Molecular cloning and expression in Escherichia coli of the cDNA coding for rat lipocortin I (calpactin II)

Lipocortins (LC) are a family of proteins that were initially described to be induced by glucocorticosteroids and to inhibit phospholipase A2 (PLA2). Using oligodeoxynucleotide probes corresponding to partial amino acid (aa) sequences of rat lipocortin I (LCI), we have isolated a cDNA clone for rat LCI from a cDNA library prepared from poly(A)+RNA of peritoneal cells of dexamethasone-treated rat. The cDNA insert (1355 bp) had an open reading frame of 1038 bp that encoded a 346-aa polypeptide (Mr 38,784). The nucleotide sequence and the amino acid sequence deduced from it showed high homology with the reported sequences of human LCI. A plasmid containing the trc promoter and cDNA sequence for 346 aa residues of the rat LCI was constructed and expressed in Escherichia coli. Antibody to human LCI crossreacted with the recombinant rat LCI, and the recombinant protein had characteristics of natural rat LCI including PLA2 inhibitory activity in vitro.     

Protein synthesis by long-lived messenger ribonucleic acid in bacteria

1. Experiments were performed to investigate two hypotheses about the function of long-lived messenger RNA in bacteria. After RNA synthesis had been stopped by the addition of actinomycin, continuing protein synthesis was used as a measure of persistent messenger RNA. 2. The hypothesis that messenger RNA responsible for the synthesis of membrane protein is exceptionally long-lived was tested in experiments with protoplasts of Bacillus megaterium. However, this messenger RNA proved to be of approximately average stability. 3. The hypothesis that long-lived messenger RNA is responsible for the synthesis of constitutive proteins was tested by comparing the synthesis of penicillinase in an inducible and a constitutive strain of Bacillus licheniformis. After the addition of actinomycin, penicillinase synthesis continued for far longer in the constitutive than in the inducible strain. This difference is attributed to a difference in stability of the penicillinase-messenger RNA in the two strains, which does not extend to all messenger RNA indiscriminately. 4. A model is tentatively proposed to account for the altered stability of messenger RNA in the constitutive mutant.     

Sequence content of oligo(uridylic acid)-containing messenger ribonucleic acid from HeLa cells

Oligo(uridylic acid)-containing [oligo(U+)] RNA was isolated from poly(adenylic acid)-containing [poly(A+)] mRNA from HeLa cells by using either formaldehyde pretreatment or poly(A) removal, both of which resulted in increased accessibility of oligo(U)-rich sequences to a poly(A)-agarose affinity column. In this report, we compared the sequence content of oligo(U+) RNA with that of molecules lacking oligo(U) [oligo(U-) RNA] by their relative hybridization to cDNA reverse-transcribed from poly(A+) mRNA and by comparison of their in vitro translation products synthesized in a rabbit reticulocyte lysate. Formaldehyde-modified poly(A+) RNA, treated to remove the formol adjuncts, was inactive as a template for in vitro protein synthesis; consequently, only depolyadenylated RNA, which retains its translatability, could be used in the translation studies. The hybridization kinetic experiments revealed that oligo(U+) RNA contained most of the sequence information present in oligo(U-) RNA but at a reduced level (ca. 25%), the majority of the oligo(U+) RNA sequences being poorly represented in the cDNA. This result was supported by one- and two-dimensional gel analysis of their in vitro translation products which showed that oligo(U+) RNA, although less effective as a template for translation than oligo(U-) RNA, coded for proteins, the most abundant of which were encoded by rare messages not highly represented in oligo(U-) RNA or the total poly(A+) RNA. Although some minor products were synthesized by both oligo(U+) and oligo(U-) RNA, at least 33 proteins were unique to or highly enriched in the pattern of products directed by oligo(U+) RNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Annexin II (calpactin I) in the mouse mammary gland: immunolocalisation by light- and electron microscopy

Summary To elucidate the putative role of annexin II (calpactin I) in the secretory function of mammary tissue its immunolocalisation in the mammary gland of pregnant and lactating mice was investigated by light- and electron microscopy using the immunoperoxidase technique. A low level of fairly uniform annexin II staining was evident throughout the gland despite its mixed composition during pregnancy. In lactating tissue it was revealed that apparently mature alveoli contained a concentration of annexin II staining outlining their epithelium. The staining was localised by immuno-electron microscopy to the apical membrane of these alveolar epithelial cells and their microvillar extentions. There was also an apparent association of annexin II with vesicles of a range of sizes located near, or actually fused with, the apical membrane. Many of the small, stained vasicles could clearly be identified as casein-containing vesicle while the large vesicles were apparently associated with either casein granules or possibly lipid. The appearance of a selective concentration of annexin II in apparently actively secreting mammary epithelial cells, as revealed in this study, is consistent with a possible structural and/or functional role for this protein at the membranes participating in the secretion of protein and possibly lipid from these secretory cells.     

Regulation of aspartate aminotransferase messenger ribonucleic acid level by testosterone

The effect of testosterone on precursor mitochondrial aspartate aminotransferase (pmAAT) mRNA was studied in rat ventral prostate and primary cell cultures of mini-pig prostate. Testosterone induced a 2-3-fold increase in pmAAT mRNA level in both rat ventral prostate and mini-pig prostate cultures. The pmAAT mRNA induction occurred 30 min after testosterone treatment and was maximal by 1.5 h. Prostatic mAAT activity was also induced by testosterone with a 1-2 h lag period. The time-course of induction of pmAAT mRNA, pmAAT activity and mAAT activity was consistent with stimulation of mRNA synthesis followed by increased synthesis and import of pmAAT into mitochondria. The effect of testosterone on pmAAT mRNA was specific because the increase in pmAAT mRNA was at least 2-fold greater than the increase in poly (A+) RNA. These results suggest that testosterone stimulated mAAT activity by induction of pmAAT mRNA. This continues to support our proposal that a major physiological effect of testosterone is increased pmAAT mRNA steady-state levels which result in increased pmAAT synthesis and increased mAAT activity. These changes ultimately result in increased citrate production by prostate epithelial cells.     

Location of oligo(uridylic acid) sequences within messenger ribonucleic acid molecules of HeLa cells

A significant fraction of the polyadenylated mRNAs of HeLa cells contain an oligo(uridylic acid) [oligo(U)] sequence of 15-30 nucleotides. Several different experimental approaches were used to determine if these oligo(U)'s occupied similar sites within all mRNAs. In one approach, poly(adenylic acid)-containing mRNAs [poly(A+) mRNAs] averaging 2800 nucleotides in length were reduced to an average size of 500 nucleotides by controlled alkaline hydrolysis. Over 20% of the oligo(U)-containing fragments isolated from the hydrolysate retained a poly(A) sequence, showing that oligo(U)'s were not exclusively located near 5' ends of mRNA although 20% were apparently close to 3' ends. To confirm these observations, oligo(U)-containing mRNA [oligo(U+) mRNA] was exposed to the 3'-exonucleolytic activity of polynucleotide phosphorylase to produce fragments containing the 5' regions of mRNA. Each of a set of fragments of decreasing length generated by increased times of exposure of the mRNAs to the enzyme was found to have about the same oligo(U) content, including the shortest that averaged 550 nucleotides. These data not only eliminated an exclusive location for oligo(U) in either 3' or 5' ends of mRNA but also suggested that oligo(U)'s might be close to the 5' ends of some mRNAs. To verify this last observation, periodate-oxidized poly(A+) mRNA was labeled at the 5' caps and at 3'-adenosine residues by sodium [3H]borohydride reduction before it was nicked 3-5 times with alkali to produce 5' and 3' end-labeled pieces that could be separated with oligo(thymidylic acid)-cellulose.(ABSTRACT TRUNCATED AT 250 WORDS)     

5'-Terminal cap structures of oligo(uridylic acid)-containing messenger ribonucleic acid from HeLa cells: comparison with other ribonucleic acid subpopulations

The 5'-terminal cap structures of 32P-labeled oligo(uridylic acid)-containing messenger ribonucleic acid [oligo(U+)mRNA] isolated from HeLa cell polyadenylated [poly(A+)] mRNA were analyzed and compared to those of the poly(A+) mRNA. A method employing P1 nuclease, alkaline phosphatase, and adsorption to activated charcoal showed that the types of cap core (m7 GpppXm) in oligo(U+) mRNA were essentially identical with those in poly(A+) mRNA. Analysis of RNase T2 digestion products of oligo(U+) mRNA demonstrated the presence of both cap 1 (m7GpppXmpYp) and cap 2 (m7GpppXmpYmpZp) in this subpopulation, confirming its cytoplasmic location. The base compositions of these two types of caps were different from each other and nonrandom but did not differ significantly between oligo(U+) and poly(A+) m RNA. The only observed difference between the mRNA populations was a higher ratio of cap 1 and cap 2 in the former. Possible implications of these findings for the relationship between oligo(U+) mRNA and poly(A+) mRNA are discussed.     

A method for isolation of oligo(uridylic acid)-containing messenger ribonucleic acid from HeLa cells

When cytoplasmic polyadenylated ribonucleic acid [poly(A+)RNA] from HeLa cells was treated with ribonuclease H (RNase H) and oligodeoxythymidylate [oligo(dT)] to remove its 3'-poly(A) tail, an increased binding to poly(A)-agarose was observed. The bound material, which comprised 4-6% of the initial RNA, contained 65-80% of the oligo(uridylic acid) [oligo(U)] sequences generated by RNase T1 digestion. Oligo(U) isolated from the bound fraction was shown to be 83% U and to have a U/G ratio of 33. In contrast, oligo(U) from the unbound material was 77% U and had a U/G ratio of 13, suggesting that it is shorter and less U rich than the oligo(U) in the bound fraction. On sucrose gradients, oligo(U+)RNA consistently sedimented with a larger s value than oligo(U-) RNA. The oligo(U) content of oligo(U+) RNA suggests one oligo(U) tract of 33 nucleotides per RNA molecule of 2000-3000 residues.     

Isolation and characterization of poly(adenylic acid)-containing messenger ribonucleic acid from rat liver polysomes

Undegraded rat liver polysomes were obtained after homogenizing the tissue in a medium containing NH4Cl, heparine, and yeast tRNA. Purification of poly(A)-containing RNA from polysomal RNA was accomplished by affinity chromatography on oligo(dT)-cellulose columns. Poly(A)-containing RNA molecules were monitored by the formation of ribonuclease-resistant hybrids with [3H]poly(U). To improve the separation of messenger RNA and ribosomal RNA by oligo(dT)-cellulose it was found essential to dissociate the aggregates formed between both molecular species by heat treatment in the presence of dimethylsulfoxide (Me2SO) prior to chromatography. Sucrose gradient analysis under denaturing conditions showed that the preparations obtained were virtually free of ribosomal RNA. Poly(A)-containing RNA constituted approx. 2.2% of the total polysomal RNA and the number average size was 1500--1800 nucleotides, as judged by sedimentation analysis on sucrose density gradients containing Me2SO. Approximately 8.2% of the purified preparation obtained was able to anneal with [3H]poly(U); the number average nucleotide length of the poly(A) segment of the RNA population was calculated to be 133 adenylate residues. Based on these values, our preparations appear to be greater than 90% pure. The RNA fractions obtained after oligo(dT)-cellulose chromatography were used to direct the synthesis of liver polypeptides in a heterologous cell-free system derived from wheat-germ. The system was optimized with respect to monovalent and divalent cations, and presence of polyamines (spermine). More than 65% of the translational activity present in the unfractionated polysomal RNA was recovered in the final poly(A)-containing RNA fraction. However, about 25% of the activity was found to be associated with the unbound fraction which was essentially free of poly(A)-containing RNA. Immunoprecipitation analysis with a specific antiserum to rat serum albumin demonstrated that about 6--8% of the labeled synthetic products translated from the poly(A)-containing RNA sample corresponded to serum albumin. Analysis of the translation products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a heterogeneous distribution of molecular sizes ranging from 15 000 to greater than 70 000 daltons. Spermine not only increased the overall yield and extent of protein synthesis, but also resulted in higher yields of large protein products. Under optimal translation conditions a discrete peak representing about 7% of the total radioactivity was observed to migrate with rat serum albumin.