Synthesis and degradation of termination and premature-termination fragments of beta-galactosidase in vitro and in vivo.

A number of abnormal polypeptides which are products of the lacZ gene in Escherichia coli have been characterized. A variety of experiments indicates that one class of at least nine different fragments arises from premature termination of protein synthesis. They have been detected as products of protein synthesis both in vitro and in vivo, although the percentage of fragments made in vitro is greater than that in vivo. The percentage of total Z gene-encoded protein which is found as fragments is estimated to be roughly 23% in vivo. This means that about 31% of the total number of β-galactosidase monomers synthesized are prematurely terminated molecules. The average molecular weight of the polypeptides produced is 91,000. This corresponds to a termination event occurring on the average once every 3200 codons. The sites at which termination occurs appear to be specific and are located primarily near the 3′-end of the gene.Polypeptides synthesized in vitro and in vivo from templates containing mutations in the Z gene have also been compared. Most of the mutationally generated fragments synthesized in vitro are stable, unlike their in vivo counterparts, which are often rapidly degraded. One fragment, however, generated by an early amber mutation in the Z gene, is degraded in the in vitro system. The mechanism of degradation appears to be specific for small abnormal polypeptides. Internal reinitiation polypeptides generated by nonsense mutations, which have been found in vivo, are not detected in the in vitro protein synthesis system under the conditions used here.     

Hfq variant with altered RNA binding functions

The interaction between Hfq and RNA is central to multiple regulatory processes. Using site-directed mutagenesis, we have found a missense mutation in Hfq (V43R) which strongly affects2 the RNA binding capacity of the Hfq protein and its ability to stimulate poly(A) tail elongation by poly(A)-polymerase in vitro. In vivo, overexpression of this Hfq variant fails to stimulate rpoS–lacZ expression and does not restore a normal growth rate in hfq null mutant. Cells in which the wild-type gene has been replaced by the hfqV43R allele exhibit a phenotype intermediate between those of the wild-type and of the hfq minus or null strains. This missense mutation derepresses Hfq synthesis. However, not all Hfq functions are affected by this mutation. For example, HfqV43R represses OppA synthesis as strongly as the wild-type protein. The dominant negative effect of the V43R mutation over the wild-type allele suggests that hexamers containing variant and genuine subunits are presumably not functional. Finally, molecular dynamics studies indicate that the V43R substitution mainly changes the position of the K56 and Y55 side chains involved in the Hfq–RNA interaction but has probably no effect on the folding and the oligomerization of the protein.     

Requirement for Phe36 for DNA binding and mismatch repair by Escherichia coli MutS protein

The MutS family of DNA repair proteins recognizes base pair mismatches and insertion/deletion mismatches and targets them for repair in a strand-specific manner. Photocrosslinking and mutational studies previously identified a highly conserved Phe residue at the N-terminus of Thermus aquaticus MutS protein that is critical for mismatch recognition in vitro. Here, a mutant Escherichia coli MutS protein harboring a substitution of Ala for the corresponding Phe36 residue is assessed for proficiency in mismatch repair in vivo and DNA binding and ATP hydrolysis in vitro. The F36A protein is unable to restore mismatch repair proficiency to a mutS strain as judged by mutation to rifampicin or reversion of a specific point mutation in lacZ. The F36A protein is also severely deficient for binding to heteroduplexes containing an unpaired thymidine or a G:T mismatch although its intrinsic ATPase activity and subunit oligomerization are very similar to that of the wild-type MutS protein. Thus, the F36A mutation appears to confer a defect specific for recognition of insertion/deletion and base pair mismatches.     

Synthesis, transport and localization of a nuclear coded 22-kd heat-shock protein in the chloroplast membranes of peas and Chlamydomonas reinhardi

The synthesis, transport and localization of a nuclear coded 22-kd heat-shock protein (HSP) in the chloroplast membranes was studied in pea plants and Chlamydomonas reinhardi. HSPs were detected in both systems by in vivo labeling and in vitro translation of poly(A)⁺RNA, using the wheat-germ and reticulocyte lysate systems. Heat-shock treatment of pea plants for 2 h at 42-45°C induces the expression of ˜10 nuclear coded proteins, among which several (18 kd, 19 kd, 22 kd) are predominant. A 22-kd protein is synthesized as a 26-kd precursor protein and is localized in a chloroplast membrane fraction in vivo. Following post-translational transport into intact chloroplasts in vitro of the 26-kd precursor, the protein is processed but the resulting 22-kd mature protein is localized in the chloroplast stroma. If, however, the in vitro transport is carried out with chloroplasts from heat-shocked plants, the 22-kd protein is preferentially transported to the chloroplast membrane fraction. In C. reinhardi the synthesis of poly(A)⁺RNAs coding for several HSPs is progressively and sequentially induced when raising the temperature for 1.5 h from 36°C to 42°C, while that of several preexisting RNAs is reduced. Various pre-existing poly(A)⁺RNAs endure in the cells at 42°C up to 5 h but are no longer translated in vivo, whereas some poly(A)⁻RNAs persist and are translated. As in pea, a poly(A)⁺RNA coded 22-kd HSP is localized in the chloroplast membranes in vivo, although it is translated as a 22-kd protein in vitro. The in vitro translated protein is not transported in isolated pea chloroplast which, however, processes and transports other nuclear coded chloroplast proteins of Chlamydomonas. The poly(A)⁺RNA coding for the 22-kd HSP appears after 1 h at 36°C. Its synthesis increases with the temperature of incubation up to 42°C, although it decreases after ˜2 h of heat treatment and the already synthesized RNA is rapidly degraded. The degradation is faster upon return of the cells to 26°C. None of the heat-induced proteins is identical to the light-inducible proteins of the chloroplast membranes.     

Translation of bacteriophage R17 and Qbeta RNA in a mammalian cell-free system

The polycistronic RNAs from both bacteriophage R17 and Qβ are translated in a mammalian cell-free system of purified and partially purified components. The requirement of one of the partially purified initiation factors (IF-E₃ from rabbit reticulocytes) for the phage RNA translation is strikingly different from that for rabbit globin messenger RNA translation. The phage RNA-directed products are characterized by acrylamide gel electrophoresis and compared with those synthesized in an Escherichia coli cell-free system. There is good agreement between the respective coat proteins and the presumptive synthetase proteins. R17 RNA directs the synthesis of two additional defined polypeptides. However, their possible relationship with the A-protein cistron has not yet been investigated. The RNA from the amB2 mutant of R17, which carries an amber triplet at position 6 in the coat protein cistron, directs the synthesis of the same polypeptides as the wild-type RNA with the exception of the coat protein which is completely abolished. This identifies the product made with wild-type RNA as coat protein and provides a direct in vitro assay for the suppression of nonsense mutations in eukaryotic cells.     

An rne-1 pnp-7 Double Mutation Suppresses the Temperature-Sensitive Defect of lacZ Gene Expression in a divE Mutant

A divE mutant, which has a temperature-sensitive mutation in the tRNA₁^Ser gene, exhibits differential loss of the synthesis of certain proteins, such as β-galactosidase and succinate dehydrogenase, at nonpermissive temperatures. In Escherichia coli, the UCA codon is recognized only by tRNA₁^Ser. Several genes containing UCA codons are normally expressed after a temperature shift to 42°C in the divE mutant. Therefore, it is unlikely that the defect in protein synthesis at 42°C is simply caused by a defect in the decoding function of the mutant tRNA₁^Ser. In this study, we sought to determine the cause of the defect in lacZ gene expression in the divE mutant. It has also been shown that the defect in lacZ gene expression is accompanied by a decrease in the amount of lacZ mRNA. To examine whether inactivation of mRNA degradation pathways restores the defect in lacZ gene expression, we constructed divE mutants containing rne-1, rnb-500, and pnp-7 mutations in various combinations. We found that the defect was almost completely restored by introducing an rne-1 pnp-7 double mutation into the divE mutant. Northern hybridization analysis showed that the rne-1 mutation stabilized lacZ mRNA, whereas the pnp-7 mutation stabilized mutant tRNA₁^Ser, at 44°C. We present a mechanism that may explain these results.     

Specific protein synthesis in isolated epithelium of guinea-pig seminal vesicle. Effects of castration and androgen replacement

Four intrinsic soluble secretory proteins are synthesized in vitro by isolated seminal-vesicle mucosa from sexually mature guinea pigs. Newly synthesized specific proteins labelled with [¹⁴C]glycine and [¹⁴C]lysine were precipitated by using double-antibody immunoprecipitation techniques and their radioactivity was assessed. Rates of synthesis were determined on each of 5 days after castration. By 5 days after castration the wet weight of the epithelium decreased to 42% of intact control values; the absolute amount of specific protein synthesized in vitro after 60min incubation decreased to 28% and the 27500g cytoplasmic protein content decreased to 31%. Thus androgen deprivation leads to a decrease in general protein synthesis in vivo, as well as to a decrease in specific protein synthesis in vitro. Specific protein synthesis comprised 76% of the total protein formed in isolated tissue from animals 5 days after castration as compared with 99–100% in tissue from intact animals. At 72h after an injection of testosterone or dihydrotestosterone, seminal-vesicle epithelium wet weight, cytoplasmic protein content and capability for synthesizing specific proteins in vitro were restored to approx. 70% of normal values. At 72h after onset of therapy with 3α-androstanediol, both epithelium wet weight and cytoplasmic protein content had increased significantly, but without a corresponding increase in the capability of the isolated tissue to synthesize specific proteins. The soluble labelled proteins synthesized in vitro by isolated epithelium from intact animals during 60 or 120min incubation were essentially entirely immunoprecipitable, i.e. specific. In contrast, approx. 29% of all soluble protein newly synthesized by isolated epithelium from animals 5 days after castration was acid-precipitable, but not immunoprecipitable, i.e. `non-specific'. The injection of testosterone into castrated animals inhibited the synthesis of the non-specific fraction by isolated tissue. The effects of castration on the ultrastructure of guinea-pig seminal-vesicle epithelium are also presented.     

Plant Desiccation and Protein Synthesis : VI. Changes in Protein Synthesis Elicited by Desiccation of the Moss Tortula ruralis are Effected at the Translational Level

Upon rehydration of the moss Tortula ruralis following desiccation at a rapid or slow rate, there is increasing utilization of newly synthesized-poly(A)⁺ RNA for protein synthesis. Initially, poly(A)⁺ RNA conserved in the dry moss is associated with polysomes, but by 2 hours of rehydration there is an overwhelming recruitment of newly synthesized poly(A)⁺ RNA, at the expense of conserved messages. In rehydrated moss, there is a marked synthesis in vivo of new proteins, which are separable by two-dimensional electrophoresis, and identifiable by fluorography. These new proteins, termed rehydration proteins, are synthesized after both rapid and slow desiccation, but their synthesis persists longer after rapid desiccation. The protein patterns obtained following in vitro translation of bulk RNA from hydrated, desiccated, and rehydrated moss were qualitatively identical. Thus the differences in protein patterns observed in vivo must result from preferential selection of specific mRNAs from the same pool, which is indicative of control of protein synthesis at the translational level. The implications of these observations in relation to the response of the moss to drying in its natural environment are discussed.     

The synthesis of hemolymph proteins by the larval midgut of an insect Calpodes ethlius (Lepidoptera:Hesperiidae)

Ligated tubes of Calpodes ethlius (Lepidoptera:Hesperiidae) larval midgut with normal (i.e. apical secretions into the lumen and basal into the hemocel or medium) or inverted orientation (i.e. apical secretions into the hemocoel or medium and basal into the lumen) were incubated in artificial hemolymph in the presence of [³⁵S]methionine to investigate protein synthesis and vectorial secretion. The midgut synthesizes and secretes at least eight polypeptides basally and seven apically. The tissue also synthesizes many other polypeptides that are not released at either surface. Two dimensional analysis of proteins labeled in vitro and in vivo showed that (a) proteins synthesized in vitro are the same as those synthesized in vivo, (b) different proteins are secreted on apical and basal surfaces, (c) in vitro apical secretions are the same as in vivo luminal proteins, (d) at least two of the basal secretions can be demonstrated in the hemolymph labeled in vivo. Almost all basal secretions showed immunological similarity with hemolymph proteins as observed by immunoprecipitation and fluorography. Arylphorin is a main hemolymph protein synthesized by the midgut. Midgut arylphorin has been identified by its precipitation by antibodies to hemolymph arylphorin. We conclude that insect midgut has bi-directional secretion. Luminal proteins (presumably digestive enzymes and perhaps goblet cell luminal contents) are carried to the apical face. A different set of proteins are carried basally to the hemolymph.     

Analysis of specific misreading in Escherichia coli

The pattern of specific misreading by nonsense suppressors has been investigated using nonsense mutants in the rIIB gene of phage T4 and in the lacZ gene of Escherichia coli. It is shown that a su⁺ transfer RNA which reads UAG also misreads UAA but not UGA, a su⁺ tRNA which reads UAA (while it also reads UAG by wobble) misreads UGA and a su⁺ tRNA which reads UGA also probably misreads UAA but not UAG.These specific types of errors in translation occur in the absence of streptomycin. The addition of the drug raises their level without altering the pattern described. A ribosomal mutation str A reduces the level of specific misreading; by contrast, a ram mutation strongly increases this level. In all cases the specific pattern is not affected.The rate of specific misreading of nonsense codons in different cases ranges from less than 0.001% to more than 3%. Since the frequency of misreading is sitespecific (unpublished observations), the rates obtained cannot be extrapolated to any other codon at any other site.     

Pre-selection of integration sites imparts repeatable transgene expression

Variable gene expression amongst transgenic lines occurs due to copy number and to random associations of incoming DNA with chromosomal elements at the site of integration. Here we describe a method of identifying sites permissive for transgene expression and their use for efficient introduction of single copy transgenes by homologous recombination. ES clones were selected in HAT medium for expression of a randomly integrated HPRT marker lying 5′ to an Oct4/lacZ transgene. 794 clones were assessed in vitro for appropriate down-regulation of lacZ following differentiation. Two clones were chosen for further analysis which displayed appropriate and inappropriate gene regulation (clones 710 and 91, respectively). Three developmental promoters (thyroglobulin, Hox2.6 and Myf5) were then sequentially introduced into the original insertion sites in each clone (710 and 91) by homologous recombination, to drive expression of lacZ. Transgenic embryos were assessed for their ability to direct lacZ expression to tissues in which the respective promoter sequences are normally active. The site which appropriately down-regulated lacZ in vitro (710) also showed appropriate in vivo regulation of lacZ from the three developmental promoters. Site 91, however, directed an additional pattern of ectopic expression, which was common to all four promoters. Pre-selection of genomic sites for the introduction of transgenes by gene targeting improves the repeatability of transgene expression and provides an efficient means of single copy transgene introduction by homologous recombination.     

Development of preimplantation rabbit embryos in vivo and in vitro

Qualitative patterns of protein synthesis in preimplantation rabbit embryos grown in vivo and in vitro were examined by SDS polyacrylamide gel electrophoresis followed by autoradiography. The results demonstrate that (1) most qualitative changes in the pattern of protein synthesis occur during cleavage, (2) the blastocyst period of development is characterized by a remarkably uniform and constant pattern of protein synthesis, and (3) the qualitative pattern of protein synthesis in embryos cultured in vitro from the 1-cell to the blastocyst stage is essentially identical to the pattern of protein synthesis in embryos grown to a comparable stage in vivo.These results indicate that no “special” maternal factors, such as uterine proteins, are required in vitro either for the qualitative changes in the pattern of protein synthesis during cleavage, or for the initial expression of a pattern of protein synthesis characteristic of the entire blastocyst period. From these studies we conclude that, once fertilized, the rabbit egg proceeds through cleavage and blastocyst formation on its own endogenous developmental program.     

Initiation of Mammalian Viral Protein Synthesis

CULTURED human cells (KB) infected with human adenovirus type 2 (Ad 2) provide a model system for protein synthesis in mammalian cells. Adenovirus messenger RNA molecules are transcribed from nuclear viral DNA and transported to the cytoplasm for translation¹. Late after infection (18 h) 9–10 viral mRNA species with sedimentation values of 7S to 32S are present in polysomes (Parsons, Gardner and Green, in preparation) and specify eight viral structural proteins which account for 80–90% of the polypeptides synthesized in vivo^2–4. We now describe an in vitro cell-free system, derived from KB cells infected with Ad 2, which synthesizes 8–9 viral polypeptides and can initiate protein synthesis with a special class of yeast methionyl-tRNA. In vivo and in vitro experiments suggest that methionine is the initiator amino-acid for most, if not all, adenovirus structural proteins.     

Translation in vitro of Drosophila heat-shock messages.

Polysomal RNA from heat-shocked Drosophila tissue culture cells directs the in vitro synthesis of proteins which appear to be the same as heat-shock proteins made in vivo. Each message is about 1.5 times larger than the minimum size needed to code for the protein it specifies. In situ hybridization indicates a correspondence between messages for some of the proteins and particular chromosomal sites.     

Analysis of the coding activity and stability of messenger RNA in Drosophila oocytes.

The coding activity of the messenger RNA in the ooplasm of late stage 14 (S14) oocytes of Drosophila melanogaster was analyzed by labeling the oocytes in vitro with [³⁵S]methionine and examining the labeled products by two-dimensional gel electrophoresis and fluorography. This analysis was done both with newly formed S14 oocytes from rapidly laying females and with S14 oocytes stored for about 10 days in females that were prevented from laying. Comparison of the fluorographs showed that the proteins labeled in the newly formed oocytes were also labeled in the stored oocytes. Thus, the coding activity of S14 oocyte messenger RNA appears to remain stable during prolonged storage in utero. The oocyte proteins synthesized during oogenesis and incorporated into S14 oocytes were labeled in vivo by injecting [³⁵S]methionine into newly eclosed females, and the S14 oocytes were removed 2 days later for gel electrophoresis and fluorography. Comparison of the fluorographs produced by the in vivo and in vitro labeling procedures showed that most of the oocyte proteins labeled in vivo were also labeled in vitro. The S14 oocytes, therefore, appear to contain messenger RNA for most of the oocyte proteins synthesized during oogenesis. There were also several additional proteins detected only in the fluorographs of the in vivo labeled oocytes; the most prominent of these were identified by immunoprecipitation tests as vitellogenin proteins of yolk granules, which are known to be synthesized outside the oocyte, in fat bodies. The occurrence of stable S14 oocyte messenger RNA for most of the oocyte proteins suggests that the synthesis of those proteins during oogenesis occurs in the developing oocytes, specified by a stable population of oocyte messenger RNA.