PCR-based site-directed mutagenesis using primers with mismatched 3'-ends.
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Base Sequence↗
Biomedical subjects
M Nassal
Publications and source records attributed to M Nassal.
Hepatitis B virus nucleocapsid assembly: primary structure requirements in the core protein.
As a step toward understanding the assembly of the hepatitis B virus (HBV) nucleocapsid at a molecular level, we sought to define the primary sequence requirements for assembly of the HBV core protein. This protein can self assemble upon expression in Escherichia coli. Applying this system to a series of C-terminally truncated core protein variants, we mapped the C-terminal limit for assembly to the region between amino acid residues 139 and 144. The size of this domain agrees well with the minimum length of RNA virus capsid proteins that fold into an eight-stranded beta-barrel structure. The entire Arg-rich C-terminal domain of the HBV core protein is not necessary for assembly. However, the nucleic acid content of particles formed by assembly-competent core protein variants correlates with the presence or absence of this region, as does particle stability. The nucleic acid found in the particles is RNA, between about 100 to some 3,000 nucleotides in length. In particles formed by the full-length protein, the core protein mRNA appears to be enriched over other, cellular RNAs. These data indicate that protein-protein interactions provided by the core protein domain from the N terminus to the region around amino acid 144 are the major factor in HBV capsid assembly, which proceeds without the need for substantial amounts of nucleic acid. The presence of the basic C terminus, however, greatly enhances encapsidation of nucleic acid and appears to make an important contribution to capsid stability via protein-nucleic acid interactions. The observation of low but detectable levels of nucleic acid in particles formed by core protein variants lacking the Arg-rich C terminus suggests the presence of a second nucleic acid-binding motif in the first 144 amino acids of the core protein. Based on these findings, the potential importance of the C-terminal core protein region during assembly in vivo into authentic, replication-competent nucleocapsids is discussed.
Amino Acid Sequence↗
Detection of HBV DNA in HBsAg-positive sera after amplification using the polymerase chain reaction.
The presence of hepatitis B virus (HBV) DNA in serum as detected by molecular hybridization is considered the most reliable marker for the presence of complete virions and, therefore, infectivity. This technique, however, has a lower limit of detection of 0.1 pg HBV DNA. Using the polymerase chain reaction (PCR), a technique by which DNA sequences can be amplified selectively, we investigated sera from 30 HBsAg carriers, 6 also positive for HBeAg and 24 negative for HBeAg. After PCR followed by Southern blot, 27 sera were found to be positive for HBV DNA, whereas only 7 sera were positive for HBV DNA in the conventional dot blot. PCR followed by Southern blot analysis lowered the limit of detection to 0.5 fg HBV DNA. Amplified HBV DNA fragments from some samples were directly sequenced without previous cloning. We conclude that PCR is a suitable method to amplify parts of viral genomes present in human sera, and that PCR with subsequent Southern blot analysis allows the detection of hepatitis B virions in the majority of HBsAg-positive sera.
Blotting, Southern↗
Proteaselike sequence in hepatitis B virus core antigen is not required for e antigen generation and may not be part of an aspartic acid-type protease.
The hepatitis B virus (HBV) C gene directs the synthesis of two major gene products: HBV core antigen (HBcAg[p21c]), which forms the nucleocapsid, and HBV e antigen (HBeAg [p17e]), a secreted antigen that is produced by several processing events during its maturation. These proteins contain an amino acid sequence similar to the active-site residues of aspartic acid and retroviral proteases. On the basis of this sequence similarity, which is highly conserved among mammalian hepadnaviruses, a model has been put forward according to which processing to HBeAg is due to self-cleavage of p21c involving the proteaselike sequence. Using site-directed mutagenesis in conjunction with transient expression of HBV proteins in the human hepatoma cell line HepG2, we tested this hypothesis. Our results with HBV mutants in which one or two of the conserved amino acids have been replaced by others suggest strongly that processing to HBeAg does not depend on the presence of an intact proteaselike sequence in the core protein. Attempts to detect an influence of this sequence on the processing of HBV P gene products into enzymatically active viral polymerase also gave no conclusive evidence for the existence of an HBV protease. Mutations replacing the putatively essential aspartic acid showed little effect on polymerase activity. Additional substitution of the likewise conserved threonine residue by alanine, in contrast, almost abolished the activity of the polymerase. We conclude that an HBV protease, if it exists, is functionally different from aspartic acid and retroviral proteases.
Amino Acid Sequence↗
Total chemical synthesis of a gene for hepatitis B virus core protein and its functional characterization.
We have chemically synthesized a DNA duplex of 560 nucleotides that codes for the hepatitis B virus (HBV) core protein. The synthetic gene contains 27 unique internal restriction sites. Thereby, it can easily be mutagenized by replacement of rather short restriction fragments. A number of restriction recognition sequences are in common between the synthetic and the authentic gene, thus allowing for the transfer of synthetic segments into the cloned viral genome. Several unexpected mutations in the synthetic gene were readily corrected utilizing the multiple unique restriction sites. In Escherichia coli, the expression level of the synthetic gene product amounts to about 4% of the total soluble protein. It forms particles closely resembling native HBV cores. After transfer of the synthetic gene into the viral genome, transient expression in a hepatoma cell line yields proteins indistinguishable from the native gene products. The synthetic gene thus provides a useful tool for studies on the structure and function of the isolated HBV core protein as well as the gene and its various products in the viral life-cycle.
Base Sequence↗
Structure-function studies on bacteriorhodopsin. III. Total synthesis of a gene for bacterio-opsin and its expression in Escherichia coli.
We have chemically synthesized a DNA duplex of 757 base pairs which encodes the entire protein sequence of mature bacterio-opsin of Halobacterium halobium. The main aim of the synthesis was to facilitate site-specific mutagenesis in all parts of the gene by replacement of short restriction fragments by their counterparts containing the required nucleotide changes. Therefore, 30 unique restriction sites that are fairly evenly spaced were introduced in the synthetic DNA. A total of 28 oligonucleotides ranging in size from 21 to 69 nucleotides were synthesized corresponding to both strands. The entire gene was assembled from four synthetic fragments of 25, 268, 219, and 245 base pairs. The correctness of the nucleotide sequence was confirmed by sequencing the fragments as well as the complete gene. When expressed under the control of PL promoter in Escherichia coli, the synthetic and the native genes gave similar amounts of bacterio-opsin. Attempts to increase expression of the synthetic gene by introducing codons that are preferred in E. coli or by introduction of a synthetic transcription terminator were without significant effect.
Bacteriorhodopsins↗
Structure-function studies on bacteriorhodopsin. II. Improved expression of the bacterio-opsin gene in Escherichia coli.
The aims of this work have been to express bacterio-opsin with minimal variation from the native primary structure and to improve the level of expression in Escherichia coli. We describe the construction of plasmids in which the bacterio-opsin gene contains only an additional methionine residue at the N terminus and in which the C-terminal aspartic acid encoded in the gene has been deleted to conform to the mature protein. In attempts to improve bacterio-opsin expression, a variety of expression plasmids were constructed in which the promoters and the ribosome-binding sequences were varied. Invariably, in these plasmids, translation but not transcription of the bacterio-opsin gene was limiting. A striking increase in expression of the gene occurred when the codons for several of the N-terminal amino acids were changed to increase the A = T content. Bacterio-opsin expressed in E. coli was degraded with a half-life of 8-10 min. The addition of hydrophobic signal sequences at the N terminus increased the half-life and overall yield of the protein. Bacterio-opsin thus produced regenerated the native bacteriorhodopsin-like chromophore and carried out light-dependent proton translocation at a rate comparable to that of the native bacterio-opsin prepared from the purple membrane.
Amino Acid Sequence↗
Rapid procedure for the detection of hepatitis delta virus RNA in sera of HBsAg-positive and anti-delta-positive patients.
A synthetic oligonucleotide duplex of 78 bp corresponding to part of the recently published RNA sequence of hepatitis Delta virus (HDV) was cloned into the plasmid pSBO1 and used for the detection of HDV RNA in sera of patients with chronic HDV infection by molecular hybridization. RNA containing the 78 bp sequence was synthesized in vitro and used as a positive control. For this purpose, a fragment containing the cloned oligonucleotide was transferred into the plasmid pSPT 18. HDV RNA was present in 5 out of 32 hepatitis B surface antigen (HBsAg)- and anti-HD-positive patients. It was neither found in the sera of 19 HBsAg-positive, anti-HD-negative patients, nor in the sera of 26 patients with chronic liver disease negative for both HBsAg and anti-HD. The method appears to be suitable for the detection of viruses of which either only parts of the genome or the entire sequence is known.
Antibodies, Viral↗
Total synthesis of a gene for bovine rhodopsin.
To carry out systematic structure-function studies of bovine rhodopsin by specific amino acid replacements, we have accomplished the total synthesis of its gene, which is 1057 base pairs long. The synthetic gene contains 28 unique restriction sites that are on the average 60 base pairs apart. Replacement of specific restriction fragments by synthetic counterparts containing the desired nucleotide changes permits specific mutagenesis in all parts of the gene. The synthesis of the gene involved enzymatic joining of a total of 72 synthetic oligonucleotides, 15-40 nucleotides long, to form DNA duplexes. The total gene was assembled from three synthetic fragments that were cloned. All synthetic oligonucleotides were characterized by 5'-end analysis, and the accuracy of the joining reactions was confirmed by sequencing the three fragments as well as the complete gene.
Animals↗
The phalloidin binding site of F-actin.
Tritium-containing affinity-labelling derivatives of phalloidin, an alkylating iodoacetyl compound (EAL) and a photolabile, carbene generating diazirine (PAL), have been reacted with rabbit muscle actin, the former after protection of thiol groups with N-ethylmaleimide. Labelled peptides generated by tryptic and/or thermolysin digestion were isolated by paper peptide mapping and characterized by determination of their amino acid sequences. EAL binds to methionine-119 and methionine-355; PAL binds to glutamic acid-117. These residues are located in regions with extremely conserved amino acid sequences. The cleft between the two domains of the actin monomer is suggested as the possible binding site for phalloidin.
Actins↗
Identity of hepatic membrane transport systems for bile salts, phalloidin, and antamanide by photoaffinity labeling.
Phalloidin, a bicyclic heptapeptide, and antamanide, a monocyclic decapeptide from the poisonous mushroom Amanita phalloides, interact with bile-salt-binding polypeptides of the hepatocyte membrane, as demonstrated by photoaffinity labeling using the photolabile bile salt derivative 7,7,-azo-3 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oic acid, either unconjugated or taurine conjugated. With the photolabile derivatives of phalloidin, N-delta-(4-[(1-azi-2,2,2-trifluoroethyl) benzoyl]-beta-alanyl)-delta-aminophalloin, (N epsilon-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]lys6)-anta manide, the same membrane polypeptides with apparent MrS of 54,000 and 48,000 were labeled as with the photolabile derivatives of unconjugated and conjugated bile salts. The presence of bile salts decreased markedly the extent of labeling of these phalloidin- and antamanide-binding polypeptides. These results indicate that hepatic uptake systems for bile salts, phallotoxins, and the cycloamanide antamanide are identical, thus explaining the organotropism of phallotoxins.
Affinity Labels↗
A carbene-generating photoaffinity probe for beta-adrenergic receptors.
A new radioiodinated (2.2 Ci/mumol) iodocyanopindolol derivative carrying a 4-(3-trifluoromethyldiazirino)benzoyl residue has been synthesized. The long-wavelength absorption of the diazirine permits formation of the carbene by photolysis under very mild conditions. [125I]ICYP-diazirine binds with high affinity (Kd = 60 pM) to beta-receptors from turkey erythrocyte membranes. Upon irradiation, [125I]ICYP-diazirine is covalently incorporated in a Mr 40 000 protein. Stereoselective inhibition of photolabeling by the (-)enantiomers of alprenolol and isoproterenol indicated that the Mr 40 000 protein contains a beta-adrenergic binding site. The yield of specific labeling was up to 8.2% of total beta-receptor binding sites. The Mr 40 000 protein photolabeled in the membrane could be solubilized at comparable yield with either digitonin or Triton X-100. Irradiation of digitonin-solubilized turkey erythrocyte membranes with [125I]ICYP-diazirine resulted in specific labeling of two proteins with Mr 40 000 and 50 000. In guinea-pig lung membranes, at least five proteins were photolabeled, of which one (with approximately Mr 67 000) was labeled specifically.
Affinity Labels↗
Carbon-13-N.M.R.-spectra of antamanide, several analogues and their alkali ion complexes.
Natural abundance carbon-13 Fourier transform n.m.r.-spectra were obtained of the cyclic decapeptides [Phe4Val6] antamanide (I); antamanide (II); [Tyr5] antamanide (III); [Ala1] antamanide (IV) and their ion complexes (I)-Na+, (II)-Na+, (II)-Li+, (III)-Na+ and (IV)-Na+. Based upon literature data, systematic comparisons and model compounds, a line assignment approach was performed for the majority of the aliphatic carbons. The spectra of the [Ala9Val6] analogues (V) (Bystrov et al., 1972) and II (measured in CD3CN Patel, 1973a, b) and their complexes were also assigned. Characteristic chemical shift variations observed upon complex formation were calculated (delta delta free peptide[Me+ complex) for a number of corresponding carbons, revealing shift changes up to 2.4 p.p.m. Preliminary calculations of the theta angle at selected prolines for some ion complexes are included.
Amino Acid Sequence↗
Preparative merits of the mixed anhydride (MA) method in the excess use of DDZ-amino acids in the peptide synthesis of biologically active new antamanide analogues.
The mixed anhydride (MA) method of peptide synthesis is further simplified by the repetitive excess use of Ddz-amino acids. In six comparative preparations of decapeptides this is demonstrated by the ease and speed of the synthetic manipulations, the efficiency of the monitoring of the reactions and purifications, and by the complete recycling of excess amino acid derivatives. In using of Ddz-amino acyl isobutylformate mixed anhydrides, side reactions are effectively suppressed, as proved by good coupling of Ddz-proline on to prolyl-peptides. Hydrophilic, crystalline and biological active antamanide analogues are obtained containg various functional side groups. The known cis-conformation of the antamanide prolyl-prolyl bonds is also established in the analogues by 13C-n.m.r. measurements. All new compounds are characterized by molecular peaks in the mass spectra.
Amino Acids↗