Subcellular localization of recombinant truncated Gag precursor proteins of HIV expressed in Saccharomyces cerevisiae.

OBJECTIVE: To determine signals contained in the HIV-1 Gag precursor implicated in protein transport. DESIGN: To study the localization of truncated Gag proteins expressed in Saccharomyces cerevisiae. METHODS: Thin-section immunoelectron microscopy studies were performed on S. cerevisiae cells producing myristoylated or non-myristoylated Pr55gag, the core protein (p24) and several truncated Gag proteins. RESULTS: Pr55gag and the carboxy-terminal truncated Gag proteins were myristoylated and localized at the plasma membrane. p24 was localized in the nucleus or perinuclear membrane. However, addition of a myristoyl group to p24 targeted this molecule to the plasma membrane. CONCLUSIONS: The myristoylated amino-terminal 214 amino acids are sufficient to target Pr55gag to the plasma membrane. Subcellular signals implicated in protein transport are present in the core p24 polypeptide which may become dominant or accessible in the absence of the amino-myristoyl group.

Simultaneous synthesis and assembly of various hepatitis B surface proteins in Saccharomyces cerevisiae.

Yeast transposon of class-1-based vectors, allowing integration at a series of chromosomal loci by homologous recombination with resident transposons, were constructed. Using such vectors, we have introduced several copies of an expression cassette encoding the major hepatitis B surface protein as well as expression cassettes encoding the middle (M) or/and the large (L) surface protein into Saccharomyces cerevisiae. In extracts of such strains, coassembly of the different proteins into a single lipoprotein structure is observed. This was demonstrated by immunoprecipitation of the major protein using monoclonal antibodies directed specifically against epitopes that are present only on the M or the L protein. These results show that hepatitis B surface antigen envelope proteins synthesized in yeast are able to assemble into structures composed of different polypeptides. This opens the possibility of producing in yeast a variety of particles carrying well-defined amounts of preS epitopes on their surface. Also, one can envisage the production of mixed particles containing different foreign epitopes on their surface, in defined relative abundance, which could be useful for vaccine applications.

Characterization of human haptoglobin cDNAs coding for alpha 2FS beta and alpha 1S beta variants.

A human liver library, derived from a heterozygous (Hp2-1) donor, has been used to isolate cDNA clones coding for the haptoglobin (Hp) alpha 1S beta and alpha 2FS beta variants. DNA sequencing has shown that the two variants are identical except for the alpha F duplicated segment in Hp alpha 2FS beta. Four nucleotide changes have been found between the phenotypically different F and S regions of the Hp alpha 2 gene, resulting in an Asp,Lys/Asn,Glu substitution.

High-level production and isolation of human recombinant alpha 1-proteinase inhibitor in yeast.

The cDNA coding for mature human alpha 1-proteinase inhibitor (alpha 1-PI) has been inserted into a variety of yeast expression vectors. Yeast cells transformed with these plasmids were then assayed for the production of mature, unglycosylated alpha 1-PI. The production level is optimal when the recombinant plasmid carries the TDH promoter, the complete 2mu and the leu2D selection marker. Biologically active recombinant alpha 1-PI can be purified either analytically, by affinity chromatography using a monoclonal antibody, or on a large scale, by a procedure involving precipitation of high-Mr yeast material with polyethylene glycol 3300 followed by successive chromatography on DEAE-agarose, Zn-chelate agarose, kappa-chain agarose, heparin-agarose and aminohexyl-agarose.

The large surface protein of hepatitis B virus is retained in the yeast endoplasmic reticulum and provokes its unique enlargement.

The coding sequences for each of the three envelope proteins of hepatitis B virus (HBV), the major (S), middle (M), and large (L) surface proteins, were expressed in Saccharomyces cerevisiae. Analysis by immunoelectron microscopy of thin sections of yeast cells showed that production of L protein but not of M or S protein provoked morphological changes in the yeast endoplasmic reticulum. A large accumulation of membranous structures connected with the perinuclear cysternae and specifically labeled by a monoclonal antibody directed against the amino-terminal (preS1) sequence of the L protein, was observed. The L protein was post-translationally modified by N- and O-linked glycosylation, indicative of its entry into the yeast secretory pathway and by N-myristoylation of its amino-terminal glycine residue. Deletion of this glycine residue resulted in the synthesis of a nonmyristoylated L protein. Proliferation of the endoplasmic reticulum was comparable in cells producing either the myristoylated or nonmyristoylated L protein, indicating that myristoylation alone is not responsible for the induction of the abnormal membrane morphology.

Immunoelectron microscopic detection of the hepatitis B virus major surface protein in dilated perinuclear membranes of yeast cells.

The major surface protein of hepatitis B virus produced in Saccharomyces cerevisiae can be recovered from cell lysates in the form of 22-mm lipoprotein particles. Immunoelectron microscopy was applied to investigate site and time of particle assembly. Thin sections of yeast cells revealed that production of the S protein provoked a dilation of the endoplasmic reticulum. Dilated areas were specifically labeled with a polyclonal antibody raised against glutaraldehyde-treated yeast-derived HBsAg particles. In contrast to previous postulates of particle formation during cell lysis and extract preparation, these results suggest that particle formation in yeast occurs in the endoplasmic reticulum and that transport of particles along the secretion pathway is blocked.

Intradural disc herniation associated with epidural gas.

STUDY DESIGN: A case report of a patient suffering from an intradural herniated disc associated with the presence of epidural gas. OBJECTIVE: To advise spine surgeons of the possible association of intradural disc herniation and epidural gas, to prevent overlooking intradural disc fragments during surgery. SUMMARY OF BACKGROUND DATA: Three cases of this rare association were published previously, something surprising given the relatively rare occurrence of intradural herniations and the presence of epidural gas. METHODS: A case is presented where such an association occurred, on the basis of preoperative examinations and intraoperative findings. The literature is reviewed for cases of herniated discs associated with epidural gas and for intradural herniations. RESULTS: During the open discectomy, after a negative epidural examination, an intradural examination was performed, revealing a disc herniation, which was removed. The patient's postoperative development was satisfactory. CONCLUSION: The possibility of an intradural herniated disc must always be considered when performing an open discectomy on a patient whose computed tomography scan reveals the presence of epidural gas. In the event that no clear disc herniation is found to justify the clinical symptoms or the previous radiologic findings, an intradural exploration may be indicated.

L5 root compression caused by degenerative spinal stenosis of the L1-L2 and L2-L3 spaces.

STUDY DESIGN: A severe bilateral L5 root lesion associated with spinal stenosis at L1-L2 and L2-L3 is described. OBJECTIVE: To describe clinical findings and the difficulty in obtaining a correct diagnosis of L5 Root Compression. SUMMARY OF BACKGROUND DATA: The disorder reported in this study has not been reported previously. Only one similar case has been described in the literature: an L5 root compression at L1-L2 caused by disc herniation. METHODS: Diagnosis was obtained by using computed tomography scanning, magnetic resonance imaging, and computed tomography myelography. The findings at L5-S1 were minimal to justify the patient's clinical symptoms, but a detailed study of the upper levels revealed spinal stenosis at L1-L2 and L2-L3, which could have been causing L5 and S1 root compression. A decompressive laminectomy and partial facetectomy in both levels were performed. RESULTS: The patient's pain and claudication disappeared, and clinical symptoms associated with the right L5 root improved. The left L5 root deficit remained stable. CONCLUSION: An unusual case of L5 root compression caused by degenerative stenosis of L1-L2 and L2-L3 is described.

Identification of a family of streptococcal surface proteins with extremely repetitive structure.

The group B Streptococcus (GBS) causes the majority of life-threatening bacterial infections in newborn children. Most GBS strains isolated from such infections express a surface protein, designated Rib, that confers protective immunity and therefore is of interest for analysis of pathogenetic mechanisms. Sequence analysis demonstrated that Rib has an exceptionally long signal peptide (55 amino acid residues) and 12 repeats (79 amino acid residues each) that account for >80% of the sequence of the mature protein. The repeats are identical even at the DNA level, indicating that an efficient mechanism operates to maintain a highly repetitive structure in Rib. The structure of Rib is similar to that of alpha, a previously characterized surface protein that is common among GBS strains lacking Rib. However, highly purified preparations of Rib and alpha did not cross-react immunologically, although the two proteins show extensive amino acid residue identity (47% in the repeat region). When analyzed in Western blots, Rib and alpha give rise to a regularly spaced ladder pattern, apparently due to hydrolysis of acid-labile Asp-Pro bonds in the repeats. We conclude that Rib and alpha are members of a novel family of streptococcal surface proteins with unusual repetitive structure.

Construction and characterization of a Saccharomyces cerevisiae strain (RIT4376) expressing hepatitis B surface antigen.

A host/vector system suitable for large-scale production of HBsAg has been constructed and optimized in terms of the expression plasmid and yeast host strain in order to permit fermentation to very high cell densities. The final expression plasmid contains the coding sequence of the major HBsAg protein (P24) flanked by the promoter sequences from a glycolytic gene and by the transcription-termination region of the ARG3 gene. The host/vector system was found to be genetically stable under large-scale fermentation conditions as demonstrated by nucleotide sequencing and restriction mapping experiments. The P24 protein is recovered from yeast as particles whose physiochemical properties are very similar to those of plasma-derived HBsAg.

Cooperative control of translation fidelity by ribosomal proteins in Escherichia coli. I. Properties of ribosomal mutants whose resistance to neamine is the cumulative effect of two distinct mutations.

Two spontaneous mutants of Escherichia coli strain KMBL-146 selected for resistance to the aminoglycoside antibiotic neamine show severe restriction of amber suppressors in vivo. Purified ribosomes from the mutant strains exhibit low neamine-induced misreading in vitro and a decreased affinity for the related antibiotic streptomycin. Biochemical analysis shows that the mutants each have two modified 30S ribosmal proteins, S12 and S5. In agreement with these results, genetic analysis shows that two mutations are present, neither of which confers resistance to neamine by itself; the mutation located in gene rpxL (the structural gene for protein S12) confers streptomycin dependence but this dependence is suppressed in the presence of the second mutation, located in gene rpxE (the structural gene for protein S5).

Resistance to the aminoglycoside antibiotic neamine in Escherichia coli. A new mutant whose NeaR phenotype results from the cumulative effects of two distinct mutations.

A spontaneous mutant of Escherichia coli (strain AB2847), selected for resistance to the aminoglycoside antibiotic neamine, shows severe restriction of amber suppressors in vivo. Ribosomes isolated from the mutant exhibit only low misreading in vitro in the presence of the antibiotic. Genetic and biochemical analyses indicate that the neamine-resistant phenotype is the result of two distinct mutations. The first, res3128, appears to affect the gene (strA) coding for the ribosomal protein S12. Although it leads to a restrictive phenotype it does not, however, confer resistance to streptomycin. The second mutation, X3128, is located between the sirA and AROB loci and is lethal when segregated from the res3128 mutation. It may affect the ribosome at the level of a post-translational modification.

Phage Mu-1 mediated transposition: a tool to study the organization of ribosomal protein genes in Escherichia coli.

Phage Mu-1 mediated transposition has been used to map genes coding for ribosomal proteins and elongation factor G inside transcriptional units. The data indicate that 1) the str A and fusA genes belong to the same operon, 2) the spcA and strA genes are expressed independently, 3) the spcA gene is located in a different transcriptional unit to that of the eryA and eryB genes.