives mutant yields of 40-60%, when the mutants contain about one amino acid change per protein molecule. All types of base substitution mutations can be generated and deletions are rare. The efficiency of this method permits the use of relatively elaborate screening systems to isolate mutants of either structural genes or regulatory regions.

EGIII, a new endoglucanase from Trichoderma reesei: the characterization of both gene and enzyme.

A novel endoglucanase from Trichoderma reesei, EGIII, has been purified and its catalytic properties have been studied. The gene for that enzyme (egl3) and cDNA have been cloned and sequenced. The deduced EGIII protein shows clear sequence homology to a Schizophyllum commune enzyme (M. Yaguchi, personal communication), but is very different from the three other T. reesei cellulases with known structure. Nevertheless, all the four T. reesei cellulases share two common, adjacent sequence domains, which apparently can be removed by proteolysis. These homologous sequences reside at the N termini of EGIII and the cellobiohydrolase CBHII, but at the C termini of EGI and CBHI. Comparison of the fungal cellulase structures has led to re-evaluation of hypotheses concerning the localization of the active sites.

Efficient secretion of two fungal cellobiohydrolases by Saccharomyces cerevisiae.

Two different cellobiohydrolases, CBHI and CBHII, of the filamentous fungus Trichoderma reesei both hydrolyse highly crystalline cellulose. Cellulolytic strains of the yeast Saccharomyces cerevisiae were constructed by transferring cDNAs coding for these enzymes into yeast on an expression plasmid. These cellulolytic yeasts were able to secrete efficiently the large, heterologous proteins to the culture medium. The recombinant cellulases were observed to be heterogeneous in Mr due, at least partly, to variable N-glycosylation. Recombinant CBHII was able to bind to crystalline cellulose, although slightly less efficiently than the native enzyme. Both of the two recombinant cellulases were able to degrade amorphous cellulose. In a fermenter cultivation, around 100 micrograms/ml of CBHII was secreted into the yeast growth medium.

Expression of two Trichoderma reesei endoglucanases in the yeast Saccharomyces cerevisiae.

The cDNA copies of the two endo-beta-1,4-glucanase genes, egl1 and egl3, from the filamentous fungus Trichoderma reesei were expressed in yeast Saccharomyces cerevisiae under the control of the yeast phosphoglycerate kinase gene promoter. Active EGI and EGIII enzyme was produced and secreted by yeast into the growth medium. The recombinant EGI enzyme was larger and more heterogeneous in size than the native enzyme secreted by Trichoderma, due to differences in the extent of N-glycosylation between these two organisms. The morphology of the yeast cells producing EGI or EGIII was clearly different from control strain.

Construction of cDNA libraries by blunt-end ligation: high-frequency cloning of long cDNAs from filamentous fungi.

A simplified cDNA synthesis and cloning method, suitable for efficient generation of cDNA libraries at frequencies up to 10(6) clones/micrograms mRNA, is described. Routine synthesis of transcripts of well over 4 kb is facilitated by the use of high-quality RNA template isolated from materials rich in RNases. Laborious cloning steps, like tailing or addition of linkers, can be omitted by the use of efficient blunt-end ligation to plasmid vectors, and rapid verification as well as characterization of the clones is possible by double-stranded plasmid sequencing. Using this method we have constructed several cDNA libraries of different filamentous fungi and show here the synthesis and cloning of cDNA copies larger than 1.8 kb corresponding to three Trichoderma reesei cellulases.

Homologous domains in Trichoderma reesei cellulolytic enzymes: gene sequence and expression of cellobiohydrolase II.

Fungal cellobiohydrolases are unique enzymes capable of degrading highly ordered crystalline cellulose. We present here the isolation and complete sequence analysis of the chromosomal and cDNA copies of the structural gene (cbh2) coding for one of the major cellobiohydrolases (CBH II) of Trichoderma reesei. We also present data on expression of the cbh2 gene and show that the transcription start points of the cbh2 gene are heterogeneous and are located 32 to 52 bp downstream from a putative TATA box. The derived CBH II protein sequence is 471 amino acids long and the coding region is interrupted by three short introns. Most of the CBH II protein bears no apparent resemblance to CBH I and endoglucanase I. However, a short region of extensive homology is found in all Trichoderma cellulases characterized so far, suggesting that this region is important for cellulose hydrolysis. The implications of this information with regard to the evolution of fungal cellulase genes and the enzymology of cellulose hydrolysis are discussed.

Efficient secretion of Bacillus amyloliquefaciens alpha-amylase by [corrected] its own signal peptide from Saccharomyces cerevisiae host cells [corrected].

The expression and secretion of Bacillus amyloliquefaciens alpha-amylase was studied in yeast Saccharomyces cerevisiae. The Bacillus promoter was removed by BAL 31 digestion and three forms of the alpha-amylase gene were constructed: the Bacillus signal sequence was either complete (YEp alpha a1), partial (YEp alpha a2) or missing (YEp alpha a3). Secretion of alpha-amylase into the culture medium was obtained with the complete signal sequence only. The secreted alpha-amylase was glycosylated and its signal peptide was apparently processed. The glycosylated alpha-amylase remained active. The enzyme produced by the other constructions was not glycosylated and thus probably remained in the cytoplasm.

Homology between cellulase genes of Trichoderma reesei: complete nucleotide sequence of the endoglucanase I gene.

The filamentous fungus Trichoderma reesei produces several endoglucanases (EG) and cellobiohydrolases (CBH) which are involved in cellulose hydrolysis in a complex synergistic manner. We have cloned and sequenced the gene and the full-length cDNA coding for the major endoglucanase EG-I, and compared this to the cbh1 gene sequence to clarify the relationship between the EG and CBH classes of cellulases. The deduced 437-amino acids (aa) long EG-I protein with a 22-aa long signal peptide is 45% identical in aa sequence with CBH-I. The best conserved region is found at the C terminus and shows about 70% homology. The data suggest that the two enzymes have arisen from a common ancestor by gene duplication. Despite this, the intron positions have not been conserved in these genes which both contain two short introns. The deduced EG-I sequence contains six putative N-glycosylation sites, and a putative O-glycosylated region is found near the C terminus, closely resembling a similar region at the C terminus of CBH-I. Comparison of the aa sequences suggests that the evolutionary divergence of EG-I from CBH-I has involved four separate 10-20 aa "deletions" from the ancestral protein.

Transcription and translation of foreign genes in Bacillus subtilis by the aid of a secretion vector.

Expression levels of Bacillus amyloliquefaciens alpha-amylase, Escherichia coli TEM-beta-lactamase, and Semliki Forest virus glycoprotein E1 genes were compared in Bacillus subtilis. All three model genes were expressed by using a secretion vector, constructed by joining the B. amyloliquefaciens alpha-amylase promoter and signal sequence with plasmid pUB110 (I. Palva, M. Sarvas, P. Lehtovaara, M. Sibakov, and L.Kääriäinen, Proc. Natl. Acad. Sci. U.S.A. 79:5582-5586, 1982). When transformed B. subtilis cells were grown to early stationary phase, the amount of beta-lactamase in the culture medium was ca. 10% and that of E1 was ca. 0.01% of the amount of alpha-amylase. The amounts of specific, full-length transcripts of the cloned genes were estimated by Northern blot hybridization to be roughly equal. The half-lives of these transcripts in B. subtilis were also similar. Pulse-chase experiments with [35S]methionine showed that alpha-amylase and beta-lactamase were translated and secreted at comparable rates but that beta-lactamase was degraded during the chase periods. In transformed minicells from B. subtilis, the products of alpha-amylase, beta-lactamase, and E1 genes accumulated at similar rates. We conclude that the expression of the three genes cloned in the secretion vector was similar at the levels of transcription and translation in B. subtilis. In the case of beta-lactamase, the low-yield could be explained by proteolytic degradation of the secreted product by B. subtilis exoproteases, whereas with E1 we could not determine whether the low yield was due to proteolytic degradation, inefficient secretion, or both.

In vivo transcription initiation and termination sites of an alpha-amylase gene from Bacillus amyloliquefaciens cloned in Bacillus subtilis.

The alpha-amylase gene, originally isolated by molecular cloning from chromosomal DNA of Bacillus amyloliquefaciens, is efficiently expressed from its own promoter in a Bacillus subtilis host when present in the multicopy plasmid vector pUB110. The flanking regions of this gene were sequenced and the ends of the in vivo-generated messenger RNA were mapped by the S1 procedure. Outside the coding sequence, the mRNA for alpha-amylase contains about 30 nucleotides at the 5' end and 51 nucleotides at the 3' end. The promoter region has -10 sequence TAAAAT starting eleven nucleotides upstream from the transcription start point, pppU, and the -35 hexanucleotide TTGTTA is separated from it by 16 nucleotides. As indicated by its sequence, the terminator is bidirectional and of the rho-independent kind, and the mRNA can form a long hairpin structure at the very 3' end. The 3' terminus of the transcript does not seem to include a U stretch, although the DNA template codes for U3AU6 at the 3' end of the hairpin sequence. The bulk of the amylase mRNA does not contain any 3'-terminal poly(A).

Secretion of interferon by Bacillus subtilis.

Bacillus subtilis was transformed with a hybrid gene in which the sequence encoding the alpha-amylase signal peptide was joined by a linker to the sequence encoding mature human interferon alpha 2(IFN-alpha 2). The hybrid preprotein was cleaved precisely following the last amino acid of the alpha-amylase signal sequence and was secreted at 0.5--1 mg per liter. IFN-alpha 2, preceded by either one or six amino acids, has the same specific antiviral activity as IFN-alpha 2 itself.