In a lacZ expression vector (pMC1403Plac), all 64 codons were introduced immediately 3′ from the AUG initiation codon. The expression of the second codon variants was measured by immunoprecipitation of the plasmid‐coded fusion proteins. A 15‐fold difference in expression was found among the codon variants. No distinct correlation could be made with the level of tRNA corresponding to the codons and large differences were observed between synonymous codons that use the same tRNA. Therefore the effect of the second codon is likely to be due to the influence of its composing nucleotides, presumably on the structure of the ribosomal binding site. An analysis of the known sequences of a large number of Escherichia coli genes shows that the use of codons in the second position deviates strongly from the overall codon usage in E. coli. It is proposed that codon selection at the second position is not based on requirements of the gene product (a protein) but is determined by factors governing gene regulation at the initiation step of translation.

Infection of cattle with the bovine leukemia virus (BLV) results in a strong permanent antibody response to the BLV antigens some weeks after infection. However, cattle may carry provirus and not have detectable antibody titers. To prove the occurrence of different BLV provirus variants in German cattle and to study the influence of special BLV variants on the immunoreaction, a 444-bp fragment of the env gene of 35 naturally BLV infected animals was analyzed. Seven different groups of BLV provirus variants were found on the basis of restriction fragment length polymorphism. Three BLV provirus variant groups and five additionally sequenced BLV isolates showed a high similarity to BLV provirus isolates from other geographical areas. The variation in nucleotide sequence of the five BLV isolates compared with nine previously sequenced BLV isolates ranged up to 5. 3%. While BLV provirus variant groups A, C, D, E, F, and G were clearly related to agar-gel immunodiffusion test (AGID)- and enzyme-linked immunosorbent assay (ELISA)-positive animals, BLV provirus variant group B was solely found in permanent AGID- and ELISA-negative or in transient ELISA-positive animals. Altogether, these results indicate that special BLV provirus variants may be responsible for atypical forms of BLV infection in cattle.

No abstract

An Aspergillus niger endopolygalacturonase (EC 3.2.1.15) cDNA was expressed in the yeast Saccharomyces cerevisiae. Secretion of the protein into the growth medium was efficiently directed by the fungal leader sequence, and processing occurred at the same site as in Aspergillus. The expression level was significantly enhanced by using a "short" version of the yeast ADHI promoter. An additional increase in the yield of heterologous protein was due to a higher plasmid stability and a rise in plasmid copy number. This was achieved by deleting most of the bacterial sequences from the expression vector. The yeast-derived enzyme showed the same enzymatic and biochemical properties as the fungal polygalacturonase, such as substrate specificity, pH and temperature optima and pI value. The yeast-derived enzyme, however, showed a higher degree of glycosylation and exhibited a more pronounced temperature stability than the fungal enzyme.

The mechanism of surfactant protein (SP)-A-mediated lipid uptake by rat type II pneumocytes was investigated. In the absence of SP-A, freshly isolated type II pneumocytes actively take up very little if any liposomes. Most of the increase with time is independent of energy or temperature but is most likely due to spontaneous exchange of labeled lipids between liposomes and cell membranes. With 5 micrograms/ml SP-A, type II cells actively take up liposomes (244 pmol dipalmitoylphosphatidylcholine.h-1.10(6) cells-1). The effect of SP-A on uptake is temperature dependent and can be abolished by ATP depletion of the cells. Coincubation with an auto-anti-idiotypic antibody against the SP-A-binding protein BP55 on the cell membrane of type II pneumocytes inhibits SP-A-mediated lipid uptake by type II cells. With increasing amounts of extracellular SP-A present, increasing amounts of liposomes are taken up and directed toward a nondegrading compartment. We suggest that SP-A-mediated surfactant lipid uptake is a receptor-mediated endocytotic process involving BP55.

Type II pneumocytes, which synthesize, store, and secrete pulmonary surfactant, require exogenous fatty acids, in particular palmitic acid, for maximum surfactant synthesis. The uptake of palmitate by type II pneumocytes is thought to be protein mediated, but the protein involved has not been characterized. Here we show by RT-PCR and Northern blot analysis that rat type II pneumocytes express the mRNA for fatty acid translocase (FAT/CD36), a membrane-associated protein that is known to facilitate the uptake of fatty acids into adipocytes. The deduced amino acid sequence from rat type II pneumocytes reveals 98% identity to the FAT/CD36 sequence obtained from rat adipocytes. The uptake of palmitate by type II pneumocytes follows Michaelis-Menten kinetics (Michaelis-Menten constant = 11.9 ± 1.8 nM; maximum velocity = 62.7 ± 5.8 pmol ⋅ min−1 ⋅ 5 × 105pneumocytes−1) and decreases reversibly under conditions of ATP depletion to 35% of control uptake. Incubation of cells at 0°C inhibited the uptake of palmitate almost completely, whereas depletion of potassium was without effect. Preincubation of the cells with bromobimane or phloretin decreases the uptake of palmitate significantly as does preincubation with sulfo- N-succinimidyl oleate, the specific inhibitor of FAT/CD36 (C. M. Harmon, P. Luce, A. H. Beth, and N. A. Abumrad. J. Membr. Biol. 121: 261–268, 1991). From these data, we conclude that FAT/CD36 is expressed in type II pneumocytes and mediates the uptake of palmitate in a saturable and energy-dependent manner. The data suggest that the uptake process is independent of the formation of coated pits and endocytotic vesicles.

No abstract

Using a previously described vector (pKL203) we fused several heterologous ribosomal binding sites (RBSs) to the lacZ gene of E. coli and then studied the variation in expression of the fusions. The RBSs originated from bacteriophage Q beta and MS2 genes and the E. coli genes for elongation factor EF-Tu A and B and ribosomal protein L11 (rplK). The synthesis of the lacZ fusion proteins was measured by an immuno precipitation method and found to vary at least 100-fold. Lac-specific mRNA synthesis follows the variation in protein production. It appears that there is a correlation between the efficiency of an RBS to function in the expression of the fused gene and the lack of secondary structure, involving the Shine and Dalgarno nucleotides (SDnts) and/or the initiation codon. This efficiency is context dependent. The sequence of the SD nts and the length and sequence of the spacer region up to the initiation codon alone are not able to explain our results. Deletion mutations, created in the phage Q beta replicase RBS, reveal a complex pattern of control of expression, probably involving the use of a "false" initiation site.